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HARPER 82 CLIFF ST^T.\n 1833.\n\n\n\n\n NATURAL HISTORY;\n OR,\n UNCLE PHILIP'S\n CONVERSATIONS WITH THE CHILDREN\n ABOUT\n TOOLS AND TRADES\n AMONG\n INFERIOR ANIMALS.\n\n WITH NUMEROUS ENGRAVINGS.\n\n\n NEW-YORK:\n PUBLISHED BY HARPER & BROTHERS.,\n NO. 82 CLIFF-STREET.\n\n 1835.\n\n\n\n\n Entered, according to Act of Congress, in the year 1835,\n By Harper & Brothers,\n In the Clerk's Office of the Southern District of New-York.\n\n\n\n\nADVERTISEMENT.\n\n\nWe must tell our little readers something about this number of their\nLibrary. It was sent to us by a very kind old uncle of ours, who, when\nwe were young, was so much from home, visiting various places in the\nworld, that we do not remember seeing him very often at that period. At\nlast, the old man, finding that he could not bear fatigue as he had done\nwhen young, determined to come home; and we had heard so much about him\nthat we were quite anxious to see him. He came to our house one evening,\nand appeared rather odd to us; but he was so good-natured, and told us\nso many curious things, that we soon forgot his odd appearance.\n\nThe old gentleman brought home with him a very large number of books,\nand a great many strange things which he had gathered in his travels,\nsuch as stones, and dried insects, and leaves, and flowers, and stuffed\nbirds, and animals. He did not stay with us long, but went to the\nvillage where he was born, and built a small house to which he carried\nall his books and curiosities, and said that he should spend the rest of\nhis days there.\n\nWe sometimes pay him a visit. The last time we were there, we found him\ntalking to several children around him. In the beginning of the book\nthere is a picture of the old gentleman. After you have looked at it,\nyou may read the letter which he sent us, and learn how he came to write\nthis book.\n\n Your friends,\n The Publishers.\n\n\n\n\nUNCLE PHILIP'S LETTER.\n\n\n My dear Nephews,\n\nI was very much pleased to receive the numbers of your Library for\nBoys and Girls which you sent to me. You know I am now an old man,\nand have travelled a great deal, and seen a great many strange things\nin the course of my life. I am too old to travel any more, and so I\nam quietly living in the cottage I built by the side of that pleasant\nand shady little stream where I played when I was a boy. I read my\nbooks, and especially that best of all of them, my Bible; and so am\npatiently waiting till my Heavenly Father shall call me to take my last\njourney; when I hope, for the sake of the blessed Saviour, to go to Him.\nSometimes I walk out into the village, and meet the children and have\na long talk with them. They all know me; and very often, some of them\nwill come to my house, and ask me to tell them about things which I have\nseen in my travels or read of in books: and so I spend many happy hours\nwith the little creatures; for you know how much I love children. When\nI had read the books you sent to me, I lent them to the children, who\nwere delighted; and I thought that if I should sometimes write down what\nwe here talked about, it might please the little boys and girls for whom\nyou print your books, and perhaps they might learn something from our\nconversations which would be useful: and so I determined to send them to\nyou, from time to time, to print, if you pleased.\n\nIf you think fit to print what I send, just tell your little readers who\nI am; an aged and quiet old man, who is very fond of little boys and\ngirls, and wishes them to be wise and good here, and happy hereafter,\nand that I am your\n\n Uncle Philip.\n _Newtown, Feb_. 1833.\n\nP.S. If you print what I send now, please to print the Preface to\nParents, which I also send; in order that they may, by reading it,\nsee what sort of a book Uncle Philip has been making for their dear\nchildren, and may be satisfied that it will not harm them to read it.\n\n\n\n\nPREFACE TO PARENTS.\n\n\nThe author of the following book avails himself of the opportunity\nafforded by its publication, to address a word to those who sustain the\ndelightful and responsible relation of parents.\n\nTo such of that class as may honour by a perusal this humble attempt\nto interest and instruct their offspring, the author need not say that\nthe subject of his book possesses for himself peculiar attractions: it\nwill readily be perceived that he has found a charm in the pursuits of\nthe naturalist. The votary of a favourite science would anticipate too\nmuch, should he expect every one to partake of the enthusiasm which is\napt to stimulate him; it is wisely and kindly ordered that we shall\nnot all be enthusiasts in the same direction. The author, however,\nstill ventures to hope, that in his subject there is enough to attract,\nthough it may fail to fascinate. He hopes, too, that it will be found\nnot attractive merely, but profitable also to his young countrymen.\nThere are many reasons on which to found such a hope. If to entertain\nreverence for our Maker, to admire and adore his wisdom and goodness\nin the illustrations of nature, thankfully to acknowledge and duly to\nimprove the superiority which mind confers, be exercises in which a wise\nparent would desire to train a child,--the study of natural science\nis admirably adapted to the attainment of these objects. Again, if it\nbe desirable to encourage habits of patient observation, accuracy of\ninvestigation, and soundness of thought; let the volume of nature be\nopened before the youthful mind. If to learn _things_ be better than\nto learn _words_, it is important to place things before the growing\nintellects of the young. Let it not be supposed that to present matters\nof science intelligibly to the minds of children is a hopeless task. It\nrequires not learning or maturity of understanding to perceive a _fact_;\nit needs only the ordinary senses which God has bestowed alike upon\nchildren and their parents. Natural science is emphatically the science\nof _facts_; built upon any other foundation it becomes conjecture\nmerely: and he knows but little of the mind of a child who is not aware\nof the facility with which a fact is impressed upon it. The secret of\ninstructing the young will be found to consist more in the mode of\ncommunication than in the nature of the subject.\n\nAs to the style of this work a word may be said; not, of course, for\nthe purpose of disarming criticism (for truly the writer has never\nsupposed his trifle worth the critic's labour or notice), but simply to\nremark, that the object has been to write for the minds of _children_;\nif the book be intelligible to them, the utmost ambition of Uncle Philip\nwill be attained. Truth and plainness were all he sought. The first he\nbelieves he has attained; and to determine his success in attempting\nthe last, he turns from the parents, and looks for the decision of the\nquestion to the suffrages of the children. He would rather hear the\nexpression of satisfaction from the lips of one intelligent little\nreader, than receive the words of approbation from many who are elders;\nthe first is testimony derived from experience, the last is but opinion.\nChildren always know better than any one else does what books they\nunderstand.\n\nIn conclusion, the author owes it to himself to say to the parents\nof his young countrymen, and to the patrons of the \"Boy's and Girl's\nLibrary,\" that what he has written will be found on the side of religion\nand morals. So far as these important points are concerned, the writer\nis not ashamed to avow himself a Christian; nor yet does he mean to make\nit the subject of boasting. In his simple view, Christianity is a very\nquiet and gentle thing, which eschews strife, and promotes practical\ngoodness; and truly can he say, that he has indulged in some of his\nhappiest and, as he trusts, his holiest musings when, in the solitary\npursuit of his favourite science,--to use the language of good old\nIzaak Walton, that simple-hearted lover of God, and all his works,--\"he\nhas looked upon the wonders of nature with admiration, or found some\nharmless insect to content him, and pass away a little time, without\noffence to God, or injury to man.\"\n\n\n\n\nCONTENTS.\n\n\n Page\n CONVERSATION I.\n About a Fly that can work with a Saw and a Rasp, like the Carpenter 13\n\n CONVERSATION II.\n About Grasshoppers and Bees that bore Holes with a Gimlet 19\n\n CONVERSATION III.\n About Animals that are Tailors 27\n\n CONVERSATION IV.\n About the first Paper in the World made by Wasps 41\n\n CONVERSATION V.\n A Story about Tom Smith, and of Bees with Brushes and Baskets, and\n of a Bird with a Chisel, and a Gnat with a Lancet 53\n\n CONVERSATION VI.\n About Animals that can do Mason's Work 66\n\n CONVERSATION VII.\n About Animals that throw Dirt with a Spade; and about an Animal\n with a Hook; and about one that is a Wire-drawer 80\n\n CONVERSATION VIII.\n About a Door, with a Hinge and Spring to it, made by a Spider; and\n the Difference between God's Work and Man's 94\n\n CONVERSATION IX.\n A Story about a Philosopher and his Kite; and about Ants that\n have Awls, and build Cities, and Stairs, and Bridges, and many\n other Things 104\n\n CONVERSATION X.\n More about the white Ants 120\n\n CONVERSATION XI.\n About some other Ants that are very good Masons, and build Walls\n and Ceilings; and a Story about a very sensible Ant which seemed\n to think a little 129\n\n CONVERSATION XII.\n About Ants that go to War, and fight Battles; and about some that\n are Thieves, and have Slaves 138\n\n CONVERSATION XIII.\n A Voyage; and an Animal that makes itself into a Ship; and of an\n Insect that builds a Boat, and floats about in a Canoe; and of\n another that pumps Water, and wears a Mask; and of a Spider that\n builds a Raft, and floats upon it 151\n\n CONVERSATION XIV.\n About an Insect with Tweezers, and another with Pincers; and how\n a Fly's Foot is made, so as to stick to the Wall 167\n\n CONVERSATION XV.\n How Hats are made; and about Animals that can make Felt\n like the Hatter 181\n\n CONVERSATION XVI.\n About Birds that are Weavers, and the Politician Bird; a\n Story about some Philosophers; and what may be learned\n from these Conversations 202\n\n\n\n\nNATURAL HISTORY.\n\n\n\n\nCONVERSATION I.\n\n _Uncle Philip tells the Children about a Fly that can work\n with a Saw and a Rasp, like the Carpenter._\n\n\n\"Well, boys, this is a beautiful day. The sun is shining brightly, and\nthe birds are singing, and the insects are flying about, and the grass\nis green, and every thing appears pleasant, and you feel happy too, and\nhave come, I suppose, to see old Uncle Philip.\"\n\n\"Yes, Uncle Philip, we are tired of playing now, and so we have come to\nask you to talk with us, and tell us about some of the curious things\nyou know.\"\n\n\"Well, boys, I will tell you about some very strange things. I will talk\nto you about animals that know how to work with tools like a man.\"\n\n\"Work with tools, Uncle Philip! That is strange; but we know it is so,\nif you say so; because you will not tell us any stories but true ones.\nBut where do they get the tools?\"\n\n\"Ah, boys, 'the hand that made them is _divine_!' They get them where we\nget all that is useful and good,--from God. The Bible says that He '_is\nwise in heart, and wonderful in working_;' and he has made many a poor\nlittle insect, and given it tools to work with for its comfort, as good\nand perfect as any that man can make. Yes, these poor little creatures\nhad tools long before man had. God cares for the insects, boys, as well\nas for us.\"\n\n\"But, Uncle Philip, what sort of tools do you mean? Tell us about them.\"\n\n\"Very well, I will; do you think of some kind of tools that men use:\nthink of the carpenter and his tools, and let us see if we cannot find\nsome of them among the insects.\"\n\n\"Why, the carpenter has a saw. Is there any saw among these little\nfellows?\"\n\n\"Yes indeed, there is; and a capital saw it is. Now listen, and I will\ntell you all about it. There is a kind of fly called the _saw-fly_; it\nhas four wings, and commonly its body is yellow, and its head is black;\nbut the most curious part of it is the saw. The young ones feed upon the\nleaves of rose-bushes, and gooseberries, and raspberries, and currants,\nand several other kinds of bushes; and the old ones always lay their\neggs on the branches of these bushes, so that the young ones may have\nsomething to eat as soon as they come out. It uses its saw to make a\nplace in the branch to put its egg in.\"\n\n\"Uncle Philip, what is the saw made of?\"\n\n\"It is made of something like horn, and is fixed very nicely in a case;\nit resembles what the cabinet-makers call a _tenon-saw_ more than it\ndoes the carpenter's common saw. The tenon-saw is made of a thin plate\nof steel, and has a stiff brass back, to keep it from bending. The brass\nback has a groove in it, and the saw is put in that groove, and then it\nis fastened to it. But the fly's saw is fixed in another way: there is\na back to it too, but that back is not fastened to the saw. The groove\nis in the saw, and there is a ridge all along the back-piece, which\njust fits in the groove, and so the saw slides backwards and forwards,\nand the ridge always keeps it in its place. Besides all this, boys, the\nfly is better off than the cabinet-maker, for he uses only one saw at\na time; but our little workman has two exactly alike, and they are so\nfixed that the creature first pushes out one, and when it is drawing\nthat back, pushes out the other; so that it is all the time cutting, and\ndoes double work. I think the fly's saw is the best, too, for another\nreason. The saws of the carpenter and cabinet-maker have their teeth\nbent; first, one a little on one side, and then the next to it a little\non the other side, and so on to the end of the saw; so that when sawing,\nthe cut may be wide enough for the blade to move easily. Now the fly's\nsaw has the teeth a little bent, or twisted, too; but it has something\nelse: on the outside of every tooth there are a great many very small\nteeth, so that the outside of every one is just like a _rasp_, or\n_file_.\"\n\n\"But, Uncle Philip, it must take them a great while to saw a very little\ncut; they are so small.\"\n\n\"Yes, it does; but they persevere. It takes them more than an hour and a\nhalf to make one groove, and sometimes they will go on and make as many\nas six without stopping. That shows, boys, what perseverance will do.\"\n\n\"And when it is done sawing, Uncle Philip, where does it keep its saws?\"\n\n\"Oh, I told you they fitted in a case; but when the fly is done sawing,\nit uses the saws to put the egg in the place cut for it, and then it\ndraws the saws almost entirely into the case, and drops upon the egg a\nsort of frothy stuff like a drop of soap-lather.\"\n\n\"What is that for?\"\n\n\"I suppose it is to glue the egg fast, or else to keep the juices in the\nbush from hurting it.\"\n\n\"Well, this is a curious fly, Uncle Philip.\"\n\n\"It is strange, boys, because you never heard of it before; but it is a\ncunning fly, as well as a curious one.\"\n\n\"What does it do, Uncle Philip?\"\n\n\"Why, when it is frightened, it will fold up its case and saws under its\nbody, and draw up its legs, and pretend to be dead; and then it will not\nmove, even if you stick a pin through it.\"\n\n\"Can you tell us any thing more about this fly?\"\n\n\"Nothing very strange, boys; but we have found out _two_ tools, I think,\na saw and a rasp, and that is enough for one poor little fly to give us.\nHere, boys, are pictures of these saws; I have made them a great deal\nlarger than they are in the fly, so that you can see them plainly.\"\n\n[Illustration: Saw of the Saw-fly, with Rasps shown in the Cross-lines.]\n\n[Illustration: Portion of the Saw-fly's comb-toothed Rasp, and Saw.]\n\n\n\n\nCONVERSATION II.\n\n _Uncle Philip tells the Children about Grasshoppers and Bees,\n that bore Holes with a Gimlet._\n\n\n\"Well, Uncle Philip, here we are again, to hear more about the tools\nthat animals work with; we have seen in the bark of trees, and old\nwooden posts, little holes as round as a gimlet could make, and we\nhave been thinking whether any of these little creatures have augers\nand gimlets, as well as saws. Do you know of any of them that can bore\nholes?\"\n\n\"Oh yes, boys; I know of more than one that can bore as smooth and round\na hole as any carpenter you ever saw. There are some of the grasshoppers\nthat have an excellent gimlet. The contrivance has five pieces in it;\ntwo of the pieces make a case to keep the augers in, two more are the\naugers or borers, and the other is a piece between the two borers on\nwhich they slide; this piece has a ridge on each side of it, and the\naugers have a groove which exactly fits the ridge. Besides this, each\nauger ends in a knob, and that knob has teeth all around it. Here is a\npicture of it.\"\n\n[Illustration: Ovipositors, with files, of the Grasshopper, magnified.]\n\n\"But, Uncle Philip, what is the piece with the ridge for?\"\n\n\"Ah, boys, that piece shows the wisdom and the goodness of God. '_His\ntender mercies are over all his works_:' he has placed that piece there\nto keep the borers stiff, so that they cannot get out of joint, or be\nbroken, when the little workman is boring.\"\n\n\"Well, this is very curious.\"\n\n\"Yes; but there are some of these insect workmen more curious still.\nDid you ever see a spy-glass? You know it is a round, hollow piece of\nwood, with brass tubes in it, which are made smaller and smaller, so\nas to slide into one another, when the glass is not used. Now there\nis a sort of gadfly (she is a little creature, too) which has exactly\nsuch a contrivance to keep her gimlet in. It is in four pieces, and the\nsmallest piece ends in five sharp points, three of which are longer than\nthe other two: she twists these five sharp points into one piece, and\nas some are longer and some shorter, when they are all put together,\nthey make a sharp edge running all around, and are almost exactly like\nan auger or gimlet. When she wants to use it, she just shoots out the\ndifferent tubes, so as to make a stem for the gimlet; and when she is\ndone, she puts all back into its case again.\n\n\"Here is a drawing of it, and I think that by looking at it you will\nunderstand what I have been telling you: I do not know whether men\nlearned from this part of the fly how to make the case of a spy-glass;\nbut I know they might have learned.\n\n[Illustration: Ovipositor or Gimlet of the Gadfly, greatly magnified,\nwith a claw and part of the tube, distinct.]\n\n\"There is also a bee, boys, which is called the _carpenter-bee_, because\nit is such an excellent wood-borer. It commonly looks for some old post,\nor dry plank, or withered part of a tree, to work in. It never works in\nwood that is green and has the sap or juices in it; for the bee knows,\njust as well as any carpenter does, that it is very hard to get tools\nthrough such wood. I expect that you have seen sometimes, when an old\npost or dry board was split, a long hollow groove in the middle of it,\nwith little round thin pieces of something like paper, about as thick as\na wafer, fastened in it by their edges, one above the other, all the way\nthrough. These show the work of the carpenter-bee: she bored the hole,\nand she put those little partitions like paper in it, to separate the\ncells; and more than that, she made the partitions out of the dust she\ngot by boring. She always likes, too, to get a piece of wood in a place\nwhere the sun can shine on it; and when she has made her choice, she\nbegins to bore at first into the post in a slanting direction, and as\nsoon as she has gone far enough in, she then turns and bores straight,\nwith the grain of the wood.\"\n\n\"Does she do it quickly, Uncle Philip?\"\n\n\"Not very quickly, for sometimes the wood is very hard; I have seen\none of these holes nearly twelve inches long in a very hard oak board.\nSometimes she has to work at it for months; but she works steadily,\nboys, and that does a great deal. What makes it more tiresome is, that\nthe poor little creature has to bring out all the dust she makes by\nboring.\"\n\n\"How large is the hole?\"\n\n\"Oh, large enough to put my forefinger in, and sometimes fifteen inches\nlong. After she has bored it as deep as is necessary, she begins to\ndivide it into separate cells. So she commences at the bottom, and puts\nin a quantity of what is called bee-bread, until it reaches about an\ninch in height; on the top of this she lays an egg, and the bread is put\nthere to feed the young one as soon as it comes out of the egg. She then\nmakes a floor over it out of the dust, as I told you; she knows how to\nglue this dust together, and she brings it grain by grain from the heap\nin which she put it when she first brought it out: and she always begins\nby gluing the dust around the outside of the hole she has bored, and\nthen glues another ring to that, and then another, and another, making\neach ring smaller and smaller, until she has it all filled; so that her\nfloor, when it is done, appears like a parcel of rings of smaller and\nsmaller sizes placed within each other. On the top of this floor she\nputs bee-bread, as before, and places another egg on it, and then covers\nit with a floor again; and so she goes on making cells and filling them\nwith bread, and covering each with a floor, until she has filled up the\nhole.\"\n\n\"Uncle Philip, how do the young bees get out when the egg is hatched? It\nseems as if they were shut up for ever in prison.\"\n\n\"No, boys; there is a way for them to get out, and it shows the\nwonderful wisdom of God in teaching this poor bee how to contrive the\nmatter. The egg which is put in the lowest cell being the oldest, the\nlittle worm that is afterward to be a bee will come out of that one\nfirst: now, you know, he never could get through all the cells over his\nhead, filled as they are with bee-bread, so as to come out at the top\nof the hole. If he gets out at all, then, it must be at the bottom. The\nold bee knows this, and she so arranges these eggs that when the worm\ncomes out it will be with his head pointed downwards; he falls to eating\nhis bread, and so eats himself down to the bottom of his cell, and there\nhe finds that his mother has bored a hole from his cell to the outside,\nand through that he comes out. When his brother in the cell above him\nhas eaten his way down to the bottom of his cell, he just eats through\nthe floor and gets into the cell below, which is then empty, you know,\nand walks out at the same hole which his older brother used before him.\nAnd so all the rest one after another eat their way downwards into the\nempty cells below them, and get out at the same back-door, which their\nmother made by what we call her _instinct_, which just means the share\nof wisdom which God gives to the lower animals to show them how to take\ncare of themselves.\"\n\n[Illustration: =A=, represents a part of a post, tunnelled in several\nplaces by the violet carpenter-bee; the stick is split, and shows the\nnests and passages by which they are approached. =C=, a piece of thin\nstick, pierced by the carpenter-bee, and split, to show the nests. =D=,\nperspective view of one of the partitions. =E=, carpenter-bee. =F=,\nteeth of the carpenter-bee, greatly magnified; _a_, the upper side; _b_,\nlower side.]\n\n\"Why, that instinct, as you call it, Uncle Philip, is a curious thing.\"\n\n\"Very curious, very curious indeed, boys; and at some other time, if\nyou wish, we will talk more about it, and I will tell you a great many\nstories of animals, which will show you their instinct. But for this\ntime I have told you enough to keep you thinking until we meet again. So\nnow just look at this picture of the carpenter-bee's house, and then you\nmay go home.\"\n\n\n\n\nCONVERSATION III.\n\n _Uncle Philip tells the Children about Animals that are\n Tailors._\n\n\n\"Uncle Philip, we are very glad to see you, and we think we have found\nout something to ask you, about a kind of work which men do, that no\nother animal can accomplish. As we came along this morning to visit\nyou, and were talking of what you had told us of insects that, like\ncarpenters, could saw wood and bore holes in it, we passed by the\ntailor's shop, near the church; 'and now,' said we, 'we have found out\nsomething which will puzzle good Uncle Philip: there are surely no\ntailors among the lower animals; so we will ask him to-day to talk about\ncreatures that can cut out cloth and sew it up with a needle.'\"\n\n\"Ah, my dear children, there are a great many things which would puzzle\nUncle Philip. I do not know every thing; nor do I suppose that I can\nfind _every_ trade in the world among the dumb creatures which God has\nmade. But you have made a bad choice of a puzzle this morning, my boys;\nfor there are tailors among the inferior creatures, and some pretty nice\nones, too; at any rate, they always cut so as to fit exactly.\"\n\n\"Why, Uncle Philip! You do not mean to say that they can cut out\n_cloth_, and then sew it up again with a needle and thread!\"\n\n\"No, boys; I do not think it is to be expected that they should take\na pair of shears and cut a piece of cloth, or put a piece of thread\nthrough the eye of a steel needle; any more than we expect the insect\nthat saws, to go to the cabinet-maker, and borrow his tool to work\nwith. But with the instruments which God has given to them, they will\ncut what is cloth to them, the leaves of trees and flowers, and will sew\nthem together too: and, now I think of it, there is one that will cut\nhis garments out of our cloth.\"\n\n\"Pray let us hear about them, Uncle Philip.\"\n\n\"Softly, boys, softly. I have two things to say to you before I begin.\nIn the first place, I am very glad to hear that you think and talk among\nyourselves about the things which I tell you: and in the next place, I\nknow that you love _me_, and, therefore would not wish, by _puzzling_\nme, as you call it, to produce mortification or vexation; nor do I\nthink that I should have felt either vexed or mortified had I not been\nable to find tailors among the lower animals; but I do not wish you to\ntake pleasure in puzzling people; for it is very apt to produce in you\na feeling of triumph, and to make you vain: and you must remember that\nfor _one_ of your questions which cannot be answered, a _thousand_ might\nbe put to you, of the answer to which you would be ignorant. No man, my\ndear boys, knows every thing. Wise men talk with each other, that they\nmay learn from each other; and the wisest are not ashamed to acknowledge\ntheir ignorance of some things; and I believe they take very little\npleasure in puzzling. It is our duty to learn all that we can, and to be\nalways willing at a proper time to teach others what we know.\"\n\n\"Thank you, dear Uncle Philip, for your advice. We did not mean to\ntriumph over _you_, if you had not been able to tell us of tailors among\nthe animals. But we see that you are right. We might get a foolish\nhabit, which would do us harm.\"\n\n\"Exactly what I meant, boys; and now let us begin. And first we will\ntalk of the cutting out, as the tailor always does that before he sews.\nThere is a kind of bee[1] which, like some of the insects we have\nalready spoken of, is furnished with a borer. With this she forms a\nround hole, like that made with an auger or gimlet, in a hard-trodden\npath, or sometimes in a piece of soft decayed wood. It is in making her\nnest in this hole that she plays the part of a tailor, for the nest is\nmade of leaves, sometimes taken from the rose, at others from the birch,\nash, or other trees. The little creature cuts them commonly, and I\nbelieve always, into two shapes. They are either half-oval, that is,\nhalf the shape of the bowl of a spoon, or round, and are of different\nsizes. Sometimes she makes a mistake in the size, and when she finds it\nout, she alters it. These leaves are prepared to line the hole which\nshe has bored, and she begins with the largest pieces; taking them into\nthe hole, she winds around in it, until she has spread very smoothly a\ntube of leaves the whole length of it; she then closes up one end of it\nby rounding it off and doubling the pieces of leaf one over another.\nIn this case she sets about making her _cells_. She takes three of her\nhalf-oval pieces which have been cut to fit, and contrives to roll them,\nso that the edge of one piece will just lap over the edge of the next;\nthese, when she has finished rolling them, make the hollow of the cell,\nwhich is not quite an inch high. She next turns up the ends of these\npieces, which are cut to fit, so as to form the bottom: she then sets to\nwork with three other pieces rolled in the same way inside of the cell\njust finished, turning up their ends as before to form the bottom; and\nwithin these she again works three others, so that her cell, when it is\ndone, is of nine thicknesses of leaves. And you see why, though she cuts\nthe pieces of the same shape, they are not all of one size: they are\nof three sizes, so as to make the cells within each other smaller and\nsmaller.\"\n\n\"But, Uncle Philip, you have not said any thing about the round pieces\nwhich she cuts; how does she use them?\"\n\n\"I will tell you: after she has finished one cell she lays an egg in it,\nand fills it all round with food nearly liquid; now as the cell is lying\ndown on its side, all this liquid food would run out if it were not\ncorked up, and the bee therefore uses her circular pieces to stop up the\ncells.\"\n\n\"And does she really make these round pieces to fit the cell?\"\n\n\"Yes, boys, exactly; and they are cut too as regularly as if they had\nbeen first measured and marked with a pair of compasses. And, more than\nthis, the little creature will fit one in in less than a minute. But the\nmost curious thing is, that sometimes she will fly off to a distance\nto get this round piece, and bring back one which will exactly suit;\nso that it really seems as if she carried the size in her head. After\nfinishing one cell she will make another, until she has completed as\nmany as she wants; and then, as she always builds them one upon another,\nthey appear like a parcel of thimbles stuck into each other and put into\na case: and here is a picture of it.\"\n\n[Illustration: Rose-leaf-cutter Bees, and Nest lined with Rose-leaves]\n\n\"This is very wonderful, Uncle Philip; and it does seem like cutting out\npieces to fit.\"\n\n\"Very true: but this is not the only cutter-out of leaves among the\nbees. There is another kind, called the poppy-bee,[2] because it uses\nthe scarlet leaves of the poppy-flower to line its cell. It makes its\nhole in the ground, as smooth and regular and polished as can be, and\nthen proceeds to line it all around with pieces of the leaves, and cuts\nthem to fit as she goes on. If a piece is too large she will trim it\ndown to the proper size and shape, and always carries away the scraps.\nNow if you should take a pair of scissors and try to cut the leaf of a\npoppy-flower, you would wrinkle it, but this little workman will spread\nout what she cuts as smooth as glass. When she has lined this hole\nthroughout, and carried the lining out beyond the entrance, she fills it\nwith honey and _pollen_, or bee-bread, as it is called, about half an\ninch high, lays an egg, then folds down the leaves on it, and finally\nfills the upper part with earth.\"\n\n\"Then she was not working for herself?\"\n\n\"No; she was providing a house for her young, and God has taught her\nthus to take care of it.\n\n\"I will now tell you of another little workman, which I have heard\ncalled the cloak-maker, because it makes for itself a mantle which\nreally appears very much like a cloak; and, stranger still, this cloak\nis lined throughout with silk.\"\n\n\"Can it be possible, Uncle Philip?\"\n\n\"Listen, and you shall hear. These mantle-looking cases are made by\nthe _larva_, as it is called, or grub of a little moth which forms a\ncovering of pure silk; this silk it spins from itself; it is not woven\nso as to appear like our silk, but still it is real silk, and is worked\ninto a great many thin scales, which lap over one another like the\nscales of a fish. But this is only the lining of the cloak. This little\ntailor is the field-moth, which first eats what it wants from a green\nleaf, and then, from the thin membranes left, sets about making its\nmantle: and it makes it of two pieces cut out and joined together with a\nseam, just as a tailor would make it.\"\n\n\"How does it go to work, Uncle Philip?\"\n\n\"Why, I will give you the account as it was given by a gentleman[3] who\nwas very fond of observing insects, and who watched one of these little\ncreatures. He says that from the thin membrane of the leaf it first\ncut two pieces just equal in size and of exactly the same shape; each\nof these pieces was to form one-half of the cloak, and this he says was\ndone wonderfully fast. He noticed, too, that one end of each piece, that\nwhich was meant for the bottom of the cloak, was just twice as long as\nthe other end, which was the top. The insect then placed itself between\nthe two pieces while they were lying flat; it afterward brought the two\nsides where the seam was to be, together, and fastened them at certain\nplaces, still leaving, however, considerable spaces open. It then began\nto turn and twist its body about in all directions, until it moulded\nthe pieces into a hollow form to fit. When it found that it would fit\nits body, it brought the edges of the seam close together through the\nwhole length, and contrived to sew or fasten them so neatly together,\nthat when the gentleman looked, even with a magnifying-glass, he said he\ncould hardly find the seam. The whole was lined with the silk spun from\nitself, and was finished in about twelve hours.\"\n\n\"Why, this little workman is the strangest of all: but, Uncle Philip,\nyou said there was one of these animal tailors that cut his garment out\nof _cloth_: pray tell us of him.\"\n\n\"When I said that, boys, I was thinking of the clothes-moth.[4] They\nmake their coats of wool commonly taken from our cloth, and silk drawn\nfrom their own mouths; and the strangest thing concerning them is,\nthat when they outgrow their clothes they will piece them to make them\nlarger. Suppose the insect wants it longer, it adds a new ring of wool\nto the end: suppose it wants it wider, it slits the case or garment, not\nfrom one end to the other, for this would leave it naked, but it splits\nit half-way down the sides, and when it has filled it in with proper\npieces, it splits the remaining half, and puts other pieces in them.\nThere is another curious thing about this tailor: it always makes its\ncoat of the same colour with the cloth from which it takes the wool; so\nthat if it has first made its garment of a piece of blue cloth, and is\nplaced on a bit of red cloth when it wishes to enlarge it, you will see\nits work exactly, for the pieces which it puts in will be red. This is\nthe little fellow, boys, which does so much mischief to our clothes.\"\n\n\"Well, Uncle Philip, one can almost forgive his mischief for the sake\nof his ingenuity. But you have said nothing yet about _needles_; how do\nthese little creatures sew?\"\n\n\"Why, they have what serves as a needle to them: but I can tell you of\nanother animal which sews with a needle a great deal plainer to be seen\nthan that of these little insects.\"\n\n\"Pray let us hear of him, Uncle Philip.\"\n\n\"I must go among the birds to find this workman. There is a kind of\nstarling, called the orchard starling,[5] about which, Mr. Wilson, a\ngentleman who has written a great deal concerning the birds of our\ncountry, gives a very curious account. He says that this bird commonly\nhangs its nest from the twigs of an apple-tree, and makes it in a very\nsingular manner. The outside is made of a particular kind of long\ntough grass, that will bend without breaking, and this grass is knit\nor sewed through and through in a thousand directions, just as if done\nwith a needle. The little creature does it with its feet and bill. Mr.\nWilson says that he one day showed one of these nests to an old lady,\nand she was so much struck with the work that she asked him, half in\nearnest, if he did not think that these birds could be taught to _darn\nstockings_? Mr. Wilson took the pains too to draw out one of these\ngrass threads, and found that it measured thirteen inches, and in that\ndistance the bird who used it had passed it in and out thirty-four\ntimes.\"\n\n\"Why, this was sewing, sure enough.\"\n\n\"Yes; and I saw, when I was in the West Indies, another kind of\nstarling[6] which will cut leaves into a shape like the quarter of\nan orange-rind, and sew the whole very neatly to the under side of a\nbanana-leaf, so as to make one side of the nest. But, boys, there is\nanother most beautiful little bird, which is called the tailor-bird,\nbecause it sews so well.[7] It first picks out a plant with large\nleaves, then it gathers cotton from the shrub, and with the help of\nits fine long bill and slender little feet it spins this cotton into a\nthread, and then using its bill for a needle, it will sew these large\nleaves together to hide its nest, and sew them very neatly, too.\"\n\n\"Why, dear Uncle Philip, this is the most wonderful tailor of them all.\"\n\n\"He is, indeed: but, my children, what do we learn from all that I have\nbeen telling you? Who made these little creatures with such curious\nskill, and taught them to work so well? It was the same God who made us;\nfor such wonderful things never came from what people call _chance_.\nChance, boys, never made any thing: and how very wise he must be to\nform such nice little workmen; and how very good thus to teach them how\nto take care of themselves. The Bible says, truly, that '_his tender\nmercies are over all his works_.' And I think, boys, we may learn\nanother thing: it is, not to be so very proud of what we know; for I\nrather suppose that we shall often find that the lower creatures around\nus understood many of our trades long before we found them out.\"\n\n\"Yes, Uncle Philip, it is likely that these little fellows you have been\ntelling us of this morning were the first tailors in the world.\"\n\n\"Very likely, very likely indeed, boys. But now I must bid you good\nmorning; for here comes our good clergyman, and I am going with him to\nsee a poor sick woman.\"\n\n\"Good morning, Uncle Philip; we will come again on Saturday.\"\n\n\nFOOTNOTES:\n\n[1] Megachile centuncularis.\n\n[2] Osmia papaveris.\n\n[3] Reaumur.\n\n[4] Tinea sarcitella.\n\n[5] Icterus mutatus.\n\n[6] Icterus bonana.\n\n[7] Sylvia sutoria.\n\n\n\n\nCONVERSATION IV.\n\n _Uncle Philip tells the Children about the first Paper in the\n World, made by Wasps._\n\n\n\"Ah, boys! how do you do? This is Saturday, and I have been expecting to\nsee you come for some time.\"\n\n\"Why, Uncle Philip, we should have been here sooner, but we went round\nby the old mill; because we thought that perhaps we might find in some\nof the old timbers, holes bored by some of those industrious little\ncarpenters you told us about.\"\n\n\"Well; and did you find any?\"\n\n\"No; but we found something else, which we have brought to show you: and\nwe have been talking about it all the way. We have not discovered any\nnew _tools_ among the animals, but we think we have found out a _trade_\nthat some of them work at; and we wish you to tell us if we are right.\"\n\n\"Oh, that I will do, with pleasure, if I can. What is the trade that\nyou think you have discovered?\"\n\n\"It is paper-making, Uncle Philip. We have found this part of a wasp's\nnest, which we have brought along; and as you told us it was always best\nto notice every thing closely, we examined this, and it appeared so much\nlike coarse paper that we thought (for we knew it was made by wasps)\nthat man did not make the first paper in the world.\"\n\n\"Well, boys, that was not a bad thought. Now you see the advantage of\ntaking notice of things, and of thinking about what you see. You are\nperfectly right in supposing that wasps make paper; and, if you please,\nwe will talk this morning about the wasps.\"\n\n\"Oh yes, yes, by all means, Uncle Philip; and we will thank you, too.\"\n\n\"I must first tell you, then, that of the wasps there are several kinds.\nSome build their nests under ground, and some hang theirs in the air to\nthe limb of a tree. This part of a nest which you have found belonged to\nthe last kind; but I will tell you something about both. But before I\nbegin let me get some drawings I have, which will help us to understand\nbetter. I have them. And now, of the wasps which build under ground.\nAs soon as the warm season begins, the first care of the mother-wasp\nis to look for a fit place in which to build; and in the spring of the\nyear she may very often be seen flying about a hole in the bank of a\nditch, and looking into it. These holes which she examines are the old\nhouses of field-mice or moles, and some persons have thought, what I\nexpect is true, that she likes to take such old holes, because they save\nher a great deal of hard work. But still, as the holes are not large\nenough for her use, she has a great deal of labour to make them do. So\nshe goes at once to work, digging in the hole she has chosen, and makes\na winding, zigzag gallery, about two feet long, and about an inch in\nwidth. She digs out the earth, and carries it out, or pushes it out\nbehind her as she goes on. This gallery ends in a large chamber or hole\nfrom one to two feet across when it is done: and now she is ready to\nbegin her nest.\"\n\n\"Now then, Uncle Philip, she will begin to make paper, will she not?\"\n\n\"Yes; but here I ought to tell you that it was a long time before men\nfound out what she made it of. Do you remember my telling you of a\ngentleman who watched the little cloak-maker to see how he made his\ngarment? Well, this gentleman, whose name was Reaumur, was trying for\ntwenty years, he says, to find out how the wasp made paper, before he\nsucceeded. At last, one day, he saw a female wasp alight on the sash\nof his window and begin to gnaw the wood; he watched her, and saw that\nshe pulled off from the wood fibre after fibre, about the tenth part of\nan inch long, and not so large as a hair. She gathered these up into a\nknot with her feet, and then flew to another part of the sash, and went\nto work, stripping off more fibres or threads, and putting them to the\nbundle she had already. At last he caught her, to examine the bundle,\nand found that its colour was exactly like that of a wasp's nest; but\nthe little ball was dry; she had not yet wetted it to make a pulp of it\nwhich could be spread out. He noticed another thing, that this bundle\nwas not at all like wood gnawed by other insects; it was not sawdust,\nbut threads of some little length bruised into lint. He then set to work\nhimself with his penknife, and very soon scraped and bruised some of the\nwood of the same window-sash, so as to make a little ball exactly like\nthe wasp's. Mr. Reaumur thought that this was the stuff out of which the\nwasp made paper, and it has since been found out that he was right. The\nanimal wets its little bundle of bruised wooden fibres or threads with\na kind of glue that it has, and this makes it stick together like pulp\nor paste; and while it is soft, the wasp walks backwards, and spreads it\nout with her feet and her tongue, until she has made it almost as thin\nas the thinnest paper. With this she lines the top of the hole in which\nshe is going to build her nest, for she always begins at the top. But\nthis is so thin that it would be too weak to keep the dirt from falling\nin; and therefore she goes on spreading her papers one upon the other\nuntil she has made the wall nearly two inches thick. These pieces are\nnot laid exactly flat on each other like two pieces of pasteboard, but\nwith little open spaces between, being joined at the edges only. This\nis the ceiling; and when it is finished she begins to build what may be\ncalled the highest floor of the nest; this she makes of the same paper\nin a great number of little cells all joined together at the sides; and\ninstead of fastening this floor to the sides of the nest, she hangs it\nto the ceiling by rods made also out of this paper: these rods are small\nin the middle, and grow larger towards the ends, so as to be stronger.\nHere is a drawing of one.\n\n[Illustration: The Cut represents one of the Rods from which the Floors\nare suspended.]\n\nShe then makes a second floor, and hangs it under the first by rods as\nbefore; and the whole of it, when finished, if it should be cut straight\nthrough the middle, would appear something like the following picture of\none which I made some years ago.\"\n\n[Illustration: _Section of the Social-Wasp's Nest._--_aa_, the outer\nwall; _b_, _cc_, five small terraces of cells for the neuter wasps;\n_dd_, _ee_, three rows of large cells for the males and females.]\n\n\"This is a very ingenious little paper-maker. Uncle Philip.\"\n\n\"Yes, boys, it is so. This of which I have been telling you is the\nground-wasp. The tree-wasp makes its nest of paper prepared in the same\nway; and the nests are of different shapes. One makes it in a round\nflattened ball, not much larger than a rose, and when cut open it shows\nlayer upon layer of leaves of the same thin grayish-looking paper. This\nkind is not so common, however. Here is one of their nests.\n\n[Illustration: Wasp's Nest.]\n\n\"Another makes its nest of cells placed in separate floors, but without\nany outer wall to keep off the rain; and the most curious thing in this\nnest is, that it is not placed in a horizontal way; that is, it is not\nplaced with the floors level, because then the cells would catch the\nrain, and the nest would be spoiled; but it is always placed slanting,\nso that the rain may run off. It is always placed, too, so as to face\nthe north or the west, and I suppose it is because the wasp knows that\nit is in more danger of rain from the south and the east. Here is a nest\nof this kind.\"\n\n[Illustration: Wasp's Cells attached to a branch.]\n\n\"Ah, Uncle Philip! this must be a kind of lazy wasp. It does not choose\nto take the trouble to cover up the house, and so it hangs it slanting,\nto make the rain run off.\"\n\n\"It may be so, boys; but I think that in making this wasp lazy, you make\nit a very sensible wasp; else how should it know that water would run\ndown a slanting surface? But I cannot believe that it is so lazy; for,\nthough it does not cover up the whole house in a paper shell, yet it\ndoes what no other wasp does, it covers its nest with a complete coat of\nshining, water-proof varnish, to prevent the rain from soaking into the\ncells. And putting on this varnish, I can tell you, is no trifling work.\nIt forms a pretty large part of the labour of the whole swarm belonging\nto the nest; and sometimes you may see some of them at work for hours at\na time, spreading it on with their tongues. No, my lads, he who wants an\nexample of laziness, will not find it among the wasps.\n\n\"But let us come back to the paper. Hornets make paper for their nests\nmuch in the same manner as the wasps do, only it is coarser. There is,\nhowever, one kind of wasp which makes a sort of paper more curious than\nthis which you have found. It is not a wasp found in this country at\nall. It is the Cayenne wasp, and so smooth, strong, and white is the\noutside of his nest that it appears like a card, and he is for that\nreason sometimes called the card-maker wasp. He hangs his nest on the\nbranch of a tree, and it is so hard and polished on the outside that\nthe rain rolls off from it, as if it were glass. A little hole in the\nlower end is left for the animal to pass in and out; and in this picture\nof it, which I have, a piece is left out of the side to show how the\ncells within are fixed.\"\n\n[Illustration: Nest of the Card-maker Wasp, with part removed to show\nthe arrangement of the Cells.]\n\n\"Well, then, Uncle Philip, we were right in thinking that wasps were the\nfirst paper-makers; and very glad we are that we saw this old piece of\na wasp's nest. Who would have thought that so much could be learned by\npicking up this old scrap of a wasp's work!\"\n\n\"Very good sense, boys, in that thought. A wise man will learn something\nfrom almost any thing. Use your eyes, and think of what you see. Now in\nthis very trade of paper-making. I think that man would have found it\nout a great deal sooner if he had watched the wasps at their work. They\nhave been excellent workmen at this business from the beginning; and man\nhas gone on learning little by little of this very trade, as I will tell\nyou at some other time, when he might have made a long step at once,\nhad he but noticed wasps and hornets. We go on very slowly sometimes in\nlearning to make a trade as perfect as it can be: the poor animal, with\nits knowledge such as God gave it, is often our superior. These dumb\ncreatures cannot teach us every thing; there is a point to which they\ncan go, and no further: but as far as they do know, their knowledge is\nperfect; and I make no doubt that a great many useful things not now\nknown will hereafter be found out by watching dumb animals.\"\n\n\n\n\nCONVERSATION V.\n\n _Uncle Philip tells the Children a Story about Tom Smith; and\n of Bees with Brushes and Baskets, and of a Bird with a Chisel, and\n a Gnat with a Lancet._\n\n\n\"Uncle Philip, as the day is fine, instead of sitting here, will you\nwalk with us, this morning?\"\n\n\"Yes, boys; let me get my cane and hat, and we will take a ramble;\nperhaps we may see something, if we will use our eyes. Where do you wish\nto go?\"\n\n\"Oh, we do not care much, if you are with us, which way we walk; any\ncourse will be pleasant.\"\n\n\"Come on, then; we will cross the river, and go down on the other side\nbeyond the old mill, where you found the wasp's paper. And now, such of\nyou as will, may keep a look-out for curious things, while the rest of\nus will talk together.--Boys, do any of you know Tom Smith?\"\n\n\"Know him! Why, Uncle Philip, everybody in this part of the country\nknows him; he is such a shocking drunkard, and swears so horribly, that\nnobody can forget him; and what makes it worse, he is an old man, too.\nHis hair is almost as white as yours, Uncle Philip.\"\n\n\"Yes; he is just about my age. We were both born here, and I have known\nhim ever since we were boys; and when we played together as children,\nover this very field which we are now crossing, or caught fish in\nthe river down yonder by the rocks, there was not a more decent,\nwell-behaved, handsome boy among us than was Tom Smith. Poor Tom lost\nhis father when he was about twelve years old, and his mother, having\nno other child, indulged him, until he was sent to the city to go into\na store. But Tom then, boys, had good principles; he neither swore nor\ngot drunk. In a little time he fell into bad company, and they led him\nastray by degrees. He was so good-natured (as they call it), boys,\nthat he had never the firmness to say _no_ to the proposals of his\ncompanions. He went with them to places of amusement; and instead of\nspending his evenings in his own room, reading, he was at the theatre,\nor dancing in some place, or at a supper with his young companions;\nand finally he began to play cards and billiards with them; while the\ninside of the church was a place which he never saw. He was cheated by\nhis companions; and too honest he was then not to pay what he lost by\ngaming: he wrote to his poor mother, and told her the truth, as to his\nlosses, and she sent him money to pay his debts, and told him to come\nhome. He did come home; and even after all that had happened, poor Tom\nmight have been respectable and happy; for his friends were all willing\nto forget the past, and encourage him for the future. For a time he went\non pretty well, and married an affectionate and good young woman, and\nhis prospects were bright enough: but one thing, boys--one single thing,\nruined his comfort for ever. In the city he had learned _to drink strong\nliquors_.\n\n\"I remember, too, soon after he came home and married, that a man was\nhung not far from here for murdering his wife. The man was a drunkard,\nthough he was quite sober when he killed the poor woman; and drunkenness\nhad hardened his heart. I have no doubt, as it will the heart of any\nman. Tom was talking to me about that man, and I remember he said then\nthat when a man _began_ to drink, he could never say where it would end,\nnor what he would do: 'therefore,' said Tom, 'beware of the _first_\ndrink.' But Tom, though he talked like a Christian and a man about it,\ndid not act like one: for it was not long before he began to follow his\nbad habit, and he soon killed his poor mother; for she died of grief\nand sorrow, I think. His excellent wife speedily followed her to the\ngrave; and Tom Smith left the village, a perfect vagabond, whom no one\ncared for. Where he went, or what he did for a long time, no person\nhere knows. I went to other countries, and neither heard of nor saw Tom\nSmith until my return home, when I found him wandering about here, a\ngray-headed swearer and drunkard. He did not know me, and I never should\nhave known him, had not some one told me who he was. And last night I\nreceived a letter from one of my nephews in the city, which informed\nme that Tom Smith had been tried in the court, and found guilty of\nstealing, and was sent to the state prison for ten years to hard work.\nThere I suppose he will die for he is now old; and it is awful to think\nof what is then to become of his soul. Ah, my dear boys! I could not\nhelp thinking, when I read my letter, of what that man said to me years\nago--and I have told you his story, hoping that you will remember his\nwords, 'Beware of the _first_ drink.' The man who does that will never\nbe a drunkard. And when old Uncle Philip is laid in the grave, boys,\nwhich must be before many years, remember, as you look upon the place,\nthat he told you the story of Tom Smith, and charged you to 'beware of\nthe _first_ drink.'\n\n\"But here come some of the boys, running towards us; I suppose they have\nfound something.\"\n\n\"Oh, Uncle Philip! Uncle Philip! Do come with the boys this way. Under\nthat fence yonder there are a great many beautiful wild flowers, and a\nnumber of bees are as busy as they can be about them; pray come and see\nthem.\"\n\n\"Well, I will; but not so fast, boys; you forget that I am an old\nman, and cannot run as you do.--So, here are, indeed, a great many\nindustrious little workmen.\"\n\n\"What are they doing, Uncle Philip?\"\n\n\"These are _workers_ among the bees, and they are gathering the dust out\nof the flowers, to work it up into what is commonly called bee-bread.\nMore tools here, boys!\"\n\n\"Tools, Uncle Philip! Ah, we like that: pray let us hear of them; what\nare they?\"\n\n\"Why, there is a brush and a basket in the legs of these little fellows;\nbut they are so small that you cannot see them without a microscope.\"\n\n\"What is a microscope?\"\n\n\"It is an instrument, made by fixing glasses in such a way to look\nthrough, that small things will seem to be very large. Do you not see\nhow some of these little fellows are rolling themselves over in the\ninside of the flowers, so that the yellow dust is sticking to them?\nNow their breasts, and legs, and many other parts of their bodies are\ncovered with very short hairs, which catch the dust. The last joint but\none of each leg is made exactly like a brush, the hairs being longer\nthere than on any other part; and with these they brush off the dust,\nand get it into two little heaps. The bags into which they put it, or\nrather the baskets, are in the thighs of the last pair of legs. These\nare hollow, so as to form a three-sided basket. The bottom of it is\nsmooth and shining, and appears like horn, and all around the edges are\nplaced very strong, thick-set hairs, like bristles.\"\n\n\"What are these for?\"\n\n\"To keep things from falling out of the basket; and these bristles are\nso strong that even if they heap up more than the basket will hold, the\nbristles will keep it from falling. Here is a drawing of these legs.\n\n[Illustration: Structure of the legs of the Bee for carrying propolis\nand pollen, magnified.]\n\nBesides carrying this dust, they also carry what is called _propolis_.\"\n\n\"What is propolis, Uncle Philip?\"\n\n\"It is a gum which is found upon some trees. This they work up into\nlittle balls, and knead it until it is a little dry, so as not to stick.\nThis takes the bee sometimes as much as half an hour. When the balls\nare ready, she passes them backwards with her feet to the basket, puts\nthem in, and gives them a pat or two to make them lie close; and when\nshe adds more, she pats it still harder, and when the basket is full,\naway she goes to the hive. But there is another curious instrument about\nthe bee. I mean its sting: this is like the head of a barbed or bearded\narrow. There is a sheath for it when the bee does not wish to use it;\nand here is a picture of it.\n\n[Illustration: _a_, The sting of a Bee, magnified to show the barbed\ndarts; _b_, the last ring of the abdomen of a Bee opened, showing the\nsting in its sheath.]\n\nBut let us now continue our walk.\"\n\n\"Well, Uncle Philip, it is really very pleasant to walk with you: it\nseems as if you met nothing which could not teach us things worth\nknowing.\"\n\n\"Why, my dear boys, there are, as I told you once before, a great many\nthings which I do not know; and what I do know I am very willing to tell\nyou. But you may learn just as I did,--by reading, by taking notice of\nthings around you, and by thinking for yourselves. And I do not know any\nthing more pleasant to notice than the works of God. I see his wisdom\nand his goodness in every thing which he has made. I see them in the\ninsects, and the birds, and the larger animals; I see them in the grass,\nand the flowers, and the trees; and I see them in the rocks and the\nstones upon the ground. All these things are well worth our attention,\nboys; the study of all these things around us is called the study of\n'Natural History;' and I think it is apt to make him who loves it a\nbetter man; at any rate, I believe that there have been very few who\nhave been fond of it, who have not been amiable and benevolent men. But,\nhark! Do you hear that noise?\"\n\n\"Yes, Uncle Philip; it is the sound of men chopping wood in that clump\nof trees.\"\n\n\"No, boys; it is like the sound of a wood-cutter; and it is a\nwood-cutter, but he does not use one of our hatchets.\"\n\n\"What is it that he uses, then?\"\n\n\"He uses the tool which God gave him. It is a bird, boys, which you\nhear: it is the woodpecker. See, there it is on yonder tree, and look,\nat the foot of it, there is something like a bushel of the bird's chips\nor dust. Its bill is a complete chisel; it is straight, hard, and sharp,\nwith edges too upon the sides. It is not a very broad chisel, but still\nit is one, and used as we use ours. But the chisel is not the only\ninstrument of that workman. Its tongue is worth examining. It bores a\nhole into a tree that is dead or decaying, to look for insects whose\nnests are in the tree; and when it reaches the cell where the young\ninsect is, it uses its tongue to get it out, and it suits exactly for\nthe business. In the first place, it is so long that the bird can shoot\nit out three or four inches longer than the bill is; in the next place\nthe end of it is tipped with a stiff, sharp, long thorn; and in the\nlast place, that thorn has little teeth on both sides of it, like that\nwhich you see on the point of a fish-hook: these teeth are to keep the\ninsect from falling off when it puts its tongue in the hole and sticks\nits sharp point into it to draw it out for food. So that besides the\nchisel, the woodpecker has a spear, or lance, or arrow, barbed (as it is\ncalled) or bearded at the point.\n\n\"But we are some distance, boys, beyond the old mill: suppose we now\nturn back towards home; I find the gnats rather troublesome.\"\n\n\"So do we, Uncle Philip; they have been biting us for some time: it\nwould be well if there were no such tormenting things in the world.\"\n\n\"I am not sure of that, boys. We may not always be able to find out the\nexact use of some of these little animals; but that only shows that we\nare ignorant, not that they are of no use. God would never have made\nthem if he had not some wise purpose in doing so: I do not believe he\never wastes his power in making useless things. But what will you say\nabout gnats, when I tell you that they have a tool to work with, and a\nvery perfect one, too?\"\n\n\"Why, we will almost forgive them for biting us.\"\n\n\"Biting you! They have not been biting with teeth: they are doctors,\nboys; they have only been bleeding you, and cupping you.\"\n\n\"And what have they been bleeding us with?\"\n\n\"Why, with a lancet, to be sure; what should a doctor use but a lancet\nto let blood?\"\n\n\"And has the gnat really a lancet?\"\n\n\"Yes, it has: this instrument forms a part of what you may call the\ntongue of the gnat: it is made up of five pieces, which are shut up in\na case, split from one end to the other; these give steadiness to the\nlancet when it is used. But the reason of the pain is not so much the\nwound of the lancet, as it is the fluid or poisonous juice which the\ngnat puts into the wound to make the blood thin enough for the insect to\nsuck it up through a tube or case, which makes part of its mouth. Here\nis a drawing of part of a gnat's mouth.\n\n[Illustration]\n\nAnd here is a picture of the lancet or knife of a horse-fly.\n\n[Illustration]\n\n\"We have now reached the bridge,--and here we must part; your homes are\nin one direction, and mine is in the opposite. I hope, however, that you\nhave learned something in our morning's walk.\"\n\n\"We have, Uncle Philip, and we thank you much, and bid you, good day.\"\n\n\"Good day, boys.\"\n\n\n\n\nCONVERSATION VI.\n\n _Uncle Philip tells the Children about Animals that can do\n Mason's Work._\n\n\n\"Uncle Philip, we saw a very strange thing just now; as we were coming,\nwe saw a great many bees flying by us, and each one was carrying a\nlittle stone.\"\n\n\"That was strange, indeed. Did you find out any thing about them?\"\n\n\"We asked a man who was near what they did it for, and he said that they\ncarried the stones to prevent the wind, which is blowing pretty fresh,\nfrom tossing them about too much.\"\n\n\"That is a very silly story, boys, though it is a very old one: for I\nhave seen them carrying what you call stones when it was quite calm, and\nthere was no wind to blow them away. The man was very ignorant, or he\nwould have told you another story, which would have been both strange\nand true.\"\n\n\"Will you have the goodness, Uncle Philip, to tell us what it meant?\"\n\n\"Very willingly, boys. What you saw I presume were bees. You remember\nthat I told you there were several kinds of bees; and this one is called\nthe mason-bee. This kind builds his nest of mortar, and was therefore\ncalled the mason-bee by Mr. Reaumur first, I believe.\"\n\n\"Where does it get the mortar, Uncle Philip?\"\n\n\"It makes it, boys. This kind of bee may be seen flying about, picking\nup sand, grain by grain, putting it into a heap, gluing them together\nwith a sort of gum out of her own mouth, and building with them a\nfoundation for her house. This little workman commonly builds against\nthe side of a wall between two bricks where the mortar has fallen out;\nand if you should see one of the nests, it appears exactly like a lump\nof dry mud which has been thrown wet upon the wall out of a cart-rut:\nbut when you examine it closely, you may see a great many small stones\nin it, more than is common in mud: a hundred people, though, might pass\nby it, and never think it was any thing more than a lump of dirt, which\nhad been thrown upon the wall when it was wet, and had afterward dried\nthere. Here is a picture of one of these nests.\n\n[Illustration: Exterior wall of Mason-bee's Nest.]\n\nYou see there is a small hole in it; this leads to a cell inside about\nan inch deep, and shaped exactly like a lady's thimble; the inside of\nthis cell is polished smooth, and appears like a wall of plaster, except\nthat it has little yellow stains upon it. Here is the bee that makes it.\n\n[Illustration: Mason-bee.--Natural size.]\n\nIn making this mortar to build with, the bee will sometimes add earth\nthat is soft to its grains of sand, and when the lump is about the size\nof a small shot, it takes it up and flies away with it, to work it into\nthe wall.\"\n\n\"Does it always use sand, Uncle Philip?\"\n\n\"Not always: sometimes it takes wet clay, and will dig into a bank of\nclay baked hard by the sun on the outside, so as to get that which is\nwet.\n\n\"Mr. Rennie, a gentleman in England who is very fond of watching\ninsects, and has found out a great many curious things about them, has\ngiven an account of some of these bees which he noticed at work. Every\none was carrying out of a hole in the clay-bank a small lump of clay;\nand on catching one of them, he found that this lump was wetter than\nthe clay in the hole, so that the bee had moistened it, and worked it\ntogether, to make it stick like good mortar. These lumps too were larger\nthan a shot; they were as large as a garden-pea.\"\n\n\"How long did it take them to work up the lump?\"\n\n\"About half a minute, Mr. Rennie says. He watched one of these little\ncreatures, and found that she was building on the inside wall of a\ncoal-house, where the bricks and mortar were left rough: she was at work\nbetween two of the bricks where the mortar had fallen out, or where the\nbee had taken it out. And the conduct of the bee at her house was very\ndifferent from what it was at the clay-bank. She was not frightened,\nbut went on working when any one came near the clay-bank; but at her\nhouse she seemed afraid that it should be found out where it was. She\nwould alight first on the roof outside, as if she merely wished to rest\nherself; and when she flew into the coal-house she would not go directly\nto her nest, but would settle on a shelf, and sometimes pretend to be\nexamining a great many places in the wall between different bricks; and\nat last, when she supposed there was no risk, or when there was nothing\nto alarm her, she would fly to her nest, and go to work with all her\nmight in fixing her piece of clay to the wall.\"\n\n\"Why did she wish her house not to be found out, Uncle Philip?\"\n\n\"Mr. Rennie supposed it was her instinct: she had seen probably some of\nthe insects which would destroy her young, watching her to see where\nshe was building; and sometimes after flying nearly to her nest with a\nload, she would turn back and fly towards the clay-bank, or take a large\nsweep off in another direction, and so come to her house.\n\n\"Besides the mason-bee, boys, there is the mason-wasp, which I have\nheard some persons call the _dirt-dauber_: it is very common, especially\nin the southern part of the United States. It works very much like the\nmason-bee, only it is apt to fix its nest under the eaves of old houses,\nwhich I think the mason-bee never does.\n\n\"There is also another kind of mason-wasp which will actually break a\nhard brick. Mr. Rennie says that he saw one at work on a brick of a hard\nyellow kind. Whether the wasp found a hole in the brick to begin with,\nhe did not know; but if he did he was hard at work making it larger: he\nwould break off a piece as large as a mustard-seed at a time. Here is\na drawing of that kind of wasp; and the insect is no larger than the\npicture.\n\n[Illustration: Mason-wasp.--Natural size.]\n\nIt seems wonderful that so small an insect should have so much strength.\nHere is a picture of its jaws, seen through a microscope, so as to\nappear a great deal larger than they really are.\n\n[Illustration: Jaws of Mason-wasp.--Greatly magnified.]\n\n\"I do not know, boys, that the masons we have been talking of, show us\nany _tools_ like those with which men who are masons work; but they show\nus, at any rate, how to make mortar by kneading or working it together;\nand they certainly show us that we were not the first who built walls.\nBut there is another kind of mason who works in stone. He picks out\nthe stones which are of proper size, and he fastens them together with\nmortar really as men do.\"\n\n\"Pray tell us of him, Uncle Philip.\"\n\n\"I will. The insect I mean is the caddis-worm, which is to be found\nsometimes in ponds, and very often in springs of fresh water. There\nare several sorts of them, but the one I am thinking of now, builds a\nstone house to live in. These worms are in the habit of making a little\ntube, sometimes of sand, or shell, or wood, or leaves, or stones, to\nlive in; and their skill consists in joining these perfectly, and\nmaking them stick together. But we are talking now of the caddis-worm\nthat uses stone. What the worm has to do is to make a tube out of small\nstones, that shall have a hollow about as large as a wheat straw, and be\nperfectly smooth inside. This is a pretty hard task--at least it would\nbe very hard to us. When the stone-mason wishes a stone of a particular\nsize or shape, and cannot find it, he takes his hammer and breaks one\nuntil it will suit; but the caddis-worm has no hammer, and must take\nthe stones just as it finds them. The little insect then has to pick\nout a great many stones before he gets the right one, because they have\nso many little rough points about them that it is very difficult to\nget those which will make the tube perfectly smooth inside. Remember,\ntoo, that the bottom or lower side of this stone case has to be pretty\nnearly smooth, so that the worm can drag it along on the bottom of the\nspring or pond (for it never comes out of it), and you will see that the\npicking out of the stones alone is no trifle. But besides this, it has\nto fasten them together with mortar.\"\n\n\"And can the worm really do this, Uncle Philip? Will not the water wash\nthe mortar all away?\"\n\n\"It certainly would if it were like common mortar. It was a long time\nthat men lived before they found out a mortar that would remain, and\ngrow hard under water. When they want to build a wall that is to be\nunder the water, they use a cement which is called pozzolana; it is\nmade of lava out of a volcano, and is water-proof. Our caddis-worm has\na cement too, which is better than pozzolana, and though it has been\ntried, it cannot be melted or dissolved in water. Here is a drawing of\nthe stone nest of a caddis-worm.\"\n\n[Illustration: Stone Nest of Caddis-worm.]\n\n\"Uncle Philip, you said that sometimes these worms built their nests\nof other things besides stones; let us hear something of them, if you\nplease.\"\n\n\"Very willingly, boys. Some build of shells: here are pictures of their\nnests.\n\n[Illustration: Shell Nests of Caddis-worms.]\n\nSome build of leaves, and others of pieces of reed or light bark.\n\n[Illustration: Reed Nest of Caddis-worm.]\n\nAnd a curious thing about those which build of light pieces of bark\nor reed is this, that they will make the top-piece come over so as to\nhide their heads, and prevent you from seeing them. Some build of sand;\nand then as the house would be so light that the water running from\nthe spring might wash it down and carry it away, the wonderful little\ncreature takes care to anchor it by fastening a pretty large stone to it\nwhen it has nearly finished it. And as the worm anchors it when it is\ntoo light, so it lightens it when it is too heavy, by fixing a bit of\nlight wood or hollow straw to it to buoy it up.\"\n\n[Illustration: Sand Nest, balanced with a Stone.]\n\n[Illustration: Nest of Caddis-worm, balanced with Straws.]\n\n\"Well, this is truly a wonderful insect. Uncle Philip.\"\n\n\"Truly so indeed, boys. In all these cases it uses its water-proof\ncement, and if you break its house to pieces, and will patiently watch,\nyou may see it build another. The insect always lives with its head out\nof doors, and its body inside; so that its head is firm and hard, while\nits body is soft.\"\n\n\"Uncle Philip,\" said one of the larger boys, \"there is one thing I have\nbeen thinking about, as you have been talking: these little masons have\nno trowel, but I believe I know of one animal that uses something like\nthat tool.\"\n\n\"Ah! What animal is it?\"\n\n\"Why, I was reading the other day something about the beavers building\ntheir dams and their houses, and the book said that they built their\nhouses of logs first, and then plastered them with mud, and that they\nused their tails for trowels.\"\n\n\"I am very glad to find that you remember what you read; but I am sorry\nthat your book did not tell you the truth. There have been very strange\nstories told about the beaver; and these stories have been taken from\none book and printed in another, so that an untrue account has gone down\nfor a great many years. The beaver is very ingenious, but is not quite\nso much of a mason as you suppose.\"\n\n\"Well, Uncle Philip, will you tell us the truth about it?\"\n\n\"Yes, boys, I will, so far as I know it myself. I have seen these\nanimals, for they were once a great deal more common in our country than\nthey are now; and many of the stories told of them are not true. But\nbefore I begin, let me tell you of one book which I think does tell the\nplain truth about them; and the truth is curious enough.\"\n\n\"What book is it, Uncle Philip?\"\n\n\"It is a book written on American Natural History, by Doctor John\nGodman. I knew him, boys, and a most excellent man he was. He is now\ndead--and he died a Christian. The book he wrote you will find worth\nreading, when you get old enough to understand it. But now for the\nbeaver.\n\n\"His tail is very broad and flat at the end, and might be used very well\nfor a trowel; but when he builds his house he does not cut down trees,\nand place them first, and then fill up the cracks with mud-mortar; but\nall the sticks and mud (and stones too when the beaver can get them),\nare first mixed up together, and the beaver builds his house with this\nfrom the very foundation. As soon as he has placed a lump of this stuff\nupon the wall, he turns round and gives it a blow with his flat tail;\nand that, boys, is all he does with his tail for a trowel. Sometimes\nhe slaps his tail upon the water when he is swimming; and some persons\nhave supposed that this was done by the king, or ruler, to call his\nworkmen. It may be so, but I do not believe it, because they almost\nalways dive as soon as they have slapped the water; and I think it is\nprobably a part of their motion in diving. In the autumn they cover the\noutside of their houses with mud, and they walk over it as they are at\nwork, and their tails drag along upon it; and this I expect made persons\nfirst suppose that they were plastering it, with the tail for a trowel.\nWhen they are caught and kept, boys, they still keep up this fashion of\nslapping with the tail; so that I rather think it is part of the nature\nof the animal.\n\n\"At another time, perhaps, I will tell you more about the beaver; but it\nis now late, and I must bid you good morning.\"\n\n\"Good day, Uncle Philip.\"\n\n\n\n\nCONVERSATION VII.\n\n _Uncle Philip talks to the Children about Animals that throw\n Dirt with a Spade; and about an Animal with a Hook; and about one\n that is a Wire-drawer._\n\n\n\"Boys, I have some men at work digging a small ditch for me, and I wish\nto see them; will you go with me?\"\n\n\"Oh, yes--very gladly, Uncle Philip; for you will be sure to tell us of\nsomething curious before we come home.\"\n\n\"Come on, then: yonder are the men at work; they have been very\nindustrious, I see.\"\n\n\"But, Uncle Philip, look! There is one of the men putting a bottle to\nhis mouth. Is that right?\"\n\n\"Yes, boys, right enough; for the bottle has nothing but molasses and\nwater in it; and the man is thirsty, I suppose. I would not employ him\nif he brought a bottle of spirits out with him, for two reasons. In the\nfirst place, I think that I ought not to encourage a man who gets drunk,\nby employing him; for I would rather give my money to a sober man who\nwill not spend it for rum and brandy, but will take care of his family:\nand, in the second place, a drunkard would not work faithfully without\nbeing watched all the time. I never knew a drunkard who was really and\nhonestly an industrious man.\"\n\n\"See, Uncle Philip, how strong that man is; what a large spadeful of\ndirt he throws out!\"\n\n\"Yes, I see, boys: do you think that men had the first spades in the\nworld?\"\n\n\"Ah! now you are going to tell us something about tools among animals:\nthat is good; we like to hear of that. What animal is it that has a\nspade?\"\n\n\"Oh, a very common animal indeed in some parts of our country. The\ncountry people call it a _woodchuk_, and sometimes a _ground-hog_:\nits right name is the marmot; and as there are several sorts, ours is\ncalled the Maryland marmot, to distinguish it; but it is in New-York,\nConnecticut, New-Jersey. Pennsylvania, Virginia, and some of the other\nstates, as well as in Maryland. This is rather a mischievous animal,\nand does harm to the clover-fields; but it is in making his house that\nhe uses his spade.\"\n\n\"Then he digs his house in the ground. Uncle Philip?\"\n\n\"Yes; he burrows, or digs his nest in banks of earth, or on the sides\nof hills; and he has sense enough to make the passage to the inside\nupwards, instead of downwards, so that water cannot run in. In digging\nsoft earth he uses his fore-paws to loosen the dirt, for his fore-legs\nare very strong; and if the ground is hard he will use his teeth too.\nAs he gets farther in, he throws the dirt with his fore-paws under his\nbelly, and when he has a heap gathered, he balances himself on his\nfore-feet, and begins to throw it out with his spades.\"\n\n\"What are his spades, Uncle Philip?\"\n\n\"His hinder feet, boys, which are very broad, and just fit to take up\nthe dirt as a spade does, and to throw it from him: there is a skin\nwhich grows between the toes of his hinder feet, so that he can spread\nthem out when he chooses, like a duck's foot.\"\n\n\"But, Uncle Philip, perhaps they are made so for the sake of _swimming_;\nthe duck's are.\"\n\n\"That is a sensible thought, boys. Always think for yourselves; and when\nyou make a mistake, try again: everybody is mistaken sometimes. Let it\nteach you to be modest and humble; but do not be afraid to think again.\nA person who is always thinking cannot _always_ think wrong. Now you\nsuppose the marmot's feet may be made like a duck's for swimming: let me\ntell you something else, and we shall see what you will think then. The\nmarmot hates a rain as much as you would if you had no umbrella; he very\nseldom even drinks water, and then only a little; and you cannot drive\nhim into a stream or pond; he is afraid of it. What do you think now?\"\n\n\"Why, Uncle Philip, we think that he is no swimmer.\"\n\n\"Very true, boys: so his feet, then, you now think, were made for\nspades, and not for paddles?\"\n\n\"We do. Can you tell us any thing more about this animal, Uncle Philip?\"\n\n\"Oh yes. The burrows or holes of the marmot run a great distance under\nground, and end in several chambers or rooms, according to the number\nthat is to live in them. They make beds in them of dry leaves, or\ngrass, or any thing soft and dry which they can find. They cram their\nmouths full of it to carry, when they are making their nests. As soon as\ncold weather begins, the animal goes into his house, and stops up the\nhole on the inside; and there he stays till the warm weather has come\nagain.\n\n\"He is quite a thief at times. I saw one once which a gentleman had\ntamed, and he played about the yard; but every thing that he could get\nhold of which was fit to make his bed of, he was sure to steal, and\ncarry into his hole under ground. When clothes were hung out to dry\nhe would take them off the line, and as soon as any were missed the\nwasherwoman knew very well where they were. She kept a long stick with\na hook at the end of it, and with this she drew them out of the burrow.\nHe soon found out what it meant, and whenever she used the stick, it was\nnecessary first to tie him up; for he did not choose to have his bed\nspoiled, and would run to the hole and try to get in, and prevent the\nclothes from being drawn out. One day he stole eight pairs of stockings,\na towel, and a little girl's frock; and he carried them into his burrow\nas far as six feet from the entrance.\n\n\"But, boys, as we have begun this morning upon the old subject of tools\namong animals. I will tell you of something which, though not exactly\na tool, is a very useful instrument, and is found belonging to a very\ncommon creature. Did you ever take notice of a bat?\"\n\n\"Oh yes, Uncle Philip, often, as they were flying about in a room at\nnight, but not nearer.\"\n\n\"Then you never saw what I mean, I expect. Our common bat, boys, has\ntwo very excellent hooks; one on each of what you call its wings: I\nsay what you call its wings, because the bat is not really a bird, but\na quadruped; that is, an animal with four feet: and when it is on the\nground, any one may see that it is a four-footed animal. If a monkey's\npaw should be flattened out very much, it would be something like a\nbat's paw or hand. The long finger-bones are just like the sticks of an\numbrella; there is a thin skin between them, and they stretch it out, so\nthat the air underneath will keep them up. When they are on the ground\nall this is folded up. Their hinder feet have five toes, all small, and\nending in sharp claws. On the fore-feet there is but one finger which\nthe bat can use much, because the others are like umbrella-sticks, as I\ntold you; and the end of that one is a hook. Here is a picture, in which\nyou can see it plainly.\n\n[Illustration]\n\nWhen the bat is on the ground, it is hard work for it to get along. At\nfirst it will reach forward a little to one side the hooked end of its\nfore-leg, and stick it in the ground; then it draws its hind-legs under\nits belly, and raising itself up, just tumbles forward its whole body.\nAt the next step it stretches out the other fore-leg, and hooks it, as\nit did before, and drawing itself up, tumbles forward again. The bat\ndoes not like a level place, because it cannot raise itself in the air\nfrom it. When they rest, they hang by the hooks; and here is a drawing\nof one, resting. In the other picture which I showed you just now the\nbat was flying.\"\n\n[Illustration]\n\n\"Uncle Philip, we did not know before that bats were such curious\nthings; we always thought that they were birds; but if these pictures\nare like them, these hooks are as good as fish-hooks.\"\n\n\"The pictures, boys, are very much like the animal, and the hooks are\njust as plain as they seem to be in the drawings. But how often do you\nsuppose that you have seen a bat?\"\n\n\"Oh, many hundreds of times; for they are very common.\"\n\n\"True, boys; and yet you never knew before that they had hooks about\nthem. Suppose that everybody had done as you have, just passed by the\nbats, without taking notice of them; I could not have told you then\nwhat strange creatures they are, for no person would have known any\nthing about them. You see, then, that men may have eyes, and yet not see\nthings; because they will not look for them. Use your eyes, boys; God\nmade them to be used.\"\n\n\"But, Uncle Philip, bats are such ugly things! and they can bite, too.\nWe are afraid of them.\"\n\n\"Ugly, boys! And what of that? Will you look at nothing but what is\nhandsome? If the bat could think and speak, I expect he would call\nyou very ugly. But it is foolish, boys, to be afraid of these smaller\nanimals. There are many creatures which might hurt you, and I would\nadvise you to keep out of their way: but it is silly to be afraid of\nevery poor little insect or animal which you see. I have seen a large\nboy cry when he saw a poor little caterpillar or bug near him. Now\nthere are very few insects, indeed, which can or will hurt you; and a\ngreat many of them you may watch without touching them at all. And I\nthink that he is a wicked and cruel boy who kills every poor bug that he\nsees, merely because he is stronger than the bug. It would be a great\ndeal kinder and wiser in the boy to notice what the bug was doing, for\nthen he might learn something worth knowing.\"\n\n\"But, Uncle Philip, is it wrong to kill _spiders_?\"\n\n\"Spiders! Why, boys, the spider is one of the very last of these little\ncreatures that I should wish to kill. There is not a more curious little\nanimal in the world, nor one that will pay a man better for watching its\nmotions. At some other time I will tell you all about spiders and ants,\nfor I have noticed them a great deal: but now, just to show you how much\nyou would lose if you should kill all the spiders, I will talk with you\nabout a tool which man uses, and which he might have learned to make\nfrom a spider.\"\n\n\"Oh, do tell us; what is it?\"\n\n\"The next time you go to Mr. Brown's, the silversmith, ask him to show\nyou his plate for drawing out wire. Tell him that I told you to ask\nhim, and he will show it to you. You will see a flat piece of steel with\nholes made through it in regular lines, beginning with a large size, and\ngrowing smaller and smaller until the last is very small indeed.\n\n\"Now the wire is drawn through these holes; beginning at the larger\nones, and passing every time through the next smaller one, it stretches\nthe wire out, until it becomes as small as the workman wishes it to be.\n\n\"The spider is a wire-drawer, too; for it has a contrivance to draw\nout its threads, and make them smaller or larger, as it pleases. If\nyou will look at a very large spider, you can see with your naked eye,\njust at the end of its body, four, and sometimes six, little knobs like\nteats, with a circle around them. These are its spinners. Each one of\nthese small knobs, inside of that circle, is so full of little holes or\ntubes, that Mr. Reaumur (of whom I told you before, you will recollect)\ncalculated that a place no larger than the point of a pin had a thousand\nof these little holes in it. These holes are sometimes so very small,\nthat another gentleman,[8] who looked at spiders through a microscope\nvery often, thought it would take four millions of the threads which\ncame through those holes to make one thread as thick as a hair of his\nbeard. Here is a picture of a spider hanging by a thread coming out of\nits spinner, or, as it is sometimes called, its spinneret.\"\n\n[Illustration]\n\n\"Then, Uncle Philip, the spider does not spin its thread all at once?\"\n\n\"No, boys. Fine as you see that thread to be, it is not one single line,\nbut it is made of many thousands joined together. The spiders have\nlittle bags of gum within their bodies, near their spinners, and out of\nthese they draw the threads: when they have come out about the tenth\npart of an inch, they join them all into one with their claws; and they\ncan shut their spinners when they please, so as to make the threads\nlonger or shorter; and they can break them off, too, when they wish.\"\n\n\"But, Uncle Philip, we do not see why there should be so many threads to\nmake up one.\"\n\n\"I cannot exactly tell you, boys, why there are so many; but probably\nto make the thread dry quicker, by letting the air touch so many parts\nof it: and I expect, too, the thread is stronger, because we know that\nin two pieces of cord of one size, if one is made of several smaller\ncords put together, it will be stronger than the other, which was spun\nall at once. The following is a picture of the spider's spinnerets, and\nsome of the threads as it appears through the microscope; only you must\nrecollect that _all_ the threads are not drawn: there are a great many\nmore than you see in the picture.\"\n\n[Illustration: Spider's Threads coming from the Spinnerets.]\n\n\nFOOTNOTE:\n\n[8] Leuwenhoek.\n\n\n\n\nCONVERSATION VIII.\n\n _Uncle Philip tells the Children of a Door, with a Hinge and\n Spring to it, made by a Spider; and shows them Pictures to let them\n see the Difference between God's Work and Man's._\n\n\n\"I was thinking, boys, last night, of what you said about killing the\npoor spiders; and I was sorry that I did not then recollect one thing\nabout a spider which I could have told you, and which would have made\nyou like the poor little creatures better. However, I determined that\nwhen you came to see me again, it should be the first thing I would tell\nyou, if you wished to hear it.\"\n\n\"Wish to hear it! Why, Uncle Philip, we always wish to hear you tell us\nof any thing that you please to talk about. You have told us of a great\nmany strange things, about which we knew nothing before; and we will\nthank you to tell us the story about the spider.\"\n\n\"Very well, boys; you shall hear it. Pray, do you not think that it is\na piece of difficult work to make a door to a house, and to make hinges\nto hang it with, and to fit it so nicely that when it is done you cannot\nsee the joints where the door is shut?\"\n\n\"Indeed it is a piece of very hard work. Uncle Philip, and it takes\nthe carpenter a long time to do it; and it is hard work, too, for the\nblacksmith to make the hinges. But what has that to do with the story\nabout the spider?\"\n\n\"Patience, boys, patience: you shall know presently. Never be in too\ngreat a hurry: it is a bad plan. I have always noticed that those\npersons who hurried most, went slowest in the end. Another question I\nwish to ask you is this,--do you not think it was hard work for the\nfirst man who ever made a spring, and put it on a door, to make it shut\nitself again when it had been opened?\"\n\n\"Yes, it was so: and the man who does it now gets well paid for it.\"\n\n\"Very good, boys. And now what will you say when I tell you that a poor\nlittle spider did all these things long before man did?\"\n\n\"What, Uncle Philip! A spider make a door with a hinge and a spring to\nmake it shut itself!\"\n\n\"Yes, boys; a spider. Do you think he deserves to be killed for doing\nit?\"\n\n\"Oh no, no! But pray tell us all about it. Uncle Philip.\"\n\n\"This kind of spider, then, boys, I saw in Jamaica, and I saw its house,\ntoo. It is called the mason-spider. The nest or house which I saw was a\ntube made of very hard clay, about six inches long, and an inch across,\nand was a little bent at one end. The inside of this tube was lined\nall the way through with a kind of soft silky stuff, something like\nsilk-paper, but stronger, and it was of a yellowish colour; but the\ncurious part was the door. I never saw any thing which an insect had\nmade more strange than that. This door was round, about as large as a\nquarter of a dollar, and was a little hollowed on the upper side like\na saucer; the inside of it was rounded like the outside of the saucer.\nIt was of the same stuff with the lining of the nest, and seemed to be\nmade of more than a dozen pieces of that lining, put one on the top of\nanother: it was shaped so, too, that the inside layers or pieces were\nthe broadest, and the outside ones became smaller and smaller, except\nat the hinge, which was about an inch long. All the pieces in the door\nwere joined into this hinge, and then the hinge was joined and worked\ninto the lining in the tube. That made the hinge the thickest and\nstrongest part of the whole work. How the spider made it so, boys, I\ncannot tell; but so it was, that this hinge not only was a hinge, but\nwas so good a spring, that whenever the door was opened it would shut\nitself immediately: and when shut, it fitted so nicely that it was very\ndifficult to see the place of joining.\"\n\n\"Well, Uncle Philip, this is most wonderful! But will not the hinge wear\nout at last?\"\n\n\"Wonderful as it is, boys, it is all true. As to its wearing out, I\ncannot tell you; but I know that a gentleman who had one, said that his\nfriends were very anxious to see it; and there were so many of them,\nthat he had to open the door and let it shut itself many hundreds of\ntimes to satisfy them; and it did not hurt the spring at all.\"\n\n\"Uncle Philip, we shall not kill the poor spiders any more.\"\n\n\"A good resolution, boys: only let them alone, and they will not hurt\nyou. There is another kind of mason-spider, which I never saw, but I\nhave read of it. It is found in the south of France; I did not happen,\nhowever, to meet with one while I was in that beautiful country. This\nkind digs a gallery or hole under ground as much as a foot deep. She\nlines it with a sort of silk glued to the walls, and makes her door,\nwhich is round also, with many layers of mud or earth all kneaded and\nbound together with some of her silk. On the outside, the door is flat\nand rough, to make it appear like the dirt around it, and hide it; on\nthe inside it is shaped like the inside of the door of the other spider\nI have told you about; and all covered with a coat of fine silk. The\nthreads of this silk are left long on one side, and fastened to the\nupper part of the hole; and these make the hinge. There is no spring to\nthis; but when the spider pushes its door open and comes out, it shuts\nagain by its own weight. If this door is forced open by any one when\nthe spider is at home, she will catch hold of it and pull it in; and\nsometimes even when it is half-opened; she will snatch it out of the\nhand. Here is a picture which shows the nest open, and another of it\nshut; and there is a drawing of the spider, too.\n\n[Illustration: A, the Nest shut; B, the Nest open; C, the Spider; D, the\nEyes, magnified; E, F, Parts of the Foot and Claw magnified.]\n\nA gentleman says, in a book which he wrote about insects, that he once\nbroke one of these doors off, to see what the spider would do.\"\n\n\"And what did she do, Uncle Philip?\"\n\n\"She made another door; but took very good care not to put any hinge\nto it, for fear she should be disturbed again. But when she thought\nall danger was gone, she could then put a hinge to it, you know; and\nprobably she did.\"\n\n\"Well, Uncle Philip, we thank you again for this account of the spiders,\nand shall always look at them hereafter with more pleasure. Who would\nhave thought that we should ever find doors and hinges among such little\ncreatures, and these too so very well made and fitted!\"\n\n\"Why, boys, I have noticed the works of God very often; and I will now\ntell you one thing which I always found. It is this: a piece of the very\nbest work which man can make is really coarse when you compare it with\nthe work of God. The poor spider that we have talked about, when she\nmakes her door, makes it to fit perfectly; because in doing that one\nthing, God made her to know perfectly how to do it. The knowledge is\nGod's, boys; but the work is the spider's: but in making any thing else,\nexcept about her house, the spider knows nothing.\"\n\n\"Uncle Philip, you told us once that you were very fond of watching all\nsorts of dumb animals, and we think now that we know the reason.\"\n\n\"Well, what is it?\"\n\n\"It is because you see so much of God's knowledge in them; is it not?\"\n\n\"Yes, my dear boys, it is. When I look at many things which man makes\nor does, I think to myself, 'Now this thing is likely to have a mixture\nof sense and nonsense in it; the sense is God's, and the nonsense is\nman's.' But when I look at a thing made by one of the dumb creatures for\nits own comfort and safety, like the spider's house, with its door and\nhinge, for instance, I say to myself, 'Now here is the wisdom of God,\nwithout any of man's nonsense.' And yet, boys, men are far wiser than\nany other animal in this world.\"\n\n\"But, Uncle Philip, you said that a piece of man's best work was really\ncoarse: some things must be neat, we should think. Is the point of a\nneedle coarse? It does not seem so.\"\n\n\"Boys, you have mentioned the very thing which was in my mind when I\nspoke. The point of the smallest needle is very coarse. You have heard\nme talk of the microscope. I told you it was a set of glasses, so\nfixed that when you looked through them, it made small things appear\nvery large: on some other day, perhaps, I will let you look through\nmy microscope for yourselves; but now, I just wish to show you the\ndifference between the work of God and that of man. Let us go home,\nand I will show you some pictures I made, and you can see in them the\ndifference. Last winter, you know, was very cold, and there was a great\ndeal of snow: one day, while the snow was falling pretty fast, I was\nobliged to go out; and as the flakes of snow fell upon the sleeve of my\ncoat (which was black), I thought they had a curious shape, and did not\nall appear alike; so when I returned home I caught some of the flakes,\nand looked at them through my microscope. They were so beautiful that I\nmade pictures of them; and as we have now reached home, just let me step\ninto my study, and I will bring them to you. Here they are, boys.\"\n\n[Illustration: Snow-flakes, seen through a Microscope.]\n\n\"Oh, Uncle Philip! these are very pretty; they are all so different,\ntoo!\"\n\n\"Yes, boys, I picked out different ones to draw: when I was done, I\nbegan to look into my books to find out what others had written about\nthis thing; and I found that a gentleman named Dr. Hook had seen more\nthan a hundred different shapes and sizes of these flakes. This is God's\nwork, boys.\n\n\"Now I have brought you out another picture: it is the point of a very\nsmall needle, seen through the very same microscope which showed me the\nsnow-flakes. Just look at it, boys. This is _man's_ work.\"\n\n[Illustration: The Point of a very small Needle, seen through the\nMicroscope.]\n\n\n\n\nCONVERSATION IX.\n\n _Uncle Phillip tells the Boys a Story about a Philosopher and\n his Kite.--He tells them, too, about Ants that have Awls, and build\n Cities, and Stairs, and Bridges, and many other things._\n\n\n\"So, boys, you have come again to see me. I am very glad of it; for as\nthis is a leisure day, we shall have time enough to talk: but what is\nthat you have there? Oh, I see now; it is a kite.\"\n\n\"Yes, Uncle Philip; it is a French kite that we have been making, and we\nhave come to ask you to go out with us this morning and see us fly it.\"\n\n\"Very good, boys; I will go. I am an old man now; but I remember that I\nwas a boy once, and loved to make a kite sail. It always makes me happy\nto see boys and girls playing about in health, provided they are not\nwasting time, and their play is not to do harm to anybody or any thing.\nSo come on; we will go out upon the green common yonder, behind the\nchurch, and I think we shall have a grand kite-flying, for the wind is\nabout right.\n\n\"There, boys! up she goes! Let out the string. I think she behaves very\nwell; there, she is done pitching about: now she is steady; see how she\nmounts. Ah, that is a very good kite.\"\n\n\"Uncle Philip, I was reading a book yesterday which said, 'A philosopher\nonce found great help from a kite.' What did it mean?\"\n\n\"Do you know what a philosopher is?\"\n\n\"Yes, Uncle Philip; a philosopher is the same thing with a very wise\nman, is it not?\"\n\n\"That is pretty near the meaning. Philosopher means a person who loves\nwisdom; and such a person, you know, will always be trying to get\nknowledge; and a person who is always trying to get knowledge is apt to\nbe a wise and learned man.\"\n\n\"Well, how could a kite help a man to get learning? Did he read the old\nnewspapers it was made of? I cannot see any other way.\"\n\n\"But there may be another way without your seeing it, you know. The\nphilosopher whom your book meant was Dr. Franklin. Did you ever hear of\nhim?\"\n\n\"Oh, yes; he was born in Boston, and was a printer, and afterward became\na very great man.\"\n\n\"That is true. He was a man of excellent sense, who both read and\nthought a great deal; and in the war which the people of this country\nhad with England to get their freedom. Dr. Franklin's sound sense was of\ngreat use to his countrymen.\"\n\n\"But, Uncle Philip, how did the kite help him?\"\n\n\"I will tell you. If you take a proper piece of glass, or sulphur, or\nsealing-wax, or rosin, and rub it for some time, and then hold it near\nto small bits of thread or paper, the thread or paper will fly towards\nthe glass or sealing-wax, and stick to it for a short time. That which\nmakes them fly to the glass and stick there, is called _electricity_.\nAfter this was found out, men went on slowly finding out more and more,\nuntil at last a man named Hawksbee made a large machine with a glass\nbarrel, which could be turned around by a handle like that which you see\nto a grindstone; and with this machine (which I will show you at some\nother time) he managed to get a great deal of this electricity, and it\nwould shoot off in sparks, which appeared like little lightning.\n\n\"And now, boys, what I am going to tell you will show you the use of a\nman's eyes. Dr. Franklin knew all about the electrical machine, and was\nvery fond of drawing off the sparks from it, to see what he could find\nout about it. And when he saw it appeared so much like lightning, and\ncould feel too a spark strike his hand a smart blow, he began to think\nthat perhaps it was exactly the same thing with the lightning which came\nfrom the clouds; so he determined, if he could, to find out whether it\nwas or not. He was a great deal troubled for some time to know how he\nshould get down any of the lightning from the clouds; until at last,\none day, he saw a boy flying a kite; and that showed him the way. So\nhe took a large silk handkerchief, and stretching it upon sticks, soon\nmade his kite; and not long afterward, when he saw a black thunder-cloud\ncoming up, he took his kite and walked out of Philadelphia (where he\nthen lived) into the fields, and sent his kite up. He had a string like\nyours, made of hemp, and to the end of this he tied an iron key, and\nthen fastening it to the post of a shed by a silk cord, which he tied\nto the end of the hemp string, he got under the shed, and waited a long\ntime. Now, boys, if he had been impatient, all his work would have been\nof no use. But he even waited, after a very heavy cloud had passed over\nhis kite without giving it any of its lightning. At last, when he was\nalmost ready to give it up and go home, he saw some loose threads on the\nhemp string rise and stand up straight, just as he knew the electrical\nmachine would make them do. He directly put his knuckle to the iron key,\nand off came the spark, which he knew at once was exactly like the spark\nwhich he could get from the machine. And so, boys, he found out what\nhe wished to know; and this was the way in which the kite helped the\nphilosopher.\"\n\n\"Well, Uncle Philip, this is really a very pretty story about Dr.\nFranklin and his kite. Was anybody with him?\"\n\n\"Nobody but his son; he took him out to help him raise the kite. But,\nboys, I see the other lads are walking on towards the bridge with our\nkite; let us follow them, and as we go, I will tell you of an electric\n_animal_.\"\n\n\"Oh, we shall be very glad to hear of him.\"\n\n\"Listen, then. There is a kind of eel, which when it is touched will\ngive a very hard blow, just like an electric shock, to the person who\ntouches it.\"\n\n\"Is there any spark, Uncle Philip?\"\n\n\"No, boys; there is no spark,--but the blow is tremendous. I remember\nreading of one of these fish which was caught in a net, and a foolish\nsailor would take it up, though he was told it would hurt him. The fish\nshocked him so violently that he fell down in a fit, and it was a long\ntime before he came to his senses; and his story was, that the moment he\ntouched the fish, 'the cold ran swiftly up his arm into his body, and\npierced him to the heart.' The fish has this power to defend itself, and\nto kill other fish for food.\"\n\n\"But, Uncle Philip, how do they ever manage to catch them alive? I\nshould think they would be shocked to death.\"\n\n\"I will tell you. A very sensible traveller and learned man[9] gives an\naccount of the manner in which they catch them, by a way called, by the\nSouth American Indians, 'fishing with horses.'\"\n\n\"Fishing with horses! What does that mean?\"\n\n\"The savannas, or large open plains, in South America have a great many\nwild horses and mules running over them. M. Humboldt says that the\nIndians caught about thirty of these, and drove them into the pond where\nthe electrical eels were. The horses made a great noise, and stirred up\nthe mud with their hoofs, and this brought up the eels from the bottom\nin a great rage. They were very large, and looked more like water-snakes\nthan like eels; and rising to the top of the water, they crowded under\nthe bellies of the horses and mules, and began to shock them. The poor\nhorses would try to get out of the pond; but the Indians, with spears\nand long reeds, would stand around to hinder them from coming out: some\nof them, too, would climb upon the trees around the banks, and get out\nupon the branches which were over the pond, and by crying out aloud,\nand using their long reeds, kept the horses in the pond. The eels would\ncontinue to shock them with tremendous blows, and a great many of the\npoor creatures were either stunned or killed, and would sink. Those not\nkilled would pant and raise their heads, while their eyes would show\ntheir pain, and they would try to get out. The Indians still drove them\nback, but some few escaped, and reached the shore, stumbling at every\nstep, and would stretch themselves on the bank, tired out, and benumbed\nin their limbs by the shocks they had received. M. Humboldt says that in\nless than five minutes after the fight began, two horses were drowned;\nand he thought that the end of it would be, that every horse which did\nnot get out of the water would be killed: but at last the eels became\ntired, and began to disperse. This is just what the Indians wish. They\nknow that the eels have spent so much of their electrical power that\nthey will need a long rest. It takes them a great while to get back\ntheir strength; so that if, the next day after such a fight, you send in\nmore horses, they cannot kill one. When the eels, tired out in this way,\nbegin to separate, they will swim to the edge of the pond, and there the\nIndians take them with small harpoons fastened to long cords. When the\ncords are dry, the Indian feels no shock in raising the eel out of the\nwater. In this way M. Humboldt got five, all alive, and very little\nhurt. But here we are at the bridge.\"\n\n\"Uncle Philip, suppose we sit down under the shade of this tree, and\nrest.\"\n\n\"I am willing, boys; but take care of the damp ground: there is an old\npiece of timber that the men have taken out of the bridge, for I see\nthey have been mending it; we will sit on that.\"\n\n\"But, Uncle Philip, what shall we do with the kite? shall we draw it\ndown?\"\n\n\"Do with it! Why, just tie the end of your string to that root, and it\nwill take care of itself in this wind.\"\n\n\"What a monstrous piece of timber this is. Uncle Philip! It must have\ntaken a great many men to move it; and see--there are some larger ones\nstill in the bridge. It must be a difficult work to build a bridge.\"\n\n\"Yes, it is so: but there are bridges much harder to build than our\nlittle wooden one here. Some are built of stone, and it takes years to\nfinish them. None but a good workman can plan and build a good stone\nbridge: but I know a little fellow that can make as good a bridge as\nanybody; and yet no man ever taught him the trade.\"\n\n\"Ah! now we know what is coming. You are going to tell us of a dumb\nanimal that can make a bridge.\"\n\n\"Yes, I am: and a small animal it is, too, for it is an ant.\"\n\n\"What sort of an ant is it?\"\n\n\"It is called the white ant; and as there is a great deal that is very\ncurious about this insect. I think that I had better tell you all about\nit at once.\"\n\n\"Pray do, Uncle Philip; you know you promised to tell us about ants.\"\n\n\"True, boys; and I like to keep my promises. In my story about these\nants, I think, if you attend, you will find more tools; and besides\nthat, you will hear of a great many things which man makes, and which\nshow matters quite as strange as any of which I have yet told you.\"\n\n\"What are they, Uncle Philip?\"\n\n\"They are the building of something like a city, and bridges, and\nstairs, and roads, and tunnels under ground, and--\"\n\n\"Oh, let us hear--let us hear! We have heard nothing equal to this yet.\"\n\n\"Well, then, I begin by telling you that these insects are very common\nin Africa, and in the East Indies, and are troublesome enough, for they\neat almost every thing but metal and glass. They love wood, though,\nbetter than any thing else; and they are so numerous that they destroy\nit wonderfully fast. They are very cunning, too; they never eat the\noutside of the wood first, but will work upon the inside, so as to leave\nthe outer part not thicker than a piece of pasteboard. But the curious\nthings I meant to tell you were about their city; so I will go on to\nthat. When they first begin to build you will see little hills shaped\nlike a sugar-loaf, and rising up above the ground about a foot, or a\nlittle more. Here is a picture of them.\n\n[Illustration]\n\nThe highest of these little hills is always in the middle; and they go\non building more and more, and making them all higher, still keeping\nthe tallest one in the centre. When they have made them as high as\nthey wish, then they fill in the spaces between the tops of all these\nsugar-loaf hills, so as to make one roof over all. Here is a picture of\none finished.\n\n[Illustration]\n\nAfter this is done, they take down nearly all of the little sugar-loaf\nhills inside; for they only wanted them for a scaffold to support the\ntop while they were building it.\"\n\n\"Uncle Philip, what is all this built of?\"\n\n\"It is built of clay, which the ant makes almost as hard as stone.\"\n\n\"Are they strong, Uncle Philip?\n\n\"So strong, boys, that five men may stand on them; and it is a common\nthing for the wild bulls to get upon them and look out, while the rest\nof the herd are feeding below.\"\n\n\"Why, how high are they?\"\n\n\"Oh, of different heights; some as many as five or six feet, others are\ntwelve, and the largest are as high as twenty feet, and would easily\nhold a dozen men.\"\n\n\"And how large are the ants, Uncle Philip?\"\n\n\"Not above a quarter of an inch high as they stand. Now, boys, just\ncompare the size of one of these nests with the size of the ant that\nmade it; and it is quite as large in proportion as the city of New-York\nis when compared to a man's size; yes, it is a great deal larger in\nproportion. These nests are sometimes five hundred times as high as the\nants which build them: now suppose that men built their houses five\nhundred times as high as themselves, and as large at the foundation in\nproportion to their height, what monstrous buildings they would be! But\nlet me go on. This outside shell, which I have been telling you how they\nmake, is nothing but the wall of the city; the buildings are all inside\nof that.\"\n\n\"But, Uncle Philip, there is one thing I have been thinking of which\nwould make it more like a city still. But I hardly think they can have\nthat.\"\n\n\"What is it?\"\n\n\"Why in a city, you know, where there are a great many people, there are\na great many trades: some do one thing and some do another to get money.\"\n\n\"Yes, that is true; and I am glad that you mentioned it, because it\nreminds me of one thing I intended to tell you about these ants. Now,\nit would not be reasonable, you know, to expect the ants to have many\ndifferent kinds of business to do, as the people in New-York have; but\nstill, boys, they are not all alike, and they do have work of different\nkinds. There are in the city of the white ants a king and a queen, and\nsoldiers, and labourers, or workmen, and all these are different. Here\nare pictures which will show them all to you. This is the king.\n\n[Illustration: King of the Termites.]\n\nAt first he has four wings, but soon loses them. He never grows any\nlarger after he loses them. The king may be known by his having two\nlarge eyes. Here is the queen. She is the mother of the whole city;\nand you see what a large body she has. It is full of eggs, and eighty\nthousand will come from her in twenty-four hours. She also has two eyes.\n\n[Illustration: Queen of the Termites.]\n\n\"Here is a soldier: he has a large head, armed with two hooks, shaped\nlike a crooked awl, and very sharp.\n\n[Illustration: Soldier of the Termites.]\n\n\"For every one of these soldiers there are about one hundred labourers.\nThe soldiers do the fighting; and though they are perfectly blind, they\nfight well, and are very brave. The following is a picture, too, of the\nsoldiers' awls, seen through the microscope, to show you how sharp they\nare.\n\n[Illustration]\n\n\"And here is a picture of the labourer; the largest part of the city is\nmade up of the labourers,--which shows us, I think, boys, that there is\nmore need of working than there is of fighting.\n\n[Illustration: Labourer of the Termites.]\n\nThis class, like the soldiers, is blind, and scarcely ever go into the\nopen air; their work is mostly under ground or in the inside of wood.\nBoth, however, do come out when their city is attacked and broken: but I\nwill tell you of that presently.\"\n\n\nFOOTNOTE:\n\n[9] M. Humboldt.\n\n\n\n\nCONVERSATION X.\n\n _Uncle Philip tells the Children more about the White Ants._\n\n\n\"Now, boys, as I have told you about the kinds of different work which\nthese ants do, we will go on, and I will tell you about the inside\nof the city. The first thing to be done is to build a house for the\nking and queen. This is the first house built in the city, and always\nstands in the centre, directly under the point of the roof or top of\nthe outside wall. It is built nearly on a level with the ground, and is\nshaped very much like a long oven, or the half of an egg split through\nthe long way. The floor is exactly level, and about an inch thick; the\nroof is about the same thickness. The doors are on a level with the\nfloor, and just large enough to let one of the labourers go in.\"\n\n\"Why, Uncle Philip, how do the king and queen ever get out then.\"\n\n\"They never do get out, boys; they live in that house always, and they\nare not the first kings and queens who have found that a palace is\nsometimes a prison. Just around this house of the king and queen are\nother houses built of clay, arched at the top, and of different shapes.\nThese are for the servants or labouring ants, who remove the eggs of\nthe queen as fast as she discharges them. The soldiers also live in\nthese houses. Next to these are the magazines, that is, the houses where\nthey keep their food, such as dry juices of trees and gums; and mixed\nup with these are the nurseries. These are made by the labourers, and\nare different from all the other buildings, for they are made of wood\ngnawed or broken into fine threads, and joined together with some kind\nof gum, and around each of them there is a case of clay. These nurseries\nare to carry the eggs into for the young ants to be hatched. Between\nall these different houses or parts there are thousands of galleries\nor ways, which run among them and separate them from each other, and\nthese may be called the streets of the city. These streets run in all\ndirections, and extend as far as the outside wall; and houses are built\non top of houses, and streets run over streets, until they reach up\nas high as two-thirds of the inside wall. But under the top of their\noutside case they always leave a large open place that is never filled\nup with houses. And around this space they will build three or four\nlarge arches, sometimes two or three feet high; these I suppose are to\nprevent the houses from falling in towards the centre of the city, which\nis an open space, and on the other side they are fastened to the outside\nwalls, so that these houses are very firm.\"\n\n\"And what is all this made of, Uncle Philip?\"\n\n\"All of clay, except the nurseries, which I told you were made of wood\nand gum. Over the house of the king and queen there is a sort of flat\nfloor, some distance above it, with nurseries and magazines between the\nunder side of it and the top of the queen's house. This floor will not\nlet the water through it, so as to wet the palace where the king and\nqueen live, but will turn it off into large trenches or gutters under\nground, of which I will speak directly. The bridge I told you of they\nbuild from this floor in the open space, directly under the top or dome\nof the outside wall; it rises up and is joined to some hole in the side\nwall of the houses above it.\"\n\n\"How large is it, Uncle Philip?\"\n\n\"Why, sometimes it is half an inch broad, a quarter of an inch thick,\nand ten inches long; all made of clay, so that it is very strange how\nthey manage to join it to the wall without its falling down by its own\nweight while they were building it.\"\n\n\"And what do you suppose this bridge is for, Uncle Philip?\"\n\n\"Why, I think there can be no doubt what it is for. When the city has\nbeen growing for some time, some of the nurseries will be very high up\nabove the queen's house; but the labourers have to carry her eggs into\nthem, no matter how far off they may be. If they carry them through all\nthe streets, they will have to walk as many as fifteen or twenty feet,\nfor it would be five or six feet in a perfectly straight line, and these\nstreets are very crooked; but if they make a bridge in the open space\nin the centre, they can then go from the queen's house over the bridge,\nand get to the upper nurseries without travelling more than two feet. So\nthey made the bridge to shorten the way, to be sure.\"\n\n\"This is very wonderful: but you said something about large trenches or\ngutters underground; what are they, Uncle Philip?\"\n\n\"These galleries lead from the city under ground, and are as large as\nthe bore of a large cannon; they are thirteen inches across, and more\nthan a hundred yards long. I have already told you that the labourers\nnever come out into the light, when they can help it; and these\nunderground ways are the great roads to the city, to fetch in clay, or\nwood, or water, or provisions: and now I will tell you another thing\nwhich shows a great deal of sense. As some of their houses are very high\nup, you know they would find it very hard to climb up through all the\nstreets with a heavy load in a straight line; so when these large ways\nunderground reach the outside wall, they just come through and keep on\nwinding around the inside of it like a corkscrew all the way to the top;\nand there are other galleries opening from it at different places into\nthe city. One thing has been noticed about these ants; they can scarcely\nclimb at all up a perpendicular wall. Therefore on the upright side of\nany part of the city you may see a road made, standing out from the wall\nlike a ledge; it is flat on the top, and half an inch wide, and goes\nup gradually like a stair-case, or like a road cut out on the side of a\nmountain.\n\n\"Here is a picture of one of their cities cut straight down through the\nmiddle. At the bottom, in the centre, is the queen's house; over it is a\nfloor, and the two crooked things you see rising up from the floor, are\nbridges.\"\n\n[Illustration]\n\n\"But, Uncle Philip, you said you would tell us about the soldiers and\nlabourers coming out when the city is attacked.\"\n\n\"Yes, I did. As soon as a hole is made in the outside wall, you will\nsee a soldier run out, and walk about as if to look around; but as he\nis blind, it cannot be to see what the danger is. He may have some way\nthough of finding out without seeing. Presently he will go in, as if\nto tell the others, and then out pour the soldiers in great numbers,\nas fast as the hole will let them; and just as long as you strike the\noutside wall, they will continue to rush out. They seem to be in a\nterrible passion. They are in such a hurry that sometimes they slip, and\nroll down the outside of the hill; but they jump up again instantly,\nand begin to bite every thing they run against, for they are blind and\ncannot tell a friend from an enemy.\"\n\n\"Do they bite hard, Uncle Philip?\"\n\n\"Very hard indeed. They make their hooked jaws, which are like awls, you\nknow, meet at every bite; and if it should happen to be a man's leg they\nget hold of, you would see upon his stocking a spot of blood an inch\nlong. At every bite too you may hear their jaws snapping together and\nmaking quite a noise. Some of them too (perhaps they are the officers)\nare constantly beating with their awls upon the outside wall, and make a\nsound something quicker and sharper than the ticking of a watch. You may\nhear it at a distance of three or four feet. When these biters lay hold,\nnothing will make them let go; you must tear them away by pieces. After\nyou stop striking the wall, in about half an hour they seem to get over\ntheir rage and go back into the city, and then out come the labourers.\nWhile the noise continues you will not see one of them; they all fled\nat the first appearance of danger. But now they come, each one with a\nbundle of mortar in his mouth, ready made; and they stick it on the hole\nso fast, and with such order, that though thousands and thousands are at\nwork they never interrupt each other. And while the labourers are busy,\nif you look you may commonly see a soldier or two walking about; but\nthey never touch the mortar, nor help in any way to mend the hole. One\nof these soldiers always stands near the spot where the labourers are at\nwork, and every now and then turns slowly around, and frequently lifts\nup his head, and with his awls beats upon the building, and makes a\nsort of hissing noise. As soon as that is done you may hear a loud hiss\nfrom all the labourers, both on the outside of the wall and from the\ninside of the city, and then the labourers run faster, and work as quick\nagain. If you attack the nest again, away run all the labourers as fast\nas their legs will carry them, and out pour the soldiers as before; and\nthe same thing is always seen upon every attack, of soldiers to fight\nand labourers to work.\"\n\n\"Well, Uncle Philip, this is a very strange story; much more interesting\nthan any we have yet heard.\"\n\n\"I thought it would please you, boys; but there are still stranger\nthings among animals, and perhaps you will say so when you know more.\n\n\"But it is now time to return home; so take in the kite and let us be\ngoing.\"\n\n\nFOOTNOTE:\n\n[10] Mr. Huber the younger.\n\n\n\n\nCONVERSATION XI.\n\n _Uncle Philip tells the Children about some other Ants, that\n are very good Masons, and build Walls and Ceilings; and a Story\n about a very sensible Ant, which seemed to think a little._\n\n\n\"Good morning, good morning, Uncle Philip; we have been so much\ndelighted with the stories about the white ants, that we hope you have\nmore of the same kind to tell us.\"\n\n\"Why, as to that matter, boys, I can tell you much more about ants, for\nthere are many kinds of them; but I am not sure that I can show you any\nof the _tools_ with which they work, though a large portion of their\nwork is like that of man; and they have, too, several customs which our\nfellow-creatures have. I can tell you, however, of several other animals\nwhich do use tools shaped like those used by men, if you would rather\nhear of them.\"\n\n\"If you please, Uncle Philip, we will hear of them at another time; but\nnow we would rather have you tell us of the other kinds of ants.\"\n\n\"Oh, very well, boys; it shall be as you wish. All that I desire is to\ninstruct and amuse you, and I am sure that the ants can furnish a good\nlesson to us. I shall begin with the mason ants. They always build their\nnests either of clay which is damp, and dug from the inside of their\ncity under ground, or which has been made wet by the rain; and a part of\ntheir building is always above the ground, so that you can easily see\nit. There is no fixed rule for the ants to build by. Their cities are\nnot all alike in the inside. Sometimes the walls are larger and coarser,\nand the ways and galleries are higher, than at other times. The rooms,\ntoo, are different in shape and size, so that this industrious little\ninsect seems to have sense enough to work in the best way according to\ncircumstances. There is only one general rule which they seem to have,\nand that is always to build in a number of different stories, one above\nthe other. If you examine one of these stories you may see a number\nof large places or halls, some smaller rooms, and some long galleries\nwhich serve as passage-ways. The tops of these large halls are covered\nwith an arched ceiling, and this ceiling is held up, sometimes by little\ncolumns, sometimes by very thin walls, or by props built against the\nside walls, just like buttresses. There are also chambers which have but\none door, which opens into the lower story, and large open places in the\ncentre of the nest something like a cross road, and all these little\ngalleries or streets come into that open place.\"\n\n\"Any bridges, Uncle Philip?\"\n\n\"No, boys; no bridges among these ants, so far as I know. There will\nsometimes be as many as twenty stories above ground in the ant-hill, and\nas many below. The best time to see these little fellows at work is in a\ngentle shower of rain, or directly after.\"\n\n\"Why do they work in the rain, Uncle Philip?\"\n\n\"I suppose, boys, it is because the earth is then better for them; and\none thing that has been noticed about their work is, that the rain, when\nit is not too violent, seems to make it solid, for these ants have no\ngum or glue about them like some other insects, to make the earth stick\ntogether. As soon as the rain begins, if you watch the brown ants, you\nmay see them come out of the ground in great numbers; and then running\nin again, they will soon return, each one with a little piece of dirt\nin his mouth, which he puts down upon the roof of the nest. A gentleman\nwho watched them very closely[10] says, that at first he could not think\nwhat this was done for, but at last he saw little walls begin to rise up\nwith spaces left between them. In some places pillars were begun, placed\nat regular distances, and he knew that these were to support ceilings;\nso he found out that they were going to build another story to their\nhouse, and they were laying the foundations.\"\n\n\"How I should have liked to see them. Uncle Philip. I would not have\ncared for the rain.\"\n\n\"Ah, I see you are fit to be a naturalist. You know what that word\nmeans, do you not?\"\n\n\"It means, Uncle Philip, a man who loves to study about the animals and\ninsects, does it not?\"\n\n\"It means a man or a woman either, boys, who loves to study the things\nin nature no matter whether they are animals, or stones, or grass, or\nflowers, or any of the things which God has made. Mark, boys, I said\nto study the things, and you said to study _about_ the things. Now a\nperson may read a great deal that is interesting and true about all\nthese things in books; and it is very well to do so; but I think that\nthe real naturalist will never be satisfied with books only; he will\nbe looking to see things for himself. And I said a woman might be a\nnaturalist, because some ladies have been fond of natural history, and\nhave proved themselves to be very good naturalists. But let us go back\nto the ants. Mr. Huber, in the account which he gives of his having\nwatched these little workmen, never thought of telling us whether he\ngot wet or not, because he was too busy to think or care much about it.\nHe had an opportunity of seeing what he might never see again, and a\nlittle rain was not to spoil it. He says that each ant, as it brought\nout its little lump of dirt, would place it on the spot where it wished\nit to be, and press against it with its teeth, so as to make it fit\nclosely. It then rubbed its feelers all over it, and after that pressed\nupon it lightly with its fore-feet. The walls went on very rapidly, and\nit often happened that two little walls, which were to make a passage\nor gallery, would be raised opposite and at a small distance from each\nother. When they were about a quarter of an inch high, the ants would\nset to work and cover them with an arched ceiling. After they had raised\nall the walls as high as they wished, on the inside of each wall at the\ntop they began to put in pieces of wet dirt almost level, and in such a\nway as to make a ledge; and by joining on more dirt to it, it would meet\nthe ledge made from the opposite wall so as to make a roof: these roofs\nover the galleries were about a quarter of an inch across. The ceilings\nover the large halls were sometimes as much as two inches in breadth,\nand to support these they raised pillars; and beginning in the corner\nwhere two walls joined, they would commence the ceiling with a ledge,\nwhile from the top of each pillar they would also build out a layer of\nearth a little rounded on the top; these they continued to add to until\nall met and made a complete cover for the hall.\"\n\n\"I wonder it did not fall, Uncle Philip, before they could join it\ntogether.\"\n\n\"Mr. Huber says that he thought several times it would fall, from the\nrain which was dropping upon it all the time; but he found that the\npieces held together, and that the rain, instead of hurting it, only\nmade it more solid. All that it wants, when the ants have done, is a\nlittle heat from the sun to bake it hard, and then it appears like a\npiece of solid dirt. Sometimes, if the rain be violent, the apartments\nwill be destroyed, especially if the arches are not built strongly; and\nwhen this happens, the little creatures go to work very patiently and\nrebuild them.\"\n\n\"And how long, Uncle Philip, did it take the ants to put another story\non their house?\"\n\n\"Between seven and eight hours; and they had hardly finished one before\nthey began another, but they had not time to finish it, for the rain\nstopped before they had built much: however, they kept on, taking\nadvantage of the wet earth; but a cold drying wind soon sprang up, and\nthe earth would not stick; so they stopped: but what do you think they\ndid with the new story which they had not time to finish?\"\n\n\"What _did_ they do, Uncle Philip?\"\n\n\"Pulled every part of it to pieces, and scattered the dirt here and\nthere over the roof of the story which they had finished.\n\n\"I will tell you another story about these ants, boys, which I think is\nmost wonderful, because it appears so much like reason. These insects\nall seem to work separately,--I mean without attending to the work of\nothers: of course sometimes the work done by different ants on opposite\nsides of the same gallery or hall will not suit: one wall will be higher\nthan the other, so that the ceilings will not meet. Mr. Huber saw just\nsuch a case; the ceiling which was begun from one wall would just have\nreached the other wall about half-way up; and while he was wondering\nhow the ants would cure the fault, one of them came, and looking at the\nwork, seemed to know that it was wrong, and immediately began by taking\ndown the ceiling from the lower wall; he then raised it to the same\nheight with the opposite wall, and made a new ceiling in Mr. Huber's\npresence with the pieces of the old one.\"\n\n\"Uncle Philip, if that ant did not know how to think, I am mistaken.\"\n\n\"I must confess, boys, it does seem very much like thinking; and if it\nwas not thinking, we must at any rate own that it was something which,\n_in this case_, did quite as well; for no thought of man could have hit\nupon a better plan. But if the ant knew how to think as a man does, do\nyou suppose it would ever have made the mistake? Would not the workmen\nhave all agreed beforehand what they were to do, and how it should be\ndone, so that there might be no need of pulling down any of the work\nbecause it would not suit? I think that this story, boys, while it shows\nus something like reason in _one_ particular instance, shows us also\nthat _in general_ the ant has not reason like ours. But I am tired now,\nand can tell you no more this morning. When you come again I shall be\nready to talk with you about some other kinds of ants. But before you\ngo, tell me--can any of you inform me what the Bible says about the ant?\"\n\n\"Oh yes, Uncle Philip: it says, 'Go to the ant, thou sluggard, consider\nher ways, and be wise.'\"\n\n\"Well, another question. Have you any lessons to say when you go into\nschool on Monday?\"\n\n\"Yes, Uncle Philip; we have.\"\n\n\"Have you learned them, boys?\"\n\n\"No, sir.\"\n\n\"Then remember what the Bible says to the sluggard, and go and learn\nthem at once. Good-by, children.\"\n\n\"Good-by, Uncle Philip: we will learn our lessons.\"\n\n\n\n\nCONVERSATION XII.\n\n _Uncle Philip tells the Boys about Ants that go to War and\n fight Battles; and about some that are Thieves, and have Slaves._\n\n\n\"Well, my lads, how do you do to-day?\"\n\n\"Very well, Uncle Philip, we thank you. And we wish to let you know that\nwe kept our promise to learn our lessons. Our teacher was very well\nsatisfied with every one of us.\"\n\n\"That is well, boys. I am truly glad to hear this from you: and I make\nno doubt that you also felt a great deal happier than you would have\ndone had you neglected to learn your lessons. Did you not?\"\n\n\"Oh yes, Uncle Philip, much happier; and far more cheerful and\ngood-natured.\"\n\n\"Such are apt to be the feelings, boys, of those who have done their\n_duty_. I am verily persuaded that there is no such thing as real, solid\nhappiness in this world, but in that man who acts from a sense of duty.\nHis is true peace, because it is 'the peace of God.' I do not say, boys,\nthat a man, even when he does his duty, _always_ feels comfortable or\nhappy _at once_; but he will be more apt to feel so than if he did not\ndo his duty: and I do say that no man who does not act from a sense of\nduty, is likely to feel any thing like happiness very often or very\nlong.\"\n\n\"Then, Uncle Philip, a man who wishes to be happy will try in the first\nplace to find out what his duty is.\"\n\n\"To be sure, he will; and he need not try very long either, if he really\nwishes to know it. The will of God, boys, is at the bottom of all our\nduties; and an honest man, yes, or boy either, can commonly tell what\nGod will think to be right or wrong in his conduct. You know where a\ngreat many of our duties are very plainly written down for us; do you\nnot?\"\n\n\"Oh yes, sir; in the New Testament, which tells us of what our Saviour\nsaid and did.\"\n\n\"True. And what our Saviour commanded, boys, God commanded; for He is\nGod. But besides this, when it is not exactly written down in the New\nTestament what we should do, still if we will think, we shall very often\nfind out what to do, from what is written.\"\n\n\"Uncle Philip, we almost always know what you mean; but now, we do not\nquite understand you.\"\n\n\"Thank you, boys, for telling me that you do not know what I mean:\nalways tell any person who is trying to teach you something, when you\ndo not understand what is said to you. Now I will try to make what I\nsaid plainer to you. The New Testament does not say any thing about your\ngoing to school; does it?\"\n\n\"No, sir.\"\n\n\"Who sends you to school, boys, and pays your teachers for instructing\nyou?\"\n\n\"Our parents, Uncle Philip.\"\n\n\"Very well. Now suppose that John Carter here, should wish, instead of\ngoing to school, to do, what I am very sure he never did do: suppose he\nshould determine to play the truant. The Bible does not say a boy shall\nnot play the truant, does it?\"\n\n\"No, Uncle Philip.\"\n\n\"But if John Carter should play the truant, he would, in doing so,\ndisobey what God has commanded in the Bible just as much as if the Bible\ndid say 'A boy shall not play the truant;' for the Bible does say,\n'_Children obey your parents_,' and he could not be a truant without\ndisobeying his parents, who bade him go to school.\"\n\n\"Uncle Philip, we understand you very well now.\"\n\n\"There is another thing I wish you to understand, boys. John Carter, as\nyou see, would not only disobey his parents, which is wicked, but he\nwould also commit a _sin_ against God. That is always the thing to look\nat first. When we are going to do something that we are not very sure\nis right, we should always stop to ask ourselves whether God will be\npleased with it. But I have said enough to you about our duty for this\ntime. Now for the ants I promised to tell you of. And the first sort I\nshall mention are great fighters.\"\n\n\"Fighters, Uncle Philip! What do they fight about?\"\n\n\"About trifles, boys, just as men do. They have terrible wars, and\nwill dispute with and kill each other for a few inches of dirt, when\ncertainly this world is large enough for them all. But animals wiser\nthan ants, boys, act in the same foolish way. Men sometimes go to war\nand kill each other to determine who shall have a river, or a small\ntown, or a fort, or some little spot of ground; while the poor creatures\nwho do the fighting, and get all the wounds, and lose their lives, had\nthey been let alone, would have lived on in peace, and never cared a\nstraw who had the miserable little spot they fight for. But let me go\non with the account of these ants. In the forests, where the fallow\nants live, you may see these wars. The battle will be between the ants\nof different hills, but they are all ants of the same sort. Thousands\nand thousands of them will meet on the ground between their hills, and\nthe battle begins by two ants, who seize each other by the claws (or\n_mandibles_, as they are called), and rising up on their hind-legs,\nthey bring their bodies near together, and spirt a sort of venomous or\npoisonous juice upon each other. These will be followed by thousands of\nothers on both sides, who seize each other in the same way, and fight\nin pairs--ant to ant. Sometimes they will get so wedged together that\nthey fall down upon their sides, but they do not let go on that account;\nthey keep on fighting in the dust until they rise on their feet again.\nSometimes, too, a third ant will come in, and joining whichever of\nthem belongs to his nest, the two will begin to drag the third, until\nsome of his friends come to his help; and in this way, others joining\non both sides, they will form strings of six, or eight, or ten on a\nside, pulling with all their strength. And while some are fighting,\nyou will see others leading off prisoners towards their hills, while\nthe prisoners are trying to escape. The field of battle is not more,\nperhaps, than three feet square; multitudes of dead ants covered with\nvenom may be seen upon it, and there is a very strong scent which comes\nfrom it. When night comes they go off to their hills. Before dawn the\nnext day they are at it again in still larger numbers, and they fight\nwith greater fury than before, until at last one side or the other\ngives way. They are so busy that even if you stand near them they take\nno notice of you, and not one stops fighting, or crawls up your legs.\"\n\n\"Do all of them that belong to the hill go out to fight, Uncle Philip?\"\n\n\"No; near the hills all is peace and order, and work seems to be going\non as usual. Only on the side next to the battle, crowds may be seen\nrunning backwards and forwards from both hills; some as messengers, I\nsuppose, and some to fight, or carry back prisoners.\"\n\n\"But, Uncle Philip, you said that these ants were all of one sort; how\nthen do they know one another so as to tell which party each one belongs\nto? I should think that sometimes they would make a mistake, and fight a\nfriend instead of an enemy.\"\n\n\"This, boys, is one of the most wonderful things concerning them. They\nare alike in form, and size, and weapons, and strength; and sometimes\nit happens that they do make a mistake, but it is very seldom; and when\nthey do, Mr. Huber, who watched one of their battles, says that they\nfind it out directly, and caress each other with their feelers, and\nmake up the difficulty at once.\n\n\"Are you tired, boys, or do you wish to hear more?\"\n\n\"Oh, let us hear more, by all means: we are not at all tired.\"\n\n\"I will then tell you of another kind of ants called legionary ants,\nand sometimes amazons; but I am sorry to say that they are unlike other\nants, for they are lazy; and yet they live very comfortably.\"\n\n\"How is that, Uncle Philip? Can they be comfortable without working?\"\n\n\"Yes, boys, if they can get others to work for them; and these have\ntheir work mostly done by their slaves.\"\n\n\"By their slaves! what are their slaves, and where did they get them?\"\n\n\"As to your first question, boys, their slaves are ants of another kind;\nas to the second question--where they get them--they _stole_ them when\nthey were young.\"\n\n\"Why you surprise us, Uncle Philip.\"\n\n\"I dare say I do. There are persons much older than you are who have\nnever attended at all to the doings of insects, who would be very much\nastonished by the history of the legionary ants; and probably would\nlaugh at the whole account as an idle story; and yet it is all true,\nand those who have read and seen, know it to be true; and they know,\ntoo, that to deny it shows nothing but ignorance. However, I always let\nsuch persons alone. I can do them no good; for they are apt to be very\nconceited, and will not be convinced. And now for the legionary ant.\nThis is a fighting ant, as well as the last I mentioned; and it actually\nsteals the young of another kind, rears them, and puts all the work on\nthem, so as to be idle itself. This curious fact was first found out\nby Mr. Huber; another gentleman, named Latreille, afterward saw the\nsame thing; and now a great many naturalists know it, because they have\nsought for and seen it. The ant which it steals is of a dark ash colour;\nthe legionary is of light colour. The dark- ant is now called\nthe ant, and is a very industrious, peaceable insect, without any\nsting. The legionary is a strong, brave ant, with a sting, but very\nlazy. I shall relate to you the account which Mr. Huber gives of the\nlegionary. He was walking near the city of Geneva during an afternoon\nin the summer of 1804, when he saw quite an army of these legionary\nants crossing the road; they passed through a thick hedge, entered a\npasture, and kept on through the grass without separating; and Mr. Huber\nfollowed them until he saw them come near a nest of ants. Some of\nthese ants seemed to be guarding the holes into their nest; but as\nsoon as they saw the legionaries, they, with a great many more from the\ninside of the nest, attacked the thieves. The legionary ants, however,\nwere too powerful for them, and after a short but severe fight they\nconquered the s, who ran into the lower part of their nests. The\nlegionaries then mounted their ant-hill, some entered it by the holes\nalready made, and others began with their teeth to break other holes,\nso that all the army might get into the hill. They went in and remained\nbut a few minutes, when they came out, each one having in his mouth a\nyoung ant, and off they scampered, without any order among them,\nevery one going his own way, until Mr. Huber lost sight of them. The\nnext day he set out to go back and examine further, and on his way he\nfound a large ant-hill full of legionaries, and saw an army start from\nit, which he followed. They made the attack as before, and each one\ncame off with a young ant in his mouth, and on going back to their\nhill, from which Mr. Huber saw them start, he had an opportunity of\nseeing them return, and was very much surprised to find all around the\nnest of the legionaries a great many full-grown ants. At first he\nthought that perhaps they had gone there to fight the legionaries, but\nhe soon saw that instead of fighting, the ants went out to meet\nthe legionaries returning, and would caress them, and give them food,\nand finally take the young ants and carry them within the nest.\"\n\n\"But, Uncle Philip, why do the legionaries always take the young ones?\"\n\n\"Because, boys, they know, I suppose, that the old ants would never be\nsatisfied to remove from their homes; and therefore they take the young.\nThese legionaries could work if they would, I think, but they depend\nupon the ants for house and home, and food too; and nothing can be\nmore faithful and affectionate than these poor slaves are. To try them,\nMr. Huber took thirty of the legionaries, and put them with some of\nthe larvae, or grubs of their own young, into a glass box with a thick\ncoat of earth at the bottom of it, and he put honey also in the box,\nthat they might not want food. At first the legionaries paid a little\nattention to their young; but they soon stopped; and they neither tried\nto make a house, nor took any food, so that in two days half of them\ndied. Mr. Huber then put in _one_ ant, and this little creature\nset to work alone, made a chamber of the earth in the box, gathered the\nyoung together, fed the old, and put every thing into complete order.\n\n\"At another time Mr. Huber broke one of the ant-hills of these\nlegionaries, to see how they would act, and in doing it, he, of course,\naltered their galleries and chambers. The legionaries seemed to be\nlost, and went wandering about, without knowing where to go; but the\n ants appeared to understand very well where they were: they could\nfind such of the galleries as were not broken, and would take up the\nlegionaries in their mouths and carry them into them. If the \nsometimes seemed for a short time to be lost, and not to know where it\nwas, it laid down its master, ran round and examined until it knew, and\nthen would come back, and pick up the legionary ant, and carry it off.\nIn one case Mr. Huber saw that the entrance to a gallery was stopped up\nby a small lump of earth; the ant laid his master down, took away\nthe piece of earth, and then carried him in.\"\n\n\"Why, these poor ants are sensible as well as faithful, Uncle\nPhilip.\"\n\n\"Yes, boys, they are so; and I think it is likely that both kinds depend\nin some way upon each other, but we have not yet found all about it. I\nexpect that in some things the legionary does for the ant what it\ncould not do for itself. God has made them necessary to each other, and\nthis is the reason they live together so kindly.\n\n\"But I think it is time now to leave the ants, and go back to our\nbusiness of seeking for something like man's inventions and tools among\nthe lower animals. Perhaps hereafter I may tell you more about ants; but\nat present I must bid you good morning.\"\n\n\"Good morning, Uncle Philip.\"\n\n\n\n\nCONVERSATION XIII.\n\n _Uncle Philip and the Boys make a Voyage, and he tells them\n of an Animal that makes itself into a Ship; and of an Insect that\n builds a Boat, and floats about in a Canoe; and of another that\n pumps Water, and wears a Mask; and of a Spider that builds a Raft,\n and floats upon it._\n\n\n\"Well, boys, I have a most delightful plan for us to-day.\"\n\n\"What is it, what is it, Uncle Philip?\"\n\n\"Why, I have a little voyage to make, and my boat is on the river just\nabove the mill. I have the men there to row it, and every thing is\nready.\"\n\n\"Oh! dear Uncle Philip, this is charming! we shall be so happy!\nBut--but--\"\n\n\"But what, boys?\"\n\n\"Why, Uncle Philip, we have not asked leave at home. Now our parents are\nvery happy to have us visit you, and say that they are very much obliged\nto you for telling us so many things; but they have told us, too, never\nto get into a boat without asking their permission first. Uncle Philip,\nwe are sure they will let us go, if they know that you are going; only\nlet us run home and ask them.\"\n\n\"My dear boys, I am very much pleased with your conduct; and, what\nis far better, my children, God is pleased; for he has commanded you\nto honour your father and mother: but you need not go home to ask\npermission, for you may depend upon it I would not take one of you upon\nthe water without the consent of your parents: so I went yesterday,\nwhile you were all at school, and have got permission from your friends\nfor every one of you to go--only I asked them to tell you nothing about\nit.\"\n\n\"Oh dear, Uncle Philip, you are so very, very good: thank you, thank\nyou, a thousand times over.\"\n\n\"Once is enough, boys. There is but one Being who deserves a thousand\nthanks, and he, in truth, deserves a great many more than a thousand;\nbut I fear that from a great many he is just the Being who gets the\nfewest,--it is our Heavenly Father: but come on, boys, let us be going\nto the boat. We shall soon reach her. Ah, yonder she is; I see her\nthrough the trees.\"\n\n\"Oh, what a beauty she is, Uncle Philip, with her green sides and white\nbelt near the top. We shall have a charming voyage.\"\n\n\"Come, then; get in, my little sailors, and seat yourselves yonder in\nthe stern. Now we are all ready; shove off, men, and use your oars. I\nwill take care of the helm.\"\n\n\"Oh, Uncle Philip, how smoothly we go along! this is charming. Is this\nthe way a ship goes, Uncle Philip?\"\n\n\"A ship floats, boys, just as the boat does; but she is not rowed with\noars; she has sails, and the wind blowing upon them sends her along.\"\n\n\"Uncle Philip, there are no ships among animals, are there?\"\n\n\"Oh no; but there is a very curious little animal which lives in the\nwater, and manages to rig out something like a ship, and to sail.\"\n\n\"What is it, Uncle Philip? pray let us hear of it.\"\n\n\"It is called the nautilus, and I saw a great many of them in the\nMediterranean sea. The shell is nearly round, and six or eight inches\nacross, not much thicker than paper, and of a whitish colour: it\nhas, too, a keel or ridge upon each side. When it wishes to sail, it\nstretches upwards two of its legs: these have a very thin skin at the\nend, which the nautilus spreads out for sails, and the other legs hang\nover on each side of the shell for oars or rudders. When the sea is\ncalm, a great many of them may be seen playing about; but as soon as a\nstorm arises, or they are disturbed, they take in their sails and sink\nto the bottom. But, boys, the most curious boat that I know, made by one\nof the dumb creatures, is the work of the little insect that played the\ndoctor the other day, and stuck his lancet into us. Do you remember what\ninsect that was?\"\n\n\"Oh yes, very well, Uncle Philip, it was the gnat.\"\n\n\"True, boys, it was the gnat, which is an insect that spends the first\npart of its life in the water, and the latter part in the air. The grub\nof the gnat lives in water, and I will give you the whole history of\nthis curious insect. We will first speak of the eggs, for out of these\nit is that the boat is made. In order to see this boat made, you must\ngo early in the morning, between five and six o'clock, to a bucket, or\npond of stagnant water, where gnats are to be found: if you go later\nyou will not see it. The gnat's eggs are shaped something like a pocket\npowder-flask, and it is by putting a great many of these together that\nshe makes the boat. To do this, the mother gnat stands by her fore-legs\nupon the side of the bucket, or on a leaf or stick in the pond, and her\nbody is on a level with the water, and rests upon it, except the last\nring of her tail, which she raises a little. She then crosses her two\nhind-legs in the shape of the letter X, and begins to put her eggs in\nthat part of the X nearest to her body. So she brings her legs, crossed\nin this way, near to her body, and puts an egg in the angle, covered\nwith a kind of glue, which will make the eggs stick together. On each\nside of the first egg she puts another in this shape .*., and here is a\ndrawing of the insect at this part of her work.\n\n[Illustration: A Gnat making her Boat of Eggs.]\n\n\"She then goes on adding eggs, which are all put in the water with their\nends downwards, until she has got her boat half-finished; she then\nuncrosses her legs, and just keeps one on each side of the boat as she\ngoes on, until she has completed it.\"\n\n\"And how many eggs, Uncle Philip, will she put together in this way?\"\n\n\"From two hundred and fifty to three hundred and fifty, and when all are\nlaid they make quite a good boat, sharp, and raised at both ends, and\nfloating on the water. Then the mother gnat leaves it. Here is a picture\nof one of these boats.\n\n[Illustration]\n\n\"Now I will tell you of what becomes of the young ones in these eggs.\nThey come out of the lower part of the egg, and commonly swim, with\ntheir heads downward, near to the top of the water.\"\n\n\"With their heads downward, Uncle Philip! what is that for?\"\n\n\"Why, they have a tube at the end of their bodies, near the tail,\nthrough which they breathe; and that part must, you know, be at the top\nto get air. Besides this, its tail and its breathing tube both end in a\nsort of funnel, made up of hairs placed somewhat in the form of a star,\nand covered with oil, so as to keep off water, and these buoy or float\nit up. When it wishes to sink, it just folds up its funnels, and shuts\nup in them a little bubble of air, which it breathes under the water;\nand when it wishes to rise, it opens its funnels, and they float it to\nthe top again. Here is a drawing which will show it to you.\n\n[Illustration: Larva of the common Gnat floating in water, greatly\nmagnified. _aa_, the body and head of the larva; _b_, the respiratory\napparatus, situated in the tail; _c_, the larva, not magnified.]\n\n\"They are hatched in a few days, and then the boat of empty eggs floats\nabout until it is destroyed by the weather. And to show you how good a\nboat it is, I will tell you what a gentleman did to prove it. Mr. Kirby,\nwho is very fond of natural history, and has written a great deal about\ninsects, says that he put half a dozen of these gnat-boats in a tumbler\nhalf full of water, and then poured upon them a stream from the mouth of\na quart bottle, held up a foot above them, and he could not sink them.\nMore than that, the water would not stay in them. If you push one to the\nbottom with your finger, it will come up to the top directly, and you\ncannot see any water in it.\"\n\n\"Why, this is a noble boat, Uncle Philip, that will never sink.\"\n\n\"True, boys; but listen, and you will find that before it can use its\nwings the gnat has to sail in another boat still, much more dangerous\nthan this is. After it is hatched, it has to pass through several shapes\nbefore it gets to be such an insect as you see. Here is a picture which\nwill show you its different shapes.\n\n[Illustration]\n\n\"The first is the same which you saw in the last picture, only in this\ndrawing the head is uppermost. But its last change, when it becomes\nan insect with wings, is the most curious part of the whole. When it\nis about to get its wings, and become a perfect gnat, it raises its\nshoulders just above the top of the water, and its skin cracks, so that\nthe head of the gnat immediately comes through. The shoulders come next,\nand make the crack larger; but it has yet all its body to get out, and\nits legs and wings are as yet all shut up in its case. Now is the time\nof danger for the gnat. It raises itself nearly straight out of the\ncrack, and by wriggling works its body along: and if a particle of water\nshould get upon the case, or touch its wings, it would be overset, and\nmust perish. Thousands and thousands die in this way. It is so very\nlight, too, that the wind will drive it about, and whirl it round and\nround upon the top of the water; and when it is almost out, the insect\nis tossed about in a canoe or boat of the very weakest sort, while its\nbody is a mast, which appears much too large for so small a boat. At\nlast it gets far enough out of the case to stretch its fore-legs, and\nput them down upon the water (which will bear a gnat's weight), and then\nit is safe; it spreads its wings, and soon leaves the little boat which\nwas so dangerous. Here is a picture of the gnat getting out of its case.\"\n\n[Illustration]\n\n\"Well, Uncle Philip, all this is very strange; we never knew before that\nthe gnat was a sailor.\"\n\n\"I suppose that you did not, boys. But as we are talking of boats, pray\ncan you think of any way of making a boat move through water without\noars, or paddles, or sails, or something to pull it along?\"\n\n\"No indeed.--Oh yes, Uncle Philip, by steam.\"\n\n\"Ah, I mentioned paddles, boys, and a steamboat is forced along by them.\"\n\n\"No; Uncle Philip, we do not know.\"\n\n\"Well, I will tell you then of another way in which I have no doubt a\nboat might be made to move. If there were any contrivance by which a\nlarge quantity of water could be kept in the boat, and if this water\nwere forced out of tubes or holes at one end very violently, it would\npush against the water in which the boat was floating, and force her\nalong. Some years ago a plan was thought of to make a steam engine throw\nthe water out of the stern of the boat, and thus to force her along; and\nbefore that, Dr. Franklin tried some schemes for the same purpose, but\nthey never succeeded. Now there is an insect which adopts precisely this\nplan, and perhaps some of those who thought of it got the notion from\nthe insect.\"\n\n\"What insect is it, Uncle Philip?\"\n\n\"It is the grub of the dragon-fly. If you catch one of these grubs and\nput it into a saucer of water with some of the dead leaves or sticks\nit had for a covering, you will see these leaves or sticks floating\ntowards the tail of the grub, and afterward driven off again. This is\nbecause the insect is pumping in water, and then throwing it out. If you\ntake one of them out of the water, and hold it with its head down, and\nlet a drop of water fall upon its tail, it instantly sucks it in, and\nyou can see it grow larger; and when it throws it out again it becomes\nsmaller.\"\n\n\"But, Uncle Philip, how can you see it suck the water in?\"\n\n\"Very easily, boys. When it is in the water, if you will colour some\nother water with indigo, or ink, or any thing else, and then hold a\nglass tube just over the tail of the insect, and very carefully put some\nof the water into the tube, you will soon see the grub spirt\nout a stream of it to the distance of several inches: or if you will put\nthe insect in a saucer of water, and then suddenly move it, and\nput it into one of clean water, you will see it spirt out the \nstream plainer still.\"\n\n\"Why, Uncle Philip, it must have a pump inside of it.\"\n\n\"It has, boys, something very like one. This stream of water is forced\nout to help the insect along; for though it has six feet, it uses\nthem very little except for catching food. It drives the water out so\nstrongly against the still water behind it, that it sends it forward,\nwith a dart, very rapidly. Here are two pictures; one shows the pump\nopen, and the other shows it shut.\"\n\n[Illustration]\n\n\"Uncle Philip, is there any thing else curious about this insect?\"\n\n\"There is, boys, something well worth attention; did you ever see a\nmask?\"\n\n\"Do you mean, Uncle Philip, a face made of pasteboard, very frightful\ncommonly, which you can wear over your own face?\"\n\n\"That is a mask, boys; but so is any thing which is made to wear over\nthe face, and hide it. Now this little insect has a mask, not made\nlike a man's face, but which completely hides its mouth, and it is\nexceedingly curious.\"\n\n\"How is it made, Uncle Philip?\"\n\n\"Why, boys, I am not sure that I can tell you, so that you will\nunderstand me; but I will try. Suppose your under-lip was horn, instead\nof being flesh; and suppose it hung straight down until it reached the\nbottom of your chin, so as to cover the whole of it, and that at the\nbottom there was a large three-sided plate which was hollowed out, and\nfastened by a joint or hinge to the bottom of your long lip, so that it\ncould turn up on the hinge and cover your face as high up as your nose,\nand hide your long lip and your mouth and part of your cheeks: suppose,\ntoo, that at the upper end of this long face-cover there were two other\npieces, so broad that they would cover all your nose and your temples,\nand could open sidewise like jaws, and show your nose and mouth, so that\nwhen they were opened they would appear like the blinders to a horse's\nbridle; and then suppose that these jaws, upon their inner edges, were\ncut into a great many sharp teeth, which fitted into each other, and you\nwill have some notion of this curious mask. Do you think you understand\nme?\"\n\n\"Why, pretty well, Uncle Philip, we think.\"\n\n\"Well, boys, here are some pictures, and with their help I hope what I\nhave been saying will be plain enough. In one picture the mask is shut;\nand in the other, one of the jaws, like a blinder to a bridle, is open.\nWhile the insect is at rest, it keeps the mask over its face; when it\nwishes to use it, it unfolds it, and catches its food, and holds it to\nits mouth. A gentleman once saw one of them holding and eating a large\ntadpole.\"\n\n[Illustration: Mask of the Dragon-fly, shut and open.]\n\n\"Uncle Philip, this mask is any thing but handsome.\"\n\n\"Very true; but you know we agreed when we were talking about the bats\nto look at animals even if they were not handsome. And there is your\npoor little ugly insect that you thought it right to kill, the spider;\ndid you know that the spider was a sailor, too?\"\n\n\"No, indeed, Uncle Philip! Pray tell us of it, will you?\"\n\n\"Yes; but wait a little, until we bring the boat's head right, for we\nare near the landing-place. So--now, boys, I am ready. There is a very\nlarge spider, about which not much is yet known, which actually builds\na _raft_, for the purpose of getting its food more easily. You may see\nit sailing about upon the water, on a ball of weeds about three inches\nacross, which is held together probably by small silk cords spun from\nitself; and the moment it sees an insect drowning, it leaves the raft,\ngets the insect, and then returns to eat it at leisure. If you frighten\nit, or it thinks danger is near, in an instant it is under the raft out\nof sight.\"\n\n\"Ah, this is a cunning spider, Uncle Philip.\"\n\n\"Not half so cunning, boys, as the one we talked of which built a door\nto its house. But here we are at land. Jump ashore, my lads, and give my\nrespects to your fathers and mothers, when you get home.\"\n\n\"We will. Good day, Uncle Philip.\"\n\n\"Good day, boys. I shall be glad to see you next Saturday.\"\n\n\n\n\nCONVERSATION XIV.\n\n _Uncle Philip tells the Boys about an Insect with Tweezers,\n and another with Pincers; and shows them how a Fly's Foot is made,\n so as to stick to the Wall._\n\n\n\"How do you do, Uncle Philip, this morning?\"\n\n\"Very well, boys, I thank you. You are all well, I suppose, or I should\nnot see you here.\"\n\n\"Yes, we are all well, thank you, Uncle Philip. But one of us would be\nvery glad to have your help.\"\n\n\"Why, what is the matter?\"\n\n\"Charles Walker has run a splinter into his hand, and he wishes you to\nget it out for him.\"\n\n\"Oh, certainly, I will do that, if I can. Let me see: but stay--I must\nfirst put on my spectacles. Ah, now I see it; I can get it out, but I\nmust take my tweezers to it. There, it is out.\"\n\n\"Uncle Philip, those tweezers are very useful. If you had not had them,\nyou could not have taken hold of the splinter with your fingers; and\nwhat would you have done then?\"\n\n\"Tried to cut it out with the point of my penknife; but the tweezers are\nbetter for such work; and that reminds me, boys, to tell you that there\nare insects with tweezers.\"\n\n\"Why, what tool is it that you cannot find among them, Uncle Philip? It\nreally seems as if you found almost every kind among the lower animals.\"\n\n\"Oh, no--no, boys. There are a great many which I cannot find; but there\nare several, too, which, as you know, we have discovered.\"\n\n\"And, Uncle Philip, we suppose that men learned to make their tools and\nwork at many of their trades from these dumb creatures.\"\n\n\"Stay, boys--I never said that, because I think that it is not true. We\nknow that in some things men did not learn from the insects, though they\nmight have done so. There is paper, for instance. How could men learn\nto make it from the wasps, when it was a thins: in common use a long\ntime before Mr. Reaumur, of whom I told you, found out how the wasp made\nit? So, too, with a great many tools; men invented them, and afterward,\nperhaps, it was found out that insects had instruments like them:\nand at other times the insects did show men how to make some things.\nI will tell you of one which I think of just now. The city of London,\nin England, is on the river Thames. Some time since a plan was adopted\nto make what is called a tunnel under the river. This tunnel is a road\ndug out of the earth, under the bottom of the river, across it; and of\ncourse to keep the water from pressing in the earth as fast as it was\nhollowed out, it was propped up by walls built on each side, with a very\nstrong arch at the top. The work has now stopped; but about half of it\nwas made. In building this arched road under the water, the workmen used\nwhat they called a shield, to keep the water from coming through upon\nthem: and the gentleman who invented it, says that he first thought of\nit, from examining a little animal named Taret, which will bore holes in\nlarge pieces of timber under the water. This little animal has upon its\nhead a kind of shield, by which it keeps off the force of the water, and\nworks without being disturbed. So here was a case in which the insect\ntaught the man.\"\n\n\"Uncle Philip, that gentleman was a sensible man, in the first place to\nwatch the Taret and examine its head, and in the next place not to be\ntoo proud to learn from it. I expect he was a naturalist; was he, Uncle\nPhilip?\"\n\n\"I do not know, boys; but I should think his discovery of the shield\nwould make him an attentive observer, if he was not so before.\"\n\n\"Now, Uncle Philip, will you tell us of the tweezers?\"\n\n\"Very willingly, boys. This instrument or tool belongs to the moths\nwhich you see flying about at times. The tails are covered with a down,\nwhich grows in the form of a thick brush or tuft, and has a shining\nsilky gloss, different in colour from the short hair upon the rest\nof the body. The moth pulls off this hair to cover its eggs, and the\ntweezers are used for that purpose. Here is a picture of the moths.\"\n\n[Illustration: Females of the brown and gold-tailed Moths, showing the\nbunch of down on the tails.]\n\n\"Uncle Philip, you said that the moth pulled this hair off to cover its\neggs; are they easily frozen?\"\n\n\"Not very easily, boys; but you are mistaken in thinking that the moth\ncovers these eggs to keep off the cold; for as she lays them in July and\nAugust, and covers them at that time, it cannot be to keep off the cold.\"\n\n\"What is it for, then, Uncle Philip?\"\n\n\"To keep off the summer heat, boys.\"\n\n\"Why, Uncle Philip! who ever heard of covering a thing up in hair or\nwool to keep off heat?\"\n\n\"I have heard of it, and seen it too, boys. It may seem strange, but\nit is true, that down and wool, and such things, are nearly as good to\nprotect an animal from very great outward heat as they are to keep off\nvery severe cold. When I was at Naples, in Italy, it was summer;--the\nclimate is a very warm one.--The country people were in the habit of\nbringing snow into the city from Mount Vesuvius, and every morning I\ncould see them coming in with their snow, which they sell to the rich\nto use for cooling things: and they kept it from melting with straw\nand wool. And in our own country, especially at the south, it is very\ncommon when a large lump of ice is brought to the house to be used\nthrough the day in midsummer, to wrap it up in a thick blanket until it\nis wanted.\n\n\"But I have not yet told you of the tweezers. The moth has no jaws, like\nbees and wasps, so that it cannot pull off these hairs as the bee would;\nbut, as I told you, it performs the work with its tweezers, which are\nplaced in its tail, and are like the points of a pair of sugar-tongs.\nThe insects, too, will use them very rapidly, and pull off a little of\nthe down, spread the egg upon it, and then cover it with more down, and\nsmooth it very neatly. Here are pictures of these tweezers.\"\n\n[Illustration: Tweezers of the brown and gold-tailed Moths, magnified.]\n\n\"This is a curious instrument for the insect to have, Uncle Philip.\"\n\n\"True, boys, but a very useful one. I will tell you, however, of another\nstrange thing concerning moths with their tweezers; I mean the way\nin which they will sometimes place their eggs. The kind of moth that\ndoes this work is not exactly known, but naturalists think that the\neggs are moth's eggs, because they are covered with the down, exactly\nlike those which are known to be moth's eggs. These eggs are twisted\nround a branch, like the thread of a screw, or like the curled end of\na corkscrew put over a small stick. Here is a picture of some of these\neggs.\"\n\n[Illustration: Spiral group of Eggs of an unknown Moth.]\n\n\"Ah, this is wonderful work indeed for a moth, Uncle Philip.\"\n\n\"As you seem to like this, boys, I will just mention to you that there\nis another moth, called the lackey-moth, which winds its eggs also\naround a branch. They are hard, however, and not covered with any down,\nand are put on in the strongest possible way. If men wish to make an\narch of stone, you know that the stones will be more narrow at the\nbottom than at the top, so that the bottom of the arch may make a small\ncircle, and the top a larger one: thus--\n\n[Illustration: A, Key-stone of an arch; B, Arch completed.]\n\nNow the moth goes on this principle. Its eggs are shaped like the bowl\nof a wine-glass, and the smaller end is put next to the branch. They are\nall glued together, too, with a kind of gum, which will not dissolve or\nmelt in water; so that the rain cannot injure them. Here is a picture of\nthese eggs.\n\n[Illustration: Eggs of the Lackey-moth, wound spirally round a twig of\nhawthorn; natural size, and magnified.]\n\n\"There is another insect, boys, which has something like tweezers;\nthough I think they resemble pincers most.\"\n\n\"What is it, Uncle Philip?\"\n\n\"The boys call it father long-legs, and I dare say you have often seen\nit. It is the crane-fly, and its pincers are used for putting its egg in\nthe hole it has made for it.\"\n\n\"Where does it put its eggs, Uncle Philip?\"\n\n[Illustration: Ovipositor and Eggs of the Crane-fly.]\n\n\"In the earth, boys; and to enable the insect to do this, the female has\nthe pincers I spoke of: they are made of something like horn, and are\nsharp at the point. With these she first bores a hole in the ground, and\nthen puts the egg in. The egg is like a grain of gunpowder, and she puts\nherself in a very curious posture to bore the hole. Here, boys, you may\nsee a picture of the pincers as they appear through a microscope, for\nthey are not near as large as the picture. And here is a drawing of one\nboring.\"\n\n[Illustration: Crane-fly ovipositing, and the larva beneath, in the\nearth, feeding upon grass roots.]\n\n\"What good pincers those are, Uncle Philip: but will you tell us one\nthing which we wish to know? Talking about the crane-fly has put me in\nmind of it: the other day we were sitting together in school, and the\nwall over our heads was covered with common flies; and when we came out,\nwe were talking about the way in which the fly stuck to the wall without\nfalling down; and as we could not tell what kept him up, we agreed to\nask you about it.\"\n\n\"I will tell you, boys, very willingly. I do not wonder that you were\nunable to tell how the fly stuck to the wall; for you never tried to\nfind out, and therefore could only guess at it.\"\n\n\"And that is not a good way to find out any thing, Uncle Philip?\"\n\n\"No, boys; though some persons much older than you are, did nothing but\nguess about this very thing, and guessed very far from the truth too.\nSome thought that the fly had a sponge in its foot, and squeezed a sort\nof glue out of it which made it stick fast; others said that the glass\nor wall was so rough that the fly's feet would catch hold of the little\npoints upon it; but both were wrong.\"\n\n\"How does it hold on, Uncle Philip?\"\n\n\"Did you ever see what the boys call a sucker, made of a piece of soft\nsole leather? That will show you how the fly's foot sticks fast. This\nleather is cut round, and has a string through the centre; the boys wet\nit, and then put it upon a board or something smooth, and stamp on it,\nand try to raise it up from the board by the string; and it requires\nsome strength to pull it up: sometimes they put it on a small smooth\nstone, and then lift up the stone by it. The reason why the leather\nsticks so fast is because the air is pressing on it upon the outside,\nand there is very little or no air between it and the board, to press\nthe other way.\"\n\n\"Why, Uncle Philip, is the air heavy?\"\n\n\"Oh yes, boys, when there is so much of it as there is above the earth,\nit presses down very heavily. Now the fly's foot is like the sucker;\nwhen he puts it down he has a contrivance to drive out the air from\nunder it, so that there will be little or none between it and the wall;\nand then the outer air presses upon it, and holds it fast.\"\n\n\"But, Uncle Philip, how does he get it up again?\"\n\n\"Why, boys, by another contrivance, he can let air in under his foot\nagain, and then he can easily move it; for we do not feel the weight of\nair when it presses upon both sides of us. The reason why you stand up\nstraight is because the air is pressing all around you; if it were on\none side of you only, it would press you down on the other side. Here is\na picture of the fly's foot, as it appears through the microscope. You\nwill see it has three suckers with the edges all like saws; these are\nto make it stick the closer. This picture, boys, is sixty-four hundred\ntimes as large as the fly's foot is.\"\n\n[Illustration: Fly's foot magnified.]\n\n\"But, Uncle Philip, there is one thing yet hard to understand.\"\n\n\"What is it?\"\n\n\"Why, the fly walks on the wall over our heads; now the air cannot press\ndown upon his feet there.\"\n\n\"Very true, boys: it cannot press _down_, but it can and does press\n_up_ against his feet; for the air presses up and down and sidewise all\nalike.\"\n\n\"Ah, now it is plain enough, and we are much obliged to you, Uncle\nPhilip, for telling us what we wished to know.\"\n\n\"You are quite welcome, my dear boys, to all that I can teach you: if it\nmakes you to be wiser and better men when you grow up, I shall be very\nthankful to God that I have been able to do you any good.\"\n\n\"Good morning, Uncle Philip.\"\n\n\"Good day, boys; I shall expect to see you all in church to-morrow.\"\n\n\"We shall be there, Uncle Philip.\"\n\n\n\n\nCONVERSATION XV.\n\n _Uncle Philip tells the Boys how Hats are made; and then talks\n to them about Animals that can make Felt like the Hatter._\n\n\n\"Boys, do you remember my telling you of a remarkable bird, called the\ntailor-bird, which sews very neatly?\"\n\n\"Oh yes, Uncle Philip; it is not easy to forget such an excellent little\nworkman; but why do you ask--have you any thing more to tell us about\nthat bird?\"\n\n\"No, boys, not any thing of that bird; but I was thinking last night\nof the work done by several other kinds of birds, some of them quite\nas good workmen as our little tailor; and I thought that, perhaps, you\nmight like to hear of them.\"\n\n\"We would, Uncle Philip, be very happy to hear of them, if you will have\nthe kindness to tell us about them. But what kind of work is it they do?\"\n\n\"Various kinds, boys. There are some which make what is called _felt_,\njust as the hat-maker does; and some are weavers, others basket-makers;\nsome build platforms to live on; and I assure you some birds' nests are\nas curious as any of the things of which I have yet told you.\"\n\n\"Pray let us hear of them, Uncle Philip.\"\n\n\"Very well, you shall. I will begin with birds that make felt like the\nhatter. Do you know how a hat is made?\"\n\n\"Not exactly, Uncle Philip; but we know what it is made of.\"\n\n\"What is it, boys?\"\n\n\"Of sheep's wool, and the hair of other animals: is it not?\"\n\n\"Yes, commonly of these things; and to understand what I am going to\ntell you, I think it will be necessary first to say something about the\nhatter's trade. The business of the man who makes a hat is to mix up\nwool or hair in such a way that it will stick together and make felt;\nor something like a piece of thick, strong cloth. To do this, he does\nnot weave the hairs together, for they are of different kinds, and of\ndifferent lengths, and it would be endless work to weave every one in;\nbesides the cloth or felt would not be thick enough when it was done.\"\n\n\"How do they stick together then, Uncle Philip?\"\n\n\"Why, boys, their sticking together is owing to something in the hairs\nthemselves. I will show you. Pull a hair out of your head; now hold it\njust between the ends of your two fore-fingers, and rub the fingers\ngently against each other.\"\n\n\"Why, Uncle Philip! see, the hair is moving towards my body.\"\n\n\"Very true; and if you will turn it with the other end towards you, and\nrub your fingers as before, you will see it move from your body.\"\n\n\"This is very strange, Uncle Philip: the hair is smooth; how can my\nfingers make it move so?\"\n\n\"No, that is a mistake, boys, the hair is not smooth. If some kinds of\ncoarse hair are seen through the microscope, each one will seem to be,\nnot one hair, but ten or twelve smaller ones, which are joined at the\nroot, and form a hollow tube, like a straw; and sometimes it will have\njoints just like some kinds of grass or straw. In some sorts of finer\nhair you cannot see this even with the microscope; but you can feel it,\nas you did just now when you moved your fingers. These joints overlap\none another, just as if you should take several pieces of straw and\nstick them into each other. I will show you some pictures of hairs as\nseen through the microscope, and then these joints will be plain enough.\"\n\n[Illustration: Hairs of (_a_) the Bat, (_b_) the Mole, and (_c_) the\nMouse.]\n\n\"These are strange-looking hairs, Uncle Philip.\"\n\n\"Yes, they are curious; but now you may see why, when hairs are worked\ntogether, they may be made to stick to each other. These rough parts\ncatch into each other, and hook themselves; and the more you press them\nor move them, the more closely you work them into one solid mass, which\nyou cannot easily pull to pieces. Besides, you must remember that the\nhairs will work only one way, as you found out just now when your finger\nends caught upon the little joints and moved them along. Now, suppose\nthat a very large heap of hairs, or wool, or fur, after it is made\nready, should be put upon a table, and covered with a linen cloth, and\npressed down in different directions. Each hair would begin to move in\nthe direction of its root, just as it did between your fingers, and so\nall would be joined together at last into one solid piece.\"\n\n\"We understand you, Uncle Philip.\"\n\n\"Then you understand, boys, the way in which a hat is made. These hairs\nare all worked together by the hands of the hat-maker, and to make them\nwork more easily (for curled hair, such as wool, does not move easily)\nthe hatter uses hot water, and dips his hat in it while he is working\nit. After it is done, it is died, and then put upon a wooden block to\ngive it shape, and is ironed smooth.\"\n\n\"And this is the way, then, Uncle Philip, to make hats: it is curious,\nis it not?\"\n\n\"Yes, boys; but plain enough when you come to examine into it. And the\nbest stuff for the hatters is such hair as has most joints ready to\ncatch into each other: the rabbit's hair is very good, and for that\nreason.\"\n\n\"And is it possible, Uncle Philip, that any bird can do such work as\nthis?\"\n\n\"Not only possible, boys, but true. There are several birds very expert\nat making felt, and their nest appears like a piece of hatter's felt, or\ndouble-milled woollen cloth. I do not mean to say that it is as close\nand solid as the hat or cloth; it would feel in your fingers looser than\neither, still it is quite close; and when you examine it, you will find\nit put together in the same way; it is all carded into one mass, and not\nwoven together thread by thread, or hair by hair.\"\n\n\"And are there many birds able to do such work, Uncle Philip?\"\n\n\"I told you, boys, that there were several. The chief article which\nthey use is wool, but with this many other things will be found\nmixed--sometimes, upon the outside, fine moss--sometimes pieces of a\nspider's web rolled up into a little bundle--sometimes, when cotton can\nbe had, they will use small bunches of cotton-wool; but sheep's wool\nthey must have, and by means of that, they contrive to make, with the\nother things I have mentioned, a felt wonderfully smooth.\"\n\n\"Is it smooth on the outside, Uncle Philip?\"\n\n\"Sometimes quite so; but always as smooth on the inside, when it is\nfirst made, as if it had been felted together by the hat-maker. There\nis another thing curious enough in some of these nests. The hatter, you\nknow, binds the rim of his hat to make it stronger; and some of these\nfelt-making birds will make their nests stronger by a binding all around\nthem of dry grass stems, and sometimes of slender roots, and they take\ncare to cover these grass stems, or roots, with their felt-work of moss\nand wool. But there is something else not less strange, I think, than\nthe binding. It is this: they will build their nests in the fork of a\nshrub or tree; and to keep them from falling, they will work bands of\nthis felt round all the branches which touch the nest, both below and at\nthe sides. And those parts of the nest which touch the large branches\nare always thinner than the other parts, which have no support; in those\nparts the nest is nothing but a thin wall of felt, fixed around to\nfit the shape of the branch, and that is enough to make that part of\nthe nest warm and soft. Here is a picture of one of these felt-nests,\nfastened in the way of which I have been telling you.\"\n\n[Illustration: Chaffinch's Nest on an Elder-tree.]\n\n\"This, boys, is the nest of the chaffinch. The goldfinch makes a nest\nof the same kind, only rather neater and smoother than that of the\nchaffinch; for it takes pains to show nothing but the wool, and covers\nup all the other materials which it uses.\"\n\n\"Uncle Philip, do these birds all use the same things to make their\nnests?\"\n\n\"All use wool, boys; but the truth is, that birds will commonly take\nfor their nests that article which they can get most easily, if it will\nsuit. A gentleman, named Bolton, tried this with some goldfinches. He\nsaw a pair of these birds beginning to build in his garden; they had\nlaid the foundation of their nest with moss, and grass, and such things,\nas they commonly use: he scattered some wool about in different parts of\nthe garden; the birds took the wool: afterward he scattered cotton; they\ntook the cotton: on the next day he gave them some very fine down; they\ntook that, and finished the nest with it, and a very handsome nest it\nwas.\"\n\n\"How long were they in making it, Uncle Philip?\"\n\n\"Three days. The canary-bird, boys, which you sometimes see in cages,\nwhen free, builds a nest of the same kind. But the most curious\nfelt-makers among the birds, are in Africa. There is the Cape-tit,\na bird in the southern part of Africa, which builds a very strange\nnest: it is shaped like a bottle of India-rubber, as thick as a coarse\nworsted stocking, and made of cotton, and down, and other things felted\ntogether. On one side of the nest there is something like a pocket, and\nhere is a picture of it.\"\n\n[Illustration: Nest of the Cape-tit, from Sonnerat.]\n\n\"Uncle Philip, what is that pocket for?\"\n\n\"Why, boys, some have supposed that it was for the male bird to sit on\nand keep watch, while the female was inside of the nest sitting on the\neggs; but I think this is a mistake. And some have said, that when the\nfemale leaves the nest, and the male wishes to go too, he sits in this\npocket, and beats against the side of the nest with his wing until he\nhas made the edges of the top meet, and thus shuts up the mouth of the\nnest, and keeps off insects and other animals that would eat the young\nones; but I do not believe this story.\"\n\n\"Then what do you think the pocket is for. Uncle Philip?\"\n\n\"I think, boys, that it is nothing but a perch, or place for the bird\nto sit on before going into the nest. If the bird had no such place for\nstopping, it might be troubled to get into its nest. The mouth is small,\nand the bird could not enter it with its wings spread; and if it should\nalight on the edge of the nest constantly, it would injure it, for it is\nbut slightly made. And I will tell you another reason why I think this\nis the use of the pocket. There is another bird in South Africa, called\nthe pinc-pinc, which is the same species of bird as the Cape-tit; and\nthis bird we know uses its little nest built upon the side of the other\nmerely as a resting-place before going into the nest.\"\n\n\"Uncle Philip, does the pinc-pinc build its nest like a bottle, as the\nCape-tit does?\"\n\n\"No, boys, not so smooth, but felted in the same way. The nest is\nmade mostly of the down of plants, and is either snowy white or\nbrownish, according to the colour of the down. On the outside it is a\nclumsy-looking thing, but fastened, like the nest of the chaffinch,\nvery firmly to the branches near it, so that you cannot take it away\nwithout breaking it to pieces. But rough as the outside is, you would be\nastonished, if you were to look at the inside, and see how a bird, and a\nsmall one too, with nothing but its wings, and tail, and feet, and bill\nfor tools, could ever have worked the down of plants together, so as to\nmake of it a piece of fine cloth. It has a narrow neck, something like\na chimney, at the top of it. This is the entrance; and at the lower end\nof it there is a lump, which appears something like a small nest stuck\non to the larger one; sometimes there will be three or four of these\nsmall-looking nests, and sometimes when there is a branch near the mouth\nof the nest which makes a good resting-place, there will be none. Here\nis a picture, boys, of the outside of one of these nests.\n\n[Illustration: Nest of the Pinc-pinc.]\n\nThese birds are easily watched; and a French gentleman, who has written\nthe best account of the birds of Africa,[11] says that he has found\nat least a hundred of these nests, and watched the birds for a whole\nmorning together, and never saw one sitting on the small nest as a\nwatch-bird; but has seen both the male and female arrive at the nest\ntogether, perch upon the nearest branch, hop from this upon the edge of\nthe little nest, and then putting their heads into the hole, dart into\nthe large nest. And now, boys, what do you think about the use of these\nlittle pockets?\"\n\n\"Oh, Uncle Philip, we think that what you tell us is always right,\nbecause you know a great deal more than we do.\"\n\n\"But, boys, you do not understand me. I may be mistaken, though I do\nknow more than you. I have been telling you my reasons for thinking\nthese little pockets are nothing but perches. Do you think the reasons\nare good ones?\"\n\n\"Why, yes, Uncle Philip, we do. The French gentleman who watched the\nbirds so much would have seen some of them using the pockets for a place\nto keep watch in, if they were made for that.\"\n\n\"Right, boys. What I wish to teach you is to think for yourselves.\nWhenever any one gives you a reason for a thing, just ask yourselves,\n'Is this a good reason?'\"\n\n\"But, Uncle Philip, how did it happen that the other people who saw\nthese birds should have said that these pockets were for the male bird\nto sit in and watch?\"\n\n\"I suppose, boys, that they really thought so; but then they had not\nnoticed the birds enough to find out the truth. It requires a great\ndeal of time and patience to find out the truth about animals: and this\nis the reason why so many mistakes have been printed about them. It is\na pity that such mistakes have been made; for really there is enough\nthat is very curious about them, without men's making stories to appear\nstrange. But I think that there will be fewer mistakes made in future.\"\n\n\"Why so, Uncle Philip?\"\n\n\"Because, boys, men are taking more pains to see for themselves. There\nare more naturalists now than there were formerly; and I hope there\nwill be more still, especially in our own large and beautiful country,\nwhere there have not yet been many. I hope that natural history will be\nstudied in all our schools before a great while. But let us go back to\nour African birds.\n\n\"There is another kind which Mr. Vaillant speaks of, and I will tell you\nof that. He calls it the capocier, and he had a very fine opportunity to\nwatch two of them. It is a bird easily made gentle, and he had managed\nby feeding two of them to make them so tame that they would come into\nhis tent and hop about several times in a day, though he never had them\nin a cage. When it became time for them to build a nest, they staid away\nfor some time, and would come to the tent once only in four or five\ndays. At last they began to come regularly, as before, and Mr. Vaillant\nsoon found out what they came for. They had seen upon his table cotton\nand moss and flax, which he used to stuff the skins of birds, and which\nwere always lying there; and the capociers had come for these things,\nto build their nest of them. They would take up large bunches of them\nin their bills and fly away. Mr. Vaillant followed and watched them to\nsee the nest built, and found them at work in the corner of a garden, by\nthe side of a spring, in a large plant which grew under the shade of a\ntree. They were building in the fork of the branches, and had laid the\nfoundation, which was about four inches high and six inches across. This\npart was made of moss and flax, mixed with grass and tufts of cotton.\nThe next day this gentleman never left the side of the nest: the female\nwas at work building, and the male brought the materials. In the morning\nthe male bird made twenty-nine journeys to Mr. Vaillant's table for flax\nand cotton and moss; and in the afternoon he made seventeen. He would\nhelp his mate to trample down and press the cotton with his body, so\nas to make it into felt. Whenever he came with a load, he would put it\neither upon the edge of the nest or upon some branch within reach of the\nfemale.\n\n\"After he began to help the female at her work, he would often break\noff, and begin to play; and sometimes, as if in mischief, he would pull\ndown a little of her work. She would get angry, and peck him with her\nbill: but he still continued to vex her, until at last, to save her work\nfrom being pulled down, she would stop working, and fly off from bush\nto bush, to tease him. They would then make up the quarrel, and she\nset about her work, while he would sing most delightfully for several\nminutes. After his song was finished, he would go to work again, until\nhe got into a new fit of mischief and frolic, and then he would torment\nher as before.\n\n\"On the third day the birds began to build the walls, after having\nrepeatedly pressed the bottom, and turned themselves round upon it in\nall directions, to make the nest solid. They first made a plain border\nall around; this they trimmed, and on it they piled up tufts of cotton,\nwhich they felted in by beating and pressing with their breasts and\nwings; and if any part stuck out, they worked it in with their bills,\nso as to make all perfectly smooth and firm. And they worked their nest\nround the branches near it, just as the chaffinch does.\n\n\"In seven days they finished it. It was as white as snow, and on the\noutside it was nine inches high, and not smooth or regular in its shape;\nbut in the inside it was shaped exactly like a hen's egg, with the small\nend up: the hollow was five inches high, and between four and five\ninches across; and it was so neatly felted together that it might have\nbeen taken for a piece of fine cloth a little worn; and so close that\nyou could not take away any part without tearing the nest in pieces.\nHere is a picture of the nest, boys, and it is wonderful work for a\nsmall bird.\"\n\n[Illustration: Nest of the Capocier, from Vaillant's figure.]\n\n\"Oh, Uncle Philip! we like the capociers very much. When they were tired\nof working, they were ready to play; and when they had played enough,\nthey went back to work. Do not you think there was good sense in that?\"\n\n\"Yes, boys, I do: it will not do, either to work all the time or to play\nall the time. All that we have to do is to take care that we do not\nspend more time than we should at either. But there is a sweet little\nbird, boys, quite common in our own country, which makes felt: would you\nlike to hear of it?\"\n\n\"Oh yes, Uncle Philip. What bird is it?\"\n\n[Illustration: Nest of the Humming-bird.]\n\n\"It is the humming-bird. Here is a drawing of its nest. It is about an\ninch deep, and an inch across; and from a little distance, appears more\nlike a small knot upon the branch than like a bird's nest. The outside\nof the nest from which this picture is made, was covered with a kind of\nbluish-gray lichen, that grows in scales upon old trees and fences: this\nseemed to be glued on by the bird in some way or other. The inside was\nthe felt, and was made of the fine down from seeds that float about in\nthe air, mixed with the down from mullein-weed and stalks of fine grass.\nThis, boys, is the smallest nest made by a bird, I believe; and some\ninsects make larger houses for themselves than this bird does.\n\n\"But I have not time at present to talk with you any longer, as I have\nletters to write; and therefore I must bid you good morning.\"\n\n\"Farewell, Uncle Philip.\"\n\n\nFOOTNOTE:\n\n[11] M. Vaillant.\n\n\n\n\nCONVERSATION XVI.\n\n _Uncle Philip tells the Boys about Birds that are Weavers; and\n about the Politician-bird; and a Story about some Philosophers; and\n what may be learned from these Conversations._\n\n\n\"Well, boys, were you pleased enough with our last conversation to wish\nto hear more about birds' nests?\"\n\n\"Yes, if you please, Uncle Philip. You said something about birds that\nwere weavers; we should like to hear something of them.\"\n\n\"Very well, then; I will talk about the weavers this morning. And the\nfirst thing I have to say is that this is no uncommon trade among birds.\nTake the nest of any of the common small birds that use hair for a\nlining, and you will be apt to find some part of it woven.\"\n\n\"But, Uncle Philip, you do not mean that birds weave as smoothly and\nregularly as people do!\"\n\n\"Not quite, boys; but still it is very fair weaving, and done as our\nweaving is, by working a hair or thread in and out between other hairs\nand threads, or roots, or bits of stick and grass. The best way to see\nit, is to remove the outside work of hay or roots very carefully, or to\ntake away the felt-work of wool or moss, and you may see a round piece\nof hair-cloth, sometimes finer, and sometimes coarser, according to the\nbird that made it, and the things of which it is made. In the common\nsparrow's nest the hair-cloth is very thin, so that you can see through\nit easily; but still every hair is woven in singly, and always bent,\nso as to lie smooth in the bottom of the nest. And there are no ends\nof hairs left sticking out; they are always worked into the moss which\nmakes the outside of the nest.\"\n\n\"Uncle Philip, how do the birds make the hairs lie smooth in their\nplaces?\"\n\n\"About that, boys, there is some uncertainty. Some persons think that\nthe birds have a kind of glue in their mouths by which they make them\nstick; and others suppose that they wet the hairs, so as to make them\nbend. But there are much better weavers than the common sparrow. The\nred-breast and the yellow-hammer are both better workmen.\"\n\n\"Where do they get hairs. Uncle Philip?\"\n\n\"They find bunches of them sticking in the cracks of a fence or post\nwhere a horse or cow has been rubbing; and some of these little\ncreatures, when they find such a bunch, will pull it to pieces, and work\nit in, hair by hair.\"\n\n\"Are there many of these weaver-birds. Uncle Philip?\"\n\n\"Yes, boys, a great many: our country is quite full of them. There is\nthe mountain ant-catcher,[12] which will weave a nest of dry grass, and\nwind the blades round the branches of a tree; and the king-bird,[13]\nwhich first makes a basket frame-work of slender sticks, and afterward\nweaves in wool and tow, and lines it with hairs and dry grass. There is\nanother, too, the white-eyed fly-catcher, which some have called the\npolitician. This bird builds its nest and hangs it up by the upper edge\nof the two sides on a vine. The outside is made of pieces of rotten\nwood, threads of dry stalks or weeds, pieces of paper, commonly old\nnewspapers; and all these are woven together with caterpillar's silk,\nand lined with fine dry grass and hair.\"\n\n\"Uncle Philip, why do they call it the politician? What is a politician?\"\n\n\"What is commonly called a politician, boys, is a person who is always\nreading in newspapers about the government of the country, and talking\na great deal about the President and Congress, and the laws that are\nmade, and all such things: but the real politician is one who studies\nthe different kinds of government which have been in the world, and\nendeavours to find out which is good and which is bad, and why they are\ngood or bad. He reads, too, a great deal of history, to learn how other\nnations have done, what kind of laws they made, and why they made them,\nhow they became great nations, or how they became very poor; and he\n_thinks_, too, a great deal, that he may find out what will be best for\nhis own nation. It requires hard study and thought, boys, to make a good\npolitician.\"\n\n\"Then, Uncle Philip, a man cannot learn how to be one out of the\nnewspapers.\"\n\n\"No, boys; not out of newspapers alone: but still he will read them,\nand very often learn from them things very useful to him in his\nbusiness. Newspapers are valuable things, and I think it is always best\nfor a country to have a great many of them spread about in it. But they\nwill not, of themselves, make a man a politician; and if you should ask\nthe persons who print them, whether they expect them to teach men all\nabout governments, they will tell you, No: but they will teach people\nwhat is doing in all the governments in the world. No good government,\nboys, will ever be afraid to let the people have newspapers. They are\nalways fewest where the government is hardest upon the people. But let\nus go back to the birds. Can you tell me now why some people call the\nfly-catcher a politician?\"\n\n\"Oh, yes; because he has so many bits of old newspapers about his nest.\"\n\n\"That is the reason, boys. There is another kind of fly-catcher, called\nthe hooded fly-catcher, and it weaves its nest of flax and strings\npulled from the stalks of hemp: but the best weaver in this country\nis the Baltimore starling. This bird chooses the ends of high bending\nbranches for his nest, and he begins in a forked twig, by fastening\nstrong strings of hemp or flax around both branches of the fork, just\nas far apart as he means the width of his nest to be: he then with\nthe same kind of strings, mixed in with pieces of loose tow, weaves\na strong, firm kind of cloth, which is like the hatter's felt in\nappearance, only that you can see that the nest is woven, not felted.\nIn this way he makes a pouch, or purse, six or seven inches deep, and\nlines it on the inside with several soft things, which he weaves into\nthe outside netting, and finishes the whole with horse-hair. Mr. Wilson\ndescribes one of these nests which he had. He says that it was round\nlike a cylinder. Do you know what a cylinder is?\"\n\n\"No, Uncle Philip.\"\n\n\"A smooth round pillar to hold up a porch is a cylinder; my walking-cane\nis a cylinder; so is the straight body of a tree. When these are of the\nsame size all through their whole length, they are perfect cylinders;\nand any thing in that shape is a cylinder.\"\n\n\"We understand you, Uncle Philip; a gun-barrel is a cylinder, and there\nis a cylinder in your garden.\"\n\n\"What is it?\"\n\n\"The heavy stone roller that you let us pull over the walks.\"\n\n[Illustration: Baltimore Starling, and Nest.]\n\n\"Right. Well, this nest was like a cylinder, about five inches across,\nand seven inches long. At the top the bird had worked a level cover, so\nas to leave a hole only two inches and a half across; at the bottom it\nwas round. It was made of flax, tow, hemp, hair, and wool, and was woven\ninto a complete cloth; it was also tightly sewed through and through\nwith long horse-hairs, some of which when drawn out measured two feet.\nHere is a picture of this nest. In the bottom it had bunches of cows'\nhair, and these were also sewed down with horse-hairs. This bird, boys,\nis a thief.\"\n\n\"A thief, Uncle Philip! What does it steal?\"\n\n\"When I say it is a thief, boys, I mean that it takes what does not\nbelong to it: but it is not a thief as man is. When a man takes\nsomething which belongs to another person, he _knows_ that it is not\nhis; and therefore he steals: but the poor bird does not know, and that\nmakes a difference. You asked me what it steals: I will tell you. At\nthe time for building its nest, it will take whatever suits for that\npurpose; and therefore the country women are obliged to watch their\nthread that they have put out to bleach: the farmer, too, who has cut\noff young grafts from his fruit-trees and tied them up in bundles, must\nbe careful, or the bird will pull at the string till he gets it off; and\nsometimes, when the bunch is not too large, he will fly off with the\nwhole. In autumn, when the leaves have fallen, you may sometimes see\nskeins of silk and hanks of thread hanging about the starling's nest,\nbut so woven up and entangled in it that they are good for nothing. Now,\nboys, before this country was settled by people from Europe, where do\nyou suppose the starling got silk and thread for his nest?\"\n\n\"Why, Uncle Philip, are you sure he got them at all?\"\n\n\"A very sensible question, boys. When you are asked _why_ a thing is so,\nit is always well, first to be satisfied that it is so, before you begin\nto look for a reason. I have read a story about this very thing: would\nyou like to hear it?\"\n\n\"Oh yes, Uncle Philip.\"\n\n\"Well, then, I have read that there were once several philosophers\n(I told you what a philosopher is, you know), who were in the habit\nof meeting together to put questions to each other, and to make new\ndiscoveries. At one of these meetings, one of them asked the others,\n'_Why_ a fish weighed more _in_ the water than he did _out_ of it?'\nSeveral of them gave very wise reasons, as they thought; and all the\nreasons were different: so they could not agree. There was among them,\nhowever, a very sensible old gentleman, who listened to them all, but\nsaid nothing. When he went home, he got a fish and weighed it, out of\nthe water, and wrote down its weight; he then took a bucket of water,\nand weighed that; and when he dropped the fish in the bucket, he found\nthat it increased the weight of the whole, precisely as many pounds as\nthe fish had weighed out of the water; so he found out that there was\nno reason why a fish weighed more in the water than he did out of it,\nbecause it was not true: his weight was the same either in or out of it.\"\n\n\"Ah, Uncle Philip, that is a pleasant story: he was a sensible old\ngentleman.\"\n\n\"Yes, boys, he was; and it was sensible in you to ask first whether the\nstarling _did use_ silk and thread before Europeans came here; and after\nthat is answered, it is time enough to ask where he got such things. Now\nthe truth is, that he _did not_ use them until after Europeans brought\nthem here; because there were no such things in this country: for the\nIndians who lived here could not make thread. I think; and I am sure\nthey could not spin silk: but I will tell you, boys, what it shows us;\nand it is that I wish you to notice.\"\n\n\"What is it, Uncle Philip?\"\n\n\"It is the wisdom of this bird in taking advantage of circumstances. No\ndoubt he built very good nests long before silk and thread were in the\ncountry; but he had sense enough to know that they were exactly what\nsuited him, and he used them as soon as he could get them.\"\n\n\"Then, Uncle Philip, you think that the bird has reason?\"\n\n\"No, boys, I do not: but you have reason, and I have something to say\nto you about it. It is this: as God has given you reason, and so made\nyou better than the poor dumb animals, he expects more from you. That is\nfair, is it not?\"\n\n\"Yes, Uncle Philip; very fair.\"\n\n\"Then what I wish you to remember is this: that you must use your reason\nin such way as to glorify God. He gave it to you to learn his will and\nhis commandments, and to live accordingly. So now you see the things\nwhich our conversations about the animals can teach us. In the first\nplace, we see the goodness of God; in the second place, we see the\npower of God; in the third place, we see the wisdom of God: and we see\nin ourselves that God has done more for us than he has done for them,\nand therefore we ought to love and serve him: we ought to believe what\nhe says in his Word; we ought to pray to him for his blessed help; we\nought, _first of all_, to seek the salvation of our souls, through our\nLord Jesus Christ.\n\n\"Now, my dear children, to-morrow I must leave home for a few weeks; but\nwhen I come back we will talk together again: and as I am going to see\nmy nephews, I will get a book which they printed about insects; it is\ncalled the History of Insects,[14]--and I will bring it to you; and some\nof the largest boys among you may read it aloud, and I will explain to\nyou what you cannot understand. If you are pleased with what I have been\ntelling you, that book will tell you a great deal more.\"\n\n\"Oh, thank you, Uncle Philip. We shall like it very much.\"\n\n\"Farewell, boys.\"\n\n\"Good-by, dear Uncle Philip.\"\n\n\nFOOTNOTES:\n\n[12] Myiothera obsoleta of Bonaparte.\n\n[13] Tyrannus intrepidus.\n\n[14] Family Library, No. VIII.--_Publishers._\n\n\n\n\nTHE END.\n\n\n\n\n _Now republishing, on good paper and large type, in 18mo. volumes_,\n\n\n SOCIAL EVILS\n AND\n THEIR REMEDY.\n\n A SERIES OF NARRATIVES TO BE PUBLISHED QUARTERLY.\n\n BY THE\n REV. CHARLES B. TAYLER, M.A.\n\n No. I.\n\n THE MECHANIC.\n\n IS NOW REPUBLISHED, AND FOR SALE BY THE BOOKSELLERS.\n\n \"Other foundation can no man lay than that is laid,\n which is Jesus Christ.\"\n\nAUTHOR'S ADDRESS\n\nNo doubt can be felt as to the fact, that there are at present many\ncrying evils in all ranks of society--perhaps there never was a time\nwhen more remedies were proposed. It is, however, a melancholy truth,\nthat the only remedy is too generally over-looked, or despised.\nRemedies, selfish in principle, and selfish in their proposed end, are\nheld forth and confided in by those who profess to be Christians, and,\nas such, dependent on the Great Head of the church. Man is taught how\nto live in time, and to be wise for time; but it has become unusual\nto refer to that fine old scriptural prayer, \"So teach us to number\nour days that we may apply our hearts unto wisdom.\" Indeed, the\nwisdom desired by too many is that which is so forcibly described by\nan apostle's pen, as \"earthly, sensual, devilish;\" not that wisdom\nthe attributes of which form the graces of man's new and regenerate\ncharacter, which is first pure, then peaceable, gentle, and easy to\nbe entreated; \"full of mercy and good fruits, without partiality, and\nwithout hypocrisy.\"\n\nIt is intended, in the series of narratives now advertised, to set\nforth, faithfully and simply, the one great principle on which\nChristians profess to act. This principle should never be lost sight\nof, in any publication addressed by a Christian author to Christian\nreaders. \"Other foundation can no man lay, than that is laid,\" laid by\nInfinite Wisdom himself--\"which is Christ Jesus.\" My illustrations will\nextend to every class of society; from the highest to the lowest. When\nit is found necessary to introduce the subject of political economy, I\nshall endeavour to give what seem to me the right views of the subject;\nand I shall take care to show, that when political economy cannot be\nidentified with Christian economy, it ought to occupy a subordinate\nplace. If it enters society as the servant of Christian principle, it\nmay be very useful as a servant; but, if it is to teach a man to walk in\nthe counsel of the ungodly, to speak of its usefulness in a Christian\ncommunity is absurd.\n\nFalse principles, however taking they may be, for a while, with the\nignorant, or with those who are not deep thinkers, can never stand for\nany length of time; and as for the ungodly, we know _Who_ has told us\nthey are \"like the chaff which the wind driveth away.\" I have undertaken\nthis work in a spirit of prayer to God for His assistance, and His\nblessing. Many of my readers. I am sure, will unite their prayers to\nmine, that it may be continued in the same spirit. Some few may object\nto this address from a minister of Christ to a Christian community,\nand say that it is according to the puritanical cant of the day. I\nanswer, that such cant (if mere cant) is quite as offensive to me as to\nthemselves; almost as offensive as the cant of ungodliness; but I cannot\nforget those words of solemn warning, from One who, alas, is still the\ndespised and rejected of many men: \"Whosoever shall be ashamed of me and\nof my words, in this adulterous and sinful generation, of him shall the\nSon of man be ashamed, when he cometh in the glory of his Father with\nthe holy angels.\"\n\nThe second number of \"Social Evils,\" entitled \"_The Lady and the Lady's\nMaid_,\" will be republished about the 1st of February, 1834.\n\n * * * * *\n\nTHEOLOGICAL LIBRARY.\n\nNo. I. The Life of Wiclif. By Charles Webb Le Bas, A.M.\n\nII. The Consistency of the whole Scheme of Revelation with Itself and\nwith Human Reason. By Philip Nicholas Shuttleworth, D.D.\n\nIII., IV. Luther and the Lutheran Reformation. By John Scott, A.M.\n\nV., VI. The Life of Archbishop Cranmer. By Charles Webb Le Bas, A.M.\n\nVII., VIII. History of the Reformed Religion in France. By Rev. Edward\nSmedley, M.A. _In Press._\n\n\n\n\n +----------------------------------------------------------------- +\n | Transcriber's Note: |\n | |\n | * Obvious punctuation and spelling errors repaired. |\n | |\n | * Original spelling and its variations were not standardized. |\n | |\n | * Original use of quotation marks was left unchanged. |\n | |\n | * The word \"scattered is missing between pages 135 and 136. |\n | |\n | * \"... have got permission from your friends....\" This should |\n | be \"permission from parents,\" as the context suggests. |\n | |\n | * Italicized words are surrounded by underline characters, _like |\n | this_. Words in bold characters are surrounded by equal signs, |\n | =like this=. |\n | |\n | * Footnotes were moved to the end of the paragraphs to which |\n | they applied and numbered in one continuous sequence. |\n +------------------------------------------------------------------+\n\n\n\n\n\n\nEnd of the Project Gutenberg EBook of Natural History, by Anonymous\n\n*** ","meta":{"redpajama_set_name":"RedPajamaBook"}} +{"text":"\n\n\n\nProduced by Steven desJardins and the Online Distributed\nProofreading Team at http:\/\/www.pgdp.net\n\n\n\n\n\n\nMAURUS JOKAI\n\nTHE LION OF JANINA\nOR\nTHE LAST DAYS OF THE JANISSARIES\n\nA Turkish Novel\n\nTRANSLATED BY\nR. NISBET BAIN\n\n\nHARPER & BROTHERS PUBLISHERS\nNEW YORK AND LONDON\n1898\n\n\n\n\n BY THE SAME AUTHOR.\n\n THE GREEN BOOK; or, Freedom Under the Snow. A Novel.\n Translated by Mrs. Waugh. 16mo, Cloth, Ornamental,\n $1 50. (In \"The Odd Number Series.\")\n\n BLACK DIAMONDS. A Novel. Translated by Frances A.\n Gerard. With a Photogravure Portrait of the Author.\n 16mo, Cloth, Ornamental, $1 50. (In \"The Odd Number\n Series.\")\n\n HARPER & BROTHERS, PUBLISHERS,\n NEW YORK AND LONDON.\n\n\n\n\nCopyright, 1897, by Harper & Brothers.\n\nAll rights reserved.\n\n\n\n\nTHE LION OF JANINA\n\n\n\n\nPREFACE\n\n\nThe first edition of _Janicsarok vegnapjai_ appeared forty-five years\nago. It was immediately preceded by the great historical romance,\n_Erdely aranykora_ (_The Golden Age of Transylvania_), and the still\nmore famous novel of manners, _Egy Magyar Nabob_ (_A Hungarian\nNabob_), which Hungarians regard as, indisputably, Jokai's\nmasterpiece, while only a few months separate it from _Karpathy\nZoltan_ (_Sultan Karpathy_), the brilliant sequel to the _Nabob_. Thus\nit belongs to the author's best literary period.\n\nIt is also one of the most striking specimens of that peculiar group\nof Turkish stories, such as _Toeroekvilag Magyarorszagon_ (_Turkey in\nHungary_) and _Toeroek mozgolmak_ (_Turkish Incursions_), _A ketszarvu\nember_ (_The Man with the Antlers_), and the extremely popular _Feher\nrozsa_ (_White Rose_), which form a genre apart of Jokai's own\ncreation, in which his exuberant imagination revels in the rich colors\nof the gorgeous East, as in its proper element, while his ever alert\nhumor makes the most of the sharp and strange contrasts of Oriental\nlife and society. The hero of the strange and terrible drama, or,\nrather, series of dramas, unfolded with such spirit, skill, and\nvividness in _Janicsarok vegnapjai_, is Ali Pasha of Janina,\ncertainly one of the most brilliant, picturesque, and, it must be\nadded, capable ruffians that even Turkish history can produce.\nManifold and monstrous as were Ali's crimes, his astonishing ability\nand splendid courage lend a sort of savage sublimity even to his\nblood-stained career, and, indeed, the dogged valor with which the\noctogenarian warrior defended himself at the last in his stronghold\nagainst the whole might of the Ottoman Empire is almost without a\nparallel in history.\n\nWith such a hero, it is evident that the book must abound in stirring\nand even tremendous scenes; but, though primarily a novel of incident,\nit contains not a few fine studies of Oriental character, both Turkish\nand Greek, by an absolutely impartial observer, who can detect the\nworth of the Osmanli in the midst of his apathy and brutality, and\nwho, although sympathetically inclined towards the Hellenes, is by no\nmeans blind to their craft and double-dealing, happily satirized in\nthe comic character of Leonidas Argyrocantharides.\n\nFinally, I have taken the liberty to alter the title of the story.\n_Janicsarok vegnapjai_ (_The Last Days of the Janissaries_) is too\nglaringly inapt to pass muster, inasmuch as the rebellion and\nannihilation of that dangerous corps is a mere inessential episode at\nthe end of the story. I have, therefore, given the place of honor on\nthe title-page to Ali Pasha--the Lion of Janina.\n\nI have added a glossary of the Turkish words used by the author in\nthese pages.\n\nR. NISBET BAIN.\n\n\n\n\nContents\n\n Chapter Page\n I. THE CAVERNS OF SELEUCIA 1\n II. EMINAH 19\n III. A TURKISH PARADISE 45\n IV. GASKHO BEY 62\n V. A MAN IN THE MIDST OF DANGERS 72\n VI. THE LION IN THE FOX'S SKIN 78\n VII. THE ALBANIAN FAMILY 105\n VIII. THE PEN OF MAHMOUD 110\n IX. THE CIRCASSIAN AND HIS FAMILY 129\n X. THE AVENGER 160\n XI. THE FLOWERS OF THE GARDEN OF BEGTASH 187\n XII. THE SHIPWRECK OF LEONIDAS 198\n XIII. A BALL IN THE SERAGLIO 213\n XIV. KURSHID PASHA 238\n XV. CARETTO 244\n XVI. EMINAH 252\n XVII. THE SILVER PEDESTAL IN FRONT OF THE SERAGLIO 262\n XVIII. THE BROKEN SWORDS 275\n GLOSSARY OF TURKISH WORDS 293\n\n\n\n\nThe Lion of Janina\n\n\nCHAPTER I\n\nTHE CAVERNS OF SELEUCIA\n\n\nA savage, barren, inhospitable region lies before us, the cavernous\nvalley of Seleucia--a veritable home for an anchorite, for there is\nnothing therein to remind one of the living world; the whole district\nresembles a vast ruined tomb, with its base overgrown by green weeds.\nHere is everything which begets gloom--the blackest religious\nfanaticism, the darkest monstrosities of superstition--while an\neternal malediction seems to brood like a heavy mist over this region,\ncreated surely by God's left hand, scattering abroad gigantic rocky\nfragments, smiting the earth with unfruitfulness, and making it\nuninhabitable by the children of men.\n\nMan rarely visits these parts. And, indeed, why should he come, or\nwhat should he seek there? There is absolutely nothing in the whole\nregion that is dear to the heart of man. Even the wild beast makes no\nabiding lair for himself in that valley. Only now and then, in the\nburning days of summer, a lion of the wilderness, flying from before\nthe sultry heat, may, perchance, come there to devour his captured\nprey, and then, when he is well gorged, pursue his way, wrangling as\nhe goes with the echo of his own roar.\n\nSolitary travellers of an enterprising turn of mind do occasionally\nvisit this dreary wilderness; but so crushing an impression does it\nmake on all who have the courage to gaze upon it, that they scarce\nwait to explore the historic ground, but hasten from it as fast as\ntheir legs can carry them.\n\nWhat is there to see there, after all? A battered-down wall, as to\nwhich none can say who built it, or why it was built, or who destroyed\nit. A tall stone column, the column of the worthy Simon Stylites, who\npiled it up, stone upon stone, year after year, with his own hands,\nbeing wont to sit there for days together with arms extended in the\nshape of a cross, bowing himself thousands and thousands of times a\nday till his head touched his feet. The northern and southern sides of\nthe valley are cut off from the rest of the world by gigantic masses\nof rocks as steep and solid as the bastions of a fortress; only\ntowards their summit, at an elevation of some three to four hundred\nyards, is a little strip of green vegetation visible.\n\nDarkly visible at intervals in this long and steep rocky wall are the\nmouths of a series of caverns, of various sizes, all close together.\nIt looks as if some monstrous antediluvian race had cut two or three\nstories of doors and windows into the living rock, in order to make\nthemselves palaces to dwell in.\n\nThe walls of these caverns are so rugged, their bases are so\nirregular, that it is scarcely conceivable that they could be the\nwork of human hands, unless, indeed, the arched concavities of the\nchasms and the regular consecutiveness of the series may be assumed to\nbear witness to the wonder-working power of finite forces.\n\nThree of the entrances to these caverns have all the loftiness of\ntriumphal arches; nay, one of them, carved in the base of the rock, is\nso exceptionally vast that it rather resembles the nave of a huge\nchurch, and is said to penetrate the whole mountain to the sea beyond.\nIt is said that if any one has the courage to attempt the journey, he\nwill discover mysterious hieroglyphics carved on the walls. Who could\nhave been the authors of this unknown runic language? The Chaldeans\nperhaps, or the worshippers of Mithra. What hidden secrets, what human\nmemorials are enshrined in these symbols? That question must remain\nforever without an answer.\n\nMost probably this valley was used as a burial-place by some\nlong-vanished nation, whose tombs have survived them, making the whole\nregion still more dreadful; the gaping crevices of the rocks seem to\nproclaim, as from a hundred open throats, that here an extinct race\nhas found its last resting-place.\n\nMoreover, the largest cavern of all has the unusual property of\nsometimes emitting whistling sounds like interrupted human voices. The\nshepherds on the mountain summits listen terror-stricken to this\nbellowing of its rocky throat. At first it resembles the buzzing of\nimprisoned wasps, but the din gradually gathers force and volume till\nit seems as if the demons of the wind had lost their way within the\ncavern, and were roaring tumultuously in their endeavors to find an\nexit. This noise is generally followed by the blast of the simoon,\nwhich no doubt penetrates into the cavern through a gap on the other\nside, and thus gives rise to the mysterious voices of the valley.\n\nBut not on these occasions only; at other seasons also the cavern is\nwont to speak. It happens now and then that a shepherd, more foolhardy\nthan his fellows, ventures into the hollow of the cavern to light a\nfire, and, full of bravado, provokes the _dzhin_ of the cavern to\nappear, till the cavern suddenly re-echoes his voice; but it does not\nre-echo the words he utters, but replies in a soft, low accent to the\ninsolent youth, bidding him withdraw and cease to mock God's\ncreatures.\n\nOn another occasion an adulterous woman and her paramour strolled\ntowards the spot with the intent of using the deep darkness as the\ncloak for their sinful joys; but what terror filled the guilty lovers\nwhen their sweet whispering was interrupted by a voice which was\nneither near nor far, and belonged neither to man nor spirit, but\nwhose cold sigh turned their hot blood into ice as it whispered,\n\"Allah is everywhere present!\"\n\nOnce, too, some robbers were lying in wait for their comrades, whom\nthey intended to murder in that place, when a roaring began in the\ncave which seemed to make the very welkin ring, and the murderers\nclearly distinguished the terrible words: \"The eye of Allah is upon\nyou, and the flames of Morhut are burning for your souls!\" whereupon,\ninsane with fright, they rushed from the cave.\n\nEvery one who lived near the place knew of, and believed in, the\n_dzhin_ of the cavern, who, they said, harmed not the good, but\npersecuted evil-doers.\n\nBut it was not only terror-stricken hearts who knew of the voice of\nthe invisible _dzhin_--crushed and bleeding hearts likewise repaired\nthither. And the invisible _dzhin_ read their secrets; they had no\nneed to acquaint him with their griefs, and he gave them good counsel,\nand, for the most part, sent them away comforted. Doubtless anybody\nelse might have given them similar counsels; but if the advice had\ncome from ordinary men, the suppliants would not perhaps have welcomed\nit with such enthusiasm, or have turned it to such good account.\n\nAnd people often came thither to inquire into the future; and the\ninvisible being, it was found, could distinguish between those who\ncame to him in real anguish of mind and those whom only curiosity had\nattracted thither, or who merely wished to prove him. To the latter he\nmade no answer, but to the former he often spoke in prophetic\nparables, whose deeply figurative meaning was frequently fulfilled\nword for word.\n\nThe superstitious common folk made a merit of sacrificing to this\nunknown being. The dwellers round about made a point of living on good\nterms with him, took care not to provoke him with vain words, did not\nfly to him at every trifle; nay, on one occasion, the Kadi[1] of\nSeleucia even laid by the heels a couple of wanton rascals who were\ncaught throwing stones into the cavern.\n\n[Footnote 1: For this and all other Turkish words see the glossary at\nthe end of this book.]\n\nFrom the mouth of the cave inward extended a sort of staircase\nconsisting of about forty steps, terminating at a point whither the\nlight of day scarcely ever reached. Here stood a huge stone, not\nunlike a rude altar, in the midst of which was a slight hollow. This\nhollow the pious inhabitants of the district used to fill with rice or\nmillet, and on returning next day they would see that the _dzhin_ had\nremoved it from thence, and, by way of payment, had left a small\nsilver coin in this natural basin--a coin belonging to that old silver\nmoney which had been struck in the brilliant days of the Turkish\nEmpire, and was worth thrice as much as the present coinage. Thus the\n_dzhin_ would take nothing gratis, but paid for everything in ready\nmoney.\n\nThose who wished to speak with him had to penetrate into the depths of\nthe cave where no daylight was visible, for he was only to be found\nwhere the darkness was complete. If any one went with sword or dagger\nhe got no answer at all. And a visitor standing alone there in the\ndarkness was as plainly visible to the _dzhin_ as if the glare of\nnoonday were beating full upon him; not a change of countenance was\nhidden from this mysterious being. So they more readily believed that\nhe who could thus see through the darkness of earth could also see\nthrough the darkness of human hearts and the darkness of the\nunrevealed future.\n\nThis marvel had now been notorious for fifty years, the ordinary span\nof human life, and princes, pashas, generals, wise men, priests,\nulemas, were in the habit of visiting the abode of the _dzhin_, who\nseemed to know about everything that was going on in the world above.\nTo many he prophesied death, and to those who pleased him not he\nforetold the Nemesis that was to come upon them as a reward for their\niniquities.\n\n * * * * *\n\nIn the year one thousand eight hundred and nineteen, at the season\nimmediately following the raging of the simoon, it chanced that a\npirate ship sailed into the haven of Suda, whence the magnificent\nruins of the ancient Seleucia are still to be seen. The corsair\ncarried the French flag, but her crew consisted entirely of Albanians.\nThe deck was encumbered with wreckage, cast down upon it by the\nhappily weathered tempest, and this the crew were energetically\nengaged in removing; but every one on shore was astounded to see her\nthere at all, much more in such trim condition, for she had lost\nneither mast nor sail. But then, after the manner of corsairs in\ngeneral, she was very much better equipped with both masts and sails\nthan ships of ordinary tonnage are wont to be. In the same hour that\nthe ship cast anchor the largest of her boats was lowered, and manned\nby four and twenty well-armed Trinariots. Every one of these stout\nfellows carried orders of merit on his cheek, the scars of many a\nbattle, which accentuated the savage sternness of their weather-beaten\nfaces.\n\nA little old man descended after them into the boat; presently his\nhorse was also let down by means of a crane. This was the officer in\ncommand. He was a middling-sized but very muscular old fellow, already\nbeyond his seventieth and not very far from his eightieth year; but he\nwas as vigorous now both in mind and body as he had been when his\nbeard, which now swept across his breast like the wing of a swan, was\nas dark as the raven's plume.\n\nHis broad shoulders spoke of extraordinary strength, while the firm\nexpression of his face, the flashing lustre of his eyes, and his calm\nand valiant look, testified to the fact that this strength was\nsquandered upon no coward soul.\n\nSome stout rowing brought the boat at last near to the shore, but not\nall the efforts of the men could bring her to land; the wash of the\nsea was so great that the foam-crested waves again and again drove the\nboat back from the shore.\n\nAt a sign from the old man three of the ship's crew leaped into the\nwaves in order to drag after them the boat's hawser, but the sea tore\nit out of the hands of all three as easily as a wild bull would toss a\npack of children.\n\nThen the old man vaulted upon his steed, kicking the stirrups aside,\nand leaped among the churning waves. Twice the horse was jostled back\nby the assault of the foaming billows, but at the third attempt the\nshore was reached. The people on the shore said it was a miracle; but\nhe, wasting no words upon any one, directed his way all alone along\nthe shore of the haven, and leaving behind him the lofty turreted row\nof bastions--which crowns the edge of the rocky promontory, encircles\nthe town, and hangs upon the shoulders of the hill like an ancient and\ngigantic necklace--picked his way among the lofty, scattered bowlders,\nand, unescorted as he was, quickly disappeared from view amid the\nwilderness.\n\nHe had scarcely proceeded more than half an hour among the fig and\nolive trees which covered the s of the hills, and whose scorched\nand withered leaves marked the passage of the burning wind, when he\narrived at the place he sought. It was a crazy, tumble-down hut, whose\nshapeless mass was so clumsily compounded of wood, stone, and mud,\nthat a swallow would have been ashamed to own it, let alone a beaver,\nwhose ordinary habitation is an architectural masterpiece compared\nwith it. Nature, however, had been gracious to this shanty, and\nclothed it with creeping plants, which nearly hid away all the\nsuperfluous cracks and crevices which the architect had left behind\nhim.\n\nIt was here that the new-comer dismounted from his horse, tied it to a\ntree, and, proceeding to the latchless door, amused himself by reading\nthe scrawl which had been written on the outside of it, and was, as\nusual, one of those sacred texts which the Turks love to see over\ntheir door-posts: \"Accursed be he who disturbs a singing-bird!\"\n\nThe stranger fell a listening. Surely there was no singing-bird here,\nhe thought. Then he went on reading what followed: \"He who knocks at\nthe gate of him who prays will knock in vain at the gate of Paradise.\"\n\nThe stranger did not take the trouble to knock; he simply kicked the\ndoor down.\n\nWithin was kneeling an anchorite of the order of Erdbuhar on a piece\nof matting. He was naked to the girdle, and before him stood a wooden\ntub full of fresh water. He was just finishing his ablutions.\n\nHe did not seem to observe the violent inroad of the stranger, but\nconcluded his religious exercises with great fervor. First of all he\nwashed his hands, reciting thirty times the sacred words, \"Blessed be\nGod, Who hath given to water its purifying power, and hath revealed\nthe true faith to us!\" Next he thrice conveyed water to his mouth in\nhis right palm, and prayed, \"O Lord! O Allah! refresh me with the\nwater Thou didst give to Thy Prophet Muhammad in Paradise, which is\nmore fragrant than balm, whiter than milk, and sweeter than honey, and\nsatisfies eternally those who pine with thirst!\" Then, with the palm\nof his hand, he cast water upon his nostrils, and exclaimed,\nfervently, \"O Lord! cause me to smell the perfume of Paradise, which\nis sweeter than musk and ambergris, and suffer me not to inhale the\naccursed fumes of hell!\" Then, filling both palms with water and well\nwashing his face, he said these words, \"Purify my face, O Lord, like\nas Thou wilt purify the faces of Thy prophets and servants on the\ngreat Day of Judgment!\" But even this did not suffice, for now he put\nwater in his right palm again, and, letting it run down his elbows, he\nsighed, \"Lord, suffer me at the last day to hold in my right hand,\nwhich is the hand of Thine elect, the book of my good deeds, and admit\nme to Thy Paradise!\" With that he dipped his head into the tub of\nwater, but so as to keep his mouth clear of it, and spake in this\nwise, \"O Lord, when I appear before Thee, encompass me with Thy\nmercies, and crush not my head beneath the fiery wreath of my sins,\nbut adorn it with the golden crown of my merits!\" Then came the turn\nof his ears, the worthy man crying the while, with unction, \"Grant, O\nLord, that mine ears may hear, for ever and ever, those joyous sounds\nwhich are written in the Kuran!\" This accomplished, he sprinkled his\nneck and throat, suitably exclaiming, \"O Lord, deliver me from those\nfetters which will be cast upon the necks of the accursed!\" After\nwhich pious ejaculation he sat down on the ground, and, reverently\nwashing his right foot, exclaimed, \"O Lord, suffer not my feet to slip\non the bridge of Alserat which leads across hell to heaven!\" Then he\ncleansed thoroughly his left foot also, and sighed, \"May the Lord\nforgive me my trespasses and listen to my supplications!\"\n\nAnd the honest dervish did not utter all these pious ejaculations in a\nlow mumble, but in an intelligible, exalted voice, as becomes an\northodox Mussulman, who does not consider it a shameful thing to pray\nto God in the presence of men.\n\nAfter that he took up the tub and, carrying it out, sprinkled the\nwater it contained over the wild flowers growing there, blessing them\nseverally and collectively; then he filled it full again with fresh\nwater from the spring, and bringing it back into the hut and turning\nthe mat over, placed the tub full of water on it, whereupon the\nstranger immediately divested himself of his slippers and upper\nkaftan, unwound his turban, removed his red fez from his head, and\nproceeded to perform his ablutions also in the self-same manner.\n\nWhen he had finished he kissed the hand of the dervish, and when the\nlatter drew from his girdle a long manuscript reaching to the very\nground, and began, from its eighty sections, to laud and magnify the\neighty properties of Allah, the stranger repeated them after him with\ngreat unction, and, at the end of each one of them, intoned with him\ntwice over the verse, \"La illah, il Allah, Muhammad roszul Allah!\"--in\nthe chanting of which he was as practised as any muezzin.\n\nAll these pious practices were accomplished with the utmost devotion;\nbut when the new-comer arose from his place, the expression of\nlowliness vanished from his features and he reassumed his former\ncommanding look, while the dervish now humbly bowed down before him to\nthe very earth and murmured:\n\n\"What are my lord's commands to his servant?\"\n\nThe stranger let him lie there and slowly raised his sword.\n\n\"Art thou,\" cried he, \"that dervish of Erdbuhar[2] to whom I\ndespatched a fakir of the Nimetullahitas, who dwelleth in Janina?\"\n\n[Footnote 2: The orders of Erdbuhar and Nimetullahita are the severest\nof all the Turkish religious fraternities: the former fast so\nrigorously twice a week that they do not even swallow their saliva;\nthe latter observe the fast only during their year of probation, after\nwhich they are free to return to the joys of this world.]\n\n\"Thy servant is that man.\"\n\nThe stranger thereupon, with his right hand, drew a dagger from his\ngirdle, and with his left hand a purse.\n\n\"Dost thou see this dagger and this purse?\" said he. \"In the purse are\na thousand sequins; on the blade of this sword is the blood of at\nleast as many murdered men. I ask thee not--Dost thou recognize me? or\ndost thou know my name? Maybe thou dost know--for thou knowest all\nthings--and, if so, thou dost also know that none hath ever betrayed\nme on whom I have not wreaked my vengeance. If, therefore, thou dost\nwant a reward, listen; but if chastisement, speak!\"\n\nThe dervish raised his hand to his ear to signify that he would prefer\nto listen.\n\n\"Arise, then! take my horse's bridle, and lead me to that cavern where\ndwelleth the _dzhin_ of prophecy. Dost thou know him?\"\n\n\"I know him, my master, but go to him I will not, for he is wroth with\nme. He loves not the dervishes, because they would always be teaching.\nIf I go to him he throws stones at me from out of the cavern, or leads\nme into deep pitfalls. Therefore, if thou so desire it, I will lead\nthee thither; but I would not go with thee if I had as many heads upon\nmy shoulders for thy sword to sever as there are sequins in that\npurse.\"\n\n\"There is no need of that. Thou canst remain outside and hold my\nhorse.\"\n\nAnd with that the herculean old man flung himself haughtily on his\nhorse, and the dervish, seizing the steed's bridle, began to lead him\nalong the mountain path among the rugged rocks and bowlders.\n\nThe moon was already high in the heavens when they reached the mouth\nof the cavern.\n\nLooking back upon the country whence they came, the region seemed more\ndesolate than ever. In front, the savage, natural ruins; behind, the\nblack cedar forests, where thick foliage cast night-black shadows even\nat noonday; on each side, the endlessly sublime masses of rocks, which\nstood out still vaster in the moonlight. The caverns looked still\nblacker at night, and the rock and ruins more sterile; but, night and\nday alike, the place was deserted.\n\nOn reaching the cavern of the _dzhin_, the old man dismounted from his\nhorse and, bidding the dervish stand and hold it till he returned,\ndisappeared in the cavern without the slightest hesitation.\n\nHe could only grope his way, step by step, through the blinding\ndarkness; cautiously he advanced, but without fear. He tested the\nground in front of him as he advanced, with one hand over his eyes and\nthe other on the hilt of his sword. It must, indeed, be a resolutely\nwicked spirit that would venture to attack him.\n\nEvery now and then a bat sped rapidly past him, close to his ears,\nwith a sound like a mocking titter; at other times he trod upon some\ncold, moving body. But what cared he for these? The deep silence which\nencircled him was far more terrible than all the voices of hell; and\nnot even the darkness terrified him, for his powerful voice now\npierced that subterranean stillness as with a sword.\n\n\"I summon thee, thou spirit, whether thou art good or evil, whom Allah\npermits to hold discourse with living men--I summon thee to speak with\nme!\"\n\n\"I am even now beside thee,\" a voice suddenly whispered. It was low\nand hollow, just as if the atmosphere of the cavern were speaking.\n\nThe stranger made a clutch after the voice, as if his audacious hand\nwould have seized the spirit; but he found nothing. It was a voice\nwithout a shape.\n\n\"Speak to me!\" cried the old man, in a voice that never quavered.\n\"Dost thou know my fate?\"\n\n\"I know it,\" answered the invisible voice; \"thou art a poor man who\nhast lost what thou hadst, and what thou now hast is not thine.\"\n\n\"Thou art a senseless spirit,\" growled the stranger. \"Go back to thy\ntomb and slumber; I will inquire nothing more of thee. Thou dost not\neven know my present fate; how canst thou know my future? Go back to\nthy hole, I say, and sleep in peace.\"\n\n\"I know thee,\" continued the voice, \"and I have spoken the truth. Do\nnot they call thee Ali Tepelenti?\"\n\nThe stranger was amazed. \"That is indeed my name,\" he answered.\n\n\"Wert thou not a fugitive yesterday, and wilt thou not be dust and\nashes to-morrow?\"\n\n\"True; but that yesterday was eighty years ago; and who shall say when\nto-morrow will be?\"\n\n\"Thou knowest that here there is neither morning nor evening,\"\nanswered the voice. \"To me yesterday was when I last saw the sun, and\nto-morrow will be when I see it again. Ali Tepelenti, Lord of Janina,\nthou art poorer than the lowliest Mussulman who girds himself with a\ngirdle of hair, for thou hast lost everything which thou didst account\nprecious. Thy kinsmen, who were for thy defence, thou hast slain; thy\nmother, who loved thee, thou hast strangled; thy right hand has pulled\ndown the house which thou didst build up; thy glory, in which thou\ndidst exalt thyself, has become a curse to thee; and thou hast made\nbitter haters of those who loved thee best.\"\n\n\"So it is. I know what I have done. I repent me of nothing. The hare\nnibbles the flower, the vulture seizes the hare, the hunter slays the\nvulture, the lion fells the hunter, the worm devours the lion. All of\nus turn to earth. Allah is mighty, and He orders it so. What am I?\nOnly a bigger worm than the rest. Who shall strive with God? What is\nmy fate in the future?\"\n\n\"But yesterday thou wert younger than thy newborn son, to-morrow thou\nshalt die older than thy oldest ancestors.\"\n\n\"Speak more plainly. I perceive the meaning of thy words as little as\nI perceive thyself.\"\n\n\"'He who sins with the sword shall perish with the sword,' saith\nAllah. He who sins with love, shall perish by love. Thou hast two\nhands, the right and the left; thou hast two swords, one covered with\ngold and one with silver; thou hast three hundred wives in thy harem,\nbut only one in thy heart; thou hast twelve sons, but only one whom\nthou lovest. Look, now! Take good heed of thy life, for thy death\nlieth in what is nearest to thee; thine own weapon, thine own child,\nthine own property, thine own two hands, shall one day slay thee.\"\n\n\"Mashallah! Death is inevitable. Tell me but one thing. Shall I one\nday pass in triumph through the gates of the seraglio at Stambul?\"\n\n\"Thou shalt. Thou shalt stand there on a silver pedestal in the face\nof the rejoicing multitude.\"\n\n\"When?\"\n\n\"That day will come when thou shalt be in two places at the same time,\nin Janina and in Stambul; the days to come will explain it.\"\n\n\"One word more. Wherefore didst thou mention that woman whom I love\nbest?\"\n\n\"She will be the first to betray thee.\"\n\n\"Accursed one!\" roared Ali, drawing his sword and madly striking in\nthe direction of the voice.\n\nThe sword hissed fiercely through the vacant air, and the next moment\nthe voice replied from a respectable distance:\n\n\"It has happened already.\"\n\n\"This is a dream, all a dream!\" moaned Ali.\n\n\"'Tis no dream; thou art wide awake,\" cried the mysterious voice.\n\n\"If it be no dream, give me a sign that I may know before I depart\nhence that I have not been dreaming.\"\n\n\"First put thy sword into its sheath.\"\n\n\"I have done so,\" said Ali; but he lied, for he had only slipped it\ninto his girdle.\n\n\"Into the sheath, I say,\" cried the voice.\n\nIt was with a tremor that Ali felt that this being could distinguish\nhis slightest movement in the dark.\n\n\"And now stretch forth thy hand!\" cried the voice. It was now quite\nclose to him.\n\nAli stretched forth his hand, and the same instant he felt a vigorous,\nmanly hand seize his own in a grasp of steel; so strong, so cruel was\nthe pressure that the blood started from the tips of his fingers.\n\nAt last the invisible being let go, and said in a whisper as it did\nso:\n\n\"Not a muscle of thy face moved under the pressure of my hand; only\nTepelenti could so have endured.\"\n\n\"And there is but one man living who could press my hand like that,\"\nreplied Ali. \"His name was Behram, the son of Halil Patrona,[3] who,\nforty years ago, was my companion in warfare, and has since\ndisappeared. Who art thou?\"\n\n[Footnote 3: The extraordinary adventures of this Mussulman reformer\nare recorded in another of Jokai's Turkish stories, _A feher rozsa_\n(_The White Rose_).]\n\n\"Aleikum unallah!\"[4] said the voice, instead of replying.\n\n[Footnote 4: \"God be with thee!\"]\n\n\"Who art thou?\" again cried Ali, advancing a step.\n\n\"Aleikum unallah!\" was the parting salutation of the already\nfar-distant voice.\n\nThe mighty pasha turned back in a reverie, and when he got back into\nthe moonlight, he still saw plainly on his hand the drops of blood\nwhich that powerful grasp had caused to leap forth from the tips of\nhis fingers.\n\n\n\n\nCHAPTER II\n\nEMINAH\n\n\nAnd now for a story, a marvellous story, that would not be out of\nplace in a fairy tale! Away to another clime where the very sunbeams\nand blossoms, where the very beating of loving hearts, differ from\nwhat we are accustomed to.\n\n * * * * *\n\nIn whichever direction we look around us, we shall see the land of the\ngods rising up before us in classical sublimity, the mountains of\nHellas, the triumphal home of sun-bright heroes. There is the mountain\nwhence Zeus cast forth his thunderbolts, the grove where the thorns of\nroses scratched the tender feet of Aphrodite, and perchance a whole\nolive grove sprung from the tree into which the nymph, favored and\npursued by Apollo, was metamorphosed. The sunlit summits of snowy OEta\nand Ossa still sparkle there when the declining sun kindles his\nbeacons upon them, and Olympus still has its thunderbolts; yet it is\nno longer Zeus who casts them, but Ali Tepelenti, Pasha of Albania and\nmaster of half the Turkish Empire, and the rose which the blood of\nVenus dyed crimson blooms for him, and the laurel sprung from the love\nof Apollo puts forth her green garlands for him also.\n\nThe poetic figures of the bright gods are seen no more on the quiet\nmountain. With a long gun over his shoulder, a palikar walks hither\nand thither, who has built his hut in a lurking-place where Ali Pasha\nwill not find it. The high porticos lie level with the ground; the\npaths of Leonidas and Themistocles are covered with sentry-boxes, that\nnone may pass that way.\n\nFrom the summit of the mighty Lithanizza you can look down upon the\nfairy-like city which dominates Albania. It is Janina, the\nhistorically renowned Janina.\n\nBeside it stands the lake of Acheruz, in whose green mirror the city\ncan regard itself; there it is in duplicate. It is as deep as it is\nhigh. The golden half-moons of the minarets sparkle in the lake and in\nthe sky at the same time. The roofless white houses, rising one above\nanother, seem melted into a compact mass, and they are encircled by\nred bastions, with exits out of eight gates.\n\nBut what have we to do with the minarets, the bazaars, the kiosks of\nthe city? Beyond the city, where Cocytus, rippling down from the\nwooded mountain, forms, with the lake into which it flows, a\npeninsula, there, on an isthmus, stands the strong fortress of Ali\nPasha, with vast, massive bastions, a heavy, iron-plated drawbridge,\nand a ditch in front of the walls full of solid sharp-pointed stakes\nin two fathoms of water. From the summits of the ramparts the throats\nof a hundred cannons gape down upon the town--iron dogs, whose barking\ncan be heard four miles off. On the walls an innumerable multitude of\narmed men keep watch, and in front of the gate the guns look out upon\neach other from the port-holes of the steep bastions on both sides of\nit. Woe to those who should attempt to make their way into the citadel\nby force! The gate, fastened with a huge chain, is defended by three\nheavy iron gratings, and from close beneath the lofty projecting roof\ncircular pieces of artillery shine forth, in front of which are\npyramidal stacks of bombs.\n\nThe court-yard forms a huge crescent, in which nothing is visible but\ninstruments of warfare, engines of destruction. In the lower part of\nthe semicircular barracks stand the sentry-boxes, while in the\nopposite semicircle a long pavilion cuts the fortress in two,\nextending from the end of one semicircle to the end of the other, and\nhere are three gates, which lead into the heart of the fortress.\n\nIn all this long building there are no windows above the court-yard,\nonly two rows of narrow embrasures are visible therein. All the\nwindows are on the other side overlooking the garden, and there dwell\nthe odalisks of Ali Pasha's three sons. The three sons, Omar, Almuhan,\nand Zaid, inhabit the building with the three gates. The back of this\nbuilding looks out upon the garden, in which the harems of the pasha's\nsons are wont to disport themselves.\n\nHere again a long bastion barricades the garden, a bastion also\nprotected by trenches full of water, across whose iron bridge you gain\nadmission into the pasha's inmost fortress.\n\nAnd what is that like? Nobody can tell. The brass gates, covered with\nsilver arabesques, seem to be eternally closed, and none ever comes in\nor goes out save Ali and his dumb eunuchs, and those captives whose\nheads alone are sent back again. The bastion surrounding this central\nfortress is so high that you cannot look into it from the top of the\ncitadel outside; but if any one could peep down upon it from the\nsummit of the lofty Lithanizza he would perceive inside it a fairy\npalace, with walls of marble protected by silver trellis-work,\nwith blue-painted, brazen cupolas, with golden half-moons on their\npointed spires. One tower there, the largest of all, has a roof of red\ncast-iron, and this one roof stands out prominently from among all the\nother buildings of the inner fortress. The kiosks are\neverywhere wreathed with garlands of flowers, and the spectator\nperched aloft would plainly discern cradles for growing vines on the\ntop of the bastion. He might also, in the dusk of the summer evenings,\ndistinguish seductive shapes bathing in the basins of the fountains,\nand lose his reason while he gazed; or it might chance (which is much\nmore likely) that Ali Pasha's patrols might come upon him unawares and\ncast him down from the mountain-top.\n\nThis wondrous retreat was Ali's paradise. Here he grouped together the\nmost beautiful flowers of the round world--flowers sprung from the\nearth or from a human mother. For maidens also are flowers, and may be\nplucked and enjoyed like other flowers. But the most beautiful among\nso many beautiful flowers was Eminah, Tepelenti's favorite damsel, the\nsixteen-years-old daughter of the Pasha of Delvino, who gave her to\nAli just as so many eminent Turks are wont to give their daughters. On\nthe day of their birth they promise to give them to some powerful\nmagnate, and by the time the _fiancee_ is marriageable the _fiance_\nhas already one foot in his grave.\n\nA pale, blue-eyed flower was she, looking as if she had grown up\nbeneath the light of the moon instead of the light of the sun; her\nshape, her figure, was so delicate that it reminded one of those\nsylphs of the fairy world that fly without wings. Her voice was\nsweeter, more tender, than the voices of the other damsels; and, wiser\nthan they, she could speak so that you felt rather than heard what she\nsaid. Ali loved to toy with her light hair, unwind the long folds of\nher tresses, cover his face with their silken richness, and fancy he\nwas reposing in the shades of paradise.\n\nAnd the child loved the man. Ali was a handsome old fellow. His beard\nwas as glossy and as purely white as the wing of a swan; the roses of\nhis cheeks had not yet faded; when he smiled he was no longer a tiger,\nbut revealed a row of teeth even handsomer than her own. And, in\naddition to that, he was valiant--a hero. Even in old men love is no\nmere impotent desire when accompanied with all the vigorous passion of\nyouth.\n\nAnd Eminah knew not that there were such beings as youths in the\nworld. Excepting her father and her husband, she had never seen a man,\nand therefore fancied that other men also had just such white beards\nand silvery eyelashes as they. Brought up from the days of her\nchildhood in the midst of a harem, among women and eunuchs, she had\nnot the remotest idea of the romantic visions which the hearts of\nlove-sick girls are wont to form from the contemplation of their\nideals; to her her husband was the most perfect man for whom a\nwoman's heart had ever beaten, and she clung to him as if he had been\na supernatural being.\n\nIn her heart Eminah pictured Ali as one of those beneficent genii who\nin the marvellous tales of the Arabs rise up from the bowels of the\nearth and the depths of the sea, a hundred times greater than ordinary\nmen, ten times younger, and a thousand times more powerful, who are\nwont to give talismanic rings to their earthly favorites, appearing\nbefore them when they turn this ring in order to instantly gratify\ntheir desires, their wishes; to transport them from place to place\nwith their huge muscular hands, to make them ride a cock-horse on\ntheir middle fingers, play hide-and-seek with them in the thousand\ncorners of their vast palaces, watch over them when they sleep,\noverwhelm them with heaps and heaps of gifts and treasures, and yet\nare gentle and complacent in spite of their immense power. They need\nbut take one step to crush the towers and bastions of the mightiest\nfortress in the dust, and yet they walk so warily as not even to graze\nthe tiny ant they meet upon their path. Why, once Ali had waded into\nthe lake up to his waist to rescue two amorously fluttering\nbutterflies that had fallen into it! Oh! Ali has such a sensitive soul\nthat he weeps over the bird that has accidentally beaten itself to\ndeath against the bars of its cage; whenever he plucks a flower from\nits stalk he always raises it to his lips to beg its pardon; and when\nthey told him how at the siege of Kilsura all the poor doves were\nburned, the tears sparkled in his eyes!\n\nEminah does not fully know the meaning of a siege; she only grieves\nfor the poor doves. How they would hover above the burning town in\nwhite clusters amid the black smoke, and fall down into the fire\nbelow!\n\nIn reality the matter stood thus: Ali was besieging Kilsura, but could\nnot take it; the besiegers fought valiantly, and the natural\nadvantages of the place prevented him from drawing near enough to it.\nSo he signified to the inhabitants that he would make peace with them\nand depart from their town, and desired them, in earnest of their\npacific intentions, to send him a number of white doves. The besieged\nfell in with his proposal, and collecting together all the white doves\nin the town they could lay they hands upon, sent them to Ali. He\nimmediately withdrew his siege artillery, with which he had already\nwrought no small mischief, but at night, when every one was asleep, he\nfastened fiery matches by long wires to the feet of the doves, and\nthen set them free. The natural instincts of the doves made them fly\nback to their old homes, the familiar roofs where their nests were,\nand in a moment the whole town was in flames, the doves themselves\ncarrying the combustible material from roof to roof and perishing\nthemselves among the falling houses.\n\nAli wept sore as he told to Eminah the story of the doves of Kilsura;\nyes, Ali was certainly a sensitive soul!\n\nThe beautiful woman had everything that eye could covet or heart\ndesire. In her apartments were mirrors as high as the ceiling,\nmasterpieces of Venetian crystal, and the floor was covered with\nPersian carpets embroidered with flowers. Blossoming flowers and\nsinging birds were in all her windows, and a hundred waiting-women\nwere at her beck and call. From morn to eve Joy and Pleasure were her\nattendants, and each day presented her with a fresh delight, a fresh\nsurprise.\n\nThirty rooms, opening one into another, each more magnificent than the\nlast, were hers, and hers alone. The eye that feasted on one splendid\nobject quickly forgot it in the contemplation of a still more splendid\nmarvel, and by the time it had taken them all in was eager to begin\nagain at the beginning.\n\nBut there was one thing which did not please Eminah. When one had got\nto the end of all the thirty rooms, it was plain that they did not end\nthere, for then came a round brass door; and this door was always\nclosed against her--never was she able to go through it. Now this door\nled into that huge tower with the red cast-iron roof, which could be\nseen such a distance off.\n\nThe inquisitive woman very much wanted to know what was inside this\ndoor through which she was never suffered to go, though Ali himself\nused it frequently, always closing it most carefully behind him, and\nwearing the key of it fastened to his bosom by a little cord.\n\nNow and then she had asked Ali what was in this tower that she was not\nallowed to see, and what he did when he remained there all night\nalone? At such times Ali would reply that he went there to consort\nwith spirits who were teaching him how to find the stone of the wise,\nhow to become perpetually young, how to foresee the future, and make\ngold and other marvels--all of which it was easy to make a woman\nbelieve who did not even know that all men do not wear white beards.\n\nAfter all such occasions Eminah, when she was alone again, would\nconjure up before her all sorts of marvellous blue and green denizens\nof fairyland appearing before Ali in the elements of air, fire, and\nwater, to teach him how to make gold. And Ali always proved to Eminah\nthat what he told her was no idle tale, for whenever he returned the\nnext day he was followed by a whole procession of dumb eunuchs\ncarrying baskets filled with gold and precious stones. Thus Ali not\nonly knew how to make gold, but also those things that are made of\ngold--that is to say, coined money and filigreed ornaments, which he\npiled up before her; and to Eminah it seemed a very nice thing, and\nquite natural that if these peculiar spirits could manufacture gold\nfrom nothing, they should also be able to make necklaces and bracelets\nout of smoke, as Ali told her they did without any difficulty at all.\n\nNow any one would have been curious to get to the bottom of such\nmysteries, especially if they were close at hand; how much more, then,\na spoiled and pampered young woman, who frequently was not able to\nsleep for the joy which the presents heaped upon her by Ali excited in\nher breast. How much she would have loved to see these benevolent\nspirits who had given her so much pleasure!\n\nFrequently she implored Ali to take her with him when he went into the\nred tower; but the pasha always tried to frighten her by saying that\nthese spirits were most cruel to strangers in general, and women in\nparticular, whom they would be ready to tear limb from limb, so that\nEminah always had to abandon her desire.\n\nBut when once a woman has made up her mind to do a thing, do it she\nwill, though a seven-headed dragon were to stand in the way; and if\nfear is a great power in this world, curiosity is a still greater.\n\nOne evening Eminah accompanied Ali right up to the brass door, and as\nhe went in she dexterously thrust a little pebble between the door and\nthe threshold. Thus the door not being completely closed, the catch of\nthe lock, despite a double turn of the key, shot back again; so\ninstead of closing the door behind him, as Ali fondly imagined, he\nleft it ajar.\n\nEminah waited till the sound of her husband's footsteps had quite\nceased. Then she softly opened the door, and at first contented\nherself with peeping in. Perceiving nothing to frighten her back, she\nventured right in, cautiously peering around at every step lest any\nangry spirit should suddenly rise up before her.\n\nBefore her lay a long corridor, and she went right to the very end of\nit. Then she came upon a spiral staircase, which was so dark that she\nhad to painfully grope her way along. A fatal curiosity goaded her on\nin spite of the darkness, and presently she found herself in a large,\nround room, dimly lit by a hanging lamp.\n\nAll round the walls of this room were arranged marble benches,\npitchers of water, funnels, and curious instruments of iron, leather,\nand wood, of all shapes and sizes, looking all the more\nincomprehensible in the semi-darkness. These were, no doubt, the\nimplements with which Ali was in the habit of making gold, thought\nEminah to herself, and, discovering a convenient niche at the head of\nthe staircase, she squeezed herself into it so that she could see\neverything from thence without being seen herself.\n\nA few moments afterwards the door at the opposite end of the room\nopened, and Ali and twelve dumb eunuchs entered with torches. The room\nwas illuminated at once, the eunuchs thrusting the torches into large\niron sconces; one of them then proceeded to light the fire and pile up\nvarious instruments around it; some sort of liquid also began bubbling\nin a caldron. Ali meanwhile was sitting down on a camp-stool and\ndistributing his commands in a low voice. \"Now we shall see how Ali\nmakes gold,\" thought Eminah.\n\nBut now at a sign from Ali two of the eunuchs entered a trap-door, and\na few moments afterwards the rattling of chains was audible; the\ntrap-door opened again, and in came two old men, peculiar-looking\ncreatures, with long gray hair, closely cropped beards, and strange\ngarments, the like of which Eminah had never seen before.\n\n\"Ah! no doubt these are the spirits which help Ali to make gold,\"\nthought Eminah to herself. \"Well, at any rate, they are in chains, so\nI need not be afraid of them.\" And, like the timid spectator of some\nstrange drama, she looked out from her hiding-place at the scene which\nfollowed.\n\nThe two old men were led up to Ali, who, smiling and rubbing his\nhands, stood up before them, and for a long time did not speak, but\nonly smiled. At last he gently stroked the face of the younger of the\ntwo.\n\n\"Merchant of Naples, thou still dost not know, then, where thy\ntreasures lie hidden?\" said he, gently.\n\n\"My lord,\" replied the other, with desperate obsequiousness, \"I have\ngiven up everything that was mine. I am indeed a beggar.\"\n\n\"Merchant of Naples! how canst thou say so? Let me refresh thy memory!\nThou didst go to Toulon with a full cargo of Indian goods, and there\nsold it all. When we met together on thy return journey thou didst\noffer me a thousand ducats, which I also took. But where is the\nremainder? A profit of twelve thousand ducats appears entered in thy\ntrading-books.\"\n\n\"Those books are false, my lord,\" said the merchant, in a tearful\nvoice. \"I made those totally fictitious entries simply to preserve my\ncredit.\"\n\n\"Merchant of Naples, thou dost calumniate thyself. Thou dost want to\nmake me believe that thou art not an honest man. Forgive me if I\nenliven thy memory a little.\"\n\nWith that he beckoned to the eunuchs, and they, undressing the\nmerchant, laid him on the torturing slab and tortured him for two\nmortal hours. It would be too horrible to say what they did to him.\nOh, that curious woman amply atoned for her curiosity! She was obliged\nto look upon tortures which made her limbs shake and shiver as if she\nwere in the grip of an ague. She covered her face, but the howls of\nthe tortured wretch penetrated to her very soul, and her sensitive\nnerves suffered almost as much as if she had felt these torments\nherself. Gradually, however, a curious sort of torpor seemed to stop\nthe beating of her heart; her limbs ceased to tremble, she opened her\neyes and, motionless as a statue, watched the hellish scene to the\nvery end.\n\nAli was evidently a past-master in this horrible science. He himself\nelaborately graduated the whole process, indicating briefly when and\nhow long the thumb-screws, the Spanish boot, the boiling oil, and the\nwater funnel were to be used. Last of all came the culminating\ntorment. They wrapped the merchant round in a raw buffalo-skin and\nlaid him down before the fiercely blazing fire. As the fire began to\ncompress the raw hide, and slowly press together the tortured limbs,\nthe limit of the poor wretch's endurance was reached, and he confessed\nthat his treasures were concealed in an iron chest, fastened by a\nchain to the bottom of the ship.\n\nThen they freed him from the torturing hide; in a state of collapse,\nwith foaming lips, a bleeding body and dislocated limbs, he flopped\ndown upon the cold marble.\n\n\"Thou seest now, my dear,\" observed Ali, gently, \"what trouble thou\nmightest have saved thyself and me also.\" Then he beckoned to the\neunuchs to remove the merchant.\n\nSo this was the way in which Ali made gold! A very simple sort of\nalchemy, certainly!\n\nAnd now it was the turn of the second man. And a haughty,\nbroad-shouldered fellow he was, who had regarded the torments of his\ncomrade without moving a muscle of his face.\n\n\"Then thou wilt not tell me thy name, valorous warrior?\" inquired Ali.\n\n\"I will tell thee thine--Devil, Belial, Satan!\"\n\n\"I thank thee! Thou dost me too much honor. But it is thy name I\nshould like to know. I suppose thou art some wealthy Venetian noble,\nwhose whereabouts his kinsmen are rather anxious to discover, and who\nwould not be ungrateful if any one sent thee back to them. For I\nvalue thee very highly.\"\n\n\"Know, then, that I _am_ a rich noble, and that at home I have a\npalace and treasures, but not a para of my property shalt thou ever\nsee, for I have taken poison. Dost thou not see the blue spots upon my\nhand? Presently thou wilt see them on my face. In five minutes' time I\nshall be dead.\"\n\nAnd so indeed it fell out. The haughty noble died, while Ali, furious\nwith passion, cursed the Prophet.\n\nAnd Eminah, from her hiding-place, looked intently upon Ali's face.\nWhat must have been her thoughts at that moment?\n\nThe eunuchs removed the dead body, and Ali beckoned once more to them,\nwhereupon they brought in through the opposite doors a wondrously\nbeautiful damsel and a handsome youth. When the youth and the damsel\nbeheld each other the tears gushed from their eyes. They were lovers,\nand lovers meet for each other.\n\nEminah now perceived with amazement that there were other kinds of men\nbesides those who wore gray beards. The captive youth, with his frank\nand comely countenance and long black locks, so rejoiced her eyes that\nshe could not take them off him. She had never seen anything of the\nsort before.\n\nAli approached the pair and smiled upon them both, and each of them\nsaid to him, \"I curse thee!\"\n\nHe said to the youth, \"Renounce thy bride and thou shalt live!\" and\nthe youth replied, \"I curse thee!\"\n\nHe said to the damsel, \"Love me, be mine, and thy betrothed shall\nlive!\" and the girl replied, \"I curse thee!\"\n\nAnd Eminah unconsciously murmured after them each time, \"I curse\nthee!\" without knowing what she was saying.\n\nThen Ali forced the youth down on his knees, and the eunuchs stripped\noff his robe. One of them then seized him by his beautiful long black\nhair, and raised him up into the air thereby, while the other stood\nbehind him with a large sharp sword.\n\n\"Thy beloved shall die this instant,\" roared the infuriated Ali, \"if\nthou dost not set him free! Embrace either me or his headless body.\"\n\nEminah turned her loathing eyes from the vile face of Ali, which, in\nthat moment, was deformed out of all recognition.\n\nAnd the young couple replied with one voice, \"We curse thee!\" It was\nas though they had taken an oath to say nothing else. The same instant\nthe sword flashed around the youth. His beautiful head bounded into\nthe air, then rolled along the floor to the foot of the spiral\nstaircase, and stood still before the very niche where Eminah was\nconcealed--at her very feet, in fact. The headless body, convulsed by\na final spasm, rent its fetters in twain, and then falling prone,\nstretched out its hands towards the terror-stricken girl, while the\nsevered head, which had rolled up to Eminah's feet, seemed to be\nmurmuring something--anyhow the lips moved. Eminah bending down\ntowards it, put her ears close to the quivering mouth and whispered,\n\"I hear! I hear what thou sayest!\" And she really believed she heard\nsomething. Perhaps it was only her heart that was speaking.\n\nAfter that she wrapped the head in her shawl, and hastened away from\nthe tower back into her own room, concealing the ghastly but still\nbeautiful trophy beneath the pillows of her sofa. Then she commanded\nher odalisks to appear before her, that they might dance and sing.\n\nDawn was now not far distant, and still the entertainment was going\non. Then Ali returned from the red tower--his face was gentle and\nsmiling--and after him came two eunuchs carrying gold and treasure in\nlarge baskets; and they emptied them all at Eminah's feet. The damsel\nrejoiced, laughed at the sight of the treasures, and, throwing herself\non Ali's neck, repaid him with kisses, and dragged him down to her on\nthe sofa.\n\n\"Behold, the _dzhins_ have sent thee treasures,\" said Ali. \"But a\nstrange thing hath befallen me; one of my treasures rolled away upon\nthe floor, and, search where I will, I cannot find it.\"\n\nEminah laughed, and fell a-teasing him. \"Perchance the _dzhins_ have\nstolen it from thee,\" cried she. Suppose she had said, \"Thou art\nsitting upon it, Ali Pasha?\"\n\nAli Pasha took the damsel upon his lap, and rejoiced in her innocent,\nartless eyes and her childlike smile. He fancied he could look through\nthose eyes down to the very depths of her heart. If only he _could_\nhave seen into it!\n\nAnd while he was thus toying with her, the kadun-keit-khuda entered\nthe room of the odalisks, bringing with him a veiled damsel.\n\n\"Gracious lady,\" said he to Eminah, \"I bring thee a Greek maiden, who\nhath heard the fame of thy benevolence, and hath come of her own\naccord to bask in the light of thy countenance, and gather fresh\nstrength from my smiles;\" and he drew the maiden forward towards\nEminah, who immediately recognized the girl whose lover Ali Pasha had\ndecapitated, and said, playfully, to the guardian of the harem:\n\n\"Lo, kadun-keit-khuda, the damsel is trembling! If thou dost not\nsupport her she will fall!\"\n\n\"It is by reason of her great shyness, gracious lady.\"\n\n\"But how pale she is!\"\n\n\"Thy beauty casteth a shadow upon her.\"\n\n\"But look!--she weeps!\"\n\n\"They are tears of joy, lady.\"\n\nEminah gave the guardian of the harem a handful of ducats for his good\nanswers, and allowed the bashful damsel to stand before her. Then she\nsent for sweetmeats, golden bread-fruits, wine with the lustre of\ngarnets, and her opium narghily; and, cradling Ali's gray head in her\nbosom, seized her mandolin and sang to him Arab love-songs--hot,\nburning, rose-scented, dew-besprinkled love-songs--and the pasha drew\nover his face the long silken tresses of the damsel, as if he would\nenvelop himself in the cool shade of Paradise, and sleep a sleep of\nsweet melody, intoxicating rapture, and soothing opium.\n\nWhen the ivory stem of the narghily dropped from the hands of the\npasha, Eminah sent from the room all the damsels; only the newly\narrived Greek maiden remained behind. She made her sit down before her\non a cushion, and, putting into her hands a large silk fan to fan the\npasha with, she asked the damsel her name.\n\nThe damsel shook her head--she would not say.\n\n\"Why wilt thou not tell me?\"\n\n\"Because I have still a sister at home.\"\n\nEminah understood the answer. \"Come nearer,\" said she. \"Last night I\nhad a dream. Methought I was in a large tower, the interior of which\nwas illuminated by twelve torches. Whichever way my eyes turned they\nlit upon horrors--strange, terrifying objects appeared before me; and,\nalthough, twelve torches were burning, darkness was still all around.\nAnd it seemed to me as if this darkness was not vapor or thick smoke,\nbut a black mass of human beings all wedged together, who raised their\neyelids every now and then. After that I saw Ali Pasha sitting in a\nred velvet chair with golden tiger feet, and as he sat cross-legged,\nafter the Turkish manner, it looked as if the tiger feet were his own\nfeet. Many terrifying shapes passed before me, and at last a young man\nand a young woman were all who remained in the room, and to every\nquestion put to them they replied, 'I curse thee!' Ali Pasha said to\nthe damsel, 'Love me!' and she replied, 'I curse thee!' And\nimmediately the head of the youth began rolling from one end of the\nmarble floor to the other, right up to my feet; and a drop of blood\ndripped from it on to my slipper, and, strange to say, the drop of\nblood was still there when I awoke. Look, is that really a drop of\nblood, or is it only my imagination?\"\n\nAnd therewith Eminah put out her pretty little foot, which hitherto\nshe had kept hidden beneath the folds of her garment, and showed it to\nthe Greek girl. Then the girl fell weeping at her feet and kissed the\nslipper. But it was not the foot of her mistress that she kissed--no,\nno; what she kissed was the drop of blood that had dropped upon the\nslipper.\n\n\"Look! that drop of blood has burned right through the morocco leather\nof my shoe! What will it do, then, to the soul on which it has\nfallen?\"\n\nAnd with that she withdrew her hair from the pasha's face and looked\nat him with loathing. Yet he slept as calmly as if he were sleeping\nthe sleep of the just.\n\nFor nine and seventy years he had lived happily, joyously,\ntriumphantly, beloved by angels; and all the curses, all the murders,\nthat were upon his aged head were unable to carve one wrinkle on his\nforehead, or distort a feature of his face, or cut off one day of his\nlife, or even to disturb one of his dreams; and there he lies on one\nand the same couch with the head of his victim, the only difference\nbeing that his head lies on the pillow, while the head of the murdered\nman lies beneath it.\n\nEminah bent over him and bared the breast of the sleeper, who slept\ncalmly and regularly all the time.\n\n\"On that table lies an enamelled dagger,\" said she to the girl; \"bring\nit hither.\"\n\nThe girl darted away for the dagger, and came back with it. There she\nstood, grasping it convulsively in her hand, as if she only awaited a\nsignal to drive it home.\n\n\"No, not so,\" said Eminah. \"Cut not off his life, but cut through this\ncord!\" and, taking the key which Ali wore round his neck, she cut it\nfrom its cord with the dagger. \"This key opens the red tower. When\nthey pitched the dead bodies through the trap-door I heard the roar\nof falling water. It is certain, therefore, that one can get through\nthe torture-chamber to the lake of Acheruz. We can get down to it by\nropes. I can swim, and thou canst also, I am sure; for art thou not a\nHydriot girl?[5] When we have reached the heights of Lithanizza we\nshall find a safe refuge in the midst of the forests. Wherever it is,\nit will be all one to me. Better to be among wolves and lynxes than\nnear Ali Pasha. Will you do what I say?\"\n\n[Footnote 5: An inhabitant of the isle of Hydra. The Hydriots were\nremarkable for their enterprise and daring.]\n\nThe damsel's bosom heaved violently; she hid her head on Eminah's\nshoulder and kissed her.\n\n\"Freedom!\" she whispered, full of rapture; \"freedom above all things!\nIt is now my only joy.\"\n\n\"Nobody will observe us,\" said Eminah, spurning aside the jewels,\nwhich she loathed now that she knew whence they came. \"It is the last\nnight of the Feast of Bairam. Every one is hastening to compensate\nhimself for the privations of the Fast of Ramadan, every one is\nsleeping or enjoying himself; the greater part of the garrison is\nmaking merry in the apartments of the beys; even the sons of Ali\nPasha, all three of them, are feasting with Mukhtar Bey. We shall be\nable to escape them, and then the whole world lies before us.\"\n\nThe Greek girl pressed the lady's hand. \"We will go together!\" she\ncried. \"My brother dwells among the mountains of Corinth; he is a\nvaliant warrior, and will give us an asylum.\"\n\n\"Then go thither! I shall seek refuge with my kinsmen at Stambul. Now\ngo into the apartments of the odalisks and ask for apparel. I have\nalready hatched a good plan. If they are all asleep come softly back\nwith thy clothes. The kadun-keit-khuda only sleeps with half an eye;\nbeware of him! If he ask thee whither thou art going, show him the\npasha's handkerchief, and he will fancy Ali awaits thee.\"\n\nThe face of the Greek girl blushed purple at these words; even to lie\non such a subject was a horrible thought to her. But Eminah beckoned\nto her to be gone, and when she found herself alone she drew forth the\nhead she had concealed beneath the pillow and placed it on a round\ntable in front of her. For a long time she gazed at the sunken eyes,\nthe gaping mouth, and the long black tresses which rolled over the\ntable on both sides. The lady smoothed the raven-black tresses with\nher soft hand, and passed her fingers right across the noble features\nwithout a shudder at their icy coldness.\n\nThere she sat an hour long opposite the dead head; and beside her Ali\nTepelenti, the terror of the whole region, lay prone in a deep,\nmotionless slumber. It was a strange sight, this young girl alone\nthere between these two horrors. She had resolved to quit Ali and set\nthe Greek damsel free; but what she meant to do after that she herself\ncould not have said.\n\nIn an hour's time the Greek damsel returned. She came so softly that\nnobody could have heard her; even Eminah did not perceive her till the\ndamsel stood before the severed head and uttered a cry of terror. Only\nfor an instant, only for the duration of a lightning-flash did this\ncry last; the damsel stifled it at once, and if it awoke any one in\nthe palace he must have fancied he was dreaming or had dreamed it, and\nwould go on sleeping again. Then the damsel, in an agony of speechless\ngrief, bent over the head of her betrothed, and her tears flowed in\nstreams, though not a word escaped her lips.\n\nAt last Eminah grasped the girl's hand and bade her make haste. So she\ndried her tears, and after placing the severed head in front of that\nof the sleeping pasha so that they confronted each other, and cutting\noff one of the locks from its temples, she covered the cold eyes with\nbitter, burning kisses, and then, taking up her things, rapidly\nfollowed Eminah through the long suite of rooms.\n\nA few minutes later they were in the torture-chamber. It was quite\nempty; the blood stains had been washed away, there was nothing to\nrecall the horrors of the night before.\n\nThey opened the trap-door through which the dead bodies were wont to\nbe cast. At the bottom of the deep black void there was a roaring\nsound as if the lake were in a commotion. No doubt a tempest was\nraging outside. How were these girls to escape by way of the\nsubterranean stream? Perhaps some of the headless corpses were also\nswimming down yonder amidst the foaming waves. Would those who\nventured down into those depths ever see the light of day again? But\nto them it was all one. Better to perish in the deep void than be\ncondemned to the embraces of Ali Pasha. How the two girls abominated\nhim!--the one because he had murdered her love, the other because he\nhad loved her.\n\n\"Don't be afraid,\" they said to each other; and fastening their\nbundles to a long rope which was used in torturing, they let it down\ninto the deep well, with a lamp at the end of it, and when the water\nput out the light they fastened the other end of the rope to the hinge\nof the door, and each in turn let herself down by it.\n\nAnd whether they lived or whether they died, Ali Pasha lost on that\nday two talismans which he should have guarded more jealously than the\nlight of his eyes: one was the spirit of blessing, the other the\nspirit of cursing, both of which he had held fast bound, and both of\nwhich had now been let loose.\n\n * * * * *\n\nAt the moment when the two damsels plunged into the lake of Acheruz\nthe slumber of tranquillity disappeared from the eyes of Ali Pasha,\nand he began to see spectres.\n\nA peculiar feeling came over him. He whom phantoms avoided even when\nhe slept, he who had never even dreamed of fear, he whom the angel of\nsleep had never known to be a coward, now began to experience a\npeculiar sensation which was worse than any sickness and more painful\nthan any suffering. He was afraid!\n\nHe dreamed that the head of the young Suliot, which had been cut off\nby his order, and which had rolled away and disappeared so that nobody\ncould find it, was now standing face to face with him on a table,\nstaring at him fixedly with stony eyes, and repeatedly addressing the\nsleeper by name: \"Ali Pasha! Ali Pasha!\"\n\nThe limbs of the sleeper shook all over in a strange tremor.\n\n\"Ali Pasha!\" he heard the head call for the third time.\n\nGroaning, writhing, and turning himself about, he contrived to knock\nthe head off the cushion, smearing all the bed with blood. And now he\nsaw and heard more terrible things than ever.\n\n\"One, two,\" said the severed head. And Ali understood that this was\nthe number of the years he had still to live. \"Thy head hath no longer\neither hand or foot,\" continued the head; and Ali was obliged to\nlisten to what it said. \"Two severed heads now stand face to face,\nmine and thine. Why dost thou not reply to me? Why dost thou not look\ninto my eyes? Two headless trunks stand before the throne of God, mine\nand thine. How shall the Lord recognize thee? He inquires which is\nAli. For every soul there is a white garment laid up. And thou deniest\nthy name, with thy right hand on thy heart. Thou _art_ Ali, for on thy\nwhite garment are five bloody finger-prints.\"\n\nAli writhed in his sleep, and covered with his hand that part of his\ncaftan which lay over his heart. And all the time the head never\ndisappeared from before his eyes and its lips never closed. Presently\nit went on again.\n\n\"Listen, Ali! Mene, mene, tekel, upharsin! The hand which guided thee\nin the performance of thy mighty deeds is also bringing thine actions\nto an end, and thou shalt no longer be a hero whom the world admires,\nbut a robber whom it curses. Those whom thou lovedest will bless the\nday of thy death, but thine enemies will weep over thee. Moreover, God\nhath ordained that thou shalt be the ruin of thine own nation.\"\n\nAli tossed, sighing and groaning, upon his couch, and could not awake;\na world of crime lay upon his breast. He felt the earth shake beneath\nhim, and the sky above his head was dark with masses of black cloud,\nand the thought of death was a terror to him.\n\nThe head went on speaking. \"Two birds quitted thy rocky citadel at the\nsame hour, a white dove and a black crow. The white dove is Peace,\nwhich has departed from thy towers; the black crow is Vengeance, which\nwill return in search of carcasses at the scent of thy ruin. The white\ndove is thy damsel, the black crow is mine; and woe to thee from them\nboth!\"\n\nAli, in the desperation of his rage, roared aloud in his sleep, and\nhis violent cry tore asunder the light fetters of sleep. He sprang\nfrom his couch and opened wide his eyes--and lo! the severed head was\nstanding before him on the table.\n\nThe pasha looked about him in consternation; he was not sufficiently\nmaster of himself at first to tell how much of all this was a dream\nand how much reality. He still seemed to hear the terrible words which\nhad proceeded from those open lips, and his hand involuntarily\nclutched at his breast as if he would have covered there the five\nbloody finger-marks. Then the cut cord from which the key was missing\nfell across his hand, and immediately his presence of mind returned.\nDrawing his sword, he rushed towards the brazen door, and discovered\nthat the fugitives had had sufficient forethought to close the door\nand leave the key in the lock outside, so that it could only be opened\nby force. He turned back and rushed to the end of the dormitories.\nSome of the odalisks were awakened by the sound of his heavy\nfootsteps, and perceiving his troubled face, plunged underneath their\nbedclothes in terror; in front of the doors stood the dumb eunuch\nsentries, leaning on their spears like so many bronze statues.\n\nHe rushed down into the garden to the end of the familiar walks, and\nwhen he came to the gate was amazed to perceive that the drawbridge\nwhich separated his palace from the dwellings of his sons had been let\ndown and nobody was guarding it. The topidshis, the s, knowing\nthat Ali always turned into his harem on the Feast of Bairam, had gone\nacross to the palace of Mukhtar Bey, who was giving a great banquet in\nhonor of Vely Bey and Sulaiman Bey, his brothers. All three had\nbrought together their harems to celebrate the occasion, and while the\nmasters were diverting themselves upstairs, their servants were making\nmerry below. Music and the loud mirth of those who feast resounded\nfrom the house; every gate of the citadel was open; slaves and guards\nlying dead drunk in heaps, victims of the forbidden fluid, cumbered\nthe streets. A whole hostile army, with drums beating and colors\nflying, might easily have marched into the citadel over their\nprostrate bodies.\n\nWrath and the cold night air gradually gave back to Ali his soul of\nsteel. Wary and alert, he entered the palace of Mukhtar Bey.\n\n\n\n\nCHAPTER III\n\nA TURKISH PARADISE\n\n\nAli Pasha himself had built the whole citadel of Janina, and had been\nwise enough, as soon as the fortress was finished, to at once and\nquietly remove out of the way all the builders and architects who had\nhad anything to do with it, so that he only knew all the secrets of\nthe place. There were secret exits and listening-galleries in every\npart of the building, and each single group of redoubts which, viewed\nfrom the outside, seemed quite isolated, was really so well connected\ntogether by means of subterranean passages, that one could go backward\nand forward from one to the other without being observed in the least.\nAt a later day Ali Pasha's enemies were to have very bitter experience\nof these architectural peculiarities.\n\nOne could go right round the palace of the three Beys, both above and\nbelow, by means of a secret corridor, and not one of the inhabitants\nof the building had the least idea of the existence of this corridor.\nIt was in the midst of the fathom-thick wall between two rows of\nwindows, and within this space invisible doors opened into every\napartment, either between windows, or behind mirrors, or beneath the\nceiling between two stories, and these doors could not be opened by\nkeys, but turned upon invisible hinges set in motion by hidden\nscrews, and they closed so hermetically as to leave not the slightest\norifice behind them.\n\nAli Pasha stood there in the banqueting-chamber unobserved by any one.\nHe stood beside a huge Corinthian column, and here hung a black board\nindicating the direction in which Mecca lay. He had no fear that any\none would look thither. That place, towards which every truly\nbelieving Mussulman must turn when he prays, was carefully avoided by\nevery eye, for fear it should encounter the golden letters which\nsparkle on the walls of the Kaaba.[6]\n\n[Footnote 6: The chief sanctuary of the Mussulmans standing in the\nmidst of the great mosque at Mecca.]\n\nFor now is the time for enjoyment. There is no need of a heavenly\nParadise, for Paradise is already here below. There is no need to\ninquire of either Muhammad or the angel Izrafil concerning the wine\nwhich flows from the roots of the Tuba-tree; far more fiery, far more\nstimulating, is the wine which flashes in glass and goblet. The houris\nmay hide their white bosoms and their rosy faces, for what are they\ncompared with the earthly angels whose mundane charms intoxicate the\nhearts of mortals? Truly Muhammad was but an indifferent prophet, he\ndid not understand how to arrange paradise; let him but regard the\narrangements of Mukhtar Bey--they will show him how that sort of thing\nought to be managed.\n\nMuhammad imagined that the embraces of seven and seventy houris would\nmake an enraptured Moslem eternally happy. Why, the bungler forgot the\nbest part of it. Would it not be more satisfactory if now and then,\nsay once in a thousand years or so, the Moslems were to exchange their\nown houris for those of their neighbors? In this way the aroma of\nbrand-new kisses would prevent their raptures from growing stale, and\nthe Paradise of Muhammad would be worth something after all. With all\neternity before him, a man would scarcely mind waiting for his own\nwives for a paltry millennium or two while he enjoyed the wives of his\nneighbors, and when he returned to his seven and seventy original\ndamsels again, what a pleasant reunion it would be!\n\nNow the Prophet had forgotten to introduce this novelty into his own\nParadise, and Mukhtar Bey was the happy man to whom the fairy Malach\nTaraif whispered the idea during the fast preceding the Feast of\nBairam while he slept, and he immediately proceeded to discuss the\nmatter with his kinsmen.\n\nAll three brothers lived under one roof, each of the three had his own\nspecial harem, and each of them possessed in their harems beauties far\nsurpassing what the angels Monkar and Nakir could promise them in the\nnext world. After the Feast of Bairam, when Mukhtar Bey had well plied\nhis brethren with good wine, he said to them, \"Let us exchange\nharems!\"\n\nSulaiman Bey immediately gave his hand upon it; Vely Bey laughed at it\nas a good idea at first, but afterwards drew back. The other two\nworthies laughed uproariously at his simplicity, made fun of him, and\nproceeded at once to transfer to each other their respective damsels,\nand on the morrow and the following days aggravated Vely by extolling\nbefore him the exchanged odalisks, each of them confiding to him what\nnovel attractions he had discovered in this or that bayadere. Thus\nSulaiman could not sufficiently extol the extraordinary brilliance of\nthe eyes of Mukhtar Bey's favorite damsel, while Mukhtar protested\nthat the languishing Jewish maiden he had got in exchange from\nSulaiman quivered in his arms like a dancing flame.\n\nVely laughed a good deal over the business, but still continued to\nshake his head, confessing at last that the reason why he did not\nexchange his harem was because it contained an Albanian damsel whom he\nhad neither purchased nor captured, but who had come to him of her own\naccord, and whom he had promised long ago never to abandon, and her he\nwould not give for both their harems put together; nay, he said he\nwould not give her up for a whole world full of damsels. The two\nbrethren thereupon assured Vely that if he loved this particular\ndamsel so very much, he might exclude her from the others and keep her\nfor himself, and it need make no difference. Then Vely Bey also\nacceded to this fraternal division of delights, and transferred his\nharem also, with the exception of Xelianthe.\n\nMukhtar Bey had fixed the last night of the great Bairam feast for the\nentertainment that was to rival Paradise, inviting his brethren and\nthe Prophet Muhammad himself, in order that he might learn from them\nhow to be happy, and might regulate heaven accordingly. To this end\nthey had a fourth divan added to their three, with its own\nwell-appointed table in front of it, and bade the attendant odalisks\nbe diligent in keeping the fourth goblet well filled, and do their\nbest to entertain the invited guest. Mockery of religious subjects was\nno unusual thing with Turkish magnates in those days. Blasphemy had\ngone so far as to become an open scandal; popular fanaticism and\nofficial orthodoxy made it all the more glaring.\n\nSo the sons of Ali Pasha invited the Prophet to be their guest, and\nhad made up their minds that if he did appear among them he would not\nbe bored.\n\nAll the odalisks danced and sung before them in turn, and the brethren\ndiverted themselves by judging which of the damsels was the sweetest\nand loveliest.\n\nIn every song, in every dance, Rebecca, Mukhtar Bey's beautiful Jewish\ndamsel, and the blue-eyed bayadere Lizza, who was Sulaiman Bey's\nfavorite, equally excelled. It was impossible to decide which of the\ntwain deserved the palm. At last they were made to dance together.\n\n\"Look!\" cried Mukhtar, his eyes sparkling with delight, \"look! didst\never behold a more beautiful figure? Like the flowering branch of the\nBan-tree she sways to and fro. How proudly she throws her head back,\nand looks at thee so languishingly that thou meltest away for very\nrapture! Would that her light feet might dance all over me; would that\nshe might encompass every part of me like the atmosphere!\"\n\n\"She really is charming,\" admitted Sulaiman, \"and if the other were\nnot dancing by her side, she would be the first star in the firmament\nof beauty. But ah! one movement of the other one is worth all the life\nin her body. She is but a woman, the other is a sylph. She kills you\nwith rapture, the other raises you from the dead.\"\n\n\"Thou are unjust, Sulaiman,\" said Mukhtar; \"thou dost judge only with\nthine eyes. If thou wouldst take counsel of thy lips, they would speak\nmore truly. Taste her kisses, and then say which of them is the\nsweeter.\"\n\nWith that he beckoned to the two odalisks. Rebecca, the lovely Jewish\ndamsel, sank full of amorous languor on Sulaiman's breast, while\nLizza, with sylph-like agility, sat her down upon his knee, and the\nintoxicated Bey, in an access of rapture, kissed first one and then\nthe other.\n\n\"Rebecca's lips are more ardent,\" he cried, \"but the kisses of Lizza\nare sweeter. The kiss of Rebecca is like the poppy which lulls you\ninto sweet unconsciousness, but Lizza's kiss is like sweet wine which\nmakes you merry.\"\n\n\"Lizza's kiss may perchance be like sweet wine,\" interrupted Mukhtar,\n\"but Rebecca's kiss is like heavenly musk which only the Blessed may\npartake of, and those who partake thereof _are_ blessed.\"\n\nAnd with that Mukhtar caught up both the odalisks in his arms, that he\nmight pronounce judgment as to the sweetness of their lips. It was an\nenviable process. The contending parties themselves were in doubt as\nto which of themselves should obtain a verdict. At length they called\nupon Vely Bey to decide--Vely, who was now lying blissfully asleep\nbeside them on the divan, overcome with wine, his head in Xelianthe's\nbosom. His two brethren awoke him that he might judge between them as\nto the sweetness of rival kisses.\n\nIt took a good deal of trouble to make the stupidly fuddled Bey\nunderstand what was required of him, and when he did understand, the\nonly answer he made was, \"Xelianthe's kisses are the sweetest;\" and\nwith that he embraced his favorite damsel once more and, reclining his\nhead on her bosom, went off to sleep again.\n\nThen cried Mukhtar, \"Wherefore dost thou ask for _his_ judgment, when\namongst us sits the Prophet himself? Let him judge between us.\"\n\nWith these words he pointed to the empty place which had been left for\na fourth person. Rich meats were piled up there on gold and silver\nplate, and wine sparkled in transparent crystal.\n\n\"Come, Muhammad!\" exclaimed Mukhtar, addressing the vacant place;\n\"thou in thy lifetime didst love many a beauteous woman, and in thy\nParadise there is enough and to spare of beauty. I summon thee to\nappear before us. Here is a dispute between us two as to whose damsel\nis the sweeter and the lovelier. Thou hast seen them dance, thou hast\nheard them sing; now taste of their kisses!\"\n\nWith that he beckoned to the two damsels, and they sat down, one on\neach side of the empty divan, and made as if they were embracing a\nshape sitting between them, and filled the air with their burning,\nfragrant kisses.\n\n\"Well, let us hear thy verdict, Muhammad!\" cried Mukhtar, with drunken\nbravado; and, taking the crystal goblet from the empty place and\nraising it in the air, looked around him with a flushed, defiant face,\nand exclaimed, \"Come! drink of the wine of this goblet her health to\nwhom thou awardest the prize!\"\n\nAli Pasha, shocked and filled with horror at the shamelessly impudent\nwords he heard from his hiding-place, drew a pistol from his girdle\nand softly raised the trigger.\n\n\"Drink, Muhammad!\" bellowed Mukhtar, raising the goblet on high,\n\"drink to the health of the triumphant damsel! Which shall it be,\nRebecca or Lizza?\"\n\nAt that same instant a loud report rang through the room, and the\nupraised crystal goblet was shivered into a thousand fragments in\nMukhtar's hand. Every one leaped from his place in terror. But\nwhichever way they looked there was nothing to be seen. The only\npersons in the room were the three brothers and the damsels. Only at\nthe spot from whence the shot had proceeded a little round cloud of\nbluish smoke was visible, which sluggishly dispersed. Nobody present\ncarried weapons, and there was no door or window there by which any\none could have got in.\n\nFrom the minarets outside the muezzins proclaimed the prayer of dawn:\n\"La illah il Allah! Muhammad razul Allah!\"--\"There is no God but God,\nand Muhammad is His Prophet!\"\n\n * * * * *\n\nAli Pasha did not pursue the fugitives. That day he was praying all\nthe morning. He locked himself up in his inmost apartments, that\nnobody might see what he was doing. He now did what he had not done\nfor seventy years--he wept. For a whole hour his inflexible soul was\nbroken. So that woman whom he had loved better than life itself, she\nforsooth had given the first signal of approaching misfortune, the\nfirst sign of the coming struggle! Let it come! Let her veil be the\nfirst banner to lead an army against Janina! Tepelenti would not\nattempt to stay her in her flight. For one long hour he thought of\nher, and this hour was an hour of weeping; and then he bethought him\nof the approaching tempest which the prophetic voice had warned him\nof, and his heart turned to stone at the thought. Ali Pasha was not\nthe man to cringe before danger; no, he was wont to meet it face to\nface, and ask of it why it had tarried so long. He used even to send\noccasionally for the _nimetullahita_ dervish who had been living a\nlong time in the fortress, and question him concerning the future. It\nmust not be supposed, indeed, that Tepelenti ever took advice from\nanybody; but he would listen to the words of lunatics and soothsayers,\nand liked to learn from magicians and astrologers, and their sayings\nwere not without influence upon his actions.\n\nThe dervish was a decrepit old man. Nobody knew how old he really was;\nit was said that only by magic did he keep himself alive at all. Every\nevening they laid him down on plates of copper and rubbed invigorating\nbalsam into his withered skeleton, and so he lived on from day to day.\n\nTwo dumb eunuchs now brought him in to Tepelenti, and, bending his\nlegs beneath him, propped him up in front of the pasha.\n\n\"Sikham,\" said Ali to the dervish, \"I feel the approach of evil days.\nMy sword rusted in its sheath in a single night. My buckler, which I\ncovered with gold, has cracked from end to end. A severed head, which\nhid itself away from me so that I could not find it, came forth to me\nat night and spoke to me of my death; and in my dreams I see my sons\nmake free with the Prophet. I ask thee not what all these things\nsignify. That I know. Just as surely as in winter-time the hosts of\nrooks and crows resort to the roofs of the mosques, so surely shall\nmy sworn enemies fall upon me. I am old compared with them, and it is\na thing unheard of among the Osmanlis that a man should reach the age\nof nine and seventy and still be rich and mighty. Let them come! But\none thing I would know--who will be the first to attack me? Tell me\nhis name.\"\n\nThe dervish thereupon caused a wooden board to be placed before him on\nwhich meats were wont to be carried; then he put upon it an empty\nglass goblet, and across the glass he laid a thin bamboo cane. Next he\nwrote upon the wooden board the twenty-nine letters of the Turkish\nalphabet, and then, thrice prostrating himself to the ground with\nwide-extended arms, he fixed his eyes steadily upon the centre of the\ngoblet.\n\nIn about half an hour the goblet began to tinkle as if some one were\nrubbing his wet finger along its rim. This tinkling grew stronger and\nstronger, louder and louder, till at last the goblet moved up and down\non the wooden board, and began revolving along with the light cane\nplaced across it, revolving at last so rapidly that it was impossible\nto discern the cane upon it at all.\n\nThen, quite suddenly, the dervish raised his fingers from the table,\nand the goblet immediately stopped. The point of the cane stood\nopposite the letter _ghain_--G.[7]\n\n[Footnote 7: The marvels of our modern table-turning and table-tapping\nspirits, and all the wonders of this sort, were known to the Arab\ndervishes long ago.--JOKAI.]\n\n\"That signifies the first letter of his name,\" said the dervish--\"G!\"\n\nAnd then the mysterious operation was repeated, and the magic stick\nspelled out the name letter by letter: \"G--a--s--k--h--o B--e--y.\" At\nthe last letter the goblet stopped short and would move no more.\n\n\"I know no man of that name,\" said Ali, amazed that he whose name was\nso world-renowned was to tremble before one whose name he had never\nheard before.\n\n\"Where does the fellow live?\" he inquired of the dervish.\n\nThe magic jugglery was set going again, and now the dancing goblet\nspelled out the name, \"Stambul.\"\n\nThat was enough. Ali beckoned to the eunuchs to take the dervish away\nagain.\n\nAli thereupon summoned forty Albanian soldiers from the garrison, and\ngave to each one of them twenty ducats.\n\n\"This,\" said he, \"is only earnest money. I want a man put to death\nwhose name and dwelling-place I know. His name is Gaskho Bey, and he\nlives in Stambul. This man's head is worth as many gold pieces as\nthere are miles between him and me. He who brings the head can measure\nthe distance and be paid for it. The first who brings but the report\nof his death shall receive two hundred ducats; he who slays him, a\nthousand.\"\n\nThe Albanians consulted together for a brief moment, and then\nintimated that if a bey of the name of Gaskho really existed, he was\nas good as dead already.\n\nTowards mid-day Ali sent for his sons. He said not a word to them of\nthe anxieties, the visions, and the apparitions of the night before,\nbut made them, after they had respectfully kissed his hands, sit down\nall around him. Mukhtar Bey he invited to sit down on his left hand,\nVely on his right, and Sulaiman directly opposite.\n\nHe addressed himself first of all to Sulaiman.\n\n\"Thou art the youngest and boldest,\" said he. \"To-morrow thou must go\nto sea and take three ships with thee. These ships thou must take to\nSicily, load them there with sulphur, and return without losing an\ninstant.\"\n\n\"Oh, my father!\" replied Sulaiman, \"the tempest is now abroad upon the\nsea. Who would venture now with a ship upon the billows? All the\nmonsters of the ocean are now running upon the surface seeking whom\nthey may devour, and the phantom ship, with her shadowy rigging and\nher shadowy crew, pursues her zigzag course across the waters.\"\n\nAli Pasha said no more, but turned towards Mukhtar Bey.\n\n\"Thou art the most crafty,\" said he; \"go then to the captains of the\nSuliotes and invite them to assemble with their forces at Janina with\nall despatch. Spare neither promises nor assurances nor fair winds.\"\n\nMukhtar Bey's face turned quite angry, and, wagging his head, still\nheavy from his overnight debauch, he answered, sullenly: \"In the\nmountains the snow is now thawing; every stream is swollen into a\nriver; naught but a bird can find a place for its foot on the dry\nground; how, then, can armies move hither and thither? Wait for a\nweek, till the inundations have subsided. Truly there is no enemy on\nthy borders. In thy whole realm there is not so much as a rat to\nnibble at thy walls. What dost thou want now with chariots and armed\nmen?\"\n\nAli now turned to Vely, who was sitting on his right hand. \"Go thou\nover to Misrim,\" said he, \"and purchase for me two thousand horses; a\nthousand of them shall be meet for war-chargers, and a thousand for\ndrawing guns.\"\n\n\"Oh, my father!\" answered Vely, who was the eldest and wisest of Ali's\nsons, \"I will not object to thy command that the simoon has now begun\nin Misrim, before whose burning, suffocating breath every living\ncreature is forced to fly. I reck little of that, but the horses, thy\nprecious horses, will perish. And, moreover, I would ask of thee one\nquestion. Wherefore dost thou get together a host, and horses and\nguns, without cause, and with no danger threatening thee? Will not all\nthese warlike preparations excite the rage of the Padishah against\nthee, and so thy preparing against an imagined peril will saddle thee\nwith a real war?\"\n\nAli Pasha laughed aloud--a very unusual habit with him.\n\n\"Well,\" said he, \"it is for me to prove to you, I suppose, that you\nare all wrong in your calculations. Dine with me and be merry. After\ndinner you shall see that the sea is not stormy, that the rivers are\nnot in flood, and that the simoon is not suffocating. I have a\ntalisman which will convince you thereof.\"\n\nSo he entertained his sons till late in the evening, and immediately\nafter dinner he whispered to one of the dumb eunuchs, and then he took\nhis sons with him into the red tower, the doors of which were left\nwide open. He stopped short with them in one of the rooms, the\nsolitary semicircular window of which looked out upon the lake of\nAcheruz. The window was guarded by an iron grating. Here he sat down\nwith them to smoke his narghily and sip his coffee. The sons would\nhave preferred to mount upon the roof of the tower, where the fresh\nair and the fine view would have made their siesta perfect; but Ali\nfacetiously observed that in the open air cold and hot winds were just\nthen blowing together at the same time, and he did not want the simoon\nto make them sweat or the trade-winds to make them shiver.\n\nAs they were sipping their coffee there the splashing of oars was\naudible beneath the tower, and the sons beheld three large,\nflat-bottomed boats propelled upon the surface of the water, in which\nsat the damsels of their harems; the boats were rowed by muscular\neunuchs.\n\nThe faces of the three beys lighted up when they saw the damsels being\nrowed on the water, and Mukhtar Bey whispered roguishly in Sulaiman's\near, \"Shall we make the old man also one of our party?\"\n\nAli overheard the whisper, and replied, with a smile, \"Truly your\ndamsels are most beauteous\"--here he stroked his white beard from end\nto end--\"I am not surprised, therefore, that you like to stay at home\nhere and call the wind hot and cold, though it is nothing but the\nbreath of Allah, and what comes from God cannot be bad. But your\ndamsels _are_ beautiful, of that there can be no doubt. Now, last\nnight I dreamt a dream. Before me stood the Prophet, and he told me\nhow you had challenged him to say which of your damsels was the\nsweeter and the more beautiful.\" (Here the sons regarded each other,\nfull of fear and amazement.) \"The Prophet replied,\" continued Ali,\n\"that it was not meet that he should come to your damsels; they should\nrather go to him. So I mean to send them to Paradise.\"\n\n\"What doest thou?\" cried all three sons, horror-stricken.\n\nThe only answer Ali gave was to give a long shrill whistle, at which\nsignal the eunuchs drew out the plugs from holes secretly bored at the\nbottom of the three boats, leaping at the same time into the water,\nand leaving the boats in the middle of the lake.\n\nThe damsels shrieked with terror as the water began to rush into the\nboats from all sides. The air was filled with cries of agony.\n\nMukhtar rushed madly to the door and found it locked. With impotent\nviolence he attempted to burst it open. Sulaiman meanwhile tore away\nat the iron window-grating with both hands, as if he fancied himself\ncapable of pulling down the whole of the vast building by the sheer\nstrength of his arms. The blue-eyed Albanian girl and the languishing\nJewish damsel, with the fear of death in their eyes, looked up at the\nclosed window; the waves had already begun to swallow their beautiful\nlimbs.\n\nOnly Vely Bey remained motionless. He, at any rate, had not sinned. He\nhad not angered the Prophet in that orgie of amorous rivalry. He had\nloved one only, by her only had he been loved, and she, yes, she was\nperishing there among the others!\n\nThe boats sank deeper and deeper; nothing could be heard but the\ncries of the drowning wretches in all the accents of despair. The two\nsons saw their damsels dying before their eyes, and were unable to\nrush out and save them; not even one could be rescued. One more shriek\nof woe, and then the boats sank. For a few moments the surface of the\nwater was covered with bright gauze veils and shiny turbans and white\nlimbs and dishevelled tresses, and then a few solitary turbans floated\non the water.\n\nSulaiman, sobbing in despair, fell down in a heap close by the window,\nwhile Mukhtar fell madly on the door and kicked it with all his might,\nas if he would drown in the din the cries for help of the perishing\ndamsels. Only Vely Bey looked in bitter silence upon the detestable\nwaves, which within a minute had swallowed three heavens.\n\nFar, far away on the crest of the rising waves a black object appeared\nto be swimming. What was it? Perhaps one of the damsels. One moment it\nvanished in the wave-valleys, the next it appeared again on the top of\na high ridge of water. What could it be? But farther and farther it\nreceded. Perchance some one had escaped, after all. Greek girls are\ngood swimmers.\n\nAnd now Ali Pasha arose from his place and said, with a smile, to his\nsons:\n\n\"Methinks that neither the storms of ocean, nor the swollen waters,\nnor the breath of the simoon will now appear so terrible to you as\nthey did a few hours ago. Depart now with all speed. When you return\nyou will find new harems here, which will make you forget the old\nones.\" And with that he quitted them.\n\nSulaiman and Mukhtar immediately went their way. Woe to whomsoever\nshall now give them a pretext for wreaking their vengeance upon him!\n\nBut Vely Bey remained there looking out upon the water, and as the\nevening grew darker he thought upon Ali Pasha. His brothers had loaded\ntheir father with curses; he had not said a word. They will soon make\ntheir peace with their father--he never will.[8]\n\n[Footnote 8: It is a fact that Ali drowned the harems of his\nsons in the lake of Acheruz because he feared their excessive\ninfluence.--JOKAI.]\n\n\n\n\nCHAPTER IV\n\nGASKHO BEY\n\n\nThe lightning strikes to the earth the man that flies from it. Ill\nluck is a venomous dog, which runs after him who would escape it.\n\nAli Pasha's band of Albanians, on arriving at Stambul, began to make\ninquiries about Gaskho Bey.\n\nHe turned out to be a good honest man, by profession an inspector of\nthe ichoglanler of the Seraglio, and a particularly mild and peaceful\nMussulman to boot. In temperament he was somewhat phlegmatic, with a\nleaning to melancholy. A palmist would have told you that the\nsympathetic line on the palm of his hand was so little prominent as to\nbe scarcely visible, whereas on Tepelenti's palm there was such an\nabundant concourse of sympathetic lines that they even ran over on to\nthe back of the hand. In those days the Mussulmans frequently diverted\nthemselves with such superstitious games as palmistry.\n\nAs to his figure--well, Gaskho Bey might have stood for a perfect\nmodel of the Farnese Hercules; his huge shoulders were almost out of\nproportion with the rest of his body. He could stop the wing of a\nwindmill with one hand; on the birthday of the Sultan's heir he\nhoisted a six-pound cannon on his shoulders and fired it off, and he\ncould break a hard piastre in two when he was in a good humor.\n\nIt could not be said that he had hitherto used this terrible strength\nto injure any one; on the contrary, he was universally known as the\nmost forbearing of men. The pages of the court, whom he taught to\nfence, would sometimes in the midst of a lesson, as if by accident,\nbut really from sheer petulance, batter him with their blunt swords\ntill they rang again, and Gaskho Bey would always reprimand them, not\nfor striking him but for striking so clumsily. He had never gone to\nwar, and those who did not send him thither flattered themselves not a\nlittle on their humanity, for if it came to a serious tussle there was\nreally no knowing what damage he might not do.\n\nAt home he was the gentlest paterfamilias conceivable. You would\nfrequently find him on all-fours, with his little four-year-old son,\nSidali, riding on his back, and persecuting his father with all sorts\nof barbarities. He did nothing all day but teach the pages of the\nSeraglio games and exercises, and at home he made paper birds for his\nown little boy, flew kites for and played blind man's buff with him.\nWhatever time he could spare from these occupations he would spend in\nleaning out of the window of the Summer Palace overlooking the\nGoekk-sue, or Sweet Waters, and looking about him a bit with a pipe in\nhis mouth, the stem of which reached to the ground, and if any one had\nasked him while so engaged what he was looking at, he would assuredly\nhave answered, \"Nothing at all.\"\n\nNow there were always the liveliest goings-on in the Goekk-sue Park of\nan evening. The harems of the beys and pashas who dwelt on its banks\ntook the air there under the plantain-trees, and swung and danced and\nsang; the wandering Persian jugglers exhibited their hocus-pocus, and\nthe magnificent Janissaries resorted thither to fight with one\nanother. Every Friday afternoon whole bands of these rival warriors\nflocked thither as if to a common battle-field, and frequently left\ntwo or three corpses on the scene of their diversions.\n\nGaskho Bey appeared to take very little notice of all these things,\nhis chibook curled comfortably on the ground beneath him. At every\npull at it large light-blue clouds of smoke rolled upwards from its\ncrater, taking all manner of misty shapes and forms till they\ndisappeared through the window, and Gaskho Bey buried himself in the\ncontemplation of these smoky phantasms as deeply as if he were intent\non writing a dissertation on the philosophy of pipe-smoking, oblivious\nof the fact that below the very house in which he was sitting two\nAlbanian soldiers, in high-peaked, broad-brimmed caps and coarse black\nwoollen mantles, who seemed to be taking the greatest possible\ninterest in him and trying to get as near him as they could, had\nalready strolled past for the third time, always separating and going\nin different directions, somewhat nervously, if they perceived any one\ncoming towards them.\n\nOnly now and then a sly expression on Gaskho's face betrayed the fact\nthat he was conscious of something going on behind his back. There\nlittle Sidali was amusing himself, while Gaskho Bey was leaning out of\nthe window, by kneeling on the ottoman behind, and tickling the\nuplifted naked soles of his father's feet with a blunt arrow.\nSometimes the arrow would slip and come plumping down on Gaskho's\nhead, and then the bey would smile indulgently at the naughtiness of\nhis little son.\n\nAnd now the evening was falling, and the crowd beneath the\nplantain-trees grew thinner. The two Albanians, side by side, again\ncame towards Gaskho Bey, who now puffed forth such clouds of smoke\nfrom his chibook that one could see neither heaven nor earth because\nof them. But the two Albanian mercenaries could make him out very\nwell, and both of them standing a little way from the window drew\nforth their pistols, and one of them standing on the right hand and\nthe other on the left, they both aimed at Gaskho Bey's temples at a\ndistance of three paces.\n\nBut little Sidali was too quick for them, for he now gave his father\nsuch a poke with the arrow that the latter, provoked partly by the\npain and partly by the tickling, sharply turned his head, and the same\ninstant there was the report of two shots, and two bullets--one on the\nright hand and one on the left--buried themselves in the window-sill.\n\nGaskho's movement was so unexpected that the two Albanian braves, who\nhad imagined that their bullets must of necessity have met each other\nin the middle of the bey's brain, were so terrified when they saw him\nstill sitting there unwounded, that they stood as if nailed to the\nearth. Indeed, before they could make up their minds to fly, Gaskho\nwas already outside the window, upon them with a single bound, and\nimmediately seizing the pair of them with his terrible fists, flung\nthem to the ground as if he were playing with a couple of dummies,\nand without wasting so much as a word upon them, tied them together\nwith their own leather belts, so that on the arrival of the members of\nhis own family, who flew to the spot, alarmed by Sidali's shrieks, the\ntwo hired assassins lay half dead and all of a heap upon the ground,\nfor Gaskho Bey's grip had wellnigh broken all their bones.\n\nThey were conveyed at once to the Kapu-Kiaja, and Gaskho Bey went too.\nFor a long time he was unable to contain himself, and bellowed out all\nalong the road, \"I never heard of anything like it--never!\"\n\n\"It is an unheard-of case, sir,\" said he, on arriving at the\nKapu-Kiaja's. \"To furtively shoot at a peaceful Mussulman when he is\nsmoking his pipe and amusing himself with his children, I never heard\nthe like. If any one wants to kill me, he might at least, I think, let\nme know beforehand, so that I may perform my ablutions, say my\nprayers, and take leave of my children. But just when I am smoking my\nchibook!--I never heard of such a thing!\"\n\nIt was plain that what he took to heart the most was that they should\nhave tried to shoot him while he was smoking his chibook.\n\nThe Kapu-Kiaja, on the other hand, looked upon the case from another\npoint of view. To him it was a matter of comparative indifference\nwhether the deed was attempted before or after prayers. Why, he wanted\nto know, should these madmen run amuck of their fellow-men at all? He\ntherefore asked the assassins who had set them on to murder Gaskho\nBey. They, at the very first stroke of the bamboo, made a clean breast\nof it, and threw the blame on Tepelenti.\n\nAt first the Kapu-Kiaja regarded this confession as incredible. Why,\nindeed, should Tepelenti be wrath with Gaskho Bey, who knew nothing at\nall of Ali except by report? Nay, he greatly revered him as a valiant\nwarrior, and had never said a single word to his discredit.\n\nNevertheless, the two assassins not only stuck to their confession,\nbut maintained that besides themselves eight and thirty other soldiers\nhad been sent to Stambul by Ali on the self-same mission.\n\nCiauses were immediately sent to every quarter of the city to seize\nthe described Albanians. Five or six of them hid or escaped, but the\nrest were captured.\n\nThe confessions of these men were practically unanimous. Every\ncircumstance of the affair, the amount of the promised reward, the\nwords spoken on the occasion--everything, in fact, corresponded so\nexactly that no doubt could possibly remain that Tepelenti had\nactually sent them out to murder Gaskho Bey.\n\nThe affair made a great stir everywhere. Ali Pasha was as well known\nin Stambul as Gaskho Bey. The former was as famous for his power and\nriches, his envy and revengefulness, as was the latter for his\nstrength and gentleness, his sympathy and tenderness.\n\nThe great men of the palace, jealous for a long time of Ali's\ngreatness, brought the matter before the Divan, and great debates\nensued as to what course should be taken against this mighty protector\nof hired assassins. And for a long time the opinions of the\ncounsellors of the cupolaed chamber were divided. Some were for taking\nAli by the beard and despatching him there and then. Others were for\nadvising Gaskho Bey to be content with seeing the heads of the Arnaut\nassassins rolling in the dust before the Pavilion of Justice, and at\nthe same time privately informing Ali that if he were wise he would\nwaste neither his money nor his powder on such quiet, harmless men as\nGaskho Bey, who had never done, and never meant in future to do, him\nany harm.\n\nThe latter alternative was the opinion of the wiser heads, and among\nthese wiser ones was the Sultan himself.\n\n\"Ali is my sharp sword,\" said Mahmud. \"If my sword wounds any one\naccidentally, and without my consent, is that any reason for snapping\nit in twain?\"\n\nNevertheless, the enemies of the pasha kept goading Gaskho on to\ndemand satisfaction of Ali personally. The worthy giant, hearing his\nown name on everybody's lips for weeks together, grew as wild as a\nbaited heifer, and began to believe that he was a famous man, that he\nalone was ordained to clip the wings of the tyrant of Epirus, and at\nlast was so absorbed by his dreams of greatness that when he had to\ngive the usual lessons to the youths of the Seraglio he trounced them\nall, in his distraction, as severely as if they had been the soldiers\nof Ali Pasha.\n\nThe pacific Viziers promised him a house, a garden, beautiful horses,\nand still more beautiful slaves. But all would not do; what he did\nwant, he said, was the head of Tepelenti, and he cried to Heaven\nagainst them for their procrastination.\n\nBut Sultan Mahmud was a wise man. He had no need to consult\nstar-gazers or magicians, or even the caverns of Seleucia, as to the\nfuture, in order to discover and discern the storm whose signs were\nalready visible in the sky.\n\n\"Ye know not Ali, and ye know not me also,\" he said to those who urged\nhim to pronounce judgment against Ali. \"If I were to say, 'Ali must\nperish!' perish he would, even if my palaces came crashing down and\nhalf the realm were destroyed in consequence. If, on the other hand,\nAli said 'No!' he would assuredly never submit, and would rather turn\nthe whole realm upsidedown, till not one stone remained upon another,\nthan surrender himself. Therefore ye know not what ye want when ye\nwish to see Ali and me at war with one another.\"\n\nThe conspirators, however, were not content with this, but distributed\nsome silver money among the Janissaries, and egged them on to appear\nbefore the palace of the Kapu-Kiaja and demand Ali's head.\n\nThe Kiaja, warned in good time of the approaching storm, took refuge\nin the interior of the Seraglio, which was speedily barricaded against\nthe Janissaries, and the mouths of the cannons attached to the gates\nwere exhibited for their delectation. As it did not meet the views of\nthe Janissaries just then to approach any nearer to the cannons, they\ngratified their fury by setting fire to the city and burning down a\nwhole quarter of it, for they considered it no business of theirs to\nput out the blazing houses.\n\nThe next day, however, the tumult having subsided as usual, when the\nSultan and his suite were trotting out to inspect the scene of the\nconflagration, and had got as far as the fountain in front of the\nSeraglio, the figure of a veiled woman cast herself in front of the\nhorse's hoofs, and with audacious hands laid hold of the bridle of the\nsteed of the Kalif.\n\nThe Sultan backed his horse to prevent it from trampling upon the\nwoman, and, thinking she was one of those who had been burned out the\nday before, ordered his treasurer--who was with him--to put a silver\npiece in her hand and bid her depart in the name of the Prophet.\n\n\"Not money, my lord; but blood! blood!\" cried the woman; and, from the\nring of her voice, there was reason to suspect that she was a young\nwoman.\n\nThe Sultan in amazement asked the woman her name.\n\n\"I am Eminah, the daughter of the Pasha of Delvino, and the wife of\nAli Tepelenti.\"\n\n\"And whose blood dost thou require?\" asked the Sultan, scandalized to\nsee the favorite wife of so powerful a man prostrate in the dust\nbefore his horse's feet.\n\n\"I demand death upon his head!\" cried the woman, with a firm\nvoice--\"on the head of Ali Tepelenti, from whose gehenna of a fortress\nI have escaped on the waters of a subterranean stream in order that I\nmight accuse him to thee; and if thou dost not condemn him, I will go\nto the judgment-seat of God and accuse him there!\"\n\nThe Sultan was horrified.\n\nIt is a terrible thing when a woman accuses her own husband, who has\nloaded her with benefits. He must, indeed, be an evil-doer whom\nturtle-doves, the gentlest of all God's creatures, attack!\n\nThe Sultan listened, full of indignation, to the woman's accusations.\n\nAfter happily escaping from the fortress of Ali Pasha with the Greek\ngirl, she learned, during her short sojourn among the Suliotes, of all\nAli's cruelties, and learned also, at the same time, that in Delvino\nhad just died a rich Armenian lady, who had been the flame of Gaskho\nBey in his younger days, and had left him all the property she owned\nin Albania. Of this nobody as yet knew anything. What more natural\nthan that every one should immediately fancy he had found the key to\nthe riddle of the mysterious attempt at assassination? Why, of course,\nAli wanted to slay Gaskho Bey in order that he might take possession\nof his Albanian property.\n\n\n\n\nCHAPTER V\n\nA MAN IN THE MIDST OF DANGERS\n\n\nThe Pasha of Janina, for thirty successive days, received nothing but\nill tidings; and twice within the period of two waxing moons did his\nown power as steadily wane.\n\nThe first Job's-messenger which reached him was the Arnaut horseman,\nwho had escaped from Stambul, and whom the Sultan's Tartars had\npursued as far as Adrianople. This man told him that the attempt on\nthe life of Gaskho Bey had failed, and that the captured assassins had\nrevealed the name of their employer.\n\n\"Behold, I have wounded myself with my own sword,\" exclaimed Ali. \"The\nprophetic voice of Seleucia spoke the truth; yea, verily, it spoke the\ntruth.\"\n\nAnd still more of the prophecy was to be accomplished.\n\nA few days later the report reached him that Eminah had cast herself\nat the feet of the Sultan and demanded judgment on the head of her\nhusband.\n\n\"I knew it beforehand,\" sighed Ali. \"The Prophet told it all to me.\nNevertheless, I shall stand at the gates of the Seraglio on a silver\npedestal.\"\n\nNext day he heard that Gaskho Bey had been appointed Pasha of Janina.\n\n\"They act as if I were dead already,\" murmured the veteran, with as\nbitter a feeling as if he already saw his youthful supplanter standing\non his threshold. \"They bury me before I am dead, they divide my\nproperty before I have made my will. Nevertheless, one day I shall\nstand in the gates of the Seraglio on a richer pedestal.\"\n\nAnd with that Tepelenti sent forth his ciauses to all the towns within\nhis domains, and to all the local governors, commanding all who had\nsons to send their sons and all who had brothers to send their\nbrothers to him without delay. Then he ordered that every beast of\nburden that could be spared should be driven into the mountains, and\nthat every barque they could lay their hands upon should be brought\nfrom the sea-coast into the Gulf of Durazzo. The arsenal of Janina\nbristled with terrific rows of cannons and bombs, and the commanders\nof the various army corps received instructions to concentrate their\nforces under the walls of Janina. At any rate, he was determined not\nto be taken unawares. At least, he would have time to unfurl the red\nflag before the dread message arrived from Stambul that the Padishah\ndemanded his head.\n\nAh, ha! Ali Tepelenti would not surrender his gray beard so easily.\nThe hunters shall find out what manner of lion they are pursuing. A\nfirman of the Grand Signior nominated the banished Pehlivan Pasha,\nLord of Lepanto; Sulaiman Pasha was made Governor of Trikala, and the\ntwo mountain passes guarding it; Muhammad Bey, whose father Ali had\nslain, was proclaimed Lieutenant-General of Durazzo. Thus they had\ndivided his territories beforehand among his most bitter and most\ndangerous enemies. Ah! this will, indeed, be a magnificent chase.\n\nAli called together his sons, of whom Vely was Lord of Lepanto,\nSulaiman of Trikala, and Mukhtar Pasha of Durazzo. He showed them on\nthe map where their territories lay, and pointed out that if they lost\nthem they would have nothing left. Let all three of them, therefore,\ngird upon their thighs the swords he intrusted to them and fight like\nmen. The two younger sons swore fervently that they would conquer\nFortune with their weapons, but Vely Bey preserved a gloomy silence.\n\n\"Art thou not my son?\" asked the veteran.\n\n\"Allah hath so willed it,\" answered Vely, \"and I also will fight, not\nfor thee but for myself, not for life nor for what is on the other\nside of death, but because I have a little child in Lepanto, and the\nenemy is besieging that fortress. That little child is all the world\nto me. I will fight as only a father can fight for his son. I will\nrescue him if possible. Thy glory or thy ruin is alike indifferent to\nme. If the report reach thee that the enemy hath taken Lepanto and\nslain my son, then count no more upon the sword which thou hast\nintrusted to me.\"\n\nAnd with these words Vely turned his back on his father and softly\nwithdrew.\n\nAs Ali saw his son quietly pass before him, it occurred to him whether\nit would not be as well to draw his pistol from his belt and shoot\ndown the waverer before he quitted Janina. It is true that he had\nknown all this beforehand. His own wife, his own sons, his own\nweapons, were to turn against him; but then, on the other hand, was\nhe not to stand at the gate of the Seraglio on a silver pedestal?\n\nA host of more than twenty thousand men stood under arms at his\ndisposal, Albanians and Suliotes. A gallant host, if only it would\nfight. But for whom would it fight?--for him or for the Sultan? And\nthese soldiers, when they saw him besieged, would they forget their\nmurdered kinsfolk, their plundered fields, their burned villages? Did\nnot every man of them know that Ali Tepelenti had been amassing\ntreasures all his life, but had never troubled himself about good\ndeeds? And now these treasures would surely be his ruin.\n\nTime brought the answer. While his enemies were still afar off, the\nSuliotes arose, under the leadership of a girl among the mountains of\nBracori, where one of Ali's grandsons, Zaid, was recruiting soldiers,\nand massacred Ali's men to the very last one. The last one, however,\nthey suffered to escape and convey to Ali Zaid's severed head, at the\nsame time informing him that it was sent by that girl the head of\nwhose betrothed he had cut off before her very eyes, and she meant to\nsend him still more.\n\nThis was the Greek's declaration of war. There at Janina, under his\nvery nose, the Greek captain, Zunga, deserted the Albanian camp, and\nwhen the Grand Signior's army reached Trikala, and Gaskho Bey's herald\ngalloped between the two armies with the imperial firman hanging round\nhis neck, and summoned the vassals to take up arms against the Pasha,\nthe whole camp went over to Gaskho Bey. Alone, without the smallest\nescort, Sulaiman, Ali Pasha's youngest son, fled without having had\nthe opportunity of testing his father's sword, and they captured him\non the road.\n\nStill he had the other two. Mukhtar Bey, with a powerful fleet, lay in\nthe Gulf of Durazzo, and Vely Bey, wroth though he might be with his\nfather, was a valiant warrior, and his son was in Lepanto, and save\nhim he must and would.\n\nBut not only his son, some one else was there also. On that cruel,\nmurderous day when Ali Pasha drowned the harems of his sons in the\nlake, one person among so many escaped, and this was Xelianthe. The\ndamsel loved Vely as much as he loved her, and contrived to let him\nknow that she was alive. Vely Bey sent her to Lepanto, and kept her in\nhiding there with his little son in order that she might be far from\nhis father.\n\nAnd now the bey himself hastened to Lepanto, arrived at night in the\nneighborhood of the town, and perceived already from afar that the\ncitadel in which he had concealed his darlings was in flames.\n\nWhat if he had arrived too late!\n\nWith the fury of a savage wild tiger he flung himself upon the\nbesieging Pehlivan, and in a midnight battle routed him beneath the\nwalls of Lepanto, the Albanians fighting desperately by the side of\ntheir leader. But what was the use of it? The fortress was saved,\nindeed, but it was already in flames. Vely, roaring with grief and\npain, flung himself on the gate, scarcely recognizing again the place\nhe had quitted so short a time ago.\n\nHe reached the pavilion where he had concealed his wife and child. It\nwas built entirely of wood, except the roof, which was of copper. A\ncurious mass of molten dark-red metal gleamed among the fire-brands.\nVely rushed bellowing to the spot, and his soldiers, tearing aside the\ncharred beams and rafters, came upon two skeletons burned to cinders.\nA coral necklace lying there, which the fire had been unable to\ncalcine, told him that these were the remains of his wife and son.\n\nNot a word did Vely say to a living soul; but he plunged his sword\ninto its sheath, and that same night he rode unarmed into the camp of\nthe discomfited Pehlivan Pasha and surrendered himself to the enemy.\n\nHis army, utterly demoralized, immediately fled back to Janina,\nbringing the tidings to his father that Vely Bey, immediately after\nhis victory, had surrendered of his own accord to the Sultan.\n\nSo every one abandoned Ali. His cities opened their gates to his\nenemies, his best friends betrayed, his two sons forsook, him. Still\nthe third son remained. And Mukhtar Bay was the best man of the three.\nHe was the bravest, and he loved his father the best.\n\nTwo days later came the tidings that Mukhtar Bey with his whole fleet\nhad surrendered before Durazzo to the Kapudan Pasha.\n\n\"The soothsayer foretold it all to me,\" said Ali, calmly, when the\nnews was brought to him. \"So it was written beforehand in heaven.\nNevertheless, at the last, I shall stand at the gates of the Seraglio\non a silver pedestal!\"\n\n\n\n\nCHAPTER VI\n\nTHE LION IN THE FOX'S SKIN\n\n\nBlow upon blow rain down upon thee, thou veteran warrior! Thine armies\ngo over to the enemy, thy friends leave thee desolate, thy sons betray\nthee, they capture thy cities without unsheathing their swords, thine\nallies turn their arms against thee, and with thine own artillery, of\nthe best French manufacture, the Suliotes from the walls of Janina\nshoot down thine Albanian guards!\n\nAh, those Suliotes! How they can fight! If only now they would raise\ntheir swords on thy behalf, how thine enemies would fall in rows! But\nnow it is thy soldiers that fall before _them_! A brother and a sister\nlead them on--a youth and a girl; the youth's name is Kleon, the\ngirl's name is Artemis. Every time thou dost hear their names, it is\nas if a sword were being plunged into thy heart, for the girl is she\nwhom thou wouldst have sacrificed to thy lust, and with whom thy wife\ndidst escape; and thou never dost hear that name without hearing at\nthe same time of the loss of thy bravest warriors!\n\nLike the destroying angel Azrael, she fares through the din of battle,\nwaving her white banner amidst the showers of bullets, and not one of\nthem touches her. Before thy very eyes she plants the triumphant\nbanner on thy bastions, and thou hast not strength of mind enough left\nto wish her to fall; nay, rather, when thou dost see her appear before\nthee, thou dost forbid thy gunners to fire upon her!\n\nDanger approaches Janina from all sides. Thou must drain the cup,\nTepelenti, to the very last drop, to the last bitter drop; and what\nthen? Why, then thou wilt stand before the Seraglio on a silver\npedestal!\n\n * * * * *\n\nOne night there was a rolling of drums before the seven gates of\nJanina, and a bomb flying down from the heights of Lithanizza exploded\nin the market-place of the town. Up, up, ye Albanians! up, up, ye who\nhave any martial blood in your veins, the enemy has seized the guns on\nthe seven gates! Ali throws himself on his prancing steed, and in his\nhand is the good battle-sword which has befriended him in so many a\ndanger. How many times has it not been the lot of Ali to lose\neverything but this one sword, and then to win back everything by\nmeans of it?\n\nIn a moment the army of the besieged stood in battle-array. Ali\ncontemplated the ranks of the enemy, and a smile passed across his\nface. That worthy captain, Gaskho Bey, was leading his troops to the\nshambles. In an hour's time Ali will so completely have annihilated\nthem that not even the rumor of them will remain behind. It will be a\nbattle-field worthy of the veteran general. Every one who sees it will\nsay--there is no escaping from him! Only let them advance, that is\nall! And again he was disappointed. At the first shot, before a sword\nhad been drawn, his army surrendered to the enemy. If only they had\nfired once, the victory would have been his; but no, the army laid\ndown its arms and the cunningly concealed gunners turned his own\nartillery against him.\n\nIt was all over! Only seven hundred Albanian horsemen remained with\nAli, the rest either went over to the enemy or allowed themselves to\nbe taken.\n\nThe old lion waved his sword above his head, and turning to his\nhandful of heroes exclaimed, with a voice that rang out like a brazen\ntrumpet, \"Will ye behold Ali die?\"\n\nAnd with that he galloped towards the market-place of Janina, the\nfaithful seven hundred following closely upon his heels.\n\nThe enemy poured into the town through every gate, but the\nmarket-place cut off one part of the town from the others, and the\ntriumphant hordes came upon some very evil-looking trenches bristling\nwith _chevaux de frise_, and the long narrow streets were swept by\nAli's last twelve cannons, ably handled by the pasha's dumb eunuchs,\nwho stood at their posts like the symbols of constancy on a tomb.\n\nAli Pasha put down his foot in the middle of Janina. Of his ten\nthousand horsemen only seven hundred remained with him. The enemy had\ntwenty thousand men and two hundred guns, and yet all the skill of\nGaskho Bey was incapable of dislodging Ali from the market-place of\nJanina, and although the enemy held one portion of the city, it was\nunable to take the other portion. If only they could have come to\nclose quarters with him, they would have crushed him with one hand;\nbut get at him they could not--that required skill, not strength.\n\nAt last the besiegers set the town on fire all around him, but still\nAli did not budge from his place, and the wind blew the flames in the\nface of Gaskho Bey, who began to look about him uncomfortably when the\ntwo Suliote kinsfolk, Kleon and Artemis, at the head of their\nsquadrons, urged him to boldly assault the market-place.\n\nTepelenti saw the girl with her white banner, and as her troops filled\nthe broad space at the head of the square, he himself, at first, drew\nnear to her. Four cannons were pointed at the Suliotes, loaded with\nchain-shot and broken glass. Ali looked towards them with a gloomy\ncountenance, then stuck his sword in its sheath, bade his gunners turn\nthe guns round, harness the horses to them, and take refuge in the\ncitadel. He would not let a single shot be fired at the Suliotes.\n\nThe moment Ali turned his back, the besieging host captured the field\nof battle. They followed hard upon the heels of the retreating band\nall the way, and when Ali reached the bridge, the Spahis and\nTimariots, like two swarms of bees mingled together, gained the head\nof the bridge at the same time, and swarmed after him with a shout of\ntriumph. The real struggle began on the bridge itself. Man to man they\nfought at close quarters with their shorter weapons (they could use no\nother), and clubs and dirks did bloody work in the throng which poured\nfrom two different quarters, along and over the overcrowded bridge\nlike ants coming out of a slender reed. Six hundred of the Albanians\nsucceeded in escaping into the citadel, and then, at Ali's command,\nthe iron gates were clapped to, leaving the remaining hundred to\nperish on the bridge, where the overwhelming crowd swallowed them up.\nEach single Albanian fought against ten to twenty Timariots. The\nbridge rang with the din of combat, and trembled beneath the weight of\nthe heavy crowd. Then suddenly the guns on both sides of the bastions\nwhich were attached to the bridge began to roar, the supports of the\ncaptured bridge collapsed, and the bridge itself, with its load of\nfighting Turks and Albanians, plunged down into the deep trenches\nbelow.\n\nDown there were sharp-pointed stakes beneath the deep waters, and\nthose of the besiegers who remained on the bank were horrified to\nperceive that not one of the fallen crowd reappeared on the surface of\nthe water, while the water itself gradually grew redder and redder,\ntill at last it was a bright crimson, painted by the blood of the\ncorpses below.\n\nAnd opposite to them stood the fast-barred gate.\n\nAh--ha! 'Tis not so easy to capture Tepelenti as ye thought.\n\nEverywhere else ye have triumphed; ye have triumphed up to the very\nlast point. And now ye _have_ come to the last point, and your\nvictories are worth nothing, for the last point is still to be won.\n\nThe fortress is unapproachable. The bastions are built in the middle\nof the lake, and from their dark quadrangular cavities rows of guns\n(each one of them a sixty-pounder) sweep the surface of the water, so\nthat it is impossible to draw near in boats. On the land side one\nhundred cannons defend the bastions, and who can surmount the triple\nditch?\n\nYe will never capture Ali there. He has sufficient muniments of war\nto last him for an indefinite period, and to show them how determined\nhe was, he caused the solitary gate of the fortress to be filled with\nmasonry and walled up. So the fortress has no longer a gate. Even\ndesertion is now an impossibility.\n\nThere he will remain, then, walled up as in a tomb, buried alive! The\nonly roads from thence lead to heaven or hell; the exit from the land\nside is guarded by the Suliotes; even if he could fly he could not\nescape from them.\n\nThe campaign is ended. The victorious Gaskho Bey proclaims himself\nPasha of Janina. The whole of Epirus does homage to him, and deserts\nthe fallen Vizier. In Stambul thanksgivings are offered up in the Ejub\nmosque and the church of St. Sophia for the accomplished victory,\nwhich is proclaimed, amidst the roaring of the cannons, by heralds in\nthe great market-place; and all the newspapers of Europe amazedly\nreport that the mighty and terrible adventurer, the ever-victorious\nveteran of seventy-nine, the party-leader who grew to such a height\nthat it was doubtful whether he or the Sultan were the real ruler of\nTurkey, the man who had been the ally of the great Napoleon, who a few\nmonths before had sent as a present to England a precious\ndinner-service of pure gold worth 30,000 thaler, who had heaped up\nmore treasures than any Eastern nabob--is suddenly crushed,\nannihilated, shut up in a fortress! It now only remains for him to\ndie.\n\nAnd not very long afterwards he did die. One night a couple of bold\nAlbanian horsemen descended the bastions by means of a long rope, and,\ncrossing the lake of Acheruz on a pine log, sought out Gaskho Bey in\nhis camp that very night.\n\nAli Tepelenti was dead. They were the first to bear the joyful tidings\nto the bey. He died in his grief, in his wretchedness. Perhaps also he\nhad taken poison. On the morrow, at three o'clock, they had arranged\nto bury him in the fortress! Before his death he had called together\nhis lieutenants, and taken an oath of them that they would defend the\nfortress to the very last gasp of the very last man. His treasures\nwere piled up in the red tower--more than thirty millions of piastres.\nHe had left it all to them. But what was the use of all this treasure\nto them if they could not get out of this eyrie? They would not\nsurrender themselves, for Ali had made them swear by every Turkish\nsaint that they would defend the fortress to the death. But the rank\nand file were of a different opinion; they would joyfully retire from\nthe fortress if they were assured of a free forgiveness. Gaskho Bey\nhad only to stretch out his hand and the fortress of Janina, the\nimpregnable fortress with its two hundred cannons and its enormous\nmass of treasure, would be his.\n\nEarly in the morning the gray moonless flag, the sign of death, was\nwaving on the red tower of Janina, and the guns overlooking the water\nfired three and thirty volleys, whose echo proclaimed among the\nmountains that Ali Tepelenti was dead. Within the fortress sounded the\nroll of the muffled drums, and it was also possible to distinguish the\ndirges of the imams.\n\nGaskho Bey and his staff, from the top of the Lithanizza hills,\nwatched the burial of the pasha. There was an observatory here from\nwhose balcony they could look down into the court-yard, and the\nsplendid telescopes, which the sultan had got from Vienna, rendered\npowerful assistance to the onlookers, who through them could observe\nthe smallest details of what was going on in the court-yard of the\nfortress; one telescope in particular brought the objects so near that\none could read the initial letters of the verses of the Kuran which\nthe imams held in their hands.\n\nIn the midst of a simple coffin lay Ali Pasha. It was really he; of\nthat there could be no doubt. Let every one look for himself! There he\nlay--dead, cold, motionless. His lieutenants and his servants stood\naround him weeping. Those who walked along by his side stooped down to\nkiss his hands.\n\nIn the town outside the Suliotes knew of Ali's death, and by way of\ncompliment they fired a bomb into the citadel. But the match of the\nbomb was too short, and it exploded in the air.\n\nFrom the observatory they could see very well the fright of the crowd\nassembled in the court-yard at the whizzing of the bomb over their\nheads, and how every one looked anxiously at the little round white\ncloud there; only he who lay dead in the midst of them remained cold\nand tranquil. He will never again be disturbed by the roar of an\nexploding bomb.\n\nThe imams raised him on their shoulders, and, amidst the melancholy\ndirges of the mourners and the muffled roll of the drums, they carried\nhim away to his open tomb, for his grave was already dug.\n\nThe Moslems do not put their dead in a closed coffin; they only half\nboard the tomb up in order that the angels of death may have room to\nplace the corpse in a sitting posture when they come to take an\naccount of his actions.\n\nThey really did lower Ali Tepelenti into his tomb.\n\nThe garrison fired a triple salute, the imams thrice sang their sacred\nverses, and then came the gravediggers and cast the earth upon the\ncorpse. A large marble slab was standing there, and with it they\npressed down the earth on the tomb, at the same time placing two\nturbaned headstones, one at each end of the tomb.\n\nThey really did bury Ali.\n\nWhen the imams and the officers had departed from the covered tomb,\nGaskho Bey summoned the keepers of the observatory to the summit of\nLithanizza and laid this command upon them:\n\n\"Let a man stand in front of this telescope from morning to evening\n(and mind that he is relieved every four hours), and never withdraw\nhis eye from that tomb. At night, when the moon goes down, a rocket is\nto be fired every five minutes, that the watchers may see the tomb and\nnever leave it out of sight, and report upon it every hour.\"\n\nWhat? Is Gaskho Bey actually afraid that old Ali, a veteran of\nseventy-nine, will be able to arise from his tomb and hurl away that\nheavy marble slab with his dead hands? There are men of whom it is\nimpossible to believe that they are dead, and whom people are afraid\nof even when they are buried.\n\nEvery hour till late in the evening they reported to Gaskho Bey that\nthe tomb remained unchanged, and all the night through not a soul\napproached it.\n\nTepelenti, then, was really dead--totally dead.\n\nEarly next morning Gaskho Bey heard a very curious story.\n\nIn the artillery barracks, where the round guns stood, a drummer had\nlaid down his drum close beside him, with the drumsticks leaning over\nit, when he suddenly perceived the two drumsticks begin to move of\ntheir own accord over the tightly drawn skin of the drums as if some\ninvisible hand wished to beat a tattoo. The drummer cried out at this\nmarvel, and fancied that a _dzhin_ was in the drum.\n\nGaskho Bey would not believe it till he had himself gone to the\nbarracks and seen with his own eyes how the two drumsticks vibrated\nwith sufficient force to tap the drum pretty loudly, moving in a\nspiral line backward and forward across it, tap-tap-tapping as they\nwent.\n\n\"It is very marvellous!\" cried the bey; and he immediately summoned\nthe imams to drive the _dzhin_ out of the drum.\n\nThe imams set to work at once. They fetched their fumigators and their\nsacred books, and they fumigated the drum with nose-offending odors\nand recited over it drum-expelling exorcisms in a shrill voice. And\ncertainly if the devil was in that drum, and had anything of a nose or\nears, he would have been obliged to escape from that noise and stink.\nSo long as the drum was in any one's hand the drumsticks did not move,\nbut when it was put down on the ground the mysterious tap-tapping\nbegan again.\n\nThe imams went on howling, and horribly they howled.\n\nThe chief of the observatory was present during this scene. As a\nFrench renegade he was a man of some education, and therefore he did\nnot accept the theory of the _dzhins_. When he perceived that the\nimams were not successful in expelling the evil spirits, he called\nGaskho Bey aside and whispered in his ear:\n\n\"I know nothing about your _dzhins_, and don't understand what you are\ndriving at with all this noise and stench, but I can tell you that\nthis beating of the drum is a sign that invisible hands are at work\nhere.\"\n\n\"What?\"\n\n\"It means that we ought to get away from here, for they are digging\nmines beneath us, and that is why the ground trembles and the\ndrumsticks vibrate.\"\n\nGaskho Bey began smiling. He had as little idea of sapping and mining\nas the French renegade had of Turkish monsters.\n\n\"How superstitious thou art, my brave moosir!\" said he, shrugging his\nshoulders and looking down upon the Frenchman.\n\nThe latter, however, did not remain there much longer, but hastened as\nquickly as he could to the summit of the Lithanizza.\n\nAfter about an hour and a half's more hubbub the imams succeeded in\nexpelling the _dzhin_. The drum grew quiet, the excitement subsided,\nand the soldiers were instructed to lay two swords crosswise in front\nof the gate, so that the spirit might not be able to come back any\nmore; and with that termination of the affair every one was satisfied.\n\n * * * * *\n\nOpposite the gate of the fortress of Janina, at the head of the\ncollapsed bridge, stood a stone building, fenced about with redoubts\nand palisades, which had now fallen into the hands of the Suliotes.\nThis building had been chosen by the two Greek kinsfolk for their\ndwelling-place. They wanted to get as close to Ali as possible; they\nwould not suffer him to escape even in the shape of a bird or a\nspirit; their large siege-guns were pointed at the walled-up gate. Let\nhim surrender or find his tomb in the fortress.\n\nAnd lo! he _had_ found his tomb without consulting them about it. In\nvain they had sharpened their weapons against him--the sword of Death\nis quicker and cuts down sooner. They had not been able to reach him\non the field of battle; they had not been able to plunge their\navenging swords into his heart; they had not been able to bring his\ngray head to the block; it had been reserved for him to pass quietly\naway--to die in his bed, untroubled, unmolested, to die the death of\nthe righteous.\n\nKleon and Artemis were sitting sullenly in a room of the fort by the\nlight of a flickering candle. The girl had absently divested herself\nof her cuirass and was walking up and down the room with folded arms.\nThere was not a single womanly trait in her face. It was as cold as\nthe face of a statue.\n\n\"So he is dead, then--dead!\"\n\nThis phrase she repeated to herself again and again. She seemed unable\nto get away from it.\n\n\"Ali has died, and not by my hand.\"\n\nKleon was strikingly like his sister; indeed, his young face scarcely\ndiffered at all from hers, but in his eyes quite another sort of flame\nsparkled. Her face, full of dark thoughts, was much more terrible;\nhis was free and open, and full of radiant hope.\n\n\"My triumph has lost its worth if Ali is dead,\" she said, with a sigh.\n\"The old fox has dodged my steel by taking refuge in hell. Oh, would\nthat I might follow him thither also, that I might tear his gray\nbeard, which he has bathed in my kinsman's blood!\"\n\n\"Behold! here is my gray beard!\" cried a voice at that instant from\nthe other end of the room, and the brother and sister beheld Ali\nTepelenti standing before them.\n\nThe terror-stricken young people involuntarily crossed themselves.\nHorror nailed them to the ground and petrified all their limbs, when\nthey saw what they imagined to be a spectre standing there before them\nin the self-same gray robe in which he had been buried two days\nbefore.\n\n\"Behold, here I am, Ali Tepelenti!\"\n\nWith that the spectre clapped his hands, and from every corner of the\nroom rushed forth Albanians armed to the teeth, and before the brother\nand sister could approach their weapons, they were overpowered and\ntied together.\n\nIt was really Ali Tepelenti who stood before them.\n\nThey had put him away underground, it is true, but underground there\nwere paths and passages only too well known to him. The whole\nspectacle of the interment had been arranged by himself, and there was\nan exit from the bottom of his tomb into subterranean corridors. When\nthe general joy and satisfaction at the victory was at its height, he\nwas abroad and at work.\n\nA strongly built subterranean trench had been constructed below the\nditches encircling the redoubts, and its ramifications extended to the\nfort at the head of the bridge. Ali had so completely surprised the\ngarrison that they had not been able to fire a shot; the Suliotes had\nbeen surprised and disarmed while in their dreams.\n\nUp, up, Gaskho Bey! Arise, Muhammad Aga! To horse, ye captains! Seize\nthy sword, Pehlivan Pasha! Danger is at hand! This is a bad night for\nsleeping!\n\n * * * * *\n\nSuddenly a frightful explosion shook the ground, just as if the earth\nwas being wrenched from its hinges, and amidst a flame brighter than\nthe light of day, which seemed to leap up to the very stars, huge\nround cannons were seen flying. The gunners in the barracks were also\npitched into the air. The minarets tottered and fell before the\nterrific shock, every building round about crumbled into ruins. In a\nmoment one-half of the town was reduced to a rubbish-heap, and the\nnext moment a hail of burning beams and lacerated human limbs fell\nback upon the ruins from the blood and fire besmudged heavens.\n\nIt was thus that Ali Pasha signified his resurrection to his enemies!\nHe had gone underground, and now from underground he began the war\nanew.\n\nGaskho Bey, his gigantic body half undressed (he had just leaped out\nof bed), rushed to the end of the street, and was so confused that he\nasked all whom he met where he was. The suddenly aroused soldiers,\nhalf mad with terror, rushed hither and thither in confusion, crying\nout, one for his horse, another for his weapons. And above their\nheads, more terrible than heaven's thunder-bolts, resounded the dread\ncry, \"Ali, Ali!\" There comes the entombed pasha on a white horse, with\nhis white beard; who will dare to look him in the face? The\npanic-stricken throng falls in thousands beneath the swords of the\nAlbanians, blood flows in streams in the streets of Janina, and Ali\nPasha, the dead man, the buried captain, fills the hearts of their\nwarriors with the fear of death. There is none who can stand against\nhim.\n\nOnly Pehlivan, the stalwart hero, was able to prevent the vast\nbesieging army from being scattered altogether by a handful of\nArnauts. He rallied the fugitives outside the town, and, while Ali's\nmen-at-arms were murdering every one inside, he quickly seized all the\ngates, advanced in battle-array, and stayed the triumph of the veteran\ncaptain.\n\nAnd enough had surely been done.\n\nThree thousand of the besiegers lay dead, the guns were spiked or\noverthrown, and the leaders of the Suliote band were prisoners--and\nall this the result of Ali's nocturnal rally! It was time for him to\nreturn.\n\nPehlivan thus recaptured the town and marshalled his men in the\nmarket-place, without pursuing Ali any further. But he had reckoned\nwithout Gaskho Bey, who now came rushing up and furiously accosted\nhim:\n\n\"Why hast thou not pursued him right into the citadel?\"\n\n\"It would not do to press Ali too closely,\" replied the practised\ngeneral; \"let him fly, if fly he will.\"\n\nAt this, Gaskho Bey, foaming with rage, tore the sword out of\nPehlivan's hand (where he had left his own sword he could not have\nsaid for the life of him), and, placing himself at the head of a band\nof Spahis, began to pursue the retreating foe.\n\nAli was proceeding quite leisurely towards the fortress, as if he did\nnot trouble himself about his pursuers, although they were six times\nas numerous as his forces.\n\nWhen Gaskho Bey had got within ear-shot, Tepelenti shouted back to\nhim:\n\n\"Thou hast come to a bad place, brave Bey. This ground is mine, and\nwhat is beneath it is mine also, dost thou not know that yet?\"\n\nGaskho Bey naturally did not understand a word of this till, at a\ngesture from Ali, a rocket flew up into the air, at which signal those\ninside the fortress suddenly exploded all the mines which had been dug\nunder all the streets of the town. Tepelenti had prepared these during\nhis fortunate days by piercing water conduits and making subterranean\nvaults large enough to hold great stores of gunpowder.\n\nAli rallied his own bands at the head of the bridge, and when,\nsuddenly, the explosion burst forth along the whole length of the\nstreet, and the destroying flame tossed the pursuing squadrons into\nthe air one after the other, he amused himself by contemplating the\nruin from the top of the fort, and was the last who disappeared in the\nhidden tunnel. For a long time those in the fortress could hear the\nagonized cries of the vanquished. One-third of the besieging army had\nbeen destroyed in a single night. The rest quitted the accursed town,\nwhich seemed to have been built over hell itself, and took up a\nposition in the fields outside and on the heights of Lithanizza.\n\nThe rising sun revealed a horrible spectacle. The town of Janina no\nlonger existed, the beautiful tall houses, the cupolaed mosques, the\nslender white minarets, the imposing barracks--where were they?\nInstead of them, all that could be seen was a shapeless mass of\npiled-up ruins; here and there, on a dark background, scorched by\nflickering flames, a huddle-muddle of broken rafters, mangled corpses,\ncharred black or gaping hideously open, lay scattered about amongst\nthe rubbish, and from the mouth of a conduit at the side of the\nbastion there trickled sadly down into the lake a dark red stream,\nwhich wound its way in and out amongst the ruins.\n\n * * * * *\n\n\"Poor children, how sweetly they are sleeping!\"\n\nThus spoke Ali.\n\nIn a corner of the red tower, sleeping side by side, were the two\nSuliote kinsfolk, Artemis and Kleon. They slept in each other's\nembrace, and not even the gaze of Ali awoke them.\n\n\"Don't arouse them,\" said Ali to his dumb eunuchs; \"let them sleep\non!\"\n\nAnd again he regarded them with a smile--they slept so soundly. And\nyet they knew not when they fell asleep whether they would ever awake\nagain.\n\nAli did not arouse the slumberers. Thrice he sent to see if they had\nawakened, but he would not have them disturbed. At last the hand of\nthe youth made his chain clank, and both of them opened their eyes at\nthe sound.\n\n\"I was on my way to Akro-Corinth,\" said he, rubbing his large dreamy\neyes with his hands, \"and I saw them rebuilding the Parthenon.\"\n\n\"I stood at Thermopylae,\" said the girl, \"and the enemy fell before me\nby thousands.\"\n\n\"And now we shall go to the block,\" sighed Kleon, listening as the\niron doors of his dungeon slowly opened.\n\n\"Be strong!\" whispered the girl, pressing the hand of her brother\nwhich was enlaced in hers.\n\nThe dumb eunuchs surrounded them, and led them before Ali Pasha.\n\nThe pasha was sitting on a divan, and still wore his funeral robe; all\nthe furniture was shrouded with cinder- cloth; there was\nnothing golden, nothing that sparkled in the room.\n\nThe brother and sister stood before him, pressing each other's hands.\n\n\"My dear children,\" said the pasha, in a voice that trembled with\nemotion, \"don't look into each other's eyes, but look at me!\"\n\nAt this unusual tone, at these kindly words, the brother and sister\ndid look at him, and perceived that the old man was looking at them\nsadly, doubtfully, and that his eyes were full of tears.\n\nAli beckoned to the eunuchs, and they freed the brother and sister\nfrom their chains.\n\n\"Behold, ye are free, and may return to your homes,\" said Ali.\n\nThese words had the effect of an electric shock upon the youth, and\nhis face lit up with a flush of joy.\n\n\"Why dost thou rejoice?\" cried Artemis, casting a severe look upon\nhim; \"dost thou not perceive that the monster is mocking us? He only\nwants to excite joy within us that he may kindle our hopes, and then\nmake death all the more bitter to us. Why dost thou make sport of us,\nthou old devil? Slay us quickly, or slay us with lingering torments,\n'tis all one to us, but do not mock us!\"\n\nTepelenti devoutly raised his eyes to heaven.\n\n\"My soul is an open book before you. Ye are free. Ye free Suliotes, we\nunderstand one another. I have sinned grievously against you, but ye\nhave revenged yourself upon me. I burned your villages, ye, in return,\nhave destroyed my fortresses. I have pillaged your lands, and ye have\ntaken my possessions from me. I have slain your bridegroom and\nsnatched thee from thy parent's house; thou hast cut off the head of\nmy favorite grandson, and ravished from me my favorite wife. Now we\nare quits, and owe each other nothing. Go in peace!\"\n\nThere was so much sincerity, so much repentant, contrite grief in the\nwords of Ali, that the watchful maid began to regard him with curious\nsympathy.\n\n\"Thou art amazed at my change of countenance,\" said Ali, observing the\nimpression his words had produced on Artemis. \"Thou hast not seen me\nlike this before! That other Ali is no more. He died, and was buried.\nA penitent kneels before thee who has a horror of his past sins, and\nbegs thy forgiveness, kissing the hem of thy garment.\"\n\nAnd, indeed, Ali fell down on his knees before Artemis, in order that\nhe might kiss the border of her robe, and breaking forth into moans,\nshed tears at the girl's feet, so that she involuntarily bent down and\nraised him up.\n\nShe was a woman, after all, and could not bear to see any one weeping\nbefore her.\n\n\"Listen now to what I say,\" continued the pasha, \"and do not fancy\nthat Ali has gone mad. This night I saw a vision. A beauteous and\nradiantly majestic maiden descended at my threshold from the midst of\nthe bright, open heavens, surrounded by a company of winged children's\nheads. The maiden looked at me so gently, so kindly. A divine light\nshone from her countenance, and, on the earth beneath, all the flowers\nturned their faces towards her as if she were the sun. In the arms of\nthis heavenly maid sat a child, but what a child! At the sight of him,\neven I, old man as I am, trembled with joy. Round about the head of\nthis child was a wreath of stars, and the smile upon his face was\nsalvation itself. And when I raised my trembling hands towards her,\nthe heavenly lady and the child extended their arms towards me, and\nfrom the lips of the maiden, in a sweet, inexpressibly sweet voice,\ncame these words: 'Ali Tepelenti, I call thee!' And I, all trembling,\nfell down on my knees before her.\"\n\nThe brother and sister involuntarily knelt down beside Ali and\nstammered, full of devotion, \"Blessed be the most holy Virgin!\"\n\nAli Pasha continued the recital of his vision.\n\n\"With my face covered, I listened to the words of the bright\napparition, and now she addressed me once more in a dolorous voice,\nwhich pierced my very heart, 'Ali Tepelenti, behold me!' And when I\nraised my face, lo! I beheld seven swords pointing towards the heart\nof the heavenly maid, and I felt my hand grow numb with fright. 'Ali\nTepelenti,' said the lady for the third time, 'these swords _thou_\nhast thrust into my wounds, and my blood be upon thy head!' And I,\ngroaning, made answer, 'How could I have done so when I do not know\nthee?' And she replied, 'He who persecutes mine, persecutes me, and\nwho robs my temples, robs me; didst thou not pull down the churches of\nTepelen, Turezzo, and Tripolizza?' 'I swear that I will build them up\nagain,' I replied, raising my hand to give solemnity to my vow; and as\nI spoke one of the seven swords fell from the heart of the lady.\n'Didst thou not rob the Suliotes of their children,' inquired the\nheavenly vision anew, 'in order to bring them up as Moslems?' 'I swear\nthat I will make them Christians again!' and at these words the second\nsword fell out of her heart. 'Didst thou not carry off their maidens\nfor thine own harem?' 'I swear that I will give them back to the\nSuliotes!' and with that the third sword fell from her heart. 'Didst\nthou not gather together immense treasures from the heritage of widows\nand orphans?' And, smiting the ground with my head, I answered: 'All\nmy treasures shall be dedicated to thy service.' And thus she recorded\nmy mortal sins one by one, and thus I swore to make rigorous\nreparation for them with an irrefragable oath, and as many times as I\nso swore a sword fell at my feet. Finally but one sword remained in\nher bleeding heart, and then she asked me, 'Hast thou not sought the\ndeath of that Suliote brother and sister who were the most faithful\ndefenders of my altars? Hast thou not plunged them into thy dungeon,\nand is not their death already resolved upon in thy heart?' And,\nterrified, I laid my hand upon my heart, for verily that thought was\nin it, and not without a fierce struggle, I stammered, 'Oh, heavenly\nvision! these two young people are my mightiest enemies, and they\nhave sworn to kill me; yet if thou dost command it I will lay my gray\nhead in their hands, and I will be in their power, not they in mine.'\nAt these words the last sword also fell from her heart, and she\nanswered, 'Ali Tepelenti, take these swords in thy hand, and do as\nthou hast said.' And with that she reascended into heaven, the clouds\nclosed behind her, and I remained alone with the seven swords in my\nhand, on which seven vows were written. This vision I saw in the night\nthat has just past; and now reflect upon my words.\"\n\nThe minds of the brother and sister were deeply agitated. The old\nMoslem before them had spoken with such devotion, with such enthusiasm\nof his vision, that it was impossible to question its reality. The\nemotion visible in his countenance, the tears in his eyes, the tremor\nin his voice, proved that he really felt what he said. While they were\nstanding there pondering over the old man's vision, he took them by\nthe hand and led them into his treasure-chamber, and showed them the\nheaps and heaps of gold and silver, the coins piled up in vats, and\nthe steel which had been melted into bars and stacked up there.\n\n\"My treasures are at your disposal--use them as you will.\" Then,\nselecting from amongst his choicest diamonds two stones, worth a\nhundred thousand sequins, he placed them in the hands of Kleon and\nArtemis, and said, \"These I will send to the war-chest of the\nHetaeria!\"\n\nWhy, what does Ali mean by mentioning this secret society, which had\nalready undermined the whole Turkish Empire--just as he had undermined\nJanina? Perhaps he would fire these mines also! Of a truth the arm of\nAli reached as far as Stambul! aye, and as far as Bucharest also.\n\nAnd now he led the brother and sister into his armory, and there they\nsaw whole chests full of firearms from the manufactories of the best\nEnglish and French makers.\n\n\"You see, I could arm a whole realm with the weapons I have in\nJanina.\"\n\nThe brother and sister sighed; one and the same thought suddenly\noccurred to them both.\n\n\"Tepelenti,\" said the girl.\n\n\"Command me!\"\n\n\"Thou hast done much harm to us, we also have done much harm to thee;\nlet us act as if we now saw each other for the first time.\"\n\n\"I forgive you.\"\n\n\"I will forget that thou didst put to death my betrothed in this room,\nand thou forget that we killed thy grandson. Call to mind, moreover,\nthat not only are we captives in this fortress, but thou art also\nsurrounded by the hosts of thine enemies.\"\n\n\"I alone am a captive,\" said Ali, humbly. \"I swear by Allah, as I have\npromised the holy Virgin, that I will let you and all your companions\nfree! What may happen to you after that I care not. Ali has not long\nto live now. But your days of combat are yet to be, and if ever the\ntime should come when your plans need the help of arms and treasures,\nremember that there is enough of both at Janina.\"\n\nArtemis was constrained to believe in the sincerity of Ali's words.\n\nAnd now the pasha, with his own hand, selected two beautiful Damascus\nblades from among his store of weapons, and bound them to the girdles\nof the brother and sister. What a warmth of self-confidence came over\nthem when they felt once more that they had swords by their sides!\n\nThen he led them down to their companions, who were assembled in the\ncourt-yard of the fortress, and informed them that they were free to\ngo whither they would. And then he put wine and pilaf before the\njubilant crowd of captives, and left them to eat and drink with his\nown Arnauts; and, beneath the peace-making influence of the good wine,\nit was not very long before they fell to kissing one another and\nswearing eternal fellowship like brothers.\n\nThen Ali produced his best long-range rifles, with bayonets attached,\nand distributed them amongst the captive Suliotes; he had not the\nleast fear now that they would turn these arms against him. Then he\nkissed the brother and sister on their foreheads, and, giving them his\nblessing, let them through that secret tunnel which led into the town.\n\n * * * * *\n\nMeanwhile, in Gaskho Bey's camp outside curious reports began to\ncirculate. A pair of captured Albanians, who had been surprised\namongst the ruins of the town when Ali retreated, began to make the\nmost astounding revelations before their judges; amongst other things\nthey maintained that the Suliotes, in the camp of the bey, had a\nsecret understanding with the Pasha of Janina--their former master.\nAnd, as a matter of fact, every one had observed that Ali had quitted\nthe field of battle rather than fire upon the Suliotes.\n\nBut the captives confessed still more. They said that Artemis and\nKleon had had secret meetings with Ali in the subterranean tunnel,\nand had surrendered to him voluntarily. It must have been so, argued\nthose who had survived the last sally. Ali had made his assault from\nthe tower at the head of the bridge, and yet the Suliotes there had\nnot so much as fired a gun to signify his approach.\n\nThe captives also insisted that Ali was going to make another sally on\nthe following night against the besieging army, and then all the\nChristians in the camp of the bey would join him.\n\nThese reports, with still more terrible variations, began to extend\nthroughout the whole army, and here and there slight _melees_ even\ntook place between Christians and Moslems. The Osmanlis began to\nthreaten the foreign soldiers, and the latter began to everywhere form\nthemselves into independent little bands for mutual protection.\n\nGaskho Bey and Pehlivan Pasha hastily summoned a council of war at\nthis disquieting symptom, and it was there resolved that the Greeks\nshould be disarmed. For this purpose they assembled them together in\nthe midst of the camp, surrounded them with Turkish veterans, and\nthen, pointing the guns at them, summoned them to instantly lay down\ntheir arms or they should all be shot down like dogs.\n\nThe Suliotes and Albanians listened to this summons with terror. They\nbeheld the bloodthirsty masses around them, and reflected how many\ntimes men had lost their lives by surrendering the very weapons\nwherewith they might have defended themselves, and, in their\nhesitation, they chose out twelve youths from amongst their ranks to\ngo to the general and ask the reason of this alarming demonstration.\n\nGaskho Bey was still in a towering passion, and the bold speech of the\nyoung men irritated him still further. He had them dragged into the\nmidst of the camp, in front of the assembled battalions, and commanded\nthat their heads should be cut off, proclaiming at the same time that\nany who dared to disobey this order should meet with the same fate.\n\nThe garments of the twelve young men were stripped from off them in\nthe presence of their comrades, and the usual head severing giant\nstood behind them, ready to force them down upon their knees and\ndecapitate them one by one. But he had not yet cut off a single head\nwhen a loud noise was heard coming from the direction of Janina; it\nwas the liberated sister and brother. Artemis and Kleon, at the head\nof their bands. They had beheld from the tower of Janina the danger\nwhich threatened their comrades, and arrived just as the executioners\nwere preparing to carry out Gaskho Bey's commands.\n\nThe Suliotes scattered here and there looked at each other. A\ntremendous roar filled the air--a roar of grief and rage and\nterror--breaking forth into despair. Those from before, those from\nbehind, fell upon the ranks of the Moslems. In a moment Gaskho Bey's\nwhole camp was converted into a chaotic mob, where Albanians and\nSpahis. Suliotes and Timariotes, fought together without any fixed\nplan, and, in utter defiance of all military science, recognizing\nneither friend nor foe. In vain the standard-bearers raised their\nbanners, in vain the officers of the Spahis roared themselves hoarse,\nand the Sorbadzhis and the gigantic Gaskho Bey himself did the same.\nThe army was so completely disorganized that not even the victorious\nenemy could make head or tail of it. Towards evening the Suliotes,\nunder Kleon and Artemis, captured Lithanizza; while Gaskho Bey, in his\ndespair, fled all the way to Durazzo. When he got there he discovered\nthat of all his army only twelve ciauses remained with him. The whole\nhost had fled higgledy-piggledy along the first road it came across,\nleaving behind it all its artillery, baggage, and ammunition wagons.\n\nBut Ali Pasha, sweetly smiling, calmly looked on from the red tower of\nJanina, while the enemy worried itself to death, and the besieging\nthousands scattered in every direction without his having to waste a\nsingle cannon-shot upon them.\n\nBut as I have already said. Ali was often so reduced as to possess\nnothing but his sword, and with this same sword he would win\neverything back again.\n\n\n\n\nCHAPTER VII\n\nTHE ALBANIAN FAMILY\n\n\nAnd now we will let the rumor of great deeds rest a while; we will\nclose our eyes to the wars that followed upon the siege of Janina; we\nwill shut our ears against the echoes of the names of a Ulysses,\nTepelenti, a Kolokotrini, those heroes who shook the throne of the\nSultan, and all of whom the Pasha of Janina called his very dear\nfriends. While these bloody wars are raging we will turn into the\ngrove of Dodona, where formerly the ambiguous utterances of sacred\nprophecies were always resounding in the ears of contemplative\ndreamers. Let us go back eighty years! Let us seek out that quiet\nlittle glen whither neither good report nor evil report ever comes\nflying, whose inhabitants know of nothing but what happens amongst\ntheir own fir-trees; why, even the tax-collecting Spahi only light\ndown amongst them to levy contributions once in a century!\n\nThe house of Halil Patrona's consort no longer stands beside the\nrippling stream. Nobody even knows the tomb in which the beautiful,\nthe elfin Guel-Bejaze now lies; Guel-Bejaze, the White Rose,[9] blooms\nno longer anywhere in that valley. Nobody knows the name even; only\nthe oldest old grandmother in the circle of the spinning maidens can\ntell them tales, which she also has heard from her mother or her\ngrandmother, of a mad lady who used to dwell in this valley and lay a\ntable every evening and prepare a couch every night for an invisible\nspirit, whom she called her husband, and whom nobody saw but herself.\n\n[Footnote 9: The heroine of another Turkish tale of Jokai's, _A feher\nrozsa_ (_The White Rose_).]\n\nThis old woman had a son called Behram, a brave, honest, worthy youth;\nmany a time with his comrades he would pursue the Epirot bandits, who\nswooped down upon their valley and carried off their cattle.\n\nNear to him dwelt the widow Khamko, whose husband had been shot at\nTepelen, and who, with her son, little Ali, in her bosom, had sought\nrefuge amongst these mountains.\n\nFormerly Khamko was a gentle creature, but when they began to talk to\nher about the mad lady she also grew as crazy as ever the other was.\nShe was ready to destroy the whole world, and over and over again she\nwould utter the wildest things; she would like, she said, to see the\nwhole four corners of the world set on fire so that the flames might\nshoot up on all four sides of it, and every living man within it, good\nas well as bad, might be burned. Listen not to such words. O Allah!\n\nBehram was a very quiet fellow, not more than six and twenty years\nold; little Ali was scarce sixteen. But this wild, restless lad was\nalready wont to wander for days together amongst the glens and\nmountains, and whenever he came home he invariably brought his mother\nmoney or jewels. And nobody knew whence he got them save Behram, to\nwhom the youth confessed everything, for he loved him dearly.\n\nAli joined the company of the Epirot adventurers and with them he\nwould go sacking villages, waylaying rich merchants, and shared with\nthem the easily gotten booty.\n\nAnd whenever he returned home without money, his mother. Khamko, would\nrail upon and chide him, and let him have no peace until he had\nengaged in fresh and more lucrative robberies.\n\nBehram looked askance at the perilous ways of his young comrade, and\nas often as he was alone with him did his best to fill his mind with\nhonest, noble ideas, which also seemed to make some impression on Ali,\nfor he gradually began to abandon his marauding ways, and in order\nthat he might still be able to get money for his mother, he fell to\nselling his sheep and his goats, and even parted with his long,\nsilver-mounted musket. At last he had nothing left but his sword. Dame\nKhamko, meanwhile, scolded Ali unmercifully. If he wanted to eat, let\nhim go seek his bread, she said. And the lad wandered through the\nwoods and thickets, and lived for a long time on the berries of the\nforest. At last, one day, when he was wellnigh famished and in the\ndepths of misery, he came upon an Armenian inn-keeper standing in the\ndoorway of his lonely little tavern. Ali rushed upon him, sword in\nhand, like a wolf perishing with hunger. The Armenian was a worthy old\nfellow, and when he saw Ali he said to him:\n\n\"What dost thou want, my son?\"\n\nThe honest, open look of the old man shamed Ali, and casting down his\neyes, he replied: \"I want to give thee this sword.\" Yet the moment\nbefore he had determined to slay him with it.\n\nThe Armenian took the sword from him, and gave him ten sequins in\nexchange for it, besides meat and drink. So Ali returned home without\nhis sword.\n\nWhen Dame Khamko saw her son return home disarmed she was greatly\nincensed and exclaimed:\n\n\"What hast thou done with thy sword?\"\n\n\"I have sold it,\" answered Ali, resolutely.\n\nAt this the mother flew into a violent rage, and catching up a\nbludgeon, belabored Ali with it until she was tired. The big, muscular\nlad allowed himself to be beaten, and neither wept nor said a word,\nnor even tried to defend himself.\n\n\"And now dost see that spindle?\" cried Dame Khamko. \"Learn to spin the\nthread and turn the bobbins quickly; thou shalt not eat idle bread at\nhome, I can tell thee. A man who can sell his sword is fit for nothing\nbut to sit beside a distaff.\"\n\nSo Ali sat down to spin.\n\nFor a couple of days he endured the insults which his mother heaped\nupon him, and on the third day he returned to the Armenian, to whom he\nhad sold his sword, robbed him of and slew of him with it, plundered\nand burned down his house, and from thenceforth became such a famous\nrobber that the whole countryside lived in mortal terror of him.\n\nDame Khamko lived a long time after this event, and ruined her son's\nsoul altogether by urging him to kill and slay without mercy, till one\nfine day her son murdered her likewise, and thus added her blood also\nto the blood of those whom, at his mother's instigation, he had\ncruelly murdered.\n\nAnd this lad became the Pasha of Janina. Ali Tepelenti!\n\nThrough what an ocean of treachery, perjury, robbery, and homicide he\nhad to wade before he attained to that eminence! How often was he not\nso reduced as to have nothing left but his sword and his crafty brain?\nBut many a time, in the midst of his most brilliant successes, in the\nvery plenitude of his power, he would bethink him of the two quiet\nlittle huts where he and Behram had been wont to dwell. He never heard\nof Behram now, but he used frequently to think in those days and\nwonder what would have become of himself if he had listened to\nBehram's words and lived a quiet, contented life. 'Tis true he would\nnot have been so mighty a man as he was now, but would he not have\nbeen a much happier one?\n\nOnce, when he was a very great potentate, he had visited the little\nvillage in the glen in which they had hidden away together. But nobody\nwould tell him anything of Behram. He had disappeared none knew\nwhither. Perhaps he had died since then!\n\n\n\n\nCHAPTER VIII\n\nTHE PEN OF MAHMOUD\n\n\nWhen, during the reign of Mahmoud II., the caravan of Meccan pilgrims\nwas plundered by the Vechabites, lying in ambush, the Sultan ordered\nthe rulers of Mecca and Medina to immediately send to the lair of the\nVechabites and buy back the dervishes with ready money.\n\nThe Vechabites gave up the captives in exchange for the ransom sent\nthem, but they adhered so rigidly to the terms of the bargain whereby\nthey were to surrender the captives only, that they even kept for\nthemselves the garments that happened to be on the captives, and let\nnothing go but their bare bodies, on which account Mahmoud was obliged\nto give his rescued subjects raiment as well as freedom.\n\nAmongst those who were so liberated was a dervish of the Nimetullahita\norder, who, after this incident was over, arose, sought out the Sultan\nand said to him, \"Thou art a poor potentate. Thou art the most sorry\nof all the caliphs. Thou art the greatest son of suffering[10] among\nall the sultans who have gone before thee, or shall come after thee. I\nthank thee for delivering me from the hands of the Vechabites,[11]\nand as a reward, therefore, I bring thee a gift which, even when they\nleft me without any raiment, I was still able to conceal from them.\"\n\n[Footnote 10: _I.e._, patient of insult.]\n\n[Footnote 11: The Vechabites are accounted heretics by the orthodox\nMussulmans.]\n\nAnd with that he produced a writing-reed and gave it to the Sultan,\nand when Mahmoud asked him in what way he had concealed it from the\neyes of the robbers, he explained how he had cunningly thrust it into\nhis thick black beard, where nobody had perceived it.\n\nMahmoud accepted the gift of the dervish, and put it where he put his\nother curiosities; but he did not think of it for very long, and\ngradually it escaped his memory altogether.\n\nOne day, however, when one of his favorite damsels, moved by\ncuriosity, had induced him to show her the treasures of his palace,\nand they came to the spot where lay the pen of the dervish, the damsel\nsuddenly cried out, and said that she had seen the pen move.\n\nThe Sultan looked in that direction, and, observing nothing, treated\nthe whole affair as a joke, and went on showing the damsel the\naccumulated relics and curiosities of centuries which thirteen\nsuccessive Sultans had stored up in the khazne or treasury, and then\ngave the damsel permission to choose for herself whichever of these\ntreasures might please her most.\n\nMany costly things were there covered with gems, and worth, each one\nof them, half a kingdom; there were also rare and precious relics, and\nantiquities rich in historical associations. But the Sultan's pet\ndamsel chose for herself none of these things; to the amazement of the\nPadishah, she only asked for this simple black pen.\n\nMahmoud was astonished, but he granted the damsel her wish, and making\nlight of it, he gave her the writing-reed which was fashioned out of a\nsimple bamboo cane, and was nothing very remarkable even at that.\n\nThe odalisk took the pen away with her to her room, and waited from\nmorning to night to see it move. But the pen calmly rested where she\nhad placed it all day long and all night too, and the odalisk began to\nbe sorry that she had not rather selected for herself some other more\nprecious thing instead of the object of her curiosity; but one\nevening, when the Sultan was visiting her in her flowery chamber, and\nthey were holding sweet converse together, they suddenly heard in the\nroom, where nobody was present but themselves, a faint sound as if\nsome one were writing in great haste, the scratching of a pen on the\nextended parchment was distinctly audible.\n\nThey both looked in the direction of the sound, and words failed them\nin their astonishment, for behold! the writing-reed was half raised in\nthe air, just as when one is holding it in his hand, and it seemed to\nbe writing of its own accord on the parchment extended beneath it.\n\nThe damsel trembled for terror, while the Sultan, who was a stranger\nalike to fear or superstition, imagining that perhaps a spider had got\ninto the upper part of the reed, and consequently made it move up and\ndown, and anxious to convince his favorite thereof, approached the\ntable, and took up the pen in order to shake the spider out of it.\nBut there was nothing at all there, and the pen went on writing of its\nown accord.\n\nThe Sultan himself began to be astonished at this phenomenon. What the\npen seemed to be so diligently writing remained a hidden script,\nhowever, for its point had not been dipped in ink. Wishing, therefore,\nto put it to the test, the Sultan dipped the point of the reed in a\nlittle box full of that red balsamic salve with which Turkish girls\nare wont to paint their lips, and then placed it on a smooth, clean\nsheet of parchment, whereupon it again arose, and wrote in bright,\nplainly intelligible letters these words, \"Mahmoud! Mahmoud!\"\n\nThe Sultan's own heart began to beat when he saw his own name written\nbefore his eyes, and he inquired with something like consternation,\n\"What dost thou want of me?\"\n\nThe pen immediately wrote down again these two words, \"Mahmoud!\nMahmoud!\" and then lay still.\n\n\"That is my name,\" said the Sultan; \"but who then art thou. O\ninvisible spirit?\"\n\nThe pen again arose and wrote beneath the name of Mahmoud this name\nalso, \"Halil Patrona!\"\n\nMahmoud trembled at this name. It was the name of a man who had been\nmurdered by one of his ancestors, and if the apparition of a spirit be\nterrible in itself, how much more the spirit of a murdered man!\n\n\"What dost thou want here?\" exclaimed the terrified Sultan.\n\nThe pen answered, \"To warn thee!\"\n\n\"Perchance a danger threatens me, eh?\" inquired the Sultan.\n\n\"'Tis near thee!\" wrote the pen.\n\n\"Whence comes this danger?\"\n\nAnd now the pen wrote a long row of letters, and this was the purport\nthereof, \"A great danger from the East, a greater from the West, a\ngreater still from the North, and here at home the greatest of all.\"\n\n\"Where will the Faithful fight?\" asked the Sultan.\n\n\"In the whole realm!\" was the reply.\n\n\"Near which towns?\"\n\n\"Near every town and within every town.\"\n\n\"How long will the war last?\"\n\n\"Nine years.\"\n\nIt was now the year eighteen hundred and twenty, and there was not a\nsign of danger at any point of the vast boundaries of the Turkish\nempire.\n\nThe Sultan permitted himself one more question: \"Tell me, shall I\ntriumph in these wars?\"\n\nThe pen replied, \"Thou wilt not.\"\n\n\"Who will be my enemies?\"\n\nThere the pen stopped short, as if it were reflecting on something; at\nlast it wrote down, \"Another time.\"\n\nThe Sultan did not understand this answer, so he repeated his\nquestion, and now the pen wrote, \"Ask in another place!\"\n\n\"Where?\"\n\n\"Alone.\"\n\nEvidently it would not answer the question in the presence of the\nSultan's favorite. It did not trust her.\n\nThe Sultan almost believed that he was dreaming, but now his favorite\ndamsel also drew near and, leaning on Mahmoud's shoulder, stammered\nforth, \"Prithee, mighty spirit, wilt thou answer me?\"\n\nAnd the pen replied, \"I will.\"\n\nThe woman asked, \"Tell me, will Mahmoud love me to the death?\"\n\nThe Sultan was somewhat offended. \"By the prophet!\" cried he, \"that\nthou shouldst put such a question!\"\n\nBut what is not a living woman capable of asking?\n\nThe pen quivered gently as it wrote down the words, \"He will love thee\ntill thou diest.\"\n\n\"And when _shall_ I die?\"\n\nTo this the pen gave no answer.\n\nIn vain the favorite pressed her question. How many years, how many\nmonths, how many days had she to live? The spirit answered nothing.\n\n\"And how shall I die?\" asked the woman.\n\nThe Sultan shivered at this senseless question, and would have made\nthe girl withdraw; but, in an instant, the pen had written out the\nanswer, \"Thou shalt be killed.\"\n\nThe woman grew as pale as a wax figure, and stammered, \"Who will kill\nme?\"\n\nBoth of them awaited in terror and with baited breath what the pen\nwould answer, and the pen, taking good care not to form a single\nillegible letter, wrote on the parchment, \"Mahmoud!\"\n\nThe favorite fell unconscious into the arms of the Sultan, who,\ncarrying her away, laid her on the divan, watching over her till she\ncame to herself again, and then comforting her with wise saws.\n\nAn evil, mocking spirit dwelt in the reed, he said, consolingly, who\nonly uttered its forebodings to agitate their hearts. \"Did it not say\nalso that I should love thee to the death? How then could I slay thee?\nA lying spirit dwelleth in that reed!\"\n\nAnd yet the Sultan himself was trembling all the time.\n\nThat night no sleep visited his eyes, and early in the morning he took\nthe reed from his favorite by force, telling her that he was going to\nthrow it into the fire.\n\nBut he did _not_ throw it into the fire. On the contrary, the Sultan\nfrequently produced it, and, inasmuch as he sometimes convicted the\nspirit of a false prophecy, he began to regard the whole thing as a\nsort of magic hocus-pocus, invented by the kindly Fates to amuse\nmankind by its oddity, and he frequently made it serve as a plaything\nfor the whole harem, gathering the odalisks together and compelling\nthe enchanted pen to answer all sorts of petty questions, as, for\ninstance, \"How old is the old kadun-keit-khuda?\" \"How many sequins are\nin the purse of the Kizlar-Agasi?\" \"At what o'clock did the Sultan\nawake?\" \"When will the Sultan's tulips arrive?\" \"How many heads were\nthrown to-day into the sea?\" \"Is Sadi, the poet, still alive?\" etc.,\netc. Or they forced the pen to translate the verses of Victor Hugo\ninto Turkish, Arabic, and Persian. And the pen patiently accomplished\neverything. At last it became quite a pet plaything with the odalisks,\nand the favorite Sultana altogether forgot the evil prophecy which it\nhad written down for her.\n\nNow it chanced one day that the famous filibusterer Microconchalys,\nwho had for a long time disturbed the archipelago with his cruisers,\nand defied the whole fleet of the Sultan, encountered in the open sea,\noff Candia, a British man-of-war, which he was mad enough to attack\nwith three galleys. In less than an hour all three galleys were blown\nto the bottom of the sea, nothing of them remaining on the surface of\nthe water but their well-known flags, which Morrison, the victorious\nEnglish captain, conveyed to Stambul, and there presented them to the\nDivan.\n\nBoundless was the joy of the Sultan at the death of the vexatious\nfilibusterer, and there was joy in the harem also, for a feast of\nlamps was to be held there the same night, and Morrison was to be\npresented to the Divan on the following day to be loaded with gifts\nand favors.\n\nAt night, therefore, there was great mirth among the odalisks. The\nSultan himself was drunk with joy, wine, and love, and the hilarious\nSultana brought forth the magic pen to make them mirth, and compelled\nit to answer the drollest questions, as, for instance, \"How many hairs\nare there in Mahmoud's head?\" \"How many horses are there in the\nstable?\" and \"How many soldiers are there on the sea?\" And, finally,\nlaughing aloud, she commanded it to tell her how many hours she had to\nlive.\n\nAh, surely a life full of joy lay before her! But the Sultan shook his\nhead; one ought not to tempt God with such questions.\n\nThe pen would not write.\n\nThen the favorite cried angrily, \"Answer! or I will compel thee to\ncount all the drops of water in the Black Sea, from here to Jenikale\nin the Crimea!\"\n\nAt these words the pen, with a quivering movement, arose, and\nscratching the paper with a shrill sound, as if it would weep and\nmoan, wrote down some utterly unintelligible characters, with the\nnumber \"8\" beneath them, and surrounded the whole writing with a\ncircle to signify that there was nothing more to come.\n\nEverybody laughed. It was plain that the spirit also loved its little\njoke, and was angry with the Sultana for torturing it with so many\nsilly questions.\n\nIt was then the third hour after midnight, all the clocks in the room\nhad at that moment struck the hour. After that the odalisks fell\na-dancing again, and the eunuch-buffoons exhibited a puppet show on a\ncurtained stage, which greatly diverted the ladies of the harem. But\nthe number \"8\" would not go out of the head of the favorite, and as\nall the clocks in the room, one after the other, struck four, she took\nout the pen, and with an incredulous, mocking smile on her face, but\nwith horror in her heart, she asked, \"Come, tell me again, if thou\nhast not forgotten, how many hours have I got to live?\"\n\nThe pen wrote down the number \"7.\"\n\nThose who stood around now began to tremble. But Mahmoud treated the\nwhole affair as a joke, and assured them that the pen was only making\nthem sport. And again they went on diverting themselves.\n\nAn hour later the clocks, in the usual sequence, struck the hour of\nfive. And now the favorite stole aside, and placing the reed on a\ntable repeated her former question. And the pen wrote down the number\n\"6.\"\n\nThus, with each hour, the number indicated was lesser by one than the\nprevious number. The Sultan observed the gloom of his favorite, and to\ndrive away her sad thoughts, compelled her to retire to her\nbedchamber, where she enjoyed two hours of sweet repose, leaning on\nthe Sultan's breast; whereupon the Sultan arose and went into his\ndressing-room, for he had to hold a divan, or council.\n\nThe first thing the favorite did on awaking was to look at the time,\nand she perceived that it was now seven o'clock. She immediately\nhastened to interrogate the pen, and asked the question of it with\nfear and trembling; and now the pen wrote down the number \"4.\"\n\n * * * * *\n\nThe Sultan himself sent for Morrison.\n\nThe English sailor was proudly conscious of owning no master but the\nsea. During his long roamings in the East and South he had always made\nit a point of visiting all the barbarous chiefs and princes who came\nin his way. He regarded them simply as freaks of nature, whose absurd\nrites and customs he meant to thoroughly investigate in order that he\nmight make a note of them in his diary, and he even went the length of\nadopting for a time their manners and customs, if he could not get\nwhat he wanted in any other way.\n\nA summons to appear before the divan was scarcely of more importance\nin his eyes than an invitation to a wild elephant hunt, or initiation\ninto the mysteries of Mumbo Jumbo, or an ascent in the perilous aerial\nship of Montgolfier. He donned a dark-blue- garment and a\nplumed three-cornered hat, and condescended to allow himself to be\nconducted by the ichoglanler specially told off to do him honor to the\nsplendid canopied, six-oared pinnace, which was to take him to the\npalace.\n\nThey escorted him first to the Gate of Fountains, and left him waiting\nfor a few moments in the Chamber of Lions, allowing him in the\nmeanwhile to draw a pocket-book from his breast-pocket and make a\nrapid sketch of all the objects around him. They then relieved him of\nhis short sword, as none may approach the Sultan with arms, and threw\nacross his shoulders an ample caftan trimmed with ermine. He did not\nreflect for the moment what a distinction this was. His only feeling\nwas a slight surprise that he should be dressed in green down to his\nvery heels, as, with the dragoman on his left hand, he was conducted\ninto the Hall of the Seven Viziers, where the Sultan sat in the midst\nof his grandees.\n\nMorrison greeted the Padishah very handsomely, just as he would have\ngreeted King George IV. or King Charles X., perhaps.\n\n\"Bow to the ground--right down to the ground, milord!\" whispered the\ndragoman in his ears.\n\n\"I'll be damned if I do!\" replied Morrison. \"It is not my habit to go\ndown on my knees in uniform!\"\n\n\"But that was why they put the caftan on you,\" whispered the dragoman,\nhalf in joke. \"'Tis the custom here.\"\n\n\"And a deuced bad custom, too,\" growled Morrison; and, after\nreflecting for a moment or two, he hit upon the idea of letting his\nhat fall to the ground, and then bent down as if to pick it up again.\nBut, by way of compensation, immediately after righting himself he\nstood as stiff and straight as if he were determined never to bend his\nhead again, though the roof were to fall upon him in consequence.\n\nThe Sultan addressed a couple of brief words to the sailor,\nmetamorphosed by the dragoman into a floridly adulatory rigmarole,\nwhich he represented to be a faithful version of the Sultan's\nineffable salutation. In effect he told the sailor that he was a\nterrible hippopotamus, an oceanic elephant, who had ground to death\ncountless crocodiles with his glorious grinders, trampled them to\npieces with his mighty hoofs, and torn them limb from limb with his\ntrunk, and had therefore merited that the sublime Sultan should cover\nhim with the wings of his mantle. Let him, therefore, ask as a reward\nwhatever he chose, even to the half of the Padishah's kingdom. I may\nadd that if any one had in those days actually asked for half of the\nSultan's kingdom, he would probably have got that part of it which\nlies underground.\n\nMorrison thanked the Sultan for his liberal offer, and asked that he\nmight see the favorite wife of the Grand Signior.\n\nAt these words the dragoman turned pale, but the Sultan turned still\npaler. The convulsive twitching of the muscles of his face betrayed\nhis strong revulsion of feeling, and, lowering his heavy, shaggy\neyebrows, he dashed at the sailor a look of deadly rage, while a heavy\nsigh escaped from his deep chest.\n\nThe Englishman only regretted that he could not acquit himself as\ncreditably in this play of eyebrows. His own were small, of a bright\nblonde color, and somewhat pointed.\n\nThe dragoman, however, could read an ominous meaning in this deep\nsilence.\n\n\"O glorious giaour, rosebud of thy nation!\" whispered he, \"fleet\nwater-spider of the ocean, ask not so senseless a thing from the Grand\nSignior! Behold his wrathful eyes, and ask for something else; ask for\nhis most precious treasure; ask for all his damsels, if thou wilt, but\nask not to see the face of his favorite. Thou knowest not the meaning\nthereof.\"\n\nMorrison shrugged his shoulders. \"I want neither his treasure nor his\ndamsels. I only want to see his favorite wife.\"\n\nMahmoud trembled, but not a word did he speak. Two tear-drops twinkled\nin his dark eyes and ran down his handsome, manly face.\n\nAt this the Viziers leaped to their feet, and it was evident from\ntheir agitated cries that they expected the Sultan to order the\npresumptuous infidel to be cut down there and then.\n\nThe dragoman, in despair, flung himself at the seaman's feet.\n\n\"O prince of all whales!\" he cried. \"O unbelieving dog! Thou seest me,\na true believer, lying at thy feet. O wine-drinking giaour! Why wilt\nthou entangle me with the words which the Sultan said to thee through\nme? Art thou not ashamed to place thy foot on the neck of the lord of\nprinces? Ask some other thing!\"\n\nIn vain. The sailor changed not a muscle of his face. He simply\nrepeated, with imperturbable _sang-froid_, the words:\n\n\"I want to see his favorite wife.\"\n\nThe Viziers rushed at him with a howl of fury, but Morrison merely\nthrew back the caftan which had been folded across his breast,\nrevealing his dreaded uniform and the decorations appended\nthereto--memorials of his services at Alexandria and Trafalgar. That,\nhe thought, would quite suffice to preserve him from any violence.\n\nBut the Sultan leaped down from his throne, beckoned with his hand to\nthe Viziers, and whispered some words in the ear of the Kislar-Agasi,\nwho thereupon withdrew. This whispered word went the round of the\nViziers, who straightway did obeisance and disappeared in three\ndifferent directions through the three doors of the room, their places\nbeing taken by two black slaves in red fezes and white robes, with\nbroad-bladed, crooked swords in their hands. Only the Sultan remained\nbehind there with the sailor.\n\n * * * * *\n\nThe clocks in the rooms of the Seraglio struck a quarter to ten. The\npen of the dervish in reply to the question of the favorite as to how\nmany hours she had to live now wrote down \"1\/4.\"\n\nAt that moment the Kislar-Agasi entered. The favorite went to meet\nhim, trembling like a lost lamb coming face to face with a wolf.\n\nThe Kislar-Agasi bowed deeply, and beckoned to the serving-women of\nthe Seraglio standing behind him to come forward.\n\n\"Has the Sultana accomplished the prescribed ablutions?\" said he.\n\n\"Yes, my lord!\"\n\n\"Gird her round the body with a triple row of pearls; fasten on her\nturban the bird of paradise with the diamond clasp. Put on her gold\nembroidered caftan.\"\n\nThe favorite let them do what they would with her without saying a\nword.\n\nThe waiting-woman, covering the favorite's face with a light fan,\nthickly sewn with tiny gold stars, conducted her to the door which led\nto the Porcelain Chamber, and there the Kislar-Agasi left her, after\nindicating whither they had to go next.\n\nGuards stood in couples before each one of the doors; the last door\nthey came to was only protected by a curtain. This was the door of the\ncupola chamber where the Sultan had received the sailor.\n\nThe favorite could not see the sailor because of the lofty projecting\nwings of the throne; she only saw the Sultan sitting on a divan. She\nhastened up to him, and when she stood before him she suddenly caught\nsight of the stranger regarding her with coldly curious eyes.\nShrinking away with terror, she screamed out \"Giaour!\" and, wrapping\nher veil more closely around her, turned to the Sultan for protection.\nThen Mahmoud seized the damsel's trembling hand with one of his, and\nwith the other raised the veil from the face of his dearest wife in\nthe presence of the stranger.\n\nThe girl shrieked as if her face had been bitten by a serpent; then\nshe fell at the knees of the Sultan, and looked at the face of the\nGrand Signior with an appealing glance for mercy. In the eyes of the\ncaliph of caliphs the moisture of human compassion sparkled. Poor\nSultana! who would not have pitied her?\n\nMorrison made a courtly bow, and the dragoman not being present, he\nexpressed his thanks by using the well-known Turkish salutation,\n\"Salam alakuem!\" The extraordinary charms of the damsel made no more\nimpression upon him than the sight of any ordinarily pretty lady at a\ncourt presentation at home would have done.\n\nThe damsel meanwhile writhed in torments at the feet of the Sultan,\nwho, having had enough of it himself, covered her with her veil, and\nbeckoned to the Kislar-Agasi. He raised the damsel, and carried her\nbehind the curtains that surrounded the throne; the same instant the\ntwo eunuch guards standing beside the throne also disappeared.\n\nThe Sultan listened and covered his eyes.\n\nAfter a few moments of deep silence, it seemed to the sailor as if he\nheard a long sigh behind the curtains. The Sultan shivered in every\nlimb, and immediately afterwards the clocks in the Seraglio began to\nstrike; they struck eleven.\n\nThen the Sultan arose from his place and said, with a deep sigh:\n\n\"'Twas the will of Allah!\" Then he descended from the divan and said\nto Morrison in the purest Italian, \"Thou didst see her; was she not\nbeautiful?\"\n\nMorrison, astonished to hear Italian spoken by the Sultan, who, as a\nrule, never spoke a word save through an interpreter, in his amazement\ncould not find an answer to this question quick enough.\n\n\"Come now and see her once more,\" continued the Grand Signior, and\nwith these words he went towards the curtains.\n\nMorrison fell back confounded. The rosy-red damsel of a few moments\nbefore lay there pale, lifeless, at full length, her lips and eyes\nclosed, her bosom motionless. A thin red line was visible round her\nbeautiful white neck--the mark of the silken cord!\n\n\"But this is brutal!\" exclaimed the sailor, beside himself with\nindignation.\n\nThe Sultan coldly replied, \"Whenever a Christian man beholds the face\nof one of our women, that woman must die.\" He then signified to the\nsailor that he was dismissed.\n\nMorrison hastened from the room, immediately hoisted his anchor, and\nthe same night sailed out of the Golden Horn, everywhere pursued by\nthe memory of the beautiful Sultana, whom he had killed with a glance\nof his eyes.\n\n * * * * *\n\n\"Behold, behold!\" cried the Sultan, pressing the cold, murdered limbs\nto his bosom; \"the _dzhin_ told the truth. Mahmoud loved thee to the\ndeath, and yet Mahmoud slew thee!\"\n\nThese words he repeated two or three times to the dead woman, and\nthen, descending the steps of the throne, rent his garments across his\nbreast, and looking up to heaven with tearful eyes, exclaimed:\n\n\"And now let the rest come too!\"\n\nAnd the rest did come. It came from the east and from the west, from\nthe north and from the south--four empire-subverting tempests, which\nshook the strong trunk of Osman to its very roots, and scattered its\nleaves afar.\n\nAli Pasha of Janina was the first to kindle the blood-red flames of\nwar in the west, and soon they spread from the Morea to Smyrna. In the\nnorth the crusading banners of Yprilanti raised up a fresh foe\nagainst Mahmoud, and the cries of \"the sacred army\" re-echoed from the\nwalls of Athens and the banks of the Danube and the summits of\nOlympus. In Stambul the unbridled hosts of the Janissaries shed\ntorrents of blood among the Greeks of the city on the tidings of every\ndefeat from outside. And when the peril from every quarter had reached\nits height, the Shah of Persia fell upon the crumbling realm from the\neast, and captured the rich city of Bagdad.\n\nAnd still Mahmoud had the desire to live--to live and rule. A pettier\nspirit would have fled from the Imperial palace and taken refuge among\nthe palm-trees of Arabia Felix when it recognized that an endless war\nencompassed it on every side, that to conquer was impossible, and that\nthe nearest enemy was the most dangerous. A mine of gunpowder had been\ndug beneath the throne, and around the throne a mob of madmen were\nhurrying aimlessly to and fro with lighted torches. And yet it was\nMahmoud's pleasure to remain sitting on that throne.\n\nFrequently he would steal furtively at night from his harem. Alone,\nunattended, he would contemplate the flight of the stars from the roof\nof the Seraglio, and would listen to the nocturnal massacres and the\nshrieks of the dying in the streets of Stambul. He would watch how the\nconflagrations burned forth in two or three places at once, both in\nPera and Galata their lordships the Janissaries were working their\nwill. And he felt that cruelly cold piercing wind which began to blow\nfrom the north, so that in the rooms of the Seraglio the shivering\nodalisks began to draw rugs and other warm coverings over their\ntender limbs. Never had any one in Stambul felt that cold wind before.\nWhence came it, and what did it signify?\n\nMahmoud knew whence it came and what it signified, and he had the\ncourage to look steadily in the face of the future, in which he\ndiscerned not a single ray of hope.\n\n\n\n\nCHAPTER IX\n\nTHE CIRCASSIAN AND HIS FAMILY\n\n\nIn those days Kasi Mollah did not go by the name of Murstud--_i.e._, a\npillar of the faith. He was a simple sheik at Himri, in the northern\npart of the land of Circassia, a remote little place, where the\nMuscovite was no more than a rumor from afar.\n\nNature herself had fashioned a strong fortress around Himri. Immense\nmountain-chains enclosed it within massive walls on both sides, rising\nbleak, interminable, and ever upwards into the dim distance.\n\nIn the midst of this valley of eternal shadows arose a third rocky\nmass, forming--on both sides--a steep, ladder-like wall; and, after\nextending far among the other mountains, terminating in a\nragged-looking, concave hill, defended by the junction of the\nimpetuous mountain streams, which dug a deep hollow among the\nexcavated rocks. Along this channel, running like a spinal cord\nthroughout the backbone of the mountain, extended some few thousands\nof acres of luxuriant corn--a long but narrow strip.\n\nAt the head of an opening in the chain a rocky scaffolding was\nvisible, about one hundred feet in height, as regularly disposed as if\na number of gigantic dice had been designedly placed there one on the\ntop of another. By a marvellous freak of Nature, this rocky\nconglomeration was provided apparently with towers, bastions, and\nbuttresses; so that, viewed from afar, it looked like a gigantic\nfortress, and, on the very first glance at it, the thought\ninvoluntarily occurs to one that if but four guns were planted on\nthose summits a few hundred men might defend themselves against an\narmy-corps. At the rear of the hill, moreover, where the cataracts\nmake any approach impossible, the flocks and herds of the defending\narmy could go on contentedly browsing for years together.\n\nA foolish idea! To whom would it ever occur to attack Himri, that tiny\nCircassian village with scarcely five hundred inhabitants, who have\nnothing in the world but their kine, their goats, and their pretty\ngirls? Who would ever come against Himri with guns and an\narmy--against those most worthy men who all their life long have never\ndone anything but make cheese and tan hides, who only exercise their\nvalor against the devastating bands of bears, and only extirpate with\ntheir long, far-reaching muskets the wild goats of the rocks?\n\nThey do not even build their houses on the summit of this wondrous\nfortress of Nature, but among the rocks below, constructing them\nprettily of regularly disposed logs, with roofs like dove-cots,\nsurrounding them with linden-trees and flower-gardens. And so far from\nkeeping a visitor at bay with cannon-shots, they go forth to meet him,\nconduct him into their villages, hospitably entertain him, insist on\nhis tarrying long with them; and if the visitor be a handsome young\nfellow, the loveliest eyes that ever smiled and wept grow moist at\nhis departure. Who amongst those who have been lulled to sleep in\nHimri by the songs of the lovely and bewitching Circassian girls could\never have dreamed that the time would come when these mountain walls\nall round about would be dyed red with the blood of thousands and\nthousands of strangers, who came thither to seek death, and found what\nthey sought?\n\nThe house of the meritorious sheik differed in no respect from the\ndwellings of the other inhabitants. It also was entirely built of\ntimber, consisted of four rooms leading one out of another, and two\nvenerable nut-trees stood in front of it.\n\nKasi Mollah sits outside, leaning tranquilly against the door-post\nbeneath the projecting eaves, both sides of which are covered by large\nscarlet-runners, plaiting with great care and solemnity a whip out of\ntwelve fine thongs of kid-skin hanging on a crooked nail.\n\nSquatting on the ground beside him on a bear-skin sits a\npeculiar-looking stranger. Even if you had not seen it in his features\nand clothing, his mules standing before the door would have told you\nthat he did not belong to these parts. He was, indeed, a Greek\nmerchant from Smyrna, who visited Circassia every year to purchase\nkid-skins--or, so he said. He had three palaces in Smyrna; but it is\nscarcely credible that he could have acquired them by his kid-skins\nonly. At any rate, his mules were laden now with whole bundles of furs\nand pelts, and the merchant was toasting his host in a sour beverage,\nmade by the Circassian from horse's milk, the evil odor of which he\nwas striving to dispel with the smoke of good Latakia tobacco.\n\nIt was for him also that the Circassian was making that long\nmule-driving whip of thongs of twelve different colors, serpentine in\nshape, and plaited at the ends with beautiful white horse-hair; and\nwhen it was ready he smacked it so vigorously, by way of showing it\noff, that the merchant could scarce save his eyes from it.\n\n\"A pretty whip, and a good whip,\" he said, at last, in order that its\nowner might leave off cracking it.\n\n\"I'll very soon prove whether it is a good whip or not,\" said the\nCircassian, without moving a muscle of his brown, oval-shaped,\napathetic face; and with that he began to make the handle of the whip\nout of fine copper wire of a fantastically ornate pattern nicely\nstudded with leaden stars.\n\n\"How will you prove that it is a good whip?\" asked the merchant.\n\n\"Stop till my children come home.\"\n\n\"Your _children_?\"\n\n\"Yes, naturally. I should not think of proving it on other people's\nchildren.\"\n\n\"You are surely not going to prove the whip on your own?\"\n\n\"On whom else, then? Children should be whipped in order that they may\nbe good, that they may be kept in order, and that they may not get\nnonsense into their heads. 'Tis also a good thing to train them\nbetimes to endure greater sorrow by giving them a foretaste of lesser\nones, so that when they grow up to man's estate, and real misfortune\novertakes them, they may be able to bear it. My father used always to\nbeat me, and now I bless him for it, for it made a man of me. Children\nare always full of evil dispositions, and you do well to drive such\nthings out of them with the whip.\"\n\nA peculiar smile passed across the long, olive- face of the\nGreek at these words; he seemed to be only smiling to himself. Then he\nfixed his sly, coal-black eyes on the sheik, and inquired,\nsceptically:\n\n\"But surely you don't beat your children without cause?\"\n\n\"Oh, there's always cause. Children are always doing something wrong;\nyou have only to keep an eye on them to see that, and whoever neglects\nto punish them acts like him who should forbear to pull up the weeds\nin his garden.\"\n\n\"Kasi Mollah,\" said the Greek, puffing two long clouds of smoke\nthrough his nostrils, \"I tell you, children are not your speciality,\nfor you do not understand how to bring them up. In the whole land of\nCircassia there is none who knows how to bring up children.\"\n\n\"Then how comes it that our girls are the fairest and our youths the\nbravest on the face of the earth?\"\n\n\"Your girls would be still more beautiful and your lads still more\nvaliant if you brought them up in the land where dwell the descendants\nof white-bosomed Briseis and quick-footed Achilles. O Hellas!\"\n\nThe Greek began to grow rapturous at the pronunciation of these\nclassical names, and in his excitement blew sufficient smoke out of\nhis chibook to have clouded all Olympus.\n\n\"I tell you. Kasi Mollah,\" continued he, \"that children are the gifts\nof God, and he who beats a child lifts his whip, so to speak, against\nGod Himself, for His hands defend their little bodies. You do but sin\nagainst your children. Give them to me!\"\n\n\"You are a Christian; I am a Mussulman. How, then, shall you bring up\nmy children?\"\n\n\"Fear nothing. I do not want to keep them for myself; I mean rather to\nget them such positions as will enable them to rise to the utmost\ndistinction. I would place them with some leading pasha, perhaps with\nthe Padishah himself, or, at any rate, with one of his Viziers, all of\nwhom have a great respect for Circassians.\"\n\n\"Thank you. Midas, thank you; but I don't mean to give them up.\"\n\n\"Prithee, prithee, call me not Midas; that is an ominous name which I\ndo not understand. You might have learned any time these ten years,\nwhen I first came to buy pelts from you, that my name is Leonidas\nArgyrocantharides, and that I am a direct descendant of the hero\nLeonidas, who fell at Thermopylae with his three hundred valiant\nSpartans. One of my great-great-grandfathers, moreover, fell at Issus,\nby the side of the great Alexander, from a mortal blow dealt to him by\na Persian satrap. If you do not believe me, look at this ancient coin,\nand at these others, and at this whole handful which are in my purse,\nall of which were struck under Philip of Macedon, or else under Michel\nKantakuzenos or Constantine Porphyrogenitus, all of whom were powerful\nGreek emperors in Constantinople, which now they call Stambul, and\nbuilt the church of St. Sophia, where now the dervishes say their\nprayers; and then look at the figures which are stamped on these\ncoins, and tell me if they do not resemble me to a hair. It is so.\nNo, you need not give me back the money; give me rather the two\nlittle children.\"\n\nThe Circassian, who had taken the purse with the simple intention of\ncomparing the figures on the coins with the face of the merchant, drew\nthe strings of the purse tight again at this offer, and thrust it back\ninto the merchant's bosom.\n\n\"Thank you,\" said he, dryly. \"I deal in the skins of goats, not in the\nskins of men.\"\n\nThe face of the merchant showed surprise in all its features. Not\nevery man possesses the art of controlling his countenance so quickly,\nespecially when his self-command is put to so sudden and severe a\ntest. The Georgians, more to the south, were a much more manageable\nrace of men. With them one could readily drive a bargain for their\ndaughters and give them a good big sum on account for their smallest\nchildren. One could purchase of them children from two to three years\nof age at from ten to twenty golden denarii a head, and sell them in\nten years' time for just as many thousands of piastres to some\nillustrious pasha. This was how Leonidas was able to build himself\npalaces at Smyrna.\n\n\"You talk nonsense, my worthy Chorbadzhi,\" said the merchant, when he\nhad somewhat recovered himself. \"Shall I prove it to you? Well, then,\nin the first place, you do not sell your children, and, in the second\nplace, why shouldn't you sell them? If a Circassian wrapped in a\nbear-skin comes to you and asks you for your daughter, would you not\ngive her to him? And at the very outside he would only give you a\ndozen cows for her, and as many asses. I, on the other hand, offer you\na thousand piastres for them from good, worthy, influential beys, or\nperhaps from the Sultan himself, and yet you haggle about it.\"\n\nThe sheik's face began to show wrath and irritation. He was well aware\nthat the merchant was now dealing in sophisms, though his simple\nintellect could not quite get at the root of their fallacy. It was\nplain that there was a great difference between a Circassian dressed\nin bear-skin, who carries off a girl in exchange for a dozen cows, and\nthe Captain-General of Rumelia, who is ready to give a thousand ducats\nfor her--and yet he preferred the gentleman in bear-skins.\n\nThe Greek, meanwhile, appeared to be studying the features of the\nCircassian with an attentive eye, watching what impression his words\nhad produced, like the experimenting doctor who tries the effects of\nhis medicaments _in anima vili_.\n\n\"But I know that you will give them. Kasi Mollah,\" he resumed, filling\nup his chibook. \"No doubt you have promised them to another trader.\nWell, well! you are a cunning rogue. Merchants of Dirbend or Bagdad\nhave no doubt offered you more for them. They can afford it, they do\nsuch a roaring business. Those perfidious Armenians! They buy the\nchildren for a mere song, and sell them when they are eight or nine\nyears old to the pashas, so that not one of them lives to see his\ntwentieth year, but all die miserably in the mean time. I don't do\nsuch things. I am an honest man, with whom business is but a labor of\nlove, and who is just to all men. It is sufficient for me to say that\nI was born where Aristides used to live. Numbers and numbers of my\nancestors were in the Areopagus, and one of my great-great-uncles was\nan archon. Do not imagine, therefore, that I would do for every\nfoolish fellow what I offer to do for you. I only do kindnesses to my\nchosen friends; the ties of friendship are sacred to me. Castor and\nPollux, Theseus and Pirithous are to me majestic examples of that\nexcellent brotherhood of kindred spirits which I constantly set before\nme. Wherever I have gone people have always blessed me; nay, did I but\nlet them, they would kiss my feet. The daughter of a Georgian peasant\nwhose father trusted me is now the first waiting-woman of the wife of\nthe Governor of Egypt. Is that glory enough for you! The daughter of a\npoor goatherd, whom I picked up from the mire, is now the premier\npipe-filler of the Pasha of Salonica. A high office that, if you like!\nWhat Ganymede was to Jove in those classical ages-- Ah! the tears gush\nfrom my eyes at the sound of that word. O Hellas!\"\n\nThe Circassian allowed his good friend to weep on, considering it a\nsufficient answer to let his dark bushy eyebrows frown still more\nfiercely, if possible, over his downcast eyes. Then he caught up a\nhammer and hammered away with great fury at the handle he had prepared\nfor the whip, riveting the wire with copper studs.\n\n\"Kasi Mollah, hitherto I have only been joking, but now I am going to\nspeak in earnest,\" resumed Leonidas Argyrocantharides, raising his\nvoice that he might be heard through the hammering. \"You should\nbethink you seriously of your children's destiny. I am your old\nfriend, your old acquaintance; my sole wish is for your welfare. I\nlove your children as much as if they were my own, and the tears gush\nfrom my eyes whenever I part from them. What will become of them when\nthey grow up? I know that while you are alive it will be well with\nthem, but how about afterwards? You may die to-morrow, or the next\nday; who can tell? We are all in the hands of God. Now I'll tell you\nsomething. Mind. I'm not joking or making it all up. I know for\ncertain that Topal Pasha has been informed that you have two lovely\nchildren. Some flighty traders of Erzeroum revealed the fact to him.\nThey are wont to trade with you here, and he has paid them half the\nstipulated sum down on condition that they bring the children to him.\nNow this pasha is a filthy, brutal, rake-hell sort of fellow, the\npressure of whose foot is no laughing matter, I can tell you; a\nhorrible, hideous, cruel man. I can give you proofs of it. And these\nmerchants have made a contract with him, and have engaged, under the\npenalty of losing their heads, to deliver your children to him within\na twelvemonth. What do you say? You'll throw them down into the abyss,\neh? Ah! they are not as foolish as I am. They will not openly profess\nthat they have come here for your children, as I do, but they will lie\nin wait for them when they go to the forest, and when nobody perceives\nit they will clap them on the back of a horse and off they'll go with\nthem, so that nobody will know under what sky to look for them. Or,\nperhaps, when you yourself are going along the road with them, they'll\nlay a trap for you, shoot you neatly through the head, and bolt with\nyour children. Well, that will be a pretty thing, won't it? You had\nbetter not throw me over.\"\n\nThe Circassian did not know what to answer--words were precious things\nto him--but he thought all the more. While the merchant was speaking\nto him, his reflections carried him far. He saw his children in the\ndetested marble halls, he saw them standing in shamefully gorgeous\ngarments, waiting upon the smiling despot, who stroked their tender\nfaces with his hands, and the blood rushed to his face as he saw his\nchildren blush and tremble beneath that smile. Ah, at that thought he\nbegan to lash about him so vigorously with the whip that was in his\nhand, that the Greek rolled about on the bear-skin in terror, holding\nhis hands to his ears.\n\n\"Do not crack that whip so loudly, my dear son,\" said he, \"or you'll\ndrive away all my mules. I really believe your whip is a very good\none, but you need not test it to the uttermost. I thank you for making\nit; but now, pray, put it down. I must go. It is a good thing you have\nnot knocked out one of my eyes. You certainly have a vigorous way of\nenjoying yourself. But let us speak sensibly. Do you believe that I am\nan honest man, or not?\"\n\nAt this the Circassian did _not_ nod his head.\n\n\"Very well, then. It is natural that you should believe, you ought to\nbelieve it. Since Pausanias there has not been a sharper among my\nnation. He was the last faithless Greek, and they walled him up in the\ntemple. I am a man without guile, as you are well aware. But I am more\nthan that, more than you suspect. Oho! in this shabby, worn-out caftan\nof mine dwells something which you do not dream of. Oho! I know what I\nreally am. I am on friendly terms with great men, with many great\nmen, standing high in the empire, whose fame has never reached your\nears. In the palm of this hand I hold Hellas, in the other the realm\nof Osman. I shake the whole world when I move. Why do I take all this\ntrouble? Oh, for the sake of your holy shades, Miltiades,\nThemistocles, Lysippus, and Demosthenes! for the sake of your shades,\nO Solon, O Lycurgus, O Pythagoras, and a time is coming in which I\nwill prove it! It is thy memory, Athene, which inspires me to heap up\ntreasures for the future! Thou, O holy Goddess of Liberty, hath\nwhispered in my ear that thou canst make use of the lowly as well as\nof the mighty to promote thy cause!\" Here the merchant leaped to his\nfeet in his enthusiasm, and, extending his hand towards the Circassian\nexclaimed, \"Kasi Mollah, you groan beneath the yoke just as much as we\ndo; let us join hands against our oppressors, and let us gradually\nmelt the hearts of their leaders by the strongest of fires, by the\nfire of the eyes of the Greek and Circassian maidens, and we shall\ncatch them in a flowery net!\"\n\nKasi Mollah did not clasp the hand of the enthusiastic Greek; and,\nwithout turning towards him, replied, coldly, \"I do not grudge you the\ndrink which I put before you, worthy merchant, but I perceive that it\nhas begun to mount into your head, or else you would not talk such\nrubbish as selling free people to your enemies from motives of\nfreedom. Nor do you say well in saying that we are under the yoke, for\nthat is not true. Nobody has ever made the Circassian do homage, nor\nwould any try to conquer us for the sake of the eyes of our poor\ndamsels. Say no more about my children. I will not give them up. If\nany one comes to visit me, I'll send him about his business; if any\none tries to deceive me, I'll cudgel him; and if any one tries to rob\nme, I'll slay him. And tell that to the merchants of Erzeroum also.\nAnd now say no more about it.\"\n\nAt these words the face of the merchant grew very long indeed. In his\nspite he began pulling at the stem of his chibook with such force that\nhis face was furrowed right down the middle, and his eyebrows ascended\nto the middle of his forehead. From time to time he kept on wagging\nhis head, and his scarlet, mortar-shaped fez along with it, and burned\nthe tips of his fingers by absently poking the red-hot bowl of his\npipe. But his indignation did not go beyond a shaking of the head, and\nthere he wisely let the matter rest.\n\n\"Very well, Kasi Mollah. You are an honest fellow. We shall see--we\nshall see.\"\n\nThe sun was now setting, and from among the hills the bells of the\nhome-returning cattle resounded across the level plain which extended\nin front of the rocky heights of Himri. Fifteen head of snow-white\nkine strolled leisurely towards the house of Kasi Mollah, passing one\nby one through the gate of their enclosure; behind the last of them\ncame the children of the sheik, who guarded the herd in the forest.\n\nThe boy appeared to be about twelve, and the girl a year younger, and\nso closely did they resemble each other that, viewed in profile, it\nwas impossible to distinguish one from the other. Both had the same\nlong, black hair, which flowed in wondrous ringlets down their\nshoulders, the same soft complexion of a naive maturity, and as smooth\nas velvet, just as if they never walked in the sunlight, and yet they\nhad no head-coverings. The youth's face revealed so much girlish\ntenderness, and the girl's so much vigor and expression, that by\nchanging their clothes it would have been possible to substitute one\nfor the other; and, but for the well-known, tight-fitting corset,\npeculiar to the Circassian maidens, which caused her figure, slender\nas a delicate flower-stalk, to bend somewhat backwards, throwing into\nrelief the contours of her childlike breasts, it would have been\nscarcely possible to have distinguished her from her brother,\nespecially when, as now, they walked side by side, half embracing. The\nsnow-white arm of the girl was round her brother's neck, and her\nhumidly glittering black eyes seemed to be sucking the virile courage\nfrom his face; the boy held the slim figure of his sister encircled by\none of his arms, tapping her, from time to time, caressingly on the\nshoulder, while his eyes rested, full of tenderness, on her beloved\nface.\n\n\"What a majestic pair of children!\" exclaimed Leonidas\nArgyrocantharides, in his enthusiasm. \"What a shame it is to lock them\nup in this corner of the world! But what the deuce is the lad dragging\nalong with his left hand while he embraces his sister with his right?\nWhat _is_ it, my pretty children? Nay, don't bring it here. What sort\nof unclean animal is it?\"\n\nThe lad, with a triumphant smile, stood before the merchant while his\nsister ran to her father, climbed on to his knees, and throwing her\narms shamefacedly round his neck hid her face from the stranger.\n\n\"Do you not recognize the bear-skin?\" cried the youth, in a strong,\nclear voice; and as he spoke you became aware of the light black down\nwhich shaded his upper lip and revealed the man, and with one of his\nhands he raised up the beast he was dragging after him on to its hind\nlegs. It was a young bear, about a year and a half old, whose head was\nbattered and smashed in a good many places, thus showing what a severe\nstruggle it had cost to bring it down.\n\n\"Where did you find that monster? Who gave it to you?\" cried Leonidas,\nholding his hand before him as if he believed that the hideous\nmonster, even when dead, could clutch hold of his thin drumsticks of\nlegs.\n\n\"Where did I find it? Who gave it me?\" cried the youth, proudly, and\nwith that he pointed to his sister, and, as if ashamed to speak of his\nheroic deed himself, he said, \"Tell him, Milieva!\"\n\nThe old Circassian looked attentively at the two children. Neither of\nthem perceived that their father was angry.\n\n\"We were in the forest,\" began the girl--her voice was like a silvery\nbell. \"Thomar was carving a fife, and I was twining a garland for his\nhead, because he pipes so prettily, when all at once a little kid with\nits mother came running towards us, and the little kid hid itself\nclose to me--it trembled so, poor little thing! but its mother only\nbleated and kept running round and round, just as if it wanted to\nspeak. Thomar looked all about, and not far from us perceived two\nyoung bears running off, and one of them had another little white kid\non its back, which was certainly the young one of the little she-goat\nthat was trying to talk to us. 'Thomar,' said I, 'if I were a boy, I\nwould go after that young bear and take away the poor little kid from\nit.' 'And dost thou think I will not do it?' replied Thomar, and with\nthat he caught up his club and went after the two young bears. One of\nthem perceived him and quickly ran up a tree, but the other would not\ngive up his prey, but turned to face Thomar. Ah! you should have seen\nhow Thomar banged the wild beast on the head with his club till the\nblood ran down its shoulders, and suddenly it let go the white kid,\nwhich ran bleating after its mother.\"\n\nThe child clapped her little hands for joy, while her father softly\nstroked her long hair.\n\n\"But now the young bear, gnashing its teeth, rushed upon Thomar and\nseized the club in Thomar's hands with its teeth and claws. 'Thomar,\ndon't let him have it!' cried I. But, indeed, he had no fear of the\nwild beast, for he drew his knife from his girdle and thrust it with\nall his might into the head of the furiously charging wild beast.\"\n\n\"Oho!\" interrupted Thomar, \"don't forget that you also rushed upon it,\nand gave me time to draw out my knife by seizing the ears of the bear\nin both hands and dragging it off me.\"\n\nThe father looked at the two children with an ever-darkening face, but\nthe merchant solemnly shook his head and raised his hands aloft with\nan expression of horror. \"O foolish--O mad children!\" cried he.\n\n\"The bear had now had enough,\" continued Milieva, trying to give her\ntalkative little mouth an earnest expression befitting her serious\nnarration; \"it tore itself out of our hands, and with a great roar\ntook refuge from us in a subterranean cave, taking along with it\nThomar's knife, buried in its head. Now this knife we had got from\nHassan Beg, so we could not afford to lose it. So what do you think\nThomar did? He dived into the narrow hole after the bear, and, seizing\nit there by the throat, throttled it, and dragged it out.\"\n\nCold drops of perspiration trickled down the foreheads of the two men.\n\n\"Then he caught the young bear by the foot, and as it was heavy we\nboth dragged it along together. We had to make haste, for the old bear\nhad scented our trail and was after us, and pursued us as far as the\nherds, where the herd-keepers shot it down, but its young one we\nbrought along with us.\"\n\n\"O ye senseless children!\" cried the merchant in his terror. \"O\nblockheads! Suppose the bear had clawed your faces, you would have\nbeen disfigured forevermore. It would really serve you right if your\nfather gave you a good thrashing with this new whip.\"\n\nAnd that is what really did happen.\n\nIn his wrath Kasi Mollah seized the freshly made, mule-driving whip,\nand cannot one imagine the fury, begotten of fear, which would take\npossession of a father's heart on hearing such a hair-bristling\nnarrative from the lips of his children? To poke their noses into a\nbear's den, forsooth! The old bear would have torn the pair of them to\npieces had she been able to catch them! They had certainly well\ndeserved a thrashing, and a good thrashing too! Thomar would not have\nwept or groaned however many stripes he might have got; he only\nclinched his teeth, and, standing upright, bore with tearless eyes the\nlashing of the whip on his back and shoulders without a cry, without a\nsob.\n\nBut Milieva cast herself, shrieking, on her father's breast, and the\ntears began to pour abundantly from her radiantly bright eyes. She\ncaught hold of the Circassian's chastising right arm with both her\nhands, and begged so sweetly, \"Do not hurt Thomar; do not hurt him,\nfather! It was indeed not his fault. I assure you I set him on. I told\nhim to go after them. Thomar only went because I asked him.\"\n\nKasi Mollah tried to push the child aside, whereupon she flung her\narms round Thomar's neck and protected her brother's body, exclaiming,\nher face all aglow, \"'Tis my fault, beat me, but don't hurt Thomar!\"\n\nThe lad would have disengaged her arms, and, clinching his teeth for\npain, said:\n\n\"'Tis not true! Milieva did not urge me to do it. Milieva was looking\non from a distance. Milieva was not there. Don't hit Milieva.\"\n\nBut the girl threw her arms so tightly round her father that he was\nnot able to tear himself loose. At last, in sheer desperation, he was\nobliged to lift the paternal instrument of admonition against the girl\nalso. But now the youth snatched at the whip, and exclaimed, with\nsparkling eyes:\n\n\"Strike her not, for she has done no wrong! Beat me as much as you\nlike, but do not strike Milieva. If you do I will leave your house,\nand you shall never see me more!\"\n\n\"What, you ragged cub, you!\" cried the old Circassian, infuriated by\nthe opposition of his son, and forcibly tearing away the whip from his\nhand, he struck the girl a violent blow across the shoulders with it.\n\nMilieva ceased to weep, she only pressed her lips together, as her\nbrother had already taught her to do, and cast down her eyes; but\nThomar perceived a tremor run through her tender, maidenly bosom at\nthe torture.\n\nThe old Circassian himself felt sorry for the poor thing, though he\nwas too proud to show it; but it was plain he had put his wrath behind\nhim from the fact that he now began to wind the whip round its handle.\n\nThomar bent over the girl's shoulder, and wherever he saw one of the\npainful bruises which she had got on his account he kissed it softly,\nand after that he kissed the girl's face, and those kisses were\nparting kisses.\n\nHe said not a word to anybody in the house, but taking up his\nshepherd's staff and his rustic flute, he went forth from his father's\ndwelling without once looking behind him.\n\n\"Father,\" cried the girl, sobbing, \"Thomar is going away forever!\"\n\nThe old Circassian made no reply. His son did not look back at him,\nand he did not cast a glance after his son, and yet they were both\nheart-broken on each other's account.\n\n\"He'll soon be back,\" thought the father to himself. \"Hunger and want\nwill bring him back.\"\n\nIt was late evening, and still the youth had not returned. The sun had\nset long ago. A violent storm with thunder and lightning arose. The\nwind roared among the trees of the distant woods, and the wolves\nhowled in the mountains.\n\n\"Father, let me go and bring back Thomar,\" pleaded the girl, gazing\nsorrowfully into the dark night through the window.\n\n\"He will come back of his own accord,\" replied the Circassian, and he\nwould not let the girl go.\n\n\"Listen, how the rain pours, and how the wild beasts are howling!\nThomar is all alone there in the tempest, and it is so dark.\"\n\n\"'Tis a good night for a son who forsakes his father,\" replied the\nsheik. But within himself he thought, \"Some neighbor is sure to take\nthe lad in and give him shelter.\"\n\nAt midnight the tempest abated, and the moon shone forth brightly.\nFrom the distant woods came floating back to the village the notes of\na rustic flute. Neither father nor daughter had had any sleep.\n\n\"Listen, father!\" said Milieva. \"Thomar is piping in the wood; let me\ngo and bring him back!\"\n\n\"That is not a flute, but a nightingale,\" replied the stony-hearted\nCircassian. \"Lie down and sleep!\"\n\nYet he himself could not sleep.\n\nIn the morning both the tempest and the song had ceased. The old\nCircassian pretended to be asleep. Milieva softly raised her head and\nlooked at her father, and seeing that his eyes were closed, stealthily\nput on her clothes and went out of the house on tiptoe. Her father did\nnot tell her not to go. He had already forgiven his son, and resolved\nnever to be angry with him any more. After all, it had only been an\nebullition of fatherly affection that had made him punish his son for\njeopardizing his life so blindly.\n\nShortly afterwards the jingling of the asses' bells told him that the\nGreek, who slept on the floor outside, was getting ready to depart.\nThe merchant seemed to be in great haste. He piled his boxes on the\nbacks of his beasts higgledy-piggledy, even overlooking a parcel or\ntwo here and there, and all the time he kept talking to himself,\nstopping short suddenly when he caught sight of the Circassian.\n\n\"I was just going to take leave of you, Chorbadzhi. Why do you get up\nso early? Go to sleep! What a nice day it is after the storm! Salam\nalakuem! Peace be with you! Greet my kinsmen, your sweet children. No,\nI will speak no more of your children. I will do as you desire, I\npromise you, and what I have once promised-- So our business is at an\nend? You are a worthy man, Kasi Mollah! . . . You are a good father--a\nvery good father. I only wish every man was like you. The only thing\nthat grieves me is that you cannot join our holy covenant. The Hellene\nand the Circassian groan together beneath the yoke of a common tyrant.\nAnd then you don't reflect who are on our side. Our northern neighbor\nis always ready to liberate us. I say no more. To a wise man a hint is\na revelation. But do you not long for glory? You have no glorious\nancestors. With you there are no memories of a Marathon, a Plataea.\n. . . God bless you, Kasi Mollah! Go on shooting lots of antelopes,\nand I'll come back and buy the hides from you; mind you let me have\nthem cheap! Take this kiss for yourself, this for your son, and this\nthird one for your daughter. Then you won't give them to me, eh? Well,\nGod bless you, Kasi Mollah!\"\n\nThe sheik felt as if a great stone had rolled off his breast when at\nlast he saw his guest depart, though even from afar the Greek turned\nback and shouted all manner of things about Leonidas and the other\nheroes. But the Circassian did not listen to him. He went back into\nhis house again, lest he should seem to be moping for his children.\n\nLeonidas Argyrocantharides, on the other hand, whistling merrily,\nproceeded with his asses on his way to the forest, and, when he found\nhimself quite alone there, began to sing in a loud voice the song of\nfreedom of the Hetairea, which put him into such a good humor that he\neven began to flourish his weapon in the most warlike manner, though,\nunfortunately, there was nobody at hand whom he could smite.\n\nIt would be doing a great injustice to the worthy merchant, however,\nto suppose that he was fatiguing his precious lungs without rhyme or\nreason, for during this melodious song he kept on looking continually\nabout him, now to the right and now to the left. He knew what he was\nabout.\n\nYes, he had calculated well. Any one who might happen to be hidden in\nthe forest was bound to hear the great blood-stirring song. He had not\nadvanced more than a hundred yards or so when a well-known suppliant\nvoice struck his ear. It came from among the thick trees.\n\n\"Oh, please! listen, please!\"\n\nAt first he pretended not to know who it was, and, shading his eyes\nwith his hand, made a great pretence of looking hard.\n\n\"Oho, my little girl! so 'tis you, eh? Little Milieva, by all that's\nholy! Come nearer, child.\"\n\nThe girl was not alone. She had found her brother, and was shoving and\npushing the lad on in front of her, who, sulkily and with downcast\neyes, was skulking about among the trees as if he were ashamed to\nappear before the Greek, who had been a witness of his flogging.\n\nMilieva had insisted on his returning home and begging his father's\npardon, and the lad had consented, not for his own sake, but for his\nsister's.\n\n\"What a good job I've met you! Come here, little girl. Don't be afraid\nof me. I want to whisper something in your ear that your brother must\nnot hear.\"\n\nAnd he bent down towards the girl from the back of the ass and\nwhispered in her ear, it is true, but quite loud enough for her\nbrother to hear also:\n\n\"My dear child, don't take your brother home now, for your father is\nfurious with the pair of you, and is coming after you straightway.\nThat is why I have been singing so loudly, for I thought you had come\nhither and might hear; and let me tell you that it will be just as\nwell for Thomar to hide himself for a time, for your father, when I\nleft him, had shouldered his musket, and he swore in his wrath that he\nwould hunt his runaway son with the dogs, and shoot him down wherever\nhe found him.\"\n\n\"Let him shoot me down!\" cried the lad, defiantly. He had heard the\nwhole of the whisper.\n\nThe good-hearted merchant shook his head reprovingly.\n\n\"Keep your temper, my son; anger is mischievous. It would be much\nbetter if you left these parts for a little while, and Milieva can go\nback in the mean time and pacify her father. I should mention,\nhowever, that Kasi Mollah is preparing a rope in salt-water, with\nwhich he intends to beat her.\"\n\n\"What!\" cried Thomar, with flashing eyes. \"He would whip her again,\nand with a rope?\"\n\nHe could say no more. The two children fell upon each other's necks\nand wept bitterly.\n\n\"Poor children! orphans worthy of compassion!\" cried the sympathetic\nLeonidas, stroking their pretty heads. \"It is plain that they have no\nmother. Willingly would I shed my blood for you. But it is vain to\nspeak to that savage madman. The last thing he said was that your\nmother had been faithless to him, and that was why he was so furious\nagainst you.\"\n\n\"Then he shall never see us again,\" said the lad, tenderly embracing\nhis sister. \"I will go away, and I will take you with me.\"\n\n\"Where?\" said his sister, trembling.\n\n\"The world is wide,\" said the lad. \"I have often seen from the summits\nof the mountains how far it stretches away. I will go away as far as\never I can.\"\n\n\"But what provision have you got?\" inquired the worthy merchant.\n\nAt this idea the lad seemed to hesitate, and for a moment his face\nflushed red; but he soon recovered his _sang-froid_.\n\n\"You complained the other day that your ass-driver had run away, and\nthat you had all the trouble of looking after the beasts yourself.\nTake me for your ass-driver. I will do all your work for you, and I\nwill ask nothing except that Milieva may come with me without doing\nany hard work. I will work extra in her stead.\"\n\nThe merchant was quite overcome by these words.\n\n\"O children, what words must I hear! Thou art the pearl of youths, my\nson. What a pity thou wast not born in Samos, the isle of heroes! Thou\nshalt be no ass-driver of mine; no, thou shalt be my own son, and thy\nsister shall be my own daughter, and ye shall both sit on my asses,\nnot follow after them. In the neighboring village I shall get\nass-drivers and to spare. I will share my last crumb with you, and ye\nshall dwell at home within my palace as if ye were my own children.\"\nAnd with that he embraced them both.\n\nAs for the children, they were overpowered by so much unexpected\ngoodness, and did not hesitate to accept the offer, although Milieva\nsaid, somewhat tremulously:\n\n\"But you will take us back afterwards to our father, won't you?\"\n\n\"Certainly; is he not my good friend? When we get to my house I will\nlet him know that you are with me, and he will be very glad. But first\nwe will go from here to splendid cities by the sea, where edifices\nthree stories high float on the surface of the water. There my great\npalaces are--you could put the whole of your father's house inside the\nhall of any one of them--and my gardens are full of those beautiful\nfruits which I have so often brought for you in my sack. Thomar shall\nhave a beautiful steed. You would like to ride a horse, my son, eh?\nWell, don't be afraid, and it shall fly away with you like the wind.\nAnd it shall have a mane as white as a swan's--or perhaps you'd like a\nblack one? I have got both, and you shall sit on which you like, with\na sword dangling at your side. And when you draw that sword? Ah, ha!\nIt shall be a bright Damascus blade, and you will be able to make it\nspan your body right round without breaking. I will bet anything that\namong five hundred Turkish youths you will carry off the wreath of\npearls in the sports. How nicely that wreath of pearls will become\nMilieva's head! How beautifully the folds of the silken robe\nembroidered with flowers will sweep around her slim figure! And then\nthe palm-leaf shawl when she dances! Eh, children?\"\n\n\"When will you take us back to our father?\" inquired the girl,\nsorrowfully.\n\n\"Why, at once, of course. As soon as Thomar has become a famous man;\nas soon as half the world recognizes him as a valiant bey, and the\nfame of him spreads to the huts of Himri likewise. Then will Thomar go\nwith you to your father. He will sit on a proudly prancing horse,\ntossing its head impatiently beneath its gold trappings. A grand\nretinue will come riding behind him--valiant heroes, all of them, with\nglittering shields and lances. And after them will follow a litter on\ntwo white asses, with curtains of cloth of gold, and in this litter\nwill sit a wondrously bright and beautiful maiden, and men will stand\nat all the gates and cry, 'Make way for the valiant lord and the\nmajestic lady!'\n\n\"But, meanwhile, old Kasi Mollah will be sitting at his door, and,\nperceiving the splendid magnates, will do obeisance to them; then you\nwill leap from your horse, assist Milieva to descend from her litter,\nand will go to meet him. He, however, will not recognize you. Milieva\nwill be so much rosier, and her figure so much more lovely; and as for\nyou, you will be wearing a beard and mustache, and without doubt you\nwill be scarred with wounds received upon the field of glory. So Kasi\nMollah will conduct you into his house with the utmost respect and\nmake you sit down; but you will have victuals and sherbet brought from\nyour carriages, and will constrain him to eat and drink with you. Then\nyou will fall a-talking, and you will ask him whether he has any\nchildren, and thereupon the tears will start to his eyes.\"\n\n\"Oh,\" sighed the girl, melting at the thought.\n\n\"No, no; it would not do at all to make yourself known all at once.\nThe joy would be too much for him; he might even have a stroke. You,\nlittle Milieva, would be content to sit and listen, leaving Thomar to\nspeak. And Thomar will say that he has heard tidings of Kasi Mollah's\nlost children, gradually leading him on from hope to joy, and at last\nyou will throw yourselves on his neck, and say to him, 'I am thy son\nThomar! I am thy daughter Milieva!' How beautiful that will be!\"\n\nThe heads of the children were completely turned by this conversation,\nand they followed the merchant joyfully all the way to the next\nvillage. There Leonidas Argyrocantharides rested for a little while,\nand made the children dismount and have some lunch in a hut. Then he\nproduced a gourd full of strong, sweet wine, and the children drank of\nit. The wine removed whatever of sadness was still in their hearts,\nand they then resumed their journey. The asses he left behind, but two\nwell-saddled horses were awaiting them in front of the hut. On these\nthe children mounted, and leaving the asses to stroll leisurely on by\none road, under the charge of the hired ass-drivers, they themselves\ntook another. How delighted the children were with their fine steeds!\n\nThe sheik, meantime, was still awaiting the return of his children,\nand as they did not come back by the evening he began to make\ninquiries about them. Some of his neighbors, who had been in the\nforest, informed him that they had seen the children with the Greek\nmerchant; they were riding on his asses. At this Kasi Mollah began\nroaring like a wild beast.\n\n\"He has stolen my children!\" he groaned in his despair, and flew back\nhome for his horse and his weapons, not even waiting for his comrades\nto take horse also. One by one they galloped after him, but could not\neasily overtake him.\n\nRiding helter-skelter he soon reached the neighboring village, but\nhere the track of the asses led him off on a false scent, for only\nwhen he overtook them did he realize that the merchant with his\nchildren had gone far away in another direction.\n\nWith the rage of despair in his heart he galloped back again. Not till\nevening did he dismount from his horse; then he watered his horse in a\nbrook and rushed on again. Through the whole moonlit night he pursued\nthe Greek, and as towards dawn Argyrocantharides looked behind him he\nsaw a great cloud of dust on the road rapidly approaching him, and the\nbright points of lances were in the midst of it.\n\n\"Well, children,\" said he, \"here we must all die together, for your\nfather is coming and will slay the three of us. But whip up your\nhorses.\"\n\nThen, full of terror, they bent over their horses' necks, and the\ndesperate race began.\n\nThe Circassian perceived the merchant and the children, and rushed\nafter them with a savage howl. They had better horses, but the\nCircassian's horses were more accustomed to mountainous paths and had\nbetter riders.\n\nThe distance between the two companies was visibly diminishing. The\nmerchant flogged with his whip the horses on which the children were\nriding. They dared not look back.\n\nTheir father shouted to them to turn their horses' reins. He called\nThomar by name, and bade him tear the merchant from his saddle. The\nson heard his father's voice, he heard his own name mentioned; but he\nfancied his father was threatening him, and clung to his horse still\nmore tightly.\n\nA steep mountain torrent ran across the road in front of them. If only\nthe Greek could succeed in getting across it with but two minutes to\nspare, so that he might pitch the little wooden bridge over it down\ninto the abyss below, he would be saved, for the space between the two\nsteep mountain-sides was much too wide for a horse to leap, and a ford\nwas not to be found within an hour's ride.\n\nBy the time they came to the bridge the pursuing Circassians were\nscarcely distant more than three gunshots, and Kasi Mollah was riding\nwell in advance of the rest. He must needs overtake them before the\nGreek could push the bridge over.\n\nAt that instant the horse on which Milieva sat slightly stumbled, and\nplunging forward on to its knees, fractured its leg.\n\n\"Hah!\" cried the sheik, with wild delight, \"I have got back one of my\nchildren, at any rate.\"\n\nBut how amazed was he when he saw Milieva, instead of running to him\nor even remaining in the road, cry out in terror to her brother and\nraise her arms towards him, and Thomar, never expecting to save her,\nbent down from his horse, and grasping his sister round the waist with\na swift hand, placed her in the saddle in front of him, casting a wild\nlook behind him, and then galloping on farther.\n\nKasi Mollah suddenly reined in his flying horse and stopped short,\nallowing them to escape. Not a step farther did he pursue them. By the\ntime his comrades had joined him the Greek was well on the other side\nof the bridge, and they could all see Thomar helping the merchant to\ncast it down.\n\nTwo burning tear-drops stood in Kasi Mollah's eyes. They really\nburned, and he felt the pain. And yet--and yet, when the two children\nsat in the saddle again, Milieva extended her hands towards her father\nas if in most ardent supplication. What was the meaning of it?\n\nThe good Greek shortly afterwards arrived safely in Smyrna with the\nchildren, and had them taught singing, riding, and how to walk about\nin nice clothes, and some years after he sold them to the Seraglio of\nthe Grand Vizier for two thousand sequins.\n\nAnd all that he had said at random to the children during the journey,\nto cheer their spirits, actually came to pass, as we shall presently\nsee.\n\nWhen Sultan Mahmoud lost his favorite damsel so strangely, Milieva\nwas brought into the Seraglio instead. The girl was then about\nfourteen years old. The Circassian girls at that age are fully mature,\nand the bloom of their beauty is at its prime. Milieva, from the very\nfirst day when she entered the harem, became the Sultan's favorite\ndamsel.\n\nThomar joined the ranks of the ichoglanler, a band of youths who are\nbrought up in the outer court and form the Sultan's body-guard.\n\nIt was in this year that Mahmoud instituted the Akinji corps,\nselecting its members from amongst the Janissaries, and formed them\ninto a small regular army. Thomar very soon won for himself the\ncommand of a company, and continued to rise higher and higher till at\nlength he reached the eminence which the merchant had foretold to him;\nand when the course of time brought with it the day on which he was to\nsee Kasi Mollah again, he had become Derbend Aga, one of the Sultan's\nvery highest officials, and his name was mentioned respectfully by all\ntrue believers. And in the village of Himri his name was also\nmentioned. Kasi Mollah often heard it attached to the title of \"bey,\"\nand Thomar also heard a good deal of the village of Himri and of Kasi\nMollah, for they now called his father \"murshid,\" and the name\n\"murshid\" is full of mournful recollections for both Moscow and\nPetersburg.\n\nBut of all these things we shall know more at another time.\n\n\n\n\nCHAPTER X\n\nTHE AVENGER\n\n\nAnd what now is old Ali Tepelenti about in his nest at Janina? Is he\ncontent with a state of things which results in this--that he must\neither perish or pass the brief remainder of his days in constant\nfighting? Is he satisfied with this sea of blood over which the\ntempest rages, and whose shores he cannot see?\n\nNot yet has he surrendered to fate. His country has declared war\nagainst him, the Sultan has pronounced his death-sentence, his family\nhave abandoned and turned against him; but Ali has not suffered his\nsword to be broken in twain. For eight and seventy years he has been\nthe scourge of his enemies, the defence of his country, the Sultan's\nright hand, the patriarch of his family, and in his nine and\nseventieth year the Sultan and his relations say to him, \"Die! thou\nhast lived long enough!\" And he, by way of reply, set his country in\nflames, shook the throne of the Sultan, and extirpated his own\nkinsfolk.\n\nThe Greeks, whose tyrant he once was, are now his allies. Tepelenti\nprovides them with arms and money, and with good and bad counsel,\nwhichever they want most.\n\nThree armies were sent out against him, and he has annihilated all\nthese.\n\nHis enemy, Gaskho Bey, has lost his army in a battle against the\nrebels without anything to show for it, and now only holds the\nfortresses round about Janina, to wit: Arta, Prevesa, Lepanto,\nTripolizza, and La Gulia. The Hellenes are besieging every one of them\nday by day. One day Ali proclaims that in Tripolizza there are five\nhundred eminent Greeks whom the Turks compel to fight along with them.\nAt this report the besiegers attack the fortress with redoubled fury.\nNow these five hundred Greeks Ali himself got together while\nTripolizza was still in his possession. When he was obliged to leave\nthe fortress, he cast these Greeks down into a well, placed three\nloads of stones upon them, and covered the spot with grass. This he\ndid himself.\n\nExhausted by furiously fighting against superior numbers, the Turks\nsurrendered in three days to Kleon, who conducted the siege, simply\nstipulating that they might be allowed to go free, and this was\npromised them. When, however, the fortress was surrendered to the\nGreeks, their first question was, \"Where are the hostages, our\nbrethren?\" The Turks were amazed. They knew not what to reply, for\nthey had no hostages in their hands.\n\nThen a Suliote warrior discovered the pit which had been sown over\nwith grass, and what a sight presented itself when they broke it open!\n\nThirsting for blood and vengeance, the Greeks flung themselves\nforthwith on the disarmed garrison, and despatched them to the very\nlast man, nay, they did not leave a living woman or child remaining\nin the fortress--they threw them all down headlong from the bastions.\n\nBut Ali Pasha smiled to himself in the fortress of Janina.\n\nHe himself had destroyed more Turks than the whole Greek host had\ndone.\n\nWhen Demetrius Yprilanti captured Lepanto, he allowed the garrison a\nfree exit from the citadel. Demetrius himself signed the terms of the\nsurrender. But when the Turks emerged from the fortress, Ali Pasha's\nSuliotes rushed upon them and cut them all to pieces. Yprilanti, full\nof indignation, threw himself in the midst of them, exhibiting the\ndocument in which he had promised the Turks their lives. But Kleon\nonly laughed--he had learned that brutal, scornful laugh from Ali.\n\n\"Don't trouble yourself about them,\" cried he. \"We are only killing\nthose whose names are not written in the agreement.\"\n\nYprilanti turned from the butchery in disgust, and immediately\nembarking his army, set sail for Chios again.\n\nAh, the Greeks had learned a great deal from Ali. Woe to those\nMussulmans who fall alive into their hands, or who are not so brave or\nso cunning as they themselves are! The Turkish general, Omar Vrione,\nalong his whole line of advance, marched between rows of high gibbets\non which bleached the bones of horribly tortured Turks. Here and\nthere, by way of variety, nailed by the hands to upright planks, were\nthe bodies of dead Jews, half flayed and singed--a ghastly spectacle.\n\nVerily the descendants of the heroes of Marathon have diverged very\nfar indeed from their forefathers, and the experienced Turkish\ncommander knew right well that he is a bad soldier who even descends\nto cutting off the head of his slain foe on the battle-field.\n\nAt Pulo, Omar Vrione encountered the army of Odysseus. Now Omar was at\none time one of the best of Ali Pasha's lieutenants. Ali promoted him\nto the rank of general, and he had begun life as a shepherd-boy. Ali\nhad taught him how to use his weapons, and now he turned them against\nhis master.\n\nThe Sultan had intrusted to him a fine army with which he had assisted\nGaskho Bey to beleaguer Ali. It consisted of eight thousand gallant\nAsiatic infantry, two thousand Spahis, and eight guns. The leader of\nthe Spahis was Zaid, the Bey of Kastorid, Ali's favorite grandson,\nwhom, twenty years before, he had rocked upon his knee, and whom,\nwhile still a child, he had carried in front of him on his saddle, and\ntaught him to ride. Zaid himself had asked, as a favor, that he might\nlead a division of cavalry against his grandfather. He had promised\nhis mother to seize that sinful old head by its gray beard and bring\nit home to her.\n\nA precious grandson, truly!\n\nSo Omar Vrione reached Pulo. Looking down from the hill-tops there, he\ndiscerned the army of Odysseus. He saw him planting his white banners\nin rows upon the heights, and without giving his forces a moment's\nrest, he set his own martial chimneys a-smoking and attacked the\nGreeks with all his might.\n\nAfter an hour's combat, in which they fought man to man, the Greeks\nwere driven from their intrenchments, and began slowly descending into\nthe valley.\n\nThe Timariotes remained behind, and Zaid began to send forward his\nSpahis to attack the retreating army in the rear. Odysseus slowly\nretraced his steps till he came to Pulo. There his war-path stopped.\nHis banner was no longer white, but red; it was sprinkled with the\nblood of the many heroes who had died in its defence.\n\nSuddenly, from the heights of Pindus above them resounded the\ntempestuous melody of the \"Marseillaise,\" which the Greeks had adopted\nas their war-song, and rapid as a storm-swollen mountain torrent the\nSuliotes, with Kleon and Artemis in the van, hurled themselves upon\nthe Turks.\n\nOmar Vrione was caught between two fires. It was too late to turn\nback, too late to reform his order of battle. His guns were useless,\nhis cavalry could not move forward, and his infantry columns were so\ncompletely isolated that they could not render each other any\nassistance.\n\nThe general saw that he could not save his army, but he was at least\ndetermined not to save himself, so he hastened to where the fight was\nraging most furiously.\n\nA wild, merciless _melee_ was proceeding between the inextricably\nintermingled foes. Forcing his way along, Omar Vrione suddenly\nencountered, in the midst of reeking powder and streaming blood, a\ntall youth with a blackened face, whom he at once recognized as Kleon.\nThere, then, they stood, face to face. Three years before, when Ali\nhad sent Omar Vrione to threaten the Suliotes, Kleon fled before him,\nand then he had called after the fugitive, \"Stand, I would send thy\nhead to Ali Tepelenti!\"\n\nAnd there, indeed, Omar Vrione fell, combating, and Kleon cut off his\nhead.\n\nHow strange is fate!\n\nThe fall of Omar Vrione sealed the fate of his army. The Turks fled\nwherever they saw the chance, leaving all their guns, all their flags,\nand all their officers in the lurch. The cavalry had no chance of\nescaping. Half of it fell, the other half surrendered.\n\nZaid, in the moment of extremest danger, took his silver aigrette out\nof his turban and threw it away; then he changed caftans with his\nservant, and mingled with the rank-and-file, so that none might\nrecognize him. It would have been much better for a child like him to\nhave remained at home than to have gone hunting that old lion, his\naged grandfather.\n\nThe Suliotes surrounded Zaid's company. \"Dismount from your horses!\"\nexclaimed the clear voice of Kleon.\n\nThe Spahis, full of shame, dismounted.\n\n\"Which is your leader, Zaid?\" cried Kleon, advancing. The edge of his\nsword was dripping with blood.\n\n\"I am,\" said the servant who had changed clothes with Zaid, and he\napproached Kleon.\n\n\"Bow down before me, thou slave!\" cried Kleon, kicking him.\n\nThe servant bowed his head before the victor, and he never raised it\nagain, for Kleon chopped it off with his bloody sword, and sticking it\non the point thereof, raised it on high and cried to his bloodthirsty\ncomrades: \"Here is their second general, Zaid, who came to subdue us!\nHallelujah!\" and the victorious host repeated after him, \"Hallelujah!\nHallelujah!\"\n\nAnd then they stuck the heads of the two generals on the points of two\nlances, and carried them through the streets of Pulo in the sight of\nthe crowds of women and children on the housetops, bellowing, \"We have\nconquered! We have conquered! These are the heads of the enemy's\nleaders: one of them is Omar Vrione, and the other is Zaid Bey! Kyrie\neleison?\"\n\nAnd what face was ever so pale as Zaid's when he heard his name called\nout and saw how they mocked and jeered at the head they took for his?\n\nThe Suliotes returned to Janina with the captives and the emblems of\nvictory. Tepelenti, hearing that they had decapitated Zaid, went down\ninto the camp and demanded his head.\n\nKleon was sitting in front of his tent _en deshabille_. He was not\ndisposed to part with the symbol of victory, but wanted it to dazzle\nthe eyes of the host for some little time longer.\n\nBut Ali was ready at once with a good idea: \"Cut off the head of\nanother prisoner,\" said he, \"in its stead; none will notice the\ndifference.\"\n\nKleon acted upon the advice, and immediately sent forth his\nmen-at-arms to take the exhibited head to Ali. But Ali shook his own\nhead when he saw it, and wagging his finger at Kleon, he said: \"Thou\nart over-young, my son, to try and impose upon Ali. Thou wouldst turn\nmy counsel to my own hurt, and give me the head of another instead of\nZaid's!\"\n\nKleon leaped to his feet. \"Do you mean to say that is not Zaid's\nhead?\"\n\n\"Of a truth it is not. Dost thou suppose I do not know the youth--I\nwho used to dandle him on my knee ever since he was a child, and was\nthe first to place a sword in his hand?\"\n\n\"But, indeed, he himself told me,\" cried Kleon, pointing at the head,\n\"that he was Zaid, and he was wearing a general's uniform.\"\n\n\"'Tis a slave,\" said Tepelenti, regarding the head more closely. \"Dost\nthou not see? His ears have been cropped, so that he may not wear\near-rings in them, which only great lords may do.\"\n\n\"Then Zaid has gone free!\"\n\n\"Zaid will be among the captives,\" said Tepelenti. \"I would recognize\nhim amongst a thousand. He was my favorite grandson. His image even\nnow is engraved in my heart.\"\n\nThen they went down amongst the captives. Ali had scarce cast a glance\nat them when he pointed with his finger.\n\n\"There he is! Dost thou not perceive how much paler his face is than\nthe faces of the others?\"\n\nKleon wrathfully drew his sword and would have rushed upon the person\nindicated, but Ali held his hand.\n\n\"What doest thou? Wouldst thou slay my grandson before my very eyes?\"\n\n\"Thou didst ask for his head, and it shall be thine.\"\n\n\"But now I ask for his life, Kleon. Zaid is my favorite grandson. I\nbrought him up. I loved him better than his dear mother--better than\nall my children. Look now, I share with thee all the booty, and all I\nask of thee is mine own--flesh of my flesh.\"\n\nThe unhappy youth, hearing these words, fell at Ali's feet and\nembraced his knees, wept, covered his hands with kisses, and implored\nhim to release him--he would be a good and dutiful son to him ever\nafterwards.\n\n\"Thou seest, too, how much he loves me,\" said Ali, looking with\ntearful eyes at Zaid and covering the cowering fugitive with his long\ngray beard. \"Well, Zaid,\" said he, \"so thou dost now fly for refuge\nbeneath the shadow of that same gray beard, by grasping which thou\nwert minded to take Ali's head to thy mother, eh?\"\n\nKleon looked at Ali Pasha with a contemptuous smile. Then Ali was\ntender, Ali had a heart, Ali's heart ached at the slaying of his\nkinsfolk! The Greek felt a cruel satisfaction in tormenting the pasha.\n\n\"If thou dost not wish to see Zaid die,\" said he, \"depart from hence.\nAlive thou shalt not have him!\"\n\n\"What!\" cried Ali, and, standing erect, he drew his sword. \"Because my\nbeard is long dost thou think thou canst trample upon me? I will\ndefend my blood with my blood, and will perish myself rather than let\nhim be slain. Let us see, mad youth, wouldst thou lop off thine own\nright hand?\"\n\nKleon was so surprised that he did not know what to do. It was in his\npower to slay Ali; but then that would be a greater triumph for\nStambul than all the victories of the campaign.\n\nAt that moment a herald arrived from Odysseus with a command for Kleon\nto send all the Turkish officers captured at the battle of Pulo to\nPrevesa, that they might be exchanged against the youths of the\nsacred army who had been captured in Moldavia.\n\nKleon's pride was wounded by this direct command. He considered\nhimself just as good a general as Odysseus or Yprilanti, and did not\nrecognize orders sent from them.\n\nTurning from the herald to Tepelenti, he thus replied:\n\n\"Tell Odysseus that I and my soldiers are in the habit of killing the\nenemy's officers on the battle-field. Only one of them, and he in\ndisguise, remains. He, however, is Tepelenti's grandson, who has\nrecognized him and ransomed him from me for a hundred thousand\npiastres, which he has engaged to pay me within an hour. Is it not so,\nTepelenti?\"\n\n\"It is so,\" said Ali; \"within an hour the hundred thousand piastres\nshall be in thy hands.\"\n\nZaid, with a shriek of joy, kissed the hem of his grandfather's robe,\nand Kleon gave his hand upon the bargain. An hour later the money\narrived in little hogsheads, and he had it weighed in the presence of\nhis captains. Ali, however, binding his grandson by the left arm, and\ngiving him his own caftan, had him conducted into the fortress of\nJanina.\n\nKleon looked contemptuously after him. So the old man had become\nsoft-hearted! How he had wept and supplicated and paid for this youth,\nwho was his favorite grandson!\n\nAn hour later the roll of drums was heard on the bastions of Janina,\nand when the Greeks looked in that direction they saw the stake of\nexecution erected there. Four black executioners were carrying Zaid,\nwho had his hands tied behind his back, and was wearing the self-same\ncaftan which Ali had given him. Ali himself, mounted on a black horse,\nrode right up to the stake. At a signal from him the executioners\nhoisted Zaid into the air, and a moment later Tepelenti's favorite\ngrandson, whom he had dandled so often on his knee, was done to death\nby the most excruciating torments!\n\nAli watched his death-agony with the utmost _sang-froid_, and, when\nall was over, he shouted down from the bastions with a strong, firm\nvoice, \"So perish all those of Tepelenti's kinsfolk who draw the sword\nagainst him! For them there is no mercy!\"\n\nKleon felt his heart's blood grow cold. Ah! he had much, very much to\nlearn from the agonized cries of the dying before he could overtake\nAli, that old man who weeps, prays, and pays, in order to rescue his\nfavorite grandson for the sole purpose of killing him himself with\nrefined tortures!\n\nOf all Ali's large family only two sons now remained, Sulaiman and\nMukhtar. They were the first who had betrayed their father, and it was\ntheir treachery that had wounded him most. For a whole year Ali\ncarried that wound about in his heart. During that time nobody was\nallowed to mention the names of his sons in his presence. Everything,\nabsolutely everything, which reminded him of them was removed from the\nfortress. If any one was weary of life, he had only to mention the\nname of Mukhtar before Ali, and death was a certainty.\n\nMeanwhile the two apostate sons were living in great misery at\nAdrianople; for the Sultan, though he paid them for their treachery,\nwould have nothing more to do with them. The first instalment of the\nmoney which they were to receive as the price of their father's blood\nmelted away very rapidly in merry banquets, pretty female slaves, fine\nsteeds, and precious gems; and when it was all gone the second\ninstalment never made its appearance. Far different and far more\nimportant personages had still stronger claims upon the Sultan's\npurse. Tepelenti's vigorous resistance, the innumerable losses\nsuffered by the Sultan's armies, buried in forgetfulness the services\nof the good sons whose betrayal of their father had profited the\nSultan nothing. They were already beginning to bitterly repent their\noverhasty step when the rumor of Ali's victories reached them; and as\nthe days of necessity began to weigh heavily upon them, as money and\nwine began to fail them, as they found themselves obliged to sell, one\nby one, their horses, their jewels, and, at last, even their beautiful\nslave-girls, it became quite plain to them that no help could be\nlooked for from any quarter, unless perhaps it was from wonder-working\nfairies, or from the genii of the _Thousand and One Nights_.\n\nBut let none say that, in the regions of the merry Orient, fairies and\nwonders do not still make their home among men.\n\nJust when the beys had consumed the price of the last slave they had\nto sell, such wealth poured in upon them, in heaps, in floods, as we\nonly hear of in old fairy tales; and fairy tales, as we all know very\nwell, have no truth in them at all.\n\n * * * * *\n\nOne day, as Ali Pasha was walking to and fro on the bastions of\nJanina, he perceived among the garden-beds in the court-yard below a\ngardener engaged in planting tulips.\n\nTepelenti knew all the servants in the fortress thoroughly, down to\nthe very lowest. He not only knew them by name, but he knew what they\nhad to do and how they did it.\n\nThe name of this gardening slave was Dirham, and he was so named\nbecause, many years before Mukhtar had purchased him when a child from\na slave-dealer for a dirham, and although his master often plagued\nhim, he nevertheless cared for him well, and brought him up and\nprovided him with all manner of good things. Thus Dirham, whenever his\nmaster's name was mentioned, bethought him how little he was worth\nwhen Mukhtar Bey bought him, and how many more dirhams he was worth\nnow, and for all this he could not thank Mukhtar enough.\n\nAli Pasha for a long time watched from the bastions this man planting\nhis tulips. Some of them he pressed down into the ground very\ncarefully, strewing them with loose powdery earth, preparing a proper\nplace for the bulbs beforehand, and moistening them gently with watery\nspray; others he plumped down into the earth anyhow, covering them up\nvery perfunctorily, and never looking to see whether he watered them\ntoo much or too little.\n\nAli carefully noted those bulbs which Dirham had bestowed the greatest\npains upon, and then went down and entered into conversation with him.\n\n\"What are the names of these tulips?\"\n\nDirham ticked them all off: King George, Trafalgar, Admiral\nGruithuysen, Belle Alliance, etc., etc. But at the same time he\nskipped over one or two here and there, and these were the very ones\nwhich he had covered up with the greatest care.\n\n\"Then thou dost not know the names of those others?\" inquired Ali.\n\n\"I have lost my memoranda, my lord, and I cannot remember all the\nnames among so many.\"\n\n\"Look, now, I know the names of these flowers. This is Sulaiman, that\nover there is Mukhtar Bey.\"\n\nDirham cast himself on his face before the pasha. Ali had guessed\nwell. Dirham remembered the two gentlemen just as a good dog remembers\nhis master--they were ever in his mind.\n\nThe wretched man fully expected that Ali would immediately tear these\nbulbs out of the ground and plant his own head there in their place.\n\nInstead of that Ali graciously raised him from the ground and said to\nhim in a tender, sympathetic voice, \"Fear not, Dirham! Thou hast no\nneed to be ashamed of such noble sentiments. Thou art thinking of my\nsons. And dost thou suppose that I never think of them? I have\nforbidden every one in the fortress to even mention their names; but\nwhat does that avail me if I cannot prevent myself from thinking of\nthem? What avails it to never hear their names if I see their faces\nconstantly before me? The world says they have betrayed me; but I do\nnot believe, I cannot believe it. What says Dirham? Is it possible\nthat children can betray their own father?\"\n\nDirham took his courage in both hands and ventured to reply:\n\n\"Strike off my head if you will, my lord, but this I say--they were\nnot traitors, but were themselves betrayed; for even if it were\npossible for sons to betray their father, Tepelenti's children would\nnot betray Tepelenti.\"\n\nAli Pasha gave Dirham a purse of gold for these words, commanding him,\nat the same time, to appear before him in the palace that evening, and\nto bring with him, carefully transplanted into pots, those tulips\nwhich bore the names of Sulaiman and Mukhtar.\n\nDirham could scarcely wait for the evening to come, and the moment he\nappeared in Ali's halls he was admitted into the pasha's presence.\nThen Ali bade every one withdraw from the room, that they twain might\nremain together, and began to talk with him confidentially.\n\n\"I hear that my sons are living in great poverty at Adrianople. As to\ntheir poverty, I say nothing; but, worse still, they are living in\ngreat humiliation also. Nobody will have anything to do with them. The\nwretched Spahis, who once on a time mentioned their names with\nchattering teeth, now mock at them when they meet them in the street,\nand when they go on foot to the bazaar to buy their bread, the women\ncry with a loud voice, 'Are these, then, the heroes at whom Stambul\nused to tremble?' Verily it is shameful, and Ali Pasha blushes\nthereat. I know that if once I ever place in their hands those good\nswords which I bound upon their thighs they would not surrender them\nso readily to the enemies of Ali Pasha. What says Dirham?\"\n\nDirham was only able to express his approval of Ali's words by a very\naudible sigh.\n\n\"Hearken, Dirham! I have known for a long time a secret, which I will\nventure to confide to thee.\"\n\n\"'Twill be as though you buried it under the earth, my master.\"\n\n\"In the Gulf of Durazzo there lies at anchor an English vessel, under\nthe command of Captain Morrison. On that ship I have deposited five\nmillions of piastres in gold--not less than five millions. A large\namount, eh! At any moment I like I can blow the fortress of Janina\ninto the air, embark on board that ship, and sail away to England or\nSpain, and there I can live in a lordly fashion without care, just as\nI please. But to what purpose? My remaining days are but few. Why\nshould I try to save them? Here I must perish. Here, where I have\ngrown great, it becomes me to die, and it is not for me to retreat\nbefore the advancing sword. This money must serve another design of\nmine, which has been in my mind long since, but I seek a man capable\nof executing it.\n\n\"Thou shalt be that man. Falter not. Fate does great things with\nlittle ones. Thou shalt go from Janina and pass through Gaskho Bey's\narmy. When thou dost arrive at Durazzo, show Morrison this ring. When\nhe sees it he will do everything thou sayest to him, for he will know\nthat these are my commands. Thou wilt have the anchor raised and sail\nwith the first favorable wind to Stambul. Sail not into the Golden\nHorn, for it will be more difficult to get out of it again, but cast\nthy anchor hard by Anadoli Hissar. There thou wilt land, and, taking\nwith thee a hundred thousand piastres, thou wilt put them in sacks of\nchaff, the chaff being on the top, and lading sundry asses with the\nsacks, thou wilt take them to Adrianople. There thou wilt seek out my\nsons, and, humbly kissing the hem of their garments, give them to\nunderstand that I have sent thee. Then thou wilt tell them of the\nwarfare waged around Janina, all that thou thyself hast seen and\nheard. If from their faces thou seest that they receive thy words\ncoldly, and show no ardor of soul, then measure out to them the\nhundred thousand piastres, and bid them buy and keep shop therewith,\nstart a large wholesale business if they feel any disposition that\nway, and apply themselves diligently to heap up riches upon riches, as\nit becomes honest men to do who have long years to live. But if thou\nseest their face aflame and the heroes' love of glory sparkle in their\neyes; if they listen to thy words with parted lips and throbbing\nhearts; if they press thy hand warmly and frequently clutch the hilts\nof their swords; if they ask thee to tell them again and again what\nthou hast told them already--then tell them that the path of glory and\nTepelenti's arms are always open before them, that those one hundred\nthousand piastres are only for buying horses and weapons. I have five\ntimes as much on board the English ship, and five hundred times as\nmuch in the red tower of Janina. With the five millions of piastres\nthey must get ships, and these ships they must fully equip in secret.\nAnd this will not be difficult, for all the Greek seamen have deserted\nthe Turkish fleet. These Greeks will offer their services gratis. When\nthe ships are ready, let them, through thee, inform thereof Bublinia,\nthe heroic Greek amazon, who is cruising off Crete with thirty vessels\nto divert the attention of the Turkish fleet, and then row out to\nBeikos. With favorable weather thou shouldst get to Durazzo in ten\ndays. Simultaneously, I from one quarter, Kleon from a second, and\nOdysseus from a third will attack the army of Gaskho Bey, and if my\nsons are victorious at sea, in the evening of the same day we shall be\nable to rest in one another's arms.\"\n\nDirham wept like a child.\n\nThe pasha continued his directions:\n\n\"At every step be cautious. Accomplish everything amidst the greatest\nsecrecy. Don't let my sons scatter their money right and left, lest\ntheir wealth be suspected and give rise to envy and jealousy. It would\nbe better if they left the bulk of it on board ship, and only drew\nfrom it whatever may be necessary for the time being. When thou dost\ncommunicate with Bublinia, write on the parchment all sorts of\ndifferent things higgledy-piggledy. Say, for instance, that thou art\ndisembarking wool in Crete, and will consign it to Argyrocantharides,\nwho is friendly with the Sultan and all the pashas, and, at the same\ntime, an intermediary between us and the Greeks. But in the empty\nspaces between the lines let Mukhtar write the message for Bublinia in\nspecial characters with oil of vitriol; then, when thou dost hand over\nthe documents, moisten these special rows of letters with a piece of\ncitron. But stay, I will give thee a still better counsel. Melt some\nlunar caustic in water, and write therewith thy message on the shell\nof hard-boiled eggs. Then boil the eggs again; and when thou dost\nbreak them open thou wilt find the writing visible on the white\nmembrane inside. Do that. Eggs are the least suspicious of cargoes.\"\n\nDirham made a careful mental note of all that was told him, secretly\namazed that Ali Pasha should have extended his attention to the\nsmallest details.\n\n\"One thing more,\" said Ali, and his voice trembled with emotion. \"I\nknow right well that I am giving my sons dangerous parts to play, and\nthe issue thereof is uncertain. Take, therefore, this ring; the stone\nset in it contains a talisman. Give it to Mukhtar. Let him wear it on\nhis finger, and if ever he finds himself environed by a great danger,\na very great danger--which Allah forfend!--then let him open the stone\nof the ring and read the talisman engraved therein. But this he is\nonly to do if a great danger be at hand, when he trembles for his\nlife, when the lowest slave would not change heads with him; for when\nonce it has been read the talisman loses all its virtue. And now\ndepart, and bethink thee of all I have told thee.\"\n\nDirham kissed the hem of the pasha's garment and promised that he\nwould carefully perform everything. Ali accompanied him down into the\ngarden. On their way back to the place they had to cross the spot\nwhere Zaid was buried. As the hollow earth resounded beneath Ali's\nfeet, he stopped for a moment and murmured to himself, \"H'm! thou\nshalt not be the only one!\"\n\n * * * * *\n\nTwo weeks later Dirham met the sons of Ali in Adrianople. Morrison's\nship had taken him on the way thither, and during the voyage Dirham\nhad countless opportunities of convincing himself that the money\ndeposited by Ali was safely guarded in the hold of the vessel. There\nhe said everything which Ali had confided to him, and as it seemed to\nthe poor servant, through the medium of his tearful eyes, as if the\nbeys grew enthusiastic at the tidings of the war which their aged\nfather was waging, he told them, in this persuasion, that Ali had sent\nthem five million piastres, that they might buy ships and collect arms\nand unite their forces to his.\n\nThe beys rejoiced greatly at the tidings of the five millions, and\nembraced Dirham, who did his best to attribute all the merit of the\ndeed to Tepelenti for sending the money so magnanimously.\n\n\"The old man might have sent us still more,\" said Sulaiman. \"What does\nhe want with it in Janina? Sooner or later it will become the prey of\nhis enemies.\"\n\n\"Pardon me, my lord!\" objected Dirham. \"It will become nobody's prey\nif only you unite with him.\"\n\n\"Ugh!\" said Sulaiman; and at that moment the two brothers caught each\nother's eye, and it was as though the same thought suddenly occurred\nto them both.\n\nWhen Dirham delivered the ring to Mukhtar, the latter asked,\nsuspiciously:\n\n\"Is there any poison in this ring?\"\n\n\"What are you thinking of, my lord? I wore it on my finger the whole\nway hither. There is a talisman in it.\"\n\nAt this both the brothers burst out laughing. They had often ridiculed\nAli for his absurd superstition. Nevertheless, Mukhtar kept the ring,\nfor there was a splendid emerald in it.\n\nBut the secret of the eggs completely won the favor of the brothers.\nThat was really a capital idea of Ali's. In this way the pashas could\nsend secret messages even in their harems. Who would ever suspect an\negg? They would put it to the proof at once. They would send a\ndeclaration of love to the odalisks of the Seraskier, written in an\negg.\n\nDirham shook his head and spoke seriously, and entreated the beys to\nfirst of all enter into a league with Bublinia, the amazon of Chios,\nwho was even bold enough on occasions to make a dash at the\nDardanelles; for if they did not hasten, the money that had been sent\nto them would be of no use. It would be dangerous, he urged, to show\nthe people of Adrianople that they had received money. The English\ncaptain, moreover, was not disposed to render any other service than\nthat of keeping safe custody of the money confided to him; but if any\nharm happened to them because of it, he would neither defend them nor\neven convey them out of Turkish waters.\n\nThese wise remonstrances made some impression upon the beys. Just as\nif their thoughts were pursuing the same course, they both hastened to\nbeg Dirham to let them have at once the eggs, the lunar caustic,\nwriting materials, and all other indispensable things. Moreover, they\nforgot to give him money for these purchases, so the poor fellow had\nto buy them out of his own purse.\n\nDirham's foot was scarcely out of the house when the two brothers\nlooked at each other and smiled.\n\n\"I have a good idea,\" began Sulaiman.\n\n\"And I also,\" said the other.\n\n\"I don't mean to return to Ali.\"\n\n\"Nor I. I bear in mind what happened to Zaid.\"\n\n\"I propose we buy a ship, on which we may hide our money.\"\n\n\"And we'll man her with a Greek crew.\"\n\n\"Then we will send Dirham with the messages written in the eggs to\nBublinia, and we'll write great things therein. We'll tell her that we\nstand ready here with our fleets, and if she will attack the Kapudan\nPasha in front we will attack him in the rear. The woman is mad. She\nwill come forth from the Archipelago and fall upon the Turkish fleet.\nThen the Kapudan Pasha will assemble his forces against her, and she\nwill engage all his attention till we have nicely set sail, nor will\nwe stop till we reach Cadiz.\"\n\n\"Admirable! for that is the land of good wine and fair women.\"\n\n\"And then Ali Pasha may wait for us till the angel Izrafil blows his\ntrumpet on the last day!\"\n\n\"And Bublinia as well--not forgetting the Sultan! Let them worry each\nother.\"\n\n\"Mashallah! Life is sweet!\"\n\nAnd so it chanced that the sons of Ali, like the princes in a fairy\ntale, suddenly and marvellously came into the possession of great\nriches, and were wise enough to profit by these riches in the merriest\nmanner in the world. The money was given to them for blood and\nweapons. They were going to lavish it on love and wine. And is not\nlife lovelier so?\n\nWhen Dirham came back they immediately boiled the eggs hard, and wrote\nupon them every sort of magnificent message that occurred to their\nminds. They promised to hasten to the assistance of the Greeks, both\nby land and by sea; to cut their way through the fleets with their\nfire-ships and blow the Turkish flag-ship into the air; to incite the\nJanissaries to rise against the Sultan and the Greeks to rise against\nthe Janissaries; in all of which there was not a single word of truth.\nOnly worthy Dirham believed these things, and trembled in body and\nsoul at the bare thought of the sublime deeds that his masters had\ndetermined to perform.\n\nHe himself hired a barge, loaded it with wool, and, hiding the eggs\nfull of secrets in a basket, set out for the Archipelago.\n\nThe good youths meanwhile laughed to their hearts' content. They\nlaughed at worthy Dirham; they laughed at the worthy Bublinia, and at\nthe wise Kapudan Pasha; they laughed at this amusing piece of good\nfortune which brought them riches in heaps. But at nobody did they\nlaugh so much as at old Tepelenti, who was believing all along that\nhis sons were collecting war-ships for him.\n\nBut did he really believe it?\n\nOn the same day that Dirham quitted Adrianople, a fakir of the\nNimetullahita Order penetrated into the Seraglio and demanded an\naudience of the Sultan. It was the self-same old soothsayer who had\nexhibited his enchantments to Ali.\n\nOn being admitted to the presence of Mahmoud, he stood audaciously\nupright before him, bending his head no lower than it was already\ncrooked by the weight of years.\n\n\"Allah hath sent me to thee,\" said the dervish, in a deep, hollow\nvoice, which had lost all its sonorousness. \"A great danger is\napproaching thee. The storm hanging over thy head is at this moment\ncompressed within the skin of an egg, and thou couldst crush it in the\npalm of thy hand; but if thou dost suffer it to come forth from the\negg, thy whole realm will not be sufficient to contain it. This,\ntherefore, is the word of Allah unto thee: This day and this night,\nand to-morrow and to-morrow night, stop every vessel which sails up\nthe narrow waters of the Golden Horn and search them, and whenever thy\nguards come upon an egg, let them seize it and bring it to thee; for\namongst them are diverse cockatrice eggs which, if once they be\nhatched, will swallow up both thee and thy realm.\"\n\nHaving said these words, the dervish turned him about, and without so\nmuch as saluting the Padishah, without even taking off his slippers\nbefore him, he withdrew, not even asking for a reward.\n\nThe Sultan was profoundly impressed by this audacity. He immediately\nsent orders to the wardens of the two watch-towers at the entrance of\nthe Golden Horn to board and search thoroughly every vessel that\npassed between them, seize every egg they found on board and bring\nthem to him, at the same time detaining all the crews of such vessels.\n\nFate so willed it that Dirham's was the first vessel that fell into\nthe hands of the searchers.\n\nWhen the unfortunate servant perceived that the guards seized the\neggs, he leaped into the sea, and although he was a good swimmer, he\nallowed himself to be suffocated in the water lest he should be\ncompelled to betray his masters.\n\nThe eggs they carried to the Sultan, and when he had opened them and\nhad read the writing written on their inner skins, he was horrified.\nTreachery and rebellion! The conspiracy was spreading from one end of\nthe empire to the other. The complicated intrigue, one of whose\nthreads was in Janina and the other in the islands of the Archipelago,\nhad its third in the very capital. This called for terrible reprisals.\n\nThe beys were seized the same night in the midst of their joys, and\ndragged from the paradise of their hopes to be thrown into a dungeon.\n\nWho could have betrayed the secret of the eggs? they asked themselves.\nWhy, who else but Tepelenti?\n\nFools! to fancy that they could make a fool of Tepelenti!\n\nSulaiman fainted when they informed him that the secret of the eggs\nwas discovered. Mukhtar felt that the moment had come of which Ali had\nsaid that the lowest slave would not then exchange heads with his two\nsons, and in that hour of peril he bethought him of the talismanic\nring which had been sent to him. Hastily he removed the emerald,\nbelieving that at least a quickly operative poison was contained\ntherein, by which he might be saved from a shameful death. There was,\nhowever, no poison inside the ring, but these words were engraved\nthereon, \"Ye have fallen into the hands of Ali!\"\n\nMukhtar dropped the ring; he was annihilated.\n\nThe hand of Ali, that implacable hand which reached from one end of\nthe world to the other, which clutched at him even out of the tomb--he\nnow felt all its weight upon his head.\n\nDie he must, and his brother also.\n\nThe Reis-Effendi examined them, and both of them doggedly denied all\nknowledge of what was written on the eggs. But there was one thing\nthey could not deny--the five million piastres on the English ship;\nthis was the most damaging piece of evidence against them, and proved\nto be their ruin.\n\nThe Sultan demanded from Morrison the money of the beys, and Morrison\nhimself appeared before the Reis-Effendi to defend his consignment,\nwhich he maintained he was only bound to deliver to its lawful owner.\n\nThe Reis-Effendi replied that in the Ottoman Empire there was only one\nlawful owner of every sort of property, and that was the Sultan. The\nproperty of every deceased person fell to the Grand Signior, and\nnobody could make a will without his permission.\n\nMorrison objected, very pertinently, that as the beys were not\ndeceased the Sultan could scarcely be looked upon as their heir.\n\nInstead of making any answer, the Reis-Effendi sent out his officers\nwith a little piece of parchment which he had previously subscribed,\nand a few moments later the severed heads of the beys stood in front\nof Morrison on a silver trencher.\n\n\"If their not being dead was the sole impediment,\" remarked the\nMinister of Foreign Affairs, \"you perceive that it has now been\nremoved.\"\n\nMorrison thereupon handed over all the gold and silver in his\npossession as rapidly as possible, and quitted Constantinople that\nvery hour; he had no great love of a place where every word cost the\nlife of a man.\n\nBut the heads of the beys were stuck on the gates of the Seraglio for\nthree days and three nights in the sight of all the people, and\nmounted heralds proclaimed, at intervals of an hour, \"Behold the heads\nof the sons of the rebellious Ali Tepelenti, who would have devastated\nStambul!\"\n\nAnd the people loaded the heads with curses each time the proclamation\nwas made.\n\n * * * * *\n\nA few days later the news reached Janina that Sulaiman Bey and Mukhtar\nBey had been beheaded at Stambul.\n\nAli Pasha thrice bowed his face to the ground and gave thanks to Allah\nfor His mercies. And he caused to be proclaimed on the ramparts,\namidst a flourish of trumpets, that his sons, the treacherous beys,\nhad been decapitated at Stambul. Such is the reward of traitors!\n\nAfter that, for three days and three nights--just as long a time as\nthe heads of the beys had been exposed on the gates of the Seraglio--a\nbanquet, with music and dancing, was given in the fortress of Janina,\nand every morning a hundred and one volleys were fired from the\nbastions--the usual ceremony after great triumphs.\n\nAnd when in the evening Ali took a promenade in his garden, and walked\nup and down among his flowers, he would now and then trample the earth\nbeneath his feet. It was the grave of Zaid that he was trampling upon.\nThere stood an old dahlia, the sole survivor of its extirpated family,\nand, levelling it to the ground with his foot, he trod it into the\ngrave, murmuring to himself, \"No longer art thou alone--no longer\nalone!\"\n\n\n\n\nCHAPTER XI\n\nTHE FLOWERS OF THE GARDEN OF BEGTASH\n\n\nAt the end of the fifteenth century, when the Turkish crescent had won\nan abiding-place among the constellations of Europe, there dwelt in\nthe Turkish dominions a worthy dervish, Haji Begtash by name.\n\nAs the overflowing armies of the newly founded empire submerged the\nsurrounding Christian kingdoms, Haji Begtash went everywhere with the\nconquering hosts, but in the intervals of peace he begged his way\nabout the empire, and scraped together a little money from the Turkish\ngrandees or from the extravagant, booty-laden Turkish soldiers.\n\nNow wherefore did this worthy dervish make it a point to collect so\nmuch money and wear himself out by travelling from the Adriatic to the\nEuxine, when he might have sat all day long at the gate of the Kaaba,\nas they call the stone on the tomb of the Prophet, and recited from\nhis long bead-string the nine properties of Allah (no very exhausting\nlabor, by-the-way), and received therefor, from the pilgrims to the\nshrine, meat, drink, and abundance of alms?\n\nWell, Haji Begtash had taken up a great work. When he accompanied the\nTurkish armies, and they, on entering a Christian village, began to\ncut down the inhabitants and tie the captives together with ropes,\nthe dervish would force his way through the bloodthirsty soldiery, and\nif he beheld any wild Bashkir or Kurdish desperado about to dash out\nthe brains of a forsaken, weeping orphan child against a wall, he\nwould lay his hand upon them, take away the child, cover it with his\nmantle, caress it, and take it away with him. And thus he would keep\non doing till he had with him a whole group of children, all of whom\nwere concealed beneath the folds of his ample cloak, where nobody\ncould hurt them; nay, frequently he would carry babies in\nswaddling-clothes in his bosom, till people began to wonder what on\nearth he meant to do with them.\n\nSubsequently he announced that any captive who brought him his\nchildren should receive a silver denarius per head for each one of\nthem. This was not much, it is true; but then there was little demand\nfor children. In the slave-market only the adult human animal had its\nprice-current. And so it came about that innumerable children were\nbrought to the worthy dervish.\n\nHe took them away with him to a mosque at Adrianople. Folks laughed at\nhim, and asked him mockingly if he was going to plant a garden with\nthem.\n\nHaji Begtash accepted the jest in real earnest, and called his\nchildren the flowers of Begtash's garden; and this name they preserved\nin the coming centuries.\n\nThese saplings (amongst them were some of the loveliest little\ncreatures of six and seven years of age) were brought up by the\nindefatigable Haji year after year. He instructed them in the Kuran;\nhe told them everything concerning the innumerable and ineffable joys\nwhich the Prophet promises to those who fall in the defence of the\ntrue Faith; and at the same time accustomed them to endure all the\nhardships and privations of this earthly life.\n\nMost of these children had never known father or mother, and those who\nhad quickly forgot all about them as they grew up. No love of home or\nkindred bound them to this world, and therefore they were all the more\nattached to one another. Their comrades were the only beings they\nlearned to love, and every one of them treated old Begtash as a\nfather. His words were sacred to them.\n\nTheir days were passed in hard work, in perpetual martial exercises,\nfighting, and swimming. A youth of twelve among them was capable of\ncoping with full-grown men elsewhere, and each one of them at maturity\nwas a veritable Samson.\n\nIn those days the Ottoman armies suffered many defeats from the\nChristian arms. Their strength lay for the most part in their cavalry,\nbut their innumerable infantry was a mere mob, two of their\nfoot-soldiers not being equal to one of the well-disciplined European\nmen-at-arms who advanced irresistibly against them in huge compact\nmasses; and they were of no use at all in sieges, except to fill up\nthe ditches and trenches with their dead bodies, and thus make a road\nfor the more valiant warriors that came after them.\n\nAnd now, as if by magic, a little band of infantry suddenly appeared\non the theatre of the war. These new soldiers were dressed quite\ndifferently from the others. On their heads they wore a high hat\nbulging outward in front, with a black, floating cock's plume on the\ntop of it; their dolmans were of embroidered blue cloth; their hose\nonly reached down to their knees, below that the whole leg was bare;\ntheir only weapon was a short, broad, roundish sword, in marked\ncontrast to the other Turkish soldiers, who loaded themselves with as\nmany weapons as if they were going to fight with ten hands.\n\nNone recognized the youths--and youths they all were. They did not\nmingle with the other squadrons, nor place themselves under any\ncaptain, nor did they ask for pay from any one.\n\nBut in the very first engagement they showed what they were made of. A\nfortress had to be besieged which was defended in front by a broad\nstream of water. The strange youths clinched their broad swords\nbetween their teeth, swam across the water, scaled the bastions amidst\nfire and flames, and planted the first horse-tail crescent on the\ntower.\n\nThese were the flowers of Begtash's garden.\n\nThe first battle established the fame of the youthful band that had\nbeen brought up by the old dervish, and by the time the second\ncampaign began, Haji Begtash was already the chief of innumerable\nmonasteries whose inmates were called the Brethren of the Order of\nBegtash. Consisting, as they did, of captive Christian children, and\nstanding under the immediate command of the Sultan, they composed a\nnew army of infantry, the fame of whose valor filled the whole world.\n\nThese were the \"jeni-cheri\" (new soldiers), which name was\nsubsequently altered into Janichary or Janissary. But for long ages to\ncome, if any Janissary warrior had a mind to speak haughtily, he would\ncall himself \"a flower from Begtash's garden.\"\n\nMany a glorious name bloomed in this garden in the course of the ages.\nThe power of the Sultan rested on their shoulders, and if they shook\nthe Sultan from off their shoulders, down he had to go.\n\nIf they were powerful servants, they were also powerful tyrants. Their\nvalor often reaped a harvest of victories, but their obstinacy again\nand again imperilled their triumphs. With the increase of their power\ntheir self-assurance increased likewise. It was not so much the\nSultans and Viziers who commanded them as they who commanded the\nSultans and Viziers. And if the rebellious Janissaries hoisted on the\nAtmeidan a kettle, the signal of revolt, it was always with fear and\ntrembling that the Seraglio asked them what were their demands; and\nthe whole Divan breathed more freely when the answer came that it was\ngold they wanted, and not blood--the blood of their officers. And\nwhen, after the great Feast of Bairam, there was the usual\ndistribution of pilaf, and the dangerous kettles were filled full with\nthis savory mess of rice and sheep's flesh, the Sultan, all trembling,\nwould anxiously watch to see how the majestic Janissaries partook of\ntheir pottage. If they devoured it voraciously, that was a sign of\ntheir satisfaction; but if they only touched it in a finiking sort of\nway, then the Sultan would fly into the Seraglio, and lock himself up\namong the damsels of the harem, for it was now certain that their\nlordships the Janissaries were displeased, and it was well if their\ndispleasure only expressed itself by reducing a whole quarter or so of\nthe city to ashes.\n\nTwo Sultans had tried to break in two this dangerous double-edged\nweapon, which inflicted as many wounds in the heart of the realm as\never it dealt outside; but the Janissaries' magic influence was so\ninterwoven with, so ingrafted in, the mind of the nation that public\nfeeling was on their side, and both rulers perished in the bold\nattempt. They dragged Sultan Osman forth from the Seraglio, and set\nhim on the back of an ass with his face to its tail, carried him in\nderision from one end of the town to the other, and then flung him\ninto the fatal Seven Towers, where the Turkish rulers and their\nrelatives are wont to be buried alive and die forgotten. Mahmoud II.'s\nfather, Selim, on the other hand, expired beneath the sword-thrusts of\nthe rebels, and those swords were still sharp and those hands were\nstill strong when the son of the man whom they had slain sat on the\nthrone, and under no other Sultan did the throne tremble so much as\nunder him.\n\nIn these days the mighty corps of the Janissaries lived only to commit\ncrimes or gigantic mistakes; its ancient glory was not renewed. During\nthe last century their arms had constantly been shattered whenever\nthey came into collision with the progressive military science of\nEurope. In the course of the ages the flowers in Begtash's garden had\nsadly faded. The flowery petals of their glory had fallen from them,\nand only the thorns remained; and even these were no longer the thorns\nof the brave thick-set hedge which defends the borders of the garden\nagainst would-be invaders, but the stings of the nettle which hurts\nthe hand of the gardener as he hoes.\n\nNeither life nor property was any longer safe from them. The Sultan\nhimself, when he sat upon the throne, was in the most dangerous place\nof all, and the Viziers--the chief officials of the realm--trembled\nevery day for their lives. The turbulence of the Janissaries was a\nperpetually recurring disease running through all the arteries of the\nrealm, and covering the once mighty empire with poisonous ulcers.\n\nThese seditious outbreaks occurred even during the deliberations of\nthe Divan, and fear on such occasions was a more urgent counsellor\nthan conviction to the palace magnates who sat in the cupolaed\nchamber.\n\nThe threats of the Janissaries had compelled Mahmoud to take up arms\nagainst Ali Pasha; and now, when Ali had kindled the flames of war all\nover the empire, and the Sultan bade the Janissaries hasten against\nthe enemy and subdue him, they replied that they would not fight\nunless the Sultan led them in person.\n\nInstead of that, they waged war within the very walls of Stambul, for\nwhenever the news of a defeat reached the capital, the Janissaries\nwould fall upon the defenceless Greeks and massacre them by thousands.\n\nFrom distant Asia, from the most savage parts of the empire, Begtash's\npriests appeared and proclaimed in the mosques death and destruction\non the heads of all the Greeks. It was they who, with torches in their\nhands, headed the rush of the fanatical Janissaries against Buyukdere,\nPera, and Galata, the quarters of the city where the Greeks resided,\nand every day they thundered with their bludgeons at the gates of the\nSeraglio, demanding ever more and more sentences of death against the\nGreek captives who were shut up in the Seven Towers. The Sultan's\nofficials, trembling with fear, wrote out the sentences demanded of\nthem, and the victims fell in hundreds; and when the Russian\nambassador, Stroganov, protested against this butchery, the\nJanissaries attacked his palace and riddled all the doors and windows\nwith bullets, which was the subsequent pretext for the long war which\nshook the empire to its base, though the Janissaries never lived to\nfeel it.\n\nMahmoud watched from the summit of the imperial palace the devastation\nof Stambul and the devastation of his empire, and he saw no help\nanywhere. He saw nothing but the melancholy examples of his ancestors\nand the disappearance of his dominions; and as he stroked the head of\nhis first-born, Abdul Mejid, a child of nine, he thought to himself,\n\"This lad will not sit on the throne, he will not be a ruler as his\nforefathers were; he will not dictate laws to half the world like the\nother descendants of Omar; but he will be a fugitive on the face of\nthe earth, the slave of strange people, as was the fugitive Dzhem,\nwhom they cast forth ages ago.\"\n\nHow miserable was the life of the Sultan! What avails it though an\nearthly paradise be open to him if life itself be closed against him?\nWhat avails it to be a god if he cannot be a man? The Sultan never\nknows what it is to have relatives. Very early, while they are still\nchildren, the latest born are shut up in the Seven Towers. The\nfirst-born son can never meet them, unless it be on the steps of the\nthrone, when the rebellious Janissaries drag one of them from his\ndungeon to raise him to the throne, and lock up the first-born in his\nstead. The Sultan cannot be said to possess a wife; all that he has\nare favorite concubines, in hundreds, in thousands, as many as he\nchooses to have, and there is no difference between them except\ndifferences of feminine loveliness and the blind chance which blesses\nsome of them with children. And he makes no more account of one than\nhe does of another. Not one of them feels it her duty to love her\nhusband; it is enough if she be the slave of his desires. If the\nPadishah be troubled or sorrowful, there is none about him to whom he\ncan open his heart. He may go from one end of the harem to the other,\nlike one who wanders through a conservatory whose flowers are all so\nbeautiful, so radiantly smiling; but in vain will he tell them of his\ngrief and trouble, for they do not understand him, they do not trouble\ntheir heads about his thoughts; and if, perchance, he tells them that\nfrom all four corners of the world mighty foes are marching against\nStambul, here and there, perchance, he may hear a sigh of longing from\nsome captive maiden, who cannot conceal her secret joy at the thought\nof the happy hour when the hand of deliverance will thunder at the\nharem door and break its bolts and give freedom, beautiful sunbright\nfreedom, to the captives.\n\nIt is slavish obsequiousness and nothing else which bends its knee\nbefore the Padishah; it is fear, not love, which obeys him. And to\nwhom shall he turn when his heart is held fast in the iron grip of\nthat numbing sensation which makes the mightiest feel they are but\nmen--fear?\n\nMahmoud's sole joy was his nine-year-old son. The child was brought\nup by his grandmother, the Sultana Valideh, herself scarce forty years\nof age. This dowager Sultana had civilized, European tastes. She had\nbeen educated in France; the young prince was passionately attached to\nher and she inspired him with all those desires and noble instincts\nunder whose influence, thirty years later, new life was to be poured\ninto the decrepit Turkish Empire.\n\nThe Sultana Valideh wished to so educate her grandson that one day he\nmight occupy a worthy position among the other rulers of Europe. She\nsowed betimes in his heart the seeds of high principles and\nenlightened tastes, and the Sultan would frequently listen to the wise\nsentences of his little lad, and, while rocking him on his knee, with\na smile upon his face, his heart would beat in an agony of fear, \"What\nif anybody got word of this?\"\n\nFor the old Turkish party lay in wait for every word that fell from\nthe Sultan's mouth, and the pointing of the little finger of one of\nBegtash's fakirs was more to be feared than the armed hand of the most\nvaliant of the Greek heroes. If any one of the Ulemas should chance to\ndiscover that the young heir to the throne listened to any other\nbookish lore than what was contained within the covers of the Kuran,\nwhich comprised within itself (so they taught) all the wisdom of the\nworld, they were capable of hounding on the Janissaries against the\nSeraglio, and slaying both sovereign and child.\n\nThe recollection of Achmed Sidi was still fresh in the memory of men.\nSidi had been one of the Chief Ulemas, and the Imam of the Mosque of\nSophia; and when, a few years ago, the warriors and the diplomatists\nof the Tsaritsa Catherine had won victory after victory over the\nOttomans, not only on every battle-field, but also in every political\narena, the unfortunate imam advised the Divan that, in view of the\nindisputable superiority of the Christians, it was necessary to teach\nthe Turkish diplomatists the Bible, the inference being that just as\nthe Moslem sages derived all their military science and all their\nadministrative wisdom from the Kuran, so also the Christians must\nneeds learn all these things from their Bible, thereby tacitly\nacknowledging the capacity of the Christians for appropriating all\nknowledge. But the well-meaning Ulema paid dearly for this good\ncounsel. They banished him to the Isle of Chios, and there, for a very\ntrivial offence, he was first degraded from his office (for it is not\nlawful to kill a Ulema with weapons), and then handed over to the\npasha of the place, who pounded him to death in a stone mortar--a\ndeterrent example for future reformers. Let them beware, therefore, of\nmoving a single stone in the ancient fabric of the Ottoman\nconstitution!\n\n\n\n\nCHAPTER XII\n\nTHE SHIPWRECK OF LEONIDAS\n\n\nNow, one fine day, when the worthy Leonidas Argyrocantharides set out\nfrom Smyrna on one of his prettiest ships, a vexatious little accident\nbefell him by the way. The ship, which had taken in a cargo of tanned\nhides at Stambul, was overtaken, _en route_, by a tempest which drove\nher upon the coast of Seleucia. There, in the darkness of the night,\nshe was thrown upon a sand-bank, from which she was unable to\nextricate herself till morning; and it was only when the land became\nvisible in the early light of dawn that the merchant began to realize\nthe awkward position into which his ship had got, despite Saint\nProcopius and Saint Demetrius, who were very beautifully painted on\nboth sides of her prow. The vessel had heeled over on one side, and\nthat side of her which lay above the waves was threatened every moment\nwith destruction by the onset of the foaming surf which broke from\ntime to time over the deck, making a pretty havoc of the masts and\nspars. The joints of the ship's timbers began to be loosened, creaking\nand shivering at each fresh shock of the waves. And if the fate of the\nship on the sand-bank was sad enough, still sadder would it have been\nif she had broken loose therefrom; for right in front of her lay the\nrocks of the Seleucian coast, whose steep crags were lashed so\nfuriously by the raging sea that the crashing waves leaped fully a\nhundred fathoms up their sides. A nice place this would have been for\nany ship to play pitch-and-toss in!\n\nThe worthy merchant sorely lamented his fate, sorely lamented, also,\nhis fine ship, which was painted in elaborate patterns with all the\ncolors of the rainbow. He lamented his many beautiful goat-skins, not\na single bundle of which he would allow to be cast into the sea for\nthe purpose of lightening the ship; rather let them all go to the\nbottom together! He mourned over himself, too, condemned at the\nbeginning of the best years of his life to be suffocated in the sea;\nbut what he lamented far more than ship, goat-skins, or even life\nitself, were the two Circassian children, the precious, beautiful boy\nand girl, Thomar and Milieva, who were worth, at the current market\nprices of the day, ten thousand ducats apiece; Leonidas would have\ngiven his own skin for them any day!\n\nFull of great hopes, he had embarked the two children at Stambul (the\ntanned hides were only a secondary consideration); and lo! now, just\nwhen he was reaching his goal, the curse of Kasi Mollah overtook him.\n\nTwo long-boats fully manned had made an attempt to reach the shore, in\norder that they might from thence haul the ship off the sand-bank, and\nboth boats had been seized before his very eyes by the breakers, and\ndashed to pieces against the steep rocks; so there was nothing for it\nbut to remain behind and perish on the sand-bank.\n\nOne wave after another drove the hulk deeper and deeper down; those\nwho still remained aboard wrung their hands and prayed or cursed,\naccording as temperament or habit urged them.\n\nAs for Leonidas, he did both--he prayed and cursed at the same time;\nfor it seemed quite clear to him that praying or cursing separately\nwas of not the slightest use. The two children, meanwhile, holding\neach other tightly embraced, sat beside the broken stump of the mast\nand seemed to mock at the terrible tempest.\n\nNot a sign of fear was visible on their faces. This roaring wind,\nthese foam-churning waves, seemed to afford them a pleasant pastime.\nThe black-and-white storm-birds sitting on the towering billows were\nswimming there all round the doomed ship, merrily flapping the water\nwith their wings. Oh, those sea-swallows were having a fine time of\nit!\n\nThe two children had agreed between themselves, some time before, that\nif the ship went down, they would fling themselves into the water and\nswim ashore. That would be a mere trifle to them, of course.\n\nFull of despair, the merchant rushed towards them, and embracing them\nwith both his arms, he exclaimed, looking bitterly at the sky,\n\"Merciful Heaven! ten thousand ducats!\"\n\nThe children fancied that terror had made the merchant mad, and they\ntried to comfort him with kind words:\n\n\"Don't distress yourself, dear foster-father; we will not perish here,\nand we will not leave you to perish either. As soon as the ship goes\ndown, we'll swim for the shore. We both of us know very well how to\ncleave the waves with our strong arms, and we will fasten you to our\ngirdles and save you along with ourselves.\"\n\nThe merchant kissed the two dear children, and embraced them tenderly.\nAn hour later the last planks of the fine ship broke away from each\nother, and the shipwrecked crew clung desperately to the floating\nspars that the waves tossed hither and thither. The greater part of\nthe ship's company was ingulfed forthwith by the waves or dashed to\npieces against the hard rocks; only three persons were saved--the\nmerchant and the two children.\n\nLeonidas, fast tied to their girdles, allowed himself to be cast among\nthe waters. The first who rose on the crest of the foaming waves was\nThomar. He perceived the rock on which a huge mountain of surf,\nrushing after him, threatened to dash him to pieces, and, watching his\nopportunity, grasped the long dangling roots of a tree which grew out\nof a cleft of the rocks and, with a tremendous effort, dragged all\nthree of them up to it. The wave rolled right over them, burying them\nfor an instant in deep water; but the next moment the surge rolled\nback again, and they were on the rocky coast.\n\nThe merchant was more dead than alive, so the children had to drag him\nwith them for a long way inland, lest the returning surge should carry\nthem back to sea again. They only ventured to rest when they had\nreached a rocky cavity where they could feel sure that they were safe.\nEven here the water, which shot up as high as a tower against the\nopposing rock, covered them every moment; but they did not feel its\nweight.\n\nThere they had to remain, crouching closely together, till the\nevening. Neither in front nor behind was there any place of refuge,\nand it was with a feeling of envy that they looked down upon the\nstormy petrels which towards evening began to sit down in long rows on\nthe edge of the rocks, whither it was impossible for them to follow.\n\nGradually, however, the storm died away, the sea subsided and grew\nsmooth, and the place where the shipwrecked group had taken refuge\nrose three ells above the surface of the water. Then they could\nventure to look around them. The whole shore was strewn with pieces of\ntimber and mangled corpses. Wreckage and dead bodies were all that the\nsea had vomited forth of the rich cargo of the fine ship.\n\nBut the merchant did not despair. Making the two children kneel down\nbeside him, he knelt down in their midst, and made them pray a prayer\nof gratitude to Heaven for their marvellous deliverance; and then,\npressing them to his bosom, he sobbed, with the tears in his eyes,\n\"What do I care, though my ship is lost and all my wares are\nsubmerged, so long as ye remain to me, my precious offspring? That is\nquite consolation enough for me.\"\n\nAnd the worthy merchant told the truth, for as soon as ever he could\nreach Stambul he was sure of getting for these two children enough to\nenable him to buy two ships and twice as many wares as he had lost at\nthe bottom of the sea.\n\nBut now the most difficult question arose--How were they to get away\nfrom that spot to any place inhabited by man? All ships gave this\ndangerous coast a wide berth; there was nothing to tempt them to the\nspot. Even fishermen did not venture as far in their barks, so that\nthe unfortunate refugees who had escaped the waters saw starvation\napproaching them.\n\nBut suddenly, while they were meditating over the misery of their\nposition, they fancied they heard human voices a little distance\noff--deep, manly voices, apparently engaged in a lively dispute.\n\nThe two children rejoiced, thinking that good men were hard by; but\nthe merchant trembled, for, thought he, \"What if they be robbers?\"\n\nThomar now bade his sister remain with Leonidas while he went in the\ndirection of the voices to discover who the speakers might be. The\nbrave boy clambered from one cliff to another, made the circuit of the\nrock-chamber behind which they were sitting, and when he came to the\nopposite side of it a spacious empty cavern yawned blackly in front of\nhim, half covered by whortleberry bushes. Probably the conversation\ncame from thence, but neither near nor far was a human creature to be\nseen, nor were there any footprints of men on the ground; the front of\nthe cavern was covered with thick green moss, on which footprints left\nno trace. Thomar shouted into the cave, and as not a word came back,\nhe boldly entered, and slowly advanced forward. He went on and on as\nfar as the light of the outside world extended, and then, as no one\nreplied to his loud challenges, turned back again by the way he had\ncome, and, making the circuit of the rock again, told the merchant\nthat he had not come upon any human beings, but had only found a\ncavern which, at any rate, would make them good night quarters.\n\nThe conversation they thought they had heard must have been a\ndelusion. Then they helped one another along the rocks and arrived at\nthe mouth of the cavern.\n\nMilieva had scarcely cast a glance into it when she exclaimed, full of\njoy: \"Look, Thomar, here are two chests among the bushes!\" And,\nindeed, there were two boxes made of boards, and Thomar wondered that\nhe had not noticed them before. No doubt the sea had cast them up\nthither out of some ship that had been wrecked there before.\n\nOne of the boxes resembled those chests in which sailors keep their\nbiscuits, but the shape of the other suggested that it was one of\nthose hermetically sealed vessels used for holding good wines. Why\nshould they not turn them to some account?\n\nThey were not long in forcing them open, and what was their\nastonishment when they perceived that the biscuits in the first box\nwere not even mouldy, but quite dry and sound, as if they had only\nbeen brought thither quite recently; while in the second box not one\nof the scores of flasks there displayed was broken or cracked, but lay\nneatly stored away in layers of straw?\n\nThe refugees did not greatly concern themselves with the question, Who\nput these boxes here? and why? Nobody who, after being tossed about on\nthe sea for three days with nothing to eat or drink all the time, and\nis then unexpectedly confronted with rich stores of bread and\nwine--nobody, I am sure, under such circumstances would think of\nconsulting the Kuran as to whether a conscientious Mussulman should\neat and drink such things, but would fall to at once, and thank Allah\nfor the chance.\n\nThe children forgot, in the twinkling of an eye, the dangers to which\nthey had been exposed, and, after the first glass or two of wine,\novercome by fatigue, lay down on the soft bed which Nature had made\nready for them with her most fragrant moss. Leonidas, however,\nremained sitting where he was, considering it his bounden duty to\ntaste all the wines which were here offered to him gratis, one after\nthe other; in consequence whereof, when he _did_ lie down at last, he\nchose a position in which his head was very low down while his feet\nwere high in the air, and so they all three slumbered peacefully\ntogether.\n\nThen the voices of men were heard once more far off in the cavern, and\nnot long afterwards there emerged from its black mouth six\ngray-haired, pale-faced human beings. He who came first was the\neldest. His white beard reached to his girdle, his mouth was hidden by\nhis mustache, and his eyes were covered by his white eyebrows.\n\nThese men were fakirs of the Omarite Order, whose rule obliges them to\nendure the most terrible of all renunciations--abstention from all\nenjoyment of the light of day. Plunging themselves into eternal\ndarkness for the glory of Allah, they make of life a long midnight,\nand the sun never beholds them on the face of the earth.\n\nThe night was well advanced when the six Omarites came forth to the\nsleepers, and while five of the fakirs stood round them in silence,\nthe sixth--the one with the long flowing beard--bent over the\nchildren and examined their features attentively in the darkness of\nthe night, which was only mitigated by the light of a few faint stars\nhalf hidden among errant clouds. At last he whispered to his comrades,\n\"It is they.\" Then, turning the tips of his thumbs downwards, he laid\nthem softly on Thomar's head. All five fakirs listened with rapt\nattention. The bosom of the sleeping lad began to heave tumultuously;\nhe clinched his fists; his face grew hot; his lips swelled. The old\nman then seemed to breathe upon his forehead, as if he would whisper\nsomething, whereupon the sleeping lad exclaimed, in a strong, audible\nvoice, \"With swords, with guns, with arms!\"\n\nThe old men shook their heads, showing thereby that they approved of\nhis words.\n\nThen the eldest old man bent over the other child and made passes over\nher face with his five fingers. The maiden's bosom expanded visibly,\nand when the old man stooped over and breathed upon her she cried out\nin an energetic, dictatorial manner, \"Down on your knees before me!\"\n\nAt this the Omarites all whispered together, and two of them lifting\nthe lad, two the girl, and two the merchant, they carried them on\ntheir shoulders into the depths of the cavern.\n\nThe mouth of this cavern was the already mentioned tunnel whose\nfarthest exit debouched upon the valley of Seleucia, half a league\nfrom the sea--that waste, barren, and savage valley.\n\nThe Omarites moved to and fro in the black cave without a torch, like\nthe blind, who do not go astray in the turnings and windings of the\nstreets, although they see them not. The sleepers had drunk a magic\npotion, which did not permit them to awake for some time, and the men\ncarried them on their shoulders to the opposite entrance of the cavern\nand there laid them down on the moss, in a place where the sunlight\nwas wont to penetrate.\n\nIt was already late in the day when the two children awoke. As soon as\nthey had opened their eyes, their first care was to kiss and embrace\neach other. Then they aroused the merchant also and, rubbing sleep out\nof their eyes, began to tell him, in childish fashion, what they had\nbeen dreaming about.\n\n\"Ah! what a lovely dream I had!\" cried Thomar, and even now his eyes\nsparkled. \"I was standing beside the Sultan, who was leaning on my\nshoulder. Before me and around me howled a rebellious multitude, and\nthe Sultan was pale and sad. Turning towards me he sighed, 'Wherewith\nshall I appease this raging sea?' For a long time I could find no\nanswer. It was as if something were weighing me down, something as\nheavy as a mountain, when suddenly the words escaped from my lips,\n'With swords, with guns, with weapons!' And then the Padishah girded\nhis own sword upon me, and I rushed among the howling mob, and I cut\nand hacked away at them till they were all consumed, and at last a\nfield that had been reaped lay before me, and it was covered with\nnothing but corpses.\"\n\n\"That is a foolish dream,\" said Leonidas. \"Why did you eat so much\nlast night?\"\n\nAnd now Milieva told her dream.\n\n\"I also must have been confused by the wine. Before me also a\nrebellious multitude appeared, and it then seemed to me as if I was\nnot a girl but a boy. Furiously they rushed upon me from every side,\nbut I feared them not, and when they were quite near to me I cried out\nto them, 'Down on your knees before me! I am the Sultan's daughter!'\nAnd everything was instantly quiet.\"\n\nThe merchant laughed till he choked at this dream. Who but children\ncould dream such rubbish?\n\n\"But at home they used to say,\" observed Thomar, with a grave face,\n\"that whatever any one dreams in a strange place where he has never\nslept before, he will see that dream accomplished.\"\n\n\"Well, I am much obliged to you,\" said the merchant, \"for in my dream\nI was hanging up in Salonika by my feet, with my head downwards.\"\n\nThen the merchant made the children leave the cavern.\n\n\"Come, my children,\" said he, \"let us see if the sea has calmed down,\nand whether a ship is approaching from anywhere.\"\n\nThomar obeyed, quitted the cavern, and exclaimed, in astonishment:\n\n\"Look, my dear foster-father! How could a ship come here when the very\nsea has vanished, and only the bottom of it remains.\"\n\nAnd indeed the district stretching out before them was quite bare and\nbarren enough to be taken for the bottom of the sea.\n\nLeonidas took the lad's words for a joke, and it was a joke he did not\nrelish.\n\n\"Keep your witticisms for another time, my son,\" said he, \"and rub\nyour eyes that they may see the better.\"\n\nBut Milieva leaped after Thomar, and when she had got up to him she\nclapped her hands together, and exclaimed, with naive amazement:\n\n\"Why, the sea has run away from us!\"\n\nAnd now the merchant himself arose from his place, went out of the\ncavern, and could scarce believe his eyes when he saw before him the\nsavage, rocky region, where not a drop of moisture could be seen, to\nsay nothing of the sea!\n\n\"God has worked wonders for us,\" sighed the merchant. \"It is plain\nthat we are in quite a different place from that wherein we went to\nsleep.\"\n\n\"No doubt the peris of the mountains of Kaf have conveyed us hither,\"\nsaid Milieva.\n\n\"Peris, no doubt,\" observed Leonidas, absently, groping for his long\nreticule, and feeling whether his diamonds were still there. If it\nwere not peris, they would certainly have searched him for his\ndiamonds.\n\nAnd now they had to find out where they were, and what was the best\nway to get out of the wilderness. The greatest anxiety had\ndisappeared; they had no longer anything to fear from the sea. On dry\nland it would be much easier to find a place of refuge.\n\nAfter a little searching they came upon footprints in the sand, and\nthese footprints led them to the mouth of the valley. Whole forests of\nthe large cochineal cactus grew among the rocks, and here and there\nthey saw a light-footed kid grazing on the dry sward. Not very long\nafterwards they fell in with the goatherd. Leonidas was rather alarmed\nthan delighted at the sight of the grim muscular figure, who, on\nperceiving them, came straight towards them, and addressed them in a\ngruff voice.\n\n\"Are ye those shipwrecked fugitives who slept at night in the Cavern\nof the _dzhin_?\"\n\n\"_Dzhin!_\" said Leonidas to himself. \"Methinks it must have been a\nspirit of evil, then.\"\n\nThe children answered the goatherd boldly, and begged him to direct\nthem to some inhabited region.\n\n\"Go straight along this gorge,\" said he; \"you cannot mistake the path.\nOn your right hand you will find a hut where dwells a fakir of the\nErdbuhar Order, who will direct you farther. Salam alek!\" And with\nthat the goatherd quitted them, to the great amazement of Leonidas,\nwho had expected nothing less of him than highway robbery.\n\nTowards evening they had arrived at the hut of the Erdbuhar hermit.\n\n\"I have been expecting you,\" said the dervish, when they came up to\nhim. \"Have you not suffered shipwreck and slept all night with the\n_dzhin_?\"\n\nEvidently one marvel after another was in store for them.\n\nThe dervish gave them meat and drink, and washed their feet, and after\nthey had enjoyed his hospitality he offered to conduct them all the\nway to the gates of Seleucia. The merchant would very much have liked\nto know something of his wondrous deliverers, but as the dervish\nanswered all his questions with quotations from the Kuran, he learned\nvery little that was definite from that holy man.\n\nWhen Seleucia came in sight, the merchant began thanking the dervish\nfor his good offices. \"Do not weary thyself any further, worthy\nMussulman,\" cried he; \"I know not how to reward thy labors, but Allah\nwill requite thee. I am a beggar. Thou dost see that I am as bare as\none of my fingers. The ocean hath swallowed up my all.\"\n\nAnd all the while his reticule was full of precious stones; but he\nwould have considered it a very great act of folly not to have made\ncapital out of his wretchedness, and paid the dervish with fine words.\n\nBut the dervish would not even accept his thanks. \"It is but my duty,\"\nsaid he, \"and I did it not for thy sake, but for the sake of others.\"\nAnd with that he quitted them, after giving a string of praying-beads\nto each of the children.\n\nThe children went on in front till they reached the gate of the city,\ntalking in a low voice together; but when they found themselves in the\npopulous streets they took Leonidas by the hand, and Thomar said, \"All\nthat was thine has been lost in the sea, and who will help us in the\ngreat strange city, where nobody knows us? Let us therefore sing in\nthe market-place and before the houses of the great men, and they will\ngive us money, and so we shall be able to go on farther.\"\n\nThe merchant was greatly affected by this naive offer, and allowed the\nchildren to sing in the market-place and in the porch of the pasha's\nhouse, and in this way they gained enough money to enable them to go\non to the next city.\n\nThus, at last, they got back to Smyrna. If they had been his own\nchildren Argyrocantharides could not have looked for greater and\nheartier affection from them. They fasted that he might feast, they\nshivered that he might be warmly clad, they denied themselves sleep\nthat he might slumber all the more tranquilly, and lowered themselves\nto singing in the market-place that he might not be compelled to beg\nat the corners of the streets.\n\nGood children! sweet children!\n\nAs soon as the merchant could get a new ship he took them with him to\nStambul, and this time no misfortune happened to them by the way.\n\nAt Stambul he exhibited them to the Kizlar-Agasi, who, after examining\ntheir limbs and satisfying himself as to their capabilities, bought\nthe pair of them from the merchant at his own price--the youth for the\nSultan's corps of pages, the girl for the harem.\n\nTo the honor of the worthy merchant, however, it must be said that\nwhen he did hand the children over he sobbed bitterly. Good, worthy\nman!\n\n\n\n\nCHAPTER XIII\n\nA BALL IN THE SERAGLIO\n\n\nIt was the birthday of the Sultana Valideh. The Sultana, Mahmoud's\nmother, was, we may remember, a Frenchwoman, whose parents, natives of\nthe Isle of Martinique, had sent her to Paris while still very young,\nand placed her, till she was sixteen, in a convent to be educated.\nThen the family sent word that she was to return to the beautiful\nisland on the farther side of Africa; but during the voyage a tempest\ndestroyed the ship, and the crew had to take to the boats. One of\nthese boats, in which was the pretty French girl, was captured by\nBarbary corsairs, who sold her to the Sultan. The rest we know, of\ncourse--\n\n \"Elle eut beau dire: Je me meurs!\n De nonne elle devient Sultane!\"\n\nThose poor flowers that are brought together from all the corners of\nthe earth to stock the Grand Signior's harem, and who know nothing\nexcept how to love, paled before the radiant loveliness and the\nsparkling wit of this damsel, who had been brought up in the midst of\nEuropean culture. She became the favorite wife of Selim, she bore him\nMahmoud, and her son loved his mother much better than all his damsels\nput together.\n\nA great surprise had been prepared for the Sultana Valideh. The Sultan\nhad arranged the whole thing himself in secret. He was going to give a\ndance, after the European fashion, in the Seraglio.\n\nTailors were brought from Vienna who set to work upon dresses in the\nlatest fashion for the odalisks; the eunuchs were taught the latest\nwaltz music, a minuet, and two French square dances; and the girls\nwere all taught how to dance these dances. The men who had admittance\ninto the harem, the Kizlar-Agasi, the Anaktar Bey, the heir to the\nthrone (Abdul Mejid), and the Sultan himself, wore brown European\ndress-suits, so that when the Sultana stepped into the magnificently\nilluminated porcelain chamber she stood rooted to the floor with\nastonishment. She imagined herself to be at a court ball at Paris,\njust as she had seen it at the Louvre when a child. A surging mob of\nhundreds and hundreds of young odalisks was proudly strutting to and\nfro in stylish dresses of the latest fashion, in long gloves and silk\nstockings. Instead of turbans, plumed hats and bouquets adorned the\nmagnificent masses of their curled and frizzled locks. They moved\nabout with bare shoulders and bosoms, in soft wavy dresses, with fans\npainted over with butterflies, freely laughing and jesting in this, to\nthem, newest of worlds, and the only thing that differentiated this\nball from our dancing entertainments was the absence of the darker\nportion of the show--the masculine element.\n\nThere were only four representatives of this _sombre nuance_--to wit,\nthe Sultan, the heir to the throne, the Kizlar-Agasi, and the Anaktar\nBey. Of these four, two were no longer and two were not yet men. All\nfour were dressed in stiff Hungarian dolmans, long black pantaloons,\nand red fezes. The Sultan, with his thick-set figure, would have\npassed very well for a substantial Hungarian deputy-lord-lieutenant,\nwith his tight-fitting, bulging dolman buttoned right up to his chin.\nThe young prince's elegant figure, on the other hand, was brought into\nstrong relief by his well-made suit; his hair was nicely curled on\nboth sides, and his genteel white shirt was visible beneath his open\ndolman. The Kizlar-Agasi, on the contrary, cut a very amusing figure\nin his unwonted garb. He was constantly endeavoring to thrust his hand\ninto his girdle, and only thus perceived that he had none, and he kept\non holding down the tails of his coat, as if he felt ashamed that they\nmight not reach low enough to cover him decently.\n\nThe Sultana Valideh was favorably surprised. The spectacle brought\nback to her her childish years, and she gratefully pressed her son to\nher bosom for this delicate attention, while he respectfully kissed\nhis mother's hands. The Sultan scattered his love among a great many\nwomen, but his mother alone could boast of possessing his respect.\n\nThe odalisks surrounded the good Sultan, rejoicing and caressing him.\nHe was never severe to any of them--nay, rather, he was the champion,\nthe defender of them all, and those whom he loved might be quite sure\nthat his affection would be constant.\n\nEvery one tried to please the Sultana Valideh by showing her their new\ngarments, but none of them found such favor in her eyes as the new\nflower, which had only recently been introduced into the Seraglio,\nand was now the foremost of them all, the beautiful Circassian damsel.\nHer light step, the dove-like droop of her neck, the charm of her\nfull, round shoulders, and her lovely young bosom, were such that one\nwas almost tempted to believe that she had been carried off bodily\nfrom some Parisian salon, where they know so well how to take the\nutmost advantage of all the resources of fashion. Her locks were\ndressed up _a la Valliere_, with negligently falling curls which gave\na slightly masculine expression to her face--an additional charm in\nthe eyes of a connoisseur. Yes, the Greek merchant was right; there\nwas no spot on the earth worth anything except the place where Milieva\nlived and moved.\n\nThe Valideh kissed the odalisk on the forehead, and led her by the\nhand to the Sultan, who would not permit her to kiss his hand (who\never heard of a lady kissing the hand of a gentleman in evening\ndress?), but permitted the young heir to the throne to take Milieva on\nhis arm and conduct her through the room. What a pretty pair of\nchildren they made! Abdul Mejid at this time was scarce twelve years\nof age, the girl perhaps was fourteen; but for the difference of their\nclothes, nobody could have said which was the boy and which the girl.\n\nAnd now the tones of the hidden orchestra began to be heard, and a\nfresh surprise awaited the Sultana. She heard once more the pianoforte\nmelodies which she had known long ago, and the height of her amazement\nwas reached when the Sultan invited her to dance--a minuet.\n\nWhat an absurd idea! The Sultana dowager to dance a minuet with her\nson, the Sultan, before all those laughing odalisks, who had never\nbeheld such a thing before? Where was the second couple? Why here--the\nprince and Milieva, of course. They take their places opposite the\nimperial couple, and to slow, dreamy music, with great dignity they\ndance together the courteous and melancholy dance, bowing and\ncourtesying to each other with as much majesty and _aplomb_ as was\never displayed by the powdered cavaliers and beauty-plastered\ngoddesses of the age of the _OEil de Boeuf_.\n\nNever had such a spectacle been seen in the Seraglio.\n\nThe Sultana herself was amazed at the triumphant dexterity which\nMilieva displayed in the dance; she was a consummate maid of honor,\nwith that princely smile for which Gabrielle D'Estrees was once so\nfamous. The good Mahmoud so lost himself in the contemplation of the\neyes of Milieva, his _vis-a-vis_, that towards the end of the dance he\nquite forgot his own part in it, folding Milieva to his breast in\ndefiance of all rule and ceremony, and even kissing her face twice or\nthrice, although he ought not to have gone beyond kissing her\nhand--nay, he ought not to have kissed her hand at all, but the hand\nof his partner, the Sultana Valideh.\n\nWhen the minuet was over the eunuch musicians played a waltz in which\nall the odalisks took part, clinging to one another in couples, and\nthus they danced the pretty _trois pas_ dance, for the _deux pas_\nrevolution was the invention of a later and more progressive age.\nLouder than the music was the joyous uproar of the dancers themselves.\nHere and there some of them tumbled on the slippery floor to which\nthey were not accustomed, and the nymphs coming after them fell\naround them in heaps. Some disliked the dance or were weary, but their\nfirier and more robust partners dragged them along, willy-nilly. The\nold Kizlar-Agasi and the bey stood in the midst of them to take care\nthat no scandal took place. Suddenly the madcap odalisk army\nsurrounded them, clung on to them in twos and threes, dragged them\ninto the mad waltz, and twisted them round and round at a galloping\npace, till the two good old gentlemen had no more breath left in them.\n\nThe Sultan and the Valideh, with the prince and Milieva, were sitting\non a raised dais, laughing and looking on at the merry spectacle. The\npipers piped more briskly, the drummers drummed more furiously, the\ncymbals clashed more loudly than ever, while the odalisks dragged\ntheir prey about uproariously.\n\nAh! Listen! What didst thou hear, good Sultan? What noise is that\noutside which mingles with the hubbub within? Outside there also is to\nbe heard the roll of drums, the flourish of trumpets, and the shouts\nof men.\n\nNonsense! 'Tis but imagination. Bring hither the glasses--not those\ntiny cups of sherbet, for this is the birthday of the Valideh. We will\nbe Europeans to-night. Bring hither wine and glasses for a toast!\n\nThe Sultan had a particular fondness for Tokay and champagne, and the\nambassadors of both these great Powers had the greatest influence with\nhim.\n\nThe odalisks also had to be made to taste these wines; and after that\nthe dance proceeded more merrily, and the boisterous music and\nsinging grew madder and madder.\n\nWhat was that?\n\nThe Sultan grew attentive. What uproar is that outside the Seraglio?\nWhat light is that which shines at the top of the round windows?\n\nThat uproar is no beating of drums; those shouts are not the shouts of\nrevellers; that din is not the beating of cymbals; no, 'tis the\nclashing of swords, the thundering of cannons, the tumult of a siege,\nand that light is not the light of bonfires but of blazing rafters!\n\nUp, up, Mahmoud, from thy sofa! Away with thy glass and out with thy\nsword! This is no night for revelry; death is abroad; insurrection is\nat thy very gate! They are besieging the Seraglio!\n\nTwelve thousand Janissaries, joined with the rabble of Stambul, are\nattacking the gates at the very time when the orchestra is playing its\nliveliest airs in the illuminated hall.\n\n\"Do ye hear that?\" exclaimed Kara Makan, the most famous orator of the\nJanissaries, who with his own hand had hung up the Metropolitan of\nConstantinople on the very threshold of the palace. \"Do ye hear that\nmusic? Here they are rejoicing when the whole empire around them is in\nmourning. Do ye know what are the latest tidings this night? The\nSuliotes have captured Gaskho Bey, and annihilated our army before\nJanina. A woman has blown up the ship of the Kapudan Pasha, and the\nShah has fallen upon Kermandzhan with an army! Destruction is drawing\nnear to us, and treachery dwells in the Seraglio. Hearken! They dance,\nthey sing, they bathe their lips in wine, and their blasphemies bring\nupon us the scourge of Allah! We shed our tears and our blood, and\nthey make merry and mock at us! Shall not they also weep? Shall not\ntheir blood also be shed? So fare it with them as it has fared with\nour brethren whom they sent to the shambles!\"\n\nThe furious mob answered these seditious words with an indescribable\nbellowing.\n\n\"If we traversed the whole empire we should not find a worse spot than\nthis place.\"\n\n\"Set fire to the Seraglio!\" cried one voice suddenly, and the others\ntook up the cry.\n\n\"And if you escape from all other enemies, would you fall into the\nclaws of the worst enemies of all?\"\n\n\"Death to the Viziers! Death to the lords of the palace!\" thundered\nthe people; and one voice close to Kara Makan, rising above the\nothers, exclaimed, \"Death to the Sultan!\"\n\nKara Makan turned in that direction and defended his master. \"Hurt not\nthe Sultan! The life of the Sultan is sacred. He and his children are\nthe last survivors of the blood of Omar; and although he be not worthy\nto sit on the throne which the heroic Muhammad erected for his\ndescendants, yet he is the last of his race, and, therefore, the head\nof the Sultan is sacred. But death upon the head of the Reis-Effendi,\ndeath to the Kizlar-Agasi and the Kapudan Pasha! They are the cause of\nour desolation. The chiefs of the Giaours pay them to destroy their\ncountry. Tear all these up by the roots, and if there be any children\nof their family, destroy them also, even to the very babes and\nsucklings, that the memory of them may perish utterly!\"\n\nThe mob thundered angrily at the gates of the Seraglio, which were\nshut and fastened with chains. The Janissaries blew the horns of\nrevolt, the drums rolled, and within there the Sultan was reposing his\nhead on the bosom of a beautiful girl. Suddenly a loud report shook\nthe whole Seraglio. An audacious ichoglan had fired his gun upon the\nmob as it rushed to attack the water-gate.\n\nThe Sultan, in dismay, quitted the harem, and hastened to the middle\ngate in order to address the mob. On his way through the corridor, his\nservants and his ministers threw themselves at his feet and implored\nhim not to show himself to the people. Mahmoud did not listen to them.\nIn the confusion of the moment, moreover, it never occurred to him\nthat he was wearing a Frankish costume, which the people hated and\nexecrated.\n\nWhen he appeared on the balcony the light of the torches fell full\nupon him, and the Janissaries recognized him. Every one at once\npointed their fingers at him, and immediately an angry and scornful\nhowl arose.\n\n\"Look! that is the Sultan! Behold the Caliph--the Caliph, the Padishah\nof the Moslems--in the garb of the Giaours! That is Mahmoud, the ally\nof our enemies!\"\n\nThe Sultan shrank before this furious uproar of the mob, and,\ninvoluntarily falling back, stammered, pale as death:\n\n\"With what shall we allay this tempest?\"\n\nHis servants, with quivering lips, stood around him. At that moment\nthey neither feared nor respected their master.\n\nSuddenly a bold young ichoglan rushed towards the Sultan, and\nanswered his question in a courageous and confident voice:\n\n\"With swords, with guns, with weapons!\"\n\nIt was Thomar.\n\nThe Sultan scrutinized the youth from head to foot, amazed at his\naudacity; then hastening back to his dressing-chamber, exchanged his\nball dress for his royal robes, and, coming back from the inner\napartments, descended into the court-yard.\n\nThe guns were already pointed at the gates, the topijis stood beside\nthem, match in hand, impatiently awaiting the order to fire.\n\nWhen the Sultan appeared in the court-yard he was at once surrounded\nby some hundreds of the ichoglanler, determined to defend him to the\nlast drop of their blood. Mahmoud again recognized Thomar among them;\nhe appeared to be the leading spirit of the band.\n\nThe Sultan beckoned to them to put back their swords in their sheaths.\nHe commanded the topijis to extinguish their matches. Next he ordered\nthat the gate of the Seraglio should be thrown open to the people.\nThen, having bidden every one to stand aside, he went alone towards\nthe gate in his imperial robes, with a majestic bearing.\n\nNo sooner was the gate thrown open than the mob streamed into the\ncourt-yard with torches and flashing weapons in their hands, standing\nfor a moment dumb with astonishment at the appearance of the Sultan.\nHe was no longer ridiculous, as he had been in that foreign garb. The\nmajestic bearing of the prince stilled the tumult for an instant, but\nfor an instant only. The following moment a hand was extended from\namong the mob of rebels which tore the Sultan's caftan from his\nshoulder.\n\nMahmoud grew pale at this audacity, and this pallor was a fresh\noccasion of danger to him, for now he was suddenly seized from all\nsides. The Sultan turned, therefore, and perceiving Thomar, called to\nhim, \"Defend my harem!\" and, at the same time freeing his sword-arm,\nhe drew his sword, waved it above his hand, and, while his foes were\nwaiting to see on whom the blow would fall, he threw the sword to\nThomar, exclaiming, \"Defend my son!\"\n\nThe young ichoglan grasped Mahmoud's sword, and, while the captured\nSultan disappeared in the mazes of the mob, he and his comrades\nreturned to the inner court-yard, and, barricading the door, fiercely\ndefended the position against the insurgents. He had now to show\nhimself worthy of that sword, the sword of the Sultan.\n\nGradually two thousand ichoglanler and three thousand bostanjis\ngathered round the young hero. The Janissaries already lay in heaps\nbefore the door, which they riddled with bullets till it looked like a\ncorn-sifter. But the youths of the Seraglio repelled every onset.\n\nAnd why did not the Sultan remain with them? They would have defended\nhim against all the world: Who knew now what had become of him?\nPerhaps they had killed him outright.\n\nThe Janissaries speedily perceived that they could not have done\nanything worse for themselves than to have brought torches with them,\nfor thereby they were distinctly visible to the defenders of the\nSeraglio, and every shot that came from thence told.\n\n\"Put out the torches!\" shouted Kara Makan, who was holding a huge\nconcave buckler in front of him, and felt a third bullet pierce\nthrough the twofold layers of buffalo-hide and graze his body.\n\nThe torches went out one after another, whereupon the spacious\ncourt-yard was darkened; only the flash of firearms cast an occasional\ngleam of light upon the struggling mass.\n\nIt might have been two hours after midnight when suddenly there was a\ncessation of hostilities. Both sides were weary, and ceased firing;\nthe Janissaries whispered amongst themselves, and at last in the midst\nof a deep silence, Kara Makan's thunderous voice made itself heard:\n\n\"Listen, all of ye who are inside the Seraglio. Ye are good warriors,\nand we are good warriors also, and it is folly for the Faithful to\ndestroy one another. We did not take up arms to slay you and plunder\nthe Seraglio, neither do we wish to kill the Padishah nor the heir to\nthe throne; but we would rescue them from the hands of the traitors\nwho surround them, and we would also deliver the realm from faithless\nViziers and counsellors. Give us, therefore, the prince, the Sultan's\nson. Of a truth no harm shall befall him, and we will thereupon quit\nthe court-yard of the Seraglio and trouble nobody within these doors.\nIf, however, you will not grant our request, then Allah be merciful to\nall who are within these beleaguered walls.\"\n\nThe Kizlar-Agasi conveyed this message into the Seraglio, and\nbesiegers and besieged awaited with rapt attention the reply of the\nValideh; for the decision lay with her--she was superior in rank to\nall four of the Asseki sultanas.\n\nAfter the lapse of a quarter of an hour the Kizlar-Agasi returned, and\nsignified to the besiegers that the prince would be handed over to\nthem.\n\nThe Janissaries received this message with a howl of triumph, while\nthe ichoglanler shrugged their shoulders.\n\n\"They are not all women in there for nothing,\" said Thomar, savagely,\nto the Kizlar-Agasi, and he remained standing in the gate, that he\nmight, at any rate, kiss the young prince's hand and whisper to him\nnot to go.\n\nThe Janissaries relit their torches and crowded towards the gate.\nInside reigned a pitch-black darkness.\n\nNot long afterwards footsteps were audible in the dark corridor, and,\nescorted by two torch-bearers, the prince descended the steps. He had\non the same garment which he wore when he went on horseback to the\nMosque of Sophia during the Feast of Bairam. How the people had then\nhuzzahed before him! He wore pantaloons of rose- silk, yellow\nbuskins with slender heels, a green caftan embroidered with gold\nflowers, and a handsome yellow silk vest buttoned up to his chin. His\nribbons and buttons were made so as to represent brilliant fluttering\nbutterflies incrusted with precious stones.\n\nOn reaching the gate he beckoned to the torch-bearers to stand still,\nsent back the Kizlar-Agasi, and, proceeding all alone to the gate,\ncommanded that it should be flung open.\n\nWhile this was being done Thomar pressed close up to him, and seizing\nthe prince's hand, kissed it, at the same time whispering in his ear,\n\"Go not; we will defend you if you remain here.\"\n\nThe prince pressed Thomar's hand and whispered back, \"I must go; you\nkeep on defending the Seraglio!\" And with that he embraced the youth\nand kissed him twice with great fervor.\n\nThomar was somewhat startled by this burning, affectionate kiss, and\nwondered what it meant. The darkness did not allow him to distinguish\nthe prince's features; and when he tried to detain him once more the\nprince hastily disengaged himself and stepped forth from under the\ndark vault among the Janissaries.\n\nThomar covered his eyes with his hands; he did not want to see the\nfate of the prince at that moment. It was quite possible that the\nblood-thirsty might cut him down on the spot in a sudden access of\nfury.\n\nThe prince stepped forth among the rebels.\n\nAt that moment a cry of unbridled joy, triumph, and blood-thirstiness\nburst from the Janissaries. It needed but one of them to raise his\nhand, and the next would speedily have completed the bloodiest deed of\nall.\n\nBut the prince stood before them haughtily and valiantly, and, with\namazing audacity, cried to them, \"Down on your knees before me, ye\nrebels!\"\n\nAt these words Thomar, with a start of terror, looked at the prince.\nThe full light of the torches fell upon his charming face. It was not\nAbdul Mejid, but--Milieva! They had dressed her inside the harem in\ngarments suitable to the Feast of Bairam, and she had come out instead\nof the prince, courageously, as if she had been born to it. Who was\nlikely to notice the change? The heart of this odalisk loved to play a\nmanly part, and it was not merely the masculine garb she wore which\ntransformed her, but the masculine soul within her.\n\nThe Janissaries, moreover, were dumfounded by this bold attitude. This\ngraceful, noble figure stood face to face with them and domineered the\nmob with a commanding look, proudly, majestically, as became a born\nruler. And yet death hovered over the head of him who dared to say, \"I\nam the prince!\"\n\nThomar, forgetting himself, seized his sword, and would have rushed to\nthe defence of his sister but his comrades held him back. \"What would\nyou do, unhappy wretch? Trust to Fate!\"\n\nKara Makan, in savage defiance, approached the false prince with a\ndrawn sword in his hand.\n\n\"On your knees before me!\" cried the odalisk, and indicating where he\nshould kneel with an imperious gesture, she looked steadily into the\neyes of the savage warrior.\n\nThe ferocious figure stood hesitatingly before her. The magic of her\nlook held the wild beast in him spellbound for an instant. His\nbloodshot eyes slowly drooped, his hand, with its flashing sword, sank\ndown by his side, his knees gave way beneath him, and, falling down at\nthe feet of the young child, he submissively murmured a salaam,\nkissing her hand and laying his bloody sword at her feet.\n\nMilieva pressed her right hand on the head of the subdued rebel,\nlooked proudly and fearlessly upon the dumb-stricken rebels, and then,\nraising the sword and giving it back to Kara Makan, she cried, \"Go\nbefore and open a way for me!\"\n\nAs if in obedience to a magic word, the crowd parted on both sides\nbefore her, and Kara Makan, with his sword over his shoulder, led the\nway along. The crowd, with an involuntary homage, made way for her\neverywhere from the Seraglio to the Seven Towers, and two\ntorch-bearers walked by her side, between whom she marched as proudly\nas if she were making her triumphal progress. Nobody perceived the\ndeception. The resemblance of the young face to that of the prince,\nthe well-known festal raiment of the Feast of Bairam, her manly\nbearing, all combined to keep up the delusion, and amongst this\n_canaille_ which held her in its power there was not a single\ndignitary who knew the prince intimately and might have detected the\nfraud.\n\nThe Sultan had just been thrust into the dungeon of the Seven Towers,\nthat place of dismal memories for the Sultans and their families in\ngeneral. In that octagonal chamber, whose round windows overlooked the\nsea, more than one mortal sigh had escaped from the lips of the\ndescendants of Omar, whom a powerful faction or a triumphant rival\nhad, sooner or later, condemned to death.\n\nIt was now morning, the uproar of the rebellion had died away outside,\nthe Seraglio was no longer besieged. It was now that Kara Makan\nappeared before the Sultan.\n\nThe Padishah was sitting on the ground--on the bare ground. His royal\nrobes were still upon him, a diamond aigrette sparkled in the turban\nof the Caliph, and there he sat upon the ground, and never took his\neyes off it.\n\n\"Your majesty!\" cried Kara Makan, addressing him.\n\nThe Padishah, as if he had not heard, looked apathetically in front of\nhim, and not a muscle of his face changed.\n\n\"Sire, I stand before thee to speak to thee in the name of the Moslem\npeople.\"\n\nHe might just as well have been speaking to a marble statue.\n\n\"Every storm proceeds from Allah, sire, and nothing which Allah does\nis done without cause. When the lightnings are scattered abroad from\nthe hands of the angel Adramelech, is not the air beneath them heavy\nwith curses? and when the living earth quakes beneath the towns that\nare upon it, shall not innocently spilled blood shake it still more?\nSo also the Moslem people rising in rebellion is the instrument of\nAllah, and Allah knoweth the causes thereof. I will guard my tongue\nagainst telling these causes to thee; thou knowest them right well\nalready, nor is it for me to reprove the anointed successor of the\nProphet. But I beg thee, sire, to promise me and the people, in the\nname of Allah, that thou wilt do what it beseemeth the ruler of the\nOttoman nation to do--promise to remedy our wrongs, and we will set\nthee again upon thy throne.\"\n\nAt these words Mahmoud fixed his eyes upon the speaker, and gazed long\nupon those dark features, as sinister as an eclipse of the sun. Then\nhe arose, turned away, and replied in a low voice, hissing with\ncontempt:\n\n\"The Sultan owes no reply to his servants.\"\n\nKara Makan's face was convulsed at these words. Scarce was he able to\nstifle his wrath, and he replied, in broken sentences:\n\n\"Sire, the lion is the king of the desert--but if he is in a cage--he\nlistens to the voice of his keeper--thou knowest this hand, which hath\nfought for thee in many engagements--and thou knowest that whatever\nthis hand seizeth it seizeth with a grasp of iron.\"\n\nThe Sultan pondered long. Then all at once he seemed to bethink him of\nsomething, for his face seemed to lose its severity, and he turned\ntowards the Janissary leader with a mild, indulgent look.\n\n\"What, then, dost thou require?\" This softened look concealed the\ngenesis of the thought--the Janissaries must be wiped off the face of\nthe earth. \"What dost thou require?\" said the Padishah, softly.\n\nKara Makan put on an important look, as of one who knows that the fate\nof empires is in his hands.\n\n\"Hearken to our desires. We are honest Mussulmans. We do not ask\nimpossibilities. If thou canst convince us that our demands are\nunlawful, we renounce them; if thou canst not convince us, accomplish\nthem.\"\n\nMahmoud's lips wore a bitter smile at this wise speech.\n\n\"I do not strive with you,\" he replied. \"Ye command me. The Caliph of\ncaliphs listens to his servants. Bring hither parchment and an\nink-horn, and dictate to my pen what ye demand. The Sultan will be\nyour scribe, great rebel!\"\n\nKara Makan was not bright enough to penetrate the irony of these\nwords; nay, rather, he felt himself flattered by the humility of the\nSultan's speech. With haughty self-assurance he bared his bosom and\ndrew forth a large roll of manuscript.\n\n\"I will save your majesty the trouble,\" said he to Mahmoud, smoothing\nout the document before him. \"Behold, it is all ready. Thou hast only\nto write thy name beneath it.\"\n\n\"Will ye allow me to read it?\" inquired the Sultan, with the same\nbitter smile; \"or is it the wish of the people that I should sign it\nunread?\"\n\n\"As your majesty pleases.\"\n\nMahmoud took up the documents one after another, and piled them up\nbeside him as he read them.\n\n\"Ah! the appointment of a new seraskier! I will read no further. I\nagree, but I would know his name. Is he whom you desire fit for the\npost?\"\n\n\"We want Kurshid,\" explained Kara Makan, perceiving that the Sultan\nhad not read the document.\n\n\"And the Janissaries demand other rewards for themselves. 'Tis only\nnatural: I grant them. They cannot be expected to storm the Seraglio\nfor nothing. The chief treasurer will pay you whatever you require.\nThis third article, too, I see, demands the capture of Janina. Be it\nso. I grant it. Most probably the whole Janissary host will want to go\nagainst Ali Pasha.\"\n\n\"So long as thou art at their head,\" said Kara Makan, somewhat\ndisturbed. \"The Janissaries are only bound to fight under the direct\ncommand of the Sultan.\"\n\n\"And all these other demands are equally reasonable, eh?\" said the\nSultan, just glancing at one or two of them.\n\nHe took up the last one, but when he had unfolded it his face\ndarkened, and he suddenly leaped to his feet, his good-natured apathy\nchanged into wrath and fierceness, and, striking the open document\nwith his fist, he exclaimed, with an access of emotion:\n\n\"What's this? Are ye so bold as to expect me to sign this paper?\"\n\nKara Makan was so well prepared for this outburst of anger on the\nSultan's part that he was not in the least taken aback. With rustic\nstolidity he replied:\n\n\"We wish it, and we demand it.\"\n\n\"Do you know what is written in this document?\"\n\n\"Yes; that thou must free the realm from foreigners; that thou must\nput the Russian ambassador Stroganov on board ship and send him home;\nrefuse to admit French and English ships into the Bejkoz; send the\nSultana Valideh far away to Damascus; and slay the Grand Vizier, the\nKizlar-Aga, the Berber Pasha, and the Kapudan Pasha, and give their\nbodies to the people.\"\n\nThe Grand Signior contemptuously threw the document to the floor and\ntrampled it beneath his feet.\n\n\"Shameless filibusterers,\" he cried; \"not blood but money is what you\nwant. Ye want permission not to deliver the realm, but to plunder it.\nAnd you expect the Padishah to sanction it! Did not you yourselves\nraise the Viziers to power? Were not you the cause of their not being\nable to make any use of that power? Whenever the arms of the Giaours\nwere triumphant, were you not always the first to fly from the field\nof battle? And when the realm was sinking, were you not always the\nlast to hasten to its assistance? You are no descendants, but the mere\nshadows of those glorious Janissaries whose names are written with\nletters of blood in the annals of foreign nations; but ye make but a\npoor and wretched figure therein. Kill me, then! I shall not be the\nfirst Sultan whom the Janissaries have murdered, but, in Allah's name\nI say it, I shall be the last. After me, either nobody will sit on the\nthrone of Omar, or, if any one sits there, he will be your ruin.\"\n\nThe opposition of his august captive only restored the Janissary\nleader to his proper element. He felt much more at home with those\nwrathful eyes than with the previous contemptuous nonchalance. He\ncould now give back like for like.\n\nHe picked up the crumpled document, in which were written the\ndeath-sentences of the Viziers, and, brushing off the dust, again\npresented it to the Sultan.\n\n\"Either sign this document or descend from the throne of the family of\nOmar, and we will seek us out from among the descendants of the\nProphet another who shall reign in thy stead.\"\n\n\"Most abject of slaves! In thy pride thou knowest not what thou\nsayest! Death comes from Allah and none can avoid it; but who amongst\nthe descendants of Omar would be powerful enough to seize the royal\nsceptre, and who would be senseless enough to desire it?\"\n\n\"Look at me.\"\n\n\"I am looking. The sun does not soil itself by shining upon a swamp,\nand therefore I may look even at thee; but I see nothing in thee that\nwould justify the adorning of thy head with a diadem so long as one\nof the descendants of Sulaiman the Magnificent is alive.\"\n\n\"Another word and thou shalt cease to live!\" cried the desperado,\nhaughtily throwing back his head before the Sultan. \"Art thou aware\nthat thy son Abdul Mejid is in our hands?\"\n\nThe Sultan shuddered. His consternation at these words was written in\nevery feature.\n\n\"My son, Abdul Mejid? Impossible!\"\n\n\"So it is. The Sultana Valideh gave him up at our request.\"\n\n\"Oh, madness!\" exclaimed the Sultan; and he began pacing to and fro.\n\nAbdul Mejid was still a mere child. The shock of such a rebellion\nmight easily make an epileptic of him. To deliver him into the hands\nof these rebels was as good as to sign his death-warrant. Even if they\ndid not kill him outright, his nerves might suffer from their\nviolence, and he might perish, as the two and twenty other children of\nSultan Mahmoud had perished, every one of whom had died of epilepsy.\nTheir delicate nervous constitutions had been shattered in their youth\nunder the influence of that perpetual terror to which the children of\nthe Caliph of caliphs had been exposed from time immemorial. What,\nthen, might not happen to Abdul Mejid if he fell into the hands of\nthis savage mob?\n\n\"Oh, ye are hell's own children! Ye are worse than the Giaours, worse\nthan the Greeks, worse than the Muscovites! Ye do place your feet on\nthe heads of your rulers!\"\n\nThe despair of the Sultan emboldened the Janissary still further.\n\n\"Sign this document, or thy son shall die in our hands!\"\n\n\"Miserable cowards!\" moaned the Sultan. \"And cowards they also who\nshould have defended him! Did not even his mother defend him? Was it\nnecessary to give him up?\"\n\n\"He is in no danger,\" said Kara Makan; \"nay, he is in a safe place. It\nrests with thee to receive him back into thy arms;\" and he shoved\ntowards him again the soiled and crumpled manuscript.\n\nThe Padishah, overcome by the shock of his own feelings, humiliated by\nthe sense of his own soft-heartedness, tottered to the wall, and when\nhis groping hands came in contact with the cold marble he collapsed\naltogether, and leaning against it, he pressed his burning temples to\nthe cold stone. The Janissary might now say whatever he would, the\nSultan neither listened to nor answered him.\n\nAt last the rough warrior, who had jumped so suddenly into power,\nshouted angrily to his comrades, who were cooling their heels outside,\n\"Bring hither the prince!\"\n\nThe Sultan heard the pattering of many footsteps in the corridor\noutside, and the clashing of swords mingled with the murmuring of\nvoices, but he did not look in that direction.\n\n\"Behold!\" cried Kara Makan, advancing towards him, \"here is thy son! A\ndrawn sword hovers above his head! Choose either to see thine own name\nat the foot of that paper or his head at thy feet!\"\n\nMahmoud trembled, but he answered nothing, nor did he turn his head.\n\n\"Write, or thy son dies!\" cried a number of the Janissaries, suddenly.\n\nThen a musical, familiar voice responded amidst the wild uproar:\n\n\"My father! hearken not unto them! Let them slay me if they be valiant\nenough, but chaffer not with thy slaves!\"\n\nMahmoud looked up in astonishment at this well-known voice, and saw\nbefore him a handsome figure in the prince's garments and with a proud\nand majestic countenance; but that face, though familiar to him and\nvery dear, was not his son's face. Ah, it was Milieva!\n\nThe odalisk perceived that Mahmoud's features softened, that he looked\ntenderly upon her; and as if she feared that the Sultan might yield\nout of compassion towards her, she hastily turned her flaming face to\nthe Janissaries and exclaimed:\n\n\"Ye blood-thirsty dogs of Samound! who bay down the sun from the\nheavens, accomplish your bloody work! Forward, ye valiant heroes, with\nwhose backs alone the enemy is familiar, fall upon me in twos and\nthrees, if any one of you has not the courage to plunge his steel\nsingle-handed into the heart of the last scion of Omar's stock! My\ndeath will not constrain the Sultan to bargain with you. Kill me while\nyou have power over me, for if ever I have power over you I will not\nweep before you, as ye have seen Mahmoud and Selim weep; but I will so\nutterly destroy you that even he who wears a garment like unto yours,\neven he who shall mention your name, shall pronounce his own doom.\"\n\nThe infuriated rebels raised their flashing swords above the head of\nthe presumptuous child at these menacing words; another moment and she\nwould have lain in the dust. But Mahmoud arose, spurned them aside\nfrom the prince, as they supposed him to be, and taking from the hands\nof Kara Makan the document and writing materials, signed his name\nbeneath it. Milieva seized the Sultan's hand to prevent him from\nwriting, but he tenderly kissed her on the forehead and gently\nwhispered, \"Rather would I lose the whole world than thee,\" and with\nthat he placed in the hands of the Janissaries the subscribed\ndeath-warrants.\n\nAfter obtaining these concessions, the rebels grew calmer, the Sultan\nproclaimed amnesty for all offenders, appointed the chief brawlers to\nhigh offices, and distributed money amongst them from the treasury.\n\nPeace was thus restored. The Sultan and the sham prince returned to\nthe Seraglio, accompanied all the way by a vast throng, and the whole\nsquare by the fountains of Ibrahim was filled by the well-known\nturbans of the Janissaries, who, in the joy of their insulting\ntriumph, shouted long life to the humiliated Padishah.\n\nMahmoud surveyed the huzzaing throng, where, man to man, they stood so\ntightly squeezed together that nothing could be distinguished but a\nsea of heads. And the Sultan thought to himself, \"What a fine thing it\nwould be to sweep all those heads away at one stroke!\"\n\n\n\n\nCHAPTER XIV\n\nKURSHID PASHA\n\n\nGaskho Bey, the incapable giant, was captured by the Suliotes in a\nnight attack, his army was scattered beneath the walls of Janina, and\nAli Pasha became once more the absolute master of Epirus.\n\nThen, like lightning fallen from heaven, unexpectedly, unforeseen, a\nman came from Thessalonica whose name was shortly to ring through half\nthe world. The name of this man was Kurshid Pasha.\n\nHe was a man of a puny, meagre frame, his features were widely\ndivergent from the characteristic Ottoman type, for he had a delicate\nprofile, a bright blond beard and mustache, and blue eyes with\nflexible eyebrows, all of which gave a peculiar character to his face,\nwhich showed unmistakable traces of a penetrating mind and cool\ncourage.\n\nTen thousand warriors accompanied the new commander to Janina, which\ngrew into thirty thousand at the very first battle. Kleon's and\nYpsilanti's armies were routed, and Gaskho Bey's scattered squadrons\nrallied around the banners of the victor.\n\nWhile Ali Pasha was defending Janina, the leaders of the Greek\ninsurgents besieged the fortress of Arta, which Salikh Bey defended\nwith a small garrison.\n\nKurshid's predecessor, Gaskho Bey, had committed the error of\nbesieging Janina and endeavoring to relieve Arta at the same time, and\nthus he came to grief at both places. The new commander acted on a\ndifferent plan. He knew well that not a head amongst all the Greek\nrebels was half so dangerous as Ali Tepelenti's; so, leaving Salikh\nPasha to his fate, he directed all his energies against Janina.\n\nA man indeed hath come against thee, O Ali Pasha! A man as valiant, as\ncrafty as thou; if thou be a fox, he is an eagle of the rocks, that\npounces down on the fox; and if thou be a tiger, he is the\nboa-constrictor which infolds and crushes the tiger.\n\nAli urged Kleon and Artemis to hasten to his assistance. His\nmessengers did not return to the fortress. The Greek leaders gave no\nreply to his summons. Anybody else would have found some consolatory\nexplanation of their remissness, but Ali divined things better. The\nGreeks said amongst themselves, \"Let the old monster tremble in his\nditch; let them close him in and hold him tight. He will be\nconstrained to make a life-and-death struggle to save his old beard.\nWhen we have captured Arta, and when our detested ally\" (for they did\ndetest him in spite of his being their good friend) \"is at the very\nlast gasp, then we will go to the rescue, relieve him, and let him\nlive a little longer.\"\n\nTepelenti was well aware that they spoke of him in this way. He knew\nwell that they hated him, and would gladly leave him to perish. The\nonly reason the Greeks had for allying themselves with Ali was that\nhis fortress was filled with an enormous store of treasure, arms, and\nmuniments of war; his gray head was the pivot of the whole rebellion.\n\nIf the fortress were taken, they would be deprived of this strong\npivot, those treasures, that gray head!\n\nOne day the Suliotes encamped before Arta heard the terrible tidings\nthat Kurshid Pasha had captured Lithanizza and La Gulia, the two\noutlying forts of the stronghold of Janina, and had driven Ali back\ninto the fortress. The tidings filled them with consternation. If\nJanina were lost, the whole Greek insurrection would lose the source\nof its supplies. The treasures which Ali had scattered amongst the\nGreeks with a prodigal hand would at once fall into the hands of the\nSultan, and then he would be able to secure Epirus at a single blow.\n\nA Greek army under Marco Bozzari immediately set out from Arta to\nrelieve Janina. Ali knew of it beforehand. Bozzari's spies had crept\nthrough Kurshid's camp into Janina, and signified to Ali that their\nleaders were on their way to \"The Five Wells,\" and that he should send\nforth an army to meet them.\n\n\"There is no necessity for it,\" replied Ali, with a cold smile. \"I am\nquite capable of defending myself in Janina for three months against\nany force that may be brought against me. It is much more necessary to\ncapture Arta. Go back, therefore, and say to Marco Bozzari, 'Come not\nto Janina, but go against Salikh Pasha. Tepelenti is sufficient for\nhimself in Janina.'\"\n\nBozzari understood the old lion's hint. He did not wish the Greek\nforces to get into Janina, he preferred to defend himself to the very\nlast bastion. All the forces he had consisted of four hundred and\nthirty Albanians, but this number was quite sufficient to serve the\nguns. Even if but a tenth of this force remained to him, that would be\namply sufficient to defend the red tower, and if the worst came to the\nworst, Ali alone would be sufficient to blow the place into the air.\n\nHere Ali had accumulated all his treasures, all his arms, his\ngarments, his correspondence with the princes of half the universe,\nhis young damsels. In the cellar below the tower were piled up a\nthousand barrels of gunpowder, a long match reached from one of these\nbarrels to Ali's chamber, and there a couple of torches were always\nburning by his side.\n\nWhoever wanted Ali's head had better come for it!\n\nSo Bozzari returned to Arta, and not very long afterward the Greek\narmy took the place by storm. In the whole fortress they did not find\npowder enough to fill a hole in the barrel; the Turkish army had, in\nfact, fired away its very last cartridge.\n\nAli had once more the satisfaction of seeing one of his enemies,\nSalikh Pasha, prostrate. Hitherto all who had fought against him had\nbeen his furious haters, personal enemies, enviers of his fortune;\nand, bitter hater as he was, it was with a strong feeling of\nsatisfaction that Tepelenti saw them all bite the dust; but this\nKurshid was quite indifferent to him, and knew nothing either of his\nfury or his intrigues. He had never been Ali's enemy, and had no\nreason for hating him. This thought made Ali uneasy.\n\nIt had often been Ali's experience that when any one who greatly hated\nhim came during a siege or a battle within shooting distance of him,\nand he then pointed a gun at him, the ball so fired seemed to fly on\nthe wings of his own savage fury, and would hit its man even at a\nthousand paces; but Kurshid often took a walk near the trenches, and\nthough they fired at him one gun after another, not a bullet went near\nhim.\n\n\"Let him alone,\" said Ali; \"we shall never be able to kill this man.\"\nAnd his old energy left him as if he had suddenly become crippled.\n\nHe invited Kurshid Pasha to intercede for him with the Sultan, that he\nmight be restored to favor, offering in such case to place his\ntreasures at the disposal of the Grand Signior, and turn his arms\nagainst the Greeks. Kurshid demanded an assurance to this effect in\nwriting, and when Ali complied, Kurshid sent the document, not to the\nSultan at Stambul but to the Suliotes at Arta, that they might see how\nready Ali was to betray them. The Greeks, in disgust, abandoned Ali.\nThis last treachery dismayed them at the very zenith of their triumph;\nthey perceived that a mighty antagonist had risen against them in\nKurshid Pasha, who was magnanimous enough not to make use of traitors,\nbut spurn them with contempt. This intellectual superiority guaranteed\nthe success of Kurshid's arms. The Turkish commander had been acute\nenough to extend the hand of reconciliation, not to Ali, but to the\nSuliotes.\n\nTepelenti waited in vain in the tower of Janina for the arrival of the\narmy of deliverance. The Suliotes returned to their villages, and\nArtemis reflected with secret joy that in the very red tower in which\nAli had decapitated her plighted lover, he himself now sat in his\ndespair, environed by foes, waiting with the foolish hope that the\nembittered Suliotes would hasten to deliver him.\n\nThe Epirote rebellion was already subdued by Kurshid Pasha, and only\none point in the whole empire now glowed with a dangerous fire--the\nhaughty Janina.\n\n\n\n\nCHAPTER XV\n\nCARETTO\n\n\nAli had now only about room enough to cover his head. His enemies had\ntwenty times as much, and they besieged him night and day. The\nfortress on the hill of Lithanizza and the Isle of La Gulia were in\nKurshid's power already.\n\nStill the old warrior did not surrender. The bombs thrown into the\nfortress levelled his palaces with the ground. His marble halls were\nreduced to rubbish heaps, his kiosks were smoking ruins, and his\nsplendid gardens lay buried, obliterated. Yet, for all that, Ali Pasha\nvomited back his wrath upon the besiegers out of eighty guns, and it\nhappened more than once that hidden mines exploded beneath the more\nforward advanced of the enemy's batteries, blowing guns and gunners\ninto the air.\n\nThe defence was conducted by an Italian engineer whom Ali had enticed\ninto his service in his luckier days with the promise of enormous\ntreasures and detained ever since. This Italian's name was Caretto. It\nwas his science that had made Janina so strong. The clumsy valor of\nthe Turkish gunners fell to dust before the strategy of the Italian\nengineer. Of late Caretto was much exercised by the thought that he\nmight be discharged without a farthing, but discharge was now out of\nthe question. If Caretto were outside the gates of Janina, then the\nfate of Janina would be in his hands, for every bastion, every\nsubterranean mine, every corner of the fortress was known to him.\n\nNow at home in Palermo was Caretto's betrothed, who, as the daughter\nof a wealthy family, could only be his if he also had the command of\nriches; and that was the chief reason why the youth had accepted the\noffer of the tyrant of Epirus. And now tidings reached him from Sicily\nthat the parents of his bride were dead, and that she was awaiting him\nwith open arms; let him only come to her, poor fellow, even if he\nbrought nothing with him but the beggar's staff. And go he could not,\nfor Ali Pasha held him fast. He had to point the guns, and send forth\nhissing bullets amongst the besiegers, and defend the fortress to the\nlast, while his beloved bride awaited him at home.\n\nOne day, as Caretto was directing the guns, a grenade fired from the\nheights of Lithanizza burst over his head and struck out his left eye.\nCaretto asked himself bitterly whether his bride would be able to love\nhim with a face so disfigured. Henceforth he went about constantly\nwith a black bandage about his wounded face, and the besiegers called\nhim \"the one-eyed Giaour.\"\n\nOne fine morning in February Kurshid Pasha again directed a fierce\nfire against the fortress. The siege guns had now arrived which the\narmy had used against Cassandra, and after a three hours' cannonade,\nthe destructive effect of the new battery was patent, for the tower of\nthe northern bastion lay in ruins. Ali Pasha galloped furiously up and\ndown the bastions, stimulating and threatening the gunners with a\ndrawn sword in his hand. Whoever quitted his place instantly fell a\nvictim beneath Ali's own hand. Caretto was standing nonchalantly\nbeside a gabion, whence he directed the fire of the most powerful of\nall the batteries, each gun of which was a thirty-six pounder. The\nguns of this battery discharged thirty balls each every hour.\n\nAll at once the battery stopped firing.\n\nTransported with rage, Ali Pasha at once came galloping up to Caretto.\n\n\"Why don't you go on firing?\" he cried.\n\n\"Because it is impossible,\" replied the engineer, coolly folding his\narms.\n\n\"Why is it impossible,\" thundered the pasha, his whole body convulsed\nwith rage, which the coolness of the Italian raised to fever heat.\n\n\"Because the guns are red-hot from incessant firing.\"\n\n\"Then throw water upon them!\" cried Ali, and with that he dismounted\nfrom his horse.\n\nCaretto, for the life of him, could not help laughing at this\nsenseless command. Whereupon Tepelenti suddenly leaped upon him and\nstruck him in the face, so that his cap flew far away, right off the\nbastion. He had struck Caretto on the very spot where Kurshid Pasha's\ngrenade had lacerated his face a few weeks before.\n\nThe Italian readjusted over his eye the bandage, which had been\nknocked all awry by the blow, and observed, with a cold affectation of\nmirth:\n\n\"You did well, sir, to strike my face on the spot where one eye had\nbeen knocked out already, for if you had struck me on the other side\nyou might have knocked out the other eye also, and then how could I\nhave pointed your guns?\"\n\nAli, however, pretended to take no notice, but directed that the guns\nshould be douched with cold water and then reloaded; he himself fired\nthe first. The cannon the same instant burst in two and smashed the\nleg of a cannonier standing close to it.\n\n\"It does not matter,\" cried Ali; \"load the others, too.\"\n\nWhen the second cannon also burst he dashed the match to the ground,\nthrew himself on his horse, and galloped off, quivering in every nerve\nas if shaken by an ague.\n\nThe Italian, however, with the utmost _sang-froid_, ordered that the\nexploded cannons should be removed and fresh ones fetched from the\narsenal and put in their places, and set them in position amidst a\nshower of bullets from the besiegers. When the battery was ready the\nenemy withdrew their siege guns, and till the next day not another\nshot was fired against Janina.\n\nTepelenti was well aware that he had mortally offended Caretto, and he\nhad learned to know men (especially Italians) only too well to imagine\nfor an instant that Caretto, for all his jocoseness on the occasion,\nwould ever forget that cowardly and ungrateful blow. For, indeed, it\nwas an act of the vilest ingratitude. What! to strike the wound which\nthe man had received on his account! To strike a European officer in\nthe face! Ali was well aware that such a thing could never be\npardoned.\n\nThe same night he sent for two gunners and ordered them not to lose\nsight of Caretto for an instant, and if he attempted to escape to\nshoot him down there and then.\n\nNext day Caretto was unusually good-humored. Early in the morning he\nwent out upon the ramparts, which were then covered with freshly\nfallen snow. The winter seemed to be pouring forth its last venom, and\nthe large flakes fell so thickly that one could not see twenty paces\nin advance.\n\n\"This is just the weather for an assault,\" said Caretto in a loud\nvoice to the Turks standing around him; \"in such wild weather one\ncannot see the enemy till he stands beneath the very ramparts. I will\nbe so bold as to maintain that Kurshid's bands are likely to steal\nupon us under cover of this thick snow-storm. I should like to fire a\nrandom shot from the ramparts to let them know we are awake.\"\n\nMany thought his anxiety just. Ali Pasha was also there, and he said\nnothing either for or against the proposal.\n\nCaretto hoisted a cannon to the level of the ramparts of Lithanizza\nand fastened a long chain to the gun whereby his group of Albanians\ncould raise and lower it.\n\n\"Leave the chain upon it,\" said Caretto, \"for we may have to turn it\nin another direction.\"\n\nNevertheless it was in a good position already. Caretto calculated his\ndistances with his astrolabe, then pointed the gun and ordered it to\nbe loaded.\n\nThe two gunners whom Ali had set to watch him never took their eyes\noff the Italian; both of them had loaded pistols in their hands.\nCaretto did not seem to observe that they were watching him; he might\nhave thought that they were there to help him.\n\nThe gun had to be turned now to the right and now to the left.\nCaretto himself took aim, but the clumsy Albanians kept on pushing the\nheavy laffette either a little too much on this side or a little too\nmuch on that, till at last he cried to the two watchers behind him:\n\n\"Just lend a hand and help these blockheads!\" They stooped\nmechanically to raise the laffette. \"Enough!\" cried the Italian, and\nwith that he put his hand on the touch-hole. \"Now fire!\" he cried to\nthe artilleryman, at the same time removing his hand.\n\nThe match descended, there was a thunderous report, and the same\ninstant Caretto seized the chain wound round the wheel of the cannon,\nand, lowering himself from the ramparts, glided down the chain.\n\nThe watchers, with the double velocity of rage and fear, rushed to the\nbreastwork of the ramparts. Caretto had got to the end of the chain\nand was grasping it with both hands; below him yawned a depth of\nthirty feet. The chain was not long enough, and there he was suspended\nbetween two deaths.\n\n\"Come back,\" cried the watchers, aiming their pistols at his head, \"or\nwe will shoot you through and through!\"\n\nCaretto cast a wild glance upward, the bandage fell from his bloody\neye, and he looked at them with the dying fury of a desperately\nwounded wild beast. Then suddenly he kicked himself clear of the wall\nby a sharp movement of his foot, and describing the arc of a circle,\nhe plunged into the depth beneath him like a rebounding bullet. The\nAlbanians fired after him, but neither of them hit him. Below, at the\nfoot of the bastion, the daring Italian lay motionless for a moment,\nbut then he quickly rose to his feet and began to clamber up the other\nside of the ditch. He could only make use of one arm, for the other\nhad been dislocated in his fall. Straining all his might, he struggled\nup; a whole shower of bullets pursued him and whistled about his head,\nbut not one of them hit him, for the heavy snowfall made it difficult\nto take aim. At last he reached the top of the opposite side of the\ntrench, and then he turned round and shook his fist at the devastating\nfortress, and disappeared in a heavy snow-drift. The gunners kept on\nfiring after him at random for some time.\n\nAli Pasha turned pale and almost fell from his horse when the tidings\nreached him that Caretto had escaped.\n\n\"It is all over now!\" cried he in despair, broke his sword in two, and\nshut himself up in the red tower. In the outer court-yard they saw him\nno more.\n\nAli knew for certain that with the departure of Caretto the last\nremains of his power had vanished; his stronghold and its resources\nwere hopelessly ruined if any one revealed their secrets to his\nenemies outside. Caretto knew everything, and \"the one-eyed Giaour\"\nwas received with great triumph in the camp of Kurshid Pasha. The next\nday Ali Pasha had bitter experience of the fact that the hand which\nhad hitherto defended him was now turned against him. Within nine\nhours a battery, constructed by Caretto, had made a breach thirty\nfathoms wide in the outworks of Janina; the other cannons of the\nbesiegers were set up in places whither Ali's mines did not extend,\nand when he made new ones they were immediately rendered inoperative\nby countermining, and at last Caretto discovered the net-work of\nhidden tunnels at the head of the bridge, although they had been\ncarefully buried, and after a savage struggle forced his way through\nthem into the fortress. The Albanians fought desperately, but Ali's\nenemies, who could afford to shed their blood freely, forced their way\nthrough and planted their scaling-ladders against the side of the\nfortress opposite the island, and where the _debris_ of the\nbattered-down wall filled up the ditch they crossed over and occupied\nthe breach. In the evening, after a fierce combat in the court-yard,\nTepelenti's forces were cut to pieces one by one, and he himself, with\nseventy survivors, took refuge in the red tower.\n\nSo only the red tower now remained to him.\n\n\n\n\nCHAPTER XVI\n\nEMINAH\n\n\nThe vanquished lion was shut up within a space six yards square; a\nnarrow tower into all four windows of which his enemies were peeping\nwas now his sole possession! There he sits in that octagonal chamber,\nin which he had passed so many memorable moments. Perhaps now, as he\nleaned his heavy head upon his hand, the remembrance of those moments\npassed before his mind's eye like a procession of melancholy shadows.\nAround him lay his treasures in shining piles; heaps of gold and\nsilver, massive gold plate, the spoils of sanctuaries, sparkling gems,\nlay scattered about the floor higgledy-piggledy, like so much sand or\ngravel.\n\nOf all his kinsfolk, of all his warriors, not one was present with\nhim; all had fallen on the battle-field, fighting either with him or\nagainst him. Of the seventy warriors who had taken refuge with him in\nthe tower, sixty-four had deserted him. Kurshid had promised a pardon\nto the renegades, and only six remained with Ali. Why did these six\nremain? Ali had not told them not to leave him.\n\nThese faithful ones were keeping guard in his antechamber, and for\nsome little time they had been whispering together.\n\nAt last they went in to Ali.\n\nTepelenti looked them every one through and through. He could read\nwhat they wanted in their confused looks and their unsteady eyes. He\ndid not wait for them to speak, but said, with a wave of his hand:\n\n\"Go! leave me; you are the last. Go where the others have gone; save\nyourselves. Life is sweet; live long and happily. I will remain here.\nTepelenti can die alone.\"\n\nSighing deeply, the soldiers turned away. They durst not raise their\neyes to the face of the gray-haired veteran. Noiselessly, without a\nword, on the tips of their toes, five of them withdrew. But the sixth\nremained there still, and, after casting about for a word for some\ntime, said, at last, to Ali:\n\n\"Oh, sir, cast the fulness of pride from thy heart, suffer not thy\nname to perish! The Sultan is merciful; bow thy head before him and he\nwill still be gracious to thee!\"\n\nThe soldier had scarce uttered the last word of this recommendation\nwhen Ali softly drew a pistol from his girdle and shot him through the\nhead, so that he spun round and fell backward across the threshold.\nThis was all the reward he got for advising Ali to ask for mercy.\n\nAnd now Ali is alone. His doors, his gates stand wide open; anybody\nwho so pleases can go in and out. Why, then, does nobody come to seize\nthe solitary veteran? why do they fear to cross the threshold of the\nvanquished foe?\n\nBut hearken! fresh footsteps are resounding on the staircase, and\nthrough the open door, guarded by the corpse of the last soldier whom\nAli slew, a strange man entered, dressed in an unusual, new-fangled\nuniform; he was Kurshid Pasha's silihdar.\n\nTepelenti allowed him to approach within five paces of where he sat,\nand then beckoned him to stop.\n\n\"Speak; what dost thou want?\"\n\n\"Ali Tepelenti,\" said the silihdar, \"surrender. Thou hast nothing left\nin the world and nobody to aid thee. My master, the seraskier, Kurshid\nPasha, hath sent me to thee that I might receive thy sword and escort\nthee to his camp.\"\n\nTepelenti, with the utmost _sang-froid_, drew forth from the folds of\nhis caftan a magnificent gold watch in an enamelled case set with\ndiamonds.\n\n\"Hearken!\" said he, in a low, soft voice. \"It is now twenty minutes\npast ten; take this watch and keep it as a souvenir of me. Greet\nKurshid Pasha from me, and point out to him that it was twenty minutes\npast ten when you spoke with me, and let him take notice that if after\ntwenty minutes past eleven I can see from the windows of this tower a\nsingle hostile soldier in the court-yard of the fortress, then--I\nswear it by the mercies of Allah!--I will blow the fortress into the\nair, with every living soul within it. Inform Kurshid Pasha of this\nwhen you give him my salutation.\"\n\nThe silihdar hastened off, and at a quarter to eleven not a soul was\nto be seen in the court-yard of the fortress of Janina. Alive in his\ncitadel sits Ali Tepelenti, the tyrant of Epirus, mighty even in his\nfall, who has nothing and nobody left, save only his indomitable\nheart.\n\nNight descended upon the fortress of Janina, but sleep did not descend\nupon the eyes of Ali.\n\nHe sat in that red tower where he had perpetrated his crimes, in that\nchamber where his victims had breathed forth the last sighs of their\ntortured lives, and all round about glittering treasures looked upon\nAli as if with eyes of fire--all of it the price of robbery, fraud,\ntreason. What if these things could speak?\n\nEverything was silent, night lay black before the eyes of men, only\nAli saw shadows moving about therein, phantoms with pale, phantoms\nwith bloody faces, who rose from the tomb to visit their persecutor\nand announce to him the hour of his death.\n\nAli trembled not before them; he had seen them at other times also. He\nhad slept face to face with the severed head that spoke to him, he had\nlistened to the enigmatical words of the _dzhin_ of Seleucia, and he\ncalled them to mind again now. Calmly he looked back upon the current\nof his past life, from which so many horrible shapes arose and glared\nat him with cold, stony eyes. He recked them not, Allah had so ordered\nit. The hare nibbles the root, the vulture devours the hare, the\nhunter shoots the vulture, the lion fells the hunter, and the worm\neats the lion. What, after all, is Ali? Naught but a greater worm than\nthe rest. He has devoured much, and now a stronger than he devours\nhim, and a still greater worm will devour this stronger one also.\n\nEverything was fulfilled which had been prophesied concerning him. His\nown sons, his own wife, his own arms had fought against him. If only\nhis wife had not done this he could have borne the rest.\n\n\"One, two,\" the decapitated head had said, and the last moments of\nthe two years were just passing away. \"The hand which wipes out the\ndeeds of the mighty shall at last blot out thy deeds also, and thou\nshalt be not a hero whom the world admires, but a slave whom it\ncurses. Those whom thou didst love will bless the hour of thy death,\nand thy enemies will weep, and God will order it so to avert the ruin\nof thy nation.\"\n\nSo it is, so it has chanced; the hazard of the die has gone against\nhim, and he has nothing left.\n\nIf only his wife had not betrayed him!\n\nAt other times also Ali had seen these phantoms of the night arise. He\nhad seen them rise from the tomb pale and bloody; but in his heart\nthere had always been a sweet refuge, the charming young damsel whose\nchildlike face and angelic eyes had robbed the evil sorcery of all its\npower. When Tepelenti covered his gray head with her long, thick,\nflowing locks, he reposed behind them as in the shade of Paradise,\nwhither those heart-tormenting memories could not pursue him. Why\nshould he have lost her? She was the first of all, and the dearest;\nbut Fate at the last would not even leave him her.\n\nEven now his thoughts went back to her. The pale light of that face,\nthat memory, lightened his solitary, darkened soul, which was as\ndesolate as the night outside.\n\nBut lo! it is as if the night grew brighter; a sort of errant light\nglides along the walls and a gleam of sunshine breaks unexpectedly\nthrough the open door of the room.\n\nThe pasha looked in that direction with amazement. Who could his\nvisitor be at that hour? Who is coming to drive the phantoms of\ndarkness from his room and from his heart?\n\nA pale female form, with a smile upon her face and tears in her eyes,\nappears before him. She comes right up to the spot where Tepelenti is\nsitting on the ground. She places her torch in an iron sconce in the\nwall and stands there before the pasha.\n\nAli looked at her sadly. He fancied that this also was only a dream\nshape, only one of those apparitions created by a fevered mind, like\nthose which walked beside him headless and bloody. It was Eminah, at\nwhose word the devastating tempest had been unchained against the\nmightiest of despots.\n\nTepelenti believed neither his eyes nor his heart when he saw her thus\nbefore him. The damsel took the old man by the hand and called him by\nhis name, and even now the pasha believed that the warmth of that hand\nand the sweetness of that voice were only part of a dream.\n\n\"Wherefore hast thou come?\" he inquired in a whisper, or perchance he\ndid not ask but only dreamed that he asked.\n\nYet the gracious, childlike damsel was sitting there at his feet as at\nother times, and she had pillowed his gray head upon her breast and\ncovered his face with the tent of her long tresses, as she had done\nlong, long ago in the happy times that were gone.\n\nOh, how sweet it would be to still live!\n\n\"Oh, Ali Tepelenti, let go the hand of Death from thy hand and grasp\nmy hand instead! See how warm it is! Oh, Ali Tepelenti, rise up from\namong these barrels of gunpowder, and rather lay thy head upon my\nbreast; hearken how it beats! Oh, Ali Tepelenti, ask mercy from the\nSultan! See, now how lovely life is!\"\n\nOnly at these words did Ali recover himself. His enemies had sought\nout this woman, the only being that he loved, and sent her to him to\nsoothe away the rage of his soul and soften his heart with her\ncaresses. Oh, how well they understood his heart!\n\n\"Kurshid Pasha swore to me that he would obtain the Sultan's favor for\nthee,\" said Eminah, in a tone of conviction. \"He wrote a letter under\nhis seal that thou shouldst never die beneath the hands of the\nexecutioner; that thy death should not be a violent one, unless it\nwere in an honorable duel or on the field of battle. Behold, here is\nthe letter!\"\n\nIf at that moment Ali had listened to his heart, he must have extended\nthe hand of submission without any letter of amnesty, but, like an\nescutcheon above a crown, pride was perched higher than his heart and\nspurned the offer.\n\n\"Allah may humble Ali, but Ali will never humble himself.\"\n\n\"Then thou wilt not live with me?\" asked Eminah, fixing her piteously\nentreating eyes upon her husband.\n\nAli shook his head in silence.\n\n\"Then I will die with thee!\" cried the damsel, with a determined\nvoice.\n\nThe pasha regarded her in amazement.\n\n\"I swear,\" cried Eminah, \"that I will either go back with thee or die\nwith thee here! Dost thou hear that noise? They are slamming to the\niron gates from the outside. At this moment every exit is closed, so\nthat even if I wished to escape from hence I could not. These doors\ncan only open at a word from Ali, and they will only open once more.\nEither thou wilt go with me from hence or I will remain here with\nthee.\"\n\nAli pressed the damsel to his bosom. She lay clinging there like a\ntender blossom. He pressed his lips to that pale brow, and covering\nher gently and gradually with his silken caftan, he whispered in a\nscarcely audible voice:\n\n\"Be it so! be it so! Here we will die together!\"\n\nEarly next morning a flourish of trumpets awoke the Lord of Janina,\nthe Lord of the last tower of Janina. The herald of Kurshid Pasha was\nstanding beneath the round windows, and delivered in a loud voice the\ngeneral's message to Ali Pasha, whereby he summoned Tepelenti to\nsurrender voluntarily on the strength of the solemn assurance\nconfirmed by oath to his wife.\n\nTepelenti appeared at the window with Eminah reclining on his bosom.\n\n\"Go back to your master,\" he cried to the messenger, \"and tell him\nthat Ali and his wife have resolved to die here together. The moment\nan armed host enters the court-yard of this fortress I will\nimmediately blow up the tower.\"\n\nIn half an hour the messenger returned and again summoned Ali to the\nwindow.\n\n\"Kurshid Pasha sends thee this message,\" cried he. \"If thou dost\nsurrender, it is well, and if thou dost not surrender, it is well\nalso. Thou hast still half an hour wherein thou mayest choose betwixt\nlife and death. After that thou mayest, if thou wilt, throw thy torch\ninto thy powder barrels and blow the fortress into the air. As to\nthyself, Kurshid Pasha troubles himself but little. As to thy\ntreasures they will not remain in the air, and when they come to the\nground it will be easy to pick them up. If, however, thou dost delay\nthy resolution beyond the half-hour, then Kurshid Pasha himself will\nhelp thee in the matter, and will blow up thy tower for thee, to save\nthee the trouble of blowing it up thyself. Do as thou wilt, then, and\nhoist either the white or the red flag as seemeth best to thee, for in\nhalf an hour the fortress of Janina shall see thee no more.\"\n\nAli listened solemnly to this ultimatum, and let the messenger depart\nwithout an answer.\n\nEminah lay down on a sofa in a corner, all trembling. Ali paced the\nvast chamber to and fro with long strides; but his strides became more\nand more uncertain. If only this woman were not here! If only he might\nbe spared seeing her before him; might be spared half an hour's\ndeliberation as to what he was to do! Nevertheless minute after minute\nsped away, and still Tepelenti could not make up his mind. Twice his\nhand seized the burning torch; he had but to bend over the nearest\nbarrel of powder and all would be over; but on each occasion his eye\nfell upon the trembling woman who lay there looking at him without a\nword, and the death-bearing match fell from his hand. No, no; he was\nincapable of doing the terrible deed. And now the hour struck; the\ntime had passed. Ali felt a pressure about his heart. Would Kurshid\naccomplish his dreadful threat?\n\nAt that instant a report sounded outside the fortress, and half a\nmoment later a red-hot steel bullet burst through the metal roof and\nthe massive vault of the tower with a violent crash. Falling heavily\non the marble floor, it rebounded thence, and, passing between the\npowder-barrels, describing a wide semicircle as it went, ricocheted\nonce more and struck the wall opposite, in which it bored a deep hole,\nwhence it flashed and gleamed with a strong red glare, forcing blue\nsparks from the nitrous humidity of the walls.\n\nAli was now convinced that the enemy was quite capable of keeping his\npromise.\n\nThe scared woman, mad with terror, flung herself at his feet, and\nsnatching the white veil from her head, forced it into the pasha's\nhand.\n\nTepelenti hastily seized the veil, and, hanging it on the point of a\nlance, hoisted it out of the round window.\n\nOutside the besiegers set up a shout of triumph. Eminah, kissing Ali's\nhands, sank down at his feet. Tepelenti had given her more than\nmanhood can bear to give: for her sake he had humbled his pride to the\ndust. If only he could have died as he had lived!\n\n\"Go, now,\" he said to the woman, with a sigh; \"go and tell my enemies\nthat they may come for me. I am theirs!\"\n\n\n\n\nCHAPTER XVII\n\nTHE SILVER PEDESTAL IN FRONT OF THE SERAGLIO\n\n\nThe emissaries of Kurshid Pasha received the veteran warrior with\ngreat respect in the gates of the fortress, whither he went to meet\nthem; they showed him all the honor due to his rank; they allowed him\nto retain his sword and all his other weapons. At the same time they\nconfirmed by word of mouth the promise which Kurshid Pasha had given\nto Eminah in writing--that the executioner should never lay his hand\non Ali's head, and that he should not die a violent death, except it\nwere in an honorable duel or on the battle-field, which is a delight\nto a true Mussulman.\n\nA former pleasure-house, a kiosk on the island of La Gulia, was\nassigned to him as a residence for the future. There they conveyed his\nfavorite horses, his favorite slaves and birds, and took abundant care\nof his personal comfort.\n\nAli allowed them to do with him as they would. Neither threatening nor\npleasant faces made any impression upon him; he merely looked from\ntime to time at his wife, who had seized his hand, and never left him\nfor an instant. At such times softer, gentler feelings were legible in\nhis face; but at other times he would gaze steadily before him into\nthe distance, into infinity. Perhaps he was now thinking within\nhimself, \"When shall I stand in front of the Seraglio on a silver\npedestal?\"\n\nThe _dzhin_ of Seleucia had prophesied this termination to his career.\nAll the other prophecies had been strictly fulfilled; this only\nremained to be accomplished.\n\nA Mussulman's promise is stronger than his oath. Who does not remember\nthe story of the Moorish chieftain in whose house a Christian soldier\nhad taken refuge, and who begged for his protection? The Moor promised\nthe man his protection. Subsequently the pursuers informed the Moor\nthat this Christian soldier had killed his son, and still the father\nwould not give up the fugitive, but assisted him to escape, because of\nhis promise.\n\n\"A great lord is the sea,\" says the Kuran; \"a great lord is the storm\nand the pestilence; but a greater lord still is a man's given word,\nfrom which there is no escape.\"\n\nThe Mussulman keeps his word, but beware of a play upon words, for\ntherein lies death. If he has sworn by the sun, avoid the moon, and if\nhe has promised to love thee as a brother, discover first whether he\nhath not slain his brother.\n\nWhen Sulaiman adopted Ibrahim as a son, he swore that so long as he\nlived no harm should befall Ibrahim. Later on, when Ibrahim fell into\ndisgrace, the wise Ulemas discovered a text in the Kuran according to\nwhich he who sleeps is not alive, and they slew Ibrahim while Sulaiman\nslept.\n\nKurshid had given his word and a written assurance that Ali should not\ndie at the hand of the executioner; the document he had given to\nAli's wife, his word he had given in the presence of his whole army;\nand he had escorted Ali Pasha with all due honor to the island kiosk,\npermitting him to retain his weapons and the jewelled sword with which\nhe had won so many victories, with which he had so many times turned\nthe tide of the battle; nay, more, they had selected fifty of Ali's\nown warriors, the bravest and the most faithful, to serve him as a\nguard of honor.\n\nNevertheless, a courier despatched in hot haste to Stambul announced\nthere, from Kurshid Pasha, that the treasures of Ali Tepelenti of\nJanina were in his hands, and that a Tartar horseman would follow in\nthree days with the head of the old pasha. And yet at this very moment\nTepelenti's head stood firmly on his shoulders, and who would dare to\nsay that that head was promised away while his good sword was by his\nside, and good comrades in arms were around him, and the sworn\nassurance of the seraskier rested upon him?\n\nEminah never quitted him for a moment. She was always with him. She\nsat beside him, with her head on his breast, or at his feet, and in\nher hand she carried the amnesty of the seraskier, so that if any one\nshould approach Ali with dangerous designs she might hold it before\nhis eyes like a magic buckler, and ward off the axe of the executioner\nfrom his head.\n\nBut there was nothing to guard against; the executioner did not\napproach Ali. He received, indeed, a great many visitors, but these\nwere all worthy, honorable men, musirs, effendis, officers of the\narmy, who treated him with all respect, and sipped their sherbet-cups\nmost politely, and smoked their fragrant chibooks, exchanging a word\nor two now and then, perhaps, and on taking their leave saluted him in\na manner befitting grave Mussulmans.\n\nHe was allowed free access to every part of the island, and never\nencountered anybody there but his own warriors.\n\nAt such times great ideas would occur to him. Perchance with these\nfifty men he might win back everything once more? And then he would\nhug himself with the thought of the silver pedestal in front of the\nSeraglio, where he was one day to stand, amidst the joyful plaudits of\nthe people; and then the night before him was not altogether dark, for\nhere and there he saw a gleam of hope.\n\nIt was only Eminah who trembled. God has created woman for this very\npurpose; she has the faculty of fearing instead of man, and can\nforesee the danger that threatens him.\n\nWhence will this danger come, and in what shape? Perchance in the\ndagger of the assassin? The woman's bosom stood between it and the\nheart of Ali; the assassin will not be able to pierce it. In a\npoisoned cup, perhaps? Eminah herself tastes of every dish, of every\nglass, before they reach the hands of Ali; the power of the poison\nwould reach her first.\n\nAnd yet danger is near.\n\nOne day they told Ali that an illustrious visitor was coming to see\nhim; Mehemet Pasha, the sub-seraskier and governor of the Morea,\nwished to pay his respects to him.\n\nThis was a great honor for the fallen general. Ali began to be\nsensible that even his enemies respected him. Who knows? he might find\ngood friends amongst his very enemies, who would not think him too\nold for use and employment even in his last remaining years.\n\nOn the day of the visit, the kiosk was swept and garnished. Tepelenti\nput on his most costly caftan, his warriors were marshalled in front\nof his dwelling, and he himself went out on horseback to meet the\nseraskier when he arrived, with an escort of one hundred mounted\nspahis.\n\nMehemet Pasha was a tall, powerful man, the hero of many a fight and\nmany a duel. He had often given proof of his dexterity, when the\nhostile armies stood face to face, by galloping betwixt them and\nchallenging the bravest warriors on the other side to single combat,\nand the fact that he was alive at the present moment was the best\npossible proof that he had been always victorious.\n\nThe two heroes exchanged greetings when they met, and returned\ntogether to the pleasure-house. Ali conducted the sub-seraskier into\nthe inner apartments; the attendants remained outside.\n\nA richly spread table awaited them, and they were waited upon by a\ngroup of young odalisks, the hand-maidens of Eminah, who sat at Ali's\nfeet on the left-hand side, and, as usual, tasted of every dish and\ncup before she gave it to Ali.\n\nPleasant conversation filled the intervals of the repast, and at the\nend of it a mess of preserved pistachios was brought in and presented\nto Mehemet Pasha.\n\n\"I thank thee,\" said he, \"and, indeed, I am very fond of them, but\npiquant, hot-spiced meats always awaken within me sinful desires and a\nlonging for wine which is forbidden by the Prophet, and, as a good\nMussulman, I would rather avoid the occasion of sinning than suffer\nthe affliction of a late repentance.\"\n\nAli laughed aloud.\n\n\"Eat and be of good cheer, valiant seraskier,\" said he, \"and set thy\nmind at rest. What I give thee shall be wine and yet not wine--the\njuice of the grape, yet still unfermented; 'tis an invention of the\nFranks. This the Prophet does not forbid.[12] I have still got a case\nof bottles thereof, which Bunaberdi[13] formerly sent me, and we will\nnow break it open in thy honor. Truly fizz is not wine, but only the\njuice of the grape which they bottle before it becomes wine. It is as\nharmless as milk.\"\n\n[Footnote 12: The Moslems do not include French \"fizz\" amongst the\ncanonically forbidden drinks.]\n\n[Footnote 13: Bonaparte.]\n\nMehemet shook his head and laughed, from which one could see that the\nproposition was not displeasing to him, whereupon Ali beckoned to the\nodalisks to fetch the bottles from the cellar.\n\nEminah, all trembling, bent over him and whispered, imploringly, \"Oh,\nput not wine on thy table; it will be dangerous to thee!\"\n\nAli smiled, and stroked his wife's head. He thought that only\nreligious scruples made her dissuade him from drinking the wine, so he\ndrew her upon his bosom and began to reassure her.\n\n\"Say now, my one and only flower, is not Moses a prophet, like unto\nMuhammad?\"\n\n\"Of a truth he is. His tent stands beside the tent of Muhammad in the\nParadise of the true Believers.\"\n\n\"And yet Moses said: Give wine to them that be sorrowful! Leave the\nmatter then to the two prophets up above there; surely, what passes\nthorough our lips does not make us sin?\"\n\nBut that was not the reason why Eminah feared the wine.\n\nThey brought the bottles, and the liberated corks popped merrily. At\nfirst Mehemet Pasha hesitated, but they filled his glass with fizz\nand, to prevent the sparkling foam from running over, he sipped a\nlittle of it, and quickly drained the glass, maintaining afterwards,\nwith a smile, that it was a similar drink to wine, but much more\npleasant.\n\nAli filled once more the glass of the seraskier, while Eminah\ntremulously watched his features, which gradually grew darker as he\ndrank. Drink has this effect on some men.\n\nSuddenly the sub-seraskier dashed his glass upon the table and\nexclaimed, with a furious expression of countenance:\n\n\"I'll drink no more! I'll drink no more! Thou art a villain, Ali! Thou\nhast made me drink wine and hast lied to me, saying it was not wine;\nbut it is wine, a frightful, burning drink, which has made my head\nwhirl.\"\n\n\"Come, come, Mehemet,\" said Ali, in the coaxing tone one uses to\ndrunken men, \"be not so wrathful.\"\n\n\"Speak not to me, thou dog!\" thundered the other, striking the table\nwith his fist. \"I might have known when I dismounted at thy door with\nwhom I had to do, thou sly, treacherous fox, thou godless renegade!\"\n\nAli leaped from his seat with flashing eyes, and clapped his hand on\nthe hilt of his sword at these words; but Eminah seized his hand, and\nsaid to him, in a terrified whisper:\n\n\"Draw not thy sword, Ali; show no weapons here! Dost thou not perceive\nthat he only came hither to fasten a quarrel upon thee?\"\n\nAli instantly recovered himself at these words. He saw now the snare\nthat had been laid for him, and calmly sat down in his place again,\ncrossing his legs beneath him, and, quietly taking up his chibook,\nbegan to smoke with an air of unconcern.\n\nMeanwhile, Mehemet played his drunken _role_ still further.\n\n\"I might have known beforehand, when I sat down at table with thee,\nthat I was sitting down with an accursed wretch, thou blood-thirsty\ndog, who hath lapped up the blood of thy kinsfolk; but I never\nventured to imagine that thou wouldst be audacious enough to make me\ndrink that abominable liquid--may its sinfulness fall back again on\nthine accursed head!\"\n\nWith these words Mehemet caught up the half full glass and pitched all\nthe wine that was in it straight between Ali's eyes, so that it\ntrickled down the full length of his long white beard.\n\nAli, with the utmost _sang-froid_, beckoned to the attendant odalisks\nto place before him a bowl of fresh water, in which he washed his face\nand beard. He did not answer the sub-seraskier a single word.\n\nMehemet planted himself in front of him with a contemptuous\nexpression.\n\n\"Wretched worm! that can wipe away such an insult so tamely! Thou wert\nnever valiant, thy heroic deeds were so many murders. Those whom thou\ndidst slay, thou didst butcher as doth a headsman. Thou couldst\nsurprise like a thief, but to fight like a man was never thy way, and\nthe blood that stains thee is the blood of fettered slaves. Thou\nabominable thing! The very victory is abominable which we have gained\nover such a writhing worm as thou art. I should pity my sword if it\never came into contact with thine. Let others say if they will that\nthey have conquered Ali, I will only say that I have struck Ali\nTepelenti in the face.\"\n\n\"By Allah, the one true God, that thou shall never say!\" thundered\nAli, leaping from his seat; and quickly drawing his sword, he whirled\nit like a glittering circle through the air.\n\nMehemet retreated a step backward, and drew his Damascus blade with a\nsatisfied air.\n\n\"Fight not, Ali; go inside!\" exclaimed Eminah, violently seizing Ali\nby the sword-arm.\n\nTepelenti shook her off and, with his sword flashing above his head,\nfell upon the sub-seraskier. Mehemet parried the stroke with his\nsword, and the next instant a huge jet of blood leaped into the air\nfrom Ali's shoulder.\n\nEminah, full of despair, flung herself between the combatants. She saw\nthat Ali was bleeding profusely, and throwing one arm around his knee,\nwith the other hand she held up before the seraskier the amnesty of\nKurshid Pasha.\n\n\"Look at that! The general swore that Tepelenti should not be slain.\"\n\n\"Not by the executioner,\" replied Mehemet; \"but he did not guarantee\nhim against the sword of a warrior. Come, thou coward! or wilt thou\nhide behind the petticoat of thy wife?\"\n\nEminah stretched out her arms towards Ali, but the old man thrust her\naside and rushed upon Mehemet Pasha once more; but before he could\nreach him another thrust pierced him through the heart. Without a sob\nhe collapsed at the feet of his foe.\n\nThe terrified odalisks rushed shrieking into the camp, whilst outside\na bloody combat began between the warriors of Mehemet and the warriors\nof Ali. The former were numerous, so it was not long before\nTepelenti's guards were cut down, and Mehemet, with a contented\ncountenance, returned to camp. A silken-net bag was hanging to his\nsaddle-bow, and in it was the head of Ali.\n\nKurshid Pasha washed his hand when the head was placed before him.\n\n\"I was not the cause of thy death!\" he cried. \"I guaranteed thee\nagainst the headsman, but not against the sword of warriors. Why didst\nthou provoke the lion?\"\n\nOn the day fixed, beforehand, the Tartar horseman arrived in Stambul\nwith the head of Ali. The hours of his life had been calculated\nexactly. An astronomer who determines the distances between\nconstellation and constellation is not more accurate in his\ncalculations than was Kurshid in determining the date of his enemy's\ndeath.\n\nOn that day the Sultan held high festival.\n\nThe Tsirogan palace, the Seraglio, all the fountains were illuminated,\nand Ali's head was carried through the principal streets of the town\nin triumphal procession, and finally exhibited on a silver salver in\nfront of the middle gate of the Seraglio in the sight of all the\npeople.\n\nSo there he stood at last, on a silver pedestal in front of the\nSeraglio. And the prophecy was fulfilled which had said, \"A time will\ncome when thou shalt be in two places at once, in Stambul and in\nJanina!\" So it was.\n\nAli's dead body was buried at Janina, and his head, at the same time,\nwas standing in front of the Seraglio. At Janina, a single mourning\nwoman was weeping over the headless corpse; at Stambul a hundred\nthousand inquisitive idlers were shouting around the bodyless head.\n\nAt that gate where the head of Ali was exhibited the throng was so\ngreat that many people were crushed to death by the gaping\nsight-seers, who had all come hither to stare at the gray-bearded\nface, before whose wrathful look a whole realm had trembled.\n\nAt last, on the evening of the third day, when the well-feasted mob\nhad stared their fill and begun to disperse, there drew nigh to the\ngate of the Seraglio an old yellow-faced fakir who, from the\nappearance of his eyes, was evidently blind. His clothing consisted of\na simple sackcloth mantle, girded lightly round the waist by a cotton\ngirdle, from which hung a long roll of manuscript; on his head he wore\na high mortar-shaped hat, the distinguishing mark of the Omarites.\n\nAll the people standing about respectfully made way for him as, with\ndowncast eyes and hands stretched forth, he groped his way along, and,\nwithout any one guiding him, made his way straight up to Tepelenti's\nhead.\n\nThere he stood and laid his right hand on the severed head, none\npreventing him.\n\nAnd lo! it seemed to those who stood round as if the severed head\nslowly opened its eyes and looked upon the new-comer with cold, stony,\nstiff, dim eyeballs. This only lasted for a moment, and then the\nOmarite took his hand off the head and the eyes closed again. Perhaps\nit was but an illusion, after all!\n\nThen the dervish spoke. His deep, grave voice sank into the hearts of\nall who heard him: \"Go to Mahmoud, and tell him that I have bought\nfrom him the head of Ali Pasha and the heads of his three sons,\nSulaiman, Vely, and Mukhtar, and a whole empire is the price I pay him\ntherefor.\"\n\n\"What empire art thou able to give?\" inquired the captain of the\nciauses who were guarding the head.\n\n\"That which is the fairest of all, that which is nearest to his heart,\nthat which he had the least hope of--his own empire.\"\n\nThese bold words were reported to the Sultan, and the Grand Signior\nsummoned the Omarite dervish to the palace, and shut himself up alone\nwith him till late at night. When the muezzin intoned the fifth\nnamazat, towards midnight, Mahmoud dismissed the dervish. What they\nsaid to each other remained a secret known only to themselves. The\nfakir, on emerging from the Sultan's dressing-room, plucked a piece of\ncoal from a censer, and wrote on the white alabaster wall this\nsentence, \"Rather be a head without a hand than a hand without a\nhead,\" and nobody but the Sultan understood that saying.\n\nMahmoud commanded that nine purses of gold should be given to the\ndervish; he gave him also the heads of Ali and of Ali's three sons.\n\nThe dervish left the Seraglio with the four heads and the nine\npurses. With the nine purses he bought an empty field in front of the\nSelembrian gate and planted it with cypress-trees, and at the foot of\nevery cypress he set up a white turbaned tombstone--there were\nhundreds and hundreds side-by-side without inscriptions. He said, too,\nthat it would not be long before the owners of these tombs arrived. In\nthe middle of this cemetery, moreover, he dug a wide grave, and in it\nhe buried the heads of Ali's three sons, with their father's head in\nthe middle. He erected four turbaned tombstones over them, two at the\nhead and two at the foot of the grave, and on the largest of these\ntombstones was written: \"Here lies the valiant Ali Tepelenti, Pasha of\nJanina, leaving behind him many other warriors who deserve death just\nas much as he.\"\n\nThe people murmured because of what was written on the tomb, but who\ndurst obliterate what is inscribed on the dwellings of the dead?\n\nThere the mysterious inscription remained on the tomb for four years,\nand in the fourth year its meaning was revealed.\n\nNow this dervish was the _dzhin_ of Seleucia.\n\n\n\n\nCHAPTER XVIII\n\nTHE BROKEN SWORDS\n\n\n \"Allah Kerim!\n Allah akbar!\n Great is God and mighty!\"\n\nWhat avails prayer if there be no longer any to hearken? What avails\nthe bright sword if there be none to wield it? What avails the open\nbook if there be none to understand what is written therein?\n\nYe nations of the half-moon! now is the time when the song of the\ndervishes, and the scimitar, and the dirk, and the Kuran, can help no\nmore! From the west and from the north strange people are coming,\narmed warriors in serried ranks, like a wall of steel, who are set in\nmotion, brought to a stand-still, expanded into an endless line,\ncontracted into a solid mass by a single brief word of command. Before\nthe charge of their bayonets the ranks of the Janissaries scatter and\ndisperse like chaff before the wind, and before their fire-vomiting\nbrazen tubes the flowers of Begtash's garden fall like grass before\nthe mower. Wise men are with them, who go about in simple black coats,\nwho know much that ye do not know; each one of whom is capable of\ndirecting a state, and who are equally triumphant on the battle-field\nand in the council-chamber.\n\nIn vain ye call upon the name of the Prophet, in vain do ye knock at\nthe gate of Paradise. It is closed. Muhammad slumbers, and the other\nprophets no longer trouble themselves about earthly affairs. Paradise\nis full already. There they look askance now at new-comers, who reach\nthe shadow of the tuba-tree without the rumor of victory. The\neternally young houris, from beyond the Bridge of Alsiroth, no longer\nsmile upon those who fall in battle, for battle has now lost its\nglory. Ye must be born again, or die forever.\n\nLook now! the more far-seeing ones among you know what to do. They\nsend their children far, far away, to the dominions of the Giaours,\nthere to learn worldly wisdom, and prepare to make great changes in\nthe empire.\n\nThe old dervishes, the friends of the Turks, are excluded from the\nSeraglio; they do but creep stealthily up and peep through the guarded\ngates, and compare notes with one another, \"Behold! within there, they\nare doing the work of the stranger, they are teaching the\ntrue-believing warriors to leap to and fro at a word of command, and\ntwirl their weapons. They have abandoned the jiride, that\never-victorious weapon, and have stuck darts at the ends of their\nmuskets, as do the unbelievers, who dare not come within\nsword-distance of the enemy. It is all over, all over with the faith\nof Osman.\"\n\nMost jealous of all these innovations were the priests of Begtash. One\ncould every moment see them in their ragged, dirty mantles, lounging\nabout in front of the gates of the Seraglio, impudently looking in\nthe faces of all who go in and out; and if an imam passed them, or one\nof those wise men who favored the innovations, they would spit after\nhim, and exclaim in a loud voice, \"Death to every one who proclaims\nthe forbidden word!\"\n\nNow this forbidden word was the name \"Neshandchi.\" The mob of Stambul\nhad murdered Mahmoud's father because of this name, which designated a\nnew order of soldiers, and his successor had been compelled to order\nthat whoever pronounced this name should be put to death.\n\nThe mob would often follow the Grand Vizier all the way to the palace,\nreviling him all the way, and shouting up at the windows, \"Remember\nthe end of Bajraktar!\"\n\nBajraktar had been the Sultan's Grand Vizier fourteen years before,\nwho had wished to reform the Turkish army, on which account a riot\nbroke out at Stambul, which lasted till the partisans of Bajraktar\nwere removed from office. As for Bajraktar himself, he was burned to\ndeath in one of his palaces, together with his wife and children.\nEvery one who took part in these mysterious and accursed deliberations\nin the Seraglio, from the lowliest soldier to the sacred and sublime\nSultan himself, carried his life in his hands.\n\nIt had long been rumored that some great movement was on foot, and the\npriests of Begtash went from town to town through all the Turkish\ndomains fanning the fanaticism of their beloved children, the\nJanissaries, and gradually collecting them in Stambul. In those days\nthere were more than twenty thousand Janissaries within the walls of\nthe capital, not including the corporation of water-carriers who\ngenerally made common cause with them in times of uproar. When their\nlordships, the Janissaries, set the place on fire, it was the duty of\nthe water-carriers to put out the flames, whereupon they plundered\ncomfortably together; hence the ancient understanding between them.\n\nWith the exception of the Ulemas, only the blind fakirs of the Omarite\norder were admitted into the council of the Divan, and their chief,\nBehram, often took counsel with the Sultan for hours together when he\nwas alone.\n\nOn the 23d May, 1826, at the invitation of the chief mufti, all the\nUlemas assembled in the Seraglio and decided unanimously that, in\naccordance with the words of the Kuran, it was lawful to fight the\nenemy with his own weapons.\n\nSix days later they reassembled, and then the Sheik-ul-Islam laid\nbefore them a fetva, by which it was proclaimed that a standing army\nwas to be raised for the defence of the realm. In order, however, that\nnobody might pronounce the accursed name of Neshandchi, three names\nwere given to the corps of the army to be raised. The first was\nakinji, or \"rushers,\" these were the young conscripts; the second was\ntaalimlueaske, \"practised men,\" these were selected from the soldiers\nof the Seraglio; the third name was khankiar begerdi, and designated\nthe corps to be chosen from amongst the Janissaries. This name meant\n\"the will of the emperor,\" yet the word \"khankiar\" means, in Turkish,\nby itself, \"effusion of blood.\"\n\nWhen the fetva came to be signed, very few of the leaders of the\nJanissaries were present, but amongst those who were was the Janissary\nAga, or colonel, and his name stood there alongside the name of the\nSheik-ul-Islam, the Grand Vizier, and Najib Effendi.\n\nEarly next morning the people of Stambul read the fetva, which was\nposted up at every corner. The decisive word had been spoken which was\nto evoke the bloody spectre to whom so many crowned heads had been\nsacrificed.\n\nThe first day a fearful expectation prevailed. Every one awaited the\ntempest, and prepared for it. The Sultan was passing the time at his\nsummer palace, Bekshishtash, so, at least, it was said. An anxious,\ntormenting, and bloody pastime it proved to be.\n\nIn one wing of his palace were the damsels of the harem, in the others\nthe chief Ulemas and councillors. Mahmoud paced from one room to\nanother, and found peace nowhere.\n\nHundreds of times he sat in a row with his wise men, and caused the\nannals of the Ottoman Empire by his favorite historian, Ezaad Effendi,\nto be read aloud to him, and yet it was a terror to him to listen. The\nwhole history from beginning to end was written in blood! The same\nprinciples always produced the same fruits! How many Grand Viziers,\nhow many Padishahs, had not fallen? Their blood had flowed in streams\nfrom the throne, which had never tottered as it now tottered beneath\nhim. And when he returned to the harem, and the charming odalisks\nappeared before him with their music and dances, and Milieva amongst\nthem, the loveliest of them all, to whom in an hour of rapture he had\ngiven the rose-garden of his realm, Damascus, he bethought him that\nperchance to-morrow, or even that very night, those sweetly smiling\nheads might all be cut off, seized by their flowing locks and cast in\nheaps, while their dear and tender bodies might be sent swimming in\nthe cold waves of the Bosphorus, to serve as food for the monsters of\nthe deep. Who knows how many hours, who knows how many moments, they\nhave still to live?\n\nEvery hour, every moment, the tidings arrive from Stambul that the\nJanissaries are assembling in menacing crowds, and now the\nconflagrations begin; every day fires break out in three or four parts\nof the town, but the heavy rains prevented any great damage from being\ndone. This was always the way in which the riots began in Stambul.\n\nThe priests of Begtash stirred up the fanaticism of the masses in\nfront of the mosques and in the public squares, incited the mob which\nhad joined the ranks of the Janissaries to acts of outrage against the\nSultan's officials and those of the Ulemas, softas, and Omarite fakirs\nwho were in favor of the reforms.\n\nOn July 14th a rumor spread that a company of Janissaries, actuated by\nstrong suspicion, had surrounded the cemetery which had been laid out\nand enclosed by the Omarite fakir, and cut down all the dervishes they\nfound there, and amongst them their chief, Behram. They found upon him\na bundle of papers which plainly revealed that a secret understanding\nexisted between him and the great men of the Seraglio. They also found\nin his girdle a metal plate, on which was the following inscription:\n\n\"I am Behram, the son of Halil Patrona, the strong man, and of\nGuel-Bejaze,[14] the prophetess. My father in his lifetime began a\ngreat work, which after his death I continued. This work will only be\naccomplished and confirmed when I am dead and there is no further need\nof me. Blessed be he who knoweth the hours of his life and of his\ndeath.\"\n\n[Footnote 14: The heroine of Jokai's _White Rose_.]\n\nThose who were acquainted with the life and the end of Halil Patrona\nknew right well what this great work was thus mentioned by Behram, who\nhad lived one hundred and eight years after his father's death, and\nhad striven all that time to develop and mature the ideas which the\nformer had vainly attempted to carry out at the point of the sword.\n\nThe mob tore the dervish to pieces and distributed his bleeding limbs\nas trophies, and then, like wild beasts who have scented blood, they\nattacked the castles of the great men. Whom should they fall upon\nfirst? That was the only question.\n\nSuddenly one of the priests of Begtash tore down from the corner of\nthe street a copy of the fetva which proclaimed the reform and showed\nit to the mob. \"Behold!\" cried he, \"here, foremost amongst the names\nof the destroyers of the Faith stands the name of the Janissary Aga!\nThe leader of the Janissaries has himself betrayed his own children.\nDeath to him!\"\n\n\"Death to him!\" howled the mob, and, seizing their torches, they\nrushed towards the palace of the Janissary Aga.\n\nThe Janissary Aga heard the tumult, and, quickly dressing a slave in\nhis robes, mingled with the crowd, and, without being noticed, reached\nthe palace of the Grand Vizier in safety.\n\nThe Grand Vizier was sitting down to supper when the Janissary Aga\nrushed in and informed him of his danger. He lost no time in\nbarricading the gates, and, slipping through his garden with his\nservants and his family, escaped across the Bosphorus to the Jali\nKiosk, on the other side of the water. The besieging mob, therefore,\nonly found empty walls upon which to wreak their fury, and these they\nlevelled with the ground.\n\nBut the Janissary Aga had left his wives and children in his palace,\nand these the rioters seized and murdered with the most excruciating\ntortures. In the evening twilight the Aga, from his place of safety on\nthe other side of the water, could see the flames of his palace\nshooting up towards the sky, and heard perchance the agonized\ndeath-cries of those he loved best.\n\nA few moments later they were joined by Nedjib Effendi, the\nrepresentative of the Viceroy of Egypt, who also took refuge with them\nand brought the tidings that the insurgents were in possession of the\nwhole of Stambul, and had wreaked their savage fury on the families of\nthe refugee magnates.\n\nThe Sultan was standing on the roof of his palace, whence he could\nview far away the spreading scarlet glow of the conflagration which\nlit up the night with a terrifying glare, whose fiery columns were\nreflected in the black Bosphorus.\n\nPanic-stricken fugitives spread the report that the Seraglio itself\nwas in flames, and indeed it looked in the distance as if the fiery\nwaves had reached its cupolaed towers.\n\nMahmoud spent the whole night in prayer. Two hours after midnight a\nhorseman arrived who had forced his way through Stambul, his good\nsteed collapsing as it reached the cypress grove of Bekshishtash. The\nhorseman himself demanded an audience of the Sultan, and was instantly\nadmitted.\n\nA bright momentary ray of hope was visible on the face of Mahmoud as\nhe recognized the horseman. It was Thomar, now the Akinji Feriki, the\nbravest warrior in the three continents of the Ottoman Empire.\n\nWhen Mahmoud had quitted the Seraglio he had picked out sixteen young\nhorsemen from amongst his retinue, and left them behind in the palace,\nwith the injunction that if a rebellion should break out in Stambul,\nwhich was pretty certainly to be anticipated, they were to cut their\nway through the enemy and bring him word thereof. Thomar alone had\narrived--the other fifteen had been killed by the rebels; he had cut\nout a road for himself and contrived to reach Bekshishtash.\n\n\"The dragon has raised all his twelve heads, my master,\" said he to\nthe Sultan; \"now is the time to cut them all off, or it will devour\nthy empire.\"\n\nThe Sultan, who greatly loved the youth, wiped the sweat from his face\nwith his own handkerchief, and bade him await him below in the\nbanqueting-chamber.\n\nAnd with that he resumed his devotions.\n\nTowards five o'clock, when the sun rose from behind the blue hills of\nAsia in all its glory, the Sultan descended from the roof of his\npalace and commanded his servants and men-at-arms to form in rank in\nfront of the palace. All the fighting-men he had with him were a\nthousand akinjis and about as many horsemen, silihdars, and bostanjis.\nHe himself first went to take leave of his womenkind.\n\nThose who had seen his face but an hour ago were amazed at the change\nthat had come over it. Its generally mild and peaceful expression had\ngiven place to a proud resentment and a death-defying audacity. He\nembraced his wife and the Sultana Asseki, and finally his son, the\nheir to the throne. Not a tear was visible on his face as he embraced\nhis beloved ones. They all noticed a new vigor flashing from his eyes;\nhe looked as if he were inspired. He had no need now for any to\nencourage him.\n\nAs he held one arm round his wife and the other round his child, he\nsaid to them, \"And now I go. My path leads me into Stambul; whether it\nwill lead me back again I know not. But I swear that if I do return it\nwill be as the veritable ruler of my realm. What will ye do if I\nperish?\"\n\nThe face of Milieva glowed at this question. She led Mahmoud aside\ninto the back part of the room. There the Sultan perceived a large\nheap of pillows and cushions.\n\n\"If Mahmoud perishes,\" said the Circassian girl, enthusiastically,\n\"those who loved him will discover a way of following him; yea, thine\nenemies, when they look for us, will only find our ashes here.\"\n\nMahmoud kissed the girl on the forehead; she was indeed worthy to sit\nat the foot of the throne.\n\nWith that he descended into the court-yard, and they led his good\nsteed in front of the arched door. The Sultan beckoned to Thomar to\nhold the reins while he mounted, then he detached an agate from the\nheron plume that waved above his turban, and fastened it on the fez of\nthe youth as he knelt before him.\n\n\"I name thee leader of the akinjis; and now whoever has a sword, let\nhim show that he is worthy of our ancestors!\"\n\nWith these words the Padishah drew his scimitar, and, galloping to the\nfront of his horsemen, took the place of command. A moment later the\nlittle host was already on its way to Stambul. In front marched the\nakinjis with glittering bayonets; in the centre was the Sultan with\nhis suite; the rear was brought up by the horsemen and the gardeners.\nEvery one of them was resolved to die honorably and gloriously.\n\nOn reaching the city the bold band met at first with but little\nopposition, for they came unawares. The rebels were weary from the\nexertions of the previous night. After putting out the conflagration\nthe mob had set to work plundering, and towards morning the greater\npart of it had dispersed amongst the coffee-houses and other places of\namusement.\n\nMahmoud and his aggressive band met with no opposition right up to the\nSeraglio. The streets indeed were thronged by a noisy mob, but it made\nway at once before the serried ranks of the akinjis. None insulted the\nSultan by so much as an offensive word; on the contrary, cries of\nadmiration were audible here and there. Men were astounded when they\nbeheld the Padishah appear with a handful of armed men amidst the\nraging tempest, and permitted him to enter the gates of the Seraglio\nin peace.\n\nThe shout bursting through all the doors, which resounded for some\nminutes from the inside of the place, announced to those outside what\ncourage the appearance of the Sultan had instilled into the hearts of\nthose of his warriors who were shut up in the Seraglio.\n\nKara Makan, full of amazement, withdrew the bulk of the rebels from\nthe Grand Signior's palace and massed the Janissaries near the\nEtmeidan, where banners were hoisted side by side with the subverted\nkettles. At the corners of the streets the wild priests of Begtash\ncontinued to incite the agitated mob with hoarse cries, and from the\nsummits of the minarets the horns of the rebels sounded continuously,\nonly ceasing at such times as the imams summoned the people of Osman\nto glorify Allah, about the fifth hour of the day. At the sound of the\nnamazat even the furious popular tempest abated, only beginning again\nwhen the last notes of the call to prayer ceased to resound.\n\nStambul was literally turned upsidedown, and the dregs were swimming\non the surface. The confraternity of porters, the water-carriers, the\nboatmen, all stood by the Janissaries and swelled enormously the bulk\nof the rebels. Every mosque, every barrack, was in their power; even\nthe towers of the Dardanelles had opened their gates to the Jamaki,\nwho were in alliance with the Janissaries. The Sultan was shut up in\nhis own palace.\n\nThe Janissaries intended to carry the edifice of the Sublime Porte by\nassault, and had, therefore, sent forth criers to the jebejis, or\ncamp-blacksmiths, who were encamped with the heavy cannons on the\ngrounds of the Mosque of Sophia, to invite them to begin the siege.\n\nThe emissaries of the Janissaries, in brief, savage harangues, called\nupon the jebejis to put their hands to the bloody work. The latter\nlistened to them, but for a long time hesitated. Suddenly a shot fired\nfrom amongst the crowd struck one of the speakers, who fell down dead,\nwhereupon the other jebejis rushed upon the envoys of the Janissaries,\ncut them down, and, flinging their severed heads into a heap, shouted,\n\"Long live the Sultan!\" and with that they proceeded in force to the\nSeraglio, took up their positions in front of it, and turned their\nguns against the rebels.\n\nTowards mid-day, amidst strains of martial music, the Kapudan Pasha\nIbrahim, whose nickname was \"The Infernal,\" arrived with four thousand\nmarines and fourteen guns. A quarter of an hour later were to be seen\nin the proximity of the Jali Kiosk the overwhelming forces of the\nGrand Vizier Muhammad, who, under the protection of the night, had got\ntogether the hosts of Asia, which had always been opposed to the\nJanissaries. The Janissary Aga was there, too, with the Komparajis\nfrom Tophana. The concentrating masses welcomed one another with\nblood-thirsty greeting. It was evident, from the faces of their\nleaders, that they were determined not to retreat a step on the path\nthey had taken. The last hour of the Janissaries, or of the Ottoman\nEmpire, had struck.\n\nAnd now the gates of the Seraglio were thrown open, and, escorted by\nthe high officers of state and the Ulemas, the Sultan came forth.\n\nThe Ulemas, the imams, and the officers of the army stood in a\nsemicircle round the gate. The Sultan remained standing on the highest\nstep. There he stood in the full regalia of the padishahs, holding in\none hand the banner of the Prophet and in the other a drawn sword.\n\n\"What do the rebels desire,\" exclaimed, with a loud, penetrating\nvoice, the Sheik-ul-Islam, \"who rise up against Allah and against the\nHead of the Faith, the Padishah?\"\n\nThe chief mufti replied with unction: \"It is written in the Kuran, 'If\nthe infidels rise against their brethren, let them die the death!'\"\n\n\"Then swear by the banner of the Prophet that ye will root out them\nwho have risen up against me!\"\n\nThe viziers kissed the holy flag and took the oath to defend it to the\nlast drop of their blood.\n\n\"And now close the gates!\" commanded the Sultan; and immediately he\nsent orders to the warders of all the gates of Stambul to let nobody\neither out or in. One of the opposing hosts was never to leave the\ncity alive.\n\n\"Long life to the Sultan! Death to the Janissaries!\" resounded from\nfifteen thousand lips in front of the Seraglio.\n\nThe Sultan would have led his army in person against the rebels, but\nhis generals fell down on their knees and implored him in the name of\nthe Prophet not to expose his life to danger. Let him at least give\nhis sword to the Grand Vizier, that he might not soil it in the blood\nof rebels.\n\nSo the gates were shut. This circumstance filled the hearts of the\nrebels with terror. They foresaw that this day would not be followed\nby another; the hand of indulgence, of reconciliation, now grasped the\nweapons of war, of massacre.\n\nThey all assembled round the Etmeidan, pulled down the buildings in\nthe street, and made barricades of them. 'Tis a bad sign for a\nrebellion when it has to look to its defence.\n\nThe forces of the Grand Vizier slowly approached amidst the roll of\nkettle-drums; the Derben Aga appeared in front of the barricades of\nthe Janissaries, with the sanjak-i-sherif in his hand, and summoned\nthe rebels to disperse and return to the allegiance of the sacred\nbanner. The rebels drowned his speech in curses, and above the curses\nrose the thundering voice of Kara Makan hounding on the fanatical mob\nagainst the destroyers of the faith of Osman.\n\n\"Wipe out these new ordinances, give up the heads of the godless ones\nwho signed their names below the khat-i-sherif--to wit the Janissary\nAga, the Grand Vizier, the chief mufti, and Nedjib Effendi! This is\nwhat the ortas of the Janissaries demand and their honest\nconfederates, the Jamaki, the Kayikjis, and the Hamaloks, who remain\nfaithful to the God of the Moslemin.\"\n\nThrice did the Derben Aga summon the rebels to surrender, and thrice\ndid he receive the same answer. They demanded the heads of the\nviziers.\n\nMahmoud's predecessor had, on a similar request, surrendered the heads\nof the viziers. Mahmoud broke his sword in two above their heads, and\nthrowing the broken pieces in the dust, exclaimed:\n\n\"Just as I now break in two this sword and nobody shall weld it\ntogether again, so also shall ye be overthrown and none shall raise\nyou up again.\"\n\nThe next moment the cannons of Ibraham the Infernal thundered forth\ntheir volleys from the Etmeidan. The bombs tore through the rickety\nwooden barriers, and through the breach thus made rushed Hussein Pasha\nat the head of the akinjis with Thomar Bey by his side.\n\nThe appearance of the detested new soldiers was greeted by the\nJanissaries with a furious howl, but the very first moment convinced\nthem that the bayonet was a very much more powerful weapon than the\ndirk. Thomar Bey headed the charge in person, making a way for himself\nwith his bayonet and clearing the ranks of the insurgents like a sharp\nwedge.\n\nOn this side there was no deliverance, so now, with the fury of\ndespair, the insurgents flung themselves on the guns of Ibraham Pasha,\nthree times charging his death-vomiting batteries, and, thrice\nrecoiling, leaving the ground covered with their corpses, the terrible\ngrape-shot mowing them down in heaps.\n\nIt was all, all over. The flowers of Begtash's garden, vanquished,\nhumbled by the new soldiers, fled for refuge to the huge quadrangular\nbarracks which occupied the ground at the rear of the Etmeidan.\n\nKara Makan did not live to experience that hour of humiliation; a\ncannon-ball took off his head so cleanly that his body could only be\nidentified by his girdle.\n\nWithin the walls of the barracks the Janissaries made ready for their\nlast desperate combat. It was now late. Ibrahim the Infernal began to\nbombard the barracks with red-hot bullets, and within an hour's time\nthe whole of the enormous building was in flames. Those who were\ninside the gates remained there, for there they were doomed to perish\ntogether. Amidst the roaring of the flames their death-cries were\naudible, but the flames grew stronger every moment and the cry of\ntheir mortal anguish waxed fainter. The generals stood around the\nbuilding, and tears glittered in more eyes than one; after all, it had\nbeen a valiant host!\n\nHad been! Those words explain their doom.\n\nOn that day twenty thousand Janissaries fell by the command of the\nPadishah. Those whom the bullet and the sword did not reach perished\nby the axe and the bowstring. Their bodies were given to the\nBosphorus, and for a long time afterwards the billows of distant seas\ncast their headless trunks on the shores of countries far away. These\nwere the flowers of Begtash.\n\nAnd so the name of the Janissaries was blotted out of the annals of\nOttoman history.\n\nThe wearing of their uniforms and their insignia was forbidden under\nsentence of death. Their barracks were levelled with the ground, their\nbanners were torn to bits, their kettles were smashed to pieces, their\nmemory was made accursed.\n\nThe order of the Priests of Begtash was abolished forever, their\nreligious homes were destroyed, their possessions confiscated.\n\nThus came to an end a soldiery which had existed for centuries, which\nthe wise Chendereli founded, and which had won so many glorious\ntriumphs for the Ottoman arms. It was now unlawful to mention its very\nname.\n\nBut when the bloody work was done, the Ottoman nation arose again full\nof fresh vigor, and it owed a new life, full of glorious days, to the\nhand which delivered the empire from its two greatest\nenemies--Tepelenti and the Janissaries.\n\n\n\n\nGLOSSARY OF THE TURKISH WORDS USED IN THIS STORY\n\n\nAGA--a military and aulic title.\n\nAKINJI--a sort of irregular cavalry.\n\nANADOLI HISSAR--eastern castle.\n\nAZAB--irregular infantry.\n\nBAIRAM--the great Muhammadan ecclesiastical feast.\n\nBAYADERE--a dancing-girl.\n\nBEY--a dignitary next below a pasha.\n\nBOSTANJI--originally the gardeners of the Seraglio, subsequently\nattendants, body-guards.\n\nCHORBAJI--a Janissary officer.\n\nCIAUS--palace officials employed as attendants, messengers, envoys.\n\nDERBEND AGA--the chief of the street watchmen.\n\nDIRHAM--a coin worth about 2-1\/2_d._\n\nDIVAN--council of state.\n\nDZHIN--a huge supernatural being.\n\nEFFENDI--a title of honor.\n\nETMEIDAN--the headquarters of the Janissaries.\n\nFETVA--the opinion or judgment of a mufti.\n\nFIRAK--bodies of troops.\n\nFIRMAN--a decree issued by the Sultan.\n\nGIAOUR--an infidel.\n\nICHOGLANLER--pages of non-Muhammadan parentage brought up at the\nSultan's palace.\n\nIMAM--a priest who recites the canonical prayers.\n\nJAMAK--the servant of a Janissary.\n\nJANISSARIES--literally, \"new soldiers\" (jeni-cheri), originally\ncaptive children brought up to be soldiers. This corps was for\ncenturies the flower of the Ottoman army.\n\nJANISSARY AGA--the chief of the Janissaries.\n\nJERID--a stick used as a dart in military exercises.\n\nKADI--a judge.\n\nKADUN-KEIT-KHUDA--guardian of the harem.\n\nKAPU-AGASI--Lord Chamberlain.\n\nKAPUDAN PASHA--Lord High Admiral.\n\nKAPUJI--gate-keeper of the Seraglio.\n\nKAPUJI PASHA--the introducer of the ambassadors.\n\nKAPU-KIAJA--chief magistrate.\n\nKHAT-I-SHERIF--a command either signed by the Sultan or issued\ndirectly through him.\n\nKHUMBARAJI--a bombardier.\n\nKIZLAR-AGASI--chief inspector of the harem.\n\nMOLLAH--the title of the highest grade of Ulemas.\n\nMUEZZIN--the caller to prayer.\n\nMUFTIS--those of the Ulemas who publish or seal the fetvas or other\npublic documents.\n\nMURSHID--a spiritual guide.\n\nNAMAZAT--the canonical prayer.\n\nODALISK--a concubine; literally, chambermaid.\n\nORTA--a company of Janissaries.\n\nPALIKAR--\"strong youth,\" a name given to themselves by the Klephts,\nfreebooters of Thessaly.\n\nPARA--a farthing.\n\nREIS-EFFENDI--Minister of Foreign Affairs.\n\nSANDJAK-I-SHERIF--the sacred banner of the Prophet.\n\nSERAGLIO }\nSERAI } The Sultan's court.\n\nSERAI-AGASI--chief inspector of the Seraglio.\n\nSERASKIER--a commander-in-chief.\n\nSHEIK-UL-ISLAM--the chief of all the muftis and Ulemas.\n\nSILIHDARS--one of the six divisions of the mercenary cavalry, also\nthe Sultan's armor-bearers.\n\nSIPAHIS }\nSPAHIS } One of six divisions of the mercenary cavalry.\n\nSULIOTES--a warlike Hellenized race of Albanian origin in the Pachalik\nof Janina.\n\nSULTANA-ASSEKI--The Sultan's consort.\n\nSULTANA-VALIDEH--the Sultan's mother.\n\nTIMARIOTES--Turkish feudal militia.\n\nTOPORABAJI--gunners.\n\nTOPIJIS--gunners.\n\nULEMAS--the learned men, including the muftis, the mollahs, the\nkadis--in short, all the legal and ecclesiastical functionaries.\n\n\n\n\nTHE END\n\n\n\n\nTranscriber's Note: The following typographical errors present in the\noriginal text have been corrected for this electronic edition.\n\nIn Chapter I, \"superflous cracks and crevices\" was changed to\n\"superfluous cracks and crevices\".\n\nIn Chapter II, \"siezed him\" was changed to \"seized him\".\n\nIn Chapter III, \"ninrethullita\" was changed to \"nimetullahita\", and\n\"It must not he supposed\" was changed to \"It must not be supposed\".\n\nIn Chapter IV, \"the besieging Pehlivan\" was changed to \"the besieging\nPehlivan\".\n\nIn Chapter VIII, \"Meccao and Medina\" was changed to \"Mecca and\nMedina\", and \"Procelain Chamber\" was changed to \"Porcelain Chamber\".\n\nIn Chapter IX, \"hill, morever\" was changed to \"hill, moreover\", \"wont\nyou\" was changed to \"won't you\", and a question mark was changed to an\nexclamation point after \"thy daughter Milieva\".\n\nIn Chapter X, \"La Gullia\" was changed to \"La Gulia\", \"to horribly\ntortured Turks\" was changed to \"of horribly tortured Turks\", and \"rank\nor general\" was changed to \"rank of general\".\n\nIn Chapter XVIII, \"silchidars\" was changed to \"silihdars\".\n\nIn the Glossary, \"Silchidars\" was changed to \"Silihdars\".\n\nSeveral names and words were spelled inconsistently in the original\ntext. Except as noted above, these variant spellings have been\nleft as they originally appeared.\n\n\n\n\n\n\nEnd of the Project Gutenberg EBook of The Lion of Janina, by Mor Jokai\n\n*** ","meta":{"redpajama_set_name":"RedPajamaBook"}} +{"text":"\n\n### Eternally Bound\n\nBy\n\nBrenda K. Davies\n\nCopyright \u00a9 2016 Brenda K. Davies\n\n# Books By The Author\n\n### Books written under the penname Brenda K. Davies\n\n### The Alliance Series\n\nEternally Bound (Book 1)\n\n### Hell on Earth Series\n\nHell on Earth (Book 1) Coming August 2017\n\n### The Road to Hell Series\n\nGood Intentions (Book 1)\n\nCarved (Book 2)\n\nThe Road (Book 3)\n\nInto Hell (Book 4)\n\n### The Vampire Awakenings Series\n\nAwakened (Book 1)\n\nDestined (Book 2)\n\nUntamed (Book 3)\n\nEnraptured (Book 4)\n\nUndone (Book 5)\n\nFractured (Book 6)\n\n### Historical Romance\n\nA Stolen Heart\n\n### Books written under the penname Erica Stevens\n\nThe Captive Series\n\nCaptured (Book 1)\n\nRenegade (Book 2)\n\nRefugee (Book 3)\n\nSalvation (Book 4)\n\nRedemption (Book 5)\n\nBroken (The Captive Series Prequel)\n\nVengeance (Book 6)\n\n### The Fire & Ice Series\n\nFrost Burn (Book 1)\n\nArctic Fire (Book 2)\n\nScorched Ice (Book 3)\n\n### The Kindred Series\n\nKindred (Book 1)\n\nAshes (Book 2)\n\nKindled (Book 3)\n\nInferno (Book 4)\n\nPhoenix Rising (Book 5)\n\n### The Ravening Series\n\nRavenous (Book 1)\n\nTaken Over (Book 2)\n\nReclamation (Book 3)\n\n### The Survivor Chronicles\n\nBook 1: The Upheaval\n\nBook 2: The Divide\n\nBook 3: The Forsaken\n\nBook 4: The Risen\n\nTable of Contents\n\nOther books by the Author\n\nChapter 1\n\nChapter 2\n\nChapter 3\n\nChapter 4\n\nChapter 5\n\nChapter 6\n\nChapter 7\n\nChapter 8\n\nChapter 9\n\nChapter 10\n\nChapter 11\n\nChapter 12\n\nChapter 13\n\nChapter 14\n\nChapter 15\n\nChapter 16\n\nChapter 17\n\nChapter 18\n\nChapter 19\n\nChapter 20\n\nChapter 21\n\nChapter 22\n\nChapter 23\n\nChapter 24\n\nChapter 25\n\nChapter 26\n\nChapter 27\n\nChapter 28\n\nChapter 29\n\nChapter 30\n\nChapter 31\n\nChapter 32\n\nChapter 33\n\nChapter 34\n\nChapter 35\n\nChapter 36\n\nChapter 37\n\nChapter 38\n\nChapter 39\n\nChapter 40\n\nChapter 41\n\nChapter 42\n\nChapter 43\n\nChapter 44\n\nChapter 45\n\nChapter 46\n\nWhere to Find the Author\n\nExcerpt from Into Hell\n\nAbout the Author\n\n# CHAPTER 1\n\nStanding on the balcony, Ronan's eyes scanned the bodies writhing below him. The only thing he found enticing about the vast amount of flesh on display was breaking the necks of every person in the club. He tried to shake the impulse off, but his fangs throbbed in his gums at the thought, and it dug deeper into him with every passing second.\n\nHis entire existence had revolved around one mission: protect the innocents of this world whether they be human or vampire. It was a mission he'd followed for over a thousand years, but with every passing day, the bloodlust growing within him dragged him closer to the edge he'd seen so many other vampires plummet over.\n\nSome of those other vampires he'd believed to be far better and stronger than him, yet they'd given into their more sinister impulses. And somehow, he remained and now stood as the oldest vampire in existence, not just within his close-knit group of men, but throughout all the vampires.\n\nEvery day he woke, he questioned if that day would be the day he fell too and became the thing he despised the most, a Savage. He didn't try to tell himself that he would never give in. He'd done that for many years, but this past year he'd come to realize it may be inevitable that he succumbed to the bloodlust beckoning him. If he didn't kill himself before that happened, he would start to kill the innocents he protected.\n\nIt was the killing himself first part that would be tricky. Savage vampires thrived on the blood of innocents, but they weren't stupid or lost to the madness of the death they delivered. No, many of them remained intelligent and calculating, and they didn't want to die. A vampire who gave himself over to their inhumanity simply saw nothing wrong with what they were doing. The blood they consumed warped them into believing a vampire's true nature was to kill and they were only living the way vampires were supposed to live.\n\nNo matter how much he despised Savages, if he gave in, he would most likely come to believe that too.\n\nPerhaps they were right, but Ronan refused to believe vampires were meant to be little more than animals who ruthlessly slaughtered the weaker masses.\n\nHowever, it didn't matter what he believed or what he didn't, he was teetering toward the Savage side. When he went over, would his men, or even multiple vampires, be capable of taking him out as he had taken out so many of those who had fallen before him?\n\nThat was a question he dreaded he would find out the answer to soon. The fine line he walked became thinner with every passing day, and with every death he delivered to the Savages amongst his kind. The thrill of killing the vampires he hunted had once satisfied him, but that was centuries ago. Now, it _barely_ kept the demon part of him at bay.\n\nThe emptiness within him would never be filled. There wasn't enough blood, wasn't enough death to begin to satisfy him anymore. He faced the same bleak concept that the many who had fallen before him also faced: an eternity of nothing, or the possibility that giving into their more savage nature would finally fill the emptiness inside.\n\nFor some vampires, it hadn't been a difficult choice.\n\nHow much more time do I have?\n\nHis hands tightened on the railing, twisting over the cool metal as his teeth clamped together and his fangs slid free to press against the back of his lips.\n\nI will not be one of them!\n\nHe told himself this every day when he opened his eyes and the emptiness greeted him, but it sounded hollow then, and it did again now. Even amongst those who were born vampires, the purebreds such as himself, he was an anomaly and stronger than the rest. The turned vampires battled their darker natures too, but not as badly as the purebreds did. Because turned vamps were human before becoming vampires, they had more humanity in them than purebreds did.\n\nIf he gave himself over to the darkness, he would never know this sensation of being torn in two again, and he would slaughter hundreds, possibly thousands, before being stopped, if he could be stopped.\n\nNo matter what, he couldn't lose control like that. He didn't like humans overly much, but there were rules, and they must be obeyed if the vampire race was to continue undetected. Vampires were far stronger than humans, but they were also vastly outnumbered by mortals.\n\nFear would have humans turning on them, slaughtering them, and studying them like lab rats. Some, if not many of the mortals, may try to become vampires, which would create more Savages. If such a thing happened, the vampire food supply would be depleted and the world would fall into chaos.\n\nThe rules had to be strictly followed.\n\nThis had been instilled into him from the moment of his birth. His parents had made sure he knew he would one day lead and keep the vampire and human races protected. That he would become a Defender, a vampire who protected the innocents and made sure the rules were obeyed. Things may be far different now than when his parents had been alive, but that mission still drove him every day.\n\nBeside him, Declan shifted his stance as he glanced at Ronan from the corner of his eye. Declan's eyes were so pure gray, they appeared silver as he surveyed the scene beneath them. He ran a hand through his dark auburn hair before tugging at the ends.\n\nAt six hundred years of age, Declan was the second oldest of their group, younger than only him. They'd been fighting together since Declan had reached maturity at twenty-four. Ronan had known Declan for his entire life, yet he still didn't know all of Declan's secrets.\n\nHe did know that he'd never seen his friend so apprehensive about a possible kill before, and he didn't understand it. He'd expected Declan to be eager to destroy Joseph. Ronan suspected Declan's apprehension was because his father had been the last Defender to give into his Savage nature, and it was stirring up old memories, rather than the fact they were now hunting one of their own.\n\nJoseph had been a powerful fighter and ally for nearly fifty years. However, he and Declan had been like two dogs circling each other whenever they were in the same room. Declan's more easygoing nature had abraded with Joseph's austere personality.\n\nIn truth, Ronan had never liked Joseph either and never considered him a friend as he did the other Defenders who worked with him, but Joseph had been a strong fighter and had made it all the way through the rigorous training every purebred that worked with him had to endure.\n\nRonan had seen little of Joseph over the years as he'd run the training facility for turned and purebred vamps who wished to hunt Savages. Months ago, Joseph had given in to his bloodlust and become a Savage. Ronan had been trying to track him down ever since, but Joseph knew how they operated and how to fly under their radar. Not even Brian, a turned vamp who sometimes helped them to track down Savage vamps, could pinpoint Joseph's location, until now.\n\nBrian had called a few hours ago to let him know he'd gotten a track on Joseph in this area of Providence, Rhode Island. This club was the most likely place to attract Joseph. A club full of drunk humans was a homing beacon for vampires on the prowl. It was easy to prey on the humans here, and if Ronan got the chance, he would feed here tonight.\n\nFeeding could wait; for now, they were on the hunt for something else.\n\nHis eyes swept the dance floor once more. Unable to take the flashing lights in places such as this, he'd put on a pair of dark sunglasses before entering, but the pulse of the flashing lights still made his head pound. The thumping beat of the music vibrated the floor beneath his feet. His gaze landed on the DJ. He ran his tongue over his fangs as he contemplated tearing the man's throat out to end the annoying beat.\n\nWhat had happened to real music like Beethoven and Chopin? He'd even take some Duke Ellington, Frank Sinatra, or Billy Joel over this. This crap was enough to drive the best of vampires over the edge, and he was far from the best. But the humans liked it as they ground against each other with a frenzy the equivalent of foreplay.\n\nMovement behind him drew his attention to Killean and Saxon as they approached from the shadows. The few humans who had been standing nearby shrank away from Killean and vanished down the stairs.\n\n\"Anything?\" Ronan demanded of them.\n\nKillean shook his head as his golden-tiger eyes went past Ronan to the dance floor. A scar sliced straight down from his deep brown hairline and over his right eye before ending halfway down his cheek. Killean had come to work with Ronan when he was fifty-two, that had been four hundred years ago, and Ronan still had no idea what had caused that scar or who had given it to him. He knew it had been inflicted on Killean before he'd become a fully mature vampire only because it remained.\n\nNext to Killean, Saxon folded his arms over his chest and his hazel eyes drifted toward the ceiling. His dark blond hair stood up in spikes around his head. \"Something else might have attracted him away from here,\" Saxon said. \"There may be another club or bar or something we missed nearby.\"\n\nOr Joseph had managed to elude them again. Ronan's teeth ground together as he released the railing. He wasn't looking forward to killing the vampire he'd once considered an ally, but he wanted this over with. With Joseph's purebred status, knowledge of the way they worked, and complete disregard of human and vampire life, he was far more lethal than many of the other Savages they'd dealt with. It had been centuries since one of their own had given in to their savage nature.\n\nAs far as Ronan could tell, going by the increased amount of disappearances, people who'd had their throats cut, and animal attacks since Joseph had given in, he'd killed over a hundred people and was averaging at least one a day. That didn't include the amount of vampires he'd slaughtered too.\n\nWith every human death, Joseph grew in power, but he also became weaker. By now, Ronan knew his old ally couldn't tolerate the sun anymore. Holy water and crucifixes would affect him, and soon enough, he wouldn't be able to cross large bodies of water, if he still could now.\n\nMovement on the dance floor drew his attention as a tall man with black hair glided through the crowd with ease. Some of the women stopped dancing to flirt with him and the three men trailing him. All the men continued through the crowd as if they didn't see the women.\n\nRonan grasped the rail again as he watched the four of them. Judging by the way they carried themselves and the thick coats they wore when the humans had all checked theirs, Ronan knew what they really were...\n\n\"Hunters,\" he murmured. \"And not human hunters, but born ones.\"\n\nThe born vampire hunters sometimes took in humans who knew of the existence of vampires. The hunters trained these humans how to kill vampires, but often those human hunters were bait for vampires. The hunters believed vampires were the monsters, but the practice of using the humans as bait was more ruthless than anything Ronan had ever done to a human.\n\n\"Bastards,\" Killean hissed so low that Ronan barely heard him.\n\n\"Well, that made this night a lot less fun,\" Declan said and leaned his hip against the rail.\n\n\"Do you think they're tracking Joseph?\" Saxon inquired.\n\n\"I don't know,\" Ronan replied as he watched the four men disappear beneath the balcony. \"Watch the stairs. If they come this way, be prepared to fight.\"\n\nSaxon and Killean slipped back toward the stairs, moving through the few humans gathered in the shadows. Most of the mortals were entangled with someone else, or multiple someone elses. A few were huddling together, seeking a fix from their drug of choice.\n\nRonan had come here to destroy Joseph, but now they may also have to take out an enemy they hadn't expected\u2014an enemy that didn't have to be an enemy, if the hunters weren't so fucking stupid. But throughout the history of vampires and hunters, and despite their common ancestors, the hunters had always believed all vamps were bad. There was no reasoning with them; there was only surviving them.\n\nHe'd done what he had to do to survive before and killed a couple of hunters in his lifetime. If it became necessary, he would do it again tonight.\n\n# CHAPTER 2\n\nKadence watched from the shadows as her brother, Nathan, disappeared down the stairs to the club with her fellow hunters, Asher, Logan, and Jayce, following him. Nathan would _kill_ her if he knew she was here, or more likely lecture her for hours on end, but she wasn't going to sit this hunt out. She was forced to sit out most of her life; she wouldn't miss this.\n\nShe just had no idea how to get into that club. Her gaze ran over the line of people snaking around the side of the building and waiting to get inside. The man at the front of the line held a red rope. He'd let her brother in as soon as Nathan approached him. Kadence had never met the man before, and if she had to wait in line, she would never make it inside in time. She didn't know how or why, but she'd always somehow known things over the years, and her instincts were telling her this would be the night and she had to get inside now.\n\nJust as her instincts had told her when her father was killed. She shuddered at the memory of the sweeping, empty feeling that had descended over her the second her father left this world. Hours later his death was confirmed, but it had been no surprise to her when she saw his body. Her fingers dug into her palms at the reminder of how ineffective she'd been. Yes, she'd known when he died, but she hadn't known in time to try to prevent that death.\n\nNow she knew that tonight was the night they would find the vamp who had murdered her father. She'd been working on trying to break out of the stronghold for a while, but she'd known tonight was the night she _had_ to go. She'd buried her anxiety over the prospect of being on the other side of the walls that had encompassed her for most of her life, and put her escape plan into motion.\n\nAs a hunter, she'd been trained on how to kill a vamp, but as a female hunter, she'd never been allowed to actually fight a vamp or to mingle with humans. This was the first time she'd been out of the hunter stronghold in twenty years, and the last time was only because they'd relocated from Virginia to Massachusetts when an upswing of vampire activity drew them to the area.\n\nMost times, she understood why she was kept so sheltered, but she resented it _all_ the time. There was so much of the world to see, so much to explore and learn outside of the thousands of books she'd poured through in her lifetime. She knew hunters were vital to keeping the human population safe from the monsters that came out at night, but did that mean her life had to be sacrificed?\n\nKadence sighed and her shoulders hunched forward. Yes, that's exactly what it meant.\n\nAs her dad had always told her, one life was nothing compared to the billions of lives relying on the hunter race to continue. Yes, they could recruit humans, and they did, but it was necessary to continue the pure hunter line as well. However, it was often difficult for a female hunter to conceive so the women were protected and the men fought.\n\nShe'd been privileged to be born into her role as a future wife and mother\u2014or at least that's what she'd been told, repeatedly, over the years. She didn't think being locked away to the point of claustrophobia and already betrothed to Logan was all that lucky, but her opinion didn't matter.\n\nNext month she would be living out her purpose as a wife. Kadence's skin crawled at the reminder of her impending nuptials. Logan was a good, strong, capable man, but he was more like a brother to her than a husband. There was no spark between them, at least not on her end.\n\nShe had no idea what a spark with a man felt like, but she'd read about it in some of the romance novels in the stronghold. In those books, a spark was _extremely_ important. But those were books and this was reality, and in her reality there were no sparks with her soon-to-be husband.\n\nBecause her father had been the leader of the hunters and her brother now was, she'd been paired with the strongest available hunter. And as Nathan's second-in-command, Logan was one of the best.\n\nKadence blew out a breath as she watched the unmoving line. She had no idea what one had to do to get inside, but she hadn't managed to escape the stronghold to be deterred now. It had taken a _lot_ of planning to break free. She hadn't been prepared for this, but she would figure it out.\n\nThrusting her shoulders back, Kadence brushed her braid over her shoulder and slipped from the shadows. The January air chilled her cheeks as she approached the mountain of a man holding the rope. At first, his chestnut eyes ran dismissively over her, but then they became more leisurely in their perusal of her. Kadence frowned at him before waving her hand after where Nathan and the others had gone down the steps.\n\n\"Nathan is my brother,\" she said.\n\n\"Is that so?\" the man inquired with a smirk.\n\n\"Yes.\"\n\n\"Then why didn't you enter with him?\"\n\nKadence grappled to come up with a response. Glancing down the line of people, she noticed most of them were focused on the phones in their hands. A few were watching her, some with interest, others with hostility.\n\n\"I forgot my phone at home and couldn't call to let him know I was running late. He probably assumed I'd changed my mind about meeting him here,\" she lied with ease and flashed the man her best smile.\n\nThe man's eyes ran over her one more time. Kadence stilled her fingers when they fidgeted with the edges of her sleeves. She was so far out of her element here, awkward in a way she'd never been before. If she'd been a human woman, she would be comfortable with talking to this man, she would know what to say, but all she knew was what she'd been raised to know, vampires, marriage, babies.\n\nFresh resentment shot through her. _Life could be worse_. She told herself this a thousand times a day, but it had yet to fully sink in.\n\nShe should be grateful for all she had, instead of resentful of everything denied her. Still, she wanted to push this guy out of her way and stroll into the club instead of standing here like a moron with a growing group of humans peering curiously at her.\n\n\"Next time, come with him,\" the man said and lifted the rope before he stepped aside.\n\n\"Oh, come on! This is bullshit! Nathan is my brother too!\" a chorus of voices shouted from the line.\n\nKadence ignored all of them as she hastened down the steps toward the thumping beat coming from behind the closed door below. Before she could open the door, it swung inward to reveal a woman holding it for her. The woman smiled at her as the music increased to the point where Kadence's head pounded in rhythm with it.\n\n\"You can check your coat,\" the woman said and waved her arm at a window behind her. A man stood there resting his elbows on the wooden windowsill, but he straightened when he saw Kadence.\n\n\"No, thank you.\" Kadence pulled her coat closer against her as she turned away from the woman. She didn't know much about humans, but her books, her family, and other hunters had told her enough about the human world to know the weapons tucked into her coat would not be welcome here.\n\nHurrying down the hall, she walked toward the music. She worried Nathan would discover her here, but she couldn't deny the thrill of excitement running through her at being free and finally seeing something of the human world.\n\nGranted, it was for the worst reason possible that she was out of the stronghold, and she had no idea how to interact with this world, but she was _free_.\n\nStepping out of the hall, she found herself standing at the edge of a dance floor. Kadence gawked at the spectacle of all the humans moving and jumping around before her. She and her friend, Simone, had spent a lot of time dancing in each other's rooms over the years, but she'd never seen anything like this.\n\nThe complete inhibition, lack of clothing, and energy of the place fascinated her as much as it unnerved her. The aromas of sweat and alcohol intermingled so completely with each other that she could barely separate the two. There was nothing like this in the stronghold. Simone would be in absolute awe when Kadence went back and told her what she'd seen here.\n\nWent back...\n\nKadence shook her head to clear it of the sadness creeping through her at the thought of returning to the stronghold\u2014a place that was a beautiful prison for her and so many other women. Granted, most of the women there didn't think of it that way, but she'd give anything to spread her wings and break free of her gilded cage.\n\n_Not the time_ , she told herself sternly. _You are here for a reason._ Her gaze scanned over the crowd as she searched for the vampire who had murdered her father. She also kept an eye out for her brother.\n\nRonan's gaze honed in on the woman who emerged from the hallway to stand at the edge of the dance floor. Her silver blonde hair, dangling in a braid over her shoulder to her left breast, reflected the colors flashing over her in an array of reds, blues, and yellows. The lights played over her delicate features and lit the awe on her face as she stared at the humans.\n\nA smile tugged at the corners of her full mouth when a group danced close by her. Many of the people below displayed more flesh than they covered, but Ronan found his gaze riveted on her fully clothed body in its form-fitting black pants, which were tucked into ankle-high, black boots. Her calf-length, black coat pushed back as she settled her hands on her hips to watch the crowd. Beneath the coat, she wore a black turtleneck that hugged her breasts, slender waist, and rounded hips. She looked to be a good five inches shorter than him at about five-seven.\n\nHer smile slid away and her hands fell from her hips as she surveyed the crowd with a far more serious eye. Then her head fell back, her gaze locked onto his, and he was treated to a full-on view of her striking beauty.\n\nThe image of her body, naked beneath his and moving in a sensuous dance against his sheets, caused his cock to harden. He felt like he had when he'd been twelve and first discovering the joys of the female body, before the enjoyment of sex had faded away over the many years of his death-filled life. He couldn't recall the last time he'd desired a woman, but it had never been with this intensity.\n\nFor the first time in centuries, he lusted after a woman, and he would have her.\n\n# CHAPTER 3\n\nThe air rushed out of Kadence's lungs when she spotted the man above. Though his eyes were shaded with sunglasses, she knew he was focused on her. Butterflies fluttered to life within her stomach as she drank in the details of the man.\n\nNot handsome, she wouldn't define him as that, but definitely intriguing. Her fingers itched to trace the contoured planes of his high cheekbones and the stubble lining his square jaw. His sable eyebrows drew together over the bridge of his roman nose as he watched her. She had no idea why he was wearing sunglasses inside, but she would give anything to pull them off and reveal the color of his eyes.\n\n_Sparks_. For the first time in her life, she understood the romance novels as she was hit with the urge to get closer to this man, to touch him like she'd never touched another. To slip that coat from his shoulders and run her hands over him before rising on her toes to kiss him. The possibility of those arms wrapping around her and drawing her close had her taking a step forward.\n\nA woman bumped into her, knocking her back and tearing her attention away from the man. Kadence blinked as she was jarred back to reality. Her gaze ran over the humans before she retreated from the dance floor to hover on the edge. She refused to look up again for fear she would be lost to the strange pull that man had over her. She'd come here for a reason, and she would not be deterred from it.\n\nIt had seemed odd the man wore sunglasses inside, but that faded when she realized some of the other men and women also wore them. Must be some human fashion thing, she decided.\n\n\"Hello, sugar,\" Declan purred from beside Ronan. \"I think she might make a _very_ tasty treat.\"\n\nRonan didn't have to look at him to know Declan had also spotted the woman. In a crowd of nearly a hundred humans, she stood out as clearly as the full moon from the stars. Declan leaned forward to inspect the woman more closely.\n\n\"Don't,\" Ronan snarled, half tempted to throw his friend over the railing.\n\nHe had no idea where the impulse came from. He was not an easy man, but he was tolerant. He'd learned centuries ago that giving into anger and having a temper were pointless. However, the way Declan looked at the woman, as if he could see straight through her clothes, had Ronan ready to punch him.\n\nDeclan's head turned toward him. \"Claiming her for yourself tonight?\"\n\n\"We have a mission,\" Ronan bit out. The thick sunglasses may be shading his eyes, but he knew Declan was aware that he remained focused on the woman.\n\n\"After the mission then?\"\n\nRonan tore his attention away from her to look at Declan. His friend's casual air vanished; he straightened away from the railing and took an abrupt step back. Ronan didn't have time to contemplate Declan's reaction to him before the rancid stench of garbage wafted through the air. His gaze returned to the dance floor as he searched for the source of the smell.\n\nThe crowd of people flowed away from the corner as Joseph glided out of the shadows from the entryway. Yet even as the humans moved away from him, some of the women and men practically tripped over themselves to get closer to him.\n\nA vampire's innate ability to lure someone closer drew the humans to Joseph like a bee to nectar. Their instincts told them this was no nectar, but a Venus flytrap set to spring and devour them whole. Unfortunately for these humans, Joseph's lure won out over their flight-or-fight instincts.\n\nRonan looked to the woman only twenty feet away from Joseph. His lips skimmed back when the woman's gaze locked on Joseph strolling through the crowd. Unlike the other females, she didn't saunter toward the vampire. Instead, her hand went to something at her side.\n\nRonan's eyes narrowed at her unusual reaction. The black-haired, male hunter emerged again at the edge of the dance floor, drawing the woman's gaze to him.\n\nKadence wanted to kick herself when Nathan spotted her from the other side of the dance floor. She'd _finally_ succeeded in breaking free of the stronghold, finally made it here, and she'd been busted within five minutes of walking into the club. So much for being a stealthy hunter.\n\nShe'd completely blown it, and now she would never have another opportunity to be free again. Nathan would make sure of that. If the monster didn't die tonight, she'd never be able to witness it.\n\nGoing by what she'd been told about him and the odor coming from him, she'd known the minute the vampire who killed her father came into eyesight. She'd also forgotten about everything else as her blood thrummed with the need to see the monster slaughtered.\n\nFor a second, Ronan watched as the hunter and woman locked eyes, and then the male was moving toward her so fast that the humans didn't register his passing. The born hunters may not be vampires, but they certainly weren't entirely human either.\n\nTen feet before the hunter reached the woman, he stopped in the middle of the dance floor. His head swiveled and his nostrils flared as his gaze locked on Joseph. The hunter's eyes darted between the woman and Joseph before he closed the distance to the woman.\n\nRonan couldn't hear what they said to each other over the thumping music, but when the man snatched the woman's arm, she yanked it away from him and planted her hands on her hips. A low growl rumbled up Ronan's throat. Joseph was right there, yet he found himself thinking about breaking the hunter's hand for daring to touch her when she obviously didn't welcome it.\n\nFor daring to touch her when it was all that _he_ wanted to do.\n\nThe woman's hands moved through the air as she spoke; the man's followed suit as they faced off. Then, the crowd parted and Joseph moved within feet of them. Joseph's attention remained on the women at his sides as he walked by the male hunter. The man and woman stopped speaking as they focused on Joseph. Their faces filled with a hatred Ronan suspected ran deeper than a hunter's normal animosity toward vampires.\n\nWhen Joseph was out of sight, the man took hold of the woman's arm and led her over to join the three other hunters standing beside the dance floor. The woman moved with the same lethal speed as the man, confirming her as what she was. Ronan shoved aside the disappointment slithering through him at the realization the woman was completely off limits.\n\n\"A female hunter,\" he murmured.\n\n\"I thought they were a myth,\" Declan stated.\n\n\"Apparently not,\" Ronan said as his gaze returned to Joseph. It didn't matter who or what the woman was, all that mattered was ending this tonight. He only hoped the hunters stayed out of his way.\n\nHe'd prefer not to have to kill them too.\n\n\"Let's go.\"\n\nHe stalked toward where Killean and Saxon stood at the top of the stairs. Lucien, one of his best fighters, was on his way to meet them, but Ronan didn't think he'd make it in time for this battle. Lucien had reluctantly agreed to take over the running of the training facility after Joseph turned Savage. Ronan had expected to destroy Joseph sooner, so he hadn't bothered to move the training facility out of New York or find someone else to run it yet, but that would change if Joseph wasn't brought down tonight.\n\nStepping off the last stair, Ronan paused to survey the crowd before following Joseph's stench through the club. Any vampire who killed a human took on the aroma of trash and decay. If they didn't kill again, eventually the smell faded away. The more a vampire killed, the more rotten they smelled. Roadkill mixed with feces and month-old bodies sometimes became preferable to the odor some Savages emitted. However, only a purebred vampire could detect the odor.\n\nJoseph had been having more fun than Ronan realized, judging by the scent of him.\n\nWinding through the crowd, Ronan caught another glimpse of the black-haired, male hunter on his left. His gaze instinctively sought the woman, who now stood with one of the other men. That man had his hand around her slender bicep while she glowered at him.\n\nKadence considered kicking Logan in the nuts to break free of her fiance's hold, but she was afraid if she made a move now, she would scare the vampire they hunted away, or worse, get Nathan killed. Logan's displeasure beat against her. She didn't care that he was mad at her; he would have to get used to her not doing what she was told once they were married.\n\nLogan had to know she wasn't the proper, well-behaved hunter she was supposed to be. Everyone in the stronghold knew that. She'd gotten in more trouble over the years than all the other women combined.\n\nAt one time, Nathan and his friends had laughed over her antics. They'd stopped laughing years ago, and they certainly weren't laughing tonight. Jayce and Asher both stared at her as if she were a ten-legged, alien cat who had dropped on their heads from a beam above. Nathan refused to look at her as he tapped his foot and ran a hand through his black hair.\n\nShe tilted her head back to look at Logan. Unlike the man on the balcony, Logan was definitely what most would consider handsome with his pine-colored eyes, light brown hair, and refined features. Unfortunately, his handsomeness did nothing for her. She tried to tug her arm free again, but his hold on her only intensified.\n\n\"Let me go,\" she commanded.\n\n\"No.\" The simple refusal set her teeth on edge.\n\nRonan watched the woman trying to break free of the man's hold on her. He almost detoured to yank her away from the hunter, but there was no time for that, and the last thing they needed was a fight with the hunters tonight. Slipping through the shadows, he tracked Joseph to one of the back doors.\n\nThe expression on Joseph's face was one of boredom as a human woman ground her hips against his while rubbing her breasts on his chest. Joseph's head came up and a smile curved his mouth when his eyes latched onto Ronan's over the sea of human heads separating them.\n\nBending low, Joseph whispered something in the woman's ear before sinking his fangs into her throat. Joseph tore a chunk out of the woman's neck and spit it out. The woman's scream was drowned out by the beat of the music as she staggered back. Her hand flew to the wound as blood poured from between her fingers.\n\n\"Shit!\" Ronan shouted. \"Saxon, take care of her!\"\n\nHe didn't care if the woman lived or died, but if she somehow survived this, she couldn't be allowed to tell the tale of the man who had torn her throat out with his teeth. If she died, she couldn't do so with the evidence of a vampire's fangs on her. It would only attract more hunters if she did.\n\nJoseph spun and crashed into the back door, flinging it open and vanishing into the alley beyond. The woman slumped to the side and fell into him. Ronan steadied her as the coppery scent of her blood hit him. Ignoring the lure of her blood, he pushed her over to Saxon.\n\nFrom the corner of his eye, he saw another door to the alley swinging closed. He glanced back to see the female hunter standing at the edge of the dance floor. Ronan hesitated when he realized they'd left her alone, but she didn't show any sign of following her brethren out the door. Then, he spotted the man who had been holding her jogging toward the other exit. The man stopped beside the door and leaned against the wall before glancing back at the woman.\n\nTurning away from them, Ronan didn't look back as he followed Joseph out the door. The hunter woman was no concern of his; her relatives in the alley were.\n\n# CHAPTER 4\n\nRonan stepped into the alley, his eyes and ears attuned to his environment as he searched for Joseph and the hunters. Garbage pushed up the lids of the dumpsters lining the brick wall of the buildings across from him. The refuse flowing over the sides of the dumpsters helped to mask Joseph's scent.\n\nDeclan's and Killean's booted feet thudded on the concrete behind Ronan as he turned to the left and started walking that way. A few lazy snowflakes spiraled from the sky. The club was close enough to the ocean that a shift in the wind brought the briny scent of low tide on the air with it. The alley was unnaturally silent; the predators lurking amid it had scared off the rats who resided within.\n\nThe end of his coat beat against his calves as the alley split off and he continued down another corridor. Tucked within the inner pockets of his coat were a couple of stakes and a small crossbow. However, he mostly relied on his hands and his fangs when in battle and didn't like to weigh himself down with weapons. He also didn't like to deny himself the pleasure of an up-close and personal kill. It was what kept the demon in him at bay after all.\n\nHe'd avoid killing the hunters if he could. He didn't want to bear the stench of garbage and the increased vulnerability to the sun that their deaths would bring him. The hunters may not be entirely human, but their blood staining a vampire's hands had the same effect a human's did.\n\nAlthough the hunters wouldn't hesitate to slaughter any of them, he didn't consider them his enemy, not completely. They were more of a nuisance that sometimes had to be stomped. The hunters meant well, but they didn't know the difference between the vampires who killed humans for amusement and those who didn't.\n\n_They've never known the difference_ , Ronan thought bitterly.\n\nThe hunters had killed many Savage vampires over the years, but they'd also destroyed some of the good ones. Thankfully, they hadn't killed as many of the good ones as they had Savages. Vampires who didn't kill tended to stay off their radar. They led peaceful lives, and unlike the Savages, they didn't draw attention to themselves by leaving a trail of bodies or missing people behind them.\n\nRonan turned another corner, stopping instantly when he spotted Joseph at the end of the alley. Joseph stood before a ten-foot-high brick wall, studying the blockade before him. More garbage than before overflowed the dumpsters and spilled onto the asphalt. Most of the bags had been torn open and picked through by the animal scavengers and probably some humans.\n\nRonan clenched and unclenched his hands as he studied his old ally. Joseph could have easily cleared the wall and been out of here by now, so why did he remain?\n\nDeclan and Killean halted beside him as Joseph glanced over his shoulder at them. The grin that split his face revealed his lethal fangs as he turned to face them. The hazel of Joseph's eyes briefly flickered through the red encompassing them as he gave a come-and-get-it gesture with his hands.\n\n\"Something's not right here,\" Ronan murmured to the others. \"He's trying to set us up for something.\"\n\n\"What though?\" Declan inquired.\n\n\"I don't know, but stay alert.\"\n\nDeclan and Killean spread out to the sides of him as the three of them prowled down the alley. Ronan didn't know why Joseph had chosen to stay, but he wasn't going to rush him until he knew what the Savage vampire had up his sleeves.\n\nThey had to approach this cautiously, but in his head, he heard the seconds of a clock ticking away. When they exited the club, the hunters must have gone the other way in the alley, but they would come this way eventually. They had to take Joseph down before the hunters arrived on the scene.\n\nAt one time, Ronan's body would have been alive with the thrill of the impending kill. Now he experienced no excitement as he closed in on the fallen Defender. Joseph's elongated canines sliced his bottom lip, and the scent of garbage grew stronger as blood trickled down his chin from the gash.\n\nJoseph crouched down before launching himself at Ronan's chest. It was a move Joseph never would have made before becoming Savage, but the increased strength he'd experienced with his kills made him far more brazen. Despite that increased strength, Ronan had no fear Joseph could take him down. It would take far more than Joseph alone to do so.\n\nRonan swung out at him, catching him squarely beneath the jaw and flinging him into one of the dumpsters. Metal dented with a loud bang. Garbage spewed onto the ground and rats screeched as they scattered into the gloomy recesses of the alley. Joseph came up spitting blood as he launched off the dumpster.\n\n\"So you're going to kill me now? You're going to kill one of your own?\" Joseph inquired. \"So much for loyalty, hey, Ronan?\"\n\nRonan circled him as Joseph moved toward the wall and Declan and Killean closed in on him. \"You're not one of us anymore, Joseph,\" Ronan replied.\n\n\"You have no right to judge me. You're closer to the edge than I ever was.\"\n\nRonan didn't flinch at the assessment; it was true after all.\n\n\"Obviously not,\" Declan replied, \"considering you're the one we're hunting.\"\n\nHatred twisted Joseph's features; Declan grinned at him in return. Joseph snarled, but this time instead of coming for Ronan, he charged straight at Declan whose smile only widened as he braced himself for Joseph's attack. Joseph lowered his shoulder and crashed into Declan, sending him reeling into the wall.\n\nDeclan didn't go down beneath Joseph. Instead, he clasped his hands together and drove them into Joseph's back. Joseph grunted as he wrapped his arms around Declan's waist, lifted him up, and bashed him into the wall again. Declan swung an uppercut that broke Joseph's hold on him and staggered Joseph back a few feet.\n\nBefore he could fully recover, Ronan grabbed Joseph by the collar of his shirt and yanked him back. His hand encircled his throat, crushing Joseph's windpipe as he pinned him to the wall. Pulling his arm back, Ronan fisted his hand in preparation to drive it through Joseph's chest and end this.\n\n\"Ronan!\" Killean's shout alerted him to the threat he'd missed while focused on the kill.\n\nA whistling reached him in time for him to turn to the side, but not in time to avoid the bolt completely. A piercing pain shot through his shoulder as the weapon impaled him from behind. Stumbling slightly forward, he nearly lost his grip on Joseph as a burning sensation spread through the thick muscle of his shoulder. Turning his head, he spotted three of the hunters at the other end of the alley. The black-haired one had an empty crossbow aimed at him.\n\nJoseph grabbed the end of the bolt and twisted it. Ronan involuntarily released his hold on him when numbness spread through his shoulder. Joseph's hands flew to his brutalized throat. Broken sounds issued from him as he clawed at his flesh and his eyes rolled in his head. Ronan had no idea what he was trying to say, and he didn't care.\n\n\"Ronan!\"\n\nDeclan's shout enabled him to dodge the next arrow one of the other hunters fired at him, an arrow aimed straight at his heart. He plucked the arrow from the air and shattered it in his fingers before leveling the hunters with a murderous stare. Two of them took a step away from him, but the black-haired one held his ground as he focused on Joseph.\n\nWithout warning, the black-haired hunter barreled down the alley with his shoulder lowered. He plowed into Joseph, running him backward as he kept his shoulder buried in Joseph's sternum. Joseph and the hunter tumbled into the dumpsters. Garbage spilled over them, momentarily burying them both beneath a wave of trash.\n\nRonan didn't move as Joseph and the hunter thrashed on the ground. He wasn't the only one startled by the uncharacteristically reckless display from a hunter. Declan and Killean remained unmoving as the hunter punched Joseph with enough force to crack a cheekbone. The next blow he delivered shattered Joseph's nose. Blood splattered Joseph's shirt and sprayed onto the concrete within inches of Ronan's boots.\n\nHe'd never seen a hunter behave like this. Whenever he'd encountered them, they'd always been methodical and careful as they worked together. They never let their emotions rule them or broke ranks. Even the hunter's partners were thrown off as they had yet to attempt moving in for their own kills.\n\nThe hunters didn't remain stunned into immobility for long. While what Ronan assumed was their leader continued to pummel Joseph, the other two inched forward. One held a crossbow at the ready, its arrow aimed straight at Declan's heart. The other trained a 9mm on Killean. Unless the gun was loaded with wooden bullets, it wouldn't kill them. However, it would still hurt them enough to slow them down and make it easier for them to be killed.\n\nThe one holding the crossbow kept glancing between Ronan and Declan, but the hunter didn't aim the crossbow at him again. The two remaining hunters must have decided Ronan's injury didn't make him much of a threat. They couldn't be more wrong.\n\nRonan scanned the alley behind them, but the woman and the other hunter they'd been with were nowhere to be seen.\n\nHe turned his attention back to the ugly situation they now faced. The black-haired hunter was dragging Joseph's beaten form up behind him as he climbed to his feet.\n\nAt one time, seeing a fellow Defender so broken and bruised would have enraged him, but Joseph wasn't one of them anymore. Ronan couldn't help feeling a grudging admiration for the damage the hunter had inflicted on the Savage. If it had been any other Savage, Ronan would have walked away and allowed the hunters to have them, but he had to make sure Joseph died this night. The fallen Defender knew far too many of their secrets to risk him continuing to live.\n\nRonan's lips skimmed back when the black-haired hunter's azure eyes fell on him. At about six four, the hunter was a good four inches taller than him, and lean in build.\n\n\"This doesn't have to happen,\" Ronan said. \"We don't have to fight each other. Give us Joseph and walk away.\"\n\nThe hunter bared his teeth and his hand clamped down on Joseph's already crushed throat. Joseph's head lulled forward on his shoulders before it snapped up. The black-haired hunter's eyes were full of antipathy as they held Ronan's. He didn't have to say a word; Ronan knew the real fight was about to begin.\n\nSignaling Declan and Killean, he moved to the side, putting himself in a better position to go after the hunter with the crossbow. Ronan charged at him just as the black-haired hunter's command to kill them rang through the air.\n\nThe hunter holding the crossbow caught his charge out of the corner of his eye. Startled, he spun toward Ronan, but he was too late. Grabbing the end of the bow, Ronan ripped it from the hunter's hands. He drove a fist into the man's nose, shattering it before he shoved the hunter into a dumpster.\n\nA gunshot rang out. Killean's grunt could be heard over the ensuing echo of the shot as it reverberated off the brick walls surrounding them. Another shot fired, but this time it cracked off brick. Either Killean or Declan had managed to subdue the hunter with the gun.\n\n# CHAPTER 5\n\n\"What were you thinking coming here?\" Logan demanded when she crept forward to stand at his side within the club.\n\nKadence barely glanced at him before focusing on the doorway Nathan and the others had exited through. Her hand slid to the stake in the inner pocket of her coat. \"I should be out there,\" she whispered.\n\nLogan grasped her shoulders, spinning her to face him. Kadence blinked at him, startled by the harshness of his handsome face as he glared at her. \"You have no business being here at all!\" he barked at her.\n\n\"You have no business telling me what to do!\"\n\n\"I have every right to tell you what to do. We're to be married soon, and you've put yourself, your brother, and our entire way of life at risk by coming here. I will not have it!\"\n\nThe one thing her instructors had always hated most about her was her willful streak, but she wouldn't be ordered around by anyone. Not her father, not her brother, and certainly not the man who she had no choice about marrying.\n\n\"I will not have you talking to me in such a way,\" she grated from between her teeth. \"We may be getting married, but no one will order me around, Logan. Not even my husband.\"\n\nShe jerked out of his grasp and gave him a scathing glance before focusing on the door again. Around them, people danced and swayed, the lights flashed over the walls and floor, but she tuned it all out to focus on hearing anything from the outside world. Her father's line of hunters had always been the strongest; it was why their line had been the leader of the hunters since the beginning. She and Nathan were faster and stronger than the others, their senses more honed.\n\nBecause she was a woman, she would not have been allowed to lead even if she were the only living descendent, but she carried the strength of that line in her blood. If Nathan were killed before he could produce an heir, her husband would lead the hunters until she produced a male heir who would one day take control. If she had only a female child, her daughter would face the same fate. Only three times in their history had someone outside of their line been a leader until an heir was born.\n\n\"I am only trying to keep you safe,\" Logan said.\n\nHe broke her concentration on the outside when he rested his hand on her shoulder. His finger slid up to stroke her cheek. She resisted cringing away from the tender touch. She loved Logan as a friend, she always had and always would, but she was well aware his feelings for her were more than friendly.\n\nHe clasped his hand possessively around her nape, drawing her a step closer. Kadence stiffened beneath his touch, but it was something she would have to get used to if she were going to survive her marriage to him.\n\nHer stomach rolled at the thought of their wedding night, and the many nights that would follow, before she blocked it out. If she pondered it too much, she would never make it through the wedding ceremony, never mind the next hundred and fifty or so years they could possibly be married, if Logan didn't get killed on a hunt.\n\nA gunshot from outside barely registered through the music, but she knew what she'd heard. Plunging forward, she slammed into the bar on the door and shoved it open. The cold air robbed her breath as the second gunshot sounded.\n\nLogan grabbed her arm, pushing her back toward the club. \"Get inside!\"\n\nKadence staggered backward, but she didn't turn toward the closing door. Instead, she followed Logan down the alley, running as fast as her legs would carry her. One vampire versus three hunters, her brother and the others should have easily taken him down. Yet, she could hear the sounds of fists hitting flesh, the twang of a bolt firing, and the scuffle of numerous feet from deeper within the alley.\n\nSomething had gone wrong.\n\n_Not my brother, please not my brother too._ She couldn't handle it if she lost her father and Nathan.\n\nHer brother was the only family she had left. Nathan wouldn't stand in the way of her marriage; as the leader, he would follow tradition, no matter how unhappy it made her. However, he would be there for her to lean on when she needed it. She hadn't complained to him about her marriage\u2014she couldn't when he had enough weighing on him right now\u2014but she knew he was aware she wasn't happy about it.\n\nJust as she knew that being the newly appointed leader wasn't something he wanted, though he'd never said it to her. As twins, they had always been close. They'd been closer when they were younger, before Nathan went into training and she was forced to learn how to be the perfect wife and mother. They followed separate courses in life, but he'd still been her best friend over the years, her rock.\n\nLegs aching, lungs straining for air, Kadence flew around a corner of the alley and skidded to a halt. The alley reeked of the coppery scent of fresh blood and garbage. Bile rolled up her throat, and it took all she had to keep from vomiting. The last time she had smelled blood so strongly...\n\nMemories of her father's broken body tried to drag her under before she locked them away.\n\nShe found Nathan instantly, unharmed and holding _the_ _vampire_ in his grasp. Hatred blurred her vision as she gazed at the creature who had murdered her father and torn her life apart. The vamp was beaten and bloody, and though she scented him on the air, it was not his odor filling her nostrils now. This scent was different, spicier, and held no hint of the evil pouring from the vamp.\n\nHer eyes latched onto a heavily muscled man shoving Asher aside. Blood poured from Asher's broken nose, but it wasn't his blood she smelled either. It was the blood of the man who had shoved Asher away. A man with lethal fangs fully extended and an overwhelming aura of power emanating from him.\n\nDisbelief screamed through her as she recognized him as the man who had been standing on the balcony watching her. The man who had finally made her understand some of what she'd read in those romance novels. A man who was _not_ a man at all, but one of the twisted freaks who thrived on killing.\n\nHow could his blood smell like that? All vampires smelled like rot! Over the years, the male hunters had captured some vampires and brought them to the stronghold for training purposes. They'd allowed the women of the camp to get close enough to detect the stench vampires emitted. It was a smell she would never forget.\n\nAs this vamp turned toward her, she saw the bolt protruding from his shoulder and the blood oozing from the puncture. His coat was pushed back, his black shirt ripped open to expose a slash of bronzed flesh across his broad chest.\n\nHis sunglasses remained in place, but she knew he watched her. Molten lava spread through her veins as those covered eyes exposed her and ripped her soul bare. He stared _into_ her in a way no other ever had. Kadence shuddered while her heart leapt into her throat. Despite the fangs and the fact he was a creature she'd been born to hate, she couldn't deny her pull toward him.\n\nShock slid through Ronan as he stared at the female hunter. He hadn't expected her to leave the club, to come out here, and he certainly hadn't expected the rapt way she watched him. He'd seen many beautiful women over the thousand years of his life, but none of them had captivated him in such a way.\n\nThe battle and his men faded away as he stared at her. The wind tugged at her silvery hair, whipping the loose strands of it around her oval-shaped face. Her azure eyes watched his every move. The look on her face said she couldn't figure him out, but then he had no idea what to make of this woman either.\n\nKadence felt time crawl by as they gazed at each other, but when Logan suddenly crashed into the vamp, she realized only mere seconds had passed. Asher barely dodged out of the way of the vamp and Logan as Logan propelled the vamp into the brick wall.\n\n\"Ronan!\" the auburn-haired man shouted.\n\n\"I'm fine,\" the one wearing the sunglasses grunted, and she realized he was Ronan.\n\nKadence took a step forward to stop Logan from attacking him, but then her heritage, her _common_ _sense_ , kicked in and disgust at herself filled her gut. He was a vampire for crying out loud! What was the matter with her?\n\nHer first time out of the stronghold and she was having the warm and fuzzies for her worst enemy. She was an idiot and a pitiful excuse for a hunter.\n\nTaking a deep breath, she struggled to control the accelerated beat of her heart as Ronan hit Logan, knocking him backward. \"Declan, go that way,\" Ronan ordered with a wave of his hand at the auburn-haired man. \"Killean, that way,\" he commanded the one with the scar.\n\nKadence's hands went to two of the stakes tucked into her inner coat pocket when she realized there were two _more_ vamps in the alley. Declan moved to the right as Jayce fired a shot at Killean, hitting him in the shoulder. Killean lunged forward, knocking the gun from Jayce's hand with one blow.\n\nKillean jerked Jayce forward, but instead of sinking his fangs into Jayce's throat and tearing it out, he threw Jayce away from him. Killean could have killed Jayce, there was no denying that. Instead, he'd pushed him away. Kadence's mind spun as she tried to understand what she'd seen, but none of it made any sense.\n\n\"Kadence! Get out of here! Now!\"\n\nNathan's bellow made her cringe, but she wouldn't follow his command. She had to witness what happened here. Kadence gasped when Logan brandished a stake and tried to plunge into Ronan's chest. Ronan grabbed Logan's hand and tore the stake away as if Logan were no more than a child. With a flick of his fingers, he tossed the stake aside.\n\nHis lethal fangs gleamed in the dim light of the alley, but instead of striking, he pushed Logan away from him like Killean had. Kadence watched as the vamps lined up on one side of the alley, with the hunters moving to position themselves on the other side. The vamp who had killed her father thrashed in her brother's grasp, his red eyes blazing like rubies in the sun. His florid face and wheezing breaths led her to believe his windpipe had been crushed at some point.\n\n\"We are at a crossroads,\" Ronan stated.\n\nHis deep voice sent shivers down her spine. Kadence shook herself. No matter how compelling she found him to be, he was a vampire, plain and simple.\n\nSo caught up in her own confusion and emotions, she failed to notice what had happened during the fight. Her brother and the hunters were lined up on their side, closest to the wall, with the vampires lined up before them.\n\nWhich would have been fine. Her brother had her father's killer. Kadence was more than happy to leave here with him, more than happy to go somewhere else to dispose of the monster, while they left the rest of the vamps alone, for now.\n\nThe real problem was that she'd been separated from the hunters and was stuck behind the vampires.\n\n# CHAPTER 6\n\n\"Do you plan to fight to the death?\" Ronan asked.\n\n\"If that's what it takes, considering it will be your deaths, not ours,\" Nathan replied.\n\nDeclan and Killean chuckled while Ronan shifted his stance. Kadence's heart leapt into her throat. \"No,\" she croaked.\n\nOnce the word was out of her mouth, she knew she'd made a mistake. Until she spoke, everyone had forgotten about her. Now, all eyes turned toward her.\n\n\"Kadence, leave!\" Nathan spat.\n\nHer eyes flew around the group. Her hand trembled as she lifted it to her treacherous mouth. However, she couldn't run from here. She couldn't lose her brother, and for some strange reason, she didn't want anything bad to happen to Ronan either.\n\nLater, when she was alone, she would kick herself in her stupid ass for finally encountering vampires in the world and turning into a complete idiot because one of them fascinated her. For now, she had to completely ignore her bizarre reaction to Ronan as the other two did absolutely nothing for her.\n\n\"The fight is a draw. No more blood has to spill,\" she said. \"We have the one we came for. There will be other nights to continue this.\"\n\n\"And more innocent deaths before then!\" Nathan sneered.\n\n\"Nathan, please, I can't lose you too!\" she cried.\n\nSo the hunter, Nathan, was her lover or husband, Ronan realized. He eyed the vein in Nathan's throat and licked his lips as he contemplated ending his competition for her.\n\n\"Get out of here, Kadence!\" Nathan yelled at her.\n\nA low growl emanated from him over the way Nathan spoke to her. Declan and Killean exchanged startled glances, but neither of them could be as surprised as he was over it.\n\nNathan tightened his hold on Joseph's throat. He seemed to think Ronan was going to charge him to take Joseph back. However, Ronan had no intention of going anywhere near Nathan, not if it meant leaving Kadence unprotected.\n\n\"Nathan\u2014\" Kadence started.\n\n\"I said _leave_!\"\n\nThe roared order echoed throughout the alley. Kadence preferred not to see the bloodbath she was certain was about to unfold, but she wouldn't let it happen if she could somehow stop it. Her fingers slid away from one of her stakes as she looked to Ronan, whose head was turned in her direction.\n\nEverything she'd been taught over the years told her he was evil. However, something within her said he wasn't. That belief might get her killed tonight. If it did, then she well deserved to die for her stupidity, but she couldn't bring herself to attack someone who hadn't come after her.\n\n\"I'm not leaving,\" she declared as she looked back to her brother.\n\nNathan's eyes widened. Declan glanced over his shoulder at her, an approving smile curving his full mouth. Before she could think about what that smile meant, a large hand enclosed on her arm. She spun to face the two men who materialized from the shadows behind her.\n\nThe one holding her stared back at her. His pitiless black eyes caused chills to run down her spine. His sandy blond hair framed his handsome face as his upper lip twisted into a sneer of disapproval.\n\nShe didn't have to see his fangs to know this was another vampire. Dread flared through her as his grip became bruising. The other vamp who had appeared with him moved forward to join Ronan and his group. Kadence's heart sank as she realized the hunters were now outnumbered.\n\nShe didn't get any murderous vibes from Ronan, but she had no doubt the vamp holding her would break her neck and step over her dead body with the same amount of consideration he gave to pulling on his socks.\n\n\"Hello, boys,\" the vamp holding her greeted, his voice as glacial as the rest of him.\n\n\"About time you showed up, Lucien,\" Killean said.\n\n\"Looks like Saxon and I are just in time, Killean,\" Lucien replied with a shrug. \"Quite the pickle we're all in. Especially Joseph there,\" he nodded to the vamp Nathan held. \"Looks like everyone's after your blood, asshole.\"\n\nJoseph opened his mouth to respond, but only a choked _ugh_ sound came out. Hatred churned in her gut as Kadence gazed at the vamp who had killed her father. She knew his name now, but to her he would always be the vamp who had torn her world apart. The one who had propelled her into breaking free of the boundaries she'd chafed against her whole life.\n\n\"Let go of her!\" Nathan snapped at Lucien.\n\n\"Is she what I think she is?\" Lucien inquired, completely ignoring her brother's command as he bent toward her to inspect her more closely.\n\n\"She is a born hunter,\" Killean answered.\n\nKadence leaned away from him and, before she could think about it, kicked him in the shin. Lucien jumped and surveyed her with more interest. \"Feisty little hunter, aren't you?\" he murmured.\n\n\"Let go of her, Lucien,\" Ronan said. \"She has no part in this.\"\n\nKadence gawked at him. Was Ronan, the _vampire_ , trying to protect _her_? Even Nathan looked confused by his words.\n\n\"From what I can tell, they have Joseph and we have her, so I think she has a big part in this,\" Lucien replied. \"Not to mention, _no_ one has seen a born, female hunter. She may be something we can use to our advantage against these do-gooder, idiotic pricks.\"\n\nRonan spun toward them. The fury emanating from his body caused her to step back. Lucien squeezed her arm, eliciting an involuntary wince from her. Adrenaline spiked through her as Ronan stalked toward them with menace exuding from every inch of him. She'd been wrong about him; he was going to drain her dry before tossing her lifeless body aside.\n\nKadence refused to cower from his approach as she defiantly lifted her chin. She'd put up one hell of a fight against him, though she was certain he could crush her skull with only one of his hands. Her fingers twitched toward the stake at her side while she awaited her opportunity to inflict as much damage as possible. Ronan would remember her years after he'd killed her, she'd make sure of it.\n\n\"Stay away from her!\" Nathan's bellow rent the air.\n\nShoving Joseph at Jayce, her brother rushed forward. Killen stepped forward to block his attack. Kadence screamed and lurched forward to help her brother as he barreled into Killean. The two of them tumbled to the ground in a frenzied heap.\n\nLucien jerked her back before she made it two steps toward them. Ronan's hand lashed out to grasp Lucien's forearm.\n\n\"I said let her go!\" Ronan ordered.\n\nKadence's jaw dropped.\n\n\"What is the matter with you?\" Lucien demanded.\n\n\"Let her go.\" His hand squeezed Lucien's arm to the point where Kadence was certain the bone would break.\n\nLucien released her, and his brows furrowed as he studied Ronan. \"I'd rather fight anyway,\" he muttered before stalking away from the two of them.\n\nIt was then that Kadence realized the battle had commenced again. The only one hanging back now was Jayce, but he'd been saddled with Joseph.\n\n\"No!\" Kadence stepped forward, determined to stop this, or at least try to help as her brother and Killean circled each other like wolves.\n\n\"No.\" Ronan stepped in front of her, his solid body blocking her way.\n\nHe became all she could see as his chest alone was three times the size of hers. He was not as tall as the others, but there was no doubt he was the strongest one here. She didn't have to see him in action to know that. Every strand of her DNA was aware of what stood before her: an old vampire with more power than she could begin to comprehend.\n\nHer lips parted on a breath as her eyes fell on his shaded ones. She had no idea what it was about him, but she couldn't find it in herself to care that he was a vampire\u2014not when her skin felt electrified by his nearness and her nerve endings tingled with her need to touch him.\n\nShe craved more of him.\n\nThousands upon thousands of hunters were turning over in their graves right now. Self-hate skittered through her, but she couldn't bring herself to pull out her stake and attack him.\n\nRonan shifted as the scent of her arousal on the air caused him to harden. She wanted him too. Her forehead furrowed in confusion over her reaction to him. He completely understood her confusion as he felt it too.\n\nHe didn't know what drove him, but he _had_ to know if her skin was as supple as it looked. Without thinking, he reached out to touch her cheek. Beneath the pads of his fingers, her skin was like fine spun silk. She didn't recoil from him, didn't look repulsed. Instead, to his amazement, she turned her cheek into his hand so that her lips brushed against his palm.\n\nA loud crash from behind him jerked them both apart and brought the world rushing back in around him.\n\n\"Shit!\" he hissed.\n\nHow could he have forgotten about the fight behind him? How could he have left his friends to fend for themselves against the hunters and Joseph?\n\n\"Go!\" he gruffly commanded her. \"Get to safety.\"\n\nHe turned away from her, knowing he would never see her again.\n\n\"Wait!\" Her pale hand on his arm seared into his flesh, causing his teeth to scrape together when his cock jumped in response. He was not some horny teen, yet this woman had somehow managed to reduce him to that when his men needed him. He glanced at her over his shoulder. \"Please, don't do this.\"\n\n\"We don't want this,\" he replied coldly, pulling his arm away from her before she distracted him further from his duties.\n\n\"I... I don't understand,\" she whispered.\n\n\"No, _your_ kind never has.\"\n\nThe anger in his voice would have deterred anyone else; it didn't deter her though.\n\n\"Please, I can't lose him,\" she whispered. Anger surged through Ronan; she was lusting over him, but still pleading for her lover's life. He contemplated killing Nathan, simply so the man couldn't continue to have what he so desperately craved for himself. \"Not my brother.\"\n\n\"Your brother?\"\n\n\"Yes.\"\n\n\"Your brother will be safe.\" If he wasn't already dead. \"Now go!\"\n\n\"Thank you.\"\n\nHis chest constricted at those words. \"Go.\"\n\nRonan didn't look back at her as he ran toward the others. A good fight and death were what he needed to purge himself of his reaction to her.\n\n# CHAPTER 7\n\nKadence rounded the corner, but went no further. She couldn't leave. Her brother was fighting for his life, and that man, that _vampire_ , was there. The warmth of Ronan's fingers still lingered on her cheek. He hadn't acted like her enemy. She supposed his strange behavior could be some sort of sick, vampire trick, but she doubted it. He'd had the perfect opportunity to kill her. He could have easily struck her down, or had his friend do so. Instead, he'd told her to run.\n\nThe monsters she'd always been taught vampires were would have killed her without a second's hesitation. Those monsters wouldn't have told her that her brother would be safe. What had just happened between them was not normal and she needed answers.\n\nShe also hadn't come all this way not to watch Joseph die.\n\nKadence crept back to the corner of the alley and poked her head out to watch the battle. She pulled her crossbow free and loaded it. Her hand squeezed on her weapon while she watched them all fight in a flurry of fists and blood, but the vampires still didn't go in for the killing blow. Now that she was watching more closely, she realized they spent most of their time deflecting the hunters' attacks. The hunters were outnumbered; this fight should be over. It wasn't.\n\nThe more she examined the vampires in action, the more she began to question if Ronan and his friends were the murderous, mindless creatures she'd been taught to hate her whole life. They seemed to be something different, something she'd never known existed when it came to vampires. Something she didn't think _any_ hunter had ever known existed.\n\nRonan cut his way through the thick of the fight. Seizing Logan and Nathan by their necks, he lifted them and threw them toward where Jayce and Asher stood with Joseph. Kadence couldn't help but marvel over the amount of strength Ronan possessed. He'd tossed them both aside as if they weighed no more than a feather. The other vampires fell into line behind him as the hunters grouped together. Blood tricked from the injuries the hunters had sustained, but none of them were mortal wounds.\n\n\"Give us Joseph,\" Ronan commanded. \"We will take care of him.\"\n\n\"Set him lose to kill more, you mean,\" Nathan said as he slid his crossbow free of his hip.\n\n\"He'll never know freedom again,\" Ronan replied. \"He's ours to destroy. Give him to me.\"\n\nNathan's crossbow dipped a bit before leveling on Ronan's broad chest, right at his heart. Kadence's breath stuck in her throat when Ronan stalked forward as if he didn't see the weapon. The resolute expression on Nathan's face was one she recognized well. The bolt fired less than a split second later.\n\nRonan didn't change his course or even blink when he plucked the bolt out of thin air and tossed it aside as if it had been an annoying fly. Kadence gaped at him. She'd never seen anyone move that fast before, had never thought it possible that someone could.\n\nBefore anyone could react, or Ronan could reach Nathan, Joseph shouted a command. \"Now! Attack now!\" The words were garbled, but his windpipe had healed enough for him to speak again.\n\nKadence frowned as she tried to figure out who he was yelling at. The others in the alley exchanged a questioning glance. Ronan leapt forward, his hand encircling Joseph's throat. He looked about to tear Joseph from Jayce's grasp just as another vampire leapt on top of the brick wall. The feral-looking vamp with the blood-red eyes was followed by a dozen more vampires.\n\nRonan froze with his hand around Joseph's throat as he gazed at the Savages perched on the wall like gargoyles ready to take flight. Their putrid odor barely pierced through the stench of the alley. It hit him that Joseph had lured them into this area of the alley because of the amount of trash and the fact that his cohorts could remain hidden on the other side of the wall until he was ready to draw them forth. If he hadn't crushed Joseph's windpipe, the Savages would have arrived sooner.\n\n\"Well, that's a heap of shit right there,\" Declan said and reached over his back to slide two swords free from under his coat.\n\nKadence bit back a shout when the vampires launched themselves off the wall. Right now, no one knew she was there; she could be the hidden weapon they may need, but only if she didn't give away her location. One of the vamps crashed into Ronan, knocking his hold on Joseph free. Another hit Nathan, sending him staggering into Jayce and Joseph.\n\nJoseph twisted in Jayce's grasp and yanked Jayce's hand to the side. The young hunter howled as his wrist broke with a snap of bone. Kadence's stomach nosedived into her toes, but she lifted her crossbow and aimed it at Joseph's heart. Joseph clutched Jayce's head as she fired the bolt.\n\nA bead of blood formed on Jayce's ear from where the bolt nicked him before driving into Joseph's chest, centimeters off his heart. Blood-red eyes briefly met hers before Joseph snapped Jayce's neck to the side. The cracking of his spine echoed down the entire alley. The scream of _No!_ burned its way up her throat and choked her as a cruel grin curved Joseph's lips.\n\nSorrow filled her as Jayce's limp body collapsed to the ground. She'd grown up with Jayce. They'd played tag and eaten ice cream cones on hot summer days. He'd once told her he would have preferred to be an astronaut, to travel into space, but instead he'd trained to be a pilot for the hunters and to kill. And now he was gone.\n\nKadence wanted nothing more than to sit down and sob for the loss of her friend. Instead, she pulled another bolt free from her pocket and reloaded the crossbow. She lifted it to end the vicious monster who had destroyed her father and her friend.\n\nShe was about to fire again when Ronan stalked through the fray and captured Joseph by the throat with one hand. With ease, he lifted Joseph high before smashing him into the pavement. The asphalt cracked and splintered beneath Joseph's body. Ronan drew his other hand back to strike Joseph when two more vamps leapt onto the top of the wall before launching themselves off and onto his back.\n\nKadence's hand quivered on her crossbow when Ronan vanished beneath their bodies. She searched for Nathan and the other hunters, but they were also nowhere to be seen amid the mass of vamps. She fired more bolts at two of the vamps on Joseph's side, successfully taking them both out.\n\nShe'd gone through a fair amount of training over the years to learn how to protect herself, but she'd never been allowed to hunt a vampire. Now, her blood hummed with excitement, her body was alive in a way it never had been before, and she couldn't deny that this felt _right_.\n\nRonan rose from the mass, shedding the vamps clinging to him. Grabbing one of them, he threw the vamp forward with enough force to shatter some of the bricks in the wall. The indent of the vamp's body was left behind as he slumped to the ground. The wall rocked on its foundation, and for a minute, Kadence thought it would topple over.\n\nShe searched for her brother, worry tearing at her insides as she spotted Asher and Declan beating back the wave of vampires coming at them. If they found it weird to be working together, they didn't show it. _The enemy of my enemy,_ she realized as they stood nearly back to back with each other. Logan was fighting near the one with the scar, Killean. Lucien and Saxon stood back to back as they fought.\n\nRelief filled her when she finally spotted Nathan next to one of the dumpsters. Blood streaked his face from a nasty gash above his right eye, but it didn't slow him as he drove a stake through the heart of a vamp.\n\nKadence reloaded her weapon as another vamp rose behind Declan. The vamp was about to grab Declan when Kadence fired a shot that went straight through the vamp's heart. Declan glanced at her over his shoulder, his silver eyes glittering with amusement when they met hers. He nodded to her before driving a sword through the stomach of another vamp and running him into the wall.\n\nThe already damaged wall swayed back from the impact and gave an ominous creaking sound.\n\n\"Look out!\" Asher shouted.\n\nAsher staggered away from the wall as it rocked precariously. Pieces of breaking brick clattered over the concrete and thumped off the dumpsters. The wall swayed back again before crumpling with a loud bang and the clatter of bricks crashing into one another. A cloud of gray dust blew all the way out to where she stood.\n\nKadence stepped forward as her view of the battle was obscured by the debris filling the air. When it finally cleared enough, she saw the vampires who had joined the fight on Joseph's side were fleeing down the street. Asher and Logan followed them, and ahead of them, she spotted Nathan turning a corner.\n\nJoseph was nowhere to be seen. She suspected he'd been the first one to tuck tail and run like the coward he was.\n\nThe other vamps remained standing in the alley, surrounded by the carcasses of the vamps who hadn't survived and Jayce. Kadence tore her attention away from her friend's body before she started to sob and didn't stop. She retreated into the shadows as Ronan wiped some of the debris from his hair. Bits of dust and brick rained down around him.\n\nHe glanced idly at the arrow protruding from his shoulder. Without so much as a flinch, he ripped it free and tossed it aside. Kadence winced for him.\n\n\"If the hunters get a hold of Joseph\u2014\" Lucien started.\n\n\"They won't,\" Ronan replied. \"And if we go after them, we'll only end up fighting them again.\"\n\n\"And then we'll inevitably kill one of them,\" Saxon said.\n\n\"I don't see a problem with that,\" Killean replied.\n\n\"The hunters are not our enemies,\" Ronan said.\n\nWhy did he keep saying that? Kadence wondered. Why did he _believe_ that?\n\n\"Maybe not, but they believe we are _theirs_ , and because of that they fucked this night all up,\" Killean said. \"Joseph would be dead right now and everyone on this planet would be a lot safer, if it wasn't for their stupidity.\"\n\n\"Enough, Killean,\" Ronan said. \"You and Saxon gather these bodies and take them somewhere the sun can get them in the morning, or where you can set them on fire.\"\n\n\"I'll get the van,\" Saxon volunteered. He climbed over the bricks and jogged toward the street.\n\nAt the end of the alley, she spotted a crowd of humans growing. The humans whispered behind their hands as they pointed down the alley at the vampires standing where the wall once had stood.\n\n\"Declan, take care of the crowd,\" Ronan ordered.\n\n\"With pleasure,\" Declan replied. He climbed over the bricks and sauntered toward the humans. \"Hello, darling,\" he purred to one. The uneasy look left the girl's face. She giggled and blushed prettily when he ran his finger under her chin.\n\nKadence watched as Declan spoke with the crowd. She couldn't hear what he said, but when he was done, they all walked away. She'd heard about a vampire's ability to change the memories of another, but she'd never witnessed it in action. She had to admit it was impressive, and scary.\n\nAs a hunter, she was immune to a vamp's ability of persuasion\u2014something she was extremely grateful for right then.\n\n\"What about the hunter's body?\" Lucien inquired.\n\nKadence almost stepped forward to scream at them to leave it be, but Ronan was already speaking, \"The hunters will come back for him.\"\n\n\"And if someone discovers it before then?\"\n\n\"That's the hunters' problem to deal with, not ours. There are no signs of a vampire attack on him, and the police will have a difficult time trying to figure this all out, but again, not our problem. What about the girl Joseph attacked in the club?\" he asked Saxon.\n\n\"I couldn't save her,\" Saxon replied. \"I obscured the wound on her neck and hid her body. They should find her by morning.\"\n\nRonan clenched his teeth as he gazed down the road where the hunters had vanished in pursuit of Joseph. The human race was a food supply to him, but he'd failed in his mission tonight and an innocent had died because of it. Even worse, more of them would die now that Joseph had gotten away again.\n\nThey should have killed the hunters and Joseph and called it a night. They all would have had to deal with aftereffects of the hunters' lives on their hands, but they would have saved countless other lives in the long run. If the hunters interfered again, he may have no choice but to take them down.\n\nKadence remained in the shadows when she was certain the vampires wouldn't be taking Jayce's body with them. Her brother would be back soon, but even if he didn't return, they would find a way to claim Jayce's body.\n\nIf the vamps left soon, she would come back and get Jayce herself. She didn't care if he weighed a good seventy pounds more than her; she would figure out a way to bring him home.\n\nA beat-up gray van pulled up at the end of the alley and backed down toward what remained of the wall. The vehicle didn't come to a full stop before Killean pulled the back doors open and they began to toss the bodies of the vampires into it.\n\nThey would all be gone soon, and she should retreat from here until they were. She should have already left this place behind. Her brother was gone, and she was in an alley full of vamps.\n\n# CHAPTER 8\n\nShe edged down the alley, her heart growing heavier with every step as she reloaded her crossbow. She'd lost a friend tonight, and now it was time for her to return to the stronghold where she would be locked away once more and married to Logan.\n\nKadence tilted her head back to stare at the night sky. She'd seen it every night from the stronghold, but this was the first time she'd seen it outside of the stronghold walls. This was her last night of freedom, the _only_ night of freedom she'd ever experienced. She didn't realize she was crying until the first drop of chilly water fell onto her hand.\n\nStartled, she wiped the tears from her cheeks. What was the matter with her? She'd always resented her lot in life, always wanted more freedom, but a part of her also accepted what was to be. Now the idea of returning to the stronghold and being married was more than she could stand.\n\nTaking a deep breath, she started walking again. Her lot in life didn't matter; it was far better than _no_ life. Fresh tears welled in her eyes as she recalled Jayce's body at the end of the alley. She had to get to him and return home; she had no other choice.\n\nShe didn't know much about this human world. However, the little she'd seen of it, and the vast quantity of reading she'd done about it, fascinated her. The humans were a weaker species, but they were brilliant and creative. Over the years, she'd spent countless hours studying the paintings and numerous pieces of art they had in the stronghold. It amazed her that she found some new detail amongst those canvases and sculptures _every_ time she looked at them.\n\nShe'd read all the thousands of books in the library of the stronghold, some of them so many times she'd memorized them. Her favorites had always been mysteries and detective novels, especially Sherlock Holmes.\n\nShe would give anything to visit all the many places she'd read about and seen pictures of, but she would never be able to do any of those things once she returned to the stronghold. However, she couldn't stay out here.\n\nHer brother would be looking for her soon. Nathan was probably just recalling that he'd left her behind to pursue the monster who had killed their father. He would go crazy if she didn't return. And she might go crazy if she did.\n\nBut where could she go? What could she do?\n\nNothing. There was nothing she could do. She had no ID, no birth certificate, no money, nothing she could use to help her navigate the human world. She was schooled in the arts, science, math, history, and other things. She may never use her education, but the hunters believed everyone should be taught. Some of the males were sent out to become pilots, doctors, nurses, plumbers, electricians, and numerous other things that would benefit their society before returning to the stronghold.\n\nShe had no real training unless the job included self-defense techniques, cooking, and being a good wife and mother. She was faster and stronger than humans, a good fighter, and a passable cook, but she'd failed on epic levels at being a good wife and mother during her schooling.\n\nShe'd spent twenty of her twenty-three years trying to learn how to sew, and she still couldn't do it. Though, she suspected her instructor, Mrs. Cranon, was right and she'd chosen not to learn the technique. No matter how good it felt to stab things sometimes, she _hated_ sewing.\n\nIn the end, she had no skills that would help her survive in the human world, a world she knew so little about other than what she'd seen on TV and read in her books. Even her glimpses of the humans on TV had been rare as there were few TVs in the stronghold, and the women were often kept away from them.\n\nEven if she could somehow survive out here on her own, it didn't matter; she had to return for Jayce's body, and she couldn't leave Nathan to worry about her.\n\nResigned anew to her lot in life, Kadence took a deep breath and continued forward. She was almost to the back door of the club when the garbage stench of the alley rose a little and a shadow moved forward, blocking the way. She froze when she recognized Joseph standing before her.\n\nHow had he gotten away and back here? She had no answer for that question, but it didn't matter. He was here.\n\nKadence took a step back as his red eyes ran leeringly over her body. The faint stench of decay emanating from him was enough to make her gag. At one time, he may have been good looking with his golden-brown hair and narrow features. Now she saw nothing but a twisted creature.\n\n\"I came back for you, beautiful,\" he purred.\n\nHer hand gripped the crossbow tighter, but she had a feeling she wouldn't get the chance to hit him with a bolt again, not now that he was prepared for it. She itched to rip his heart out with her bare hands to avenge her father's death, but her instincts screamed at her to run far and fast from here.\n\nHe took another step toward her. The dim illumination of the bulb over the club door revealed his blood-covered shirt, the bruises on his face, a jagged cut on his upper right thigh, and the hole from where her bolt had pierced him in his chest.\n\n\"So pretty.\" His insidious voice washed over her, chilling the marrow of her bones.\n\nHe moved suddenly and much faster than she'd anticipated with his injuries. She lifted the crossbow, aimed at his heart, and fired. The bolt sliced across his shirt and the front of his chest when he turned to the side to avoid taking a direct hit. His hand wrapped around her braid and he yanked her against him.\n\nLifting her hands to his chest, she shoved at him as she tried to pull herself free. His grip on her hair tightened until a sharp pain throbbed in her skull and some of the strands tore from her scalp. Giving up on trying to free herself, she squirmed against him and punched him in the stomach. Her blow against the solid wall of muscle in his abs had the same effect on him as a mouse beating on an elephant would have.\n\nTwisting to the side, she tried to get her leg in between them to knee him in the crouch. He knocked her knee to the side and bile rushed up her throat when he rubbed his erection against her stomach. She clamped her teeth against vomiting on him as she put her fingers together and drove them at his eye.\n\nHe chuckled when he swatted her hand aside. \"I love it when they fight,\" he murmured in her ear.\n\nTurning, she finally managed to land a solid punch against the underside of his chin. The monster laughed and leaned back to survey her. \"Do it again,\" he taunted, his face only inches from hers.\n\nIt hit her then that all her training in weapons and self-defense had been for nothing. Dummies didn't hit back or block her blows; they didn't laugh in her face when she gave them an uppercut, and they didn't have the strength of twenty men. The women had been taught how to defend themselves, but they'd never been prepared to actually do it against a vampire.\n\nManaging to get her hand up again, her fingers hooked into claws that she raked down the side of his face. As the skin tore away, flesh dug beneath her fingernails and blood welled forth.\n\n\"Bitch!\" he spat at her before slapping her across the face. Blood exploded into her mouth, a ringing sounded in her ears, and she was certain he'd knocked one of her teeth loose.\n\nClutching her hand, he twisted her arm behind her back. Agony tore through her shoulder and screamed up her back as he turned the joint a way it was never meant to go. \"The power,\" he murmured against her ear. \"I can smell it thrumming through your veins.\" He jabbed his erection against her again.\n\nHer shoulder popped out of place when she twisted to the side. She cried out as he bent his head to run his revolting tongue across her ear. He propelled her back against the wall, his heavy body plastering her to the cold brick. White fangs glinted when he pulled back to reveal his lethal canines seconds before he struck.\n\nFire burned through her veins, her heart stuttered in her chest, and her breath froze in her lungs as pain ripped through her, rendering her helpless. Blurred stars filled her field of vision. Tears streamed down her cheeks as her life drained unwillingly from her in slurping gulps.\n\n# CHAPTER 9\n\n\"Did you hear that?\" Ronan asked.\n\n\"Hear what?\" Declan inquired.\n\nRonan's hand fell away from the doors of the van after closing them. The alley remained hushed, but he'd heard something. Blocking out the sound of approaching sirens, the hum of the nearby traffic, and the distant beat of the music from the club. He honed all his senses onto the alley, trying to sense another presence there.\n\nThen, he smelled her vanilla scent and her _blood_. He'd assumed Kadence had run when he told her to, and in the ensuing battle with the vamps, he'd lost her scent, but it was all he could smell now.\n\n\"Get out of here, Saxon!\" he barked.\n\nDeclan, Killean, and Lucien stared at him in surprise as the red taillights of the van washed over them and Saxon pulled forward.\n\nRonan moved so fast down the alley that the walls around him were a blur. His vision became clouded with a haze of red as he turned the corner and spotted her. Spotted _them_.\n\nMoving faster than he'd ever known he could, Ronan closed the distance between him and Joseph in less than a heartbeat. He tore Joseph off her and threw him thirty feet through the air before he crashed onto the top of a dumpster.\n\nIn all his life, Ronan had never been this hell-bent on murder. It was bad enough that Joseph had touched her, but that he had _hurt_ her was intolerable. He'd tear Joseph limb from limb for this.\n\nJoseph spun back toward him as he launched off the dumpster. He braced himself to charge forward, but then he turned and fled down the alley. The muscles in Ronan's legs bunched in preparation to follow the Savage vampire, but Kadence's blood permeating the air froze him in place.\n\n\"Follow him!\" Ronan commanded, and Lucien and Killean took off after Joseph.\n\nHis gaze fell on where Kadence had slumped against the wall. Declan knelt at her side, examining her injuries. Two jagged tears marred her flesh from where Joseph's fangs had torn across the right side of her neck. The vivid red of her blood stood out starkly against her pale skin. Tears streaked her cheeks from the pain she'd endured by having her blood unwillingly taken from her body. Her right arm was twisted at an unnatural angle behind her back. He'd had his shoulders dislocated enough times over the years to recognize the damage done to her.\n\n\"She's alive,\" Declan murmured and moved his fingers to prod at the wounds on her neck.\n\nStalking forward, Ronan pushed Declan's hand aside. \"Don't touch her.\"\n\nDeclan gave him a questioning look, but he edged away from her. Kneeling at her side, Ronan stroked her soft skin in the hopes she would wake. She remained unmoving, her heart beating sluggishly in her chest. Fighting back the rage threatening to consume him over what Joseph had done to her, Ronan leaned forward and scooped her into his arms.\n\nHer head lolled to the side, coming to rest against his chest. Her silver-tipped eyelashes curled against her frightfully pale cheek. Blonde eyebrows drew together as a moan whispered past her lips.\n\nWith her slender nose, round cheeks, and full lips, she was exquisite. Despite what he knew her to be, he wanted to hold her close and shelter her from the horror of their violent world. Footsteps in the alley drew his attention as Lucien and Killean emerged from the shadows. Killean's eyes latched onto Kadence; his mouth compressed into a flat line.\n\n\"Did you get him?\" Ronan asked.\n\nLucien ran his hands through his sandy blond hair. \"No, he was gone before we made it to the end of the alley.\"\n\nA sneer curved Ronan's upper lip, but he couldn't be mad at them. He should have gone after Joseph himself. If it hadn't been for Kadence, he would have, and Joseph wouldn't have evaded him, not again.\n\n\"What are you going to do with her?\" Killean demanded.\n\nRonan glanced at Kadence as red, amber, and white lights flashed over the walls around them. The human's emergency vehicles had arrived. He didn't know when he'd come to his conclusion, but he knew exactly what he would be doing with her. \"She's coming with us.\"\n\n\"What?\"\n\nRonan strode past Killean without bothering to respond to his question. He carried Kadence toward the other end of the alley, away from the humans. \"Killean, call Saxon and make sure he takes care of those bodies. Lucien where's your vehicle?\" he inquired.\n\nKillean stared at him before slipping his phone out of his pocket and hitting a button. He walked away as he spoke into it.\n\n\"There.\" Lucien arrived at Ronan's side and pointed to a black SUV in the parking lot beside the club. \"You can't take her. The hunters\u2014\"\n\n\"What would you have me do, leave her on the street?\" he demanded.\n\n\"Ronan\u2014\"\n\n\"The human workers are here. She cannot stay, not with these wounds on her.\"\n\n\"Saxon is taking care of it,\" Killean said when he hung up the phone. His gaze focused on Kadence. \"We will obscure her wounds.\"\n\n\" _No_ one is touching her again,\" Ronan growled.\n\n\"The hunters will come back for her,\" Lucien said.\n\n\"They may not find her before the humans do, and we don't know where the hunters are. She's coming with us,\" he said. \"And I don't want to hear one more word about it.\"\n\nLucien nodded briskly. Killean opened his mouth to reply, before closing it again. Declan remained silent as he walked beside Lucien. Something about the way Declan watched Kadence caused an uneasy feeling to settle in the pit of Ronan's stomach. With every step he took, he couldn't rid himself of the feeling there would be no turning back from this decision.\n\n***\n\nRonan laid Kadence on his bed, careful not to jar her injuries. She hadn't moved since they'd left the alley, but her breathing and heartbeat remained steady. \"Get me some bandages, towels, and water,\" he ordered.\n\nDeclan rushed out of the room. Killean and Lucien stood by the doorway, their disapproval evident on their stony faces. \"The hunters are going to be really pissed off when they realize we have one of theirs. A woman no less. They'll tear this city apart looking for her,\" Lucien said.\n\n\"Then they shouldn't have left her in the alley,\" Ronan replied.\n\nKillean snorted. \" _None_ of us knew she was still there. We all assumed she'd retreated to safety.\"\n\n\"We should leave her somewhere they can find her,\" Lucien pressed.\n\n\"I'm not leaving her somewhere unprotected!\" Ronan snarled.\n\nLucien's raven eyes narrowed, but his head bowed in acquiescence and he stepped back. Declan returned with a set of towels slung over his shoulder, a pot of water, bandages, and medical supplies in hand that Ronan's friend, Marta, most likely kept somewhere for her and her husband, Baldric. Declan set the supplies down on the nightstand.\n\n\"Leave us,\" Ronan ordered gruffly. He didn't look up to make sure his command had been obeyed; he knew it would be.\n\nLifting one of the towels, he wet it in the pot of warm water. He sat beside Kadence on the bed and carefully cleaned the jagged tears on her neck. Her bleeding had stopped, but the alluring scent of her blood teased his nostrils.\n\nHe took a deep breath to calm his thirst for her and wiped away the last of her blood. She stirred only once while he worked on the wounds already showing signs of healing. Tenderly holding her chin, he sprayed some antibiotic on the injury and taped a bandage over it. Her shoulder remained out of place, but that would have to wait until she was stronger.\n\nLeaning back, he studied the healthy pink color creeping into her cheeks. Her breathing grew stronger with every passing minute, and the solid beat of her heart sounded within her chest.\n\nWhen he ran his knuckles over her cheek, her head turned slightly toward him. He held his breath as he waited for her eyes to open, but she still didn't wake. If she'd been human she never would have survived the amount of blood Joseph had taken from her.\n\nReluctantly pulling his hand away from her, he rose and walked to the end of the bed. Careful not to move her too much, he untied her boots and pulled them off. He set them beside the bed before gently removing her coat. A smile curved his lips when he spotted the small arsenal tucked within. He removed the weapons and tossed them into the hallway. If someone else didn't take them away, he would throw them away later.\n\nHe draped her coat over a chair in the corner before turning back to her. The collar of the turtleneck she wore had been ripped during her attack, but it still hugged her willowy frame and handful-sized breasts. He deliberated taking her clothes off to make her more comfortable, but it would most likely frighten her if she woke and realized he'd stripped her.\n\nReluctantly, he pulled the blankets over her slumbering form and ran his fingers over the end of her braid. He didn't understand this strange effect she had on him. Not only did he crave her body, but he also wanted to hold her throughout the night and keep her protected. However, he didn't think she would be pleased to wake up next to a vampire after everything she'd been through tonight.\n\nThe desire and tenderness she evoked in him were two things he'd believed himself long ago deadened to. He didn't know how to handle their resurgence. His life was neat and orderly; it had to be if he was going to keep himself from succumbing to the bloodlust. This woman made him feel anything but orderly. Just by being here, she'd upset his structured life. He should resent her for it; instead he found himself craving more of her.\n\nRising, he moved into the bathroom. A cold shower was exactly what he needed. After pulling his clothes off, he turned the shower on, slipped his glasses off, and stepped beneath the chilly spray. The water washed over him as he tried to drown the need for her pounding through his body.\n\nHe bent his head, pressing it against the tiled wall as blood pooled around his feet. The sight of it didn't bother him; he was well used to seeing and smelling his own blood. The puncture in his shoulder was still raw and seeping blood, but healing rapidly. He'd survived worse than this, and he would survive the many more injuries he would sustain for the rest of his life.\n\nHe turned the water off and stepped out of the shower. Padding over to the rack, he pulled a towel from it and dried himself off before examining his shoulder. The bleeding had stopped, his muscle had already closed within the hole, and before his eyes, his skin was healing over the top of it. He didn't worry about it getting infected. His vampire DNA wouldn't allow an infection or any diseases to survive in his bloodstream.\n\nHe checked to make sure Kadence still slept before tossing his towel aside and striding back into the room. He walked over to the armoire, for the first time noticing the ivy leaves etched into its solid wood surface. The hinges creaked when he opened one of the doors to remove a pair of jeans from within.\n\nHe pulled the jeans on before walking over to the picture window facing out on the pool beyond. Bars lined the outside of the window. The moon shone down across the wintry landscape as he gazed out at the night before pushing a button beside the window. Heavy metal shutters slid silently down over the glass.\n\nIt had been almost seventy years since he'd killed a hunter and felt the effects of their life on his body. However, having spent his life hunting Savages, he'd adapted to their habits and become a creature of the night himself. Usually, he slept through the day and he preferred the sunlight blocked out when he did.\n\nThe fact he'd become more and more like the creatures he hunted over the years was not lost on him. He knew what he was becoming.\n\nHis gaze returned to Kadence. He didn't feel quite so Savage around her, or maybe he did, he thought as his gaze lingered on her full lips, but he didn't feel the bloodlust as intensely. With her, he knew one thing, keep her safe. He could do that; he could focus on that.\n\nGrabbing the brown leather chair from the corner, he placed it next to the bed and settled in to watch over her.\n\n# CHAPTER 10\n\nKadence blinked against the darkness as confusion swam through her groggy mind. She normally didn't go to sleep until almost dawn, when she was sure the hunters had all returned safely, but she always had a nightlight on in her room, or the sun would be peaking around the edges of her drapes when she woke in the afternoon.\n\nStruggling to sit up, she groaned as pain tore through her neck and shoulder. She tried to raise her right hand to her neck, but it hung limply at her side. Her left hand flew up to the bandage there as memories of the night before flooded her.\n\nShe was not in her room, not in her _home_! She bit back a scream. If Joseph was around somewhere, he couldn't know she was awake.\n\nKadence forced herself to lie back as she fought against bolting out of bed and fleeing into the night. She had to learn her surroundings first. If she launched out of this bed in a panic, she was sure to draw attention to herself and wouldn't get anywhere.\n\nShe remained still while her eyes picked up the small bits of light filtering in from under the door across the room. The nightstand beside her gradually took shape, and then the chair next to it.\n\nAdrenaline shot through her. Someone sat in that chair. She could hear their shallow breathing, see the broad expanse of their shoulders. The fingers of her good arm dug into the sheets. Was it Joseph?\n\nShe had no idea why he would keep her alive, but if he had, it meant worse things than what had happened in that alley were in her future. And if it wasn't Joseph and she wasn't at home, then her future had become a whole lot more uncertain.\n\nShe pushed the blanket aside and slid her feet to the floor. A wave of dizziness assailed her, causing her to sway. She bit into her lower lip, drawing blood in an attempt to keep herself grounded in the moment. She had to remain conscious, and she had to move while whoever sat in that chair remained asleep. Her only chance of survival was to make it to the door and out of here.\n\nStaggering to her feet, another wave of dizziness caused her to lurch to the side and bang into the nightstand. Something rattled on it. Kadence's breath froze in her lungs while she waited to see what would happen. The form in the chair remained unmoving, but the hair on her nape rose as she felt eyes on her.\n\nShe saw no movement, heard no sound, but suddenly a warm hand clasped her elbow. A shrill cry escaped her as she flung herself backward. Her frantic movements caused her to knock the nightstand over. Glass shattered when whatever had been perched on the nightstand toppled onto it. Dull thuds sounded as whatever else had been on the nightstand fell onto the carpet.\n\n\"Easy,\" a voice soothed. \"It's okay; you're safe here.\"\n\nKadence took a deep breath while she strained to see the face of the man holding her. His chest brushed against her arm when he leaned toward her. A wave of heat flooded her, and before he flicked the light switch on, she knew who stood before her.\n\nKadence went completely still when the light in the ceiling blazed to life and illuminated Ronan's face. Her hand flew to her mouth as his eyes met hers, and she finally got to see what his sunglasses had been covering. She'd never seen eyes like his before. They were such a magnificent combination of red and brown that she couldn't figure out if they were the soulful color of a deer's eyes, or the deep red shade of blood.\n\nThe blood comparison should have unnerved her; she found it only fascinated her more as the colors swirled more intricately together until they became the deep hue of burgundy while he watched her.\n\nHeat crept through her cheeks when she realized she was staring at him. It didn't get any better when her gaze dropped lower and she saw he wasn't wearing a shirt. To her horror, her mouth actually watered as she drank in the chiseled muscles of his broad shoulders, chest, and abdomen.\n\nShe'd seen men's chests before, when the hunters were training, but none of them had ever affected her in this way. There was no hair on his chest, but a deep brown trail of it ran from his belly button to the edge of the jeans slung low on his hips. She barely kept her fingers restrained from following that trail before tracing the lines etching his abdomen with her tongue. As a vampire, she knew he couldn't be in the sun, but his skin was a bronzed hue that made her think of the sun-kissed beaches she'd never been to but had read about.\n\nThe numerous paintings and photos she'd seen of men had nothing on the specimen standing before her. Unable to fight the strange pull he had over her, Kadence swayed toward him. How could she so strongly want someone she barely knew, someone who could kill her with a flick of his wrist?\n\nAfter last night, she thought her teachings about vampires had been wrong, or at least incomplete, and her instincts were telling her that no matter what he was, he would not hurt her. However, she wasn't about to toss aside twenty-three years of upbringing because she found herself wanting to touch, explore, and kiss a man more than she wanted air right now.\n\nHer eyes lifted to his mouth with its stiff upper lip and full bottom one. She almost licked her lips as she contemplated what he would taste like. If he tasted as good as he smelled, he would be delicious.\n\nKadence gave herself a mental shake before jerking her gaze back up to his eyes. She bit her lip as she struggled to keep her strange attraction to him restrained. Unfortunately, the action caused his eyes to fasten on her lip and hunger sparked within his gaze. Except, this was not a hunger for her blood. Oh no, she saw and felt the desire in him while he watched her. It caused her breath to hitch as her toes curled into the plush carpet. His palm on her elbow burned into her skin; her nipples tightened and she tried to recall how to breathe from one second to the next.\n\nRonan watched as the blue of Kadence's eyes deepened in hue and her gaze fell to his mouth again. He moved closer until her arm rested against his chest. His fingers slid over her elbow when he bent over her. His eyes never left hers as his lips hovered only inches above her.\n\nHis mouth was about to touch hers when common sense returned to her and she stepped back. What was _wrong_ with her? Being attacked by a vampire and having pints of blood drained had completely rattled her intellect. She should be trying to stake every vampire she came across after everything Joseph had done to her loved ones, not thinking about kissing one of them!\n\nShe was alone with this man \u2014 _vampire\u2014_ apparently in his place. If she didn't keep her wits, she'd end up dead or a vampire blood bag.\n\nRonan reluctantly released her arm and stepped away from her when she stiffened against him. The last thing he wanted was for her to fear him, yet she edged further away from him. \"I'm not going to harm you; there's no reason to be afraid.\"\n\nHer chin rose. \"I'm not afraid.\"\n\nThe small smile curving his lips made him even more irresistible. Ugh, she was a mess, she decided. Just then, her heel connected with something on the ground. Looking down, she spotted the lamp she'd knocked off the nightstand and the scattered medical supplies on the floor\u2014the medical supplies that had been used on her, she realized as she recalled the bandage on her neck.\n\nMonsters didn't help heal their victims. Or maybe they did, what did she know? She had no experience with any of this, and she had contemplated licking the vampire, so her brain cells weren't exactly up to snuff right now.\n\n\"Where am I?\" she inquired.\n\n\"You're in our home.\"\n\nHer eyes went to the closed door behind him before coming back to him. \"Who lives here?\"\n\n\"Lucien, Saxon, Killean, Declan, and I do, along with Marta and Baldric. Though Lucien has been staying somewhere else lately.\"\n\n\"How come?\"\n\n\"Because it is necessary for him to be elsewhere. Let me put your shoulder back into place.\"\n\nRonan braced himself for her rejection when she frowned at him. Then, she glanced at her sagging shoulder as if just recalling it.\n\n\"Yes, that must be done,\" she murmured.\n\n\"It will hurt.\"\n\nA muscle in her cheek twitched as she eyed him. \"I can handle pain. Do you know how to do it?\"\n\n\"I've dislocated and broken more things than I can count. Most of them I've put back into place myself; some I've had help with. I'm sure, as a hunter, you would prefer not to go to the hospital for it.\"\n\n\"Would you let me go to the hospital?\"\n\nRonan rocked back on his heels. He wanted to put her at ease, but he couldn't lie to her. \"No. I will make sure you are safe when I let you go, but I will not leave you to the care of the humans. Even though you are more human than I, there are still differences between you and them that they would find very intriguing.\"\n\n\"I can take care of myself.\"\n\n\"I'm sure you can, but I won't risk your life in such a way.\" More, he wasn't ready to let her go, but he didn't say that. She already looked like a rabbit ready to bolt, but he couldn't tell if she was frightened of him or wary of the whole situation.\n\n\"None of this makes any sense,\" she said.\n\n\"I suppose it doesn't, to you. Perhaps it will make sense by the time you leave.\" Her lips flattened into a thin line. \"Your shoulder,\" he prodded.\n\n\"Yes, of course,\" she murmured.\n\n\"Please turn around and grip the wall.\"\n\nSwallowing heavily, Kadence turned and did as he suggested. She could handle pain, but she was not looking forward to this at all. However, her shoulder had to be back in place if she was going to defend herself. After what had happened with Joseph, she didn't delude herself into thinking she could take a vamp out on her own, and especially not this one.\n\nThe power emanating from him made every other vampire she'd seen look like a child. She had a feeling she'd only seen the surface of what Ronan could be capable of, but she would go down fighting if this was all some ruse and she really was dessert.\n\nRonan clasped her shoulder and her arm. Her head bowed so that her braid fell forward to reveal her nape. His eyes latched onto her bare skin as images of running his lips over her flooded his mind. She'd be sweet against his lips and tongue. The erotic sounds she would make would be something he'd never get enough of hearing, and he knew her blood would be a rush unlike anything he'd ever experienced before.\n\nShaking his head to clear it of his thoughts, he pulled back on her arm. A loud crack echoed through the room. The muffled cry she released made his blood boil. Releasing her, he glared at his hands for inflicting hurt on her as she rotated her shoulder back.\n\nHe almost drove a fist into the wall. He pulled it back at the last second and rested it on the wall as he battled the turmoil rolling through him. Never again would he be the one to make her issue a sound like that.\n\nHe felt too large around her suddenly, too cumbersome. She was not a petite or fragile woman, but he could break her so easily. He teetered on the edge of becoming a Savage, what would he do to her if he plummeted over that edge while she was here?\n\nShe looked at him over her shoulder, the shadows lining her eyes emphasizing their blue hue. The breath rushed out of him as the emotions battering him eased and a sense of calm stole through him while he gazed at her.\n\n\"I'm sorry I hurt you,\" he murmured.\n\nKadence was pretty sure her eyebrows were never going to come out of her hairline after those five words. _He_ had apologized to _her_.\n\n\"It needed to be done.\" She rotated her shoulder again and winced. \"It will be fine by tomorrow. I heal fast.\"\n\n\"Hmm,\" he murmured as she turned to face him.\n\n\"I know you are Ronan, but what is your full name?\" Kadence inquired. \"Or do vampires not have a last name?\"\n\nHis crooked smile revealed the tip of one of his canines. His fangs weren't extended, but those teeth were still sharper than her canine teeth. Oddly, she wasn't unnerved by the sight of that smaller fang.\n\n_I have got to get away from this man_ , she realized. _No, vampire,_ she reminded herself again. The reminder didn't have the effect of making her hate this guy like she believed it should.\n\nLess than twenty-four hours away from the stronghold and she was already forgetting her entire upbringing. She should be plotting how to escape, not pondering what his fangs would feel like running over her skin.\n\nShe moved further away until her legs hit the wooden bed frame behind her.\n\n\"We have last names,\" he said.\n\n\"What?\" she asked, completely confused by his words.\n\nHis smile widened; his eyes actually _twinkled_. She would have thought that impossible for any vampire. Not only that, but he looked young, carefree, and irresistible when he smiled like that. She was certain that smile had melted more than a few women in his lifetime; it certainly melted her.\n\n\"My original surname was Caomh\u00e1nach, a name I assumed years after my birth. No matter how small a thing it is, vampires must blend in with the humans the best we can, and I assumed the name when it was becoming more popular for the humans to have more than one name. Over the years, the name has been anglicized to Kavanagh, and that is what I go by now.\"\n\n\"Oh.\" She glanced away from him, embarrassed she'd forgotten she'd asked the question and more than a little unsettled by his answer. How old did that make him if his last name had originated and changed in such a way? Old enough to make her skin dance from the power he emanated.\n\n\"And I know you are Kadence, but do you have a last name?\" he asked.\n\nHer mind spun as she tried to figure out if there was something he could use her last name for, but she couldn't think of anything. \"It's Holter.\"\n\n\"Kadence Holter.\"\n\nHer name sounded more like a caress as it rolled off his tongue. Was that the faintest hint of an Irish accent she'd detected from him? She'd met a few hunters from Ireland when they visited the stronghold. Their accents had fascinated her, as did their tales of their homeland.\n\n\"It's a pleasure to meet you, Kadence.\"\n\n\"Are you going to kill me, Ronan?\" the question popped out before she could stop herself.\n\nHis eyes narrowed as all amusement vanished from him. \"Yes, I brought you back here, dressed your wounds, and took care of you, all so I could kill you.\"\n\nThe deep growl of his voice vibrated through the room. No matter how angry he sounded, she realized he still didn't scare her. \"I don't know what to think. Your kind\u2014\"\n\n\"My kind is _not_ your enemy,\" he interrupted. \"No matter what you were raised to believe, we never have been your enemy.\"\n\nHer mouth opened, then closed again. She didn't know what to say. She shouldn't believe him; her upbringing screamed at her he was a lying. But instinctually she _knew_ she could trust him and that he would keep her safe.\n\nIf Ronan wanted her dead, she would be already. If he intended to rape her, he would be on her now. She didn't sense any kind of mental games going on here, didn't believe he was toying with her, but maybe he was.\n\nHe might have decided to leverage her against her brother and the others. That was a good possibility, but it still didn't feel right. Maybe she'd lost more blood than she realized and her deprived mind was having a breakdown. _Best possibility yet_ , she decided.\n\n# CHAPTER 11\n\n\"I think you should rest some more. Your coloring is still off,\" Ronan said as her eyes went between him, the door, and her coat.\n\nHer fingers encircled his wrist when he reached behind her to pull back the blankets on the bed. He froze as the feel of her warm skin burned into his flesh. One of her fingers hesitatingly caressed his wrist, as if she couldn't stop herself from feeling more of him.\n\nHis eyes came back to hers. She had no idea what she did to him, no idea how badly he wanted to pick her up, put her on the bed, and bury himself inside of her. If she knew, she would run screaming from this room, and she would have every right to.\n\n\"I don't understand what you're saying. How are you not my enemy?\" she asked. \"We were born to kill each other.\"\n\nHe almost grabbed her when she swayed on her feet, but he didn't know how she would react if he did, and she should be spending her energy on recovering instead of fighting him.\n\n\"I know you don't understand, know you've been taught differently, but now is not the time to get into it,\" he told her. \"You look about ready to pass out.\"\n\n\"Is Joseph dead?\"\n\nRonan stiffened at the reminder that the one who had done this to her still lived. \"No. Lucien and Killean chased him after he attacked you, but they lost him.\"\n\nShe closed her eyes and inhaled deeply. Her hand fell away from his wrist as she hugged her middle. When her head bent, he had no idea what she was doing until the salty scent of tears drifted to him and inaudible sobs shook her.\n\n\"Hey now, hey, don't do that.\"\n\nUnsure of what to do, Ronan awkwardly patted her shoulders. In all his many years, he'd never had to deal with a crying woman, and he had no idea how to make it stop. He patted her head next, but when that did nothing to ease her, he wrapped his arms around her and drew her to him. He cradled her head against his chest as he rocked on his heels with her. Her tears dampened his skin, yet she showed no signs of slowing down or being aware of who held her.\n\nTrying to calm her, he ran his hand over her silken braid. His fingers caught in the band at the end. Unable to stop himself, he slipped the elastic away and undid the braid before spreading its silvery length. The thick mass of her hair fell freely to the middle of her back in waves.\n\nShe buried her face against his chest, her breath warming his skin as her tears continued to fall. He felt nothing sexual as he held her; all his lust had been drenched beneath the onslaught of her sorrow. All he wanted was to comfort her as he held her closer.\n\n\"He'll never hurt you again,\" Ronan vowed. \"Never get close to you again. I will make sure of it.\"\n\nFeeling completely mortified over her behavior, Kadence stifled her next sob and willed the tears to stop flowing. Her fingers had a mind of their own as they dug into his back. His skin was smooth against her, but his muscles provided a firm pillow for her head.\n\nInhaling his scent helped to calm her further. He smelled like cinnamon, and the aroma of ozone, a scent she recognized as power, emanated from his pores. She found herself fighting against pressing her lips to him. Her knees knocked together from the force of her overwhelming craving to taste him. On her next breath, she gave into the impulse and turned her head enough that the corner of her lips brushed over his flesh.\n\nHe became rigid against her, and she immediately moved her mouth away. She strained to keep the blush from her face in the hopes he would think it had been an accident and not deliberate on her part.\n\n\"Do you feel better now?\" he inquired.\n\nShe wasn't sure what she felt right now. Confused and exhausted, definitely. Irritated at herself for not being able to stop Joseph and being the reason he got away, without a doubt. Completely confused about her attraction to this creature, yep she could add that to the list of conflicting emotions battering her.\n\n\"Yes,\" she said and finally succeeded in getting her traitorous fingers to release their hold on him.\n\nShe clasped her hands before her to keep from grabbing him again. When she saw how wet his chest was from her tears, she was unable to stop her mouth from dropping. She went to wipe her tears off him, but he caught her wrist before she could touch him and held it loosely between his thumb and forefinger.\n\n\"I'm sorry!\" she blurted.\n\n\"You had a rough night.\" Reluctantly, Ronan released her wrist when she tugged at it.\n\nShe turned away from him, ashamed at herself. She wasn't a crier, she never had been, but since her father's death, she'd been constantly fighting back tears and the urge to curl into a ball and sob until she couldn't walk anymore. Now, she'd finally broken, and in front of _him_. He probably thought her a weak child now.\n\n_What does it matter what he thinks of you, you idiot_? She didn't know why it mattered, but it did.\n\nKadence had never felt more drained or humiliated in her life. Her legs gave out and she sank onto the thick mattress behind her. She should be demanding answers, looking for a weapon, planning her getaway, but she couldn't fight the incessant pull of her eyelids closing. She lay back on the mattress and sighed when her head hit the pillow.\n\nSettling the blankets back over her, Ronan tucked them around her. His hand brushed over her cheek, wiping away the wetness lingering there. He began to pull his hand away, but she captured it to stop him.\n\n\"Are you going to keep me here?\" she inquired.\n\n\"No,\" he answered in a rough voice. \"As soon as you are well enough, we will find a way to return you to your brother.\"\n\n\"I don't know what to believe, what to think,\" she whispered.\n\nHe swept the hair away from her forehead. \"I know, but for now, you should rest and heal. You are safe here. I will keep you protected.\"\n\nOn her next breath, Kadence fell asleep.\n\nRonan slipped his hand from hers and made his way around to the other side of the bed. She may not appreciate it if she found out he'd slept beside her for the rest of the day, but he would remain above the covers and he wouldn't touch her. He required sleep, and if he was honest with himself, he couldn't resist the chance to lie beside her, if only for a day. He would make sure to get out of bed before she woke up so he didn't upset her.\n\nHe stretched out on the bed and listened to her soft inhalations as he allowed her sweet scent to lure him into sleep.\n\n***\n\nKadence started awake again. Uncertain of the time or where she was, she nearly fell out of the large bed when she bolted upright.\n\n\"Easy,\" a groggy male voice said from beside her.\n\nShe squeaked and spun so fast she was certain she'd given herself whiplash.\n\n\"Easy,\" the male coaxed again.\n\nHad she gotten married? Was it Logan beside her? Then the scent of the male hit her and relief caused her shoulders to sag. Why she felt more relieved to find herself in bed with a vampire instead of her future husband was a question she would never delve into during her lifetime.\n\n\"What... what are you doing?\" she managed to ask.\n\n\"I was sleeping.\" Her pulse skyrocketed at the lazy murmur of his words and the Irish accent that slipped through more now. \"I will get up.\"\n\n\"No, I...\" _I what? No please stay and drain my blood? No stay, I like you here?_ They all made her sound pathetic and were the last things she should be saying. \"It's your bed.\"\n\n\"It is,\" he agreed. He hadn't meant to still be in it, but for the first time in as long as he could remember, his dreams hadn't been plagued with cravings for blood and death. It wasn't until she'd sat up that he'd awoken again. \"I'm more than happy to share it with you, but I will also leave it if you ask me to.\"\n\nNow that her eyes had adjusted to the dark, she could see him clearly as he lay a few feet away from her. _Why does he have to be so irresistible for a vampire?_ She wondered as she gazed at him.\n\n\"You can stay on your side,\" she said.\n\nRonan chuckled as Kadence settled against the pillow and put her feet back on the bed. She'd lain back down, but her body remained tensed to bolt.\n\n_She has to go back_ , he reminded himself when he found himself pleased by her willingness to lie beside him. For the life of him, he didn't give a fuck about that right now. All he cared about was having the opportunity to be near her while he could.\n\nShe relaxed a little more and turned her head to gaze at him. \"I shouldn't trust you,\" she whispered.\n\n\"I swear to you, Kadence, you are safe with me. The vampire who did this to you last night is as good as dead.\"\n\nShe let out a breath that blew the hair back from her forehead. \"For some reason, I find myself believing you, but we are supposed to be enemies\u2014\"\n\n\"We are not. Hunters have made us their enemies when we don't have to be,\" he cut in, more abruptly than he'd intended, but he was tired of this mistaken belief of the hunters. It had taken the lives of numerous innocent vampires over the years and cost the hunters many of their own.\n\n\"Joseph tried to kill me last night. He did kill my friend, and he has hurt my family.\"\n\nTurning on his side, he propped his head on his hand as he gazed down at her. \"Joseph is a Savage and no longer one of us.\"\n\n\"What do you mean by no longer one of you?\"\n\n\"Not all vampires kill innocents. In fact, many don't kill at all. But, there are some vamps who are swayed by the power killing can bring them and give into the bloodlust that resides in all vampires. Many vampires fight giving into that bloodlust every day of their existence. Some of them have to fight it more than others. At one time, Joseph worked with us and fought his destructive nature, but he recently gave up that fight and became what we call a Savage.\"\n\n\"Why do some give in?\" she asked.\n\n\"Vampires go wrong for many different reasons. They're seeking more power, something inside of them snaps, they are inherently evil to begin with, they are tired of constantly having to fight themselves, or they are simply bored.\"\n\n\"They're _bored_!\" she spat.\n\n\"Not the best reason, but who knows what finally causes them to lose control. When faced with living for an eternity, things inevitably get... monotonous. Perhaps it is curiosity in some, while others are simply trying to feel something new and exciting again. Whatever it is, once a vampire takes the first step toward becoming a Savage, it is much easier to take the following steps that will make it impossible for them to ever again be what they once were.\"\n\n\"Is that why you wanted Joseph, because you try to stop these Savage vampires?\"\n\nRonan placed his hands on the mattress and lifted himself to a seated position against the headboard. He crossed his ankles as he stared at the blank wall across from him. She would leave here soon, and she couldn't leave with too much knowledge of them, but better educating her about vampires might save a life in the future.\n\n\"We _do_ stop them,\" he replied.\n\n\"Why?\"\n\n\"Why does your kind hunt them?\"\n\n\"Because they're evil, and it is what hunters were created and are bred to do.\"\n\nHe knew that was most likely what the hunters had been created to do. The world had a way of balancing itself out, and it had balanced the birth of the more demon-like vampires with the birth of the more human hunters.\n\n\"What do you mean by bred?\" he inquired.\n\nShe shrugged and lifted her head onto her hand as she rolled toward him. Her fingers played with the stitching in the charcoal-colored blanket as she gazed at the wall behind him. \"The hunter line must be kept strong,\" she said. \"The best available men and women are paired together to make sure of that.\"\n\nRonan's teeth grated together as she revealed this information. She had no ring on, her brother seemed to have been the one in charge of her, but he realized that one day she would be paired off too.\n\nHe'd lived through the days when women were married off like they were no better than cattle, and he hadn't liked it then. He liked it less now. Female vampires, especially the purebred ones, always had a mind of their own and spoke it freely. Those were the type of women he'd grown up with and admired.\n\nThey hadn't been mated or in love, but his mother had agreed to bear his father a child because with her second-generation purebred status and his father being the only fourth generation to ever exist, their offspring would be the most powerful vampire created. His mother had loved power and attention, and his birth brought her both of those things. After his creation, his parents had found other lovers, but they remained friends until the day they were both killed.\n\n\"I see,\" he said.\n\n\"So not all vampires are Savage,\" Kadence prodded in the hopes of getting him off the depressing topic of marriage.\n\n\"No, they are not. I believe you are right and hunters were created to stop Savages, but you weren't given the whole story.\"\n\n\"And that is?\"\n\n\"That not all vampires are evil. It is only some who go bad, but _all_ vampires are hunted indiscriminately by your kind.\"\n\n\"We didn't know that,\" she whispered.\n\n\"I know.\"\n\n\"Why weren't we taught any of this? Why were we never told? Why would we be enemies from the very beginning if what you say is true?\" she demanded.\n\n# CHAPTER 12\n\n\"Do you know how vampires and hunters were created?\" he asked.\n\n\"At one point in time, a handful of demons escaped to freely roam the earth. Those demons were unable to tolerate the sun, were creatures of the night, immortal, and slaughtered humans to feast on their blood. When humans learned of their existence, they hunted them to the point where the greatly outnumbered demons were once again forced to seal themselves away in what many believe to be Hell,\" Kadence recited from her studies on the subject.\n\n\"While they were here, the demons also mated with some humans,\" she continued. \"The first vampires were born from those humans. Like the demons, they had a thirst for blood, eternal life, and many other demon capabilities such as the power to bend another to their will or cloak their presences. Unlike the demons, they were more human in appearance, had a heartbeat, breathed, and passed as human. Over time, they came to be known as vampires. Vampires discovered that, by sharing their blood with a human, they could create more of their kind.\"\n\n\"And hunters came about how?\" Ronan inquired.\n\n\"They were created in the same way, only hunters took after the human species even more than vampires. We are the mortal version of a vampire. We have many of your abilities, such as enhanced strength, senses, and extended lifespans, but not your thirst for blood or immortality. Your powers of persuasion and cloaking do not work on those of us who are born hunters, but we do not possess them.\"\n\nThe hunters also didn't have the restrictions vampires faced when they took too many innocent lives, but he didn't tell her that. He also wouldn't reveal to her that many vampires could walk freely through the day. If she went back and told her fellow hunters, which she most likely would, the revelation could make those of his kind who weren't Savage more vulnerable to the ignorance of the hunters.\n\n\"The hunters have also figured out a way for the human allies they recruit to not be susceptible to our ability to change their minds,\" he said.\n\nHer lips compressed into a flat line, and he knew she wouldn't confirm it. It didn't matter; he'd already encountered an entirely human hunter who resisted a vampire's ability to change her memories. A mixture of herbs and hunter blood had been given to the human to keep her mind blocked from a vampire's mind control.\n\n\"Why do you and your friends kill the vampires who go wrong?\" she asked.\n\nRonan draped his arm over his forehead as he lifted his gaze to the ceiling. \"Humans aren't the only ones who need protection from the vampires who turn Savage. The Savages attack other vampires too, for our blood gives them the most power and we are a far more thrilling kill than any mortal or hunter could ever be.\"\n\nShe inhaled sharply. \"That's awful.\"\n\nHe glanced at her out of the corner of his eyes. \"That sounded very _un_ -hunter like of you.\"\n\n\"I've never been the hunter I'm supposed to be,\" she murmured. \"I've been a thorn in my family's side since I was old enough to speak.\"\n\nHe imagined she had been, considering she was the firstborn female hunter who had ever been seen by a vampire, as far as he knew anyway. \"And why is that?\"\n\nShe shrugged absently. \"Little things here and there I guess. I was always the rebellious one amongst the women within the stronghold, and I was _never_ any good at my classes. I can't sew, I'm not much of a cook, and I've dropped a lot of the baby dolls over the years. _Far_ more than any of the other girls in my classes. I still can't pin a diaper without stabbing the doll. Though, I'll admit that's more because it's ridiculous to diaper a fake baby, and it feels good to give it a jab.\"\n\nRonan lifted an eyebrow at that admission as he tried not to laugh at the look of consternation on her face.\n\n\"Unlike the other women in the stronghold, I always dreamed about going out in the world, to see things, and _experience_ them. We're not allowed to do that. We're told from the moment we're born that we shouldn't yearn for things we can never have, and that being a good wife, mother, and continuing the hunter line is what we are meant to do. The world is so big and marvelous that I always wanted to see more of it and explore it _all_.\"\n\nThe longing on her face and the melancholy in her voice had him contemplating booking her the first flight to anywhere she asked to go in the world. She should be set free to enjoy her life, not secluded and locked away.\n\n\"Women must be protected and sheltered from the world,\" he grated out.\n\nResentment simmered in her eyes when they came back to his. \"You sound like my brother.\"\n\nHe realized she'd taken his words the wrong way, but he couldn't resist provoking her further as it brought color back to her face and more life to her than he'd seen since her attack. \"Your brother sounds like a wise man.\"\n\nHe was certain visions of strangling him were dancing through her head while she glowered at him. Her anger also made her forget her wariness of him as she leaned closer to him.\n\n\"I do not wish to be paired off for breeding purposes or want a husband,\" she said.\n\nNot married then. He ignored the relief her confirmation of that brought with it.\n\n\"I am perfectly capable of taking care of myself,\" she declared.\n\n\"Yes, you take great care of yourself. That's why you're here,\" he replied, hoping to bait her even closer.\n\nShe bolted upright, sputtering with indignation. Realizing he'd pushed her too far, he moved to calm her before she reopened her wounds, but she slapped his hand away. Ronan did a double take as he gazed between her and his hand. In over a thousand years, _no_ one had dared to rebuke him in such a way.\n\nShe swung her legs over the side of the bed. Getting over his shock, he wrapped his arm around her waist and pulled her back before she could rise. She slapped at his arm. Her attempts to get free would have made him laugh, if he hadn't known it would only piss her off more.\n\n\"Let go of me!\" she cried.\n\n\"No.\" He may not have the same beliefs as her kin, but she would learn he was the one who ruled here. \"Relax, before you hurt yourself. I pushed too far, and I am sorry for that.\"\n\nHer hair whipped into her eyes when her head spun toward him. \"That's the second time you've done that.\"\n\n\"Done what?\" he asked.\n\n\"Said you were sorry. I'm always the one apologizing for doing something wrong, for not obeying when I should or not doing as I'm told. I'm supposed to be sorry for not being overjoyed about my glorious role as soon-to-be bride. Or at least that's what my instructors tell me.\"\n\nRonan kept his arm around her waist, his fingers caressing her flat stomach while she spoke. \"Then they are idiots.\"\n\n\"Do vampires not have the same rules for women as hunters do?\"\n\n\"There would be no caging a female vampire, especially not a purebred one.\"\n\n\"What is a purebred?\"\n\n\"A vampire who is born to two vampires instead of being a human who is turned by one.\"\n\nHer breath exploded from her. \"There's such a thing?\"\n\n\"Yes. I am a purebred as are the other vampires who live here.\"\n\n\"Oh,\" she breathed as her eyes ran over him. That revelation, and his age, had to be why she sensed so much power in him. \"What about a human and a vampire? What would their offspring be?\"\n\n\"It depends on which side is stronger. I've seen some be more like hunters, but not as strong, and some be entirely human. The combination of a human and a vampire doesn't happen often, but it has in the past. Most live normal, human lives, some choose to be turned later in life. If they are turned, they can be stronger than an average turned vamp, but not as strong as a purebred.\"\n\n\"Vampires are able to conceive and bear children,\" she murmured. \"We believed they were all turned.\"\n\n\"We are part human too after all, which is why not all of us are killers. We have as much human DNA as demon. I'm not an overly big fan of the human race, they're rather annoying, but I don't like seeing them slaughtered for sport, even if they do it to each other on a daily basis.\"\n\nKadence felt as if he'd given her a combo punch that left her lying flat in the middle of the boxing ring. Never in a million years had she expected to learn these things about vampires, never would she have considered they had _any_ compassion within them.\n\n\"Do vampires have a difficult time conceiving?\" she asked.\n\n\"No. Do hunters?\" Her eyes went to the shuttered windows behind him. \"So that is why they lock their women away,\" he guessed, and knew he was right when her jaw clenched. \"The demon DNA must have reacted differently with the hunters, making it difficult for them to conceive.\"\n\nShe didn't reply.\n\n\"Lie down,\" he coaxed, knowing he would get no more out of her on the subject. He gave a tug on her waist, but she didn't budge. \"Joseph didn't take enough blood to permanently damage you, but he took enough to weaken you for a while.\"\n\n\"The permanent damage he's done to me happened before last night,\" she muttered more to herself than to him.\n\nHe frowned as he realized more than a rebellious streak had brought her out last night. \"Why were you in that alley, Kadence?\"\n\nHer gaze was fixed on the blanket as she responded. \"Nathan has been tracking Joseph for a while.\"\n\n\"Why is your brother so interested in him? Why did you disobey everything you've been raised to believe to be there?\"\n\n\"I never obey when I'm supposed to.\"\n\n\"I get that, but why risk leaving your home now?\"\n\n\"It took me a while to figure out a way to break free of the stronghold.\"\n\n\"How did you get out?\" he asked.\n\n\"I can't tell you that.\"\n\n\"Fair enough.\" He wouldn't ask her to divulge any information he wouldn't divulge himself. \"So why were you so determined to escape and see Joseph brought down?\"\n\nFire burned in her eyes when they met his again. \"He killed my father.\"\n\nNo wonder she'd been determined to achieve freedom and her brother had been so determined to destroy Joseph himself. Nathan wouldn't back down and would get in the way again, but that was something he would have to deal with later.\n\n\"I'm sorry for your loss,\" he said honestly. He knew how it felt to lose a parent to a Savage.\n\nKadence gazed at him, trying to decide if he was playing with her or not, but she saw only sincerity on his face and in his eyes. \"Thank you. I'm hoping that watching Joseph die will make me feel better. That this... this emptiness in me will ease once he's gone.\"\n\nHe didn't want to take the hope away from her, but he knew nothing completely eased the pain of losing a loved one. It had been nearly a thousand years since he'd lost his parents. He'd had such a short time with them, but their guidance had forged him and the hole of their passing had never been filled.\n\nThe young, idealistic man he'd been before their deaths never would have believed himself capable of slipping into the darkness. That man hadn't been plagued with the need to kill, as at twenty-eight, he'd just reached maturity, stopped aging, and started to come more fully into his powers.\n\nBack then, he'd been a young fool who had no idea that soon after becoming fully matured, there would be little left of the vampire race to lead. He hadn't known that centuries of delivering death, even to those who deserved it, would stain and warp his soul. He'd been told by his father that as a mature, purebred vampire he would start to crave something insatiably and that he would have to fight giving into that craving every day of his life, but he'd never fully understood it until his need for blood and death became an ever-constant companion.\n\nHe knew the truth of it all now.\n\n\"The emptiness of a lost loved one never goes away completely,\" he told her.\n\n\"Never?\" she whispered.\n\n\"No, but time makes you better capable of dealing with it. Revenge is never the salve you think it will be. It is often necessary and must be carried out, but it will not make everything better.\"\n\nWhen he realized he was still holding her and she didn't seem about to jump up and run off, he removed his arm from her waist and leaned back against the headboard. Her haunted eyes met his, her raw anguish evident in her gaze. He'd give anything to take the hurt from her, but that was impossible.\n\n\"You should lie down before you make yourself sick,\" he said.\n\n\"I don't get sick,\" she mumbled as she slid back onto the bed.\n\n\"Exhausted then.\"\n\nShe rolled toward him. \"It was a tiring night.\"\n\nKadence's heart leapt in her chest when he gave her a darling, lopsided smile. She may be exhausted, but she doubted she'd get any sleep with a half-naked Ronan lying in the bed with her.\n\nShe bit her lip as she thought about what her brother and the other hunters would say if they could see her now. They'd probably believe she went crazy and lock her away, just as they'd locked away some of the other hunters who had broken their laws or lost their minds over the years.\n\nNothing she'd revealed to Ronan could be used against her or the hunters, but she was lying in bed with him, and she found herself content to be there. He brought a strange sense of calm to her, one she hadn't experienced in years, if ever. Her kind would consider her a traitor for her actions. She kind of considered herself one by staying beside him, but she wouldn't crawl out of this bed, and she didn't ask him to leave it.\n\nShe inched subtly closer until the heat of his body warmed hers. Maybe tomorrow she would get her mind together enough to realize the situation she was in and plot a way out of it, but for now, she didn't have the energy or the will to fight this draw to him.\n\n\"I imagine that it was,\" he said.\n\n\"That what was?\" she asked as she stifled a yawn.\n\n\"A tiring night.\"\n\n\"Oh, yes.\"\n\nThe tips of his fingers briefly caressed her cheek, causing her eyelids to close as she turned into his touch. She smiled when he tucked a strand of hair behind her ear before tracing the outer shell.\n\n\"This is all so strange,\" she murmured and opened her eyes as he ran his finger over her bottom lip.\n\nHer heart rate accelerated when he leaned toward her. Would he kiss her? She wanted that so badly, yet she remained unmoving as she waited to see what he would do next.\n\nRonan heard the hitch in her breath when his finger stilled on her lip, and he lifted his gaze to hers again. She watched him as one would a predator, afraid to take her eyes away, but he sensed no fear from her. Leaning toward her, he slid his finger away to brush his lips over hers.\n\nKadence's eyes crossed as she watched him. The butterfly caress made her body melt, but she couldn't tear her gaze away from him as she waited to see what he would do next. Out of everything she'd been anticipating, it was not to have him abruptly pull away from her.\n\n\"You must sleep,\" he said gruffly. \"You are not at full strength.\"\n\nAnd if he kept touching her, he would be inside her. She may not be aware of where this would lead, but he was, and she was a temptation he wouldn't refuse. He could be a heartless prick on his best days, but not even he was so callous as to take advantage of her now.\n\nKadence refused to let her disappointment show as she gave a brisk nod. She should feel relieved, not as if she'd missed out on something. _Blood loss, it's screwed up your brain._ She told herself this, but she knew it was wrong.\n\nKadence closed her eyes and feigned sleep. It was at least an hour before sleep claimed her again; she knew he was still awake when it did.\n\n# CHAPTER 13\n\nRonan woke with the hardest erection he'd ever had. Gritting his teeth, he glanced down at the cause of his raging hard-on. Sometime during the remainder of the day, Kadence had rolled over and draped herself across his chest. Her head was tucked beneath his chin, her hand resting against the side of his cheek.\n\nShe'd hooked her leg over his waist so that her thigh rubbed against his cock, and her breasts were pressed to his chest. He realized, not only had she sprawled across him, he'd also draped his arms around her and held her against him. _Idiot!_\n\nStifling a groan, he resisted running his hands over her until she woke as desperate for him as he was for her. If he could convince himself she wasn't an innocent, he would have done just that. But, the hunters kept their women too locked away not to make sure they were virginal when they were ready for breeding.\n\nThe seconds of the clock on the wall gave way to minutes before his conscience finally won the war with his dick. Easing his way out from under her, he shifted her over a little to get free. She settled in with a murmur that caused his heart to constrict. Swinging his legs over the side of the bed, he launched to his feet in his rush to be away from her. She whimpered and curled her hand into the pillow his head had been on.\n\nStepping back, he ran his hand over his face and winced when he realized his fangs had also extended with their need to be inside her. With wooden movements, he turned and strode into the bathroom. A cold shower, some blood, and he would regain control of himself. Having a woman in his bed again, and the unfinished fight with Joseph, had him on edge.\n\nTurning the shower on, he tugged off his jeans and stepped beneath the icy spray. While Kadence remained here, he had a feeling he and cold showers would be getting to know each other a _lot_ better. Leaning his forearm against the wall, he rested his head on the tile as he grappled to get himself under control.\n\nThe more he tried to shut them out, the more images of Kadence moving over his body, kissing him, and sucking him off flooded his mind. The feel of her breasts against him had been burned into his flesh. He found his hand wrapping around his shaft and giving it a long stroke as he recalled the way her breath had warmed his skin.\n\nIt had been years since he'd jerked off, centuries maybe, he couldn't really recall. He still got erections, but he'd felt no compulsion to ease them with a woman or with his own hand. A long time ago, his need for sex had been eclipsed by his never-ending need for death, and he hadn't missed it.\n\nUntil now. With images of Kadence in mind, he found himself becoming more aroused than he'd been in years. His hips thrust as he imagined it was the muscles of her sheathe gripping him. Throwing his head back, the cold water hit him in the face as he found his release.\n\nHis muscles quaked, his head tilted back down, and he gazed at his still hard dick in his hand. He'd just come, yet he craved more. He wanted _her_ holding him now, not himself. His mind whispered at him to go to Kadence, to wake her and claim her. She didn't know what to make of everything, but she wouldn't turn him away.\n\nThrough sheer strength of will, he remained in the shower. After another half-an-hour passed, and he jerked off again, he shut the water off and stepped out of the shower. He still felt as tense as a wild, caged animal as he strode over to pull a towel from the rack and dried himself off, but at least he trusted himself not to crawl back into bed with her and take her.\n\nStepping before the mirror, he wasn't at all surprised to find his eyes more red than brown right now. That demon part of him was still close to the surface, and like him, it wanted _her_. Neither of them could have her though.\n\nShe was a hunter, but she'd grown up sheltered from the true brutality of the world her brother lived in. Sheltered from the world _he_ lived in. She deserved better than someone who had killed more vampires and a couple of hunters than he cared to recall. He wasn't proud of any of the deaths he'd delivered, but he was proud he'd kept many innocents safer throughout his life.\n\nTurning away from the mirror, he ran a hand over the stubble lining his jaw before grabbing his jeans off the floor. He hastily pulled them on and stepped from the room. His heart clenched when he found Kadence still sleeping. What would it be like to find her like this every day for the rest of his life?\n\nBefore he could think about the fact that thought had even entered his mind, never mind how much he found himself wanting to know the answer to it, a faint knock sounded on the door. He hurried to open it before it woke Kadence. Declan stood on the other side, looking far more relaxed than Ronan felt right now. Declan's hair was still damp from a recent shower, one Ronan was certain had been nice and hot.\n\n\"What is it?\" Ronan demanded as he slipped into the hallway and quietly closed the door behind him.\n\n\"Aren't we testy. Did you not get enough beauty rest?\"\n\n\"I slept fine. Why are you here?\"\n\nDeclan flashed a grin as his eyes danced with merriment. Out of everyone, Declan could get away with pushing him the farthest. He'd earned that right after their many centuries together, but he was grating on Ronan's nerves.\n\nAll amusement vanished from Declan's face as he glanced at the door. \"We were curious about what was going to happen with the hunter.\"\n\nRonan ran a hand through his disheveled hair. He'd prefer not to leave her, it might scare her to wake up alone, but he had to deal with this. \"I'll meet you and the others in the dining room after I speak with Marta about Kadence.\"\n\nDeclan's eyes took on an odd gleam as he appraised Ronan. \"Kadence, is it?\"\n\n\"It is her name,\" Ronan bit out.\n\n\"You like this girl.\"\n\n\"She will be taken care of while here. She's been through a lot.\"\n\n\"We've been together a long time. I've never seen you like this with a woman.\"\n\n\"You don't know what you're talking about.\"\n\nDeclan held up his hands and backed away. \"My mistake then. I'll see you in a few minutes.\"\n\nRonan noted the amusement in Declan's eyes before he turned and strolled down the hall.\n\n***\n\nNathan paced the grounds of the stronghold. His tracks taking him from the front gate to Kadence's window. He scowled at the rose trellis beside her window. Surveillance video revealed nothing of her, but he suspected she'd climbed down it last night. Someone most likely would have at least seen her exit the house otherwise.\n\nSomehow, she'd managed to take one of the cars from the garage, punch in the gate code, and leave the stronghold all before he'd left to go hunting for the night. He only knew that because he recalled the white Ford sedan she'd taken already missing from its space when he left, but he'd assumed one of the other hunting teams had taken it.\n\nThey'd located the car near the club and brought it back. He'd still been here when Kadence left the stronghold, and he'd never known it. She must have parked somewhere and waited for them to exit before following them.\n\nOver the years, he'd taken her driving around the stronghold and taught her how to use a vehicle. Those drives had been the only times he'd really seen her laugh or smile as they'd traveled through the hundreds of acres of woods and dirt roads crisscrossing the property.\n\nMost of the women weren't taught how to drive, there was no real reason for them to be, but when Kadence had asked him to teach her five years ago, he couldn't resist. There were so many things she was denied; he saw no reason to deny her that too.\n\nNow he was kicking himself in the ass for it. He didn't think she'd asked him to teach her with escape in mind, but now she was in danger, possibly dead, because of his lessons.\n\nWhat he didn't know was how she had discovered what the gate code was and managed to stay off camera? Those were questions only she could answer, but he was curious about them. If Kadence had managed to do it, then others could get past their defenses too.\n\n_No, not true,_ he realized. No matter what her instructors said about her, Kadence had always been extremely intelligent. She'd always been eager to learn and more inquisitive than most of the more submissive females in the stronghold.\n\nBecause of her spirit, he'd believed Logan to be a fine match for her\u2014one he suspected she didn't want\u2014but she had remained silent about the arrangement. Having grown up together and gone through all their training together, Logan was one of his best friends. Logan would be strong enough to keep her safe. He also cared for her and knew her well enough that Nathan didn't worry he would want to break her spirit. Kadence had to be married, but he'd be damned if it was to someone who would try to crush her.\n\nBut then, after this, he may throw her in a cage himself. If he ever found her!\n\nThe idea of her out there, completely unprepared for the world and all its horrors, was a lead weight around his heart. He'd failed to prepare her for what resided beyond their twenty-foot-high walls and sprawling, secluded property, but then he'd never thought she'd be out there _alone_.\n\nHad she fallen into the hands of the vampires? He couldn't think of a more horrifying and cruel fate for her. She was willful, but she also had one of the biggest hearts he'd ever encountered. The vampires would abuse her until there was nothing left of the Kadence he knew.\n\nAnd she was alive. They'd always been close; he'd _know_ if she were gone.\n\nHe kicked himself in the ass again for taking off after Joseph like he had and leaving her behind. He'd just been so determined to destroy the monster himself, that it had been his only thought. When he'd recalled that Kadence had unexpectedly arrived in the club, he'd abandoned his pursuit of Joseph and returned to the alley.\n\nBy the time he made it back, the humans were swarming all over it and there had been no sign of Kadence in the alley or club. The humans had also already roped off Jayce's body. It had taken some maneuvering, but they'd been able to steal his body this afternoon. Thankfully, Kadence's body hadn't been in the morgue with him.\n\nHe spun on his heel and stalked back the other way. No matter how pissed off and concerned he was about her, he had to admit Kadence was resourceful. She was also more determined than he'd realized to see their father's killer brought down. If she'd been born a man, she would have been a strong asset out in the field.\n\nHe glared at the wall as he marched toward it. Why hadn't he known what she'd been planning? He should have known the show of obedience she'd been giving recently was an act. He should have known she was up to something.\n\nThey had shared the same womb, they had spent the early part of their childhood playing together, and they were the closest of friends as well as siblings. Then, he'd turned ten and left his boyhood behind to embrace his hunter training.\n\nKadence, on the other hand, had resisted her training every step of the way. She'd been a constant thorn in her instructors' sides. She'd never wanted to accept her lot as a woman within the stronghold. Their father had believed she'd outgrow it; Nathan hadn't been as convinced, but he'd kept that opinion to himself.\n\nWhen they were younger, Nathan was amused by her antics of skipping classes, setting her cooking on fire, catching and putting a couple dozen frogs and snakes into the schoolroom, and flat out refusing to do as she was told. He'd believed she wasn't ready to leave her childhood behind. That amusement faded to sadness as it became increasingly obvious his sister was truly unhappy with her designated future.\n\nThen, their father had been killed. Over the years, Kadence had always known things, so he hadn't been surprised to find her waiting for him when he returned with their father's body. Many had tried to keep her from seeing it, but she succeeded in getting past all of them to stand beside their father as he was carried into the mourning chamber.\n\nShe hadn't cried or demanded revenge. She'd simply... retreated was the only way he could think to describe it. Her docility had been out of place. He'd believed it was because of the weight of her grief, but he realized now she'd been plotting.\n\nHe should have known better. The loss of their father wouldn't have broken Kadence's willful nature. However, he'd been so happy she finally settled into her role, that he hadn't looked past the fa\u00e7ade. And he'd lost her because of it.\n\nNathan slammed his fist into the brick wall running the entire length of the nearly three square miles that made up the stronghold property in this rural area of Massachusetts. Pain lanced through his hand and up to his elbow, but he barely acknowledged the cracking of one of his knuckles. The broken bone would heal soon enough.\n\nThat rapid healing ability, along with many of his other enhanced senses, had been what made him the leader upon his father's passing. There were other male hunters here with more experience than him, but his bloodline had led from the beginning and would continue to do so while it survived.\n\nThe older hunters accepted this, just as they had accepted he would be bringing his own men with him on the hunts. The men who had fought closely with his father continued to hunt, but they were no longer the seconds-in-command to their leader. He still asked them for their guidance and advice, but the new generation was rising, taking over now.\n\nBesides, his father had been over a hundred years old when he'd been killed, as were many of his friends. They all still had many good years in them, but it was time for the older ones to start stepping aside. Sometimes, Nathan took some of the ones in their fifties and sixties with him on hunts, but last night he hadn't expected to encounter so many vamps, or to have Kadence show up.\n\nHe wanted to wring her neck as badly as he wanted to hug her.\n\n\"Nathan.\"\n\nNathan turned to find Logan crossing the grounds toward him. \"Has anything new been learned?\" he demanded.\n\nLogan stopped before him. \"No, nothing.\"\n\n\"She's not dead.\"\n\nLogan nodded, but his eyes were distant. There had been so many vamps in that alley. Yes, they'd chased many of them away, but there had also been those _others_. He still couldn't explain them, and he didn't care to. They were vamps and they would die, especially if they had hurt his sister.\n\n\"I would _know_ if she were dead,\" he insisted. Where was she though? What had happened to her? If she wasn't being held by vampires, then why hadn't she called? Nathan paced faster. \"Get everyone together; we're going out in full force tonight to find her.\"\n\n\"Nathan\u2014\"\n\n\"She's alive!\" he snapped.\n\n\"I believe you, but I think we should leave extra enforcements here, just in case.\"\n\nNathan closed his eyes; he should have considered that. Kadence would never willingly disclose any information about them, but the strongest man could crack under torture. The gate code had been changed and men stood guard outside, watching for her in case she came back and couldn't get in. However, even with their numerous security measures, there was still a chance the vamps could get in if Kadence was coerced into disclosing their location.\n\n\"Double the guard for tonight and increase the electricity on the wall. Day break tomorrow, we'll move the women to the mission to be on the safe side.\"\n\n\"I will have them start preparing for the move,\" Logan said.\n\nHe wasn't concerned that Kadence would reveal the location of the mission; she didn't know where it was. All the history of the hunters was housed within the mission, an underground bunker in the center of the stronghold. There were enough supplies within for the women and children to live there for at least a month, but they would be blindfolded before they were taken to it.\n\nNone of their enemies could ever know where the mission was. It had been designed to provide shelter in times of a crises as well as protect the documentation of the hunters' history. Only four people knew its location, himself and three of the eldest hunter men. All of them would kill themselves before ever revealing where it was.\n\nNathan resumed pacing as Logan turned back toward the numerous cabins laid out in a circular pattern around the large brick house in the middle. The brick house had been home to his family since they'd owned this property. It had also been the original building here. The cabins were all added over the years.\n\nOver three hundred hunters lived on the grounds. At one time there had been nearly four hundred, but some had left to work with other strongholds or marry women there, and others were killed. They were in a drought of sorts when it came to children with only a handful of them being under ten.\n\nThere were fifteen more strongholds such as this throughout the world. Some of them had more hunters, others less. All of them were run by hunters who were nominated by the elders within that stronghold and who his father had agreed to appoint. When the next new leader was required in one of those strongholds, Nathan would have to travel there in order to agree or disagree with the nomination. It was not something he looked forward to, but it was his role now.\n\nHe stopped pacing when his gaze fell on the archway the women had recently been decorating for Kadence's wedding. The white ribbon covering it stood out starkly against the growing dusk.\n\nNo one had asked Kadence if this was what she wanted, he realized. But they'd never asked any of the women or men who were paired together when the time came. It was simply accepted that it would happen, as it had always happened over the years.\n\nHe spent enough time in the human world to know it was an archaic tradition. He'd never questioned if things should be different though. When a woman was ready to breed, they were to be married off as soon as possible, with the hopes that within the next seventy years, while she was still capable of doing so, she would conceive a child.\n\nSome did not.\n\nNathan knew the elder men and women of the stronghold would choose a woman for him soon. Mostly, it was elder women who made the choice as few men lived to a hundred, never mind the two hundred that gave one the lauded status of an elder. If there were no acceptable women here, they would choose one from another location, but he suspected his wife would be Kadence's friend Simone.\n\nHe would be happy with Simone. She was beautiful and docile. She excelled in her classes and would make a suitable mother and wife. She was everything he'd ever wanted in a woman.\n\nHe told himself this, but he could feel the noose cinching around his neck and he realized what Kadence was going through. When he married, he would at least be able to keep hunting and have freedom beyond these walls. Kadence would simply be locked away here for the rest of her life.\n\nNo wonder Kadence had rebelled; he certainly would have. However, none of that mattered right now. The most important thing was getting her back, and he was hell-bent on doing that.\n\n# CHAPTER 14\n\nEmerging from the bathroom after her shower, Kadence tied the belt of the robe she'd discovered hanging over the back of the chair around her waist. Not only had someone left the robe for her, but they'd also brought her a change of clothes and a toothbrush.\n\nShe didn't know who the clothes belonged to, and she didn't care. Her clothes weren't so ruined she couldn't wear them again, but the idea of climbing back into them after Joseph's attack made her skin crawl.\n\nShe glanced at the bedroom door before retreating to the bathroom again. Ronan had been gone when she'd woken. She didn't know when he'd come back, and the last thing she wanted was to be caught naked. Releasing the towel, she slipped on the white bra set out for her. The bra was a little too big on her, but fit well enough. Opening the package of underwear, she removed a white, cotton pair and pulled them on before sliding the yellow sundress over her head.\n\nWandering over to the sink, she lifted her wet hair to inspect the wound on her neck. It was mostly healed with only the two original puncture marks from Joseph's fangs remaining. Her stomach turned at the reminder of the degradation she'd endured from his attack. She didn't care what they all believed, female hunters should be trained better.\n\nShe discovered a hairbrush amongst the sparse toiletries set beside the sink. Her fingers trembled as she ran her fingers over the brush before the shaving cream can and a disposable razor that wouldn't be much of a weapon. These were _Ronan's_ personal things. Standing here, touching his things, seemed almost as intimate as sleeping beside him all night had been\u2014something she'd known he'd done, judging by the still warm dent in his pillow she discovered upon waking.\n\nHer fingers settled on the hairbrush. She turned it over in her hand, admiring the fine ivory handle before using it to work out the tangles in her hair. Placing the brush down, she left the bathroom again.\n\nHer stomach grumbled, but she ignored the food on the platter as she inspected the room. From its gray walls to the large armoire and chestnut head and footboard of the bed, there was no sign of any softness in the personality of the man who slept in this room or of anything personal.\n\nShe had more signs of her personality on her small nightstand at home\u2014with its ever-present Shasta daisy from the greenhouses and the only photo she had of her entire family together, taken when she was five, before her mother had died from a fall off a horse\u2014than this entire room possessed. Looking around, she would assume the man who slept here was cold and stern, that he was nothing like the man who had talked with her during the day and who stared at her with such heat in his gaze. _Which side is the real him?_\n\nIt didn't matter; she wouldn't get to know him well enough to learn the answer to that.\n\nPadding over to the window, she ran her hands over the metal shutters covering them before spotting the button beside them. Pushing it, she stepped back as the shutters rolled up and folded themselves neatly above the picture window. Bars covered the outside of the glass.\n\nKadence stepped closer to the window to stare down at the large pool below. The puddles on the pool cover were frozen, and the shrubs encircling it were all weighed down with a layer of ice. It had rained sometime last night and frozen over again. Even the black, wrought iron fence surrounding the pool was covered in a sheet of ice.\n\nThe fence was high enough that it blocked her view of what lay beyond the pool area. Frustration filled her as she stepped back to survey the room once more. Nothing about the room or the outdoors gave any indication as to where she was, other than she hadn't been transported to a tropical climate while she'd been unconscious.\n\nInspecting the walls, she frowned as she noted the patches of darker gray paint on them. It took her a minute to realize the patches with their outlines were where photos or paintings had once hung. Now that she was looking for them, she also saw the holes in the walls from the nails that had been removed. There were at least ten places where something had been taken off the walls.\n\nWhat had happened to it all? She didn't think Ronan was planning to redecorate\u2014she had a feeling he didn't have much time for color schemes\u2014but then why had all the paintings or photos been removed?\n\nIn the long run, it really didn't matter what had happened to them all. However, her curiosity was piqued.\n\nWhen her stomach rumbled again, she walked over to the tray and picked up a piece of bacon. Why vampires had food in their house was another thing she didn't know how to explain, but she happily ate the bacon before grabbing another piece and biting into it.\n\nShe doubted any weapons remained in her coat, but she still searched its pockets to confirm it. Dropping her coat back over the chair, she rotated her shoulder, relieved to find she no longer felt any discomfort.\n\nLifting the navy blue cardigan that was set out with the dress, she slid it around her shoulders and buttoned it. She _really_ didn't want to go through Ronan's things, but with nothing else of use in view, she saw no other option.\n\nShe pulled open the heavy wood doors on the armoire. The crisp scent of cedar met her as she gazed at the clothes hanging neatly within. Most of them were jeans and button down shirts in an assorted array of colors. There were some black pants of the cargo variety and a black, three-piece suit she bet Ronan looked striking in.\n\nThe idea of peeling that suit off him to reveal all the ridges and carved muscles of his chest and abdomen made her mouth water.\n\n_Stop it_! She was no longer missing at least two pints of blood and could no longer blame her strange attraction to a vampire on that. She had to plan her escape, not stand here wondering what it would be like if Ronan kissed her. She'd broken free of the stronghold; she could figure this place out too.\n\nAlthough, it had taken a lot of plotting before she'd succeeded in breaking out of the stronghold. In a book she'd once read, the detective used baby powder to uncover fingerprints, so she'd decided to give that a shot. One night, shortly after Nathan and the hunters had gone out, she snuck out to use the baby powder on the keypad by the gate. All the numbers had fingerprints on them, as the code was changed once a month, but she'd used the four most visible fingerprints to figure out the right combination of numbers.\n\nOver the years, she'd spent a lot of time with Roland, the man who ran the security system at the stronghold. She'd never sat with Roland with the intention of escaping. She resented the plans for her life, but she'd never thought she'd do anything other than what had been laid out for her. She'd spent time with Roland because he was one of the few elder men alive, and his stories were fascinating.\n\nHowever, during all the time she'd spent with him, she'd also watched the cameras as they talked. There were no cameras on the homes or the massive garage with all the vehicles. No one saw any need for that. All the cameras were focused on the outside world.\n\nEventually, Kadence had realized that the three cameras covering the gate and roadway had a minute, every hour, at the twenty-three mark when none of them were focused on the gate. That minute had been enough time for her to run up and punch two new combinations into the pad every night after the hunters left.\n\nOver her years in the stronghold, she'd learned that three wrong combinations in a twenty-four-hour period set the entire system off. Two wrong tries went by unnoticed though. She had hoped and prayed no other hunters entered the wrong combination on their exits and entrances after her; they would have known something was wrong then.\n\nIt had taken her weeks of patience and wrong tries before she finally lucked out on the right combination. She'd closed the gate before it could open more than an inch and fled.\n\nShe'd broken free the next night and ended up in the hands of a vampire.\n\nWith a sigh, she closed the armoire and turned back to the sparse room. She wanted to know where Ronan was and what was going to happen to her, but she couldn't walk around this place without some kind of weapon.\n\n_If I'm not locked in_...\n\nHer gaze went to the door as the possibility occurred to her, but she'd leap that obstacle if she came to it. She could only handle one thing at a time right now. Walking away from the armoire, she bent to peer under the bed. There wasn't even a dust bunny under there.\n\nSitting back, she rested her hands on her knees as she surveyed the room again. Her eyes fell on the metal serving cart and the platter of food before returning to the room. Unless she planned on destroying some furniture, there was nothing she could use in this room to defend herself with against a vampire. The only thing she'd be able to break anyway was the nightstand, and that would defeat the whole purpose of being secretly armed.\n\nRising, she walked over to the cart and snatched the butter knife laid out neatly next to the fork. The silver knife may not kill a vamp, but she could at least inflict some damage with it. She slipped the knife up the sleeve of her cardigan before snatching the rest of the bacon. She greedily ate it as she walked to the door and grabbed the handle. Taking a deep breath, she braced herself for it to be locked.\n\nThe handle turned within her grasp and the door inched open. Stepping forward, Kadence pressed her eye against the crack and peered into the hall beyond. She couldn't see much of what was on the other side, but she heard no movement and detected no one out there. Feeling like a thief in the night, she slid out of the room and closed the door. Thick, dark wood doors lined the long hall before her.\n\nHer bare feet made no sound on the plush, red rug lining the hall as she walked. She was halfway down the hall when her step faltered, and she stopped to take in the bare, white walls. The walls were lit by dim, candle-flame-shaped bulbs housed within the glass sconces lining the walls. Those small bulbs illuminated the numerous places where things had once hung on the walls.\n\n_What is up with this place?_ She wondered as her fingers touched against the handle of the knife. The weapon gave her zero reassurance.\n\nDespite the electricity, she felt as if she'd stepped into the eighteenth century as she continued onward. She saw no bare spots or stains to indicate the rug was anything other than brand new, but something about it, or maybe it was the vibe of this whole place, made it feel ancient.\n\nShe crept forward until she came to a large, curving staircase at the end of the hall. The mahogany banister shone in the light spilling from the chandelier above. The hundreds of bulbs within the chandelier created a rainbow of colors amongst the crystals that danced over the white and gray marble foyer below. She had no idea who changed those bulbs when they burnt out, but she didn't envy them their job.\n\nThe dome of the ceiling had been painted with an exquisite landscape. Animals were gathered within a beautiful meadow as the sun shone down on a glistening lake. It was an outdoor scene she was certain none of the inhabitants of this place had seen, at least not by day. She hadn't asked, but she assumed since Ronan had been born a vampire that he'd never seen the sun.\n\nFeeling ridiculously saddened for his inability to feel the warmth of the sun, Kadence turned her attention to the stairs. She tiptoed down the steps so as not to make a sound.\n\nHer feet became instantly chilled when she stepped off the wood and onto the marble. Pausing with her hand on the banister, she glanced left and right, uncertain of where to go from here. Her gaze went to the front door across from her, but there would be no walking out of it as a heavy metal gate blocked the way.\n\nVoices drifted from down the hall on her left. Kadence crept toward the voices, her hand slipping into her sleeve to grip the knife handle. Arriving at a set of sliding double doors, she stopped when the voices within became louder and more distinct. The doors had been mostly closed, but a small crack ran down the middle of them to allow the light from within to spill out.\n\nMoving closer, she stepped to the side of one of the doors to peer in at the large men gathered around the ten-foot-long table within. Ronan was on his feet, his arm resting on the mantle of the gray stone fireplace at the far end of the room. His dark hair was disheveled, and an air of angry tension surrounded him.\n\n\"She cannot stay here,\" Killean said, his hands flattened on the table before him. His scar stood out more starkly in the glow of the small chandelier hanging over the table.\n\nRonan had been focused on the wall, but his head turned toward Killean when he spoke. \"I never said she could. However, I am not going to turn her out on the streets and hope the hunters find her or that she can find her own way home. She's been secluded her whole life; she may not survive on her own.\"\n\nKillean shrugged. \"I don't see anything wrong with a dead hunter.\"\n\nKadence winced at his callousness, but she knew her brother would have the same attitude if the roles were reversed. _She_ had the same attitude about Killean. She may not want to see anything bad happen to Ronan, but Killean could bite it for all she cared.\n\nRonan's eyes became redder as they narrowed on Killean. \"I do,\" he grated between his teeth.\n\nKillean bowed his head before speaking again. \"So we'll arrange a meeting with the hunters and release her somewhere.\"\n\n\"And how do you plan to do that?\" Saxon inquired.\n\n\"She has to know some way to get in touch with them,\" Killean replied. \"We should arrange something tonight. The sooner she is out of here, the better off we'll all be. The hunters will be out in force and far more of a nuisance to us while searching for her.\"\n\nKadence's heart leapt in her chest and disappointment crashed through her. She opened her mouth to shout, _no_ , before closing it again. She should be elated they didn't plan to kill her, didn't plan to use her as leverage over her brother and her kind. Instead, she felt... deflated.\n\nShe wanted to see her brother and friends again and let Nathan know she was safe. He _had_ to be told she was fine. He would go crazy with worry otherwise, and the hunters would be out looking for her. Someone could get killed because of her.\n\nHowever, she wasn't ready to go back yet. She was free\u2014well, as free as she could be while being held by her enemies...\n\n_Not my enemies._ She didn't understand how it all worked yet, and it would take her time to get used to the idea, but these vampires were not her enemies.\n\nRonan wasn't going to just turn her loose. However, she felt freer here than she'd ever felt at the stronghold. There were no expectations for her to be docile here. No looming marriage. No sequestered, endlessly boring days where she sat idly by while her brother and the others went out to make a difference in the world.\n\nAnd Ronan was here. Her eyes ran over him again. There could never be anything between them, but she couldn't help admiring the way his maroon shirt molded to his broad shoulders and chest. Her gaze lingered on the jeans hugging his powerful thighs and taut ass.\n\nHe stepped away from the mantle, drawing her attention back to his face. His gaze was focused on the crack in the doors. She gulped when she realized he knew she stood there.\n\n# CHAPTER 15\n\n\"Come in,\" he ordered.\n\nShe didn't pretend to hide or duck away. She'd been caught. Releasing the knife, she made sure it was tucked securely away before pushing open one of the doors and stepping into the dining room. Like the rest of the walls she'd seen in the house, the white walls within here were also bare.\n\nHer gaze finally settled on Ronan. Her fingers itched to touch him as she recalled the warmth of his body so near hers when he'd lain beside her and how he had nearly kissed her. She almost lifted her fingers to her lips as desire coiled within her belly. She'd thought maybe her weakened state yesterday had caused her to imagine some of her intense attraction to him; she'd been mistake. If anything, it felt stronger.\n\nWhen Kadence's heightened scent drifted to him, Ronan was unable to stop himself from taking a step toward her. He halted abruptly as he restrained himself from going any closer. If he did, he would take her in his arms and carry her from this room. She wanted him, and all he could think of was easing her need.\n\nNo one within the room spoke as their eyes traveled between Kadence and him. A blush crept through her cheeks as she fiddled with the sleeves of her sweater. Then, Declan cleared his throat and gave a discreet cough.\n\n\"How much did you hear?\" Ronan inquired, his voice more gravelly than normal.\n\n\"Enough to know you plan to release me to my brother,\" she replied.\n\nRonan stepped back to rest his arm on the mantle and focused on the wall across from him, but not before she saw a flash of pure red in his eyes.\n\n\"You must know where you came from,\" Saxon said.\n\nShe met his hazel eyes head on. \"I do, but I can't let you know that.\"\n\n\"I told you we aren't your enemies,\" Ronan growled.\n\n\"I realize that now, but you also said Joseph was once one of you. How can I trust that none of you will become a monster like him? I won't divulge our location to you. There are too many lives at stake.\"\n\nKadence braced herself as she waited to see how they would react to her refusal. Declan smiled as he sat forward and rested his hands on the table. Killean and Saxon stared at her as if she were a snake with the head of a spider, but Declan gazed at her as if he understood her, or at the very least maybe kind of liked her. She smiled back at him.\n\n\"Is there any guarantee none of you will ever become a Savage?\" she asked.\n\n\"Fate is a fickle bitch, and sometimes she takes even the best of us down, but sometimes she also intervenes to save us,\" Declan replied.\n\nKadence blinked at him, uncertain of what _that_ was supposed to mean, but she didn't have time to get into it.\n\n\"Enough, Declan,\" Ronan said as he glowered at his friend.\n\n\"He's a little testy right now,\" Declan said to her before leaning back in his seat and crossing his legs. His expression was innocent when he focused on Ronan.\n\n\"Why are you testy?\" Kadence blurted.\n\nRonan refused to answer her question or react to Declan's antics. He had no idea what his friend was trying to get at, and he didn't care. All that mattered was getting Kadence safely back to where she belonged. His nails scraped across the stone of the mantle as he balled his hands.\n\nDeclan's silver eyes ran appraisingly over her before he grinned. \"Because not only is fate fickle, but sometimes she's downright _devious_.\"\n\n\"Huh?\" she asked.\n\n\"I said _enough_!\" Ronan roared, causing her to jump as her attention swung back to him.\n\nHe'd stepped away from the mantle once more. His hands were fisted at his sides, making the muscles in his forearms and biceps bulge beneath his shirt. For a second, she thought the seams of that shirt would burst open as his eyes became almost entirely red.\n\nFear raced over her skin, not for herself, but for Declan. The vamp became as still as stone while Ronan stared at him. Before she could think about it, she stepped forward, drawing Ronan's gaze to her. She didn't know what she'd expected, but his shoulders relaxed, some of the red bled away from his eyes, and the tension in the room eased.\n\nSaxon cleared his throat. \"You must know of another way you can get in contact with your kind then.\"\n\n\"I could call my brother,\" she said.\n\n\"And was your brother one of the annoyances there last night?\" Killean inquired.\n\n\"That annoyance could kick your ass,\" she retorted.\n\nKillean lifted an eyebrow, causing his scar to pull upward with the gesture. \"Doubtful, but I will give him a go if you'd like.\"\n\n\"Would he be able to trace the call?\" Saxon asked, sending Killean a quelling look.\n\nShe'd read enough books to know what tracing a call meant, even if she'd never been around as much technology as the male hunters had. There were computers in the stronghold, but only a few of them had the Internet, and she'd never been granted access to those. If she hadn't read those books though, she would have no idea what Saxon was talking about.\n\nFor the first time, she felt not only resentful but infuriated by all she'd been denied. There was so much out in the world, so much for her to see and do. She didn't want to just learn other languages; she wanted to travel to the places where they were spoken and immerse herself in the culture and people there. She didn't want to simply stare at pictures of art in books; she wanted to breathe it in as she stood in the Sistine Chapel or the Louvre.\n\nShe yearned to stand somewhere and feel small and to learn without any limitations. There were thousands upon thousands of books in the stronghold, ones for recreation and others for learning, but all she would ever know was what was in those pages. Never would she experience it for herself.\n\nAnd it pissed her off.\n\nShe'd once asked one of her instructors why the women could read about and see photos of the outside world, but not use the computers or experience the world firsthand. Her instructor had replied it was because they had no reason to learn about computers, and as women they must be kept safe. However, as women, they also had to be educated so they were intelligent enough to carry on a conversation with their husband and not bore him.\n\nAt the time, she hadn't understood why the response had exasperated her so much that she'd walked out of the class, but she understood it now. They'd given her a taste of things she could never have to educate her for her husband, but they'd never taken into account that maybe her husband would bore _her_.\n\nKadence took a deep breath to steady herself. She could rage against her fate until she became bitter, or she could accept it for what it was. She couldn't stay here; it would only cause more problems between the hunters and vamps. It was more than obvious Killean didn't want her here, and Saxon would prefer her gone.\n\nHer temples throbbed as she tried to figure everything out, but she could feel time slipping away from her.\n\n\"Can he trace the call, Kadence?\" Declan nudged.\n\n\"I'm not sure the depth of their technology,\" she replied, \"but I know they have a lot.\"\n\n\"Then we'll give her a burner phone, take her out of here, and have her call from somewhere else,\" Killean declared.\n\nBurner phone, something untraceable, a onetime thing, she knew. Nathan had told her they used burner phones when they hunted in case one of their phones fell into the wrong hands. He gave her the number to each new phone he had, and she memorized it before he went out to hunt. He'd had the same phone for two weeks now, unless something had happened to it, and then she would have to call Logan.\n\n\"The sun will set in an hour,\" Saxon said.\n\n\"We can take her out to call then,\" Killean said.\n\nRonan ran a hand through his hair, tugging at it as his other hand gripped the edge of the mantle. Bits of rock broke off and bit into his palm, but he didn't release his hold on it. If he let it go, he may attack Killean. Ronan knew his friend was right, that she had to be returned, but did Killean have to be in such a rush to see it done?\n\nA jagged piece of rock sliced into his palm, drawing blood as he remained focused on the wall across from him. The best thing would be to get her out of here as soon as possible, but he wasn't ready to see her go.\n\nThough he didn't look at any of them, Kadence saw the pure red color of Ronan's eyes. She held her breath as she waited to hear what he would say.\n\n\"We'll leave in an hour,\" he finally said in a voice so hoarse she barely recognized it.\n\nKadence felt as if she'd been punched in the gut. Unless she planned something quick, she would be back in the stronghold tonight and married within the month.\n\nRonan's eyes had become their burgundy hue again when he finally turned to look at her. \"Are you hungry?\" he asked.\n\nThe bacon hadn't been enough to fill her, but whatever hunger she'd been experiencing was effectively swallowed by the pit in her stomach. She shook her head, not trusting her voice to speak. Without another word, she turned, left the room, and fled up the steps. By the time she made it to the top, she was running, but there was nowhere for her to run to.\n\n# CHAPTER 16\n\nA short time later, Ronan opened the door to his bedroom. He'd meant to stay completely away from Kadence until it was time for her to leave, but with every passing second he'd become increasingly compelled to return to her. Eventually, he'd given up the battle. He told himself he was going to see her to make sure she was ready to leave, but he knew he lied.\n\nKadence sat on the bed, her back to him and her head bent forward. Her pale hair cascaded down her back in thick waves that shone in the light. She'd removed the sweater and draped it over the back of the chair; her coat lay beside her on the bed. The sundress Marta had given her exposed the creamy skin of her bare shoulders and clung to her slender curves.\n\nHer head rose when the door click closed, but she didn't turn to look at him. \"It's almost time to go,\" he stated. \"I'm sure you'll be happy to see your brother again.\"\n\n\"I will,\" she replied in a clipped tone.\n\nHe frowned as he tried to puzzle out why she didn't seem more excited by the prospect of being reunited with her family. Moving around the bed, he stopped before her. Her face was completely serene and impassive, like a statue. She revealed no emotion as she clasped her hands demurely before her and stared at him.\n\nIt took him a moment to realize this was the docile, female hunter she was supposed to be. He didn't like it one bit.\n\n\"What is wrong?\"\n\nThat odd mask of composure never slipped. \"I am to be married soon.\"\n\nOut of everything he'd expected her to say, that hadn't been it. His gaze ran over her slender body and alluring breasts that would fit perfectly in the palm of his hand. His fangs lengthened at the idea of any other man knowing what it was like to feel her body against his while they slept, to be _inside_ of her.\n\nHe managed to suppress a snarl. \"When?\"\n\n\"Within the month.\"\n\nHe recalled what she'd said earlier, _\"I'm supposed to be sorry for not being overjoyed about my glorious role as soon-to-be bride.\"_ At the time, he'd assumed she meant far in the future, not within a month.\n\n\"To who?\" he demanded.\n\n\"Logan. He is an outstanding hunter from a strong family line. Our children will be fine hunters. He is a good man, a good friend, and loyal to Nathan.\"\n\n\"Your brother arranged this?\"\n\n\"The elders chose Logan for me when I became capable of reproducing.\"\n\n\"You're not a _fucking_ _horse_!\"\n\nShe blinked, but it was the only reaction she showed to his explosive response.\n\n\"Do you want to marry Logan?\" he demanded.\n\n\"It is a great honor to marry a hunter and bear him children. I must be grateful that I am of an age to do so and that a fine husband has been chosen for me.\"\n\nShe sounded as if she were reciting something drilled into her head over the years. \"You accept this?\"\n\nHer blank eyes met his as she spoke in a dull monotone. \"Of course. It is why I was born. It will be a privilege to carry on the legacy of my heritage.\"\n\nHe didn't understand this complacent person who had taken the place of the spirited woman he believed her to be, but he wanted to shake her to reveal the woman she truly was. The woman he wanted for himself alone.\n\nThe irrational urge to possess her was back again, but this time it refused to be buried in the cold recesses of his heart once more. Unthinkingly, he stroked her cheek with the tips of his fingers. She didn't nuzzle him like she had before, but at least she didn't turn away from him.\n\nCrouching before her, he grasped her chin so she had to look him in the eye. \"Do you really accept this?\"\n\nSomething flickered in her eyes, but her mask didn't change. \"I have no choice. What else am I to do?\"\n\nHe had no answer for her, no solutions. There was nothing he could give her, nothing he could say that would help. \"You could tell them you are not ready to be married.\"\n\nShe let out a harsh laugh. He winced inwardly, hating the bitterness of the sound. \"I can no more fight who I am and what is expected of me than you can. My turn has come. I have known since I was a child it would happen. There's nothing I can do to change it, and Logan is a good man. We've been friends since childhood. I know he won't abuse me, and he will make a fine father for our children.\"\n\nNot only would another man know her body, but he would also get to share the bond of raising their children with her. Ronan stilled at the realization, even as he wanted to tear the room apart.\n\nHe released her chin and rocked back on his heels. \"What happens if Logan dies?\"\n\n\"Then I will become an instructor. I will show the young our heritage, teach them our ways, and I will guard over our ancestry.\"\n\n\"What if he dies before you have a child, or six months from now?\"\n\n\"If I am still at a good breeding age, and a hunter loses his wife, then I may be considered to fill the role.\"\n\nNo wonder she'd rebelled against her life, there was no hope within it, he realized.\n\nRemoving his hand from her cheek, he rose to pace across the floor. He couldn't stand the thought of her being locked away for the rest of her life. Couldn't stand the idea of someone so beautiful and alive being caged so remorselessly and used like cattle.\n\nHe turned back to gaze at her as she remained sitting rigidly on the bed. He couldn't begin to imagine what she faced when she returned. Spinning on his heel, he paced back and forth once more. He couldn't help her. If they didn't return her, the hunters would go on a rampage, slaughtering every vampire they came across. Many innocents could become caught in the crossfire of the sudden onslaught. Many of his kind could die.\n\nHe couldn't allow that. For his entire life, it had been his duty to protect them.\n\nBeing married was her destiny, her fate. Who was he to go against the ways of the hunters, even if he didn't approve of it?\n\nStalking back, he stopped before her. \"I can't help you.\"\n\nShe showed no reaction to his callous words. \"I didn't ask you to.\"\n\nRonan ignored the fierce pounding of his heart as he pushed aside the clamoring in his head telling him to keep her, that she belonged to him.\n\n\"Maybe your brother can stop it\u2014\"\n\n\"It is my fate. It is who I am, who my people are. No one can stop that,\" she cut in.\n\n\"Shit,\" Ronan mumbled. He ran his hand through his hair as he started pacing again. A solid rap on the door drew him to an abrupt stop. \"What?\" he barked.\n\n\"It's time, Ronan,\" Declan replied in a subdued tone.\n\n\"We'll be right there.\"\n\nKadence rose from the bed with her hands still held demurely before her. Slowly, she met his gaze. \"I will tell Nathan to come alone. He may believe it's a trap, but he'll do whatever he believes is necessary to get me back.\"\n\n\"He loves you,\" Ronan realized.\n\n\"Yes.\"\n\n\"And you him?\"\n\n\"He's my twin and my best friend. Twins are very rare amongst our kind so we were exceptionally close.\"\n\n\"How rare are they?\"\n\n\"We are the first ones born in nearly seven hundred years and the first girl\/boy twins born in over a thousand years.\"\n\nRonan folded his arms over his chest. \"I see. Would he stop this marriage if you asked him to?\"\n\n\"He might try, for me, but I would never put him in such a position by asking him to do that.\"\n\n\"Is he the leader of your people?\"\n\n\"We have many different leaders in different places,\" she replied vaguely.\n\n_He is at least one of those leaders then_ , Ronan realized and she would sacrifice herself not to cause her brother any trouble. He assumed Nathan had taken over after their father was killed, if the leadership role passed with hunters the same way it used to pass with the vampires.\n\n\"Kadence.\" He moved back to her, and placing his finger under her chin, he lifted it. For the rest of his life, he would recall every detail of her. He brushed her hair back, letting the silken strands run through his fingers.\n\nStepping closer, he stopped when his chest brushed against hers. Her eyes searched his as he stood over her, but her face remained serene. He found himself unable to resist the lure of her plump, red lips as he bent his head to hers. He only meant to have a little taste, but the minute his lips touched hers, he knew he was gone.\n\n# CHAPTER 17\n\nHis hand rested on her hip before wrapping around her waist. When she didn't try to resist him, he pulled her lush body firmly against his and lifted her. Not even with his first bedding had he been concerned about coming in his pants, but he was now as she sighed when she rubbed against the rigid length of his erection and pressed her breasts against his chest.\n\nTangling his fingers in her hair, he pulled her head back and ran his tongue over her lips. Her lips parted to his prodding and he delved into her mouth. She gasped then melted against him as he tasted her. Her tongue hesitantly touched his before becoming bolder as she eagerly met each of his thrusts.\n\nHe needed to feel more of her, to know what every inch of her looked and tasted like. Releasing her hair, he ran his hand down the velvet expanse of her shoulders. Gripping the strap of her dress and bra, he pulled them both down until her entire breast fit his hand perfectly. He ran his thumb over her nipple until she moaned and the bud puckered against his flesh, branding him for eternity.\n\nKadence squirmed in Ronan's hold, wanting to get closer to him yet unable to do so. No one had ever touched her so intimately before or made her feel this alive. For the first time in her life, she didn't know uncertainty and dread. With complete clarity, she knew she didn't give a damn what this man was, she just craved more of him.\n\nHer heart beat against her ribs when he ran his thumb and forefinger over her nipple before pinching it. She didn't know how to react to the overwhelming sensations he ceaselessly stoked to life in her. It was almost too much, but nowhere near enough.\n\nHer fingers curled into his hair when he broke their kiss to leave a trail of heat over her skin with his lips and tongue as they moved over her ear and down her neck. The graze of his fangs against her flesh didn't scare her. Instead, she drew him closer when his head dipped toward her breast. He hesitated for a second before his mouth clamped onto her nipple.\n\nThe low groan he issued and his tongue laving her nipple caused her to cry out as a wet heat spread between her legs. When he drew her nipple deeper into his mouth and sucked at her, she nearly screamed as she ground her hips against him with a wildness she hadn't known she possessed until him.\n\n\"Ronan!\"\n\nKillean's yell from outside the door jolted through the haze of lust consuming Ronan. Still, he couldn't tear himself away from the inviting warmth of her body.\n\n\"It's time!\" Killean shouted and pounded once on the door.\n\nRonan's hands clenched on her. He didn't think he would ever be able to let her go now that he'd tasted her, now that he knew how responsive she could be and how ensnaring the scent of her passion was.\n\nWith a ragged breath, he succeeded in tearing his mouth away from her. Rising over her, he gazed at her face. She was flushed not from embarrassment but from unquenched desire. He couldn't leave her in this state, not when she would _let_ him ease her, but he couldn't take her innocence and ship her back to the hunters. He had no idea what they would do if they discovered she was no longer a virgin.\n\nHis head dropped down so his forehead rested against hers as she gazed at him with wide-eyed awe. She was his for the taking, and he was going to hand her over to never see her again.\n\nSuddenly feeling more Savage than he'd ever felt in his life, he was hit with the impulse to sink his fangs into her, drain her to near death, and turn her. To make her his completely. The sharp points of his fangs pressed against his lower lips. The thump of the blood beating within the vein in the pale column of her throat called to him. He would make her the thing she hated most and he didn't care, not if it meant he could be selfish and keep her for himself.\n\nThen, her hand rose to pull her dress and bra back up, covering the breast he'd exposed. The tremor in her hand pierced through the demon to his more human side. He set her on her feet and stepped away from her before he lost control.\n\n\"I'm sorry,\" he managed to get out. \"I didn't mean for that to happen.\"\n\nKadence rested her fingers against her swollen lips. She longed to have him replace her fingers with his mouth once more, but they couldn't continue this. A lump formed in her throat. If things had been different...\n\nIt didn't matter, she decided. Things weren't different and hoping for things that could never be was a fool's game.\n\n\"Don't apologize,\" she said. \"I wanted it as much as you did.\"\n\nTo hear her say she wanted him too was more than he could tolerate. There was no point in denying either of them this. He _would_ have her. He stepped toward her, not caring about anything anymore except possessing her.\n\n\"Ronan!\" Killean shouted again.\n\nHis friend's incessant yells cleared his mind, but he wouldn't mind ripping Killean's tongue out before they left here. \"We'll be right there!\" he barked.\n\nKadence flinched at his words, but she didn't move away from him. Turning on his heel, Ronan strode away from her. He removed his coat from the armoire and slid it on to cover his erection.\n\n\"Ronan.\" He turned at his name. \"Thank you.\"\n\n\"For what?\"\n\n\"For being kind to me, for showing me that not all vampires are evil. I will tell the hunters that. I will tell my brother we have it wrong. They probably won't listen to me, but I will try to make them understand. Thank you for giving me an extra day of freedom.\" She paused as her fingers touched her lips again. \"For showing me what it is like to be kissed. For letting me experience something I probably never will again.\"\n\n\"Your husband will kiss you,\" he growled, his fingernails digging into his palm and tearing into flesh at the thought.\n\n\"Yes, but he won't make me feel like you do.\"\n\nRonan let out a hiss as an invisible fist socked him in the stomach. A red haze shaded his vision as the demon part of him screamed at him to take her. When her hand fell from her lips and she edged away from him, he knew that what she saw of him rattled her.\n\nThe sight of her distress eased the turmoil of his emotions, and he was left to deal with the disbelief of what had thundered through him. He needed to get this woman out of his house and out of his life before he plummeted over the edge and became the thing they both hated most.\n\nHe couldn't stand it if he ever hurt her or if she looked at him with disgust. If he stayed with her, both those things could come to pass.\n\nTurning on his heel, he stormed across the room and threw the door open. Killean and Declan staggered away from him as the door rebounded off the wall. \"Let's go!\" he spat.\n\n# CHAPTER 18\n\nNow that they'd removed her blindfold, Kadence watched as the woods whipped by her at an extraordinary speed from the back seat of the SUV. Her skin was still electrified, the hair on her arms stood up from the lingering effects of Ronan's body pressed against hers. Her lips tingled with the reminder of his kiss. She had meant what she said, even if it upset him, no man would ever make her feel the way he did.\n\nThat was why she _almost_ felt bad about what she planned to do.\n\nDeclan sat beside her, his brow furrowed as if in deep thought. Occasionally, he would glance between her and Ronan. Every time those impossibly silver eyes swung to her, she felt as if he stared straight into her soul, and she prayed he hadn't somehow figured out what she intended.\n\nThat was impossible. He would have told Ronan if he knew, and he'd definitely try to stop her, but he didn't say a word. He probably suspected that something had transpired between them and was curious about it. She was only being paranoid.\n\nShe met his gaze head on as he peered at her again. To her surprise, his mouth quirked into a small smile. Before she could decide what to make of that smile, he turned away from her to stare at Ronan in the seat before him.\n\n\"Here,\" Ronan commanded.\n\nKillean pulled to the side of the back road. Kadence's heartbeat kicked up a notch as, before Killean could put the car in park, Ronan threw his door open and climbed out. She barely saw him through the heavily tinted windows as he stalked past Declan's door and made his way toward the back of the vehicle.\n\nShe jumped when the door next to Saxon swung open far sooner than she'd expected. Ronan stood outside of it, his large body filling the entire doorway.\n\nSaxon slid out of the vehicle and shuffled past Ronan, who only moved back an inch or two to give Saxon the room to maneuver. Ronan's eyes were more red than brown when they met hers. He stepped aside and gestured for her to climb out. Kadence clutched her coat to her as she slid across the leather seat and exited the vehicle.\n\nThe January wind howled down the lonely road, causing the barren tree branches to click against each other, and the dress to billow around her calves. The frigid air wasn't cold enough to explain the ice creeping through her veins. Her hands trembled when she reached for the phone Ronan handed her. Most likely he would attribute her shaking to the winter night, but she knew it was because she had no idea how he would react to what she was about to do.\n\nRonan's fingers brushed hers before he jerked away as if she'd burned him. Kadence studied his face, trying to find some hint of the man who had kissed her with such intensity. She was met with a wall of stone.\n\nWhat she planned to do now would infuriate the most powerful being she'd ever encountered. With one hand, he could break her neck between this heartbeat and the next. He wouldn't though, that much she knew. He might go on a tirade, but he would not harm her.\n\nKadence's eyes inadvertently darted toward Declan. He leaned against the side of the SUV as he stared at the woods with a look that said he'd rather be anywhere but here. He didn't look at her while he dipped into his pocket and pulled a lollipop free. She gawked at him as he opened it and popped the candy in his mouth before shoving the wrapper into his pocket.\n\n_What is a vamp doing eating a lollipop_? She'd never seen anything so absurd as he twirled it in his mouth while remaining focused on the woods. Kadence had the feeling he was aware of her stare, but refused to acknowledge it.\n\nKillean walked a little way down the dirt road, his eyes constantly searching the area around them. Saxon strode to the front of the vehicle and crossed his legs as he leaned against the hood. Lucien must have returned to wherever else he was staying, something she was grateful for now.\n\nFor the first time, she considered how they would _all_ react to what she was about to do. They would be as angry, if not angrier, than Ronan. She looked back to Declan again. He turned toward her and gave a barely perceptible nod.\n\nKadence nearly dropped the phone. He _did_ know! Somehow, he knew or suspected what she planned to do. That knowledge was more than a little unnerving, yet she found her confidence growing as her shoulders relaxed a little. He either believed it would all be okay, or he was trying to get her killed. Whichever it was didn't matter, she was going through with this.\n\n\"Call your brother!\" Ronan ordered.\n\nKadence scowled at him, not at all pleased with the command or the tone of his voice. \"I'm not yours to order around!\" she retorted and held his gaze when a muscle in his cheek twitched and a vein appeared in his forehead.\n\nAfter a minute of tense silence, Kadence turned away from him and flipped the phone open. Her fingers felt thick as she punched in the last number she'd had for Nathan. It took three tries before she finally got the number right. She stared at the screen as she waited for something to happen, but nothing did. Knowing she must have missed something, she searched the phone for the answer.\n\nRonan winced when he realized that Kadence wasn't familiar with cell phones. The hunters kept her so sheltered that she only had a rudimentary knowledge of things that most took for granted. Before he could intervene to show her what to do, she hit the send button and lifted the phone to her ear.\n\nHolding her breath, Kadence closed her eyes as she waited for her brother to answer. \"Who is this?\" Nathan demanded after the fifth ring.\n\nKadence winced at the raw anger coming across the phone even as love and sorrow swelled within her. She took a deep breath to steady her wire-taut nerves before plunging in. \"It's me, Nathan.\"\n\n\"Kadence! Thank God! Are you okay? _Where_ _are_ _you_?\"\n\nGuilt burrowed into her at the relief in her brother's voice. She would have given almost anything to ease the anguish she heard, to see him one more time, but she couldn't, she _wouldn't_ , give up her freedom again. One thing she'd realized after Ronan's kiss was that life was too short to continue to do as she'd always done. There were so many new experiences out there for her, and she wanted all of them.\n\nShe'd rot in the stronghold; she'd die someone far different from the woman she was now if she went back, and she couldn't do it.\n\n\"I'm fine, Nathan, really.\"\n\n\"Where are you? What happened?\"\n\nKadence glanced over her shoulder. All the vampires were staring at anything other than her, but she knew they were listening to every word she was saying. \"After what happened in the alley, I ran, and I kept running.\"\n\n\"To where?\" he demanded. \"Where are you? Tell me and I'll come get you.\"\n\n\"No.\"\n\nThe silence on the other end stretched on for a full minute before her brother spoke again. \"What do you mean, _no_?\"\n\n\"I ran as far as I could, and I'm not coming back!\" she blurted before she completely lost her nerve. She had no idea what she would do with her life now. That knowledge should terrify her; instead, she felt like a baby bird spreading its wings for the first time, and she nearly laughed aloud from the free-falling experience of plunging out of the nest.\n\nSaxon gawked at her as he turned toward her. Killean's head swiveled on his shoulder, as he leveled her with the stare of a snake ready to strike. A small smile curved Declan's lips and he tossed the lollipop aside. Ronan's breath exploded from him as he whirled toward her.\n\n\"What?\" Nathan yelled into the phone. \"Have you lost your mind? Where are you? I'm coming to get you _right_ _now_! You can't survive out there on your own!\"\n\nKadence grimaced and started moving backward when Ronan came toward her. His eyes became a more vibrant red with every step he took. She couldn't let him get his hands on the phone; he would tell her brother everything if he did. He would make her go back.\n\n\"I'm not coming back!\" Kadence gushed into the phone. \"I'm sorry, Nathan. I can't be what you need me to be, and I want to see, to learn, to _experience_. Please forgive me, and tell Logan this has nothing to do with him. I'll call you as soon as I can. I'll be all right. I love you.\"\n\n\"Kadence!\" Nathan's shout resonated through the phone as she pulled it away from her ear and snapped it in half.\n\n\" _What are you doing_?\"\n\nKadence jumped at Ronan's bellowed words. He wasn't mad. He was _irate_!\n\n\"Give me that phone.\" The steady calm of Ronan's command unnerved her more than if he'd yelled again.\n\nKadence shook her head, her hair flying around her face. \"No,\" she managed to choke out.\n\nShock briefly registered on his face before his wrath blazed back to life. He looked mad enough to strangle her.\n\n\"Give me that phone,\" he growled.\n\n\"I... I broke it,\" she stammered.\n\n\"Then you will give me his number and I will call him back.\"\n\nKadence jumped back as he grabbed at her. She spun to flee, but his hand snagged her coat and he pulled her against him. The air in her lungs rushed out of her when her back crashed into his solid chest. His grip on her instantly gentled, though she remained firmly trapped against him.\n\nShe had no idea if he'd somehow be able to get the number off the broken phone. She released the remains of the phone and stomped them beneath her feet. The satisfying crack of the material sounded seconds before he spun her to face him.\n\nRonan's face was inches from hers as he gripped her upper arms. His lips skimmed back to reveal the lethal points of his fangs. Despite his furious countenance and the lethalness of him, she still didn't believe he would attack her.\n\nRonan had no idea how to react to what she'd done. Part of him wanted to shake her for disobeying him when _no_ _one_ else did. The other part wanted to hug her close. Had she told her brother that because of him? Did she want to stay with _him_?\n\nHe shut the hope down. It wouldn't be possible, no matter what. His life was far too treacherous for her to stay with him. She had to go back, or at least go somewhere away from him.\n\n\"What are you doing?\" he demanded.\n\nKadence lifted her chin as she met his stare. She'd made her choice, she wouldn't back down from it now.\n\n\"From the second I was born, my entire life has been plotted out for me. I've resented it, I've rebelled against it, but I still followed along with that plan. I succeeded in breaking free of the stronghold, and I was going to return to it. I was going to allow myself to be married off, to be caged again, because I'm afraid of what is out here.\n\n\"However, I refuse to be a coward. I'm letting my people and family down by doing this, but I'm free, and I have to see what is out here. Once the monster who killed my father is dead, I will go out and experience all the things I've read and dreamt about my entire life. Maybe one day I'll return to the stronghold, but today is not that day. My brother has no idea I'm with you, he knows I'm alive, and believes I'm free, so the hunters won't blame vampires for my disappearance. I made sure of that.\"\n\nFor the first time in his life, Ronan had _no_ idea what to do. Forcing her back to her people would destroy her, but so would keeping her locked up with him. He couldn't just turn her loose on the streets. With her beauty and naivet\u00e9, the humans would eat her alive. He loathed all of the options, but she would be safest with the hunters.\n\nHe tore his eyes away from her and looked over her shoulder to Killean, who had retrieved the broken pieces of the phone. \"Give them to me.\"\n\nKillean's hand stretched over her shoulder and Ronan released her to take the phone. Kadence couldn't bring herself to look at the device for fear she hadn't destroyed it as badly as she'd hoped. Ronan stared at the pieces in his hand as she held her breath and waited.\n\n\"Shit!\" he exploded.\n\nKadence jumped as he spun away from her and heaved the phone into the woods. The remains shattered off a tree with a crack of mechanical bits. Kadence stiffened her spine, refusing to be intimidated by him when he turned back to her.\n\n\"I am not going back there. You can't make me, so leave me here,\" she said coldly.\n\nHis hand swallowed her bicep when he clasped her arm and dragged her toward the SUV. Declan stepped toward them, but he didn't try to intervene. Kadence dug her heels into the asphalt to stop Ronan as he pulled her onward.\n\nHer attempt was as futile as a gnat trying to stop a raging rhino. \"Stop it! Let me go!\"\n\nHe didn't stop. Without thinking, she kicked him in the back of his calf. Her mouth dropped open as he spun toward her. She couldn't believe she'd done that, but she couldn't take it back now. He stared at her with his full lips compressed into a flat line and a look that said he might splat her like a fly. Despite her possible impending splatting, Kadence ripped her arm free of his grasp and glowered back at him.\n\nRonan took a step toward her, but she didn't back away. Meeting him toe to toe, she stuck out her chin. Her insolence maddened him, yet he couldn't help admiring her. She was strong, but nowhere near as strong as the vampires standing here. _None_ of them would dare to stand up to him in such a way.\n\n\"You are going back to your brother,\" he stated.\n\n\"No. I. Am. Not,\" she enunciated clearly.\n\nShe didn't think he'd ever been denied before if the look on his face was any indication, and she'd done it twice in less than three minutes. She could almost feel the breeze of the flyswatter over her head, but she wouldn't back down from this.\n\n\"You are going to do what I tell you to do,\" he said as if this would resolve the issue.\n\n\"The hell I am! You have no say over me or my life. And yes, this is _my_ life; I finally get to have one! I don't expect you to understand, but I am not going back there. I won't give up this opportunity. I know it's selfish, I know it's insensitive to my people, but I must do this _for_ _me_. This may be the only chance I ever have to be free, and if I waste it, I will hate myself for the rest of my life.\n\n\"The hunters will not come for you; they will come for _me_. There will still be a lot of them looking for Joseph, but once he's dead, things will calm down again. I will not let you take this chance from me!\"\n\n# CHAPTER 19\n\nRonan couldn't deny the sway of her impassioned words. What awaited her at the stronghold wasn't the life he wanted for her. He would prefer to keep her with him, but that would be impossible. If any of his enemies learned of her, they would go after her to take him down.\n\nAnd then he also had to worry about himself around her. If he turned Savage, Kadence's life may be the first one he took.\n\nNo, he hated what awaited her at the stronghold, but she was safest there. She'd be a broodmare, but she'd be alive and protected. Best of all, he wouldn't be able to find her again and she would be safe from _him_.\n\n\"You have to go back to your family,\" he told her, his voice softening at the desperation in her eyes.\n\n\"No, I don't.\"\n\n\"What do you plan to do?\" he demanded. \"You have no money, no ID, and no job prospects. You have nothing.\"\n\nShe pointed a finger at him as she yelled at him. \"You are wrong. I will have my freedom!\"\n\nLowering his head into his hand, he rubbed the bridge of his nose as he tried to decide what to do with her. He couldn't leave her here as she'd asked; she'd never survive on her own. But then, maybe she'd smarten up and return to her family if he did drive away. That was if she didn't get herself killed or raped first. At least if she'd returned to her brother he would have known she was safe. He couldn't leave her here without knowing what had become of her.\n\nLifting his head, he met her steely gaze. \"I can't leave you here.\"\n\nShe grabbed his arms, squeezing them as her fingers dug into him. \"You owe me _nothing_ , Ronan. Let me do this!\"\n\n\"I will not leave you here! You'll never survive in this world. You are too much of an innocent. Some of the human race is as bad as the worst of my kind. The humans may not be as strong or fast as you, but they can still destroy you.\"\n\nKadence contained her rising temper over his condescending words. Shouting at him now wouldn't help her situation at all. Only reason would break through his stubborn fa\u00e7ade. He was right, she was over her head in many ways, but she'd made it out of the stronghold, she would figure this out too. She'd have to take it one day at a time, but she needed that first day in order to start living her own life.\n\n\"I am very strong, capable, and intelligent. I _can_ survive,\" she replied.\n\n\"This world will destroy you.\"\n\n\"No\u2014\"\n\n\"Enough! You are not staying here and that is final!\"\n\nKadence gaped at him in disbelief. \"But\u2014\"\n\nHe started dragging her forward again, completely ignoring the protests she sputtered. Killean's golden eyes narrowed to slits as she was pulled past him. Declan held the back door open, a nonchalant look plastered on his handsome face. He barely glanced at her as Ronan pushed her into the SUV, climbed in beside her, and slammed the door.\n\nKadence was about to scramble out the other side when that door opened and Saxon slid in. He looked completely baffled as he stared at her.\n\n\"Where to?\" Killean inquired gruffly when he settled into the driver's seat.\n\n\"Head back to the mansion; we'll figure something out there,\" Ronan commanded.\n\n\"Leave me here,\" Kadence said. \"I'm not your responsibility.\"\n\nRonan glowered at her. \"Never going to happen.\"\n\nShe fell back against the seat and hunched her shoulders to avoid touching him and Saxon as she silently fumed over his refusal to set her free. After ten minutes of driving, Ronan slid a blindfold over her eyes. She stiffened at his touch and fought the impulse to slap his hand away. A few minutes later, they came to a stop. Ronan pulled the blindfold off her and climbed out of the vehicle. He held the door for her, but didn't look at her.\n\nKadence glared at him as she stepped out of the SUV. Finally, she tore her attention away to focus on the building looming over her. She'd been blindfolded when she'd been led outside earlier so she hadn't seen it, and she wished she hadn't seen it now.\n\nThe place was a monstrosity. No one ever would have guessed the spartan beauty of the inside by the ugliness of the outside. The mansion towered into the night sky, its peaks and turrets looking like something straight out of a gothic novel. The huge gargoyles on the peaks and outside the doorways didn't help that image at all. Bars covered all the arched windows, the front door was painted black, and the entire place had been built using a dreary, gray stone. Long wings fanned out behind the building and to the sides of the main structure.\n\nIt was the largest building she'd ever seen, and the ugliest.\n\n\"Doesn't meet your fine standards?\" Ronan murmured in her ear.\n\n\"Really trying to uphold that Dracula image, aren't you?\" she retorted.\n\nA spark of humor crossed his face before he buried it. \"Get inside.\"\n\nKadence snorted, but did as he said, mainly because the place unnerved her. She was almost to the front door when it opened. Her hand flew to her heart as she jumped back into Ronan's massive chest.\n\nKillean's low laugh came from behind her. \"You should be scared.\"\n\nShe shot him a dirty look, straightened her shoulders, and strode forward. A tall man stood inside the foyer, his hand resting on the doorknob. Gray speckled his brown hair at his temples, and a pair of round spectacles were perched crookedly on the tip of his large nose. He offered her a small smile before sweeping low in a grand bow.\n\n\"Good evening, madam. May I take your coat?\"\n\nKadence almost tripped over her own feet when she came to an abrupt halt. They had a butler? Yes, apparently they did, and he was staring at her expectantly. \"Um, yeah.\" She slid it off and handed it over to him.\n\n\"We'll be in the poolroom, Baldric, if you could please bring some food for the human,\" Ronan instructed.\n\nHis icy tone caused a shiver to run down her spine. She'd never heard him sound so distant and reserved. He'd been kind to her before, but she might have pushed him to a breaking point with her actions tonight. Well too bad, because she wasn't exactly jumping for joy right now either.\n\n\"Of course, sir,\" Baldric replied.\n\nRonan clasped her arm, leading her across the foyer and down the hall. She allowed him to pull her along, mainly because she didn't know what else to do. She was trapped here, with them. Maybe she should have gone home after all.\n\nThe second the idea crossed her mind, she buried it. They couldn't keep her here forever, and she couldn't think of any way they could find her brother without risking a fight. Hopefully, they couldn't think of one either.\n\nRonan paused outside a set of wooden doors and slid them open to reveal the sumptuous room beyond. Red velvet couches were set on either side of a beautiful red and gold oriental rug. She'd never seen anything like these couches with their etched wood backs and arms. The dainty legs barely looked strong enough to support her weight, never mind the weight of any of the vampires spreading out to stand in the room.\n\nTwo large pool tables with their smooth green surfaces were set in the middle of the room. One had balls scattered across the top of it. The numerous arching windows in the room were covered with sheer, crimson curtains and each had a red window seat. A bar lined the entire back wall; the large mirror behind it reflected light onto a multitude of liquor bottles. Like all the other rooms, the walls in here were bare. Nothing in this room fit any of these men except for maybe the pool tables and the bar, if vampires drank liquor.\n\n\"Sit.\"\n\nKadence frowned at Ronan's rude tone, but moved toward one of the couches. She'd read about settees in a few books and imagined this is what the author must have been describing as she carefully perched on the edge of one. Settling her dress, she folded her hands demurely in her lap. She plastered the look of serenity, the one that had managed to get her through so many boring days in the stronghold, on her face.\n\nRonan strode past her, and moments later she heard the clatter of a glass behind the bar and liquid being poured into it. _Guess that answers my question about vampires and liquor_ , she thought.\n\nKillean and Saxon followed Ronan, but Declan walked over to one of the windows and leaned against the wall beside it. Baldric wheeled in a cart loaded with all sorts of food. Kadence's stomach grumbled and saliva filled her mouth. She hadn't realized how famished she was until then. Baldric swept out of the room, closing the doors behind him. Kadence licked her lips, itching to get her hands on some of the food.\n\n\"Eat,\" Ronan said from where he stood by the bar.\n\nHer pride wanted her to refuse to eat anything from him, but her common sense told her she was an idiot if she starved herself to be spiteful.\n\nClimbing to her feet, she made her way to the cart. She loaded a plate with cheeses, crackers, sliced meats, fruits, and vegetables. She munched on the food as she made her way back to her seat.\n\nKadence was finishing the last bite when Ronan started in. \"You can't stay here. It is too risky.\"\n\nPutting her plate down on the table beside her, she wiped her hands on her napkin while she bided for time to regain her mask of composure. \"I never asked to stay here. If you do recall, I was quite satisfied with staying on the side of the road. Plus, I'm sure you have enough security around here to keep anyone from getting in or me from escaping.\" Though, if she figured out the stronghold, she'd figure out this place too.\n\nRonan cursed loudly, and liquid sloshed out of his glass when he set it on the bar. Kadence remained stoic as she turned to face him. \"That is not what I meant!\" he snapped.\n\n\"Then what exactly did you mean?\"\n\nHis jaw locked, but he didn't answer her. A new, disturbing possibility occurred to her. Did he mean _he_ was the threat to her?\n\nBefore she could question him, Declan stepped forward. \"I'm sure she'll be perfectly safe here until we can figure something out.\"\n\n\"You can't be serious!\" Killean retorted. \"For all we know she could be spying on us, just waiting to give the hunters information about us.\"\n\n\"Oh come on, Killean, even you know that is ridiculous,\" Saxon drawled.\n\n\"She doesn't belong here and we all know it,\" Killean spat.\n\n\"We can't put her out on the street,\" Declan said.\n\n\"No we can't, and until she comes to her senses, she will stay here,\" Ronan said and shook his head as if he were annoyed with a child.\n\nKadence's mask of serenity slipped. \"I have come to my senses! You have no right or reason to interfere in my life!\"\n\n\"She's right,\" Killean said. \"Put her out on the street, let her get killed. It's no loss to us. Besides, she'll probably call her brother and beg him to come get her within in an hour of being on her own.\"\n\n\"Fuck you!\" Kadence shouted as she leapt to her feet.\n\nShe threw a hand over her mouth when she realized what she'd said, but his disdain had infuriated her into reacting before she could stop herself. She'd rarely heard swears from her father and brother. Occasionally, she'd said a few in private, testing out how they would feel on her tongue, but she'd never said one in front of others before. It was not appropriate for the women in the stronghold to curse, and she'd never heard another female utter one.\n\nShe had to admit it felt... _good_. Especially considering it was Killean she'd said it to. She wouldn't mind staking the guy.\n\nKillean snorted. \"I'd bet that's the first time you've ever said that going by your reaction. I was wrong, you'll be crying and calling your brother in five minutes.\"\n\nShe opened her mouth to tell him where to go with that, but Ronan spoke first. \"I won't have her death on my conscience. She'll stay.\"\n\nHer eyes swung to him, and for the first time in her life, she wanted to claw the eyes out of someone. \"I can take care of myself!\"\n\n\"It will be okay.\" Declan touched her arm as he offered her some reassurance.\n\nShe'd been so focused on the two complete asses in the room that she hadn't realized he'd approached her. She smiled hesitatingly at him, grateful for his reassurance. He was the only one who didn't seem to hate her for what she'd done.\n\nA low growl emanated through the room. Before she could comprehend where the sound had come from, Ronan stalked toward them. Declan's hand slipped away and he stepped hastily away from her.\n\nRonan inserted himself between them, his hand encircling her upper arm as he moved her away from his friend. She frowned at him as she tried to figure out where his strange aggression toward Declan had come from. Declan edged further away, his eyes twinkling with an amusement Kadence didn't understand.\n\n\"I'll have Marta and Baldric set up one of the rooms for her,\" Ronan said.\n\nDespite her continued annoyance with Ronan for denying her freedom, a twinge of disappointment went through her. She should be grateful they were going to be in separate rooms while she remained here. Grateful he was giving her at least some distance.\n\nShe didn't feel at all grateful though. She'd never admit it to him, but she'd enjoyed sleeping beside him.\n\nShe tugged on her arm to get him to release her. She had to put some distance between them if she was going to break free of this odd pull he had over her. He let her go.\n\n\"The three of you get ready to go out for the night,\" he said to the others. \"We'll go hunting as soon as our guest is situated.\"\n\nKadence scoffed. \"Let's not pretend I'm anything other than a prisoner here.\"\n\nHe shot her a dark look, which she returned. \"That was the choice you made when you pulled your little stunt earlier.\"\n\n# CHAPTER 20\n\nRonan led Kadence to the room Baldric and his wife, Marta, had readied for her. He cursed inwardly when he realized they'd prepared the room next to his. The covers on the bed were already pulled back for her. Marta had managed to find more women's clothes close to her size. They hung neatly in the wardrobe, the doors left open so Kadence would know they were there.\n\n\"I'm sure there are fresh towels and shampoo in the bathroom,\" he said to her.\n\nKadence walked around the room before stopping to examine the clothes inside the wardrobe. \"Whose are these?\"\n\nRonan shrugged. \"I don't know. I suppose various women may have left them here.\"\n\n\"You have that many women coming through here that you can't remember their names or what they wore?\" she demanded, unprepared for the flare of anger that realization brought with it. He was a vampire, he was old, he'd probably had countless women in his lifetime, but she did _not_ want to be wearing their clothes!\n\nRonan leaned against the doorway as he surveyed her. He thought he'd detected a hint of jealousy in her tone, but her face remained blank. \"I have had no women come through here, none of us have yet.\"\n\n\"Then where did the clothes come from?\"\n\n\"Left behind by the previous owners.\"\n\n\"Why would they leave their clothes behind?\"\n\n\"Because they left in a rush,\" he replied.\n\nKadence gazed at him and then the clothes in confusion. Some of the dresses and shirts were really pretty. It wasn't her style, she'd always worn simpler clothing, usually a plain skirt with a blouse, but sometimes she'd worn pants or a cotton dress. White, gray, or black were their options in the stronghold.\n\nShe had to admit, she was looking forward to trying on some of these more colorful clothes. Her fingers itched to pull the orange skirt and bright pink tank top out. It didn't matter they didn't match; they were so colorful!\n\n\"Why did they leave in such a hurry? What happened to them?\"\n\nRonan really didn't want to give her any answers, not after what she'd pulled earlier, but he found himself reluctant to leave her here.\n\nHe wasn't going to tell her that they traveled so often they had numerous bases of location, none of them as ostentatious or ugly as this one. Most were simple homes, some condos, a few cottages, and a couple of chateaus and wineries that were more investment properties. He'd spent so little time in any of those places that he could barely recall any of them.\n\nThe only problem was that Joseph knew about most of those places. It had been many years since they'd had a central base of location, but they spent most of their time on the East Coast as that was where the training facility was located. The training facility had never been the Defender's main home, but they had wanted to be near it to help train the recruits if they had the time, which they rarely did. With Joseph turning and staying near the area, this place had become their first central base of operations in nearly a century.\n\nRight now, Baldric and Marta were working on selling the rest of their numerous properties and purchasing new ones. If the new properties didn't come heavily secured, as this one had, they were having security systems installed in them. Ronan had instructed them to sell all of his personal properties too. Joseph didn't know where they were located, but it was time to make some changes.\n\nThe only property they'd considered retaining was the training compound, but that plan had changed. Before Lucien returned to the facility, Ronan had ordered him to get everyone prepared to leave it and asked Baldric to sell the old one and find a suitable new location. If Joseph was organizing the Savages, they couldn't take the risk he would be brave enough or strong enough to attack the training facility. There were a fair number of vampires who could fight there, but not enough to survive an onslaught of Savages.\n\nHe couldn't take the chance of losing their fighters now.\n\nHe hadn't told the others yet, but he was also contemplating bringing some of the turned vampire recruits into their group. The Defenders had been composed of purebreds ever since the creation of vampires, but these were precarious times and sticking with tradition may get them all killed.\n\nIt was a decision he was loathe to make, as he did not want to break with the old ways, but the world had changed vastly since the laws of the Defenders were first laid down.\n\n\"What happened to the previous owners of these clothes?\" Kadence asked again, drawing Ronan's attention back to her.\n\n\"Nothing happened to them. We simply made them an offer on this place they couldn't refuse, not even with all of their money.\"\n\nShe released the orange skirt and stepped away from the closet. \"I don't understand.\"\n\n\"We wanted this place, so we offered the previous owners far more than it was worth. Under the conditions that they vacate within two days and leave the furniture behind,\" he replied.\n\n\" _That's_ why all the walls are bare and none of the furniture or really anything here goes with any of your personalities. Why did you want this place so badly?\"\n\n\"It was heavily secured and Joseph doesn't know about it.\"\n\n\"I see,\" she murmured as she gazed at the bare walls. \"Did Baldric and Marta come with the place? And by the way, who is Marta?\"\n\n\"Marta is Baldric's wife; they've been married for almost thirty years. No, they did not come with the place. Baldric's family has been with mine for over two thousand years. When the family member who works with me passes, a new one takes over.\"\n\n\"So they're your slaves?\"\n\n\"Hardly,\" he retorted. \"It is their choice to stay and work. They are free to go at any time, as are their children. They know if they ever reveal anything, they will be slaughtered outright, but they also know few would believe them. I trust each one of them to uphold their family's history. When Marta and Baldric pass on or retire, their son Lamont has already chosen to take over for them. He has been out traveling the world, but he will join us when he's ready to learn about the estates and how things work.\"\n\n\"Why do they chose to stay and be your help?\" she asked.\n\n\"They are far more than my _help_ ,\" he grated. \"They are extremely well taken care of, as is their whole family, and they are as much my family as Killean, Lucien, Saxon, and Declan.\"\n\n\"I see,\" she murmured. \"Why has his family worked with yours for so long?\"\n\n\"The very first vampire king, my ancestor, saved Baldric's ancestor from an attack by a Savage. Instead of the king changing the man's memories, when the man asked to serve the king to repay the debt, the king agreed to it. It was only supposed to be that man, but his son stepped forward, and then the next, and so on. Over the years, many men and women have served the vampire kings who have risen and fallen. They have all had the option of moving on, but at least one member of their family has chosen to remain working with my bloodline until now they work for me.\"\n\n\"Amazing,\" she breathed. \"And your bloodline leads the vampires?\"\n\n\"That is the way it always was.\"\n\n\"So you are the vampire king?\"\n\n\"No.\"\n\n\"Why not?\"\n\nHe contemplated his answer as she stared at him curiously. He shouldn't be telling her anything after earlier, but he couldn't deny that it pleased him that she wanted to learn more about him, and none of what he would tell her could be used against them.\n\n\"At one time, there was a ruling vampire senate and a king. All turned and purebred vampires who weren't Savage were organized and ruled by that government. My father was the last king to rule. I was supposed to claim the crown when he died. However, the battle that took my parents also took most of the senate and the king's followers. After the conflict, there were few vampires left to rule over.\"\n\n\"How awful,\" she murmured. \"What happened to cause such a thing?\"\n\nHe preferred not to think about it, but he found himself having a difficult time denying her.\n\n\"It was a battle no one saw coming until it was too late. A Savage vamp, one who had been a member of the senate and a purebred vampire before giving in to the bloodlust, organized the other Savages. They prepared an attack against all those who hunted them. When they realized what was happening, the senate and my father gathered as many Defenders and other vampires as they could to counteract the growing threat, but it was already too late. The ensuing battle left the number of vampires decimated, especially the purebred ones.\"\n\nHe didn't reveal to her that those numbers had never recovered in the nearly thousand years since the battle. Few vampires reproduced, unless they were of the royal line and required an heir, and that practice had fallen away after the war with the Savages. Other vampires found their mates over the years and wanted to have children with them, but mates were a sporadic thing and sometimes even mated vamps didn't want offspring.\n\nTo this day, he only knew of a little over a hundred purebreds in existence. Their numbers were growing, but not fast enough. If there were other purebreds alive that he didn't know about, they weren't many. Ronan sighed and ran a hand through his hair as he thought back to the stark night that had forever altered his world.\n\n\"So much time has passed since then,\" he murmured, \"but I can clearly recall standing on that field and gazing out at the thousands of bodies surrounding me. Blood soaked the ground so much that the dirt no longer absorbed it, and it turned the green field into a lake of red.\"\n\nKadence realized he was stuck in the past, haunted by the memories when his unseeing gaze focused on the wall behind her. Sorrow swelled within her as she watched him. She didn't know how long ago it had been, but it was clear that battle had forged him into the man he was now, and it still haunted him.\n\n\"I found my parents amongst the carnage. My mother was decapitated, but my father had been torn to shreds, and bits and pieces of him were scattered across the earth. The stench of their blood stuck in my nostrils as I stepped over the bodies of the many vampires who raised me, yelled at me, trained me, and laughed with me.\"\n\nKadence's hand flew to her mouth as she stifled a cry over what he had witnessed and how much he had lost. He would not want her sadness, and she knew he would stop speaking if he believed she was giving him any kind of pity. And she didn't pity him; she admired him and understood him far better. He'd survived something horrific, yet he continued to fight every day for his kind. Her heart swelled with emotion. Despite her anger with him for bringing her back here, all she wanted was to hold him against her and try to soothe the lingering grief she sensed in him.\n\n\"Everything I'd ever known before then was slaughtered on that field, my life forever altered, my hatred of the Savages secured,\" Ronan recalled. Yet, he still teetered on becoming one of them no matter how much he despised them.\n\n\"Were you hurt?\" she asked when he stopped speaking.\n\nHe tore his eyes away from the wall to focus on her. \"My arm had been nearly severed by a broadsword and hung on by a thread of muscle. I had more cuts and stab wounds than I could ever count, but somehow, I survived. I left Ireland the following week, and I've never stayed longer than a week the few times I've returned to my homeland.\"\n\n\"And the Savages who attacked you, what became of them?\"\n\n\"They were all slaughtered in the war, but not before the number of vampires on both sides were wiped out to the point of near extinction.\n\n\"How many vampires survived?\" she asked.\n\n\"Myself and fifteen others. Some of the survivors were turned vamps, and some were purebreds, but only one other purebred was a Defender, and I was the only one with royal blood who survived, so most of our old ways were lost after the war.\"\n\n\"What happened to those survivors?\" Kadence inquired.\n\n\"Some turned Savage and had to be destroyed, others were taken out by hunters, and some were killed by other vampires. Before they died though, those survivors turned humans, or had children of their own to keep the vampire race from going extinct. I am the only one who remains of the original survivors.\"\n\nKadence's heart twisted and tears unwittingly sprang into her eyes. Her life in the stronghold had been lonely, but so had Ronan's. She couldn't imagine what it must be like to be the only one who remembered such an awful event, or the time before it occurred, when things had been far different for him.\n\n\"Who was the other Defender that survived?\" she asked.\n\n\"Declan's father, Aengus. He'd been my toughest and most demanding instructor while I was going through Defender training. After, he became one of my closest friends.\"\n\n\"What happened to him?\"\n\n\"He left Ireland with me and we worked together until he turned Savage about forty years after Declan was born. I killed him so that Declan wouldn't have to.\"\n\nKadence bit on her lip to keep from gasping at that revelation. Ronan's gaze was challenging as he stared at her, almost as if he dared her to condemn him for it.\n\n\"That must have been very difficult for you to do,\" she whispered, feeling like she'd uttered a _huge_ understatement.\n\nRonan clamped his teeth and gave a brisk nod as the memory of destroying Aengus played through his mind. He'd never thought it possible that Aengus would ever succumb to his darker nature. Ronan had believed him to be far stronger than that; believed Aengus to be a better man than him. He'd been wrong.\n\nKilling Aengus had been the most difficult thing he'd ever done. Aengus had been his last tie to a life all but forgotten by the other vampires, his closest advisor and friend, but his death had been necessary. The destruction Aengus wrought before his death had caused a panic amongst the humans, and Declan hadn't been physically strong enough to kill his father at the time, even if he had wanted to be the one to do it, which he hadn't.\n\n\"It was very difficult,\" he murmured.\n\n\"Declan doesn't hold any anger towards you for it.\"\n\n\"He understands it had to be done and that I didn't enjoy having to be the one who did it. Now, I have a feeling history might be trying to repeat itself and that the Savages are organizing,\" he said.\n\nHer mouth parted and her eyes widened as realization sank in. \"Joseph and those vamps who arrived to help him.\"\n\n\"Yes.\"\n\n\"Can you bring in more vampires to help fight him?\"\n\n\"I can and I will, but there are many who have remained scattered over the years. Many who don't even know their true heritage, or that there are Defenders amongst them, or that the ones who fall to the bloodlust are called Savages. As long as a vampire doesn't turn Savage, they have no reason to know we work to keep them safe from the worst of our kind.\"\n\nHe hadn't helped with the lack of knowledge most vampires possessed. Because they had become so scattered over the years, he hadn't bothered to regroup them or try to educate them. He simply hadn't seen a reason to inform the turned vampires he knew, and the purebreds who knew all their history and their ways were Defenders.\n\nHowever, those who did know of his existence looked to him as their leader. He was the eldest of their kind, the most powerful, and not just because of his age, but also his breeding and lineage. No other purebred such as himself had ever existed in the history of vampires.\n\n\"They know nothing of what you do, yet you still protect them,\" she said.\n\n\"And I will until the day I die.\" _Or have to be destroyed,_ but he kept that to himself.\n\n\"You are one of these Defenders?\"\n\n\"Yes.\"\n\n\"What does that mean?\"\n\n\"From the time I was old enough to learn how, I've been trained in fighting and war and how to protect my kind. Defenders are the best trained purebred warriors who uphold the vampire laws, and they track down the Savages in order to keep the innocents safe.\"\n\n\"So, you have sacrificed your freedom and happiness for the vampires, and they don't even know it,\" she whispered.\n\n\"I have sacrificed nothing; I have chosen my course in life. I may not be the king as my father was, but I will keep vampires and humans protected from the Savages. I don't have to be king to fulfill that duty.\"\n\n\"How old were you when the battle against the Savages happened?\"\n\n\"It was less than a year after I reached maturity, so twenty-eight.\"\n\n\"What do you mean by reached maturity?\"\n\n\"When a purebred stops aging and starts to really come into their vampire powers. I stopped aging later than most vampires do, but I was more powerful than many of them before then.\"\n\n\"I see,\" she said. \"How old are you now?\"\n\n\"I was born a vampire one thousand twenty-three years ago.\"\n\nKadence gawked at him. \"You're exactly a thousand years older than me!\"\n\n\"I feel every one of those thousand years,\" he replied.\n\n\"Amazing,\" she murmured. \"In all that time, you have never felt the sun on your skin without pain. Have you ever seen it?\"\n\nThe sadness in her tone tugged at his heart. He opened his mouth to tell her he could walk freely through the day, but stopped himself. She would most likely still be going back to her brother. \"I have seen the sun,\" he replied, unwilling to lie to her.\n\n\"Would you like me to tell you about it?\" she asked.\n\nHis heart leapt in his chest as he gazed at her. Despite the rod of steel going down her spine, she was achingly sweet and innocent. He had never imagined anyone could be like her. He didn't know what he'd done to deserve her throwing his life into a tailspin, but he was suddenly thankful she had.\n\n\"Maybe someday, but not tonight.\"\n\nShe crossed her hands before her, but he was not fooled by the demure gesture. There was nothing demure about this little hunter.\n\n\"It is only the five of you who fight?\" she inquired.\n\n\"No, there are others who are not Defenders who also fight the Savages. Some of them train with us to become better at it, and others do it on their own.\"\n\n\"Why has your family always led? Why were you supposed to lead?\"\n\n\"Like your bloodline amongst the hunters, my line is the strongest and I am also the only vampire still alive who can trace my line back to the original demon offspring. I am the first vampire in existence that is fifth generation purebred. Most are only first generation purebreds, some are two, and a couple are three, but that is all.\"\n\n\"I see,\" she murmured.\n\nKadence moved away from the wardrobe and walked over to the king-sized bed as she tried to process everything he told her. She didn't think that would ever be possible. At least now she understood better what drove him and why he could be so demanding and unbending. Now she also knew why he emanated such a vast amount of power.\n\n\"Would you let me come with you to hunt Joseph?\" she inquired.\n\n\"No.\"\n\n\"I have every right to be there.\"\n\nRonan's nostrils flared. She could see the outline of his fangs behind his lips as he watched her. \"You are a mortal. You have no training\u2014\"\n\n\"I was taught to defend myself in the stronghold, and I can expertly handle numerous weapons!\" she interrupted.\n\n\"Let me guess, you can kill a stuffed dummy with ease,\" he drawled. \"You have no training where it counts, in the field. The hunters failed you in that. You will be more of a hindrance out there than an asset, and I refuse to put my men in danger to satisfy your vendetta.\"\n\nKadence opened her mouth to protest, but he was right and she knew it. She'd left the stronghold thinking she had the training to fend off any vampire. Joseph had proved her completely wrong.\n\n\"And you also don't want me there because I'm a woman,\" she said bitterly.\n\nRonan snorted. \"I know women are just as capable as men when it comes to inflicting damage and death. This is because you are not prepared.\"\n\n\"Will you... will you teach me? Prepare me?\" she asked and held her breath as she waited for his rejection.\n\nRonan studied her as he tried to decide what to do. \"I will until we find a way to return you,\" he replied. \"I'd rather have you actually _able_ to protect yourself rather than just thinking you can. That will only get you killed faster.\"\n\n\"Gee thanks,\" she muttered.\n\n\"It's the truth. I think you'll find all you need for tonight. If you do need something else, Marta or Baldric will be happy to help you,\" he said as he turned toward the door.\n\n\"Do you have any books?\"\n\nHe stopped in the doorway. \"Books?\"\n\nShe smiled at him, an amused gleam in her azure eyes. \"Yes, you know, bound paper you read from. My sleeping habits are much like yours and I often stay up all night.\"\n\n\"There is a library downstairs. I will ask Marta to show it to you.\"\n\n\"Thank you.\"\n\n\"I'll send her up.\"\n\n\"Ronan.\" Her voice stopped him from leaving again. The light cascading over her lit her pale hair and emphasized the curves of her willowy frame. He found himself growing aroused as he gazed at her. \"Be careful tonight.\"\n\nThe words rocked him. No one had said those words to him before, and the fact it was her...\n\n_She's leaving,_ he reminded himself. His jaw locked as he stormed out the door. Stalking into his room, he slammed the door closed with so much force it shook the frame, but it did nothing to ease his frustration.\n\nChanging into a pair of black pants and a black shirt, he strapped on some stakes, a crossbow, and slipped two daggers into the holsters at his sides. He never wore so much weaponry while hunting, but after what he'd seen with Joseph and the Savages, he wasn't taking any chances.\n\nHe slipped his coat on, effectively covering the weapons before he left his room. Kadence's door was closed when he walked past it to the stairs, but her scent still floated to him.\n\nKillean and Saxon waited for him by the door. \"Where's Declan?\" he asked of them.\n\n\"Still in the poolroom the last I saw him,\" Saxon replied.\n\n\"Wait here.\"\n\nHe turned away from them and strode toward the poolroom. He found Declan leaning against the bar with a snifter of whiskey in hand. Declan rested his elbow on the bar while he gazed out the window. Ronan didn't know all of Declan's secrets, but he did know his friend had a special affinity for knowing or at least sensing things.\n\n\"Did you know she didn't intend to return home?\" Ronan demanded.\n\nDeclan didn't look at him as he responded with his usual self-assurance. \"I had my suspicions she was up to something.\"\n\nRonan grit his teeth. \"Why didn't you try to stop her, or warn me about what you suspected?\"\n\n\"It's not my place to interfere with someone else's life,\" Declan replied. \"Nor was it my place to clip her wings; everyone deserves a chance to fly.\"\n\n\"Declan\u2014\"\n\n\"It's too late now, Ronan. She's here. I thought you would be happy about it.\"\n\n\"What are you talking about?\" Ronan snarled. \"Why would I be happy a _hunter_ is still in our house?\"\n\nDeclan casually swirled the amber liquid in his glass. \"If she had told her brother a place to meet her, would you have let her go?\"\n\n\"Of course.\"\n\nDeclan gazed at him before taking a sip of his whiskey. \"You say that because she is here now, but we both know you're lying. You would have kept her.\"\n\nRonan was across the room between one heartbeat and the next. Lifting Declan by the throat, Ronan smashed his back against the bar and bent the larger man backward. \"I realize that you have your own way of doing things, but this is the first and the _last_ time you will go against me in anything, do you understand me?\"\n\nDeclan lifted his hands in a pacifying gesture. It was the first time Ronan had ever laid a hand on any of his friends in anger, yet Declan didn't seem surprised. \"Yes.\"\n\nRonan released him. Pacing away, he ran his hands through his hair as he tried to understand what had happened. He'd never believed he'd ever attack one of his friends, unless they turned Savage, but there had been a moment when he'd truly contemplated sinking his fangs into Declan's throat and feasting on him. A good kill would help to calm him.\n\n\"It's time to hunt,\" he said and turned away.\n\n\"Ronan.\" He stopped and looked over his shoulder at Declan. \"You may not believe me, but I did not go against you in this. One day I hope you will get the chance to see I did this for you. Killean, Lucien, and Saxon, they haven't been here long enough to know.\"\n\n\"To know what?\" Ronan demanded.\n\n\"That at one time your eyes were entirely brown. That they've changed over the centuries.\"\n\nIt was true. Ronan didn't just feel the madness creeping over him, he _saw_ it every time he looked in the mirror. \"What is your point, Declan?\" he bit out.\n\n\"We all fight our more malevolent side every day. Because of your lineage, you fight it more than the rest of us. I've watched you descend into the darkness more and more over the years, but for one brief second in the alley, when you first held Kadence against you, your eyes were entirely brown again.\"\n\n\"You're losing your mind.\"\n\nDeclan lifted a shoulder and finished off the rest of his whiskey. \"I think we all are, but I also think we're coming to a head with something.\"\n\n\"With what?\"\n\nDeclan set the glass on the bar, lifted his bomber jacket, and slid it on. \"That I do not know. Now, let's kill something. It will make us all feel better.\"\n\n# CHAPTER 21\n\nThe grandfather clock in the corner chimed 3:00 a.m. Kadence stared at the creeping hands, the book in her lap all but forgotten. She hadn't been able to concentrate on anything since Ronan and the others left. Setting the book on the small stand beside the chair she reclined in, she climbed to her feet. Pacing the large room, she scanned the thousands of volumes lining the floor-to-ceiling shelves of the two-story library.\n\nMarta had explained that Ronan and the others had brought the books with them from wherever they'd been before. The older woman had stayed with her for a good hour, pointing out her favorite books while she gave Kadence a tour. Marta exuded enthusiasm and warmth as she shared her love of the books and the vampires in residence. She hadn't left until Baldric came to retrieve her for help with something.\n\nNormally, being alone in a room such as this would be her idea of heaven. Reading was the way she spent most of her days. It was her only refuge from a life of boredom. Now, she found herself unable to concentrate on the endless spines lining the shelves.\n\nKadence pulled back the navy blue curtains covering the windows. She peered into the night, barely able to discern the bars on the outside. Releasing the curtain, she strode to the center of the room. She had traded one prison for another, but at least there were no expectations of perfection for her here, no one for her to marry. She may not be free to go outside or travel at will, but she still had more freedom than she'd had before.\n\nShe felt liberated. Yet, there was a constricting grip on her chest that she'd never experienced before.\n\nTurning away from the multitude of shelves, she left the library. Baldric was descending the staircase when she walked into the foyer. He smiled at her as he stepped off the last stair and onto the marble. His brown eyes were warm when they met hers.\n\n\"Can I get you anything, miss?\"\n\n\"No, I'm all set. Thank you.\"\n\nHe bowed his head to her and started walking toward where Marta had told her the kitchen was located. \"Let us know if you require anything. There is a buzzer in almost every room.\" He rested his hand beneath a small golden button on the wall and pointed to it. Kadence recalled seeing one in her room earlier too. \"It goes straight to the back hall where we can hear it even if we're sleeping.\"\n\n\"Thank you,\" she said though she suspected Ronan and his men would never bother them by hitting that button while Marta and Baldric slept, unless it was an absolute emergency, and neither would she.\n\n\"Good night, miss.\"\n\n\"Good night, Baldric.\"\n\nShe remained standing in the foyer after he'd vanished into the shadows beyond. She glanced back at the hall leading to the library as she debated returning there, going to her room, or wandering the large mansion and seeing what she could discover now that Marta and Baldric were going to sleep. She might be able to figure out how to get out of this place while no one was around.\n\nShe decided it was time to explore seconds before the front door flung open, letting in a rush of wind and fresh snow. Ronan strode into the house, his coat swirling around his calves. Declan, Saxon, and Killean followed him into the foyer. A large, ugly gash seeped blood down the side of Killean's face. Kadence let out a gasp of horror. Forgetting all about her dislike of Killean, she rushed toward him.\n\n\"What happened?\" Before she could reach him, Ronan grasped her outstretched wrist and pulled her back a step. \"I only want to help him!\" she protested as she uselessly tried to tug her arm from Ronan's grasp.\n\n\"Declan and Saxon will take care of him,\" Ronan replied.\n\n\"But I'm good at tending wounds,\" Kadence protested.\n\n\"Not this one.\"\n\n\"What happened?\" she demanded.\n\n\"Savages.\"\n\n\"Joseph?\" she managed to choke out.\n\nRonan's body vibrated with barely contained power as he stared at her. \"Joseph wasn't there.\"\n\n\"But he sent his friends,\" Saxon muttered.\n\nKadence bit her lower lip. \"Joseph sent them?\"\n\n\"I believe so,\" Ronan answered.\n\n\"Was my brother there?\"\n\n\"No.\"\n\nShe turned to look at Killean's brutalized face. The skin of his unscarred cheek hung down to reveal the fine white bone beneath, and blood continued to ooze from the injury. \"Please let me help him.\"\n\n\"No. It will heal on its own and he will be fine. I will not have you around this. You are going upstairs,\" Ronan replied.\n\n\"No, I'm\u2014\"\n\nHer protest was cut off when he tugged her toward the steps. Kadence glared at his back as he stalked up the stairs ahead of her, every muscle in his body rigid. \"What happened out there?\" she inquired as they arrived at the top and he pulled her forward.\n\n\"Nothing good,\" was the crisp reply.\n\n\"I _can_ help him.\"\n\n\"I won't have you near a vampire who has lost so much blood and is still geared for the kill.\"\n\n\"Oh,\" she said as realization dawned on her. These were not hunters returning from a battle. These vampires hadn't hurt her, but they were still lethal creatures with a lot of power and a thirst for blood. She shivered at the thought and hurried to walk beside Ronan.\n\nHe opened the door to her room and she stepped inside. \"I'm sorry for what happened tonight.\"\n\nHe took hold of her chin. \"Joseph is organizing the Savages, Kadence. After tonight, I am certain of it. It is not safe for you here.\"\n\n\"Then let me go.\"\n\nHis finger rubbed her chin, melting her bones from the inside out. Heat pooled through her body, causing her toes to curl. Like everyone else in her life, he'd taken her freedom from her, but she found she couldn't be mad at him when he was gazing at her like she was the most precious thing he'd ever seen.\n\n\"Where would you go?\" he asked.\n\n\"Everywhere,\" she breathed. \"I'd go to Machu Picchu and Venice and Rome. I'd stand on the Great Wall and try to puzzle out the mysteries of Stonehenge before kissing the Blarney Stone and drinking ale at a pub. I'd breathe in the air of history, soak in the arts, speak the languages I've learned, and immerse myself in the people and cultures. I'd _laugh_. I've done so little laughing in my life. I want to know what it is like to throw open my arms, laugh to the skies, and inhale freedom.\"\n\nRonan was stunned speechless by her impassioned words and the vision she created with them. He could well imagine her laughing and celebrating all those places. This young, vibrant woman had been caged so much she'd rarely laughed in her life, and now he was placing bars around her all over again. Now, he was the one denying her the life she deserved.\n\nWas he keeping her here because she had disobeyed him and he was concerned about her safety, or was it because Declan was right and he really didn't want to let her go?\n\nHe'd done some atrocious things in his lengthy life. Holding her captive here until she bent to his will was one of the worst of them. Self-hatred burned into him as he caressed her chin.\n\n\"All wonderful things,\" he murmured. \"So you would go Machu Picchu first then?\"\n\n\"Maybe the Grand Canyon or Niagara Falls, it doesn't matter as long as I see it _all_.\"\n\n\"And what about money?\"\n\n\"I would figure it out. I'm not stupid or as na\u00efve as you believe me to be. I know it will be difficult, but I _can_ do it. I'm not used to the human world, but I'm a hard worker and I am stronger than others. I'm sure I can find something to do while I travel.\"\n\nHe didn't remind her that she had no ID either. There were some times it was best to let things go. \"If anyone could do it, I think it would be you, Kadence.\"\n\nA beautiful smile lit her face. \"That's not what you were saying earlier.\"\n\n\"Even with my many years of experience, I am sometimes wrong.\"\n\nShe chuckled and gripped his hand on her chin. \"I imagine someone could live for all of time and still get things wrong. We were born to be flawed after all.\"\n\n\"We were. I can't let you go out into this world alone. You have no idea what it is like out there.\"\n\nHer crestfallen expression tore at his heart. \"I realize there is a lot I don't know, but no one has ever given me the chance to learn it either.\"\n\n\"I know they haven't. I wish I could take you to all of those places myself.\" He would give anything to see her face the first time she saw Ireland or the Colosseum, to hear her laughter as she looked on each new sight with delight. \"My duty is to my kind, especially now, and that means that I have to stay here, where the threat is located.\"\n\nHer delicate fingers tightened around his. \"I know.\"\n\n\"Baldric and Marta can take you.\" The words were out of his mouth before he realized he'd intended to say them. He fought against drawing her against his chest and holding her there as denial raced across his mind. He'd just found her and now he intended to send her away. No, he would set her _free_.\n\nThey may only be human, but Marta and Baldric were better trained at fighting vampires than Kadence was. They would keep her safe. They knew places to bring her that would be safe if they ever ran into trouble, and they knew vampires around the world who would help them if it became necessary.\n\nHe didn't want her out in the world without him, but that selfishness only made him a bigger monster than Joseph.\n\n\"I will give you money and they can go with you wherever you wish to go,\" he told her. \"With the condition and the understanding that you are to stay with them throughout your journey. I know it is not the complete freedom you seek, but they will help keep you safe, and they won't stand in the way of anything you do as long as they believe it's not too dangerous.\"\n\nHe'd make sure they didn't stand in her way if she found a man she loved and decided to settle down with him. His fangs ached in his gums at the thought. She was _his_. But she wasn't.\n\nKadence's heart hammered as hope bloomed in her chest. He was offering her dreams to her. \"Do you mean it?\"\n\nHe gave a brisk nod as the demon clawed at his chest, seeking to break free to claim her. It took everything he had to keep it securely locked away. \"Yes, but if you try to run away from them, they will bring you back. If you get away from them, or do anything to hurt them, I will hunt you to the ends of the earth, Kadence. Do you understand?\"\n\n\"I would never hurt them or try to escape them, I promise. Will they bring me back when I'm done exploring?\"\n\n\"That is up to you. It is your life to live now. They will only be there to help guide and protect you through your travels. If they feel you are safe to be on your own, and that is what you ask of them, they will also leave you, but _only_ if they feel you will be safe.\"\n\n\"You... you would do that? You would have them leave here with me? You would give me money and let me go?\"\n\n_No_! The denial rang throughout every part of him. \"Yes. It will take about a week to get everything organized and to get you a passport and ID that will make it through security everywhere in the world.\"\n\n\"What about Joseph? I left my home to witness his death.\"\n\n\"You stayed away from your home because you wanted your freedom. Now, you must decide what you want more; to possibly witness a death that will never fill the hole inside of you and may leave you feeling more hollow after, or to live out your dreams?\"\n\nKadence rocked back on her heels as she contemplated his words. \"I can always go after Joseph is dead.\"\n\n\"No. This is a now or never deal, Kadence.\" He may decide to keep her if she were here for more than a week. It would be difficult enough to let her go after spending that much time with her, never mind longer. \"Things may change in the future and I may be unable to offer this to you. I can't promise you that you will be there when Joseph dies either. Not with your lack of training.\"\n\n\"I see,\" she whispered.\n\nShe churned his words over in her mind. What would her dad want her to do? Easy enough to answer, he would want her back in the stronghold, but since that wasn't an option for her right now, she knew he wouldn't have her living her life for revenge.\n\nShe so badly wanted to see Joseph dead, but was witnessing his death worth giving up everything else for? Especially since she might not even get the chance to see it. Ronan would let her go travel, but he would keep her out of the fight until he believed her better prepared to be there.\n\n\"I... I will go,\" she managed to stammer out.\n\nRonan clasped his hands behind his back, nearly tearing the skin from them as he restrained himself from grabbing her. \"I will inform Marta and Baldric and have them set everything in motion.\"\n\nBefore he could change his mind, he turned and walked out of the room. His fangs extended further with every step he took away from her.\n\n# CHAPTER 22\n\nHer sleep plagued with dreams of Ronan, Kadence had risen earlier than normal and decided to explore the mansion. Maybe she should still be plotting her getaway. She felt in some way she was betraying her own kind by not doing so, but her kind had been wrong about so many things that she found herself not looking for a way out as she prowled through the rambling structure. It didn't matter anyway. After this week, she wouldn't be here anymore.\n\nShe should be overjoyed about that. She should have been kept awake with excitement, and not because she was fighting the urge to cry. She was finally going to be free; she was going to be able to do all the things she'd always dreamed about doing. Instead of being elated, all she wanted was to seek out Ronan and spend as much time with him as possible before leaving.\n\nShe kept waiting for someone to tell her she shouldn't be in this area or that one, but no one bothered her as she strolled through the place. She explored the numerous rooms on the first floor. Some of them were empty, but most had furniture to mark what they were. There was an elaborate dining room with a table that easily fit fifty, what she assumed was a parlor with its tables and chairs, another room with sofas and a fireplace that took up the entire back wall, and another with an old desk and floor-to-ceiling shelves behind it.\n\nShe only went down one wing before realizing it was mostly empty rooms. She had no idea why anyone needed a place so big, and she felt completely overwhelmed by the vastness of it while she made her way back to the main foyer.\n\nShe stopped when she spotted Ronan standing at the bottom of the steps, his elbow resting on the banister as he watched her. \"What do you think of the place?\" he inquired.\n\n\"It's huge.\"\n\n\"And?\"\n\n\"It's kind of depressing. I don't know if that's because it's so empty or because what they left behind feels like the remnants of ghosts instead of people.\"\n\nRonan gazed around the foyer as he tried to see it through her eyes. To him, it didn't matter. This place already had heavy security, was in a part of Massachusetts that was relatively peaceful, but Boston, Providence, Hartford, and New York City were all easily accessible by car.\n\nIt had been ideal for their needs. He hadn't cared what it looked like when he purchased it, never would have taken a second look at the interior or had the time to do anything about it, but seeing it through her eyes made him realize the place was bleak.\n\n\"You're right,\" he said and moved away from the stairs. \"Come with me.\"\n\n\"Where are we going?\"\n\n\"You wanted training, you're going to get it, or at least a crash course in it. Marta and Baldric will continue working with you while you're traveling. They're highly skilled fighters.\"\n\nKadence couldn't help but smile as she pictured the plump woman, with her kind smile, kicking a vampire's ass.\n\nTheir footsteps rang against the marble as she followed him to a door leading to a set of stairs. She'd opened the door earlier, but after looking down the shadowed staircase, she'd decided against exploring it.\n\nRonan flicked on a switch and descended the stairs before her. \"I've asked Marta and Baldric to make sure most of your time outdoors is during the daytime. They will also take you overseas first, anywhere you ask to go, but across the ocean.\"\n\n\"Why?\" she inquired.\n\n\"The more they kill, the more Savages have a difficult time of crossing bodies of water. The ones following Joseph will mostly be located on this continent, and it seems in this area. For now, I'd prefer to have you away from them. You can explore this continent once they've been destroyed.\"\n\nWhat if something happened to _him_ while she was gone? The idea caused a tug on her heart and a lump to form in her throat. He was ancient and powerful, but vampires could be killed. What about her brother? What if something happened to Nathan?\n\nShe tried to shake the worries off, but once they lodged in her mind, it was difficult for her to get rid of them.\n\nAt the end of the stairs, Ronan turned on another switch. Kadence's step faltered when numerous overhead lights flickered on to reveal the broad expanse of the gym. It was easily the size of the entire main floor of the house. The light shone off the weights, the stainless-steel doors at the other end of the room, and the entire back wall of weapons.\n\n\"Don't get any ideas about those weapons,\" he said to her over his shoulder as she gawked at them.\n\n\"Wouldn't dream of it,\" she replied, but her fingers itched to touch some of those knives and throwing stars.\n\nHe stepped onto one of the mats and turned to face her. He bounced on the balls of his feet as he backed a few feet away. \"So, young hunter, let's see what you've got.\"\n\nKadence glanced down at her jeans, baggy blouse, and socks. \"I'm not exactly dressed for sparring.\"\n\n\"And you most likely won't be dressed to fight if you're attacked on the street either.\"\n\n\"True,\" she admitted and stepped onto the mat across from him.\n\nHe held his hands up before her as she circled him, stretching her arms as she moved. She was about to stretch her calves when she found herself flat on her back, staring at the sparkly lights above her. Ronan leaned over her, blocking out the lights with his broad shoulders. She didn't know how he'd managed to knock her feet out from under her when she'd never seen him move, but he had.\n\n\"An attacker isn't going to give you time to stretch, Kadydid,\" he told her.\n\n\"It's a katydid.\" It was a ridiculous point to make, but it was better than glaring uselessly at him.\n\n\"Not anymore,\" he replied.\n\nThe sexy smile he gave her made her realize she didn't care what he called her when he smiled like that. He stretched his hand out to her. She sat up to clutch it and somehow found herself lying on her back, staring at the lights again. The exasperating man was faster than lightning.\n\n\"An attacker won't help you up either,\" he stated.\n\nHis infuriating chuckle had her launching to her feet and glowering at him. She adjusted her blouse and ended up on her ass again. She almost thumped her hands off the mat and yelled at him to stop it. She bit her tongue before she could speak. He may be pissing her off, but he was also exposing how woefully unprepared she was.\n\nShe pushed herself back to her feet and danced back before he could knock her down again. He circled around her, his body flowing with grace around the mat. \"You're spry,\" he told her. \"You'll never overpower a vampire, but you can outmaneuver one enough to get away.\"\n\n\"I want to kill, not run.\"\n\nHe stopped moving to face her. \"You're not strong enough for that. You need to know how to defend yourself enough to escape one, but you're not strong enough to face a vamp one on one and take them down. There's a reason hunters work in packs against us. An experienced hunter may be able to take down a turned vamp on their own, but you're not experienced.\"\n\n\"Okay, so teach me how to outmaneuver one.\"\n\nHe came at her then, a rush of muscle and male that robbed her of her breath far more than her exertion to keep away from him did. He worked her through a series of punches and kicks as he came at her again and again. She found herself staring at the lights more often than she was on her feet, but she was getting actual _experience_.\n\nShe had no idea what time it was when the sound of a throat clearing from the doorway drew both their eyes to where Declan lounged against the frame. \"What is it?\" Ronan inquired.\n\n\"Will you be hunting with us tonight?\" Declan asked.\n\nRonan glanced at the clock on the wall. \"Shit,\" he said when he realized it was almost eight. \"Yes, I'll be up shortly.\"\n\nDeclan disappeared from the doorway. Ronan shifted his attention to Kadence as she wiped a few strands of hair from her eyes. The blouse, damp with sweat, emphasized her breasts as it cleaved to them. He found his gaze locked onto them and the outline of the simple bra beneath.\n\n\"It was a good start,\" he said gruffly as his cock swelled with his need for her. \"We'll continue this tomorrow.\"\n\n\"I look forward to it.\"\n\nHe turned and started for the door before realizing she wasn't behind him. \"Are you coming?\"\n\n\"I'm going to train with some weights and weapons,\" she said. \"I think you've proven I have more work to do.\"\n\n\"Kadence\u2014\"\n\n\"It's not a bad thing,\" she broke in with a wave of her hand. \"I needed the wake-up call, and I _will_ get better. I won't be much longer, but I'm not ready to stop yet either. Go. I won't take any weapons with me. Besides, I'm sure you would know if I did.\"\n\n\"I would,\" he replied. \"Don't overdo it or you'll be useless tomorrow.\"\n\n\"I won't. Be careful out there.\"\n\nThis time, he found himself pleased by her concern instead of angered by it. \"I will,\" he vowed.\n\n***\n\nOver the following days, she continued to find herself still staring at the ceiling more often than she remained on her feet, but she was getting better. Already she had more muscle definition in her arms and legs, and she was faster. She'd even managed to get in a couple of blows against Ronan before staring at the pretty lights again.\n\nHer endurance was also better. Though, every night she still crawled into a hot bath to ease the soreness in her muscles and stayed there until she was more pruned than a raisin. Then, she would go down to the library and read until Ronan and the others returned. Her eyelids were always drooping by then, but she couldn't sleep until she knew he was safe.\n\nTonight, while waiting for him to come down to work with her, Kadence wandered into the poolroom. She was examining the chess table near one of the windows when he found her. The table had been carved from the trunk of a tree and appeared to be at least a couple hundred years old. Judging by the grain of the wood, each of the chess pieces had also been carved from the same kind of tree and polished until they shone.\n\n\"Do you play chess?\" he asked.\n\n\"I have a basic knowledge of the game,\" she replied. \"Enough so that I would be able to play with my husband, if he enjoyed the game.\"\n\nHe strode into the room, his eyes running over her lithe figure in the black yoga pants and form-fitting, pink tank top. He'd asked Marta to purchase some workout clothes for her, but he'd expected sweatpants and sweatshirts, not this temptation. He'd have kept her in jeans had he known Marta was going to torment him with this outfit.\n\n\"And what if you enjoyed the game and your husband didn't?\" he asked.\n\nShe shrugged and glanced back at the pieces. \"Then I probably wouldn't have played again, at least not often.\"\n\n\"Ridiculous,\" he muttered.\n\n\"It's the way of things with us,\" she replied. \"I was educated enough to converse with my husband without boring him.\"\n\n\"And what if he bored you?\" The radiant smile that lit her face struck straight to his heart.\n\n\"I wondered the same thing recently!\" she exclaimed.\n\nRonan had to take a minute to gather himself as she continued to beam at him. \"Well, it is not something you will have to be concerned about anymore.\"\n\n\"No, it's not,\" she agreed.\n\n\"Would you like to play now?\" he asked.\n\nShe glanced back at the table and then him. He saw the longing on her face before she shook her head. \"No, I should train more.\"\n\n\"You've done well. You could use a break.\"\n\n\"Maybe tomorrow night,\" she replied. \"After you left last night, I mastered throwing the shuriken and want to show you.\"\n\n\"Mastered it, did you?\" he inquired as they made their way out of the room and to the basement door.\n\n\"Okay, maybe not mastered, but I hit the target multiple times.\"\n\n\"Good,\" he said as he opened the basement door and followed her down to the gym. \"They're easily concealed and will allow you to impair your attacker without getting close. It may not kill them, but it will slow them down and might make them rethink coming after you.\"\n\n\"I like that,\" she said as she walked across the room to the weapons.\n\nStepping back, Ronan admired her taut ass in those pants. He was going to spend the next couple of hours trying to conceal his erection from her, but it would be completely worth it. She stretched to grab some shurikens from the wall, and he almost groaned aloud when the action exposed a patch of the creamy skin on her lower back.\n\nShe came back toward him, her breasts swaying with each step as she took up a position across from the dummy he'd set up for her to work with. Unlike the dummies the hunters had given her to train with, this one was hooked to a rope and could be jerked around by hand or by a machine. She pushed a button and the machine came to life, jerking the dummy one way and then another.\n\nThe weapons sliced through the air as she released them with deadly speed and accuracy. She may not be able to take on a vampire by herself, but she was extremely talented, and he couldn't deny that she was faster than the hunters he'd come across, except for maybe her brother. She was also stronger than they were and growing more so every day.\n\nWhen she was done, she'd hit the target with all six of the shurikens and bounced on her toes as she spun to face him. Drawn toward her, he stalked across the mat and hit the button on the machine, shutting it down.\n\n\"Much better,\" he said, his gaze dipping to her red lips before he tore his eyes away. \"Now let's try some live training with the shurikens.\"\n\nKadence stopped bouncing and frowned at him. \"What do you mean?\"\n\nHe didn't respond as he crossed over and pulled the shurikens free of the dummy. He carried them back to her and handed them over. \"See if you can hit me with them.\"\n\n\"No,\" she said and tried to shove them back at him.\n\n\"Yes.\" He folded her fingers around them before walking away from her. \"Remember, if you hit me, it won't kill me.\"\n\n\"I'm not throwing these at you.\"\n\n\"You have to know how to react against a vampire. Your hunter blood is exceptionally strong, but that doesn't matter if you're not properly trained. You can do this, and if you don't, I'm going to spend the next couple of hours knocking you on your ass until you can't sit for a week.\"\n\n\"I've spent plenty of time on my ass these past days,\" she retorted.\n\n\"I've been taking it easy on you.\"\n\nKadence gawked at him as he turned to face her, the cocky look on his face both endearing and maddening.\n\n\"Don't be afraid, Kadydid. I won't hurt you.\"\n\nShe scoffed at his words, but before she could formulate a reply, he was coming at her in a blur of speed and muscle.\n\n# CHAPTER 23\n\nKadence had gotten better at being able to discern where he was as they worked together, and her eyes adjusted to his fluid movements. Reacting on instinct, she released the shurikens in rapid fire, purposely aiming not to hit him, but making a point. When she released the last one, she darted to the side. She didn't move fast enough to avoid him though.\n\nWrapping his arm around her waist, he lifted her off her feet before taking her down to the mat. Though he'd knocked her on her ass more times than she could recall recently, he'd never gone to the ground with her. Now, she found herself staring into his burgundy eyes when he planted his fists beside her head and his thighs between hers as he knelt over her. They didn't touch, but the heat of him melted her body as if they were flesh to flesh.\n\n\"You got a little close on that last one,\" he said with a glance at the blood beading from the scratch on his corded bicep.\n\nGlancing down his body, she noted the slices in his black shirt and the one in his baggy pants. She could have hit him with each one she'd thrown, something she'd made sure he knew.\n\n\"Maybe I didn't get close enough with the others,\" she replied.\n\n\"Is that so?\" he inquired and lowered himself so that his chest barely touched her.\n\nKadence sucked in her breath, but didn't release it as his eyes dropped to her mouth and they heated with desire. All the nerve endings in her body screamed for him to touch her, to ease this need he so easily brought to life in her.\n\nJust when she thought she might pass out from lack of oxygen, he bent his head until their lips brushed against each other. He seemed to be waging a silent battle with himself as he remained unmoving above her. Then, he let out a low groan and took possession of her mouth. Her heart knocked into her ribcage as his tongue slid over her lips before she opened her mouth to his.\n\nThe scent and feel of him filled all her senses. Her legs opened further to him, and he settled himself between her thighs. He growled against her lips and thrust his hips forward. The rigid evidence of his arousal rubbing against her made it impossible to think as sensation took her over completely. The delicious sensations his body evoked in hers made it impossible to catch her breath as his tongue drove into her mouth in the same rhythm of his hips moving against her.\n\nRonan stroked his hand over her face before running it down her shoulder and lower as her hips rose to meet his. He had no idea how any of this had happened, but he lost himself to the wonder of her body as he cupped and kneaded her breast.\n\nFor nearly a week, he'd kept himself restrained from touching her in an intimate way. He hadn't planned for anything to change tonight, but he'd been unable to stop himself from bringing her down beneath him, and then there had been no denying him after.\n\nHer back arched to press her breast more firmly into his palm. His hand twisted in her shirt to tear it away from her. He wanted to shred her clothing, bare her to him, and take her with all the savagery of the demon within him.\n\n_She's an innocent!_ The reminder made him realize that, if he took her the way he planned, he would hurt her, badly. He would ruin her, but worst of all, she would hate him for it.\n\nRonan launched himself off her so fast, Kadence didn't realize he wasn't there until cold air flowed in to chill her overheated skin. She blinked at the empty space above her before turning to find him stalking across the room. His body was so rigid she thought he might break apart with the way he carried himself.\n\nKadence rolled over as he turned back to face her from fifty feet away.\n\n\"I am sorry,\" he said in a clipped tone she barely recognized.\n\n\"For what?\" she inquired as she pushed herself to her feet. When she stepped toward him, he took one back like she was a magnet repelling him.\n\n\"I did not mean for that to happen. If I hurt you\u2014\"\n\n\"You didn't.\"\n\n\"Frightened you then.\"\n\n\"You didn't,\" she said. \"I can handle myself, Ronan.\"\n\n\"You think you could stop me from hurting you?\"\n\n\"I wouldn't have to.\"\n\n\"You have no idea what I'm capable of.\"\n\n\"Yes, I do. It's _you_ who doesn't know.\"\n\nRonan couldn't stop his mouth from dropping at her words. In all his many years, no one had shocked him as much as she just had. \"I am well aware of what I am capable of doing. I have witnessed the blood staining my hands for over a thousand years.\"\n\n\"Yes, you are aware of your capability for violence, but are you also aware that you are capable of kindness and being gentle?\"\n\n\"You have no idea what I want to do to you right now, and there is nothing gentle about it!\"\n\n\"You could have done it, but you didn't.\"\n\n\"I would ruin you, do you understand that, Kadence? What I would do to you would ruin you for any other man.\"\n\n\"I don't want any other man. I only want you.\"\n\nNope, he'd been wrong, she'd just managed to shock him even more. Before he could respond to her, footsteps sounded behind him. He turned to find Declan standing in the doorway ten feet behind him.\n\n\"What is it?\" he demanded.\n\n\"It is time to hunt,\" Declan replied. \"Marta also asked me to inform you that everything is ready for them to leave tomorrow.\"\n\nThose final words hit Ronan like a punch between his eyes. \"Good,\" he somehow managed to say. He glanced at Kadence over his shoulder. \"Pack what you will take with you. Marta and Baldric will make sure to buy you whatever else you require on your travels. If you have decided where you would like to go first, let Marta know. It will mean less time for you at the airport.\"\n\n\"I have decided, and I will let her know,\" she murmured.\n\nHe followed Declan into the stairwell. \"Ronan, be careful.\"\n\nHe smashed his fist into the wall before storming up the stairs behind Declan. Tomorrow, he would have no one to tell him to be careful again. Tomorrow she would be gone. He should be relieved; instead, he knew he would make the death of any Savage they came across tonight exceptionally brutal.\n\n***\n\nKadence stirred when she felt arms slipping beneath her. Warmth enveloped her as she was braced against a solid chest and something was removed from her hands. Her eyes blinked open as Ronan set the book she'd been holding on the chair he'd lifted her from.\n\nShe hadn't meant to fall asleep while waiting for him, but after he'd left she'd worked out for another two hours in the hopes of burying the sadness that leaving him brought to her. Then she'd packed her things and spent the rest of the night pacing the library before trying to read. Her emotional and physical exhaustion had caught up with her.\n\n\"What time is it?\"\n\n\"Almost three,\" he replied.\n\nShe took in the wet hair falling across his forehead and curling at the corners of his eyes. \"You showered.\"\n\n\"Long night,\" he replied.\n\nHe refused to tell her that he'd been covered in blood when he'd returned twenty minutes ago. He'd been unwilling to see her like that, been determined not to see her at all before she left, but when he realized she wasn't in her room, he couldn't resist following the lure of her scent here.\n\n\"Is everyone okay?\" she asked as she came more awake.\n\n\"Yes.\"\n\nHe carried her from the room and down the hall to the stairs. She nestled closer to him, savoring the scent of the spiced soap on his body. Unable to resist, she rested her fingers against his chest and sighed when she felt the powerful flex of his muscles with every step he took. She slid her fingers down his flesh. The yearning to taste him grew until she turned her mouth into his chest and slid her tongue out to lick his salty skin.\n\nRonan's fangs tingled as they lengthened, and he found himself drawing her closer as her tongue slid over him again. He shifted his hold on her to grip the knob of her door and pushed it open.\n\nShe withdrew from him as he set her on her feet. Before, she would have blushed over her actions with him; now she couldn't find it in herself to be embarrassed, not if this was to be their last night together for a while, possibly forever. Resting her hand on his cheek, she stepped into him.\n\nRonan told himself to back away from her, but his feet were planted to the floor as her breasts brushed against his chest and her eyes searched his. He knew he had to look half wild with his need for her, yet she showed no fear as she rose onto her toes.\n\nHer gaze never left his when she kissed him. Her breath warmed his mouth as they stared at each other and Ronan resisted drawing her closer. If he did, if this continued, there would be no stopping, not this time.\n\n# CHAPTER 24\n\nThe heightened scent of her arousal filled his nostrils and permeated his body. He was taken off guard by the intensity of her longing for him as he remained unmoving against her. Her lashes fell to shadow her eyes, and she broke the kiss.\n\nAs she moved to pull away, his arm lashed out and drew her to him. Her eyes deepened in color when they met his again. He caressed her cheek before his hand slid back to entwine in her silken hair.\n\nHis eyes were drawn to her mouth as he waited for her to tell him no. Waited for her to pull away. She did neither of those things; instead, she leaned into him, her breath coming faster as her fingers dug into his chest.\n\nHe knew he shouldn't do this, knew it would only make it more difficult to let her go. She would fly free from here and the brutal life she'd been born into. He would remain and continue to fight until he died or succumbed to the lure of becoming a Savage.\n\nBut none of that mattered. All he wanted now was to be inside of her, moving over her, listening to her sensual moans as he claimed her for this brief period of time.\n\nWhen she kissed him again, he pulled her so firmly against him that no air flowed between their bodies. She belonged with _him_ and him alone.\n\nSomething niggled at the back of his mind, an intuition that he should acknowledge, but couldn't, as everything in him became focused on her alone.\n\nHe pulled her hair back, deepening the kiss and catching her sigh when her mouth parted and her tongue entwined with his. Lifting his mouth from hers, he ran his tongue along the slender column of her throat before he nipped at her supple flesh.\n\nHis fangs ached to pierce her skin, to taste the blood pulsing through her vein and calling to him like a siren on the rocks, but he resisted the impulse. It could drive her away if he took from her now.\n\nHer knees buckled when his other hand slid up to cup her breast. He supported her weight as she cried out and her head fell back to allow him better access to her throat. Lifting her, he swung her into his arms, kicked the door shut behind them, and carried her further into the room.\n\nStopping beside the bed, he set her on her feet and stepped back to drink in the sight of her. She stared up at him, her lips swollen, her eyes heavy lidded and seductive. She was the most beautiful thing he'd ever seen.\n\nA trickle of unease slid through her as Ronan's eyes ran ravenously over her body. His raw need for her was evident on his face and in the heavy bulge behind his pants. No matter what he believed about himself, if she asked him to stop this now, he would walk away from her, but despite her apprehension over what she knew was to come, she didn't want to stop it.\n\nAt one time, she'd believed him to be a monster simply because of what he was. Now he was the only man who had ever made her feel like she truly mattered in this world because of who she was, and not because she might be able to have children one day. He was the only man she had ever wanted to share this experience with.\n\nHe was setting her free today, and before she left, she would have the memory of this night to hold close while she was gone.\n\nSwallowing heavily, she pushed aside her fear as he remained where he was, giving her a chance to change her mind, she knew. The elder women had prepared her for her wedding night, told her what to expect and what would happen. They hadn't prepared her for her overwhelming desire for him too.\n\nHis eyes were almost entirely red when she met them. The outline of his fangs pressed against his lips, but the obvious signs of what he was didn't scare her. \"I want this. I want _you_.\"\n\nRonan released an explosive exhale as he gripped the hem of her shirt. He pulled it slowly up, giving her the time to change her mind as he revealed more of supple flesh and curves. Her eyes never left his as she lifted her arms for him. He pulled the shirt over her head and tossed it aside. The black sports bar she wore kept her handful-sized breasts concealed, but there was something incredibly sexy about this tomboyish look on her.\n\nHooking his fingers into the band of her sweatpants, he pulled her a step closer until their chests were flush against each other. He tugged her sweats and underwear lower over her rounded hips. He kept waiting for her to tell him to stop, but all he saw in her gaze was yearning as she watched every move he made.\n\nWhen he'd been in the gym with her earlier, all he could think of was tearing the clothes from her and burying himself inside her before she could stop him. Now, when he knew he would have her, all he wanted was to savor every inch of her. He bent to slide the sweats over her supple thighs and down her shapely calves. She stepped out of them as he rose to stand before her again.\n\nHe grasped the edges of her bra and slid it over her head. Her silvery hair cascaded around her shoulders when her arms settled at her sides. Her luscious curves and the small triangle of darker blonde hair shielding her sex from his view caused his mouth to water.\n\nStepping closer, he gritted his teeth against the throbbing need of his cock while he leisurely slid the backs of his knuckles up her sides. She bit on her lower lip as she raptly watched him, and he memorized her every reaction to his touch. His gaze latched onto her pert breasts with their pink nipples standing out tantalizingly, but he didn't touch them, not yet. Instead, he continued to explore her as he ran his hands over her flat stomach and round hips.\n\nSmelling her desire on the air, he knew she was most likely wet for him already, and he wanted to feel that wetness slipping over his fingers. However, if he slid his fingers between her legs now, he knew he would be lost to her, and he wanted to take this as slow as possible. Her breath hitched in when his hands cupped her firm ass.\n\n\"You're exquisite,\" he murmured.\n\nA spark lit her eyes when her gaze ran hungrily over him. \"You're not so bad yourself,\" she said as her hands gliding over his chest caused his muscles to contract beneath her gentle ministrations.\n\nNo one had ever touched him with such reverence before. When he'd still cared enough to have it, sex had always been enjoyable enough for him, but it had always been a scratching of an itch with a willing woman who had understood that was all he wanted from her. It had also been all she wanted from him too.\n\nHowever, Kadence touched him as if she cared for him.\n\nHe stiffened as that realization sank in. She cared for him when none of his other lovers ever had, and he couldn't deny that she'd become special to him too. Dipping his head, he nuzzled her ear with his lips as his hands slid up the slender curve of her spine. \"If we do this, Kadence, there will be no turning back.\"\n\n\"I don't want to turn back, ever.\"\n\n\"You could regret\u2014\"\n\n\"No. Even if for some reason I decide to return home, I will _never_ regret this night, or you.\"\n\nKadence held her breath as Ronan shifted so his forehead was against hers and his reddened eyes burned into hers. Then his mouth took hers in a kiss that burned all the way to the tips of her toes.\n\nHis hands fell to her waist, and he lifted her easily. Taking two steps forward, he placed her on the bed and stepped back to admire her. She lay before him, her skin flushed with passion and her lips swollen from his kiss. Unbuttoning his pants, he hastily pulled them off and tossed them aside.\n\nKadence's eyes widened as he revealed himself completely to her. A fresh trickle of alarm slid through her as she gazed at the erection jutting proudly out from his body. The elder women had told her what to expect, but they hadn't prepared her for how _big_ he would be. It stood a good eight inches away from him and was so thick she knew she wouldn't be able to wrap her hand completely around it.\n\nSwallowing heavily, she pushed aside her trepidation over what was to come. It would hurt, but this was Ronan, and she would deal with the pain if it meant easing this incessant ache for him.\n\nHe climbed onto the bed with her and took her into his arms to cradle her against him. The warmth and strength of his body enveloping hers eased her fear. She clasped his back when he reclaimed her mouth and moaned when her breasts rubbed against his chest. The movement sent shivers over her body. He cupped one of her breasts in his palm, running his thumb over her nipple before rolling it between his fingers.\n\nReleasing her breast, he skimmed his hand over her hip and down her thigh. \"Open your legs for me,\" he whispered against her mouth.\n\nUnable to resist, Kadence spread her legs at his command. Small electrical currents coursed through her body as his hand slid between her thighs. His fingers stroking her caused her belly to clench with need.\n\nRonan rubbed his thumb against her clit, relishing in her increased breaths as her body reacted to his touch. Already wet with her want for him, he spread that wetness over her before dipping his finger into her. Her inner heat enveloped him as her tight muscles gripped his finger and he began to move it within her.\n\nKadence's mind spun as his finger and thumb stroked her. Her hips instinctively rose and fell in rhythm to his movement within her. His finger was _inside her_ and doing such wonderfully decadent things to her body that she could barely think straight. It was all happening so fast that she thought it should be at least a little overwhelming, but all she wanted was to experience more of him.\n\nShe ran her hand over the muscles of his chest before dipping down to explore the ridges of his abdomen. She couldn't get enough of him as she traced the trail of hair running from his bellybutton to his erection. Unable to resist, she ran a finger over the taut skin of his shaft before touching the bead of liquid forming on the head of it.\n\nRonan grasped her hand when she ran her fingers tentatively over his head once more. Holding her hand within his grasp, he turned it and wrapped it around his cock. He worked her hand up and then back down the length of him.\n\n\"Like this,\" he whispered against her mouth.\n\nKadence almost jerked away when his shaft jumped in her hand as he guided her up and down him. Then, he groaned and his head tipped back until the muscles in his neck stood out. His reaction to her touch fascinated her as did the heat of him against her palm. She forgot all about the delicious sensations he evoked in her as she concentrated on the pleasure she gave him.\n\nThen, he began to move his finger in a completely different way within her and his palm rubbed against her. She ground against his hand as he pushed her closer and closer to the brink of something far beyond anything she'd experienced those times when she'd woken after a dream and felt unfulfilled. During those times, she had touched herself in an attempt to ease what those dreams had awoken in her, but her touch and those dreams were nothing compared to the passion Ronan evoked within her.\n\n\"Ronan,\" she groaned when he slid his hand away from her.\n\n\"Easy,\" he whispered in her ear and eased her hand away from his cock. \"I want to be in you the first time a man makes you come.\"\n\nShe wasn't entirely sure what his words meant, but she was impatient to find out. He moved over her, his massive body filling her field of vision and all her senses, as he nudged her legs apart with his thigh.\n\nThe corded muscles of his arms stood out as he held himself above her and he no longer tried to keep his lethal fangs concealed. She lifted her hips invitingly to him, needing him to quench the fire he'd created within her.\n\nLeaning down, he took hold of her mouth again and delved his tongue into her. He grasped her hips, holding her as he positioned the head of his dick against her inviting folds and began to inch forward. Gritting his jaw, it took all his restraint not to plunge into her as her wet heat enveloped him more and more. He stopped when he felt the barrier of her virginity. Releasing her hips, he lifted his lips from hers to take in the vision of her beneath him.\n\n\"This is going to hurt,\" he warned.\n\n\"I know,\" she murmured as she ran her fingers over his high cheekbones before slipping them around his neck. \"The elder women told me what to expect.\"\n\nKadence's breath caught when, for the briefest of seconds, she stared into a pair of eyes the same color as a deer's. She'd seen hints of this rich, deep brown in his eyes before, but never this clearly. She knew she was being gifted a glimpse of Ronan while at peace and not the brutal, unyielding warrior she'd come to know. As fast as it had come, the brown faded away to become burgundy once more.\n\nShe had no time to ponder what had caused the change before he thrust forward to penetrate deep into her. Kadence cried out, her nails dug into his nape and her thighs clamped against his sides.\n\n\"I'm sorry,\" he whispered, his hands caressing her face as he kissed her again. \"The pain will ease.\"\n\nShe nodded as he ran his lips across her collarbone before dipping lower to run his tongue over her nipple. He remained unmoving within her as he caressed her flesh, slowly evoking her desire for him again. He drew her nipple into his mouth and sucked on it as he ran his tongue around it once more.\n\nThe coiling tension built within her once more as he turned his attention to her other breast. The sight of his dark head bent over her, the heat of his mouth, and the scrape of his fangs became an overwhelming sensation she almost couldn't take, but she didn't want it to stop.\n\nInstinctively, she lifted her hips to take him deeper into her as her fingers entwined in his hair and she drew him closer to her breast. He growled against her flesh as she lowered her hips and gasped when a wave of bliss speared through her.\n\nRonan kept himself still as he waited for her to adjust to the feel of him inside her and to explore him in her own way and own time. It had been years since he'd last had sex, yet he didn't feel out of control with need. A calm had descended over him the second she gave herself to him, a tranquility he'd never expected to experience in his life.\n\nShe lifted her hips again, and he groaned as she fell and rose on his cock once more, drawing him deeper into her. He had wanted to take his time, but when she wrapped her legs around his back and arched against him, he knew he was lost. He couldn't stop himself from moving faster, harder, deeper until all he could feel was her pliant body against his and her tight muscles squeezing his cock.\n\nKadence's hands gripped his hard ass as she held him closer to her. \"Please,\" she panted as the tension coiling higher in her body felt ready to unravel.\n\nShe was close to orgasm; he could sense it in her rapid pulse and the demanding movements of her body beneath his. \"Please what?\" he asked as he nipped at her ear.\n\n\"I... I don't know.\"\n\nHe would have laughed at her words, if they hadn't unleashed something primitive within him, something he'd never experienced before. She was _his_ , and only his. She may not know what she wanted, but he did, and he would give her the answer. He would be the only one to _ever_ ease her in such a way.\n\nDriving deeper into her, he reached between them to rub his finger against her clit until she was writhing and her body was moving wantonly beneath his. Her head fell back as she arched off the bed.\n\n\"Ronan!\"\n\nThe cry erupted from her as waves of pleasure crashed through her, causing her whole body to shake. She could only cling to him as she completely unraveled. She had never felt anything like this, never thought to experience such bliss.\n\nWith a shout, he plunged into her a final time and the clenching muscles of her sheath milked his release from him. Tremors shook him as he continued to come inside her in a seemingly endless stream. Finally spent, his head fell to her shoulder and he remained buried within her as he rolled to the side with her trapped in his arms.\n\nHe savored the astonishing feelings she evoked in him. Most of them were feelings he was experiencing for the first time. He'd never felt so protective of another before, never wanted to cherish and hold someone as close as he held her.\n\nHe inhaled her sweet scent, pleased to realize he could smell himself all over her too. Others would smell him on her skin and within her as well. Her hands released their death grip on his shoulders, and she trailed her fingers leisurely up his back.\n\nHe inhaled again, drawing her essence deeper into him. Then, the scent changed. In an instant, all he could smell was her sweet blood pounding through her veins. And he wanted to have _his_ blood coursing as strongly within her as her own. He also wanted to feel her flowing through him to form an unbreakable connection to her.\n\nWithout warning, the demon part of him rushed to the forefront.\n\n# CHAPTER 25\n\nHis fangs lengthened against her skin as his instincts screamed at him to drain her blood and replace it with his own. To make it so they would be bound for eternity and she would never be able to walk away from here or return to her family. His fangs grazed her neck. The vein pulsing beneath her flesh was right there; he could pierce it before she ever knew what he intended.\n\nHe'd have her drained to the point of death within a minute and his own blood filling her in the next minute.\n\n_She'll hate you for an eternity if you cage her against her will and in such a way!_ A small sliver of reason pierced through his bloodlust.\n\nPulling himself out of her, he flung himself back so fast that he nearly toppled off the bed.\n\n\"Ronan, are you okay?\"\n\nHer hand rubbed his back. His control unraveled further at her touch. \"Don't!\" he snarled.\n\nKadence recoiled as if she'd been slapped. She had no idea what was wrong with him, but she suspected it had something to do with what had just transpired between them. Had she done something wrong?\n\nWell, if she had, then it was _his_ fault, she decided. He had a lot more experience in this than she did after all.\n\nWhat if they had both done something wrong? He may have a lot more experience in this area than her, but she could guarantee he'd never been with a hunter before. Maybe their kinds were never supposed to come together in such a way and now something was happening to him because of it.\n\nShe pulled the blanket up against her chest as he launched to his feet and stalked away from her. He didn't look at her as he prowled the room.\n\n\"Did we do something wrong?\" she asked. \"Is this because I'm a hunter and you're a vampire?\"\n\nHe gave a bitter laugh as realization settled over him and he lifted his head to find her blue eyes locked on him. Something had gone completely off the tracks wrong, but not because of what she was. It was because of _who_ she was to him. He was a train wreck waiting to happen because she was not simply a woman he'd come to care for, not a hunter, but his _mate_.\n\n_Fuck_! He tugged at his hair, trying to keep himself calm as the realization rocked him. How had he not seen it earlier?\n\nThen, Declan's words replayed in his head, _\"You say that because she is here now, but we both know you're lying._ _You would have kept her.\"_\n\nWouldn't he have let her go? He'd been determined to do so; he still was. It didn't matter she was his mate, didn't matter he'd accelerated the need to complete the mating bond by taking her, he would not chain her as she'd been chained her whole life, and he would _not_ chain her to his world. The danger of her life as a hunter was a day at the circus compared to what came with his life.\n\nThe constant fights and death that made up every day of his existence. The treacherous edge he walked. _She_ would be the first one he killed if he turned Savage. Or she could calm the demon within him and make it so he never again walked the line between the darkness and the light. It was what mates did for a vampire after all, especially a purebred vampire.\n\nBut no other vampire like him had ever existed before. It could be possible he was so completely bound to the demon part of him that she wouldn't calm him, that nothing would change within him, and he would be pushed over the edge by that realization.\n\n_Who are you trying to convince of that?_ He wondered, knowing it was a lie even as he thought it. She'd already calmed him in so many ways. Declan had said that when he first held her, his eyes had been entirely brown again. In that instant, she'd brought forth something in him that hadn't been there in centuries. If she was his, he knew, his constant drive to deliver death would not plague him so badly.\n\nBut with the bond not completed between them, she'd also made him far more explosive in other ways. He now understood why his temper had been more unstable lately, why he could barely control himself around her when no other female had ever made him lose control before. A vampire who encountered their mate was more volatile until the bond between the mates was completed.\n\nHowever, if she were bonded to him, she may never be able to live out her dreams. He could guarantee her nothing but fighting and uncertainty in his foreseeable future, maybe forever.\n\nShe deserved better, and he would make sure she had it.\n\n\"No, Kadence, this is not because you're a hunter and I'm a vampire. We did nothing wrong.\"\n\n\"Then what is the matter with you?\" Her eyes narrowed on him and she pulled the covers more firmly against her chest. \"Or is this how you treat all women after you have sex with them?\"\n\nHe closed his eyes against the misery in her voice. He was a bastard. He should be in that bed now, holding her close. Instead, he was scared if he got too close to her, he might drain her dry, change her, and never let her go.\n\n\"No, this is not the way I treat women after sex.\"\n\n\"I guess I'm the lucky one then.\" Her tone dripped acid as she spoke.\n\n\"No... This isn't _you_.\"\n\nTaking a deep breath, he braced himself to meet her gaze again. She stared back at him, her eyes glittering with fury, but also with a hurt he'd never wanted to inflict on her. Her anguish shoved the demon within him aside. He took a step toward her, and when the thirst for her blood didn't immediately rush back to the surface, he closed the distance between them.\n\nHer hair fell into one of her eyes when she tilted her head back to stare at him. He brushed the strand aside to tuck it behind her ear. Maybe he could control himself enough to crawl back into the bed with her and hold her the way she deserved to be held.\n\nHe stroked his hand over her cheek and lower to her neck. His gaze fastened on the vein running just under her skin. He dropped his hand and stepped back as his veins became as scorched as a desert.\n\n\"I can't.\"\n\nHe moved further away as he closed his eyes against the hunger tearing at him. Lifting his hands, he scrubbed his palms over his cheeks, pulling at his flesh.\n\nKadence had never seen anyone look so distraught before. She didn't understand what was going on with him, but the fact it was Ronan tore at her heart. \"Maybe I can help.\"\n\nOh, she could help, he ruminated bitterly. She was the only one who _could_ help. Lowering his hands from his face, he lifted his eyes to hers.\n\nKadence's mouth parted on a breathless _oh_ when Ronan met her gaze again. His eyes were a glittering ruby color that blazed out at her and his fangs had elongated past his lower lip. She'd seen his fangs and red eyes before, but this was somehow different.\n\n_Because he's not in control_ , she realized and her heart leapt into her throat. Before, she'd always been able to see Ronan behind the vampire traits. Now, she saw only the demon.\n\nThis was what Joseph had looked like before he'd bit her. There had been no humanity in his gaze, nothing other than evil. Her hand involuntarily went to the place on her neck where Joseph had sank his fangs. The memory of the pain he'd inflicted on her came readily back to life.\n\n\"I scare you,\" he said.\n\nKadence closed her eyes against her self-disgust over her cowardice. \"No,\" she lied.\n\n\"I can smell the fear on you.\"\n\n\"Not of you, not really. The bite, it's painful.\"\n\nRefusing to be cowed by the memory of Joseph, she opened her eyes. Her heart sank when she realized Ronan was gone.\n\n***\n\nRonan stormed down the stairs, heedless of his nudity. He moved swiftly across the marble foyer, not noticing the coldness of it beneath his bare feet as he made his way to the basement door, flung it open, and swiftly descended.\n\nHe stepped off the last stair and strode across the matted floor of the gym. He would head straight for one of the treadmills as soon as he fed. He'd run his body for so long and hard that he would be too tired to move, too tired to do anything but crawl into bed and pass out. He would be too tired to think about anything, especially _her_. Though, he suspected he'd have to be dead before he stopped thinking of Kadence.\n\n\"Some of us prefer clothes to work out in, but to each their own.\"\n\nRonan ground his teeth together as the words drifted across the room from the stairwell he'd left behind. The last thing, the last _one_ , he wanted to deal with right now was Declan. Ronan refused to look back at him as continued through the set of swinging steel doors and into the storage room beyond. A tall, stainless steel refrigerator was set against the far wall. They kept emergency supplies of blood stashed inside, and with the way he felt right now, this definitely classified as an emergency.\n\nPulling one of the doors open, he snatched a blood bag from within. He ripped the top off it, and heedless of the cold temperature, downed the contents in one gulp. He crumpled the bag, tossed it aside, and grabbed another one. He drank it down before flinging it aside.\n\nHe had never required a third bag to sate his thirst before, but fire still raged through his veins. Seizing another bag, he consumed it and was reaching for a fourth when the doors swung open behind him. Declan's footsteps sounded on the tile floor as he entered the room.\n\n\"Get out,\" Ronan commanded.\n\nHe still wouldn't look at Declan as he ripped the top off the fourth bag and drank it. The hunger continued to shred his insides with its incessant demand for more. For something _other_ than this bagged shit, or rather, _someone_ else. No matter how much he drank, he wouldn't be satisfied until it was Kadence's vein his lips were pressed against.\n\nThrowing the bag away, he cursed loudly before slamming his fist into the fridge. Metal screeched as it crumpled from the impact of the blow.\n\n\"What did the fridge ever do to you?\" Declan quipped.\n\nRonan whirled on him. \"I said _get_ _out_!\"\n\nDeclan held his hands up as he backed away. Ronan barely glanced at whatever was hanging from Declan's fingers before his friend tossed it at him. \"Easy, Ronan, I'm not here to start anything with you.\"\n\n\"Then why are you here?\"\n\nDeclan's nostrils flared as he scented the air. Ronan stiffened when he realized Declan had caught his scent, which meant he could smell what had transpired between him and Kadence.\n\n\"You're in a feeding frenzy,\" Declan said flatly, wisely choosing not to comment on his relationship with Kadence.\n\n\"Your powers of observation are astronomical!\" Ronan spat.\n\n\"When was the last time you fed?\"\n\n\"What business is that of yours?\"\n\n\"You're out of control. That makes it my business. If this were me in here, you would lock me away or destroy me.\"\n\n\"Try it,\" Ronan dared him.\n\n\"I'm not going to try anything. We both know you could kill me, just as we both know why you're like this.\"\n\nRonan turned away from him and removed another bag of blood. This time, he didn't bother to tear off the top but sank his fangs into the bottom and drained the contents. He would drink every drop of blood in the fridge, and when he was done, he would return upstairs, take her again and drain her.\n\nHe stretched a shaky hand in for the sixth bag. In all his years, he'd never been this out of control.\n\n# CHAPTER 26\n\n\"When was the last time you fed?\" Declan asked again.\n\n\"Two days ago.\" He occasionally went two days at a time, never longer. When he'd been younger, he'd been able to go for almost a week without much of a problem. Now, he tried to feed every day to keep the part of him that craved blood and death at bay.\n\n\"You slept with her,\" Declan stated.\n\nRonan's head swiveled on his shoulders toward Declan. His friend edged back until his heel connected with one of the swinging doors. \"That's none of your fucking business.\"\n\n\"I know, I know,\" Declan said quickly. \"But come on, Ronan, you must realize what she is to you.\"\n\n\"I do,\" Ronan admitted.\n\n\"What happened between you tonight has accelerated things for you. That is why you're so out of control right now.\"\n\n\"A _hunter_ for a mate. How is this possible?\"\n\n\"We don't pick our mates. A lot of us don't find them. But when we do\u2014\"\n\n\"I am well aware of what happens when we do.\" A loss of control until the bond between them was completed, and since she was mortal, Kadence would also have to be turned. Which meant she would have to die.\n\n\"Then you know what must happen now,\" Declan said.\n\nRonan closed his eyes and rubbed at the bridge of his nose. \"Even if she would agree to such a thing, I would never allow it. She deserves better than this brutal existence.\"\n\n\"She's a hunter who lost her father to a Savage. She may have led a life of seclusion, but she is no innocent to the brutality of our world, or her own.\"\n\n\"I promised her freedom, and she will have it,\" he said hoarsely.\n\nDeclan exhaled sharply. \"You know what will happen if she leaves here, leaves _you_.\"\n\n\"I will not chain her to me! What was it you said to me? _'Everyone deserves a chance to fly.'_ You wouldn't clip her wings, yet you expect me to do it. It will not happen.\"\n\nDeclan looked at him as if he'd lost his mind, and perhaps he had.\n\n\"It already has happened, Ronan. The more you fight it, the worse it's going to get, until you lose all control and turn Savage, or you end up forcing the change on her. If you take her by force, you may kill her, and you will kill the trust she has in you. You won't be able to live with yourself if you do that to her.\"\n\nRonan's skin felt as if it were drawing taut against him, strangling him. He resisted the urge to dig his fingers in and shred the skin from his body so he could breathe again.\n\n\"I've promised her the world she dreams of, and she will be leaving in mere hours. I won't be able to hurt her, because she won't be here. I'll make sure Marta and Baldric know not to tell me where they are so I can't go after her. She'll be set free to live, instead of caged here.\"\n\nDeclan gawked at him. \"That's not an option for you.\"\n\n\"It _is_ an option for me! I know what they say about mates going crazy without each other, I've witnessed it, but that doesn't mean it will happen to me. The bond is not completed between us. We have not shared blood. If anyone can control this, _I_ can.\"\n\n\"You may be the strongest and oldest vampire to ever walk this earth, but not even you can control this. You're _already_ losing control. No one has ever come back after they've given into their Savage nature. _She_ may not even be able to bring you back if you cross that line, and if you did encounter her again as a Savage, there is no telling what you would do to her.\"\n\nDeclan gestured at the bags of blood scattered across the ground at Ronan's feet. \"If you become Savage, we won't be able to stop you. Everything you've worked for will be for naught. The Savages will eventually win because, if you don't take your own life, you will take all of _ours_. You will most likely rise up to become their leader and there will be no stopping that, Ronan. Are you ready to deal with that?\"\n\n\"I have dealt with it for centuries. You felt it necessary to make a point about the color of my eyes before, so it is no secret to you that I've been battling bloodlust for centuries. It will be worse when she goes, but I will control it just as I always have.\"\n\n\"You just drank six bags of blood, yet I can still feel the hunger coming off you. You've never had to deal with finding your mate before.\"\n\nRonan eyed Declan as wrath burnt through him. \"You knew what she was to me when she pulled that little stunt with her brother.\" He stalked toward Declan as he spoke. Declan's heel pushed open one of the doors as he backed into the gym. \"You could have stopped this. She would have been gone from here before our relationship ever progressed to this point.\"\n\nHe'd regretted going after Declan in the poolroom before; now he would like nothing more than to tear his friend's arms off and beat Declan with them. If that didn't satisfy him, he would tear Declan's head off next.\n\n\"After what I saw with you in that alley, I did suspect it,\" Declan admitted, apparently unaware of how close to death he was. \"But this was going to happen no matter what. Even if she had told her brother where to find her, you never would have handed her over to be married to another man. You're unraveling fast, Ronan, faster than I thought possible; most likely because of your age, power, and lineage.\"\n\nDeclan's gaze was sad when he stopped retreating. \"You have no control over this. It will claw at you until you go mad.\"\n\nRonan turned away from him, no longer able to meet Declan's gaze. \"I _promised_ her she could see everything she ever dreamed about seeing. _I_ can't take her to see those things, I can't give her the life she deserves, not now. The traveling we do consists of: arrive, hunt, kill, return. There's no time for sightseeing and tourist traps. Even if there was time and I did take her with me, I would be dropping her off to go kill before returning to take her on a guided tour. What kind of fucked-up life is that?\n\n\"Not to mention, I don't think any of us will be going anywhere anytime soon, not with Joseph here. My life revolves around death. She's too vibrant and beautiful for hers to be the same way. She risked everything to gain freedom, and I won't be the monster who rips it away from her.\"\n\n\"You can figure all that out, and one day, maybe you will be able to take her everywhere she wants to go.\"\n\n\"That's a big maybe. She's also frightened because of what Joseph did to her in that alley.\"\n\n\"You'll get her past that. You need to change her and mark her so that every vampire will know who she belongs to and so you don't completely lose it.\"\n\n\"Mark her for death, you mean,\" Ronan replied. \"Because I will be painting a bullseye on her for all of our enemies if I do that.\"\n\n\"If anyone can keep her safe, you can, and we will all help you do so.\"\n\n\"She won't understand any of this.\"\n\n\"She will, if you explain it to her.\"\n\nRonan laughed harshly. \"She'll understand that any hope of freedom she ever had is gone?\"\n\n\"That's not true. Things can change over time.\"\n\n\"There will always be vampires who turn Savage.\"\n\n\"Yes,\" Declan said. \"But maybe one day we won't be the only ones fighting them. She gave herself to you; I would say she cares about you a great deal if she did that. In fact, I think she has already given you her heart. She's a good woman, Ronan.\"\n\n\"Too good for the likes of me,\" he muttered.\n\n\"Tell her what is going on and see what she decides.\"\n\n\"I won't put this burden on her.\"\n\n\"She's strong and she's smart. You deserve some calm in your life; deserve someone who cares for you. Tell her and let her make the choice.\"\n\nThe scent of vanilla drifting to him silenced Ronan's response. He took a step back toward the swinging doors and nudged one open with his heel as the sound of Kadence's footsteps on the stairs reached him.\n\nHe'd considered himself somewhat settled, but having her so close caused hunger to surge through him once more. \"Get her out of here, Declan.\"\n\n\"Ronan, you know what must be done.\"\n\n\"I need more control over myself before I can see her again. Get her out of here.\"\n\n\"Ronan\u2014\"\n\n\"Now,\" he hissed. \"I might hurt her otherwise.\" Declan turned to leave. \"Declan.\" He looked back, his silvery eyes troubled. \"I understand why you didn't stop her from doing what she did with her brother, and that you weren't trying to go against me, but she can't stay.\"\n\n\"She can if you give her a chance to decide.\"\n\nRonan didn't have the time to get into it with Declan again as Kadence's scent strengthened on the air. \"Keep her away from Saxon and Killean; I don't want them to know about this yet.\"\n\n\"I will. I threw a pair of sweats at you earlier.\"\n\nAs Ronan slipped back into the storage room, he vaguely recalled Declan tossing him something earlier. He spotted the blue sweats on the floor amid the bags of blood as their hushed voices drifted to him through the swinging doors.\n\nRonan returned to the fridge and took out more blood. He was determined that one of these bags would finally drench the fire within him.\n\n# CHAPTER 27\n\n\"Is he okay?\" Kadence inquired.\n\nDeclan heard the concern in her voice, saw it in the way she kept turning her head to look over her shoulder for Ronan. A spurt of jealousy went through him. He wasn't jealous of the condition Ronan was in now, for he wouldn't wish that on his worst enemy, but of the relationship he and Kadence could have together.\n\nThey already cared for each other, that much was obvious. It was only a matter of time before it deepened into love.\n\nHe shook his head to rid himself of the crazy ideas running through it. No, he was much better off the way he was. The last thing he wanted was to get shot by the bullet that was taking Ronan down. Ronan was going insane, and Declan was calmly walking the cause of his insanity away from the gym.\n\n\"Declan?\"\n\nHe returned his attention to her as he tried to recall her question. \"He'll be fine,\" he assured her.\n\n\"Are you sure?\"\n\nDeclan nodded, but he couldn't be completely sure until everything was settled between the two of them. If everything went well, within the next day or two, Ronan _would_ be fine. Hopefully, Ronan still had a day or two left in him before the impending meltdown occurred.\n\nKadence bit her lip as she twisted her hands in front of her. She was beautiful, yet there was an inner beauty and strength to her that outshone her looks. Enemy or not, he liked this little hunter and hoped she would make the right choice.\n\n\"He'll be fine,\" Declan assured her again.\n\n\"I may not know as much as I thought about vampires, but whatever this is doesn't seem like him. Is it because of me?\"\n\nShe was completely out of her depth in this whole mess and didn't understand the greedy, unpredictable nature of the demon lurking within them. She had given herself to a man she cared for, and in doing so she'd escalated a chain of events she never could have known were already starting to unfold.\n\nTo be fair, even if she hadn't given herself to Ronan, it would have only been a matter of time before Ronan recognized she was his mate.\n\n\"This isn't like him,\" he replied, uncertain of how to respond. \"But it will all work out soon.\"\n\nHer mouth pursed as they stepped into the main foyer. He guided her toward the stairs, eager to get her to her room before Killean or Saxon arrived and caught the scent of Ronan all over her. Killean would not react well to such a thing.\n\nMaybe he should tell her what Ronan was going through and how she could help. He shut the idea down as soon as he thought it. Even if he were stable, Ronan might kill him for interfering in such a way. He'd definitely kill him now for it.\n\nThis was not his place, and he knew better than to interfere in others' lives.\n\n\"What's going on?\"\n\nKadence stopped short when Killean strolled into the foyer from the hall. He held a glass of scotch in his hand and his golden eyes surveyed them with curiosity.\n\n\"What are the two of you doing?\" Killean inquired.\n\n\"I gave her a tour of the place,\" Declan lied.\n\nKillean took a sip of his drink as he walked closer to rest his elbow on the banister of the stairs. The tension in his body belied the casual gesture. \"Why, she won't be staying for much longer?\"\n\n\"It was something to do,\" Kadence replied flippantly.\n\nKillean swirled the liquid in his glass as he inhaled deeply and his upper lip twisted into a sneer. Declan braced himself for whatever was about to unfold.\n\n***\n\nKadence's stomach twisted at the look on Killean's face. She knew he didn't like her, but when he looked at her like that, she realized that she also repulsed him.\n\n\"Where's Ronan?\" Killean demanded.\n\n\"In the gym,\" Declan replied. \"I would suggest leaving him be.\"\n\n\"Leaving him be,\" Killean snorted. \"I can smell him all over her. What is he thinking screwing her?\"\n\nColor flooded her cheeks, but she managed to stop herself from gaping at Killean's blunt words. She should have known the vamps would be able to tell what had transpired between her and Ronan tonight. She didn't know if she wanted to melt through the floor or kick Killean in the nuts more. Driving his nuts into his throat would feel really, _really_ good right now, she decided.\n\n\"Watch yourself, Killean,\" Declan advised.\n\n\"It's not you I smell on her, Declan. Why are you being so protective?\" Killean demanded.\n\n\"It's not her he's protecting. It's _you_.\" The hair on Kadence's arms rose as Ronan growled the words from the doorway behind her. \"Because if you say another goddamn word, I'll rip your throat out.\"\n\n***\n\nDeclan cringed inwardly as Ronan's words filled the foyer. He released Kadence's arm, surreptitiously pushing her a little away from Killean and himself. The last thing they needed was to provoke Ronan any more by having her so close to them.\n\nA scarlet haze clouded Declan's vision as Ronan's wrath reverberated within him. He blinked the cloud away, desperate to get his vision back, but Ronan's emotions were too wild to be shoved away.\n\nHe wanted to reach out to someone, to grab them in order to stay grounded. Killean was out of the question unless he wanted to experience more anger. He couldn't touch Kadence, not with Ronan so wound up. He settled for resting his fingers against the wall, if only to keep standing. It did nothing to wash away the red tide shading his eyes.\n\n\"Are you okay?\"\n\nKadence's voice filled his ears as her hand latched onto his arm. The red cloud intensified tenfold, causing his ears to hum and his head to throb. Ronan must have seen her grab him. \"Let go!\" he ordered and tried to shake off her hand.\n\n\"You don't look so good.\" She clung to him as her voice took on a frantic tone.\n\nHe didn't feel so good. He was either going to pass out or his head was going to explode. He didn't care which happened first as long as the awful haze stopped. It had been centuries since anyone had broken through his carefully constructed barriers this bad. He'd spent years armoring himself against others, but Ronan was far more out of control than he'd realized, and he was far more powerful than Declan's walls.\n\n\"Let go of him!\" Ronan snarled.\n\n\"But he looks ill,\" Kadence protested.\n\n\"Let go!\" Declan finally succeeded in getting her to release him. \"Calm down, Ronan,\" he managed to choke out.\n\nHe couldn't see Ronan right now, but Declan knew his friend approached when the haze grew darker. _Breathe, just breathe,_ he counseled himself. The deep inhalations helped him to piece his broken walls back together and the pressure inside him eased.\n\nGradually, the red haze faded until he saw parts of the foyer once more. He took in the shadows, the stairs, and then Ronan. His eyes widened on the vamp coming at them, and he wished he couldn't see again.\n\n# CHAPTER 28\n\nKadence couldn't stop the tremor that went through her as Ronan stalked across the room. His eyes were a shade of red she would have only thought possible in Hell before she saw him like this. They were so bright they lit the shadows around him as his attention remained focused on Killean.\n\nShe'd never seen the normally distant, often asshole vampire look at all ruffled. Now, Killean straightened away from the banister and fear flashed briefly across his face before his expression became void of all emotion. Declan took a hitching breath. His cheeks had regained some color, but he still had a blank look in his eyes that troubled her.\n\nThe anger Ronan radiated was impossible to ignore as it pulsated through the room like a living entity. The veins in his arms bulged, and every muscle through his chest and arms stood out. She didn't know where they'd come from, but the pair of blue sweats he wore hung low on his hips. He looked ready to kill, yet his hand was gentle when he clasped her elbow and drew her closer to him.\n\nEver since he'd walked out of her room, she'd felt a strange sense of loss and an odd stretching of her skin, as if it didn't fit her body right. It made no sense, but the second he touched her again, the sensation eased and she found herself able to breathe easier again.\n\nRonan pierced Killean with a look that would have made most men cower. Killean's golden eyes filled with disbelief. The only reaction he showed to the hostility Ronan directed at him was a tensing of his fingers around his glass.\n\n\"You're not to talk to her like that ever again,\" Ronan said, the hint of his Irish accent more pronounced. \"You're not to do anything but treat her with respect and defend her life with yours. If I see or hear you doing anything disrespectful to her ever again, I'll beat you to within an inch of your life, and when you heal, I'll do it all over again.\"\n\nKadence gasped at Ronan's threat. She opened her mouth to deny his words, but Killean was already speaking. \"You're the boss.\"\n\nRonan took a step toward Killean, who had the good sense to back away. Killean's eyes darted toward her, but instead of his normal antipathy toward her, she sensed only curiosity.\n\n\"Don't fuck with me on this, Killean,\" Ronan stated.\n\n\"I have no intention of doing so,\" Killean replied.\n\nKadence glanced over her shoulder as Ronan nudged her forward and started leading her up the steps. Killean stared blankly back at her as he took another sip of his drink, while Declan rubbed at his temples.\n\nTurning away from them, she focused on Ronan. With every step they took, his eyes shifted back to that amazing burgundy color, but now that she knew the true depth of the brown in his eyes, she wanted to see more of it.\n\nHe pushed open the door to her room and stepped aside to let her enter first. She was instantly assailed with the lingering scent of their sex. Her heart kicked up a notch, and to her horror, she felt herself growing aroused again. After what had happened earlier, she'd been certain she'd never have sex with him again. She wanted answers about what had happened to him, but she would not be fooled twice. Unfortunately, her body seemed to have other ideas.\n\nShe couldn't meet his gaze as he stepped into the room beside her. His fingers stroked briefly over her skin before he released her arm. He strode over to her bed and pulled the blanket back. Kadence's jaw clenched as the sight of her blood staining the white sheets brought back the horrible events of him fleeing her room earlier. His hand stilled and his shoulders hunched up as if he were preparing himself for a blow when he looked at her over his shoulder.\n\n\"Did you not know I was a virgin?\" she asked coldly when the stunned look didn't leave his eyes.\n\n\"No, I knew you were,\" he said. Turning away, he spoke so softly she barely heard his following words. \"It wasn't supposed to be like this.\"\n\nYanking the sheets from the bed, Ronan wadded them into a ball. He hadn't thought it possible, but seeing her blood made him hate himself even more for the way he'd treated her tonight. Seeing her blood on the sheets slammed home the reality of what an asshole he was.\n\n\"I'll get you some new sheets,\" he said.\n\nHe couldn't meet her eyes as he walked past her and into the hallway. He opened a few doors before he found the closet with the sheets Marta kept stacked within. Staring at them, he struggled to maintain the thin thread of control he'd managed to reclaim after glutting himself on ten bags of blood.\n\nFinally feeling capable of being near her again, he removed a set of sheets from within and returned to the room. Kadence remained where he'd left her, her eyes guarded as she watched him remake the bed.\n\n\"You should probably try to get some sleep,\" he said when he was done.\n\n\"I'm not tired.\"\n\n\"You've had a rough night; you should rest.\"\n\nShe glared at him as she crossed her arms over her chest. He'd hurt her, he knew. When she should have had comfort and security, he'd abandoned her. He wasn't surprised she was mad at him; he was surprised by how much he didn't like it.\n\n\"It wouldn't be a rough night if you would tell me what is wrong with you. Besides, I don't recall having to take orders from you,\" she said.\n\nRonan gritted his teeth as he met her gaze. Her face was suffused with color; her hair cascaded around her face in waves that emphasized her striking features. She was exquisite and _his_. There was no denying it. Even after she left here, she would always be his.\n\n\"There is something we must discuss,\" he said.\n\n\"Noooo,\" she retorted sarcastically.\n\nHe lifted an eyebrow at her, but he deserved her anger. She hadn't cried and carried on about her circumstances and what he'd done to her tonight, but what would she say when he told her he wanted to bind her to him for eternity? That he wanted to turn her into something else entirely and deny her the freedom she so rightly deserved to have?\n\nIf they completed the mating bond, it would be for him; there would be nothing for her. Just as there had been nothing for her amongst her own kind. He'd thought the hunters archaic for marrying her off like they intended to, so what was he now? Because his demon DNA recognized her as his mate, he believed he had more of a right to her than her own family and her own kind?\n\nHe'd fought against losing his humanity for years; he could fight it longer, for her.\n\n\"I'm sorry for what transpired here earlier.\"\n\nHer eyes became a vivid shade of blue as fury emanated from her. \"I see.\"\n\nHe didn't understand why that had pissed her off more. He was trying to apologize. He knew it would never be enough, but it was a beginning at least. \"I must explain\u2014\"\n\n\"Don't bother,\" she cut in. \"It was a mistake, a passing fling.\"\n\n\"It was not a mistake!\" he snapped, unable to control the flare of temper her words provoked in him. Taking a deep breath, he took a second to steady himself before continuing. \"I am not sorry for what happened between us; I am sorry for my behavior afterward.\"\n\n\"Oh.\" Her arms fell to her sides as some of her ire faded. \"Okay.\"\n\nHe couldn't help but smile at her even as his insides twisted into knots at the words he knew he would be issuing soon. \"Okay.\"\n\n\"Why did you act like that then?\"\n\n_Tell her._ The words screamed across his mind, the selfish part of him roaring to life. She cared enough for him to have given herself to him; she might agree to stay with him if she knew his sanity could depend on it. However, he couldn't bring himself to place that responsibility on her, not when she was so close to being free.\n\n\"It had been a while since I fed.\" Not a lie, not a truth.\n\nHer brow furrowed then cleared when his eyes were involuntarily drawn to the vein in her neck. \"Oh,\" she said. \"And you've fed now?\"\n\nThat was not the word for what he'd done. \"I have.\"\n\n\"So you're better now?\"\n\n_No._ \"Yes,\" he lied.\n\n\"What about what happened with Killean and Declan just now? I've never seen you so angry at Killean, and I think something was wrong with Declan.\"\n\n\"No one is going to talk to you in such a way. Killean knows that now. And Declan is... well, he's different from others. Before you ask, I don't know exactly how he's different. I just know he is. I've never asked him to reveal all of his secrets to me, but his difference can be useful to him, or stressful.\"\n\n\"I see.\"\n\n\"You should rest before you leave.\"\n\nKadence blinked at him, thrown off by the abrupt change in conversation. \"Excuse me?\"\n\n\"Rest. It is going to be a tiring day for you, and you will be leaving shortly.\"\n\n\"Oh... ah... yeah,\" she muttered. She had no idea what was wrong with her. She should be jumping for joy. Soon she'd be leaving this place and _him_ behind to do everything she'd always dreamed about doing. The knowledge brought her no joy. Instead, it made that awful tightening of her skin start all over again.\n\nHer fingers bit into her arms as she held back the tears burning her eyes. She would not cry in front of him. She _refused_ to do so again, especially since she had no idea why she wanted to cry. Okay, well, she had a little bit of one. She had come to care for this man who she would have gladly staked two weeks ago, and she wasn't ready to leave him.\n\n_Do I want to go?_ she asked herself. _Of course you do, you idiot. You abandoned your whole family for a shot at freedom, and Ronan is offering you more than you ever could have hoped for._\n\nThen why did her heart feel like it was being squeezed in a vise?\n\n\"Do you want to leave, Kadydid?\" he asked.\n\nHer heart squeezed further at the nickname he'd given her; one she'd come to cherish over the past week. Was that a measure of hope she heard in his voice, or was she only trying to hear it there?\n\n\"I want... I want to see the world and learn things,\" she whispered.\n\nHe glanced to the windows, his hands fisting as on the horizon the night sky began to lighten toward dawn. \"And I will give that to you,\" he murmured. \"I must speak with Marta and Baldric now.\"\n\nHe kept his head averted from her as he strode toward the door. \"Ronan.\" He flinched away from her hand when she rested it on his arm. \"I should speak with my brother.\"\n\nHis head swiveled toward her. She couldn't stop herself from taking a step back from the burning red of his eyes. \"Why?\" he asked.\n\nKadence swallowed. \"To let him know we were wrong, that you are not all bad, that we all fight a common enemy. Maybe... maybe you could work together. Think of the good that could be done and the lives that could be saved if hunters and vampires united.\"\n\n\"And do you think he would listen to you?\"\n\nShe contemplated his question before sighing. \"Probably not, but don't you think it's worth a chance?\"\n\nHis hand briefly rested over hers, his fingers squeezing her before releasing her. \"I think it's best if you get away from here. If you leave behind the violence and death of both our worlds and live the life you are meant to live.\"\n\n\"And what life is that?\" she inquired.\n\n\"One of happiness.\"\n\n\"Ronan\u2014\"\n\nPulling her close, he kissed her forehead before releasing her. \"Let this go.\"\n\nThe lump in her throat choked her as he strode toward the doorway. \"Ronan!\" she blurted when he stepped out the door. He hesitated in the threshold, his shoulders drawn up to his ears and his back hunched. \"Thank you, for everything. No matter what, I will never regret anything that transpired between us.\"\n\n\"Neither will I.\"\n\n\"When I come back\u2014\"\n\nHe looked at her over his shoulder. \"If you come back, I will be here, waiting for you.\"\n\n\"You'll have found someone else by then,\" she said with what felt like the worst smile.\n\n\"No, Kadydid, there will never be anyone else for me, only you. Do not return here unless you're ready for an eternity with me.\"\n\nBefore she could reply, he was gone from the doorway and she was left gawking after him.\n\n# CHAPTER 29\n\nKadence stood in the foyer, her small bag in hand as Marta bustled around her. For the thousandth time, Kadence's eyes went to the hallway where the poolroom was located. Somehow, she knew Ronan was down there. She kept waiting for him to come to say goodbye, but she hadn't seen him again since he'd left her room.\n\n\"Are you ready, miss?\"\n\nKadence glanced at Marta before focusing on the hall again. That awful feeling of not belonging in her own skin hadn't eased, but now she found it nearly impossible to breathe as she kept waiting.\n\n\"Miss?\" This time it was Baldric who stepped before her.\n\nGo, or stay for an eternity? And what did he mean by an eternity with him? He couldn't have _really_ meant what she'd thought when he said that? She wasn't a vampire. There was no eternity for her.\n\nKadence looked over at where Marta stood by the door with a small duffel bag in hand. Her mostly gray hair had been pulled into a ponytail. Her round face only showed lines around her eyes and mouth when she smiled. Plump with kind hazel eyes, Marta had become someone Kadence really liked.\n\n\"Yes,\" she croaked. \"Yes. I am ready.\"\n\nBaldric stepped back and opened the door for her. Kadence glanced over her shoulder, but there was still no sign of Ronan. She took a step toward the poolroom before retreating. If she went to say goodbye to him now, she might never leave.\n\nShe forced herself to walk out the door. She'd turned against her brother and all her kind for this opportunity at freedom. She'd forsaken everything she knew; she could not change her mind now, even if she was contemplating staying for a man she'd only known for a short while.\n\nA man she had willingly given herself to. By doing so, she'd chosen a course she could never take back and didn't want to. If she tried to return to the hunters now, and they somehow learned who she had given herself to, they may label her a traitor and kill her. Even Nathan wouldn't be able to stop that from happening if they decided that's what she was.\n\nNot like she would ever willingly tell them she had given herself to a vampire, but like Declan, there were those of her kind who had gifts. She certainly did. She had the same strength, speed, and enhanced senses her father and Nathan possessed and her strange knowing of things.\n\nStepping into the day, Kadence tipped her head back and let the warmth of the sun wash over her. Baldric opened the back door of a black car with heavily tinted windows for her. Every step she took caused her shoulders to sag more, but she tossed her bag into the back seat and climbed in behind it.\n\nShe winced at the clicking sound of the door closing behind her. Huddling into herself, she watched as Baldric and Marta climbed in and Baldric started the vehicle. She couldn't look back as they drove down the tree-lined, cobblestone drive to the thirty-foot-high gate at the end.\n\n\"Put your blindfold on now, miss,\" Marta said.\n\n\"Please call me Kadence. It's going to be an extremely long time together if you keep calling me miss.\"\n\n\"Kadence then,\" Marta said with a smile. \"Do you need help with the blindfold?\"\n\n\"No,\" she whispered and slid the thick material over her eyes.\n\n***\n\n\"What have you done, Ronan?\"\n\n\"If you want to survive to hunt tonight, I would suggest leaving,\" Ronan didn't look back at his friend as he replied to Declan's question.\n\n\"You have to stop them. You can't let her go.\"\n\n\"I can and I did.\"\n\nHe kept his gaze focused on the bar as he swirled the whiskey in his glass. He was going on his third bottle of Jameson, and he still didn't feel any effects from the alcohol, which was probably a good thing. In his current mood, he might tear this entire house down if he were drunk.\n\nThe people who had lived here before them had shitty taste in d\u00e9cor, but they had fantastic taste in alcohol, he decided as he took another sip of the Jameson Vintage Reserve he'd discovered beneath the bar. At one time, he'd lived on Irish whiskey and women.\n\nHe couldn't remember the last time he'd taken a sip of alcohol, but he could clearly recall the last time he'd taken a woman. Every second of that encounter had been emblazoned on his mind, along with the fiasco of an ending. And now she was gone, set free by none other than himself.\n\nHad he hoped she'd stay? Right up until she walked out the door. He would have denied it, but he realized now, he'd been holding out hope she would decide to stay for him.\n\nNow he was trying to remember the last time whiskey had burned its way down his throat instead of thinking about who had walked out the door.\n\nThe light Declan let in when he'd slid open the doors flashed, but not because he was closing them again; it was because Declan was coming closer. Ronan had closed the shutters over all the windows, unable to handle that small amount of sun right now as the demon churned within him. He definitely couldn't deal with Declan's worry for him.\n\n\"Ronan\u2014\"\n\nThe tumbler shattered in his hand, and liquid splashed over him as shards drove deep into his flesh. He didn't bother to pull the glass from his palm before he lifted the bottle he'd set next to his chair. Declan remained mute as he padded by him to the bar where he removed a couple of bottles before walking over to him. Grabbing the back of one of the ugly chairs, Declan set it beside him, placed one of the bottles between them, and opened the other.\n\n\"Not one more word about her, Declan.\"\n\n\"Understood.\"\n\nThe light shifted again and Killean's resin scent drifted to him. Ronan's fangs lengthened in anticipation of one of Killean's remarks, but the vampire only walked by him to the bar before moving a chair to sit on Ronan's other side. Saxon joined minutes later, and when Ronan heard the click of the front door shutting, he knew Lucien had also arrived.\n\nHe'd forgotten Lucien was supposed to be coming today to discuss the moving of the training facility. _Ah well, the more the merrier,_ he thought as he lifted the bottle to his lips and drank half of it down.\n\nRonan closed his eyes and took another pull on the bottle. The bloodlust slithered through his veins like an insidious snake waiting to strike. Every beat of his heart caused his temples to pulse with it. Yet there was still so much to deal with, so many still counting on him.\n\nIf he gave in and gorged himself on the blood, he wouldn't have to deal with it ever again. He'd only have to deal with himself, and Kadence's hatred of him should she return.\n\nMate or not, she was gone and he had to accept that she may never return. He had to continue with what he'd been born to do. Rising to his feet, he drank the rest of the bottle as he made his way toward the bar.\n\nHe waited until Lucien walked in and settled into another chair before speaking. \"We have to take out Joseph before he continues to organize.\"\n\nDeclan passed his bottle to Lucien and opened the one he'd set on the floor. \"We've had no new leads on him, even Brian is coming up blank,\" Lucien said.\n\n\"How is that possible?\" Declan inquired.\n\nLucien shrugged. \"Brian said he's not a GPS. Sometimes whatever he does just doesn't work. He also said Joseph is most likely staying in a large crowd or city if he can't pinpoint him.\"\n\n\"A large crowd of Savages?\" Saxon asked.\n\n\"Perhaps,\" Lucien replied.\n\n\"Not good,\" Declan muttered.\n\n\"The trainees, do they know what is going on? Why the facility is being closed and moved?\" Saxon asked.\n\n\"I saw no reason to keep it from them,\" Lucien replied and tee-peed his hands before his face. \"If they decide to bail instead of fight, then good riddance. I hope they get butchered by a Savage.\"\n\n\"Nice,\" Declan muttered and took another drink.\n\nLucien scowled at him. \"You'd wish them well?\"\n\n\"I wouldn't wish them death.\"\n\n\"We have to discuss if we will bring the turned vamps in to work with us,\" Ronan said. Some of the glass still embedded in his hand clattered against the bar when it succeeded in working its way out of his flesh. He glanced at the blood splattered piece before focusing on the others.\n\n\"It goes against tradition,\" Saxon said.\n\n\"It does,\" Ronan agreed. \"And the turned vamps aren't as strong as we are, but they can take on a Savage. I lived through an attack from the Savages before. If Joseph comes at us with numbers like what they had back then, it will be a war the likes of which none of you have ever experienced.\"\n\nThey stared stonily back at him. He understood their reluctance to add turned vampires to their ranks and their pride. There were so few purebred vamps left, even fewer who had made it through the extreme training that elevated them to the status of Defender. The turned vamps went through a lot of training, but they didn't go through the same kind of training they had all endured and survived.\n\n\"They still don't have to be one of us,\" Lucien said. \"We can call them something else if we want to use them for this battle.\"\n\n\"I vote we call them pissants,\" Killean said.\n\n\"I like it,\" Lucien agreed. \"They many not even be willing to fight.\"\n\n\"I'm sure you've inspired no loyalty,\" Declan replied.\n\nLucien gave him the finger. \"I'm not there to inspire them. I'm there to make sure they don't get killed by a Savage, and as soon as we find a replacement for me, I'm done with training them. Joseph boned me the most when he went Savage on us, and I got stuck with all the idiot newbies.\"\n\nThankfully, Joseph had given into the demon part of him outside of the training facility. Most likely because there had been enough recruits there to possibly take him down when they realized he'd become a Savage. Joseph had known that. Despite their endless thirst for blood, Savages were far from stupid. Before Joseph, they'd had no one to organize them, to lead them, but Joseph was a purebred, something different and stronger than they were, and the Savages were falling into line with him.\n\n\"Bring the turned vamps in training, who agree to fight with us, here,\" Ronan said to Lucien. \"There will be enough room for them to stay here between the carriage house and the guest house, but make sure they have no idea where this place is located.\" His teeth ground together at the idea of having to be so close to the recruits, but there was little else that could be done about it. As long as they stayed out of his way, they'd survive. \"Cut the ones who aren't willing to fight loose.\"\n\n\"What about Aiden, the purebred in training?\" Lucien inquired. \"His sisters and Brian are still at the training facility too.\"\n\nAiden had already been in training when his sister, Vicky, was captured by vampires looking to sell off her blood to the highest bidder. She'd been chained and held with other purebreds. It had taken them a while, but Ronan and the others had hunted down and destroyed most of that remaining threat against purebreds.\n\nWhat remained of those vampires were a far smaller threat than what the vampire race faced with Joseph. But then, Joseph and the Savages were a danger to everyone who crossed their paths. For the first time, he was glad Kadence would be far from here.\n\n\"Bring Aiden and his family here too, if they agree to it,\" Ronan replied.\n\nLucien took another swig of his bottle.\n\n\"There are other turned vamps who have already gone through the training and are out there hunting Savages,\" Saxon said. \"Do we bring them in?\"\n\nHaving as much help as they could get would be the smart thing to do, but having vampires crawling over this place and the nearby cities and towns in search of food would be a sure-fire way to draw Joseph's attention, along with that of the humans.\n\nHowever, if Joseph was accruing Savages, it was only a matter of time before he caught the attention of someone he shouldn't.\n\n\"Let's get the recruits here first, and then we'll discuss bringing more turned vamps in,\" Ronan replied as the last of the glass in his hand worked its way out of his flesh and clattered onto the bar.\n\n\"When should I bring them?\" Lucien inquired.\n\n\"Now. I want this over with,\" Ronan replied.\n\n# CHAPTER 30\n\n\"I'd like to make a phone call, if I can,\" Kadence said.\n\n\"Of course, miss... ah, Kadence,\" Baldric replied with a smile. \"You're not a prisoner. We're only here to make sure you're safe.\"\n\nShe smiled at him as he held one of the cell phones out to her and she took it. \"Thank you.\"\n\n\"If you're going to call your brother, I'd keep it short. That may be a throwaway phone, but we don't want to take the chance they could find you.\"\n\n\"I will. It's... ah... it's okay if I call him?\"\n\n\"Ronan said you could do whatever you wanted once you were free, as long as it didn't put you at risk, and I am to use my judgment on that. Since you can't do anything to hurt yourself, or Ronan, I see no reason why you can't call him. I don't believe you would do anything to put Marta or I at risk either.\"\n\n\"Of course not,\" she whispered.\n\n\"Don't forget our flight leaves in ten minutes.\"\n\nKadence resisted tugging on the collar of her shirt at the reminder. \"I know.\"\n\nShe tried to tune out the crowd of people around her as she walked away from Baldric. It was quieter in this area of the airport than it had been when they were going through security, but she was ready to leave Logan Airport far behind her. Though she knew all the airports would have the hectic hustle and bustle of travelers trying to reach their destinations, she found the activity she'd assumed she'd love difficult to handle when a growing weight was bearing down on her chest.\n\nShe started dialing as Marta walked by with a stack of magazines and books in hand. The crawling in her skin had increased since she'd left Ronan, but she didn't know if that was from being away from him or from her unfamiliar surroundings. Not to mention, there were so many people; they were _everywhere_.\n\nHer head pounded from all the noise. The scents of cooking food, coffee, body odor, and one woman who had enough perfume on to drown an elephant filled her nose. Kadence couldn't stop her nose from wrinkling as she passed the woman. She returned the dirty look the woman shot her and got as far from the woman as she could, but the heavy floral scent followed her.\n\nShe hadn't expected to be this overwhelmed by the human world, but her senses were being bombarded, and her body ached. Someone bumped against her, and she nearly jumped out of her skin before she hastily sidestepped them.\n\nTaking a deep breath, she tried to steady the riotous beat of her heart as she punched in Nathan's number and held her breath in the hopes her brother still had the same phone. She knew he would have done everything he could to keep it, knowing it was her only connection to him, but sometimes things went horribly wrong.\n\n\"Kadence?\"\n\n\"Yes, it's me,\" she whispered and wiped away the tear that slid down her cheek at the much-loved voice coming through the line.\n\n\"Oh, thank God,\" Nathan breathed. \"I've been so worried about you.\"\n\nGuilt filled her at the relief in his voice. \"I'm sorry, I really am. I hope you know that.\"\n\n\"It doesn't matter. Just tell me where you are and I'll come get you. We can forget all about this when you get home.\"\n\nEven if she did go home, she could never forget about any of this. \"No, Nathan, I'm still not coming back. I... I'm doing this for me. Please understand, and don't ask me to come back again, not now.\"\n\n\"Kadence\u2014\"\n\n\"Listen to me, I didn't call you to hear a lecture or to come home. I'm not coming back. I'm not going to marry someone I don't love\u2014\"\n\n\"Then we will find you someone else!\" he exploded.\n\nShe couldn't tell him that there was no one else for her, not after Ronan. She rubbed at one of her temples as she tried to ease the headache pounding there before shaking her head to clear it of the absurd notion. Of course there could be someone else for her, eventually, maybe.\n\nShe told herself this, but her skin felt stretched so tight that she had the urge to tear it away from her, and her heart yearned to go back to him. Did she love Ronan? No, that couldn't be possible. She'd only known him for a little over a week; it was impossible to fall in love with someone so fast. She kept telling herself this, but now that the possibility had taken hold it wouldn't let go.\n\nHe was a vampire, but he was a good man. Strong and caring, he made her feel things no other man had ever made her feel. She knew vampires weren't the enemies she'd always believed them to be, but to fall in love with one, to...\n\nWhat? Settle down and marry him? She didn't exactly see Ronan as the marrying kind, and just because he'd tossed out that eternity comment to her, it didn't mean he wanted anything more with her. She was standing here after all when he could have made her stay. No, he would have never done that, she knew. But he could have _asked_ her to stay, and he hadn't.\n\nKadence kept that in mind as she inhaled a shuddery breath and focused on speaking with Nathan.\n\n\"I didn't call to be talked into coming home or because of marriage. I called because...\" She had no idea how to say to him what she wanted to say to him. She'd been trying to figure it out since she'd left Ronan behind, but it always sounded the same to her, ridiculous. \"We have it wrong, Nathan,\" she blurted.\n\nNot exactly the smooth words she'd hoped to be able to come up with, but the truth.\n\n\"We have what wrong?\" he demanded.\n\n\"The vampires, we have it wrong.\"\n\n\"What are you talking about?\"\n\nShe glanced over at Baldric when he stepped into her line of view and tapped his watch. This was never going to be elegant, it would never be received well, but she couldn't leave here without at least trying to make a difference, and she was running out of time.\n\n\"Not all vampires are evil,\" she said. \"Some of them are good. Some of them are trying to do exactly what we are. They protect innocents from those vampires who kill\u2014\"\n\n\"Kadence\u2014\"\n\n\"Listen to me! It could save so many lives if you do! They could have killed you in that alley. They didn't. They could have killed me. They didn't!\"\n\n\"You're with the vampires who were in the alley?\" he barked.\n\n\"No, not anymore. They set me free. I'm leaving, Nathan.\"\n\n\"Did they twist your mind in some way? Where are you? Tell me, and we will get you help.\"\n\n\"You know I'm not susceptible to their persuasion. No one twisted my mind. They opened it.\"\n\nSilence met her statement. Baldric tapped his watch faster.\n\n\"I have to go.\"\n\n\"Did they hurt you?\" Nathan asked.\n\n\"No. They knew who and what I was too, Nathan, and they let me go. Ronan would never hurt me.\"\n\n\" _Who_ _the fuck is_ _Ronan_?\"\n\nShe had to move the phone away from her ear as Nathan's words reverberated through it.\n\n\"He's... he's... the one who set me free to live my dreams, and gave me the means to do so,\" she finished lamely, knowing he was so much more than that to her.\n\n\"If he set you free, then he's trying to kill you, Kadence. This world\u2014\"\n\n\"I know. It's big, it's cruel, and I have no idea about it.\" Being in this airport had hammered that fact home. \"But I'll learn, just as I learned to walk and talk. Just as I've learned everything else. And I'm not alone; I have friends to help me through and money.\"\n\n\"What friends do you have with you? How do you have money? Where can you go? You have no ID; you have nothing.\"\n\n\"That's not important. What is important is that you listen to me. Not all vampires are brutal killers; not all of them are our enemies.\" A line was forming outside the gateway for her plane. Baldric had stopped tapping his watch and was coming toward her as the first person in line handed over their ticket. \"Vampires and hunters can do good, together,\" she said as she watched more people moving through the ticket line.\n\nHer gaze went to the silver airplane outside the floor-to-ceiling windows. The setting sun lit the plane in a multitude of different colors and caused the windows of the plane to reflect the growing pink of the sky. That plane would take her from here and deliver her to the first stop on her journey.\n\nBaldric stopped before her and pointed toward the shortening line. \"I have to go, Nathan. I'll be in touch again soon. Take care of yourself, and remember what I said. I love you.\"\n\nShe flipped the phone closed before he could respond. Baldric took it from her and tossed it in a trashcan. \"We have to go,\" he said.\n\nLifting her bag from the floor, she walked with him toward the line.\n\n***\n\nRonan leaned against the wall of the club to watch the humans grinding against each other. Saliva filled his mouth at the possibility of sinking his fangs into every one of their necks. Draining them all dry and leaving them nothing more than shriveled husks was the only thing he could think about doing right now.\n\nDeclan stepped in front of him, drawing his attention away from the crowd. \"Perhaps you shouldn't be here.\"\n\nRonan straightened away from the wall. \"And where should I be?\"\n\n\"There are a lot of humans here.\"\n\n\"There are, and they're all prime targets for Joseph.\"\n\n_And me_. It was what Declan was thinking too, but he was wise enough not to say it.\n\nA shifting behind Declan's shoulder drew Ronan's attention to the dance floor as Killean and Lucien cut across it toward them. Saxon followed behind, moving slower due to a woman with blue hair and another redheaded woman grinding against his sides. Saxon bestowed his charming smile on them, causing them to nearly swoon.\n\nRonan's teeth ground together so forcefully, he thought he might shatter his fangs. Now was not the time for Saxon's playboy shit.\n\nSaxon finally succeeded in extricating himself from the women. Ronan's attention was drawn away from them, and his desire to feast on their blood, when the distinct odor of garbage filled the air. Saxon's head swiveled on his shoulders seconds before a Savage crashed into his side.\n\nRonan leapt forward, shoving his way through the crowd as the Savage propelled Saxon across ten feet of dance floor and into the exit door. The impact of their weight caused the door to burst open. They both tumbled outside and vanished from view. Ronan heedlessly shoved humans out of his way as he ran forward.\n\nNever had he seen such a brazen attack from a Savage in public before. Some of the humans were still staring at the closing metal door when he pushed them out of his way. A few were creeping closer, trying to see what was going on outside. They scrambled away when he snarled at them before plunging out the door.\n\nHe didn't get a chance to look around before he was assailed with the stench of garbage and something hit him in the side of his head. Staggering to the side, the scent of his blood filled his nostrils as the glasses he'd been wearing fell to the ground. He spun, lashing out at whoever had hit him and catching them under the jaw with an uppercut that shattered bone with a crack.\n\nHe heard the scuffle of another fight, but before he could look for the source, a whistling sound came from behind him. Throwing his arm up, he caught the blade of the sword that would have sliced his head from his body. The blade bit into his palm, slicing deep as he yanked the sword from the grasp of the Savage wielding it.\n\nHis blood coating the weapon made it slippery as he spun it around and stabbed it through the Savage's throat. Lunging forward, he drove it into the wall, shattering bricks and breaking the tip of the blade off. The Savage clawed at the weapon as Ronan twisted it deeper and yanked it to the side, severing the vamp's head from his shoulders.\n\nRonan spun to face three Savages racing at him, their shoulders hunched up and their fangs fully extended. He braced his legs apart and grinned as the first one ran into him. He'd been itching for a fight, and these assholes had delivered it to him.\n\nCapturing the Savage by the shoulders, Ronan lifted him up and slammed him facedown onto the pavement as the second one launched onto him. The thin thread of control he'd been retaining over himself shattered. Red shaded his vision, and his fangs ached to be buried in someone's throat. Death was the only thing that would fill the hole Kadence's leaving had torn into him.\n\nHe didn't recognize the sound that came out of him as he stomped his foot onto the back of the one he'd driven into the ground. He wrapped his hand around the throat of the one clawing at his back and tore the vampire's head off with his other hand.\n\nThe next Savage spun two daggers through his fingers while he eyed Ronan, looking for a weakness. Blood dripped from Ronan's hand as he stepped onto the one still lying on the pavement, unable to move due to his broken back. He stalked forward, ignoring the spinning blades the Savage swung back and forth at him. The daggers whistled as they sliced through the air in front of his face, but he didn't ease in his pursuit.\n\nThe Savage backed steadily away, his eagerness for a fight lessening when Ronan grinned at him. The Savage swung at him. Ronan leaned away from the blade as it sliced so close to his nose he felt the nick of the blade across his skin. When the Savage swung at him again, Ronan clutched his arm and brought it down over his knee, snapping it in half.\n\nThe Savage howled and stumbled back as Ronan pounced on him, sinking his fangs into his throat. Putrid blood filled his mouth and he spit it out. The Savage drove the next dagger at his eye, but Ronan clasped hold of the wrist, halting it before it could blind him.\n\nHe bared down on the Savage's wrist, shattering bone and causing the knife to clatter onto the pavement. The Savage gagged when Ronan punched him in the face, knocking his fangs down his throat. Before the Savage could react, Ronan seized his throat and ripped backward, tearing it out.\n\nHe spun the vamp's head around and tore it from his shoulders before tossing it aside. He whirled to find the others entangled with a handful of Savages. Ronan raced toward the one climbing over Saxon's back and wrenched the Savage away. The vampire's startled red eyes met his before Ronan drove his fist through the vamp's chest and ripped out his heart. He crushed the still beating organ in his hand as the vamp fell before him.\n\nThe scent of the blood and the thrill of the kill had his pulse racing and his temples pulsing with the adrenaline coursing through him. _More_. He needed more death, more blood, more of this mindlessness. It was the only thing that made any sense to him anymore.\n\nHe pulled another Savage off Killean and twisted its head completely around. Killean ran a hand through his hair, pushing it away from his face and streaking more blood over him.\n\n\"Ronan\u2014\"\n\nFootsteps silenced Killean and drew Ronan's attention to the end of the alley. A crossbow bolt fired out of the darkness. He sprinted to the side to avoid taking the hit as more were fired at them. Saxon cursed as one of the bolts hit him in the upper shoulder.\n\nRonan bounded down the alley toward the half a dozen hunters emerging from the shadows. The hunter's blood wouldn't be putrid. No, it would be delicious, powerful. It would finally satisfy the unending thirst he'd been dealing with his entire life.\n\nA small part of him still recognized that once he gave into the bloodlust, it would never be eased, that it would haunt him until he was destroyed. The far larger part didn't care as he slammed into the first hunter. Ronan struck the hunter in the nose, shattering it.\n\nThe enticing aroma of the hunter's blood filled his nostrils and caused his veins to burn with hunger. The hunter's startled blue eyes met his. Ronan hesitated when he recognized it was Kadence's brother, but then he heard the release mechanism of a crossbow and a bolt slammed into his shoulder. Ronan grinned as he dragged the man beneath him.\n\n# CHAPTER 31\n\n\"What happened to you guys?\"\n\nRonan didn't bother to respond as Lucien strolled across the grounds toward them. His body language remained casual, but his gaze raked Ronan's blood-soaked frame from head to toe and back again before his black eyes darted nervously to the others.\n\n\"Savages and hunters,\" Saxon replied.\n\n\"Looks like a bloodbath.\"\n\n\"It was,\" Ronan grated.\n\n\"It was ugly,\" Declan said.\n\n\"I can tell,\" Lucien remarked.\n\n\"How are things here?\" Ronan demanded.\n\n\"I just returned with everyone,\" Lucien replied. \"The recruits are settling into the carriage house and the guest house. All of them decided to come.\"\n\n\"Good.\" Ronan turned away from him. His body still thrummed with his need to kill; he hadn't had enough of it tonight, but that would have to wait until their next hunt. He could feel the eyes of the others boring into his back as he stalked toward the hideous house that had nothing on the hideousness creeping into his veins.\n\nHe threw open the door and walked into the foyer, pausing when the aroma of vanilla drifted to him. Kadence's lingering scent caused a stabbing pain to shoot through him as he strode toward the stairs.\n\nSomething shifted in the shadows and Baldric emerged from the area of the kitchen. Ronan's hand fell onto the banister at the same time his foot hit the first stair. Baldric's eyes widened on him and he stumbled back. Ronan could only stare at him as he tried to figure out why the man was back here.\n\nHad she returned to her brother? Had she been trying to spy on them and betrayed him, or had she run away from Marta and Baldric?\n\n\"Where. Is. She?\" Ronan bit out even as he realized that none of his questions were right.\n\nHe turned away before Baldric could respond and followed the enticing lure of her scent toward the library. Every step had him feeling more keyed up than the one before it. \"Ronan\u2014\"\n\nHe didn't look back at Declan, but he heard his footsteps as Declan hurried after him.\n\n\"Ronan, you're covered in blood. Some of it is her brother's.\"\n\nRonan ignored him as he stepped into the doorway of the library to find Kadence lying on one of the couches with her head resting on her hand and her eyes closed as she slept. Her silvery hair cascaded around her shoulders in waves. The sight of her calmed him even as his body roused to her nearness and his fangs lengthened in anticipation of tasting her.\n\n\"Ronan\u2014\"\n\n\"Leave us be,\" Ronan commanded. Spinning, he grabbed the doors. Declan's troubled eyes held his as he slid the doors closed, shutting Declan out.\n\n\"Ronan?\"\n\nHe turned toward the beautiful sound of her voice to find her eyebrows in her hairline as she surveyed him. The rays of the moon spilling through the windows were the only illumination in the room as she rose. The increased beat of her heart sounded in his ears; her hand went to her mouth.\n\n\"What happened? Are you injured?\" Kadence took a step toward him and froze when he remained unmoving and staring at her as if he didn't know what to make of her. \"Ronan?\"\n\n\"What are you doing here?\" he grated out.\n\nKadence didn't know how to respond, what to say to this man standing across from her. No, not a man. Right now, he was every inch the demon that had created the vampire line. Blood streaked him from head to toe. It coated his hair, making it stand up from where he'd been running his hand through it.\n\nHis torn shirt exposed a swath of his chest that hadn't been spared any blood either. The hole in his upper chest near his shoulder oozed blood, but most of the blood covering him was not his own. He didn't have enough injuries for that to be possible.\n\nShe'd never been afraid of Ronan before, but he looked Savage, and damn if he didn't smell like one of those murderous things too. Her nose wrinkled at the stench wafting off him. She felt every beat of her heart against her ribs.\n\nWhat had happened to him while she'd been gone? What had she returned to?\n\n\"What are you doing here?\" he asked again.\n\nThe tone of his voice, the way the shadows caressed his body, and the fiery glow of his red eyes caused her to gulp and wipe her palms on her pants.\n\n\"I... I came back.\"\n\n\"Why?\" he demanded as he prowled forward.\n\nShe couldn't stop herself from stepping back as he glided across the room with lethal grace. He didn't come at her; instead, he circled her like a shark circling its prey. She was nothing more than the guppy.\n\n\"Ronan, what happened tonight?\" she asked, unable to keep the tremor from her voice.\n\n\"Why are you here, Kadence?\"\n\nThe hair on her nape rose when he uttered the words from behind her. Why did he smell like those Savages who had attacked in the alley? Was it because it was Savage blood on him, or was it something more? Had he given in? Before, she never would have believed it possible; now she shared the room with a lethal predator, and she wasn't sure she knew this man.\n\n\"I'm here because I called my brother from the airport.\"\n\n\"Did you now?\" The words were murmured in her ear, but when she spun to face him, he was ten feet away from her again, circling once more. Despite her excellent night vision, she briefly lost him in the shadows near the back of the room. \"And what did you say to him?\"\n\n\"I... I told him the truth,\" she whispered and stepped away from where she'd last seen him. \"I told him not all vampires are killers. That the hunters have it wrong.\"\n\n\"I see. And why would you do that?\"\n\n\"Because we're trying to accomplish the same thing and working against each other is just going to help Joseph and the Savages rise in power. You need help, the hunters need help, coming together to fight a common enemy will benefit both sides and prevent unnecessary deaths.\"\n\n\"And that's why you're here now?\"\n\n\"Ye-yes,\" she stammered when she felt his breath against her neck once more, and he inhaled her scent. She didn't bother to try to face him; she knew he would be gone before she could turn around. \"Maybe I can help bring both sides together.\"\n\nHis bitter laugh caused her to wince. \"I don't think your brother cared much for what you had to say.\"\n\n\"What happened tonight?\" she whispered.\n\n\"Savages and hunters. Your brother was there.\"\n\nKadence's stomach turned, and a cold sweat coated her body. \"Is... is... did you kill my brother?\" Silence stretched on so long she nearly screamed in frustration as her fingernails bit into her palms. \"Ronan?\"\n\n\"He is alive,\" he replied, and Kadence heaved a sigh of relief. \"Though I didn't want to leave him that way, not after he shot me with his crossbow.\"\n\nKadence took an involuntary step toward where she'd last seen him, but movement to her right alerted her that he'd already moved on. \"Are you okay?\"\n\n\"What do you think?\"\n\n\"No,\" she admitted. \"Something is wrong, and I don't know what it is.\"\n\n\"You sacrificed yourself, Kadence. Sacrificed your freedom because you believe you can help forge an alliance between the hunters and vampires.\"\n\n\"No, not a sacrifice. I never thought of it like that, but right now you're making me feel like one!\" she blurted, unnerved by the creature in this room with her.\n\nShe felt a brush against her nape before his hand clasped it and he held her loosely within his grasp. He had her flight-or-fight response kicked into hyperdrive, yet she couldn't help but reach out to him in some way. She tried to touch his hand, but he was gone so fast that she took a stumbling step back from where he'd been.\n\n\"But you have sacrificed yourself. I told you, don't come back here unless you're ready for an eternity with me. Yet here you are.\"\n\nShe wiped her palms on her pants again. \"Ronan\u2014\"\n\n\"Did you think I was playing with you, Kadence? Did you think my words were an idle promise? Did you think you could come back here, try to play peacekeeper, and then take off again?\"\n\n\"I... I don't know,\" she admitted. \"I didn't know what to expect.\"\n\nShe'd simply known she couldn't get on that plane and abandon her family and Ronan. No, she'd never known freedom before. Yes, there was so much in this world she dreamed of seeing and doing, but she would enjoy none of it if she left them behind. Not if there was something she could do to bring both sides together and possibly save some lives.\n\n\"I couldn't go, not while knowing you and Nathan would be here fighting. I've always been caged, but I was born into this life. How could I abandon everyone I know and love because _I_ want to see the world? How could I live with myself if something were to happen to him or to you? Your deaths would be on my hands, and I couldn't allow that. I would have been back earlier, but the humans weren't exactly pleased that we weren't going to board our flight even though we didn't check any baggage.\"\n\n\"You have no idea what you've unleashed by coming back here.\"\n\nThe words were uttered from her left, but when she turned to face him, she didn't see him there. Then, on her right, he emerged from the dark, stalking toward her like the predator he was. He stopped before her, his ruby eyes burning into hers.\n\n\"I had to come back,\" she said.\n\nHe reached for her before recalling the wound the sword had sliced across his palm and the blood splattering him. \"There are more things you don't know about vampires, Kadence. You have no idea what you are to me, but _I_ do.\"\n\n\"And what am I to you?\"\n\n\"My mate. We will be bonded. I can't and won't let you go again. It nearly drove me over the edge to do so this time; it won't happen again. I'll turn you because I must claim you as mine. If I don't, I will have to be destroyed. I gave you the chance to be free; you should have taken it and never come back here.\"\n\nHer mouth parted on a gasp when he spun away from her and stalked toward the doors. \"I'll give you time to absorb that knowledge, but know you will either become a vampire or I will be destroyed. I can't turn Savage; it would spell doom for everyone, including you.\"\n\nHe threw the doors open and strode away before she could form a response to his proclamation. What had she done by coming back here? He'd told her not to come back unless she was prepared for an eternity, but she hadn't really believed him when he'd said that, or had she and it was what she wanted too?\n\nBeing in his presence again had caused her skin to stop crawling, and her chest no longer felt like someone was trying to rip it open. He'd unnerved her with his strange behavior, and his declaration was frightening, but she finally felt almost normal again.\n\nWhen she was with Ronan, she felt like she'd finally found her home.\n\nBut become a vampire? She hadn't been expecting that.\n\nWhat else would eternity mean, you idiot?\n\nShe was staring at the doorway and trying to puzzle that out when Declan appeared. \"Are you okay?\" he inquired.\n\n\"Yes.\" Declan started to turn away from her, but she stopped him before he could leave. \"Declan?\"\n\n\"Yeah?\" he asked over his shoulder.\n\n\"Would he really have to be destroyed if I left here again?\"\n\n\"If he allowed himself to be taken down by one of us, yes, he would have to be killed. Ronan is the strongest creature in existence on this planet. If he turns Savage, we're all doomed. I told him it was a horrible mistake for him to let you go without telling you all this to begin with. I like you, Kadence, really I do, but make the right choice here, and make it fast, or I may turn you myself. Ronan will kill me for it, but if you're already a vampire, he can complete the bond with you after. I will do what must be done to save his life just as he's saved mine countless times over the years.\"\n\nKadence gawked after him when he vanished from the doorway. She'd stepped into a steaming pile of crap, but no matter what happened now, what she became, she'd made the right choice by coming back here. She could save lives, and she would. Even if she lost hers in the process.\n\n# CHAPTER 32\n\nNathan held the icepack to his nose as he paced back and forth within the living room of the brick house. His head pounded from the beating he'd taken and every breath he took whistled in and out of his nose, but he was alive.\n\nAnd for the life of him, he didn't know why. That vampire had had him dead to rights. He should be taking a dirt nap right now. Instead, he was drinking scotch in the hope of numbing the soreness of his body and trying to figure out why that vamp hadn't torn his throat out.\n\nNo, not _that_ vampire, Ronan. That was what one of the other vamps had called him when he'd thrown Nathan away from him and taken off with them. _Ronan_. The same Ronan that Kadence had mentioned?\n\nIt had to be, as it was definitely the same vampire who had been in the alley the night Jayce was killed. None of it made any sense, and trying to figure it out only made his head pound more. The guy had beaten the snot out of him, and Nathan had shot him with his crossbow, yet his throat remained intact.\n\n\"I don't get it,\" he muttered.\n\nLogan and Asher didn't say a word from where they sat on the couch, nursing their own wounds. There was nothing for them to say. None of them understood what had happened tonight or in that alley the first time they'd encountered Ronan and those other vampires. Maybe the first time he could have written it off as the vamps had decided to retreat, but he couldn't tonight. Yes, the vamps had left, but they could have easily killed them before they departed.\n\nHe'd felt the power emanating from Ronan, and it had rocked him even before the punch to his nose had. Never had he encountered anything like that. There was no denying they should all be dead, yet they remained standing, or mostly standing anyway.\n\n\"Maybe it's a game they're playing or something,\" Asher muttered.\n\nNathan hadn't told them about Kadence calling him again. It was a sensitive topic for Logan that she'd fled in the first place. Plus, his sister hadn't exactly sounded like someone they could trust when he'd spoken with her, and no matter what she'd done, he didn't want anyone thinking badly of her when she finally did come back.\n\nNow, he knew he had to tell them about the call. He couldn't figure this out on his own, and he could trust them not to go to the elders with it. If they did, Kadence may never be welcomed back here again.\n\nNathan finished off his scotch before turning to face them. \"There's something I have to tell you. It's not going to make any sense, but you have to know. Kadence called me earlier.\"\n\nLogan winced and rubbed at his chest, but he raptly listened as Nathan repeated the conversation he'd had with his sister.\n\n***\n\nRonan slid the doors to the library open and glanced around the dimly lit room. Someone had closed the shutters over the windows. He had a feeling it was Kadence, who still believed the sunlight would burn him. She was curled up on the sofa, her hand tucked beneath her head and her chest rising and falling with her soft inhalations as she slept soundly.\n\nIt had been hours since he'd last seen her, since he'd felt in control of himself enough to come anywhere near her again. His eyes were still red; he believed it might be a permanent condition until the bond between them was complete.\n\n_She came back!_ He'd been too geared up last night, too covered in death to really grasp that concept. Standing there gazing at her, the realization hit him fully now. She may not have been prepared for what she'd walked into, but she'd come back to him.\n\nHe couldn't tear his gaze away from her as he walked across the room to kneel at her side. She looked like an angel as she slept, her face serene. He stroked her cheek, relishing the satiny feel of her skin beneath his as she calmed him further.\n\nHer sweeping lashes fluttered open, her azure eyes were dazed as she stared at him. Then, a smile curved her lush mouth. \"Hello,\" she murmured.\n\nHe couldn't help but smile back at her. \"Hello.\"\n\nHe slid his arms beneath her and lifted her from the sofa. She draped her arms around his neck and nestled closer against him. Her head fell into the hollow of his shoulder as he carried her from the room and up the stairs.\n\n\"Why didn't you go up to your room?\" he asked her.\n\n\"I wasn't sure I still had one here.\"\n\n\"You'll always have a place with me, Kadydid. _Always_.\"\n\nShe rested her lips against his neck. \"You're much calmer now.\"\n\n\"Do not be fooled. I'm barely in control, but I had to see you.\"\n\n\"At least you're not covered in blood anymore.\"\n\nHe bent his head so that his lips rested against her cheek as he spoke. \"You never should have seen me like that.\"\n\nTilting her head back, her eyes searched his as she heard the self-loathing in his voice. \"I know what you are, Ronan. You've never denied it. I know you're lethal, brutal, a killer.\"\n\n\"Hmm.\"\n\n\"It was the smell that had me worried.\"\n\nHe froze in the middle of the hall. \"The smell?\"\n\n\"Yes, you smelled like them.\"\n\n\"Like who?\"\n\n\"Like the Savages.\"\n\n\"And what do they smell like?\" He knew what they smelled like to him, but to learn they may also smell to a hunter wasn't something he'd expected.\n\n\"Like garbage,\" she murmured. \"Like death. It's not a strong aroma, but it's there. I smelled it that first night in the alley, then with Joseph, and again on you last night. It must have been because you had their blood on you.\"\n\nRonan was too shocked by the revelation to continue forward for a minute. The hunters could smell the difference too, not even a turned vampire had the ability to smell the Savages, only purebreds did, and now hunters. It didn't sound as if she smelled the aroma as strongly as he did, but perhaps there was more demon in the hunters than he'd realized.\n\n\"I see. Do other hunters smell it?\"\n\n\"We assumed it was what all vampires smelled like, something else we were wrong about,\" she said as she rested her head against his chest and placed her hand over his heart. She loved the solid pulse of it beneath her palm. \"You smell like cinnamon and the ozone scent of power. I like it.\"\n\n\"I'm glad you like it,\" he said and kissed her ear. \"To smell the difference between us is an unusual ability to have. All purebred vampires can smell the foulness of the Savages, but the turned ones can't.\"\n\n\"You smell it?\"\n\nHe smiled at her as he continued walking again. \"I do. For every human life a vampire takes, the scent increases. When I was forced to kill hunters before, I acquired the smell for a while. It has faded over the years.\"\n\n\"So hunters can tell the difference between Savages and those who aren't, and we don't know it?\"\n\n\"It seems that way.\"\n\n\"I will talk to Nathan again and tell him.\"\n\n\"After last night, I'm not sure your brother wants to hear anything you have to say about vampires.\"\n\n\"Did you hurt him?\"\n\n\"I did, but he will live, and he deserved it.\"\n\nShe didn't flinch away from his blunt words. Her fingers rested against the fading red mark from where a bolt had pierced him. Her brother and her lover could have killed each other last night over a complete misunderstanding. This insanity had to stop.\n\n\"Nathan injured you too.\"\n\n\"Barely felt it.\"\n\nShe sniffed. \"Tough guy, huh?\"\n\nHe didn't tell her that he could take a bolt to the chest and barely feel it, yet the idea of losing her again could knock him to his knees. He nudged the door to her room open with his foot.\n\n\"I can talk to Nathan. I _can_ make him listen, make him see reason,\" she insisted.\n\n\"Not today, Kadence. One battle at a time.\"\n\n\"I couldn't stand it if something happened to either one of you when I might be able to stop it.\"\n\nWhether she realized it or not, she'd just admitted to caring for him. He didn't know what the strange sensation filling his chest was, didn't know how to react to it as it robbed him of his breath.\n\n\"Is there anything else about vampires we have wrong?\" she asked.\n\n\"There are other things,\" he replied.\n\n\"Such as?\"\n\n\"Not now.\"\n\n\"You still don't trust me?\"\n\nHe set her on the bed and stepped away. \"There are many things I have to tell you, but there is something else we need to get straight between us first.\"\n\n\"This mate thing?\"\n\n\"This mate thing,\" he confirmed.\n\nTaking a deep breath, she gripped the edges of the mattress. \"Tell me about it.\"\n\n\"I can't let you go again. I need you to stay here, with me.\"\n\nShe didn't understand the raw need she saw in his eyes, but it touched something primal inside her. Rising, she rested her hands against his cheeks and met the red of his eyes as she pulled him down to her.\n\nThe minute his lips touched hers, she knew what the awful tightening of her skin had been\u2014her need for _him_. Her heart swelled, and her body rejoiced as heat pooled through her limbs and she relaxed into his kiss.\n\nIt didn't matter what the future held; she never wanted to leave him again either.\n\nHis arms wrapped around her. Kadence's fingers slid through his hair to draw him closer. His hands swept over her back, bunching her shirt in his grasp and lifting it up her sides.\n\nHe pulled back suddenly, breaking the kiss. Kadence couldn't catch her breath as she gazed at him while he clasped her cheeks in his palms. He stared wordlessly down at her, his face harsh with his barely leashed restraint.\n\n\"We cannot do this, not right now.\" His voice was hoarse and his accent more noticeable again. \"I could hurt you if this continues.\"\n\n\"No, you couldn't.\"\n\nHe exhaled loudly. \"I _can_ hurt you, and it's a chance I'm not willing to take.\"\n\n\"Then I think it's time you fully explain this mate thing to me.\"\n\nHe reluctantly released her and moved as far away from her as he could. Leaning against the wall, he stared at the shuddered window as he spoke. \"Sometimes a vampire discovers what is known as their mate. We don't know why it happens, why some of us find them while others don't, but it is most likely tied in with the demon part of us.\n\n\"When a vampire encounters their mate, they experience an instantaneous connection to whoever their mate is. The vampire may not realize it is the mate bond at first, I didn't, but I was drawn to you from the second I saw you. When the bond between mates is completed, it can only be severed by death. And if it is severed by death, the remaining mate will either die or go mad. When vampires find their mates, the consequences can be volatile until the bond is completed, especially if one of them is mortal.\"\n\nShe folded her hands in front of her when he started pacing before the window. \"And what happens if one of them is mortal?\"\n\n\"They must be turned into a vampire. There is no other option. It is the only way to calm the vampire again, and it is necessary to seal the bond between them.\"\n\n\"And how is the bond sealed?\"\n\n\"One of the steps is through sex, which is what escalated the mating bond within me and brought it to the forefront.\"\n\n\"Oh,\" she murmured, unable to keep the blush from her cheeks. \"That's why you reacted like you did afterward.\"\n\n\"Yes. Not only did I want to feed from you, but I also wanted to change you, right then.\"\n\nKadence gulped at his words and clenched her hands tighter as she realized how close he'd been to hurting her as Joseph had.\n\n\"If both mates are vampires, then they will exchange blood during sex. That will finish creating the bond and opening the pathways between their minds, which will allow them to communicate with each other,\" Ronan continued. \"It will also unify them for as long as they live.\"\n\n\"What if the mortal doesn't want to be turned?\"\n\nHis eyes swung toward her and she found herself staring at the vampire who had been in the library with her last night. The muscles in his arms and chest stood starkly out as his lips skimmed back. In amazement, Kadence watched his fangs descend. She'd seen them before, but she'd never seen the lengthening of his canines in such a way.\n\n\"Then the vampire will go mad, turn Savage, and have to be killed.\"\n\n\"And what happens to the vampire when the bond is completed and the mortal becomes immortal?\"\n\n\"They regain control of themselves, for the most part, but if their mate is ever threatened, there is no control.\"\n\nKadence imagined he'd be Hell on earth in such a scenario.\n\n\"The mating bond also makes both the vampires stronger,\" he continued. \"Most likely because, not only does at least one of them continue to feed on humans, but also because they are both feeding from another vampire on a consistent basis. Plus, many mates are deeply in love with each other, and I've been told there isn't anything one won't do for love whether they are vampire or human.\"\n\n_Love,_ she thought as she watched him pacing a hole into the rug. She cared for him, deeply, but love? _Just get through one thing at a time,_ she told herself. _You can figure that out later._\n\n\"What do you mean by at least one of them continues to feed on humans?\" she asked.\n\nHe stopped pacing and turned to face her. \"At least one vampire needs to still have fresh blood from an outside food source, but the other can survive solely off the blood of their mate, if that is what they chose to do.\"\n\n\"I see. And you truly believe me to be your mate?\"\n\n\"From the moment I saw you, I wanted you, and believe me, that is not something I've experienced in years, and I've _never_ experienced it as strongly as I do with you.\"\n\n\"What?\" she blurted. \"I'm sure you've seen and desired thousands of women over the centuries.\"\n\nHe had the nerve to chuckle.\n\n\"I have,\" he confirmed. \"And when I was younger, I found a diversion in those women. As I got older and the years wore on me more, my need for death and blood started to outweigh my desire for women. Eventually, all I cared about was killing and sating the demon within me.\"\n\n\"I'm not really sure what you're telling me. All you want to do is kill?\"\n\n\"The demon DNA that lurks within all vampires is stronger in purebreds. We are more closely tied to the demon than a turned vampire is, and we have more of the demon's traits. When a male purebred becomes fully mature, the demon becomes this insatiable, clawing thing that is only caged through strength of will, or the finding of a mate. Some set it free and let it do as it will and become Savage; others fight it every day of their lives. The females can experience it too, but not on the same level or with the same intensity as the males. I am assuming this was the way of the demons too.\n\n\"In each male purebred, the insatiable demon shows itself in different ways. Some seek out pain, others copious amounts of blood, others want death, some can't have enough sex, some lock themselves away from humans, and others give in and become Savage to make it stop. Turned vampires can experience it too, but not as strongly as purebreds do.\n\n\"In all purebred males, maturity means they stop aging, their power increases and continues to do so as they age, and they hunger for things to the point of madness,\" he continued. \"Many experience a combination of heightened urges, but there is usually one that is more dominant than the others.\n\n\"For me, that dominant thing is death and blood. I am a bloodthirsty killer, and I enjoy it. I've focused my impulses on hunting Savages, and it has kept the madness at bay for a millennia, but every year it has become increasingly difficult to keep myself in check. Every year, I feel the madness seeping in more and more. And with every year, I stopped wanting women and craved the blood more, until _you_. I want you a thousand times more than I want to kill, which is something I _never_ believed I'd be able to say.\"\n\nKadence bit on her lip as she tried to understand the battle he'd been waging against himself. She couldn't imagine what it must be like to live only for death, or how hopeless some days must feel for him. Yet, she brought him some hope.\n\n\"You could never be like one of those Savages,\" she murmured.\n\n\"I could and I will, without you. I've been more out of control since we had sex, and that is because the bond isn't complete, Kadence. The rest of it must be finished, and you will become a vampire when it is done.\"\n\nHer hand instinctively flew to her neck when his eyes latched onto it. \"No,\" she said as memories of the suffering Joseph had inflicted on her burst through her mind.\n\n# CHAPTER 33\n\nRonan didn't like what he would have to do to her, didn't want to bring her any pain, but that is all he would bring to her when she went through the change. There would be no stopping that. He would give anything not to have her become a part of his world, but he couldn't change what had been set into motion, not now that she'd returned here.\n\n\"I am sorry, Kadence,\" he said. \"I would change this if I could.\"\n\nThe fear in her eyes was nearly his undoing. \"It will hurt.\"\n\nNever in his life had he felt this helpless and inadequate. \"The taking of blood isn't painful, if you're willing. It will be nothing like what you experienced with Joseph if you don't fight me. It's extremely pleasurable to those who are willing.\"\n\n\"Have you ever allowed another to do it to you?\" she demanded, not at all liking the idea that another had known him in such a way.\n\n\"I've never allowed another to feed from me, but I do know that those I've fed from have never been harmed by it. Some enjoyed it nearly as much as sex, some more so. You will be the first and only vampire who will ever take my blood.\"\n\nShe recoiled from him. \" _Me_? Drink blood?\"\n\n\"That is what vampires do.\"\n\n\"I know that's what you've meant this entire time, but I never really _thought_ about what being a vampire completely entailed.\"\n\nShe had no idea how to react to anything he was telling her. A vampire, her? Tied to him for an eternity? What if he annoyed her or treated her badly?\n\nHe was abrasive, demanding, and used to getting his way. She was certain he would annoy her numerous times over an eternity, but he would never treat her badly. That she knew with absolute certainty. But to drink blood? The idea made her stomach turn. However, when she looked at him again, she had to admit that it didn't seem as repulsive if it was _his_ blood. She found herself curious to know what it would actually taste like.\n\nShe found herself licking her lips before she shook her head to clear it of the absurd notion. \"This is all so much,\" she muttered.\n\n\"I should have told you this before you left here. You might have stayed away if you had known the consequences of your returning, but I didn't want you to have this knowledge hanging over your head. I wanted you to truly know freedom.\"\n\nHe would have sacrificed himself for her; he had, in a way. Her heart swelled with emotion as she gazed at him. Love, she loved this man. She didn't know when it had happened, but it had, and it didn't matter if she hadn't known him for long or not. She loved him, but was she ready to give up everything she'd ever known for him?\n\nShe contemplated Declan's promise to change her if she didn't allow Ronan to do it. He knew Ronan would kill him if he did it, but Declan cared enough for Ronan to die for him; everyone in this place did. That said more about him than anything else, she knew.\n\n\"If there wasn't some kind of mate connection between us, would you even like me?\" she asked.\n\nShe didn't know why she required the answer to that, but she did. She respected and admired him, and she wanted the same from him.\n\nHis mouth pursed, the lines etching the corners of his lips revealing his strain. \"What do you mean?\"\n\n\"I mean, if I had been a normal woman who you met, would you like my personality? Would you like who I am and want to get to know me better, or would you find me annoying?\"\n\nFor the briefest of seconds, brown swirled within his eyes again as he strode across the room and cupped her cheek. She turned into his hand, nuzzling his palm and the slice in his flesh from another wound he'd sustained last night.\n\n\"Yes, Kadence, even without the mate bond I'd want to get to know you better. You're unlike anyone I've ever met before. You have more courage than most of the men and women I've come across in my lifetime. Though there have been times when you've annoyed me.\"\n\nHis lips quirked at these last words and she smiled back at him. \"The feeling is mutual.\"\n\n\"I'm sure it is. You must know, I will protect you and our children with everything I am.\"\n\nShe couldn't breathe as she lifted her hand to clasp his against her cheek. \"It is difficult for a hunter to conceive. Whether it's on the male's part, the female's part, or perhaps both, we can often go a lifetime without children.\"\n\n\"Then I will protect _you_ with all that I am for every day of our lives together. I am the only purebred who has five generations of purebreds as ancestors before me. None of them were mated, they simply bred to create a stronger line. At one point, such a thing was expected of me, but that was centuries ago. Over the years, it has become an expectation that I never planned to fulfill.\"\n\n\"What changed?\" she asked. \"Why have you not fathered children?\"\n\n\"After the Savage attack that killed my parents, I was more focused on revenge, on trying to protect the vampires who remained and slowly rebuilding the Defenders, than on trying to have children. I never took the king title, so I didn't see it as necessary to have an heir. Then, as time went on, I had no interest in women or having a child, until now.\"\n\n\"But\u2014\"\n\n\"It doesn't matter if we don't have children, Kadence. All I want is you; a child would only be a bonus.\"\n\n\"I'm scared of this.\"\n\n\"I know, and you have every right to be. My bite will not hurt if you don't fight it. The transition will though. It's excruciating for a mortal to become immortal, but you will never have to experience it again once it's over. It may also be different for you, given what you are. You already have some demon DNA in you, the transition could be worse.\"\n\n\"Or it could be easier.\"\n\n\"It could.\"\n\n\"What would become of me if we are mated and you turn Savage?\" she asked.\n\n\"I will not turn Savage, not while I have you. The bond between us will keep me grounded; it will be the thing I want most in this world. Not blood, not death, _you_.\"\n\nShe sucked in a breath and bit her lip at this revelation. Then, she recalled Declan's words from before. _\"Fate is a fickle bitch, and sometimes she takes even the best of us down, but sometimes she also intervenes to save us.\"_ It suddenly made sense to her now, as did his reason for not intervening when she decided she wouldn't return to the stronghold.\n\nDeclan was hoping to save Ronan from becoming a Savage, and he believed she was the key to keeping him sane.\n\nHer fingers fiddled with the edges of her sleeves as she tried to process everything. She'd never feel the sun's warmth again, she'd have to feed on blood for the rest of her life, and the hunters may all turn against her forever. No matter what Ronan said, she thought he might let her walk away again if she asked him to let her go.\n\nAnd then what? She couldn't return to the stronghold, she wouldn't have Marta and Baldric to help her go somewhere else this time, and she would destroy Ronan if she left. She cared too much for him to inflict that on him. But was she ready to die and be reborn as the one thing she'd been raised to hate? Was she ready to give up the sun and drink blood?\n\nHer gaze went to the shuttered window. No matter what she decided, she had to feel the rays of the sun on her one more time.\n\n\"I can give you time to process this,\" he said.\n\nShe tore her eyes away from the window and back to him. \"I would really like that.\"\n\nHe hesitated for a minute before caressing her cheek with his thumb. She caught his hand when he went to pull it away.\n\n\"If we had been a normal man and woman, I would have chosen you,\" she told him.\n\nHis hand enclosed on hers. \"Take the time you need,\" he murmured, and before she could reply, he was gone.\n\n***\n\nKadence moved swiftly down the hall. She glanced behind her at Ronan's closed door, but she knew he wasn't in there. He hadn't entered his room when he left hers. She didn't know where he'd gone. She descended the stairs and hurried to the front door. The gate wasn't blocking it this time.\n\nShe glanced up at the cameras on either side of the door, but no one emerged to stop her when she continued toward it. Grasping the knob, she turned it and pulled it open. Sunlight spilled across her as she walked outside and lifted her face to absorb its warmth.\n\nA few birds sang in the barren trees lining the drive. Their melodious songs floated across the vast expanse of snow-covered lawn. The lawn rolled forth as far as she could see. Trees were spaced throughout, and though they had no leaves, she'd spent enough time wandering the stronghold and studying nature books to know that they were maples, pears, cherries, apples, and oaks. The leaves and flowers on them would be breathtaking in the spring.\n\nIf she didn't look at the house, it was actually beautiful here.\n\nA large garden was set around a giant fountain in the center of the yard. The plants within were all bare or buried beneath the snow, and the fountain was off, but she was drawn toward it. She stopped at the edge of the fountain and slid her hands into the sleeves of her coat as a breeze blew over the land, lifting snow and swirling it around her. She smiled as she studied the marble cupid in the middle of the fountain. She'd bet anything Ronan _hated_ it.\n\nShe looked at the sky again, her head falling back as she threw her arms wide and closed her eyes. Over the years, the sun had never failed to make her feel better if she was sad or lost, and this may be her very last day experiencing its rays.\n\nTears brimmed in her eyes and slid down her cheeks.\n\n***\n\n\"What is she doing?\" Declan asked as he unwrapped a lollipop and stuck it in his mouth. Ronan had no idea why he'd started sucking on the candy a couple months ago, but it had become his new thing.\n\nDeclan plopped his feet on the desk and leaned back in the desk chair. \"I'm not sure,\" Ronan replied as he focused on Kadence making her way toward that hideous fountain.\n\n\"Perhaps the mortal simply needed fresh air,\" Saxon suggested.\n\n\"Or perhaps she's going to make a run for it,\" Lucien drawled.\n\n\"Probably straight into the enemy's arms,\" Killean said.\n\n\"She's not going to make a run for it,\" Declan replied and bit down. \"Ugh.\" Grabbing the trashcan, he pulled it forward and spit the remains of the lollipop into it.\n\n\"What is it with you and those human things?\" Lucien demanded.\n\nDeclan set the can down and leaned back in his chair again. \"I'm trying to figure out how many licks it takes to get to the center, but I keep biting into it before I get there.\"\n\n\"Why on earth are you doing that?\" Saxon asked.\n\n\"Might as well solve the mysteries I can in this world,\" Declan said.\n\nRonan sensed more behind the man's words and his newfound obsession with human snacks than Declan was saying, but he let it go.\n\nTears streaked Kadence's face as the sun glistened upon her.\n\n\"Why is she crying?\" Lucien asked.\n\n\"Shit,\" Ronan hissed as realization sank in.\n\n\"What is it?\" Declan inquired.\n\n\"She doesn't know,\" he murmured. \"I didn't tell her.\"\n\n\"Tell her what?\" Saxon asked.\n\n\"That we are able to go out in the sun.\"\n\nDeclan's feet hit the floor as he spun to face Ronan. \"How could you _not_ tell her that?\"\n\nRonan scowled at him. \"I wasn't going to reveal everything to her, and then I just... forgot.\"\n\n\"You should go tell her before she decides she likes the sun more than you. Which, she might,\" Lucien said.\n\nTurning on his heel, Ronan stalked out of the security office where he'd been meeting with the others about their next step in trying to locate Joseph. He cursed himself with every step he took to the front door. The sun's rays hit him as soon as he opened the door, and the cool air brushed over his skin as he strode forward.\n\nKadence no longer stood by the fountain, but he followed her scent down a trail leading through the privet hedges lining the seashell walkway. At the end of the privet was a far larger garden. It covered at least three acres of land, though there was little to see now except for some heather, lavender, and fountain grasses.\n\nHe stopped short when he found her at the end of the walkway, gazing at the remains of the gardens. Seeming to sense him, she turned and her face lit with a smile. Her hand was stretching toward him when it fell back to her side and her mouth dropped open. Her gaze ran over him as if she expected him to burst into flames at any second, and she most likely did.\n\n\"Ronan?\" she squeaked.\n\nHe strode toward her, took her hand, and bent to kiss her. Her eyes remained on his as their lips touched and her breath rushed out of her.\n\n\"How is this possible?\" she inquired when he rose away from her.\n\n\"Only the Savages can't go out in the sun,\" he told her as her eyes ran over him again and then again. \"With each kill, they become stronger, yet they also become weaker.\"\n\n\"But... but... you can be out in the sun?\"\n\n\"I can,\" he replied. \"There was a time, after I killed those hunters, that it was more difficult for me, but that time has passed. I no longer experience any ill effect from the sun's rays.\"\n\n\"We were so wrong,\" she murmured, her skin paler than normal and her lower lip trembling. \"I don't have to give up the sun if I become a vampire?\"\n\n\"You don't,\" he assured her.\n\n\"You _have_ to let me tell Nathan about this, let me talk to him. The two of you should meet. I will arrange it.\"\n\n\"I doubt your brother would want to see me again, Kadydid. We didn't exactly part on good terms.\"\n\n\"I can help to fix that; let me do this.\"\n\nHe pondered her words as he ran his fingers over her cheek, savoring every detail of her. \"Why don't we do one thing at a time and figure us out first?\" he suggested.\n\n\"I've figured that out,\" she said and stepped closer to him. Her arms slid around his waist as she rested her head on his chest. \"Whatever this is between us scares me, but I won't lose you. You have to agree to let me speak with my brother though when this is done.\"\n\n\"Kadence,\" he breathed, kissing her neck as he held her to him. \"I will not deny you that.\"\n\n\"You also have to promise you won't lock me away. I realize we can't go traveling all over the world right now, but I won't be confined again. I want to help hunt Joseph and stop this growing threat.\"\n\n\"You are not ready for a fight like that.\"\n\n\"You will keep training me,\" she insisted. \"And as a vampire, I will probably be faster and stronger. You also said, as a mated vampire, I would be stronger than other vamps. Don't cage me, Ronan. It will ruin us if you try to lock me away. I am a hunter, and I will hunt.\"\n\n\"The last thing I want to do is ruin us or make you unhappy,\" he admitted as he ran his lips down the slender column of her throat. \"But you will agree to listen to me when it comes to Joseph and the Savages.\"\n\n\"I will,\" she promised.\n\nHe lifted her against him, and she snuggled closer to his chest as he carried her across the property toward the house. His heart hammered with every step, his cock swelling with its need to be inside her made walking difficult, but he didn't slow.\n\nHe shoved the front door open and stalked across the foyer to the stairs. He took them three at a time to the top and sped down the hall to his room.\n\n# CHAPTER 34\n\nKadence lifted her head from his shoulder to study the angles of his face. She sensed the demon simmering just beneath his surface.\n\nShe should be afraid. She wasn't. She'd made her choice; Ronan was her destiny. She wouldn't go back, and she wouldn't dread what was to come. He would protect her, and she would love him.\n\nHe set her on her feet before kicking the door shut behind him. Clasping her cheeks, he bent to kiss her. A wave of emotion built within her, warming her to her core as love burst through her. Entwining her fingers in his hair, Kadence kissed him hungrily back. Her body clamored for his as she realized that she never would have been able to leave him again. Her fate had been tied to his since she'd laid eyes on him. Perhaps it had been sealed from the moment of her birth.\n\nBreaking their kiss, he pulled off his shirt before clasping the edges of hers and sliding it up to reveal her body to his voracious gaze. The demon within him clamored to possess her as he pulled off her boots and socks before kicking off his own.\n\nConcerned he would hurt her, he kept his darker impulses leashed, but he was unable to stop himself from tearing her pants off her body. She gasped at the rending sound of the fabric, but she didn't pull away from him when he reclaimed possession of her mouth.\n\nHer hands slid across his shoulders before trailing over his chest and down to his waist. Her touch seared through him and burned its mark upon every inch of his flesh. He'd been in a rush to possess her, but he realized then that she already possessed him in every way.\n\nHe shook with his need to be within her while slaking his thirst on her blood. Her fingers gripped the button of his jeans, and she slipped it free before pulling the zipper down. Ronan groaned as she pushed his jeans lower and freed his dick from the confines of his jeans. His body jerked when she gripped his shaft and stroked him the way he had taught her to. His teeth grated together as she squeezed him, causing him to nearly come in her hand.\n\nKadence watched in awe as his head fell back against the door. The corded muscles of his neck stood out as he thrust his hips in rhythm to the motion of her hand. She couldn't help but marvel at the power she had over this man\u2014a man who was far stronger than her, or anyone she'd ever known, yet he was _hers_. The possessive urge the thought brought forth set something free within her, something she'd never known existed before, something savage. He was hers, and she would kill anyone who tried to take him from her.\n\nBending her head, she ran her tongue over his chest, tasting the saltiness of his flesh as his cinnamon and ozone scent engulfed her senses. She was wet and aching to be filled by him when she nipped at his flesh.\n\nRonan jerked against her when she bit him lightly before swirling her tongue over the bite. He growled as she bit him again. This time, hard enough to draw blood.\n\nShe licked it away before she bit again, drawing more blood forth. His fingers threaded through her hair as she mewled and her hands dug into his shoulders. She released his dick and bit him again.\n\n\"Harder, Kadence,\" he said, fisting her hair tighter as he held her closer.\n\nUnable to resist his command, she bit down more forcefully and swallowed his blood.\n\nHe felt the unraveling within her, the growing desperation as her fingernails scored him. He didn't know what had unleashed this need within her, but he was helpless to deny her what she wanted as she bit him again.\n\nOne second, he was leaning against the door, the next his hands were on her hips and he was lifting her. He kicked his jeans from around his ankles seconds before he lowered her onto the swollen length of him.\n\nKadence cried out as he stretched and filled her, completing her. She froze, her eyes locking on his while he stared raptly at her. He was the most magnificent thing she'd ever seen or experienced as he remained buried deep within her while the potent taste of his blood lingered on her lips.\n\nShe had no idea what was wrong with her; she should be repulsed to be yearning his blood in such a way, but she wanted more of it, and him. She'd never tasted anything as delicious as it was, hadn't believed it possible blood could be so sweet.\n\nHer hands cupped his face as she bent to kiss him. A thrill went through her when she ran her tongue over his fangs, feeling the sharp edges of them. He nipped at her lip before drawing it into his mouth to soothe the sting.\n\nThen, his fang pierced his lip, drawing his blood forth. She sucked on it as she laved the puncture. Being buried within her and his ability to feed her calmed the demon within him. Ronan's hands tightened on her hips and he braced his legs apart as her body moved fluidly against his, yet he sensed something more within her, a hunger he'd never expected as he fed her more of his blood.\n\nPulling away, she studied his face. Her lips, swollen from his kisses, were also tinged red with his blood. Her tongue slid out to lick it away, and she shuddered against him.\n\nShe kept her eyes on his as she rose over him again before sliding slowly back down. She was magnificent, and she was his. Completely and utterly _his_. He bent his head to one of her nipples, and rolled his tongue over it as he sucked on her. Her body bowed and her head fell back as she thrust harder against him.\n\nKadence clung to him as if nothing outside the two of them existed anymore. The building tension in her belly and between her thighs splintered apart when his fangs grazed her breast before his lips tugged at her nipple. She cried out, her fingers digging into his shoulders as she came apart.\n\nPanting for breath, her head fell into the hollow of his shoulder. The enticing scent of her orgasm filled the air, but Ronan held back his release. He intended for this to last longer.\n\nHe nipped at her breast again as he strode across the room. Each of his steps drove him in and out of her. She moaned against his shoulder when he laid her on the bed. He remained deep inside her as he climbed on top of her.\n\n\"We're not done yet, love,\" he assured her.\n\nHe braced his arms and withdrew from her before burying himself deep again. Her mouth parted, and her legs wrapped around his waist as she lifted her hips and eagerly took his next hard thrust. He knew he was unraveling, that he should be gentler with her, but he couldn't stop himself from driving harder and faster into her. She propelled him heedlessly onward as her body rose and fell to match his pace. The demon burst free of its cage as he took her with a ferocity he couldn't stop.\n\nKadence held onto him as she felt the unraveling within him and she knew she was the only one who could ease him. He plunged into her again and tossed his head back as his arms went rigid beside her head and a guttural shout escaped him. The pulsations of his release filling her brought forth an answering orgasm from her and she cried out.\n\nFalling back on the bed, she marveled over the beauty of his chiseled body, dampened with sweat, as he remained rigid above her. Slowly, his head came back down and his red eyes met hers. His hair was tussled as it fell almost boyishly across his forehead. There was nothing boyish in the vampire above her as the demon within him was on full display.\n\nWhat had transpired between them had done nothing to ease his need for her. Instead, it had only whet the demon's appetite for more.\n\nKadence braced herself for what was to come. However, he didn't strike as he stared unwaveringly down at her and kept her body caged beneath his. He didn't so much as breathe. She knew it was taking all he had not to pounce on her and take the rest of what he needed from her.\n\nShe loved him even more for it.\n\n\"It's okay,\" she murmured. \"Don't fight it anymore. I want this too.\"\n\n\"The transition is difficult, Kadence, prepare yourself for that, and I have no idea what it will be like for a hunter.\"\n\n\"I know,\" she whispered and brushed back a strand of his hair.\n\n\"Kadence\u2014\"\n\n\"I have made my decision, Ronan. There is no going back now. This can't be changed for either of us. Whatever happens, we will deal with it together.\"\n\nHe gazed wordlessly at her as some new emotion came to life within him. He wasn't sure what this feeling was, but she was the only one who had ever brought it out in him.\n\nKadence smiled at him as the red bled away from his eyes and she found herself staring into a pair of beautiful, deep brown eyes once more. \"There's the Ronan I love,\" she whispered.\n\n# CHAPTER 35\n\nHis mouth parted on an explosive breath. He stared at her as if she'd punched him in the gut. \"Love?\" he choked out.\n\nKadence took a deep breath, bracing herself against the sorrow that would follow if he rejected her. \"Yes, I think... no, I _know_ I love you.\"\n\nHis body softened around hers as one of his hands clasped the silken strands of her hair. \"I will spend forever trying to be someone who deserves your love.\"\n\n\"You already deserve my love.\"\n\nHe lowered his forehead so it rested against hers and inhaled her enticing scent. She humbled him in ways he'd never expected to be humbled in his life. He was determined she would never again feel trapped and lost. Never again be unhappy.\n\n\"I may not be able to give you everything you dreamed of right now, but one day you will have everything you've always wanted. One day, when things settle some, I will take you anywhere you ask to go. I will make you the happiest woman in the world, Kadence.\"\n\nHer hands ran over his sweat-slicked back as tears burned in her eyes. \"I am happy,\" she whispered.\n\n\"Good.\"\n\nShe shivered with delight when his arms tightened around her and she felt him harden within her once more. Her body quickened in response to his arousal. Wrapping her hand around the back of his head, she turned her head as she guided him to her neck and lifted her hips to him.\n\nHe slid out of her before burying himself within her again. He ran his tongue over the pulse pounding in the side of her neck as the rush of her blood filled his ears. Thirst burned him when he rested his fangs against her vein.\n\nTaking deep breaths, Kadence tried to stay relaxed even as her body braced for what was to come.\n\n\"Don't fight it,\" he whispered in her ear before he sank his fangs into her neck.\n\nThe piercing sting caused her body to jerk beneath his, and she winced as she waited for the excruciating agony she'd experienced with Joseph, but when Ronan gave his first pull, all she felt was joy as he took her blood while his body moved within hers.\n\nStrength flooded through Ronan as her blood filled his cells, branding him forever as hers. He'd never tasted anything as intoxicating as her, or experienced so much power from another's blood. He'd only rarely fed from other vampires, and they'd all been turned ones. It had been centuries since he last fed from another of his kind, but her blood felt far stronger than theirs ever had.\n\nFor the first time in centuries, he was free from the burning in his veins and his unquenchable need to kill. Joy swelled within him as a peace he'd never thought to find settled over him. Her fingers curled into his back and her pleasure slid through the connection opening between them.\n\nTurning her face into his neck, she sank her teeth into his shoulder and cried out when she broke through his skin and licked away his blood. Too far gone in the freedom she gave him and the ecstasy of her body, Ronan didn't think about her reaction and craving for his blood.\n\nShe arched beneath him and bit into him as she came again. With one final thrust, he found his release within her welcoming body. He pulled out of her as her blood continued to fill him and she bit him again.\n\nGathering her closer to him, he bit deeper into her vein. Her grip on his back loosened, her fingers eased their hold on him. The slowing beat of her heart sounded in his ears as the life drained from her, and he knew she was almost to the point of no return. Retracting his fangs from her, he licked the drops of blood from her neck when her head lulled to the side.\n\nPanic gripped him as her shallow breaths rattled in and out of her chest. He had to get more of his blood into her soon. He bit into his wrist. Blood welled forth, its new scent causing his nostrils to flare. Kadence was etched into his cells now as her blood flowed through his veins, making him stronger than he'd been before.\n\nHe brought his wrist to her mouth and rested it against her parted lips. His blood trickled inside, but whereas she had eagerly consumed it before, she remained limp beneath him now. \"Drink, Kadence,\" he commanded gruffly.\n\nHer dazed eyes met his before fluttering closed again. Gathering her closer to him, he cradled her in his arms. The weakness in her body caused his heart to race as he kissed her forehead, and his fingers entangled in her hair. He could feel his blood seeping into her, but he couldn't feel it making any changes within her or strengthening her.\n\nTerror clawed at his insides. Maybe a hunter couldn't become a vampire; maybe it was impossible to turn them. It had never occurred to him that might be a possibility, but maybe the fact they were already part demon would make them immune to the change.\n\nIf that were true, then they were both doomed as he could feel the life slipping from her. Madness crept steadily into the edges of his mind at the realization that he may have destroyed her.\n\nShe was his, but more than that, somewhere along the way, he'd fallen in love with her too. He should have told her that before doing this, should have told her what she meant to him, and now he might never get the chance.\n\nThen she shifted and her fingers twitched against his chest. Unexpectedly, love cascaded around him, robbing him of his breath. It took him a second to realize it wasn't his own emotions he felt, but _hers_ as her love surrounded him within its comforting embrace.\n\nExcitement hammered him when Kadence seized his arm and she drew more blood from him. Ronan's head lowered to hers while his blood strengthened her and replaced the blood he'd taken from her.\n\nThe satisfaction Ronan experienced from her drinking from him brushed against the edges of her mind, as did his concern for her and his love. That love spread in her a warmth stronger than the sun. An unfamiliar, but not unpleasant, tingling sensation started in her gums. Easing her bite on him, she ran her tongue over her teeth. Her canines lengthened when she prodded at them.\n\n\"Kadence?\" Ronan murmured as he brushed the hair back from her face. Her eyes remained closed, her skin paler than normal, but she'd released her bite on him.\n\nThen, her fangs sank into his wrist. He jerked against her, disbelief filling him as she pulled on his blood. He'd never changed another, but he knew that wasn't normal. All humans had to go through the change before they could be reborn as a vampire. It took hours, sometimes as much as a day for fangs to emerge, not minutes.\n\nHe held her tighter when she squirmed against him and bit deeper. He had no idea what he'd unleashed by changing a hunter, but he would never let her go.\n\n***\n\nRonan rolled over and climbed from the bed. He glanced back at where Kadence remained sleeping peacefully. He'd watched religiously over her for the past few hours, searching for any hint of suffering, but she hadn't so much as twitched as she lay on her side with her hair fanned out around her.\n\nEven in the easiest of transitions, there was still excruciating pain, but there had been none with her. Her body had accepted his blood as if it were her own.\n\nThe transition wasn't complete. He could still feel the changes taking place in her through the bond connecting them, the shifting of her cells and molecules as she became immortal. He suspected her transition had been easier because she was already part demon, but he didn't know what that would mean when she woke again.\n\nWhatever she became, she was his now, and they would deal with the consequences of that, if there were any.\n\nMoving away from her, he walked into the bathroom. He turned the sink on and splashed his face with water. He pulled a towel from the rack and dried himself before lifting his head to meet his haggard reflection in the mirror. His face hadn't seen a razor since Kadence left. Thick stubble lined his cheeks and jaw. He'd rubbed her skin raw earlier because of it, something he would not do again, but he couldn't spend the time away from her that it would take him to shave now.\n\nHe walked into the bedroom as a knock sounded on the door. Grabbing his pants, he tugged them on and hurried to answer it before it woke Kadence. He cracked the door open, his eyes narrowing on Declan.\n\n\"What is it?\" He slipped out and closed the door to within an inch.\n\n\"How is everything going?\"\n\nRonan glanced at the partially closed door as he sought out his bond to Kadence. \"Better than I'd expected.\"\n\n\"Good. We're getting ready to go hunting.\"\n\n\"If you come across Joseph, bring him to me if it's possible. We should try to learn what he's been up to.\"\n\n\"We will, but I have a feeling he's going to be lying low for a while.\"\n\n\"So do I.\"\n\n\"Ronan...\"\n\nDeclan's strangled voice broke off as his eyes darkened until the silver color became almost entirely black. Ronan took a step back as the color drained from Declan's face. Uncertain of what was happening to his friend, Ronan took his arm to steady him when Declan swayed.\n\nDeclan fell forward to brace his hand against the wall as tremors wracked his large frame. A blood curdling scream rent the air, piercing deep into the hushed night. Spinning, Ronan flung the door open, shattering plaster when it banged off the wall.\n\nHorror turned the blood in his veins to ice when his eyes landed on Kadence. Her entire body was lifted off the bed, her head and heels the only things touching the mattress. The scream broke off as she flopped onto the bed and began to thrash as if she were in the middle of a seizure.\n\nRonan raced across the room and gripped her shoulders in an attempt to stop the spasms wracking her. The delicacy of her bones was not lost on him as she jerked against him and he feared she would break something. Sweat rolled down her, plastering the sheets to her slender frame. She fell back, panting heavily as her fingers dug into the bed.\n\nShe jerked beneath him, nearly breaking free of his hold when another round of spasms shook her. He glanced at Declan when he slumped heavily against the doorframe. Declan's eyes were closed as he labored to breathe.\n\n\"Declan\u2014\"\n\n\"What's going on?\" Lucien burst into the room. Killean and Saxon were close on his heels as Lucien skidded to a halt inside the doorway.\n\nKadence whimpered again and her eyes fluttered open. No longer their bright blue color, they'd become a bruised blue as they stared unseeingly up at him.\n\n\"The pathways are opening,\" she whispered.\n\nHe stared at her in stunned silence when she slumped listlessly back to the bed, unconsciousness claiming her once more. His heart hammered as he caught the much too faint thump of her pulse. He'd witnessed transitions before, he knew they were never pleasant, but he'd never seen anything like this. Never seen one go so smoothly before going so horribly wrong.\n\nThrough the bond connecting them, he felt nothing from her. He turned toward Declan. \"What is going on?\"\n\nDeclan opened his eyes to reveal their still black color. \"Pain.\" His voice came out gravelly. \"She was in pain.\"\n\n\"What do you mean _was_?\"\n\n\"I don't know. It's... it's quiet now.\"\n\n\"Too quiet,\" Ronan murmured as he gazed down at her parted lips. He ran his fingers over her forehead, brushing back the hair sticking to her pale skin.\n\n\"Have any of you heard or seen anything like this before?\" he demanded of the others.\n\nKillean and Saxon shook their heads. \"No,\" Lucien answered.\n\n\"Get the records,\" he ordered.\n\n\"I'll go.\" Declan spun and fled from the room as if the hounds of Hell were on his heels.\n\n***\n\nSaxon sat in the chair beside the bed, flipping through one of the numerous books that contained much of the vampire's vast history. Declan had disappeared after returning with the records. Uncomfortable with being near Kadence in her current condition, Lucien and Killean had retreated to the hall where they were flipping through more annals, but Saxon was studying her reactions and trying to find something in the records to match.\n\nThe records were the only things Ronan had maintained full custody of after the war with the Savages. One of the selling points of this place had been the vault built into a wall in the gym. If one didn't know the vault was there, they would never see it.\n\nSome of the records were so old they were written on parchment and had to be handled with the utmost care. They'd focused on those older documents first, the ones written before Ronan was born.\n\n\"Anything?\" Ronan demanded when Kadence remained unmoving before him, her hand cold as he held it within both of his. He'd ordered the others from the room earlier and slipped a nightgown on her, but she was still far too exposed for his liking.\n\n\"Not yet,\" Saxon murmured.\n\nLucien's head emerged at the bottom of the doorway, where he sat with his back against the wall. \"No,\" he said and ducked back again.\n\nDeclan walked into the room with a book clasped to his chest.\n\n\"Declan?\" Ronan asked.\n\n\"I discovered nothing in the books I went through,\" Declan said, his eyes once again a silvery gray color as they met Ronan's. \"In all your many years, a hunter has never been turned, and it doesn't seem to have occurred before you were born either. I think if it _had_ occurred, it wouldn't have been a secret, but full-blown knowledge. We'll find nothing in these journals, Ronan.\"\n\n\"I didn't think we would,\" he murmured as he turned his attention back to Kadence.\n\nRosy color tinged her cheeks once more. Her heart continued to beat within her chest, and her breath warmed her lips when she exhaled, but she hadn't moved in the three hours since she'd collapsed onto the bed again. The transition should be over by now, but it still held her within its unrelenting grasp.\n\n\"She will survive this,\" Saxon said.\n\nAnd become what? Had he destroyed her? Turned her into something different? What pathways were opening and where would they lead her? His hands tightened around hers at the possibility of something terrible awakening within her. Closing his eyes, he bowed his head. He had no idea what she was going through as the bond remained oddly closed off on her end.\n\n\"Get the crossbow and weapons ready, in case she doesn't survive,\" he ordered.\n\n\"Ronan\u2014\"\n\n\"If you don't put me down immediately, I will kill you all,\" he interrupted Lucien.\n\n\"I'll get them,\" Killean said and Ronan listened as he rose to his feet.\n\n\"Wait,\" Declan ordered. \"She's awake.\"\n\nRonan's eyes flew open and his head turned toward Kadence. Her hand remained limp in his grasp, but her eyes were on his. Except, she didn't seem to see him, and the color of her eyes robbed him of his breath. He'd never seen anything like the white blue shade of her eyes as they glowed with an eerie light.\n\nDeclan stepped closer when her eyes shifted toward him. \"The paths,\" she croaked. \"The paths, they're all broken. They go nowhere. They go everywhere.\"\n\nHer eyes closed as fierce spasms shook her. Ronan clasped her to his chest until the spasms stopped and she fell silent once more. Her body eased against his as her shallow breaths warmed his neck.\n\nHe had done this to her; he had caused this to happen. The uncertainty surrounding her and this hideous pain was his fault. Self-hatred boiled through him, rocking him to the very core of his being.\n\n\"What paths, Kadence?\" he whispered, but she didn't respond, and he hadn't expected her to. \"You will not die.\"\n\nHe didn't know if she heard the order or not, but he willed her to survive as he sought her out through their bond. This time, he found a small opening, but he discovered only a slumber so deep that he could barely connect with her.\n\n\"She is strong,\" Declan said. \"Stronger than any of us realize.\"\n\n\"I hope so,\" Saxon muttered.\n\n\"That strength is what is making this more difficult and different from any other transition. The parts of her that have been dormant since birth, possibly since the creation of the hunters, are changing and evolving,\" Declan said. \"We will soon know what she will become.\"\n\n# CHAPTER 36\n\nKadence stirred, causing Ronan to nuzzle her forehead with his lips as he held her in his arms. Declan stepped out of the shadows by the back wall when she whimpered.\n\nHer eyelashes fluttered against his skin as she blinked. Hope clutched at Ronan's chest when he leaned away to peer down at her. Shadows circled her eyes, but the brilliant azure color of them had returned.\n\n\"Ronan,\" she whispered.\n\nHe exhaled sharply as relief filled every part of him. He kissed her as his hands ran over her face. Warmth radiated from her flesh instead of the unnatural cold she'd been emitting. Her heart beat more solidly within her chest and her breathing was stronger.\n\n\"Are you okay? How do you feel?\" he inquired.\n\nA small smile curved her lips. \"I'm fine now. That wasn't normal, was it?\"\n\n\"No.\" He brushed the lank strands of her hair away from her face. \"It shouldn't have happened.\"\n\n\"We both knew it may be different for me, but there was no choice. It had to happen. How long have I been out of it for?\"\n\n\"Almost two days.\"\n\n\"Not too bad.\" She stifled a yawn and struggled to sit up. Ronan steadied her as she pulled herself up to sit against the headboard. \"It's over now. There's only the future to look to.\"\n\nHer voice took on an odd tone as she said those last words and her eyes became distant.\n\n\"Kadence\u2014\"\n\n\"I'm fine, Ronan,\" she interrupted and her eyes came back to him. \"Truly.\"\n\nHe turned to Declan. \"Leave us.\"\n\n\"I'm glad you are well, Kadence,\" Declan said before he slipped out the door and closed it behind him.\n\nRonan gathered Kadence's hands in his. \"I was worried about you,\" he admitted.\n\nA tug pulled at Kadence's heart as she inched closer to him. \"I know.\"\n\nKadence held him as she tried to bury the memory of the murky realm of uncertainty she'd been trapped in. It felt like weeks had passed instead of days. \"I could hear you and feel you, but no matter how hard I tried, I couldn't come back to you. There were so many different things.\"\n\nShe knew she didn't make any sense, but she couldn't quite process what had happened to her yet. She rubbed her hand over his back, taking comfort in the familiar strength of his etched muscles. He was something solid when nothing had been solid for the past two days.\n\n\"What happened, Kadence? You mentioned something about pathways.\"\n\n\"There were only brief periods of pain,\" she murmured. \"But they were _so_ painful.\" She trembled as she recalled those moments when it had felt like every bone in her body was breaking and snapping back into place. \"And then there would be quiet...\"\n\n\"And pathways?\" he prodded when her voice trailed off and her eyes took on that distant look again.\n\n\"Yes, or at least they looked like pathways in my mind. They were full of vibrant blues and purples as they unrolled around me to lead me onward. They took me deeper and deeper.\"\n\n\"Toward what?\"\n\n\"Deeper inside me, I think,\" she murmured. \"It was all so confusing, yet it felt right. The pathways led me to where I was supposed to be.\" She rested her hand against his cheek. \"Here, with you, and... whole?\"\n\n\"What do you mean by whole?\"\n\nHer hand fell into her lap. \"When I left here, when I walked away from you, it didn't feel right. _I_ didn't feel right. I told myself it was nothing and that I couldn't run away from my family to live my dreams only to give them up for someone I barely knew, even if I did care for you. I tried to convince myself it was the crowd at the airport making me so uncomfortable. I told myself all those things, yet my skin felt too tight, my body ached, and all I wanted was to come back to you.\"\n\nRonan scrubbed a hand over his face at this revelation. \"That's why you came back.\"\n\n\"That was part of it. I was walking to the plane, getting ready to board and thinking about kissing the Blarney Stone when I realized that I was probably the only person who had a chance of bringing our sides together and that I couldn't go.\"\n\n\"You were going to Ireland?\"\n\nShe smiled at him as she brushed the hair away from his face, taking note of the shadows under his eyes and the exhaustion radiating from him. \"I kind of have a thing for this Irish guy I know. I wanted to see where he came from.\"\n\nRonan clasped her hand and kissed the back of it. \"You will one day,\" he promised. \"Why did you come back here instead of going to your brother?\"\n\n\"I knew you would listen to me more than he would, and the minute I decided to return to you, some of the pressure in me eased. Not completely, but it was better, and I almost felt normal again. When I finally saw you, I could breathe easier again.\"\n\n\"It sounds to me like the demon DNA in you also recognized me as your mate.\"\n\n\"I think so too. I sense the bond between us more strongly now that I'm a vampire, but it was there before too; I just didn't know what it was. You're _mine_ , Ronan. Nothing will ever change that.\"\n\n\"No, nothing will,\" he vowed. \"Do hunters have mates or something similar?\"\n\n\"Not that I know of. We are paired off, and some of those pairs come to love each other if they're lucky enough, others don't. Sometimes hunters find someone they would like to marry before they can be paired off, and sometimes they're granted permission to marry, but I've never heard anyone describe anything like what I felt when away from you. However, few love matches occur before marriage, and I've never met one, so I don't know what those couples experience with each other.\"\n\n\"I see,\" he murmured.\n\n\"My family is the strongest line.\" She stifled a yawn and nestled closer to the warmth of him. \"Maybe that has something to do with it. Maybe we are more in tune with the demon part of us.\"\n\n\"Maybe,\" he agreed.\n\nKadence lifted her head to gaze around the room and her brow furrowed. \"Ronan\u2014\"\n\n\"It will take some time to get used to your heightened senses. I'm told the sights and sounds can be especially overwhelming in the beginning.\"\n\n\"No,\" she said. \"It all looks and sounds the same to me.\"\n\nHe sat back as he studied her. \"It seems our species have a lot more in common than we'd ever realized. You were also seeking out my blood before the change.\"\n\n\"I was,\" she murmured as her eyebrows drew together over her nose. \"And I enjoyed it. I have to speak with Nathan.\"\n\n\"Soon,\" he promised. \"First you should feed and rest.\"\n\nHis words drew her attention to the blood pumping through his veins. She felt the answering pulse of his heart in every molecule of her body. For the first time, his new scent enveloped her, as did her own. The two of them had become intricately woven together.\n\nA new inner strength pulsed within her, and with the next beat of his heart, her fangs lengthened. She recalled sinking those fangs into Ronan earlier, but feeling them now startled her.\n\nShe would never change what had passed between them, but she felt thrown off now. She hadn't experienced as many changes in her body as a human would have, but there was no denying she was no longer the woman she'd been before.\n\nShe was also extremely aware of another change in her. She felt Ronan at the edges of her mind, and she could feel the edges of his. \"We will both have to get used to the mental bond,\" he said as he ran his hands over her. \"It can be shut off again, if you would prefer.\"\n\n\"No,\" she said. \"I will get used to it.\"\n\nHe smiled and kissed the tip of her nose. \"We will get through this together, and I will do what I can when it comes to your brother.\"\n\nHer breath caught in her chest as his pure brown eyes gazed back at her. \"Ronan, your eyes, they're completely brown.\"\n\n\"When I was younger, they used to be brown all the time, but as time went on and the urge to kill became more incessant, the color of my eyes changed.\"\n\n\"Why are they different now?\"\n\n\"Because you calm me more than all the blood and death in the world ever could. They may not remain brown all the time, but I didn't know they could ever be purely brown again until Declan told me they were when I first held you in the alley.\"\n\nShe drew her lip into her mouth to bite on it and winced when she pierced it with one of her fangs. \"Easy,\" he murmured and bent his head to lick the blood away. \"It will take time.\"\n\n\"I know,\" she said and sniffed at the air again. Now that she was adapting to his strong scent within her, and hers within him, she was beginning to smell herself more. \"I could really use a shower.\"\n\nHe chuckled and rose to his feet with her locked securely in his arms. \"Of course.\"\n\n\"I smell really bad.\"\n\nHe dropped his head down and inhaled deeply. \"I think you smell like me, and I smell fantastic.\"\n\nShe couldn't help but laugh as he grinned at her. She'd never seen this side of him; he was almost jovial as his brown eyes twinkled. \"Ronan, are you teasing me?\"\n\n\"I think I might be,\" he replied, sounding more surprised by it than she was.\n\n\"I like it.\" She rested her head against his chest as he carried her into the bathroom.\n\n\"Then I will try to do it more often.\"\n\n\"Good.\"\n\n# CHAPTER 37\n\nHe didn't bother with the light in the bathroom as he strode toward the large, gray tiled shower. Kadence savored the feel of his skin beneath her and the power of the muscles enveloping her. The solid beat of his heart sounded in her ears as her fangs throbbed.\n\n\"You need to feed soon to complete the change,\" he told her when he sensed her hunger.\n\n\"You mean it's not complete?\"\n\n\"Not normally no, but you're not exactly normal.\"\n\n\"No, I'm not.\"\n\nBending, he kissed her forehead. \"You're hungry.\"\n\n\"I guess I am,\" she murmured.\n\n\"You'll only ever have to feed from me,\" he assured her as he adjusted his hold on her to pull back the glass encasing the shower. He fiddled with the water until steam rose to swirl around her. Setting her carefully on her feet, he gripped her nightgown and pulled it over her head.\n\nKadence's nose wrinkled when the sweaty scent of the clothing hit her. Her gaze fell on the toilet when he threw it onto the closed seat cover.\n\n\"Will I still have to go to the bathroom?\" she asked.\n\n\"You will. Even us purebreds have at least some human in us, though you will probably have to go far less than you did before.\"\n\n\"Hmm,\" she murmured. \"So, am I now considered a turned vamp?\"\n\nHe unbuttoned the pants he wore and tugged them off. Stepping closer to her, he ran his hands over her sides, enjoying the feel of her silken skin. \"Technically, yes,\" he replied. \"However, you are so much more than that. I could always sense power within you, but now it is far stronger.\"\n\n\"Maybe that's your blood in me that you're sensing.\"\n\n\"I definitely sense that too, but this is more.\"\n\nShe stepped into him, sighing when her breasts came into contact with his chest. Even with the knots in her muscles, desire for him bloomed within her. She rested her fingers on his chest as his hand cupped her cheek.\n\nHis other arm wrapped around her waist and he lifted her against him. Kadence draped her arms around his neck. She nibbled at his bottom lip, drawing it into her mouth as he stepped into the shower with her. A blissful groan escaped her when the hot water beat against her skin and eased some of the soreness from her.\n\nRonan lowered her to the ground and reached behind her to grab the bottle of shampoo. \"Close your eyes,\" he said gruffly.\n\nDespite her displeasure over the loss of contact between them, Kadence closed her eyes as he guided her around. He worked the shampoo into her hair with a tenderness she never would have suspected from him as he massaged her scalp.\n\n\"Marta said women like conditioner,\" he said after he'd washed the shampoo from her hair and lifted another bottle.\n\nShe turned her head to look at him over her shoulder. The water running down his face and broad chest created rivulets she longed to lick away from him. \"You asked Marta about women's things?\"\n\nHis mouth curved into a smile. \"I am not well versed in what women require.\"\n\nShe laughed as she brushed the wet hair out of her eyes. \"I could argue differently about that, but yes, conditioner does help with the tangles in long hair. I doubt you have to worry about that.\"\n\n\"Not in years, and there was no conditioner the last time I had long hair.\"\n\n\"You had long hair?\"\n\n\"At one time. I cut it off the first time it got in my way during a fight and never let it grow long again.\"\n\n\"I see,\" she murmured as he worked the conditioner into her hair.\n\nShe closed her eyes as his hands kneaded her cramped muscles afterward. He stepped closer to her, his erection rubbing against the hollow of her back while he worked over her body until all the knots were out of her muscles and her entire body felt electrified with its need for him. He washed the conditioner from her hair before positioning her so that her back was flush to his chest.\n\nStretching above her head and behind her, she ran her hand over the stubble lining his cheek. His lips pressed into her palm as one of his hands skimmed down her belly while the other rubbed the underside of her breasts.\n\n\"We should get you back to bed,\" he whispered against her palm.\n\n\"You read my mind,\" she replied. She lowered her other hand to grip his rigid shaft. \"I want you, Ronan, now.\"\n\nHe should deny himself, she needed rest, but for the life of him he couldn't get the words past his constricted throat. She turned into him, and her gaze latched onto his mouth. The scent of her arousal grew stronger on the air, ensnaring him within its intoxicating depths.\n\nHer fingers threaded through his hair as she rose onto her toes. He gripped her ass as her wet breasts slid over his skin, and she rubbed enticingly against his cock.\n\n\"You've been through a lot. You should rest,\" he said.\n\n\"There is no rest for the wicked,\" she murmured as she ran her tongue over his lower lip before nipping on it. \"And I have more than one hunger right now.\"\n\nReleasing her, he reached behind her to turn the water off. He opened the shower door, lifted her up, and carried her out. He removed a towel from the rack near the shower and enclosed her within the soft, terrycloth material. Her damp hair fell about her shoulders as she wiggled against him and bit at his lip again.\n\nHer fingers glided over his chest, her gaze following everywhere she touched. The muscles of his stomach rippled as she traced them and delighted in the feel of him. He was everything she could have ever wanted and more.\n\nKadence pushed the towel away from her before gripping his shoulders and lifting herself so she could circle her legs around his waist. His eyes swirled a burgundy color as she rubbed her sex teasingly against the head of his cock. When he went to lower her onto him, she lifted herself away and shook her head as she gave him a playful smile.\n\nRonan lifted an eyebrow at her when she rubbed teasingly against him again, but didn't take him into her. Stalking forward, he set her on the marble counter near one of the sinks and pulled away from her. She went to grab him, but he caught her wrists and pinned them over her head to the mirror behind her. Her breasts heaved with her inhalations as she gazed at him from passion-darkened eyes.\n\n\"Do you enjoy teasing me, Kadence?\" he inquired.\n\nA smile curved her mouth as she slid her hips toward him. Her legs parted in invitation to him, but he held back from entering her.\n\n\"Now it's my turn to tease,\" he murmured.\n\nBending his head, he licked his way down her breast toward her nipple. Kadence's back bowed as he drew the puckered bud into his mouth and ran his tongue around it before turning his attention to her other breast.\n\nHer breath came in shallow pants, the ache in her spread from between her thighs and into her belly as he suckled on her other breast. Then, he sank his fangs into her breast just above her nipple. She cried out, her body bucking on the counter, her wrists jerking in his grasp as overwhelming pleasure speared her.\n\nHe kept her wrists firmly within his hold as his tongue laved her nipple while he drew on her blood. Shifting his hold on her wrists into one hand, he kept them pinned as his fingers skimmed over her flesh in a touch so faint that she barely felt it, but it left her skin electrified.\n\nRonan slid his hand between her thighs, spreading them wider. Her wetness slipped over his fingers as he stroked her, causing her to cry out. He kept his mouth on her breast while he dipped a finger into her.\n\nShe writhed against him, her head moving back and forth as he parted her further and slid another finger into her. Her muscles clenched around him when he rubbed his palm over her clit, stoking the fire within her higher.\n\n\"Ronan!\" she gasped. \"I can't!\"\n\nHe released his bite on her breast and lifted his head to meet her gaze. Leaning closer to her, his mouth brushed over hers as he spoke. \"Can't what?\"\n\n\"I can't take anymore.\"\n\nHe slid his fingers away from her and grinned when she moaned and pressed her hips forward on the counter. \"I thought you enjoyed teasing,\" he said and pulled her wrists down so that he held them against her belly.\n\nBefore she could respond, he trailed his fingers up the inside of her thigh in a wispy touch that she barely felt, but her body begged for more of it. She tried to pull her hands from his grasp again, but he kept them firmly in place as he pushed her thighs further apart and knelt before her.\n\nHer eyes widened when he bent his head between her legs. \"Ronan!\" Any protest she'd been about to utter died on her lips when his tongue stroked over her aching center.\n\nHer head fell back as he worked his tongue into her in penetrating thrusts. Finally, he released her hands. Kadence grasped his head, not to push him away but to pull him closer.\n\nRonan growled as her hands curled into his hair and her hips surged toward him. Grabbing her ass, he dragged her closer to his mouth as he tasted the heated wetness of her core. Her hips moved faster as she fucked his tongue with a wild abandon that had him growing impossibly harder.\n\nHe sensed her impending release in the tightening of her muscles. Pushing her closer and closer to the brink, he pulled away before she could plummet over the edge. Her hands tugged at his hair as she groaned her disappointment. He rose before her and seized her wrists again with one hand. Drawing them over her head, he pinned them to the glass once more as he met her dazed gaze.\n\n\"I love to tease too,\" he murmured before taking hold of his dick and guiding it to her entrance. He held her gaze as he buried himself within her.\n\n\"I love the way you do,\" she murmured as her eyes drifted closed.\n\nRonan pulled back to drive into her again. He lost himself to the frenzy of his need as he thrust into her again and again. He was on the brink of spilling, but he gritted his teeth and held back his release.\n\nKadence's eyes opened, and his breath caught as he found himself gazing into their startling white-blue depths once more. She most certainly was something more than a normal turned vampire, and she was starving.\n\nReleasing her wrists, he wrapped his hand around her head and pulled her face into the hollow of his throat. \"Feed,\" he commanded.\n\nHer fangs slid over his skin before sinking into his vein. His body jerked as she pulled hungrily on his blood and her body kept rhythm with the demands of his. The muscles of her sheath clenched around him and she cried out against his throat. This time, he didn't hold back his own release, but followed her over the edge.\n\n# CHAPTER 38\n\n\"My brother would never hurt me,\" Kadence said for what felt like the thousandth time that night.\n\nRonan barely glanced at her as his eyes remained focused on the trees around them. He tuned out the clacking of the branches in the breeze swirling through the park as he listened for any other sounds. The hunters would come, he had no doubt, but he didn't know what would happen when they did.\n\nKadence searched the woods for her brother, her hands twisting anxiously before her. She'd spent the past week adapting to her new vampire status and training with Ronan. She'd always been fast before, but now she moved with a speed that still startled her, and her reflexes had amped up.\n\nShe'd managed to avoid having Ronan take her down once, but it had been a big improvement. He'd reluctantly admitted to her that she was doing better than he'd expected and, with more training and her added vampire strength, she would be able to take down a Savage on her own. He'd even admitted that, if she kept working as hard as she had been and completed the training, she would make a good Defender one day.\n\nFinally, he had agreed that it was time to contact Nathan, and she'd called him earlier today. She was surrounded by over eight hundred pounds of solid vampire flesh as Ronan and the others stood close by her, yet a shiver of unease raced up her spine. The swings across the way swayed back and forth, their creaking chains adding a creepy air to the already tense night.\n\nHer brother would never hurt her, but the eerie playground, coupled with the fact she knew Nathan wasn't going to react well to what she had to tell him, made her feel like screaming. She'd told Nathan he could bring some of the other hunters with him, but no more than ten. She really hoped he'd listened to her. Otherwise, this night could end badly, and she desperately wanted everything to go right.\n\nThe two men she loved the most could die if this blew up in her face.\n\nKillean kept muttering about killing hunters, and Lucien looked like someone had taken away his favorite toy as he studied the woods with a sullen expression. Declan idly twirled a lollipop in his mouth as Saxon tapped a foot while his hand rested on one of the knives strapped to his side. Ronan stepped closer to her.\n\n\"My brother won't hurt me,\" she insisted again.\n\nHis hand settled into the small of her back. \"You are not one of them anymore, Kadence. There is no way to know how they will react to that revelation.\"\n\n\"I may be a vampire now, but I will always be one of them.\"\n\n\"They're here,\" Ronan said.\n\n\"Where?\" Kadence asked as she searched the woods. A fresh, subtle scent wafted from her right, and she turned in that direction as Nathan emerged from the woods with Asher and Logan flanking him.\n\n\"There are others still in the woods,\" Lucien said.\n\n\"Yes,\" Ronan replied.\n\n\"We didn't expect them to be brave enough to reveal all their numbers,\" Killean said.\n\nKadence glared at him before focusing on her brother. Her heart leapt as joy and love swept through her. She took a step toward Nathan, but Ronan blocked her from going any further with his arm. She turned her angry gaze on him.\n\n\"I told you before we came here that you were to stay by me,\" Ronan said.\n\n\"He's my brother.\"\n\n\"And if he wishes to come to us, he can, but you will _not_ go to him.\"\n\nShe opened her mouth to protest, but when his eyes shifted to a more volatile red color, she held it back. Everyone was on edge enough without her pushing it to a breaking point. Nathan stopped ten feet away from them; his gaze ran over the vampires before settling on her.\n\n\"Are you okay?\" he demanded.\n\n\"Yes,\" she assured him. He studied her, as if trying to figure out if she were telling the truth or not. \"Really, Nathan, I'm fine.\"\n\nLogan glanced at her before focusing on Ronan. His lips flattened when he saw the protective way Ronan stood against her. Kadence's heart twisted, and unexpected tears burned her eyes. She'd never loved Logan the way she loved Ronan, but she cared deeply for him. She'd never wanted to hurt him or her brother, but she knew she had, and she would have to live with that for the rest of her life. She could only hope that one day they would forgive her, but she didn't expect it from them.\n\n\"I'm sorry,\" she said, keeping her gaze focused on Logan as she spoke. \"Know that the last thing I ever wanted was to hurt anyone. I just...\" She trailed off as she tried to think of how to explain what had happened without making it worse. \"I broke out only to see Joseph die, but once I was free, I decided to try to see more of the world.\"\n\nLogan's eyes finally slid to her. \"You didn't get very far.\"\n\nKadence winced at the accusation in his voice. \"I was going to go to Ireland. I was there, in the airport, getting ready to board the plane, but I couldn't do it.\"\n\n\"Why not?\" Nathan demanded.\n\n\"Because we have it wrong,\" she told him again. \"And if there is a chance I can save one hunter's life, or one innocent vampire, I knew I had to try to make it happen.\"\n\n\"Innocent vampire? Sounds like an oxymoron to me,\" Logan spat.\n\n\"I'd say you're the moron,\" Killean grumbled, his face stone cold as Logan glowered at him.\n\nKadence swallowed heavily. She'd cut Logan far deeper than she'd realized. Ronan clasped her nape, drawing her closer. Nathan stiffened at the possessive gesture, and Logan looked as if murder was the only thing he wanted.\n\n\"Shit,\" Asher hissed.\n\n\"What have you done, Kadence?\" Logan demanded. \"You can't be stupid enough to trust them!\"\n\n\"Watch how you talk to her.\"\n\nThe low, lethal tone of Ronan's voice caused the hair on her neck to rise. She'd known this wouldn't be easy, but it was rapidly deteriorating into an explosive situation. \"Listen to me!\" she cried and stepped forward.\n\nRonan moved with her. He bared his fangs at the hunters as he held every one of their gazes. \"Know that if you try to harm her, I'll kill you,\" he promised.\n\nNathan's eyes darted between them.\n\n\"Animals,\" Logan scoffed.\n\nNathan held his hand up. \"Enough,\" he said before focusing on Kadence. \"Tell me what is going on. What do we have wrong?\"\n\nKadence met her brother's troubled blue eyes, so similar in hue to hers. \"Not all vampires are evil. Not all of them kill for fun. Not all of them are like Joseph.\"\n\n\"I see,\" Nathan murmured. \"And we're supposed to believe they're not all evil because...?\"\n\n\"Because you can smell it,\" she said in reply to her brother's trailing question. \"I didn't realize it until recently, but the stench we believed all vampires to have _doesn't_ affect them all. Ronan told me all purebred vampires can smell the difference between a vampire who has turned Savage and one who doesn't kill, but a vampire who has been turned from a human cannot smell the difference.\"\n\n\"What is a purebred vampire?\" Nathan inquired.\n\n\"A vampire who is born a vampire, and not a human who has been turned into one,\" Ronan replied.\n\nNathan's eyebrows shot into his hairline, Asher's mouth dropped, and Logan made a scoffing sound. \"Vampires can be born?\" Nathan inquired.\n\n\"Yes,\" Kadence replied.\n\n\"Holy shit,\" Asher muttered.\n\n\"It's a far bigger world than all you little hunters believed,\" Killean drawled, earning scowls from everyone.\n\nChoosing not to rise to Killean's baiting, Kadence focused on brother again. \"Hunters can smell the difference between the vampires who kill and those that don't too, not as strongly as a purebred, but we can detect it. I know the vampires here don't smell like Joseph did.\"\n\nNathan gazed at the men around her before giving a brief nod. \"They don't,\" he agreed. \"But just because there's no foul odor, I'm supposed to believe they're not killers?\"\n\n\"We're killers,\" Ronan said, and she almost elbowed him in the gut for it. \"But we only kill those of our kind who turn Savage and start slaughtering other vampires and humans.\"\n\n\"We've also taken out a hunter or two when they've gotten in our way,\" Lucien added with a smirk.\n\n\"Enough,\" Ronan said, and Lucien became silent.\n\n\"So you've killed an innocent hunter before?\" Logan demanded.\n\n\"You're not innocent if you're threatening our lives,\" Ronan replied. \"Your kind may have been ignorant to the truth about vampires since the very beginning, but ignorance is not an excuse when our lives are on the line. You better remember that.\"\n\n\"Ronan,\" Kadence whispered.\n\nRonan's thumb rubbed her nape reassuringly as he spoke. \"They must know the whole truth if there is to be any kind of trust.\" Ronan kept his attention focused on Nathan. The bruises he'd inflicted a week ago on the hunter had vanished. His broken nose was no longer swollen and crooked. \"I could have killed you,\" Ronan said to him. \"I didn't.\"\n\nA muscle ticked in Nathan's cheek at the reminder. Ronan waited for him to be foolish enough to deny it, but the young hunter nodded. \"True,\" he agreed.\n\n\"They saved me from Joseph. He attacked me in the alley,\" Kadence said. \"I'd be dead now if it wasn't for Ronan. He also set me free. I was _free_ , Nathan.\" Anguish flickered in her brother's eyes. \"I could have gone anywhere I wanted to, I had money and _humans_ who were going to help keep me safe, yet I'm standing here now.\"\n\n\"I know being kept in the stronghold was difficult on you. I didn't realize until recently how difficult it was and how confining. We can work on making it better for you there,\" Nathan said.\n\nRonan stiffened beside her. She rested her hand on his chest, but it did nothing to ease the tension he emitted. \"Nathan, I'm not returning to the stronghold.\"\n\n\"Then what do you intend to do, Kadence, live with the vampires?\" Nathan asked.\n\n\"Yes.\"\n\nLogan threw his arms into the air. \"They've corrupted you and you can't even see it!\"\n\n\"No, they haven't,\" she replied. \"I know it's hard to believe that they aren't our enemies, at least not all of them. I struggled with it too in the beginning, but I know the truth now.\"\n\nBefore coming here, she'd promised Ronan she wouldn't reveal to her brother that they could go out in the day, not until he deemed Nathan trustworthy enough. Now, she had little to work with in order to make them see reason, but she understood Ronan's reasons for guarding some of their secrets.\n\n\"Nathan, take her and let's get out of here,\" Logan said.\n\n\"No one will _ever_ take her from me,\" Ronan promised.\n\n\"Enough, Logan,\" Nathan said before focusing on her again. \"It seems you have traded one cage for another.\"\n\n# CHAPTER 39\n\nKadence opened her mouth to reply, but Ronan spoke before she could. \"I would never keep her caged as you did. She had no choices with you, not even on whom she would marry.\"\n\nHis eyes fixed on Logan, the man Kadence had told him she was to marry. Ronan suspected Logan's obvious love for Kadence would turn to loathing when he realized what she was now. The hunter may become an issue that would have to be dealt with later, but for now, he would continue to play nice, for Kadence.\n\n\"Every step she has taken with me has been one of her choosing, not someone else's. I tried to make her return to you. She refused. I set her free. She came back. No one will take her choices from her again, not even me.\"\n\nNathan rocked back on his heels. \"And just what is it between you two?\"\n\nHe saw the question in Kadence's eyes when she lifted her gaze to him. \"Reveal what you will about that,\" he told her.\n\nShe turned back to her brother. Part of her screamed not to say the words out loud. She was afraid he would walk away from her and she would never see him again if she did. The other part knew she couldn't lie, not to Nathan, and it would come out eventually. It was better to know if he would turn against her now, than tomorrow or a month from now.\n\n\"I... I love him, Nathan.\"\n\n\"Bullshit!\" Logan exploded. He jerked a crossbow up from his hip, and before Kadence could blink, he fired the bolt.\n\n\"No!\" Nathan shouted as Kadence leapt forward.\n\nRonan pulled her back with one hand while he snatched the bolt out of the air with the other. Lifting his head, Ronan held Logan's gaze as he broke the bolt in half. The hunter took a startled step back when the vampires closed in around Ronan and started toward Logan.\n\n\"Back off,\" Ronan commanded his men.\n\nThey moved back, but all their eyes had taken on a fiery red hue as they stared at Logan. If he allowed it, the hunter would be dead before he could take his next breath. Ronan released Kadence and strode forward. She snagged his hand and tugged on it until he stopped.\n\n\"No, this is not... This will _not_ happen!\" she yelled.\n\nReleasing Ronan's hand, she moved forward with lethal speed to yank the crossbow from Logan's grasp. She threw the weapon onto the ground and stomped it, shattering it into pieces. Her fangs lengthened as she resisted tearing Logan's throat out for trying to kill Ronan. Feeling somewhat more in control of herself, she lifted her head to meet Logan's startled gaze.\n\nRonan drew her against his chest when her eyes burned that feral, white-blue color. Her brother and the other hunters gasped when they saw her eyes.\n\n\"Don't you ever do that again!\" she yelled at Logan. \"I'm sorry I hurt you, I really am, and I will regret it for the rest of my life. But _I_ have chosen Ronan, and no matter what anyone else says or believes, it is a choice I would make over and over for the rest of my life. If you try to attack him again, I'll kill you myself, and don't think I'm not capable of it.\"\n\nShe turned to face her brother who gawked at her as if he didn't know her. She supposed he didn't know her, not really. \"I love you, Nathan. I always will no matter what happens. Maybe, one day, if you'll listen to me and accept my choices, you can get to really know _me_. However, none of that matters, not when innocent lives are on the line.\n\n\"There are vampires who have been killed that didn't deserve it, and there are hunters who have died that didn't have to. Joseph is bringing together a large group of Savage vampires that we can beat if we all work together, but if the vampires and hunters remain divided and killing each other, then Joseph will only grow stronger until he becomes unstoppable.\"\n\nHer brother succeeded in closing his mouth, but it fell open again. \"Your eyes,\" he murmured.\n\nKadence's heart sank. \"Are my eyes really what you're most concerned about after everything I just said?\"\n\nNathan closed his mouth and threw back his shoulders. \"No, they're not. What is a Savage vampire?\"\n\n\"Those of our kind who kill for pleasure and take the lives of innocents. There is no humanity left within them,\" Ronan replied. \"They are the ones we hunt.\"\n\nNathan's gaze ran over them before focusing on Ronan again. \"How big of a threat is this with Joseph and these Savages?\n\n\"It's growing stronger every day,\" Ronan said.\n\n\"You can't seriously be considering this insanity!\" Logan hissed.\n\n\"Not another word, Logan,\" Nathan commanded. \"Asher, what do you think?\"\n\n\"I think your sister has hopped on the crazy train express, but I believe there is something to what she says,\" Asher replied.\n\n\"Thanks,\" Kadence muttered.\n\n\"You gotta admit it's a little crazy, kid. I mean, we're all a little crazy in one way or another, but you're rounding out the top of the looney tier right now. I suppose the vampires could have found a way to warp her mind and turn her to their side, but I don't see why he would have set her free if that were the case.\"\n\n\"What do you think of that, Logan?\" Nathan inquired.\n\n\"He set her free as part of his manipulation to gain her trust,\" Logan replied.\n\nKadence almost stomped her foot in frustration, but she managed to stop herself from doing so. Acting like a child would get her nowhere right now.\n\n\"True,\" Nathan said as he rubbed at his jaw. \"And judging by the color of my sister's eyes and the speed with which she moved over here, I'm going to say she's not the same as when she left the stronghold. We know a vampire can control a human's mind, perhaps\u2014\"\n\n\"You know hunters are immune to a vampire's powers of persuasion,\" she interrupted brusquely.\n\n\"True,\" he said. \"But he's no normal vampire, we can all sense that, and you are not a normal hunter anymore.\"\n\nRonan didn't speak as he waited for Nathan to figure things out for himself. It wouldn't do any good to rush him or argue with him. Nathan was young, but the fact he was taking time with his decision and listening to the views of the others revealed a maturity beyond his years.\n\n\"But then, we've never been normal hunters. Our family line has always been stronger,\" Nathan continued.\n\nNathan's gaze traveled to the other vampires. \"What makes a vampire turn into a Savage?\"\n\n\"Death and blood is a temptation many vampires live with every day, and some give into it,\" Ronan replied. \"Those who kill become stronger, especially if they kill our kind or a hunter, though they enjoy slaughtering humans too. A vampire doesn't become completely Savage until the innocent deaths of the humans warp their souls.\"\n\n\"So at any time one of you could also become Savage?\"\n\n\"Yes.\" There was no reason to deny it or to lie. If they were to work together, they would have to be honest with each other. If Nathan discovered the truth later, it would sever any relationship they may have built between them.\n\n\"Many of us fight it,\" Declan said. \"And that's just it, we _fight_ it.\"\n\n\"That's fine. I'm more concerned about thinking we can trust you and having one of you turn on us,\" Nathan said.\n\n\"We could say the same to you,\" Ronan said and glanced pointedly at Logan. \"We're not asking for your secrets.\"\n\n\"I know more about them than they know about us,\" Kadence said as she silently pleaded with Nathan to believe her.\n\nNathan stared at her before looking to Ronan. \"What will happen to my sister if you turn Savage?\"\n\nRonan slid his arm around her waist. \"Because of your sister, I will not become a Savage, not unless something happens to her.\"\n\n\"I don't understand.\"\n\n\"That's something I'd like to keep between us for now at least,\" Ronan replied before focusing on Logan. \"But know I will defend her life above my own and I will kill anyone who threatens her, no matter who they are.\"\n\nLogan's nostrils flared and hatred burned from his eyes, but he kept his mouth shut.\n\n\"I see,\" Nathan said. \"And what of them?\" he asked with a wave at the other Defenders.\n\n\"I've known all of them for centuries. They battle their demons, but they're as determined to destroy Joseph and the Savages as I am.\"\n\nNathan folded his arms over his chest as he speared Kadence with his stare. \"Are you a vampire?\"\n\nKadence had hoped not to have to deal with this yet, but she'd given herself away with her eyes. \"Yes,\" she replied.\n\nLogan sucked in a breath. Before anyone could respond, he spun on his heel and stalked away. Kadence took a step after him, but Ronan held her back as Nathan put out a hand to halt her.\n\n\"Let him go,\" Nathan said. \"This is something he may never come to terms with, and you have to accept that.\"\n\nA lump lodged in her throat as Logan vanished into the woods. \"I never meant to hurt him.\"\n\n\"I know, but you did, and we're the ones who put you in that position. You shouldn't have taken off the way you did, you could have gotten killed, but we... _I_ was wrong for expecting you to accept a life I wouldn't have accepted for myself. I know that now, but that doesn't change what has happened.\"\n\n\"Nathan\u2014\" She stepped toward him and froze when he kept his hand up between them.\n\n\"I'm not ready either. I love you, but this is a lot to take in.\"\n\nTears burned her eyes as she bent her head. Ronan embraced her as he leveled her brother with an unblinking stare.\n\n\"We'll work through it in time,\" she said.\n\n\"I have to discuss this with the elders,\" Nathan said. \"They're not going to take it well.\"\n\n\"No, they're not,\" Kadence agreed.\n\n\"Call me tomorrow,\" he said to Kadence before turning and walking away.\n\n\"Crazy or not, I hope you found happiness, Kadence,\" Asher said before following her brother.\n\nKadence nestled against Ronan's chest when he lifted her and held her against him. His body remained tensed as he studied the trees. Over his shoulder, her eyes landed on Killean. The pitiless vampire stared back at her for a minute before briefly bowing his head to her.\n\nShe may have just lost everyone she had known for her whole life, but she knew then that she'd somehow managed to earn Killean's respect.\n\n# CHAPTER 40\n\nKadence put the book down, rose, and paced over to one of the bookshelves before stopping and turning back to the chair she'd vacated. Her gaze went to the window and the sun spilling inside. She strode over to pull back the curtain and reveal the covered swimming pool.\n\nShe should call Nathan, but she dreaded picking up the phone. She knew the elders would never agree to the hunters working with the vampires. They hated and resisted any kind of change, and the hunters had been set in their ways for centuries. She stalked back toward the chair, her dread growing with every step.\n\nThere had been a niggling at the back of her mind ever since she'd woken this afternoon. It was the same feeling she got when she somehow knew things. Then, there was always this nagging tug on her mind. Before the tugging had been small, but now it felt like something was digging its way into her brain, and it would not go away.\n\nBefore, there had been some sort of revelation, but there were no revelations coming to her now. She sensed nothing more than what she already knew. However, the incessant digging sensation grew with every passing hour, and she could feel it expanding within her brain and taking over.\n\nShe shook her head to clear it of the sensation, but it clung like a burr. She rubbed at her temples as they pounded with every beat of her heart.\n\n\"Kadence?\"\n\nLifting her head, she found Ronan standing in the doorway. The sweats he wore hung low on his hips, a sheen of sweat emphasized the chiseled ridges of his abs, and his damp hair had been brushed back from the rugged angles of his face. He'd been working out with the others, and the new recruits who had been moved here. Now it was time for the two of them to train together.\n\n\"What is wrong?\" he asked as he strode toward her.\n\n\"I... I don't know,\" she muttered. \"I have this feeling that something is coming, but I don't know what, and I can't shake it.\"\n\n\"You've been through a lot.\" He rested his hands on her shoulders as he stopped before her. \"It's understandable that you're unsettled.\"\n\n\"No, it's more than that. Over the years, I've always just _known_ things. This is kind of like what I experienced then, but it's more.\"\n\n\"More how?\" he inquired.\n\nShe blinked when he blurred before her until he became two and then three. She leaned toward him, inhaling his scent as she tried to stay focused on him, but as she watched, the floor vanished beneath her feet.\n\nRonan caught her when her legs gave out. Terror burst through him when he felt her distress and uncertainty battering against the bond connecting them. He tried to get deeper into her mind to see if he could soothe her, but something kept him blocked from experiencing more of what she felt.\n\n\"Declan!\" he bellowed.\n\nHe'd left the others in the gym, but seconds later the pounding of footsteps rang across the marble. Kadence hunched forward and her head snapped up. He found himself gazing into her white-blue eyes. Though she stared at him, he didn't think she saw him as she remained focused on something far beyond him.\n\n\"What the fuck?\" Lucien said from the doorway.\n\nRonan glanced over to find him standing with Declan and Killean. Lucien and Killean took a step back, but Declan winced as he lifted a hand to his head. \"Shit,\" Declan said.\n\n\"What is it?\" Ronan demanded.\n\nKadence wheezed, drawing his attention back to her. He held her cheeks in his hands when she started speaking, \"The pathways.\" Ice slid down his spine as she uttered the same words she'd spoken during her transformation, but this time she spoke in a much more alien tone of voice. \"Plymouth, rock, warehouse, bricks, Joseph.\"\n\nColor flooded back into her eyes and she met his gaze. She trembled in his arms, but determination etched her features. \"I know where Joseph is, or at least where he was.\"\n\n***\n\nRonan had originally refused to bring her with them, which had resulted in their first big fight. She'd eventually worn him down when she'd reminded him that he'd promised not to cage her, that she was stronger than most turned vamps, and that she had found a way out of the stronghold, she would find a way out of the mansion too.\n\nShe also hadn't spilled enough details for them to easily locate Joseph in Plymouth, but she would be able to locate him on her own, once she escaped. The pathway to Joseph's location had unraveled in flashing swirls of brilliant blues and purples in her mind. Each new flash had revealed a landmark she would recognize when she saw it again.\n\n\"Joseph killed my father, and you promised me that you would not dictate my life,\" she said again when Ronan's disapproval vibrated through their bond.\n\n\"You may not have seen a lot of vampires in your vision, but there will be a lot there if Joseph's managed to stay hidden from Brian this whole time,\" Ronan replied, not for the first time.\n\n\"I'm not so sure it was a vision, more like a guide or map,\" she said, refusing to rehash their argument again. \"I saw road signs, landmarks, then the warehouse, and finally Joseph.\"\n\n\"You said when you were a hunter you knew things,\" Ronan said as he turned onto another road leading deeper into the thick woods she'd directed them into. \"Becoming a vampire most likely unlocked something that has been within you. It will grow stronger with time and practice.\"\n\n_Wonderful_ , she thought. Being knocked to her knees by the burst of images flooding her mind earlier had been anything but fun. However, if those images helped them to locate and exterminate Joseph, she'd happily receive them again.\n\nShe glanced at the cell phone in her hand. She'd called Nathan earlier and explained what had happened, where they were going, and who they were hunting. She had a feeling that, if she hadn't told him it was Joseph, he wouldn't be meeting them. Her brother had been distant on the phone, and he'd sounded exhausted. She had no idea what had happened since she'd last seen him, but the elders wouldn't have made what he revealed to them easy on him.\n\nThe screen on the phone lit up as a large boulder with a tree growing through the center of it appeared ahead. \"Turn there,\" she said and pointed at the rock. She clearly recalled the strange rock from the seventh image she'd received.\n\nRonan made the turn and continued down a winding road. It was so dark the headlights barely pierced the night surrounding them. She typed a message to Nathan and closed the phone. \"We're almost there,\" she murmured.\n\n\"You're going to stay in the SUV,\" Ronan replied.\n\n\"No, I'm not.\"\n\n\"Kadence\u2014\"\n\n\"No. We're here because of me. I will not allow you to order me about and keep me locked away like the hunters did. Either accept that I will see this through to the end, or get out of my way because you won't be keeping me in this vehicle.\"\n\nThe others inhaled sharply in the back seat, but didn't say a word. Ronan's head turned toward her, and his red eyes lit the night better than the headlights as he gazed at her. Kadence held his gaze, refusing to back down.\n\n\"I may not have realized it before, but I was submissive for most of my life; I won't be again,\" she said.\n\n\"You've never been submissive around me,\" he muttered.\n\n\"What can I say? You bring out the best in me,\" she replied with a smile that didn't ease the tension from him as she'd hoped.\n\nHe glanced at her again as the knuckles in his hands turned white from his grip on the wheel. Kadence rested her hand over one of his. \"You've been training me. I'm strong and fast, and I promise I won't do anything foolish. I will do what you ask of me as long as you don't ask me to stay behind.\"\n\n\"We've barely gotten any real training in you,\" he muttered.\n\n\"And that is why I'm agreeing to stay out of the way.\"\n\nBefore he could formulate a response, she sat forward in the seat and slapped her hand on the dashboard. \"Take the next left. The warehouse is through the woods about a hundred feet beyond that.\"\n\nShe texted the last stage of the directions to her brother before tossing the phone into the glovebox. Her hand rested on the handle and her foot tapped against the floorboard as she prepared to leap from the vehicle.\n\nPulling to the side of the road, Ronan seized Kadence's arm before she could bolt. She stiffened in his grasp, her gaze swinging back to him. \"Know that I will drag you away from here if you don't listen to me. Promises or not, your safety comes first and I won't have you doing anything to risk your life or anyone else's. I'm willing to let Joseph get away again before I allow that to happen.\"\n\nKadence glanced at the vampires crowding the back seats before releasing the handle. They were all watching her and Ronan with expressions that clearly said they'd rather be toasting marshmallows in Hell than sitting where they were.\n\n\"What do we do?\" she asked Ronan.\n\n\"You stay close to me. Text your brother and tell him to park off the road behind us and continue to the warehouse on foot.\"\n\nShe retrieved the phone from the glovebox and typed the message before focusing on Ronan again. The red had faded from his eyes, but not completely. He released her arm and slid out of the vehicle. He stood there for a minute before sticking his head back in.\n\n\"Landfills smell better,\" he said.\n\n\"Now we know why he's out in the boonies,\" Lucien said and opened his door.\n\nRonan focused on Kadence as she shifted in her seat. The last thing he wanted was to have her here, but he'd promised not to cage her again. He was close to breaking that promise and screw the consequences. She could be mad forever as long as she was alive. He glanced at Declan in the back seat, who stared blankly back at him. Declan could stay with her; he'd be able to keep her restrained if it became necessary.\n\n\"Don't,\" she whispered. \"I promise, Ronan, I'll do what you say, but don't make me stay here. I won't forgive you if you do.\"\n\n\"You'll be alive.\"\n\n\"Life isn't worth living if you take my freedom away from me again.\"\n\nHe strove to maintain restraint as he inhaled a deep breath and then another. \"We'll just check things out for now,\" he finally said and closed the door.\n\nWalking to her side, he opened her door and took hold of her arm to help her out. Her nose wrinkled as soon as she exited the vehicle, and she stepped back. Her hand flew up to block her nostrils. \"It's horrible,\" she muttered.\n\n\"It is,\" he agreed. \"Come.\"\n\n# CHAPTER 41\n\nThey moved down the dirt road with rapid speed before he led her into the forest and around to the side of a dilapidated, three-story, brick building. Judging by its numerous boarded windows and sagging roof, it had been years since anyone had done any upkeep on the warehouse.\n\n\"Smart,\" Declan said as he crouched on the ground beside Kadence. \"We'd never think to look for him out here, and there are no humans around to interrupt them.\"\n\n\"He was one of us,\" Saxon said. \"He knows how we work, what we look for, but Brian should have been able to locate him out here.\"\n\n\"Not if he's been moving around a lot,\" Lucien said. \"This most likely isn't where he's staying. Judging by the smell, there are too many Savages in there for his liking.\"\n\n\"Can he smell the Savages still?\" Kadence asked.\n\n\"No,\" Ronan replied. \"Years ago, Declan and I caught a purebred who turned and questioned him. Once they give in, they no longer smell the rot of another. Probably because they are rotten too.\"\n\nKadence shuddered as her eyes drifted back to the building. Beside her, Ronan's body was coiled so tight, she thought he might snap. \"We have to see how many Savages are in there,\" he murmured. \"This way.\"\n\nHe kept her hand in his as he led her around the crumpling structure to a sliding barn door in the front. From inside, laughter and music flowed out, as did the pungent stench of blood and decay. Turning to Kadence, he clasped her shoulders.\n\n\"I need you to stay here.\"\n\n\"But\u2014\"\n\n\"You promised you would do as I say, and I need you to _stay_ _here_. I can get closer by myself, and I don't want you to see what is going on inside.\"\n\nKadence glanced at the building as she tried to keep breathing through her mouth. Even still, she could _taste_ the horrific stench of this place. She didn't think it was any worse than what she would have experienced before becoming a vampire; there were just far more Savages here than she'd ever encountered before.\n\n\"You can't go alone,\" she whispered.\n\n\"I'm only going to look, not attack.\"\n\n\"Okay,\" she relented. \"You have to be careful.\"\n\n\"I will be.\"\n\nHe drew her close, his hand sliding through her hair to cup the back of her head as he held her close and claimed her mouth. Reluctantly, he stepped back and released her before turning to look at the shadows dancing through the trees behind her.\n\n\"I'd lower those weapons if you want to live,\" he said coldly.\n\nKadence followed Ronan's gaze, as the others turned toward the woods and edged back until they were flanking her and Ronan. The shadows shifted when her brother came forward, lowering his crossbow as he walked. Behind him, Asher and Logan also emerged with half a dozen other hunters.\n\n\"Don't ever point a weapon at me again unless you're prepared to fight,\" Ronan said to Nathan. \"And if any of you think to aim one of those at her, you'll all be dead before you know you're missing your throats.\"\n\nNathan's gaze flicked to her. \"Fair enough,\" he replied. \"I smell garbage, like you said the Savages of your kind smelled, but it's worse than what I've experienced before.\"\n\nRonan edged Kadence behind his back as he spoke. He didn't trust Logan anywhere near her. \"There are a lot of them inside that building.\"\n\nNathan's gaze went to the warehouse. \"Joseph is in there?\" he asked as he slipped the crossbow onto his back.\n\n\"I think so, or at least I saw him in there, sort of,\" Kadence said.\n\n\"What do you mean by that?\"\n\nKadence sighed. \"It's tough to explain, but you know how I always kind of _knew_ things?\"\n\n\"Yeah.\"\n\n\"I kind of knew this too, but in a way I've never experienced before. Joseph was in there when I saw how to get here.\"\n\n\"Interesting,\" Nathan said. \"So what do we do now?\"\n\n\"Now, I'm going to get closer and see what we're up against,\" Ronan replied.\n\n\"I'm coming with you,\" Nathan stated.\n\nRonan opened his mouth to tell the hunter no, before deciding against it. They were here to try to work together on this. However, he didn't want to leave Kadence here with the rest of the hunters. Seeming to sense this, Declan moved closer to her while Lucien and Killean stepped into position in front of and behind her. Saxon hedged her in on the other side. Kadence scowled at them, but she didn't protest the box they'd closed her into.\n\n\"My men won't harm my sister,\" Nathan said.\n\n\"That's not a chance I'm willing to take, not with her,\" Ronan replied.\n\nNathan turned to face the remaining hunters. \"I'll be back shortly. Make sure Kadence stays safe.\"\n\n\"Oh, for crying out loud, I'm probably stronger than you now,\" Kadence muttered, earning her a dark look from her brother that she returned.\n\nThe two of them didn't look much alike, but in that moment, Ronan saw the striking similarities in the twins' obstinate personalities.\n\n\"Let's go,\" Ronan said and slipped from the woods.\n\nNathan stayed close by his side, moving noiselessly over the ground toward the back of the building. Stopping beside the warehouse, Ronan edged toward one of the broken windows. The board covering it had slipped to expose the bottom of the window. He knelt beside it, and Nathan crouched down across from him. Nathan's eyes were wary, but Ronan didn't detect a spike in the hunter's heartbeat over being alone together.\n\nRising slowly, Ronan peered into the window. His teeth clamped together when he saw the vast number of Savages within. There had to be at least a hundred of them, and probably more on the upper floors. Beside him, Nathan's breath hissed in when he rose to look inside too.\n\nMixed in with the Savages were humans. Most of the humans were dead, their bodies littering the floor, but there were still some who were being feasted on by numerous Savages at once. His gaze ran over the naked bodies covered in blood and writhing over each other as many of them feasted on each other while they screwed in the middle of the floor.\n\nRonan's forehead furrowed as his eyes were drawn to the back wall and the line of vampires there. All of them had their hands pulled over their heads and were chained to the wall. Their red eyes gleamed in the candlelight and their fangs extended as they jerked against their bonds. Their emaciated frames made it impossible for them to break free of their binds.\n\nDespite the stench of the place, Ronan didn't get an overwhelming sense of power coming from the Savages crowded within. He would bet most of them were no more than a month old.\n\nSliding away from the window, he rested his fingers on the ground as he tried to puzzle out what was going on within. Anger etched Nathan's face when he knelt in front of him again. Ronan jerked his head to the side and glided back toward the woods. An uneasy feeling grew in the pit of his stomach with every step he took. Something was wrong here; he just didn't know what it was yet.\n\n\"Why is my sister here?\" Nathan asked, breaking into his thoughts.\n\nRonan stopped walking to face him. Nathan had a good four inches on him, but he still stepped away from him. \"Because she is.\"\n\n\"She doesn't belong here.\"\n\n\"If I forced her to stay behind, I'd have done to her what you did to her, and look at how well that worked out for you. She may love me, but she'll grow to hate me and it will destroy her if I try to lock her away, even if it's what I'd like to do to keep her safe.\"\n\nSaying the words out loud made him realize how true they were. He'd agreed to allow her to come to make sure she didn't try to find Joseph on her own, but he realized he'd also agreed to it because he knew he could never crush her spirit. He loved her too much for that.\n\n\"She belongs here as much as you do. Besides, she's right, she is stronger than you are now. She's stronger than any other turned vampire who has come before her, and I've been training her, _really_ training her. She's lethal.\"\n\nA muscle twitched in Nathan's cheek, but he didn't argue with him. Ronan turned away from him and continued through the woods as his mind churned over what they'd seen in the warehouse. He pulled Nathan back when the missing piece slid into place.\n\n\"This is where Joseph is storing his recruits. His _new_ recruits,\" he said.\n\n\"Recruits?\"\n\n\"Yes.\"\n\nTurning away, he poured on the speed until he came back to the place where he'd left Kadence and the others. She pushed past Killean to come to him. Logan sneered and hatred burned in his eyes as he watched her. Stepping forward, Ronan gathered her in his arms and held her firmly against his chest. He pinned Logan with a remorseless stare that caused him to pull at his collar as he looked away.\n\n\"How bad is it?\" Kadence asked.\n\n\"Bad,\" he replied, unwilling to release her. \"There are at least a hundred Savages inside.\"\n\n\"Is Joseph in there?\"\n\n\"I don't think so. He's definitely been here, as this is where he's creating and keeping his recruits.\"\n\n\"What do you mean _creating_?\" Declan asked.\n\n\"I mean, he's turning humans and putting them in there. He's probably feeding them after they turn so they can complete the transformation, but then he's chaining them to the walls until they're so famished they ravish the first human they come into contact with. If they kill often enough, they won't fight their bloodlust anymore. The Savages who aren't chained are in the middle of a feeding and sexual frenzy. They've completely given themselves over to the bloodlust.\"\n\n\"He's also probably recruiting shitty humans who would be more than happy to live forever while continuing to be shitty vampires and killing for sport,\" Saxon said.\n\n\"Probably,\" Ronan agreed. \"The few humans still alive in there won't be for much longer.\"\n\n\"If he plans on turning humans into Savages, he'll build an endless army,\" Lucien muttered and ran a hand through his hair.\n\n\"Yes,\" Ronan agreed. \"And he may have other buildings like this out there.\"\n\n\"How do we stop them from being set loose?\" Nathan inquired.\n\n\"First things first. At sunrise, we're burning this place down and taking out as many Savages as we can.\"\n\n\"You mean _we_ are,\" Nathan replied and waved at the other hunters.\n\n\"You'll learn, young hunter, that I always mean what I say,\" Ronan replied.\n\n# CHAPTER 42\n\nNathan rested his hand on her arm and drew her away from the others. Ronan lifted his head to watch them. Through the bond connecting them, she sensed his dislike of their distance, but he didn't come after her.\n\n\"I don't understand,\" Nathan said as the rays of the sun lit the earth and fell over them. His gaze ran over her before going to the sun and then beyond her to Ronan. \"How is it possible you're all out in the sun? Are they really vampires? Are _you_?\"\n\nKadence glanced at the others as they worked to fill glass bottles with the gasoline they would be using to set the warehouse on fire. Ronan had sent Declan to retrieve the supplies a couple of hours ago; Nathan had sent Asher with him. Kadence recognized it as the first olive branch between the two factions, but it had been a very thin branch. No one had spoken while Asher and Declan were gone, and she doubted the vamp and hunter had done any bonding while on their mission.\n\n\"I wouldn't lie to you about that, Nathan,\" she said. \"I am a vampire and so are they. I know you can feel their power.\"\n\n\"I can, and I can feel... a difference in you. Something more than just a vampire.\"\n\n\"I am different than they are. Not much, but my transformation was different. My eyes don't turn red when I'm upset, and I _saw_ this place. It was the strangest thing ever, but I saw flashes of different things, which helped to guide my way here. It's like something inside of me was set free when I turned. Most likely some dormant part of our demon DNA.\"\n\n\"How is it possible all of you can be in the sun?\"\n\n\"I told you, Nathan, we've been wrong all these years. Not completely wrong, but enough that we can all work together to make a difference. Vampires who don't kill innocents can go out in the day. The more vampires kill, the more they are unable to tolerate the sun.\"\n\n\"Holy shit.\" He ran a hand through his disordered hair before tugging at the ends of it. \"Working together is easier said than done, Kadence. The elders\u2014\"\n\n\"Won't understand this, I know, but _you_ have to. You're the leader, Nathan. If we don't work together, if we don't stop fighting vampires who aren't our enemies, we'll all be destroyed. I think this warehouse proves there is something horrible in the works right now.\"\n\nNathan's eyes were haunted when they met hers. \"It's more than just the elders. Yes, we were wrong about some things, but there are other strongholds around the world. There are others with us who will not understand this or agree to it. Everyone in our stronghold wants to change our location, and I have agreed to it.\"\n\nShe ignored the twinge of sadness his words caused her. She should have known they would decide to do that once they'd learned of her fate. \"I would never reveal where it is,\" she murmured.\n\n\"I know, but it doesn't matter. They don't feel safe, and I can't have that. They're in the process of moving as we speak.\"\n\n\"I see.\"\n\n\"How did you escape, Kadence?\"\n\nShe smiled at him. \"Planning,\" she replied and gave him the shortened rundown on her method.\n\n\"Smart,\" he murmured.\n\n\"Determined,\" she replied. \"Nothing is ever easy, Nathan, not in our worlds, but the hunters have followed our family for years, and for more reasons than we were the strongest line. We are born leaders who can do anything we set our minds to. The hunters believe things are one way, and we've always accepted it, but you will make them see that things must change if we are all to survive.\"\n\nHe opened his mouth before closing it again. His head tilted to the side as he studied her. \"We've been together our whole lives, but I'm not sure I ever knew you. A part of you would have died if you stayed in the stronghold.\"\n\n\"This was the path I was meant to take in life, and I truly believe that. It's more than seeing things more clearly now, like this place, but I also see them _differently_.\"\n\n\"I can understand that,\" he said.\n\nRonan strode over to them and slid his arm around her waist as he handed Nathan a bottle of gasoline with a rag hanging out of the top. \"Let's get this over with.\"\n\nNathan took the bottle and glanced between the two of them. \"We have much to discuss when this is over.\"\n\n\"We do,\" Ronan agreed. He removed his arm from her waist and turned to face her. She'd come armed with stakes and a crossbow, but he pulled out another crossbow and a lethal-looking switchblade knife. He handed them both to her. \"I know you won't return to the vehicle, and I'd prefer to have you where I can see you, but don't you let anything that comes out of that building anywhere near you. The fire and the sun will take care of most of them before they can get close to you, and _I_ will take care of anything else.\"\n\nShe nodded and he clasped the lapel of her coat. Pulling her closer, he kissed the end of her nose before his forehead fell briefly against hers and he inhaled her fragrant scent. Every instinct he had said to take her from here before this started.\n\nHe couldn't, he knew, but allowing Kadence to embrace who she was becoming may be his undoing. She wrapped her hands around his wrists as she lifted her lips to his. _I love you_.\n\nThe words were a soothing caress that came from her mind and whispered into his. _I love you too_ , he told her.\n\nLifting his head, he kissed her again before releasing her. \"I don't want you in the shadows,\" he said as he led her toward the edge of the small clearing surrounding the crumpling building. The woods had crept in to reclaim most of their land, but there were still some open patches. \"As soon as that building is on fire, get in the sunlight.\"\n\n\"I will,\" she promised and stroked the thick muscles in his forearm when his body vibrated with tension.\n\n\"Don't get out of the sunlight.\"\n\n\"I won't.\"\n\nHis gaze remained latched on her, his eyes turning redder with every passing second. She knew that color wasn't entirely because of his concern for her, but also because of the impending battle. She could feel his growing excitement over what was about to unfold. Their bond would keep him from turning Savage, but she'd never be able to completely ease the compulsion to kill from him, and she didn't want to. She loved him for who he was, a powerful warrior.\n\nNathan stepped beside them and rested his hand on her shoulder. Kadence grasped his hand and tears burned her eyes at the connection. He squeezed her fingers before releasing her.\n\nHe moved swiftly forward with Ronan and the others. Kadence remained in the tree line with Declan and Saxon, watching as half of the hunters and three vampires approached the warehouse. A clammy sweat coated her skin and her heart raced as Ronan and Nathan crept up to the backside of the building.\n\nThe strengthening rays warmed Ronan's skin as Lucien and Killean broke off to head around the building. Nathan stayed by his side as he reached the warehouse. Turning, bricks bit into his back when he pressed it against the wall and lit the rag sticking out of the bottle he held. Nathan did the same with his bottle.\n\nRising to his feet, Ronan used his fist to smash the board out of the window and tossed the bottle inside before Nathan threw his in. The crack of more boards breaking sounded around the warehouse before the first startled cry pierced the air.\n\nRetreating from the building, Ronan fell back as the screams within rose. Declan led Kadence out of the shade of the trees to stand twenty feet behind him. Saxon moved forward to join the circle near the front of the building. From his spot, Ronan couldn't see the hunters guarding the front.\n\nFlames shot out of a lower window as the first tendril of smoke spiraled from the roof. The back doors burst open and a rush of Savages poured out to take their chances with the sun instead of the flames. Some of them didn't make it ten feet before they shrieked and fell to the ground. They wailed as the sun blistered their skin and smoke poured from them. Others made it to the circle where they were taken out by a hunter or vampire.\n\nRonan seized the first one coming at him and threw him to the ground before grabbing the next two. He tossed one of them onto the ground before tearing the head from the first. Placing his foot into the back of the one he'd thrown down, he ripped the heart out of the next Savage before finishing off the one pinned beneath him.\n\nFrom behind him, a bolt whistled past his ear and embedded into the heart of a Savage racing toward him with smoke billowing off his body. The impact of the bolt through his heart threw the Savage back and he fell to the ground, dead. Ronan glanced over his shoulder as Kadence lowered her crossbow and grinned at him.\n\nShe reloaded her weapon and spun to take out another Savage barreling toward her brother. Nathan gave her a startled look before returning to the fight.\n\nThe next Savage Ronan captured had flames shooting from his eyes when he tore its head off and tossed it aside. One of the hunters on the side of the building fell beneath the weight of three Savages who were barreling toward the woods. Saxon clasped the hunter's hand and yanked him to his feet as flames and smoke trailed behind the escapees.\n\nThe hunter turned to follow them, but Ronan's bellow froze him in place. \"Do _not_ break formation!\"\n\nThe hunter glanced after the Savages. If they made it to cover and put out the flames, they were still less of a threat than the mass fleeing the warehouse. Ronan had just grabbed another Savage when the clatter of shattering glass and wood sounded. Driving the Savage into the ground, he lifted his head to look up as vampires leapt out of the burning building from the windows above.\n\nHis head spun toward where Kadence stood behind him as the first wave of vampires leapt over him. \"Kadence!\" he roared as the Savages jumped to their feet and fled toward the woods.\n\nDeclan yanked her back and caught the first two coming at her. Kadence fired the bolt in her crossbow before releasing the weapon to let it fall against her side. She pulled her stakes free as she prepared for the next wave of Savages.\n\nAdrenaline raced through Kadence as her fangs lengthened. She bounced on the balls of her feet and spun to drive a stake through the heart of a female Savage who had been coming at her with her fangs fully extended. Yanking the stake free, Kadence dashed to the side and drove both her stakes into the hearts of two more Savages as Declan took down another. The Savages parted enough to reveal Ronan carving his way ruthlessly through them toward her.\n\nShe ducked to avoid the clawing grasp of another Savage when something hit her in the back. A foot kicked out, knocking the stake from her hand. Her other stake was yanked from her as a heavy weight fell on her, nearly driving her to her knees.\n\nBefore she could hit the ground, she was pulled back up by a hand gripping the collar of her jacket. \"What do we have here?\" a voice murmured in her ear, and she realized the bastard had jumped onto her from above.\n\nFor a minute, she thought it was Joseph who held her when an arm cinched around her neck and her head was pulled roughly back. The voice didn't match Joseph's though. She smelled death on the man holding her, but it wasn't as overwhelming as Joseph's stench had been.\n\nDeclan took a step toward her and stopped when the arm around her neck jerked, causing her to choke as she was dragged back three feet.\n\n\"Tsk, tsk, Declan,\" the man holding her murmured.\n\nDeclan's forehead creased as he gazed at the man. The Savage chuckled and pulled her back another step. \"You have no idea who I am.\"\n\n\"Not a clue,\" Declan said.\n\n\"And it doesn't fucking matter,\" Ronan replied as he stalked toward them.\n\nThe fury emanating from him in waves caused her skin to prickle. The hold on her neck tightened until there was no room between her body and the solid wall of flesh behind her.\n\n# CHAPTER 43\n\n\"Stop!\" Ronan snarled when her toes were pulled off the ground. His long fangs glinted in the sun's rays and his eyes became so intense a red that Kadence swore she felt the heat of them against her skin.\n\nKadence tried to turn to see who held her, but her head was jerked back again, making it impossible for her to move it. Gritting her teeth, she forced herself to go limp. The Savage behind her grunted and then wrapped his other arm around her waist to support her weight.\n\nRonan's eyes focused on her as the Savage dragged her further back. \"Joseph will like my little prize,\" the Savage muttered and turned his nose into her hair before inhaling deeply. \"You and Ronan are entwined with each other. You reek of him, cupcake.\"\n\nKadence stiffened when he ran his tongue over her cheek and Ronan's rage blistered across their bond. Her vision blurred when Ronan's emotions swamped her mind. Then, he retreated from her mind and a wall slammed down between them.\n\nShaking off the lingering effects of Ronan's rage, she lifted her head and gasped when her gaze landed on him once more. The staccato beat of the Savage's heart sounded in her ears and sweat now coated his forearm as he held her closer, something she hadn't considered possible until his body practically molded around hers.\n\nShe didn't blame him for being terrified. She was more than a little unnerved herself as a reddish-black hue began to seep insidiously over Ronan's skin. The only time she'd ever seen anything like that color was in pictures of demons. The color seeped over the backs of his hands and crept up from the collar of his shirt to spread across his face. The way the color moved through him reminded her of the flames spreading out to destroy the warehouse behind him. The stare he leveled on the Savage promised death, and not a merciful one.\n\nWhen something poked her in the chest, she tore her gaze away from Ronan's startling visage to the stake aimed directly at her heart. Yet, she felt no fear of the weapon. Not when she had a very pissed off purebred standing across from her, and the vamp holding her had begun to quake.\n\nThe flames of the building rose higher behind Ronan, and she became aware that the screaming had stopped and that all the hunters and Defenders were creeping toward them now. The ground was littered with bodies and drenched in blood. Some of the bodies still had flames consuming them, but most were little more than smoldering ash in the growing daylight.\n\nThe Savage holding her hissed and stepped further into the shadows of the forest as his skin blistered and smoke curled into her face. He didn't have the same intense reaction to the sun that most of the others had, leading her to believe that he hadn't killed as much as they had.\n\nRonan's face became completely encompassed by that insidious color. Though the rest of him was covered by his clothing, she _knew_ his entire body was now that reddish black hue. The rest of his men fanned out behind him, but most of the hunters had started to hang back, afraid to move any closer to Ronan. Nathan edged forward, coming toward her from the side.\n\n\"Back off, Ronan, or I'll kill her,\" the Savage said.\n\n\"No,\" Ronan said in a guttural voice she barely recognized. \"You won't.\"\n\n\"You're all over her,\" the Savage spat. \"You think I can't recognize your claim on her? A newly turned vampire could realize it, and I am no newly turned vamp!\"\n\nThe last of his words took on a hysterical tone. Ronan's unblinking stare mixed with the demon-color was the most frightening thing Kadence had ever seen.\n\n\"No,\" Ronan finally said. \"You're a purebred, and you're a dead one.\"\n\n\"You won't do anything to me while I have her, and you'll do anything we say once I give her to Joseph.\"\n\nRonan's skin swirled and pulsed with those colors as his eyes burned like hot coals. \"You won't make it one more step, let alone reach Joseph.\"\n\nKadence's fingers inched toward her side as the knees of the Savage literally knocked together. Slipping the knife Ronan had given her from her pocket, her fingers slid over it as she worked to free the blade.\n\nThe Savage gave a harsh bark of laughter. \"You think you're so strong, Ronan, so powerful, but you don't know what's coming. Not even _you_ will be able to stop it, and _I_ will be standing there laughing when you're finally taken down.\"\n\nKadence finally succeeded in opening the knife and turned it in her grasp. \"I can't wait to see you destroyed,\" the Savage growled.\n\nGripping the handle, Kadence kept her arm close to her side as she swung the blade back and drove it into the Savage's thigh. She threw her weight to the side to keep from taking a stake to her heart as she twisted the blade in his flesh and jerked it to the side. The putrid scent of his blood hit her as he grunted and fumbled to keep his hold on her. His hand twisted around her braid and he yanked her back.\n\nKadence never saw Ronan move until he was on top of the Savage, driving him into the ground like a jungle cat taking down its prey. She bit back a cry as her head jerked forward and she was brought to the ground with them. Before she could try to free herself, Ronan tore the hand holding her from the Savage with a brutal twist of bones and sinew. Bile surged up Kadence's throat when the fingers of the severed hand remained entangled in her hair as she scrambled back.\n\nFumbling awkwardly, she managed to tear the hand free and throw it aside. The Savage wailed in horror as blood sprayed from the stump of his wrist and Ronan remained perched on his chest. With methodical ruthlessness, Ronan tore the other hand away from him.\n\nKadence crab-crawled backward across the ground, looking to get away as the sour stench of urine filled the air, and she realized the Savage had pissed himself. She didn't blame him.\n\nHands seized her arms, and her head fell back to take in Nathan standing above her. His mouth fell open when Ronan's head swiveled toward them. Ronan's nostrils flared, and the red of his skin took on a scarlet hue.\n\n\"Let her go!\" Declan snapped and yanked Nathan's hands away from her.\n\nNathan released her and took two steps back, but Ronan continued to watch him. Kadence realized the Savage had pushed him too far and he was on the verge of completely snapping.\n\n\"I'm okay,\" Kadence choked out. He'd said that because of their bond he wouldn't turn Savage, but if the man before her wasn't Savage, then she didn't know what was. \"Nathan was just trying to help me!\"\n\nNathan lifted his hands in the air as if to prove her point. Ronan watched him for a second more before his attention shifted back to the Savage.\n\n\"Don't touch her again, not when he's like this. He won't hurt her, but he'll shred every one of us,\" Declan warned, and Nathan nodded.\n\n\"Where is Joseph?\" Ronan snarled.\n\n\"I... I don't know!\" the Savage wailed. \"Not here. He left me here yesterday and went somewhere else!\"\n\nRonan leaned so close to the savage that their noses almost touched. Amusement slithered through him as the man's misery battered him. This vampire had no idea what suffering was yet, but he would by the time Ronan was done with him. The vamp's eyes rolled in his head as his stumpy arms beat against Ronan's sides. Ronan slid his fingers around the vamp's throat, drawing blood as he dug into the Savage's flesh.\n\n\"What is coming?\" he demanded.\n\nSpittle flew from his mouth when the Savage violently shook his head back and forth. Ronan dug his fingers deeper in until he could feel the Savage's spine. He scraped his fingers across the bone and the Savage blurted out a reply. \"A war! Your death!\"\n\n\"Not my death, but yours. I'll make it quick if you tell me where Joseph is,\" he promised.\n\n\"I really don't know!\" the Savage cried.\n\n\"Ronan, he doesn't know,\" Kadence whispered.\n\nHis attention was drawn to her, sitting on the ground ten feet away from him. Her neck was still red from where the vamp had choked her. This _thing_ had dared to touch her. Yet, he couldn't tear this creature leisurely apart like he longed to do, not in front of her. She understood his darker side, but he wouldn't expose her to such cruelty, not when he knew she was right; this thing didn't know where Joseph was right now.\n\nWith a growl, he jerked his hands apart, tearing the savage's throat in half and rending its head from its shoulders. The head rolled away before the rays of the sun lit upon it and flames licked over its skull.\n\nHe inhaled a shuddery breath as he struggled to regain control of himself. He'd seen what could happen to a purebred when infuriated or when their mate was in jeopardy, but he'd never experienced it before. He gazed down at the red and black coloring of his skin as he wiped his blood-drenched hands on the ground.\n\nDespite the fact the Savage was dead and Kadence was safe, the color wouldn't retreat from his body. Turning, he looked to Kadence again as she gazed at him in wordless wonder. She'd witnessed him at his most brutal and out of control, yet he saw no revulsion in her eyes.\n\nScrambling across the ground, she flung herself into his arms. Ronan remained motionless for a second, unwilling to touch her with the putrid blood still on him, but he couldn't resist holding her. His arms closed around her and he drew her against him.\n\n\"Did he hurt you badly?\" he demanded.\n\n\"No, I barely felt anything,\" she whispered. \"Are you all right?\"\n\n\"There isn't a mark on me.\"\n\nShe ran her hand over the front of him. \"What is this?\"\n\n\"It will fade.\"\n\n\"What...?\" Her voice trailed off, and she bit her lip as she glanced at the others who were edging closer to them.\n\n\"Purebreds are capable of almost anything when their mate is in trouble,\" he told her and looked pointedly at the hunters nearby, making it clear that if they tried anything with her, he would joyfully tear them all apart too.\n\nKeeping his arms around her, he lifted her from the ground when he rose to face the others. The roof of the warehouse gave way with a loud crack; smoke and flames burst high into the air as bricks crumpled and fell.\n\n\"We have to get out of here before the humans arrive,\" he said and focused on Nathan. \"We have much to discuss.\"\n\n\"We do,\" Nathan replied, though he was paler than he had been as he stared at Ronan.\n\n\"I know a place if you'll follow us.\"\n\n\"We will,\" Nathan said.\n\nKadence buried her face in Ronan's neck and inhaled his scent. She slid her fingers into his hair when he started walking across the ground with her. Reluctantly, she leaned away to look at him. His eyes still glistened like rubies, but that reddish-black hue was retreating from his face.\n\n\"Why did your skin change like that?\" she asked as she ran her fingers over his face. Some of the color remained in his cheeks, but his skin felt no different to her.\n\nHer words drew his attention to the color seeping out of his hands. \"Because the demon part of me was close to the surface,\" he replied. \"Closer than it's ever been before. I've witnessed it happening to a purebred before, but I've never experienced it. When that part of a purebred is unleashed, there is little they can't do. They can even enter a house uninvited and break the rules that normally govern our kind.\"\n\n\"Does it only happen with purebreds?\"\n\n\"Yes... well, at least it only used to be purebreds. You are a new entity, so who knows what you might be capable of doing.\"\n\nHer lips flattened as her fingers played with the hair at his nape. \"I would do anything to destroy someone who attacked you,\" she replied.\n\nHe couldn't help but smile over the possessive tone of her voice. \"I believe you.\"\n\n# CHAPTER 44\n\nRonan turned on the lights within the closed hotel as he moved through it. Baldric purchased the property in Falmouth for him last month, not for the hotel part of it, but for the fifteen acres of land that came with it, a rarity on Cape Cod and an investment for Ronan. The hotel had closed for the winter months before he'd purchased it.\n\nRonan expected to add the property to his collection and forget about it, but they'd been close enough to it today for it to make a good meeting place for now.\n\nThe briny scent of the ocean filled the air as the waves crashed on the shore outside of the boarded-over windows. He kept his arm around Kadence as he walked, not to protect her, but because he needed her calming presence right now. He'd regained most of his control, but it wouldn't take much to push him over the edge again.\n\nArriving at the restaurant area, he flicked on the lights and strode over to pull a chair from the top of one of the tables. Dust floated up as he set the chair on the ground and settled Kadence onto it.\n\n\"Will you be sitting?\" he asked Nathan.\n\n\"Not unless you are,\" he replied.\n\nRonan smiled at him as he pulled down two more chairs and placed them on the ground. The others spread out around the room, but didn't bother to remove the chairs from any of the remaining tables. Ronan hadn't expected them to.\n\n\"Your injuries from last week have healed well,\" Nathan said. \"I would have thought the bolt hole from a crossbow would take longer to close.\"\n\nThe hunter was brave for digging at him in such a way, or stupid, but he'd come to realize Kadence and her brother were far from stupid. Nathan was trying to learn more about him.\n\n\"I heal faster than most,\" Ronan replied. \"As it seems, you do too. Broken noses don't often heal within a week.\"\n\n\"Lucky punch,\" Nathan replied.\n\nRonan chuckled. \"Lucky shot from you. The fact you're her brother made me hesitate in taking you down.\"\n\nKadence rolled her eyes as the two of them stared at each other. She could practically taste the amped up testosterone on the air. \"Yeah, yeah, you both heal fast and are manly men. Could you please move on so we can discuss more important things sometime today?\"\n\nSettling next to Kadence, Ronan rested his hands on the table as he waited for Nathan to sit across from him. Finally, Nathan tore his attention away from Kadence and focused on Ronan again as he settled into the chair across from him.\n\n\"What happened back there, I've never seen a vampire do what you did,\" Nathan said to him. \"What was with the weird skin change?\"\n\n\"You'll probably never see it again,\" Ronan replied. \"It's a trait of purebreds only.\"\n\n\"And that is what you are?\"\n\n\"I am fifth generation purebred, the only one of my kind to ever exist with such breeding and the only who can trace their lineage back to the first demon offspring.\"\n\n\"So, you are the leader?\" Nathan asked.\n\n\"When I was younger, I was raised to one day be king; I never took that role.\"\n\n\"Why not?\"\n\n\"Many reasons.\"\n\n\"Are the other non-Savage vamps unwilling to follow you?\"\n\n\"We would follow him to Hell and back,\" Declan said. \"You know naught of our history.\"\n\nNathan clasped his hands before him as he leaned back in his chair. \"We apparently don't know much about you at all. I didn't know there were such things as turned and purebred vampires, Savages, and well... you.\"\n\n\"He looked like a Savage to me earlier,\" Logan muttered.\n\nRonan didn't bother to acknowledge him as he remained intent on Nathan. \"Almost a thousand years ago, I chose not to become king. It is a decision I will be changing, somewhat. Because they have been so scattered over the years, most vampires don't know our history either, how we were originally created, or the full extent of what some of us can do. It is time that is rectified. I will not be king, that title died with my father as far as I'm concerned, but from now on, I will work to gather the vampires and lead them.\"\n\nDeclan grinned while Saxon, Lucien, and Killean exchanged surprised looks before nodding enthusiastically.\n\n\"Why?\" Nathan inquired.\n\n\"With or without the help of the hunters, Joseph must be taken down and what he is doing must be stopped.\"\n\nRonan didn't tell him about the Savage attack he'd survived when he'd been younger. They may end up working together against Joseph, but he couldn't have the hunters knowing about their small purebred numbers.\n\n\"The Savage that attacked Kadence, he knew you.\"\n\n\"He did,\" Ronan confirmed. On the half hour drive it had taken to get here, Declan recalled who the Savage was. \"He was a purebred who tried to make it through Defender training under Joseph.\"\n\n\"What is a Defender?\" Nathan inquired.\n\n\"The purebreds who work to keep humans and innocent vampires safe have always been known as Defenders. While he was with us, Joseph worked to train purebred vampires. He also worked with turned vampires and taught them how to differentiate a Savage from another vampire.\"\n\n\"How can they tell the difference if the turned ones can't smell them?\"\n\n\"It's easy enough to look for the signs of a Savage hunting in the area. An increase of missing people reports, unexplained deaths, animal attacks, and such. Then they focus on a Savage's favorite places to hunt, like bars and clubs. They prefer anywhere a human is more vulnerable and their defenses are lowered. Once the vampire is located, it's simply a matter of confirming that they are a Savage.\"\n\n\"By witnessing the death of a human?\" Nathan asked.\n\n\"No,\" Ronan replied. \"When we feed from humans, we do not enjoy making them suffer. It is usually the opposite, and we seek to give them pleasure, or we shut down their emotions so that they feel nothing.\" That was the way he'd fed from others, with no emotional involvement, until Kadence. \"Afterward, we change their memories of what occurred. However, a Savage prefers to experience the pain of their victim. They thrive on the fear and agony. It gives them a bigger rush, as does hunting and killing other vampires in such a way. If you come across a vampire whose victim is suffering during the blood taking, then you have come across a Savage.\"\n\nNathan looked dumbfounded by this revelation. Ronan glanced at the other hunters who were hiding their shock with far less success than Nathan as they gawked at him and exchanged looks.\n\n\"Plus, you now know you can smell the difference too. Remember that before you stake your next vamp,\" Ronan continued.\n\n\"I will,\" Nathan replied though some of the hunters scowled behind him. \"So the vamp who attacked Kadence was one of you?\"\n\n\"No, he didn't successfully make it through the training, and it seems Joseph decided to recruit him to his cause. That Savage was a new edition to Joseph's collection. He hadn't killed many, yet. Otherwise, he couldn't tolerate the sun as well as he did. By next week, he probably would have burst into flames like the others. I'd be willing to bet there are others who have also failed their training and that Joseph is looking to recruit them too.\"\n\n\"Others who have been trained to fight by one of your men?\" Nathan asked.\n\n\"One of our _ex_ men,\" Ronan replied.\n\n\"But Joseph is highly trained, they're highly trained, and there's a chance they could have a grudge against all of you.\"\n\n\"They _could_ have a grudge against us, but _every_ one of them hates hunters,\" Ronan said pointedly.\n\n\"It's hard not to, you stake happy bastards.\" Lucien smirked.\n\nRonan shot Lucien a silencing glance before continuing. \"They know they can't take us down, not without a lot of help, and they have no idea where to find us. You, on the other hand, unless you plan on calling off all your hunts until this danger is eradicated, they'll be able to take you out with far more ease than us.\"\n\nAnger flashed through Nathan's eyes before he smothered it. \"We are stronger and more capable than you think.\"\n\n\"Believe me, I know that.\" Ronan clasped one of Kadence's hands within both of his. \"But that will get you nowhere if you're outnumbered by Savages. The more they kill, the more limitations they have on them such as not being able to handle sunlight or cross large bodies of water, but they also grow physically stronger.\"\n\n\"Do you really think that Joseph organizing an army is the bigger threat the Savage was talking about, or do you think there is something more out there?\"\n\n\"I don't know,\" Ronan admitted. \"I can't think of what else it could be, but Savages are extremely cunning, and they do not want to die. They'll do everything in their power to eradicate any threat to their lives.\"\n\n\"It won't be easy to convince the elders we should work together, or the other strongholds around the world,\" Nathan replied.\n\n\"After last night and this morning, it will be easier,\" Asher chimed in. \"I know I would prefer not to kill a vampire who isn't looking to kill me or a human. That whole blood drinking thing is still disgusting, but we all do what we must to survive, and if they're not hurting someone\u2014\"\n\n\"They're draining the blood of another and changing their memories; there is something wrong with that,\" Logan sneered.\n\n\"There is a difference between that and killing, Logan, and you know it,\" Asher said.\n\n\"There is,\" Nathan agreed.\n\n\"We can continue to work in opposition and possibly all be slaughtered because of that, or we can all work together and destroy the bastard who killed Dad,\" Kadence said.\n\nNathan quirked an eyebrow at her. Kadence lifted her chin as she waited to be admonished for her language. If they'd been sitting in the stronghold, she would have been, but Nathan didn't comment on her word choice. She wasn't his to take care of anymore, and she realized her brother was coming to grips with that.\n\nHer hand tightened around Ronan's as she spoke. \"Nathan, we can do this together.\"\n\nNathan leaned back in his seat and rubbed at the black stubble lining his jaw. Dropping his hand down, he leaned forward again. \"You will be taking control of the vampires, rising to the position of leader, the one you claim was supposed to be yours,\" he said to Ronan.\n\n\"Like you, I was specifically bred and groomed to one day take control of my kind. Unprecedented events detoured my course for a while, but unprecedented events have steered me back onto it.\" Lifting Kadence's hand, he placed a kiss against her knuckles to ensure her brother understood his point.\n\n\"And how will you go about taking control?\" Nathan inquired.\n\n\"I will organize them. Most vampires live in peace, but what Joseph is doing is threatening to all our kind and the entire world. For many millennia, only purebreds have been Defenders against the Savages. I was reluctant to bring turned vampires in to become a Defender, reluctant to change our customs, but I realize it has to be done and that living in the past may be the downfall of us all. You will do what you must to protect your kind, and I will do what I must to ensure the same thing, but we are both working toward the same common goal.\"\n\n\"And these vampires you aim to bring in and organize, they won't want to destroy us too?\" Nathan asked.\n\nKillean shifted behind him, his face remained stony, but the gleam in his golden eyes told Ronan he'd happily destroy every hunter in this room and consequences be damned.\n\n\"I'm sure some of them will,\" Ronan replied honestly. \"Just as some of the hunters you bring in will still prefer to see every vampire dead. Continuing to fight and kill each other is pointless. No matter the resentment and history that is sure to boil forth on both sides, we must find a way to either work together or stay out of each other's way.\"\n\n\"I agree about that,\" Nathan said. \"But what are we going to do? Go on patrols with each other?\"\n\n\"We can start there,\" Ronan replied. \"And over time work toward more. Perhaps one day, we can train together. It would create some very lethal fighters.\"\n\n\"It is a start,\" Nathan agreed.\n\n\"And what if one of you turns Savage, what then?\" Logan demanded.\n\nRonan pierced him with a remorseless stare. \"They will be hunted as ruthlessly as any other Savage. Despite our bonds to one another, we all know that is the consequence of turning. Joseph will be located, he will be brought down, and I will be there to do it.\"\n\n\" _I_ will be the one to do it.\" Nathan's quietly spoken words were more forceful than if he'd bellowed them to the room.\n\nRonan had destroyed the prick who killed his parents; he would not deny another the opportunity for revenge, even if he knew it would bring no relief. \"If it works out that way and you are there, then you will be the one to do it, but Joseph will be taken down the first opportunity we have to do so. It is necessary he is destroyed no matter who does it.\"\n\nNathan's hands fisted on the table. \"Agreed.\"\n\n\"I will also be there to see it, if it works that way,\" Kadence said.\n\nNathan sat back in his seat as he gazed at Ronan in challenge. With that action, Ronan realized Nathan had acknowledged Kadence was now under his protection, but it was clear he didn't approve of her being anywhere near the fight.\n\n\"Will you inform the other strongholds about the alliance we will be trying to forge here?\" Ronan asked Nathan.\n\n\"Not right away. We will see how things play out before bringing them in. They haven't reported an increase in Savage attacks or missing people from their locations. For now, it seems as if this growing threat is located here in the Northeast.\"\n\n\"For now,\" Ronan agreed. \"I'm sure Joseph will be trying to establish more warehouses throughout the area in an attempt to expand. We will locate the other turned and purebred vamps he's trained over the years and either bring them in or learn if they've also turned Savage.\"\n\n\"How do you intend to find them?\"\n\n\"We know where some of them are. For the others, we have a friend who may be able to help locate them.\"\n\n\"Will this friend be joining in the fight?\" Nathan inquired.\n\nFive months ago, Brian would have happily jumped in to help kill as many Savages as possible, but it was unlikely he would now that he was mated. \"He will aid us when he can.\"\n\n\"I see. Can he locate Joseph?\"\n\n\"Not recently, but he has a couple of times in the past, and so has Kadence and she may be able to do so again.\" Nathan's jaw clenched when he realized Ronan was telling him that he would not keep Kadence out of this. \"On our way here, I called Brian and asked him to join us. He should be arriving shortly.\"\n\n# CHAPTER 45\n\nAn hour later, Brian strolled into the room with their newest purebred recruit, Aiden, at his side. \"Someone call some hookers so we can really make this a party,\" Aiden drawled as he gazed at the brooding occupants of the room.\n\n\"That would make it a regular old fuck fest,\" Lucien replied.\n\nAiden grinned at him as he hooked his fingers into his belt buckle. \"Nice one.\"\n\n\"I thought so.\"\n\n\"Enough,\" Ronan said. \"Did you bring the things I asked for?\" he inquired of Brian.\n\n\"Yes,\" Brian replied.\n\nHe turned and walked back to the door. Ronan heard whispered voices before the door closed, shutting Brian's mate on the other side. Brian walked over with a stack of folders and placed them before Ronan. The files had been moved from the training facility to the mansion with the rest of the recruits. Everyone moved closer as Ronan spread the folders across the table so that Brian could see the photos attached to the outside of each one.\n\nHe'd told Brian to leave behind the numerous files marked deceased and any over fifty years old. Joseph hadn't taken over the running of the training facility before then. They may have to go through the older files eventually, but right now they had to concentrate on the vampires they knew Joseph had direct contact with.\n\n\"Which ones do we know where they are now?\" he inquired and looked at each of his men.\n\nHands reached around him to pull out the files of those they would be able to locate without Brian's help. He wasn't entirely sure how Brian's ability worked, but he suspected Brian needed to meet someone in person to locate them again. He didn't think Brian would have met many of the unknowns remaining, but even one of them was one less that they would have to waste time trying to find.\n\n\"Which ones do you think you can locate, Brian?\"\n\nBrian studied the pictures before pulling out nearly a dozen files, more than Ronan had expected. \"These.\"\n\n\"Good,\" Ronan said and pushed the remaining folders aside before focusing on Nathan. \"We will find a more central location to meet next time and go out to hunt together from there. Neither of us will reveal where we live, or be expected to. If anyone is caught trying to find the location of the other, it is a breach of trust and the offender will be dealt with swiftly by the other side. We will send out groups of hunters and vampires together every night. If someone is wrongly injured by someone on the other side or knowingly attacked, the attacker will be dealt with by both sides.\"\n\nThese were details they had already hashed out while waiting for Brian to arrive, but he wanted to make sure they were clear to everyone.\n\n\"Agreed,\" Nathan said.\n\n\"It's a solid start,\" Kadence said.\n\n\"It is,\" Nathan replied.\n\nRonan rose with Nathan and extended his hand across the table to him. \"A good beginning to what could become a strong alliance,\" he said as he took hold of Nathan's hand.\n\n\"Yes, it is,\" Nathan agreed and shook his hand.\n\nThere was only one more thing he had to discuss with Nathan before leaving here.\n\n\"I think it is time for us to go. We will be in contact tonight.\" Nathan said.\n\nNathan went to step away, but Ronan kept his hand, holding him back as the others started to file out of the room. \"I'd like to speak with you and Kadence alone,\" he said.\n\nNathan hesitated before waving the rest of his men out the door as Ronan's followed behind them. Ronan released Nathan's hand and drew Kadence against his side.\n\n\"You're to make sure your man, Logan, stays away from Kadence,\" he said to Nathan when the door closed behind the others.\n\n\"I never had romantic feeling for Logan, if that's what you're worried about,\" Kadence said.\n\n\"No, you didn't, but he had feelings for you, and now those feelings have turned to anger.\" His eyes burned into Nathan's as he spoke.\n\n\"Logan would never hurt her,\" Nathan said.\n\n\"Maybe not before, but he's bitter and angry now. That's a deadly combination. If he does anything that I deem threatening toward her, I'll kill him, alliance between us or not.\"\n\n\"Ronan!\" Kadence gasped.\n\n\"No.\" The look he sent her caused her to hold back any further protest. \"On this, I will not budge. He won't be anywhere near you. There will be _no_ arguing about that. I have only one weakness, Kadence, and it is you. Everyone who was in this room knows that. They won't exploit it, but Logan might. Not to mention, you both think you can trust him, so your guard will be lowered around him.\"\n\n\"I'll keep him away from her,\" Nathan promised.\n\n\"Good.\"\n\n\"One more thing,\" Kadence said. \"The women in the stronghold need to be better trained. I'm not saying prepare them for going out in the streets, but you have to do more, Nathan. I believed I was prepared when I left the stronghold, but I wasn't. It nearly got me killed.\"\n\n\"Ah hmm,\" Nathan cleared his throat and gave her a pointed look.\n\n\"I know leaving in the first place put me in danger,\" she agreed, and her brother nodded. \"But I was _really_ unprepared. The women think they can defend themselves. They can't. I know you think you can keep them all sheltered, but there may come a time when you can't. If that time comes, they'll be lambs to the slaughter, and you would have allowed it to happen by not teaching them more.\"\n\nNathan stared at the wall behind her before focusing on her again. \"We will train them better.\"\n\n\"Good,\" Kadence said. \"And speaking of women, how is Simone?\"\n\n\"She is fine,\" Nathan answered.\n\n\"Will you tell her I say hi and that I miss her?\"\n\n\"I will,\" he promised.\n\n***\n\nNathan's mind spun as he tried to sort through everything that had happened in such a short time. He had no idea how he would get the elders and some of the other male hunters in the stronghold on board with this, but he had to. After what he'd seen with those Savages in the warehouse, he knew it was only a matter of time before they were all completely outnumbered.\n\nThere would be no stopping the spread of the Savages once that happened. Sighing, he ran a hand through his hair as he walked out of the room beside Ronan and Kadence. His gaze slid to his headstrong sister. He also had no idea what to make of her newfound vampire status or her relationship to this man\u2014a man who would tear the world and everyone in his way apart to protect her.\n\nHe never would have thought he'd be okay with his sister in a relationship with a vampire, and especially not okay with her _being_ one, but he had to admit, Ronan may be the only one who could keep her safe. But more than that, Ronan made her happy in a way he'd never seen Kadence happy before.\n\nHe wanted to hate Ronan, his entire life had been built on hatred for what he was, but he found he couldn't. Not when Kadence was smiling like that and practically glowing with self-confidence, happiness, and love. More than that, he couldn't deny that the vampire loved her too. He wouldn't have believed a vampire capable of feeling love, but he was beginning to realize he'd been wrong about many things throughout his life.\n\nHe never would have chosen this life for his sister, but he was glad she'd stumbled into it. She would have gradually died within the stronghold. He had a feeling she would be blossoming more with every passing day now.\n\nStepping through the doorway, his gaze traveled over the hunters and vamps standing within the hallway. They stood on opposite sides of the hall while they stared distrustfully at each other, but at least they weren't trying to kill one another.\n\n_Baby steps,_ he reminded himself. Baby steps that had the possibility of leading to a better and safer life for all of them.\n\nHis gaze drifted to the two vamps who had brought the files for Ronan. Brian stood next to two pretty blonde women. Brian's shoulders went back and he pulled the one standing closest to him back a step when Nathan's eyes fell on her. Nathan turned away from them to focus on the other woman who was the identical version of the woman with Brian. Her head canted to the side as her green eyes studied him. Nathan found his steps slowing as he gazed inquisitively back at her.\n\nThen, she scowled at him. Despite her apparent dislike of him, Nathan smiled at her before walking out of the hotel. He gave Kadence a hug and climbed into the battered truck he favored for hunting.\n\n# CHAPTER 46\n\n\"It's going to work between the hunters and the vampires,\" Kadence murmured as she trailed her fingers up Ronan's bare chest to his chin and back down again. \"It's going to take time, there will be problems, most likely _many_ problems in the beginning, but it's going to work out for the better.\"\n\n\"Yes,\" Ronan agreed as he took her hand and pinned it to his chest.\n\n\"I'm going to be there to help with that, and I'm going to be there when Joseph dies. If I get the chance, I'll kill him myself.\"\n\nShe wiggled her way up his body and sprawled across his chest. Against her thigh, she felt him hardening again, but his face remained serious as he gazed at her.\n\n\"Joseph is an old, powerful, purebred vampire,\" he said.\n\n\"And I'm a turned hunter, the first of my kind, who knows what I can do. I might be as fierce as a purebred when I'm in a rage. Perhaps my skin will turn color too.\"\n\n\"Hmm,\" he grunted.\n\nHer smile slid away at the grave look on his face. \"Don't lock me away, Ronan.\"\n\nHis hand tightened around hers as he took a deep breath. \"I promised you I wouldn't do that, and I won't, but there are some things we should discuss.\"\n\n\"Such as?\" she asked warily.\n\n\"Such as, you will continue training with me every day, and if you go into the field, it will be with me. If I don't think you're ready, or if I feel you're not working as hard as you have been, I will continue to keep you from the fight.\"\n\n\"I agree,\" she said. \"But you have to be fair and unbiased in your assessment of me.\"\n\n\"I will,\" he replied.\n\n\"Anything else?\"\n\n\"Not right now, but we'll discuss this again when Joseph is dead.\"\n\nShe smiled as she leaned forward to kiss him. \"Are you really going to lead the vampires?\"\n\n\"It is another thing that will take time, but I will work to gather the vampires again, to lead them, and to better educate them on our history, our ways, and what they are.\"\n\n\"You will make an outstanding ruler,\" she assured him.\n\n\"And you will make an impressive, stubborn ruler at my side,\" he replied and nipped at her bottom lip.\n\nKadence giggled as her fingers curled into his chest. \"I love you.\"\n\n\"You'd better because you're bound to me for an eternity, and I'm head over fucking heels for you.\"\n\n\"I'm okay with that.\"\n\nShe squealed when he flipped her over to pin her to the mattress beneath him. His beautiful brown eyes met hers before he bent his head to kiss her. Kadence forgot all about the problems waiting for them outside of this room as she lost herself to the rhythm of his body and the strength of his love enveloping her.\n\nTHE END\n\nLook for book 2 in **The Alliance Series** which will focus on Nathan and Vicky.\n\nIf you enjoyed this story you may enjoy the **Vampire Awakening Series** too and get a chance to meet some of the other characters in this book more.\n\nCheck out Book 1 of the **Awakening Series** for free everywhere ebooks are sold.\n\nSign up for the mailing list to stay up to date on future releases in this series and others from the author:\n\n**Brenda K. Davies\/Erica Stevens Mailing List:** \n\nRead on for an exclusive excerpt of _Into_ _Hell_ (The Road to Hell Series, Book 4).\n\nThis is the final book in The Road to Hell Series.\n\n# \n\n# WHERE TO FIND THE AUTHOR\n\nBrenda K. Davies\/Erica Stevens Mailing List: \nFacebook: http:\/\/bit.ly\/BKDFBpage\n\nBrenda K. Davies\/Erica Stevens Book Club: \nInstagram: http:\/\/bit.ly\/BKDInsta| \nTwitter: http:\/\/bit.ly\/BKDTw\n\nWebsite: \n\nBlog: \n\n# [ EXCERPT FROM INTO HELL \n(THE ROAD TO HELL SERIES, BOOK 4)](tmp_99c05f1d6111beba335e9a97d8fcbd63_u9pYbq.ch.fixed.fc.tidied.stylehacked.xfixed_split_003.html#ref_toc)\n\n### Kobal\n\nMy eyes narrowed on the shadows from where the voice had come, but whoever stood there remained concealed by the darkness. Scenting the air, I detected a new aroma within the cavern. Whoever it was had not been there long. I recognized the odor as the same aroma Lucifer and the other angels emitted. It brought to mind water, but whereas River made me think of fresh rain, this was more like a pond.\n\nI focused my gaze on a shifting in the shadows as my lips skimmed back to bare my fangs.\n\n\"I have not come here to fight, Kobal,\" the voice murmured. \"I would not have revealed my presence in this cave if I intended to battle you. I would have simply struck while the two of you were focused on whatever it was the oracle revealed to her.\"\n\n\"Then why not show yourself?\" I demanded.\n\nThe darkness moved and flowed before a raven swept out of it to land fifty feet away. Larger than the ravens on Earth, it stood nearly three feet tall and weighed at least a hundred pounds. It moved with the grace of its mortal counterparts as it settled its feathers against its side with a fluid ripple of motion.\n\n\"I've shown myself,\" the raven murmured and River gasped behind me. \"As you can see, I could have remained hidden from you for a lot longer. Even if you scented me, you would not have been looking for me.\"\n\n\"What the fuck is with the talking bird?\" Hawk blurted.\n\n\"It's not a bird,\" Bale said and drew her sword from her back.\n\nThe first book in this series, _Good Intentions_ , is free.\n\n_Into Hell_ **is now available.**\n\nSign up for the mailing list to stay up to date on future releases in this series and others from the author:\n\n**Brenda K. Davies\/Erica Stevens Mailing List:** \n\nAbout the Author\n\nBrenda K. Davies is the USA Today Bestselling author of the Vampire Awakening Series, Alliance Series, Road to Hell Series, Hell on Earth Series, and historical romantic fiction. She also writes under the pen name, Erica Stevens. When not out with friends and family, she can be found at home with her husband, dog, and horse.\n\n","meta":{"redpajama_set_name":"RedPajamaBook"}} +{"text":" \n# NUTRITION IN THE PREVENTION AND TREATMENT OF DISEASE\n\nANN M. COULSTON\n\nHattner\/Coulston Nutrition Associates, Palo Alto, California\n\nCHERYL L. ROCK\n\nDepartment of Family and Preventative Medicine, University of California, San Diego, La Jolla, California\n\nELAINE R. MONSEN\n\nDepartment of Nutrition and Medicine, University of Washington, Seattle, Washington\n\n# Table of Contents\n\nCover\n\nTitle page\n\nCopyright\n\nContributors\n\nPreface\n\nAcknowledgments\n\nSECTION I: Basic Principles and Concepts\n\nA.: Examining the Relationship between Diet, Nutrition, and Disease\n\nChapter 1: Dietary Assessment Methodology\n\nI. INTRODUCTION\n\nII. DIETARY ASSESSMENT METHODS\n\nIII. DIETARY ASSESSMENT IN SPECIFIC SITUATIONS\n\nIV. DIETARY ASSESSMENT IN SPECIAL POPULATIONS\n\nV. SELECTED ISSUES IN DIETARY ASSESSMENT METHODS\n\nChapter 2: Energy Requirement Methodology\n\nI. INTRODUCTION\n\nII. COMPONENTS OF ENERGY EXPENDITURE\n\nIII. TOTAL ENERGY EXPENDITURE\n\nIV. RECOMMENDED ENERGY INTAKES\n\nChapter 3: Physical Assessment\n\nI. INTRODUCTION\n\nII. COMPONENTS OF CLINICAL ASSESSMENT\n\nIII. ANTHROPOMETRIC ASSESSMENT\n\nIV. FUNCTIONAL ASSESSMENT\n\nV. CLINICAL MANIFESTATIONS IN SPECIFIC DISEASE STATES AND POPULATIONS\n\nVI. PROGNOSTIC INDICATORS\n\nVII. SUMMARY\n\nChapter 4: Overview of Nutritional Epidemiology\n\nI. INTRODUCTION\n\nII. PRINCIPLES OF EXPOSURE MEASUREMENT IN NUTRITIONAL EPIDEMIOLOGY\n\nIII. STUDY DESIGNS USED IN NUTRITIONAL EPIDEMIOLOGY\n\nIV. INTERPRETATION OF CAUSE AND EFFECT IN NUTRITIONAL EPIDEMIOLOGY\n\nV. OBSTACLES TO FINDING ASSOCIATIONS OF DIETARY INTAKE AND DISEASE RISK\n\nVI. FUTURE RESEARCH DIRECTIONS\n\nAcknowledgment\n\nChapter 5: Analysis, Presentation, and Interpretation of Dietary Data\n\nI. INTRODUCTION\n\nII. ANALYSIS OF DIETARY DATA\n\nIII. PRESENTATION OF DATA\n\nIV. INTERPRETATION OF DATA\n\nV. CONCLUSION\n\nB.: Nutrition Intervention\n\nChapter 6: Current Theoretical Bases for Nutrition Intervention and Their Uses\n\nI. INTRODUCTION\n\nII. IMPORTANCE OF UNDERSTANDING INFLUENCES ON DIETARY BEHAVIOR\n\nIII. WHAT IS THEORY?\n\nIV. EXPLANATORY AND CHANGE THEORIES\n\nV. UNIQUE FEATURES OF DIETARY BEHAVIOR TO CONSIDER WHEN USING THEORY\n\nVI. IMPORTANT THEORIES AND THEIR KEY CONSTRUCTS\n\nVII. FINDINGS REGARDING APPLICATIONS OF THEORY TO NUTRITIONAL BEHAVIOR\n\nVIII. CONSTRUCTS AND ISSUES ACROSS THEORIES\n\nIX. IMPLICATIONS AND OPPORTUNITIES\n\nChapter 7: Nutrition Intervention: Lessons from Clinical Trials\n\nI. INTRODUCTION\n\nII. CONCEPTUAL MODELS OF MOTIVATION\n\nIII. THEORIES USED IN ACHIEVING DIETARY BEHAVIOR CHANGE IN CLINICAL TRIALS\n\nIV. SUMMARY\n\nChapter 8: Tools and Techniques to Facilitate Eating Behavior Change\n\nI. INTRODUCTION\n\nII. THE TEACHING\/LEARNING PROCESS\n\nIII. NUTRITION EDUCATION TECHNIQUES\n\nIV. BEHAVIOR CHANGE TECHNIQUES\n\nV. CONCLUSION\n\nChapter 9: Evaluation of Nutrition Interventions\n\nI. INTRODUCTION\n\nII. OVERVIEW: TYPES OF NUTRITION INTERVENTION PROGRAM EVALUATIONS\n\nIII. OUTCOMES OR ENDPOINTS USED TO ASSESS INTERVENTION EFFECTIVENESS\n\nIV. DESIGN OF NUTRITION INTERVENTION EVALUATIONS\n\nV. MEASUREMENT ISSUES WHEN ASSESSING DIETARY CHANGE AND OTHER INTERVENTION OUTCOMES\n\nVI. DIETARY ASSESSMENT INSTRUMENTS AND THEIR APPLICABILITY FOR INTERVENTION EVALUATION\n\nVII. CONCLUSIONS\n\nChapter 10: Biomarkers and Biological Indicators of Change\n\nI. INTRODUCTION\n\nII. BIOMARKERS OF DIETARY INTAKE OR EXPOSURE\n\nIII. FUNCTIONAL BIOMARKERS\n\nIV. BIOMARKERS OF GENETIC SUSCEPTIBILITY\n\nV. CRITERIA FOR SELECTING AND USING BIOMARKERS\n\nVI. SUMMARY\n\nC.: Genetic Influence on Nutritional Health\n\nChapter 11: Genetic Influences on Blood Lipids and Cardiovascular Disease Risk\n\nI. INTRODUCTION\n\nII. EXOGENOUS LIPOPROTEIN PATHWAY\n\nIII. ENDOGENOUS LIPOPROTEIN METABOLISM\n\nIV. REVERSE CHOLESTEROL TRANSPORT\n\nV. CONCLUSION\n\nAcknowledgments\n\nChapter 12: Genetics of Human Obesity\n\nI. INTRODUCTION\n\nII. GENETIC EPIDEMIOLOGY OF HUMAN OBESITY\n\nIII. GENE\u2013ENVIRONMENT INTERACTIONS\n\nIV. THE OBESITY GENE MAP\n\nV. SINGLE-GENE OBESITY IN HUMANS\n\nVI. SINGLE-GENE MUTATIONS RESULTING IN ADIPOSE TISSUE ATROPHY\n\nVII. RARE GENETIC SYNDROMES WITH OBESITY AS A PROMINENT FEATURE\n\nVIII. EVIDENCE FROM LINKAGE STUDIES OF OBESITY PHENOTYPES\n\nIX. ASSOCIATION STUDIES OF CANDIDATE GENES FOR OBESITY AND OBESITY-RELATED INSULIN RESISTANCE AND HYPERLIPIDEMIA\n\nX. CLINICAL IMPLICATIONS OF THE DISCOVERY OF OBESITY GENES\n\nChapter 13: Genetic Influence on Cancer Risk\n\nI. INTRODUCTION\n\nII. BACKGROUND\n\nIII. MECHANISMS OF DIET\u2013GENETIC INTERACTIONS\n\nIV. METHODOLOGIC ISSUES IN ASSESSMENT OF DIET AND GENE INTERACTIONS RELATED TO CANCER\n\nV. DIET\u2013GENE INTERACTIONS AND CANCER\n\nVI. FUTURE DIRECTIONS\n\nChapter 14: Inborn Errors of Metabolism\n\nI. INTRODUCTION\n\nII. DIAGNOSIS\n\nIII. TREATMENT: INITIATION AND MONITORING\n\nIV. NEW TREATMENT DEVELOPMENTS\n\nV. ADULT TREATMENT\n\nVI. SUMMARY\n\nChapter 15: Role of Liquid Dietary Supplements\n\nI. MEDICAL FOODS AND NUTRITIONAL SUPPLEMENTS\n\nII. WHAT IS A MEDICAL FOOD\/SUPPLEMENT?\n\nIII. PREVALENCE AND ECONOMIC IMPACT OF MEDICAL NUTRITION THERAPY\n\nIV. BARRIERS TO USING MEDICAL NUTRITIONAL SUPPLEMENTS\n\nV. SUMMARY\n\nChapter 16: Composite Foods and Formulas, Parenteral and Enteral Nutrition\n\nI. INTRODUCTION\n\nII. ENTERAL NUTRITION\n\nIII. PARENTERAL NUTRITION\n\nIV. CONCLUSION\n\nChapter 17: Herbs and Botanical Supplements: Principles and Concepts\n\nI. INTRODUCTION: HISTORY OF BOTANICAL MEDICINE\n\nII. USE OF HERBS AND BOTANICALS\n\nIII. EVIDENCE FOR SAFETY AND EFFICACY\n\nIV. ADVERSE EFFECTS OF HERBS AND BOTANICALS\n\nV. REGULATION OF BOTANICAL AND HERBAL MEDICINALS\n\nVI. RECOMMENDATIONS FOR CLINICAL CARE\n\nVII. SUMMARY\n\nSECTION II: Disease-Specific Intervention: Prevention and Treatment\n\nA.: Cardiovascular Disease\n\nChapter 18: Dietary Macronutrients and Cardiovascular Risk\n\nI. INTRODUCTION\n\nII. DIETARY FAT\n\nIII. DIETARY CARBOHYDRATE\n\nIV. DIETARY PROTEIN\n\nV. CONCLUSION\n\nChapter 19: Other Dietary Components and Cardiovascular Risk\n\nI. INTRODUCTION\n\nII. DIETARY FIBER AND CARDIOVASCULAR RISK\n\nIII. HOMOCYSTEINE AND CARDIOVASCULAR DISEASE\n\nIV. SOY AND CARDIOVASCULAR DISEASE\n\nV. PHYTOCHEMICALS: PROPOSED ASSOCIATION WITH CARDIOVASCULAR DISEASE\n\nVI. CONCLUSION\n\nChapter 20: Nutrition, Diet, and Hypertension\n\nI. INTRODUCTION\n\nII. INDIVIDUAL NUTRIENTS AND BLOOD PRESSURE\n\nIII. OTHER DIETARY AND LIFESTYLE MODIFICATIONS\n\nIV. DIETARY PATTERNS\n\nV. SUMMARY\n\nChapter 21: Nutrition and Congestive Heart Failure\n\nI. INTRODUCTION\n\nII. PATHOPHYSIOLOGY OF HEART FAILURE\n\nIII. STANDARD MEDICAL CARE FOR HEART FAILURE\n\nIV. RESTRICTIONS IN SODIUM\n\nV. RESTRICTIONS IN FLUID INTAKE\n\nVI. RECOMMENDATIONS REGARDING FAT, FIBER, AND CHOLESTEROL\n\nVII. OTHER DIETARY RECOMMENDATIONS\n\nVIII. SPECIAL CONSIDERATIONS\n\nIX. SUMMARY\n\nB.: Cancer Prevention and Therapy\n\nChapter 22: Nutrition and Breast Cancer\n\nI. INTRODUCTION\n\nII. NUTRITIONAL FACTORS IN THE ETIOLOGY OF BREAST CANCER\n\nIII. NUTRITIONAL ISSUES FOLLOWING THE DIAGNOSIS OF BREAST CANCER\n\nIV. SUMMARY AND CONCLUSIONS\n\nChapter 23: Nutrition and Colon Cancer\n\nI. INTRODUCTION\n\nII. MODEL 1: BILE ACIDS, FAT\/FIBER, AND MEATS\/VEGETABLES\n\nIII. MODEL 2: COOKED FOODS\n\nIV. MODEL 3: INSULIN RESISTANCE\n\nV. MODEL 4: DNA METHYLATION\n\nVI. MODEL 5: CELL GROWTH REGULATORS\n\nVII. FOOD INTAKE RELATIONSHIPS\n\nVIII. PREVENTION OF COLON CANCER\n\nChapter 24: Nutrition and Prostate Cancer\n\nI. INTRODUCTION\n\nII. DESCRIPTIVE EPIDEMIOLOGY OF PROSTATE CANCER\n\nIII. STUDIES OF DIET IN RELATION TO PROSTATE CANCER\n\nIV. GENETICS AND GENE\u2013ENVIRONMENT INTERACTIONS\n\nV. DIETARY INTERVENTION TRIALS\n\nVI. CONCLUSIONS AND IMPLICATIONS FOR PREVENTION AND TREATMENT\n\nChapter 25: Nutrition and Lung Cancer\n\nI. INTRODUCTION\n\nII. DIETARY CONSTITUENTS AND PRIMARY PREVENTION OF LUNG CANCER\n\nIII. DIETARY CONSTITUENTS AND PREVENTION OF SECOND LUNG CANCER\n\nIV. CONCLUSIONS AND RECOMMENDATIONS\n\nChapter 26: Nutrition and the Patient with Cancer\n\nI. INTRODUCTION\n\nII. RATIONALE AND IMPORTANCE OF OPTIMAL NUTRITION\n\nIII. CANCER CACHEXIA\n\nIV. BASIC NUTRITION CONCEPTS FOR MANAGING PATIENTS WITH CANCER\n\nV. NUTRITION ISSUES THROUGHOUT THE CONTINUUM OF CARE\n\nVI. SPECIAL SITUATIONS\n\nVII. SUMMARY AND CONCLUSIONS\n\nC.: Diabetes Mellitus\n\nChapter 27: Obesity and the Risk for Diabetes\n\nI. INTRODUCTION\n\nII. DEFINITIONS AND CLASSIFICATIONS OF OBESITY AND DIABETES\n\nIII. WHY ARE THE OBESE AT RISK?\n\nIV. CONCLUSION\n\nChapter 28: Nutrition Management for Type 1 Diabetes\n\nI. INTRODUCTION\n\nII. DEFINITION AND BURDEN OF TYPE 1 DIABETES\n\nIII. DIABETES MEDICAL NUTRITION THERAPY\n\nIV. INSULIN REGIMENS\n\nv. CONCLUSION\n\nChapter 29: Nutritional Management for Type 2 Diabetes\n\nI. INTRODUCTION\n\nII. ENERGY INTAKE AND BODY WEIGHT MANAGEMENT\n\nIII. MACRONUTRIENT INTAKE\n\nIV. TRACE MINERAL REQUIREMENTS\n\nV. CONCLUSION\n\nChapter 30: Nutritional Management for Gestational Diabetes\n\nI. INTRODUCTION\n\nII. SCREENING AND DIAGNOSIS\n\nIII. RISKS AND COMPLICATIONS\n\nIV. TREATMENT AND MONITORING\n\nV. POSTPARTUM RECOMMENDATIONS AND CONSIDERATIONS\n\nVI. CONCLUSION\n\nD.: Obesity\n\nChapter 31: Obesity: Overview of Treatments and Interventions\n\nI. INTRODUCTION\n\nII. ASSESSMENT OF OVERWEIGHT AND OBESITY\n\nIII. LIFESTYLE MODIFICATION\n\nIV. PHARMACEUTICAL INTERVENTION\n\nV. SURGICAL TREATMENT\n\nVI. SPECIAL ISSUES IN THE TREATMENT OF PEDIATRIC OBESITY\n\nVII. ACUTE WEIGHT LOSS VERSUS MAINTAINING LONG-TERM WEIGHT LOSS\n\nVIII. THE FUTURE OF WEIGHT MANAGEMENT\n\nChapter 32: Obesity: Role of Physical Activity\n\nI. INTRODUCTION\n\nII. RELATIONSHIP OF PHYSICAL ACTIVITY TO THE PREVALENCE OF OBESITY IN POPULATIONS\n\nIII. PHYSICAL ACTIVITY AND DETERMINANTS AND ETIOLOGY OF OBESITY\n\nIV. ROLE OF PHYSICAL ACTIVITY IN TREATMENT (WEIGHT LOSS) OF OVERWEIGHT AND OBESITY: EVIDENCE FROM RANDOMIZED CONTROLLED TRIALS\n\nV. ROLE OF PHYSICAL ACTIVITY IN PREVENTING AND TREATING OBESITY-RELATED COMORBIDITIES\n\nVI. CONCLUSIONS\n\nChapter 33: Macronutrient Intake and the Control of Body Weight\n\nI. INTRODUCTION\n\nII. FAT CHANCE\n\nIII. ESTABLISHING CAUSAL LINKS\n\nIV. IS IT FAT OR ENERGY DENSITY?\n\nV. ARE CARBOHYDRATES THE CULPRIT RESPONSIBLE FOR OVERWEIGHT?\n\nVI. ENERGY COMPENSATION FOR FAT AND SUGAR SUBSTITUTES\n\nVII. PROTEIN PARADOX\n\nVIII. SUMMARY AND IMPLICATIONS OF THE RESEARCH ON MACRONUTRIENTS AND INTAKE\n\nChapter 34: Behavioral Risk Factors for Obesity: Diet and Physical Activity\n\nI. INTRODUCTION\n\nII. PHYSICAL ACTIVITY\n\nIII. DIETARY FACTORS\n\nIV. SUMMARY AND PUBLIC HEALTH RECOMMENDATIONS\n\nV. CONCLUSION\n\nChapter 35: Role of Taste and Appetite in Body Weight Regulation\n\nI. INTRODUCTION\n\nII. GENETICS AND BODY WEIGHT\n\nIII. TASTE FACTORS AND FOOD CHOICES\n\nIV. HUNGER, APPETITE, AND SATIETY\n\nV. ENERGY DENSITY OF FOODS\n\nVI. APPETITE FOR ENERGY\n\nE.: Gastrointestinal Diseases\n\nChapter 36: Nutrition in the Prevention and Treatment of Common Gastrointestinal Symptoms\n\nI. INTRODUCTION\n\nII. EXCESSIVE GAS\n\nIII. HEARTBURN AND GASTROESOPHAGEAL REFLUX DISEASE\n\nIV. DIARRHEA\n\nV. CONSTIPATION\n\nVI. NAUSEA AND VOMITING\n\nVII. CONCLUSION\n\nChapter 37: Nutrient Considerations in Lactose Intolerance\n\nI. INTRODUCTION\n\nII. LACTOSE IN THE DIET\n\nIII. DIGESTION OF LACTOSE\n\nIV. LOSS OF LACTASE ACTIVITY\n\nV. DIAGNOSIS OF LACTOSE MALDIGESTION\n\nVI. LACTOSE MALDIGESTION AND INTOLERANCE SYMPTOMS\n\nVII. LACTOSE DIGESTION, CALCIUM, AND OSTEOPOROSIS\n\nVIII. DIETARY MANAGEMENT FOR LACTOSE MALDIGESTION\n\nIX. GENE THERAPY FOR LACTOSE INTOLERANCE\n\nX. SUMMARY\n\nChapter 38: Nutrient Considerations in Inflammatory Bowel Disease and Short Bowel Syndrome\n\nI. INTRODUCTION\n\nII. INFLAMMATORY BOWEL DISEASE\n\nIII. SHORT BOWEL SYNDROME\n\nIV. CONCLUSION\n\nChapter 39: Nutrition and Liver Disease\n\nI. INTRODUCTION\n\nII. PATHOGENESIS OF MALNUTRITION\n\nIII. MAJOR LIVER DISEASES\n\nIV. NUTRITIONAL MANAGEMENT OF LIVER DISEASES\n\nV. PREPARATION FOR LIVER TRANSPLANT\n\nVI. SUMMARY AND CONCLUSIONS\n\nF.: Other Major Diseases\n\nChapter 40: Nutrition and Renal Disease\n\nI. INTRODUCTION\n\nII. CHRONIC RENAL FAILURE\n\nIII. NUTRITION FOR THE PATIENT REQUIRING RENAL REPLACEMENT THERAPY\n\nIV. NUTRITIONAL REQUIREMENTS OF THE POST-TRANSPLANT PATIENT\n\nV. CONCLUSION\n\nChapter 41: Nutritional Management of Parkinson's Disease and Other Conditions Like Alzheimer's Disease\n\nI. INTRODUCTION\n\nII. NUTRITIONAL FACTORS IN RELATION TO COGNITIVE FUNCTION\n\nIII. NUTRITIONAL FACTORS IN RELATION TO ALZHEIMER'S DISEASE AND PARKINSON'S DISEASE\n\nIV. SPECIFIC NUTRITIONAL TRIALS IN COGNITIVE, ALZHEIMER'S, AND PARKINSON'S DISEASES\n\nV. NUTRITIONAL MANAGEMENT ISSUES IN NEUROCOGNITIVE DISORDERS\n\nVI. SUMMARY\n\nChapter 42: Osteoporosis\n\nI. INTRODUCTION\n\nII. NUTRITION IN THE OSTEOPOROTIC FRACTURE CONTEXT\n\nIII. PROBLEMS IN THE INVESTIGATION OF NUTRITIONAL EFFECTS ON BONE\n\nIV. THE NOTION OF A NUTRIENT REQUIREMENT\n\nV. THE NATURAL INTAKE OF CALCIUM AND VITAMIN D\n\nVI. CALCIUM\n\nVII. VITAMIN D\n\nVIII. VITAMIN K\n\nIX. OTHER ESSENTIAL NUTRIENTS\n\nX. CONCLUSION\n\nChapter 43: Eating Disorders: Anorexia Nervosa, Bulimia Nervosa, and Binge Eating Disorder\n\nI. INTRODUCTION\n\nII. DEFINITIONS AND DIAGNOSTIC CRITERIA\n\nIII. ETIOLOGY\n\nIV. ANOREXIA NERVOSA\n\nV. BULIMIA NERVOSA\n\nVI. EATING DISORDER NOT OTHERWISE SPECIFIED\n\nVII. OUTCOME\n\nVIII. PREVENTION OF EATING DISORDERS\n\nIX. SUMMARY AND CONCLUSIONS\n\nChapter 44: Nutrition and Food Allergy\n\nI. INTRODUCTION\n\nII. DEFINITIONS\n\nIII. THE IMMUNE RESPONSE AND ALLERGY\n\nIV. PREVALENCE OF FOOD ALLERGY\n\nV. FOOD ALLERGENS\n\nVI. CLINICAL MANIFESTATIONS OF FOOD ALLERGY\n\nVII. NONALLERGIC IMMUNOLOGIC DISEASES\n\nVIII. NONIMMUNOLOGIC REACTIONS TO FOODS\n\nIX. CONDITIONS WITH NO PROVEN RELATIONSHIP TO FOODS\n\nX. DIAGNOSIS OF FOOD ALLERGY\n\nXI. DIFFERENTIAL DIAGNOSIS\n\nXII. TREATMENT\n\nXIII. PROGNOSIS OF FOOD ALLERGY\n\nXIV. PREVENTION OF FOOD ALLERGY\n\nXV. FOOD ADDITIVES\n\nXVI. GENETICALLY ENGINEERED FOODS\n\nChapter 45: Nutrition and Cystic Fibrosis\n\nI. INTRODUCTION\n\nII. OVERVIEW OF NUTRITIONAL PROBLEMS IN CYSTIC FIBROSIS\n\nIII. PREVALENCE OF MALNUTRITION\n\nIV. NUTRITIONAL ASSESSMENT AND INTERVENTION\n\nV. CONCLUSIONS\n\nChapter 46: Osteomalacia\n\nI. INTRODUCTION\n\nII. MINERALIZATION\n\nIII. OSTEOMALACIC BONE\n\nIV. CAUSES OF OSTEOMALACIA\n\nV. TREATMENT OF OSTEOMALACIA\n\nChapter 47: Nutrition and Immunodeficiency Syndromes\n\nI. INTRODUCTION\n\nII. HIV-ASSOCIATED WEIGHT LOSS\n\nIII. HIV-ASSOCIATED LIPODYSTROPHY SYNDROME\n\nIV. CAUSES OF WEIGHT LOSS IN HIV\/AIDS\n\nV. HORMONAL IMBALANCES\n\nVI. NUTRITIONAL ASSESSMENT\n\nVII. NUTRITIONAL RECOMMENDATIONS\n\nVIII. NUTRITIONAL COUNSELING\n\nIX. SUMMARY\n\nG.: Overall Disease Prevention\n\nChapter 48: Nutrition Guidelines to Maintain Health\n\nI. INTRODUCTION\n\nII. GUIDELINES FOR NUTRIENT ADEQUACY AND SAFETY\n\nIII. GUIDELINES FOR HEALTHY FOOD CHOICES\n\nIV. BEYOND FOOD AND NUTRIENT GUIDELINES: PHYSICAL ACTIVITY GUIDELINES\n\nV. SUMMARY\n\nEpilogue\n\nIndex\n\n# Copyright\n\nThis book is printed on acid-free paper.\n\nCopyright \u00a9 2001 by ACADEMIC PRESS\n\nAll Rights Reserved.\n\nNo part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.\n\nRequests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Inc., 6277 Sea Harbor Drive, Orlando, Florida 32887-6777\n\nAcademic Press\n\n_A Harcourt Science and Technology Company_\n\n525 B Street, Suite 1900, San Diego, California 92101-4495, USA\n\n\n\nAcademic Press\n\nHarcourt Place, 32 Jamestown Road, London NW1 7BY, UK\n\n\n\nLibrary of Congress Catalog Card Number: 00-111387\n\nInternational Standard Book Number: 0-12-193155-2\n\nPRINTED IN THE UNITED STATES OF AMERICA\n\n01 02 03 04 05 06 EB 9 8 7 6 5 4 3 2 1\n\n# Contributors\n\n_Numbers in parentheses indicate the page number(s) on which the contribution begins._\n\nANN ALBRIGHT, PhD, RD (429), Sacramento, California 94234\n\nNANCY ANZLOVAR, RN (325), Cleveland Clinic Foundation, Cleveland, Ohio 44195\n\nSUJATA ARCHER, Phd, RD (291), Northwestern University Medical School, Chicago, Illinois 60611\n\nAMY BALTES, RD (291), Northwestern University Medical School, Chicago, Illinois 60611\n\nPETER L. BEYER, MS, RD (577), Department of Dietetics and Nutrition, University of Kansas Medical Center, Kansas City, Kansas 66160\n\nAMY BINKOSKI (279), Department of Nutrition, Pennsylvania State University, University Park, Pennsylvania 16801\n\nBETTE J. CAAN, PhD (357), Division of Research, Kaiser Foundation Research Institute, Oakland, California 94611\n\nLAWRENCE J. CHESKIN, MD (549), Division of Gastroenterology, Johns Hopkins Bayview Medical Center, Baltimore, Maryland 21224\n\nANN M. COULSTON, MS, RD (441), Hattner\/Coulston Nutrition Associates, Palo Alto, California 94303\n\nSTACIE COVAL (279), Department of Nutrition, Pennsylvania State University, University Park, Pennsylvania 16801\n\nDEBRA COWARD-McKENZIE (31, 69), The University of Vermont, Burlington, Vermont 05405\n\nPAULA DAVIS McCALLUM, MS, RD (397), Advantage Nutrition, Ltd., Chagrin Falls, Ohio 44023\n\nLINDA DELAHANTY, MS, RD (105), Massachusetts General Hospital Diabetes Center, Boston, Massachusetts 02114\n\nWENDY DEMARK-WAHNEFRIED, PhD, RD (337), Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710\n\nADAM DREWNOWSKI, PhD (539), Nutritional Sciences Program, University of Washington, Seattle, Washington 98195\n\nBARBARA ELDRIDGE, RD (397), University of Colorado Health Sciences Center, Denver, Colorado 80123\n\nPHILIP M. FARRELL, MD, PhD (715), Medical Sciences Center, University of Wisconsin\u2013Madison, Madison, Wisconsin 53706\n\nJANIS S. FISLER, PhD (183), University of California, Davis, California 95616\n\nJO L. FREUDENHEIM, PhD, RD (199), Department of Social and Preventive Medicine, State University of New York at Buffalo, Buffalo, New York 14260\n\nJUL GERRIOR, RD (741), Department of Family Medicine and Nutrition Infection Unit, Tufts University School of Medicine, Boston, Massachusetts 02111\n\nKAREN GLANZ, PhD, MPH (83), Cancer Research Center of Hawaii, University of Hawaii, Honolulu, Hawaii 96813\n\nD. JORDI GOLDSTEIN, DSc, RD (617), IVonyx, Inc., Reno, Nevada 89509\n\nREJEANNE GOUGEON, PhD, RD (415), McGill Nutrition and Food Science Centre, Crabtree Nutrition Laboratories, Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1A1\n\nROBERT HEANEY, MD (653), Creighton University, Omaha, Nebraska 68178\n\nKARI HECKER, MS, RD (279), Department of Nutrition, Pennsylvania State University, University Park, Pennsylvania 16801\n\nJOAN M. HEINS, MA, RD, CDE (105), Washington University School of Medicine, St. Louis, Missouri 63108\n\nSTEVE HERTZLER, PhD, RD (563), School of Allied Medical Professions, Medical Dietetics Division, Ohio State University, Columbus, Ohio 43210\n\nJAMES O. HILL, PhD (465), Center for Human Nutrition, University of Colorado Health Sciences Center, Denver, Colorado 80262\n\nKARRY A. JACKSON (563), School of Consumer and Family Sciences, Purdue University, West Lafayette, Indiana 47907\n\nRACHEL K. JOHNSON, PhD, MPH, RD (31, 69), The University of Vermont, Burlington, Vermont 05405\n\nWALTER H. KAYE, MD (685), Western Psychiatric Institute and Clinic, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213\n\nJANET KING, PhD (773), USDA, Western Human Nutrition Research Center, University of California at Davis, Davis, California 95616\n\nLAURENCE N. KOLONEL, MD, PhD (373), Cancer Research Center of Hawaii, University of Hawaii, Honolulu, Hawaii 98613\n\nPENNY KRIS-ETHERTON, PhD, RD (279), Department of Nutrition, Pennsylvania State University, University Park, Pennsylvania 16801\n\nALAN R. KRISTAL, PhD (123), Cancer Prevention Research Program, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109\n\nHUI-CHUAN LAI, PhD, RD (715), Department of Biostatistics and Medical Informatics and Department of Pediatrics, Clinical Sciences Center, University of Wisconsin\u2014Madison, Madison, Wisconsin 53792\n\nJOHANNA W. LAMPE, PhD, RD (139), Cancer Prevention Research Program, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109\n\nDAVID A. LEVITSKY (499), Cornell University, Ithaca, New York 14853\n\nPAO-HWA LIN, PhD (303), Epidemiology & Surveillance Research, The American Cancer Society, Atlanta, Georgia 30329\n\nSUZANNE LUTTON, MD (325), Diagnostic Cardiology Associates, Youngstown, Ohio 44504\n\nROBERT B. LUTZ, MD (261), Arizona Cancer Center, University of Arizona, Tucson, Arizona 85719\n\nLYNNE LYONS, MPH, RD, CDE (453), Burlingame, California 94010\n\nROBERT MARCUS, MD (729), Stanford University School of Medicine, Stanford, California; and Veteran's Administration Medical Center, Palo Alto, California 94304\n\nLAURA MATARESE, MS, RD, LD, FADA, CNSD (245), The Cleveland Clinic Foundation, Cleveland, Ohio 44195\n\nKATHLEEN E. MAYER, MS, RD, LD (229), Ross Products Division, Abbott Laboratories, Columbus, Ohio 43215\n\nSUSAN T. MAYNE, PhD (387), Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut 06520\n\nMARJI MCCULLOUGH, PhD, RD (303), Epidemiology & Surveillance Research, The American Cancer Society, Atlanta, Georgia 30329\n\nBETH McQUISTON, MS, RD (617), Park Ridge, Illinois 60068\n\nDEBRA L. MILLER, PhD (549), Central Soya Co., Inc., Fort Wayne, Indiana 46818\n\nKRIS M. MOGENSEN, MS, RD, CNSD (43), Frances Stern Nutrition Center, New England Medical Center, Boston, Massachusetts 02111\n\nELAINE R. MONSEN, PhD, RD (xvii), Department of Nutrition and Medicine, University of Washington, Seattle, Washington 98195\n\nSUZANNE P. MURPHY, PhD, RD (753), Cancer Research Center of Hawaii, University of Hawaii, Honolulu, Hawaii 96813\n\nDIANNE NEUMARK-SZTAINER, PhD, MPH, RD (517), Division of Epidemiology, University of Minnesota, Minneapolis, Minnesota 55454\n\nJOSE M. ORDOVAS (157), USDA-HNRCA, Tufts University, Boston, Massachusetts 02111\n\nRUTH E. PATTERSON, PhD, RD (59), Cancer Prevention Research Program, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024\n\nDIANE READER, RD, CDE (453), International Diabetes Center, Minneapolis, Minnesota 55416\n\nSARAH H. RIGBY, MS, RD, LD (601), Johns Hopkins Children's Center, Baltimore, Maryland 21287\n\nCHERYL L. ROCK, PhD, RD (139, 337, 397, 685), Department of Family and Preventive Medicine, University of California at San Diego, La Jolla, California 92093\n\nEDWARD SALTZMAN, MD (43), Tufts University and New England Medical Center, Boston, Massachusetts 02111\n\nJESSIE A. SATIA, PhD (123), Cancer Prevention Research Program, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109\n\nDENNIS SAVAIANO, PhD (563), School of Consumer and Family Sciences, Purdue University, West Lafayette, Indiana 47907\n\nNANCY A. SCHONFELD-WARDEN, MD (183), University of California, Davis, California 95616\n\nKATHLEEN B. SCHWARZ, MD (601), Johns Hopkins Children's Center, Baltimore, Maryland 21287\n\nHELEN M. SEAGLE, MS, RD (465), Center for Human Nutrition, University of Colorado Health Sciences Center, Denver, Colorado 80262\n\nDENISE SHAFFER TAYLOR, MS, RD (279), Department of Nutrition, Pennsylvania State University, University Park, Pennsylvania 16801\n\nNANCY E. SHERWOOD, PhD (517), Division of Epidemiology, University of Minnesota, Minneapolis, Minnesota 55454\n\nMARTHA L. SLATTERY, PhD, MPH, RD (357), Oncological Sciences\/PHS, University of Utah, Salt Lake City, Utah 84108\n\nLINDA G. SNETSELAAR, PhD, RD, LD (95), Department of Epidemiology, College of Medicine, University of Iowa, Iowa City, Iowa 52240\n\nMARCIA L. STEFANICK, PhD (481), Department of Medicine, Stanford Center for Research in Disease Prevention, Stanford University, Palo Alto, California 94304\n\nMARY STORY, PhD, RD (517), Division of Epidemiology, University of Minnesota, Minneapolis, Minnesota 55454\n\nFABRIZIS L. SUAREZ, MD, PhD, FACN (563), Minneapolis Veteran's Administration Medical Center, Minneapolis, Minnesota 55417\n\nAMY F. SUBAR, PhD, MPH, RD (3), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892\n\nCHRISTY TANGNEY, PhD, CNS, FACN (637), Department of Clinical Nutrition, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612\n\nABBA I. TERR, MD (701), San Francisco, California 94108\n\nKIMBERLY THEDFORD, MS, RD (291), Northwestern University Medical School, Chicago, Illinois 60611\n\nFRANCES E. THOMPSON, PhD, MPH (3), Applied Research Program, Division of Cancer Control and Population Sciences, National Cancer Institute, Bethesda, Maryland 20892\n\nCYNTHIA THOMSON, PhD, RD, FADA (261), Arizona Cancer Center, University of Arizona, Tucson, Arizona 85719\n\nCRISTINE M. TRAHMS, MS, RD, FADA (209), Department of Pediatrics, University of Washington, Seattle, Washington 98195\n\nLINDA VAN HORN, PhD, RD (291), Northwestern University Medical School, Chicago, Illinois 60611\n\nANNE COBLE VOSS, PhD, RD (229), Ross Products Division, Abbott Laboratories, Columbus, Ohio 43215\n\nCHRISTINE WANKE, MD (741), Department of Family Medicine and Nutrition Infection Unit, Tufts University School of Medicine, Boston, Massachusetts 02111\n\nVICTORIA WARREN-MEARS, MS, RD (539), Nutrition and Food Services, Harborview Medical Center and Nutritional Sciences Program, University of Washington, Seattle, Washington 98195\n\nHOLLY WYATT, MD (465), Center for Human Nutrition, University of Colorado Health Sciences Center, Denver, Colorado 80262\n\nGUIXIANG ZHAO, MS (279), Department of Nutrition, Pennsylvania State University, University Park, Pennsylvania 16801\n\n# Preface\n\nThe purpose of this text is to provide an update of current knowledge in clinical nutrition and an overview of the rationale and science base of its application to practice in the treatment and prevention of disease. The text addresses basic principles and concepts that are central to the major clinical nutrition-related activities, such as nutritional assessment and monitoring, current theoretical base and knowledge of efficacious interventions, interactions between genetic and nutritional factors, and the use and interpretation of population-based or clinical epidemiological evidence. The various roles of clinical nutrition and current knowledge of nutrition in the prevention and treatment of major disease-specific conditions are also reviewed, with an emphasis on past and current scientific evidence that supports these roles. New areas of interest and study are also discussed, with the perspective that the application of the scientific method is by definition an evolutionary process.\n\nTreatment of the disease diabetes mellitus provides an excellent and current example of treatment evolution. In the early part of the 20th century, before the discovery of insulin by F. G. Banting and C. H. Best in 1921, the treatment of choice for individuals with diabetes mellitus was morphine for pain abatement along with a very restricted, starvation diet. When insulin injections became available, dietary protocols were developed. Initially, dietary treatment was based on food exchange lists that encouraged prescribed intakes of carbohydrate, protein, and fat. Recent research from the Diabetes Control and Complications Trial and a similar research trial in the United Kingdom has been the base for the current dietary management emphasizing blood glucose monitoring throughout the day and individualized adjustment of carbohydrate ingestion and insulin injection in individuals who require insulin therapy. Nutrition intervention plays a major role in the management of the patient with diabetes mellitus and in the treatment of the disease and the prevention or delay of complications (see Part II.C).\n\nAnother essential role for nutrition intervention is in the prevention of cancer. Cancer represents a disease continuum and at all stages, from primary prevention to treatment, nutrition is a key factor. As discussed in the section highlighting nutrition and major cancer types, an explosion of new knowledge has identified nutrition as a major factor in the etiology and progression of disease (see Part II.B).\n\nNutrition is the process by which the human body utilizes food for the production of energy, for the maintenance of health, for growth, and for the normal functioning of every organ and tissue. Clinical nutrition is the study of nutrition and diet as related to the development and treatment of human disease. Nutrition is an interdisciplinary field of study, built on a foundation of biomedical and behavioral sciences. Clinical nutrition is the aspect of nutrition science that is related to the development, progression, or management of disease, as differentiated from the issues of normal requirements, cellular functions and activities, and various topics that must be addressed in meeting basic requirements to enable normal growth and development.\n\nAreas of study that contribute to knowledge in clinical nutrition include the disease-relevant biochemistry, metabolism, and activities of nutrients and dietary factors within the tissues and cells; the bioavailability and utilization of nutrients from various food sources, as disease risk or diagnosis may influence these factors; the regulation and compartmentalization of nutrients in the body; the attitudes about food and the eating patterns and behaviors of the targeted individual or group; the technology of food science and specialized or modified food products; and the technology involved in providing adequate and appropriate nutrients or foods to individuals and various community-based or institutionalized groups. Other aspects of clinical nutrition include the development and evaluation of nutrition education efforts; the development of nutrition policies, guidelines, and practice standards that affect the goals and objectives of government and private health agencies, professional practice groups, and health-related organizations; and the design and implementation of individual, clinical, and community-based nutrition and diet interventions. Clinical nutrition interventions range in scope from efforts to maintain health during shortterm illness, to optimization of health status in individuals at risk for or diagnosed with chronic diseases, to major nutritional and diet modifications as specific or adjuvant treatments for disease. Clinical nutrition encompasses primary, secondary, and tertiary disease prevention, in addition to management of disease.\n\nDietary intake or nutritional status may be altered as a result of disease or by the treatment modalities that are utilized, such as surgical treatments, or medical management strategies, such as drugs. The altered needs must be met by dietary or nutritional interventions in order to prevent malnutrition and the associated consequences, which would contribute to overall morbidity and mortality. Also, nutrition intervention can be a critical component of disease prevention, an important aspect of disease management, or the primary treatment for disease. A complicating factor is that people generally eat food, rather than nutrients, so that the practical and psychosocial aspects of diet modification and food or food product availability must be considered in any nutrition intervention, whether individual or community based, and irrespective of whether the goal is primary prevention or disease treatment.\n\nAs in any area of the biomedical sciences, the importance of science-based activities and practices cannot be understated. Clinical nutrition concepts and practices that can become popular with either the lay public or professionals are sometimes based on the type of scientific evidence that cannot truly support the rationales and practices, regardless of how standard and common they might be. Popular theories may be generated by observational epidemiological studies, case series, or anecdotal reports, all of which lack the capability of truly demonstrating a causative or efficacious role for the nutritional factor. Such studies are useful for generating hypotheses, but the apparent associations between diets and disease may be confounded by uncontrolled or unmeasured factors and other determinants of health and disease. Unproven diet therapies exist for the treatment of numerous conditions, and many aspects of common nutrition interventions are sorely in need of testing in an appropriate research design. As in any other aspect of disease prevention and treatment, the use of nutrition interventions or diet therapies should be based on a scientific rationale and sound data, not on anecdotal experience. The scientific basis for clinical nutrition needs to expand considerably in order to fully support claims for the efficacy of many of the common activities and interventions, and progress in this area is being made.\n\nOur definitions of diseases need to further evolve to bring greater clarity and improve precision of treatment. As gene\u2013diet interactions are scientifically delineated, laser-sharp therapies may be applied to specific individuals. For the public, however, generating and analyzing data that summarize dietary intake and its association with disease will be valuable tasks in both treating disease and developing disease prevention strategies. Well-designed focused screening will be an aid in disease detection, and well-founded medical nutrition therapies can minimize disease development and related complications. Providing scientifically sound, creative, and effective nutrition interventions can be challenging. In so doing, however, we will serve the public good.\n\nIt is our goal to update our knowledge and its application through updated editions of this text. In addition, we plan to provide online access to relevant new findings and their import to nutrition in the prevention and treatment of disease. It is our goal to raise the bar for both understanding and treatment.\n\nAnn M. Coulston, Cheryl L. Rock and Elaine R. Monsen\n\n# Acknowledgments\n\nWe dedicate this book to all the women and men\u2014our colleagues, teachers, and friends\u2014who dedicated their professional lives to the pursuit of excellence in nutrition.\n\nYour intelligence, research efforts, and perseverance brought us the scientific tools and perspective to prepare this book which brings the field of clinical nutrition into the 21st century.\n\nYour work is truly evolutionary.\nSECTION I\n\nBasic Principles and Concepts\nA.\n\nExamining the Relationship between Diet, Nutrition, and Disease\nCHAPTER 1\n\nDietary Assessment Methodology\n\nFRANCES E. THOMPSON and AMY F. SUBAR, National Cancer Institute, Bethesda, Maryland\n\n## I. INTRODUCTION\n\nThis chapter reviews the major dietary assessment methods, their advantages and disadvantages, and specific issues to consider when collecting these types of data. The intent is for this chapter to lead to an understanding of alternative dietary assessment methods so that the appropriate method is chosen for a particular need. This chapter updates the \"Dietary Assessment Resource Manual\" [1].\n\n## II. DIETARY ASSESSMENT METHODS\n\nA. Dietary Records\n\nFor the dietary record approach, the respondent records the foods and beverages and the amounts of each consumed over 1 or more days. The amounts consumed may be measured, using a scale or household measures (such as cups, tablespoons), or estimated, using models, pictures, or no particular aid. Typically, if multiple days are recorded, they are consecutive, and no more than 3 or 4 days are included. Recording periods of more than 4 consecutive days are usually unsatisfactory, as reported intakes decrease [2] because of respondent fatigue. Theoretically, the recording is done at the time of the eating occasion, but it need not be done on paper. Dictaphones, computer recording, and self-recording scales have been used [3\u20135] and hold special promise for low-literacy groups and other difficult-to-assess populations because of their ease of administration and potential accuracy, although tape recording has not been shown to be useful among school-aged children [6].\n\nTo complete a dietary record, the respondent must be trained in the level of detail required to adequately describe the foods and amounts consumed, including the name of the food (brand name, if possible), preparation methods, recipes for food mixtures, and portion sizes. In some studies this is enhanced by contact and review of the report after 1 day of recording. At the end of the recording period, a trained interviewer should review the records with the respondent to clarify entries and to probe for forgotten foods. Dietary records can also be recorded by someone other than the subject. This is often done with children or institutionalized individuals.\n\nAlthough intake data using dietary records are typically collected in an open-ended form, close-ended forms have also been developed [4, 7\u20139]. These forms consist of listings of food groups; and the respondent indicates whether that food group has been consumed. Portion size can also be asked, either in an open-ended manner or in categories. In format, these \"checklist\" forms resemble food frequency questionnaires (FFQs) (see Section II.C). Unlike FFQs, which generally query about intake over a specified time period such as the past year or month, they are filled out either concurrently with actual intake (for precoded records) or at the end of a day for that day's intake (daily recall).\n\nThe dietary record method has the potential to provide quantitatively accurate information on food consumed during the recording period. For this reason, food records are often regarded as the \"gold standard\" against which other dietary assessment methods are compared. By recording foods as they are consumed, the problem of omission is lessened and the foods are more fully described. Further, the measurement of amounts of food consumed at each occasion should provide more accurate portion sizes than if the respondents were recalling portion sizes of foods previously eaten.\n\nA major disadvantage of dietary records is that they are subject to bias both in the selection of the sample and in the measurement of the diet. Dietary record keeping requires that respondents or respondent proxies be both motivated and literate (if done on paper), which can potentially limit the method's use in some population groups (e.g., low socioeconomic status, poorly educated, recent immigrants, children, and some elderly groups). The requirements for cooperation in keeping records can limit the generalizability of the findings from the dietary records to the broader population from which the study sample was drawn. Research indicates that there is a significant increase in incomplete records as more days of records are kept, and the validity of the collected information decreases in the later days of a 7-day recording period, in contrast to information collected in the earlier days [2]. Part of this decrease may occur because many respondents develop the practice of filling out the record at one time for a previous period.\n\nWhen respondents record only once per day, the record method approaches the 24-hour recall in terms of relying on memory rather than concurrent recording. More importantly, recording foods as they are being eaten can affect both the types of food chosen and the quantities consumed [10]. The knowledge that food requires recording and the demanding task of doing it, therefore, alter the dietary behaviors the tool is intended to measure [11]. This effect is a weakness when the aim is to measure unaltered dietary behavior. However, when the aim is to enhance awareness of dietary behavior and change that behavior, as in some intervention studies, this effect can be seen as an advantage. Recording, by itself, is an effective weight loss technique [12].\n\nAs is true with all quantitative dietary information, the information collected on dietary records can be burdensome to code and can lead to high personnel costs. Dietary assessment software that allows for easier data entry using common spellings of foods can save considerable time in data coding. Even with high-quality data entry, maintaining overall quality control for dietary records can be difficult because information is often not recorded consistently from respondent to respondent.\n\nThese weaknesses may be less pronounced for the hybrid method of the \"checklist\" form, since checking off a food item may be easier than recording a complete description of the food, and the costs of data processing can be minimal. The checklist can be developed to assess particular \"core foods,\" which contribute substantially to intakes of some nutrients. However, as the comprehensiveness of the nutrients to be assessed increases, the length of the form also increases, and becomes more burdensome to complete at each eating occasion. The checklist method may be most appropriate in settings with limited diets or for assessment of a limited set of foods or nutrients.\n\nSeveral studies indicate that reported energy and protein intakes on diet records for selected small samples of adults are underestimated in the range of 4\u201337% when compared to energy expenditure as measured by doubly labeled water or protein intake as measured by urinary nitrogen [12\u201320]. Because of these findings, the record is considered an imperfect gold standard. A few studies suggest that low-energy reporters compared to non-low-energy reporters have intakes that are lower in absolute intake of most nutrients [21], higher in percentage of energy from protein [21, 22], and lower in percentage of energy as carbohydrate [21\u201323]. Underreporters may also report lower intakes of desserts and sweet baked goods, butter, and alcoholic beverages but more grains, meats, salads, and vegetables [21].\n\nUnderreporting on food records is probably a result of the combined effects of incomplete recording and the impact of the recording process on dietary choices leading to undereating [12, 20]. The highest levels of underreporting have been found among individuals with a higher body mass index (BMI) [13, 15, 16, 21, 24], particularly women [13, 15, 16, 22, 25, 26]. This effect, however, may be due, in part, to the fact that heavier individuals are more likely to be dieting on any given individual day [27]. Other research shows that demographic or psychological indices such as education, employment grade, social desirability, body image, or dietary restraint may also be important factors related to underreporting on diet records [13, 20, 22, 26, 28, 29].\n\n### B. The 24-Hour Dietary Recall\n\nIn the 24-hour dietary recall, the respondent is asked to remember and report all foods and beverages consumed in the preceding 24 hours or in the preceding day. The recall typically is conducted by personal interview or, more recently, by telephone [30, 31], either computer assisted [32] or using a paper-and-pencil form. Well-trained interviewers are crucial in administering a 24-hour recall because much of the dietary information is collected by asking probing questions. Ideally, interviewers would be dietitians with education in foods and nutrition; however, non-nutritionists who have been trained in the use of a standardized instrument can be effective. All interviewers should be knowledgeable about foods available in the marketplace and about preparation practices, including prevalent regional or ethnic foods.\n\nThe interview is often structured, usually with specific probes, to help the respondent remember all foods consumed throughout the day. One study found that respondents with interviewer probing reported 25% higher dietary intakes than did respondents without interviewer probing [33]. Probing is especially useful in collecting necessary details, such as how foods were prepared. It is also useful in recovering many items not originally reported, such as common additions to foods (e.g., butter on toast) and eating occasions not originally reported (e.g., snacks and beverage breaks). However, interviewers should be provided with standardized neutral probing questions so as to avoid leading the respondent to specific answers when the respondent really does not know or remember. National dietary surveys currently employ a multiple-pass system in which intake is reviewed more than once in an effort to retrieve forgotten eating occasions, and includes a \"forgotten foods list\" of foods commonly omitted in 24-hour recall reporting [34\u201337]. A 24-hour recall interview using the multiple-pass approach typically requires between 30 and 45 minutes.\n\nA quality control system to minimize error and increase reliability of interviewing and coding 24-hour recalls is essential [31, 35, 38\u201341]. Such a system should include a detailed protocol for administration, training, and retraining sessions for interviewers, duplicate collection and coding of some of the recalls throughout the study period, and the use of a computerized database system for nutrient analysis. Data entry can be costly, but these costs can be reduced with computer software specially designed for dietary data entry.\n\nThere are many advantages to the 24-hour recall. An interviewer administers the tool and records the responses, so literacy of the respondent is not required. Because of the immediacy of the recall period, respondents are generally able to recall most of their dietary intake. Because there is relatively little burden on the respondents, those who agree to give 24-hour dietary recalls are more likely to be representative of the population than are those who agree to keep food records. Thus, the 24-hour recall method is useful across a wide range of populations. In addition, interviewers can be trained to capture the detail necessary so that the foods eaten by any population can be researched later by the coding staff and coded appropriately. Finally, in contrast to food record methods, dietary recalls occur after the food has been consumed, so there is less potential for the assessment method to interfere with dietary behavior.\n\nDirect coding of the foods reported during the interview is now possible with computerized software systems. The potential benefits of automated software include substantial cost reductions for processing dietary data, less missing data, and greater standardization of interviews [42]. However, a potential problem in direct coding of interview responses is the loss of the respondent's reported description of the food, in contrast to paper records of the interview, which are then available for later review and editing. If direct coding of the interview is done, methods for the interviewer to easily enter those foods not found in the system should be available and these methods should be reinforced by interviewer training and quality control procedures.\n\nThe main weakness of the 24-hour recall approach is that individuals may not report their food consumption accurately for various reasons related to memory and the interview situation. These cognitive influences are discussed in more detail in Section V.F. Because most individuals' diets vary greatly from day to day, it is not appropriate to use data from a single 24-hour recall to characterize an individual's usual diet. Neither should a single day's intake, be it a recall or food record, be used to estimate the proportion of the population that has adequate or inadequate diets (e.g., the proportion of individuals with less than 30% of energy from fat, or who are deficient in vitamin C intake) [43]. This is because the true distribution of usual diets is much narrower than is the distribution of daily diets (there is variation not only between people in usual diet, but also from day-to-day for each person). The principal use of a single 24-hour recall is to describe the average dietary intake of a group because the means are robust and unaffected by within-person variation. Multiple days of recalls or records can better assess the individual's usual intake and population distributions but require special statistical procedures designed for that purpose [44, 45].\n\nThe validity of the 24-hour dietary recall has been studied by comparing respondents' reports of intake either with intakes unobtrusively recorded\/weighed by trained observers or with biological markers. In general, group mean nutrient estimates from 24-hour recalls have been found to be similar to observed intakes [2, 46], although respondents with lower observed intakes have tended to overreport, and those with higher observed intakes have tended to underreport their intakes [46]. Similar to findings for food records, biological markers such as doubly labeled water and urinary nitrogen show a tendency toward underreporting of energy and protein in the range of 13\u201324% for 24-hour dietary recalls [20, 47, 48]. One study, however, found overreporting of protein from 13\u201325% depending on level of BMI [49]. In national dietary surveys, data suggest that underreporting may affect up to 15% of all 24-hour recalls [41, 50]. Underreporters compared to non-underreporters tend to report fewer numbers of foods, fewer mentions of foods consumed, and smaller portion sizes across a wide range of food groups and tend to report more frequent intakes of low-fat\/diet foods and less frequent intakes of fat added to foods [50]. Factors such as obesity, gender, social desirability, restrained eating, education, literacy, perceived health status, and race\/ethnicity have been shown in various studies to be related to underreporting in recalls [20, 27, 28, 41, 48, 50\u201352].\n\n### C. Food Frequency\n\nThe food frequency approach asks respondents to report their usual frequency of consumption of each food from a list of foods for a specific period [53\u201355]. Information is collected on frequency and sometimes portion size, but little detail is collected on other characteristics of the foods eaten, such as the methods of cooking or the combinations of foods in meals. To estimate relative or absolute nutrient intakes, many food frequency questionnaires (FFQs) also incorporate portion size questions or specify portion sizes as part of each question. Overall nutrient intake estimates are derived by summing, over all foods, the products of the reported frequency of each food by the amount of nutrient in a specified (or assumed) serving of that food.\n\nThere are many FFQ instruments, and many continue to be adapted and developed for different populations and different purposes. Among those validated and commonly used for U. S. adults are the Health Habits and History Questionnaire (HHHQ) or Block Questionnaire [56\u201362], the Fred Hutchinson Cancer Research Center Food Frequency Questionnaire (a revised HHHQ) [63], and the Harvard University Food Frequency Questionnaire or Willett Questionnaire [53, 64\u201369]. Comparisons between the widely used Block and Willett instruments have been conducted indicating differences in estimates for some nutrients [70\u201372]. A new instrument, the Diet History Questionnaire, developed and in use at the National Cancer Institute, was designed with an emphasis on cognitive ease for respondents [73\u201375] (see ). Other instruments have been developed for specific populations. Two FFQs have been developed by researchers at the University of Arizona, the University of Arizona Food Frequency Questionnaire and the Southwest Food Frequency Questionnaire, to capture the diverse diets of Latinos and Native Americans [22, 76, 77]. Investigators at the University of Hawaii have developed a questionnaire for assessing the diverse diets of Hawaiian, Japanese, Chinese, Filipino, and Caucasian ethnic groups [78, 79]. This instrument was recently adapted for use in a multiethnic cohort study conducted in Hawaii and Los Angeles [80]. In Europe, a number of FFQs have been developed within Western European countries for the European Prospective Investigation into Cancer and Nutrition (EPIC) [19, 81\u201386]. In addition, a few \"brief\" FFQs have been developed composed of shorter lists of 40\u201360 line items from the original 100 or so items [87\u201390]. Such shortened instruments may reflect distributions of usual intakes of specific nutrients\/food groups or percentage of energy from macronutrients. Because of the number of FFQs available, investigators need to carefully evaluate which best suits their research needs.\n\nThe major strength of the FFQ approach is its ability to estimate the respondent's usual intake of foods over a long period of time such as one year. It can also be used to circumvent recent changes in diet (e.g., changes due to disease) by obtaining information about individuals' diets as recalled about a prior time period. Food frequency responses can be used to rank individuals according to their usual consumption of nutrients, foods, or groups of foods. Most food frequency instruments have been designed to be self-administered, require 30\u201360 minutes to complete depending on the instrument and the respondent, and most are optically scannable to reduce data entry costs. Because the costs of data collection and processing and the respondent burden are typically much lower for FFQs than for multiple diet records or recalls, FFQs have become a common way to estimate usual dietary intake in large epidemiological studies.\n\nThe major limitation of the food frequency method is that many details of dietary intake are not measured, and the quantification of intake is not as accurate as with recalls or records. Inaccuracies result from an incomplete listing of all possible foods and from errors in frequency and usual serving size estimations. As a result, the scale for nutrient intake estimates from a FFQ may be shifted considerably, yielding inaccurate estimates of the average intake for the group. Research suggests that longer food frequency lists may overestimate, while shorter lists may underestimate intake of fruits and vegetables [91], but it is unclear as to whether or how this applies to nutrients and other food groups. In the absence of knowledge about the true usual intake of the population, it is unknown how closely the distribution of intake estimates from FFQs reflects the distribution of true intake in that population.\n\nControversy has arisen over whether it is proper to use FFQs to estimate quantitative parameters of a population's dietary intake 64, 92\u201396] (see [Chapter 4). Although some FFQs seem to produce estimates of population average intakes that are reasonable [92], different FFQs will perform in often unpredictable ways in different populations, so the levels of nutrient intakes estimated by FFQs are best regarded as only approximations [93]. FFQs are much better suited for ranking subjects according to food or nutrient intake than for estimating the levels of intake.\n\nServing size of foods consumed is difficult for respondents to evaluate and is thus problematic for all assessment instruments (see Section V.A). However, the inaccuracies involved in respondents attempting to estimate usual serving size in FFQs may be even greater because a respondent is asked to estimate an average for foods that may have highly variable portion sizes across eating occasions. The importance of whether or not to include portion size at all on FFQs has been widely debated. Because frequency is believed to be a greater contributor than typical serving size to the variance in intake of most foods, some prefer to use FFQs without the additional respondent burden of reporting serving sizes [53]. Others cite small improvements in the performance of FFQs that ask the respondents to report a usual serving size for each food [58, 59].\n\nDevelopment of the food list is crucial to the success of the food frequency method [56]. The full variability of an individual's diet, which includes many different foods, brands, and preparation practices, cannot be fully captured with a finite food list. Obtaining accurate reports for foods eaten both alone and in mixtures is particularly problematic. FFQs can ask the respondent to report either a combined frequency for a particular food eaten both alone and in mixtures, or to report separate frequencies for each food use. The first approach is cognitively complex, but the second approach may lead to double counting. Often FFQs will include similar foods in a single question (e.g., hamburger, steak, roast beef). However, such grouping can create a cognitively complex question (e.g., for someone who often eats hamburger but never eats steak). In addition, when a group of foods is asked as a single question, assumptions about the relative frequencies of intake of the foods constituting the group must be made when calculating nutrient estimates. These assumptions are often not based on information from the study population even though true eating patterns may differ considerably across population subgroups and over time.\n\nFFQs are commonly used to rank or group study subjects for the purpose of assessing the association between dietary intake and disease risk, such as in case-control or cohort studies [97\u201399]. For estimating relative risks, the degree of misclassification of subjects from their correct quantile of intake is more important than is the quantitative scale on which the ranking is made [100]. Although analyses on the extent of misclassification by the food frequency method indicate that the amount of extreme misclassification (e.g., from lowest quartile to the highest) is small, even a small amount of such misclassification can create a large bias in estimates of associations [101, 102].\n\nThe definitive validity study for a food frequency-based estimate of long-term usual diet would require nonintrusive observation of the respondent's total diet over a long time. No such studies have ever been done. One feeding study, however, with three defined 6-week feeding cycles (in which all intakes were known) showed some significant differences in known absolute nutrient intakes as compared to the Willett FFQ for several fat components, mostly in the direction of underestimation by the FFQ [103].\n\nThe most practical approach to examining the concordance of food frequency responses and usual diet is to use multiple food recalls or records over a period as an indicator of usual diet. This approach has been used in many studies examining various FFQs [5, 18, 19, 47, 53, 57, 60, 61, 63, 65, 66, 68, 80\u201386, 104\u2013115]. This type of study is more properly called a \"calibration study\" rather than \"validation study\" (see Section V.H), because recalls and records themselves may not represent the time period of interest, may contain error, and may underestimate nutrient intakes [13\u201317, 24, 47\u201349, 116]. In such studies, the correlations between the methods for most foods and nutrients are in the range of 0.4\u20130.7. Findings from calibration studies that have incorporated biological markers, such as urinary nitrogen for protein intake or doubly labeled water for energy expenditure, have shown correlations with FFQs ranging from 0.2 to 0.7 for protein [18, 19, 47, 86, 106, 114, 117, 118] and from 0.4 to 0.5 for energy [14, 47].\n\nDepending on characteristics of FFQs, such as length and detail of the food list, quality of the nutrient database, and method of querying portion size, the estimates of food and nutrient intake can be higher or lower than those from the more quantitative methods of the 24-hour dietary recall or food record. Given that there is measurement error in all self-reported methods of dietary assessment, various statistical methods employing measurement error models and energy adjustment are used to assess the validity of FFQs and to adjust estimates of relative risks for disease outcomes [119\u2013129] (see Sections V.H and V.I).\n\nIn pursuit of improving the validity of the FFQ, investigators have addressed a range of questionnaire design issues such as length, closed- versus open-ended response categories, portion size, seasonality, and time frame. Frequency instruments designed to assess total diet generally list more than 100 individual line items, many with additional portion size questions, requiring 30\u201360 minutes to complete. This raises concern about length and its effect on response rates. Though respondent burden is a factor in obtaining reasonable response rates for studies in general, a few studies have shown this not to be a decisive factor for FFQs [74, 130\u2013134]. Others suggest that adding more low-fat foods to the tool leads to better fat and energy estimates [135, 136]. This tension between length and specificity highlights the difficult issue of how to define a closed-ended list of foods for a food frequency instrument. Whether or not incorporating separate questions to assess some detail in portion size is necessary has been controversial given that the most important factor in estimating intakes is frequency. However, research has been conducted to determine the best ways to ask about portion size on FFQs [73, 137, 138]. Another design issue is the time frame about which usual intake is queried. Many instruments inquire about usual intakes during the past year [56, 64], but it is possible to ask about the past week or month [139] depending on specific research situations. Even when usual intake during the past year is asked, several studies have indicated that the season in which the questionnaire is administered has an influence on reporting over the entire year [140, 141]. Finally, optically scanned instruments have necessitated the use of closed-ended response categories, forcing a loss in specificity [142].\n\n### D. Brief Dietary Assessment Methods\n\nMany brief dietary assessment methods have been developed. These instruments can be useful in situations that do not require either assessment of the total diet or quantitative accuracy in dietary estimates. For example, a brief diet assessment might be used to triage large numbers of individuals into groups to allow more focused attention on those at greatest need for intervention or education. Measurement of dietary intake, no matter how crude, can also serve to activate interest in the respondent to facilitate nutrition education. These brief methods may, therefore, have utility in clinical settings or in situations where health promotion and health education are the goals. Brief methods have also been used to track changes in diet within an intervention setting, although there is concern that responses to questions of intake that directly evolve from intervention messages may be biased [143]. Brief methods are used often for population surveillance at the state or local level, for example, in the Centers for Disease Control and Prevention's (CDC's) Behavioral Risk Factor Surveillance System (BRFSS) (see Section III.F).\n\nSuch brief methods can be simplified FFQs or questionnaires that focus on eating behaviors other than the frequency of intake of specific foods. Complete FFQs typically must contain 100 or more food items to capture the range of foods contributing to the many different nutrients in the diet. If an investigator is interested only in estimating the intake of a single nutrient or a single type of food, however, then far fewer foods need to be included. Often, only 15\u201330 foods might be required to account for most of the intake of a particular nutrient in the diet of a population [144, 145].\n\nNumerous single-exposure short questionnaires using a food frequency approach have been developed and compared with multiple days of food records, dietary recalls, and\/or complete FFQs. In early work, Block selected 13 foods that accounted for most of the intake of fat in the diets of American women to develop a brief \"fat screener\" for use in selecting women for a dietary intervention trial; the correlation between the fat index and fat intake from multiple records was 0.58 [146]. A similar tool used in the Behavioral Risk Factor Surveillance System (BRFSS) was evaluated in five different populations relative to more extensive dietary assessment instruments [147]. Correlations between fat scores and quantified absolute fat intakes ranged from 0.22 (in the Latino population) to 0. 60, and were lower (0.26\u20130.42) between fat scores and percent energy from fat. A later adaptation of the tool, tested among men and women, indicated substantial misclassification when ranking individuals for percent energy from fat, and only moderate agreement when ranking absolute fat intakes [148]. The Block fat screener, currently composed of 17 items, has been modified to ask only about versions of the food that are not low fat. Information about this tool is found at . A recently developed 16-item percent energy from fat screener correlated 0.65 with 24-hour recalls in an older U. S. population [149]. Similar sets of questions have been developed and tested by others to briefly characterize dietary fat or percent energy from fat intake [150\u2013154]. Published validity studies of fat screeners are listed and reviewed by Yaroch _et al._ [155].\n\nA seven-item tool developed by National Cancer Institute (NCI) staff and private grantees for the NCI \"5 a Day for Better Health\" effort provides an indicator of the average number of servings of fruits and vegetables consumed per day, and has been used widely in the United States. The tool is similar to one used in the BRFSS [156]. Validation studies of various brief instruments to assess fruit and vegetable intake have suggested that they underestimate actual intake [88, 157\u2013160]. A newer tool based on cognitive interviewing methods has been developed at NCI, and its validity is currently being evaluated (see ). Single-nutrient FFQs have been developed for calcium [58, 161], iron [162], and isoflavones [163].\n\nBecause the cognitive processes for answering food frequency-type questions can be complex, some attempts have been made to reduce respondent burden by asking questions that require only \"yes\u2013no\" answers. Kristal _et al._ [164] developed a questionnaire to assess total fat, saturated fat, fiber, and percent energy from fat which contains 44 food items for which respondents are asked whether they eat the items at a specified frequency. A simple index based on the number of \"yes\" responses was found to correlate well with diet as measured by 4-day records and with FFQs assessing total diet [164]. This same \"yes\u2013no\" approach to questioning for a food list has also been used as a modification of the 24-hour recall [165, 166].\n\nOften, interventions are designed to target specific food preparation or consumption behaviors rather than frequency of consuming specific foods. Examples of such behaviors might be trimming the fat from red meats, removing the skin from chicken, or choosing low-fat dairy products. Many questionnaires have been developed in different populations to measure these types of dietary behaviors, and several have been compared with more complete dietary assessments. A 9-question instrument designed to measure high-fat food consumption behaviors of Mexican Americans was shown to correspond with fat estimates from 24-hour recalls 167]. In England brief questions on high-fat behaviors correlated with fat-intake estimates from a FFQ [168] and with blood-cholesterol change [169]. The British instrument has been adapted to reflect North American eating habits; the Northwest Lipid Research Clinic Fat Intake Scale has been evaluated [170] and is available at [http:\/\/depts.washington.edu\/\u223cnwlrc\/fis.html. In rural North Carolina, an 8-item questionnaire was correlated with fat intake from 3-day food records [171]. A 33-item fat and fiber-related behavior questionnaire correlated with food frequency measures [172] among participants from a health maintenance organization in Seattle, Washington. Among white middle-class volunteers in Oregon, changes in individual responses over time to a 32-item eating behavior questionnaire were correlated with changes in lipid profiles [173].\n\nThe brevity of these methods and their correspondence with dietary intake as estimated by more extensive methods create a seductive option for investigators who would like to measure dietary intake at a low cost. Although brief methods have many applications, they have several limitations. Most measures are not quantitatively meaningful and, therefore, estimates of dietary intake for the population cannot be made. Even when measures aim at providing estimates of total intake, the estimates are not precise and have large measurement error. Generally, brief methods are only designed to capture information about a single nutrient, so the entire diet cannot be assessed. Finally, the specific dietary behaviors found to correlate with dietary intake in a particular study may not correlate similarly in another population, or even in the same population in another time period. For example, behavioral questionnaires developed and tested in middle-class, middle-aged U.S. women [174] were found to perform very differently when applied to Canadian male manual laborers [175] and to a low-income, low-education adult Canadian population [176]. Investigators should carefully consider the needs of their study and their own population's dietary patterns before choosing an \"off-the-shelf\" instrument designed to briefly measure either food frequency or specific dietary behaviors.\n\n### E. Diet History\n\nThe term _diet history_ is used in many ways. In the most general sense, a dietary history is any dietary assessment that asks the respondent to report about past diet. Originally, as coined by Burke, the term _dietary history_ referred to the collection of information not only about the frequency of intake of various foods but also about the typical makeup of meals [177, 178]. Many now imprecisely use the term _dietary history_ to refer to the food frequency method of dietary assessment. However, several investigators have developed diet history methods that provide information about usual food intake patterns beyond simply food frequency data [179\u2013182]. Some of these methods characterize foods in much more detail than is allowed in food frequency lists (e.g., preparation methods and foods eaten in combination), and some of these methods ask about foods consumed at every meal [181, 183]. The term _diet history_ is therefore probably best reserved for dietary assessment methods that are designed to ascertain a person's usual food intake in which many details about characteristics of foods as usually consumed are assessed in addition to the frequency and amount of food intake.\n\nThe Burke diet history included three elements: a detailed interview about usual patterns of eating, a food list asking for amount and frequency usually eaten, and a 3-day diet record [177, 178]. The detailed interview (which sometimes includes a 24-hour recall) is the central feature of the Burke dietary history, with the food frequency checklist and the 3-day diet record used as cross-checks of the history. The original Burke diet history has not often been exactly reproduced, because of the effort and expertise involved in capturing and coding the information if it is collected by an interviewer. However, many variations of the Burke method have been developed and used in a variety of settings [179\u2013182, 184\u2013186]. These variations attempt to ascertain the usual eating patterns for an extended period of time, including type, frequency, and amount of foods consumed; many include a cross-check feature [187, 188]. One such diet history has been automated, is self-administered, and eliminates the need for an interviewer to ask the questions. The software incorporates sound, orally delivered questions and dialogue, and pictures of foods to improve communication and motivation [181]. Other diet histories have been automated but still continue to be administered by an interviewer [189]. Short-term recalls or records are often used for validation or calibration rather than as a part of the tool.\n\nThe major strength of the diet history method is its assessment of meal patterns and details of food intake rather than intakes for a short period of time (as in records or recalls) or only frequency of food consumption. Details of the means of preparation of foods can be helpful in better characterizing nutrient intake (e.g., frying vs. baking), as well as exposure to other factors in foods (e.g., charcoal broiling). When the information is collected separately for each meal, analyses of the joint effects of foods eaten together is possible (e.g., effects on iron absorption of concurrent intake of tea or foods containing vitamin C). Although a meal-based approach often requires more time from the respondent than a food-based approach, it may provide more cognitive support for the recall process. For example, the respondent may be better able to report total bread consumption by reporting bread as consumed at each meal.\n\nA weakness of the approach is that respondents are asked to make many judgments both about the usual foods and the amounts of those foods eaten. These subjective tasks may be difficult for many respondents. Burke cautioned that nutrient intakes estimated from these data should be interpreted as relative rather than absolute. All of these limitations are also shared with the food frequency method. The meal-based approach is not useful for individuals who have no particular eating pattern and may be of limited use for individuals who \"graze,\" i.e., eat small bits throughout the day, rather than eat at defined meals. The approach, when conducted by interviewers, requires trained nutrition professionals.\n\nThe validity of diet history approaches is difficult to assess because we lack independent knowledge of the individual's usual long-term intake. Nutrient estimates from diet histories have often been found to be higher than nutrient estimates from tools that measure intakes over short periods, such as recalls or records [108, 190\u2013194]. However, results for these types of comparisons depend on both the approach used and study characteristics. Validation studies that estimate correlations between reference data from recalls, records, or observations and diet histories are limited and show correlations in ranges similar to those for FFQs [182, 193, 195]. There are few validations of diet history questionnaires using biological markers as a basis of comparison. One study showed that, on average, 12 adults completing a diet history underreported by 12% in comparison to energy expenditure (measured by doubly labeled water) [196]; another showed that, in comparison to protein intake as measured by urinary nitrogen, 64 respondents completing a diet history questionnaire underreported by 3% [197].\n\nTable 1 summarizes the various advantages and disadvantages of the dietary assessment instruments.\n\nTABLE 1\n\nAdvantages and Disadvantages of Dietary Assessment Instruments\n\n## III. DIETARY ASSESSMENT IN SPECIFIC SITUATIONS\n\nThe primary research question must be clearly formed and questions of secondary interest should be recognized as such. Projects can fail to achieve their primary goal because too much attention is paid to secondary goals. The choice of the most appropriate dietary assessment tool depends on many factors. Questions that must be answered in evaluating which dietary assessment tool is most appropriate for a particular research purpose include [98]: (1) Is information needed about foods, nutrients, other food constituents or about specific dietary behaviors? (2) Is the average intake of a group or the intake of each individual needed? (3) Is absolute or relative intake needed? (4) What level of accuracy is needed? (5) What time period is of interest? (6) What are the research constraints in terms of money, time, staff, and respondent characteristics?\n\n### A. Cross-Sectional Surveys\n\nOne of the most common types of studies is the simple cross-sectional survey, a \"snapshot\" of the dietary practices of a population at a particular point in time. The population can be variously defined as the entire country (as in the National Health and Nutrition Examination Survey (NHANES) and Continuing Survey of Food Intakes by Individuals (CSFII)], the residents of a state (as in the BRFSS), or individuals who attend a particular facility such as a health clinic. In most dietary surveys, 24-hour recalls are used, allowing for quantitative accuracy in estimating average daily food and nutrient intake in the population studied. At least two independent days of recalls or records have to be collected from each respondent (or at least a sample of respondents) if the intent is to describe the true distribution of usual food and nutrient intake of the population. Otherwise, the prevalence of high or low intakes in the population will be overestimated. New statistical models and supporting software have been developed and are required to estimate the true distribution of nutrient intake with as few as 2 days of recall or record data [44, 45]. Food frequency instruments that are designed to measure usual individual diet also have been used in surveys, but they are limited by their lack of quantitative accuracy. Brief methods designed to measure specific diet behaviors may also be useful in some dietary surveys.\n\n### B. Case-Control (Retrospective) Studies\n\nA case-control study design classifies individuals with regard to disease status currently (as cases or controls) and relates this to past (retrospective) exposures. For dietary exposure, the period of interest could be either the recent past (e.g., the year before diagnosis) or the distant past (e.g., 10 years ago or in childhood). Because of the need for information about diet before onset of disease, dietary assessment methods that focus on current behavior, such as the 24-hour recall, are not useful in retrospective studies. The food frequency and diet history methods are well suited for assessing past diet and are therefore the only good choices for case-control (retrospective) studies.\n\nIn any food frequency or diet history interview, the respondent is not asked to call up specific memories of each eating occasion, but to respond on the basis of general perceptions of how frequently he\/she ate a food. In assessing past diet, an additional requirement is to orient the respondent to the appropriate period. In case-control studies, the relevant period is often the year before diagnosis of disease or onset of symptoms, or even a time many years in the past. Cognitive factors may greatly affect the performance of this method.\n\nLong-term reproducibility of various FFQs has been assessed in various populations by asking participants from past dietary studies to recall their diet from that earlier time [198]. Correspondence of retrospective diet reports with the diet as measured in the original study has usually been greater than correspondence with diet reported by subjects for the current (later) period. This observation implies that if diet from years in the past is of interest, then it is probably better to ask respondents to recall it than to simply consider current diet as a proxy for past diet. The current diets of respondents may affect their retrospective reports about past diets. In particular, retrospective diet reports from seriously ill individuals may be biased by recent dietary changes [198, 199]. Studies of groups in whom diet was previously measured indicate no consistent differences in the accuracy of retrospective reporting between those who recently became ill and others [200, 201].\n\n### C. Cohort (Prospective) Studies\n\nIn a cohort study design, exposures of interest are assessed at baseline in a group (cohort) of people, and disease outcomes occurring over time (prospectively) are then related to the baseline exposure levels. In prospective dietary studies, dietary status at baseline is measured and related to later incidence of disease. In studies of many chronic diseases, large numbers of individuals need to be followed for years before enough new cases with that disease accrue for statistical analyses. A broad assessment of diet is usually desirable in prospective studies, since many dietary exposures and many disease endpoints will ultimately be investigated.\n\nTo relate diet at baseline to the eventual occurrence of disease, a measure of the usual intake of foods by study subjects is needed. Although a single 24-hour recall or a food record for a single day would not adequately characterize the usual diet of study subjects in a cohort study, such information could be later analyzed at the group level for contrasting the average dietary intakes of subsequent cases with those who did not acquire the disease. Multiple dietary recalls, records, diet histories, and food frequency methods have all been used effectively in prospective studies. Cost and logistic issues tend to favor food frequency methods, as many prospective studies require thousands of respondents.\n\nEven in large studies using FFQs, it is desirable to include multiple recalls or records in subsamples of the population (preferably before beginning the study) to construct or modify the food frequency instrument and to calibrate it (see Section V.H). Information on the foods consumed could be used to ensure that the questionnaire includes the major food sources of key nutrients, with reasonable portion sizes. Because the diets of individuals change over time, it is desirable to measure diet throughout the follow-up period rather than just at baseline. One study showed that data from annual administrations of FFQs showed only small dietary changes over time and that repeat administrations more than 5 years apart would be acceptable to assess dietary change over time [202]. If diet is measured repeatedly over the years, repeated calibration is also desirable. Information from calibration studies can be used for three purposes: to give design information, e.g., the sample size needed [203]; to show how values from the food frequency tool (or a brief food list thus derived) relate to values from the recalls\/records [96, 100]; and to determine the degree of attenuation\/measurement error in the estimates of association observed in the study (e.g., between diet and disease) [122, 123, 125, 127, 129, 204\u2013206] (see Section V.H).\n\n### D. Intervention Studies\n\nMeasurement of the dietary changes resulting from an intervention requires a valid measure of diet before, during, and after the intervention period. Very little work has been done on the development of valid methods to measure dietary change in individuals or in populations [207\u2013210]. Measurement of specific dietary behaviors in addition to (or even in place of) dietary intake should be considered in intervention evaluations when the nature of the intervention involves education about specific behaviors. If, for instance, a community-wide campaign to choose low-fat dairy products were to be evaluated, food selection and shopping behaviors specific to choosing those items should be measured. Intentional behavior change is a complex and sequential phenomenon, however, as has been shown for tobacco cessation [211]. A complex sequence of events may also lead to dietary change [212]. The effects of educational interventions might also be assessed by measuring knowledge, attitudes, beliefs, barriers, and perceptions of readiness for dietary change, although the reliability of these types of questions has not been well assessed.\n\nWhether an intervention is targeting individuals or the entire population, repeated measures of diet among study subjects can reflect reporting bias in the direction of the change being promoted. Even though not intending to be deceptive, respondents tend to want to tell the investigators what they think they want to hear. Though there has been little methodological research in measuring dietary change, behavioral questions and the food frequency method, because of their greater subjectivity, may be more susceptible to reporting biases than the 24-hour recall method [30, 143]. Because all subjective reports are subject to bias in the context of an intervention study, an independent assessment of dietary change should be considered. One such method useful in community-wide interventions is monitoring food sales. Often, cooperation can be obtained from food retailers [213]. Because of the large number of food items, only a small number should be monitored, and the large effects on sales of day-to-day pricing fluctuations should be carefully considered. Another method to consider is measuring changes in biomarkers of diet in the population.\n\n### E. Dietary Screening in Clinical Settings\n\nAccurate measurement of intake is not always required in clinical settings. For some goals, a crude indication of dietary habits to screen for probable dietary risk is adequate. The brief \"fat screener\" of Block _et al._ [146] was originally developed as a screening tool to crudely classify women for entry into a low-fat intervention trial. Another screening questionnaire was developed as a crude instrument intended only to identify a group of respondents who might be in need of nutritional and\/or medical counseling [214, 215].\n\nIn clinical settings, the caregiver is generally interested in assessing an individual's usual dietary practices but has only limited time. Accurate information may be needed, such as for counseling on medically prescribed diets. Qualitative information about usual dietary practices and behaviors is, however, usually sufficient. Dietary recalls, diet histories, and food frequency methods are useful as methods to crudely classify (\"screen\") individuals in clinical settings. While 24-hour dietary recalls can provide useful quantitative information, there is a danger in interpreting yesterday's recalled diet as the individual's usual intake. Food frequency approaches may provide adequate information to qualitatively assess usual dietary practices. Brief questionnaires can serve to identify individuals who may be at dietary risk from, for example, frequent consumption of high-fat foods [146]. Short forms that measure specific dietary behaviors (e.g., choosing low-fat salad dressings or dairy products) may provide useful information about specific intervention points for counseling [164, 174, 210].\n\n### F. Dietary Surveillance or Monitoring\n\nNutritional surveillance is increasingly acknowledged as important at the national and state levels as an activity for problem recognition, policy making, and evaluation. In addition to assessing food and nutrient intakes in the U. S. population, the components of nutritional surveillance can include the regular monitoring of mortality, morbidity, risk factors, knowledge of information sources, and knowledge levels of the populations of interest [216]. Although it is important that the data are rapidly analyzed and reported, an important factor is that the methods for collecting data, including the sampling procedures, must be similar through time [217] to assess trends. Food composition databases must remain comparable for this purpose but also reflect true changes in actual food composition over time. The status of efforts to monitor diet in the United States is summarized in reports on the National Nutrition Monitoring and Related Research Program [218\u2013225] and elsewhere [207, 226, 227].\n\nTwo major surveillance surveys, the National Health and Nutrition Examination Surveys (NHANES) and the Continuing Survey of Food Intakes by Individuals (CSFII), conducted by the National Center for Health Statistics (NCHS) and the U. S. Department of Agriculture (USDA), respectively, have been conducted periodically to survey the health and nutritional status of representative samples of Americans [221, 228\u2013246]. Information about these surveys is available at their web sites: and . National surveys of knowledge and attitudes about nutrition and health have been conducted periodically. Examples of such surveys are the Food and Drug Administration's Health and Diet Survey, and USDA's Diet and Health Knowledge Survey, which has been administered in conjunction with the CSFII [35]. Other nutrition monitoring activities sponsored by federal and state agencies are listed in _The Directory of Federal and State Nutrition Monitoring Activities_ [221].\n\nThe type of information required for a surveillance or monitoring system can vary. For some purposes, quantitative estimates of intake are needed, whereas for other purposes, only qualitative estimates of intake, like food frequency or behavioral indicators, are needed. There is a particular need to monitor dietary trends at the local level. To help provide local data, the Centers for Disease Control and Prevention (CDC) has developed brief FFQs for administration on the telephone to assess the intake of dietary fat (13 questions) and fruits and vegetables (6 questions) within their Behavioral Risk Factor Surveillance System. Information about the survey is available at the CDC web site: .\n\nTable 2 summarizes the dietary methods commonly used in different study designs.\n\nTABLE 2\n\nDietary Assessment in Different Study Situations\n\nStudy situation | Methods commonly used \n---|--- \nCross-sectional | 24-Hour recall; FFQ; brief methods \nCase-control (retrospective) | FFQ; diet history \nCohort (prospective) | FFQ; diet history; 24-hour recall; record \nIntervention | FFQ: brief methods; 24-hour recall \nClinical screening | 24-Hour recall; brief methods; diet history \nSurveillance | 24-Hour recall; brief methods\n\n## IV. DIETARY ASSESSMENT IN SPECIAL POPULATIONS\n\nA. Surrogate Reporters\n\nIn many situations, respondents are unavailable or unable to report about their diets. For example, in case-control studies, surrogate reports may be obtained for cases who have died or who are too ill to interview. Although the accuracy of surrogate reports has not been examined, comparability of reports by surrogates and subjects has been studied, in hopes that surrogate information might be used interchangeably with information provided by subjects [247]. Common sense indicates that the individuals who know the most about a subject's lifestyle would make the best surrogate reporters. Adult siblings provide the best information about a subject's early life, and spouses or children provide the best information about a subject's adult life. When food frequency instruments are used, the level of agreement between subject and surrogate reports of diet varies with the food and possibly with other variables such as number of shared meals, interview situation, case status, and sex of the surrogate reporter. Mean frequencies of use computed for individual foods and food groups between surrogate reporters and subject reporters tend to be similar, but agreement is much lower when detailed categories of frequency are compared. Several studies have shown that agreement is better for alcoholic beverages, coffee, and tea than for other foods.\n\nAlthough subjects reporting themselves in the extremes of the distribution are seldom reported by their surrogates in the opposite extreme, many subjects who report they are in an extreme are reported by their surrogates in the middle of the distribution [248]. This may limit the usefulness of surrogate information for analyses at the individual level that rely on proper ranking. Furthermore, there may be a substantial difference in the quality of surrogate reports between spouses of deceased subjects and spouses of surviving subjects [249]. For these reasons, use of surrogate respondents should be minimized for obtaining dietary information in analytical studies. When used, analyses excluding the surrogate reports should be done to examine the sensitivity of the reported associations to possible errors or biases in the surrogate reports.\n\n### B. Ethnic Populations\n\nSpecial modifications are needed in the content of dietary assessment methods when the study population is composed of individuals whose cuisine or cooking practices are not mainstream [250]. If the method requires an interview, interviewers of the same ethnic or cultural background are preferable so that dietary information can be more effectively communicated. If dietary information is to be quantified into nutrient estimates, examination of the nutrient composition database is necessary to ascertain the number of ethnic foods already included and those to be added. It is also necessary to examine the recipes and assumptions underlying the nutrient composition of certain ethnic foods. Some very different foods may be called the same name, or similar foods may be called by different names [251]. For these reasons, it may be necessary to obtain detailed recipe information for all ethnic mixtures reported. For Latino populations, the U. S. Department of Agriculture Nutrient Database for Standard Reference is a good starting point because foods reported in the Hispanic HANES have now been incorporated. Databases developed for the Hawaiian cancer studies include many foods consumed by Hawaiian natives and by Japanese, Chinese, and Polynesian groups [250].\n\nTo examine the suitability of the initial database, baseline recalls or records with accompanying interviews should be collected from individuals in the ethnic groups. These interviews should focus on all the kinds of food eaten and the ways in which foods are prepared in that culture. Recipes and alternative names of the same food should be collected, and interviewers should be familiarized with the results of these interviews. Recipes and food names that are relatively uniform should be included in the nutrient composition database. Even with these modifications, it may be preferable to collect records and recalls in the field by detailed records (not the preselected lists most common in computer-assisted methods) when special ethnic foods are common. This would prevent the detail of food choice and preparation from being lost by _a priori_ coding.\n\nUse of food lists developed for the majority population for FFQs may be inappropriate for many individuals with ethnic eating patterns. Many members of ethnic groups consume both foods common in the mainstream culture and foods that are specific to their own ethnic group. Development of the food list can be accomplished either by modifying an existing food list based on expert judgment of the diet of the target population or, preferably, by examining the frequency of reported foods in the population from a set of dietary records or recalls. Food frequency questionnaires for Navajos, Chinese Americans, and individuals in Northern India have been developed using these approaches [252\u2013254].\n\nBesides the food list, however, other important issues must be considered when adapting existing FFQs for use in other populations. The relative intake of different foods within a food group line item may differ, thus requiring a change in the nutrient database associated with each line item. For example, Latino populations may consume more tropical fruit nectars and less apple and grape juice than the general U. S. population and therefore would require a different nutrient composition standard for juices. In addition, the portion sizes generally used may differ. For example, rice may be consumed in larger quantities in Latino and Asian populations; the amount attributed to a large portion for the general population may be substantially lower than the amount typically consumed by Latino and Asian populations. Adaptation of an existing FFQ considering all of these factors is illustrated by Tucker _et al._ [255] for an elderly Puerto Rican population.\n\nPerformance of FFQs varies across ethnic groups [256]. Questionnaires aimed at allowing comparison of intakes across multiple cultures have been developed; however, the limited number of studies done thus far indicate that there are validity differences among the various cultural groups [255, 257\u2013259]. Understanding these validity differences is crucial to the appropriate interpretation of study results.\n\n### C. Children\n\nThe 24-hour dietary recall, food records, and food frequency instruments have all been used to assess children's intakes, which is considered to be even more challenging than assessing the diets of adults [260\u2013265]. Children tend to have diets that are highly variable from day to day, and their food habits can change rapidly. Younger children are less able to recall, estimate, and cooperate in usual dietary assessment procedures, so much information by necessity has to be obtained by surrogate reporters. Adolescents, while more able to report, may be less interested in giving accurate reports. Baranowski and Domel [266] have posited a cognitive model of how children report dietary information.\n\nFor preschool-aged children, information is obtained from surrogates, usually the primary caretaker(s), who may typically be a parent or an external caregiver. If information can be obtained only from one surrogate reporter, the reports are likely to be less complete. Even for periods when the caregiver and child are together, foods tend to be underestimated [267]. A consensus recall method, in which the child and parents report as a group to give responses on a 24-hour dietary recall, has been shown to give more accurate information than a recall from either parent alone [268]. For older children, a blended instrument, the record assisted 24-hour recall (in which the children record only the names of foods and beverages consumed throughout a 24-hour period, serving as a cue for the later 24-hour recall interview), has been developed and tested. While foods were generally reported accurately, accurate reporting of portion sizes was difficult, creating only modest accuracy in overall nutrient intake estimates [269].\n\nAdaptation of food frequency instruments originally developed for adults requires consideration of the food list and portion sizes. Food frequency instruments have been especially developed and tested for use in child and adolescent populations [8, 270]. Generally, correlations between the criterion instrument and food frequency instruments have been lower in child and adolescent populations than in adult populations.\n\n### D. Elderly\n\nMeasuring diets among the elderly can, but does not necessarily, present special problems [271, 272]. Both recall and food frequency techniques are inappropriate if memory is impaired. Direct observation in institutional care facilities or shelf inventories for elders who live at home can be useful. Even when memory is not impaired, other factors can affect the assessment of diet among the elderly. Because of the frequency of chronic illness in this age group, special diets (e.g., low sodium, low fat, high fiber) are often recommended to these individuals. Such recommendations could not only affect actual dietary intake, but could also bias reporting, as individuals may report what they should eat rather than what they do eat. Alternatively, respondents on special diets may be more aware of their diets and may more accurately report them. When dentition is poor, the interviewer should probe for foods that are prepared or consumed in different ways. Elderly individuals are also more likely to be taking multiple types of nutritional supplements, which present special problems in dietary assessment [273] (see Section V.G).\n\nAdaptations of standard methods have been suggested and evaluated, including use of memory strategies, prior notification of a dietary interview [274], combining methods [272], and developing new methods [275]. Research suggests that under many circumstances the validity of dietary information collected from the elderly is comparable to that collected from younger adults [105].\n\nTable 3 summarizes optimal assessment strategies for special populations.\n\nTABLE 3\n\nOptimal Strategies for Special Populations\n\n## V. SELECTED ISSUES IN DIETARY ASSESSMENT METHODS\n\nA. Estimation of Portion Size\n\nResearch has shown that individuals have difficulty estimating portion sizes of foods, both when examining displayed foods and when reporting about foods previously consumed [276\u2013282]. In general, consumers are not skilled at estimating the weights of foods, and there is further confusion about the term _ounces,_ which is interpreted as indicating either volume or weight. Furthermore, volume amounts of foods may have limited meaning, as respondents appear to be relatively insensitive to changes made in portion size amounts shown in reference categories asked on questionnaires [283]. Portion sizes of foods that are commonly bought and\/or consumed in defined units (e.g., bread by the slice, pieces of fruit, beverages in cans or bottles) may be more easily reported than irregularly shaped foods (e.g., steak, lettuce).\n\nPortion size aids are commonly used to help respondents estimate portion size. The National Health and Nutrition Examination Surveys use an extensive set of three-dimensional models; the CSFIIs have used common household measures, such as cups and teaspoons. In one study, which compared these two approaches among men, there was little difference in the frequency of over- and underreporting [284]. However, those using the household measures had greater overestimates than did those using the food models. Studies indicate that accuracy of reporting using either models or household measures can be improved with training in that method [285, 286], but the effects of training deteriorate with time [287].\n\nTwo-dimensional and three-dimensional pictures have been developed and used in 24-hour dietary recalls, diet records, and FFQs. One study, which examined the comparability of portion size reports for the same foods using food models and equivalent two-dimensional pictures of those same models, found that although some respondents reported differently, no apparent bias in the direction of reporting was evident [288]. Another study using only photographs showed that, in general, small portion sizes were overestimated and large portion sizes underestimated, and that older subjects overestimated portion size more often than younger subjects [278].\n\n### B. Mode of Administration\n\nOne way to reduce the costs of collecting dietary information is to administer the instrument by telephone or by mail. Both telephone and mail surveys are less invasive than face-to-face interviews. The use of telephone surveys to collect dietary information has been reviewed [289]. Telephone surveys have higher response rates than do mail surveys [290], and have been used in a variety of public health research settings [291]. However, there is increasing concern about response rates in telephone surveys given increasing telemarketing and current technology, which allows for screening of calls via caller identification and message machines. For example, in the Behavioral Risk Factor Surveillance Surveys, response rates declined by approximately 10% between 1994 and 1998 (). Nevertheless, interviews by telephone can be substantially less expensive than face-to-face interviews, but cost comparisons vary with the research setting. The difficulty of reporting serving sizes by telephone can be eased by mailing picture booklets or other portion size estimation aids to the participants prior to the telephone interview.\n\nThere is current interest in evaluating the quality of data from telephone versus in-person 24-hour dietary recall interviews. Several studies have found substantial but imperfect agreement between dietary data collected by telephone and dietary data estimated by other methods, including face-to-face interviews [31, 36, 292\u2013294], expected intakes [295], or observed intakes [296]. Recognition of the potential economic advantages of telephone interviewing led to its adoption in the CSFII survey conducted in 1985 and 1986, and in the Third NHANES (NHANES III), with collection of one in-person and two telephone follow-up 24-hour recalls for respondents aged 50 years and older in phase I [297]. However, some segments of the population do not have telephones, and some persons will not answer their telephones under certain circumstances. Therefore, it is important to consider a dual sampling scheme or another scheme that accounts for this concern so potential respondents who do not have telephones are included [291].\n\n### C. Multiple Days of Diet Information\n\nWhen recalls or records are being used to estimate usual intake of individuals for surveillance purposes or to examine relationships between diet and disease, more than 1 day of dietary information is needed. Eating patterns vary between weekdays and weekends and across seasons, so multiple observations for individuals should include days in all parts of the week and in all seasons of the year. Nonconsecutive days are preferable to capture more of the variability in an individual's diet, since eating behaviors on consecutive days are correlated [298]. The number of days of information needed depends on which dietary parameter is being estimated, the extent of variability in the population, the research objectives, and the variability of the nutrient or food to be measured [299, 300]. For most nutrients, as many as 7\u201314 independent days of diet information may be necessary to characterize the individual's usual intake [298].\n\nIf the intent is only to generate a population distribution of a nutrient's intake (i.e., to separate within-person variability from total observed variability in order to estimate between-person variability), then only two days of recalls are needed on a sample. As part of an effort to estimate the distribution of usual diet in a particular population (e.g., the proportion eating diets with fewer than 30% of energy from fat), the National Center for Health Statistics collected three independent 24-hour recalls on a sample of older respondents in phase 1 of NHANES III [228]. The U. S. Department of Agriculture (USDA) has been collecting multiple days of dietary intake data since 1977. Various statistical methods to estimate the distribution of usual intakes from two or more days of dietary information have been suggested. One of these methods, developed by a National Academy of Sciences subcommittee [43], has been further developed by researchers at Iowa State University [44, 45] using computer software now available.\n\nWhile studies using records or recalls often use one or the other, a combined dietary recall and record approach, in which an initial interviewer-administered 24-hour recall is followed by self-administered 2- or 3-day records, was used by USDA in its 1977\u201378 and 1987\u201388 Nationwide Food Consumption Surveys and its Continuing Survey of Food Intakes by Individuals 1989\u201391 [221] and by others [301].\n\nThe costs of collecting and processing additional days of information include not only increased financial costs, but also reduced response rates as individuals tire of participating. All costs should be carefully considered relative to the benefits of collecting such information for the hypothesis of interest.\n\n### D. Choice of Nutrient Database\n\nIt is necessary to use a nutrient composition database when dietary data are to be converted to nutrient intake data. Typically, such a database includes the description of the food, a food code, and the nutrient composition per 100 g of the food. The number of foods and nutrients included varies with the database.\n\nSome values in nutrient databases are obtained from laboratory analysis; however, because of the high cost of laboratory analyses, many values are estimated based on conversion factors or other knowledge about the food [302]. In addition, accepted analytical methods are not yet available for some nutrients of interest [303], the analytical quality of the information varies with nutrient [223, 303], and the variances or ranges of nutrient composition of individual foods are in most cases unknown [304]. Rapid growth in the food processing sector and the global nature of the food supply add further challenges to estimating the mean and variability in the nutrient composition of foods.\n\nOne of the U.S. Department of Agriculture's (USDA) primary missions is to provide nutrient composition data for foods in the U.S. food supply, accounting for various types of preparation [305]. USDA produces and maintains the _Nutrient Database for Standard Reference,_ which includes information on up to 82 nutrients for 6200 foods. Interest in nutrients and food components potentially associated with diseases has led to development of databases for limited numbers of foods. These include databases for isoflavones, carotenoids, selected trans-fatty acids, sugar, and vitamin K. Values for selenium and vitamin D have been incorporated into the current Release 13 of the _Nutrient Database for Standard Reference._ Information regarding all of USDA's nutrient databases is available at the USDA's Nutrient Data Laboratory home page: . The International Network of Food Data Systems (INFOODS) [306] maintains an international directory of nutrient composition data, which is available at .\n\nResearch on nutrients (or other dietary constituents) and foods to improve current estimates is ongoing, and there is constant interest in updating current values and providing new values for a variety of dietary constituents of interest. Methods for converting dietary intake information into number of servings recommended in the U.S. Food Guide Pyramid [307] have been developed using the Minnesota Nutrition Data System [308] and the U.S. Department of Agriculture pyramid servings database [309]. One limitation in all nutrient databases, particularly for fatty acids, is the variability in the nutrient content of foods within a food category [310, 311]. For example, one study found that among 17 brands of crackers, _trans_ -fatty acid content varied from 1 to 13 g per 100 g of crackers [23]. Depending on the level of detail queried on the dietary assessment instrument, the respondent's knowledge of specific brand names, and the specificity of a particular nutrient database, estimating accurate fatty acid intake can therefore be problematic. For FFQs, this problem is compounded by the collapsing of foods into categories (such as crackers) that might have highly variable nutrient content.\n\nMany other databases are available in the United States for use in analyzing records, recalls, or FFQs, but most are based fundamentally on the USDA database, often with added foods and specific brand names. Estimates of nutrient intake from dietary recalls and records are often affected by the nutrient composition database that is used to process the data [312\u2013314]. Differences are due to the number of food items in the database, the recency of nutrient data, and the number of missing or imputed nutrient composition values. Therefore, before choosing a nutrient composition database, a prime factor to consider is the completeness and accuracy of the data for the nutrients of interest. For some purposes, it may be useful to choose a database in which each nutrient value for each food also contains a code for the quality of the data: e.g., analytical value, calculated value, imputed value, or missing. Investigators need to be aware that a value of zero is assigned to missing values in some databases. The nutrient database should also include weight\/volume equivalency information for each food item. Many foods are reported in volumetric measures (e.g., 1 cup) and must be converted to weight in grams. The number of common mixtures (e.g., spaghetti with sauce) available in the database is another important factor. If the study requires precision of nutrient estimates, then procedures for calculating the nutrients in various mixtures must be developed and incorporated into nutrient composition calculations. Another key consideration is how the database is maintained and supported.\n\nResearch or guidance on how to best compile a nutrient database for a FFQ or diet history is limited [56, 75, 315]. However, it is clear that an approach that is primarily data driven, using national or other carefully collected dietary data connected to a trustworthy nutrient database, is critical.\n\n### E. Choice of Computer Software\n\nCurrent computerized data processing requires creating a data file that includes a food code and an amount consumed for each food reported. Computer software then links the nutrient composition of each food on the separate nutrient composition database file, converts the amount reported to multiples of 100 g, multiplies by that factor, stores that information, and sums across all foods for each nutrient for each individual. Many computer packages have been developed that include both a nutrient composition database and software to convert individual responses to specific foods and, ultimately, to nutrients. Information on computer software packages can be found at the USDA's Food and Nutrition Information Center of the National Agricultural Library, which maintains a Food and Nutrition Software and Multimedia Programs database of more than 200 nutritional software programs. The programs cover dietary analysis, nutrition education, and other subjects. While the center does not loan the programs, visitors can come to the center and preview the software. Information regarding the center and other resources on computer software can be found on their web site, .\n\nSoftware should be chosen on the basis of the research needs, the level of detail necessary, the quality of the nutrient composition database, and the hardware and software requirements [316]. If precise nutrient information is required, it is important that the system be able to expand to incorporate information about newer foods in the marketplace and to integrate detailed information about food preparation (e.g., homemade stew) by processing recipe information. Sometimes the study purpose requires analysis of dietary data to derive intake estimates not only for nutrients, but also for food groups (e.g., fruits and vegetables), food components other than standard nutrients (e.g., nitrites), or food characteristics (e.g., fried foods). These additional requirements limit the choice of acceptable software.\n\nThe automated food coding system used for the USDA CSFII and upcoming NHANES is the SURVEY NET, developed by USDA's Agricultural Research Service and the University of Texas\u2013Houston School of Public Health [35]. SURVEY NET is a network dietary coding system that provides on-line coding, recipe modification and development, data editing and management, and nutrient analysis of dietary data with multiple-user access to manage the survey activities. It is available to government agencies and the general public only through special arrangement with USDA. However, most of the elements of SURVEY NET are available in a commercial software program called the Food Intake Analysis System available from the University of Texas at .\n\nMany diet history and food frequency instruments have also been automated. Users of these software packages should be aware of the source of information in the nutrient database and the assumptions about the nutrient content of each food item listed in the questionnaire.\n\n### F. Cognitive Research Related to Dietary Assessment\n\nNearly all studies using dietary information about subjects rely on the subjects' own reports of their diets. Because memory of these events is based on cognitive processes, it is important to understand and take advantage of what is known about how respondents remember dietary information and how that information is retrieved and reported to the investigator. The implications of such cognitive processes for dietary assessment have been researched and discussed by several investigators [183, 198, 266, 283, 317\u2013319].\n\nThere is an important distinction between episodic memory and generic memory. Episodic memory relies on particular memories about episodes of eating and drinking, while generic memory relies on general knowledge about the respondent's typical diet. A 24-hour recall relies primarily on episodic memory of all actual events in the very recent past, whereas a FFQ in which the respondent is asked to report the usual frequency of eating a food during the previous year relies primarily on generic memory. As the time between the behavior and the report increases, respondents may rely more on generic memory and less on episodic memory [318].\n\nWhat can the investigator do to enhance retrieval and improve reporting of diet? Research indicates that the amount of dietary information retrieved from memory can be enhanced by the context in which the instrument is administered and by use of specific memory cues and probes. For example, on a 24-hour dietary recall, foods that were not initially reported by the respondent can be recovered by interviewer probes. The effectiveness of these probes is well established and has been part of the interviewing protocols in two major national dietary surveys, the NHANES and the CSFII [34, 35]. Probes can be useful in improving generic memory, too, when subjects are asked to report their usual diets from periods in the past. Such probes can feature questions about past living situations and related eating habits.\n\nSocial scientists have long known that the way in which questions are asked affects responses. Certain characteristics of the interviewing situation may affect reporting according to social desirability for foods seen as \"good\" or \"bad.\" For example, the presence of other family members during the dietary interview probably enhances biases related to social desirability, especially for certain foods like alcoholic beverages. An interview in a health setting such as a clinic may also enhance biases related to social desirability for foods tied to compliance with dietary recommendations previously made for health reasons. In all instances, interviewers should be trained to refrain from either positive or negative feedback about good or bad dietary habits and should repeatedly encourage subjects to accurately report all foods.\n\n### G. Assessment of Nutritional Supplements\n\nIt is often important or useful in dietary assessments to assess the intake of nutritional supplements, such as vitamin and mineral pills. Intake of herbal, botanical, or other biologic supplements may also be of interest [320]. Recent data from the NHANES III 1988\u201394 show that about 40% of the population reported taking a vitamin or mineral supplement in the past month [321]. There has yet to be a nationally representative survey specifically designed to estimate the use of nonvitamin, nonmineral supplements in the United States, but estimates range from 8 to 20% [321\u2013323]. In addition, there are now a wide variety of botanical products also containing vitamins and\/or minerals; the array of these products and their use are changing rapidly. Their composition and bioavailability have not been documented, nor do we know how these products alter nutrient metabolism.\n\nSupplement intake can be assessed for the past 24 hours, or for several days, by including supplement use in 24-hour dietary recalls or food records. Alternatively, the research question may require assessment of usual supplement use over a longer period of time. This requires food frequency-type supplement questions, which provide less detail and are more prone to error [324].\n\nOf primary interest is the type of supplement taken. Hundreds of different products are available, both over the counter and by prescription. While it is possible to ask respondents to report in an open-ended manner which supplements they take, this information is difficult to code. An alternative approach is to ask close-ended questions about specific types that are of interest. Additional parameters need to be assessed for each brand of supplement taken: the amount per pill, the frequency of use, and the duration of use. A particular complexity in assessing supplement use is that many individuals take supplements inconsistently, in patterns that are hard to characterize (e.g., frequently for a while until they feel better or the bottle runs out, then not at all for a while, then irregularly). Usage can be so variable that assessment at one point in time may not reflect either long- or short-term usage [325, 326]. In many studies subjects are asked to have available all medications they take, including supplements, in order to better account for the types and amounts taken.\n\n### H. Validation\/Calibration Studies\n\nIt is important and desirable that any new dietary assessment method be validated or calibrated against other more established methods [126, 127, 129, 327]. The purpose of such studies is to better understand how the method works in the particular research setting and to use that information to better interpret results from the overall study. For example, if a new food frequency questionnaire or brief assessment questionnaire is to be used in the main study, results of that questionnaire should be compared to results from another dietary assessment method, for example, 24-hour dietary recalls or a more detailed food frequency questionnaire, conducted on the same individuals, and a biological marker such as doubly labeled water or urinary nitrogen, if possible. The National Cancer Institute maintains a register of validation\/calibration studies and publications on the web at [328].\n\nValidation studies yield information about how well the new method is measuring what it is intended to measure, and calibration studies use the same information to relate (calibrate) how one method of dietary assessment compares to a reference method. Validation\/calibration studies are challenging because of the difficulty and expense in collecting independent dietary information. Some researchers have used observational techniques to establish true dietary intake [52, 264, 267, 329]. Others have used laboratory measures such as the 24-hour urine collection to measure protein, sodium, and potassium intakes and the doubly labeled water technique to measure energy expenditure [13\u201319, 48, 49, 86, 106, 117, 118, 330]. However, the high cost of this latter technique can make it impractical for most studies. The over-all validity of energy intake estimates from the dietary assessment can be roughly checked by comparing weight data to reported energy intakes, in conjunction with use of equations to estimate basal metabolic rate [16, 21\u201323, 26, 29, 41, 51, 330\u2013332].\n\nBecause they are relatively expensive to conduct, validation\/calibration studies are done on small samples compared to the size of the main study. However, the sample should be sufficiently large to estimate the relationship between the study instrument and a reference method with reasonable precision. Increasing the numbers of individuals sampled and decreasing the number of repeat measures per individual (e.g., two nonconsecutive 24-hour recalls on 100 people rather than four recalls on 50 people) can often help to increase precision without extra cost [299]. To the extent possible, the sample should be chosen randomly, perhaps within strata, defined by either dietary or other variables.\n\nThe resulting statistics, which quantify the relationship between the new method and the reference method, can be used for a variety of purposes. Because, in most cases, both the reference method (usually records or recalls) and the new method are imperfect and subject to bias and error, measures such as correlation coefficients may underestimate the level of agreement with the actual usual intake. This phenomenon, referred to as _attenuation bias,_ can be estimated and the measure of agreement can be \"corrected\" to more nearly reflect the correlation between the diet measure and true diet [203, 206]. This information also gives guidance about the sample size required, because the less precise the diet measure, the more individuals will be needed to attain the desired power [203]. The estimated regression relationship between the new method and the reference method can also be used to adjust the relationships between diet and outcome as assessed in the larger study [100]. For example, the mean amounts of foods or nutrients, and their distributions, as estimated by a brief method, can be adjusted according to the calibration study results [160]. In addition, methods to adjust estimates of relationships measured in studies (e.g., relative risk estimates for disease relative to low nutrient intake) have been described [122, 123, 206, 333, 334]. Many of these adjustments require the assumption that the reference method is unbiased [122, 204]. Much evidence, however, indicates that intakes from recalls and records are underreported, violating this assumption. The possible existence of correlated person-specific biases between the reference and test instruments requires a rethinking of measurement error models in the future [125, 129, 335, 336].\n\n### I. Energy Adjustment\n\nMany researchers have suggested that when relationships between nutrient intakes and diseases are analyzed, nutrient intake should be adjusted for total energy intake. The rationale for this recommendation involves considering biological mechanisms, statistical confounding, and imprecision of measurements of nutrient intake. Biological mechanisms of the etiological relationship may present a compelling reason for the use of energy adjustment. Unfortunately, however, our understanding of these basic biological processes for many disease processes is limited.\n\nIn the absence of clear biological reasons, the potential for confounding is the most commonly cited reason for energy adjustment. Confounding would occur if energy intake is related both to the nutrient of interest and the outcome variable. Relationships between energy intake and various outcome variables may be unknown. However, total energy intake is related to many other dietary patterns, e.g., macronutrient intakes, meat intake, total grams of food (which might reflect exposure to contamination), and the intake of fiber, fruits, and vegetables. Because energy is derived from many nutrients, the adjustment of energy can camouflage a true nutrient effect [126]. This potential for overadjustment may be particularly important for macronutrients such as dietary fat.\n\nSeveral statistical methods for incorporating energy and other nutrients in the same model have been proposed [53, 123, 124, 337, 338]. These models have been reviewed and evaluated [119, 123, 124, 128, 339, 340]. Each approach is appropriate for addressing a different study question [340, 341]. Interpretation of each of the models requires assumptions about the relative importance of other variables. 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HFS-727. 3-12-1997Roe B.E., Derby B.M., Levy A.S. _Demographic, lifestyle and information use characteristics of dietary supplement user segment: Prepared for the Commission on Dietary Supplement Labeling_. Hyattsville, MD: U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition; 1997.\n\n323. Wood L. Herbal supplements attract new users. _Health Foods Busi_. 1997;43:101.\n\n324. Patterson R.E., Kristal A.R., Levy L., McLerran D., White E. Validity of methods used to assess vitamin and mineral supplement use. _Am. J. Epidemiol_. 1998;148:643\u2013649.\n\n325. Patterson R.E., Neuhouser M.L., White E., Kristal A.R., Potter J.D. Measurement error from assessing use of vitamin supplements at one point in time. _Epidemiology_. 1998;9:567\u2013569.\n\n326. Bates C.J., Prentice A., van der Pols J.C., Walmsley C., Pentieva K.D., Finch S., Smithers G., Clarke P.C. Estimation of the use of dietary supplements in the National Diet and Nutrition Survey: People aged 65 years and over. An observed paradox and a recommendation. _Eur. J. Clin. Nutr_. 1998;52:917\u2013923.\n\n327. Buzzard I.M., Sievert Y.A. Research priorities and recommendations for dietary assessment methodology. _Am. J. Clin. Nutr_. 1994;59:275s\u2013280s.\n\n328. Thompson F.E., Moler J.E., Freedman L.S., Clifford C.K., Stables G.J., Willett W.C. Register of dietary assessment calibration-validation studies: A status report. _Am. J. Clin. Nutr_. 1997;65:1142S\u20131147S.\n\n329. Baranowski T., Dworkin R., Henske J.C., Clearman D.R., Dunn J.K., Nader P.R., Hooks P.C. The accuracy of children's self-reports of diet: Family Health Project. _J. Am. Diet. Assoc_. 1986;86:1381\u20131385.\n\n330. Bingham S.A. The use of 24-h urine samples and energy expenditure to validate dietary assessments. _Am. J. Clin. Nutr_. 1994;59:227s\u2013231s.\n\n331. Samaras K., Kelly P.J., Campbell L.V. Dietary underreporting is prevalent in middle-aged British women and is not related to adiposity (percentage body fat). _Int. J. Obes. Relat. Metab. Disord_. 1999;23:881\u2013888.\n\n332. Eye Disease Case-Control Study GroupAjani U.A., Willett W.C., Seddon J.M. Reproducibility of a food frequency questionnaire for use in ocular research. _Invest. Ophthalmol. Vis. Sci_. 1994;35:2725\u20132733.\n\n333. Rosner B., Spiegelman D., Willett W.C. Correction of logistic regression relative risk estimates and confidence intervals for measurement error: The case of multiple covariates measured with error. _Am. J. Epidemiol_. 1990;132:734\u2013745.\n\n334. Paeratakul S., Popkin B.M., Kohlmeier L., Hertz-Picciotto I., Guo X., Edwards L.J. Measurement error in dietary data: implications for the epidemiologic study of the diet-disease relationship. _Eur. J. Clin. Nutr_. 1998;52:722\u2013727.\n\n335. Plummer M., Clayton D. Measurement error in dietary assessment: An investigation using covariance structure models. Part II. _Stat. Med_. 1993;12:937\u2013948.\n\n336. Plummer M., Clayton D. Measurement error in dietary assessment: An investigation using covariance structure models. Part I. _Stat. Med_. 1993;12:925\u2013935.\n\n337. Willett W., Stampfer M.J. Total energy intake: Implications for epidemiologic analyses. _Am. J. Epidemiol_. 1986;124:17\u201327.\n\n338. Howe G.R. Re: \"Total energy intake: Implications for epidemiologic analyses\" (letter). _Am. J. Epidemiol_. 1989;129:1314\u20131315.\n\n339. Kipnis V., Freedman L.S., Brown C.C., Hartman A., Schatzkin A., Wacholder S. Interpretation of energy adjustment models for nutritional epidemiology. _Am. J. Epidemiol_. 1993;137:1376\u20131380.\n\n340. Kushi L.H., Sellers T.A., Potter J.D., Nelson C.L., Munger R.G., Kaye S.A., Folsom A.R. Dietary fat and postmenopausal breast cancer [see comments]. _J. Natl. Cancer Inst_. 1992;84:1092\u20131099.\n\n341. Brown C.C., Kipnis V., Freedman L.S., Hartman A.M., Schatzkin A., Wacholder S. Energy adjustment methods for nutritional epidemiology: The effect of categorization. _Am. J. Epidemiol_. 1994;139:323\u2013338.\n\n* * *\n\nAdapted with permission from Thompson, F. E., and Byers, T. (1994). Dietary assessment resource manual. _J. Nutr._ **124** , 2245S-2318S. \u00a9 Journal of Nutrition, American Society for Nutritional Sciences.\nCHAPTER 2\n\nEnergy Requirement Methodology\n\nRACHEL K. JOHNSON and DEBRA COWARD-McKENZIE, The University of Vermont, Burlington, Vermont\n\n## I. INTRODUCTION\n\nKnowledge of energy requirements throughout the life cycle and during various physiological conditions and disease states is essential to the promotion of optimal human health. In the past, the measurement of energy intake served as an important tool from which to base energy requirements among all age groups [1]. The heavy reliance on such subjective forms of measurement, however, prompted research for more valid techniques to estimate energy needs [2]. The aim of this chapter is to familiarize the reader with contemporary techniques to measure energy expenditure. These include the highly sophisticated technique of doubly labeled water which allows nutrition scientists to accurately establish energy requirements based on the measurement of energy expenditure in free-living subjects.\n\n## II. COMPONENTS OF ENERGY EXPENDITURE\n\nEnergy is expended by the human body in the form of resting energy expenditure (REE), the thermic effect of food (TEF), and energy expended in physical activity (EEPA) [3]. These three components make up a person's daily total energy expenditure (TEE). Except in extremely active subjects, the REE constitutes the largest portion (60\u201375%) of the TEE [4]. The TEF represents approximately 10% of the total daily energy expenditure. The contribution of physical activity is the most variable component of TEE, which may be as low as 100 kcal\/day in sedentary people or as high as 3,000 kcal\/day in very active people (Fig. 1).\n\nFIGURE 1 The components of total energy expenditure. [From Poehlman, E. T., and Horton, E. S. (1988). Energy needs: Assessment and requirements in humans. _In_ \"Modern Nutrition in Health and Disease.\" William & Wilkins, Baltimore, MD.]\n\n### A. Resting Energy Expenditure\n\nResting energy expenditure is the energy cost of the physiological functions necessary to maintain homeostasis. These involuntary functions include respiration, cardiac output, body temperature regulation, and other functions of the sympathetic nervous system.\n\nThe term basal energy expenditure (BEE) is also used to describe this component of daily energy expenditure. BEE can be defined as the minimal amount of energy expended that is compatible with life. BEE is the amount of energy used in 24 hours by a person who is lying at physical and mental rest, at least 12 hours after the last meal, in a thermoneutral environment that prevents the activation of heat-generating processes, such as shivering. Basal metabolic rate (BMR) measurements are made early in the morning, before the person has engaged in any physical activity, and with no ingestion of tea or coffee or inhalation of nicotine-containing tobacco smoke for at least 12 hours before the measurement. If any of the conditions for BMR are not met, the energy expenditure should be termed the resting metabolic rate (RMR). For practical reasons, the BMR is rarely measured. In its place, RMR measurements are used, which in most cases, are higher than the BMR.\n\n#### 1. DETERMINANTS OF RESTING ENERGY EXPENDITURE\n\nThe determinants of resting energy expenditure are well established in both adults and children. The principal factors contributing to individual variation in REE include body size and composition, gender, age, physical fitness, hormonal status, and environmental influence [5\u20137].\n\n##### a. Body Size.\n\nLarger people have higher metabolic rates than do people of smaller size. A difference in weight of 10 kg would lead to a difference in RMR of approximately 120 kcal\/day in adult men or women, or a difference in total daily energy expenditure of approximately 200 kcal\/day in people with low levels of physical activity.\n\n##### b. Body Composition.\n\nFat-free mass (FFM) or lean body mass is the primary determinant of TEE in all age groups [8]. FFM is the metabolically active tissue in the body. Hence, most of the variation in REE between people can be accounted for by the variation in their FFM. FFM is in turn affected by other factors such as age, gender, and physical fitness. FFM can be accurately measured using a number of techniques. These include underwater weighing, measuring total body water using stable isotopes of deuterium or oxygen-18, and dual-energy x-ray absorptiometry (DXA). DXA is a novel scanning technique that accurately estimates fat-free mass, lean body mass, and bone mineral mass. Subjects lie supine on a padded table for 20\u201345 minutes during which time two very low energy (6.4 and 11.2 fJ) x-ray beams are passed through the body (Fig. 2; see color plate at the back of the book). Calculation of fat mass and lean body mass using these data are discussed in detail by Svendsen _et al._ [9]. The total x-ray dose is less than 1 mrem, on the order of a single day's background radiation, for a whole-body composition analysis. Thus, DXA provides an accurate measure of lean body mass and fat mass in a short time with a minimal radiation dose. Pregnancy tests should be performed before administering the DXA to further ensure safety.\n\nFIGURE 2 Dual-energy x-ray absorptiometry is a scanning technique that accurately estimates bone mineral, fat, and fat-free soft tissue.\n\nOften, due to the expense or impractical nature of research techniques for body composition, other, less accurate methods are often used in practice to estimate body composition. These include skinfold anthropometry, bioelectrical impedance, and near-infrared interactance. See Chapter 3 for a detailed description of body composition measurement techniques.\n\nIt has long been questioned whether people who are obese or at risk of developing obesity have reduced metabolic rates. Several studies have produced no evidence that children at high risk of obesity have lower metabolic rates than lean children when the measurements are adjusted for lean body mass [10, 11]. Hence, there is little evidence that a greater metabolic efficiency leads to the development of obesity.\n\n##### c. Sex\n\nSex is another factor that affects REE. The values for REE and 24-hour energy expenditure in a metabolic chamber are lower in female than in male subjects by approximately 50 kcal\/day [12, 13]. The reduced energy expenditure in female subjects is not explained by the confounding effects of the menstrual cycle because the effect is consistent in post-menopausal women as well as prepubertal girls [5, 7]. Women typically have more fat in proportion to muscle (FFM) than men and in general women have metabolic rates that are 5\u201310% lower than men of the same weight and height.\n\n##### d. Age.\n\nThere is a well-documented age-related reduction in resting metabolic rate [14]. Researchers have shown that older healthy people have RMRs that are significantly lower than younger people, even when adjusted for differences in fat mass and fat-free mass. This decline in REE can be partly explained by a reduction in the quantity, as well as the metabolic activity, of lean body mass [14].\n\n##### e. Physical Fitness.\n\nAthletes with greater muscular development show approximately a 5% increase in RMR over that in nonathletic individuals owing to their greater FFM. Habitual exercise has been shown to cause an 8\u201314% higher metabolic rate in men who were moderately and highly active, respectively, owing to increased FFM [15].\n\n##### f. Hormonal Status.\n\nHormonal status can impact metabolic rate, particularly in endocrine disorders, such as hyper-thyroidism and hypothyroidism, when energy expenditure is increased or decreased, respectively. Stimulation of the sympathetic nervous system, such as occurs during emotional excitement or stress, increases cellular activity by the release of epinephrine, which acts directly to promote glycogenolysis. Other hormones, such as cortisol, growth hormone, and insulin, also influence metabolic rate. Researchers have shown that serum leptin concentrations are a positive determinant of RMR [16].\n\nThe metabolic rate of adult females fluctuates with the menstrual cycle. An average of 359 kcal\/day difference in the BMR has been measured between its low point, about 1 week before ovulation at day 14, and its high point, just before the onset of menstruation. The mean increase in energy expenditure is about 150 kcal\/day during the second half of the menstrual cycle [17]. During pregnancy, RMR decreases in the early stages, whereas later in pregnancy, the metabolic rate is increased by the processes of uterine, placental, and fetal growth and by the mother's increased cardiac work [18].\n\n##### g. Environmental Influences.\n\nREE is affected by extremes in environmental temperature. People living in tropical climates usually have RMRs that are 5\u201320% higher than those living in a temperate area. Exercise in temperatures greater than 86\u00b0F also imposes a small additional metabolic load of about 5% owing to increased sweat gland activity. The extent to which energy metabolism increases in extremely cold environments depends on the insulation available from body fat and protective clothing.\n\n#### 2. MEASURING RESTING ENERGY EXPENDITURE: INDIRECT CALORIMETRY\n\nThe technique of indirect calorimetry has become the method of choice in most circumstances when a measurement of REE is needed. The term _indirect_ refers to the fact that energy (heat) production is determined by measuring O2 consumption and CO2 production rather than directly measuring heat transfer. The equipment varies but a ventilated hood system is most commonly used. A clear plastic hood is placed over the subject's head and made airtight around the neck (Fig. 3; see color plate at the back of the book). Indirect calorimetry has the advantage of mobility and low equipment cost and is frequently used in clinical settings to assess patients' energy requirements. Indirect calorimetry also provides quantitative information about the types of substrates that are oxidized [19]. The pretesting environment impacts the measurement of RMR. Outpatient-test experimental conditions have been shown to overestimate RMR by approximately 8% compared with inpatient measurements of RMR [20]. This factor should be taken into account when results are compared between laboratories and when daily energy requirements based on measures of REE are being evaluated.\n\nFIGURE 3 Measurement of resting metabolic rate using indirect calorimetry.\n\n#### 3. ESTIMATING RESTING ENERGY EXPENDITURE: HARRIS\u2013BENEDICT EQUATIONS\n\nThe Harris\u2013Benedict equations remain the most commonly used tool by clinicians when estimating people's REE. The equations are often used as a basis for prescribing energy intake for hospitalized patients and to formulate energy intake goals for weight loss. A review of the data used in the formulation of the Harris\u2013Benedict equations in the early 1930s deduced that the methods and conclusions of Harris and Benedict appear valid but not error free [21]. Only between 50 and 75% of the variability in REE is explained by the equations, but subsequent equations have not generally improved on this level of error [21]. The equations in use today are shown in Table 1. For obese people, the Harris\u2013Benedict equation can be applied up to a body mass index of 35\u201340 [21].\n\nTABLE 1\n\nPredicting Resting Energy Expenditure Using the Harris\u2013Benedict Equations\n\n_Note:_ Weight is in kilograms, height is in centimeters, and age is in years.\n\n_Source:_ Frankenfield, D. C., Muth, E. R., and Rowe, W. A. (1998). The Harris\u2013Benedict studies of human basal metabolism: History and limitations. _J. Am. Diet. Assoc._ **98,** 439\u2013445.\n\nREE has been characterized for a variety of various disease states and physiological conditions. These include burns [22], anorexia nervosa [23], severe central nervous system impairment [24], cerebral palsy [25], pregnancy [26], and lactation [27]. In addition REE has been studied in both children [5] and the elderly [28, 29]. Clinicians should not assume that prediction equations such as the Harris\u2013Benedict equation, which was developed in normal, healthy people, are valid in these special populations.\n\n### B. Thermic Effect of Food\n\nThe thermic effect of food (TEF) is the increase in energy expenditure associated with the consumption of food. It accounts for approximately 10% of TEE [30]. TEF is also termed _diet-induced thermogenesis._ TEF can be divided into obligatory and facultative thermogenesis. Obligatory thermogenesis is the energy needed to digest, absorb, and metabolize nutrients. Facultative thermogenesis is the excess energy expended above the obligatory thermogenesis attributable to metabolic inefficiency. TEF is not measured in clinical settings, but rather estimated as 10% of TEE. Hence, the equation:\n\nTEE = (RMR + EEPA) \u00d7 1.10\n\nwhere TEE = total energy expenditure, RMR = resting energy expenditure, EEPA = energy expended in physical activity, and the factor of 1.10 accounts for the TEF in the equation.\n\n#### 1. DETERMINANTS OF THERMIC EFFECT OF FOOD\n\nThe composition of the diet affects the TEF, with TEF being greater after carbohydrate and protein consumption in comparison with fat. This is accounted for by the differences in metabolic efficiency when metabolizing carbohydrate and protein versus fat. Fat is efficiently metabolized and stored with only 4% wastage, compared with 25% inefficiency when carbohydrate is converted to fat for storage. These differences have been hypothesized to contribute to the obesity-promoting characteristics of high-fat diets [31].\n\nOther dietary factors are known to impact TEF. For example, spicy foods such as chili and mustard have been shown to increase the metabolic rate significantly in comparison with unspiced meals. The effect was prolonged, lasting up to 3 hours [32]. Caffeine has been shown to increase TEF by 8\u201311% [33]. Other factors such as nicotine and cold also stimulate the TEF [34].\n\n### C. Energy Expended in Physical Activity\n\nEnergy expended in physical activity is the most variable component of total energy expenditure. EEPA includes energy expended in voluntary exercise, which include activities of daily living (bathing, feeding, and grooming, for example), sports and leisure, and occupational activities. EEPA also includes the energy expended in nonexercise activity thermogenesis (NEAT), which is associated with fidgeting, maintenance of posture, and other physical activities of daily life. The share of total energy expenditure accounted for by physical activity is obviously greater for active individuals [35]. It can vary from 10% in a bedridden person to as high as 50% or more of TEE in athletes. Because of the alarmingly high rates of obesity in the United States, increasing EEPA through voluntary physical activity is being stressed as an effective way of achieving healthy weight [36].\n\nNEAT can add significantly to a person's energy expenditure. Researchers found that when nonobese volunteers were fed 1000 kcal\/day in excess of weight-maintenance requirements for 8 weeks, two-thirds of the increase in total daily energy expenditure was due to increased NEAT. Changes in NEAT accounted for 10-fold differences in fat storage that occurred and directly predicted resistance to fat gain with overfeeding. These results suggest that as humans overeat, activation of NEAT dissipates excess energy to preserve leanness and that failure to activate NEAT may result in fat gain [37].\n\nA novel study suggested that gum chewing is sufficiently exothermic that it can lead to a meaningful increase in energy expenditure over time. Chewing gum led to a mean increase in energy expenditure of 11 \u00b1 3 kcal\/hour in seven nonobese volunteers with stable weight. The researchers speculated that if a person chewed gum during waking hours and changed no other components of energy balance, a yearly loss of more than 5 kg of body fat could be realized [38].\n\n#### 1. DETERMINANTS OF ENERGY EXPENDED IN PHYSICAL ACTIVITY\n\nDifferences in EEPA are due both to patterns of activity (both voluntary and involuntary) as well as the body composition that results from that pattern of activity [39]. Hence, the level of a person's fitness will affect EEPA, due to the increased lean body mass and metabolically active tissue.\n\nIn general, EEPA declines with age. Because the decline in EEPA is often disproportionately greater than the decline in energy intake, positive energy balance results. This often leads to increased total and central body fatness, a loss of muscle mass, and a greater predisposition to comorbidities associated with obesity and physical inactivity [40]. Fortunately, it has been demonstrated that regular aerobic exercise can successfully increase EEPA in middle-aged men and hence raise the daily energy requirements for weight maintenance [41]. Thus, regular exercise may counter the age-related tendency toward obesity\n\n#### 2. MEASURING ENERGY EXPENDED IN PHYSICAL ACTIVITY\n\nObtaining a valid and appropriate measurement of EEPA is a challenging task. There are subjective measures, such a questionnaires, which are widely used in epidemiological studies to assess physical activity in populations [35]. Objective activity assessment tools are also often used to validate the subjective activity measures. These include the doubly labeled water technique, movement counters, heart rate monitoring and graded exercise testing.\n\n##### a. Objective Measures of EEPA.\n\ni.: Doubly labeled water. EEPA can be estimated by determining the difference between total daily energy expenditure as measured by doubly labeled water and resting metabolic rate and thermic effect of food measured by indirect calorimetry. The advantages of the doubly labeled water (DLW) approach are that it requires little subject cooperation, is unobtrusive, and measures free-living activity throughout a person's daily routine. Unfortunately, the high price of the isotopes, mass spectrometer instrumentation, and the need for technical expertise in sample preparation and measurement have limited its widespread application. Nevertheless, DLW has been used as a gold standard for validating other methods to measure EEPA in free-living individuals [42]. DLW measurement of total energy expenditure is discussed in detail in the TEE section of this chapter.\n\nii.: Movement counters. Accelerometers are movement counters that can measure the occurrence of body movement and its intensity. Accelerometers cannot be used to measure the static component in exercises, like weight lifting or carrying loads. However, in normal daily life, it is assumed that the effect of static exercise on the total level of physical activity is negligible [43]. Currently, several accelerometers are available for the assessment of physical activity. The most widely available are the Caltrac (Hemokinetics, Madison, WI) and the Tritrac-R3 D accelerometer (Hemokinetics).\n\nThe Caltrac is a one-axial accelerometer for vertical movement. Energy expenditure is estimated by entering the subject's age, height, weight, and gender. Activity counts are displayed when predetermined constants are entered in place of the subject's personal data. The Caltrac was not found to be a meaningful predictor of EEPA in a group of free-living, school-aged children [44] or in older men and women [42]. The Caltrac may be useful, however, in supervised settings in which researchers want to assess children's levels of physical activity [45].\n\nThe triaxial accelerometer measures movement on three planes and possesses the ability to store extensive data within its internal computer for retrieval at a later time. The evidence to date suggests that a triaxial accelerometer provides a better estimate of usual physical activity or energy expenditure than a single-plane accelerometer. Studies have demonstrated that these instruments can be used to distinguish differences in activity levels between individuals and to assess the effect of interventions on physical activity within individuals [43].\n\niii.: Heart rate monitoring. The use of heart rate monitoring as a proxy measure for EEPA is based on the principle that heart rate and oxygen consumption (Vo2) tend to be linearly related throughout a large portion of the aerobic work range. When this relationship is known, the exercise heart rate can be used to estimate Vo2 [46]. Heart rate monitoring is relatively inexpensive, can assess people in a free-living state, and has the potential for providing information on the pattern as well as the total level of energy expenditure [47]. The technique has been successfully applied in small groups of children, lactating women, normal adults, athletes, and in remote indigenous populations [46]. Wareham and colleagues have confirmed its feasibility for assessing the pattern and total level of energy expenditure in the epidemiological context [47]. However, users of the technique must be cognizant of the uncertainties and likely sources of error in its use.\n\n##### b. Physical Activity Questionnaires.\n\nPhysical activity questionnaires have been used in many studies because they are easy to administer to large numbers of people and do not intrude on people's everyday activities. Although questionnaires do not provide precise estimates of EEPA, they may be helpful in ranking groups of subjects from the least to the most active. The ranking can then be used to correlate activity levels with disease outcomes [37]. An accurate questionnaire is both reliable and valid. A reliable questionnaire consistently provides similar results under the same circumstances while a valid questionnaire truly measures what it was designed to measure. The validity of a physical activity questionnaire should be determined by comparing it with an objective measure of EEPA such as the doubly labeled water method. Starling and colleagues [42] found that the Yale Physical Activity Survey estimates of EEPA compared favorably with DLW on a group basis. However, its use as a proxy measure for individual EEPA is limited. In the same study, the Minnesota Leisure Time Physical Activity Questionnaire significantly underestimated EEPA in free-living older men and women [42]. This points out the importance of ascertaining the validity of a questionnaire before applying it in large epidemiological studies. The latest versions of most of the popular physical activity questionnaires, along with descriptions for their use, are available [48].\n\n## III. TOTAL ENERGY EXPENDITURE\n\nTotal energy expenditure is composed of the energy required for both unconscious and conscious activities. As already mentioned, it consists of three distinct parts: resting energy expenditure, thermic effect of food, and the energy expended in physical activity. TEE estimates energy requirements, assuming that people are in energy balance [6, 49]. The state of energy balance requires that, to maintain a specific weight, body energy stores must remain constant, that is, energy intake must match total energy expenditure. Thus, in significant weight gain or loss, when body composition is changing, metabolizable energy intake is not an accurate predictor of energy expenditure. Therefore, changes in body composition must be monitored for the accurate determination of energy expenditure.\n\n### A. Measuring Total Energy Expenditure\n\n1. DIRECT CALORIMETRY\n\nDirect calorimetry obtains a direct measurement of the amount of heat generated by the body within a structure large enough to permit moderate amounts of activity. These structures are called whole-room calorimeters. Direct calorimetry provides a measure of energy expended in the form of heat. The technique of direct calorimetry has several disadvantages. The structure is costly, requires complex engineering, and appropriate facilities are scarce around the world. Subjects must remain in a physically confined environment for long periods. In addition, direct calorimetry does not provide any information about the nature of substrates that are being oxidized to generate energy within the body [19].\n\n#### 2. DOUBLY LABELED WATER\n\nThe introduction of the DLW technique for human use in 1982 by Schoeller and van Santen [50] provided a scientific breakthrough in the measurement of TEE in free-living humans. The method was originally described by Lifson _et al._ [51] in the 1940s. It is based on the principle that carbon dioxide production can be estimated by the difference in elimination rates of body hydrogen and oxygen. Through his observations, Lifson concluded that the oxygen in expired carbon dioxide was derived from total body water [52]. This results from the equilibrium between the oxygen in body water and the oxygen in respiratory carbon dioxide [51]. With this finding, Lifson and colleagues predicted that carbon dioxide production could be indirectly measured by separately labeling both the hydrogen and oxygen pool of the body water with naturally occurring, stable isotopes. Based on the theory that the hydrogen of body water will exit as water and the oxygen of body water as carbon dioxide and water, the differential elimination rates of hydrogen and oxygen will indicate the amount of carbon dioxide expired during a given period. Carbon dioxide production can then be equated with energy expenditure under the assumption that carbon dioxide is an endproduct of substrate metabolism. Therefore, carbon dioxide production can be converted to an estimate of oxygen production using a food quotient estimated from total dietary intake.\n\nThe term _doubly labeled water_ indicates that the water consists of isotopic forms of hydrogen and oxygen. Deuterium oxide (2H2O) is the isotope that labels the hydrogen component of body water, and oxygen\u201318 (H218O) is the isotope that labels the oxygen component of body water. Together, these isotopes label the hydrogen and oxygen of body water and trace their path within the body during the course of the study. The levels of the isotopes can be determined by periodic sampling of body water through urine, saliva, or plasma. Before a preweighed loading dose of DLW is orally administered, a baseline measurement of body water is obtained to detect the amount of deuterium oxide and oxygen\u201318 already present within the body water. After the baseline sample collection and administration of the DLW, two samples of body water are collected several days apart (usually 10\u201314 days) to determine the elimination rates of the isotopes. This two-point method has been shown to minimize the errors encountered from daily variations in the rate of carbon dioxide production [53].\n\n##### a. Assumptions of the Doubly Labeled Water Method.\n\nAs with all scientific methodology, assumptions are made that need to be understood when using the DLW technique. A few of the fundamental assumptions are examined here. First, it is assumed that there is a constant total body water pool in which the isotopes turn over. However, it is known that with certain conditions such as infancy or intense exercise, the body water pool may change because of increases or decreases in body mass. This change in body mass must be greater than 15%, however, to produce an error in the total energy expenditure measure of less than 1\u20132% [54].\n\nSecond, the rate of flux for carbon dioxide and water should remain constant under steady-state kinetics. However, because water intake and physical activity are episodic in nature, this rate of flux is not constant. By implementing the previously mentioned two-point sample collection method, an average carbon dioxide production rate can be obtained over the measurement period without encountering the daily fluctuations that will contribute to error [54].\n\nAnother assumption states that no carbon dioxide or water enters the body via the skin or lungs. However, when a subject is exposed to higher-than-normal levels of carbon dioxide, such as cigarette smoke, there may be an apparent increase in carbon dioxide production. Nonetheless, to produce a 3\u20136% error in the estimation of true carbon dioxide production, the person would have to smoke roughly three packs of cigarettes a day [54]. In addition, water vapor can easily penetrate the lungs and skin to be directly absorbed into the body. However, Pinson and Langham [55] demonstrated that this type of exchange does not affect the calculation of carbon dioxide production, because the elimination rates of hydrogen and oxygen are proportional. Other assumptions of the method are associated with the isotopic dilution spaces and isotopic fractionations. These are beyond the scope of this chapter but are thoroughly reviewed by Schoeller [54] and Wolf [56].\n\n##### b. Advantages and Disadvantages of Doubly Labeled Water.\n\nThe DLW technique has numerous advantages that make it the ideal method for measuring total daily energy expenditure in a variety of populations. The advantages to the researcher and the subject are the ease of administration and the ability of the subject to engage in free-living activities during the measurement period. This is extremely advantageous in infants, young children, the elderly, and populations with disabilities that cannot be subjected to the rigorous testing involved in the measurement of oxygen consumption during various activities. The DLW technique provides the objective criterion measure necessary to validate more subjective estimates of energy expenditure such as activity logs and diet recalls. Most important, the method is accurate and has a precision of between 2 and 8% [54].\n\nAlthough there are many advantages to the DLW technique, there are also drawbacks, namely, the expense of the stable isotope (approximately $500 to dose an average weight adult), periodic worldwide shortages of oxygen\u201318, and the expertise required to operate the highly sophisticated and costly mass spectrometer for analysis of the enrichments (Fig. 4; see color plate at the back of the book). The advantages and disadvantages of the DLW technique are highlighted in Table 2.\n\nTABLE 2\n\nAdvantages and Disadvantages of the Doubly Labeled Water Technique to Measure Total Energy Expenditure\n\nAdvantages | Disadvantages \n---|--- \nNoninvasive, unobstrusive, and easily administered | Availability and expense of oxygen-18 (approximately $900 for 70-kg adult) \nMeasurement performed under free-living conditions over extended time period (7\u201314 days) | Reliance on isotope ratio mass spectrometry for analysis of samples \nAccurate and precise (2\u20138%) | A direct measure of CO2 production, and not energy expenditure, does not measure substrate oxidation \nCan be used to estimate activity energy expenditure when combined with measurement of resting metabolic rate | Not suitable for large-scale epidemiological studies\n\nFIGURE 4 Mass spectrometer used for the analysis of isotope enrichments in the DLW method.\n\n### B. Estimating Total Energy Expenditure\n\nThe introduction of the DLW technique in humans has produced a large and robust database of TEE measurements in a variety of populations. A meta-analysis of 574 DLW measurements helped to establish the average and range of habitual energy expenditures in different age and sex groups [57]. The data shown in Table 3 are compiled from healthy, free-living people aged 2\u201395 years. These data provide a frame of reference for energy needs in the general population and can be used to evaluate other estimates of energy expenditure. Special circumstances such as illness or enforced exertion were excluded from this database.\n\nTABLE 3\n\nTotal Energy Expenditure across Age and Sex Groups from a Meta-Analysis of 574 Free-Living DLW Measurements\n\nDue to the high cost of and small numbers of laboratories with the capacity to do DLW studies, clinicians continue to rely heavily on prediction equations to estimate energy requirements. As more measurements of TEE are conducted using doubly labeled water, increasingly accurate tools to estimate TEE should become available. In the meantime, clinicians and researchers typically use prediction equations to estimate energy requirements. The steps used to calculate total energy expenditure using prediction formulas are shown in Table 4.\n\nTABLE 4\n\nEstimating Total Energy Expenditure Using Prediction Equations\n\n_Source:_ Adapted from Shetty, P. S., Henry, C. J. K., Black, A. E., and Prentice, A. M. (1996). Energy requirements of adults: An update on basal metabolic rates and physical activity levels. _Eur. J. Clin. Nutr._ **50** (suppl 1), S11\u2013S23.\n\n### C. Total Energy Expenditure in Special Populations\n\nThe number of studies using DLW to examine TEE in various disease states, physiological conditions, and across the life cycle have proliferated during the past decade. Hence, data are now available on energy expenditure during infancy [58], childhood [59], adolescence [60], and in the elderly [61, 62]. TEE during pregnancy and lactation has been well characterized in some elegant longitudinal studies [18, 63]. In addition TEE has been examined in adults and children with obesity [64, 65], burns [66], cerebral palsy [67], Down syndrome [68], HIV\/AIDs [69], and Alzheimer's disease [70]. The effects of these various conditions on energy requirements are highlighted in Table 5.\n\nTABLE 5\n\nEffects of Disease States and Physiological Conditions on Energy Requirements: Results from Doubly Labeled Water Studies\n\nDisease or condition | Effect on energy requirements | Explanation \n---|---|--- \nObesity | Increased | Increased fat-free mass coupled with decreased physical activity \nBurned children | No change | Increased resting metabolic rate counteracts decreased physical activity \nAnorexia | Increased | To counteract increased physical activity with underweight and hypometabolism \nHIV\/AIDS | No change | Energy expenditure not elevated, reduced energy intake causes weight loss \nCerebral palsy | Relative to individual | High interindividual variation in energy expended in physical activity; ambulation status an important predictor. \nAlzheimer's | No change | Energy expenditure not elevated, low-energy intake predisposes to weight loss \nSpinal cord injury | Decreased | Lower energy expended in physical activity, resting metabolic rate, and thermic effect of food \nPregnancy | Relative to individual | No prediction of metabolic response \nLactation | Increased | Energy cost of milk production partially offset by reduced physical activity\n\n## IV. RECOMMENDED ENERGY INTAKES\n\nClassically the Recommended Dietary Allowances (RDAs) have been used as a guide to determine energy intakes for groups of normal, healthy people. The last RDA for energy was set in 1989. RDAs for all nutrients except energy are set at levels well above those estimated to minimize the occurrence of deficiency syndromes. For energy, this obviously is not the case. Recommendations for energy have always been set as an average of energy requirements for a population group. In the past recommended energy intakes have relied heavily on data from dietary surveys that estimate energy intake. This is based on the assumption that people are in energy balance at the time of measurement and that the estimates of energy intake are valid. A large body of evidence now demonstrates that self-reported estimates of food intake do not provide accurate or unbiased estimates of people's energy intake and that underreporting of food intake is pervasive in dietary studies 71]. Underreporting is discussed in detail in [Chapter 5. It is anticipated that the forthcoming dietary reference intakes for energy will rely heavily on objective measurements of energy expenditure in free-living people to provide more accurate recommendations for energy intake in different age and sex groups and during various physiological conditions (e.g., pregnancy and lactation). In addition, as the prevalence of obesity has reached epidemic proportions in the United States, people's energy expended in physical activity has become so low that it may become increasingly difficult to meet micronutrient needs on the energy intakes required to keep people in energy balance. Hence, it will become increasingly imperative that emphasis be placed on increased energy expended in physical activity for the maintenance of optimal health.\n\nReferences\n\n1. World Health Organization. Energy and protein requirements. _WHO Tech. Rep. Ser_. 1985:724.\n\n2. Schoeller D.A., Racette S.B. A review of field techniques for the assessment of energy expenditure. _J. Nutr_. 1990;120:1492\u20131495.\n\n3. Hildreth H.G., Johnson R.K. The doubly labeled water technique for the determination of human energy requirements. _Nutr. Today_. 1995;30:254\u2013260.\n\n4. Poehlman E.T. Regulation of energy expenditure in aging humans. _Geriatr. Biosci_. 1993;41:552\u2013559.\n\n5. Goran M.I., Kaskoun M., Johnson R.K. Determinants of resting energy expenditure in young children. _J. Pediatr_. 1994;125:362\u2013367.\n\n6. Poehlman E.T. A review: Exercise and its influence on resting energy metabolism in man. _Med. Sci. Sports Exerc_. 1989;21:515\u2013525.\n\n7. Ravussin E., Lilloja S., Anderson T.E., Christin L., Bogardus C. Determinants of 24-hour energy expenditure in man. _J. Clin. Invest_. 1988;78:1568\u20131578.\n\n8. Weinsier R.L., Schutz Y., Bracco D. 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Krahn D.D., Rock C., Dechert R.E., Nairn K.K., Hasse S.A. Changes in resting energy expenditure and body composition in anorexia nervosa patients during refeeding. _J. Am. Diet. Assoc_. 1993;93:433\u2013438.\n\n24. Bandini L.G., Puelzi-Quinn H.M., Morelli J.A., Fukagawa N.K. Estimation of energy requirements in persons with severe central nervous system impairment. _J. Pediatr_. 1995;126:828\u2013832.\n\n25. Johnson R.K., Goran M.I., Ferrar M.S., Poehlman E.T. Athetosis increases resting metabolic rate in adults with cerebral palsy. _J. Am. Diet. Assoc_. 1996;96:145\u2013148.\n\n26. Goldberg G.R., Prentice A.M., Coward W.A., Davies H.I., Murgatroyd P.R., Wensing C., Black A.E., Harding M., Sawyer M. Longitudinal assessment of energy expenditure in pregnancy by the doubly labeled water method. _Am. J. Clin. Nutr_. 1993;57:494\u2013505.\n\n27. Spaaij C., van Raaij J., de Groot L., van der Heijden L., Boeholt H.A., Hautvast J.C. Effect of lactation on resting metabolic rate and on diet- and work-induced thermogenesis. _Am. J. Clin. Nutr_. 1994;59:42\u201347.\n\n28. Arciero P.J., Goran M.I., Gardner A.M., Ades P.A., Tyzbir R.S., Poehlman E.T. A practical equation to predict resting metabolic rate in older females. _J. Am. Geriatr. Soc_. 1993;41:389\u2013395.\n\n29. Arciero P.J., Goran M.I., Gardner A.M., Ades P.A., Poehlman E.T. A practical equation to predict resting metabolic rate in older men. _Metab. Clin. Exper_. 1993;42:950\u2013957.\n\n30. Poehlman E.T., Horton E.S. Energy need: Assessment and requirements in humans. In: Bloch A.S., Shils M.E., eds. _Modern Nutrition in Health and Disease_. Baltimore: Williams & Wilkins, 1998.\n\n31. Prentice A.M. All calories are not equal. _Int. Dialogue Carbohydr_. 1995;5(4):1\u20133.\n\n32. McCrory P. Energy balance, food intake and obesity. In: Hills A.P., Wahlqvist M.L., eds. _Exercise and Obesity_. London: Smith-Gordon and Co., 1994.\n\n33. Dulloo A.G., Geissler C.A., Horton T., Collins A., Miller D.S. Normal caffeine consumption: Influence on thermogenesis and daily energy expenditure in lean and post-obese human volunteers. _Am. J. Clin. Nutr_. 1989;49:44\u201350.\n\n34. Hofstetter A. Increased 24-hour energy expenditure in cigarette smokers. _N. Engl. J. Med_. 1986;314:79\u201382.\n\n35. Kriska A.M., Caspersen C.J. Introduction to a collection of physical activity questionnaires. _Med. Sci. Sports Exer_. 1997;29:S5\u2013S9.\n\n36. Pate R.R., Prat M., Blair S.N., Haskell W.L., Macera C.A., Bouchard C., Buchner D., Ettinger W., Heath G.W., King A.C., Kirska A., Leon A.S., Marcus B.H., Morris J., Paffenbarger R.S., Patrick K., Pollack M.L., Rippe J.M., Sallis J., Wilmore J.H. Physical activity and public health\u2014A recommendation from the Centers for Disease Control and Prevention and the American College of Sports Medicine. _J. Am. Med. Assoc_. 1995;273:402\u2013407.\n\n37. Levine J.A., Eberhardt N.L., Jensen M.D. Role of nonexercise activity thermogenesis in resistance to fat gain in humans. _Science_. 1999;283(5399):212\u2013214.\n\n38. Levine J., Baukol P., Pavlidis I. The energy expended in chewing gum. _N. Engl. J. Med_. 1999;341(27):2100.\n\n39. Food and Nutrition Board, National Research Council, National Academy of Sciences. _Recommended Dietary Allowances_ , 10th ed. National Academy Press; 1989.\n\n40. Poehlman E.T. Effect of exercise on daily energy needs in older individuals. _Am. J. Clin. Nutr_. 1998;68:997\u2013998.\n\n41. Bunyard L.B., Katzel L.I., Busby-Whitehead J., Wu Z., Goldberg A.P. Energy requirements of middle-aged men are modifiable by physical activity. _Am. J. Clin. Nutr_. 1998;68:1136\u20131142.\n\n42. Starling R.D., Matthews D.E., Ades P.A., Poehlman E.T. Assessment of physical activity in older individuals: A doubly labeled water study. _J. Appl. Physiol_. 1999;86(6):2090\u20132096.\n\n43. Westerterp K.R. Physical activity assessment with accelerometers. _Int. J. Obes_. 1999;23:S45\u2013S49.\n\n44. Johnson R.K., Russ J., Goran M.I. Physical activity related energy expenditure in children by doubly labeled water as compared with the Caltrac accelerometer. _Int. J. Obes_. 1998;22:1046\u20131052.\n\n45. Sallis J.F., Buono J.F., Roby J.L., Carlson D., Nelson J. The Caltrac accelerometer as a physical activity monitor for school-age children. _Med. Sci. Sports Exer_. 1989;22:698\u2013703.\n\n46. Livingstone M.B.E. Heart-rate monitoring: The answer for assessing energy expenditure and physical activity in population studies? _Br. J. Nutr_. 1997;78:869\u2013871.\n\n47. Wareham N.J., Hennings S.J., Prentice A.M., Day N.E. Feasibility of heart-rate monitoring to estimate total level and pattern of energy expenditure in a population-based epidemiological study: The Ely young cohort feasibility study 1994\u20135. _Br. J. Nutr_. 1997;87:889\u2013900.\n\n48. Kriska A.M., Caspersen C.J., eds. A collection of physical activity questionnaires for health-related research. _Med. Sci. Sports Exer_. 1997;29:S3\u2013S205\n\n49. Poehlman E.T. Energy expenditure and requirements in aging humans. _J. Nutr_. 1992;122:2057\u20132065.\n\n50. Schoeller D.A., van Santen E. Measurement of energy expenditure in humans by the doubly labeled water method. _J. Appl. Physiol_. 1982;53:955\u2013959.\n\n51. Lifson N., Gordon G.B., Visscher M.B., Nier A.O. The fate of utilized molecular oxygen and the source of heavy oxygen of respiratory carbon dioxide, studied with the aid of heavy oxygen. _J. Biol. Chem_. 1949;180:803\u2013811.\n\n52. Lifson N., Gordon G.B., McClintock R. Measurement of total carbon dioxide production by means of D2O18. _J. Appl. Physiol_. 1955;7:704\u2013710.\n\n53. Welles S. Two-point vs. multipoint sample collection for the analysis of energy expenditure by use of the double labeled water method. _Am. J. Clin. Nutr_. 1990;52:1134\u20131138.\n\n54. Schoeller D.A. Measurement of energy expenditure in free-living humans by using doubly labeled water. _J. Nutr_. 1988;118:1278\u20131289.\n\n55. Pinson E.A., Langham W.H. Physiology and toxicology of tritium in man. _J. Appl. Physiol_. 1957;10:108\u2013126.\n\n56. Wolf R.R. Radioactive and Stable Isotope Tracers in Biomedicine Principles and Practice of Kinetic Analysis. Washington, DC: Wiley-Liss; 1992:207\u2013234.\n\n57. Black A.E., Coward W.A., Cole R.J., Prentice A.M. Human energy expenditure in affluent societies: An analysis of 574 doubly-labeled water measurements. _Eur. J. Clin. Nutr_. 1996;50:72\u201392.\n\n58. Davies P.S.W. Energy requirements for growth and development in infancy. _Am. J. Clin. Nutr_. 1998;68:939S\u2013943S.\n\n59. Goran M.I., Gower B.A., Nagy T.R., Johnson R.K. Developmental changes in energy expenditure and physical activity in children: Evidence for a decline in physical activity in girls before puberty. _Pediatrics_. 1998;101:887\u2013891.\n\n60. Bratteby L.E., Snadhagen B., Fan H., Enghardt H., Samuelson G. Total energy expenditure and physical activity as assessed by the doubly labeled water method in Swedish adolescents in whom energy intake was underestimated by 7-d records. _Am. J. Clin. Nutr_. 1998;67:905\u2013911.\n\n61. Goran M.I., Poehlman E.T. Total energy expenditure and energy requirements in healthy elderly persons. _Metabolism_. 1992;41:744\u2013753.\n\n62. Roberts S.B., Young V.R., Fuss P., Heyman M.B., Fiatarone M., Dallal G.E., Cortiella J., Evans W.J. What are the dietary energy needs of elderly adults? _Int. J. Obes_. 1992;16:969\u2013976.\n\n63. Goldberg G.R., Prentice A.M., Coward W.A., Davies H.L., Murgatroyd P.R., Wensing C., Black A.E., Harding M., Sawyer M. Longitudinal assessment of energy expenditure in pregnancy by the doubly labeled water method. _Am. J. Clin. Nutr_. 1993;57:494\u2013505.\n\n64. Lichtman S.W., Krystyna P., Berman E.R., Pestone M., Dowling H., Offenbacher E., Weisel H., Heshka S., Matthews D.E., Heymsfield S.B. Discrepancy between self-reported and actual caloric intake and exercise in obese subjects. _N. Engl. J. Med_. 1992;327:1893\u20131998.\n\n65. Goran M.I., Shewchuk R., Bower B.A., Nagy T.R., Carpenter W.H., Johnson R.K. Longitudinal changes in fatness in white children: No effect of childhood energy expenditure. _Am. J. Clin. Nutr_. 1998;67:309\u2013316.\n\n66. (Pt 1)Goran M.I., Peters E.J., Herndon D.N., Wolfe R.R. Total energy expenditure in burned children using the doubly labeled water technique. _Am. J. Physiol_. 1990;259:E576\u2013E585.\n\n67. Johnson R.K., Hildreth H.G., Contompasis S.H., Goran M.I. Total energy expenditure in adults with cerebral palsy as assessed by double labeled water. _J. Am. Diet. Assoc_. 1997;97:966\u2013970.\n\n68. Luke A., Roizen N.J., Sutton M., Schoeller D.A. Energy expenditure in children with Down syndrome: Correcting metabolic rate for movement. _J. Pediatr_. 1994;125:829\u2013838.\n\n69. Macallan D.C., Noble C., Baldwin C., Jebb S.A., Prentice A.M., Coward A. Energy expenditure and wasting in human immunodeficiency virus infection. _N. Engl. J. Med_. 1995;333(2):83\u201388.\n\n70. Toth M.J., Goran M.I., Carpenter W.H., Newhouse P., Rosen C.J., Poehlman E.T. Daily energy expenditure in free-living non-institutionalized Alzheimer's patients: A doubly labeled water study. _Am. Acad. Neurol_. 1997;48:997\u20131002.\n\n71. Johnson R.K. What are people _really_ eating? _Nutr. Today_. 2000;35:40\u201346.\nCHAPTER 3\n\nPhysical Assessment\n\nEDWARD SALTZMAN12 and KRIS M. MOGENSEN2\n\n1Tufts University, Boston, Massachusetts\n\n2New England Medical Center, Boston, Massachusetts\n\n## I. INTRODUCTION\n\nThe purpose of clinical evaluation is to assess current health and nutritional status, to identify those nutritional and health factors that determine current status, and to provide prognostic data for risk of further nutrition-related morbidity. The mutual influence of nutrition and disease on each other is critical, and at times distinguishing between the ill effects of disease and malnutrition may be difficult or impossible.\n\nAssessment is guided by the symptoms and signs of disease. Risk for disease and etiologic factors is often revealed by demographic, occupational, and socioeconomic conditions as well as by family histories. On a population level, or in apparently healthy persons with low risk, screening may be used in lieu of a more detailed and resource-intensive assessment. The results of screening may indicate the need for further assessment, and several screening tools have such staged approaches, as discussed later in this chapter. Individuals with existing, or at risk for, nutritional compromise or disease should undergo a more detailed assessment. Indications and tools for screening in contrast to detailed assessment have been reviewed in detail elsewhere [1].\n\nThe term _malnutrition_ is often used to describe protein energy malnutrition, but micronutrient malnutrition occurs in multiple disease states and must also be assessed. Nutritional disorders of excess should also be considered malnutrition and are of increasing importance. Such disorders include obesity, diets characterized by imbalanced macronutrient intake, and micronutrient toxicity induced by food faddism or supplement use.\n\n## II. COMPONENTS OF CLINICAL ASSESSMENT\n\nClinical assessment of nutritional status includes the clinical and nutritional history, assessment of anthropometric parameters, physical examination, and assessment of functional status. Although some elements of the clinical evaluation are relatively insensitive or nonspecific indicators of nutritional disorders, it is these clinical observations that stimulate further confirmatory measures, such as detailed assessment of diet and appropriate diagnostic tests. Indices that combine historical, anthropometric, functional, and biochemical parameters have been developed to improve sensitivity and specificity in the diagnosis of malnutrition.\n\nThe nutritional and medical histories address details of the present complaint or illness, body weight change, and the past medical history. A review of organ systems to elicit relevant clinical factors not directly related to the present illness should also be conducted. Medication use should be reviewed, and in hospitalized patients, prehospitalization medications should also be included. The nutritional history must also address the spectrum of behaviors and physiologic functions necessary to maintain adequate nutritional status, including appetite and thirst, and the abilities to procure, prepare and ingest food. Socioeconomic and psychosocial factors may figure prominently in nutritional status and should not be neglected.\n\nRelevant medications include prescription and over-the-counter medications, vitamin and mineral supplements, and herbal preparations. Although it is estimated that 40% of the U. S. population takes at least one vitamin or mineral supplement [2], many do not consider nutritional supplements to be medications and direct questioning may be necessary to elicit this history. Medications interfere with nutritional status by multiple mechanisms, including drug-related alterations in appetite, taste, thirst, nutrient absorption, nutrient metabolism, or excretion. Many medications induce changes in bowel function, which in turn may influence intake. Table 1 describes nutritional effects of some commonly used medications.\n\nTABLE 1\n\nCommon Medications and Potential Effects on Nutrient Status\n\nClass (specific example) | Effect \n---|--- \nAmphetamines | \u2193 Appetite\/weight \nAnorexiants | \u2193 Appetite\/weight \nAntibiotics ( _N_ -methylthiotetrazole sidechains) | \u2193 Vitamin K function \nAnticonvulsants (phenytoin, phenobarbitol) | \u2193 calcium absorption, \u2193 vitamin D, bone loss, \u2193 folate levels \nAntipsychotics (clozapine) | \u2191 Appetite\/weight \nBile acid sequestrants | \u2193 Vitamins A, D, E, K absorption \nCorticosteroids | \u2191 Appetite\/fat mass\/weight, hyperglycemia, \u2193 lean mass, \u2193 vitamin D, \u2193 calcium, bone loss, \u2193 vitamin B6 levels (significance unclear) \nDiuretics | \u2193 Potassium, \u2193 magnesium, \u2193 thiamin, \u2193 sodium, \u2191 calcium (thiazides) \nEthanol (abuse) | \u2193 Thiamin, \u2193 folate, \u2193 riboflavin, \u2193 vitamin B6, \u2193 vitamin D, \u2193 vitamin A \nInsulin | \u2191 Appetite\/weight \nIsoniazid | \u2193 Vitamin B6, \u2193 niacin, \u2193 vitamin D (significance unclear) \nLithium | \u2191 Appetite\/weight \nMethotrexate | \u2193 Folate \nOrlistat | \u2193 Vitamins A, D, E, K absorption \nProton pump inhibitors | \u2193 Vitamin B12 \nSelective serotonin reuptake inhibitors | \u2193 or \u2191 Appetite\/weight \nSulfasalazine | \u2193 Folate \nSulfonylureas | \u2191 Appetite\/weight \nTheophylline | \u2193 Appetite\/weight \nTricyclic antidepressants | \u2191 Appetite\/weight\n\nPhysical examination may reveal etiologic factors contributing to malnutrition or may reveal manifestations of malnutrition. Unfortunately, significant physical examination findings often occur only late in the course of malnutrition. Also, many findings associated with micronutrient malnutrition such as angular stomatitis, glossitis, and dermatitis are nonspecific. The reason for this lack of specificity is twofold. First, deficiencies of several micronutrients may result in similar signs; second, micronutrient deficiencies often do not occur in isolation, such that physical findings may reflect multiple deficiencies. Use of biochemical tests can be used to diagnose specific deficiencies if indicated.\n\nAnthropometric parameters can be used to describe the body as a whole or to subdivide the body into compartments. Anthropometric data can be utilized directly (such as body weight) to estimate lean or fat mass, or to predict energy and protein needs.\n\nFunctional assessment, which is based on measures of strength, mobility, or function, allows semiquantitative or quantitative evaluation of processes that are indicative of current nutritional status and risk for nutritional compromise. As discussed below, functional parameters correlate with other measures of nutritional status and may predict morbidity in ill or malnourished patients.\n\n## III. ANTHROPOMETRIC ASSESSMENT\n\nAnthropometric measurements describe or quantify basic physical characteristics, and include height, weight, circumferences of certain body parts, and skinfold thickness. Assessment of these parameters allows comparison to values in the same individual to monitor change over time, or to reference values, which can be used to classify nutritional status.\n\n### A. Height\n\nMeasurement of height is necessary to estimate ideal body weight or desirable body weight, body mass index (BMI), and is used in the calculation of body composition and energy requirements. Height should be measured when possible and is best obtained by a stadiometer. Vertical height decreases with aging as a result vertebral bone loss, vertebral compression fractures, and thinning of intervertebral discs and weight-bearing cartilage. Height begins to decline at approximately age 30 years for both men and women and accelerates with age; in one longitudinal series, between the ages of 30 and 80 years, women lost 8 cm and men lost 5 cm [3]. Loss of vertebral mass and disc compression may induce kyphosis (curvature with backward convexity of the spine), which will reduce measured height.\n\nWhen height cannot be accurately measured, such as in acutely ill or immobilized patients, or in those with severe osteoporotic changes, alternatives include self-reported height, estimated height, or surrogate methods to estimate height. Self-reported heights are less accurate than measured heights, as men tend to overreport and women tend to underreport height [4]. Self-reported height is more accurate, however, than clinicians' estimates of height. Coe and co-workers [5] have reported that height tends to be overestimated by visual exam of supine patients. In this study, accuracy of visual estimation of height was better for taller patients compared to shorter patients, and the authors propose that the taller patients were closer to the length of the beds in which they were lying, thus making it easier to approximate height in those cases [5].\n\nArm span and knee height correlate with vertical height, but are subject to fewer age-related changes in stature and are less influenced by impediments to the measurement of vertical height such as disability or frailty [6\u20139]. Equations to predict vertical height from measured arm span or knee height have been derived by regression analysis, and these specific formulas are available for use in several age, gender, and ethnic groups (Table 2). Arm span, which is the entire distance from the tip of the middle finger of one hand to the other, can be measured with arms stretched at right angles to the body, with the measuring tape crossing in front of the clavicles. Frail or debilitated persons may require assistance to maintain the correct position for measurement [6, 8]. Half arm span (the distance from the sternal notch to the tip of the middle finger of one hand) can also be measured and then doubled to determine arm span. Knee height is best measured with specialized calipers, and can be performed either in sitting or recumbent patients, making this useful in most ambulatory and hospital settings. Use of knee height or arm span to estimate vertical height may be useful in clinical as well as research situations [3, 7] for individuals who cannot stand, who are debilitated, or who have experienced loss of height.\n\nTABLE 2\n\nRecommended Equations for the Prediction of Stature\n\n_Source:_ Adapted from Heymsfield, S. B. (1999). Nutritional assessment of malnutrition by anthropometric methods. _In_ \"Modern Nutrition in Health and Disease\" M. E. Shils, J. A. Olson, M. Shike, and A. C. Ross, Eds.), pp. 903\u2013921. Williams and Wilkins, Baltimore, MD.\n\n### B. Weight\n\nLoss of body weight in the setting of starvation or illness is a marker of protein energy malnutrition and is associated with increased risk of morbidity and mortality [10\u201316]. In patients with cancer undergoing chemotherapy, loss of 5% or more of usual body weight was associated with impaired functional status and significantly decreased median survival lengths compared to patients without weight loss [11]. In hospitalized patients with a variety of gastrointestinal, infectious, and neoplastic diseases, protein energy malnutrition at admission was associated with an approximately twofold risk of subsequent complications [17]. More than 60 years ago, Studley [12] recognized that unintentional weight loss of 20% or more of usual body weight prior to surgery for peptic ulcer significantly increased risk of postoperative mortality. Others have confirmed that protein energy malnutrition preceding surgery increases risk of postoperative complications [13, 14, 18, 19]. Patients who have lost 10\u201320% of initial body weight over 6 months and have associated physiological defects, or those who have lost 20% or more over 6 months, should be considered at high risk [20, 21]. Involuntary change in weight may better predict risk for protein energy malnutrition than does absolute weight [14\u201316, 20, 22].\n\nAdequate store of lean and fat mass may act as buffers against chronic protein energy malnutrition, but will not prevent protein energy malnutrition in acute illness. Despite high levels of energy reserves and expanded lean body mass, obese patients still experience protein energy malnutrition when acutely ill [23]. In acutely ill obese patients, body weight may be \"adjusted\" or used in prediction equations to guide provision of energy and medications [23\u201325].\n\nIdeally, body weight should be measured by use of calibrated beam-type or electronic scales. However, clinical situations at times dictate use of alternatives, such as a calibrated bed scale, chair scale, or wheelchair scale. To monitor changes in weight over time, use of the same scale is highly recommended given variability between scales.\n\nSelf-reported weights are often inaccurate; overweight women and men tend to underestimate weight, while men of lesser weight tend to overestimate [4]. Use of a single self-reported weight is also an insensitive measure of weight change in ill patients, because weight loss in approximately one-third of patients may be missed [26]. In cases where a person cannot be weighed, the clinician may estimate weight, an inaccurate practice that, although improving with experience [5], should be discouraged.\n\nGains and losses in weight should be documented, and contemporaneous factors should be sought to explain weight change. Recognizing trends in weight with intake and the behavioral or emotional determinants is essential to effect lasting change in intake patterns. Temporally demarcated gains in weight often reveal illness, psychosocial, or life changes, or may be associated with medication initiation or cessation. Precipitous changes in weight are usually due to alterations in body water due to conditions such as heart failure, cirrhosis, or renal failure. Thus, the presence of edema or ascites should be noted, as should clinically relevant data such as the presence of dehydration or volume overload.\n\n### C. Weight for Height\n\nFor comparison to population norms, weight must be expressed relative to height. Historically, ideal body weight or desirable body weight has been defined based on actuarial data of weight for height, often with adjustment for frame size. As with all predictive methods, these tables will best apply to members of the population from which the data were derived, and thus may have reduced applicability to diverse ethnic groups, older adults, or those with chronic illnesses [22, 27]. Another limitation of some of these tables is that frame size must be determined. Frame size can be determined by measurement of elbow or wrist breadth or of wrist circumference, which requires use of specialized calipers or measuring tape.\n\nCalculation of percent ideal body weight allows determination of the relative degree of over- or underweight. In practice, relative weight outside the range of 90\u2013120% of ideal body weight is considered clinically important. Ideal body weight is also utilized in the setting of obesity or volume overload for the calculation of adjusted body weight, on which energy needs or drug dosing may be estimated.\n\nBody weight may be expressed as a function of height and takes the general form: weight\/height _x_ [28]. Now widely used is the body mass index (BMI) or Quetelet's index, kg\/m2. Use of the BMI to assess weight for height in individuals, with the classifications found in Table 3, is consistent with current recommendations of the National Institutes of Health [29] and World Health Organization [16].\n\nTABLE 3\n\nClassification of Weight by Body Mass Index\n\n_Source:_ Adapted from Ferro-Luzzi, A., Sette, S., Franklin, M., and James, W. P. (1992). A simplified approach of assessing adult chronic energy deficiency. _Eur. J. Clin. Nutr._ **46** , 173\u2013186; and Nation Heart, Lung, and Blood Institute. (1998). Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults\u2014The evidence report. _Obes. Res._ **6** (Suppl 2), 51S\u2013209S.\n\nBMI correlates with body fat [28, 30], although a linear relationship is not observed throughout the range of BMI (Fig. 1). While BMI correlates well with body fat for populations, interpretation in individuals must include consideration of clinical and other factors. For example, BMI may be elevated despite relatively low levels of body fat in those with edema or in bodybuilders. Similarly, the same low BMI may be observed in a patient who has experienced significant loss of weight and in a long-distance runner who is healthy and weight-stable.\n\nFIGURE 1 Relationship between BMI and fat mass in women. [Reproduced with permission from Heysmfield, S. B., Tighe, A., and Wang, Z.-M. (1994). Nutritional assessment by anthropometric and biochemical methods. _In_ \"Modern Nutrition in Health and Disease\" (M.E. Shils, J.A. Olson, and M. Shike, Eds.), p. 824. Lea and Febiger, Malvern, PA.]\n\nBoth low and high BMI correlate with morbidity and mortality [16, 31\u201333]. Low levels of BMI are also associated with lethargy and diminished work productivity [16]. The lowest survivable levels of BMI, as suggested by observations in starvation, famine, and anorexia nervosa, or by theoretical models, have been estimated to be 12\u201313 kg\/m2 [28, 34]. When weight loss is rapid or associated with illness, morbidity and mortality can occur at any level of BMI.\n\nA potential problem with use of BMI is that it does not account for body composition changes associated with aging, as body fat content may vary considerably in older and younger persons with the same BMI. Also, ethnic differences in body composition will not be reflected by BMI. Despite these potential problems, BMI remains an easily calculated and clinically useful method of classifying weight relative to height. In individuals, BMI can be used as a rough indicator of nutritional status, and at its extreme ranges indicates nutritional compromise.\n\n### D. Circumferences and Skinfold Thickness Measurements\n\nMeasurement of circumferences of the trunk or limbs provides descriptive information about underlying lean and fat mass [28]. Skinfold measurements describe the amount of subcutaneous fat when the skin at various sites is \"pinched\" by specialized calipers. The sites at which these measurements are conducted are illustrated in Fig. 2. Measurements of circumference or skinfold thickness from a single body site have been used to assess protein energy status [16], but are likely to be influenced by the interindividual variability in body composition. Measurements at multiple sites reduce the potential contribution of this variability. As such, single site measurements are better used to follow trends over time in an individual than for comparison to normative standards. A number of investigators have related combinations of circumference or skinfold thickness measurements at multiple sites to other measures of body composition such as hydro-densitometry, dual energy x-ray absorptiometry (DXA), or calculated axial tomogrophy scanning to develop prediction equations for lean and fat mass for the whole body or specific segments [28, 35, 36].\n\nFIGURE 2 Body sites for measurement of circumferences, skinfold thickness, and widths. [From Wang, J., Thornton, J. C., Kolesnik, S., and Pierson, R. N., Jr. (2000). Anthropometry in body composition. An overview. _Ann. N.Y. Acad. Sci._ **904,** 317\u2013326.]\n\nMeasurement of circumferences and skinfold thickness may be influenced by several factors, including age, sex, gender, ethnicity, and under-or overhydration [37]. Specific reference data for the individual or population should be used when possible. Most reference data have been developed in healthy populations. Limited reference data exist for hospitalized patients; however, acute illness is associated with widely fluctuating perturbations in body water such that generalizable reference data may never be obtainable. Upper body measurements may be preferable in those individuals with evidence of edema or ascites, because the upper body is less likely to accumulate excess body water. Measurements obtained in the reduced massively obese are likely to be of diminished predictive value due to error introduced by redundant skin and other factors.\n\n### E. Body Fat Distribution\n\nCentral distribution of body fat increases risk for multiple cardiovascular risk factors such as diabetes, hypertension, and dyslipidemia [38, 39]. The traditional measure of central adiposity has been the ratio of waist to hip circumference (waist:hip ratio, WHR). However, debate exists as to the optimal landmarks to define waist and hip circumferences, a situation exacerbated by the variable geometry of the abdominal panniculus in obesity. Current guidelines suggest measurement of waist circumference only as marker of abdominal adiposity, which is measured at the level of the top of the iliac crest (Fig. 3) [29]. Use of this single measure correlates well with visceral adiposity, which is thought to contribute the risk of developing diseases associated with central fat distribution [38, 39]. Because increased waist circumference is associated with increased disease risk at all levels of BMI except extreme obesity (see Table 3), measurement of waist circumference should be performed routinely.\n\nFIGURE 3 Landmarks for measurement of waist circumference. [From Nation Heart, Lung, and Blood Institute. (1998). Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults\u2014The evidence report. _Obes. Res._ **6** (Suppl 2), 51S\u2013209S.]\n\n## IV. FUNCTIONAL ASSESSMENT\n\nDeficits in function may arise from malnutrition, predict risk for future malnutrition, and be associated with increased risk for malnutrition-related comorbidity. Functional assessment is based on the premise that protein energy malnutrition or other forms of malnutrition will be associated with impaired strength, mobility, or function [19, 21]. Tools developed for functional assessment vary in complexity from simple measures of handgrip strength or respiratory muscle strength to batteries of multistage tasks requiring complex physical and cognitive processes. Some functional assessment tools are useful for general populations, while others have been developed and validated for specific disease states or in specific groups such as the elderly, hospitalized patients, or community dwellers.\n\nA common simple functional test is handgrip strength. Handgrip strength as measured by handgrip dynamometry has been shown to positively correlate with lean body mass and correlate negatively with protein energy malnutrition [21, 40\u201342]. In patients undergoing surgery, preoperative handgrip strength has also been found to predict risk of postoperative complications [43\u201345]. Handgrip dynamometry requires the cooperation of a conscious patient and can be hampered by factors such as neuromuscular disease or arthritis. Handgrip strength is useful in the serial assessment of an individual, but can also be used for reference to age-and sex-specific norms [44, 46]. Comparison of individual values to these norms should be approached with caution because these investigators employed different types of devices (strain-gauged vs. mechanical). Also, in the study by Bassey and Harries [46], it is interesting and of some concern that longitudinal changes in grip strength exceeded cross-sectional differences with age. Early improvement in grip strength in malnourished patients after refeeding has been observed [41, 47]. It is likely that repletion of intracellular energy, ions, and hydration contributes to this functional improvement long before significant accretion of protein [41, 47].\n\nNon-volitional measures of muscle function have also been developed. Electric stimulation can be used to measure contraction and relaxation characteristics of the adductor pollicis muscle, and similar to handgrip strength, these measures are associated with loss of lean mass and have predicted postoperative complications [13, 19, 47]. Improvement in muscle function parameters has also been noted early in refeeding, and is likely explained by mechanisms similar to that in handgrip strength [19, 47]. Nonvolitional testing is potentially useful in those who cannot cooperate to the extent necessary for handgrip strength or other testing, such as in critically ill, comatose, or sedated individuals. However, Finn _et al._ [48] have noted that several factors may limit use of nonvolitional muscle function testing in the critically ill, such as concomitant neuromuscular blockade and illness-associated heightened sensitivity to discomfort associated with testing. Another recent report suggests that the day-to-day variability of nonvolitional muscle tests significantly exceeded that of other assessment measures such as body weight, midarm muscle circumference, triceps skinfold thickness, and handgrip strength [49].\n\n## V. CLINICAL MANIFESTATIONS IN SPECIFIC DISEASE STATES AND POPULATIONS\n\nThe following sections describe important historical, examination, anthropometric, and functional findings in specific organ systems and in populations at high risk. This chapter generally focuses on common disorders and findings, and discussion of disease-specific assessment and treatment is found later in this text and elsewhere as noted. Table 4 summarizes selected physical findings found in nutrient deficiency or excess, some of which are depicted in Fig. 4 (see color plate at the back of the book).\n\nTABLE 4\n\nSigns of Nutrient Deficiency and Excess\n\nFIGURE 4 Physical signs associated with nutrient deficiencies. (A) Muscle wasting in severe protein energy malnutrition. (B) Tenting of skin in dehydration; the skin retains the tented shape after being pinched. (C) Glossitis and angular stomatitis associated with multiple B vitamin deficiencies. (D) Dermatitis associated with zinc deficiency. (E) Cheilosis, or vertical fissuring of the lips, associated with multiple B vitamin deficiencies. (F) Bitot's spot accompanying vitamin A deficiency. (Photos courtesy of Dr. Robert Russell and Dr. Joel Mason.)\n\n### A. Oral and Dental Health\n\nDental health influences food choices and eating enjoyment [50]. Surveys in the elderly reveal a direct relationship between degree of edentulousness and measures of nutritional status or dietary intake [50, 51]. Those who are edentulous and without dentures, or with only one denture, appear to be at higher risk [51] than those with two dentures. Acutely ill edentulous patients are often admitted without dentures, which may predispose to in-hospital malnutrition by limiting ability to chew.\n\nMultiple deficiency states, as well as disease states and their treatments, have oropharyngeal manifestations. A sore, atrophied, red, or magenta tongue (glossitis), cracking or ulceration of the lips (cheilosis), or cracking or ulceration at the corners of the mouth (angular stomatitis) are seen in several B vitamin and other deficiencies. Bleeding from the gums may indicate coagulopathy due to vitamin K deficiency or scurvy. Cancer chemotherapy may result in pain or ulcers in the mouth and throat (mucositis), a common cause of poor intake in treated patients with cancer (see Chapter 26).\n\n### B. Skin, Hair, and Nails\n\nLoss of subcutaneous fat is evident by simply pinching the skin. Tenting of the skin, seen with dehydration, may persist after pinching. The skin should be examined for evidence of rash or scaling, decubitus ulcer, and open wounds. Dermatitis accompanies many micronutrient deficiencies, as well as essential fatty acid deficiency (Table 4 and Fig. 4), but may only occur in advanced states of deficiency. Dermatitis herpetaformis is an itchy eruptive rash that results from gluten sensitivity. Dermatitis herpetaformis may accompany celiac sprue; thus, even if diarrhea and weight loss are absent, consideration should be made of subclinical intestinal disease and screening for vitamin deficiency. Yellow or orange discoloration of the skin may indicate carotenoid excess, which spares the sclerae and thus may be distinguished from jaundice.\n\n### C. Cardiovascular System\n\nCardiovascular risk factors such as dyslipidemia, hypertension, and diabetes mellitus are associated with multiple dietary factors, as well as with obesity (see Chapter 18). A newly identified risk factor for atherosclerotic disease is hyperhomocysteinemia [52], which may be elevated in inadequate intake or poor status of folate, vitamin B12, and vitamin B6 [53]. Trials are currently ongoing to assess the effect of supplementation with these nutrients on primary or secondary prevention of cardiovascular disease.\n\nCardiac cachexia is a syndrome associated with chronic heart failure, characterized by progressive loss of weight and lean mass, and mediated by diminished appetite and increased resting energy expenditure [54]. Loss of lean mass is likely to exacerbate symptoms because exercise tolerance will be further reduced.\n\nAdvanced thiamin deficiency may result in congestive heart failure (\"wet\" beriberi). High-output heart failure may occur and is characterized by rapid heart rate and pulmonary and peripheral edema.\n\n### D. Pulmonary System\n\nPulmonary muscle strength may be diminished in protein energy malnutrition and predisposes to respiratory complications in those with chronic pulmonary disease or in acutely ill patients. Acute respiratory failure can occur due to hypophosphatemia, which may accompany protein energy malnutrition, and alcohol abuse, and occurs during correction of diabetic ketoacidosis [55].\n\nChronic obstructive lung disease (COPD) is associated with protein energy malnutrition due to diminished intake and increased resting energy expenditure [56]. In patients with severe COPD, eating itself may induce dyspnea. Also, acute exacerbations or severe COPD may be accompanied by aerophagia (or swallowing air), which may cause gastric distention, abdominal bloating, and decreased intake. Chronic treatment of COPD with corticosteroids predisposes to further loss of lean mass, gains in fat mass, glucose intolerance, and bone loss.\n\nPatients with cystic fibrosis now frequently live into adulthood, where the associated maldigestion can result in protein energy malnutrition and fat-soluble vitamin deficiencies [57, 58]. Protein energy malnutrition and these vitamin deficiencies may in turn diminish pulmonary function and increase risk for infection. Advanced cystic fibrosis may also involve the endocrine pancreas, resulting in diabetes mellitus [57].\n\nObesity is among the most potent risk factors for obstructive sleep apnea, characterized by disturbed sleep and daytime fatigue. Effective treatment of obstructive sleep apnea was observed to facilitate weight loss efforts [59]. Extreme obesity is also associated with other disorders including pulmonary hypertension, cor pulmonale, hypercapnea, hypoxemia, and increased risk for pulmonary embolism.\n\n### E. Gastrointestinal System\n\nGastrointestinal illness promotes protein energy and micronutrient malnutrition. Common diseases and mechanisms are briefly discussed below; details can be found in other chapters of this text. Dietary factors may result in gastrointestinal symptoms, some of which are uncomfortable but harmless and time limited, whereas others may progress to serious or life-threatening disease. Included in this spectrum are lactose intolerance, consumption of osmotic cathartics such as sorbitol (in food, candy, or medications) and gluten-sensitive enteropathy (celiac disease). Following gastric resection or gastric bypass for obesity, dumping syndromes may occur in response to hyperosmolar meals [60] and are characterized by varying degrees of lightheadedness or near-syncope, nausea, diaphoresis, chest pain, and abdominal pain or cramps.\n\nDigestive and absorptive processes may be site specific and vary by nutrient. Accordingly, the extent and location of both disease activity and resection should be noted. Inflammatory bowel disease, celiac disease, resection of the intestinal tract, and short bowel syndrome are associated with protein energy malnutrition and deficiencies of water-and fat-soluble vitamins, as well as iron, calcium, other minerals, and essential fatty acids [47, 61\u201364]. Recently, Geerling _et al._ [62] found that patients with Crohn's disease in remission had persistent deficiencies in several water-and fat-soluble vitamins as well as zinc. Thus the absence of current symptoms in those with chronic or remitting disease should not be taken as a guarantee of adequate nutritional status.\n\nEnd-stage liver disease or cirrhosis is frequently associated with protein energy malnutrition. In the acutely ill cirrhotic individual, changes in mental status may indicate hepatic encephalopathy, which may dictate specific dietary recommendations. Chronic pancreatitis or pancreatic insufficiency may lead to maldigestion of macronutrients. In both chronic liver disease (especially cholestatic disease) and pancreatic disease, fat-soluble vitamin deficiency may occur [65]. Manifestations of deficiency states may be masked by malaise associated with chronic illness or be asymptomatic, and may only become apparent when symptoms such as night blindness, bone pain, or easy bleeding are elicited [65].\n\nAtrophic gastritis, which predisposes to vitamin B12 deficiency, increases in prevalence with advancing age. Not surprisingly, more than 12% of a free-living elderly population was found to be deficient in vitamin B12 [66]. The common chronic use of acid-suppressing drugs (proton pump inhibitors and H2 antagonists) may also predispose to vitamin B12 deficiency [67].\n\n### F. Musculoskeletal\n\nProtein energy malnutrition may result in reductions in muscular size and strength, as well as in functional changes such as diminution of work capacity or endurance. Generalized weakness is a highly nonspecific symptom and is observed in dehydration, iron deficiency (even prior to the development of significant anemia), hypophosphatemia, and in multiple vitamin deficiencies. Generalized or proximal weakness, as well as frank myopathies, due to vitamin D deficiency have recently been described [68]. Muscle wasting may be most apparent at the temporalis muscle (temporal atrophy or wasting), the shoulder girdle, and between the bones of the dorsum of the hand (interosseus wasting).\n\nA history of fractures and bone pain may indicate metabolic bone disease or osteoporosis, and should stimulate evaluation of the skeleton, as well as of calcium and vitamin D status. Persons with diseases known to influence calcium and vitamin D metabolism (such as those with malabsorptive disorders, chronic renal failure, and the institutionalized elderly) are at high risk for metabolic bone disease, and appropriate monitoring and treatment should be undertaken.\n\nObesity is a potent contributor to the development of osteoarthritis, the symptoms of which may be ameliorated with weight loss.\n\n### G. Hematologic System\n\nClassical nutritional anemias result from deficiencies of vitamin B12, folate, and iron. Populations at increased risk include alcoholics (vitamin B12 and folate), the elderly (vitamin B12), women with menometorrhagia (iron), and vegans (vitamin B12). However, anemia has also been reported due to protein energy malnutrition and deficiencies of vitamin C, vitamin B6, riboflavin, and copper [69]. The absence of anemia, or of specific findings in red and white blood cell morphology, should not be used to assess nutrient sufficiency, as deficiency states may exist without these hematologic changes.\n\nAbnormal bleeding may be observed due to vitamin K deficiency. Most vitamin K-related bleeding usually occurs in the setting of oral anticoagulant use, which antagonizes vitamin K. Frank vitamin K deficiency with coagulopathy may be observed in alcoholics or in association with medications or starvation (see Table 1).\n\n### H. Cancer\n\nDietary factors that may prevent or predispose to neoplasm are beyond the scope of this chapter, but are discussed elsewhere in this text. Cancer may influence nutritional status by its effects on appetite, on energy expenditure, and on processes that are directly due to tumor burden (such as dysphagia with head and neck cancer, or bowel obstruction in advanced colon cancer) or due to pain. In addition, chemotherapy or radiation therapy may result in mucositis, nausea, vomiting, diarrhea, and alterations in taste. The implications for cancer treatment are discussed in Chapter 26.\n\n### I. Renal System\n\nNutritional issues in acute and chronic renal failure share common elements, but also may differ considerably depending on the etiology, the acuity and (in)stability of disease, and treatment modality. Uremia is commonly associated with anorexia and nausea, which may also occur due to conditions underlying renal failure. In addition to effects on intake, uremia-associated metabolic acidosis may perturb protein metabolism by increasing protein catabolism and limiting synthesis [70]. Nutritional issues associated with acute renal failure include fluid management, adapting protein delivery to minimize uremia, and preventing electrolyte imbalance. Chronic renal failure and dialysis patients are at increased risk for protein energy malnutrition, and, as in other chronic diseases, an inverse relationship exists between body mass index and mortality [71]. Protein-restricted diets may be prescribed to slow progression of chronic renal disease in predialysis patients, a strategy that may require monitoring to ensure adequacy of energy and other nutrients necessary to prevent protein energy malnutrition during reduced protein intake [72]. Micronutrient issues in chronic renal failure include deficiency of vitamin D, prevention of hyperphosphatemia and related bone disease, deficiencies of several water-soluble vitamins, and toxicity of vitamin A. A newer concern is hyperhomocysteinemia, which has been observed in dialysis and renal transplant patients [73\u201375], and may increase risk for cardiovascular disease.\n\n### J. Neurologic and Psychiatric\n\nNeurologic disorders, such as stroke, Parkinson's disease, and head injury, may impair ability to feed oneself or to swallow effectively (dysphagia) (see Chapter 41). A history of difficulty initiating a swallow, choking or gagging, wet cough, and retained food in the mouth are among the signs that should stimulate further evaluation for dysphagia [76]. A large number of non-neurologic conditions may also predispose to dysphagia including prolonged endotracheal intubation or absence of oral intake, as well as chronic diseases such as head and neck or esophageal cancer [76].\n\nAnorexia nervosa, bulimia, and binge eating disorder may be suspected on the basis of eating patterns and attitudes, by low body weight or BMI, and by excessive exercise. An up-to-date clinical approach to the assessment of disordered eating is available elsewhere [77]. Signs of bulimia nervosa may include dental erosions and parotid hyperplasia. Nutritional problems associated with anorexia nervosa and bulimia nervosa include protein energy malnutrition, electrolyte abnormalities, vitamin and mineral deficiencies, and, in the longer term, osteopenia [78].\n\nPatients suffering from advanced dementia are likely to experience malnutrition due to loss of the cognitive as well as the motor skills necessary for adequate intake. Depression may also be associated with decreased or increased intake. Medications for the treatment of depression, bipolar disease, and psychosis, as well as anticonvulsants, are often associated with changes in intake and body weight (see Table 1).\n\nMultiple psychiatric or neurologic syndromes related to nutrient deficiency (e.g., thiamin, niacin, vitamin B6, vitamin E, essential fatty acids) or excess have been described (Table 4). In the United States, common predisposing factors to deficiency syndromes are alcoholism and malabsorptive disorders [79, 80], as well as aging in the case of vitamin B12. Of particular importance is that deficiency of vitamin B12 may be manifest by neurologic or psychiatric symptoms in the absence of anemia or macrocytosis [81]. Syndromes associated with micronutrient excess may result from supplementation or food faddism or may be associated with underlying diseases.\n\n### K. Infectious Disease and AIDS\n\nImmune competence depends on adequate protein energy nutrition as well as multiple micronutrients. In hospitalized patients, the complications of protein energy malnutrition often are manifest as infection. Infectious complications are not only due to compromise of immune function per se, but also reflect other functional deficits. For example, aspiration pneumonia may be precipitated by poor ability to expectorate or cough and be facilitated by impaired ciliary clearance.\n\nMalnutrition in acquired immunodeficiency syndrome (AIDS) may be secondary to reduced intake, malabsorption, medications, or increased nutrient needs (see Chapter 47). Reduced intake can be associated with generalized anorexia due to disease or medications, oropharyngeal or esophageal lesions, and diarrhea. The human immunodeficiency virus (HIV) wasting syndrome may result from decreased intake, increased resting energy expenditure, and HIV-induced loss of lean mass [82]. Chronic protein energy malnutrition in AIDS will likely be exacerbated by acute losses associated with infection or other complications. Multiple vitamin and mineral deficiencies have been observed in HIV infection and AIDS and have been linked to increased rates of mortality [83].\n\n## VI. PROGNOSTIC INDICATORS\n\nAttempts have been made to improve the sensitivity and specificity in assessment of nutrition-related risk by combining various assessment parameters. Table 5 illustrates some prognostic indexes, and some popular examples are discussed below. Recent reviews of these indexes have been compiled [84, 85].\n\nTABLE 5\n\nSelected Prognostic Index\n\n_Source:_ Adapted from Schneider, S. M., and Hebuterne, X. (2000). Use of nutritional scores to predict outcomes in chronic diseases. _Nutr. Rev._ **58,** 31\u201338.\n\n### A. Prognostic Nutritional Index\n\nThe Prognostic Nutritional Index uses biochemical, anthropometric, and measures of immunocompetence to calculate an index of risk of developing postoperative complications. A prognostic nutritional index greater than 40% is indicative of presence of malnutrition, and has been predictive of in-hospital complications, sepsis, length of hospital stay, and postoperative death [84, 86, 87].\n\n### B. Subjective Global Assessment\n\nSubjective Global Assessment combines historical elements, along with examination for edema and loss of subcutaneous fat, to stratify individuals into three categories: well nourished, moderately (or suspected of being) malnourished, or severely malnourished [88] (Table 6). Patients with a variety of illnesses who have been classified as moderately or severely malnourished have been shown to have greater postoperative complications, longer hospital stays, and accrued greater hospital charges compared to well-nourished patients [84]. Covinsky and colleagues [89] used Subjective Global Assessment to assess 369 hospitalized older adults (\u226570 years old), and then followed patient outcomes at 3 months and 1 year. Patients classified as severely malnourished were more likely to be dependent in activities of daily living at 3 months after discharge and were more likely to have spent time in a nursing home during the year after hospitalization. Both moderately and severely malnourished patients were more likely to have died at the 3-month and 1-year follow-up after discharge compared to the well-nourished group [89].\n\nTABLE 6\n\nComponents of Subjective Global Assessment\n\nPatient history:\n\nWeight change\n\nOverall loss in past 6 months\n\nChange in past 2 weeks (increase, stable, or decrease)\n\nDietary intake change relative to normal\n\nNo change\n\nChange\n\nDuration (number of weeks)\n\nTypes of change\n\nSuboptimal solid diet\n\nFull liquid diet\n\nHypocaloric liquids\n\nStarvation\n\nGastrointestinal symptoms (that persisted >2 weeks)\n\nNo symptoms\n\nNausea\n\nVomiting\n\nDiarrhea\n\nAnorexia\n\nDisease and its relation to nutritional requirements\n\nPrimary diagnosis\n\nMetabolic demand (stress)\n\nNo stress\n\nMild stress\n\nHigh stress\n\nPhysical exam:\n\nFor each trait specify: 0 = normal, 1+ = mild, 2+ = moderate, 3+ = severe\n\nLoss of subcutaneous fat (triceps, chest)\n\nMuscle wasting (quadriceps, deltoids)\n\nAnkle edema\n\nSacral edema\n\nAscites\n\n_Source:_ Adapted from Detsky, A. S., McLaughlin, J. R., Baker, J. P., Johnston, N., Whittaker, S., Mendelson, R. A., and Jeejeebhoy, K. N. (1987). What is subjective global assessment of nutrition status? _JPEN_ **11,** 8\u201313.\n\n### C. Screening Tools for Older Adults\n\nThe elderly are the fastest growing segment of our population, and they are at particular risk for malnutrition because of illness, changes in body composition, diminished mobility and function, social isolation, and economic factors. Special attention to these issues is required to identify problem areas and institute early intervention [85, 90].\n\n#### 1. NUTRITION SCREENING INITIATIVE\n\nThe Nutrition Screening Initiative is a multistage screening and assessment device. The initial stage is the DETERMINE Your Nutritional Health Checklist [91], which is designed to be self-administered, but also can be administered by a caretaker. It includes 10 yes\/no statements covering dietary assessment, general assessment, and social assessment. Each statement has a weighted score, which is then tallied to a final score for stratification to low, moderate, or high nutritional risk [85, 91]. Posner _et al._ [91] surveyed 749 Medicare beneficiaries using a 14-point checklist (the predecessor of the currently used checklist) and found that 24% of this population was at high nutritional risk. Of this group, 38% had dietary intakes below 75% of the recommended dietary intakes for three or more nutrients.\n\nThose at high nutritional risk require further assessment by a health care professional by using the level 1 or level 2 screening tools. The level 1 screening tool includes information about body weight, BMI, weight change, eating habits, living environment, and functional status. The level 2 screening tool includes all of the level 1 information, but also anthropometric measurements, biochemical measurements, cognitive and emotional status assessments, medication use, and signs and symptoms of nutrient deficiencies.\n\n#### 2. MINI NUTRITIONAL ASSESSMENT\n\nThe Mini Nutritional Assessment was initially developed for the frail elderly, but has been validated for use in other elderly populations [90]. Designed to be administered by a health care professional, the mini nutritional assessment consists of 18 questions relating to anthropometrics (including weight loss, BMI, midarm circumference, and calf circumference), dietary intake (including change in appetite, number of meals\/day, and autonomy of feeding), global assessment (including mobility, lifestyle, and medication use), and subjective assessment (self-perception of health and nutritional status). Each area of assessment receives a score, which is tallied at the end of the assessment, to a maximum score of 30. A score of 24\u201330 points indicates no risk of malnutrition, 17\u201323.5 indicates risk of malnutrition, and a score of <17 points indicates existing malnutrition [85, 90].\n\n## VII. SUMMARY\n\nClinical and physical assessment of nutritional status is integral to the detection of present or potential malnutrition. Because individual components may lack sensitivity or specificity, a combination of these components is recommended. Basic medical and historical data such as present illnesses, weight change, and medications, as well as anthropometric data such as height and weight, should always be collected, regardless of the clinician's perception of risk. Populations usually at increased risk include those with weight loss greater than 10% of usual weight, those with certain chronic diseases, alcoholics, and the elderly. When indicated, more detailed assessment, including use of functional parameters and prognostic indexes can then be performed.\n\nReferences\n\n1. Hensrud D.D. Nutrition screening and assessment. _Med. Clin. N. Am_. 1999;83:1525\u20131546.\n\n2. Balluz L.S., Kieszak S.M., Philen R.M., Mulinare J. 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Nutr_. 1992;46:173\u2013186.\nCHAPTER 4\n\nOverview of Nutritional Epidemiology\n\nRUTH E. PATTERSON, Fred Hutchinson Cancer Research Center, Seattle, Washington\n\n## I. INTRODUCTION\n\nNutritional epidemiology is the study of dietary intake and the occurrence of disease in human populations. One factor that distinguishes this discipline is the extraordinary challenge of the exposure assessment. In epidemiology, exposure is defined as participant characteristics or agents (e.g., food, medications, sun) with which a participant comes into contact that may be related to disease risk (see Table 1) [1]. Measuring dietary intake is unusually complicated for many different reasons. For purposes of illustration, we can compare the assessment challenges for two common exposures: cigarette smoking and diet. Because it is a single (yes\/no) activity, participants usually can accurately report whether or not they smoke. Because smoking is addictive, it tends to be a consistent long-term behavior rather than one that individuals stop and start. Because it is a habit, most people smoke roughly the same number of cigarettes per day; and because of the expense, most people know how many cigarettes they smoke per day. In comparison, over the course of even 1 week, an individual can consume hundreds, even thousands of distinct food items, making it difficult for respondents to accurately report on their intake. Meals can be prepared by others (e.g., in a restaurant, by a spouse, as prepackaged food) so that the respondent is not knowledgeable about what, or how much, he is eating. Food choices typically vary with seasons and other life activities (e.g., weekends, holidays, vacations). In fact, the day-to-day variability in food intake can be so large that it is difficult to identify any underlying consistent pattern. In addition, foods themselves are often a surrogate for the exposure of interest (e.g., dietary fat), which means that investigators must rely on food composition databases to calculate the exposure variable. Given even this superficial summary of the problems inherent in assessing dietary intake, it is not surprising that it has been difficult to obtain consistent and strong evidence regarding how diet affects disease risk.\n\nTABLE 1\n\nExamples of Exposures Relevant to Nutritional Epidemiologic Studies\n\nExposure | Diet-related example | Other example \n---|---|--- \nAgent that may cause or protect from disease | Vegetable consumption may be protective for colon cancer. | Physical activity may be protective for colon cancer. \nConstitutional host factors | Genetic predisposition to nutrition-related disease. | Older adults are more predisposed to chronic disease. \nOther host factors | Food preferences that determine food choices. | More educated adults may have better disease screening. \nAgents that may confound the association between another agent and disease | Correlation between dietary constituents (e.g., a diet high in fruits and vegetables is usually low in fat). | Smokers are less likely to engage in physical activity. \nAgents that may modify effects of other agents | Fruits and vegetables may protect against lung cancer among smokers. | Alcohol results in increased risk of lung cancer from smoking. \nAgents that may determine outcome of disease | Malnutrition. | Medical treatment.\n\nThe vast majority of nutritional epidemiologic research in the past 20 years has focused on the identification of foods and\/or constituents in foods (i.e., nutrients) that cause, or protect against, the occurrence of chronic diseases. Therefore, the tools and methods of nutritional epidemiology were developed to address scientific issues unique to the biology of chronic diseases. In particular, epidemiologic methods were designed to take into account the following: (1) the extensive time for disease development, (2) the multifactorial nature of chronic diseases, and (3) research conducted in human beings, which precludes direct observation of cause and effect. Below, each of these issues in relation to nutritional epidemiology is discussed.\n\n### A. Extensive Time for Disease Development\n\nChronic diseases develop over many years and even decades. This fact has important implications for the field of nutritional epidemiology. For example, the currently accepted model of colon cancer assumes that it is a multistep pathogenic pathway beginning with mutations (germline or somatic) leading to growth of polyps (preneoplastic growths), which become adenomas and progress to carcinoma [2]. Even after an adenoma finally develops, many years may elapse before it is clinically detected. Upon clinical detection of colon cancer, it is clear that a meal consumed that day, or the month before, could have no significant effect on the disease for two reasons. First, the critical period of cancer initiation and promotion occurred many years ago. Second, it is accepted that the biologically relevant exposure is the long-term or usual diet, rather than any single eating occasion. Therefore, the exposure of interest in the development of cancer (and other chronic diseases) occurred throughout the past 10\u201320 years.\n\nThis time lag between dietary exposure and disease occurrence presents significant difficulties in the study of diet and chronic disease. These difficulties have been addressed in two major ways. First, dietary assessment instruments described as food frequency questionnaires were developed to capture information on usual, long-term dietary intake. Second, study designs were developed that either ask about diet in a retrospective manner (e.g., case-control studies) or assess current diet and follow participants over time for the development of disease (e.g., cohort studies, intervention trials).\n\n### B. Multifactorial Nature of Chronic Diseases\n\nIn addition to dietary intake, there are many other determinants of chronic diseases, including participant constitutional factors (e.g., genetic susceptibility) and other behaviors (e.g., cigarette smoking). These other factors may \"confound\" our ability to find an association of dietary intake with disease risk. For example, individuals with an interest in health may eat a diet high in fruits and vegetables (which also tends to be low in fat) and have high physical activity levels. If vegetable intake is found to be associated with reduced risk of colon cancer, it can be very difficult to determine whether it is high vegetable intake or low-fat intake that is affecting disease risk. It is also possible that diet itself is not related to disease risk, but is merely serving as a marker for some other healthful behavior (such as physical activity). Statistical techniques and study designs that deal with these problems of confounding are covered in detail below.\n\n### C. Research in Human Beings\n\nConducting research in healthy people significantly limits the types of nutritional studies that can be performed. For example, humans cannot (knowingly) be exposed to potentially dangerous dietary regimes, they will not follow extremely rigid or unpalatable diets for years, and they cannot undergo hazardous procedures (e.g., liver biopsies) for purposes of providing biologic samples. For these reasons, nutritional epidemiology is a discipline that consists largely of (1) measuring an exposure (e.g., dietary intake), (2) measuring an outcome (e.g., disease occurrence), and (3) using statistical techniques to quantify the magnitude of the association between these two observations.\n\nEpidemiology encompasses three major topic areas: (1) exposure measurement, (2) study design, and (3) interpretation of cause and effect. Below we discuss each of these in relation to nutritional epidemiology.\n\n## II. PRINCIPLES OF EXPOSURE MEASUREMENT IN NUTRITIONAL EPIDEMIOLOGY\n\nBecause chronic disease develops over many years, the biologically relevant exposure is usual or long-term diet, consumed many years prior to disease diagnosis. Therefore, assessment instruments that only capture data on current or short-term dietary intake (e.g., food records or recall) are not often useful in nutritional epidemiology. Food frequency questionnaires (FFQs) are generally regarded as the dietary assessment instrument best suited for most epidemiologic applications [3]. Although the design of FFQs can vary somewhat, they typically contain the following sections: (1) adjustment questions, (2) the food checklist, and (3) summary questions.\n\n### A. Food Frequency Questionnaires\n\nFFQs often contain _adjustment questions_ that assess the nutrient content of specific food items. For example, participants are asked what type of milk they usually drink and are given several options (e.g., whole, skim, soy), which saves space and reduces participant burden compared to asking for the frequency of consumption and usual portion sizes of many different types of milk. Adjustment questions also permit more refined analyses of fat intake by asking about food preparation practices (e. g, removing fat from red meat) and types of added fats (e.g., use of butter vs. margarine on bread).\n\nThe main section consists of a _food_ or _food group checklist,_ with questions on usual frequency of intake and portion size. The foods are selected to capture data on (1) major sources of energy and nutrients for most people, (2) between-person variability in food intake, and (3) major hypotheses regarding diet and disease. To allow for machine scanning of these forms, responses are typically categorized from \"never or less than once per month\" to \"2+ per day\" for foods and \"6+ per day\" for beverages. Portion sizes are often assessed by asking respondents to mark \"small,\" \"medium,\" or \"large\" in comparison to a given medium portion size. However, some questionnaires only ask about the frequency of intake of a \"usual\" portion size (e.g., 1 cup milk). In the latter instances, respondents are asked to calculate the frequency of the amount given, rather than actual serving size consumed.\n\n_Summary questions_ ask about usual intake of fruits and vegetables because the long lists of these foods (needed to capture micronutrient intake) leads to overreporting of intake [4].\n\nNote that development of a FFQ is a daunting, complex task requiring considerable understanding of exposure measurement in nutritional epidemiology, food composition knowledge, formatting and questionnaire design expertise, and programming resources.\n\n### B. Vitamin\/Mineral Supplement Assessment\n\nConsiderably less attention has been paid to measuring vitamin\/mineral supplement use compared to food intake. However assessing vitamin\/mineral supplement use is important, because supplement use per se is an exposure of interest for the risk of several chronic diseases [5, 6]. In addition, supplements contribute a large proportion of total (diet plus supplement) micronutrient intake [7]. Epidemiologic studies typically use personal interviews or self-administered questionnaires to obtain information on three to five general classes of multiple vitamins, single supplements, the dose of single supplements, and sometimes frequency and\/or duration of use.\n\nIn a validity study comparing this self-administered assessment method to label transcription among 104 supplement users, we found correlation coefficients ranging from 0. 1 for iron to 0. 8 for vitamin C [8]. The principal sources of error were investigator error in assigning the micronutrient composition of multiple vitamins and respondent confusion regarding the distinction between multiple vitamins and single supplements. These results suggest that commonly used epidemiologic methods of assessing supplement use may incorporate significant amounts of error in estimates of some nutrients.\n\nIn a marketplace that is becoming rapidly more complex, with vitamins, minerals, and botanical compounds combined in unusual mixtures at highly variable doses, the association of dietary supplements with disease risk is becoming increasingly difficult to assess.\n\n### C. Use of Biomarkers in Nutritional Epidemiology\n\nDietary biomarkers are critical to the advancement of nutritional epidemiology. Recent studies using doubly labeled water to estimate energy expenditure have found significant underreporting and person-specific biases in nutrient estimates, such as the tendency for obese women to underestimate dietary intake [9]. Identification and understanding of the effect of these person-specific biases are the major challenges now facing nutrition studies. Movement in this field, however, has been hampered by the lack of practical and available biomarkers. For example, there is no established biomarker for total fat intake. In addition, many biomakers (e.g., serum \u03b2-carotene for total fruits and vegetables) are not on the same metric as the food or nutrient being assessed and, therefore, are of limited usefulness in assessing overall or person-specific biases in self-report.\n\nHowever, there is a growing awareness of the importance of biomarker substudies for the interpretation of observational nutritional epidemiologic studies. For example, the American Assocation of Retired Persons (AARP) cohort study being coordinated by the National Cancer Institute has a sizable substudy that includes doubly labeled water and urinary nitrogen assessment of protein intake 15]. These types of studies are an important step toward strengthening the reliability and interpretability of epidemiologic studies of diet and disease. [Chapter 10 discusses biomarkers in more detail.\n\n## III. STUDY DESIGNS USED IN NUTRITIONAL EPIDEMIOLOGY\n\nEpidemiologic studies can be roughly divided into two types: observational and experimental. The primary observational study designs include ecologic, cohort, and case-control studies. In studies of humans, the main experimental study design is the intervention trial, also called a randomized clinical trial. An overview of these study designs is given below in relation to nutritional epidemiology.\n\n### A. Observational Studies\n\n1. ECOLOGIC AND MIGRANT STUDIES\n\nImportant hypothesis-generating studies have examined the relationship, between countries, of national estimates of per capita supply of foods (e.g., dietary fat) with time-lagged rates of cancer or heart disease incidence or mortality [10\u201313]. These correlational analyses strongly suggest that dietary fat intake is related to these major diseases. However, it must be noted that (1) the estimate of per capita intake from food disappearance data is extremely imprecise, (2) it is generally not feasible to control for other differences between the countries (e.g., differences in physical activity levels), and (3) it is unknown whether the individuals within the countries that are exposed to specific dietary factors are the same individuals experiencing the disease. Migrant studies have often shown that with a single move from Eastern to Western countries, large and significant increases occur in risk of several chronic diseases, such as breast cancer [14]. These studies point to the importance of lifestyle and environment in disease causation; however, few such studies have included pertinent dietary data [15].\n\n#### 2. CASE-CONTROL STUDIES\n\nIn a case-control study, individuals are identified and studied according to a single disease outcome. Specifically, individuals who have recently been diagnosed with a disease (e.g., colon cancer) are asked about their past exposure to diet and other risk factors and often provide a blood sample. A matched set of control individuals, usually drawn from the same population, is also enrolled in the study and the individuals are asked about their past exposures. The two groups (those with and without the disease) are compared for differences in dietary intake and other exposures. The major advantage of this design is that an entire study can be completed in 4 years with as few as 400 cases and 400 controls. However, this study design can only answer questions about a single disease outcome. In addition, these studies can introduce potentially serious biases.\n\nTwo major concerns with case-control studies are recall bias and selection bias. In studies of chronic disease, investigators typically ask participants to recall behavior and other exposures (e.g., dietary intake) from 5\u201310 years in the past. Bias can occur when cases recall exposure to potential risk factors differently than controls. Selection bias occurs when controls agree to join the study because of an interest in health and are therefore more likely to exhibit healthy behavior (e.g., eat healthful diets). The higher prevalence of healthy behavior in the controls appears to be associated with reduced risk of disease when actually it is associated with willingness to participate in a research study on health. Another problem with case-control studies is that biomarkers (e.g., serum micronutrient concentrations) are potentially affected by the disease process and therefore may not be reliable indicators of long-term status (e.g., risk) in cases.\n\n#### 3. COHORT STUDIES\n\nThe cohort study typically enrolls people who are free of disease, assesses baseline risk factors, and follows the participants over time to monitor disease occurrence. The major advantage of cohort studies is that exposure to potential risk factors is assessed before the development of disease. Therefore, exposures such as self-reported dietary intake or serum micronutrient concentrations cannot be influenced by the disease process. In addition, cohort studies can examine many different exposures in relation to many different disease outcomes. A cohort study is generally a large enterprise because most diseases affect only a small proportion of a population, even if the population is followed for many years. These studies typically have sample sizes exceeding 50, 000, can have a total cost in excess of $100 million, and require that the cohort be followed for 10 or more years [16].\n\nBecause of the large size of these studies, the analysis of biologic markers (e.g., serum micronutrient concentrations) for all participants is prohibitively expensive. Therefore, cohort studies often archive (e.g., bank) serum, white blood cells, DNA, or other biologic specimens for the purpose of conducting nested case-control studies in the future. In a nested case-control study, a sample of cohort participants who develop a disease such as breast cancer (e.g., cases) are matched to other individuals in the cohort who do not develop the disease (e.g., controls). Biologic samples from cases and controls are retrieved and analyses are performed to determine whether there are differences in prevalence of exposures between the cases and the controls. This can be an efficient and powerful study design that avoids many of the pitfalls of the classic case-control studies.\n\n#### 4. INTERVENTION TRIALS\n\nIntervention trials prospectively examine the effect of a randomly assigned exposure, such as a low-fat diet, on an outcome such as disease occurrence, risk factor for a disease, or a biomarker. An important consideration when designing these studies is the degree of dietary control needed: controlled diet provided by the investigators vs. vitamin supplementation vs. dietary counseling. The stringency of dietary control is determined in part by the expected size of the response (e.g., change in disease risk) and the length of the treatment period required. For example, if the required dietary treatment period exceeds several months, a controlled feeding study is usually not logistically or financially viable.\n\nIn an intervention trial, the random assignment of participants to the control vs. the intervention group means that participants with predisposing conditions or unmeasured factors that might influence the outcome are equally likely to be randomized into the intervention or the control group. In addition, random allocation of the exposure eliminates the possibilities of selection bias and recall bias. However, these projects are usually expensive and labor intensive and, therefore, are only conducted for important public health questions where the observational data are suggestive, but considerable controversy remains in the scientific community.\n\n## IV. INTERPRETATION OF CAUSE AND EFFECT IN NUTRITIONAL EPIDEMIOLOGY\n\nGiven that nutritional epidemiology is the study of dietary intake and its association with disease risk, we must use scientific judgment in determining when the strength of the evidence supports a causal link between the exposure and the outcome. When assessing causality, important considerations include (1) the main measure of association used in epidemiologic studies; (2) the major alternative explanation for an observed association in observational studies, which is confounding; and (3) methods for assessing causality in studies of associations.\n\n### A. Measures of Association\n\nThe most commonly used measure of association between dietary intake and disease risk is relative risk. The relative risk (RR) estimates the magnitude of an association between the dietary exposure and disease and indicates the likelihood of developing the disease in the exposed group relative to those who are not exposed [17]. For example, an RR of 1.0 indicates that the incidence of disease in both the exposed and nonexposed groups is the same. An RR greater than 1.0 indicates a positive association. For example, an RR of 2.0 between dietary fat and colon cancer indicates that individuals eating a high-fat diet are twice as likely to develop colon cancer as those eating a low-fat diet. RRs less than 1.0 are typically considered protective. An RR of 0.5 for the association of vegetable intake with colon cancer risk indicates that among individuals with diets high in vegetables, the risk of colon cancer is approximately half compared to those with diets low in vegetables. Often RRs are given for the highest category of intake (e.g., highest quartile of fat or vegetable intake) in comparison to the lowest category of intake.\n\nGiven the degree of error present in dietary intake estimates, RRs in nutritional epidemiology rarely exceed 3.0. RRs are typically presented with their associated confidence interval (e.g., RR 2.0, 95% confidence interval of 1.3\u20132.9), which provides information on the precision of the point estimate (e.g., the RR). Specifically, it is the range within which the true point estimates lies with a certain degree of assurance. Typically 95% confidence intervals (CIs) are given, which corresponds to the traditional test of statistical significant, _p <_ 0.05. A 95% CI that does not include the null value (1.0) is, by definition, statistically significant at the _p =_ 0.05 level. The width of the CI also provides information about the variability in the point estimate, which is a function of sample size. Therefore, the wider the CI, the more variability in the measure, the smaller the sample size, and the less confidence we can have that the observed point estimate is the true point estimate.\n\nIt is important to separate the strength of an RR from its public health significance. For example, a large RR (e.g., RR 5. 0) might be observed between a certain food and a risk of disease. However if consumption of that food is rare in a population, then its overall impact on morbidity or mortality will be minimal. Conversely, an RR of 1. 5 might be very important from a public health perspective if the dietary exposure is common. Once RR estimates are used to ascertain causality, estimates of the impact of an exposure on public health (termed population attributable risk) become important in the development of policy and allocation of resources.\n\n### B. Confounding\n\nConfounding is the possibility that an observed association between the dietary intake and disease is actually due to other differences in the exposed vs. the nonexposed groups. Confounding is a critical concept in nutritional epidemiology because it is plausible that people who choose one behavior (e.g., healthful diets) might differ from those who did not choose that behavior with regard to other exposures (such as physical activity).\n\nFor example, a population-based study of participant characteristics associated with supplement use among 1,449 adults confirmed that supplement users were more likely to be female, older, better educated, nonsmokers, regular exercisers, and to consume diets higher in fruits and vegetables and lower in fat [18]. We also found previously unreported associations of supplement use with cancer screening, use of other chemopreventive agents (hormone replacement therapy and aspirin), and a psychosocial factor: belief in a diet\u2013cancer connection. These relationships could confound studies of supplement use and cancer risk in complex ways. For example, male supplement users were more likely to have had a prostate specific antigen test, which is associated with increased diagnosis of prostate cancer [19]. Therefore, supplement use could spuriously appear to be associated with increased incidence of prostate cancer. Alternatively, if early diagnosis of prostate cancer by because of a prostate specific antigen test reduces mortality, supplement users could spuriously appear to have lower prostate cancer mortality.\n\nThe observed relationship between supplement use and belief in a connection between diet and cancer is especially interesting. Health beliefs influence cancer risk through behavior such as diet and exercise. For example, in a previous prospective study, we found that belief in a connection between diet and cancer was a statistically significant predictor of changes to more healthful diets over time [20]. In cohort studies, the increasing healthfulness of supplement users' diets and other health practices over time could result in a spurious positive association between supplement use and chronic disease risk.\n\nIt is important to note that in studies in which nutrient intake is summed from foods and supplements, the intake of micronutrients in the highest exposure category often appears too high to be obtained from food alone and probably reflects supplement use [5]. Therefore, studies of nutrient intake may also be confounded by the relationship between supplement use and healthful lifestyle. In these studies, consistency of findings for the nutrient from foods and vitamin supplements separately would increase our confidence that an observed association was not confounded by supplement users' healthful lifestyles.\n\nIn theory, statistical adjustment in analyses for participant characteristics and major health-related behavior can control for some of the effects of confounding factors. However, absence of residual confounding cannot be assured, especially if other important confounding factors are unknown, not assessed, or not included in the analyses.\n\n### C. Evidence of Causality\n\nEpidemiology is the study of associations, and statistical methods provide the means for \"testing\" the association. However, it is important to note that the existence of a statistically significant association does not indicate that the observed relationship is one of cause and effect. For any observed association, the following questions should be considered:\n\n\u2022 How likely is it that the observed association is due to chance?\n\n\u2022 Could this association be the result of poor study design, poor implementation, or inappropriate analysis?\n\n\u2022 How well do these results meet other criteria of causality, as given in Table 2 [17]? Specifically, is the association weak or strong? Is there a plausible biologic mechanism? Did the exposure precede the outcome? Is there a dose\u2013response relationship?\n\nTABLE 2\n\nCriteria for Judging whether Observed Associations between Diet and Disease Risk Are Causala\n\naFrom Ref. 17.\n\n\u2022 How well do these results fit in the context of all available evidence on this association? Causality is supported when a number of studies, conducted at different times, using different methods, among different populations, show similar results.\n\nIn a field characterized by as much uncertainty as nutritional epidemiology, it is rare for a cause-and-effect relationship to be considered unequivocal. However, lack of complete certainty does not mean that we should ignore the information that we have or postpone action that appears needed at a given time [21]. It merely means that we exercise prudence and thoughtful consideration before acting on epidemiologic evidence.\n\n## V. OBSTACLES TO FINDING ASSOCIATIONS OF DIETARY INTAKE AND DISEASE RISK\n\nHere we review the major obstacles to epidemiologic research, including error in exposure assessment and limitations of study designs.\n\n### A. Sources of Error in Food Frequency Questionnaires\n\nTable 3 gives a summary of the potential sources of error and bias in estimating dietary intake using an FFQ. Many of these errors are respondent based, including problems with memory, errors in frequency judgments and portion size estimation, and social desirability bias. The form itself is a major source of error because of limitations inherent in closed-ended scannable response options, the use of a limited food list (generally about 100 items) to minimize respondent burden, inadequate food composition information, and the requirement that respondents average intake over long periods of time. Finally research indicates that dietary interventions themselves introduce reporting bias toward the more desirable responses [22].\n\nTABLE 3\n\nSources of Error and\/or Bias in Dietary Intake Estimates from a Food Frequency Questionnaire\n\n#### 1. ASSESSING THE RELIABILITY AND VALIDITY OF FOOD FREQUENCY QUESTIONNNAIRES\n\nReliability is generally used to refer to reproducibility, or whether an instrument will measure an exposure (e.g., nutrient intake) in the same way twice on the same respondents. Validity, which refers to the accuracy of an instrument, is a considerably higher standard. Generally a validity study compares a practical, epidemiologic measurement method (e.g., an FFQ) with a more accurate but more burdensome method (e.g., food records). Reliability and validity are typically investigated by means of statistical measures of bias and precision.\n\nIn a reliability study, reproducibility is assessed by comparing mean intake estimates from two administrations of the FFQ in the same group of respondents. If an instrument is reliable, the mean intake estimates should not vary substantially between the two administrations. In a validity study, bias is generally assessed by comparing the mean estimates from an FFQ to those from food records or recalls in the same respondents. This comparison allows us to determine whether nutrient intake estimates from an FFQ appear to be generally under-or overreported in comparison to the criterion measure. Bias is especially important when the objective is to measure absolute intakes for comparison to dietary recommendations or some other objective criteria. For example, bias is critical when estimating how close Americans are to meeting the dietary recommendation to eat five servings of fruits and vegetables per day.\n\nPrecision is concerned with whether an FFQ accurately ranks individuals from low to high nutrient intakes, which is typically the analytic approach used to assess associations of dietary intake with risk of disease. In this situation, bias in the estimate of absolute intake is not important as long as precision is good. In a reliability study, reproducibility is assessed as the correlation coefficient between nutrient intakes estimated from two administrations of the FFQ in the same group of respondents. In a validity study, precision is the correlation coefficient between nutrient intake estimates from the FFQ in comparison to a criterion measure (usually dietary recalls or records). Often FFQ studies also assess validity by ranking nutrient intake estimates, dividing them into categories (e.g., quartiles) and comparing these to similar categories calculated from another instrument. However, classifying a continuous exposure into a small number of categories does not reduce the effects of measurement error [1] and, therefore, this analysis does not provide additional information above correlation coefficients.\n\nIt is important to know that an instrument can be reliable without being accurate. That is, it can yield the same nutrient estimates two times and be wrong (e.g., biased upward or downward) both times. Alternatively, an instrument can be very reliable and consistently yield an accurate group mean (e.g., unbiased), but have poor precision such that it does not accurately rank individuals in the group from low to high in nutrient intake. Reliability is easy to measure, and nutrient correlation coefficients between two administrations of the same FFQ are generally in the range of 0.6\u20130.7. Estimates of reliability give an upper bound to the accuracy of an instrument. Whereas a high reliability coefficient does not imply a high validity coefficient, a low reliability coefficient clearly means poor validity.\n\nStudies comparing FFQs with records or recalls are often called validation studies. The theory behind this type of study is that the major sources of error associated with FFQs are independent of those associated with short-term dietary recall and recording methods, which avoids spuriously high estimates of validity resulting from correlated errors. As summarized by Willett 3], the errors associated with FFQs are the restrictions imposed by a fixed list of foods, perception of portion sizes, and the cognitive challenge of assessing frequency of food consumption over a broad time frame. These sources of error are only minimally shared by diet records, which are open ended, do not depend on memory, and permit measurement of portion sizes. Biases in food records result from coding errors and changes in eating habits while keeping the records. Like food records, dietary recalls are open ended. However, recalls are usually collected without advance notification. Therefore, participants cannot change what they eat retroactively and the instrument itself should not affect food intake. Bias in recalls results from estimation of portion sizes, participant memory, and coding errors. Nonetheless, it is apparent that there are correlated errors between FFQs and records or recalls. Social desirability could influence how participants record or recall food intake across all types of dietary assessment instruments [22, 23]. Participant error in estimating portion sizes could bias recall and FFQ estimates of intake in similar ways. There are also correlated errors in nutrient databases. For example, estimates of selenium intake from FFQs and food records are correlated, which is merely the result of correlated errors in the nutrient database. Finally, research using doubly labeled water to determine energy requirements has demonstrated significant underreporting of energy intakes from food records that may vary by participant characteristics [9]. (See [Chapter 2.) It is important to be aware of limitations of records and recalls as criterion measures of dietary intake and to interpret cautiously results based on these measures.\n\nA final important consideration is that an FFQ cannot, in and of itself, be validated. Only individual nutrient intake estimates can be validated by comparison of a nutrient estimate from an FFQ to a more accurate measure.\n\n#### 2. EFFECTS OF ERROR IN FOOD FREQUENCY QUESTIONNAIRES\n\nError in dietary assessment can be of two types, with markedly different consequences. Random error refers to mistakes such as inadvertently marking the wrong frequency column, skipping questions, and lapses in judgment. These errors introduce noise into nutrient estimates such that our ability to find the \"signal\" (e.g., an association of dietary fat and breast cancer) is masked or attenuated (biased toward no association).\n\nSystematic error refers to under-or overreporting of intake across the population (e.g., bias), but also person-specific sources of bias. For example, studies indicate that obese women are more likely to underestimate dietary intake than normal-weight women [9]. Systematic error may result in either null associations or spurious associations. In one report, Prentice [24] used data from FFQs collected in a low-fat dietary intervention trial to simulate the effects of random and systematic error on a association of dietary fat and breast cancer, where the true RR was assumed to be 4.0. Assuming only random error exists in the estimate of fat intake, the projected (i.e., observed) RR for fat and breast cancer would be 1.4. Assuming both random error and systematic error exists, the projected RR would be 1.1, similar to that reported in a recent meta-analysis on dietary fat and breast cancer [25]. These results clearly suggest that FFQs may not be adequate to detect many associations of diet with disease, even if a strong relationship exists. Therefore, it is not surprising that results from diet\u2013disease studies are often null or conflicting, given the error in our dietary assessment methods.\n\n### B. Limitations in Research Designs\n\n1. OBSERVATIONAL STUDIES\n\nIn studies of nutritional epidemiology, unique obstacles exist to finding clear and interpretable relationships between dietary intake and disease risk [15]. In roughly increasing order of importance, these obstacles include the following:\n\n\u2022 Current or recent dietary intake may differ from intake over the time frame relevant to the development of disease, which will reduce our ability to find associations between diet and disease.\n\n\u2022 Certain nutrient intakes within a population may not be highly variable. For example, energy from dietary fat in a population of postmenopausal women may only vary from 25\u2013 40%, resulting in inadequate range of disease risk to find an association with breast cancer. This situation is akin to assessing whether smoking causes cancer by studying men who smoke 1 pack per day in comparison to men who smoke 1.5 packs per day.\n\n\u2022 Diet is a complex mixture of foods and nutrients, including many highly correlated compounds, making it difficult to separate the effects of any one compound from other dietary factors.\n\n\u2022 Dietary intake may relate in a complicated manner to other risk factors such as hormonal status, obesity, or hypertension. These relationships (some of which may be in the causal pathway) make it difficult to appropriately control for confounding factors.\n\n\u2022 Measurement properties of existing dietary self-report instruments are largely unknown, although it is clear that there are many sources of random error and systematic error, both of which obscure our ability to find associations between dietary intake and disease risk.\n\nAn important point to consider is that most of the obstacles listed above will limit or attenuate our ability to find associations between dietary intake and disease. For example, as shown in Table 4, an observed association of dietary fat intake with body mass index (BMI) might appear too small to be clinically important. However, if we assume that significant measurement error exists in our estimate of fat intake (e.g., a correlation of 0.30 between our measure and \"true\" intake), then the real association would be 4.0 BMI points per 10 g of fat intake, which is considerably more important. Therefore, studies showing weak or no associations between dietary intake and disease (e.g., null results) need to be interpreted cautiously.\n\nTABLE 4\n\nEstimates of the Observed Associationa between Dietary Fat Intake (Grams Fat\/10) and BMI after Adjustment for Random Measurement Error in the Measure of Dietary Fat\n\nCorrelation coefficientb between the FFQ estimate and \"true\" fat intake | Observed increase in BMI for every 10 g of fat consumedc \n---|--- \n1.00 (FFQ is perfect measure of fat intake) | 4.0c \n0.70 (FFQ is a good measure of fat intake) | 2.8 \n0.50 (FFQ is a weak measure of fat intake) | 2.0 \n0.30 (FFQ is a poor measure of fat intake) | 1.2\n\na\u03b2observed = \u03b2true \u00d7 validity coefficient.\n\nbCorrelation coefficient from validity study comparing FFQ to multiple 24-hour recalls.\n\ncAssume true regression coefficient from a multivariate model predicting BMI equals 4.0.\n\nEven this cursory review of the obstacles to interpretation of observational studies of diet and disease makes it clear that these studies alone may not provide reliable information on the associations of dietary intake and disease, regardless of their size or duration.\n\n#### 2. LIMITATIONS OF CLINICAL TRIALS OF DIETARY INTAKE AND DISEASE RISK\n\nIn spite of the many desirable features of dietary intervention trials, unique obstacles are present in these types of studies [15], as summarized below.\n\nThe costs of the dietary intervention itself can be formidable. For example, the National Institutes of Health-sponsored Women's Health Initiative (WHI) is testing the impact of a \"low-fat eating pattern\" on the incidence of breast cancer, colorectal cancer, and coronary heart disease among 48, 837 postmenopausal women in the United States [26]. The dietary intervention requires participants to attend monthly sessions (run by specially trained nutritionists) for the first 18 months and then quarterly classes for the remainder of the trial, which will average 8.5 years. In addition, new intervention components are being added to the trial to encourage adherence. The costs of implementing this type of intervention far exceed those required for comparatively simple pill\u2013placebo trials.\n\nMaintenance of dietary adherence for a sufficient period of time to be able to ascertain clinical outcomes (e.g., disease risk) can be a formidable task. On one hand, the greater the difference in dietary intake between the intervention and control groups, the more likely the study will be able to detect an effect on the outcome. However, it is clearly more difficult to get participants to adhere to very strict or limited regimes, which can result in such poor adherence that the trial becomes futile.\n\nMonitoring of dietary adherence typically requires use of self-reported dietary instruments, with their attendant weaknesses (see above).\n\nIntervention trials can only test a specific intervention or intervention program, so that the effects of specific elements of the intervention may be unclear. For example, the WHI intervention tests a dietary pattern low in fat and high in fruits, vegetables, and grains. If this intervention is shown to reduce risk of breast cancer, the trial itself cannot separate out the effects of these dietary exposures without relying on dietary self-report.\n\nGiven the difficulties of delivering an effective dietary intervention and assessing the degree of dietary adherence, null trial results can be ambiguous.\n\n## VI. FUTURE RESEARCH DIRECTIONS\n\nAs is apparent from this overview of nutritional epidemiology, the biggest challenge is that of addressing random, systematic, and person-specific sources of error in dietary assessment. Only when well-designed, true validity studies clarify these sources of error will we be able to markedly improve our ability to draw valid inferences from epidemiologic studies of diet and disease.\n\nAn exciting area of future research concerns diet\u2013gene interactions in the etiology and pathogenesis of many chronic diseases 27]. Despite the vigorous investigation of environmental causes of disease, it has long been recognized that not all persons exposed to the same risk factors will develop the associated disease [28]. For example, although it is well accepted that smoking causes lung cancer, only 10\u201315% of smokers will be diagnosed with the disease in their lifetime [29]. More and more, we are beginning to understand the impact of differential genetic susceptibility in the etiology and pathogenesis of common diseases such as coronary heart disease and cancer. If only a subgroup of individuals is sensitive to certain dietary exposures, the effect will be diluted and the association will be undetectable when the entire population is the focus of study. Better understanding of these individual susceptibilities has the potential to bring considerable clarity to nutritional epidemiologic research. (See [Chapters 11\u2013 on genetics and nutritional health.)\n\nTo summarize, in spite of all the historical difficulties in obtaining consistent evidence of associations between diet and chronic diseases, considerable optimism remains that nutrition offers great hope in reducing morbidity and mortality from many chronic diseases. While the elimination of error in dietary assessment methods is probably not a realistic objective, a better understanding of these errors (based on objective biomarkers), combined with statistical methods to address these errors, may be a reachable goal. It is the combined contribution of many different study types (e.g., observational, intervention, biomarker, mechanistic feeding studies, genetic susceptibility studies) that offers the greatest potential for identification of lifestyle strategies for disease prevention.\n\n## Acknowledgment\n\nThe author thanks Ann Shattuck for her careful review and thoughtful comments on this chapter.\n\nReferences\n\n1. Armstrong B.K., White E., Saracci R. Principles of exposure measurement in epidemiology. _Monographs in Epidemiology and Biostatistics_. Baltimore, MD: Oxford University Press; 1994;Vol. XXI:22\u201348\n\n2. Potter J.D. Colorectal cancer: Molecules and populations. _J. Nat. Cancer Inst_. 1999;91:916\u2013932.\n\n3. Willett W. _Nutritional Epidemiology_ , 2nd ed. Oxford: Oxford University Press; 1998.\n\n4. Kristal, A. R., Vizenor, N. C., Patterson, R. E., Neuhouser, M. L., and Shattuck, A. L. Validity of food frequency based measures of fruit and vegetable intakes. _Cancer Epidemiol. Biomarkers Prevent._ (in press).\n\n5. Patterson R.E., White E., Kristal A.R., Neuhouser M.L., Potter J.D. Vitamin supplements and cancer risk: A review of the epidemiologic evidence. _Cancer Causes Control_. 1997;8:786\u2013802.\n\n6. Suzukawa M., Ayaori M., Shuge H., Hisada T., Ishikawa T., Nakamura H. Effect of supplementation with vitamin E in LDL oxidizability and prevention of atherosclerosis. _Biofactors_. 1998;7:51\u201354.\n\n7. Patterson R.E., Kristal A.R., Carter R.A., Fels-Tinker L., Bolton M.P., Agurs-Collins T. Measurement characteristics of the Women's Health Initiative food frequency questionnaire. _Ann. Epidemiol_. 1999;9:178\u2013197.\n\n8. Patterson R.E., Kristal A.R., Levy L., McLerran D., White E. Validity of methods used to assess vitamin and mineral supplement use. _Am. J. Epidemiol_. 1998;148:643\u2013649.\n\n9. Black A.E., Prentice A.M., Goldberg G.R., Jebb S.A., Livingstone M.B., Coward W.A. Measurement of total energy expenditure provide insights into the validity of dietary measurements of energy intake. _J. Am. Diet. Assoc_. 1993;93:572\u2013579.\n\n10. Armstrong B., Doll R. Environmental factors and cancer incidence and mortality in different countries with special reference to dietary practices. _Int. J. Cancer_. 1975;15:617\u2013631.\n\n11. Gray G.E., Pike M.C., Henderson B.E. Breast cancer incidence and mortality rates in different countries in relation to known risk factors and dietary practices. _Br. J. Cancer_. 1979;39:1\u20137.\n\n12. Prentice R.L., Sheppard L. Dietary fat and cancer: Consistency of the epidemiologic data and disease prevention that may follow from a practical reduction in fat consumption. _Cancer Causes Control_. 1990;1:81\u201397.\n\n13. Roberts D.C. Dietary factors in the fall in coronary heart disease mortality. _Prostag. Leukotr. Essent. Fatty Acids_. 1991;44:97\u2013101.\n\n14. Ziegler R.G., Hoover R.N., Pike M.C., et al. Migration patterns and breast cancer risk in Asian-American women. _J. Natl. Cancer Inst_. 1993;85:1819\u20131827.\n\n15. Prentice R.L. Fat and fiber and breast cancer research\u2014where is the field going? _Breast Cancer Res_. 2000;2:268\u2013276.\n\n16. Rothman K.J. Types of epidemiologic studies. In: _Modern Epidemiology_. Oxford: Little, Brown and Company; 1986.\n\n17. Hennekens C.H., Buring J.E. Epidemiology in Medicine. Mayrent S.L. Boston: Little, Brown and Company; 1987.\n\n18. Patterson R.E., Neuhouser ML., White E., Hunt J.R., Kristal A.R. Cancer related behavior of vitamin supplement users. _Cancer Epidemiol. Biomarkers Prev_. 1998;7:79\u201381.\n\n19. Gann P.H. Interpreting recent trends in prostate cancer incidence and mortality. _Epidemiology_. 1997;8:117\u2013120.\n\n20. Patterson R.E., Kristal A.R., White E. Do beliefs, knowledge, and perceived norms about diet and cancer predict dietary change? _Am. J. Public Health_. 1996;86:1394\u20131400.\n\n21. Hill A.B. The environment and disease: Association or causation?. _Proc. R. Soc. Med_. 1965;65:58\u2013295\n\n22. Kristal A.R., Andrilla C.A.H., Koepsell T.D., Dieht P.H., Cheadle A. Dietary assessment instruments are susceptible to intervention-associated response set bias. _J. Am. Diet. Assoc_. 1998;98:40\u201343.\n\n23. Hebert J.R., Clemow L., Pbert L., Ockene I.S., Ockene J.K. Social desirability bias in dietary self-report may compromise the validity of dietary intake measures. _Int. J. Epidemiol_. 1995;24:389\u2013398.\n\n24. Prentice R.L. Measurement error and results from analytic epidemiology: Dietary fat and breast cancer. _J. Natl. Cancer Inst_. 1996;88:1738\u20131747.\n\n25. Hunter D.J., Speigelman D., Adami H.O., Beeson L., van den Brandt P.A., Folsom A.R., Fraser G.E., Goldbohm R.A., Graitam S., Howe G.R., Kushi L.H., Marshall J.R., McDermott A., Miller A.B., Speizer F.E., Wolk A., Yaun S-S., Willett W. Cohort studies of fat intake and the risk of breast cancer\u2014a pooled analysis. _N. Engl. J. Med_. 1996;334:356\u2013361.\n\n26. Women's Health Initiative Study Group. Design of the Women's Health Initiative clinical trial and observational study. _Control. Clin. Trials_. 1998;1:61\u2013109.\n\n27. Patterson R.E., Eaton D.L., Potter J.P. The genetic revolution: Change and challenge for the profession of dietetics. _J. Am. Diet. Assoc_. 1999;99:1412\u20131420.\n\n28. Khoury M.J. Genetic epidemiology. In: Rothman K., Greenland S., eds. _Modern Epidemiology_. 2nd ed. Boston: Little, Brown and Company; 1997:609\u2013621.\n\n29. American Cancer Society. _Cancer Facts and Figures_. Boston: American Cancer Society; 1995.\nCHAPTER 5\n\nAnalysis, Presentation, and Interpretation of Dietary Data\n\nDEBRA COWARD-McKENZIE and RACHEL K. JOHNSON, The University of Vermont, Burlington, Vermont\n\n## I. INTRODUCTION\n\nNutritional epidemiological studies, while not an exact science, play a critical role in relating dietary intake to risk of disease. These investigations often require the gathering of dietary intake data from various samples, which must then be translated into a usable form. This chapter discusses what is done with the dietary data once they have been collected. This includes _analysis,_ the examination of the dietary data to determine the nutritional composition of the subjects' diet; _presentation,_ presenting the data from the research in a logical form, often done by comparing the analyzed data to a standard; and _interpretation,_ the translation of the data\u2014what do the data really tell us?\n\n## II. ANALYSIS OF DIETARY DATA\n\nThe methods most often used to obtain dietary intake information for research investigations include dietary recalls, food records or diaries, and food frequency questionnaires (FFQs). Dietary recalls and food records provide detailed descriptions of the types and amounts of food and beverages consumed throughout a specified period of time, normally 1 (24-hour recall) to 7 days. The FFQ provides a less detailed list of selected foods and the frequency of their consumption in the past. (See Chapter 1 for further description of these methods.) The data received must then be analyzed to determine the total intake of nutrients or food components consumed by each subject.\n\n### A. Computer-Based Analysis\n\nIn the past, analysis was performed manually. This process was painstakingly tedious and expensive, requiring a highly trained person to code and enter data. Coding included looking up every food in a table to find a code number to be entered. Amounts were entered by unit and a multiplier. These coding techniques required many calculations to be performed by hand, leaving numerous possibilities for error. Now, a variety of computer-based food composition databases and nutrient computation systems are available in which the foods can be directly entered by name and computation of nutrient values are automated. The accuracy of the data obtained from these systems will differ, depending on several factors.\n\n1.: Updating of the database. New foods are constantly being added to the market, so the best databases are updated often to keep up with the changes. Most databases use the U.S. Department of Agriculture (USDA) Nutrient Database for Standard Reference (SR) as their primary source of nutrient data. The SR contains information once published in the Agriculture Handbook 8, but it is no longer available in the printed form [1]. The most recent release of the SR, number 13 [2], was issued in November 1999 and contains more than 6000 items. The USDA receives information from industry, scientific literature, government agencies, and contracted laboratories and universities [3]. Although the information is not complete, specific criteria have been established for evaluating foods to ensure the data are as accurate as possible [4]. Many databases also add information from specific food manufacturers to provide information on name brand foods not available in the SR.\n\n2.: The number and types of food items available. This is particularly important for recalls and food records or for FFQs containing \"Write-in\" sections where all foods must be given nutrient values. In regions with ethnocultural diversity, special care must be taken when selecting databases [5]. Databases that contain a variety of ethnic foods will provide greater accuracy and will require less manual entry of nutrient values for foods.\n\n3.: The ability to add foods or nutrients. This is most important for those investigations in areas with multiethnicity or when there is a high tendency for the subjects to include restaurant foods that may not be included in the database. The ability to adapt or add recipe information should also be available. For example, if a subject had homemade beef stew for lunch, the database should allow the coder to either add or delete ingredients from an existing recipe or add a new recipe to the file.\n\n4.: The ease of data entry and analysis. It is no longer necessary to code diets manually. Systems should be easy to use to avoid unnecessary coding errors that can occur. Entry of products by name, particularly brand names, should be available. Some research databases, such as the Food Intake Analysis System [6], offer default options. These choices provide average estimations for foods for which exact information is not known. For example, if a subject had chicken breast, but was not sure of the cooking method or the serving size, the coder has the default option to choose from instead of making guesses. These options can help decrease differences in nutrient intake values caused by multiple coders or data entry technicians.\n\n5.: The nutrients available. Not every database contains all nutrients and some contain more accurate data for particular nutrients. Systems should be evaluated for the accuracy of the nutrient values that are being studied. Analysis should include the option of choosing nutrient calculations for each food as well as summaries for an individual meal or day.\n\n6.: The handling of missing nutrient values. If a specific nutrient value is unknown for a particular food, the way the database handles the missing information may affect the accuracy of the data. Some systems intelligently impute values, while others simply use a value of zero. An imputed value is almost always a better estimation [7]. However, imputing nutrient values is a labor-intensive task and requires nutritionists with knowledge of data evaluation and imputing procedures. Therefore, caution must be taken when using databases with imputed values, because few database developers have access to qualified nutrition-trained personnel required for accurately estimating values [8].\n\nWhen computing nutrient intake from food consumption data, it is assumed that the nutrient quality and content of certain foods are virtually constant, and that what is consumed is available for use. However, we know that this assumption is not totally correct. There are various reasons why the actual value of a consumed nutrient may differ from the calculated value. The level of certain nutrients in foods may be affected by differences in growing and harvesting conditions (i.e., selenium [9, 10]), storage, processing, and cooking (i.e., vitamin C [11, 12]). Databases have tried to account for some of the differences by increasing the databanks to include preparation methods, cuts of meat, and specific manufacturers for processed food. For example, if chicken is entered into the database, the coder may have approximately 455 items from which to choose. This large number includes name brand foods, particular pieces of chicken available (i.e., breast or thigh), and cooking methods (i.e., baked or fried, cooked with skin on or off, skin eaten or not). Because so many choices are offered, recalls should be as detailed as possible to provide enough information to make an accurate selection.\n\nThe use of controlled feeding trials in a study, such as the Dietary Approaches to Stop Hypertension (DASH) trial [13], can help alleviate some of the differences between the calculated and actual nutrient values of food. The DASH trial was a multicenter study designed to compare the effects of dietary patterns on blood pressure. The subjects were asked to consume only foods prepared by the centers. Menu items could then be analyzed in a laboratory to obtain nutrient values [14]. Food procurement, production, and distribution guidelines were set and strictly adhered to at all sites to ensure that menus consistently met nutrient goals. For example, food items were given specific purchasing sizes, detailed descriptions, and\/or defined brand names to ensure that all site recipes were of uniform composition [15]. When possible, foods can be obtained from central suppliers to further eliminate any differences in nutritional content of foods due to regional variations in a study of this type.\n\nThe diet as a whole can also affect the availability of some nutrients. For example, high-fiber diets may decrease the availability of certain nutrients, such as zinc and iron [16, 17], generally due to the binding effects of phytate. Computer-based analysis programs do not generally examine the overall diet and cannot determine how nutrient\u2013nutrient interactions may affect availability. Iron, for example, is a mineral for which intake is not a good marker for availability. The absorption of iron is subject to many components: (1) the source of iron (heme is more easily absorbed than nonheme); (2) the iron status of the individual (decreased stores increase absorption); and (3) the overall composition of the meal. All of these components play a role in determining how much of the iron consumed is available to the body [17]. In turn, iron consumption can also affect the absorption of other nutrients, such as zinc. Nutrient\u2013nutrient interactions can greatly determine how well a calculated nutrient value represents the actual available amount of a nutrient.\n\nOther factors that should be taken into account are drug\u2013nutrient interactions and those people who may be malnourished or suffer from malabsorption. The elderly, for example, have decreased ability to absorb vitamin B12. This group is also at higher risk for drug\u2013nutrient interactions because they are often prescribed many medications. Researchers must be aware of any illnesses or medications taken by subjects that could interfere with nutrient absorption.\n\nThe development of fat-blocking drugs, i.e., Orlistat [18], can decrease absorption of not only the fat, which will decrease actual fatty acids and calories absorbed, but also other fat-soluble nutrients. Orlistat is an intestinal lipase inhibitor approved for the treatment of obesity [18]. This drug has been shown to decrease absorption of fat consumed, which also reduces fat-soluble vitamins, vitamins A, D, E, and K, and other compounds.\n\nOlestra, a sucrose esterified with fatty acids, has been approved by the U.S. Food and Drug Administration (FDA) as a fat replacer in savory snacks [19]. Studies concluded that when Olestra was eaten with foods containing fat-soluble vitamins it decreases the absorption of these nutrients. Although these vitamins are added to the Olestra-containing foods, actual absorption may still be affected for constituents not fortified in olestra-containing foods such as carotenoids.\n\nAlthough food composition databases are increasingly becoming more accurate and may be closer to actual values of energy intake than laboratory analysis 14], they cannot provide exact measurements for all nutrient intakes. Furthermore, even if these values are determined to be accurate, intake does not necessarily mean the nutrient is available for use. To obtain more accurate information on nutrient status, other methods, such as biomarkers (see [Chapter 10), should be employed. Also, familiarity with the subjects being investigated is essential for more accurate calculations. This includes, but is not limited to, considering factors such as supplementation, medications used, specific diseases or illness, as well as special diets (i.e., vegetarian).\n\n### B. Total Diet Analysis\n\nBecause accurate measures of nutrient values based on food intake are difficult to calculate, some investigators have begun to use innovative ways to evaluate the overall diet. Although using these techniques will not provide exact numbers for specific nutrients, they will give investigators a better picture of overall diet quality and health risk. With the increasing evidence that other non-nutrient constituents, such as phytochemicals, may play a role in disease prevention [20], these indexes could prove to be very useful.\n\nIn reviewing the indexes of overall diet quality, Kant [21] found that there were three major approaches to the development of indexes: (1) derived from nutrients only, (2) based on foods or food groups, and (3) based on a combination of nutrients and foods. The definition of \"diet quality\" differs based on the attributes chosen by the investigators of each index [21], so the index chosen will depend on the needs of the study. Those indexes based on nutrients only tend to look at the percentage of the Recommended Daily Allowance (RDA) consumed as a marker for diet quality. Those based on foods and food groups examine the intake patterns of foods to identify patterns associated with adequacy [21].\n\nAlthough numerous tools are available for examining the overall diet quality [21] those most commonly applied are based on the combination of nutrients and foods. These indexes, including the Healthy Eating Index (HEI) [22] and the Diet Quality Index (DQI) [23, 24] use the Dietary Guidelines for Americans [25] and the Food Guide Pyramid [26] to score the overall diet. These are based on the premise that if the USDA Food Guide Pyramid and guidelines are followed, including a variety of foods within each food group, the resulting diet will be adequate in nutrients and promote optimal health [27].\n\nPatterson _et al._ [23] were among the first to relate diet quality to the Dietary Guidelines for Americans. The DQI included measures of eight food groups and the recommendations from the Committee on Diet and Health of the National Research Council Food and Nutrition Board, published in 1989 [28]. The index was revised by Haines _et al.,_ called the Dietary Quality Index\u2013Revised (DQI-R) [24], to reflect the updated guidelines and Food Guide Pyramid. The DQI-R incorporates both nutrients and food components to determine diet quality. It is based on 10 components, with a 100-point scale, each component worth 10 points (Table 1). Components are based on total fat and saturated fat as a percentage of energy, milligrams of cholesterol consumed, recommended servings for fruit, vegetables, and grains, adequacy of calcium and iron intake, dietary diversity and dietary moderation. The dietary diversity, score was developed to show differences in intake across 23 broad food group categories including seven grain-based products, seven vegetable components, two fruit and juice categories, and seven animal-based products [24]. Dietary moderation scores added sugars, discretionary fat, sodium intake, and alcohol intake. The DQI-R was designed to monitor dietary changes in populations, but can provide an estimate of diet quality for an individual relative to the national guidelines and can note improvement or decline of diet quality with multiple calculations [24].\n\nTABLE 1\n\nDiet Quality Index\u2013Revised\n\naScoring range for each component is 0 (min) to 10 (max).\n\n_Source:_ Adapted from Haines, P. S., Siega-Riz, A. M., and Popkin, B. M. (1999). The Diet Quality Index revised: A measurement instrument for populations. Copyright \u00a9 The American Dietetic Association. Reprinted by permission from Journal of The American Dietetic Association, Vol. 99:697\u2013704.\n\nThe HEI [22, 27] was first developed by the USDA Center for Nutrition Policy and Promotion (CNPP) to assess and monitor the dietary status of Americans using the 1989 data from the Continuing Survey of Food Intake of Individuals (CSFII) [27], and again in 1994\u20131996 [22]. It also uses both nutrients and food components to determine overall diet quality. Like the DQI-R, it is based on a 10-component, 100-point scale (Table 2). Components are based on recommendations of servings for fruits, vegetables, grains, milk, and meat; percentages of energy from total fat and saturated fat; cholesterol and sodium intake; and a food variety score. Dietary variety is assessed by totaling the number of different foods eaten in a day in amounts that contributed at least one-half of a serving for any food group [22].\n\nTABLE 2\n\nHealthy Eating Indexa\n\nComponent | Max score (10) criteria | Min score (0) criteria \n---|---|--- \nGrains | 6\u201311 servings | 0 servings \nVegetables | 3\u20135 servings | 0 servings \nFruit | 2\u20134 servings | 0 servings \nMilk | 2\u20133 servings | 0 servings \nMeat | 2\u20133 servings | 0 servings \nTotal fat (% energy intake) | \u226430% | \u226545% \nSaturated fat (% energy intake) | >10% | \u226515% \nCholesterol intake | \u2264300 mg | \u2265450 mg \nSodium intake | \u22642400 mg | \u22654800 mg \nFood variety | \u22658 different items\/day | \u22643 different items\/day\n\naEach component has a score range of 0 to 10. The number of food group servings is based on recommended energy intake.\n\n_Source:_ Adapted from Kennedy, E. T., Ohls, J., Carlson, S., and Fleming, K. (1995). The Healthy Eating Index: Design and applications. _J. Am. Diet. Assoc._ **95,** 1103\u20131108.\n\nCategorizing foods into appropriate groups, particularly combination foods, can be a problem when using these analysis techniques. Cleveland _et al._ [29] developed a method for assessing food intakes in terms of food servings. These guidelines help overcome two major obstacles when assessing food intake with respect to the dietary guidelines, including dealing with food mixtures and differing units of measurement used. Because many foods are eaten as mixtures and difficult to categorize into food groups, Cleveland _et al._ [29] developed a recipe file that helps break down food mixtures into ingredients so they can be assigned their respective groups more easily (see Fig. 1). Standard serving sizes were assigned gram weights to help overcome the units problem, allowing for the use of only one unit of measure. The 1994 USDA-CSFII database data file [30] incorporates these guidelines. Although combined foods must still be separated and coded before entering, this file does provide a more refined method for developing reproducible data [24].\n\nFIGURE 1 Example of a cascaded recipe file showing multiple levels of disaggregation. Italics indicates level of disaggregation required for classification according to food guidance definitions. [Reprinted with permission from Cleveland, L. E., Cook, D. A., Krebs-Smith, S. M., and Friday, J. (1997). Methods for assessing food intakes in terms of servings based on food guidance. _Am. J. Clin. Nutr._ **65** (suppl), 1254S\u20131263S. \u00a9 _Am. J. Clin. Nutr._ American Society for Clinical Nutrition.]\n\n## III. PRESENTATION OF DATA\n\nOnce the data gathered from the dietary assessment methods have been analyzed, they must then be presented in a useful fashion. This is often done by comparing the analyzed data to a standard. These standards may include the Dietary Reference Intake (DRI) or comparison to a national average, such as the National Health and Nutrition Examination Surveys (NHANES) or the USDA (CSFII) data.\n\nThe DRIs are a set of nutrient-based reference values. This set includes an estimated average requirement (EAR), a Recommended Dietary Allowance (RDA), and an adequate intake (AI), which are defined by nutrient adequacy and may relate to the reduction of the risk of chronic disease [31]. Once the EARs have been established they are used to set the new RDAs, which should be used as a daily intake goal by healthy individuals, and should be sufficient to meet the needs of 97\u201398% of all healthy individuals. If there is not sufficient evidence to determine an EAR, then an AI is set, once again based on groups of healthy individuals. A tolerable upper intake level (UL) is also set where information is available as an indicator of excess for nutrients [31].\n\nEach value has a specific goal and use [31] (Table 3). For example, the EAR is the estimate that is believed to meet the nutrient needs of half of the healthy individuals in a gender or life-stage group. When assessing nutrient intake of healthy groups, it is highly recommended that the EAR be used instead of the RDA [31]. However, because DRIs have not been set for all nutrients at this time, the old RDAs can be used for these nutrients until new guidelines have been established.\n\nTABLE 3\n\nUses of Dietary Reference Intakes for Healthy Individuals and Groups\n\n_Key:_ EAR, estimated average requirement; RDA, recommended dietary allowance; AI, adequate intake; UL, tolerable upper intake level.\n\naRequires statistically valid approximation of usual intake.\n\n_Source:_ Standing Committee on the Scientific Evaluation of Dietary Reference Intakes; Food Nutrition Board, Institute of Medicine. (1997). \"Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride.\" National Academy Press, Washington, DC. Reprinted with permission.\n\nDRIs are set for specific subgroups based on age and gender. They are to be applied to healthy populations and may not be adequate for those who are or have been malnourished, or have certain diseases or conditions that increase nutrient requirements. For individuals, the RDA and AI can serve as a goal for nutrient intake.\n\nSome researchers use national surveys as a standard when presenting dietary data. The NHANES and CSFII are both surveys that regularly collect data on the nation's nutritional status. The NHANES are conducted approximately every 5 years, while the CSFII is conducted annually. NHANES provides medical history, physical measurements, biochemical evaluation, physical signs and symptoms, and diet information from 24-hour recalls. The CSFII provides diet information based on two 24-hour recalls. Researchers may wish to compare their subjects' data to the information obtained from these surveys to determine how their sample compares to the national average. Although the data from these surveys may be applied to certain subgroups, such as specific age groups, gender, socioeconomic levels, education levels and some ethnic groups, they cannot be used as guides for others, such as malnourished or specific disease states.\n\nResearchers who choose to use the HEI or the DQI-R report their data by using the 100-point scale. For example, the HEI scores are based on the assumption that a \"good\" diet has a score greater than or equal to 80, a diet that \"needs improvement\" has a score of 51 to 80, and those diets that score 50 or below are considered \"poor\" [22]. When this method was applied to the CSFII 1994\u20131996 data, the average mean score for the 3 years was 63 [22]. The CSFII researchers found that 70% of the population had diets that needed improvement, 12% had good diets, and 18% had poor diets. Researchers may also compare the scores from their subjects' data to the national scores for that time period, when available, to determine if their study population falls in line with the national average.\n\n## IV. INTERPRETATION OF DATA\n\nOnce the dietary intake data have been analyzed and compared to a standard, researchers must then look at the results to determine what the data really mean. How the data are interpreted can depend on the assessment method used and the nutrient being studied, the study type, and the accuracy of the subject's responses.\n\n### A. Assessment Methods\n\nThe assessment method chosen for use in a study can determine how the data collected can be interpreted. Recalls and records gather present intake data, while FFQs provide data based on past intake. It is known that an individual's nutrient and energy intake varies not only from day to day, but season to season as well. So if past intake is needed, FFQs may be the better choice.\n\nThe number of days of food intake records or recalls available can also affect the interpretation of the data. If high levels of accuracy are needed for an individual's intake of a nutrient, a greater number of days will be required than if a group average could be used. Care must be taken when determining the number of days to use in a study. For example, researchers using data from a single 24-hour recall from 832 men estimated their fat intake and determined that eating saturated fat prevents strokes [32]. Because of the day-to-day variability, dietary changes or recommendations for an individual should not be based on a single day's intake. Basiotis _et al._ [33] determined the number of days of food intake data needed to estimate individual intake as well as group intake for food energy and 18 nutrients. They found that for a female, an average of 35 days is needed to determine a true average of energy intake for each individual, while 3 days of food records from each subject are required to determine a group average.\n\nIt is also important that data obtained from dietary assessment methods be utilized properly. The FFQ was developed to rank nutrient intakes from low to high. It was not intended to be used to determine and develop levels of nutrient intake to prevent disease. However, many researchers have chosen to use the FFQ for this purpose. For example, studies based on FFQ data recommended levels of vitamin E intake to prevent heart disease [34, 35]. Recently, studies have concluded that these levels may not have the effect on heart disease development as previously suggested [36]. When choosing a method for dietary assessment, care must be taken to ensure that the data are properly interpreted.\n\n### B. The Hierarchy of Scientific Evidence\n\nTo make a judgment about dietary changes, the hierarchy of scientific evidence should be kept in mind (Fig. 2). The most rigorous evaluation of a dietary hypothesis is the randomized feeding trial, which should ideally be conducted as a double-blind experiment. Large, randomized feeding trials may eventually provide definitive answers to some of the questions we have regarding the relationships between dietary factors and the major illnesses of our society. Unfortunately, these are extremely costly, and may be of long duration if the disease studied has a long latency period. For now, our knowledge of many of these relationships has been derived mainly from cohort studies.\n\nFIGURE 2 Hierarchy of scientific evidence.\n\n### C. Data Validity\n\nOne major concern when interpreting dietary data is the accuracy of the information reported (see Chapter 4). Because most of the information gathered is self-reported, the reliability and validity of the data depend on the reporter's motivation, memory, ability to communicate, and awareness of the foods consumed. Most methods have been proven to be generally reliable; that is, they will provide the same estimate on different occasions. However, do the techniques gather true and accurate measurements, or valid measurements, of what people are really eating? In the past, assumptions were made that the information was indeed valid. The validity of the techniques was often verified by comparing the different methods to each other. For example, a FFQ may be \"validated\" by comparing results to food records. If the food records are 100% accurate, this method of validation would be fine. However, because both methods require self-reported intake, the validity of both methods should be questioned. Therefore, external independent markers of intake are needed for true validation [37].\n\nDuring the 1980s, the search began for biochemical indicators that closely reflect dietary intake. These indicators, a type of biomarker, measure specific variables in body fluids or tissues to reflect intake of a food component 38]. Doubly labeled water (DLW) is the most widely used biomarker for energy intake at this time (see [Chapter 2 for further details). Although this method can only determine the accuracy of a dietary intake method with respect to total energy, it is reasonable to assume that if that method is accurate (or not) for energy, it will also be so for specific macro- and micronutrients [39, 40]. Therefore, it is accepted that if group estimates of energy intake are found to be truly valid using a method such as DLW, the estimates of macro- and micronutrient intakes can also be considered valid [41].\n\nOther biomarkers that have been used to validate nutrient intake (also discussed in Chapter 4) include fatty acid patterns in blood to reflect fatty acid intake [42], urinary nitrogen to validate protein intake [43\u201345], and serum carotenoids and vitamin C concentrations as markers of fruit and vegetable consumption [46, 47]. With the increased use of these biomarkers, particularly DLW, to determine the accuracy of dietary intake data in a variety of subjects, it has become clear that the traditional dietary assessment methods are not completely accurate. It is now well accepted that misreporting of food intake\u2014over- or underreporting\u2014is widespread and that no data gathering method is immune [37].\n\n#### 1. OVERREPORTING\n\nOverreporting occurs when reported intakes are higher than the measured energy expenditure levels. Overreporting has not been found to be as big of a problem as underreporting, but still has the potential to interfere with research data. Johansson _et al._ [48] found that individuals who overreport tend to be younger, and with lower body mass indexes, often considered lean. The highest proportion of overreporters was found among those subjects who wanted to increase body weight. While overreporters are not common, care should be taken when obtaining data from subjects that are at highest risk.\n\n#### 2. UNDERREPORTING\n\nUnderreporting occurs when reported intakes are much lower than measured energy expenditure levels. These reports are often so low that basal metabolic needs could not be met and are not biologically plausible. Depending on the age, gender, and body composition of a given sample, underreporters may compose anywhere from 10\u201345% of the total sample. It is now understood that underreporting tends to be associated with certain groups. It is well accepted that the obese are at greatest risk of underreporting dietary intake. Many studies have shown that the obese underreport more often and to a greater degree\u201430\u201347%\u2014than the lean [49, 50]. Women have also been found to underreport more often than men [51\u201353]. Other characteristics of low energy intake reporters include those associated with low-socioeconomic status, that is, low-income and low-education levels [51\u201353]. Table 4 provides a summary of groups who have been found to be underreporters. These factors are further complicated due to the fact that they are also risk factors for many chronic diseases.\n\nTABLE 4\n\nSummary of Underreporting\n\naU.S. Survey from: Kreb-Smoth, S. M., Graubard, B., Cleveland, L., Subar, A., Ballard-Barbash, R., and Kahle, L. (1998). Low-energy reporters vs others: A comparison of reported food intakes. _Eur. J. Clin. Nutr._ **52** (2), S18.\n\nbBritish Survey from Bingham, S. A., Cassidy, A., Cole, T. J., Welch, A., Runswick, S. A., Black, A. E., Thurnham, D., Bates, C., Khaw, K. T., Key, T. J. A., Day, N. E. (1995). Validation of weighed records and other methods of dietary assessment using the 24 h urine nitrogen technique and other biological markers. _Br. J. Nutr._ **73,** 531\u2013550.\n\n#### 3. REASONS FOR UNDERREPORTING\n\nOnce underreporting became well accepted and documented, researchers began, and continue, to look for reasons why people underreport. We know that being obese is not the cause of underreporting, but it is most likely the psychological and behavioral characteristics associated with obesity that lead to underreporting [54, 55]. A need for social acceptance, a desire to be liked or accepted by the interviewer, may cause the subject to underreport \"sinful\" foods. A high level of body dissatisfaction, that is, if a person sees a leaner physique as being healthier or more desirable than their own, may cause him or her to misreport foods. Also, researchers have found that women who scored higher on restrained eating scales, those who feel they are making a conscious effort to avoid certain foods, tend to underreport as well [54, 55].\n\n#### 4. FOODS MOST OFTEN UNDERREPORTED\n\nIf underreporting occurred in all foods and nutrients to the same degree, the solution would be to add a correction factor to the data of the underreporters. This would bring their reported intake of all nutrients into line with that of the valid reporters. Unfortunately, it has become clear that underreporters often fail to report those foods that are seen as \"bad\" or \"sinful\" [40]. In a U. S. survey of 8334 adults, 1224 were found to be low energy reporters [56]. Some of the foods that were found to be most often underreported included cakes\/pies, savory snacks, cheese, white potatoes, meat mixtures, regular soft drinks, fat-type spreads, and condiments [56]. British researchers found that little difference was seen between underreporters and normal energy reporters with regard to bread, potatoes, meat, vegetables, or fruit, but a significant difference was seen with cakes, sugars, fat, and breakfast cereal [44]. Table 4 gives a summary of underreported foods. Some researchers have found that underreporters tend to report lower intakes of fat, and higher intakes of protein and carbohydrates as a percentage of total energy [57, 58], while others show that reports of added sugar intake are significantly lower [59]. No agreement has been reached as to how much, if at all, specific macronutrients are misreported. Therefore, adding a correction factor would not be effective.\n\n#### 5. THE PROBLEMS WITH UNDERREPORTING\n\nThe major problem with underreporting occurs when researchers begin to classify dietary intake information to determine diet and disease associations. This is often done by ranking nutrient intakes from low to high, and then looking for any associations between nutrient intake and occurrence of disease. There is a very real danger of misclassification of subjects if this ranking is based on false or underreported intake. As pointed out earlier, those who tend to be at higher risk for underreporting are also those who are at greater risk for many chronic diseases. For example, obesity is a known risk factor for coronary heart disease as well as underreporting. Because bias in measuring dietary intake has the potential of removing as well as creating associations, it can generate misleading conclusions about the impact of diet and disease [60, 61].\n\nUnderreporting is now understood to be a very real and potentially misleading problem in nutrition research. Although the use of biomarkers can help validate and interpret dietary intake data and, ideally, should become routine in nutritional epidemiology, they cannot totally replace the collection of estimated dietary intakes. Food is made up of many components, not simply the nutrients that we are aware of and that can be found in a database. Records of foods consumed may help researchers find non-nutrient constituents that help reduce risks. More and more evidence is accumulating to support the belief that the entire food, not just specific components, leads to increased health [62, 63]. Also, there is not a biomarker for every dietary constituent that is of interest to researchers. Finally, collection methods can be performed on very large populations at a much lower cost than the use of biomarkers and requires less technology and fewer skills.\n\n#### 6. IDENTIFYING UNDERREPORTERS\n\nTo help identify underreporters, researchers can apply methods such as the Goldberg cutoff, extensively described by Goldberg and colleagues 64] and Black and colleagues [65]. The Goldberg cutoff identifies the most obviously implausible intake values by evaluating the energy intake against estimated energy requirements. Basal metabolic rate (BMR) can be measured using methods described in [Chapter 3, or height and weight measurements can be used to predict BMR from a standard formula (the Schofield equation is recommended by Goldberg _et al._ [64]). A ratio of the estimated energy intake (EI) to measured or predicted BMR is calculated as EI\/BMR. This ratio can then be compared with a study-specific cutoff value (provided in Goldberg _et al._ [64]). This cutoff represents the lowest value of EI\/BMR that could reasonably reflect the energy expenditure if the person led a sedentary lifestyle. A summary of Goldberg cutoffs can be found in Table 5. Studies using the Goldberg cutoff classified 28\u201339% of the women and 18\u201327% of the men as low energy reporters [52, 57].\n\nTABLE 5\n\nGoldberg Cutoff\n\nSource: Data found in Goldberg, G. R., Black, A. E., Jebb, S. A., Cole, T. J., Murgatroyd, P. R., Coward, W. A., and Prentice, A. M. (1991). Critical evaluation of energy intake data using fundamental principles of energy physiology: 1. Derivation of cut-off limits to identify under-recording. _Eur. J. Clin. Nutr._ 45, 569\u2013581.\n\nThe Goldberg cutoff has certain limitations. Because the cutoff assumes that everyone leads a sedentary lifestyle, it tends to underestimate the underreporters. If researchers can gather information on lifestyle, occupation, leisure time, and particularly information regarding physical activity of the subjects, calculations can be more specific and improve the chances of identifying the underreporters [66].\n\n#### 7. HANDLING UNDERREPORTING IN DIETARY DATA\n\nResearchers are still not sure how to handle databases containing large numbers of underreporters. Several approaches have been suggested, but none are ideal. One technique would be to exclude anyone who is found to report implausible energy intakes. The problem with this method is that the underreporters tend to fall into specific subgroups (i.e., obese, smokers), and as stated previously, will seriously alter the sample. Some investigators have analyzed their data with all the subjects, and then again after the underreporters have been removed [67]. If there are no significant discrepancies between the finding, this can improve the confidence in the results and conclusions.\n\nSome have suggested that once the difference between the low reporters and valid reporters is noted, upward adjustments of all the nutrients could be made. Because underreporting does not appear to occur equally for all nutrients, this method is not advisable. Research has shown that underreporters tend to report micronutrient-rich diets when compared to valid reporters [51]. Adjusting would give a false impression of the nutrient status, and could mask possible risks of disease.\n\nOther researchers have suggested adjusting nutrient intakes for energy intake using the regression of nutrient vs. energy [68]. This would be feasible only if portion sizes were underestimated, yet the actual foods were all reported accurately. Otherwise, this method could make the reports worse [69]. As noted earlier, it is most likely that foods are systematically omitted from recalls. So if, for example, fat-containing foods, such as desserts, are often underreported, while vitamin A containing foods, i.e., cantaloupe, are not, energy adjustments would provide lower than actual measures of fat intake, but a higher measure of vitamin A intake. It has been recognized by many researchers that adjustments cannot eliminate bias caused by selective underreporting [70].\n\n## V. CONCLUSION\n\nAlthough dietary assessment methods do not provide true nutritional intake values, it is not feasible to eliminate them from nutritional research studies. Nutritional health cannot be evaluated without also examining the total diet composition. Improving dietary intake methodology is critical to the credibility of nutrition research. 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The Heart Outcomes Prevention Evaluation Study Investigators. _N. Engl. J. Med_. 2000;342(3):154\u2013160.\n\n37. Black A.E., Prentice A.M., Goldberg G.R., Jebb S.A., Bingham S.A., Livingstone B.E., Coward A.W. Measurements of total energy expenditure provide insights into the validity of dietary measurements of energy intake. _J. Am. Diet. Assoc_. 1993;33:572\u2013579.\n\n38. (abstract)Katan M.B. Biochemical indicators of dietary intake. _Eur. J. Clin. Nutr_. 1998;52:S5.\n\n39. Schoeller D.A., Van Santen E. Measurement of energy expenditure in humans by the doubly labeled water method. _J. Appl. Physiol_. 1982;53:955\u2013995.\n\n40. Mertz W. Food intake measurements: Is there a 'gold standard'? _J. Am. Diet. Assoc_. 1992;92:1463\u20131465.\n\n41. Johnson R.K., Driscoll P., Goran M.I. Comparison of multiple-pass 24-hour recall estimates of energy intake with total energy expenditure determined by the doubly labeled water method in young children. _J. Am. Diet. 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Cancer Prev_. 1997;6:118\u2013124.\n\n46. 1994Le Marchand L., Hankin J.H., Carter F.S., Essling C., Luffey D., Franke A.A., Wilkens L.R., Cooney R.V., Kolonel L.N. A pilot study on the use of plasma carotenoids and ascorbic acid as markers of compliance to a high fruit and vegetable dietary intervention. _Cancer Epidemiol. Biomarkers Prev_. 1994;3(3):245\u2013251.\n\n47. Pierce J.P., Faerber S., Wright F.A., Newman V., Flatt S.W., Kealey S., Rock C.L., Hryniuk W., Greenberg E.R. Feasibility of a randomized trial of a high-vegetable diet to prevent breast cancer recurrence. _Nutr. Can_. 1997;289(3):282\u2013288.\n\n48. Johansson L., Solvoll K., Bjorneboe G.-E.A., Drevon C.A. Under- and overreporting of energy intake related to weight status and lifestyle in a nationwide sample. _Am. J. Clin. Nutr_. 1998;68:266\u2013274.\n\n49. Prentice A.M., Black A.E., Coward W.A., Davies H.L., Goldberg G.R., Murgatroyd P.R., Ashford J., Sawyer M., Whitehead R.G. High levels of energy expenditure in obese women. _Br. Med. J_. 1986;292:983\u2013987.\n\n50. Lichtman S.W., Pisarska K., Berman E.R., Pestones M., Dowling H., Offenbacher E., Weisel H., Heshka S., Matthews D.E., Heymsfield S.B. Discrepancy between self-reported and actual caloric intake and exercise in obese subjects. _N. Engl. Med. J_. 1992;327:1893\u20131898.\n\n51. Price G.M., Paul A.A., Cole T.J., Wadsworth M.E.J. Characteristics of the low-energy reporters in a longitudinal national dietary survey. _Br. J. Nutr_. 1997;77:833\u2013851.\n\n52. Pryer J.A., Vrijheid M., Nichols R., Kiggins M., Elliot P. Who are the 'low energy reporters' in the dietary and nutritional survey of British adults? _Int. J. Epidemiol_. 1997;26:146\u2013154.\n\n53. Johnson R.K., Soultanakis R.P., Matthews D.E. Literacy and body fatness are associated with underreporting of energy intake in U.S. low-income women using the multiple-pass 24-hour recall: A doubly labeled water study. _J. Am. Diet. 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J. Clin. Nutr_. 1998;52:119\u2013126.\n\n59. Poppitt S.D., Swann D., Black A.E., Prentice A.M. Assessment of selective underreporting of food intake by obese and non-obese women in a metabolic facility. _Int. J. Obes_. 1998;22:303\u2013311.\n\n60. Johnson R.K., Black A.E., Cole T.J. Letter to the editor. _N. Engl. J. Med_. 1998;338:917\u2013919.\n\n61. Livingstone M.B.E., Prentice A.M., Strain J.J., Coward E.A., Black A.E., Barker M.E., McKenna P.G., Whitehead R.G. Accuracy of weighed dietary records in studies of diet and health. _Br. Med. J_. 1990;300:708\u2013712.\n\n62. Thomson C., Bloch A.S., Hasler C.M. Position of The American Dietetic Association: Functional foods. _J. Am. Diet. Assoc_. 1999;99:1278\u20131285.\n\n63. (suppl)Craig W.J. Health-promoting properties of common herbs. _Am. J. Clin. Nutr_. 1999;70:491S\u2013499S.\n\n64. Goldberg G.R., Black A.E., Jebb S.A., Cole T.J., Murgatroyd P.R., Coward W.A., Prentice A.M. Critical evaluation of energy intake data using fundamental principles of energy physiology: 1. Derivation of cut-off limits to identify under-recording. _Eur. J. Clin. Nutr_. 1991;45:569\u2013581.\n\n65. Black A.E., Goldberg G.R., Jebb S.A., Livingston M.B.E., Cole T.J., Prentice A.M. Critical evaluation of energy intake data using fundamental principles of energy physiology: 2. Evaluating the results of published surveys. _Eur. J. Clin. Nutr_. 1991;45:583\u2013599.\n\n66. Black A.E. Poor validity of dietary assessment: what have we learnt? _Eur. J. Clin. Nutr_. 1998;52(2):S17.\n\n67. Munoz K., Krebs-Smoth S., Ballard-Barbash R., Cleveland L. Food intakes of U.S. children and adolescents compared with recommendations. _Pediatrics_. 1997;100(3):323\u2013329.\n\n68. Willett W., Stampfer M.J. Total energy intake: Implicationis for epidemiologic analyses. _Am. J. Epidemiol_. 1986;124(1):17\u201326.\n\n69. Carter L.M., Whiting S.J. Underreporting of energy intake, socioeconomic status, and expression of nutrient intake. _Nutr. Rev_. 1998;56(6):179\u2013182.\n\n70. Stallone D.D., Brunner E.J., Bingham S.A., Marmot M.G. Dietary assessment in Whitehall II: The influence of reporting bias on apparent socioeconomic variation in nutrient intakes. _Eur. J. Clin. Nutr_. 1997;51:815\u2013825.\nB.\n\nNutrition Intervention\nCHAPTER 6\n\nCurrent Theoretical Bases for Nutrition Intervention and Their Uses\n\nKAREN GLANZ, University of Hawaii, Honolulu, Hawaii\n\n## I. INTRODUCTION\n\nThis chapter discusses contemporary theoretical bases for nutrition intervention for disease prevention and management and their applications in practice. Other chapters in this text provide specific recommendations regarding dietary advice for disease prevention and for nutritional management of patients. In this chapter we (1) introduce key concepts related to the application of theory in understanding and improving dietary behavior, (2) review behavioral issues related to healthful diets, (3) describe several current theoretical models that can be helpful in planning and conducting nutrition intervention, (4) highlight important issues and constructs that cut across theories, and (5) provide a summary of current thinking about the determinants of dietary behavior and change processes.\n\nNutritional intervention is a central component of disease prevention and management. Health professionals' roles in nutritional intervention are pivotal because of their centrality in health care and their credibility as patient educators [1]. Current public health recommendations in the United States give high priority to including nutrition education in all routine health care contacts [2]. People who report receiving advice about dietary change during health care visits report more health-enhancing diet changes than those who received no such advice [3].\n\n## II. IMPORTANCE OF UNDERSTANDING INFLUENCES ON DIETARY BEHAVIOR\n\nSuccessful nutrition interventions take many forms. Interventions to yield desirable changes in eating patterns can be best designed with an understanding of relevant theories of dietary behavior change and the ability to use them skillfully [4]. Whereas many early reports of nutrition interventions did not cite a particular theory or model as the basis for the strategies they employ [5, 6], the application of sound behavioral science theory in nutrition interventions is becoming increasingly common [7]. Also, emerging evidence suggests that interventions developed with an explicit theoretical foundation are more effective than those lacking a theoretical base [8].\n\nSix theoretical models are in current use and can be particularly useful for understanding the processes of changing eating patterns in health care and community settings: social cognitive theory, the stages of change construct from the transtheoretical model, consumer information processing, the health belief model, multiattribute utility theory, and diffusion of innovations [4, 7, 9]. The central elements of each theory and how they can be used to help formulate nutrition interventions are described in this chapter.\n\n### A. Multiple Determinants of Food Choice\n\nMany social, cultural, and economic factors contribute to the development, maintenance, and change of dietary patterns. No single factor or set of factors has been found to adequately account for why people eat as they do. Physiologic and psychological factors, acquired food preferences, and knowledge about foods are important individual determinants of food intake. Families, social relationships, socioeconomic status, culture, and geography are also important influences on food choices. A broad understanding of some of the key factors and models for understanding food choice can provide a foundation for well-informed clinical nutrition intervention, help identify the most influential factors for a particular patient, and enable clinicians to focus on issues that are most salient for their patients.\n\n### B. Multiple Levels of Influence\n\nCommon wisdom holds that nutrition interventions are most likely to be effective if they embrace an ecological perspective for health promotion [10, 11]. That is, they should not only be targeted at individuals but should also affect interpersonal, organizational, and environmental factors influencing dietary behavior [7, 12]. This is most clearly illustrated when one thinks of the context of selecting and purchasing food. Consumers learn about foods through advertising and promotion in the media, by labels on food packages, and via product information in grocery stores, cafeterias, and restaurants [13]. Their actual purchases are influenced by personal preferences, family habits, medical advice, availability, cost, packaging, placement, and intentional meal planning. The foods they consume may be further changed in the preparation process, either at home or while eating out. The process is complex and clearly determined not only by multiple factors but by factors at multiple levels. Still, many food choices can be represented by routines and simple, internalized rules.\n\nTraditionally, health educators focus on intra-individual factors such as a person's beliefs, knowledge, and skills. Contemporary thinking suggests that thinking beyond the individual to the social milieu and environment can enhance the chance of successful health promotion and patient education. Health providers can and should work toward understanding the various levels of influence that affect the patient's behavior and health status. This will be discussed and illustrated with examples later in this chapter.\n\n## III. WHAT IS THEORY?\n\nA theory is a set of interrelated concepts, definitions, and propositions that presents a systematic view of events or situations by specifying relations among variables, in order to explain and predict the events or situations. The notion of generality, or broad application, is important [14]. Even though various theoretical models of health behavior may reflect the same general ideas, each theory employs a unique vocabulary to articulate the specific factors considered to be important. Theories vary in the extent to which they have been conceptually developed and empirically tested.\n\nTheory can help us during the various stages of planning, implementing, and evaluating interventions [4, 14]. Theories can be used to guide the search for reasons why people are or are not consuming a healthful diet or adhering to a therapeutic dietary regimen. They can help pinpoint what you need to know before working effectively with a client, group, or patient. They also help to identify what should be monitored, measured, and\/or compared in evaluating the efficacy of nutrition intervention.\n\n## IV. EXPLANATORY AND CHANGE THEORIES\n\nTheories can guide the search to understand _why_ people do or do not follow medical advice, help identify _what_ information is needed to design an effective intervention strategy, and provide insight into _how_ to design an educational program so it is successful [14]. Thus, theories and models help _explain_ behavior and also suggest how to develop more effective ways to influence and _change_ behavior. These types of theory often have different emphases but are quite complementary [4]. For example, understanding why someone chooses the foods they eat is one step toward successful nutrition management, but even the best explanations will not be enough by themselves to fully guide changes to improve health. Some type of change model will also be needed. All of the theories and models described here have some potential as both explanatory and change models, although they might be better for one or the other purpose. In particular, the health belief model was originally developed as an explanatory model, whereas in contrast the stages of change construct was conceived to help guide planned change efforts.\n\n## V. UNIQUE FEATURES OF DIETARY BEHAVIOR TO CONSIDER WHEN USING THEORY\n\nDietary behavior changes are most likely to be effective for preventing or managing disease when they are sustained over the long term and in people's natural environments, outside the clinical setting. To be effective in nutritional intervention, health care providers need to understand both the principles of clinical nutrition management and a variety of behavioral and educational issues [15].\n\nSeveral core issues about nutritional change should be recognized. First, most diet-related risk factors are asymptomatic and do not present immediate or dramatic symptoms. Second, health-enhancing dietary changes require qualitative change, not just modification of the amount of food consumed, and cessation is not a viable option (as with smoking or other addictive behaviors). Third, both the act of making changes and self-monitoring require accurate knowledge about the nutrient composition of foods. Thus, information acquisition and processing may be more complex for dietary change than for changes in some other health behaviors, such as smoking and exercise [16]. Because of this, consumer information processing models (described below) are more important for nutrition intervention than for other types of health-related behaviors. Other important issues include longterm maintenance, the format of dietary advice, nutritional adequacy, options for initiating the change process, the changing food supply, fad diets, and special populations.\n\n### A. Long-Term Dietary Change\n\nBecause nutritional intervention leads to meaningful improvements in health only when long-term change is achieved, both providers and patients need to \"look down the road\" when formulating expectations and setting goals [17]. For example, for most patients who follow recommended dietary changes for cholesterol reduction, significant reductions are seen within 4\u20136 weeks, and cholesterol reduction goals can be reached within 3\u20136 months [18]. Even after goals are achieved, new dietary habits must be maintained. Thus, if it takes several weeks or even months to adjust to the new dietary regimen, patience and persistence by both physician and patient may be worthwhile in the long run. And different skills are required to make initial changes and to maintain them over the long term, so follow-up consultations and advice should address new issues, not merely repeat or rehash old information.\n\n### B. Restrictive and Additive Recommendations: Typical Reactions\n\nTraditionally, nutrition intervention has focused on advice to _restrict_ intake of certain foods or nutrients, e.g., reduce fat and saturated fat intake, limit energy intake, or limit sodium intake. Yet the most often-mentioned obstacle to achieving a healthful diet is not wanting to give up the foods we like [19]. Basic psychological principles hold that when people are faced with a restriction, or loss of a choice, that choice or commodity becomes more attractive. In other words, focusing mainly on what _not_ to eat, or on eating less of some types of foods, may evoke conscious or unconscious negativism in some people. In contrast, emphasizing _additive_ recommendations, such as increasing intake of fruits and vegetables, or eating more fiber-rich foods, often appeals to people because it sanctions their doing more of something. The challenge is to make these recommendations attractive to patients, and to ensure that they are presented in the context of an overall healthful diet.\n\n### C. Implications of Counseling for Gradual Change or Very Strict Diets\n\nCommon wisdom holds that the chances of long-term dietary compliance are greater when efforts to change are guided in a gradual, stepwise manner. This might involve attempting changes within specific food groups one at a time, until the total diet comes close to recommendations. A basic principle involved is that small successes (i.e., recognition of each successful behavioral change) increase confidence and motivation for each successive change. While this is effective for many people, others become impatient or even lose their enthusiasm for changes that are minimally recognizable. An alternative is to begin with a highly restrictive diet such as a very-low-fat diet or a very-low-energy diet for weight loss. These types of programs, with very strict dietary regimens, may be useful for patients who are highly motivated, post-surgically, after a coronary incident, or for those who have not been successful in making gradual changes. In some cases, a strict diet for an initial short time period will yield visible and\/or clinical changes that help motivate patients to continue adhering to a less extreme regimen. Such diets require careful supervision, however.\n\n### D. Special Populations\n\nIdeally, each patient should be treated as an individual with unique circumstances and health history. Still, epidemiological research indicates that certain demographic subgroups differ in terms of both risk factors and diet. Understanding these population trends can help prepare a provider to work with various types of patients. Data from the National Health and Nutrition Examination Survey show that most cardiovascular disease risk factors are higher among ethnic minority women (African-American and Mexican-American) than among white women [20]. Women of lower socioeconomic status tend to have higher body mass index (BMI) than more affluent women, across all ethnic groups [20]. White women also tend to have more positive attitudes toward the impact of diet on health than African-American women, even when accounting for differences in age, education, and income [21]. Women experience gender-specific differences in cardiovascular risks related to hormones, contraceptives, and hormone replacement therapy [22]. Younger persons may feel invulnerable to coronary events, and older adults may be managing multiple chronic conditions and using both prescribed and over-the-counter medications that could interact with foods. These are just a few examples of how population subgroups may differ, and they serve as a reminder to be sensitive to group patterns, but to avoid stereotyping in the absence of firsthand evidence about an individual.\n\n## VI. IMPORTANT THEORIES AND THEIR KEY CONSTRUCTS\n\nSeveral of the available and widely used models and theories of behavior change are applicable to nutrition intervention. This section describes six theoretical models that are in current use and make unique contributions to the interventionist's tool kit. They are social cognitive theory, the stages of change construct from the transtheoretical model, consumer information processing, the health belief model, multiattribute utility theory, and diffusion of innovations [4, 7, 9, 23]. The central elements of each theory and how they can be used to help formulate nutrition interventions are described in this chapter. Table 1 provides illustrative statements showing the application of each theory.\n\nTABLE 1\n\nStatements Representing Theoretical Approaches to Understanding and Changing Dietary Behavior\n\n_Source:_ Adapted in part from Glanz, K., and Rudd, J. (1993). Views of theory, research, and practice: A survey of nutrition education and consumer behavior professionals. _J. Nutr. Educ._ **25,** 269\u2013273.\n\n### A. Social Cognitive Theory\n\nSocial cognitive theory, the cognitive formulation of social learning theory that has been best articulated by Bandura [24, 25], explains human behavior in terms of a three-way, dynamic, reciprocal model in which personal factors, environmental influences, and behavior continually interact. Social cognitive theory synthesizes concepts and processes from cognitive, behavioristic, and emotional models of behavior change, so it can be readily applied to nutritional intervention for disease prevention and management. A basic premise is that people learn not only through their own experiences, but also by observing the actions of others and the results of those actions [14]. Key constructs of social cognitive theory that are relevant to nutritional intervention include observational learning, reinforcement, self-control, and self-efficacy [7].\n\nPrinciples of behavior modification, which have often been used to promote dietary change, are derived from social cognitive theory. Some elements of behavioral dietary interventions based on social cognitive theory constructs of self-control, reinforcement, and self-efficacy include goalsetting, self-monitoring and behavioral contracting [7, 15].\n\n_Self-efficacy,_ or a person's confidence in his or her ability to take action and to persist in that action despite obstacles or challenges, seems to be especially important for influencing health behavior and dietary change efforts [25]. Health providers can make deliberate efforts to increase patients' self-efficacy using three types of strategies: (1) setting small, incremental, and achievable goals; (2) using formalized behavioral contracting to establish goals and specify rewards; and (3) monitoring and reinforcement, including patient self-monitoring by keeping records [14]. In group nutrition programs, it is possible to easily incorporate activities such as cooking demonstrations, problem-solving discussions, and self-monitoring that are rooted in social cognitive theory.\n\nThe key social cognitive theory construct of reciprocal determinism means that a person can be both an agent for change and a responder to change. Thus, changes in the environment, the examples of role models, and reinforcements can be used to promote healthier behavior.\n\n### B. Stages of Change\n\nLong-term dietary change for disease prevention or risk reduction involves multiple actions and adaptations over time. Some people may not be ready to attempt changes, while others may have already begun implementing diet modifications. The construct of \"stage of change\" is a key element of the transtheoretical model of behavior change, and proposes that people are at different stages of readiness to adopt healthful behaviors [26, 27]. The notion of readiness to change, or stage of change, has been examined in dietary behavior research and found useful in explaining and predicting eating habits [16, 28\u201332].\n\nStages of change is a heuristic model that describes a sequence of steps in successful behavior change: precontemplation (no recognition of need for or interest in change), contemplation (thinking about changing), preparation (planning for change), action (adopting new habits), and maintenance (ongoing practice of new, healthier behavior) [26]. People do not always move through the stages of change in a linear manner. They often recycle and repeat certain stages; for example, individuals may relapse and go back to an earlier stage depending on their level of motivation and self-efficacy.\n\nThe stages of change model can be used both to help understand why patients might not be ready to undertake dietary change and to improve the success of nutrition intervention [14, 15]. Patients can be classified according to their stage of change by asking a few simple questions: Are they interested in trying to change their eating patterns, thinking about changing their diet, ready to begin a new eating plan, already making dietary changes, or trying to sustain changes they have been following for some time? By knowing their current stage, you can help determine how much time to spend with the patient, whether to wait until he or she is more ready to attempt active changes, whether referral for in-depth nutritional counseling is warranted, and so on. Knowledge of the patient's current stage of change can also lead to appropriate follow-up questions about past efforts to change, obstacles and challenges, and available strategies for overcoming barriers or obstacles to change [15].\n\n### C. Consumer Information Processing\n\nPeople require information about how to choose nutritious foods in order to follow guidelines for healthy eating. A central premise of consumer information processing theory is that individuals can process only a limited amount of information at one time [14, 33]. People tend to seek only enough information to make a satisfactory choice. They develop heuristics, or rules of thumb, to help them make choices quickly within their limited information-processing capacity [33]. The nutrition information environment is often complex and confusing, especially when programs rely heavily on print nutrition-education materials that may be written at too sophisticated a level in terms of wording and concepts. Recently, introduction of the new food \"Nutrition Facts\" labels have simplified generally available nutrition information somewhat and contributed to an increase in usual label use and satisfaction with their content [34].\n\nThere are several implications of consumer information processing theory for nutrition intervention. Information that is provided should be made easily accessible, not confusing, and processable with limited effort. Messages that are food focused rather than nutrient focused may be particularly helpful [35]. Nutrition information should be tailored to the comprehension level of the audience, matched to their lifestyles and experience, and either be portable or available at or near the point of food selection [33].\n\nKnowledge about which foods to choose and how much to consume on a therapeutic diet are the _sine qua non_ of dietary adherence. However, knowledge of how to use nutrition information and the skills to choose or prepare healthful foods are not enough without motivation and support. Further, patients with low literacy skills may require more explanations and fewer printed materials, thus posing important challenges [36]. One community-based dietary fat intervention that used few print materials, emphasized interactive experiences, and was targeted to the cultural backgrounds of participants was successful in promoting desirable dietary changes [37].\n\n### D. Health Belief Model\n\nThe health belief model was one of the first models to adapt theory from the behavioral sciences to health problems, and it remains one of the most widely recognized conceptual frameworks of health behavior. It emerged in the 1950s, during a time in history when a modest number of preventive health services were available, such as flu vaccines and chest X rays for tuberculosis screening [38]. The model was based on an assumption that people fear diseases, and that health actions are motivated in relation to the degree of fear (perceived threat) and expected fear-reduction potential of actions, as long as that potential outweighs practical and psychological obstacles to taking action (net benefits) [14].\n\nThe four key constructs of the health belief model are identified as perceived susceptibility and perceived severity (two dimensions of \"threat\"), and perceived benefits and perceived barriers (the components of \"net benefits\"). More recent adaptations have added the concepts \"cue to action,\" a stimulus to undertake behavior; and self-efficacy, or confidence in one's ability to perform an action [39]. While the health belief model was originally conceived as an explanatory model, it has some applications for planning change, as well. The most promising use of the health belief model in designing interventions is as a foundation for developing messages that may persuade individuals to make healthy decisions.\n\nTo what extent does the health belief model fit well with nutrition intervention? Does it help us understand how people view their eating habits and can it motivate them to make healthy changes? In fact, the health belief model is of limited use for _primary prevention_ of chronic diseases such as cardiovascular disease and cancer. However, it can play an important role in interventions for persons with clinical nutrition-related risk factors, such as high blood cholesterol or diabetes. Such individuals are faced with the important and often overriding concern about health. For practitioners, health concerns\u2014emphasized by applying the health belief model\u2014are most likely to be influential when they are emphasized in a clear and specific manner, placed in the context of overall risk for diseases, and when dietary change recommendations can be linked prospectively to tangible risk reduction [15]. Symptomatic patients also tend to be more motivated [40].\n\n### E. Multiattribute Utility Theory\n\nBoth health professionals and marketers recognize that people seek the things they like and that give them pleasure, and that they take action to obtain these things. Identifying those concerns that are most important to a person's decision about performing a specific behavior can lead to the development of effective interventions and decisions aids to promote desirable behaviors [4]. Multiattribute utility theory is a form of value expectancy theory that aims to specify how people define and evaluate the elements of decision making about performing a specific behavior. Key elements of value expectancy theory are the valence, or importance, of a particular feature of a behavior or product; and the expectancy, or subjective probability, that a given consequence will occur if the behavior is performed [41].\n\nMultiattribute utility theory is a form of value expectancy theory with particular relevance to understanding influences on food choice and changes in eating habits. Multiattribute utility theory posits that people evaluate decisions based on multiple attributes, and somehow consciously or unconsciously weigh the alternatives before deciding what actions to take. The literature on food choice has identified several key factors that appear to be important in food selection: taste, nutrition, cost, convenience, and weight control [42]. For the general public, taste has been reported to be the most important influence on food choice, followed by cost [9]. Understanding the relative importance of various concerns to individuals can guide the design of nutrition counseling and nutrition education programs. For example, by designing and promoting a nutritious diet as tasty, an intervention might be more successful than if it is presented primarily as nutritious or inexpensive.\n\n### F. Diffusion of Innovations\n\nThe last conceptual model is the diffusion of innovations, which provides guidance both for developing successful programs and ensuring that they are optimally communicated through social environments in health care settings, homes, and the community. Diffusion concepts emphasize the macro level of social, and dietary, change [43]. A key implication of diffusion theory is that mediated information sources\u2014that is, sources that rely on media rather than interpersonal communication (including brochures, mass media, etc.)\u2014are most important in the early stages of adoption, such as awareness and interest building. Interpersonal communication grows more important during active evaluation, trial, and adoption of new habits [43]. Diffusion principles are consistent with social cognitive theory concepts that suggest initially creating an environment conducive to change and disseminating the program and new ideas through successful examples [24]. However, it also appears that special efforts are required to sustain the effects of nutrition interventions after an initial active period.\n\n### G. Selecting an Appropriate Theoretical Model or Models\n\nEffective nutrition intervention depends on marshaling the most appropriate theory and practice strategies for a given situation [14]. Different theories are best suited to different individuals and situations. For example, when attempting to overcome a patient's personal barriers to changing his diet to reduce his cholesterol level, the health belief model may be useful. The stages of change model may be especially useful in developing diabetes education interventions. When trying to teach low-literacy patients how to choose and prepare healthy foods, consumer information processing may be more suitable. The choice of the most fitting theory or theories should begin with identifying the problem, goal, and units of practice [4, 14], _not_ with simply selecting a theoretical framework because it is intriguing or familiar.\n\nWhen it comes to practical application, theories are often judged in the context of activities of fellow practitioners. To apply the criterion of _usefulness_ to a theory, most providers are concerned with whether it is consistent with everyday observations [4]. In contrast, researchers usually make scientific judgments of how well a theory conforms to observable reality _when empirically tested._ Patient educators should review the research literature periodically to supplement their firsthand experience and that of their colleagues. A central premise in applying an understanding of the influences on health behavior to patient education is that you can gain an understanding of a patient through an interview or written assessment, and better focus on that individual's readiness, self-efficacy, knowledge level, and so on. Clearly, it is necessary to select a \"short list\" of factors to evaluate, and this may differ depending on clinical risk factors or a patient's history. Once there is a good understanding of that person's cognitive and\/or behavioral situation, the intervention can be personalized, or tailored. Tailored messages and feedback have been found to be promising strategies for encouraging healthful behavior changes in primary care, community, and home-based settings [44\u201346].\n\nThe challenge of successfully applying theoretical frameworks in nutrition programs involves evaluating the frameworks and their key concepts in terms of both conceptual relevance and practical value [4]. Also, the integration of multiple theories into a comprehensive model tailored for a given individual or community group requires careful analysis of the audience and frequent reexamination during program design and implementation.\n\n## VII. FINDINGS REGARDING APPLICATIONS OF THEORY TO NUTRITIONAL BEHAVIOR\n\nIn the past 5\u201310 years, published research has increased in the area of applying theoretical models to the study of nutritional behavior [6, 7]. Numerous studies have examined the determinants of eating behavior using coherent theoretical frameworks and constructs [16, 31, 47]. Most of this research has been somewhat limited by the use of cross-sectional designs. There continues to be a need for more studies using longitudinal designs; studying families and changing food roles in families; and studying the relationships among various eating behaviors and not just a few nutrients or types of foods [6].\n\nDuring the 1990s, a substantial increase has been seen in research applying the stages of change model to dietary behavior [16, 48, 49]. Several intervention trials have explicitly used this model to help shape their nutrition promotion programs [50\u201352]. Prospective intervention research examining employees' readiness to change their eating patterns has revealed \"forward movement\" across the stage continuum in work site nutrition studies, and shown that changes in stage of change for healthy eating are significantly associated with dietary improvements [32, 53].\n\nAlso, people's initial stage of change may influence their participation in nutritional intervention. People who are initially in the later stages of change (preparation, action, and maintenance) tend to spend more time on dietary change [32] and to report making more healthful changes in their food choices [45].\n\nSeveral recent large work site nutrition programs have applied constructs from social cognitive theory [46, 51, 54]. Another multisite study, the Working Well Trial, used an intervention rooted in social cognitive theory, consumer information processing, stages of change, and the diffusion of innovations [7, 55]. Several of these nutrition interventions have been found effective when compared to a control condition, although they have done little to test the elements of social cognitive theory that might be most associated with observed changes.\n\nA recent report used multiattribute utility theory as the framework for analyzing surveys of a national sample of 2967 adults. The study examined the relative importance of taste, nutrition, cost, convenience, and weight control on personal eating choices. Taste was reported to be the most important overall influence on food choices, followed by cost. The importance of nutrition and weight control were best predicted by respondents being within a particular health lifestyle cluster [9]. No published reports have explicitly applied multiattribute utility theory to design a nutrition intervention, but some leading nutrition professionals have suggested that professionals in the field should invest effort in designing food plans (including menus and recipes) that are good tasting and meet health guidelines [56].\n\nThere is a need for further research on diffusion of effective intervention models, because few studies have addressed how best to disseminate tested interventions [57].\n\n## VIII. CONSTRUCTS AND ISSUES ACROSS THEORIES\n\nIt is important to bear in mind that the various theories that can be used for nutrition intervention are not mutually exclusive. Not surprisingly, they share several constructs and common issues across theories. It is often challenging to sort out the key issues in various models. This section focuses on important issues and constructs across models. The first of these is that successful dietary behavior change depends on a sound understanding of the patient's, or consumer's, view of the world.\n\n### A. Patient's View of the World: Perceptions, Cognitions, Emotions, and Habits\n\nFor health professionals who work with patients and provide them with advice on health and lifestyle, adherence to treatment is often disappointingly poor, even in response to relatively simple medical advice. Such poor adherence often arises because patients do not have the necessary behavioral skills to make changes to their diet. Following a heart attack, for example, patients might well understand the importance of adopting such changes to their lifestyle, but be unable to make those changes. In other circumstances, patients might not understand the importance of such changes, and may even believe that such changes pose an additional risk to their health. In yet other circumstances, a patient might be experiencing depression or anxiety, such that emotional dysfunction will be a major barrier to compliance.\n\nTraditionally, it has been assumed that the relationship among knowledge, attitudes, and behavior is a simple and direct one. Indeed, over the years, many prevention and patient education programs have been based on the premise that if people understand the health consequences of a particular behavior, they will modify it accordingly. Moreover, the argument goes, if people have a negative attitude toward an existing lifestyle practice and a positive attitude toward change, they will make healthful changes. However, we now know from research conducted during the past 30 years that the relationships among knowledge, awareness of the need to change, intention to change, and an actual change in behavior are very complex indeed.\n\nIdeally, each patient should be treated as an individual with unique circumstances and health history. Still, epidemiological research indicates that certain demographic subgroups differ in terms of risk factors and health behaviors. Understanding these population trends can help prepare a provider to work with various types of patients. For example, younger persons may feel invulnerable to coronary events, and older adults may be managing multiple chronic conditions. An active middle-aged professional may place returning to his previous level of activity above important health protective actions. These examples serve as a reminder to be sensitive to group patterns, yet to avoid stereotyping in the absence of firsthand evidence about an individual. Within this general context, various theories and models can guide the search for effective ways to reach and positively motivate patients.\n\n### B. Behavior Change as a Process\n\nSustained health behavior change involves multiple actions and adaptations over time. Some people may not be ready to attempt change, some may be thinking about attempting change, and others may have already begun implementing behavioral modifications. One central issue that has gained wide acceptance in recent years is the simple notion that _behavior change is a process, not an event._ Rather, it is important to think of the change process as one that occurs in stages. It is not a question of someone deciding one day to change her diet and the next day becoming a low-fat eater for life! Likewise, most people will not be able to dramatically change their eating patterns all at once. The idea that behavior change occurs in a number of steps is not particularly new, but it has gained wider recognition in the past few years. Indeed, various multistage theories of behavior change date back more than 50 years to the work of Lewin, McGuire, Weinstein, Marlatt and Gordon, and others [4, 58\u201361].\n\nThe notion of readiness to change, or stage of change, has been examined in health behavior research and found useful in explaining and predicting a variety of types of behaviors. Prochaska, Velicer, DiClemente, and their colleagues have been leaders in beginning to formally identify the dynamics and structure of change that underlie both self-mediated and clinically facilitated health behavior change. The construct of stage of change (described earlier in this chapter) is a key element of their transtheoretical model of behavior change, and proposes that people are at different stages of readiness to adopt healthful behaviors [26, 27].\n\nWhile the stages of change construct cuts across various circumstances of individuals who need to change or want to change, other theories also address these processes. Here we look across various models to illustrate four key concerns in understanding the process of behavior change: (1) motivation vs. intention, (2) intention vs. action, (3) changing behavior vs. maintaining behavior change, and (4) the role of biobehavioral factors.\n\n#### 1. MOTIVATION VS. INTENTION\n\nBehavior change is challenging for most people even if they are highly motivated to change. As has already been noted in this chapter, the relationships among knowledge, awareness of the need to change, intention to change, and an actual change in behavior are very complex indeed. For individuals who are coping with disease and illness, and who are often having to make very significant changes to their lifestyle and other aspects of their lives, this challenge is even greater. According to the transtheoretical model, people in precontemplation are neither motivated nor planning to change, those in contemplation intend to change, and those in preparation are acting on their intentions by taking specific steps toward the action of change [27].\n\n#### 2. INTENTION VS. ACTION\n\nThe transtheoretical model makes a clear distinction between the stages of contemplation and preparation, and that of overt action [26, 27]. A further application of this distinction comes from one of the most researched models of the relationship between cognitive-attitudinal factors and health behavior change, the health belief model. This model proposes that three constellations of factors or determinants are associated with the likelihood of change at the individual level: socioenvironmental and demographic factors, the individual's perception of the threat of disease, and the individual's perception of the potential value of treatment [39]. If all these factors point in the direction of favorably perceiving change, a person is considered \"predisposed to action,\" or _intending_ to act. It is only when a \"cue to action\" sets a further process in motion that he or she actually moves into action.\n\n#### 3. CHANGING BEHAVIORS VS. MAINTAINING BEHAVIOR CHANGE\n\nEven where there is good initial compliance to a lifestyle change program, such as changing diet, relapse is very common. It is widely recognized that many overweight persons are able to lose weight, only to regain it within a year. Thus, it has become clear to researchers and clinicians that undertaking initial behavior changes and maintaining behavior change require different types of strategies. The transtheoretical model distinction between \"action\" and \"maintenance\" stages implicitly addresses this phenomenon [26, 27]. Another model that is not described in detail here, Marlatt and Gordon's relapse prevention model, specifically focuses on strategies for dealing with maintenance of a recently changed behavior [61]. It involves developing self-management and coping strategies, and establishing new behavior patterns that emphasize perceived control, environmental management, and improved self-efficacy. These strategies are an eclectic mix drawn from social cognitive theory [24], applied behavioral analysis, and the forerunners of the stages of change model.\n\n#### 4. BIOBEHAVIORAL FACTORS\n\nThe behavioral and social theories described thus far have some important limitations, many of which are only now beginning to be understood. Notably, for some health behaviors\u2014especially addictive or addiction-like behaviors\u2014there are other important determinants of behavior, which may be physiological and\/or metabolic. Among the best known are the addictive effects of nicotine, alcohol, and some drugs. Physiologic factors increase psychological cravings and create withdrawal syndromes that may impede even highly motivated persons from changing their behaviors (e.g., quitting smoking, not consuming alcoholic beverages). Some behavior changes, for example, weight loss, also affect energy metabolism and make long-term risk factor reduction an even greater challenge than it would be if it depended on cognitive-behavioral factors alone. Research into the psychobiology of fat appetite and the role of metabolic factors, including opioid peptides, as promoters of fat and protein intake offers intriguing possibilities for understanding biobehavioral models of food intake [62].\n\n### C. Barriers to Actions, Pros and Cons, and Decisional Balance\n\nAccording to social cognitive theory [24], a central determinant of behavior involves the interaction between individuals and their environments. Behavior and environment are said to continuously interact and influence one another, which is known as the principle of _reciprocal determinism._ The concept of barriers to action, or perceived barriers, can be found in several theories of health behavior, either explicitly or as an application. It is part of social cognitive theory [24] and the health belief model [39]. An extension of the concept of barriers to action involves the net benefits of action, also referred to as the \"benefits minus barriers,\" in the health belief model [39]. In the transtheoretical model, parallel constructs are labeled as \"pros\" (the benefits of change) and \"cons\" (the costs of change) [27]. Taken together, these constructs are known as \"decisional balance,\" or the pros minus cons, similar to the net benefits of action in the health belief model.\n\nThe idea that individuals engage in relative weighing of the pros and cons has its origins in Janis and Mann's model of decision making, published in their seminal book more than 20 years ago [63], although the idea had emerged much earlier in social psychological discourse. Lewin's idea of force field analysis [59], the health belief model's exposition of psychological risk\u2013benefit analysis [39], and other work on persuasion and decision counseling by Janis and Mann predated that important work. Indeed, this notion is basic to models of rational decision making, in which people intellectually think about the advantages and disadvantages, obstacles and facilitators, barriers and benefits, or pros and cons of engaging in a particular action.\n\n### D. Control over Behavior and Health: Control Beliefs and Self-Efficacy\n\nSometimes, \"control beliefs\" and self-efficacy hold people back from achieving better health. These deterrents to positive health behavior change are common, and can be found in several models of health behavior, including social cognitive theory, the health belief model, and relapse prevention. One of the most important challenges for these models\u2014and ultimately for health professionals who apply them\u2014is to enhance perceived behavioral control and increase self-efficacy, thereby improving patients' motivation and persistence in the face of obstacles.\n\n## IX. IMPLICATIONS AND OPPORTUNITIES\n\nTheory and research suggest that the most effective nutrition interventions are those that use multiple strategies and aim to achieve multiple goals of awareness, information transmission, skill development, and supportive environments and policies [64]. The range of nutrition intervention tools and techniques is extensive and varied. Programs will differ based on their goals and objectives, the needs of clients, and the available resources, staff, and expertise. Nutrition programs can stand alone or be part of broader, multicomponent and multiple-focus health promotion [65] and patient education programs.\n\nWhat can be expected? Program design relates closely to what one can expect in terms of results. Generally speaking, minimally intensive intervention efforts such as one-time group education sessions can reach large audiences, but seldom lead to behavior changes. More intensive programs typically appeal to at-risk or motivated groups, cost more to offer, and can achieve relatively greater changes in knowledge, attitudes, and eating patterns.\n\nNutrition interventions must be sensitive to audience and contextual factors. Food selection decisions are made for many reasons other than just nutrition: taste, cost, convenience, and cultural factors all play significant roles [9]. The design and implementation of dietary change strategies must take these issues into consideration. The health promotion motto \"know your audience\" has a true and valuable meaning.\n\nFurther, change is incremental. Many people have practiced a lifetime of less than optimal nutrition behaviors. It is unreasonable to expect that significant and lasting changes will occur during the course of a program that lasts only a few months. Programs need to pull participants along the continuum of change, being sure to be just in front of those most ready to change with attractive, innovative offerings.\n\nIn population-focused programs, it appears to be of limited value to adopt a program solely oriented toward modifying individual choice (e.g., teaching and persuading individuals to choose low-fat dairy products). A more productive strategy would also include environmental change efforts, e.g., expanding the availability of more nutritious food choices [12]. When this is done in conjunction with individual skill training, long-lasting and meaningful changes can be achieved.\n\nFinally, when planning interventions we should strive to be creative. Nutrition interventions should be as entertaining and engaging as the other activities with which they are competing. People will want to participate if they can have fun with the nutrition programs. Emerging communication technologies are opening up new channels for engaging people's interest in better nutrition. E-mail support and motivation systems, \"Internet buddies,\" and interactive web-based approaches can be used creatively to promote healthful eating. The communication of nutrition information, no matter how important it is to good health, is secondary to attracting and retaining the interest and enthusiasm of the audience.\n\nPractitioners at once benefit from and are challenged by the eclectic nature of their endeavor. For the unprepared, the choices can be overwhelming, but for those who understand the commonalities and differences among theories of health behavior, the growing knowledge base can provide a firm foundation on which to build. Theories and models can be\u2014and _are_ \u2014useful because they enrich, inform, and complement the practical technologies of health promotion and education.\n\nReferences\n\n1. Glanz K., Gilboy M.B. Physicians, preventive, care, and applied nutrition: Selected literature. _Acad. Med_. 1992;67:776\u2013781.\n\n2. U.S. Preventive Services Task Force. _Guide to Clinical Preventive Services_ , 2nd ed. Washington, DC: U. S. Department of Health and Human Services, Office of Disease Prevention and Health Promotion; 1996.\n\n3. Hunt J.R., Kristal A.R., White E., Lynch J.C., Fries E. Physician recommendations for dietary change: Their prevalence and impact in a population-based sample. _Am. J. Public Health_. 1995;185:722\u2013726.\n\n4. Glanz K., Lewis F.M., Rimer B.K., eds. Health Behavior and Health Education: Theory, Research and Practice, 2nd ed., Washington, DC: Jossey-Bass, 1997.\n\n5. (Supplement)Glanz K., Seewald-Klein T. Nutrition at the worksite: An overview. _J. of Nutr. Educ_. 1986;18:S1\u2013S12.\n\n6. Glanz K., Eriksen M.P. Individual and community models for dietary behavior change. _J. Nutr. Educ_. 1993;25:80\u201386.\n\n7. Glanz K. Behavioral research contributions and needs in cancer prevention and control: Dietary change. _Prev. Med_. 1997;26:S43\u2013S55.\n\n8. Contento I. The effectiveness of nutrition education and implications. _J. Nutr. Educ_. 1995;27:279\u2013418.\n\n9. Glanz K., Basil M., Maibach E., Goldberg J., Snyder D. Why Americans eat what they do: Taste, nutrition, cost, convenience, and weight control as influences on food consumption. _J. Am. Diet. Assoc. Association_. 1998;98:1118\u20131126.\n\n10. McLeroy K., Bibeau D., Steckler A., Glanz K. An ecological perspective on health promotion programs. _Health Educ. Q_. 1988;15:351\u2013377.\n\n11. Sallis J., Owen N. Ecological models. In: Glanz K., Lewis F.M., Rimer B.K., eds. _Health Behavior and Health Education: Theory, Research, and Practice_. 2nd ed. San Francisco: Jossey-Bass; 1997:403\u2013424.\n\n12. Glanz K., Lankenau B., Foerster S., Temple S., Mullis R., Schmid T. Environmental and policy approaches to cardiovascular disease prevention through nutrition: Opportunities for state and local action. _Health Educ. Q_. 1995;22:512\u2013527.\n\n13. Glanz K., Hewitt A.M., Rudd J. Consumer behavior and nutrition education: An integrative review. _J. Nutr. Educ_. 1992;24:267\u2013277.\n\n14. Glanz K., Rimer B.K. Theory at a glance: A guide for health promotion practice. _NIH Publication No. 95-3896. National Cancer Institute, Bethesda, MD_. 1995.\n\n15. Glanz K. Nutritional intervention: A behavioral and educational perspective. In: Ockene I.S., Ockene J.K., eds. _Prevention of Coronary Heart Disease_. San Francisco: Little, Brown; 1992:231\u2013236.\n\n16. Glanz K., Patterson R., Kristal A., DiClemente C., Heimendinger J., Linnan L., McLerran D. Stages of change in adopting healthy diets: Fat, fiber and correlates of nutrient intake. _Health Educ. Q_. 1994;21:499\u2013519.\n\n17. Glanz K. Patient and public education for cholesterol reduction: A review of strategies and issues. _Patient Educ. Couns_. 1988;12:235\u2013257.\n\n18. Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults. _Arch. Int. Med_. 1988;148:36\u201369.\n\n19. Morreale S.J., Schwartz N.E. Helping Americans eat right: Developing practical and actionable public nutrition education messages based on the ADA Survey of American Dietary Habits. _J. Am. Diet. Assoc_. 1995;95:305\u2013308.\n\n20. Winkleby M.A., Kraemer H.C., Ahn D.K., Varady A.N. Ethnic and socioeconomic differences in cardiovascular risk factors: Findings for women from the Third National Health and Nutrition Examination Survey, 1988\u20131994. _JAMA_. 1998;280:356\u2013362.\n\n21. Gates G., McDonald M. Comparison of dietary risk factors for cardiovascular disease in African-American and white women. _J. Am. Diet. Assoc_. 1997;97:1394\u20131400.\n\n22. Rao A.V. Coronary heart disease risk factors in women: Focus on gender differences. _J. La. State Med. Soc_. 1998;150:67\u201372.\n\n23. Glanz K., Rudd J. Views of theory, research, and practice: A survey of nutrition education and consumer behavior professionals. _J. Nutr. Educ_. 1993;25:269\u2013273.\n\n24. Bandura A. _Social Foundations of Thought and Action: A Social Cognitive Theory_. Boston: Prentice-Hall; 1986.\n\n25. Bandura A. _Self-Efficacy: The Exercise of Control_. Englewood Cliffs, NJ: W. H. Freeman; 1997.\n\n26. Prochaska J.O., DiClemente C.C., Norcross J. In search of how people change: Applications to addictive behaviors. _Am. Psychol_. 1992;47:1102\u20131114.\n\n27. Prochaska J.O., Redding C., Evers K. The transtheoretical model of behavior change. In: Glanz K., Lewis F.M., Rimer B.K., eds. _Health Behavior and Health Education: Theory, Research, and Practice_. 2nd ed. New York: Jossey-Bass; 1997:60\u201384.\n\n28. Curry S.J., Kristal A.R., Bowen D.J. An application of the stage model of behavior change to dietary fat reduction. _Health Educ. Res_. 1992;7:97\u2013105.\n\n29. Greene G.W., Rossi S.R., Reed G.R., Willey C., Prochaska J.O. Stage of change for reducing dietary fat to 30% of energy or less. _J. Amer. Diet. Assoc_. 1994;94:1105\u20131110.\n\n30. Brug J., Glanz K., Kok G. The relationship between self-efficacy, attitudes, intake compared to others, consumption, and stages of change related to fruits and vegetables. _Am. J. Health Promotion_. 1997;12:25\u201330.\n\n31. Glanz K., Kristal A.R., Tilley B.C., Hirst K. Psychosocial correlates of healthful diets among male auto workers. _Cancer Epidemiology Biomarkers Prev_. 1998;7:119\u2013126.\n\n32. Glanz K., Patterson R.E., Kristal A.R., Feng Z., Linnan L., Heimendinger J., Hebert J.R. Impact of work site health promotion on stages of dietary change: The Working Well Trial. _Health Educ. and Behav_. 1998;25:448\u2013463.\n\n33. Rudd J., Glanz K. How consumers use information for health action: Consumer information processing. In: Glanz K., Lewis F.M., Rimer B.K., eds. _Health Behavior and Health Education: Theory, Research, and Practice_. San Francisco: Jossey-Bass; 1990:115\u2013139.\n\n34. Kristal A., Levy L., Patterson R., Li S., White E. Trends in food label use associated with new nutrition labeling regulations. _Am. J. Public Health_. 1998;88:1212\u20131215.\n\n35. Hunt M.K., Stoddard A.M., Glanz K., Hebert J.R., Probart C., Sorensen G., Thomson S., Hixson M.L., Linnan L., Palombo R. Measures of food choice behavior related to intervention messages. _J. Nutr. Educ_. 1997;29:3\u201311.\n\n36. Macario E., Emmons K.M., Sorensen G., Hunt M.K., Rudd R.E. Factors influencing nutrition education for patients with low literacy skills. _J. Am. Diet. Assoc_. 1998;98:559\u2013564.\n\n37. Howard-Pitney B., Winkleby M., Albright C.L., Bruce B., Fortmann S.P. The Stanford Nutrition Action Program: A dietary fat intervention for low-literacy adults. _Am. J. Public Health_. 1997;87:1971\u20131976.\n\n38. Rosenstock I.M. Historical origins of the health belief model. _Health Educ. Monogr_. 1974;2:328\u2013335.\n\n39. Strecher V.J., Rosenstock I.M. The health belief model. In: Glanz K., Lewis F., Rimer B., eds. _Health Behavior and Health Education: Theory, Research and Practice_. 2nd ed. San Francisco: Jossey-Bass; 1997:41\u201359.\n\n40. Van Horn L., Kavey R.E. Diet and cardiovascular disease prevention: What works? _Ann. Behav. Med_. 1997;19:197\u2013212.\n\n41. Carter W.B. Health behavior as a rational process: Theory of reasoned action and multiattribute utility theory. In: Glanz K., Lewis F., Rimer B., eds. _Health Behavior and Health Education: Theory, Research and Practice_. San Francisco: Jossey-Bass; 1990:63\u201391.\n\n42. Food Marketing Institute. _Trends in the United States: Consumer Attitudes and the Supermarket_. San Francisco: Food Marketing Institute; 1996.\n\n43. Rogers E.M. _Diffusion of Innovations_ , 3rd ed. Washington, DC: The Free Press; 1983.\n\n44. Campbell M.K., DeVellis B.M., Strecher V.J., Ammerman A.S., DeVellis R.F., Sandler R.S. Improving dietary behavior: The effectiveness of tailored messages in primary care settings. _Am. J. Public Health_. 1994;84:783\u2013787.\n\n45. Beresford S.A., Curry S.J., Kristal A.R., Lazovich D., Feng Z., Wagner E.H. A dietary intervention in primary care practice: The Eating Patterns Study. _Am. J. Public Health_. 1997;87:610\u2013616.\n\n46. Brug J., Glanz K., Van Assema P., Kok G., Van Breukelen G. The impact of computer-tailored feedback and iterative feedback on fat, fruit, and vegetable intake. _Health Educ. Behav\/_. 1998;25:517\u2013531.\n\n47. Kristal A., Patterson R., Glanz K., Heimendinger J., Hebert J., Feng Z., Probart C. Psychosocial correlates of healthful diets: Baseline results from the Working Well Study. _Prev. Med_. 1995;24:221\u2013228.\n\n48. Kristal A., Glanz K., Curry S.J., Patterson R.E. How can stages of change be best used in dietary interventions? _J. Am. Diet. Assoc_. 1999;99:679\u2013684.\n\n49. Campbell M.K., Reynolds K.D., Havas S., Curry S., Bishop D., Nicklas T., Palombo R., Buller D., Feldman R., Topor M., Johnson C., Beresford S., Motsinger B., Morrill C., Heimendinger J. Stages of change for increasing fruit and vegetable consumption among adults and young adults participating in the National 5 A Day for Better Health Community Studies. _Health Educ. Behav_. 1999;26:513\u2013534.\n\n50. Sorensen G., Thompson B., Glanz K., Feng Z., Kinne S., DiClemente C., Emmons K., Heimendinger J., Probart C., Lichtenstein E. Working well: Results from a worksite-based cancer prevention trial. _Am. J. Public Health_. 1996;86:939\u2013947.\n\n51. Tilley B., Glanz K., Kristal A., Hirst K., Li S., Vernon S., Myers R. Nutrition intervention for high-risk auto workers: Results of the Next Step Trial. _Prev. Med_. 1999;28:284\u2013292.\n\n52. Sorensen G., Stoddard A., Macario E. Social support and readiness to make dietary changes. _Health Educ. Behav_. 1998;25:586\u2013598.\n\n53. Kristal A., Glanz K., Tilley B.C., Li S. Mediating factors in dietary change: Understanding the impact of a worksite nutrition intervention. _Health Educ. Behav_. 2000;27:112\u2013125.\n\n54. Sorensen G., Hunt M.K., Cohen N., Stoddard A., Stein E., Phillips J., Baker F., Combe C., Hebert J., Palombo R. Worksite and family education for dietary change: The Treatwell 5 A Day Program. _Health Educ. Res_. 1998;13:577\u2013591.\n\n55. Abrams D., Boutwell W.B., Grizzle J., Heimendinger J., Sorensen G., Varnes J. Cancer control at the workplace: The Working Well Trial. _Prev. Med_. 1994;23:15\u201327.\n\n56. Hess M.A. Taste: The neglected nutrition factor. _J. Am. Diet. Assoc_. 1997;97(suppl 2):S205\u2013S207.\n\n57. Sorensen G., Emmons K., Hunt M.K., Johnston D. Implications of the results of community intervention trials. _Ann. Rev. Public Health_. 1998;19:379\u2013416.\n\n58. Weinstein N.D. Testing four competing theories of health-protective behavior. _Health Psychol_. 1993;12:324\u2013333.\n\n59. Lewin K. _A Dynamic Theory of Personality_. New York: McGraw Hill; 1935.\n\n60. McGuire W.J. Public communication as a strategy for inducing health promoting behavioral change. _Prev. Med_. 1984;13:299\u2013313.\n\n61. Marlatt A.G., Gordon J.R. _Relapse Prevention: Maintenance Strategies in the Treatment of Addictive Behaviors_. New York: The Guilford Press; 1985.\n\n62. Drewnowski A. Nutritional perspectives on biobehavioral models of dietary change. In: Roos K.K., ed. _Proceedings: Promoting Dietary Change in Communities\u2014Applying Existing Models of Dietary Change to Population-Based Interventions_. New York: Fred Hutchinson Cancer Research Center; 1992:96\u2013109.\n\n63. Janis I., Mann L. _Decision Making: A Psychological Analysis of Conflict_. Seattle: Free Press; 1977.\n\n64. Glanz K., Sorensen G., Farmer A. The health impact of worksite nutrition and cholesterol programs. _Am. J. Health Promotion_. 1996;10:453\u2013470.\n\n65. Heaney C.A., Goetzel R.Z. A review of health-related outcomes of multi-component worksite health promotion programs. _Am. J. Health Promotion_. 1997;11:290\u2013308.\nCHAPTER 7\n\nNutrition Intervention: Lessons from Clinical Trials\n\nLINDA G. SNETSELAAR, University of Iowa, Iowa City, Iowa\n\n## I. INTRODUCTION\n\nThe modification of dietary patterns to the degree necessary to prevent chronic disease and to optimize management of disease has been traditionally perceived as a difficult and challenging task. However, much has been learned during the past two decades about how to successfully modify eating patterns. For example, several diet intervention studies aimed toward the prevention of cancer or cardiovascular disease have demonstrated the feasibility of reducing dietary fat intake in targeted groups, and complex dietary modifications testing the effect of diet on progression of renal disease have also been successfully achieved in clinical trials.\n\n## II. CONCEPTUAL MODELS OF MOTIVATION\n\nA. Self-Regulation Theory\n\nThis theory, originally described by Kanfer, states that behavior is regulated by cycles that involve the monitoring of one's own status, comparing that status with expectations, and correcting the course of action when status does not match the goal or the expectancy. [1, 2] To change dietary behavior, a person seeks to increase knowledge of the discrepancy between current status and the identified goal. Two ways to accomplish this are (1) to increase the awareness of current status (e.g., through feedback such as dietary self-monitoring) or (2) to change the goal to make it more attainable. In conflict situations, when a goal is desired and yet not seen as important enough to strive to attain, ambivalence (feeling at least two different ways about something or wanting mutually exclusive goals) is a normal, key obstacle to be changed.\n\n### B. Rokeach's Value Theory\n\nStudies in persons who have undergone sudden transformation shifts in behavior show that personality is hierarchically organized [3]. An individual's attitudes, numbering in the thousands, represent an organizational series of steps inward. More central are our beliefs and even more central are our core personal values. The most central is the sense of personal identity. The more central the shift occurs, the more sweeping is the resulting change.\n\n### C. Health Belief Model\n\nThe health belief model is a psychological model that attempts to explain and predict health behaviors by focusing on the attitudes and beliefs of individuals. The key variables of the health belief model are as follows [4]:\n\n1. _Degree of perceived risk of a disease._ This variable includes perceived susceptibility of contracting a health condition and its perceived severity once contracted.\n\n2. _Perceived benefits of diet adherence._ A second benefit is the believed effectiveness of dietary strategies designed to help reduce the threat of disease.\n\n3. _Perceived barriers to diet adherence._ This variable includes potential negative consequences that may result from taking particular health actions, including physical (weight gain or loss), psychological (lack of spontaneity in food selection) and financial demands (cost of new foods).\n\n4. _Cues to action._ Events that motivate people to take action in changing their dietary habits are crucial determinants of change.\n\n5. _Self-efficacy._ A very important variable is the belief in being able to successfully execute the dietary behavior required to produce the desired outcomes [5, 6, 7].\n\n6. _Other variables._ Demographic, sociopsychological, and structural variables affect an individual's perceptions of dietary change and thus indirectly influence his ability to sustain new eating behaviors.\n\nMotivation for change depends on the presence of a sufficient degree of perceived risk in combination with sufficient self-efficacy. Perceived risk without self-efficacy tends to result in defensive cognitive coping, such as denial, rationalization, and projection, rather than behavior change. The first element of this change model can easily be converted to a degree of perceived promise (for a positive goal), being the cross-product of perceived probability of obtaining the eventual reward.\n\n### D. Decisional Balance\n\nThe classic Janis and Mann decisional balance model [8] is a rational view, describing decision as a process of weighing cognitively the pros and cons of change. Change depends on the pros of change outweighing the cons.\n\n### E. Interaction\n\nAccording to Miller and Rollnick [9], motivation can be thought of not as a client trait, but as an interpersonal process between nutrition counselor and participant. Rather than seeing motivational change as something the participant achieves, this process is one that is experienced by both the nutrition counselor and the participant.\n\n## III. THEORIES USED IN ACHIEVING DIETARY BEHAVIOR CHANGE IN CLINICAL TRIALS\n\nThe nutrition components of clinical trials require skills in long-term dietary maintenance. These skills go beyond educating participants, and instead involve strategies designed to reinitiate participants who no longer comply with the recommended eating plan. The studies described below provide research data collected when the theories presented above are initiated in a clinical trial setting.\n\n### A. Women's Health Initiative\n\nThe Women's Health Initiative (WHI) [10] is a randomized controlled clinical trial designed to look at prevention of cancer, heart disease and osteoporosis. The dietary arm of this study focuses on a diet with 20% energy from fat, five servings of fruits and vegetables and six servings of grain products per day.\n\nTo accomplish this change in dietary habits, nutritionists in the study use a variety of behavior change techniques based on the models discussed above. Much of the stages of change model drives efforts to increase compliance in the WHI. The Prochaska\u2013DiClemente model includes six designated stages of change: precontemplation, contemplation, determination, action, maintenance, and relapse [11]. In an effort to simplify and accommodate different levels of adherence, WHI investigators chose to use only three levels of readiness to change: ready to change, unsure, and not ready to change. The decision to simplify levels is based on work with study participants showing that strategies to modify behavior fall within these three categories.\n\nTo test the effectiveness of using motivational strategies targeted at the above three levels of change, a small research study was devised. Results of that study show a positive change in dietary behavior following its implementation [12]. In this pilot study, researchers evaluated an intensive intervention program with diet. The basis of the program was use of motivational interviewing with participants in the WHI. The goal was to meet study nutrition goal of 20% energy from fat.\n\nWHI dietary intervention participants ( _n_ = 175) from three clinical centers were randomized to intervention or control status. Those randomized to the intensive intervention program participated in three individual motivational interviewing contacts from a nutritionist, plus the usual WHI dietary intervention. Those randomized to continue continued with the usual WHI dietary management intervention. Percent energy from fat was estimated at intensive intervention program baseline and intensive intervention program follow-up (1 year later) using the WHI food frequency questionnaire (FFQ).\n\nThe change in percent energy from fat between intensive intervention program baseline and intensive intervention program 1-year follow-up was \u20131.2 percentage points for intensive intervention program intervention participants and + 1.4 percentage points for intensive intervention program control participants. The result was an overall difference of 2. 6 percentage points ( _p_ \u2264 .001).\n\nTable 1 presents summary statistics on the intensive intervention program effects comparing baseline levels of fat consumption. The changes in fat consumed varied by intensive intervention program baseline fat intake as a percentage of energy intake. Participants having the highest intensive intervention program baseline fat intake (\u226530% energy) showed the largest overall change in percent energy from fat between intensive intervention program baseline and intensive intervention program follow-up. As might be expected, the smallest change was found in participants who consumed between 25% and 30% of energy from fat at intensive intervention program baseline. These participants were closer to their goal of 20% energy from fat at baseline, allowing for less overall change.\n\nTABLE 1\n\nEffect of IIP Intervention on FFQ Percent Energy from Fat Stratified by Baseline Percent Energy from Fata\n\naParticipants with missing FFQ data were excluded.\n\nb _p_ <0.05 using paired _t_ -test.\n\n_Source:_ Modified from Bowen, D., Ehret, C., Pedersen, M., Snetselaar, L., et al. (2001). Results of an adjunct dietary intervention program in the Women's Health Initiative. _J. Am. Diet. Assoc._ (in press).\n\nThe results of this study show that a protocol based on motivational interviewing and delivered through contacts with trained nutritionists is effective. Those subjects who participated in the intervention arm of the study further lowered their dietary fat intake to achieve study goals.\n\n### B. Diet Intervention Study in Children\n\nA similar protocol was used in the Diet Intervention Study in Children (DISC) [13]. DISC was a randomized, multi-center clinical trial assessing the efficacy and safety of lowering dietary fat to decrease low-density lipoprotein cholesterol in children at high risk for cardiovascular disease [14, 15]. Children began this study between ages 7 and 10 and participated in group dietary intervention programs. As they moved into adolescence (ages 13\u201317) and encountered added obstacles to dietary adherence and retention, researchers in the study designed and implemented an individual-level motivational intervention. The diet prescription in the DISC study required providing 28% energy from total fat, less than 8% energy from saturated fat, up to 9% energy form polyunsaturated fat, and less than 75 mg\/1000 kcal\/day of cholesterol. The diet met age- and sex-specific Recommended Dietary Allowance for energy, protein, and micronutrients.\n\nResearchers used a pre- to postintervention design among a subset of the total intervention cohort ( _n_ = 334). The first 127 participants who appeared for regularly scheduled intervention visits after implementation of the new intervention method were considered part of the study. These participants ranged from 13 to 17 years of age with equal numbers of boys and girls. Nutrition interventionists asked all of the 127 participants to return in 4\u20138 weeks for a follow-up session. Initial sessions were conducted in person, and follow-up sessions were conducted either in person or by telephone.\n\nThree 24-hour dietary recalls were collected within 2 weeks after the follow-up session. These dietary data were compared to three baseline 24-hour dietary recalls collected in the year preceding initial exposure to the motivational intervention method.\n\nSelf-reported data were also collected. At initial and follow-up intervention sessions, participants were shown \"assessment rulers\" (see Fig. 1) numbered 1\u201312, and asked to rate their adherence to dietary guidelines and their readiness to make new or additional dietary changes.\n\nFIGURE 1 Assessment rulers. [Data from Berg-Smith, S. M., Stevens, V. J., Brown, K. M., VanHorn, L., Gernhofer, N., Peters, E., Greenberg, R., Snetselaar, L., Ahrens, L., and Smith, K., for the Dietary Intervention Study in Children (DISC) Research Group. (1999). A brief motivational intervention to improve dietary adherence in adolescents. _Health Educ. Res._ **14,** 399\u2013410.]\n\nResults from the study show that the mean energy from total fat decreased from 27.7% to 25.6% ( _p_ < 0.001) (Table 2) and the mean energy from saturated fat decreased from 9.5% to 8.6% of total energy intake ( _p_ < 0.001). Additionally, dietary cholesterol decreased from 182.9 to 157.3 mg\/1000 kcal ( _p_ < 0.003). A comparison of males and females showed no differences in gender relative to study results. Note that for this preliminary test, no control group was randomly assigned or examined. Therefore, the researchers cannot predict if significant reductions in consumption of dietary fat and cholesterol are attributable to the intervention.\n\nTABLE 2\n\nChanges in Total Fat Intake, Saturated Fat Intake, and Dietary Cholesterol after Two Intervention Sessions\n\n_Source:_ Data from Berg-Smith, S. M., Stevens, V. J., Brown, K. M., VanHorn, L., Gernhofer, N., Peters, E., Greenberg, R., Snetselaar, L., Ahrens, L., and Smith, K., for the Dietary Intervention Study in Children (DISC) Research Group. (1999). A brief motivational intervention to improve dietary adherence in adolescents. _Health Educ. Res._ **14,** 399\u2013410.\n\nThe self-reported adherence rating score and readiness to change score increased by approximately 1 point on a scale from 1 to 12 (both _p_ < 0.001). To help accomplish goals, action plans were also made. The study results show that 94% of the participants made action plans and 89% successfully implemented them.\n\nThis study also examined counselor satisfaction. The results showed that nearly three-quarters of the nutrition counselors were satisfied or very satisfied with using the motivational intervention methods (Table 3).\n\nTABLE 3\n\nNutrition Counselor Satisfaction with the Motivational Intervention Method\n\n_Source:_ Data from Berg-Smith, S. M., Stevens, V. J., Brown, K. M., VanHorn, L., Gernhofer, N., Peters, E., Greenberg, R., Snetselaar, L., Ahrens, L., and Smith, K., for the Dietary Intervention Study in Children (DISC) Research Group. (1999). A brief motivational intervention to improve dietary adherence in adolescents. _Health Educ. Res._ **14,** 399\u2013410.\n\n### C. Motivational Intervention Method\n\nFigure 2 provides a method for establishing rapport prior to tailoring the intervention to the readiness to change level: ready to change, unsure, and not ready to change. Figure 3 provides specific strategies for each level of change.\n\nFIGURE 2 Motivational intervention model. [From Berg-Smith, S. M., Stevens, V. J., Brown, K. M., VanHorn, L., Gernhofer, N., Peters, E., Greenberg, R., Snetselaar, L., Ahrens, L., and Smith, K., for the Dietary Intervention Study in Children (DISC) Research Group. (1999). A brief motivational intervention to improve dietary adherence in adolescents. _Health Educ. Res._ **14,** 399\u2013410.]\n\nFIGURE 3 Motivational intervention components for three specific levels. [Modified from Berg-Smith, S. M., Stevens, V. J., Brown, K. M., VanHorn, L., Gernhofer, N., Peters, E., Greenberg, R., Snetselaar, L., Ahrens, L., and Smith, K., for the Dietary Intervention Study in Children (DISC) Research Group. (1999). A brief motivational intervention to improve dietary adherence in adolescents. _Health Educ. Res._ **14,** 399\u2013410.]\n\n#### 1. FIRST LEVEL\u2014NOT READY TO CHANGE\n\nThe main goal for this level of intervention is to raise awareness of the need to continue meeting goals (e.g., fat grams, carbohydrate grams, energy intake). Additionally to achieve this goal it is necessary to reduce resistance and barriers to meeting goals (e.g., decreasing cues to eat high fat foods). Also, very importantly, focus is placed on increasing interest in considering behavioral steps toward meeting the goals above.\n\nThroughout the initial interview, when working with a patient in this level, it is important to ask open-ended questions, listen reflectively, affirm, summarize, and elicit self-motivational statements. Figure 3 provides examples of questions designed to facilitate the participant's ability to make motivational statements.\n\n##### a. Open-Ended Questions.\n\nInitially for a participant at this level, it is important to ask questions that require explaining or discussing. Questions focus on requiring more than one-word answers. The goal is to guide the participants to talk about their dietary change progress and difficulties. Figure 2 provides some opening questions. Other questions and statements are presented below:\n\n\"Let's discuss your experience with diet up to now. Tell me how changing your diet has been for you.\"\n\n\"What things would you like to discuss about your experiences with dietary change and your progress with changes? What do you like about these changes? What don't you like about these changes?\"\n\n##### b. Listen Reflectively.\n\nListening goes beyond hearing what a person has said and acknowledging those words. Crucial in responding to a patient or participant is understanding what is meant beyond the words. Reflective listening involves a guess at what the person feels and is phrased as a statement rather than a question. Stating the feeling behind the statement serves two purposes. It allows the participant to tell you if your judgment of the feeling is on target. It also shows that you really are trying to understand more than just words and do care about feelings also. Below are some participant\u2013nutritionist interactions that illustrate reflective listening:\n\n_SCENARIO 1_\n\nParticipant : \"There are times when I do a wonderful job of meeting my fat gram goal, but sometimes I don't do so well. I keep trying though.\"\n\nNutritionist: \"You seem to feel badly that you don't always meet your fat gram goal.\"\n\n_SCENARIO 2_\n\nParticipant: \"I am so tired of trying to follow this diet. It seems that I have put hours into following it, and I have little to show for it. I certainly have not lost weight.\"\n\nNutritionist: \"You feel frustrated and angry about trying so hard and still getting nowhere.\"\n\n_SCENARIO 3_\n\nParticipant: \"When I don't fill in a food diary, I am not sure that I am doing well or not.\"\n\nNutritionist: \"You are worried on days when you do not fill in a food diary.\"\n\n_SCENARIO 4_\n\nParticipant: \"I really don't want to continue following this diet. I have other things that are more of a priority now.\" Nutritionist: \"You seem hassled by these other competing desires and feel that following a new eating pattern is getting in the way.\"\n\n##### c. Affirm.\n\nCommunicating support to participants is an excellent way of letting them know that you appreciate what they are doing. Affirmations are statements that indicate alignment and normalization of the participant's issues. Alignment means telling participants that you understand them and are with them in their difficulties.\n\nNormalization means telling the participants that they are perfectly within reason and \"normal\" to have such reactions and feelings.\n\nExamples of affirmations include:\n\n\"It is very hard to struggle with competing priorities. You've done amazingly well.\"\n\n\"That is an insightful idea.\"\n\n\"Thank you for telling me that. It must have been hard for you to tell me.\"\n\n\"I can see why you would have this difficulty. Many people have the same problem.\"\n\n##### d. Summarize.\n\nPeriodically, and at the point when you begin to elicit self-motivational statements, summarize the content of what the participant has said. Cover key points even if they involve negative feelings. You can discuss conflicting ideas that the participant has brought up by using the strategy \"on the one hand you... and on the other hand you...\". This reminds both of you about the issues and ensures clarity.\n\n##### e. Elicit Self-Motivational Statements.\n\nThe most important part of self-motivational statements is that they help participants realize that a problem exists, that they are concerned about the problem, that they intend to correct the problem, and that they think they can do better in the future. Figure 4 provides questions to elicit self-motivational statements. These statements fall in to four categories: problem recognition, concern, intention to change, and optimism.\n\nFIGURE 4 Examples of questions designed to elicit self-motivational statements. [Modified from Bowen, D., Ehret, C., Pedersen, M., et al. (2000). Results of an adjunct dietary intervention program in the Women's Health Initiative. _J. Am. Diet. Assoc._ (in press).]\n\nIt is important to respectfully acknowledge decisions that participants make. These decisions may mean that a participant has decided not to make changes immediately (see Fig. 3).\n\nIt is appropriate to offer professional advice, but still leave the actual decision to make a change up to the participant. Figure 3 provides some ideas on how to approach the participant.\n\nClose the discussion with another summary. Concentrate on any self-motivational statements that the participant has made. End the session with the idea that both of you should think about what has been discussed and that you can revisit the issues next time.\n\n#### 2. SECOND LEVEL\u2014UNSURE ABOUT CHANGE\n\nThe main goal for this intervention is to tip the balance toward working to meet the goals. Four steps are important in meeting the goals: (1) regroup, (2) ask key questions, (3) negotiate a plan, and (4) conclude the work.\n\n##### a. Regroup.\n\nThe first step in dealing with a participant who is unsure about changing dietary habits is regrouping to focus on the transition from not being ready to deal with the problems of change to moving toward a reinitiating of behavior adjustment. This process of regrouping can serve as a reminder of what has happened in previous sessions. Below are four ways to regroup:\n\n1. _Summarize_ the participant's perceptions of what is going on. The summary might include self-motivational statements that the participant has made.\n\n2. _Identify_ ambivalence or other conflicting issues.\n\n3. _Review_ any self-monitoring related to dietary intake.\n\n4. _Restate intentions or plans to change or do better in the future._\n\n##### b. Ask Key Questions.\n\nAsk questions that focus on the participant's statements regarding future plans to make dietary changes. The goal is to ask open-ended questions that cause the participant to think about what you have just summarized and come to the conclusion that action is necessary. The goal is for the participant to provide a statement showing the desire to change. Here are some examples of questions that facilitate positive participant statements:\n\n\"How might we work together to proceed from here?\"\n\n\"Hearing my summary of how things have gone in the past, what do you want to do?\"\n\n\"How might you become more involved in dietary change? What are the good parts and the bad parts about continuing to change?\"\n\n\"You are currently unsure of what to do. How might we work together to resolve the issue?\"\n\n##### c. Negotiate a Plan.\n\nThere are three parts to the negotiation process. The first involves setting goals, the second, considering the options, and finally, arriving at a plan.\n\n1. _Set goals._ Past wisdom dictated that setting goals meant being very specific and behavioral. \"I will eat candy bars only one time per week on Sunday.\" Motivational interviewing dictates that goal setting may start out very broadly at first and then move to behavioral goals that are very specific. To elicit broadly stated goals, the following questions might be used:\n\n\"What about your diet would you like to change?\"\n\n\"How would you like things to be different from how they are now?\"\n\n2. _Consider options._ Make a list of things that might be changed to bring the participant closer to the dietary goal. Ask the participant to choose among the options. If the first one does not work, the participant has many choices as backups.\n\n3. _Arrive at a plan._ Ask the participant to arrive at a plan. Include in the plan the specific behavioral goals with potential problems that may serve as barriers to making these changes.\n\n##### d. Conclude the Work.\n\nAlways end the session with an encouraging statement and a reflection on the participant's resourcefulness in identifying the plan. Follow this statement with the idea that he is the best expert relative to behavior change. Indicate that you will stay in touch to check on how the behavior change is going.\n\n#### 3. THIRD LEVEL\u2014READY TO CHANGE\n\nThe main goal for this third intervention is to reengage the participant in meeting the dietary goals.\n\n##### a. Review.\n\nCover the previous discussions with the participant. Focus on the statements made by the participant that show interest and willingness to change. Use statements that show that it was the participant's idea to meet dietary goals previously set. For example, \"You said that you were interested in trying again.\"\n\n##### b. Encourage Choices and Activities.\n\nAsk the participant what she would like to do to get reengaged. Encourage the participant to make her own choice. Collaborate and negotiate short-term, easily attained goals initially. For example, \"I will drink 1% milk for a week in place of 2% milk and gradually reduce to fat-free milk.\"\n\n##### c. Summarize.\n\nReview the discussion, the issues, and difficulties on both sides. Remind the participant to keep trying and to believe in herself.\n\n### D. Modification of Diet and Renal Disease Study(MDRD)\n\nThe Modification of Diet and Renal Disease Study (MDRD) [16] used the self-management approach to modify dietary behavior. In this population of persons diagnosed with renal disease, research nutritionists counseled participants on diets low in protein and phosphorus. Although the diets were difficult to follow, the study participants showed great motivation based on their desires to avoid renal dialysis [17]. The following strategies were used successfully in the MDRD [18]:\n\n1. _Single-nutrient approach to dietary behavior change._ The focus in this study was on reducing protein content of the diet. With this reduction, dietary phosphorus was also reduced. When other nutrients were modified, specific food groups were identified. Even with changes in other nutrients, protein was still a primary focus.\n\n2. _Self-monitoring._ The participant's ability to self-monitor was crucial to keeping protein intake down to goal levels. Weighing and measuring was used as a means of matching dietary change on a daily basis with the biological marker of urinary nitrogen. Further study in self-monitoring matched with the biological marker showed that problems occur in knowing how to best represent dietary intake [19]. Often it is difficult to closely mirror the exact amount of protein in a cut of meat if that cut is not precisely specified.\n\n3. _Staging changes._ In the MDRD, nutritionists also staged changes in dietary protein by gradually reducing the dietary protein intake. This gradual change made day-to-day modifications easier.\n\n4. _Modeling._ Nutritionists modeled dietary changes by offering both recipes low in protein and taste-testing sessions. Group sessions were held at which a special meal was offered with food preparation techniques modeled.\n\n### E. Diabetes Control and Complications Trial (DCCT)\n\nThe Diabetes Control and Complications Trial (DCCT) [20] used techniques similar to those of the MDRD study [21]:\n\n1. _Single-nutrient approach._ Investigators focused on carbohydrate as a single nutrient, where it was matched with insulin to achieve normalized blood glucose.\n\n2. _Self-monitoring._ Monitoring consisted of following blood glucose concentrations and dietary intakes to verify where problems might be occurring. If dietary intake was high along with blood glucose concentrations, dietary intake and\/or insulin was modified to achieve normal blood glucose levels.\n\n3. _Staging changes._ Changes were staged by working on specific times of day that were most problematic. If lunchtimes were most often high, we focused on dietary intake modifications to alter blood glucose levels. Insulin and exercise also often played a role.\n\n4. _Modeling._ Dietary modifications were facilitated by providing recipes, modeling, and going to restaurants to identify and anticipate blood glucose levels after eating a favorite lunch or other meals out of the home.\n\n## IV. SUMMARY\n\nConsiderable experience in clinical trials suggests that dietary modification requires a process of making changes on an individual basis with constant negotiation with the patient or participant. Working as a team, the nutritionist and participant can achieve dietary change that alters biological markers and may reduce disease risk and optimize management.\n\nReferences\n\n1. Agostinelli G., Brown J.M., Miller W. Effects of normative feedback on consumption among heavy drinking college attendants. _J. Drug Educ_. 1995;25:31\u201340.\n\n2. Brown J.M., Miller W.R. Impact of motivational interviewing on participation in residential alcoholism treatment. _Psychol. Addict. Behav_. 1993;7:211\u2013218.\n\n3. Rokeach M. _The Nature of Human Values_. New York: Free Press; 1973.\n\n4. Rosenstock I., Strecher V., Becker M. The health belief model and HIV risk behavior change. In: DiClemente R.J., Peterson J.L., eds. _Preventing AIDS: Theories and Methods of Behavioral Interventions_. New York: Plenum Press; 1994:5\u201324.\n\n5. Bandura A. Perceived self-efficacy in the exercise of control over AIDS infection. In: _Primary Prevention of AIDS: Psychological Approaches_. New York: Sage Publications; 1989:128\u2013141.\n\n6. Bandura A. Self-efficacy: Toward a unifying theory of behavioral change. _Psychol. Rev_. 1977;84:191\u2013215.\n\n7. Bandura A. Self-efficacy mechanism in physiological activation and health-promoting behavior. In: Madden J., ed. _Neurobiology of Learning, Emotion and Affect_. London: Raven Press; 1991:229\u2013270.\n\n8. Janis J.L., Mann L. _Decision-Making: A Psychological Analysis of Conflict, Choice and Commitment_. New York: Free Press; 1977.\n\n9. Miller W.R., Rollnick S. _Motivational Interviewing: Preparing People to Change Addictive Behavior_. New York: Guilford Press; 1991.\n\n10. Women's Health Initiative. _WHI Protocol for Clinical Trial and Observational Components, NIH Publication no. N01-WH-2-2110_. New York: Fred Hutchinson Cancer Research Center; 1994.\n\n11. Prochaska J., DiClemente C. Transtheoretical therapy: Toward a more integrative model of change. _Psychother. Theory Res. Prac_. 1982;19:276\u2013288.\n\n12. Bowen, D., Ehret, C., Pedersen, M., et al. Results of an adjunct dietary intervention program in the Women's Health Initiative. _J. Am. Diet. Assoc._ (in press).\n\n13. Berg-Smith S.M., Stevens V.J., Brown K.M., VanHorn L., Gernhofer N., Peters E., Greenberg R., Snetselaar L., Ahrens L., Smith K., for the Dietary Intervention Study in Children (DISC) Research Group. A brief motivational intervention to improve dietary adherence in adolescents. _Health Educ. Res_. 1999;14:399\u2013410.\n\n14. DISC Collaborative Research Group. Efficacy and safety of lowering dietary intake of fat and cholesterol in children with elevated low-density lipoprotein cholesterol: The Dietary Intervention Study in Children (DISC). _JAMA_. 1995;273:1429\u20131435.\n\n15. DISC Collaborative Research Group. Dietary Intervention Study in Children (DISC) with elevated LDL cholesterol: Design and baseline characteristics. _Ann. Epidemiology_. 1993;3:393\u2013402.\n\n16. Klahr S., Levey A.S., Beck G.J., Caggiula A.W., Hunsicker L., Kusek J.W., Striker G., for the Modification of Diet in Renal Disease Study Group. The effects of dietary protein restriction and blood-pressure control on the progression of chronic renal disease. _N. Engl. J. Med_. 1994;330:877\u2013884.\n\n17. Milas C., Norwalk M.P., Akpele L., Castaldo L., Coyne T., Doroshenko L., Kigawa L., Korzec-Ramirez D., Scherch L., Snetselaar L. Factors associated with adherence to the dietary protein intervention in the modification of diet in renal disease. _J. Am. Diet. Assoc_. 1995;95(11):1295\u20131300.\n\n18. Snetselaar L. Dietary compliance issues in patients with early stage renal disease. _Clin. Appl. Nutr_. 1992;2(3):47\u201352.\n\n19. Snetselaar L., Chenard C.A., Hunsicker L.G., Stumbo P.J. Protein calculation from food diaries underestimates biological marker. _J. Nutr_. 1995;125:2333\u20132340.\n\n20. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. _N. Engl. J. Med_. 1993;329:977\u2013986.\n\n21. Greene T., Bourgorgnie J., Hawbe V., Kusek J., Snetselaar L., Soucie J., Yamamoto M. Baseline characteristics in the modification of diet in renal disease study. _J. Am. Soc. Neprhol_. 1993;3(11):1819\u20131834.\nCHAPTER 8\n\nTools and Techniques to Facilitate Eating Behavior Change\n\nJOAN M. HEINS1 and LINDA DELAHANTY2\n\n1Washington University School of Medicine, St. Louis, Missouri\n\n2Massachusetts General Hospital, Boston, Massachusetts\n\n## I. INTRODUCTION\n\nThe potential effect of medical nutrition therapy to improve health outcomes from cardiovascular, cancer, diabetes, obesity, gastrointestinal, and other health conditions is clearly described in other chapters in this text. However, the process of implementing medical nutrition therapy is not as straightforward and is based on thorough assessment of each client's lifestyle, capabilities, and motivation to change.\n\nAt a glance, one might perceive the nutrition education and counseling process to be routine, where nutrition information and recommended food choices are discussed as they pertain to particular health concerns of a client. For a patient with a high cholesterol level, teach the National Cholesterol Education Program (NCEP) Step 1 diet and possibly the NCEP Step 2 diet; for high blood pressure, teach weight loss and a Dietary Approaches to Stop Hypertension (DASH) diet; for type 2 diabetes, teach weight loss, exercise and possibly carbohydrate counting. If only changing eating behavior were that straightforward! The truth is that each person who is referred for nutrition counseling presents with varying levels of knowledge and motivation for changing eating habits. Today's nutrition counselor must draw on knowledge from the biomedical and behavioral sciences to define the nutrition prescription and design an intervention that will truly impact eating.\n\nThe traditional model for delivery of nutrition interventions has been individual consultations in health care settings. This paradigm is changing, however. Increased focus on chronic disease prevention has resulted in an enhanced need for nutrition education at a time when economic constraints in health care have led to a decline in traditional hospital-based nutrition counseling services. Thus, dietitians are providing counseling services in shopping centers, offering cholesterol education classes in health clubs, and communicating with clients via telephone and the Internet. Although individualized counseling will continue as an important component of clinical nutrition, use of alternative methods, such as group sessions or guided self-study, has been shown to be both efficient and effective. Regardless of the setting, nutrition counseling has a common goal: to help people make healthy changes in eating behaviors. This chapter will discuss tools and techniques for applying education and behavior change theories in the practice of medical nutrition therapy.\n\n## II. THE TEACHING\/LEARNING PROCESS\n\nKnowledge is not sufficient but is essential for behavior change [1]. To be effective, nutrition counselors need to understand the factors that influence learning and the different domains in which people learn.\n\n### A. Factors Influencing Learning\n\nLearning is influenced by many factors including age, literacy, culture, and individual learner style.\n\n#### 1. AGE\n\nMuch of our understanding of the differences in the way adults and children learn comes from the work of Knowles [2], who identified concepts of _need to know, performance centered,_ and _experiential learning_ as important to the adult learner. These concepts have been expanded by the research of others who describe self-directed learning [3] and critical thinking techniques [4] as elements that enhance adult learning. A common theme that emerges across learning theories and studies of adult learning is the importance of active involvement of the individual in the learning process [5].\n\n#### 2. LITERACY\n\nThe term _literacy_ includes not only the ability to read and write but also to process information. With the wide use of printed materials to support nutrition education, client literacy and the reading level of teaching materials are important issues. Assessing level of formal education is easy, but unfortunately not always an accurate indicator of literacy. Tests such as ABLE [6], WRAT [7], and the Cloze procedure [8] can be used to evaluate an individual's literacy level. These tests, however, take time to administer and can be fatiguing. Their best use may be to assess reading levels in targeted groups prior to development of educational materials rather than for individual assessment in clinical practice. The steps for evaluating nutrition education materials are relatively simple and can be used by dietitians when information on the reading level of material is not provided. Methods such as the SMOG readability formula [9], Gunning's FOG Index [10], and the Fry Readability Graph [11] are described in the reading and health education literature [12\u201314]. These formulas use the number of words, the number of syllables, and similar criteria to assign a grade or reading level. Scoring can be done by hand or with computer programs. One of the difficulties of using readability formulas in clinical nutrition is that core words such as _calories, carbohydrate, vitamins, minerals,_ and _cholesterol_ contain three or more syllables and consequently raise the reading level of materials. These words may be sufficiently common, however, so that people with reading abilities lower than the assigned grade level can process and comprehend the materials based on their familiarity with the words as well as the organization of the written text [15].\n\n#### 3. CULTURE\n\nEthnicity and culture influence the way people learn. Addressing cultural difference is not simply translating educational materials into the primary language of the client. Fundamentals of values, health beliefs, and communication styles also vary by culture. The changing demographics of the American population have resulted in an increased need for nutrition education materials appropriate for a wide range of cultures. One response to this need is the _Ethnic and Regional Food Practice_ series developed by the Diabetes Care and Education practice group of The American Dietetic Association [16]. The series, written for health professionals, currently includes 11 modules ranging from Jewish to Hmong cuisine. The modules discuss traditions and beliefs influencing health behaviors such as eating habits, provide nutrient analysis of foods specific to the culture, outline sample recipes, and include a reproducible master of a client teaching tool. Other sources for ethnically appropriate teaching materials include government printing agencies, volunteer health organizations, ethnic special interest groups, and vendors of health education materials. Even when specific guidelines are not available, an appreciation that food and health hold different meanings to people of different cultures can aid dietitians in counseling individuals from diverse ethnic backgrounds.\n\n#### 4. LEARNER STYLE\n\n_Learner style_ is a phrase that has been used to describe the way people cognitively process information [17] and the interaction of the individual with the learning environment [18]. Drawing from the work of Carl Jung, Osterman has identified four types of learners: _feelers, thinkers, sensors,_ and _intuitors_ [19]. Table 1 summarizes key characteristics that differentiate these styles. Teaching strategies can be selected to match the learning style or characteristic of the individual. When working with groups, an education session can be constructed to include components that will appeal to all learning styles [19]. Walker, in her review of adult learner characteristics [18], contrasts different environmental methods of learning, such as group vs. individual, computer vs. print vs. video, didactic vs. emotional appeal in messages, and directed learning vs. self-directed approaches. Although comparison studies of environmental methods are limited, she found that research on group vs. individual sessions for diabetes education showed some benefits for group education beyond potential cost savings. A review of research evaluating nutrition education methods found that small groups plus a follow-up telephone call were the most effective, with one-on-one sessions ranking next, followed closely by the small group (no follow-up) approach [5]. The authors concluded that in terms of cost efficiency and cost effectiveness, groups showed a clear advantage. A comparison of group vs. self-directed education for blood cholesterol reduction found that the self-directed approach was a viable alternative to group diet instruction [20]. These findings support use of alternatives to individual counseling sessions to match client learning styles as well as to provide efficient methods for serving more people.\n\nTABLE 1\n\nLearning Styles\n\n_Source:_ Adapted from Osterman, D. N. (1984). The feedback lecture: Matching teaching and learning styles. _J. Am. Diet. Assoc._ **84,** 1221\u20131222.\n\n### B. Domains of Learning\n\nLearning includes knowledge, attitude, and skill. The education literature describes these areas of learning as domains: cognitive (knowledge), affective (attitude), and psychomotor (skill) [21, 22]. Within each domain there is a range or level of learning that can be achieved.\n\nThe cognitive domain concentrates on knowledge outcome and includes sixhierarchical levels: knowledge, comprehension, application, analysis, synthesis, and evaluation. The affective domain encompasses an individual's feelings or attitudes associated with a particular topic. The five levels of the affective domain progress from receiving to responding, valuing, organizing, and characterizing by a value or a value complex. The psychomotor domain looks at skill development including perception, set (prepared for learning), guided response (performance), mechanism (response more habitual), complexovert response (response is effective and routine), and adaptation (response continues in new situations.)\n\nDetailed classification systems or taxonomies have been developed that include action verbs to define levels of learning [23, 24]. The taxonomies are used to set learning objectives. Objectives can be written for any domain, and for any level within that domain, depending on the desired outcome of the counseling session. A learning plan can be developed for an individual that systematically advances him or her from a low level to a high level of competence.\n\nApplication of the taxonomies of learning to nutrition counseling offers a framework for integrating learning and behavior change theories. Learning objectives are written in language that clearly identifies _who_ will do _what, when, where,_ and _how. Who_ is the client, _what_ is the information, _how_ is the measurable behavior, and _when_ and _where_ define the situation. For example, a behavioral objective for a patient with hyperlipidemia, targeted at the application level of the cognitive domain, could be as follows: At the end of the counseling session ( _when_ ), the client ( _who_ ) will be able to identify (how) low-fat entr\u00e9e options ( _what_ ) from sample menus ( _where_ ).\n\nThe value of setting behavioral objectives for nutrition interventions is well supported in the literature. A review found that programs that were more behaviorally focused were more effective [5]. This extensive review identified education and behavior change strategies that were successful in changing eating habits. Educational strategies of self-evaluation or self-assessment and active participation worked well for individual or group interventions. Effective behavioral strategies included use of a systematic behavior change process, tailoring the intervention to the specific needs of the individual, involvement of others to provide social support and an empowerment approach that enhances personal control.\n\nIn summary, for nutrition counseling to be successful, learning principles suggest that as much attention must be given to selecting the most effective way to communicate the diet to the individual as is given to assessing what would be the most effective diet for the individual. Most health professionals have strong grounding in the biological sciences, but less exposure to the behavioral sciences. For this reason, the comfort level for determining the appropriate nutrient intake is greater than for evaluating learner needs and setting behavioral objectives. The current demand in all areas of health care to measure effectiveness in terms of patient outcomes requires clinicians to look beyond the diagnosis and intervention phases of care and evaluate the ultimate results. Nutrition interventions that do not show measurable improvement in patient status are not effective. The argument that treatment failure is due to patient non-compliance does not negate the lack of effect. Dietitians and other health care providers must be adept at combining nutrition, learning, and behavior change principles in the process of nutrition education.\n\n## III. NUTRITION EDUCATION TECHNIQUES\n\nLength of time since diagnosis, acuity of disease condition, complexity of the nutrition intervention, preferred client learning style, and readiness to change behaviors are factors to consider in developing a client's education plan. The counselor needs to assess learning needs, decide on the level of education, select an optimal method, and choose appropriate nutrition education tools.\n\n### A. Assessing Learning Needs\n\nAssessment of learning needs should be an integral part of nutrition counseling. Table 2 provides a basic list of variables that should be assessed. Additional items are added to gather information pertinent to the individual and the clinical condition. The amount of time spent in assessment can limit time spent in counseling. Some studies report up to 55% of a counseling session devoted to the assessment phase [25]. However, time spent on a comprehensive assessment is regained by the effectiveness of a nutrition counseling session that has been tailored to the individual's needs.\n\nTABLE 2\n\nNutrition Educational Assessment\n\nA variety of approaches can be used to reduce time spent on assessment. Before the counseling session, data can be collected from medical records, and patients can be asked to submit information. Questionnaires can be sent and received by mail, via fax, through the Internet, or completed by the client in the waiting room before the visit. If advanced data collection has not been successful, ask the patient. Although clients may not recount a comprehensive description of their referring physician's intent, their perception of what the interaction should achieve provides a basis for assessing learning needs and willingness to make behavior changes.\n\n### B. Levels of Education\n\nNutrition education should be planned as a continuum of learning that starts with fundamental guidelines, then incrementally adds more complex information as basic applications are mastered. The terms _initial\/survival, practical,_ and _continuing_ have been used to differentiate three levels of education [25].\n\n#### 1. SURVIVAL LEVEL\n\nThe first level focuses on essential information that the client needs in order to make important fundamental adjustments in health behaviors. Ideally, initial education will occur shortly after diagnosis. The extent of information included at the survival level differs by disease condition and learner characteristics. A person with diabetes treated with insulin needs enough information to understand the association between food, activity, and insulin so that he or she can select appropriate meals to avoid hyper- and hypoglycemia. For the patient with congestive heart failure with frequent hospital admissions, the survival information would focus on the sodium content of foods and avoidance of fluid retention. Survival education needs to be simple and directive; the dietitian serves as a teacher providing concrete guidelines on what the patient should or should not do.\n\n#### 2. PRACTICAL LEVEL\n\nThe practical level of education can occur as follow-up to initial counseling or as a new encounter, with the patient having had initial instruction some time before. Information should expand on the fundamentals learned for survival by applying them to a variety of situations. New topics can be introduced as well. Clients often will identify \"need to know\" information they have found important to learn, such as how to eat in restaurants, modify recipes, and\/or interpret food labels. At this level the dietitian serves as a counselor by providing guidelines for patients to use in making decisions.\n\n#### 3. CONTINUING EDUCATION\n\nOnce a client has mastered the basic skills and can apply them successfully in his or her life, continuing education can be used to reinforce learning, update information, and achieve higher levels of knowledge. In-depth knowledge of the relationship between nutrition and the disease process, nutrition principles, food preparation, and eating behaviors can enable patients to \"take charge\" of their disease management. The dietitian at this level serves as a consultant helping the client synthesize and personalize information.\n\n### C. Educational Methods\n\nIn addition to the content of the educational intervention, the process or method offers different techniques to make nutrition education more effective. In-person individual or group sessions are the most common formats used for nutrition counseling. While decisions on group or individual counseling sessions may be made by feasibility criteria (i.e., time, money), the client's learning preference should be considered as well. Using Osterman's classification of learning styles (Table 1) _feelers_ would like group sessions that have discussion opportunities, whereas _thinkers_ could be frustrated by this method because they \"just want the facts.\" _Sensors_ would respond to either method as long as an application exercise is included. A combination of individual and group sessions offers practical advantages. Information presented during group sessions can be tailored to the individual in a one-on-one discussion either in-person, by telephone or e-mail.\n\nAnother method for nutrition education is the use of self-study materials. This approach has been used more often in population or community nutrition interventions than in group or individual counseling sessions. Self-study modules offer advantages in terms of convenience, pace of learning, active involvement of the individual, and economics. Modules range in size and scope. Two popular examples are the 28-page pamphlet of the Shape Up America program [26] and the 312-page Learn Program manual [27]. Self-study modules generally include self-assessment exercises, general information on the health topic, steps for identifying personal behaviors to modify, guidelines for making behavioral changes, methods for monitoring behavior change, and tips on sustaining the new behaviors in special situations (e.g., eating out, stressful days). This method of learning offers something for all learning styles, especially _intuitors_ who will appreciate the opportunity for self-discovery.\n\nA model for using patient assessment to guide selection of education methods for weight management has been developed by Caban and colleagues (Fig. 1) [28]. They use a comprehensive set of physical and psychological indicators to classify individuals by risk status, and then select interventions with an intensity that matches individual needs. People who are in stable clinical condition, have few behaviors to change, and are highly motivated are classified as low risk and are candidates for self-directed learning methods. Those with clinical conditions targeted for improvement, several behaviors to change, and less motivation to make changes require more structure and support. Group interaction, periodic monitoring, and structured activities are appropriate for this type of client. The high-risk client, who has multiple health problems that need to be addressed, has little motivation, and requires a great deal of support, will need individualized care provided by clinicians. In their program, computers are used to access assessment data, tailor questionnaires to individual profiles, and provide feedback to the client. The authors recognize that all practice settings do not have these resources, but encourage counselors to use even a brief assessment to collect information that will determine the best type of program for the individual.\n\nFIGURE 1 Model for individualizing treatment approaches. [Adapted with permission from Caban, A., Johnson, P., Marseille, D., and Wylie-Rosert, J. (1999). Tailoring a lifestyle change approach and resources to the patient. _Diab. Spectrum_ **12,** 33\u201338.]\n\n### D. Nutrition Educational Tools\n\nNutrition counseling does not suffer from a lack of tools. Information on nutrition education materials can be found in the catalogues of publishing houses, volunteer health and professional organizations, government agencies, reviews published in professional journals, and by professional networking. Education materials also are available from companies manufacturing health products (e.g., pharmaceutical companies) and associations that promote a food or food group (e.g., National Dairy Council). Although there may not be a teaching tool that matches all aspects of a patient's learning profile, availability of educational materials is not the prominent problem. Resources for acquisition and storage are more common deterrents to dietitians and other health care providers being able to use a teaching tool tailored to a client's specific needs.\n\n#### 1. MEAL PLANNING TOOLS\n\nSeveral formats for nutrition education tools have been applied in multiple areas of clinical nutrition. These include guidelines, menu approaches, counting methods, and exchange systems.\n\nGuidelines, such as the Food Guide Pyramid, are a tool to provide basic information to help people make healthy food choices. They may include some information on servings and food preparation but not the specificity of nutrient information that can be found with other methods. Guidelines work well as a tool for initial education if precision in nutrient intake is not required. They may contain sufficient information for some people to be able to make eating behavior changes that reduce health risks and improve clinical indicators.\n\nMenus are a tool to give clients specific direction on what to eat, including food type, preparation method, and serving size. For survival education, several days of menus can be written using familiar foods that ensure appropriate nutrition. The number of patients who carefully collect their tray menus during a hospital stay attests to the popularity of this approach. Menu planning tools generally rely on input from the patient so that food likes and dislikes can be taken into consideration. Menu planning can be combined with other instructional formats and used at the practical and ongoing education levels as well. Computer programs are becoming available that plan menus taking into account the nutrient prescription and individual food preferences.\n\nCounting methods have become popular in recent years although one tool, _The Point System,_ was introduced as early as 1944 [29]. Calorie counting is a standard approach for weight management. Fat gram counting is utilized in teaching materials for cholesterol reduction, cancer prevention, and weight management. Carbohydrate counting, initially considered a technique for intensive insulin therapy, is now being used with all types of diabetes for initial as well as continuing education.\n\nExchange approaches focus on food groups versus individual foods to teach nutrition principles. A popular example is the _Exchange Lists for Meal Planning_ [30] that has been used for decades to instruct patients with diabetes. The system provides a tool for teaching patients how to select a diet that meets a macronutrient prescription. The exchange lists concept has been adapted for weight management education and is sometimes applied to nutrient information provided with recipes and manufactured food products. The Plate Model, a common teaching tool in Europe, has been used in the Diabetes Atherosclerosis Intervention Study (DIAS) [31]. The method uses a visual tool of a plate divided into three sections: one covers one-half of the plate and the other two are one-quarter sections. The guidelines place vegetables including salads in the one-half plate section, grains in one of the one-quarter sections and meat and alternatives in the other one-quarter section. Other food groups are included as side dish servings. DIAS has established target nutrient intakes for study participants that are evaluated by annual 24-hour recalls. Both the Plate Model and the more quantitative exchange lists have been used to help DIAS participants achieve the study goals.\n\nAdaptations of the above tools for nutrition education can be found to match many idiosyncratic learner characteristics. Simplified versions appropriate for initial education or for low-literacy clients and versions that have been translated into Spanish are the most common tools. A recent trend in nutrition education materials is to provide \"copy masters\" for client education tools that can be photocopied without copyright infringement. Purchasing reproducible tools offers nutrition counselors an efficient way to have a wide range of teaching materials available to meet individual client needs. Even more promising is the emerging option of computer software providing nutrition education tools. Computer programs allow the provider to tailor materials to patient characteristics and to change content if it becomes obsolete.\n\n#### 2. SKILLS TRAINING\n\nMeal planning tools, even those carefully selected and matched to individual learner characteristics, are not sufficient for nutrition education. People may learn what they need to do, but lack the \"skill\" or ability to translate knowledge into practice. Teaching an individual how to read a nutrition label provides the knowledge necessary to interpret the information. Applying that knowledge to decisions made at the point of purchase or in meal preparation requires skill. Comparison of the nutrition labels on two products requires analytical skills if the products have different serving sizes. After using the nutrition label to select a product, additional skills are required to integrate the selected food into the total diet. Tools are available in print, video, CD-ROM, and three-dimensional models for teaching clients a wide range of nutrition skills.\n\nSome clients, however, cannot or choose not to master certain skills. Meal planning is a prime example. No matter how well educated and trained, there are some people who find meal planning a real barrier to following their diet. \"Just tell me what to eat\" is their common complaint. Preprinted menus offer a solution in some cases. The American Diabetes Association introduced the first of their _Month of Meals_ publications in 1989 and quickly added four more editions in response to the demand [32]. Books offering low fat and\/or low calorie menus are abundant, as well.\n\nMenus, however, do not meet the needs of people who lack the skills and interest to prepare meals from menus. The Cardiovascular Risk Reduction Dietary Intervention Trial addressed the difficulty many people have in translating complex dietary recommendations into meals. The trial compared a nutritionally complete prepared meals program with (1) a diet prescribed to meet the same nutrient levels established for the prepared meals [33] and (2) usual-care dietary therapy [34]. The prepared meals program resulted in significantly greater weight loss and reduction in blood pressure than the other two interventions, and improvement in lipid and glucose levels compared with the usual-care therapy.\n\nThis \"need\" for more specific information that will help people choose healthy meals is not limited to clinical nutrition. The food industry, recognizing that each generation of Americans spends less time cooking, has responded with carry-out and order-in meals from restaurants, and deli, salad bar, and complete \"meals to go\" in supermarkets. Today's nutrition counselor must be just as adept at helping clients develop the skills to select healthy preprepared meals as teaching the fundamentals of reducing dietary fat intake.\n\nTo change eating habits, nutrition counseling needs to include more than well-selected education techniques. A review has found that many studies based on dissemination of information and teaching of skills were not effective in changing eating behavior [5]. Even studies supposedly following the knowledge, attitude, behavior communications model were not successful if the behavioral components were actually skills training (e.g., label reading, food preparation). Only in studies where participants were self-selected and already motivated were they able to show changes in eating patterns with an intervention that did not have a true behavior change technique. Unfortunately, diagnosis of a disease or health risk does not automatically result in a motivated patient, therefore, dietitians need to include behavior change techniques in their practice.\n\n## IV. BEHAVIOR CHANGE TECHNIQUES\n\nTheories for behavior change are extensive and are described in Chapter 6. Certain theories offer techniques that are practical for use in nutrition counseling. Some techniques such as self-monitoring, behavioral contracting, and goal setting are patient focused. They allow the individual to \"personalize\" the nutrition intervention, to be an active participant in the process, and to receive feedback on progress made toward identified goals. Other techniques such as motivational interviewing, consciousness raising, and assessing readiness for change are implemented by the counselor. The stages of change transtheoretical model [35] offers dietitians a systematic framework for applying behavior change techniques to nutrition education. While the model can be used with all education methods, the following section will describe application in nutrition counseling sessions.\n\n### A. Stages of Change\n\nThe stages of change model, discussed also in Chapter 7, was described by Prochaska & colleagues. It postulates that both cessation of high-risk behaviors and the acquisition of healthy behaviors involves progression through five stages of change: precontemplation, contemplation, preparation, action and maintenance (see Table 3) [35]. The precontemplation stage is characterized by having no intention of changing the behavior in question in the foreseeable future. People in this stage tend to be unaware that they have a problem and are resistant to efforts to modify the behavior in question. Contemplation is characterized by awareness that the particular behavior is a problem, and serious consideration about resolving the problem but having no commitment to take action in the near future. The preparation stage is the stage of decision making. The person has made a commitment to take action within the next 30 days and is already making small behavioral changes. In the action stage, clients make notable overt efforts to change. Maintenance involves working to stabilize the particular behavior change and avoid relapse for the next 6 months. People do not simply progress through the stages in a straight line; they may recycle by relapsing and repeating stage progressions and they can enter or exit at any point [36, 37]. This transtheoretical model has been shown to generalize across a broad range of problem behaviors (including diet, exercise and weight control) and populations [36].\n\nTABLE 3\n\nStages of Change\n\n_Source:_ Reprinted with permission from HealthPartners, Inc., Bloomington, MN. \u00a9 2000. All other rights reserved. For more information, contact HealthPartners, Center for Health Promotion.\n\nIntegral to the stages of change model is a \"standard\" or goal for the behavior that has been proven to produce results. For example, the goal of the NCEP Step 1 diet for a dietary fat intake of less than 30% of energy was established based on research showing associated reduction in serum cholesterol concentration. Stages of change techniques help individuals evaluate current behaviors against a defined standard then assess their readiness to change. The objective is to help patients achieve the standard for the behavior; however, incremental goals are often required. Fortunately, many nutrition interventions in clinical nutrition show benefits associated with stepwise progression to the goal.\n\nIf dietitians use the stages of change model as a basic technique for approaching nutrition counseling sessions, it often requires a modification in their teaching and counseling style [38]. The focus of attention shifts away from an agenda to merely educate the individual and shifts toward an approach that assesses the status of the individual as a basis for tailoring the counseling session. For example, instructing a client with hypercholesterolemia who is clearly in the precontemplation stage on an NCEP Step 1 diet will be less effective than discussing the risks associated with high cholesterol levels and the benefits of reducing the fat content of the diet. While the counselor may feel an obligation to impart specific information, mismatching stages and interventions could break rapport and may lead the client to avoid further follow-up sessions.\n\n### B. Effective Use of Stages of Change Technique in Nutrition Counseling\n\nNutrition assessments are typically done at the initial counseling session. It is important for dietitians to use the stages of change model in each session to assess attitudes toward nutrition and health, readiness to learn, and willingness to change. Assessment of these attitudes requires proficient use of open-ended questions and listening skills.\n\nOpen-ended questions begin with what, how, why and could. The first clue about a client's stage of change is in the response to opening questions such as \"How can I help you?\" \"What are your goals for our meeting today?\" Attentive listening is an important tool to assess the client's responses to these questions. Attentive listening involves not only allowing sufficient silence to hear the client's verbal response, but also paying attention to facial expression, voice tone, and body language as the person is speaking. The nutrition counselor can use the listening technique of paraphrasing to see if he or she has accurately understood the content of the client's statements or the listening technique of reflection to find out if he or she understands the emotional feeling that the client is trying to convey [39]. It is the combination of open-ended questioning and proficient listening skills that builds rapport.\n\nAs the counselor collects assessment information in the first part of the counseling session, there are repeated opportunities to evaluate a client's stage of change through the use of open-ended questions and effective listening skills.\n\n### C. Consciousness Raising\u2014A Technique for Precontemplators\n\n1. A CASE OF PRECONTEMPLATION\n\n_Dietitian:_ \"How can I help you?\" \"What are your goals for today's session?\"\n\n_Patient:_ \"I don't know. The doctor sent me.\" \"I feel fine.\" \"I already have so many problems, so why bother.\" (arms crossed, sitting back in the chair, stiff body posture, voice tone may display resignation, anger, upset, or denial concerning necessity for changing habits or coming to see the dietitian)\n\n_Dietitian:_ \"Did your doctor say why he wanted you to come?\" \"What did your doctor tell you about your _____ (e.g., cholesterol levels\/diabetes control\/ blood pressure)?\"\n\nPatients in the precontemplation stage tend to focus more on the difficulties or disadvantages of changing eating behavior\u2014the diet restriction, the inconvenience, or the expense of making healthy food choices or keeping food records. They tend to put much less emphasis on the benefits of changing eating behavior. Patients can be in the precontemplation stage for various reasons including lack of knowledge, lack of skills, lack of resources, distorted health beliefs, or competing priorities [40]. The counselor needs to explore and assess the reasons particular to each client before proceeding further with the session.\n\n_Questions to Assess Knowledge, Skills, Resources, Health Beliefs and Competing Priorities:_\n\n\"What did your doctor tell you about your ____ (e.g., cholesterol level\/diabetes control\/blood pressure)?\"\n\n\"What did your doctor tell you about your laboratory results?\"\n\n\"Do you know what the goals are for ____ (cholesterol levels\/hemoglobinA1c levels\/blood glucose levels?\"\n\n\"What are your personal goals?\"\n\n\"What do you know about how your diet affects your ____\n\n____ (e.g., cholesterol level\/hemoglobin A1c\/blood glucose patterns?\"\n\n\"What do you think is most important when reading a nutrition label if you have ____ (e.g., diabetes\/high cholesterol)?\"\n\n\"Do you have a blood glucose meter?\"\n\n\"Do you test your blood glucose levels at home?\" How often and what times of day?\"\n\n\"What do you think that you would need to do to improve your ____ (e.g., cholesterol levels\/hemoglobinA1c levels\/blood glucose levels\/activity level)?\"\n\n\"Are there any factors that you feel make it difficult for you to focus on improving your ____ (e.g., cholesterol levels\/hemoglobinA1c levels\/blood glucose levels\/activity level)?\"\n\n#### 2. CONSCIOUSNESS RAISING\n\nConsciousness raising is an important strategy for dietitians to use if a client presents to a session in the precontemplation stage [37]. The following steps can be used to facilitate consciousness raising (Table 4):\n\nTABLE 4\n\nStage Matched Counseling Techniques\n\n_Source:_ Reprinted with permission from HealthPartners, Inc., Bloomington, MN. \u00a9 2000. All other rights reserved. For more information, contact Health Partners, Center for Health Promotion.\n\n1. Discuss the medical problem or condition of concern.\n\n2. Review lab results related to the condition vs. target\/normal values for the test results.\n\n3. Review the relationship of diet, exercise, and other self-care habits to the medical condition and personal lab data.\n\n4. Use visual aids (e.g., test tubes of fat, 1-lb or 5-lb fat models), audiovisuals, and personalized profile sheets to enhance the message.\n\n5. Elicit feedback from the client regarding this information.\n\nAs the interaction between the counselor and the patient proceeds, the counselor can watch for changes in body language (leaning forward, changing voice tone, arms uncrossing) and in level of interaction (asking more questions). Once this information sharing process is complete, the counselor has provided the client with the information necessary to make an informed choice about changing eating behavior. The clients who are in the precontemplation stage because of lack of knowledge or skills may move toward contemplation or preparation stages once these issues are addressed. On the other hand, some clients may be fully informed about their medical condition, lab data, and the impact of eating, activity, and self-care habits on their health and still do not view changing eating habits as a priority. These clients are less likely to progress to contemplation or preparation within one session. In these cases, it is important to avoid judgment and show understanding by acknowledging their feelings and choices. For example, a client may feel too depressed to consider change at the time of the session and a competing priority may be to seek counseling and treatment for depression first. The counselor's responsibility is to provide information and then reassess the client's stage of change. It's the client's responsibility to make an informed choice.\n\n### D. Motivational Interviewing\u2014A Technique for Contemplators\n\n1. A CASE OF CONTEMPLATION\n\n_Dietitian:_ \"How can I help you?\" \"What would you like to accomplish at our meeting today?\"\n\n_Patient:_ \"I'm not sure. I know what to do. I just need motivation.\" \"I know that I should ____ (e.g., lose weight\/eat less fat), but _____.\"\n\nPatients in the contemplation stage view the pros and cons of changing eating, exercise or self-care habits as about equal, with the cons slightly greater than the pros. They may focus on the short-term costs of changing eating behaviors (e.g., limiting food choices, adjusting food purchases when shopping or menu selections when eating out, investing time in keeping food records or nutrition appointments) and pay less attention to the long-term health benefits [40]. Clients can be in the contemplation stage due to limited knowledge about the problem, wishing that someone else would fix the problem or that the problem would solve itself, competing priorities, or low self-efficacy regarding ability to change eating habits.\n\nIf limited knowledge is the problem, then the counselor can use the technique of consciousness raising to provide information, clarify any misconceptions, and respond to specific questions. Before proceeding further with the session, the counselor needs to develop a clear understanding of the client's ambivalence about changing eating behavior. To do this, the counselor needs to explore the client's concerns and perceived barriers to making changes as well as the perceived benefits. This technique of exploring the pros and cons of behavior change is called _decisional balance._\n\nThe counselor also needs to evaluate each client's self-efficacy for changing eating behavior because perceived self-efficacy influences the acquisition of new behaviors and the inhibition of existing behaviors. It also affects people's choices of behavioral settings, the amount of effort they will expend on a task, and the length of time they will persist in the face of obstacles. Finally, self-efficacy affects people's emotional reactions and thought patterns [41].\" The process of using decisional balance and other techniques to increase motivation and self-efficacy is often referred to as _motivational interviewing._\n\nFor clients to move from the contemplation stage toward the preparation stage of change, they must believe that the advantages of changing their eating behavior outweigh the disadvantages. The counselor can help reduce or minimize barriers to change by assisting the client with practical problem solving (some barriers may be related to access to treatment and resources and other barriers will be more attitudinal, such as fear of change or the results of change). It is also helpful to identify the client's positive incentives for continuing with current behaviors (i.e., the payoff for staying the same) and use strategies to decrease the perceived desirability of the behavior (e.g., increase awareness of the negative consequences of the behavior using facts and audiovisuals) [40].\n\nMotivational interviewing is a client-centered technique designed to build commitment and reach a decision to change. It involves an interviewing process that assesses decisional balance and self-efficacy beliefs and focuses on increasing the client's intrinsic motivation to change and self-confidence in ability to do so. The following types of questions can be used to assess decisional balance [40].\n\n#### 2. DECISIONAL BALANCE QUESTIONS\n\nExplore the advantages of not changing (staying the same):\n\n_Dietitian:_ \"What are some of the positive aspects of not _____ (e.g., reducing your fat intake\/losing weight)?\"\n\n_Patient:_ \"I can eat what I want and enjoy my food and not feel deprived.\"\n\n_Dietitian:_ \"What else is good about it?\"\n\n_Patient:_ \"I don't have to risk failing at this again.\"\n\n_Dietitian:_ \"What else?\"\n\n_Patient:_ \"Those are the main things.\"\n\nExplore the disadvantages of not changing\n\n_Dietitian:_ \"What are some of the not so good things about\n\n____ (e.g., eating a high fat diet\/maintaining your current weight)?\"\n\n_Patient:_ \"My cholesterol is too high. My clothes don't fit and I'm sluggish.\"\n\n_Dietitian:_ \"Anything else?\"\n\n_Patient:_ \"I have a higher risk for a heart attack.\"\n\n_Dietitian:_ \"Any other disadvantages?\"\n\n_Patient:_ \"My family worries about me.\"\n\nSummarize by paraphrasing\n\n_Dietitian:_ It sounds like you like to eat and enjoy your food without feeling deprived. I also get the sense that it's important to you not to feel like a failure when you try to change your eating habits. On the other hand it sounds as though there are some disadvantages to your current way of eating\u2014your high cholesterol level, the risk for heart disease and feeling sluggish. Also, it sounds as though you think your family worries about you and your health.\"\n\n_Patient:_ \"That's right.\"\n\n_Dietitian:_ \"How do you feel about this now? What would you like to do about this?\"\n\n_Patient:_ \"I'm not sure.\"\n\n_Dietitian:_ \"It's important to enjoy your food and not feel deprived. If we could come up with some ideas to help lower your cholesterol that are do-able and that won't make you feel deprived, would you be interested? If you'd like, we can explore some of the possibilities together.\"\n\nEach client's motivation to change is influenced by two components\u2014importance and confidence [42]. Assessing importance and confidence is a technique that dietitians can use to help decide what steps to take with patients who are in the contemplation stage of change.\n\n#### 3. IMPORTANCE AND CONFIDENCE TECHNIQUE*\n\n**Introduce the Discussion**\n\n\"I'm not really sure how you feel about _____ (e.g., reducing your fat intake\/losing weight\/exercising more). Can you help me by answering two simple questions and then we can see where to go from there?\"\n\n**Assess Importance and Confidence**\n\n\"How do you feel _right now_ about _____ (eating less fat\/losing weight\/exercising more)? On a scale from 0 to 10, with 0 meaning 'not important at all' and 10 meaning 'very important,' how _important_ is it to you personally to _____ (eat less fat\/lose weight\/exercise more)?\"\n\n\"If you decided _right now_ to _____ (eat less fat\/lose weight\/exercise more), how _confident_ do you feel that you would succeed? If 0 is 'not confident at all' and 10 is 'very confident,' what number would you give yourself?\"\n\nSummarize the answers:\n\n**Selecting the Focus**\n\n\u2022 If _importance_ is low (<3), focus on _importance_ first.\n\n\u2022 If both are the same, focus on _importance_ first.\n\n\u2022 If one number is distinctly lower than the other, focus on the _lower_ number first.\n\n\u2022 If both are very low (<3), explore feelings about participating further in the session.\n\n**Exploring Importance**\n\n\"This is (very\/pretty\/somewhat\/a little bit) important to you. What made you choose _____ and not 1 or 2?\"\n\n\u2022 Reflect reasons given (self-motivational statements).\n\n\u2022 Ask for elaboration. (\"What makes that important to you?\" \"Tell me more about that.\")\n\n\u2022 Ask for more reasons. (\"What else makes it [very\/pretty\/somewhat\/a little] important?\")\n\n\u2022 Summarize. (\"So what makes this [very\/pretty\/somewhat\/a little] important right now is...\". \"What would have to happen for you to move up to a _____ [score plus 3\u20134]?\" \"What stops you from being at a _____ [score plus 3\u20134]?\")\n\n\u2022 Reflect, ask for elaboration, ask \"What else.\"\n\n\u2022 Summarize. (\"So what makes this [very\/pretty\/somewhat\/a little] important is...\". \"It would become more important to you if...\". \"What has kept it from being more important is...\".)\n\n**Exploring Confidence**\n\n\"You feel (very\/pretty\/somewhat\/a little) confident that you could do this if you tried. What made you choose _____ and not 1 or 2?\"\n\n\u2022 Reflect reasons given (self-motivational statements).\n\n\u2022 Ask for elaboration. (\"Tell me more about that.\")\n\n\u2022 Ask for more reasons. (\"What else makes you feel [very\/pretty\/somewhat\/a little] confident?\")\n\n\u2022 Summarize (\"So what makes you [very\/pretty\/somewhat\/a little] confident right now is....\") \"What would help you move up to a _____ [score plus 3\u20134]?\" \"What stops you from being at a _____ [score plus 3\u20134]?\")\n\n\u2022 Reflect, ask for elaboration, ask \"What else?\"\n\n\u2022 Summarize. (\" So what makes you [very\/pretty\/somewhat\/a little] confident is....\" \"You would feel more confident if...\". \"What has kept you from feeling more confident is...\".)\n\nThe following types of questions can be used in the motivational interviewing process to reinforce confidence\/self-efficacy. These types of questions are referred to as _competency\u2013focused interviewing_ [39].\n\n_Questions That Are Competency Focused and Success Oriented:_\n\n\"What do you know about _____ (e.g., diabetes, high cholesterol)?\"\n\n\"What kind of changes have you made in your diet so far?\"\n\n\"How have you fit exercise into your schedule in the past?\"\n\n\"What is the most important thing that you were able to learn?\"\n\n\"What do you think you would do differently this time to enable you to _____ (lose weight\/lower your cholesterol\/improve your blood glucose levels?)\"\n\n\"How do you think you would feel when you reach your goal?\"\n\n\"What do you see as the next step?\"\n\n#### 4. FRAMES STRATEGY\n\nMiller and Rollnick suggest that the following specific motivational techniques can be combined to achieve an effective motivational interviewing strategy (FRAMES) [40].\n\n1. **F** eedback. Clearly discuss the client's current health situation and risks and explain results of objective tests, share observations based on food records and weight trends. Clarify goals by comparing feedback on patient's current situation to some standard and set goals toward that standard that are realistic and attainable.\n\n2. **R** esponsibility. Emphasize that it is the client's responsibility to change.\n\n3. **A** dvice. Clearly identify the problem or risk area, explain why change is important and advocate specific change.\n\n4. **M** enu. Offer the client a menu of alternative strategies for changing eating habits. Offering each client a range of options allows the individual to select strategies that match his or her particular situation and enhance the sense of perceived personal choice.\n\n5. **E** mpathy. Show warmth, respect, support, caring, concern, understanding, commitment, and active interest through attentive listening skills.\n\n6. **S** elf-efficacy. Reinforce the client's self-efficacy via competency-based questions and statements. Research has shown that the counselor's belief in the client's ability to change can be a significant determinant of outcome [40].\n\nThe dietitian's responsibility in counseling patients in the contemplation stage is to help the client reduce the barriers to making changes, focus more on the benefits, simplify the \"to do\" steps, and enhance self-efficacy. Once the dietitian has completed this process, it is the client's responsibility and choice to make a decision about changing eating habits.\n\n### E. Goal Setting\u2014A Technique for the Preparation Stage\n\n1. A CASE OF PREPARATION\n\n_Dietitian:_ \"How can I help you?\" \"What are your goals for today's session?\"\n\n_Patient:_ \"I want to _____ (improve my blood glucose control\/lower my cholesterol level\/lose weight).\"\n\nPatients in the preparation stage feel that the advantages of changing their eating habits outweigh the disadvantages. They may have already tried making small changes, but are looking for more specific guidance and support. Their ambivalence may not have disappeared, however they show more interest in change by making self-motivational statements, asking more questions about change and experimenting with small changes [40]. The dietitian's responsibility is to strengthen the client's commitment to change and to assist him\/her in making realistic plans to modify his\/her lifestyle and eating habits. It is the client's responsibility to participate in goal setting by considering the options and selecting a strategy that provides sufficient direction to prevent floundering, but not so many goals that it undermines self-efficacy and success.\n\n#### 2. GOAL SETTING\n\nFor clients to move toward the action stage, they must resolve their ambivalence and establish a firm commitment to a plan of action. The counselor can use the FRAMES technique to guide the client toward a specific action plan. In addition, the counselor can ask questions and make statements that imply competency and build self-efficacy. It is important for the dietitian to accentuate and reinforce behaviors that the client is doing right and then set goals from there. Goals that are specific, realistic, positive, short term, and measurable are best. It is also important to help the client anticipate obstacles to success and problem-solve strategies to deal with those barriers (Fig. 2). Finally, the counselor can ask questions to be sure that the client has set reasonable achievable goals. If clients are not at least 80% confident in their ability to achieve their goals, it is important to help them reset their goals to a level at which they feel they can be successful.\n\nFIGURE 2 Weekly goals form.\n\n_Questions to facilitate goal setting:_\n\n\"What do you think you would like to do?\"\n\n\"What would you say is the first step that you need to take?\"\n\n\"What other changes would you like to make?\"\n\n\"What options would you consider trying?\"\n\n\"What obstacles can you anticipate that might interfere with your ability to accomplish your goals?\"\n\n\"How do you think you will handle these obstacles?\"\n\n\"How confident are you that you will be able to accomplish your goals?\"\n\n_Patient:_ \"I'd like to start to increase my activity to improve my blood sugars.\"\n\n_Dietitian:_ \"That's great! It's good that you have so many options for exercise\u2014a treadmill, outdoor walking, an exercise bike. You've also already got a great start on increasing your activity by walking with your coworker at lunch two times per week for 20 minutes each time. If you could increase your activity minutes toward 150 minutes per week, that is a level that would be a good target. What would you say would be a reasonable next step to increase your activity?\"\n\n_Patient:_ \"I'd like to try to walk four times per week for 20 minutes at lunch.\"\n\n_Dietitian:_ \"Are there any problems or roadblocks that you can anticipate that would get in the way of accomplishing this goal?\"\n\n_Patient:_ \"If it rains, we might not go out.\"\n\n_Dietitian:_ \"How will you handle that situation?\"\n\n_Patient:_ \"I'll either suggest that we walk indoors or I'll make a plan to walk on my own on the weekend.\"\n\n_Dietitian:_ \"Any other barriers or obstacles that you can anticipate?\"\n\n_Patient:_ \"No, not right now.\"\n\n_Dietitian:_ \"Out of 100% confidence, how confident are you that you can do 80 minutes of activity in the next week?\"\n\n_Patient:_ \"I'm 80% confident.\"\n\n_Dietitian:_ \"Good, because if you were less than 80% confident, that would mean that we should consider changing the goal so that you feel that the likelihood of success is fairly high.\"\n\n### F. Self-Management Skills Training\u2014Techniques for Action\n\n1. A CASE OF ACTION\n\n_Dietitian:_ \"How can I help you?\" \"What would you like to accomplish in our meeting today?\"\n\n_Patient:_ \"I'm doing well with my food choices and exercise during the week, but on weekends it can be harder if I go out to eat.\"\n\nPatients in the action stage have started making changes in their environment to support changes in eating habits. They need positive reinforcement for making behavioral changes and assistance strengthening self-management skills. The counselor's responsibility is to provide continued praise and support for positive behavioral changes and offer ongoing information and advice to enhance self-management skills. The client's responsibility is to actively participate in the session by sharing feelings about changes that he or she has made and discussing questions and concerns about maintaining the behavior changes.\n\n#### 2. SELF MANAGEMENT SKILLS TRAINING\n\nThe core techniques that dietitians can combine to help clients in the action stage to strengthen self-management skills are concrete nutrition information and advice, self-monitoring, stimulus control, and exercise [1].\n\n1. _Concrete nutrition information and advice._ Dietitians expertise in translating nutrition recommendations into food choices that are meaningful and satisfying is key in supporting clients in the action stage. In particular, dietitians can assist patients in trying new recipe ideas\/modifying favorite recipes, finding healthier food choices, and suggesting new and interesting food combinations at meals and snacks. In this way, dietitians can help patients learn that dietary change can occur without disrupting family food patterns or personal enjoyment of food.\n\n2. _Self-monitoring._ Dietitians can encourage clients to use self-monitoring as a tool to enhance behavior change. When clients keep track of their eating habits by recording the amounts and types of food eaten, they become more aware of how their food choices affect their health outcomes (e.g., weight, cholesterol, blood sugar). Patients who are overweight or have hypercholesterolemia may focus on food records that track fat gram and\/or energy intake, whereas patients with diabetes may focus on self-monitoring food, carbohydrate intake, activity, and blood sugar patterns and then use the data to learn a problem-solving approach for understanding food\u2013activity\u2013blood sugar relationships.\n\n3. _Stimulus control._ Dietitians can discuss with patients how to set up their environment for success. If patients remove problem foods from their environment and follow a shopping list that includes only healthy food choices, then they can create an environment conducive to successful dietary change. Clients can also learn strategies to reduce the temptations for undesired foods by minimizing exposure to these food items at parties or buffets.\n\n4. _Exercise._ Increasing exercise is a positive lifestyle change that can improve high density lipoprotein (HDL) cholesterol, blood glucose control, blood pressure, weight loss and weight maintenance, self-esteem, and motivation. Increasing physical activity therefore can enhance the impact of dietary change on health outcomes and may also strengthen the self-esteem, self-efficacy, and motivation necessary to maintain diet behavior change.\n\n### G. Problem-Solving Skills and Coping Strategies\u2014Techniques for Maintenance\n\n1. A CASE OF MAINTENANCE\n\n_Dietitian:_ \"How can I help you?\" \"What are your goals for today's session?\"\n\n_Patient:_ I feel good about the way that I'm eating now. It has become more of a habit to _____ (eat less fat\/exercise more\/count carbohydrates).\"\n\nPatients in the maintenance stage have been actively working on changing eating habits for at least 6 months. Although the changes in eating habits may have become part of a patient's routine, there is still a risk of lapse or relapse. The dietitian's responsibility is to help clients plan ahead for high-risk situations and develop the problem solving and coping skills necessary to avoid relapsing. The client's responsibility is to share any feelings or concerns related to the changes that they have made, discuss the particular situations that challenge his or her ability to continue with eating behavior change and actively participate in the problem-solving process.\n\n#### 2. PROBLEM-SOLVING SKILLS AND COPING STRATEGIES\n\nSome examples of the high-risk situations that can lead to lapse or relapse of eating behavior change are eating out, stress and other emotions (feeling anxious or depressed), hunger, and vacations. If clients do not develop coping strategies to deal with high-risk situations, then they are likely to interpret an experience of overeating as a failure. This can diminish self-efficacy and undermine long-term success. Alternatively, if clients can respond to high-risk situations with effective coping strategies, then the experience of managing the situation improves self-efficacy and increases the likelihood of sustaining behavior change [1].\n\nThe best way to help clients prevent relapse is to focus on both cognitive and behavioral techniques to appropriately respond to lapses. The behavioral steps to help clients include the following: (1) Anticipate and identify high-risk situations, (2) facilitate a problem-solving approach to determine possible solutions, (3) select a coping strategy, and (4) evaluate the effectiveness of the plan. The cognitive techniques that are important in preventing relapse are directed at how clients think and feel in response to a relapse. Cognitive restructuring techniques include the following: (1) Listen to self-talk associated with a lapse and evaluate if thoughts are logical, reasonable, and\/or helpful; (2) counter any negative self-talk with positive statements; and (3) stay focused on the progress so far and the advantages of making changes in eating behavior [27].\n\nIn the process of discussing lapses in high-risk situations, it is important for dietitians to remind clients that lapses are normal and to ask open-ended questions about how clients feel regarding changes they have made in eating habits and respond to them with empathy not judgment [40].\n\n_Questions to Facilitate Discussion of High-Risk Situations_\n\n\"How are you feeling about the changes that you have made in your eating habits so far?\"\n\n\"Are there any situations that make it more challenging for you to sustain your _____ (activity level\/reduced fat intake \/weight loss)?\"\n\n\"What strategies have you tried so far to deal with the situation?\"\n\n\"How did they work?\"\n\n\"Are there any other ideas that you might try?\"\n\n\"Would you like to hear about some strategies that have worked for other people in the same situation?\"\n\n\"If we take a moment to review the various options that we have discussed, are there any that you would like to try?\"\n\n\"Which ones?\" (See Fig. 2.)\n\nThe problem-solving skills and coping strategies that are important for working with clients in the maintenance stage often incorporate the techniques used to help move the client forward through the stages of change. Note that patients can recycle by relapsing and repeating stage progressions, and that they can exit and reenter at any point. In fact, many new clients that dietitians see are relapsers who are coming to reenter and recycle through the eating behavior change process. In these cases, there are some important questions that the counselor can ask to assess each client's experience.\n\n_Questions to Assess Prior Experience of Relapsers_\n\n\"What were your three most important reasons for _____ (losing weight\/ eating less fat\/ exercising more)?\"\n\n\"How long did you sustain the behavior change?\"\n\n\"Who supported you at that time?\"\n\n\"How did you handle temptations?\"\n\n\"What coping strategies did you use?\"\n\n\"What was going on in your life and how were you feeling when you started slipping?\"\n\n\"What did you learn from that experience?\"\n\nIn sum, the stages of change model is a useful technique for approaching nutrition counseling sessions. At first glance, however, adding a new component to counseling sessions may appear difficult when allocation of time already is an issue [25]. Fisher uses a three-phase model to show that stages of change techniques can be applied in sessions as brief as 15 minutes (Fig. 3). The model expedites identification of the behavior, clarification of patient's readiness to change, and selection of stage-appropriate tools and techniques. The time the dietitian allocates to tailor counseling to information that the patient is receptive to learning will result in a more productive session.\n\nFIGURE 3 Three-phase model within 15-minute encounter. (Adapted with verbal permission from E. B. Fisher, Jr.)\n\n### H. Social Support\n\nSocial support influences all stages of change. A number of studies have shown the benefit of support from family, friends, and coworkers [1, 5, 43]. When spouses or significant others understand the nutritional advice given and the importance of self-monitoring, stimulus control, and exercise in managing eating behaviors, then they are better able to provide support. Support might include cooking and shopping for appropriate food items, keeping tempting food out of sight, modeling a slower eating speed, exercising together, showing a positive attitude, and offering praise.\n\nSupport from clinical staff (e.g., nurses, dietitians) was studied in the very successful Diabetes Control and Complications Trial (DCCT) [44]. The success of the DCCT was due in part to participants in the intensively treated group being able to make multiple behavior changes that resulted in their achieving improved glycemic control [45]. A study examining the effect of diet behaviors found that adherence to meal plans and appropriate treatment of hypoglycemia were associated with better diabetes control [46]. After the close of DCCT, telephone interviews were used to ask a sample of participants about the types of staff support that helped them adhere to all aspects of their treatment plan. Nondirective types of support (suggests, willing to help but does not take over) were mentioned more often than directive types of support (tells, assumes responsibility) [43]. Studies of staff support conducted with patients with acute as well as chronic illnesses indicate that stage or phase of the patient's clinical condition may influence the type of staff support that is most helpful [47]. Patients newly diagnosed or in an acute phase of their illness appear to appreciate directive support (gives me great solutions) while those in stable conditions value nondirective support (gives me suggestions but lets me make up my own mind). Applying type of staff support to stages of change techniques, nondirective support is most appropriate for the precontemplation, contemplation, and maintenance phases, whereas directive support would be appropriate for problem solving in the preparation and action stages.\n\n## V. CONCLUSION\n\nThe challenge in clinical nutrition is to prevent or treat disease by changing people's eating habits. Research on nutrition education is extensive but not conclusive. Although a definitive model has not been identified, a variety of strategies have shown success and can be incorporated into nutrition education programs. There is consensus on two elements: (1) Education needs to be tailored to the individual's learning needs and (2) both education and behavior change techniques are necessary. Understanding factors that influence the learning and behavior change processes enables better selection of educational tools and techniques to make counseling more effective. The stages of change model is a useful technique for tailoring education to the individual and applying behavior change strategies.\n\nReferences\n\n1. Brownell K.D., Cohen L.R. Adherence to dietary regimens 2: Components of effective interventions. _Behav. Med_. 1995;20:155\u2013164.\n\n2. Knowles M. _The Adult Learner: A Neglected Species_. Seattle, WA: Gulf Publishing Co.; 1990.\n\n3. Tough A. How adults learn and change. _Diabetes Educator_. 1985;11:12\u201325.\n\n4. Brookfield S.D. _Developing Critical Thinkers: Challenging Adults to Explore Alternative Ways of Thinking and Acting_. Houston: Jossey-Bass; 1987.\n\n5. Contento I., Bronner Y.I., Paige D.M., Gross S.M., Bisignani L., Lytle L.A., Maloney S.K., White S.L., Olson C.M., Swadener S.S. The effectiveness of nutrition education and implications for nutrition education policy, programs, and research: A review of research. _J. Nutr. Educ_. 1995;27:355\u2013364.\n\n6. Karlsen B., Gardner E.F. _Adult Basic Learning Examination Norms Booklet_. San Francisco: Harcourt Brace; 1986.\n\n7. Wilkinson G.S. _Wide Range Achievement Test Administration Manual_. San Antonio: Wide Range; 1993.\n\n8. Taylor S.C. Cloze procedure: A new test for measuring readability. _Journalism Q_. 1953;10:425\u2013433.\n\n9. McLaughlin G.H. SMOG grading: A new readability formula. _J. Reading_. 1969;12:639\u2013646.\n\n10. Gunning R. _The Technique of Clear Writing_. Wilmington, DE: McGraw-Hill; 1952.\n\n11. Fry E.B. Fry's readability graph: Clarifications, validity, and extension to level 17. _J. Reading_. 1977;21:242\u2013252.\n\n12. Powers R.D., Summer W.A., Kearl B.E. A recalculation of four readability formulas. _J. Educ. Psychol_. 1958;48:99\u2013105.\n\n13. Vaughn J., Jr. Interpreting readability assessments. _J. Reading_. 1976;19:635\u2013639.\n\n14. Pitchert J., Elam P. Readability formulas may mislead you. _Patient Educ. Couns_. 1985;7:181\u2013191.\n\n15. Nitzke S.V.J. Overview of reading and literacy research and applications in nutrition education. _J. Nutr. Educ_. 1992;24:261\u2013265.\n\n16. \"Ethnic and Regional Food Practices: A Series (1989\u20131999).\" The American Dietetic Association, Chicago.\n\n17. Achterberg C. Factors that influence learner readiness. _J. Am. Diet. Assoc_. 1988;88:1426\u20131429.\n\n18. Walker E. Characteristics of the adult learner. _Diabetes Educator_. 1999;25:16\u201324.\n\n19. Osterman D.N. The feedback lecture: Matching teaching and learning styles. _J. Am. Diet. Assoc_. 1984;84:1221\u20131222.\n\n20. Johnston J.M., Jansen G.R., Anderson J., Kendell P. Comparison of group diet instruction to a self-directed education program for cholesterol reduction. _J. Nutr. Educ_. 1994;26:140\u2013145.\n\n21. Mager R.F. _Preparing Instructional Objectives_. New York: Fearon; 1975.\n\n22. Houston C., Haire-Joshu D. Application of health behavior models. In: Haire-Joshu D., ed. _Management of Diabetes Mellitus: Perspectives Across the Lifespan_. Belmont, CA: Mosby-Year Book, 1995.\n\n23. Bloom B.S. _A Taxonomy of Educational Objectives. Handbook 1: Cognitive Domain_. St. Louis: David McKay Co.; 1956.\n\n24. Harrow A. _A Taxonomy of the Psychomotor Domain_. New York: David McKay Co.; 1971.\n\n25. Pichert J.W. Teaching strategies for effective nutrition counseling. In: Powers M.A., ed. _Handbook of Diabetes Nutritional Counseling_. New York: Aspen Publishers, 1987.\n\n26. Glass W. _On Your Way to Fitness_. Rockville, MD: Shape Up America; 1996.\n\n27. Brownell K.D. _The Learn Program for Weight Control_. Bethesda, MD: American Health Publishing; 1994.\n\n28. Caban A., Johnson P., Marseille D., Wylie-Rosett J. Tailoring a lifestyle change approach and resources to the patient. _Diab. Spectrum_. 1999;12:33\u201338.\n\n29. Green J.A. Meal planning approaches. In: Powers M.A., ed. _Handbook of Diabetes Nutritional Counseling_. Dallas, TX: Aspen, 1987.\n\n30. Exchange Lists for Meal Planning. Rockville, MD: The American Dietetic Association, Chicago, and American Diabetes Association; 1995.\n\n31. Camelon K.M., Hadell K., Jamsen P.T., Ketonen K.J., Kohtamaki H.M., Makimatilla S., Tormala M.L., Valve R.H. The plate model: A visual method of teaching meal planning. _J. Am. Diet. Assoc_. 1998;98:1155\u20131162.\n\n32. Month of Meals series. Alexandria, VA: American Diabetes Association; 1989\u20131994.\n\n33. Metz J.A., Kris-Etherton P.M., Morris C.D., Mustad V.A., Stern J.S., Oparil S., Chait A., Haynes R.B., Resnick L.M., Clark S., Hatton D.C., McMahon M., Holcomb S., Snyder G.W., Pi-Sunyer F.X., McCarron D.A. Dietary compliance and cardiovascular risk reduction with a prepared meal plan compared to a self-selected diet. _Am. J. Clin. Nutr_. 1997;66:373\u2013385.\n\n34. Haynes R.B., Kris-Etherton P., McCarron D.A., Oparil S., Chait A., Resnick L.M., Morris C.D., Clark S., Hatton D.C., Metz J.A., McMahon M., Holcomb S., Snyder G.W., Pi-Sunyer F.X., Stern J.S. Nutritionally complete prepared meal plan to reduce cardiovascular risk factors: A randomized clinical trial. _J. Am. Diet. Assoc_. 1999;99:1077\u20131083.\n\n35. Prochaska J.O., DiClementi C.C. Stages and processes of self-change in smoking: Toward an integrative model of change. _J. Consult. Clin. Psychol_. 1983;51:390\u2013395.\n\n36. Prochaska J.O., Velicier W.F., Rossi J.S., Goldstein M.G., Marcus B.H., Rakowski W., Fiore C., Harlow L.L., Redding C.A., Rosenbloom D., Rossi S.R. Stages of change and decisional balance for 12 problem behaviors. _Health Psychol_. 1994;13:39\u201346.\n\n37. Prochaska J.O., DiClementi C.C., Norcross J.C. In search of how people change: Applications to addictive behaviors. _Diab. Spectrum_. 1993;6:25\u201333.\n\n38. Gehling E. The next step: Changing us or changing them? _Diab. Care Educ. Newsflash_. 1999;20:31\u201333.\n\n39. Powers M.J. Counseling skills for improved behavior change. In: Powers M.A., ed. _Handbook of Diabetes Nutritional Counseling_. Alexandria, VA: Aspen Publishers, 1996.\n\n40. Miller W.R., Rollnick S. _Motivational Interviewing: Preparing People to Change Addictive Behavior_. Gaithersburg MD: The Guilford Press; 1991.\n\n41. Strecher V.J., McEvoy Devellis B., Becker M.H., Rosenstock I.M. The role of self-efficacy in achieving health behavior change. _Health Educ. Q_. 1986;13:73\u201391.\n\n42. Rollnick S., Mason P., Butler C. _Health Behavior Change: A Guide for Practitioners_. New York: Churchill Livingstone; 1999.\n\n43. Fisher E.B., Jr., La Greca A.M., Arfken C., Schneiderman N. Directive and nondirective support in diabetes management. _Int. J. Behav. Med_. 1997;4:131\u2013144.\n\n44. Davis K., Heins J., Fisher E.B., Jr. Types of social support deemed important by participants in the DCCT. _Diabetes_. 1997;46(suppl 1):89A.\n\n45. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. _N. Engl. J. Med_. 1993;329:977\u2013994.\n\n46. Delahanty L.M., Halford B.N. The role of diet behaviors in achieving improved glycemic control in intensively treated patients in the Diabetes Control and Complications Trial. _Diab. Care_. 1993;16:1453\u20131458.\n\n47. AprilFisher E.B., Bickle C., Harber K., Hughes C.R., Jeffe D.B., Kahl L., LaGreca A.M. Benefits of directive and nondirective support are moderated by severity of circumstances. Paper presented at the 18th Annual Scientific Sessions of the Society of Behavioral Medicine. San Francisco. 1997.\n\n* * *\n\n*This section is reprinted with permission of Allan Zuckoff, M. A., University of Pittsburgh Medical Center; adapted from S. Rollnick, P. Mason, and C. Butler (1999). \"Health Behavior Change: A Guide for Practitioners.\" London: Churchill Livingstone.\nCHAPTER 9\n\nEvaluation of Nutrition Interventions\n\nALAN R. KRISTAL and JESSIE A. SATIA, Fred Hutchinson Cancer Research Center, Seattle, Washington\n\n## I. INTRODUCTION\n\nNutrition interventions include a broad array of programs and activities, with many different goals. Interventions may be designed for treatment of acute or chronic disease, prevention of specific diseases, or simply improvement of nutritional status. Interventions can focus on changing an individual's dietary behavior, both directly or indirectly, or can target the composition, manufacture, and availability of food. Research in dietary intervention can be either behavioral, to test whether a dietary intervention program can promote dietary change, or clinical\/epidemiological, to test whether dietary change can affect a disease endpoint or disease risk. Nutrition intervention programs can be delivered as services to individual clients, groups, or entire communities. The evaluation needs of each nutrition intervention program will differ, based on the program content, design, and goals.\n\nThe optimal way to evaluate a nutrition intervention is to complete a hypothesis-driven, randomized trial. This means that an _a priori_ hypothesis should be used to evaluate whether or not the intervention was effective, an experimental design created, and careful attention paid to factors that contribute to the overall validity of a scientific experiment, such as protocol development, measurement, and statistical analysis. While expertise from many scientific disciplines is required to complete such a trial, it is important for nutritional scientists to understand the methodological issues that underlie the design of a valid intervention evaluation.\n\nIn this chapter, we give a general overview of quantitative evaluation, with an emphasis on those aspects likely to be the responsibility of a nutritional scientist. We focus on quantitative _outcome_ evaluation and, in particular, on whether or not an intervention is effective in achieving change in dietary behavior.\n\n## II. OVERVIEW: TYPES OF NUTRITION INTERVENTION PROGRAM EVALUATIONS\n\nA well-designed and clearly articulated evaluation plan is a key aspect of a successful nutrition intervention program. An evaluation plan will require an intervention program to have clearly defined and realistic objectives. An evaluation plan can also give timely feedback at each stage of program implementation, allowing modifications to improve program effectiveness. The three types of evaluation that are most suitable for nutrition intervention programs are (1) formative evaluation, focusing on program design; (2) process evaluation, emphasizing program implementation, quality assurance, and participant reaction; and (3) outcome evaluation, measuring the achievement of program objectives.\n\n### A. Formative Evaluation\n\nOne challenge for nutrition intervention programs is matching the content of the interventions to the interests and needs of the intended audience. Nutrition information is inherently complex, and it must balance between being scientifically correct and still comprehensible and useful to the intended audience. Intervention activities should also be reasonable for the context in which they are delivered. At the stage of formative evaluation, a nutritionist assesses whether or not materials and programs are appropriately intensive, scientifically coherent, convenient, and otherwise consistent with their intended use.\n\n### B. Process Evaluation\n\nOnce in place, it is important to know if the intervention program is reaching its audience and how it is being received. There is a tendency for persons who are already interested in nutrition and motivated to change to participate in intervention trials. Thus, if an intervention is to be generalizable, it is important to ensure that program components successfully reach men, younger persons, racial and ethnic minorities, and other groups less likely to be drawn to programs in nutrition. This is also the stage at which to evaluate whether the audiences' reactions to the program are favorable, or whether changes are needed to have a broader impact.\n\n### C. Outcome Evaluation\n\nUltimately, the effectiveness of a nutrition intervention will be judged based on its ability to achieve program objectives. The authors judge the specification and collection of outcome measures, and their correct analysis, to be essential to any nutrition intervention evaluation.\n\n## III. OUTCOMES OR ENDPOINTS USED TO ASSESS INTERVENTION EFFECTIVENESS\n\nThe most obvious intervention outcomes or endpoints are based on changes in nutrient intake, but for comprehensive evaluations of dietary interventions, this is too limited. Often indirect measures of intervention effectiveness, for example, changes in supermarket sales or implementation of a work site catering policy, can serve as meaningful outcomes. In addition, to understand how an intervention did or did not work, it is necessary to measure intermediate or mediating factors for dietary change, such as beliefs, attitudes, or nutrition knowledge.\n\nBefore discussing outcomes in detail, we make two overarching points. First, the single most important consideration for selecting outcomes is that they must have clear interpretations that relate directly to the intervention you are evaluating. Examples of poor evaluation outcomes include \"compliance with the USDA Food Pyramid,\" \"dietary adequacy,\" \"compliance with the Recommendation Dietary Allowances,\" or even the well-characterized \"Diet Quality Index\" [1, 2]. These types of outcomes are not useful as evaluation endpoints, because they are too multidimensional, cannot be precisely defined, or their interpretation is too subjective. Second, if you will not measure dietary change per se, then the outcomes you select should have a known and reasonably strong relationship to dietary behavior. For example, increased nutrition knowledge or awareness of relationships between diet and disease are not sufficient in themselves as intervention endpoints, because they have low or no predictive value for dietary behavior change. Experience suggests that it is best to carefully formulate and define intervention outcomes as a part of the overall intervention design. This results in a more focused intervention program and yields evaluation data that are optimally informative.\n\n### A. Types of Outcomes or Endpoints Used to Assess Intervention Effectiveness\n\nOutcomes can be classified most broadly into four types: (1) physiologic or biological measures, (2) behavioral measures based on self-report, (3) diet-related psychosocial measures, and (4) environmental or surrogate measures of dietary behavior.\n\n_Biological or physiologic measures_ are objective indicators of dietary change. For well-funded and relatively small clinical intervention trials, measures based on serologic concentration of nutrients or metabolic changes associated with dietary change are optimal approaches to evaluation. The strength of biological measures is that they are objective and unbiased. Their weaknesses are that they do not exist for many outcomes of interest (e.g., reduced percentage of energy from fat in the diet) and they are rarely sufficiently sensitive to detect the relatively modest dietary changes one can expect from low-intensity health promotion interventions. It can also be procedurally difficult and prohibitively expensive to add biological measures to large, health-promotion intervention trials.\n\n_Self-reported dietary behavior_ is the most often used basis for intervention evaluation. There are two conceptually distinct types of measures based on self-reported diet. The most common measures are the intakes of specific nutrients in an individual's diet, such as percentage of energy from fat or milligrams of beta-carotene, or measures of food use, such as servings per day of fruits and vegetables. One can also characterize dietary habits, for example, removing the skin before eating chicken or using low fat instead of regular salad dressings. The primary weakness of all self-reported behavioral outcomes is that persons exposed to an intervention may bias their reports of behavior to exaggerate true behavior change. Their strengths are that they are easy to interpret and often reflect an intervention's specified goals.\n\n_Psychosocial outcomes_ consist of theoretical constructs that relate to diet or dietary change. These include nutrition knowledge, attitudes, and beliefs about diet, and intentions and self-efficacy to change diet. These constructs are best interpreted in the context of structured theoretical models of behavior change. For example, an intervention based on the Precede\/Proceed model [3] would assess changes in predisposing, enabling and reinforcing factors for dietary change. The primary weakness of psychosocial outcomes is that they do not measure dietary change, rather they measure factors that relate, often quite weakly, to dietary behavior. Their strength is that they are often the actual target or focus of the intervention. For example, an intervention designed to increase awareness of the benefits of eating more fruits and vegetables could be evaluated by measuring changes in perceived benefits. Psychosocial measures are best considered as mediating factors for dietary change, that is, factors that explain how an intervention ultimately results in changed behavior [4\u20136]. Collecting information on psychosocial factors can yield valuable insights into how to improve the design and content of dietary interventions, and we believe they should be given high priority in research designs.\n\n_Environmental measures_ assess characteristics of communities, organizations, or the physical environment that in some way reflect dietary behavior or dietary change. Measures some researchers have used are the percentage of supermarket shelf space used for healthful versions of staple foods (e.g., low-fat milk or whole-grain breads) and percentage of foods in vending machines that are low in fat. The weakness of these measures is that there is relatively little research to support their validity as measures of dietary change, and the available evidence suggests that associations between change in environmental measures and individual dietary behavior are modest. However, the strengths of these measures are that they are objective and unbiased, and frequently very inexpensive to collect.\n\nSelection from among these various types of intervention outcomes is based on many criteria. However, the primary criterion is that they should be meaningful measures of the desired intervention outcome. This requires judgment and thoughtful consideration of both the goals of the intervention and of the evaluation. The following examples serve to illustrate the diversity of options available for evaluating different types of interventions. For an intensive, clinical intervention with a goal of lowering fat intake from 35% to 20% of energy, outcomes could include self-reported diet and body weight. For a work site-based intervention to increase the availability of healthful foods to workers during the workday, outcomes could include foods offered at cafeterias and in vending machines. For a work site-based intervention designed to test different approaches to promoting healthy dietary patterns, outcomes could include self-reported diet as well as mediating psychosocial factors such as knowledge of fat in foods and stage of change to adopt a low-fat diet. For a public health campaign to increase the use of lower fat milk products, outcomes could include supermarket sales data and random digit dial surveys to assess consumption of low fat milk.\n\n## IV. DESIGN OF NUTRITION INTERVENTION EVALUATIONS\n\nEvaluation design encompasses the protocols for participant recruitment, measuring intervention delivery and outcomes, and analyzing and presenting results. Choices made during the development of an evaluation design are primarily guided by two considerations: (1) the content, type, or design of the nutrition intervention; and (2) the purpose of the evaluation.\n\nBy type of intervention, the broadest distinctions are between _clinical_ interventions and _public health_ interventions. Clinical interventions target high-risk individuals and generally consist of intensive, multiple individual or group sessions that address both dietary behavior (nutrition education) and psychological support for maintaining dietary change. In contrast, public health interventions target large groups of individuals, usually not limited to persons at high risk, and generally consist of a broad range of low-intensity and low-cost components such as media messages and self-help materials. Some interventions fall between these two extremes (e.g., work site-based health promotion programs may offer a series of intensive nutrition education classes) and some may fall outside of this classification altogether (e.g., the decision to fortify cereal-grain products with folate). For purposes of evaluation, the two most important distinctions between clinical and public health interventions are the timing and amount of expected change: Intensive clinical interventions produce rapid and dramatic change, whereas public health interventions yield slow change over long periods of time.\n\nBy purpose of the evaluation, we make a broad distinction between _research_ that contributes to scientific knowledge and _documentation_ that serves the needs of practitioners and administrators. Comprehensive scientific evaluations are generally beyond the financial means of any but the most well-funded research trials, because the costs of evaluation generally far exceed those for intervention design and delivery. Practitioners and administrators must assume that the interventions being delivered are at least somewhat effective, and evaluation to serve their needs is focused on evidence that the program is reaching the intended target population and is benefiting those who participate.\n\n### A. Design Components of Nutrition Intervention Evaluations\n\nFive components characterize the design of an intervention evaluation:\n\n1. A representative sample of persons who would be likely program participants or targets of an intervention\n\n2. One or more measures of the evaluation outcome preintervention\n\n3. One or more comparison groups, most often a control group not receiving an intervention\n\n4. Randomized assignment to treatment (intervention or control) groups\n\n5. One or more measures of the evaluation outcome postintervention.\n\nThe most robust research evaluations include all of these design characteristics. However, not all of these components need be present to have a scientifically valid design. The only necessary characteristics are that there be postintervention outcome measures and a comparison group. Intervention evaluations not based on a randomized trial can be either quasi-experimental, in which treatment is assigned in a fashion other than randomization, or observational, in which epidemiological methods are used to statistically model differences between those receiving and not receiving an intervention. Due to their complexity, nutritionists should consult with appropriate experts before choosing such design alternatives.\n\nAdministrative evaluation of program effectiveness should strive to incorporate as many of these evaluation components as feasible. In practice, however, an administrative evaluation may simply consist of documenting the number of persons receiving the intervention and measuring changes among those exposed. One improvement in this design would be to calculate participation rates, based, for example, on the number of persons offered the intervention or the number eligible. This would give administrators insight into the acceptability and penetration of the intervention into eligible populations. Another improvement would be to document level of exposure to or participation in the intervention, and to correlate level of exposure to changes in outcome. This type of dose\u2013response analysis can suggest whether more intensive intervention would be cost effective.\n\n#### 1. REPRESENTATIVE SAMPLE\n\nEvaluation of a representative sample means that evaluation participants include persons from demographic and socioeconomic groups who would be targets for the intervention. Representativeness in dietary interventions is often difficult, because participation in nutrition interventions tend to be higher among women and older people [7]. However, representativeness is not always important or even desirable. If the purpose of the evaluation is to test whether an intervention can work at all (e.g., in the best of all possible circumstances), it will be preferable to recruit only highly motivated volunteers using highly selective enrollment criteria. It may even be appropriate to require participants to complete a prerandomization run-in activity, for example, completing a 4-day food record, to eliminate participants not likely to complete the trial [8]. Alternatively, if an evaluation is designed to examine how an intervention will work as it is to be delivered in practice, then participants are best recruited from representative samples from defined populations, attempting to achieve as high a recruitment rate as possible [9].\n\n#### 2. PREINTERVENTION MEASURES\n\nMeasures before intervention are desirable for two reasons. First, even in randomized experiments, there might be differences in baseline measures across treatment groups. Second, pre- and postintervention measures allow evaluators to calculate change from baseline. Basing evaluation on differences in change between treatment groups rather than simply on differences in outcome measures postintervention almost always yields superior statistical power. If possible, preintervention assessments should be completed before treatment group assignment, so that neither the evaluation staff nor the participant can be biased by knowing which intervention they will receive.\n\n#### 3. COMPARISON GROUP\n\nThe choice of comparison group(s) depends on the goal of the intervention. The most common design is to compare outcomes in a group receiving an intervention to one not receiving an intervention. Options include comparisons between a new and a standard intervention, or between groups receiving different levels of the same intervention. It is rarely satisfactory to have no comparison group, because it is not possible to determine whether any observed changes can be attributed to the intervention or to other factors outside of an investigator's control or not directly associated with the intervention itself.\n\n#### 4. RANDOMIZED ASSIGNMENT TO TREATMENT GROUPS\n\nRandomization is the best way to ensure comparability between treatment groups, but it is not always possible. In this case, it may be feasible to devise an unbiased approach to assign individuals to contrasting treatments, for example, based on health care practitioner, day of the week, or hospital clinic. One entirely unacceptable design is to offer an intervention and compare results in self-selected participants to those refusing to participate. Participation in a nutrition intervention is strongly associated with characteristics that _a priori_ predict dietary change, such as sex, age, and interest in nutrition and health. With this approach, comparisons between participants and nonparticipants are so strongly biased that they should be excluded from consideration for any type of evaluation design. In our judgment, examination of change among participants alone is a superior design, because no inferences can be made beyond documentation that change occurred in those who received the intervention.\n\n#### 5. POSTINTERVENTION ASSESSMENTS\n\nIt is optimal to complete two postintervention measures, one after the intervention is complete and one delayed by at least several months. The first measure assesses the immediate impact of the intervention, while the latter gives insight into whether effects are durable and whether there is continued change over time. Basing an evaluation on an early measure alone can be misleading: Some interventions may have no long-term effect, because behavior change is not sustained. Some interventions may yield continual, gradual change over time, in which case only a long-term assessment will demonstrate intervention effectiveness.\n\n### B. Analysis of Intervention Effects\n\nStatistical analysis of nutrition interventions can pose many challenges. Below we describe some of these statistical issues and give nontechnical recommendations for the most commonly used designs and statistical models.\n\n#### 1. LEVEL OF MEASUREMENT AND UNITS OF ANALYSIS\n\nThere are many choices for how outcomes are assessed and analyzed. Unit of measurement describes whether the outcomes are assessed on individual participants (e.g., self-reported diet) or on a group-level or environmental characteristic (e.g., supermarket sales or availability of fresh fruit in work site cafeterias). Unit of analysis describes whether analyses of outcomes are based on individual observations (e.g., individual changes in fat intake), on aggregated measures of individual observations (e.g., percent of population drinking low-fat milk in the previous day), or on group-level or environmental outcomes. For clinical interventions and for public health interventions in which outcome assessments are based on measurements of individuals, the units of measurement and analysis are almost always at the individual level. For some public health interventions, especially those that target large groups or communities, there are many options for combinations of unit of measurement and unit of analysis. The most common is to aggregate measures on a sample of individuals and interpret these as measures of the community. One can also measure outcomes at the community level, such as availability of healthful foods in supermarkets and restaurants, existence of nutrition programs in the community, or media coverage of nutrition-related information.\n\nIt is of utmost importance to match the evaluation design, in particular, the randomization scheme, to the unit of analysis. In grouped randomized designs, for example, a trial randomizing work sites or schools to different intervention treatments, the analysis must be based on the unit of randomization, not the individuals participating in the intervention. Unlike individually randomized designs, the sample size for analysis of grouped randomized designs is a function both of the number of groups and the number of participants in each group. Consider an intervention trial that is evaluating whether a school nutrition curriculum can affect students' lunch choices. If 12 schools with 2000 students were each randomized such that 6 implemented the curriculum and 6 did not, the number of experimental units is 12, not 24, 000. Readers will find an excellent and nontechnical overview of these issues in reviews by Koepsell [10, 11].\n\n#### 2. CALCULATING THE SIMPLE INTERVENTION EFFECT\n\nThe best and most comprehensible measure of intervention effectiveness is the difference between the change in intervention group participants minus the change in control (or alternative treatment) group participants. We call this measure the _intervention effect._ As an example, in an intervention with a goal to decrease percent energy from fat the intervention effect is defined as:\n\nWhere Fat (%en) is the percent of energy from dietary fat, subscripts _b_ and _f_ refer to baseline and follow-up, and subscripts _I_ and _C_ refer to intervention and control groups. The statistical test of whether or not the intervention effect is different from zero is based on the standard error of the intervention effect, which is defined as:\n\nwhere _n 1_ is the sample size in the intervention group and _n C_is the sample size in the control group.\n\nMost measures of dietary intake and serum nutrient concentrations have log-normal or other non-normal distributions; however, changes in these measures are characteristically normally distributed. It is therefore rarely necessary to transform measures before analysis, and for simplicity of interpretation it should be avoided.\n\n#### 3. CALCULATING INTERVENTION EFFECTS ADJUSTED FOR SOCIODEMOGRAPHIC AND CONFOUNDING FACTORS\n\nThe intervention effect is best estimated after adjustment for demographic and other characteristics that are associated with dietary behavior. This is because (1) randomization may not have resulted in these characteristics being evenly divided between treatment groups and (2) there may be increased statistical power to detect a statistically significant intervention effect if variance associated with these factors is controlled for in the analyses.\n\nHere are two approaches to calculating adjusted intervention effects, which differ depending on the scale of measurement of the outcome variable. Most outcome measures are either continuous (e.g., percent energy from fat) or ordered categories (e.g., motivation to change measured on a 1\u201310 scale), and thus multiple linear regression can be used to calculate adjusted intervention effects. For a simple, two-treatment randomized design, the best approach is to build a regression model as follows: The dependent variable is calculated as the change from baseline to follow-up in the outcome measure; the covariates are the baseline value of the outcome measure plus the demographic and diet-related measures you wish to control for in the analysis; the independent variable representing treatment group is an indicator variable coded 0 = control and 1 = treatment. In this model, the regression coefficient for the treatment indicator variable is the adjusted treatment effect, and the standard error of this regression coefficient is used to test the statistical significance of the adjusted intervention effect. Table 1 gives an example of how this approach is used for the primary analysis of a two-group randomized trial of a dietary intervention.\n\nTABLE 1\n\nExample of Statistical Analyses Used to Report Outcome of a Dietary Intervention to Reduce Fat and Increase Fruit and Vegetable Intakes\n\naScore from 21-item scale, scored from 1.0 (low fat) to 4.0 (high fat).\n\nbAdjusted for baseline value, age, sex, race, body mass index, and income.\n\n_Source:_ From Kristal, A., Curry, S., Shattuck, A., Feng, Z., and Li, S. (2000). A randomized trial of a tailored, self-help dietary intervention: The Puget Sound Eating Patterns Study. _Prev. Med._ **31,** 380\u2013389.\n\nIf an outcome measure is categorical, for example, whether or not a participant lost 10 pounds or more, it is then appropriate to use logistic regression models. For a simple, two-treatment design, the following model would be appropriate: The dependent variable is an indicator variable, coded 0 or 1, representing whether or not the outcome is absent or present; the covariates are variables you wish to control in the analysis; and the independent variable representing treatment group is an indicator variable coded 0 = control and 1= treatment. The exponentiated regression coefficient of the treatment group indicator variable ( _e_ \u03b2) is the relative odds of the outcome comparing the control to treatment group. The standard error of the regression coefficient is used to determine whether the relative odds are statistically different from 1. 0. Multiple categorical outcomes, for example, movement through stages of dietary change, pose considerable statistical challenges [12, 13]. Consultation with a biostatistician is important in modeling these types of outcomes, and some approach to simplifying the analysis may prove to yield more interpretable and useful results.\n\n### C. Statistical Power\n\nA final aspect of an evaluation design is to choose an appropriate sample size. This requires making a judgment on what size intervention effect is worth detecting. Even a trivially small intervention effect can be found statistically significant given a large enough sample size, and a clinically meaningful intervention effect may not reach statistical significance if the sample size is to small. For clinical interventions, it is reasonable to choose an effect size that is meaningful for an individual. For a public health intervention the effect size will be much smaller, and needs only be meaningful in terms of changes in population distributions of the outcome measure. When choosing minimum effect sizes, it is worthwhile to review what other interventions have achieved. The general recommendation is to never set a minimum detectable intervention effect larger than 1.5 times that observed in other interventions, unless there is very strong reason to believe that the new intervention being evaluated is far superior.\n\n## V. MEASUREMENT ISSUES WHEN ASSESSING DIETARY CHANGE AND OTHER INTERVENTION OUTCOMES\n\nOnce outcomes are well defined and an evaluation design is established, one must select, modify, or develop measures. The two standard characteristics of dietary assessment methods, validity and reliability, are described in detail elsewhere in this text. Here we extend this discussion to cover measures of psychosocial factors and aspects of measurement that are relevant to measuring dietary change and, in addition, discuss practical considerations that have important implications for trial design and feasibility. Section VI describes how these measurement issues influence selection from among alternative measures of intervention outcomes.\n\n### A. Validity\n\nSimply stated, validity is the extent to which your assessment tool measures what you want it to measure. Validity is not necessarily an intrinsic aspect of a particular tool or assessment instrument, because validity can vary as a function of method of administration, participants, or time. There are many types of validity, each having implications for intervention evaluation. Content validity is the extent to which you have sampled the domain of what you are trying to measure. For example, if your intervention goal were to lower total fat intake, high content validity would mean that you have measured all foods with meaningful amounts of fat. A limited but nevertheless important part of content validity is commonly referred to as _face validity._ Face validity is a judgment made by experts about whether or not a completed questionnaire measures what it is supposed to measure [14]. Construct validity is primarily a consideration for psychosocial measures. In this context, high construct validity means that the items used to form a scale are a good measure of some meaningful, underlying or latent construct. An example is a scale that measures enabling factors for dietary change, consisting of six items on barriers, norms, and social support [15]. Criterion validity considers how well one measure correlates with another measure of the same construct. A type of criterion validity most important for nutrition intervention evaluations is predictive validity, which is based on whether a measure made at one time point predicts change at later point in time. For example, based on the theory of reasoned action [16], one would validate a measure of intention to eat more fruits and vegetables by examining how it predicts increased intake.\n\n### B. Reliability\n\nTwo types of reliability are important for evaluating intervention trials. Test\u2013retest reliability measures agreement between multiple assessments. In practice, this means that a measure taken on one day would be strongly correlated with a measure taken on another day. Although no measures have perfect reliability, measures of daily nutrient intake or specific dietary behavior have particularly low reliability due to the variability in the amounts and types of foods people eat from day to day. This type of variability, termed intra-individual or within-persons variability, makes even a perfect assessment of a single day's diet not very informative for evaluating whether or not a person has changed their usual diet as a response to an intervention.\n\nA second and entirely different type of reliability, which is relevant primarily to measures of psychosocial factors, is internal consistency reliability. Most psychosocial factors cannot be assessed directly (e.g., social support for eating low-fat foods), and they are generally measured using a set of items that taken together characterize the construct (e.g., \"How much support do you get from your co-workers to select healthy foods from the cafeteria at lunch?\"). The statistic called Cronbach's alpha, which ranges from 0 to 1, is a measure of how well the mean of scale items measures an underlying construct. High internal consistency reliability is a function of two factors, the average correlation among items in the scale _and_ the number of items in the scale. Most scientists suggest a minimum of 0.7 for internal consistency; however, this ignores the practical problem that in applied evaluations it is not feasible to use lengthy scales. When scales are restricted to three or four items, a Cronbach's alpha of 0.50 is quite satisfactory.\n\n### C. Intervention-Associated Recall Bias\n\nBias is a measure of the extent to which an instrument under-or overestimates what it is attempting to measure. Ample evidence indicates that person-specific biases exist in self-reports of diet; for example, overweight persons tend to systematically underestimate energy intake [17]. If person-specific biases are constant, they will have little impact on evaluation, because analyses will be based on differences between measures assessed at baseline and follow-up. However, two unique sources of bias are introduced whenever evaluation of a dietary intervention is based on self-reported behavior. First, repeated monitoring results in changed responses to assessment instruments. For example, the number of different foods reported on a 4-day diet record decreases from day 1 to day 4, suggesting that study participants simplify their diets [18]. There is also some evidence that the quality of dietary intake data improves with repeated assessments, and this may differ by intervention treatment group [18]. Second, although well-designed dietary intervention trials randomize participants to intervention and control groups, the delivery of the behavioral intervention cannot be blinded as in conventional placebo-controlled trials. Thus, if intervention group participants report eating diets that match the goals of the intervention program rather than what they actually ate, there will be a bias toward overestimating intervention effectiveness. This bias can be substantial [20], and it appears to be larger in women then men [21\u201323].\n\n### D. Responsiveness\n\nA measure only recently introduced into the nutrition literature, related specifically to measuring change, is termed _responsiveness._ Conceptually, responsiveness is a measure of whether an instrument captures information on intervention-related dietary patterns [24]. In intensive clinical interventions, in which a successful intervention results in large changes in foods consumed and food-preparation techniques, a sensitive instrument will capture information on both the most common foods and dietary practices of the sample at baseline and on new practices adopted due to the intervention. In public health interventions, in which there are only modest changes in dietary behavior, a sensitive measure will detect very small changes in behavior targeted by the intervention. Statistically, responsiveness is the ratio of the intervention effect divided by the standard deviation of the intervention effect. Responsiveness is thus a function of several aspects of a measure: (1) how well an instrument measures the intervention outcome both pre- and postintervention, (2) the magnitude of the intervention effect, and (3) the variance in the dietary or other measure used as the intervention outcome.\n\n### E. Participant Burden\n\nIf outcome assessments are too long or complicated, require biological samples, or must be repeated many times, high participant burden results. High participant burden can significantly compromise an evaluation. During recruitment, participation rates will be poor and participants will be less representative of the intervention target population. Once entered into the study, long or repeated follow-up assessments will contribute to high drop-out rates. Once recruited and assigned to a treatment group, all participants should be included in the evaluation whether or not they complete the intervention or subsequent follow-up assessments. Thus, high drop-out rates (larger than 25%) make an evaluation suspect.\n\n### F. Instrument Complexity\n\nAccurate assessment of nutrient intake requires complex instrumentation, regardless of whether instruments are interviewer- or self-administered. Some diet-related psychosocial factors may also require many questions with complicated skip patterns. It is important to remember that nutrition knowledge and literacy may be poor in some populations. Intervention participants may have difficulty answering detailed questions about food preparation or serving sizes, or may not be able to understand complex questions about attitudes and beliefs [25].\n\nComplexity of analysis is an issue that is often overlooked. Before using an evaluation instrument, understand how it will be analyzed, evaluate the underlying nutrient database and associated software, and make sure that these analyses produce the variables you wish to measure. Dietary records, food frequency questionnaires, or scales to measure diet-related psychosocial factors are not useful unless there are means to transform data from these instruments into interpretable measures of the evaluation outcomes. Similarly, while it is simple to collect blood for serologic outcomes, analysis of many diet-related serologic measures is possible only in specialized research laboratories. These laboratories are primarily in academic research centers, and they are rarely capable or interested in processing the large numbers of samples required for evaluating a dietary intervention.\n\n### G. Costs\n\nRandomized trials are expensive, and the costs of evaluating a nutrition intervention are often far greater than the costs of intervention delivery. A large proportion of evaluation cost can be attributed to outcome assessments, especially if they are based on dietary records or recalls or on serologic measures of micronutrient concentrations. Not surprisingly, the high cost of dietary assessments strongly motivates the use of alternative measures.\n\n## VI. DIETARY ASSESSMENT INSTRUMENTS AND THEIR APPLICABILITY FOR INTERVENTION EVALUATION\n\nMost research on dietary assessment is motivated by the needs of nutritional epidemiologists, and is focused on how to best understand relationships between diet and health outcomes or on surveillance of population-level nutritional status. Dietary intervention studies have tended to use dietary assessment methods developed for other purposes, with the hope that they will serve the current intended purpose. However, as described earlier in this chapter, evaluating dietary interventions and measuring dietary change have many special nuances. Here, we review the available tools for measuring intervention effectiveness, focusing on the characteristics most relevant for outcome evaluation.\n\n### A. Anthropometric and Biochemical Measures\n\nMany anthropometric and biochemical measures correlate with nutrient intake, but relatively few of these are useful or appropriate for the evaluation of interventions to change dietary behavior (Table 2). The exceptions will be interventions that are designed to increase intake of a specific micronutrient, for example, the fortification of cereal-grain products in the United States with folate or the fortification of sugar with vitamin A in Guatemala [26].\n\nTABLE 2\n\nAnthropometric and Biochemical Measures Suitable for Intervention Evaluation\n\nThe most useful anthropometric measure is change in body weight, which can either be the goal of the nutritional intervention or a marker of decreased energy intake relative to expenditure, or fat intakes. Given the lack of objective markers of dietary fat reduction, this relationship with weight deserves more comment. Randomized trials of fat reduction show intervention effects for weight of about 3 kg associated with a 10 percentage point decrease in percent energy from fat [8, 27], and of about 0. 25 kg with decreases of 1 percentage point [9]. Weight loss associated with fat reduction is likely due to incomplete substitution of nonfat sources of energy in a fat-restricted diet [28].\n\nBiochemical measures are useful for assessing changes in foods containing unique constituents that can be measured easily in blood or urine. The most often used measure is change in total serum carotenoids, which reflect usual intakes of carotenoid-containing fruits and vegetables [29\u201331], however total carotenoids minus lycopene is a superior measure [32] due to the low correlation of serum lycopene with other serum carotenoids. Biochemical measures are also useful to assess metabolic changes that result from dietary change. Serum cholesterol can be used as a measure of polyunsaturated and decreased saturated fatty acid intake in some target groups [33]. Because biochemical measures are expensive and require invasive collection procedures, they are impractical to use as primary outcome measures in public health interventions. An alternative is to collect biochemical measures on a small subsample of volunteers, and use these data as secondary outcomes to confirm results based on dietary self-report.\n\n### B. Self-Reported Dietary Behavior\n\nTable 3 gives an overview of measures used to collect data on self-reported diet. Selection from among these many choices requires balancing the costs and participant burden of \"gold standard\" measures such as multiple 24-hour dietary recalls with the practical benefits of using short, self-administered questionnaires that assess specific dietary patterns.\n\nTABLE 3\n\nMeasures of Self-reported Diet Suitable for Intervention Evaluation\n\nNutrients from foods consumed\n\n24-hour dietary recalls\n\nNutrients from \"usual diet\"\n\nFood frequency questionnaires\n\nDietary patterns\n\nDiet behavior questionnaires\n\nShort food frequency questionnaires\n\n\"Focused\" 24-hour dietary recalls\n\nThe primary distinctions among types of instruments are based on whether diet is measured based on foods actually consumed, foods \"usually\" consumed, or on patterns of food consumption. Note that there is an inherent hierarchy across these types of instruments; one can measure dietary patterns based on any dietary assessment instrument or foods usually consumed based on foods actually consumed. Measuring nutrients from foods actually consumed is a \"gold standard\" for intervention evaluation, because any changes in foods, portion sizes, and preparation techniques can be captured. However, because many days of foods must be assessed to characterize an individual's usual diet, any measure based on actual foods consumed will be very expensive and have high participant burden. The benefit of assessing nutrients from foods usually consumed is that only a single measure is needed at each time point, but it is not possible to capture details on all foods and their preparation methods in precise detail. The benefits of assessing dietary patterns alone are that it is a less burdensome task and is often a more direct measure of whether new dietary behaviors were adopted. Its limitation is that changes in dietary patterns cannot be directly interpreted as changes in nutrient intake and is thus less well accepted in the nutritional science community.\n\n#### 1. NUTRIENTS FROM FOODS ACTUALLY CONSUMED\n\nThe best approach to measuring nutrients from foods actually consumed are unannounced (unscheduled), interviewer-administered 24-hour dietary recalls. Unannounced recalls are administered by telephone, which in practice is facilitated by collecting information at the beginning of an evaluation on convenient days, places, and times to call. Participants can be given serving size booklets, so that they can refer to specific pictures when reporting amounts of foods consumed. The protocol or script used for collecting 24-hour recalls can also be modified to focus on assessing the intervention outcomes (e.g., for an intervention to decrease saturated fat, type of margarine will be important) and deem-phasize details that are uninformative (e.g., for a fruit and vegetable intervention, probes for added salt are not relevant). Finally, it is important to consider the limitations to the accuracy of 24-hour recalls, because respondents often do not know answers to questions about food composition, preparation, or portion size.\n\nHigh costs (between $35 and $55 per day) and participant burden are serious drawbacks to any study wishing to evaluate intervention effectiveness using dietary recalls. This is especially true when the evaluation is at the individual level and therefore many days of intake must be captured for each participant. For randomized designs, it may be sufficient to calculate intervention effects based on one pre- and one postintervention recall per participant. This measure of the intervention effect will be unbiased, but there are problems due to the very low reliability of a single day's measure of nutrient intake. Most importantly, because the group-level intervention effect will have very high variance there will be low statistical power to detect differences across treatment groups. Further, it is statistically inappropriate to adjust this measure of the intervention effect for individuals' demographic characteristics or other diet-related factors (33). Intervention effects calculated from a single 24-hour recall can be used in much the same way as biochemical measures as a secondary outcome used to corroborate a more practical but less valid measure of nutrient intake.\n\nOther methods of capturing foods actually consumed, including food records and scheduled 24-hour recalls, are inappropriate for intervention evaluation. This is because both record keeping and anticipating a dietary recall can substantially change dietary behavior. While unannounced 24-hour recalls may be subject to intervention-associated bias through differential recall of foods consumed, food records or announced recalls are much more likely to be biased due their effects on food choice.\n\n#### 2. NUTRIENTS FROM FOODS USUALLY CONSUMED\n\nDuring the past 20 years, food frequency questionnaires (FFQs) have become a standard measure in nutritional epidemiology. They have also become quite common for intervention evaluations, because they can be self-administered, processed using mark-sense technology, and cost less than $15 per administration.\n\nTwo characteristics of any FFQ detract from their use for measuring dietary change. First, whereas FFQs are convenient for investigators, they are burdensome to participants, requiring time, high literacy, and knowledge about food. Second, the cognitive processes required to complete an FFQ might make them highly subject to intervention-associated bias. Respondents must construct answers to FFQ items using knowledge about their characteristic diet, because memory about actual eating and drinking episodes erodes after only a few days [35]. Thus, not knowing the true answer to most questions, it is likely that perceptions of behavior deemed desirable due to the intervention would bias FFQ responses [23]. Nevertheless, practical considerations often require using FFQs. For these evaluations, it is necessary to incorporate methods to minimize response burden and increase response rates, such as limiting total questionnaire length and giving incentives.\n\nStandard FFQs developed for epidemiological studies are not necessarily good for intervention evaluation. An intervention-focused FFQ must usually collect detailed information on food choices that reflect the intervention goals. This may require any of the following: (1) regrouping foods into categories relevant to the intervention (e.g., grouping soups into \"creamed,\" \"vegetable,\" \"bean,\" and \"broth\" to capture fat, carotenoids, and fiber); (2) assessing relatively fine distinctions between similar foods (e.g., nonfat vs. regular mayonnaise, refried vs. baked beans, or plain lettuce salads vs. mixed salads with carrots and tomatoes); (3) assessing preparation methods (e.g., chicken with or without skin, shellfish boiled or fried); and (4) collecting information on portion sizes. It is also important to examine the nutrient database and computer algorithms underlying analysis of the FFQ. Several approaches are available for assigning nutrient values to FFQ items [36, 37], and some may not produce a nutrient database that reflects substantial differences in relevant nutrients between foods targeted by the intervention. There are also different approaches in how FFQ analysis software incorporates information about types of foods (e.g., type of milk) or preparations (e.g., chicken with or without skin) when computing nutrients. It may be necessary to modify these algorithms to better reflect changes targeted by an intervention. Unfortunately, developing a new FFQ is very time consuming and complex, and most evaluations must choose wisely from among those available based on how well they capture dietary behavior of interest. Some commercially available FFQ software packages do allow minor modifications that can be helpful in developing FFQs for intervention trials [38], and some FFQs are available that were developed specifically for evaluating interventions [19, 36, 39].\n\n#### 3. DIETARY PATTERNS\n\nThe many practical and scientific benefits to using short, simple instruments to measure dietary patterns targeted by an intervention were described earlier in this chapter. A modest amount of research has been done in this area that has yielded four approaches to short dietary assessment: (1) short FFQs; (2) prediction equations, based on regression models; (3) diet-habits questionnaires; and (4) focused 24-hour recalls. These approaches differ substantially in their underlying statistical assumptions and their use of behavioral theory related to dietary behavior change. Understanding these differences can help nutritionists to select and modify available instruments or to develop instruments suited to the intervention under evaluation.\n\n##### a. Short Food Frequency Questionnaires.\n\nThese instruments typically contain between 10 and 15 FFQ items, and are designed to assess intake of a specific nutrient or frequency of eating a specific group of foods. Examples in the literature include instruments for fat [40], calcium [41], and fruits and vegetables [42\u201345]. For intervention evaluation, a short FFQ should be based on knowledge of dietary patterns in the sample receiving the intervention [46, 47] and the behavioral targets of the intervention. There is modest evidence that an approach based solely on statistical criteria can be valid in epidemiological studies [37], but this approach lacks face validity when evaluating an intervention. Short FFQs are appropriate for intervention evaluation only if a small number of foods is being targeted by the intervention or if the outcome is a nutrient that is concentrated in very few foods. Thus, short FFQs are best for nutrients such as calcium or for foods groups such as fruits and vegetables, and suspect for nutrients such as fat or sodium that are spread throughout the food supply.\n\n##### b. Prediction Equations.\n\nMany nutritional scientists have proposed using regression models to predict nutrient intake from a short set of questions. These models are built typically on data from FFQs, in which the frequencies of eating specific foods are entered into a stepwise linear regression model predicting the nutrient of interest. A relatively small number of foods will predict a large amount of variance in most nutrients [48], and on the surface this appears to be a useful way to simplify dietary assessment. However, these models have very poor face validity, because these prediction equations assign coefficients (weights) to food items that often have little to do with the nutrient of interest [49]. The models also have poor criterion validity, because models developed from one sample typically do not predict nutrient intake in a different, independent sample [50, 51].\n\nA related approach is based on using factor analysis to identify patterns of association among foods on an FFQ. These patterns are given descriptive names (e.g., \"junk food\" or \"plain home cooking\") based on the interpretation of the foods in the factor, and these factors are treated as meaningful measures of an underlying dietary pattern. These patterns lack both face validity and criterion validity and are not reproducible between samples. One of the reasons both regression and factor analysis do not yield useful measures is that they are based on correlations among FFQ items in a specific sample. There is little consistency in the correlations among FFQ items across time or across samples and even less consistency if a dietary intervention changes dietary patterns. Measures based on regression models or factor analysis are entirely inappropriate for intervention evaluation.\n\n##### c. Diet-Habits Questionnaires.\n\nDiet-habits questionnaires are unique because they have been developed specifically to measure intervention outcomes. These questionnaires typically contain between 15 and 30 questions, with response options that are qualitative (e.g., rarely\/never to usually\/always) or ordered frequency categories. The most robust of these measures is based on theoretical or at least explicit models of dietary behavior change [52, 53], and their development is facilitated by an understanding of nutrition, psychometric theory, and cognitive psychology, as well as skill in item construction and questionnaire design. There is ample evidence that these measures can be valid measures of dietary change [52\u201360], and they tend to have higher responsiveness than alternatives such as short FFQs, full FFQs, or repeated 24-hour recalls [7, 9, 24, 61, 62].\n\nDiet behavior questionnaires are well suited to evaluations of public health interventions, because of their low participant burden and high sensitivity to change, and less well suited for clinical interventions requiring a measure of changes in nutrient intake. Before beginning an evaluation, it is important to pilot these measures in the target population, to make sure that the questionnaire format language and diet patterns being measured are appropriate. Nutritionists should take the liberty of modifying existing questionnaires to better suit their target population, as long as the basic structure and content of the instrument remains intact.\n\n##### d. Focused 24-Hour Recalls.\n\nA focused recall uses techniques similar to those used to collect standard 24-hour recalls, but collects only information related to the evaluation outcome. For example, if the intervention goal is to increase servings of fruits and vegetables, then the only information captured during the interview is about fruits and vegetables. The amount of detail collected can also vary. A focused recall could collect details about the type and serving size of all targeted foods, or an interviewer could simply read a list of foods and ask respondents whether of not they ate them in the previous day [63]. Focused recalls are far simpler to administer and analyze than standard 24-hour recalls, yet they share the characteristic of low bias due to reliance on episodic rather than general memory [35]. Similar to a 24-hour recall, only repeated assessments can be used to characterize behavior of an individual, but a single measure is fine for characterizing a group. For example, one could contrast the percentages of participants in intervention and control groups who drank skim milk or ate french fried potatoes on the previous day. More complex focused recalls have been developed to measure carotenoid intake [64], and more research on developing these measures is warranted. Nutritionists should consider developing instruments based on this approach when the 24-hour recalls are the desired evaluation tool but cannot be used due to costs or participant burden.\n\n#### 4. DIET-RELATED PSYCHOSOCIAL FACTORS\n\nTable 4 gives an overview of psychosocial factors that can be used to assess intervention outcomes. Comprehensive behavioral models are built from many constructs [65], but in the context of an intervention evaluation it is generally both feasible and necessary to measure only those constructs that are central to the model. Thus, for each of the behavioral models used to design nutrition interventions, those psychosocial factors that are key to their evaluation are selected.\n\nTABLE 4\n\nDiet-Related Psychosocial Factors Suitable for Intervention Evaluation\n\nVery little research exists on the validity and responsiveness of diet-related psychosocial measures. Measures developed and validated for one behavioral domain, for example, for smoking cessation, are often adapted for dietary intervention research without consideration of their face or construct validity. For some diet-related psychosocial constructs, measurement is quite simple and little validation is necessary. For example, one could measure intention to change diet with a single item such as \"In the next 6 months, how likely is it that you will change your diet to eat less fat?\" Alternatively, measuring a construct such as stage of dietary change can be quite complex and requires considerable developmental research. When adopting psychosocial measures from other behavioral domains, it is important to remember that dietary behavior is unique. First, dietary behavior is not a single behavior but a composite of many behaviors consisting of food choice, preparation, and frequency of consumption. Psychosocial factors related to one aspect of dietary behavior may differ from those relating to another. Second, dietary behavior is not discrete, such a smoking, rather it occurs on a continuum in which any discrete definition of desirable dietary behavior is necessarily arbitrary (e.g., <30% energy from fat). Thus, diet-related psychosocial factors related to a discrete criterion, such as eating five or more servings a day of fruits and vegetables, may not capture the intent of an intervention to simply increase servings regardless of baseline intake.\n\nOne of the most popular approaches to organizing dietary intervention programs is based on using the \"stage of change\" construct from the transtheoretical model [66]. Interventions are designed to move participants from preaction stages of dietary change (precontemplation, contemplation, and decision) into action and then maintenance. There is increasing evidence that interventions can move people through stages of dietary change [67], and that movement through stages of change is associated with dietary behavior change [12, 13]. However, much controversy surrounds the conceptualization of stages of change when applied to dietary behavior, and thus little agreement in the literature of how it should be assessed [67\u201369].\n\nA second popular approach for organizing nutrition interventions is PRECEDE\/PROCEDE, which is not a behavioral model but rather a planning model for intervention development and delivery. Practical measures of the main constructs from this model, predisposing, enabling and reinforcing factors, have been developed and validated [15, 70]. One study has demonstrated that change in these factors, in addition to stage of dietary change, explained up to 55% of the intervention effect in a large randomized trial [13].\n\n### C. Environmental and Surrogate Measures of Diet\n\nTable 5 gives an overview of environmental indicators and other surrogate measures that can be used to assess outcomes of dietary interventions. With the exception of household food inventories, these measures are only useful to evaluate intervention outcomes at the group or community level, and thus are best suited to evaluate environmental-level interventions.\n\nTABLE 5\n\nSurrogate and Environmental Measures Suitable for Intervention Evaluation\n\nHousehold food inventories consist of asking a set of questions about whether or not specific foods are currently available in the household. Characteristically the list consists of 10\u201315 foods that relate to the intervention. Examples of foods that could be included are types of staple foods (e.g., regular vs. low-fat mayonnaise) or the types of foods available for snacks (potato chips, fresh fruit, or baby carrots). There are no studies formally evaluating household inventories as intervention outcome measures, but there is evidence that study participants can recall foods in their households accurately [71], and that the types of foods correlate with individually assessed nutrient intake [72, 73]. Due to the simplicity of this approach, further efforts to evaluate these measures are well motivated.\n\nMonitoring food sales can be used as a direct measure of an intervention effect, but it is extraordinarily challenging [74\u201377]. Relating changes in supermarket sales to nutrition interventions is very difficult, and most interventions attempting this approach have failed. Reasons are many, including the complexity of the food supply, the large number of food items that are sold in typical supermarkets, business confidentiality of sales data, and poor match between the data needs for business and the needs of nutrition researchers. Very careful planning, pilot studies, and ongoing contact between researchers and persons responsible for data collection are necessary to make supermarket sales data useful for outcome evaluation. Monitoring sales in food services such as work site and school cafeterias or vending machines is much simpler and has been used successfully to evaluate interventions [78\u201383]. One good approach is to devise a simple scheme for unobtrusively observing and recording food choices as customers move through a cafeteria line [84].\n\nChanges in the food environment, such as supermarket signage or distribution of supermarket shelf space, are also potential surrogate measures of intervention outcomes. A series of studies has shown that it is possible to reliably measure supermarket environments, that measures of shelf space correlate with community-level measures of diet, and that changes in supermarket shelf space correlate weakly with changes in community-level diet [85\u201389]. It is possible that changes in supermarket signage or foods offered in restaurants could reflect an effective community-level intervention, as businesses adjust to demands from consumers for information about and access to healthier foods. These are not likely to be very sensitive measures, because the supermarket environment is saturated with signage, and restaurant menus are difficult to evaluate objectively.\n\n## VII. CONCLUSIONS\n\nEvaluations of nutrition interventions can be both scientifically and operationally challenging. Nutritionists can and should take the lead in conceptualizing and interpreting the evaluation of a nutrition intervention, but they should also seek collaborations or consultations with scientists in other disciplines to ensure that methods are optimal. When planning an evaluation, make sure that the time line allows you to test, pilot, and if necessary, refine measures and procedures, even if you are using previously developed instruments and methods. 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Perlmutter C., Canter D., Gregoire M. Profitability and acceptability of fat- and sodium-modified hot entrees in a worksite cafeteria. _J. Am. Diet. Assoc_. 1997;97:391\u2013395.\n\n84. Mayer J., Brown T., Heins J., Bishop D. A multi-component intervention for modifying food selection in a worksite cafeteria. _J. Nutr. Edu_. 1987;6:277\u2013280.\n\n85. Cheadle A., Psaty B., Wagner E., Diehr P., Koepsell T., Curry S., Von Korff M. Evaluating community-based nutrition programs. Assessing the reliability of a survey of grocery store product displays. _Am. J. Public Health_. 1990;80:709\u2013711.\n\n86. Cheadle A., Psaty B., Curry S., Wagner E., Diehr P., Koepsell T., Kristal A. Community-level comparisons between the grocery store environment and individual dietary practices. _Prev. Med_. 1991;20:250\u2013261.\n\n87. Cheadle A., Wagner E., Koepsell T., Kristal A., Patrick D. Environmental indicators: A tool for evaluating community-based health-promotion programs. _Am. J. Prev. Med_. 1992;9:78\u201384.\n\n88. Cheadle A., Psaty B., Curry S., Wagner E., Diehr P., Koepsell T., Kristal A. Can measures of the grocery story environment be used to track community-level dietary changes? _Prev. Med_. 1993;22:361\u2013372.\n\n89. Cheadle A., Psaty B., Diehr P., Koepsell T., Wagner E., Curry S., Kristal A. Evaluating community-based nutrition programs: Comparing grocery store and individual-level survey measures of program impact. _Prev. Med_. 1995;24:71\u201379.\n\n90. Karr S., Lampe J., Hutchins A., Slavin J. Urinary isoflavonoid excretion in humans is dose-dependent at low to moderate levels of soy protein consumption. _Am. J. Clin. Nutr_. 1997;66:46\u201351.\n\n91. Seow A., Shi C., Franke A., Hankin J., Lee H., Yu M. Isoflavonoid levels in spot urine are associated with frequency of dietary soy intake in a population-based sample of middle-aged and older Chinese in Singapore. _Cancer Epidemiol. Biomarkers Prev_. 1998;7:135\u2013140.\n\n92. Stanford J.L., King I., Kristal A.R. Long-term storage of red blood cells and correlations between red cell and dietary fatty acids: Results from a pilot study. _Nutr. Cancer_. 1991;16:183\u2013188.\n\n93. Agren J., Hanninen O., Julkunen A., Fogelholm L., Vidgred H., Schwab U., Pynnonen O., Uusitupa M. Fish diet, fish oil and docosahexaeonic acid rich oil lower fasting and postrandial plasma lipid levels. _Eur. J. Clin. Nutr_. 1996;50:765\u2013771.\n\n94. Burr M., Fehily A., Gilbert J., Rogers S., Holliday R., Sweetnam P., Elwood P., Deadman N. Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: Diet and reinfarction trial (DART). _Lancet_. 1989;2:757\u2013761.\n\n95. Chung F., Jiao D., Getahun S., Yu M. A urinary biomarker for uptake of dietary isothiocynates in humans. _Cancer Epidemiol. Biomarkers Prev_. 1998;7:103\u2013108.\n\n96. Seow A., Shi C., Chung F., Jioa D., Hankin J., Lee H., Coetzee G., Yu M. Urinary total isothiocynate (ITC) in a population-based sample of middle-aged and older Chinese in Singapore: Relationship with dietary total ITC and glutothione S-transferase M1\/T1\/P1 genotypes. _Cancer Epidemiol. Biomarkers Prev_. 1998;7:775\u2013781.\n\n97. McDougall J., Litzau K., Haver E., Saunders V., Spiller G. Rapid reduction of serum cholesterol and blood pressure by a twelve-day, very low fat, strictly vegetarian diet. _J. Amer. Coll. Nutr_. 1995;14:491\u2013496.\n\n98. Howell W., McNamara D., Tosca M., Smith B., Gaines J. Plasma lipid and lipoprotein responses to dietary fat and cholesterol: A meta-analysis. _Am. J. Clin. Nutr_. 1997;65:1747\u20131764.\n\n99. Green L.W., Kreuter M.W. _Health promotion planning: an educational and environmental approach_. Mountain View, CA: Mayfield Publishing; 1991.\n\n100. Paradis G., O'Loughlin J., Elliot M., Masson P., Renaud L., Sacks-Silver G., Lampron G. Coeur en sante St-Henri\u2014A heart health promotion programme in a low income, low education neighborhood in Montreal, Canada: theoretical model and early field experience. _J. Epidemiol. Comm. Health_. 1995;49:503\u2013512.\n\n101. Richardson N., Shepherd R., Elliman N. Current attitudes and future influences on meat consumption in the U.K. _Appetite_. 1993;1993:41\u201351.\n\n102. Brewer J., Blake A., Rankin S., Douglass L. Theory of reasoned action predicts milk consumption in women. _J. Am. Diet. Assoc_. 1999;99:33\u201344.\n\n103. Kloeblen A., Batish S. Understanding the intention to permanently follow a high folate diet among a sample of low-income pregnant women according to the health belief model. _Health Educ. Res_. 1999;14:327\u2013338.\n\n104. Schafer R., Keith P., Schafer E. Predicting fat in diets of marital partners using the health belief model. _J. Behav. Med_. 1995;18:419\u2013433.\n\n105. Ling A., Howarth C. Self-efficacy and consumption of fruit and vegetables: Validation of a summated scale. _Am. J. Health Promotion_. 1999;13:290\u2013298.\n\n106. Shannon J., Kirkley B., Ammerman A., Keyserling T., Kelsey K., DeVellis R., Simpson R. Self-efficacy as a predictor of dietary change in a low-socioeconomic-status southern adult population. _Health Educ. Behav_. 1997;24:357\u2013368.\n\n107. Glanz K., Patterson R.E., Kristal A.R., Di Clemente C.C., Heimendinger J., Linnan L., McLerran D.F. Stages of change in adopting healthy diets: Fat, fiber, and correlates of nutrient intake. _Health Educ Q_. 1994;21:499\u2013519.\n\n108. Campbell M., Symons M., Demark-Wahnefried W., Polhamus B., Bernhardt J., McClelland J., Washington C. Stages of change and psychosocial correlates of fruit and vegetable consumption among rural African-American church members. _Am. J. Health Promotion_. 1998;12:185\u2013191.\n\n109. Nitzke S., Auld G., McNulty J., Bock M., Bruhn C., Gabel K., Lauritzen G., Lee Y., Medeiros D., Newman R., Ortiz M., Read M., Schutz H., Sheehan E. Stages of change for reducing fat and increasing fiber among dietitians and adults with a diet-related chronic disease. _J. Am. Diet. Assoc_. 1999;99:728\u2013731.\n\n110. Graves K., Shannon B. Using visual plate waste measurement to assess school lunch behavior. _J. Am. Diet. Assoc_. 1983;83:163\u2013165.\nCHAPTER 10\n\nBiomarkers and Biological Indicators of Change\n\nJOHANNA W. LAMPE1 and CHERYL L. ROCK2\n\n1Fred Hutchinson Cancer Research Center, Seattle, Washington\n\n2University of California at San Diego, La Jolla, California\n\n## I. INTRODUCTION\n\nA biomarker or biological indicator is a characteristic that is measured and evaluated as a marker of normal biological processes, pathogenic processes, or responses to an intervention. In theory, almost any measurement that reflects a change in a biochemical process, structure, or function can be used as a biomarker. In addition, an exogenous compound that, as a result of ingestion, inhalation, or absorption, can be measured in tissues or body fluids can also be considered a biomarker.\n\nBiomarkers can be classified broadly into markers of exposure, effect, and susceptibility and have numerous applications in nutrition. They can be used to assess dietary intakes (exposure); biochemical or physiologic responses to a dietary behavior or nutrition intervention (effect); and predisposition to a particular disease or response to treatment (genetic susceptibility).\n\nAlthough certain biologic markers, such as serum cholesterol and glucose, have been used by clinicians for generations, use of biomarkers has taken on new importance with the dramatic advances in various fields of biology and desire for objective measures in large-scale, population-based descriptive and intervention nutrition research. Exquisitely sensitive laboratory techniques can detect subtle alterations in molecular processes that reflect events known or believed to occur along the continuum between health and disease.\n\nThis chapter presents the basic concepts and key issues related to the various uses of biomarkers in nutrition; it is not intended to be a comprehensive review. Identification and use of biomarkers is continuously evolving with the growing understanding of biologic processes and the improved sensitivity of laboratory assays. Consequently, our examples of existing biomarkers are mere snapshots in the greater scheme of biomarker development and application.\n\n## II. BIOMARKERS OF DIETARY INTAKE OR EXPOSURE\n\nBiomarkers are used to monitor dietary exposure and for nutritional assessment for several reasons. One reason is to provide biochemical data on nutritional status by generating objective evidence that enables evaluation of dietary adequacy or ranking of individuals on exposure to particular nutrients or dietary constituents. Biochemical or biological measurements may also be collected to provide objective evidence of a dietary pattern, such as overall fruit and vegetable consumption, or to validate dietary assessment instruments or self-reported dietary data. Another possible purpose for obtaining these biological measures is to establish the biological link between the nutritional factor and a physiological or biochemical process, when the concentration of the micronutrient or dietary constituent is measured in a peripheral tissue.\n\n### A. Biomarkers of Nutrient Intake\n\nBiochemical measures of nutrients can be a valuable component of nutritional assessment and monitoring. Overall, the usefulness of biochemical indicators of nutritional status or exposure is based on knowledge of the physiologic and other determinants of the measure. For several micronutrients, the concentration of the nutrient in the circulating body pool (i.e., serum) appears to be a reasonably accurate reflection of overall status for the nutrient (Table 1). In contrast, the amount of some micronutrients in the circulating pool may be homeostatically regulated when the storage pool is adequate, or may be unrelated to intake, and thus has little relationship with total body reserves or overall status. Figure 1 illustrates the relation between various compartments or body pools that may be sampled in the measurement of biological indicators.\n\nTABLE 1\n\nBiomarkers of Nutrient Intake _a_\n\n_Key:_ FAD, flavin adenine dinucleotide; EGRAC, FAD-dependent erythrocyte glutatione reductase activation coefficient; NAD, nicotinamide adenine dinucleotide; LDL, low-density lipoprotein; GSH, glutathione; SOD, superoxide dismutase; TSH, thyroid-stimulating hormone; T3, triiodothyronine; T4, thyroxine.\n\naDirect measures of dietary exposure and nutrient-specific functional markers. This table includes both established markers and additional markers that show promise.\n\nbIn approximate order of increasing severity of iron shortage [104].\n\nFIGURE 1 The relation between body compartments and biological specimens that can be assayed for dietary indicators.\n\nKnowledge of the influencing nondietary factors is particularly important for accurate interpretation of the nutrient concentration in tissues. For example, tocopherols and carotenoids are transported in the circulation nonspecifically by the cholesterol-rich lipoproteins [1, 2], so higher concentrations of these lipoproteins are predictive of higher concentrations of the associated micronutrients in the circulation, independent of dietary intake or total body pool. Smoking and alcohol consumption need to be considered in the interpretation of serum and other tissue concentrations of several micronutrients, particularly compounds that may be subject to oxidation (e.g., vitamin C, tocopherols, carotenoids, folate). Knowledge of the relationship between the indicator and the risk of nutrient depletion, in addition to the responsiveness of the indicator to interventions or change, is also necessary [3]. For some nutrients, such as calcium and zinc, a specific sensitive exposure marker of diet simply has not yet been identified.\n\nTable 1 lists examples of biochemical measures of nutrients that may serve as useful biomarkers in nutritional assessment or monitoring of dietary intake. For more details, the reader is referred to in-depth reviews addressing the use of biomarkers for assessing nutrient exposure [4\u20137]. Unfortunately, a static measurement (i.e., a tissue concentration) is typically not as sensitive as a functional marker in the assessment of status; however, a good functional measure is still lacking in many instances.\n\n### B. Biomarkers of Other Dietary Exposures\n\nNumerous dietary constituents, particularly of plant origin, although not recognized as essential for life, have demonstrated biologic activity and are thought to play an important role in prevention of chronic disease [8, 9]. These phytochemicals are absorbed to various degrees, often metabolized in the intestinal epithelium and liver, and excreted; thus, the metabolites can be monitored in serum or plasma and\/or urine.\n\nSome classes of compounds such as flavonoids are found in many plant foods, where as others such as isoflavones are limited to select sources (Table 2). The isoflavones, daidzein and genistein, are highly concentrated in soybeans and soy products [10, 11]. Urinary isoflavone excretion is associated strongly and directly with soy protein intake under controlled dietary conditions [12]. In observational studies of populations that usually consume soy, soyfood intake and urinary isoflavonoid excretion also are positively correlated [13\u201315]. Because the plasma half-lives of the isoflavones genistein and daidzein are short (6\u20138 hours) [16], intermittent soy consumption may be under- or overestimated if isoflavone exposure is monitored in plasma or spot urine specimens. Metabolism of isoflavones is also inextricably linked to the health of colonic bacterial populations, and therefore plasma and urinary levels may be influenced by the effects of diet and drugs on the colonic environment.\n\nTABLE 2\n\nPhytochemical Content of Plant Food Families and Select Plant Foods _a_\n\naSome phytochemicals are present in most plant foods; others are restricted to particular botanical families or even particular plant species.\n\nbCabbage family\n\nccitrus\n\ndonion family\n\netomato family\n\nfcarrot family\n\ngsquash family.\n\nhU, urine; P, plasma or serum; S, stool; B, breath.\n\n_Source:_ Adapted from Caragay, A. B. (1992). Cancer-preventive foods and ingredients. _Food Technol._ **46,** 65\u201368; and Fahey, J. W., Clevidence, B. A., and Russell, R. M. (1999). Methods for assessing the biological effects of specific plant components. _Nutr. Rev._ **57,** S34\u2013S40.\n\nDietary exposure to flavonoids and other polyphenols can be monitored by measuring parent compounds and metabolites in urine or plasma [17, 18]. Several compounds in cruciferous vegetables, such as sulforaphane and other isothiocyanates, have been of interest due to their potential chemopreventive effects. Although, with present technologies, levels of these compounds are typically too low to measure directly in plasma, dithiocarbamates (metabolites of isothiocyanates) can be quantified in urine, following extraction and measurement by high-performance liquid chromatography; these provide a measure of cruciferous vegetable exposure [19].\n\nBiomarkers also exist for monitoring exposure to less desirable food constituents, such as mycotoxins (e.g., aflatoxin) in mold-contaminated grain products and pyrolysis products that result from cooking meat at high temperatures (e.g., heterocyclic amines). Because of the nature of these compounds, exposure to potentially carcinogenic compounds can be determined by measuring the presence of adducts\u2014the result of covalent binding of the chemicals to proteins or to nucleic acids in DNA. The rationale for using measurements of carcinogen-DNA adducts is based on the assumption that DNA adducts formed _in vivo_ are responsible for genetic alterations in genes critical for carcinogenesis and that protein adducts formed through the same processes reflect the formation of DNA adducts [20]. Because adducts represent an integration of exposure and interindividual variability in carcinogen metabolism and DNA repair, they may provide a more relevant measure of exposure, i.e., a biologically effective dose [20]. Some adducts are specific for dietary exposure; aflatoxin-albumin adducts result from ingestion of aflatoxin. Other adducts, such as benzo[ _a_ ]pyrene-DNA adducts, are nonspecific because benzo[ _a_ ]pyrene comes from a variety of sources besides diet, including air pollution, tobacco, and occupational exposures. Adducts can be used to monitor exposure within individuals. They can also serve as early markers of the efficacy of interventions designed to prevent exposure to genotoxic agents or to modify the metabolism of procarcinogens once exposure has occurred. An example of this latter use is an intervention to reduce aflatoxin-albumin adducts using the drug oltipraz [21].\n\n### C. Biomarkers of Energy Intake\n\nTo date, few biological measures are available that objectively monitor energy intake, and those that are available are cumbersome in free-living populations and\/or expensive. Under steady-state conditions, indirect calorimetry provides an estimate of energy expenditure and some insight about intake. Indirect calorimetry estimates the rate of oxidation or energy expenditure from the rate of oxygen consumption (VO2) and the rate of carbon dioxide production (VCO2). This technique is relatively inexpensive and portable, although some participant cooperation is required. These traits make the technique attractive primarily for clinical applications [22].\n\nEnergy expenditure can also be measured using a doubly labeled water technique 23], as discussed in detail in another chapter (see [Chapter 2). This method uses nonradioactive isotopes of hydrogen (2H) and oxygen (18O) to measure free-living total energy expenditure by monitoring urinary isotope excretion. Energy expenditures determined by room calorimetry, indirect calorimetry, and doubly labeled water measures are not significantly different within the calorimeter environment; however, in free-living subjects, doubly labeled water-derived energy expenditures are found to be 13\u201315% higher than those for other methods [24]. The doubly labeled water method has the distinct advantage of allowing the subjects to go about their usual activities with energy expenditure calculated after a study period of 7\u201314 days. Unfortunately, the 18O isotope required to conduct doubly labeled water studies is expensive and is often in short supply. Although doubly labeled water methodology is suited to nutrition research aimed at quantifying total energy expenditure for specific groups, the cost for large samples limits broad use.\n\n### D. Biomarkers as General Dietary Indicators\n\nMonitoring overall dietary patterns or changes in patterns in response to dietary interventions presents additional challenges. The goal in this case is to assess and monitor the intake of certain types of foods or food groups, rather than specific nutrients; therefore, these dietary indicators ideally should be distributed generally within certain types of foods.\n\nPlasma carotenoids provide a good example of the use of biomarkers as a dietary indicator when the goal is to assess and monitor dietary patterns. Vegetables and fruits contribute the vast majority of carotenoids in the diet, and plasma carotenoid concentrations have been shown to be useful biomarkers of vegetable and fruit intakes in cross-sectional descriptive studies, controlled feeding studies, and clinical trials [25\u201328]. The consistency of this relationship across diverse groups and involving various concurrent diet manipulations (with differences in amounts of dietary factors that could alter carotenoid bioavailability) is notable, although considerable interindividual variation in the degree of response is typically observed. Also, nondietary factors that are among the determinants of plasma carotenoid concentrations (e.g., body mass, plasma cholesterol concentration) will influence the absolute concentration that is observed in response to dietary intake, so these characteristics must be used as adjustment factors.\n\nAlthough vitamin C also is provided predominantly by fruits and vegetables in the diet, this measure is much less useful as a biomarker of this dietary pattern because the relationship between vitamin C intake and plasma concentration is linear only up to a certain threshold [29]. The use of vitamin C supplements (which is common in the U. S. population) often increases the intake level beyond the range in which linearity between intake and plasma concentration occurs, and thus obscures the relationship between food choices and tissue concentrations.\n\nLignans are a group of compounds present in high-fiber foods, particularly cereals and fruits [30]. These compounds are not found in animal products and, similar to carotenoids, may be useful markers of a plant-based diet [31]. Lignans provide an example of how using dietary constituents as biomarkers requires an understanding of the metabolism of the compounds. Lignans in plant foods are altered by intestinal microflora, so that the specific compounds, enterodiol and enterolactone, monitored in plasma or urine are actually bacterial metabolites. Because of this bacterial conversion, lignan concentrations in urine or plasma in response to a similar dietary dose will vary significantly among individuals. In addition, nondietary factors (e.g., use of oral antibiotics) will reduce enterolactone and enterodiol production [32].\n\nAs another example, the fatty acid composition of membrane phospholipids is in part determined by the omega-6 and omega-3 fatty acid composition of the diet. Thus, the fatty acid pattern of serum phospholipids or plasma aliquots has been used as a biomarker of compliance with omega-3 fatty acid supplementation in clinical trials [33, 34]. Although enzyme selectivities and other physiologic factors are also important determinants of the fatty acid composition of phospholipids, a diet high in omega-3 polyunsaturated fats will result in increased amounts of eicosapentaeneoic and docosahexaenoic acids in circulating tissue pools.\n\nSpecific fatty acids also can be associated with certain types of foods. Pentadecanoic acid (15:0) and heptadecanoic acid (17:0) are fatty acids produced by bacteria in the rumen of ruminant animals. These fatty acids, with uneven numbers of carbon atoms, are not synthesized by humans; therefore, their presence in human biologic samples can be indicative of dietary exposure to milk fat. Proportions of 15:0 and 17:0 in adipose tissue and concentrations of 15:0 in serum have been found to correlate with milk fat intake in men and women [35, 36].\n\n## III. FUNCTIONAL BIOMARKERS\n\nIf a nutrient or dietary constituent has an identified impact on physiologic, biochemical, or genetic factors, measuring markers of those functional effects can be extremely useful. Such functional indices can be classified as those which are measures of _discrete_ functions of a nutritional factor and those which are measures of more _general_ functions [37]. A discrete functional index often relates to the first limiting biochemical system, for example, a particular enzymatic pathway [38]. These markers can be used to identify the dosage or concentration of a nutritional factor necessary to achieve a clinically meaningful response or to define optimum nutrient status (Table 1). Unfortunately, for many nutritional factors, the first limiting biochemical system is unknown or not readily measured or accessible in humans. A general functional index is less specific, but may be more directly linked to the pathogenesis of disease or ill health. Often a panel of markers, rather than one specific measure, provides a better picture. Examples of general functional indices or markers are oxidative stress, immune function, bone health, and cell turnover\u2014processes that have been shown to play roles in the risk of various diseases.\n\nFor a functional index to be an effective nutritional biomarker, a cause-and-effect relationship must be established between (1) nutritional status and the functional index,(2) between the functional index and ill health, and (3) between nutrient status and ill health. Such an undertaking is a daunting and time-consuming task, but is especially important if a functional biomarker is going to be used as a proxy, or surrogate, for a clinical endpoint or disease outcome. A clinical endpoint is a characteristic or variable that measures how a patient feels, functions, or survives. A surrogate endpoint biomarker is an index whose modulation has been shown to be indicative of progression or reversal of the disease process; it is a biomarker that is intended to substitute for a clinical endpoint. In an intervention trial, the use of surrogate endpoint biomarkers (rather than the frank diagnosis of disease) requires substantially less time and fewer resources in the evaluation of efforts aimed toward reducing risk for chronic diseases such as cancer, cardiovascular disease, and osteoporosis [39].\n\nTo date, very few markers have been established as true surrogate endpoint biomarkers; i.e., they cannot accurately substitute for a clinical endpoint [40]. The evidence supporting the linkage of a biomarker to a clinical endpoint may be derived from epidemiologic studies, clinical trials, _in vitro_ analyses, animal models, and simulated biologic systems. Many biomarkers have been proposed as potential surrogate endpoints, but relatively few are likely to achieve this status because of the complexity of disease mechanisms and the limited capability of a single biomarker to reflect the collective impact of multiple therapeutic effects on ultimate outcome.\n\n### A. Biomarkers of Enzyme Function\n\nUnderstanding how diet influences enzyme systems is important in developing strategies for disease prevention and treatment. For example, dietary modulation of enzymes involved in carcinogen metabolism may be important in reducing cancer risk, and dietary intervention that reduces expression of rate-limiting enzymes in cholesterol synthesis may alter cardiovascular disease risk. Enzymes that require micronutrients as cofactors are also used as biomarkers of nutritional status (Table 1).\n\nComponents of diet have the capacity to modulate protein synthesis and function. An ideal discrete functional marker would be one that reflects the direct effect of a dietary constituent\u2014for example, mRNA amount when the dietary factor regulates gene expression or level of enzyme activity when the factor acts as a competitive inhibitor of the enzyme (Fig. 2). Unfortunately, at present, monitoring at these levels in the pathway in an intact human is not always feasible. Often, we rely primarily on a downstream marker, whose measurement may be influenced by subsequent or parallel pathways and may give a diluted signal.\n\nFIGURE 2 Direct functional markers of dietary exposure.\n\nOften the enzymes of interest are located primarily in tissues that are not readily accessible (e.g., liver, intestine, lung). One approach to meeting this challenge is to measure the enzymes in more accessible tissue; for example, enzymes that are present in high levels in the liver can often be measured in plasma or serum as a result of normal hepatocyte turnover. Enzyme activity of glutathione _S_ -transferase (GST), a biotransformation enzyme important in carcinogen detoxification, can be measured spectrophotometrically in serum [41] or concentrations of the enzyme itself can be determined in serum by immunoassay [42]. Serum concentration of the GST isoenzyme GST-\u03b1 has been shown to increase when cruciferous vegetables are added to the diet [42]. A limitation of using serum measures of a hepatic enzyme is that the assumption is made that liver function is normal. Thus, including other measures of liver function in the data collection is important to verify that no underlying hepatic disease is resulting in spurious GST values. Additionally, some enzymes are present in isoforms in various tissues. GST-\u03bc, another GST isoenzyme, is present in lymphocytes as well as in liver; therefore, for this isoenzyme, GST activity or protein concentration can be measured in cells extracted from blood samples.\n\nAnother approach to monitor enzyme activity _in vivo_ is to use a drug probe. Many of the same xenobiotic metabolizing enzymes that metabolize carcinogens also metabolize and are modulated by commonly used drugs. The metabolites of these drugs can be monitored in serum, plasma, or urine and used to determine enzyme activities. For example, caffeine metabolites measured in urine samples collected 4 hours after consumption of 500 mg caffeine allows determination of cytochrome P450 1A2, _N_ -acetyltransferase, and xanthine oxidase activities [43], and urinary concentrations of the glucuronide and sulfate conjugates of acetaminophen (paracetamol) are used to measure UDP-glucuronosyltransferase and sulfotransferase activities [44]. Drugs can be administered as probes during a nutrition intervention to determine the degree of change in enzyme activity in response to diet or in population-based studies to examine gene\u2013environment interactions [45\u201347].\n\nMeasurement of arachidonic acid metabolism, which involves measuring the concentration of prostaglandins or leukotrienes (metabolic products) or enzymes in the eicosanoid metabolic pathway (i.e., cyclooxygenase), provides another example. Altered arachidonic acid metabolism is among the biochemical activities of nonsteroidal anti-inflammatory agents and may also be influenced by antioxidant micronutrients, such as vitamin E [48], and quantitative changes in these products or enzymes in tissues serve as biomarkers of this activity [49]. A reasonable amount of biological evidence suggests some role for this enzymatic pathway in colon carcinogenesis [50], but the overall relationship with disease process is still under investigation.\n\nSimilarly, endogenous compounds can serve as probes to monitor enzyme activity. Serum concentrations of the amino acid homocysteine, compared to serum and red blood cell folate concentration, are a more sensitive systemic measure of cellular folate depletion [51, 52]. Serum concentrations of homocysteine increase with folate inadequacy because the remethylation of homocysteine requires _N_ -5-methyltetrahydrofolate as a cosubstrate [53], which therefore provides a functional marker for folate status. Nonetheless, because homocysteine is at the intersection of two metabolic pathways, remethylation and transsulfuration, deficiencies in other nutrients in these pathways, namely, vitamin B12, vitamin B6, and possibly riboflavin, can contribute to elevated serum homocysteine concentrations [51].\n\n### B. Biomarkers of Oxidative Stress\n\nOxidative stress has been suggested to play a role in the pathophysiologic disease process in cancer, atherosclerotic cardiovascular disease, and many other acute and chronic conditions [54], although the specific relationship with the disease process remains to be established in most instances. Cellular damage caused by reactive oxygen species, which are generated from cellular respiration, co-oxidation during metabolism, and the activity of phagocytic cells of the immune system, is controlled by antioxidant defense mechanisms that involve several micronutrients. Oxidative stress describes the condition of oxidative damage resulting when the balance between free-radical generation and antioxidant defenses is unfavorable. Direct measurement of active oxygen and related species in biological samples is very challenging, mainly because these compounds have very short half-lives. Thus, the oxidative stress biomarkers used in human studies are typically adducts or endproducts that reflect reactions that have occurred between free radicals and compounds such as lipids, proteins, carbohydrates, DNA, and other molecules that are potential targets [55].\n\nOne frequently described assay used as an oxidative stress biomarker is the thiobarbituric acid reactive substances (TBARS) assay. The TBARS assay basically quantifies a product of malondialdehyde, which presumably reflects lipid hydroperoxides in the sample. However, this assay has some serious limitations in specificity, and the product measured cannot be interpreted as directly reflecting lipid peroxidation _in vivo_ [55]. Direct measurement of malondialdehyde in biological samples using high-performance liquid chromatography (HPLC) has also been examined as an alterative approach, although the specificity of this more direct HPLC measurement has also not been established to the level desirable.\n\nMeasurement of breath pentane is another biomarker of oxidative stress that has been utilized in human studies [56]. The approach basically involves collecting exhaled air for the measurement of the products of peroxidation of unsaturated fatty acids, a portion of which are volatile and released in the breath, using gas chromatography methods. However, the specific measurement methodologies vary a great deal and are not always reliable, and standardization of the procedure and knowledge of various influencing factors are needed to improve the usefulness of this approach [55].\n\nAnother biomarker of oxidative stress is the measurement of urinary 8-hydroxydeoxyguanosine (8OHdG) using HPLC and electrochemical detection [57]. The 8-hydroxylation of the guanine base is a frequent type of oxidative DNA damage, and 8OHdG is subsequently excreted without further metabolism in the urine after repair _in vivo_ by exonucleases. In previous studies, certain demographic factors and physiologic characteristics such as gender and body mass [58] have been observed to influence urinary 8OHdG concentration, so these factors may need to be considered in interpretation. Urinary 8OHdG is increased in association with conditions known to be characterized by increased oxidative stress, such as smoking, whole-body irradiation, and cytotoxic chemotherapy [57\u201359]. Urinary 8OHdG (unadjusted or adjusted for urinary creatinine) has also been observed to decline in response to a high-vegetable and fruit diet intervention in human subjects [60], which is particularly interesting because this type of diet has been suggested to promote reduced oxidative stress [8, 60, 61].\n\nProstaglandin-like compounds produced by nonenzymatic free-radical-catalyzed peroxidation of arachidonic acid, termed F2 isoprostanes, are currently of great interest as useful biomarkers of oxidative damage [62]. Specific gas chromatography\/mass spectrometry assays for the measurement of some of these compounds, such as iPF2a-III (also called 8-iso-PGPF2) and iPF2-VI, have been developed and used to quantify the compounds in urine and blood samples. These markers have been shown to be less variable than 8OHdG [63], and elevated levels have been observed in plasma and urine samples from subjects under a wide variety of conditions of enhanced oxidative stress [64, 65].\n\nAnother approach to measuring DNA oxidative damage that appears to be useful in human nutrition research is the measurement of 5-hydroxymethyluracil levels in DNA in blood. 5-Hydroxymethyluracil is produced when DNA is exposed to oxidants, is relatively stable when compared to other oxidation products, and can be quantified with gas chromatography\/mass spectrometry [66, 67]. In a small cross-sectional study, 5-hydroxymethyluracil concentration was observed to be inversely associated with cooked vegetables and directly related to beef and pork intakes in the diets of women enrolled in a low-fat diet intervention trial [68].\n\nOxidative damage to low-density lipoprotein (LDL) has been specifically linked to atherogenesis, and in an application of this biological activity, measurement of LDL oxidation _ex vivo_ has been used in clinical studies as a biomarker of oxidative stress [69]. Basically, this process involves isolating the LDL fraction from a blood sample, exposing this fraction to oxidants such as Cu2+, and measuring the lag time before oxidation. Although this biomarker might appear to be specific to cardiovascular disease risk, results from this assay have not yet been specifically linked with risk for disease, so results should be interpreted as simply another approach to the assessment of oxidative stress [55]. Also, various specific methodologies are used across laboratories, and the lack of standardization in the approaches in use constrains the ability to make comparisons across studies.\n\nSeveral other approaches to measuring biomarkers of oxidative stress have been proposed and are under study, and the reader is referred to a review of this topic [55]. Because of their inherent variability, and uncertain responsiveness to dietary manipulations, clinical researchers often employ a panel of potential measures of oxidative stress, rather than relying on a single indicator [60].\n\n### C. Biomarkers of Immune Function\n\nThe human immune system is a complex and highly interactive network of cells and their products that has a central role in protecting against various external disease-promoting factors and perhaps against malignant cells. Many of the components of the immune system can serve as biomarkers and are monitored _in vivo_ or _ex vivo_ [70, 71]. Because of the complexity of the system, the selection of assays should be closely aligned with the research question being asked. Furthermore, multiple parameters need to be measured; one single biomarker is inadequate to monitor immune function.\n\nCell-mediated immune variables include the absolute amounts or ratios of various white blood cell (WBC) types (e.g., total counts, WBC differentials, T-cell subsets) and measures of T-cell function (e.g., lymphocyte proliferation, cytokine release from mitogen-stimulated cultures, cytotoxic capacities, delayed-type hypersensitivity). Both number and activity of natural killer cells (one of the cell types that plays an important role in immune surveillance) are used as biomarkers in nutrition intervention trials [72, 73]. Cytokines (e.g., the interleukins) are soluble factors released by immune cells, which control and direct the function of other immune effectors. Some of these have been used as markers of immune response in randomized trials of vitamin supplementation [73, 74].\n\nAn _in vivo_ functional test, the delayed-type hypersensitivity (DTH) skin test is widely used to monitor the immune system in humans, including in studies of dietary modulation of immune function [75]. It measures the capacity of an individual's immune system to mount a response to antigenic stimulation. The DTH test typically involves the simultaneous intradermal application of one or several DTH antigens. These antigens elicit an immunologic reaction involving the release of lymphokines by antigen-sensitized T cells. These compounds, in turn, activate macrophages, which release inflammatory mediators, resulting in measurable skin induration.\n\n### D. Biomarkers of Bone Health\n\nBone mass measurements and biomarkers of bone turnover are used as functional indices of bone health and, to a certain extent, can also be used as markers of the adequacy of calcium intake. Measures of bone mass include bone mineral content (BMC; i.e, the amount of mineral at a particular skeletal site, such as the femoral neck, lumbar spine, or total body) and bone mineral density (BMD; i.e., bone mineral content divided by the area of the scanned region); both are strong predictors of fracture risk 76\u201378]. Controlled calcium intervention trials that measure change in BMD provide evidence for the intake requirement for calcium [79] (see [Chapter 42 for more details).\n\nBiochemical markers of bone turnover predict bone mass changes and fracture risk, and respond to dietary calcium intake [79]; thus, they provide some promise for a biochemical indicator of calcium status. Unlike BMD, they reflect more subtle changes in bone metabolism. Bone turnover is the cyclical process by which the skeleton undergoes renewal by a coupled, but time-separated, sequence of bone resorption and bone formation [80]. Markers of bone turnover rely on the measurement in serum or urine of enzymes or matrix proteins synthesized by osteoblasts or osteoclasts that spill over into body fluids, or of osteoclast-generated degradation products of the bone matrix [79]. Currently, serum levels of skeletal alkaline phosphatase and osteocalcin are used as markers of bone formation, and products of collagen degradation measured in urine are used to measure bone resorption. These markers exhibit substantial shortterm and long-term fluctuations related to time of day, phase of menstrual cycle, and season of the year, as well as other factors that alter bone remodeling (e.g., exercise) [81].\n\n### E. Biomarkers of Cell Turnover\n\nCellular markers of proliferation, differentiation, and apoptosis (i.e., programmed cell death) can be useful as biomarkers in research focused on nutritional factors and cancer, although the measured effect is a general indicator of an altered cell growth regulation effect. Use of such markers is severely restricted by the difficulty accessing the tissue of interest. Consequently, research in this area has been limited primarily to tissue available via endoscopic or fine-needle biopsy procedures, e.g., gastrointestinal tract, breast, prostate.\n\nAs a general rule, increased proliferation of undifferentiated cells defines one aspect or characteristic of carcinogenesis, and in colon cancer, this relationship has been well established. For example, cell proliferation occurs at the base of the colonic crypts, and as cells migrate from the crypts to the luminal surface, they become increasingly differentiated and mature and lose their proliferative capabilities [82]. The shift in which the proliferative zone extends to the surface, so that cells on the luminal surface retain proliferative capabilities and are immature and underdifferentiated, may be considered a field defect that sets the stage for current and future neoplastic changes [82\u201384]. Early work in this area relied on the incorporation of tritiated-thymidine or bromo-deoxyuridine into the DNA of dividing cells during incubation of a biopsy specimen. These methods required that the tissue be freshly obtained, so the cells were viable and replicating. Often, label incorporation was incomplete. Now, with increased sensitivity in immunohistochemical techniques, proteins present in proliferating cells (e.g., proliferating cell nuclear antigen [PCNA] and Ki67) are used more widely to quantify proliferative activity in tissue specimens. Labeling indices involving tritiated thymidine and PCNA have been used to quantify the proliferative activity in colonic mucosal samples from human subjects [85] and have been used successfully as endpoints in several nutrition intervention studies to prevent colon cancer [86]. These indices are being further refined by staining for proteins present during apoptosis (e.g., Bax, Bcl\u20132) and in differentiated cells, in order to provide a more complete picture of cell dynamics.\n\nAdoption of aberrant crypt foci (early morphologic changes in colonic epithelium that are considered potential precursors of adenomatous polyps) as biomarkers in humans is an example of how improvements in technology have led to the adoption of a biomarker that until recently could only be used in animal studies. Development of magnifying en-doscopes with improved resolution now allows investigators to monitor aberrant crypt foci in colon tissue samples from healthy humans [87].\n\n## IV. BIOMARKERS OF GENETIC SUSCEPTIBILITY\n\nThe health of individuals and the population in general is the result of interaction between genetic and environmental factors. For the great majority of human diseases, purely genetic or purely environmental etiologies are insufficient to explain individual variability in occurrence, prognosis, or outcome [88]. This is especially the case with chronic diseases, such as heart disease and cancer. Genetically determined susceptibility factors alter disease frequency or treatment response through variations in DNA coding sequences of genes. As a result, genetically susceptible individuals produce proteins that are structurally different, or produce them in greater or lesser amounts than individuals who are not at increased risk of disease.\n\nBy genetic standards, traits with a frequency of between 1\/100 and 1\/10,000 in a population are considered uncommon, and rare traits are those with a frequency of less than 1\/10,000 [89]. Typically, these are low-prevalence and high-penetrance genes (e.g., genes associated with familial cancers). Common genetic traits are those in which the least common allele is present in at least 1% of the population [89]. Traits with this characteristic are known as genetic polymorphisms. They include the high-prevalence, low-penetrance genes\u2014\"susceptibility genes\"\u2014thought to contribute to disease risk.\n\nIn cases where specific genetic mutations or variations may be indicative of disease risk or progression or may be modified by nutritional factors, genetic markers can also be useful biomarkers. Various molecular techniques have been developed to help characterize genetic abnormalities or differences. Genetic factors are important to consider in nutrition research for several reasons. One reason is that it is increasingly evident that genetic polymorphisms may contribute substantially to differences in the response to environmental and dietary exposures 90]. For example, genetic variations in the expression of the xenobiotic metabolizing enzymes may mediate the potentially carcinogenic effect of heterocyclic amines (obtained from meat cooked at high temperature) [91] (see also [Chapter 13). Also, results from laboratory animal studies suggest that dietary modifications can promote alterations in genetic factors [92], so that measuring genetic abnormalities may be considered an approach to demonstrating a biological link between dietary factors and disease risk.\n\nSome polymorphic traits may only be important in the presence of a particular dietary exposure. For example, carrying the 5, 10-methylene-tetrahydrofolate reductase (MTHFR) thermal-labile variant has been shown to be a risk factor for colorectal adenomatous polyps, but only in the context of low-folate, vitamin B12 and vitamin B6 intake [93].\n\nGiven that a goal of the field of nutrition is the prevention and treatment of disease, genetic markers may aid in this effort by identifying population subgroups at high risk of disease in the presence of particular dietary exposures. Genetic susceptibility markers may also strengthen our understanding of disease by focusing attention on possible pathways of disease causation and progression. There is considerable heterogeneity in disease risk within populations; thus, markers of susceptibility may also help to clarify associations between dietary exposures and diseases within population subgroups [94].\n\n## V. CRITERIA FOR SELECTING AND USING BIOMARKERS\n\nWhen a candidate biomarker is identified, certain basic considerations need to be addressed before it can be adopted for use in research or in a clinical setting [95]. These considerations relate to the reliability of the laboratory assay itself, the biologic relevance of the marker, and the characteristics of the marker within a population. Whether or not a particular established marker is used also depends on the purpose the marker will serve.\n\nDevelopment of a biomarker usually builds on scientific knowledge from various types of laboratory studies, including tissue culture and animal studies. In the laboratory, an initial priority is to determine a marker's reliability or reproducibility. Assay performance can be evaluated using coefficients of variation [CV%; (SD\/mean) x 100] to estimate within- and between-batch precision; these are measures of analytic, laboratory performance and do not reflect intra- or interindividual variation. The within-batch precision is determined by dividing single samples into multiple aliquots and analyzing them together. Between-batch precision is determined by analyzing multiple aliquots on separate days. It is difficult to generalize about acceptable numerical values for the laboratory CV% because the degree of error acceptable depends on the use of the biomarker data. For epidemiological studies, if the goal is to establish a stable estimate of the group mean, an acceptable CV% may depend on the number of samples available, the mean concentration of the biomarker, and between-individual variation [96]. If a biomarker is being used in a clinical setting to diagnose and monitor treatment in an individual, day-to-day quality performance of an assay is imperative. Techniques of quality control [97, 98] and statistical methodologies for managing quality control data [99] have been established and are used in clinical laboratories.\n\nBiomarkers should be relatively easy to measure and require relatively noninvasive techniques of tissue sampling. This requires that the biologic relevance or validity\u2014i.e., the relationship between biomarkers in tissues readily available for human monitoring (e.g., peripheral blood) to those in target tissue (e.g., lung, liver) and the relationship between the biomarker and the disease or exposure being studied\u2014of the measure be established. One example is the use of serum ferritin as a marker of body iron stores. Serum ferritin was validated as a measure of iron stores against bone-marrow examination for stainable iron, the criterion method for\u2014but very invasive way\u2014of measuring iron stores [100]. As a result, serum ferritin has been adopted as a simple, quantitative biomarker of iron stores in otherwise healthy individuals.\n\nIf a particular biomarker is to be used as a measure of dietary exposure, it must be evaluated with respect to its sensitivity to that intake. Several approaches, both observational and interventional, can be used to define the relationship between long-term dietary intake and biologic levels. Investigators can rely on geographic differences in exposure: Tissue samples from areas of known nutrient deficiency of a specific nutrient can be compared with samples from average and high-exposure areas. This approach has the advantage that it can reflect the long-term intake of a settled group of individuals; however, identifying and controlling confounding factors is a major challenge [96]. Another observational approach is to establish within individuals the relation between a dietary exposure and the biochemical marker. Participants for such a study can be selected randomly or can be selected specifically in order to maximize the range of intakes. For example, in one study designed to test the use of plasma carotenoids as markers of vegetable and fruit intake, study participants were selected on the basis of their reported vegetable and fruit intake\u2014only those who had intakes <2 or \u22655 of servings per day were recruited into the study [26]. Rigorous testing of the relationship between intake of the dietary factor and a biomarker under controlled dietary conditions is also valuable to establish dose\u2013response relations; however, these trials are usually limited to weeks or months and, if they involve extensive changes to usual diet, blinding of participants may not be feasible.\n\nDepending on the biomarker, significant variability can be seen in a biomarker. Sources of variation can be internal (e.g., age, sex, genetics, body build, biologic rhythm) or environmental (e.g., diet, season, time of day, immobilization, exercise, drugs). These can contribute to both within- and interindividual variation. Additional external sources of variation, beyond laboratory accuracy and precision, can include an individual's posture during sample collection and sample handling and storage; protocols should be established to minimize these latter sources of variability.\n\nSelection of a biomarker is dependent in part on its use. A biologic indicator that is going to be used as a measure of a dietary exposure in an epidemiological study needs to be a valid representation of long-term intake [96]. Repeated sampling and measurement of a biomarker over time can provide some estimate of the within-individual variability and, therefore, the likelihood that the biomarker is a stable estimate of long-term intake. If repeated measures of a biochemical indicator vary substantially over time in the same individuals, then a single measure will not reflect true, long-term intake [96]. This lack of consistency may occur because diet has changed over the sampling interval or because the measure is overly sensitive to short-term influences, such as recent intake. When using dietary constituents or their metabolites as biomarkers, an understanding of the metabolism and pharmacokinetics of the compound and the frequency of exposure will help to establish the utility of the measure as a biomarker of long-term intake.\n\nA nutritional intervention study may require a biomarker that is a short-term measure of response to treatment. A biomarker that is to serve as a short-term measure of response needs to change within the time frame of the intervention. For example, serum folate provides a measure of recent folic acid exposure; however, erythrocyte concentrations are dependent on the life span of the cells and therefore will not reflect short-term changes in dietary folate. Serum folate concentrations decline within 3 weeks after initiation of a low-folate diet, whereas erythrocyte folate concentration remains in the normal range for at least 17 weeks [101].\n\nAdditional practical considerations in the use of established biomarkers include the ability to conveniently access the body compartment for measurement, the procedures necessary to collect and process the sample, burden to study participants or patients, and the resources for laboratory analysis. For example, multiday collections of feces or urine can be a major burden for many individuals. In addition, they can result in incomplete collections, which also compromise the final results. An accurate quantification of vitamin C or folate in a circulating body pool requires processing steps that must be conducted immediately after blood collection to preserve the sample appropriately and prevent degradation that would otherwise make the resulting measurement inaccurate. These extra steps can add time and effort to the labor of blood processing. Furthermore, the complexity of an assay method can vary from the ability to analyze hundreds of samples a day at a cost of a few dollars per sample to a labor-intensive, week-long process that cost hundreds of dollars per sample.\n\nThe ability to measure particular biomarkers is also often linked to technological challenges and existing capabilities. For example, the development of HPLC in the 1970s, and improved separation and detection technologies that are currently emerging, facilitates the quantification of many micronutrients and other dietary constituents that are present in very low concentrations in biological samples. Similarly, immunoassays allow for quantitation of phytochemicals, proteins, etc. in small volumes of serum or plasma, where previous methods required substantial quantities of sample. The recent development of microarray technology has provided the ability to analyze the expression profiles for thousands of genes in parallel [102, 103]. This technique will rapidly advance knowledge regarding the mechanisms by which nutrition and diet affect disease risk; however, its application in intact humans will still be limited by access to the tissue of interest.\n\n## VI. SUMMARY\n\nThe use of biomarkers in humans is an integral component of nutrition research, as well as of nutritional care. Biochemical measurements of dietary constituents in blood or other tissues can provide a useful assessment of the intake of certain dietary factors. However, for some nutrients, functional markers, or direct functional indices, provide a better estimate of the significance of the true status for a nutrient. More general functional indices relating to processes associated with disease risk are important for establishing the relationships between diet and disease prevention and response to treatment.\n\nThe development of biomarkers continues at a rapid pace. New types of markers are being proposed constantly and analytic techniques for existing markers are improved. With this advancement comes the need to establish accuracy, reliability, and interpretability of the biomarkers; to obtain data on marker distributions within different age and sex groupings in normal populations; to determine the extent of intraindividual variation in markers with respect to tissue localization and persistence; and to assess the contribution of genetic and acquired susceptibility factors to interindividual variability.\n\nReferences\n\n1. Clevidence B.A., Bieri J.G. Association of carotenoids with human plasma lipoproteins. _Methods Enzymol_. 1993;214:33\u201346.\n\n2. 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Gene expression profiling in human peripheral blood mononuclear cells using high-density filter-based cDNA microarrays. _J. Immunol. Methods_. 2000;239:167\u2013179.\n\n104. Cook J.D. Defining optimal body iron. _Proc. Nutr. Soc_. 1999;58:489\u2013495\nC.\n\nGenetic Influence on Nutritional Health\nCHAPTER 11\n\nGenetic Influences on Blood Lipids and Cardiovascular Disease Risk\n\nJOSE M. ORDOVAS, USDA-HNRCA, Tufts University, Boston, Massachusetts\n\n## I. INTRODUCTION\n\nThe major public health concerns in the developed world (i.e., cardiovascular disease, cancer, and diabetes) have both genetic and environmental causes. The interface between public health and genetics consists of working toward an understanding of how genes and the environment act together to cause these diseases and how the environment (e.g., diet), rather than genes, might be manipulated to help prevent or delay the onset of disease.\n\nCardiovascular disease (CVD), the leading cause of mortality in most industrialized countries, is a multifactorial disease that is associated with nonmodifiable risk factors, such as age, gender, and genetic background, and with modifiable risk factors, including elevated total and low-density lipoprotein (LDL) cholesterol levels, as well as reduced high-density lipoprotein (HDL) cholesterol levels. Convincing evidence shows that lowering serum lipid levels will slow the progression or even induce regression in atherosclerotic lesions [1\u20134]. Based on this evidence, the National Cholesterol Education Program (NCEP) has recommended that all adult Americans reduce their serum total cholesterol values to less than 5.18 mmol\/L (200 mg\/dL) and their LDL cholesterol values to less than 3.37 mmol\/L (130 mg\/dL) [5]. Although several types of drug therapies have been developed and have been shown to be highly effective in lowering serum LDL cholesterol values and CVD, the NCEP has emphasized that lifestyle modification should be the primary treatment for lowering cholesterol values, with drug therapies reserved for cases in which lifestyle modification is ineffective. The modifications advocated include dietary changes, regular aerobic exercise, and normalization of body weight. The recommended dietary changes include restriction in the amount of total fat (\u226430% of energy), saturated fat (<10% of energy), and cholesterol to <300 mg\/day. For individuals with elevated LDL cholesterol levels or cardiovascular disease, the saturated fat target should be much lower (<7% of energy) and the dietary cholesterol reduced to < 200 mg\/day. Also recommended is an increase in the consumption of carbohydrate and dietary fiber, especially water-soluble fiber. However, it is not known how many individuals can achieve the recommended levels of serum lipids using this approach [5], one of the major reasons being our current inability to predict individual plasma lipid response to dietary changes.\n\nSeveral studies demonstrated, during the first half of the 20th century, that serum cholesterol could be modified by the composition of dietary fat [6, 7], and studies by Keys _et al._ [8] and Hegsted _et al._ [9] provided the first quantitative estimates of the relative effects of the various classes of dietary fatty acids and amount of cholesterol on serum cholesterol changes. Other predictive algorithms have been developed in recent years, including predictions of response for LDL and HDL cholesterol [10\u201312]. These relationships between dietary changes and serum lipid changes are well founded and predictable for groups; however, a striking variability in the response of serum cholesterol to diet between subjects was reported as early as 1933 [13], and this variability has been the subject of multiple reports in recent years [12, 14\u201317]. In some individuals, plasma total and LDL cholesterol levels dramatically decrease following consumption of lipid-lowering diets, while they remain unchanged in others [12, 14, 15, 18]. It has been shown in elegant studies in nonhuman primates that the serum lipoprotein response to dietary manipulation has a significant genetic component [19\u201321]. Such genetic variability could have a significant impact on the success of public health policies and individual therapeutic interventions. At this point, the summary of a Scientific Conference on Preventive Nutrition: Pediatrics to Geriatrics, convened by The Nutrition Committee of the American Heart Association, specifically states that \"theoretically, genetic differences can render a particular set of dietary conditions more harmful or beneficial in one ethnic group than in another. This is one explanation for why individuals of different ethnic groups who consume similar diets might have varying disease profiles.\" Moreover, the statement underscores the need to \"identify specific genes and genetic variations that affect risk directly and indirectly by the way they interact with nutrients.\" [22]\n\nAs indicated above, we have traditionally measured the success of CVD risk-reducing strategies based on their effect on plasma lipids and, more specifically, lipoprotein levels. Lipoproteins are macromolecular complexes of lipids and proteins that originate mainly from the liver and intestine and are involved in the transport and redistribution of lipids in the body. Lipid and lipoprotein metabolism can be viewed as a complex biological pathway containing multiple steps. Lipid homeostasis is achieved by the coordinated action of a large number of nuclear factors, binding proteins, apolipoproteins, enzymes, and receptors involving hundreds of genes. Lipid metabolism is also closely linked with energy metabolism and is subjected to many hormonal controls that are essential for adjustment to environmental and internal conditions. Genetic variability has been demonstrated in humans for most of these proteins and some of these mutations can result in abnormal lipid metabolism and plasma lipoprotein profiles that may contribute to the pathogenesis of atherosclerosis. This complex picture can be dissected into three pathways (see Fig. 1). The exogenous lipoprotein pathway describes the metabolism of lipoproteins synthesized in the intestine following dietary fat intake; the endogenous pathway deals with the metabolism of lipoproteins involved in the transport of liver lipids to peripheral tissues; and finally the reverse cholesterol transport depicts the process by which excess peripheral lipids (primarily cholesterol) are transported to the liver for catabolism. Our current knowledge about how variants at candidate genes involved in each of these three interrelated processes affect dietary response and cardiovascular risk is presented below.\n\nFIGURE 1 Simplified scheme of lipoprotein pathways showing the exogenous (____), endogenous (.......) and reverse cholesterol (-----) pathways. Arrows indicate that following lipolysis by lipoprotein lipase, the FFA are released from the lipoproteins for utilization as a source of energy by tissues or for storage in the adipose tissue.\n\n## II. EXOGENOUS LIPOPROTEIN PATHWAY\n\nThe exogenous lipoprotein pathway has its origin in the enterocyte with the synthesis of chylomicron particles. Dietary fats absorbed in the intestine are packaged into large, triglyceride-rich chylomicrons for delivery to sites of lipid metabolism or storage. During their transit to the liver, these particles interact with lipoprotein lipase (LPL) and undergo partial lipolysis to form chylomicron remnants. These chylomicron remnants pick up apoE and cholesteryl ester from HDL, and are taken up by the liver via a process mediated by the interaction of apoE with hepatic receptors. For most people, this is a fast process and chylomicrons are not usually present in the blood after a prolonged fasting period. However, dramatic individual variability is seen in postprandial lipoprotein metabolism, which is in part determined by genetic factors and this variability could be highly relevant to achieving a more precise definition of individualized CVD risk. The most relevant candidate genes involved in this metabolic pathway and their known associations with plasma lipid levels and dietary response are described below.\n\n### A. Apolipoprotein B\n\nApoB is an essential protein component of intestinal chylomicron particles. In humans, the form of apoB synthesized by the intestine is apoB\u201348, produced by a mRNA editing mechanism [23, 24]. However, some apoB\u2013100 synthesis may occur also in the intestine [25, 26].\n\nThe _Apob_ gene has been mapped to the region 2p24-p23 on chromosome 2 [27]. Because apoB is the major protein of chylomicrons, as well as of LDL and very low-density lipoprotein (VLDL), it is reasonable to expect that genetic variation at this locus could influence plasma cholesterol and\/or triglyceride levels. More than 20 polymorphisms have been reported in this locus (). Some of these polymorphic sites have been utilized as markers in population or case-control studies in an attempt to correlate individual alleles or haplotypes with lipid levels and CVD risk. In general, the outcome of these studies has not been unanimous [28\u201337]. The findings and controversies associated with some of these polymorphisms are presented below.\n\nA silent mutation causing a cytosine to thymidine (ACC\u2192ACT) change characterizes the well-studied apoB _Xba_ I restriction fragment length polymorphism (RFLP). It involves the third base of the threonine codon 2488 in exon 26 without changing the amino acid sequence [38]. This RFLP has been associated with variability in plasma lipid levels [29\u201331, 39, 40]. The allele lacking the _Xba_ I recognition site (X\u2212) has been associated with lower total cholesterol, LDL cholesterol, and triglyceride concentrations. Paradoxically, this same X\u2013 allele has been found to be more common among coronary artery disease cases than in controls [41\u201343]. It has been speculated that the causal mutation associated to the _Xba_ I polymorphism might result in a structural change in apoB that affects the egression of LDL from the arterial wall [44].\n\nThe association between this polymorphism and variability in dietary response has been studied by several investigators and the results are quite controversial. Some reports show that subjects carrying the X+X+ or X+X\u2014genotypes respond to a low-fat, low-cholesterol diet with greater reductions in plasma total cholesterol, LDL cholesterol, apoB [45, 46], and, surprisingly, HDL cholesterol levels than subjects with the X\u2013X\u2013 genotype [45]. A recent meta-analysis suggests that this polymorphism plays a minor role in determining individual variability in response to dietary intervention [46a].\n\nWe have evaluated whether the apoB _Xba_ I polymorphism is associated with the interindividual variability observed during postprandial lipemia [47]. For this purpose, we carried out an oral fat load study involving 51 healthy young male volunteers [20 X\u2013\/X\u2013(X\u2013), and 31 X+\/X\u2013 or X+\/X+ (X+)], homozygous for the apoE3 allele. Our data showed that subjects with the X\u2014genotype had significantly greater retinyl palmitate (RP) and apoB\u201348 postprandial responses on both the large and the small triglyceride-rich lipoprotein fractions compared with X+ subjects, suggesting that the X\u2013\/X\u2014genotype is associated with greater postprandial response as compared with the X\u2013\/X+ or the X+ \/X+ genotypes. These differences observed in postprandial lipoprotein metabolism could explain some reported associations of this polymorphism to coronary artery disease risk. As indicated above, this mutation does not result in an amino acid change at the affected codon and it cannot have a direct functional effect. However, it is in strong linkage disequilibrium with the apoB Val591\u2192Ala polymorphism (Ag al\/d), which may be the functional sequence change. An alternative hypothesis relates to the position of this mutation near the apoB\u201348 editing site at position 2153. The mechanism of this editing involves a form of cytidine deaminase. The \"mooring\" sequence model for the editosome suggests that the recognition and binding sequences of nuclear factors that identify the specific site for editing and \"moor\" the editing activity are distal to and different from the editing site. It is possible that the _Xba_ I mutation, close to the editing site, could affect these \"mooring\" sequences, thus modifying the editing activity on the apoB mRNA. This might result in changes on apoB\u201348 synthesis and intestinal triglyceride-rich lipoprotein secretion as shown in our study.\n\nAnother potentially interesting polymorphism is the three-codon (leu-ala-leu) I\/D polymorphism within the apoB signal peptide [48]. This mutation has been found to be associated with variation in total and LDL cholesterol and\/or triglyceride levels [37, 49] as well as CVD risk [32]; however, these effects have not been confirmed by other investigators [50, 51]. Xu _et al._ [37] reported that subjects with the I\/I genotype had the highest triglyceride levels and D\/D subjects had the lowest, while consuming a high-fat, high-cholesterol diet. This effect disappeared when the subjects were consuming a low-fat, low-cholesterol diet. These results were not confirmed by Boerwinkle _et al._ [52] in a study in which subjects received two levels of dietary cholesterol without modification of dietary fat. In a more recent study, the D allele was found to be associated with reduced postprandial lipid response as compared with individuals homozygous for the I allele, suggesting that this mutation in the signal peptide may affect apoB secretion during the postprandial state [53]. Moreover, this polymorphism has been reported to be involved with postprandial lipoparticles' responses [54], and with the association between free fatty acid concentrations and triglyceride-rich lipoproteins in the postprandial state [55]. A plausible mechanism to explain the observed interaction between this I\/D polymorphism, postprandial free fatty acids, and triglyceride-rich lipoproteins has been proposed [55]. _In vitro_ studies have shown that apoB is synthesized and then either assembled into lipoproteins and secreted or degraded intracellularly. Oleate may increase secretion by protecting intracellular apoB from degradation. Using a yeast expression system, it has been shown that the 24-amino-acid signal peptide mediates apoB translocation into the endoplasmic reticulum less efficiently than the 27-amino-acid signal peptide, which may result in reduced apoB secretion. If this mechanism held _in vivo,_ then in subjects homozygous for the 27-amino-acid signal peptide, increased free fatty acids might result in increased protection of apoB from degradation and increased VLDL production. However, in subjects carrying the 24-amino-acid signal peptide, increased free fatty acids fail to regulate VLDL production because of accelerated intracellular degradation of apoB.\n\nThe _Msp_ I (CGGG\u2192CAGC) polymorphism in exon 26 causes an adenine to guanosine change that results in an amino acid change (Arg3611\u2192Gln) [56]. We have previously found a significant association between the less common allele (M2) and the presence of premature CVD [28], with this allele being nearly twice as frequent in CVD (0.105) as in the control population (0.057). However, no associations of this allele with alterations in plasma apoB or LDL cholesterol levels in subjects with coronary artery disease were noted [28]. The association between this RFLP and variability in dietary response has been examined and the results of a recent meta-analysis suggest that this polymorphism may play a minor role as a determinant of dietary response [46a].\n\nAn _Eco_ RI RFLP described in exon 29 consists of a single base pair (bp) mutation (GAA\u2192AAA) [57], which results in an amino acid change from Gln4514\u2192Lys. This RFLP is in linkage disequilibrium with the _Msp_ I described above and it should reveal similar phenotype associations.\n\nA 3\u2032-VNTR region approximately 300-bp distal to the 3' end of the apoB gene results in approximately 17 different alleles. Some initial reports suggested that larger number of repeats were associated with increased CVD risk [28, 41, 58]. However, other studies did not observe such association [59].\n\nA comprehensive analysis of these polymorphisms and the Bsp 1261I RFLP in exon 4 (the second base in codon 71 causing a cytosine to thymidine change and changing threonine to isoleucine) has been recently reported by Rantala _et al._ [60]. Moreover, the authors conducted a meta-analysis on the available data. The controlled dietary intervention study was conducted in 44 healthy, middle-aged subjects, consisting of a 3-month baseline, a 1-month fat-controlled, a 1-month high-fat, and a 1-month habitual diet period. In this dietary study, the apoB _Xba_ I restriction-site polymorphism affected the responsiveness to diet of the plasma LDL-cholesterol concentration, especially during the high-fat diet. The X\u2013\/X\u2013 subjects had a greater increase in LDL cholesterol (44 \u00b1 5%) than X+\/X+ (27 \u00b1 7%) or X+\/X\u2014(40 \u00b1 5+) subjects. The high-fat diet also induced a larger increase in plasma LDL cholesterol in subjects with the R\u2013\/R\u2013 genotype (59 \u00b1 10%) than in those with the R%\/R\u2013 (39 \u00b1 6%) or R+\/R+ (36 \u00b1 4%) genotypes. M+\/M+ subjects were also more responsive (41 \u00b1 3% increase in LDL cholesterol) than the M+ \/M\u2013subjects (27 \u00b1 10% increase). Their meta-analysis supported the finding of the significant role of the _Eco_ RI and _Msp_ I polymorphisms, but not that of the _Xba_ I polymorphism [60].\n\nThe results of the meta-analysis support the association between the apoB _Eco_ RI and _Msp_ I genotypes and responsiveness to diet. However, the percent of variability in response due to this locus is very small and this information does not yet provide enough information to be used as a clinical tool in dietary counseling.\n\n### B. Apolipoprotein A-IV\n\nIn humans, apoA-IV is synthesized primarily in the intestine as a 46-kDa glycoprotein [61]. The experimental evidence from familial apolipoprotein A-I\/C-III\/A-IV deficiency [62] suggests that it plays a role in dietary fat absorption and chylomicron synthesis. _In vitro_ studies have shown that the activation of lipoprotein lipase by apoC-II is mediated by apoA-IV [63], and that apoA-IV can serve as an activator of lecithin cholesterol acyltransferase (LCAT) [64]. ApoA-IV-containing lipoproteins promote cholesterol efflux from cultured fibroblasts and adipose cells _in vitro,_ and there is evidence that apoA-IV may be one of the ligands for the HDL receptor [65\u201367]. A report by Duverger _et al._ [68] demonstrated that overexpression of human apoA-IV in atherosclerosis-prone apoE-deficient mice resulted in an increased atherogenic lipid profile, with no significant effects on HDL cholesterol concentrations. Despite these findings, mice expressing human apoA-IV were more resistant to atherosclerosis. This was also true for those mice that were not knockouts for the apoE gene, but expressed the human apoA-IV. These data suggest that apoA-IV may protect against atherosclerosis by mechanisms that do not involve increased HDL cholesterol concentrations, and also may explain the absence of atherosclerosis in the apoA-I-deficient mice [69]. However, this hypothesis have been partially challenged by data generated in normocholesteroleic mice. In these animals, high plasma levels of human apoA-IV did not enhance cholesterol mobilization _in vivo_ in normolipemic mice [70]. Overall, the data suggest that apoA-IV plays a role in fat absorption and reverse cholesterol transport.\n\nIt has been shown that apoA-IV expression is regulated by fat feeding and recent data provide evidence that isolated fatty acids regulate gene expression and the production of apoA-IV in the enterocyte. Specifically, in confluent Caco\u20132 cells incubated with 1 m _M_ oleic (n\u20139), linoleic (n\u20136), alpha-linolenic (n\u20133), or docosahexaenoic (n\u20133) acids for a long-term period, both apoA-IV protein levels and _de novo_ synthesis were increased. This induction resulted from the upregulation of apoA-IV mRNA transcripts. In contrast, an inhibitory effect was evident with short-term incubation. Fatty acid chain length and degree of unsaturation had little effect in altering apoA-IV transcript and biogenesis. [71].\n\nGenetically determined isoforms of apoA-IV have been detected in humans and in other mammalian species (http:\/\/gdbwww.gdb.org\/gdb-bin\/genera\/accno?accessionNum= GDB:119000). The most common isoform detected using iso-electric focusing is A-IV-1, with an allele frequency in Caucasians ranging from 88\u201395%. ApoA-IV\u20132 (Gln360\u2192His) is the second most common isoform with an allele frequency in the range of 5\u201312% in Caucasians [72, 73]. Additional variation within these isoforms has been detected using the polymerase chain reaction (PCR). A relatively common mutation (Thr347 \u2192 Ser) has been documented within subjects with the apoA-IV-1 isoform. The effect of apoA-IV genetic variation on plasma lipid levels has been studied in several populations [73\u201377]. In some Caucasian populations, the apoA-IV-2 allele has been associated with higher levels of HDL cholesterol and\/or lower triglyceride levels [72, 77, 78], but no associations have been observed in other studies [79\u201384].\n\nThe effect of genetic variation at this locus on dietary response has been examined [85, 86]. Our own data show that the apoA-IV-2 (Gln360\u2192His) allele (apoA-IV-2) is associated with hyporesponsiveness of LDL cholesterol to dietary therapy consisting of reduction in total fat and cholesterol intakes. McCombs _et al._ [86] have demonstrated that this effect may be due to the reduction in dietary cholesterol. We also observed that subjects with the apoA-IV-2 allele tended to have greater decreases in HDL cholesterol concentrations following a low-fat and low-cholesterol diet. To followup on this observation, we have examined the effect of this polymorphism on HDL cholesterol response in 41 healthy males subjects [87]. They were fed three consecutive diets (high-saturated, low-fat and high-monounsaturated fat diets) for 4 weeks each. After consuming the high-saturated fat diet, carriers of the apoA-IV-2 allele had a greater decrease in HDL cholesterol and apoA-I. In these subjects, replacement of a high carbohydrate diet by monounsaturated fat resulted in a greater increase in HDL cholesterol and apoA-I as compared with homozygotes for the apoA-IV-1 allele. These data suggest that in apoA-IV-2 subjects, a high-carbohydrate diet may induce an apparently increased atherogenic lipid profile (LDL cholesterol does not decrease, but HDL cholesterol does decrease). Therefore, these subjects may benefit specially from a diet relatively high in monounsaturated fat.\n\nThe mechanism by which this mutation may exert the observed effect is still unknown. The apoA-IV-2 isoform binds to lipoproteins with higher affinity than apoA-IV-1, which may result in delayed hepatic clearance of chylomicron remnants, as shown in metabolic studies [88]. Given the important role that this apolipoprotein may have in lipid absorption, it is possible that mechanisms involving intestinal fat absorption and\/or the metabolism of triglyceride-rich lipoproteins may be differentially affected by each isoform.\n\nThe apoa-IV-2 allele has been also studied in relation to the changes in CVD risk factors associated with urbanization in developing countries. We demonstrated in a population-based study in Costa Rica that lifestyles associated with an urban environment, such as increased smoking and saturated fat intake, elicit a more adverse plasma lipoprotein profile among subject carriers of the apoA4-IV-2 allele than in apoA-IV-1 homozygotes, which could make them more susceptible to CVD [89]. These results may be difficult to reconcile with those from the dietary metabolic studies. However, note that the changes associated with \"modernization\" (i.e., increased fat and cholesterol intakes, smoking, and decreased physical activity) are more complex gene\u2013environment interactions than those taking place during a well-controlled dietary protocol carried out in a metabolic unit.\n\nWe have also examined the association between the apoA-IV(Thr347\u2192Ser) variant observed within the apoA-IV-1 allele and the LDL cholesterol response to dietary intervention [90]. Our data show that carriers of the less common Ser347 allele have a greater decrease in total cholesterol, LDL cholesterol and apoB concentrations when they are switched from a low-fat and cholesterol diet to a high-monounsaturated fat diet as compared with homozygous carriers of the Thr347 allele.\n\nSimilar studies in subjects heterozygous for familial hypercholesterolemia [91] show that the apoA-IV-2 allele was associated with lower LDL cholesterol and apoB levels independent of diet effects. No differences in total cholesterol, LDL cholesterol, HDL cholesterol, and apoB levels were observed between subjects homozygous for the apoA-IV Thr347 allele and those carriers of the apoA-IV Ser347 allele. After dietary intervention, Ser\/Ser subjects showed significant reductions in plasma triglycerides and VLDL cholesterol levels, but no changes were found in carriers of the Ser allele.\n\nThe combined information for the Thr347\u2192Ser and the Gln360\u2192His suggests that the responsiveness of LDL cholesterol to changes on dietary fat could follow this pattern: 347Ser\/360Gln>347Thr\/360Gln>347Thr\/360His. The mechanisms by which these mutations may exert the observed effects are still unknown. The apoA-IV-2 allele binds to lipoproteins with higher affinity than apoA-IV-1, which may result in delayed hepatic clearance of chylomicron remnants as shown in metabolic studies. The substitution of Ser for Thr at position 347 induces changes in the secondary structure and a slight increase in hydrophilic profile at this position, which could result in a decrease in its affinity for lipids on the triglycerite-rich lipoprotein particles. This could facilitate the exchange with apoC-II, thereby increasing LPL activity over those particles, which would in turn accelerate clearance of remnants. The increased influx of dietary cholesterol would downregulate the LDL receptors, with consequent increases in LDL cholesterol concentrations. Therefore, consumption of fat-rich diets would produce a greater increase in LDL cholesterol in Ser347 carriers.\n\n### C. Apolipoprotein E\n\nApoE in serum is associated with chylomicrons, VLDL, and HDL, and serves as a ligand for the LDL receptor and the LDL receptor-related protein [92, 93]. When apoE deficiency is present, there is marked accumulation of cholesterol-enriched lipoproteins of density <1.006 g\/mL containing apoB\u201348 and apoA-IV, as well as apoB\u2013100 [94]. Moreover, in this disorder there is delayed clearance of both apoB\u2013100 and apoB\u201348 within triglyceride-rich lipoproteins. Genetic variation at the apoE locus results from three common alleles in the population, E4, E3 and E2, with frequencies in Caucasian populations of approximately 15, 77, and 8%, respectively [95]. Population studies have shown that plasma cholesterol, LDL cholesterol, and apoB levels are highest in subjects carrying the apoE4 isoform, intermediate in those with the apoE3 isoform, and lowest in those with the apoE2 isoform [96, 97]. It has been suggested that apoE allelic variation may account for up to 7% of the variation in total and LDL cholesterol levels in the general population [95]. This relationship between LDL cholesterol levels and apoE genetic variation is not independent of environmental and ethnic factors. Thus, it appears that the association of the apoE4 isoform with elevated serum cholesterol levels is greater in populations consuming diets rich in saturated fat and cholesterol than in other populations. These data indicate that the higher LDL cholesterol levels observed in subjects carrying the apoE4 isoform are manifested primarily in the presence of an atherogenic diet characteristic of certain societies, and that the response to dietary saturated fat and cholesterol may differ among individuals with different apoE phenotypes. Many studies have been conducted to prove this hypothesis [98\u2013100]. Some investigators reported greater plasma lipid responses in subjects carrying the apoE4 allele, while others failed to find significant associations between apoE genotype and plasma lipid response [101\u2013103]. Important differences exist among these studies that could account for some of the discrepancies observed. Studies differed in subject gender, age, and baseline lipid levels, and all of these variables are known to play an important role in the variability of dietary response. Dreon _et al._ [104] have shown that the apoE-dependent mechanism may be specific for large, buoyant LDL particles. Consequently, baseline LDL particle distribution will also play a significant role on the outcome of different studies and this variable should be controlled in future studies. In addition, Lehtim\u00e4ki _et al._ [105] have demonstrated that the association between serum lipids and the apoE phenotype is influenced by diet in a population based sample of free-living children and young adults.\n\nOverall, a significant diet by apoE gene interaction has been shown in studies with men alone. In those studies including men and women, significant effects were noted only in men, suggesting a significant gene\u2013sex interaction. Another difference between the negative studies and those reporting significant apoE gene\u2013diet interactions related to the baseline lipid levels of the subjects. Positive findings were frequently observed in those studies reporting significant associations that included subjects who were moderately hypercholesterolemic and\/or had significant differences in baseline total cholesterol and LDL cholesterol among the apoE genotype groups, suggesting that the significant gene by diet interaction is apparent only in subjects who are susceptible to hypercholesterolemia. Concerning differences in dietary interventions, significant interactions were more commonly observed among studies in which total dietary fat and cholesterol were modified. It is possible that dietary cholesterol may play a significant effect in this gene\u2013diet interaction. Note also that some reports have shown that cholesterol absorption is related to apoE genotype.\n\nOther less traditional dietary interventions have been also examined in terms of gene\u2013diet interactions. Thus, Loktionov _et al._ [106] examined the effects of the apoE alleles and tea drinking on blood lipids and blood coagulation factors on healthy men and women. In this study, apoE4 subjects had significantly elevated total cholesterol, LDL cholesterol and triglyceride levels. Moreover, mean plasminogen activator inhibitor (PAI-1) activity was higher in apoE4 subjects than in E3\/3 or E3\/2 subjects. These findings suggest that elevated PAI-1 activity may be an additional factor involved in the increased cardiovascular risk associated with the apoE4 allele. Tea drinking was associated with significant decreases in HDL cholesterol levels of E3\/3 subjects as well as decreases in triglyceride levels and PAI-1 activities of apoE2\/3 subjects. These results indicate that tea drinking has a beneficial effect on plasma lipid levels and coagulation factors, especially in subjects carrying the E2 allele. Note that fruits and vegetables also contain polyphenols similar to those in tea, and high consumption of fruits and vegetables have been reported to decrease PAI-1 activity. The molecular mechanisms involved in the interaction between the apoE gene and PAI-1 are still unknown, but one possible link lies on the LDL receptor-related protein receptor, which is known to bind several ligands including apoE, tPA, and PAI-1. Per the hyperresponse observed for the apoE2 allele and triglyceride levels, it should be noted that previous studies using dietary manipulation that included type of carbohydrates and fiber rather than type and amount of dietary fat and cholesterol demonstrated that apoE2 subjects were more responsive to these dietary modifications than apoE3\/3 and apoE4 subjects. Moreover, apoE2 carriers are significantly more responsive to HMG CoA reductase inhibitors than E3\/3 and E4 subjects [107, 108]. However, these studies were carried out in small groups and they should be confirmed in larger studies.\n\nAfter several decades of research, it is becoming evident that postprandial lipemia is a major determinant of blood lipoprotein concentrations and CVD risk [109]. The postprandial response is highly heterogeneous, and multiple factors such as age, exercise levels, body weight, fasting lipid levels, diet, and genetics have been noted to be responsible for this variability. The apoE gene has been implicated as one of the genetic factors mediating these effects. The apoE2 isoform is considered to decrease remnant clearance because of impaired affinity for the receptors. Conversely, the apoE4 isoform should induce a faster clearance. However, studies that have compared postprandial triglyceride responses across different apoE genotypes have produced conflicting results, especially regarding the effects associated with the apoE4 allele [110\u2013114]. Postprandial response was examined at 4 and 8 hours by Boerwinkle _et al._ [115] in a sample of individuals taking part in the Atherosclerosis Risk in Communities Study (ARIC), following a single high-fat meal containing vitamin A (used as a marker for intestinal lipoprotein synthesis). Postprandial plasma retinyl palmitate response was significantly different among apoE genotypes, with delayed clearance in subjects carrying the apoE2 allele, compared with E3\/3 and E3\/4 subjects; however, measurements of other lipid variables, such as triglyceride concentration and triglyceride-rich lipoprotein were not sensitive enough to detect these effects. Another study by Nikkil\u00e4 _et al._ [113], carried out in CVD cases and controls, showed that in CVD patients with the apoE2\/3 phenotype triglyceride levels were highest and still increasing after 7 hours, reflecting delayed chylomicron remnant clearance. The same effect was observed also in normotriglyceridemic non-insulin-dependent diabetic patients [116] and in nondiabetic normolipemic subjects [117], although in this report, the delayed chylomicron remnant was observed only on E2\/2 individuals. The findings associated with the apoE4 allele have been more discordant. In an earlier report, heterozygosity for this allele was associated with lower lipemic response in relation to other phenotype groups [111]; however, in a more recent study the E4 allele was associated with prolonged postprandial responses of lipids and apolipoproteins in triglyceride-rich lipoproteins [118]. This subject has been recently revisited by Wolever _et al._ [119]. These investigators examined the longterm effect of soluble fiber-containing foods on postprandial fat metabolism in dyslipidemic subjects, 16 with apoE3\/3 and 17 with E3\/4 genotypes. These subjects consumed low-fat (20% of energy), high-fiber (> 24 g\/1000 Kcal) diets for two 4-month periods separated by a 2-month washout period according to a randomized crossover design. One diet contained foods rich in insoluble fiber and the other foods rich in soluble fiber. They carried out a 1-day postprandial study during the last 2 weeks of each diet. Subjects ingested a standard, fiber-free, fatty liquid meal containing retinyl palmitate as a marker of intestinally derived lipoproteins. Blood samples were obtained at hourly intervals for 10 hours. Their results suggested that a long-term increase in dietary soluble fiber does not affect postprandial fat metabolism in subjects with the apoE4 allele; however, soluble fiber enhanced fat absorption in E3\/3 subjects, which could be due to an increased bile acid pool and increased micelle formation.\n\nSeveral mechanisms have been proposed to explain these apoE-related differences in individual response to dietary therapy. Some studies have shown that intestinal cholesterol absorption is related to apoE phenotype, with apoE4 carriers absorbing more cholesterol than non-apoE4 carriers. Other mechanisms, such as different distribution of apoE on the lipoprotein fractions, LDL apoB production, bile acid and cholesterol synthesis, and postprandial lipoprotein clearance, may also be involved.\n\n### D. Apolipoprotein C-III (APOC3)\n\nPlasma apoC-III is a component of chylomicrons, VLDL, and HDL. This protein is synthesized primarily in the liver and to a lesser extent in the intestine [120]. _In vitro,_ apoC-III inhibits LPL activity [121] and the binding of apoE-containing lipoproteins to the LDL receptor, but not to the LDL receptor-related protein [122, 123]. In agreement with the observations _in vitro,_ the overexpression of the human apoC3 gene in transgenic mice resulted in severe hypertriglyceridemia [124]. The apoC3 gene is closely linked to the apoA1 and apoA4 genes on the long arm of chromosome 11 (region 11q13) [125]. Several RFLPs have been described at this locus. The S2 allele of the SstI RFLP (C3238G), 3' to the apoC3 gene, has been associated in some studies with hypertriglyceridemia and increased coronary artery disease risk [126\u2013128]. A PvuII RFLP located in the first intron of the apoC3 gene has also been associated with variation in HDL cholesterol levels. In addition, several polymorphisms have been identified in the promoter region of the gene (C\u2212641\u2192A, G\u2212630\u2192A, T\u2212625\u2192deletion, C\u2212482\u2192 T, and T\u2212455\u2192 C) [129]. These mutations are in linkage disequilibrium with the _Sst_ I site in the 3' untranslated region [129, 130]. An insulin response element has been mapped to a 42-nucleotide-long fragment located between \u2013 490 and \u2013 449 relative to the transcription start site and _in vitro_ studies demonstrated that transcriptional activity of the apoC3 gene was downregulated by insulin only in the construct bearing the wild-type promoter, but not in those constructs containing the C\u2013482\u2192 T, and T\u2013455\u2192C variants [130, 131]. These results may provide the molecular basis to understand the increased levels of apoC3 found in subjects carrying the S2 allele and its association with hypertriglyceridemia. Our own studies show that following an increase in dietary monounsaturated fat intake, S1S1 subjects responded with an increase in LDL cholesterol levels, whereas S1S2 subjects experienced a significant decrease [132], suggesting that the apoC3 locus is involved in LDL cholesterol responsiveness to dietary fat. This interaction could begin to explain the different effects associated with this polymorphism that have been reported in the literature. Along those lines, Waterworth _et al._ [133] examined the association of the S2 allele and several other polymorphisms at this locus on postprandial lipid levels following an oral fat load test. The population consisted of young, healthy male offspring whose fathers had had a myocardial infarction before the age of 55 years, compared with age-matched controls. The apoC3 variations examined were C3238G (SstI) in the 3\u2032-UTR, C1100T in exon 3, C-482T in the insulin response element, and T-2854G in the apo-CIII-AIV intergenic region. The postprandial response was regulated by variation at the T-2854G and C3238G sites. After the oral fat tolerance test, carriers of the rare alleles had significantly delayed clearance of triglyceride levels; G-2854 carriers showed the largest effect on triglycerides, and G3238 carriers showed a lesser response. However, after adjustment for fasting triglyceride levels, only the effect with the T-2854G remained significant.\n\nThe reported association of the S2 allele with elevated triglyceride and cholesterol concentrations, and with high blood pressure, all of which are characteristic of an insulin-resistant state, and the presence of an insulin response element in the promoter region of the apoC3 gene, suggests that this polymorphism could also be involved in dysfunctional glucose metabolism. Therefore, we have examined the effect of the C3238G polymorphism on carbohydrate metabolism in healthy subjects. We gave 41 males three consecutive diets [134]. The first was rich in saturated fat, the second was an NCEP Step 1 diet, and the last was rich in monounsaturated fat. At the end of each dietary period, subjects received an oral glucose tolerance test. _APOC3_ genotype significantly affected basal glucose concentrations and insulin concentrations after the test. Carriers of the S2 allele (n = 13) had higher insulin concentrations after the test than S1\/S1 subjects ( _n_ = 28) in the three periods. Multiple regression analysis showed that this polymorphism predicted the insulin response to the oral glucose tolerance test and the difference between basal insulin concentrations and insulin concentrations in response to the saturated fat-rich diet.\n\nAn association between the apoC3 locus and carbohydrate metabolism was also reported by Waterworth _et al._ [133] on the EARSII study. Variation at the C-482T insulin response element determined response to the oral glucose tolerance test, with carriers of the rare T-482 having significantly elevated glucose and insulin concentrations.\n\nThese data suggest that specific genetic variants at the apoC3 gene locus differentially affect postprandial lipids [133] and response to oral glucose [133, 134], which could result in reduced sensitivity to insulin, especially when persons consume diets rich in saturated fat.\n\n### E. Lipoprotein Lipase\n\nLipoprotein lipase is a heparin-releasable enzyme, bound to glycosaminoglycan components of the capillary endothelium. It plays a key role in lipoprotein metabolism by catalyzing the hydrolysis of 1,3 ester bonds of triglycerides in chylomicrons and VLDL. The active form of LPL is constituted by two identical subunits each of approximately 60,000 daltons, which require apoC-II as a cofactor, whereas apoC-III acts as an inhibitor. LPL is synthesized in the adipose and muscle tissue, as well as in macrophages [135]. The gene for LPL has been located to the short arm of chromosome 8 (region 8p22) [136, 137]. Several common RFLPs have been reported at this locus, including a _P_ _vu_ II RFLP located in the intron between exons 6 and 7 [138], a _Hin_ dIII located in the intron between exons 8 and 9 [139], and a C-to-G transversion at nucleotide 1595 of the cDNA sequence. Unlike the _P_ _vu_ II and _Hin_ dIII RFLPs, the later mutation alters the structure of the protein and it results in the production of a truncated protein (Ser447-Stop) [140]. In population studies, these variants have been found associated with variability in plasma lipid levels and with CVD [141\u2013143]. The _Hin_ dIII RFLP has also been reported to be associated with the variability in lipid response to changing from a high saturated to a low-saturated fat diet [144].\n\nOther relatively common mutations at this locus have been recently reported to be associated with mild alteration in lipid profiles. A missense mutation (Asn291Ser) in exon 6 has been found with relative high frequency (2\u20134%) in Western populations, and appears to be enriched in familial combined hyperlipidemia [145, 146] and low HDL cholesterol [147]. _In vivo_ and _in vitro_ measurements of LPL activity indicate that this mutation is associated with approximately 50\u201370% of normal lipase catalytic activity. Pimstone _et al._ [148] have shown that normolipidemic Asn291Ser carriers exhibited a more pronounced postprandial response compared with noncarriers as shown by higher chylomicron triglyceride and retinyl palmitate peaks. It is possible that carriers of this mutation may be unable to respond to a high-fat diet by an increase in their LPL activity as normal subjects do. Thus, an oral fat challenge may unmask a hidden defect in lipolysis in these subjects that may not be evident in the fasting state. Another common variant (Asp9Asn) has been associated with elevated triglyceride levels and with increased progression of coronary atherosclerosis [149\u2013151]; however, to date no reports have appeared regarding the response of these subjects to a fat challenge.\n\nWe have examined these LPL variants and their potential associations with lipid levels and CVD risk on the Framingham Heart Study [152, 153]. Carrier frequency of the S447X allele was 17%; in men, carrier status was associated with higher total cholesterol and HDL cholesterol, and lower triglyceride levels. Moreover, in men, the S447X allele conferred protection against CVD. These effects on lipids and CVD were not seen in women. We have also assessed in this population the effect of the D9N and N291S alleles, and we extended our study by calculating weighed means of lipids and lipoproteins in carriers and noncarriers for these mutations in patients with genetic dyslipidemias, CVD patients and healthy controls. In the Framingham Offspring sample, the D9N and N291S alleles were associated with lower HDL cholesterol and a trend towards increased triglycerides. In women, a trend toward a high triglyceride, low HDL cholesterol lipid pattern was evident. Cumulative analysis of other studies of male carriers of the D9N and N291S alleles revealed higher levels of triglycerides and lower HDL cholesterol. In females, results differed with higher triglyceride levels for the N291S and D9N carriers, and lower HDL cholesterol levels only for N291S carriers. These data provide evidence that these LPL variants are significant modulators of lipid levels in both men and women.\n\n### F. Microsomal Triglyceride Transfer Protein\n\nMicrosomal triglyceride transfer protein (MTP) is a lipid transfer protein that is required for the assembly and secretion of VLDL by the liver and chylomicrons by the intestine. MTP is a heterodimer composed of a 55-kDa multifunctional protein, protein disulfide isomerase, and a unique large subunit with an apparent molecular weight of 88 kDa [154]. The gene has been localized to 4q22-q24. Wetterau _et al._ [155] demonstrated that MTP activity and the large subunit of MTP were present in intestinal biopsy samples from eight control persons but were absent in four abetalipoproteinemic subjects. They suggested that the findings proved that MTP is the site of the defect in abetalipoproteinemia and that MTP is required for lipoprotein assembly.\n\nA common G\u2013493T polymorphism at the MTP gene promoter has been shown to be associated with decreased plasma LDL cholesterol and apoB content of VLDL [156]. We have investigated the association of this mutation with variations in lipid and apoprotein levels, lipoprotein subclass profiles, and CVD risk in Framingham Heart Study participants [157]. In men and women, no significant association was found between the G\u2013493T MTP polymorphism and variations of plasma levels of total cholesterol, LDL cholesterol, apoB, HDL cholesterol, apoA-I, and triglyceride. To further investigate potential relationships with variations of lipoprotein phenotypes, lipoprotein subclass profiles were measured using automated nuclear magnetic resonance (NMR) spectroscopy. Each NMR profile yielded information on lipid mass of VLDL, LDL, and HDL subclasses. In both genders, there was no significant association between the G\u2013493T polymorphism and variability of lipoprotein subclass distributions or lipoprotein particle size. Furthermore, no significant association was found between the polymorphism of the MTP promoter and prevalence or the age of onset of CVD. Thus, our results suggest that the G\u2013493T mutation in the MTP promoter is unlikely to have significant implications for CVD in men and women. No reports are available regarding potential associations between this locus and dietary response.\n\n### G. Intestinal Fatty Acid Binding Protein\n\nThe intestinal fatty acid binding protein (IFABP) gene ( _FABP2_ ) codes for the IFABP. The IFABP is a member of a family of small (14\u201315 kDa) intracellular lipid binding proteins. Besides the IFABP, this family also includes the heart, epidermal, brain, and liver fatty acid binding proteins, testicular, myelin, adipocyte, and ileal lipid binding proteins, and cellular retinol and retinoic acid binding proteins [158]. The gene located in 4q28-q31[159] has the conserved 4 exons and 3 introns characteristic of this family of genes [160, 161]. It has been found that the expression of IFABP mRNA is under dietary control [162].\n\nIFABP plays important roles in several steps of fat absorption and transport, including uptake and trafficking of saturated and unsaturated long-chain fatty acids, targeting free fatty acids toward different metabolic pathways, protecting the cytosol from the cytotoxic effects of free fatty acids, and modulating enzyme activity involved in lipid metabolism [163, 164]. Besides free fatty acids, the IFABP may bind other ligands, such as phenolic antioxidants. The IFABP is abundant in the enterocyte and represents 2\u20133% of an enterocyte's cytoplasmic mass [165]. Sacchettini _et al._ [166] found that the fatty acid binding proteins each have 10 anti-parallel \u03b2 strands, which form two orthogonal \u03b2 sheets. There are two short \u03b1 helices, which are connected to the first two \u03b2 strands. This structure has been characterized as a \u03b2 clam because it resembles a clamshell. It is hypothesized that fatty acids enter the fatty acid binding protein by passing a portal that includes three structures; the portal is comprised of one of the \u03b1 helices, and two sharp turns between \u03b2 strands, which include residues 54\u201355 and 73\u201374 [166, 167]. A mutation in the IFABP at any of these amino acid residues that surround the portal may alter fatty acid entry into the protein. This may result in abnormal binding of fatty acids, which can then influence lipid profiles and consequently disease risk.\n\nBaier _et al._ [168] reported a new G\u2192A mutation, which results in an amino acid substitution in IFABP at residue 54, alanine54 (wild-type) \u2192threonine (mutant-type). This polymorphism is very common, with a thr54 allelic frequency of around 29% in most populations. To this date, the ala54thr mutation in the IFABP is the only functional mutation found in humans in a member of the cytoplasmic fatty acid binding protein family [169]. This amino acid substitution was found to be associated with elevated fasting insulin levels, insulin resistance, and increased fatty acid binding in Pima indians [168, 170]. Associations of this polymorphism with several biochemical and anthropometric variables have been subsequently carried out in Japanese, Mexican-American, Native Americans, and Caucasian populations. In general, the presence of the thr54 allele has been associated in some, but not all [171\u2013175], studies with higher fasting insulin concentrations [168, 176] and insulin resistance. On the other hand, a study of Keewatin Inuit (Eskimos) found the thr54 allele to be associated with lower 2-hour glucose concentrations [175]. It is well known that Eskimos have large dietary intakes of omega\u20133 fatty acids from fish. This finding in Eskimos suggests that differences in the type of fatty acid consumed interact with the functional differences in the gene products to produce phenotypic differences.\n\nSeveral studies have utilized the euglycemic, hyperinsulinemic clamp for determining insulin resistance. Using this approach, the _FABP2_ mutation was associated with a lower mean insulin-stimulated glucose uptake rate in Pima indians. However, no significant findings were noted in subjects with familial combined hyperlipidemia [177] and in overweight, Finnish subjects [178]. In a separate study, Stern _et al._ [179] found that the IFABP was not significantly linked to the major gene for the age of onset for non-insulin-dependent diabetes mellitis (NIDDM) [179].\n\nOther studies have found an association between the presence of the thr54 mutation and higher mean fat oxidation rate [168, 178] and greater fasting plasma triglycerides [180]. However, Vidgren _et al._ examined a small group of Finnish subjects after an overnight fast and found no differences in the proportion of long-chain fatty acids in serum lipids among the three genotypes.\n\nRegarding differences in dietary response, Hegele _et al._ [182] found that the ala54thr mutation was associated with variation in the response of plasma lipoproteins to dietary fiber. Subjects with the mutant thr54 allele, compared to those homozygous for the ala54 allele, had significantly greater decreases in plasma total and LDL cholesterol and apoB when consuming a high soluble fiber diet than when consuming a high insoluble fiber diet. Moreover, Agren _et al._ [183] have shown that the triglyceride response to an oral fat load test was greater in Thr homozygotes. In addition, the classical correlation between fasting and postprandial triglyceride levels was present in Ala carriers, but not so in Thr individuals, suggesting that the delayed triglyceride-rich lipoprotein clearance may affect insulin action (or vice versa).\n\n_In vitro_ studies have found that the thr54 IFABP differs from the ala54 IFABP, and these studies offer a biologically plausible mechanism for thr54\u2032s role in altered lipid metabolism and disease states. Baier _et al._ [168] found, through titration microcalorimetry, that the thr54 IFABP had a twofold greater affinity for long-chain fatty acids than the ala54 IFABP. In addition, in another study, Baier's group created permanently transfected Caco\u20132 cells that express the ala54 or thr54 IFABP [184]. The cells expressing the mutant thr54 protein were found to have greater transport of long-chain fatty acids and greater secretion of triglycerides than the ala54 protein. A recent study with mouse L-cells transfected with wild-type IFABP found that the increase in secretion of triglycerides over nontransfected cells was due to increased synthesis in the cell [185]. The results of these studies support Baier _et al._ 's findings in which the thr54 protein has a higher affinity for long-chain fatty acids than the ala54 protein [168, 184]. The three-dimensional structure of the wild-type IFABP has been studied by NMR and it was shown that amino acid 54 in the IFABP is important in the stabilization of the portal region, where the fatty acid enters the protein [167, 186]. Amino acid 54 interacts with \u03b1-helix II and helps to stabilize the portal region. Hodsdon and Cistola [187] hypothesize that since threonine can hydrogen bond and alanine cannot, then thr54 will be better able to interact with \u03b1-helix II and stabilize the portal, which may lead to increased binding of fatty acids [167].\n\nIn summary, the physiological function of the protein coded by this gene and the preliminary findings reported regarding the only common variant described warrant further research to assess the full impact of this locus on the interindividual variability in dietary response.\n\n## III. ENDOGENOUS LIPOPROTEIN METABOLISM\n\nThe hepatocyte synthesizes and secretes triglyceride-rich VLDL, which may be converted first to intermediate-density lipoproteins and then to LDL through lipolysis by a mechanism involving LPL, similar to that described for the exogenous lipoprotein pathway. The excess surface components are usually transferred to HDL. Some of these remnants may be taken up by the liver, whereas others are further lipolyzed to become LDL, which, in humans, contains apoB as their only apolipoprotein. This pathway shares many of the genes described already for the exogenous pathway. This section will describe briefly some of the studies related to the LDL receptor gene and coding for the LDL receptor, which has major responsibility for catabolism of the LDL particles in the liver and peripheral tissues.\n\n### A. LDL Receptor\n\nThe LDL receptor gene has been localized to the 19p13. 1 region. Mutations at this locus have been found to be responsible for familial hypercholesterolemia, an autosomal dominant disorder characterized by elevation of LDL cholesterol and premature CVD. The frequency of homozygosity is estimated to be 1 in 1 million, and the frequency of heterozygotes approximately 1 in 500. Several hundred different mutations have been already described in the LDL receptor gene and many of them have been reviewed in detail [188, 189]. In addition to those mutations associated with familial hypercholesterolemia, several highly informative (maximum heterozygosity ranging from 0.4 to 0.5) deletions [190], point mutations [191], dinucleotide repeats [192], and RFLPs [192\u2013200] have been described in this locus.\n\nSeveral lines of LDL receptor transgenics have been generated. In one of them, plasma concentrations of apoB and apoE decreased by more than 90% [201]. In another line, expression of the human LDL receptor gene in the mouse liver was able to completely obliterate the diet-induced hypercholesterolemia in these mice [202].\n\nThe targeted disruption of the LDL receptor gene resulted in hypercholesterolemia due primarily to increases in intermediate-density lipoproteins and LDL with no changes in HDL levels [203]. This LDL-receptor-deficient mouse is a good model for the human familial hypercholesterolemia, and it has been extensively used as a background to generate additional transgenic models and to test gene replacement therapy.\n\nAlthough we have not directly tested the effect of different mutations at the LDL receptor locus and variability in dietary response, we have carried out several studies examining other loci in heterozygote subjects. In general, our data suggest that the strong effect of the LDL receptor mutations tend to obscure other minor effects associated with other loci in normolipemic subjects [91, 204].\n\n### B. 3-Hydroxy\u20133-Methylglutaryl-Coenzyme A Reductase\n\n3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA) catalyzes a rate-limiting step in cholesterol biosynthesis and also participates in the production of a variety of other compounds. This enzyme has been an extremely successful drug target in the form of HMG-CoA reductase inhibitors. _In situ_ hybridization permitted the precise regionalization of this gene to chromosome 5. The findings from our own genome scan [205] suggest that this chromosomal region may be important in determining plasma lipid variability in this same population.\n\nDespite its important biological role and the fact that it has been a very successful drug target, this gene has been barely examined in terms of variability in dietary response. Boucher _et al._ [206] have studied the effects of cholesterol feeding in healthy subjects on monocyte mRNA levels for the LDL receptor, HMG-CoA receptor, and LRP genes. These authors found coordinated regulation of these genes involved in the synthesis and removal of cholesterol. Briefly, during cholesterol restriction phase, LDL receptor and HMG-CoA mRNA levels increased, whereas during the cholesterol-rich phase, the levels of these mRNAs decreased significantly. Conversely the levels of LRP mRNA increased during this phase. This research demonstrates for the first time that the LRP gene is also responsive to dietary cholesterol.\n\n## IV. REVERSE CHOLESTEROL TRANSPORT\n\nHDL is synthesized by both the liver and the intestine. Its precursor form is discoidal in shape and matures in circulation as it picks up unesterified cholesterol from cell membranes (see ATP binding cassette-1 below), other lipids (phospholipids and triglycerides), and proteins from triglyceride-rich lipoproteins as these particles undergo lipolysis. The cholesterol is esterified by the action of LCAT and the small HDL3 particle becomes a larger HDL2 particle. The esterified cholesterol is either delivered to the liver or transferred by the action of the cholesterol ester transfer protein to triglyceride-rich lipoproteins in exchange for triglyceride. The liver may then take up this cholesterol via receptors specific for these lipoproteins, or it can be delivered again to the peripheral tissues. The triglycerides received by the HDL2 are hydrolysed by hepatic lipase and the particle is converted back to HDL3, completing the HDL3 cycle in plasma. In the liver, cholesterol can either be excreted directly into bile, converted to bile acids, or reutilized in lipoprotein production.\n\nSome of these factors involved in reverse cholesterol transport have common genetic variants. Their associations with plasma lipid levels, CVD risk, and variability in response to diet are described below.\n\n### A. Apolipoprotein A-I (APOA1)\n\nApoA-I, the major protein of HDL, plays several crucial roles in the metabolism of these particles as (1) structural component, (2) activator of the enzyme LCAT [207], and (3) key component of the reverse cholesterol transport process [208]. The gene for apoA-I is clustered with the apoC-III and apoA-IV genes on the long arm of human chromosome 11 [125, 209]. This DNA region has been extensively analyzed, resulting in the identification of about 16 common RFLPs with a heterozygosity value ranging from 0.499 to 0.095. Multiple studies have examined some of these genetic variants in relation to variability in plasma lipids or CVD risk in populations. Several investigators have found statistically significant associations between some of these RFLPs and lipid abnormalities as well as increased CVD risk [127, 128, 210, 211], but others have failed to do so [212].\n\nIn addition to the common variants, more than 20 different rare genetic abnormalities have been reported within this locus (). Some have been associated with severe HDL deficiency and premature coronary atherosclerosis [62, 213, 214]; however, at least one of these mutations shows a protective effect despite its association with low HDL [215, 216].\n\nThe common variant most intensively studied is the one resulting from an adenine (A) to guanine (G) transition (G\/A), 75-bp upstream from the apoA-I gene transcription start site. Several studies have reported that individuals with the A allele, which occurs at a frequency of 0.15\u20130.20 in Caucasian populations, have higher levels of HDL cholesterol than those subjects homozygous for the most common G allele. The magnitude and the gender distribution of this effect have differed among studies. These data are summarized in a recent meta-analysis [216a]. The reasons for these discrepancies are not known. Our own data [217, 218] support the notion that in well controlled dietary studies performed in normolipidemic subjects, the A allele of this G\/A polymorphism appears to be associated with hyperresponse to changes in the amount and saturation of dietary fat. Moreover, other investigators have shown that the significance of the associations may depend on cigarette smoking. Therefore, these data suggest an interaction between this polymorphism and environmental factors such as dietary and smoking habits [217\u2013219].\n\nIt is not clear whether the putative effect of this variant on HDL cholesterol levels is due to the G to A substitution per se, or to linkage disequilibrium between the A locus and a distinct and as yet unidentified effector locus. _In vitro_ analysis of the effects of this polymorphism on transcription has yielded conflicting results. Smith _et al._ [220] reported that the A allele decreased _in vitro_ transcription by 30%, consistent with their own _in vivo_ turnover studies that showed decreased apoA-I synthetic rates in individuals with the A allele, although plasma HDL cholesterol did not differ between GG and GA individuals. Tuteja _et al._ [221] reported that substitution of A for G decreased transcription about two-fold, and Jeenah _et al._ [222] reported a four-fold increase in transcription. Angotti _et al._ [223] reported a five-to sevenfold increase in transcription associated to the A allele, and demonstrated that this may be due to reduced binding affinity of a nuclear factor to the A allele that results in increased transcription efficiency of the apoA-I promoter. More recent research has demonstrated that the induction of apoA-I mRNA level in response to gemfibrozil is mediated at the transcriptional level and this effect is mediated by two copies of the \"drug-responsive element\" in the apoA-I promoter region in the same area as the \u2013 75 G\/A polymorphism. These data suggest that this apoA-I variant may be a functional mutation responsible for some of the individual variability in HDL cholesterol response to gemfibrozyl therapy [224].\n\nIn summary, the mechanisms responsible for the observed effect are still unknown. This mutation may have a direct effect on liver and\/or intestinal apoA1 gene expression as suggested in previous studies or it may be in linkage disequilibrium with a functional mutation in either of the neighboring genes (apoC3 and apoA4). Further studies are needed to clarify these results.\n\n### B. Cholesteryl Ester Transfer Protein\n\nCholesteryl ester transfer protein (CETP) mediates the exchange of neutral lipid core constituents (cholesteryl ester and triglycerides) between plasma lipoproteins. The facilitation of the transfer of cholesteryl ester from HDL to triglyceride-rich lipoproteins results in a reduction in HDL cholesterol levels, but CETP may also promote the reverse cholesterol transport. Therefore, the overall effect of CETP expression on atherogenesis is uncertain. The gene has been located on chromosome 16 adjacent to the LCAT gene (16q21). A common _TaqI_ polymorphism has been identified in intron 1 ( _TaqIB_ ) [225\u2013229]. The presence of the cutting site has been referred to as B1 and its absence as B2. The B2 allele has been shown to be associated with lower lipid transfer activity [230] and higher HDL cholesterol concentrations [231]. Fumeron _et al._ [232] found that alcohol intake modulates the effect of the _Taq_ IB polymorphism on plasma HDL and the risk of myocardial infarction. They found that HDL cholesterol was increased in subjects with the B2B2 genotype only when they ingested at least 25 g alcohol per day. In that study, the cardioprotective effect of the B2B2 CETP genotype was restricted to subjects who consumed the highest amounts of alcohol.\n\nSeveral reports have demonstrated a significant gene-smoking interaction associated with this RFLP, but only one study has examined the relationship between this polymorphism and dietary response. Dullaart _et al._ [233], in a study of patients with insulin-dependent diabetes, demonstrated that the ratio of VLDL cholesterol plus LDL cholesterol to HDL cholesterol fell in response to a linoleic acid-enriched, low-cholesterol diet in B1B1 homozygotes but not in B1B2 heterozygotes.\n\nAn interesting gene\u2013drug interaction has been reported [234] in men with angiographically documented coronary atherosclerosis who were participants in a cholesterol-lowering trial designed to induce the regression of coronary atherosclerosis and were randomly assigned to treatment with either pravastatin or placebo for 2 years. The presence of the _Taq_ IB polymorphism was associated with both higher plasma CETP concentrations and lower HDL cholesterol concentrations. In addition, they observed a significant association between this marker and the progression of coronary atherosclerosis in the placebo group. This association was abolished by pravastatin. Pravastatin therapy slowed the progression of coronary atherosclerosis in those homozygous for this polymorphism but not in those were homozygous for the most common allele. This common DNA variant appeared to predict whether men with coronary artery disease will benefit from treatment with pravastatin to delay the progression of coronary atherosclerosis.\n\nThe evidence of the asociation of the CETP _Taq_ IB polymorphism with HDL cholesterol levels and its interaction with smoking is solid. Similar evidence for gene\u2013diet interaction is beginning to emerge. However, the mechanism is unknown. The _Taq_ IB polymorphism is within the noncoding region of the CETP, thus suggesting that the _Taq_ IB may be in linkage disequilibrium with another polymorphism that may affect CETP activity and, consequently, plasma HDL cholesterol levels.\n\n### C. Hepatic Lipase\n\nHepatic lipase is a lipolytic enzyme that is synthesized in the hepatocytes, secreted, and bound extracellularly to the liver [235]. Hepatic lipase participates in the metabolism of intermediate density lipoproteins and large LDL to form smaller, denser LDL particles and in the conversion of HDL2 to HDL3. In addition, hepatic lipase can mediate the unloading of cholesterol from HDL to the plasma membrane in the liver [236]. It has also been suggested that hepatic lipase may act as a ligand protein with cell surface proteoglycans in the uptake of lipoproteins by cell surface receptors. Lipid profile of individuals with complete hepatic lipase deficiency is characterized by elevated plasma cholesterol and triglyceride levels, triglyceride enrichment of lipoprotein fractions with a density > 1.006 g\/Ml, the presence of a \u03b2-VLDL, and an impaired metabolism of postprandial triglyceride-rich lipoproteins [237, 238]. Four polymorphism in the 5\u2032-flanking region of hepatic lipase gene (G \u2192 A at position \u2013250, C \u2192 T at \u2013514, T \u2192 C at \u2013710, and A \u2192 G at \u2013763), with respect to the transcription site were observed to be in total linkage disequilibrium and were found to be associated with a lowered hepatic lipase activity and higher HDL cholesterol levels. A number of recent studies have also shown an association between low hepatic lipase activity and more buoyant, less atherogenic LDL particles and suggest that variants in the hepatic lipase promoter may contribute significantly to the prevalence of the atherogenic small, dense, LDL particle phenotype associated with an increased CVD risk [239].\n\nIn recent studies, the C\u2013514T polymorphism in the promoter region of the hepatic lipase gene has been shown to be associated with significant variations in hepatic lipase activity, plasma HDL cholesterol levels and LDL particle size [240, 241]. We have investigated the association of this polymorphism to lipoprotein levels in the Framingham Heart Study [242]. Our data show that in men and women, carriers of the \u2013 514T allele had higher HDL cholesterol and ApoA-I concentrations compared with noncarriers. The higher HDL cholesterol levels associated with the \u2013 514T allele were due to an increase in the HDL2 cholesterol subfraction and this association was stronger in women compared with men ( _P_ = 0.0517 vs. 0.0043). We also measured HDL and LDL subclass profiles using automated NMR spectroscopy and gradient gel electrophoresis, respectively. The association of the \u2013 514T allele with higher HDL cholesterol levels seen in men and women was primarily due to significant increases in the large HDL subfractions (size range: 8.8\u201313.0 nm). In contrast, there was no relationship between hepatic lipase polymorphism at position \u2013 514 and LDL particle size distribution after adjustment for the familial relationships, age, body mass index, smoking, alcohol intake, use of beta-blockers, and ApoE genotype, and also menopausal status and estrogen therapy in women. Moreover, multiple regression analyses suggested that the C\u2013514T polymorphism contributes significantly to the variability of HDL particle size in men and women ( _P_ < 0.04). Thus, our results show that the C\u2013514T polymorphism in the hepatic lipase gene is associated with significant variations in the lipoprotein profile in men and women.\n\n### D. Cholesterol 7 Alpha-Hydroxylase\n\nThe first reaction of the catabolic pathway of cholesterol is catalyzed by _CYP7_ and serves as the rate-limiting step and major site of regulation of bile acid synthesis in the liver. The cholesterol catabolic pathway, exclusive to the liver, is comprised of several enzymes, which convert cholesterol into bile acids. Previous studies show that _CYP7_ is regulated by bile acid feedback, cholesterol, and hormonal factors [243]. In addition, dietary fats modulate the regulatory potential of dietary cholesterol on _CYP7_ gene expression [244]. The human _CYP7_ has been localized to chromosome 8q11-q12 [245]. A common A-to-C substitution at position \u2013 204 of the promoter of the _CYP7_ gene is associated with variations in plasma LDL cholesterol [246]. Moreover, the study of the molecular mechanisms of the transcriptional regulation of _CYP7_ by sterols and bile acids has revealed that the promoter region between \u2013 432 and \u2013 220 contains several cell-specific enhancer elements whose activity is controlled, in part, by hepatocyte nuclear factor 3 [247]. Therefore, it is conceivable that the A\u2013204C polymorphism might modulate transcription of the _CYP7_ gene and, consequently, the rate of cholesterol catabolism. The balance between the rates of hepatic sterol acquisition and excretion largely determines whether an individual is a hypo- or hyperresponder to dietary cholesterol. Therefore, even though there is no published evidence of the involvement of this locus in dietary response, the current evidence suggests that this should be examined in future studies [248].\n\n### E. Scavenger Receptor B Type I\n\nScavenger receptor B type I (SRBI) is a multilipoprotein receptor found in the liver and steroidogenic glands of mice [249] and humans [250\u2013252]. The cDNA for human SRBI [also known as LIMPII analagous 1 (CLA-1)] was originally cloned by homology to human CD36 and rat lysosomal integral membrane protein II (LIMPII), members of a family of transmembrane proteins [253]. Another study identified the hamster homolog by its ability to mediate the binding of modified LDL, and it was also shown to bind native LDL [254]. Subsequently, murine SRBI was shown to mediate the uptake of lipid, but not apoprotein, from HDL into cells [255], a process described as selective uptake [256]. This finding established SRBI as the first HDL transmembrane receptor to be identified and cloned. Further studies of the human homolog demonstrated that it also is a multilipoprotein receptor that binds HDL, LDL, and VLDL [250, 257]. Preliminary evidence from our studies indicates that the SRBI gene plays a significant role in determining lipid concentrations and body mass index [258]. Moreover, the observation that SRBI mediates absorption of dietary cholesterol in the intestine [259] suggests that it may also play a role in the postprandial response and insulin metabolism. Our preliminary data regarding association of variants at this locus with lipid levels and anthropometric variables, as well as the differential gender effects, warrant a more detailed study of this locus.\n\n### F. ATP-Binding Cassette 1\n\nFor several decades the elucidation of the reverse pathway involving the cholesterol efflux from cells and its transport and internalization by the liver has been intensively studied. However, these mechanisms have been poorly understood. The identification of SRBI as an HDL receptor was a step forward in our understanding of reverse cholesterol transport. However the role of this receptor within the scheme of human lipoprotein metabolism is still unknown. We may have reached a breaking point in our understanding of reverse cholesterol transport with the elucidation of a biological mystery that began almost four decades ago in the small Tangier Island located in Chesapeake Bay in the United States. Two brothers living on the island presented with orange tonsils, peripheral neuropathy and, more interesting for the present focus, deficiency of HDL cholesterol. Scores of investigators have studied families affected with this rare disease with the notion that a better understanding of this disease could provide the key to the molecular basis of reverse cholesterol transport, therefore providing with new mechanistic tools to prevent the development of atherosclerosis and the treatment of CVD.\n\nPatients with Tangier disease are characterized by their almost complete absence of HDL and they accumulate cholesteryl esters in tissues, resulting in enlarged orange tonsils, hepatosplenomegaly, peripheral neuropathy, and deposits in the rectal mucosa [260]. After 40 years of research, several groups have reported, almost simultaneously, the gene locus and several specific mutations responsible for Tangier disease [261\u2013263, 265] and for some forms of familial hypoalphalipoproteinemia [266]. These investigations clearly demonstrate that mutations in the ATP-binding cassette transporter 1 (ABC1) gene are responsible for the Tangier phenotype in several kindred from a variety of ethnic backgrounds. ABC1 belongs to the ABC family of genes. These genes encode for proteins involved in vectorial movement of substrates across biological membranes. The ABC1 transporter consists of two symmetric halves, each including six membrane-spanning domains and a nucleotide-binding fold. Moreover, a long charged region and a highly hydrophobic segment link these two halves of the molecule. The transporter activity depends on its interaction with ATP. This specific transporter appears to be involved in free cholesterol transport (and perhaps phospholipids), and the alteration of this important cellular function results in impaired efflux of free cellular cholesterol and onto HDL particles, thus preventing the formation of normal nascent HDL particles. Consequently, the lipid-poor HDL is quickly removed from the circulation without being able to accomplish its function. This abnormality clearly prevents normal reverse cholesterol transport and patients with Tangier disease tend to develop premature CVD, despite relatively lowlevels of LDL cholesterol.\n\nIt is important to remark that all these reports have found different mutations in the different kindred studied. These data support the concept that Tangier disease may be very heterogeneous, a situation similar to that of familial hypercholesterolemia, where several hundred mutations have been reported for the LDL receptor locus. This may also explain the heterogeneous phenotypic expression of this disease, with pedigrees showing a high incidence of premature CVD, whereas in other families, the risk does not appear to be much higher than in normal subjects.\n\nThese findings point toward a new physiological mechanism controlling cellular cholesterol metabolism and its efflux to HDL. However, given the rarity of Tangier disease, mutations at the ABC1 gene could have very limited impact in modulating HDL levels in the population at large. However, the findings reported by Marcil _et al._ [266] are of great relevance, because they demonstrate that the ABC1 locus is also responsible for a much more common HDL deficiency known as familial hypoalphalipoproteinemia that shares with Tangier's disease the low levels of HDL cholesterol but not some of the other phenotypic characteristics (such as neuropathy and orange tonsils). Familial hypoalphalipoproteinemia is one of the most common genetic abnormalities in subjects with premature CVD [267].\n\nThe identification of ABC1 as the gene for Tangier disease and for some cases of familial hypoalphalipoproteinemia opens enormous opportunities for the lipoprotein field and for CVD therapy. The metabolic pathways determined by the product of the ABC1 gene will contribute to our understanding of reverse cholesterol transport and its regulation by dietary fat and cholesterol. It remains to be elucidated whether common mutations at this locus are associated with altered HDL cholesterol levels in the population. This research is being carried out by several groups and it will determine the full impact of the ABC1 in controlling cholesterol efflux and HDL metabolism in the general population, beyond those affected by Tangier disease or familial hypoalphalipoproteinemia. This information will be applied to the genetic screening of subjects at high risk for CVD and it could provide us with new therapeutic targets thus increasing our ability to prevent and treat CVD.\n\n## V. CONCLUSION\n\nThe mechanisms involved in the regulation of plasma lipoprotein levels by dietary factors, such as intakes of cholesterol and fatty acids, have been partially elucidated during the past century, thanks to the research efforts of scores of investigators. This research has also brought up a number of candidate genes that regulate the homeostasis of blood lipids. Considering the significant effect of diet on plasma lipids, genetic variation at those loci is expected explain some of the dramatic interindividual variations in lipoprotein response to dietary change that have been shown to exist among individuals. In fact, the current evidence supports the importance of gene\u2013diet interactions in humans. Multiple candidate genes have been examined under different experimental conditions and the major findings have been highlighted in this chapter. However, because of conflicting results, more studies will be required to increase our predictive capacity and to reconcile the multiple discordances found in the literature.\n\nAt this regard, we have to keep in mind that lipoprotein response to dietary factors is extremely complex, as illustrated by the multiple interrelated pathways and genes discussed herein. Therefore, the effects of individual gene variants can be difficult to identify. In fact, it is quite possible that the concerted action of differences in \"gene families\" may be required to elicit significant interindividual differences in responsiveness to diet. Moreover, until the information generated by the Human Genome Project is available and analyzed, we currently have an incomplete list of the genes potentially involved in these processes.\n\nWe should also be cautious concerning the interpretation of studies of association between allelic variants and common phenotypes [268]. We should direct attention to the population admixture, which can cause an artificial association if a study includes genetically distinct subpopulations, one of which coincidentally displays a higher frequency of disease and allelic variants. Consideration of the ethnic backgrounds of subjects and the use of multiple, independent populations can help avoid this problem. The most robust tests involve family-based controls such as the transmission disequilibrium test. In this case, if a given allele contributes to disease, then the probability that an affected person has inherited the allele from a heterozygous parent should vary from the expected Mendelian ratio of 50:50; the association of a neutral polymorphism due to admixture displays no such deviation.\n\nAnother source of concern is multiple-hypothesis testing, which is aggravated by publication bias. Authors who test a single genetic variant for an association with a single phenotype base statistical thresholds for significance on a single hypothesis. But many laboratories search for associations using different variants. Each test represents an independent hypothesis, but only positive results are reported, leading to an overestimate of the significance of any positive associations. Statistical correction for multiple testing is possible, but the application of such thresholds results in loss of statistical power.\n\nAn additional caveat about the published literature relates to the fact that most studies were not initially designed to examine gene\u2013diet interactions, but they are reanalysis of previously obtained data using new information from genetic analysis carried out _a posteriori._ Future studies need to be carefully designed in terms of sample size, taking into consideration the frequencies of the alleles examined. Moreover, we do not really know yet the specific dietary factors responsible for most of the effects already reported. Therefore, baseline and intervention diets should be carefully controlled in terms of dietary cholesterol, individual fatty acids, levels of fat, as well as fiber and other minor components of the diet such as phytosterols. It is also important to emphasize that some allele effects may be apparent primarily during situations of metabolic \"stress,\" such as the postprandial state. Therefore, studies should be designed to test gene\u2013diet interactions, both in the fasting and fed states. As indicated above, the most plausible scenario is that multiple genes will determine the response to dietary manipulation. Consequently, attention should be paid to gene\u2013gene interactions. However, the large number of study subjects required and subsequent costs involved may make such studies infeasible. Two alternatives to examine these complex interactions in humans are possible: One would be to select study participants based on their genetic variants; the second would be to make use of the large cohort studies for which dietary information has been collected. The latter approach would take the concept of gene\u2013diet interactions beyond the metabolic unit into the real world.\n\nThe use of animal models will play a crucial role for mapping new genes involved in dietary responsiveness and atherogenesis. Furthermore, future studies will need to include siblings and families with the dual purpose of getting a more accurate measure of heritability and the performance of wide genome scans to search for new responsiveness loci.\n\n## Acknowledgments\n\nThis work was supported by grant HL54776 from the NHLBI and contract 53-K06\u20135\u201310 from the U.S. Department of Agriculture Research Service.\n\nReferences\n\n1. Blankenhorn D.H., Johnson R.L., Mack W.J., el Zein H.A., Vailas L.I. The influence of diet on the appearance of new lesions in human coronary arteries. _JAMA_. 1990;263:1646\u20131652.\n\n2. Scandinavian Simvastatin Survival Study Group. 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INTRODUCTION\n\nComplex and incompletely defined interactions between environment and genetics determine each individual's height and weight, as well as many other human traits. The result is a population in which individuals vary widely for height and weight, but no one factor can be identified as controlling either trait. In humans, long-term adult weight is relatively stable, as evidenced by the difficulty of sustaining intentional weight loss and the automatic return to previous weight following brief periods of overeating. This drive to constancy of body weight is due to both behavioral and physiological alterations that accompany weight change.\n\nFurther convincing evidence of the biological basis of the regulation of body fat stores comes from the identification of single-gene mutations that result in spontaneous massive obesity or in adipose tissue atrophy. There are also Mendelian disorders in which obesity or abnormalities of fat distribution are a prominent feature and for which the chromosomal locations, but not the genes or their functions, are known.\n\nMost human obesity, however, is not due to mutations in single genes but exhibits a complex, non-Mendelian inheritance. Obesity is usually dependent on a permissive environment. Fat deposition can occur uniformly or there can be preferential deposition of fat, for example, in the abdominal area. There are also likely to be interactions among genes such that some alleles of one gene will not cause obesity unless specific alleles of another gene are present. Although animal models clearly demonstrate that gene\u2013Sgene interactions are common and can have substantial effects on many traits, technical difficulties have made it more difficult to identify such interactions in human studies. Expression of an obesity gene may also be age or gender dependent. Thus, identification of all the genes promoting human obesity is not a trivial task.\n\nGenetics is a rapidly progressing field, and knowledge of the genetic basis for obesity is expanding exponentially. Therefore, the reader should use this chapter only as the starting point for an understanding of this exciting body of knowledge.\n\n## II. GENETIC EPIDEMIOLOGY OF HUMAN OBESITY\n\nGenetic epidemiology of human obesity is the study of the relationships of the various factors determining the frequency and distribution of obesity in the population. Such studies of obesity are limited in that they do not examine DNA and rarely directly measure the amount or location of body fat. However, genetic epidemiology studies do provide information as to whether there is a genetic basis for the trait, whether inheritance is maternal or paternal, and whether expression of the trait is gender or age dependent.\n\nGenetic epidemiology studies of human obesity employ a variety of designs and statistical methods, each giving somewhat different heritability estimates for obesity. For a discussion of genetic epidemiology methods employed in the study of obesity, see Bouchard _et al._ [1].\n\nThe heritability estimates for human obesity are derived from a large number of studies of adoptees, twins, and families. Heritability of human obesity may be as low as 10%, as estimated from some adoption studies, or as high as 80%, as estimated from some twin studies [1] (Table 1). Two studies that incorporated twins, adoptees, and nuclear families into the analyses yielded heritability estimates for obesity of approximately 25\u201340%. These studies indicate that familial environment has only a minor impact on obesity.\n\nTABLE 1\n\nOverview of the Genetic Epidemiology of Human Body Fat\/Obesitya\n\naBased on the trends in about 50 different studies. In most of the studies, the BMI was the phenotype considered. In some cases, skinfolds or estimates of percentage body fat or fat mass were used.\n\nbMaternal or paternal effect indicates whether transmission through mother or father alters heritability.\n\n_Source:_ Reprinted from Bouchard, C., P\u00e9russe, L., Rice, T., and Rao, D. C. (1998). The genetics of human obesity. _In_ \"Handbook of Obesity\" (G. A. Bray, C. Bouchard, and W. P. T. James, Eds.), p. 166, by courtesy of Marcel Dekker, Inc., New York.\n\nThe number of genes involved in human obesity has not been estimated, primarily because such estimates are complicated by the conclusion that genes implicated in obesity have major, minor, and polygenic effects. A major gene is a single gene that has a large effect on the phenotype. Polygenic effects are due to many genes, each with a small effect on the phenotype. Segregation analyses1 indicate that the percentage of minor gene transmission ranges from 25 to 42% and that there is a single major gene for high body mass segregating from the parents to their offspring. These data do not mean that there is only one major gene contributing to obesity in humans. Rather, the specific obesity gene may vary from person to person, such that there may be several major obesity genes in the entire population. However, no major obesity genes have been identified in any population.\n\n## III. GENE\u2013ENVIRONMENT INTERACTIONS\n\nWhy some people in modern societies become obese, despite considerable effort and expense to avoid this condition, whereas others stay lean without such effort, appears to have a genetic basis [2]. The chronic overfeeding studies by Sims and colleagues beginning in the 1960s showed interindividual differences in weight gain [3, 4]. More recently, Bouchard and colleagues determined the response to changes in energy balance by submitting pairs of monozygotic twins either to positive energy balance induced by overeating or to negative energy balance induced by exercise training. During 100 days of overfeeding by 1000 kcal\/day, significant intrapair resemblance was observed for changes in body composition and was particularly striking for changes in regional fat distribution and amount of visceral fat, with six times as much variance among as within twin pairs [5]. During long-term energy deficit induced by exercise training, intrapair resemblance was observed for changes in body weight, fat mass, percent fat, and abdominal visceral fat [6]. One explanation for these differences is that some twin pairs were found to be better oxidizers of lipid, as evidenced by reduced respiratory quotient, during submaximal work than were other twin pairs [6].\n\nAn important component of the interindividual difference in response to overeating may be individual differences in spontaneous physical activity or ''fidgeting\" [7, 8]. A large portion of the variability in total daily energy expenditure, independent of lean body mass, is due to fidgeting, which varies by more the sevenfold among subjects [7], is a familial trait, and is a predictor of future weight gain [9]. In a very elegant study of the fate of excess energy during overfeeding, Levine _et al._ [8] again demonstrated the considerable interindividual variation in susceptibility to weight gain. Two-thirds of the increases in total daily energy expenditure in nonobese subjects overfed by 1000 kcal\/day for 8 weeks was due to increased spontaneous physical activity associated with fidgeting, maintenance of posture, and other daily activities of life independent of volitional exercise [8].\n\n## IV. THE OBESITY GENE MAP\n\nA genetic map is a representation of the distribution of a set of genetic loci or markers. (For a discussion of genetic maps, see [10].) The three types of genetic maps are linkage, chromosomal, and physical maps. Genetic maps provide many kinds of information, from overall chromosomal views to more detailed molecular information, but all genetic maps place items (usually genes or clones) in an order, from top to bottom or left to right.\n\nThe Human Obesity Gene Map 11] incorporates information from all three types of maps\u2014linkage, chromosomal, and physical. The map and its associated summary provide an overview of the data reported in peer-reviewed journals on human obesity genes and markers as well as published evidence from rodent obesity models. Five types of data were used to generate the map, all of which are represented in Fig. 1: (1) single-gene obesity mutations, e.g., _POMC_ ; (2) Mendelian disorders exhibiting obesity as a clinical feature, e.g., _ALMS1_ ; (3) quantitative trait loci (QTLs)2 identified in linkage studies in animals, e.g., _Mob 6_ and _Pfat1,_ and in humans; (4) linkage studies of candidate genes in humans, e.g., _ACP1_ ; and (5) association studies of candidate genes, e.g., also _ACP1._ The most up-to-date compilation of obesity gene information, covering all 22 autosomal chromosomes as well as the X and Y chromosomes, is available in the Human Obesity Gene Map, published each January in the journal _Obesity Research._ The same information is available on-line at [http:\/\/www.obesity.chair.ulaval. ca\/genes.html. The current update [11] of the map includes many obesity-related phenotypes, including body mass index, percent body fat, fat mass, skinfolds, abdominal fat, macronutrient intake, metabolic rate, energy expenditure, and fat-free mass.\n\nFIGURE 1 Chromosome 2 demonstrates each of the types of putative obesity loci found in the Human Obesity Gene Map. Mutation of the _POMC_ gene causes single-gene obesity in humans. Mutations of _ALMS_ and _BBS5_ cause the rare Mendelian syndromes, Alstrom syndrome and Bardet-Beidl-5 syndrome. _IRS1,_ the insulin receptor substrate-1, is a candidate gene for type 2 diabetes, obesity, and hyperinsulinemia in humans. _D2S165_ is an anonymous marker linked to obesity in humans and _Obq3_ is a QTL linked to adiposity in a mouse model. [Reprinted with permission from Chagnon, Y. C., P\u00e9russe, L., Weisnagel, S. J., Rankinen, T., and Bouchard, C. (2000). The Human Obesity Gene Map: The 1999 update. _Obes. Res._ **8,** 89\u2013117.]\n\n## V. SINGLE-GENE OBESITY IN HUMANS\n\nCloning of the rodent obesity genes,3 _Lep ob, Leprdb, Cpefat, Tub,_ and _A y,_ between 1992 and 1996 led to an explosion of knowledge, summarized below, of the genetic causes of obesity. In addition to severe obesity, most of these models are characterized by insulin resistance and infertility. The function of only one of these rodent obesity genes, _Tub,_ remains unknown. The human gene, _TUB,_ 4 located at 11p15.5, encodes a novel protein of unknown function, the C terminus of which is highly conserved across species. Recent papers provide hints of _Tub_ function. One reports that _Tub_ codes for a protein that functions in intracellular signaling by insulin [12]. The other shows that _Tub_ binds to DNA and may thus influence transcription [13]. To date, no studies implicate _TUB_ in human obesity or insulin resistance.\n\nObesity in these rodent models exhibits Mendelian segregation, indicating that the obesity is inherited as a single-gene mutation. With one exception, these mutations result in the loss of gene or protein function and are expressed only when both copies of the gene in an individual are defective. Therefore, obesity due to these mutations in humans would not be common. Although most human obesity is not believed to be due to a single-gene defect but exhibits a complex, non-Mendelian inheritance, these genes are of great interest, since subtle mutations may contribute to common forms of obesity. Also, study of these genes has identified many new pathways for investigation of the physiology of obesity and provided many therapeutic targets for newan-tiobesity drugs [14].\n\nAs of early 2000, five genes have been shown to cause spontaneous Mendelian obesity in humans: leptin (gene abbreviation, _LEP_ ), leptin receptor ( _LEPR_ ), proopiomelanocortin ( _POMC_ ), melanocortin-4 receptor ( _MC4R_ ), and proprotein convertase subtilisin\/kexin type 1 ( _PCSK1_ ) (Table 2).\n\nTABLE 2\n\nGenes Known to Cause Spontaneous Mendelian Obesity in Humans\n\n### A. Leptin Deficiency\n\nCloning and characterization of the mouse _Lep ob_ gene identified its protein product, leptin, a hormone that is secreted from adipose tissue [15] and to a lesser extent from placenta [16] and gastric epithelium [17]. Leptin circulates in the blood [18], crosses the blood\u2013brain barrier [19], and binds to its receptor in the hypothalamus to regulate food intake and energy expenditure. Thus, leptin functions as an afferent signal in a negative feedback loop to maintain constancy of body fat stores. Because leptin is expressed in, and secreted from, adipose tissue, circulating levels of leptin closely match the total amount of fat stores [20] and decrease with weight loss [21]. Peripheral or central administration of leptin reduces food intake and body fat in several mouse models of obesity (those with a functional leptin receptor), while preserving lean tissue mass, although relatively high doses are required in certain models [18, 22\u201324]. A recent clinical trial indicates that the same is true for both lean and obese humans treated with recombinant leptin [25].\n\nLeptin clearly has a broader physiological role than just the regulation of body fat stores. Leptin deficiency results in many of the abnormalities seen in starvation, including reduced body temperature, reduced activity, decreased immune function, and infertility. Leptin levels, for equivalent body fat mass, are higher in women than in men [20]. In prepubertal females, leptin levels are highly correlated with body fat, and the increase in serum leptin is associated with a younger age of menarche [26]. Leptin reverses the starvation-induced suppression of T cells even in the presence of continued energy deficit [27]. For a reviewof the physiological role of leptin see Friedman and Halaas [28].\n\nKnown mutations in leptin (Table 2) causing spontaneous massive obesity in humans are autosomal recessive and are rare in the population.5 However, two highly consanguineous families were identified that carry mutations in _LEP._ Two severely obese children of one family have very low serum leptin levels despite massive obesity [29]. One of the children weighed 86 kg at the age of 8 years, with 57% body fat. The other child weighed 29 kg at the age of 2 years. Both children had normal birth weight but were markedly hyperphagic and gained weight rapidly in the early postnatal period. Four massively obese members of another family are homozygous for a different mutation in _LEP_ and have very lowleptin levels. Three of these individuals are adults: The females have primary amenorrhea and the male never entered puberty. Seven obese members of this pedigree, presumably also carrying the mutation in _LEP,_ died of infectious diseases during childhood (no normal weight family members have died). All others in the family are either heterozygous for the mutation or homozygous for the wild-type allele and have normal body weight and serum leptin levels [30, 31]. Recombinant leptin therapy in doses sufficient to raise serum concentrations of leptin to the normal range in a 9-year-old girl with congenital leptin deficiency corrected many aspects of the obese phenotype. Over a 12-month period, this patient, with a baseline weight of 94 kg, lost 16 kg, primarily as fat mass [32].\n\n### B. Leptin Receptor Deficiency\n\nLeptin acts through the leptin receptor, a single-transmemSbrane-domain receptor of the cytokine-receptor family [33]. The leptin receptor is found in many tissues in several alternatively spliced forms, raising the possibility that leptin affects many tissues in addition to the hypothalamus [34]. For additional discussion of the leptin receptor, see [28].\n\nAn autosomal recessive mutation in the human leptin receptor gene ( _LEPR_ ) that results in a truncated leptin receptor was discovered in homozygosity in a consanguineous family. Three of nine siblings had severe hyperphagia and developed early morbid obesity despite normal birth weight [35]. Individuals homozygous for this mutation have no pubertal development and their secretion of growth hormone and thyrotropin is reduced. This phenotype is similar to that seen in individuals with mutation of the leptin gene.\n\n### C. Proopiomelanocortin Deficiency\n\nSequential cleavage of the precursor protein proopiomelanocortin (POMC) generates the melanocortin peptides adreno-corticotrophin (ACTH), the melanocyte-stimulating hormones (\u03b1-and \u03b2-MSH), and the opioid-receptor ligand beta-endorphin. \u03b1-MSH plays a central role in the regulation of food intake by the activation of the brain melanocortin-4 receptor (Section V. E) [36]. The dual role of \u03b1-MSH in regulating food intake and influencing hair pigmentation predicts that the phenotype associated with a defect in _POMC_ function would include obesity, alteration in pigmentation, and ACTH deficiency. The observations of these symptoms in two probands6 led to the identification of three separate mutations within their _POMC_ genes [37] (Fig. 2, see color plate at the back of the book). One individual is a compound heterozygote7 for two mutations that interfere with appropriate synthesis of ACTH and \u03b1-MSH. The other patient is homozygous for a mutation that abolishes _POMC_ translation. These findings define a new monogenic endocrine disorder resulting in early-onset obesity, adrenal insufficiency, and red hair pigmentation.\n\nFIGURE 2 Photo of 5-year-old boy with early-onset obesity, adrenal insufficiency, and red hair caused by mutation of the _POMC_ gene. This patient had early hypoglycemia and hyponatriaemia due to ACTH deficiency. Birth weight was normal but, due to hyperphagia, obesity was apparent by 5 months of age. [Reprinted with permission from Krude, H., Biebermann, H., Luck, W., Horn, R., Brabant, G., and Grtlters, A. (1998). Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by _POMC_ mutations in humans. _Nature Genet._ **19,** 155\u2013157.]\n\n### D. Proprotein Convertase Subtilisin\/Kexin Type 1 Deficiency\n\nA wide variety of hormones, enzymes, and receptors are initially synthesized as large inactive precursors. To release the active hormone, enzyme, or receptor, these precursors must undergo limited proteolysis by specific convertases. Examples are the conversion of proinsulin to insulin by the combined actions of proprotein convertase 1 and 2 and the clipping of _POMC_ by proprotein convertase 1. A recessive mutation of carboxypeptidase E, an enzyme active in the processing and sorting of prohormones, causes obesity in the _Cpe fat_ mouse. A mutation in a homologous enzyme was found in a woman with extreme childhood obesity, abnormal glucose homeostasis, hypogonadotrophic hypogonadism, hypocortisolism, and elevated proinsulin and proopiomelanocortin concentrations but a very low insulin level [38]. This woman is a compound heterozygote for mutations in proprotein convertase subtilisin\/kexin type 1 (PCSK1; also known as prohormone convertase-1), which acts proximally to carboxypeptidase E in the pathway of post-translational processing of prohormones and neuropeptides. Since the proband and the _fat_ mouse share similar phenotypes, it can be inferred that molecular defects in prohormone conversion represent a generic mechanism for obesity.\n\n### E. Mutation in the Melanocortin-4 Receptor Gene\n\nThe agouti protein, identified in the yellow obese ( _A y_) mouse, inhibits binding of \u03b1-MSH to melanocortin receptors, including Mc4r, which is located in the hypothalamus, and Mc1r, which is located in the skin. Obesity and yellow coat color in the _A y_ mouse result from expressing agouti in all tissues, not just in skin, which is the normal condition. The melanocortin-4 receptor, a G-protein-coupled receptor, is highly expressed in the hypothalamus, a region of the brain intimately involved in appetite regulation. It is a receptor for \u03b1-MSH, a product of the _POMC_ gene (Section V.C), which inhibits feeding. Inactivation of _Mc4r_ by gene targeting in mice results in a maturity-onset obesity syndrome associated with hyperphagia and impaired glucose tolerance [39]. Mc4r-deficient mice do not respond to a \u03b1-MSH-like agonist, suggesting that \u03b1-MSH inhibits feeding primarily by activating Mc4r [36]. Mice heterozygous for a null _Mc4r_ allele exhibit phenotypes intermediate to that seen in wild-type and homozygous littermates [36].\n\nIn screening children that were severely obese from an early age, mutations in _MC4R_ resulting in haploinsufficiency8 as well as several missense9 mutations were identified [40\u201343]. The functional significance of the missense mutations [42] is uncertain. The haploinsufficiency mutations were present in the heterozygous state, and other members of each family were obese in a pattern consistent with autosomal dominant inheritance. Adrenal function is not impaired in the MC4R-deficient subjects. Sexual development and fertility are normal. Affected subjects are tall, similar to the increased linear growth that occurs in heterozygous Mc4r-deficient mice. Female haploinsufficiency carriers are heavier then male carriers in their families, a pattern also seen in Mc4r-deficient mice. These data are strong evidence for dominantly inherited obesity, not associated with infertility, due to haploinsufficiency mutations in _MC4R._\n\n## VI. SINGLE-GENE MUTATIONS RESULTING IN ADIPOSE TISSUE ATROPHY\n\nThe lipodystrophies are characterized by the absence or reduction of subcutaneous adipose tissue. Patients with familial partial lipodystrophy (FPLD) are born with normal fat distribution, but close to puberty, they experience regional adipose tissue atrophy that is often associated with insulin resistance, diabetes, and hyperlipidemia. The gene coding FPLD was mapped to 1q21. 2-q21. 3 and found to be _LMNA,_ which codes for a polypeptide, lamin A, found in the nuclear lamina of the cell. Three different amino acid substitutions at one position in the lamin A polypeptide that result in heritable partial lipodystrophy were identified in a dozen families [44, 45]. Different mutations in _LMNA_ result in specific muscular dystrophy and cardiomyopathy disorders as well.\n\n## VII. RARE GENETIC SYNDROMES WITH OBESITY AS A PROMINENT FEATURE\n\nThere are at least 26 rare Mendelian syndromes, in which obesity is a prominent clinical feature, described in the Online Mendelian Interitance in Man (OMIM) database10 [46]. Among the better known of these syndromes are the Prader-Willi syndrome, the Bardet-Biedl syndromes, and Alstrom syndrome. Currently, the pathophysiologic mechanisms leading to obesity in these syndromes are not known. The identification of the underlying genes will likely help define the mechanisms controlling appetite, satiety, and obesity. The characteristic features of these syndromes were recently reviewed [47].\n\nWith an incidence of about 1 in 25, 000 births, the most common of the Mendelian syndromes is the Prader-Willi syndrome, which results from a microdeletion of paternal chromosome 15q11-q13 or, more rarely, as a result of maternal disomy11 of chromosome 15. In addition to obesity, the Prader-Willi syndrome is characterized by hypotonic musculature, mental retardation, hypogonadism, short stature, and small hands and feet (for review, see [48, 49]). Aberrant behavior, including hyperphagia and aggressive food seeking, makes management of these patients difficult.\n\nAutosomal recessive Mendelian obesity syndromes include the Bardet-Biedl syndromes (BBS1\u2013BBS5) and the Alstrom syndrome (ALMS1). The Bardet-Biedl syndromes are associated with variants on chromosomes 11q13 (BBS1), 16q21 (BBS2), 3p13-p12 (BBS3), 15q22. 3\u201323 (BBS4), and 2q31 (BBS5). The Bardet-Biedl syndromes are all characterized by mental retardation, pigmentary retinopathy, polydactyly, obesity, and hypogonadism. It is, therefore, apparent that mutation in at least five separate genes can result in the same phenotype. The gene on chromosome 16 coding for BBS2 may predispose males carrying only one copy of the gene to obesity and may explain approximately 3% of severely overweight males [50]. Of interest, BBS2 parents of both sexes were significantly taller than U. S. individuals of comparable age [50].\n\nIn addition to obesity, Alstrom syndrome is characterized by retinitis pigmentosa leading to blindness, insulin resistance, diabetes, and deafness, but does not involve mental retardation or polydactyly (for review, see [51, 52]). Onset of obesity is usually between 2 and 10 years of age and can range from mild to severe. The gene for Alstrom syndrome (ALMS1) was narrowed down to a small region on chromosome 2p13-p12 [53].\n\n## VIII. EVIDENCE FROM LINKAGE STUDIES OF OBESITY PHENOTYPES\n\nA. Mapping of Loci in Animals\n\nAnimal models have been very important in the dissection of complex traits [54]. Quantitative trait locus mapping is a method for mapping Mendelian factors that underlie complex traits, in virtually any animal model, by using genetic linkage maps12 (for discussion, see [10, 54]). As of October 1999, 98 animal QTLs were linked variously to body weight, body fat, energy expenditure, food intake, leptin levels, or weight gain [11]. A number of these QTLs identified in separate crossbreeding experiments of different strains are overlapping and it is likely that the same underlying gene is responsible for these overlapping QTLs. Many of these QTLs have pleiotropic effects.13\n\n### B. From Mouse to Human\n\nQTLs are valuable for identifying candidate genes to be further evaluated by gene targeting experiments in mice or by linkage studies or association studies of the candidate genes in humans. Because of the evolutionary relationship between mice and humans, many ancestral chromosomal segments have been retained where the same genes occur in the same order within discrete regions of chromosome (homology)14 [55]. These regions of homology may include many hundreds to thousands of genes in the same orders, although some regions of homology are made more complex by chromosomal rearrangements within the region of homology. Because regions of homology between mouse and human chromosomes are well defined, the identification of a gene in the mouse frequently gives the chromosomal location of the same gene in the human. This relationship was used to map a gene for obesity to human chromosome 20 [56]. To test whether an obesity QTL on mouse chromosome 2 contributes to human obesity, linkage analysis between markers located within the homologous region on chromosome 20 and measures of obesity was performed in a large study of more than 150 French Canadian families; a locus on 20q13 that contributes to body fat and fasting insulin was found [56]. This locus was later confirmed in a second population group [57]. A polymorphism within this region in the candidate gene, adenosine deaminase ( _ADA_ ), was recently associated with obesity in a population of subjects with non-insulin-dependent diabetes (NIDDM) [58] (Section IX.B).\n\n### C. Linkage Studies in Humans\n\nLinkage studies in humans are conducted with large extended families or with nuclear families. A conceptually simple and practical method is the nonparametric sib-pair linkage method that provides statistical evidence of linkage between a quantitative phenotype and a genetic marker [1, 59]. The method is based on the concept that siblings who share a greater number of alleles (1 or 2) identical by descent15 at a linked marker locus should also share more alleles at the phenotypic locus of interest and should be phenotypically more similar than siblings who share fewer marker alleles (0 or 1). The method has been expanded to use data from multiple markers, allowing higher resolution mapping [60]. Linkage studies do not identify any specific gene but are useful in identifying candidate genes for further study.\n\nA number of whole genome scans and linkage studies covering smaller chromosomal regions, published as of October 1999, identified 56 QTLs for various measures of adiposity, respiratory quotient, metabolic rate, and plasma leptin levels in humans (for details, see [11]). Many of these chromosomal loci contain candidate genes for obesity, including genes known to cause single-gene obesity (Section V). Linkage studies suggest that the _LEP_ gene or a gene very near it on 7q31. 3 contributes to obesity in several different populations although the monogenic syndrome of leptin deficiency is rare [61\u201365]. One group linked both the _LEPR_ [66] and _MC4R_ [67] genes to multigenic obesity-related phenotypes in French Canadians. Candidate genes first identified through linkage studies include the adrenergic receptors [68, 69], _UCP2\/UCP3_ [70], and _ADA_ [56].\n\n## IX. ASSOCIATION STUDIES OF CANDIDATE GENES FOR OBESITY AND OBESITY-RELATED INSULIN RESISTANCE AND HYPERLIPIDEMIA\n\nAssociation studies examine the correlation of a genetic variant (polymorphism) within a gene with the phenotype of interest. It is assumed that variants within a gene's coding region alter gene function, although proof of that requires gene\u2013targeting experiments in cell or animal models. Association studies are generally carried out in unrelated individuals and are frequently designed as case-control studies. Although case-control studies remain a powerful tool in some areas, they are less powerful for genetic studies due to methodological issues that complicate analyses in complex populations such as those found in the United States. Therefore, most association studies are nowconducted in isolated populations, such as occur in Finland and Quebec. A positional candidate gene is identified both by its location in a chromosomal region that has significant linkage to obesity in family studies and because its biological functions are generally consistent with a role in body weight regulation. Forty candidate genes have been associated with varying degrees of confidence with obesity phenotypes to date [11]. The complete physical map of the human genome should be available by 2003 providing many new candidate genes for investigation.\n\n### A. Role of Single-Gene Obesity in Common Forms of Human Obesity\n\nSeveral of the genes causing spontaneous massive obesity in humans have been implicated in polygenic obesity using association studies. The _LEP_ gene is associated with body weight, weight loss, or leptin levels [71\u201373]. The _LEPR_ gene is associated with obesity in three studies, including one of severe obesity in children [66, 74, 75]. Populations from the Pacific Island of Nauru have some of the highest rates of obesity and NIDDM in the world. In Nauruan males, specific combinations of alleles in the _LEP_ and _LEPR_ genes are associated with increased risk for development of insulin resistance [76]. _POMC_ is associated with variation in leptin levels [77]. _MC4R_ is associated with fat mass in one study [67]. These data suggest that mutations of the genes causing Mendelian obesity also contribute to common (polygenic) obesity in humans.\n\n### B. Candidate Genes with Variants Causing Altered Function\n\nA number of other genes, with variants that may alter gene product function, are believed to contribute to common obesity and its comorbidities. Obesity frequently clusters with insulin resistance, hyperlipidemia, and hypertension [78]. This clustering might arise by any of several mechanisms: Obesity might promote comorbidities, comorbidities might promote obesity, or some genes might promote development of both obesity and its comorbidities. Several association studies have reported evidence for genes that influence obesity and one or more comorbidities. However, the demonstration that these variants result in single-gene obesity in any case remains to be established. The more promising candidates are described in Table 3.\n\nTABLE 3\n\nGenes Associated with Obesity and Comorbidities in Humans\n\nPeroxisome-proliferator-activated receptor \u03b3 (PPAR\u03b3)is a member of the nuclear hormone receptor subfamily of transcription factors that includes T3 and vitamin D3 receptors. PPARs regulate expression of genes involved in, among other things, lipid metabolism and energy balance (for review, see [79]). Due to combined effects on both fat and muscle, activation of PPAR\u03b3 improves insulin sensitivity and glucose metabolism, and decreases blood triglyceride levels. Three association studies have implicated _PPAR\u03b3_ in obesity and insulin resistance phenotypes. Four of 121 obese German subjects had a missense mutation in the _PPAR\u03b3_ gene compared to none in 237 normal weight individuals [80]. All of the subjects with the mutant allele were severely obese. The mutant gene was then overexpressed in mouse fibroblasts, which led to the accelerated differentiation of the cells into adipocytes with greater accumulation of triglyceride than seen with the wild-type _PPAR\u03b3_ gene [80]. A different mutation of the _PPAR\u03b3_ gene was identified in Finnish populations and was found to be associated with lower body mass index (BMI), lower fasting insulin levels, and greater insulin sensitivity [81].\n\nThe \u03b2-2-adrenergic receptor (ADR \u03b22) is a major lipolytic receptor in human adipose tissue, and, thus, plays a significant role in lipid mobilization. Several polymorphisms have been identified and their frequencies compared between lean and obese subjects. These variants were associated with body mass index and blood triglycerides in both Swedish and Japanese populations [82\u201384]. Swedish women with two copies of a common polymorphism, Glu27, were about 10 times more likely to be obese than those with the wild-type _ADR\u03b22_ gene, with approximately 20 kg excess body fat and a 50% increase in fat cell size [82]. Another variant of the _ADR\u03b22_ gene, Gly16, was associated with improved adipocyte ADR\u03b22 function. Thus, genetic variability in the human _ADR\u03b22_ gene may be a significant contributor to human obesity.\n\nThe \u03b2-3-adrenergic receptor (ADR\u03b23) is expressed in adipose tissue and is involved in the regulation of lipolysis and thermogenesis. Disruption of the _Ad_ \u03b2 _3_ gene in mice results in moderate obesity [85]. This potential relevance to human obesity led to an initial positive report in 1995 of an association between _ADR\u03b23_ and obesity [86] followed by numerous studies with conflicting results (for review, see [87]). A paired sibling design that aimed to detect effects of the variant by accounting for background genes examined 45 non-diabetic sibling-pairs discordant for the variant who were identical by descent at another marker that is known to be associated with obesity in this population. Presence of the variant was significantly associated with increases in BMI, fat mass, and waist circumference [88]. However, a meta-analysis16 combining 23 studies and 7399 subjects concluded that the _ADR\u03b23_ gene variant is not significantly associated with BMI [87]. The possible association of the variant with diabetes phenotypes was not examined in the meta-analysis. Thus, whether the _ADR\u03b23_ gene contributes to obesity or diabetes phenotypes is still subject to debate.\n\nAdenosine deaminase (ADA) was identified as a positional candidate gene for obesity by linkage studies in both mice and humans [56]. ADA is an \u03b1-adrenergic agonist with potent lipolytic and vasodilator effects that regulates both lipolysis and insulin sensitivity in human adipose tissue. Thus, variants in the _ADA_ gene could theoretically explain the effects of this locus on both energy balance and insulin levels. A recent association study of _ADA_ reported that one _ADA_ variant was more commonly observed in subjects with NIDDM who were obese [58].\n\nUncoupling proteins 2 and 3 (UCP2\/UCP3) are structurally related to UCP1, a mitochondrial protein found in brown fat that plays an important role in generating heat and burning calories without the production of adenosine triphosphate. UCP1 is critical in the maintenance of body temperature of newborn humans, but is unlikely to be significantly involved in weight regulation because brown fat is normally atrophied in adult humans. _UCP2_ and _UCP3_ are recently identified genes [89, 90] located very near each other on chromosome 11 that, like _UCP1,_ encode mitochondrial transmembrane carrier proteins. _UCP2_ is widely expressed in human tissues, whereas _UCP3_ is expressed only in skeletal muscle. In a large French Canadian study, _UCP2_ and _UCP3_ were linked with resting metabolic rate, BMI, percentage of body fat, and fat mass [70]. Several groups have, therefore, examined polymorphisms within the coding regions of both _UCP2_ and _UCP3._ Polymorphisms in _UCP2_ were associated with metabolic rate during sleep in older, but not younger, Pima Indians [91]. A stronger association between a _UCP2_ exon variant and body mass index was found in South Indian subjects [92]. The same variant was not associated with obesity in a British population, but was correlated with fasting serum leptin concentrations in the presence of extreme obesity [92]. Two other studies failed to find a relationship between _UCP2_ variants and energy expenditure, obesity, or insulin resistance [93, 94]. To determine whether _UCP3_ mutations could contribute to human obesity, the nucleotide sequence of coding exons was determined in obese and\/or diabetic Africans, African-Americans, and Caucasians. A mutation and two missense polymorphisms in the _UCP3_ gene were identified in two severely obese probands of African descent [95]. The gene variants were not found in the Caucasian population. The variants were transmitted in a Mendelian fashion; however, they were not consistently associated with obesity in other family members. Individuals who carried one copy of the exon 6-splice polymorphism were found to have only 50% the capacity to oxidize fat and had elevated respiratory quotients (RQs), even though they were not obese. These data indicate that _UCP3_ could alter the availability in the cell of fatty acids for oxidation, promoting fat storage. High RQ and low fat oxidation were previously identified risk factors for future weight gain in Pima Indians [96] and African-Americans [97]. Thus, _UCP3_ is a potentially important obesity gene in certain population groups.\n\nAnimal models of obesity demonstrate the importance of glucocorticoid receptor ( _GRL_ ) activity in the etiology and maintenance of the obese state. In humans, glucocorticoid excess (i.e., Cushing syndrome) results in central fat distribution. The _GRL_ gene was weakly linked with BMI in a study of French obese families [98]. Therefore, polymorphisms of _GRL_ were examined for association with obesity. A variant of the BC1I polymorphism of _GRL_ was associated with increased abdominal fat measured by computerized tomography in middle-aged Canadians [99] and hyperinsulinemia in British Caucasian women [100]. In an elderly population in the Netherlands, 6% carried a polymorphism resulting in altered sensitivity to glucocorticoids [101]. Although healthy, these subjects had higher BMI and higher insulin response to dexamethasone suppression [101].\n\nIntestinal fatty acid-binding protein 2 (FABP2) is thought to facilitate the uptake, intracellular metabolism, and\/or transport of long-chain fatty acids. Linkage between measures of insulin action and a region on chromosome 4q near the _FABP2_ locus was found in Pima Indians of Arizona [102]. Therefore, an Ala54Thr polymorphism in _FABP2_ was examined in Pima Indians and in an isolated population of native Canadians. The variant was associated with insulin resistance and increased fat oxidation rate in Pima Indians [103] and obesity and higher fasting plasma triglyceride levels in the Canadians [104]. The variant was also associated with insulin resistance and greater intra-abdominal fat in Japanese men [105].\n\nLipoprotein function has been associated with several obesity phenotypes. Apolipoprotein D is a protein component of high-density lipoprotein (HDL). Variants of _APOD_ were associated with obesity, elevated fasting insulin [106], and NIDDM [107]. Apolipoprotein B is the main apolipoprotein of chylomicrons and low-density lipoproteins (LDLs) and occurs in two main forms, apoB\u201348 and apoB\u2013100. A polymorphism of the _APOB-100_ gene was associated with abdominal fat and LDL particle size in obese hyperinsulinemic men [108, 109]. Lipoprotein lipase (LPL) deficiency reduces clearance of chylomicrons and other triglyceride-rich lipoproteins. _LPL_ polymorphisms are associated with BMI and hypertriglyceridemia [110].\n\n## X. CLINICAL IMPLICATIONS OF THE DISCOVERY OF OBESITY GENES\n\nThe recent discoveries of human obesity genes have broad implications for clinical practice. Most human obesity genes have only been identified in the last few years. For most of the obesity gene mutations there is no information on the physiological impacts of these mutations, except for obesity. Thus, methods for their diagnosis and the implications of their discovery for treatment have not been discussed in depth. The discovery of human obesity genes will influence several areas of clinical practice including diagnosis and therapy.\n\n### A. Diagnosis of Obesity Disorders 2000\n\nUntil recently, only the rare Mendelian (single-gene) mutations, such as Prader-Willi and Bardet-Biedl syndromes, caused known heritable obesity (Section VII) [46]. These disorders are easily recognized, both by a wide spectrum of phenotypes and by the use of cytogenetics assays that are widely available. However, the new molecular Mendelian obesity disorders (Section V) are not so easily diagnosed, because obesity is often the only apparent phenotype and molecular assays for known obesity gene mutations are currently not practical. It has been estimated that 2\u20137% of morbidly obese patients have mutations in _MC4R_ [40\u201343], 3% have mutations in _PPAR\u03b3_ [80, 111], and an unknown, but smaller, percent have mutations in other obesity genes, including _POMC_ [77]. Thus, only about 1 in 10 morbidly obese patients has a known mutation that explains the obesity, and molecular assays for the currently known Mendelian obesities would be negative in the majority of morbidly obese patients. Also, there are several known distinct mutations in each of these genes. Thus, no clinical laboratories yet provide diagnosis of these mutations, rather they have only been diagnosed by research laboratories, which are not licensed to provide patient information. However, inability to make specific molecular diagnosis does not mean that one cannot identify people with increased risk for genetic obesity, and this may influence choices or approaches to treatment.\n\nSeveral criteria can be used to estimate individual risks for genetic obesity. At the present time, due to the lack of data, these estimates do not produce any quantitative values revealing individual risk that obesity is genetic.\n\n1. The earlier the age of onset and the more extreme the obesity, the more likely that there is a genetic basis for the obesity. Extreme trait values are more likely to be genetic for many complex diseases, simply because extremes tend to result from the actions of severe mutations or from mutations in genes that have larger effects [112]. Children with single-gene obesity are normal weight at birth but severe early hyperphagia, often associated with aggressive food-seeking behavior, results in rapid weight gain, usually beginning in the first year of life.\n\n2. Prader-Willi, Bardet-Biedl, and other Mendelian syndromes can be diagnosed by a variety of characteristic phenotypes as well as by cytogenetic assays. Thus, one should rule out these diagnoses by phenotype determination and by absence of characteristic chromosomal abnormalities.\n\n3. A strong family history of obesity is consistent with the presence of an obesity gene shared among family members.\n\n4. _POMC_ defects can cause red hair and obesity [37], although most red hair results from mutations in melanocortin receptor-1 ( _MC1R_ ) [113], which does not influence obesity. Thus, red hair is only informative when red hair and obesity cosegregate within a family.\n\n5. At present, few diagnostic tools are available for the medical evaluation of patients suspected of having Mendelian obesity. The only screening tests available are for endocrine abnormalities. Leptin should be measured. Very low or very high serum leptin levels will indicate mutation in _LEP_ or _LEPR,_ respectively. A subset of obese individuals has inappropriately low leptin levels for their fat mass, suggesting a less severe defect in leptin regulation [72]. ACTH and proinsulin should be measured to indicate defects in _POMC_ or in prohormone processing.\n\n### B. Implications of Obesity Genes for Obesity Treatment\n\nThe recent discovery of human obesity genes may have broad future implications for diet, behavioral, and drug therapy of genetically obese humans, and perhaps of all obese people. The identification and characterization of gene products associated with obesity have provided novel pathways that can be targeted for pharmaceutical intervention. A significant new drug is the hormone leptin, which, as of this writing, is still in clinical trials. However, an early study suggests that exogenous leptin induces weight loss even in some obese subjects with elevated endogenous serum leptin levels [25]. Leptin therapy may, therefore, be effective even for obese individuals without defects in leptin production. Another potential drug target identified by the cloning of obesity genes is the melanocortin receptor. Development of safe and effective drugs such as an \u03b1-MSH-like agonist for the melanocortin receptor to inhibit food intake or stimulators of the expression of _UCP2_ or _UCP3_ to enhance energy expenditure are certainly goals of the pharmaceutical industry [14].\n\nPatients with monogenic obesity will probably be more difficult to treat than those with polygenic obesity, because individuals with monogenic obesity will likely have strong food-seeking behavior and may have physiological resistance to fat loss. Certainly, the primary therapy for individuals with documented leptin deficiency is recombinant leptin. Specific pharmaceutical treatment of other single-gene obesities will have to await development of drugs targeted further along the pathway of the mutated gene. Meanwhile, lifestyle changes that may promote weight loss and improve metabolic fitness and quality of life should be recommended. 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Mammes O., Betoulle D., Aubert R., Giraud V., Tuzet S., Petiet A., Colas-Linhart N., Fumeron F. Novel polymorphisms in the 5\u2032 region of the LEP gene: Association with leptin levels and response to low-calorie diet in human obesity. _Diabetes_. 1998;47:487\u2013489.\n\n74. Thompson D.B., Ravussin E., Bennett P.H., Bogardus C. Structure and sequence variation at the human leptin receptor gene in lean and obese Pima Indians. _Hum. Mol. Genet_. 1997;6:675\u2013679.\n\n75. Roth H., Korn T., Rosenkranz K., Hinney A., Ziegler A., Kunz J., Siegfried W., Mayer H., Hebebrand J., Grzeschik K.H. Transmission disequilibrium and sequence variants at the leptin receptor gene in extremely obese German children and adolescents. _Hum. Genet_. 1998;103:540\u2013546.\n\n76. de Silva A.M., Walder K.R., Aitman T.J., Gotoda T., Goldstone A.P., Hodge A.M., de Courten M.P., Zimmet P.Z., Collier G.R. Combination of polymorphisms in OB-R and the OB gene associated with insulin resistance in Nauruan males. _Int. J. Obes. Relat. Metab. Disord_. 1999;23:816\u2013822.\n\n77. Hixson J.E., Almasy L., Cole S., Birnbaum S., Mitchell B.D., Mahaney M.C., Stern M.P., MacCluer J.W., Blangero J., Comuzzie A.G. Normal variation in leptin levels is associated with polymorphisms in the proopiomelanocortin gene, POMC. _J. Clin. Endocrinol. Metab_. 1999;84:3187\u20133191.\n\n78. Must A., Spadano J., Coakley E.H., Field A.E., Colditz G., Dietz W.H. The disease burden associated with overweight and obesity. _JAMA_. 1999;282:1523\u20131529.\n\n79. Clarke S.D., Thuillier P., Baillie R.A., Sha X. Peroxisome proliferator-activated receptors: A family of lipid-activated transcription factors. _Am. J. Clin. Nutr_. 1999;70:566\u2013571.\n\n80. Ristow M., Muller-Wieland D., Pfeiffer A., Krone W., Kahn C.R. Obesity associated with a mutation in a genetic regulator of adipocyte differentiation. _N. Engl. J. Med_. 1998;339:953\u2013959.\n\n81. Deeb S.S., Fajas L., Nemoto M., Pihlajamaki J., Mykkanen L., Kuusisto J., Laakso M., Fujimoto W., Auwerx J. A Pro12Ala substitution in PPARgamma2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity. _Nature Genet_. 1998;20:284\u2013287.\n\n82. Large V., Hellstrom L., Reynisdottir S., Lonnqvist F., Eriksson P., Lannfelt L., Arner P. Human beta-2 adrenoceptor gene polymorphisms are highly frequent in obesity and associate with altered adipocyte beta-2 adrenoceptor function. _J. Clin. Invest_. 1997;100:3005\u20133013.\n\n83. Ishiyama-Shigemoto S., Yamada K., Yuan X., Ichikawa F., Nonaka K. Association of polymorphisms in the beta2-adrenergic receptor gene with obesity, hypertriglyceridaemia, and diabetes mellitus. _Diabetologia_. 1999;42:98\u2013101.\n\n84. Mori Y., Kim-Motoyama H., Ito Y., Katakura T., Yasuda K., Ishiyama-Shigemoto S., Yamada K., Akanuma Y., Ohashi Y., Kimura S., Yazaki Y., Kadowaki T. The Gln27Glu beta2-adrenergic receptor variant is associated with obesity due to subcutaneous fat accumulation in Japanese men. _Biochem. Biophys. Res. Commun_. 1999;258:138\u2013140.\n\n85. Susulic V.S., Frederich R.C., Lawitts J., Tozzo E., Kahn B.B., Harper M.E., Himms-Hagen J., Flier J.S., Lowell B.B. Targeted disruption of the beta 3-adrenergic receptor gene. _J. Biolog. Chem_. 1995;270:29483\u201329492.\n\n86. Clement K., Vaisse C., Manning B.S., Basdevant A., Guy-Grand B., Ruiz J., Silver K.D., Shuldiner A.R., Froguel P., Strosberg A.D. Genetic variation in the beta 3-adrenergic receptor and an increased capacity to gain weight in patients with morbid obesity. _N. Engl. J. Med_. 1995;333:352\u2013354.\n\n87. Allison D.B., Heo M., Faith M.S., Pietrobelli A. Meta-analysis of the association of the Trp64Arg polymorphism in the beta-3 adrenergic receptor with body mass index. _Int. J. Obes. Relat. Metab. Disord_. 1998;22:559\u2013566.\n\n88. Mitchell B.D., Blangero J., Comuzzie A.G., Almasy L.A., Shuldiner A.R., Silver K., Stern M.P., MacCluer J.W., Hixson J.E. A paired sibling analysis of the beta-3 adrenergic receptor and obesity in Mexican Americans. _J. Clin. Invest_. 1998;101:584\u2013587.\n\n89. Fleury C., Neverova M., Collins S., Raimbault S., Champigny O., Levi-Meyrueis C., Bouillaud F., Seldin M.F., Surwit R.S., Ricquier D., Warden C.H. Uncoupling protein-2: A novel gene linked to obesity and hyperinsulinemia. _Nature Genet_. 1997;15:269\u2013272.\n\n90. Boss O., Samec S., Paoloni-Giacobino A., Rossier C., Duloo A., Seydoux J., Muzzin P., Giacobino J.P. Uncoupling protein-3: A new member of the mitochondrial carrier family with tissue-specific expression. _FEBS Lett_. 1997;408:39\u201342.\n\n91. Walder K., Norman R.A., Hanson R.L., Schrauwen P., Neverova M., Jenkinson C.P., Easlick J., Warden C.H., Pecqueur C., Raimbault S., Ricquier D., Silver M.H.K., Shuldiner A.R., Solanes G., Lowell B.B., Chung W.K., Leibel R.L., Pratley R., Ravussin E. Association between uncoupling protein polymorphisms (UCP2-UCP3) and energy metabolism\/obesity in Pima Indians. _Hum. Mol. Genet_. 1998;7:1431\u20131435.\n\n92. Cassell P.G., Neverova M., Janmohamed S., Uwakwe N., Qureshi A., McCarthy M.I., Saker P.J., Albon L., Kopelman P., Noonan K., Easlick J., Ramachandran A., Snehalatha C., Pecqueur C., Ricquier D., Warden C., Hitman G.A. An uncoupling protein 2 gene variant is associated with a raised body mass index but not Type II diabetes. _Diabetologia_. 1999;42:688\u2013692.\n\n93. Urhammer S.A., Dalgaard L.T., Sorensen T.I., Moller A.M., Andersen T., Tybjaerg-Hansen A., Hansen T., Clausen J.O., Vestergaard H., Pedersen O. Mutational analysis of the coding region of the uncoupling protein 2 gene in obese NIDDM patients: Impact of a common amino acid polymorphism on juvenile and maturity onset forms of obesity and insulin resistance. _Diabetologia_. 1997;40:1227\u20131230.\n\n94. Klannemark M., Orho M., Groop L. No relationship between identified variants in the uncoupling protein 2 gene and energy expenditure. _Eur. J. Endocrinol_. 1998;139:217\u2013223.\n\n95. Argyropoulos G., Brown A.M., Willi S.M., Zhu J., He Y., Reitman M., Gevao S.M., Spruill I., Garvey W.T. Effects of mutations in the human uncoupling protein 3 gene on the respiratory quotient and fat oxidation in severe obesity and type 2 diabetes. _J. Clin. Invest_. 1998;102:1345\u20131351.\n\n96. Ravussin E. Metabolic differences and the development of obesity. _Metabolism_. 1995;44:12\u201314.\n\n97. Jakicic J.M., Wing R.R. Differences in resting energy expenditure in African-American vs Caucasian overweight females. _Int. J. Obes. Relat. Metab. Disord_. 1998;22:236\u2013242.\n\n98. Clement K., Philippi A., Jury C., Pividal R., Hager J., Demenais F., Basdevant A., Guy-Grand B., Froguel P. Candidate gene approach of familial morbid obesity: Linkage analysis of the glucocorticoid receptor gene. _Int. J. Obes. Relat. Metab. Disord_. 1996;20:507\u2013512.\n\n99. Buemann B., Vohl M.C., Chagnon M., Chagnon Y.C., Gagnon J., P\u00e9russe L., Dionne F., Despr\u00e9s J.P., Tremblay A., Nadeau A., Bouchard C. Abdominal visceral fat is associated with a BclI restriction fragment length polymorphism at the glucocorticoid receptor gene locus. _Obes. Res_. 1997;5:186\u2013192.\n\n100. Weaver J.U., Hitman G.A., Kopelman P.G. An association between a Bc1I restriction fragment length polymorphism of the glucocorticoid receptor locus and hyperinsulinaemia in obese women. _J. Mol. Endocrinol_. 1992;9:295\u2013300.\n\n101. Huizenga N.A., Koper J.W., De Lange P., Pols H.A., Stolk R.P., Burger H., Grobbee D.E., Brinkmann A.O., De Jong F.H., Lamberts S.W. A polymorphism in the glucocorticoid receptor gene may be associated with and increased sensitivity to glucocorticoids in vivo. _J. Clin. Endocrinol. Metab_. 1998;83:144\u2013151.\n\n102. Prochazka M., Lillioja S., Tait J.F., Knowler W.C., Mott D.M., Spraul M., Bennett P.H., Bogardus C. Linkage of chromosomal markers on 4q with a putative gene determining maximal insulin action in Pima Indians. _Diabetes_. 1993;42:514\u2013519.\n\n103. Baier L.J., Sacchettini J.C., Knowler W.C., Eads J., Paolisso G., Tataranni P.A., Mochizuki H., Bennett P.H., Bogardus C., Prochazka M. An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance. _J. Clin. Invest_. 1995;95:1281\u20131287.\n\n104. Hegele R.A., Harris S.B., Hanley A.J., Sadikian S., Connelly P.W., Zinman B. Genetic variation of intestinal fatty acid-binding protein associated with variation in body mass in aboriginal Canadians. _J. Clin. Endocrinol. Metab_. 1996;81:4334\u20134337.\n\n105. Yamada K., Yuan X., Ishiyama S., Koyama K., Ichikawa F., Koyanagi A., Koyama W., Nonaka K. Association between Ala54Thr substitution of the fatty acid-binding protein 2 gene with insulin resistance and intra-abdominal fat thickness in Japanese men. _Diabetologia_. 1997;40:706\u2013710.\n\n106. Vijayaraghavan S., Hitman G.A., Kopelman P.G. Apolipoprotein-D polymorphism: A genetic marker for obesity and hyperinsulinemia. _J. Clin. Endocrinol. Metab_. 1994;79:568\u2013570.\n\n107. Baker W.A., Hitman G.A., Hawrami K., McCarthy M.I., Riikonen A., Tuomilehto-Wolf E., Nissinen A., Tuomilehto J., Mohan V., Viswanathan M., Snehalatha C., Ramachandran A., Dowse G.K., Zimmet P., Serjeantson S. Apolipoprotein D gene polymorphism: A new genetic marker for type 2 diabetic subjects in Nauru and south India. _Diabet. Med_. 1994;11:947\u2013952.\n\n108. Pouliot M.C., Despr\u00e9s J.P., Dionne F.T., Vohl M.C., Moorjani S., Prud'homme D., Bouchard C., Lupien P.J. ApoB-100 gene EcoRI polymorphism. Relations to plasma lipoprotein changes associated with abdominal visceral obesity. _Arterioscler. Thromb_. 1994;14:527\u2013533.\n\n109. Vohl M.C., Tchernof A., Dionne F.T., Moorjani S., Prud'homme D., Bouchard C., Nadeau A., Lupien P.J., Despr\u00e9s J.P. The apoB-100 gene EcoRI polymorphism influences the relationship between features of the insulin resistance syndrome and the hyper-apoB and dense LDL phenotype in men. _Diabetes_. 1996;45:1405\u20131411.\n\n110. Jemaa R., Fumeron F., Poirier O., Lecerf L., Evans A., Arveiler D., Luc G., Cambou J.P., Bard J.M., Fruchart J.C., Apfelbaum M., Cambien F., Tiret L. Lipoprotein lipase gene polymorphisms: Associations with myocardial infarction and lipoprotein levels, the ECTIM study (Etude Cas Temoin sur l'Infarctus du Myocarde). _J. Lipid. Res_. 1995;36:2141\u20132146.\n\n111. Valve R., Sivenius K., Miettinen R., Pihlajamaki J., Rissanen A., Deeb S.S., Auwerx J., Uusitupa M., Laakso M. Two polymorphisms in the peroxisome proliferator-activated receptor-gamma gene are associated with severe overweight among obese women. _J. Clin. Endocrinol. Metab_. 1999;84:3708\u20133712.\n\n112. Lander E.S., Schork N.J. Genetic dissection of complex traits. _Science_. 1994;265:2037\u20132048 Lander E.S., Schork N.J. Genetic dissection of complex traits. _Science_. 1994;266:353.\n\n113. Palmer J.S., Duffy D.L., Box N.F., Aitken J.F., O'Gorman L.E., Green A.C., Hayward N.K., Martin N.G., Sturm R.A. Melanocortin-1 receptor polymorphisms and risk of melanoma: Is the association explained solely by pigmentation phenotype? _Am. J. Hum. Genet_. 2000;66:176\u2013186.\n\n* * *\n\n1Segregation analysis is used to determine whether the trait is segregating in families according to Mendelian expectations.\n\n2A quantitative trait is one that varies over a continuous range, such as body weight and height, and is generally controlled by more than one gene. A quantitative trait locus (QTL) is a chromosomal region in which alleles are linked to variation of a quantitative trait.\n\n3The designations _Lep ob, Leprdb,_ and _Cpe fat_ represent mutations of the genes coding for leptin, the leptin receptor, and carboxypeptidase E that occur in the _obese_ mouse, the _diabetes_ mouse, and the _fat_ mouse, respectively.\n\n4Capital letters, e.g., _TUB,_ indicate the human gene, whereas lowercase letters, e.g., _Tub,_ indicate the mouse gene.\n\n5If an allele is rare and if two copies (homozygosity) of a mutation are required for the phenotype to be expressed, then homozygotes are usually found only in highly consanguineous (inbred) families.\n\n6A proband is the index case, the person through whom the pedigree (family) was acertained.\n\n7A compound heterozygote would have two different mutations in the gene, one on each chromosome.\n\n8Haploinsufficiency occurs when a gene, or a group of genes, is present in too fewcopies or too many copies. In autosomes (all but the X and Y chromosomes) one copy of each gene is inherited from each parent. The presence of extra chromosomal material or lack of chromosomal material alters gene dosage, causing abnormalities in gene function.\n\n9A missense mutation is one that changes amino acid sequence, but does not produce a stop codon (nonsense mutation).\n\n10The Online Mendelian Inheritance in Man (OMIM) [46] database is available at . This database is a catalog of human genes and genetic disorders authored and edited by Dr. Victor A. McKusick and his colleagues at Johns Hopkins and elsewhere, and developed for the World Wide Web by the National Center for Biotechnology Information (NCBI).\n\n11Maternal disomy is inheritance of an extra maternal chromosome or part thereof.\n\n12A locus is any segment of DNA that is measurable in genetic analysis. A locus may be within a gene or may be an alternative DNA sequence of no known function. A linkage map represents a set of loci on a single chromosome in which all members of the set are linked either directly or indirectly with all other members of the set. Linkage in humans refers to the cosegregation of a genetic marker and a trait together in families.\n\n13Pleiotropy means that one gene has a primary effect on more than one phenotype.\n\n14The Davis Human\/Mouse Homology Map is a table comparing genes in homologous segments of DNA from human and mouse sources, sorted by position in each genome. A total of 1793 loci are presented, most of which are genes, in 201 homology groupings [55]. Current homology data are available at .\n\n15Identical by descent is in contrast to identical by state. Two siblings sharing the same allele are identical by descent if you know that it is the same allele inherited from the same parent. They are identical by state if they have the same allele, but you do not know if they are derived from the same parental haplotype.\n\n16Meta-analysis is a statistical tool for pooling data from many studies into a single analysis, thus greatly increasing the statistical power of the analysis.\nCHAPTER 13\n\nGenetic Influence on Cancer Risk\n\nJO L. FREUDENHEIM, State University of New York at Buffalo, Buffalo, New York\n\n## I. INTRODUCTION\n\nCancer leads to the death of about 5.2 million people in the world each year with more than 8 million new cases of cancer each year. In the United States alone, there are about 1 million new cases annually [1]. Therapies to cure cancer are useful but prevention is clearly a superior strategy. For most kinds of cancer, only 50% or fewer of patients survive more than 5 years after diagnosis [2]. It is generally agreed that most common cancers are caused by environmental factors with the potential to be controlled [3]. It has been estimated that about 30% of cancer cases could be eliminated with dietary changes [4]; it appears that for most kinds of cancer dietary factors play a role in etiology.\n\nIt is important to note that a cancer may have more than one cause. For example, both occupational exposures and diet could contribute to the etiology of a cancer, and either a change in the occupational exposure or a change in diet might prevent that cancer. Genetic factors likely play a role in many if not all cancers. Even when an exposure is clearly required for a particular kind of cancer, genetic factors often play a role in determining who, among exposed individuals, develops that cancer. Also for dietary risk factors, evidence is now accumulating that genetic factors may interact with diet in the etiology of the disease.\n\n## II. BACKGROUND\n\nCancer is a genetic disease. That is, cancer is characterized by the accumulation of changes in the structure of the DNA of the cell such that there are significant changes in the functioning of the cell. Genes are the basic unit of heredity with each gene having its own location in a particular chromosome [5]. A mutation is a structural change in the DNA [5]. _Genotype_ refers to the chemical composition of the DNA, it is the nucleic acid sequence of the DNA, inherited from both parents. From knowledge of the genotype, it is possible to infer information about an individual's characteristics. That is, the genetic code can be translated into information about the amino acid sequence of a protein. From that information, it would be possible to determine, for example, whether an individual had the gene for the faster or slower version of a particular enzyme. _Phenotype,_ on the other hand, refers to the appearance of the individual, the expression of the genotype [5]. Agreement between genotype and phenotype often is not perfect. A person may have the genotype for a less active enzyme, but other exposures (e.g., dietary factors) may induce a high level of expression of that enzyme. A measurement of phenotype might be based on the rate of metabolism of a particular compound, and might not be able to distinguish high activity due to genotype from that related to enzyme induction.\n\nPenetrance of a genetic factor refers to the likelihood that those with a particular gene will exhibit a particular phenotype under particular environmental circumstances [5]. A gene with high penetrance would be one where virtually everybody with the gene has the expressed trait while a gene with low penetrance would have low likelihood of expression of the gene. The measurement of phenotype is of interest because it gives an indication as to the true level of exposure, while the measurement of genotype is of interest because it gives some indication of exposure over the lifetime and is not biased by factors such as disease and recent exposures.\n\nSome individuals are at greater risk for certain types of cancer. This increased risk can be related to differences in exposure to cancer-inducing or -protecting agents, and\/or to the individual's genetic makeup, or to a combination of these factors. Genetic factors vary widely in the magnitude of their effects. Some inherited mutations greatly increase the risk of cancer; others cause smaller increases in risk. Some mutations may only increase risk in the presence of a particular exposure. An example of an inherited mutation that causes a substantial increase in risk is the _BRCA1_ gene. Particular mutations in this gene have been shown to be related to risk of breast and ovarian cancer. Current estimates are that carriers of this genetic mutation have a 50% risk of breast cancer by age 50 and an 85% risk by age 70. However, relatively few individuals in the population carry this factor (estimates range between 1 in 2000 and 1 in 500). Therefore, athough those with the gene are at high risk, only about 1\u20135% of women with breast cancer have this mutation [6, 7]. There are also other genetic variants that are more common and that have weaker effects on risk. These are referred to as _genetic polymorphisms._ Genetic polymorphism means that the gene has more than one form; generally these differences entail the alteration of a single nucleic acid in the sequence or the deletion of a section of the gene. Genetic polymorphisms can be quite common; they are found in up to 50% of the population. Generally these factors have a much smaller effect on cancer risk, increasing risk two to four times. However, because they are so common, they can have a significant impact on the rate of disease in a population. Also, in general, these factors affect the response to an exposure; that is, they affect risk only when that exposure is present, referred to as a gene\u2013environment interaction. For these types of gene\u2013environment interactions, an understanding of both the genetic factor and the environmental exposure is important in order to understand disease etiology and prevention. In terms of diet and genetics in relation to risk of cancer, at this time it appears that the second category of genetic effects is of primary importance. Risk of cancer is affected by an interaction of commonly occurring genetic variants and diet. These interactions can occur in any of several ways.\n\n## III. MECHANISMS OF DIET\u2013GENETIC INTERACTIONS\n\nA. Carcinogen Metabolism\n\nThe bulk of the research on the epidemiology of gene\u2013environment interactions at this time has focused on genetic factors that modulate the metabolism of carcinogens. These carcinogens include compounds occurring naturally in foods or contaminating foods. If an individual has a genetic variant that results in slower metabolism of a carcinogen, then they will be more affected by a given dose of that carcinogen than would someone who could metabolize and excrete the carcinogen more rapidly. The enzymes related to carcinogen metabolism and excretion are generally divided into two groups: phase I and phase II. The phase I enzymes activate the compound; phase II enzymes then attach polar groups to the activated compound so that it will be more water soluble and can be excreted in the urine. Chief among the phase I enzymes are the cytochrome P-450 family (CYP). Important phase II enzymes are glutathione- _S_ -transferase (GST) and _N_ -acetyltransferase (NAT). In some cases, phase I activation leads to the production of compounds with greater carcinogenic potential. Often rather than carcinogens, foods contain procarcinogens, compounds that are carcinogenic after metabolic activation. The process of metabolic activation may vary depending on genetic factors. For example, heterocyclic amines are a group of compounds found in meat cooked at high temperatures, primarily in ''well-done\" meat [8]. Metabolism and excretion of heterocyclic amines involve CYP1A2 and NAT2. Preliminary epidemiologic evidence indicates that intake of well-done meat may be related to risk of colorectal adenomas, and that the effect depends in part on which of the genetic variants of CYP1A2 and NAT2 is carried by an individual [9]. Figure 1 illustrates the relationships between these enzyme systems [10, 11].\n\nFIGURE 1 Interrelationship between the biotransformation enzyme systems. [From Patterson, R. E., Eaton, D. L., and Potter, J. D. The genetic revolution: Change and challenge for the dietetics profession. Copyright the American Dietetic Association. Reprinted by permission from _Journal of The American Dietetic Association,_ vol. 99, pp. 1412\u20131420, 1999.]\n\n### B. Enzyme Induction\n\nAnother mechanism of interaction for genetic factors and diet in relation to cancer risk is that factors in foods may also induce some of the genetic factors involved in carcinogen metabolism. That is, food components may affect the activity of the enzyme related to that gene so that there is more rapid metabolism by that enzyme. For example, CYP1A2 can be induced by indole\u20133-carbinol found in cruciferous vegetables, by heterocyclic amines in meat, and by polycyclic aromatic hydrocarbons found in grilled meat. CYP1A2 is also inhibited by a compound found in grapefruit, narin-genin [9]. Therefore, metabolism of heterocyclic amines may depend not only on the genetic variant of CYP1A2 that is carried by an individual, but also on their intake of these other food components that affect CYP1A2 induction.\n\n### C. Genetic Variation in Vitamin Pathways\n\nA third mechanism by which genes and diet may interact in relation to risk of cancer is that there can be genetic variation in vitamin pathways. There are commonly occurring genetic differences in vitamin receptors. If a vitamin plays a role in either carcinogenesis or in cancer prevention, differences in the receptor for the vitamin could also affect the susceptibility of an individual to cancer. An example of this type of relationship is found in research on prostate cancer. There is evidence that genetic variation in the vitamin D receptor can affect prostate cancer risk [12\u201314].\n\n### D. Oxidative Stress\n\nA fourth example of a mechanism for gene and dietary interaction in relation to cancer risk is with regard to oxidation. There is evidence that reactive oxygen species from both exogenous and endogenous sources affect the rate of DNA mutations. Energy consumption is correlated with the number of oxidized DNA bases [3]. Antioxidant vitamins and other enzymes affected by genetic variation are part of the body's defense against these oxidative processes. Those who have genetically weaker defense systems against oxidation may benefit more from increased intake of antioxidant vitamins, such as vitamin C and vitamin E. For example, superoxide dismutase is important in the control of endogenously produced reactive oxygen species; manganese superoxide dismutase (MnSOD2) is found in the mitochondria, an important source of oxidative species. MnSOD2 is polymorphic and there is a study suggesting that one variant of this gene may be associated with increased risk of premenopausal breast cancer, but primarily among women with a low intake of antioxidant vitamins from fruits and vegetables [15].\n\nAnother example of a gene\u2013environment interaction related to oxidation is evidence that relates intake of antioxidant vitamins to smoking-related DNA adducts. DNA adducts are the chemical bonding of molecules to DNA; such adducts may interfere with DNA replication and result in mutations or deletions in the DNA structure [3]. There is evidence of an inverse association between formation of these adducts and \u03b2-carotene intake among smokers. However, this association was limited to those individuals who carried homozygous deletions for the gene for glutathione _S_ -transferase ( _GSTM1null_ ) a detoxification enzyme [16]. The _GSTM1 null_ genotype is found in about 50% of Caucasian populations.\n\n### E. One-Carbon Metabolism\n\nOne further mechanism for an interaction of diet and genetic factors in cancer etiology is an effect of diet on one-carbon methyl metabolism. Folate, vitamin B12, vitamin B6, and methionine are all involved in the metabolism of methyl groups. Alcohol consumption also adversely affects folate status and therefore methyl status. Methylation is important in the synthesis of purines and pyrimidines for DNA [17], and methylation of DNA can play a role in regulation of DNA transcription [18].\n\nIn animal studies, it has been shown that a diet deficient in these nutrients can lead to changes in the available nucleotides for DNA synthesis [19] and DNA strand breaks [20]. In humans, there is some evidence that hypomethylation of DNA is a step in the carcinogenic process [18, 21, 22]. In particular, there is evidence that a diet low in one or more of these factors related to methyl metabolism may be associated with risk of colon cancer [23, 24]. Further, at least one of the enzymes in the methyl pathway, methylenetetrahydrofolate reductase (MTHFR), has been identified as polymorphic. This enzyme catalyzes the conversion of folate from the form that is most common intracellularly to the form most common in the plasma. One study showed significant differences in plasma homocysteine levels depending on the MTHFR genotype [25].\n\nIn another study, risk for colon cancer was higher for one MTHFR variant than for the other. The protective effect was less pronounced when dietary sources of methyl groups were lower, that is for higher intake of alcohol and lower intake of folate and methionine [26, 27]. It appears that these dietary factors and these genetic factors may have an interactive effect on risk of colon cancer.\n\nThe field of genetics is changing rapidly; numerous discoveries are leading to major shifts in our understanding of the role of genetic factors in cancer risk and of the interaction of environment with those genetic factors. At this time, the several pathways detailed above are the major ones identified for diet and gene interactions. However, in the next several years, other pathways of interaction may be identified and some of the ones detailed here may be determined to be of lesser importance to humans. Because much of our understanding of gene\u2013environment interactions in relation to cancer is based on relatively new information, it is important to recognize that much of the data is subject to reinterpretation and that an understanding of the methodologic issues involved is key.\n\n## IV. METHODOLOGIC ISSUES IN ASSESSMENT OF DIET AND GENE INTERACTIONS RELATED TO CANCER\n\nAn individual's genotype can be determined from DNA extracted from cells. The DNA is typically obtained from white blood cells, from sloughed cells in saliva, or from cells brushed from the inside of the mouth. For polymorphic genes, laboratory methods are used to identify which of the variants for a particular gene are carried by an individual based on the sequence of the DNA. Depending on the gene, one or more variants is possible. The variants may constitute the deletion of the whole gene, other mutations leading to a gene without function, or mutations leading to a change in the amino acid structure of the protein for which the gene is coding. Some variations may not affect function. For example, there may be two codes for the same amino acid, or there may be a genetic change in a part of the DNA that is not related to the function of the enzyme. Generally, epidemiologic studies should be limited to examination of polymorphisms that affect function.\n\nAn individual's phenotype may also be measured. Typically, to measure phenotype, an individual ingests a measured amount of a substance (e.g., caffeine) and then blood or urine samples are collected for several hours or days to determine the rate of metabolism of that substance. Measurement of phenotype is advantageous in that it provides an indication of the sum of the processes involved and therefore an indication of the true level of exposure. Phenotype may depend on more than one gene; it may reflect processes involved in absorption and excretion as well as metabolism. However, when comparisons are being made of individuals with and without a disease, the phenotype may be affected by the disease state and might not be reflective of the diseased individual's lifetime exposure. In studies of this kind, a determination of phenotype may be less useful than determination of a genotype, unless it can be shown that the phenotype does not change for those with the disease.\n\nIn epidemiology, the interaction of genes and diet may be assessed in case-control studies, in nested case-control studies, or in clinical trials. In case-control studies, the cases are people with recently diagnosed disease who are compared to healthy controls from the same population. Statistical methods are used to determine whether there are systematic differences between those people who get the disease and those who do not. In a cohort study, a population of exposed and unexposed individuals is identified and their exposure status measured. Because cancer is relatively rare, these studies need to include thousands or even hundreds of thousands of individuals in order to examine diet in relation to cancer.\n\nIf blood has been collected from the study participants, it is possible to determine genotype for the participants. In a cohort study, the measurement of phenotype for metabolizing enzymes is rarely used because of the logistic difficulties. Generally, a nested case-control study is conducted for analysis of gene\u2013environment interactions using genotype within a cohort. For this kind of study, those individuals in the cohort with incident cancer (cases) are compared to a group of controls selected from the cohort. The controls are chosen to be similar to the cases for relevant characteristics such as age, race, and gender. Genotypes are then determined, and the gene\u2013environment interactions analyzed for this subset of the whole cohort.\n\nFor both case-control and nested case-control studies, the examination of gene\u2013diet interactions generally involves a few steps. These include (1) the examination of the association between risk of a particular cancer and the genetic factor alone, (2) the examination of the association between risk of that cancer and the dietary factor alone, and then (3) the examination of the gene\u2013diet interaction. The third step generally involves the analysis of the association between diet and cancer in strata defined by genotype or else the analysis of the association between gene and cancer in strata defined by level of dietary exposure. In some cases, there will be further examination of interaction with a third factor (e.g., exposure to hormone replacement therapy, body weight).\n\nAlthough this is a relatively new area of research, in some clinical trials, the interaction of genes and environment are studied. The effect of a dietary intervention might be examined within groups defined by strata to determine if one group is more susceptible to the intervention.\n\nFor all of these study designs, there are some important methodological considerations. Many of these are the same considerations as for any epidemiologic study of diet and cancer [28]. A significant one is that, as in all epidemiology, conclusions about causality need to come from a synthesis of epidemiologic findings in a number of populations, animal research, and metabolic studies. No single epidemiologic study can be considered definitive and used to establish causality. In case-control and cohort studies, there is always concern that the findings may not be causal but rather the result of confounding. That is, another factor may be correlated with the exposure under study and with the disease, and may be the causative agent. If the investigators are unaware of this confounding factor or they are unable to control for it sufficiently well, it might appear that the exposure under study is associated with disease simply because it is correlated with the second factor.\n\nFor example, in a study of diet and lung cancer, if individuals who smoke are also more likely to drink alcohol, unless smoking is measured well and controlled for in analysis, one might incorrectly assess the relation of alcohol to lung cancer because of the correlation between these two behaviors. However, when studies are done in different populations in different cultures, the likelihood of confounding is diminished; it is less likely that the same correlations exist between behaviors. Additionally, other sources of error may differ among studies making a consistent finding more believable. Only with a randomized trial can a causal link be identified with certainty. However, even among clinical trials, differences can result because of differences in participant populations, so results need to be carefully interpreted.\n\nBecause the availability of the technology to measure genotypes in large studies is relatively new, there are few hypotheses related to the interaction of diet and genetics in cancer etiology that have been examined in numerous studies. Many questions have been examined only in a single study. A large number of studies are just starting or are ongoing. With the development of this field, it will be possible to begin to identify what appear to be causal relations. At all times, it is important to examine findings critically and to look for consistent findings from well-conducted studies.\n\nA major concern in evaluating diet and genetic interactions in relation to risk of cancer is that studies need to have large numbers of participants in order to have sufficient power to examine risk within strata defined by genotype or by diet. At the present time, stored blood and interview data from previously conducted studies are often used to examine these questions. Because these studies were designed to address other questions, the number of participants may be relatively small, and analysis of gene\u2013environment interactions may be based on samples of 10 or fewer individuals. Even if the results are statistically significant, such findings can be unstable. That is, if the study were redone, the findings might be very different.\n\nAs more and more studies are conducted, especially to address gene\u2013environment interactions, this problem will become less severe for studies planned with sufficient statistical power. Nonetheless, for genes with low frequencies, the number of participants required can be very large. Further, to examine the interactions of more than one gene and more than one dietary or other exposure, the number of participants required may be exceedingly large. In evaluation of any study, it is important to look at the number of participants in each cell to make a determination of the likely stability of the findings.\n\nAt this time, most of the research regarding interactions of diet and genetics focus on a single dietary factor and a single nutrient. Such a focus is of considerable interest. Nonetheless, cancer is a multifactorial disease, and it is likely that several genetic factors are of importance even within a single causative pathway. Analysis of a single polymorphism might not capture the total picture of variation in risk. Similarly, several nutrients are likely to be important even within a single causal pathway. As noted above, the examination of gene\u2013gene and gene\u2013environment interactions can be seriously hampered by the required sample size. Further, diet is also a complex set of exposures. A single food may include different factors with both carcinogenic and anti-carcinogenic properties. Intakes of different nutrients are generally highly correlated, so that an association attributed to intake of one nutrient may in fact be the result of a causal relationship with a different nutrient. Finally, when there is evidence that a particular nutrient is related to a decrease in risk, it cannot be assumed that larger quantities of that nutrient would be even more protective. For example, while vitamin C has antioxidant properties at the level found in most foods, at higher intake levels, it may have pro-oxidant properties [29].\n\nA final challenge in this field is in the measurement of dietary exposure. Much literature is available on the problems of measurement of diet for epidemiologic purposes [30]. Beyond those concerns, the study of gene\u2013diet interactions in relation to cancer risk has led to an interest in a number of new dietary factors. For many of these compounds, the dietary instruments used in the past may not provide sufficient detail for assessment of intake of that compound. For example, the study of heterocyclic amines has led to the development of a questionnaire that specifically addresses the sources of these compounds, because the information regarding intake on the existing questionnaires did not include the necessary detail to assess intake of heterocyclic amines [31]. Further, nutrient composition databases may be limited and may need work in order to determine the composition for compounds that may relate to disease risk but that have not been previously analyzed.\n\n## V. DIET\u2013GENE INTERACTIONS AND CANCER\n\nIn this section, the current information regarding diet and gene interactions for particular types of cancer is reviewed. As noted above, for most of these associations, only a few studies have been done examining a particular association. Because of the paucity of data, much of our understanding of these relationships is likely to change with further investigation. This review is provided to give some sense of the state of the field, but needs to be regarded as very preliminary. Further, only diet and gene interactions for some cancer sites will be included. Exclusion of a site does not indicate that diet and\/or genetic factors do not play a role in the etiology of that disease, but rather, that there is little research at this time on any possible relations. Also, because the field is a relatively new one and because there are few studies, this review does not include studies that show no association between a diet and genetic factor. The review is limited to those studies showing some association.\n\n### A. Cancer of the Colon and Rectum\n\nConsiderable epidemiologic evidence indicates that dietary factors are associated with cancer of the colon and rectum. There is also some evidence that genetic factors modify those associations. Red meat has been found to be associated with risk with some consistency [3]. One possible mechanism for the effect of red meat on risk is through the intake of heterocyclic amines. Heterocyclic amines are formed in the cooking of meats at high temperature; they can be activated to forms that make them mutagenic and carcinogenic. _N_ -acetyltransferase is an enzyme involved in the activation of heterocyclic amines. Two forms of this enzyme are polymorphic (NAT1 and NAT2), and there is some evidence that those who are rapid acetylators are at increased risk of colorectal cancer. In a few of the studies that have been done, there is also evidence that those individuals with the highest meat consumption who are rapid acetylators are at the highest risk [32]. In a small metabolic study, individuals were fed fried meats and their urine was tested for mutagenicity. Those who were rapid acetylators, or those with the rapid NAT2 phenotype, had significantly more urinary mutagenicity than the slow acetylators over the 6 weeks of this study [33]. Further, as noted above, preliminary evidence indicates that intake of heterocyclic amines is related to risk of polyps of the colon and rectum, and that the association depends both on intake of these substances and on genetic susceptibility based on assessment of the NAT2 and CYP1A2 variants [9].\n\nThere is also evidence that a high intake of fruits and vegetables is protective for colon and rectum cancers [3]. Folate intake may explain part of this effect [23, 24]. As noted above, there is a common genetic polymorphism in the gene for MTHFR. Those with the T\/T genotype have elevated levels of plasma homocysteine [25], lower levels of plasma folate in some but not all studies [25, 34], and different response to folate given as a supplement than do those with the more common C\/C or C\/T genotypes [34]. About 12% of Caucasians and Asians are heterozygous T\/T; the prevalence is lower among African-Americans [34]. In two nested case-control studies, risk was decreased for those with the C\/C genotype. For those with high alcohol intake, low methionine intake, or low plasma folate, the effect of the C\/C genotype was less strong [26, 27]. All of these nutrients are involved in the one-carbon cycle that includes MTHFR.\n\nOne study looked at family history of colorectal cancer in first-degree relatives (parents, siblings, or offspring). They found that for men with a family history of colorectal cancer, the association of risk with intake of beef and alcohol was much higher than for those men who did not report a family history. No such interactions were observed for the women in the study [35]. These findings suggest that other genetic factors interact with diet in the etiology of this disease. Clearly considerable research remains to be done in this area, and our understanding of the etiology of colon cancer may change as these relationships are elucidated.\n\n### B. Breast Cancer\n\nThe evidence linking dietary factors to breast cancer is much less consistent than that for cancer of the colon and rectum. There is quite consistent evidence of a higher risk associated with alcohol intake; vegetable and fruit intake also appear to be related to lower risk [3]. Some preliminary evidence suggests that genetic factors modify both of these dietary risk factors.\n\nAs described above, manganese superoxide dismutase is an important enzyme in the modulation of oxidative stress. There is a relatively common polymorphism of the gene for this enzyme; probably about 20% of Caucasians in the United States are homozygous for the variant. One study indicates that among premenopausal women, those with the variant are at increased risk of breast cancer. Further, it appears that the increased risk is primarily for women with low intakes of fruits and vegetables, or vitamins C or E. For those women with higher intakes, there was less difference in risk by genotype. Among postmenopausal women, some increase in risk was observed with the variant genotype but little modification of risk by reported dietary intake [13].\n\nFairly consistent evidence links regular alcohol consumption to a modest increase in the risk of breast cancer. It may be that this increase in risk is limited to women with a particular genotype. There is a family of enzymes, the alcohol dehydrogenases (ADH), that catalyzes the rate-limiting step in the metabolism of alcohol to acetaldehyde. Acetaldehyde dehydrogenase (ALDH) catalyzes the further oxidation of acetaldehyde. Polymorphisms have been identified for two of the ADH enzymes, ADH2 and ADH3, and for ALDH. The frequencies of those polymorphisms vary by population. In the United States, the ADH3 variant is relatively common among those of European descent, the ADH2 variant is relatively common among those of African descent, and the ALDH variant is common among those of Asian descent.\n\nIn one case-control study, ADH3 genotype was examined in relation to alcohol consumption and risk of premenopausal breast cancer. Those with the variant metabolize alcohol to acetaldehyde more rapidly; acetaldehyde has been identified as a probable carcinogen. The researchers found that women who were above the median for alcohol consumption and who were homozygous for the gene coding for the more rapid form of the enzyme were at increased risk of breast cancer. Women who drank less or who had the other genes were not at increased risk. There was also no apparent gene\u2013alcohol effect for postmenopausal women [36].\n\nAgain, as for colorectal cancer, there is evidence that dietary risk factors may differ for individuals with a family history of breast cancer compared to those with no such history. In one study, among premenopausal women with a first degree relative with breast cancer (mother, sister and\/or daughter), intake of \u03b1-tocopherol was protective, while the association was less strong for women without a family history. There was also evidence that \u03b2-carotene was more strongly related to risk among women without a family history of disease than among those reporting a family history [37]. There are most likely a number of genetic factors that are related to risk of breast cancer and that are modified by dietary intake. These factors remain to be identified. Their identification may not only suggest interventions for women at higher risk, but also elucidate a mechanism for an effect of diet on risk of breast cancer.\n\n### C. Prostate Cancer\n\nProstate cancer rates are high, particularly among older men [38]. There are considerable differences in the rates of this cancer around the world, and the evidence is strong that environmental factors can explain a large part of this variation. Individuals who migrate from regions where the rate of disease is low to regions where the rate is high show higher rates of disease than their counterparts who remain in the region with a low rate [39]. There is not a great deal of evidence regarding the etiology of the disease, although among possible risk factors is vitamin D exposure [38]. Vitamin D plays a role in the control of cell division in the prostate. The vitamin D receptor has genetic variants that have been shown to have functional effects on receptor expression, and preliminary evidence suggests that these vitamin D receptor polymorphisms are related to risk of prostate cancer [12\u201314]. At this point, no studies have examined both vitamin D exposure and genetic variation in relation to risk.\n\n### D. Other Cancers\n\nAlcohol and smoking have been identified as strong risk factors for oral and nasopharyngeal cancers. In general, the levels of alcohol consumption that are associated with risk are much higher than those that have been related to increased risk of breast cancer. As for breast cancer, an interaction of alcohol consumption and ADH3 has been identified. Evidence exists that those individuals who are heavier drinkers and who have the genotype coding for the more active form of the enzyme are at increased risk [40]. Another possible interaction has also been identified. CYP2E1 is related to the metabolic activation of nitrosamines. In one study, a CYP2E1 polymorphism was shown to be associated with risk of nasopharyngeal cancer [41]. However, these researchers did not assess exposure to nitrosamines to determine if there were an interaction.\n\nLittle research has been done related to diet and gene interactions in the etiology of lung cancer. One report stated that the association between smoking and risk of lung cancer was stronger among those individuals who also had the GSTM1null genotype [42]. However, this interaction was found only among those individuals who did not take \u03b1-tocopherol supplements. Among those using \u03b1-tocopherol supplements, there was no modification of the relationship between smoking duration and lung cancer risk by _GSTM1_ genotype. \u03b2-carotene supplement use did not affect the _GSTM1_ and smoking interaction [42].\n\nAs another example, the foodborne mold aflatoxin has been hypothesized to be related to the etiology of cancer of the liver. Epoxide hydrolase is a polymorphic enzyme involved in the metabolic deactivation of aflatoxin. One case-control study indicates that the genetic variant of this enzyme is related to increased risk of liver cancer [43]. DNA adducts of aflatoxin have been shown to be higher in individuals with both the epoxide hydrolase variant and the _GSTM1null_ genotype [43, 44]. There is also evidence of an increased risk of liver cancer associated with the ADH2 variant in a Japanese population [45].\n\n## VI. FUTURE DIRECTIONS\n\nThis review makes it clear that this field is a very new one, and that most of the available evidence is very preliminary. However, the field is also rapidly expanding. New genotypes are being identified at a very rapid pace; many of these have implications for our understanding of the role of dietary factors in cancer etiology. Clearly, as the field progresses, extremely large studies will be needed to allow for the examination of multiple levels of interaction. As in the example given above of an effect of _GSTM1_ genotype on the association between smoking and lung cancer only among men who were not receiving an \u03b1-tocopherol supplement [42], the likelihood exists that multiple layers of interaction will need to be considered to fully understand the relevant etiological pathways.\n\nIn the future, genotype is likely to be examined in metabolic studies to determine the short-term effects on intermediate outcome measures in healthy individuals. A great deal remains to be understood about the relation between genotype and phenotype of the genes that appear to be important in terms of risk. Further, we will need to understand the factors that affect gene induction and the etiologic pathways. More work is going to have to be done to identify the relevant genes that are likely to be both rate limiting and also polymorphic.\n\nThere is little doubt that a large portion of the etiology of cancer can be attributed to environmental factors. Twin studies indicate that genetic factors alone are not sufficient to explain who will get the disease and who will not [46]. Important among the likely environmental factors are dietary factors. With increased understanding of genetic susceptibility as additional risk factors, it may eventually become possible to identify individuals with higher or lower requirements for particular nutrients or to identify individuals with greater sensitivity to agents like alcohol. 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Interaction of family history of breast cancer and dietary antioxidants with breast cancer risk (New York, United States). _Cancer Causes Control_. 1995;6:407\u2013415.\n\n38. Ekman P. Genetic and environmental factors in prostate cancer genesis: Identifying high-risk cohorts. _Eur. Urol_. 1999;35:362\u2013369.\n\n39. Mettlin C. Recent developments in the epidemiology of prostate cancer. _Eur. J. Cancer_. 1997;33:340\u2013347.\n\n40. Harty L.C., Caporaso N.E., Hayes R.B., Winn D.M., Bravo-Otero E., Blot W.J., Kleinman D.V., Brown L.M., Armenian H.K., Fraumeni J.F., Shields P.G. Alcohol dehydrogenase 3 genotype and risk of oral cavity and pharyngeal cancers. _J. Natl. Cancer Inst_. 1997;89:1698\u20131705.\n\n41. Hildesheim A., Chen C.-J., Caporaso N.E., Cheng Y.-J., Hoover R.N., Hsu M.-M., Levine P.H., Chen I.-H., Chen Y.-Y., Daly A.K., Idle J.R. Cytochrome P4502E1 genetic polymorphisms and risk of nasopharyngeal carcinoma: Results from a case-control study conducted in Taiwan. _Cancer Epidemiol. Biomarkers Prev_. 1995;4:607\u2013610.\n\n42. Woodson K., Stewart C., Barrett M., Bhat N.K., Virtamo J., Taylor P.R., Albanes D. Effect of vitamin intervention on the relationship between GSTM1, smoking and lung cancer risk among male smokers. _Cancer Epidemiol. Biomarkers Prev. 1999_. 1999;8:865\u2013970.\n\n43. McGlynn K.A., Rosvold E.A., Lustbader E.D., Hu Y., Clapper M.L., Zhou T., Wild C.P., Xia X.-L., Bafoe-Bonnie A., Ofori-Adjei D., Chen G.-C., London W.T., Shen F.-M., Buetow K.H. Susceptibility to hepatocellular carcinoma is associated with genetic variation in the enzymatic detoxification of aflatoxin B1. _Proc. Natl. Acad. Sci. USA_. 1995;92:2384\u20132387\n\n44. Rebbeck T.R. Molecular epidemiology of the human glutatione S-transferase genotypes GSTM1 and GSTT1 in cancer susceptibility. _Cancer Epidemiol. Biomarkers Prev_. 1997;6:733\u2013743.\n\n45. Lai, C., and Shields, P. G. The role of interindividual variation in human carcinogenesis. _J. Nutr._ 552S\u2013555S.\n\n46. Greenwald P. Cancer risk factors for selecting cohorts for large-scale chemoprevention trials. _J. Cell. Biochem_. 1996;25S:29\u201336.\nCHAPTER 14\n\nInborn Errors of Metabolism\n\nCRISTINE M. TRAHMS, University of Washington, Seattle, Washington\n\n## I. INTRODUCTION\n\nRecent dramatic advances in genetics at the laboratory and clinical levels have presented opportunities for understanding and promoting health and decreasing the morbidity and mortality associated with metabolic disorders. The treatment of inborn errors of metabolism is, in fact, fairly new to medicine and nutrition. Forty years ago, some services for children with inborn errors of metabolism were offered through child development clinics; however, the children were always neurologically compromised because of late diagnosis. At that time, the diagnosis of an inborn error of metabolism was based on clinical symptoms, which often occurred after the initial neurological damage. By the late 1960s, state newborn screening programs were under way and infants were identified before neurological damage occurred. It was at this point that the impact of nutrition intervention in the treatment of inborn errors of metabolism could be appreciated and is particularly beneficial to the affected individual, family, and society.\n\n### A. Public Health Services\n\nAlthough inborn errors of metabolism are individually rare, as a group they have a significant impact on the health care system. The greatest savings to health care services occurs when individuals are identified before physical and neurological damage occurs and when they are consistently treated so that damage is prevented. Thus, the backbone of an effective management program for inborn errors of metabolism is an effective newborn screening program.\n\nMaternal and child health agencies often provide services to meet the health care needs of infants and children with metabolic disorders. Essential public health functions related to inborn errors of metabolism can be described as follows:\n\n1. _Infrastructure planning services._ Policy development, needs assessment, evaluation of systems, quality of care, and coordination of services are functions to which health care providers for children with metabolic disorders must ascribe to ensure the best possible health outcomes.\n\n2. _Population-based services._ Newborn screening, oral health, nutrition education, genetic counseling, and public outreach are essential components of early identification and treatment of children with metabolic disorders.\n\n3. _Enabling services._ Care coordination, transportation, translation, and family support services are needs that many families with fragile children with metabolic disorders require.\n\n4. _Direct health care services._ Direct provision of basic health services is mandatory if these children are to survive.\n\n### B. Definition of Inborn Errors of Metabolism\n\nInborn errors of metabolism make up a large group of rare disorders caused by an inherited deficiency or absence of proteins that have enzymatic, carrier, receptor, or structural roles. For many of these disorders, the molecular nature of the disorder is known and the chromosomal locus of the gene has been mapped. It is not yet possible to cure any of these disorders with gene replacement or gene repair. However, as knowledge of the pathophysiology of each disorder is more completely understood, plans of management that mitigate the effect of the disorder can be instituted.\n\nThe most frequently identified disorders are those in which the absent or defective protein serves an enzymatic function. This diverse group of disorders responds to nutritional therapy intervention and will be discussed in this chapter.\n\nMuch work has been done to improve diagnosis, nutritional intervention, and medical intervention for inborn errors of metabolism. The goal of intervention is to prevent the ravages of the missing or defective enzyme. For most disorders, a \"shotgun\" approach is currently available, whereas a \"laser\" approach to treatment is needed to completely prevent the impact of the disorder. The two discrete parts to effective intervention for inborn errors of metabolism are (1) an immediate diagnosis and initiation of treatment, which require the support of an effective newborn screening program; and (2) a long-term approach to care, in which treatment is continued to avoid symptoms of the enzyme deficiency and to try to normalize the biochemical parameters caused by the disorder.\n\n### C. Newborn Screening Programs\n\nThe evolution of newborn screening program (mass populations screening) efforts has resulted in a more positive outcome of treatment for many metabolic disorders. Newborn screening is a preventive public health measure available to all neonates. Population-based newborn screening is a major factor in making effective nutrition intervention possible. If infants are identified in the first few days of life, the diagnostic process can be completed and treatment started before damage occurs.\n\nThe American Academy of Pediatrics recommends that a newborn screening blood sample be collected before the infant is discharged from the nursery. If the blood sample is collected before the infant is 24 hours of age, it is essential that a second screening sample be obtained [1]. A general newborn screening (NBS) timetable is shown in Table 1.\n\nTABLE 1\n\nGeneral Timetable for Collecting Newborn Screening (NBS) Samples\n\nSuccessful newborn screening, of course, depends on reliable, valid, and timely laboratory results. Many states have established a central newborn screening laboratory, which processes a large enough number of samples to maintain quality control. A qualitative newborn screening report offers a presumptive positive result that must be quantitatively confirmed. Laboratory confirmation of diagnosis is mandatory.\n\nAn effective newborn screening program involves laboratory, education, administration, follow-up, management, and evaluation components [2, 3]. Each state has developed individual legislative newborn screening mandates. However, many commonalities are seen, as shown in Table 2. Most states provide newborn screening for phenylketonuria, congenital hypothyroidism, galactosemia, and the hemoglosbinopathies. These disorders all meet the criteria for newborn screening shown in Table 3 [1].\n\nTABLE 2\n\nNumber of States Screening for Selected Disorders in the United States\n\nDisorder | Number of states screeninga | Incidence per 1000 live newborns\u2014general U.S. population \n---|---|--- \nPhenylketonuria | 53\/53 | 1:10,000\u20131:25,000 \nCongenital hypothyroidism | 53\/53 | 1:3,600\u20131:5,000 \nGalactosemia | 46\/53 | 1:60,000\u20131:80,000 \nHemoglobinopathies | 45\/53 | 1:385\u20131:835 (African-American) 1:40,000\u20131:60,000 (non-African-American) \nMaple syrup urine disease | 25\/53 | 1:250,000\u20131:400,000 \nHomocystinuria | 22\/53 | 1:50,000\u20131:150,000 \nBiotinidase deficiency | 21\/53 | 1:72,000\u20131:126,000 \nCongenital adrenal hyperplasia | 11\/53 | 1:12,000 \nTyrosinemia | 7\/53 | 1:120,000 (Swedenb) \nCystic fibrosis | 4\/53 | 1:2,000 (northern European extraction)\n\naIncludes District of Columbia, Puerto Rico, and Virgin Islands.\n\nbNo current U.S. data.\n\n_Source:_ Adapted from Committee on Genetics. (1996). Newborn screening fact sheets. _Pediatrics_ **98,** 473\u2013501.\n\nTABLE 3\n\nGenerally Accepted Criteria for Newborn Screening for a Specific Disorder\n\n\u2022 Symptoms usually absent in newborns.\n\n\u2022 Disease results in developmental impairment, serious illness, or death.\n\n\u2022 Sensitive, specific laboratory tests available on a mass population basis.\n\n\u2022 Disease occurs frequently enough to warrant screening.\n\n\u2022 Successful treatment procedures are available.\n\n\u2022 Benefits of screening justify the cost.\n\n\u2022 Follow-up and treatment programs are available.\n\n_Source:_ Adapted from Committee on Genetics. (1996). Newborn screening fact sheets. _Pediatrics_ **98,** 473\u2013501.\n\nMost newborn screening programs use a blood sample dried on filter paper to complete a bacterial inhibition assay. This type of sample requires a very small amount of blood, is easily transported to the central laboratory (after the blood has dried), and several tests can be completed on a single blood sample. Newer technologies, such as tandem mass spectrometry, have the potential to increase the number of disorders that can be effectively screened [4].\n\nMany of the disorders included in newborn screening programs are autosomal recessive. This means each parent carries the affected gene and the incidence is 1:4 for each pregnancy. Thus affected infants identified by newborn screening usually do not have a positive family history of the disorder.\n\nWith newborn screening programs providing an opportunity for earlier diagnosis, and with recent laboratory developments and increased specificity of treatment modalities, the long-term outcome for persons with inborn errors of metabolism is brighter than in the past. The contrast between outcome without early treatment and expected outcomes with early identification and treatment is shown in Table 4. For many disorders, however, treatment is a recent development, and no long-term outcome data are available.\n\nTABLE 4\n\nOutcome for Inborn Errors of Metabolism with and without Early Diagnosis\n\nDisorder | Outcome without treatment | Expected outcome with early identification and treatment \n---|---|--- \nPhenylketonuria | Mental retardation (IQ < 40) | Normal IQ with lifelong treatment \nMaple syrup urine disease (MSUD) | Encephalopathy \u2192 death | Variable; may be cognitively compromised \nTyrosinemia, Type I | Liver failure \u2192 death | With NTBC treatment, encouraging data on preventing neurologic crises, renal and hepatic failure, rickets \nGlycogen storage diseases | Hypoglycemia, liver CA \u2192 death | Typical growth, development \nGalactosemia | Sepsis \u2192 severe delays, death | Often learning disabilities \nUrea cycle disorders | Hyperammonemia \u2192 severe delays or death | Variable; may be cognitively compromised \nFatty acid oxidation disorders, e.g., long-chain acyl-CoA dehydrogenase deficiency (LCAD), medium-chain acyl-CoA dehydrogenase deficiency (MCAD), short-chain acyl-CoA dehydrogenase deficiency (SCAD) | Encephalopathy, hepatomegaly, often death which is attributed to SIDS | No long-term data; encouraging short-term data \nOrganic acidurias, e.g., methylmalonic aciduria, propionic acidemia | Metabolic acidosis \u2192 coma \u2192 death | Variable; may be cognitively compromised \nGlutaric acidemia I | Impaired movement, dystonia, vomiting, seizures, coma | No long-term data \nHomocystinuria | Cardiac problems, organ damage, psychiatric disturbances, death | Variable; may be physically and cognitively compromised \nIsovaleriac acidemia | Metabolic acidosis \u2192 coma \u2192 death | Variable; may be cognitively compromised\n\n## II. DIAGNOSIS\n\nAlthough all states in the United States have a newborn screening program and screen for a variety of disorders, many disorders are not currently part of newborn screening programs. The treatment of all metabolic disorders, whether tested by newborn screening programs or not, requires a well-organized system of laboratory analysis and monitoring. There are two phases of management: (1) the initial, acute diagnostic phase, and (2) the long-term management phase.\n\nThe acute diagnostic phase requires attention to the symptoms presented by the infant. Most toxic metabolites cross the placenta and are cleared by the mother. Thus the severity of the defect and the initial feeding may affect the timing of the onset of symptoms. Depending on the disorder, the onset of symptoms can range from hours to months. If infants do not become ill in the early postnatal period, it is common for the metabolic disorder to be manifested when the child is weaned from breast milk or when high-protein solids are added to the diet.\n\nIn the newborn period, there are general, nonspecific symptoms that warrant further investigation. The most frequent signs of \"trouble\" are lethargy, poor feeding, and vomiting. Apnea and respiratory distress are significant symptoms that may indicate the presence of a metabolic disorder, as do jaundice and increased liver enzymes. These general symptoms usually move quickly into a more precarious situation for the infant.\n\nInfants may frequently present with encephalopathy as a result of accumulating toxic metabolites in the central nervous system. Examples of this are organic acid disorders, urea cycle disorders, and amino acid disorders. Blood ammonia levels should be checked if the infant is lethargic, vomits, or has signs of encephalopathy. Metabolic acidosis with an increased anion gap is another sign of an infant with a metabolic disorder, as are jaundice or increased liver enzyme activity [5]\n\nAppropriate and aggressive treatment before confirmation of the diagnosis may save the life of the infant and reduce the possibility of neurological sequelae. The first step is to remove offending metabolites. If a disorder of amino acid metabolism or protein intolerance is suspected, protein intake should immediately be discontinued. Hemodialysis may be necessary to decrease blood ammonia and\/or amino acids levels [6]. The second step is to prevent catabolism. This may require administration of intravenous glucose.\n\n## III. TREATMENT: INITIATION AND MONITORING\n\nThe approach to treatment for each disorder depends on the enzyme(s) affected and the metabolic consequences of the diminished enzyme(s). The goal of treatment for inborn errors of metabolism is to strive for correction of the biochemical abnormality. Although the clinical and biochemical picture of each disorder is unique and ranges from mild to life-threatening illness, metabolic disorders can be thought of as a group in which the absence or inactivity of a specific enzyme or cofactor causes the buildup of the substrate and\/or deficiency of the product. The outcome of treatment for metabolic disorders is variable and depends on early diagnosis and intensive and continuous intervention. Nutritional therapy is the primary mode of treating metabolic disorders.\n\nIn this section, the concepts, principles, and strategies of treatment of disorders of protein, carbohydrate, and fat metabolism will be discussed. A detailed approach to management of disorders amenable to treatment or a catalog of all possible disorders will not be provided. A complete discussion of diagnosis and management of the vast array of metabolic disorders can be found in Scriver _et al._ [7], McKusick [8], or the Online Mendelian Inheritance in Man (OMIM): [9]. Each disorder has been assigned a specific Mendelian Inheritance in Man (MIM) number, which will be used in this chapter to differentiate similar disorders from each other and to avoid confusion. Table 5 outlines the essential components of nutritional therapy for treatment of metabolic disorders. Table 6 outlines some metabolic disorders that respond to nutritional therapy.\n\nTABLE 5\n\nComponents of Nutritional Therapy for Metabolic Disorders\n\n\u2022 Identify precise diet appropriate to the problem.\n\n\u2022 Provide nutritional surveillance to ensure that diet is adequate.\n\n\u2022 Provide a mechanism for follow-up of child for symptoms of nutritional deficiency or toxicity.\n\n\u2022 Provide emotional and educational support for child and family.\n\nTABLE 6\n\nSelected Metabolic Disorders That Require Nutritional Therapy\n\na2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione that is an inhibitor of 4-hydroxy-phenylpyruvate dioxygenase.\n\nbSodium benzoate and sodium phenylbutyrate are chemicals administered to enhance waste ammonia excretion; other compounds producing the same effect are also used.\n\n### A. Principles of Treatment\n\nThe two major principles of treatment are (1) to mitigate the effect of the altered gene by modifying components of diet to adjust the environment at the cellular level, and (2) to provide protein, energy, and nutrients to support growth and development.\n\nFor many disorders, the treatment is determined by the identification of the missing or inactive enzyme. In an effort to modify the detrimental effect of the decreased or absent enzyme activity, the paradigm of \"working around\" the enzyme is used. In many cases, decreasing the substrate available for the reaction and supplementing the product to \"normal\" levels prevent or decrease the deleterious effects of the disorder. An example of this strategy is in the treatment of phenylketonuria (MIM 261600): Phenylalanine (substrate) is restricted because of the absence or inactivity of phenylalanine hydroxylase (enzyme), and tyrosine (product) is supplemented. In some disorders, the defective enzyme is further down the amino acid degradation pathway and may affect the metabolism of two or more amino acids [lysine and tryptophan in glutaric acidemia (MIM 231670) or leucine, isoleucine, and valine in maple syrup urine disease (MSUD, MIM 248600)]. The amino acids in most aminoac-idopathies are essential, that is, they cannot be synthesized by the body and therefore must be provided in the diet. This is critical because these essential amino acids must be provided at the requirement level so the child can grow and develop, but they must also be limited so there is no toxic build-up of amino acid(s) that cannot be metabolized.\n\nIn some disorders, the additional step of enhancing enzyme activity by supplying its co-factor can be helpful. An example of this is the provision of pharmacological doses of biotin in biotinidase deficiency (MIM 253260) [10] or of vitamin B6, on which cystathionine \u03b2-synthase (MIM 236200) is dependent, in some forms of homocystinuria [11].\n\nIn metabolic disorders of carbohydrate metabolism, such as galactosemia (MIM 230400), the nutrient of concern, galactose, is not essential. Therefore, the goal of effective therapy is to eliminate as much of the exogenous component as possible from the diet. Other sources of nourishment need to be provided to compensate for the omitted foods and nutrients.\n\nDisorders of fatty acid metabolism require a source of energy other than fat because fat cannot be metabolized to meet energy needs. Fatty acids of specific carbon lengths are often minimized or eliminated, depending on whether or not the fatty acid is essential for growth and development. Most fats contain a mixture of fatty acids of varying carbon lengths, making it difficult to eliminate specific fatty acids. In the cases of long-chain acyl-CoA dehydrogenase deficiency (MIM 210460) or very long chain acyl-CoA dehydrogenase deficiency (MIM 201475), shorter chain fatty acids can be metabolized [12]. Frequently, the larger problem is providing enough energy for these children to avoid oxidation of long-chain fatty acids to medium-chain derivatives. The shorter chain fatty acids are often supplemented, for example, medium-chain triglycerides as MCToil. However, in medium-chain acyl-CoA dehydrogenase deficiency (MIM 201450), sources of medium-chain fatty acids can be eliminated without problems. Other essential components of treatment are (1) supplementation of L-carnitine, an amino acid that functions in transport of fatty acid acyl-CoA esters during mitochondrial beta-oxidation; and (2) avoidance of fasting, to reduce the accumulation of partially oxidized metabolites associated with impaired energy production [13].\n\nIn all metabolic disorders, nutritional therapy addresses the symptoms of the disorder but not the disorder itself. Perhaps when gene therapy matures as a science, this option will be available.\n\n### B. Specialized Metabolic Team\n\nAs a group, children with metabolic disorders comprise a small percentage of the pediatric population, and even the most common of these disorders is rare in the general population. However, the health care needs of these children are specific and urgent. The American Academy of Pediatrics has recommended that a team experienced in management supervise the therapy of children with metabolic disorders [1]. Effective treatment requires the expertise of a team of health care professionals, generally comprised of a geneticist, nutritionist (registered dietitian), nurse, genetic counselor, psychologist, and neurologist. This team of experts is familiar with the nuances of current treatment for metabolic disorders and will be able to incorporate new research innovations as they are deemed appropriate. The complex nutritional and medical management of these children cannot occur effectively without the follow-up and support of the community. Communication between the team at the tertiary center, the community, and the family is crucial for supporting the best possible medical, nutritional, and intellectual outcome for these children.\n\n### C. Role of the Nutritionist\n\nThe nutritionist who works with families whose children have inborn errors of metabolism must be meticulous in the understanding of metabolic disorders and should develop a collaborative support system with team members and the family.\n\n### D. Providing a Metabolic Diet\n\nGeneral principles of protein and energy management address the issues of biochemical control specific to the disorder. These principles also address the needs of overall nourishment: (1) The child should be provided adequate amino acids, total protein, and nitrogen to support growth; (2) the child should be provided adequate energy to support growth and prevent growth failure; (3) energy needs may be increased when L-amino acids provide the protein equivalent; (4) maintaining an adequate energy intake is essential in preventing catabolism; and (5) suppression of destructive metabolites can produce a striking biochemical and clinical improvement [14, 15].\n\n### E. Nutritional Assessment\n\nThree components of nutritional assessment are necessary for the evaluation of children with metabolic disorders: (1) assessment of growth and comparison to appropriate guidelines, (2) assessment of nutrient intake and comparison to requirements for treatment of the specific disorder and the recommended intakes for all children, and (3) assessment of biochemical parameters specific to the disorder and those of appropriate nourishment.\n\n### F. Assessment of Growth\n\nNormal parameters of physical growth are expected for many children with metabolic disorders, provided that all of the components of nourishment appropriate to the diagnosis are provided [16]. Optimal nutritional support is required to meet these goals. As for all children, appropriate growth reflects an adequate intake of protein, energy, vitamins, and minerals. For children with metabolic disorders, appropriate growth also reflects the achievement of metabolic balance. For growth to proceed in a normal fashion, energy and all required nutrients must be provided in adequate amounts, while the nutrients that the individual cannot effectively metabolize must be limited to the requirement level or entirely eliminated.\n\nGrowth differences are typical of children with metabolic disorders. Poor growth is often a reflection of several factors. Infants who have endured a severe neonatal illness may require an extended time of appropriate nourishment before they catch up in growth. Frequent febrile illness may interfere with achieving expected physical growth. Children with repeated episodes of hyperammonemia or metabolic crisis are not able to grow. Slowed or altered growth patterns often reflect poor metabolic control or an inadequate protein or energy intake. However, slowed or altered growth in a child with a metabolic disorder may not be the result of noncompliance with treatment on the part of the family. Many of these children with little or no enzyme activity are medically fragile and are difficult to bring into and maintain in metabolic balance.\n\nThe disorder itself may affect growth and growth achievement. Older children with glycogen storage disease, for example, are often shorter than their age peers. Younger children with glycogen storage disease who have had the benefit of intensive treatment with cornstarch are more likely to achieve their genetic potential for physical growth. Children with galactosemia are usually shorter and lighter than their peers without galactosemia. There is no clear explanation for this altered growth pattern.\n\n### G. Growth Assessment Parameters\n\nGender- and age-appropriate parameters for growth assessment are weight, length or stature, weight for length or stature, skinfold measures, and increments of growth in weight and length or stature. Assessment of growth or size is the basis for many therapeutic recommendations for children with metabolic disorders. These measurements, therefore, need to be completed by someone who is properly trained to conduct them, who has a knowledge of accurate and reliable techniques, and who knows how to operate the standardized equipment [17].\n\n### H. Assessment of Nutrient Intake\n\nThe welfare of the infant or child with a metabolic disorder depends on accurate and minute adjustments of one or more dietary components. Parents are urged to provide an accurate record of food and formula intake so that, in cooperation with the metabolic nutritionist, an accurate assessment of intake of critical nutrients as well as total protein and energy intakes can be made.\n\n### I. Assessment of Biochemical Parameters\n\nMaintaining metabolic balance for children with metabolic disorders requires frequent and intensive monitoring of biochemical parameters specific to the disorder. It also requires monitoring parameters that reflect general nutritional status. The goal of treatment for all of these disorders is to achieve biochemical levels at or near the normal range. Laboratory parameters that are frequently monitored include plasma amino acids, urine organic acids, hematologic status, protein status, electrolytes, and blood lipid and ammonia levels. A general plan for biochemical assessment is shown in Table 7.\n\nTABLE 7\n\nMonitoring for Children with Metabolic Disorders Biochemical, Growth, and Nutritional Parametersa\n\naThe frequency of monitoring biochemical parameters depends on the age and health status of the child. Many parameters are monitored monthly if the infant or child is well; more frequently if the child is ill. Other parameters are monitored at each clinic visit. Parameters of general nutritional status may be monitored biannually or as needed.\n\n### J. Providing Adequate Nutrition\n\n1. PROTEIN\n\nFor some disorders, total protein is not restricted, but the composition of the protein may be adjusted. The treatment of phenylketonuria, for example, requires the restriction on phenylalanine intake but not total protein. This is accomplished with the use of a specially designed semisynthetic formula. For other disorders, total protein intake is restricted. For the treatment of ketone utilization disorders (MIM 203750), for example, total protein is often restricted to 1.5 g\/kg\/day. Infants and children with disorders of amino acid metabolism must obtain most of their protein from specialized metabolic formulas. Natural foods seldom provide more than 25% (often 10%) of the protein requirements of infants and children with amino acid, organic acid, or urea cycle disorders. It is generally not possible to provide an adequate intake of total protein without providing an excess of the offending amino acids. Some disorders, such as the organic acidopathies, require an overall total protein restriction as well as provision of amino acids from a specialized formula. Table 8 indicates the strategies of protein restriction for infants and children with metabolic disorders.\n\nTABLE 8\n\nExamples of Restricted Protein Diets for the Treatment of Metabolic Disorders\n\nProtein adjustment needed | Disorder \n---|--- \nNo restriction in protein | Fructose intolerance \nProvide total protein intake at recommended level + eliminate specific protein-containing foods, e.g., all lactose, galactose containing proteins | Galactosemia \nProvide total protein intake at recommended level + eliminate specific protein-containing foods, e.g., all high-fat protein foods | Glycogen storage diseases, fatty acid oxidation defects \nRestrict protein intake to physiological tolerance; provide small amounts of HBVa proteins | Mild urea cycle and organic acid disorders, ketone utilization disorders \nRestrict protein intake to physiological tolerance; provide small amounts HBV protein + specific amino acids | Severe urea cycle and organic acid disorders \nProvide total protein intake at recommended level + restrict one or more amino acids to requirement level | Phenylketonuria, maple syrup urine disease, isovaleric acidemia, glutaric adidemia type I\n\naHBV, high biological value.\n\nProtein in the specialized formulas is provided as individual amino acids (excluding those amino acids, which are contraindicated for each condition). Amino acids are more readily oxidized than intact protein, thus the requirement for protein when amino acids are the protein source is greater than usual. The overall diet for the treatment of these disorders is not low in total protein, but low in specific amino acids. The infant or child will not thrive without the use of specialized formulas and the amino acids they contain. Adequate protein and energy are required to maintain an anabolic state. If adequate formula is not prescribed or consumed, a catabolic state will develop, causing both high plasma amino acids levels and clinical problems.\n\n#### 2. ESTABLISHING AMINO ACID REQUIREMENTS FOR THE INDIVIDUAL INFANT OR CHILD\n\nThe requirements for essential amino acids have been established [18] and are used as clinical guidelines. Frequently, these guidelines are used to establish the diet for a newly diagnosed infant or child and are adjusted as necessary to stabilize critical plasma levels. Table 9 describes situations that may arise when components of protein nutriture are adjusted inappropriately. Specialized metabolic formulas have been designed to meet the unique protein, carbohydrate, and fat needs of these infants and children. Sources of metabolic formulas are shown in Table 10.\n\nTABLE 9\n\nPotential Problems with Amino Acid Prescriptions\n\naHBV, high biological value.\n\nTABLE 10\n\nSources of Formulas and Low Protein Foods for the Treatment of Metabolic Disorders\n\nThe urea cycle disorders require the restriction of overall protein intake because excess nitrogen from any source can be toxic. However, it is also imperative to provide enough protein and energy to support growth. This fine line requires extensive collaboration between the family and the metabolic team. The formulas for the treatment of urea cycle disorders have a high concentration of amino acids to ensure ready incorporation into protein. Most children with these disorders also require medications to enhance the excretion of excess nitrogen through secondary pathways other than the urea cycle. For infants and children with urea cycle disorders, catabolism from excess protein, weight loss, illness, or infection are dangers. Hyperammonemia can occur rapidly (in several hours) and can be life threatening. Effective management of children with urea cycle disorders requires frequent and intense monitoring. The total amount of protein tolerated by the individual child depends on residual enzyme activity, age, and growth rate. The proper amount of protein is determined by considering growth rate, plasma amino acids, ammonia, ketones, and urine organic acids specific to the disorder.\n\n#### 3. SUPPLYING RESTRICTED AMINO ACIDS\n\nSmall amounts of the \"toxic\" essential amino acids specific to each disorder are required for growth and development. These essential amino acids are provided to the infant by including small amounts of proprietary infant formula in the metabolic formula mixture. During infancy, adding small amounts of cow's milk or soy formula can provide an infant with these essential amino acids. In older children, the addition of fruits, vegetables, and grains can provide these amino acids.\n\n#### 4. BREASTFEEDING\n\nSome infants with metabolic disorders are able to maintain low and stable plasma amino acid levels with a combination of breast and metabolic formula feedings. Many infants, however, are too metabolically fragile to maintain appropriate plasma levels with breastfeeding. Infants with phenylketonuria are most likely to be able to tolerate partial breastfeeding and maintain low plasma phenylalanine levels. Breastfeeding is overtly contraindicated for infants with some disorders, such as galactosemia.\n\n### K. Energy\n\nProtein and energy needs are intertwined. If sufficient energy is not provided in the diet, the body will use protein as an energy source to meet essential energy needs. When protein is used as an energy source, it is not available for incorporation into protein-containing substances such as muscle, enzymes, and hormones. Protein catabolism can have significant metabolic consequences in some metabolic disorders, such as the organic acid and urea cycle disorders where catabolism is problematic.\n\nInfants and children with metabolic disorders have increased energy needs, often as great as 120\u2013140 kcal\/kg in infancy [14]. Energy needs vary from disorder to disorder and from child to child. Therefore, it is essential to carefully monitor growth, biochemical parameters, and nutrient intake to evaluate the adequacy of treatment.\n\nAdditional protein-free energy sources are an important adjunct to dietary management of many metabolic disorders. Foods that have been modified to be low in protein, such as flours, pastas, and baked products, are an appropriate and necessary source of energy and variety in the diet. Sources are listed in Table 10.\n\n### L. Vitamins and Minerals\n\nWhenever a significant group of foods is eliminated or severely restricted from the diet of an infant or child, a plan for replacement of essential nutrients must be in place. For example, children whose diets are restricted in protein must eliminate muscle meats and most dairy products from their diets. Thus, they eliminate sources of vitamin B12, iron, zinc, calcium, and other nutrients. Generally, the most reliable method of nutrient replacement for these nutrients is from a specially formulated and supplemented formula.\n\n### M. Fats\n\nWhenever a specific nutrient or dietary component is eliminated or severely restricted, a plan for replacement of that component must be in place. The essential fatty acids cannot be endogenously synthesized and so must be provided from external sources. Deficiency of essential fatty acids is avoided by providing 2\u20134% of total energy intake as linoleic acid.\n\n### N. Notes on Management Strategies\n\n1. HYDRATION\n\nStrict attention must be paid to fluid status, because dehydration in children with metabolic disorders can precipitate a metabolic crisis. Fluid intake and fluid requirements must be carefully monitored. Constipation can also cause metabolic imbalance for these infants and children. Both the osmolality and renal solute load of the formula mixtures should be in the safe ranges for age and health status.\n\n#### 2. CHILDHOOD ILLNESSES\n\nThe usual childhood illnesses often pose a medical crisis for the child with a metabolic disorder. Frequently, children require hospitalization and the administration of intravenous fluids to prevent metabolic crisis. During an illness that results in catabolism, sources of protein are often refused. Continued administration of some form of fluid and energy assists in rehabilitation.\n\n#### 3. FEEDING\n\nSome children with metabolic disorders who have neurological difficulties develop oral-motor problems that interfere with the intake of adequate nourishment. A hyperactive gag reflex is the most frequent problem. Some providers use nasogastric or gastrostomy tubes to support an adequate intake of nourishment and medications.\n\nPoor appetite is not uncommon for children with metabolic disorders, especially urea cycle disorders, MSUD, and the organic acidemias. Poor appetite and poor feeding may be organic, behavioral, or both. Buildup of toxic metabolites is frequently the offender. Many children with metabolic disorders who have had acute episodes may also have a history of vomiting, which may lead to feeding avoidance. Families are fully aware that a child who does not eat may face metabolic disaster, and this stress intensifies the situation. Many families have found specialized feeding teams to be helpful.\n\nChildren who have age-appropriate oral-motor skills should be encouraged to use them. Foods should be offered that provide a variety of textures and encourage biting, chewing, and the movement of the bolus of food in the mouth.\n\n#### 4. ORAL HEALTH\n\nChildren with metabolic disorders may be at increased risk for dental caries and dental erosion due to the nature of their therapeutic diets. The combination of frequent intake of carbohydrates and the acidic nature of the protein formulas appear to put these children at increased risk for oral health problems [19]. It is important that oral hygiene and regular tooth brushing be an important part of everyday health management.\n\n#### 5. FOOD-RELATED BEHAVIORS\n\nThe requirement of the ingestion of a specialized semisynthetic formula and accompanying food restrictions appropriately cause parents to be anxious. Parents and children may then be predisposed to the development of adverse interactions around food. A thorough knowledge of typical food patterns and the development of feeding skills and behaviors around food will enable the nutritionist and other team members to appropriately support and guide the family as they work to provide an appropriate feeding environment for their child.\n\n#### 6. PARENT\u2013CHILD INTERACTION\n\nThe rigorous requirements of nutritional therapy may place stresses on the parent\u2013child relationship. Families should be supported as they strive to better understand the metabolic disorder in their family, to adjust personal schedules to meet the rigor of nutritional and medical therapies for the child, and to maintain a healthy family environment for all family members.\n\n_Bright Futures: Guidelines for Health Supervision of Infants, Children, and Adolescents_ is an important guideline for health supervision for all children. Families whose children have metabolic disorders will find these developmentally focused guidelines to be particularly helpful. The _Bright Futures_ materials can be found at .\n\n## IV. NEW TREATMENT DEVELOPMENTS\n\nA. Maple Syrup Urine Disease or Branched-Chain Ketoaciduria\n\nMSUD is an autosomal recessive disorder with a metabolic block at the decarboxylation of branched-chain alpha keto-acids derived from leucine, isoleucine, and valine. The accumulation of branched-chain amino acids (BCAA) and other compounds exerts neurotoxic effects. MSUD is heterogeneous and ranges from severe to mild. Infants with the severe manifestation usually present in the emergency room of the hospital with encephalopathy that quickly escalates to coma. If untreated, the disease is lethal. L-Alloisoleucine is the most specific and sensitive diagnostic marker for all forms of MSUD [20].\n\nTreatment comprises dietary management, with strictly reduced quantities of protein and BCAA, as well as aggressive intervention during acute neonatal and subsequent metabolic complications. Recently, hyperalimentation solutions without the offending amino acids have been successfully used to curtail acute episodes [21].\n\nMSUD is regarded as a metabolic disorder that has a potentially favorable outcome with carefully supervised longterm therapy. Liver replacement has been considered because of the difficulty of careful chronic supervision of dietary treatment. With successful liver transplant, patients no longer require protein restriction, and the risk of metabolic decompensation during catabolic events is eliminated. However, it is not known whether the overall risks of liver transplantation outweigh the benefits and\/or if outcome is significantly different than that with dietary management [22].\n\n### B. Tyrosinemia\n\nTyrosinemia is an autosomal recessive disorder with a metabolic block at fumarylacetoacetate hydrolase. It is a devastating metabolic disorder that presents as severe liver disease at less than 6 months of age. If tyrosinemia is untreated, affected infants will die from liver failure within a few weeks or months after the onset of symptoms. Deficiency of fumaryl-acetoacetic hydrolase allows the accumulation of fumarylacetoacetate and the subsequent conversion to succinylacetone. It is believed that the accumulated succinylacetone is toxic and responsible for the liver damage.\n\nThe three treatment strategies are (1) dietary intervention that limits the precursor amino acids phenylalanine and tyrosine, and (2) metabolic inhibition of the tyrosine pathway, or (3) liver transplantation. Although dietary restriction alone slows the process of the disorder, it does not prevent liver damage. Liver transplantation has been the treatment of choice for infants and children who were healthy enough for the procedure [23]. Even with a successful transplantation procedure, succinylacetone continues to be detected in the urine. The drug 2-(2-nitro-4-trifluoromethylbenzoyl)\u22121, 3-cyclohexanedione (NTBC) is used to block the formation of fumarylacetoacetate and succinylacetone by inhibiting the tyrosine pathway. Currently in clinical trials, NTBC therapy plus dietary phenylalanine and tyrosine restrictions look very promising for significantly improving the outcome for these children [24].\n\n### C. Phenylketonuria\n\nDietary treatment of phenylketonuria is recognized as one of the first effective treatments of genetic disease. Phenylketonuria is an autosomal recessive disorder with a metabolic block at phenylalanine hydroxylase that allows the accumulation of phenylalanine and prevents the conversion to tyrosine. The abnormal metabolites phenylpyruvate and phenylacetate are produced. Infants show no signs or symptoms of metabolic derangement. Prior to newborn screening, all individuals with phenylketonuria were identified because of significant development delays at 6\u20139 months of age.\n\nPhenylalanine levels of 10\u201320 times normal are toxic to the developing brain. There has been great debate about treatment for phenylketonuria on two counts: (1) the degree to which plasma phenylalanine levels need to be lowered to prevent neurological damage and (2) the duration of treatment. There is now consensus that (1) restricted phenylalanine therapy must be continued on a lifetime basis, and (2) plasma phenylalanine levels 1\u20136 times normal provide a better environment for normal intellectual development than higher levels [25\u201327]\n\nAlthough fewer than 2% of infants with elevated phenylalanine levels will have defects in dihyropteridine reductase (MIM 261630), it is recommended that infants with elevated phenylalanine levels be further evaluated for this disorder, which results in progressive neurological deterioration [1].\n\n### D. Galactosemia\n\nGalactosemia is an autosomal recessive disorder with a metabolic block at galactose-1-phosphate uridyl transferase leading to a buildup of galactose and galactose-1-phosphate. Galactose-1-phosphate is believed to be the neurotoxic compound. Infants appear unaffected at birth and generally become ill during the first week of life after ingestion of galactose. Blood levels of galactose-1-phosphate are elevated and thought to be toxic to the liver, kidneys, and central nervous system. Treatment with a low-galactose diet promotes clinical improvement. However, many children develop learning disabilities. Although it is understood that endogenous production of galactose contributes significantly to blood galactose-1-phosphate levels [28], it is not understood how this contributes to clinical outcome or how stringent the dietary restriction of galactose needs to be to prevent further damage [29, 30]. Soy-based infant formula is the sole source of nourishment for the infant with galactosemia. Recent data suggest that other (nondairy) foods may also contribute dietary galactose and possibly affect plasma galactose-1-phosphate levels and outcome [31].\n\n### E. Glycogen Storage Disease\n\nThe most common types of glycogen storage disease (types Ia, MIM 232200; Ib, MIM 232330; and III, MIM 232400) are autosomal recessive disorders with metabolic blocks that prevent the utilization of glycogen for energy. The glycogen storage diseases are characterized by severe hypoglycemic episodes. Type Ia glycogen storage disease is the most severe because both gluconeogenesis and glycogenolysis are impaired. Children develop severe hepatomegaly and frequently develop liver adenoma and glomerular sclerosis [32, 33]. Glycogen storage disease type Ib is clinically and biochemically similar to type Ia. However, individuals with this disorder are susceptible to bacterial infections because of neutropenia. This impaired neutrophil function increases the mortality rate for this group of children. Glycogen storage disease type III is less severe than type I forms. Risk of cardiomyopathy increases with age [34].\n\nIngestion of raw cornstarch to prevent hypoglycemia and glycogen storage has improved the outcome for these children [35\u201337]. The treatment protocol is rigorous and requires ingestion of cornstarch at regular intervals day and night or a nasogastric tube at night. With cornstarch therapy, the growth of the children has improved, hypoglycemic episodes are prevented, and the consequent neurological damage is also prevented. Children with glycogen storage disease type Ib are now treated with granulocyte stimulating factor for neutrophil stimulation to prevent severe neutropenia [38]. Although these recent therapies have significantly improved the short-term status of these children, limited longterm outcome data are available.\n\n### F. Homocystinuria\n\nHomocystinuria is an autosomal recessive disorder of homocystine metabolism involving the enzyme cystathionine \u03b2-synthase or methylenetetrahydrofolate reductase (MTHFR, MIM 236250). Levels of methionine and homocystine are elevated with cystathionine \u03b2-synthase deficiency; methionine is usually not elevated with MTHFR. The elevated levels of homocystine interfere with collagen cross-linkage, and the result is dislocated lenses, osteoporosis, scoliosis, a long, thin body, and a high-arched palate. Elevated homocystine levels also interfere with the vascular epithelium and lead to thrombus formation. Many individuals with homocystinuria have behavioral or psychiatric disturbances. Recently, betaine has been added to the treatment protocol for homocystinuria [39, 40]. Betaine decreases the plasma homocystine levels and diminishes behavioral and psychiatric effects.\n\n## V. ADULT TREATMENT\n\nMost diagnostic and treatment services are pediatric based. As diagnosis and treatment improve, persons with inherited metabolic disorders not only survive, but also thrive. Pediatric health services are inappropriate for this group of adults. However, these surviving adults, whether competent or compromised, require continued support and effective treatment to maintain their health and nutritional status.\n\nFew, if any, adult providers are trained to meet the needs of fragile adults with metabolic disorders. These health care needs are highlighted by the need for lifelong treatment and changing service needs with age. If the precise treatments of specific metabolic disorders are not meticulously followed, biochemical status deteriorates. This leads to poor judgment and decision making and can lead to a permanent decrease in neurological function and even life-threatening illness. Young adults with inherited metabolic disorders require a specialized system of health care delivery to maintain optimal status.\n\n### A. Transition\n\nTransition from pediatric to adult care is a major step for most families. Table 11 illustrates the differences between pediatric and adult-based care. The transition from pediatric to adult care highlights significant concerns for this group. These concerns include (1) the need for complex nutritional therapy, (2) ingestion of special metabolic formula, (3) complex schedule of medication intake, (4) the need for lifelong treatment, (5) reproductive concerns, (6) changing medical needs with age, and (7) monitoring of health status by providers who understand inherited metabolic disorders.\n\nTABLE 11\n\nCharacteristics of Pediatric- and Adult-Based Care\n\nPediatric care | Adult care \n---|--- \nParents in charge | Self-directed \nCare is monitored | Self-monitored \nAppointments are scheduled | Schedule own appointment \nSupport services offered for financial and emotional issues | Must seek support services for financial and emotional issues \nParents responsible for finances\/payment | Responsible for own finances\/payment \nParents have insurance | Provide own insurance \nTransportation provided by parents | Provide own transportation \nParents request information on treatment | Must request own information \nParents request information on outcome | Must request own information \nSpecialty care program | Generic adult care \nPublic health nurse services | No public health nurse services \nPediatric specialty | General medicine \nTeam approach to services | No team approach \nInterdisciplinary approach | Disciplinary approach \nUsually has built-in support from parent\/caregiver | Different sort of support for emotional needs \nReproduction generally not an issue | Reproductive\/contraceptive decisions must be made\n\nSome families need to organize specialized health care for their college-bound student, while others are struggling to find programs that support daily living skills. Regardless of the competence or status of the young adult with a metabolic disorder, transition to adult-based care is medically and socially appropriate.\n\n#### 1. BARRIERS\n\nBarriers do exist, however, to effective transition from pediatric to adult-based health care. Often, pediatric health care providers feel that there are no knowledgeable providers in the adult health care system for persons with metabolic disorders. Pediatric providers may be hesitant to \"let go\" because they have established long-term relationships with both patient and family. Patients and their families are similarly ambivalent about transitioning out of a familiar situation. Often, adult care providers are reluctant to accept patients who have rare and chronic metabolic disorders and who often require extraordinary services from the health care system.\n\n#### 2. TRANSITION NEEDS\n\nAn organized approach to adult-based health care, with each step and expectation carefully outlined, is essential. Table 12 provides an example of the commitment required by family and providers to make transition a successful process.\n\nTABLE 12\n\nExample of Transition Services from Pediatric to Adult Medical Services\n\nCurrent status | Goal \n---|--- \nGeneral health monitored by pediatrician | Supervision by family practice physician \nMedical supervision by pediatric geneticist | Consultation from university internal medicine and genetics \nNutritional status monitored by pediatric metabolic nutritionist | Monitored by community-based nutritionist with consultation from metabolic nutritionist \nNeurological status monitored by pediatric neurologist | Neurologist \nGynecological supervision by pediatrician | Consultation by specialty gynecologist to primary care physician \nMedically stable | Maintenance of medical stability \nParents supervise medications | Self-manages medications with support \nIn high school | Employed \nLives at home | Lives independently \nParents supervise diet | Self-manages diet with support\n\n### B. Adulthood\n\nLittle is known about the natural history of those with early diagnosed and treated metabolic disorders. Two examples illustrate this point. It is clear that lifelong treatment is necessary for phenylketonuria to prevent neuropsychological problems [41]. Table 13 outlines the neuropsychological aspects of discontinued treatment for persons with phenylketonuria (PKU). However, there is no long-term outcome data to indicate how rigorous this lifelong treatment must be. In the case of tyrosinemia I (fumaryl-acetoacetate hydrolase deficiency), treatment with NTBC appears to be successful in preventing early liver failure and death.\n\nTABLE 13\n\nPhenylketonuria Outcome Data\n\n**Results of long-term neglect of management of PKU**\n\nPoor short-term memory\n\nDeteriorating physical state\n\nAgoraphobia\n\nPoor judgment\n\nFragile emotional state\n\nPoor impulse control that leads to loss of job, inability to maintain interpersonal relationships, and problems with the law\n\n**Results of long-term effective management of PKU and general health care**\n\nCollege students\n\nSuccessful employment\n\nStable interpersonal relationships\n\nReliable memory\n\nEmotional equilibrium\n\nMany disorders have secondary long-term effects. Many of these secondary effects are now being fully realized, for example, renal disease in methylmalonic aciduria [42], neuropsychological disorders in phenylketonuria [43], and ovarian failure in galactosemia [44].\n\n### C. Pregnancy\n\nPregnancy and its management in women with metabolic disorders have also recently been undertaken as standard care. Women with metabolic disorders face special risks to themselves and to their fetuses. There are several aspects of the impact of pregnancy on inborn errors of metabolism: (1) Maternal metabolism negatively affects the development of the fetus, e.g., maternal PKU syndrome; (2) the pregnancy poses health risks for the mother in the postpartum period, e.g., urea cycle disorders, and (3) the disorder diminishes reproductive capability, e.g., galactosemia.\n\nIn the case of maternal phenylketonuria, the neurotoxic effects of elevated maternal phenylalanine levels damage the fetus. There is a dose\u2013response effect related to maternal plasma phenylalanine concentrations. The higher the maternal phenylalanine level, the greater the risks to the developing fetus for microcephaly and cardiac anomalies [45]. Some success has been reported with early, rigorous prepregnancy treatment. Although the children are young and the longterm outcome is not known, it is believed that maintaining maternal phenylalanine concentrations at near-normal physiological levels prior to conception and during pregnancy prevents structural and neurological damage to the fetal brain and heart [46].\n\nCase reports of women with urea cycle disorders indicate rigorous management is needed to maintain a pregnancy. The postpartum reabsorbtion of uterine material causes an increase in ammonia levels and neurotoxic problems, which can be fatal. Case reports suggest that women who have a mild manifestation of the disorder fare better postpregnancy than those women who are severely affected. A woman with mild argininosuccinate lyase deficiency, for example, who had careful clinical and biochemical monitoring during her pregnancy is believed to have reduced the risk of maternal metabolic compensation in the perinatal period. Arginino-succinate did not appear to be teratogenic to the fetus [47].\n\nClassic galactosemia diminishes reproductive capability. About 80% of women with galactosemia suffer ovarian failure. Those few women who successfully complete a pregnancy should be instructed about the risks of self-intoxication with galactose during breastfeeding [48, 49].\n\nA case report of a pregnant woman with MSUD suggested that maintaining BCAA plasma levels between 100 and 300 \u03bcmol\/L was compatible with the delivery of a normal infant. Careful monitoring of the mother after delivery can minimize the risk of metabolic decompensation in the postpartum period [50].\n\n## VI. SUMMARY\n\nProgress in the diagnosis and treatment of genetic diseases has been fast paced. The goal for treatment of all disorders is to provide normal biochemical plasma concentrations. This goal is difficult to achieve [51]. Knowledge of genetics and use of diagnostic procedures in health care have the potential to prevent symptoms and manifestations of the disorders by providing interventions at the presymptomatic stage of the disorder. At this point, treatment must await diagnosis that is based on symptoms of a metabolic disorder. There is hope that further progress in the understanding of genetic disorders will promote earlier and more effective treatment and prevent the symptomatic sequelae of metabolic disorders.\n\nReferences\n\n1. Committee on Genetics. Newborn screening fact sheets. _Pediatrics_. 1996;98:473\u2013501.\n\n2. Pollitt R.J., Green A., McCabe C.J., Booth A., Cooper N.J., Leonard J.V., Nicholl J., Nicholson P., Tunaley J.R., Virdi N.K. Neonatal screening for inborn errors of metabolism: cost, yield, and outcome. _Health Technol. Assess_. 1997;1:i\u2013iv.\n\n3. Thomason M.J., Lord J., Bain M.D., Chalmers R.A., Littlejohns P., Addison G.M., Wilcox A.H., Seymour C.A. A systematic review of evidence for the appropriateness of neonatal screening programmes for inborn errors of metabolism. _J. Public Health Med_. 1998;20:331\u2013343.\n\n4. Naylor E.W., Chace D.H. Automated tandem mass spectrometry for mass newborn screening for disorders in fatty acid, organic acid and amino acid metabolism. _J. Child Neurol_. 1999;14:S4\u2013S8.\n\n5. Burton B.K. Inborn errors of metabolism in infancy: A guide to diagnosis. _Pediatrics_. 1998;102:69\u201378.\n\n6. Schaefer F., Straube E., Oh J., Mehls O., Mayatepek E. Dialysis in neonates with inborn errors of metabolism. _Nephrol. Dial. Transplant_. 1999;14:910\u2013918.\n\n7. Scriver C.R., Beaudet A.L., Sly W.S., Valle D., eds. The Metabolic and Molecular Basis of Inherited Disease. New York: McGraw-Hill, 2000.\n\n8. McKusick V.A. _Mendelian Inheritance in Man. Catalogs of Human Genes and Genetic Disorders_ , 12th Ed. New York: Johns Hopkins University Press; 1998.\n\n9. Available atMcKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University, Baltimore, MD, National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD. Online Mendelian Inheritance in Man, OMIM. 2000. .\n\n10. Hymes J., Wolf B. Biotinidase and its roles in biotin metabolism. _Clin. Chim. Acta_. 1996;15:1\u201311.\n\n11. Anonymous. Homocystinuria. _BMJ_. 1996;313:1025\u20131026.\n\n12. Tyni T., Pihko H. Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. _Acta Pediatr_. 1999;88:237\u2013245.\n\n13. Saudubray J.M., Martin D., de Lonlay P., Touati G., Poggi-Travert F., Bonnet D., Jouvet P., Boutron M., Slama A., Vianey-Saban C., Bonnefont J.P., Rabier D., Kamoun P., Brivet M. Recognition and management of fatty acid oxidation defects: A series of 107 patients. _J. Inher. Metab. Dis_. 1999;22:488\u2013502.\n\n14. Acosta P.B., Yanicelli S. Nutrition support of inherited disorders of amino acid metabolism: Part 1. _Topics Clin. Nutr_. 1993;9:65\u201382.\n\n15. Acosta P.B., Yanicelli S. Nutrition support of inherited disorders of amino acid metabolism: Part 2. _Topics Clin. Nutr_. 1995;10:48\u201372.\n\n16. Acosta P.B., Yannicelli S., Marriage B., Mantia C., Gaffield B., Porterfield M., Hunt M., McMaster N., Bernstein L., Parton P., Kuehn M., Lewis V. Nutrient intake and growth of infants with phenylketonuria undergoing therapy. _J. Pediatr. Gastroenterol. Nutr_. 1998;27:287\u2013291.\n\n17. Lohmann T.G., Roche A.F., Martorell R. _Anthropometric Standardization Reference Manual_. Baltimore, MD: Human Kinetics Books; 1988.\n\n18. Garza C., Scrimshaw N.S., Young V.R. Human protein requirements: The effects of variations in energy intake within the maintenance range. _Am. J. Clin. Nutr_. 1976;29:280\u2013285.\n\n19. Kilpatrick K.M. The implications of phenylketonuria on oral health. _Pediatr. Dentistry_. 1999;21:433\u2013437.\n\n20. Schadewaldt P., Bodner-Leidecker A., Hammen H.W., Wendel U. Significance of L-alloisoleucine in plasma for diagnosis of maple syrup urine disease. _Clin. Chem_. 1999;45:1734\u20131740.\n\n21. Wajner M., Vargas C.R. Reduction of plasma concentrations of large neutral amino acids in patients with maple syrup urine disease during crises. _Arch. Dis. Child_. 1999;80:579.\n\n22. Wendel U., Saudubray J.M., Bodner A., Schadewaldt P. Liver transplantation in maple syrup urine disease. _Eur. J. Pediatr_. 1999;158:S60\u2013S64.\n\n23. Mohan N., McKiernan P., Preece M.A., Green A., Buckels J., Mayer A.D., Kelly D.A. Indications and outcome of liver transplantation in tyrosinaemia type 1. _Eur. J. Pediatr_. 1999;158:S49\u2013S54.\n\n24. Holme E., Lindstedt S. Tyrosinemia type I and NTBC (2(2-nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione). _J. Inherit. Metab. Dis_. 1998;21:507\u2013517.\n\n25. Seashore M.R., Wappner R., Cho S., de La Cruz F. Development of guidelines for treatment of children with phenylketonuria: Report of a meeting at the National Institute of Child Health and Human Development held August 15, 1995, National Institutes of Health, Bethesda, Maryland. _Pediatrics_. 1999;104:6\u20139.\n\n26. Medical Research Council Working Party on Phenylketonuria. Recommendations on the dietary management of phenylketonuria. _Arch. Dis. Child_. 1993;68:426\u2013427.\n\n27. Wappner R., Cho S., Kronmal R.A., Schuett V., Seashore M.R. Management of phenylketonuria for optimal outcome: A review of guidelines for phenylketonuria management and a report of surveys of parents, patients, and clinic directors. _Pediatrics_. 1999;104:6\u201312.\n\n28. Berry G.T., Nissim I., Lin Z., Mazur A.T., Gibson J.B., Segal S. Endogenous synthesis of galactose in normal men and patients with hereditary galactosemia. _Lancet_. 1995;346:1073\u20131074.\n\n29. Walter J.H., Collins J.E., Leonard J.V. Recommendations for the management of galactosemia. U.K. Galactosemia Steering Group. _Arch. Dis. Child_. 1999;80:593\u2013596.\n\n30. Schweitzer S., Shin Y., Jakobs C., Brodehl J. Long-term outcome in 134 patients with galactosemia. _Eur. J. Pediatr_. 1993;152:36\u201343.\n\n31. Gropper S.S., Weese J.O., West P.A., Gross K.C. Free galactose content of fresh fruits and strained fruit and vegetable baby foods: More foods to consider for the galactose-restricted diet. _J. Am. Diet. Assoc_. 2000;100:573\u2013575.\n\n32. Restaino I., Kaplan B.S., Stanley C., Baker L. Nephrolithiasis, hypocitraturia, and a distal renal tubular acidification in type 1 glycogen storage disease. _J. Pediatr_. 1993;122:392\u2013396.\n\n33. Wolfsdorf J.I., Holm I.A., Weinstein D.A. Glycogen storage diseases. Phenotypic, genetic, and biochemical characteristics, and therapy. _Endocrinol. Metab. Clin. N. Am_. 1999;28:801\u2013823.\n\n34. Greene H.L., Swift L.L., Knapp H.R. Hyperlipidemia and fatty acid composition in patients treated for type 1a glycogen storage disease. _J. Pediatr_. 1991;119:398\u2013403.\n\n35. Gremse D.A., Bucuvalas J.C., Balisteri W.F. Efficacy of cornstarch therapy in type III glycogen storage disease. _Am. J. Clin. Nutr_. 1990;52:671\u2013674.\n\n36. Goldberg T., Slonim A.E. Nutrition therapy for hepatic glycogen storage diseases. _J. Amer. Diet. Assoc_. 1993;93:1423\u20131430.\n\n37. Wolfsdorf J.I., Crigler J.F., Jr. Effect of continuous glucose therapy begun in infancy on the long-term clinical course of patients with type 1 glycogen storage disease. _J. Pediatr. Gastroenterol. Nutr_. 1999;29:136\u2013143.\n\n38. McCawley L.J., Korchak H.M., Douglas S.D., Campbell D.E., Thornton P.S., Stanley C.A., Baker L., Kilpatrick L. In vitro and in vivo effects of granulocyte colony-stimulating factor on neutrophils in glycogen storage disease type Ib: Granulocyte colony-stimulating factor therapy corrects the neutropenia and the defects in respiratory burst activity and Ca2% mobilization. _Pediatr. Res_. 1994;35:84\u201390.\n\n39. Wilcken D.E., Wilcken B., Dudman N.P., Tyrrell P.A. Homocystinuria-The effects of betaine in the treatment of patients not responsive to pyridoxine. _N. Engl. J. Med_. 1983;309:448\u2013453.\n\n40. Sakura N., Ono H., Nomura S., Ueda H., Fujita N. Betaine dose and treatment intervals in therapy for homocystinuria due to 5, 10-methylenetetrahydrofolate reductase deficiency. _J. Inherit. Metab. Dis_. 1998;21:84\u201385.\n\n41. Schiaffino C.K., DiStefane S., Veneselli E. Phenylketonuria: Diet for life or not? _Acta Paediatr_. 1999;88:664\u2013666.\n\n42. Van Calcar S.C., Harding C.O., Lyne P., Hogan K., Banerjee R., Sollinger H., Rieselbach R.E., Wolff J.A. Renal transplantation in a patient with methylmalonic acidemia. _J. Inherit. Metab. Dis_. 1998;21:729\u2013737.\n\n43. Pietz J., Dunckelmann R., Rupp A., Rating D., Meinck H.M., Schmidt H., Bremer H.J. Neurological outcome in adult patients with early-treated phenylketonuria. _Eur. J. Pediatr_. 1998;157:824\u2013830.\n\n44. Kaufman F., Kogut M.D., Donnell G.N., Koch H., Goebelsmann U. Ovarian failure in galactosemia. _Lancet_. 1979;II, Oct. 6; 2:737\u2013740.\n\n45. Rouse B., Matalon R., Koch R., Azen C., Levy H., Hanley W., Trefz F., de la Cruz F. Maternal phenylketonuria syndrome: Congenital heart defects, microcephaly, and developmental outcomes. _J. Pediatr_. 2000;136:57\u201361.\n\n46. Koch R., Friedman E., Azen C., Hanley W., Levy H., Matalon R., Rouse B., Trefz F., Waisbren S., Michals-Matalon K., Acosta P., Guttler F., Ullrich K., Platt L., de la Cruz F. The International Collaborative Study of Maternal. Phenylketonuria Status report 1998. _MRDD Res. Rev_. 1999;5:117\u2013121.\n\n47. Worthington S., Christodoulou J., Wilcken B., Peat B. Pregnancy and argininosuccinic aciduria. _J. Inherit. Metab. Dis_. 1996;19:621\u2013622.\n\n48. Brivet M., Raymond J.P., Konopka P., Odievre M., Lemonnier A. Effect of lactation in a mother with galactosemia. _J. Pediatr_. 1989;115:280\u2013281.\n\n49. DeJongh S., Vreken P., Ijst L., Wanders R.J., Jakobs C., Bakker H.D. Spontaneous pregnancy in a patient with classical galactosemia. _J. Inherit. Metab. Dis_. 1999;22:754\u2013755.\n\n50. Grunewald S., Hinrichs F., Wendel U. Pregnancy in a woman with maple syrup urine disease. _J. Inherit. Metab. Dis_. 1998;21:89\u201390.\n\n51. Treacy E., Childs B., Scriver C.R. Response to treatment in hereditary metabolic disease: 1993 survey and 10-year comparison. _Am. J. Hum. Genet_. 1995;56:359\u2013367.\nCHAPTER 15\n\nRole of Liquid Dietary Supplements\n\nANNE COBLE VOSS and KATHLEEN E. MAYER, Abbott Laboratories, Columbus, Ohio\n\n## I. MEDICAL FOODS AND NUTRITIONAL SUPPLEMENTS\n\nChanging demographics and the current health care environment have created the opportunity for growth of medical foods and medical nutritional supplements (MNSs). Older adults are the largest consumers of health care accounting for nearly 30% of all health care expenditures. It is estimated that by 2030, adults over the age 85 will have doubled and those over the age of 65 will account for 22% of the population [1]. These consumers, the \"Baby Boomers,\" are expected to take a very active role and interest in their health care as it relates to extending longevity and quality of life [2]. Nutrition is a modifiable, lifestyle factor that has a prominent role in the health care model of the twenty-first century.\n\nOlder adults are particularly at risk for malnutrition for a variety of reasons including functional and cognitive decline, increased nutritional needs, chronic disease conditions and co-morbidities, economic limitations, and social factors. It has been well documented in the literature that malnutrition is associated with poor outcomes such as increased morbidity and mortality [3\u20138], impaired immunity and wound healing [9], increased length of stay [10], decreased function [11], hospital readmissions [3], and reduced quality of life [12, 13]. Studies demonstrate that medical nutrition therapy (MNT), including supplementation with medical foods, can improve clinical and economic outcomes associated with the conditions listed in Table 1.\n\nTABLE 1\n\nConditions in Which Medical Nutrition Therapy May Improve Outcomes\n\nAIDS | Cancer \n---|--- \nCerebral vascular accident | Congestive heart failure \nChronic obstructive pulmonary disease | Dementia \nDiabetes mellitus | Hip fracture \nGeneral medicine\/surgery | Pneumonia \nPressure ulcers | Renal disease\n\nThis chapter will address the increasing need for supplemental MNT, differentiate between medical foods and medical nutritional supplements, and provide examples of benefits and barriers to their use.\n\n### A. Prevalence of Malnutrition across the Continuum of Care\n\nThe negative impact of malnutrition has been highlighted by early classic reports such as \"The Skeleton in the Hospital Closet\" [14]. Since then, researchers and health care professionals have learned much more about nutritional screening and assessment, nutritional biomarkers, and the prevention and treatment of malnutrition. Despite these advances in knowledge, little has changed regarding the prevalence of malnutrition in acute care. It is estimated that malnutrition in hospital\/acute care ranges from 10% to 55% [15\u201317] and is often dependent on diagnoses and medical procedures.\n\nUnfortunately, malnutrition is not confined to the hospital setting. As the provision of health care has emerged into other points of service, malnutrition has been identified in these areas as well. Reports have indicated that malnutrition, or risk for malnutrition, ranges from 39% to 74% [18\u201320] in long-term care (LTC) facilities. In addition, LTC residents often have nutrient intakes that are less than 50% of the Recommended Dietary Allowances (RDA) [20]. Risk for malnutrition is also abundant among older adults living in the community. A study of general practice patients in England indicated that the prevalence of malnutrition was 10% [21], but a prevalence rate as high as 43% has been reported elsewhere [15]. Poor energy and nutrient intakes have also been reported among independently living older adults with studies indicating that many eat less than two-thirds of the RDA [22\u201324]. With increased longevity, trends indicate that more adults will remain in their homes for longer periods of time. Therefore, health care professionals will need to develop and implement ways of preventing and treating the nutritional decline that is common in the population of independently living older adults.\n\n## II. WHAT IS A MEDICAL FOOD\/SUPPLEMENT?\n\nAccording to the Food and Drug Administration (FDA), \"A medical food is prescribed by a physician when a patient has special nutrient needs and the patient is under the physician's ongoing care. The label must clearly state that the product is intended to be used to manage a special medical disorder or condition\" [25]. Medical foods can be separated into four categories according to the FDA: (1) nutritionally complete formulas that can be used as sole-source nutrition; (2) nutritionally incomplete formulas, i.e., modular products of fat, protein, or carbohydrates; (3) oral rehydration products that are electrolyte\/fluid replacements for dehydration; and (4) disease-specific nutritional formulas, which are specifically developed to help manage conditions such as phenylketonuria.\n\nOne of the specifications by the FDA definition is that a medical food must be used for special dietary needs and must be managed under the care of a physician. In recent years, oral nutritional beverages have become commercially available due to increasing demand by consumers who use them as meal replacements and supplements as well as for the management of special dietary needs. However, if they are consumed without the supervision of a physician then they do not meet the FDA's definition of a medical food. This definition is important from a regulatory standpoint. For clinical application, the main question is whether a product meets an individual's medical needs. Therefore, to avoid confusion all oral nutritional beverages will be referred to as medical nutritional supplements. Table 2 provides some examples of MNSs and the indicated uses.\n\nTABLE 2\n\nExamples of Medical Nutritional Supplements and Indicated Uses\n\n_Note:_ This list provides examples of some of the more common categories. It is not intended to be inclusive of all medical nutritional supplements available on the market.\n\naRecommended for oral consumption only.\n\nClinicians often think of MNSs as a standard or disease-specific formula. Standard MNSs contain appropriate levels of carbohydrates, protein, and fat and usually at least 25% of the Recommended Dietary Intakes (RDI) for vitamins and minerals per serving. Some can be used as the sole source of nourishment, while others can be used only as a supplement. Disease-specific, or specialized, formulas may have varying levels of macronutrients and micronutrients as appropriate for the indicated disease state. These formulas may also contain unique ingredients such as structured lipids, special fatty acid blends, prebiotics (e.g., fructooligosaccharides), peptides, protein hydrolysates, and free amino acids (e.g., L-arginine, L-glutamine).\n\n## III. PREVALENCE AND ECONOMIC IMPACT OF MEDICAL NUTRITION THERAPY\n\nDespite increasing knowledge of the prevention and treatment of malnutrition, it remains a problem across the continuum of care. The demand for MNT is increasing as more studies indicate that MNT can reduce the costs associated with patient care by decreasing complications [4], mortality and morbidity [26], length of stay in the hospital, readmissions, and recovery time [27]. Improved outcomes result in cost reductions ranging from 35% to 75% [28]. Medical nutrition therapy has become so notably effective that managed care plans are beginning to increase and expand their coverage of MNT interventions [29]. In addition, the Institute of Medicine recently recommended that Congress expand Medicare coverage of nutrition therapy for patients in the home care setting upon referral from a physician [30]. While the significance of MNT is certainly becoming more recognized, it is important for clinicians and researchers to be cognizant of all the demonstrable outcomes of MNT. The following sections will highlight the benefits of oral supplementation in different points of service as well as different disease states.\n\n### A. Standard Medical Nutritional Supplements\n\n1. GENERAL MEDICINE\/SURGERY\n\nMalnutrition, particularly protein energy malnutrition (PEM), is a common confounding variable in the surgical [16] and general medical patient [17] for reasons such as poor intake as a result of stress, anxiety and NPO status; elevated metabolism and increased nutrient needs required for healing; negative nitrogen balance; and limited activity [16]. Patients who are malnourished have significantly greater mortality and morbidity [5, 31] and are more likely to be readmitted to the hospital [31]. Furthermore, studies suggest that many hospitalized patients do not receive adequate dietary intake [31] and are at risk for further nutritional decline. The continued prevalence of malnutrition in this setting indicates the crucial need for MNSs as a first line of defense in combating malnutrition.\n\nBenefits of postoperative supplementation with a high-protein MNS (1.5 kcal\/mL, 0.05 g protein\/mL) include improved dietary intake; less weight loss; improved hand grip strength, which is an indicator of functional status; and fewer postoperative complications [32]. Improvements in weight and lean body mass result in improved functional status and quality of life [13]. A study by Larrson and colleagues [8] reported that the administration of MNSs twice per day to geriatric patients in the medical ward resulted in less weight loss, improved immunocompetence, and improved functional and mobility status. Patients who benefited the most were those who were well nourished at admission.\n\nBeing at nutritional risk preoperatively is also associated with poorer outcomes [33]. In a study of patients undergoing total hip replacement, presurgical albumin was an independent indicator of postoperative recovery time. Patients with serum albumin concentrations less than 3.9 g\/dL were two times more likely to have longer hospitalizations. It is much easier to prevent nutritional deficiencies rather than correct them along with all of the associated sequelae. However, it is not always feasible to improve nutritional status before a major surgery. But when the surgery is an elective procedure, such as hip or total knee replacements, it is recommended that patients use high-calorie, high-protein MNSs prior to their scheduled surgery to improve outcomes such as recovery and rehabilitation time.\n\n#### 2. HIP FRACTURE\n\nHip fractures are a common consequence of osteoporosis in older adults. Patients who have had hip fractures have a higher risk of mortality, complications, and rehospitalizations [7, 34]. Hip fractures are a very costly problem for older adults and the health care industry. Protein energy malnutrition often coincides with hip fractures and is thought to contribute to the incidence and complications of hip fractures [34, 35].\n\nSupplementation with MNSs has proven beneficial for patients who are recovering from hip fractures [35\u201338]. Hip fracture patients who received a daily MNS (254 kcal, 20.4 g protein) experienced a rapid improvement in protein status and had significantly fewer complications ( _p_ <0.05), reduced mortality ( _p_ <0.02) and length of stay ( _p_ <0.02). Research has also demonstrated that high-protein MNSs are more advantageous to this population than standard MNSs. Two studies comparing a high-protein MNS to a standard MNS reported improved laboratory indicators such as serum prealbumin and insulin-like growth factor one; increased bone mineral density; reduced complications; and reduced length of stay in the hospital and rehabilitation in the patients receiving the high-protein MNS [35, 36]. High-protein MNSs in this population improve clinical outcomes and are also cost effective.\n\n#### 3. CEREBRAL VASCULAR ACCIDENT\n\nMobility and functional and cognitive changes, as a result of a stroke, often impair an individual's ability to eat. Unosson _et al._ [6] reported that patients who had a recent cerebral vascular accident consumed only 72% of the food served. Furthermore, many stroke patients often have low serum albumin and body mass index [6, 39]. Research indicates that serum albumin concentration is an independent predictor of mortality among stroke patients and is associated with a greater risk of infections and poor functional outcome [39]. Supplementation with high-protein MNSs improved nutritional intake and nutritional parameters such as albumin and hemoglobin, and also resulted in decreased length of stay and mortality in patients with a recent stroke [40]. Poor nutritional status is also related to longer length of stay in the rehabilitative setting. Malnutrition should be a primary factor addressed in this setting because it is the most likely factor that could be modified to improve functional outcomes [11].\n\n#### 4. LONG-TERM CARE AND HOME CARE\n\nMedical nutritional supplements are often administered to residents in LTC in an effort to combat two of the most serious and prevalent problems: unintentional weight loss and pressure ulcers [41, 42]. It is estimated that more than 1 million individuals develop a pressure ulcer annually [43] with estimated costs as high as $1.3 billion per year to the health care industry [44]. The prevalence of pressure ulcers in LTC and home care is 2.4\u201323% [45, 46] and 8.7\u201312.9%, respectively [47]. Nutritional status is a critical factor in both the prevention and treatment of pressure ulcers [9, 20, 43, 48\u201350]. Low body weight, serum albumin, hemoglobin, and reduced food intake are all correlated positively with the incidence of pressure ulcers [50]. Poor nutritional status also has deleterious implications on healing times of pressure ulcers. A study by Van Rijswijk and Polansky [51] indicated that residents with a suboptimal nutritional status took 20 days longer to heal a pressure ulcer than residents who were well nourished. Many efforts have been made to improve the nutritional status of LTC residents; however, it has been demonstrated that poor intake and unintentional weight loss remain major problems. In a recent study Thomas _et al._ [52] indicated that as many as 40% of LTC residents had poor meal intake and more than 50% had unintentional weight loss.\n\nEvidence exists to support the use of MNSs in LTC residents. Studies demonstrate that MNSs improve dietary intake [20, 22] weight gain\/maintenance [20, 53], and wound healing [54]. A multicenter intervention trial demonstrated that patients who received a standard 1800-kcal diet and oral MNSs had a lower incidence of pressure ulcer development [22]. One of the most successful programs in recent years has been the Medication Pass Supplement Program (MPSP). In the MPSP, approximately 2\u20134 ounces of a nutrient-dense MNS (1.5\u20132.0 kcal\/mL, 13\u201320 g protein) are distributed 3 times per day with the resident's medications. The consumption of MNSs is documented similar to other medications. Residents do not usually refuse the MNS because they perceive it as part of their medication distributed by their health care professional. Facilities that have implemented this program have demonstrated weight gain or weight maintenance and reduced weight loss [55\u201357]. Clinicians anticipate that the MPSP can result in significant cost savings associated with the reduction of the cost of treating a pressure ulcer, savings associated with preventing a pressure ulcer, and savings associated with the minimized waste of MNSs [58].\n\nDelivering a MNS in smaller amounts between meals in programs such as the MPSP is particularly favorable for residents who have a reduced appetite and early satiety. A MNS delivered between meals is less likely to affect the resident's meal intake, which is often a concern of dietitians and physicians in LTC facilities. Turic and colleagues [20] demonstrated that supplementation with MNSs between meals helped residents meet or exceed the RDA without replacing nutrients consumed at meals.\n\n#### 5. ALZHEIMER'S DISEASE\n\nIndividuals with Alzheimer's disease commonly have disturbed hunger, thirst, and satiety cues as well as altered sensory perceptions and an impaired ability to feed themselves. Because of these changes, many patients with Alzheimer's disease experience progressive weight loss [59]. Research has also demonstrated that these patients may have low serum levels of specific nutrients, such as folate and vitamin B12 [60]. This is significant because deficiencies of these vitamins have been associated with cognitive impairment [60]. Many patients with Alzheimer's disease are often easily distracted and have a tendency to \"wander,\" which may result in increased energy needs and missed meals. While some of these patients are prone to hyperphagia, studies suggest that they do not typically differ in weight from patients who do not overeat, which may indicate that this subset has increased energy needs [61]. It is advantageous to provide MNSs between meals to these patients to help compensate for the dietary intake they may miss at other times. In recent years, some companies have developed alternative forms of supplements such as puddings and bars (Ensure Bars, Nubasics). These alternative forms are useful because these handheld snacks can be easily carried and eaten quickly by the patient who is likely to be distracted or wander.\n\n#### 6. COMMUNITY\n\nRecent studies have indicated that independently living older adults with a body mass index (BMI) less than 22 kg\/m2 are at a much higher risk for all-cause mortality [62] and increased health care expenditures [63, 64]. Those who were at the least risk had a BMI of 24\u201326 kg\/m2, which is slightly higher than recommended for the general population. Researchers have also demonstrated that low BMI and inadequate dietary intake are associated with diminished functional status among older adults in the community [65, 66]. Low serum albumin has also been implicated as an independent predictor of mortality in this population [67]. In fact, albumin levels lower than 43 g\/L (4.3 g\/dL), a value considered to be clinically normal, were associated with increased mortality compared to individuals who had higher albumin levels. These studies indicate that older adults living in the community may benefit from improving their nutritional status to a higher standard than what is recommended for the general public.\n\nMany older adults are involved in meal programs that are delivered to their homes (e.g., Meals on Wheels) or served in a congregate meal setting [68]. Meals provided by such programs provide at least one-third of the RDA for energy and key nutrients. Nevertheless, as many as 33% [69] to 70% [70] of recipients of Meals on Wheels remain at nutritional risk. One way to improve the dietary and nutritional status of these recipients is to add a standard or high-calorie, high-protein MNS (250\u2013355 kcal, 7\u201313 g protein) to each of the delivered meals. Studies have demonstrated that the addition of a MNS resulted in improved energy and nutrient intakes [23, 66, 71] and weight gain or stabilization [72]. One of these studies also indicated that individuals who received oral MNSs experienced fewer falls than those who did not receive MNSs [72]. This is a great benefit to a population who is at increased risk for falls and hip fractures. Beyond the nutritional benefits of the MNS-enriched meals, the interaction with other individuals provides relief from some of the loneliness and isolation experienced by older adults, both of which are additional risk factors for poor nutritional status [73].\n\n### B. Disease-Specific Medical Nutritional Supplements\n\nMany different specialized formulas have been developed for individuals with certain dietary needs. Many of the disease-specific formulas are intended for enteral tube feeding use, however, several can be used as an oral supplement to help manage dietary needs. For some of the disease-specific MNSs, examples of outcomes will be reported from studies of enteral tube feeding.\n\n#### 1. HIV\/AIDS\n\nProgressive weight loss, or wasting, is very common in persons with HIV\/AIDS. Malnutrition among individuals with human immunodeficiency virus\/acquired immunodeficiency syndrome (HIV\/AIDS) has been associated with a poor prognosis and increased hospitalizations and costs [74, 75]. The wasting associated with HIV\/AIDS is multifactorial and is attributed to anorexia, altered digestion, malabsorption and metabolism, taste alterations, diarrhea and oral lesions.\n\nPatients with HIV-related wasting lose proportionately more lean body mass than fat mass, which is associated with poor survival [75]. Studies of total parenteral nutrition (TPN) and appetite stimulation with megestrol acetate have helped patients gain weight; however, the weight gained is predominantly fat mass [76\u201379]. Studies have suggested that early enteral tube feeding and supplemental nutrition is beneficial in patients with HIV\/AIDS. However, because many patients suffer from nutrient malabsorption, it is possible that some standard MNSs may exacerbate bowel symptoms in the mal-absorbing patient.\n\nElemental formulas and fat-modified formulas have been proposed as nutrition therapies for these patients. In a study using a specialized medium-chain triglyceride (MCT) formula versus dietary counseling alone, there were trends for improvement in lean body mass and hand-grip strength in the group receiving the specialized MNS [80]. A study by Salomon _et al._ [81] determined that an elemental formula with hydrolyzed proteins and 70% of fat from MCT was able to help manage patients' diarrhea as evidenced by reduced number of stools and fecal fat. The reduction in diarrhea had major implications on quality of life and activities of daily living.\n\nA study by Chlebowski _et al._ [82] compared a standard MNS to a disease-specific MNS with hydrolyzed protein, MCT oil, and eicosapentaenoic acid. After 6 months of supplementation, the group receiving the disease-specific MNS had improved weight gain and anthropometric measurements as well as significantly fewer hospitalizations. Patients in both groups had similar dietary intakes, therefore suggesting that those who received the disease-specific MNS were able to utilize the energy and nutrients, whereas those receiving the standard MNS remained in a hypermetabolic state [82].\n\nEach of these studies demonstrated the positive benefits of a disease-specific MNS versus a standard MNS in patients with HIV\/AIDS. The malnutrition associated with HIV\/AIDS has a variety of different components, and therefore not all specialized MNSs are appropriate. It is important to critically assess the primary symptoms that are responsible for the patient's weight loss and use a MNS that addresses these issues.\n\n#### 2. CANCER\n\nInvoluntary weight loss occurs in as many as 50\u201380% of patients with cancer at some stage in the course of their disease [83]. Poor nutritional status in patients with cancer is associated with increased morbidity and mortality [83, 84], reduced functional status and quality of life [85], reduced response to therapy [86\u201389] and increased length of stay in the hospital. Similar to HIV\/AIDS, weight loss and malnutrition in patients with cancer is multifactorial and may result from diminished intake, increased losses, and altered metabolism. Weight loss in patients with cancer can be grouped into four major categories as outlined in Table 3.\n\nTABLE 3\n\nTypes and Causes of Weight Loss in Patients with Cancer\n\nA retrospective chart review of patients with head and neck cancer receiving nutrition support indicated that those who received nutrition support had enhanced immunocompetence, which was positively associated with a 2-year survival [90]. Enteral feeding has also been associated with better adherence to radiation regimens and less weight loss, according to Zogbaum and colleagues [88]. Patients who did not receive enteral supplementation in a study of patients with cancer of the esophagus had significant decreases in body weight and visceral protein status, while those receiving enteral supplementation maintained their nutritional status [91]. These studies demonstrated that enteral supplementation, in patients who would otherwise have reduced or obstructed intake, improved clinical outcomes.\n\nThe clinical efficacy of standard oral MNS in the management of patients with cancer remains uncertain [82, 85, 87, 92\u201394]. A study by Ovesen and colleagues [85] demonstrated that aggressive dietary counseling and oral supplementation in patients with cancer improved nutritional intake but not weight gain. A study of lung and colorectal cancer patients undergoing chemotherapy and radiation reported that nutritional indicators such as serum albumin concentration, and body weight were independently associated with survival. However, there were no differences in outcomes between the groups receiving the supplement or control diet [92]. The authors suggested that the underlying metabolic abnormalities were responsible for the failure of the oral MNS to improve outcomes and that effective interventions could only be developed once the underlying mechanisms were more clearly understood and treated.\n\nThe metabolic abnormalities of cancer cachexia distinguish it from other types of weight loss associated with cancer. Due to these abnormalities, patients with cancer cachexia do not respond to standard MNS. Therefore, disease-specific MNSs that target some of the underlying mechanisms are necessary. In recent years, studies have investigated MNSs enriched with novel ingredients such as omega-3 fatty acids, protein hydrolysates, and single amino acids. Omega-3 fatty acids, such as eicosapentaenoic acid, are thought to have immunomodulating and anti-proinflammatory properties.\n\nCachexia is often accompanied by a prolonged acute phase protein response [95]. This response is characterized by elevated synthesis of C-reactive protein (CRP), haptoglobin, caeruloplasmin, \u03b1-1 acid glycoprotein (A1AGP) and \u03b1-1 antitrypsin (A1ATP) and decreased synthesis of albumin, prealbumin, and transferrin [94\u201398]. The acute phase protein response is an important part of the inflammatory response because it provides proteins that are useful in systemic immunity and tissue repair [95, 99]. Proinflammtory cytokines such as tumor necrosis factor (TNF-\u03b1), interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), and leukemia inhibitory factor (LIF) are all probable mediators of the response. In the inflammatory state, these cytokines are produced at the site of malignancy or injury and by a variety of cells throughout the body resulting in local and systemic physiological alterations. Of particular interest in cachexia, the acute phase protein response has been associated with elevated resting energy expenditure among cachectic patients with lung [100] and pancreatic [94] cancer.\n\nStudies of ecosapentaenoic acid (EPA) supplementation have indicated an attenuation of the acute phase protein response [98], which is thought to be modulated by the proinflammatory cytokines. Studies have also shown that EPA supplementation is associated with weight stabilization in patients with pancreatic cancer [101, 102]. Additionally, omega-3 fatty acids have immunomodulatory properties, which are also potentially beneficial to patients with cancer who are undergoing surgical resection [101, 103]. Supplemental amino acids such as arginine and glutamine are thought to improve wound healing and help preserve nitrogen balance [104]. A meta-analysis by Heys _et al._ [105] indicated that a specialized enteral formula enriched with essential fatty acids, RNA, and arginine was associated with reduced infections and hospital length of stay in patients with gastrointestinal cancer, but was not associated with reduced mortality. A study by Barber _et al._ [94] indicated that an EPA-enriched MNS [1.09 g EPA, 0.46 g docosahexanoic acid (DHA)] improves markers and outcomes in cachectic patients with pancreatic cancer. Prior to diagnosis, patients lost an average of 3.2 kg\/month. However, after supplementation with the EPA-enriched supplement, a mean weight gain of 1.1 kg was realized at 3 weeks and 2.5 kg at 7 weeks. Analysis of body composition by bioelectrical impedance analysis revealed that the weight gained was predominantly lean body mass. In contrast, other cachexia therapies, such as appetite stimulation by megestrol acetate or medroxyprogesterone, increase nutrient intake and weight but the weight gain is primarily adipose tissue. An improvement in lean body mass is significant because restoration of this compartment has been associated with improved quality of life [12, 13]. Supplementation with an EPA-enriched MNS also significantly reduced resting energy expenditure and improved functional status and appetite [94]. Supplementation with EPA alone, via fish oil or pure EPA capsules, has only resulted in weight stabilization, thereby indicating that it is the combination of these factors that is effective in treating cachexia.\n\nSimilar to patients with HIV\/AIDS, many standard MNSs may exacerbate some gastrointestinal symptoms or fail to address underlying mechanisms of weight loss. Novel ingredients, such as dipeptides, may be better tolerated in some patients with gastrointestinal symptoms [106]. Enteral and oral supplementation with standard MNSs can improve nutritional status if there are not underlying metabolic abnormalities. However, in cachectic patients, underlying metabolic abnormalities must be ameliorated in conjunction with supplying adequate nutrition.\n\n#### 3. CHRONIC OBSTRUCTIVE PULMONARY DISEASE\n\nAccording to the Centers for Disease Control [107], chronic obstructive pulmonary diseases (COPD) are the fourth largest cause of death in the United States. Projections indicate that mortality from COPD will continue to rise globally over the next 20 years [108]. Malnutrition, ranging from 19% to 74% in patients with COPD, is dependent on the severity of illness and whether the patient is hospitalized [109,110]. Factors contributing to malnutrition include reduced intake due to dyspnea and early satiety and anorexia, elevated resting energy expenditure, metabolic abnormalities, and the effects of pharmacologic agents [111, 112]. Poor nutritional status in the COPD patient further compromises pulmonary structure and function. Malnutrition in COPD patients also has adverse consequences on clinical outcomes such as ventilator requirements [110, 113], intensive care unit days, decreased functional status, quality of life, and mortality [114].\n\nStudies of nutrition support in patients with COPD have produced mixed results. A summary of studies indicated that nutrition support resulted in improved pulmonary function only in the studies where weight also improved [111]. A study by Efthimiou _et al._ 115] indicated that oral supplementation resulted in weight gain and improved walking distance and dyspnea scores. Individuals who responded to nutritional support were also more likely to be weaned from ventilators sooner than those with a poorer nutritional status [116, 117].\n\nPatients with COPD have impaired gas exchange resulting in hypercapnia, high levels of circulating CO2 in the blood, which requires increased ventilation, or work from the lungs, to expel it. Research has demonstrated that hypermetabolism in patients with pulmonary diseases is associated with increased ventilation. Disease-specific MNSs target impaired gas exchange and try to regulate it. The composition of pulmonary disease-specific formulas are depicted in Table 4.\n\nTABLE 4\n\nMacronutrient Compositions of Pulmonary Products\n\nDisease-specific formulas for patients with COPD are typically higher in fat because fat yields the lowest respiratory quotient (RQ). Studies of both normal subjects and patients with COPD have indicated that pulmonary ventilation, VCO2, and oxygen consumption are all elevated after a high carbohydrate load [118]. Therefore, a MNS higher in fat is more advantageous for these patients.\n\nComparison studies between low carbohydrate\/high-fat MNSs and standard MNSs have shown improved respiratory function [119] and reduced VCO2 and RQ [120\u2013122], and reduced use of mechanical ventilation [123]. A study by Frankfort and associates [122] also suggested that low-carbohydrate\/high-fat MNSs may help manage postprandial dyspnea, which is sometimes associated with pulmonary diseases.\n\nIn addition to the macronutrient distribution, it is also important not to overfeed patients with COPD because excessive energy intake can lead to lipogenesis, which results in large amounts of CO2 production. A study by Talpers _et al._ [124] compared three isocaloric TPN formulas of varying carbohydrate content (40%, 60%, and 75% of energy) in patients with ventilators. There were no differences in CO2 production among the three groups. In a second group of mechanically ventilated patients receiving a TPN formula providing 60% energy from carbohydrate, 20% energy from protein, and 20% energy from fat at varying caloric levels, those who received the highest caloric level (2.0 times the estimated resting energy expenditure) had the highest CO2 production. A similar study by Van den Berg and Stam [123] demonstrated that pulmonary function parameters were significantly better in patients receiving enteral formulas at 1.5 times versus 2.0 times the measured resting energy expenditure. Clinical opinions vary on the most appropriate feeding regimens for patients with COPD. However, studies have demonstrated that high-fat\/low-carbohydrate MNSs improve biochemical parameters in COPD patients. While similar results may be achieved by varying the caloric content, it is important to recognize that many patients with COPD are malnourished and would benefit from nutritional repletion. Therefore feeding at 1.5\u20132.0 times the measured resting energy expenditure is sometimes necessary.\n\nDisease-specific pulmonary MNSs have also been beneficial for patients with cystic fibrosis (CF). Nutritional status is correlated with survival among patients with CF. A study by Kane and Hobbs [125] demonstrated that resting energy expenditure in CF patients increases as pulmonary disease progresses. In this study, the low-carbohydrate\/high-fat MNS was compared to a high-carbohydrate\/low-fat commercial supplement. Patients who received the low-carbohydrate\/high-fat MNS experienced smaller increases in VCO2, RQ, and minute ventilation. Similar results were also demonstrated when the disease-specific MNS was compared to a medium-carbohydrate and a high-carbohydrate MNS during nighttime enteral feedings [126]. Because patients with CF often experience pancreatic insufficiency as their disease progresses, pancreatic enzymes were provided to reduce the incidence of steatorrhea with the higher fat MNS. In addition, the higher proportion of MCT oil (20\u201340%) in these formulas is beneficial for more effective fat absorption in patients with CF.\n\n#### 4. DIABETES MELLITUS\n\nDiabetes mellitus (DM) is the sixth leading cause of death in the United States and a major contributing factor to microand macrovascular conditions including cardiovascular diseases, vision problems, nerve damage, and renal disease. Diabetes is classified as either type 1, resulting from inadequate insulin production and secretion, or type 2, resulting from cellular resistance to the action of insulin. Although diabetes is a condition characterized by multiple metabolic abnormalities, results from the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) indicated that glucose control is the most important factor with respect to short- and longterm complications [127, 128]. Glucose regulation can be achieved through nutrition therapy, exercise, and medications such as oral hypoglycemic agents, antihyperglycemic agents, and insulin. Successful lifestyle modifications for patients with type 2 diabetes can delay or ameliorate the need for more expensive and complicated medical therapies including pharmaceutical treatments and interventions related to diabetes complications.\n\nStandard MNSs administered enterally to individuals with DM can have adverse effects on glycemic control because they have rapid gastric emptying and absorption, which can lead to unpredictable glucose excursions in the patient with diabetes. Therefore, DM specialty products have been specifically designed to manage the occurrence of hyperglycemia. Characteristics of many DM-specific formulas include a reduced carbohydrate level, especially simple sugars (except fructose); high monounsaturated fatty acids (MUFA); high fiber; and elevated antioxidants. Special ingredients such as fructose, MUFAs, and fiber all reduce glycemic response and therefore help control blood glucose levels. One of the main criticisms is the high level of fat, which typically ranges from 40% to 50% of total calories (Table 5). Individuals with DM are at risk for cardiovascular diseases (CVD), therefore the concern over elevated lipid levels is justified. However, many of the products contain a high level of MUFAs, which have been demonstrated to help improve lipoprotein levels [129\u2013132]. Furthermore, the fatty acid profiles of many of the formulas comply with the American Heart Association guidelines of \u226430% total energy from total fat and \u226410% energy from saturated fat.\n\nTABLE 5\n\nMacronutrient Composition of Common Specialty Products for Diabetes\n\nThe clinical benefits of DM-specialty MNSs have been demonstrated in both type 1 and type 2 DM [133\u2013135]. These studies demonstrated controlled glucose responses and less urinary glucose loss among patients receiving the disease-specific formulas. Reduced carbohydrate formulas have also been beneficial in patients with an acute head injury who were likely to develop stress-induced hyperglycemia. The administration of a DM-specialty product helped maintain normal glucose and eliminated the need for insulin administration [136].\n\nThe DM-specific MNS has also been studied as a replacement to typical solid-food snacks served to LTC residents. In this study the DM-specific MNS was compared to typical snacks and standard MNS. Blood glucose was much higher in the residents who received the standard MNS, but was not different between the DM-specific MNS and a typical snack [137], indicating that DM-specific MNS can be a good alternative for residents who have diminished dietary intake. The MSPS can also be implemented in patients with DM. Many of the 2.0 kcal\/ ml MNSs are high in fat and actually have a similar composition to DM-specific MNSs.\n\nStudies have been conducted to compare the clinical efficacy of the various DM-specific formulas. When the formulas were compared as enteral feedings or oral MNS, there was no difference observed between glucose or insulin curves [134, 135]. Similar results were also observed when different formulas were used for glucose tolerance tests [138]. The predominant difference in these formulas is that they vary in total fat content from 40% to 50% of energy. To determine whether the high-fat formula has any deleterious effects on serum lipid levels, a comparison was made between a standard MNS and a DM-specific MNS. There were no significant differences in the lipid levels between the two groups [139], which may be attributed to the high level of MUFA.\n\nUntil recently, none of the MNSs was designated to be specifically administered as snacks or meal replacements. Therefore, enteral supplements were often consumed as supplements. These formulas were clinically effective; however, they were not as palatable as those that have been developed for oral consumption. Recently a number of DM-specific MNSs, specifically intended for oral consumption, have become available in liquid and bar forms. The liquid MNSs have modified carbohydrate levels, are high in MUFAs, meet current guidelines for fatty acid profile, and have moderate to high levels of dietary fiber. Clinical studies indicate that these oral MNSs lower blood glucose compared with standard MNSs [140]. Bars that are specifically formulated to control postprandial blood glucose contain moderate levels of fiber and\/or resistant starch. These ingredients help to delay glycemic excursions by delaying gastric emptying. Additional benefits of these bars and oral MNS include a known nutrient content and ready-to-use packaging.\n\nThe clinical efficacy of disease-specific products for individuals with DM has been demonstrated effectively in the aforementioned studies. However, limited studies have demonstrated improved outcomes, which are of much interest in the current health care environment. One study has demonstrated the clinical outcomes of a disease-specific MNS versus a standard enteral formula. Residents who received the disease-specific MNS had a reduced incidence of infections (e.g., cellulitis, pneumonia, urinary tract, pressure ulcers) and reduced utilization of insulin and oral hypoglycemic agents as well as better glucose and lipid levels [129]. More studies such as this one need to be performed to help indicate the benefit of disease-specific MNSs.\n\n#### 5. RENAL DISEASE\n\nIndividuals with renal disease are likely to become malnourished in the course of their disease. Similar to other disease states, malnutrition is associated with an increased risk of morbidity and mortality among these patients. Dietary modifications are often imposed to help prevent the progression of renal failure and\/or manage symptoms of renal failure. Dietary modifications include high energy and protein, fluid, and electrolyte restrictions, as well as maintaining a strict balance between calcium and phosphorus intake. Renal patients often have an inadequate dietary intake [141] that may be attributed to their limited diets. Furthermore, dietary intake may be affected by symptoms of the disease such as nausea, vomiting, anorexia, and fatigue.\n\nThe goal of MNT for the renal patient is dependent on disease stage, whether they are undergoing dialysis, and which type of dialysis (hemodialysis or peritoneal dialysis). For patients with chronic renal failure, the goal is to maintain nutritional status and manage uremia without compromising renal function. Patients who are unable to meet their dietary needs may benefit from a disease-specific MNS intended for the predialysis patient. Formulas are energy dense (2.0 kcal\/mL); low in protein, fluids, and electrolytes; and have vitamin and mineral profiles specifically developed for renal patients. Patients who are receiving dialysis have a more liberalized diet with respect to protein; however, these individuals would still benefit from MNS. Formulas intended for patients on dialysis are also energy dense (2.0 kcal\/mL), have a moderate level of protein, contain restricted fluids and vitamin and mineral profiles specifically for dialysis patients.\n\nThere have been very few clinical trials of enteral or oral supplementation among renal patients for several reasons including very few patients per site and a preference of the patient and physician for TPN. In one study, clinical benefits were demonstrated in patients on hemodialysis using an orally administered disease-specific formula versus a standard formula. In this study, all of the MNSs were well tolerated and there was a significant improvement in serum calcium and phosphorus with disease-specific versus standard MNSs. Hyperphosphatemia is often a limiting symptom to MNS administration. Therefore, this benefit is clinically important because it indicated a way to improve nutritional status without compromising other biochemical parameters [142]. Cockram _et al._ [143] also investigated the effects of a nutrient-dense MNS on patients with chronic renal failure (CRF) who were not undergoing dialysis. Patients were randomized to receive the disease-specific MNS or an isocaloric supplement of glucose polymers. At baseline the dietary intake was well below recommendations for patients with CRF. Supplementation with a disease-specific MNS improved energy intake by 54\u201358%. Food intake did not change, indicating that the MNS did not displace meal intake.\n\nRenal MNSs have been developed to meet very specific nutrient levels within very selected ranges.\n\n## IV. BARRIERS TO USING MEDICAL NUTRITIONAL SUPPLEMENTS\n\nA. Cost\n\nCost can be a barrier to MNS; however, many individuals who require MNSs as part of their therapy can be reimbursed by third-party carriers. In the acute or LTC settings, MNSs are covered in room and board costs. Reimbursement for specialty products are even stronger under the diagnosis-related groups and the prospective payment system. Home care patients and consumers are expected to pay for MNSs as they would any other food. There are exceptions to this policy, which include patients who have special medical or financial needs. These individuals are reimbursed through local and state programs such as Medicaid.\n\nThe cost of an MNS typically varies and is dependent on whether it is a standard or disease-specific MNS. Disease-specific MNSs are typically more expensive because of specialized ingredients, and often more extensive clinical research is undertaken to validate their use. Studies referenced in this chapter have demonstrated the cost effectiveness of MNSs in terms of improved outcomes, such as decreased complications, medications (i.e., insulin, phosphate blockers), readmissions, and length of stay. Therefore, although cost is a concern for many individuals and health care plans, medical nutrition therapy should not be an area of neglect.\n\n### B. Taste\n\nTaste is a very important consideration because it influences patient and consumer compliance with MNSs. Nutritional companies are aware of how important the taste of MNSs is and therefore strive to formulate and reformulate their products to deliver the most palatable product. In addition, many oral MNSs are available in a variety of flavors to prevent flavor fatigue, a common complaint among patients. While taste is a very important factor, some MNSs may not always be palatable to patients. Disease-specific formulas often contain unique, novel ingredients that contribute to their efficacy but may detract from their taste. As health care professionals, it is important to help patients understand that this is an important part of their therapy. Recognizing MNT as part of a therapeutic regimen helps patients adhere to their treatment because they view it as something they should be taking, similar to other medications. Table 6 contains some suggestions that may help with patient compliance.\n\nTABLE 6\n\nTips for Consuming Medical Nutritional Supplements\n\n## V. SUMMARY\n\nMedical nutritional supplements cost effectively improve outcomes across the continuum of care as well as in different disease states. Disease-specific MNSs are specifically formulated and scientifically studied to effectively help manage symptoms and improve outcomes. Concerns that MNSs displace energy and nutrients from meals have not been substantiated by the research reviewed in this chapter. Medical nutritional supplements are convenient and provide an alternative to meals when time or medical symptoms are an issue. Demonstration of improved outcomes with MNSs is an exciting area of research and can add to the health care provider's arsenal of effective disease management options.\n\nReferences\n\n1. White J.V. _The Role of Nutrition in Chronic Disease Care_. Champaign, IL: The Nutrition Screening Initiative; 1997.\n\n2. Dychtwald K. 'Age power': How the new-old will transform medicine in the 21st century. _Geriatrics_. 1999;54:22\u201327.\n\n3. Sullivan D.H. Risk factors for early hospital read-mission in a select population of geriatric rehabilitation patients: The significance of nutritional status. _J. Am. Geriatr. Soc_. 1992;40:792\u2013798.\n\n4. Woo J., Ho S.C., Mak Y.T., Law L.K., Cheng A. 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Chlebowski R.T., Beall G., Grosvenor M., Lillington L., Weintraub N., Ambler C., Richards E.W., Abbruzzese B.C., McCamish M.A., Cope F.O. Long-term effects of early nutritional support with new enterotropic peptide-based formula vs. standard enteral formula in HIV-infected patients: Randomized prospective trial. _Nutrition_. 1993;9:507\u2013512.\n\n83. DeWys W.D., Begg C., Lavin P.T., Band P.R., Bennett J.M., Bertino J.R., Cohen M.H., Douglass H.O., Engstrom P.F., Ezdinli E.Z., Horton J., Johnson G.J., Moertel C.G., Oken M.M., Perlia C., Rosenbaum C., Silverstein M.N., Skeel R.T., Sponzo R.W., Tormey D.C. Prognostic effects of weight loss prior to chemotherapy in cancer patients. _Am. J. Med_. 1980;69:491\u2013497.\n\n84. Chlebowski R.T., Palomares M.R., Lillington L., Grosvenor M. Recent implications of weight loss in lung cancer management. _Nutrition_. 1996;12:S43\u2013S47.\n\n85. Ovesen L., Allingstrup L., Hannibal J., Mortensen E.L., Hansen O.P. Effect of dietary counseling on food intake, body weight, response rate, survival and quality of life in cancer patients undergoing chemotherapy: A prospective randomized study. _J. Clin. Oncol_. 1993;11:2043\u20132049.\n\n86. Menck H.R., Jessup M., Eyre H.J., Cunningham M.P., Fremgen A., Murphy G.P., Winchester D.P. _CA Cancer J. Clinicians_. 1997;47:161\u2013170.\n\n87. (suppl.)VanEys J. Effect of nutritional status on response to therapy. _Cancer Res_. 1982;42:747\u2013753.\n\n88. OctoberZogbaum A.T., Farkas S., Dufy V.B. Enteral feedings are associated with improved adherence to radiation treatment prescription and weight maintenance in head and neck cancer patients. _Abstract presented at The American Dietetic Association 79th Annual Meeting and Exhibition, San Antonio, Texas_. 1996;96:A\u201335\n\n89. Andreyev H.J., Norman A.R., Oates J., Cunningham D. Why do patients with weight loss have a worse outcome when undergoing chemotherapy for gastrointestinal malignancies? _Eur. J. Cancer_. 1998;34(4):503\u2013509.\n\n90. Lopez M.J., Robins P., Madden T., Highbarger T. Nutritional support and prognosis in patients with head and neck cancer. _J. Surg. Oncol_. 1994;55:33\u201336.\n\n91. Bozzetti F., Cozzaglio L., Gavazzi C., Bidoli P., Bonfanti G., Montalto F., Parra H.S., Valente M., Zucali R. Nutritional support in patients with cancer of the esophagus: Impact on nutritional status patient compliance to therapy and survival. _Tumori_. 1998;84:681\u2013686.\n\n92. Evans W.K., Nixon D.W., Daly J.M. A randomised study of oral nutrition support versus ad lib nutritional intake during chemotherapy for advanced colorectal and non-small cell lung cancer. _J. Clin. Oncol_. 1987;5:113.\n\n93. McCarter M.D., Gentilli O.D., Gomez M.E., Daly J.M. Preoperative oral supplement with immunonutrients in cancer patients. _J. Parenteral Enteral Nutr_. 1998;22:206\u2013211.\n\n94. Barber M.D., Ross J.A., Voss A.C., Tisdale M.J., Fearon K.C.H. The effect of an oral nutritional supplement enriched with fish oil on weight-loss in patients with pancreatic cancer. _Br. J. Cancer_. 1999;81:80\u201386.\n\n95. Falconer J.S., Fearon K.C.H., Plester C.E., Ross J.A., Carter D.C. Cytokines, the acute phase protein response, and resting energy expenditure in cachectic patients with pancreatic cancer. _Ann. Sur_. 1994;219(4):325\u2013331.\n\n96. Hyltander A., Korner U., Lundholm K.G. Elevation of mechanisms behind elevated energy expenditure in cancer patients with solid tumors. _Eur. J. Clin. Invest_. 1993;23:46.\n\n97. Fearon K.C.H., McMillan D.C., Preston T., Winstanley P., Cruickshank A.M., Shenkin A. Elevated circulating interluekin-6 is associated with an acute phase response, but reduced fixed hepatic protein synthesis in patients with cancer. _Ann. Surg_. 1991;213:26.\n\n98. Wigmore S.J., Plester C.E., Richardson R.A., Fearon K.C.H. Changes in nutritional status associated with unresectable pancreatic cancer. _Br. J. Cancer_. 1997;75(1):106\u2013109.\n\n99. Falconer J.S., Fearon K.C.H., Ross J.A. Acute-phase protein response and survival duration of patients with pancreatic cancer. _Cancer_. 1995;75(8):2077\u20132082.\n\n100. Staal-van den Brekel A.J., Schols A.M.W.J., ten Velde G.P.M., Buurman W.A., Wouters E.F.M. Analysis of the energy balance in lung cancer patients. _Cancer Res_. 1994;54:6430\u20136433.\n\n101. Wigmore S.J., Ross J.A., Falconer J.S., Plester C.E., Tisdale M.J., Carter D.C., Fearon K.C.H. The effect of polyunsaturated fatty acids on the progress of cachexia in patients with pancreatic cancer. _Nutrition_. 1996;12:S27\u2013S30.\n\n102. Wigmore S.J., Fearon K.C.H., Maingay J.P., Ross J.A. Down-regulation of the acute-phase response in patients with pancreatic cancer cachexia receiveing oral eico-sapentaenoic acid is mediated via suppression of interleukin-6. _Clin. Science_. 1997;92:215\u2013221.\n\n103. Gogos C.A., Ginopoulos P., Slasa B., Apostolidou E., Zoumbos N.C., Kalfarentzos F. Dietary omega-3 polyunsaturated fatty acids plus vitamin E restore immunodeficiency and prolong survival for severely ill patients with generalised malignancy. _Cancer_. 1998;82(2):395\u2013402.\n\n104. Daly J.M., Lieberman M.D., Goldfine J., Shou J., Weintraub F., Rosato E.F., Lavin P. Enteral nutrition with supplemental arginine, RNA, and omega-3 fatty acids in patients after operation: Immunologic, metabolic, and clinical outcome. _Surgery_. 1992;112:56\u201367.\n\n105. Heys S.D., Walker L.G., Smith I., Eremin O. Enteral nutritional supplementation with key nutrients in patients with critical illness and cancer. _Ann. Surg_. 1999;229:467\u2013477.\n\n106. Chlebowski R.T., Dugan W., Laufman L., Whie L., Brotherton T., Reeves J., Sher H., Badolato C., Forte F., Sambuco S., Courtney K., Ceh S., McCamish M., Green D., Cope F. A novel peptide-based enteral formula and 5-FU\/leucovorin related adverse experiences in colorectal cancer patients: An interim evaluation. Abstract presented at American Society of Clinical Oncology Annual Meeting. 1997:922.\n\n107. National Center for Health Statistics Fact Sheet (1996). Centers for Disease Control, Fact Sheet (1996). Centers for Disease Control. Atlanta, Georgia.\n\n108. Murray C.J.L., Lopez A.D. Alternative projections of mortality and disability by cause 1990\u20132020: Global burden of disease study. _Lancet_. 1997;349:1498\u20131504.\n\n109. Schols A., Moster R., Souters P., Gruieve H., Wouters E.F.M. Inventory of nutritional status in patients with COPD. _Chest_. 1989;96:247\u2013249.\n\n110. Laaban J.P., Kouchakli B., Dore M.F., Orvoen-Frija E., Rochmaure J. Nutritional status of patients with chronic obstructive pulmonary disease and acute respiratory failure. _Chest_. 1993;103:1362\u20131368.\n\n111. Donahoe M., Rogers R.M. Nutritional assessment and support in chronic obstructive pulmonary disease. _Clin. Chest Med_. 1990;11:487\u2013504.\n\n112. Hogg J. Wasting and chronic obstructive pulmonary disease. _Support Line_. 1998;20:13\u201318.\n\n113. Ambrosino N., Foglio K., Rubini F., Clili E., Nava S., Vitracca M. Noninvasive mechanical ventilation in acute respiratory failure due to chronic obstructive pulmonary disease: Correlates for success. _Thorax_. 1995;50:755\u2013757.\n\n114. Gray-Donald K., Gibbons L., Shapiro S.H., Mackelm P.T., Martin J.G. Nutritional status and mortality in chronic obstructive pulmonary disease. _Am. J. Respir. Crit. Care Med_. 1996;153:961\u2013966.\n\n115. Efthimiou J., Fleming J., Gomes C., Spiro S.G. The effect of supplementary oral nutrition in poorly nourished patients with chronic obstructive pulmonary disease. _Am. Rev. Respir. Dis_. 1988;137:1075\u20131082.\n\n116. Larca L., Greenbaum D.M. Effectiveness of intensive nutritional regimen in patients who fail to wean from mechanical ventilation. _Crit. Care. Med_. 1982;10:297\u2013300.\n\n117. Bassili H.R., Deitel M. Effect of nutritional support on weaning patients off mechanical ventilators. _J. Parenteral Enteral Nutr_. 1981;5:161\u2013163.\n\n118. Saltzman H.A., Salzano J.V. Effects of carbohydrate metabolism upon respiratory gas exchange in normal men. _J. Appl. Physiol_. 1971;30:228\u2013231.\n\n119. Garfinkle F., Robinson S., Price C. Replacing carbohydrate calories with fat calories in enteral feeding for patients with impaired respiratory function. _J. Parenteral Enteral Nutr_. 1985;9:3.\n\n120. Angelillo V.A., Bedi S., Durfee D., Dahl J., Patterson A.J., O'Donohue W.J. Effects of low and high carbohydrate feedings in ambulatory patients with chronic obstructive pulmonary disease and chronic hypercapnia. _Ann. Intern. Med_. 1985;103:883\u2013885.\n\n121. Goldstein S., Thomashow B., Askanazi J. Functional changes during nutritional repletion in patients with lung disease. _Clin. Chest Med_. 1986;7:141\u2013151.\n\n122. Frankfort J.D., Fisher C.E., Stansbury D.W., McArthur D.L., Brown S.E., Light R. Effects of high- and low- carbohydrate meals on maximum exercise performance in chronic airflow obstruction. _Chest_. 1991;100:792\u2013795.\n\n123. Van den Berg B., Stam H. Metabolic and respiratory effects of enteral nutrition in patients during mechanical ventilation. _Intens. Care Med_. 1988;14:206\u2013211.\n\n124. Talpers S.S., Romberger D.J., Bunce S.B., Pingleton S.K. Nutritionally associated increased carbon dixoide production. _Chest_. 1992;102:551\u2013555.\n\n125. Kane R.E., Hobbs P. Energy and respiratory metabolism in cystic fibrosis: The influence of carbohydrate content of nutritional supplements. _J. Pediatr. Gastroenterol. Nutr_. 1991;12:217\u2013223.\n\n126. Kane R.E., Hobbs P.J., Black P.G. Comparison of low, medium and high carbohydrate formulas for nighttime enteral feedings in cystic fibrosis patients. _J. Parenteral Enteral Nutr_. 1990;14:47\u201352.\n\n127. DCCT Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin dependent diabetes mellitus. _N. Engl. J. Med_. 1993;329:977\u2013986.\n\n128. UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). _Lancet_. 1998;352:837\u2013853.\n\n129. Craig L.D., Nicholson S., Silverstone F.A., Kennedy R.D. Use of a reduced-carbohydrate, modified-fat enteral formula for improving metabolic control and clinical outcomes in long-term care residents with type 2 diabetes: Results of a pilot trial. _Nutrition_. 1998;14:529\u2013534.\n\n130. Garg A., Bonanome A., Grundy S.M., Zhang Z., Unger R. Comparison of a high-carbohydrate diet with a high-monounsaturated fat diet in patients with non-insulin-dependent diabetes mellitus. _N. Engl. J. Med_. 1988;319:829\u2013834.\n\n131. Garg A., Grundy S.M., Koffler M. Effect of high carbohydrate intake on hyperglycemia, islet function, and plasma lipoproteins in NIDDM. _Diabetes Care_. 1992;15:1572\u20131580.\n\n132. Garg A., Bantle J.P., Henry R.R., Coulston A.M., Griver A., Raatz S.K., Brinkley L., Chen I., Grundy S.M., Huet B.A., Reaven G.M. Effects of varying carbohydrate content of diet in patients with non-insulin-dependent diabetes mellitus. _JAMA_. 1994;271:1421\u20131428.\n\n133. Peters A.L., Davidson M.B., Isaac R.M. Lack of glucose elevation after simulated tube feeding with a low-carbohydrate, high-fat enteral formula in patients with type 1 diabetes. _Am. J. Med_. 1989;87:178\u2013182.\n\n134. (Suppl.)Reader D.M., Fish L., Franz M. Plasma glucose and insulin response to isocaloric quantities of three nutritional formulas in persons with non-insulin-dependent-diabetes. _Diabetes_. 1996;2:45.\n\n135. Reader D.M., Fish L.H., Franz M.J. Response to isocaloric quantities of enteral feedings in persons with non-insulin-dependent diabetes mellitus (NIDDM) [abstract]. _J. Am. Dietet. Assoc_. 1994;94:A\u201339.\n\n136. Grahm T.W., Harrington T.R., Isaac R.M. Low carbohydrate (CHO) with fiber enteral formula impedes development of hyperglycemia in patients with acute head injury. _Clin. Res_. 1989;37:138A.\n\n137. Galkowski J., Silverstone F.A., Brod M., Isaac R.M. Use of a low carbohydrate with fiber enteral formula as a snack for elderly patients with type 2 diabetes. _Clin. Res_. 1989;37:89A.\n\n138. Clintec Sales Aid. Deerfield, IL, 1999.\n\n139. McCargar L.J., Innis S.M., Bowron E., Leichter J., Toth E., Wall K. Effect of enteral nutritional products differing in carbohydrate and fat on indices of carbohydrate and lipid metabolism in patients with NIDDM. _Mol. and Cell Biochem_. 1998;188:81\u201389.\n\n140. Kipnes M., Shade S., Geraghty M., Craig L., Bossetti B. Effect of a liquid nutritional designed for oral supplementation on glucose tolerance in subjects with type 2 diabetes. _Diabetes_. 1998;47:A90.\n\n141. Kopple J.D. Abnormal amino acid metabolism in uremia. _Kidney Int_. 1978;14:340\u2013348.\n\n142. Cockram D.B., Hensley M.K., Rodriguez M., Agarwal G., Wennberg A., Ruey P., Ashbach D., Hebert L., Kunau R. Safety and tolerance of medical nutritional products as sole sources of nutrition in people on hemodialysis. _J. Ren. Nutr_. 1998;8:25\u201333.\n\n143. Cockram D.B., Moore L.W., Acchiardo S.R. Response to an oral nutritional supplement for chronic renal failure patients. _J. Ren. Nutr_. 1994;4:78\u201385.\nCHAPTER 16\n\nComposite Foods and Formulas, Parenteral and Enteral Nutrition\n\nLAURA MATARESE, The Cleveland Clinic Foundation, Cleveland, Ohio\n\n## I. INTRODUCTION\n\nOral consumption of a standard diet is the best way to meet nutrition needs. But when oral feeding with regular food is not possible, nutrients may be delivered as a supplement or as total nourishment through nonvolitional feedings. This chapter will describe composite foods and formulas, as well as the general principles of enteral and parenteral nutrition.\n\n## II. ENTERAL NUTRITION\n\nThe use of enteral feedings dates back to Egypt many centuries before Christ. The earliest recorded evidence of enteral nutrition employed the use of rectal feedings. These formulas were often composed of wine, whey, milk, and barley and were used to coat inflamed intestines and to provide nutrients. Eventually, nutrient substances were introduced into the upper gastrointestinal (GI) tract, but this was not until the 1600s. Early enteral feeding formulas were generally composed of regular foodstuffs, which were pulverized and administered with syringes. Now, sophisticated nutrient formulas are commercially available along with advanced equipment to infuse them.\n\nThe decision to feed enterally or parenterally depends on the integrity and functional status of the GI tract. If the GI tract is functional, accessible, and safe to use, enteral nutrition is always the preferred method of nutrition support. Enteral nutrition (feedings by tube) promotes comparable or better outcomes [1\u20133] and is less expensive [4] than parenteral nutrition. The benefits of enteral nutrition over parenteral nutrition appear to be largely due to a reduction of septic complications. It is believed that delivery of nutrients through the GI tract helps to maintain the gut mucosal barrier and prevent bacterial translocation into systemic circulation [5]. Enteral nourishment is generally considered to be safer than parenteral nutrition. But enteral nutrition therapy does possess its own set of potential complications [6].\n\n### A. Indications\/Contraindications\n\nGeneral indications for enteral nutrition include insufficient oral intake, nutrient repletion, and support during the transition phase of parenteral nutrition. Contraindications include intractable vomiting, intestinal obstruction, upper GI tract hemorrhaging, high risk for aspiration, and severe intractable diarrhea. The decision to initiate enteral feedings begins with an assessment of GI tract function. Signs of adequate function include normal upper GI tract or small bowel X ray, flatus or bowel movement, bowel sounds, hunger, absence of vomiting, absence of obstruction or ileus, and no uncontrolled diarrhea.\n\n### B. Enteral Access\n\nWhen initiating enteral feedings it is important to consider the route, access device, method of administration, and type of feeding formula [7]. The route of access is determined by the anticipated length of therapy and the risk of aspiration. Nasoenteric and oroenteric feeding tubes are used when the anticipated length of therapy is short, generally less than 4 weeks, or for interim access before the placement of a long-term feeding tube. If the risk of aspiration is low, the tip of the feeding tube can terminate in the stomach. Feeding into the stomach is more physiologic and allows feeding to be administered without a feeding pump. However, if there is an increased risk of aspiration due to mental obtundation, an incompetent lower esophageal sphincter with gastroesophageal reflux or gastroparesis, the tip of the feeding tube should be placed beyond the pylorus. This may be accomplished intraoperatively, by active bedside placement, with the use of prokinetic agents or a fluoroscope or endoscope. Spontaneous transpyloric passage of feeding tubes through peristalsis can lead to successful placement in 36\u201356% of weighted tubes and in 84\u201392% of unweighted tubes, but may take several days before feeding can be initiated [8]. Zaloga [9] has developed an active bedside technique for placing tubes transpylorically that involves careful advancement of the feeding tube while rotating it around its long axis. The rate of successful tube placement using this technique ranges from 40% to 85%. Prokinetic agents (metoclo-praminde, cisapride, erythromycin) have been used to aid in tube placement [8]. In some settings (i.e., intensive care) it may be appropriate to use transpyloric feeding with simultaneous gastric decompression in order to minimize the risk of aspiration [10]. This may be accomplished with the use of specialized tubes that perform both functions or the use of two separate tubes.\n\nLong-term (permanent) access requires a percutaneous or surgically placed feeding enterostomy. If the risk of aspiration is low, a percutaneous endoscopic gastrostomy (PEG) can be placed [11, 12]. A surgical gastrostomy should be considered when access to the stomach is anticipated during a major abdominal procedure or when PEG placement is contraindicated. If the risk of aspiration is high, a PEG with a jejunal extension can be used [13]. Another technique for providing long-term access into the small bowel is direct endoscopic jejunostomy (DEJ) [14]. The DEJ tube is placed endoscopically, as is a PEG, except that the endoscope is passed through the duodenum, past the ligament of Treitz, into a loop of jejunum adjacent to the abdominal wall. If an endoscope cannot be passed, a surgical jejunostomy may be indicated.\n\n### C. Formula Composition: Nutrient Components\n\nThe composition and nutritional value of commercially prepared formulas vary considerably. The composition of the formula determines the use and has a tremendous bearing on patient tolerance.\n\n#### 1. PROTEIN\n\nProtein generally comprises 15\u201320% of the total energy provided in an enteral feeding solution but may range from 8% to 25% of energy. Protein sources for enteral formulas include [1] intact protein from eggs, milk, or meat; [2] protein isolates from milk (casein, whey), soybean, or egg white; [3] hydrolyzed protein from casein, fish, meat, and soy and may contain added amino acids; [4] short-chain peptides; and [5] free amino acids. The protein source must be considered when selecting a formula for a patient, particularly when there is impaired ability to digest or absorb nutrients. The products containing hydrolyzed protein tend to be more expensive and therefore should be limited to use with patients with appropriate indications.\n\nThe quality of a protein is dependent on the amino acid composition. Casein and soy protein, which are usually the protein sources found in enteral formulas, do not have as high a biological value as albumin, and therefore more may be required to achieve positive nitrogen balance [15]. Many enteral formulas have altered amino acid composition designed to support patients in various disease states. Traditionally, a factor of 6.25 is used when converting grams of protein to grams of nitrogen. With the recent proliferation of new specialty formulas containing supplemental and altered amino acids, conversion factors ranging from 5 to 7.5 may be used depending on the amino acid and nitrogen source. This information can be obtained from the manufacturer of the specific enteral formula.\n\nThe use of short-chain peptides of varying lengths or single amino acids as the protein source in enteral feedings remains controversial. Data from animal studies suggest that peptide-based diets are associated with improved absorption, lower mortality, and decreased incidence of bacterial translocation [16\u201318]. Data from human studies also demonstrate more rapid and efficient absorption of peptides than for free amino acids in both the healthy and diseased gut [19\u201323]. However, other studies have not confirmed the beneficial effects of peptides over free amino acids or intact proteins [24\u201326]. It does appear that the use of peptide-containing solutions may be beneficial for those patients who have impaired digestion or absorption capabilities [27, 28].\n\n#### 2. CARBOHYDRATE\n\nCarbohydrate comprises the major source of energy, generally 40\u201350%, but as high as 90% of total energy in an enteral formula. It also has the greatest effect on tolerance because the carbohydrate source exerts the greatest effect on the osmolality of the solution. The carbohydrate may be in the form of monosaccharides, disaccharides, oligosaccharides, or starch. Monosaccharides, such as glucose, have a greater osmotic effect than complex carbohydrates, such as starches. The use of these hyperosmolar solutions may result in abdominal discomfort, diarrhea, and dumping. However, these smaller molecules require fewer pancreatic enzymes and intestinal mucosal disaccharidases for adequate digestion and may be useful for patients with an impaired ability to digest and absorb. The predominant form of carbohydrate found in enteral formulas is from large molecules, such as glucose oligosaccharides, maltodextrin, and hydrolyzed cornstarch. Most commercially available enteral formulas are lactose free.\n\n#### 3. FAT\n\nDietary fat is necessary to supply essential fatty acids (EFA). It acts as a carrier of fat-soluble vitamins and is a concentrated energy substrate. The addition of fat to enteral formulas also enhances palatability and flavor, factors of importance when enteral formulas are used as oral supplements. The fat content of enteral formulas varies considerably in both quantity and type. In general, 30\u201345% of total energy is from fat but may range from 1.3% to 55% of energy. Sources of fat commonly found in enteral formulas include long-chain triglycerides (LCT) from corn, safflower, sunflower, or soybean oil. These sources are high in omega-6 fatty acids which are thought to suppress immune function [29\u201332].\n\nSome formulas contain medium-chain triglycerides (MCT), which are more rapidly hydrolyzed and absorbed than LCTs. MCT oil does not require significant bile salt or lipase activity for digestion and is transported directly to the liver for metabolism by the portal circulation instead of the lymphatic system following absorption [33]. MCT oil is derived from coconut oil and does not contain essential fatty acids.\n\nMany products now contain omega\u20133 fatty acids for immune enhancing properties. Recently, there has been interest in the use of structured lipids, which are formed by the random transesterification of MCT with LCT. This compound contains both MCT and LCT on the same glycerol backbone, thus offering the advantage of enhanced absorption and EFA content [29, 34].\n\n#### 4. VITAMINS AND MINERALS\n\nVitamins, minerals, and trace elements are all included in standard tube feeding formulas. Most formulas meet the U.S. Recommended Dietary Allowances (RDA) for these nutrients in 1.5\u20132 L of formula. Some otherwise complete enteral formulas do not contain vitamin K. Vitamin K deficiency is rare, theoretically, because it is synthesized by bacteria in the gut. Vitamin K may also interfere with anticoagulation therapy (e.g., warfarin). Alternatively, vitamin K supplementation may be necessary for patients receiving antibiotics for prolonged periods. Some of the vitamins and minerals have been fortified beyond the RDA to meet the requirements imposed by illness and injury. Patients receiving enteral formulas for more than 6 months have been shown to have normal or high blood levels of certain vitamins [35]. Certain disease-specific formulas may not have vitamins added due to their potential toxicity in various clinical conditions.\n\n#### 5. FIBER\n\nFiber has many beneficial effects such as increasing stool bulk, serving as an energy source for colonocytes, and normalization of bowel function [36\u201338]. Fiber has been promoted for treating diarrhea, constipation, and blood glucose control [36, 38\u201340]. There are basically two types of fiber of interest in enteral nutrition: cellulosic and noncellulosic. Cellulosic fibers are of high molecular weight and are non-soluble. Examples include cellulose, and wheat bran is a rich source. These contribute to fecal mass and water content and reduce stool mean transit time. Noncellulosic fibers are more water soluble and include hemicelluose, pectin, gums, and mucilages. They are rapidly degraded by anaerobic micoflora of the cecum and colon to yield short-chain fatty acids.\n\n#### 6. PHYSICAL CHARACTERISTICS\n\nThe osmotic activity of an enteral formula is generally measured as osmolality, the concentration of particles (solutes) that affects the osmotic balance across a semipermeable membrane. It is expressed as mOsm\/kg H2O. Because the osmolality of an enteral formula is based on the number of dissolved particles in solution, the smaller the particle size, the greater the number of particles per unit weight of solute and the higher the osmolality. Thus, complex high molecular weight macronutrients, such as starch and proteins, contribute less to the osmolality of the solution than a lower molecular weight compound such as glucose oligosaccharides or short- chain peptides. Fats do not form a solution in water and therefore have no osmotic effect. Electrolytes added as salts, such as sodium chloride or potassium chloride, dissociate in solution and increase the osmolality of the formula.\n\nThe significance of osmolality is related to the clinical tolerance of the enteral formula. In general, the closer a formula is to isotonic concentration, the lower the potential for GI complications such as diarrhea, dumping, nausea, and vomiting. However, administration of isotonic solutions does not guarantee tolerance. Although many clinicians associate diarrhea with enteral feedings, controlled trials have failed to demonstrate this relationship [41\u201344].\n\nIt is common for manufacturers to report the renal solute load of the formula. This is a measure of the concentration in a feeding solution of the particles that the kidney must work to excrete. Protein, yielding urea as its endproduct, and the electrolytes sodium, potassium, and chloride are the main contributors to renal solute load. The renal solute load is of particular concern in geriatric and pediatric populations. High renal solute loads can predispose a patient to dehydration.\n\nThe hydrogen ion concentration (pH) of enteral formulas is also of interest. Gastric motility is decreased when solutions have a pH lower than 3.5. Most enteral formulas have a pH above 3.5. The pH of a formula can also contribute to tube occlusion. Most intact protein formulas coagulate when acidified to a pH of less than 5.0 [45]. However, attempts have also been made to acidify enteral feedings in order to reduce gastric colonization and the incidence of aspiration pneumonia [46]. Although acidifying enteral feedings has been shown to reduce gastric colonization in critically ill patients, larger studies are needed to examine the effect of this strategy on morbidity, such as pneumonia, and mortality.\n\n### D. Formula Classification\n\nEnteral formulas can be divided into three major categories: polymeric, hydrolyzed, and modular and disease specific. Polymeric formulas contain intact protein generally from casein and soy protein isolates, polyscaccharides and glucose polymers, and a mixture of LCTs and MCTs in proportions that mimic a standard diet. These products tend to be relatively inexpensive and are generally well tolerated. The caloric content may vary between 1.0 and 2.0 kcal\/mL; protein generally provides 15\u201320% of the total energy and fat provides 30\u201350% of energy. The osmolality of these formulas varies from 300 to 600 mOsm\/kg.\n\nHydrolyzed formulas contain macronutrients that have been partially or completely hydrolyzed to the component oligopeptides, short-chain peptides, free amino acids, and glucose oligosaccharides. They also tend to be very low in fat. These formulas are useful for patients who have an impaired ability to digest or absorb nutrients.\n\nModular formulas are used to supplement individual nutrients in an existing commercial formula or to compose a specific new formula to meet the unique requirements of a particular patient. They usually consist of one or more macronutrients.\n\n#### 1. DISEASE-SPECIFIC FORMULAS\n\nRenal failure formulas are generally low in protein and contain essential amino acids (EAA) or a combination of EAA and nonessential amino acids (NEAA). The rationale for the use of these products is that urea can be recycled as a nitrogen source in the GI lumen by bacterial urease. Fragments are then reabsorbed and converted to NEAA in the liver [47]. These products may also be low in electrolytes and fat-soluble vitamins. Early studies demonstrated lowered blood urea nitrogen levels and a reduction of uremic symptoms with the use of EAA [48, 49]. Subsequent trials, however, did not demonstrate a clear benefit of EAA alone compared to a mixture [50, 51]. Now with newer dialysis techniques, the trend is to provide patients with a full complement of amino acids and then treat with dialysis. Standard amino acid formulas containing EAA and NEAA should be used during dialysis and chronic renal failure. EAA may be useful in promoting improved nutritional status in acute renal failure or to delay the initiation of treatment.\n\nPatients with liver disease generally present in a malnourished state. Administration of protein to improve nutritional status often results in hepatic encephalopathy [52]. Patients may have abnormal plasma amino acid patterns, characterized by elevated concentrations of methionine and the aromatic amino acids (AAA), phenylalanine, tyrosine, tryptophan, and decreased levels of the branched chain amino acids (BCAA), valine, leucine, and isoleucine [53]. Hepatic failure formulas generally contain enriched levels of BCAA and low AAAs. The rationale is to normalize amino acid patterns and improve or reverse hepatic encephalopathy. The efficacy of these specialized products remains controversial [54\u201356]. Many of the studies involving the use of BCAAs have failed to show any benefit other than improved nitrogen balance [57\u201360]. Most patients with hepatic encephalopathy can tolerate low doses of standard amino acids. However, if the patient becomes encephalopathic or the encephalopathy worsens, a BCAA-enriched formula may be useful.\n\nPatients with pulmonary disease may suffer from carbon dioxide (CO2) retention and oxygen deprivation. This is of particular concern when attempting to wean patients off ventilators. Specialty pulmonary failure formulas tend to be high in fat. The rationale is to decrease the CO2 production from oxidation of carbohydrate. Several studies have demonstrated lowered CO2 production, lowered respiratory quotients, lowered PaCO2, and decreased ventilatory time with the use of these products [61\u201364]. But most of these studies did not control for the use of sedatives and other muscle relaxants affecting mechanical ventilation. It is more important to avoid overfeeding these patients since CO2 production seems to be more affected by the amount of energy provided than by the level of carbohydrate [65].\n\nMetabolic stress is a phrase used to describe patients who are catabolic, hypermetabolic, and critically ill. A number of products, designed for these patients, have been enriched with BCAA, glutamine, arginine, nucleotides, and\/or structured lipids. The rationale has been to blunt the hypermetabolic response and improve immune function. Products have been enriched with BCAA to stimulate protein synthesis and minimize protein degradation [66]. The profile of the BCAA for stress formulas does differ from those used in hepatic encephalopathy. The use of products enriched with BCAA for metabolically stressed patients has resulted in improvement of nitrogen retention, total lymphocyte count, and visceral protein levels [67\u201371]. Unfortunately none of these studies has been able to demonstrate any difference in morbidity or mortality.\n\nVarious NEAAs such as glutamine and arginine, have been added to enteral formulas designed to support patients with metabolic stress because it is hypothesized that these amino acids may be conditionally essential during these periods. Glutamine exerts a tropic effect on the bowel and stimulates nutrient absorption [72\u201374]. Glutamine also functions as a precursor of nucleic acids, nucleotides, amino sugars, and proteins and is the preferred fuel for rapidly proliferating cells such as enterocytes, macrophages, and lymphocytes [75]. Glutamine is an abundant amino acid and found in all enteral formulas as protein bound. It is unclear whether glutamine must be present as a free amino acid to exert a pharmacologic effect.\n\nArginine may also be conditionally essential during periods of metabolic stress. It has been shown to improve weight gain and increase nitrogen retention during recovery from injury in experimental animals [76]. Arginine has been shown to increase collagen deposition and improve wound healing [77, 78]. But supplemental arginine has been added to enteral formulas mostly for its reported immune-enhancing properties [79\u201382].\n\nSeveral enteral formulas are now available that have combined supplemental levels of amino acids, omega-3 fatty acids, and enhanced vitamin levels for use in metabolically stressed patients. Many of the studies suggest promising results on various outcome parameters, such as improved immune response, better nitrogen balance, reduction in infections, decreased length of stay, and decreased days on a ventilator [83\u201395]. Although absolute efficacy has not been clearly demonstrated, the data suggest that these products may be helpful in reducing complications in specific patient populations.\n\nSpeciality products are available for patients with diabetes mellitus and impaired glucose tolerance. Patients with diabetes mellitus requiring long-term enteral feeding should have a formula that mimics the dietary guidelines of the American Diabetes Association [96]. Some of the specialty enteral formulas designed for use with these patients are very high in fat, providing up to 50% of energy to assist with blood glucose control. Some success has been seen with the use of these products in preventing elevation of blood glucose levels in nonhospitalized persons consuming formula by mouth [97, 98]. The fiber-containing products are useful in glucose only if fed into the stomach to delay gastric emptying.\n\n### E. Formula Selection\n\nThe clinician now has a vast array of composite foods and enteral formulas from which to choose. When choosing a formula it is important to consider a few basic points. First, what is the patient's digestive capability? If there is any impaired ability to digest or absorb nutrients, a predigested formula may be indicated. These formulas typically have macronutrients which are present as hydrolyzed components; protein in the form of short-chain peptides or amino acids; carbohydrate in the form of glucose oligosaccharides; and fat in varying combinations of LCTs and MCTs. Secondly, does the patient have severe organ dysfunction that may benefit from a disease-specific enteral formula? Third, does the patient require a fluid restriction or have increased metabolic needs necessitating the use of a nutrient-dense formula? Finally, the feeding route should be considered. If the formula is to be administered orally, it must be palatable. If the formula is to be infused through a small-bore feeding tube, it should be of an appropriate viscosity so as to prevent occlusion of the tube.\n\n### F. Administration Techniques\n\nThe method selected for delivery of tube feeding depends on the location of the feeding tube, the emptying ability of the stomach, the risk of gastroesophageal reflux, the digestive capability of the GI tract, and patient mobility. With bolus administration, 240\u2013400 mL of enteral formula is provided over a 20- to 30-minute period. It is easy to do and does not require the use of a pump. This method is best for gastric feedings. However, this technique may result in nausea, vomiting, and diarrhea. Many of the gastrointestinal complications associated with bolus delivery can be alleviated with the use of a timed intermittent infusion. With this technique, a similar amount of formula is delivered over 1\u20132 hours. This may be done by gravity or with an enteral feeding pump. Feedings may also be delivered continuously over 12\u201324 hours by gravity or pump. This method is generally well tolerated and is usually chosen for feedings into the small bowel [99]. Finally, patients can be cycled on tube feedings whereby the feeding is delivered over an 8- to 12-hour period. This is generally used for enteral feeding in the home setting or while transitioning patients onto oral intake.\n\n### G. Complications\n\nComplications can be divided into three general categories: mechanical, gastrointestinal, and metabolic. Most of these problems can be avoided by using the proper technique during enteral access, administration, and close monitoring.\n\nThe frequency of mechanical complications, such as nasopharyngeal\/mucosa erosion, sinusitis, and otitis media, has markedly decreased with the development of small-bore, pliable feeding tubes [100]. Tube misplacement, however, is a very serious potential complication associated with the use of these tubes. It is most likely to occur in patients who are uncooperative, obtruded, or seriously ill. The presence of an endotracheal tube does not prevent its occurrence. Because tube misplacement can lead to esophageal or pleural perforation and the inadvertent administration of feeding into the mediastinum or lung, a chest radiograph is mandatory after tube placement and prior to initiation of feeding [101, 102].\n\nAspiration pneumonia is a serious and potentially life-threatening complication of enteral feeding. It is important to differentiate the aspiration of tube feeding from the aspiration of oropharyngeal secretions since patients with the latter problem will aspirate while receiving any form of nutrition support [103, 104]. A decrease in the rate of gastric emptying is the most common risk factor for aspiration and is due to a wide variety of medical conditions including sepsis, trauma, head injury, electrolyte abnormalities, and anti-cholinergic and narcotic drugs. Gastroesophageal reflux can occur when a tube is placed through the lower esophageal sphincter; however, this risk is greatest when the patient is supine [105]. Rapid bolus feeding can lead to transient relaxation of the lower esophageal sphincter [106]. Intragastric feeding should be given with the patient's head raised 30 degrees or more, and if intermittent feeding is administered, the patient should remain in this position for at least 1 hour after the feeding [107]. The risk of aspiration can be further minimized by feeding beyond the pylorus with simultaneous gastric decompression.\n\nObstruction of feeding tubes is another potential mechanical complication of enteral feedings. This is generally the result of placing medications and incompletely dissolved formula through the tubes. Failure to routinely irrigate tubes will also increase the risk of tube occlusion [108\u2013110].\n\nGastrointestinal intolerance is the most common complication of tube feeding. Symptoms include nausea, vomiting, diarrhea, constipation, abdominal pain, and distention. In many cases, the patient's disease or medications used to manage it contribute to these symptoms. If nausea and vomiting are due to gastric distention or gastroesophageal reflux, a slow continuous infusion of the formula may reduce these symptoms. A prokinetic agent, such as metoclopraminde or cisapride, may also be used. Diarrhea is perhaps the most common gastrointestinal complication of tube feeding. Causes of diarrhea include concomitant antibiotic treatment, _Clostridium difficile_ toxin with or without pseudomembranous colitis, sorbitol-based liquid medication use, hypoalbuminemia, and magnesium content of the enteral formula [111\u2013113]. Diarrhea can be controlled by decreasing the infusion rate and using antidiarrheal agents. In some instances, it may be necessary to change the enteral feeding formula. Fiber-containing formulas may decrease diarrhea by increasing transit time and by supplying the colon with fermentable fiber, which produces short-chain fatty acids, the primary fuel of the colon [114].\n\nPatients receiving enteral feedings may also experience a variety of metabolic complications ranging from glucose intolerance, hyperkalemia, hypokalemia, hyponatremia, hypernatremia, hypophosphatemia, and dehydration [115]. Electrolytes can be repleted by supplementing the tube feeding or by providing them parenterally. Oral potassium salts can cause gastrointestinal upset while magnesium and phosphate salts can result in diarrhea. Although metabolic complications are relatively uncommon in association with enteral feeding, patients should be monitored carefully, especially at the beginning of therapy.\n\n## III. PARENTERAL NUTRITION\n\nThe infusion of nutrients via the parenteral route is a fairly recent technique. During the 1960s when techniques became available to access central circulation, people began to infuse concentrated forms of dextrose into patients. Finally in 1968, a child was supported by nutrients exclusively by vein. Since that time, this area has seen tremendous growth, with sophisticated techniques and specialized nutrient formulas that have saved many lives.\n\nWhen the GI tract is not functional and nutrition support is indicated, nutrients can be delivered via the parenteral route. Parenteral nutrition supplies protein in the form of amino acids, carbohydrate as dextrose, fat, vitamins, and minerals. It may be infused through peripheral or central veins. The exact route will depend of the length of therapy, availability of intravenous access, fluid status, nutritional requirements, goal of nutrition therapy, and severity of illness.\n\n### A. Indications\/Contraindications\n\nIn general, parenteral nutrition is indicated for patients who are malnourished and do not have a functional GI tract [54]. More specific indications include perioperative support, inflammatory bowel disease, short bowel syndrome, severe pancreatitis, mechanical intestinal obstruction or pseudo-obstruction, severe malabsorption, and hyperemesis gravidarum. Parenteral nutrition is not indicated when the GI tract is functional, when the patient's prognosis is not consistent with aggressive nutrition support, or when the risks of parenteral nutrition outweigh the benefits.\n\n### B. Parenteral Access\n\nThe composition of the parenteral nutrition solution is dependent on the location of the vein in which it is delivered. Peripheral parenteral nutrition (PPN) is usually reserved for patients requiring short-term therapy who are not markedly hypermetabolic or fluid restricted and have adequate peripheral venous access. Hypertonic solutions may contribute to phlebitis; therefore, the osmolarity of the parenteral nutrition solution should be less than 900 mOsm\/L [116]. This generally necessitates the use of a three-in-one or total nutrient admixture (TNA), where the amino acids, dextrose, and intravenous lipid emulsion are compounded in one container.\n\nCentral parenteral nutrition is indicated for patients requiring long-term parenteral nutrition who have increased nutritional and metabolic requirements and\/or are fluid restricted. Because the parenteral nutrition solution is delivered centrally, where there is high and rapid blood flow, osmolarity is not a consideration. The solutions may be dextrose based (dextrose and amino acids) or TNA.\n\n### C. Parenteral Macronutrients\n\nProtein is provided in the form of synthetic crystalline amino acids. The standard amino acid solutions contain the eight EAAs plus histidine and six NEAAs. Commercial amino acid solutions are available in concentrations ranging from 3.5% to 15% (w\/vol). Most pharmacies use a 10% solution to compound parenteral nutrition. Parenteral amino acids provide 4.0 kcal\/g.\n\nAs with enteral formulas, disease-specific parenteral formulas are available. The modifications are generally in the protein component. Amino acid solutions designed for renal failure contain the eight EEAs plus histidine [51]. It is indicated for short-term use only in patients with acute renal failure who are not on dialysis. Patients with acute renal failure who have been started on dialysis should use the standard amino acid preparation. Amino acid solutions designed for hepatic failure contain higher levels of the BCAAs and lower levels of the AAAs. These solutions are indicated during periods of grade III and IV hepatic encephalopathy after a trial of standard medical treatment fails [117, 118].\n\nDextrose is the most commonly used energy substrate. Intravenous dextrose is available in the monohydrate form, which provides 3.4 kcal\/g. Dextrose solutions are available in concentrations ranging from 2.5% to 70% (w\/vol). Minimum requirements are estimated to be 1 mg\/kg\/minute or approximately 100 g\/day for a 70-kg man. The maximal amount of carbohydrate tolerated is approximated 5\u20137 mg\/kg\/minute [119]. When glucose oxidation rates are exceeded, fat synthesis will occur. This may also result in excess CO2 production. In stressed and septic patients, ability to tolerate glucose is likely to be impaired. A carbohydrate intake of less than 5 mg\/kg\/minute may be necessary in the critically ill patient.\n\nParenteral lipid emulsions are administered as a source of EFA and calories. They contain soybean or a combination of soybean and safflower oil as a source of polyunsaturated fatty acids, egg phospholipid as an emulsifier, and water. Glycerol is added to make the emulsion isotonic. A 10% lipid emulsion provides 1.1 kcal\/mL, a 20% emulsion provides 2.0 kcal\/mL, and a 30% emulsion provides 3.0 kcal\/mL. These emulsions have a pH range of 5.5\u20138.0 and an osmolarity range of 260\u2013300 mOsm\/L. The emulsified fat particles range from 0.1 to 0.5 \u03bcm in diameter. Two to 4% of total calories should come from linoleic acid to prevent EFA deficiency. This translates into 500 mL of a 10% lipid emulsion or 250 mL of a 20% lipid emulsion administered 3 times a week. The lipids should be administered slowly, generally over 8\u201310 hours to avoid reduced lipid clearance and impaired reticuloendothelial function and pulmonary gas exchange [120].\n\n### D. Fluid and Electrolytes\n\nOnce tolerance to the macronutrients has been established, the day-to-day management of parenteral nutrition evolves around fluid and electrolytes (Table 1). Fluid needs can be estimated from the sum of urine output, gastrointestinal losses, and insensible water losses from the skin and respiratory tract (approximately 500 mL). In general, young adults need 30\u201340 mL\/kg\/day and the elderly require 30 mL\/kg\/day. When starting a patient on parenteral nutrition it is often helpful to note the maintenance IV solution the patient had been receiving and use this as a guide to prescribe the parenteral nutrition formula (Table 2). Patients administered parenteral nutrition should have strict intake and output records. It is important to note losses through urine, stool, and nasogastric tubes and other drains. Body fluids contain electrolytes that should be replaced in the parenteral nutrition [121, 122] (Table 3).\n\nTABLE 1\n\nDaily Electrolyte Requirements in Patients with Adequate Renal Function Receiving Parenteral Nutrition\n\naMinimum requirements of healthy persons.\n\nTABLE 2\n\nElectrolyte Composition of Common Intravenous Fluids (mEq\/L)\n\nTABLE 3\n\nApproximate Electrolyte Composition of Various Body Fluids\n\n_Sources:_ Data from Farber, M. D., Schmidt, R. J., Bear, R. A., and Narins, R. G. (1987). Management of fluid, electrolyte, and acid-base disorders in surgical patients. _In_ \"Clinical Disorders of Fluid and Electrolyte Metabolism,\" 5th ed. (R. G. Naris, Ed.), pp. 1407\u20131436. McGraw-Hill, New York; and Grant, J. P. (1992). \"Handbook of Total Parenteral Nutrition,\" 2nd ed., p. 174. W. B. Saunders Co., Philadelphia, PA.\n\nCalcium, an extracellular cation is usually added to parenteral nutrition solutions in dosages of 9\u201322 mEq\/day. This is generally added as calcium gluconate, which yields 4.65 mEq\/g. Parenteral nutrition causes no real deficiency in calcium due to the buffering capacity of bone. However, supplemental calcium is required with phosphate administration to maintain serum calcium levels and avoid hypocalcemic tetany.\n\nMagnesium functions in enzyme reactions, such as glycolysis, and in all reactions involving adenosine triphosphate. As parenteral nutrition is initiated, serum magnesium levels may decrease as the patient becomes anabolic. Hypomagnesemia causes an increase in central nervous system and neuromuscular hyperactivity and has a significant effect on potassium, calcium, and phosphorus metabolism. Magnesium deficiencies are common in patients with GI diseases, protein energy malnutrition, and prolonged intravenous fluid therapy. Magnesium is added to parenteral nutrition solutions in the form of magnesium sulfate in dosages of 8\u201324 mEq\/day. Magnesium sulfate yields 8.12 mEq\/day.\n\nPhosphorus is a major intracellular anion and functions in the metabolism of carbohydrate, fat, and protein. Phosphorus is also a constituent of nucleic acids, phospholipid membranes, and nucleoproteins. The usual dose of phosphorus is 15\u201330 m _M_ \/day and is generally supplied as either potassium or sodium. A 1 mEq potassium phosphate injection provides 0.68 m _M_ of phosphate or 21 mg of elemental phosphorus. A 1 mEq sodium phosphate injection provides 0.75 m _M_ of phosphate or 23 mg of elemental phosphorus.\n\nPotassium is the major cation of intracellular fluid with 75% of body potassium located within muscle mass. The usual adult daily dosage of potassium for patients receiving parenteral nutrition is 60\u2013120 mEq\/day. Potassium is supplied as either chloride or acetate depending on acid-base balance. Potassium losses are in a fixed ratio with nitrogen losses. For every 1 g of nitrogen lost, a deficiency of 3.5 mEq of potassium exists. Hypokalemia may result from the use of diuretics, amphotericin B, nasogastric suction, or vomiting. Acute deficits of potassium should be corrected outside of the parenteral nutrition solution.\n\nSodium is a major extracellular ion and functions in the maintenance of osmotic pressure and in acid\u2013base balance. Maintenance sodium requirements range from 100 to 150 mEq\/day. Adjustments may be required for patients with renal and cardiac disease, patients with ascites and liver disease, and patients with altered fluid status. A low serum sodium may result from high blood glucose, generalized sodium deficiency, or water excess. Each increase of 100 mg\/dL serum glucose above the normal value of 100 results in a decrease of serum sodium of 1.6\u20132 mEq\/L. Hyponatremia generally results from fluid overload and not a sodium deficit (Table 4).\n\nTABLE 4\n\nEvaluation and Treatment of Hyponatremia in Parenteral Nutrition Patients\n\n_Source:_ Reprinted with permission from Matarese, L. E. (Ed). (1997). \"Nutrition Support Handbook,\" p. 77. The Cleveland Clinic Foundation, Cleveland, OH.\n\nChloride is a major extracellular anion and functions in the maintenance of osmotic pressure and acid\u2013base balance. Sodium and chloride are generally added in a one-to-one molar ratio to prevent hyperchloremic acidosis, although some patients may need a higher concentration of acetate. Chloride is added to parenteral nutrition solutions as sodium or potassium salts. Additionally, some of the amino acid solutions contain chloride.\n\nAcetate salts should be included to prevent metabolic acidosis from amino acid metabolism. Acetate is rapidly converted to bicarbonate in the liver. Sodium bicarbonate is not used and should never be added to parenteral nutrition solutions because this product is incompatible with other additives. For example, the addition of sodium bicarbonate to a parenteral nutrition solution containing the parenteral equivalent of the RDA for elemental calcium readily forms insoluble calcium carbonate.\n\n### E. Parenteral Micronutrients\n\nThe addition of vitamins to the parenteral nutrition solution is essential to the utilization of the nutrient components. Deficiencies of the water-soluble vitamins can occur rapidly. When vitamins are given via the intravenous route there is a greater amount of elimination through the kidney. Therefore, though exact requirements of intravenous vitamins are unknown, a higher dose, generally 2 to 3 times the RDA, is recommended for water-soluble vitamins [123]. Fat-soluble vitamins are also a component of the multivitamin injections. Vitamin K is not present in the multivitamin injections and must be added separately (Table 5).\n\nTABLE 5\n\nDaily Parenteral Vitamin Supplementation\n\nIntravenous trace element injections are available that contain zinc, copper, chromium, selenium, and manganese [124]. These should be added to the parenteral nutrition solution on a daily basis (Table 6).\n\nTABLE 6\n\nDaily Parenteral Trace Element Supplementation\n\n### F. Medications and Other Additives\n\nVery few medications are added to parenteral nutrition solutions due to potential incompatibilities. Additionally, if the parenteral nutrition solution should have to be suddenly discontinued for any reason, the medication is discontinued as well. The few exceptions are noted: Heparin is sometimes added to help prevent subclavian vein thrombosis and to decrease thrombus formation at the catheter tip. It is also added to peripheral parenteral nutrition solutions to improve peripheral vein tolerance to the solution. Regular insulin is added to parenteral nutrition solutions for blood glucose control. Histamine H2 antagonists such as famotidine, ranitidine, or cimetidine are used to decrease gastric output, particularly in patients with massive small bowel resection or to prevent stress ulcers. Octreotide is a somatostatin analog that is used to decrease gastric and other intestinal secretions and has been shown to decrease the time needed for gastrointestinal fistula closure [125, 126]. Intravenous methylprednisolone can be added to parenteral nutrition solutions for those patients who need long-term corticosteroid therapy and cannot take oral corticosteroids.\n\n### G. Methods of Administration\n\nParenteral nutrition is generally administered continuously over 24 hours for most hospitalized patients. However, some patients, particularly those receiving parenteral nutrition at home, may cycle the parenteral nutrition so that it infuses over 10\u201316 hours. This allows a patient to be free of the infusion for a period of time during the day. Cycling also reverses hepatic steatosis and liver enzyme changes associated with continuous infusion [127].\n\n### H. Complications\n\nAlthough parenteral nutrition has been a life-saving therapy, it is not totally benign and carries with it an extensive set of potential complications. Recognition, prevention, and treatment of the potential metabolic complications are imperative. These complications can be divided into four main categories: technical, septic, metabolic, and gastrointestinal.\n\n#### 1. TECHNICAL\n\nMost of the technical complications associated with parenteral nutrition center around the placement of the catheter. Central venous access can be obtained via subclavian, jugular vein, or peripherally inserted central catheters. Hickman or Broviac catheters or implantable ports are used for longterm access. Mechanical complications of catheter insertion may include pneumothorax, hydrothorax, and vessel injury [128]. These risks are minimized when catheters are placed by experienced personnel. A chest X ray to verify venous placement should be obtained before infusing central parenteral nutrition formulas. The maximum osmolarity tolerated by a peripheral vein is 900 mOsm\/L in order to prevent phlebitis. Therefore, these solutions must be carefully calculated (Table 7). Femoral lines are generally considered to be central lines. However, when administering parenteral nutrition solutions it is best to keep the concentration the same as a peripheral formula because the blood flow in the femoral vein is less than that of the subclavian or jugular. Solutions are therefore not diluted as quickly.\n\nTABLE 7\n\nOsmolarity Calculations for Parenteral Nutrition\n\n#### 2. SEPSIS\n\nInfectious complications associated with parenteral nutrition can be life threatening. Many hospitalized patients receiving parenteral nutrition have multiple other reasons for elevations in temperature and sepsis. However, a sudden change in the patient's usual temperature may be an indication of possible catheter sepsis. Sudden increases in blood glucose concentration in a patient who was previously normoglycemic may be an early indication of sepsis. The parenteral nutrition catheter can be seeded by a remote source or it can be the primary source of infection. Blood cultures should be drawn, the catheter changed over a guidewire, and the tip sent for culture. If there is obvious exudate coming from the catheter exit site, the catheter should be removed and a new one placed on the opposite side. If the catheter tip removed by a guidewire change is infected, the new catheter should be removed and another catheter placed on the opposite side [129].\n\n#### 3. METABOLIC\n\nSeveral hepatobiliary and GI complications have been associated with parenteral nutrition. Abnormalities in liver function tests, namely, SGOT, SGPT, bilirubin, or alkaline phosphatase, have been associated with the infusion of parenteral nutrition [130]. The etiology of these changes is unclear, but the changes are transient and generally not associated with adverse clinical sequelae in adults. Hepatic steatosis has also been associated with the use of parenteral nutrition as exhibited by elevation of serum aminotransferase values, alkaline phosphatase, and bilirubin levels. But this, too, is a benign, transient, reversible phenomenon in patients on short-term parenteral nutrition and generally resolves in 10\u201315 days. The parenteral nutrition solution should be tailored to the individual patient's energy requirements and in some cases it may be necessary to provide a solution that contains dextrose, amino acids, and lipids. Intrahepatic cholestasis can occur within 2\u20136 weeks of initiation of parenteral nutrition. This may be manifested by a progressive increase in the total bilirubin and an elevation of serum alkaline phosphatase. The lack of intraluminal nutrients to stimulate hepatic bile flow by cholecystokinin is thought to lead to cholestasis [131]. Ursodeoxycholic acid has been used as therapy for patients with cholestasis [132]. The risk can be further reduced with the use of cyclic parenteral nutrition, restriction of dextrose, avoidance of overfeeding, and early enteral stimulation when feasible. The lack of enteral stimulation is also associated with villus hypoplasia, colonic mucosal atrophy, decreased gastric function, impaired gastrointestinal immunity, bacterial overgrowth, and bacterial translocation in experimental models [133]. Therapy should be aimed at providing some enteral nutrition as soon as possible whenever feasible.\n\nParenteral nutrition has also been associated with various macronutrient-related complications. There are risks associated with over- and underfeeding. Providing calories, especially in the form of hypertonic dextrose, above requirements, can result in hyperglycemia, hepatic abnormalities from fatty infiltration, potential respiratory acidosis from increased CO2 production, and difficulty in weaning from ventilators. Minimum requirements for dextrose are estimated to be 1 mg\/kg\/minute or approximately 100 g\/day for a 70-kg man. The maximal amount of dextrose tolerated is approximately 5\u20137 mg\/kg\/minute [119]. Underfeeding may result in depressed ventilatory drive, decreased respiratory muscle function, impaired immune function, and increased infection. Thus, it is important to tailor the parenteral nutrition prescription to the patient's needs.\n\nHyperglycemia is the most common complication associated with the administration of parenteral nutrition. Patients receiving parenteral nutrition often have numerous reasons for elevated blood glucose such as sepsis or administration of steroids. Serum glucose concentration should be maintained below 200 mg\/dL. This may be achieved with the addition of insulin and\/or substitution of lipid for dextrose. Hypoglycemia may occur from excess insulin administration, either in the parenteral nutrition solution or subcutaneously. Treatment may include initiation of a 10% dextrose infusion or administration of D50.\n\nThe delivery of calories, particularly in the form of dextrose, may induce refeeding syndrome in a patient who has been chronically starved or is extremely cachectic. Refeeding syndrome refers to the intracellular shift of electrolytes and minerals, especially phosphorus, magnesium, and potassium, as a result of aggressive nutrition support [134, 135]. This is a direct result of increased plasma insulin concentration when dextrose is administered. This shift of electrolytes and minerals can result in life-threatening low serum values. Thus, for patients who are at risk for refeeding, calories should be administered at 20 kcal\/kg\/day and advanced slowly.\n\nThe crystalline amino acid solutions used in parenteral nutrition are generally well tolerated in patients with normal hepatic and renal function. The amino acids should be adjusted according to the patient's tolerance, clinical response and monitoring of visceral protein status, nitrogen balance, blood urea nitrogen, and creatinine.\n\nIntravenous lipid emulsion is administered as a source of essential fatty acids and calories. Hyperlipidemia may result with excess or rapid administration. Serum triglyceride levels should be checked in any patient with a known history of hyperlipidemia prior to infusion of intravenous lipid emulsion. Acceptable serum triglyceride levels are less than 250 mg\/dL 4 hours after lipid infusion for piggybacked lipids, and less than 400 mg\/dL for continuous lipid infusion.\n\nElectrolyte and mineral imbalances may occur in severely stressed patients both before and after parenteral nutrition infusion begins. It is best to correct any electrolyte abnormalities before parenteral nutrition is initiated. Once parenteral nutrition is initiated, it is important to monitor electrolytes, particularly magnesium, potassium, and phosphorus, carefully. Abnormally low values of electrolytes should be corrected promptly with intravenous replacements to avoid serious complications such as seizures, arrhythmias, or even death (Tables 8 and 9).\n\nTABLE 8\n\nDosing and Intravenous Replacement of Minerals in Adult Patients Receiving Parenteral Nutrition\n\na22\u201344 mEq of potassium phosphate or 20\u201340 mEq of sodium phosphate.\n\nb1 mEq of potassium phosphate = 0.68 m _M_ phosphorus; 1 mEq of sodium phosphate = 0.75 m _M_ phosphorus.\n\n_Source:_ Adapted from Matarese, L. E. (Ed). (1997). \"Nutrition Support Handbook,\" p. 76. The Cleveland Clinic Foundation, Cleveland, OH.\n\nTABLE 9\n\nDosing and Intravenous Replacement of Electrolytes in Adult Patients Receiving Parenteral Nutrition\n\naMinimum requirements of healthy persons.\n\n_Source:_ Adapted from Matarese, L. E. (Ed). (1997). \"Nutrition Support Handbook,\" p. 79. The Cleveland Clinic Foundation, Cleveland, OH. The Cleveland Clinic Foundation\n\nDehydration and fluid overload are also potential complications of parenteral nutrition. In general, fluid requirements can be estimated at 30\u201335 mL\/kg\/day. Patients who are dehydrated may have fluid requirements as high as 40\u201350 mL\/kg\/day. The problem can be further exacerbated by excessive losses from nasogastric tubes, surgical drains, high output ostomies, and diarrhea. The volume can be increased in the parenteral nutrition formula to accommodate these needs and\/or separate supplemental intravenous fluids can be employed. Fluid overload, edema, and anasarca may be present in critically ill patients and in patients with renal or hepatic failure, congestive heart failure, and hypoalbuminemia. In these situations, it is best to concentrate the parenteral nutrition solution.\n\n## IV. CONCLUSION\n\nEnteral and parenteral nutrition are effective methods of providing nutrients to patients who cannot or will not eat. 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Diarrhea in tube-fed patients: Feeding formula not necessarily the cause. _Am. J. Med_. 1990;88:91\u201393.\n\n113. Eisenber P.G. Causes of diarrhea in tube-fed patients: A comprehensive approach to diagnosis and management. _Nutr. Clin. Prac_. 1993;8:119\u2013123.\n\n114. Clausen M.R., Bonnan H., Tvede M., Mortensen P.B. Colonic fermentation to short-chain fatty acids is decreased in antibiotic-associated diarrhea. _Gastroenterology_. 1991;101:1497\u20131504.\n\n115. Vanlandingham S., Simpson S., Daniel P., Newmark S.R. Metabolic abnormalities in patients supported with enteral tube feeding. _J. Parenteral Enteral Nutr_. 1981;5:322\u2013324.\n\n116. Skipper A. Principles of parenteral nutrition. In: Matarese L.E., Gottschlich M.M., eds. _Contemporary Nutrition Support Practice_. Philadelphia, PA: W. B. Saunders Co.; 1998:227\u2013242.\n\n117. Freund H., Dienstag J., Lehrich J., Yoshimura N., Bradford R.R., Rosen H., Atamian S., Slemmer E., Holroyde J., Fischer J.E. Infusion of branched-chain enriched amino acid solution in patients with hepatic encephalopathy. _Ann. Surg_. 1982;196:209\u2013219.\n\n118. Naylor C.D., O'Rourke K., Detsky A., Baker J.P. Parenteral nutrition with branched-chain amino acids in hepatic encephalopathy. A meta-analysis. _Gastroenterology_. 1989;97:1033\u20131042.\n\n119. Wolfe R.R., O'Donnell T.F., Jr., Stone M.D., Richmand D.A., Burke J.F. Investigation of factors determining the optimal glucose infusion rate in total parenteral nutrition. _Metabolism_. 1980;29:892\u2013900.\n\n120. Seidner D.L., Mascioli E.A., Istfan N.W., Porter K.A., Selleck K., Blackburn G.L., Bistrian B.R. Effects of long-chain triglyceride emulsions on reticuloendothelial system function in humans. _J. Parenteral Enteral Nutr_. 1989;13:614\u2013619.\n\n121. Farber M.D., Schmidt R.J., Bear R.A., Narins R.G. Management of fluid, electrolyte, and acid-base disorders in surgical patients. In: Naris R.G., ed. _Clinical Disorders of Fluid and Electrolyte Metabolism_. 5th ed. Philadelphia, PA: McGraw-Hill; 1987:1407\u20131436.\n\n122. Grant J.P. Handbook of Total Parenteral Nutrition, 2nd ed., New York: W. B. Saunders Co.; 1992:174.\n\n123. A statement by the Nutrition Advisory GroupAmerican Medical Association Department of Foods and Nutrition. Multivitamin preparations for parenteral use. _J. Parenteral Enteral Nutr_. 1979;3:258\u2013262.\n\n124. AMA Department of Foods and Nutrition. Guidelines for essential trace element preparations for parenteral use: A statement by an expert panel. _J. Am. Med. Assoc_. 1979;241:2052\u20132054.\n\n125. Lembycke B., Creutzfeldt W., Schleser S., Ebert R., Shaw C., Koop I. Effect of the somatostatin analogue sandostatin SMS 201-995 on gastrointestinal, pancreatic, and biliary function and hormone release in man. _Digestion_. 1987;36:108\u2013124.\n\n126. Torres A.J., Landa J.I., Moreno-Azcoita M., Arguello J.M., Silecchia G., Gastro J., Hernandez-Merlof F., Jover J.M., Moreno-Gonzales E., Balibrea J.Z. Somatostatin in the management of gastrointestinal fistulas. _Arch. Surg_. 1992;127:97\u201399.\n\n127. Maini B., Blackburn G.L., Bistrian B.R., Flatt J.P., Page J.G., Bothe A., Benotti P., Rienhoff H.Y. Cyclic hyperalimentation: An optimal technique for preservation of visceral proteins. _J. Surg. Res_. 1976;20:515\u2013525.\n\n128. Fuhrman M.P. Management of complications of parenteral nutrition. In: Matarese L.E., Gottschlich M.M., eds. _Contemporary Nutrition Support Practice_. Philadelphia, PA: W. B. Saunders Co.; 1998:243\u2013263.\n\n129. Bozzetti F., Terno G., Bonfanti G., Scarpa D., Scotti A., Ammatuna M., Bonalumi M.G. Prevention and treatment of central venous catheter sepsis by exchange via a guidewire. _Ann. Surg_. 1983;198:48\u201352.\n\n130. Clarke P.J., Ball M.J., Kettlewell M.G. Liver function tests in patients receiving parenteral nutrition. _J. Parenteral Enteral Nutr_. 1991;15:54\u201359.\n\n131. Quigley E.M.M., Marsh M.N., Shaffer J.L., Markin R.S. Hepatobiliary complications of total parenteral nutrition. _Gastroenterology_. 1993;104:286\u2013301.\n\n132. Beau P., Labat-Labourdette J., Ingrand P., Beauchant M. Is ursodeoxycholic acid an effective therapy for total parenteral nutrition-related liver disease? _J. Hepatol_. 1994;20:240\u2013244.\n\n133. Illig K.A., Ryan C.K., Hardy D.J., Rhodes J., Locke W., Sax H.C. Total parenteral nutrition-induced changes in gut mucosal function: Atrophy alone is not the issue. _Surgery_. 1992;112:631\u2013637.\n\n134. Sacks G.S., Walker J., Dickersons R.N., Kudsk K.A., Brown R.O. Observations of hypophosphatemia and its management in nutrition support. _Nutr. Clin. Prac_. 1994;9:105\u2013108.\n\n135. Brooks M.J., Melnik G. The refeeding syndrome: An approach to understanding its complications and preventing its occurrence. _Pharmacotherapy_. 1995;12:713\u2013726.\nCHAPTER 17\n\nHerbs and Botanical Supplements: Principles and Concepts\n\nCYNTHIA THOMSON and ROBERT B. LUTZ, University of Arizona, Tucson, Arizona\n\n## I. INTRODUCTION: HISTORY OF BOTANICAL MEDICINE\n\nA. Historical Background\n\nThe use of herbal and botanical products to prevent or treat disease has been in existence for thousands of years. Asians and Native Indian tribes throughout the world have been acknowledged for their contribution to botanical medicine. It is estimated that more than 30,000 herbs and botanicals have been studied for medicinal qualities, but fewer than 300 are currently used in Western medicine. The use of botanicals to prevent or treat disease is likely innate. Given a world without prescription medications, humans explored their surroundings to find the answers to ailments. Documentation of the virtues of herbs is available as early as the eighth century B.C. through the writings of Hesiod and Hippocrates who described their usage in the fourth century B.C. [1]. The National Institute of Medical Herbalists, established in 1864, provided the first professional identity for the study of botanical medicine.\n\n### B. Definitions and Related Terminology\n\nThe study of botanical medicine warrants an understanding of the terminology used by herbal medicine practitioners. It is clear that this is an area of medicine for which consensus has generally not yet been established and legal and practical definitions vary. Table 1 provides a list of definitions of the terms commonly used. An understanding of the selected terminology is the foundation for studying any science\u2014new or old\u2014and is essential to the advancement of the science. Yet, with botanical medicine, there remain inconsistencies in defining the terminology, which may at least partially contribute to the unease most scientists have with the use of these products. To begin, even the word _herb_ is difficult to define. Botanists would define any herb as a nonwoody, seed-producing plant that regresses to its root structure after each growing season and is generally administered as a whole plant [2]. Botanicals, in contrast to herbs, include all trees and shrubs as well as herbs, including those that thrive year round [3]. Most practitioners of botanical medicine describe herbs and botanicals based on the medicinal qualities or the ability of the plant to induce a pharmacological effect in humans. Herbs are generally administered as whole plants, while botanicals may be derived from parts of plants such as the root, stem, or leaf. Varro Tyler [4], a recognized expert in the field of botanical medicine, has defined herbs as ''crude drugs of vegetable origin utilized for the treatment of disease states, often of a chronic nature, or to attain or maintain a condition of improved health.\" The Food and Drug Administration uses the term _botanical_ in its legal definition of an herb [5].\n\nTABLE 1\n\nBotanical Medicine Terminology\n\nTerm | Definition \n---|--- \nBotanicals | Herbs, trees, and shrubs or components thereof that exert a pharmacologic effect on the body \nElixir | Hydroalcoholic combination extract sweetened with sugar or glycerol \nEthnobotanist | Person who studies the use of plants as food and medicine in a variety of cultures throughout the world \nExtract | Concentrated (1:1 dilution) ground herb that has been treated with alcohol and heat until 1 mL is equivalent to 1 g of herb powder \nFunctional food | Any modified food or food ingredient that may provide a health benefit beyond the traditional nutrients it contains \nGuaranteed potency herb (GPH) | Herbal product tested throughout the manufacturing and harvesting processes to ensure that the endproduct has the proper percentage of active ingredient \nHerb | \"Crude drugs\" of vegetable origin utilized for the treatment of disease states, often of a chronic nature, or to attain or maintain a condition of improved health \nHerbalism | Use of crude plant-based products to treat or prevent disease \nHerbal medicinal | Product derived from plants or parts of plants that elicits a pharmacological effect \nPhytopharmaceuticals | Plant-based medicines that have been standardized based on the pharmacologically active constituent(s) \nStandardized | Use of a particular or selected chemical or active constituent as an indicator or marker of biological activity\/effectiveness \nSynergy | Influence or effect that occurs when constituents of a botanical act together in biological systems to elicit an effect that is greater biologically than the effect obtained by ingestion of any of the individual constituents alone \nTincture | Less concentrated (1:3, 1:5, 1:10) liquid botanical remedy that contains alcohol and water solvents \nTonic | Agent that restores the activity of an organ or organ system within the body \nPhytotherapy | Used in European literature to describe herbalism that uses only standardized products\n\nAlthough the exact definition may seem a trite issue, it is in fact an issue of significant relevance in that the definition selected may determine whether botanicals are drugs or food. If the product is used to treat or mitigate a disease, it should be considered a drug. However, if the herb\/botanical is used as a food flavoring or dietary component it would be considered a food [6]. The confusion evolves as more and more food products are used for multiple purposes and cross the line between food and drug. Examples include garlic, used to treat hypertension and to flavor Italian food; cranberry, used to prevent or treat urinary tract infections and a base for juice; or peppermint, used to relax smooth muscle in patients with irritable bowel syndrome and as a flavoring agent. The line between food and drug becomes even more obscure when foods are enhanced with botanicals with the intent to augment the health-promoting value of the food. These foods are generally referred to as functional foods [7]. Examples of functional foods include soups with added echinacea, snack chips with kava kava, and beverages with added ginseng.\n\n## II. USE OF HERBS AND BOTANICALS\n\nA. Reported Use\n\nAlthough there are no universally accepted statistics on herbal and botanical use in the United States, several surveys have been completed by private industries during the past several years that provide insight into the trends in herb and botanical use. What is clear is that the use of botanical remedies in the United States has risen significantly during the past decade with 1500\u20131800 products currently on the U.S. market [8]. Internationally, it has been estimated that 80% of adults use herbal remedies [9]. A recent survey published by Aarts [10] estimated that dietary supplement sales were $12 billion annually with approximately 34% of Americans consuming herbal supplements. Specifically, U.S. expenditures for herbal supplements ranged from $2 billion in 1996 to an estimated $5 billion in 2000 [11]. This reflects a 15% annual growth rate with no clear plateau in sales in sight.\n\nRecently, the National Institutes of Health Office of Dietary Supplements and the U. S. Department of Agriculture reported plans to expand the National Health and Nutrition Examination Survey to include more specific data regarding dietary supplement use [12]. This is an essential first step to more clearly ascertain the depth and breadth of herbal and botanical use among Americans. Secondly, there is growing interest in establishing dietary supplement databases, which allow for more accurate scientific investigation into the safety and efficacy of these products. Several reports on the use of alternative medicine, including herbal product use, have been published in recent years [13\u201316] and indicate that the use of herbal product is one of the leading forms of alternative medicine selected by patients. Most recently, Eisenberg _et al._ [13] cited a 380% increase in botanical use among Americans.\n\n### B. Factors Associated with Increased Use of Botanicals\n\nSeveral factors have been identified as contributing to the increase in botanical use [17]. First, America's population is aging and thus looking for cost-effective, convenient, and low-risk ways to reduce risk for chronic disease. In the case of those experiencing chronic disease, the goal is to reduce symptoms and improve quality of life. In addition, Americans are taking a greater responsibility for their health and are less satisfied with conventional medicine. Many believe herbals are a more natural and, therefore, less risky approach to treating disease.\n\nIn recent years, the National Institutes of Health established the National Center for Complementary and Alternative Medicine and the Office of Dietary Supplements. These offices have provided new financial and research support for this area of practice, resulting in expanded interest in this growing area of health care. Finally, the computer age has significantly increased access to information on the potential benefits of botanicals, leading to increased supply and demand. Certainly, the etiology of the recent emergence of botanical medicine is multifactorial and complex but its expansion cannot be overlooked.\n\n### C. Assessing Patient Use of Botanical Medicines\n\nOne challenge facing health care professionals is developing and maintaining open communication with patients concerning the use of botanical supplements [18]. Research by Eisenberg and others [13, 19, 20] indicates that patients are reluctant to share information regarding alternative medicine including botanical supplement use or adverse effects with their physicians. Clearly, this information is essential to providing optimal patient care. Table 2 lists the patient interview process for assessing and implementing appropriate botanical (and other) supplement use.\n\nTABLE 2\n\nAssessment of Botanical Supplement Use\n\n1. What supplements are you currently taking? Include over-the-counter, self-selected, and those prescribed by a health care practitioner.\n\n2. Dose and frequency\n\n\u2022 Regularity\n\n\u2022 With meals or other supplements?\n\n\u2022 What compound or formulation?\n\n\u2022 What route of administration?\n\n3. Why?\n\n\u2022 Perceived benefits\n\n\u2022 Prevention or treatment\n\n\u2022 Label claims\n\n4. Patient education\n\n\u2022 Potential\/proven benefits\n\n\u2022 Interactions with medications, other nutrients, or botanicals\n\n\u2022 Appropriate dosage\n\n\u2022 Duration of use\n\n\u2022 Reliable resources\n\n5. Documentation\n\n\u2022 Product, dose, frequency\n\n\u2022 Clinical application\n\n\u2022 Efficacy\n\n\u2022 Education provided and patient understanding\n\n\u2022 Expected adherence\n\n## III. EVIDENCE FOR SAFETY AND EFFICACY\n\nMany consumers of herbal medicines believe that herbs are safer, more effective and more ''natural\" than pharmaceutical medications [21]. The long history of herbal\/botanical use serves to substantiate the safety of these products. Yet, modern usage of herbs and botanicals differs from traditional use in that botanicals are consumed in an extracted form, at higher dosage and in higher frequency, and with the expectation of an almost immediate response. Safety and efficacy, particularly under the current circumstances for consumption, are not a ''given,\" and both should be adequately studied for each botanical product before standards for safety and efficacy are established.\n\n### A. Safety\n\nAlthough historical evidence would support that botanical products are generally safe, a lack of consistent report mechanisms for adverse effects makes it difficult to reach this conclusion. Certainly, dosage is central to safety, including both absolute dose and frequency. For a large percentage of the currently available botanical supplements, dosage recommendations may not be available or vary considerably depending on the disease or symptom under treatment and the form of botanical selected. Normal adult dosage is based on a standard 70-kg male and is generally divided into three equal doses spread throughout the waking hours. There is considerable variation to this dosage approach, but it is not unlike that used for conventional, over-the-counter medications. In addition, even if the label provides dosage standards, many consumers either fail to adhere to the recommendations or decide that more can only be better and, therefore, consume quantities beyond those recommended.\n\nPrerequisite to dosage determinations is the issue of quality regarding content of active ingredients and absence of contaminants. A Canadian study, analyzing the active component in North American feverfew, reported that none of the products analyzed contained the required amount of active ingredient to achieve therapeutic efficacy, despite quality claims of label information [22]. A study by Liberti and DerMarderosian [23], using spectrodensitromic thin-layer chromotography, showed that there was considerable variation in the active constituents of panax ginseng, with tablet formulations containing no detectable panaxosides. Clearly, more stringent guidelines for dosage and integrity in quality labeling of dosage are needed to ensure safety and efficacy for botanical supplements.\n\n### B. Efficacy\n\nAs with safety, efficacy of botanical products will depend on the presence of active constituents, dosage, adherence to the recommended dosage regime, product formulation, bioavailability, and biological activity of the botanical product. Botanical products are uniquely different from pharmaceutical agents in that they are comprised of a variety of plant constituents combined in a single plant. This makes application of the scientific process to identify the biologically active ingredient that is associated with improved health outcome more complex and difficult. In fact, ethobotanists would argue that it is the synergy among constituents that naturally occurs in the whole plant that provides the health-promoting attributes of the botanical. Thus, establishing bioavailability standards may be difficult. However, even complex botanical medicinals can be subjected to the scrutiny of well-designed research in order to ''prove\" the efficacy for the treatment of a specific disease or symptom.\n\n### C. The Scientific Process\n\nThe scientific process is well described in the chemoprevention literature [24]. The randomized, controlled clinical trial remains the gold standard for testing efficacy. Prior to clinical trials, products undergo phase I and II testing to determine toxicity and efficacy standards for use in clinical trials. Phase I studies are considered after epidemiological and experimental data are available. In the case of botanicals, one could consider their historical use as ''epidemiological evidence\"; however, this should not preclude the establishment of _in vitro_ evidence. Once historical and _in vitro_ data have been collected, phase I studies can be performed to establish an appropriate dosage for clinical use. The objective of phase I studies is to establish the highest dose (and frequency) that can be administered without toxicity. This is the risk:benefit ratio. The lower the risk:benefit ratio, the more desirable the treatment. Generally, botanicals are considered to have a low risk:benefit ratio; however, few have undergone well-controlled phase I trials.\n\nOnce phase I data have been collected, the dosage can be established for use in Phase II trials. Phase II studies should be randomized, double-blind, placebo-controlled studies. Phase II studies provide an opportunity to refine toxicity data in a larger population, document side effects, establish appropriate biomarkers of clinical efficacy, and study the pharmacokinetics of the botanical. Because botanicals are comprised of multiple bioactive constituents, establishing pharmacokinetics properties for each active ingredient could become quite burdensome. The phase II study would be appropriately employed to study the efficacy of a botanical to reduce clinical symptoms in studies of moderate sample size; for example, for the study of garlic as an agent to reduce cholesterol levels or ginger as an antiemetic for patients undergoing chemotherapy.\n\nFinally, with the completion of adequate phase Iand II research, the scientific community can initiate phase III clinical intervention trials. These trials are large, randomized, placebo-controlled studies that generally last several years. The primary endpoint is reduction in the incidence of a given disease. Multiple botanicals and dosages may be included in a multi-factorial study design. This type of study would be warranted for the investigation of botanicals as potential agents for chemoprevention or cardiovascular disease prevention.\n\nTo date, only a small percentage of currently available botanical products have under gone such rigorous scientific testing. This accounts for the reluctance by many health care professionals to employ botanicals in medical practice. However, many have been investigated in controlled, clinical trials and may warrant consideration for use clinically.\n\nTable 3 provides a listing of select herb\/botanical products and their proposed health benefits, side effects, and contradictions. The scientific evidence for these select botanicals is variable [25].\n\nTABLE 3\n\nCommon Botanicals: Purported Uses, Contraindications, and Resource Information\n\naWHO, World Health Organization.\n\nbUSPNF, United States Pharmacopea nonprescription formulary.\n\ncGerman Comm E, \"The Complete German Commission E Monographs: Therapeutic Guide to Herbal Medicines (Blumenthal, M., _et al.,_ Eds). German Federal Institute for Herbal Medicine, American Botanical Councils, Austin, TX, 1999.\n\n_Source:_ Developed by Laswell, A., and Thomson, C. (1999). American Dietetic Association Physician Nutrition Education Project.\n\n### D. Botanicals Use Supported by Scientific Evidence\n\nAs discussed above, the effectiveness of botanical and herbal remedies can be evaluated according to pharmacological principles and the scientific methodology. In particular, the use of randomized, controlled clinical trials allows for evidence-based outcome assessment to establish the efficacy of herbal\/botanical products. A brief review of the evidence for eight commonly used botanicals is presented here.\n\n#### 1. ST. JOHN'S WORT FOR THE TREATMENT OF DEPRESSION\n\nSt. John's wort or _Hypericum perforatum_ is licensed in Germany for the treatment of anxiety, mild to moderate depression and sleep disorders. Biologically active ingredients include naphthodianthroms, flavonoids, xanthose, and bioflavonoids [26]. A phase II study to determine the effect of St. John's wort on neurophysiological function demonstrated that the two extracts studied were able to induce electrochemical changes consistent with improved mentation [27]. A randomized controlled clinical trial conducted in Germany of 260 patients with mild to moderate depression showed St. John's wort to be equivalent to standard drug therapy in clinical efficacy [28]. Additional randomized trials by Vorbach _et al._ [29] and also by Wheatley and colleagues [30] also demonstrated clinical effectiveness. A recent meta-analysis of 1757 patients conducted by Linde et al. [31] established that _Hypericum_ extracts were 33. 2% more effective than placebo or prescription medications for treating depression. A recent study of drug interaction revealed that the use of St. John's wort with indinavir reduces expected plasma drug levels. It may be that St. John's wort induces cytochromes P-450 so that all drugs metabolized by this route are affected [32].\n\n#### 2. ECHINACEA FOR RESPIRATORY AND INFLUENZA\n\nEchinacea or _Echinacea purpurea_ is extracted from the juice of the purple coneflower and has been widely used and studied throughout Europe for the past century. _In vitro_ and animal studies have shown that the arabinogalactan-containing glycoproteins and chichoric acid are capable of inducing B-lymphocyte and macrophage proliferation, TNF-\u03b1, and interleukin 1 [33\u201335]. Recent randomized, controlled clinical trials, one by Melchart et al. [36] and another by Barnes [37] were unable to demonstrate the efficacy of echinacea to reduce upper respiratory tract infections. However, the majority of randomized controlled trials, as well as an earlier review of 26 clinical trials conducted by Barrett, have supported the use of echinacea to reduce the symptoms or duration of upper respiratory tract infections (38\u201344) with minimal adverse effects (33, 45). Nonetheless, results from the recent studies showing no benefit need to be considered [36, 37].\n\n#### 3. GARLIC FOR HYPERLIPIDEMIA OR HYPERTENSION\n\nGarlic or _Allium sativum_ has been studied for a variety of potential health benefits, based on activities ranging from antiviral to anticoagulant. Epidemiological evidence from more than 20 studies worldwide supports the potential for garlic (and onion) as a chemopreventive agent against gastrointestinal cancers [46]. The strongest scientific evidence is for the use of garlic as a lipid-lowering or antihypertensive agent [47]. Animal data demonstrate the hypotensive effects of garlic (48\u201350). Controlled clinical trials have shown reductions in systolic blood pressure of between 5.5% and 21% [51, 52]. Studies have shown reductions in cholesterol and triglycerides to range from 10% to 15% [53]. Other double-blind, controlled clinical trials have failed to demonstrate the efficacy of garlic for the treatment of hyperlipidemia [54\u201357]. However, two meta-analyses support the use of garlic as a lipid-lowering agent and an antihypertensive [53, 58, 59]. Poor study design and few subjects is a significant concern in the literature in this area [60]. Studies conducted using garlic are difficult to blind and have a relatively high frequency of side effects, such as gastrointestinal upset, dermatitis, and allergic reactions [61].\n\n#### 4. GINGER FOR NAUSEA OR EMESIS\n\nGinger or _Zingiber officinalis_ as been extensively used as an antiemetic and antispasmodic for more than 2500 years in China. Although _in vitro_ research appears to be lacking, randomized, controlled clinical trials have shown efficacy for motion sickness [62\u201364] although negative results have also been published [64, 65]. Ginger has been demonstrated to reduce postoperative nausea in gynecological patients [66] and ambulatory surgery patients [67]. One other study was unable to confirm this effect [68]. Ginger is one of the few botanicals prescribed for pregnant women because it has been shown to be efficacious for the treatment of hyperemesis gravidarum [69]. However, data supporting the safety of consumption and appropriate dosage during pregnancy are lacking [70], especially considering the potential for ginger to inhibit thromboxane synthetase, thus prolonging bleeding time [71].\n\n#### 5. GINKGO FOR DEMENTIA\n\nGingko or _Ginkgo biloba_ is among the most popular herbs used by Americans. It has been approved for the treatment of dementia in Germany where prescriptions now exceed 5 million [72]. Bioactive components, including flavonoids, terpenoids, ginkgolides, and organic acids, have been isolated from the ginkgo plant and are thought to contribute to its antioxidant and antiplatelet activity. _In vitro_ data documenting the biological activity are available and have led to an extensive study of the clinical efficacy of ginkgo in human trials. In an intent-to-treat analysis conducted by LeBars _et al._ [73] in 1997, gingko was shown to significantly reduce scores for the Alzheimer's Disease Assessment Scale and the Cognitive subscale. Other studies in patients with Alzheimer's have also demonstrated positive effects [74, 75], discussed in a review paper by Kleijnen and Knipschild [76]. Clinical efficacy for dementia has also been demonstrated for the elderly with mild to moderate memory impairment [77, 78]. Adverse effects during ginkgo administration are limited, but include mild gastrointestinal upset, headache, and more significantly spontaneous subdural hematoma [79]. These more significant effects have been reported in patients taking concomitant aspirin, nonsteroid anti-inflamatory drugs (NSAIDS), or anticoagulants [80].\n\n#### 6. VALERIAN FOR INSOMNIA\n\nValerian or _Valeriana officinalis,_ an extract of the valerian root, has been used as a mild tranquilizer and for the treatment of insomnia. Valerian use can result in hypotensive, anticonvulsant, sedative, and hypnotic effects. Animal studies demonstrate biological activity from several of the root components, including valerinic acid, valeranone, valepotriates and gamma-aminobutyric acid, but it remains unclear if these specific compounds are responsible for promoting sleep [81]. Randomized, controlled clinical trials evaluating the use of valerian to treat insomnia are few and have been criticized for the limited number of subjects enrolled. However, one study of eight patients with mild insomnia showed sleep was induced with valerian in an average of 9 minutes compared to 15. 8 minutes for the placebo group [82]. No additional benefit was demonstrated by doubling the dose of valerian from 450 to 900 mg. In a follow-up randomized, controlled study of 128 patients with and without insomnia, Leathwood and Chauffard [83] demonstrated reduced insomnia for those given valerian with the effect being most significant for those with sleep difficulties. The effects of valerian on sleep stages or electroencephalograph spectra have not been studied [84]. A study by Schultz and colleagues [85] showed increased slow-wave sleep and another by Lindahl and Lindwall [86] showed subjective improvement of sleep quality. Valerian root is generally well tolerated; however, some patients have complained of difficulty awakening and some reports suggest a detrimental interaction with alcohol and barbiturates [87].\n\n#### 7. SAW PALMETTO FOR BENIGN PROSTATIC HYPERTROPHY\n\nSaw palmetto, also known as _Serenoa repens,_ is a fruit-derived botanical used for the treatment of benign prostatic hypertrophy. The hexane ring isolated from saw palmetto has shown antiandrogenic and estrogenic effects _in vitro_ [88]. Two mechanisms have been demonstrated as likely to account for the antihypertrophic effects. First, saw palmetto inhibits dihydrotestosterone and, second, it inhibits 5-\u03b1-reductase activity [89]. Two randomized, controlled double-blind studies have been published that showed increased urine flow, decreased residual urine, reduced prostate size, and reduced frequency of urination [90, 91]. In one study, saw palmetto was shown to be more effective in reducing urine residual and improving urine output than a commonly prescribed medication, finasteride [92]. A review of well-controlled studies has been provided by Wilt and colleagues [93]. Saw palmetto is well-tolerated with minimal side effects that are limited to gastrointestinal complaints [94].\n\n#### 8. FEVERFEW FOR MIGRAINE HEADACHES\n\nFeverfew or _Tanacetum parthenium_ has received approval by the Canadian Health Protection Branch for use in the treatment of migraine headaches. Both fresh leaf and dried leaf preparations are available and both contain sesquiterpene lactones, which inhibit arterial contraction and induce arterial smooth-muscle contraction. Feverfew, a serotonin antagonist, has been shown to be an inhibitor of prostaglandins [95]. Double-blind, placebo-controlled studies are limited but support an approximate 25% reduction in migraine incidence among migraine patients who take feverfew as a preventive agent [96, 97].\n\nA summary of the randomized, controlled clinical trial data published in the scientific literature is shown in Table 4.\n\nTABLE 4\n\nHighlighted Herbs and Evidence of Efficacy\n\naDB RCT, double-blind randomized control trial; Meta, meta-analysis; Review, review article. Individual studies are referenced in the original source.\n\n_Source:_ Reproduced with permission from Barrett, B., Kiefer, D., and Rabago, D. (1999). Assessing the risks and benefits of herbal medicine: An overview of scientific evidence. _Altern. Ther. Health Med._ **5,** 40\u201349.\n\n## IV. ADVERSE EFFECTS OF HERBS AND BOTANICALS\n\nA. Incidence\n\nThe incidence of adverse effects of herbs and botanicals in the United States is not well documented, but has been reported to be low except when taken in excess of label recommendations [99, 100]. Because the majority of patients do not discuss herbal medicinal use with their physicians, many adverse reactions to these products are thought to go unreported [20]. Physicians use the FDA MedWatch system for reporting adverse drug reactions, but may be less aware of the need to use this same system to report adverse reactions to over-the-counter herbal and botanical supplements. Internationally, studies in Hong Kong [101], Taiwan [102], and the Philippines [103] have documented the extent of adverse reactions seen among users of herbal supplements. A 1995 report from the World Health Organization (WHO), focused on international drug monitoring, indicated that more than 5000 reports of herbal-induced adverse effects have been documented [104]. In contrast, a meta-analysis suggested that pharmaceuticals accounted for more than 100,000 deaths per year in American hospitals [105].\n\n### B. Botanicals Associated with Adverse Reactions\n\nAllergic adverse reactions, from dermatitis to anaphylactic shock, are commonly associated with yohimbine [106] and camphor [107]. Toxic reactions have also been documented, including fatal reactions, to pennyroyal [108]. Liver failure has also been reported and is associated with the use of such herbs as germander [109], chaparral [110], comfrey [111], and bajiaolian [112]. An excellent review of adverse reactions associated with herbal medicine use is provided by Ernst [113].\n\n### C. Herbal\u2013Medication Interactions\n\nKnowledge of the potential interactions between herbs, botanicals, and prescription medications is critical to their safe application in health care. The review provided focuses on several common botanical\u2013medication interactions. However, for a more in-depth review of the topic, several recently published books are available including one from Brinker [114] and the publishers of _Pharmacy Letters_ [115].\n\nTable 5 lists common botanical\u2013medication interactions. The interactions between herbal medicinals and conventional pharmacological agents has been grossly understudied in the scientific literature.\n\nTABLE 5\n\nHerb\/Botanical Medication Interactions\n\n## V. REGULATION OF BOTANICAL AND HERBAL MEDICINALS\n\nThe regulation of dietary supplements, including botanicals and herbs, changed substantially in 1994 when congress passed the Dietary Supplement Health and Education Act (DSHEA) [116]. This legislation provides an extremely flexible and permissive approach to dietary supplement labeling and excluded dietary supplements from the more stringent approach used in the labeling and marketing of foods. As a result of this legislation, dietary supplement product labels, including herbs and botanicals, can make \"structure\u2013function\" claims to promote product sales. A structure\u2013function claim would include statements such as ''improves prostate health\" or ''promotes a healthy heart.\" On the other hand, the regulation requires that no dietary supplement make claims to prevent or treat disease. For example, claims such as ''reduces risk for prostate cancer\" or ''prevents heart disease\" are not permissible. For the consumer, this differentiation is not clearly distinct and therefore leads to confusion as to the scientific evidence for efficacy. The label claim must also include the FDA disclaimer, ''This statement has not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, mitigate, cure or prevent any disease\".\n\nThe DHSEA also required that manufacturers provide label information to consumers regarding all of the ingredients contained in the product. The provision of ingredient information provides the FDA with a legal opportunity to challenge product manufacturers who were not reporting impurities, and as a basis for determining the presence of active ingredients. It is the manufacturer's responsibility to provide safe products. It is the responsibility of the FDA to investigate any reported cases of harm, to prove harm was done, and to take the necessary corrective action to ensure public safety.\n\n## VI. RECOMMENDATIONS FOR CLINICAL CARE\n\nAre botanical supplements an appropriate therapeutic modality? This is a complex question that warrants careful consideration. Issues of safety, efficacy, ethical behavior, cost effectiveness, and alternative dietary approaches must also be considered in the decision to recommend a certain botanical supplement.\n\n### A. Legal and Ethical Issues\n\n1. DIETETICS PROFESSIONALS\n\nProfessionals who are registered to practice dietetics must adhere to the American Dietetic Association Code of Ethics as the primary source of ethical guidance. State licensure laws also guide ethical decisions for health care professionals. The Code of Ethics for the American Dietetic Association states that dietetics professionals must not ''promote or endorse products in a manner that is false or misleading\" [117]. Thus, when recommending botanical supplements, it is important that dietetics professionals have a sound knowledge of the subject area and the scientific evidence for efficacy and patient safety. In addition, the American Dietetic Association (ADA) and other professional nutrition science organizations have peer-reviewed position and\/or policy statements that each member is expected to uphold. The ADA's position on dietary supplements states: ''It is the position of the American Dietetic Association that the best nutritional strategy for promoting optimal health and reducing the risk for chronic disease is to obtain adequate nutrients from a wide variety of foods. Vitamin and mineral supplementation is appropriate when well accepted, peer-reviewed, scientific evidence shows safety and effectiveness\"[118]. Standards of professional practice also serve to guide dietetics professionals in the provision of quality nutrition service [119], particularly as dietetics professionals expand their traditional practice roles to include complementary therapeutics including the use of herbs and botanicals [120].\n\n#### 2. MEDICAL PROFESSIONALS\n\nPhysicians, unlike dietetics professionals who are guided by the ADA Code of Ethics, have no central governing body that provides such guidance. Many physicians are members of the American Medical Association (AMA) and reference this organization's policies and recommendations. The AMA has an established Code of Ethics that may provide guidance for those who wish to reference it. The AMA has yet to forward a specific policy statement related to herbal and botanical products and the use of these products in clinical practice. The organization has called on the federal government to empower the FDA to place more strict requirements on the supplement industry with respect to DSHEA. It has also recommended that the AMA itself study DSHEA to determine if it is satisfactory in its present form and to determine whether or not the operations of the regulatory bodies have been appropriate [121].\n\nPhysicians, on receiving state licensure, are subject to the state's regulatory authority under police power [122]. This serves to protect a citizen's health and safety by determining who may practice medicine through the establishment of licensing boards. Within this framework, the state will often define the practice of medicine through the use of such language as _diagnosis, treatment_ and _cure._ This provides a broad definition of the act of practicing medicine (scope of practice). Scope of practice limitations serve to ensure that providers will only offer services within their level of training and expertise. For physicians, the area of botanical medicine, including the prescribing of herb and botanical supplements, may or may not be an area of formal training and expertise and therefore prescribing botanicals may at times border on licensing limits.\n\nPhysicians are not taught botanical medicine in medical school, nor are they routinely educated in these practices during postgraduate training. The proper prescription of herbals and botanicals may be as complicated as knowing the proper pharmaceutical to recommend and comes with experience. Knowledge in this area is often obtained from conferences and\/or references rather than through formal educational training. A growing number of medical schools provide some training in complementary and alternative medicine. However, these are usually elective courses [123]. It is therefore necessary for physicians to approach this information with a critical eye. It is also imperative that physicians make every attempt to either educate themselves about this rapidly growing complementary practice, or partner with a practitioner who is well versed in herbal\/botanical medicine. Herbal medicine is the fastest growing component of complementary and alternative medicine [13] and an area not without potential problems (medical contraindications, herb\u2013drug interactions). As long as providers either remain uninformed or biased against herbal\/botanical medicine, patients will continue to hide their use, thus increasing the possibility of harm. By affording themselves an opportunity to expand their knowledge, physicians will be able provide the quality of care that patients are requesting of them.\n\n### B. Cost Effectiveness of Botanical Medicine\n\nCurrently, no published reports exist that compare the cost of treating symptoms or chronic disease with botanical versus conventional pharmaceutical-based medicine. The assumption is that botanicals will be more cost effective and have fewer side effects. Clinicians must consider the cost in recommending any botanical supplement. The out-of-pocket cost for botanical therapies may be significantly higher than that of prescription medications where medical insurance is available. In addition, the use of a botanical or herbal should be considered augmentation of a well-founded dietary (or other) approach to treating the disease or disease manifestations. Without a foundation in dietary intervention, it is unlikely the botanical will be efficacious at the optimal level. Many patients are without adequate dietary advice and may be consuming several botanicals on a daily basis. To ensure efficacy, botanical supplements should be limited to products that have been demonstrated to be effective using scientifically sound methodology. Without evidence for efficacy, expenditures for botanicals cannot be considered cost-effective care.\n\n### C. Resources for Medical and Dietetics Professionals\n\nMedical and dietetic professionals must expand their knowledge of botanical use, safety, and therapeutic efficacy and remain informed in this rapidly expanding area of practice. Many professionals may not have been adequately educated in the area of herbal\/botanical medicine during their formal training and therefore must rely on self-education to develop this expertise. Developing fundamental knowledge in this area is essential prior to making recommendations to patients. In addition, working collaboratively with physicians, pharmacists, ethnobotanists, and other practitioners trained in the area of botanical medicine is essential to providing optimal care. Table 6 provides a list of reliable, scientifically based resources that can be used to develop expertise in this dynamic area of health care.\n\nTABLE 6\n\nResources for Botanical Product Use\n\nOrganizations\/Web sites:\n\nOffice of Dietary Supplements\n\nNational Institutes of Health\n\nBuilding 31, Room 1B25\n\n31 Center Drive MSC 2086\n\nBethesda, MD 20892\n\n\n\n301-435-2920\n\nInternational Bibliographic Information on Dietary Supplements (IBIDS)\n\n\n\nCouncil for Responsible Nutrition\n\n1875 Eye Street NW\n\nSuite 406\n\nWashington, DC 20006\n\n\n\n202-872-1488\n\nFood and Drug Administration\n\nDepartment of Health and Human Services\n\nFood and Drug Administration\n\nRockville, MD 20857\n\n\n\nNational Center for Complementary & Alternative Medicine\n\nNCCAM Clearing House\n\nPO Box 8281\n\nSilverspring, MD 20907-8218\n\n\n\n1-888-644-6226\n\nNational Council Against Nutrition Fraud, Inc.\n\n300 E. Pink Hill Road\n\nIndependence, MO 64057\n\n816-228-4595\n\nExecutive Director: William Jarvis\n\n909-824-4690\n\nAlternative Medicine Foundation, Inc.\n\nBethesda, MD\n\nHerbalMed: \n\nBooks\/Reference Text\n\n\u2022 American Dietetic Association. (1999). \"Clinician's Guide to Vitamins, Minerals and Other Dietary Supplements.\" American Dietetic Association, Chicago, IL.\n\n\u2022 Gruenwald, J., Brendler, T., and Jaenicke, C. Eds. (1998). \"PDR for Herbal Medicines.\" Medical Economics Company, Montvale, NJ.\n\n\u2022 Blumenthal, M., Goldberg, A., Gruenwald, J., Hall, T., Riggins, C. W., and Rister, R. S., Eds. (1998). \"The Complete German Commission E Monographs: Therapeutic Guide to Herbal Medicines.\" American Botanical Councils, Austin, TX.\n\n\u2022 Cohen, M. H. (1998). \"Complementary and Alternative Medicine: Legal Boundaries and Regulatory Perspectives.\" John Hopkins University Press, Baltimore, MD.\n\n\u2022 Tyler, V. E. (1994). \"Herbs of Choice.\" Haworth Press, Inc., New York.\n\n\u2022 Miller, L. G., and Murray, W. J. (1998). \"Herbal Medicinals: A Clinician's Guide.\" Pharmaceutical Products, Press, Binghamton, NY. Available at: .\n\n\u2022 Therapeutic Research Faculty, Pharmacist's Letter (1999). \"Natural Medicines Comprehensive Database.\" Available at .\n\n## VII. SUMMARY\n\nThe use of botanical and herbal medicinals to reduce the symptoms of and to treat or prevent chronic disease is on the rise. Medical and dietetic professionals are uniquely positioned to provide patients with reliable, scientifically sound advice on the use of these products in the context of a varied, nutritionally sound diet. Medical and dietetics professionals must be knowledgeable of safety, efficacy, and potential harm for botanical supplements used by their patients. Awareness of ethical and legal considerations when recommending the use or discontinuation of specific botanical supplements is paramount. It is inappropriate for medical and dietetics professionals to recommend any herbal or botanical product to their patients if they stand to achieve financial support for doing so, regardless of the scientific evidence. The use of botanicals and herbal supplements to enhance health is not new to medicine; however, the growing use of these products dictates that dietetics professionals must expand their knowledge and understanding of the growing body of scientific evidence so that optimal nutritional care can be provided to all patients.\n\nReferences\n\n1. Huxtable R.J. Safety of botanicals: Historical perspective [editorial]. _Proc. West. Pharmacol. Soc_. 1998;41:1\u201310\n\n2. Miller L.G., Murray W.J., eds. Herbal Medicinals: A Clinician's Guide. Philadelphia, PA: Pharmaceutical Products Press, 1998.\n\n3. Duke J.A. _The Green Pharmacy_. New York: Rodale Press; 1997.\n\n4. Tyler V.E. _Herbs of Choice: The Therapeutic Use of Phytomedicinals_. 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Saw palmetto extracts for treatment of benign prostatic hyperplasia: A systematic review. _JAMA_. 1998;280:1604\u20131609.\n\n94. Glisson J., Crawford R., Street S. The clinical applications of Ginkgo biloba, St. John's wort, saw palmetto, and soy. _Nurse Pract_. 1999;24:28.\n\n95. Collier H.O., Butt N.M., McDonald-Gibson W.J., Saeed S.A. Extract of feverfew inhibits prostaglandin biosynthesis. _Lancet_. 1980;2(8200):922\u2013923.\n\n96. Johnson E.S., Kadam N.P., Hylands D.M., Hylands P.J. Efficacy of feverfew as prophylactic treatment of migraine. _Br. Med. J. (Clin. Res. Ed.)_. 1985;291:569\u2013573.\n\n97. Murphy J.J., Heptinstall S., Mitchell J.R. Randomised double-blind placebo-controlled trial of feverfew in migraine prevention. _Lancet_. 1988;2(8604):189\u2013192.\n\n98. Barrett B., Kiefer D., Rabago D. Assessing the risks and benefits of herbal medicine: An overview of scientific evidence. _Altern. Ther. Health Med_. 1999;5:40\u201349.\n\n99. Farnsworth N.R. 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Assoc_. 1999;99:109\u2013113.\n\n118. Available atAmerican Dietetic Association. Position of the American Dietetic Association: Vitamin and Mineral Supplementation. 2000. .\n\n119. American Dietetic Association. The American Dietetic Association standards of professional practice for dietetics professionals. _J. Am. Diet. Assoc_. 1998;98:83\u201387.\n\n120. Practice and Policy Guidelines Panel, National Institutes of Health Office of Alternative Medicine. Clinical practice guidelines in complementary and alternative medicine, an analysis of opportunities and obstacles. _Arch. Fam. Med_. 1997;6:149\u2013154.\n\n121. American Medical Association, House of Delegates. \"Proceedings of the House of Delegates, Resolution 510, I-99.\" American Medical Association, Chicago.\n\n122. Cohen M.H. _Complementary and Alternative Medicine; Legal Boundaries and Regulatory Perspectives_. Stockton, CA: The Johns Hopkins University Press; 1998.\n\n123. Wetzel M.S., Eisenberg D.M., Kaptchuk T.J. Courses involving complementary and alternative medicine at U.S. medical schools. _JAMA_. 1999;280(9):778\u2013784.\nSECTION II\n\nDisease-Specific Intervention: Prevention and Treatment\nA.\n\nCardiovascular Disease\nCHAPTER 18\n\nDietary Macronutrients and Cardiovascular Risk\n\nPENNY KRIS-ETHERTON, KAI HECKER, DENISE SHAFFER TAYLOR, GUIXIANG ZHAO, STACIE COVAL and AMY BINKOSKI, Pennsylvania State University, University Park, Pennsylvania\n\n## I. INTRODUCTION\n\nCardiovascular disease (CVD) is the leading cause of death in the United States accounting for more deaths than all other causes combined. Numerous risk factors for CVD have been identified, many of which are modifiable by diet and lifestyle practices. Major modifiable risk factors include cigarette smoking, elevated total and low-density lipoprotein (LDL) cholesterol levels, overweight and obesity, hypertension, diabetes mellitus, and a sedentary lifestyle. Other important risk factors that are modifiable by diet are a low level of high-density lipoprotein (HDL) cholesterol, elevated levels of triglycerides (TGs), lipoprotein (a), insulin, hypertension, altered hemostatic factors, and small, dense LDL particles.\n\nDiet continues to be an important cornerstone in the prevention and treatment of CVD. Current recommendations are to reduce saturated fat (SFA) and _trans_ fatty acids (TFAs) by decreasing total fat and replacing SFA calories with carbohydrate (CHO) resulting in a lower fat, higher CHO diet with protein held constant. An alternative approach is to replace SFA calories with monounsaturated fatty acids (MUFAs), resulting in a diet higher in total fat. Polyunsaturated fatty acids (PUFAs), and omega\u20133 fatty acids in particular, have been a focus of attention recently because of their striking beneficial effects on CVD risk. The rapid increase in our understanding of fatty acid biology has clearly established the remarkable diversity of the effects of fatty acids on CVD risk factors. These findings underscore the importance of targeting fatty acids to maximally decrease CVD risk. For example, decreasing SFA is an absolute requisite, whereas maintaining or even increasing intake of other fatty acid classes is important for CVD risk reduction.\n\nHistorically the emphasis of dietary recommendations has been on modifying the type and amount of fat; however, modifying the type and amount of CHO and protein to lower CVD risk factors has attracted recent attention of scientists. With respect to dietary CHO, researchers are exploring how semi- and nondigestible carbohydrates and the glycemic index of CHO-rich foods affect risk factors for CVD. Likewise, studies are ongoing to unravel the different physiological effects that animal and plant proteins appear to elicit on CVD risk. We are transiting an exciting era in which we are gaining a better understanding of how all macronutrients affect CVD risk, and thus, it is not unreasonable to speculate that we will identify more effective dietary approaches for reducing CVD risk.\n\nThis chapter will review our present understanding of how changes in the macronutrient profile of the diet affect CVD risk status. We describe various low SFA and cholesterol diets that differ in macronutrient content and present the plasma lipid and lipoprotein responses that have been reported for these diets (Table 1). In addition, there is exciting new information about how diet affects emerging CVD risk factors. Thus, we describe the effect of various low-SFA, low-cholesterol diet options with different macronutrient profiles on newly defined CVD risk factors (Table 2).\n\nTABLE 1\n\nDiets Low in Saturated Fat and Cholesterol with Varying Macronutrient Contents\n\na9\u201313 g\/day fish oil (1.1\u20137 g\/day n-3 fatty acids).\n\nbCompared with an average Western style diet.\n\ncNC, no change.\n\nTABLE 2\n\nCardiovascular Risk Factors Modifiable by Diets Low in Saturated Fat and Cholesterol\n\nRisk factors | Beneficial diet strategies _a_ | Potentially adverse diet strategies \n---|---|--- \nHypercholesterolemia | LF-HC; Hi-MUFA; HP; Soy; Hi-sFib: FOS; Lo-GI | Hi-n-3 (fish oil) \nHypertriglyceridemia | Hi-MUFA; Hi-n-3; HP; Soy; FOS | LF-HC \nLow HDL | Hi-MUFA | LF-HC \nSmall, dense LDL | Hi-MUFA | LF-HC \nInsulin resistance | Hi-sFib; Hi-MUFA; Lo-GI; RS; FOS | LF-HC \nPlatelet aggregation | Hi-n-3; Soy | Unknown \nLipid peroxidation | LF-HC; Soy; Hi-MUFA | Hi-PUFA \nClotting | Hi-n-3; Soy | Unknown \nVascular reactivity | Hi-n-3; Soy | Unknown\n\na _Abbreviations:_\n\nLF-HC low-fat, high-carbohydrate\n\nHi-MUFA high monounsaturated fat\n\nHP high protein\n\nSoy diet high in protein with 25 or more grams of soy protein\n\nFOS fructooligosaccharides\n\nHi-sFib high soluble fiber\n\nHi-n-3 high omega-3 fatty acid content\n\nLo-GI low-glycemic index\n\nRS resistant starch\n\nHi-PUFA high polynsaturated fat\n\n## II. DIETARY FAT\n\nA. Total Fat\n\nDiscussions are ongoing about what the ideal quantity of total fat should be in the diet. Inherent to the discussion about total fat is that SFA and cholesterol should be reduced (i.e., <10% of calories and <300 mg\/day, respectively) to most favorably affect risk of CVD and promote weight control. The central question is whether MUFA or CHO should replace SFA calories. A growing body of evidence suggests that replacing SFA with CHO leads to a decrease in HDL cholesterol and an increase in TG [1\u20133], both of which increase risk of CVD. In contrast, when MUFAs replace SFAs, plasma TGs are decreased and HDL cholesterol is either not decreased or decreased less compared with a high-CHO diet [2\u20134]. Thus, a high-MUFA, low-SFA diet is thought to result in a more favorable overall CVD risk profile than a low-SFA, high-CHO diet. Advocates of a high-CHO, low-SFA diet, however, argue that this diet will promote weight loss, because fewer calories are consumed and, consequently, elicit a beneficial effect on HDL cholesterol and TGs [5].\n\nIntertwined in the ongoing discussion is the question of whether a high-MUFA, low-SFA diet (a higher fat diet) leads to an increase in energy consumption, resulting in weight gain and perhaps predisposing one to overweight\/obesity. Scientists who favor the position that increasing dietary fat leads to weight gain because of the higher caloric value of fat [6] suggest that we eat a constant volume of food regardless of caloric or macronutrient content. Thus, increasing dietary fat leads to overconsumption of energy [7\u201311]. In contrast, scientists who oppose this conclusion cite epidemiologic evidence showing little association between total fat intake and the incidence of overweight\/obesity [12]. They also argue that fat plays a role in satiety and, thus, helps control calorie intake and body weight [13].\n\n### B. Saturated Fatty Acids\n\nThe Seven Countries Study [14], a landmark epidemiologic investigation, demonstrated that diet affected serum cholesterol levels and that an elevation in cholesterol increased risk of coronary disease. SFA intake (as a percent of calories) was positively correlated with serum cholesterol levels as well as with 5-year incidence of coronary heart disease (CHD). Many well-controlled clinical studies followed, resulting in the development of blood cholesterol predictive equations for estimating the changes in total cholesterol in response to changes in type of fat and amount of dietary cholesterol. The original equations developed by Keys _et al._ [15] and Hegsted _et al._ [16] demonstrated that SFA was twice as potent in raising blood cholesterol levels as PUFA was in lowering them. MUFA was shown to have a neutral effect and dietary cholesterol raised the blood cholesterol level but less so than SFA. More recently, predictive equations have been developed for LDL and HDL cholesterol [17\u201320]. The LDL cholesterol response mimics that for total cholesterol. All fatty acid classes and dietary cholesterol increase HDL cholesterol; SFAs are most potent, PUFAs are least potent, and MUFAs have an intermediate effect.\n\nRecent studies have evaluated the effects of individual fatty acids on plasma lipids and lipoproteins [21]. The effects reported are quite divergent when comparisons are made among the different SFAs. Myristic acid (C14:0) is twice as potent as lauric acid (C12:0) in raising total and LDL cholesterol. However, stearic acid (C18:0) is uniquely different; it has a neutral cholesterol-lowering effect.\n\nThere is some epidemiologic evidence from the Atherosclerosis Risk in Communities Study [22] that a high intake of total fat, SFA, and cholesterol is associated with higher levels of factor VII and fibrinogen, two hemostatic factors that play a role in blood clot formation and are considered risk factors for CVD. Likewise, in the Dietary Effects on Lipoproteins and Thrombogenic Activity Study, a well-controlled multicenter feeding study, a reduction in SFA decreased factor VII [23, 24]. Fibrinogen levels were increased in response to a reduction in total fat [23, 24]. Of note, however, was that the magnitude of the response was modest (i.e., 2\u20133%).\n\nThe evidence is overwhelming that SFA, specifically lauric, myristic, and palmitic acids, have potent total and LDL cholesterol-raising effects. Irrespective of the total amount of fat in the diet, it is imperative that these fatty acids be reduced in the diet to decrease CVD risk.\n\n### C. Unsaturated Fatty Acids\n\n1. MONOUNSATURATED FATTY ACIDS\n\nMUFAs provide great flexibility in diet planning because they can be used to replace SFAs, carbohydrate, or calories from both. Depending on the substitution, there can be a variable change in the total fat content of the diet (i.e., from 15% to 40% of energy), varying from essentially no or little change to an approximate twofold increase. On average, MUFAs provide about 15% of energy to the diet, while a high-MUFA diet typically provides about 20\u201322% of energy. Currently, there is great interest in MUFAs as a substitute for dietary CHO because of their beneficial effects on CVD risk factors [2, 3]. Diets high in MUFAs (that are low in SFAs and cholesterol) will lower plasma total and LDL cholesterol and TGs and minimize any potential decrease in HDL cholesterol [25]. There is limited evidence indicating that MUFAs may decrease susceptibility of LDL particles to oxidative modification, which is an important initiating event in the development of atherosclerosis, thereby reducing their atherogenic potential [26, 27]. Moreover, a diet higher in total fat has been shown to maintain a higher LDL particle diameter size [28, 29], which is important because small, dense LDLs increase CVD risk [30, 31].\n\nIn individuals with type 2 diabetes, a high-MUFA\/low-CHO diet decreased both postprandial glucose and plasma insulin by 13.1% (for both) compared with a high-CHO diet [32]. Another study conducted by Garg _et al._ [33], compared a 55% CHO, 30% fat diet with a 40% CHO, 45% fat diet, in which the increase in fat was accomplished with addition of MUFA, on risk of CVD in patients with type 2 diabetes. They reported a significant decrease in a day-long glucose and insulin concentrations with the high-MUFA diet, although there was no change in fasting glucose or insulin in two diet groups. In addition, in obese patients with type 2 diabetes, substitution of MUFA for CHO in two hypocaloric, weight-loss diets resulted in a greater decrease in both fasting and 24-hour glycemia, although the weight loss was similar in two diet groups [34]. In this same study, postprandial glycemia deteriorated after refeeding subjects with the CHO-enriched but not the MUFA-enriched formula. Collectively, these data suggest that MUFAs may improve the glycemic profile (plasma glucose and insulin levels) in individuals with type 2 diabetes.\n\n#### 2. TRANS FATTY ACIDS\n\nNumerous controlled feeding studies demonstrated that TFAs or hydrogenated fats elicited a blood cholesterol response that was intermediate to that observed for unhydrogenated oils and saturated fats [35]. More recently, studies have been conducted to evaluate the plasma lipid and lipoprotein effects of TFAs. These studies have consistently shown that TFAs increase plasma total and LDL cholesterol relative to unsaturated fatty acids [35]. Compared with SFA, TFAs elicit a similar or perhaps slightly less cholesterol-raising effect [35]. In addition, TFAs lower HDL cholesterol resulting in a worsening of the TC:HDL and LDL:HDL ratios, which, in turn, increase CHD risk [36]. Studies have also reported that TFA intake increases lipoprotein(a) levels [37], which is associated with an increased risk of CVD. Consequently, consumption of products that are low in TFAs and SFAs showed more beneficial effects on serum cholesterol levels [38]. While data from epidemiologic studies related to TFA intake and risk of developing heart disease are inconsistent because of the potential confounding dietary factors, substitution of hydrogenated fat with unhydrogenated fat is recommended in food processing and preparation [39].\n\n### D. Polyunsaturated Fatty Acids\n\n1. OMEGA\u20136 FATTY ACIDS\n\nAs a result of the early studies by Keys _et al._ [15] and Hegsted _et al._ [16] that demonstrated the hypocholesterolemic effects of PUFAs, clinical trials were conducted to evaluate the effects of a low-SFA diet that was very high in PUFAs (\u223c16\u201320.7% of energy) on incidence of CVD. These studies demonstrated a marked cholesterol-lowering effect of a high-PUFA diet (17.6\u201320.0% reduction in serum cholesterol compared with baseline values) [40, 41]. Importantly, the cholesterol-lowering response was associated with a reduction in the incidence of CVD (16\u201334%). However, the perception by some that high-PUFA diets may increase risk of certain cancers, despite the lack of strong evidence to support this relationship, diminished support for this diet strategy. This has led to the present recommendation that PUFA calories not exceed 10% of energy.\n\nRecent studies have reported that PUFAs have a slightly greater total and LDL cholesterol-lowering effect versus MUFAs [42]. Thus for practical purposes, MUFAs or PUFAs will elicit effects that are quite similar when incorporated in a diet that meets current recommendations for total fat (30% of energy) and PUFAs (<10% of energy). Some experts advocate, however, that PUFAs not exceed approximately 7% of total energy [43], based on some evidence that PUFAs increase _in vitro_ LDL oxidative susceptibility (compared with MUFAs), thereby possibly increasing CVD risk. Thus, a PUFA recommendation of <10% of energy is, at present, prudent.\n\n#### 2. OMEGA\u20133 FATTY ACIDS\n\nThe 1970s marked the beginning of extensive scientific evaluation of the role of omega\u20133 fatty acids in the development of CVD. The seminal studies of Dyerberg _et al._ [44] noted that coronary atherosclerotic disease was rare in Greenland Eskimos and prevalent in a Danish population. These scientists attributed this difference in the incidence of CHD to the high intake of marine oils by the Eskimos and, in particular, eicosapentanoic acid (EPA, C20:5) and docosahexanoic acid (DHA, C22:6). During the past 30 years numerous studies have demonstrated that these fatty acids may confer cardioprotective effects via multiple mechanisms that involve antiarrhythmic actions, sudden death, thrombosis and hemostasis, growth of atherosclerotic plaques, and lipids and lipoproteins.\n\nThere is impressive evidence that the omega\u20133 fatty acid alpha-linolenic acid, reduces coronary morbidity and mortality in patients with heart disease. The Lyon Diet Heart Study [45] reported that any AHA Step 1 Mediterranean dietary pattern (high in alpha-linolenic acid) reduced all cardiac death and nonfatal myocardial infarction by approximately 70% and all coronary events by about 50% despite no improvement in lipids, lipoproteins and adiposity.\n\nFish oil has a marked hypotriglyceridemic effect in both normotriglyceridemic and hypertriglyceridemic (\u22652 mmol\/L) individuals. The addition of approximately 9\u201313 g\/day of fish oil (e.g., 1.1\u20137 g\/day of omega-3 fatty acids) resulted in a TG decrease of about 20\u201325% in normotriglyceridemic individuals and a decrease of about 26\u201333% in hypertriglyceridemic individuals [46]. However, fish oil elevates LDL cholesterol levels modestly (e.g., 4\u20135%) in normotriglyceridemic individuals and more so (\u223c5\u201311%) in hypertriglyceridemic individuals, and even more so (30%) in some individuals with familial hyperlipidemia (type IV\/V) [47]. Thus, fish oil supplements can be an effective treatment for some patients with hypertriglyceridemia, although close monitoring by a physician is essential to ensure that there is no concurrent significant increase in LDL cholesterol.\n\n## III. DIETARY CARBOHYDRATE\n\nAs discussed in the previous section on dietary fat, there is an ongoing debate about whether it is better to replace saturated fat with calories from MUFAs or CHO to most favorably impact CVD risk. While both diets lower LDL cholesterol, a low-fat, high-CHO diet, when not accompanied by weight loss, decreases HDL cholesterol and increases TG [48]. This is particularly problematic for people with insulin resistance, whose dyslipidemia often presents as decreased HDL cholesterol and elevated TG [49]. A low-fat, high-CHO diet has also been shown to increase plasma glucose and insulin in individuals with type 2 diabetes [32] and healthy women [50]. On the other hand, it can be argued that a low-fat, high-CHO diet facilitates a reduction in energy intake [51] and effectively reduces and maintains body weight [6], which is a key factor in lowering CVD risk, especially in overweight\/obesity.\n\nAmong the proponents of high-CHO diets, the National Cholesterol and Education Program (NCEP) [52] recommends that 55% or more energy come from CHO, because total fat is reduced to facilitate a decrease in SFA and help control energy intake. Others advocate a more extreme reduction in fat (10% energy) and thus a much higher CHO (70\u201380%) diet [53]. This latter approach has been shown to be effective, when combined with other intensive lifestyle changes (e.g., stress management and aerobic exercise), resulting in a reversal of coronary heart disease as measured by percent change in diameter stenosis [54].\n\nWhile the broader issue is still unresolved as to whether CHO or MUFA should replace SFA calories to lower CVD risk, many scientists are turning their focus to the type of CHO used in a low-fat, high-CHO diet. They argue that low-fat, high-CHO diets have not been adequately evaluated in terms of their effects on the overall CVD risk profile. It has been suggested that in studies comparing high-MUFA to high-CHO diets, part of the explanation for the beneficial effects of MUFA could be due to the specific type of CHO used in the low-fat diets.\n\nWhen discussing different types of carbohydrate, scientists historically used the terms _complex_ versus _simple CHO._ As we gain a better understanding of how CHO affects CVD risk, this terminology is not useful and thus is being phased out. However, there are no well-established categories for CHO as it relates to CVD risk, such as SFA, MUFA, PUFA, and TFA for fat and animal versus plant for protein. This is due in part to the complexity of carbohydrate food sources and their impact on CVD risk. Many foods that are included in a high-CHO diet, such as fruits and vegetables, breads and cereals, and legumes, contain multiple compounds that could favorably affect CVD risk. In this section, we will address a few of the actively researched areas associated with CHO and reduction of CVD risk: the role of glycemic index, dietary fiber, resistant starch, and fructooligosaccharides.\n\n### A. Glycemic Index\n\nCarbohydrates differ in terms of their effects on glucose metabolism [55]. Carbohydrates can be classified according to their blood glucose-raising effects using the glycemic index. Low glycemic index foods, such as mature beans and peas, elicit less of a glycemic response than high glycemic index foods, such as potatoes and ready-to-eat cereals. Low glycemic index diets have been shown to increase insulin sensitivity [56, 57] and decrease total serum cholesterol [56] and LDL cholesterol [57] in people with type 2 diabetes. These findings illustrate the importance of considering not only the macronutrient composition of the diet, but also the type of CHO consumed within the context of a low-fat diet.\n\nIn a recent editorial, Grant [58] illustrates the point that in one study, half of the increase in CHO in a low-fat diet was achieved with the addition of sugars [59]. Similar substitutions are evident in another study comparing the effects of high-MUFA and low-fat diets in postmenopausal women. The sample menus reported by the authors show that the low-fat diet contained more sugars or refined foods with a high glycemic index, such as bread, cookies, and potato, than the high-MUFA diet [50].\n\nIn general, foods high in soluble fiber have a low glycemic index; however, this is an oversimplification because food preparation and consumption of a specific food in a mixed meal can alter glycemic index. A mixed meal can have a low glycemic index with the selection of certain foods. Because of the complexity of implementing the glycemic index, it likely will be difficult for consumers to adopt at the present time with currently available foods and contemporary lifestyle practices. Furthermore, the concept of glycemic index must also address the role of other nutrients in a way that is consistent with current dietary recommendations. For example, some low glycemic index foods are high in total fat, saturated fat, and sugar and therefore should be limited. Thus, it is apparent that many questions remain about glycemic index. Clearly, additional evidence is needed to refine glycemic index of foods, snacks, meals, and diets and to establish the health effects of glycemic index and how it can be implemented in the population.\n\n### B. Dietary Fiber\n\nAn abundance of evidence supports a negative association between dietary fiber intake and risk of CVD [60]. Dietary fiber is found naturally in fruits, vegetables, whole-grain cereals, and legumes, and has been supplemented to foods and beverages for several recent studies [61, 62]. Numerous epidemiologic studies support the cardioprotective effect of dietary fiber. For example, data from two large U.S. prospective studies, one including male health professionals [63] and another with female nurses [64], has shown that dietary fiber (most notably cereal fiber) was associated with a reduced risk of fatal and nonfatal myocardial infarction. In addition to epidemiologic evidence, many clinical trials have confirmed the favorable effects of dietary fiber intake on CVD risk factors [65\u201370].\n\nSoluble fiber, including oat bran, psyllium, guar gum, and pectin, has been shown to reduce CVD risk through its action on lipids and lipoproteins and glucose metabolism. A recent meta-analysis of 67 controlled human trials [71] determined that various soluble fibers (2\u201310 g\/day) modestly reduced total and LDL cholesterol (2%), and did not affect HDL cholesterol and TGs. In addition, soluble fiber has been shown to lower glucose and insulin levels in healthy individuals [69] and favorably affect insulin sensitivity in individuals with diabetes [68] and moderate hypercholesterolemia [70].\n\n### C. Resistant Starch\n\nIn recent years, it has become appreciated that the rate of starch hydrolysis can vary from quickly to quite slow. In some instances, the hydrolysis occurs so slowly that some starch may pass into the large intestine undigested. This resistant starch has been defined as ''the sum of starch and products of starch hydrolysis not absorbed in the small intestine of healthy individuals\" by the European FLAIR Concerted Action on Resistant Starch (euresta) [72].\n\nThe beneficial effects of resistant starch on CVDare not well understood. Several animal studies have shown a positive effect on blood lipids and lipoproteins when resistant starch was added to the diet [73, 74]. However, in human studies [75, 76] where free-living normolipidemic subjects were supplemented with two types of resistant starch (retrograded and chemically modified), no effect on serum lipids was observed.\n\nThis lack of effect was also observed when examining the effects of resistant starch in hypertriglyceridemic subjects [77]. Although there were no significant effects on serum lipids in the study by Noakes _et al._ [77], a 17% reduction in postprandial plasma insulin concentrations was observed when high-amylose starch comprised 33% of the carbohydrate content of a test meal. Further research is necessary in various experimental conditions to explore whether resistant starch has a beneficial effect on blood lipids and insulin sensitivity.\n\n### D. Fructooligosaccharides\n\nFructooligosaccharides (FOSs) are indigestible, highly fermentable CHO that occur naturally in foods such as onions, bananas, tomatoes, garlic, and wheat. FOSs can also be produced commercially and have been added to many food products and nutriceuticals [78]. FOSs pass through the intestinal tract undigested and are fermented in the large intestine by bacteria into lactate and short-chain fatty acids [78]. The short-chain fatty acids produced by FOS fermentation ultimately yield 1\u20132 kcal\/g rather than the 4 kcal\/g of digestible carbohydrates [79].\n\nA growing body of research has described numerous beneficial effects of FOSs including improvements in plasma lipids and lipoproteins. Evidence from animal studies report that FOSs, as part of a high-CHO, fiber-free, or high-fat diet, reduce total cholesterol and especially TGs [80, 81]. The TG-lowering action of FOSs occurs as a consequence of a reduction in _de novo_ fatty acid synthesis secondary to modification of gene expression of lipogenic enzymes [82, 83]. Additionally, FOS has been found to reduce serum insulin and glucose concentrations in animal models [82].\n\nStudies of the effects of FOSs on lipids and lipoproteins in humans are few in number and have been conflicting. Williams [84] speculates that this inconsistency is due to the significantly lower doses of FOSs administered in human studies compared to the doses used to elicit effects in animals. Most subjects do not tolerate FOS levels greater than 30 g\/day due to adverse gastrointestinal symptoms [84]. Some studies have not observed any changes in blood lipids and lipoproteins with FOS intakes ranging between 9 and 20 g\/day [85, 86], while others report significant reductions in total and LDL cholesterol or TGs [87, 88]. TGs have been reported to decrease by as much as 27% [87]. It is interesting to note that in hypertriglyceridemic subjects, the TG-lowering action of FOSs may be more pronounced if the diet is high in CHO rather than fat [84]. Furthermore, as observed in animal studies, FOSs have been reported to alter glucose metabolism. Roberfroid [89] and others have demonstrated that the increase of blood glucose and insulin is very low compared to fructose [85, 90, 91].\n\nFuture research is needed to assess whether FOSs have a consistent lipid-lowering effect in humans and to assess the dose necessary to achieve beneficial effects. Furthermore, the lipid-lowering mechansims of FOS remain to be elucidated. However, present data indicate that FOSs reduce total cholesterol and may exert a potentially potent TG-lowering action.\n\n## IV. DIETARY PROTEIN\n\nIn the attempt to define the optimal ratio of dietary macronutrients for CVD risk reduction, nutrition research has recently turned its attention to the role of protein in cardiovascular health. Available evidence at this time is somewhat limited, but indicates that dietary protein of animal and, particularly, plant (soy) origin may beneficially affect several CVD risk factors.\n\n### A. Animal Protein\n\nEarly studies reported that hypercholesterolemia and atherosclerosis could be induced in rabbits and other animals by feeding low-fat, cholesterol-free diets containing milk casein as the source of protein [92, 93]. Interestingly, the cholesterol-raising effect of animal protein has not been consistently observed in other animals or in humans [94].\n\nEpidemiologic studies in the 1950s showed strong associations between dietary (animal) protein and mortality from CVD [95]. However, animal protein intake was also significantly correlated with SFA and cholesterol intake\u2014dietary factors known to be hypercholesterolemic and atherogenic [96]. More recent epidemiologic data, from the Nurses' Health Study, showed that high protein intakes (up to 24% of total energy intake), including animal and plant proteins, significantly reduced the risk of CVD(RR = 0.75; 95% CI: 0.61, 0.92) [97]. In addition, the Cholesterol Lowering Atherosclerosis Study reported a reduction in new coronary artery lesions with increasing dietary protein (low-fat meat and dairy) in place of fat, while persons developing new lesions showed a decrease in mean protein intake [98].\n\nSeveral controlled feeding studies have found that replacing CHO with low-fat animal protein, such as lean beef, poultry, fish, cottage cheese, and skim milk, can elicit favorable changes in lipid risk factors. In a crossover study conducted by Wolfe _et al._ [99, 100], subjects with moderate hypercholesterolemia were randomly assigned to either a high-protein diet containing 27% protein (79% animal) and 53% CHO or a low-protein diet (11% protein; 65% carbohydrate). Dietary fat (25%), cholesterol (less than 200 mg\/day), and fiber were held constant. Compared to subjects on the low-protein diet, moderately hypercholesterolemic subjects on the high-protein diet experienced a 6% reduction in total and LDL cholesterol, 23% and 28% reductions in TG and very low-density lipoprotein (VLDL), and a 12% increase in HDL cholesterol [99, 100]. Subjects with familial hypercholesterolemia (receiving cholestyramine) experienced similar beneficial changes in plasma lipids with increased dietary protein intake [101]. In another study, normolipidemic subjects consumed either a higher fat (35%), high-protein diet containing 22% protein (70% of animal origin) or a low-protein (12%) diet. Those on the high-protein diet sustained a 6% reduction in total cholesterol, a 9% reduction in LDL cholesterol, an 18% decrease in TG, and a 28% reduction in VLDL compared to subjects who consumed a low-protein diet [102]. In a considerably longer-term study (36 weeks), NCEP Step 1 diets containing primarily lean red meat (beef, veal, pork) or lean white meat (poultry and fish) produced similar reductions in LDL-C and elevations in HDL-C in hypercholesterolemic men and women for the duration of the study [102a].\n\nOne recent controlled feeding study, Dietary Approaches to Stop Hypertension (DASH), investigated the effect of dietary patterns, rather than specific compounds on hypertension, a major CVD risk factor. DASH showed that a dietary pattern including animal protein from low-fat dairy products improved blood pressure. Specifically, a diet rich in fruits, vegetables, and low-fat diary products and reduced in saturated fat (18% protein) significantly improved both systolic (decreased by 5.5 mmHg) and diastolic (decreased by 3.0 mmHg) blood pressure beyond that of a lower protein diet (10%) rich in fruits and vegetables [104]. The DASH combination diet is in general accordance with the dietary guidelines established by the American Heart Association and other organizations. It has been reported by Harsha _et al._ [103a] that if the DASH combination diet were strictly adopted by the general American population and resulted in a nationwide blood pressure reduction of the magnitude found in DASH, a 15% decrease in coronary heart disease and about a 27% decrease in stroke incidence would be expected.\n\nMechanisms in which animal protein may reduce lipid risk factors are uncertain at this time. As opposed to high-CHO diets, high protein diets have been shown to reduce TG and increase HDL cholesterol, and some propose that substituting protein for CHO may increase catabolism or decrease production of VLDL and LDL cholesterol [100]. In rats, a high-protein diet has been reported to significantly decreased hepatic VLDL secretion [104]. Additionally, mild hypercholesterolemia due to increased hepatic HMG-CoA reductase activity was observed in rats fed a low-protein diet (8%) [105].\n\nThough current research suggests that increasing intake of animal protein (20\u201325% of total energy) as part of a low-fat diet may reduce CVD risk factors, caution may be warranted in recommending a high-protein diet for several reasons. Available evidence supporting the cholesterol-lowering potential of protein is limited and will require more research. Increasing protein intake from animal sources such as meat and dairy can result in increased intakes of saturated fat and cholesterol if lean meats and low-fat dairy products are not chosen. Thus, plasma cholesterol may be adversely affected and override any benefit gained from increasing protein in the diet. Also, high-protein diets may promote the development of renal disease and osteoporosis. Findings in these areas are conflicting and further research to assess the long-term safety of a high-protein diet is needed.\n\n### B. Soy (Vegetable) Protein\n\nA multitude of animal studies has reported that blood cholesterol levels are lowered by consumption of soy protein rather than animal protein [106, 107]. In humans, epidemiologic studies report that Asian populations, which consume 30\u201350 times more soy than Western populations, have a lower prevalence of many chronic diseases, including CVD [108]. A strong correlation between increasing intakes of soy foods and decreasing total cholesterol concentrations in men and women of Asian populations has also been observed [109]. Though many clinical studies reported that substituting soy protein, such as tofu, tempeh, and soy milk, for animal protein reduced plasma cholesterol levels, the results were somewhat inconsistent. However, in 1995, a meta-analysis of 38 controlled clinical trials concluded that soy intakes ranging from 31 to 47 g\/day significantly reduced total cholesterol by 9.3%, LDL cholesterol by 12.9%, and TG by 10.5%, with a greater response observed in subjects with higher baseline cholesterol levels [110]. HDL cholesterol was increased modestly (i.e., 2.4%; nonsignificant).\n\nFactors responsible for the antiatherogenic effect of soy remain relatively unknown. Some investigators suggest that soy protein upregulates LDL receptors depressed by hypercholesterolemia [111, 112]. This may explain, in part, why normolipidemic subjects do not experience significant reductions in total cholesterol subsequent to soy intake [111]. Others hypothesize that the antiatherogenic properties of soy protein are due to its amino acid profile, specifically the low lysine-to-arginine ratio [92, 113]. Arginine is thought to be less hypercholesterolemic than lysine and thus higher concentrations may reduce serum cholesterol levels. Sanchez _et al._ [114] report that the higher amount of arginine in soy protein, roughly twice that of casein, induces a low postprandial insulin-to-glucagon ratio, and decreases serum cholesterol levels by suppressing the lipogenic functions of insulin. In addition to lowering plasma cholesterol levels, the higher concentration of arginine in soy foods is also suggested to improve vascular reactivity. Arginine is the physiological substrate for nitric oxide synthesis, and nitric oxide functions as a potent vasodilator [115].\n\nVarious bioactive components in soy, particularly isoflavones, are thought to contribute to its hypocholesterolemic and antiatherogenic properties as well [116]. Several studies have shown that soy protein without isoflavones has little or no effect on serum cholesterol levels [117, 118]. Crouse _et al._ [117] demonstrated a significant dose\u2013response effect where 25 g soy protein with 62 mg isoflavone significantly reduced plasma total and LDL cholesterol compared to soy protein containing 3\u201327 mg isoflavone. Conversely, other studies have demonstrated that isoflavones alone have very little effect on serum cholesterol levels [119]. Investigators thus propose that synergy between soy protein and its naturally occurring isoflavones is necessary to reduce cholesterol levels [117].\n\nIn addition to the effects on plasma cholesterol, soy protein with soy isoflavones is reported to inhibit platelet activation and aggregation [120], LDL oxidative susceptibility [121], and smooth muscle cell migration and proliferation [122]. Soy isoflavones, with and without soy protein, have been shown to improve vascular function [119, 123]. Furthermore, soy isoflavones, also referred to as phytoestrogens, bind to estrogen receptors and may offer cardioprotective effects similar to that of estrogen [116].\n\nThe evidence is fairly convincing that consuming 25 g or more of soy protein (with isoflavones) a day beneficially affects other CVD risk factors besides lipids and lipoproteins, particularly in hypercholesterolemic individuals. Whether soy protein or its various bioactive components exert greater cardioprotective action is currently under investigation. However, it is probable that the combination of soy protein and its naturally occurring isoflavones incur the greatest benefit on cardiovascular health.\n\nResearch on the effects of protein and CVD risk is promising. Low-fat animal protein (lean meats and low-fat dairy), and especially soy protein, appears to exert many benefits on cardiovascular health. Of specific interest are their effects on TG and HDL cholesterol, as well as hemostasis, platelet function, and vascular reactivity. Therefore, a low-fat, high-protein (20\u201325% energy from protein) diet may be considered as an alternative to more traditional low-fat, high-CHO diets, particularly in the case of hypertriglyceridemia. Incorporating soy protein (25 g\/day) in the diet will improve multiple CVD risk factors.\n\n## V. CONCLUSION\n\nRemarkable progress has been made in our knowledge of how diet affects CVD risk. The early studies focused on lipids and lipoprotein risk factors in response to the type of fat in the diet. However, as we have gained a better understanding of how diet affects CVD risk, it has become evident that other risk factors are integral to the progression of CVD. Moreover, it is clear that the other macronutrients, i.e., carbohydrate and protein, affect CVD risk. 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AHA science advisory: Monounsaturated fatty acids and risk of cardiovascular disease. _Circulation_. 1999;100(11):1253\u20131258.\nCHAPTER 19\n\nOther Dietary Components and Cardiovascular Risk\n\nLINDA VAN HORN, SUJATA ARCHER, KIMBERLY THEDFORD and AMY BALTES, Northwestern University Medical School, Chicago, Illinois\n\n## I. INTRODUCTION\n\nConclusive data have long established a strong positive association between saturated fat, dietary cholesterol, and elevated levels of total and low-density lipoprotein (LDL) cholesterol [1, 2]. Most national dietary guidelines aimed at reducing cardiovascular risk are based on reductions in saturated fat, dietary cholesterol, and weight loss for those patients who are overweight [3]. The Step 1 and 2 diets recommended by the National Cholesterol Education Program (NCEP) and the American Heart Association (AHA) are based on reduced intakes of total fat, saturated fat, and cholesterol to achieve reductions in population mean blood cholesterol level [1, 4]. These campaigns have been instrumental in achieving the Healthy People 2000 goal mean blood cholesterol level of 200 mg\/dL and contributing to the overall reduction in cardiovascular mortality that has occurred in this country during the past three decades [5, 6].\n\nRecent studies have reaffirmed the primary focus on reduced saturated fat and dietary cholesterol intake, but there is also mounting evidence that other dietary factors beyond fat may play a significant supporting role in prevention of heart disease. These include dietary fiber, especially soluble fiber, whole grains, soy, vitamins B6, B12, and folate, plant stanols and sterols, and a host of phytochemicals. The purpose of this chapter is to summarize current knowledge regarding these dietary factors and their impact on blood lipids and cardiovascular risk. Most of these factors have not merited national dietary guideline status, but as dietary adjuncts they warrant further consideration. The evidence implicating cause and effect surrounding these other factors pales by comparison to that of fatty acid research. This chapter summarizes the quality and quantity of current evidence to place the factors in proper scientific context before considering their potential clinical applications.\n\n## II. DIETARY FIBER AND CARDIOVASCULAR RISK\n\nA growing number of prospective studies report a protective benefit of dietary fiber on risk of cardiovascular disease [7\u201314]. In 1997, the American Dietetic Association published a position statement that ''the public should consume adequate amounts of dietary fiber from a variety of plant foods\" [15]. General recommendations for intake range between 20\u201335 g\/day or 10\u201313 g of dietary fiber per 1000 calories [16]. Current population mean intake of dietary fiber is no more than half of the recommended amount [16, 17]. Because nationwide increases in dietary fiber have not accompanied reductions in total and saturated fat intake, other processed fat-modified but non-fiber-rich foods appear to have been substituted instead [16, 18].\n\nThe benefits of consuming dietary fiber from carbohydrate foods, including whole grains, cereals, legumes, fruits, and vegetables are many. There is increasing evidence that a dietary pattern rich in these foods not only reduces risk of high blood cholesterol, but also stroke, hypertension, and cancer [14, 19\u201321]. The AHA, NCEP, and other national health-related organizations emphasize the importance and nutritional benefits of these foods beyond their fiber contributions (1\u20134). Conversely, diets high in refined carbohydrates and sugars have been linked with adverse effects on high-density lipoprotein (HDL) cholesterol, triglycerides and insulin resistance [18, 22, 23].\n\n### A. Definition and Sources of Fiber\n\nDietary fiber is defined as the storage and cell wall polysaccharide of plants that cannot be hydrolyzed by human digestive enzymes [24, 25]. Dietary fiber includes cellulose, hemicelluloses, pectin, and lignin. Difficulties in perfecting the chemical analysis of fiber had confounded assessment of specific amounts of dietary fiber in previous studies [16]. More reliable methodology for estimating total dietary fiber as well as quantifying soluble fiber content now facilitates research testing specific types of fiber and relationships with lipid metabolism, glucose metabolism, and other biological outcomes [24, 25]. Current nutrient databases document amounts of total dietary fiber as well as soluble and insoluble components making it possible to evaluate biological impacts from different food sources. Table 1 provides data on common sources of dietary fiber [26].\n\nTABLE 1\n\nSources of Dietary Fiber\n\nag\/sv = grams per serving.\n\n_Source:_ Adapted from Marlett, J. A., and Cheung, T.-F. (1997). Database and quick methods of assessing typical dietary fiber intakes using data for 228 commonly consumed foods. _J. Am. Diet. Assoc._ **97,** 1139\u20131148.\n\n### B. Mechanisms Related to Fiber and Serum Cholesterol Reduction\n\nThe exact mechanisms involved in lipid lowering and\/or overall risk reduction remain inconclusive. Viscous polysaccharides act in the gastrointestinal tract to reduce blood cholesterol levels by decreasing absorption of cholesterol or fatty acids as well as by decreasing absorption of biliary cholesterol or bioacids [16, 24]. Fiber may also alter serum concentration of hormones and short-chain fatty acids that influence lipid metabolism [25, 27, 28]. \u03b2-glucan, the water-soluble fiber prevalent in oats and barley, has been identified in animal models as the active agent influencing cholesterol metabolism reduces LDL cholesterol concentration in human studies [25, 27]. In addition to lipid-lowering benefits, studies on oats and barley have further suggested that \u03b1-tocotrienols and other antioxidant components may inhibit HMG-CoA, thereby contributing a weak statin-like influence [25]. The amino acid content of oats or the arginine-to-lysine ratio may also favor the hypocholesterolemic response. Based on more than 40 clinical trials, animal experimental data and meta-analyses, the Food and Drug Administration (FDA), for the first time, adopted a health claim regarding intake of oats as part of a fat-modified diet for reducing risk of coronary heart disease [29]. All prior health claims had been based on specific nutrients or dietary factors, but the oats health claim carried precedent-setting significance for future research on whole foods or food patterns.\n\n### C. Psyllium and Lipids\n\nSoon after the oats health claim was established, the FDA approved another health claim for psyllium and cardiovascular disease based on a similar premise [30]. Psyllium, a form of soluble fiber found in a type of grass, can be used as a food supplement. Inherently, psyllium offers no other nutritional benefits compared to foods like oats, barley, beans, or fruits, but studies report LDL cholesterol reductions of 5\u201315% with 7 or more grams per day [27, 31]. As noted in most other fiber intervention studies, the higher the baseline blood cholesterol level, the greater the LDL cholesterol reduction with psyllium [31, 32]. A meta-analysis of eight studies including psyllium as part of a low-saturated-fat diet reported that consumption of 10.2 g of psyllium per day lowered serum total cholesterol by 4% ( _p <_ 0.0001) with no effect on serum HDL or triacylglycerol concentrations [32].\n\n### D. Summary\n\nIntake of dietary fiber, especially soluble fiber, decreases LDL cholesterol with little or no effect on HDL cholesterol [24, 27, 33\u201335]. Based on recent epidemiologic evidence, dietary fiber intake is also inversely related to body mass index (BMI) and insulin levels [36]. While impact of soluble fiber on LDL cholesterol lowering is modest, approximately 5% beyond what can be achieved on a fat-modified diet [25], there are other possible health benefits such as increased insulin sensitivity, decreased triglycerides, and improved weight control, that support the dietary recommendations for increased intake of fiber-rich foods [37].\n\n## III. HOMOCYSTEINE AND CARDIOVASCULAR DISEASE\n\nHomocysteine is an intermediary amino acid in the pathway of methionine metabolism. Intracellular homocysteine is metabolized by either transsulfuration or remethylation to methionine [38]. Approximately 50% of homocysteine enters the transsulfuration pathway. Condensation of homocysteine with serine is catalyzed by cystathionine-\u03b2-synthase, which requires pyridoxal 5\u2032-phosphate, the biologically active form of vitamin B6 [39, 40]. In the remethylation pathway, methylcobalamin and methlytetrahydrofolate serve as cofactors and cosubstrates for the enzyme 5-methlytetrahydrofolate-homocysteine methlytransferase (methionine synthase) [39]. The second remethylation pathway requires the enzyme betaine-homocysteine methlytransferase [41]. Disruption in the transsulfuration or remethylation pathways results in accumulation of circulating homocysteine, a phenomenon linked with a higher risk for cardiovascular disease [42\u201344].\n\nElevated serum homocysteine concentrations are considered an independent risk factor for coronary artery disease (CAD) [39, 42]. The role of homocysteine in vascular disease is postulated to be caused via collagen destabilization, platelet accumulation, or a lowering of serum antithrombin activity [42]. Boushey _et al._ [45] reported that for women, the odds ratio (OR) for CAD, with a 5 \u03bcmol\/L increase in total homocysteine was 1.8 (95% CI, 1.3\u20131.9). The Physicians' Health Study reported that the risk of myocardial infarction even among those who had no history of vascular disease was 3.4-fold greater within 5 years among those with elevated plasma homocysteine concentrations compared to those with lower plasma homocysteine concentrations [46].\n\nHyperhomocysteinemia is designated as moderate, intermediate and severe corresponding to concentrations of homocysteine between 16\u201330, 31\u2013100, and greater than 100 nmol\/mL, respectively [42]. The normal concentration of homocysteine in plasma is about 10 nmol\/mL [42]. Some studies report that elevated plasma homocysteine levels do not pose a risk until levels reach the upper 95th percentile in control subjects [46], whereas others report an increased risk even at lower concentrations [45].\n\nGenetic studies have also identified abnormal thermolabile variants in enzymes such as methlyenetetrahydrofolate reductase, which leads to hyperhomocysteinemia [47]. Only recently has the relationship between circulating vitamins B12, B6 and folate and plasma homocysteine been addressed.\n\n### A. Vitamins and Homocysteine\n\nHigh levels of plasma homocysteine (> 100 nmol\/L) can occur due to nutritional deficiencies of vitamins B12, B6 and folate [42]. Several studies have shown that vitamin B12 or folate deficiencies produce elevations in fasting plasma homocysteine levels up to 20 times the upper limits of the normal level (48\u201350). The influence of vitamin B6 deficiency with plasma homocysteine is unclear. Some studies have shown tissue depletion of vitamin B6 with no increase in plasma homocysteine concentrations, whereas others have shown that plasma homocysteine concentrations may be elevated in those who have a vitamin B6 deficiency (51\u201353).\n\nIn normal metabolism, greater than 50% of homocysteine is recycled into methionine by a transmethylation reaction requiring folate and vitamin B12, whereas vitamin B6 is a cofactor for the enzyme cystathionine b-synthase in the transsulfuration pathway. Deficiency of this enzyme results in homocystinuria. Several studies have shown an inverse association between blood homocysteine levels with plasma or serum levels of folate, B6, and B12 [54\u201356].\n\nSelhub _et al._ [56] reported from the Framingham Study that mean homocysteine levels were significantly higher in the lowest two deciles for folate (15.6 and 13.7 \u03bcm\/L, respectively) compared with the highest deciles (11.0 \u03bcm\/L) [57]. It is estimated that there can be a 25% reduction in homocysteine levels with supplementation of 0.5\u20135.7 mg\/day of folate [58]. The Institute of Medicine recommends no more than 1 mg\/day of folate supplementation due to the risk of higher amounts masking signs of a vitamin B12 deficiency [58].\n\nIn patients with a vitamin B12 deficiency who may have intermediate or severe hyperhomocysteinemia, B12 can normalize homocysteine levels in 70% of the cases [49]. Vitamin B12 intakes of 0.02\u20130.5 mg\/day along with 0.5\u20135.7 mg\/day of folate can lower homocysteine levels up to 7% [58]. Rasmussen _et al._ [59] administered 2 mg of B12 along with 10 mg of folate to 126 women and 109 healthy men and reported a decrease of 4.8% in circulating homocysteine levels due to the B12 intake. Bronstrup _et al._ [60] provided a supplement with 400 mcg of folate with 6 \u03bcg of vitamin B12 vs. 400 \u03bcg of folate and 400 \u03bcg B12 and reported a greater reduction in homocysteine levels with the higher B12 concentrations vs. the lower dose (\u201318% vs \u201311%). Ubbink _et al._ reported a 56.8% prevalence of hyperhomocysteinemia among South African men with suboptimal B12 levels [61]. If a patient has a B12 deficiency, folate supplementation may be ineffective in reducing homocysteine levels [62].\n\nSupplementation with vitamin B6 has been shown to enhance the activity of the transsulfuration pathway, which leads to a decrease in elevated circulating homocysteine levels [63]. It is reported that in populations with intakes of less than 1.92 mg\/day of B6 there was a prevalence of high total homocysteine [64]. Some studies report a reduction in homocysteine levels with a supplement of 50 mg B6 combined with folate and vitamin B12. However not all studies have shown a decrease in plasma homocysteine levels even with high-dose (70\u2013300 mg) supplementation of vitamin B6 [62]. Vitamin B6 deficiency is primarily associated with hyperhomocysteinemia after methionine loading. Levels of vitamin B6 used to treat moderately elevated plasma homocysteine levels range from 25 to 50 mg\/day. A concern of vitamin B6 supplementation relates to sensory neuropathy, which is usually seen in patients treated with dosages greater than 400 mg\/day [65].\n\nLong-term supplementation with vitamins to reduce homocysteine levels has not been studied extensively. Ubbink _et al._ [65] studied 22 men who had plasma homocysteine concentrations of >16.3 \u03bcmol\/L and were given a multivitamin supplementation with 1.0 mg folate, 50 \u03bcg B12 and 10 mg B6 for 6 weeks. A decline in circulating homocysteine concentrations was observed from 30.9 to 14.0 \u03bcmol\/L. Vitamin supplementation was discontinued for 18 weeks and an increase in homocysteine was observed. Treating the patients again with the vitamin formulation for 6 weeks resulted in a decline of homocysteine. Discontinuation of the vitamin therapy and dietary intake aimed at getting foods high in these vitamins resulted in elevated plasma homocysteine concentrations after 18 weeks. The authors concluded that some individuals with hyperhomocysteinemia may be unable to lower plasma homocysteine levels with diet alone [65].\n\nSome other vitamins have been reported to influence plasma homocysteine levels. An intake of 0.6 mg of riboflavin has resulted in a modest decline in plasma homocysteine levels (0.475 mmol\/L). Riboflavin functions as a cofactor for methylenetetrahydrofolate reductase [66]. High doses of niacin (3 g\/day) have caused an elevation in plasma homocysteine levels [67].\n\nIt is unclear if homocysteine should be reduced by diet and\/or vitamin therapy [68, 69]. The AHA recommends that intakes of folate, B12, and B6 be met by intake of vegetables, fruits, legumes, meat, fish, and fortified grains and cereals [68]. The Food and Nutrition Board of the National Academy of Sciences and Institute of Medicine recommends the following RDAs for nonpregnant and nonlactating adults: folate 400 \u03bcg, 2.4 \u03bcg B12, and 1.7 mg B6 [58]. Because most of the population does not meet these recommendations, an increase of foods rich in these vitamins is advised. For those who may have malabsorption of some of these vitamins, such as the elderly, foods fortified with these vitamins or a supplement with these vitamins is recommended [59]. An increase in intake of vitamin fortified foods and\/or a daily supplement with 0.4 mg folate, 2 mg B6 and 6 \u03bcg B12 is recommended when there is a family history of cardiovascular disease, malabsorption, use of certain pharmaceutical agents such as niacin, bile acid-binding resins or L-dopa [70].\n\n### B. Summary\n\nIn summary, the B vitamins folate, B12, and B6 play an important role in methionine metabolism and in determining circulating homocysteine levels. However, many questions remain regarding the association of folate, B12, and B6 with homocysteine levels. Folate supplementation with or without B12 and B6 has been reported to reduce homocysteine levels in hyperhomocysteinemia and in those with normal homocysteine levels. However, data from randomized clinical trials regarding the benefits of folate on cardiovascular disease are lacking [70]. Until such data are available it is recommended that reduction of risks of CAD be achieved via proven methods such as consuming a diet based on the Dietary Guidelines for Americans.\n\n## IV. SOY AND CARDIOVASCULAR DISEASE\n\nIt has long been recognized that vegetable protein has beneficial health effects compared to animal protein, especially related to serum cholesterol status [1, 2, 71]. Countries with high soy intake have reduced rates of cardiovascular mortality compared with countries consuming primarily animal protein products [3]. During the past 35 years, much research has focused on the relationship between soy intake and reduced risk for cardiovascular and other chronic disease. In a meta-analysis of 38 clinical studies, Anderson _et al._ [72] reported that substitution of soy protein for animal protein was associated with significant decreases in total cholesterol, LDL cholesterol and triglyceride concentration. HDL cholesterol was unchanged in most studies. These data contributed to the 1999 approval by the FDA of a health claim for reduced cardiovacular risk related to intake of at least 25 g of soy per day [73]. No isoflavone concentration was included in the health claim. Clinical trial data further document that the reductions in LDL cholesterol are greatest among those with the highest baseline levels. This section will briefly review some of these data and the possible mechanisms involved.\n\n### A. Is It the Isoflavones?\n\nSoybeans are rich in the isoflavones genistein, daidzein, and to a lesser degree glycitein [74]. These occur primarily as glycoside forms genestin, daidzin, and glycitin [71]. Bacteria in the gut are required to produce the enzymes needed to hydrolyze these glycosides and convert them to the aglycone forms [75]. The isoflavones possess estrogenic activity and binding to estrogen receptors. Levels of these isoflavones vary by the type of product and the degree of processing [76]. Data in animal and human studies report that isoflavones have an independent effect on blood cholesterol [76\u201380], but dose may dictate response. Cassidy _et al._ [76] reported that 45 mg of isoflavonoids, but not 23 mg of isoflavonoids, resulted in a significant reduction in total and LDL cholesterol concentrations in young females. In an animal study, comparison of soy protein with or without isoflavones lower total cholesterol and LDL cholesterol and higher HDL cholesterol were reported with isoflavones. The prevalence of atherosclerotic lesions was also lower in monkeys fed the soy plus isoflavones [81]. Human data confirming these associations are needed, and national guidelines regarding favorable isoflavone levels have yet to be developed.\n\n### B. Possible Mechanisms: Soy Impact on Lipid Lipoproteins\n\nSoy may reduce total cholesterol and LDL cholesterol through various mechanisms. The most commonly accepted hypothesis is that soy may cause possible interruption in hepatobiliary circulation, thereby increasing LDL receptor activity [71]. Another possibility involves hormonal changes in thyroid and insulin-to-glucagan ratio but these remain speculative. Table 2 summarizes possible mechanisms.\n\nTABLE 2\n\nSoy Proteinand Decreased Cardiovascular Risk: Possible Mechanisms\n\n1. \u2193Plasma cholesterol levels, possibly due to:\n\na. \u2191 bile excretion\n\nb. \u2191 LDL receptor activity\n\nc. \u2191 thyroxine and thyroid-stimulating hormone\n\nd. \u2193 cholesterol absorption\n\ne. \u2191 soy globulins\n\n2. \u2193 Susceptibility to LDL oxidation\n\n3. \u2191 Arterial compliance\n\n4. Estrogenic activity of soy isoflavones may improve blood lipids\n\n_Source:_ Adapted from Lichtenstein, A. (1998). Soy protein, isofla-vones, and cardiovascular disease risk. _J. Nutr._ **128,** 1589\u20131592.\n\n### C. Increased Apolipoprotein E Receptor Activity\n\nSoy's impact on increased LDL receptor expression in humans [82] may be related to lipid-lowering benefits through an up-regulation of LDL receptors [82\u201384]. Genetic influences on response may also play a role. Subjects with the apoE2 genotype have less hypercholesterolemia, but the E2 protein is an inefficient regulator of LDL receptor activity. When the plasma cholesterol responses in individuals with apoE2 versus apoE3 and E4 were compared, the apo E2 carriers responded weakly to the diet, but not so the E3 and E4 patients. Patients with higher LDL receptor activity seem less likely to show a favorable response to this dietary change as compared to patients whose LDL receptor activity may be relatively suppressed [83].\n\n### D. Oxidative Status\n\nAs summarized by Lichtenstein [84], the oxidation of LDL increases atherogenicity. Isoflavones appear to inhibit the oxidative modification of LDL by macrophages [85], enhance the resistance of LDL to oxidation [86, 87], and exhibit antioxidant activities in an aqueous phase [88, 89]. Isoflavonoids also appear to scavenge free radicals [90].\n\n## V. PHYTOCHEMICALS: PROPOSED ASSOCIATION WITH CARDIOVASCULAR DISEASE\n\nPhytochemicals are biologically active plant compounds found in fruits, vegetables, and whole grains [91]. Interest in phytochemicals has increased due to various epidemiological and clinical studies that report on the benefits of consumption of these compounds for lowering risks for cardiovascular disease and cancer [92\u201394]. A summary of some of the most frequently reported substances is presented in Table 3. Three classes of phytochemicals\u2014flavonoids, plant sterols, and plant sulfur compounds\u2014have been studied most extensively and will be discussed briefly as they may relate to cardiovascular disease.\n\nTABLE 3\n\nPhytochemicals with Possible Health Benefits\n\nActive substance | What it may help do | Where it is found \n---|---|--- \nAllylic sulfides | Inhibit cholesterol synthesis | Aged garlic extract, onions, leeks, chives \nAlpha-linolenic acid | Reduce inflammation and stimulate the immune system | Flaxseed, soy products, purslane, walnuts \nAnthocyanidins | Antioxidant, supports cardiovascular function, may have significant application in the field of ophthalmology | Bilberry, blueberries, grapes \nCapsaicin | May support healthy cardiovascular function and digestive function | Cayenne pepper \nCarotenoids | Antioxidant properties that may help reduce the accumulation of arterial plaque | Parsley, carrots, winter squash, sweet potatoes, yams, cantaloupe, apricots, spinach, kale, turnip greens, citrus fruit \nCatechins | May help the immune system and lower cholesterol | Green tea, berries \nCoumarins | Prevents blood clotting | Parsley, carrots, citrus fruit \nCurcumins | Antioxidant, modulates prostaglandin | Turmeric, curcumin \nFlavonoids | Block receptor sites for certain hormones involved in cancer promotion, antioxidant, supports liver detoxification | Parsley, carrots, citrus fruits, broccoli, cabbage, cucumbers, green tea, squash, yams, tomatoes, eggplant, peppers, soy products, berries, milk thistle, onion, garlic \nGamma-glutamyl allyic cysteines | May have a role in lowering blood pressure and increasing immune system activities | Aged garlic extract \nGinsenoside | Adaptogen, may help to improve the body's ability to adapt to physical or mental stress | Siberian and panax ginseng \nIndoles | Induce protective enzymes that deactivate estrogen | Cabbage, brussels sprouts, kale, cauliflower, broccoli \nIsoflavones | May inhibit cholesterol production | Soybeans, tofu, soy milk \nIsothiocyanates | Powerful inducers of protective enzymes | Mustard, horseradish, radishes, cruciferous vegetables \nLignans | May lower LDL cholesterol, inhibit smooth-muscle proliferation, plaque formation, and activity of thrombin | Soy, whole grains, oats, oat bran, barley, legumes, prunes, apples, carrots, grapefruit, psyllium seed \nLimonoids | Powerful inducer of protective enzymes | Citrus fruits \nLycopene | Antioxidant | Tomatoes, red grapefruit, red peppers \nMonoterpenes | May inhibit cholesterol production and aid protective enzyme activity | Parsley, carrots, broccoli, cabbage, cucumbers, squash, yams, tomatoes, eggplant, peppers, mint, basil, citrus fruits \nPhenolic acids | May inhibit nitrosamine formation | Parsley, carrots, broccoli, cabbage, cucumbers, tomatoes, eggplant, peppers, citrus fruits, whole grains, berries, nuts \nPhenylalkylketones | Antioxidant, supports healthy digestive function, modulates prostagladin metabolism | Ginger \nPhthalides | Stimulates the production of beneficial enzymes that detoxify carcinogens | Parsley, carrots, celery \nPlant sterols | Block estrogen promotion of breast cancer activity; help the absorption of cholesterol | Broccoli, cabbage, cucumbers, squash, yams, tomatoes, eggplant, peppers, soy products, whole grains \nPolyacetylenes | Protect against certain carcinogens found in tobacco smoke and help regulate prostagladin production | Parsley, carrots, celery \nProanthocyanidins | Antioxidant, supports healthy capillary integrity, modulates prostagladin metabolism | Grapes, green tea, wine \nPolysaccharides | Supports immune function | Aloe, astragalus \nSchisandrins | Adaptogen, may help to improve the body's ability to adapt to physical and mental stressors, supports live function, supports healthy vision | Schisandra \nTriterpenoids | May prevent dental decay and act as an antiulcer agent | Citrus fruits, licorice-root extract, soy products\n\n_Source:_ Adapted from Bravo, L. (1998). Polyphenols: Chemistry, dietary sources, metabolism and nutritional significance. _Nutr. Rev._ **56,** 317\u2013333.\n\n### A. Flavonoids\n\nFlavonoids are polyphenolic compounds found in fruits, vegetables, nuts, grains, tea, and wine [24, 95]. Flavonols and flavones are subclasses of flavonoids. The presence of polyphenols in plant foods is influenced by genetic factors, environmental conditions, germinations, degree or ripeness, processing, and storage [96]. The average daily intake of dietary flavonoids in the United States is approximately 1.0 g\/day, depending on the season [97]. Epidemiological studies have indicated a reduced risk for ischemic heart disease and stroke with the intake of some of the flavonoids [98\u2013100]. However, not all epidemiological and experimental studies have reported an inverse association between flavonoid intake and cardiovascular disease [95, 101]. Different flavonoids exert their influence on reducing CAD risk by varying mechanisms. Soy proteins, rich in isoflavones, have a hypocholesterolemic effect [72]. Quercetin, a flavonol found in broccoli, red grapes, and cereals, is reported to inhibit LDL oxidation [102]. Red wine and grape juice have phenolic flavonoids that act as antioxidants, thereby preventing LDL oxidation [103]. It is also reported that flavones are potent inhibitors of cyclooxygenase activity, thereby inhibiting platelet aggregation [104].\n\nWhile these potential associations between flavonoids and health are compelling, randomized controlled clinical trials are needed before dietary recommendations and guidelines can be established. The AHA's Scientific Advisory Board recommends that additional research is needed on the classification of flavonoids, their efficacy, and any adverse effects they may have [105]. More specifically, experimental data demonstrating a direct association between flavonoids and platelet aggregation, serum lipoproteins, and blood pressure are needed before cause and effect can be established.\n\n### B. Plant Sterols\n\nPlant sterols are another class of phytochemicals that differ from dietary cholesterol due to their side chains. Plant sterols or phytosterols, including sitosterol, stigmasterol, and campesterol, are reported to have serum cholesterol-lowering effects [105]. All vegetable foods have some plant sterol content [106]. The Western diet includes approximately 200\u2013400 mg\/day of plant sterols [107].\n\nDuring the 1950s and 1960s soy sterols were studied extensively and were reported to reduce cholesterol by 10% [108, 109]. The decrease in plasma cholesterol was thought to be via an increase in LDL receptor activity. Animal experimental studies done _in vitro_ reported that in isolated rat livers perfused with very low density lipoprotein (VLDL), LDL, and HDL particles obtained from patients with hereditary phytoesterolemia rich in plant sterols showed that only HDL particles provided a vehicle for unesterified cholesterol elimination in bile [110]. Studies with margarines enriched with plant stanol esters reported that an intake of 2\u20133 g\/day reduced total cholesterol by 10% and LDL cholesterol by 15% due to increased cholesterol elimination [111]. Studies on the effects of margarines enriched with different vegetable oil sterols, i.e., margarines with sterol esters from soybean oil (sitosterol, campesterol, and stigmasterol), report them to be as effective as margarines with sitostanol esters in lowering blood total and LDL cholesterol without influencing HDL cholesterol levels [112].\n\nPlant sterols in products other than margarines may show further benefits. Mice fed a diet supplemented with 2% phytoesterols for 20 weeks increased HDL cholesterol and decreased hepatic lipase activity and plasma fibrinogen concentrations, but clinical studies are needed [113]. Because most studies reporting the benefits of plant sterols on cardiovascular disease have been conducted on hypercholesterolemic patients, data on efficacy and safety long term in normocholesterolemic or mildly hypercholesterolemic people are needed [114]. Also, whether significant decreases in total cholesterol or LDL cholesterol will occur if low doses of these compounds are consumed by individuals is not fully known with no CAD [115].\n\n### C. Sulfur-Containing Plant Foods\n\nSulfur-containing plant foods come from the allium family of vegetables, which includes garlic, onions, and leeks. Garlic and onion have many sulfur-containing active components mainly in the form of cysteine derivatives. These decompose into thiosulfinates and polysulfides by action of allinase on extraction, producing volatile products on decomposition [116]. Onion and garlic have been reported to decrease cholesterol levels, lower risk of thrombosis, and suppress platelet aggregation [117\u2013119].\n\nGarlic is the most widely studied sulfur-containing food. Garlic's putative cardioprotective effects include antithrombotic activity and blood pressure and lipid lowering [120]. It is reported that one-half to one clove of garlic a day reduces hypercholesterolemia by approximately 0.59 mmol\/L (23 mg\/dL) [116]. However, not all studies report hypocholesterolemic effects of garlic [121, 122]. Some of the concerns about the reported benefits of garlic usage are related to the pharmaceutical dosages required to see results, such as 4\u20135 g of fresh garlic cloves per day; side effects such as abdominal pain or anemia; differences in garlic manufacturing processes, such as dry powders, oils, freeze-dried preparations and aged extracts; and study designs [120, 121]. Further well-designed clinical studies are required to demonstrate the effect of the sulfur-containing compounds on cardiovascular disease risk factors [94].\n\n### D. Summary\n\nPhytochemicals found in fruits and vegetables may play an important cardioprotective role, but well-designed randomized controlled studies are needed to characterize these products and their mode of action. Until such results are available, it is recommended that traditional risk lowering factors such as decreased fat intake, weight loss, and increased physical activity should be addressed. Table 4 summarizes the potential impact of a cumulative beneficial approach.\n\nTABLE 4\n\nPortfolio of Dietary Factors for Cholesterol Reduction\n\naDepending on the sterol and stanol.\n\nbReduce _trans_ fatty acids as close to zero as possible.\n\ncAssuming the effects are additive.\n\n_Source:_ Adapted from Jenkins, D. J., Kendall, C. W., Axelsen, M., Augustin, L. S., and Vuksan, V. (2000). Viscous and nonviscous fibres, nonabsorbable and low glycaemic index carbohydrates, blood lipid and coronary heart disease. _Curr. Opin. Cardiol._ **11,** 49\u201356.\n\n## VI. CONCLUSION\n\nThere is no question that saturated fat, dietary cholesterol, and obesity are major diet-related risk factors for cardiovascular disease. Building from this base, a host of other nonlipid, dietary factors may also provide important benefits. Dietary fiber, soy, antioxidant vitamins, decreased plasma homocystine levels, phytochemicals, and flavonoids appear to offer advantages that enhance and complement the lipid-modified aspect of the anthiatherogenic diet. Fruits, vegetables, and whole grains are major contributors of these dietary factors and can safely be recommended for other health promoting attributes as well. Future studies will help quantify and validate these associations and also provide evidence of possible long-term benefits.\n\nReferences\n\n1. The Expert Panel. Summary of the second report of the National Cholesterol Education Program (NCEP) Expert Panel on detection, evaluation and treatment of high blood cholesterol in adults (Adult Treatment Panel II). _JAMA_. 1993;269:3015\u20133023.\n\n2. Ginsberg H.N., Kris-Etherton P.M., Dennis B., Elmer P.J., Ershow A., Lefevre M., Pearson T., Roheim P., Ramakrishnan R., Reed R., Stewart K., Stewart P., Phillips K., Anderson N. 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College Physicians_. 1996;30:329\u2013334.\nCHAPTER 20\n\nNutrition, Diet, and Hypertension\n\nMARJI McCULLOUGH1 and PAO-HWA LIN2\n\n1The American Cancer Society, Atlanta, Georgia\n\n2Duke University Medical Center, Durham, North Carolina\n\n## I. INTRODUCTION\n\nApproximately 25% of U.S. adults have hypertension,1 a major risk factor for coronary heart disease, stroke, and premature death [1\u20133]. Even high normal blood pressure is associated with graded, increased risk of cardiovascular disease [3, 4] (Table 1). In industrialized societies, blood pressure increases with age: by age 60, 50\u201380% of Americans will be diagnosed with hypertension. It is also more common in people of African-American descent [2]. The cause of hypertension is largely unknown, except that in about 5% of cases it is secondary to underlying pathophysiologic correctable conditions.\n\nTABLE 1\n\nBaseline Systolic Blood Pressure and Age-Adjusted 10-Year Mortality from Cardiovascular Disease from the Multiple Risk Factor Intervention Trial\n\n_Note:_ Men free of history of myocardial infarction at baseline ( _N_ = 347,978); Multiple Risk Factor Intervention Trial primary screenees [4].\n\n_Source:_ Reprinted with permission from Stamler, J. (1991). Blood pressure and high blood pressure: Aspects of risk. _Hypertension_ **18** (suppl 1), I95\u2013I107.\n\nRecent reports suggest that more than two-thirds of individuals with hypertension are not well controlled (blood pressure <140 mm Hg systolic\/<90 mm Hg diastolic). The full potential of diet modification for treatment of hypertension has likely not yet been realized, because researchers have incompletely identified the dietary causes of hypertension, and because of poor adherence by both the clinicians and the public alike to the established medical and dietary guidelines [5]. Long-standing principal dietary factors thought to be important for blood pressure regulation include salt, alcohol, and body weight, and data to support these are strong. Other nonpharmacologic recommendations include increasing potassium intake and aerobic exercise and smoking cessation [1]. These factors are all recommended as part of the first-line therapy for low-risk individuals, defined as those without diabetes or cardiovascular disease and with systolic blood pressure less than 160 mm Hg and diastolic blood pressure less than 100 mm Hg [1], and as part of the combination therapy program for high-risk individuals. Magnesium, calcium, fiber, fat, protein, and certain carbohydrates have also been hypothesized to affect blood pressure, though information linking these dietary factors to blood pressure reduction is less consistent. Researchers continue to search for additional dietary strategies to prevent and reduce high blood pressure, and to clarify some of the inconsistencies from earlier studies in this area.\n\nIn this chapter, we provide an overview of epidemiologic and clinical evidence for established and suspected dietary risk factors for hypertension. Results from the recent Dietary Approaches to Stop Hypertension (DASH) trial [6] and the DASH-Sodium trial [7], both of which demonstrated effective food-based approaches to blood pressure reduction, are also included. We conclude with a summary of qualitative and quantitative recommendations for prevention and control of high blood pressure through dietary means.\n\n### A. Methodologic Considerations\n\nSeveral approaches have been used to study the relationship between diet and blood pressure, and the strengths and limitations of each are worth noting. Cross-sectional observational studies have generated a myriad of hypotheses. However, because these studies cannot determine whether the dietary factor preceded the disease, or whether the diet changed in response to diagnosis or symptoms, they cannot be used to determine causality [8]. Cross-cultural comparisons, another variant of cross-sectional studies, cannot be used to confirm causality because of potentially inadequate control for other confounders of the diet\u2013blood pressure relationship (e.g., body weight, physical activity) that vary among populations [9]. These comparisons are further complicated by possible genetic differences in response to environmental stimuli across populations [10]. Prospective cohort studies provide stronger evidence of cause and effect because the dietary factors of interest are measured at the start of the study, prior to disease diagnosis. The population is then followed over time, and disease risk is estimated according to level of the dietary factor. However, the potential for uncontrolled confounding variables still exists in this type of observational study. Particular methodological issues have the potential to affect the validity of blood pressure studies. For example, because blood pressure varies widely throughout the day, and tends to decrease with repeated measurements, multiple standardized measurements are required, and attention to study design is crucial [11]. Although intervention trials are often of relatively short duration, they offer the most powerful evaluation of cause and effect because they can address these limitations more fully [12].\n\nBecause a comprehensive review of all individual trials in this area is beyond the scope of this chapter, when applicable, we review meta-analyses. These analyses quantitatively pool results from several studies according to specific inclusion criteria and are useful for evaluating consistency in the literature. Some aspects and limitations of this method (especially for pooling observational studies) should be noted [13, 14]: (1) large studies are weighted more heavily, (2) many studies do not control for potential confounders, (3) it is rarely feasible to conduct subanalyses on potentially important modifying factors, and (4) when specific biological markers are not available, accounting for variable dietary adherence among studies is rarely possible.\n\n### B. Populations with Low Prevalenee of Hypertension\n\nIn contrast to the prevalence in the United States, many non-Westernized, remote populations have a low prevalence of hypertension and do not experience an increase in blood pressure with age [10, 15]. Their protection from hypertension is often attributed to a very low salt intake [16\u201318], though a salt intake similar to U.S. levels has also been documented [19, 20]. Potassium intake tends to be ample or high [18, 20], and these groups tend to be physically active [10]. Low alcohol consumption has been reported in some [18, 21] but not all [22] studies. In most of these societies, food is obtained primarily from subsistence agriculture, fishing and hunting, and the diet is often high in plant foods and fish.\n\nMigration studies of indigenous populations report increasing prevalence of hypertension with urbanization, providing additional evidence for a role of environmental factors [22, 23]. With urbanization, access to processed food increases, and fresh foods that were previously readily available become less affordable. In addition to other lifestyle changes, increases in body weight, sodium intake, dietary fat, and the ratio of urinary sodium to potassium have been observed during the process of acculturation [21\u201327]. Assessment of nutrient intake in unacculturated populations may be challenging if there are language barriers and cultural differences in food intake and social norms, and also because nutrient data on commonly consumed local foods may not be available. It is imperative that studies of these groups involve members of the community to facilitate data collection and interpretation [28, 29]. Populations undergoing transition from a traditional to a Westernized lifestyle present a unique opportunity to study the etiology of hypertension. Unfortunately, these opportunities are quickly disappearing.\n\nVegetarian groups in the United States and abroad have been observed to have lower blood pressure than their non-vegetarian counterparts in many [30, 31] but not all [32] studies. The term _vegetarian_ comprises several heterogeneous groups [33], but, in general, the diet tends to be high in whole grains, beans, vegetables, and sometimes fish, dairy products, eggs, and fruit [31]. Aspects of the vegetarian diet suggested to be protective include a low intake of animal products [31], and a high potassium, magnesium, fiber, and (sometimes) calcium [33, 34] intake. Controlled intervention studies of vegetarian diet patterns report significant reductions in blood pressure in both normotensive [36] and mildly hypertensive [34] groups.\n\nA common dietary pattern among vegetarians and unacculturated groups emerges: plant foods (grains, nuts, seeds, vegetables, and sometimes fruit) provide the bulk of the diet, animal products are limited, and (in some cases) fish consumption is high. Available quantitative nutrient data suggest that these diets are moderate to high in potassium, magnesium and fiber, have a high polyunsaturated-to-saturated fat ratio, and are often\u2014but not always\u2014low in sodium. Clearly, differences in physical activity, stress, alcohol consumption, and other unmeasured factors may also contribute to a lower blood pressure in these groups, but their relative importance is not clear. These observations have nevertheless provided much of the foundation for generating hypotheses for a role of diet in modifying blood pressure.\n\n## II. INDIVIDUAL NUTRIENTS AND BLOOD PRESSURE\n\nA. Micronutrients\n\nMicronutrients associated with blood pressure tend to be highly correlated with each other because of their distribution in food sources [37], thus limiting the interpretability of observational studies. For example, foods high in magnesium (e.g., nuts) are also high in fiber and potassium, making it virtually impossible to attribute associations with blood pressure to the effects of a single nutrient. To isolate and test the effect of individual nutrients, intervention studies typically use dietary supplements. In this way, any changes in blood pressure with intervention can be attributed to the nutrient being examined. However, supplements may not be absorbed as well or have similar physiologic effects as when they are consumed in natural form. Varying levels of other components in the diet may also modify the effectiveness of supplements.\n\n#### 1. SODIUM\n\nThe potential for sodium reduction to lower blood pressure is supported by a broad range of data, and it is also clear that the association is influenced by genetics [38] and other components in the diet [39, 40]. Whether salt reduction should be broadly recommended to lower blood pressure in individuals without hypertension has been the subject of much controversy [41, 42]. Advocates propose that population-wide sodium reduction of 50\u2013100 mmol\/day (1150\u20132300 mg\/day) would substantially lower the incidence of cardiovascular disease in the general population [43, 44]. Some express concern that sodium reduction may raise vasoconstrictive hormones and lipid levels [45], and increase blood pressure in certain individuals [46, 47], while yet others have concluded that sodium reduction to recommended levels has no adverse effects [48].\n\nDietary sodium intake is not easily measured by standard dietary assessment methods, because salt added at the table and during cooking is difficult to quantify, and because processed foods vary widely in sodium content. Most often, 24-hour urine collections are used to assess daily sodium intake. Under stable conditions (e.g., adequate health, hydration, no excessive sweating), 90\u201395% of dietary sodium is excreted in the urine [49]. Wide variations in day-to-day sodium excretion within individuals will weaken the correlation of single 24-hour urinary sodium levels with blood pressure. This weakness may be minimized by collecting multiple samples in individuals or by increasing sample size in group analyses. Several investigators now employ statistical methods to correct for this source of error by using data from repeated collections in a subset of the study population [50, 51]. Improperly collected urine samples and varying geographic conditions (e.g., climate) among populations [10] can introduce additional error.\n\n##### a. Observational Studies.\n\nObservations of a direct relationship between sodium intake and blood pressure across populations support a causal role for sodium in hypertension [52, 53]. The INTERSALT study measured the relationship between 24-hour urinary sodium excretion and blood pressure in 10,079 men and women from 52 centers around the world [54]. A positive relationship between mean urinary sodium excretion by center and blood pressure was noted (Fig. 1a, solid line). When excluding data from the four isolated, traditional populations in whom other unmeasured potentially relevant factors were of particular concern, the association disappeared (dashed line). After adjusting for alcohol and body mass index (Figs. 1b and 1c), a slightly positive relationship was again noted. These investigators also reported a strong positive relationship between sodium intake and the slope of blood pressure increase with age across populations [54], suggesting a role for sodium in age-related blood pressure increase. In a recent reanalysis of the original INTERSALT data, corrected for measurement error due to use of single 24-hour urine collections, results were stronger: a 100 mmol\/day (2300 mg sodium) increase in urinary sodium was associated with an increase in systolic blood pressure of 3\u20136 mm Hg and diastolic blood pressure of 0\u20133 mm Hg [55]. In a meta-analysis of observational studies, Law _et al._ [56] reported somewhat stronger findings than INTERSALT, especially in the elderly and those with higher baseline blood pressures, but diet and other confounders were not assessed in a standard manner across studies.\n\nFIGURE 1 Relation between age and sex-adjusted 24-hour urinary sodium excretion and systolic blood pressure in 52 centers from the INTERSALT study. (a) With and without including four remote populations, (b) with additional control for alcohol intake and body mass index in 52 centers, and (c), with control for alcohol intake and body mass index in 48 centers. * _p_ < 0.05; ** _p_ < 0.01; *** _p_ < 0.001. [Reprinted with permission from Intersalt Cooperative Research Group. (1988). Intersalt: An international study of electrolyte excretion and blood pressure: Results for 24-hour urinary sodium and potassium excretion. _Br. Med. J._ **297,** 319\u2013328.]\n\nFew prospective epidemiologic studies (where sodium intake is measured prior to development of hypertension) have been published. In an analysis of National Health and Nutrition Examination Survey I (NHANES I) data, a lower sodium intake, unadjusted for energy intake, was associated with higher rates of stroke and all-cause mortality [57]. Similarly paradoxical results were reported in the Scottish Heart Health Study [58]. Limitations in dietary assessment methods, uncontrolled confounding, and a correlation of sodium intake with energy [46, 59] (with higher energy intake often representing higher physical activity) limit the interpretability of these prospective studies. In an analysis of the first NHANES study, He _et al._ [60] found that a 100-mmol increase in dietary sodium was associated with a significant increase in risk of total mortality, a 32% increase in risk of stroke, and an 89% increase in stroke mortality in overweight individuals.\n\n##### b. Intervention Studies.\n\nThe early observation of a large blood pressure reduction in patients with severe hypertension consuming the Kempner rice diet [61] is often attributed to its low sodium content (7 mmol or 150 mg\/day), though the diet was also high in fruit, low in fat and protein, and supplemented with vitamins. The many trials of sodium reduction conducted since then have varied in design, population age, blood pressure status, race and gender composition, amount and form of sodium provided, quantity and control of other nutrients in the diet, trial procedures (inpatient vs. outpatient), and in degree of adherence to the diets. Several meta-analyses of clinical trials of sodium reduction have been published in the last 10 years [43\u201345, 62]. In a meta-analysis of 28 trials published by Midgely _et al._ [62], a reduction in urinary sodium by 100 mmol\/day was associated with a 3.7\/0.9 mm Hg lower systolic and diastolic blood pressure, respectively, in individuals with hypertension and 1.0\/0.1 mm Hg blood pressure reduction in normotensives. A meta-analysis by Cutler and others [43] in 1997 of 78 published trials reported stronger results. In people with hypertension, a 100 mmol\/day reduction in sodium intake was associated with a 5.8 mm Hg systolic and 2.5 mm Hg diastolic reduction in blood pressure; for normotensives, these reductions were 2.3\/1.4 mm Hg in systolic and diastolic blood pressure. Statistical approaches used to quantify the relationship between sodium and blood pressure reduction may partly explain differing conclusions of these investigators.\n\nIn the study of Cutler _et al._ [43], the regression line was forced through zero, which assumes that nothing else changed blood pressure during intervention. The regression line was not forced through the origin in the study by Midgley _et al._ [62]; the negative intercept suggested that blood pressure was reduced even without sodium reduction. The recent meta-analysis by Graudal and others [45] included 58 trials in individuals with hypertension and 56 trials in normotensives. Despite greater mean reductions in sodium intake used to estimate the blood pressure-lowering effect (a 118 mmol sodium reduction in individuals with hypertension, and 160 mmol reduction in normotensives), their findings of a 3.9\/1.9 mm Hg and 1.2\/0.26 mm Hg blood pressure reduction, respectively, were closer to those of Midgley _et al._ [62]. Graudal and colleagues [45] also found that blood pressure regulating hormones, cholesterol and low-density lipoprotein (LDL) cholesterol levels increased on a low-salt diet, more so in studies with extreme sodium reduction.\n\nGraudal _et al._ [45] and Midgely _et al._ [62] included studies of acute sodium reduction (which stimulates blood pressure regulating hormones), while Cutler _et al._ [43] did not. Nevertheless, all meta-analyses reported a direct relationship between sodium and blood pressure, which was consistently significant in hypertensives. The estimate of the magnitude of the reduction varied, at least partly because of statistical methods used, and the choice of studies included in the meta-analyses.\n\nThe recently completed DASH-Sodium multicenter trial [7] may help to resolve many issues surrounding the sodium controversy. The study, funded by the National Heart Lung and Blood Institute, compared the effect of three sodium levels (resulting in urinary sodium excretion of 65, 107, and 142 mmol\/day) on blood pressure in those with greater than optimal blood pressure or stage 1 hypertension. The sodium intervention was provided in conjunction with a typical U.S. (control) diet or a diet pattern previously shown to lower blood pressure (DASH diet, Appendix 2). The investigators found that the lowest sodium intake (65 mmol\/day), superimposed on the already effective DASH diet, provided the most effective blood pressure lowering combination. Compared to the control diet with the highest sodium level, the DASH diet with the lowest sodium level reduced systolic blood pressure by 8.9 mm Hg in participants with hypertension and 7.1 mm Hg in normotensives. The reduction in blood pressure due to sodium was stronger in those consuming the typical American diet than the DASH diet, but was significant in both [7]. Results in the DASH-Sodium trial were seen in hypertensives as well as nonhypertensives, suggesting that a 65 mmol\/day general sodium guideline for prevention and treatment of hypertension is warranted [7].\n\nSeveral long-term, randomized clinical trials have provided evidence that moderate sodium reduction with or without weight loss reduces the incidence of hypertension and cardiovascular events, especially in overweight participants [63]. In the Trial of Nonpharmacologic Interventions in the Elderly (TONE), a 10-pound weight loss and dietary sodium reduction of 40 mmol\/day were independently associated with about a 40% reduced risk of hypertension or cardiovascular event after medication withdrawal, compared with those on standard care. In these obese subjects, the combined intervention was associated with a 53% reduced risk of hypertension or CVD event [64]. In the Trials of Hypertension Prevention (TOHP) Phase II, overweight adults who were counseled to reduce sodium achieved a 2.9\/1.6 mm Hg blood pressure reduction from sodium reduction alone, and 4.0\/2.8 mm Hg when coupled with weight loss, after 6 months. Although effects on average blood pressure declined over time with recidivism, 20% reductions in hypertension incidence were still noted after 48 months of follow-up in each intervention group [65].\n\n##### c. Salt Sensitivity.\n\nApproximately 50% of individuals with hypertension and 25% of normotensives are considered by some to be salt sensitive [66\u201368], defined arbitrarily as a mean arterial pressure reduction of at least 10 mm Hg or 10% with salt restriction. It is more common in the elderly and in African-Americans [69]. Various protocols have been employed to diagnose salt-sensitivity, including feeding low- and high-sodium diets, and a more rigorous protocol of saline infusion (to expand blood volume), followed by volume contraction with a low-sodium diet and administration of a diuretic [66]. The reasons why certain individuals respond differently to a sodium load are unclear but may be due to differences in the ability of the vascular system to adjust to a change in circulating blood volume, and to blunted sodium excretion [70]. Renal damage will also reduce the ability of the kidney to handle a sodium load. The renin-angiotensin system, activated by many factors, including sodium restriction, stimulates aldosterone secretion, which conserves total body sodium. Physiologic abnormalities, such as low renin hypertension and abnormal modulation (\"nonmodulation\") of these hormones, predispose to salt sensitivity [71]. Sodium-chloride-induced increases in blood pressure may also be enhanced by a low calcium or potassium intake [40, 72, 73]. Unfortunately, no method is available yet that can be applied clinically to diagnose salt sensitivity. Genetic markers offer promise [74], but much work remains to explain the magnitude of heterogeneity observed. Ideally, future studies will identify markers of susceptibility to aid in targeting interventions.\n\nSodium is not the only factor that affects blood pressure, and it appears to interact with body weight and levels of other nutrients in the diet. However, sodium reduction is likely to benefit most people, and ultimately the 65 mmol\/day recommendation of the DASH-Sodium investigators may prove most effective. Because sodium is ubiquitous in the U.S. food supply, large reductions in intake are not easily attainable. For example, the TOHP Phase I research group reported a mean decrease in sodium excretion of 44 mmol\/day after 18 months of intensive dietary counseling in free-living populations [75]. Only 20% of the population met the teaching goal of 60 mmol\/day, but 56% were able to reduce sodium to less than 100 mmol\/day. Counseling was least effective in men and in African-Americans [75]. Seventy-five percent of sodium intake is derived from processed foods [76], so significant reductions in sodium intake will not be easy to achieve unless sodium is reduced during processing for most foods, including staples such as bread.\n\n#### 2. POTASSIUM\n\nThe evidence for a role of potassium in lowering blood pressure is consistent across study types and is biologically plausible. Potassium may lower blood pressure through a direct vasodilatory role, alterations in the renin-angiotensin-aldosterone axis and renal sodium handling, and by natriuretic effects [77].\n\n##### a. Observational Studies.\n\nEpidemiologic observations support a role of dietary potassium in blood pressure control. Both potassium alone (inversely) and the sodium-to-potassium ratio (directly) have been associated with blood pressure in cross-cultural studies [54]. Ophir _et al._ [78] noted that urinary potassium was the strongest discriminating feature related to low blood pressure in vegetarian Seventh Day Adventists in Australia, compared to nonvegetarians; however, their descriptive study also revealed differences in other dietary and lifestyle factors among groups.\n\n##### b. Intervention Studies.\n\nIn 1997, Whelton _et al._ [79] published a meta-analysis of randomized clinical trials of potassium and blood pressure. The 33 studies included mostly individuals with hypertension, some of whom were receiving antihypertensive medication. In all studies, potassium was provided as a supplement (median 75 mmol), either superimposed on a controlled research diet or added to participants' usual diets. After excluding one outlier, systolic and diastolic blood pressure were 3.11 and 1.97 mm Hg lower, respectively, with a high potassium intake. Interestingly, greater blood pressure reductions occurred in those with progressively higher urinary sodium excretion during follow-up (measured at the end of the study). This suggests that potassium is more effective at higher levels of sodium intake. In addition, results were significantly stronger in studies that included >80% African-Americans. This meta-analysis reached qualitatively similar conclusions to those of a 1995 analysis by Cappuccio _et al._ [80], with a roughly 50% overlap in studies included.\n\nIn a large intervention trial of U.S. female nurses (Nurses Health Study II), Sacks and colleagues [81] administered supplemental potassium [40 mmol (1560 mg)], calcium [30 mmol (1200 mg)], magnesium [14 mmol (336 mg)], all three minerals, or placebo to women who reported habitually low intakes of these nutrients, for a 6-month period. Potassium, administered alone, was the only intervention that reduced blood pressure [81]. The mild, yet significant reduction occurred even though the women were nonhypertensive (116 \u00b1 8 systolic blood pressure, 73 \u00b1 6 diastolic blood pressure), and were young (mean age 39 years).\n\nConsistent findings in the epidemiologic and clinical literature support a role for potassium in the reduction and prevention of high blood pressure. Considering the markedly lower blood pressure of vegetarians and those living in remote populations compared to industrialized societies, dietary factors other than potassium are also likely to be protective. Other nutrient or non-nutrient factors in a high potassium diet may work additively or interactively with potassium to reduce blood pressure. In the DASH Study [6], the combination dietary pattern lowered blood pressure more than the fruit and vegetable intervention\u2014even though both contained equal levels of potassium and sodium.\n\n#### 3. CALCIUM\n\nAlthough a modest blood pressure lowering effect of calcium is noted in some observational studies, results from intervention trials have been inconsistent. Studies showing the greatest effect have tended to use dietary sources of calcium (e.g., dairy products), in which several dietary factors change [6]. A blood pressure lowering effect of calcium in certain subgroups (e.g., African-Americans) has been suggested.\n\n##### a. Observational Studies.\n\nThe higher calcium and magnesium content of ''hard\" water, and its inverse relation to cardiovascular mortality [82] initially sparked epidemiologic investigation into the relation of both minerals to blood pressure. Cutler and Brittain [83] reviewed 25 observational studies of calcium and hypertension, 19 of which were cross-sectional, and some of which involved repeated analyses of the same data sets. As a whole, these studies showed only modest associations between calcium and blood pressure. Because most studies used 24-hour recall methods to assess diet, random day-to-day variation may have obscured any relation with blood pressure [83]. It is also worth noting that some low blood pressure populations have minimal calcium intakes [20]\u2014more counter evidence to the link between calcium and blood pressure. Cappuccio and others [84] conducted a meta-analysis of 23 observational studies, and found negligible associations for calcium with both systolic blood pressure and diastolic blood pressure [84], though a reanalysis of this data resulted in a somewhat stronger inverse association [13].\n\n##### b. Intervention Studies.\n\nIn 1924, Addison and Clark [85] recorded the blood pressure response to repeated administration and discontinuation of oral calcium supplements in a convenience sample of hospital outpatients and inpatients. They noted that blood pressure decreased after 2 weeks of calcium supplementation and immediately rose with calcium discontinuation. It was not until much later in the century that the calcium hypothesis was tested in a more methodologically rigid fashion in a number of intervention studies.\n\nSeveral meta-analyses of calcium-intervention trials have been published since 1989 [86\u201389], all showing only a slight blood pressure reduction, primarily of systolic blood pressure, with calcium supplementation of about 1000 mg. Intervention studies using calcium from food sources have sometimes [90], but not always [91], been more effective, though these studies involve changes in several other nutrients. An interaction between calcium and sodium has been observed [39, 72, 92\u201394], such that calcium supplementation may prevent a salt-induced rise in blood pressure in susceptible individuals. Inability to either control for sodium intake, or stratify by level of sodium intake in most meta-analyses, may obscure a relationship. Although the interpretation of these studies differs [90], it seems that the modest blood pressure reduction observed with calcium supplementation does not warrant public health recommendations to increase calcium intake specifically for blood pressure control.\n\n#### 4. MAGNESIUM\n\n##### a. Observational Studies.\n\nAs with calcium, one of the early suggestions for a role of magnesium in hypertension came from reports that water hardness (increased calcium and magnesium) was associated with lower cardiovascular mortality [82], a finding recently corroborated in Taiwan [95]. Several cross-sectional [96] and prospective observational analyses [97, 98] have found high-magnesium diets to be associated with lower blood pressure. Here again, the major limitation is that high-magnesium diets tend to be high in other beneficial dietary factors as well.\n\nAdequate magnesium is required for the Na\/K-ATPase pump, which regulates intracellular calcium\u2014one of the critical determinants of vascular smooth-muscle contraction [99]. Magnesium deficiency is recognized only rarely, being seen usually in the severely malnourished, chronic alcoholics, and in association with malabsorption [99].\n\n##### b. Intervention Studies.\n\nAt least 13 randomized intervention trials of magnesium supplementation have been conducted [81, 100\u2013111]. In all but 1 of these studies, volunteers had high blood pressure, and in 5 of these trials, they were receiving antihypertensive medication (primarily diuretics). Magnesium lowered blood pressure in 5 of these studies [100, 101, 107, 108, 110]. Patients were magnesium depleted (due to diuretic treatment) in 2 of the studies [100, 101], and in 1 study an effect was only seen in those with a low baseline intake of dietary magnesium [111]. Kawano _et al._ [110] found the greatest blood pressure reduction in older men on antihypertensive medications [110]. The majority of studies, however, found no blood pressure lowering effect with magnesium supplementation.\n\n### B. Macronutrients\n\nStudies of macronutrients and blood pressure are often subject to various design limitations and thus results can be difficult to interpret. For example, when a study is designed to examine the effect of the amount of fat intake on blood pressure, alteration in fat intake under isocaloric conditions inevitably will change intake of protein and\/or carbohydrate and may change the intake of other nutrients as well. As a result, it may be difficult to attribute the effect to only the change in fat intake. In addition, the impact of macronutrients on blood pressure potentially involves the aspect of absolute quantity and the type of macronutrients. Both aspects can affect blood pressure independently, but they are not always distinguishable in research designs.\n\n#### 1. PROTEIN\n\nA high-protein diet has long been suggested to increase kidney load unfavorably and raise blood pressure [112] and may have been the underlying rationale for the Kempner rice diet treatment [61]. However, many epidemiological studies have shown an inverse relationship between dietary protein and blood pressure [113\u2013115]. A detailed review of studies investigating the relationship between dietary protein and blood pressure from 1988 to 1994 has been published [116] and very little new research has been conducted since then.\n\nOverall, high-protein intake, as indicated by urinary nitrogen or dietary protein, was found to be associated with lower diastolic and systolic blood pressures in two large cross-sectional studies in humans [114, 115]. Animal studies also demonstrated that low-protein diets increase blood pressure and stroke, while high-protein diets are protective [117]. However, most intervention trials in humans do not support findings from observational studies. Most human intervention trials use protein supplements and many do not control for other nutrients. Supplements of 7 grams of rice protein to 93 grams of meat protein for 2 weeks to 3 months did not affect blood pressure (118\u2013125). In an 8-week intervention trial, skim milk supplementation reduced blood pressure significantly among 82 normotensive participants [126]. Note that skim milk supplements provided not only additional protein, but also other nutrients, including calcium and magnesium, and both may affect blood pressure. Thus, the blood pressure responses may be caused by multiple factors including protein.\n\nDespite findings from several epidemiological studies demonstrating that blood pressure is associated with only animal protein or only vegetable protein [117, 127, 128], most intervention trials have not shown such differences [123\u2013125]. The only exception was that vegetable protein did not affect blood pressure in a group of normotensive vegetarians [122], while animal protein raised systolic blood pressure in another group of normotensive vegetarians [121]. The animal protein supplements were given as 250 g of beef (approximately 8 ounces), which contains protein, fat, and other nutrients that may all have contributed to the increases in blood pressure. On the contrary, an epidemiological study demonstrated that animal protein was inversely related to blood pressure [129].\n\nThe underlying mechanism by which dietary protein may affect blood pressure is not clear. Specific amino acids, such as arginine, tyrosine, tryptophan, methionine, and glutamate, have been suggested to affect neurotransmitters or humoral factors that affect blood pressure [116]. Soy protein was thus hypothesized to reduce blood pressure because it is rich in arginine, a potential vasodepressor and a precursor for the vasodepressor nitric oxide [130]. In a group of peri-menopausal women [131], soy protein supplementation for 6 weeks reduced diastolic blood pressure significantly. However, intakes of other nutrients including calcium, magnesium, and potassium were also increased in the soy protein group. Thus, the blood pressure response may not be attributable to the increase in arginine or soy protein alone. A protein load can increase renal blood flow [132], but the effect of habitual diets on renal blood flow, or the role of renal blood flow in hypertension is not clear [133]. More research is needed to understand the effects of various amino acids in humans, and the mechanisms underlying the relationship between dietary protein and blood pressure if such a relationship exists.\n\n#### 2. DIETARY FAT\n\nNumerous studies have investigated the relationship between dietary fat and blood pressure. However, because of discrepancies in study design, lack of adequate sample size, and other design limitations, the issue is still controversial. Both the absolute total intake of dietary fat and the relative fatty acid composition may be independently related to blood pressure control.\n\nMost observational studies have not found consistent association between total fat intake and blood pressure [134\u2013136]. However, two large European studies [135, 136] but not others [137] show a positive relationship between saturated fatty acids and blood pressure. In clinical intervention trials, lowering total fat intake from 38\u201340% to 20\u201325% of energy and\/or increasing the polyunsaturated-to-saturated fat ratio from 0.2 to 1.0 reduced blood pressures in several studies [138\u2013141], but not all [142, 143]. As mentioned earlier, any change in total fat intake often introduces changes in other dietary factors as well, so the blood pressure responses may not be attributed solely to the change in fat intake. In addition, it is important to recognize that previous trials of dietary fat have had small sample sizes, and lacked the sensitivity to detect 3\u20134 mm Hg effects.\n\nLittle is known about the relationship between dietary cholesterol and blood pressure. In the prospective analyses of the Western Electric Study, a significant positive independent relationship was found between dietary cholesterol and systolic blood pressure change during an 8-year period [128, 144]. Stamler _et al._ [114] also reported that dietary cholesterol was significantly and positively related to blood pressure among the 11,342 participants of the Multiple Risk Factor Intervention Trial. However, in a short-term intervention study, dietary cholesterol at low to moderate levels of intake was found to have no significant effects on blood pressure [145].\n\nMany short-term intervention trials have been undertaken to determine if supplementation of either fish or fish oil lowers blood pressure. Because of variations in research design, participant criteria, dosage and type of supplements, and length of intervention, the results have been inconsistent. Recently, pharmacological dosages of dexapentaenoic acid (DPA) were shown to lower blood pressure, while eicosapentaenoic acid (EPA) was not [146]. It is suggested that the blood pressure lowering effect of fish oil may be strongest in individuals with hypertension and in those with clinical atherosclerotic disease or hypercholesterolemia [147]. In another study, both fish oil and corn oil supplements were shown to reduce blood pressure to similar degrees among elderly hypertensives [148]. Long-term studies are required to confirm if fish oil can lower blood pressure. Until such information is available, it is more advisable to encourage greater fish consumption as part of a healthy diet than taking fish oil supplementation. This is in agreement with the recommendation of the DASH dietary pattern (see later section) to prevent and treat high blood pressure.\n\nA high monounsaturated fatty acid (MUFA) Mediterranean type of diet is also suggested to lower blood pressure mainly based on observational studies. However, little research has been conducted investigating the specific effects of MUFA on blood pressure. A small-scale clinical trial found that a high-MUFA diet (17% of energy) lowered daily antihypertensive medications as compared to a high polyunsaturated fatty acid (PUFA) diet [149]. Another intervention study showed that blood pressure was significantly reduced when 16 persons with diabetes were provided olive oil [150]. Other studies, nevertheless, did not find an effect of a high-MUFA diet on blood pressure [140, 151]. It is important to note that most of these trials of dietary fats and blood pressure have had a small sample size, and lacked sensitivity to detect even 3\u20134 mm Hg effects in systolic blood pressure. It has been shown that a MUFA-enriched diet produced changes in erythrocyte membrane composition in participants with hypertension [152]. It was hypothesized that the changes in membrane cholesterol modifies the fluidity and the transmembrane fluxes of Na% and K%, which may then indirectly affect blood pressure [153].\n\nIt is also possible that fatty acids such as the n-6 and n-3 series may affect blood pressure through the prostaglandin (PG) pathway [154]. Animal studies have shown that n-6 fatty acids increased the tissue and circulating levels of PGs, PGI2, and PGE2, all of which may act as vasodilators [155, 156]. Little research is available in humans examining how fat intake may affect blood pressure through this pathway.\n\nIn summary, it is still unclear how fat intake and various fatty acids affect blood pressure and if an interaction exists among these factors. Even though a majority of interventions demonstrate that lowering fat intake reduces blood pressure, the effect may not be attributed to fat alone.\n\n#### 3. CARBOHYDRATES\n\nVery few studies have been designed specifically to investigate the impact of the quantity of carbohydrate intake on blood pressure. Nevertheless, studies of the relationship between fat intake and blood pressure often alter intakes of fat and carbohydrate while keeping protein intake constant. Thus, interpretation of the effect of fat intake on blood pressure is potentially confounded by the effects of carbohydrate intake. An impact of the type of carbohydrate on blood pressure, on the other hand, has been examined in a limited number of studies. In an early study using Wistar rats, Ahrens _et al._ [157] showed that increasing the proportion of sucrose in the diet to 10\u201315% of energy for 14 weeks increased blood pressure significantly. These sucrose-fed rats also retained more sodium than the controls, which may have contributed to the blood pressure responses. In addition, the blood pressure of these rats returned to baseline levels when sucrose was replaced with maltose. Other animal studies [158, 159] also showed similar hypertensive effects of sucrose.\n\nResults from human studies have not been consistent. In one study [160], systolic blood pressure rose significantly 1 hour after ingestion of a sucrose solution. However, in an earlier study of patients with coronary artery disease, both systolic and diastolic blood pressures decreased after 4 days of a sucrose load at 4 g\/kg\/day [161]. In another study that was designed to examine the metabolic effect of sucrose in a group of overweight women, blood pressure levels were not changed after consuming a hypocaloric diet with sucrose as the main source of carbohydrate for 6 weeks [162]. These results are confounded by the concomitant weight reduction diet discussed in the next section.\n\nIn the study by Palumbo _et al._ [161], fructose loading in these patients also reduced blood pressures significantly, but glucose loading had no effect. Similar hypotensive effects of fructose were observed in other studies [163, 164], yet oral glucose loading was found to be either hypertensive [160, 163] or hypotensive [165]. Such inconsistencies in the study of various simple carbohydrates on blood pressure may be due to the differences in study design, study population, inadequate sample size, or length of feeding. More research is needed to clarify the effects of various carbohydrates on blood pressure using well-controlled designs under longterm situations.\n\n#### 4. FIBER\n\n##### a. Observational Studies.\n\nBoth cross-sectional [37, 166, 167] and prospective analyses [97] have demonstrated inverse associations of fiber with blood pressure, but have also noted the high correlation of fiber with other nutrients. In a prospective cohort analysis, Witteman and colleagues [98] found that the protective association with fiber was reduced when magnesium and calcium were used as adjustment factors in the statistical models. Conversely, in the study of Ascherio _et al._ [97], only dietary fiber remained significantly and inversely related to hypertension when the association was adjusted for other nutrients. Different sources of fiber, including fruit fiber [97] and cereal fiber [166], have been associated with blood pressure reduction in prospective studies.\n\n##### b. Intervention Studies.\n\nSeveral intervention studies have examined the effect of fiber on blood pressure, with most adding cereal fiber to the diet, and are reviewed by He and Whelton [166]. With an average supplementation of 14 g fiber, systolic and diastolic blood pressure is reduced by about 1.6\/2.0 mm Hg [166]. In some studies, fiber has been provided as a combination of soluble and insoluble fibers. Only soluble fiber influences gastrointestinal function and, indirectly, insulin metabolism, a possible mechanism by which fiber may lower blood pressure [166]. The weak effects of fiber in these studies may also be due to small study size, and because many were conducted in young, normotensive individuals, in whom large changes in blood pressure are more difficult to detect. Further studies with adequate power to detect smaller differences in blood pressure are needed to clarify the role of fiber.\n\n## III. OTHER DIETARY AND LIFESTYLE MODIFICATIONS\n\nA. Weight Reduction\n\n##### a. Observational Studies.\n\nObservational studies report a positive relationship between several indices of body weight or body fatness and high blood pressure [54, 168\u2013172]. Mechanisms that may be involved in weight loss include suppression of sympathetic nervous system activity, lowered insulin resistance, normalization of blood pressure regulating hormones [173], decreased body sodium stores, decreased blood volume and cardiac output, and higher salt sensitivity in overweight individuals [60, 174, 175].\n\n##### b. Intervention Studies.\n\nSeveral intervention trials of weight reduction in those with high-normal blood pressure [176\u2013178], often combined with dietary sodium reduction [64, 65], report lower blood pressures with sustained weight reduction of about 3\u20137 kg. Of several nonpharmacologic interventions examined in the Trials of Hypertension Prevention Phase I [179], weight loss was found to be the most successful at lowering blood pressure. At the 6-month follow-up visit, men and women in the intervention group had lost 6.5 and 3.7 kg, respectively. This level of weight loss was achieved with a fairly rigorous counseling approach aimed at simultaneously reducing energy intake and increasing exercise [178]. At study termination, blood pressure fell an average of 2.9\/2.3 mm Hg overall (after subtracting the blood pressure change in the control group) [178]. In this study, some recidivism occurred, and at 18 months, men had maintained a 4.7-kg reduction, and women, 1.6 kg. After a 7-year follow-up in a subset of study participants, the odds of developing hypertension were reduced by 77% in the weight loss group [180], even though their long-term weight loss was nearly identical to that of the control group (4.9 and 4.5 kg, respectively). This raises the intriguing possibility of long-term effects from previous interventions and deserves further study. Additionally, significant weight loss (5%) is suggested to reduce the need for antihypertensive medication [173].\n\n##### c. Summary.\n\nThe high prevalence of overweight and obesity in U.S. adults (54.9%), defined as a body mass index >25 and 30 kg\/m2, respectively [181], is likely to contribute importantly to hypertension, as well as to other chronic diseases and their risk factors. The relative contributions of lower energy intake and exercise [182] in affecting blood pressure change is not completely understood. Weight loss is an effective method to reduce blood pressure but is difficult to implement and maintain [183], particularly among African-Americans, who have a higher prevalence of high blood pressure.\n\n### B. Alcohol\n\n##### a. Observational Studies.\n\nExcessive alcohol consumption is associated with higher blood pressure and higher prevalence of hypertension in observational studies [184]. Men who consume \u22653\u20135 drinks\/day [185], and women who consume 2\u20133 drinks\/day [186] may be at particularly higher risk, but levels below this are not associated with increased risk. The relation of alcohol type to risk is inconsistent, and chronic, habitual intake may be more related to blood pressure than recent intake [185]. In one study, men who reduced their alcohol consumption over a 20-year period experienced less age-related increase in blood pressure than those who did not [187].\n\n##### b. Intervention Studies.\n\nThe relatively few intervention studies of alcohol and blood pressure have tended to be small and of short duration, and are reviewed by Cushman _et al._ (188). In 9 of 10 studies examined, systolic blood pressure was significantly reduced after a reduction of 1\u20136 alcoholic beverages per day [188]. The Prevention and Treatment of Hypertension Study (PATHS) was designed to evaluate the long-term blood pressure lowering effect of reducing alcohol consumption in nondependent moderate drinkers (those who consumed >3 alcohol-containing drinks\/day) [189]. The goal of intervention in this study was either 2 or fewer drinks daily or a 50% reduction in intake (whichever was less). After 6 months, the intervention group experienced an insignificant (1.2\/0.7 mm Hg) reduction in blood pressure compared with the control group, and among individuals with hypertension, this reduction was even more modest. In this study, the intervention group reduced their intake by 2 alcoholic drinks per day, but the control group also lowered their alcohol intake during intervention, so that the difference in intake between the groups was 1.3 drinks\/day. Perhaps a greater reduction is necessary to see a stronger effect. However, this level of reduction appears realistic in moderate alcohol drinkers, and is similar to the absolute reduction achieved in an earlier study [190]. In two studies, 6\u201318 weeks in duration, replacing alcohol with nonalcoholic substitutes resulted in greater reductions [191, 192], suggesting that certain behavioral approaches may promote adherence. In one of these studies, alcohol cessation (from 5 drinks to 0) and energy restriction (mean 9. 6 kg weight loss) combined showed additive effects, and resulted in a reduction in systolic and diastolic blood pressure of 14 and 7 mm Hg, respectively [192].\n\nThe exact mechanism for an alcohol\u2013blood pressure association is not clear, but possibilities include a stimulation of the sympathetic nervous system, inhibition of vascular relaxing substances, calcium or magnesium depletion, and increased intracellular calcium in vascular smooth muscle [184, 188]. Variations in study design preclude arriving at a specific cutoff for benefit, but limiting alcohol consumption to two or fewer drinks per day may improve blood pressure control in heavy drinkers. Moderate alcohol consumption also has well-known benefits for overall cardiovascular disease risk [193, 194].\n\n## IV. DIETARY PATTERNS\n\nAs previously noted, lower blood pressure is often observed among populations who consume a vegetarian-like diet. However, studies on individual nutrients as outlined in previous sections of this chapter, have shown inconsistent results. Explanations for such inconsistencies may include these: (1) The effect of individual nutrients may be too small to be detected, particularly when trials do not contain sufficient sample size and thus statistical power; (2) most intervention studies employed supplements of nutrients, which may function differently from naturally occurring nutrients in foods; (3) other dietary factors naturally occurring in foods that are not hypothesized to affect blood pressure may also have an impact on blood pressure; and (4) nutrients occurring in foods simultaneously may exert synergistic effects on blood pressure. Thus, the DASH multicenter trial was designed to test the impact of whole dietary patterns on blood pressure while controlling for multiple nutrients and dietary factors simultaneously [195, 196].\n\n### A. The Dietary Approaches to Stop Hypertension Trial\n\nThree dietary patterns varying in amounts of fruits, vegetables, and dairy products were developed for the DASH trial [197]. The first dietary pattern was a control diet that mimicked the typical American diet, and contained fewer fruits, vegetables, and dairy products, and was high in total and saturated fats, cholesterol, and low in dietary fiber, calcium, potassium, and magnesium (Table 2). The ''fruits and vegetables\" dietary pattern had more fruits and vegetables, and thus higher levels of dietary fiber, potassium, and magnesium as compared to the control diet. The DASH ''combination\" dietary pattern (also referred to as the DASH diet) emphasizes fruits, vegetables, and low-fat dairy products, includes whole grains, poultry, fish, and nuts, and is reduced in fats, red meat, sweets, and sugar-containing beverages. The DASH diet had reduced amounts of total and saturated fat and cholesterol, and increased amounts of potassium, calcium, magnesium, dietary fiber, and protein. Sodium intake, body weight, and alcohol consumption were kept constant throughout the intervention.\n\nTABLE 2\n\nNutrient Targets, Menu Analysis, and Average Daily Servings of Foods, According to Intervention Diet in DASH _a_\n\naValues are for diets designed to provide an energy level of 2100 kcal.\n\nbValues are the results of chemical analyses of the menus prepared during the validation phase and during the trial. NA denotes not available.\n\n_Source:_ Reprinted with permission from Appel, L. J., Moore, T. J., Obarzanek, E., _et al._ (1997). A clinical trial of the effects of dietary patterns on blood pressure. _N. Engl. J. Med._ **336,** 1117\u20131124 [6].\n\nA total of 459 participants consumed the control diet for the first 3 weeks, and then were randomly assigned to one of the three dietary patterns for an additional 8 weeks. The DASH diet reduced blood pressure by 5.5\/3.0 mm Hg (systolic\/diastolic) more than the control group (both systolic blood pressure and diastolic blood pressure _p <_ 0.001) [6]. The fruits and vegetables diet reduced blood pressures by 2.8\/1.1 mm Hg more than the control diet ( _p <_ 0.001 and _p =_ 0.07). The reductions in blood pressures were significant after participants consumed the diets for 2 weeks and were sustained for the following 6 weeks (Fig. 2). In addition, blood pressure lowering was similarly effective in men and women, younger and older persons, and particularly effective among minorities and those who had high blood pressure. These reductions occurred while body weight, sodium intake, alcohol consumption, and exercise patterns remained stable. Among the 133 participants with hypertension (systolic blood pressure \u2265140 mm Hg; diastolic blood pressure \u226590 mm Hg; or both), the combination diet lowered systolic and diastolic blood pressure by 11.4 and 5.5 mm Hg, respectively. These effects in people with hypertension are similar to reductions seen with single drug therapy.\n\nFIGURE 2 Mean systolic and diastolic blood pressures at baseline and during each intervention week, according to diet, for participants in the DASH study. [Reprinted with permission from Ref. [6], Appel, L. J., Moore, T. J., Obarzanek, E., _et al._ (1997). A clinical trial of the effects of dietary patterns on blood pressure. _N. Engl. J. Med._ **336,** 1117\u20131124.]\n\nEven though the DASH trial was not designed to identify specific nutrient(s) responsible for the blood pressure lowering effect, data from the fruits and vegetables group support the hypothesis that increasing potassium, magnesium, and dietary fiber intake reduces blood pressure. In addition, by further lowering total and saturated fat and cholesterol, and increasing low-fat dairy products in the DASH diet, blood pressure reduction was nearly doubled. Because whole-food items rather than single nutrients were manipulated in this trial, other nutrients that were not controlled for in the study, or other beneficial factors as yet unrecognized, may also have contributed to the blood pressure responses. Further research is needed to analyze the specific nutrients or factors responsible for the effect. The nutrient profile of the DASH diet is included in Table 2. Further details on DASH can be found in Appendix 2 and on the following web site: http:\/\/dash.bwh.harvard.edu.\n\n## V. SUMMARY\n\nThe evidence that diet modification can prevent and lower blood pressure is strong, and recommendations are summarized in Table 3. In some cases, the effective intervention strategy and mechanisms involved are still being clarified. Due to various design limitations, inadequate statistical power and measurement issues, studies of single nutrients have generally been inconsistent, except for potassium. However, when multiple nutrients or dietary factors are combined in the same intervention strategy as seen in the DASH study, blood pressure was significantly and effectively reduced. Nutrients may have additive or interactive effects when provided together in the diet. In addition to the DASH pattern, important dietary factors include sodium reduction, weight loss, and avoidance of excessive alcohol. Concurrent adherence to several recommendations is likely to hold the greatest promise for preventing and lowering blood pressure. In addition to addressing unresolved nutritional hypotheses, future research should focus on methods to motivate and maintain dietary changes for blood pressure control. At both the population and individual levels, success in dietary intervention relies on multiple levels of support ranging from clinicians to government agencies to private institutes and industries. In particular, partnering with industry to improve the nutritional value of the food supply, such as reducing the sodium and fat content of processed foods, plays a critical role in implementing dietary changes. Consistent efforts to educate and promote adherence to nutritional guidelines by dietetic and other health care professionals are also instrumental to the prevention and management of hypertension.\n\nTABLE 3\n\nSummary of Evidence Relating Dietary Factors with Blood Pressure\n\na\\+ + +, strong; ++, somewhat consistent and\/or likely to benefit certain subgroups; +, data suggestive; \u2013, inconclusive.\n\nAPPENDIX 1\n\nClassification of Blood Pressure in Adults Age 18 and Older _a_\n\naNot taking antihypertensive drugs and not acutely ill. When systolic and diastolic blood pressures fall into different categories, the higher category should be selected to classify the individual's blood pressure status. For example, 160\/92 mm Hg should be classified as stage 2 hypertension, and 174\/120 mm Hg should be classified as stage 3 hypertension. Isolated systolic hypertension is defined as systolic blood pressure of 140 mm Hg or greater and diastolic blood pressure below 90 mm Hg and staged appropriately (e.g., 170\/82 mm Hg is defined as stage 2 isolated systolic hypertension).\n\nbOptimal blood pressure with respect to cardiovascular risk is below 120\/80 mm Hg. However, unusually low readings should be evaluated for clinical significance.\n\ncBased on the average of two or more readings taken at each of two or more visits after an initial screening.\n\n_Source:_ The Sixth Report of the Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure, National Institutes of Health, NHLBI, NIH Publication No. 98\u20134080, November, 1997.\n\nAPPENDIX 2 National Institutes of Health\n\nThe DASH Diet\n\nThis eating plan is from the ''Dietary Approaches to Stop Hypertension\" (DASH) clinical study. The research was funded by the National Heart, Lung, and Blood Institute (NHLBI), with additional support by the National Center for Research Resources and the Office of Research on Minority Health, all units of the National Institutes of Health. The final results of the DASH study appear in the April 17, 1997, issue of the _New England Journal of Medicine._ The results show that the DASH ''combination diet\" lowered blood pressure and, so, may help prevent and control high blood pressure.\n\nThe ''combination diet\" is rich in fruits, vegetables, and low-fat dairy foods and low in saturated and total fat. It also is low in cholesterol; high in dietary fiber, potassium, calcium, and magnesium; and moderately high in protein.\n\nThe DASH eating plan shown below is based on 2,000 calories a day. Depending on your caloric needs, your number of daily servings in a food group may vary from those listed.\n\nThe DASH Diet \u2022 Sample Menu \u2022 based on 2,000 calories\/day\n\nTo learn more about high blood pressure, call 1-800-575-WELL or visit the NHLBIWeb site at . DASH is also online at .\n\n_Source:_ The Sixth Report of the Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure, National Institutes of Health, NHLBI, NIH Publication No. 98-4080, November, 1997.\n\n Start small. Make gradual changes in your eating habits.\n\n Center your meal around carbohydrates, such as pasta, rice, beans, or vegetables\n\n Treat meat as one part of the whole meal, instead of the focus.\n\n Use fruits or low-fat, low-calorie foods such as sugar-free gelatin for desserts and snacks.\n\nREMEMBER! 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Alcohol, hypertension and cardiovascular disease. _J. Hypertens_. 1995;13:939\u2013942.\n\n194. Rimm E.B., Giovannucci E.L., Willett W.C., Colditz G.A., Ascherio A., Rosner B., Stampfer M.J. A prospective study of alcohol consumption and the risk of coronary disease in men. _Lancet_. 1991;338:464\u2013468.\n\n195. Sacks F., Obarzanek E., Windhauser M., Svetkey L.P., Vollmer W.M., McCullough M., Karanja N.M., Lin P-H., Steele P., Proschan M.A., Evans M.A., Appel L.J., Bray G.A., Vogt T.M., Moore T.J. Rationale and design of the Dietary Approaches to Stop Hypertension trial. _Ann. Epidemiol_. 1995;5:108\u2013118.\n\n196. (Suppl.)Vogt T., Appel L., Moore T., Obarzanek E., Vollmer W.M., Svetkey L.P., Sacks F.M., Bray G.A., Cutler J.A., Windhauser M.M., Lin P-H., Karanja N.M. Dietary Approaches to Stop Hypertension: Rationale, design, and methods. _J. Am. Diet. Assoc_. 1999;99:S12\u2013S18.\n\n197. (Suppl.)Karanja N., Obarzanek E., Lin P.-H., McCullough M.L., Phillips K.M., Swain J.F., Champagne C.M., Hoben K.P. Descriptive characteristics of the dietary patterns used in the Dietary Approaches to Stop Hypertension trial. _J. Am. Diet. Assoc_. 1999;99:S19\u2013S27.\n\n* * *\n\n1The operational definition of hypertension is a systolic blood pressure of 140 mm Hg or greater, a diastolic blood pressure of 90 mm Hg or greater, or current use of an antihypertensive medication [1]. Finer classifications of high blood pressure, used to guide treatment, are included in Appendix 1.\nCHAPTER 21\n\nNutrition and Congestive Heart Failure\n\nSUZANNE LUTTON1 and NANCY ANZLOVAR, Cleveland Clinic Foundation, Cleveland, Ohio\n\n## I. INTRODUCTION\n\nMore than 4.8 million Americans are afflicted with congestive heart failure and another 555,000 new cases are diagnosed annually [1]. As the population ages and with improved treatment of other cardiovascular disorders such as myocardial infarction, hypertension, and valvular heart disease, the prevalence of heart failure is anticipated to increase even more. Congestive heart failure (CHF) is actually a constellation of signs and symptoms resulting from impairment of systolic and\/or diastolic functioning of the myocardium. Patients with CHF experience difficulties such as shortness of breath, chest discomfort, limitations of exercise capacity, peripheral edema, anorexia, and become fatigued easily.\n\nMany individuals incorrectly interchange the terms _congestive heart failure_ and _cardiomyopathy._ Cardiomyopathies are diseases of the myocardium that are associated with cardiac dysfunction and result in the syndrome of congestive heart failure. Cardiomyopathies are divided into several types as defined by the World Health Organization [2] and include the broad categories of dilated, restrictive, hypertrophic, right ventricular, specific, or unclassified. When a patient presents with CHF, it is important to try to identify the underlying cause in order to direct therapy and potentially reverse the disease process. For instance, ventricular dysfunction can be the result of hypertensive cardiomyopathy and aggressive blood pressure lowering can reduce symptoms, diminish left ventricular hypertrophy, and frequently improve the ejection fraction. The symptoms of CHF are often divided into New York Heart Association functional classes (see Table 1). Functional class I is defined as being asymptomatic, even with exertion. Class II patients develop difficulties with moderate exertion such as shortness of breath with walking up hill or climbing stairs. Class III patients have symptoms with mild exertion such as performing the activities of daily living like showering or dressing. Functional Class IV implies symptoms of CHF while at rest.\n\nTABLE 1\n\nNew York Heart Association Functional Classes\n\nFunctional class | Description \n---|--- \nClass I | Asymptomatic; even with heavy exertion \nClass II | Slight limitations with exertion; no symptoms at rest \nClass III | Marked limitations with exertion; no symptoms at rest \nClass IV | Symptoms at rest\n\n## II. PATHOPHYSIOLOGY OF HEART FAILURE\n\nThe cause of the initial insult to the individual myocyte and myocardium as a whole can vary widely from myocardial infarction to exposure to a toxin such as adriamycin. However, once the injury has occurred, regardless of cause, the heart and cardiovascular system can only respond in a limited number of ways. A decrease in cardiac output from ventricular dysfunction leads to hypoperfusion of vital organs such as the kidneys, with the body inappropriately perceiving a hypovolemic state. This results in activation of the sympathetic nervous, renin-angiotensin-aldosterone, and arginine-vasopressin systems. Consequently, many neurohormones are increased in heart failure, including aldosterone, angiotensin II, atrial natriuretic factor, endothelin, epinephrine, growth hormone, norepinephrine, prostaglandins, renin, tumor necrosis factor-alpha and vasopressin.\n\nAngiotensin II has numerous effects aimed at protecting against hypovolemia and hypotension. Its primary actions are vasoconstriction of the systemic and renal arterioles and stimulation of the production of the potent mineralocorti-coid aldosterone by the adrenal glands. Aldosterone and angiotensin II increase sodium reabsorption in the proximal and distal tubules of the kidney. Vasopressin release is also triggered by the changes in sodium and water homeostasis, which further exerts antidiuretic and direct, systemic vasoconstrictor effects. The sympathetic nervous system induces systemic vasoconstriction, stimulates additional renin release, and directly and indirectly increases sodium reabsorption in the proximal tubule of the kidney [3].\n\nAlthough many of these neurohormones are beneficial in acute settings such as cardiogenic shock, in chronic conditions they have detrimental effects on the myocardium and play a significant role in the progressive decline of ventricular function [4, 5]. The chronic actions of all of these neurohormones result in worsening hemodynamics, progressive ventricular dilation and unfavorable remodeling, myocardial fibrosis, and increased morbidity and mortality.\n\n## III. STANDARD MEDICAL CARE FOR HEART FAILURE\n\nThe main goal in the management and treatment of heart failure is to reduce morbidity and mortality. Heart failure accounts for more than 1 million hospital discharges annually and a 3-month readmission rate of 20\u201350%. It is essential to try to identify the cause of the cardiomyopathy and any factors that may have contributed to an episode of decompensation. Numerous outstanding articles and guidelines have been previously published that review the standard approach to and medications for the treatment of both acute and chronic heart failure [6\u201311]. With a better understanding of the pathophysiology of heart failure, including new information from the molecular and cellular levels, the approach to its treatment has evolved dramatically during the last few decades.\n\nCurrently, the treatment of CHF involves quadruple therapy, of angiotensin-converting enzyme inhibitors, beta blockers, digoxin, and diuretics as needed. The first aim of therapy for CHF is to relieve the symptoms of vital organ congestion and improve hemodynamics. Diuretics are the mainstay of this therapy. Although diuretics work very well to relieve volume overload and congestion, no studies have demonstrated that diuretics actually improve survival. In fact, there is evidence to suggest that diuretics can be toxic to the myocardium over time through activation of the renin-angiotensin-aldosterone and sympathetic nervous systems. The current recommendations are to use diuretics as needed for symptoms of fluid overload, such as peripheral edema, weight gain, abdominal bloating, increasing cough, or worsening shortness of breath. Once symptomatic improvement occurs and a euvolumic state is achieved, often the diuretic can be decreased or used only on an intermittent basis. Angiotensin-converting enzyme inhibitors are the cornerstone to the long-term treatment of heart failure. They act by preventing the conversion of angiotensin I to angiotensin II and have both hemodynamic and neurohormonal actions.\n\nLarge clinical trials such as SOLVD (Studies of Left Ventricular Dysfunction), CONSENSUS (Cooperative North Scandinavian Enalapril Survival Study), SAVE (Survival and Ventricular Enlargement), and V-HeFT-II (Veterans Administration Cooperative Vasodilator Heart Failure Trial II) have provided evidence that angiotensin-converting enzyme inhibitors offer relief from heart failure symptoms and, most importantly, reduce morbidity and mortality [12\u201315]. Digoxin has been investigated recently in several trials [16\u201318]. This medication promotes an increase in contractility and also increases baroreceptor sensitivity. It is useful in patients with severe CHF to decrease morbidity, although it does not improve mortality.\n\nOne of the newer directions in the treatment of heart failure involves the use of beta-adrenergic receptor blockers to suppress the neurohormonal substances described above in an attempt to improve long-term outcome. Beta-blockers, once contraindicated in heart failure, are now one of the standard medications following their demonstration of safety and improved mortality [19\u201325]. Aldosterone antagonists, endothelial antagonists, TNF-\u03b1 antagonists, and several other drug classes are currently being used or are under investigation for the treatment of CHF. In the future, therapy may be directed at altering the cardiovascular system at the genetic and cellular levels before heart failure ever develops.\n\nAnother key step in the management of CHF involves modifying any other risk factors for cardiovascular disease. Some of the most important risk factors to control include diabetes, obesity, hypertension, tobacco use, and hyperlipidemia. These risk factors for cardiovascular disease further adversely affect patients already afflicted with CHF. For example, obesity increases systemic vascular resistance, thus increasing the workload of an already failing heart. Weight loss decreases blood pressure, improves lipid and glucose metabolism, and promotes regression of left ventricular hypertrophy [26], all of which improve CHF. These areas are covered in detail in the other chapters on cardiovascular diseases and also under disease-specific interventions in this book. In addition to following standard medical therapy, patients with CHF are encouraged to make lifestyle modifications regarding regular exercise programs and stress management and to follow dietary guidelines. Because of the sodium and water retention present in heart failure, the most important dietary modification is restriction of sodium and water intake.\n\n## IV. RESTRICTIONS IN SODIUM\n\nAs described above, acute and chronic heart failure results in the activation of the renin-angiotensin-aldosterone, sympathetic nervous, and arginine-vasopressin systems. The kidneys respond to these neurohormones by inappropriately retaining too much sodium and fluid. This leads to further decompensation. Although the measured serum sodium level is often normal and sometimes low, the total body store of sodium is markedly elevated. This is a difficult concept for most to understand. A low serum sodium level is an independent predictor of increased mortality. A common response of patients and healthcare providers to a low serum sodium level, unfortunately, is that they may try to increase the level by increasing sodium intake, which only results in further fluid retention. It is essential in CHF to restrict both sodium and water to treat congestion, maintain compensation, and avoid electrolyte derangements.\n\nMost Americans already consume over three times the daily requirement of sodium, sometimes taking in as much as 10 g\/day. (One teaspoon is the equivalent of 2.3 g.) In those with mild heart failure, sodium consumption should be limited to 3.0 g a day. Those with moderate to severe heart failure are advised to limit sodium even further to 2.0 g a day. On occasion, patients floridly decompensated may need to temporarily decrease the sodium intake to 0.5\u20131.0 g\/day, although this becomes very difficult to do. The first step in decreasing sodium intake for patients is to completely discontinue use of added salt. To flavor food, a variety of salt substitutes and salt-free seasonings are available on the market. Many of the salt substitutes are high in potassium, which may or may not be desirable for an individual patient. This is usually dependent on the use of other heart failure medications (potassium-wasting vs. potassium-sparing diuretics) and renal function. Patients should consult with their physician regarding use of potassium-containing salt substitutes. Patients should completely avoid high-salt foods (see Table 2). Obvious foods to be avoided are most processed foods, smoked or cured meats, vegetables pickled in brine, tomato sauces, and canned soups, unless they are specifically labeled as containing low or no sodium. The vast majority of fast foods should be eliminated because both the sodium and fat content far exceed recommended intakes.\n\nTABLE 2\n\nHigh-Salt Foods _a_\n\nBeverages\u2014soft drinks, tomato juice\n\nCanned foods and soups, bouillon cubes\n\nCheeses (some)\u2014cottage cheese, American cheese\n\nCondiments and sauces\u2014barbeque sauce, olives, relish, soy sauce, catsup\n\nFast foods and many ethnic foods\n\nFrozen main dishes\n\nPickled vegetables\n\nShellfish\n\nSmoked, cured, processed meats\u2014hotdogs, ham, bacon, sausage, corned beef, bologna\n\nSnack foods\u2014salted nuts, potato chips, salted popcorn, pork rinds\n\naUnless product is specifically labeled lowsodium or no sodium.\n\nLabel reading is one of the most important means of helping to control sodium intake. The patient and family need to be aware of hidden salts that are used in the preparation, processing, and packaging of foods (see Table 3). Additionally, many medications contain sodium (see Table 4). Generally acceptable foods are those in which the sodium content is less than 0.14 g per serving. Many tables, charts, and books are available to guide patients in food selections.\n\nTABLE 3\n\nHidden Salts in Foods\n\nTABLE 4\n\nSodium in Medications\n\nAlkalizers\u2014sodium citrate\n\nAntacids\u2014sodium bicarbonate\n\nAntibiotics\u2014certain IV forms\n\nExchange resins\u2014sodium polystyrene sulfonate\n\nLaxatives\u2014sodium phosphate, sodium sulfate\n\nWhen patients present with congestion, it is quite common to discover that they have markedly exceeded their sodium intake limits. Routinely obtaining a history of what was consumed in the preceding 24\u201348 hours can be quite revealing and educational. Often patients think they are following a low-salt diet, yet, unknowingly, their food selections are rather high in salt content. Frequently, patients with mild peripheral edema can be adequately treated with vigilant salt restriction, and thus avoid diuretic therapy.\n\nA common complaint of patients with heart failure is that they are no longer obtaining a brisk response to their diuretic treatment. This may reflect excess sodium intake. Unfortunately, the first response of many practitioners is to simply increase the dosage or change to a different diuretic brand or class. In reality, many cases of apparent ''diuretic resistance\" are actually due to the fact that the patient has been indiscriminate in sodium consumption. Careful diet control will usually return the urine output to the expected levels.\n\nThis apparent resistance can be explained as follows. When a diuretic is first administered and reaches a therapeutic level in the system, naturiesis occurs. However, once the drug level falls out of the effective range, a rebound in sodium resorbed can occur. The amount of sodium reabsorbed by the kidney during this time and until the next dose of diuretic is given can negate the previous losses. Thus, the net sodium loss may be minimal or zero through the entire dosing interval [27]. To maximize the diuretic effects, it is essential that sodium intake be restricted to prevent the kidneys from compensating for the initial losses.\n\n## V. RESTRICTIONS IN FLUID INTAKE\n\nThe pathophysiology of CHF, as described above, results in chronic fluid and sodium retention. Patients may range from having minimal or no congestion to a tremendous amount of fluid excess. As mentioned earlier, symptoms of fluid overload can include complaints of leg and\/or hand swelling, weight gain, abdominal bloating or distention, nausea, early satiety, cough, dyspnea with exertion or at rest, orthopnea, and paroxysmal nocturnal dyspnea. By the time peripheral edema is present and detectable, the patient usually has at least 5 kg of excess fluid. The standard recommendation is to limit total liquid intake to 2 liters (2000 cc) a day or about 64 ounces. Patients who are severely decompensated may require further restrictions to 1000\u20131500 cc\/day. Restricting below this level can be very uncomfortable for patients because of unrelenting thirst, and also increases the risk of kidney failure. Many patients have the incorrect belief that they must consume large quantities of fluid in order to keep the kidneys functioning normally, prevent infections, and help the body ''flush out\" toxins. Education about not deliberately drinking fluids and providing general guidelines about fluid restriction are some of the first steps in preventing fluid excess and correcting volume overloaded conditions.\n\nFluid restriction is also of particular importance in the management of hypervolemic hyponatremia. Another common cause for why a patient's diuretic ''is not working,\" besides consuming too much sodium, is that patients will drink as much (or more) volume than they urinate. Total daily input must be less than daily output in order to produce a net diuresis. Excessive fluid consumption can increase the risk for severe electrolyte abnormalities, especially in the setting where a diuretic is being used.\n\nWeight monitoring is very important in the management of congestive heart failure and should be performed after the first morning elimination dressed in a minimal amount of clothing. Patient education focuses on recording body weight and knowledge that a weight increase of more than 2\u20133 pounds in a day or 5 pounds in a week usually signals fluid retention. Early excess fluid retention frequently can be detected by the scale before edema is physically noted. On these days, patients are encouraged to carefully maintain their prescribed fluid and sodium restriction and even decrease total fluid intake by 1\u20131.5 cups. Additional diuretics may be necessary in order to reestablish the dry weight if this intervention is unsuccessful. It should be stressed that diuretic therapy alone is insufficient to control and\/or prevent excess fluid accumulation.\n\nPatients are encouraged to ''visualize\" their fluid consumption by using an empty 2-liter bottle. Each time they take a drink, the corresponding amount of fluid is to be placed into the bottle. This enables one to gauge total volume in relation to various household containers. A common misconception is that water is the only fluid that matters. Patients should be reminded that fluid consumption includes foods that become liquid at room temperature, such as ice cream, yogurt, and gelatin desserts. Ice chips or crushed ice are an alternatives to drinking a large glass of water. To accurately assess volume in relation to total fluid allotment, ice chips should be allowed to melt at room temperature. Psychologically, this may have some benefit since a full glass of ice chips is approximately only half the volume of the container once it melts. Placing grapes or strawberries in the freezer or using sugar-free hard candies may also provide relief from excessive thirst and dry mouth. On hot days or where there is excessive perspiration, patients may slightly liberalize their fluid and sodium restrictions. Maintaining a fluid restriction is challenging and there are times that intake exceeds recommendations. If this should occur, the patient should be counseled to be particularly vigilant the following day and decrease intake by at least 1 cup.\n\n## VI. RECOMMENDATIONS REGARDING FAT, FIBER, AND CHOLESTEROL\n\nPatients with cardiovascular disease in general should reduce their fat and cholesterol consumption. The current consensus is that total fat intake should be less than 65 g\/day and saturated fats should be less than 20 g\/day. Overall, fats should be approximately 35% of the total daily energy intake, and cholesterol intake should be less than 0.3 g\/day. These general guidelines are reviewed in other chapters of this book. Patients with CHF are generally prescribed the standard cardiovascular prevention guidelines as they apply to their other risk factors such as obesity and hyperlipidemia.\n\nPatients with CHF are typically advised to consume a minimum of 25\u201335 g of fiber per day and preferably more. The rationale is that patients with heart failure are quite prone to constipation for several reasons, such as from diuretic use and decreased physical activity. Straining can precipitate dysrhythmias and pulmonary edema and must be avoided. An adequate fiber intake can help minimize constipation. High-fiber foods include vegetables, cooked dried peas and beans, whole-grain foods, bran, cereals, pasta, rice, and fresh fruit.\n\n## VII. OTHER DIETARY RECOMMENDATIONS\n\nA. Tobacco\n\nAll patients with heart failure are advised to quite smoking and discontinue the use of all nicotine products, including snuff and chewing tobacco. Nicotine can increase heart rate and blood pressure by increasing the levels of norepinephrine. Platelets can also be adversely affected, leading to an increased risk of thrombosis, which is particularly problematic in patients with underlying coronary atherosclerosis. A healthy endothelium in the blood vessels is needed to maintain proper vasodilation and vascular compliance. Endothelial dysfunction is already common in heart failure and it can be further aggravated by smoking, resulting in cell membrane abnormalities, lipid accumulation in the blood vessel walls, increased vascular resistance, and impaired regional blood flow. Finally, cigarette smoking is associated with reduced intake of nutrients such as vitamins E and C [28].\n\n### B. Alcohol\n\nAlcoholic cardiomyopathy accounts for about one-third of all the nonischemic, dilated cardiomyopathies [29]. When the cardiovascular system is exposed to ethanol, acute and chronic changes occur in both systolic and diastolic functioning, with the most significant being a depression in contractility. If alcohol consumption continues, 40\u201350% of patients will die within 3\u20136 years [30], because continued consumption leads to further myocardial damage and fibrosis. The metabolites of alcohol, acetaldehyde and acetate, can have direct toxic effects on the heart as well. Complete abstinence may stop the progression of heart failure or even allow for recovery in early stages [31\u201333]. All patients with severe ventricular dysfunction, regardless of cause, should avoid alcohol because of the risk for worsening of their heart failure.\n\nAlcohol can exacerbate many other underlying problems common in patients with CHF. For example, it can have a pressor effect, cause hypertension, and increase left ventricular mass [34]. Those who drink heavily may experience substantial increases in their blood pressure. Alcohol consumption may also lead to deficiencies in magnesium, potassium, phosphorus, and thiamine, which may further exacerbate existing dysfunction. Alcohol can also worsen hyperlipidemia, primarily by elevating the triglyceride levels, although it can also increase both the total cholesterol and low-density lipoprotein concentration [35]. In smaller quantities (less than 1\u20132 ounces a day), the high-density lipoprotein levels typically increase, thus alcohol may exert a slightly favorable effect. Overall, the risks of continued alcohol consumption outweigh this small benefit of this lipid improvement. The risk of atrial and ventricular dysrhythmias and sudden cardiac death are also increased in this population already prone to these adverse events [36, 37]. Consequently, alcohol consumption should be avoided in all patients with substantial heart failure and in those whose cardiomyopathy is suspected to be primarily from alcohol regardless of severity.\n\n### C. Caffeine\n\nThere are conflicting studies regarding the effects of caffeine on the cardiovascular system. Caffeine is known to antagonize adenosine receptors and cause phosphodiesterase inhibition. The results of acute ingestion are increases in catecholamine and renin levels and an increase in blood pressure. Tolerance quickly develops to caffeine, so it is unclear whether or not there are any long-term harmful effects in patients with CHF. Many are concerned about the potential for arrhythmias; however, studies suggest that moderate amounts of caffeine do not precipitate dysrhythmias [38]. The current recommendations are to limit caffeine to the equivalent of 4\u20135 cups of coffee in patients with coronary artery disease [39].\n\n## VIII. SPECIAL CONSIDERATIONS\n\nA. Nutritional Supplements\n\nCoenzyme Q10, also known as ubiquinone, is a coenzyme for oxidative phosphorylation. It can be found naturally in red meats, fish, soybeans, and vegetable oils. It is present in large quantities in organs that require a fair amount of energy to function adequately, such as the heart, liver, and lung. Coenzyme Q10 also has antioxidant properties and acts as a free radial scavenger [40]. The quantity of this coenzyme has been demonstrated to be low in myocardial biopsy specimens of patients with heart failure. Further, the extent of the deficiency correlates with the severity of heart failure [41]. It has been suggested that supplementation with this mitochondrial enzyme might improve energy mechanics, cardiac contractility, and clinical outcomes. Several small studies have suggested possible benefits [42, 43]; however, most of these studies had major design flaws limiting the interpretation and applicability of the results. Two recent, well-conducted studies showed no significant differences in left ventricular ejection fraction, hemodynamics, exercise duration, peak oxygen consumption, cardiac volumes, or quality of life indices after 3\u20136 months of treatment [44, 45]. It is important to note that coenzyme Q10 can adversely interact with warfarin and aspirin and should be avoided when patients are taking these medications [45a\u201345c].\n\nTaurine is an amino acid found in animal foods such as poultry, beef, pork, fish, and milk. It participates in cell membrane stabilization through modulation of cellular calcium levels, antioxidation [46], and brain and retinal development and it has high concentrations in cardiac tissue. Experimentally, taurine can increase myocardial contractility and potentiate the inotropic response to digoxin presumably by regulating the intracellular calcium concentrations. In laboratory animals with heart failure, taurine has been shown to prevent a reduction in cardiac function, delay the onset of symptoms, and improve survival [47]. Results from very small studies suggest that there may be a beneficial effect in humans [48]. Others suggest that supplementation with this drug, like other oral inotropes studied to date, may actually increase mortality. Investigations with rodents have proposed antiatherosclerotic and hypocholesterolemic effects [49, 50]. Similar to coenzyme Q10, evidence to suggest a benefit of this supplement is lacking. The current Food and Drug Administration recommended daily intake of taurine is 13 mg\/kg\/day [51], although supplements as high as 3 g have been suggested for cardiac benefit.\n\nOther nutrients have gained popularity in the field of alternative medications for CHF. Carnitine, L-arginine, and creatine are other supplements that have theoretical benefits in the treatment of CHF. It is known and accepted that carnitine deficiency may result in a dilated or occasionally restrictive cardiomyopathy. Deficiency may also worsen a preexisting cardiomyopathy from other causes. Oral therapy with L-carnitine can be quite effective in this condition [52]. L-Arginine is a precursor or nitric oxide, which induces vasodilation. Heart failure is often associated with endothelial dysfunction, with resultant impairment of nitric oxide-dependent vasodilation. It is postulated that L-arginine, through conversion to nitric oxide, may prevent or correct endothelial dysfunction [53]. However, there have been no proven mortality benefits in large-scale clinical trials of any of these substances. Patients should be reminded that these supplements could be considered in addition to their standard therapy, rather than replacements.\n\n### B. Food and Drug Interactions\n\nMany medications commonly used today can be affected by the contents of the gastrointestinal tract. Often the presence of food can delay or decrease the absorption of a medication. The interactions between food and cardiovascular medications are clinically important to understand (see Table 5). Depending on the desired effect, patients need to be instructed when to take their pills in relationship to meals. For instance, food slows down the absorption of carvedilol. If patients take this medication on an empty stomach, they often absorb it too rapidly and, consequently, can experience dizziness and hypotension. In this case, patients are advised to take carvedilol with food to diminish side effects. Finally, some cardiac medications such as amiodarone or furosemide are known to cause nausea or abdominal discomfort and are best taken with a light meal or snack.\n\nTABLE 5\n\nEffect of Food on Common Heart Failure Medications\n\naDelayed with high bran fiber meals.\n\nbTake 1 hour before meals.\n\ncFood increases bioavailability, but clinical significance unknown.\n\n### C. Cardiac Cachexia\n\nCardiac cachexia is common in those suffering from chronic, severe CHF. The syndrome is characterized by a drastic reduction in both lean body muscle mass and adipose tissue, with a subsequent decrease in activity level, functional capacity, and strength. Cardiac cachexia is associated with decreased survival. Information gleaned from nutritional surveys obtained on those requiring hospitalization for heart failure have demonstrated that 50\u201368% of patients are malnourished as determined by total body weight, plasma protein status, and anthropometric measurements [54\u201356]. The exact mechanism of cachexia is unknown, but most likely many factors contribute to the profound loss (see Table 6).\n\nTABLE 6\n\nPossible Causes of Cardiac Cachexia\n\nAbdominal discomfort, bloating\n\nAltered taste\n\nAnorexia, nausea, and vomiting\n\nCytokines such as tumor necrosis factor-\u03b1, interleukin-1, interferon-\u03b3\n\nDecreased absorption of food\n\nDifficulty eating from shortness of breath\n\nFluid restriction\n\nIncreased caloric expenditure\u2014work of breathing, fever, increased basal metabolic rate\n\nNutrient losses\u2014renal protein loss, protein-losing enteropathy\n\nUnpalatable diet from restrictions\n\nMany cytokines have been identified that probably play a significant role in the catabolism and weight loss, as reviewed recently by Tisdale [57]. Studies performed in other wasting states, such as malignancies, have shown that increasing nutrient intake alone is unable to reverse this process. Therapies are generally directed toward reversing the possible factors known to impair the ability to eat, such as treating nausea, reducing fluid retention as much as possible, and improving hemodynamics. Small, frequent meals are helpful. At times, some of the restrictions placed on the diet will need to be relaxed so patients can increase their energy intake. An alteration in food composition to be 35% energy from fat, 15% energy from protein and 50% energy from carbohydrate has been suggested to combat the cachexia, although not all agree. Perhaps by decreasing neurohormonal activation, some of the cytokines contributing to the wasting syndrome will be diminished and patients will stabilize or even increase their muscle mass. Ongoing studies with the use of tumor necrosis factor antagonists and fish oil may reveal interesting outcomes and lead to further therapies directed at this devastating complication of heart failure.\n\n### D. Right-Sided Heart Failure\n\nWhen patients develop CHF, it is sometimes easier to describe their symptoms as either ''left sided\" or ''right sided.\" Often, both are present simultaneously, although one side typically predominates. The term _right-sided heart failure_ implies that the symptoms a patient is experiencing are attributable to dysfunction of the right ventricle. Symptoms can include abdominal discomfort, bloating, leg swelling, early satiety, and nausea. On physical exam, patients can have prominent jugular venous distention, hepatomegaly, splenomegaly, ascites, and pitting edema. The gastrointestinal symptoms occur from both gut edema and organ enlargement. As the liver expands, the capsule around it stretches and becomes quite uncomfortable. It is not uncommon to have right-sided heart failure be confused as an acute abdomen, cholecystitis, appendicitis, or mesenteric ischemia.\n\nPatients with right-sided CHF tend to have difficulties eating an adequate amount. Small, frequent meals are better tolerated than large volumes. Often patients will naturally switch to more liquid meals, which may interfere with fluid and sodium restriction. For example, soups are easier to consume, however, they contribute a high sodium load. Many of the nutritional supplemental drinks on the market can be used, but patients should do so cautiously because some contain large quantities of sodium or are high in sugar and may worsen glucose control in patients with diabetes. Patients again should be encouraged to read labels closely.\n\nFinally, patients with significant edema of the intestinal wall may have decreased absorption of some of their heart failure medications, thus precipitating further decompensation and starting a vicious cycle of deterioration. Hospitalization and conversion of medications to intravenous forms may be necessary until adequate functioning returns.\n\n## IX. SUMMARY\n\nCongestive heart failure is the only cardiovascular disorder that is increasing in prevalence. This is mostly due to improved survival from other cardiac conditions such as hypertension and myocardial infarction. Once the initial insult occurs, the renin-angiotensin-aldosterone and sympathetic nervous systems become activated. These neurohormones and other cytokines lead to adverse ventricular remodeling and progressive ventricular dysfunction. Patients are treated routinely now with angiotensin-converting enzyme inhibitors, beta blockers, digoxin, and diuretics. In addition to these medications, diet modification and exercise are also important in controlling the high morbidity and mortality associated with CHF. Sodium restriction to approximately 2 g\/day is the most important dietary modification, and this alone will frequently keep patients euvolemic and diminish the need for diuretics.\n\nExcess sodium intake is often the underlying cause for decompensation in a previously stable state. Fluid restriction to 2 L\/day is also advisable to decrease fluid retention and assist in net diuresis. This may need to be reduced even further in times of decompensation. Patients with heart failure are also advised to follow the standard guidelines for prevention of cardiovascular disease regarding fat, carbohydrate, and cholesterol intakes. Because patients with CHF are prone to constipation, adequate fiber intake is encouraged.\n\nA number of alternative therapies, such as coenzyme Q10 and taurine, have been touted as beneficial in heart failure. These are yet to be scientifically proven to improve morbidity or mortality in large, randomized, placebo-controlled trials. Patients should not use these therapies in place of the standard heart failure medications, which are clearly beneficial. Finally, certain patients with CHF present with further difficulties in obtaining adequate energy and nutrient intakes. As the pathophysiology of the cardiomyopathies becomes better understood and elucidated, medical and nutritional interventions are likely to be even more helpful in the future.\n\nReferences\n\n1. Statistical SupplementAmerican Heart Association. _2000 Heart and Stroke Statistical Update_. New York: American Heart Association; 2000.\n\n2. World Health Organization\/International Society and Federation of Cardiology Task Force. Definition and classification of cardiomyopathies. _Circulation_. 1996;93:841\u2013842.\n\n3. Davis J.O. The mechanism of salt and water retention in cardiac failure. _Hosp. Prac_. 1970;5:63\u201376.\n\n4. Brunier M., Brunner H.R. Neurohormonal consequences of diuretics in different cardiovascular syndromes. _Eur. Heart J_. 1992;13(Suppl. G):28\u201333.\n\n5. Kaye D.M., Lefkovits J., Jennings G.L., Bergin P., Broughton A., Esler M.D. Adverse consequences of high sympathetic nervous activity in the failing human heart. _J. Am. College Cardiol_. 1995;26:1257\u20131263.\n\n6. ACC\/AHA Task Force. Guidelines for the evaluation and management of heart failure. _J. Am. College Cardiol_. 1995;26:1376\u20131398.\n\n7. Dracup K., Baker D.W., Dunbar S.B., Dacey R.A., Brooks N.H., Johnson J.C., Oken C., Massie B.M. Management of heart failure II: Counseling, education and lifestyle modifications. _JAMA_. 1994;272:1442\u20131446.\n\n8. Haas G.J., Young J.B. Acute heart failure management. In: Topol E.J., ed. _Textbook of Cardiovascular Medicine_. Dallas, TX: Lippincott-Raven, 1998.\n\n9. Young J.B. Chronic heart failure management. In: Topol E.J., ed. _Textbook of Cardiovascular Medicine_. Philadelphia, PA: Lippincott-Raven, 1998.\n\n10. Konstam M., Dracup K., Baker D., Bottorff M., Brooks N., Dacey R., Dunbar S., Jackson A., Jessup M., Johnson J., Jones R., Luchi R., Massie B., Pitt B., Rose E., Rubin L., Wright R., Hadorn D. Heart Failure: Evaluation and Care of the Patient with Left-Ventricular Systolic Dysfunction. Clinical Practice Guideline. _AHCPR Publication No. 94-0612. Agency for Health Care Policy and Research, U.S. Department of Health and Human Services, Rockville, MD_. 1994.\n\n11. Packer M., Cohn J. Consensus recommendations for the management of chronic heart failure. _Am. J. Cardiol_. 1999;82:2A\u201338A.\n\n12. The SOLVD Investigators. Effect of enalapril on mortality and on the development of heart failure in asymptomatic patients with reduced left ventricular ejection fractions. _N. Engl. J. Med_. 1992;327:685\u2013691.\n\n13. CONSENSUS Trial Study Group. Effects of enalapril on mortality in severe congestive heart failure: Results of the Cooperative North Scandinavian Enalapril Survival Study (CONSENSUS). _N. Engl. J. Med_. 1987;316:1429\u20131435.\n\n14. Pfeffer M., Braunwald E., Moye L., Basta L., Brown E., Cuddy T., Davis B., Geltman E., Goldman S., Flaker G., Klein M., Lamas G., Packer M., Rouleau J., Rouleau J.L., Rutherford J., Wertheimer J., Hawkins C., on behalf of the SAVE Investigators. Effect of captopril on mortality and morbidity in patients with ventricular dysfunction after myocardial infarction. Results of the survival and ventricular enlargement trial. _N. Engl. J. Med_. 1992;327:669\u2013677.\n\n15. Cohn J.N., Johnson G., Ziesche S., Cobb F., Francis G., Tristani F., Smith R., Dunkman W., Loeb H., Wong M., Bhat G., Goldman S., Fletcher R., Doherty J., Hughes C., Cardon P., Cintron G., Shabetai R., Haakenson C. A comparison of enalapril with hydralazine-isosorbide dinitrate in the treatment of chronic congestive heart failure (VeHEFT-II). _N. Engl. J. Med_. 1991;325:303\u2013310.\n\n16. Digitalis Investigation Group. The effect of digoxin on mortality and morbidity in patients with heart failure. _N. Engl. J. Med_. 1997;336:525\u2013533.\n\n17. Uretsky B.F., Young J.B., Shahidi F.E., Yellen L.G., Harrison M.C., Jolly M.K. Randomized study assessing the effect of digoxin withdrawal in patients with mild to moderate chronic congestive heart failure: Results of the PROVED trial: PROVED Investigative Group. _J. Am. College Cardiol_. 1993;22:955\u2013962.\n\n18. Packer M., Gheorghiade M., Young J.B., Costantini P.J., Adams K.F., Cody R.J., Smith L.K., Van Voorhees L., Gourley L.A., Jolly M.K. On behalf of the RADIANCE Study. Withdrawal of digoxin from patients with chronic heart failure treated with angiotensin-converting-enzyme inhibitors. _N. Engl. J. Med_. 1993;329:1\u20137.\n\n19. CIBIS Investigators and Committees. A randomized trial of \u03b2-blockade in heart failure: The Cardiac Insufficiency Bisoprolol Study (CIBIS). _Circulation_. 1994;90:1765\u20131773.\n\n20. Waagstein F., Bristow M.R., Swedberg K., Camerini F., Fowler M.B., Silver M.A., Gilbert E.M., Johnson M.R., Goss F.G., Hjalmarson A. for the Metoprolol in Dilated Cardiomyopathy (MDC) Trial Study Group. Beneficial effects of metoprolol in idiopathic dilated cardiomyopathy. _Lancet_. 1993;342:1441\u20131446.\n\n21. Australia-New Zealand Heart Failure Research Collaborative Group. Randomised, placebo-controlled trial of carvedilol in patients with congestive heart failure due to ischaemic heart disease. _Lancet_. 1997;349:375\u2013380.\n\n22. Bristow M.R. \u03b2-adrenergic receptor blockade in chronic heart failure. _Circulation_. 2000;101:558\u2013569.\n\n23. for the U.S. Carvedilol Heart Failure Study GroupPacker M., Bristow M.R., Cohn J.N., Colucci W.S., Fowler M.B., Gilbert E.M., Shusterman N.H. The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. _N. Engl. J. Med_. 1996;334:1349\u20131355.\n\n24. CIBIS-II Investigators and Committees. The Cardiac Insufficiency Bisoprolol Study II (CIBIS-II): A randomized trial. _Lancet_. 1999;353:9\u201313.\n\n25. MERIT-HF Study Group. Effect of metoprolol CR\/XL in chronic heart failure: Metoprolol CR\/XL Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF). _lancet_. 1999;353:2001\u20132007.\n\n26. Albert M.A., Terry B.E., Mulekar M., Cohen M.V., Massey C.V., Fan T.M., Panayiotou H., Mukerji V. Cardiac morphology and left ventricular function in normotensive morbidly obese patients with and without congestive heart failure, and effect of weight loss. _Am. J. Cardiol_. 1997;80:736\u2013740.\n\n27. Wilcox C.S., Mitch W.E., Kelly R.A., Skorechi K., Meyer T.W., Friedman P.A., Souney P.F. Response of the kidney to furosemide. I. Effects of salt intake and renal compensation. _J. Lab. Clin. Med_. 1983;102:450\u2013458.\n\n28. Dallongeville J., Marecaux N., Fruchart J.C., Amouyel P. Cigarette smoking is associated with unhealthy patterns of nutrient intake: A meta-analysis. _J. Nutr_. 1998;128:1450\u20131457.\n\n29. Schwarz F., Mall G., Zebe H., Schmitzer E., Mauthey J., Scheusten H. Determinants of survival in patients with congestive cardiomyopathy: Quantitative morphologic findings and left ventricular hemodynamics. _Circulation_. 1984;70:923\u2013928.\n\n30. Kinney E.L., Wright R.J., Caldwell J.W. Risk factors in alcoholic cardiomyopathy. _Angiology_. 1989;40:270\u2013275.\n\n31. Jacob A.J., McLaren K.M., Boon N.A. Effects of abstinence on alcoholic heart muscle disease. _Am. J. Cardiol_. 1991;68:805\u2013807.\n\n32. M\u00f8lgaard H., Kristensen B.\u00d8., Baandrup U. Importance of abstention from alcohol in alcoholic heart disease. _Int. J. Cardiol_. 1990;26:373\u2013375.\n\n33. Pavan D., Nicolosi G.L., Lestuzzi C., Burelli C., Zardo F., Zanuttini D. Normalization of variables of left ventricular function in patients with alcoholic cardiomyopathy after cessation of excessive alcohol intake: An echocardiographic study. _Eur. Heart J_. 1987;8:535\u2013540.\n\n34. Regan T.J. Alcohol and the cardiovascular system. _JAMA_. 1990;264:377\u2013381.\n\n35. Gaziano J.M., Manson J.E. Diet and heart disease: The role of fat, alcohol and antioxidants. _Cardiol. Clin_. 1996;14:69\u201383.\n\n36. Sharper A.G., Wannamethee G. Alcohol and sudden cardiac death. _Br. Heart J_. 1992;68:443\u2013448.\n\n37. Koskinen P., Kupari M. Alcohol and cardiac arrhythmias. _Br. Med. J_. 1992;304:1394\u20131395.\n\n38. Myers M.G. Caffeine and cardiac arrhythmias. _Ann. Intern. Med_. 1991;114:147\u2013150.\n\n39. Lynn L.A., Kissinger J.F. Coronary precautions. Should caffeine be restricted in patients after myocardial infarction? _Heart Lung_. 1992;21:365\u2013370.\n\n40. Beyer R.E., Emster L. The antioxidant role of coenzymes Q. In: Lenaz G., Barnabeli O., Rabbi A., Battino A., eds. _Highlights in Ubiquinone Research_. Philadelphia, PA: Taylor and Francis; 1990:191\u2013213.\n\n41. Folkers K., Vadhanavikit S., Mortensen S.A. Biochemical rationale and myocardial tissue data on the effective therapy of cardiomyopathy with coenzyme Q10. _Proc. Natl. Acad. Sci. USA_. 1985;82:901\u2013904\n\n42. Langsjoen P.H., Langsjoen P.H., Folkers K. Long-term efficacy and safety of coenzyme Q10 therapy for idiopathic dilated cardiomyopathy. _Am. J. Cardiol_. 1990;65:521\u2013523.\n\n43. Lampertico M., Comis S. Italian multicentre study on the efficacy and safety of coenzyme Q10 as adjuvant therapy in heart failure. _Clin. Invest_. 1993;71:S129\u2013S133.\n\n44. Watson P.S., Scalia G.M., Galbraith A., Burstow D.J., Bett N., Aroney C.N. Lack of effect of coenzyme Q on left ventricular function in patients with congestive heart failure. _J. Am. College Cardiol_. 1999;33:1549\u20131552.\n\n45. Khatta M., Alexander B.S., Krichten C.M., Fisher M.L., Freudenberger R., Robinson S.W., Gottlieb S.S. The effect of coenzyme Q10 in patients with congestive heart failure. _Ann. Intern. Med_. 2000;132:636\u2013640.\n\n45a. Spigset O. Reduced effect of warfarin caused by ubidecarenone. _Lancet_. 1994;344:1372\u20131373.\n\n45b. Landbo C., Almdal T.P. International between warfarin and coenzyme Q10. _Ugeskrift for Laeger_. 1998;160(22):3226\u20133227.\n\n45c. Heck A.M., DeWitt B.A., Lukes A.L. Potential interactions between alternative therapies and warfarin. _Am. J. Health-System Pharmacy_. 2000;57:1221\u20131230.\n\n46. Cunninham C., Tipton K.F., Dixon H.B.P. Conversion of taurine into N-chlorotaurine (taurine chloramine) and sulphoacetaldehyde in response to oxidative stress. _Biochem. J_. 1998;330:939\u2013945.\n\n47. Takihara K., Azuma J., Awata N., Ohta H., Hamaguchi T., Swamura A., Tanaka Y., Kisimoto S., Sperelakis N. Beneficial effect of taurine in rabbits with chronic congestive heart failure. _Am. Heart J_. 1986;112:1278\u20131284.\n\n48. Azuma J., Sawamura A., Awata N. Usefulness of taurine in chronic congestive heart failure and its prospective application. _Jpn. Circ. J_. 1992;56:95\u201399.\n\n49. Kamata K., Sugiura M., Kojima S., Kasuya Y. Restoration of endothelium-dependent relaxation in both hypercholesterolemia and diabetes by chronic taurine. _Eur. J. Pharmacol_. 1996;303:47\u201353.\n\n50. Yokogoshi H., Mochizuki H., Nanami K., Hida Y., Miyachi F., Oda H. Dietary taurine enhances cholesterol degradation and reduces serum and liver cholesterol concentrations in rats fed a high-cholesterol diet. _J. Nutr_. 1999;129:1705\u20131712.\n\n51. National Research Council. _Recommended Dietary Allowances_ , 10th Ed. London: National Academy Press; 1989.\n\n52. Tripp M.E., Katcher M.L., Peters H.A., Gilbert E.F., Arya S., Hodach R.J., Shug A.L. Systemic carnitine deficiency presenting as familial endocardial fibroelastosis: A treatable cardiomyopathy. _N. Engl. J. Med_. 1984;310:142\u2013148.\n\n53. Hambrecht R., Hilbrich L., Erbs S., Gielen S., Fiehn E., Schoene N., Schuler G. Correction of endothelial dysfunction in chronic heart failure: Additional effects of exercise training and oral 1-arginine supplementation. _J. Am. College Cardiol_. 2000;35:706\u2013713.\n\n54. Carr J.G., Stevenson L.W., Walden J.A., Heber D. Prevalence and hemodynamic correlates of malnutrition in severe congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. _Am. J. Cardiol_. 1989;63:709\u2013713.\n\n55. Mancini D.M., Walter G., Reichek N., Lenkinski R., McCully K.K., Mullen J.L., Wilson J.R. Contribution of skeletal muscle atrophy to exercise intolerance and altered muscle metabolism in heart failure. _Circulation_. 1992;85:1364\u20131373.\n\n56. Blackburn G.L., Gibbons G.W., Bothe A., Benotti P.N., Harken D.E., McEnany T.M. Nutritional support in cardiac cachexia. _J. Thor. Cardiovasc. Surg_. 1977;73:489\u2013495.\n\n57. Tisdale M.J. Wasting in cancer. _J. Nutr_. 1999;129:243S\u2013246S.\n\n* * *\n\n1Current address: Diagnostic Cardiology Associates, Youngstown, Ohio.\nB.\n\nCancer Prevention and Therapy\nCHAPTER 22\n\nNutrition and Breast Cancer\n\nCHERYL L. ROCK1 and WENDY DEMARK-WAHNEFRIED2\n\n1University of California at San Diego, La Jolla, California\n\n2Duke University Medical Center, Durham, North Carolina\n\n## I. INTRODUCTION\n\nBreast cancer is the most common invasive cancer in women in the United States, accounting for approximately 30% of new cancer cases in women. An estimated 192,200 U.S. women will be diagnosed with breast cancer in 2001 [1]. Caucasian women in the United States are more likely to be diagnosed with breast cancer than women of other racial\/ethnic groups, but rates of survival for African-American women following diagnosis are lower than for Caucasians [2]. Incidence rates have decreased very slightly during the past several years, following a steady increase since the 1930s. Breast cancer mortality has been declining in recent years, a trend that has been attributed to earlier diagnosis and improvements in initial treatments [3]. As a result, an increasing number of women in the population are breast cancer survivors and are considered at risk for breast cancer recurrence. Recurrence is an important issue in breast cancer management, because the yearly rate of secondary cancer events, even for women who have been diagnosed with very early stage cancers, does not return to the level of similarly aged women who have not been diagnosed with breast cancer [4].\n\nA very large body of scientific evidence on the relationship between nutritional factors and the risk for breast cancer has been accumulated and reported during the past several decades. Notably, research findings in this area are generally characterized by inconsistencies in the evidence for specific dietary factors and divergent views on the interpretation of these data. Much less data on the relationship between factors that may influence risk for recurrence or improve prognosis after the diagnosis of breast cancer (compared with data on primary prevention) have been collected and reported. In addition to dietary and other environmental factors, risk factors that have been linked to risk for breast cancer are gender (women have a 100-fold increased risk compared to men), age (mean age of onset is 66 years), family history (including specific genetic factors), and reproductive history, such as the ages of women at menarche, the birth of the first child, and menopause [1, 5].\n\n### A. Mammary Cell Biology and Genetic Factors\n\nMuch has been learned about the biology of breast cancer in the past several years. An important and recognized feature of breast cancer is the heterogeneity on a molecular level. As is true of all cancers, mammary carcinogenesis is a multistep process, involving the accumulation of genetic and epigenetic changes that result from the interaction between genetics and the environment [6]. On a cellular level, each case is characterized by a unique disease pathway, with associated genetic changes that may or may not be influenced by different etiological factors or interventions. As discussed by Slattery _et al._ [7], this heterogeneity likely constrains the ability to identify specific links between dietary factors and risk for breast cancer. Variable cellular characteristics also should be anticipated to be among the determinants of response to diet interventions that may reduce risk for breast cancer or cancer recurrence.\n\nIn addition to the cellular factors and mitogens that can affect growth regulation and apoptosis and thus influence carcinogenesis in all cell types, an important characteristic of normal cell proliferation and differentiation in the breast is the responsiveness of these cells to ovarian steroids. Estrogens have a direct effect on the mammary gland during various stages of development, such as growth, puberty, pregnancy, and lactation. For example, early full-term pregnancy, a characteristic in the reproductive history that is known to be associated with reduced risk for breast cancer, promotes increased degree of cellular differentiation, a cellular characteristic that is inversely related to the initiation of the neoplastic process [8]. Estrogens also stimulate cell proliferation in breast tissue, which increases the likelihood of random genetic errors and risk for the formation of a malignant phenotype [9]. In laboratory animal experiments, systemic ovarian hormones have been demonstrated to promote breast tumorigenesis [9]. The evidence to support a relationship between serum concentrations of estrogens and risk for breast cancer in human studies is much more inconsistent, which is usually attributed to methodological problems, and laboratory imprecision, and the fact that people are more genetically diverse than laboratory animals. For example, in premenopausal women, concentrations of hormones in the circulation vary substantially across the menstrual cycle, the timing of which is only crudely assessed in most studies. Breast cancer is considered a hormone-dependent cancer, so nutritional factors that may influence the biosynthesis, metabolism, and inactivation of ovarian hormones, throughout development and adulthood, are suspected to influence risk for breast cancer or recurrence (see Fig. 1). Epidemiological studies have identified some apparent differences in risk factors for breast cancer in premenopausal and postmenopausal women, and menopausal status influences the treatment recommendations on diagnosis, so these subgroups are often (but not always) separated in the analysis of nutrition-related risk factors or response to dietary interventions.\n\nFIGURE 1 Some of the proposed hormonal mechanisms and their associations with mammary cell proliferation.\n\nIn addition to the ovarian steroids, other growth factors and mitogens that may be modified by nutritional factors and dietary patterns also appear to play an important role in the initiation and promotional phases of breast cancer, based on epidemiological and laboratory studies. Two examples that are currently of intense scientific interest are insulin and insulin-like growth factor 1, and the interactions of these factors with adiposity and weight gain [10, 11], which will be addressed in the sections that follow.\n\nA small proportion of breast cancers can be linked to a specific inherited susceptibility, the most well-known being the highly penetrant, dominant gene mutations, breast cancer gene 1 ( _BRCA1_ ) and _BRCA2_ [12]. Both are currently thought to be tumor suppressor genes. The presence of _BRCA1_ increases the individual breast cancer risk by approximately 200-fold, but the mutation is rare, accounting for only 5.3% of breast cancer cases in women aged <40 years, 2.2% of cases in women aged 40\u201349 years, and 1.1% of cases in women aged 50\u201370 years [12]. The majority of women who are diagnosed with breast cancer do not have this or other specific mutations that have been identified. Inherited variations in other biochemical or metabolic pathways relevant to breast cell biology, such as those involved in estrogen metabolism, are other sources of genetic susceptibility currently under study [13], and their contributions to genetic risk are not yet known.\n\nAs discussed in an earlier chapter (see Chapter 13), interactions between genetic and dietary factors also are likely to be among the determinants of risk for breast cancer. Examples of gene\u2013diet interactions currently under study in breast cancer are interactions between polymorphisms of _N_ -acetyltransferase and meat consumption [14, 15] and between glutathione _S_ -transferase and dietary sources of anti-oxidants [16].\n\n### B. Staging of Breast Cancer and Other Histopathologic and Prognostic Factors\n\nStaging is important in understanding the management of breast cancer because stage at diagnosis determines the choice of recommended treatments and also is a predictive factor in the overall prognosis. Staging in breast cancer is based on an internationally recognized system [17]. Categorization from stages I through III is based on tumor size and the presence and degree of involvement of axillary lymph nodes and other regional tissues, but no evidence of distant metastases is present in these stages. Stage IV includes the presence of distant metastases. Ductal carcinoma _in situ_ and lobular carcinoma _in situ_ are not invasive cancers, although these conditions are often treated like invasive breast cancer, because the lesions may progress to invasive breast cancer or be a marker of increased risk for the development of overt carcinoma [18].\n\nThe presence (and degree of involvement) of axillary lymph nodes at diagnosis has been found to be the most important predictor of prognosis [19]. However, several other tumor-related factors have been found useful in predicting the prognosis, and these characteristics also may influence the choice of treatments and the response to pharmacological and other interventions. For example, expression of estrogen (and also progesterone) receptors by tumor tissue indicates that normal cellular uptake and response to estrogen has been retained by the cancer cells. In follow-up studies, patients with estrogen receptor-positive tumors have been observed to have better long-term survival than patients with estrogen receptor-negative tumors [19]. Other cellular characteristics that appear to be of prognostic importance include DNA ploidy and S-phase fraction, oncogene amplification, or overexpression of the epidermal growth-factor receptor or _erbB2_ ( _HER2\/neu_ ), and\/or expression of mutant _p53_ [18, 19].\n\n## II. NUTRITIONAL FACTORS IN THE ETIOLOGY OF BREAST CANCER\n\nA. Primary Cancer Risk\n\nEarly interest in the relationship between nutrition and breast cancer was stimulated in large part by data from international comparisons that show a fivefold difference in breast cancer mortality rates across countries that is not explained by different treatment modalities [5]. More importantly, the risk for breast cancer among migrant populations who originate from low-risk countries has been observed to change with relocation (e.g., migration of Japanese women to the United States), subsequently reflecting the rate of the adopted culture and regions with higher breast cancer rates [20\u201322]. This pattern suggests that environmental factors, such as diet, are likely to play an important etiological role.\n\n#### 1. DIETARY AND NUTRIENT INTAKES AND RISK FOR BREAST CANCER\n\nClearly, the most well-known hypothesis that has been examined in the investigation of the link between nutrition and risk for breast cancer is that total dietary fat intake may increase breast cancer risk. More data and resulting reports on this proposed relationship have been collected and published than on any other nutritional or dietary factor and breast cancer. Several eloquent and comprehensive reviews on this topic were published during the 1990s, and the reader is referred to these publications for detailed review and discussion of the evidence collected to date and the challenges in interpreting these data [23\u201327]. In addition to these comprehensive reviews, several authors have specifically addressed current theories, evidence for the proposed mechanisms, and relevant findings from laboratory animal and cell culture studies [28, 29].\n\nThe totality of the evidence suggests great inconsistency, at best, and is not very supportive, particularly when an effort is made to separate the effect of fat intake from the effect of total energy consumption. Although cross-country comparisons from ecological studies demonstrate a striking association between dietary fat intake and incidence of breast cancer [30], data from observational studies conducted within populations generally do not show this relationship, particularly in cohort studies (which exhibit less reporting bias than do simple case-control studies). This can be illustrated by results presented in recent reports. In a pooled analysis involving 4980 cases that arose from 337,819 U.S. women [31], the multivariate pooled relative risk of breast cancer was 1.05 (95% confidence interval [CI] 0.94\u20131.16), when comparing women in the highest quintile of energy-adjusted total fat intake compared with women in the lowest quintile. Diet was assessed by means of food frequency questionnaires in all of the studies included in this pooled analysis. Relative risks for intakes of saturated, monounsaturated, and polyunsaturated fat were also were nonsignificant. In a single large cohort study of 88,795 women, of whom 2956 were diagnosed with breast cancer over a follow-up period of 14 years, women consuming 30\u201335% energy from fat had a relative risk of 1.15 (95% CI 0.73\u20131.80), compared to women consuming \u226420% energy from fat [32]. In this study by Holmes _et al._ [32], specific types of fat also were not found to be associated with risk of breast cancer, although a small but significant increase in risk was associated with intake of omega-3 fatty acids from fish (relative risk 1.09, 95% CI 1.03\u20131.16), and _trans_ fatty acid intake had a slightly protective effect (relative risk 0.92, 95% CI 0.86\u20130.98).\n\nInterpretation of these epidemiological data is complicated, of course, by known limitations in dietary assessment methodologies and confounding variables, as discussed in Chapter 4. Measurement error is particularly likely when the evidence relies solely on self-reported dietary data that are collected by instruments with inherent inaccuracy in quantifying nutrient consumption or capturing long-term dietary intakes [33]. It also has been noted that the range of fat intake among the women in these within-population studies is too limited to enable the identification of a relationship with breast cancer risk, and that the number of women who consume diets that provide \u226420% of energy from fat, even in larger studies, is very small [24].\n\nWhen testing the relationship between dietary fat and the promotion of breast tumorigenesis in laboratory animal studies, the interpretation of findings also is often difficult, due to the inextricable link between fat intake and energy balance. High-fat diets are energy dense, promoting increased overall growth of the animals and increased adiposity, two factors that independently affect number and size of tumors. However, specific fatty acids do appear to have differential effects on mammary cancer and tumor growth in animal studies. Omega\u20136 fatty acids have been shown to promote tumor development when fed to laboratory animals, particularly in comparison to omega\u20133 fatty acid-rich diets [28, 29], theoretically by influencing tumor eicosanoid production and possibly other factors (e.g., protein kinase C, membrane permeability, immune response). The practical significance of these findings from laboratory studies, however, is unclear, because examination of dietary patterns based on food choices does not consistently reveal a protective pattern of specific fatty acids. Also, marked changes in phospholipid and tissue composition of fatty acids may not be readily achieved in humans without fish oil supplements, and fish oil supplements carry some adverse metabolic risk in addition to any possible benefits. In comparison, regular consumption of fish and other food sources of omega-3 fatty acids (e.g., walnuts) could have a more favorable overall risk-to-benefit ratio. This is an area of interest, but consensus does not exist.\n\nA mechanism by which a reduction in dietary fat intake is hypothesized to influence breast cancer risk is that of reducing serum estrogen concentrations. Various feeding and intervention studies have examined the effect of dietary fat reduction on serum estrogen levels. As summarized in a review of these studies by Wu _et al._ [34], estrogen levels generally decreased with fat restriction. Note, however, that significant weight loss occurred in 6 of the 13 studies. Also, intake of dietary fiber was concurrently (and usually substantially) increased in the majority of these studies. Therefore, the interpretation that dietary fat restriction per se promotes a reduction in serum estradiol concentrations can be challenged, because an energy deficit, weight loss, and increased fiber intake could be the true or primary factors that promoted a reduction of hormone levels in these studies.\n\nA relevant theory is that a high-fat (and thus energy-dense) diet during the developmental years may be a determinant of breast cancer risk in adulthood, rather than adult dietary fat intake (for which the evidence has not generally been supportive) [27]. This is a difficult relationship to test and would likely be confounded by the effect of energy density of the diet through childhood and adolescence on anthropometric characteristics (discussed in following sections).\n\nIn comparison with dietary fat intake, the evidence is somewhat more consistent for a protective effect of vegetables and fruit intake on risk for breast cancer. Because vegetables and fruits contribute the overwhelming majority of vitamin C and carotenoids in the diet, examination of the relationship between these compounds and breast cancer risk overlaps with investigation of an effect of vegetables and fruit. However, vegetables and fruit provide numerous phytochemicals and also fiber, in addition to the known micronutrients, that may contribute to a protective effect. In a meta-analysis based on 26 studies published from 1982 to 1997, the relationships between risk for breast cancer and intakes of vegetables, fruit, beta-carotene, and vitamin C were examined [35]. High (versus low) consumption of vegetables was found to convey the strongest protective effect (relative risk 0.75, 95% CI 0.66\u20130.85), with protective effects also evident for beta-carotene and vitamin C intakes as independent variables, but the relationship with fruit consumption was not significant (relative risk 0.94, 95% CI 0.74\u20131.11) [35].\n\nRecent U.S. case-control and cohort studies provide examples of the degree of risk reduction that has been attributed to these foods. Freudenheim _et al._ [36] examined the association between breast cancer risk and intakes of vegetables, fruit, vitamin C, folate, individual carotenoids, alphatocopherol, and dietary fiber in premenopausal women (297 cases and 311 controls) in an analysis that separated the effect of food sources and dietary supplements for the micronutrients. Dietary (but not supplemental) beta-carotene, lutein + zeaxanthin, vitamin C, folate, alpha-tocopherol, and dietary fiber were all found to provide significant protective effects. Most notable was the odds ratio for total vegetable intake, which was 0.46 (95% CI 0.28\u20130.74), and adjusting only for beta-carotene and lutein + zeaxanthin attenuated this effect. In a large U.S. case-control study that included both premenopausal and postmenopausal women (3543 cases and 9406 controls), eating carrots or spinach more than twice weekly (compared with no intake) was found to be associated with an odds ratio of 0.56 (95% CI 0.34\u20130.91) [37]. In a U.S. cohort study involving 83,234 women with 2697 incident cases of invasive breast cancer over a 14-year follow-up period [38], strong inverse associations with risk were found for intakes of alpha-carotene, beta-carotene, lutein % zeaxanthin, vitamin C from foods, and total vitamin A. Premenopausal women who consumed \u22655 servings of vegetables and fruit per day had a relative risk of 0.77 (95% CI 0.58\u20131.02), compared to those consuming <2 servings per day ( _P_ = 0.05 for trend), and the effect was stronger among those with a family history or if consumption of \u226515 g alcohol per day was reported. Results of studies in which plasma or adipose tissue concentrations of carotenoids (a marker of vegetable and fruit intake) were quantified have often (but not always) suggested a protective effect in association with higher amounts of these compounds in the tissue [39\u201341], which would support the observations based on self-reported dietary intakes.\n\nBiologically feasible mechanisms and\/or supportive laboratory evidence for several constituents of vegetables and fruits have been reported. In cell cultures, both retinoids and carotenoids have been shown to enhance cellular differentiation and to have marked inhibitory effects on mammary cell growth [42, 43]. Several studies using breast epithelial cells and cell lines have demonstrated that both provitamin A and nonprovitamin A carotenoids, similar to retinoids, promote growth inhibition and apoptosis in transformed cells [43\u201345]. Vitamin A and carotenoids have a biochemical overlap, because both can be a source of retinoids and exhibit retinoid-like activities. However, the food sources, tissue uptake, regulation, and metabolism of the carotenoids differ from those of preformed vitamin A. Carotenoids can be delivered to peripheral tissue in direct association with intakes across a wide range, with resulting retinoid-like activities in those tissues. In contrast, dietary preformed vitamin A is correlated with biological activity in peripheral tissue only in the lower range of intakes. Another mechanistic theory, which is more limited and not as well supported by current evidence, would group carotenoids with vitamin C and vitamin E, and then assume their mechanistic relationship with breast cancer to be due to an antioxidant effect.\n\nAntioxidants could theoretically reduce the risk for breast cancer by protecting against DNA damage and other free-radical-induced cellular changes that would promote mammary carcinogenesis. However, the relationship between risk for breast cancer and status for vitamin E, which has a well-established function as a biological antioxidant, has been examined in numerous studies, and the results are generally not very supportive [23, 46]. In a large European case-control study among postmenopausal women, adipose tissue concentrations of alpha-tocopherol and beta-carotene and concentration of selenium in toenails were examined in 347 incident cases of breast cancer and 374 controls [47]. Mean levels of these compounds did not differ between the study groups, and analysis did not reveal significant trends suggesting a beneficial effect. U.S. cohort studies also have not generally found an association between vitamin E intake or serum concentrations and risk for breast cancer [48\u201351].\n\nAnother micronutrient of current interest as possibly influencing the risk for breast cancer is folate. Historically, vegetables and fruits have been the major sources of folate in the diet, prior to folic acid fortification. Studies with laboratory animals have demonstrated that folate inadequacy promotes DNA strand breaks and hypomethylation of the _p53_ gene [52], indicating a potential biological role in cancer initiation and promotion.\n\nResults from one large prospective study suggest that folate intake, in addition to a possible independent effect, may influence breast cancer risk through an interaction with alcohol [53]. Quite consistently, alcohol intake has been positively associated with risk for breast cancer in epidemiological studies reported during the 1980s and 1990s. The relationship between alcohol intake and breast cancer risk across the range of alcohol consumption among women in developed countries was examined in a pooled analysis of data from six prospective studies that included a total of 322,647 women [54]. Pooled analysis of data from these cohort studies indicates that alcohol intake exhibits a dose-response relationship with risk for breast cancer, at least up to 60 g\/day. The multivariate adjusted relative risk for total alcohol intake of 30\u201360 g\/day (approximately 2\u20135 drinks\/day), compared to nondrinkers, was 1.41 (95% CI 1.18\u20131.69). Source of alcohol (e.g., wine, beer, spirits) did not influence risk estimates in this pooled analysis. Alcohol has well-known effects on folate status, by interfering with the metabolism of folate and thus increasing the requirement if normal folate-related functions are to be maintained at an optimal level. In a prospective cohort study involving 88,818 women with 3483 incident breast cancer cases over a 16-year follow-up period [53], folate intake \u2265600 \u03bcg\/day compared to 150\u2013299 \u03bcg\/day was associated with a multivariate relative risk of 0.55 (95% CI 0.39\u20130.76, _P =_ 0.01 for trend) among women who consumed \u2265 15 g\/day of alcohol. These data would suggest that one mechanism by which alcohol influences risk for breast cancer is through an effect on folate status, because that risk appears to be attenuated by increased folate intake. The prevailing hypothesis has been that alcohol intake promotes increased serum estrogen levels, which could explain the link to breast cancer risk. As reviewed by Ginsburg [55], results from controlled studies suggest that the interaction between alcohol and estrogen is more complicated than has been suggested. In premenopausal women, acute and chronic alcohol consumption appears to increase serum estrogen. In postmenopausal women, acute alcohol consumption increases serum estradiol in users of estrogen replacement therapy, but does not increase circulating estrone or estradiol levels in postmenopausal women who do not use estrogen replacement therapy [55].\n\nAs noted above, dietary fiber has been suggested to reduce risk for breast cancer, by perhaps mediating levels of circulating estrogens. This proposed biological mechanism is based on potential interference with enterohepatic estrogen circulation, which is a normal aspect of estrogen metabolism. In this process, about one-third to one-half of the circulating estrogens are secreted in the bile, followed by reabsorption of about 80% of these estrogens [56]. In animal models, dietary fiber has been shown to promote a reduction in circulating estrogen levels as a result of binding in the intestinal lumen, which results in a decreased efficiency of estrogen reabsorption. Among the types of cereal grains and bran that have been tested, those with the highest percentage of lignin, such as wheat bran, exhibit the highest binding affinity for estrogen [57]. However, case-control and cohort studies that have examined the relationship between fiber intake and risk for breast cancer have generally not found a significant protective effect [23, 25]. Limited support for a protective effect of dietary fiber in epidemiological studies, in spite of an evident biological effect in laboratory animal studies, has been attributed to methodological limitations in diet assessment, which may be greater for fiber than for many other dietary factors, including imprecise food content data and limitations of assessment instruments in categorizing subjects by fiber intake.\n\nIn ecological studies, countries that consume greater amounts of soy and soy products, mainly native Japanese and Chinese populations, have historically exhibited the lowest breast cancer mortality rates, when compared with the United States and most European countries [58]. Soy is the richest source of phytoestrogens in the diet, and these soy isoflavones have been shown to exert hormonal effects in cell culture systems and laboratory animal models [59]. Thus, a role in breast cancer prevention for soy and soy isoflavones, as well as other phytoestrogens, has been investigated in numerous laboratory and epidemiological studies. As reviewed by Herman _et al._ [60], both lignans, found in whole grains, fruits, vegetables, and seeds, and the isoflavone phytoestrogens, found in soy and most soy products, have been shown to bind to estrogen receptors and exhibit weak estrogenic activity. Cell culture studies have further shown that genistein, which is the principal isoflavone in soy, has additional biological activities that could be anticarcinogenic in the human biological system, such as an inhibitory effect on tyrosine kinases and related signaling pathways and angiogenesis, and thus could promote differentiation [61]. Because phytoestrogens can act as estrogen agonists as well as antagonists, however, it is possible that phytoestrogens could potentially promote cancer-related changes in mammary cells. This would be most relevant for postmenopausal women, who do not have high levels of endogenous estrogens in the circulation that would compete for estrogen receptor binding sites. Indeed, results from cell culture studies have shown both beneficial and also adverse effects on the growth of mammary epithelial cells, and these observations may be due in part to the concentration of phytoestrogens in the cell culture medium [62].\n\nThe reader is referred to Murkies _et al._ [63] for a comprehensive review of the clinical evidence linking phytoestrogens to chronic disease, including breast cancer. Overall, evidence for an effect of soy isoflavones and flaxseed (a source of lignans) on serum hormone concentrations in premenopausal women is inconsistent [59, 64\u201367]. Clinical evidence for a modest estrogenic effect in response to soy feeding has been observed in studies involving postmenopausal women, as reviewed by Kurzer [59]. Results from case-control studies that have examined the relationship between dietary soy intake and breast cancer risk among Asian and Asian-American populations have been inconclusive [68]. Thus, more clinical studies of the biological effects that might increase or decrease risk for breast cancer are clearly needed before conclusions or consensus opinions on soy or other sources of phystoestrogens can be formulated.\n\nTable 1 lists nutrients and other dietary constituents that have been the focus of research on the link between dietary intakes and risk for breast cancer.\n\nTABLE 1\n\nNutrients, Foods, and Other Dietary Factors Suggested to Be Associated with Risk for Primary Breast Cancer\n\n#### 2. ANTHROPOMETRIC CHARACTERISTICS AND RISK FOR BREAST CANCER\n\n##### a. Associations with Skeletal Markers.\n\nPreviously thought to be a predictive factor only in populations where energy restriction was significant enough to affect skeletal growth, adult height has emerged as one of the more consistent risk factors for breast cancer in both pre- and postmenopausal women [69\u201371]. Large cohort and case-control studies conducted among American, European, and Asian women consistently suggest that adult height is a significant risk factor for this disease [71\u201375]. Among U.S. women ( _N_ = 3430), Brinton and Swanson [72] found that height greater than 68 inches was associated with breast cancer risks that were 50\u201380% higher than height \u226462 inches. It has been hypothesized that growth factors that drive skeletal development also play a role in stimulating the proliferation of mammary stem cells and thereby increase mammary mass [70]. Although many of these growth factors may be more under the control of heredity rather than diet, recognition of height as a risk factor may be of some benefit by allowing identification of high-risk women who then could be targeted for interventions. Other skeletal indices, such as increased elbow breadth (a marker of frame size), femur length, and bone density also have been associated with increased risk [72, 76]. To date, however, studies have failed to show any association between skeletal markers and disease progression or mortality, in contrast to anthropometric markers of adiposity (discussed in the following section).\n\n##### b. Associations with Adiposity.\n\nHippocrates was the first to report an association between obesity and breast cancer. The classic studies by Tannenbaum in the 1940s confirmed this premise, and in 1975 deWaard put forth the hypothesis that body nutriture affects risk\u2014an effect mediated by hormonal levels. Despite these observations, it should be noted that a clear discrepancy exists with regard to adiposity and its ability to portend risk for premenopausal versus postmenopausal disease, with relative body leanness serving as a risk factor for disease occurring before menopause and obesity serving as a risk factor for postmenopausal breast cancer [69, 77, 78]. Many have speculated about the reasons for these differences, with some theorizing that body leanness may enhance early detection among younger women, whereas others adhere to the belief that premenopausal breast cancer is distinctly different from postmenopausal disease, being governed more by genetic predisposition and growth factors rather than by long-term exposure to the interacting effects of ovarian steroid hormones and lifestyle factors [70, 79].\n\nIn addition to obesity, body fat distribution also appears to play a role in predicting risk for obesity. A majority of studies support central or visceral obesity (primarily assessed via waist:hip ratio) as an additional risk factor [69, 73, 80]. Results from a large cohort study by Sonnenschein _et al._ [81] ( _N_ = 8157) suggest that waist:hip ratio serves as an independent risk factor for premenopausal breast cancer, in which the increased presence of abdominal fat may be linked to increased levels of insulin and related growth factors, whereas among postmenopausal women, waist:hip ratio may serve as another indicator of obesity. Studies by Sellers _et al._ [82] and London _et al._ [83] suggest that risk conferred by either obesity or waist:hip ratio may be further increased by a positive family history.\n\nGiven that weight is not a static measure and fluctuates throughout life, it is conceivable that risk may be modified by the presence of obesity or body weight status at differing ages. Although there is a dearth of data, some researchers have speculated that the _in utero_ experience, the hormonal milieu of the host mother and her weight gain during pregnancy, may establish a \"gonadostat\"\u2014a hormonal thermostat that governs the hormonal levels of progeny after birth and thereby influences risk for hormonally linked cancers [78]. Further research is necessary to either support or refute the presence of hormonal setpoints and the importance of maternal host factors. Indeed, one of the more severe limitations to such research, as well as any studies that assess lifelong exposure, is the reliance on case-control studies and data that for the most part are collected retrospectively.\n\nStudies that have assessed weight prior to or during early adulthood suggest that obesity may actually be protective [84, 85]. This relationship appears logical for premenopausal breast cancer, in which relative obesity is already an acknowledged protective factor. However, given the relationship between increased weight and early menarche, it is difficult to reason why early obesity would be protective for postmenopausal breast cancer. Recent studies, however, indicate that although increased body weight during childhood is predictive of early menarche (among both tall and obese girls), obese girls have significantly fewer ovulatory cycles and thereby have lower circulating levels of both estrogen and progesterone\u2014hormones that are known to stimulate breast cancer growth [86, 87].\n\nOn attainment of adulthood, especially during later adulthood, there appears to be a consistent finding of weight gain and increased body weight being highly associated with increased risk for postmenopausal cancer [69, 71, 77, 88, 89]. The reader is referred to reviews by Ballard-Barbash [69] and Ziegler [71], who provide detailed information and interpretation of body weight and other anthropometric markers in relation to breast cancer risk. In the case of risk associated with adult weight gain, this increased risk has been largely explained by the fact that as women age, their circulating levels of estrogen become more influenced by estrogens produced by adipose tissue rather than those produced by the ovary [79, 89]. Weight gain after age 40 years is also is more likely to be deposited in an android versus gynoid pattern and hence may foretell insulin resistance and the increased production of insulin and insulin-like growth factors that may act synergistically with estrogen to confer risk [69, 77, 88].\n\nAdditionally, increased body weight at diagnosis has been recognized as a poor prognostic factor for breast cancer for more than 25 years [90\u201392]. While obese women are more likely to exhibit advanced stage disease at the time of diagnosis, more than 13 large cohort and case-control studies suggest increased body weight as a risk factor for recurrent disease even after controlling for menopausal status, age, stage, tumor size, and nodal involvement [71, 91\u201394]. Furthermore, weight gain after diagnosis also increase risk for recurrent disease and mortality (discussed below).\n\nBecause body weight is a modifiable risk factor, diet and physical activity may be beneficial for the prevention and management of breast cancer. Few data, however, currently exist regarding weight loss relative to risk. Results are conflicting and confounded by the inability of studies to discern whether reported weight loss was the result of voluntary or involuntary efforts [69].\n\n#### 3. PHYSICAL ACTIVITY AND RISK FOR BREAST CANCER\n\nThe first associations between cancer and physical activity were reported by Taylor _et al._ [95] in 1962. They found that all-cancer mortality rates were significantly increased among sedentary office workers when compared to workers who had more physically demanding positions. During the past 30 years, several investigations have specifically explored the role of physical activity in relation to colon cancer\u2014investigations in which consistent inverse associations have been found [96, 97]. In comparison, far fewer studies that have addressed associations between physical activity and breast cancer have been conducted or reported, and these have been fairly recent. Frisch _et al._ [98] were the first to report a protective effect of exercise for breast cancer. In a study of 5398 college alumni (2622 of whom were former college athletes and 2766 were nonathletes), they found that nonathletes had a relative risk of 1.86 (95% CI 1.00\u20133.47) compared to athletes. The investigators credited risk reduction to the ability of exercise to delay the onset of menarche [98]. Research in this area remained fairly fallow until Bernstein _et al._ [99] reported findings of a large case-control study (545 breast cancer cases and 545 age, race, parity, and neighborhood-matched controls) that found women who spent 3.8 hours or more per week in physical exercise activities were significantly less likely to develop breast cancer (odds ratio 0.42, 95% CI 0.27\u20130.64). During the late 1990s, more than two dozen case-control and cohort studies explored associations between physical activity and breast cancer risk. Although findings of these studies have not detected protective effects as strong and consistent as those obtained in studies of colon cancer, a majority of the studies (18 of 26) suggest that increased physical activity is inversely related to breast cancer risk [96, 97, 100, 101]. The reader is referred to comprehensive reviews by Friedenreich and Rohan [100] and Gammon _et al._ [101]. Because results from animal studies suggest that exercise decreases tumor burden across cancers [102], some speculate that the odds ratios observed in breast cancer studies may be attenuated due to an inability to control for reproductive factors (in many studies, these data were unavailable) or a limitation of instruments to assess physical activity related to housework and childrearing [97]. The protective effects of physical activity are hypothesized to be due to the ability of exercise to mediate levels of endogenous hormones and ovulatory cycles, affect energy balance, and enhance immune response [103]. Studies by Thune _et al._ [104] and Latikka _et al._ [105] suggest that exercise may be even more protective among lean women, because they are more likely to become anovulatory with increased physical activity.\n\n### B. Nutritional Factors, Prognosis, and Risk for Cancer Recurrence\n\nFactors associated with risk for primary breast cancer are generally assumed to have a high likelihood of association with risk for recurrence following the diagnosis, although actual reported cohort studies addressing this issue are still limited in number (i.e., less than a dozen that examined a role for dietary factors have been published to date). Notably, much of the evidence for biological activity that is cited to support a link between nutritional factors and risk for primary breast cancer is based on studies using cell lines derived from breast cancers or transformed cells (i.e., cells that already have molecular changes leading toward breast cancer). Most of the epidemiological studies that have addressed whether nutritional factors might be associated with improved prognosis or longer breast cancer-free survival time have relied on data that were collected at the time of diagnosis. This approach assumes that spontaneous, substantial changes in food choices or other factors would be unlikely or difficult for individuals following the diagnosis, which may or may not be true. Risk for recurrence and\/or overall survival (i.e., risk of death) are the dependent variables in these investigations, and associations with nutritional factors are typically similar for these two outcomes because they are highly correlated. Another important characteristic that affects interpretation of results from the studies that attempt to link diet with survival is the adjustment for factors that are known to influence survival, such as stage at diagnosis. If not considered in the analysis, these characteristics could confound the associations and may even influence factors such as body weight and food choices at diagnosis.\n\nAs noted above, the most consistent association with survival, among the various nutritional factors, is the link between degree of overweight at diagnosis and risk for recurrence. For example, in 557 women diagnosed with negative-node-negative breast cancer, those with body weight greater than 25% of desirable weight for height at diagnosis had significantly reduced rates of disease-free survival compared with nonobese women (hazard ratio 1.93, 95% CI 1.29\u20132.88) [106]. Among 735 women with lymph node-positive breast cancer who received adjuvant chemotherapy, the adjusted proportional-hazards regression model indicates that risk for disease recurrence over a 10-year period for patients more than 20% over desirable weight for height was 1.33 times that of patients not more than 20% overweight (95% CI 1.05\u20131.68) [107]. In the Iowa Women's Health Study, women in the highest tertile of body mass index at diagnosis of breast cancer had an age-adjusted 1.9-fold higher risk (95% CI 1.0\u20133.7) of dying during a median follow-up time of 2.9 years after diagnosis, when adjusted for other prognostic variables [108].\n\nSeveral studies have associated decreased survival with dietary fat intake at diagnosis, adjusted [109\u2013111] or unadjusted [108, 112, 113] for energy, whereas others [114\u2013116] did not detect this association. The issue of energy adjustment is illustrated in the findings from a study by Saxe _et al._ [117], in which both energy (hazard ratio 1.58, 95% CI 1.05s\u20132.38) and total fat (hazard ratio 1.46, 95% CI 1.05\u20132.01) intakes were directly associated with risk for recurrence, but the significance of the association with fat intake disappeared when energy-adjusted. In a study that used a food-based approach to describing dietary intake, risk for recurrence was associated with intakes of butter, margarine, and lard (servings\/day) with a risk ratio of 1.67 (95% CI 1.17\u20132.39) among premenopausal women, adjusted for disease stage and age [118]. In a prospective study by Holmes _et al._ [119] involving 1982 women diagnosed with breast cancer, in which dietary data (from food frequency questionnaires) were collected preceding and following the diagnosis, and numerous adjustments were performed to separate the influence of dietary factors, no apparent association between fat intake and mortality was observed. However, protein intake, mainly linked to poultry, fish, and dairy food sources, was found to exert a protective effect (relative risk 0.65, 95% CI 0.47\u20130.88).\n\nProtective effects for vegetables and fruits and the associated micronutrients, vitamin C and the carotenoids, also have been observed in several of these follow-up cohort studies [109, 111, 119, 120], with more consistency than has been observed for dietary fat. Rohan _et al._ [111] found that women consuming \u22658058 mg beta-carotene per day at diagnosis, compared to women consuming \u22643051 mg\/day, had a 32% reduction in risk of death over a mean of 5.5 years follow-up, although this difference was only marginally significant ( _P_ = 0.075 for trend) when adjusted for energy and other risk factors. Ingram [120] found total fruit and vegetable ( _P_ = 0.04), beta-carotene ( _P_ = 0.001), and vitamin C ( _P_ = 0.03 for trend) intakes at diagnosis to be associated with increased likelihood of survival in a cohort of 103 women diagnosed with breast cancer and followed for 6 years, although this analysis was not adjusted for stage at diagnosis. Similar findings were reported from a cohort of 678 women diagnosed with breast cancer followed for nearly 8 years [109]. The hazard ratio for dying of breast cancer in this study was 0.48 (95% CI 0.23\u20130.99) for the highest compared to the lowest quartile of beta-carotene intake, and was 0.43 (95% CI 0.21\u20130.86) for vitamin C intake, adjusted for stage, tumor characteristics, age, and menopausal status. In the largest cohort of this type reported to date (described above), increased vegetable and fruit intake (\u22654.2 versus \u22642.1 servings\/day) was associated with decreased mortality in the women without metastatic disease at diagnosis [119]. This association was generally supported by data on the relevant individual nutrients provided by these foods. For example, the multivariate relative risk of death for lutein + zeaxanthin intake was 0.87 (95% CI 0.62\u20131.21, _P =_ 0.04 for trend), comparing the highest to the lowest quintile.\n\nAlthough alcohol intake has been identified as a risk factor for primary breast cancer, as discussed above, follow-up studies in which this variable has been examined have rather consistently not found a relationship between alcohol intake and overall survival [110, 111, 117, 119]. Earlier cohort studies of women diagnosed with breast cancer also did not find an association between dietary fiber intake and likelihood of survival [109\u2013111, 120], although data from more recent reports are not in agreement. Energy-adjusted bread and cereal consumption was found to be protective against recurrence, although quantified dietary fiber intake per se was not significantly associated, in a recent cohort study of 149 women followed for >5 years following diagnosis [117]. In the largest of this type of cohort followed to date, relative risk of death was significantly linked to dietary fiber intake (relative risk 0.69, 95% CI 0.50\u20130.97), comparing the highest to the lowest quintile [119].\n\n## III. NUTRITIONAL ISSUES FOLLOWING THE DIAGNOSIS OF BREAST CANCER\n\nDuring the 1980s and 1990s, the development and use of effective screening technologies have resulted in an increased ability to detect cancers at earlier stages when treatments are most effective. With increased breast cancer awareness and the use of screening mammography, a majority of all breast cancers are now diagnosed at a localized stage, with 96% 5-year relative survival rates [121]. These successes have resulted in a paradigm shift, because breast cancer is now managed largely as a chronic disease, rather than treated as an acute fatality [122]. Therefore, although the next section will address nutritional concerns that may emerge in the months that immediately follow diagnosis, nutrition interventions for patients with breast cancer have become largely focused on promoting long-term health via healthful eating, a physically active lifestyle, and the avoidance of obesity [123\u2013125]. However, little is known about what constitutes an optimal diet during the initial treatment for breast cancer, no diets or dietary supplements have yet to be proven to be helpful, and little is currently known about nutrient-treatment modality interactions [126]. Indeed, further research is sorely needed in this area.\n\n### A. Issues and Interventions in the Initial Treatment of Breast Cancer\n\n1. SURGERY-RELATED NUTRITIONAL ISSUES\n\nAlmost all patients with breast cancer, except those with inflammatory breast cancer, receive surgery (i.e., either mastectomy or lumpectomy) as a primary treatment for their disease. Some receive axillary node dissection or sentinel node biopsy to provide additional information that can be used to help guide decision making for adjuvant forms of treatment. In some cases, such as when tumors are excessively large, diffuse, or difficult to excise, neoadjuvant therapy is given prior to surgery with the expectation that this will reduce the tumor and thus make it easier to remove. In most cases, however, surgery is usually the first, and sometimes the only, treatment received [127].\n\nIn contrast to other cancers, such as head and neck, in which patients often are malnourished on diagnosis, or in some gastrointestinal cancers in which prolonged postsurgical recovery may impinge upon nutritional status, patients with breast cancer are unlikely to have nutritional deficiencies and are also unlikely to manifest them either at diagnosis or during the postsurgical period [128]. This includes any additional surgeries that might be pursued for reconstructive purposes. Therefore, there is seldom a need to promote any particular diet other than one that is healthful. Exceptions to the rule, however, do occur and standard procedures to assess nutritional status and to intervene, if necessary, are still warranted (discussed below).\n\nGiven the important role of obesity in influencing risk for breast cancer among postmenopausal women, and the role it plays as an unfavorable prognostic factor in both pre- and postmenopausal disease, patients with breast cancer should be encouraged to achieve a desirable weight [69, 77, 129]. Interventions that promote the avoidance of weight gain are prudent during surgical recovery, and also during the completion of any adjuvant chemotherapy and\/or radiation therapy [129]. Active efforts toward weight loss, however, are most often delayed until wound healing and adjuvant treatment are complete, given concern that energy restriction may adversely affect immune function and healing [130].\n\n#### 2. ADJUVANT CHEMOTHERAPY\n\nChemotherapy is usually offered to breast cancer patients who are either at increased risk for systemic disease or among those in whom metastases have been identified. Side effects of chemotherapy include hair loss, neutropenia, mu-cositis, dysphagia, dysgeusia, nausea, vomiting, and, surprisingly, weight gain [91, 92].\n\nIn recent years, the development and use of antiemetic agents have greatly relieved the side effects of nausea and vomiting. However, a substantial number of patients with breast cancer still report these effects. Small and frequent feedings that are low in fat and comprised primarily of carbohydrates are often effective in relieving symptoms. Altered sense of taste also is frequently reported, with many patients complaining that foods do not taste as they used to, ''sweet foods taste too sweet,\" and ''meats taste metallic.\" In responding to these taste alterations, some patients may gravitate toward bland foods, such as rice, potatoes, and gelatin, whereas others seek out salty, spicy, or tart foods [131].\n\nA smaller number of patients develop mucositis, which generally appears in mid- to late therapy [127]. Mouth sores may be exacerbated by foods with high acidity, such as salad dressings and fruit juices, as well as flinty foods such as tortilla chips. Difficulty swallowing is even more rare, and appropriate intake usually can be achieved with cool soothing foods such as gelatins and yogurts, as well as the use of sauces or gravies on solid food items. Fortunately, these symptoms are often short lived, usually lasting for a portion of the week following chemotherapy administration.\n\nSeemingly in direct contrast to these side effects is the side effect of weight gain. Previous studies suggest that weight gain is especially pronounced among premenopausal women, and among those who have nodal involvement and who receive multiagent regimens [91, 92]. Gains in weight commonly range from 2.5 to 6.2 kg; however, greater gains are not unusual. Boyd reports that one out of four premenopausal patients with breast cancer gains more than 11 kg during the course of adjuvant chemotherapy [132].\n\nThis weight gain is undesirable for several reasons. First, it may negatively affect quality of life. Previous studies indicate that a majority of patients with breast cancer find weight gain distressing and report it as a major concern [133\u2013136]. Second, this weight gain may predispose women to other diseases and weight-related problems, such as hypertension, cardiovascular disease, gallbladder disease, orthopedic disturbances, and diabetes mellitus [122, 137]. Such disease can cause significant morbidity and mortality, which continues once management of breast cancer is achieved. Finally, although not conclusive, there is evidence to suggest that weight gain adversely affects disease-free survival. Camoriano _et al._ [133] followed 646 patients with breast cancer for a median of 6.6 years and found that premenopausal patients who gained more than the median amount of weight (5.9 kg) were 1.5 times more likely to relapse and 1.6 times more likely to die of their breast cancer. Results from studies conducted by Chlebowski _et al._ [138] and Goodwin _et al._ [139] parallel these findings and suggest a need for interventions that prevent the prevalent problem of weight gain in this population.\n\nTo date, only a handful of interventions to prevent gains in weight that specifically occur during adjuvant chemotherapy have been tested, and these have been met with mixed results. In a randomized trial of 104 early stage breast cancer patients, Loprinzi _et al._ [140] found that patients who received intensive diet counseling on energy-restricted diets failed to gain significantly less weight than those assigned to the control arm. In contrast, a phase II study by Goodwin _et al._ [141], who tested a structured program of diet (an energy-restricted, low-fat, and high-fiber diet) and exercise on 61 newly diagnosed, loco-regional breast cancer patients, found this multifaceted intervention to be successful. Follow-up analyses revealed that the strongest predictor of this program's success was increased physical activity, not diet. A cross-sectional study by Rock _et al._ [142] also points to the role of physical activity as an important factor governing weight gain in women following the diagnosis of breast cancer.\n\nAlthough much more research is needed, results of these studies provide support for interventions to include, if not center on, physical activity. This premise is further supported by findings of Aslani _et al._ [143], Cheney _et al._ [144], Demark-Wahnefried _et al._ [145, 146], and Kutynec _et al._ [147], who by differing methods (i.e., dual energy X-ray absorptiometry, computer-assisted tomography, _in vivo_ neutron capture analysis) found that the weight gain experienced by patients with breast cancer occurs either in the absence of any gains in lean tissue or in the presence of lean tissue loss. This distinctive weight gain pattern, increased gains in adipose tissue with concurrent with losses of muscle mass, is a condition usually associated with the chronic use of corticosteroids, hypopituitarism, neuromuscular diseases, hypogonadism, prolonged inactivity or bed rest, and menopause, and one in which resistance training and aerobic activity represent the cornerstones of treatment. Because most physical activity interventions directed toward this population have either been inadequately controlled or focused on other endpoints (i.e., improvement in quality of life, reduction of nausea), much more research is necessary to determine optimal interventions for this population with regard to both content and delivery [148]. Given that most well-controlled studies do not support increased energy intake as a cause of weight gain in this population, it is doubtful that energy restriction should play a major role in these interventions. Also, an overemphasis on dieting or energy restriction may exacerbate eating pathology and psychological distress in these patients [149]. However, these patients may benefit from programs that promote plant-based diets that are high in calcium and low in fat [92, 131, 146, 147, 150]. Furthermore, there may be a need to develop discrete interventions that respond to the specific needs of both premenopausal and postmenopausal patients.\n\n#### 3. NUTRITIONAL ISSUES ASSOCIATED WITH RADIATION THERAPY\n\nRadiation therapy is often used to establish local control of breast cancer. Because radiation fields rarely include the alimentary tract, there are usually no direct nutrition-related effects. Indeed, side effects appear to be limited to mild erythema and fatigue [151]. As with chemotherapy, patients actually may feel energized if they pursue routine physical activity during the period that they receive these treatments (usually a time period of 6 weeks). Several physical activity interventions have been pursued within this population as a means to improve quality of life and increased functional capacity; however, for the most part, these efforts have been uncontrolled and small in size [148].\n\n#### 4. NUTRITIONAL ISSUES ASSOCIATED WITH HORMONAL THERAPY\n\nMegestrol acetate is a long-standing hormonal agent used in the treatment of breast cancer [91]. Today, the use of megestrol acetate is usually confined to women who have later stage disease. One of the more noteworthy side effects of this agent is weight gain, an effect so reliable that megestrol acetate is marketed as an anticachexic agent for other cancers. Although one might assume that the issue of weight gain would be less of a concern among women with later stage disease, a study by Kornblith _et al._ [152] suggests that most patients still find this side effect distressing. To date, no interventions aimed at weight control have been reported in this select patient population.\n\nTamoxifen and next-generation selective estrogen receptor modulators represent hormonal agents that have been offered to women with earlier stage disease. In the past, these were only offered to women with estrogen receptor-positive tumors; however, recently their use has become more widespread, as agents for cancer control and as agents for cancer prevention [153]. Reports of weight gain with tamoxifen have been mixed, with some studies reporting gains [154] and some not [153]. A body composition study by Ali _et al._ [155], however, suggests that irrespective of weight gain, tamoxifen is associated with increases in body fatness. Thus, women prescribed tamoxifen and\/or next-generation estrogen receptor modulators also may benefit from interventions that include increased physical activity; however, no such studies have been reported to date.\n\nAn additional concern with these hormonal agents is their potential interaction with foods or supplements that are rich in phytoestrogens, such as soy foods and soy supplements. Speculation exists that specific phytoestrogenic compounds may interfere with the action of hormonal agents and reduce their effect, or perhaps stimulate the growth of initiated breast cancers by themselves [156]. Further research is necessary to determine potential interactions or direct effects, but until then, the risks and\/or benefits of supplemental genistein, daidzein, or other related compounds are unknown and they cannot be recommended. Likewise, the use of soy foods above moderate levels also cannot be supported without further research.\n\n#### 5. ADDITIONAL CONCERNS\n\nIn 1993, Monnin _et al._ [136] reported the results of a descriptive study of 143 early stage breast cancer patients who were participating in a community-based Reach to Recovery Program. Respondent interest was high for diets aimed at cancer prevention (60%), reduced fat (56%), and weight reduction (48%). Although somewhat dated, this study suggests that patients with breast cancer are interested in nutrition-related programs, findings that were supported in a study by Demark-Wahnefried _et al._ [157], who found high levels of interest in both diet (57% were ''very\" to ''extremely\" interested) and exercise (56% were ''very\" to ''extremely\" interested) programs among 531 breast cancer survivors. Fifty-two percent of these respondents indicated a preference for interventions to be offered within 3 months of diagnosis. These findings suggest that interventions are likely to be greeted with good reception and high levels of interest; however, again more research is necessary to truly discern the unique barriers, the specific content, and the methods of delivery in order to promote optimal efficacy.\n\nPrevious studies also suggest that the majority of patients with breast cancer use either vitamin or mineral or herbal supplements 136, 157, 158]. While general concerns relating to herbal supplements (see [Chapter 17) and vitamin and mineral supplements [159] have been previously addressed, there is a paucity of well-controlled research studies that have either supported or refuted the use of supplements among women who have already been diagnosed with breast cancer. Concern exists, however, with regard to supplement use during treatment, because of speculation that high-dose supplemental nutrients may stimulate cell repair of neoplastic cells, which are the intended targets of both chemotherapy and radiation therapy, while others argue that supplements may render cancer cells more chemosensitive by enhancing cell cycling [160]. Again, further research is necessary to determine the potential benefits and\/or risks of nutritional and herbal supplements with respect to their use during treatment.\n\n### B. The Long-Term Perspective: Issues and Interventions under Study\n\nNutrition and breast cancer is currently an active area of research, with several studies testing the relationship between dietary intake and cancer incidence. In addition to studies aimed at primary prevention, two large ongoing multicenter studies are examining whether diet modification can influence the risk for recurrence and the overall survival following the diagnosis of breast cancer.\n\nIn the Women's Health Initiative, postmenopausal women aged 50\u201379 years are enrolled at 40 clinical centers nationwide into a clinical trial ( _N =_ 64,000) or an observational study ( _N_ = 100,000) [161]. One arm of the randomized clinical trial is testing whether a low-fat eating pattern (\u226420% of energy from fat, \u22655 servings\/day vegetables and fruits, \u22656 servings\/day grain products) may help to prevent breast cancer, with colorectal cancer and cardiovascular disease as additional study outcomes under evaluation in that arm. The Canadian Diet and Breast Cancer Prevention Trial, a multi-center randomized controlled study, is testing whether a low-fat (15% of energy), high-carbohydrate diet intervention can reduce the incidence of breast cancer over a 10-year period among women aged 30\u201365 years who have increased mammographic density [162]. Studies suggest that mammographic density is a marker of histopathologic changes in breast tissue associated with increased risk for cancer [163], and preliminary studies indicate that a reduction in dietary fat intake may promote a reduction of mammographic density in the breast [164, 165]. Other studies are targeting women with a family history of breast cancer, testing whether a low-fat diet (<15% of energy) and\/or nine daily servings of vegetables and fruits is feasible in this group and is associated with changes in oxidative DNA levels [166, 167].\n\nTable 2 summarizes the key features of two ongoing multicenter randomized clinical trials that are testing whether diet intervention can reduce risk for recurrence in women who are breast cancer survivors. In the Women's Intervention Nutrition Study, the primary dietary goal is a reduction in dietary fat intake (\u226415% energy from fat) [168]. Preliminary data indicate good adherence, with an average reduction from 33% to 20% energy from fat at 6 months into the study [168]. In the Women's Healthy Eating and Living Study, the primary emphasis is on increased vegetable and fruit intake, with daily dietary goals of five vegetable servings, 16 ounces of vegetable juice, three fruit servings, 15\u201320% energy from fat, and 30 g dietary fiber. Feasibility study reports and preliminary trial data from this study also indicate excellent adherence [169\u2013171]. Key outcomes of interest in both of these studies are recurrence-free survival and overall survival, and study participants are followed for an average of 6 years.\n\nTABLE 2\n\nKey Features of Two Large Ongoing Multicenter Randomized Clinical Trials Testing Whether Diet Intervention Can Reduce Risk for Recurrence of Breast Cancer in Women Diagnosed with Early Stage Disease\n\nUntil more definitive evidence becomes available, published dietary guidelines, such as those from the American Cancer Society [172, 173] and the American Institute for Cancer Research [174], form the basis of current recommendations. Among the published dietary guidelines, those that appear to be most relevant to the prevention and control of breast cancer, based on current knowledge, emphasize plant-based diets with increased intake of vegetables and fruit, the promotion of increased physical activity to promote healthy weight management, and limited consumption of alcohol (for primary prevention).\n\nThe risk for morbidity and mortality from causes other than breast cancer should be considered in dietary recommendations for breast cancer survivors, especially those diagnosed with early stage cancers 124]. Due to heightened health concerns, patients with breast cancer often are highly motivated to adhere to dietary recommendations that may improve overall health. For example, even though the current evidence to support a link between fat intake and risk for breast cancer (or the prognosis following diagnosis) is not strongly supportive, limiting saturated fat intake is a well-established strategy to promote a plasma lipoprotein and lipid profile that is associated with reduced risk for cardiovascular disease (see [Chapter 18). Similarly, eating a diet with adequate dietary fiber has been associated with decreased risk of coronary heart disease in women [175] and may contribute to overall health, irrespective of a possible link between fiber and breast cancer.\n\nA long-term effect of chemotherapy that can occur in women who are premenopausal at diagnosis is ovarian dysfunction. In women at risk for breast cancer recurrence, whatever their age, estrogen replacement therapy may not be an option, so dietary factors and lifestyle behaviors that may reduce risk for osteoporosis can be important in long-term care. Although tamoxifen and other selective estrogen receptor modulators have been observed to promote maintenance of bone density in postmenopausal women [176], many women are not prescribed these drugs because they are not considered good candidates for this type of pharmacologic treatment and, when prescribed, the treatment duration is typically \u22645 years. Sufficient dietary calcium, adequate vitamin D intake, and increased physical activity are particularly appropriate recommendations to maintain bone health in this population.\n\n## IV. SUMMARY AND CONCLUSIONS\n\nBreast cancer is the most common cancer among women in the United States and is a disease associated with multiple genetic, hormonal, environmental, and lifestyle factors. Diet is presumed to play a significant role in both the risk of breast cancer and the progression of disease. To date, however, given the dearth of findings from randomized, controlled clinical trials, which represent the ultimate test of the effects of nutritional factors on disease, there is little that can be definitively stated. Fortunately, within the next decade, results from large diet intervention trials should be able to provide solid information that can be used to develop evidence-based interventions and guidelines for both the prevention and treatment of breast cancer. Until then, findings from epidemiologic and laboratory studies provide some insight. Currently, good support exists for women to choose diets that are plant based (i.e., rich in vegetables and fruits) and limited in alcohol, which may reduce risk for primary breast cancer. While support for fat restriction in the prevention of breast cancer is conflicting, it must be borne in mind that although breast cancer is a major health concern for U.S. women, cardiovascular disease remains the largest cause of mortality. 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Med_. 2000;343:191\u2013198.\nCHAPTER 23\n\nNutrition and Colon Cancer\n\nMARTHA L. SLATTERY1 and BETTE J. CAAN2\n\n1University of Utah, Salt Lake City, Utah\n\n2Kaiser Foundation Research Institute, Oakland, California\n\n## I. INTRODUCTION\n\nIn the United States, approximately 130,000 new cases and 55,000 deaths are attributed to colorectal cancer yearly, making it one of the most common cancers for both men and women [1]. Worldwide, an estimated 875,000 cases of colorectal cancer occurred in 1996, accounting for 8.5% of all incident cancers [2]. A 20-fold variation in reported incidence of colorectal cancer has been noted around the world, with developed countries having high incidence rates and developing countries having much lower incidence rates. Furthermore, migrant populations who move from countries of low to high incidence adopt the rates of the host country [3]. These facts suggests that changes in diet and lifestyle that correlate with changes in populations as they become more developed are associated with colorectal cancer. Much research attention has been focused on diet\u2014with the expectation that identification of specific dietary factors that contribute to the observed variation and to changing incidence rates will provide avenues for prevention of colorectal cancer.\n\nOne way to explore dietary links to colorectal cancer is through ecological studies, which correlate differences in colorectal cancer mortality with differences in population intakes of nutrients such as fat, fiber, and calcium [4\u20136]. Although far from definitive, these studies suggest that populations with diets low in fat and high in fiber or calcium have lower mortality from colorectal cancer. Another approach is analytic epidemiological studies, which have produced inconsistent findings regarding the relationship between many dietary factors and colorectal cancer.\n\nCase-control studies comparing recalled dietary differences in those with and without cancer, and cohort studies linking reported diet to disease development have also singled out many suspect dietary components. More than 75 case-control and cohort studies have been conducted, often with inconsistent findings. Conflicting results can stem from many sources, including study design, methods used to collect and analyze data, age and sex of the population being studied, range of dietary exposures captured from the questionnaire, the referent period for which the dietary data were collected, and possibly the tumor site itself. In the case-control study, participants are asked to recall past dietary intake; it is possible that recalled dietary intake might be influenced by changes in diet as a result of disease. Cohort studies frequently have limited ranges of dietary exposure, because they are often based on a more select population than population-based case-control studies. If dietary factors are only linked at high levels of intake, associations might be missed in cohort studies because of a truncated range of dietary intake. Data are usually obtained from self-administered questionnaires in cohort studies, therefore less detailed information on diet and other possible exposures is obtained than with the interviewer-administered questionnaires usually used in case-control studies. These differences can lead to misclassification of levels of dietary intake. For more information on strengths and weaknesses of epidemiological research see Chapter 4.\n\nResults from controlled clinical trials of diet and colonic polyps are providing credence to associations for some dietary factors. Although polyps are precursor lesions to colon tumors, not all polyps progress to tumors. Thus some differences in dietary associations could be expected for studies that focus on adenomas versus adenocarcinomas.\n\nSeveral major hypotheses for mechanisms have emerged in research on nutrition and adenocarcinoma of the colon (Table 1). These hypotheses are overlapping and complementary. They stress the complexity of colon cancer and the difficulty of identifying individual mechanisms, and also elucidate the diverse approaches being used to examine the links between nutrition and colon cancer.\n\nTABLE 1\n\nSummary of Some of the Hypothesized Mechanisms Related to a Diet and Colon Cancer Relationship\n\nModel | Hypotheses \n---|--- \nBile acid\/volatile fatty acid | Bile acid metabolism is central to colon cancer. A high-fat diet increases levels of bile acids, and bile acids damage cells in the colon and induce proliferation. \nFat and fiber | International correlation studies suggest that dietary fat increases risk of colon cancer; fat is associated with bile acid metabolism. Colon cancer was rare among Africans who ate a diet high in whole grains; early case-control studies supported this. \nMeats and vegetables | A variation of the fat and fiber hypothesis. Data suggest variability in cancer risk that correlates with the spectrum of meat and vegetables in the diet [31]. \nCooked foods | Meat prepared at high temperatures increases heterocyclic amines and polycyclic aromatic hydrocarbons. These carcinogens increase risk of colon cancer. \nInsulin resistance | Insulin resistance hypothesis links many of the previously identified risk factors for colon cancer. Increased insulin levels influence cell proliferation and stimulate growth of colon tumors. \nDNA methylation | Dietary factors such as folate, vitamin B6, vitamin B12, methionine, and alcohol are involved in DNA methylation pathways. Levels of intake of these nutrients that lead to availability of low levels of methyl groups cause hypomethylation of DNA. DNA damage leads to colon cancer. \nGrowth medium | Dietary factors act as initiators and promoters of the transition from normal to malignant cell. Dietary factors are involved in apoptosis and cell growth and regulation.\n\nIn this chapter we discuss five different models describing mechanisms by which diet may either promote or prevent colon cancer and review the supporting epidemiologic data (Table 2) for each.\n\nTABLE 2\n\nSummary of Epidemiological Studies of Diet and Colon Cancer Associations\n\n_Note:_ Nutrients fit into multiple models. They are listed with the model with which they have been most aligned.\n\n## II. MODEL 1: BILE ACIDS, FAT\/FIBER, AND MEATS\/VEGETABLES\n\nPerhaps the oldest theory that has guided epidemiologic studies on diet and colon cancer has focused on bile acids [7]. This model and closely related ones focus on a variety of nutrients and form the bulk of the epidemiologic literature. This hypothesis suggests that high-fat consumption stimulates bile acid output and higher concentrations of conjugated bile acid in the colonic contents. In the colon, bile acids are deconjugated and dehydroxylated to form secondary bile acids that, in turn, damage cells and induce proliferation. In more recent years, this model has incorporated both fat and fiber. Fat stimulates bile acids and fiber binds bile acids. Dietary fiber also reduces transit time, increases stool bulk, and helps ferment volatile fatty acids\u2014all of which may help reduce the conversion of primary to secondary bile acids in the colon [8]. The products of fermentable volatile fatty acids include butyrate, which, in addition to being a major colonic epithelial cell fuel, may play a role in apoptosis and cell replication [9]. Pectin, a water-soluble fiber, reduces the rate of glucose absorption and decreases the rate of absorption and\/or availability of lipids.\n\nMeat and vegetable intake is also relevant to this hypothesis [7]. Meat contains high levels of fat and protein that may be harmful to the colon, while vegetables are rich sources of vitamins, minerals, fiber, and phytochemicals (plant substances) that may protect against the development of colon cancer.\n\n### A. Dietary Fat\n\nEcological studies have reported that fat in diets varies directly with rates of colon cancer deaths [6]. While many early case-control and cohort studies detected an increased risk of colon cancer with increasing intake of dietary fat [4], more recent cohort, large case-control, and a pooled analysis of 13 case-control studies [10\u201312] have failed to find an association between dietary fat and colon cancer. The Nurse's Health Study is one of the few cohort studies that has detected an association between dietary fat and colon cancer [13]. In that study, a twofold increased risk of colon cancer was associated with high fat intake. In contrast, a cohort study of colorectal cancer in Hawaiian-Japanese men reported an inverse association, higher intakes of total fat associated with lower colon cancer risk. [11].\n\nStudies examining specific types of fat in the diet as potential risk factors for colon cancer have similarly been inconclusive. The Nurse's Health Study and Male Professional Study found an increased risk of colon cancer associated with high intakes of animal fat [12\u201313]. On the other hand, the Netherlands cohort identified no increased risk associated with types of dietary fat [14], as have other studies attempting to look at specific fatty acids in the diet [15\u201317].\n\nAlthough the data only weakly support an association between dietary fat and colon cancer, there are plausible biological explanations in addition to its role in bile acid production that support an association between specific types of fat and colon cancer. Specific fatty acids such as butyric acid have been shown to influence apoptosis [9], eicosanoids derived from 20-carbon polyunsaturated fatty acids have been shown to regulate cell proliferation and immune response [18], and linoleic acid has been shown to be involved in prostaglandin synthesis as well as acting synergistically with other growth factors [19, 20]. Early studies linking dietary fats to colon cancer could have been confounded by the effect of total energy, because fats are major contributors to variability in total energy in the diet, and statistical methods to sort these factors were often not employed [21\u201323]. Studies that have tried to separate the effects have generally not shown an association for dietary fat [24]. It is possible that fat is only important at high levels of intake and, because fat consumption is decreasing, important associations are no longer detected. It is also possible that other factors that modify the harmful effects of dietary fat are changing in the population.\n\n### B. Meat\n\nGiven the inconsistent associations between dietary fat and colon cancer, it is possible that red meat, a major source of animal fat, may actually be the agent responsible for the increased risk attributed to dietary fat. However, associations between red meat and colon cancer are also inconsistent for both case-control and cohort studies. While two cohort studies found increased risk with higher consumption of red meat, processed meat, or other types of meat [12, 13], 10 other cohort studies did not find an association [25]. Only with a Norwegian cohort was there an increased risk associated with sausage intake as a main meal in women, but not in men [26]. Case-control studies are likewise inconsistent [4, 27, 28]. Some studies detected associations [29], while others did not [30]. Manousos _et al._ [31] has shown an eightfold variation in colon cancer rates based on the combined intake of meat and vegetables. In a study of Seventh Day Adventists, meat was associated with colon cancer only among those who did not eat legumes [32].\n\n### C. Dietary Fiber\n\nFiber has been associated with reduced risk of colon cancer in numerous studies [4, 33\u201337]. Types of fiber may be important, although a recent study with the power to examine specific types of dietary fiber did not observe differences in risk from soluble and insoluble fiber [38]. In that study it was observed that the strongest associations were for older people and those with proximal tumors, possibly explaining the lack of association observed in some cohorts of younger people [39]. Level of fiber intake may also be an important determinate of risk. Some research based on a wide range of intake shows protection only at high levels of fiber intake [38]; other studies may fail to identify the association because of truncated levels of fiber intake [39].\n\nDespite the relatively consistent association between dietary fiber and colon cancer in both case-control and cohort studies, two randomized intervention studies have failed to detect reduced polyp recurrence between groups that consumed high intakes of fiber and those that did not. In the Polyp Prevention Trial [39a], a randomized study of 2079 men and women with recently detected colorectal adenomas, an increase in dietary fiber intake (from approximately 10\u201317 g\/1000 kcal) combined with reduction in dietary fat (from 35% to 24% of energy) and increased consumption of fruits and vegetables did not reduce the likelihood of having recurrent adenomatous polyps during the 4-year follow-up. Similarly, Alberts and colleagues [39b] did not observe an effect on risk for recurrence of polyps at 3-year follow-up with dietary supplementation of 13.5 g wheat bran fiber\/day in a randomized study of 1303 men and women. These studies point out the limitations of clinical trials. Nutrition intervention studies generally use polyps as the endpoint instead of cancer. While polyps are generally considered precursors to cancer, the majority of polyps do not advance to cancer. In intervention trials, such as these, many of the initial polyps were small. The critical time for inervention is possibly earlier than after the development of polyps. Participants in clinical trials often change other behaviors, some of which may directly influence the effect of the dietary intervention itself. Thus, the interpretation of these nutrition intervention studies is not always clear and straightforward.\n\n### D. Plant Foods: Vegetables, Fruits, Legumes, and Grains\n\nPlant foods, in addition to fiber, are the custodians of numerous dietary constituents, including vitamins, minerals, and other potentially anticarcinogenic factors. Vegetables, specific types of vegetables, fruits, legumes, whole grains, and refined grains have all been examined in relationship to colon cancer. Of these plant foods, vegetables are perhaps one of the most consistently identified factors associated with a reduced risk of colon cancer [4]. Most studies report a 30\u201340% reduction in risk in those with the highest level of vegetable intake relative to those with the lowest level of intake [33, 38, 40\u201342]. According to the World Cancer Research Fund report, \"Evidence that diets rich in vegetables protect against cancer of the colon and rectum is convincing\" [42].\n\nCruciferous vegetables are the most widely studied type of vegetable, with inconsistent results associated with high levels of intake [4]. Other specific types of vegetables reported to reduce risk of colon cancer are dark yellow vegetables [odds ratio (OR) 0.7; 95% confidence interval (CI) 0.6\u20131.0] and tomatoes (OR 0.6; 95% CI 0.4\u20130.8) [38]. La Vecchia [43], for example, reported a significant 20\u201360% reduction in colon and rectal cancer risk for high consumption of tomatoes. Levi _et al._ [29] noted a significant inverse association between garlic consumption and colorectal cancer (OR 0.3; 95% CI 0.2\u20130.6).\n\nAssociations between fruits and colon cancer are much less consistent than those found for vegetables. While some studies report inverse associations [40], others report null associations, or even a slight increase in risk for canned fruits and juices (OR 1.2; 95% CI 0.9\u20131.5) [38]. Levi and colleagues [29] observed a halving of risk of colorectal cancer associated with high levels of citrus fruit intake (OR 0.5; 95% CI 0.3\u20130.8).\n\nWholegrain and refined grain products have also been evaluated, with some studies showing that wholegrain products reduce risk of colon cancer while more refined grains are associated with an increased risk (34, 35, 38, 44). Levi _et al._ [29] observed an 80% increased risk (95% CI 1.1\u201352.9) of colorectal cancer associated with refined grains, whereas a halving of risk was observed for high levels of wholegrain intake (OR 0.5; 95% CI 0.3\u20130.9). A study conducted in France similarly found a twofold increase in colorectal cancer associated with high intake of refined grains [30].\n\nStudies that have tried to ferret out the association between the different compounds present in plant foods and colon cancer have shown that, even when controlling for dietary fiber, trace minerals, antioxidants, and other nutrients found in plant foods, vegetables still remain significantly inversely associated with colon cancer [38]. This implies that other unmeasured compounds contained in vegetables are protective. However, these bioactive compounds are unavailable in standard nutrient databases, leaving assessment of foods that contain these compounds as a surrogate for their intake.\n\n### E. Dietary Antioxidants\n\nVitamins with antioxidant properties, including beta-carotene, vitamin E, and vitamin C, are associated with decreased colon cancer risk in some but not all studies [4, 41, 45\u201347]. Enger and colleagues [48] observed that beta-carotene intake was inversely associated with polyp development. However, several clinical trials [45, 49\u201351] have found that high-dose beta-carotene supplementation increases risk of colorectal cancer and adenomas among smokers. In addition, the Australian Polyp Study [45] associated higher levels of beta-carotene with greater risk of recurrence of large polyps. On the other hand, a study in the Netherlands [14] linked a high vitamin C intake with a marked decrease in colon cancer risk (OR 0.5; 95% CI 0.3\u20130.9).\n\nFew investigators have examined the relationship between colon cancer risk and individual carotenoids other than beta-carotene [52]. Carotenoids, long recognized for their antioxidant properties, are of increasing interest in relation to cancer because of their effect on regulation of cell growth and modulation of gene expression, as well as their possible effect on immune response [53]. Data from a large case-control study revealed an inverse relationship between lutein intake and colon cancer (40% to 10% lower risk depending on subgroup). The association was strongest for proximal tumors and in those diagnosed when younger. Lycopene-rich tomatoes have also been associated with reduced risk of colon cancer [38, 54], while another study examining lycopene failed to show an association [52].\n\nVitamin E has also been inversely associated with colon cancer in some but not all studies [4, 41, 55, 56]. This vitamin represents a group of tocol and tocotrienol derivatives that may have specific biological mechanisms of action [57]. Alpha-tocopherol is the most biologically active tocopherol and has been the major tocopherol considered in dietary calculations of vitamin E intake. Despite the lower biological activity of other tocopherols, such as beta-, gamma-, and delta-tocopherol, their presence in the diet in amounts two to four times that of alpha-tocopherol makes them potentially important chemopreventive agents [57].\n\nThe association between colorectal cancer and vitamin E, mainly in the form of alpha-tocopherol, is mixed among analytic epidemiologic studies [55, 56, 58, 59] and clinical trials [60, 61]. The work of Bostick and colleagues [58] provides the strongest evidence of an association between vitamin E and colon cancer. In that cohort of women living in Iowa, a strong protective effect was seen in women categorized in the upper quintile of vitamin E, after adjusting for other confounding factors. Women younger than 59 years old were the most protected by vitamin E; and supplemental vitamin E appeared to be more protective than vitamin E from dietary sources. On the other hand, clinical trials have not supported the observation that vitamin E supplements decrease risk of colorectal polyps, a precursor to colon cancer [60, 61], nor have data from a large case-control study of colon cancer, after adjusting for related dietary and lifestyle factors [62]. In work by Longnecker and colleagues [59], the association between serum vitamin E and cancers of the colon and rectum was not statistically significant after adjusting for serum cholesterol. Examination of various forms of Vitamin E in conjunction with colon cancer risk also failed to show an inverse association [62].\n\n## III. MODEL 2: COOKED FOODS\n\nMore recently it has been suggested that the methods used to cook meat and other foods are more important than the foods themselves [63\u201365]. Diets high in fat and protein\u2014high-meat diets, for example\u2014contain greater amounts of heterocyclic amines when cooked at high temperatures. Greater exposure to these carcinogens increases cancer risk. Cooked sucrose may also be harmful in that it has been shown to promote aberrant crypt foci in rodents [66].\n\nUsing data from a case-control study, Gerhardsson de Verdier _et al._ [67] reported an increased risk of colon cancer (OR 2.7; 95% CI 1.4\u20135.9), comparing most frequent consumers of fried meat with a heavily browned surface to those who did not use these cooking methods; associations in this study were higher for rectal cancer than for colon cancer(OR 6.0; 95% CI 2.9\u201312.6). Schiffman and Felton [68] reported more than a threefold increase in risk associated with consuming well-done meat. Other studies failed to detect these associations or reported much weaker associations (OR 1.3; 95% CI 1.0\u20131.7), using an index based on frequency of meat consumption and temperature of cooking [28].\n\n## IV. MODEL 3: INSULIN RESISTANCE\n\nAn emerging and potentially important hypothesis relates nutrition to colon cancer through dietary contributions to insulin resistance. McKeown-Eyssen [69] first proposed this theory and Giovannucci [70] provided additional support in a literature review. The insulin resistance hypothesis pulls together many risk factors into a central biological mechanism. Physical activity, being protective for colon cancer [71], is inversely associated with blood glucose levels [72]; body size is directly associated with glucose levels as well as with colon cancer [73, 74]; and diets high in fiber and low in sucrose are inversely associated with both glucose levels and colon cancer [75, 76]. Moreover, increased levels of blood glucose and\/or triglycerides can result in increased insulin levels that may in turn influence cell proliferation and stimulate growth of colon tumors.\n\n### A. Energy and Energy Balance\n\nEnergy intake, energy expenditure, and body size are factors that have been repeatedly studied in relation to colon cancer. Briefly, high levels of energy intake are associated with increased risk of colon cancer in many case-control studies, although cohort studies generally have not observed the association [4, 42]. In contrast, high levels of physical activity are consistently associated with reduced risk of colon cancer (this association has not been detected in studies of rectal cancer) [4]. Having a large body mass has also been associated with a greater risk of colon cancer, especially in men [74].\n\nGiven the connection of these factors to each other, the importance of energy balance has also been examined in relation to colon cancer [77]. High energy intake and high body mass both become highly significantly associated with colon cancer when levels of physical activity are low. Similarly, being physically active is most critical when energy intake is high. Thus, the underlying activity level of the population studied may be important to detect associations between colon cancer and energy intake.\n\n### B. Sugar and Glycemic Index\n\nLevels of simple sugar consumption also vary from country to country and may typify a Western-style diet. High consumption of simple sugars may result in increased triglyceride and plasma glucose levels, especially among those who are insulin resistant [78].\n\nIn addition to examining dietary sugar, many studies have focused on high sucrose-containing foods [79\u201383]. Bostick and colleagues [79], for example, in a cohort of older women, observed that sucrose-containing foods showed stronger associations with colon cancer than did sucrose itself, with the strongest associations being observed when dairy foods that were high in sugar, such as ice cream, were excluded. Others have demonstrated links to specific foods [81, 82], although the high sugar content by itself may not be driving the association [81, 83]. Desserts or dairy products with a high-sugar content, for instance, have other components, such as fat or calcium, that may account for the observed associations with colon cancer. Findings reported by Slattery and colleagues [84] and Bostick and colleagues [79] suggest that the strongest associations are among older women. Findings regarding sucrose itself have been mixed [84\u201390]. Of the studies reported, only two found a significant association between sucrose and colon or colorectal cancer [8, 85]. Some of these discrepancies could be accounted for by age of study participants.\n\nAlthough most studies in this area have focused on sugars themselves and foods high in sugars and starches, few attempts have been made to estimate a metabolic response based on consumption of foods. The variation in reported risk estimates between studies for high-sugar foods and\/or sugar may be the result of different metabolic responses to specific foods. The glycemic index was proposed to provide an indication of plasma glucose response to diet [91, 92]. One study noted a 40\u201360% increase in colon cancer risk among those with the highest dietary glycemic index [84]. Glycemic index, however, does not correlate with simple sugar intake.\n\n## V. MODEL 4: DNA METHYLATION\n\nMethylation of DNA is one step in the regulation of gene activity [93\u201396]. Disturbances in DNA methylation are thought to result in abnormal expression of oncogenes and tumor suppressor genes [96\u201398]. In the case of colorectal tumors, for example, both generalized genomic hypomethylation and hypermethylation of usually unmethylated sites occur frequently [97\u201399]. Several dietary components, including folate, methionine, vitamin B12, and vitamin B6, are involved either directly or indirectly in DNA methylation [99, 100]. Alcohol may also alter DNA methylation patterns indirectly by affecting the intestinal absorption, hepatobiliary metabolism, and renal excretion of folate [101, 102]. Dietary involvement in the DNA methylation process has become a focus of research into the nutrition and colon cancer relationship\n\n### A. Folate, Vitamin B6, and Methionine\n\nFreudenheim and colleagues [103] originally observed that high intakes of dietary folate in women, but not men, were inversely associated with colon cancer, with stronger associations observed as the tumor site became more distal. In that study, the energy-adjusted risk estimate for the highest quartile of intake relative to the lowest quartile of intake (relative risk, RR) for women was 0.69 (95% CI 0.36\u20131.30), and for men was 1.03 (95% CI 0.56\u20131.89). In a large case-control study, folate intake was associated with a slight decrease in risk, with risk estimates for upper quintile of intake for women ranging from 0.7 to 0.9 and ORs for men ranging from 0.8 to 1.1, depending on age and site-specific subgroup [100]. Adjustment for dietary fiber attenuated the association in that study, although Freudenheim and colleagues [103] did not observe an alteration in risk after adjustment for fiber from vegetables. Giovannucci _et al._ [104] observed that men in the highest quintile of dietary folate intake were not at reduced risk (RR 0.85; 95% CI 0.54\u20131.39).\n\nInclusion of vitamin supplement data had little effect in findings reported either by Giovannucci _et al._ [104] or Slattery _et al._ [100]. One study noted that people taking folate supplements for 15 or more years were at reduced risk of colon cancer, but there was no effect from folate supplements taken for less than 15 years [105]. It is not clear if this finding is one of chance, if it points to a time when folate supplementation may have had important protective influences, or if it points to other characteristics that may be linked to reduced colon cancer risk in a small subset of the population.\n\nOther nutrients involved in this pathway have been examined less extensively than dietary folate. Methionine was not associated with risk of colon cancer in a large case-control study [10], although Giovannucci and colleagues [104] observed that those with the highest intake were at reduced risk (RR 0.65; 95% CI 0.42\u20131.02) in their cohort study. Vitamin B6 intake has also been shown by some investigators to reduce risk of colon cancer [101].\n\n### B. Alcohol\n\nThe association of alcohol with colon cancer has been inconsistent, with approximately half of all studies showing an increase in risk [4, 106] and the rest showing no association [107\u2013109]. Evidence linking alcohol and colorectal cancer has usually been more consistent and stronger for more distal or rectal tumors, and some investigators have pointed to cigarette smoking as a confounding variable [109].\n\nIt is possible that interaction between nutrients is needed to create a situation that can lead to disturbances in DNA methylation. For instance, in the study by Freudenheim and colleagues [103], men at highest risk for rectal cancer were those who consumed large amounts of alcohol and had low intakes of dietary folate; similar results were not reported for colon cancer. Giovannucci _et al._ [104] found the greatest increase in risk of colon cancer when a combination of factors was present: high alcohol and low folate or low methionine. However, the study by Slattery and colleagues [100], in which a large sample size provided the most power to detect an association, found no significant increase in risk of colon cancer with a dietary profile that could be considered \"high risk\" with respect to alcohol and folate intakes [100]. Because several genetic variants have been identified that may impact this pathway, these potential interactions are being investigated and will be discussed later in this chapter.\n\n## VI. MODEL 5: CELL GROWTH REGULATORS\n\nGiven that dietary factors act as initiators and promoters of tumors that advance along the continuum from polyp, the common precursor lesion, to colon cancer, this model of cell growth regulation encompasses several dietary factors because the proliferative activity of colon mucosa is modified by different nutrients [110, 111]. Newmark and colleagues [112] have suggested that calcium may induce saponification of free fatty acids and bile acids, thereby diminishing the proliferative stimulus of these compounds on the colon mucosa. Calcium might also directly influence proliferation by inducing cell differentiation [113]. Few data are available for vitamin D, but animal and human experimental studies suggest inhibition of cell proliferation by vitamin D, either directly or by way of its effect on calcium absorption [112\u2013114].\n\n### A. Calcium\n\nAs concluded in a meta-analysis conducted in 1996 [115] and in a recent review [116], epidemiological data on calcium and colon cancer are not conclusive. However, results published subsequently add support to the hypothesis that calcium has a protective role. A cohort study in Finland, for example, found a significant reduction in risk at high levels of calcium intake [15], as did a large multicenter case-control study from the United States [117] and a case-control study conducted in Wisconsin [118]\n\nAlthough several studies support reduced risk of colon cancer with calcium, with risk estimates for high levels of intake being 0.5 [117\u2013121], other studies do not confirm these results [115, 116]. A prospective study among Iowa women, including 241 colon cancer cases after 10 years of follow-up, suggested an inverse association between calcium and colon cancer only among those without a family history of colorectal cancer among first-degree relatives [122]. Support for an association between calcium and colon cancer also comes from controlled studies which show that epithelial proliferation of the colon mucosa, a presumed intermediate marker of colorectal carcinogenesis, is diminished by calcium supplementation in humans [123]. Additionally, a double-blind, placebo-controlled clinical trial of calcium in sporadic adenoma patients showed that, although the overall proliferation rate was unchanged, the distribution of proliferating cells was favorably altered [124]. And recently, a large controlled clinical trial demonstrated that polyp recurrence was reduced by 15\u201320% with calcium supplementation [125].\n\nInconsistent findings in epidemiologic studies examining the relationship between calcium and colon cancer may be explained by methodological issues related to an observational epidemiologic study design. For instance, the variation in calcium intake in some studies may have been insufficient to detect differences in risk across categories. In studies that observed a weak inverse association, the differences in median dietary calcium values between the lowest and the highest category of intake ranged between 500 and 700 mg\/day, whereas in a larger multicenter study of colon cancer [117], in which an inverse association was detected, the median dietary calcium values between the lowest and highest quintile of intake were 1601 mg for men and 1236 mg for women. That study noted that the protective effect was essentially limited to the highest quintile of intake and that looking only at the fourth quintile of intake one might consider the results as null or only weakly protective.\n\n### B. Vitamin D\n\nAs reviewed by Martinez and Willett [116], epidemiological data on vitamin D and colorectal cancer are sparse and suggest protective effects of vitamin D on colon cancer risk. In large studies\u2014both case control and cohort\u2014the inverse association between dietary vitamin D and colorectal cancer ranges between 0.5 and 0.9, although not always reaching statistical significance after multivariate adjustment [117\u2013121, 126\u2013128]. An inverse association was detected in the Iowa Women's Health Study among those with a negative family history of colorectal cancer but not among those with a positive family history [122]. Supplemental vitamin D has been significantly inversely related to colon cancer [118]. Most studies where an inverse association with vitamin D intake was found are prospective cohort studies in which exposure is assessed many years before diagnosis. Vitamin D may have a protective effect on early stages of tumor development that is not captured in case-control studies.\n\n## VII. FOOD INTAKE RELATIONSHIPS\n\nA. Dietary Patterns\n\nDiet is complex. Patterns of consumption are not focused on a single food or nutrient, despite the fact that our assessment of risk generally takes a one nutrient or specific food approach. It is possible that the overall pattern of intake is more important than any single food. To assess this possibility, several groups have identified dietary patterns and evaluated their association with risk of developing colon cancer. For example, when a large case-control study identified eating patterns using factor analyses [129], two major dietary patterns emerged.\n\nAlthough the descriptive labels were arbitrarily given, foods clustering together represent what could be described as the \"Western diet\" and the \"prudent diet.\" The Western diet represented an eating pattern characterized by high intakes of red meat, fast foods, high-fat dairy foods, refined grains, and foods with a high sugar content. The prudent diet, on the other hand, was typified by high intakes of fruits, vegetables, whole grains, and fish and poultry. Subsequent to the initial study, these same dietary patterns were identified and validated using data from a large cohort study [130].\n\nAssessment of these dietary patterns revealed that the Western dietary pattern was associated with increased risk of colon cancer, while the prudent dietary pattern was inversely associated with colon cancer. While the link with individual food items is not consistent between studies, the data are unified on the more broadly focused eating patterns. The dietary pattern can also modify the risk associated with a family history of colorectal cancer [131].\n\n### B. Dietary and Genetic Interactions\n\nGenetic susceptibility can take two forms: inheritance of high-penetrance genes, such as the adenomatous polyposis coli ( _APC_ ) gene 132], or mismatch repair genes leading to hereditary nonpolyposis colon cancer ( _HPNCC_ ) [133], or low-penetrance genes, such as the ones discussed below. Susceptibility associated with inheritance of low-penetrance genes, while carrying a much lower independent risk than high-penetrance genes, is relatively common. Thus, low-penetrance genes may be associated with a much higher population attributable risk than high-penetrance genes [133], especially when coupled with dietary factors that may serve as modulators. (See [Chapter 13.) Table 3 summarizes some of the suspected relationships between diet and genetic factors.\n\nTABLE 3\n\nInterrelationship between Diet and Genetic Factors\n\nNAT1 = N-acetyltransferase 1\n\nCYP1A1 = cytochrome P450 1A1\n\nGST = glutathione-S-transferase\n\nMTHFR = methylenetetrahydrofolate reductase\n\nIGF = Insulin-like growth factor\n\nTGF\u03b2 = Transforming growth factor beta\n\nVDR = Vitamin D receptor\n\nApoE = Apolipoprotein E\n\naNAT2 = N-acetyltransferase 2\n\nCarcinogens in meat prepared at high temperatures contain heterocyclic amines and polycyclic aromatic hydrocarbons that are metabolized by enzymes such as N-acetyltransferase and glutathione S-transferase (GST) [134]. The internal dose and actual DNA exposure to heterocyclic amines, polycyclic aromatic hydrocarbons, and _N_ -nitrosocompounds seems to be particularly important for those who are rapid metabolizers of these compounds. Refined grains and fats also contain polycyclic aromatic hydrocarbons and heterocyclic amines and may also interact with variants of these low-penetrance genes [135, 136]. Increased risk of colon cancer among rapid acetylators who consume high levels of meat has been observed in some populations [137\u2013139] or population subsets, i.e., only older men [139], but not in other studies [28].\n\nSeveral constituents of cruciferous vegetables, including isothiocyanates and indoles, have been hypothesized to reduce risk of cancer through activation of GST [140, 141]. Sulforaphane, an isothiocyanate compound found predominately in broccoli, is one of the most potent inducers of GST [142\u2013144]. Differences in observed associations between cruciferous vegetables and colon cancer could be the result of genetic variants that alter susceptibility from exposure to these vegetables. One study has examined the interaction between colon cancer and cruciferous vegetable consumption and _GST_ \u03bc _-1_ genotype [145]. In that study those most influenced by the combination of cruciferous vegetable consumption and _GST_ \u03bc _-1_ genotype were younger individuals who smoked cigarettes. A study linking colonic adenomas with cruciferous vegetables found that those with the _GST_ \u03bc _-1_ null genotype received more protection from ingestion of large amounts of broccoli than did those with _GST_ \u03bc _-1_ positive genotype [146].\n\nKahweol palmitate and cafestol palmitate are two other potent inducers of GST found in green coffee beans and roasted coffee beans [144]. Mice fed green coffee beans have a six- to sevenfold increased level of GST activity [147], and similar effects have been observed for roasted coffee beans. Epidemiologic studies have generally reported an inverse association between high consumption of coffee and risk of colon cancer [148], although the mechanism is not well known.\n\nMethylenetetrahydrofolate reductase ( _MTHFR_ ) is a key enzyme in the conversion to 5-methyltetrahydrofolate, the major circulating form of folate in the body and the primary methyl donor for the methylation of homocysteine to methionine. This enzyme is also key in the methylation process of DNA. A polymorphism of the human _MTHFR_ gene that leads to reduced _MTHFR_ activity resulting in elevated plasma homocysteine levels has been described [149].\n\nInvestigators have evaluated the interaction between folate, vitamin B12, vitamin B6, alcohol and the presence of _MTHFR_ polymorphisms. One large case-control study did not observe significant interaction between these dietary factors and _MTHFR_ genotype [150]. Others have shown more variation in risk estimates [151, 152] by _MTHFR_ genotype, although estimates of association have generally been imprecise because of small sample sizes [153]. Other genes involved in methylation processes, such as methionine synthase [154] and alcohol dehydrogenase, may also be influenced by dietary factors. However, one study evaluating methionine synthase did not find variation in risk by genotype in conjunction with either alcohol or folate intake [155].\n\nAnother way in which dietary factors may influence colon cancer risk is by interacting with genetic variants that influence insulin levels, apoptosis, and cell growth. For example, variants of genes for the insulin-like growth factor ( _IGF_ ) [156, 157], vitamin D receptor ( _VDR_ ) [158], apolipoprotein E ( _apoE_ ) [159], and the transforming growth factor ( _TGF_ \u03b2) [160] may be associated with colon cancer.\n\nPotentially important diet and genetic interactions may occur for each hypothesized model of the relationship between diet and colon cancer [161]. Because we are only beginning to explore and understand various disease pathways, our knowledge of such interactions is in its infancy. As we gain a better grasp of the genes involved in disease pathways, possible dietary interactions with those genes will undoubtedly become clearer.\n\n### C. Diet and Specific Mutations in Tumors\n\nA spectrum of mutations occurs in colon cancer tumors, implying that multiple pathways to disease exist. The primary genetic mutations observed in colon tumors are mutations in the _APC_ gene, _Ki-ras_ gene, _p53,_ and microsatellite instability [162]. Studies have reported approximately 85\u201390% of colon tumors with an _APC_ mutation. Most population-based studies estimate that 30\u201340% of colon tumors have _Ki-ras_ mutations [163, 164], while microsatellite instability has been found to occur in between 14 and 18% of cases [165]. The prevalence of _p53_ mutations, the most commonly mutated gene in most cancers including colon cancer, varies by the method used to detect the abnormality [166, 167]. While immunohistochemistry studies estimate the prevalence of _p53_ overexpression at 50\u201375% of colon tumors, studies attempting to identify mutations provide much lower estimates of closer to 40% of tumors [167, 168].\n\nFew studies have examined the association between diet and these distinct tumor mutations. _Ki-ras_ mutations have been evaluated in two studies of cancer and one of colonic polyps. The first study found that calcium intake reduced risk of _Ki-ras_ mutations, while monounsaturated fat intake increased risk of these mutations [169]. The second study, being much larger, detected mutation-specific associations [163]. Whereas dietary fat intake increased risk of G \u2192 T mutations, low levels of dietary factors associated with DNA methylation actually reduced the risk of having a G \u2192 A mutation. This information is inconsistent with that presented by Martinez and colleagues [164] in their study of adenomas. They observed that supplemental folate reduced the risk of having a _Ki-ras_ mutation, while dietary intake of folate was unrelated to _Ki-ras_ mutations.\n\nDietary associations with _p53_ mutations have been reported for two studies. In one study [168], fat intake was associated with a greater likelihood of a _p53_ mutation, especially transversions. In another study, beef intake was more strongly associated with _p53_ negative cases than _p53_ positive cases [170]. Associations between dietary factors and microsatellite instability have not been reported.\n\n## VIII. PREVENTION OF COLON CANCER\n\nThe link between diet and colon cancer is undoubtedly complex. Given that the effects of individual nutrients and overall dietary composition will vary depending on genetic predisposition and other environmental influences, it is somewhat understandable that attempts to identify consistent associations between dietary factors and colon cancer are difficult. 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Biomarkers Prev_. 1996;5:285\u2013291.\nCHAPTER 24\n\nNutrition and Prostate Cancer\n\nLAURENCE N. KOLONEL, University of Hawaii, Honolulu, Hawaii\n\n## I. INTRODUCTION\n\nThis chapter discusses the epidemiologic evidence for associations of dietary factors with prostate cancer risk, and the potential for diet to play a role in prostate cancer prevention. Some findings from animal and _in vitro_ studies, as well as possible mechanisms for the carcinogenic effects, are presented in support of the epidemiologic findings.\n\n### A. Normal Prostate Anatomy and Function\n\nThe normal adult prostate gland is a walnut-sized organ that surrounds the urethra and the neck of the bladder. The gland is composed of three distinct zones: peripheral, central, and transition. The peripheral zone is comprised of left and right lobes that can be palpated during digital rectal examination. The transition zone is the region that enlarges in benign prostatic hyperplasia, which is common in older men [1]. The prostate gland is a male secondary sex organ that secretes one fluid component of semen. Prostatic fluid is essential for male fertility.\n\nNormal growth and activity of the prostate gland is under the control of androgenic hormones. Circulating testosterone, primarily produced in the testes, diffuses into the prostate where it is irreversibly converted by the enzyme steroid 5\u03b1-reductase type II to dihydrotestosterone (DHT), a metabolically more active form of the hormone. Dihydrotestosterone binds to the androgen receptor, and this complex then translocates to the cell nucleus where it activates selected genes [2].\n\n### B. Pathology and Diagnosis of Prostate Cancer\n\nAlmost all prostate tumors are classified as adenocarcinomas (i.e., they arise from the glandular epithelial cells), and occur most commonly in the peripheral zone of the gland. Accordingly, they can often be felt by the physician during digital rectal examination. A unique feature of human prostate cancer is the high frequency of small, latent tumors in older men. A clear relationship between these occult tumors and those which become clinically apparent has not been established, although it is commonly assumed that the latter evolve from the former as a consequence of additional genetic mutations.\n\nGenerally, prostate cancer in its early stages is asymptomatic. Enlargement of the prostate gland, (benign prostatic hyperplasia or BPH) commonly begins after the age of 45, ultimately leading to urinary tract symptoms (difficult and frequent urination). Many cases of prostate cancer are diagnosed as a result of digital rectal examination performed when a man visits his physician for relief of these symptoms. (Suspicious lesions on examination may be confirmed by transrectal ultrasound, followed by a biopsy of the gland.) In recent years, the prostate-specific antigen (PSA) test has come into widespread use. This test is not specific for prostate cancer, however, and gives an abnormal result if there is any increased tissue growth in the gland, such as occurs in BPH. Because of its sensitivity, the PSA test can lead to the diagnosis of very early, microscopic tumors. Although such lesions might never progress to clinical disease, surgical removal carries a risk of major complications (notably incontinence and\/or impotence), leading to controversy regarding the proper use of PSA as a screening test for early prostate cancer [3].\n\n## II. DESCRIPTIVE EPIDEMIOLOGY OF PROSTATE CANCER\n\nA. Incidence and Mortality Trends\n\nProstate cancer is a common cancer among men in many Western countries, and is the leading male incident cancer in the United States, where 180,400 new cases are projected for the year 2000 [4, 5]. Incidence trends in the United States show a rather slow increase over most of the last 50 years, with a rather striking increase between 1989 and 1992, attributable in large measure to the widespread adoption of the PSA screening test, which first became available in the early 1980s [6]. Since 1992, the incidence has declined, reflecting an end to the surge in cases due to the introduction of this new screening procedure [7], as well as, perhaps, to the more judicious application of PSA screening. Moreover, mortality from prostate cancer is low relative to its incidence. This is because prostate cancer is generally well controlled by treatment (surgery, radiation, and androgen ablation) and occurs at relatively late ages, so that even men who are not cured of the disease often die from other causes. Interestingly, a parallel increase in prostate cancer mortality did not occur during the period 1989\u20131992, presumably because most of the additional cases diagnosed would not otherwise have led to fatal outcomes.\n\n### B. Risk Factors for Prostate Cancer\n\nFewrisk factors for prostate cancer have been established. Proposed factors are listed in Table 1. Age is the strongest risk factor. Prostate cancer incidence increases more sharply with age than does any other cancer; more than 50% of cases in the United States are diagnosed in men above the age of 70 [4, 8].\n\nTABLE 1\n\nProposed Risk Factors for Prostate Cancer\n\nCategory | Characteristic or Exposure \n---|--- \nDemographic | Age, ethnicity, geography \nGenetic | Family history (father, brothers); major predisposing genes \nOccupational | Cadmium products, rubber industry, agricultural chemicals \nHormonal | Androgens (testosterone, dihydrotestosterone) \nLifestyle | Sexually transmitted agent, smoking, alcohol, vasectomy, physical activity, diet\n\nRace\/ethnicity is a second risk factor for prostate cancer. In the United States, the lowest incidence rates are seen among Korean and Vietnamese men, both relatively recent immigrant groups from Asia; the rates are somewhat higher among Chinese, American Indian, Alaska Native, and Native Hawaiian men. Caucasian men have very high rates, but by far, the highest incidence of this cancer is among African-American men [9].\n\nThe incidence of prostate cancer varies more than 60-fold in populations around the world (Fig. 1). Indeed, of all common cancers, this site shows the widest variation between low- and high-risk countries or populations. High rates are seen in developed, especially Western, countries, including the United States, Canada, parts of Europe, and Australasia. Low rates tend to occur in Asia, particularly China [4, 10]. The highest reported rates in the world are among African-Americans, whereas the lowest reported rates are among Chinese men in Shanghai. Interestingly, Chinese men in more developed areas of Asia (Singapore and Hong Kong) and Chinese men in the United States have much higher incidence rates than men in mainland China (see cross-hatched populations in Fig. 1). Furthermore, immigrants from Japan to Brazil and the United States have higher rates than do men in Japan [8]. Although the incidence of prostate cancer in Japan increased about threefold between 1970 and 1990, which was similar to the rate of increase in the United States during the same period, the actual incidence in Japan remains very low.\n\nFIGURE 1 Prostate cancer incidence in selected populations, 1988\u20131992 (rates age adjusted to the world standard population).\n\nMen with a first-degree male relative who has had prostate cancer are at a two- to threefold increased risk; whether this reflects an inherited predisposition for the disease or a shared environmental exposure has not been confirmed [11, 12]. The search for major predisposing genes for prostate cancer has identified some candidates, though none has yet been confirmed (discussed below).\n\nApart from these few established risk factors, the etiology of prostate cancer is unknown. Among the several potential causal agents, apart from diet, that have been proposed are (1) occupational exposures (rubber industry; manufacture of products containing cadmium, such as paints and batteries; use of agriculture chemicals); (2) sexually transmitted agents (e.g., cytomegalovirus); (3) smoking; (4) alcohol use; (5) vasectomy; and (6) physical activity [11, 13\u201315]. However, the evidence is not yet convincing for any of these exposures.\n\nAlthough it is suspected that most exogenous factors affecting prostate cancer risk exert their influence by altering endogenous androgen levels [16], epidemiologic studies have not yet clearly established the role of androgens in prostate cancer. Studies of prediagnostic circulating levels of individual androgens generally showed no clear association with prostate cancer [11, 17\u201319]. However, two studies showed weak positive relationships between the ratio of testosterone to dihydrotestosterone and prostate cancer [20, 21]. A third study showed a strong positive association of plasma testosterone with prostate cancer only after adjustment for the sex hormone binding globulin level [22]. The latter finding suggests that the level of free (unbound) testosterone may be most relevant.\n\nThe most promising area of research on the etiology of prostate cancer pertains to diet.\n\n## III. STUDIES OF DIET IN RELATION TO PROSTATE CANCER\n\nA. Origin of the Diet\u2013Prostate Cancer Hypothesis\n\nThe descriptive patterns of prostate cancer, especially data showing very different rates of the disease in the same ethnic\/racial group living in different geographic settings, as well as changing rates in migrants and their offspring [8], prompted investigators to seek environmental risk factors for this cancer. Diet became an important focus of this research because (1) geographic variations in food and nutrient intakes are known to be large [10]; and (2) components of the diet can influence the levels of circulating androgens [23, 24], which, as noted above, are thought to play a role in prostate cancer risk. Many different dietary factors, including both foods and particular constituents of foods, have been proposed and studied. Some of these appear to increase risk, while others are possibly protective. These factors are listed in Table 2, and the supporting evidence is discussed in the following sections of this chapter.\n\nTABLE 2\n\nProposed Dietary Risk Factors for Prostate Cancer\n\n### B. Dietary Factors That Increase Risk\n\n1. FOODS AND BEVERAGES\n\n##### a. Red Meat.\n\nMany epidemiologic investigations of different designs, including ecologic [25\u201327], case-control [28\u201333], and cohort [34\u201337] studies, have reported positive associations between the consumption of meat, especially red meat, and prostate cancer. However, not all studies reproduced this finding [38\u201343].\n\nExplaining the association with meat is not straightforward. Initially, the finding was thought to reflect a high exposure to dietary fat, especially saturated fat, because meat and dairy products are the major contributors to fat intake in the Western diet. However, because the findings on dietary fat per se and prostate cancer are equivocal (discussed below), other explanations for the association should be considered. There are several possibilities: (1) In the American diet, red meat is a major source of zinc, which is essential for testosterone synthesis and may have other effects in the prostate (discussed below). (2) Diets high in meat and other animal products may be relatively deficient in certain anti-carcinogenic constituents found primarily in plant foods. (3) Most intriguingly, many meats are cooked at high temperatures, such as by pan-frying, grilling, or barbecuing. Cooking meats at high temperatures can result in the formation of heterocyclic amines, which are potent carcinogens in animals, including the rat prostate [44]. Furthermore, when meats are cooked on charcoal grills, rendered fat is pyrolized by the coals, leading to the deposition of polycyclic aromatic hydrocarbons, which are also carcinogenic in animals, on the outer surface of the meat [45]. Few epidemiologic studies have been able to examine the relationship of such exposures to prostate cancer risk, because their levels in the diets of individuals cannot be easily and precisely assessed. An epidemiologic study which estimated heterocyclic amine intake from cooked meat and risk of prostate cancer did not lead to a clear result [46].\n\n##### b. Dairy Products.\n\nSeveral case-control [28, 31, 32, 47, 48] and cohort [34, 37, 49] studies found positive associations between the consumption of milk and other dairy products and the risk of prostate cancer. Nevertheless, other studies did not find this association [33, 38, 41, 43, 50]. One explanation for the positive association could be an adverse effect on the prostate of the high fat, especially saturated fat, content of dairy products. Another prominent constituent of these foods is calcium, which has also been proposed as a risk factor for prostate cancer (discussed below).\n\n##### c. Alcoholic Beverages.\n\nMost case-control studies showed no association of prostate cancer with either total alcohol intake or with the intake of specific types of alcoholic beverages [30, 38, 39, 51\u201353], although a study among U.S. black and white males found a positive association for total alcohol intake in both groups [54]. Although some cohort studies also found no association [34, 37, 55], a few did see an effect, including a cohort study in Japan [56] that found an increased risk for alcohol drinkers in general, a U.S. cohort [43] that found an elevated risk for drinkers of beer, and a European cohort that found some evidence of an elevated risk for drinkers of white (but not red) wine [57]. Some general mechanisms by which alcohol might enhance carcinogenesis have been proposed, including the activation of environmental nitrosamines, production of carcinogenic metabolites (acetaldehyde), immune suppression, and secondary nutritional deficiencies [54, 57].\n\n#### 2. NUTRIENTS AND OTHER FOOD CONSTITUENTS\n\n##### a. Energy.\n\nTotal energy intake was not reported in early epidemiologic studies of diet and prostate cancer, because the dietary assessment methods used at the time were incomplete. However, several studies in recent years have examined this variable, particularly in relation to the effects of dietary fat intake. The findings have been very inconsistent. Some studies reported a positive association for energy intake and no independent association with dietary fat [58, 59]. Others reported an effect of dietary fat, but no independent effect of energy [32, 50, 60, 61]. One study reported a positive association with both total energy and total fat [33], and two studies found no association with either total energy or fat [39, 62]. An experimental study in rodents (rats and mice) found that energy restriction reduced prostate tumor growth, possibly by inhibiting tumor angiogenesis [63].\n\n##### b. Fat.\n\nDietary fat has been the most studied nutrient with regard to effects on prostate cancer risk. This topic was recently reviewed in detail [64]. Total fat intake has been associated with the risk of prostate cancer in several case-control studies [30, 51, 65\u201367], although most found the association to be strongest for saturated or animal fat [29, 60, 65\u201368]. In two cohort studies that examined total and saturated fat, one [62] found no evidence of an effect on prostate cancer, and the other [36] found an elevated risk for total, but not saturated, fat. When limited to studies that controlled for energy intake, however, the findings from both case-control and cohort studies are more equivocal, with many investigations showing no independent effect of fat [39, 58, 59, 61, 62]. In some studies, the association with total and saturated fat was stronger for the advanced cases [36, 60, 67].\n\nSome epidemiologic studies examined intakes of monounsaturated and polyunsaturated fat as well. Most of these studies found no association of either of these classes of fat with prostate cancer [62, 64], although a few case-control studies reported positive associations [41, 50, 67].\n\nA few studies in recent years also examined specific fatty acids (including several omega-3 and omega-6 polyunsaturated fatty acids, as well as monounsaturated fatty acids), based either on dietary intake data or biochemical measurements in blood or adipose tissue [62, 64]. Most studies found no significant associations, although the data are not consistent, and no conclusions as to the role of specific fatty acids on the risk of prostate cancer can be reached on the basis of current data.\n\nSome animal experiments have tested the fat-prostate cancer hypothesis. For example, a high-fat diet increased prostate cancer incidence and shortened the latency period in Lobund Wistar rats treated with exogenous testosterone to induce the tumors [69]. Conversely, prostate tumor growth rate was reduced by a fat-free diet in Dunning rats [70] or by lowering dietary fat intake in athymic nude mice injected with LNCaP cells (a human prostate cancer cell line) [71]. With regard to specific types of fat, fish oils containing high levels of omega-3 fatty acids, such as eicosapentaenoic and docosahexaenoic acids, generally suppressed prostate tumor growth in rodents, whereas omega-6 polyunsaturated fatty acids, such as linoleic and linolenic acids, promoted tumor growth [72, 73]. However, because most animal studies have been conducted in rodents, whose prostate glands differ anatomically from that of the human, extrapolation of these findings to humans is particularly tenuous.\n\nA number of plausible mechanisms by which dietary fat could increase cancer risk have been proposed. These include the formation of lipid radicals and hydroperoxides that can produce DNA damage, increased circulating androgen levels, decreased gap-junctional communication between cells, altered activity of signal transduction molecules, effects on eicosanoid metabolism, and decreased immune responsiveness [64].\n\n##### c. Calcium.\n\nAs noted above, a number of studies that have examined the relationship of dairy product consumption to prostate cancer risk found a positive association. Dairy products could be a marker of exposure to calcium as well as saturated or animal fat, because this food group is a major source of both nutrients in the Western diet. Data on calcium specifically are more limited, but one case-control study [48] and a cohort study [74] both reported statistically significant positive associations, especially with advanced or metastatic cancer. However, a case-control study among U.S. whites and blacks did not show an effect of calcium on prostate cancer risk [32].\n\nA mechanism for an adverse effect of calcium on prostate carcinogenesis has been proposed, based on the observation that a high intake of calcium decreases the circulating levels of 1,25(OH)2 vitamin D, which may inhibit cell proliferation and promote differentiation in prostatic tissue [75]. The role of vitamin D in prostatic carcinogenesis is discussed below.\n\n##### d. Zinc and Cadmium.\n\nThe trace elements zinc and cadmium are considered together because they act as antagonists in biological systems. Few epidemiologic studies have examined dietary zinc in relationship to prostatic cancer. Most of the studies found no effect of zinc, especially after energy adjustment [39, 50, 58, 67], although a positive association was suggested in two reports [66, 76]. The frequent association of prostate cancer risk with high intake of red meat (discussed above) could also be explained by a higher intake of zinc, rather than animal fat, because meat, especially red meat, is an important source of zinc in the American diet (other sources are shellfish, whole-grain cereals, nuts, and legumes) [77]. Reports based on zinc levels in serum or prostatic tissue of patients with cancer and controls have not been consistent [78, 79], but such studies are unreliable, because the levels of zinc measured after diagnosis in the cases may reflect physiologic changes in the prostate as a result of the cancer.\n\nAs a major constituent of prostatic fluid [80, 81], zinc is essential for normal prostate function. Zinc is also essential for normal testicular function, and high levels of zinc have been proposed to increase the production of testosterone, leading to enhanced tissue growth in the prostate. Plasma levels of zinc have been positively correlated with testosterone and dihydrotestosterone levels in men [82, 83]. Furthermore, in the rat prostate, zinc has been shown to increase 5\u03b1-reductase activity [84] and to potentiate androgen receptor binding [85]. Thus, one might speculate that higher intake of zinc could partially offset the normal decline in testosterone levels with age [86\u201388], thereby contributing to prostate cancer risk.\n\nEpidemiologic evidence for cadmium as a risk factor for prostate cancer is also limited. Only two studies attempted to assess dietary exposure [89, 90], and neither found very convincing evidence for an effect of diet alone, although both studies found some evidence for a positive association when combined cadmium exposure from multiple sources (diet, cigarette smoking, occupation) was considered. Most studies of cadmium and prostate cancer have been among exposed workers in industries that utilize cadmium, and the findings of those investigations are only weakly suggestive of an adverse effect [11]. Cadmium is a competitive inhibitor of zinc in enzyme systems and accumulates in the body throughout life, because no mechanism exists for excreting it. Thus, the hypothesis that cadmium may be carcinogenic for the prostate has biologic plausibility. This hypothesis is further supported by studies showing that cadmium is carcinogenic in animals, and that the effect can be blocked by simultaneous injection of zinc [91, 92].\n\n#### 3. DIET-ASSOCIATED RISK FACTORS\n\n##### a. Obesity.\n\nProstate cancer is sometimes considered a male counterpart to breast cancer in women, for which there is clear evidence of a positive association with obesity, especially in postmenopausal cases. However, evidence for a similar association of adult obesity with prostate cancer is limited. Although a few epidemiological studies reported a significant positive association [28, 49, 93], most studies found no clear relationship of measures of obesity to prostate cancer risk [35, 37, 51, 59, 60, 65\u201367, 94\u201396]. Even when limited to prospective cohort investigations, the findings have been inconsistent, and some studies even showed inverse associations [e.g., 97\u2013100]. The influence of obesity at younger ages is also unclear. One study suggested that childhood obesity may be protective against adult prostate cancer [99], but another study found that obesity at age 20 years was associated with an increased risk [97].\n\nThe basis for an association between obesity and prostate cancer could involve endocrine factors, because adult obesity in men has been associated with decreased circulating levels of testosterone and increased levels of estrogen [101]. This mechanism would suggest an inverse rather than a direct association between obesity and this cancer.\n\n### C. Dietary Factors That Decrease Risk\n\n1. FOODS AND BEVERAGES\n\n##### a. Vegetables.\n\nIntake of vegetables has been inversely associated with cancer risk at many sites. This has led to strong recommendations to consume significant quantities of these foods as part of a healthful diet. However, the evidence for a beneficial effect of vegetables on prostate cancer risk is not overwhelming. Although some case-control studies showed inverse associations with selected vegetables or vegetable groupings, including green and yellow vegetables, cruciferous vegetables, and carrots [30, 33, 102\u2013105], others showed no significant associations [28, 31, 32, 40, 41, 106, 107]. The findings from prospective cohort studies have been mostly null [35, 43, 49, 108\u2013110]. The findings for legumes, a vegetable subgroup, are considered separately (discussed below), and the findings for tomatoes are included in the later discussion of carotenoids.\n\nBecause vegetables contain numerous compounds that can act through a variety of mechanisms to inhibit carcinogenesis [111], an inverse association between vegetables and prostate cancer is plausible. Some of these mechanisms are discussed with respect to specific food constituents below.\n\n##### b. Fruits.\n\nThe epidemiologic data on fruits and prostate cancer are also inconsistent, but interestingly, several studies, both case-control [40, 66, 102, 103] and cohort [34, 110], showed a direct (positive) association. Other case-control [31\u201333, 41, 50, 104, 105] and cohort [35, 43, 49, 108, 109] studies showed either no association or a statistically nonsignificant inverse relationship to prostate cancer risk. In one cohort study [74], a statistically significant inverse association was found for fruit intake and advanced prostate cancer; this finding did not persist after adjustment for total fructose intake.\n\nWhy intake of fruits might have an adverse effect on the prostate is not clear. Fruits contain many of the same compounds with anticarcinogenic properties that are found in vegetables, such as various carotenoids and vitamin C [111]. Although this finding is not yet established firmly, it does appear that fruit intake has no particular benefit with regard to the risk of prostate cancer.\n\n##### c. Legumes, Including Soy Products.\n\nLegumes, and particularly soy products, have recently become a focus of research on prostate cancer. Prostate cancer rates have traditionally been low in populations, such as those of Japan and China, where the intake of soy products is relatively high. A few case-control [39, 40, 103, 104] and cohort [34, 35, 110, 112] studies have reported inverse associations between intake of legumes and prostate cancer, including soyfoods specifically [34, 40, 104, 112]. At present, the data are suggestive of a beneficial effect of legumes, but not soy products uniquely. Additional research may clarify this issue.\n\nIn the past, legumes were of interest in nutritional epidemiology primarily because of their important contribution to fiber intake. However, these foods also contain phytoestrogens, plant constituents that have mild estrogenic properties. Because estrogens are associated with lower risk of prostate cancer and are used in prostate cancer therapy, there is a good rationale for the hypothesis that phytoestrogen intake can protect against prostate cancer. Soybeans and many products made from soy, such as tofu, are rich in a class of phytoestrogens known as isoflavones (other classes of phytoestrogens include the coumestans and lignans). The main isoflavones found in soy include genistein, daidzein, and glycetein [113, 114]. Although the mechanism for a protective effect of soy products on prostate carcinogenesis could entail the estrogenic effects of isoflavones, other actions of these compounds, such as inhibition of protein tyrosine phosphorylation, induction of apoptosis, and suppression of angiogenesis, have also been proposed [115]. Laboratory data based on human tissue as well as animal models offer support for the hypothesis that soy products may protect against prostate cancer [115]. For example, in a xenograft model for prostate cancer in mice, a low-fat diet combined with soy protein and isoflavones produced lower tumor growth rates and reduced final tumor weights compared with mice on a high-fat diet (with or without the soy\/isoflavone supplement), or a low-fat diet without the soy\/isoflavone supplement, suggesting that both low-fat and high-soy diets may be important in reducing the risk of prostate cancer [116].\n\nAlthough soy products and isoflavones are of particular interest, legumes contain other bioactive microconstituents, including saponins, protease inhibitors, inositol hexaphosphate, \u03b3-tocopherol, and phytosterols; mechanisms by which each of these compounds can inhibit carcinogenesis have been proposed [111, 115, 117\u2013119].\n\n##### d. Tea.\n\nOnly a few studies have examined the relationship between tea consumption and prostate cancer risk, and the findings have been inconsistent. One cohort study in Hawaii showed an inverse relationship between daily tea consumption and prostate cancer risk [120], but other cohort [34, 121] and case-control [39, 40] studies did not reproduce this finding.\n\nTea contains polyphenols that are potentially anticarcinogenic because of their antioxidant properties, effects on signal transduction pathways, inhibition of cell proliferation, and other actions in the body [122].\n\n#### 2. NUTRIENTS AND OTHER FOOD CONSTITUENTS\n\n##### a. Vitamin D.\n\nEvidence for a protective effect of vitamin D against prostate cancer is limited. In one cohort study [74] and two case-control studies [33, 48], estimates of dietary vitamin D intake, whether from foods or supplements, were not associated with risk. Four cohort studies that examined the relationship of prediagnostic 1,25-dihydroxyvitamin D (1,25(OH)2D), the biologically active form of the vitamin, in serum to subsequent development of prostate cancer produced discrepant results. One study found a clear inverse association [123], but the other three studies did not reproduce this finding [124\u2013126].\n\nVitamin D reduces cell proliferation in the prostate (and other tissues) and enhances cell differentiation, both of which would be expected to lower the risk of cancer [75]. In a mouse model, a 1,25(OH)2D analog inhibited the growth of prostate tumors [127].\n\n##### b. Vitamin E.\n\nThe intake of vitamin E is not adequately assessed with dietary histories, because much of the vitamin is obtained from fats and oils added during food preparation; estimating the amounts of added fats and oils is especially difficult. Nevertheless, a few epidemiologic studies attempted to determine vitamin E intake, with variable results. Two case-control studies [58, 59] found no association with prostate cancer risk. Another case-control study [41] found an inverse association with risk. Use of vitamin E supplements was not associated with prostate cancer risk overall in a cohort of U.S. men [128], although a reduced risk for advanced cancers was seen among current smokers and recent ex-smokers. Some cohort studies reported on findings for vitamin E and prostate cancer based on prediagnostic serum levels. In one report, an inverse association was seen [129], whereas in three others, no association was found [130\u2013132]. In an intervention trial among male heavy smokers in Finland, an incidental finding was a reduced risk of prostate cancer associated with intake of vitamin E supplements [133, 134]. Because the study was not specifically designed to test hypotheses related to prostate cancer, these results need confirmation. Furthermore, neither dietary nor serum vitamin E level at baseline was associated with subsequent development of prostate cancer in the non-vitamin E supplementation group [135].\n\nVitamin E inhibits the growth of human prostate tumors in nude mice [136]. As a powerful antioxidant, one mechanism for a protective effect of vitamin E against carcinogenesis in the prostate could be inhibition of lipid peroxidation [137].\n\n##### c. Carotenoids (\u03b2-Carotene and Lycopene).\n\nThe epidemiologic evidence related to carotenoids and prostate cancer is inconsistent. The results of case-control studies on \u03b2-carotene intake are mixed. Although some studies reported an inverse association [47, 51, 67, 102], most offered no support for a protective effect of this nutrient [33, 39, 50, 58, 59, 61, 66, 105, 107]. Furthermore, the findings often differed between younger and older men. Cohort studies have reported that dietary intake of \u03b2-carotene decreased risk [43] or had no effect on risk [108, 109], but, like the case-control studies, the findings sometimes differed between younger and older men [43]. One of these studies also showed no effect of \u03b1-carotene, \u03b2-cryptoxanthin, or lutein on the risk of prostate cancer [109]. Studies based on prediagnostic serum have reported both an increased risk [138] and no association [130, 139] with elevated \u03b2-carotene levels. (A recent study showed that circulating levels of \u03b2-carotene are significantly correlated with the levels in prostatic tissue [140].) Finally, in an intervention trial in Finland, \u03b2-carotene supplementation was associated with a decreased risk of prostate cancer among nondrinkers of alcohol, but an increased risk among drinkers [133].\n\nA carotenoid of current interest with regard to prostate cancer is lycopene, found primarily in tomatoes and tomato products (other food sources include watermelon, grapefruit, and guava). Although a few case-control studies showed an inverse association between tomato consumption, particularly cooked tomatoes [32, 41, 103], most studies have found no association [39, 40, 104\u2013107]. Of the studies that estimated actual lycopene intake, none showed a clear inverse relationship to prostate cancer risk [33, 39, 103, 107]. One study [32] found a weak inverse association with raw tomatoes but not cooked tomatoes (which is surprising, because the lycopene should be more bioavailable in the cooked tomatoes due to enhanced absorption as a result of heat processing and the presence of lipids [141]). The findings from cohort studies are also inconsistent: Two found a significant inverse association [35, 109], while a third found no association [110]. The results of studies based on prediagnostic circulating levels of lycopene are also inconsistent, two showing an inverse relationship [139, 142], and one, no association [130].\n\n\u03b2 \u2013Carotene, lycopene, and other carotenoids are widely distributed in human tissues, including the prostate [140, 143], where, as potent antioxidants, they help protect cell membranes, DNA, and other macromolecules from damage by reactive oxygen species. Other biological activities of carotenoids, such as the upregulation of gap-junctional communication [144] may also contribute to their anticarcinogenic effects. In three human prostate cancer cell lines (PC-3, DU 145, and LNCaP), \u03b2-carotene significantly inhibited _in vitro_ growth rates [145].\n\n##### d. Fructose.\n\nIntake of fructose was inversely associated with the risk of prostate cancer in one cohort study [74], which found a similar relationship for the intake of fruit, a major source of fructose in the diet. Although other epidemiologic studies have not assessed fructose intake per se, several, as noted earlier, examined the relationship of fruit intake to prostate cancer and most did not find an inverse association. However, the metabolism of calcium, phosphorus, fructose, and vitamin D are interrelated, and unless all components are considered simultaneously in an analysis, their individual effects could be missed [74].\n\nThe hypothesis for the protective effect of fructose is that it reduces plasma phosphate levels, resulting in increased levels of circulating 1,25(OH)2 vitamin D, which in turn may reduce the risk of prostate cancer [74, 146] (discussed above).\n\n##### e. Selenium.\n\nOnly a few epidemiologic studies have examined selenium and prostate cancer. Two case-control studies [67, 103] found no association between estimated selenium intake and prostate cancer risk, and baseline selenium intake was not associated with subsequent development of prostate cancer in a Finnish intervention trial [135]. In addition, prediagnostic serum selenium was not related to prostate cancer in a prospective cohort in Finland [147]. In contrast, prediagnostic selenium levels in toenails were inversely associated with advanced prostate cancer [148] and, in a selenium intervention trial to prevent skin cancer, an incidental finding was a reduced incidence of prostate cancer [149]. Because prostate cancer was not the primary endpoint of the trial, this finding needs to be confirmed.\n\nSelenium is a component of glutathione peroxidase, an important enzyme in certain antioxidative pathways. In an _in vitro_ experiment, selenium was shown to inhibit the growth of human (DU-145) prostate carcinoma cells [150]. Selenium may exert its anticancer effects through any of several proposed mechanisms, such as antioxidation, enhanced immune function, inhibition of cell proliferation, and induction of apoptosis [151].\n\n##### f. Isoflavonoids.\n\nThe potential role of isoflavonoids in prostate carcinogenesis was discussed above in the section on legumes.\n\n#### 3. DIET-ASSOCIATED PROTECTIVE FACTORS\n\n##### a. Physical Activity.\n\nThe role of physical activity in human prostate caricinogensis is at present quite unclear. Several epidemiologic studies have examined this relationship, but the findings have been inconsistent. Although some studies showed an inverse association, others showed no association or a positive association with prostate cancer risk [13, 152, 153]. These discrepancies may be resolved if future studies distinguish better between different types of physical activity, and can establish the time of life (e.g., young adulthood versus older ages) that may be most relevant.\n\nBecause exercise influences androgen levels in the body, an effect of physical activity on prostate cancer risk is biologically plausible. Exercise lowers testosterone in the blood, and also raises the level of sex hormone binding globulin, which reduces the circulating free testosterone levels; both effects would be expected to lower prostate cancer risk [13, 154].\n\n## IV. GENETICS AND GENE\u2013ENVIRONMENT INTERACTIONS\n\nAs noted above, prostate cancer shows a familial association (i.e., men whose fathers or brothers have had prostate cancer are at a two- to threefold risk of getting the disease as men without such a family history). The search for one or more highly penetrant genes that predispose to prostate cancer and that may explain at least part of this familial association has not yet been successful. Several candidate genes have been identified thus far, including a promising locus at q24\u201325 on chromosome 1 [155\u2013157].\n\nHowever, genetic predisposition may have a more indirect relationship to prostate cancer. Cell growth and differentiation in the prostate are regulated by androgens and various growth factors. It has been shown that several genes related to these cellular constituents are polymorphic (i.e., exist in variant forms) [158]. Some of these variants may have functional effects, leading to altered activity of the gene product. For example, a variant form, A49T, of the gene _SRD5A2_ (steroid 5\u03b1-reductase type II) that encodes the enzyme responsible for converting testosterone to dihydrotestosterone in the prostate, was recently found to have lower activity in an _in vitro_ assay [159]. Such functional consequences of genetic polymorphisms in constitutional DNA could lead to differing susceptibilities to prostate cancer. Indeed, men with the less active variant form (A49T) of the _SRD5A2_ gene were found to be at a greater risk for prostate cancer [159]. Variant forms of the androgen receptor gene ( _AR_ ), the vitamin D receptor gene ( _VDR_ ), and the insulin-like growth factor 1 gene ( _IGF-1_ ) have also been examined in relation to prostate cancer risk [160\u2013164]. Such polymorphisms may not only affect the risk for prostate cancer, but also the pathological characteristics of the tumors and hence prognosis [165].\n\nBecause diet (and other behaviors, such as physical activity [166]) can influence androgen, vitamin D, and IGF-1 levels in the body, interactions may occur between dietary exposures and inherited susceptibilities in determining actual risk for prostate cancer. Thus, men who consume an unhealthful diet and who also carry the high-risk variant of one or more of the genes mentioned above may be at especially high risk for prostate cancer. Such gene\u2013environment interactions offer considerable potential for elucidating the etiology of prostate cancer and will no doubt be explored in future research.\n\n## V. DIETARY INTERVENTION TRIALS\n\nA randomized intervention trial is the closest method to an experimental design that can be implemented in human subjects. Such a trial should be conducted only when there is considerable evidence for a beneficial effect, without apparent harm, based on observational studies and supported by animal and _in vitro_ studies. As the preceding review indicates, the data in support of most dietary factors are either limited or inconsistent, and would not justify the expense and risks of an intervention trial specifically for prostate cancer. However, two recent randomized intervention trials that were conducted for other purposes have shown unexpected reductions in prostate cancer incidence in the intervention groups. One of these studies [149, 167] was designed to test the potential of a daily selenium supplement (200 \u03bcg) to reduce the occurrence of basal and squamous cell skin cancers in men with a prior history of such lesions. The second trial [133, 134] tested the effects of daily \u03b2-carotene (20 mg) and\/or vitamin E (50 mg _d_ \/-\u03b1-tocopherol acetate) on the risk of lung cancer in a group of male smokers. In both trials, the incidence of prostate cancer was significantly lower in the men who received the intervention compared with the placebo groups. In the lung cancer trial, the reduced incidence was in the men who received vitamin E rather than \u03b2 \u2013carotene. However, because protection against prostate cancer was not a prespecified hypothesis in either trial, the results cannot be taken as definitive. Based on these findings, and the supportive evidence from other epidemiologic and laboratory research, a double-blind randomized trial to test the potential benefit of supplemental selenium (200 \u03bcg\/day) and vitamin E (400 mg\/day), alone and in combination, is about to begin in the United States [168]. However, the results will not be available for several years.\n\n## VI. CONCLUSIONS AND IMPLICATIONS FOR PREVENTION AND TREATMENT\n\nConsidering the combined evidence from descriptive epidemiologic studies (especially the remarkable changes in migrant populations), analytic epidemiologic studies in widely varying populations, experimental studies in animals, and _in vitro_ studies, the likelihood that certain dietary components or general patterns of eating influence the risk of prostate cancer remains high. However, at the present time, no specific relationships have been established conclusively. Continued research on this topic should be a high priority because diet is a modifiable risk factor and because prostate cancer incidence is extremely high in many populations. In addition, further research on genetic polymorphisms that affect susceptibility to prostate cancer should contribute to better identification of high-risk groups of men who can be targeted for future preventive dietary interventions.\n\nCurrently, the primary treatment modalities for prostate cancer consist of surgery, radiation, and hormonal therapy. The fact that the findings for many dietary factors were stronger in advanced or metastatic cases of prostate cancer (e.g., saturated fat, calcium) indicates that dietary effects can occur very late in the disease process. This suggests that dietary interventions have the potential not only to reduce the incidence, but also to improve the survival rates of the disease, providing another possible form of therapy. Indeed, a recent study showed significantly worse survival for prostate cancer patients in the upper tertile of saturated fat intake (> 13.2% of calories) compared with men in the lower tertile (<10.8% of calories) [169]. A possible mechanism to explain this result is reduced circulating androgen levels in the men with lower fat intake [23, 24], since prostate tumors are androgen sensitive, at least early in their clinical course.\n\nBased on current knowledge, it is not prudent to make very specific dietary recommendations to prevent or treat prostate cancer. However, taken as a whole, the evidence offers reasonably strong support for a diet that emphasizes vegetables, including legumes, and is moderate or low in the consumption of meat, especially red meat, and dairy products.\n\nReferences\n\n1. Bostwick D.G., Amin M.B. Prostate and seminal vesicles. Damjanov I., Linder J., eds. Anderson's Pathology, 10th ed., Vol. 2. Washington, DC: Mosby, 1996;2197\u20132230.\n\n2. Partin A.W., Coffey D.S. The molecular biology, endocrinology and physiology of the prostate and seminal vesicles. Walsh P.C., Retik A.B., Vaughan E.D., Jr., Wein A.J., eds. Campbell's Urology, 7th ed., Vol. 2. St. Louis, MO: W. B. 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Tymchuk C.N., Tessler S.B., Aronson W.J., Barnard R.J. Effects of diet and exercise on insulin, sex hormone-binding globulin, and prostate-specific antigen. _Nutr. Cancer_. 1998;31:127\u2013131.\n\n167. Clark L.C., Dalkin B., Krongrad A., Combs G.F., Jr., Turnbull B.W., Slate E.H., Witherington R., Herlong J.H., Janosko E., Carpenter D., Borosso C., Falk S., Rounder J. Decreased incidence of prostate cancer with selenium supplementation: Results of a double-blind cancer prevention trial. _Br. J. Urol_. 1998;81:730\u2013734.\n\n168. Klein E.A., Thompson I.M., Lippman S.M., Goodman P.J., Albanes D., Taylor P.R., Coltman C. SELECT: The selenium and vitamin E cancer prevention trial-rationale and design. _Prostate Cancer Prostat. Dis_. 2000;3:145\u2013151.\n\n169. Meyer F., Bairati I., Shadmani R., Fradet Y., Moore L. Dietary fat and prostate cancer survival. _Cancer Causes Control_. 1999;10:245\u2013251.\nCHAPTER 25\n\nNutrition and Lung Cancer\n\nSUSAN T. MAYNE, Yale University School of Medicine, New Haven, Connecticut\n\n## I. INTRODUCTION\n\nLung cancer is the most common cause of cancer death in both men and women in the United States. An estimated 171,600 persons were diagnosed with lung cancer in the United States in 1999 (94,000 men and 77,600 women), and 158,900 persons died of lung cancer during the same time [1]. Cigarette smoking is established as the dominant risk factor for lung cancer. With progress in tobacco cessation in American men, mortality from lung cancer has declined significantly in this group (\u20131.6% per year from 1991 to 1995 [1]). The gains made with regard to declining rates in men are encouraging, but must be considered against the rates for women, which continue to increase, although the rate of increase has begun to slow [1]. Also, tobacco use in youth is increasing, indicating that tobacco prevention and cessation continue to be public health priorities as the primary strategies for reducing lung cancer morbidity and mortality.\n\nAnother possible approach for reducing lung cancer deaths is detection of lung cancer when the disease is still localized. Early detection of lung cancer has great potential; the 5-year relative survival rate for lung cancer diagnosed when the disease is still localized is 50% [1]. In contrast, the 5-year relative survival rate for all stages of lung cancer combined is only 14%. A variety of approaches for early detection are being investigated; one newer approach that has been suggested to have potential is low-dose computed tomography screening [2].\n\nWhile tobacco is clearly the predominant risk factor for lung cancer, other risk factors have also been identified, including exposure to certain industrial and organic chemicals; radon and asbestos exposure; radiation exposure; air pollution; personal history of tuberculosis and other prior lung diseases; family history of lung cancer; and exposure to environmental tobacco smoke for nonsmokers [1, 3]. Also, as detailed below, dietary factors have been associated with lung cancer risk. Many of these risk factors are potentially modifiable, particularly certain occupational and environmental exposures and diet, offering opportunities to further reduce risk of lung cancer. However, it cannot be overly emphasized that tobacco prevention and cessation remain the foundation of public health efforts for lung cancer prevention; all other approaches should be viewed as adjunct approaches to tobacco control.\n\n## II. DIETARY CONSTITUENTS AND PRIMARY PREVENTION OF LUNG CANCER\n\nThere are some excellent, comprehensive reviews on the topic of nutrition in the etiology of lung cancer [4, 5]. This chapter will attempt to summarize and interpret the current literature on the topic of nutrition and lung cancer, drawing primarily from observational epidemiology studies and human intervention trials.\n\n### A. Fruit and Vegetable Consumption\n\nLung cancer is a highly prevalent disease in many countries of the world, therefore, it is not surprising that numerous observational studies, both case-control and cohort, have examined the association between consumption of various foods and food groups and lung cancer risk. In reviewing this literature, the most consistent observation seen is that increased consumption of fruits and\/or vegetables is associated with a lower risk of lung cancer. This inverse association has been noted for decades, has been observed in studies from many different countries, has been seen in both smokers as well as nonsmokers, is observed in both men and women, and has been observed for all histologic types of lung cancer [4]. The World Cancer Research Fund, in association with the American Institute for Cancer Research, recently concluded that \"seven cohort and 17 case-control studies are almost entirely consistent with a protective effect of vegetables and fruits against lung cancer. The evidence that diets high in vegetables and fruits decrease the risk of lung cancer is convincing\" [5]. In some studies, the magnitude of the inverse association for fruits exceeds that of vegetables, while in others, the converse appears to hold. An inverse association has been seen with many different types of fruits and vegetables; the evidence is the most abundant for green vegetables and carrots [5]. The majority of studies that examined the association between consumption of cruciferous vegetables and lung cancer risk also find strong or moderate decreases in risk at the highest consumption levels [5].\n\nThese results from observational studies are reasonably consistent; however, observational studies cannot prove that there is a causal relationship between low fruit and vegetable intake and lung cancer risk. That is, observational studies are susceptible to confounding, and thus can only demonstrate associations (see Chapter 4). Confounding occurs if some other factor is associated with both low fruit and vegetable consumption and lung cancer risk. One such possible confounder is tobacco use itself, in that it is clearly associated with lung cancer risk, and studies also demonstrate that cigarette smokers report consuming significantly less vitamin C and beta-carotene (markers of fruit and vegetable consumption [6]) as compared to nonsmokers [7]. In most of the epidemiologic studies of fruit and vegetable consumption and lung cancer risk, investigators have appropriately controlled or adjusted their estimates for tobacco use. However, residual confounding, due to imperfect measurement of tobacco, remains a concern.\n\nOne approach for minimizing the potential for confounding by smoking in studies of diet and lung cancer risk is to examine the association between diet and lung cancer risk in nonsmokers. Several case-control studies of lung cancer in nonsmokers and some cohort studies with nonsmoking lung cancer cases are available. For example, Kalandidi _et al._ [8] examined passive smoking and diet as risk factors for lung cancer among Greek women, the majority of whom were lifelong nonsmokers. In this study, high consumption of fruits was associated with a significantly lower lung cancer risk. The associations of lung cancer risk with passive smoking and reduced fruit intake were independent and did not confound each other. Inverse associations between fruit intake and lung cancer risk were also noted in a study of Chinese women in Hong Kong who never smoked [9]. Candelora _et al._ [10] also examined the association between diet and lung cancer risk in women who never smoked; increased consumption of vegetables was associated with a significantly lower lung cancer risk. Mayne _et al._ [11] studied both men and women nonsmokers with lung cancer, and reported inverse associations of lung cancer risk with increased consumption of fruits and vegetables; the association was strongest for raw fruit and vegetables. The previous studies in nonsmokers were all case-control studies, but the results are supported by cohort data as well. For example, Knekt _et al._ [12] reported that nonsmokers who consumed the lowest tertile of fruits and berries had a relative risk for lung cancer sevenfold greater than nonsmokers who consumed the highest tertile of fruits and berries ( _p =_ 0.001 for trend). Thus, these studies of lung cancer risk in nonsmokers appear to indicate that protective associations observed for fruits and\/or vegetables do not appear to be due to confounding by tobacco, although confounding by other lifestyle factors still remains a possibility.\n\nWhile most of the studies of diet and lung cancer have emphasized the role of diet in the etiology of lung cancer, some literature suggests that diet could be important in lung cancer prognosis. For example, Goodman _et al._ [13] used dietary data collected as part of two case-control studies on lung cancer to examine a possible association between diet 1 year prior to diagnosis and lung cancer survival. The results suggested that increased consumption of all vegetables combined was associated with a significant reduction in the risk of death in women but not men. Fruit intake was also associated with increased survival among women. The authors concluded that certain components of fruits and vegetables may prolong survival in some lung cancer patients.\n\nFruits and vegetables are complex mixtures of many different compounds, several of which might have cancer-preventive properties [14]. Much research has been targeted toward identifying the components of fruits and vegetables that might have cancer-preventive activity. Although several components, such as carotenoids and vitamin C, have received considerable study as discussed below, it is presently unknown which components\/combinations of components in fruits and vegetables are thought to influence human lung carcinogenesis.\n\n### B. Vitamins and Minerals\n\n1. CAROTENOIDS AND RETINOL\n\nCarotenoids are plant pigments that are found primarily in a variety of fruits and vegetables. The most commonly ingested carotenoids in human populations include beta-carotene, alpha-carotene, lycopene, lutein and zeaxanthin, and beta-cryptoxanthin. Intake of carotenoids is highly correlated with intake of fruits and vegetables; carotenoids as measured in blood or other tissues are considered one of the best biomarkers for fruit and vegetable intake [15, 16]. Given this, it is not surprising that epidemiologic studies generally find inverse associations between consumption of carotenoids or blood levels of carotenoids and lung cancer risk, as reviewed elsewhere [4, 17].\n\nNumerous studies have examined the association between the carotenoid beta-carotene, in diet or in serum\/plasma, and lung cancer risk. A recent review of this literature by the International Agency for Research on Cancer [17] concluded that the association of lower serum or plasma beta-carotene with lung cancer risk was remarkably consistent. For example, three of three cohort studies, six of seven nested case-control studies, and five of five case-control studies reported an inverse association between plasma or serum beta-carotene concentrations and lung cancer risk. There was evidence of a dose\u2013response in 12 of the 15 total studies reviewed. Considering estimated intake of carotenoids in diet, in nearly all studies reviewed, risk for lung cancer was lower among people with a high dietary intake of beta-carotene or carotenoids [17]. Some studies have used recently available food composition databases for carotenoids to estimate consumption of the major dietary carotenoids. In these studies, no particular carotenoid has consistently emerged as being associated with reduced risk of lung cancer. Some studies have suggested that higher intake of alpha-carotene, in particular, seems to be more strongly associated with lower lung cancer risk [18, 19]. Other studies have reported comparable inverse associations for alpha-carotene, beta-carotene, and lutein [20]. However, other studies have observed no association with alpha-carotene but inverse associations with beta-cryptoxanthin and lutein + zeaxanthin [21], or with lycopene [22]. The majority of studies, however, found stronger inverse trends with vegetable and fruit intake than with estimated carotenoid intake [17]. Le Marchand _et al._ [20], for example, concluded that their data supported greater protection afforded by consuming a variety of vegetables compared to only foods rich in a particular carotenoid.\n\nCertain carotenoids, known as provitamin A carotenoids, can be metabolically converted into retinol. Provitamin A carotenoids include beta-carotene, alpha-carotene, and beta-cryptoxanthin. Many of the early epidemiologic studies of diet and lung cancer examined the association of total vitamin A in the diet with lung cancer risk [4]. In 1975, for example, Bjelke [23] noted an association between dietary vitamin A and human lung cancer risk. As investigators began to examine associations separately for carotenoids versus retinol (preformed vitamin A), it became apparent that inverse associations with vitamin A were largely being driven by provitamin A carotenoids [4]. That is, the evidence from observational studies linking retinol with reduced lung cancer risk is inconsistent and weak at present.\n\nGiven the consistency of the results of epidemiologic studies on beta-carotene, coupled with chemopreventive efficacy of beta-carotene in animal models of skin carcinogenesis and buccal pouch carcinogenesis [17], several intervention trials of beta-carotene for the prevention of lung and other cancers were implemented in the 1980s and early 1990s. The first lung cancer prevention trial involving beta-carotene to be completed was the Alpha-Tocopherol Beta-Carotene (ATBC) Study [24], which involved 29,133 Finnish males ages 50\u201369 who were heavy cigarette smokers at entry (average one pack\/day for 36 years). The study design was a 2 \u00d7 2 factorial with participants randomized to receive either supplemental beta-carotene (20 mg\/day), alpha-tocopherol (50 mg\/day), the combination, or placebo for 5\u20138 years. Unexpectedly, participants receiving beta-carotene (alone or in combination with alpha-tocopherol) had a statistically significant 18% increase in lung cancer incidence and 8% increase in total mortality relative to participants receiving placebo. Supplemental beta-carotene did not appear to affect the incidence of other major cancers occurring in this population.\n\nThe Carotene and Retinol Efficacy Trial (CARET) was a multicenter lung cancer prevention trial of supplemental beta-carotene (30 mg\/day) plus retinyl palmitate (25,000 IU\/day) versus placebo in asbestos workers and smokers [25]. CARET was terminated nearly 2 years early in January 1996, because interim analyses of the data indicated that, should the trial have continued for its planned duration, it is highly unlikely that the intervention would have been found to be beneficial. Furthermore, the interim results indicated that the supplemented group was developing more lung cancer, not less, consistent with the results of the ATBC trial. Overall, lung cancer incidence and total mortality were significantly increased by 28% and 17%, respectively, in the supplemented subjects. The increase in lung cancer following supplementation with beta-carotene and retinyl palmitate was observed for current, but not former, smokers.\n\nIn contrast to these findings are the results of the Physicians' Health Study (PHS) of supplemental beta-carotene versus placebo in 22,071 U.S. male physicians [26]. There was no effect\u2014positive or negative\u2014after 12 years of supplementation with beta-carotene (50 mg every other day) on total cancer, lung cancer, or cardiovascular disease. The relative risk for lung cancer was reduced by a nonsignificant 10% in current smokers randomized to beta-carotene and a nonsignificant 22% in nonsmokers randomized to beta-carotene as compared to placebo. The apparent lack of an effect of long-term supplementation of beta-carotene on lung cancer incidence, even in baseline smokers who were administered the supplements for up to 12 years, is noteworthy. A similar lack of effect of supplemental beta-carotene on overall cancer incidence was seen in the Women's Health Study [27], although the duration of intervention was short (median 2.1 years).\n\nA clear mechanism to explain the apparent enhancement of lung carcinogenesis by supplemental beta-carotene, alone or in combination with retinol, in smokers has yet to emerge. As detailed elsewhere [28], it should be noted that the two trials that observed this enhancing effect [24, 25] had higher median plasma beta-carotene concentrations in their intervention groups relative to trials that did not observe an enhancing effect on lung cancer [26, 29]. Thus, it is possible that high tissue concentrations of beta-carotene in the presence of strongly oxidative tobacco smoke cause an interaction that promotes carcinogenesis. A recent animal study has suggested that this effect might be mediated by altered retinoid signaling [30].\n\nThe surprising results of the intervention trials involving beta-carotene and lung cancer prevention emphasize the value of results from randomized intervention trials prior to establishing public policy on the basis of observational data. Many have interpreted the observational data as being contradictory with the intervention trial results, but they really are not contradictory when it is recognized that the observational data that are derived from fruits and vegetables reflect relatively low doses of carotenoids in a complex matrix involving many other compounds, and generally reflect dietary patterns that may have been in existence for decades. The trials, in contrast, reflect one specific carotenoid given in a highly bioavailable preparation for a relatively short time period, and administered relatively late in the carcinogenic process to a high-risk group of subjects. The intervention trial data involving high-dose supplemental beta-carotene should not be interpreted as evidence against possible benefits of fruits and vegetables; there are currently no data to suggest that fruits and vegetables might have adverse effects with regard to lung cancer.\n\n#### 2. VITAMIN C\n\nMost of the early studies on dietary factors and lung cancer were designed to examine the association of carotenoids or vitamin A with lung cancer risk. Consequently, many of these studies did not use food frequency questionnaires designed to estimate intake of vitamin C, omitting foods such as potatoes that are important sources of vitamin C but are not important sources of carotenoids or retinol. Also, relatively few studies on diet and lung cancer performed plasma vitamin C measurements, because samples were not specifically preserved at the time of sample collection, which is necessary for vitamin C measurements. Given this, the literature on vitamin C and lung cancer risk is not as extensive as that on carotenoids and lung cancer; however, several studies are available.\n\nThe available literature on vitamin C and lung cancer risk has been reviewed elsewhere [4, 5, 31]. In general, most studies indicate that persons who consume more vitamin C in the diet are at reduced risk of lung cancer, although some studies find no association between vitamin C and lung cancer risk. According to the most recent review [5], \"most of six cohort and eleven case-control studies have found some degree of decreased risk of lung cancer with higher vitamin C intake, although these associations were in many cases only weak or moderate; no studies found a strong increase in risk.\" Some of the studies examined associations of both vitamin C and fruit\/vegetable intake in relation to lung cancer risk; the majority found more consistent and\/or stronger inverse associations with fruit and\/or vegetable consumption [4]. Thus, it seems probable that the observed decrease in risk is really attributable to some other component of vitamin C-containing foods (vegetables and fruits), and not to vitamin C itself [5].\n\nRandomized trials of supplemental vitamin C for lung cancer prevention have not been initiated, although large trials are ongoing that include supplemental vitamin C as one of several factors being studied for chronic disease prevention. One such trial is the Women's Antioxidant Cardiovascular Study (WACS), which is randomizing 8000 female health professionals with a prior cardiovascular event or with three or more coronary risk factors to antioxidant nutrients [32]. The nutrients included are beta-carotene (50 mg), alpha-tocopherol (600 IU), or vitamin C (500 mg; all given every other day) in a 2 \u00d7 2 \u00d7 2 factorial design. Another such trial is Physicians' Health Study II (PHS II), an extension of the original Physicians' Health Study of aspirin and beta-carotene. PHS II is a randomized trial enrolling 15,000 eligible male physicians aged 55 years and older to a 2 \u00d7 2 \u00d7 2 \u00d7 2 factorial design of antioxidant vitamins in the prevention of total and prostate cancer, cardiovascular disease, and cataract and macular degeneration [33]. The antioxidant nutrients being studied in this trial are vitamin E (400 IU every other day), beta-carotene (50 mg every other day), vitamin C (500 mg every day), and a multivitamin (Centrum Silver daily). While neither of these trials is specifically designed nor powered to evaluate an effect of vitamin C on lung cancer, they are some of the largest trials yet under way to investigate the effects of vitamin C as a single agent on chronic disease outcomes.\n\n#### 3. VITAMIN E\n\nSome epidemiologic studies have attempted to quantitate intake of vitamin E from foods, but the intake estimates are generally considered to be somewhat unreliable [34]. Vitamin E is concentrated in many vegetable oils and fats and the amount of fats and oils added during food preparation is difficult to assess. Also, different oils have different concentrations of tocopherols, and food labels often do not provide the specific fat or oil in the product, and manufacturers may substitute fat sources depending on availability and cost. Given this situation, epidemiologic studies of vitamin E and cancer generally rely on biochemical markers of exposure, such as plasma concentrations of alpha-tocopherol. In studies that rely on biochemical markers, greatest weight should be given to cohort studies, where bloods are obtained prior to lung cancer diagnosis, rather than case-control studies, where bloods are obtained postdiagnosis.\n\nA recent review of this literature [35] indicates that results are somewhat mixed, with some studies suggesting an inverse association with plasma vitamin E but other studies failing to observe such an association. For example, a Finnish study of male smokers participating in the ATBC trial (discussed above) examined the association between prediagnostic serum vitamin E and lung cancer risk [36]. Those in the highest quintile of serum alpha-tocopherol had 19% lower lung cancer incidence compared to those in the lowest quintile [relative risk (RR) = 0.81, 95% confidence interval (CI) = 0.67 \u2013 0.97]. This suggestion of a protective effect was especially strong among men younger than 60 years and men with less cumulative tobacco exposure. Results from a cohort of Chinese tin miners [37] also suggested an inverse association between serum alpha-tocopherol and lung cancer risk among men less than 60 years of age ( _p_ = 0.002 for trend). Although an early analysis of members of the Washington County, Maryland, cohort revealed significantly lower serum vitamin E levels among cases compared to controls [38], this result was not replicated in a subsequent analysis [39]. Several other prospective studies suggested nonsignificant inverse associations in a variety of different populations; however, inverse associations were not observed in other studies, as reviewed elsewhere [35].\n\nResults from a large intervention trial of vitamin E for lung cancer prevention are available. That is, the ATBC Trial discussed previously investigated both beta-carotene and alpha-tocopherol in the primary prevention of lung cancer [24]. The trial was conducted in Finnish male smokers. The RR of lung cancer in men randomized to receive vitamin E (50 mg\/day) for 5\u20138 years was 0.98 [95% CI, 0.86\u20131.12]. The dose of vitamin E is relatively modest; trials with higher doses of vitamin E are under way [32, 33]. However, lung cancer is not the primary endpoint for ongoing trials involving vitamin E, thus their statistical power with regard to lung cancer endpoints is likely to be limited.\n\n#### 4. SELENIUM\n\nSelenium is widely present in the food supply, although the selenium content of food varies, depending on the selenium content of the soil where the plant was grown or where the animal (which ingests local forage crops) was raised. The amount of selenium in soil can vary substantially, and the same food items may have more than a 10-fold difference in selenium content [34]. For these reasons, estimation of selenium exposure through dietary assessment is generally considered unreliable, with most epidemiologic studies relying on biomarkers for assessing selenium status, such as blood-based measures or toenail clippings.\n\nAs reviewed elsewhere [35], several observational studies have examined the association between selenium status and lung cancer risk with mixed results. Van den Brandt and colleagues [40] detected a strong inverse relationship between toenail selenium and lung cancer risk in a large Dutch cohort followed for 3.3 years ( _p_ = 0.006 for trend). An inverse association of serum selenium and lung cancer risk was also observed in a large cohort of Finnish subjects [41, 42]. However, at least five other studies have not observed an inverse association between selenium status and lung cancer risk (reviewed in [35]).\n\nResults of one clinical trial involving selenium are pertinent to the issue of selenium and lung cancer prevention. The Nutritional Prevention of Cancer Trial was a placebo-controlled, randomized clinical trial, designed to test the ability of high-selenium yeast supplements (200 \u03bcg\/day) to reduce second skin cancer in 1312 men and women who had a recent history of nonmelanoma skin cancer [43]. Participants were selected from dermatology clinics located in regions of the United States with high rates of nonmelanoma skin cancer and with low levels of soil selenium. While selenium supplementation was of no benefit in terms of preventing new skin cancers, persons randomized to selenium supplementation as compared to the placebo group had statistically significant decreases in risk of total cancer ( _p_ = 0.002) and cancers of the lung ( _p_ = 0.04), colorectum ( _p_ = 0.03), and prostate ( _p_ = 0.002). A total of 17 lung cancers occurred in the selenium group as compared to 31 in the placebo group (RR = 0.54, 95% CI = 0.30\u20130.98). The possibility that supplemental selenium might help reduce the risk of lung cancer is provocative, but it should be noted that the findings with regard to lung and other cancers were unexpected, based on small numbers, and require confirmation in subsequent trials. Also, participants in the selenium trial were selected from low-selenium regions of the United States, so the results may not be generalizable. Finally, selenium is a trace mineral with a relatively narrow range of safety. That is, the tolerable upper limit for selenium as set by the National Academy of Sciences is 400 \u03bcg\/day [34]. Usual selenium intake from foods averages around 100 \u03bcg\/day in the United States [34]. Supplementation can thus readily elevate total selenium intake to levels approaching or exceeding the upper limit, increasing the probability of encountering adverse effects of selenosis (most notably hair and nail brittleness).\n\n#### 5. FOLATE\n\nFolate has recently been of considerable interest with regard to a possible role in cancer prevention. Folate is crucial to the synthesis of nucleic acids and thus to normal cell replication. Also, folate is involved in methyl transfer reactions; abnormalities in DNA methylation are known to occur during carcinogenesis. Folate is also concentrated in fruits and vegetables, particularly green leafy vegetables, leading to the suggestion that folate might account for some of the lower risks observed with regard to green vegetables, for example.\n\nOnly a few epidemiologic studies have specifically reported on associations between dietary folate and lung cancer risk. In the Netherlands Cohort Study on Diet and Cancer [21], the association between dietary folate and risk of lung cancer was evaluated. After 6.3 years of follow-up in the cohort, a total of 939 male lung cancer cases were identified. Using a case-cohort analysis, the authors reported that higher dietary folate was associated with a lower risk of lung cancer overall (adjusted RR = 0.70, 95% CI = 0.51\u20130.95 comparing fifth quintile to first quintile of intake). Inverse trends were noted in both former and current smokers, with a statistically significant trend in the latter ( _p_ = 0.0001 for trend). The authors also stratified by histologic subtype; higher dietary folate was inversely associated with risk of small cell carcinomas, squamous cell carcinomas, and adenocarcinomas. As expected, dietary folate was correlated with intake of many other micronutrients ( _r_ = 0.66 for vitamin C, _r =_ 0.62 for beta-carotene). The authors concluded that folate and vitamin C had stronger protective associations with lung cancer risk than the carotenoids, but noted that the inverse associations might reflect a generalized fruit and vegetable effect rather than effects of specific nutrients. Another cohort study also noted a highly significant inverse association between dietary folate and lung cancer risk [44]. In this analysis, the protective effect of folate was stronger for heavy smokers, and was limited to cases with squamous cell carcinomas. In contrast, no association between total folate consumption (diet and supplements) was noted in the Nurses' Health Study cohort [45].\n\nNo trials using folate for the primary prevention of cancer have been completed; however, one randomized and blinded trial of folate investigated effects on an intermediate end-point for lung cancer. In this trial, smoking men with squamous metaplasia of the lung were randomized to either high-dose supplements of folate (10 mg\/day) and vitamin B12 (500 \u03bcg\/day) or placebo for 4 months [46]. The authors noted a significantly greater reduction in atypical cells in sputum in the intervention group as compared to the placebo group ( _p_ = 0.02). However, the authors cautioned overinter-pretation of their findings given the small study population ( _n_ = 73 total), the supraphysiological doses used, and the substantial spontaneous variation in sputum cytologies.\n\n### C. Macronutrients and Related Substances\n\n1. ENERGY BALANCE\/ADIPOSITY\n\nObesity has been shown to increase the risk for many cancers, such as postmenopausal breast cancer, endometrial cancer, and colon adenomas and adenocarcinomas, as discussed elsewhere [47]. Suggestive positive associations have also been noted for prostate cancer. However, lung cancer (along with premenopausal breast cancer) stands out in that most of the current literature suggests that obesity, as measured by the body mass index, is associated with a lower risk [47]. The interpretation of an inverse association between obesity and lung cancer risk, however, is complicated in that this relationship has been observed in smoking populations, where the well-known impact of smoking on both body fatness and on lung cancer risk may obscure true relationships (i.e., confounding). Most investigators adjust their estimates for the effects of smoking, but it is not clear that statistical adjustment can fully compensate for the effects of smoking.\n\nStudies of the relationship of obesity to lung cancer risk in nonsmokers thus afford another approach for examining this relationship while minimizing potential confounding by tobacco. One large, population-based case-control study is now available on the association between obesity and lung cancer risk in nonsmokers [47]. This analysis of 412 case-control pairs indicated a positive relationship between body mass index [weight (kg)\/height (m2)] and lung cancer risk for those who had never smoked and those who formerly smoked. The risk was particularly noted at high levels of adiposity; a body mass index >30.84 kg\/m2 (highest octile) was associated with a threefold greater risk of lung cancer as compared to a body mass \u2264 21.26 kg\/m2 (lowest octile). These results require replication in other large population-based case-control studies of nonsmokers or pooled cohort studies of nonsmokers, but if replicated, they suggest that lung cancer may well be added to the lengthy list of chronic diseases in which risk increases with obesity.\n\n#### 2. DIETARY FAT, SATURATED FAT, AND CHOLESTEROL\n\nPossible associations between total fat, saturated fat, cholesterol, and lung cancer risk have been reviewed comprehensively elsewhere [4, 5]. Approximately six cohort and six case-control studies have examined some aspects of dietary fat or cholesterol in relation to lung cancer risk. Overall, the evidence regarding total fat and lung cancer risk is somewhat inconsistent, with some studies indicating higher risk with higher intake levels, but other studies failing to observe any association. Associations with dietary fat might reflect residual confounding by smoking; again, studies of nonsmokers can be informative in this regard. One of the strongest associations noted for dietary fat and lung cancer risk comes from a study of nonsmoking women in Missouri [48]. Risk of lung cancer increased with increasing levels of total fat; the effects seemed to be particularly attributable to saturated fat. The adjusted risk estimate for saturated fat was 6-fold greater for the highest quintile of fat intake and was even more pronounced among women with adenocarcinoma, reaching 11-fold for this histologic type. However, following this initial publication, the authors reported that the odds ratios for saturated fat were highly sensitive to the method used for energy adjustment, due to the high correlation between saturated fat and total energy [49]. Adjustment using the nutrient residual method reduced the magnitude of the odds ratios to 1.78 for the highest quintile of saturated fat as compared to the lowest quintile. The same investigators did another case-control study of lung cancer in Missouri women, most of who were smokers [50]. In this analysis, an association between dietary fats, frequency of meat consumption, and lung cancer risk was observed. However, after adjusting for potential confounders and removing data from proxy respondents, dietary fat and consumption of red meat were no longer associated with lung cancer risk. These studies illustrate the difficulties in analyzing and interpreting effects of dietary fats on risk of lung and other cancers.\n\nA tangential link between fats and lung cancer risk comes from some interesting studies on cooking practices and lung cancer risk in Chinese women. Chinese cooking often involves frying ingredients in oil at high temperatures, which produces fumes to which the cook is exposed. In Taiwanese women nonsmokers, lung cancer risk increased with the number of meals cooked per day [51]. The risk was also greater if women usually waited until fumes were emitted from the cooking oil before they began cooking. Shields _et al._ [52] examined the production of potentially mutagenic substances emitted from a variety of cooking oils heated to the temperatures typically encountered in wok cooking. Their results suggested that unrefined Chinese rapeseed oil, in particular, produced significant quantities of mutagenic compounds. Lowering the cooking temperature or adding an antioxidant to the oil reduced volatile emissions.\n\n#### 3. HETEROCYCLIC AMINES\n\nAs described above, some epidemiologic studies suggest that increased consumption of fat, saturated fat, and cholesterol increases the risk of lung cancer. Meat is a rich dietary source of these nutrients, and meats cooked at high temperatures are known to contain various pyrolysis products, such as heterocyclic amines. Heterocyclic amines are known to be highly mutagenic, and their production in meats is a function of cooking method. Pan-frying and grilling\/barbecuing, in particular, promote heterocyclic amine formation. Given this, some recent epidemiologic studies have investigated a possible association of meat intake and meat cooking practices as determinants of lung cancer risk. In a case-control study of women with lung cancer, Sinha _et al._ [53] reported that lung cancer risk was increased with higher consumption of total meat, red meat, well-done meat, and fried meat. Not all types of red meat were associated with an increased risk; for example, higher consumption of red meat cooked by microwave was associated with a significantly lower risk of lung cancer. The authors suggest that variable effects of meat-cooking practices on lung cancer risk may explain some of the inconsistent results in prior epidemiologic studies of meat consumption and lung cancer risk [53], and suggest a need for future studies to collect detailed information on meat cooking practices.\n\n## III. DIETARY CONSTITUENTS AND PREVENTION OF SECOND LUNG CANCER\n\nEarly stage lung cancers can be surgically resected, but patients remain at risk for second lung cancers. Thus, interest has been shown in interventions, nutritional or other, aimed at reducing the risk of second lung cancers. Two adjuvant chemoprevention trials involving retinyl palmitate have now been completed. In the first trial, patients with stage I non-small-cell lung cancer ( _n=_ 307) who had been treated surgically were assigned randomly to treatment with retinyl palmitate (300,000 IU) for 1 year or observation [54]. At a median follow-up of 46 months, survival trends favored retinyl palmitate over no therapy in estimated 5-year disease-free survival (64% vs. 51%, _p =_ 0.054) and overall survival (62% vs. 54%, _p_ = 0.44). Eighteen patients in the retinyl palmitate arm developed a second primary tumor compared with 29 patients developing 33 second primaries in the control group. Retinyl palmitate toxicity was frequent, occurring in the majority of treated patients.\n\nThese promising results, however, were not replicated in a much larger trial, known as Euroscan [55], a multicenter trial employing a 2 \u00d7 2 factorial design to test 2 years of intervention with retinyl palmitate and _N_ -acetylcysteine in preventing second primary tumors in 2592 patients. Patients in the Euroscan trial had completed definitive therapy for non-small-cell lung cancer (40% of those randomized) or for early stage head and neck cancer (60% of those randomized). The dose of retinyl palmitate in this trial was 300,000 IU daily for the first year followed by 150,000 IU daily for a second year. After a median follow-up of 49 months, there was no benefit for either retinyl palmitate or _N_ -acetylcysteine with regard to survival, event-free survival, or second primary tumors. Second primary tumors developed in 54 patients in the combined intervention group, 61 patients in the retinyl palmitate only group, 61 patients in the _N_ -acetylcysteine only group, and 32 in the no intervention group (not significant). Thus, current evidence does not support a role for high-dose vitamin A in second primary lung cancer prevention.\n\n## IV. CONCLUSIONS AND RECOMMENDATIONS\n\nThe available literature on diet and lung cancer can be readily summarized into the following general statement: Dietary patterns characterized by higher consumption of fruits and vegetables and the nutrients concentrated in these foods (carotenoids, vitamin C, folate) are associated with a lower risk of lung cancer, and diets characterized by higher consumption of meats, total fat, and saturated fat are associated with greater lung cancer risk. Nutrient supplements have not been shown to be of value in the primary or secondary prevention of lung cancer. As noted by others [56], the evidence of a protective effect of fruits and vegetables is more consistently observed in case-control studies compared to cohort studies. Moreover, the magnitude of a possible protective effect of fruits and vegetables in the cohort studies is generally less than what one would have expected based on ecologic evidence and case-control studies [56]. Koo [57] has noted that much of the available literature is based on studies of white male smokers in North America and Europe; these populations are known to be low consumers of fruit and vegetables (relative to nonsmokers) and are less likely to pursue perceived healthier lifestyles. Koo thus suggests that some of the epidemiologic findings on diet and lung cancer are artifacts due to inadequate adjustment for behavioral correlates of smoking. While this may be true in part, it must be noted that fruits and\/or vegetables have been observed to be protective in other racial and ethnic populations [58]. Also, several studies of diet and lung cancer risk in nonsmokers, including those who have never smoked, suggest protective effects of fruits and vegetables. It remains possible that protective effects are due to behavioral correlates of increased fruit and vegetable consumption (i.e., confounding), but this can only be evaluated by randomized trials. To date, no trials of fruit and vegetable intake for lung cancer prevention have been initiated, but ongoing dietary intervention trials such as the Women's Health Initiative [59] and the Women's Healthy Eating and Living Study [60] can certainly examine lung cancer incidence by treatment arm, although with uncertain statistical power.\n\nGiven the enormous costs and difficulties of conducting large-scale intervention trials, observational studies remain the primary approach for elucidating diet and chronic disease relationships. With a disease as frequent as lung cancer, cohort studies are clearly viable and will continue to be used in etiologic research. A new emphasis, however, on more diverse cohorts is clearly needed. As discussed by Riboli and Kaaks [56], multicenter cohorts offer one possible approach in that they allow for the selection of populations with varying dietary habits within the same cohort. One such cohort is the multiethnic cohort in Hawaii and Los Angeles, which includes subcohorts from different ethnic groups in these areas [61]. Another large, multicenter cohort is the European Prospective Investigation into Cancer and Nutrition [62]. Utilization of large, diverse cohorts such as these, along with biochemical markers of nutrient status, where appropriate, may increase confidence in diet and disease relationships observed.\n\nIn the meantime, however, the optimal approach for lung cancer prevention should emphasize the proven benefits of tobacco cessation and prevention, in addition to what can be considered \"prudent\" recommendations to increase consumption of fruits and vegetables of all types, and to reduce intake of meat, total fat, and saturated fat. This dietary pattern is associated not only with a lower risk of lung cancer, but also with lower risk of many other chronic diseases, as described elsewhere in this volume.\n\nReferences\n\n1. 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Biomarkers Prev_. 1993;2:183\u2013187.\n\n21. Voorrips L.E., Goldbohm R.A., Brants H.A.M., van Poppel G.A.F.C., Sturmans F., Hermus J.J., van den Brandt P.A. A prospective cohort study on antioxidants and folate intake and male lung cancer risk. _Cancer Epidemiol. Biomarkers Prev_. 2000;9:357\u2013365.\n\n22. Garcia-Closas R., Agudo A., Gonzalez C.A., Riboli E. Intake of specific carotenoids and flavonoids and the risk of lung cancer in women in Barcelona. _Nutr. Cancer_. 1998;32:154\u2013158.\n\n23. Bjelke E. Dietary vitamin A and human lung cancer. _Int. J. Cancer_. 1975;15:561\u2013565.\n\n24. The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. _N. Engl. J. Med_. 1994;330:1029\u20131035.\n\n25. Omenn G.S., Goodman G.E., Thornquist M.D., Balmes J., Cullen M.R., Glass A., Keogh J.P., Meyskens F.L., Jr., Valanis B., Williams J.H., Barnhart S., Hammar S. Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. _N. Engl. J. Med_. 1996;334:1150\u20131155.\n\n26. Hennekens C.H., Buring J.E., Manson J.E., Stampfer M., Rosner B., Cook N.R., Belanger C., LaMotte F., Gaziano J.M., Ridker P., Willett W., Peto R. Lack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease. _N. Engl. J. Med_. 1996;334:1145\u20131149.\n\n27. Lee I.M., Cook N.R., Manson J.E., Buring J.E., Hennekens C.H. Beta-carotene supplementation and incidence of cancer and cardiovascular disease: The Women's Health Study. _J. Natl. Cancer Inst_. 1999;91:2102\u20132106.\n\n28. Mayne S.T., Beta-carotene, carotenoids, and cancer prevention. DeVita V.T., Jr., Hellman S., Rosenberg S.A., eds. Principles and Practice of Oncology (PPO) Updates. Carey, North Carolina: Lippincott Raven Healthcare; 1998;Vol. 12:1\u201315.\n\n29. Blot W.J., Li J.-Y., Taylor P.R., Guo W., Dawsey S., Wang G.-Q., Yang C.S., Zheng S.-F., Gail M., Li G.-Y., Yu Y., Liu B.-Q., Tangrea J., Sun Y.-H., Liu F., Fraumeni J.F., Jr., Zhang Y.-H., Li B. Nutrition intervention trials in Linxian, China: Supplementation with specific vitamin\/mineral combinations, cancer incidence, and disease-specific mortality in the general population. _J. Natl. Cancer Inst_. 1993;85:1483\u20131492.\n\n30. Wang X-D., Liu C., Bronson R.T., Smith D.E., Krinsky N.I., Russell R.M. Retinoid signaling and activator protein-1 expression in ferrets given beta-carotene supplements and exposed to tobacco smoke. _J. Natl. Cancer Inst_. 1999;91:60\u201366.\n\n31. Block G. Vitamin C and cancer prevention: The epidemiologic evidence. _Am. J. Clin. Nutr_. 1991;53:270S\u2013282S.\n\n32. Manson J.E., Gaziano J.M., Spelsberg A., Ridker P.M., Cook N.R., Buring J.E., Willett W.C., Hennekens C.H. A secondary prevention trial of antioxidant vitamins and cardiovascular disease in women: Rationale, design and methods. _Ann. Epidemiol_. 1995;5:261\u2013269.\n\n33. Christen W.G., Gaziano J.M., Hennekens C.H. Design of Physicians' Health Study II\u2014a randomized trial of beta-carotene, vitamins E and C, and multivitamins, in prevention of cancer, cardiovascular disease, and eye disease, and review of results of completed trials. _Ann. Epidemiol_. 2000;10:125\u2013134.\n\n34. Institute of Medicine, National Academy of Sciences, Food and Nutrition Board, Panel on Dietary Antioxidants and Related Compounds. _Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids_. Cedar Knolls, NJ: National Academy Press; 2000.\n\n35. Mayne S.T., Vogt T.M., Antioxidant nutrients in cancer prevention. DeVita V.T., Jr., Hellman S., Rosenberg S.A., eds. Principles and Practice of Oncology (PPO) Updates. Washington, DC: Lippincott Williams & Wilkins Healthcare; 2000;Vol. 14:1\u201312.\n\n36. Woodson K., Tangrea J.A., Barrett M.J., Virtamo J., Taylor P.R., Albanes D. Serum alpha-tocopherol and subsequent risk of lung cancer among male smokers. _J. Nat. Cancer Inst_. 1999;91:1738\u20131743.\n\n37. Ratnasinghe D., Tangrea J.A., Forman M.R., Hartman T., Gunter E.W., Qiao Y.L., Yao S.X., Barett M.J., Giffen C.A., Erozan Y., Tockman M.S., Taylor P.R. Serum tocopherols, selenium and lung cancer risk among tin miners in China. _Cancer Causes Control_. 2000;11:129\u2013135.\n\n38. Menkes M.S., Comstock G.W., Vuilleumier J.P., Helsing K.J., Rider A.A., Brookmeyer R. Serum beta-carotene, vitamins A and E, selenium, and the risk of lung cancer. _N. Engl. J. Med_. 1986;315:1250\u20131254.\n\n39. Comstock G.W., Alberg A.J., Huang H.-Y., Wu K., Burke A.E., Hoffman S.C., Norkus E.P., Gross M., Cutler R.G., Morris J.S., Spate V.L., Helzlsouer K.J. The risk of developing lung cancer associated with antioxidants in the blood: Ascorbic acid, carotenoids, alpha-tocopherol, selenium, and total peroxyl radical absorbing capacity. _Cancer Epidemiol. Biomarkers Prev_. 1997;6:907\u2013916.\n\n40. van den Brandt P.A., Goldbohm A., van't Veer P., Bode P., Dorant E., Hermus R.J., Sturmans F. A prospective cohort study on selenium status and the risk of lung cancer. _Cancer Res_. 1993;53:4860\u20134865.\n\n41. Knekt P., Aromaa A., Maatela J., Alfthan G., Aaran R.K., Hakama M., Hakulinen T., Peto R., Teppo L. Serum selenium and subsequent risk of cancer among Finnish men and women. _J. Natl. Cancer Inst_. 1990;82:864\u2013868.\n\n42. Knekt P., Marniemi J., Teppo L., Heliovaara M., Aromaa A. Is low selenium status a risk factor for lung cancer? _Am. J. Epidemiol_. 1998;148:975\u2013982.\n\n43. Clark L.C., Combs G.F., Turnbull B.W., Slate E.H., Chalker D.K., Chow J., Davis L.S., Glover R.A., Graham G.F., Gross E.G., Krongrad A., Lesher J.L., Jr., Park H.K., Sanders B.B., Jr., Smith C.L., Taylor J.R. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. _JAMA_. 1996;276:1957\u20131963.\n\n44. Bandera E.V., Freudenheim J.L., Marshall J.R., Zielezny M., Priore R.L., Brasure J., Baptiste M., Graham S. Diet and alcohol consumption and lung cancer risk in the New York State cohor (United States). _Cancer Causes Control_. 1997;8:828\u2013840.\n\n45. Speizer F.E., Colditz G.A., Hunter D.J., Rosner B., Hennekens C. Prospective study of smoking, antioxidant intake, and lung cancer in middle-aged women (USA). _Cancer Causes Control_. 1999;10:475\u2013482.\n\n46. Heimburger D.C., Alexander C.B., Birch R., Butterworth C.E., Bailey W.C., Krumdieck C.L. Improvement in bronchial squamous metaplasia in smokers treated with folate and vitamin B12: Report of a preliminary randomized, double-blind intervention trial. _JAMA_. 1988;259:1525\u20131530.\n\n47. Rauscher G.H., Mayne S.T., Janerich D.T. Relation between body mass index and lung cancer risk in men and women never and former smokers. _Am. J. Epidemiol_. 2000;152:506\u2013513.\n\n48. Alavanja M.C.R., Brown C.C., Swanson C., Brownson R.C. Saturated fat intake and lung cancer risk among nonsmoking women in Missouri. _J. Natl. Cancer Inst_. 1993;85:1906\u20131916.\n\n49. Swanson C.A., Brown C.C., Brownson R.C., Alavanja M.C.R. Re: Saturated fat intake and lung cancer risk among nonsmoking women in Missouri. _J. Natl. Cancer Inst_. 1997;89:1724\u20131725.\n\n50. Swanson C.A., Brown C.C., Sinha R., Kulldorff M., Brownson R.C., Alavanja M.C. Dietary fats and lung cancer risk among women: The Missouri Women's Health Study (United States). _Cancer Causes Control_. 1997;8:883\u2013893.\n\n51. Ko Y.-C., Cheng L.S.-C., Lee C.-H., Huang J.-J., Huang M.-S., Kao E.-L., Wang H.-Z., Lin H.-J. Chinese food cooking and lung cancer in women nonsmokers. _Am. J. Epidemiol_. 2000;151:140\u2013147.\n\n52. Shields P.G., Xu G.X., Blot W.J., Fraumeni J.F., Jr., Trivers G.E., Pellizzari E.D., Qu Y.H., Gao Y.T., Harris C.C. Mutagens from heated Chinese and U.S. cooking oils. _J. Natl. Cancer. Inst_. 1995;87:836\u2013841.\n\n53. Sinha R., Kulldorff M., Curtin J., Brown C.C., Alavanja M.C.R., Swanson C.A. Fried, well-done red meat and risk of lung cancer in women (United States). _Cancer Causes Control_. 1998;9:621\u2013630.\n\n54. Pastorino U., Infante M., Maioli M., Chiesa G., Buyse M., Firket P., Rosmentz N., Clerici M., Soresi E., Valente M., Belloni P.A., Ravasi G. Adjuvant treatment of stage I lung cancer with high-dose vitamin A. _J. Clin. Oncol_. 1993;11:1216\u20131222.\n\n55. van Zandwijk N., Dalesio O., Pastorino U., de Vries N., van Tinteren H., for the European Organization for Research and Treatment of Cancer, Head and Neck and Lung Cancer Cooperative Groups. EUROSCAN, a randomized trial of vitamin A and _N_ -acetylcysteine in patients with head and neck cancer or lung cancer. _J. Natl. Cancer Inst_. 2000;92:977\u2013986.\n\n56. Riboli E., Kaaks R. Invited commentary: The challenge of multi-center cohort studies in the search for diet and cancer links. _Am. J. Epidemiol_. 2000;151:371\u2013374.\n\n57. (Suppl.)Koo L.C. Diet and lung cancer 20% years later: More questions than answers? _Int. J. Cancer_. 1997;10:22\u201329.\n\n58. Pillow P.C., Hursting S.D., Duphorne C.M., Jiang H., Honn S.E., Chang S., Spitz M.R. Case-control assessment of diet and lung cancer risk in African Americans and Mexican Americans. _Nutr. Cancer_. 1997;29:169\u2013173.\n\n59. The Women's Health Initiative Study Group. Design of the Women's Health Initiative clinical trial and observational study. _Controll. Clin. Trials_. 1998;19:61\u2013109.\n\n60. The Women's Healthy Eating and Living Study GroupRock C.L., Newman V., Flatt S.W., Faerber S., Wright F.A., Pierce J.P. Nutrient intakes from foods and dietary supplements in women at risk for breast cancer recurrence. _Nutr. Cancer_. 1997;29:133\u2013139.\n\n61. Kolonel L.N., Henderson B.E., Hankin J.H. A multiethnic cohort in Hawaii and Los Angeles: Baseline characteristics. _Am. J. Epidemiol_. 2000;151:346\u2013357.\n\n62. Riboli E., Kaaks R. The EPIC project: Rationale and study design. _Int. J. Epidemiol_. 1997;26:S6\u2013S14.\nCHAPTER 26\n\nNutrition and the Patient with Cancer\n\nBARBARA ELDRIDGE1, CHERYL L. ROCK2 and PAULA DAVIS McCALLUM3\n\n1University of Colorado Health Sciences Center, Denver, Colorado\n\n2University of California at San Diego, La Jolla, California\n\n3Advantage Nutrition, Ltd., Chagrin Falls, Ohio\n\n## I. INTRODUCTION\n\nNutrition is an important component of the management of individuals diagnosed with cancer. Whether undergoing active therapy, recovering from cancer treatment, or in remission and aiming to avoid recurrence, the potential importance of optimal energy and nutrient intake has been examined in numerous studies, with general agreement that there is a role for nutrition intervention specific to the situation and needs of the patient [1, 2]. The primary goals for nutrition intervention in cancer are to prevent or reverse nutrient deficiencies, to preserve lean body mass, to minimize nutrition-related side effects, and to maximize the quality of life. Consistent screening practices to identify patients at risk for malnutrition are essential. Nutrition therapy is an integral part of cancer care from diagnosis through treatment and recovery. Moreover, patients view diet and nutrition as a way to participate in their care. Providing sound, individualized nutritional guidance can be challenging due to the vast and often contradictory amount of information that is currently available. This chapter discusses nutrition issues that are pertinent for patients with cancer throughout the continuum of care and also presents practical nutrition management strategies for dealing with treatment-related side effects.\n\n## II. RATIONALE AND IMPORTANCE OF OPTIMAL NUTRITION\n\nFrom an historical perspective, providing nutritional care to the patient with cancer has not always been believed to be wise or useful to incorporate in overall management. A few decades ago, fear that nutritional repletion would stimulate tumor growth, based on early studies with animal models, was a barrier to pursuing nutrition interventions in the patient with cancer [3]. In cell culture and animal studies, starvation has indeed been observed to slow tumor growth and proliferation of cancer cells [4, 5]. However, the health status of the host is concurrently adversely affected by nutritional depletion, and the malnutrition that results when nutrition intervention is not provided to the patient with cancer was historically a major cause of cancer-related mortality [6].\n\nMore recent laboratory studies have shown that when cancer is being treated, providing adequate energy and essential nutrients may possibly improve the efficacy and reduce the toxicity of chemotherapy and other cancer treatments [7], suggesting a potential beneficial role for maintaining adequate intake given the current improved approaches to cancer management. A number of clinical research studies have examined whether improving nutritional status can improve tolerance to oncologic therapies and reduce surgical complications in patients with cancer, and important factors determining benefit include the degree of risk for malnutrition that is present and the aggressiveness (with associated risk) of the nutrition intervention (discussed below).\n\nThe presence of cancer and cancer therapy can alter nutritional status significantly. Substantial weight loss and poor nutritional status have been documented in more than 50% of patients at the time of cancer diagnosis in clinical series reports [8, 9], although the prevalence of weight loss and malnutrition varies widely across cancer types. This point can be illustrated by results from a classic study of the prevalence and prognostic effect of weight loss prior to chemotherapy [10], in which data from 3,047 patients enrolled in 12 chemotherapy protocols of the Eastern Cooperative Oncology Group were examined and grouped according to weight loss as a percent of pre-illness weight. Patients with non-Hodgkin's lymphoma subtypes with favorable tumor tissue characteristics, breast cancer, acute nonlymphocytic leukemia, and sarcomas had the lowest prevalence of weight loss, with 60\u201369% of patients with those cancer types having no weight loss. Intermediate prevalence of weight loss, which was defined as a frequency of 48\u201361%, was observed in those with unfavorable non-Hodgkin's lymphoma subtypes, colon cancer, prostatic cancer, and lung cancer. The highest frequency of weight loss (83\u201387%) was observed in patients with pancreatic or gastric cancer, with approximately one-third of these patients reporting a loss of > 10% of prediagnosis body weight. Also, these investigators found that when grouped by cancer type, tumor extent, and activity level, median survival time was shorter in those who had experienced weight loss compared with those who had not experienced weight loss [10].\n\nWeight loss and poor nutritional status also have been suggested to be associated with lower scores on quality-of-life measures in patients with cancer. For example, in a case series of 119 patients with gastrointestinal cancer [11], weight-stable patients (compared to weight-losing patients) had significantly higher scores in response to queries addressing physical, cognitive, and social functioning, although a causal relationship cannot be assumed in this cross-sectional study. However, depression and the loss of independence that result from nutritional depletion and debilitation are well-known clinical sequelae of malnutrition in patients with cancer [12], and the response of family and friends to cancer-related weight loss can add additional stress and anxiety to the patient's situation [4].\n\n## III. CANCER CACHEXIA\n\nThe predominant nutritional problem in patients diagnosed with cancer is a variant of protein energy malnutrition, which is commonly noted as a secondary diagnosis. Termed _cancer cachexia,_ the syndrome is characterized by weight loss, anorexia, muscle wasting, increased basal metabolic rate, immunosuppression, and abnormalities in fluid status and energy metabolism. It differs from simple starvation-related protein energy malnutrition primarily because the compensatory mechanisms that would promote the preservation of muscle mass and adaptation to alternate fuels are apparently not operating.\n\nThe etiology of cancer cachexia is not entirely understood, and it can manifest in patients with metastatic cancer, as well as in patients with localized disease. Energy imbalance in these patients can be due to reduced intake and\/or increased expenditure, with metabolic alterations contributing to the depletion of lean muscle mass.\n\n### A. Anorexia and Inadequate Dietary Intake\n\nInadequate dietary intake may be caused by anorexia or altered taste perceptions, or may be the result of systemic effects of the neoplastic process or tumor-related symptoms (especially if gastrointestinal tract malignancies are present) [13, 14]. Psychological problems such as anxiety, depression, and the emotional stress of diagnosis and treatment can also alter eating patterns and cause reduced intake. Anorexia is basically defined as loss of appetite or loss of the desire to eat [15]. Alterations in taste acuity and taste preferences also are often reported by patients diagnosed with cancer and may contribute to reduced dietary intake [16]. However, observations of taste perception abnormalities in patients with cancer must be interpreted cautiously if not adjusted for age or other factors known to influence this characteristic [17], and malnutrition itself is associated with changes in taste perception and preferences. An additional causal factor that can reduce the spontaneous intake of many patients with cancer is chemotherapy-induced changes in taste perceptions.\n\nIf the tumor involves the gastrointestinal tract, obstructions or tumor-related gastrointestinal symptoms, such as pain or altered sensations when swallowing, can cause intake to be markedly reduced [18]. Ovarian cancer and metastatic melanoma tumors can cause external impingement on the gastrointestinal tract, so that eating is uncomfortable [14]. Treatments for cancer, including surgery, radiation therapy, and chemotherapy (discussed below), have associated side effects that can influence appetite and ability to eat comfortably.\n\nCurrent evidence suggests that anorexia in patients with cancer also is likely caused in large part by systemic appetite-suppressing factors, such as cytokines, that are produced by the tumor cells and the immune cells [4]. These are the same factors that promote the metabolic alterations observed in cancer cachexia that result in the depletion of lean muscle mass (discussed below). Additionally, these factors also increase resting energy expenditure, so many patients experience weight loss despite normal or even above-normal dietary intake. The primary mechanism believed to be responsible for both anorexia and the metabolic characteristics of cancer cachexia is the interaction between the growing tumor and the immune system, which results in the production of cytokines (tumor necrosis factor, interleukin 1, interleukin 6, and alpha and gamma interferon), in addition to the secretion of hormonal factors that promote wasting [8, 9, 13].\n\n### B. Metabolic Abnormalities in Cancer Cachexia\n\nThe reader is referred to several comprehensive reviews of the metabolic alterations characteristic of cancer cachexia and the evidence linking the syndrome to the various circulating factors currently believed to be the primary causal agents [4, 8, 14, 19]. The overall features of cancer cachexia are similar to an acute metabolic stress response, in that the usual energy-conserving and protein-sparing metabolic adjustments to starvation are paradoxically absent in cancer cachexia.\n\nIncreased basal metabolic expenditure is often, but not always, observed in association with cancer cachexia [4]. Inconsistency in the findings in this area are likely due to patients being studied at various stages of illness and with differing nutritional status, as well as the methodological limitations in getting accurate measures in acutely ill and often hospitalized patients. In one clinical study involving patients with small cell lung cancer, a reduced or normalized level of basal energy expenditure was observed to occur in those who responded to chemotherapy, whereas patients who were nonresponders did not exhibit changes in basal energy expenditure [20], which supports the hypothesized causal role of tumor-host interactions. However, patients with cancer who are symptomatic or involved in treatments typically have lower amounts of physical activity, so their total energy expenditure may be similar to healthy subjects, despite increased levels of basal energy expenditure.\n\nIn contrast, increased protein turnover is quite consistently observed in patients with cancer cachexia, with skeletal muscle wasting resulting from increased glycolysis and decreased muscle protein synthesis [21]. Also typically present are increased lipolysis and decreased fat synthesis, due to decreased lipoprotein lipase activity, which can result in elevated serum lipids [8]. Abnormalities of carbohydrate metabolism include decreased glucose tolerance, increased glucose uptake, and increased lactate production. The fundamental problem is cytokine-induced gluconeogenesis and the increasing utilization of protein stores for fuel, which also has been hypothesized to be linked to increased tumor uptake of glucose as a preferential substrate [22]. However, it has been well established in animal models that this metabolic problem is caused by the presence of the tumor and the associated circulating factors [4, 8, 19], so limiting the intake of carbohydrate (either sources or amounts), which has been suggested among various cancer-fighting strategies in popular literature aimed toward patients with cancer, would be unlikely to alter the prognosis or progression. With or without exogenous glucose and regardless of carbohydrate sources in the diet, endogenous glucose production (from protein stores) would continue to be stimulated by the cytokines and other tumor factors until the tumor is resected or the cancer is in remission.\n\nIn summary, the anorexia-cachexia syndrome is multifactorial. In addition to metabolic aberrations, patients with cancer may experience a gamut of nutrition-related symptoms that affect their ability to eat. It is important to note that many of these symptoms are interrelated and may be relieved by a combination of nutritional counseling or support and pharmaceutical management.\n\n### C. Pharmaceutical Management of Cancer Cachexia\n\nSome patients suffer quite specifically from a lack of appetite as a primary factor leading to poor intake and weight loss. Those patients may benefit from appetite stimulants, which are ideally administered in combination with nutritional counseling and, in some cases, an anabolic agent. Published reviews on this topic include one on clinical studies of appetite-stimulating medications by Ottery _et al._ [23] and reviews on the rationale and research on pharmaceutical approaches to the management of cancer cachexia by Barber _et al._ [19] and Chlebowski [24].\n\nTable 1 provides a list of common nutrition-related symptoms and the supportive drugs therapies that are used to manage them. Many of these medications can be administered concomitantly and are ideally accompanied by nutritional counseling.\n\nTABLE 1\n\nCommon Supportive Drug Therapies for Nutrition-Related Symptoms\n\n_Source:_ Data drawn from [13, 71\u201373].\n\n## IV. BASIC NUTRITION CONCEPTS FOR MANAGING PATIENTS WITH CANCER\n\nWhatever the phase of treatment or recovery, individuals with cancer should strive to consume a nutritionally adequate diet. Eating well means including a variety of foods daily and consuming a diet that contains recommended amounts of the essential nutrients, including protein, carbohydrate, fat, vitamins, minerals, and water, necessary to maintain health (see Chapter 48). During times of illness and recovery from cancer treatments, nutritional requirements are often increased and individuals may benefit from encouragement to consume nutrient-dense foods to ensure adequate nutrition and weight maintenance. As noted above, individuals that maintain body weight and nutrient stores may be able to better tolerate treatment-related side effects and recover from therapy more quickly. Improved nutrition status helps to maintain strength and energy, enhance quality of life, and repair and rebuild tissues that have been affected by cancer therapy [11, 25].\n\nThe American Cancer Society's Guidelines on Diet, Nutrition and Cancer Prevention (shown in Table 2 [26]) also provides sound advice regarding healthy eating for cancer prevention for all individuals, including cancer survivors [11]. An additional resource from the American Cancer Society summarizes current knowledge on nutrition for cancer survivors [27].\n\nTABLE 2\n\nAmerican Cancer Society Guidelines on Diet, Nutrition, and Cancer Prevention\n\n1. Choose most of the foods you eat from plant sources.\n\n\u2022 Eat five or more servings of fruits and vegetables each day.\n\n\u2022 Eat other foods from plant sources, such as breads, cereals, grain products, rice, pasta, or beans several times each day.\n\n2. Limit your intake of high-fat foods, particularly from animal sources.\n\n\u2022 Choose foods low in fat.\n\n\u2022 Limit consumption of meats, especially high-fat meats.\n\n3. Be physically active\u2014achieve and maintain a healthy weight.\n\n\u2022 Be at least moderately active for 30 minutes or more on most days of the week.\n\n\u2022 Stay within your healthy weight range.\n\n4. Limit alcoholic beverages, if you drink at all.\n\n_Source:_ From American Cancer Society (1996). Advisory Committee on Diet, Nutrition, and Cancer Prevention (1996). Guidelines on diet, nutrition, and cancer prevention: Reducing the risk of cancer with healthy food choices and physical activity. _CA Cancer J. Clin._ **46,** 325\u2013342.\n\n### A. Intakes of Energy and Protein and Body Weight\n\nNutrition intervention for patients with cancer is basically supportive therapy, regardless of whether the current goal of care is curative or palliative. Patients often need encouragement to consume sufficient energy and protein to maintain their stores. Achievement and maintenance of an appropriate weight for their height can be another important issue. During the course of therapy and recovery, some individuals may need to increase their energy consumption for weight maintenance or desired weight gain, while others may need to decrease their energy intake for desired weight loss. However, weight lost during initial cancer therapy is more likely due to the loss of muscle (lean body mass) than fat stores. The loss of lean body mass can contribute to fatigue, adversely impact the immune system, and delay and lengthen recovery. Thus, even if patients are overweight, the maintenance of lean tissue and body cell mass should be encouraged during treatment and recovery. The reader is referred to a review of approaches to estimating energy requirements in Chapter 2.\n\nCurrently, the most widely used tool to estimate desirable body weight for height is the body mass index (see Chapter 31). Body mass index adjusts for height and has been shown to be highly correlated with actual adiposity. Based on available evidence, a body mass index range for best health is currently defined as 20\u201325 kg\/m2. Because more precise measures of body composition are generally not available for use in usual patient care settings, due to their high cost and limited accessibility, bioelectric impedance analysis is often used in this setting to estimate body composition, although there are some known limitations to this methodology. Even skinfold measurements and waist and hip circumferences can provide some insight about body composition in the patient with cancer.\n\nProtein needs are also increased in times of illness, therapy, and recovery. Extra protein is needed by the body to repair and to rebuild tissues affected by treatment and to maintain a healthy immune system. However, nitrogen and energy balance are inextricably linked, and if adequate energy is not consumed during times of stress, the body will use its protein reserves (lean body mass) as a fuel substrate.\n\n### B. Micronutrients\n\nSome patients with cancer take large amounts of vitamin and mineral supplements because they are led to believe that these products can help to combat cancer or enhance their immune systems. Although essential micronutrients are necessary for optimal health, controlled clinical trials have not shown that vitamin and mineral supplements, even at high dosages, are useful in promoting cancer remission or improving cancer-related symptoms [27\u201329]. In fact, large doses of some vitamins can cause adverse side effects such as gastrointestinal discomfort [30], which can exacerbate eating problems in patients with cancer. Another current issue relates specifically to the antioxidant micronutrients, which are promoted for their ability to protect cells from free radical damage or oxidation. Radiation therapy and alkylating chemotherapy agents kill cancer cells by oxidative damage, so some researchers have expressed concern that large doses of antioxidants, especially single nutrients, just before and during therapy may actually interfere with the action of prescribed anticancer therapeutic regimens [31]. Results from other studies using cell cultures, however, have suggested that antioxidants may enhance the effects of standard radiation and chemotherapy [32]. Thus, this is a controversal issue that is not fully resolved at this time.\n\nWhen patients are able to consume a reasonably nutritious diet with adequate energy and protein, supplementation with vitamins and minerals is usually not necessary [27, 33]. Because non-nutrient constituents of foods may provide many health benefits, supplements should never replace the intake of regular foods and the goal of eating a healthy diet. However, if patients are experiencing difficulty eating and treatment-related side effects, the use of a multivitamin and mineral supplement that provides no more than 100% of the U.S. Recommended Dietary Allowances, is generally considered to be safe [34\u201337]. Patients should also be encouraged to communicate with their health care providers regarding their use of vitamin and mineral supplements. Developing and maintaining a good rapport and avoiding a judgmental attitude and demeanor can help to promote good communications and reduce the likelihood of harm from self-treatment with high-dose micronutrient supplements.\n\n### C. Importance of Regular Physical Activity\n\nAlthough information is limited regarding the role of physical activity in the management of cancer patients and survival [27], regular physical activity is known to be associated with numerous health benefits. Increased physical activity has been shown to increase energy, improve of quality of life, and reduce fatigue and anxiety in patients with cancer [38]. Other benefits include improved maintenance of lean body mass, maintenance of mobility, and stress reduction. However, before participating in any type of physical activity or exercise program, patients with cancer should be advised to obtain an evaluation by qualified professionals for an individualized physical assessment and plan specific to their capabilities and needs.\n\n### D. Complementary and Alternative Medicine\n\nSurvey studies suggest that more than half of all patients with cancer use some form of complementary or alternative therapies [39]. Patients seek complementary and alternative medicine for a variety of reasons: to promote health and prevent cancer; to replace conventional therapies that have been exhausted, shown to be ineffective, or are associated with side effects and significant risk; or to gain control over their own care [40]. The American Cancer Society defines complementary therapies as supportive methods that are used to complement evidence-based treatment, to help control symptoms, and to improve well-being and quality of life [41]. Alternative therapies refer to treatments that are promoted as cancer cures and are unproven or scientifically disproved methods. Categories of nutrition-related complementary and alternative therapies include vitamin and mineral supplementation, single- or multinutrient preparations; herbals (leaves of a plant) and botanicals (seeds, stem, roots); and nutrition (diet) and metabolic therapies. Examples of complementary and alternative medicines commonly used by patients with cancer are listed in Table 3.\n\nTABLE 3\n\nCommon Complementary and Alternative Medicines for Patients with Cancer\n\n_Source:_ Data from [74\u201381].\n\nCurrently, the U.S. Food and Drug Administration does not regulate herbal and botanical products, and patients need to be aware that just because products maybe \"natural\" they may not be safe (see Chapter 17). General guidelines for selecting herbals and botanicals include using only standardized extracts or products that list plant components and their quantities; buying products manufactured by reputable companies that are prepared, standardized, and manufactured with quality control; and using low, conservative doses. Patients should also be advised that many of the alternative nutrition and diet therapies advocated require substantial changes in regular eating habits and may be nutritionally inadequate. As noted above, patients need to be encouraged to discuss their use of complementary and alternative therapies with their health care professionals.\n\n## V. NUTRITION ISSUES THROUGHOUT THE CONTINUUM OF CARE\n\nCancer and cancer therapy can increase nutritional needs significantly, as well as affect normal digestion, absorption, and metabolism. Many individuals experience treatment-related side effects such as fatigue, loss of appetite, taste alterations, nausea, vomiting, and changes in bowel function that can further impair their ability to consume adequate energy and essential nutrients. Side effects of therapy are usually temporary, although some individuals may experience lasting changes that affect their ability to eat and maintain optimal nutritional status.\n\n### A. Nutritional Screening and Assessment of the Patient with Cancer\n\nScreening and nutritional assessment should be interdisciplinary; the health care team (e.g., physicians, nurses, registered dietitians, social workers) should all be involved in the nutritional management of patients throughout the continuum of care. Basic concepts and components of nutritional assessment are described in Part 1 of this text and are reviewed in detail elsewhere [42]. In the clinical setting, indices that incorporate several aspects of nutritional assessment are often useful. An example of one of these nutritional screening and assessment tools is the Scored Patient-Generated Subjective Global Assessment. This global assessment tool is adapted from the original Subjective Global Assessment, developed by Detsky, to include the patient responses and more cancer-specific symptoms [43, 44]. The scored version is a further adaptation by Ottery and colleagues that also includes guidelines for the triage of nutritional therapy [45].\n\nThe Scored Patient-Generated Subjective Global Assessment is an easy-to-use, inexpensive approach that can be used in multiple clinical settings and is ideal for interdisciplinary use. Patients and\/or caregivers complete sections on weight history, food intake, symptoms, and functioning. A health care team member evaluates weight loss, disease, and metabolic stress and performs a nutrition-related physical examination. A score is generated from the information collected. The need for nutrition intervention is determined according to the score.\n\n### B. Routes of Providing Nutritional Support and Care\n\nIdeally, all patients with cancer should be screened and assessed for nutritional risk at the time of diagnosis and reevaluated throughout the course of their treatment and recovery. In general, care of the patient with cancer involves individualized guidance on the appropriate routes of intake and on how to best manage treatment-related side effects. Prompt determination and early intervention of nutrition-related problems are vital for improving outcomes.\n\nBecause of the well-established adverse effect of poor nutritional status on morbidity and mortality, and the high prevalence of malnutrition in this population, numerous clinical trials of the effect of nutrition intervention on various outcome measures in patients with cancer have been conducted and reported during the 1980s and 1990s. However, the overwhelming majority of these intervention trials have involved total parenteral nutrition, which carries risk for infection and other complications. In contrast, few research efforts have tested the efficacy of lower risk nutrition interventions, such as nutritional counseling and dietary manipulations, and more research on these interventions in this patient population is sorely needed.\n\n#### 1. ORAL INTAKE\n\nIf at all possible, oral nutrition is the preferred route for feeding. Specifically, several strategies have been proposed for modifying the diet according to patients' specific needs as dictated by their disease processes and treatment-related side effects (see Table 4). Also, the use of liquid nutritional supplements may be necessary for patients unable to consume sufficient energy and protein to maintain their weight and optimum nutritional status (see Chapters 15 and ).\n\nTABLE 4\n\nNutrition Management Strategies for Patients with Cancer\n\nA telephone-based follow-up study of nutrition counseling was reported by Schiller _et al._ [46], with a study population consisting of 400 patients who had been referred for nutrition counseling, of whom 4% of the sample were patients with cancer. Overall, the counseling interaction was found to be well received by the patients, with 85% reporting at follow-up that they knew what to eat after talking with the dietitian, and 62% reporting that they had changed their diets following the counseling while 44% reported health-related changes. In a study of diet modification specifically relevant to patients with cancer, Menashian _et al._ [47] tested the effect of a modified diet to help control nausea and vomiting in 19 patients receiving cisplatin chemotherapy. Patients in the study group who were advised to consume the modified diet, which consisted of foods observed clinically to be better tolerated during chemotherapy, experienced fewer episodes of emesis when compared to the control subjects and 57% less volume of emesis on the first day of treatment.\n\n#### 2. ENTERAL NUTRITION\n\nWhen patients are unable to meet their nutritional needs orally, other routes of nutrition support need to be considered. Enteral nutrition is indicated when patients cannot eat due to mechanical obstruction or prolonged anorexia, or if they cannot consume adequate oral intake due to treatment-related toxicities (i.e., odynophagia, mucositis, esophagitis) 48]. Feeding enterally, rather than parenterally, helps to reduce infectious complications such as bacterial translocation, by preserving gut barrier and immune function. Enteral nutritional formulas may be administered via nasoenteric tube for short-term use. A nasoenteric tube may be advanced through the stomach to the proximal duodenum to reduce the risk for aspiration. More permanent feedings may be administered via gastrostomy or jejunostomy directly into stomach or small intestine, respectively (see [Chapter 16 for a more detailed discussion). This approach has been demonstrated to be an efficient method of providing long-term enteral nutrition support and is generally more acceptable to patients than using a nasoenteric tube [48].\n\nBased on nutritional status at presentation, existing comorbidity, and prescribed cancer treatments, many patients may benefit from prophylactic placement of percutaneous endoscopic gastrostomy feeding tubes to deliver enteral nutrition support [49]. Feeding tubes usually need to remain in place through recovery to ensure normalization of oral intake and weight. One instance in which enteral (tube) feeding may be superior to oral feeding is when elemental diets are used. These diets may help to maintain energy and nutrient intakes in some circumstances, for example, during pelvic radiation therapy, when absorptive capabilities are limited.\n\n#### 3. PARENTERAL NUTRITION\n\nThe reader is referred to several recent [4, 8, 14, 19, 48] and early [24, 50, 51] reviews of the numerous clinical trials of total parenteral nutrition in the care of patients before and during treatments for cancer. This approach, when applied without specific inclusion criteria, has not been shown to improve nutritional measures in the average patient with cancer and is actually associated with increased risk of complications such as infections. In 1989, the American College of Physicians published a position paper concluding that parenteral nutrition support was associated with net harm in patients with cancer [52].\n\nHowever, parenteral nutrition support may be appropriate for some patients with cancer for whom oral intake or enteral nutrition is not an option. The main concern is with the indiscriminate use in patients who are undergoing routine cytotoxic treatments and who do not have preexisting malnutrition. For example, in the classic Veterans Affairs Total Parenteral Nutrition Cooperative Study [53], 395 patients who required laparotomy or noncardiac thoracotomy, with the majority having a diagnosis of cancer, were randomly assigned to receive either total parenteral nutrition for 7\u201315 days prior to surgery and 3 days afterward or no perioperative parenteral nutrition support. Overall, there were more infectious complications in the parenteral nutrition group than in the controls. However, among those who were severely malnourished based on global assessment score, patients who received total parenteral nutrition had fewer noninfectious complications than controls (relative risk 0.12, 95% confidence interval 0.02\u20130.91) with no increase in infectious complications [52]. Another situation in which total parenteral nutrition is currently believed to be beneficial in the management of patients with cancer is in bone marrow transplantation (discussed below) [14]. As summarized by Mercadante [48], the use of parenteral nutrition support should be considered adjuvant treatment and support during therapy for malnourished patients or in those with severely impaired gastrointestinal function who are otherwise expected to survive.\n\nIn the care of patients who are managed with parenteral nutrition, complications include potential for fluid overload in patients receiving blood products, chemotherapy and medications via peripheral or central access; hyperglycemia secondary to the high concentration of dextrose present in parenteral mixtures and insulin resistance associated with severe illness; electrolyte imbalance; and increased triglycerides in patients receiving lipid emulsions. Intense monitoring of parenteral nutrition is required to minimize possible complications and to ensure that nutritional needs are being met appropriately.\n\n### C. Nutritional Effects of Cancer Therapies\n\nConventional methods of cancer treatment include surgery, radiation therapy, and chemotherapy. In many instances, patients receive multimodality therapy to most effectively treat their cancers. Prompt nutritional screening and assessment followed by appropriate pharmaceutical intervention and nutritional counseling to manage treatment-related side effects may help improve tolerance to therapy and enable patients to recover more quickly. Patients receiving aggressive cancer therapy (e.g., bone marrow transplantation) typically need aggressive nutrition support. The following sections review common treatment-related side effects and possible nutrition implications.\n\n#### 1. SURGERY\n\nAfter surgery, sufficient energy and protein are required for wound healing and recovery. Many patients experience pain, fatigue, and loss of appetite, and are unable to consume their regular diet because of surgery-related side effects. The surgical removal or resection of any part of the alimentary tract can impair digestion (including mastication) and absorption substantially. Most side effects are temporary and dissipate within a few days of the surgical procedure. Some surgical interventions, however, may have long-lasting nutritional implications. Medications, adherence to postsurgery discharge instructions, and changes in diet can help reduce the side effects experienced. Most patients benefit from some type of a diet progression (i.e., clear liquids, then easy-to-digest foods, then regular diet) in the first few days postoperatively. Table 5 lists common side effects of cancer surgery.\n\nTABLE 5\n\nCommon Side Effects of Cancer Surgery\n\n#### 2. CHEMOTHERAPY\n\nChemotherapy involves the use of chemical agents and medications to treat cancer and is a systemic therapy that affects the whole body [14]. The action of chemotherapeutic agents can be cytotoxic to normal cells as well as malignant cells, in particular cells with a rapid turnover such as bone marrow, hair follicles, and mucosa of the alimentary tract. Common side effects with nutritional implications include loss of appetite, fatigue, nausea, vomiting, mucositis, taste alterations, xerostomia, dysphagia, and changes in bowel function. These agents can also adversely impact renal and hepatic function, as well as cause damage to the gastrointestinal tract, which can result in the impairment of digestion and absorption, further compromising nutritional status. Chemotherapy can also cause immunosuppression, resulting in neutropenia, thrombocytopenia, and anemia. The severity of the side effects experienced is related to the prescribed treatment regimen: single or combination agent therapy, dose administration, planned number of cycles, individual response, and current health status. The timely and appropriate use of supportive therapies and medications are important to the effective management of treatment-related side effects.\n\n#### 3. RADIATION THERAPY\n\nRadiation therapy can be delivered from an outside source into the body (external beam therapy) or by placing a radioactive source inside the body (brachytherapy or internal therapy). Whereas chemotherapy is a systemic treatment, radiation therapy and surgery only affect the tumor and the area surrounding it. Some chemotherapy agents may be given in combination with radiation therapy due to their radiation-enhancing effects. Patients receiving multimodality therapy may manifest side effects sooner and with greater toxicity. Side effects are most commonly site specific (i.e., limited to the area being treated). Patients generally begin to notice the effects of external beam radiation therapy after 8\u20130 days of treatment, and side effects usually resolve within 2\u20134 weeks after treatment is complete. Regardless of the site of radiation therapy, patients usually experience some degree of fatigue, loss of appetite, and skin changes and hair loss in the treated area. Use of supportive therapies and medications are important in the management of treatment-related side effects. Table 6 lists common side effects of radiation therapy.\n\nTABLE 6\n\nCommon Side Effects of Radiation Therapy\n\nSite of radiation therapy | Commonly experienced side effects \n---|--- \nBrain | Nausea; vomiting; elevated blood glucose due to steroid administration; fatigue; loss of appetite \nHead and neck | Xerostomia; sore mouth; dysphagia; odynophagia; taste alterations; mucositis; fatigue; loss of appetite \nLung, esophagus, and breast | Dysphagia; heartburn; fatigue; loss of appetite \nAbdomen | Nausea; vomiting; fatigue; loss of appetite \nPelvis | Changes in bowel function\u2014diarrhea, gas, bloating, cramping; milk intolerance; fatigue; loss of appetite \nBone | Fatigue; loss of appetite; constipation due to pain medication\n\n### D. Nutrition Concerns during and after Recovery from Cancer Therapy\n\nAfter treatment ends, optimal nutrient intake and physical activity are vital to the recovery process. Lingering effects of treatment may require continued attention and appropriate nutrition intervention. Patients should be encouraged to strive for weight stabilization or increased energy and protein intake to achieve and maintain normal body weight. The ideal is to return to normal eating and to achieve a weight that is appropriate for height. For many patients, their precancer diagnosis diets may have been poor, and due to increased health concerns, they may be interested in improving their eating habits.\n\nWhereas the acute toxicities of surgery, chemotherapy, and radiation therapy may become apparent during the course of treatment, some side effects may not manifest until several weeks, months, or even years after the treatment has been completed. Examples of treatment-related late-occurring side effects with nutrition implications are listed in Table 7. Patients living with chronic treatment-related side effects face challenges with eating. Patients need to be reassessed during follow-up for late effects of treatment and receive appropriate nutrition intervention.\n\nTABLE 7\n\nLate-Occurring Side Effects with Nutritional Implications\n\nOsteoradionecrosis of the mandible and increased incidence of dental caries in patients following head and neck irradiation\u2014Difficulty with chewing and swallowing.\n\nFibrosis of the esophagus after head and neck irradiation\u2014Dilation of the esophagus may be necessary and\/or a speech pathology consultation for swallowing evaluation and management is indicated.\n\nPneumenitis after lung irradiation\u2014Difficulty with breathing and increased fatigue.\n\nChemotherapy-induced neuropathies of the fingers and toes\u2014Patients may experience decreased sensation, causing difficulty with food procurement and food preparation.\n\nRadiation enteritis after pelvic irradiation\u2014Alterations in digestion and absorption; patients may need to restrict (or limit) lactose, fiber, and\/or fat.\n\nBowel obstruction following pelvic irradiation for gynecological cancer\u2014Often requires the use of enteral nutrition to bypass the obstructed area, or parenteral nutrition may be indicated.\n\n### E. Nutritional Care for Advanced Cancer and Palliative Care\n\nManagement of nutrition-related symptoms continues in the care of patients living with advanced cancer. Emphasis should be placed on enhancing quality of life. Assisting patients in symptom management eases feelings of anxiety and fear, and helps them to maintain their sense of independence. The reader is referred to previous reviews on the ethical and feeding issues in advanced disease [54, 55].\n\n## VI. SPECIAL SITUATIONS\n\nA. Bone Marrow Transplantation\n\nBone marrow and stem cell transplant procedures can impact nutrition status significantly, and nutrition is an important part of the management of patients who are recipients of bone marrow transplantation. To eradicate malignant cells, cytotoxic conditioning regimens are given with or without total body irradiation, followed by an intravenous infusion of bone marrow or stem cells. Effects of therapy include immunosuppression lasting 2\u20134 weeks post-transplant, anorexia, xerostomia, dysgeusia, stomatitis, oral and esophageal mucositis, and diarrhea. Immunosuppressive medications also have significant nutrition implications that can result in multiple problems with eating. Using isotopic dilution technology to assess body composition, Cheney _et al._ [56] documented decreased body cell mass and negative nitrogen balance in patients receiving bone marrow transplantation for acute lymphocytic leukemia. Thiamin deficiency, a reduction in the plasma pool of vitamin E, and electrolyte and trace element deficiencies also have been observed in patients receiving bone marrow transplantation [57\u201359]. A majority of these patients currently receive much of their care outside of the hospital, so nutritional assessment and monitoring is critically important in their care.\n\nAwareness of the actions and the nutrition implications of cytotoxic and immunosuppressive agents can help health care professionals be proactive in patient care in this circumstance. Because the majority of patients undergoing bone marrow transplantation are unable to eat sufficient quantities of food to achieve the estimated energy requirement (or maintain the level of intake reported at admission), alternative routes to nutrition support are usually considered and have become a standardized component of care at most transplant centers.\n\nHistorically, parenteral nutrition has been considered the method of choice for ensuring nutritional adequacy in bone marrow transplantation, mainly due to the likelihood that gastrointestinal symptoms of considerable severity may be present. Because of the high level of risk for nutritional problems in these patients, parenteral nutrition support may even be initiated and utilized as prophylaxis, and is usually not discontinued until the patient demonstrates the ability to consume a sufficient amount of the oral diet to achieve a predetermined goal level of nutritional requirements (ranging from 50% to 70%). The usefulness of tube feeding is often limited by the presence of mucosal and esophageal ulceration, nausea and vomiting, and local infection of the mouth and esophagus, even when gut function appears normal. In clinical trials, total parenteral nutrition has been shown to result in significantly higher energy and protein intake when compared with allowing patients to consume an oral diet _ad libitum_ [60]. However, parenteral nutrition has not been consistently associated with favorable effects on duration of hospitalization, episodes of bacteremia, or total number of complications in these patients, and it has been suggested that intestinal mucosal integrity is better maintained by using enteral rather than parenteral nutrition support [57, 61]. In practice, aggressive parenteral nutrition support is also more likely to result in fluid overload, hyperglycemia, and catheter-related complications in these patients [62]. The goal is always to promote the progression toward achieving regular oral intake as a component of normal functioning and recovery, which may be impeded or delayed when patients are discharged on parenteral nutrition. In a controlled trial in which outpatient parenteral nutrition was compared with intravenous hydration, parenteral nutrition was associated with delayed resumption of oral intake after transplantation, while failing to provide any substantial improvement in patient outcome [63].\n\nIn another prospective randomized study [64], total parenteral nutrition was compared with an individualized enteral feeding program that involved close monitoring, snacks, and\/or tube feeding. Approximately one-quarter of the patients assigned to the enteral nutrition group ultimately had to receive parenteral nutrition due to intolerance of the tube feeding, but parenteral nutrition did not shorten the duration of marrow aplasia and was associated with more complications related to feeding route (i.e., fluid overload requiring diuretics). Also worth noting is that the cost of the enteral nutrition intervention (which included individualized attention and snacks) for the 28-day feeding period was less than half that of the parenteral nutrition approach. Results from other studies of enteral nutrition support suggest that, when tolerated, this approach is effective, probably underutilized, and may even impart some special protective effects [57, 61].\n\nModifications in the parenteral nutrition solutions used in the nutrition support of bone marrow transplant patients, such as the addition of certain amino acids (i.e., glutamine, arginine) or omega\u20133 fatty acids, are also currently under study. The addition of glutamine, which is not considered an essential amino acid, is based on the rationale that it may improve intestinal mucosal integrity, reduce the incidence of bacterial translocation, and improve nitrogen balance. In a small clinical trial, Ziegler _et al._ [65] demonstrated that glutamine-supplemented parenteral nutrition after bone marrow transplantation was associated with improved nitrogen balance, reduced incidence of clinical infection, lower rates of microbial colonization, and reduced hospital stay compared with patients receiving standard parenteral nutrition support. However, no differences in the incidence of fever or time of recovery from myelosuppression were observed. This is currently an area of great interest.\n\nA major complication of allogenic transplant (related or unrelated donor) is graft-versus-host disease, in which donor cells react against the tissues of the host. Graft-versus-host disease can manifest in up to two-thirds of bone marrow transplant recipients and may occur as early as 7\u201310 days after the bone marrow infusion [66]. Although rare, graft-versus-host disease has also been documented in patients receiving autologus (patient's own bone marrow) and stem cell transplants. Major organs affected by graft-versus-host disease are the intestinal tract, skin, and liver. Side effects of graft-versus-host disease include skin rash, nausea, vomiting, abdominal cramping and\/or pain, secretory diarrhea, malabsorption, and liver dysfunction. Patients with acute gut involvement require nutritional management that consists of bowel rest and parenteral nutrition followed by a diet progression for reintroducing foods.\n\nFollowing transplantation, patients become immunocom-promised, so supportive therapy to prevent infection is essential. In general, patients need to be instructed on food safety practices including avoidance of eating foods that contain unsafe levels of bacteria (raw meats, spoiled or moldy foods, and unpasteurized beverages); importance of thorough hand washing; special handling of raw meats, fish, poultry, eggs, utensils, cutting boards and countertops; and storage of foods at appropriate temperatures (below 40\u00b0F and above 140\u00b0F.\n\nA low-microbial or low-bacteria diet, which is based primarily on empiric knowledge of the distribution of microorganisms in the food supply, is often prescribed for these patients. These diets consist mainly of cooked foods, because the major limitation imposed is on fresh or uncooked food items. In a few previous studies, the microbial content of several food items has been actually evaluated [67, 68]. Only a few previous studies have evaluated the acceptability and efficacy of low-microbial diets in patients receiving bone marrow transplantation. The large variability in how these diets are actually defined across institutions contributes to the difficulty in examining the clinical impact. In an early review of studies on immunosuppressed patients with cancer who were treated with sterile, low-microbial, or regular diets, it was concluded that the question of whether infection, morbidity, mortality, and response to therapy are affected by this strategy cannot be adequately addressed, due in part to variability across patients, interventions, and other influencing factors [69]. Because many food restrictions are imposed with this strategy if strictly followed, the nutrient adequacy of actual intake of patients who are prescribed the low-microbial diet should be monitored.\n\n### B. Nutritional Care for Pediatric Patients with Cancer\n\nWorking with pediatric cancer patients can be challenging, due to their fears of cancer treatment and their apprehension regarding unfamiliar routines and caregivers. In addition, parents and family members may feel overwhelmed by the demands of their child's therapy, as well as the treatment-related side effects that their child may experience. The overall goals for nutritional care of the child with cancer include the management of nutrition-related symptoms and side effects, and the provision of adequate energy and nutrients for proper growth and development. The reader is referred to Andrassy and Chwals [70] for a review of the issues and scientific basis of nutritional support of the pediatric patient with cancer.\n\n## VII. SUMMARY AND CONCLUSIONS\n\nNutrition plays an important role in the management of the patient with cancer. Metabolic alterations caused by the presence of cancer and numerous adverse effects of treatments for cancer increase the risk for malnutrition in this patient population. Appropriate nutritional care may improve the patient's tolerance of treatments and also the quality of life during treatment and recovery. 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Courneya K., Friedenreich C. Physical exercise and quality of life following cancer diagnosis: A literature review. _Ann. Behav. Med_. 1999;21:1\u20139.\n\n39. Brigden M. Unproven (questionable) cancer therapies. _West. J. Med_. 1993;163:463\u2013469.\n\n40. Eisenberg D. Advising patients who seek alternative medical therapies. _Ann. Intern. Med_. 1997;127:61\u201369.\n\n41. American Cancer Society. _American Cancer Society Operation Statement on Complementary and Alternative Methods of Cancer Management_. Washington, DC: American Cancer Society; 1999.\n\n42. Simko M.D., Cowell C., Gilbride J.A. _Nutrition Assessment_. Atlanta, GA: Aspen; 1995.\n\n43. Detsky A., McLaughlin J., Baker J., Johnston N., Whittaker S., Mendelson R.A., Jeejeebhoy K.N. What is subjective global assessment of nutritional status? _J. Parenteral Enteral Nutr_. 1987;11:8\u201313.\n\n44. (Suppl.)Ottery F. Definition of standardized nutritional assessment and interventional pathways in oncology. _Nutrition_. 1996;12:S15\u2013S19.\n\n45. McCallum P. Patient-generated subjective global assessment. In: McCallum P.D., Polisena C.G., eds. _Clinical Guide to Oncology Nutrition_. Gaithersburg, MD: The American Dietetic Association; 2000:11\u201323.\n\n46. Schiller M.R., Miller F., Moore C., Davis E., Dunn A., Mulligan K., Zeller P. Patients report positive nutrition counseling outcomes. _J. Am. Diet. Assoc_. 1998;98:977\u2013982.\n\n47. Menashian, L., Flam, M., Douglas-Paxton, D., and Raymond, J. Improved food intake and reduced nausea and vomiting in patients given a restricted diet while receiving cisplatin chemotherapy. _J. Am. Diet. Assoc._ **92,** 58\u201361.\n\n48. Mercadante S. Parenteral versus enteral nutrition in cancer patients: Indications and practice. _Support Care Cancer_. 1998;6:85\u201393.\n\n49. (Suppl.).Eldridge B. Effectiveness of percutaneous endoscopic feeding tubes placed in head and neck radiation therapy patients. _J. Parenteral Enteral Nutr_. 16, 1992.\n\n50. Lipman T.O. Clinical trials of nutritional support in cancer. _Hematol. Oncol. Clin. North Am_. 1991;5:91\u2013102.\n\n51. Chen M.K., Souba W.W., Copeland E.M. Nutritional support of the surgical oncology patient. _Hematol. Oncol. Clin. North Am_. 1991;5:125\u2013145.\n\n52. American College of Physicians. Parenteral nutrition in patients receiving cancer chemotherapy. _Ann. Intern. Med_. 1989;110:734\u2013736.\n\n53. The Veterans Affairs Total Parenteral Nutrition Cooperative Study Group. Perioperative total parenteral nutrition in surgical patients. _N. Engl. J. Med_. 1991;325:525\u2013532.\n\n54. American Dietetic Association. Position of the American Dietetic Association: Issues in feeding the terminally ill adults. _J. Am. Diet. Assoc_. 1992;92:996\u20131002.\n\n55. Burck R. Feeding, withdrawing, and withholding: Ethical perspectives. _Nutr. Clin. Prac_. 1996;11:2143\u20132153.\n\n56. Cheney C.L., Abson K.G., Aker S.N., Lenssen P., Cunningham B.A., Buergel N.S., Thomas E.D. Body composition changes in marrow transplant recipients receiving total parenteral nutrition. _Cancer_. 1987;59:1515\u20131519.\n\n57. Hermann V.M., Petruska P.J. Nutrition support in bone marrow transplant recipients. _Nutr. Clin. Prac_. 1993;8:17\u201319.\n\n58. Rovelli A., Bonomi M., Murano A., Locasciulli A., Uderzo C. Severe lactic acidosis due to thiamine deficiency after bone marrow transplantation in a child with acute monocytic leukemia. _Haematologica_. 1990;75:579\u2013581.\n\n59. Clemens M.R., Ladner C., Ehninger G., Einsele H., Renn W., Buhler E., Waller H.D., Gey K.F. Plasma vitamin E and \u03b2-carotene concentrations during radiochemotherapy preceding bone marrow transplantation. _Am. J. Clin. Nutr_. 1990;51:216\u2013219.\n\n60. Weisdorf S.A., Lysne J., Wind D., Haake R.J., Sharp H.L., Goldman A., Schissel K., McGlave P.B., Ramsay N.K., Kersey J.H. Positive effect of prophylactic total parenteral nutrition on long-term outcome of bone mineral transplantation. _Transplantation_. 1987;43:833\u2013838.\n\n61. Papadopoulou A., MacDonald A., Williams M.D., Darbyshire P.J., Booth I.W. Enteral nutrition after bone marrow transplantation. _Arch. Dis. Child_. 1997;77:131\u2013136.\n\n62. Taveroff A., McArdle A.H., Rybka W.B. Reducing parenteral energy and protein intake improves metabolic homeostasis after bone marrow transplantation. _Am. J. Clin. Nutr_. 1991;54:1087\u20131092.\n\n63. Charuhus P.M., Fosberg K.L., Bruemmer B., Aker S.N., Leisenring W., Seidel K., Sullivan K.M. A double-blind randomized trial comparing outpatient parenteral nutrition with intravenous hydration: Effect on resumption of oral intake after marrow transplantation. _J. Parenteral Enteral Nutr_. 1997;21:157\u2013161.\n\n64. Szeluga P.J., Stuart R.K., Brookmeyer R., Utermohlen V., Santos G. Nutritional support of bone marrow transplant recipients: A prospective, randomized clinical trial comparing total parenteral nutrition to an enteral feeding program. _Cancer Res_. 1987;47:3309\u20133316.\n\n65. Ziegler T.R., Young L.S., Benfell K., Scheltinga M., Hortos K., Bye R., Morrow F.D., Jacobs D.O., Smith R.J., Antin J.H., Wilmore D.W. Clinical and metabolic efficacy of glutamine-supplemented parenteral nutrition after bone marrow transplantation. _Ann. Intern. Med_. 1992;116:821\u2013828.\n\n66. Charuhas P. Dietary management during antitumor therapy of cancer patients. _Topics Clin. Nutr_. 1993;9:42\u201353.\n\n67. Pizzo P.A., Purvis D.S., Water C. Microbiological evaluation of food items. _J. Am. Diet. Assoc_. 1982;82:272\u2013279.\n\n68. Moe G. Enteral feeding and infection in the immuno-compromised patient. _Nutr. Clin. Prac_. 1991;6:55\u201364.\n\n69. Aker S.N., Cheney C.L. The use of sterile and low microbial diets in ultraisolation environments. _J. Parenteral Enteral Nutr_. 1983;7:390\u2013397.\n\n70. Andrassy R.J., Chwals W.J. Nutritional support of the pediatric oncology patient. _Nutrition_. 1998;14:124\u2013129.\n\n71. Murphy S., Von Roenn J. Pharmacological management of anorexia and cachexia. In: McCallum P.D., Polisena C.G., eds. _Clinical Guide to Oncology Nutrition_. Chicago, IL: The American Dietetic Association; 2000:127\u2013133.\n\n72. Eldridge B. Chemotherapeutic and nutrition implications. In: McCallum P.D., Polisena C.G., eds. _Clinical Guide to Oncology Nutrition_. Chicago, IL: The American Dietetic Association; 2000:61\u201369.\n\n73. Levy M. Constipation and diarrhea in cancer patients. _Cancer Bull_. 1991;43:412\u2013422.\n\n74. Blumenthal M., Busse W.R., Goldberg A., Hall T., Riggins C.W., Rister R.S., eds. The Complete German E Monographs: The Therapeutic Guide to Herbal Medicines. Chicago, IL: American Botanical Council, 1998.\n\n75. Cassielith B. _The Alternative Medicine Handbook_. Austin, TX: W.W. Norton; 1998.\n\n76. Montbriand M. An overview of alternative therapies chosen by patients with cancer. _Oncol. Nurs. Forum_. 1994;21:1547\u20131554.\n\n77. (Suppl.)Spaulding-Albright N. A review of some herbal and related products commonly used in cancer patients. _J. Am. Diet. Assoc_. 1997;97:S208\u2013S215.\n\n78. Tyler V., Foster S. _The Honest Herbal_ , 4th Edition. New York: Haworth Press; 1999.\n\n79. Tyler V. _Herbs of Choice_. Binghamton, NY: Haworth Press; 1994.\n\n80. _Physician's Desk Reference on Herbal Medicine_. Binghamton, NY: Medical Economics; 1998.\n\n81. Molseed L. Alternative therapies in oncology. In: McCallum P.D., Polisena C.G., eds. _Clinical Guide to Oncology Nutrition_. Montvale, NJ: The American Dietetic Association; 2000:150\u2013159.\nC.\n\nDiabetes Mellitus\nCHAPTER 27\n\nObesity and the Risk for Diabetes\n\nREJEANNE GOUGEON, McGill University, Montreal, Quebec, Canada\n\n## I. INTRODUCTION\n\nIt has long been recognized that one of the medical consequences of obesity is development of type 2 diabetes mellitus. Epidemiological studies, both cross sectional [1\u201312] and prospective [8, 13\u201322], show a positive relationship between degree of obesity, notably that of central adiposity [7, 11, 15, 16], and the risk for diabetes. Two large prospective studies have examined the impact of obesity on the incidence of diabetes in women [23] and in men [22] and calculated that 77% of new cases in women and 64% in men could be prevented if body mass index [BMI, weight (kg)\/height (m2)] were maintained below 25 kg\/m2. An increase in the prevalence of obesity in certain countries may well explain the concurrent increase in diabetes prevalence [24, 25]. Furthermore, body fat mass has also been shown to be associated with an increase in the risk of prediabetic conditions such as glucose intolerance and insulin resistance [6].\n\nDespite these strong associations with diabetes, obesity does not appear to be an essential condition for type 2 diabetes to express itself in a genetically predisposed person. Indeed, 20\u201325% of persons with type 2 diabetes are not obese [23, 26, 27], and 80% of individuals with elevated BMI and indicators of high intra-abdominal adiposity do not develop diabetes [23]. Still, the improvement in diabetes control observed with weight loss [27\u201330] indicates that obesity has an impact on diabetes and its prevention and management. This chapter will examine some of the mechanisms that relate increased adiposity to diabetes, and the independent contribution of risk factors for obesity such as low physical activity level and unhealthy diets to the development of diabetes. This chapter (1) provides the current definitions and diagnostic criteria of obesity and diabetes, (2) reexamines some of the evidence that suggests a major role for excess adiposity in the etiology of diabetes and its complications, and (3) describes the contributions of weight loss and energy restriction in the management of the obese persons with diabetes.\n\n## II. DEFINITIONS AND CLASSIFICATIONS OF OBESITY AND DIABETES\n\nA. Obesity\n\nObesity has been explained as the result of an imbalance between the intake of energy substrates and energy utilization [31]. This imbalance promotes the shunting of substrates into anabolic pathways for synthesis and storage of fat [31]. Obesity is referred to as a condition when fat accumulation is excessive to an extent that it increases risk of ill health [32], especially if it is stored in the abdominal region [33]. Obesity may well represent a heterogeneous group of conditions. Because obesity has been poorly defined, it has been difficult to establish what role it plays in the etiology of diabetes. A World Health Organization (WHO) expert committee has proposed cutoff points for the classification of overweight and obesity [34] such that it would be possible to identify individuals or groups at risk and to compare weight status across populations. A classification can also provide a basis for the evaluation of interventions. Body mass index values (using BMI calculated as the weight in kilograms divided by the square of height in meters) have been used to classify obesity in populations because they correlate with percentage of body fat and with mortality and morbidity. Furthermore, the values are the same for both sexes because the relationship between BMI and mortality is similar in men and women. Because women have a higher percentage body fat for a comparable weight than men, this indicates that women can carry fat better than men. It has been suggested that they do so because their excess fat is mainly subcutaneous and peripherally distributed (thighs, buttocks, breasts) compared with men in whom fat is stored in the abdominal region [35].\n\nThe classification proposed by WHO [Table 1 [34]) is based on the relationship between BMI and mortality. BMIs between 18.5 and 24.9 kg\/m2 are defined as normal, between 30 and 39 as obese, and >40 as severely obese [35]. A BMI >30 does not always correspond to excess adiposity. A muscle builder may have a BMI of 30 that is associated with a large muscle mass. Ethnic groups with deviating body proportions, such as being very tall and thin, have healthy BMIs ranging from 17 to 22 kg\/m2 and have excessive fat mass at a BMI of 25 [36].\n\nTABLE 1\n\nCutoff Points for Body Mass Index\n\nBMI (kg\/m2) | WHO classification of obesity \n---|--- \n<18.5 | underweight \n18.5\u201324.9 | normal range \n25.0\u201329.9 | grade 1 overweight \n30.0\u201339.9 | grade 2 overweight \n\u226540.0 | grade 3 overweight\n\n_Source:_ World Health Organization (1997). Obesity: Preventing and managing the global epidemic. Report of a WHO Consultation presented at the World Health Organization, June 3\u20135, 1997. Publication WHO\/NUT\/NCD\/98.1. World Health Organization, Geneva, Switzerland.\n\nCutoff points have also been defined for waist circumference, which is another simple measure for predicting excess visceral adipose tissue [37]. It also predicts risk of cardiovascular disease and relates to both BMI and the waist: hip ratio.\n\nWaist circumference \u226594 cm in men [37 inches] and \u226580cm [32 inches] in women indicates a need for concern [38]and \u2265102 cm [40 inches] in men and \u226588 cm [35 inches] in women are critical levels of intra-abdominal fat [37] that indicate a need for action and intervention (Table 2).\n\nTABLE 2\n\nCutoff Points for Waist Circumferences\n\nWaist circumference | Men | Women \n---|---|--- \nNeed for concern | \u226594cm | \u226580cm \nCritical level | \u2265102cm | \u226588cm\n\n_Source:_ National Heart, Lung, and Blood Institute, Obesity Education Initiative Expert Panel (1998). Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults: The evidence report. _Obes. Res._ **6** (Suppl. 2), 51S\u2013209S.\n\nA decrease in waist circumference in response to intervention is an indication of a decrease in risk for chronic disease [39]. The waist circumference measured at the narrowest part of the torso, at midpoint between the lower border of the rib cage and the iliac crest, alone, is considered a useful tool for initial screening and follow-up assessment of change.\n\nVisceral fat prediction can be obtained by more complex methods such as computed tomography scans [40]. These can measure sagittal diameters from which abdominal subcutaneous fat measured by ultrasound can be subtracted for more precision [41].\n\n### B. Diabetes Mellitus\n\nDiabetes mellitus is a heterogeneous group of metabolic diseases characterized by elevated blood glucose concentration and disturbances of carbohydrate, fat, and protein metabolism secondary to absolute or relative deficiency in insulin action and\/or secretion [42, 43].\n\nAn untreated person with diabetes will present the following symptoms: excessive thirst, polyuria, pruritus, blurred vision, and unexplained weight loss. Asymptomatic diabetes, particularly type 2 diabetes mellitus, may be diagnosed when abnormal blood or urine glucose levels are found during routine testing. Exposure to chronic hyperglycemia is associated with pathologic and functional changes in organs such as the eyes, blood vessels, heart, kidneys, and nerves [43].\n\nThe classification of diabetes mellitus is based primarily on its clinical description and comprises four major types: type 1, type 2, other specific types, and gestational diabetes mellitus. All differ in their etiology. For example, type 1 diabetes, which is prone to ketoacidosis, is due to autoimmune or idiopathic destruction of the \u03b2 cells of the pancreas, destruction that results in a deficiency in insulin secretion. Type 2 diabetes is due to metabolic abnormalities leading to a diminished response to the action of insulin, along with a defect in insulin secretion. Other types relate to genetic defects, pancreatopathy or are first diagnosed during pregnancy (such as gestational diabetes), and may or may not subsequently develop into diabetes after parturition [44].\n\nThe classification also includes impaired glucose tolerance and impaired fasting glucose because of their association with increased risk of developing diabetes and cardiovascular disease [45].\n\nIn a previous classification [42], subgroups were identified to distinguish obese and non obese persons within the type 2 diabetes and impaired glucose tolerance categories; insulin resistance was reported to be accentuated in subjects who have central obesity and are physically inactive, implicating body weight and fat distribution as risk factors for glucose intolerance.\n\n#### 1. WHO HAS DIABETES?\n\nAlthough diabetes is characterized by alterations of fat [46] and of protein metabolism [47, 48], glucose impairment remains the hallmark of diabetes diagnosis and control.\n\nThe diagnosis of diabetes mellitus is made using one of three methods recommended by the American Diabetes Association:\n\n1. A person shows symptoms of diabetes (polyuria, polydipsia, unexplained weight loss) and has a plasma glucose concentration, measured at any time of day, regardless of time of meal, greater than 11.1 mmol\/L (\u2265200 mg\/dL).\n\n2. A person has a fasting plasma glucose concentration \u22657.0 mmol\/L (\u2265126 mg\/dL), with fasting meaning at least 8 hours after food consumption.\n\n3. A person has a plasma glucose concentration \u2265 11.1 mmol\/L (\u2265200 mg\/dL) two hours after an oral intake of 75 g anhydrous glucose dissolved in water; this procedure is called an oral glucose tolerance test (OGTT). The 11.1 mmol\/L cutoff point for the 2-hour postload glucose of the OGTT has been chosen because in large population studies, it is approximately at that point that the prevalence of complications specific to diabetes, such as retinopathy and nephropathy, increases significantly and dramatically [43]. Studies in Pima Indians, in Egyptians, and in the National Health and Nutrition Examination Survey (NHANES) III (reported in [43]) show strong associations between retinopathy and both fasting plasma glucose and 2-hour postload glucose concentrations. Furthermore, the prevalence of arterial disease strongly relates with fasting plasma glucose and 2-hour postload glucose concentrations [49], and their thresholds for increased risk of macrovascular disease correspond with the values recommended for the diagnosis of diabetes.\n\nFasting plasma glucose values have been established to define normality: fasting plasma glucose < 6.1 mmol\/L (< 110 mg\/dL) and to define an intermediate category that is too high to be considered normal but too low for a diagnosis of diabetes. That category is called impaired fasting glucose and corresponds to glucose values between 6.1 and 7.0 mmol\/L (110 and 126 mg\/dL).\n\nThe results of an OGTT are classified within one of three categories:\n\n1. One of normal glucose tolerance when the 2-hour postload glucose is <7.8 mmol\/L (<140 mg\/dL).\n\n2. One of impaired glucose tolerance when the 2-hour postload glucose is \u22657.8 mmol\/L (\u2265140 mg\/dL) but <11.1 mmol\/L (<200 mg\/dL).\n\n3. One of a provisional diagnosis of diabetes that needs to be confirmed on a subsequent day, when the 2-hour postload glucose is \u2265 11.1 mmol\/L (\u2265200 mg\/dL)\n\nCriteria have been established [43] to determine when testing for diabetes should be done in order to optimize the prevention of the disease and its complications. These criteria recommend that testing be done in all individuals over 45 and, when results are normal, be repeated every 3 years thereafter. However, testing should be done at a younger age and more frequently in individuals who are obese or have a BMI >27 kg\/m2. This indicates that obese individuals are considered more at risk of having undiagnosed diabetes and experiencing chronic hyperglycemia, a factor known to contribute to microvascular and macrovascular diseases. The early detection and treatment of this disease could decrease mortality and minimize complications, especially those related to renal disease, peripheral vascular disease, and cardiovascular disease [42]. Thus testing individuals at high risk becomes highly cost effective and has implications in the prevention of diabetes [42]. Obese individuals are such individuals at high risk [43].\n\n## III. WHY ARE THE OBESE AT RISK?\n\nA. Epidemiological Evidence\n\nObesity has been implicated as a risk factor for diabetes in cross-sectional [50\u201352] and longitudinal studies [53\u201361]. Population-based studies have shown strong associations between central adiposity, assessed by measurements of skinfolds on the trunk, and type 2 diabetes [59, 62, 63]. Ratios of waist-to-hip circumferences have been reported to be highly predictive of not only abnormal blood lipids and lipoproteins, but of glucose intolerance as well [1\u201321, 64\u20139]. Prospective studies have provided evidence that, as overall adiposity and upper-body adiposity increased, so did the risk of developing diabetes [69, 70]. Elevated glucose concentrations were likewise associated with a greater risk for diabetes [70, 71]. One prospective study of Mexican-Americans, a population at high risk for this disorder [72], reported that the subjects who developed diabetes after a follow-up of 8 years had initially higher BMIs and central adiposities and were characterized by higher fasting glucose and insulin concentrations [17]. However, although BMI and ratio of subscapular-to-triceps skinfolds related to onset of diabetes in an univariate analysis, they no longer were significant predictors of diabetes in a multivariate analysis that included glucose and insulin concentrations. Such results indicated that obesity per se was not essential for the development of diabetes, but that diabetes most likely resulted from alterations in insulin metabolism.\n\nThe hyperinsulinemia, an indirect indicator of insulin resistance, mediated the development of diabetes in subjects characterized by an unfavorable body fat distribution [72]. However, in other population studies carried out in whites [56], Nauruans [73], and Japanese [74], obesity remained an independent predictive factor after adjusting for glucose and insulin, indicating that it may act through other pathways than insulin action and secretion. Even if insulin and glucose concentrations are better predictors of diabetes and their screening identifies the subjects who could benefit from intervention, it remains true that more obese than lean persons are at risk of being resistant to insulin and are candidates who can benefit from weight loss and improved physical fitness. Oral glucose tolerance tests were used to assess type 2 diabetes mellitus and IGT in a large multiethnic population in Mauritius. In that population, age, family history, overall body mass, abdominal fat, and low physical activity scores were independent risk factors for diabetes [5]. However, there were ethnic differences in the relative importance of some factors. For example, physical inactivity had a lower impact in the Chinese of both sexes and so did the waist-to-hip ratio, than in Muslim women. In other ethnic groups, the waist-to-hip ratio was a stronger predictor for glucose intolerance than BMI in women compared to men, for whom it was the reverse.\n\nThe data analyzed from a large cohort of 51, 529 U.S. male health professionals, 40\u201375 years of age, and followed during 5 years, show that as BMI increased beyond 24 kg\/m2, the risk for diabetes increased. It was 77 times greater in men whose BMI was >35 kg\/m2 than in lean men. This study provides strong evidence that weight gain during adult life increases the risk of developing diabetes. In men who had gained more than 11 kg, their relative risk was amplified according to their BMI at age 21 years by 6.3 with a BMI <22, by 9.1 with a BMI of 22\u201323 and by 21.1 with a BMI >24 kg\/m2 [22].\n\nThe data also suggested that waist circumference was a better predictor of diabetes than waist:hip ratio [22]. The reliability of waist and hip measurements is limited by their difficulty and the fact that they are taken at different sites of the body in various studies. Nevertheless, population data report strong associations between waist-to-hip ratio and glucose intolerance [8], accounting for the associations with body mass index. A high prevalence of diabetes has been found in populations that have a tendency for central adiposity and have been exposed to the Westernization of their lifestyle and its consequence, weight gain [75].\n\n### B. Fetal Origins of Type 2 Diabetes and Obesity\n\nSome individuals may be more prone to insulin resistance and diabetes with weight gain if they have been malnourished _in utero._ Malnutrition during pregnancy forces the fetus to adapt during its development to an extent that permanent changes occur in the structure and the physiology of its body [76]. These changes seen in low-birth-weight infants have been identified as contributing factors in adult life to chronic diseases like type 2 diabetes mellitus, coronary heart disease, stroke and hypertension [76\u201378]. It was first reported in England at the beginning of the 1990s that middle-aged adults who had low weights at birth and during infancy were at greater risk of type 2 diabetes and insulin resistance [78]. This association was confirmed in later studies in Europe and the United States [79\u201381] suggesting that endocrine and metabolic adaptations induced by malnutrition could explain insulin resistance in skeletal muscle.\n\nThese associations only applied to infants born of mothers without gestational diabetes. The infants of the latter, by contrast, tended to be of high birth weight (macrosomia), but also at risk for developing type 2 diabetes as adults [81].\n\nPrenatal exposure to the Dutch famine in 1944 was associated with lower glucose tolerance and insulin resistance in the offsprings once adults, even if the famine had little effect on their birth weights [82]. These associations support a role for prenatal nutrition in type 2 diabetes.\n\nIn the Nurses' Health Study [81], the inverse association between birth weights and diabetes remained significant even after adjustment for adult adiposity. The higher relative risk was seen among lean, moderate, and obese women, indicating that _in utero_ growth had independent effects from adult body weight on the risk for developing type 2 diabetes. The greatest risk remained, however, in women of low birth weight who developed obesity as adults [81].\n\nData obtained from rat studies confirm human observations. Pregnant rats fed isoenergetic protein restricted diets gave birth to offsprings with low birth weights, reduced pancreatic \u03b2-cell mass and islet vascularization, and an impaired insulin response [83, 84], conditions that were not restored by normal nutrition after birth [84]. These offspring experienced diabetic pregnancies, exposing their fetus to hyperglycemia, and increasing their risk of becoming diabetic adults. These observations indicated that _in utero_ environment affects endocrine function and when deficient, contributes to insulin resistance and \u03b2-cell dysfunction that may lead to type 2 diabetes, especially in the presence of obesity [75].\n\nMost prospective studies of prediabetic individuals demonstrate that they are hyperinsulinemic [85], or have impaired insulin secretion, subtle abnormalities of \u03b2-cell function that may be present before overt diabetes develops. Absence of rapid oscillations of insulin has been reported in first degree relatives of patients with type 2 diabetes [86]. Other abnormalities include a greater proportion of proinsulin in plasma of patients with type 2 diabetes [87].\n\n### C. Visceral Obesity and Insulin Resistance: Major Predictors of Diabetes\n\nType 2 diabetes is a syndrome of diseases with different causes. For a minority of patients, these include mutations in some genes, such as the insulin receptor gene, that appear to cause insulin resistance, or in other genes that impair \u03b2-cell functions, compromise the glucose-sensing mechanism in the \u03b2 cell and appear to be a mild form of the autoimmune disease [88, 89]. Not all individuals with identified mutations express the diabetic phenotype [90]. It is conceivable that other gene mutations not yet identified and environmental factors, including nutrition and physical activity modulate its expression [90]. But for the majority of patients, the best predictor of the future onset of type 2 diabetes is insulin resistance. Insulin resistance is closely related to abdominal adiposity, contributing to glucose intolerance also in the aged [91]. Insulin resistance is common in obesity [91\u201394] and is characterized by hyperinsulinemia. Thus, visceral adiposity is considered a risk factor for diabetes because of its associated insulin resistance. A prospective study [95] that followed second-and third-generation Japanese-Americans for up to 10 years confirms that the amount of intraabdominal fat plays an important role in the development of diabetes. In this study, visceral adiposity, measured by computed tomography, was predictive of diabetes incidence, regardless of age, sex, family history of diabetes, fasting insulin, insulin secretion, glycemia, and total and regional adiposity. Incremental insulin response to an oral glucose challenge, an assessment of insulin secretion, was depressed in the older generation, suggesting that a failure in \u03b2-cell function preceded the onset of diabetes [96]. Impaired insulin secretion may reflect a genetic susceptibility to impaired glucose tolerance and type 2 diabetes [97, 98]. By increasing the demand for insulin, insulin resistance becomes a risk factor for diabetes, causing glucose intolerance in subjects who have impaired insulin secretory capacity and a reduction in the glucose potentiation of insulin secretion [99]. Insulin resistance was also associated with an impaired suppression of glucagon secretion by glucose in impaired glucose tolerance, suggesting that \u03b2-cell dysfunction and local insulinopenia may exaggerate glucagon secretion because the \u03b1 cell becomes less sensitive to glucose [99].\n\n### D. Parallel in the Increasing Prevalences of Obesity and Diabetes\n\nObesity is considered a risk factor for diabetes also because its reported increasing prevalence parallels that of type 2 diabetes [100]. Two massive cross-sectional surveys carried out from 1984 to 1986 and from 1995 to 1997 revealed that mean BMI increased substantially from 27.2 to 29 kg\/m2 in diabetic and from 25.1 to 26.3 kg\/m2 in nondiabetic subjects, and that the proportion with BMI >30.0 kg\/m2 went from 8 to 14% of nondiabetic men and from 13 to 18% of nondiabetic women, in all ages combined [100]. It was in the younger age group (20\u201339 years) that the increase in obesity was the greatest, paralleled by the greatest increase in known diabetes. Although mean physical activity did not change between surveys, the data indicated that the most overweight persons reported the least amount of leisure time spent on physical activity [100]. The authors concluded that this rapid increase in obesity in the younger population may worsen the predicted increase in the prevalence of diabetes as that cohort of the population ages, and reaches an age when diabetes becomes more prevalent. This distressful prediction is highly probable unless strategies are implicated to radically change lifestyles and body weight.\n\n### E. Insulin Insensitivity in Nonobese with Visceral Adiposity\n\nThe effects of nutrition and physical inactivity on body composition and metabolic fitness are becoming the burden of those who are not obese as well. Normal weight individuals also display a cluster of characteristics that predispose them to type 2 diabetes. Thirteen young women were identified in a cohort of 71 healthy nonobese women as being insulin insensitive to glucose [101]. The same women had a higher percentage of body fat and higher subcutaneous and visceral adiposity compared to the insulin-sensitive group. The energy expended in physical activity, measured by doubly labeled water methodology and indirect calorimetry, was less (2.7 \u00b1 0.9 vs. 4.4 \u00b1 1.5 MJ\/day, _p =_ 0.01) than in the insulin-sensitive group. This study indicates that body fat and inactivity override body weight in determining glucose metabolism. Furthermore, there is evidence that there is a high prevalence of these individuals in the general population [102]. Although assessed from self-reported questionnaires, physical activity has been shown to be inversely related to the incidence of type 2 diabetes [103].\n\n### F. Metabolic Alterations in Obesity That Predispose to Type 2 Diabetes Mellitus\n\nTotal fatness and a body fat distribution that favors visceral fat cell hypertrophy [65, 66] alter the metabolism of glucose (see Fig. 1 and Table 3). Both are associated with more insulin resistance than observed in individuals who are obese with lower body adiposity [104, 105], greater breakdown of the stored triacylglycerol in adipose cells [106], and elevated plasma nonesterified free fatty acid concentrations, which can lead to impaired glucose tolerance [107\u2013110].\n\nTABLE 3\n\nMetabolic Alterations in Obesity Related to Diabetes\n\n\u03b12 adrenoceptor activity in abdominal tissue of men that favors greater visceral adiposity\n\n\u03b23-adrenoceptor sensitivity in visceral fat that increases the lipolity response to catecholamine\n\nStrong relationship between insulin resistance and visceral adiposity\n\nOverproduction of tumour-necrosis factor \u03b1 by specific adipocytes, altering insulin action\n\nReduction of hepatic insulin clearance in upper-body obesity leading to hyperinsulinemia\n\nDefects in intracellular glucose transporters (GLUT 4)\n\nLess suppression of fat mobilization by insulin in visceral fat\n\nHypercortisolism in upper body obesity that favors lipolysis\n\nMetabolic inflexibility of oxidation fuel selection in skeletal muscle\n\nEndothelial dysfunction\n\nFIGURE 1 As fat mass and abdominal obesity increase, insulin sensitivity in skeletal muscle falls, leading to a decrease in glucose uptake, glucose oxidation (especially postprandially), and glycogen stores. In the liver, insulin clearance contributes to hyperinsulinemia, and gluconeogenesis increases as does glucose output. Enlarged visceral adipose tissue is associated with less response to the antilipolytic action of insulin, and more response to the lipolytic action of catecholamines is associated with increased nonesterified fatty acids (NEFA) in circulation.\n\n#### 1. VISCERAL FAT ALTERATIONS IN OBESITY\n\nThe extent of fat storage as triacylglycerol in adipocytes will depend on the balance between its formation and its mobilization. Both processes are under hormonal and nervous system regulations. Insulin plays a major role in suppressing the breakdown of triglycerides or lipolysis, by lowering cyclic AMP concentrations [111], which causes a disphosphorylation of hormone-sensitive lipase and, especially after food intake, by activating the enzyme lipoprotein lipase. Lipoprotein lipase releases fatty acids from chylomicrons and very low density lipoproteins (VLDL) in the circulation. Half of those are taken up for storage [112]. Catecholamines can suppress lipolysis [113] via their action on \u03b12 adrenoceptors, but they mostly have the opposite effect of insulin and by acting on \u03b2 adrenoreceptors, they increase cyclic AMP and the phosphorylation of hormone-sensitive lipase, stimulating lipolysis and the release of fatty acids in the circulation [113].\n\nBody fat distribution may be influenced by differences in lipoprotein lipase activity. In premenopausal women, lipoprotein lipase activity is higher in femoral and gluteal compared with abdominal regions, where fat cells are larger. These differences are not seen in men nor in postmenopausal women [114]. Furthermore it has been suggested that a greater \u03b12 activity in the abdominal tissue of men may explain greater adiposity in that location [115].\n\nBody fat distribution also affects the lipolytic response to the catecholamine norepinephrine. It has been reported that in men and women, the response is greater in abdominal compared with gluteal and femoral adipose tissues, abdominal adipocytes having greater \u03b23-adrenoceptor sensitivity. Receptor numbers are reported to be increased in obese subjects [116]. Lipolysis being relatively more elevated in visceral fat cells, subjects with upper-body obesity would be exposed to a greater release of free fatty acids into the portal system.\n\n#### 2. INSULIN SENSITIVITY IN OBESITY\n\nStudies measuring insulin sensitivity showed a positive correlation between the degree of upper body adiposity and the steady-state plasma glucose, an index of the capacity a subject has to dispose of a glucose challenge under insulin stimulus [65]. The correlation between abdominal fat and insulin resistance has been shown to be independent of total body fatness [65, 117, 118]. Overall glucose disposal by skeletal muscle in premenopausal women was lower with greater upper body fatness [119]. Insulin resistance of glucose in skeletal muscle is seen in both obesity and type 2 diabetes mellitus. The decreased efficiency in glucose disposal has been related to the reduction in insulin-stimulated activity of the glucose-6-phosphate independent form of glycogen synthase. Other factors that may explain why insulin resistance worsens in some obese individuals and not in others as their fat mass increases are the overproduction by specific adipocytes of proinflammatory cytokines such as tumor necrosis factor \u03b1(TNF-\u03b1). These cytokines block the effect of insulin on glucose transport in skeletal muscle by producing nitric oxide in excess via the induction of the expression of inducible nitric oxide synthase [120, 121]. TNF-\u03b1 mRNA expression in adipose tissue and skeletal muscle correlates with BMI and plasma insulin levels. It inhibits the tyrosine kinase activity of the insulin receptor, altering its action and possibly leading to insulin resistance [122].\n\nFractional hepatic clearance of insulin, postabsorptive and during stimulation by intravenous glucose or an oral glucose load, is reduced in upper body obesity compared with lower body obesity, despite indications that the portal plasma insulin levels do not differ [123].\n\nLess hepatic insulin extraction leads to greater peripheral insulin concentrations and hyperinsulinemia. This effect may be a consequence of elevated levels of free testosterone and decreased sex hormone-binding globulin [124] that characterize upper-body obesity [67].\n\nThe transport of glucose into the adipose cell was reduced in obesity and more so in type 2 diabetes because of a depletion of intracellular glucose transporters and of carriers for their recruitment to the plasma membrane of the cell [125]. Although the content in intracellular glucose transporters did not differ in the skeletal muscle of subjects who were obese compared with controls, the defect associated with impaired responsiveness to insulin was a loss in the functional activity of the transporters or a decrease in their translocation to the cell surface. The latter may be a consequence of long-term exposure to hyperglycemia in type 2 diabetes [126].\n\nVisceral adipose tissue shows a greater response to lipolytic stimuli but is less sensitive to suppression of fat mobilization by insulin than subcutaneous adipose tissue. The elevated concentrations in insulin in upper body obesity will have a greater inhibitory effect on lipolysis in subcutaneous adipose fat and proportionally a greater amount on nonesterified fatty acids will be mobilized from visceral fat [127]. It is conceivable that more portal nonesterified fatty acids be conducive to greater hepatic and skeletal muscle insulin resistance [128].\n\nUpper-body obesity is associated with hypercortisolism, characterized by increased degradation and clearance rates of cortisol that are compensated by increased production rates. Cortisol favors lipolysis by inhibiting the action of insulin and permitting that of catecholamines [129]. Because visceral fat shows a higher density of glucocorticoid receptors compared to subcutaneous fat, hypercortisolism in obesity may contribute to more portal nonesterified fatty acids and hepatic and skeletal muscle insulin resistance.\n\nCirculating nonesterified fatty acids concentrations increase as fat mass increases [128]. Furthermore, as insulin action diminishes with obesity, suppression of nonesterified fatty acids is decreased and their concentrations in plasma increased [130]. The larger supply of fatty acids to the liver, particularly if they are not totally suppressed after meals, is associated with accumulation of acetyl CoA and inhibition of pyruvate carboxylase altering glucose utilization and stimulating gluconeogenesis and inappropriate hepatic glucose production. In muscle, maintaining or increasing plasma nonesterified fatty acids concentrations decreased insulin-stimulated glucose uptake.\n\nUsing the euglycemic-hyperinsulinemic clamp and indirect calorimetry, lipid infusions produced a decrease in insulin-stimulated glucose oxidation and a greater decrease in glycogen synthesis [131]. These results suggested that in insulin-resistant states with increased lipid availability and oxidation, glucose oxidation was reduced. However, by contrast, in fasting conditions, hyperglycemia in type 2 diabetes was shown to override the effect of increased lipid availability and be associated with increased glucose oxidation in leg muscle. Normalization of the leg muscle hyperglycemia with a low dose of insulin increased fat oxidation [132]. There is evidence (reviewed in [133]) to suggest that, postabsorptively, skeletal muscle fat oxidation in insulin-resistant states is decreased. This decrease is explained by the presence in muscle of carbohydrate-derived malonyl CoA, which inhibits carnitine palmitoyl transferase blocking the entry of free fatty acids into the mitochondria [134]. Carnitine palmitoyl transferase activity has been shown to be reduced in the vastus lateralis muscle of insulin-resistant individuals who are obese [135]. The excess free fatty acids may increase long-chain acyl CoA concentrations and diacylglycerol, leading to the accumulation of lipids in muscle, an effect conducive to alterations of insulin signaling and insulin action.\n\nHuman studies suggest that muscle lipid content in obesity could be a determinant of insulin insensitivity [136, 137], independently of visceral fat [136]. Lipid accumulation in muscle is observed in trained athletes who are insulin sensitive but who have a great metabolic capacity for lipid utilization, which is not the case in muscles of sedentary individuals who are obese. Obese subjects appear to display metabolic inflexibility of oxidative fuel selection [133]. Compared with insulin-sensitive lean individuals, obese subjects have shown lower fat oxidation after an overnight fast, as indicated by higher leg respiratory quotient (RQ), and less capacity to switch to glucose in insulin-stimulated conditions, as indicated by an absence of increase in leg RQ. Furthermore, high levels of fatty acids may suppress insulin secretion and their effect may be toxic to the \u03b2 cells of the pancreas, becoming an important contributor to the pathogenesis of obesity-dependent type 2 diabetes [45]. Excess nonesterified fatty acids reduces hepatic insulin extraction, further increasing its serum concentration [128].\n\nThe prevalence of visceral fat measured by computer tomography correlated with endothelial dysfunction, independently of BMI in otherwise healthy women who are obese [138]. Endothelial dysfunction was also closely associated with a marker of insulin sensitivity. The authors attributed these early alterations involved in the atherosclerotic process to high nonesterified fatty acids levels.\n\nForty percent of the insulin secreted by the pancreas is removed by the liver. Defects in hepatic insulin clearance can contribute to hyperinsulinemia, a downregulation of insulin receptors and aggravation of insulin resistance [130]. As long as the pancreas maintains sufficient insulin secretion to compensate for insulin resistance, glycemia remains within the normal range. Once the \u03b2 cells fail to compensate, insulin response is decreased and glucose uptake by muscle diminished. Then, the liver oxidizes preferentially fatty acids and gluconeogenesis is stimulated. The consequences are a conversion from normal to impaired glucose tolerance and, in genetically susceptible individuals, to diabetes [139].\n\n### G. Psychological Stress, Diabetes, and Visceral Adiposity Associations\n\nBj\u00f6rntorp [140] has formulated a theory that links stress and diabetes because of the endocrine abnormalities that result from psychological stress-induced activation of the hypothalamo-pituitary-adrenal axis. Stress can be associated with high cortisol and low sex steroid concentrations. These antagonize insulin action and cause visceral adiposity, which may contribute to insulin resistance and the onset of type 2 diabetes mellitus. Psychological stress is known to aggrevate glycemia in diabetes [141]. The effect of major stressful life events over 5 years on the prevalence of type 2 diabetes was assessed in a Caucasian population aged 50\u201374 years. The results support Bj\u00f6rntorp's theory by showing a greater prevalence of previously undetected diabetes with more stressful events reported [142]; the age-and sex-adjusted association between major life changes and diabetes was independent of family history of diabetes, physical activity, heavy alcohol use, or low level of education. There was no association between work-related stressful events and type 2 diabetes in this cross-sectional study. A weak positive association was found with waist-to-hip ratio, but visceral fat was not the main mediating factor between stress and diabetes. It has been suggested that the link between stress and diabetes may be through other factors such as the chronic stimulation of the autonomic nervous system and its resulting hyperglycemia [142, 143]. Whether central obesity is a response to psychological stress, and both in turn increased the risk of glucose intolerance over time, remains to be evaluated in longitudinal studies [142].\n\nAlthough it is not an obligatory factor, obesity may increase the risk of developing diabetes in susceptible individuals. The majority of persons with type 2 diabetes are obese [42]. Data from a prospective study in men aged 40\u201359 years at screening and followed for a period of 12 years showed that the risk increased progressively with increasing BMI. Weight gain of >10% over time increased the risk even in overweight individuals with initial BMI of 25.0\u201327.9 kg\/m2. Both the magnitude of BMI and the duration of obesity increased the risk of diabetes. The risk in men who had a BMI > 30 kg\/m2 for more than 5 years was eight times that of men who were not overweight. Weight fluctuation did not affect the risk of diabetes but there was a trend for weight loss to be associated with a decrease in risk [144].\n\n### H. Improvement in Insulin Sensitivity with Weight Loss and Energy Intake Restriction\n\nIn obesity characterized by diminished insulin action, any treatment associated with weight loss improves insulin sensitivity and fasting plasma glucose; these improvements are related to losses of abdominal fat [145, 146]. Fujioka _et al._ [145] measured the changes in body fat distribution and those in metabolic disorders after an 8-week energy intake restricted diet (800 kcal\/day) in 40 women aged 38 \u00b1 9 (mean % SD) years with uncomplicated obesity. Body fat was determined by the CT scan method and 14 women were characterized as having visceral fat obesity rather than subcutaneous fat obesity with a visceral\/subcutaneous measurement of >0.4. They found that at week 8 of diet, the decrease in visceral fat volume was more sharply correlated with the changes in plasma glucose and in lipid metabolism than the decrease in body weight, total fat or subcutaneous fat volume, and that these correlations were independent of total fat loss. The reduction in visceral fat was more pronounced compared with subcutaneous fat.\n\nAnother study [146] reported that the improvements in fasting plasma glucose and insulin sensitivity with weight loss were related to losses of abdominal fat. That study was designed to distinguish the effects of energy intake restriction before substantial weight loss from those of a weight loss of 6.3 \u00b1 0.4 kg on glycemia, hepatic glucose production, and insulin action and secretion in 20 overweight subjects with or without type 2 diabetes. At day 4 of reducing energy intake by 50%, a significant decrease was observed in basal hepatic glucose output with a greater insulin suppression of endogenous glucose production in all subjects, measured during a hyperinsulinemic euglycemic clamp. Although the decrease in glucose output was counteracted by a decrease in metabolic clearance rate of glucose, it resulted in a fall in fasting plasma glucose. This fall related to the decrease in carbohydrate intake and possibly an associated reduction in hepatic glycogen content. At day 4 of energy intake restriction, fat oxidation was increased as were the nonesterified fatty acids levels. With substantial weight loss (at day 28 of the energy intake restricted diet), plasma glucose was further decreased in the diabetic subjects only, a decrease associated with increased metabolic clearance rates. Fasting insulin concentrations were reduced compared with those preceding weight loss and insulin sensitivity was improved in both groups. These improvements were associated with a reduction in the abdominal fat depot. These data support a role for central adiposity in the alterations of glucose metabolism observed in obesity. Pascale _et al._ [147] also reported a significant decrease in the waist-to-hip ratio of 69 overweight type 2 diabetic subjects after a loss of more than 13 kg at 6 months of a behavioral weight-loss program. Waist-to-hip ratio had decreased by 2.6 and 2.0% in men and women, respectively. These subjects were characterized by upper-body obesity. Waist circumferences also decreased from 121 \u00b1 17 to 106\u00b1 11 cm in women and from 120\u00b1 10 to 108\u00b1 16 cm in men. Changes in waist circumferences related significantly with weight loss ( _p_ \u2265 0.01) Baseline waist-to-hip ratio correlated significantly with change in waist-to-hip ratio at 6 months. Although the improvements in glycemic control indicated by decreases in hemoglobin A1c, plasma glucose, and insulin concentrations correlated with weight loss, they did not with changes in waist-to-hip ratio.\n\nThe improvement in glycemia observed with weight loss underlines the impact of obesity in the development of type 2 diabetes. Pories _et al._ [148] reported that 90% of the glucose intolerant or diabetic obese subjects who had lost 50 kg, 12 months after gastric bypass surgery, achieved normo-glycemia and maintained it for up to 14 years.\n\n### I. Obesity and Treatment of Diabetes\n\nThe conclusions of the UK Prospective Diabetes Study [149] are that optimal diabetic control aiming at a hemoglobin A1c level of <7. 0% must be achieved if morbidity is to be prevented significantly. To do so, intensive therapy using insulin alone or combined with oral hypoglycemic agents may be required. Results from the Finnish Multicenter Insulin Therapy Study, in which 100 insulin-treated type 2 diabetic patients were followed for 12 months, showed that good glycemic control started to deteriorate after 3 months, and more so in the obese subjects, the latter being attributed to the greater insulin resistance in these subjects. Control was best achieved in the nonobese subjects whether with insulin alone or in combination with other therapeutic agents. However, the combination therapy was associated with less weight gain [150].\n\nObesity in persons with type 1 diabetes can affect their insulin requirements by aggravating their insulin resistance. The consequences of increasing the insulin dosages can be further weight gain associated with the lipogenic effects of insulin and the additional eating in reaction to the hypoglycemic events often seen with intensive therapy [151]. Weight gain with insulin therapy is often associated with a higher waist-to-hip ratio [149]. Given the associations between obesity and dyslipidemia, atherosclerosis, and hypertension, intensive therapy of diabetes should aim at not producing weight gain [149].\n\n## IV. CONCLUSION\n\nFamily history of diabetes and age are recognized risk factors for diabetes [18], but they cannot be controlled. Recommendations for prevention of diabetes address factors that can be controlled. Because the hallmark of diabetes control is glycemia, the goals of the treatment of type 2 diabetes have been primarily to normalize blood glucose. Tight control of glycemia is necessary to prevent microvascular complications [149]. However, the increased cardiovascular risk associated with the disease commands that therapeutic strategies be devised to correct the factors related to that risk. These include elevated blood pressure, smoking, abnormal lipid profile, low physical activity and obesity. 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Res_. 1994;2:587\u2013599.\nCHAPTER 28\n\nNutrition Management for Type 1 Diabetes\n\nANN ALBRIGHT, Sacramento, California\n\n## I. INTRODUCTION\n\nDiabetes is one of the most common diseases that nutrition and other health professionals encounter. Both health professionals and people with diabetes have identified medical nutrition therapy as an extremely challenging aspect of diabetes care [1]. While nutrition has long been viewed as a cornerstone in the treatment of diabetes, the information on which many of the nutrition recommendations are based is evolving. During the last decade, landmark research has been conducted that has significantly changed how diabetes is treated. The patient is the key decision maker in diabetes care. The meal plan must be individualized and based on the patient's usual eating habits and preferences. The insulin regimen is then integrated into the patient's lifestyle. This is a significant shift from previous diet plans that were based primarily on energy intake and structured to meet the insulin regimen.\n\nThe role of the nutrition professional is to work with the patient and other members of the health care team to set and help patients achieve their nutrition goals. Once the goals have been established, the nutrition professional works with patients to select tools for making nutrition decisions based on the most current information and guides them in their decision making. To provide the most accurate information to patients, it is imperative that health professionals have the most current information available on medical nutrition therapy for diabetes care. In addition, nutrition professionals must also understand and use self-monitoring of blood glucose results and have a thorough understanding of medications and of physical activity. Optimal management of type 1 diabetes requires the health care provider to integrate all of this information into patient care.\n\n## II. DEFINITION AND BURDEN OF TYPE 1 DIABETES\n\nType 1 diabetes accounts for approximately 10% of the diabetic cases and is characterized by an inability to produce insulin, resulting in hyperglycemia [2]. Insulin, a hormone produced by the beta cells of the pancreas, is needed by muscle, adipose tissue, and the liver to utilize glucose. The hyperglycemia of diabetes places individuals with this disease at risk for developing microvascular complications, including retinopathy (eye disease) and nephropathy (kidney disease), macrovascular disease (including heart disease and stroke), and various neuropathies (both autonomic and peripheral).\n\nThe two subgroups within type 1 diabetes are immune-mediated and idiopathic diabetes. Type 1 immune-mediated diabetes was formerly known as juvenile-onset or insulin-dependent diabetes. This form of the disease usually occurs before the age of 30 with most cases occurring in childhood or adolescence. However, it is now estimated that 10\u201320% of Caucasians developing diabetes in adulthood may have immune-mediated beta cell destruction [3]. The immune destruction of the beta cells may occur over several months to years. In young people, the onset of symptoms is usually abrupt and rapid treatment is necessary to correct ketoacidosis and prevent death. The beta cell destruction in adults that develop type 1 diabetes may be much more gradual and an absolute requirement for insulin may not be necessary for many years [2].\n\nType 1 immune-mediated diabetes is considered an autoimmune disease in which the immune system attacks the body's own tissues. It is currently unclear what triggers the immune process leading to type 1 diabetes, but the process is identified by the presence of islet-cell antibodies, insulin autoantibodies, and autoantibodies to glutamic acid decarboxylase. Type 1 diabetes does not develop in all patients with these antibodies, but at least one of these antibodies is present in 85\u201390% of patients at the time of diagnosis [4]. The predisposition to type 1 immune-mediated diabetes is inherited as a multigenic trait with low penetrance. Genetic markers have been identified which indicate the potential vulnerability to this subgroup of type 1 diabetes [5]. At least one human leukocyte antigen (HLA) class II DR3 or DR4 antigen is found in 95% of type 1 patients. Also linked to type 1 diabetes are DR1, DR16, and DR8, while DR15 and DR11 are considered to confer protection. Class II DQ genes are also linked to type 1 diabetes and, although these HLA types are necessary, they are not sufficient for developing type 1 diabetes.\n\nType 1 idiopathic diabetes is a new subgroup and represents only a small number of people with beta-cell destruction. These patients have variable insulin deficiency and only intermittently require insulin treatment. This form of diabetes is strongly inherited, but does not have HLA association or evidence of autoimmunity. When seen, it is more often found in those of African and Asian background [4].\n\n## III. DIABETES MEDICAL NUTRITION THERAPY\n\nFour steps have been identified that are necessary for accomplishing diabetes medical nutrition therapy: (1) a comprehensive nutrition assessment that addresses metabolic, nutrition, and lifestyle factors; (2) setting practical, achievable goals with the patient that have been mutually agreed on by the patient and the dietitian; (3) identifying nutrition interventions that the patient can understand and is most likely to use; and (4) evaluating progress toward meeting the goals and identifying areas in need of future attention [6]. These steps have been identified in and are supported for diabetes by the Diabetes Control and Complications Trial (DCCT) [7], the American Diabetes Association nutrition recommendations [1], and the nutrition practice guidelines for type 1 diabetes developed and tested by the Diabetes Care and Education Practice Group of the American Dietetic Association [8].\n\nThe DCCT was designed to compare intensive with conventional diabetes treatment on the development and progression of early vascular and neurologic complications of type 1 diabetes [7]. This multicenter clinical trial randomized 1441 patients between the ages of 13 and 39 with type 1 diabetes into two treatment groups, conventional or intensive. Within each of the treatment groups, there were patients with no sign of diabetic retinopathy at baseline (primary prevention) and patients with mild diabetic retinopathy at baseline (secondary prevention). Intensive therapy consisted of three or more insulin injections per day or insulin pump therapy and self-monitoring of blood glucose at least four times per day. Insulin dose adjustments were made according the results of self-monitoring of blood glucose, food intake, and anticipated exercise. These patients also had frequent contact with members of the health care team. Conventional therapy consisted of one or two daily insulin injections, daily self-monitoring of blood glucose or urine testing for glucose, and education about diet and exercise. Daily adjustments in insulin were not usually made and the goal of therapy was the absence of symptoms of hyperglycemia, normal growth and development, and no severe or frequent hypoglycemia. These patients had standard follow-up with health care professionals. The DCCT conclusively demonstrated that intensive blood glucose management can significantly reduce the risk for microvascular complications of type 1 diabetes (Table 1).\n\nTABLE 1\n\nDiabetes Control and Complications Trial Results\n\nIntensive management\/blood glucose control made a difference\n\n76% reduction in retinopathy\n\n60% reduction in neuropathy\n\n54% reduction in albuminuria\n\n39% reduction in microalbuminuria\n\nImplication: Improved blood glucose control also applies to persons with type 2 diabetes.\n\n_Source:_ Reprinted with permission from Diabetes Control and Complications Trial Research Group (1993). The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. _N. Engl. J. Med._ **329,** 977\u2013986.\n\nThe DCCT showed that medical nutrition therapy is essential in achieving optimal glucose control [9, 10]. The nutrition interventions used in the DCCT were tailored to fit the patient's lifestyle, motivation, and learning level. Insulin adjustments were made to fit the selected nutrition plan. The dietitians implemented a variety of nutrition strategies to assist patients in the intensive therapy groups attain near euglycemia. The nutrition approaches ranged from the general recommendations of the Healthy Food Choices to the more detailed approach of total available glucose (discussed below). Recognizing the need for flexibility in the nutrition plan for patients with type 1 diabetes, and working with the patient to select the approach used, contributed to the reduction in hemoglobin A1c in the DCCT.\n\nHistorically, nutrition recommendations for those with diabetes have spanned everything from starvation diets to high-fat diets. In 1994 the American Diabetes Association developed nutrition guidelines that presented a major change in the philosophy of nutrition care for those with diabetes [1]. An individually developed dietary plan based on metabolic, nutrition, and lifestyle requirements replaced the defined caloric prescription. These nutrition recommendations acknowledged that a single diet does not appropriately treat all types of diabetes. The goals of medical nutrition therapy in diabetes care are (1) maintenance of near-normal blood glucose levels by balancing food intake with insulin (exogenous or endogenous) and physical activity, (2) achievement of optimal lipid levels, (3) provision of adequate energy for maintaining or attaining reasonable weight and normal growth, (4) prevention and treatment of acute and chronic complications of diabetes, and (5) improvement of overall health through optimal nutrition.\n\nThe recommendations for protein, fat, and carbohydrate are designed to achieve the goals of medical nutrition therapy. They emphasize the need to individually assess the patient and make macronutrient selections based on patient outcome goals and not just generic percentages.\n\n### A. Protein\n\nVariations in dietary protein intake can influence blood glucose levels in diabetes by modifying the availability of gluco-neogenic substrates, as well as insulin and counterregulatroy hormone secretion. There are limited scientific data on which to establish firm recommendations for protein intake in diabetes. Because evidence does not support that protein requirements for type 1 patients are higher or lower than the general population, the recommended protein intake for those with diabetes is 10\u201320% of total energy. In a study of 12 patients with type 1 diabetes, glucose response and insulin requirements were measured by a glucose controlled insulin infusion system for 5 hours after ingestion of three different meals: 450 kilocalories standard meal, the standard meal with 200 kilocalories (equivalent to 7 ounces of lean meat) of added protein, and the standard meal with 200 kilocalories of added fat [11]. The glucose response to the protein meal was greater ( _p =_ 0.005) than the response to either the standard or fat-added meals due to an increase in the late (last 150 minutes) glucose response. The late insulin response was also greater ( _p_ < 0.005) for the protein-added meal. This effect was seen with 7 ounces of protein. Whether people with diabetes who eat 1\u20132 ounces of protein with a snack to prevent subsequent hypoglycemia (especially hypoglycemia during the night) will have the same results remains unknown. If protein does affect blood glucose levels, it is likely due to stimulation of glucagon by protein [12]. The increase in glucagon exerts a transient increase in hepatic glucose release so it is not likely to provide protection against subsequent hypoglycemia.\n\nExcessive protein intake has been implicated in the pathogenesis of diabetic nephropathy since high protein intake increases glomerular filtration rate (GFR) [13]. Some studies conducted in people with diabetes suggest that the use of low-protein diets may modify the underlying glomerular injury of diabetes and delay the progression of nephropathy [14, 15]. There is evidence that the protein source may influence the progression of renal disease. Amino acids from meat proteins may adversely affect GFR and the progression of renal disease compared to vegetable, egg white, cheese, or cooked soybean proteins [16\u201318]. Most data support some restriction of protein intake to prevent or delay diabetic nephropathy, but more data are necessary to determine the optimal protein content and the stage of renal disease that responds to protein restriction. Currently, a protein intake similar to the adult Recommended Dietary Allowances (0.8 g\/kg), approximately 10% of the daily energy, is considered sufficiently restrictive for individuals with evidence of diabetic nephropathy [1].\n\n### B. Fat\/Carbohydrate\n\nThe remaining 80\u201390% of energy must be distributed between fat and carbohydrate [1]. The decisions about amount of fat and carbohydrate are guided by the patient's metabolic goals, eating habits, weight issues (if any), and the elevated risk for cardiovascular disease (CVD) in those with diabetes. Modification of CVD risk factors is especially important because diabetes is a strong independent risk factor for CVD [19]. It is recommended that saturated fat intake be less than 10% of energy, polyunsaturated fat intake be up to 10% of energy, and cholesterol be limited to 300 mg or less daily. The remaining 60\u201370% of energy comes from monounsaturated fats and carbohydrate.\n\nPeople with type 1 diabetes who are taking adequate amounts of insulin usually have plasma cholesterol, very low density lipoprotein (VLDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride concentrations similar to those without diabetes of the same age and gender, but have higher than average high-density lipoprotein (HDL) cholesterol levels [20, 21]. Uncontrolled type 1 diabetes is associated with elevated lipid levels, but adequate insulin usually normalizes them. Many studies, but not all, suggest that blood glucose control directly influences the levels of several plasma lipid concentrations [22]. Abnormalities in size and density of lipoprotein composition can exist in these patients even when the plasma lipid measurements are normal.\n\nIf elevated LDL cholesterol is the primary problem, the National Cholesterol Education Program (NCEP) Step 2 guidelines (7% of total energy from saturated fat and 200 mg dietary cholesterol) should be implemented. If elevated triglycerides and VLDL cholesterol are primary concerns, a moderate increase in monounsaturated fat and a moderate intake of carbohydrate may be implemented [6]. Some studies have shown that an increase in monounsaturated fat lowered triglycerides and blood glucose more than a high-carbohydrate intake in some individuals [23, 24].\n\nThe percentage of carbohydrates is individualized based on eating habits, glucose and lipid goals, and the presence of other medical conditions. Relative differences in plasma glucose responses to various carbohydrates have been reported and published in a tool called the glycemic index. The clinical utility of this information has raised many questions, however. If a patient is encouraged to only eat low glycemic index foods, food choices will be severely limited. Priority should first be given to the total amount of carbohydrate consumed rather than the source of the carbohydrate. The form of carbohydrate (liquid or solid) is also an important consideration since liquids are absorbed more quickly.\n\nThe most widely held belief about the dietary treatment of diabetes is that simple sugars must be avoided and replaced with complex carbohydrates. This belief is based on the assumption that sugars are more rapidly digested and absorbed than starch and, hence, aggravate hyperglycemia. There is no scientific evidence to support this assumption. When examined as a single nutrient, sucrose produces a glycemic response lower than that of bread, rice, and potatoes [25]. Fruits and milk are reported to have lower glycemic responses than many starches [1]. At least 10 studies have been conducted that show isocaloric amounts of sucrose and starch produce similar blood glucose responses (Table 2). In particular, a crossover design study in children with type 1 diabetes compared the glycemic effect of isocaloric diets containing 2% total calories as sucrose and 10% total calories from sucrose [26]. Both diets provided 50% energy from carbohydrate, 30% from fat, and 20% from protein in three meals and three snacks. Glucose, fructose, and dietary fiber content of the diets were identical. Sucrose isocalorically replaced part of the complex carbohydrate at each meal and for the afternoon snack. Insulin doses remained constant. The blood glucose responses are depicted in Fig 1.\n\nTABLE 2\n\nStudies Comparing Glycemic Effects of Isocaloric Amounts of Sucrose and Starch in Diabetic Subjects _a_\n\naMeals were provided to subjects by the investigator\n\n_Source:_ Reprinted with permission from Franz, M. J., Horton, E. S., Bantle, J. P., Beebe, C. A., Brunzell, J. D., Coulston, A. M., Henry, R. R., Hoogwerf, B. J., and Stacpoole, P. W. (1994). Nutrition principles for the management of diabetes and related complications. _Diabetes Care_ **17,** 490\u2013518.\n\nFIGURE 1 Blood glucose concentrations (mean \u00b1 SEM) from 8 A.M. to 9:30 P.M. for sucrose-free and sucrose-containing diet periods (2 days per diet period; 10 children). [From Loghmani, E., Richard, K., Wash-burne, L., Vandagriff, H., Fineberg, N., and Golden, M. (1991). Glycemic response to sucrose-containing mixed meals in diets of children with insulin-dependent diabetes mellitus. _J. Pediatr._ **119,** 531\u2013537. Reprinted with permission from Mosby, Inc.]\n\nWhen making decisions about sucrose-containing foods in the meal plan, it is important to remember that one of the goals of the nutrition recommendations is improvement of health through optimal nutrition. Sucrose can be substituted for other carbohydrates, gram for gram in the context of a healthy diet. Foods containing sucrose are usually high in fat and contain minor amounts of vitamins and minerals. It is also important to clearly articulate to the patient that sucrose and sucrose-containing foods must be substituted for other carbohydrates, not just added to the meal plan, and these choices should be made with attention to a healthy diet.\n\n### C. Nutrition Practice Guidelines\n\nThe Nutrition Practice Guidelines for Type 1 Diabetes (practice guidelines) were developed by the Diabetes Care and Education Practice Group of the American Dietetic Association [8]. The practice guidelines are intended to serve as a guide for provision of consistent, quality care to those with type 1 diabetes. The objectives of establishing the practice guidelines were to define responsibilities of dietetics professionals who work with patients who have type 1 diabetes, guide practice decisions, promote self-management training, and define state-of-the-art medical nutrition therapy. Four principles were used to guide the development and testing of the practice guidelines and are summarized as follows: The practice guidelines (1) guide practitioners about appropriate health care decisions for specific circumstances; (2) are based on the best available research and professional judgment; (3) are comprehensive, specific, and manageable; and (4) are thoroughly researched and validated through field testing by a reasonable pool of practitioners.\n\nThe practice guidelines are summarized in Fig. 2. A referral by the patient's primary care provider initiates the process. At this point, the dietitian must make a decision about the appropriateness of the referral. For example, the patient may require an intervention such as psychological counseling before referral to the dietitian can be productive. At a minimum, data that include the reason for referral, current medical condition, pertinent laboratory data, and diabetes management goals should accompany the referral. Follow-up with the referring provider should summarize the assessment findings, recommendations, intervention provided, and treatment plan.\n\nFIGURE 2 Flowchart for Nutrition Practice Guidelines for Type 1 Diabetes Mellitus. [From Kulkarni, K., Castle, G., Gregory, R., Holmes, A., Leontos, C., Powers, M., Snetselaar, L., Splett, P., and Wylie-Rosett, J. (1997). Nutrition practice guidelines for Type 1 diabetes: An overview of the content and application. _Diabetes Spectrum_ **10** (4), 248\u2013256. Reprinted with permission from the American Diabetes Association, Inc.]\n\nThe second area of the practice guidelines is a thorough assessment of the patient. Table 3 provides sample questions that guide the dietitian through this assessment. In addition to the topics identified in Table 3, it is important to assess the patient's beliefs and attitudes about chronic disease and his or her readiness to change. Once an assessment is completed, it is followed by setting realistic goals. Short- and long-term, clinical, and behavioral goals can be set. The goals must be set mutually by the clinician and the patient. The patient should not feel overwhelmed by the goals and, if necessary, goals should be adjusted to help the patient experience success. Once realistic goals have been established, the level of nutrition care needed is identified, a nutrition prescription developed, and a meal plan approach (discussed below) selected.\n\nTABLE 3\n\nMinimum Assessment Data for Type 1 Diabetes\n\n_Source:_ Kulkarni, K., Castle, G., Gregory, R., Holmes, A., Leontos, C., Powers, M., Snetselaar, L., Splett, P., Wylie-Rosett, J. (1997). Nutrition practice guidelines for Type I diabetes: An overview of the content and application. _Diabetes Spectrum_ **10** (4), 248\u2013256.\n\nThe dietitian must also determine which educational strategies (e.g., visual learner) are most effective for the patient (see discussion in Chapter 8). These educational strategies should be used to help the patient most effectively apply the interventions. Finally, evaluation of the patient's progress must occur regularly. This will allow adjustments to be made as necessary and assist the patient in achieving goals. A schedule for follow-up with the patient must also be developed. The frequency for follow-up can range from daily when initiating or intensifying therapy to quarterly for patients who are in the maintenance phase. Throughout the entire implementation of medical nutrition therapy it is important to document and communicate information about the assessment, the goals, the selected interventions, the nutrition prescription, and the follow-up plan to all members of health care team.\n\nThe practice guidelines were field tested in a variety of practice settings to determine the impact on patients' glycemic levels and perceptions about quality of care [27]. The results showed that dietitians in the practice guidelines group spent 63% more time with patients and were more likely to do an assessment and discuss the results with patients than dietitians in the usual care group. The patients who were receiving care according to the practice guidelines achieved greater reductions in hemoglobin A1C that were statistically and clinically significant.\n\n## IV. INSULIN REGIMENS\n\nInsulin is required for the treatment of type 1 diabetes. Several researchers contributed to the discovery of insulin, but credit is given to Banting and Best for first isolating insulin in 1921. At the time, insulin was considered a cure for diabetes because, without it, certain death was in store for those with type 1 diabetes. Soon after its discovery, processes were developed for isolation and commercial production of insulin from beef and pork pancrease. Over time, a number of modifications in the production of insulin have occurred, resulting in improved purity, varying action profiles (rapid-acting, short-acting, intermediate-acting, and long-acting), and structure identical to native human insulin [28]. In the intervening years since its discovery, we have clearly learned that insulin is a lifesaving medication, but it is not a cure for diabetes.\n\nIn an effort to duplicate the normal physiologic functions of insulin, it is important to understand the onset, peak, and duration of the various insulins. Table 4 summarizes this information for the insulins currently available.\n\nTABLE 4\n\nTime Course of Action of Insulin Preparations _a_\n\naBased on doses of 0.1\u20130.2 U\/kg, in the abdomen, for human insulin.\n\n_Source:_ Reprinted with permission from Skyler, J. S. (1998). _In_ \"Insulin Therapy in Type I Diabetes Mellitus,\" Vol. I, pp. 36\u201349. Mosby-Year Book, St. Louis, MO.\n\nOther factors that must be considered in the use of insulin include pharmacokinetics of insulin absorption, injection site, and timing of premeal insulin injections. An important determinant of insulin availability is its absorption from subcutaneous tissue. There is intra-individual variation in insulin absorption of approximately 25% from day to day and interindividual variation of up to 50% [28]. Although this percentage variation is the same for all insulins, in absolute terms (minutes or hours) there will be much less variation in the absorption of rapid-acting insulins and greater variation in absorption of longer-acting preparations.\n\nThe injection site also affects absorption. Absorption is usually fastest from the abdomen, followed by the arms, buttocks, and thighs. The difference in absorption rates among these sites is likely due to blood flow [28]. The variability is great enough that random rotation of injection sites should be avoided. It is better to rotate inject sites within a region rather than between regions. For example, any particular injection (e.g., prebreakfast) should be rotated within a site such as the abdomen rather than rotating it among the abdomen, arm, and thigh.\n\nBlood flow, skin thickness, ambient temperature, smoking, and massage of injection site also influence insulin absorption rate [28]. Because physical activity increases blood flow to an exercising part of the body, it can accelerate the absorption of insulin and contribute to hypoglycemia. The greater the time interval between the injection and the exercise (>60\u201390 minutes), the less effect injection site has on absorption rate [29]. The timing of an insulin injection relative to exercise must be evaluated to decrease the likelihood of hypoglycemia.\n\nAs seen from the information in Table 4, there is variability in the onset and peak of insulin action. In an effort to match blood glucose and insulin levels following a meal, it is important to consider the timing of premeal insulin injections. For example, regular insulin should optimally be administered 30\u201360 minutes before eating, allowing the insulin level in the blood to better match the glucose level. The purpose of insulin is to keep the blood glucose from getting too high postprandially. This means that insulin should be appropriately used to prevent an abnormal rise in blood glucose and not be used to chase high blood glucose levels. It is important to remember that the current insulin preparations and regimens do not guarantee perfect blood glucose control. Support and instruction need to be provided to the patient to help them most effectively use insulin and not feel defeated when the blood glucose results are not always consistently normal.\n\nPatients with type 1 diabetes may be using conventional or intensive insulin therapy. In conventional therapy, insulin administration usually consists of twice daily injections of short-acting and intermediate-acting insulin before breakfast and dinner. This method of therapy provides less flexibility for the patient and it requires that the patient be more consistent with timing of insulin administration and food intake. In intensive therapy, insulin administration is by multiple daily injections or by continuous subcutaneous insulin infusion, also called insulin pump therapy. Use of intensive insulin therapy provides more flexibility for the patient, but does require more injections and blood glucose monitoring. Intensive therapy increases the likelihood of hypoglycemia and can result in weight gain. It is essential that before patients are started on intensive therapy they have detailed information about food, physical activity, and insulins. They must also be willing to test blood glucose levels four to six times a day to make treatment plan adjustments and be able to gauge the success of these adjustments [8].\n\nThe DCCT has shown that better blood glucose control is achieved with intensive therapy, but there may some patients for whom intensive therapy is too risky and they will be better managed by conventional therapy. Patients for whom intensive therapy may be inappropriate include those with hypoglycemia unawareness and\/or history of severe hypoglycemia, advanced complications, or psychological limitations [30]. Other nutrition-related risks include eating disorders or fear of weight gain [8].\n\nInsulin can be administered by syringe or insulin pen, jet injector, or continuous subcutaneous insulin infusion. Appropriate instruction is required to teach patients how to fill the syringe and inject properly. An insulin pen makes dosage accuracy much easier since the dose is made by turning a knob. The jet injector forces insulin into the skin through pressure and not a needle. The continuous subcutaneous insulin infusion or insulin pump is a device about the size of a pager that is connected to a cannula inserted under the skin; it delivers rapid-acting insulin (Regular or Lispro) continually. The amount of insulin needed by the patient in the fasted state, known as the basal rate, must be determined first. This information is programmed into the insulin pump and the patient will receive these microliter amounts continuously. The pump can be reprogrammed to change the basal rate(s) if necessary. Prior to eating, the patient must determine how much rapid-acting or short-acting insulin will be required to cover the meal. This insulin dose (called a bolus) is delivered by pushing a button on the pump. The pump does not make insulin dosage decisions for the patient, but it does allow flexibility in eating and exercise.\n\n### A. Self-Monitoring of Blood Glucose\n\nSelf-monitoring of blood glucose is also an important part of managing diabetes. A variety of blood glucose monitoring devices is now available that allows those with diabetes to test their own blood glucose. These devices became widely available in the 1980s and have improved in accuracy and sophistication in the intervening years. All people with diabetes should test their blood glucose regardless of whether or not they use insulin, although those who use insulin are likely to have to test more frequently. There is no standard frequency for self-monitoring, but it should be done frequently enough to help the patient meet treatment goals. Increased frequency of testing is often required when initiating an exercise program to assess blood glucose response to exercise and allow safe exercise participation. Patients must be given instruction on how to operate, clean, and calibrate the device, and\u2014most importantly\u2014guidance on how to use the information to make food, exercise, and medication adjustments.\n\nHealth professionals must use patient blood glucose monitoring results to help the patient assess and make adjustments in their meal plan. It is not possible to appropriately assist patients with diabetes in developing and adjusting their meal plan without using blood glucose records. All nutrition and other health professionals who work with diabetic patients must develop the necessary skills to interpret and use blood glucose monitoring information. Reviewing these records with the patient reinforces the need to keep records. The reecord allows the patient and the health care professional to identify blood glucose patterns to make adjustments in therapy.\n\n### B. Meal Planning Approaches\n\nSeveral meal planning approaches are used in diabetes care and they vary in complexity and the ability to make insulin adjustments. When practicing intensive therapy, the meal planning approach used must provide enough information to allow for accurate adjustments in insulin. None of the meal planning approaches has been scientifically validated. The plan selected should be one that the individual with diabetes can understand and which helps them select appropriate foods and achieve their glycemic goals.\n\nCarbohydrate counting is not a new meal planning approach, but has recently gained renewed interest. This technique is based on the premise that the carbohydrate found in foods is the major factor influencing postprandial blood glucose excursions [31]. The focus is on total carbohydrate eaten, not the source of carbohydrate. Many consider carbohydrate counting to be a simpler method because it focuses on one macronutrient. When using this approach, patients must still learn about protein and fat intake, even though they have minimal direct effect on blood glucose when consumed in usual amounts. They do contribute to energy intake and weight gain may become an issue when dietary protein and fat are ignored. In addition, large amounts of fat can delay the postprandial peak in blood glucose and this can have an impact on glucose control [11].\n\nThree levels of carbohydrate counting have been developed that progress from basic (level 1) to intermediate (level 2) to advanced (level 3) [32\u201334]. Level 1 introduces the concept of carbohydrate counting, identifies foods that contain carbohydrate, sources of carbohydrate information (e.g., label reading), how to count grams of carbohydrate in food, and consistent carbohydrate intake. When introducing this phase, the patient records their usual carbohydrate intake and, with the dietitian, target carbohydrate goals are determined.\n\nLevel 2 focuses on pattern management, which is used to identify blood glucose patterns that are affected by food, physical activity, and insulin. These patterns are important to identify since they allow more logical adjustments to be made in insulin, carbohydrate, or physical activity. In the intermediate level of carbohydrate counting, the emphasis is on identifying blood glucose patterns, interpreting the data, and determining the possible actions necessary to alter blood glucose levels. In addition, information is given about subtracting the carbohydrate contribution from fiber if the food contains more than 5 g per serving amount and helping the patient avoid hypoglycemia and weight gain.\n\nLevel 3 is for those patients on intensive therapy who use carbohydrate-to-insulin ratios to make insulin dosage decisions and choose to use more detailed information. In the advanced level of carbohydrate counting, the concept of carbohydrate-to-insulin ratio, the amount of carbohydrate that is covered by 1 unit of fast-acting or rapid-acting insulin, is introduced. For example, a patient who eats 75 g of carbohydrate and requires 5 units of insulin has a ratio of 15 g of carbohydrate to 1 unit of insulin. The dietitian assists the patient in calculating the ratio for each meal and snack using food, insulin, and blood glucose records.\n\nAll levels of carbohydrate counting require that the person with diabetes practice estimating portion sizes and develop a reasonable skill level. This technique helps those with diabetes understand the relationships among food, insulin, physical activity, and blood glucose level. Armed with this information, patients are often better able to make appropriate adjustments in their management plan [35].\n\nTotal available glucose is another meal planning approach that some have found useful [9]. This system is based on the premise that 100% of carbohydrate content, 58% of the protein content, and 10% of the fat content of foods become glucose. In this system a total available glucose-to-insulin ratio is established that requires counting the glucose contribution of all macronutrients. More recent data have shown, however, that although 50\u201360% of protein has the potential to be used for gluconeogenesis, it has little effect on post-meal blood glucose [11, 36]. It is speculated that this system probably works because both protein and fat require insulin for metabolism although they have minimal effect on post-meal glucose level [11].\n\nFor many years, the most widely used method in diabetic meal planning was the exchange system [37]. This system was developed in 1950 and represented the first organized meal approach that was agreed to by the organizations involved in diabetes and nutrition care. Because of the historical contribution of this technique, it has been taught as the standard meal planning tool for people with diabetes. It is a useful technique, but it is not necessarily the best tool for all patients.\n\nFoods are organized into three main groups: carbohydrate, meat and meat substitutes, and fat. Foods are further divided into specific exchange lists that contain a listing of weighed or measured foods of approximately the same nutritional value. Because of this arrangement, foods on each list can be substituted or \"exchanged\" with other foods on the same list. The meal plan for a patient is devised in terms of the number of exchanges they should consume at a meal or snack.\n\nThere are also more simplified meal planning approaches such as the food guide pyramid or Healthy Food Choices [39]. These options provide basic information about healthy eating and are very visual, but they do not provide enough detail for most patients with type 1 diabetes to make premeal insulin adjustments.\n\n## v. CONCLUSION\n\nThe management of type 1 diabetes is challenging for both the patient and health care professional. Over the years it has become much clearer that the management of this complex disease is more successfully accomplished when the patient is at the center of the team and when the various health care professionals necessary to treat diabetes work as a team. The question in diabetes care is no longer whether blood glucose control is important for decreasing the risk for complications of diabetes, but it is now a question of how best to achieve appropriate blood glucose control. Several meal planning approaches can be utilized depending on patient needs and preferences. Patients with type 1 diabetes must be given adequate information, coaching, and support so they can make their own decisions about food choices. In addition to using current information about medical nutrition therapy, nutrition and other health professionals must be knowledgeable about and make use of blood glucose records and information on insulin to more appropriately assist the patient with nutrition and exercise decisions in their diabetes management.\n\nReferences\n\n1. Franz M., Horton E., Bantle J., Beebe C., Brunzell J., Coulston A., Henry R., Hoogwerf B., Stacpoole P. Nutrition principles for the management of diabetes and related complications. _Diabetes Care_. 1994;17:490\u2013518.\n\n2. page numbers to comeExpert Committee on the Diagnosis and Classification of Diabetes Mellitus. Report of the expert committee on the diagnosis and classification of diabetes mellitus. _Diabetes Care_. 30, 1997.\n\n3. Zimmet P.Z., Tuomi T., Mackay R., Rowley M.J., Knowles W., Cohen M., Lang D.A. Latent autoimmune diabetes mellitus in adults (LADA): The role of antibodies to glutamic acid decarboxylase in diagnosis and prediction of insulin dependency. _Diabet. Med_. 1994;11:299\u2013303.\n\n4. Branson R., Davis J., Remington P. Chronic Disease Epidemiology and Control. Amsterdam: United Book Press; 1998;Vol. II:421\u2013464.\n\n5. Atkinson M.A., Maclaren N.K. The pathogenesis of insulin dependent diabetes. _N. Engl. J. Med_. 1994;331:1428\u20131436.\n\n6. Holler H., Pastors J. Management of Diabetes: Medical Nutrition Therapy. Frederick, Maryland: American Dietetic Association; 1997;Vol. I:15\u201328.\n\n7. Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. _N. Engl. J. Med_. 1993;329:977\u2013986.\n\n8. Diabetes Care and Education, a practice of group of The American Dietetic AssociationKulkarni K.D., Castle G., Gregory R., Holmes A., Leontos C., Powers M.A., Snetselaar L., Splett P.L., Wylie-Rosett J. Nutrition practice guidelines for Type I diabetes: An overview of the content and application. _Diabetes Spectrum_. 1997;10:248\u2013256.\n\n9. DCCT Research Group. Nutrition interventions for intensive therapy in the Diabetes Control and Complications Trial. _J. Am. Diet. Assoc_. 1993;93:768\u2013772.\n\n10. Delahanty L., Halford B. The role of diet behaviors in achieving improved glycemic control in intensively treated patients in the Diabetes Control and Complications Trial. _Diabetes Care_. 1993;16:1453\u20131458.\n\n11. Peters A.L., Davidson M.B. Protein and fat effects on glucose responses and insulin requirements in subjects with insulin-dependent diabetes mellitus. _Am. J. Clin. Nutr_. 1993;58:555\u2013560.\n\n12. Ahmed M., Nuttall F.Q., Gannon M.C., Lamusga R.F. Plasma glucagon and alpha-amino acid nitrogen response to various diets in normal humans. _Am. J. Clin. Nutr_. 1980;33:1917\u20131924.\n\n13. Brenner B.M., Meyer T.W., Hostetter T.H. Dietary protein intake and the progressive nature of kidney disease: The role of hemodynamically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in aging, renal ablation and intrinsic renal disease. _N. Engl. J. Med_. 1982;307:652\u2013659.\n\n14. Zeller K.R. Low-protein diets in renal disease. _Diabetes Care_. 1991;14:856\u2013866.\n\n15. Friedman E.A. Diabetic nephropathy: Strategies in prevention and management. _Kidney Int_. 1982;21:780\u2013791.\n\n16. Kontessis P., Jones S., Dodds R., Trevisan R., Nosadini R., Fioretto P., Brosato M., Sacerdoti D., Viberti G.C. Renal metabolic and hormonal responses to ingestion of animal and vegetable proteins. _Kidney Int_. 1990;38:136\u2013144.\n\n17. Jibani M.M., Bloodworth L.L., Foden E., Griffiths K.D., Galpin O.P. Predominantly vegetarian diet in patients with incipient and early clinical diabetic nephropathy: Effects on albumin excretion rate and nutritional status. _Diab. Med_. 1991;8:949\u2013953.\n\n18. Nakamura H., Ito S., Ebe N., Shibata A. Renal effects of different types of protein in healthy volunteer subjects and diabetic patients. _Diabetes Care_. 1993;16:1071\u20131075.\n\n19. Stamler J., Vaccaro O., Neaton J.D., Wentworth D., for the Multiple Risk Factor Intervention Trial Research Group. Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial. _Diabetes Care_. 1993;16:434\u2013444.\n\n20. Nikkila E.A., Hormila P. Serum lipids and lipoprotein in insulin-treated diabetes: demonstration of increased high density lipoprotein concentrations. _Diabetes_. 1978;27:1078\u20131086.\n\n21. Kern P. Lipid disorders in diabetes mellitus. _Mt. Sinai J. Med_. 1987;54:245\u2013252.\n\n22. Dunn F.L. Plasma lipid and lipoprotein disorders in IDDM. _Diabetes_. 1992;41:102\u2013106.\n\n23. Garg A., Bonanome A., Grundy S.M., Zhang A.J., Unger R.H. Comparison of a high-carbohydrate diet with high-monounsaturated-fat diet in patients with non-insulin-dependent diabetes mellitus. _N. Engl. J. Med_. 1988;391:829\u2013834.\n\n24. Parillo M., Rivellese A.A., Ciardullo A.V., Capaldo B., Giacco A., Genovese S., Ricardi G. A high-monounsaturated-fat\/low-carbohydrate diet improves peripheral insulin sensitivity in non-insulin-dependent diabetic patients. _Metabolism_. 1992;41:1371\u20131378.\n\n25. Jenkins D.J.A., Woever T.M.S., Jenkins A.L., Josse R.G., Wong G.S. The glycaemic response to carbohydrate foods. _Lancet_. 1984;2:388\u2013391.\n\n26. Loghmani E., Richard K., Washburne L., Vandagriff J., Fineberg N., Golden M. Glycemic response to sucrose-containing mixed meals in diets of children with insulin-dependent diabetes mellitus. _J. Pediatr_. 1991;119:531\u2013537.\n\n27. Kulkarni K., Castle G., Gregroy R., Holmes A., Leontos C., Powers M., Snetselaar L., Splett P., Wylie-Rosett J., for the Diabetes Care and Education Dietetic Practice Group. Nutrition practice guidelines for type 1 diabetes mellitus positively affect dietitian practices and patient outcomes. _J. Am. Diet. Assoc_. 1998;98:62\u201370.\n\n28. Skyler J.S. Insulin therapy in Type I Diabetes Mellitus. Chicago, IL: Mosby Year Book; 1998;Vol. I:36\u201349.\n\n29. Berger M. Adjustment of Insulin Therapy. St. Louis, MI: American Diabetes Association, Inc.; 1995;Vol. I.:117\u2013132.\n\n30. Diabetes Control and Complications Trial Research Group. Implementation of treatment protocols in the Diabetes Control and Complications Trial. _Diabetes Care_. 1995;18:361\u2013376.\n\n31. Nuttall F.Q. Carbohydrate and dietary management of clients with insulin- requiring diabetes. _Diabetes Care_. 1993;16:1039\u20131042.\n\n32. and American Dietetic Association, Chicago, ILAmerican Diabetes Association and American Dietetic Association. _Carbohydrate Counting: Getting Started_. Alexandria, VA: American Diabetes Association; 1995.\n\n33. and American Dietetic Association, Chicago, ILAmerican Diabetes Association and American Dietetic Association. _Carbohydrate Counting: Moving On_. Alexandria, VA: American Diabetes Association; 1995.\n\n34. and American Dietetic Association, Chicago, ILAmerican Diabetes Association and American Dietetic Association. _Carbohydrate Counting: Carbohydrate\/Insulin Ratios_. Alexandria, VA: American Diabetes Association; 1995.\n\n35. Gillespi S., Kulkarni K., Daly A. Using carbohydrate counting in diabetes clinical practice. _J. Am. Diet. Assoc_. 1998;98:897\u2013899.\n\n36. Franz M.J. Protein: Metabolism and effect on blood glucose levels. _Diabetes Ed_. 1997;23:651\u2013663.\n\n37. and American Dietetic Association, Chicago, ILAmerican Diabetes Association and American Dietetic Association. _Exchange Lists for Meal Planning_. Alexandria, VA: American Diabetes Association; 1995.\n\n38. and American Dietetic Association, Chicago, ILAmerican Diabetes Association and The American Dietetic Association. _The First Step in Diabetes Meal Planning_. Alexandria, VA: American Diabetes Association; 1995.\n\n39. and The American Dietetic Association, Chicago, ILAmerican Diabetes Association and The American Dietetic Association. _Healthy Food Choices_. Alexandria, VA: American Diabetes Association; 1986.\n\n40. Bantle J.P., Laine D.C., Castle G.W., Thomas J.W., Hoogwerf B.J., Goetz F.C. Postprandial glucose and insulin responses to meals containing different carbohydrates in normal and diabetic subjects. _N. Engl. J. Med_. 1983;309:7\u201312.\n\n41. Slama G., Haardt M.J., Jean-Joseph P., Costagliola D., Goicolea I., Bornet F., Elgrably F., Tchobroutsky G. Sucrose taken during mixed meal has no additional hyperglycemic action over isocaloric amounts of starch in well-controlled diabetics. _Lancet_. 1984;2:122\u2013125.\n\n42. Bornet F., Haardt M.J., Costagliola D., Blayo A., Slama G. Sucrose or honey at breakfast have no additional acute hyperglycemic effect over an isoglucidic amount of bread in type ii diabetic patients. _Diabetologia_. 1985;28:213\u2013217.\n\n43. Forlani G., Galuppi V., Santacroce G., Braione A.F., Giangiulio S., Ciavarella A., Vannini P. Hyperglycemic effect of sucrose ingestion in IDDM patients controlled by artificial pancreas. _Diabetes Care_. 1989;12:296\u2013298.\n\n44. Peters A.L., Davidson M.B., Eisenberg K. Effect of isocaloric substitution of chocolate cake for potato in type I diabetic patients. _Diabetes Care_. 1990;13:888\u2013892.\n\n45. Bantle J.P., Laine C.W., Thomas J.W. Metabolic effects of dietary fructose and sucrose in types I and II diabetic subjects. _JAMA_. 1986;256:3241\u20133246.\n\n46. Wise J.E., Keim K.S., Huisinga J.L., Willmann P.A. Effect of sucrose-containing snacks on blood glucose control. _Diabetes Care_. 1989;12:423\u2013426.\n\n47. Loghmani E., Richard K., Washburne L., Vandagriff H., Fineberg N., Golden M. Glycemic response to sucrose-containing mixed meals in diets of children with insulin-dependent diabetes mellitus. _J. Pediatr_. 1991;119:531\u2013537.\n\n48. Abraira C., Derler J. Large variations of sucrose in constant carbohydrate diets in type II diabetes. _Am. J. Med_. 1988;84:193\u2013200.\n\n49. Bantle J.P., Swanson J.E., Thomas W., Laine D.C. Metabolic effects of dietary sucrose in type II diabetic subjects. _Diabetes Care_. 1993;16:1301\u20131305.\nCHAPTER 29\n\nNutritional Management for Type 2 Diabetes\n\nANN M. COULSTON, Woodside, California\n\n## I. INTRODUCTION\n\nDiabetes mellitus is a group of metabolic disorders characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The hyperglycemia of diabetes increases the risk of a variety of complications including cardiovascular disease, stroke, visual impairment and blindness, nephropathy leading to renal failure, and neuropathy. Diabetes imposes a major public health burden in the United States and is associated with more than 300,000 deaths and about $100 billion in total costs annually [1].\n\nBroadly, diabetes is classified into two major forms. Type 1 diabetes is characterized by a complete inability of the beta cells of the pancreas to produce insulin. It most commonly occurs during childhood and young adulthood and accounts for about 10% of all persons diagnosed with diabetes. Type 2 diabetes is a combination of a defect in insulin secretion and insulin resistance at the site of insulin action in the muscle, liver, and adipose tissue [2]. It is the most common form of the disease, affecting 90% of individuals with diabetes [3]. Most people with type 2 diabetes are obese, and obesity itself causes some degree of insulin resistance. Insulin secretion improves with weight loss but hyperglycemia seldom returns to normal. Type 2 diabetes has a gradual onset usually beginning with an impairment of glucose tolerance that frequently goes undiagnosed. It is estimated that, at diagnosis, most adults with type 2 diabetes have had the disease for an average of 7 years [4].\n\nOnly recently have individuals with type 2 diabetes been treated aggressively. This is partially accounted for by the fact that we are witnessing a trend toward an increase in younger individuals developing type 2 diabetes in association with an increase in childhood obesity. Also, because of the gradual disease onset, many patients, at diagnosis, have had the disease for several years and the ravages of chronic hyperglycemia may already be damaging vital body tissues [5]. Results of the United Kingdom Prospective Diabetes Study (UKPDS) [6] demonstrated that the hyperglycemia in type 2 diabetes is no less deadly than that in type 1 diabetes [7].\n\nThe management of type 2 diabetes is complex. Patients require a combination of medications and lifestyle changes to achieve blood glucose control [8]. They are frequently older and may have associated disease states that also require medical management, such as hypertension. Many are obese or overweight and this problem must also be addressed. Larger amounts of fat in the abdominal area are most closely associated with insulin resistance and type 2 diabetes [9]. Duration and degree of obesity can affect insulin secretion rates. Once diabetes is diagnosed, time continues to be a factor as beta-cell exhaustion progresses and eventually necessitates the use of insulin secretagogues or exogenous insulin therapy. Early detection followed by early intervention might slow this progress and reduce complications [4]. Reduced energy intake and moderate weight loss improve insulin sensitivity and lower blood glucose levels in type 2 patients [10]. Glycemic improvement, as a result of energy restriction, is due to the combined effects of reduced calories and carbohydrate intake.\n\nNutrition therapy is an important part of the overall management of diabetes. Current nutrition recommendations to achieve and maintain glucose, lipid, and blood pressure goals are simple to state, but difficult to initiate and even more difficult to maintain. Nutrition therapy can be summarized as follows: Lose weight if overweight (more than 30 kg\/m2), restrict saturated fat intake, and divide total nutrient intake throughout the day [11, 12]. These nutrition recommendations are necessarily vague due to the heterogeneous nature of type 2 patients, which makes it impossible to recommend one dietary pattern. Available research data to substantiate more precise guidelines are lacking. As with all treatment regimens for chronic diseases, behavior modification and lifestyle change are essential.\n\nIn addition to providing guidelines to correct blood glucose and blood lipid levels, patients with type 2 diabetes need to receive information about basic nutrition needs [12a]. The traditional approach to medical management of type 2 diabetes, consisting of monotherapy, a weight-loss or \"no sugar\" diet, and advice to \"get some exercise,\" will not yield desired medical outcomes. Current recommendations are for patients to have targeted blood glucose levels, whereby patients know and attempt to achieve their blood glucose goals. This kind of therapy is made possible by feedback from daily self-monitoring of blood glucose and routine laboratory evaluations. Ongoing training programs for educators of patients with diabetes are essential [12b]. Table 1 defines glycemic control goals for type 2 diabetes.\n\nTABLE 1\n\nGlycemic Control for Type 2 Diabetes\n\n_Source:_ Adapted from Zimmerman, B. R. (Ed). (1998). \"Medical Management of Type 2 Diabetes,\" 4th ed., p. 35. American Diabetes Association, Alexandria, VA. American Diabetes Association\n\nThis chapter will address the nutritional management of adults with type 2 diabetes. Nutrition issues for people with type 1 diabetes and with gestational diabetes are discussed in other chapters in this volume (Chapters 28 and ).\n\n## II. ENERGY INTAKE AND BODY WEIGHT MANAGEMENT\n\nApproximately 80\u201390% of people with type 2 diabetes are overweight [4]. Exercise and restriction of energy intake can improve glucose tolerance, diminish insulin resistance, and improve coronary risk factors in many patients with established type 2 diabetes, especially those who are hyperinsulinemic and insulin resistant. On the other hand, the therapeutic efficacy of diet and exercise in such patients has been limited because most individuals with this disorder are more than 40 years old at the time of diagnosis and many tend to be resistant to lifestyle changes [13].\n\nObesity, upper-body obesity in particular [9], aging, and a sedentary lifestyle are independent environmental factors that contribute to insulin resistance [14]. Weight loss improves blood glucose control by decreasing insulin resistance, which improves glucose uptake and reduces hepatic glucose output [15]. Studies have evaluated blood glucose control during weight loss and demonstrate that metabolic changes occur with as little as a 10 kg weight loss [10, 16]. Blood glucose levels and insulin sensitivity continue to improve as weight loss progresses on an energy-restricted constant diet [16]. Patients with hyperinsulinemia respond most dramatically to weight loss, thus early intervention before beta-cell exhaustion occurs provides the best possibility for improving blood glucose control with weight loss. Because we know that 30\u201340% of patients diagnosed with type 2 diabetes already have clinically significant ischemic heart disease, microvascular disease, and neuropathy at the time of diagnosis [5], diet and exercise may be more beneficial if instituted earlier in life, before the onset of hyperglycemia [17]. Glycemic control begins to improve within 24 hours of initiating a hypocaloric diet, even before any weight is lost [10]. In fact, within 10 days of a controlled hypocaloric diet 87% of the eventual drop in blood glucose occurs. These metabolic changes are temporary and require continuation of an energy intake that maintains a decreased body weight.\n\nMany suggestions have been proposed for the most effective macronutrient composition to achieve weight loss in patients with diabetes. Traditionally, weight-loss diets are high in carbohydrate and low in fat. However, because of the carbohydrate intolerance of patients with diabetes, scientists have questioned the wisdom of this approach. At the same time there is concern that higher fat diets might promote obesity. Two reports in the literature have shown equal amounts of weight loss on hypocaloric, low-fat or higher fat diets [18, 19]. When metabolic parameters were examined during weight loss, investigators found that energy restriction and weight loss improved glycemic control, lipid profiles, and blood pressure independent of diet composition. In another report, the cardiovascular risk profile was improved when monounsaturated fatty acids or carbohydrate replaced saturated fatty acids on weight loss diets [20]. Very-low-calorie diet therapy consisting of 400\u2013800 kcal\/day has been shown to be safe in obese individuals with diabetes [21]. However this is a weight reduction method that can only be used for 3\u20134 months and then weight maintenance or moderately restricted diets must be initiated. To determine how this technique might work for the long-term, Wing and colleagues [21, 22] studied short periods of energy restriction, ranging from 1 day\/week to 1 week\/month to enhance weight loss efforts. One characteristic that all current weight loss methods have in common is that actual long-term weight loss is difficult and results are not dramatically different between weight loss regimens. Unfortunately, weight loss is very difficult to achieve and maintain in obese patients in general, and even more so in patients with type 2 diabetes, especially those on oral medications [23]. The metabolic explanation for increased difficulty with weight loss in diabetics is yet to be fully explained. The complexity of weight management has led clinicians to adopt the philosophy that absence of weight gain is itself a reputable goal.\n\n## III. MACRONUTRIENT INTAKE\n\nA. Protein\n\nHistorically, the protein content, both type and amount, of the diet in patients with type 2 diabetes has played a secondary role to carbohydrate and fat content. General concerns for dietary protein adequacy have been to maintain lean body mass and nitrogen balance whether people have diabetes or not.\n\nBody proteins are continuously being synthesized and degraded. The estimated turnover is about 280 g\/day [24]. The amino acids resulting from protein degradation can be recycled, but this process is incomplete. Current data indicate that the efficiency with which amino acids are recycled may be regulated by the amount of protein in the diet [24]. The lower the protein content of the diet, the more efficiently amino acids are utilized. The current recommended amount, 0.8 g\/kg body weight, represents an intake of about 11% of energy from protein. The estimated amount of protein ingested by the general population in the United States represents 15\u201320% of energy intake or 1.1\u20131.4 g\/kg body weight based on 2000 kcal\/day. This is considerably more than the minimum protein necessary even in people with diabetes. Campbell and associates [25] evaluated the protein requirements for older adults (56\u201380 years of age) and found that 1.0\u20131.25 g\/kg is necessary to maintain lean body mass [25].\n\nSpecific dietary protein recommendations for people with diabetes have not been established. An increased loss of body protein with severe insulin deficiency has been known for years [26, 27]. This is especially striking in patients with type 1 diabetes in whom withdrawal of insulin results in a marked increase in protein loss [28]. When insulin is replaced in the management of type 1 diabetes, the protein requirement is considered to be similar to that for nondiabetic individuals. More recent studies indicating increased leucine turnover suggest that the minimum protein requirement is likely to be increased in most people with type 1 diabetes [29]. If so, the reason protein malnutrition is uncommon is probably that the amount of dietary protein ingested by the average adult regularly exceeds the required amount.\n\nIn people with established renal insufficiency, with or without diabetes, a restriction in dietary protein has been considered desirable to modify the progression of the disease. This is controversial and there is concern that frank protein deficiency may result [30, 31]. At the present time, there is no evidence that restricting protein in the diet of people with diabetes will prevent or delay the onset of renal insufficiency.\n\nFrom reports in the literature, we know that the minimum amount of protein necessary to replace body stores is relatively small, and the amount of protein that can be tolerated without toxic effects is high [32]. Data to support beneficial effects of protein in the diet lower or higher than the typical Western diet, which contains approximately 15\u201320% of calories from protein, are lacking. The position of the American Diabetes Association recommends 10\u201320% of calories from protein, with the lower amount being recommended for patients with overt nephropathy [11].\n\nFrom anecdotal information, diets composed mainly of lean meat in quantities to meet energy requirements are not readily acceptable. Even isocaloric diets containing 40\u201345% of energy from protein are not acceptable. This was confirmed by Nuttall and colleagues [33] feeding normal young volunteers diets of 41% energy from protein. Although these diets were isocaloric, subjects commented that this amount of food was more than they were comfortable eating. They also complained of malaise and lethargy after ingesting the high-protein diet. Keep in mind that the so-called \"high-protein diets\" being promoted in the popular weight loss diet literature recommend 30% of energy from protein in the context of a hypocaloric, not isocaloric, energy level. Thus, the actual recommended amount of protein in grams in such diets is not likely to be more than \"high normal\" for an isocaloric diet.\n\nIn the liver, amino acids that are not required to replace body proteins, particularly nonessential amino acids, are deaminated [34]. The amino group is condensed with carbon dioxide to form urea, which is then carried to the kidneys and excreted in the urine. The amount of urea excreted per day is an index of the amount of protein metabolized, although about 14% of newly synthesized urea is utilized by bacteria in the colon [35]. Small amounts of amino acids are also metabolized (deaminated) in the kidney [36]. The carbon skeletons remaining after deamination can be converted to glucose. The resulting glucose may contribute to plasma glucose concentration. Despite the conversion of amino acids to glucose, controlled feeding studies of known amounts of protein do not result in the predicted (calculated) increase in peripheral glucose concentration in normal or type 2 subjects [37, 38]. Further studies designed to elucidate these findings are described below.\n\nIt is known that the carbon skeleton of all amino acids derived from protein digestion, with the exception of leucine, can be used to synthesize glucose. Theoretically, ingestion of 100 g protein can yield 50\u201380 g glucose depending on the amino acid composition of the protein. Using isotope dilution techniques combined with determination of urea formation rates, it was calculated that ingestion of 50 g of cottage cheese protein would result in 34 g being deaminated over the 8-hour period of the study in normal subjects. However, the amount of glucose entering the circulation was only 9.7 g [39]. Thus, the amount of glucose produced was considerably less than the amount theorized by about 25 g. A similar technique, applied to patients with type 2 diabetes following the ingestion of beef protein, found that of the 50 g protein ingested only \u223c2 g could be accounted for by the appearance of glucose in the circulation over an 8-hour period [40]. Thus, in patients with diabetes, the amount of glucose appearing in the circulation was even less than in normal subjects. From the beef protein load, 28 g glucose was predicted. The fate of the remaining carbon skeletons is unknown.\n\nIn healthy people, protein is a weak insulin stimulator compared with glucose; however, in patients with type 2 diabetes, protein and glucose are equipotent in stimulating insulin secretion. After the ingestion of protein, the circulating insulin concentration is increased in both healthy subjects and patients with type 2 diabetes [41, 42]. When protein is ingested with glucose, healthy subjects have an additive insulin response [43], whereas obese subjects with type 2 diabetes exhibit a synergistic insulin response [44].\n\nProtein-containing foods are generally classified as plant or animal based. Studies comparing animal to plant proteins are confounded by the fact that ingestion of plant protein diets is accompanied by additional dietary fiber, while diets based on animal protein are associated with an increased ingestion of dietary fat. Animal studies have demonstratedan atherogenecity of animal protein diets as compared to plantor soy protein-based diets [45]. However, it is not possible with the data currently available to draw conclusions as to the benefits or hazards of either plant or animal proteins in the diets of people with type 2 diabetes.\n\n### B. Carbohydrate\n\nFasting blood glucose is determined by the overproduction of glucose from the liver and the body's ability to remove glucose from the blood stream. The amount of glucose absorbed from a meal largely determines the blood glucose response in the postprandial state. Patients with type 2 diabetes require from 3 to 4 hours for blood glucose to return to fasting or premeal levels after eating. However, gastric emptying rate, intestinal motility, and factors that affect glucose removal from the circulation, such as insulin response or insulin resistance, modify the absolute plasma glucose response. Thus, plasma glucose concentration at any given time is the result of the action of medication, glucose absorption from meals, and endogenous production of glucose mainly from the liver.\n\nPrior to the availability of insulin and other pharmaceutical agents, both glucose overproduction and the impaired ability to metabolize absorbed glucose were shown to be treatable by dietary manipulations. In the early 1900s semi-starvation or low-calorie diets controlled overproduction of glucose. The amount of carbohydrate in the diet depended on the severity of diabetes and was tailored to the individual utilization rate [46]. Dietary patterns were low in carbohydrate and very high in fat as an energy fuel. Adhering to these diets was difficult, and semi-starvation could only be followed for a limited time.\n\nFollowing the availability of insulin and oral agents for the treatment of diabetes, food energy restriction and the carbohydrate content of the diet have been greatly relaxed. In fact, there has been a universal trend toward advocating a higher and higher carbohydrate content in the diet, not only for people with diabetes, but also for the general population [47, 48]. An increase in carbohydrate intake allows a decrease in fat intake to meet overall energy needs. The protein content of the diet remains relatively constant. This high-carbohydrate dietary recommendation was driven by the concern that dietary fat, especially saturated fat, in the diet was responsible for the increasing incidence of cardiovascular disease.\n\nBecause cardiovascular disease is more common in people with diabetes and accounts for the majority of deaths in patients with diabetes [49\u201351] early dietary recommendations followed the American Heart Association and recommended a high-carbohydrate, low-fat diet [47]. However, the American Heart Association guidelines were aimed primarily at people with hypercholesterolemia. The most frequent lipid abnormalities in patients with type 2 diabetes are hypertriglyceridemia, increased very low density lipoprotein (VLDL) cholesterol, and reduced HDL cholesterol [52]. Plasma concentrations of total and low-density lipoprotein (LDL) cholesterol are similar to those in the general population. However, about 40% of patients with type 2 diabetes have an elevated LDL cholesterol [53]. The hypertriglyceridemia of type 2 diabetes is believed to be due in part to increased hepatic production of triglyceride-rich VLDL particles induced by increased dietary carbohydrate intake. An increase in small, dense LDL particles along with decreased HDL cholesterol and hypertriglyceridemia appear to be sequelae of insulin resistance, although a complete understanding of the mechanism remains to be elucidated [52, 54]. The insulin resistance of type 2 diabetes results in hyperinsulinemia in the face of hyperglycemia. Epidemiological studies have demonstrated that, in addition to the dyslipidemia of type 2 diabetes, hyperglycemia and hyperinsulinemia contribute to an increased risk for cardiovascular disease in people with diabetes [55, 56].\n\nDietary intervention studies of high-carbohydrate, low-fat diets as compared to moderate-carbohydrate, higher fat diets have indicated that the risk factors for cardiovascular disease in people with type 2 diabetes are exacerbated with high-carbohydrate, low-fat diets [57, 58]. Diets with relatively high fat (40\u201345% of energy from fat) in which monounsaturated and polyunsaturated fatty acids predominate demonstrate an improved lipid profile, and the increase in dietary fat does not hinder glucose disposal [59]. As a result of studies that demonstrate that higher intakes of carbohydrate accentuate plasma glucose, insulin, and triglyceride response, general nutrition recommendations currently are to individualize the amount of carbohydrate in the diet to optimize patient blood glucose and lipid goals [12].\n\nUnresolved questions about the best type of dietary carbohydrate for people with type 2 diabetes remain. For many years, it was felt that refined sucrose (i.e., table sugar) should be eliminated from the diets of people with diabetes despite the lack of convincing scientific data. More recent evidence indicates that the amount of sucrose typically found in the American diet does not have an adverse effect on blood glucose control [60, 61]. Consequently, current dietary recommendations from the American Diabetes Association do not prohibit sucrose or any type of dietary carbohydrate [11]. Rather the focus is on the amount of all dietary carbohydrate at each meal and for the total day because the amount of carbohydrate at meals and throughout the day has a major impact on daylong blood glucose control.\n\nIn recent years, interest in dietary fiber has increased. The American Diabetes Association has suggested 20\u201335 g dietary fiber\/day for people with diabetes [12]. This amount represents an increase from the recent reports of dietary fiber intake of 17 g\/day for the general population and 16 g\/day for people with diabetes [62]. Increasing consumption of soluble fiber has been shown to reduce serum cholesterol and improve colonic function [63]. Whether or not patients with type 2 diabetes can achieve improved glycemic control by consuming diets high in dietary fiber, and especially soluble fiber, has been debated. Studies have shown that mixing viscous, nonstarch polysaccharides with carbohydrate-rich foods reduces the postprandial plasma glucose response, presumably by slowing the intestinal absorption of glucose [64, 65]. However, the amounts required to achieve these one-meal test results are too high to be acceptable in a routine diet. Nevertheless, high-fiber diets had been encouraged for people with diabetes with the rationale that dietary fiber would delay or reduce the absorption of glucose from the intestine.\n\nIn early studies of mixed diets, which varied in total dietary fiber from a variety of food sources, fed to patients with type 2 diabetes, no improvement in plasma glucose response was noted [66]. In this same study, higher fiber diets did not show any benefit to plasma cholesterol levels beyond that which can be achieved with a reduction in saturated fatty acid intake [66, 67]. A more recent report led to different conclusions. Diets for patients with type 2 diabetes that contained 50 g total dietary fiber, of which 25 g were from soluble fiber and all fiber from nonfortified foods, found significant improvement in both plasma glucose control and decreased fasting plasma lipid concentrations [68]. This report is very convincing; however, the lifestyle change to consume 50 g dietary fiber\/day may be difficult for a population with reported usually intake of 16 g\/per day as reported by the National Health and Nutrition Examination Survey (NHANES III) [62].\n\nFinally, dietary fiber has been encouraged for all to prevent colon cancer. However, two recent intervention trials in normal glycemic individuals found no association between dietary fibers from fruits, vegetables, and whole grains on the recurrence of adenomatous polyps, thought to be the early signs of developing colon cancer [69, 70]. Whether the subjects were able to consume the level of fiber-rich foods recommended is not substantiated in the research report. In addition, many scientists believe that dietary fiber is only one risk factor in the development of colon cancer. It would be na\u00efve, however, to suggest that dietary fiber has no role in colorectal cancer from these reports.\n\nDuring the past century, investigators have reported differences in the plasma glucose response to the ingestion of different carbohydrate-containing foods despite the foods being matched for total carbohydrate content [71, 72]. Dietary carbohydrate foods are composed of a variety of mono-, di-, and polysaccharide structures. These sugars and starches are hydrolyzed into constituent glucose molecules by a combination of pancreatic amylase and intestinal mucosal glucosidase and maltase enzymes. These enzymes are present in excess so the rate-limiting process is not related to hydrolysis but rather the physical state of the starch. Uncooked starch in general is poorly digested and, thus, not well absorbed. Another hydrolysis factor is how readily the starch granules, which differ in size and structure depending on the plant source, undergo gelatinization. As a result, starch in many foods is not completely hydrolyzed or digestible.\n\nSugars, on the other hand, are readily digested and absorbed in the intestine. However, the monosaccharide fructose does not cause an elevation in blood glucose in normal glycemic or in patients treated for diabetes. Recently Gannon and colleagues [73] verified this concept by comparing the daylong plasma glucose and insulin response to identical carbohydrate content meals in patients with type 2 diabetes. One day the meals were high in starch foods and the other day in sugars from fruit, vegetables, and dairy products. On the day of the higher sugars diet, plasma glucose and insulin were significantly lower following each meal. Although these findings are interesting and help explain the glycemic impact of foods, dietary patterns are a combination of starches and sugars. Consequently, when individual carbohydrate-containing foods are tested for their glycemic response, foods differ.\n\nJenkins and colleagues [74, 75] tested a large number of foods and compared them to the plasma glucose response for pure glucose or white bread and developed a \"glycemic index.\" Of concern for the nutritional management of patients with diabetes is to determine whether or not these differences in glycemic response have clinical relevance. Recent reports from the Health Professionals Follow-up Study cohort and the Nurses' Health Study cohort indicate that over time, diets high in glycemic load and low in cereal fiber significantly increase the risk of type 2 diabetes [76, 77]. These observational studies need to be addressed in research settings with careful controls on potential confounding factors and in settings where the mechanisms of disease can be studied. Controlled intervention studies have failed to show clinically significant differences between so-called high and low glycemic index foods in the context of a total day's meal [78, 79]. As additional studies have appeared in the literature, generally conducted with intervention techniques relying on dietary advice and lists of foods to choose and avoid, the use of the glycemic index has been criticized and its utility in nutritional management of people with diabetes questioned [80, 81].\n\n### C. Fat\n\nBecause diabetes is associated with a marked increase in coronary artery disease, there is a strong focus on dietary fatty acid intake [51]. Several studies have shown that increased cardiovascular risk factors, including hypertension, precede the onset of type 2 diabetes [82]. In most studies the excess risk of cardiovascular disease is relatively greater in women with diabetes than men [51].\n\nPeople with type 2 diabetes have a dyslipidemia, as mentioned earlier, consisting of increased triglyceride concentrations and decreased HDL cholesterol levels, and small dense LDL cholesterol particles that do not normalize with improved glycemic control [54, 83]. This dyslipidemia is strongly associated with increased central (visceral) obesity, insulin resistance, and cardiovascular disease [54, 84\u201386]. Much has been written about the association of hyperinsulinemia and\/or insulin resistance with cardiovascular disease [87]. The study by Despres and colleagues [88] provides the strongest evidence that hyperinsulinemia is associated prospectively with the development of cardiovascular disease. LDL cholesterol is usually not different in people with or without diabetes, but a number of metabolic and compositional changes in LDL particles have been described. A preponderance of smaller denser LDL particles (subclass pattern B) has been identified as a risk for cardiovascular disease [86]. Although this has not been studied extensively in people with type 2 diabetes, small, dense LDL particles are associated with increased triglyceride and decreased HDL cholesterol levels, male sex, hyperinsulinemia, and insulin resistance [54]. People with type 2 diabetes and hypertriglyceridemia usually have both overproduction and impaired catabolism of VLDL triglyceride. Lipoprotein lipase (LPL) activity can be reduced in patients with poor glucose control and profound hypertriglyceridemia [89]. Decreased HDL cholesterol levels may be due to increased catabolism, or to decreased production of HDL due to impaired catabolism of VLDL and decreased LPL activity [90]. For additional information, Ginsberg [91] has reviewed the pathophysiology of dyslipemia in diabetes and Haffner [92] has recently reviewed the effectiveness of medical therapy for the dyslipidemia of adults with type 2 diabetes.\n\nBefore 1971, most advice on nutritional therapy for diabetes was to restrict dietary carbohydrate and increase dietary fat to meet daily caloric needs [93]. However, epidemiological studies in the 1960s showed a cardiovascular disease risk associated with increased intake of total and saturated fat [94]. As it became apparent that cardiovascular disease was a leading cause of death in people with diabetes [50], nutrition recommendations for people with diabetes called for reduction in total fat and saturated fats to <30% and <10% of calories, respectively [95]. Controversy has arisen over recommending a low-fat, high-carbohydrate diet to these patients because of concern that this may worsen triglyceride and HDL abnormalities and ultimately increase cardiovascular risk [96]. Another concern is whether overweight and obese people with type 2 diabetes will be able to lose weight on a higher fat diet.\n\nCurrent American Diabetes Association and American Dietetic Association dietary guidelines permit either a high-carbohydrate, low-fat diet, or a moderate carbohydrate, higher fat diet enriched with polyunsaturated or monounsaturated fat. The issue of whether a high-monounsaturated-fat diet is preferable to a high-carbohydrate diet remains controversial [97]. In short term studies, a high-carbohydrate diet has been associated with higher triglyceride levels and lower HDL levels than a higher fat diet [98, 99]. The nutrition practice guidelines of the American Dietetic Association suggest a 4- to 6-month trial of nutrition intervention before lipid-lowering pharmacological treatment is initiated [100]. However if the patient has preexisting cardiovasculardisease or already has made all of the lifestyle changes he or she is willing or able to make, pharmacological intervention may be initiated sooner.\n\nIt has been demonstrated in numerous studies that glycemic control and insulin action are improved\u2014or at least not made worse\u2014when the diet is restricted in saturated fatty acids [101\u2013103]. The debate, therefore, is not whether saturated fat should be limited to 10% or less of total caloric intake, as is currently recommended by the National Cholesterol Education Program (NCEP) for the prevention of cardiovascular risk, but what is the best alternative energy source for the patient with diabetes. Dietary protein is generally \u221210\u201320% of total daily energy intake. This leaves roughly 70\u201380% of energy intake to be derived from a combination of polyunsaturated fats, monounsaturated fats, and carbohydrates.\n\nSeveral short-term studies have been conducted that have explored diets of greater than 30% energy from fat with the extra fat coming from monounsaturated fatty acids [58, 98, 99]. In these isocaloric studies, saturated fatty acids were maintained at 10% of energy. At the end of each diet phase, fasting glucose and insulin concentrations, and total cholesterol and LDL cholesterol concentrations, did not differ between the two diets. However, fasting triglyceride levels, VLDL cholesterol, and postprandial excursions of glucose, insulin, and triglycerides were higher while fasting HDL cholesterol levels decreased during the high-carbohydrate diet period. The authors emphasize that the lower total glucose load keeps postprandial glucose and insulin concentrations reduced, low dietary saturated fat keeps LDL cholesterol in check, and the higher monounsaturated and polyunsaturated content of the diet prevents HDL cholesterol decrease and triglyceride concentration increase. With attention to food choices, it is possible to keep the saturated fat content of the diet low without decreasing the total dietary fat intake. The addition of unsaturated fatty acids to the diet to maintain energy needs appears to be the optimal approach to nutrition management, especially for patients with the typical dyslipidemia of type 2 diabetes and with insulin resistance.\n\n## IV. TRACE MINERAL REQUIREMENTS\n\nA. Magnesium\n\nMagnesium levels remain remarkably constant in people without diabetes, due to regulatory mechanisms. However, those with diabetes appear to be prone to low serum magnesium levels [104, 105]. The clinical significance of this finding remains undefined, because only about 0.3% of total body magnesium is in the blood. Low serum levels may be related to increased urinary magnesium losses secondary to glycosuria-induced renal wasting [106], although short-term improvement in glycemic control has not been shown to restore serum magnesium levels [107].\n\nMagnesium is intimately involved in a number of important biochemical reactions, particularly processes that involve the formation and use of high-energy phosphate bonds. As a cofactor in more than 300 enzyme reactions, it modulates glucose transport through membranes and is a cofactor in several enzymatic systems involving glucose oxidation [108]. Some believe that magnesium deficiency may increase or cause insulin resistance [109].\n\nLow levels of magnesium have been associated with hypertension, cardiac arrhythmia, congestive heart failure, retinopathy, and insulin resistance [110\u2013112]. Magnesium depletion in a few studies has been shown to result in insulin resistance as well as impaired insulin secretion and thereby may worsen control of diabetes [112]. Nadler and colleagues [113] conducted a magnesium depletion study. Men were fed 12 mg magnesium daily for 3 weeks. Intravenous glucose tolerance tests performed at the beginning and end of the 3-week period revealed a significant increase in insulin sensitivity. These findings raise the possibility that insulin resistance and abnormal glucose tolerance might be due to inadequate magnesium. In fact, Paolisso and colleagues [114] have shown magnesium supplementation to improve glucose tolerance in nondiabetic elderly, and improve insulin response in elderly, type 2 patients with diabetes. It is possible that decreased magnesium levels may represent a marker rather than a cause of the disease. In a recent report on the role of magnesium deficiency in the pathogenesis of type 2 diabetes from the large (12, 218 adults) Atherosclerosis Risk in Communities (ARIC) Study, the authors demonstrated in white, but not black, adults a strong relationship between type 2 diabetes and serum magnesium levels [115]. Findings in this study suggest that modification of magnesium intake by dietary means alone is insufficient to achieve an effect to prevent type 2 diabetes. Whether pharmacological doses of magnesium coupled with other risk factors for diabetes can reduce long-term risk remains to be studied.\n\nUntil magnesium depletion studies conducted in normal individuals can relate specific dietary intake levels with abnormal glucose tolerance testing or other indicators of glucose metabolism, it is premature to consider the prevalence of diabetes mellitus as a functional indicator of adequacy for magnesium [116]. The revised Recommended Dietary Allowance (RDA) value for magnesium for women over 50 years of age is 320 mg\/day and 420 mg\/day for men in the same age group [116]. A review of the literature by the Standing Committee resulted in a gradual increase in the RDA for magnesium with age. Thus, it makes sense, at the least, to assess the dietary intake of older patients with type 2 diabetes for magnesium adequacy. The American Diabetes Association consensus statement on magnesium supplementation for patients with diabetes concluded that until accurate indexes of magnesium deficiency are available, routine evaluation of magnesium status in otherwise healthy individuals with diabetes is not recommended [107].\n\n### B. Chromium\n\nIn laboratory animals chromium deficiency is associated with an increase in blood glucose, cholesterol, and triglyceride concentrations. This observation has led to the investigation of chromium status in humans. The biologically active elemental chromium product is called glucose tolerance factor [117]. Glucose tolerance factor is composed of nicotinic acid, elemental chromium, and the amino acids glutamic acid, glycine, and cysteine. It acts as a cofactor for insulin and may facilitate insulin-membrane receptor interaction. However, glucose tolerance factor lowers plasma glucose only in the presence of insulin (postprandial state) and not in overnight fasted animals [118].\n\nMost chromium supplements are poorly absorbed, but when combined with picolinate, absorption is improved. Because no method exists to determine chromium deficiency, the prevalence of a deficiency is unknown. Rabinowitz and colleagues [119] studied chromium in hair, serum, urine, and red blood cells and could not identify a deficiency of chromium in people with or without diabetes. In three double-blind crossover studies in patients with diabetes, chromium supplementation failed to improve glucose and lipid levels [120\u2013122].\n\nRecent evidence from China suggests a role for chromium supplementation in people with type 2 diabetes. Individuals with type 2 diabetes were randomly divided into three groups and supplemented with 1000 \u03bcg\/day or 2000 \u03bcg\/day chromium or placebo. In all three groups, fasting, 2-hour glucose concentrations and glycosylated hemoglobin values decreased but the decreases in the subjects receiving supplemental chromium were much larger. Fasting and 2-hour insulin concentrations were also lowered in the two supplemented groups. Total plasma cholesterol concentration decreased in the group receiving supplements, but there was no impact of supplementation on HDL cholesterol, triglyceride concentration, or body weight [123].\n\nHowever, before chromium supplementation can be recommended, double-blind crossover studies of the effect of chromium supplementation in people with diabetes with known dietary intake of chromium need to be conducted. Until proven otherwise, it is assumed that chromium functions as a nutrient, not as a therapeutic agent, and that it benefits only individuals with marginal glucose intolerance whose signs and symptoms are due to marginal or overt chromium deficiency [124].\n\n## V. CONCLUSION\n\nWe are witnessing a surge in public and media attention on type 2 diabetes mellitus. This is due in part to the increasing worldwide incidence of type 2 diabetes and to the reports of disease management studies that continue to show marked improvement in disease outcome with improved glycemic control. Nutrition intervention remains a key component of type 2 diabetes management.\n\nIn this chapter, nutrient components of the diet are discussed in relation to the impact they have on diabetes treatment. The entire disease management process has been enhanced due to the advances in oral medications for glucose and lipid control. In addition, with an increased focus on near-normal glycemic control in this population, all health care team members are more closely involved in assisting the patients with disease management.\n\nThe use of herbal products (commonly viewed as a component of nutrition therapy) in disease prevention and treatment is gaining in popularity [125]. Despite the tremendous recent growth in popularity, carefully controlled studies are few. 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Schatzkin A., Lanza E., Corle D., Lance P., Iber F., Caan B., Shike M., Weissfeld J., Burt R., Cooper M.R., Kikendall W., Cahill J., the Polyp Prevention Trial Study Group. Lack of effect of a low-fat, high-fiber diet on the recurrence of colorectal adenomas. _N. Engl. J. Med_. 2000;342:1149\u20131155.\n\n70. Alberts D.S., Martinez M.E., Roe D.J., Guillen-Rodriguez J.M., Marshall J.R., van Leeuwen B., Reid M.E., Rittenbaugh C., Vargas P.A., Bhattacharyya A.B., Earnest D.L., Sampliner R.E., the Phoenix Colon Cancer Prevention Physicians' Network. Lack of effect of a high-fiber cereal supplement on the recurrence of colorectal adenomas. _N. Engl. J. Med_. 2000;342:1156\u20131162.\n\n71. Labbe M. Tolerame comparee des divers hydrates de carbone part l'organisme des diabetiques. _Bull. Mem. Soc. Med. Hosp_. 1907;24:221\u2013234.\n\n72. Otto H., Bleyer G., Pehhartz M. Kehlenhydrataustausch nac biologischen Aquivalenten. In: _Diatetik bei Diabetes Mellitus_. Washington, DC: Verlag Has Huber, Bern; 1983:41\u201351.\n\n73. Gannon M.C., Nuttall F.Q., Westphal S.A., Fang S., Ercan-Fang N. Acute metabolic response to high-carbohydrate, high-starch meals compared with moderate-carbohydrate, low-starch meals in subjects with type 2 diabetes. _Diabetes Care_. 1998;21:1619\u20131626.\n\n74. Jenkins D.J.A., Wolever T.M.S., Taylor R.H. Glycemic index of foods: A physiological basis for carbohydrate exchange. _Am. J. Clin. Nutr_. 1981;34:362\u2013366.\n\n75. Wolever T.M.S., Katzman-Relle L., Jenkins A.L., Vuksan V., Josse R.G., Jenkins D.J.A. Glycemic index of 102 complex carbohydrate foods in patients with diabetes. _Nutr. Res_. 1994;14:651\u2013669.\n\n76. Salmeron J., Mason J.E., Stampfer M.J., Colditz G.A., Wing A.L., Willett W.C. Dietary fiber, glycemic load, and risk of non-insulin-dependent diabetes mellitus in women. _JAMA_. 1997;277:472\u2013477.\n\n77. Salmeron J., Ascherio A., Rimm E.B., Colditz G.A., Spiegelman D., Jenkins D.J., Stamfer M.J., Wing A.L., Willett W.C. Dietary fiber, glycemic load, and risk of NIDDM in men. _Diabetes Care_. 1997;20:545\u2013550.\n\n78. Laine D.C., Thomas W., Levitt M.D., Bantle J.P. Comparison of predictive capabilities of diabetic exchange lists and glycemic index of foods. _Diabetes Care_. 1987;10:387\u2013394.\n\n79. Coulston A.M., Hollenbeck C.B., Swislocki A.L.M., Reaven G.M. Effect of source of dietary carbohydrate on plasma glucose and insulin responses to mixed meals in subjects with NIDDM. _Diabetes Care_. 1987;10:395\u2013400.\n\n80. Calle-Pascual A.L., Gomez V., Leon F., Bordiu E. Foods with a low glycemic index do not improve glycemic control of both type 1 and type 2 diabetic patients after one month of therapy. _Diabetes Metab_. 1988;14:629\u2013633.\n\n81. Coulston A.M., Reaven G.M. Much ado about (almost) nothing. _Diabetes Care_. 1997;20:241\u2013243.\n\n82. Haffner S.M., Stern M.P., Hazuda H.P., Mitchell B.D., Patterson J.K. Cardiovascular risk factors in confirmed prediabetic individuals: Does the clock for coronary heart disease start ticking before the onset of clinical diabetes? _JAMA_. 1990;263:2893\u20132898.\n\n83. Stewart M.W., Laker M.F., Dyer R.G., Game F., Mitcheson J., Winocour P.H., Alberti K.G.M.M. Lipoprotein compositional abnormalities and insulin resistance in type 2 diabetic patients with mild hyperlipidemia. _Arterioscler. Thromb_. 1993;13:1046\u20131052.\n\n84. Kissebah A.H., Krakower G.R. Regional adiposity and morbidity. _Physiol. Rev_. 1994;74:761\u2013811.\n\n85. Gardner C.D., Fortmann S.P., Krauss R.M. Association of small low-density lipoprotein particles with the incidence of coronary artery disease in men and women. _JAMA_. 1996;276:875\u2013881.\n\n86. Stampfer M.J., Krauss R.M., Ma J., Blanche P.H., Holl L.G., Sacks F.M., Hennekens C.H. A prospective study of triglyceride level, low-density lipoprotein particle diameter, and risk of myocardial infarction. _JAMA_. 1996;276:882\u2013888.\n\n87. Reaven G.M. 1988 Banting Lecture: Role of insulin resistance in human disease. _Diabetes_. 1988;37:1595\u20131607.\n\n88. Despres J.P., Lamarch B., Mauriege P., Cantin B., Dagenais G.R., Moorjani S., Lupien P.J. Hyperinsulinemia as an independent risk factor for ischemic heart disease. _N. Engl. J. Med_. 1996;334:952\u2013957.\n\n89. Taskinen M.-R., Beltz W.F., Harper I., Fields R.M., Schonfeld G., Grundy S.M., Howard B.V. Effects of NIDDM on very-low-density lipoprotein triglyceride and apolipoprotein B metabolism: Studies before and after sulfonylurea therapy. _Diabetes_. 1986;35:1268\u20131277.\n\n90. Golay A., Zech L., Shi M.Z., Chiou Y.A., Reaven G.M., Chen Y.D. High-density lipoprotein (HDL) metabolism in non-insulin dependent diabetes mellitus: Measurement of HDL turnover using tritiated HDL. _J. Clin. Endocrinol. Metab_. 1987;65:512\u2013518.\n\n91. Ginsberg H. Lipoprotein physiology in non-diabetic and diabetic states: Relationship to atherogenesis. _Diabetes Care_. 1991;14:839\u2013855.\n\n92. Haffner S.M. Management of dyslipidemia in adults with diabetes (a technical review). _Diabetes Care_. 1998;21:160\u2013178.\n\n93. Bierman E.L., Albrink M.J., Arky R.A., Conner W.E., Dayton S., Spritz N., Steinberg D. Principles of nutritional and dietary recommendations for patients with diabetes mellitus. _Diabetes_. 1971;20:633\u2013634.\n\n94. Keys A. Coronary heart disease in seven countries. _Circulation_. 1970;41(Suppl. 1):1\u2013211.\n\n95. American Diabetes Association. Nutritional recommendations and principles for individuals with diabetes melitus. _Diabetes Care_. 1987;10:126\u2013132.\n\n96. Garg A. High-monounsaturated fat diet for diabetic patients: Is it time to change the current dietary recommendations? _Diabetes Care_. 1994;17:242\u2013246.\n\n97. Berry E.M. Dietary fatty acids in the management of diabetes mellitus. _Am. J. Clin. Nutr_. 1997;66:991S\u2013997S.\n\n98. Garg A., Bonanome A., Grundy S.M., Zhang A.J., Unger R.H. Comparison of high-carbohydrate diet with high monounsaturated-fat diet in patients with non-insulin-dependent diabetes mellitus. _N. Engl. J. Med_. 1988;391:829\u2013834.\n\n99. Coulston A.M., Hollenbeck C.B., Swislocki A.L.M., Reaven G.M. Persistence of hypertriglyceridemic effect of low-fat high-carbohydrate diets in NIDDM patients. _Diabetes Care_. 1989;12:94\u2013101.\n\n100. Monk A., Barry B., McClain K., Weaver T., Cooper N., Franz M.J. Practice guidelines for medical nutrition therapy provided by dietitians for persons with non-insulin dependent diabetes mellitus. _J. Am. Diet. Assoc_. 1995;95:999\u20131006.\n\n101. Stone D.B., Conner W.E. The prolonged effects of a low cholesterol, high carbohydrate diet upon the serum lipids in diabetic patients. _Diabetes_. 1963;12:127\u2013132.\n\n102. Anderson J.W., Ward K. High-carbohydrate, high fiber diets for insulin-treated men with diabetes mellitus. _Am. J. Clin. Nutr_. 1979;32:2312\u20132321.\n\n103. Kolterman O.G., Greenfield M., Reaven G.M., Saekow M., Olefsky J.M. Effect of a high carbohydrate diet on insulin binding to adipocytes and on insulin action in vivo in man. _Diabetes_. 1979;28:731\u2013736.\n\n104. Nadler J.L., Malayan S., Luong H., Shaw S., Natarajan R.D., Rude R.K. Intracellular free magnesium deficiency plays a key role in increased platelet reactivity in type II diabetes mellitus. _Diabetes Care_. 1992;15:835\u2013841.\n\n105. Lima J.D.L., Cruz T., Pousada J.C., Rodrigues L.E., Barbosa K., Cangucu V. The effect of magnesium supplementation in increasing doses on the control of type 2 diabetes. _Diabetes Care_. 1990;21:682\u2013686.\n\n106. Rude R.K. Magnesium metabolism and deficiency. _Endocrinol. Metab. Clin. North Am_. 1993;22:377\u2013395.\n\n107. American Diabetes Association. Magnesium supplementation in the treatment of diabetes (consensus statement). _Diabetes Care_. 1992;15:1065\u20131067.\n\n108. [technical review]Mooradian A.D., Faila M., Hoogwerf B., Isaac R., Maryniuk M., Wylie-Rosett J. Selected vitamins and minerals in diabetes mellitus. _Diabetes Care_. 1994;17:464\u2013479.\n\n109. Alzaid A., Dinneen S., Moyer T., Rizza R. Effects of insulin on plasma magnesium in non-insulin dependent diabetes mellitus: Evidence for insulin resistance. _J. Clin. Endocrinol. Metab_. 1995;80:1376\u20131381.\n\n110. Whelton P.K., Klag M.J. Magnesium and blood pressure: Review of the epidemiologic and clinical trial experience. _Am. J. Cardiol_. 1989;63:26G\u201330G.\n\n111. Shattock M.J., Hearse D.J., Fry C.H. The ionic basis of anti-ischemic and anti-arrhythmic properties of magnesium in the heart. _J. Am. College Nutr_. 1987;6:27\u201333.\n\n112. Paolisso G., Scheen A., D'Onofrio E., Lefebvre P. Magnesium and glucose homeostasis. _Diabetologia_. 1990;33:511\u2013514.\n\n113. Nadler J.L., Buchanan T., Natarajan R., Antonipillai I., Bergman R., Rude R.K. Magnesium deficiency produces insulin resistance and increased thromboxane synthesis. _Hypertension_. 1993;21:1024\u20131029.\n\n114. Paolisso G., Sgambato S., Gambardella A., Pizza G., Tesauro P., Varricchio M., D'Onofrio F. Daily magnesium supplements improve glucose handling in elderly subjects. _Am. J. Clin. Nutr_. 1992;55:1161\u20131167.\n\n115. Kao W.H.L., Folsom A.R., Nieto F.J., Mo J.-P., Watson R.S., Brancati F.L. Serum and dietary magnesium and the risk for type 2 diabetes mellitus: The Atherosclerosis Risk in Communities Study. _Arch. Intern. Med_. 1999;159:2151\u20132159.\n\n116. Food and Nutrition Board, Institute of Medicine, National Academy of Sciences. _Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride_. Switzerland: National Academy Press; 1997.\n\n117. Schwarz K., Mertz W. A glucose tolerance factor and its differentiation from factor 3. _Arch. Biochem. Biophys_. 1957;72:515\u2013518.\n\n118. Truman R.W., Doisy R.J. Metabolic effects of the glucose tolerance factor (GTF) in normal and genetically diabetic mice. _Diabetes_. 1977;26:820\u2013826.\n\n119. Rabinowitz M.B., Levin S.R., Gonick J.E. Comparisons of chromium status in diabetic and normal men. _Metabolism_. 1980;29:355\u2013364.\n\n120. Rabinowitz M.B., Gonick H.C., Levin S.R., Davidson M.B. Effect of chromium and yeast supplements on carbohydrate and lipid metabolism. _Diabetes Care_. 1983;6:319\u2013327.\n\n121. Sherman L., Glennon J.A., Brech W.J., Klomberg G.H., Gordon E.S. Failure of trivalent chromium to improve hyperglycemia in diabetes mellitus. _Metabolism_. 1968;17:439\u2013442.\n\n122. Usitupa M.I.J., Kumpulainen J.T., Voutilainen E. Effect of inorganic chromium supplementation on glucose intolerance, insulin response and serum lipids in non-insulin-dependent diabetics. _Am. J. Clin. Nutr_. 1983;38:404\u2013410.\n\n123. Anderson R.A., Cheng N., Bryden N.A., Polansky M.M., Cheng N., Chi J., Feng J. Elevated intakes of supplemental chromium improve glucose and insulin variables in individuals with type 2 diabetes. _Diabetes_. 1997;46:1786\u20131791.\n\n124. Liu V.J.K., Morris J.S. Relative chromium response as an indicator of chromium status. _Am. J. Clin. Nutr_. 1978;31:972\u2013976.\n\n125. Eisenberg D.M., Davis R.B., Ettner S.L., Appel S., Wilkey S., Van Rompay M., Kessler R.C. Trends in alternative medicine use in the United States, 1990\u20131997: Results of a follow-up national survey. _JAMA_. 1998;280:1569\u20131575.\n\n126. Margolin A., Avants S.K., Kleber H. Investigating alternative medicine therapies in randomized controlled trials. _JAMA_. 1998;280:1626\u20131628.\nCHAPTER 30\n\nNutritional Management for Gestational Diabetes\n\nLYNNE LYONS1, 1Burlingame, California\n\nDIANE READER2, 2International Diabetes Center, Minneapolis, Minnesota\n\n## I. INTRODUCTION\n\nGestational diabetes mellitus (GDM), carbohydrate intolerance of variable severity with onset or first recognition during pregnancy [1], occurs on average in 4% of all pregnancies in the United States. The incidence of GDM varies throughout the United States, due to the diversity of ethnic groups and maternal ages. The risk of GDM increases with age, obesity, and genetic predisposition or family history of diabetes. It is interesting to note that the incidence of diabetes in certain ethnic groups may be different in the country of origin compared to the same ethnic group in their country of immigration. Ethnic groups with high rates of type 2 diabetes have a high rate of GDM. Because GDM is more likely to occur in overweight women who lead a sedentary life and are older, immigrants with these characteristics and who have adopted such a lifestyle will, in turn, develop GDM at the same rate as the adopted country [2].\n\n## II. SCREENING AND DIAGNOSIS\n\nIn 1952, Jackson [3] observed a perinatal (fetal and newborn) mortality rate of 8% in pregnant women who later developed diabetes in midlife compared to a perinatal mortality rate of 2% in the control group. These observations led to the development of the 50 g oral glucose screening test. At Boston City Hospital and Lying-In-Hospital in 1954, the first major prospective study of abnormal carbohydrate metabolism in pregnancy began. [4] The 50 g glucose load was taken without regard to the previous meal or time of day. A subsequent 1-hour blood glucose sample was obtained. From this work, it was determined that an upper limit blood glucose value of 140 mg\/dL would fail to detect approximately 10% of GDM cases. In contrast, lowering the value to 130 mg\/dL would increase by 15\u201325% the number of women requiring a 100-g oral glucose tolerance test (OGTT). However, if the 50 g glucose load is taken in the fasting state, the 140 mg\/dL upper limit detects approximately the same number of elevations as the 130 mg\/dL upper limit. When the result of the 50 g glucose is elevated, the next step is the 100-g, 3-hour OGTT.\n\nIn 1964, O'Sullivan and Mahan [5] introduced the first set of diagnostic criteria to predict future diabetes. As part of this series of studies, they found an increased rate of fetal macrosomia and stillbirth in pregnant women with elevated blood glucose values. A controlled trial in which 50% of the pregnant women were treated with insulin therapy demonstrated a lower rate of these complications [5]. These values were retrospectively applied to 1013 women who were tested in pregnancy and followed for 5\u201310 years to monitor the development of diabetes. These data were based on the predictive value of the pregnant woman developing diabetes later in life rather than a focus on the effect to the fetus [6].\n\nThe method of venous whole-blood glucose analysis employed during the time of O'Sullivan and Mahan was the Somogyi-Nelson technique [6]. This method of blood glucose analysis has been replaced by the glucose oxidase method, which utilizes venous plasma instead of whole blood. In 1979, the National Diabetes Data Group suggested that the O'Sullivan criteria should be adjusted downward by 15% to approximate the values in plasma to account for the higher glucose concentration [7]. In 1982, Coustan and Carpenter [6] proposed a new set of criteria developed using the glucose oxidase method of plasma glucose analysis.\n\nControversy continues over which diagnostic criteria are most clinically appropriate. As recently as the 1997 Fourth International Workshop\u2013Conference on Gestational Diabetes Mellitus, consensus on glucose testing criteria had not been reached. However, the American Diabetes Association recommends use of the Coustan\u2013Carpenter modified criteria. Most countries outside the United States use the World Health Organization criteria for the diagnosis of GDM, a 75-g 2-hour OGTT [6]. However, this test is not as well validated as the 100-g OGTT [8] and controversy remains [9]. Many clinical scientists believe that the establishment of a definition and method of detection for GDM will not be agreed on worldwide until a worldwide, multicenter trial is conducted.\n\nThe American Diabetes Association Clinical Practice Recommendations 2000 suggests that for high-risk women, a 100-g OGTT alone should be utilized to diagnose GDM [8]. Risk factors include marked obesity, GDM in a prior pregnancy, glucosuria, or a strong family history of diabetes. This \"one-step approach\" to diagnosing GDM is more cost effective in high-risk patients or populations. The one-step approach deletes the 50-g glucose screen and moves directly to the 100-g OGTT. The \"two-step approach\" is employed for women at average risk for GDM. The first step is the 50-g glucose screening test. If this test is elevated, the 100-g OGTT, the second step, is used to diagnose the presence of GDM (Table 1).\n\nTABLE 1\n\nScreening and Diagnosis of GDM\n\naDM if, symptoms of DM and random plasma glucose concentration \u2265200 mg\/dL.\n\n_Source:_ Adapted from American Diabetes Association (2000). Gestational diabetes mellitus. _Diabetes Care_ **23** (Suppl. 1), S77\u2013S79.\n\n## III. RISKS AND COMPLICATIONS\n\nThe risk of fetal complications parallels the rise in maternal blood glucose levels. If maternal blood glucose is maintained at normal levels, the risk of complications decreases. The most common complication associated with GDM is fetal macrosomia, weight of \u22654500 g (2 standard deviations above the normal for the reference population). Maintenance of maternal blood glucose levels close to the upper end of normal are necessary to prevent the overstimulation of fetal insulin, which can, in turn, cause deposition of fetal adipose tissue, most importantly in the trunk and shoulders. Macrosomia is associated with increased birth trauma (shoulder dystocia, clavicular fracture, brachial palsy) and higher rates of Cesarean section.\n\nIf maternal blood glucose is elevated during the latter stages of pregnancy, the newborn is at higher risk for hypoglycemia. With maternal hyperglycemia, the fetal beta cells are stimulated to produce excess insulin. If maternal glucose levels have been elevated, high fetal insulin is present at delivery. This is one of the causes of newborn hypoglycemia. Newborns of GDM mothers have their blood glucose tested a short time after delivery, and if the blood glucose is low, oral dextrose is administered. In some cases, the newborn is unable to maintain normal blood glucose and must receive dextrose intravenously. If blood glucose levels remain low, coma and brain damage can occur. Persson and Hanson [10] suggest that sustained hyperglycemia and hyperinsulinemia may also cause fetal acidemia and hypoxia _in utero._\n\nAnother possible consequence of fetal hyperinsulinemia is cardiac hypertrophy with impaired circulatory function and pulmonary maturation; this may cause respiratory distress. Respiratory distress syndrome is one of the leading causes of admission into the neonatal intensive care unit for babies of GDM mothers. Polyhydramnios (excess amniotic fluid), hyperbilirubinemia, polycythemia, and hypocalcemia are also related to GDM.\n\nAdditionally, congenital anomalies are generally associated with poor glucose control during organogenesis, which occurs in the first trimester of pregnancy. GDM develops in midpregnancy; therefore, blood glucose levels have been normal during organogenesis. Fetal anomalies are usually associated with pregnancy in women with type 1 or 2 diabetes.\n\n## IV. TREATMENT AND MONITORING\n\nNutrition therapy is the primary treatment for GDM. If the blood glucose levels cannot be normalized with nutrition therapy, insulin therapy is added to the treatment regime. Self-monitoring of blood glucose provides information to determine the success of the therapies and predict risks to the fetus.\n\n### A. Blood Glucose Monitoring\n\nSelf-monitoring of blood glucose allows women to apply self-management skills to control hyperglycemia and prevent hypoglycemia [11]. Time points most commonly tested are fasting and postprandial. Patients receive immediate feedback on the impact of food intake, exercise, or insulin on blood glucose levels, and health professionals are better able to individualize the treatment plan. Frequent (2\u20135 times daily) blood glucose monitoring provides adequate information to direct the treatment plan. Levels and frequency of elevated blood glucose values indicate fetal risk. Postprandial peak blood glucose values correlate better to fetal risk than preprandial glucose values, and also indicate the need for insulin therapy or insulin dose adjustment. In one study, a group of women in the postprandial monitoring group had lower rates of neonatal hypoglycemia, had less large-for-gestational-age infants, and lower rates of Cesarean section than the women in the preprandial testing group [12].\n\nSeveral reliable blood glucose meters are available. One feature that should be considered when selecting a blood glucose meter for pregnancy is the hemoglobin range. Pregnant women have lower hematocrit values, and some meters are not accurate with a hematocrit of \u2264 30%. Additional features to consider are a memory with time and date and data downloading capabilities. These features are helpful if the patient's record keeping is inconsistent, inaccurate, or disorganized. A percentage of patients chooses to falsify their data in an attempt to avoid insulin therapy, hide dietary indiscretions, please clinicians, or simply hide the fact that they did not test their blood glucose levels. A comparison of patient hand-written blood glucose records to values in the meter memory revealed that 25% of the values were omitted from the record and 28% of the values were added to the record [13]. The majority of clinical management decisions are based on the information provided by blood glucose testing; therefore, it is imperative that the information be reliable.\n\n### B. Nutrition Therapy\n\nIt is important to determine the desired clinical outcome before appropriate nutrition therapy can be prescribed. Clinical outcomes will guide the nutrition prescription for each specific patient, such as changes in nutrition therapy at follow-up appointments and the initiation of insulin therapy.\n\nThree clinical outcomes are generally agreed on: blood glucose values within target range before and after eating, adequate nutrient intake for pregnancy, and appropriate weight gain based on body mass index (BMI). An additional clinical outcome used by some practitioners is negative fasting urinary ketone levels. The first clinical outcome is specific to GDM and seeks to produce the blood glucose levels of a pregnant woman without GDM. The second and third outcomes are common to all pregnant women. The reason they are highlighted is that weight gain or energy and nutrient intake can be adversely affected in an effort to achieve normal blood glucose levels. If all three clinical outcomes cannot be achieved with nutrition therapy alone, then insulin is indicated (Table 2).\n\nTABLE 2\n\nClinical Outcomes for Gestational Diabetes\n\n### C. Self-Management or Behavioral Outcomes\n\nManagement of any type of diabetes, including GDM, requires the patient to learn self-management techniques. The two self-management tools that help the provider guide decision making are the blood glucose monitoring record and the food intake record.\n\nThe health care provider or dietitian needs to ensure that the patient can test her blood glucose level with a self-monitoring meter. This tool provides valuable information on the impact of specific foods and meals on blood glucose values. Blood glucose values are tested before breakfast (fasting) and after each meal. The after-meal test can be done at 1 or 2 hours postprandial. The blood glucose standard is different for each time point (Table 2). Patients should test according to a consistent time schedule.\n\nAnother self-monitoring tool that is essential to the management of GDM is the daily food record. The food record provides information to determine the energy intake; carbohydrate, protein, and fat intake; the frequency and timing of meals and snacks; and the nutritional adequacy for vitamins, minerals, and protein. Although a daily food record often provides an incomplete picture of a woman's actual intake, it is the only tool that allows the diet counselor to make specific nutrition recommendations.\n\nAs a group, pregnant women are usually motivated to do what is necessary to deliver a healthy baby and readily comply with nutrition and testing recommendations. A sample daily food record is shown in Table 3.\n\nTABLE 3\n\nSample Daily Food Record\n\n### D. Referral\n\nFor most women, the initial treatment for GDM is nutrition therapy. Therefore, a consultation with a dietitian is essential as soon as possible following the diagnosis. Unlike other types of diabetes, GDM is a short-lived condition lasting only until the baby is born. With this in mind, it is important to implement nutrition therapy quickly and make the nutrition prescription easy to follow.\n\n### E. Achieving Clinical Outcomes with Nutrition Therapy\n\nDietary carbohydrates are the main nutrient affecting blood glucose levels. Therefore, nutrition therapy is primarily a carbohydrate-controlled food plan. The goal of carbohydrate control is to achieve specific blood glucose values after eating. The problem to overcome is insulin resistance or a relative insulin deficiency. In the nonpregnant state, a woman is able to make adequate amounts of insulin to keep blood glucose in a normal range. As the pregnancy progresses, hormone levels associated with pregnancy increase, which increases insulin resistance and, thus, a need for additional insulin. Most pregnant women can compensate and maintain normal glucose levels. About 4% of pregnant women cannot compensate for this state of relative insulin inadequacy and develop gestational diabetes. By controlling the carbohydrate content of the diet, the insulin requirements are controlled and minimized. Carbohydrate control is achieved in the many ways (Table 4).\n\nTABLE 4\n\nDietary Carbohydrate Guidelines\n\nTotal carbohydrate intake | 40\u201345% of energy \n---|--- \nDistribution of carbohydrate | 3 meals and 2\u20134 snacks \nMorning carbohydrate | Restrict to about 30 g \nType of carbohydrate | Use blood glucose monitoring to identify \nSugars | Eliminate and\/or evaluate foods such as juice, desserts, or cereal with blood glucose monitoring \nConsistency of carbohydrate intake | Balance with insulin therapy and prevent carbohydrate undereating or overeating\n\n#### 1. TOTAL CARBOHYDRATE\n\nThe ideal amount of carbohydrate is unknown, but generally comprises about 40\u201345% of total daily calories, which is usually a reduction from the prepregnancy diet. Major and colleagues [14] determined that GDM women, following a diet less than 42% of energy from carbohydrate, had lower postprandial blood glucose values, reduced incidence of insulin treatment, and better perinatal outcomes as compared to GDM women following a diet 45\u201350% energy from carbohydrate. Typically for the pregnant woman, this reduction in carbohydrate is stated in per-meal terms rather than in per-day terms, such as limit dinner to 60 g carbohydrate, rather than limit carbohydrate to 42% of total energy.\n\nWhy not eliminate all carbohydrate foods entirely due to their blood glucose elevating effect? The human body prefers carbohydrate for energy needs. If the carbohydrate content of the diet dips too low, the body uses fat for energy and ketone production results. High levels of the ketone, beta-hydroxybutyrate, in the maternal blood have been correlated to lower scores on Stanford-Binet intelligence tests in children [15]. Carbohydrate-rich foods also provide essential nutrients, such as calcium from milk and milk products and vitamin C from fruit. Blood glucose control is not the only goal; adequate nutrition for the growing fetus is also a high priority.\n\n#### 2. DISTRIBUTION OF CARBOHYDRATE\n\nIn the meal plan, carbohydrate is distributed throughout the day, into 3 meals and 2\u20134 snacks. Smaller, more frequent feedings help decrease postprandial hyperglycemia and maintain the necessary energy intake. Some women find that the frequent meals and snacks help in the prevention of hunger, ketone production, heartburn, and nausea. Blood glucose testing guides the distribution of carbohydrate. For example, a high blood glucose level after lunch may indicate the need to decrease the planned lunch carbohydrate and compensate for the decrease in energy by adding a protein-rich food.\n\n#### 3. MORNING CARBOHYDRATE\n\nCarbohydrate is not as well tolerated at breakfast compared to other meals. During pregnancy, increased nocturnal levels of cortisol and growth hormone appear to contribute to morning glucose intolerance. Excess carbohydrate at breakfast may aggravate this situation and contribute to even higher blood glucose values. Some clinicians have suggested that the carbohydrate consumption at the breakfast meal be limited to 15\u201330 g [16, 17]. The food record and blood glucose record are used to evaluate postprandial control. If the woman desires more carbohydrate-rich foods and postprandial glucose goals can be maintained, more carbohydrate can be added to the diet. Continued blood glucose monitoring and vigilance with dietary recommendations are needed throughout pregnancy, because placental lactogen levels increase insulin resistance. Tolerance to carbohydrate can change as the pregnancy progresses. Protein-rich foods should be added to the breakfast meal in place of carbohydrate to satisfy hunger. Protein-rich foods also stimulate insulin secretion from the pancreas and, thus, do not contribute significantly to postprandial glucose elevations.\n\nContinued blood glucose monitoring is needed throughout pregnancy. Determination of the best distribution and type of carbohydrate-containing foods tolerated at the various times of day is achieved with the information provided by the food intake record and blood glucose monitoring. This information will also help prevent unnecessary food restrictions.\n\n#### 4. TYPE OF CARBOHYDRATE\n\nMany clinicians find that certain foods give a higher glycemic response, causing a higher elevation in postprandial blood glucose. Many studies in nonpregnant populations of people with diabetes have shown that the type and amount of carbohydrate in the diet influences blood glucose levels [18, 19]. Women with gestational diabetes often observe great variation in postprandial blood glucose values, even though the carbohydrate amount is consistent.\n\nClinical observation in women with GDM indicates that ready-to-eat cereal, fruit juices, and other highly refined products tend to result in a higher postprandial blood glucose level [20]. Some health care providers or dietitians recommend that their patients consume unrefined, whole-grain breads, old-fashioned oatmeal, nuts, legumes, and lentils because of their lower glycemic response. Clapp and colleagues [21] compared two isocaloric, high-carbohydrate diets on the whole-blood glucose and insulin response to mixed energy intake and exercise in healthy nonpregnant and pregnant women. They reported a difference following exercise and postprandial blood glucose response between low-glycemic and high-glycemic index diets. Thus, instead of decreasing the amount of carbohydrate, the type or form of the carbohydrate could be altered to improve postprandial glycemia. Instead of labeling foods as acceptable or unacceptable for GDM, the clinician and the patient with GDM can evaluate her glycemic response to particular foods.\n\na.: Simple Carbohydrate. Eliminating foods high in sucrose and other simple carbohydrates helps attain blood glucose goals. Elimination of these foods usually results in an overall reduction of carbohydrate intake. Sweet foods, such as soft drinks, lemonade, juices, candy, and desserts, are usually eliminated in the nutritional management of women with GDM to allow intake of carbohydrate-rich foods with essential nutrients.\n\nMany research studies in nonpregnant patients with type 1 and type 2 diabetes report that refined carbohydrates can be included in the diet without compromising glucose control [21a\u2013S21c]. In the GDM population these foods are eliminated because they provide no nutrient value, the glucose control is more stringent, a usual portion provides too much carbohydrate, and the food restriction is for a short period of time [22]. Many women with GDM will have a very difficult time completely avoiding all \"sweets.\" In some situations, small portions containing about 15 g of carbohydrate per serving may be consumed as long as glucose control is not compromised.\n\n#### 5. CONSISTENCY OF CARBOHYDRATE\n\nCarbohydrate consistency is encouraged, specifically to prevent undereating and to assist with postprandial blood glucose evaluation. If insulin therapy is added to nutrition therapy, it becomes essential to maintain carbohydrate consistency at meals and snacks to facilitate making insulin dose adjustments.\n\nRecommendations are usually made for a specific carbohydrate amount at meals and snacks, with emphasis on consistency. An example might be 30 g carbohydrate at breakfast and 45 g carbohydrate at lunch and dinner, with remaining carbohydrate, approximately 30 g each, at snacks. Some women are taught to adjust short or rapid-acting insulin based on their preprandial blood glucose level and the amount of carbohydrate they plan to eat at that meal [11].\n\n#### 6. PROTEIN\n\nProtein inclusion in meals and snacks does not significantly affect blood glucose excursions and, thus, can provide additional energy in place of carbohydrate. Blood glucose monitoring will determine if protein is helpful in controlling blood glucose for a particular individual. Generally, paring of carbohydrate and protein at meals and bedtime provides a better postprandial blood glucose due to the delayed absorption of the carbohydrate caused by the protein and fat. The Recommended Dietary Allowance (RDA) for protein in pregnancy is 60 g\/day [23]. The RDA for protein is easily met in meal plans for GDM, because protein generally comprises 20\u201325% of energy.\n\n#### 7. FAT\n\nIn the treatment of GDM, the percentage of energy from fat may be higher than generally recommended. The increase in fat is due to a decrease in carbohydrate intake and increased animal protein intake, which inherently adds fat. Fat usually ranges from 30% to 40% of total energy in GDM management. Because GDM is a short-term condition, the total amount of fat is not designed to be a long-term recommendation. Large amounts of fat beyond total energy needs should, however, be avoided to prevent excessive weight gain, which, in turn, can result in further insulin resistance.\n\nBecause GDM is a condition of insulin resistance, some scientists suggest that reducing fat intake may decrease insulin resistance and improve blood glucose levels. Preliminary research indicates that a higher polyunsaturated-to-saturated fat (P:S) ratio may protect against the development of GDM [24]. In one study, women with a higher saturated fat intake developed GDM independent of their weight status [24]. This has implications for women that are at high risk of developing GDM as well as women who have GDM. Further research is needed to evaluate this theory. Low-fat diets would need to be closely evaluated for use in GDM. Adequate fat intake is needed during pregnancy to provide essential fatty acids needed for fetal brain development [25].\n\n#### 8. ENERGY INTAKE AND WEIGHT GAIN\n\nEnergy intake should be sufficient to promote appropriate weight gain while preventing hyperglycemia and ketonuria or ketonemia. Energy recommendations are monitored by weight gain patterns, ketone testing, appetite assessment, and review of food records.\n\nNo agreement has been reached on a minimum energy requirement for women with GDM [26]. Total energy expenditure and basal metabolic rate do not differ between GDM and control pregnant women [27]. Individual needs for energy intake vary according to body size and physical activity. A minimum of approximately 1700\u20131800 kcal\/day of carefully chosen food choices has been shown to prevent ketosis [28]. Intakes below this level are generally not advised and often result in inadequate weight gain, weight loss, and\/or ketosis [15].\n\nThe specific level of energy intake should be individualized, based on assessment of prepregnancy weight, physical activity level, and pregnancy weight gain to date.\n\nMost clinicians prefer to base energy intake recommendations on a woman's actual intake rather than energy prediction formulas. However, if someone is not adept in assessing energy intake, lacks the time to make that assessment, or prefers to start with a standard calculation, the Harris-Benedict formula is recommended. Some clinicians use an adjusted, prepregnancy body weight for obese women (adjusted body weight = [(actual body weight \u2013 desirable body weight) \u00d7 0.25] + desirable body weight). Add 300 kcal for the second and third trimester of pregnancy [29].\n\nThe use of energy restriction or hypocaloric diets has been proposed as a method to improve blood glucose control in obese women. While some studies report a reduction in macrosomia, concerns with this treatment are for the safety of the fetus. Restrictive energy intake can lead to ketone production, which may harm the fetus and is to be avoided for all pregnant women [15]. Maggee and colleagues [30] showed that a 50% energy reduction (1200 kcal\/day) resulted in ketone production; however, a follow-up study, reducing energy by 33%, did not produce ketonuria [31].\n\n#### 9. ARTIFICIAL SWEETENERS\n\nNon-nutritive sweeteners available in the United States have been deemed safe for consumption during pregnancy [32]. Moderation is encouraged and defined on an individual basis. A maximum of 3 servings of non-nutritive sweetener per day is often considered reasonable. The primary concern is that high consumption of artificially sweetened foods replaces more nutritious choices.\n\nAspartame, made of the amino acids aspartic acid and phenylalanine, has shown to pose no risk to the fetus when ingested in amounts at least three times the accepted daily intake. Women with phenylketonuria must monitor their intake of all sources of phenylalanine. Phenylalanine does cross the placenta; however, foods sweetened with aspartame contain less phenylalanine than protein foods in a meal [33]. Similarly, acesulfame potassium also has been shown to pose no risk. Multigenerational studies show no effect on fertility, number of offspring, birth weight, or mortality [34].\n\nThere is no current evidence that saccharin is harmful to the fetus, but, because it crosses the placenta, the use of saccharin during pregnancy is not recommended [32].\n\n### F. Assessing Clinical Outcomes\n\nThe individual who provides nutrition therapy is often the person who recognizes when the limits of nutrition therapy have been met and insulin therapy is needed. Follow-up appointments are needed in order to determine if the patient is achieving clinical outcomes and using self-management techniques correctly. It is generally considered appropriate to schedule a follow-up appointment 1 week after the initial consult and plan to have at least a second follow-up appointment in 2 or 3 weeks [29].\n\nA variety of factors interfere with achieving clinical outcomes. Some factors relate to patient behaviors and knowledge level and others relate to hormonal and metabolic factors beyond patient control. At each follow-up visit, the following should be assessed: records of food intake, ketone and blood glucose monitoring; body weight change; patient's ability to understand and follow regimen; and patient questions and concerns. Changes to the nutrition plan or further education to improve implementation may be recommended. Some women will not be able to achieve blood glucose targets despite their best efforts. At this point, insulin therapy is needed to supplement nutrition therapy (see Table 5).\n\nTABLE 5\n\nAssessment of Clinical Outcomes and Solutions\n\n_Source:_ Adapted from American Dietetic Association (2001). Nutrition practice guidelines for gestational diabetes. www.eatright.org.\n\n### G. Insulin Therapy\n\nIf postprandial blood glucose levels are higher than the clinical goal at the same meal on two or more occasions during the week, insulin therapy is recommended to control the blood glucose at that particular meal. This decision is made regardless of the cause of the elevations. Despite the patient's best effort to follow the nutrition therapy, some women demonstrate an extreme sensitivity to excess or simple carbohydrate intake. Insulin is the drug of choice. Oral hypoglycemic medications used in the treatment of type 2 diabetes have not been tested in pregnancy and are not recommended during pregnancy.\n\nPostprandial blood glucose is best controlled with premeal rapid or short-acting insulin. The ratio of rapid or short-acting insulin to grams of carbohydrate intake will vary depending on the level of insulin resistance. Generally, more insulin is required at breakfast to cover carbohydrate intake than at lunch and dinner. For example, the rapid or short-acting insulin-to-carbohydrate ratio at breakfast may be 1 unit to 8 g carbohydrate, compared to 1 unit to 10 g carbohydrate at lunch and dinner. To avoid three to four injections daily, the prebreakfast injection may be a combination of rapid or short-acting and intermediate-acting insulins. This regime would eliminate a prelunch injection because the lunchtime carbohydrate would be controlled by the intermediate-acting insulin given in the morning [35]. The lunch meal carbohydrate may need to be limited to 45 or 60 g because a higher dose of prebreakfast intermediate-acting insulin may cause midmorning hypoglycemia. An estimate of total daily insulin dose can be calculated as follows: present body weight (kg) \u00d7 0.8 to 1.0 units (during the second and third trimester) = total daily insulin units (units\/kg body weight). These units are then divided between prebreakfast and predinner treatments. Two-thirds of the total daily insulin units are administered before breakfast, with a further division of two-thirds administered as intermediate-acting insulin and one-third as rapid or short-acting insulin. The predinner dose is one-third of the total daily insulin dose, with further division between one-half as intermediate-acting insulin and one-half as rapid or short-acting insulin [36].\n\nWhen fasting blood glucose levels are elevated predinner or bedtime, intermediate-acting insulin is needed. Diet and exercise have little effect on fasting glucose levels. There is general consensus that one should not delay insulin initiation, because glucose intolerance and insulin resistance only worsen as the pregnancy continues.\n\nWomen who require insulin therapy vary according to ethnicity, clinical goals, and testing regimens. A 1990 survey of obstetricians and maternal\u2013fetal medicine physicians, showed the following treatment pattern: forty percent treated \u2264 10% with insulin, 31% treated 20%, 23% treated 20\u201330%, 5% treated \u2265 50%, and 2% treated 100% of their patients with insulin [35]. In contrast, 20 years of national and international research effort have resulted in reports of 30\u201360% of GDM treated with insulin [35].\n\n## V. POSTPARTUM RECOMMENDATIONS AND CONSIDERATIONS\n\nWomen with a history of GDM have a 17\u201363% risk of developing type 2 diabetes 5\u201316 years after the first pregnancy with GDM. The risk of diabetes is particularly high in women who have marked hyperglycemia during or soon after pregnancy, women who are obese, and women whose gestational diabetes was diagnosed before 24 weeks of gestation [37]. These risk factors are markers that correspond to insulin resistance, diminished beta-cell function, and increased endogenous glucose production. Maternal postpartum testing with a 75-g OGTT should be administered at 6 weeks' postpartum in order to assess glycemic status. Repeat 75-g OGTT tests should be conducted at 3-year intervals. Impaired glucose tolerance or impaired fasting glucose are intermediate stages between normal glucose metabolism and diabetes. Women that appear to have impaired glucose tolerance or impaired fasting glucose should receive testing at more frequent intervals [8]. Efforts to prevent diabetes should be directed at maintenance of a normal body weight, regular physical exercise, and avoidance of drugs that increase insulin resistance, such as steroids and high-dose combination or progestin-only oral contraceptives.\n\n_In utero_ exposure to hyperglycemia may have far-reaching consequences. The offspring of women with GDM have a higher risk of obesity and abnormal glucose metabolism when they are children and young adults. Large-for-gestational-age offspring of GDM mothers have a higher BMI at 4\u20137 years when compared to average for gestational age offspring of GDM mothers [38]. A high maternal prepregnant BMI is a significant indicator for large-for-gestational-age infants [38]. Even though obesity usually resolves during the first year of life, it reappears by 14\u201317 years of age with a mean BMI of 24.6 kg\/m2. Excess fetal insulin production is a predictor for both obesity and impaired glucose tolerance in adolescence. Aberrant maternal metabolism as measured by hemoglobin A1c, fasting plasma glucose levels, and blood ketone or beta-hydroxybutyrate concentration is associated with poorer intellectual and psychomotor development in offspring [39]. The female offspring of GDM mothers have a higher risk of GDM during their pregnancies. Once again, measures to reduce insulin resistance and obesity will benefit the offspring as well as the parents in prevention of type 2 diabetes.\n\nThe method of infant feeding also seems to have an influence on later development of diabetes. The best source of these retrospective correlation studies comes from populations with high incidences of diabetes. The Pima Indians have a 50% rate of GDM. The offspring of GDM mothers had a lower incidence of type 2 diabetes if they were breast fed for at least 2 months and their birth weight was between 2500 and 3500 g. Birth weights of <2000 g or >4000 g are associated with higher incidence of diabetes as the offspring age (25\u201335 years old) [40].\n\n## VI. CONCLUSION\n\nIn summary, gestational diabetes mellitus is a type of diabetes first diagnosed during pregnancy. The primary treatment for GDM is nutrition therapy. Generally, the diet plan is carbohydrate controlled, with 40\u201345% of total energy from carbohydrate. Nutrition therapy provides all of the nutrient requirements for pregnancy as well as adequate energy to meet the weight gain goal based on prepregnant weight status. If blood glucose levels cannot be maintained at acceptable values, insulin therapy is implemented to supplement nutrition therapy. Insulin therapy is individualized based on the hyperglycemia patterns revealed with blood glucose monitoring.\n\nGenerally, GDM resolves immediately postpartum. However, a postpartum 75-g OGTT should be performed at 6\u20138 weeks' postpartum to determine the resolution of GDM. Women who develop GDM are at a high risk of developing type 2 diabetes due their genetic predisposition. GDM is a window into the woman's metabolic future. To reduce the risk or delay the onset of type 2 diabetes, women should be advised to maintain a normal weight, exercise regularly, and eat a diet low in saturated fats.\n\nReferences\n\n1. Metzger B.E. ADA Workshop-Conference Organizing Committee: Summary and recommendations of the Third International Workshop-Conference on Gestational Diabetes Mellitus. _Diabetes_. 1990;40(Suppl. 2):197\u2013201.\n\n2. King H. Epidemiology of glucose intolerance and gestational diabetes in women of childbearing age. _Diabetes Care_. 1998;21(Suppl. 2):B9\u2013B13.\n\n3. Jackson W.P.U. Studies in pre-diabetes. _Br. Med. J_. 1952;3:690\u2013696.\n\n4. Wilkerson H.L.C., Remein Q.R. Studies of abnormal carbohydrate metabolism in pregnancy. _Diabetes_. 1957;6:324\u2013329.\n\n5. O'Sullivan J.B., Mahan C.B. Criteria for the oral glucose tolerance in pregnancy. _Diabetes_. 1964;13:278\u2013285.\n\n6. Coustan D.R., Carpenter M.W. The diagnosis of gestational diabetes. _Diabetes Care_. 1998;21(Suppl. 2):B5\u2013B8.\n\n7. Ramus R.M., Kitzmiller J.L. Diagnosis and management of gestational diabetes. _Diabet. Mellit. Rev_. 1994;2(1):43\u201352.\n\n8. American Diabetes Association. Gestational diabetes mellitus. _Diabetes Care_. 2000;23(Suppl. 1):S77\u2013S79.\n\n9. Gabbe S.G. The gestational diabetes mellitus conferences. _Diabetes Care_. 1998;21(Suppl. 2):B1\u2013B4.\n\n10. Persson B., Hanson U. Neonatal morbidities in gestational diabetes mellitus. _Diabetes Care_. 1998;21(Suppl. 2):B79\u2013B84.\n\n11. Kitzmiller J.L. Sweet success with diabetes. The development of insulin therapy and glycemic control for pregnancy. _Diabetes Care_. 1993;16(Suppl. 3):107\u2013121.\n\n12. DeVeciana M., Major C., Morgan M., Asrat T., Toohey J., Lien J., Evans A. Postprandial versus preprandial blood glucose monitoring in women with gestational diabetes mellitus requiring insulin therapy. _N. Engl. J. Med_. 1995;333(19):1237\u20131283.\n\n13. Langer O., Mazze R. Diabetes in pregnancy: Evaluating self-monitoring performance and glycemic control with memory-based reflectance meters. _Am. J. Obstet. Gynecol_. 1986;155:635\u2013637.\n\n14. Major C., Henry J., DeVeciana M., Morgan M. The effects of carbohydrate restriction in patients with diet-controlled gestational diabetes. _Obstet. Gynecol_. 1998;91:600\u2013604.\n\n15. Rizzo T., Metzger B., Burns W., Burns K. Correlations between antepartum maternal metabolism and intelligence of offsprings. _N. Engl. J. Med_. 1991;316:911\u2013916.\n\n16. Fagen C., King J., Erick M. Nutrition management in women with gestational diabetes mellitus: A review by ADA's Diabetes Care and Education dietetic practice group. _J. Am. Diet. Assoc_. 1995;95:460\u2013467.\n\n17. Lerner R.A. Studies of glycemia and glucosuria in diabetics after breakfast meals of different composition. _Am. J. Clin. Nutr_. 1976;29:716\u2013725.\n\n18. Ahern J.A., Gatcomb P.M., Held N.A., Pettit W.A., Tamborlane W.V. Exaggerated hyperglycemia after a pizza meal in well-controlled diabetes. _Diabetes Care_. 1993;16:578\u2013580.\n\n19. Collier G.R., Wolever T.M.S., Wong G.S., Josse R.G. Prediction of glycemic response to mixed meals in non-insulin dependent diabetic subjects. _Am. J. Clin. Nutr_. 1988;3:85\u201388.\n\n20. Lock D.R., Bar-eyal A., Voet H., Madar Z. Glycemic indices of various foods given to pregnant diabetic subjects. _Obstet. Gynecol_. 1988;71:180\u2013183.\n\n21. Clapp J.F. Effect of dietary carbohydrate on the glucose and insulin response to mixed caloric intake and exercise in both caloric intake and exercise in both nonpregnant and pregnant women. _Diabetes Care_. 1998;21(Suppl. 2):B107\u2013B112.\n\n21a. Peterson D.B., Lambert J., Gerring S., Darling P., Carter R.D., Jelfs R., Mann J.I. Sucrose in the diet of diabetic patients\u2014just another carbohydrate? _Diabetologia_. 1986;29:216\u2013220.\n\n21b. Bantle J.P., Laine D.C., Castle G.W., Thomas J.W., Hoogwerf B.J., Goetz F.C. Postprandial glucose and insulin response to meals containing different carbohydrates in normal and diabetic subjects. _N. Engl. J. Med_. 1983;309:7\u201312.\n\n21c. Bantle J.P., Swanson J.E., Thomas W., Laine D.C. Metabolic effects of sucrose in type 2 diabetic subjects. _Diabetes Care_. 1993;16:1301\u20131305.\n\n22. Lyons L., Flanagan G. Do the new nutrition guidelines apply to pregnancy? _Perinatal Nutrition Report_. 1994.\n\n23. Food and Nutrition Board. Recommended Dietary Allowances. 10th ed. Washington, DC: National Academy of Sciences; 1989:33\u201334.\n\n24. Wang Y., Storlien L., Jenkins A., Tapsell L., Jin Y., Pan J., Shao Y., Calvert G., Moses R., Shi H., Zhu X. Dietary variables and glucose tolerance in pregnancy. _Diabetes Care_. 2000;23(4):460\u2013464.\n\n25. (Suppl.)Conner W.E. Importance of M-3 fatty acids in health and disease. _Am. J. Clin. Nutr_. 2000;71:171S\u2013175S.\n\n26. Metzger B., Coustan D.C., the Organizing Committee. Summary and recommendations of the Fourth International Workshop-Conference on Gestational Diabetes Mellitus. _Diabetes Care_. 1998;21(Suppl. 2):xxx.\n\n27. (Suppl.)Butte N. Carbohydrate and lipid metabolism in pregnancy: Normal compared with gestational diabetes mellitus. _Am. J. Clin. Nutr_. 2000;71:1256S\u20131261S.\n\n28. Jovanovic-Peterson L., Peterson C.M. Nutritional management of the obese gestational diabetic pregnant woman (editorial). _J. Am. College Nutr_. 1992;11(3):246\u2013250.\n\n29. American Dietetic Association. Nutrition practice guidelines for gestational diabetes. 2001. .\n\n30. Maggee M.S., Knopp R.H., Benedetti T.J. Metabolic effects of 1200 kcal diet in obese pregnant women with gestational diabetes. _Diabetes_. 1990;39:234\u2013240.\n\n31. Knopp R.H., Magee M.S., Raisys V., et al. Hypocaloric diets and ketogenesis in the management of obese gestational diabetic women. _J. Am. College Nutr_. 1991;10:649\u2013667.\n\n32. American Dietetic Association. Position of the American Dietetic Association: Use of nutritive and non-nutritive sweeteners. _J. Am. Diet. Assoc_. 1993;93(17):816\u2013821.\n\n33. Pitkin R.M. Aspartame ingestion during pregnancy. In: Stegink L.D., Filer J.F., eds. _Aspartame_. Washington, DC: Marcel Dekker; 1984:555\u2013563.\n\n34. No. 16World Health Organization Expert Committee on Food Additives. Toxicological Evaluation of Certain Food Additives and Food Contaminants. New York: World Health Organization; 1983:11\u201327. No. 18, World Health Organization Expert Committee on Food Additives. Toxicological Evaluation of Certain Food Additives and Food Contaminants. Geneva: World Health Organization; 1983:12\u201314.\n\n35. Langer O. Management of gestational diabetes. Pitkin R.M., Scott J.R., eds. _Clinical Obstetrics and Gynecology_. 2000;Vol. 43:106\u2013115\n\n36. Sweet Success Guidelines for Care. Geneva: California Diabetes and Pregnancy Program; 1998.\n\n37. Kjos S.L., Buchanan T.A. Gestational diabetes mellitus. _N. Engl. J. Med_. 1999;341(23):1749\u20131756.\n\n38. Vohr B.R., McGarvey S.T., Tucker R. Effects of maternal gestational diabetes on offspring adiposity at 4\u20137 years of age. _Diabetes Care_. 1999;22(8):1284\u20131291.\n\n39. Silverman B.L., Rizzo T.A., Cho N.H., Metzger B.E. Long term effects of the intrauterine environment. _Diabetes Care_. 1998;21(Suppl. 2):B142\u2013B149.\n\n40. Pettitt D.J., Knowler W.C. Long term effects of the intrauterine environment, birth weight, and breast-feeding in Pima Indians. _Diabetes Care_. 1998;21(Suppl. 2):B138\u2013B141.\nD.\n\nObesity\nCHAPTER 31\n\nObesity: Overview of Treatments and Interventions\n\nHELEN M. SEAGLE, HOLLY WYATT and JAMES O. HILL, University of Colorado Health Sciences Center, Denver, Colorado\n\n## I. INTRODUCTION\n\nObesity is a pervasive disease in our world: Currently an estimated 1.2 billion of the global population are overweight [1]. In the United States, more than half of the population is either overweight or obese, and this disease affects all age, sex, and race-ethnic groups [2]. The prevalence of overweight and obesity made a dramatic leap upward toward the end of the twentieth century; in 1960 only about one-third of the U.S. population was overweight [2]. Because excess weight is associated with a higher incidence of diseases such as diabetes, cardiovascular diseases, osteoarthritis, and some cancers [3], the burden of obesity is high. In the United States alone, the total economic cost of this disease is estimated to be about $117.73 billion per year (2000 dollars) with more than half of that cost being derived from direct medical care expenses [4, economic costs updated from 1995 costs reported in source]. Health care providers and policymakers alike are faced with the dilemma of managing this obesity epidemic and preventing this situation from worsening.\n\nObesity is a complex disease of multifactorial origins. However, there is simplicity in the underlying model of body weight change. The energy balance equation dictates that in order for body weight to change, there must be an energy imbalance. Either a change in energy intake or a change in energy output must occur so that body stores of energy are altered, causing a change in total body weight [5]. Therefore, treatments of obesity must either focus on diminishing energy intake (e.g., diet, medications, surgery) or increasing energy output (e.g., physical activity) or a combination of both (e.g., behavior modification addressing changes in dietary intake and physical activity). However, it is imperative that any intervention for overweight and obesity address both weight gain prevention and weight maintenance as well as body weight reduction [6].\n\nThe primary care physician is often the first person to assess an individual's need for obesity treatment. Ideally, the physician would then refer the client to a comprehensive weight management program or obesity specialist, or recommend a commercial weight loss program and provide follow-up care to monitor progress. If the primary care physician decides to treat the client, referrals should be made for additional services, e.g., psychologist, exercise physiologist, or registered dietitian [7]. Determining the appropriate treatment approach for an individual requires a careful assessment of an individual's needs [6]. An obese person with two or three comorbidities clearly requires a more aggressive approach than an overweight individual suffering no medical impact from excess body fat. The use of a tailored approach to obesity treatment will ensure that more people become successful in their weight management efforts. Obesity treatment is one arena where a one-size-fits-all approach does not ensure success.\n\n## II. ASSESSMENT OF OVERWEIGHT AND OBESITY\n\nObesity is characterized by the accumulation of excess body fat. Although accurate methods to measure body fat do exist, they are expensive and impractical for general use. Body weight has traditionally been used as a surrogate measure of excess body fat. In the past, an obese or overweight body weight was based on ideal body weight tables established by The Metropolitan Life Insurance Company [8]. These insurance tables estimated an \"ideal\" weight for a given height, frame size, and gender based on collected mortality data. Overweight and obesity were then defined as some percentage above the estimated ideal body weight. Although widely used, these tables were criticized for being derived from populations with body fat contents that did not reflect those of the general public, for using frame size (an arbitrary assessment), and for being based on mortality outcomes alone without evaluating morbidity data [6].\n\n### A. Clinical Assessments of Body Fat\n\n1. BODY MASS INDEX\n\nIn recent years, the use of a body mass index (BMI) has replaced the insurance tables and has become the recommended method to estimate body fat and to define both overweight and obesity in a clinical setting [1, 6]. BMI is determined by weight in kilograms divided by height in meters squared (BMI = kg\/m2). The formula: weight (pounds)\/height (inches)2 \u00d7 704.5 can be used to convert height in inches and weight in pounds directly into BMI units [9]. Figure 1 converts measures of height and weight into BMI units.\n\nFIGURE 1 Body mass index using height in feet and inches and weight in pounds.\n\nBMI is a better estimate of body fat than body weight [6, 10] and has advantages over the ideal body weight estimation that preceded it. Unlike the ideal body weight tables that were based on mortality data alone, BMI correlates with morbidity. The relationship between a given BMI and the risk of both mortality and morbidity has been assessed in several large epidemiological studies [11\u201313]. BMI does not require a subjective assessment for frame size and the same formula is used for both men and women (unlike the insurance tables, which were specific for gender). However, there are limitations to the usefulness of the BMI. BMI may overestimate total body fat in persons who are very muscular (such as elite athletes) and may underestimate body fat in persons who have lost muscle mass (such as the elderly). Additionally, BMI will inaccurately reflect body fat in edematous states or in individuals who are less than 5 feet tall. Clinical judgment must always be used in the interpretation of BMI on an individual case basis.\n\nDespite the above limitations, the correlation between BMI and excess body fat in the general population is good [14]. Both the National Institutes of Health and the World Health Organization have defined overweight as a BMI of 25.0\u201329.9 kg\/m2, and obesity as a BMI of 30 kg\/m2 or greater [1, 6]. These BMI cutoffs were determined using studies evaluating the relationship between BMI and mortality and morbidity risk [11, 12]. In general, morbidity and mortality risk increases as BMI rises, but this relationship is curvilinear. Increases in BMI between 20 and 25 kg\/m2 alter the morbidity and mortality risk less than increases in BMI above 25 kg\/m2. For example, in the Nurses' Health Trial, relative to a woman with a BMI < 21 kg\/m2, heart disease risk was 1.8 times greater in women with a BMI between 25 and 29 kg\/m2 but 3.3 times greater for women with a BMI greater than 29 kg\/m2 [11, 15]. Gender does not alter this relationship; therefore, the same cutoff points are used to define obesity (BMI \u2265 30 kg\/m2) and overweight (BMI 25\u201329.9 kg\/m2) in both men and women [16].\n\n#### 2. WAIST CIRCUMFERENCE\n\nThe waist circumference is an assessment tool that can complement the BMI measurement for assessment of disease risk. Excess fat located in the upper abdominal region (visceral fat) is associated with a greater risk than fat located in other areas [17]. Abdominal fatness is an independent risk factor (even when BMI is not increased) and is predictive of comorbidities and mortality [17, 18]. Sex-specific cutoffs for waist circumference can be used for adults with a BMI less than 35 kg\/m2. In individuals with a BMI above 35 kg\/m2, a waist circumference does not confer additional disease risk and therefore it is not necessary to measure waist circumference in patients with BMI > 35 kg\/m2.\n\nHigh risk is defined by a waist circumference > 40 inches (102 cm) for men and > 35 inches (88 cm) for women [6]. The power of waist circumference to predict disease risk may vary by ethnicity and age [14]. For example, waist circumference is a better indicator of disease risk than BMI in Asian-Americans and in older individuals. For this reason, waist circumference cutoffs may need to be adjusted, in the future, based on age and ethnicity.\n\n### B. Assessment of Risk Factors\n\nPatients should be assessed for the presence of concomitant cardiovascular risk factors or comorbidities. Some obesity-associated diseases and cardiovascular risk factors will place the patient in a very high risk category for morbidity and mortality and, therefore, the aggressiveness of the obesity intervention or treatment should be increased.\n\nIf a patient has three or more cardiovascular risk factors, they can be classified as being at a _high risk_ for obesity-related disorders. Cardiovascular risk factors include cigarette smoking, hypertension, a low-density lipoprotein cholesterol \u2265160 mg\/dL (4.1 mmol\/L), a high-density lipoprotein cholesterol <35 mg\/dL (0.9 mmol\/L), an impaired fasting glucose (fasting glucose of 110\u2013125 mg\/dL, or 6.1\u20136.9 mmol\/L), a family history of premature heart disease, and age \u2265 45 years for men and age \u2265 55 years or postmenopausal for women. The presence of these risk factors heightens the need for obesity treatment and concomitant lipid lowering therapy and blood pressure management when appropriate. Patients are considered to be at _very high risk_ if they have existing coronary heart disease, atherosclerotic diseases, type 2 diabetes mellitus, or sleep apnea. This would include patients that have had a myocardial infarction, angina pectoris, a history of heart surgery, or angioplasty. Table 1 summarizes additional risk factors that increase the need for weight reduction and place the individual in a very high risk or high-risk category [6].\n\nTABLE 1\n\nRisk Factors That Increase the Need for Weight Reduction\n\n### C. Assessment of Readiness\n\nAfter assessing the patient's need for weight reduction, the health care provider must assess the patient's readiness to participate in treatment. Even when a patient is seriously overweight, he or she may not be ready to make a commitment to weight reduction [7]. Providers need to determine if the patient recognizes the need for weight loss and is willing (and able) to sustain a weight loss effort. A series of questions developed by Brownell can be used to help assess a patient's readiness to accept and participate in a long-term treatment plan [19, 20].\n\n### D. Selecting Treatment Options\n\nMany options are available for treating overweight and obese individuals. For each patient, the risks of each treatment option must be weighed against the benefit of the potential weight loss produced by that treatment. This risk\/benefit assessment must take into account a patient's BMI, waist circumference, and the presence of comorbidities and cardiovascular risk factors. Patients at a higher BMI or with existing obesity-related diseases are at more risk from their excess weight and, therefore, more aggressive treatments such as pharmacotherapy and surgery become appropriate options. For each patient, there is a level of obesity where the risk of the treatment is outweighed by the benefit the patient would receive from a long-term reduction in weight. Each treatment plan must be tailored to meet the BMI and risk\/benefit assessment for each patient. Table 2 shows recommended treatment options based on BMI and the presence or absence of a serious health complication [21].\n\nTABLE 2\n\nSelecting Treatment Options Based on BMI and Comorbidities\n\n\\+ indicates appropriate treatment option; \u2212 indicates inappropriate treatment option.\n\naComorbidities include hypertension, sleep apnea, dyslipidemia, coronary heart disease, and type 2 diabetes.\n\nbPrevention of weight gain with diet, exercise, and behavioral therapy is indicated.\n\n_Source:_ Based on \"NIH Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults: The Evidence Report\" (1998). National Institutes of Health, Bethesda, MD. National Institutes of Health\n\n### E. Appropriate Goal Setting\n\nThe evidence-based NIH clinical guidelines for obesity treatment set the following general goals for weight loss and management: (1) to prevent further weight gain, (2) to reduce body weight, and (3) to maintain a lower body weight long term [6]. Traditionally, the goal of obesity treatment was to achieve an ideal body weight, and for many people this meant losing extremely large amounts of weight. However, a reduction to ideal body weight is not necessary for health improvement and risk reduction. Clinical studies indicate that moderate weight reduction (i.e., 5\u201310% of the initial body weight) can correct or ameliorate many of the metabolic abnormalities associated with obesity and that small weight losses are associated with improvements in hypertension, dyslipidemia, and type 2 diabetes mellitus [22\u201324]. Prescribing a weight loss goal of 5\u201310% sets a reasonable and achievable goal that may be more easily maintained. Unfortunately, many patients are not satisfied with weight reduction in this range and the provider must work closely with the patient to help set realistic expectations and provide guidance in this area.\n\n## III. LIFESTYLE MODIFICATION\n\nLifestyle modification, in particular, the modification of a person's diet and physical activity, is the cornerstone of most obesity treatment programs. For most people this treatment incurs no side effects, has minimal cost, and, if the lifestyle changes can be maintained, has great potential for long term effectiveness [25]. In addition to creating changes in diet and physical activity patterns, the lifestyle modification component of obesity interventions usually also includes some form of behavioral treatment to enhance the long-term effectiveness of the program.\n\n### A. Dietary Modification\n\nAt any one time, about 70% of U.S. adults are trying to lose or maintain their weight [26] and almost all report modifying their diet in some way to achieve their goals [26, 27]. Because the prevalence of overweight and obesity is also at an all-time high, this interest in dieting and attempts to modify diet appear to be contradictory. This contradiction underscores the difficulty people face in making seemingly simple changes to their dietary intake in an environment that encourages easy overconsumption of energy.\n\n#### 1. CREATING AN ENERGY DEFICIT\n\nThe universal component of dietary interventions for weight loss is a creation of an energy deficit [1, 6]. Most recommendations encourage a slow rate of weight loss through an energy deficit (energy output minus energy intake) of 500\u20131000 kcal\/day [1, 6]. This recommendation is based on the energy cost of burning 1 pound of excess body weight per week. Typically, the composition of the weight loss is about 25% fat-free mass and 75% fat mass. Metabolically, the more obese person can handle a greater energy deficit, as demonstrated by a lower protein oxidation rate during fasting, than a lean person [28]. It is important to monitor the rate of weight loss during the active weight loss phase. Initially, particularly at the greater energy deficits, diuresis may occur and weight will be dropped quickly. However, after this initial drop in weight, the rate of weight loss will slow and should not be greater than 1% body weight per week [29]. An energy deficit of 500\u20131000 kcal\/day should produce about a 10% body weight reduction over 6 months [6].\n\nMany healthcare providers prescribe a standard weight loss diet of a preset calorie level, e.g., 1200 or 1500 kcal\/day. This approach has the advantage of allowing the health care provider to give out preprinted diet plans already designed to achieve the calculated energy level. The disadvantages of this approach include inappropriately low-energy intakes (i.e., >1000 kcal\/day deficits) in very large individuals with high energy requirements, and diet plans that are not tailored to an individual's lifestyle. To avoid inappropriate energy deficits, it makes more sense to estimate a person's energy expenditure and subtract 500\u20131000 kcal\/day (the greater energy deficit should be reserved for the heavier individuals). Estimating a person's energy expenditure based on sex, body weight, and age [30] is preferable to using self-reported food intakes, which are notoriously unreliable, particularly among obese subjects [31, 32]. Alternatively, as a general rule, an appropriate energy level can be determined by assigning 12 kilocalories per pound of current body weight and then subtracting 500\u20131000 calories to create an energy deficit. The NIH Clinical Guidelines recommend 1000\u20131200 kcal\/day for women and 1200\u20131500 kcal\/day for men [6]. However, they emphasize the need for dietary education and tailoring the diet plan to accommodate individual food preferences.\n\nDecreasing one's food consumption without ensuring an intake of a variety of foods may compromise a person's nutrient intake, particularly of calcium, iron, and vitamin E. At energy intakes below 1200 kcal\/day, it is difficult to consume an adequate intake of essential vitamins and minerals. Multivitamin and mineral supplements are recommended for intakes below 1200 kcal\/day and for individuals whose food choices limit their ability to consume a satisfactory nutrient intake.\n\n#### 2. VERY LOW CALORIE\/ENERGY DIETS\n\nSome dietary regimens, such as very low-calorie diets (VLCDs), establish a greater than 1000 kcal\/day energy deficit. A VLCD is typically a liquid formulation that contains up to 800 kcal\/day (3350 kJ\/day) [33]. VLCDs are enriched in protein of high biologic value (0.8\u20131.5 g\/kg of ideal body weight per day), and are supplemented with essential vitamins, minerals, electrolytes, and fatty acids. A typical VLCD program lasts for 12\u201316 weeks and the liquid formula completely replaces all usual foods. A structured period of refeeding usually occurs after a VLCD, with solid foods slowly being reintroduced into the patient's diet.\n\nVLCDs are effective in producing a weight loss quickly. The mean weight loss for a 12- to 16-week program (including the long-term weight loss of dropouts) is about 20 kg [34]. Weight losses on VLCDs are greater for men than women [34], and heavier people lose more than lighter people [35]. It has been hypothesized that, beyond the greater than usual energy deficit, the form of these diets is an important factor in the effectiveness of a VLCD to produce a weight loss [34, 35]. That is, the structured feeding regimen of a VLCD encourages excellent adherence to the low-calorie plan; a patient does not have to make food choices and refrains from making impulsive high-calorie selections.\n\nAlthough effective at producing a quick weight loss, VLCDs have not proven to be effective for long-term weight loss maintenance. Now, many programs combine VLCD with behavior modification and this combination has slightly improved the long-term weight maintenance outcomes [36]. Other disadvantages of VLCDs include the expense of the programs and the side effects of the quick weight loss. The most serious side effects include hyperuricemia, gout, gallstones, and cardiac complications. These serious side effects underscore the need for (1) appropriate medical assessment for patients entering a program; (2) use of VLCDs in only obese patients (i.e., BMI > 30), especially those individuals with co-morbid conditions that would be responsive to weight loss, e.g., diabetes, sleep apnea, or presurgery; and (3) ongoing medical supervision during the course of the program [1, 33]. VLCDs are not recommended for use in obesity treatment by the NIH Clinical Guidelines [6].\n\n#### 3. MEAL REPLACEMENTS\n\nMeal replacement drinks are sometimes used in weight loss programs. These differ from VLCDs in that they are designed to replace only one or 2 of the day's meals rather than the whole day's intake, and they are sold over the counter. The advantage of a meal replacement drink is that it can replace a person's most problematic meal of the day with a drink of a known energy and macronutrient content, thus helping to achieve a targeted energy intake goal. Few studies have specifically evaluated the efficacy of meal replacement drinks for weight loss [37]. A recent 27-month study [38] showed a significantly greater weight loss in people following a low-calorie plan utilizing two meal replacement drinks per day versus people following a conventional low-calorie plan (11.3 \u00b1 6.8% vs. 5.9 \u00b1 5.0% of initial body weight, _p <_ 0.0001). Additionally, meal replacement drinks may be useful for weight maintenance or weight gain prevention. A study conducted in rural Wisconsin evaluated the efficacy of meal replacement drink treatment against the backdrop of weight gain experienced by matched controls over a 5-year period [39]. A total of 134 men and women participated in a self-management meal replacement drink program that included a 3-month active weight loss phase (two meal replacement drinks per day with weekly weigh-ins) and a maintenance phase (self-monitoring of weight and use of a meal replacement drink if weight gain occurred). During the same 5-year period, 86% of the meal replacement drink participants were _at least_ weight stable (<0.8 kg weight gain), while only 25% of the matched controls had prevented weight gain [39]. However, further research is required to verify the efficacy of this weight loss treatment.\n\n#### 4. MACRONUTRIENT COMPOSITION\n\nA negative energy balance is the most important factor affecting the amount, rate, and composition of weight loss. The macronutrient composition of the diet has no significant additive effect on these weight loss parameters during the course of a usual obesity treatment program [40]. However, it is apparent that the macronutrient composition of a weight loss diet may have an important effect on behavioral compliance with a dietary regimen.\n\nIn feeding experiments, it has been shown that people typically eat a constant daily weight of food regardless of the type of diet consumed 41]. Because fat contains more kilocalories per gram than either protein or carbohydrate (9 vs. 4 kcal\/g), more energy is likely to be consumed when high-fat versus low-fat foods are eaten [41]. Additionally, fat has been proposed to have a weak effect on both satiation (process controlling meal size) and satiety (process controlling subsequent hunger and eating) [42] (see [Chapter 35). \"Passive overconsumption\" is a term used to describe the likelihood of increased energy intake that occurs during high-fat feeding due to the combination of these two factors: high energy value of fat and its weak effect on both satiation and satiety [42]. For these reasons a low-fat diet (less than 30% energy from fat) is currently the typical recommended macronutrient guideline for weight loss [1, 6, 43]. Because diabetes and cardiovascular disease are frequent comorbidities of obesity, it is also recommended to modify the fat content of the diet to be low in saturated and _trans_ fatty acids, and higher in monounsaturated fats [43].\n\nManufacturers have responded to consumer demand for lower fat versions of foods, and the number of fat-modified foods has increased dramatically in the marketplace [44]. Some fat-modified foods are still high in calories or may be consumed in larger than usual portions and therefore may not promote an energy deficit in an individual trying to lose weight. Mistakenly, people have assumed that a low-fat, high-carbohydrate diet can promote weight loss regardless of energy intake. Although weight loss has occurred in studies that have evaluated the use of a low-fat diet with _ad libitum_ feeding [45], it is possible for a positive energy balance to occur. High-carbohydrate diets consumed in excess of energy requirements will promote fat storage despite higher carbohydrate oxidation rates [46].\n\nReplacing some of the fat in the diet with protein may be an effective way to promote adherence to a weight loss plan. Acute appetite studies indicate that, calorie for calorie, protein exerts a more powerful effect on satiety than either fat or carbohydrate and may increase adherence to a reduced-energy, low-fat diet [42]. In a recent study, increasing the protein content to 25% of total energy intake of an _ad libitum_ fat-reduced diet significantly improved weight loss over an _ad libitum_ fat-reduced diet with a protein content of 12% [47]. Studies like this must be repeated in other populations and under different experimental conditions before specific recommendations can be made for protein content of a weight loss diet.\n\nMore recently, energy density (energy\/food weight) of foods and diets has been evaluated as a potential dietary attribute that could be manipulated to influence energy intake [48]. However, the role that energy density plays in weight regulation is unclear, and currently there are no recommendations regarding the appropriate energy density of a weight loss diet.\n\n#### 5. NOVEL DIETS\n\nMany people become frustrated with their perceived inability to change their body weight in a timely manner and are enticed by quick and painless weight loss promised by popular diet programs and books. These diets tend to be very limiting in food choices (e.g., grapefruit diet), restrictive of at least one macronutrient (e.g., Atkins diet), or rely on novel food combinations (e.g., the zone diet). If a person is able to adhere to one of these diets (which are typically hypocaloric), then weight loss usually occurs, and in that respect these diets do work. However, despite the anecdotal evidence provided by these programs and books, there are currently no carefully controlled trials evaluating the efficacy of any of these diets for weight loss or for weight loss maintenance. The initial dramatic weight loss that occurs on many of these diets is often due to the diuresis that occurs as glycogen stores are depleted in response to a low-carbohydrate intake. This weight loss is temporary and if carbohydrate intake is increased, the glycogen stores are repleted and body weight increases accordingly. On very low carbohydrate diets, the body goes through a period of ketogenesis to provide fuel that can be utilized by the brain cells. Ketones are potent appetite depressants and lack of appetite may be one reason why individuals, at least temporarily, follow such restrictive diets.\n\nThe major criticism of these restrictive diets is that they promote rapid weight loss but do not address long-term weight maintenance. Because these diets tend to be very restrictive, many people may not be able to adhere to the regimen long term. Typically there is no education component on how to make appropriate healthful food choices after returning to a normal diet to promote long-term weight maintenance. In addition, these diets may be associated with the overconsumption of foods rich in saturated fat, a risk factor for the development of coronary artery disease.\n\n#### 6. THE NONDIET APPROACH\n\nRecently, there has been a movement toward replacing restrictive diet approaches and unrealistic weight goals with promotion of healthful food choices and size acceptance. Proponents cite two rationales for adopting this approach: (1) there is no long-term effective strategy for treating obesity and (2) pressure to be thin impairs the psychological and social well-being of both people who are overweight and those who are not [49]. The focus of the nondiet approach is to encourage people to improve their self-acceptance (self-image) regardless of their current weight and to adopt healthy practices to promote physical well-being, e.g., promoting fitness and healthful food choices. Very few studies have carefully evaluated the result of the nondiet approaches on weight loss or the reduction of comorbidities associated with obesity [50]. Repeated weight loss (also termed weight cycling) does not appear to be associated with psychopathology or changes in weight- and eating-related constructs [51]. However, it is apparent that more research is needed to conclusively determine the psychological effect of repeated episodes of dieting and weight loss.\n\n### B. Physical Activity Modification\n\nIn general, physical activity has been used as a key component of obesity treatment. However, studies looking at physical activity per se for weight loss have found only modest reductions in body weight using this strategy [52]. Weight losses in the range of 0.09\u20130.1 kg\/week have been reported when exercise is used alone compared to a no-treatment control group [52, 53]. Exceptions to this trend have been reported with extreme levels of exercise as is seen in military-type training. Combining exercise with dietary restriction produces only a slight increase in weight loss over dietary restriction alone [52]. In general, groups using diet restriction plus exercise lost more weight than the diet-alone condition, but the magnitude of the difference was not significant in most studies.\n\nA possible explanation for the relatively modest effect of physical activity on weight loss is that the energy cost of exercise is minimal compared to potential changes in energy intake. Subjects who exercise for 30 minutes 5 days a week may only burn 1000 kcal more per week depending on their size, fitness level, and intensity of exercise. In comparison, subjects may consume an extra 1000 kcal in one or two unplanned snacks and easily negate the energy expended in exercise for the entire week.\n\nAlthough its impact on weight loss may be minimal, physical activity appears to have a crucial role in the longterm maintenance of a weight loss (i.e., the prevention of weight regain). Many correlation studies show a strong association between self-reported exercise at follow-up and maintenance of a weight loss [52]. Studies using doubly labeled water suggest physical activity in the range of 11\u201312 kcal\/kg\/day may be necessary to prevent weight regain following a weight loss [54].\n\nData from the National Weight Control Registry (NWCR) also support the concept that high levels of physical activity are crucial in preventing weight regain following a weight loss. The NWCR is a registry of more than 2000 individuals who have maintained a minimum of a 30-pound weight loss for at least 1 year. These individuals report using a variety of methods to lose weight initially, but more than 90% report exercise as a key element in maintaining the loss long term. They report expending, on average, 2682 kcal\/week in physical activity 25]. This is approximately the equivalent of walking 4 miles 7 days a week, and many report much higher levels. This suggests that while physical activity may not have been essential for weight loss in these subjects, they believe it to be essential in prevention of weight regain. A complete review of the role of physical activity in obesity intervention appears in [Chapter 32.\n\n### C. Behavior Modification\n\nGroup behavioral programs, conducted on a weekly basis in university or hospital clinics, have been reported to produce average reductions of 8\u201310% of initial body weight over 16\u201326 weeks [7]. The key behavioral modification components utilized in obesity treatment include self-monitoring, stimulus control, and relapse prevention strategies [55]. Self-monitoring commonly includes the systematic recording of food intake, exercise activities, and\/or weight change. The available scientific literature on obesity suggests that consistent self-monitoring, particularly of food intake, is associated with improved treatment outcome [56, 57], even during high-risk periods such as holidays [58].\n\nUse of stimulus control involves the identification and modification of environmental cues associated with overeating and sedentary activity and is widely accepted as clinically effective [59].\n\nRelapse prevention strategies involve training patients to prepare for lapses in the weight loss process and to utilize coping strategies to prevent complete relapse of behavior change efforts [60].\n\nFinally, group-based behavior modification training is used in a variety of obesity treatment formats, including university-based programs, commercial weight management programs (e.g., Weight Watchers, Jenny Craig), and self-help programs (e.g., Take Off Pounds Sensibly, Overeaters Anonymous). Although preliminary research indicates that group-based interventions may be equally as effective as individual interventions for a variety of problem behaviors, there has been no systematic assessment to date on the group process specifically for obesity intervention [61]. As such, a variety of important questions including optimal number of group participants, frequency and length of meetings, critical group leader skills, and strategies to identify the optimal candidates for group-based interventions remain unanswered [61]. Most behavioral modification techniques utilized in a group setting can be effectively incorporated into weight loss counseling provided to an individual [7, 62].\n\n## IV. PHARMACEUTICAL INTERVENTION\n\nA. Background\n\nWhile diet, physical activity and behavior modification remain the cornerstone of obesity management, pharmaceutical intervention has become a legitimate therapeutic option for obesity treatment in the last 10 years. The increasing interest in pharmaceutical interventions has primarily been fueled by the realization that obesity in the United States has reached epidemic proportions with no sign of slowing down. Additionally, the old perception that obesity is caused by a lack of willpower or gluttony has been replaced with a better scientific understanding of the genetics and biology that predispose certain individuals to gain weight in our current environment [63].\n\nThe discovery of leptin in the mid-1990s established the existence of a genetically controlled complex biological system for food intake and weight regulation. The view of obesity has shifted away from that of a behavioral problem occurring in people lacking in self-control and discipline to that of a chronic disease model with genetic, biological, and behavioral roots. This critical shift in thinking also changed the view of drug therapy for obesity from that of a shortterm quick fix for a social problem to a long-term treatment for a chronic disease. It is now acknowledged that obesity, like other chronic diseases such as hypertension or diabetes, will require long-term treatment.\n\nWeight loss medications were previously viewed as unsuccessful because weight was always regained following the withdrawal of the drug. Now it is recognized that medications need to be given long term to be effective in obesity treatment [64]. A landmark long-term weight loss trial using the combination of phentermine and fenfluramine was published by Weintraub and coworkers in 1992 [65]. This trial showed these medications' efficacy in maintenance of a weight loss over 4 years. This long-term pharmaceutical treatment data, along with the view of obesity as a chronic disease, marked the acceptance of pharmacotherapy as legitimate intervention in obesity treatment.\n\nIn the mid-1990s, dexfenfluramine alone and fenfluramine in combination with phentermine were used for longterm treatment of obesity. In September 1997, reported concerns about serious unacceptable side effects, such as valvular heart lesions, led to the withdrawal of dexfenfluramine and fenfluramine from the market. This left no longterm FDA-approved medications for obesity treatment. Since that time, the FDA has approved two new medications for long-term use in the treatment of obesity. Neither of these drugs (to date) has been associated with heart valve lesions. All other weight loss medications approved by the FDA for weight loss are approved only for short-term use (Table 3). Because obesity is a chronic disease and must be managed for long periods of time, if not a lifetime, only medications approved for long-term use are of real interest in obesity treatment.\n\nTABLE 3\n\nPharmaceutical Interventions Used in Obesity Treatment\n\n_Key:_ po = by mouth; qd = once a day; tid = three times a day; SR = slow release capsule; NE = norepinephrine.\n\n### B. Medications Approved for Short-Term Use in Weight Loss\n\nThe FDA has approved several medications for short-term use (less than 3 months). These drugs include mazindol, phentermine, and diethylpropion. These are noradrenergic or sympathomimetic drugs that either stimulate release or block reuptake of norepinephrine. In general, these medications have produced more weight loss than placebo in most shortterm clinical trials, but the magnitude of the weight loss was variable [66, 67]. Large clinical trials evaluating these drugs for efficacy and safety in long-term obesity treatment are scarce and the FDA has not approved their use for longer than 3 months.\n\nPhenylpropanolamine (PPA), available over the counter, is classified by the FDA as \"possibly effective\" in weight loss treatment and has provisional approval for weight loss [66, 67]. PPA produces more weight loss than placebo in short-term studies [68, 69]. In the one controlled trial of PPA that lasted 20 weeks, at 6 weeks the PPA-treated group had lost 2.4 kg compared with a 1.1 kg loss in the placebo group. In the optional 14-week extension to the study the PPA group lost 5.1 kg (6.5%) and the placebo group lost 0.4 kg (0.5%) of initial body weight [67]. Safety is an issue, because PPA is an over-the-counter medication and may be associated with slight elevations in blood pressure. Maximum dosage for PPA should not exceed 75 mg\/day [64, 70].\n\n### C. Medications with No Approval for Use to Promote Weight Loss\n\nFluoxetine and ephedrine\/caffeine have been evaluated in several weight loss trials [23, 71, 72], but do not have FDA approval for weight loss. Patients taking fluoxetine in doses greater than 60 mg\/day have shown more weight loss than placebo in clinical trials. However, the long-term efficacy of fluoxetine is questionable, because weight regain, while _continuing_ the medication, was also demonstrated [23, 72].\n\nThe ephedrine\/caffeine combination has also been evaluated as a potential weight loss medication. The effects of ephedrine are amplified by caffeine and this combination is more efficacious than either compound alone in safe doses. Patients receiving the ephedrine\/caffeine combination (caffeine 600 mg\/day and ephedrine 60 mg\/day) showed significantly more weight loss than patients receiving placebo (16.6 vs. 13.2 kg at 24 weeks, _p <_ 0.01) [71]. Side effects of this combination include tremor and insomnia.\n\nEphedrine is derived from a natural ephedra herb (also known as _ma huang_ ), and many herbal preparations exploit the ephedrine\/caffeine combination. More than 800 reports of side effects, including arrhythmias, increases in blood pressure, and death, have been reported to the Food and Drug Administration (FDA) by consumers taking herbal weight loss medications containing the ephedra herb. It is unclear what dosages, combinations, and circumstances may have lead to or caused the reported side effects since the herbal or so-called \"natural\" preparations are not governed by the FDA. Studies are under way to determine whether _ma huang_ and other herbal products are safe and effective for weight loss, but currently there is no evidence to support their safety or use. The NHLBI Guidelines do not recommend the use of herbal preparations for weight loss at this time [6].\n\n### D. Medications Approved for Long-Term Use\n\n1. SIBUTRAMINE\n\nSibutramine was approved in 1997 for long-term treatment of obesity. Sibutramine is a combination serotonin and norepinephrine reuptake inhibitor. Unlike the fenfluramine\u2013phentermine combination, sibutramine does not stimulate the release of either of these neurotransmitters and this may be why valvular heart lesions have not been associated with the use of this drug [73]. Sibutramine's main mechanism of action is a reduction in food and energy intake. In laboratory animals, sibutramine also increases total metabolic rate by an increase in thermogenesis but this effect remains in question in humans and is small in magnitude if present [74\u201376].\n\nThe long-term efficacy of sibutramine has been evaluated in several 6- and 12-month double-blind randomized studies [77\u201379]. Sibutramine-treated patients lose significantly more weight, on average, than placebo-treated patients in these studies. For example, a greater than 10% weight loss was achieved by 30\u201339% of the sibutramine-treated patients (on 10 and 15 mg\/day, respectively) versus only 8% of the placebo group achieving this level of weight loss in a 12-month study [77].\n\nSide effects for sibutramine include headache, dry mouth, constipation, and insomnia. Increases in heart rate and blood pressure have been reported and, therefore, blood pressure should be monitored before and regularly after starting the medication. A few patients (<5%) may have a significant increase in blood pressure and, therefore, uncontrolled hypertension, a history of heart disease, heart failure, stroke, and arrhythmia are contraindications to the use of this drug. Sibutramine is also contraindicated with monoamine oxidase inhibitors and other serotonin uptake inhibitors, which include medications for depression and migraines. Sibutramine is available in 5-, 10-, and 15-mg tablets and is taken once a day. Weight loss is dose related with the 10- and 15-mg dose. Most patients should be started on the 10-mg dose and adjustments made as necessary.\n\n#### 2. ORLISTAT\n\nThe FDA approved the use of orlistat for the long-term treatment of obesity in 1999. Orlistat is a minimally absorbable agent (< 1%) that works in the gastrointestinal tract by blocking gastrointestinal lipases and reducing the subsequent absorption of ingested fat by approximately 30% [80]. Orlistat has been studied in 1- and 2-year clinical trials [81\u20133]. In general, the orlistat-treated groups lost more weight and had a higher percentage of subjects able to achieve a 10% weight loss than subjects in the placebo-treated group [84]. In a large study evaluating over 600 patients, 38.8% of the patients in the orlistat group achieved a 10% or greater weight loss compared to only 17.7% in the placebo group [85]. Orlistat's use for weight loss maintenance has been evaluated in 2-year studies. In these studies, a hypocaloric diet in combination with orlistat was used for weight loss in the first year, and then a eucaloric diet with orlistat or placebo was used in the second year to evaluate weight loss maintenance. In a large study by Davidson and coworkers [82], almost twice as many patients (34.1%) in the orlistat group were able to maintain a \u226510% weight loss for the 2-year period compared to the placebo group (17%) [82]. Similar results indicate that mean body weight regain is less with orlistat treatment than placebo (32.4 versus 56.0%; orlistat and placebo respectively) [81]. In these clinical trials, orlistat was used in combination with either a low-fat hypocaloric diet for weight loss or a eucaloric diet for weight maintenance, and a portion of the benefit seen in these trials must be attributable to that diet.\n\nThe optimal dosing of orlistat is 120-mg po tid with meals that contain fat. Higher doses do not have any additional efficacy in weight loss and may result in more adverse events [86]. Orlistat is minimally absorbed; therefore, any systemic adverse events would be expected to be negligible. Orlistat should be used with a diet that is less than 30% energy from fat to prevent adverse side effects that include oily stools, oily spotting, flatus with discharge, fecal urgency, and fecal incontinence [86]. These events are due to the drug inhibiting fat absorption rather than a direct effect of the drug itself. Patients should be advised to maintain a low-fat diet while using the medication, because these side effects increase with diets over 30% energy from fat. In general, these events tend to decrease over time.\n\nMean plasma levels of vitamins A, D, E, and \u03b2-carotene were monitored during the trials. In general, plasma levels of these vitamins decreased but remained in the reference range [85, 86]; however, in the United States a multivitamin supplement is recommended for patients prescribed orlistat and should be taken 2 hours before or after the dose of orlistat.\n\n### E. Risk\/Benefit Ratio\n\n1. SELECTING PATIENTS FOR PHARMACOTHERAPY\n\nAfter the degree of obesity has been assessed and the presence of comorbidities determined, the potential risks and benefits of a particular pharmacotherapy should be determined for each patient. The risk of the potential side effects of the medication must be carefully weighed against the benefits of the weight loss as a result of the treatment. The risk of not losing weight or even gaining weight must also be considered in the decision-making process. Additionally, many clinicians require that patients attempt weight loss in a structured program of diet, exercise, and behavioral modification before being considered for pharmacotherapy [7]. Severe obesity (BMI > 35 kg\/m2) carries a high risk of morbidity and mortality [87], and intensive therapeutic approaches are indicated for most individuals. The lower BMI threshold for pharmaceutical intervention is not as easily defined, and other factors such as a high waist circumference, recent weight gain, family history and the presence of co-morbidities become important considerations in the decision to treat with weight loss medications. Cosmetic weight loss attempts for patients who want to lose a few pounds are not appropriate. The risk of the medication in this case is not outweighed by the benefit since a reduction in weight at a lean weight (BMI \u2264 25 kg\/m2) is not associated with significant improvements in health risks. Currently, the evidence and the NHLBI guidelines justify the use of a weight loss medication in patients with BMIs \u2265 30 kg\/m2 or \u2265 27 kg\/m2 if comorbidities such as hypertension or diabetes are present [6].\n\n#### 2. PREDICTORS OF EFFICACY\n\nNot every patient responds to drug therapy. Therefore, it is important to monitor patients on weight loss medications, not only for potential side effects, but also for the efficacy of the medication itself. Clinical trials have shown that initial responders tend to continue to lose weight, whereas initial nonresponders continue to be nonresponders. The initial rate of weight loss has been frequently noted to predict subsequent weight loss. If the patient does not lose weight or maintain a previous weight loss with the medication, the medication should be discontinued. In this case, the risk of the medication is not outweighed by the weight loss, because there has not been a reduction in weight. As a general guideline, patients who do not lose 1% of body weight during their first month of treatment should discontinue treatment [74].\n\n### F. Medications Combined with Lifestyle Modification\n\nIt is important to emphasize that weight loss medications should be prescribed in combination with a diet, exercise, and behavioral modification program. Medications are not a substitute for, but an adjunct to, lifestyle intervention, providing additional benefit by helping patients adhere to the necessary diet and exercise changes. For some individuals, diet and exercise alone may be enough to produce a 10% weight loss and long-term maintenance. For others, medications may provide a necessary additional intervention to allow-weight loss success.\n\n## V. SURGICAL TREATMENT\n\nIn general, surgical intervention is considered the mosteffective treatment for weight loss and long-term weight maintenance. Surgery, with its inherent permanence, clearly has an advantage in long-term success [88]. It is reserved for patients with severe disease that have failed less invasive interventions and are at a very high risk for obesity-related morbidity and mortality. In the past, practitioners have used a rough guide of an excess weight of 100 pounds (45.5 kg) as an indication to consider a surgical intervention. In 1991, The National Institute of Health Consensus Development Conference on gastrointestinal surgery for severe obesity set the patient selection criterion for surgery as a BMI exceeding 40 kg\/m2 [89]. In certain instances, an obese patient with a BMI between 35 and 40 kg\/m2 may be considered for a surgical procedure if a severe comorbidity is present [89]. These life-threatening comorbidities commonly include sleep apnea, hypertension, diabetes, heart failure, and vertebral disc herniation [90]. There is no recommendation at this time for children or adolescents. As the procedures for obesity surgery improve, it is expected that the BMI criteria for surgical candidates will be reduced.\n\nSimilar to pharmacotherapy, the use of a surgical intervention in obesity requires a risk\/benefit analysis for each case. Those patients that have a low probability of success with nonsurgical interventions and who meet BMI criteria may be considered for surgery. Patients who have been determined to have acceptable operative risks should be well informed and motivated. They must understand how their lives may change after the operation and be able to participate in treatment and long-term follow-up. A surgeon experienced with the procedure (and working in a clinical setting with adequate support) should carry out the procedure.\n\n### A. Surgical Procedures\n\nTwo principal types of procedures, vertical banded gastroplasty and a Roux-en-Y gastric bypass, have become the current standard in weight loss surgery [88]. The vertical banded gastroplasty is a gastric resection procedure that consists of constructing a small pouch with a restricted outlet along the lesser curvature of the stomach. A Roux-en-Y gastric bypass procedure involves constructing a small gastric pouch whose outlet is a Y-shaped limb of small bowel. The procedure results in ingested food bypassing the majority of the stomach and varying lengths of the small intestine.\n\n#### 1. AMOUNT OF WEIGHT LOSS\n\nOf the two major types of surgical procedures, the vertical banded gastroplasty and the gastric bypass, the gastric bypass produces a slightly greater weight loss. This increase in weight loss has to be balanced against a higher risk of nutritional deficiencies associated with the Roux-en-Y gastric bypass procedure. On average, weight losses produced by these procedures result in a 20\u201335% reduction in initial weight or 40\u201350% of excess weight [88, 90, 91]. Inadequate weight loss is between 20% and 25% with gastric restriction procedures and 5% with a gastric bypass [88]. In both procedures weight loss occurs over a period of 18\u201324 months and weight loss is typically well maintained for as long as 14 years [91].\n\n#### 2. REDUCTION IN OBESITY-RELATED COMORBID CONDITIONS\n\nMost patients experience substantial improvements in obesity-related comorbidities such as sleep apnea, glycemic control, and hypertension [91]. Studies looking at quality of life also report improvements following the surgical procedure and weight loss [92, 93]. The Swedish Obesity Study, a large prospective randomized study, has provided encouraging interim data that weight reduction following surgery reduced the 2-year incidence of diabetes, hypertension, and other health risks by 3- to 32-fold [94].\n\n#### 3. COMPLICATIONS AND SIDE EFFECTS\n\nFor centers that specialize in obesity surgery, the immediate operative mortality for both the vertical banded gastroplasty and Roux-en-Y gastric bypass procedures are relatively low-and usually in the range of 0.5\u20131% [88, 90]. Morbidity in the early postoperative period may be as high as 10\u201320% [88]. These postoperative complications include wound infections, wound dehiscence, leaks from the staple line breakdown, stomal stenosis, marginal ulcers, pulmonary problems, and deep venous thrombosis. Other problems may arise in the later postoperative period. These include pouch and distal esophageal dilation, incisional hernias, strictures, persistent vomiting, cholecystitis, and diarrhea.\n\nIn the long-term, micronutrient deficiencies of vitamin B12, folate, and iron are common and must be treated. Nutritional deficiencies are more likely in the bypass versus the gastric restriction procedures. Patients must be willing to take supplements postoperatively and must be monitored closely. Nutritional deficiencies are of particular concern in women of childbearing years because nutrient deficiencies carry a high risk of fetal damage.\n\n\"Dumping syndrome,\" a side effect of a gastric bypass procedure, is characterized by tachycardia, palpitations, diaphoresis, and nausea. Ingestion of energy-dense high-carbohydrate food, which is rapidly emptied into the small intestine, causes the release of vasoactive gastrointestinal polypeptides that may lead to the syndrome. Up to 70% of patients undergoing a gastric bypass procedure will experience some degree of a \"dumping syndrome.\"\n\nVomiting and intolerance to solid food are the most common side effect of a gastric restrictive procedure. Most vomiting following a restrictive procedure is behavioral, but stricture and stenosis must be ruled out.\n\n## VI. SPECIAL ISSUES IN THE TREATMENT OF PEDIATRIC OBESITY\n\nData from national surveys indicate a significant increase in the prevalence of overweight and obesity in children and adolescents [95]. Obesity now affects one in five children and is considered to be the most prevalent pediatric nutrition disease in the United States [96]. Because obese children tend to become obese adults and face an increased risk of chronic diseases, e.g., diabetes and cardiovascular diseases, pediatric obesity is considered a major public health problem. However, even during childhood and adolescence, disorders such as hyperlipidemia, hypertension, and abnormal glucose tolerance occur with increased frequency in overweight and obese children [96]. In addition to the metabolic sequelae, significant negative psychosocial consequences of obesity are observed in this population [96].\n\nTreatment goals for pediatric obesity are different than those for adult obesity. Because children are still growing with increases in lean body mass, treatment focuses on preventing weight gain rather than the weight loss focus of adult treatment [1]. Additionally, any treatment aimed at regulating body weight and body fat must also provide adequate nutrition for the growth and development of the child. It is also important that the health care provider evaluate the physiological and psychological impact of an obesity treatment for children and adolescents. Pharmacological and surgical obesity treatments have not been well evaluated in the pediatric population and are currently not recommended interventions [1, 6]. Therefore, the only recommended treatments involve changes in dietary intake, increases in physical activity and the utilization of behavior modification techniques. For a more complete review of all pediatric obesity treatments the reader is referred to Epstein _et al._ [97]. A summary of the special issues of pediatric obesity interventions is presented below.\n\nAny dietary intervention to regulate a child's body weight must provide sufficient energy for growth and development. In particular, micronutrients such as iron and calcium must be adequately provided by the diet. Dietary advice and modification are most likely to be effective if there is parental involvement because parental attitudes, purchase, and presentation of food as well as modeling of eating behavior can impact a child's intake [1]. Some child-feeding practices can have negative (and unintended) effects on a child's food preferences and ability to control food intake [98]. For example, stringent parental control can increase a child's preference for high-energy-dense foods and limit a child's acceptance of a wide variety of foods [98]. Parental support involves being a positive role model for healthful feeding behavior as well as providing a wide array of healthful food choices in a supportive eating context [99]. Finally, because the potential for emergence of eating disorders is high in both children and adolescents, dietary modifications must occur in the context of the promotion of realistic body weight goals, positive self-esteem, and body image satisfaction.\n\nIt has been stated that our current environment facilitates underactivity through increased opportunity for sedentary activities [100]. This statement is just as true for children as it is for adults with sedentary activities (e.g., television viewing, video game playing) frequently taking prominence in children's lives. Physical activity interventions in the treatment of pediatric obesity often include decreasing these sedentary behaviors [101, 102]. Studies have shown this approach to be effective both in increasing physical activity and producing relative changes in BMI [101, 102]. In addition, because children are more likely to continue being active if they are able to choose their own activity, providing a choice of activities appears to be superior to providing a specific exercise prescription [101].\n\nThe fact that children routinely attend school, an environment that has continuous and intensive contact with children, creates a unique opportunity for pediatric obesity intervention. Schools have the necessary resources to promote physical activity (e.g., gym equipment, playing fields), they provide at least one meal a day where children can be exposed to healthful food choices, and many schools have access to school nurses or health clinics, which could potentially provide services to overweight children [103]. Obese children in treatment groups in school-based interventions have shown greater reductions in percentage overweight than untreated obese controls [1]. The effectiveness of school-based programs for the treatment of obesity has been modest but the results are encouraging and are worthy of more research.\n\n## VII. ACUTE WEIGHT LOSS VERSUS MAINTAINING LONG-TERM WEIGHT LOSS\n\nUntil recently, obesity was viewed as an acute disorder and so typical treatment was a single short-term intervention. However, weight loss can rarely be maintained for long periods, e.g., longer than 1 year, with only one short-term intervention. Therefore the real challenge of the obesity epidemic lies in the _prevention_ of weight regain, not in the accomplishment of weight loss itself.\n\nTo address the chronic nature of the disease, a treatment plan for obesity must be long term and focused on both achieving and maintaining a weight loss. It may be useful to view weight loss separately from weight loss maintenance, and to consider different strategies for each. Weight loss requires creating a negative energy balance (intake < expenditure) for a relatively short period of time while weight loss maintenance requires a patient to be in energy balance (intake = expenditure) for a very long period of time. In the weight maintenance period the patient can maintain a weight loss at any level of intake, but must match expenditure to that level for long-term success. Treatments that produce the largest deficit in intake will produce the most weight loss but these treatments may not be easy to maintain chronically. Similarly, the treatments that most effectively maintain a weight loss may not be very effective in producing weight loss.\n\nFor example, VLCDs are a well-established method for safely achieving a substantial weight loss but individuals treated with VLCDs frequently regain the majority of their weight loss. They are very successful at producing a weight loss but do not easily nor safely continue for very long periods of time. Regular physical activity, on the other hand, does not produce large amounts of weight loss as an acute intervention by itself, but it has proven to be a critical strategy in long-term weight loss maintenance [25, 54]. The best overall treatment for an obese individual may incorporate treatments for acute weight loss paired with a different treatment in the weight maintenance phase. Potentially, weight loss medications may have their greatest role in helping obese individuals maintain a weight loss achieved by different interventions (such as diet and exercise) that may be very difficult to maintain at the necessary level for long periods of time.\n\n## VIII. THE FUTURE OF WEIGHT MANAGEMENT\n\nPrevention of obesity is the true future of weight management. Preventing excess weight gain from ever occurring should be an easier task than treating obesity once it is present. However, there has been surprisingly little research into obesity prevention and, to date, prevention efforts have met with modest success at best. While obesity prevention can be targeted as a high priority, there is much to learn about how to do it effectively.\n\nThe prevention of obesity should not focus exclusively on the behavior of an individual. Prevention should address the larger environmental and societal factors that ultimately influence an individual's behavior. The current environment in the United States promotes weight gain and obesity by encouraging excessive food consumption and discouraging physical activity. We live in an environment that has an abundance of cheap, good-tasting, energy-dense foods but requires little physical exertion for day-to-day living. 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Astrup A., Hansen H.L., Lundsgaard C., Toubro S. Sibutramine and energy balance. _Int. J. Obes_. 1998;22(Suppl. 1):S30\u2013S35.\n\n75. Seagle H.M., Bessesen D.H., Hill J.O. Effects of sibutramine on resting metabolic rate and weight loss in overweight women. _Obes. Res_. 1998;6:115\u2013121.\n\n76. Hansen D.L., Toubro S., Macdonald I., Stock M.J., Astrup A. Thermogenic properties of sibutramine in humans. _Int. J. Obes_. 1997;22(Suppl. 2):102A.\n\n77. Jones S.P., Smith I.G., Kelly F., Gray J.A. Long-term weight loss with sibutramine. _Int. J. Obes_. 1995;19(Suppl. 2):41.\n\n78. Apfelbaum M., Vague P., Ziegler O., Hanotin C., Thomas F., Leutenegger E. Long-term maintenance of weight loss after a very-low calorie diet: A randomized blinded trial of the efficacy and tolerability of sibutramine. _Am. J. Med_. 1999;106:179\u2013184.\n\n79. Bray G.A. Health hazards of obesity. _Endocrinol. Metab. Clin. N. Am_. 1996;25:907\u2013919.\n\n80. Zhi J., Melia A.T., Funk C., Viger-Chougnet A., Hopfartner G., Lausecker B., Wang K., Fulton J.S., Gabriel L., Mulligan T.E. Metabolic profiles of minimally absorbed orlistat in obese\/overweight volunteers. _J. Clin. Pharmacol_. 1996;36:1006\u20131011.\n\n81. Hill J.O., Hauptman J., Anderson J.W., Fujioka K., O'Neil P.M., Smith D.K., Zavoral J.H., Aronne L.J. Orlistat, a lipase inhibitor, for weight maintenance after conventional dieting: A 1-y study. _Am. J. Clin. Nutr_. 1999;69:1108\u20131116.\n\n82. Davidson M.H., Hauptman J., Di Girolamo M., Foreyt J.P., Halsted C.H., Heber D., Heimburger D.C., Lucas C.P., Robbins D.C., Chung J., Heymsfield S.B. Weight control and risk factor reduction in obese subjects treated with orlistat: A randomized, controlled trial. _JAMA_. 1999;281:235\u2013242.\n\n83. (Suppl.)James W.P.T., Avenell A., Broom J., Whitehead J. A one-year trial to assess the value of orlistat in the management of obesity. _Int. J. Obes. Metab. Disord_. 1997;21:24\u201330.\n\n84. Hill J.O., Wyatt H.R. The efficacy of orlistat (xenical) in promoting weight loss and preventing weight regain. _Curr. Prac. Med_. 1999;2(11):228\u2013231.\n\n85. Sjostrom L.M., Rissanen A., Andersen T., Boldrin M., Golay A., Koppeschaar H.P., Krempf M., for the European Multi-Center Orlistat Study Group. Randomised placebo-controlled trial of orlistat for weight loss and prevention of weight regain in obese patients. _Lancet_. 1998;352:167\u2013172.\n\n86. Van Gaal L.F., Broom J.I., Enzi G., Toplak H., for the Orlistat Dose-Ranging Group. Efficacy and tolerability of orlistat in the treatment of obesity: A 6-month dose ranging study. _Eur. J. Clin. Pharmacol_. 1998;54:125\u2013132.\n\n87. Sjostrom L.V. Mortality of severely obese subjects. _Am. J. Clin. Nutr_. 1992;55:516S\u2013523S.\n\n88. Greenway F.L. Surgery for obesity. _Endocrinol. Metab. Clin. N. Am_. 1996;25(4):1005\u20131027.\n\n89. National Institutes of Health, Consensus Development Conference. Gastrointestinal surgery for severe obesity. _Am. J. Clin. Nutr_. 1992;55:487S\u2013619S.\n\n90. Kral J. Surgical treatment of obesity. Bray G.A., Bouchard C., James W.P., eds. _Handbook of Obesity_. Newtown, PA: Marcel Dekker; 1998:977\u2013993.\n\n91. Poiries W.J., Swanson M.S., MacDonald K.G., Long S.B., Morris P.G., Brown B.M., Barakat H.A., de Ramon R.A., Israel G., Dolezal J.M., Dohm L. Who would have thought it? An operation proves to be the most effective therapy for adult onset diabetes mellitus. _Ann. Surg_. 1995;222:339\u2013352.\n\n92. (Suppl.)Castelnuovo-Tedesco P. Psychiatric complications of surgery for superobesity: A review and reappraisal. _Clin. Nutr_. 1986;5:163\u2013166.\n\n93. Rand S.W., Macgregor M.C. Successful weight loss following obesity surgery and the perceived liability of morbid obesity. _Int. J. Obes_. 1991;15:577\u2013579.\n\n94. Sjostrom L. What does SOS teach us in 1998? _Int. J. Obes_. 1998;22(Suppl. 3):S93.\n\n95. Troiano R.P., Flegal K.M. Overweight children and adolescents: Description, epidemiology, and demographics. _Pediatrics_. 1998;101:497\u2013504.\n\n96. Dietz W.H. Health consequences of obesity in youth: Childhood predictors of adult disease. _Pediatrics_. 1998;101:518\u2013525.\n\n97. Epstein L.H., Myers M.D., Raynor H.A., Saelens B.E. Treatment of obesity. _Pediatrics_. 1998;101:554\u2013570.\n\n98. Birch L.L., Fisher J.O. Development of eating behaviors among children and adolescents. _Pediatrics_. 1998;101:539\u2013549.\n\n99. Satter E.M. Internal regulation and the evolution of normal growth as the basis for prevention of obesity in children. _J. Am. Diet. Assoc_. 1996;96:860\u2013864.\n\n100. Hill J.O., Peters J.C. Environmental contributions to the obesity epidemic. _Science_. 1998;280:1371\u20131374.\n\n101. Epstein L.H., Paluch R.A., Gordy C.C., Dorn J. Decreasing sedentary behaviors in treating pediatric obesity. _Arch. Pediatr. Adolesc. Med_. 2000;154:220\u2013226.\n\n102. Robinson T.N. Reducing children's television viewing to prevent obesity. _JAMA_. 1999;282:1561\u20131567.\n\n103. Story M. School-based approaches for preventing and treating obesity. _Int. J. Obes_. 1999;22:S43\u2013S51.\nCHAPTER 32\n\nObesity: Role of Physical Activity\n\nMARCIA L. STEFANICK, Stanford University, Palo Alto, California\n\n## I. INTRODUCTION\n\nThe prevalence of obesity, defined as having a body mass index (BMI) greater than or equal to 30 kg\/m2 [1], increased markedly in the United States between the second (1976\u20131980) and third (1988\u20131994) National Health and Nutrition Examination Surveys (NHANES II and III). Specifically, the percent of the U.S. population aged 20 years or older that is obese increased from 14.5% to 22.5% between surveys, while the prevalence of overweight (i.e., BMI of 25\u201329.9 kg\/m2 [1]), persisted at 32.0% across surveys, to total 54. 5% of U.S. adults who are overweight or obese [2]. This information prompted the NHLBI Obesity Education Initiative (OEI) Expert Panel to develop evidence-based \"Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults,\" which specifies three general goals of weight loss and management: to prevent further weight gain, to reduce body weight, and to maintain a lower body weight over the long term [3].\n\nThe guidelines include the recommendation that physical activity be \"part of a comprehensive weight loss therapy and weight control program, because it: modestly contributes to weight loss in overweight and obese adults, may decrease abdominal fat, increases cardiorespiratory fitness, and may help with maintenance of weight loss\" [3]. The exercise prescription is the same as that recommended by the Centers for Disease Control and Prevention and the American College of Sports Medicine (ACSM) in 1995 [4], then endorsed by the NIH Consensus Development Panel on Physical Activity and Cardiovascular Health [5] and incorporated into the Report of the Surgeon General [6]. The prescription is that moderate levels of physical activity for 30\u201345 minutes, 3\u20135 days a week should be encouraged initially, with a longterm goal of accumulating at least 30 minutes or more of moderate-intensity physical activity on most, and preferably all, days of the week [3].\n\nSubsequent to the release of the OEI guidelines, an ACSM roundtable was convened to review more carefully the literature pertaining to the role of physical activity in the prevention and treatment of obesity and its comorbidities, using a similar evidence-based approach [7]. Drawing from the wealth of information assembled for the development of the ACSM consensus statement [8], and other sources, the role of physical activity in adult obesity, as a population level characteristic and at the level of the individual, will be reviewed here. Although it is recognized that the prevalence of obesity in U.S. children and adolescents is increasing, the complex issues underlying the relationship of physical activity and body composition changes in children and prepubertal and postpubertal adolescents are beyond the scope of this review; therefore, the reader is referred to a recent review by Epstein and Goldfield on physical activity and childhood obesity [9]. Note that _obesity_ refers to excess body fatness, which is not always present with a high BMI, such as in bodybuilders; however, the percent of the population that is lean at a high BMI is relatively small and has only a small impact on population data.\n\n## II. RELATIONSHIP OF PHYSICAL ACTIVITY TO THE PREVALENCE OF OBESITY IN POPULATIONS\n\nDespite methodological issues that preclude accurate assessment of the relationship of physical activity to body weight and adiposity, an inverse association between physical activity or exercise and body weight has been reported in many cross-sectional epidemiologic studies [10]. In the 1996 U.S. Behavioral Risk Factor Surveillance System (BRFSS), the prevalence of inactivity was high in both normal weight (defined as BMI < 25 kg\/m2) and overweight men (26.8% and 25.6%, respectively) and women (27.8% and 31.7%, respectively), but was considerably higher in obese men (32.7%) and women (40.9%), while participation in recommended physical activity was low in both normal weight and overweight men (29.3% and 29.2%, respectively) and women (30.7% and 26.0%, respectively), but was substantially lower among obese men (23.5%) and women (18.9%) [11].\n\nThe observation has often been made that the level of physical activity is a better predictor of weight gain than estimates of energy or fat intake [8]. A decrease in physical activity has been inferred in several studies that have reported secular decreases in energy intake concurrent with increases in weight and\/or fatness; and in one study that had both energy intake and physical activity data, an analysis of secular trends in obesity were accompanied by changes in transport to work, work-related activity, and\/or changes in leisure-time activity [12]. Several large-scale observational studies have also justified a relationship between higher physical activity at baseline or improvements in physical activity and either attenuated weight gain or a lower odds of significant weight gain [13]. An inverse relationship between physical activity and weight change was reported in a 4- to 7-year (median 5.7-year) study involving more than 12,500 Finnish adults, aged 25\u201364 years [14] and across 4 years in about 10,000 U.S. male health professionals aged 45\u201364 years [15], as well as over a 2-year period in approximately 3500 men and women in Minnesota, of a mean age of 38 years [16], and in nearly 2000 Chinese women, aged 20\u201345 [17]. In addition, prospective data from the Aerobics Center Longitudinal Study (ACLS) [18] and the biracial CARDIA cohort [19] showed that improved cardiorespiratory fitness over 7.5 and 7 years, respectively, relates inversely to weight change.\n\nThe longitudinal evidence suggests that habitual physical activity plays a greater role in attenuating age-related weight gain, rather than in promoting weight loss [13]. Recent data from a cross-sectional analysis from the National Runner's Health Study suggest that substantial increases in activity level are necessary to maintain body weight with age [20], findings that have been corroborated by the Male Health Professional Follow-up [15] and ACLS [18] cohort data. Results from more than 9000 adults responding to the NHANES I Epidemiologic Follow-up Study (1971\u20131975 to 1982\u20131984) suggest that even relative to people who stay very active over time, sedentary people who increase activity can minimize the risk of major weight gain compared to people who decrease their activity, thus supporting the notion that increasing physical activity over time reduces the risk of becoming overweight [21].\n\n## III. PHYSICAL ACTIVITY AND DETERMINANTS AND ETIOLOGY OF OBESITY\n\nIt is generally accepted that the development of obesity in any given individual requires that energy intake exceed energy expenditure over a period of time. To prevent weight gain, one must balance energy intake and energy expenditure long term. To lose weight, one must expend more energy than is consumed. Energy expenditure is composed of the resting metabolic rate (RMR), a measurement of the energy expended to maintain normal body functions and homeostasis at rest, in the postabsorptive state; the thermic effect of food (TEF), which includes the energy costs of food absorption, metabolism, and storage of substrates; and physical activity, which includes the energy expended above RMR and TEF, for both voluntary exercise and involuntary activity such as shivering, fidgeting, and postural control. In general, RMR accounts for about 60\u201375% of daily energy expenditure, TEF for 5\u201310%, and physical activity for 15% in sedentary individuals and up to 30% or more in highly active individuals [22].\n\nThe amount of fat-free mass (FFM) in the body is the primary determinant of RMR, which is also influenced by age, gender, and genetics [22]. There is no indication that RMR has declined during the past few decades [23], so it is unlikely that RMR is a major factor in the increased prevalence of obesity in the population; however, small perturbations in RMR can substantially impact the regulation of body weight in a given individual. RMR decreases as adults age, about 2\u20133% per decade, primarily due to loss of FFM [22]; thereby, slowly shifting the energy balance in favor of fat weight gain if energy intake and physical activity are held constant. In older individuals, aerobic exercise does not appear to increase FFM; however, it may provide an adequate stimulus to maintain FFM with age [24]. In contrast, resistance exercise increased FFM (1.1\u20132.1 kg) in 15 out of 29 studies in older adults [24], while also reducing fat mass similar to changes induced by aerobic exercise (resistance exercise: \u22121.7 \u00b1 0.4; aerobic exercise: \u22121.9 \u00b1 0.8 kg).\n\nA modest suppression of RMR is also generally seen in response to energy restriction (i.e., dieting), ranging between 5% and 25%, with an average maximum change of about 15% [25], largely due to a decrease in FFM, which often accompanies diet-induced weight loss. Several studies have reported that aerobic exercise and strength training may help preserve metabolically active, lean body mass during dieting, and prevent diet-induced reductions in RMR [6, 25, 26].\n\nControversy persists over whether the thermic effect of food ingestion increases if physical activity follows a meal and whether physical activity before a meal reduces appetite [6, 27]. The evidence suggests that physical activity programs do not necessarily produce a compensatory increase in food intake in obese individuals [6] and, overall, the literature points to a rather weak coupling between energy intake and physical activity-induced energy expenditure, suggesting that increasing energy expenditure through physical activity (or maintaining an active lifestyle) can cause weight loss or prevent weight gain [27].\n\nPhysical activity includes activities of daily living, work-related physical activity, and leisure-time physical activity. The first two have declined considerably during the past several decades and may be primary factors contributing to the increased prevalence of obesity [23]. In contrast, leisure-time physical activity has not decreased appreciably, but is very low, as the 1996 BRFSS data verify [11]. Adopting the exercise prescription presented in the OEI guidelines [3] would result in an energy expenditure of approximately 1000 kcal (4184 kJ) per week; however, the amount of physical activity that protects against obesity has not been established and it is unclear whether this prescription is adequate.\n\nFat weight loss and preservation of lean body mass should be the goals of a weight loss program. Fat weight loss may be slow, relative to weight loss strategies aimed at total weight, in which loss of water and stored carbohydrate may occur quickly. Fat, the most abundant energy reserve, is a very efficient fuel source. It has been estimated that 1 kg of body fat supplies enough energy to support about 100 km of walking or jogging, or comparable activities. Nonetheless, as little as a 2.5-km walk or jog (a 20- to 30-minute effort) five times a week, which conforms to the exercise prescription recommended in the OEI guidelines, would bring about a loss of 6.5 kg of fat over the course of a year, assuming one does not consume excess energy or expend less energy in the remaining 23.5 hours of the day. If fat loss were to be achieved solely through the direct energy cost associated with increased physical exercise, a person must be committed to expending the energy over a reasonable period of time, with respect to his or her weight loss goal.\n\nConsiderable research has been conducted during the past several decades on adaptations of muscle and fat tissue to exercise training; body composition changes associated with exercise-induced weight loss; differences between lipolytic responses of subcutaneous and intra-abdominal (visceral) fat in response to norepinephrine, which may influence differential mobilization of fat stores during exercise; and interactions between exercise and diet (both energy intake and relative contributions from fat, carbohydrate, and protein) during weight loss [26]. A clear physiological rationale supports the notion that exercise could bring about loss of excess body fat and\/or serve to maintain weight loss and\/or prevent accumulation of excess adipose tissue over time. Metabolic rate during exercise can increase up to 20 times above resting levels; therefore, it seems obvious that if a person were to engage in exercise regularly without increasing energy intake appreciably, weight loss should occur.\n\nIt is well known, of course, that within any given environment, there is considerable variation in body fatness among people who are in energy balance, due to genetic factors, which have been estimated to contribute anywhere from 25% to 70% of the variability in adiposity [28\u201331]; however, because the U.S. genotype has not changed substantially enough during the past few decades to explain the increased prevalence of obesity, it seems that a focus on increasing physical activity remains appropriate. A recent study of nearly 1000 female twins showed that current physical activity was a stronger predictor of total-body and central adiposity of healthy middle-aged women than dietary intake or smoking; furthermore, participants with a predisposition to adiposity did not show a lesser effect of physical activity on body mass [32].\n\n## IV. ROLE OF PHYSICAL ACTIVITY IN TREATMENT (WEIGHT LOSS) OF OVERWEIGHT AND OBESITY: EVIDENCE FROM RANDOMIZED CONTROLLED TRIALS\n\nBoth the OEI Panel [3] and the consensus committee assembled for the ACSM Roundtable [7, 8] considered data arising from a substantial number of randomized controlled trials, involving a substantial number of participants, as providing the highest quality evidence. After reviewing the literature, four sets of tables were developed for this review, each of which presents the basic study design (randomized intervention groups); the number of subjects (N) completing the trial versus, in parentheses, the number randomized; key subject recruitment criteria; length of training period; initial obesity status; and weight changes. Studies were included if they were of at least 3 months' duration, had at least 20 participants completing the trial, and enabled assessment of the role of the intervention on body weight.\n\nThe first two tables present randomized controlled trials of aerobic exercise versus control and trials of aerobic exercise plus diet versus diet only. Although an attempt was made to identify as many studies that met the basic criteria as possible, it is likely that many small studies that would meet these criteria were not identified; however, the reader should take others that they identify into account in relationship to those that appear here. In these tables, the studies are separated by BMI categories for normal weight, overweight, and obese, using initial mean BMI. Earlier trials often reported baseline weight, without height, and did not report BMI, thereby making it difficult to determine the initial obesity status of the participants, especially when data are not separated by gender. For the purpose of categorizing these studies, an average height of 178 cm was assumed for men and 166 cm for women. It is important to note that many studies reported significant weight changes within groups, that is, baseline versus post-treatment weights, without specifying whether there were significant differences between groups, thereby making poor use of the control group. Furthermore, in studies with more than two treatment groups, pairwise comparisons are often made without an initial analysis of variance for all randomized participants; therefore, the significance of differences between the aerobic exercise only group versus control is not always clear.\n\nDetails regarding the exercise prescriptions for the majority of the trials presented in these tables were provided in a recent review that appeared in the American Heart Association monograph on \"Obesity: Impact on Cardiovascular Disease\" [33]. For the most part, these prescriptions conform to the OEI guidelines [3]; however, adherence to the prescriptions is generally not reported, so it is not clear what percent of participants randomized to the aerobic exercise or resistance training programs were achieving the recommended levels of activity. Although it is assumed that most of the randomized controlled trials published \"intention-to-treat\" analyses on all who completed the trial, this is often not clarified.\n\n### A. Randomized Controlled Trials of Aerobic Exercise Only\n\nTable 1 presents the randomized controlled trials that included an aerobic exercise only and a control group. Noting the caveats discussed above, regarding some uncertainties in assuming significant differences between groups, one [34] of two [34, 35] trials of normal weight men, two [36, 37] of five [36\u201340] trials of overweight men, and one [41] of two [41, 42] trials of obese men reported significant, albeit modest, weight reduction with aerobic exercise versus control. It is worth noting that the study design of the second trial of obese men [42] required that exercisers not lose weight, as discussed below. The one trial that included aerobic exercise only in premenopausal women [43] was in normal weight women who were assigned to control or one of three exercise programs, only one of which was reported to reduce weight versus control. This trial is discussed below. Neither of the large trials of overweight, postmenopausal women [38, 40] reported weight loss by exercise only, despite significant increases in VO2max; however, a small trial of borderline obese postmenopausal women reported a modest weight reduction in women assigned to walking versus control [44]. Two similarly sized studies that mixed obese men and women with type 2 diabetes found no effect of exercise on weight [45, 46].\n\nTABLE 1\n\nRandomized Trials with Aerobic Exercise Only versus Control Groups\n\naN post = number of subjects completing the trial; in parentheses, number of randomized subjects.\n\nbNS = not significant.\n\nThe two trials of normal weight men [34, 35] differed primarily in their length. The Stanford Exercise Study [34] involved a 9- to 12-month intervention, compared to only 4 months for the Finnish study [35], but also, the Finnish men were assigned to individualized training programs, albeit under the supervision of an exercise physiologist at least once a month. The Stanford study provided supervised exercise sessions, with choices as to exercise volume and intensity, three times a week. Another difference was that the Stanford men were provided no dietary instruction, whereas the both exercisers and controls in the Finnish study were instructed to reduce dietary saturated fat intake, simple carbohydrate-rich foods, and alcohol, but not encouraged to lose weight; a small, but significant reduction in mean body weight occurred within each group, with no differences seen between groups. In the Stanford study, secondary analyses [34], which separated exercisers into four \"treatment-dose\" groups (based on weekly jogging\/walking distance: 0\u20136.2, 6.3\u201312.6, 12.7\u201320.6, and 20.8% km), showed that distance correlated significantly with body fat changes (r = \u22120.49; _p =_ 0.002).\n\nIn one of the trials of overweight men who lost weight with aerobic exercise versus control [36], the exercisers were advised to increase exercise gradually until they could do regular aerobic type exercise (e.g., walking, jogging) 2\u20133 times a week at an intensity of 60\u201380% of maximal heart rate and lasting 30\u201345 minutes. In the second, smaller trial of overweight men who lost weight with exercise versus control [37], exercisers selected their own aerobic exercise regimen, with a minimum participation set at three sessions of 30 minutes per week at 65\u201375% of maximum heart rate. Of the 21 exercisers, 11 walked, 4 jogged (2 alternated jogging with swimming), 3 attended a gymnasium (45 minutes of aerobic workout, 15 minutes resistance, anaerobic) and 3 rode an exercise bike. Dual-energy X-ray absorptiometry (DXA) scans revealed that more than 80% of weight loss by exercisers was fat; whereas 40% of weight lost in a third, diet only group, was lean tissue.\n\nIn one of the three trials of overweight men that reported no significant weight loss with exercise [38], participants were assigned to control or one of three different exercise programs: group-based, higher intensity (73\u201388% peak heart rate); home-based, higher intensity; home-based, lower intensity (60\u201373%). Those assigned to group-based, higher intensity were encouraged to attend an exercise program consisting primarily of walking and jogging 3 times a week during which they were expected to have at least 40 minutes of higher intensity exercise. Participants assigned to home-based exercise were expected to do either higher intensity exercise 3 times per week or lower intensity exercise 5 times per week, depending on assignment, with the goal of achieving an equal volume of exercise in all groups. All three exercise conditions improved VO2max ( _p <_ 0.03), by approximately a 5% increase, compared to controls.\n\nThe Oslo Diet and Exercise Study (ODES) investigated exercise and diet effects on blood pressure in normotensives and mild hypertensives [39], and BMI changes were reported by diastolic blood pressure tertiles (<84, 84\u201391, >91 mm Hg) for each treatment group, rather than by treatment assignment, requiring some manipulation of the data to estimate an overall mean BMI change for each treatment group. Statistical comparisons were inferred from those reported for each treatment group within blood pressure tertiles. Participants were offered a supervised program of 1 hour 3 times per week, with a focus on endurance-type exercise at an intensity of 60\u201380% of peak heart rate, such as aerobics, circuit training, and fast walking\/jogging.\n\nThe Diet and Exercise for Elevated Risk (DEER) trial [40] involved randomization to an aerobic exercise consisting of 45 minutes of walking, jogging, or comparable activity, at 60\u201380% of maximum heart rate, at least 3 times per week, which resulted in a significant increase in aerobic capacity of 2.7 mL\/kg\/min. Closer scrutiny of the energy intake data, determined through five unannounced 24-hour dietary recall telephone interviews, revealed increases in the exercise only men (on the order of about 100 kcal per day) compared to other groups, and although these were not significant, this would certainly counteract the 700\u2013900 kcal\/week energy expenditure achieved by most exercisers. Lean mass loss, assessed by hydrostatic weighing, was significantly greater in DEER men assigned to a low-fat diet, with or without the addition of exercise (1.3 kg for both), compared to exercise only men.\n\nSpecific exercise-related weight goals were integral to the study design in the two major trials of borderline obese men in Table 1. The exercise prescription for the first Stanford Weight Control Project, SWCP-I [41], consisted of approximately 45 minutes of supervised walking or jogging 3 times per week and was integrated into a goal of reducing body fat, rather than total weight, by one-third over the 9- to 12-month intervention period. Energy intake, assessed by 7-day food records, did not differ between exercisers and controls at either 7 months or 1 year. The greater initial adiposity of the exercisers in the SWCP-I trial may partially explain the greater weight loss seen in this group compared to the studies of overweight men; however, of greater interest is the fact that exercise-induced weight loss was successfully achieved. It is worth noting that the OEI Panel [3] unknowingly included two additional publications from this trial among its set of randomized controlled trials that reported successful weight loss by aerobic exercise only [33], thereby inappropriately strengthening the data in support of the role of aerobic exercise in producing weight loss. Meta-analyses reviewed by the panel may have made similar errors.\n\nIn the Katzel _et al._ [42] study, exercisers were instructed for 3 months prior to baseline tests on an isoenergetic reduced-fat diet, which they were encouraged to continue, without losing weight, throughout the trial. Their exercise consisted of 45 minutes of treadmill and cycle ergonometer workouts, 3 times per week. Of those who completed the trial, exercisers increased VO2max 17% above baseline ( _p <_ 0.001), but did not change average weight; however, percent body fat was decreased 0.8% ( _p_ < 0.005), compared to controls ( _p <_ 0.001).\n\nAn earlier metabolic study by Sopko _et al._ [47], designed to tease apart the relationships of diet- and exercise-induced weight loss on lipid changes, involved 40 initially sedentary men, aged 19\u201344, who were slightly overweight (110% of standard weight). Men were randomly assigned for 12 weeks to control, weight loss by energy restriction, weight loss by exercise, and exercise with weight maintenance. Meals were prepared in a metabolic kitchen for all groups and were fixed at 40% energy from fat. Energy intake was adjusted to maintain weight every 3 days, according to group assignment. Exercisers engaged in supervised treadmill walking with an energy expenditure of 700 kcal per session, 5 times a week, to total 3500 kcal per week, at a pace of 5.6\u20136.4 km\/hr for most subjects. Significant weight was lost by men assigned to weight loss by exercise (\u20136.2 kg), but not to control (\u20130.5 kg) or exercise with weight maintenance (\u20130.5 kg). Together with the SWCP-I [41] and Katzel _et al._ [42] trials, these data highlight the role of energy intake in determining the effectiveness of exercise on weight loss in overweight and obese men.\n\nThe trial of exercise in normal weight premenopausal women [43] required assignment to control or three different exercise intensities: aerobic walking (8.0 km\/hr), brisk walking (6.4 km\/hr) or strolling (4.8 km\/hr), with each group walking a distance of 2.4 km, 5 days per week initially, and increasing this to 4.8 km by the seventh week, which was maintained through the remainder of the 24-week trial. Weight loss was reported for women who were doing brisk walking versus control, but not by those doing an equal amount of exercise as aerobic walking (8.0 km\/hr), which resulted in the greatest increase in VO2max, or strolling (4.8 km\/hr), and it is unclear that the ANOVA was significant across the four groups.\n\nIn the small trial of borderline obese women, those assigned to walking attended supervised sessions twice a week for the first 2 weeks and once a week thereafter. Average walking speed increased from 5.5 to 6.0 km\/hr. The estimated weekly energy expenditure was 6367 kJ. The 6-month program resulted in an increase in VO2max of 11% in walkers ( _p_ < 0.01 versus controls). Analysis of 3-day food records indicated that there were no significant changes in energy intake or diet composition throughout the program. Walkers lost a small but significant amount of fat mass (\u20131.7 kg) compared with controls ( _p_ < 0.05).\n\nIn summary, the randomized controlled trials reviewed here, which involved randomization of about 1000 men and 300 women to exercise only or control, generally show only modest (or no) weight loss with aerobic exercise programs that meet the recommended exercise prescription, even though these result in improved cardiorespiratory fitness. Due to an absence of trials, the evidence that exercise without dietary change will reduce body weight in overweight or obese women is extremely weak, particularly in premenopausal women. Men seem more likely to lose weight with exercise than postmenopausal women, but this is clearly dependent on attention to energy intake. It is important to point out, however, that these studies do not exceed 1 year, and as pointed out earlier, considerable time is necessary to achieve fat loss with exercise; furthermore, if lean mass is increased, total weight will not reflect the improved body composition achieved.\n\nOf considerable interest is a review by Toth _et al._ [24] of aerobic exercise and resistance exercise in older adults, which included data from hydrostatic (underwater) weighing or DXA and revealed that aerobic exercise was effective in reducing body fat stores in 20 of 22 studies and that the changes in fat were related to the total number of exercise sessions. The initial obesity status of the participants in these trials was not included in the review; however, this report suggests that older adults may benefit more from aerobic exercise than those studied in the trials reviewed here.\n\n### B. Trials of Aerobic Exercise Plus Diet versus Diet Only for Weight Loss\n\nThe Expert Panel stated that a combination of a reduced-energy diet and increased physical activity produces greater weight loss than diet alone or exercise alone, based on results from 12 studies [36, 39, 40, 48\u201356] which are listed in Table 2, with seven additional trials that seem to meet the criteria of the Expert Panel [57\u201363]. These studies include fairly equal numbers of overweight men and women, but nearly twice as many obese women as men, including women who are considerably more obese than those who are included in studies listed in Table 1. None of the five trials of overweight individuals, which included more than 100 premenopausal women, 300 postmenopausal women, and about 550 men, reported greater weight loss in men or women assigned to aerobic exercise plus diet versus to diet only [36, 39, 40, 48, 49]; however, both treatments resulted in significant weight loss compared to controls in all five trials. Only one [48] of the nine trials [48, 50\u201352, 57\u201362] of obese class 1 (BMI = 30.0\u201334.9 kg\/m2) individuals, which included more than 300 women, mostly premenopausal, and approximately 300 men, reported greater weight loss in the exercise plus diet group versus the diet only group. The one trial that showed greater weight loss with the addition of exercise to diet versus diet only, the second Stanford Weight Control Project (SWCP-II), found this outcome in men, but not in the women, whose BMI fell within the overweight group [48]. Only one [53] of five trials [53\u201356, 63] in obese class 2 (BMI = 35\u201339.9 kg\/m2) individuals, which also included more than 300 women but only about 25 men, reported greater weight loss in the exercise plus diet group versus the diet only group, and this was a small study that mixed 9 men and 21 women and did not have a control (no weight loss treatment) group [53].\n\nTABLE 2\n\nRandomized Trials with Aerobic Exercise Plus Diet versus Diet Only Groups\n\naN post = number of subjects completing the trial; in parentheses, number of randomized subjects.\n\nbNS = not significant.\n\nIn summary, only 2 of 19 studies involving about 2000 overweight or obese women and men showed a significant benefit of the addition of aerobic exercise to a weight-reducing diet for weight loss. These data provide little support for the contention that the addition of exercise to an energy-restricted diet will result in significant additional weight loss in a majority of overweight or obese individuals; however, the majority of these studies included fewer than 20 participants per treatment assignment, whereas the SWCP-II trial [48] included more than twice that number.\n\nOne additional trial, not shown on the table, compared a dietary intervention with and without the addition of exercise in individuals with mean BMI < 25.0 kg\/m2 from a South Asian population. The Diet and Moderate Exercise Trial [64] randomly assigned 419 men and 44 women, who had one or more coronary heart disease (CHD) risk factors, to one of two groups for 24 weeks:\n\nGroup A: American Heart Association (AHA) Step I (reduced-fat) diet + fruits and vegetables + exercise, consisting of brisk walking, 3\u20134 km\/day, or spot running, 10\u201315 min\/day ( _N_ = 231)\n\nGroup B: AHA Step I diet only ( _N_ = 232).\n\nAt 24 weeks, after 20 weeks of exercise in addition to the dietary regimen, group A had lost 6.5 kg (9.8% reduction from baseline), while group B had lost only 0.3 kg ( _p <_ 0.01 versus A). Whether this difference should be attributed primarily to the exercise or to the addition of fruits and vegetables to the diet may be worthy of further investigation in overweight or obese individuals.\n\n### C. Role of Resistance Exercise in Weight Control\n\nConsiderably less information is available on the potential role of resistance exercise or strength training, which presumably favors retention of lean body mass, in managing weight control than has accumulated for aerobic exercise. The most recent ACSM recommendations for the quantity and quality of exercise to be achieved by adults includes a progressive resistance training component that provides a stimulus to all major muscle groups, with the plan to complete 10\u201315 repetitions of a set of 8\u201310 exercises, 2\u20133 days\/week [65]. Although these guidelines do not address obesity management, researchers have begun to assess the possible role of such activity in bringing about weight loss. Two of the trials of obese class 1 women, Sweeny _et al._ [59] and Marks _et al._ [52], and three of the trials of obese class 2 women, Donnelly _et al._ [63], Andersen _et al._ [54], and Wadden _et al._ [56], that appear on Table 2, compare aerobic exercise with resistance exercise or strength training. None of these show a difference in weight loss between these forms of exercise when combined with the diets; however, the greater interest would be in the maintenance of lean body mass. In a trial that included measurement of adipose tissue by magnetic resonance imaging [66], 24 women with BMI over 27 kg\/m2 and waist-to-hip ratio greater than 0.85 were randomly assigned to aerobic exercise or resistance exercise for 16 weeks, with both groups instructed on an energy-restricted diet. The aerobic exercise group, which started at a mean BMI of 34.4 kg\/m2, lost 10.9 kg, while the resistance exercise group started at mean BMI of 31.8 kg\/m2 and lost 10.1 kg. These differences were not significant among groups, and both groups reduced their volume ratio of visceral to subcutaneous fat, with no differences between groups [66].\n\nIn a 12-week strength training study of 22 obese women (mean BMI of 31.4 kg\/m2 for the resistance exercise group and 32.8 kg\/m2 for the controls), total weight was increased 1.4 kg in resistance exercise and 0.4 kg in controls, but there were no significant differences between groups [67]. The fact that weight was increased, rather than decreased, emphasizes the need to separate possible lean body mass gain and fat weight loss in such studies. As reported above, resistance exercise increased fat-free mass in 15 out of 28 studies of older adults, and also reduced fat mass and total mass in a majority of these studies [24], suggesting that resistance training may have a role to play in weight control.\n\n### D. Role of Physical Activity in the Maintenance of Weight Loss\n\nIt is generally believed that exercise is important for preventing weight regain; however, there are few randomized controlled trials that include long-term follow-up. A meta-analysis [68] noted that 1-year follow-up patients in the diet only group maintained a weight loss of 6.6 kg, whereas those in the diet plus exercise group maintained a weight loss of 8.6 kg; however, neither the overall weight loss nor the percent of weight loss retained differed significantly between conditions. Table 3 presents several long-term follow-up studies from randomized controlled trials of exercise plus diet versus diet only.\n\nTABLE 3\n\nFollow-up Studies of Trials with Aerobic Exercise Plus Diet versus Diet Only\n\n_b_ NS = not significant.\n\naN post = number of subjects completing the trial; in parentheses, number of randomized subjects.\n\nWing _et al._ [53] published two small trials and their follow-up studies in one report. The 2-year weight change data for the study with aerobic exercise plus diet (\u20137.9 kg) versus diet only (\u20133.8 kg) were similar to the data for the study of aerobic exercise plus diet (\u20137.8 kg) versus a low-intensity, flexibility (\"placebo\") exercise plus diet group (\u20134.0 kg); however, the differences among groups reached significance in the first study, but not the second.\n\nPavlou _et al._ [69] conducted a complicated study involving randomization of 160 men to four different diets and to exercise or no exercise (eight groups). To present the weight loss phase data by exercise status, tabulated data for the four diet groups were combined by exercise treatment arm to determine the mean initial weight and first-year weight loss; however, the follow-up data were provided only as a graph for all eight groups, requiring some gross estimation of weight change. However, it was fairly clear that the four groups assigned to exercise maintained weight loss quite successfully compared to the non-exercising groups.\n\nSvendsen _et al._ [70] published a 6-month follow-up to the 12-week study, shown on Table 2. Both the exercise plus diet and diet only women gained about 2 kg during this period, and although weight loss was still significant versus control, it remained not significant between the combined and diet only women.\n\nSkender _et al._ [71] published a one-year follow-up to a trial in which obese men and women had been randomly assigned to a Help Your Heart Eating Plan (HYHEP) or to aerobic exercise, primarily walking, for 3\u20135 45-minute periods per week, at an intensity that subjectively was perceived as \"vigorous\" but not \"strenuous,\" without changing diet; or to exercise plus the eating plan. At 1 year, the exercise only group (\u20132.9 kg) had lost less weight than the diet only (\u20136.8 kg) or exercise plus diet group (\u20138.9 kg), ANOVA _p_ < 0.09; however, both the diet only and exercise plus diet groups regained weight during the second year, while the exercise only group maintained its weight loss. Weight change from baseline to year 2 for 86 (of an original 127) women who returned was diet only (+ 0.9), exercise only (\u20132.7 kg), and diet plus exercise (\u20132.2 kg).\n\nWadden _et al._ [72] reported 1-year follow-up data for the study shown on Table 2. All groups, regardless of assignment to aerobic exercise, strength training, a combination, or no exercise gained back a substantial portion of the weight they had lost in the initial year and there were no differences among groups.\n\nThese few studies provide only minimal support for the notion that the addition of exercise to diet is effective for maintenance of weight loss over time; however, the data are clearly very limited. The Skender _et al._ study [71] is particularly intriguing because it suggests that exercise only may be a better approach for long-term benefits, despite the modest weight loss that is observed initially. Long-term follow-up studies are needed to evaluate the real value of any given weight loss strategy, particularly aerobic exercise, before drawing any conclusions.\n\nAlthough the randomized controlled trial data do not provide strong evidence, there is considerable anecdotal support for the belief that exercise is important in weight maintenance after losses of substantial weight. In a study involving interviews of 44 obese women who regained weight after successful weight reduction (relapsers), 30 formerly obese, average-weight women who maintained weight loss (maintainers), and 34 women who had always remained at the same average, nonobese weight ( _n_ = 34), 90% of maintainers and 82% of controls reported exercising regularly, compared to only 34% of relapsers [73]. The National Weight Control Registry, a large study of successful long-term maintainers of weight loss, includes 629 women and 155 men who lost an average of 30 kg and maintained a required minimum weight loss of 13.6 kg for 5 years [74]. These men and women report using both diet and exercise to maintain weight loss, and report a very high activity level of approximately 11,830 kJ being expended through physical activity per week. This exceeds the ACSM recommendation of 2000 kcal\/week (8368 kJ\/week) energy expenditure as optimal physical activity, a goal that was achieved by 52% of the registry sample (50% of women, 62% of men) [74].\n\n### E. Weight Loss in Trials Designed to Study Adherence to Different Exercise Programs\n\nTable 4 presents a set of behaviorally based exercise studies, in order of increasing obesity level of the participants, which were designed to determine better adherence strategies, including determination of optimal exercise dose and volume (i.e., frequency, duration, intensity) and whether breaking up daily exercise into multiple short bouts, rather than employing single long bouts, will improve adherence and thus outcomes. It is unclear whether the Duncan _et al._ study [43] demonstrated that a brisk walking pace is more likely to bring about weight loss than strolling or going at a race-walking speed, due to possible errors in the analytic approach, but this is an intriguing question. The King _et al._ [38] study, which focused on adherence issues surrounding group- versus home-based exercise as well as optimal intensity of exercise, did not identify a better approach to weight loss during the initial year; however, although groups did not differ in weight changes during a second year, the higher intensity, group-based men and women increased BMI slightly (0.1 and 0.2 kg\/m2 respectively) from baseline to 24 months, whereas, the higher intensity, home-based men and women decreased BMI slightly (\u20130.1 kg\/m2 each), as did the lower intensity, home-based men and women (\u20130.2 and \u2013 0.4 kg\/m2, respectively), which is generally not what happens to older adults over a 2-year period [75].\n\nTABLE 4\n\nRandomized Trials of Different Exercise Programs\n\naN post = number of subjects completing the trial; in parentheses, number of randomized subjects.\n\nbNS = not significant.\n\nThe effect of accumulating activity over the course of the day through \"lifestyle activity\" versus structured aerobic exercise has received interest from exercise researchers in recent years. Dunn _et al._ [76] demonstrated that a lifestyle physical activity intervention was as effective as a structured exercise program in improving physical activity, cardiorespiratory fitness, and blood pressure in a moderately overweight cohort of men and women; however, neither group changed their weight, which is not surprising considering the modest effect on weight loss associated with structured aerobic exercise. Andersen _et al._ [77] combined these exercise options with a low-fat energy-restricted diet in a 16-week randomized trial of obese women. Weight loss was similar between groups, with no difference between them; however, the aerobic group lost less fat-free mass. During a 1-year follow-up, the aerobic group regained 1.6 kg, while the lifestyle group regained 0.08 kg, suggesting that lifestyle intervention may be better for weight maintenance.\n\nJakicic _et al._ [78, 79] have published two studies of effects of doing exercise in multiple short bouts. In the first [78], obese women, aged 25\u201350 years, were randomly assigned to a behavioral weight loss program consisting of an energy-restricted diet combined with 5 days per week of either single aerobic exercise bouts per day, starting as 20-minute bouts (weeks 1\u20134), increasing to 30-minute bouts (weeks 5\u20138) and to 40-minute bouts (weeks 9\u201320); or multiple 10-minute bouts per day, starting as 2 bouts per day, increasing to 3, then 4, respectively [23]. Both groups reduced energy intake and percent energy from fat significantly. Women performing multiple short bouts lost 8.9 kg in the 20-week period, while those exercising in single long bouts lost 6.4 kg (n.s., not significant) Because the dietary changes (energy restriction) probably contributed the most to weight loss, it was not possible to determine the independent contribution of the exercise components; however, exercising in multiple short bouts was shown to improve adherence to exercise and to result in significantly greater improvement in aerobic capacity, as well as a trend for greater weight loss. In the second trial [79], obese women were assigned to one of three treatment groups, all of which included an energy-restricted diet: long-bout exercise, multiple short-bout exercise, or multiple short-bout exercise with home exercise equipment using a treadmill. Total and fat weight loss was significantly greater in the short-bout exercise with home exercise equipment group compared to multiple short-bout exercise, but did not differ between longbout exercise and either multiple short-bout exercise or short-bout exercise with home exercise equipment.\n\nAnother study that compared a 15-month group-based exercise behavioral weight loss program to a home-based program, involving moderate intensity walking (30 min\/day, 5 days\/week) revealed that obese women in the home-based program lost significantly more weight than those in the group program, presumably due to greater adherence to exercise [80].\n\n## V. ROLE OF PHYSICAL ACTIVITY IN PREVENTING AND TREATING OBESITY-RELATED COMORBIDITIES\n\nThe evidence for a relationship of overweight and obesity and central or upper body fat distribution with type 2 diabetes, hypertension, dyslipoproteinemias, in particular low high-density lipoprotein (HDL) cholesterol and elevated plasma triglyceride concentrations, and CHD is reasonably strong [81]. In addition, obesity is related to gallbladder disease, respiratory disease, certain cancers (in particular, colorectal and prostate, endometrial, cervical, and breast), and osteoarthritis [81]. The role of physical activity in treating these obesity-related disorders is therefore of great interest.\n\n### A. Type 2 Diabetes Mellitus\n\nThe evidence is reasonably strong that physical activity has favorable effects on reducing insulin resistance in obesity and among patients with type 2 diabetes mellitus [82]. Improvement in glucose tolerance is less consistently observed and is related to exercise intensity, changes in adiposity, the interval between exercise and glucose tolerance testing, and severity of glucose intolerance before initiating an exercise training program [82].\n\n### B. Hypertension\n\nEpidemiological studies show an inverse relationship between the incidence of high blood pressure and physical activity or fitness, which is either more pronounced in the overweight or independent of body size [83]. Based on a review of 68 study groups with normotensive and hypertensive subjects of both sexes, the weighted net reduction of blood pressure in response to dynamic physical training was determined to average 3.4\/2.4 mm Hg ( _p_ < 0.001), unrelated to initial BMI [83]. Exercise seems to be less effective than diet in lowering blood pressure ( _p_ < 0.02) and adding exercise to diet yields no further benefit [83].\n\n### C. Dyslipoproteinemia\n\nEvidence to support a role of physical activity in raising HDL cholesterol or lowering plasma triglyceride concentrations independent of weight loss is weak, particularly at the recommended levels of moderate intensity exercise [42]; however, if sufficient weight is lost by an energy-restricted diet or exercise without dietary change, without concomitant reductions in dietary fat, HDL cholesterol is likely to be increased and triglyceride levels decreased, particularly in overweight or obese men [35, 41, 42, 84]. The addition of exercise is also effective in preventing the diet-induced reduction in HDL cholesterol that accompanies a reduction in dietary fat, in both men and women [48, 84]. Finally, the addition of exercise to a low-fat, low-cholesterol diet enhances the low density lipoprotein lowering effect of the diet in men and postmenopasual women with unfavorable lipoprotein profiles [40, 84]\n\n### D. Coronary Heart Disease\n\nThe report of the surgeon general [6] provided strong evidence for a role of physical activity in reducing the risk of CHD, which is an obesity-related comorbidity. A prospective study of walking compared to vigorous exercise revealed that even moderate levels of activity are associated with significant reduction in CHD risk for both nonobese and obese women; furthermore, women who were physically active were leaner and had a lower prevalence of hypertension, diabetes, and hypercholesterolemia [85].\n\n### E. Cancers\n\nThe evidence to support a role for physical activity in preventing colon cancer is reasonably convincing [86]. Though not as strong, there is also considerable evidence of a protective role of physical activity on hormone-dependent cancers, particularly endometrial, and gallstones [86]. Unfortunately, the data are not clear on whether physical activity in overweight and obese adults is associated with benefits compared to sedentary overweight adults.\n\n### F. Central Adiposity\n\nThere is insufficient evidence to support the belief that physical activity will bring about preferential reduction in visceral fat and central adiposity, due to a paucity of studies that have adequately assessed this issue. Exercise intervention trials have not shown a reduction in waist circumference in the absence of weight loss; however, a modest reduction (about 3 cm) has been reported in response to exercise-induced weight loss of approximately 3 kg [87]\n\n## VI. CONCLUSIONS\n\nEpidemiological studies suggest that physical inactivity is related to obesity and that active adults are less likely to be obese or to gain weight as they age; however, it is unclear whether a sedentary lifestyle actually causes obesity or that physical activity prevents weight gain or promotes normal weight. There is ample biological rationale to support the contention that being more physically active and maintaining lean body mass, through resistance exercise and possibly aerobic exercise, will facilitate weight control. The value of exercise may be best seen in older adults, for whom preserving lean body mass may be as important as reducing body fat.\n\nUnfortunately, the evidence from clinical trials to support a strong role for adopting a more active lifestyle to bring about weight loss in overweight or obese adults is weak, particularly in women. However, it should be recognized that changes in body composition derived solely from aerobic exercise, without dietary change, will occur slowly, because fat is a very efficient fuel. The addition of resistance exercise might increase lean body mass, which would result in even slower changes in total body weight. In general, neither aerobic exercise nor resistance exercise seem to be powerful enough to cause major weight loss, without the addition of restriction of energy intake, nor do the available studies provide strong evidence that exercise will bring about long-term maintenance of weight loss. Unfortunately, the studies to date have been small and have not provided enough information on adherence to the exercise protocols to enable one to assess the true value of aerobic or resistance exercise. Because diet-induced weight loss is not associated with long-term success, it is important to work toward improving adherence to exericise programs and to recognize that patience may be required to accept the slow weight loss achieved through exercise.\n\nIn the meantime, the evidence that exercise improves other disease risk factors associated with obesity continues to accumulate. 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Physical activity in the management of obesity: Issues and implementation. In: Flecther G.F., Grundy S.M., Hayman L.L., eds. _Obesity: Impact on Cardiovascular Disease_. Atlanta, GA: Futura Publishing Co., Inc.; 1999:261\u2013293.\n\n34. Huttunen J.H., Lansimies E., Voutilainen E., Ehnholm C., Hietanen E., Pentila I., Siitonen O., Rauramaa R. Effect of moderate physical exercise on serum lipoproteins: A controlled clinical trial with special reference to serum high-density lipoproteins. _Circulation_. 1979;60:1220\u20131229.\n\n35. Wood P.D., Haskell W.L., Blair S.N., Williams P.T., Krauss R.M., Lindgren F.T., Albers J.J., Ho P.H., Farquhar J.W. Increased exercise level and plasma lipoprotein concentrations: A one-year, randomized, controlled study in sedentary, middle-aged men. _Metabolism_. 1983;32:31\u201339.\n\n36. Hellenius M.L., de Faire U.H., Berglund B.H., Hamsten A., Krakau I. Diet and exercise are equally effective in reducing risk for cardiovascular disease. Results of a randomized controlled study in men with slightly to moderately raised cardiovascular risk factors. _Atherosclerosis_. 1993;103:81\u201391.\n\n37. Pritchard J.E., Nowson C.A., Wark J.D. A worksite program for overweight middle-aged men achieves lesser weight loss with exercise than with dietary change. _J. Am. Diet. Assoc_. 1997;97:37\u201342.\n\n38. King A.C., Haskell W.L., Taylor C.B., Kraemer H.C., DeBusk R.F. Group- vs home-based exercise training in healthy older men and women: A community-based clinical trial. _JAMA_. 1991;266:1535\u20131542.\n\n39. Anderssen S., Holme I., Urdal P., Hjermann I. Diet and exercise intervention have favourable effects on blood pressure in mild hypertensives: The Oslo Diet and Exercise Study (ODES). _Blood Press_. 1995;4:343\u2013349.\n\n40. Stefanick M.L., Mackey S., Sheehan M., Ellsworth N., Haskell W.L., Wood P.D. Effects of diet and exercise in men and postmenopausal women with low levels of HDL cholesterol and high levels of LDL cholesterol. _N. Engl. J. Med_. 1998;339:12\u201320.\n\n41. Wood P.D., Stefanick M.L., Dreon D.M., Frey-Hewit B., Garay S.C., Williams P.T., Superko H.R., Fortmann S.P., Albers J.J., Vranizan K.M., Ellsworth N.M., Terry R.B., Haskell W.L. Changes in plasma lipids and lipoproteins in overweight men during weight loss through dieting as compared with exercise. _N. Engl. J. Med_. 1988;319:1173\u20131179.\n\n42. Katzel L.I., Bleecker E.T., Colman E.G., Rogus E.M., Sorking J.D., Goldberg A.P. Effects of weight loss vs aerobic exercise training on risk factors for coronary disease in healthy, obese, middle-aged and older men: A randomized controlled trial. _JAMA_. 1995;274:1915\u20131921.\n\n43. Duncan J.J., Gordon N.F., Scott C.B. Women walking for health and fitness: How much is enough? _JAMA_. 1991;266:3295\u20133299.\n\n44. Ready A.E., Drinkwater D.T., Ducas J., Fitzpatrick D.W., Brereton D.G., Oades S.C. Walking program reduces elevated cholesterol in women postmenopause. _Can. J. Cardiol_. 1995;11:905\u2013912.\n\n45. Ronnemaa T., Mattila K., Lehtonen A., Kallio V. A controlled randomized study on the effect of long-term physical exercise on the metabolic control in type 2 diabetic patients. _Acta Med. Scand_. 1986;220:219\u2013224.\n\n46. Raz I., Hauser E., Bursztyn M. Moderate exercise improves glucose metabolism in uncontrolled elderly patients with non-insulin-dependent diabetes mellitus. _Isr. J. Med. Sci_. 1994;30:766\u2013770.\n\n47. Sopko G., Leon A.S., Jacobs D.R., Foster N., Moy J., Kuba K., Anderson J.T., Casal D., McNally C., Frantz I. The effects of exercise and weight loss on plasma lipids in young obese men. _Metabolism_. 1985;34:227\u2013236.\n\n48. Wood P.D., Stefanick M.L., Williams P.T., Haskell W.L. The effects on plasma lipoproteins of a prudent weight-reducing diet, with or without exercise, in overweight men and women. _N. Engl. J. Med_. 1991;325:461\u2013466.\n\n49. Svendsen O.L., Hassager C., Christiansen C. Effect of an energy-restrictive diet with or without exercise on lean tissue, resting metabolic rate, cardiovascular risk factors, and bone in overweight postmenopausal women. _Am. J. Med_. 1993;95:131\u2013140.\n\n50. Hammer R.L., Barrier C.A., Roundy E.S., Bradford J.M., Fisher A.G. Calorie-restricted low-fat diet and exercise in obese women. _Am. J. Clin. Nutr_. 1989;49:77\u201385.\n\n51. Blonk M.C., Jacobs M.A.J.M., Biesheuvel E.H.E., Weeda-Mannak W.L., Heine R.J. Influences on weight loss in type 2 diabetic patients: Little long-term benefit from group behaviour therapy and exercise training. _Diab. Med_. 1994;11:449\u2013457.\n\n52. Marks B.L., Ward A., Morris D.H., Castellani J., Rippe J.M. Fat-free mass is maintained in women following a moderate diet and exercise program. _Med. Sci. Sports Exerc_. 1995;27:1243\u20131251.\n\n53. Wing R.R., Epstein L.H., Paternostro-Bayles M., Nowalk M.P., Gooding W. Exercise in a behavioural weight control programme for obese patients with Type 2 (non-insulin-dependent) diabetes. _Diabetologia_. 1988;31:902\u2013909.\n\n54. Andersen R.E., Wadden T.A., Bartlett S.J., Vogt R.A., Weinstock R.S. Relation of weight loss to changes in serum lipids and lipoproteins in obese women. _Am. J. Clin. Nutr_. 1995;62:350\u2013357.\n\n55. Gordon N.F., Scott C.B., Levine B.D. Comparison of single versus multiple lifestyle interventions: Are the antihypertensive effects of exercise training and diet-induced weight loss additive? _Am. J. Cardiol_. 1997;79:763\u2013767.\n\n56. Wadden T.A., Vogt R.A., Andersen R.E., Bartlett S.J., Foster G.D. Exercise in the treatment of obesity: Effects of four interventions on body composition, resting energy expenditure, appetite, and mood. _J. Consult. Clin. Psychol_. 1997;65:269\u2013277.\n\n57. Hill J.O., Schlundt D.G., Sbrocco T., Sharp T., Pope-Cordle J., Stetson B., Kaler M., Heim C. Evaluation of an alternating-calorie diet with and without exercise in the treatment of obesity. _Am. J. Clin. Nutr_. 1989;50:248\u2013254.\n\n58. Bertram S.R., Venter I., Stewart R.I. Weight loss in obese women\u2014exercise v. dietary education. _S. Afr. Med. J_. 1990;78:15\u201318.\n\n59. Sweeney M.E., Hill J.O., Heller P.A., Baney R., DiGirolamo M. Severe vs moderate energy restriction with and without exercise in the treatment of obesity: Efficiency of weight loss. _Am. J. Clin. Nutr_. 1993;57:127\u2013134.\n\n60. Dengel D.R., Hagberg J.M., Coon P.J., Drinkwater D.T., Goldberg A.P. Effects of weight loss by diet alone or combined with aerobic exercise on body composition in older obese men. _Metabolism_. 1994;43:867\u2013871.\n\n61. Fox A.A., Thompson J.L., Butterfield G.E., Gylfadottir U., Moynihan S., Spiller G. Effects of diet and exercise on common cardiovascular disease risk factors in moderately obese older women. _Am. J. Clin. Nutr_. 1996;63:225\u2013233.\n\n62. Evans E.M., Saunders M.J., Spano M.A., Arngrimsson S.A., Lewis R.D., Cureton K.J. Effects of diet and exercise on the density and composition of the fat-free mass in obese women. _Med. Sci. Sports Exerc_. 1999;31:1778\u20131787.\n\n63. Donnelly J.E., Pronk N.P., Jacobsen D.J., Pronk S.J., Jakicic J.M. Effects of a very-low-calorie diet and physical-training regimens on body composition and resting metabolic rate in obese females. _Am. J. Clin. Nutr_. 1991;54:56\u201361.\n\n64. Singh R.B., Rastogi S.S., Ghosh S., Niaz M.A., Singh N.K. The Diet and Moderate Exercise Trial (DAMET): Results after 24 weeks. _Acta Cardiol_. 1993;48:543\u2013557.\n\n65. American College of Sports Medicine. The recommended quantity and quality of exercise for developing and maintaining cardiorespiratory and muscular fitness, and flexibility in healthy adults. _Med. Sci. Sports Exerc_. 1998;30:975\u2013991.\n\n66. Ross R., Rissanen J. Mobilization of visceral and subcutaneous adipose tissue in response to energy restriction and exercise. _Am. J. Clin. Nutr_. 1994;60:695\u2013703.\n\n67. Manning J.M., Dooly-Manning C.R., White K., Kampa I., Silas S., Kesselhaut M., Ruoff M. Effects of a resistive training program on lipoprotein-lipid levels in obese women. _Med. Sci. Sports Exerc_. 1991;23:1222\u20131226.\n\n68. Miller W.C., Koceja D.M., Hamilton E.J. A meta-analysis of the past 25 years of weight loss research using diet, exercise or diet plus exercise intervention. _Int. J. Obes_. 1997;21:941\u2013947.\n\n69. Pavlou K.N., Krey S., Steffee W.P. Exercise as an adjunct to weight loss and maintenance in moderately obese subjects. _Am. J. Clin. Nutr_. 1989;49:1115\u20131123.\n\n70. Svendsen O.L., Hassager C., Christiansen C. Six months' follow-up on exercise added to a short-term diet in overweight postmenopausal women\u2014Effects on body composition, resting metabolic rate, cardiovascular risk factors and bone. _Int. J. Obes_. 1994;18:692\u2013698.\n\n71. Skender M.L., Goodrick G.K., Del Junco D.J., Reeves R.S., Darnell L., Gotto A.M., Foreyt J.P. Comparison of 2-year weight loss trends in behavioral treatments of obesity, diet, exercise, and combination interventions. _J. Am. Diet. Assoc_. 1996;96:342\u2013346.\n\n72. Wadden T.A., Vogt R.A., Foster G.D., Anderson D.A. Exercise and the maintenance of weight loss: 1-year follow-up of a controlled clinical trial. _J. Consult. Clin. Pschyol_. 1998;66:429\u2013433.\n\n73. Kayman S., Bruvol W., Stern J.S. Maintenance and relapse after weight loss in women: Behavioral aspects. _Am. J. Clin. Nutr_. 1990;52:800\u2013807.\n\n74. Klem M.L., Wing R.R., McGuire M.T., Seagle H.M., Hill J.O. A descriptive study of individuals successful at long-term maintenance of substantial weight loss. _Am. J. Clin. Nutr_. 1997;66:239\u2013246.\n\n75. King A.C., Haskell W.L., Young D.R., Oka R.K., Stefanick M.L. Long-term effects of varying intensities and formats of physical activity on participation rates, fitness, and lipoproteins in men and women aged 50\u201365 years. _Circulation_. 1995;91:2596\u20132604.\n\n76. Dunn A.L., Marcus B.H., Kampert J.B., Garcia M.E., Kohl H.W., III., Blair S.N. Comparison of life-style and structured interventions to increase physical activity and cardiorespiratory fitness: A randomized trial. _JAMA_. 1999;281:327\u2013334.\n\n77. Andersen R.E., Wadden T.A., Bartlett S.J., Zemel B., Verde T.J., Franckowiak S.C. Effects of lifestyle activity vs structured aerobic exercise in obese women: A randomized trial. _JAMA_. 1999;281:335\u2013340.\n\n78. Jakicic J.M., Wing R.R., Butler B.A., Robertson R.J. Prescribing exercise in multiple short bouts versus one continuous bout: Effects on adherence, cardiorespiratory fitness, and weight loss in overweight women. _Int. J. Obes_. 1995;19:893\u2013901.\n\n79. Jakicic J.M., Winters C., Lang W., Wing R.R. Effects of intermittent exercise and use of home exercise equipment on adherence, weight loss, and fitness in overweight women: A randomized trial. _JAMA_. 1999;282:1554\u20131560.\n\n80. Perri M.G., Marin A.D., Leermakers E.A., Sears S.F., Notelovitz M. Effects of group- versus home-based exercise in the treatment of obesity. _J. Consult. Clin. Psychol_. 1997;65:278\u2013285.\n\n81. Pi-Sunyer F.X. Comorbities of overweight and obesity: Current evidence and research issues. _Med. Sci. Sports Exerc_. 1999;31:S602\u2013S608.\n\n82. Kelley D.E., Goodpaster B.H. Effects of physical activity on insulin action and glucose tolerance in obesity. _Med. Sci. Sports Exerc_. 1999;31:S619\u2013S623.\n\n83. Fagard R.H. Physical activity in the prevention and treatment of hypertension in the obese. _Med. Sci. Sports Exerc_. 1999;31:S624\u2013S630.\n\n84. Stefanick M.L. Physical activity for preventing and treating obesity-related dyslipoproteinemias. _Med. Sci. Sports Exerc_. 1999;31:S609\u2013S618.\n\n85. Manson J.E., Hu F.B., Rich-Edwards J.W., Colditz G.A., Stamper M.J., Willett W.C., Speizer F.E., Hennekens C.H. A prospective study of walking as compared with vigorous exercise in the prevention of coronary heart disease in women. _N. Engl. J. Med_. 1999;341:650\u2013658.\n\n86. Rissanen A., Fogelholm M. Physical activity in the prevention and treatment of other morbid conditions and impairments associated with obesity: Current evidence and research issues. _Med. Sci. Sports Exerc_. 1999;31:S635\u2013S645.\n\n87. Ross R., Janssen I. Is abdominal fat preferentially reduced in response to exercise-induced weight loss? _Med. Sci. Sports Exerc_. 1999;31:S568\u2013S572.\nCHAPTER 33\n\nMacronutrient Intake and the Control of Body Weight\n\nDAVID A. LEVITSKY, Cornell University, Ithaca, New York\n\n## I. INTRODUCTION\n\nScholars of the control of food intake and the regulation of body weight have given little attention to the role played by macronutrients until fairly recently. Most researchers in this field were convinced by the dictum issued by the famous physiologist, Adolph [1], who declared that \"Within limits, rats eat for calories,\" a conclusion readily transferred to humans as well. Although Mayer [2] and Mellinkoff _et al._ [3] tried to argue particular roles for their pet nutrients (glucose and amino acids, respectively), the domination of the field by the set-point theories of body weight [4\u201317] shifted the focus of research from dietary variables to physiology in the search for those mechanisms that controlled body weight through the control of food intake.\n\nMeanwhile, the public was being fed (sold) great dreams of easy weight reduction and magical cures by eating certain macronutrients and avoiding others. In 1972, Robert Atkins was one of the first of a series of diet gurus to pontificate the virtues of eating a high-protein, low-carbohydrate, high-fat diet as a means of losing weight and curing obesity. He was followed by a rash of other \"non-nutritionist\" experts who pushed their own versions of the single macronutrient theory (carbohydrate is the culprit) and made lots of money and converts of the public on the way to their bank.\n\nFortunately, in the last 20 years the medical\/nutritional establishment has begun to listen to the public's questions and have generated a considerable amount of research on the role macronutrients play in the control of food intake and body weight. In some areas, there is almost complete agreement. In others, the answers are split almost down the middle. The purpose of this chapter is to review this literature and reveal what we do and do not know about the role played by macronutrients in determining energy intake and, ultimately, body weight.\n\n## II. FAT CHANCE\n\nThe macronutrient that has received the most attention in the scientific appetite and body weight literature is dietary fat. The relationship between dietary fat and body weight has been examined almost at every level of analysis from molecular to epidemiological. The epidemiological literature on the relationship between fat intake and body weight was critically reviewed by Lissner and Heitmann [18]. Although the data are not entirely consistent, they concluded that the greater the amount of dietary fat humans consume, the greater their body weight. Although the preponderance of the data indicate a direct relationship between fat consumption and body weight, they probably _underestimated_ the effect for two reasons. First, poor measures of food consumption lead to an underestimation of the magnitude of the effect, and most measures of food consumption in the home environment are quite poor. Even more serious, though, is the problem that the underestimation of fat intake and energy is directly related to body weight. The larger the person, the greater they underestimate their energy intake [19\u201324]. Expressing fat intake as a percent of energy ingested reduces this problem somewhat, but not entirely. Despite these limitations, the literature supports the conclusions reached by Lissner and Heitmann [18]. As depicted in Fig. 1, the preponderance of studies that have addressed this issue have found that a positive and significant relationship exists between dietary fat and body weight and\/or fat composition. The greater the amount of fat consumed, the greater the amount of body fat.\n\nFIGURE 1 Distribution of ecological studies relating body composition to dietary fat.\n\n## III. ESTABLISHING CAUSAL LINKS\n\nHowever, because the epidemiological data reported above are correlational, they do not prove that eating a diet rich in fat causes an increase in body weight. It is possible that having a body composed of a high percent of fat causes an increased preference for dietary fat. Establishing a causal link between dietary fat and body weight requires an experimental design where the amount of fat that people consume must be experimentally manipulated and its effects on food intake or body weight (fat) measured. Two kinds of studies have been used to examine the causal links between macronutrient intake and body weight: laboratory studies and clinical studies.\n\n### A. Laboratory Studies\n\nUnfortunately, studies of the effects of macronutrients in the laboratory rarely are of long enough duration to observe changes in body weight or fat composition. The dependent measure in most of these studies is the amount of food consumed. In many ways, this is a far more accurate measure of energy balance than body weight or even body composition measures, particularly if the design utilizes each subject as his or her own control. Any inaccuracy in estimating the energy or nutrient content of the food is nullified because each subject is tested under all conditions.\n\nFigure 2 illustrates the results of studies on the effects of alterations of dietary fat on energy consumption. Although the weight of the evidence appears to support the epidemiological studies (people consume fewer calories when the diet contains less fat) important and interesting methodological differences exist that are cause for concern.\n\nFIGURE 2 Distribution of experimental studies on the effects of dietary fat on food intake.\n\nMost of the studies that failed to find that humans change the amount of food they consume (compensate) for changes in dietary fat had altered the fat content of all the foods that were accessible to the subjects. For most of these studies, all foods offered to the subjects were approximately of the same relative fat content. Therefore, when the fat content was reduced, the fat content of all foods was decreased. Consequently, the only way subjects could avoid reducing their daily energy intake when faced with a low-fat diet was to increase the amount of lower fat foods that they consumed.\n\nMost of those studies that did find that subjects do compensate for alterations in dietary fat manipulated the fat content only of a single meal such as lunch or changed the fat content of only particular foods in the diet. Poppitt and Swann [25] provided a direct comparison of the two methodologies within a single study. Their results are replotted in Fig. 3. Under conditions where the fat content of only the lunch was manipulated (left panel), subjects appear to energetically compensate by changing the amount of food they consumed. However, as the right panel indicates, when the fat content of all the food is altered, humans do not compensate for the reduced energy content and, therefore, energy intake is diminished.\n\nFIGURE 3 Energy intake as a function of level of dietary fat. (Left) Only the fat content of the lunch was varied. (Right) Fat content of all foods were varied. [Adapted from Poppitt, S. D., and Swann, D. L. (1998). Dietary manipulation and energy compensation. _Int. J. Obes. Relat. Metab. Disord._ **22,** 1024\u20131031.]\n\nThis dependence of finding energetic compensation on specific methodology is quite important, but not understood. More importantly, if energy compensation for low-fat foods does exist when subjects have free access to foods of different fat composition, as suggested by these studies, then eating low-fat foods in the \"real world\" will never succeed as a strategy to chronically suppress in body weight, unless one ate only low-fat foods. The examination of clinical studies, however, indicates that people do lose weight when they use some low-fat foods, suggesting that the laboratory conditions or procedures may produce an artifact making it difficult to extrapolate the results to the \"real world.\" It is also possible that if conditions are sufficiently well controlled in the laboratory, people will demonstrate physiological regulation, but that the external variables associated with eating behavior in humans are so powerful as to easily obscure its demonstration.\n\n### B. Clinical Studies\n\nClinical studies are more realistic than laboratory studies. However, it is usually quite difficult to obtain weighed food intake measures from subjects in a clinical study as a measure of energy intake. Therefore, such studies require subjects to prepare their own meals and record what they eat. Such records, unfortunately, involve large measurement errors. As a consequence, clinical studies rely on physiological measures to corroborate their energy intake measures such as body weight or blood lipid concentrations.\n\nFigure 4 shows the results of 21 published clinical studies in which the fat content of the diet was changed and body weights of subjects in the community were measured at the beginning and the end of the study. This figure clearly illustrates that changes in dietary fat in the \"real world\" are not totally compensated for by an accurate adjustment of energy intake. A reduction of 1% in the fat composition of the diet results in a average weight loss of 0.25 kg. It is important to note that the study by Kendall _et al._ [26] is the only study in which food intake was actually dispensed and measured throughout the study. In this study the amount of fat consumed was also measured. The amount of weight loss observed falls exactly on the regression line generated by the other studies, indicating that the change in fat intake and body weight of the other studies must have been fairly accurate.\n\nFIGURE 4 Maximum weight loss as a function of percent calories from fat. a. Marckmann* (190); b. Simon _et al._ (191); c. Bloomberg _et al._ (192); d. Stefanick _et al._ (193); e. Singh _et al._ (194); f. Kendall _et al._ (26); g. Lee-Han _et al._ (195); i. Raben _et al._ (196), Jeffrey _et al._ (197); j. Siggaard _et al._ (198); k. Pritchard _et al._ (199); l. Shah _et al._ (200); m. Hunninghake _et al._ (201); n. Boyd _et al._ (202); o. Insull _et al._ (203); p. Buzzard _et al._ (204); q. Toubro & Astrup (205); s. Kasim _et al._ (206), Schlundt _et al._ (207); t. Sheppard _et al._ (208); u. Ornish _et al._ (209). Data adapted from Hill _et al._ (210). *Data point derived from Marckmann (190).\n\nThese data confirm the majority of laboratory studies and the epidemiological studies that have indicated that humans do not accurately compensate for changes in the fat content of the diet and either gain or lose weight depending on the amount of fat in the diet.\n\n## IV. IS IT FAT OR ENERGY DENSITY?\n\nThe fact that high-fat diets cause an increase in energy intake and obesity in animals has been evident for a long time in the animal feeding literature (see West and York [27] for a thorough review). Whereas this effect was thought to occur because of some unique property relating to the chemical structure of fat, Ramirez and Friedman [28] performed a series of interesting studies demonstrating that the excessive energy intake was due to an increase in the energy density of the diet rather than because of the fat content per se.\n\nIn one of the few human studies that failed to observe an increase in energy intake with increasing dietary fat, van Stratum _et al._ [29] examined the effects of introducing liquid diets that varied in the amount of fat and carbohydrate they contained, but were of equal energy density. They failed to find a difference in total energy intake between the two liquid diets either in the amount of liquid diet consumed or the amount of solid food eaten during the rest of the day. Stubbs _et al._ [30] reported similar results using a more elegant experimental design in which the amount of dietary fat was varied (20, 40, and 59%), but where the energy density of the three diets was maintained constant. A 3-day menu rotation was used for 14 days and was repeated three times; each time all the foods contained one of the three amounts of dietary fat. Subjects were free to eat as much or as little as they desired. The results of this extraordinary study are shown in Fig. 5 and are very consistent with the work of Ramirez and Friedman [28] in animals and with van Stratum _et al._ [29] in humans. Total energy intake did not differ between any condition despite the large differences between fat and carbohydrate. The study quite clearly demonstrates that the reason humans (and animals) overeat on a high-fat diet is because of the high energy density of fat and not because of any unique metabolic or physical property of high dietary fat.\n\nFIGURE 5 Intake of energy and macronutrients as a function of dietary fat when the energy density of the diet is held constant. [Adapted from Stubbs, R. J., Harbron, C. G., and Prentice, A. M. (1996). Covert manipulation of the dietary fat to carbohydrate ratio of isoenergetically dense diets. _Int. J. Obes. Relat. Metab. Disord._ **20,** 651\u2013660.]\n\nThis idea that energy density is the cause of overeating on a high-fat diet has been firmly confirmed by more recent studies [30\u201336]. These studies make it increasingly clear that humans not only fail to adjust the volume of food they consume to compensate for varying levels of fat in their food, but are equally as imprecise in adjusting the volume of food they consume when the energy density of the diet is altered with carbohydrates, water, or fiber. Daily energy intake is a direct function of the energy density (calories per weight) of the diet [33\u201338].\n\nAlthough this finding is of major theoretical and applied significance, in reality, the major determinant of energy density is the amount of fat in food. Figure 6 shows the relationship between the fat content and energy density for 125 randomly selected foods. The foods were consumed by subjects in the 1994 Continuing Survey of Food Intakes of Individuals conducted by the U. S. Department of Agriculture (USDA) [39]. A similar function was observed by Poppitt [37]. Thus, the easiest and most practical way to reduce energy density is to choose to eat foods low in fat content. Unless there is a reduction in energy expenditure, such a strategy must result in a chronic reduction in body weight.\n\nFIGURE 6 Relationship between energy density and fat concentration of 125 randomly selected foods. [Data from USDA (1996). \"Continuing Survey of Food Intakes by Individuals (CSFII): Diet and Health Knowledge Survey, 1994. Department of Agriculture, Beltsville Human Nutrition Research Center\/ARS.]\n\n## V. ARE CARBOHYDRATES THE CULPRIT RESPONSIBLE FOR OVERWEIGHT?\n\nAlthough Fig. 4 clearly indicates that energy density is determined primarily by dietary fat, there is a popular notion that carbohydrates are mainly responsible for overweight and obesity. Carbohydrates have been blamed as the culprit for obesity in such best selling books as \"Dine Out and Lose Weight: The French Way to Culinary 'Savoir Vivre' '\" by Michel Montignac [40], \"Sugar Busters! Cutting Sugar to Trim Fat\" by H. Leighton Steward _et al._ [41], \"Dr. Atkins' New Diet Revolution\" by Robert C. Atkins [42] (actually an evolution from his previous book, \"Dr. Atkins' Diet Revolution: The High Calorie Way to Stay Thin Forever\" [43]), \"The Zone\" by Barry Sears and Bill Lawren [44]; \"The Carbohydrate Addict's Diet: The Lifelong Solution to YoYo Dieting\" by Rachael and Richard Heller [45], and \"Protein Power: The High-Protein\/Low-Carbohydrate Way to Lose Weight, Feel Fit, and Boost Your Health\u2014in Just Weeks\" by Rachael and Michael Eades [46].\n\nThe basic premise behind all of these money-making books is that despite the fact that Americans are more concerned about fat than any other aspect of food, as is depicted in Fig. 7, and have been reducing their consumption of fat [47], Americans are still getting fatter [48]. Therefore, these nutrition gurus would argue that it is not dietary fat that is causing the overweight and obesity; it is the carbohydrate content that is the culprit.\n\nFIGURE 7 Nutritional concerns of supermarket consumers. [Adapted from Food Marketing Institute (1999). \"Trends in the United States\u2014Consumer Attitudes and the Supermarket.\" Food Marketing Institute, Washington, DC.\n\nThere is little doubt that daily carbohydrate intake has been increasing during the past 20 years. The data displayed in Fig. 8 are taken from USDA disappearance data [39] and show this trend quite well. However what is the evidence that carbohydrates actually cause overweight and obesity?\n\nFIGURE 8 U.S. per-capita intake of carbohydrate, 1988\u20131995. [Data from USDA (1996). \"Continuing Survey of Food Intakes by Individuals (CSFII): Diet and Health Knowledge Survey, 1994. Department of Agriculture, Beltsville Human Nutrition Research Center\/ARS.] Beltsville, Maryland.\n\nFigure 9 shows the number of studies that have examined the relationship between carbohydrate intake and body weight and\/or fat composition. The overwhelming majority of these studies find an _inverse_ relationship between carbohydrate intake and body weight, not a direct one. Larger people are associated with eating less carbohydrate, not more. Only two studies failed to find a relationship. No study has found a positive relationship.\n\nFIGURE 9 Correlational studies on the relationship between carbohydrate consumption and BMI or fat composition.\n\nBut what about sugar? Doesn't the consumption of sugar lead to increased body weight? Apparently not. Figure 10 shows the number of studies that have examined the relationship between sugar consumption and body weight. The data are quite similar to the study relating total carbohydrate to body weight\u2014the more sugar consumed, the smaller the body weight. These studies shed considerable doubt on the major premise promulgated by the popular diet books: Body size is not related to the consumption of carbohydrate, but rather to dietary fat or the concentration of energy.\n\nFIGURE 10 Distribution of ecological studies on the relationship between sugar consumption and body weight.\n\n## VI. ENERGY COMPENSATION FOR FAT AND SUGAR SUBSTITUTES\n\nIf we can generalize from the majority of studies cited above that humans appear to demonstrate very little energy compensation for reductions in either dietary fat or carbohydrate, then using palatable, low-calorie fat or sugar substitutes should be an effective way to reduce daily energy intake. Indeed, in many respects, the use of fat substitutes is a better test of the role of dietary fat on energy balance than merely reducing the amount of fat, because it replaces the food with a product of similar properties. The results of studies that have examined the relationship between fat and sugar substitutes and energy intake are presented in Figs. 11 and 12. Similar to the studies on the effect of decreasing fat intake, the majority of published studies demonstrate that humans do not accurately compensate for the reduced energy when the fat in the diet is replaced by a fat substitute, as can be seen in Fig. 11. The study by Cotton _et al._ [49] is particularly important because although they found about 72% energy compensation, the level of dietary fat was reduced to about 20% of energy from 34%. This high degree of fat restriction is substantially more than what was observed in other studies. The study by Kelly _et al._ [50] failed to show a difference in energy intake between a group receiving sucrose polyester in place of dietary fat for a 3-month trial. However, it was very possible that the measure of energy intake (dietary records) was too variable to allow accurate assessment. This interpretation is supported by the fact that the dietary change was sufficient to cause a statistically significant reduction in blood lipid and in body weight of the group receiving the fat substitute. A reduction in energy may have occurred in this group as a function of eating the fat substitutes, but the intake measure could have been too insensitive to detect it.\n\nFIGURE 11 Distribution of studies of energy compensation in response to the consumption of fat substitutes.\n\nFIGURE 12 Distribution of studies of energy compensation in response to the consumption of sugar substitutes.\n\nThe experimental studies on the effects of sugar substitutes on energy intake are even clearer than those for fat substitutes. Figure 12 shows the number of studies where sugar substitutes are used in place of sugars and its effects on intake are measured. As is evident from this figure, the vast majority of studies fail to find energy compensation for the energy lost when the sugar in foods is replaced by noncaloric sweeteners regardless of the particular food in which the sweetener was added, for example, water, soft drinks, yogurt, and cheese. With such a preponderance of studies showing lack of energy compensation when noncaloric sweeteners are substituted for sugar in the diet, one would expect it to be easy to demonstrate that the use of these sweeteners should facilitate weight loss. Unfortunately, the data are not clear and are riddled with controversy.\n\nTwo papers that fueled the controversy appeared in 1986 and caused a stir in the popular press and in the scientific community. In a fascinating, but brief, letter to _The Lancet,_ Blundell and Hill [51] reported that the ingestion of aspartame caused an increase in hunger ratings in human subjects, and in another paper, Stellman and Garfinkel [52] reported that women who consumed the sweetener saccharine were more likely to gain body weight than nonusers of saccharine. Rogers and Blundell [53] extended their finding by demonstrating that following the ingestion of saccharine, not only are subjects hungrier, but they increased their food intake. Thus, rather than being an aid to weight reduction, these studies were suggesting that artificial sweeteners may actually cause a gain in body weight.\n\nConflicts in science usually resolve themselves with replication and increased scrutiny. As evidence in Figure 12 indicates, the effect observed by Rogers and Blundell [53] was never replicated by any other investigator and may be attributed to a type 2 statistical error. Most researchers fail to find energy compensation for sweetener consumption. In a thorough critic of the Stellman and Garfinkel [52] paper, Renwick [54] pointed out major weaknesses of this study that shed considerable doubt as to its conclusion: (1) The data were based on the subject's memory of their body weight, not measured body weight and (2) anyone who actively tried to lose body weight was eliminated from the analysis. Either of these conditions would have led to the _false_ conclusion that weight gain is _related_ to sweetener use.\n\nOther published papers in the literature also shed doubt that low-calorie sweeteners could be a significant aid to weight control. No difference in body weight was observed between low-calorie sweetener users and nonusers [55, 56]. Colditz _et al._ [57] reported a _positive_ relationship between the use of the sweetener saccharine and a gain in body weight. These results would suggest that sweeteners are not effective in reducing body weight. However, it might be argued that those subjects with the largest body weight had the greatest need for sweeteners to reduce their caloric intake, but apparently, this is not the case. Richardson [58] found no difference in sweetener use between people who restrict their sugar intake compared to those who do not. Consequently, the \"need\" to use sweeteners for weight reduction explanation cannot explain these results.\n\nThese studies clearly raise serious doubts as to whether humans use low-calorie sweeteners as substitutes for sugar as originally intended. Even more disturbing from an energetic perspective is that if consumers did substitute low-calorie sweeteners for sugar, then the proportion of their total energy intake derived from fat would increase [59\u201361]. As is evident from the data presented above, increasing the percentage of energy from fat may result in an increase in body weight, not a decrease.\n\nOne of the few studies that examined the issue of how consumers were using sugar substitutes was provided by Chen and Parham [62]. They found that consumers, college students, were not using the sugar substitutes to substitute for carbohydrates, but rather they were consuming foods containing the sweeteners in addition to the sugars consumed in their diet. This pattern of behavior was not seen when low-calorie sweeteners were substituted covertly for sugar in the diet [60]. It seems that the knowledge of the contents of foods drastically changes the decision to consume it or not, an effect well documented in the laboratory [63\u201365], although not universally [66]. Miller _et al._ [67] found that information about the caloric content of potato chips significantly affected restrained eaters, but had no effect on unrestrained eaters.\n\nNevertheless, the relationship between the effectiveness of artificial sweeteners as a weight reduction aid is tenuous at best, particularly in light of the data presented in Fig. 13. This figure shows that the per-capita consumption of low-calorie sweeteners has been increasing, yet so has the incidence of obesity. Despite the fact that a reduction in body weight is the ultimate test of the effectiveness of sweeteners, remarkably few studies have examined this question. An early study by Porikos _et al._ [68] suggested that lean and obese subjects lose weight when low-calorie sweeteners are substituted for sugar and gain weight when the substitutes are replaced by sugar, but several methodological problems leave this conclusion guarded.\n\nFIGURE 13 Prevalence of obesity and consumption of low-calorie sweeteners, 1978\u20131998. [Adapted from 1Mokdad, A. H., Serdula, M. K., Dietz, W. H., Bowman, B. A., Marks, J. S., and Koplan, J. P. (1999). the spread of the obesity epidemic in the United States, 1991\u20131998. _JAMA_ **282,** 1519\u20131522; and 2Calorie Control Council (2000). Most popular low-calorie, sugar free products: Trends and statistics, Calorie Control Council national consumer survey. Available at .]\n\nTordoff and Alleva [69] showed that drinking sodas only sweetened with aspartame for 3 weeks produced a significant loss in weight at the end of a 3-week period. However, the magnitude of the weight changes were less than 1 kg in 3 weeks. Perhaps the best evidence that the use of low-calorie sweeteners can produce weight loss was published by Blackburn and his associates [70] who performed one of the only long-term studies of the effectiveness of sugar substitutes in a weight reduction program. Their results can be seen in Fig. 14. All subjects in this study were prescribed a 4180-kJ diet for 16 weeks, then observed for 2 years. The left panel illustrates that the weight loss in the group who used aspartame was not different from the group that did not use aspartame. However, the right panel shows the typical weight recovery that follows diet-induced weight loss programs. The group that had used aspartame during the dieting phase continued to use it during the follow-up phase and showed less rapid relapse than the group that did not use asparatme. Unfortunately, linear regression analysis of the weight loss at 77 weeks indicated that the sustained weight loss was unrelated to aspartame use. Most probably, the subject's measures of aspartame use were too inaccurate to observe such a relationship. Nevertheless, at present no clear demonstration exists in the literature that sustained use of low-calorie sweeteners produces a significant weight loss.\n\nFIGURE 14 Effect of asparatame on the change in body weight during weight reduction and recovery. [Adapted from Blackburn, G. L., Kanders, B. S., Lavin, P. T., Keller, S. D., and Whatley, J. (1997). The effect of aspartame as part of a multidisciplinary weight-control program on shortand long-term control of body weight. _Am. J. Clin. Nutr._ **65,** 409\u2013418.]\n\n## VII. PROTEIN PARADOX\n\nAlthough all the popular books cited above blame carbohydrates in our diet as being the major culprit responsible for our expanding waistlines, the solution they propose is a diet that is not only low in carbohydrate, but high in protein (30% of energy or greater). Figure 15 shows the relationship between the protein and fat content of 500 randomly selected foods from the USDA food consumption survey [39]. This figure shows clearly that a diet high in protein is very likely to be high in dietary fat.\n\nFIGURE 15 Relationship between protein content and fat content of 500 randomly selected foods. [Data from USDA (1996). \"Continuing Survey of Food Intakes by Individuals (CSFII): Diet and Health Knowledge Survey, 1994.\" Department of Agriculture, Beltsville Human Nutrition Research Center\/ ARS.] Beltsville, Maryland.\n\nThe problem is that the scientific evidence cited above indicates that a high-fat diet would not only be ineffective as a weight reduction tool, but should actually cause a weight gain. Do the gurus of the lay nutrition press know something about macronutrients and body weight that the scientific community does not?\n\nOne possible resolution to this paradox is that protein may cause a better suppression of appetite and food intake than carbohydrate and therefore adhering to a high-protein, high-fat, low-carbohydrate diet is easier than reducing the consumption of high-fat foods. There is reason for suspecting this to be true. It has been known for almost a century that feeding high-protein diets to animals produces a very significant reduction in spontaneous food intake [71\u201379]. Because of the robustness of this phenomenon, many attempts have been made to identify the critical aspects of the protein metabolism that may be responsible for the dramatic suppression in food intake caused by high protein diets [3, 76, 80, 81].\n\nUnfortunately, the literature on the possible appetite-suppressing effects of protein in humans is not clear. Figure 16 indicates that regardless of whether one measures appetite ratings or grams of food consumed, slightly more studies indicate that protein has a greater satiety effect than carbohydrate, but the number of studies is evenly split on this issue when energy intake is measured.\n\nFIGURE 16 Distribution of studies of the relative \"satiety effect\" of protein in relation to carbohydrate on appetite (top) and energy intake (bottom).\n\nSeveral classic, but long forgotten, nutritional studies of chronic feeding of a high-protein diet may shed some light on this protein paradox. McClellan and DuBois [82] fed two (and monitored a third) \"normal\" men who consumed solely a meat diet for a period of 1 year. All three subjects lost body weight. Yudkin and Carey [83] challenged the conclusion of an earlier and provocative paper by Kekwick and Pawan [84] who argued, as do their modern-day counterparts, that diets composed primarily of protein and fat have a particular \"metabolic effect\" that facilitates weight loss. Their subjects maintained exhaustive, weighed dietary records of everything they ate during a 2-week control period, then again during the next 2 weeks, during which time they were instructed to minimize their consumption of carbohydrate and eat as much protein and fat as they desired. The results for all six of their study subjects are shown in Fig. 17. There are several remarkable features about this work that should give us insight as to why the population is buying into the high-protein, high-fat diets, the most important of which is that all the subjects lost weight. The reason for the weight loss is shown in the top left panel of this figure. Restricting the carbohydrate in the diet reduced the total calories consumed. All six subjects did what they were told by reducing the amount of carbohydrate they ate as seen in the top right panel, but their consumption of protein and fat (lower two panels) was unaffected by their restriction of carbohydrate. The average reduction in energy intake was quite large, approximately 37%. In a subsequent replication, Stock and Yudkin [85] examined 11 more subjects in a very similar experimental paradigm. They observed a 33% decrease in energy intake and, again, no change was observed in the amount of protein or fat consumed, only a reduction in carbohydrate intake.\n\nFIGURE 17 Daily caloric and macronutrient intake of six subjects consuming a high-protein, low-carbohydrate diet. [Adapted from Yudkin, J., and Carey, M. (1960). The treatment of obesity by the \"high-fat\" diet: The inevitability of calories. _Lancet_ **2,** 939\u2013941.]\n\nThese two older studies may be criticized because they had subjects record their own data and did not use a crossover design. However, similar observations were made by Skov _et al._ [86], who also observed a reduction in energy intake on a low-carbohydrate, high-protein diet using a more experimentally sophisticated design, more subjects, and measured over a longer period of time (26 weeks). They observed an approximately 18% reduction in energy intake in a group of overweight and obese subjects who ate _ad libitum_ from a menu composed of high-protein (22%), low-carbohydrate (46%) foods prepared by their staff compared to a matched group of subjects who consumed a high-carbohydrate (59%), low-protein (12%) diet.\n\nOne reason why the Skov _et al._ study [86] observed a smaller difference in energy intake than the Yudkin [85] group may be due to the greater degree of experimental control. Skov _et al._ controlled the composition of the diet consumed by their subjects. The reduction in energy derived from dietary carbohydrate was offset by an increase in the amount of energy derived from protein. In the Yudkin studies, the subjects determined the composition of the diet and did not increase the amount of protein in response to the decrease in carbohydrate. This control allowed Skov and his associates to rule out palatability and energy density as factors contributing to their results. What we don't know is what changes the public makes when they try to follow these high-protein, low-carbohydrate diets.\n\nIt appears, therefore, that at least two processes are operating to make the low-carbohydrate, high-protein diet appealing to the public as an easy and effective means of losing weight. Protein appears to have a slight, but significant, satiating property compared to carbohydrate or fat when it is eaten in large amounts consistently. Even more intriguing is the possibility that a large reduction in carbohydrate foods does not result in energy compensation in a free-living population. Thus, without a change in energy expenditure, energy intake will be low, and body weight will be lost. For how long, we don't know. Clearly, this area deserves serious research by the scientific community.\n\n## VIII. SUMMARY AND IMPLICATIONS OF THE RESEARCH ON MACRONUTRIENTS AND INTAKE\n\nIn viewing the long list of studies in macronutrients and food intake, it becomes apparent that humans do not eat for energy. In fact, it appears that humans possess very poor mechanisms to adjust the volume of food they consume in response to alterations in energy density of the foods they consume. If these observations are true, then the use of \"artificial\" sweeteners and fats should successfully cause a sustained reduction in the body weight in our population. Why sweeteners and fat substitutes do not cause a greater weight loss than they do requires more research as to how consumers use these products. If energy concentration is the major determinant of human energy intake, as suggested by this research, then the major thrust of any programs aimed at weight reduction or obesity prevention should concentrate on the consumption of energy-dilute foods such as soups, salads, and casseroles.\n\nFrom a more theoretical perspective, the plasticity of body weight, rather than the constancy of body weight, that occurs when macronutrients are manipulated should evoke a reevaluation of the theory that energy intake in humans is well controlled and that body weight is well regulated. It is quite possible that humans maintain a vestigial process that \"controls\" our behavior of eating, but that this system is easily dominated by more powerful environmental determinants. 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Randomised comparison of diets for maintaining obese subjects' weight after major weight loss: Ad lib, low fat, high carbohydrate diet v fixed energy intake. _Br. Med. J_. 1997;314:29\u201334.\n\n206. Kasim S.E., Martino S., Kim P.N., Khilnani S., Boomer A., Depper J., Reading B.A., Heilbrun L.K. Dietary and anthropometric determinants of plasma lipoproteins during a long-term low-fat diet in healthy women. _Am. J. Clin. Nutr_. 1993;57:146\u2013153.\n\n207. Schlundt D.G., Hill J.O., Pope-Cordle J., Arnold D., Virts K.L., Katahn M. Randomized evaluation of a low fat ad libitum carbohydrate diet for weight reduction. _Int. J. Obes. Relat. Metab. Disord_. 1993;17:623\u2013629.\n\n208. Sheppard L., Kristal A.R., Kushi L.H. Weight loss in women participating in a randomized trial of low-fat diets. _Am. J. Clin. Nutr_. 1991;54:821\u2013828.\n\n209. [see comments]Ornish D., Brown S.E., Scherwitz L.W., Billings J.H., Armstrong W.T., Ports T.A., McLanahan S.M., Kirkeeide R.L., Brand R.J., Gould K.L. Can lifestyle changes reverse coronary heart disease? The Lifestyle Heart Trial. _Lancet_. 1990;336:129\u2013133.\n\n210. Hill J.O., Melanson E.L., Wyatt H.T. Dietary fat intake and regulation of energy balance: Implications for obesity. _J. Nutr_. 2000;130:284S\u2013288S.\n\n211. Food Marketing Institute. _Trends in the United States\u2014Consumer Attitudes and the Supermarket_. Ithaca, NY: Food Marketing Institute; 1999.\n\n212. Mokdad A.H., Serdula M.K., Dietz W.H., Bowman B.A., Marks J.S., Koplan J.P. The spread of the obesity epidemic in the United States, 1991\u20131998. _JAMA_. 1999;282:1519\u20131522.\n\n213. Available atCalorie Control Council. Most popular low calorie, sugar free products: Trends and statistics, Calorie Control Council national consumer survey. , 2000.\nCHAPTER 34\n\nBehavioral Risk Factors for Obesity: Diet and Physical Activity\n\nNANCY E. SHERWOOD, MARY STORY and DIANNE NEUMARK-SZTAINER, University of Minnesota, Minneapolis, Minnesota\n\n## I. INTRODUCTION\n\nObesity is a significant problem that affects children, adolescents, and adults across gender, race, and socioeconomic strata. Dramatic increases in the prevalence of obesity in recent years have focused attention on this important public health problem. Comprehensive data of trends in the prevalence of obesity provided by national surveys (NHES I, 1960\u20131962; NHANES I, 1971\u20131974; NHANES II, 1976\u2013980; NHANES III, 1988\u20131994) show that the percentage of obese adults has increased over time, particularly during the past two decades (Fig. 1) [1]. The percentage of adults classified as obese, that is, those with a body mass index (BMI) \u2265 30 kg\/m2, increased from 14% in NHANES II to 23% in NHANES III. An additional 32% of adults in NHANES III were classified as overweight according to the BMI \u2265 25 kg\/m2 standard. According to these national data, an estimated 97 million adults (or 55% of the adult population) are classified as overweight. Prevalence rates of obesity and overweight among children and adolescents have also increased dramatically since the mid-1960s and appear to be on the rise. NHANES data indicate that the prevalence of overweight (BMI \u2265 95th percentile) among youth doubled from 1976\u20131980 to 1988\u20131994, increasing from 8% to 14% for 6\u201311 year olds and from 6% to 12% for 12\u201317 year olds [2]. Currently about 11% of U.S. children and adolescents are overweight, and an additional 14% have a BMI between the 85th and 95th percentiles, indicating risk of overweight (Fig. 2). The higher prevalence of obesity among children is of particular concern given that childhood-onset obesity often tracks with adult obesity [3].\n\nFIGURE 1 Proportion of overweight (BMI 26\u201329.9 kg\/m2) and obese (BMI \u2265 30 kg\/m2) adult men and women (by gender for selected years, United States 1960\u20131994). [Adapted from National Institutes of Health, National Heart, Lung and Blood Institute (1999). \"Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults.\" NHLBI, National Institutes of Health, Bethesda, MD.]\n\nFIGURE 2 Proportion of overweight children and adolescents (by age for selected years, United States, 1963\u20131994). Overweight is defined as at or above the gender- and age-specific 95th percentile of BMI based on a preliminary analysis of data from the year 2000 growth charts. [Adapted from Troiano, R. P., and Flegal, K. M. (1998). Data reported in: Overweight children and adolescents: Description, epidemiology, and demographics. _Pediatrics_ **101** (Suppl. 1), 497\u2013504. Reproduced with permission from _Pediatrics,_ Vol. 101, Pages S497\u2013S504, Table 4, copyright 1998.]\n\nThe alarming increase in the prevalence of obesity during the last few decades has raised concerns about associated health risks for children, adolescents, and adults. Persistence of this trend could lead to substantial increases in the number of people affected by obesity-related health conditions and premature mortality. The health risks associated with obesity are numerous and include hypertension, type 2 diabetes mellitus, dyslipidemia, stroke, gallbladder disease, osteoarthritis, sleep apnea, respiratory problems, and certain cancers (e.g., endometrial, breast, prostate, and colon) [3, 4]. Obesity is also associated with psychosocial problems such as binge eating disorders and depression for some individuals [5]. Individuals who are obese are also adversely impacted by social bias and discrimination [6\u20138]. The economic burden of obesity is sizable because of its impact on individual health, costs to society due to lost productivity, and premature mortality and treatment costs [9\u201311]. Estimates of the economic burden of obesity are as high as $100 billion per year [4].\n\nObesity and overweight are multidetermined chronic problems resulting from complex interactions between genes and an environment characterized by energy imbalance due to sedentary lifestyles and ready access to an abundance of food [3]. Research suggests that obesity runs in families and that some individuals are more vulnerable than others to weight gain and developing obesity [12]. Various mechanisms through which genetic susceptibility to weight gain have been proposed include low resting metabolic rate, low level of lipid oxidation rate, low fat-free mass, and poor appetite control [3]. Genetic research holds considerable promise for understanding the development of obesity and identifying those at risk for obesity. However, the rapid increase in rates of obesity has occurred over too brief a time period for there to have been significant genetic changes in the population. Although body weight is primarily regulated by a series of physiological processes, it is also influenced by behavioral and environmental factors [3]. Recent epidemiological trends in obesity have been linked to behavioral and environmental changes that have occurred in recent years. The higher proportion of fat and the higher energy density of the diet in combination with reductions in physical activity levels and increases in sedentary behavior have been implicated as significant contributors to the obesity epidemic [3, 4]. Importantly, these dietary and activity behavioral risk factors are modifiable and can be targets for change in obesity prevention and treatment efforts.\n\nUnderstanding the determinants of obesity and developing appropriate prevention and treatment strategies requires an in-depth examination of behavioral risk factors for obesity. The goal of this chapter is to review available data regarding behavioral and environmental determinants of dietary intake and physical activity and to discuss implications for future public health research and intervention. The review will focus on behavioral risk factors for obesity in both children and adults. Figure 3 presents a conceptual model for understanding how behavior risk factors influence weight regulation and obesity.\n\nFIGURE 3 Influences on energy balance and obesity. This model shows the direct and indirect influence of biological susceptibility on energy regulation and obesity. This model also shows that both biological susceptibility and environmental factors influence dietary intake and physical activity, which, in turn, directly affect energy regulation and obesity. [Adapted with permission from World Health Organization (1997). \"Obesity: Preventing and Managing the Global Epidemic,\" Report of a WHO Consultation on Obesity, Geneva, June 3\u20135, 1997. World Health Organization, Geneva.]\n\n## II. PHYSICAL ACTIVITY\n\nThis section examines the role of physical activity in the development of obesity. Multiple factors that influence physical activity levels will be discussed, including sedentary behavior, psychological variables such as self-efficacy, social support, and environmental and societal influences.\n\nProminent among the health benefits associated with a physically active lifestyle is the protective effect of physical activity on obesity. An abundance of cross-sectional research shows that lighter individuals are more active than heavier individuals and prospective research indicates that changes in physical activity level are associated with changes in body weight in the direction predicted by the energy balance equation [13\u201322]. The majority of studies conducted in children also find that physical activity levels and body weight are negatively associated [23]. Exercise has also been shown to improve short- and long-term weight loss in experimental studies in both children and adults [19, 23].\n\n### A. Prevalence of Leisure-Time Physical Activity in Adults\n\nDespite the benefits of physical activity for body weight regulation and health, we are in the midst of a sedentary behavior epidemic. Only 15% of U.S. adults engage regularly in vigorous physical activity during leisure time, defined as 3 times a week for a minimum of 20 minutes [24]. Only 22% of adults report engaging in regular physical activity, defined as a minimum of 30 minutes of moderate to vigorous activity on most days of the week [24]. Twenty-five percent of adults report that they never engage in physical activity during leisure time [24]. Demographic differences in physical activity levels have also been observed, with men more likely to be physically active than women [25]. Approximately 60% of American women do not participate in any form of regular physical activity [26, 27]. Physical activity also declines with age, with women experiencing a greater decline in older age groups than men [27]. African-American and Hispanic adults are less physically active than Caucasians [24, 26]. Education and income are also both positively associated with physical activity level [25, 28\u201330].\n\n### B. Prevalence of Leisure-Time Physical Activity in Youth\n\nAlthough estimates of physical activity levels among youth are slightly higher than self-reports by adults, the prevalence of regular physical activity is still surprisingly low. According to data from the Youth Risk Behavior Survey (YRBS) and the National Health Interview Survey\u2013YRBS, about half of young people in the United States (age 12\u201321) regularly participate in vigorous physical activity, with one-fourth reporting no vigorous physical activity and 14% reporting no vigorous or light to moderate physical activity [24]. Data from the National Children and Youth Fitness studies [31, 32] indicate that about 60% of children in elementary through the high school years reported engaging in moderate to higher intensity physical activities throughout the year. Similar to activity patterns with adults, girls tend to be less active than boys and declines with age are more striking for girls compared to boys [24]. African-American girls, in particular, appear to be at greater risk for inactivity [24]. Representative survey data on the physical activity patterns of young children are not available, in part because of methodological difficulties in collecting such data from children. Young children are limited in their ability to accurately recall their activity patterns and the unplanned, unstructured nature of children's physical activity patterns does not lend itself well to the self-report format employed in large-scale surveys.\n\n### C. Sedentary Behavior\n\nAlthough low levels of leisure-time physical activity likely contribute to the epidemic of obesity, it is noteworthy that among adults, leisure-time activity has remained stable or increased since the mid-1980s, the time period during which the prevalence of obesity increased [24]. The past century, however, has produced dramatic changes in physical activity patterns in the United States. Machines with motors have replaced human labor in virtually every aspect of life, so that the energy expenditure now required for daily life is a fraction of what it was a generation or two ago. The consequences of this dramatic change are far reaching and only now are beginning to be carefully studied. It is likely that increases in sedentary activities such as television watching and computer use and decreases in lifestyle, household, and occupational activity that have been less carefully measured have contributed to reductions in overall energy expenditure at the population level. Television viewing is a major source of inactivity and has received considerable attention as a risk factor for obesity [33\u201335]. In addition to potentially contributing to lower energy expenditure by displacing time potentially spent in more active pursuits, television viewing has been hypothesized to contribute to excess energy intake. Television watching can serve as a cue for eating given the numerous references to food and commercials for food, often high-fat, high-calorie foods, on television [35, 36].\n\nAccording to data provided by A.C. Nielsen Company, the average household television set was turned on for more than 7 hours per day in 1998 [37]. Survey data estimating the frequency of television watching are necessary, however, because television viewing is not necessarily the primary activity when the television set is turned on. Data from the Americans' Use of Time study show that free time spent watching television increased from about 10.4 hours per week in 1965 to about 15.1 hours in 1985 [38]. Survey data from adults also indicate that time spent watching television averages about 2\u20133 hours a day [39\u201341]. Dietz and Gortmaker [35] report that children in the United States spend, on average, as much time watching television each year as they do attending school. Data from the 1990 YRBS showed that more than 70% of students in grades 9 through 12 reported watching at least 1 hour of television each school day and more than 35% reported watching television 3 or more hours per day [42]. Other national data show that the average amount of television viewing is about 5 hours per day among 10- to 15-year-old children [33]. About one-third of these children watched more than 5 hours per day and only 1% watched 0\u20132 hours per day.\n\nCross-sectional research has shown that there is generally a positive relationship between television watching and obesity in children [33, 35, 43\u201355] and adults [39, 40, 56, 57]. Prospective relationships between television watching and weight are unclear with some studies showing that frequency of television watching is weakly or moderately associated with future weight gain [35, 58, 59], and other studies not detecting an association [39]. Although it has been hypothesized that television watching influences obesity by replacing time that could otherwise be spent engaging in more active pursuits, only some studies have shown that children and adults who watch more television are less physically active [43, 48, 52, 56, 57]. Crawford _et al._ [39] caution that the link between obesity and television watching appears to be complex and that targeting television viewing alone would not be an adequate public health strategy for addressing the obesity epidemic. They also suggest that better ways of conceptualizing and measuring sedentary behavior are necessary to fully understand its impact on the development of obesity.\n\nDespite the fact that relationships between sedentary behavior and obesity are somewhat unclear, intervention research with children and adolescents that has focused on reducing television watching shows promise as an obesity prevention and treatment strategy. The work of Epstein _et al._ [36, 60\u201362] has figured prominently in the literature on decreasing sedentary activity as a strategy for promoting higher levels of physical activity. According to Epstein, the principles of behavioral economics or behavioral choice theory can be applied to sedentary individuals who, given the opportunity to choose between sedentary and physically active alternatives, will consistently choose the sedentary alternative. Choice of a given alternative, in this case, sedentary behavior, depends on the behavioral \"cost\" of that choice. Epstein argues and has demonstrated empirically in the laboratory that reducing the accessibility of sedentary behaviors or increasing the cost of being sedentary are both methods for reducing sedentary behavior. Epstein and colleagues [60] have also demonstrated that obese children participating in family-based weight control programs show the best changes when they are reinforced for being less sedentary as opposed to being reinforced for being more active.\n\nSchool-based research targeting change in sedentary behaviors also shows promise. Robinson [63] conducted a small randomized trial in which one elementary school received an 18-less on, 6-month classroom curriculum to reduce television, videotape, and video game use, and one school served as the control group. At follow-up, children in the intervention school had lower BMIs, tricep skinfold thicknesses, waist circumferences, and waist-to-hip ratios relative to children in the control school. Significant differences were also observed in children's reported television viewing and meals eaten in front of the television although no significant differences were observed for moderate-to-vigorous physical activity levels, cardiorespiratory fitness, or high-fat food intake. The Planet Health Program, another school-based obesity prevention trial, found that the decreasing television component of their multicomponent program appeared to contribute most to the decrease in prevalence of obesity observed among girls [64]. The effectiveness of reducing sedentary behaviors in preventing and treating obesity clearly deserves further exploration. One reason for the success of such strategies may be the simplicity or clarity of the intervention message.\n\n### D. Self-Efficacy for Physical Activity\n\nExercise self-efficacy is one of the strongest and most consistent predictors of exercise behavior. Self-efficacy predicts both exercise intention and several forms of exercise behavior [18, 65\u201375]. Self-efficacy is an individual's beliefs in his or her ability to successfully engage in a given behavior. It is theorized to influence the activities that individuals choose to approach, the effort expended on such activities, and the degree of persistence demonstrated in the face of failure or aversive stimuli [76]. Exercise self-efficacy is the degree of confidence an individual has in his or her ability to be physically active under a number of specific\/different circumstances or, in other words, efficacy to overcome barriers to exercise [67]. Among adults, self-efficacy is thought to be particularly important in the early stages of exercise [71]. In the early stage of an exercise program, exercise frequency is related to one's general beliefs regarding physical abilities and one's confidence that continuing to exercise in the face of barriers will pay off. Self-efficacy has also been shown to be highly related to physical activity in youth. Children with higher perceived self-efficacy for physical activity and for overcoming barriers to physical activity were more active [77\u201380]. Gender differences in self-efficacy have also been observed, with boys reporting higher self-efficacy than girls [80].\n\nGiven that self-efficacy for exercise is such a strong predictor of exercise behavior, enhancing self-efficacy should figure prominently in physical activity intervention programs [81]. Strategies for enhancing self-efficacy could include teaching individuals how to overcome barriers to exercise, creating opportunities for positive experiences with physical activity, and providing positive feedback regarding exercise performance [72, 81, 82]. Self-monitoring can be also be used as a tool for those who exercise on their own to monitor performance and provide evidence regarding physical activity accomplishments [81, 83]. Emphasis should also be placed on increasing girls' self-efficacy for exercise, given observed gender differences in exercise self-efficacy and physical activity levels.\n\n### E. Exercise History\n\nPrior history of physical activity should positively influence future physical activity behavior by promoting and shaping self-efficacy for exercise and by developing physical activity skills. The observed relationship between exercise history and exercise behavior varies, however, depending on how exercise history is defined and the time period over which physical activity behavior is \"tracked.\" Physical activity has been shown to track in early childhood [84]. Recent exercise history is generally predictive of future exercise behavior [85]. Childhood exercise history, however, is inconsistently related to physical activity in adulthood [68, 86]. Childhood physical activity experiences are also only modestly predictive of adult self-efficacy and exercise behavior [86]. The perception of the exercise experience as a child may be as important as amount of childhood exercise. One recent study found that recalling being forced to exercise as a child was associated with lower levels of physical activity in adulthood [87]. A child's enjoyment of physical activity [78, 80] and enjoyment of physical education experiences [88] are significant predictors of physical activity levels. Creating positive environments for physical activity for youth is likely a key factor in promoting higher levels of physical activity as a lifestyle habit.\n\n### F. Social Support for Physical Activity\n\nSocial support is another strong correlate of physical activity for both youth and adults. Adults who engage in regular exercise report more support for activity from people in their home and work environments [18, 68, 89, 90]. Adults who are initiating exercise programs are more likely to perceive their families as being supportive of their desire to maintain good health [91]. Additionally, individuals who joined a fitness program with their spouse had higher rates of adherence at 12 months compared to those who joined without a spouse [92]. Carron _et al._ [93] examined six major sources of social influence on physical activity, including important others such as physicians or work colleagues, family member, exercise instructors or other in-class professionals, co-exercisers, and members of exercise groups in a comprehensive review. The authors concluded that social influence generally has a small to moderate effect on exercise to behavior. Moderate to large effect sizes were found for family support and attitudes about exercise, important others and attitudes about exercise, and family support and compliance behavior.\n\nFamily support for physical activity is a robust correlate of physical activity for both boys and girls [77\u201379, 88]. Parental prompts for children to play outdoors instead of watching television or playing video games have been shown to positively influence children's activity levels [94]. Parental prompts to be active have also been shown to be related to young children's activity levels in some [95\u201397], but not all studies [98, 99]. Taylor _et al._ [100] argue that age of the child needs to be taken into account when understanding the impact of parental behavior on children's activity levels. Specifically, as children make the transition to school and spend more time in broader social contexts, other social influences may become more prominent. Not surprisingly, social support for physical activity from peers is also positively correlated with physical activity levels [31, 79]. Taylor _et al._ [100] also suggest that attention should be paid to children's perception of parents' social influence and potential adverse forms of social influence such as nagging, discouragement, or excessive pressure to be physically active.\n\n### G. Barriers to Physical Activity\n\n1. TIME\n\nAmong adults, time constraints are the most frequent barriers to exercise and are reported by both sedentary and active individuals [89, 101]. Even among regular exercisers, scheduling efficacy remains an important and significant predictor of adherence [67]. Therefore, to maintain exercise adherence, regular exercisers have to become adept at dealing with time as a barrier. The time barrier may be a particular problem for certain population subgroups. For example, Schmitz _et al._ [102] reported that becoming a parent is associated with reductions in physical activity for mothers. Time spent caring for children may make it difficult for parents to maintain a regular physical activity program.\n\nSeveral physical activity intervention approaches geared toward addressing the time barrier have been developed in recent years. Although group exercise programs can provide support and structure for participants, these advantages may be outweighed by the long-term costs involved in traveling to exercise sites at specific times for physical activity involvement [103]. Home-based physical activity programs appear to be a positive option for many adults [89, 104\u201308]. In the context of a weight loss program, Perri and colleagues [105] compared the effects of two exercise regimens, a group-based program versus a home-based program, on exercise participation, adherence, and fitness. Their results indicated that participants in the home-based program demonstrated better adherence and exercise performance data, particularly at 12-month follow-up. Two recent studies, which compared structured exercise program to a lifestyle approach, reported that the lifestyle program was as effective as the structured exercise program in improving physical activity and health outcomes including body weight and cardiorespiratory fitness [107, 108]. The recent focus on the health benefits of short bouts versus long bouts of activity given the public health recommendation to accumulate 30 minutes of physical activity on most days of the week also has implications for addressing the problem of time as a barrier to activity [109\u2013113]. Short bouts of activity may be easier for people to incorporate into their schedules and may be particularly suitable for those initiating physical activity. Research suggests that multiple short bouts of exercise have been effective at promoting adherence to exercise programs [107, 108, 112, 113]. Interestingly, Epstein and colleagues [62, 114] have also reported that a lifestyle approach to exercise is associated with better adherence and greater weight loss in children.\n\n#### 2. ACCESS\n\nAnother barrier that has received some attention in the determinants literature is access to exercise facilities. Distance between individuals' homes and exercise facilities has been shown to be negatively correlated with exercise behavior in adults [115]. Depending on an individual's activity preference, access to exercise facilities may or may not be related to exercise levels. For those individuals who prefer exercises such as walking or running, which can be done anywhere, access to facilities may be less relevant. Additionally, for those who exercise with home equipment, which could include stationary bikes, treadmills, and even exercise videos, access to facilities may also not affect exercise adherence. Regardless, the extent to which environments are conducive to physical activity (i.e., walking\/biking paths, safe streets) likely has a strong impact on population activity levels. One recent study examining the association between neighborhood safety and sedentary behavior in a population-based sample found that there was a higher prevalence of physical inactivity among persons who perceived their neighborhoods as unsafe [116]. Better measurement of environmental resources for physical activity and strategies for improving access to physical activity facilities are needed.\n\n#### 3. ENVIRONMENTAL FACTORS\n\nPhysical activity among youth appears to be particularly strongly influenced by environmental factors. First of all, the amount of time children spend playing outside has been shown to be a strong correlate of physical activity levels [98, 117]. Clearly, children who live in neighborhoods where play spaces are not adequate are going to have more difficulty achieving recommended levels of physical activity. Use of after-school time for sports and physical activity [88], access to community sports activities [80], and frequency of parents transporting children to activity locations have all been shown to be correlates of physical activity in boys and girls [99, 118]. The extent to which families have time and resources to support their children in physical activity pursuits will also have a strong impact on children's activity levels. Anecdotal reports suggest that children spend less time in unstructured physical activities (e.g., neighborhood pick-up games, hide and seek, tag) than in previous years. In contrast, there appears to have been an increase in community-organized sports (e.g., traveling soccer and basketball teams) that require increased parental time, involvement, and financial resources. These factors may potentially contribute to decreases in physical activity and increased socioeconomic differences in physical activity and obesity risk among youth and is an area worthy of further exploration.\n\n#### 4. OVERWEIGHT AND\/OR DISCOMFORT WITH PHYSICAL ACTIVITY\n\nClearly, body weight and physical activity are inextricably linked. Although it is clear that increasing physical activity is an important factor in regulating body weight, weight status may serve as a barrier to physical activity. Sedentary individuals may be heavier than those who initiate exercise programs [91]. This is due in part to physical activity being less pleasurable (i.e., it is uncomfortable for people to exercise when they are heavier), and in part because of embarrassment (i.e., individuals report feeling embarrassed about being seen in public in exercise clothes, at gyms, etc., due to weight status). However, weight status can also be a motivator for initiating exercise. One of the most common reasons adults give for exercising is weight control. Also, dieting to control weight is positively associated with frequency of participation in both high and moderate intensity physical activity [16]. Physical activity promotion programs, however, need to be modified to address the needs of overweight youth and adults.\n\nAnother important aspect of physical activity promotion for weight control is how much exercise to recommend. Data from studies of successful weight loss maintainers suggest that the optimal exercise recommendations for weight control likely exceed current public health recommendations. Optimal exercise levels for weight maintenance may be close to 1 hour of moderate to vigorous activity on most days of the week [119, 120]. This higher level of activity may be perceived as intimidating and difficult to achieve for those who have a history of sedentary behavior and are overweight. Strategies and support for helping individuals gradually increase their physical activity level while remaining injury free are needed.\n\n## III. DIETARY FACTORS\n\nThis section examines the role of dietary factors in the development of overweight and obesity. Multiple factors that influence food intake will be discussed, including the macronutrient composition of the diet, environmental and societal influences, and specific eating patterns.\n\n### A. Macronutrient Composition\n\n1. ENERGY INTAKE\n\nThe relationship between trends in obesity and in greater fat or energy intake has not been clearly established. Laboratory experiments in animals and human clinical studies have repeatedly shown that the level of fat and energy intake in the diet is strongly and positively related to excess body weight. In contrast, population-based surveys of diet and obesity have reported inconsistent results [3].\n\nIn adults, data from two national surveys suggest that energy intakes have increased slightly [121, 122], while in one national survey energy intake declined [123]. Findings from the USDA 1977\u20131978 Nationwide Food Consumption Survey (NFCS) and the USDA 1994\u20131996 Continuing Survey of Food Intakes of Individuals (CSFII) indicate that reported daily energy intakes increased from 2239 to 2455 calories in men and from 1534 to 1646 in women [122, 124]. Findings from NHANES I (1971\u20131974) and NHANES II (1976\u20131980) found that daily mean energy intakes were approximately 100\u2013300 calories higher in NHANES III (1988\u20131994) compared to NHANES II [121]. Dietary data from the 1987 and 1992 National Health Interview Surveys suggest that energy intake declined slightly between 1997 and 1992 (on average about 100 calories\/day) for adults [123].\n\nSecular trends in energy intakes of youth aged 2\u201319 have also been examined. Data from NHANES found that mean energy intake changed little from the 1970s to 1988\u20131994, except for an increase among adolescent girls [125]. Between NHANES II and NHANES III, mean energy intake increased 1\u20134% among most age groups under age 20; mean intakes declined 3% for ages 6\u201311 years and increased 16% in females ages 16\u201319 years [126]. Among adolescent girls, energy intake increased by 225 calories. The increase between surveys for black females aged 12\u201319 was even larger at 249 calories. Mean energy intakes from the USDA's national food consumption surveys showed little change for young children and slightly lower mean intakes for adolescents in 1989\u20131991 compared to 1977\u20131978 [124].\n\nThe inconsistencies in secular-trend surveys have been attributed to a number of factors, including weaknesses in the study design, methodological flaws, confounders, and random or systematic measurement error in the dietary data [3]. For example, the procedural changes between NHANES II and III in dietary survey methodologies, and survey food coding and nutrient composition databases make comparisons between the two surveys difficult [121]. Some evidence suggests that people participating in nutrition surveys underreport the food they eat, either by completely omitting food items or by inaccurately estimating the amount eaten [122]. Underreporting of food intake is discussed below.\n\nThe mixed dietary results of national surveys and the paradoxical findings that energy and fat intakes have decreased or stayed about the same, despite an increase in overweight prevalence, has led some researchers to conclude that the observed trend in obesity is not related to a shift in energy and fat intake but rather a decline in physical activity. Secular trends in energy intake suggest that increased intake over time is not the major contributor to the increased prevalence of overweight among Americans and that decreased physical activity may play a more important role in overweight population prevalence 125]. However, given the problems inherent in measuring dietary intakes, and methodological survey issues, it should be noted that under more controlled laboratory conditions consistent findings show a strong association between dietary factors and obesity [3] (see [Chapter 2, Energy Requirement Methodology).\n\nIn understanding the development of obesity, an important question is whether obese individuals eat more than leaner individuals. In most studies with both adults and children, energy intake has not been found to correlate with the degree of obesity [24]. However, this may be due, in part, to underreporting of food intake by the obese.\n\n#### 2. UNDERREPORTING OF FOOD INTAKE\n\nIn contrast to measures of body weight, dietary intake is difficult to measure accurately. Underreporting must be considered when interpreting dietary survey data. Studies have documented that food consumption is underreported by about 20\u201325% by people participating in dietary studies and occurs more often in women, overweight persons, and weight-conscious persons 121, 127, 128]. Discrepancies-between reported energy intakes and measured energy expenditures (with the doubly labeled water method) of 20\u201350% have been described in overweight individuals [127, 129]. The systematic bias of underreporting in both overweight and nonoverweight individuals may be due to socially desirable responses, poor memory for foods consumed, lack of awareness of food consumed, difficulty with portion size estimation, or undereating (consuming less food than usual because of the requirement to record food intake). A recent study of underreporting of habitual food intake in obese men found that about 70% of the total underreporting was due to a diminished intake of food over the reporting period; that is, subjects changed their food patterns during the recording period [129]. Selective undereporting of fat intake was also found. The magnitude of underestimations observed in various studies indicates the considerable error in dietary intake data and highlights the need for improved techniques of data collection [130] (see discussion in [Chapter 2).\n\n#### 3. FAT INTAKE\n\nDietary fat has a higher energy density than either protein or carbohydrates. Controversy remains about whether the percentage of dietary fat plays an important role in development of obesity and in its treatment once it has developed [131, 132]. There is evidence that consumption of high-fat diets increases total energy intake and that excess dietary fat is stored with a greater efficiency than similar excesses of dietary carbohydrate or protein [131, 133]. On the other hand, it has been argued that in short-term studies only a modest reduction in body weight is typically seen in individuals assigned to diets with a lower percentage of dietary fat, which suggests that dietary fat does not play a role in the development of obesity [132]. Results of studies in laboratory animals and metabolic studies clearly and strongly show that a high percentage of fat in the diet relative to other macronutrients contributes to the development of obesity. These kinds of research, however, do not prove causality in humans [131, 133, 134]. It has also been pointed out that the focus on dietary fat may have been overemphasized at the expense of total energy intake. Total energy balance is what matters most and the focus on dietary fat intake must be viewed through its effects on total energy intake [133]. Reduction of dietary fat is one of the most practical ways to reduce energy density of the diet.\n\nNational data show that Americans have dramatically lowered the percent of energy intake from total fat during the last three decades. The reduction is from about 45% of energy in 1965 to about 34% in 1995 [135]. Levels, though, continue to be higher than the 30% recommended. Interestingly, the percent of calories from fat continued to decrease from 1990 to 1995 even as the daily grams of fat intake remained steady or increased. The explanation for this paradox is that although daily fat consumption was increasing or remained unchanged, the total caloric intake was increasing at a faster pace. A higher number of calories consumed will reduce the percentage of calories from fat even when there is no decrease in total fat consumption. Therefore, the decrease in percent of calories from fat observed recently may be a result of increased total caloric intake and not necessarily due to decreased fat consumption [135].\n\n#### 4. SUGAR INTAKE\n\nAmericans are consuming record high amounts of caloric sweeteners, mainly sucrose and corn sweeteners. Per-capita consumption increased 45 pounds or 41% between 1950\u20131959 and 1997 [136]. In 1997, Americans consumed on average 154 pounds of caloric sweeteners and when adjusted for losses this is 33 teaspoons of added sugar per person per day [137]. Regular (nondiet) soft drinks are the major contributor of added sweeteners in the American diet and account for one-third of the intake of added sweeteners [138]. There has been a 47% increase in annual per-capita consumption of regular carbonated soft drinks, from 28 gallons per person in 1986 to 41 gallons in 1997.\n\nGuthrie and Morton [138] found increased intake of regular soft drinks to be one of the major changes in children's diets between 1989\u20131991 and 1994\u20131995. High soft drink consumption may lead to excessive energy intake (a 12-ounce soft drink contains approximately 150 kcal), which theoretically may contribute to obesity. Harnack and colleagues [139] found that energy intake was positively associated with consumption of regular soft drinks. Energy intake was higher for those in the highest soft drink consumption category. In this study the association of obesity and soft drink consumption was not assessed. However, using NHANES III data, Troiano _et al._ [125] reported that soft drink energy contribution was higher among overweight children and adolescents.\n\nThere is no consensus regarding the role of sugar intake on body weight regulation. A preference for sweet-fat foods has been observed in obese individuals, which may be a factor in promoting excess energy consumption [3]. However, the notion that a sensory \"sweet tooth,\" that is, a heightened preference for sweet taste, is a direct cause of obesity is not well supported [140].\n\n### B. Environmental Influences\n\n1. U.S. FOOD SUPPLY\n\nData of dietary levels of individuals provide key information on energy and nutrient intakes. However, food supply data can also provide a measure of changes in food consumption over time and estimated nutrient content of the food supply. Food supply data, also known as food disappearance data, reflect the amount of food commodities entering the market, regardless of final use. USDA's Economic Research Service (ERS) has developed methods to adjust the food supply for spoilage, plate waste, and other losses. USDA's Center for Nutrition Policy and Promotion provides food supply nutrient estimates derived from the ERS data [137]. These data are used as a proxy to estimate human consumption. Adjusted food supply data suggest that average daily calorie intake increased 14.7%, or about 340 calories, between 1984 and 1994.\n\nUSDA food supply data indicate that Americans are consuming record amounts of some high-fat dairy products (e.g., cheese) and caloric sweeteners and near record amounts of added fats, including salad and cooking oils and baking fats. The hefty increase in grain consumption reflects higher consumption of mostly refined rather than high-fiber, whole-grain products [136]. On the positive side, fruit and vegetable consumption continues to rise. Americans consumed about a fifth (22%) more fruit and vegetables in 1997 than in the 1970s [137]. Supermarket produce departments carry more than 400 produce items today, up from 250 in the late 1980s and 150 in the mid-1970s. Also, the number of ethnic and natural foodstores that offer fresh produce continue to increase [137].\n\nThe food industry has responded to consumer demand for lower fat products. For example, 2076 new food products introduced in 1996 claimed to be reduced in fat or fat free\u2014nearly 16% of all new food products introduced that year, and more than twice the number just 3 years earlier. The number dropped in 1997 and it is unclear whether that represents a backlash to health concerns [141]. The Calorie Control Council reports a notable rise in the percentage of the U.S. population consuming low-calorie products: 19% of the population in 1978, 29% in 1984, and 76% in 1991 [142]. Still, the use of these products has not prevented the progression of obesity in the population. It should be pointed out that many commercially available low-fat or fat-free foods are not lower in energy density than their full-fat counterparts [143].\n\n#### 2. EATING OUT\n\nDuring the past 20 years, one of the most noticeable changes in eating patterns of Americans has been the increased popularity of eating out [144]. The proportion of meals and snacks eaten away from home increased by more than two-thirds between 1977\u20131978 and 1995, rising from 16% of all meals and snacks in 1977\u20131978 to 27% in 1995 [145]. Almost half of all adults (46%) were restaurant patrons on a typical day during 1998 [146]. Currently, almost half (47%) of a family's food budget is spent on away-from-home food [147]. In 1970, the family food expenditure on away-from-home eating was only 26% of total food spending [145]. The restaurant industry projects that in the 2000s, 53% of the food dollar will be spent away from home [146]. Food away from home includes foods obtained at restaurants, fast-food places, school cafeterias, and vending machines. A number of factors account for the increasing trend in eating out, including a growing number of working women (75% of women 25\u201350 years old are in the work-force), more two-earner households, higher incomes, a desire for convenience foods because of busy lifestyles and little time for preparing meals, more fast-food outlets offering affordable food, smaller families, and increased advertising and promotion by large food service chains and fast-food outlets [144].\n\nThe trend in eating out may be related to the observed increase in caloric intake among Americans, because food away from home is generally higher in calories and fat than food consumed at home [137, 145]. Many table-service restaurants provide 1000\u20132000 calories per meal, amounts equivalent to 35\u2013100% of a full day's energy requirement for most adults [148]. Data from USDA's food intake surveys conducted during the past 20 years have been analyzed to compare the nutritional qualities of at-home and away-from-home foods and changes over time. In 1995, the average total fat and saturated fat content of away-from-home foods, expressed as a percentage of calories, was 38% and 13%, respectively, compared with 32% and 11% for at-home foods [145]. Foods eaten away from home provided 34% of total food energy consumption in 1995, up from 19% in 1977\u20131978. The 1995 data also suggest that, when eating out, people tend to eat larger amounts, eat higher calorie foods, or both [137]. Lin and colleagues [144] at the USDA calculated that if away-from-home food had the same average nutrient densities as food at home in 1995, Americans would have consumed 197 fewer calories per day, and reduced their fat intake to 31.5% of calories from fat (instead of 33.6%).\n\nConsumers may view food differently when eating out than when eating at home. Consumers may view eating away from home as an exception to their usual dietary patterns, regardless of how frequently it occurs, and an opportunity to \"splurge\" [149]. Consumers also may not be aware of the fat or calorie content of prepared foods because nutrition information is generally not provided in restaurants and other eating places. Restaurants generally do not provide detailed nutritional profiles of foods served, although some fast-food restaurants have this information available on request. New restaurant regulations mandate that menu items labeled as low-fat must comply with defined standards. However, consumers by and large must rely on their own knowledge to identify healthful menu options [149]. Because the trend of eating out is expected to increase even more in the next decade, nutrition interventions to improve the nutritional quality of food choices made away from home are needed. Such interventions should include both environmental changes (e.g., nutrition information on foods) and efforts to change consumer attitudes toward eating out and increase motivation to make healthier choices [145, 149].\n\n#### 3. FAST FOODS\n\nFast food has become a significant part of the American diet. In the United States, more than 200 people are served a hamburger every second of the day [3]. The number of fast-food outlets in the United States has risen steadily during the past 25 years, increasing from roughly 75,000 outlets in 1972 to almost 200,000 in 1997 [150]. Fast-food sales in the United States rose 56% to $102,387 million between 1988 and 1998 [151]. Many fast foods are high in fat. The fat density of fast foods is about 40% of total calories. Recently, several fast-food restaurants have introduced large-size burgers that are exceptionally high in fat and calories. For example, McDonald's Big Xtra with Cheese burger has 810 calories and 46 grams of fat; Burger King's Double Whopper with Cheese burger has 960 calories and 63 grams of fat; and Hardee's Monster Burger has 970 calories with 67 grams of fat. In addition, large soft drinks containing substantial amounts of sugar are often consumed as part of the fast-food meal and are high in calories [3, 145].\n\nSeveral fast-food chains have tried to introduce reduced-fat entrees, but later withdrew them because of slow sales. For example, in 1991, McDonald's introduced the McLean Deluxe, which used a 91% fat-free beef patty, but due to slow sales and poor public acceptance it was taken off the market after a few years. Taco Bell introduced a line of low-fat menu items in 1994, called Border Lights, but these were also largely removed because of sluggish sales [150]. Many fast-food chains offer other low-fat items, such as grilled chicken sandwiches, wraps, and salads.\n\nOne notable trend in fast-food outlets is toward larger portion sizes or super sizes that give the impression of \"better value\" for money and encourage overeating. Increasing the portion size on beverages and french fries, two of the most profitable fast-food items, and offering a \"trade-up\" is a common menu option. For example, what used to be a large portion on the standard combo menu at one fast-food restaurant, that is, a 21-ounce beverage and 5 ounces of fries, has been renamed medium. Customers can pay 40 cents to trade up to a 32-ounce beverage and 6-ounce fries, and another 40 cents to go up to the new top tier of 42-ounce soft drink and 7-ounce fries [152]. A 42-ounce soft drink has 410 calories and 7 ounces of fries has 610 calories.\n\nDespite these food service trends, direct evidence that increased fast-food consumption leads to obesity is lacking. One recent study examined the relationship between fast-food eating and body mass index in 1059 adult men and women over 1 year. The researchers found that the number of meals eaten at fast-food restaurants per week was positively associated with energy intake and body mass index in women but not in men. The strongest relationship was observed among low-income women [58]. Another recent study assessed energy intake and the frequency of consuming food from seven fast-food and table-service restaurants in 73 adults [153]. The researchers found that the frequency of consuming restaurant food was positively associated with body fatness, and total daily energy intake and fat intake. More such studies are warranted to assess whether frequency of fast-food consumption and eating out is related to the secular trends in obesity prevalence in the United States and globally.\n\nFast-food outlets are receiving increasing competition from supermarkets and other establishments offering fully or partially prepared entrees or multicourse meals for eat-in or carry-out. On an average day in 1998, 21% of U.S. households used some form of takeout or delivery [146]. Home meal replacements, as they are called, are intended to be easily reheated in the oven or microwave and are designed to eliminate the need to cook at home by providing a wide variety of higher quality foods that are as convenient and affordable as fast food [150]. The widespread adoption of microwave ovens by U.S. households (now in nearly 90% of homes) contributes to the convenience of home meal replacements for takeout [150]. Driven by consumer demand for convenience due to hectic schedules, the market for home meal replacements will continue to rise. It is speculated that miniaturized outlets offering hot fast-food meals might one day be as common in public buildings as soft drink machines are today [150]. This speaks to the need for consumer education and nutrition labeling of these products.\n\n#### 4. CHANGING PORTION SIZES\n\nIt has been suggested that food portion sizes in food service establishments have become larger and thereby have increased energy intake, which may lead to obesity. However, empirical data to support this association are lacking. It is noticeable, though, that many restaurants, especially fast-food restaurants, in recent years are offering large and extra-large portion sizes of products and meals at low cost. A comparison of food service portion sizes during the past 30 years is remarkable. Putnam [137] reports that the typical fast-food outlet's hamburger in 1957 contained a little more than 1 ounce of cooked meat, compared with up to 6 ounces in 1997. Soda pop was 8 ounces in 1957, compared with 32 ounces to 64 ounces in 1997. A theater serving of popcorn was 3 cups in 1957, compared with 16 cups (medium size popcorn) in 1997. A muffin was less than 1.5 ounces in 1957, compared with 5\u20138 ounces in 1997.\n\n#### 5. FOOD ADVERTISING\n\nAdvertising and promotion are central to the marketing of the American food supply. Although the food industry is certainly not to be blamed for the epidemic of obesity, because the food market is so competitive, advertising encourages Americans to eat and buy more palatable food. The U.S. food marketing system is the second largest advertiser in the economy (after the automobile industry), and a leading supporter of network, spot, and cable television, newspapers, magazines, billboards, and radio. The food industry spends about $11 billion annually on advertising; of this, about $7 billion was spent by food manufacturer's and $3 billion by food establishments, mainly fast-food restaurants [154]. Most of the advertising focuses on highly packaged and processed foods. Advertising expenditures on fruits and vegetables are negligible. In 1997\u20131998, advertising expenditures for candy and gum were $765 million, soft drinks $549 million, and McDonald's spent just over $1 billion [148, 154]. In contrast, the USDA spent $333.3 million on nutrition education, evaluation, and demonstrations. The food industry advertising expenditures also dwarf the National Cancer Institute's annual budget of $1 million for the educational component of the 5-a-Day campaign to promote fruit and vegetable consumption [148].\n\nTelevision is the favorite medium used by the food industry [154]. Children are exposed to much television advertising. One study examined food advertising during children's Saturday morning television programming and found that over half (56%) of all advertisements were for food. The foods promoted were predominantly high in fat or sugar, and many were low in nutritional value. As such, the diet presented on Saturday morning television is the antithesis of what is recommended for healthful eating for children [155].\n\nThere is also a growing trend toward food commercialism and marketing in schools. Corporations are interested in the youth market to build brand loyalty and because they represent a large and growing market. A study by the Consumers Union Education Services [156] found that direct advertising in schools has mushroomed. Examples include school bus advertising for soft drinks and fast-food restaurants; \"free\" textbook covers advertising candy, chips, and soft drinks; ads for high-sugar, high-fat products on wall boards and in hallways, in student publications such as newsletters and yearbooks, and on sports scoreboards; and product giveaways in coupons. In addition, Channel One, the daily news program that is broadcast to millions of students in grades 6\u201312 in thousands of schools, has 2 minutes out of each daily 12-minute program devoted to paid commercials for products that include candy bars, snack chips, and soft drinks [156]. A worrisome trend in schools is exclusive marketing agreements for soft drinks in vending machines. Some school districts have signed exclusive contracts and expect to generate as much as $11 million over a 10-year contract [157].\n\nThere is no evidence that food advertising is linked to the epidemic of obesity. It is unlikely that food advertising would have a direct influence on obesity. However, advertising does affect food choices. In halting the epidemic of obesity, the food industry needs to be involved with other sectors of society in developing public health strategies to prevent obesity and creating an environment that supports healthy eating. Policies to create more healthful environments for children are discussed later.\n\n### C. Eating and Dietary Practices\n\nThe majority of research examining the potential role of diet in the etiology of obesity has focused on associations between obesity and dietary intake (e.g., intake of energy, macronutrients, and food groups). As previously discussed, findings from this large body of research leave many questions unanswered. Fewer studies have examined associations between obesity and eating practices such as the pace of eating, meal patterns, dieting, and binge eating. In this section, we highlight some of the research that examines associations between specific eating practices and obesity, identifies questions that remain unanswered, and makes some recommendations based on this body of research for future studies and for interventions aimed at obesity prevention\/treatment.\n\n#### 1. PACE OF EATING\n\nEating practices aimed at eating at a slower pace are often encouraged in obesity treatment programs. Efforts to slow down the pace of eating may assist certain individuals to consume smaller amounts of food. However, research findings comparing the pace of eating among overweight and nonoverweight individuals have not been consistent. Some studies have shown that overweight individuals do have a rapid eating pace [158\u2013160]. However, other studies have not found a characteristic eating style among overweight individuals [161, 162]. Based on findings from their work and the work of others, Terri and Beck [162] have concluded that differences in eating behaviors between overweight and nonoverweight individuals are \"extremely variable and cannot be presumed.\" Therefore, they suggest that individual assessment is required before embarking on a treatment program that attempts to modify eating behaviors in overweight individuals [162]. For overweight individuals who tend to eat large amounts of food at a quick pace, behavioral strategies aimed at slowing the pace of eating may be helpful, whereas for others these strategies may not be suitable.\n\n#### 2. MEAL PATTERNS\n\nConcerns about skipping meals exist in that meals are important for ensuring an adequate nutrient intake, socializing (if done with family and\/or friends), and for avoiding hunger, which may then lead to binge-eating episodes. Meal skipping has been found to be higher among overweight adolescents than among their nonoverweight peers. In a cross-sectional study of more than 8000 adolescents, usual breakfast consumption was reported by 53% of nonoverweight youth, 48% of moderately overweight youth (85th\u201395th percentile), and 43% of very overweight youth (BMI > 95th percentile) [163]. Due to the cross-sectional nature of these findings, it is not clear whether breakfast skipping leads to obesity (i.e., in that it may be associated with higher energy intake at later times in the day) or rather that breakfast-skipping was a consequence of obesity (i.e., meals are being skipped for weight control purposes).\n\nMeal skipping is frequently used as a weight control method. Among adolescents and adults trying to control their weight, Neumark-Sztainer _et al._ [164] found that skipping meals was commonly reported; 18.6% of adult males and females, 22.8% of adolescent females, and 14.1% of adolescent males trying to control their weight reported skipping meals. An important question relates to the impact of skipping meals on overall energy and nutrient intake. In a study of women participating in a weight gain prevention study, skipping meals for weight control purposes was not associated with overall energy intake [165]. However, meal skippers reported higher percentages of total energy intake from fat and from sweets, lower percentages of total energy intake from carbohydrates, and lower fiber intakes than women who did not report meal skipping [165].\n\nIn summary, existing research suggests that meal skipping is associated with a poorer nutrient intake and with obesity status [163, 165, 166]. However, it is not clear whether meal skipping plays an etiological role in the onset of obesity or rather is a consequence of obesity. Prospective studies are needed to assess causality. However, in light of the inverse associations between meal skipping and nutrient intake, and the potential for leading to uncontrolled eating due to hunger, meal skipping should not be recommended as a weight control strategy. Rather, careful planning of meals with nutrient-dense foods that are low in fat and calories should be encouraged.\n\n#### 3. DIETING BEHAVIORS AND DIETARY RESTRAINT\n\nResearch findings clearly indicate that overweight individuals are more likely to report engaging in dieting and other weight control behaviors than nonoverweight individuals. For example, in a large cross-sectional study of adolescents, dieting behaviors were reported by 17.5% of underweight girls (BMI \u2264 15th percentile), 37.9% of average weight girls (BMI: 15th\u201385th percentile), 49.3% of moderately overweight girls (BMI: 85th\u201395th percentile), and 52.1% of very overweight girls (BMI \u2265 95th percentile) [167]. Due to the cross-sectional nature of this study, it is not clear whether dieting led to higher BMI values or rather that overweight status led to increased dieting behaviors. The latter assumption is usually taken to be the more probable one in that social norms emphasize the importance of thinness, and overweight girls may be trying to modify their body weight to better fit these norms.\n\nIn a prospective study on adolescent girls by Stice and his colleagues [168], baseline dieting behaviors and dietary restraint were found to be associated with obesity onset 4 years later. After controlling for baseline BMI values, the hazard for obesity onset over the 4-year study period was 324% greater for baseline dieters than for baseline non-dieters. For each unit increase on the restraint scale, there was a corresponding 192% increase in the hazard for obesity onset [168]. These findings suggest that for some individuals self-reported dieting may be associated with a higher energy intake, and not a lower energy intake as intended. One explanation for this is that self-reported \"dieting\" may represent a temporary change in eating behaviors, which may be alternated with longer term eating behaviors that are not conducive to weight control. Another explanation is that self-reported dieting and dietary restraint may be associated with increased binge-eating episodes resulting from excessive restraint, control, and hunger. Indeed, Stice and his colleagues did report positive, albeit modest, associations between binge eating and both dieting behaviors ( _r =_ 0.20) and dietary restraint ( _r_ = 0.20). Other researchers have also suggested that dietary restraint may lead to binge-eating behaviors [169, 170], thereby placing individuals at risk for weight gain, rather than the intended weight loss or maintenance.\n\nIt is noteworthy that retrospective studies of adults who have been successful in losing weight and in maintaining their weight loss over extended periods of time suggest that modifications in eating and physical behaviors are helpful strategies. The National Weight Control Registry is a large study of individuals who have been successful at long-term maintenance of weight loss [119]. The most common dietary strategy used by these successful weight loss maintainers was limiting intake of certain types or classes of foods. Other commonly used dietary strategies included limiting quantities of food eaten, limiting the percentage of daily energy from fat, and counting calories. Meal patterns tended to be regular; on average, subjects reported eating nearly 5 times per day and only a small proportion ate less than 2 times per day. Also, most members of the registry reported increasing physical activity levels as part of their weight loss effort [119].\n\n#### 4. BINGE EATING\n\nOverweight individuals are more likely to engage in binge-eating behaviors than their nonoverweight counterparts [5]. In a nonclinical sample of adult women enrolled in a weight gain prevention program, binge eating was reported by 9% of nonoverweight women and 21% of overweight women [171]. Furthermore, binge eating tends to be more prevalent among overweight individuals seeking treatment for weight loss. Based on a review of the literature, Devlin _et al._ [172] have estimated that between 25% and 50% of overweight people seeking treatment for weight loss engage in binge eating. Factors contributing to the higher rates of binge eating among overweight individuals, as compared to nonoverweight individuals, may include the following: greater appetites brought on by higher physiological needs of a larger body size, greater emotional disturbances (e.g., depressive symptoms) or different responses to stressful situations, greater exposure to stressful situations (e.g., related to weight stigmatization), increased weight preoccupation and dieting behaviors, and stronger dietary restraint.\n\nIn working with overweight individuals within health care and other settings, it is essential to be sensitive to the daily struggles they may face within thin-oriented societies. Overweight clients may be reluctant to share their binge-eating experiences; therefore a nonjudgmental attitude on the part of the health care provider is critical. For some individuals, hunger resulting from dieting or meal skipping may be a major cause of binge eating while for others binge eating may be a response to stress. Some individuals may be experiencing cyclical patterns; for example, emotional stress leads to binge eating, which leads to further emotional stress, which leads to further binge eating. Strategies for avoiding binge eating should be linked to factors that appear to be leading to binge eating for each individual.\n\n#### 5. FAMILY INFLUENCES ON DIETARY INTAKE AND EATING PRACTICES\n\nResearch has demonstrated that familial factors contribute to the etiology of obesity via genetic and shared environmental factors [173, 174]. A considerable amount of research has been devoted to the role of the family in the etiology, prevention, and treatment of obesity [175\u2013177]. With regard to dietary intake and eating practices, questions arise as to how the family environment influences individual family members' eating behaviors and what the family can do to improve eating behaviors of its members. The aim is clearly to provide for an environment in which healthful food is available, eaten in an enjoyable manner, and consumed in appropriate amounts. Most of the research in this area has focused on the influence of parents on their children's eating behaviors.\n\nParents\/caretakers may influence their children's dietary intake and eating practices via numerous channels. Some of the key channels include food availability within the home setting (including food purchasing, food preparation, and food accessibility), family meal patterns, infant and child feeding practices, role modeling of eating behaviors and body image attitudes, and verbal encouragement of specific eating practices.\n\nIn focus group discussions, adolescents reported that their parents influence their food choices in different ways: parental eating and cooking behaviors, parental food purchasing patterns, parental concern about foods their children eat, family meal patterns, overall parent\u2013child relations, and family cultural\/religious practices [167]. Adolescents also perceived that they were more likely to eat healthier foods when eating meals with their families, than when eating in other situations [178, 179].\n\nResearch in the arena of adolescent health indicates that general family context variables are strongly associated with the emotional well-being of adolescents and with eating and other health-related behaviors [180]. For example, Neumark-Sztainer and her colleagues [181, 182] found that low family connectedness (i.e., perceived level of caring and communication within the family) placed adolescents at increased risk for inadequate intake of fruit, vegetables, and dairy foods. Furthermore, among overweight youth, Mellin and her colleagues [183] found that high family connectedness was associated with more regular breakfast eating, increased fruit and vegetable consumption, and lower rates of unhealthy dieting behaviors. These findings indicate the importance of a positive familial environment for adolescents and, in particular, for overweight youth who may be experiencing social stigmatization and need additional support.\n\nAn important question relates to how involved parents should be in their children's eating practices; that is, what is an appropriate parental role? Birch and her colleagues [184, 185] have examined associations between child-feeding practices and children's ability to regulate their energy intake. Birch and Fisher [184] have suggested that individual differences in self-regulation of energy intake are associated with differences in child-feeding practices and with children's adiposity. They state that \"initial evidence indicates that imposition of stringent parental controls can potentiate preferences for high-fat, energy-dense foods, limit children's acceptance of a variety of foods, and disrupt children's regulation of energy intake by altering children's responsiveness to internal cues of hunger and satiety.\" In a study of 77 three- to five-year-old children, Johnson and Birch [185] found that children with greater body fat stores were less able to regulate energy intake accurately. The strongest predictor of children's ability to regulate energy intake was parental control in the feeding situation; mothers who were more controlling of their children's food intake had children who showed less ability to self-regulate energy intake ( _r =_ \u22120.67). They concluded from this study that \"the optimal environment for children's development of self-control of energy intakes is that in which parents provide healthy food choices but allow children to assume control of how much they consume.\"\n\nParents of overweight children and adolescents are often in a difficult situation in that they want to be supportive of their children, yet also want to help them to modify behavioral patterns that may increase their risk of obesity. Furthermore, they may feel as though they are being blamed for their child's obesity. Existing research suggests that parental involvement and support is important [177, 183], but efforts to control a child's intake may be counterproductive [184, 185], Further studies are needed to explore how parents can best help their overweight children develop a positive self-image and healthy eating and physical activity behaviors.\n\n## IV. SUMMARY AND PUBLIC HEALTH RECOMMENDATIONS\n\nWe have reviewed the literature on key physical activity and diet-related risk factors for obesity in children and adults. Highlights of the review from the physical activity domain include (1) the importance of addressing the influence of both leisure-time physical activity and sedentary behavior to total energy expenditure, (2) the importance of fostering both self-efficacy and social support for physical activity to promote higher levels of physical activity, and (3) the influence of environmental factors on physical activity levels. Highlights related to dietary intake include (1) recognizing the contribution of both dietary fat intake and total energy intake to energy regulation; (2) the importance of accurately assessing portion size; (3) the influence of eating practices such as eating out, meal skipping, restrained eating and binge eating on obesity; and (4) social and environmental factors that promote excess energy intake.\n\nTo effectively combat the public health problem of obesity, interventions that target change in dietary intake and physical activity are necessary. Intervention efforts must take into account that dietary intake and physical activity are complex, multidetermined behaviors influenced by individual, social, and environmental factors. Although obesity intervention approaches have traditionally focused primarily on an individual's change, both individual- and population-level approaches are essential. Community-based interventions including school-based programs, after-school programs and work-site programs as well as clinic-based programs focused on diet, physical activity and weight management all have the potential to effectively address the problem of obesity. Environmental and policy interventions to prevent obesity have also begun to receive greater attention [103, 148, 186].\n\n### A. School and Community-Based Youth Programs\n\nThe increasing prevalence of obesity in adults and children, coupled with difficulty in successfully treating adult obesity, highlight the urgent need for prevention approaches geared toward children. Table 1 provides an overview of essential components for obesity prevention programs in schools. School-based programs must encompass both educational and environmental strategies to promote health eating and activity levels among students. A number of school-based programs targeting physical activity and eating behaviors have shown that is possible to modify school environments and show improvements in diet and physical activity levels [64, 187\u2013190].\n\nTABLE 1\n\nStrategies for School Programs to Promote Healthy Eating and Physical Activity\n\nAlthough the majority of health promotion programs for children are provided in school settings [191], community-based programs have considerable potential for helping children acquire positive health behaviors. The school environment confers many advantages including the reduction of barriers of cost and transportation and the provision of access to a large, already-assembled population. Community-based, after-school programs have the potential to complement health promotion and education efforts made by schools for several reasons. Although children spend a large proportion of time in school each day, after-school hours constitute a substantial amount of time each week. Often children do not have opportunities to spend this time constructively, particularly in at-risk communities. According to the National Education Longitudinal Survey, the average eighth grader spends between 2 and 3 hours a day at home alone after school [192]. These hours have been shown to be those when many youth engage in high-risk behaviors due to lack of supervision. When young people are provided with safe and healthy activities in which to participate during critical gap periods (e.g., after school, on weekends), they are less likely to have time to participate in the high-risk, unhealthy activities that can delay or derail positive development [193\u2013195].\n\nCommunity-based, after-school programs provide multiple opportunities for teaching and reinforcing healthy patterns of physical activity and eating. Through their involvement in such programs, children can be educated about healthful eating habits and active lifestyles, but also have ample opportunity to practice these new habits and skills. Classroom-based eating habit programs that emphasize the behavioral skills needed for planning, preparing, and selecting healthy foods can be easily adapted for this setting. Children may have the opportunity to practice new skills in such programs by engaging in activities such as planning and preparing healthy snacks. According to Sallis and McKenzie [196], physical activity programs for children should include (1) activities and skills that have the potential for carryover into adult life,(2) moderate intensity activities, and (3) a focus on maximizing the participation of all children. Community based, after-school programs are ideal for achieving such goals.\n\n### B. Policy Recommendations\n\nTable 2 provides a thorough overview of policy recommendations for obesity prevention across a number of domains. These policy recommendations acknowledge the multiple levels at which change must occur from city, state, and federal tax programs to fund campaigns to promote healthy eating and physical activity and curb unhealthy patterns to developing better walking\/biking paths for the purposes of both recreation and transportation to work [148]. Recommendations for work-site programs are also made including environmental changes in work setting such as promoting healthy eating by increasing the availability of healthy food choices in cafeterias, instituting work-site campaigns to promote physical activity and healthy eating, and providing tax incentives to employers for providing weight management programs.\n\nTABLE 2\n\nReducing the Prevalence of Obesity: Policy Recommendations\n\n**Education**\n\n\u2022 Provide federal funding to state public health department for mass media health promotion campaigns that emphasize healthful eating and physical activity patterns.\n\n\u2022 Require instruction in nutrition and weight management as part of the school curriculum for future health education teachers.\n\n\u2022 Make a plant-based diet the focus of dietary guidance.\n\n\u2022 Ban required watching of commercials for foods high in calories, fat, or sugar on school television programs (for example, Channel One).\n\n\u2022 Declare and organize an annual National \"No-TV\" Week.\n\n\u2022 Require and fund daily physical education and sports programs in primary and secondary schools, extending the school day if necessary.\n\n\u2022 Develop culturally relevant obesity prevention campaigns for high-risk and low-income Americans.\n\n\u2022 Promote healthy eating in government cafeterias, Veterans Administration medical centers, military installations, prisons, and other venues.\n\n\u2022 Institute campaigns to promote healthy eating and activity patterns among federal and state employees in all departments.\n\n**Food labeling and advertising**\n\n\u2022 Require chain restaurants to provide information about calorie content on menus or menu boards and nutrition labeling on wrappers.\n\n\u2022 Require that containers for soft drinks and snacks sold in movie theaters, convenience stores, and other venues bear information about calories, fat, or sugar content.\n\n\u2022 Require nutrition labeling on fresh meat and poultry products.\n\n\u2022 Restrict advertising of high-calorie, low-nutrient foods on television shows commonly watched by children or require broadcasters to provide equal time for messages promoting healthy eating and physical activity.\n\n\u2022 Require print advertisements to disclose the caloric content of the foods being marketed.\n\n**Food assistance programs**\n\n\u2022 Protect school food programs by eliminating the sale of soft drinks, candy bars, and foods high in calories, fat, or sugar in school buildings.\n\n\u2022 Require that any foods that compete with school meals be consistent with federal recommendations for fat, saturated fat, cholesterol, sugar, and sodium content.\n\n\u2022 Develop an incentive system to encourage food stamp recipients to purchase fruits, vegetables, whole grains, and other healthful foods, such as by earmarking increases in food stamp benefits for the purchase of those foods.\n\n**Health care and training**\n\n\u2022 Require medical, nursing, and other health professions curricula to teach the principles and benefits of healthful diets and exercise patterns.\n\n\u2022 Require health care providers to learn about behavioral risks for obesity and how to counsel patients about health-promoting behavior change.\n\n\u2022 Develop and fund a research agenda focused on behavioral as well as metabolic determinants of weight gain and maintenance, and on the most cost-effective methods for promoting healthful diet and activity patterns.\n\n\u2022 Revise Medicaid and Medicare regulations to provide incentives to health care providers for nutrition and obesity counseling and other interventions that meet specified standards of cost and effectiveness.\n\n**Transportation and urban development**\n\n\u2022 Provide funding and other incentives for bicycle paths, recreation centers, swimming pools, parks, and sidewalks.\n\n\u2022 Develop and provide guides for cities, zoning authorities, and urban planners on ways to modify residential neighborhoods, workplaces, and shopping centers to promote physical activity.\n\n**Taxes**\n\n\u2022 Levy city, state, or federal taxes on soft drinks and other foods high in calories, fat, or sugar to fund campaigns to promote good nutrition and physical activity.\n\n\u2022 Subsidize the costs of low-calorie nutritious foods, perhaps by raising the costs of selected high-calorie, low-nutrient foods.\n\n\u2022 Remove sales taxes on, or provide other incentives for, purchase of exercise equipment.\n\n\u2022 Provide tax incentives to encourage employers to provide weight management programs.\n\n**Policy development**\n\n\u2022 Use the National Nutrition Summit to develop a national campaign to prevent obesity.\n\n\u2022 Produce a _Surgeon General's Report on Obesity Prevention._\n\n\u2022 Expand the scope of the President's Council on Physical Fitness and Sports to include nutrition and to emphasize obesity prevention.\n\n\u2022 Develop a coordinated federal implementation plan for the Healthy People 2010 nutrition and physical activity objectives.\n\n_Source:_ Nestle, M., and Jacobson, M. F. (2000). Halting the obesity epidemic: A public health policy approach. _Public Health Rep._ **115,** 12\u201324.\n\n## V. CONCLUSION\n\nThe recently released goals of Healthy People 2010 are to reduce the prevalence of obesity among adults from 23% to 15% and to reduce the prevalence of obesity among children and adolescents from 11% to 5% [197, 198]. The etiology of obesity is complex and encompasses a wide variety of social, behavioral, cultural, environmental, physiological, and genetic factors. To achieve these ambitious goals, considerable effort must be focused on helping individuals at the population level modify their diets and increase their physical activity levels, key behaviors involved in the regulation of body weight. Educational and environmental interventions that support diet and exercise patterns associated with healthy body weight must be developed and evaluated. 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Devlin M.J., Walsh B.T., Spitzer R.L., Hasin D. Is there another binge eating disorder? A review of the literature on overeating in the absence of bulimia nervosa. _Int. J. Eating Disord_. 1992;11:333\u2013340.\n\n173. Teasdale T.W., Sorensen T.I., Stunkard A.J. Genetic and early environmental components in sociodemographic influences on adult body fatness. _Br. Med. J_. 1990;300:1615\u20131618.\n\n174. Bouchard C. Genetic factors in obesity. _Med. Clin. N. Am_. 1989;73:67\u201381.\n\n175. Nader P.R. The role of the family in obesity prevention and treatment. _Ann. NY Acad. Sci_. 1993;699:147\u2013153.\n\n176. Golan M., Fainaru M., Weizman A. Role of behaviour modification in the treatment of childhood obesity with the parents as the exclusive agents of change. _Int. J. Obes. Relat. Metab. Disord_. 1998;22:1217\u20131224.\n\n177. Epstein L.H. Family-based treatment for preadolescent obesity. _Adv. Devel. Behav. Pediatr_. 1985;6:1\u201339.\n\n178. Neumark-Sztainer D., Story M., Ackard D., Moe J., Perry C. The \"family meal\": Views of adolescents. _J. Nutr. Educ_. 2000;32:329\u2013354.\n\n179. Neumark-Sztainer D., Story M., Ackard D., Moe J., Perry C. Family meals among adolescents: Findings from a pilot study. _J. Nutr. Educ_. 2000;32:335\u2013340.\n\n180. Resnick M.D., Bearman P.S., Blum R.W., Bauman K.E., Harris K.M., Jones J., Tabor J., Beuhring T., Sieving R.E., Shew M., Ireland M., Bearinger L.H., Udry J.R. Protecting adolescents from harm. Findings from the National Longitudinal Study on Adolescent Health. _JAMA_. 1997;278:823\u2013832.\n\n181. Neumark-Sztainer D., Story M., Resnick M.D., Blum R.W. Correlates of inadequate fruit and vegetable consumption among adolescents. _Prevent. Med_. 1996;25:497\u2013505.\n\n182. Neumark-Sztainer D., Story M., Dixon L.B., Resnick M., Blum R. Correlates of inadequate consumption of dairy products among adolescents. _J. Nutr. Educ_. 1997;29:12\u201320.\n\n183. Mellin, A., Neumark-Sztainer, D., Story, M., Ireland, M., and Resnick, M. (submitted). Unhealthy behaviors and psychosocial difficulties among overweight youth: The potential impact of familial factors.\n\n184. Birch L.L., Fisher J.O. Development of eating behaviors among children and adolescents. _Pediatrics_. 1998;101:539\u2013549.\n\n185. Johnson S.L., Birch L.L. Parents' and children's adiposity and eating style. _Pediatrics_. 1994;94:653\u2013661.\n\n186. Sallis J.F., Bauman A., Pratt M. Environmental and policy interventions to promote physical activity. _Am. J. Prevent. Med_. 1998;15:379\u2013397.\n\n187. Nader P.R., Stone E.J., Lytle L.A., Perry C.L., Osganian S.K., Kelder S., Webber L.S., Elder J.P., Montgomery D., Feldman H.A., Wu M., Johnson C., Parcel G.S., Luepker R.V. Three-year maintenance of improved diet and physical activity: The CATCH cohort. Child and Adolescent Trial for Cardiovascular Health. _Arch. Pediatr. Adolesc. Med_. 1999;153:695\u2013704.\n\n188. Lytle L.A., Stone E.J., Nichaman M.Z., Perry C.L., Montgomery D.H., Nicklas T.A., Zive M.M., Mitchell P., Dwyer J.T., Ebzery M.K., Evans M.A., Galati T.P. Changes in nutrient intakes of elementary school children following a school-based intervention: Results from the CATCH Study. _Prevent. Med_. 1996;25:465\u2013477.\n\n189. McKenzie T.L., Nader P.R., Strikmiller P.K., Yang M., Stone E.J., Perry C.L., Taylor W.C., Epping J.N., Feldman H.A., Luepker R.V., Kelder S.H. School physical education: Effect of the Child and Adolescent Trial for Cardiovascular Health. _Prevent. Med_. 1996;25:423\u2013431.\n\n190. Sallis J.F., McKenzie T.L., Alcaraz J.E., Kolody B., Faucette N., Hovell M.F. The effects of a 2-year physical education program (SPARK) on physical activity and fitness in elementary school students. Sports, Play and Active Recreation for Kids. _Am. J. Public Health_. 1997;87:1328\u20131334.\n\n191. Contento L., Blach G., Bronner Y., Lytle L., Maloney S., Olson C., Swadener S. The effectiveness of nutrition education and implications for nutrition education policy, programs and research: A review of research. _J. Nutr. Educ_. 1995;27:284\u2013418.\n\n192. U.S. Department of Education, Office of Education Research and Improvement NCFES. _A Profile of the American Eighth Grader: NEL: 88 Description Summary_. Washington, DC: U.S. Government Printing Office; 1990.\n\n193. Carnegie Council on Adolescent Development. _Matter of Time: Risk and Opportunity in the Nonschool Hours_. Washington, DC: Carnegie Council on Adolescent Development; 1992.\n\n194. Carnegie Council of Adolescent Development. _Great Transitions: Preparing Adolescents for a New Century_. Waldorf, MD: Carnegie Corporation; 1995.\n\n195. Mulhall P.F., Stone D., Stone B. Home alone: Is it a risk factor for middle school youth and drug use? _J. Drug Educ_. 1996;26:39\u201348.\n\n196. Sallis J.F., McKenzie T.L. Physical education's role in public health. _Res. Q. Exerc. Sport_. 1991;62:124\u2013137.\n\n197. U.S. Department of Health and Human Services. Healthy People 2010: Conference Edition. New York: U.S. Department of Health and Human Services; 2000;Vol I.\n\n198. U.S. Department of Health and Human Services. Healthy People 2010: Conference Edition. Washington, DC: U.S. Department of Health and Human Services; 2000;Vol II.\nCHAPTER 35\n\nRole of Taste and Appetite in Body Weight Regulation\n\nADAM DREWNOWSKI and VICTORIA WARREN-MEARS, University of Washington, Seattle, Washington\n\n## I. INTRODUCTION\n\nMore than 50% of all adults in the United States are classified as being either overweight or obese [1]. The prevalence of obesity has increased dramatically in the last two decades, especially among ethnic minorities and the poor [1\u20133]. Given that human physiology and genetics have changed little during that time, the explanation must lie in reduced physical activity or altered eating habits. Although the mainstream of obesity research continues to focus on genetic, metabolic, and physiological factors in weight gain [1], the obesity epidemic is most likely caused by a profound change in dietary behaviors. Changes in the global food supply may also play a part [4, 5]. Dietary Guidelines for Americans 2000 now suggest that rising obesity rates are tied to the wide availability of cheap and palatable foods [6].\n\nTaste and palatability are among the key influences on food choice [7\u20139]. According to consumer studies [9], adults' food choices are determined by food taste, cost, and convenience, rather than by concerns with nutrition or health. The best tasting and most palatable foods are often those that provide the maximum amount of energy per unit weight [9]. Many such foods contain fat, sugar, or both. Inaccessible to most people on a daily basis until the last century, corn sugars and vegetable oils are now among the cheapest food commodities available [10\u201311]. The question is whether sensory mechanisms that have evolved to maintain energy supply when food was scarce can effectively adapt to dietary excess. Examining taste responses and appetite for sweet and high-fat foods in relation to body weight is the main focus of this chapter.\n\n## II. GENETICS AND BODY WEIGHT\n\nObesity is said to be a heritable trait [12, 13]. The fact that obese parents tend to have obese children [14\u201315] was taken as evidence that obesity had a familial origin. Studies of Danish adoptees showed that obesity, or more correctly body mass, was an inherited variable [16]. Another line of evidence was provided by studies of monozygotic and dizygotic twins [17]. Identical twins had the same patterns of weight gain in response to overfeeding and a similar distribution of body fat [17]. Twin studies now suggest that genes may account for as much as 75\u201380% of the variance in percent body fat among children aged 3\u201317 years [18].\n\nSome people may be more vulnerable than others to excess weight gain. According to the \"thrifty gene\" hypothesis [10], evolutionary selection may have favored those people who were best able to store body fat when food became available. Because gene expression is modulated by nutrients, the heritable trait may not be obesity per se, but rather a vulnerability or predisposition to excessive weight gain [19, 20]. The expression of obesity among susceptible individuals must involve some aspect of dietary behavior, not to mention exposure to an obesity-promoting diet. The continuing search for an obese phenotype has focused on those \"obese\" behaviors, including taste-related ones, that might show a genetic component.\n\nBody metabolism, physical activity, and dietary choices are each influenced by genetic traits [12]. Studies explored whether infants born to obese mothers had lower metabolic rates [21], distinct eating behaviors, or showed an elevated sensory response to sweet [22]. Most of those studies were inconclusive. Sweet taste responses of obese adults have also been investigated [23\u201325]. For the most part, studies failed to show a consistent \"obese\" pattern of sweet taste responses, eating behaviors, or eating styles. While there was a great deal of individual variation in taste responsiveness, few systematic differences by body-weight category were observed. Moreover, there was no evidence that any of the taste-related behaviors had a genetic component.\n\nThe fact that obesity rates have increased so sharply during the past two decades suggests that changing dietary choices and increasingly sedentary lifestyles\u2014rather than genetics\u2014are the key factors in the obesity epidemic. If obesity is multiply determined, then the search for a single obese genotype is likely to be fruitless. Contrary to the notion that obesity is a random \"genetic lottery,\" the distribution of obesity in the United States varies in a predictable manner with age, race\/ethnicity, education, and income. With obesity rates reaching 90% in some populations [26\u201328], it would appear that most people are vulnerable in some degree to excess weight gain. Instead of actively promoting obesity, genetic factors may provide an inadequate protection against an energy-rich dietary environment. Instead of being a rare and abnormal state, obesity may represent a normal adaptive response to the prevailing environmental and dietary conditions.\n\n## III. TASTE FACTORS AND FOOD CHOICES\n\nThe chief role of the taste system is to promote and maintain eating behavior. The pleasure response to sweet taste is present at birth. A newborn's reaction to sweet has been assessed using facial expressions captured during the first few hours of life [29]. In 3-day-old infants, the taste response to sucrose causes relaxation, a slight smile, and licking of the upper lip. Three-day-old infants prefer sucrose solutions to water, prefer sweeter over less sweet sugars, and selectively consume the most concentrated sugar solutions [29]. This sensory reflex serves to orient and maintain the feeding response, assuring a constant supply of dietary energy. The key sensory characteristic of mother's milk is its sweet taste. Sugar is a major source of energy. Studies on the development of food preferences in young children suggest that novel flavors are accepted most readily when they are paired with a concentrated source of energy, most often sugar or fat [30]. Taste behaviors, orienting the newborn toward sugar and fat, most likely evolved because they were crucial to survival [31, 32].\n\nFoods that are described as good tasting are often concentrated sources of energy and contain fat, sugar, or both [7\u20139]. The concept of food palatability includes taste, aroma, and texture. Sweetness is a basic taste, perceived through receptors located in taste buds, distributed through the oral cavity, and concentrated on the dorsal area of the tongue. The sensation of fatness is mediated through the perception of texture and mouthfeel. Such texture attributes as smoothness, creaminess, crunchiness, or tenderness are all associated with the fat content of foods. In addition, many of the volatile flavor molecules are fat soluble such that the characteristic aroma of many foods is linked to their fat content. Studies in humans and rats suggest that sensory preferences for fat are either innate or acquired very early in life.\n\nThere is no question that the enjoyment of sweetness and fat has a physiological basis [31]. Placing a sweet substance on the tongue of a crying newborn has a remarkable calming effect. This persists for several minutes and can be used to calm the infant during blood draws and other painful procedures. Studies have linked taste responses to sweetness to the endogenous opiate peptide system. Further evidence that taste responses to sweetness and fat are mediated by endorphins is provided by clinical studies in adult women. Infusions of the opiate agonist naloxone suppressed sensory preferences for sweetened dairy products and selectively reduced the consumption of sweet high-fat foods such as cookies or chocolate. This effect was strongest among women diagnosed with the binge-eating disorder [33], suggesting that some food cravings may be mediated by endorphins. Although naloxone is not an effective agent for weight loss, opiate blockade may prevent palatability-induced overeating [34].\n\nThe pleasure response to foods is thought to serve the physiological needs of the organism [31]. According to some theories, sweet taste preferences are linked to both short- and long-term energy needs (see [23] for review). In laboratory studies, taste response to sweetness declined after a meal, or following the ingestion of sweet glucose solutions [31]. There was some question whether this satiety response was linked to energy content, because a similar suppression in sweet taste preference was also obtained with noncaloric aspartame solutions [23]. Other studies attempted to link sweet taste preferences with the physiological set point of body weight. In that view, a drop in body weight below set point would lead to increased sweet preferences. Conversely, an increase in body weight above set point should lead to an observable decrease in sweet preferences. However, given that the set point was a theoretical construct as opposed to a measurable variable, that hypothesis could never be tested appropriately [23]. While preferences for sweet solutions vary across individuals, they have not been linked to any particular body weight.\n\nAs shown in Fig. 1, obese male patients showed a diversity of responses to milk and cream sweetened with different amounts of sucrose, up to 16% sucrose wt\/wt. Each panel represents data for a different patient ( _n_ = 9). Hedonic ratings measured along a five-point category scale either rose (top five panels) or declined with increasing sucrose concentrations (bottom four panels), consistent with previous results. Individual patterns of response to sucrose were much the same before and after weight loss. Even a very substantial loss in body weight in excess of 40 kg failed to alter the type of taste response to sweetness.\n\nFIGURE 1 Hedonic ratings for milk and cream sweetened with different amount of sucrose. Data are presented separately for nine obese males before and after weight loss. Initial (wt 1) and final (wt 2) weights (kg) are indicated.\n\nStudies on obesity and sensory response to fat were more convincing, showing that obese women patients gave the highest hedonic ratings to energy-rich mixtures of cream and sugar. In some studies, taste preferences were associated with food preferences and selective consumption of sweet and high-fat foods. There was also evidence that the diets of obese men and women tended to be higher in fat than those of lean controls. However, such diets were probably more energy dense, providing more energy per unit weight or volume.\n\nSensory preferences for dietary fats have been linked to body mass index and body fatness in children, adolescents, and adults [35, 36]. In clinical studies using sweetened dairy foods, obese women selected taste stimuli that were high in fat over those that were intensely sweet [25]. In contrast, emaciated females with anorexia nervosa liked sweet taste but showed an aversion to dairy fat. These studies were later confirmed by the observation that fatter persons tended to select higher-fat foods [35]. In a laboratory study, preferences for fat in foods were linked with the respondents' own body fatness [35]. A related study conducted with 3- to 5-year-old children showed that the children's fat preferences were influenced by the body mass of the parents, suggesting that fat preferences may be a heritable trait [36]. It is as yet unclear whether the preferences were for fat per se, or for those fat-rich stimuli that provided maximal energy per unit weight or volume. Because fats provide a concentrated source of dietary energy, they have an impact on hunger, appetite, and satiety [37\u201340].\n\n## IV. HUNGER, APPETITE, AND SATIETY\n\nPeople eat food several times per day. These eating occasions can be divided into larger meals and smaller snacks. People begin to eat when they are hungry and stop when they are satiated. While _hunger_ is defined as a generalized state of energy depletion, the term _appetite_ often denotes a desire to eat a given food [8, 9]. In studies with rats, the onset of eating was preceded by a transient drop in plasma glucose.\n\nSeveral theories have been developed to account for the seeming day-to-day control of food intake [41\u201343]. The glucostatic theory held that food consumption was triggered by a drop in the availability of glucose to cells and tissues. The lipostatic theory held that a drop in body fat stores would provoke feeding behavior, while the aminostatic theory suggested that feeding occurred to maintain amino acid homeostasis. What these early theories shared was the common premise that hunger and appetite triggered food consumption, primarily in response to energy needs. In contrast, the current focus is on mechanisms that may halt food consumption in response to energy surplus. Instead of hunger and appetite, the current research emphasis is on the physiological mechanisms of satiety [44, 45].\n\nSatiety refers to reduced hunger and faster termination of eating [8, 9]. Foods that deliver fewer kilocalories per meal are, by definition, more satiating [7\u20139]. In contrast, foods that tend to be overeaten have, by definition, less of an effect on satiety. There has been some argument whether fat-rich foods are overeaten because of their high energy density or because of their fat content [45]. If satiety is macronutrient driven, fat may be overeaten because it affects satiety less than a corresponding amount (in kcalories) of carbohydrate or protein [46].\n\nSome researchers separate the concepts of satiation, measured by meal size, and the state of postmeal satiety, as measured by the time delay until the next meal [38\u201340]. The distinction was meant to account for the puzzling phenomenon that though energy-dense fat ought to promote satiety, high-fat foods were often eaten to excess. The favored explanation was that fats had a weak effect on satiation but a much stronger effect on satiety [38\u201340]. Satiety is typically associated with delayed gastric emptying, elevated plasma glucose and insulin levels, and a postmeal elevation in plasma lipids.\n\nBoth hunger and satiety are thought to be under neural regulatory control [41]. In animal studies, the initiation and termination of feeding can be achieved by manipulating central neurotransmitters and peptides [41, 42]. Studies of such mechanisms have led to the development of pharmacological agents for obesity treatment. Again, the focus has shifted from reducing appetite to promoting satiety. Whereas amphetamine-like substances that reduce hunger and appetite are no longer in common use, selective serotonin reuptake inhibitors that promote satiety are the current drugs of choice [43]. However, centrally acting pharmacological agents are only sporadically effective, have potential for causing side effects, and lead to only minor weight loss. Research has also addressed the role of gut peptides such as bombesin and cholecystokinin in promoting satiety; however, they do not appear to be effective in most human studies. Similarly, leptin, a hormone secreted by the adipose tissue, is largely ineffective in promoting satiety, reducing food intakes, or causing weight loss in humans.\n\nSome researchers have argued that taste factors override normal satiety signals, leading to overeating and overweight [46, 47]. Sweet taste has been singled out for special blame. The addition of sugars to foods was reported to stimulate appetite and increase food consumption [48, 49]. Identical claims were made for noncaloric sweeteners, suggesting that sweet taste, and not the presence of sugar, was the key factor. Curiously, similar claims regarding fat were based not on taste but on its energy density. Energy density of fats, the argument went, suppressed satiety signals and led to \"passive overeating\" [46] of fat-rich foods. Of course, energy-dense foods tend to be palatable and vice versa.\n\nIn principle, consumption of more satiating foods ought to lead to lower energy intakes and therefore weight loss. However, palatability and satiety are mirror-image terms that are linked, moreover, to the energy density of foods. Palatability, sometimes measured in terms of hedonic response or predisposition toward a given food, is said to heighten appetite and so increase the amount of food consumed. In contrast, satiety, often measured in terms of fullness or reduced liking for a given food, is said to reduce the amount of food consumed [7\u20139, 43\u201346]. Given that palatability and satiety are measured in terms of actual consumption, their relationship is by definition, reciprocal. Palatable foods that are overeaten are thereby, by definition, the least satiating [8]. Conversely, the highly satiating foods are, also by definition, the least palatable. The creation of palatable and yet satiating foods, the avowed goal of the weight loss industry, is a contradiction in terms.\n\n## V. ENERGY DENSITY OF FOODS\n\nEnergy density is said to be the key factor in the regulation of food intake [50\u201352]. Under _ad libitum_ conditions in the laboratory, young people tend to consume a constant weight of food rather than a constant amount of energy [53]. Foods with high energy density, that is, more kilocalories per unit weight, deliver more kilocalories per eating occasion than do energy-dilute foods [53, 54]. In contrast, lower energy density is associated with lower energy intakes per meal.\n\nEnergy-dilute foods are more bulky and so provide fewer kilocalories per eating occasion. By definition then, energy-dilute foods are more satiating than are energy-dense foods [51\u201353]. Chocolate cake, hamburgers, and French fries provide from 3 to 5 kcal\/g. Chocolate candy and peanut butter provide 5 to 6 kcal\/g. In contrast, most raw vegetables and salad greens are energy-dilute foods, providing between 0.1 and 0.5 kcal\/g.\n\nBecause energy-dense foods deliver more energy per eating occasion, they are\u2014by definition\u2014less satiating. A study of 38 common foods [55] measured satiety ratings every 15 minutes for 2 hours following the consumption of equicaloric (240 kcal) amounts of food. That amount of energy can be provided by less than 50 g of chocolate or by more than 1000 g of boiled spinach. The satiety index was calculated by dividing the area under the satiety response curve for the test foods by the mean satiety curve for white bread and multiplying by 100%. Satiety indices correlated most highly with the weight of foods consumed that ranged from a low of 38 g for peanuts to a high of 625 g for oranges [60].\n\nAnalyses of satiety ratings showed that cake, cookies, and chocolate were less satiating than were porridge, potatoes, and boiled fish [55]. However, porridge and boiled fish were rated as significantly less palatable than cookies and chocolate [55]. It would appear that energy-dense foods are palatable but not satiating, whereas energy dilute foods are satiating but not palatable [7\u20139]. Although studies [51, 52, 56] have sometimes contrived to separate energy density, nutrient composition, and palatability, these factors are rarely separable in real life. Generally, higher fat diets are more enjoyable and varied and tend to provide more energy per unit weight than do the more bulky low-fat or fat-free diets. Factors that influence food choices in real life, such as cost, accessibility, and convenience [57], are generally ignored in laboratory studies where a variety of foods is provided to the respondent at no cost.\n\nThe energy-density approach to dieting has been the topic of many articles and popular books. If people consume a constant weight of food each day, then choosing energy-dense foods will lead to higher energy intakes. Conversely, selecting foods with low energy density should lead\u2014in principle\u2014to lower energy intakes and eventual weight loss [53]. The idea that fat and protein had different effects on satiety has already been exploited by the weight loss industry. Most liquid formula diets are simply mixtures of protein, fiber, and water, together with small amounts of carbohydrate. Alhough they represent a major commercial success, their availability has not reduced the growing rates of overweight and obesity. [58]\n\nTwo main problems are associated with the energy-density approach to weight loss. First, energy density of foods is almost completely determined by their water and fat content [8, 9]. Generally, energy-dense foods are dry, high in fat, and sometimes high in sugar (e.g., chocolate). In contrast, energy-dilute foods are high in water, fiber, and sometimes protein (e.g., spinach). Multiple regression analyses of some 100 common foods in the U.S. diet showed that water content alone accounted for 85% of the variance, while water and fat accounted for 99% of the variance. Sugar, fiber, and protein content played a decidedly lesser role [9].\n\nSecond, the more energy-dense foods are usually more palatable. Classic studies on U.S. Army food preferences [59] showed that steak, milkshakes, and cake were preferred over broccoli and yogurt. A study of food preferences of obese men and women likewise showed that cakes and desserts were preferred over fresh vegetables and fruit. Whereas obese men listed steak and meat dishes among their favorite foods, obese women selected foods that were both sweet and rich in fat. Such foods included chocolate, ice cream, doughnuts, cookies, and cake [25]. Only women with anorexia nervosa have been observed to consistently list energy-dilute vegetables and salads among their favorite foods [60].\n\n## VI. APPETITE FOR ENERGY\n\nObesity is the long-term outcome of excess energy intake relative to energy expenditure [61]. Studies using doubly labeled water techniques have established that obese patients expend, and also consume, more energy than do lean controls [62]. Though energy imbalance is the chief\u2014and maybe the only\u2014reason for body weight gain, studies have sought to link obesity rates with excess consumption of a single nutrient or a given food. In some studies, obesity was linked with excess consumption of simple sugars, including sweets, snacks, desserts, and carbonated soft drinks [63\u201365]. Obesity has also been linked with excess fat consumption, including frequent consumption of fast foods [66, 67]. Studies have even linked obesity with greater dietary variety [68] and with higher frequency of food consumption away from home [69].\n\nThe once-popular externality hypothesis held that obese persons were more attuned to external variables, such as the taste of food, as opposed to the internal signals of hunger and satiety [8]. Subsequent research showed the obese were no more sensitive to sweet taste, did not necessarily prefer sweet solutions, and showed the same satiety response to sweetness as did normal-weight controls [8].\n\nAt this time, there is no convincing evidence to link obesity with specific macronutrient appetites. Although obesity has been blamed on excessive consumption of fast foods or carbonated soft drinks, no study has shown that obese persons have a selective appetite for either sugar or fat. It may be that obesity is associated with an appetite for energy-dense foods, whatever their nutrient content may be. For the most part, given the nature of the food supply, such foods will contain either sugar or fat.\n\nThe role of dietary fat in promoting weight gain has proven to be controversial [67, 70\u201372]. Noting that obesity rates in the United States have risen, while the proportion of fat energy in the diet has dropped, some scientists [71] have questioned the connection between fat consumption and obesity. However, the proportion of fat in the diet is not as important as the total amount of energy consumed. As shown by National Health and Nutrition Examination Survey III data, both energy intakes and fat consumption in grams per day actually increased between 1976\u20131980 and 1988\u20131994, this for both women and men [73]. Total energy intakes, and not percentage of fat in the diet, are likely to be more predictive of weight gain.\n\nWhy the energy imbalance is not redressed by the physiological mechanisms of hunger, appetite, or satiety is a good question. From the evolutionary standpoint, sensory mechanisms regulating food intake were meant to protect the organism against starvation and respond primarily to energy needs. A plentiful food supply is a very recent phenomenon [10, 74]. The question is whether taste mechanisms, geared toward the acceptance of sugar and fat, can also protect us from the potential ill effects of dietary excess now that sugar and fat are the most common foodstuffs available [10]. It is possible that they cannot. Far from being an abnormality or a disease, obesity may represent an adaptive response to the current environmental conditions\n\nReferences\n\n1. National Heart, Lung, Blood Institute, Obesity Initiative Task Force. Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults\u2014The evidence report. _Obes. Res_. 1998;6(Suppl. 2):1S\u2013210S.\n\n2. Flegal K.M., Carroll M.D., Kuczmarski R.J., Johnson C.L. Overweight and obesity in the United States: Prevalence and trends, 1960 to 1994. _Int. J. Obes_. 1998;22:39\u201347.\n\n3. Mokdad A.H., Serdula M.K., Dietz W.H., Bowman B.A., Marks J.S., Koplan J.P. The spread of the obesity epidemic in the United States 1991\u20131998. _JAMA_. 1999;282:1519\u20131522.\n\n4. Harnack L.J., Jeffery R.W., Boutelle K.N. Temporal trends in energy intake in the United States: An ecologic perspective. _Am. J. Clin. Nutr_. 2000;71(6):1478\u20131484.\n\n5. Cavadini C., Siega-Riz A.M., Popkins B.M. U.S. adolescent food intake trends from 1965 to 1996. _Arch. Dis. Child_. 2000;83(1):18\u201324.\n\n6. Available atUSDA, Dietary Guidelines for Americans. 2000. .\n\n7. Drewnowski A. Taste preferences and food intake. _Annu. Rev. Nutr_. 1997;17:237\u2013250.\n\n8. Drewnowski A. Palatability and satiety: Models and measures. _Ann. Nestle_. 1998;56:32\u201342.\n\n9. Drewnowski A. Energy density, palatability, and satiety: Implications for weight control. _Nutr. 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Foods with different satiating effects in humans. _Appetite_. 1990;15:115\u2013126.\n\n46. Blundell J.E., Lawton C.L., Cotton J.R., Macdiarmid J.I. Control of human appetite: Implications for the intake of dietary fat. _Annu. Rev. Nutr_. 1996;16:285\u2013319.\n\n47. Gibson S.A. Are high-fat, high-sugar foods and diets conducive to obesity? _Int. J. Food Nutr. Sci_. 1996;47:405\u2013415.\n\n48. Rogers P.J., Carlyle J., Hill A.J., Blundell J.E. Uncoupling sweet taste and calories: Comparison of the effects of glucose and three intense sweeteners on hunger and food intake. _Physiol. Behav_. 1988;43:547\u2013552.\n\n49. Rogers P.J., Blundell J.E. Separating the actions of sweetness and calories: Effects of saccharin and carbohydrates on hunger and food intake in human subjects. _Physiol. Behav_. 1989;45:1093\u20131099.\n\n50. (Suppl.)Poppitt S.D. Energy density of diets and obesity. _Int. J. Obes_. 1995;19:S20\u2013S26.\n\n51. Stubbs R.J., Harbron C.G., Prentice A.M. Covert manipulation of the dietary fat to carbohydrate ratio of isoenergetically dense diets: Effects on food intake in feeding men ad libitum. _Int. J. Obes_. 1996;20:651\u2013660.\n\n52. (Suppl.)Prentice A.M., Poppit S.D. Importance of energy density and macronutrients in the regulation of food intake. _Int. J. Obes_. 1996;20:S18\u2013S23.\n\n53. Rolls B.J., Bell E.A. Intake of fat and carbohydrate: Role in energy density. _Eur. J. Clin. Nutr_. 1999;53:S166\u2013S173.\n\n54. (Suppl.)Seagle H.M., Davy B.M., Grunwald G., Hill J.O. Energy density of self-reported food intake: Variation and relationship to other food components. _Obes. Res_. 1997;5:S87.\n\n55. Holt S.H.A., Brand Miller J.C., Petocz P., Farmakalidis E. A satiety index of common foods. _Eur. J. Clin. Nutr_. 1995;49:675\u2013690.\n\n56. Rolls B.J., Kim-Harris S., Fischman M.W., Foltin R.W., Moran T.H., Stoner S.H. Satiety after preloads with different amounts of fat and carbohydrate: Implications for obesity. _Am. J. Clin. Nutr_. 1994;60:476\u2013487.\n\n57. Glanz K., Basil M., Maibach E., Goldberg J., Snyder D. Why Americans eat what they do: Taste, nutrition, cost, convenience and weight control concerns as influences on food consumption. _J. Am. Diet. Assoc_. 1998;98:1118\u20131126.\n\n58. Drewnowski A. Low-calorie foods and the prevalence of obesity. In: Altschul A.M., ed. _Low-Calorie Foods Handbook_. New York: Marcel Dekker; 1993:513\u2013534.\n\n59. Meiselman H.L., Waterman D., Symington L.E. Armed Forces Food Preferences. _U.S. Army Natick Development Center Technical Report TR-75-63-FSL_. 1974.\n\n60. Drewnowski A., Pierce B., Halmi K.A. Fat aversion in eating disorders. _Appetite_. 1988;10:119\u2013131.\n\n61. Goran M.I. Energy metabolism and obesity. _Med. Clin. N. Am_. 2000;84(2):347\u2013362.\n\n62. Schoeller D.A. Recent advances from application of doubly labeled water to measurement of human energy expenditure. _J. Nutr_. 1999;129(10):1765\u20131768.\n\n63. Wildey M.B., Pampalone S.Z., Pelletier R.L., Zive M.M., Elder J.P., Sallis J.F. Fat and sugar levels are high in snacks purchased from student stores in middle schools. _J. Am. Diet. Assoc_. 2000;100:319\u2013322.\n\n64. Farris R.P., Nicklas T.A., Myers L., Berenson G.S. Nutrient intake and food group consumption of 10-year-olds by sugar intake level: The Bogalusa Heart Study. _J. Am. College Nutr_. 1998;17:579\u2013585.\n\n65. Harnack L., Stang J., Story M. Soft drink consumption among U.S. children and adolescents: Nutritional consequences. _J. Am. Diet. Assoc_. 1999;99:411\u2013436.\n\n66. Jeffery R.W., French S.A. Epidemic obesity in the United States: Are fast foods and television viewing contributing? _Am. J. Public Health_. 1998;88:277\u2013280.\n\n67. Bray G., Popkin B.M. Dietary fat intake does effect obesity. _Am. J. Clin. Nutr_. 1998;68:1157\u20131173.\n\n68. McCrory M.A., Fuss P.J., McCallum J.E., Yao M., Vinken A.G., Hays N.P., Roberts S.B. Dietary variety within food groups: Association with energy intake and body fatness in men and women. _Am. J. Clin. Nutr_. 1999;69(3):440\u2013447.\n\n69. McCrory M.A., Fuss P.J., Hays N.P., Vinken A.G., Greenberg A.S., Roberts S.B. Overeating in America: Association between restaurant food consumption and body fatness in healthy adult men and women ages 19 to 80. _Obes. Res_. 1999;7(6):564\u2013571.\n\n70. Hill J.O., Melanson E.L., Wyatt H.T. Dietary fat intake and regulation of energy balance: Implications for obesity. _J. Nutr_. 2000;130(Suppl. 2):284S\u2013288S.\n\n71. Willett W.C. Dietary fat and obesity: An unconvincing relation. _Am. J. Clin. Nutr_. 1998;68:1149\u20131150.\n\n72. Willett W.C. Is dietary fat a major determinant of body fat? _Am. J. Clin. Nutr_. 1998;67(Suppl. 3):556S\u2013562S.\n\n73. McDowell M.A., Briefel R.R., Alaimo K., Bischof A.M., Caughman C.R., Carroll M.D., Loria C.M., Johnson C.L. Energy and macronutrient intakes of persons ages 2 months and over in the United States: Third National Health and Nutrition Examination Survey, Phase 1, 1988\u201391. _Vital Health Statis. NCHS_. 1994;255:1\u201324.\n\n74. Trichopoulou A., Lagiou P. Worldwide patterns of dietary lipids intake and health implications. _Am. J. Clin. Nutr_. 1997;66:961S\u2013964S. (Suppl.)\nE.\n\nGastrointestinal Diseases\nCHAPTER 36\n\nNutrition in the Prevention and Treatment of Common Gastrointestinal Symptoms\n\nLAWRENCE J. CHESKIN1, 1Johns Hopkins Bayview Medical Center, Baltimore, Maryland\n\nDEBRA L. MILLER2, 2Central Soya Co., Inc., Fort Wayne, Indiana\n\n## I. INTRODUCTION\n\nThe prevalence of episodic gastrointestinal complaints in the general population is quite high and can take many forms. In a recent national survey in which respondents were asked about digestive symptoms (pain, bloating, loose stools), 40.5% reported having one or more digestive complaints in the month prior to the interview [1]. Gastrointestinal complaints include acute symptoms such as abdominal pain, bloating, excessive gas or burping, diarrhea, heartburn, constipation, and nausea. These symptoms may occur for varying amounts of time and with varying degrees of severity. Other gastrointestinal problems are syndromes or diseases and are thus more chronic in nature; the most common include irritable bowel syndrome and gastroesophageal reflux disease. Others are less common but important causes of morbidity, for example, inflammatory bowel diseases (ulcerative colitis and Crohn's disease) and celiac disease.\n\nSome of these conditions are attributed directly to the ingestion of certain foods or are potentially aggravated by specific foods. Another recent survey, which assessed the prevalence of various gastrointestinal complaints (cramps, nausea, excessive burping and gas, bloating, constipation, diarrhea, heartburn), found that individuals who reported having these complaints frequently attribute them to foods and beverages consumed. Table 1 shows the percentages of attribution of gastrointestinal complaints to foods and beverages in general and to specific foods [2]. While it is noteworthy that a large fraction of people surveyed attribute gastrointestinal symptoms to diet, in many cases this is anecdotal information and there are no scientific data to support a causative link [3].\n\nTABLE 1\n\nAttribution of Gastrointestinal Symptoms (in Past Month) to Foods or Beverages by American Adults (947 Interviews)\n\naRespondents could report multiple events.\n\nbRespondents could attribute symptoms to multiple foods and\/or beverages.\n\nIt is thought that this predilection toward gastrointestinal distress from foods is a rather recent phenomenon in human history. In prehistoric time, our ancestors rarely experienced such ailments as constipation, heartburn, gas, or irritable bowel syndrome [4]. This seems surprising given that early humans consumed nearly everything in their environment including most of the plants, roots, animals, and insects. Because early humans consumed all components of foodstuffs including husks, skins, pit, and seeds, the digestive tract needed to be an efficient processor of food and, indeed, this is what it evolved to be. However, since the 19th century, milling and processing of food (outside of the gastrointestinal tract) have become commonplace. Through milling procedures, raw foods are processed into flour, sugar, and oils. Much dietary fiber and other nutrients are lost in this process. Nutrition scientists have recently begun to recognize the importance of fiber and other natural components of foods in gastrointestinal health. Some foods, however, are indeed more likely to cause negative digestive consequences than others. The goals of this chapter are to focus on the common acute gastrointestinal complaints\/symptoms: excessive gas, heartburn, diarrhea, constipation, and nausea\/vomiting; to discuss specific foods that are associated with such symptoms; and, finally, to identify nutritional and other practices that may be therapeutic for such ailments.\n\n## II. EXCESSIVE GAS\n\nA. Prevalence and Causes\n\nHaving intestinal gas is normal; however, having excess gas can be uncomfortable or even embarrassing. Gas in the intestines comes from three sources: swallowed air, intralumenal production resulting from the normal breakdown of certain undigested foods by the harmless bacteria that are naturally present in the large intestine, and from diffusion from the blood [5]. While the self-reported frequency of excessive intestinal gas varies, humans have a relatively consistent amount of gas (\u223c200 mL) in their small and large bowel in both the fasting state and after a meal [6]. However, the rate of excretion of gas varies greatly between individuals, ranging from 476 to 1491 mL\/day (mean 705 mL\/day) [7]. In one British study, participants who ingested their usual diet passed gas per rectum an average of 8 times\/day [7], whereas a study of healthy American participants (aged 20\u201360) passed gas on an average frequency of 10 times per day with an upper limit of normal (mean \u00b1 2 SD) of 20 times per day [8].\n\n### B. Production and Composition of Intestinal Gas\n\nThree gases\u2014CO2, H2, and CH4\u2014are produced in appreciable quantity in the bowel lumen. Carbon dioxide (CO2)is thought to account for 50\u201360% of flatus volume, and such levels are usually associated with high concentrations of H2. Because the only bowel source of H2 is bacterial metabolism, flatus CO2 similarly appears to be derived from fermentation reactions. Intestinal bacteria liberate H2 during fermentation of either carbohydrate or protein; however, H2 production from amino acids is appreciably less than that from sugars.\n\nComprehensive tests of why some individuals have increased rectal gas have not been done; however, it has been determined that neither age nor sex correlated with the frequency of the passage of flatus [8]. Increased gas is often attributed to dietary factors. In individuals with intestinal disorders, carbohydrates and proteins, which are completely absorbed by the normally functioning intestine, may be malabsorbed and provide a substrate for colonic H2 production.\n\nLike H2, the sole source of CH4 (methane) in humans is the metabolism of the colonic bacteria. A person's tendency to produce methane is usually a fairly consistent trait over a period of several years. This tendency appears to be related more to genetic factors than to diet or other environmental factors. People who consistently produce large quantities of CH4 have stools that float in water due to a decreased fat-togas ratio in the density of the stool [9]. Although there has been some speculation that persons who produced larger amounts of CH4 may be at higher risk of colon cancer, this has not been substantiated by recent studies [10].\n\nInterestingly, the three main gases in flatus or intestinal gas are not those that produce noxious or unpleasant odors. Rather, odor is attributed to trace amounts of sulfur-containing compounds such as methanethiol, dimethylsulfide, and hydrogen sulfide [11]. The majority of ingested sulfur is from the amino acids: methionine, cystine, and cysteine. Specific foods that contain these compounds are high-protein foods like meats and eggs. Sulfur is also a component of the vitamins thiamin and biotin.\n\n### C. Excess Gas and Specific Foods\n\nFoods particularly associated with the production and excretion of excess gas include fruits and vegetables (particularly legumes [12]), which contain high concentrations of oligosaccharides that cannot be digested by the enzymes of the normal small bowel. Instead, the oligosaccharides are fermented by the colonic bacteria, resulting in the production of gas. Of the numerous oligosaccharide-containing foods alleged to increase the excretion of rectal gas, baked beans, which contain a large amount of the complex sugar raffinose, are the only natural food that has been carefully studied. In one study, a diet containing 51% of its calories as baked beans increased flatus elimination from a basal level of 15 to 176 mL\/hour [12a]. Table 2 includes a list of foods associated with intestinal gas [5].\n\nTABLE 2\n\nFoods Associated with Excessive Intestinal Gas\n\nOther foods and food components that have been shown to increase H2 production in healthy participants after ingestion include flours made with wheat, oats, potatoes, and corn (see Table 2), because these foods are rich in polysaccharides (not oligosaccharides). Complex carbohydrates other than oligosaccharides may not be completely absorbed [13].\n\nExcess gas production is not limited to complex carbohydrates; some commonly used sweeteners and fibers have been associated with increased H2 excretion. Fructose, present in exceptionally large quantities in sodas, juices, and other soft drinks, is malabsorbed by a sizable proportion of the healthy population [14]. Also, sorbitol, widely used as a low-calorie sugar substitute, is also poorly absorbed, and is readily fermented by colonic bacteria, causing gas production. Studies of the gas-producing effects of various fiber sources have shown that only pectin (found in apples and other fruits and vegetables) and hemicelluloses (the main structural fiber in cereals) appreciably increase H2 excretion [15].\n\nLactose intolerance is caused by a deficiency in the intestinal enzyme, lactase, which is necessary for the digestion of the disaccharide, lactose, a sugar found almost exclusively in dairy products. Lactase deficiency is the most common enzyme deficiency in humans. This common condition is thought to contribute to gastrointestinal complaints of gas and bloating, among other symptoms (see Chapter 37 on lactose intolerance). Lactose intolerance contributes to excessive gas production when unabsorbed lactose provides a substrate for H2 production by colonic bacteria.\n\nAbdominal distention and bloating, thought to be caused by \"too much gas,\" are two of the most frequently encountered gastrointestinal complaints. However, there is no evidence that excessive bowel gas is the primary cause of bloating and distention. The frequent complaint that certain foods \"give me gas\" or \"make me bloated\" may actually be more related to a hypersensitivity to bowel distention. Specific foods may actually stimulate abnormal motility rather than cause increased production of gas.\n\n### D. Therapy for Excess Gas\n\nIn trying to eliminate foods from the diet that may cause excess gas [e.g., cauliflower, brussels sprouts, dried beans (legumes), broccoli, cabbage and bran], foods that are high in fiber and food for the overall health of your digestive system may also be eliminated. Because of this, these foods may best be consumed in moderation rather than completely eliminated from the diet. For individuals who experience excess gas production as a result of eating foods containing wheat or corn flours, experimenting with rice flour as an alternative may be of benefit. Rice flour is the only complex carbohydrate that has been shown to be almost completely absorbed [13].\n\nDigestive aids such as Beano\u00ae reduce gas by facilitating the digestion of raffinose, the sugar in beans and many gas-producing vegetables. Simethicone-containing products, such as Mylicon\u00ae and Gas X\u00ae, reduce bloating by reducing surface tension and coalescing bubbles of gas. Table 3 contains a list of medications for gas. Other foods that are thought to produce gas that may be eliminated from the diet include sorbitol-containing chewing gum and hard candy (such foods may also cause excess air swallowing because of constant chewing) and carbonated beverages. Exercise can also aid in stimulating the passage of gas through the digestive tract.\n\nTABLE 3\n\nMedications for Symptomatic Relief of GI Symptoms\n\nFor individuals with lactase deficiency, restriction of lactose-containing foods (milk and milk products), ingestion of oral lactase enzyme preparations (Lactaid\u00ae), or the substitution of yogurt for milk has been thought to be of benefit to lactose maldigesters with gas problems. However, a controlled, blinded study of participants with self-diagnosed, severe lactose intolerance showed that symptoms following ingestion of 1 cup of regular milk and 1 cup of lactose-free milk were not different 16] (see [Chapter 37).\n\n## III. HEARTBURN AND GASTROESOPHAGEAL REFLUX DISEASE\n\nA. Prevalence and Causes\n\nHeartburn is the most common specific gastrointestinal complaint in the Western Hemisphere. Nearly every middle-aged American adult has had one or more episodes of heartburn. Surveys have found that about 40% of U.S. adults experience heartburn at least once a month [17, 18], while roughly 10% suffer with heartburn daily [19]. Heartburn is also common during pregnancy, particularly in the third trimester, with 90% of pregnant women experiencing some type of reflux symptoms [20].\n\nGastroesophageal reflux disease (GERD) occurs when the muscle connecting the esophagus with the stomach does not function properly, allowing stomach acids and contents to enter the esophagus. Heartburn begins as a burning pain that starts behind the breastbone and radiates upward toward the back of throat. The description of \"burning\" or \"hot\" or \"acidic\" sensations is typically used. Often there is a sensation of food coming back into the mouth, accompanied by an acid or bitter taste. Heartburn can become intense enough to cause pain that radiates throughout the chest region upward toward the neck and throat and occasionally to the back and arms.\n\nHeartburn, the classic manifestation of GERD, is a commonly used but frequently misunderstood term. It has many synonyms, including \"indigestion,\" \"acid regurgitation,\" \"sour stomach,\" and the more general \"chest pain.\" The precise physiologic mechanisms that produce heartburn are, surprisingly, poorly understood. Although the reflux of gastric acid is most commonly associated with heartburn, the same symptoms may be elicited by other gastrointestinal events (esophageal distention, reflux of bile salts, and acid-induced motility disorders).\n\nThe pain associated with heartburn seems best explained by the stimulation of chemoreceptors due to the sensitivity of the esophagus to the presence of acid, as demonstrated during perfusion studies or by monitoring the esophageal pH of people with GERD. These receptors, however, are not superficial and their specific location is not known [21]. Correlations between discrete episodes of acid reflux and actual pain are poor; therefore, painful symptoms seem to require more than just the presence of acid in the esophagus [22]. One contributing factor may be the disruption of the mucous membrane in the esophagus, although most symptomatic patients do not show such a disruption. Another possibility is hydrogen ion concentrations in the esophagus that alter its pH. One study found that 25 participants with reflux disease experienced heartburn-like symptoms when infused with solutions of pH 1.0 and 1.5, but only one-half had such symptoms with solutions of pH 2.5\u20136.0 [23]. Other factors may include inflammation with increased polymorphonuclear leukocytes, acid clearance mechanisms, salivary bicarbonate concentration, volumes of refluxed acid, and the interaction of pepsin with acid [5].\n\n### B. Heartburn and Food\n\nHeartburn is predictably aggravated by many factors, food in particular. Symptoms usually present within an hour of eating, particularly after consuming the largest meal of the day. Foods high in fat, chocolate, onions, or carminatives (peppermint and spearmint) may aggravate heartburn by decreasing lower esophageal sphincter pressure [24, 25]. While the lower esophageal sphincter is not an actual sphincter, but rather a muscular structure that acts as a sphincter, it normally remains contracted, closing the entrance to the stomach and preventing reflux of the stomach contents into the lower esophagus [5]. When lower esophageal sphincter pressure is lowered, stomach contents and acid may flow upward into the esophagus. Diminished lower esophageal sphincter pressure may result from hormonal (gastrin, secretin, cholecystokinin, and glucagon) action, muscle abnormalities, or impaired nerve innervation [5].\n\nIntake of foods such as peppermint, spearmint, chocolate, alcohol, and fats (especially fat in whole milk) may alter the hormonal milieu of the esophagus and reduce lower esophageal sphincter pressure [26]. In contrast, coffee contains the methylxanthines caffeine and theophylline, which may increase lower esophageal sphincter pressure by inhibiting phosphodiesterase, in turn, increasing intracellular concentrations of cyclic adensosine monophosphate in the lower esophageal sphincter [26]. Other studies, however, have found that theophylline decreases lower esophageal sphincter pressure and that caffeine had no effect [26]. Another study found that both caffeinated and decaffeinated coffee decreased lower esophageal sphincter pressure [26], indicating that there may an unknown substance in coffee other than caffeine that affects lower esophageal sphincter pressure. How peppermint and spearmint increase lower esophageal sphincter pressure is unknown.\n\nOther foods commonly associated with heartburn include highly acidic foods such as citrus products and tomato-based foods, and spicy foods. These foods do not affect lower esophageal sphincter pressure. Rather, they directly irritate the inflamed esophageal mucosa by increasing the presence of acid, lowering pH, or increasing osmolarity [5]. Many beverages, including citrus juices, soft drinks, coffee, and alcohol, also cause heartburn by more than one mechanism. Wine drinkers may have heartburn after hearty red wines, but not after delicate white wines. The exact reason for this is not known.\n\nIn addition, generalized chest pain, characterized as a squeezing or burning substernal sensation, radiating to the back, neck, jaw, or arms (sometimes indistinguishable from angina), is associated with the ingestion of very hot or very cold liquids. The mechanism for this is not well understood. One possible cause may be esophageal distention resulting from the activation of stretch receptors in response to a cold substance [24]. Such distention and pain are also experienced with acute food impaction, the drinking of carbonated beverages (in some individuals), and dysfunction in the belch reflex.\n\nOther factors that may aggravate heartburn include running; however, riding a stationary bicycle or using other exercise machines does not seem to have this effect and, thus, may be good exercise for those with GERD. Cigarette smoking exacerbates heartburn because nicotine lowers lower esophageal sphincter pressure. Air swallowing, which often accompanies inhalation of smoke, also tends to relax the lower esophageal sphincter. A number of medications may also cause or exacerbate heartburn by decreasing lower esophageal sphincter pressure. Aspirin may directly irritate the inflamed esophagus. Finally, emotions such as fear, anxiety, and worry may aggravate heartburn symptoms, probably through the amplification of symptoms rather than by increasing the amount of acid reflux.\n\n### C. Therapy\n\nGERD can be lessened by following a few guidelines. In addition to limiting the foods known to precipitate heartburn symptoms, decreasing the amount eaten at meals, and not eating 2\u20133 hours prior to lying down or going to bed may also help reduce symptoms. Foods to avoid due to heartburn include high-fat foods, peppermint\/spearmint, caffeinated foods and beverages (coffee, tea, chocolate, colas), alcohol, citrus fruits and juices, tomatoes and tomato products, and pepper. However, citrus fruits and tomatoes are good sources of vitamin C.\n\nBecause heartburn is such a common gastrointestinal complaint, many of those who suffer its effects do not consult a physician and seek relief from over-the-counter preparations such as antacids and H2 receptor antagonists (e.g., Pepcid AC\u00ae, Zantac\u00ae, Tagamet\u00ae) (Table 3). In a recent study of patients who use antacids daily, more than half had evidence of erosive esophagitis. Individuals with frequent or daily heartburn and pregnant women are advised to consult their physician for evaluation and individualized therapies.\n\n## IV. DIARRHEA\n\nA. Prevalence and Causes\n\nDiarrhea is best defined as an increased liquidity or decreased consistency of stools [5]. There are four mechanisms of diarrhea. The first is osmotic diarrhea, which is caused by the presence of unusually large amounts of poorly absorbed, osmotically active solutes\u2014such as dietary carbohydrate or laxative\u2014in the gut lumen. In osmotic diarrhea, the ingested solute or solutes exert an osmotic force across the intestinal mucosa causing an increase in water in the large intestine. Hallmarks of osmotic diarrhea include the following:\n\n1. Fecal water output is directly related to the fecal output of the solute or solutes exerting osmostic pressure across the intestinal mucosa.\n\n2. Diarrhea stops with fasting or the termination of ingesting the poorly absorbed solute (carbohydrate or laxatives)\n\n3. Stool analysis reveals an osmotic gap that is much more than the normal osmolarity of fecal fluid that exits the rectum (290 mOsm\/kg).\n\nThe second mechanism is secretory diarrhea, which refers to the diarrhea that is caused by abnormal ion transport in intestinal epithelial cells. This condition is not marked by an osmostic gap, but may occur in tandem with osmotic diarrhea. Infectious diseases of the gastrointestinal tract often result in secretory diarrhea.\n\nThe third mechanism of diarrhea is altered gut motility. Gut motility is the process and pace by which food is physically transported through the gastrointestinal tract. Enhanced motility may result in diarrhea by propelling boluses of fluid quickly through the gut, thereby decreasing the contact time with the absorptive epithelial-sometimes called \"intestinal hurry\" [5]. Conversely, abnormally slow motility may allow bacteria to \"overgrow\" in the small intestine and cause diarrhea or steatorrhea (an excess amount of fat in the feces).\n\nThe final mechanism of diarrhea is exudation. Exudation, as it relates to diarrhea, is usually the result of inflammation or ulceration and causes the escape of mucus, fluid, serum proteins, and blood from the intestinal mucosa [5]. These components are deposited in the gut lumen and expelled in the feces. This type of diarrhea is associated with dysentery and ulcerative colitis [5].\n\nBecause secretory, altered motility and exudation mechanisms for diarrhea are associated with infectious diseases of the gastrointestinal tract or more chronic types of diarrhea, this discussion will focus on osmotic diarrhea, the mechanism most associated with acute bouts of diarrhea related to the ingestion of food.\n\n### B. Diarrhea and Food\n\nAs noted, malabsorbed carbohydrates, such as starches, lactose, and fructose, can contribute to osmostic diarrhea. When carbohydrates are not absorbed in the small intestine, they remain in the lumen and are a potential source of osmotically active particles. Two main factors determine the severity of diarrhea that results from carbohydrate malabsorption in sensitive individuals: (1) the type and amount of carbohydrate ingested and (2) the rate at which carbohydrate reaches the colon. If relatively large amounts of malabsorbed carbohydrate are present in the gut (e.g., lactose intolerance), diarrhea is likely to result. Smaller amounts of malabsorbed carbohydrate, such as after ingestion of a sorbitol-containing food, may result only in bloating, distention. and excessive flatus (see Section II on excessive gas).\n\nMalabsorbed dietary fat, which can be normal with high-fat diets, can also contribute to diarrhea. When dietary fatty acids not properly absorbed by the small intestine reach the colon, a portion of them is hydroxylized by bacterial enzymes [5]. Under experimental conditions, fatty acids and hydroxy-fatty acids have been shown to inhibit fluid absorption from both the small intestine and colon [27\u201329]. This is the proposed mechanism by which steatorrhea causes excessive fecal fluid losses [5]. Controversy has arisen over whether the nonabsorbable, lipid-based fat replacer, olestra, causes diarrhea. In a placebo-controlled trial, consumption of olestra-containing potato chips did not result in increased reports of diarrhea or other gastrointestinal complaints compared to triglyceride-containing potato chips [3]. Unpublished data from the olestra postmarketing surveillance study also indicate no dose\u2013response relationship between diarrhea and olestra intake [2].\n\nDiarrhea is also a common complaint in individuals who consume large amounts of alcohol (acutely or chronically). Ethanol (the alcohol found in alcoholic beverages) has numerous effects on the gastrointestinal tract that contribute to diarrhea. Ethanol ingestion alters gut motility, causing rapid transit and heightened propulsive movements, resulting in food malabsorption [30, 31]. Alcohol also decreases the activity of enzymes that break down disaccharides, decreases bile secretion (when liver disease is present), and can cause malabsorption of fat, thiamin, vitamin B12, folate, glucose, amino acids, water, and sodium [5]. Many binge drinkers are also at higher risk of steatorrhea due to decreased pancreatic exocrine function from chronic pancreatitis [5]\n\nAnother common food-related cause of diarrhea is so-called, \"traveler's diarrhea,\" which refers to the gastrointestinal infections often acquired when an individual travels from industrialized to developing areas of the world. The condition is usually defined, in the setting of recent travel, as the passage of three or more unformed stools in a 24-hour period in association with one or more of the following: nausea, vomiting, cramps, fever, fecal urgency, and bloody stools [32]. Traveler's diarrhea is acquired when fecally contaminated foods or water are ingested. Infectious agents present in fecal matter such as bacteria, viruses, and parasites are the primary cause of the disorder [32]. Table 4 provides a list of high- and low-risk foods to avoid when traveling to high-risk areas and what areas of the world represent the greatest risk for travelers.\n\nTABLE 4\n\nHigh- and Low-Risk Foods and Risk Areas of the World for Traveler's Diarrhea\n\nDiarrhea is one of the most common side effects of drugs. Antibiotics are especially associated with diarrhea. A major mechanism of this effect is through antibiotic interference with the ecology of gut bacteria, suppressing growth of natural bacteria and favoring the growth of other bacteria (such as _Clostridium difficile_ ). This causes inflammation and injury of the gut lining and stimulates the secretion of fluid [4].\n\nLaxative abuse is a self-induced cause of diarrhea that is seen in young women with eating disorders in an attempt to control body weight by decreasing food absorption; however, even large doses of Correctol\u00ae have been shown to decrease energy absorption by only about 5%. Weight loss from this practice is primarily fluid loss [33]. Finally, diarrhea may also occur in runners, \"runners trots,\" especially those beginning a running training program [34]. Causes for this are speculative, but may include osmotic activity due to excess intake of fiber or carbohydrate, or increased motility from the physical impact of running [35, 36].\n\n### C. Therapy\n\nBecause a multitude of foods may contribute to diarrhea in different people, individuals must try to eliminate \"suspected\" food and experiment with others. During an episode of diarrhea, it may be helpful to briefly limit food intake to cooked food (no raw foods) and, depending on the severity, consume clear broths, teas, gelatin, bottled non-carbonated water, and drinks that replenish electrolytes such as sports drinks. Low-fat high-protein foods are also usually tolerated (i.e., skinless chicken or fish, egg whites) and should be added to improve protein content of the diet. Dairy products may be added as symptoms and time pass. Raw fruits and salads should be added last. Extended dietary restrictions to manage diarrhea (e.g., beyond a few days) can delay the healing process due to impaired nutritional status. Table 3 provides a list of medications for symptomatic relief of diarrhea.\n\nIf lactose intolerance is suspected, intake of dairy products should be greatly reduced. If this results in the disappearance of diarrhea, lactose intolerance is likely and treatment with restriction or avoidance of dairy products, supplemental lactase tablets (such as Lactaid), or lactose-reduced dairy products is helpful.\n\nFor traveler's diarrhea, the best treatment for mild symptoms without fever includes rehydration from a safe source (bottled water, soda, juice, or canned soup) coupled with a source of sodium such as salted crackers, and avoiding other solid foods until symptoms dissipate [32]. Subsalicylates or loperamide can provide symptom relief. With moderate to severe symptoms plus fever or dysentery, antibacterial agents may be necessary, in which case, travelers must seek medical treatment [32].\n\nWhen diarrhea results from running, moderating dietary fiber, waiting several hours after eating before running, and attempting to have a bowel movement before running may be helpful. The diarrhea produced by laxative abuse should improve once such abuse ceases; however, edema may develop following more severe or prolonged abuse. This edema, however, usually spontaneously resolves (see Chapter 43 on eating disorders). Finally, if diarrhea is caused by antibiotic use, the general recommendations listed above are appropriate, in addition to consulting the prescribing physician and considering stopping the possible offending agent or changing to another, less problematic antibiotic if further treatment is needed. Because antibiotic use and other infections can deplete the gut of bacteria necessary for digestion, consuming yogurt with live or active cultures (lactobacillus) may help replenish these bacteria.\n\nIt is also noteworthy that, even when there is an underlying, non-food-related cause for diarrhea (such as chronic pancreatitis), avoidance of large amounts of certain foods (such as fatty foods in chronic pancreatitis) is useful nutritional therapy for reducing diarrhea.\n\n## V. CONSTIPATION\n\nA. Prevalence and Causes\n\nThe concept of constipation differs greatly between individuals, and between the medical and lay definitions. Difficult defecation with straining and hard or infrequent stools are symptoms most often associated with constipation by the lay public. Other symptoms sometimes associated with constipation include no urge to have bowel movements, a sense of incomplete evacuation, anal or perineal pain, soiling of clothes, and discomfort (pain\/bloating). Abdominal pain relieved by defecation, a hard stool, straining at defecation, incomplete evacuation and abdominal discomfort are each reported by 5\u201330% of individuals in surveyed populations in developed nations [5]. Bowel frequency of less than three stools per week, the requirement for meeting the medical definition of constipation, is reported by about 4% of respondents, and fewer than two stools per week by 1\u20132% of respondents.\n\nIndividual characteristics associated with constipation include increased age and nonwhite ethnicity (living in Western society). Also, constipation is more prevalent in women than men [37]. Specific symptoms associated with constipation are more common [38] and infrequent stools (1\u20132\/week) tend to be reported almost entirely by women [39]. In one study of 220 healthy participants consuming their normal diet, 17% of women, but only 1% of men, passed less than 50 g of stool per day [40]. Also, many women (about 40%) experience some type of constipation during pregnancy [41]. The reason for this sex difference is unknown and does not seem to be related to circulating levels of sex hormones or other hormones [42]. Elderly persons report more straining during defecation rather than decreased bowel frequency [5]. In the United States, constipation is 30% more common among nonwhites than whites, with similar increases in prevalence with age [37, 43]. In native populations of Africa and India, however, constipation appears to be quite rare, likely because differences in the diet between native and Americanized populations [40].\n\nThis discussion will focus primarily on diet-related causes of constipation; however, there are other factors that may cause or exacerbate constipation. Any factor that increases water absorption from the colon and leads to smaller, harder stools could theoretically cause constipation. Just as reduced contact time between gut contents and the colonic mucosa favors decreased water absorption and diarrhea, increased contact time increases water absorption and can cause constipation. Individuals with wide and\/or long colons are likely to have slower transit rates and increased likelihood of constipation [44]. Also, in some individuals the frequency and duration of peristaltic colonic waves are reduced or nonexistent. This condition causes very slow transit time and the passage of one or fewer bowel movements per week [5]. Other physiological factors that may contribute to constipation are failure of relaxation of the anal sphincter, diminished rectal sensation, and diseases such as hypothyroidism, diabetes mellitus, Parkinson's disease, multiple sclerosis, and spinal cord injuries or lesions [5].\n\nInterestingly, some psychological factors also seem related to constipation. In one study of 21 healthy males, stool weight and bowel frequency were significantly correlated with personality traits such as being socially outgoing, energetic, and optimistic [45]. Patients with the diagnoses of anorexia or bulimia nervosa often complain of constipation, and prolonged whole-gut transit time has been shown in patients with both conditions [46].\n\n### B. Constipation and Food\n\nIn contrast to previously discussed gastrointestinal complaints such as diarrhea, constipation is not commonly aggravated by the ingestion of specific foods. Rather, constipation is most commonly associated with the omission of fiber in the diet. Fiber is often defined as a plant food component that cannot be digested. Many forms of fiber are polysaccharides made of sugar units joined in unique linkages that render the molecule resistant to degradation by digestive enzymes. Thus, fiber is, by and large, undigestable carbohydrate.\n\nIncreased intake of fiber helps to prevent the development of constipation in two ways: (1) fiber increases stool volume and weight and (2) fiber decreases colonic transit time. Fiber adds to stool weight both directly and indirectly. Because fiber is not degradable, this nondigested bulk contributes directly to stool weight. In addition, some types of fiber can hold water within their cellular structure, adding further bulk to the stool as well. Fiber ingestion contributes indirectly to stool bulk by increasing the proliferation of intestinal bacteria, which, in turn, increases stool weight.\n\nIngested dietary fiber has been clearly shown to decrease colonic transit time (i.e., increase the speed at which undigested material moves through the colon). A meta-analysis of 20 studies (including normal control participants, those with irritable bowel syndrome, and those with constipation) revealed that the addition of fiber to the diet consistently resulted in decreased transit time [47]. The mechanism by which this occurs is not fully understood, but it is thought that the increased bulk created by ingested fiber in the colonic lumen stimulates propulsive motor activity [5]. However, other factors may be involved. In a study comparing coarse and fine bran given twice daily, the coarse bran reduced colonic transit time while the fine bran had no effect [48]. Another study found that inert plastic particles of the same size as coarse bran increased fecal output and decreased transit time similar to coarse bran [49]. Thus, it is possible that colonic stimulation is dependent on the size and\/or nature of the particulate fiber.\n\n### C. Therapy\n\nWhen an individual with normal colonic function adds fiber to the diet, stool weight increases in proportion to its baseline weight [5]. When an individual with frequent constipation (those who pass small stools) increases fiber intake, the resulting stool weight may still be below normal. In a study of 10 constipated women given a 20 g\/day supplement of wheat bran, stool weight increased from an initial 30 to 60 g\/day and bowel movement frequency increased from a mean two to three times weekly [50]. However, this output is still only about half as large as the average stool weight of those with normal function. Thus, increased fiber intake as the treatment for frequent constipation is often disappointing. However, increased fiber intake is the simplest, least expensive, and most natural treatment for constipation. Thus, most constipated individuals are advised to increase dietary fiber intake by about 30 g of nonstarch polysaccharide per day [5]. Increased intake of fiber has also been shown helpful in decreasing risk of colorectal cancer, diverticular disease, irritable bowel syndrome, and hemorrhoids.\n\nMethods to increase fiber intake include:\n\n\u2022 Adding more fruits and vegetables to the diet (especially carrots, apples, oranges, celery)\n\n\u2022 Substituting whole-grain foods for refined products (e.g., whole wheat bread instead of white bread, brown rice for white rice)\n\n\u2022 Add wheat or oat bran to muffin or baked good recipes\n\n\u2022 Add legumes (beans and nuts) in side dishes or in casseroles\n\nIt is best to introduce additional fiber to the diet gradually, so that the gastrointestinal tract can adapt to fiber-related changes in bacterial proliferation, gas production, and transit time. For some, especially women, added fiber may aggravate abdominal distention and discomfort [51]. Symptoms usually subside within 1\u20132 weeks. Bran fiber treatment may not be appropriate for young people with congenital megacolon (Hirschprung's disease), and, in some elderly individuals, if it exacerbates incontinence [5].\n\nFluid intake is another important component of therapy for constipation. The combination of added water and fiber increases stool bulk, while dehydration or salt depletion is likely to lead to increased salt and water absorption by the large intestine, resulting in small, hard stools. Drinking eight, 8-ounce glasses of liquids per day is recommended. While any liquid is better than no liquid, some are better than others. Water is best, followed by vegetable juice, fruit juice (but these are often high in energy), milk, broth (although often high in sodium), and beverages such as coffee, tea (iced or hot), and soft drinks.\n\nFor more chronic forms of constipation, a bulk laxative may be helpful (see Table 3). Bulk laxatives are a concentrated form of nonstarch polysaccharides useful for patients who cannot consume dietary fiber in adequate quantities. These products are based on wheat, plant seed mucilage, plant gums, or synthetic methylcellulose derivatives [5]. Bulk laxatives are appropriate as treatment for constipation over the long-term and should not be expected to provide immediate relief of constipation. Bulking agents may be quite useful in constipation experienced during pregnancy.\n\nFor more severe forms of constipation or impaction, a stepped care approach is usually employed. This approach would first address lifestyle factors such as low dietary fiber and fluid intake and then add progressively more potent laxatives. A physician should be consulted to determine the proper sequence and choice of treatment options.\n\n## VI. NAUSEA AND VOMITING\n\nA. Causes and Prevalence\n\n#### 1. COMPONENTS OF VOMITING\n\nThere are three components of vomiting: nausea, retching (dry heaves), and emesis (vomiting) [5].\n\n1. _Nausea_ is an extremely unpleasant, uneasy sensation that is difficult to describe and may or may not precede vomiting. A variety of stimuli may contribute to nausea (labyrinthine stimulation, visceral pain, negative psychological issues) [5]. The neural pathways mediating nausea are not known, but are thought to be the same as those for vomiting. It is possible that mild stimulation of this pathway results in nausea, while more intense activation results in vomiting [5].\n\n2. _Retching_ is the sensation of \"dry heaves.\" It consists of spasms and abortive respiratory movements with the glottis (visible part of the larynx in the rear of the throat) closed. During an episode of retching, muscular mechanisms of breathing become opposed. The chest wall and diaphragm are coordinated in an inhalation muscular contraction (motions of breathing in) while abdominal muscles are in expiratory contractions (motions associated with breathing out). During retching, the distal portion of the stomach (antrum) contracts, whereas the upper portions (the fundus and cardiac) relax. The mouth is closed. Retching may or may not be followed by vomiting.\n\n3. _Vomiting_ occurs when the gastric contents are forced up and out of the mouth, \"throwing up.\" This occurs by forceful and sustained contraction of the abdominal muscles and diaphragm at a time when the cardia of the stomach is raised and open and the pylorus is contracted [5]. This elevation of the cardia eliminates the intra-abdominal portion of the esophagus, which, if present, would tend to prevent the high intragastric pressure from forcing gastric contents into the esophagus [5]. The mechanism by which the cardia opens during vomiting is not clear.\n\n#### 2. MECHANISMS OF NAUSEA AND VOMITING\n\nThe neurophysiology of nausea and vomiting has been extensively studied in cats by Borison and colleagues [52, 53]. Vomiting involves a complex set of neurologic activities that suggests some central \"vomiting center\" in the dorsal part of the medulla [52, 53]. Electrical stimulation of this area in cats has induced vomiting [52, 53]. Other studies have suggested that there is a certain portion of the medulla that is associated with vomiting, but does not respond to electrical stimulation. Rather, this area responds to chemical stimulation and has been called the _chemoreceptor trigger zone_ [52\u201356]. However, because most of this work has been done in cats, caution must be exercised when extrapolating these results to human causes and controls of vomiting.\n\n#### 3. OTHER PHENOMENA ASSOCIATED WITH NAUSEA AND VOMITING\n\n1. _Hypersalivation_ is an increase in saliva production, is often experienced prior to vomiting, and is thought to result from the proximity of the proposed vomiting center in the medulla to the medullary control center for salivation.\n\n2. _Tachycardia_ (increased heart rate) usually accompanies nausea, and retching is accompanied by _bradycardia_ (decreased heart rate). Retching and vomiting have been associated with the onset of an arrhythmia (atrial fibrillation) and termination of other arrhythmias (e.g., ventricular tachycardia) [57, 58].\n\n3. _Defecation,_ the passing of stool, may also occur with vomiting. Similar to the association with salivation, the central control of defecation is also thought to be mediated in the medulla. Increased stimulation of the medulla, thus, may also cause these associated physiologic responses [52].\n\n### B. Conditions Causing Nausea and Vomiting\n\n1. INFECTIONS OF THE GASTROINTESTINAL TRACT\n\nInfection of the gastrointestinal tract may result in sudden outbreaks of vomiting, usually early in the morning. The urge to vomit may be so intense that individuals may vomit in bed and also experience headaches, muscle aches, giddiness, diarrhea, sweating, and fever. Many viruses and other infectious agents may cause such symptoms; however, for most gastrointestinal infections, recovery is generally fast.\n\n#### 2. MEDICATIONS\n\nTaking medications, especially new medications, is also associated with nausea and vomiting. It is thought that many medications act on the chemoreceptor trigger zone to induce nausea and vomiting [5]. Major offenders include dopamine agonists, opiate analgesics, antibiotics, and cancer chemotherapy agents, though virtually all medications are sometimes associated with this side effect. Sensitivity to the effects of medications on nausea and vomiting may be predicted if an individual has a history of motion sickness [59].\n\n#### 3. MOTION SICKNESS\n\nSweating, dizziness, headache, and nausea that may or may not lead to vomiting while in motion in cars, trains, and airplanes is referred to as motion sickness. It is thought to be caused by conflicting sensory activation of the vestibular system (which regulates balance) in the inner ear and visual sensory input. In other words, the eyes do not detect motion to the same degree as the balance mechanism in the inner ear, leading to a stress-producing phenomenon that activates the nausea and\/vomiting center of the brain.\n\n#### 4. MORNING SICKNESS\n\nNausea and vomiting during early pregnancy are common and often occur soon after waking. Nausea occurs in 50\u201390% of pregnancies and vomiting in 24\u201355% [60, 61] and is more common in first pregnancies, younger women, nonsmokers, obese women, those with <12 years of education, and women whose corpus luteum is primarily on the right side of the uterus. The onset of these symptoms is generally at 4\u20135 weeks of pregnancy [20] and may reach a peak of severity and frequency around 12 weeks [20]. The exact cause of morning sickness is not known. The two main theories are that women who are prone to morning sickness may have (1) abnormal hormone levels (human chorionic gonadotropin, progesterone, and andogrens) and\/or (2) unstable gastric electrical activities and a reduced response to the ingestion of food. A severe form of morning sickness, hyperemesis gravidarum, occurs in 3.5 per 1000 pregnancies [5]. This condition is marked by severe vomiting, which results in nutritional and\/or electrolyte disturbances in early pregnancy. Causes of this condition are not known.\n\n#### 5. PSYCHOGENIC VOMITING\n\nPsychological factors are also associated with nausea and vomiting.\n\n1. _Food aversions._ It is also possible that the mere smell, taste, or even sight of a specific food can produce a psychologically associated nausea. When individuals eat foods prior to an episode of gastrointestinal infection or other nausea\/vomiting causing condition, such foods may become associated with nausea or vomiting. This may lead to the development of a food aversion. Although such associations were important evolutionary survival aids, teaching that eating certain foods may result in illness, modern humans can inappropriately develop these aversions, even with long delays between foods and symptoms, or even when the individual is certain that the food did not cause the illness [62].\n\n2. _Self-induced vomiting._ This behavior is most often seen in young women with eating disorders such as anorexia or bulimia nervosa who regurgitate ingested food and beverages in an attempt to lose or control body weight. These individuals may become very skilled at concealing their vomiting.\n\n3. _Erotic vomiting._ Some women with underlying psychological aberrations induce vomiting to obtain sexual gratification.\n\nOther conditions associated with nausea and vomiting may be of a more chronic nature. Causes for chronic symptoms may include mechanical obstruction of the gastrointestinal tract, brain tumors, or metabolic or endocrine disorders.\n\n### C. Vomiting and Food\n\nThe timing of nausea and vomiting following a meal may partially indicate its cause. Nausea or vomiting occurring within 1 hour of a meal may indicate a peptic ulcer or a psychological disorder [5]. Nausea or vomiting occurring 1\u20138 hours after a meal may indicate food poisoning [5].\n\nThe main food-related problem that may cause nausea and vomiting is food-borne illness or \"food poisoning.\" Most cases of food poisoning result from the ingestion of food containing bacteria such as salmonella, _Campylobacter jejuni, Escherichia coli,_ or many others. These different bacteria and even different strains of similar bacteria can cause a variety of symptoms that range in severity from traveler's diarrhea (in developing countries) to death (as witnessed in 1993 in children who ate bacteria-infested ground beef from a fast-food chain).\n\nMajor nutritional and metabolic consequences of acute and chronic vomiting may occur. Acute vomiting can result in metabolic derangements such as these:\n\n\u2022 _Potassium deficiency._ This deficiency may lead to overall muscle weakness, poor intestinal tone with gastrointestinal bleeding, heart rhythm and conduction abnormalities, and weakness of respiratory muscles.\n\n\u2022 _Sodium depletion._ When the serum and body stores of sodium are depleted rapidly, as can occur with intense vomiting episodes, extreme lethargy can result. An individual may also become irritable, confused, weak, and sometimes hostile. Sodium depletion can also lead to alkalosis [a condition in which the blood has an excess of base-forming elements (pH>7.4)] and further nausea and vomiting if not repleted.\n\nChronic vomiting may lead to other nutritional concerns such as reduced energy intake, malnutrition, and deficiency states for various nutrients.\n\n### D. Therapy\n\nMost vomiting episodes subside within 6\u201324 hours of onset and can be treated at home [5]. A number of pharmaceutical products (Table 3) are available for the treatment of the nausea and vomiting associated with motion sickness, medication usage and gastrointestinal infections. See Table 5 for guidelines suggesting when a comprehensive examination and work-up should be conducted for vomiting. Avoidance of nausea and vomiting associated with stomach-irritating medications can sometimes be improved by taking the medication with food.\n\nTABLE 5\n\nGuidelines for Conducting a Thorough Medical Work-Up for Nausea or Vomiting\n\n**Contact a physician or seek medical attention if:**\n\nFor adults and children:\n\n\u2022 Vomitus contains blood or \"coffee grounds\"\n\n\u2022 Stools contain blood or are black or tarry\n\n\u2022 Nausea lasts for more than 1 week (in women, pregnancy may be a possibility)\n\n\u2022 Severe headache\n\n\u2022 Lethargy\n\n\u2022 Confusion\n\n\u2022 Decreased alertness\n\n\u2022 Severe abdominal pain\n\nFor children 6 years and younger:\n\n\u2022 Vomiting lasts more than a few hours\n\n\u2022 Diarrhea is also present\n\n\u2022 There are signs of dehydration (dry mouth, eyes, skin, and\/or sunken eyes and confusion)\n\n\u2022 Accompanied by a fever >100\u00b0F\n\nFor children older than 6:\n\n\u2022 Vomiting lasts for more than 1 day\n\n\u2022 There are signs of dehydration (see above)\n\n\u2022 Accompanied by a fever of >102\u00b0F\n\nA number of dietary practices have been suggested traditionally to ease nausea. These include drinking cold beverages slowly, eating salty foods, eating slowly, and eating smaller, more frequent meals. Eating light meals of low-fat, starchy foods and avoiding strong-smelling or tasting foods has been shown to lessen the nausea associated with motion sickness. Avoiding certain foods such as fried, greasy, or very sweet foods may also help prevent nausea. Strong smelling or strong tasting foods are often those associated with food aversions and nausea.\n\nIf an individual feels nauseated, vomiting may be prevented by drinking small amounts of clear, sweetened liquids such as carbonated beverages or fruit juices. Resting is also advised, because activity may worsen nausea. Mechanisms for why these therapies prevent or ease nausea have not been extensively studied.\n\nOne food that has been studied in regard to its effects on nausea is ginger root. Ginger root and ginger-containing foods have long been regarded as digestive aids in Chinese medicine [63]. A number of studies have investigated ginger as an antiemetic [64, 65] and an antinausea treatment [66\u201368]. In one study, participants given 1 g of powdered ginger prior to surgery had significantly fewer incidents of nausea than the group receiving a placebo dosage [65]. Other studies have found similar results under different experimental conditions (postoperative dosage [64], prior to sea voyage [69], and prior to administration of certain nausea-causing medications [67]. Thus, ginger powder may be of aid in nausea.\n\nTo avoid vomiting and other gastrointestinal symptoms that are caused by food poisoning or contamination, standard food preparation and storage precautions should be used. The reader is referred to current guidelines and summaries on this topic [70, 71].\n\nIf persistent vomiting results in potassium or sodium loss, it is best to replace these nutrients through dietary sources to minimize the possibility of excess intake. Good sources of dietary potassium include spinach, bananas, mushrooms, broccoli, and milk. Salty crackers, broths, or other low-fat, salt-containing foods can help replace sodium. Drinking plenty of water is important to prevent or treat dehydration.\n\n## VII. CONCLUSION\n\nGiven the preceding discussion, it is evident that diet plays a large role in managing common gastrointestinal complaints. Both the inclusion and exclusion of specific foods and food categories can contribute to gastrointestinal symptoms. Although there may not be one \"diet\" per se to prevent all gastrointestinal ills, there are common characteristics of a diet appropriate for general health that may be applicable. Tenets of such a healthy diet to prevent gastrointestinal symptoms include:\n\n1. Consume an adequate, but not excess, amount of energy. Maintaining normal body weight is important.\n\n2. Reduce dietary fat intake, especially saturated fat. High-fat foods, such as fried foods, are often linked to gastrointestinal complaints of heartburn, gas, diarrhea, and constipation, as well as gallstones and exacerbation of symptoms such as bloating in chronic pancreatitis and other causes of malabsorption. Excessive fat intakes can also contribute to bloating due to steatorrhea.\n\n3. Increase fiber intake. Daily intake of 30\u201340 g of fiber can improve gastrointestinal health by increasing gut motility and adding to stool weight. This can be accomplished by increasing the number of daily servings of fruit and vegetables and choosing natural and not processed grains. Fiber sources should be added to the diet incrementally to avoid producing excess gas. If certain healthy foods are problematic, they may be able to be consumed in moderation or in small amounts.\n\n4. Eat slowly and chew food thoroughly. When food is consumed slowly the mechanisms that regulate hunger and satiety are able to communicate appropriate messages for terminating a meal. Eating slowly also allows for improved gastric emptying and lessens the sensation of bloating and risk of developing heartburn.\n\n5. Consume alcohol and caffeine in moderation (<2 alcohol- or caffeine-containing drinks per day. Excess alcohol intake causes general irritation of the upper gastrointestinal tract especially.\n\n6. Exercise regularly. Running or jogging may not be advisable for those who are sensitive to diarrhea, but other more stationary forms of aerobic exercise are advisable for both gastrointestinal and cardiovascular health.\n\n7. Do not assume that a specific food is problematic in causing gastrointestinal complaints. If a specific food is suspected to be not tolerated, the use of commercially available supplemental enzymes (such as lactase for lactose intolerance) may allow individuals to consume foods that provide important nutrients.\n\nFollowing such guidelines may bring modern food intake and behavior into better balance with a gastrointestinal tract that evolved to suit the diet of Paleolithic ancestors. In addition, being aware of potential hazards to gastrointestinal health such as traveling in developing countries and proper food safety procedures can also help prevent troubling gastrointestinal symptoms.\n\nReferences\n\n1. Sandler R.S., Stewart W.F., Liberman J.N., Ricci J.A., Zorich N.L. Abdominal pain, bloating and diarrhea in the United States: Prevalence and impact. _Dig. Dis. Sci_. 2000;45:166\u2013171.\n\n2. Miller, D. L., and Cheskin, L. J. GI symptoms associated with specific ingestion of specific foods. Unpublished data from the Olestra Post-Marketing Surveillance Study.\n\n3. Cheskin L.J., Midday R., Zorich N., Filloon T. Gastrointestinal symptoms following consumption of olestra or regular triglyceride potato chips. _JAMA_. 1998;279:150\u2013152.\n\n4. Janowitz H.D. _Good Food for Bad Stomachs_. New York: Oxford University Press; 1997.\n\n5. Feldman M., Scharschmidt B.F., Sleissinger M.H. _Gastrointestinal and Liver Disease: Pathophysiology, Diagnosis and Management_. New York: W. B. Saunders; 1998.\n\n6. Lasser R.B., Bond J.H., Levitt M.D. The role of intestinal gas in functional abdominal pain. _N. Engl. J. Med_. 1975;293:524\u2013526.\n\n7. Tomlin J., Lowis C., Read N.W. Investigation of normal flatus production in healthy volunteers. _Gut_. 1991;32:665\u2013669.\n\n8. (Suppl.)Olsson S., Furne L., Levitt M.D. Relationship of gaseous symptoms to intestinal gas production: Symptoms do not equal increased production. _Gastroenterology_. 1995;108:A28.\n\n9. Levitt M.D., Duane W.C. Floating stools\u2014flatus versus fat. _N. Engl. J. Med_. 1972;286:973\u2013975.\n\n10. Karlin D.A., Jones R.D., Stroeleim J.R., Mastromarino A.J., Potter G.D. Breath methane excretion in patients with unresected colorectal cancer. _J. Natl. Cancer Inst_. 1982;69:573\u2013576.\n\n11. Moore J.G., Jessop L.D., Osborne D.N. A gas chromatographic and mass spectrometric analysis of the odor of human feces. _Gastroenterology_. 1987;93:1321\u20131329.\n\n12. Steggerda F.R. Gastrointestinal gas following food consumption. _Ann. N.Y. Acad. Sci_. 1968;150:57\u201366.\n\n12a. Steggerda F.R. Gastrointestinal gas following food consumption. _Ann. N.Y. Acad. Sci_. 1968;150:57\u201369.\n\n13. Levin M.D., Hirsch P., Fetzer C.A., Sheehan M., Levine A.S. H2 excretion after ingestion of complex carbohydrates. _Gastroenterology_. 1987;92:383\u2013389.\n\n14. Ravich W.J., Bayless T.M., Thomas M. Fructose: Incomplete intestinal absorption in humans. _Gastroenterology_. 1983;84:26\u201329.\n\n15. Tadesse K., Eastwood M.A. Metabolism of dietary fiber components in man assessed by breath hydrogen and methane. _Br. J. Nutr_. 1978;40:393\u2013396.\n\n16. Suarez F., Savaiano D.A., Levitt M.D. A comparison of symptoms with milk or loacrose-hydrolyzed milk in people with self-reported severe lactose intolerance. _N. Engl. J. Med_. 1995;333:1\u20134.\n\n17. Gallup Organization. _Survey on Heartburn across America_. Philadelphia, PA: Gallup Organization; 1988.\n\n18. Nebel O.T., Fornes M.F., Castell D.O. Symptomatic gastroesophageal reflux: Incidence and precipitating factors. _Dig. Dis. Sci_. 1976;21:953\u2013956.\n\n19. Thompson W.A., Heaton K.W. Heartburn and globus in apparently healthy people. _Can. Med. Assoc. J_. 1982;126:46\u201348.\n\n20. Johnson R.V. _The Mayo Clinic Complete Book of Pregnancy and Baby's First Year_. Pinceton, NJ: William Morrow & Company, Inc.; 1994.\n\n21. Hookman P., Siegel C.I., Hendrix T.R. Failure of oxethazaine to alter acid induced espohageal pain. _Am J. Dig. Dis_. 1966;11:811\u2013813.\n\n22. Johnson D.A., Winters C., Spurling T.J., Chobanian S.J., Cattau E.L. Esophageal acid sensitivity in Barrett's esophagus. _J. Clin. Gastroenterol_. 1989;9:23\u201326.\n\n23. Smith J.L., Opekum A.R., Larkai E., Graham D.Y. Sensitivity of the esophageal mucosa to pH in gastroesophageal reflux disease. _Gastroenterology_. 1989;96:683\u2013689.\n\n24. Tradifilipoulos G. Nonobstructive dysphagia in reflux esophagitis. _Am. J. Gastroenterol_. 1989;84:614\u2013618.\n\n25. Feldman M., Barnett C. Relationship between acidity and osmolarity of popular beverages and reported post-prandial heartburn. _Gastroenterology_. 1995;108:125\u2013131.\n\n26. Zeman F.J., Clinical Nutrition and Dietetics. New York: Macmillan Publishing Company; 1991:197.\n\n27. Bright-Asare P., Binder H.J. Stimulation of colonic secretion of water and electrolytes by hydroxy fatty acids. _Gastroenterology_. 1973;64:81\u201388.\n\n28. Ammon H.V., Phillips S.F. Inhibition of colonic water and electrolyte absorption by fatty acids in man. _Gastroenterology_. 1973;65:744\u2013749.\n\n29. Ammon H.V., Phillips S.F. Inhibition of ileal water absorption by intraluminal fatty acids. Influence of chain length, hydroxylation and conjugation of fatty acids. _J. Clin. Invest_. 1974;53:205\u2013210.\n\n30. Martin J.L., Justus P.G., Mathias J.A. Altered motility of the small intestine in response to ethanol (ETOH): An explanation for the diarrhea associated with the consumption of alcohol. _Gastroenterology_. 1980;78:1218.\n\n31. Wegener M., Schaffstein J., Dilger U., Coenen C., Wedmann B., Schmidt G. Gastrointestinal transit of solid-liquid meal in chronic alcoholics. _Dig Dis Sci_. 1991;36:917\u2013923.\n\n32. [review]De Las Casas C., Adachi J., Dupont H. Travelers' diarrhea. _Aliment. Pharmacol. Ther_. 1999;13:1373\u20131378.\n\n33. Bo-Linn G.W., Santa Ana C.A., Morawski S.G., Fordtran J.S. Purging and calorie absorption in bulimic patients and normal women. _Ann. Intern. Med_. 1983;99:14\u201317.\n\n34. [letter]Sullivan S.N. The gastrointestinal symptoms of running. _N. Engl. J. Med_. 1981;304:915.\n\n35. Sullivan S.N. Exercise-associated symptoms in triathletes. _Phys. Sports Med_. 1987;15:105\u2013108.\n\n36. Cheskin L.J., Crowell M.D., Kamal N., Rosen B., Schuster M.M., Whitehead W.E. The effects of acute exercise on gastrointestinal motility. _J. Gastrointest. Motil_. 1990;4:173\u2013177.\n\n37. Johanson J.F., Sonnenberg A., Koch T.R. Clinical epidemiology of chronic constipation. _J. Clin. Gastroenterol_. 1989;11:525\u2013536.\n\n38. Talley N.J., Weaver A.L., Zinmeister A.R., Melton L.J. Functional constipation and outlet delay: A population-based study. _Gastroenterology_. 1993;105:781\u2013790.\n\n39. Heaton K.W., Radvan J., Cripps H., Mountford R.A., Braddon F.E., Hughes A.O. Defecation frequency and timing, and stool form in the general population. _Gut_. 1992;33:818\u2013824.\n\n40. Cummings J.H., Bingham S.A., Heaton K.W., Eastwood M.A. Fecal weight, colon cancer risk and dietary intake of nonstarch polysaccharides (dietary fiber). _Gastroenterology_. 1992;103:1783\u20131789.\n\n41. Andersen A.S. Dietary factors in the aetiology and treatment of constipation during pregnancy. _Br. J. Obstet. Gynaecol_. 1986;93:245\u2013249.\n\n42. Kamm M.A., Farthing M.J.G., Lennard-Jones J.E., Perry L.A., Chard T. Steroid hormone abnormalities in women with severe idiopathic constipation. _Gut_. 1991;32:80\u201384.\n\n43. Talley N.J., O'Keefe E.S., Zinmeister A.R. Prevalence of gastrointestinal symptoms in the elderly: A population-based study. _Gastroenterology_. 1992;102:895\u2013901.\n\n44. Preston D.M., Lennard-Jones J.E., Thomas B.M. Towards a radiologic definition of idiopathic megacolon. _Gastrointest. Radiol_. 1985;10:167\u2013169.\n\n45. Tucker D.M., Sandstead H.H., Logan G.M., Kelvay L.M., Mahalko J., Johnson L.K., Inman L., Inglett G.E. Dietary fiber and personality factors as determinants of stool output. _Gastroenterology_. 1981;81:879\u2013883.\n\n46. Kamal N., Chami T., Andersen A., Rosell F.A., Schuster M.M., Whitehead W.E. Delayed gastrointestinal transit times in anorexia nervosa and bulimia nervosa. _Gastroenterology_. 1991;101:1320\u20131324.\n\n47. Muller-Lissner S.A. Effect of wheat bran on weight of stool and gastrointestinal transit time: A meta-analysis. _Br. Med. J_. 1988;296:615\u2013617.\n\n48. Kirwas W.O., Smith A.N., McConnell A.A., Mitchell W.D., Eastwood M.A. Action of different bran preparations on colonic function. _Br. Med. J_. 1974;4:187\u2013189.\n\n49. Tomlin J., Read N.W. Laxative properties of indigestible plastic particles. _Br. Med. J_. 1988;297:1175\u20131176.\n\n50. Graham D.Y., Moser S.E., Estes M.K. The effect of bran on bowel function in constipation. _Am. J. Gastroenterol_. 1982;77:599\u2013603.\n\n51. Preston D.M., Lennard-Jones J.E. Severe chronic constipation of young women: \"Idiopathic slow transit constipation\". _Gut_. 1986;27:41\u201348.\n\n52. Borison H.L., Borison R., McCarthy L.S., Role of the area postrema in vomiting and related functions. Fed. Proc. 1984;43:2955\u20132958.\n\n53. Borison H.L., Wang S.C. Physiology and pharmacology of vomiting. _Pharmacol. Rev_. 1953;5:193.\n\n54. Baker P.C.H., Bernat J.L. The neuroanatomy of vomiting in man: Tegmentum of the pons and middle cerebellar peduncle. _J. Neurol. Neurosurg. Psychiat_. 1985;48:1165\u20131168.\n\n55. Miller A.D. Neuroanatomy and physiology. In: Sleisenger M.H., ed. _The Handbook of Nausea and Vomiting_. New York: Parthenon; 1993:1\u20139.\n\n56. Carpentar D.O. Neural mechanisms of emesis. _Can. J. Physiol. Pharmacol_. 1990;68:230\u2013236.\n\n57. Wilson C.L., Davis S.J. Recurrent atrial fibrillation with nausea and vomiting. _Aviat. Space. Environ. Med_. 1978;49:624\u2013625.\n\n58. Lyon L.J., Nevins M.A. Retching and termination of ventricular tachycardia. _Chest_. 1978;74:110\u2013113.\n\n59. Morrow G.R. The effect of susceptibility to motion sickness on the side effects of cancer chemotherapy. _Cancer_. 1985;55:2766\u20132770.\n\n60. Baron T., Ramirez B., Richter J.E. Gastrointestinal motility disorders during pregnancy. _Ann. Intern. Med_. 1993;118:366\u2013375.\n\n61. Deuchar N. Nausea and vomiting in pregnancy: A review of the problem with particular regard to psychological and social aspects. _Br. J. Obstet Gynaecol_. 1995;102:6\u20138.\n\n62. Logue A.W., The Psychology of Eating: An Introduction. 2nd ed. New York: W. H. Freeman and Company; 1986:108.\n\n63. Bone F. Ginger. _Br. J. Physiother_. 1997;4:110\u2013120.\n\n64. Bone M.E., Wilkinson D.J., Young J.R., McNeil J., Charlton S. Ginger-root, a new anti-emetic: The effect of ginger root on postoperative nausea and vomiting after major gynaecological surgery. _Anaesthesia_. 1990;45:669\u2013671.\n\n65. Phillips S.R., Ruggier S.E., Hutchison S.E. _Zingiber officinale_ (ginger)\u2014An anti-emetic for day case surgery. _Anaesthesia_. 1993;48:715\u2013717.\n\n66. Afreen A. A double-blind randomized controlled trial of ginger for the prevention of postoperative nausea and vomiting. _Anaesth. Intens. Care_. 1995;23:449\u2013452.\n\n67. Meyer K., Schwarz J., Crater D., Keyes B. _Zingiber officinale_ (ginger) used to prevent 8-MOP associated nausea. _Dermatol. Nurs_. 1995;7:242\u2013244.\n\n68. Pace J.C. Oral ingestion of encapsulated ginger and reprted self-care actions for the relief of chemotherapy-associated nausea and vomiting. _Dis. Abstr. Int. (Sci.)_. 1987;47:3297.\n\n69. Grontved A., Brask T., Kambskard J., Hentzer E. Ginger root against seasickness: A controlled trial on the open sea. _Acta Otolaryngol_. 1988;105:45\u201349.\n\n70. .\n\n71. .\nCHAPTER 37\n\nNutrient Considerations in Lactose Intolerance\n\nDENNIS SAVAIANO1, STEVE HERTZLER2, KARRY A. JACKSON1 and FABRIZIS L. SUAREZ3\n\n1Purdue University, West Lafayette, Indiana\n\n2Ohio State University, Columbus, Ohio\n\n3Minneapolis Veteran's Administration Medical Center, Minneapolis, Minnesota\n\n## I. INTRODUCTION\n\nIngestion of a large, single dose of lactose (e.g., 50 g, the quantity in a quart of milk) by lactose maldigesters commonly results in diarrhea, bloating, and flatulence [1]. The wide dissemination of this information has led some of the lay population and a fraction of the medical community to attribute common gastrointestinal symptoms to lactose intolerance, independent of the dose of lactose ingested. As a result, a segment of the population avoids dairy products due to the belief that even trivial doses of lactose will induce diarrhea or gas. However, multiple factors affect the ability of lactose to induce perceptible symptoms. These factors include residual lactase activity [2], gastrointestinal transit time [3], lactose consumed with other foods [4], lactose load [5], and colonic fermentation [6]. In the United States, approximately 72 million individuals are lactose maldigesters, many of whom are Asian-Americans, African-Americans, and Hispanics (Table 1). These minority groups are rapidly growing segments of the population. Thus, the overall number of lactose maldigesters will grow in the United States in coming years.\n\nTABLE 1\n\nProjections of Lactose Maldigestion in the United States\n\n_Source:_ Estimates based on U.S. Department of Commerce, 1990 Census.\n\nThis chapter will (1) review the pathophysiology of lactose maldigestion, (2) attempt to correct common misconceptions concerning the frequency and severity of lactose intolerance symptoms, and (3) provide dietary strategies to minimize symptoms of intolerance.\n\n## II. LACTOSE IN THE DIET\n\nLactose is the primary disaccharide in virtually all mammalian milks. It is unique among the major dietary sugars because of the (\u03b2-1\u21924 linkage between its component monosaccharides, galactose and glucose. Lactose production in nature is limited to the mammalian breast, which contains the enzyme system (lactose synthase) necessary to create this linkage [7]. Human milk contains approximately 7% lactose by weight, which is among the highest lactose concentrations of all mammalian milks [5]. Cow's milk contains 4\u20135% lactose. Lactose, being water soluble, is associated with the whey portion of dairy foods. Thus, hard cheeses (with the whey removed from the curds) contain very little lactose compared to fluid milk.\n\nIn addition to food sources of lactose, small amounts of lactose are found in a wide variety of medications due to the excellent tablet-forming properties of lactose [5]. However, lactose is usually present in milligram, rather than gram, quantities in most medications and the amount is biologically insignificant.\n\n## III. DIGESTION OF LACTOSE\n\nThe small intestine is normally impermeable to lactose. Lactose must first be hydrolyzed to glucose and galactose, which are subsequently absorbed. Inability to digest lactose is referred to as _lactose maldigestion._ Lactose digestion is dependent on the enzyme lactase-phlorizin hydrolase (LPH), a microvillar protein that has at least three enzyme activities: galactosidase, phlorizin hydrolase, and glycosylceramidase [8, 9]. Synthesis of LPH occurs in enterocytes, with the highest and most uniform synthesis being in the jejunum in humans [10]. The LPH gene is located on chromosome 2 and directs the synthesis of a pre-proLPH that is processed intracellularly (and possibly by pancreatic proteases) into the mature form that is anchored in the cell membrane at the brush border [11, 12]. Lactase activity develops late in gestation compared to other disaccharidases. Lactase activity in a fetus at 34 weeks is only 30% that of a full-term infant, rising to 70% of the full-term activity by 35\u201338 weeks [13].\n\n## IV. LOSS OF LACTASE ACTIVITY\n\nFull-term infants possess high lactase activity, except for _congenital lactase deficiency,_ in which lactase is completely absent at birth. Holzel _et al._ [14] first described congenital lactase deficiency in 1959. A very rare condition even in Finland (where it is most common), only 42 cases were diagnosed from 1966 to 1998 [11]. Lactase activity in jejunal biopsy specimens from infants with congenital lactase deficiency is reduced to 0\u201310 IU\/g protein, and severe diarrhea results from unabsorbed lactose [11]. Treatment with a lactose-free formula eliminates symptoms and promotes normal growth and development [15].\n\n_Primary acquired hypolactasia,_ in which there is up to a 90\u201395% reduction in lactase activity, is much more common than congenital lactase deficiency (alactasia) [16]. The preferred term for this type of hypolactasia is _lactase nonpersistence_ (LNP). It is estimated that approximately 75% of the world's population are LNP (see Table 2), with the exception of Northern Europeans and a few pastoral tribes in Africa and the Middle East that maintain infantile levels of lactase throughout life [17]. Thus, LNP is not a \"lactase deficiency\" disease, but is the normal pattern in human physiology, similar to the physiology of other mammalian species. This permanent loss of lactase occurs sometime after 3\u20135 years of age [9, 18]. It is hypothesized that _lactase persistence_ is the result of a genetic mutation 3000\u20135000 years ago in populations where dairy foods had become an important component of the adult diet [19]. A gene mutation may have conferred a selective evolutionary advantage in these populations [20]. Lactase persistence is inherited as an autosomal dominant characteristic [17].\n\nTABLE 2\n\nProjections of Lactose Maldigestion around the World\n\n_Source:_ Population estimates from the United Nations.\n\nThe genetic regulation of LPH has been studied extensively. Most evidence supports reduced levels of lactase mRNA in lactose maldigesters, suggesting that regulation is primarily at the level of transcription [21\u201324]. However, hypolactasia is sometimes present even when lactase mRNA is abundant, suggesting that post-transcriptional factors play a role [10, 25, 26]. One potential reason for conflicting results is the intestinal segment examined (duodenum versus jejunum). Lactase expression is higher and more uniform in the jejunum compared to the duodenum [27, 28]. Another potential discrepancy is the age of the subjects studied. A poor correlation between lactase mRNA and lactase activity was reported in intestinal biopsies from children, although the biopsy specimens in this study were duodenal [26]. Lactase activity in the jejunal enterocytes is found in a \"mosaic\"-type pattern [29]. In hypolactasic individuals, some jejunal enterocytes produce high amounts of lactase while others, even those sharing the same villus, do not produce lactase [10]. Thus, rather than a uniform reduction in lactase production among all enterocytes, a hypolactasic individual may have a \"patchy\" distribution of lactose-producing enterocytes that are low in number relative to the non-lactase-producing enterocytes. In lactase persistent individuals, all villus enterocytes may produce lactase. Current evidence suggests that the regulation of lactase is accomplished primarily at the level of transcription, although post-transcriptional factors (e.g., degradation of mRNA and post-translational processing of the LPH protein) could be important in some individuals.\n\n_Secondary hypolactasia_ occurs as the result of damage to the enterocytes via disease, medications, surgery, or radiation to the gastrointestinal tract (see Table 3) [5, 30, 31]. For example, the prevalence of microsporidiosis, which is associated with hypolactasia, can be as high as 50% in HIV-infected patients [32]. Seventy percent of HIV-infected patients showed evidence of lactose maldigestion compared to only 34% of controls [33]. In addition, the severity of lactose maldigestion increases in the more advanced stages of the disease. In general, secondary hypolactasia is reversible once the underlying cause is treated, but this reversal may require 6 months or more of diet therapy [5].\n\nTABLE 3\n\nPotential Causes of Secondary Hypolactasia\n\n_Sources:_ Adapted with permission from: Srinivasan, R., and Minocha, A. (1998). When to suspect lactose intolerance: Symptomatic, ethnic, and laboratory issues. _Postgrad. Med._ **104** (3), 109\u2013123; Scrimshaw, N. S., and Murray, E. B. (1998). The acceptability of milk and milk products in populations with a high prevalence of lactose intolerance. _Am. J. Clin. Nutr._ **48,** 1083\u20131159; and Savaiano, D. A., and Levitt, M. D. (1987). Milk intolerance and microbe-containing dairy foods. _J. Dairy Sci._ **70,** 397\u2013406.\n\n## V. DIAGNOSIS OF LACTOSE MALDIGESTION\n\nA. Direct Assessment Methods\n\nLactose digestion can be assessed directly or indirectly. The direct method involves obtaining a biopsy specimen of intestinal tissue and assaying for lactose activity or by intestinal perfusion studies [34]. While these tests can accurately measure lactase activity, they are invasive and seldom used clinically.\n\n### B. Indirect Assessment Methods\n\nSeveral indirect methods for assessing lactose digestion are available, including blood, urine, stool, and breath tests. Blood tests involve feeding a standard 50-g lactose dose and measurement of plasma glucose every 15\u201330 minutes over a period of 30 minutes to 2 hours. A rise in blood glucose of at least 25\u201330 mg\/dL (1.5\u20131.7 mmol\/L) is indicative of normal lactose digestion [34]. Unfortunately, blood glucose levels are subject to a variety of hormonal influences, reducing the reliability of this test. A blood test for galactose has been developed to correct this problem. The lactose dose is administered with a 500 mg\/kg dose of ethanol (to prevent conversion of galactose to glucose in the liver) [34]. The galactose test is more reliable than the glucose test, but the ethanol exposure and invasive blood sampling are disadvantages.\n\nA commonly used urine test involves the measurement of galactose in the urine, rather than the blood, during the lactose tolerance test with ethanol. Another urine test is conducted by simultaneously administering lactose and lactulose (a nonabsorbable disaccharide) [34]. Small amounts of lactose (up to 1% of the ingested dose) and lactulose diffuse unmediated across the intestinal mucosa and are excreted in the urine. The ratio of lactose to lactulose in the urine (collected over 10 hours) is determined by the hydrolysis of lactose. A value of less than 0.3 indicates normal lactose digestion and a ratio approaching 1.0 is observed in hypolactasia [34].\n\nThe measurement of stool pH and reducing substances in the stools has been used to assess lactose digestion in children. The analyses are easy to perform and convenient for the patient. However, stool pH has been shown to be unreliable in the diagnosis of hypolactasia in children and adults [34]. Furthermore, changes in gut motility and water excretion can alter the level of reducing substances in the stool. Thus, diagnosis of hypolactasia should not be based on stool tests alone [34].\n\nBreath tests are most widely used to diagnose maldigestion. The principle behind breath tests is that lactose, which escapes digestion in the small intestine, is fermented by bacteria in the colon, producing short-chain fatty acids and hydrogen, carbon dioxide, and methane (in some individuals) gases. One breath test measures the amount of 13CO2 excreted in the breath following administration of 13C-lactose [34]. This stable isotope test has the advantage over older tests employing radioactive 14C-lactose, but the high cost of the equipment prohibits widespread use of this method.\n\nThe current \"gold standard\" for diagnosis of carbohydrate maldigestion is the breath hydrogen test. Bacterial fermentation is the only source of molecular hydrogen in the body. A portion of the hydrogen produced in the colon diffuses into the blood, with ultimate pulmonary excretion [35]. The hydrogen breath test is widely used because it is noninvasive and easy to perform. Typically, a subject is given an oral dose of lactose following an overnight (\u226512 hours) fast. Breath samples are collected at regular intervals for a period of 3\u20138 hours. In early studies, 50 g of lactose was used as a challenge dose. Almost all lactose maldigesters will experience intolerance symptoms following a dose of lactose this large [30], and yet many will be able to tolerate smaller, more physiologic doses of lactose. Doses of lactose that are in the range of 1\u20132 cups (240\u2013480 ml) of milk (12\u201324 g lactose) have recently been more frequently used [36]. The dose of lactose used in the breath hydrogen test influences the diagnostic criterion for lactose maldigestion. Early studies with 50 g lactose showed perfect separation of lactose digesters from maldigesters using a rise in breath hydrogen of greater than 20 parts per million (ppm) above the fasting level [37]. More recently, Strocchi _et al._ [38] evaluated different criteria for diagnosis of carbohydrate maldigestion, using small doses of carbohydrate (10 g lactulose). Using a cutpoint of \u226510 ppm rise in breath hydrogen above fasting over an 8-hour period resulted in improved sensitivity (93% vs. 76%) and only a slight decrease in specificity (95% vs. 100%) compared to the 20-ppm cutoff. Further, it was shown that using a sum of hydrogens from hours 5, 6, and 7 and a \u226515-ppm above fasting cutpoint resulted in 100% sensitivity and specificity.\n\nDespite the advantages of breath hydrogen testing, care must be taken to ensure an accurate test. First, it is important to establish a low baseline breath hydrogen value, to which subsequent values are compared. This is accomplished by fasting before and after consumption of the lactose dose. In addition, it has been shown that a meal low in nondigestible carbohydrate (e.g., white rice and ground meat) the evening before the test results in lower baseline hydrogen [39]. Second, it is possible that some individuals may have a colonic microflora that is incapable of producing hydrogen. However, these individuals are rare and the possibility of a non-hydrogen-producing flora can be ruled out by the administration of lactulose [38]. Third, approximately 40% of adults harbor significant numbers of methane-producing bacteria in the colon [40]. Because methanogenic bacteria consume four parts of hydrogen to produce one part methane [40], some authors have suggested that simultaneous measurement of methane will improve the accuracy of breath hydrogen testing in methane-producing subjects [41]. The availability of gas chromatographs that can analyze both hydrogen and methane in breath samples eliminates this potential problem. Finally, a number of factors (sleep, antibiotics, smoking, bacterial overgrowth of the small intestine, and exercise) may complicate the interpretation of breath hydrogen tests [34]. Therefore, standardization of the breath test protocol and appropriate controls are important.\n\n## VI. LACTOSE MALDIGESTION AND INTOLERANCE SYMPTOMS\n\nA positive breath hydrogen test is indicative of lactose maldigestion. However, reduced lactase levels do not necessarily lead to intolerance symptoms. Symptoms of intolerance occur when the amount of lactose consumed exceeds the ability of both the small intestine and colon to effectively metabolize the dose. Unhydrolyzed lactose passes from the small intestine to the large intestine where it is fermented by enteric bacteria, producing the gases that are partially responsible for causing intolerance symptoms. The intensity of symptoms varies with the amount of lactose consumed [31, 42\u201344], the degree of colonic adaptation [45, 46] and the physical form of the lactose-containing food [47].\n\nThe correlation between lactose maldigestion and reported intolerance symptoms is unclear. Most maldigesters can tolerate the amount of lactose in up to 1\u20132 cups of milk without experiencing severe symptoms. However, some lactose maldigesters believe that small amounts of lactose, such as the amount used with coffee or cereal, cause gastrointestinal distress [48]. Individual differences observed in symptom reporting may reflect learned behaviors, cultural attitudes, or other social issues.\n\nLactose maldigesters, unselected for their degree of lactose intolerance, tolerated a cup of milk without experiencing appreciable symptoms [49\u201351]. However, the results of these studies did not gain general acceptance, in part because of failure to utilize subjects with \"severe\" lactose intolerance. In 1995, Suarez _et al._ [48] conducted a study in 30 self-described \"severely lactose intolerant individuals.\" Initial breath hydrogen test measurements indicated that approximately 30% (9 of 30) of the subjects claiming severe lactose intolerance were digesters and, thus, had no physiological basis for intolerance symptoms. These findings further demonstrate how strongly behavioral and psychological factors influence symptom reporting. Additional research is necessary to evaluate the psychological component of symptom reporting in lactose maldigesters.\n\n## VII. LACTOSE DIGESTION, CALCIUM, AND OSTEOPOROSIS\n\nIndividuals who are lactose intolerant can tolerate moderate amounts of lactose with minimal to no gastrointestinal discomfort [48, 52], however, some lactose maldigesting individuals may unnecessarily restrict their intake of lactose-containing, calcium-rich dairy foods, thus compromising calcium intake. Milk and milk products contribute 73% of the calcium to the U.S. food supply [53]. Lactose maldigestion is associated with lower calcium intakes and is more frequent in osteoporotic patients than in controls [54\u201357]. For example, Newcomer _et al._ [54] found that 8 of 30 women with osteoporosis were lactose maldigesters compared to only 1 of 30 controls. In addition, calcium intakes of LNP postmenopausal women in this study (530 mg\/day) were significantly lower than in the lactase persistent women (811 mg\/day). Interestingly, in this report, and another by Horowitz _et al._ [55], few of the LNP subjects reported a history of milk intolerance and yet they still restricted milk intake. The lower milk intakes in these subjects may have been due to factors other than lactose intolerance. However, it is also possible that these subjects restricted their milk intakes due to lactose intolerance in childhood, forgot that they had done so, and simply maintained that pattern of milk intake throughout life.\n\nAnother potential explanation for the increased prevalence of osteoporosis among lactose maldigesters is that maldigestion of lactose decreases absorption of calcium. Human and animal studies suggest that lactose stimulates the intestinal absorption of calcium [53]. However, there is considerable disagreement regarding the influence of lactose and lactose maldigestion on calcium absorption in adults. This disagreement results from a number of factors including the dose of lactose given, the choice of method for assessing calcium absorption (single isotope, double isotope, balance methods), prior calcium intake of the subjects, and the form in which the calcium is given (milk vs. water).\n\nKocian _et al._ [58], using a single-isotope (47Ca) method, demonstrated improved absorption of a 972-mg calcium dose from lactose-hydrolyzed milk as compared to milk containing lactose in lactose maldigesters. Conversely, the regular milk resulted in increased calcium absorption versus the lactose-hydrolyzed milk in lactose digesters. Another study, using dual-isotope methods, a 50-g lactose load, and 500 mg of calcium chloride in water found similar results [59]. Total fractional calcium absorption was decreased in maldigesters and increased in digesters with lactose feeding. However, the doses of lactose given in these studies (39\u201350 g or the equivalent of 3\u20134 cups of milk) were unphysiologic and may have resulted in more rapid intestinal transit than would be observed with more physiologic amounts of lactose.\n\nSeveral studies have been conducted with physiologic doses of lactose. Griessen _et al._ [60], using dual-isotope methods, found that lactose maldigesters ( _n_ = 7) had a slightly, but not statistically significantly, greater total fractional calcium absorption from 500 mL of milk compared to 500 mL of lactose-free milk. They also observed a nonsignificant decline in fractional calcium absorption in normal subjects ( _n_ = 8) when comparing lactose-free milk with regular milk. In another dual-isotope study, lactose maldigesters absorbed more calcium from a 240-mL dose of milk than did digesters (about 35% vs. 25%), which was thought to be due to lower calcium intakes in the lactose maldigesting group [61]. Most importantly, however, no difference was observed in fractional calcium absorption between lactose-hydrolyzed and regular milk in either group of subjects. Finally, calcium absorption from milk and yogurt, each containing 270 mg of calcium, was studied in our laboratory using a single-isotope method [62]. No significant differences were observed in calcium absorption between milk and yogurt in either the lactose maldigesting or digesting subjects. Interestingly, yogurt resulted in slightly, but significantly ( _p_ < 0.05), greater calcium absorption in lactose maldigesters when compared to lactose digesters.\n\nDifferences in study methodology (milk vs. water, dose of lactose, and the choice of method for determining calcium absorption) may explain contrasting results. Physiologic doses of lactose (e.g., amounts provided by up to 2 cups of milk) are not likely to have a significant impact on calcium absorption. The increased prevalence of osteoporosis in lactose maldigesters is most likely related to inadequate calcium intake rather than impaired intestinal calcium absorption.\n\n## VIII. DIETARY MANAGEMENT FOR LACTOSE MALDIGESTION\n\nIt is difficult for lactose maldigesters to consume adequate amounts of calcium if dairy products are eliminated from the diet. Fortunately, lactose intolerance is easily managed. Dietary management approaches that effectively reduce or eliminate intolerance symptoms are discussed below and shown in Table 4.\n\nTABLE 4\n\nDietary Strategies for Lactose Intolerance\n\n### A. Dose Response to Lactose\n\nThere is a clear-cut relationship between the dose of lactose consumed and the symptomatic response. Small doses (up to 12 g of lactose) yield no symptoms [1, 48, 50\u201352], whereas high doses (>20\u201350 g of lactose) produce appreciable symptoms in most individuals [1, 63\u201365]. In a well-controlled trial, Newcomer _et al._ [1] demonstrated that >85% of lactose maldigesters developed intolerance symptoms after consuming 50 g of lactose (the approximate amount of lactose in 1 quart of milk) as a single dose. The frequency of reported symptoms may be attributed to the nonphysiologic nature of the lactose dose and the physical form of lactose load administered. A physiologic dose containing 15\u201325 g of lactose is adequate to produce appreciable symptoms in some subjects [30, 66]. The incidence of symptom reporting generally remains above 50% with intermediate doses. However, the frequency varies from less than 40% to greater than 90% [30]. In a double-blind protocol, Suarez _et al._ [48] demonstrated that feeding 12 g of lactose with a meal resulted in minimal to no symptoms in maldigesters. Interestingly, in unblinded studies [66, 67], lactose maldigesters more frequently reported intolerance symptoms after consuming lactose loads similar to that given by Suarez _et al._ Recently, Suarez _et al._ [52] provided further evidence that individuals who are lactose intolerant can consume lactose-containing foods without experiencing appreciable symptoms by feeding lactose maldigesters 2 cups of milk daily. One cup of milk was given with breakfast, and the second was given with the evening meal. The symptoms reported by maldigesters after consumption of 2 cups of milk were trivial.\n\nSymptoms from excessive lactose in the intestine may increase out of proportion to dosage, which raises the possibility that the absorption efficiency decreases with increased loads. Fractional lactose absorption is most likely influenced by dosage, with more effective absorption of small loads and less effective utilization of larger doses. Hertzler _et al._ [43], using breath hydrogen as an indicator, suggested that 2 g of lactose is almost completely absorbed, whereas there was some degree of maldigestion when a 6-g load was ingested. The only study directly measuring the lactose absorption efficiency in lactose maldigesting subjects is that of Bond and Levitt [68], who intubated the terminal ileum and then fed the subjects 14C lactose mixed with polyethylene glycol, a nonabsorbable volumetric marker. Analysis of the ratio of 14C lactose to polyethylene glycol passing through the terminal ileum allowed researchers to calculate the percentage of lactose absorbed. On average, maldigesters absorbed about 40% of a 12.5-g lactose load, whereas the other 60% passed to the terminal ileum. However, sizable differences were seen in absorption efficiency among lactose maldigesters. These differences could represent differences in residual lactase efficiency and\/or gastric emptying and intestinal transit time.\n\n### B. Factors Affecting Gastrointestinal Transit of Lactose\n\nConsuming milk with other foods, rather than alone, can minimize symptoms from lactose maldigestion [4, 69, 70]. A probable explanation for these findings is that the presence of additional foods slows the intestinal transit of lactose. Slowed transit allows more contact between ingested lactose and residual lactase in the small intestine, thus improving lactose digestion. It is also possible that additional foods may simply slow the rate at which lactose arrives in the colon, because a delay in peak breath hydrogen production, rather than a significant decrease in total hydrogen production has been reported [4]. The slower fermentation of lactose might allow for more efficient disposal of fermentation gases, reducing the potential for symptoms.\n\nThe energy content, fat content, and added components such as chocolate may influence gastrointestinal transit of lactose and subsequent lactose digestion. Leichter [71] showed that 50 g of lactose from whole milk (1050 mL) resulted in fewer symptoms (abdominal discomfort, bloating, and flatulence) compared to 50 g of lactose from either skim milk (1050 mL) or an aqueous solution (330 mL). However, only blood glucose was measured to determine lactose digestion and no statistical evaluation of symptoms was done in this study. Recent studies have demonstrated that higher fat milk may slightly decrease breath hydrogen relative to skim milk [70], but not improve intolerance [70, 72, 73]. Further, increasing the energy content or viscosity of milk has not been effective in improving lactose digestion or tolerance [74, 75].\n\nChocolate milk has been recommended for individuals who are lactose intolerant. Apparently, chocolate milk empties from the stomach more slowly than unflavored milk, possibly due to its higher osmolality or energy content [3]. Two reports have demonstrated improved lactose digestion (i.e., reduced breath hydrogen) from chocolate milk [70, 76], with fewer symptoms reported in one of these studies [76].\n\nClearly, consumption of milk with other foods results in improved tolerance compared to milk alone. Therefore, consuming small amounts of milk routinely with meals is a recommended approach for individuals who are lactose intolerant to obtain sufficient calcium from dairy products. These individuals might also try chocolate milk to improve tolerance.\n\n### C. Yogurts\n\nThe lactose in yogurt with live cultures is digested better than lactose in milk and is well tolerated by those who are lactose intolerant [77]. Prior to fermentation, most commercially produced yogurt is nearly 6% lactose due to the addition of milk solids to milk during yogurt production. However, as the lactic acid bacteria ( _Lactobacillus delbrueckii_ subsp. _bulgaricus_ and _Streptococcus salivarius_ subsp. _thermophilus_ ) multiply to nearly 100 million organisms per milliliter, 20\u201330% of the lactose is utilized, decreasing the lactose content of yogurt to approximately 4% [78]. During fermentation, the activity of the \u03b2-galactosidase enzyme substantially increases. Casein, calcium phosphate, and lactate in yogurt act as buffers in the acidic environment of the stomach, thus protecting a portion of the microbial lactase from degradation and allowing the delivery of intact cells to the small intestine [79, 80]. In the duodenum, once the intact bacterial cells interact with bile acids, they are disrupted allowing substrate access to enzyme activity.\n\nYogurt consumption results in enhanced digestion of lactose and improved tolerance [47, 77, 81\u201383]. In 1984, Kolars _et al._ [77] and Gilliland and Kim [83] reported enhanced lactose digestion from yogurt in lactose maldigesters. In both studies, breath hydrogen excretion was significantly reduced with the consumption of live culture yogurt. Furthermore, Kolars _et al._ [77] found that an 18-g load of lactose in yogurt resulted in significantly fewer intolerance symptoms reported by subjects as compared to the other forms of lactose given. Also in 1984, Savaiano _et al._ [47] demonstrated that yogurt feeding resulted in one-third to one-fifth less hydrogen excretion as compared to other lactose-containing dairy foods with no symptoms. Shermak _et al._ [82] reported that a 12-g load of lactose in yogurt resulted in lower peak hydrogen in children with a delay in the time for breath hydrogen to rise when compared to a similar lactose load given in milk. Moreover, the children experienced significantly fewer intolerance symptoms with yogurt consumption.\n\nYogurt pasteurization following fermentation has been somewhat controversial [83]. One advantage of pasteurizing yogurt is a longer shelf life. However, removing the active cultures that are partly responsible for improved lactose digestion may increase lactose maldigestion and intolerance symptoms and cause lactose maldigesters to avoid yogurt products. Pasteurizing yogurt increases the maldigestion of lactose [47, 82, 83]. However, pasteurized yogurt is moderately well tolerated, producing minimal symptoms [47, 81, 82]. Because pasteurized yogurt is relatively well tolerated, other factors such as the physical form, or gelling, and the energy density of yogurt may play a role in tolerance. The level of the \u03b2-galactosidase enzyme in yogurt may not be the limiting factor for improving lactose digestion because not all yogurts have the same level of lactase activity [84]. Martini _et al._ [84] fed yogurts with varying levels of microbial \u03b2 \u2013galactosidase. The remaining characteristics of the test yogurts (pH, cell counts, and lactose concentrations) were similar. Despite the different levels of \u03b2-galactosidase activity, all yogurts equally improved lactose digestion and minimized intolerance symptoms.\n\n### D. Unfermented Acidophilus Milk\n\nIndividuals who are lactose maldigesters [85\u201387] may consume unfermented milk containing cultures of _Lactobacillus_ _acidophilus_ in an effort to consume adequate amounts of calcium and avoid intolerance symptoms. Various strains of _L. acidophilus_ exist, however strain NCFM has been most extensively studied and used in commercial products. Unfermented acidophilus milk tastes identical to unaltered milk because the NCFM strain does not multiply in the product, provided that the storage temperature is below 40\u00b0F (5\u00b0C) [85, 86, 88]. _Lactobacillus acidophilus_ strain NCFM is derived from human fecal samples [79] and contains \u03b2-galactosidase (lactase). The effectiveness of acidophilus milk on improving lactose digestion and intolerance symptoms has been evaluated. Most evidence suggests that unfermented acidophilus milk does not enhance lactose digestion or reduce intolerance symptoms [47, 88\u201390] primarily due to the low concentration of the species in the milk. Improved lactose digestion has been observed by some [91]; however, the test milk in this study contained a much higher concentration of _L. acidophilus_ than is normally used to produce commercial acidophilus milks.\n\nFurther, the microbial lactase from _L. acidophilus_ may not be available to hydrolyze the lactose _in vivo_ [84, 86, 90]. _Lactobacillus acidophilus_ is not a bile-sensitive organism [79, 88]. Therefore, once the intact bacterial cells reach the small intestine, bile acids may not disrupt the cell membrane to allow the release of the microbial lactase. However, sonicated acidophilus milk improved lactose digestion by reducing breath hydrogen [92]. Thus, if less bile-resistant strains were developed and used in adequate amounts, these strains may allow the \u03b2-galactosidase to be released, possibly yielding an effective approach to the dietary management of lactose maldigestion.\n\n### E. Lactase Supplements and Lactose-Reduced Milks\n\nThe use of lactase supplements and lactose-reduced dairy products is steadily growing in the United States. The leading brand in this industry, \"Lactaid,\" reported $126 million in sales for fiscal 1997. The number of new dairy product introductions categorized as low- or no-lactose rose 50% from 1992 to 1997 [93].\n\nLactase pills, capsules, and drops contain lactase derived from yeast ( _Kluyveromyces lactis_ ) or fungal ( _Aspergillus niger, A. oryzae_ ) sources. Dosages of lactase per pill or caplet vary from 3000 to 9000 FCC units [94, 95]. Since 1984, these over-the-counter preparations have been generally recognized as safe by the U.S. Food and Drug Administration [96]. Additionally, milk that has been treated with lactase, resulting in a 70\u2013100% reduction in lactose, is commercially available [95].\n\nA number of studies have evaluated the effectiveness of these products. Doses of 3000\u20136000 Food Chemicals Codex (FCC) units of lactase administered just prior to milk consumption decrease both breath hydrogen and symptom responses to lactose loads ranging from 17 to 20 g [94, 97, 98]. The decrease in breath hydrogen and symptoms is generally dose dependent. Doses up to 9900 FCC units may be needed for digestion of a large lactose load, such as 50 g of lactose [94, 99, 100].\n\nLactose-hydrolyzed milks also improve lactose tolerance in both children and adults [64, 65, 101\u2013111]. A by-product of lactose-hydrolyzed milk is increased sweetness, due to the presence of free glucose [48]. This increased sweetness may increase its acceptability in children [104].\n\n### F. Colonic Fermentation and Colonic Bacterial Adaptation of Lactose\n\nThe colonic bacteria ferment undigested lactose and produce short-chain fatty acids (SCFA) and gases. Historically, this fermentation process was viewed as a cause of lactose intolerance symptoms. However, it is now recognized that the fermentation of lactose, as well as other nonabsorbed carbohydrates, plays an important role in the health of the colon and impacts the nutritional status of the individual.\n\nThe loss of intestinal lactase activity in lactose maldigesters is permanent. Studies from Israel, India, and Thailand have reported that feeding 50 g of lactose or more per day for periods of 1\u201314 months has no impact on jejunal lactase activity [17, 112, 113]. Despite this fact, milk has been used successfully in the treatment of malnourished children in areas of the world where lactose maldigestion is common. In Ethiopia, for example, 100 schoolchildren, aged 6\u201310 years, were fed 250 mL of milk per day for a period of 4 weeks [114]. While the children initially experienced some degree of gastrointestinal symptoms, the symptoms rapidly abated and returned to pretrial levels within 4 weeks. Similar results were observed with schoolchildren in India [112]. Finally, a study of African-Americans, who were lactose maldigesting and lactose intolerant aged 13\u201339 years, showed that 77% of the subjects could ultimately tolerate \u226512 g of lactose if lactose was increased gradually and fed daily over a period of 6\u201312 weeks [115]. Approximately 80% of the subjects (18 of 22) had rises in breath hydrogen of at least 10 ppm above baseline at the maximum dose of lactose tolerated, suggesting that improved digestion of lactose in the small intestine was not responsible for the increased tolerance. Therefore, the authors proposed that colonic bacterial adaptation was a likely explanation for these findings.\n\nEvidence for colonic bacterial adaptation to disaccharides (lactulose, lactose) is substantial. Perman _et al._ [116] fed adults 0.3 g\/kg lactulose per day for 7 days and observed a decrease in fecal pH from 7.1 \u00b1 0.3 to 5.8 \u00b1 0.6. The breath hydrogen response to a challenge dose of lactose (0.3 g\/kg) fell significantly after lactulose adaptation. Employing the same experimental design and doses of lactulose, Florent _et al._ [46] measured fecal \u03b2-galactosidase, colonic pH, breath hydrogen, fecal carbohydrates, SCFA, and 14C-lactulose catabolism in subjects before and after the 7-day lactulose maintenance period. Fecal \u03b2-galactosidase was six times greater after lactulose feeding and breath hydrogen fell significantly. Breath 14CO2 (indicating catabolism of 14C-lactulose) increased and fecal outputs of lactulose and total hexose units were low after the lactulose feeding. Symptoms were not measured; however, a follow-up study showed that adaptation to lactulose (40 g\/day for 8 days) reduced symptoms of diarrhea induced by a large dose (60 g) of lactulose [117]. Breath hydrogen decreased significantly and fecal \u03b2-galactosidase activity increased as in the previous study.\n\nFinally, two feeding trials adapting lactose maldigesters to lactose have been reported. The first was a blinded, crossover study from our laboratory at the University of Minnesota [45]. Feeding increasing doses of lactose (from 0.3 up to 1.0 g\/kg\/day) for 16 days resulted in a threefold increase in fecal \u03b2-galactosidase activity, which returned to baseline levels within 48 hours after substitution of dextrose for lactose. Further, 10 days of lactose feeding (from 0.6 up to 1.0 g\/kg\/day), compared to dextrose feeding, dramatically decreased the breath hydrogen response to a lactose challenge dose (0.35 g\/kg) (see Fig. 1). In fact, after lactose adaptation, the subjects no longer appeared to be lactose maldigesters, based on a 20-ppm rise in breath hydrogen above fasting. The large doses of lactose fed during the adaptation period (averaging 42\u201370 g\/day) resulted in only minor symptoms. Additionally, the severity and frequency of flatus symptoms in response to the lactose challenge dose were reduced by 50%.\n\nFIGURE 1 Breath hydrogen response to a lactose challenge after lactose ( ) or dextrose ( ) feeding periods. Data are the means \u00b1 SEM, _n =_ 20. [Reprinted with permission from Hertzler, S. R., and Savaiano, D. A. (1996). Colonic adaptation to daily lactose feeding in lactose maldigesters reduces lactose intolerance. _Am. J. Clin. Nutr._ **64,** 232\u2013236.]\n\nThe second study was a double-blind, placebo-controlled trial conducted in France with a group of 46 subjects who were lactose intolerant [118]. Following a baseline lactose challenge with 50 g of lactose, subjects were randomly assigned to either a lactose-fed group ( _n_ = 24) or a sucrose-fed control group ( _n_ = 22). Subjects were fed 34 g of either lactose or sucrose per day for 15 days. Fecal \u03b2-galactosidase increased and breath hydrogen decreased as the result of lactose feeding. Clinical symptoms (except diarrhea) were 50% less severe after lactose feeding. However, the sucrose-fed control group also experienced a comparable decrease in symptoms, despite no evidence of metabolic adaptation. Thus, these authors concluded that the improvements in symptoms resulted from familiarization with the test protocol rather than from metabolic adaptation.\n\nColonic bacteria develop an increased ability to ferment lactose (indicated by increased fecal \u03b2-galactosidase) following prolonged lactose feeding. Because hydrogen gas is an endproduct of fermentation, one might expect that the increased ability to ferment lactose would result in an increase, rather than the observed decrease, in breath hydrogen. However, breath hydrogen excretion represents the net of bacterial hydrogen production and consumption in the colon [40]. A decrease in net production of hydrogen could result from either decreased bacterial production or increased consumption. To examine the mechanism for decreased breath hydrogen after lactose adaptation, we employed metabolic inhibitors of bacterial hydrogen consumption (methano-genesis, sulfate reduction, and acetogenesis) to obtain measures of absolute hydrogen production [119]. Subjects were fed increasing amounts of lactose or dextrose in a manner similar to previous studies. Fecal samples were assayed _in vitro_ for absolute hydrogen production and hydrogen consumption. Absolute hydrogen production after 3 hours of incubation with lactose was threefold lower after lactose adaptation (242 \u00b1 54 \u03bcL) compared to the dextrose feeding period (680 \u00b1 79 \u03bcL, _p =_ 0.006). Fecal hydrogen consumption was unaffected by either feeding period. These findings tend to support the hypothesis that prolonged lactose feeding favors the growth or metabolic activity of bacteria (e.g., bifidobacteria, lactic acid bacteria) that can ferment lactose without the production of hydrogen. Feeding lactose, lactulose, and nonabsorbable oligosaccharides stimulates the proliferation of lactic acid bacteria in the colon [120\u2013122]. Additionally, high populations of bifidobacteria inhibit the growth of known hydrogen-producing organisms, such as clostridia or _Escherichia coli_ [123].\n\nColonic bacterial adaptation to lactose does occur. Although the role of colonic adaptation in improving symptoms is not firmly established, it is clear that many individuals who are lactose intolerant can develop a tolerance to milk if they consume it regularly. This may represent a simpler and less expensive solution than the use of lactose digestive aids.\n\n## IX. GENE THERAPY FOR LACTOSE INTOLERANCE\n\nAlthough conventional dietary therapies for lactose intolerance exist, the possibility of gene therapy for lactase nonpersistence was examined by During _et al._ [124]. An adeno-associated virus vector was orally administered to hypolactasic rats to increase lactase mRNA. The adeno-associated virus vector is a defective, helper-dependent virus and the wild type is nonpathogenic in humans and other species. Following a single administration of a recombinant adeno-associated virus vector expressing \u03b2-galactosidase, all rats treated with this vector ( _n_ = 4) were positive for _lacZ_ mRNA in the proximal intestine within 3 days. There was no lactase mRNA in the rats treated with the control vector. On day 7, following vector administration, the rats were challenged with a lactose solution. The treated rats had a rise in blood glucose from 114 \u00b1 4 to 130 \u00b1 3 mg\/dL after 30 minutes, while the control rats had a flat blood glucose curve. Further, the treated rats still displayed similar lactase activity when challenged with lactose 6 months later. Thus, the potential of gene therapy for lactose intolerance exists.\n\n## X. SUMMARY\n\nA majority of the world's population and approximately 25% of the U.S. population are lactose maldigesters. Milk and milk products not only contain lactose, but are also important sources of calcium, riboflavin, and high-quality protein. Some maldigesters may avoid dairy products due to the perception that intolerance symptoms will inevitably follow dairy food consumption. Avoiding dairy products may limit calcium intake and bone density, thus increasing the risk for osteoporosis. Avoidance of milk and milk products is unnecessary since moderate lactose consumption does not produce a symptomatic response in maldigesters. Additionally, various dietary strategies effectively manage lactose intolerance by reducing or eliminating gastrointestinal symptoms. Dairy food consumption is possible for individuals who are lactose intolerant if simple dietary management strategies are incorporated into daily living.\n\nReferences\n\n1. Newcomer A.D., McGill D.B., Thomas P.J., Hofmann A.F. Tolerance to lactose among lactase deficient American Indians. _Gastroenterology_. 1978;74:44\u201346.\n\n2. Ravich W.J., Bayless T.M. Carbohydrate absorption and malabsorption. _Clin. Gastroenterol_. 1983;12:335\u2013356.\n\n3. Welsh J.D., Hall W.H. Gastric emptying of lactose and milk in subjects with lactose malabsorption. _Am. J. Dig. Dis_. 1977;22:1060\u20131063.\n\n4. Martini M.C., Savaiano D.A. Reduced intolerance symptoms from lactose consumed with a meal. _Am. J. Clin. Nutr_. 1988;47:57\u201360.\n\n5. Scrimshaw N.S., Murray E.B. The acceptability of milk and milk products in populations with a high prevalence of lactose intolerance. _Am. J. Clin. Nutr_. 1988;48:1083\u20131159.\n\n6. Hertzler S.R., Levitt M.D., Savaiano D.A. Colonic adaptation to the daily lactose feeding in lactose maldigesters reduces lactose tolerance. _Am. J. Clin. Nutr_. 1996;64:1232\u20131236.\n\n7. Kretchmer N., Sunshine P. Intestinal disaccharidase deficiency in the sea lion. _Gastroenterology_. 1967;53:123\u2013129.\n\n8. 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Peroral gene therapy of lactose intolerance using an adeno-associated virus vector. _Nat. Med_. 1998;4:1131\u20131135.\nCHAPTER 38\n\nNutrient Considerations in Inflammatory Bowel Disease and Short Bowel Syndrome\n\nPETER L. BEYER, University of Kansas Medical Center, Kansas City, Kansas\n\n## I. INTRODUCTION\n\nThe two primary forms of idiopathic inflammatory bowel disease, Crohn's disease and ulcerative colitis, are characterized as chronic, inflammatory diseases of varying severity. The cause is unknown but the disease appears to involve significant interaction of the intestinal wall with intestinal microbes, ingested foods and beverages, gastrointestinal secretions, local and systemic immune components, and genetic factors. The disease may result in malabsorption of nutrients, obstruction of the gastrointestinal tract, decreased oral intake, increased nutrient requirements, and adverse response to ingested foods. Malnutrition is common. Medications and other therapies employed in treating inflammatory bowel disease can further compromise nutritional status.\n\nSpecific dietary factors may enhance or attenuate the underlying inflammatory processes involved in the disease, and certain dietary habits can worsen or reduce the severity of symptoms of inflammatory bowel disease. Surgery is common in the life of patients with inflammatory bowel disease and, in some cases, may significantly affect the ability of the patient to maintain adequate nutritional status without specialized nutrition care. Consequently, dietary interventions have a significant role in the management of inflammatory bowel disease, its symptoms, and its consequences. In the first section of this chapter, characteristics of inflammatory bowel disease, the nature of the inflammatory process, and the potential role for nutrition interactions are reviewed. In the second section, nutrition and short bowel syndrome, one of the potential complications of Crohn's disease and other gastrointestinal maladies, are discussed.\n\n## II. INFLAMMATORY BOWEL DISEASE\n\nInflammatory bowel disease refers to any type of inflammatory disorder involving the small and\/or large intestine including those caused by infectious, immunologic, or toxic agents. Crohn's disease and ulcerative colitis are the two most common forms of idiopathic inflammatory bowel disease and will be the only forms considered here. Although Crohn's disease and ulcerative colitis share many clinical, genetic, and pathologic features, they also exhibit distinguishing characteristics.\n\n### A. Characteristics of Inflammatory Bowel Disease\n\nIn both Crohn's disease and ulcerative colitis, risk of weight loss, growth failure, anemia, diarrhea, fever, arthritic and dermatologic manifestations, nutrient deficiencies, and colon cancer are increased. Although malnutrition can occur in both disorders, it is more likely to occur in Crohn's disease primarily due to the involvement of the small bowel and as a result of small intestinal resections. See Table 1 for common disease characteristics.\n\nTABLE 1\n\nCharacteristics of Crohn's Disease and Ulcerative Colitis\n\n**Features common to both Crohn's disease and ulcerative colitis**\n\nChronic episodes of relapse and remission with varying degrees of duration and severity\n\nDiarrhea\n\nExtraintestinal manifestations to varying degrees\n\nFever\n\nIncreased risk of colorectal cancer\n\n**Characteristics more common in Crohn's disease**\n\nAny area of the gastrointestinal tract but more commonly involves the distal small bowel and colon, with a smaller percentage involving just the small bowel or just the colon\n\nSkipped areas\n\nTransmural, thickening of the intestinal wall\n\n70% have surgery in their lifetime; surgery does not guarantee a cure\n\nAbscesses, sinus tract, fistulas\n\nStrictures\n\nHyperoxaluria, oxalate stones\n\n**Characteristics more common in ulcerative colitis**\n\nMucosal layer only\n\nConfined to the colon\n\nSurgical resection of the colon eliminates the disease\n\nNormally originates in the distal colon and migrates proximally\n\nGastrointestinal bleeding\n\nIn Crohn's disease, malabsorption, abdominal pain, mucosal thickening, strictures, obstruction, abscesses and fistula formation and nephrolithiasis are more common. (See Figs. 1 and 2 for representation of normal small intestine and colon, Fig. 3 for Crohn's disease, and Fig. 4 for ulcerative colitis; for Figs. 3 and 4, see color plate at the back of the book.) Crohn's disease may appear anywhere along the gastrointestinal tract although it most commonly occurs in the ileum and colon together (in about 40\u201355% of the cases), in the colon alone (15\u201330% of cases), or in the small intestine alone (about 30%). Diseased bowel may be adjacent to \"skipped\" areas that appear normal. In contrast to ulcerative colitis, the disease is transmural rather than mucosal. Over the course of the disease, about 70% of persons with Crohn's disease will eventually have at least one surgery. Surgery does not guarantee a cure and it is not uncommon to encounter persons with Crohn's disease who have had numerous surgical procedures.\n\nFIGURE 1 Normal small intestine. Photo courtesy of Dr. Got-tumukkala Raju, Kansas University Medical Center, Division of Gastroenterology.\n\nFIGURE 2 Normal colon (cecum). Photo courtesy of Dr. Gottu-mukkala Raju, Kansas University Medical Center, Division of Gastroenterology.\n\nFIGURE 3 Crohn's disease. Photo courtesy of Ossama Tawfik, MD, PhD, Director, Surgical Pathology, University of Kansas Medical Center.\n\nFIGURE 4 Ulcerative colitis. Photo courtesy of Ossama Tawfik, MD, PhD, Director, Surgical Pathology, University of Kansas Medical Center.\n\nIn ulcerative colitis, only the colon is involved. In about 30% of patients, the disease remains confined to the rectum.\n\nIn about 25\u201350% of those who present with proctitis, the disease progresses eventually to more extensive colonic involvement. The disease begins distally in the rectum and moves proximally when it progresses. Diarrhea is the most common symptom but crampy abdominal pain and rectal bleeding are also common presentations. One-third or fewer of persons with ulcerative colitis eventually require colectomy during their lifetime. After colectomy, however, the disease does not recur anywhere else in the intestinal tract [1\u20134].\n\n### B. Incidence, Prevalence, and Course\n\nInflammatory bowel disease may occur at any age, but the onset most frequently occurs between the ages of 15\u201330 years; a smaller rise in occurrence is seen after the sixth decade [2, 4]. Neither Crohn's disease nor ulcerative colitis is common but because of the frequency of clinic and hospital visits associated with the diseases, health care professionals tend to have a distorted sense of the prevalence. Worldwide, the incidence of Crohn's disease ranges from less than 1 new case per 100,000 annually to 12 per 100,000 annually. The broad range perhaps reflects the genetic influence and, to varying degrees, the sophistication of the systems in place for identifying and reporting the disease throughout the world. In the United States, the incidence of new cases of Crohn's disease is about 5\u20137 per 100,000 and for ulcerative colitis is about 10\u201312 per 100,000. According to the Crohn's Colitis Society [5], the prevalence of inflammatory bowel disease is approximately 150 cases per 100,000 persons and the risk of developing Crohn's disease in an individual who had a relative with Crohn's disease is increased 10-fold. If siblings or both parents have Crohn's disease, the risk increases to 30 times normal. Because of the variance in the prevalence, severity, and course of the disease, several genes are suspected to be involved. Initial research appears to be consistent with the concept that Crohn's disease and ulcerative colitis are polygenic in nature [4, 6, 7].\n\nThe course of inflammatory bowel disease can vary from tranquil to severe and intermittent to unrelenting. In persons with more severe and active forms of disease, symptoms, health care requirements, dietary changes, and anxiety (regarding procedures, symptoms, normal aberrations in bowel habit, concern about surgery, social embarrassment) can significantly affect the quality of life [3, 4, 8, 9]. The factors involved in the onset, course, and outcomes of the disease are multifactorial and include the interactions of genetic susceptibility, environmental influence and therapeutic interventions. These factors taken together affect the overall course, severity, and outcomes of the disease [4, 6]. Nutrition appears to play a role in the clinical profile, treatment, and perhaps onset of the disease, but the impact of nutrition is not as well understood in some aspects of the disease as in others.\n\n### C. Inflammatory Events in Crohn's Disease and Ulcerative Colitis\n\nIn any discussion of nutritional aspects of an inflammatory disease, a review of the pathologic events in the inflammatory process is appropriate. Several dietary factors may be involved in the exacerbation, modulation, or resolution of the inflammatory state.\n\nThe inflammatory process is typically initiated by microbial invasion, physical trauma, exposure to toxic agents, or autoimmune reactions and subsequently a cascade of events occurs [10\u201312]. Events include recruitment and proliferation of leukocytes; release of eicosanoids, proinflammatory and regulatory cytokines; release of proteases and generation of oxygen radicals; activation of clotting mechanisms; and production of fibrous tissue. The normal inflammatory response is directed toward containment of the injury, destruction of invading microbes, inactivation of toxins, and repair of the injured tissue. Overzealous inflammatory response, which may result from extreme or chronic injury, however, can result in serious damage to tissues and organs.\n\nThe term _inflammatory bowel_ is an appropriate descriptor for Crohn's disease and ulcerative colitis because the classic events of acute and chronic inflammation are exhibited with each disease. In inflammatory bowel disease, sufficient evidence exists that the factors that drive the inflammatory response and\/or the inappropriate reaction to the initiating agent are responsible for the symptoms and the physical and pathological changes seen in the disorders. Dietary lipids, antioxidants, and dietary antigens have the potential to influence the inflammatory response and, consequently, the inflammatory response affects nutrient intake, nutrient processing, and requirements. A brief overview, therefore, of the inflammatory process in inflammatory bowel disease and the potential role of nutrients is appropriate.\n\nSeveral features of active immune and inflammatory processes have been demonstrated in both Crohn's disease and in ulcerative colitis (see Table 2). Most of the inflammatory events are localized to the bowel, but at least in active states, signs of systemic activity are also apparent. Infiltration and proliferation of macrophages, neutrophils, lymphocytes, and\/or fibroblasts are seen in the intestinal crypts, wall, lamina propria, and mesentery [3, 6, 13]. Evidence also exists that eosinophils are involved in the active disease [14]. Proinflammatory eicosanoids (e.g., leukotriene B4) are released from cell walls of leukocytes and somatic cells. Eicosanoids may be viewed as metabolic endproducts of arachidonic acid, which is produced endogenously from dietary n-6 fatty acids and incorporated into membrane phospholipids [15]. Proinflammatory cytokines, for example, tumore necrosis factor (TNF), interleukin-1 (IL-1), IL-6, and IL-8, are released in increased amounts and appear to reflect, at least to some degree, the severity of the disease [15\u201319]. Regulatory or anti-inflammatory cytokines (e.g., IL-10, and IL-11) are also increased but at levels insufficient to effectively counteract the overall proinflammatory state. Levels of peroxidation and generation of oxygen radicals are increased [20], and circulating levels of anti-oxidants may be decreased [21]. Consistent with the inflammatory process, platelets, prothrombin, fibrinogen, and collagen formation are increased, resulting in increased risk of systemic thromboembolic episodes, local fibrosis, and thickening of the bowel wall (most notably in Crohn's disease) [22].\n\nTABLE 2\n\nInflammatory Events Identified in Patients with Active Inflammatory Bowel Disease\n\nEntry and proliferation of macrophages, neutrophils, and lymphocytes into the area of active disease\n\nRelease of inflammatory metabolites of arachidonic acid (e.g., leukotriene B4)\n\nRelease of proinflammatory cytokines (e.g., TNF, IL-1, 6, 8, and 12) and anti-inflammatory cytokines (e.g., 4, 10, 11)\n\nInvolvement and hypersensitivity of mucosal T cells\n\nIncreased peroxidation, generation of oxygen radicals, and release of proteolytic enzymes\n\nIncreased permeability (gut edema, pro loss and entry)\n\nIncreased fibrin\/collagen formation; increased thickness of the gut wall\n\nDamage\/dysfunction in secretion, digestion, absorption, and the barrier against microbes\/proteins\n\nIncreased synthesis of acute phase proteins and activation of the clotting cascade\n\nFatigue, anorexia, malaise, and arthritic and other systemic complications\n\nAs a result of the release of histamine, cytokines, and eicosanoids, increased gut permeability, local edema, ulceration, and damage to the intestinal architecture may occur. Gastrointestinal secretion, digestion, absorption, and motility may be abnormal, especially in active stages, and the normal barrier to microbial fragments, toxins and large peptides may be compromised. As in any significant inflammatory response, acute phase proteins are found in increased levels and may serve, along with specific cytokine and leukotriene levels, as markers of the disease. Fatigue, malaise, anorexia, and disturbances in sleep patterns and mood can be seen in active disease [23].\n\n### D. Etiology of Inflammatory Bowel Disease\n\nThe cause of Crohn's disease and ulcerative colitis is unknown, but several possibilities have been evaluated (see Table 3). Under normal circumstance the gastrointestinal tract is continuously exposed to potential antigens and toxins in the form of microbes, dietary components, and medications. Normally, physical and immunologic barriers prevent undue host interaction with the luminal environment. The combination of some form of exogenous luminal stimuli and dysregulated endogenous response appears to bring on and allow the disease process to takes its course. One of the most popular hypotheses is an abnormal immune response to normal gastrointestinal flora or hyperreactivity to common pathogens [24].\n\nTABLE 3\n\nPossible Causes of Inflammatory Bowel Disease\n\nVirus or bacterium\n\nDefective mucosal barrier functions\n\nIncreased permeability to microbes, toxins, and antigens\n\nAbnormal response to pathogens\/antigens\n\nAbnormal response to normal flora\n\nImbalance of proinflammatory\/anti-inflammatory cytokine homeostasis\n\nAutoimmune component\n\nDietary (e.g., sugar\/refined diet, lipids, dietary antigens)\n\nIn the pathology of inflammatory bowel disease, bacteria and viruses have long been etiologic candidates because of (1) associations between the incidence of inflammatory bowel and community exposure to microbial infections, (2) antibodies in tissues to specific microbial fragments, or (3) gastrointestinal damage provoked by infectious agents. Measles virus, strains of _Eschericia coli_ and _Mycobacterium paratuberculosis_ have all been implicated, but not proven as causal factors [1, 3, 6]. Other hypotheses include a defective mucosal barrier, which allows increased interaction with luminal contents of the gut [25]; an imbalance of proinflammatory to anti-inflammatory cytokines [26]; downregulation of glucocorticoid receptors [27]; hyperresponsiveness of mucosal T cells [28]; increased nitric oxide production [29]; and increased production of oxidative compounds or decreased protection by antioxidants [20, 21].\n\nSeveral forms of genetic susceptibility appear to play a role in the endogenous response to whatever environmental stimuli may be involved [6, 7], but the disease process and the associated inflammatory response combine to confound the ability to identify clear-cut relationships. Identifying the initial trigger for disease onset is difficult. A number of years may elapse between the first symptoms of the disease and its diagnosis, and the initiation of the disease may occur long before symptoms become apparent. The disease process and the inflammatory response result in pathologic changes (e.g., increased gut permeability and antigen exposure), which creates the problem of identifying whether the disease or the antigen came first.\n\n#### 1. DO DIETARY FACTORS CAUSE OR PROMOTE INFLAMMATORY BOWEL DISEASE?\n\nAlthough certain foods or food types have been associated with changes in the incidence of inflammatory bowel disease, no specific foods have been proven to _cause_ inflammatory bowel disease. Also, no single food has consistently and objectively been identified in the onset of an acute inflammatory episode or, when withdrawn, consistently has shown to induce remission. This is not to say that patients with inflammatory bowel disease might not have food allergies, that foods might not serve to heighten the immunologic activity, or that certain food types might not aggravate symptoms [29a].\n\nRussel and colleagues [30] attempted to associate the rising incidence of inflammatory bowel disease with changes in dietary consumption patterns. The authors showed a positive risk for inflammatory bowel disease with increases in consumption of cola beverages, chocolate, and chewing gum, but negative risk for consumption of citrus fruits. As noted by these investigators, these nutritional items could be true risk factors or they could be coincidental dietary patterns adopted during the same time period. Joachim [31] reported subjective responses to foods of 60 patients with inflammatory bowel disease. Patients were asked to report whether specific foods made them feel better or worse. Patients reported an increased number of problems with chocolate, dairy products, fats, and artificial sweeteners. Unfortunately, only 122 foods were included on the questionnaire and the author did not report the number of problem foods in matched healthy controls or patients with other forms of gastrointestinal illness.\n\nSeveral investigations have been centered on the lipid content of the diet [32, 33]. High-fat diets and an increased ratio of n-6 to n-3 fatty acids have been associated with the increased occurrence of inflammatory bowel disease and perhaps the duration of remission. Consumption of predominantly n-6 lipid increases the potential to produce leukotrienes and prostaglandins from arachidonic acid released from cell membrane phospholipids during inflammation. The eicosanoids produced from n-6 fatty acids are more inflammatory and aggregatory than those from n-3 fatty acids. Leukotriene B4, for example, induces recruitment and activation of neutrophils, monocytes, and eosinophils. It also stimulates the production of a number of proinflammatory cytokines and chemical mediators [34, 35]. Reducing consumption of dietary n-6 fatty acids relative to n-3 fatty acid intake may attenuate synthesis of proinflammatory cytokines secreted by leukocytes, such as TNF-\u03b1 and IL-1 mediators.\n\nGeerling and associates [33] examined the fatty acid intake and the fatty acid composition of plasma phospholipids and adipose tissue in 20 newly diagnosed patients with Crohn's disease, 32 patients with long-standing Crohn's disease in remission, and matched controls. Fatty acid intake was not different between cases and controls but a lower percentage of the sum of n-3 fatty acids and other changes in the lipid profile were seen in the patients. Kuroki and colleagues [36] measured serum fatty acid levels in 20 patients with Crohn's disease without major resections and 18 healthy controls. Unexpectedly, they found adequate amounts of both essential n-6 and n-3 fatty acids. Increasing levels of n-3 fatty acids, however, were associated with a decreased Crohn's disease activity index. Other dietary lipids, such as gamma linolenic acid, may play anti-inflammatory roles by modifying ratios of chemical mediators [37]. As is the case of other dietary factors examined thus far, lipids appear to be an associated rather than an independent etiologic factor.\n\n#### 2. DO FOOD ALLERGIES BRING ON OR CAUSE THE DISEASE?\n\nThe lumen of the gastrointestinal tract is normally filled with antigens that have the potential to initiate at least part of the local immune and inflammatory processes. However, the role of food allergies as a major player in the cause of inflammatory bowel disease has not been established. For example, Bichoff and associates [38] studied 375 adults with gastrointestinal disorders, including inflammatory bowel disease, for objective evidence of intestinal allergic reactions. Thirty-two percent of the individuals complained of adverse reactions to foods as a cause of their abdominal symptoms, 14% had indirect evidence of allergic manifestations, but only 3% of the cases could be confirmed by allergen provocation, elimination, and rechallenge testing. The authors concluded that food antigens could be a factor in a subgroup of persons with inflammatory and functional disease. The incidence of allergies in the gastrointestinal patients (3.2%) was only slightly greater than the approximately 1.5% occurrence seen in the overall adult population [39].\n\nIn several earlier reports, milk allergy was suggested as a cause or trigger in the relapse of ulcerative colitis. Increased antibodies to milk protein have been reported [1], but their presence did not correlate with other markers of allergy or severity of symptoms. Withdrawal of milk or any other specific food does not consistently result in remission of active disease. After studying the sera of patients with Crohn's disease, Huber and colleagues [40] detected no food-specific IgE. Local allergic response to foods in the gastrointestinal tract does not appear to be a primary component of the pathology of inflammatory bowel disease [41]. However, if mucosal permeability is compromised, food antigens may contribute to the disease process. Additional work is needed to clarify the role of allergies to specific foods in the overall disease process.\n\nIn earlier reports, consumption of sucrose, refined cereals, fast foods, and fat have been positively associated with either the incidence of inflammatory bowel disease or with its onset. Intake of dietary fiber, fruits and vegetables, and specific nutrients, on the other hand, were negatively associated with the onset of inflammatory bowel disease [42, 43]. In more recent reviews, however, no consistent relationship of such foods with either the onset or management of inflammatory bowel disease could be demonstrated [44, 45]. The association between intake of foods and inflammatory bowel disease may simply reflect food intake patterns when the initial onset or relapse of the disease occurs. Establishing that specific foods may trigger the onset of a disease lare is difficult, partly because of the fact that the delay between specific dietary behaviors and notable symptoms may be lengthy. The heightened inflammatory state associated with the disease process and the resulting increased intestinal permeability may simply allow entry and reaction to certain foods that may otherwise be well tolerated. Tolerance of dietary components may be compromised simply as a result of changes in secretion, motility, digestion, absorption, or intestinal flora. Intolerance to some foods in patients with inflammatory bowel disease may also be easily predicted by their effects in normal individuals, such as consumption of excess amounts of lactose, caffeine, dietary fiber, or legumes.\n\n### E. Nutrition-Related Problems in Inflammatory Bowel Disease\n\nPatients often share concerns regarding their symptoms and potential outcomes, as outlined in Table 4. Patients with Crohn's disease may have experienced prolonged episodes of bloating, cramping, nausea, and vomiting as a result of intestinal obstruction. Patients with either Crohn's or ulcerative colitis disease may have experienced prolonged periods of diarrhea, with 15 or more stools daily. Healthy persons normally endure various types of minor abdominal pains and changes in bowel habit with little thought. Patients with inflammatory bowel disease, however, associate gastrointestinal signals with symptoms that previously preceded exacerbation of disease, surgery, diagnostic procedures, or prolonged hospitalizations. Patients with Crohn's disease who have already had significant small bowel resections may worry about their ability to absorb sufficient food and liquid, and persons with more severe forms of both Crohn's disease and ulcerative colitis may worry about ostomy surgery and its sequelae. Because neither the cause nor the triggers for the onset of the disease are clear, frustration is common among patients attempting to resolve or temper their disease activity [4, 8, 9]. Moreover, anxiety itself may worsen the manifestations of the disease.\n\nTABLE 4\n\nConcerns Expressed by Patients with Inflammatory Bowel Disease in Surveys, Internet Newsgroups, and Support Groups\n\nNeed for procedures, surgery\/ostomy\/hospitalization\n\nSources\/significance of pain and other symptoms\n\nDiarrhea, incontinence\n\nOccurrence of obstruction\/fistulae\n\nPossibility that symptoms will lead to initial or repeat surgery\n\nSymptoms or appliances interfering with daily activity, quality of life, interactions with others\n\nConcern whether nutrient intake and digestion\/absorption are adequate\n\nSide effects of medications\n\nIncomplete understanding of symptoms; no definite etiology for the disease\n\nInconsistent advice\/answers from health care professional and others\n\nDiarrhea, with or without malabsorption, and weight loss are the most common nutritional problems seen in inflammatory bowel disease [1\u20133]. In flares of inflammatory bowel disease and after small bowel resections in Crohn's disease, diarrhea may be severe enough to cause dehydration and electrolyte disturbances. Incontinence, increased stool frequency, and the occurrence of anal burning may make the diarrhea even more unpleasant.\n\nSeveral forms of anemia may occur as a result of inadequate intake, malabsorption, increased requirements, and drug\u2013nutrient interactions. Microcytic anemia in ulcerative colitis is more commonly related to blood loss than poor iron intake or malabsorption. Macrocytic anemia due to vitamin B12 deficiency is more likely to be seen in Crohn's patients who have had resections of the terminal ileum, the site of active absorption of vitamin B12 [46]. Anemia related to folate deficiency can occur as a result of poor intake, poor absorption, or drug\u2013nutrient interactions [47, 48]. See Table 5 for a list of nutrition-related problems.\n\nTABLE 5\n\nNutrition-Related Problems in Inflammatory Bowel Disease\n\nDiarrhea\n\nAnemias (from blood loss, decreased intake, or malabsorption of iron, vitamin B12, or folate)\n\nAnorexia\n\nWeight loss\n\nHypoalbuminemia\n\nGrowth failure in children\n\nMalnutrition (macro- and micronutrients)\n\nIncomplete and\/or limited diet\n\nMalabsorption\n\nVitamin and mineral depletion\n\nBone demineralization (due to poor intake and malabsorption of vitamin D, calcium)\n\nFood intolerances, food aversions, fear of eating\n\nNumerous reports of micronutrient deficiencies in persons with inflammatory bowel disease have been published during the last two or three decades [46\u201350], especially when literature from around the world is reviewed. However, today, the likelihood of micronutrient deficiency is less likely. Widespread use of enteral nutrition formulas, vitamin and mineral supplements, and fortified foods reduces the risk of deficiencies.\n\nDespite the availability of micronutrients, some vitamin and mineral deficiencies do occur. Vitamin B12, folate, zinc, calcium, iron, magnesium, selenium, copper, and vitamin A, D, and E inadequacies have all been reported in patients with inflammatory bowel disease [46\u201352]. Fear of eating, food aversions, self-imposed or iatrogenic diet restrictions, surgical resections, and drug\u2013nutrient interactions are likely to increase the risk of micronutrient deficiencies. Because the cells lining the gastrointestinal tract are especially metabolically active, macro- and micronutrient deficiencies have the potential to contribute to the pathology of the disease and compromise its resolution.\n\nAnorexia may be related to the patient's symptoms of abdominal pain, bloating, nausea, or diarrhea. Increased levels of several cytokines may also contribute to anorexia [23, 53]. Patients may be afraid to eat specific foods or food types either because they have heard that specific foods may worsen symptoms or they have had adverse experiences with specific foods. Food aversions and questionable associations of foods with subsequent symptoms are not uncommon. The pattern is not unlike that seen in cancer patients who undergo aggressive chemotherapy, become ill as a result of the side effects of therapy, and associate the illness with ingestion, taste, or smell of foods.\n\nPatients may associate adverse symptoms with consumption of specific foods or food types and may be on restricted diets imposed by health professionals, by themselves, or on the advice of \"self-proclaimed\" nutrition experts. The reason for food restrictions may be to prevent symptoms, maintain remission, or treat the disease.\n\nWeight loss, growth failure, and delayed maturation are some of the most common problems in inflammatory bowel disease. Although the inflamed gastrointestinal tract may not be as efficient at absorbing macronutrients, compared to the healthy gastrointestinal tract, most investigators and experienced clinicians attribute the nutritional inadequacy to lack of sufficient food intake [54\u201356]. In severe stages of either Crohn's disease or ulcerative colitis, varying degrees of mal-absorption may occur, but the primary cause of malabsorption is major intestinal resection in Crohn's disease and, to a lesser extent, the disease process itself. Decreased levels of transport proteins such as albumin, transferrin, or prealbumin may be related to the shift in priority in protein synthesis in the liver that occurs with physiologic stressors. Also, increased intestinal loss of nitrogen with disease relapse, or prolonged or acute periods of decreased protein and total energy intake, may worsen the level of transport proteins.\n\n### F. Treatment of Inflammatory Bowel Disease\n\nTreatment of inflammatory bowel disease usually involves several strategies, including medications, surgery, nutrition, and psychosocial support (see Table 6). The goals of treatment are essentially to promote remission (or at least resolve symptoms), treat and prevent complications, restore and maintain nutrition status, and improve quality of life. Because the etiology of inflammatory bowel disease is unknown and no single therapy is completely effective in accomplishing the overall goals, therapies are used in combination and are based on individual needs.\n\nTABLE 6\n\nPrimary Treatment Options for Inflammatory Bowel Disease\n\nMedications\u2014Anti-inflammatory agents, immune modulators, antibiotics\n\nSurgery\u2014Repair strictures, resections of small and large bowel\n\nPsychosocial\u2014Counseling, education, support\n\nNutrition\u2014Restore deficiencies, reduce symptoms, prevent relapse (induce remission?)\n\n#### 1. MEDICAL MANAGEMENT\n\nOver the course of a lifetime, patients with inflammatory bowel disease are likely to be treated with several medications. Drugs may be used for inducing remission, maintaining remission, and treating specific symptoms of the disease. Medications may be used in different combinations depending on the severity and presentation of the disease. General categories of medications commonly used to treat the primary events of inflammatory bowel disease include anti-inflammatory agents, immunomodulator\/suppressive drugs, and antibiotics. Several other medications, however, may also be used specifically to treat symptoms at various stages of the disease such as antidiarrhea or antiemetic medications [1\u20133, 57\u201360].\n\nAlthough most of the agents are used for both Crohn's disease and ulcerative colitis, some differences in application and effectiveness do occur. Drugs may be required for prolonged periods of time and at high doses to cause remission. Sometimes even combinations of approaches may fail. No single medication is consistently effective at quenching active disease or maintaining continuous remission. Use of medications brings additional costs, potential short-term and long-term side effects, and drug\u2013drug and drug\u2013nutrient interactions. They invariably alter daily life patterns. See Table 7 for a listing of commonly used medications.\n\nTABLE 7\n\nMedical Management of Inflammatory Bowel Disease\u2014Common Agents\n\nAnti-inflammatory drugs\u2014Corticosteroids, sulfasalazine, 5-aminosalicylic acid (5-ASA or Mesalamine)\n\nImmune modulators or suppressants\u2014Methotrexate, azathioprine, 6-mercaptopurine, cyclosporine, monoclonal antibody to TNF-\u03b1 (Infliximab)\n\nAntibiotics\u2014Metronidazole, ciprofloxacin\n\nTo treat moderate to severe disease in both Crohn's disease and ulcerative colitis, several classes of drugs may be used singly or in combination, but corticosteroids remain the principal drug of choice. Because of the metabolic, cosmetic, and psychogenic complications associated with use of corticosteroids, however, large doses and prolonged treatment are avoided when possible. Corticosteroids are not considered a valuable alternative in maintaining remission in either form of inflammatory bowel disease. Cyclosporine has been used in severe cases and as an alternative to surgery when the disease is resistant to steroid therapies [60]. Infliximab, a monoclonal antibody to TNF-\u03b1, has recently been approved for treatment of chronic active Crohn's disease and for the treatment of fistulas [59]. Its use has not been extended to the management of ulcerative colitis. Infliximab has been shown to have some benefit in the treatment of pouchitis [61], a complication in some patients who have had ileorectal pouches created as fecal reservoirs after colectomy.\n\nAgents used as primary treatment of mild to moderate active disease include topical corticosteroids, sulfasalazine, 5-aminosalicylic acid, azathioprine, 6-mercaptopurine, methotrexate, and the antibiotics ciprofloxacin and metronidazole. The medications can be used in combination with steroids. To maintain remission, sulfasalazine, 5-aminosalicylic acid, metronidazole, azathioprine, or 6-mercaptopurine are normally used. All of the medications may be justified in the management of various stages of inflammatory bowel disease, but mild to significant adverse side effects occur in approximately 5\u201360% of percent of patients [2, 59, 60].\n\nA number of new agents are being evaluated for treatment of inflammatory bowel disease, including anti-inflammatory cytokines, such as IL-10 and IL-11 [62]; growth hormone [62a]; neutralizing antibodies of specific proinflammatory cytokines [63]; inhibitors of, and antibodies to, inflammatory leukotrienes or their precursors [64]; nicotine (for patients with ulcerative colitis) [57]; bismuth compounds [65]; intravenous heparin [66], and a protease inhibitor from soy [67]. Other approaches also include use of other forms of antibiotics, adrenocorticotropic hormone (ACTH) [68], and leukocytapheresis [69].\n\n#### 2. SURGICAL TREATMENT\n\nIndications for surgery typically include severe, unrelenting disease, strictures, obstruction, hemorrhage, increased risk of cancer, repair of fistulas, and failure of medical therapy [70, 71]. In ulcerative colitis, colectomy \"cures\" the disease in that the disease does not subsequently occur elsewhere in the gastrointestinal tract. Approximately half of patients with ulcerative colitis have surgery in the first 10 years after diagnosis [70]. The most common form of surgery is colectomy with the creation of an ileoanal pouch (commonly called a J-pouch or W-pouch), using folds of ileum pulled into the rectal canal and anastomosed to the rectum. The pouch develops a flora and serves, to some degree, as a colonic\/rectal reservoir. Another form is the continent ileostomy or Kock pouch in which folds of ileum are used to create an internal reservoir, which is drained with a catheter several times per day [1, 2]. No appliance is worn. The traditional colectomy involves the creation of an ileostomy and the use of an external stool collection appliance. Patient problems or concerns associated with the ileostomy may include stomal irritation, irritation of the skin surrounding the ostomy, leakage or dislodgment of the ostomy bag, odors, and social stigmata associated with the use of an external appliance. Most patients appear to do well with the ostomy and feel, in general, that being disease-free outweighs the inconvenience of an ostomy [72].\n\nWith Crohn's disease, surgical resection of severely involved segments of small or large bowel does not, unfortunately, bring resolution of the disease. Endoscopic evidence of disease recurrence is seen in half or more of patients within a year after surgery. Duration of clinical remission varies greatly. In some patients, relapse may occur in month, and in other patients, apparent remission may last for years. Seventy to 90% of patients with Crohn's disease eventually have at least one surgery during their lifetime. Strictureplasty has been used in relatively uncomplicated cases of Crohn's disease to relieve narrowed segments of bowel [70]. In some cases, patients with prolonged severe courses of Crohn's disease have multiple resections resulting in short bowel syndrome and the attendant nutritional and medical consequences.\n\n#### 3. NUTRITIONAL TREATMENT\n\nNutrition and dietary patterns may play several roles in the management of inflammatory bowel disease. Nutritional rehabilitation may be required after acute or prolonged reduction in the quantity or quality of dietary intake. Dietary modifications may be required to increase the tolerance of foods during exacerbation of the active disease. Changes in nutritional practices may be needed to be able to provide adequate nourishment with complications such as short bowel syndrome, strictures, or fistulas. Refeeding protocols may be appropriate after severe malnutrition, after severe bouts of the disease, or after surgical procedures. Nutrition may also have some role in the regulation of the inflammatory response and inducing or maintaining remission. Restoration of nutrition status may include a carefully selected oral diet, vitamin and mineral supplements, or special enteral and\/or parenteral nutrition. See Table 8 for a summary of factors impacting nutrition.\n\nTABLE 8\n\nPotential Roles of Nutrition in Inflammatory Bowel Disease\n\nRestore nutrition status after prolonged or acute illness; prevent malnutrition lifelong\n\nProvide nutrients and fuels for enterocytes, colonocytes\n\nProvide fuel for and alter gastrointestinal flora\n\nReduce gastrointestinal symptoms during acute stages or prevent complications\n\nModify the immune and inflammatory response\n\nInduce and maintain remission\n\nHelp patient understand roles of diet\/foods and digestive and absorptive functions; sort out food associations and intolerances\n\nAs a result of extensive resections and severe stages of the disease, malabsorption of macro- and micronutrients could occur. Medications may also limit appetite, produce gastrointestinal-related symptoms, and result in decreased intake, decreased absorption, and\/or increased requirement for nutrients.\n\n### G. Nutrition Assessment in Inflammatory Bowel Disease\n\nA comprehensive assessment is required when evaluating nutrition risk and considering nutritional interventions in inflammatory bowel disease. Nutrition assessment for the person with inflammatory bowel disease would likely include (1) consideration of several elements of the patient's medical and surgical history (e.g., duration and severity of the disease, presence of strictures, fistulas, resections, ostomies); (2) presence of nutrition-related symptoms, such as diarrhea (e.g., stool volume, frequency) or malabsorption (increased fecal fat, abdominal cramping, bloating, or distension); (3) physical measures such as growth rate for age, body mass index, weight changes; (4) a diet history that includes habitual food intake, quantity and quality of the diet, intolerance and aversions to various foods, perceived and documented food allergies; (5) use of herbal and nutritional supplements, alternative therapies, prescription and over-the-counter medications; and (6) pertinent laboratory data that reflect protein energy and micronutrient status. The nutrition assessment should also include an evaluation of the patient's understanding of his or her nutrition status, needs, problems, and therapeutic options.\n\nEnergy requirements of persons with inflammatory bowel disease are not usually increased except with sepsis and fever, but protein requirements are increased. Energy requirements may be increased to restore weight, to return to normal growth curves, or to compensate for malabsorption. The presence of active disease by itself, however, does not appear to raise energy requirements appreciably [54, 55]. Protein needs may be increased more significantly due to gastrointestinal nitrogen losses, the inflammatory response, and the need for new tissue for weight gain and growth.\n\n### H. Modification of Specific Dietary Factors in Management\n\nPatients may be taught to help themselves manage nutrition-related symptoms such as abdominal bloating, gas and diarrhea, strictures, malabsorption, and nutritional deficiencies. Current evidence suggests that dietary practices might also alter the severity of the disease, increase the effectiveness medications, or prolong remission.\n\n#### 1. LIPIDS\n\nThe amount and nature of the fatty acids consumed in the diets may play a role in the management of several inflammatory diseases, including inflammatory bowel. The ratio of n-6 polyunsaturated fatty acids consumed to n-3 unsaturated fatty acid in the Western diet greatly favors n-6 fatty acids. Estimates of the ratio of n-6 to n-3 fatty acids consumed in various parts of the world range from approximately 9:1 [73] to 30:1 [74]. Consumption of oils such as corn, cottonseed, safflower, and sunflower were originally encouraged for their cholesterol-lowering effect and as a source of linoleic acid, an n-6 essential fatty acid. Most vegetable oils are rich in n-6 fatty acid and low in n-3. In the last two or three decades, consumption of polyunsaturated n-6 fatty acids largely replaced a significant portion of saturated fats that had been habitually consumed. Consumption of marine and terrestrial sources of preformed and precursor n-3 fatty acids is now receiving more attention because of their potential to impact several forms of inflammatory and chronic disease. Recently the ratio of fatty acid consumption has begun to shift slightly more toward n-3 fatty acids primarily as a result of increased consumption of soy and canola oils [73]. When dietary lipids are ingested, the fatty acids serve not only as a fuel but also become components of the phospholipids in virtually every cell membrane in the body. The nature of the fatty acids in the cell membranes affects the physical, chemical, and functional properties of the cell and cell wall. Lipids also serve as precursors for potent mediators in many physiologic reactions, including the immune and inflammatory response.\n\nConsumption of a diet that contains predominantly n-6 fatty acids, for example, results in adipose lipid and cell membrane phospholipids that are high in n-6 fatty acyl groups. When macrophages, neutrophils, lymphocytes, and other cells are activated, they release membrane lipids that are precursors of potent regulators of the inflammatory response. Their release and synthesis can affect recruitment and proliferation of other leukocytes and fibroblasts, alter the permeability of tissues, increase adherence of leukocytes and platelets, increase the generation of harmful oxygen metabolites, and increase the synthesis of proinflammatory eicosanoids. Eicosanoids produced from the metabolism of polyunsaturated fatty acids of the n-6 family (linoleic acid) tend to be proinflammatory and aggregatory, whereas those from n-3 fatty acids in general tend to be anti-inflammatory and decrease cellular adherence. In animal and human studies, supplemental feeding of n-3 fatty acids in the form of linolenic acid and preformed eicosapentaenoic and docosahexaenoic acids has been shown to reduce the synthesis and levels of proinflammatory cytokines, such as TNF-\u03b1 and IL-1, and reduce the generation of potent leukotrienes and prostaglandins, which enhance leukocyte chemotaxis and membrane permeability [74\u201376]. In clinical trials the value of n-3 fatty acids in the management of inflammatory bowel disease has been considered promising but more as a complementary approach rather than single independent treatment [77].\n\nIn general, n-3 fatty acid content of the diet may be especially low in some patients with inflammatory bowel disease. The n-3 fatty acids appear to be reasonably safe in the amounts required to alter membrane eicosanoids and may have value in either sparing other therapies or independently tempering the inflammatory process and its consequences.\n\nGamma-linolenic acid (C18-3 n-6) and dihomo-gamma-linolenic acid, the elongase catalyzed product of gamma-linolenic acid, have also been shown to alter membrane lipids in inflammatory cells [78, 79]. These n-6 fatty acids have been evaluated in animals and in humans in the attenuation of inflammatory and proliferative disorders, but not to the same extent as n-3 fatty acids. Initial reports have shown reduced levels of proinflammatory cytokines, and later leukotriene synthesis and modulated autoimmune activity. Sources of the fatty acids include borage oil and evening primrose oil. Conjugated linoleic acids are a group of iso-mers of linoleic acid found in bovine lipids and in other ruminant animals that have been shown to have antiproliferative properties against several cancer cell lines [80]. The lipids also tend to reduce cell adhesion, decrease platelet aggregation, and alter several chemical mediators in the immune and inflammatory processes [81]. Their role in inflammatory bowel disease, however, has not been evaluated.\n\nMedium-chain triglycerides may have little to do with the inflammatory process, but they may be valuable as an energy source in patients with lipid malabsorption. Absorption of long-chain fatty acids may be compromised during prolonged intestinal disease because of bile salt malabsorption that accompanies ileal resection. Medium-chain triglycerides are known to enter the portal route without the more extensive processing that long-chain fats undergo, but preliminary studies suggest that medium-chain triglycerides may also serve as an energy substrate for colonocytes in the same manner as short-chain fatty acids [82].\n\n#### 2. DIETARY FIBER, RESISTANT STARCHES, AND SHORT-CHAIN FATTY ACIDS\n\nConsumption of dietary fiber and carbohydrates that are incompletely digested or absorbed have the potential to alter colonic flora and affect several gastrointestinal functions. The malabsorbed polysaccharides and sugars serve as substrates for microbes in the colon and the endproducts of microbial fermentation can, in turn, serve as a primary fuel for cells lining the colon and modify their metabolic activity. Inulin, fructose oligosaccharides, pectin, banana, oat and soy fiber, and several resistant carbohydrates contain indigestible carbohydrates that have been shown to alter microbial populations in the colon [83\u201385]. Several have been shown to increase, at least transiently, bifidobacteria and lactobacillus members that are viewed as healthful microbes. Various saccharides have also been shown to reduce the growth of potentially pathogenic microbial populations such as _Clostridium difficile_ [83\u201387].\n\nThe endproducts of fiber and carbohydrate fermentation in the colon are short-chain fatty acids and gases. Short-chain fatty acids and, in particular, butyrate serve as a primary and preferred fuel for colonocytes and appear to be semi-essential for their normal function. In several animal and human studies, addition of various fermentable saccharides or short-chain fatty acids has been reported to increase colonic blood flow, maintain normal mucosal barrier function, enhance fluid and electrolyte absorption (at least at physiologic doses), prevent antibiotic-associated diarrhea, provide protection from toxins, reduce the potential for inflammatory activity, and maintain normal proliferation and differentiation of colonocytes [83\u201387]. In several trials, addition of dietary fiber sources or topical administration of short-chain fatty acids has been shown to have some value in treating mild forms of ulcerative colitis or in maintaining remission\u2014either as single agents or when used in combination with more traditional therapies. The effect is modest, but further work with the sources of fermentable saccharides and combinations of short-chain fatty acids may bring more effective therapies [88, 89]. Most of the work with dietary fiber has appropriately been focused toward ulcerative colitis, but use of fiber sources has been considered in the management of Crohn's disease.\n\nDietary fiber intake is relatively low in the U.S. diet, and patients with inflammatory bowel disease may consume even less. Fear of obstruction, increased gas, or concern about coarseness of fibrous foods may result in very low intakes, making the patient more susceptible to the reported effects of insufficient colonic substrate. Use of dietary fiber sources, even if in powdered or blenderized form, may be helpful in the diets of persons with inflammatory bowel disease and especially in ulcerative colitis. Because some fibrous foods are not greatly changed until they reach the colon, caution is advised in patients with strictures. Boluses of powdered sources have been shown to cause obstruction in the upper gastrointestinal tract. Thus the best approach is to use whole foods as the fiber source. Fruits, vegetables, and whole grains are excellent sources of fiber, vitamins, minerals, trace elements, and numerous potentially protective phytochemicals that may favorably affect the health of the gastrointestinal tract and the individual.\n\n#### 3. DIETARY SUGARS\n\nHigh sugar intake and consumption of refined foods, at least in some countries, has been linked to the preillness diet or rise in the incidence of inflammatory bowel disease [42\u201344]. No causal association, however, has been demonstrated [3, 45]. On the other hand, consumption of large amounts of sugars could displace foods that provide other nutrients. Certain sugars (e.g., lactose, fructose, alcohol sugars) are not as well absorbed as other carbohydrates and may contribute to symptoms such as bloating, gas, and diarrhea. Malabsorption and fermentation of relatively modest amounts of small molecular weight sugars can result in osmotic diarrhea [90\u201392].\n\n#### 4. VITAMINS, MINERALS, AND ANTIOXIDANT NUTRIENTS\n\nMicronutrient and antioxidant status may be compromised in patients with inflammatory bowel disease, by a combination of an incomplete diet, malabsorption, and the inflammatory process. When in remission, persons with inflammatory bowel disease can consume a good diet as a source of essential micronutrients, antioxidants, and protective phytochemicals not found in nutrient supplements. If, however, the patient is unable or unwilling to consume a complete diet, then certainly vitamin-mineral supplements and\/or liquid nutrition supplements may be appropriate. Numerous oral supplements are available with different lipid mixtures, protein sources, fiber, and other additives to meet patient needs. Some patients will have selective nutritional problems, such as vitamin B12 malabsorption due to ileal resection or calcium and vitamin D inadequacy due to avoidance of lactose. (Refer to Chapter 16 on enteral and parenteral nutrition.)\n\n### I. Parenteral and Enteral Nutrition Support\n\nAlthough the cause of inflammatory bowel disease is unknown, current information suggests that inflammatory bowel disease occurs when genetic aberrations and environmental triggers interact. The primary environmental factors to which the gastrointestinal tract is exposed are foods and microbes. Both dietary factors and elements of the gastrointestinal flora have been scrutinized, but no specific etiologic agent(s) have been identified as yet. Regular diets, however, are a potential source of antigens, contain elements that may serve as inflammatory or suppressive agents, and provide a major source of fuels for both cells lining the gastrointestinal tract and microbes residing in the gastrointestinal tract.\n\n#### 1. DOES NUTRITION SUPPORT INDUCE REMISSION?\n\nThe extent to which nutritional measures can induce and maintain remission remains an interesting and unresolved issue. Parenteral nutrition was initially considered an appropriate approach because one could restore the individual's nutritional status and provide sufficient energy for growth and weight gain. Use of parenteral nutrition and withdrawal of oral nutrition was seen as \"bowel rest\" and provided an opportunity to reduce both the antigenic load from food and reduce microbial populations. Use of parenteral nutrition has not, however, been as successful as hoped as a primary therapy in treating Crohn's disease and has been even less effective in treating ulcerative colitis [2, 94]. Although a small percentage of patients may benefit with a reasonable length of remission, short-term remission is the more common outcome. Withholding enteral nutrition may also be considered undesirable because it may compromise gut integrity.\n\nAlthough not entirely nutritionally complete, parenteral nutrition has served as a valuable tool for providing nutritional support for patients with gastrointestinal obstruction, perforation, and toxic megacolon, and it can provide a primary source of nutrition in patients with malabsorption after major small bowel resection. Parenteral nutrition does not obviously contain the phytonutrients and wide range of lipids and microelements found in a normal diet. Additional study with other supportive additives such as specific amino acids, lipids, and selected micronutrients may in the future provide more effective parenteral solutions.\n\nThe effectiveness of various forms of enteral feedings as a primary treatment for inflammatory bowel disease has been evaluated in several reviews and meta-analyses [94\u2013103]. In almost all the reviews, Crohn's disease appeared to be more amenable to enteral feeding treatment than ulcerative colitis, although in a few individual studies, such interventions were reported to have some value in managing ulcerative colitis. In ulcerative colitis, therapeutic trials with fermentable fibers, short-chain and medium-chain fatty acids, and n-3 fatty acids may ultimately achieve success more than the use of standard parenteral and enteral formulas [88, 89]. With few exceptions, enteral nutrition appears to be more effective than parenteral nutrition, but whether enteral nutrition as a sole therapy is as effective as or better than medications is less clear.\n\nIn many of the reports, corticosteroids and other medical treatments were considered clear-cut choices and enteral nutrition seen as supportive care. In other reports, enteral diets were said to be the preferred route and were considered to be at least as effective as medications. For example, Messori _et al._ [95] performed a meta-analysis of the effectiveness of chemically defined diets versus corticosteroids from seven randomized clinical trials. The authors concluded that steroids were significantly more effective than diet in inducing remission in active Crohn's disease. The authors also separated patients who failed to complete the enteral trial because they did not tolerate the formula and concluded that steroids were still superior.\n\nIn another meta-analysis, Schwab _al al._ [96] reviewed 18 studies, which included 295 patients treated with elemental diets, 214 with oligopeptide diets, and 62 with polymeric diets. When the data were adjusted for patients who completed the course of enteral diet treatment, 73, 70, and 67%, respectively, of the patients achieved remission with the three types of diets. The authors considered the rates of remission with diet to be comparable to rates achieved with corticosteroids. Heuschkel and Walker-Smith [97] reviewed several enteral diet studies and their own experiences with use of enteral nutrition in children. These authors concluded that the studies reviewed suggested that enteral nutrition may promote healing of the intestinal mucosa better than corticosteroids, but admitted that 40\u201360% of children will still become symptomatic within a year of treatment.\n\nExamination of similarities and differences in the design of the dozens of individual studies in which enteral diet or medications were considered more efficacious does not entirely clarify the situation. Numerous criteria were used to describe the severity of the patient's disease both when the therapy started and when remission was achieved. Initial disease activity ranged from mild to severe. Severity of disease and\/or resolution of disease have been measured using clinical measures such as diarrhea, weight change, and ratings of well-being. In other studies, more objective indicators, such as endoscopic and histologic evidence, levels of inflammatory mediators, or measures of gastrointestinal structure\/permeability, were used. Duration of the patient's disease prior to initiating enteral nutrition nutrition or medical therapy may or may not have been reported. In some of the study designs, diet therapy may have been evaluated with and without various medications to determine whether diet improved the overall management, and dose and route of administration of medications varied greatly. Duration of treatment with enteral therapy ranged from 2 to 8 weeks or longer and the diet may have been fed orally, by tube, or by combinations of routes. Indeed, one of the problems identified by even the proponents of nutritional therapy was that some of the diets were unpalatable or it was difficult for some patients to take the majority of their diet in the form of an enteral formula. Improved nutritional status was considered one of the measures of success in many of the studies but nutritional status may not have been well described or was described in a variety of ways. Severity of disease, degree of control required, and type of health care system employed in the various countries may have influenced the need for and duration of hospitalization.\n\nIn earlier studies, \"elemental\" or chemically defined type diets were preferred because they tended to be low in fat and the nitrogen was in the form of amino acids and small peptides. More recently, use of \"polymeric\" and whole-protein nitrogen sources and even regular foods have claimed to be as effective as elemental diets in allowing remission of Crohn's disease. In addition to differences in the approaches to comparisons with diet and medications, it should be noted that the natural course of inflammatory bowel disease includes unexplained exacerbations and remission. In the history of therapeutic interventions with medications, 30\u201360% of patients may improve with placebo alone [3, 94].\n\n#### 2. HOW MIGHT DIETS WORK AS THERAPY?\n\nSelected enteral feedings could play several roles in the management of the disease. Patients with inflammatory bowel disease are often malnourished and have inadequate intake of protein, energy, and micronutrients. This malnutrition may create changes in gastrointestinal barrier functions and digestive functions and alter immune mechanisms. Individual dietary components may act as antioxidants, serve as precursors to inflammatory mediators, and alter the relative types of microbes existing in the gastrointestinal tract. Dietary excesses may also bring about undue symptoms even though they may have nothing to do with the pathology of the disease. Many of the formulations used in past studies did not include many of the newer individual nutrient additives or modifications, yet many appeared to achieve some level of success in treatment of inflammatory bowel disease or at least tolerance. Continued trials, for example, with lipids, amino acids, short-chain fatty acids and fiber sources, antioxidant nutrients, and phytochemicals, may make enteral diets more effective in managing inflammatory bowel disease.\n\nWhether or not nutrition support is effective as a primary therapy, both parenteral and enteral feedings are valuable in restoring nutritional reserves and allowing growth and maturation in children. Enteral nutrition would appear to be an appropriate first choice of nutrition therapy. Appropriate selection and use of enteral nutrition has the potential to provide enterocytes and colonocytes with a direct source of preferred fuels, and allow normal proliferation and differentiation of those cells, increase gut barrier functions, and suppress the proliferation of unhealthy microbes. If diet does serve as a source of antigens, inflammatory stimulants, or toxins, enteral formulas may be designed specifically to reduce the offending agents yet provide reasonable nutrition support. Sufficient success with nutrition as a sole therapy alone and in combination with other treatments warrants additional study in inducing and maintaining remission in both Crohn's disease and ulcerative colitis.\n\n## III. SHORT BOWEL SYNDROME\n\nA. Definition\n\nShort bowel syndrome refers to the set of symptoms and complications that occurs after significant small bowel resection. Resection of the colon or stomach does not constitute or result in short bowel syndrome, but loss of either can compromise the patient who has had a small intestinal resection. The syndrome and its consequences are primarily related to the inability to absorb digested foods and to reabsorb endogenous secretions. Malabsorption of macronutrients, micronutrients, fluids, and electrolytes occurs but the severity varies considerably. Weight loss, growth retardation, diarrhea, dehydration, electrolyte disturbances, bone loss, renal oxalate stones, gallstones, lactic acidosis, and bacterial overgrowth may occur depending on the extent and nature of the resection [104, 105]. The syndrome may be permanent, as is the case with severe shortening of the gastrointestinal tract, or temporary with less drastic resections. The symptoms will appear immediately after the resection and continue unless sufficient adaptation occurs and\/or medical, nutritional, or surgical interventions are successful. Typically, maximal adaptation of the remaining intestine takes months or years to occur.\n\n### B. Causes\n\nIn adults, Crohn's disease is the principal cause of short bowel syndrome, accounting for about 60\u201370% of the cases. Mesenteric infarct, radiation enteritis, and volvulus are less common etiologies. In infants and children, atresia, volvulus, gastroschisis, and necrotizing enterocolitis are more common reasons for significant resections leading to short bowel syndrome [104]. The incidence and prevalence are not known but the number of persons supported on total or supplemental parenteral nutrition number in the tens of thousands.\n\n### C. Predictors of the Severity and Duration of the Syndrome\n\nThe severity and duration of symptoms, the patient's need for nutrition support, and the survival of the patient all depend on the length and function of the remaining gastrointestinal tract and the health of the host. Patients who survive without parenteral nutrition tend to adapt more completely, have fewer adverse symptoms, and are at decreased nutritional risk. They tend to be patients who are very young, have remaining distal ileum and ileocecal valve, have retained colon, and are otherwise healthy and well nourished. Alternatively, predictors of poor survival include dependence on parenteral nutrition support, increased nutrition risk, more complications, and compromised quality of life. These patients tend to have loss of terminal ileum and ileocecal valve, advanced age at resection, loss of the colon in addition to small bowel, and presence of residual gastrointestinal disease [104\u2013109].\n\n### D. Variants of Short Bowel Syndrome\n\nRequirements for nutritional, medical, and surgical care of the patient after bowel resections can be predicted somewhat by the normal physiologic role and capacity of segments removed and remaining. See Table 9 for predictors of severity of short bowel syndrome and nutrition risk. Figure 5 illustrates normal events in gastrointestinal secretion, digestion, absorption, and fermentation processes for comparison with changes following bowel resections.\n\nTABLE 9\n\nPredictors of Severity of Nutrition Risk in Short Bowel Syndrome\n\nLoss of distal ileum\n\nLoss of ileocecal valve\n\nLoss of colon in addition to small bowel\n\nAdvanced age\n\nResidual disease\n\nMesenteric infarct as cause for bowel resection\n\nFIGURE 5 Normal gastrointestinal tract.\n\n#### 1. JEJUNAL RESECTIONS\n\nDespite the fact that most gastrointestinal fluids are secreted into the proximal small intestine and the jejunum is normally the site of intense digestive activity, loss of the entire jejunum in infants or adults typically results in only transient and relatively mild malabsorption as long as the patient and the remaining gastrointestinal tract are healthy (see Fig. 6 for depiction of jejunal resection). Initially, the patient will require parenteral nutrition support but will be able to begin consuming small, frequent, simple meals as soon as bowel motility returns. Adaptation of the remaining intestine will likely to occur during the next several months. The patient will likely be able to consume regular foods with only limited restrictions and minimal compromise in digestive and absorptive capacity. The remaining gastrointestinal tract has sufficient reserve to compensate for the decreased surface area. Because lactase enzyme is produced primarily in the cells of the proximal small intestine, jejunal resection typically results in lactose malabsorption. The patient may also have slightly more rapid overall transit through the gastrointestinal tract and may not tolerate consumption of large quantities of hyperosmolar or highly concentrated foods and beverages. Net protein and energy intake, and fluid and electrolyte utilization, are not seriously threatened. The efficiency of absorption of certain nutrients such as iron, calcium, magnesium, and some lipid-soluble nutrients may be slightly compromised.\n\nFIGURE 6 Jejunal resection.\n\n#### 2. ILEAL RESECTIONS\n\nMajor resections of the ileum create several problems in maintaining nutrition [104, 110\u2013113] (see Fig. 7 for depiction of ileal resection). A summary of the consequences of ileal resection can be found in Table 10. Normally, adults consume about 2 liters of fluid per day and the proximal gastrointestinal tract secretes approximately 7\u20139 liters of fluid per day into the intestinal tract. A significant amount of fluid is normally absorbed in the distal small intestine and normally only 1\u20131.5 liters of liquid chyme per day enters the colon. Only about 100 mL of water is lost in the feces daily. When ileal resections occur, the remaining small and large intestine must absorb the residual fluid. If colon is resected in addition to the ileum, maintaining adequate hydration and electrolyte status is difficult.\n\nTABLE 10\n\nConsequences of Ileal Resection, Especially with Loss of Ileocecal Valve\n\nIncreased fluid loss\n\nShorter transit\n\nBile acid malabsorption; insufficient _de novo_ bile salt synthesis\n\nFat, fatty acid malabsorption\n\nMalabsorption of fat-soluble nutrients\n\nDecreased calcium, zinc, magnesium absorption\n\nIf colon remains:\n\n\u2014Increased colonic oxalate absorption\n\n\u2014Increased secretion from malabsorbed bile acids and fatty acids\n\n\u2014Increased risk of small bowel overgrowth\n\nFIGURE 7 Ileal resection.\n\nThe last meter of ileum is also the primary site for the absorption of vitamin B12 intrinsic factor complex and is the only site for reabsorption of bile acids entering the intestine from the biliary tract. Healthy adults usually have sufficient stores of vitamin B12 to last for 3 years or so and after ileal resection receive periodic injections of vitamin B12. More importantly, loss of the distal ileal segment, where 90% or more of bile salts are reabsorbed, results in a significant malabsorption cascade. The role of bile salts is to provide surfactant action to help reduce the particle size of lipid droplets and to participate in the formation of micelles. Micelles are the polar particles in which free fatty acids, monoglycerides, and fat-soluble vitamins are absorbed into cells of the small intestine. When bile salts are no longer \"recycled\" in the distal ileum, _de novo_ synthesis in the liver from the sterol pool cannot supply sufficient bile salts for adequate absorption of lipids and lipid-soluble nutrients. In addition, unabsorbed bile acids entering the colon are considered \"irritants\" and serve as secretagogues at a time when colon is already burdened with the task of absorbing extraordinary amounts of fluid. Increased amounts of unabsorbed fatty acids in the lumen of the bowel can result in the formation of fatty acid soaps with calcium, magnesium, and zinc, which interfere with the absorption of these minerals. The malabsorbed fatty acids may also serve as secretagogues in the colon.\n\nDietary oxalate normally binds with calcium and other divalent cations to form insoluble complexes, but free oxalate becomes more available for absorption in the colon. In both ileal resections and long-term Crohn's disease involving the ileum, hyperoxaluria and renal stone formation are not uncommon. In an attempt to bind bile acids, improve colonic fluid and electrolyte absorption, and reduce absorption of oxalate colestyramine, supplemental calcium, and modest dietary oxalate restriction are often prescribed [104, 110].\n\nAnother potential complication of distal ileal resections, especially involving loss of the ileocecal valve, is bacterial overgrowth in the small intestine. The small intestine is normally relatively sterile, but with loss of the one-way barrier provided by the ileocecal valve, microbes from the colon may more readily mix with small bowel contents. Bacterial overgrowth is usually treated with antibiotics but more recently use of small bowel irrigation, probiotics, and prebiotic foods have been tried with some success [114].\n\n#### 3. LOSS OF ILEUM AND COLON\n\nIf both the ileum and colon are resected (see Fig. 8), maintenance of adequate hydration and energy balance becomes more difficult. Although only 1\u20131.5 liters of fluid normally enters the colon, a healthy colon is capable of absorbing 3\u20134 liters. With only a jejunum, patients have difficulty absorbing ingested fluid and endogenous gastrointestinal secretions. Fecal outputs from jejunostomies may exceed 3\u20135 liters daily and patients may lose 100\u2013200 mEq sodium in the effluent. In addition to increased risk of hypovolemia, patients are also more at risk for hypokalemia and hypomagnesemia and other forms of nutrient depletion [104, 110, 112].\n\nFIGURE 8 Resection of both ileum and colon.\n\nThe presence of foodstuffs or partially digested foods in the distal intestine normally stimulates the release of neuroendocrine secretions, including glucagon-like peptide-1 (GLP-1) [115], peptide tyrosine tyrosine (peptide YY), or neuropeptide-Y [116], which may downregulate the secretory and motor activity of the stomach and proximal small intestine. Short-chain fatty acids produced in the colon from fermentation of carbohydrate and fiber may also slow gastric emptying [117]. The net effect of the ileal or ileocolonic \"brake\" in the normal, intact gastrointestinal tract is to retard gastric emptying, reduce secretions from the proximal gastrointestinal tract, and decrease motility. The mechanism is not completely understood and current hypotheses are likely oversimplified. However, the colonic brake mechanisms are reasonable explanations for the normal regulation of the volume of food released and propagated through the gastrointestinal tract and for the observed clinical maladjustment in short bowel syndrome. Regardless of the exact mechanism, the loss of distal ileum and colon appears to result in the loss of the ileocolonic brake phenomenon that slows entry of food and secretions into the intestine.\n\nThe colon also serves to salvage energy from malabsorbed substrates. In humans, the role of the colon in extracting energy from foodstuffs is normally relatively minor. In short bowel syndrome, however, more carbohydrate, lipids, and nitrogenous compounds enter the colon than usual. The amount of energy the colon can extract after small bowel resections from malabsorbed substrates varies with the length of small intestine remaining. In normal individuals consuming a typical diet, only about 5\u201310% of energy is salvaged by colonic fermentation and absorption [110]. Nordgaard and colleagues [118] demonstrated using bomb calorimetry the fecal energy losses that occur with and without the colon. Patients who had 150\u2013200 cm of small intestine without a colon excreted only 0.8 MJ\/day (110 kcal) more than those with an intact colon. But when patients had only 100 cm or less small bowel remaining, preservation of the colon for energy salvage from malabsorbed substrates became much more important. Colectomy resulted in an extra loss of 4.9 MJ\/day (about 1150 kcal).\n\nColonocytes cannot absorb long-chain fatty acids to any significant degree, but short-chain fatty acids produced from microbial fermentation of fiber, carbohydrate, and, to a lesser extent, amino acids can be used by the colonocytes and the host. Recently, investigators have shown that medium-chain fatty acids can also be used by colonocytes and absorbed systemically [119, 120]. Loss of the colon and ileum not only results in loss of the energy salvage mechanism, but also decreased fsluid and electrolyte absorption, more rapid transit, and overall decreased efficiency of absorption of foodstuffs.\n\n#### 4. MAJOR INTESTINAL RESECTIONS\n\nVolvulus, gastroschisis, or atresias often result in a small bowel length of only a 100 cm or less. The same risk factors for increased morbidity apply in infants including loss of ileum, ileocecal valve, and colon, except infants appear to adapt significantly better after resections than adults. In normal healthy infants and children, intestinal growth occurs from preterm until early adulthood. Weaver and associates [121] studied 1010 necropsy specimens from preterm fetuses and subjects up to 20 years of age. Small bowel length more than doubled from 20 to 40 weeks' gestation and almost doubled again from term (275 cm) to age 10 years (500 cm). From 10 to 20 years intestinal length increased only an additional 75 cm. The extent to which lengthening and other forms of adaptation occur after resection (beyond normal growth) is not clear, but infants appear to function reasonably well with very short intestinal remnants. In infants, survival without parenteral nutrition has been reported with as little as approximately 20\u201360 cm of small bowel if the children have retained ileocecal valve and as little as 30\u2013100 cm without the ileocecal valve [105\u2013109]. If the ileocecal valve is lost, retained colon becomes more important for maintenance of electrolyte balance and adequate hydration, and to salvage malabsorbed substrates [109, 110, 118]. Adults typically require at least 100\u2013200 cm of small intestine to avoid at least partial dependence on parenteral nutrition and adaptation may be more prolonged and less complete [104, 112\u2013113, 122]. For children with extremely short small bowel lengths who cannot adapt, several options remain. They can remain on parenteral nutrition, undergo small bowel lengthening procedures, or undergo small bowel transplantation. Parenteral nutrition imposes on the quality of life for the patient and caretakers and carries increased risk of infection, hepatic failure, gallstone formation, and nutrient deficiencies [123, 124].\n\nThe ability to tolerate major bowel resections in adults is considerably less than in children. If the patient has residual disease and has had other changes in gastrointestinal pathology due to radiation enteritis, atherosclerosis, diabetes, or inflammatory bowel disease, then bowel resections pose greater risk. In patients with radiation enteritis, even relatively small resections of the ileum, especially if the resection includes the ileocecal valve, can create short bowel syndrome.\n\nPatients who suffer mesenteric infarct may lose almost the entire small intestine and most of the colon. Because mesenteric infarct typically occurs in older patients as a result of arteriosclerotic vascular disease, adaptation of the remaining segment may not be sufficient. The patient may require lifelong support with at least supplemental parenteral nutrition. Persons with massive resections and their caretakers will need to be patient and cautious with oral and enteral feedings. Maximal adaptation may require up to 2 years. Reintroduction and advancement of foods should be gradual. Unfortunately, some patients may never be able to absorb sufficient amounts of nutrients by way of the gastrointestinal tract. Overeating or overfeeding, especially with hypertonic and large volumes of foods, may result in net loss of endogenous fluid and electrolyte [110, 125].\n\n### E. Principles of Nutrition Care and Feeding in Short Bowel Syndrome\n\nOne of the primary principles of managing patients with short bowel syndrome is to start using the gut as soon as possible. Foods and the gastrointestinal secretions are trophic to the small bowel and malnutrition compromises the adaptive process [126\u2013128]. In laboratory animal studies, the gastrointestinal tract has been shown to atrophy markedly during starvation and parenteral nutrition. In humans, the degree of gut atrophy during parenteral nutrition may not be as striking as in animal models, but it appears to occur. Biasco _et al._ [129] described the gastrointestinal response in a patient fed by parenteral and oral nutrition after a subtotal resection for mesenteric obstruction. The patient had 30 cm of proximal jejunum anastomosed to his descending colon. The patient was fed exclusively by parenteral nutrition for 30 days, and then oral nutrition was gradually introduced during the next 30 days. Jejunal biopsies were taken during parenteral and oral periods and examined for cell kinetic studies. Hypoplasia was demonstrated soon after parenteral nutrition was initiated and remained until oral nutrition was started. Hyperplasia occurred soon after oral feeding, and after 14 days crypt depth and villous height had increased by approximately 50%.\n\nA significant portion of the energy supplied to the small intestine comes from sugars, keto acids, and amino acids (notably glutamine) from the lumen of the gastrointestinal tract. An even greater percent of the energy for colonocytes come from substrates in the lumen, in this case primarily short-chain fatty acids produced in fermentation of saccharides. Enteral nutrition then provides fuels for both the intestine and the host.\n\nIn short bowel syndrome, the desired and observed response to feeding after major bowel resection is vigorous hyperplasia, hypertrophy, and increased length and diameter of the remaining small bowel. If oral or enteral feeding is not provided, atrophy occurs. Even small amounts of foodstuffs provide sufficient stimulus for intestinal growth. Initial feedings after bowel resections may include small frequent meals, sips of liquid supplements, or dilute enteral feedings to gradually restore gastrointestinal function and stimulate adaptation.\n\nAdaptation in the gastrointestinal tract is not as complete as that seen in other organ systems, and when adaptation is maximal, the patient may still have inadequate absorptive function. The extent to which adaptation takes place depends on the age of the individual, the health and vascular patency of the remaining intestine, and nutritional factors. Relatively few studies have been reported in which changes in intestinal length and diameter of the bowel following intestinal resection have been measured. Shin _et al._ [130] studied the adaptive response in dogs with massive resections after receiving nutrition support in the sequence normally recommended for human patients. Seven mongrel dogs had surgical resections, leaving 20 cm of proximal jejunum anastomosed to the mid-transverse colon. The dogs were initially fed by parenteral nutrition and, after 4 days, oral food and water were reintroduced. Parenteral nutrition was continued in addition to the oral diet for 30 days and was then stopped. Parenteral nutrition proved to be essential initially because oral feeding induced massive stool outputs. Six weeks after the resection, the five dogs that survived were reexplored and the remaining small bowel was examined. The length and circumference of the jejunum increased by 30%, and the crypt depth, number of villi and the width of the villi, had each increased by approximately 20%. Stool volume and frequency gradually decreased but were still abnormal.\n\nSolhaug [131] studied five female patients who had undergone jejuno-ileal bypass procedures for treatment of obesity. Patients had end-to-end anastomosis of their proximal jejunum to their distal ileum and had undergone a second operation because of complications or unsuccessful weight loss. The remaining segments of small intestine increased during a 1- to 2-year period from a mean of 52 to 82 cm while the subjects consumed an _ad libitum_ diet. The bypassed segments essentially remained unchanged. Mucosal thickness was approximately 20\u201340% greater than the bypassed segment, villous height was 25\u201350% greater and small intestinal circumference was double that of the bypassed segment.\n\nWhen starting feedings in patients with short bowel, the diet is normally provided in very small meals or in sips of dilute formula diets [110, 113, 132]. During the advancement of feeding, the patient is normally supported at least in part by parenteral feeding so that oral feeding proceeds gradually to allow the gut to adapt and to prevent diarrhea and malabsorption. If the patient is unable or unwilling to eat consistently in a controlled or scheduled manner, enteral feedings can be provided in frequent mini meals or delivered by tube feeding. Tube feedings can be scheduled at night or between meals as needed, then decreased as the patient is able to take more foods and beverages orally. Many patients are anorectic initially and may later become hyperphagic [125]. Overfeeding foods and beverages, especially at the beginning of refeeding, can easily result in loss of more fluid than was originally consumed. Patients sometimes express extreme hunger and weakness. They may subsequently consume large volumes of food and beverages only to experience malabsorption of foodstuffs and large luid effluent. Typically, dietary modifications are included to assure adequate nutriture without overwhelming digestive and absorptive capacity.\n\nPrinciples of managing nutritional aspects of short bowel syndrome are based on the sites and capacities of digestion and absorption [110, 126, 128, 132, 133]. The mainstays of the diet are starches and lean animal products with modest amounts of fat. Patients with retained colon may do better with relatively higher amounts of starches and lower amounts of fat (20\u201320% of energy from fat) than those without. Sources of both n-6 and n-3 fatty acids should be provided. Medium-chain triglyceride-containing products can be helpful in providing a source of energy, especially in patients without the distal ileum. Poorly absorbed sugars (e.g., fructose, alcohol sugars) and consumption of large volumes of all hyperosmolar sugar sources should be limited. The lactase enzyme may be lacking in some patients but reasonable amounts of lactose can be tolerated especially when supplied in foods such as cheeses, yogurts, or mixed foods rather than milk as a beverage [134]. Commercially available lactase enzyme can also be used to improve the tolerance to foods high in lactose.\n\nSome foods are discouraged simply because they provide little or no nutrient value and may displace better sources of nutrients in the diet. Caffeinated and alcoholic beverages tend to increase gastrointestinal secretions, alter motility, or increase permeability and contribute little to nutritional value. The diet need not be devoid of fiber; in fact, modest amounts of dietary fiber may help to regulate gastric emptying. If the patient has a residual colon, fiber may be fermented to short-chain fatty acids to enhance gastrointestinal function and increase absorption of fluid and electrolytes. In patients with strictures, dietary fiber may need to be in the form of small particulate matter and added to juices or other foods.\n\nA typical diet in the patient with short bowel may include small but frequent portions of foods such as eggs, meats, poultry, fishes, breads and cereals, rice, potatoes, pastas, vegetables, and vegetable juices. Specially tailored oral electrolyte replacement fluids [135] may be used, as needed, but low-calorie, caffeine-free beverages or flavored waters can also be used, preferably taken frequently in small quantities. Use of vitamin and mineral supplements should be tailored to the patient's dietary habits, tolerances, and absorptive capacity. Because transit through the remaining gastrointestinal tract may be rapid, tablets may not disintegrate in time for maximal absorption, so supplements may need to be crushed or provided in liquid form.\n\n### F. Nutrients, Growth Factors, and Medical and Surgical Therapies\n\nSeveral specific nutrients and neuroendocrine agents are being evaluated to hasten or enhance the adaptive process including supplemental glutamine, growth hormone, epidermal growth factor, insulin-like growth factor 1, n-3 fatty acids, nucleotides, fermentable fibers, or short-chain fatty acids [128, 132, 136\u2013139].\n\nWilmore [132] has described the use of a combination of growth hormone, supplemental dietary glutamine, and a low-fat diet high in complex carbohydrate in managing more than 300 patients who were dependent on parenteral nutritional support or considered to be at significant risk for nutritional inadequacies. Wilmore and his group report that the combined therapy has been successful in increasing body weight and lean mass, reducing dependence on parenteral nutrition, and improving overall well-being of patients with short bowel syndrome. Other investigators [140, 141] have not been as enthusiastic about this approach. Scolapio [140], for example, in a randomized, placebo-controlled, double-blind crossover trial, studied growth hormone and supplemented glutamine in eight patients who had been dependent on parenteral nutrition for 3\u201319 years. While receiving the treatment, patients gained fluid weight and lean mass but only as long as the therapy was continued and the patients did not demonstrate increased fluid and macronutrient absorption during the 21-day trial. Based on their study with animal models, Vanderhoof _et al._ [141] concluded that any adaptive response was more likely to be the result of enteral nutrition rather than the combined glutamine-growth hormone treatment.\n\nDietary fiber or short-chain fatty acids have been studied in the regulation of normal gastrointestinal function as a therapeutic agent in the management of gastrointestinal disease and as a regulatory agent in short bowel syndrome. Dietary fiber may help regulate gastric emptying by increasing the viscosity of gastric contents or as a result of a feedback mechanism when dietary fiber and resistant starch are fermented to short-chain fatty acids in the colon [120, 139, 142]. Short-chain fatty acids and certain dietary fibers may also regulate colonic cellular differentiation and serve to prevent the overgrowth of potentially pathogenic organisms [142].\n\nPatients with short bowel syndrome may be also treated with several medications to improve symptoms, decrease gastrointestinal secretions, delay gastric emptying, slow intestinal transit, or bind bile acids. Histamine-2 receptor blockers or proton pump inhibitors may be used in the initial stages of gastric acid hypersecretion; loperamide, atropine, opiates, and octeotide may be used for management of diarrhea; and cholestyramine can be used to bind bile acids [104, 112]. Other agents are being studied in the management and prevention of complications of short bowel symptoms.\n\nSeveral surgical procedures have been considered in the management of extreme short bowel and intestinal failure, including intestinal tapering and lengthening procedures, intestinal loops, reversal of intestinal segments, insertion of colonic segments (which normally exhibit slower transit), insertion of reversed segments colon, creation of intestinal loops and valve-type passages, and, most recently, intestinal transplant [143\u2013148]. Small bowel transplant may include small intestinal segments alone, small intestine with colon, and intestine with liver transplant. Transplant may be the only alternative when prolonged treatment of gut failure with total parenteral nutrition leads to liver failure. The treatments that appear to be considered the best for improving existing short bowel are small intestinal lengthening [145, 146] and, for gut failure, total parenteral nutrition and intestinal transplant [147, 148]. Transplant procedures and immunosuppresive therapy are improving and with time may provide an option for more patients in the future.\n\n## IV. CONCLUSION\n\nBoth inflammatory bowel disease and short bowel syndrome are gastrointestinal disorders that may lead to serious nutrition-related consequences, including diarrhea and mal-absorption, weight loss, growth retardation, dehydration, micronutrient deficiencies, electrolyte disturbances, and bone loss. Each disorder requires thorough evaluation of the patient's nutrition status and each requires that nutrition intervention be tailored to the individual. Nutrition interventions typically take the form of dietary modifications, enteral and parenteral nutrition support, nutrition supplements, and education of the patient and his or her caregivers. Newer medical, surgical, and nutritional approaches to prevention and treatment continue to improve the survival and quality of life in patients with inflammatory bowel disease and short bowel syndrome. However, despite the numerous investigations already performed related to nutrition in inflammatory bowel disease and short bowel, the knowledge and science base for practice of nutrition in the treatment of these disorders is still in its infancy.\n\nReferences\n\n1. Jewel D.P. Ulcerative colitis. In: Feldman M., Sleisenger H.H., Scharschmidt B.F., eds. _Gastrointestinal and Liver Disease_. 6th ed. Copenhagen, Denmark: W. B. Saunders; 1998:1735\u20131760.\n\n2. Brown M.O. Inflammatory bowel disease. _Primary Care_. 1999;26:141\u2013170.\n\n3. Kornbluth A., Sachar D.B., Salomon P. 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Mancuso P., Whelan J., DeMichele S.J., Snider C.C., Guszcza J.A., Karlstad M.D. Dietary fish oil and fish and borage oil suppress intrapulmonary proinflammatory eicosanoid biosynthesis and attenuate pulmonary neutrophil accumulation in endotoxic rats. _Crit. Care. Med_. 1997;25:1198\u20131206.\n\n80. Parodi P.W. Cows' milk fat components as potential anticarcinogenic agents. _J. Nutr_. 1997;127:1055\u20131060.\n\n81. Sugano M., Tsujita A., Yamasaki M., Noguchi M., Yamada K. Conjugated linoleic acid modulates tissue levels of chemical mediators and immunoglobulins in rats. _Lipids_. 1998;33:521\u2013527.\n\n82. (Suppl.)Jeppeson P.B., Mortensen P.B. Colonic digestion and absorption of energy from carbohydrates and medium-chain triglycerides in small bowel failure. _J. Parenteral Enteral Nutr_. 1999;23:S101\u2013S105.\n\n83. Bengmark S., Jeppsson B. Gastrointestinal surface protection and reconditioning. _J. Parenteral Enteral Nutr_. 1995;19:410\u2013415.\n\n84. Gibson G.R. Dietary modulation of the human gut microflora using prebiotics. _Br. J. Nutr_. 1998;80:S209\u2013S212.\n\n85. Emery E.A., Ahmad S., Koethe J.D., Skipper A., Peerlmutter S., Paskin D.L. Banana flakes control diarrhea in enterally fed patients. _Nutr. Clin. Prac_. 1997;12:72\u201375.\n\n86. Oli M.W., Petschow B.W., Buddington R.K. Evaluation of fructooligosaccharide supplementation of oral electrolyte solutions for treatment of diarrhea: Recovery of the intestinal bacteria. _Dig. Dis. Sci_. 1998;43:138\u2013147.\n\n87. Andoh A., Bamba T., Sasaki M. Physiologic roles of dietary fiber and butyrate in intestinal functions. _J. Parenteral Enteral Nutr_. 1999;23:S70\u2013S73.\n\n88. Spanish Group for the Study of Crohn's Disease and Ulcerative ColitisFernandez-Banares F., Hinojosa J., Sanchez-Lombrana J.L., Navarro E., Martinez-Salmeron J.F., Garcia-Puges A., Gonzales-Huix F., Riera J., Gonzolez-Lara V., Dominguez Abascal F., Gine J.J., Moles J., Gomollon F., Gassull M.A. Randomized cllinical trial of plantago ovata seeds (dietary fiber) as compared to mesalamine in maintaining remission in ulcerative colitis. _Am. J. Gastroenterol_. 1999;94:427\u2013433.\n\n89. Breuer R.I., Soergel K.H., Lashner B.A., Christ M.L., Hanauer S.B., Vanagunas A., Harig J.M., Keshavarzian A., Robinson M., Sellen J.M., Weinberg D., Vidican D.E., Flemal K.L., Rademaker A.W. Short chain fatty acid rectal irrigation for left-sided ulcerative colitis: A randomized, placebo controlled trial. _Gut_. 1997;40:485\u2013491.\n\n90. Clausen M.R., Jorgensen J., Mortensen P.B. Comparison of diarrhea induced by ingestion of fructo-oligosaccharide Idolax and disaccharide lactulose: Role of osmolarity versus fermentation of malabsorbed carbohydrate. _Dig. Dis. Sci_. 1998;43:2696\u20132707.\n\n91. Hyams J. Sorbitol intolerance: An unappreciated cause of functional gastrointestinal complaints. _Gastroenterology_. 1983;84:30\u201333.\n\n92. Smith M.M., Lifshitz F. Excess fruit juice consumption as a contributing factor in nonorganic failure to thrive. _Pediatrics_. 1994;93:438\u2013443.\n\n93. United States Department of Agriculture. Food and Nutrient Intake by Individuals in the United States by Sex and Age, 1994\u20131996. _NSF Report No. 96-2, NTIS Accession No. PB99-117251. National Technical Information Service, Springfield, VA_. 1999.\n\n94. Sitrin M.D. Nutrition support in inflammatory bowel disease. _Nutr. Clin. Prac_. 1992;7:53\u201360.\n\n95. Messori A., Trallori G., D'Albasio G., Milla M., Pacini F. Defined-formula diets versus steroids in the treatment of active Crohn's disease: A meta-analysis. _Scand. J. Gastroenterol_. 1996;31:267\u2013272.\n\n96. Schwab D., Raithel M., Hahn E.G. Enteral nutrition in acute Crohn's disease. _Z. Gastroenterol_. 1998;36:983\u2013995.\n\n97. Heuschkel R.B., Walker-Smith J.A. Enteral nutrition in inflammatory bowel disease of childhood. _J. Parenteral Enteral Nutr_. 1999;23:S29\u2013S32.\n\n99. Polk D.B., Hattner J.T., Kerner J.A. Improved growth and disease activity after intermittent administration of a defined formula diet in children with Crohn's disease. _J. Parenteral Enteral Nutr_. 1992;16:499\u2013504.\n\n100. Han P.D., Burke A., Baldassano R.N., Rombeau J.L., Lichtenstein G.R. Nutrition and inflammatory bowel disease. _Gastroenterol. Clin. N. Am_. 1999;28:423\u2013443.\n\n101. Lewis J.D., Fisher R.L. Nutrition support in inflammatory bowel disease. _Med. Clin. N. Am_. 1994;78:1443\u20131456.\n\n102. Lindor K., Fleming R., Burnes J., Nelson J., Ilstrup D., A randomized prospective trial comparing a defined formula diet, corticosteroids, and a defined formula diet plus corticosteroids in active Crohn's disease. Mayo Clin. Proc. 1992;67:328\u2013333.\n\n103. (Suppl.)Fukada Y., Kosaka T., Okui M., Hirakawa H., Shimoyama T. Efficacy of nutritional therapy for Crohn's disease. _J. Gastroenterol_. 1995;8:83\u201387.\n\n104. Westergaard H. Short bowel syndrome. In: Feldman M., Sleisenger M.H., eds. _Gastrointestinal and Liver Disease_. 6th ed. Philadelphia, PA: W. B. Saunders; 1998:1548\u20131560.\n\n105. Vanderhoof J.A., Langnas A.N. Short-bowel syndrome in children and adults. _J. Gastroenterol_. 1997;115:1767\u20131778.\n\n106. Mayer J.M., Schober P.H., Weissensteiner U., Hollwarth M.E. Morbidity and mortality of the short-bowel syndrome. _Eur. J. Pediatr. Surg_. 1999;9:121\u2013125.\n\n107. Vanderhoof J.A., Matya S.M. Enteral and parenteral nutrition in patients with short-bowel syndrome. _Eur. J. Pediatr. Surg_. 1999;9:214\u2013219.\n\n108. Goulet O. Short bowel syndrome in pediatric patients. _Nutrition_. 1998;14:784\u2013787.\n\n109. Carbonnel F., Cosnes J., Chevret S., Beaugerie L., Ngo Y., Malafosse M., Rolland P., Quintrec Y., Gendre J.P. The role of anatomic factors in nutritional autonomy after extensive small bowel resection. _J. Parenteral Enteral Nutr_. 1996;20:275\u2013279.\n\n110. Beyer P.L. Short-bowel syndrome. In: Skipper A., ed. _Dietitian's Handbook of Enteral and Parenteral Nutrition_. 2nd ed. Philadelphia, PA: Aspen Publishers; 1998:418\u2013436.\n\n111. Purdum P.P., III., Kirby D.F. Short-bowel syndrome: A review of the role of nutrition support. _J. Parenteral Enteral Nutr_. 1991;15:93\u2013101.\n\n112. Nightingale J.M. Management of patients with a short bowel. _Nutrition_. 1999;15:633\u2013637.\n\n113. Vanderhoof J.A., Langnas A.N. Short-bowel syndrome in children and adults. _Gastroenterology_. 1997;113:1767\u20131778.\n\n114. Vanderhoof J.A., Young R.J., Murray N., Kaufman S.S. Treatment strategies for small bowel bacterial overgrowth in short bowel syndrome. _J. Pediatr. Gastroenterol. Nutr_. 1998;27:155\u2013160.\n\n115. Wojdemann M., Wettergren A., Sternby B., Holst J.J., Lawsen S., Rehfeld J.F., Olsen O. Inhibition of human gastric lipase secretion by glucagon-like peptide-1. _Dig. Dis. Sci_. 1998;43:700\u2013805.\n\n116. Chen C.H., Stephens R.L., Jr., Rogers R.C. PYY and NPY: Control of gastric motility via action on R1 and Y2 receptors in the DVC. _Neurogastroenterol. Motil_. 1997;9:109\u2013116.\n\n117. Cuche G., Malbert C.H. Ileal short chain fatty acids inhibit transpyloric flow in pigs. _Scand. J. Gastroenterol_. 1999;34:149\u2013155.\n\n118. Nordgaard I., Hansen B.S., Mortensen P.B. Importance of colonic support for energy absorption as small-bowel failure proceeds. _Am. J. Clin. Nutr_. 1996;64:222\u2013231.\n\n119. Jeppesen P.B., Mortensen P.B. The influence of a preserved colon on the absorption of medium chain fat in patients with small bowel resection. _Gut_. 1998;43:478\u2013483.\n\n120. Jorgensen J.R., Clausen M.R., Mortensen P.B. Oxidation of short and medium chain C2\u2013C8 fatty acids in Sprague-Dawley rat colonocytes. _Gut_. 1997;40:400\u2013405.\n\n121. Weaver L.T., Austin S., Cole T.J. Small intestinal length: A factor essential for gut adaptation. _Gut_. 1991;32:1321\u20131323.\n\n122. Messing B., Crenn P., Beau P., Boutron-Rualt M.C., Rambaud J.C., Matchuansky C. Long-term survival and parenteral nutrition dependence in adult patients with the short bowel syndrome. _Gastroenterology_. 1999;117:1043\u20131050.\n\n123. (Suppl.)Suita S., Masumoto K., Yamanouchi T., Nagano M., Nakamura M. Complications in neonates with short bowel syndrome and long term parenteral nutrition. _J. Parenteral Enteral Nutr_. 1999;23:S106\u2013S109.\n\n124. Jeppesen P.B., Langholz E., Mortensen P.B. Quality of life in patients receiving home parenteral nutrition. _Gut_. 1999;44:844\u2013852.\n\n125. Cosnes J., Lamy P.H., Beaugerie L., LeQuintrec M., Gendre J.P., LeQuintrec Y. Adaptive hyperphagia in patients with postsurgical malabsorption. _Gastroenterology_. 1990;99:1814\u20131819.\n\n126. Bragg L.E., Thompson J.S., Rikkers L.F. Influence of nutrient delivery on gut structure and function. _Nutrition_. 1991;7:237\u2013243.\n\n127. Firmansyah A., Penn D., Lebenthal E. Isolated coloncyte metabolism of glucose, glutamine, n-butyrate and beta-hydroxybutyrate in malnutrition. _Gastroenterology_. 1989;97:622\u2013629.\n\n128. Ziegler T.R., Estvariz C.F., Jonas C.R., Gu L.H., Jones D.P., Leader L.M. Interactions between nutrients and peptide growth factors in intestinal growth, repair and function. _J. Parenteral Enteral Nutr_. 1999;23:S174\u2013S182.\n\n129. Biasco G., Callegari C., Lami F., Minarini A., Miglioli M., Barbara L. Intestinal morphological changes during oral refeeding in a patient previously treated with total parenteral nutrition for small bowel resection. _Am. J. Gastroenterol_. 1984;79:585\u2013588.\n\n130. Shin C.S., Chaudhry A.G., Khaddam M.H., Penha P.D., Dooner R. Early morphologic changes in the intestine following massive resection of the small intestine and parenteral nutrition therapy. _Surg. Gynecol. Obstet_. 1980;151:246\u2013250.\n\n131. Solhaug H. Morphometric studies of the small intestine following jejunoileal shunt operation. _Scand. J. Gastroent_. 1976;11:155\u2013160.\n\n132. Wilmore D.W. Growth factors and nutrients in the short bowel syndrome. _J. Parenteral Enteral Nutr_. 1999;23:S117\u2013S120.\n\n133. Lykins T.C., Stockwell J. Comprehensive modified diet simplifies nutrition management of adults with short-bowel syndrome. _J. Am. Diet. Assoc_. 1998;98:309\u2013315.\n\n134. Arrigoni E., Marteau P., Briet F., Pochart P., Rambaud J.C., Messing B. Tolerance and absorption of lactose from milk and yogurt during short-bowel syndrome in humans. _Am. J. Clin. Nutr_. 1994;60:926\u2013929.\n\n135. Beaugerie L., Cosnes J., Verwaerde F., Dupas H., Lamy P., Gendre J.P., LeQuintrec Y. Isotonic high-sodium oral rehydration solution for increasing sodium absorption in patients with short-bowel syndrome. _Am. J. Clin. Nutr_. 1991;53:769\u2013772.\n\n136. Thompson J.S. Epidermal growth factor and the short bowel syndrome. _J. Parenteral Enteral Nutr_. 1999;23:S113\u2013S116.\n\n137. Iijima S., Tsujinaka T., Kido Y., Hayashida Y., Ishida H., Homma T., Yokoyama H., Mori T. Intravenous administration of nucleosides and a nucleotide mixture diminishes intestinal mucosal atrophy induced by total parenteral nutrition. _J. Parenteral Enteral Nutr_. 1993;17:265\u2013270.\n\n138. Murphy M.S. Growth factors and the gastrointestinal tract. _Nutrition_. 1998;14:771\u2013774.\n\n139. (Suppl.)Bengmark S. Colonic food: Pre- and probiotics. _Am. J. Gastroenterol_. 2000;95:S5\u2013S7.\n\n140. Scolapio J.S. Effect of growth hormone, glutamine, and diet on body composition in short bowel syndrome: A randomized, controlled study. _J. Parenteral Enteral Nutr_. 1999;23:309\u2013313.\n\n141. Vanderhoof J.A., Kollman K.A., Griffin S., Adrian T.E. Growth hormone and glutamine do not stimulate intestinal adaptation following massive small bowel resection in the rat. _J. Pediatr. Gastroenterol. Nutr_. 1997;25:327\u2013331.\n\n142. (Suppl.)Scheppach W. Effects of short chain fatty acids on gut morphology and function. _Gut_. 1994;35:S35\u2013S38.\n\n143. Thompson J.S., Langnas A.N. Surgical approaches to improving intestinal function in the short-bowel syndrome. _Arch. Surg_. 1999;134:706\u2013709.\n\n144. (Suppl.)Goulet O., Jan D., Lacaille F., Colomb V., Michel J.L., Damotte D., Jouvet P., Brousse N., Faure C., Cezard J.P., Sarnacki S., Peuchmaur M., Hubert P., Ricour C., Revillon Y. Intestinal transplantation in children: Preliminary experience in Paris. _J. Parenteral Enteral Nutr_. 1999;23:S121\u2013S125.\n\n145. Bianchi A. Longitudinal intestinal lengthening and tailoring: Results in 20 children. _J. R. Soc. Med_. 1997;90:429\u2013432.\n\n146. Figueroa C.R., Harris P.R., Birdsong E., Franklin F.A., Georgeson K.E. Impact of intestinal lengthening on the nutritional outcome for children with short bowel syndrome. _J. Pediatr. Surg_. 1996;31:912\u2013916.\n\n147. Amii L.A., Moss R.L. Nutritional support of the pediatric surgical patient. _Curr. Opin. Pediatr_. 1999;11:237\u2013240.\n\n148. Thompson J.S. Intestinal transplantation. Experience in the United States. _Eur. J. Pediatr. Surg_. 1999;9:271\u2013273.\nCHAPTER 39\n\nNutrition and Liver Disease\n\nSARAH H. RIGBY and KATHLEEN B. SCHWARZ, Johns Hopkins Children's Center, Baltimore, Maryland\n\n## I. INTRODUCTION\n\nThe liver plays a central role in the metabolism of carbohydrates, proteins, lipids, vitamins, and minerals. As a result, diseases of the liver can disrupt this metabolism and adversely affect nutritional status. Acute and chronic liver diseases occur in both children and adults, and malnutrition often accompanies the disease. Many known and suspected factors contribute to malnutrition, and they will be discussed in the following sections. Liver transplantation is a viable option for many children and adults with end-stage liver disease, and good nutritional status is of utmost importance for these patients. The nutritional management of liver disease is an exciting field in which much research still needs to be done to address the many questions surrounding adequate and appropriate nutrition intervention for each disease and for the different stages of liver disease.\n\n## II. PATHOGENESIS OF MALNUTRITION\n\nA. Mechanisms of Malnutrition\n\n#### 1. ACUTE LIVER DISEASE\n\nAcute liver disease can result in weight loss due to nausea, vomiting, anorexia, and diarrhea. Increases in energy expenditure could also affect weight loss; however, it is unclear whether or not energy expenditure is altered in acute liver disease [1, 2]. Malnutrition is not normally a sequela of acute disease if premorbid nutritional status was normal. Acute liver disease may resolve or advance to chronic liver disease.\n\nFulminant hepatic failure is an acute condition that results in hepatic encephalopathy and coagulopathy. Hepatic encephalopathy must occur for the diagnosis of fulminant hepatic failure. With the development of hepatic encephalopathy, there are elevated levels of ammonia, a decrease in the plasma concentrations of the branched-chain amino acids, and an increase in the concentrations of the aromatic amino acids [3, 4]. Malnutrition can occur as a result of disordered metabolism of carbohydrates, proteins, and fats in addition to possible iatrogenic restrictions on nutrient intake.\n\n#### 2. CHRONIC LIVER DISEASE\n\nChronic liver disease can progress through stages of fibrosis to the end-stage disease, cirrhosis. Cirrhosis is irreversible and is defined by fibrosis, damage and regeneration of hepatocytes, altered hepatic architecture, and decreased hepatic function. Patients with chronic liver disease may present with cholestasis or with anicteric cirrhotic liver disease.\n\nMany patients with chronic liver disease are predisposed to malnutrition as a result of many factors, including inadequate intake and malabsorption [5, 6]. Decreased appetitite, nausea, vomiting, and restrictive diets can result in inadequate nutrient intake. Ascites itself can also affect intake, because the ascites may result in increased intra-abdominal pressure, which leads to early satiety. Cholestasis, cirrhosis, portal hypertension, and pancreatic insufficiency can all contribute to malabsorption [7]. Elevated energy expenditure and altered protein metabolism may also contribute to the development of malnutrition [1, 5, 8].\n\n### B. Specific Nutritional Issues and Inadequacies\n\n1. CARBOHYDRATES\n\nThe metabolism of carbohydrates in the liver includes glycogenesis, glycogenolysis, and gluconeogenesis. Glycogenesis is the process of storing excess glucose for use by the body at a later time. Glycogenolysis occurs when the body, which prefers glucose as an energy source, needs energy. The glycogen previously stored by the liver is broken down to glucose and dispersed throughout the body. When glycogen stores are depleted and glucose is required by the body, the liver initiates the production of glucose from amino acids, lactate, and glycerol in a process called gluconeogenesis.\n\nMost studies of energy metabolism in liver disease have focused on adults with cirrhosis. Results of such studies indicate that glucose intolerance and insulin resistance are common findings in this population; however, for most patients, these findings do not appear to be clinically significant [9, 10]. The production of glucose may be subnormal and the liver's capacity to store glycogen may be decreased as a result of the fibrosis that occurs with cirrhosis [11].\n\n#### 2. PROTEIN\n\nThe synthesis of certain proteins, including albumin, protease inhibitors, acute-phase reactants, storage and iron-binding proteins, and coagulation factors, occurs predominantly in the liver. Protein synthesis may not be adversely affected until the liver disease is late stage or in fulminant hepatic failure [12]. On the other hand, protein degradation is possibly increased even in earlier stages of disease [5, 13]. As a result, the provision of adequate protein to ensure positive nitrogen balance is imperative, because this will allow for regeneration of hepatocytes.\n\nIncreased plasma concentrations of aromatic amino acids and decreased plasma concentrations of branched-chain amino acids are seen in many patients with chronic liver disease [3, 4, 14]. The utilization of branched-chain amino acids to correct these abnormalities appears to be indicated only in fulminant hepatic failure and patients with chronic protein-induced encephalopathy [9, 15]. In patients with liver disease, standard amino acids in amounts sufficient to prevent negative nitrogen balance are recommended except in those patients who are truly protein intolerant [9].\n\n#### 3. FAT\n\nLipids are also synthesized and catabolized in the liver. The synthesis of lipids, lipogenesis, occurs with an influx of excess carbohydrate. The catabolism of fatty acids produces ketone bodies, which can be used by the brain and heart as a source of energy when glucose is not available or in short supply.\n\nPatients with cirrhosis derive a greater than normal percentage of their energy from the catabolism of fat, which may be attributed to lower stores of glycogen [11, 16]. This utilization of fat as an energy source resembles a body's reaction in a state of starvation [2, 11]. Steatorrhea, the mal-absorption of dietary fat, can occur in liver disease when bile acid flow to the intestines is insufficient. Biliary insufficiency can result in inadequate absorption of fat and other nutrients and decreased energy intake, which may lead to malnutrition.\n\n#### 4. FAT-SOLUBLE VITAMINS\n\nFat-soluble vitamins are absorbed after undergoing mi-cellar solubilization, a bile salt-dependent process, and are metabolized and\/or stored in the liver. Deficiencies occur with steatorrhea, because malabsorption of fat results in concomitant malabsorption of fat-soluble vitamins. Vitamin A deficiency can result in growth failure, anorexia, decreased resistance to infections, keratinization of epithelium, and night blindness. Vitamin D deficiency can result in rickets in children and osteomalacia in adults. Vitamin E deficiency can result in neurological disorders including peripheral neuropathy and myopathy. Vitamin K deficiency can impair clotting factor synthesis, which may result in a propensity for bleeding, bruising, and hemorrhage. The impaired synthesis of clotting factors may also result from hepatic failure. Supplementation with water-miscible fat-soluble vitamins may help correct these deficiencies; however, patients must be closely monitored for toxicity.\n\n#### 5. WATER-SOLUBLE VITAMINS\n\nChildren with chronic liver disease may be at risk for water-soluble vitamin deficiencies due to decreased intake and malabsorption; however, the incidence of these deficiencies has not been widely studied. Adults with alcoholic liver disease, and some with nonalcoholic liver disease, have been observed to exhibit deficiencies of folate, thiamin, niacin, and vitamin B12.\n\n#### 6. TRACE ELEMENTS\n\nAdults and children with liver disease may be at risk for metabolic derangements in zinc, selenium, chromium, and copper. Zinc deficiency may occur in liver disease as a result of poor dietary intake, increased urinary losses, decreased absorption, and altered metabolism [17, 18]. This deficiency may present as impaired night vision, macular degeneration, anorexia, changes in taste, skin lesions, depressed immune function, decreased protein synthesis, and slow wound healing [19]. Supplementation has been recommended and appears to be effective in correcting the deficiency [19].\n\nSelenium deficiency may exist in patients with liver disease [20, 21]; however, methods that have been used to assess deficiency are questionable [19]. If a true selenium deficiency is identified, supplementation should be administered because selenium deficiency may result in cardiomyopathy.\n\nCopper deficiency is unlikely in liver disease because the liver excretes copper in bile. Patients with cholestasis could potentially have copper toxicity as a result of decreased excretion. Wilson's disease, which causes liver disease due to increased copper levels, is discussed below.\n\n#### 7. IRON AND CALCIUM\n\nThe potential causes of malnutrition in adults and children with liver disease, as discussed above, may predispose to iron and calcium deficiencies. In addition, those patients with recurrent esophageal variceal bleeding are prone to be iron deficient. Calcium deficiency may occur with apparent vitamin D deficiency as a result of disordered metabolism rather than impaired calcium absorption [22, 23]. Supplementation with 25 (OH) vitamin D may be beneficial.\n\n## III. MAJOR LIVER DISEASES\n\nDiseases of the hepatobiliary system may be subdivided according to the location of the initial insult\u2014either in the liver (hepatocellular) or in the biliary system. However, as the disease process advances, both hepatocellular and biliary tract disease may result, regardless of the site of initial insult.\n\n### A. Hepatocellular\n\n1. ALCOHOLIC LIVER DISEASE\n\nThe intake and metabolism of alcohol play a major role in the development of alcoholic liver disease and associated malnutrition. Intake of alcohol may replace other foods in the diet and thus decrease the total intake of nutrients from other sources, thereby predisposing a person to inadequate nutrient intake. Alcohol oxidation takes precedent over lipid oxidation in the liver; as a result, lipids accumulate in the liver causing fatty liver. It has been suggested that acetaldehyde, a product of alcohol oxidation, may exert a hepatotoxic effect at high levels [24].\n\nAlcoholic liver disease is a progressive disease that is defined by three stages: hepatic steatosis (fatty liver), alcoholic hepatitis, and cirrhosis. Hepatic steatosis symptoms include fatigue, anorexia, nausea, vomiting, and hepatomegaly. Some patients in this stage may present with portal hypertension, bleeding esophageal varices, and low serum albumin [25, 26]. Abstinence from alcohol with adequate nutrient intake may reverse hepatic steatosis.\n\nAlcoholic hepatitis is an inflammation of the liver secondary to the toxic effect of alcohol. Hepatitis occurs in many, but not all, patients who initially present with hepatic steatosis. The incidence of excessive alcohol intake often precipitates hepatitis. Symptoms are similar to those for hepatic steatosis with the addition of fever and possibly acute weight loss. Low serum albumin and prolonged prothrombin time may also be present. The acute symptoms of alcoholic hepatitis can resolve with abstinence and adequate nutrition; alternately, it may progress to liver failure, hepatic encephalopathy, or cirrhosis.\n\nCirrhosis develops in only 10\u201320% of chronic alcoholics. Symptoms include fatigue, anorexia, nausea, and sometimes hepatic encephalopathy and bleeding esophageal varices. Malnutrition is common in patients with alcoholic hepatitis and cirrhosis, because anorexia, nausea, and vomiting lead to decreased intake. The substitution of alcohol for food results in decreased intake of nutrients [5, 27\u201329]. Malabsorption and reduced vitamin intake also adversely affect nutritional status. Ascites can cause premature satiety, thereby decreasing intake. Increased energy expenditure may also play a role in the development of malnutrition [1, 30]. Malnutrition has been associated with increased mortality in alcohol-related liver disease [31]; therefore, recognition of the disease and initiation of appropriate nutritional therapy is imperative.\n\nSpecific vitamin and mineral deficiencies occur in alcoholic liver disease. Thiamin deficiency (i.e., beriberi, Wernicke-Korsakoff syndrome) occurs as a result of decreased intake and the interference of alcohol with thiamin metabolism. Supplementation is not effective unless there is abstinence from alcohol. Beriberi is characterized by either peripheral neuropathy (dry beriberi) or edema and heart failure (wet beriberi). Severe thiamin deficiency, Wernicke's encephalopathy, is characterized by mental confusion, ataxia, and coma. Wernicke-Korsakoff syndrome, a progressive degenerative disease affecting memory, can be a sequela to Wernicke's encephalopathy. Patients with niacin deficiency, pellegra, present with psychosis, dermatitis, and diarrhea. Alcohol also interferes with the metabolism of vitamin B6 and folate, which can result in neurological abnormalities and anemia, respectively.\n\nNutritional therapy involves abstinence from alcohol and provision of adequate nutrients. Intake may be affected by symptoms mentioned previously, as well as by the palatability of the diet provided in a hospital setting [9]. Restrictions on protein and sodium may be initiated; however, protein restriction is not recommended unless a patient has chronic protein-induced encephalopathy [9]. For those patients unable to take adequate nutrition, the initiation of supplemental feedings is recommended.\n\n#### 2. VIRAL HEPATITIS\n\nViral hepatitis is an inflammation of the liver secondary to direct or indirect damage due to viral infection, including but not limited to hepatitis A, B, C, D, and E; Epstein-Barr virus; and cytomegalovirus. Viral hepatitis can result in acute and chronic disease states. Chronic hepatitis is defined by hepatic inflammation that persists for greater than 6 months in combination with abnormally elevated liver enzymes. The severity of chronic hepatitis ranges from elevated liver enzymes without other symptoms to hepatic failure.\n\nHepatitis A virus is usually transmitted via a fecal\u2013oral pathway from contaminated water or food. Nausea, vomiting, anorexia, and abdominal pain may occur, thereby placing the patient at risk for inadequate nutritional intake. Hepatitis A causes acute hepatitis without subsequent chronic disease or carrier state.\n\nHepatitis B virus is transmitted through the exchange of blood or other body fluids. This virus can cause acute or chronic hepatitis, cirrhosis, hepatic failure, or a carrier state. Hepatitis D virus occurs only in the presence of hepatitis B and is therefore thought to be transmitted via similar routes. Hepatitis D can present as a coinfection with hepatitis B or as a superimposed infection in a patient who is a viral hepatitis B carrier. Patients may become carriers of D virus, have acute and chronic disease, or present with fulminant hepatic failure.\n\nHepatitis C virus is transmitted via blood and blood products. Hepatitis C virus can also cause acute or chronic hepatitis, cirrhosis, or a carrier state. Hepatitis E virus may be transmitted via a fecal\u2013oral pathway from contaminated water and may only cause acute infection. Non-A, non-B hepatitis is transmitted via contaminated water and transfusions. It can cause acute and chronic hepatitis, fulminant hepatic failure, or a carrier state.\n\nEpstein-Barr virus causes infectious mononucleosis, and hepatitis is often a symptom of this disease. The hepatitis usually resolves without progression. Cytomegalovirus is usually asymptomatic in adults but children often are affected by complications. Transmission can occur during delivery from mother to infant or later to the infant via breast milk. Transfusions have also been indicated in transmission of this virus. It can cause jaundice and hepatitis in the newborn; however, progression to cirrhosis is uncommon.\n\nMedically, the treatment goal is to reduce hepatic inflammation using steroids or antiviral drugs if needed. Patients with end-stage liver disease due to hepatitis B virus or hepatitis C virus may be candidates for liver transplant; however, reccurrence of the virus is common [32]. Nutritionally, provision of adequate intake for all stages is the primary goal because symptoms may predispose patients to decreasing intake. Patients with chronic hepatitis are more at risk for developing malnutrition due to long-term inadequate intake and hepatic dysfunction. Cirrhosis may develop and cause cholestasis, which occurs when bile flow is obstructed and bile salts accumulate in the liver. As previously discussed, malabsorption occurs with the potential for the development of malnutrition. Chronic hepatitis may also lead to portal hypertension and ascites. Ascites can cause early satiety in patients, thereby decreasing nutritional intake. Sodium-restricted diets are often initiated for patients with ascites in an effort to decrease fluid retention, and sodium restriction may decrease the palability of the diet, further reducing intake [9].\n\nFulminant hepatic failure and occasionally chronic hepatitis result in hepatic encephalopathy. This is a state in which plasma ammonia levels are elevated, plasma branched-chain amino acids are decreased, and plasma aromatic amino acids are elevated. Nutritional goals in hepatic encephalopathy include provision of adequate energy to prevent catabolism and provision of sufficient protein for positive nitrogen balance. Patients with hepatic encephalopathy as a result of fulminant hepatic failure may better tolerate branched-chain amino acids as a source of protein; however, the majority of cirrhotic patients with hepatic encephalopathy can tolerate normal levels of standard protein [9].\n\n#### 3. AUTOIMMUNE HEPATITIS\n\nAutoimmune hepatitis is an inflammation of the liver with unknown etiology. It is defined by the presence of periportal hepatitis, elevated gammaglobulin levels, and autoantibodies, and on the exclusion of viral hepatitis and other hepatic disorders [33]. Autoimmune hepatitis typically presents in young women and its etiology is unknown. Symptoms may include nausea, vomiting, jaundice, fatigue, and hepatomegaly. Weight loss is not a common finding. Treatment involves the use of prednisone and azathioprine. Long-term side effects of prednisone in children include stunting, and in adults, osteopenia. Supplementation with calcium and vitamin D is recommended. Liver transplantation is considered for patients who have failed medical therapy; however, the disease may recur after transplant [34, 35].\n\n#### 4. NONALCOHOLIC STEATOHEPATITIS\n\nNonalcoholic steatohepatitis is a disease that presents with similar features of alcoholic liver disease in patients who are not alcoholics. Patients are commonly obese females who have hyperlipidemia and hyperglycemia [36, 37]. Symptoms may include fatigue, abdominal pain, and hepatomegaly. Nutritional therapy focuses on weight loss and control of hyperlipidemia and hyperglycemia. Unfortunately, improvement of nutritional status may not affect progression of the disease.\n\n#### 5. PREGNANCY-RELATED HEPATIC DISORDERS\n\nIntrahepatic cholestasis of pregnancy usually presents later in pregnancy with pruritis and increased levels of bile acid [38]. Steatorrhea as a result of cholestasis may, as previously discussed, lead to malabsorption. Maternal longterm health is not affected by intrahepatic cholestasis of pregnancy and the disease usually resolves after delivery. Conversely, intrahepatic cholestasis of pregnancy appears to cause increased risk of prematurity, fetal distress, and stillbirths [38, 39]. Nutritional goals are aimed at providing sufficient energy to promote growth and development of the fetus and at preventing fat-soluble vitamin deficiencies.\n\nLiver disease often occurs as a complication of preeclampsia, which is diagnosed by the presence of hypertension and proteinuria. A related entity is the HELLP syndrome, characterized by hemolysis, elevated liver enzymes, and low platelets. Symptoms include nausea, vomiting, and fatigue. Management includes close monitoring of the mother and fetus and provision of adequate nutrition; however, preeclampsia may prevent fetal growth secondary to hypertension-induced placental damage [40, 41]. This syndrome can progress to a fatal outcome as a result of a spontaneous rupture of the liver.\n\nAcute fatty liver of pregnancy occurs during the third trimester and patients may present with nausea, vomiting, ascites, hypoglycemia, preeclampsia, coagulopathy, and encephalopathy. Hepatic failure can lead to maternal and fetal death, therefore early diagnosis and subsequent delivery are imperative. Complete recovery is expected after delivery [42].\n\n#### 6. NEONATAL HEPATITIS\n\nNeonatal hepatitis is a cholestatic disease for which all other known causes of cholestasis, including infectious, metabolic, genetic, and toxic, have been ruled out. Patients present with hyperbilirubinemia and many present with hepatosplenomegaly and failure to thrive. Cholestasis is a result of decreased bile flow and the accumulation of substances normally excreted in bile. The decreased availability of bile in the intestines can lead to growth failure because bile is needed for the formation of micelles, which aid in the absorption of long-chain triglycerides. Treatment for growth failure includes administration of medium-chain triglycerides, which are absorbed directly into the portal vein without the aid of bile. Medium-chain triglyceride oil does not contain linoleic acid nor does it carry fat-soluble vitamins, so a source of essential fatty acids must be supplemented. Two to 4% of total energy intake from a source of essential fatty acids is recommended to prevent a deficiency [43].\n\n#### 7. GENETIC DEFECTS IN BILE ACID METABOLISM\n\nCholestasis and chronic degeneration of the liver can occur in patients with genetic defects in bile acid metabolism. Enzyme deficiencies result in an inability to produce primary bile acids, which leads to the accumulation of toxic intermediaries in the pathway producing bile acids. Medical treatment includes attempted inhibition of bile production and stimulation of bile secretion to decrease toxin accumulation. Nutritional therapy includes use of medium-chain triglyceride oil and supplementation of essential fatty acids, if needed.\n\n#### 8. METABOLIC LIVER DISEASE\n\na. Glycogen Storage Diseases.: Glycogen is the primary storage form of glucose, and patients with glycogen storage diseases have deficiencies of various enzymes involved in the metabolism of glycogen. There are at least 12 different types of glycogen storage disease but only three predominantly involve the liver.\n\nGlycogen storage disease I (von Gierke's disease) is the most common and is due to inadequate glucose-6-phosphatase activity. This enzyme hydrolyzes glucose-6-phosphate to produce free glucose. Infants present with hypoglycemia, hepatomegaly, and growth failure. Hypoglycemia occurs in the fasting state because glycogen cannot be converted to glucose. Hyperlipidemia, lactic acidemia, and metabolic acidosis occur in many patients as well.\n\nNutritionally, the goal is to provide an almost continuous supply of glucose to prevent hypoglycemia. Infants and young children with glycogen storage disease I require frequent (every 2\u20133 hours) daytime feedings and continuous feeds overnight. The provision of a fructose- or galactose-free formula for infants may be beneficial because these sugars are converted to glucose-6-phosphate [44]. Older children and adults can include cornstarch with meals to avoid frequent feedings. Cornstarch, highly branched chains of glucose, is slowly broken down into glucose and provides a steady supply of glucose to the body during fasting periods. Glycogen storage disease I does not normally result in hepatic failure or cirrhosis; however, hepatic adenomas develop in many patients. Glycogen storage disease I is an indication for liver transplant and does not appear to recur in patients with transplants [45\u201348].\n\nGlycogen storage disease III (Forbes' disease) is a deficiency of amylo-1,6-glucosidase glycogen debranching enzyme, which leads to glycogen accumulation and decreased glucose release. Patients present with hypoglycemia, hepatomegaly, and growth failure and may have muscle weakness and myopathy. This disease is less severe than glycogen storage disease I in that gluconeogenesis can occur via other pathways; however, glycogen storage disease III may eventually progress to fibrosis and cirrhosis. Some patients may need cornstarch therapy for better glycemic control, but in general, a high-protein, low-carbohydrate diet is recommended [49, 50]. This will provide usable substrates for gluconeogenesis and will decrease the storage of glycogen. The presence of cirrhosis may indicate a need for liver transplantation, which corrects the metabolic disorder [51, 52].\n\nGlycogen storage disease IV (Andersen's disease) is a deficiency of the branching enzyme 1,4-glucan-6-glycosyl-transferase (amylopectinosis) in which glycogen and amylo-pectin accumulate in the liver and other organs. Hypoglycemia is not common, but symptoms include hepatomegaly, growth failure, and hypotonia. Glycogen storage disease IV commonly progresses to cirrhosis. Appropriate dietary therapy is unclear, and both high-carbohydrate and high-protein diets have been suggested [50]. Liver transplantation has been performed successfully in patients with glycogen storage disease IV [45].\n\nb. Tyrosinemia.: Tyrosinemia is caused by a deficiency of fumarylacetoacetate hydrolase, an enzyme in the tyrosine degradation pathway. Metabolic intermediaries that accumulate in this disease are toxic. Infants may present with vomiting, cirrhosis, ascites, hypophosphatemic rickets, and coagulopathy. Older patients may experience renal and neurological involvement.\n\nNutritional therapy consists mainly of restriction of tyrosine and phenylalanine to prevent accumulation of toxic intermediaries. Sufficient energy is required to prevent catabolism and promote growth. A high-carbohydrate, low-protein diet is recommended; however, appropriate dietary management does not slow the progression of liver disease [53, 54]. Liver transplantation corrects the enzyme deficiency and can reverse any renal or neurological damage [48, 53\u201358].\n\nc. Urea Cycle Defects.: Urea cycle defects are a result of absolute or relative deficiencies in enzymes required for protein degradation. These defects can result in hyperammonemia with intake of protein, which may be exacerbated by metabolic stress. Infants present with vomiting, respiratory distress, lethargy, and coma secondary to hyperammonemia. Permanent damage to the nervous system occurs if hyperammonemia persists.\n\nTreatment includes reducing ammonia levels with drugs and alteration of diet. In the acute stage when ammonia levels are high, the goal is to reduce those levels. Tube feedings with minimal protein, high energy, and additional arginine are recommended. If unable to feed enterally, parenteral nutrition without amino acids should be initiated. Amino acids or protein can be reintroduced slowly after ammonia levels have decreased.\n\nChronically, therapy is aimed at maintaining levels of ammonia less than 50 \u03bcml and to provide sufficient protein for growth and development. A low-protein diet, in addition to sodium benzoate, which increases excretion of nitrogen, are standard therapies. Supplementation with arginine, a conditionally essential amino acid, is necessary except in patients with arginase deficiency because it also increases excretion of nitrogen [59\u201361]. Liver transplantation has been successfully performed in patients with urea cycle disorders [62].\n\nd. Byler's Syndrome.: Byler's syndrome, also known as progressive familial intrahepatic cholestasis, is a disease in which there is apparently a defect in the canicular secretion of bile acids. These children have normal synthesis of bile acids but a decreased bile acid secretion in bile [63]. The accumulation in the liver of the bile acids is hepatotoxic. Cholestasis, pruritis, and failure to thrive in the first few months of life are typical presentations of this disease. Rickets may develop as a result of steatorrhea secondary to decreased bile acids. Fibrosis, cirrhosis, and hepatic failure develop in most patients. If cirrhosis has not developed, surgical therapy may be considered. A partial biliary diversion allows bile to drain into a stoma and can improve symptoms [64, 65]; however, liver transplant is needed if cirrhosis develops. Recurrence of Byler's disease post-transplant has not been reported [66, 67].\n\ne. Cystic Fibrosis.: Cystic fibrosis is a multisystem disease caused by altered sodium chloride transport through certain endothelial-lined organ systems, including the lungs, pancreas, and hepatobiliary system. Dysfunction of the hepatobiliary system can occur when thickened secretions cause obstructions. Many patients with cystic fibrosis can present with hepatobiliary complications including fatty liver, cirrhosis, cholelithiasis, common bile duct stenosis, portal hypertension, and hepatic failure 68, 69]. Decreased bile acids in the intestines will compound the malabsorption that is frequently associated with pancreatic deficiencies. Pancreatic enzymes are given as standard therapy for patients with cystic fibrosis and pancreatic dysfunction; however, the enzymes may not be as effective for patients with decreased bile flow. In the event of liver failure, transplantation is an option for patients with stable pulmonary function [70\u201373]. Cystic fibrosis is discussed in more detail in [Chapter 45.\n\nf. Wilson's Disease.: In Wilson's disease, copper metabolism is disrupted, resulting in accumulation of copper in the liver, brain, and kidneys. The mechanism of accumulation is unknown, but it is suggested that it is a result of a decrease in the amount of copper excreted in bile. Wilson's disease usually presents after the first few years of life as acute or chronic hepatitis. Cholestasis, cirrhosis, or liver failure may also occur along with neurological and psychiatric symptoms.\n\nA copper chelating drug, D-penicillamine, is the major therapy for this disease; however, a low-copper diet is also recommended. Foods with high copper levels such as shellfish, legumes, nuts, mushrooms, chocolate, and liver should be avoided.\n\nIf the disease progresses to liver failure, transplantation is recommended. Liver transplant has been performed successfully with post-transplant improvement in metabolic and neurological symptoms [67, 74\u201378].\n\ng. Galactosemia.: Galactosemia is an inherited disorder of galactose metabolism in which there is a deficiency of galactose-1-phosphate uridyltransferase. Galactose-1-phosphate accumulates in the liver and other organs and can cause end-stage liver disease and death if not treated. Newborns present with vomiting, diarrhea, growth failure, cataracts, and cholestasis when fed formulas containing galactose or lactose. Lactose, the predominant carbohydrate in mammalian milk, is a disaccharide formed from glucose and galactose. Treatment includes eliminating galactose from the diet. If galactose is excluded early enough, acute symptoms may resolve and long-term complications may be avoided.\n\nh. Hereditary Fructose Intolerance.: Hereditary fructose intolerance is the result of a deficiency of the enzyme fructose-1-phosphate aldolase, which causes fructose-1-phosphate to accumulate in the liver. Fructose-1-phosphate is a competitive inhibitor of phosphorylase, an enzyme that regulates the conversion of glycogen to glucose. With high levels of fructose-1-phosphate, the conversion of glycogen to glucose is decreased, resulting in lactic acidosis and hypoglycemia, which may precipitate seizures. Infants with hereditary fructose intolerance present with vomiting, diarrhea, cholestasis, and hepatomegaly. Excluding fructose and sucrose (the dissacharide of glucose and fructose) from the diet is standard treatment. Early treatment results in resolution of symptoms with a normal life expectancy.\n\ni. Alagille Syndrome.: Alagille syndrome is defined in part by the decreased number of interlobular (intrahepatic) bile ducts found in the liver. Patients typically present with congenital heart disease, bone defects, opthalmologic defect, distinct facial features, chronic cholestasis, pruritis, hypercholesterolemia, and xanthomas. Fat and fat-soluble vitamin malabsorption often occurs, which can lead to growth failure. The use of formulas containing medium-chain triglyceride oil may be required in addition to vitamin supplementation. The severity of fat malabsorption may necessitate supplemental tube feedings for infants or older children to provide sufficient energy to meet nutritional needs. Liver transplantation is an option for children who progress to end-stage liver disease [56, 79].\n\n### B. Biliary Tract\n\n1. BILIARY ATRESIA\n\nBiliary atresia results from a progressive degeneration of the biliary tract. The destruction results in obstructed bile flow, which leads to cholestasis, fibrosis, and cirrhosis. Surgical therapy consists of a Kasai procedure (hepatic portoenterostomy), in which there is a direct anastamosis of the liver with the small intestines in the hopes of creating an alternative path for bile drainage. This should be done soon after diagnosis, around 6\u20138 weeks of age, prior to development of advanced fibrosis. Biliary atresia is the leading cause of liver transplant in children [56, 80]. Transplant is standard therapy for patients in which the Kasai procedure has failed [80, 81].\n\nBiliary atresia results in decreased intraluminal bile acids, which leads to fat and fat-soluble malabsorption. Nutritional therapy includes infant formulas containing medium-chain triglyceride oils to optimize fatty acid absorption and, if needed, supplemental feeds to provide sufficient energy for growth. Maintenance of nutritional status is of utmost importance for patients awaiting liver transplantation [82, 83].\n\n#### 2. CHOLEDOCHAL CYSTS\n\nCholedochal cysts are formed by dilatation of the common bile duct and if untreated may result in biliary obstruction and cirrhosis. Infants can present with jaundice, vomiting, and acholic stools. Older patients may present with jaundice, abdominal pain, and abdominal mass. Definitive therapy is surgical, and complete excision of the cyst is recommended, both to prevent biliary cirrhosis and because the cyst is premalignant.\n\n#### 3. PRIMARY SCLEROSING CHOLANGITIS\n\nPrimary sclerosing cholangitis is characterized by inflammation and progressive destruction of the biliary tract. Patients may present with abdominal pain, jaundice, pruritis, steatorrhea, and hepatomegaly. The majority of patients with primary sclerosing cholangitis also have inflammatory bowel disease [84, 85]. Primary sclerosing cholangitis progresses despite medical therapy and liver transplantation is usually indicated [81, 86]; however, primary sclerosing cholangitis may recur after transplant [35].\n\n#### 4. CHOLEDOCHOLITHIASIS\n\nCholedocholithiasis occurs when gallstones obstruct the common bile duct. Obstruction of the flow of bile results in malabsorption of fat and fat-soluble vitamins and can result in cholecystitis and secondary biliary cirrhosis if untreated.\n\n#### 5. PRIMARY BILIARY CIRRHOSIS\n\nPrimary biliary cirrhosis is a chronic degenerative liver disease found predominantly in middle-aged women. The etiology of primary biliary cirrhosis is unknown, but it has been suggested that it may be secondary to an autoimmune process [87]. Thus, an inflammatory process causes gradual destruction of bile ducts, which leads to fibrosis and then cirrhosis. With progressive destruction of the bile ducts, there is a decrease in the secretion of bile into the intestinal lumen. This decrease, as mentioned previously, can result in fat and fat-soluble vitamin malabsorption. Other symptoms of primary biliary cirrhosis include fatigue, osteopenia, pruritis, hypercholesterolemia, and hyperlipidemia. Patients may be asymptomatic for years; however, most patients experience a slow progression of the disease with gradual development of symptoms [88, 89]. There is some evidence that resting metabolic rate increases as the disease progresses [90]. The use of medium-chain triglyceride oil can improve nutritional status when fat malabsorption is present [91]. Fat-soluble vitamin supplementation may be required to prevent deficiencies. Liver transplantation is an option for patients with advanced primary biliary cirrhosis; however, primary biliary cirrhosis may recur after transplant [35, 92\u201395].\n\n## IV. NUTRITIONAL MANAGEMENT OF LIVER DISEASES\n\nA. Assessment of Nutritional Status\n\nConsistent and careful monitoring of nutritional status is necessary to identify and treat malnutrition. However, this monitoring can be challenging, because many parameters routinely used for assessment are affected by the liver disease itself (Table 1). Body weight may be elevated due to the fluid retention of ascites, edema, and hepatosplenomegaly, whether or not these symptoms are appreciable [96, 97]. True body weight decreases, therefore, may not be evident [98]. A better parameter for assessment in children may be height, because a reduced height for age can be indicative of chronic malnutrition [99]. However, use of height may be misleading because liver disease can affect skeletal development resulting in baseline short stature [96, 100].\n\nTABLE 1\n\nEvaluation of Nutritional Assessment Parameters in Childhood Liver Disease\n\n_Source:_ Reprinted from Novy, M. A., and Schwarz, K. B. (1997). Nutritional considerations and management of the child with liver disease. _Nutrition_ **13 (3)** 177\u2013184, with permission from Elsevier Science.\n\nSome of the more accurate parameters for assessment may be triceps skinfold and arm muscle and fat measurements, despite the possible existence of unappreciable edema [96]. The upper extremities are less prone to fluid accumulation, and measurements may be more accurate if done regularly by the same person.\n\nVisceral proteins, including albumin, transferrin, prealbumin, and retinol-binding protein, are typically used to assess nutritional status because they are synthesized in the liver. Alterations in serum levels can occur with liver disease as a result of malnutrition or as a result of decreased hepatic function [101, 102]. Albumin synthesis may be preserved until end-stage liver disease [12], but synthesis will be affected by inadequate protein intake [7].\n\nNitrogen balance, measured using 24-hour urine collection and diet recall, can be inaccurate due to inability of the liver to detoxify ammonia, which results in decreased production of urea secondary to hepatic dysfunction. A creatinine\u2013height index may be useful in assessing muscle mass if renal function is normal [103].\n\nCell-mediated immunity as a marker for nutritional status is assessed using the total lymphocyte count and delayed cutaneous hypersensitivity [104, 105]. However, total lymphocyte count can be decreased and hypersensitivity tests can be abnormal in patients with liver disease [106].\n\nSubjective global assessment, if combined with diet history, calorie intake records, physical examination with assessment for vitamin deficiencies, and arm anthropometric measurements may be the most useful method of determining malnutrition in patients with liver disease [106, 107].\n\n### B. Acute Liver Failure\n\nNutritional management of acute liver disease, assuming normal nutritional status prior to the injury, is primarily supportive. Malnutrition does not normally develop; therefore, maintenance of current nutritional status is the focus. Provision of the Recommended Dietary Allowances (RDA) for energy and protein should be adequate in most cases. Fulminant hepatic failure is an exception, because hepatic encephalopathy with hyperammonemia makes nutritional management more challenging. The provision of adequate protein to prevent catabolism is the goal, and recommended protein amounts for infants and adults are 1.0\u20131.5 g\/kg\/day [7, 108]. Protein recommendations for children and teenagers are 0.5\u20131.0 g\/kg\/day [7]. If standard protein intake worsens the encephalopathy, it may be necessary, in addition to medical therapy, to utilize branched-chain amino acid administration to provide sufficient protein. Vegetable protein is another alternative that is sometimes better tolerated; however, intake of adequate protein is difficult due to the volume of foods that must be consumed to meet needs. Energy recommendations for adults range from 25 to 35 kcal\/kg\/day with all stages of encephalopathy [108]. For children, at least the RDA level for energy should be provided, with higher amounts given as needed to prevent hypoglycemia and protein catabolism [7]. Ideal body weight or dry weight should be used in determining energy and protein needs when fluid overload is evident or suspected. Maintenance of nutritional status and prevention of deterioration are important aspects of the supportive care given to patients with fulminant hepatic failure as transplantation may be the only option for many patients.\n\n### C. Chronic Liver Diseases\n\n1. CHOLESTATIC LIVER DISEASE\n\nNutritional goals of chronic liver disease are to provide adequate energy and protein to prevent energy deficit and protein catabolism and to promote regeneration of hepatic cells [109]. Infants and children with cholestatic liver disease require at least 125\u2013150% of the RDA for adequate energy intake, and adults with cholestatic liver disease may also have increased energy requirements [43, 90, 96, 110, 111]. Infants need at least 3 g\/kg\/day of protein [43], while the minimum protein intake for children and adults should be the RDA level for age. For both children and adults, energy from fat is often provided in the form of medium-chain triglyceride oil to optimize absorption [91, 112, 113]. As previously mentioned, medium-chain triglyceride oil does not contain linoleic acid or transport fat-soluble vitamins, so a source of essential fatty acids must be supplemented. Two to 4% of total energy intake from a source of essential fatty acids is generally needed to prevent deficiency [43].\n\nSupplementation of water-miscible fat-soluble vitamins is standard therapy for patients with cholestatic disease; however, close monitoring of serum levels is needed to prevent toxicity. Doses for correcting deficiencies will vary, but, in general, children should receive 5000\u201325,000 IU vitamin A\/day, 3\u20135 \u03bcg 25-hydroxyvitamin D\/kg\/day, 15\u201325 IU vitamin E\/day given as alpha tocopherol polyethylene glycol-succinate, and 2\u20135 mg vitamin K twice a week [114]. Supplements of calcium and phosphorus may also be indicated using 25\u2013100 mg\/kg\/day of elemental calcium and 25\u201350 mg\/kg\/day of phosphorus [114]. Copper and manganese are excreted by the liver in bile; therefore, supplementation should be avoided in cholestatic patients.\n\nFor adults with cholestatic liver disease and deficiencies, supplementation with 5000\u201325,000 IU vitamin A\/day and 5\u201310 mg\/day of oral vitamin K are recommended [114a]. An intake of 1000\u20131500 mg calcium\/day, and, in patients with deficiencies, 400 IU of vitamin D\/day is recommended to help prevent osteoporosis [115]. Zinc, selenium, and iron should be supplemented if deficiencies are found [116].\n\nSodium and fluid restrictions may be necessary if ascites and edema are problematic, and this often decreases the palability and therefore the intake of the diet. If patients are unable to consume adequate energy and protein by mouth, supplemental enteral feedings are beneficial [117\u2013120]. Providing enteral feedings at night and allowing patients to eat during the day is one option that can help meet estimated nutritional needs. If patients are unable to eat or are eating poorly, bolus feeds can be given at meal times or after attempts to eat. Patients with ascites or severe glycemic changes may benefit from continuous feedings to decrease the sensation of fullness and better maintain blood glucose levels.\n\n#### 2. ANICTERIC CIRRHOTIC LIVER DISEASE\n\nPatients with noncholestatic liver disease should not be supplemented with vitamins or minerals unless a deficiency has been documented. Sodium and fluid restrictions may be necessary, with the potential for a resultant decrease in dietary intake, as mentioned above.\n\n### D. End-Stage Liver Disease\n\nCirrhosis is the end stage of liver disease, and almost all liver diseases may potentially progress to this stage. Cirrhosis can be stable or unstable, with varying presentations. Energy and protein recommendations for adults with uncomplicated cirrhosis are 25\u201330 kcal\/kg\/day and 0.8 g protein\/kg\/day [9, 108]. Adults with complicated cirrhosis require 25\u201335 kcal\/kg\/day and 1.0\u20131.2 g protein\/kg\/day [9, 108]. Infants and children with end-stage liver disease need an estimated 150% of the RDA for energy and at least 3 g protein\/kg\/day [43, 111, 121].\n\nFat-soluble vitamin deficiencies should be identified prior to supplementation in patients with noncholestatic liver disease [9]. Water-soluble vitamin deficiencies are common in patients with alcoholic liver disease and may be present in patients with other liver diseases as a result of malabsorption or hepatic dysfunction. Supplementation of these vitamins is recommended to correct the deficiency.\n\nHepatic encephalopathy may occur in patients with end-stage liver disease and some patients may benefit from supplementation with branched-chain amino acids [122, 123]. Formulas with branched-chain amino acids may be more beneficial than partially hydrolyzed protein in improving nutritional status of children with end-stage liver disease [116]. However, for both children and adults, standard protein formulations are appropriate unless a patient is protein intolerant [9, 121].\n\n## V. PREPARATION FOR LIVER TRANSPLANT\n\nIndications for liver transplant include fulminant hepatic failure, progressive hepatic degenerative diseases, metabolic diseases, and end-stage liver disease. Several surgical options are available for transplant candidates. Orthotopic whole-organ liver transplant is possible when the donor organ is size compatible with the recipient. Split-liver transplants allow for two patients to receive reduced-sized grafts. Living-related transplants involve transplanting the donor's left lateral segment, often to a small child [56, 67, 80, 124, 125].\n\nPatients awaiting liver transplantation are often malnourished as a result of the many disease conditions previously discussed. Improving nutritional status prior to transplant is imperative because malnutrition affects morbidity and mortality post-transplant [124, 126\u2013129]. Identification of degree of malnutrition is useful in determining aggressiveness of nutritional support [99]; however, as previously discussed, parameters of assessing nutritional status are affected by the liver disease itself and limitations must be recognized [97]. Optimal nutritional support may not completely reverse malnutrition prior to transplant [130], but can improve nutritional status [131]. Improvement of nutritional status may improve immune system response and decrease the possibility of pre- and post-transplant infections and complications [98].\n\nPost-transplant nutritional support may also be necessary to reverse malnutrition or continue pre-transplant improvement of nutritional status [121, 132]. Studies have shown that children do exhibit catch-up growth post-transplantation, yet linear growth resumes at a slower rate [130, 132\u2013135]. In addition to increased survival, post-transplant quality of life improves in comparison to life pre-transplant [136, 137].\n\n## VI. SUMMARY AND CONCLUSIONS\n\nThe liver is an important organ in the metabolism and utilization of all macro- and micronutrients. The presence of liver diseases can have a profound effect on nutritional status. Nutrition interventions play a key role in the overall management of liver diseases.\n\nReferences\n\n1. Shanbhogue R.L., Bistrian B.R., Jenkins R.L., Jones C., Benotti P., Blackburn G.L. 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Holt R.I., Miell J.P., Jones J.S., Mieli-Vergani G., Baker A.J. Nasogastric feeding enhances nutritional status in paediatric liver disease but does not alter circulating levels of IGF-I and IGF binding proteins. _Clin. Endocrinol. (Oxf.)_. 2000;52:217\u2013224.\n\n119. Charlton C.P., Buchanan E., Holden C.E., Preece M.A., Green A., Booth I.W., Tarlow M.J. Intensive enteral feeding in advanced cirrhosis: Reversal of malnutrition without precipitation of hepatic encephalopathy. _Arch. Dis. Child_. 1992;67:603\u2013607.\n\n120. Kearns P.J., Young H., Garcia G., Blaschke T., O'Hanlon G., Rinki M., Sucher K., Gregory P. Nutritional therapy for alcoholic hepatitis: Are we there yet [abstract]? _Hepatology_. 1992;16:846.\n\n121. Protheroe S.M. Feeding the child with chronic liver disease. _Nutrition_. 1998;14:796\u2013800.\n\n122. O'Keefe S.J., Ogden J., Dicker J. Enteral and parenteral branched chain amino acid-supplemented nutritional support in patients with encephalopathy due to alcoholic liver disease. _J. Parenteral Enteral Nutr_. 1987;11:447\u2013453.\n\n123. The Italian Multicenter Study GroupMarchesini G., Dioguardi F.S., Bianchi G.P., Zoli M., Bellati G., Roffi L., Martines D., Abbiati R. Long-term oral branched-chain amino acid treatment in chronic hepatic encephalopathy. A randomized double-blind casein-controlled trial. _J. Hepatol_. 1990;11:92\u2013101.\n\n124. Kelly D.A. Current results and evolving indications for liver transplantation in children. _J. Pediatr. Gastroenterol. Nutr_. 1998;27:214\u2013221.\n\n125. Alonso M.H., Ryckman F.C. Current concepts in pediatric liver transplant. _Semin. Liver Dis_. 1998;18:295\u2013307.\n\n126. Beath S., Brook G., Kelly D., McMaster P., Mayer D., Buckels J., Improving outcome of liver transplantation in babies less than 1 year. Transplant. Proc. 1994;26:180\u2013182.\n\n127. Halliday A.W., Benjamin I.S., Blumgart L.H. Nutritional risk factors in major hepatobiliary surgery. _J. Parenteral Enteral Nutr_. 1988;12:43\u201348.\n\n128. Shepherd R.W., Chin S.E., Cleghorn G.J., Patrick M., Ong T.H., Lynch S.V., Balderson G., Strong R. Malnutrition in children with chronic liver disease accepted for liver transplantation: Clinical profile and effect on outcome. _J. Paediatr. Child Health_. 1991;27:295\u2013299.\n\n129. Moukarzel A.A., Najm I., Vargas J., McDiarmid S.V., Busuttil R.W., Ament M.E., Effect of nutritional status on outcome of orthotopic liver transplantation in pediatric patients. Transplant. Proc. 1990;22:1560\u20131563.\n\n130. van Mourik I.D., Beath S.V., Brook G.A., Cash A.J., Mayer A.D., Buckels J.A., Kelly D.A. Long-term nutritional and neurodevelopmental outcome of liver transplantation in infants aged less than 12 months. _J. Pediatr. Gastroenterol. Nutr_. 2000;30:269\u2013275.\n\n131. Chin S.E., Shepherd R.W., Cleghorn G.J., Patrick M., Ong T.H., Wilcox J., Lynch S., Strong R. Preoperative nutritional support in children with end-stage liver disease accepted for liver transplantation: An approach to management. _J. Gastroenterol. Hepatol_. 1990;5:566\u2013572.\n\n132. Shepherd R.W. Pre- and postoperative nutritional care in liver transplantation in children. _J. Gastroenterol. Hepatol_. 1996;11:S7\u2013S10.\n\n133. Rodeck B., Melter M., Hoyer P.F., Ringe B., Brodehl J., Growth in long-term survivors after orthotopic liver transplantation in childhood. Transplant. Proc. 1994;26:165\u2013166.\n\n134. Sarna S., Sipila I., Jalanko H., Laine J., Holmberg C., Factors affecting growth after pediatric liver transplantation. Transplant. Proc. 1994;26:161\u2013164.\n\n135. Holt R.I., Broide E., Buchanan C.R., Miell J.P., Baker A.J., Mowat A.P., Mieli-Vergani G. Orthotopic liver transplantation reverses the adverse nutritional changes of end-stage liver disease in children. _Am. J. Clin. Nutr_. 1997;65:534\u2013542.\n\n136. Burdelski M., Nolkemper D., Ganschow R., Sturm E., Malago M., Rogiers X., Brolsch C.E. Liver transplantation in children: Long-term outcome and quality of life. _Eur. J. Pediatr_. 1999;158(Suppl. 2):S34\u2013S42.\n\n137. Zitelli B.J., Gartner J.C., Malatack J.J., Urbach A.H., Miller J.W., Williams L., Kirkpatrick B., Breinig M.K., Ho M., Pediatric liver transplantation: Patient evaluation and selection, infectious complications, and lifestyle after transplantation. Transplant. Proc. 1987;19:3309\u20133316.\nF.\n\nOther Major Diseases\nCHAPTER 40\n\nNutrition and Renal Disease\n\nD. JORDI GOLDSTEIN1, 1IVonyx, Inc., Reno, Nevada\n\nBETH McQUISTON2, 2Park Ridge, Illinois\n\n## I. INTRODUCTION\n\nHistorically, nutrition science research in renal disease has been mainly directed toward determining if nutrients modulate progressive renal disease, influence metabolic abnormalities associated with established disease, and serve as a therapy for the nutritional consequences associated with chronic renal failure. While initially descriptive in nature, current studies are mechanistic in scope, providing insight as to how nutrients interact with cellular functions. These data allow a sophistication in understanding the interaction between nutrients and how they might modulate mechanisms of renal disease. Clinical research completed in the past decade has established a relationship between nutritional status and outcome (i.e., morbidity and mortality) in patients who require dialysis therapy. These data impact the clinical arena through increased attention to nutrition assessment, monitoring, and intervention as components of medical care.\n\nIt is the intent of this chapter to summarize the past and present nutrition science base underlying current nutrition recommendations and treatment for the patient with chronic renal insufficiency, the patient requiring renal replacement therapy, and care during post-transplantation.\n\n## II. CHRONIC RENAL FAILURE\n\nAs of 1997, there were 304,083 patients in the United States receiving treatment for end-stage renal disease (ESRD) [1]. The approximate rate of new growth for the period of 1992\u20131997 was 6% per year [2]. The financial burden for ESRD in 1997 by all payers was estimated at $15.64 billion, an increase from $14.55 billion in 1996 [2]. Chronic renal failure is a disease syndrome where progressive, irreversible losses of the excretory, endocrine, and metabolic capacities of the kidney occur due to kidney damage. The mechanisms as to how and why kidney disease progresses to end stage, even following treatment of the initial insult, continue to be studied. Examples of primary diseases leading to chronic renal failure include diabetes, hypertension, glomerulonephritis and cystic disease. The nature of the disease and the portion of the nephron that becomes damaged determines the symptoms, diagnosis, and treatment [3]. Chronic renal failure (CRF) progresses slowly over time [4]. Once the disease progresses to end stage, continuance of life requires the initiation of renal replacement therapy or kidney transplantation.\n\nConsideration of the role that certain nutrients might have in causing or contributing to progressive renal disease has been an active area of research for more than 20 years. In the study of a relationship between nutrients and progressive disease, dietary protein [5\u20139] has been the most extensively studied, and more recently fatty acids [10\u201312].\n\nIt has long been recognized in rats with experimental CRF that high-protein diets lead to increased proteinuria, mortality, and renal damage, whereas restriction of dietary protein results in improvement [4]. In humans, low-protein diets initiated early in chronic renal failure have been shown to ameliorate clinical uremic symptoms by decreasing the formation of nitrogenous compounds [13\u201315]. However, the beneficial effect of dietary protein restriction with or without ketoacid analogues or essential amino acid supplementation on human renal disease progression [13\u201320] has not been consistently reproducible [4, 8].\n\nDuring the 1970s, implementation of dietary protein restriction as low as 0.3 grams of dietary protein per kilogram body weight per day (g\/kg\/day) with supplementation of ketoacid analogue or essential amino acid for the patient with chronic renal insufficiency was not uncommon [13\u201320]. The rationale for the use of these preparations was the observation that ammonium nitrogen derived from urea hydrolysis in the gut was capable of aminating the ketoacid analogue into essential amino acid that could then be used as protein by the body [13, 14, 16\u201319]. This not only exploits a valuable source of nitrogen [18], but permits daily essential amino acid requirements to be met despite the adherence to an almost nitrogen-free diet [19]. The advantage was to avoid the side effects of malnutrition, which were often observed in patients trying to adhere to a low-protein diet alone [13\u201315, 17]. However, these diets were difficult to adhere to and patients often became malnourished. The benefits of these diets had not been demonstrated, and there was no information related to how dietary protein restriction might be a beneficial intervention for the failing kidney.\n\nIn 1982, Brenner and colleagues [21] reported that ingestion of protein-rich meals in animals, and infusion of high amino acid nutrient solutions in humans, led to renal hemodynamic changes indicative of increased renal workload. They observed elevated total renal blood flow, glomerular pressures, and glomerular filtration rate. This led to the hypothesis that high-protein diets increase renal workload and might be detrimental for the patient with chronic renal failure. The role of the renin-angiotensin system in modulating renal hemodynamics is now well established. There is increasing evidence that protein intake may alter the activity of the renin-angiotensin system and the renal production and urinary excretion of eicosanoids [5, 22]. Ongoing studies have determined that an increase in protein intake stimulates the secretion of several hormones including glucagon, vasopressin, eicosanoids, dopamine, and renin.\n\nIt has been postulated that renal progression may result from increased glomerular perfusion and elevated intraglomerular capillary pressures once a critical reduction in renal mass has occurred. In this hypothesis, global sclerosis causes diseased glomeruli to stop functioning. Remaining glomeruli undergo compensatory hyperperfusion, which results in intraglomerular hypertension leading to injury, progressive sclerosis, and total loss of glomeruli. How intraglomerular hypertension develops and modifies glomerular cell functions is not clear. In rats with subtotal nephrectomy, protein restriction has been found to suppress the early hemodynamic changes and minimize later glomerular changes. In rats with a remnant kidney, a low-protein diet reduces glomerular hypertension and glomerulosclerosis. The reverse is observed with high-protein diets. In addition, the class of eicosanoids secreted with high-protein feeding promotes renal vasoconstriction [4, 5, 21, 22].\n\nDue to the inconsistent nature of results of clinical studies, the utility of dietary protein restriction for the treatment of progressive renal disease remains controversial. In 1990, a multicenter trial, the Modification of Diet in Renal Disease (MDRD) study [21, 22] was implemented to clarify the effectiveness of protein-restricted diets. Two groups of patients were studied. Group A patients had glomerular filtration rates (GFRs) in the range of 25\u201355 mL\/min\/1.73 m2 and were randomized to a diet of \u22651 g protein\/kg\/day or 0.6 g protein\/kg\/day. Mean blood pressures were maintained at 105 or 92 mm Hg. Group B patients had GFRs in the range of 13\u201324 mL\/min\/1.73 m2 and assigned diets that were 0.6 g\/kg\/day or 0.3 g protein\/kg\/day plus a ketoacid supplement. Blood pressure goals were the same. Renal function was monitored for 2.2 years by measuring 125I-iothalamate renal clearance.\n\nStudy results demonstrated that neither the low-protein diet or blood pressure control decreased the loss of GFR. Therefore, prescription of a low-protein diet alone is not sufficient to slow progression. Trends that were observed included a beneficial affect of low-protein diet for patients with proteinuria that exceeded 1 g\/day. Also, patients with polycystic kidney disease did not demonstrate any response to therapy [4]. A recent study [23] reports that a very low protein diet, providing 0.3 g protein, 35 kcal, 5\u20137 mg inorganic phosphorus\/kg\/day, supplemented with ketoacid analogues or essential amino acid, calcium carbonate, iron, and multivitamins, preserved nutritional status, corrected uremic symptoms, and deferred initiation of dialysis in 239 patients followed for 13 years. There was no correlation found between patient outcome and treatment with the low-protein, supplemented diet. To date, dietary protein restriction has not been demonstrated to stop the progression of chronic renal failure. However, the majority of studies suggest that at the least, protein restriction offers clinical benefits for the metabolic consequences of uremia.\n\nResearch interest has also been directed toward dietary fatty acids, in response to a growing body of evidence implicating eicosanoid metabolism in the pathophysiology of progressive renal disease [24]. It has been demonstrated that altering the availability of essential fatty acids can influence the natural course of several important diseases in the mammalian organism [25]. For example, epidemiological studies of the Dutch, Japanese, and native Greenland Eskimo populations attribute their low incidence of heart disease to a fish diet high in omega-3 fatty acids [25]. Several investigations exploring the effects of polyunsaturated fatty acid supplemented diets in experimental immune and nonimmune models of renal disease have been reported. Beneficial observations have included an improved lipid profile [26, 27], prolonged survival [28, 29], improved renal function [29], improved proteinuria [27, 30], and delay in progression [31].\n\nDietary essential fatty acids are the direct precursors to the biologically diverse and potent class of compounds, the eicosanoids. Several chronic inflammatory and renal diseases are characterized in part by an overproduction of eicosanoids [24]. These facts suggest that manipulation of dietary fatty acids might contribute a therapeutic influence by altering gene expression and proinflammatory and other activated pathways in disease processes [32\u201334]. Fatty acid manipulation has been reported to modify macrophage function, production of vasoactive substances, and membrane signal transduction. Alteration of phospholipid fatty acid composition in cell membranes achieved by manipulation of cell medium fatty acid content has been reported to affect many cellular properties. Examples include cell membrane fluidity, receptor binding, cell-mediated transport, ion channels, eicosanoid formation, and intracellular calcium concentration [24, 33, 35, 36].\n\nDespite the encouraging experimental results that have been reported, human trials have had inconsistent results [37\u201344]. The exception is in patients with IgA nephropathy. Renal disease progression was reduced when patients were administered a fish oil supplement enriched with eicosapentaenoic acid and docosahexaenoic acid [10, 11]. A recent report indicates that docosahexaenoic acid, not eicosapentaenoic acid, inhibits mesangial cell proliferation and may offer a clinical benefit during acute phases or relapses of glomerulopathies [12].\n\nThere are not enough data at present to justify any specific dietary recommendations regarding fatty acid modification for patients with chronic renal insufficiency. However, due to the controversial and suggestive nature of the literature on dietary protein, current recommendations include dietary protein restriction (see Table 1).\n\nTABLE 1\n\nNutrition Recommendations for Patients with Chronic Renal Failure Not Undergoing Dialysis _a_ , _b_ , _c_\n\naGFR above 4\u20135 mL\/min\/1.73 m2 and less than 25 mL\/min\/1.73 m2.\n\nbThe protein intake is increased by 1.0 g\/day of high-biologic-value protein for each gram per day of urinary protein loss.\n\ncWhen recommended intake is expressed per kilogram body weight, this refers to the patient's normal weight, as determined from the NHANES data, or adjusted body weight.\n\ndRefers to percentage of total energy intake (diet plus dialysate). If triglyceride levels are very high, the percentage of fat in the diet may be increased to about 40% of total calories; otherwise, 30% of total calories is preferable.\n\neThese dietary recommendations are considered less crucial than the others. They are only emphasized if the patient has a specific disorder that may benefit from this modification or has expressed interest in this dietary prescription and is complying well to more important aspects of the dietary treatment.\n\nfShould be primarily complex carbohydrates.\n\ngCan be higher in patients who have greater urinary losses.\n\nhPhosphate binders (aluminum carbonate or hydroxide, or calcium carbonate, acetate, or citrate) often are needed to maintain normal serum phosphorus levels.\n\niDietary intake usually must be supplemented to provide these levels. Higher daily calcium intakes are commonly ingested because of the use of calcium binders of phosphate.\n\nj\u2265 10 mg\/day for males and nonmenstruating females; \u2265 18 mg\/day for menstruating females.\n\nkVitamin K supplements may be needed for patients who are not eating and who receive antibiotics.\n\n_Source:_ Reprinted with permission from Kopple, J. (1997). Nutrition management of nondialyzed patients with chronic renal failure. _In_ \"Nutritional Management of Renal Disease\" (J. D. Kopple and S. G. Massey, Eds.), p. 495.\n\n## III. NUTRITION FOR THE PATIENT REQUIRING RENAL REPLACEMENT THERAPY\n\nA. Renal Dialysis\n\nSince the introduction of dialysis in the 1960s, significant advances have dramatically changed the way ESRD patients are managed and treated [45, 46]. The pioneering efforts by Scribner, Boen, Tenckhoff, and Popovich in the late 1950s to the mid-1960s led to what we know today as dialysis therapy. Scribner, the forefather of hemodialysis, established an acute dialysis program at the University of Washington in Seattle in the late 1950s. He later developed the Scribner shunt, and with Babb, succeeded in building the first home hemodialysis machine. Similarly, the efforts by Boen, Tenckhoff, and Popovich developed peritoneal dialysis access and paved the way for this treatment modality [46].\n\nDialysis can be defined as a process whereby the solute composition of a solution is altered by exposure to another solution through a semipermeable membrane [46, 47]. Small to mid-molecular weight solutes and water molecules can pass through the membrane, whereas larger molecular solutes cannot [47]. Utilization of hemodialysis or peritoneal dialysis is based on clinical condition, lifestyle, and psychosocial considerations. Psychosocial considerations include quality-of-life factors, depression [48\u201350], and compliance with dietary, fluid, and treatment modalities [51].\n\nOf paramount importance is the viability of a patent dialysis access. Access to the bloodstream is both the lifeline and the ''Achilles heel\" of dialysis. A well-functioning access is crucial to provide adequate dialysis and optimal medical care of the ESRD patient regardless of treatment modality. If hemodialysis is selected, blood is removed from the arterial side of the patient's access, via an arteriovenous fistula, arteriovenous graft, or catheter. It is processed through a dialyzer and then returned through the venous side of the access. This process occurs repeatedly over the course of several hours. Whenever possible, the order of preference for placement of different blood vessel access is fistula, graft, and, as a temporizing measure or if the aforementioned accesses are not viable options, a cuffed tunneled central venous catheter [52]. A fistula, the anastomosis of an artery to a vein, is the preferred and best form of hemodialysis access due to longevity and low incidence of complications. If creating a fistula is not a viable option, synthetic tubing, such as polytetrafluoroethylene, can be used to create an arteriovenous graft in the arm or the thigh [46].\n\nIn peritoneal dialysis, the body's own peritoneal membrane serves as the semipermeable membrane across which solutes and water pass. Generally, 1\u20133 liters of a dextrose salt solution is infused into the peritoneal cavity. The rate of solute transport, the type of number of peritoneal dialysis exchanges, and solution dwell times vary among patients. A method to determine membrane function and the optimal peritoneal dialysis method for each patient is the peritoneal equilibration test [53]. Rate of solute transport ranges from low to high and can be measured via this test. Peritoneal solute and solvent movement rates vary among patients and, over time, may vary even within the same patient [54]. A variety of types of peritoneal dialysis exist with the two main types being continuous ambulatory peritoneal dialysis and continuous cycling peritoneal dialysis. Similar to the decision to choose hemodialysis or peritoneal dialysis, which type of peritoneal dialysis is performed is dependent on lifestyle and clinical considerations.\n\nWhether a patient receives hemodialysis or peritoneal dialysis, adequacy of dialysis is of paramount importance to optimize patient nutritional status and, ultimately, morbidity and mortality. The landmark National Cooperative Dialysis Study provided a longitudinal evaluation of hemodialysis patients dialysis regimen over 1 year's time. Participants were divided into four treatment groups in order to assess the minimum dialysis time that would provide adequate outcome [55]. Ultimately, inadequate protein intake was associated with inadequate dialysis (as defined by an elevated blood urea nitrogen level) and increased mortality [56]. These results have since been confirmed and expanded [57\u201363]. Similar results were obtained in a larger, retrospective study conducted by Owen _et al._ [64]. The records of 13,473 patients were evaluated to determine the effects of blood urea nitrogen reduction during dialysis and nutritional adequacy on mortality. The odds ratio for death increased dramatically with poor nutritional status (as assessed by a low serum albumin) and inadequate dialysis (as assessed by urea reduction ratio). Specifically, patients with a serum albumin of 3.5\u20133.9 and 3.0\u20133.4 had odds ratios for death of 1.48 and 3.13, respectively. Patients with a urea reduction ration less than 60% also had a higher odds ratio for death compared with a urea reduction ratio of 65\u201369%. Hakim _et al._ [65] conducted a prospective study on 130 hemodialysis patients over 3 years. Differences in delivered dose of dialysis were evaluated with a 25% increase in delivered dialysis dose being correlated with a decrease in annual mortality rate. In addition, increased protein intake, as measured by protein catabolic rate, was demonstrated with increased dose of dialysis.\n\nThe methods and recommendations with how dialysis is performed as well as dietary recommendations for ESRD patients have been transformed by these results. Historically, dialysis prescriptions were empirically prescribed (Table 2). Currently, however, dialysis prescriptions are individualized and closely monitored. Although in the past, blood urea nitrogen levels or urea reduction ratios have been used to assess adequacy of dialysis, with the advent of computerized programs, urea kinetic modeling techniques should be used to assess, measure, and individualize the adequacy and quality of dialysis [66, 67]. _Kt\/V_ is a measure of the dose of dialysis given in a single treatment where _K_ is the dialyzer urea clearance, _t_ is the total treatment time, and _V_ is the total volume within the body that urea is distributed [68]. _Kt\/V_ and creatinine clearance are two methods to determine adequacy of dialysis and should be calculated using formal urea kinetic modeling. Hemodialysis patients should receive a single pool _Kt\/V_ of at least 1.2 per treatment [66]. Continuous ambulatory peritoneal dialysis, nightly intermittent dialysis, and continuous cycling peritoneal dialysis patients need to obtain a weekly Kt\/Vurea of 2.0, 2.2, and 2.3 with a weekly creatinine clearance of 60, 66, and 64 L\/1.73 m2, respectively [67]. One notable exception is for continuous ambulatory peritoneal dialysis patients that are low transporters. For this subgroup of patients, creatinine clearance should be at least 50 L\/week\/1.73 m2 with a weekly creatinine clearance of 2.0 or greater [69]. Because nutritional status and morbidity and mortality are closely linked with adequacy of dialysis, these guidelines should be rigorously followed.\n\nTABLE 2\n\nHistory of Diet Changes for Hemodialysis Patients\n\n_Source:_ Adapted from Jacobs, L., Rubens-Kenler, S., and Dwyer, J. (1996). _Topics Clin. Nutr._ **12,** 6\u201317.\n\n### B. Comorbid Conditions Associated with ESRD\n\nESRD patients have a higher mortality compared with age-matched controls in the general population [70]. Moreover, most ESRD patients have comorbid conditions that further increase their relative risk of death such as cardiovascular disease, bone disease, secondary hyperparathyroidism, anemia, and malnutrition.\n\n#### 1. CARDIOVASCULAR DISEASE\n\nCardiovascular disorders have a high prevalence in the ESRD patients and account for approximately 50% of deaths for this patient population [70\u201371]. Common cardiovascular abnormalities in these patients include heart disease, congestive heart failure, left ventricular hypertrophy, hypertension, and peripheral vascular disease [70]. Malnutrition, oxidative stress, and genetic components may play a synergistic effect on rapid development of atherosclerosis [72]. While a variety of comorbid conditions contribute to cardiovascular disease (CVD) in this population, renal failure also adds independent cardiovascular risk factors such as derangements in lipoprotein metabolism and elevated homocysteine levels [70, 73].\n\n#### 2. ABNORMALITIES IN LIPOPROTEIN METABOLISM\n\nHypertriglyceridemia with decreased high-density lipoprotein (HDL) cholesterol occurs in 50\u201370% of ESRD patients [74]. Due to the high prevalence of CVD in ESRD, these patients need to be screened for hyperlipidemia and treated [75]. Even in patients having normal HDL cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride levels, very low density lipoprotein (VLDL) cholesterol levels may be elevated due to impaired catabolism. Moreover, ESRD patients tend to have increased lipoprotein(a) levels and increased small dense LDL particles [74, 76, 77]. Furthermore, these lipoprotein particles are subject to increased oxidation due to the effect of uremic toxins and may cause vascular injury [78]. Thus, even in the appearance of normal lipoprotein levels, significant lipoprotein derangements may increase the risk of CVD. In addition to pharmacological interventions, dietary modification to lower lipid levels may be attempted; however, dietary restriction of fat\/cholesterol has not been definitely shown to improve CVD risk in this patient population, and ensuring adequate energy and protein intake may take precedence.\n\n#### 3. HYPERHOMOCYSTEINEMIA\n\nHomocysteine may directly damage vascular endothelium [47], and hyperhomocysteinemia has been found to increase the odds ratio for vascular events [79] and has been linked with an increased mortality rate in ESRD patients. The landmark study by Wilcken _et al._ [80] and confirmed by others [81\u201384], demonstrated higher plasma homocysteine levels in ESRD patients compared with the general population. With kidney failure, there is decreased metabolism of homocysteine by the renal parenchyma. In addition, increased losses of the necessary cofactors for homocysteine metabolism, such as folate, vitamin B6, and vitamin B12, may occur due to the dialysis process itself. Moreover, decreased intake of these vital nutrients may occur due to the dietary restrictions.\n\nVascular damage occurs due to generation of hydrogen peroxide and free radicals when homocysteine is oxidized to its disulfide form [85\u201387]. Other proposed mechanisms for vascular damage include impairment of endothelial-derived relaxing factor on nitric oxide production, damage to the endothelium by the inhibition of prostacyclin production (a prostaglandin antagonist of platelet aggregation), and the oxidative effects on LDL by homocysteine-induced hydrogen peroxide production [88\u201392]. Optimizing intake of folate, vitamin B12, and vitamin B6 intake has been demonstrated to decrease serum homocysteine levels by 30\u201350% [93] and may have cardioprotective effects [80, 83, 84, 91\u201393]. Supplementation of folate, vitamin B6, and B12 should be considered [80, 91, 92].\n\nIn addition to diabetes and blood pressure control, management of hyperlipidemia and homocystemia may help decrease CVD risk [70\u201372, 91, 93\u201395].\n\n#### 4. BONE DISEASE\n\nBone disease diagnoses in ESRD include osteitis fibrosa, osteomalacia, osteosclerosis, and osteoporosis [96] and are associated with increased mortality. Contributors to renal osteodystrophy include decreased conversion of vitamin D to the active form, 1,25-dihydroxycholecalciferol, by the kidney, resulting in low serum calcium and elevated parathyroid hormone levels. In addition, serum phosphorus levels are typically elevated due to decreased renal excretion. Alterations in vitamin D metabolism and increased phosphorus levels result in the development of secondary hyperparathyroidism and contribute to increased mortality [97\u2013100].\n\nA landmark study by Block _et al._ [98] clearly delineated the adverse consequences of elevated parathyroid hormone, phosphorus, and the product of the serum calcium level multiplied by the serum phosphorus level (Ca \u00d7 P). Retrospective analysis of data from more than 6400 hemodialysis patients demonstrated that elevated serum phosphorus, Ca \u00d7 P, and parathyroid hormone levels were independently associated with increased morbidity and mortality. After adjustment for comorbid conditions, patients with a phosphorus level > 6.5 mg\/dL had a 27% higher death risk than patients with a serum phosphorus between 2.4 and 6.5. Of note, 39% of these patients had a serum phosphorus > 6.5, thus placing them at increased risk of mortality. Similarly, elevated Ca \u00d7 P was associated with increased mortality, reaching statistical significance with Ca \u00d7 P >72. Specifically, patients with a Ca \u00d7 P >72 had a 34% higher risk of death relative to those with a Ca \u00d7 P between 42 and 52. Parathyroid hormone levels > 975 pg\/mL also were associated with an increased mortality rate. Studies conducted by Lowrie and Lew [57] found that patients with a serum phosphorus between 7 and 11 mg\/dL had a twofold higher relative risk of death than patients with phosphorus levels between 5 and 7 mg\/dL.\n\nThus, reduction of serum phosphorus, Ca \u00d7 P, and parathyroid hormone levels is imperative to prevent or improve many pathological consequences, such as left ventricular hypertrophy, anemia, immune suppression, hypertriglyceridemia, vascular complications, and renal osteodystrophy [71, 97, 98].\n\nHistorically, the emphasis has been placed on preventing high bone turnover in this patient population; however, a recent study by Atsumi _et al._ [101] illustrates negative consequences of low bone turnover. Male patients with parathyroid hormone levels in the lowest tertile (mean parathyroid hormone 32.9 pg\/mL) had a 31% prevalence of vertebral fractures compared with only a 13% and 17% incidence in those patients in the middle (mean parathyroid hormone 116 pg\/mL) and highest (mean parathyroid hormone 502 pg\/mL) tertile, respectively. Thus, both high and low bone turnover are associated with adverse outcomes.\n\nMost ESRD patients will require management of secondary hyperparathyroidism with dietary phosphorus restriction, phosphate binder use, and oral or intravenous supplementation of vitamin D or vitamin D analogues. Serum parathyroid hormone, calcium, and phosphorus levels should be closely monitored for ESRD patients, especially those who are administered vitamin D therapy.\n\nA variety of oral and intravenous vitamin D or vitamin D analogs are available. The dose must be titrated based on individual patient need to prevent over- or undersuppression of parathyroid hormone. Phosphate binders, such as calcium acetate, calcium carbonate, or sevelamar hydrochloride, should be individualized and prescribed with meals in order to be effective in controlling serum phosphorus levels.\n\n#### 5. ANEMIA\n\nThe majority of ESRD patients have decreased hematocrit (Hct) and hemoglobin (Hgb) levels because they are unable to synthesize adequate amounts of erthyropoietin, a hormone produced by renal tubular cells [102, 103]. They also have increased blood loss from dialysis procedures. The resulting anemia is treated with recombinant human erythropoietin (rHuEPO) and iron [104]. Prior to 1989, the main therapeutic treatment option for treating anemia in this patient population repeated blood transfusions. This was not ideal and contributed to iron overload [105]. With the advent of rHuEPO, renal anemia can be effectively treated and alleviated in the majority of ESRD patients [106\u2013108]. If left untreated, a wide variety of adverse events occur, such as cardiac enlargement, ventricular hypertrophy, angina, congestive heart failure, malnutrition, and impaired immunological response [109\u2013118]. Uncorrected, anemia is associated with increased mortality [119, 120]. Thus, the development of rHuEPO dramatically and significantly improved the health and well-being of ESRD patients.\n\nRecommended hematocrit and hemoglobin target levels for the ESRD population are 33\u201336% and 11\u201312 g\/dL, respectively [121]. Some studies suggest higher levels may be desirable in certain patients [105, 122\u2013125]. Subcutaneous administration is preferred over intravenous for pre-ESRD and ESRD patients because lower doses of rHuEPO can be used to maintain hematocrit levels > 33% [106, 121, 126\u2013129]. Generally, for adult patients, 80\u2013120 units rHuEPO\/kg\/week in two to three doses is recommended for subcutaneous administration. If administered intravenously, generally 120\u2013180 units\/kg\/week rHuEPO should be given over three dialysis sessions [121]. However, the absolute dose depends on the individual patient response.\n\nMost patients on dialysis will receive recombinant human erythropoietin to promote synthesis of red blood cells and will require iron supplementation [104, 130\u2013132]. Although many factors may contribute to rHuEPO hyporesponsiveness, such as infections, chronic inflammation, secondary hyperparathyroidism, and occult blood loss, among others [108], the most frequent contributor is iron deficiency [108, 133, 134]. Current recommendations to assess iron stores and appropriate iron therapy are used to determine if serum transferrin saturation levels are greater than 20% and if serum ferritin levels are greater than 100 units [121]. Although various oral iron preparations are available, intestinal iron absorption may be impaired in dialysis patients [135, 136]. A trial of oral iron may be tried, particularly in peritoneal dialysis patients [137\u2013140]; however, for most hemodialysis and many peritoneal dialysis patients, intravenous iron supplementation is more effective in treating iron deficiency anemia [121, 130, 132, 141\u2013143]. If oral iron is to be used, 200 mg of elemental iron should be provided per day in two to three divided doses, preferably between meals and not with phosphate binders [121, 131]. If intravenous iron is to be used, a maintenance dose of 25\u2013100 mg per week is recommended [121, 131]. Prior to maintenance, a typical course of intravenous iron therapy is 1 g over 10 dialysis treatments (i.e., 100 mg per treatment). A variety of intravenous iron preparations are available in the United States and include iron dextran, iron gluconate, and\u2014soon to be available\u2014iron sucrose, also known as iron saccha-rate [144].\n\n#### 6. MALNUTRITION\n\nMalnutrition is an important and modifiable risk for mortality in the ESRD population [57, 64, 145\u2013151]. Causes of inadequate nutritional status in this population include anorexia, catabolic state, loss of amino acids during the dialysis process, and complement activation due to exposure to various dialyzer membranes [152].\n\nThe landmark National Cooperative Dialysis Study found the number of hospitalizations per year was highest in patients with the lowest protein intake as determined by protein catabolic rate [153]. Similarly, work conducted by Lowrie and Lew [57] on more than 14,000 hemodialysis patients found a strong association between low serum albumin and mortality. Of note, nearly 70% of patients had serum albumin levels of less than 4.0 g\/dL, and 15% had serum albumins less than 3.5 g\/dL. Patients with a serum albumin <2.5 g\/dL had a 20 times higher risk of death than patients with an albumin >4.0 g\/dL. Somewhat surprisingly, patients with albumin levels of 3.5\u20134.0 g\/dL still had a two times higher risk of death than patients with albumin levels >4.0 g\/dL. The importance of nutritional status was not universally accepted and recognized until these outcome studies were reported. Both individualized dialysis prescription and dietary guidelines are essential to optimize nutritional status of ESRD patients.\n\nNutritional assessment, monitoring, and interventions are critical to prescribe appropriate clinical nutrition therapies and, ultimately, to improve patient outcomes. Improvements in quality of life may be demonstrated with improved nutritional status [49, 154]. Similarly, poor nutritional status may be associated with decreased quality-of-life scores [50].\n\n### C. Nutrition Assessment\n\nBecause malnutrition is so strongly correlated with mortality, nutrition assessment and monitoring are vital. Registered dietitians are especially skilled in this area and should be consulted [155]. A complete nutrition assessment will include a number of parameters including clinical findings, body composition, weight, dietary assessment, and biochemical indices (Table 3).\n\nTABLE 3\n\nData Categories and Constituents Inherent to Nutritional Assessment\n\n_Source:_ Reprinted with permission from Goldstein, D. J. (1998). Assessment of nutritional status in renal disease. _In_ \"Handbook of Nutrition and the Kidney\" (W. E. Mitch and S. Klahr, Eds.), pp. 45\u201386. Lippincott Williams & Wilkins, Philadelphia, PA.\n\n#### 1. SUBJECTIVE GLOBAL ASSESSMENT\n\nSubjective global assessment is a method of history taking and physical examination that can be quickly and effectively performed to assess and monitor the nutritional status of patients [152, 156\u2013160]. The subjective global assessment was originally developed for use in surgical patients, but has since been validated for use in ESRD patients. Patients are assigned to a risk category based on subjective global assessment ratings. A low or downward trend in an subjective global assessment rating would suggest compromised nutritional status and require intervention.\n\n#### 2. ANTHROPOMETRICS\n\nCommon anthropometric measurements obtained in this population include height, weight, triceps skinfold, subscapular skinfold, arm circumference, abdominal circumference, calf circumference, knee height, and elbow breadth [161, 162]. Obtained measurements are compared to standardized percentiles; however, no current standards are available that are specific to the ESRD population.\n\nAnthropometric measurements can also be combined in the evaluation of nutritional status. For example, weight for height has been demonstrated to be a strong predictor of 12-month mortality in hemodialysis patients [163]. Mortality rate appears to decrease as a patient's weight for height increases. Similarly, patients with a higher body mass index (BMI) may have a lower mortality rate than those with a lower BMI [164]. Use of correct body weight is essential for patient assessment and for determination of dietary needs. A variety of definitions of weight have been used for nutritional assessment such as usual body weight, standard body weight, and ideal body weight. The current recommendation of the Nutrition Dialysis Outcomes Quality Initiative committee suggests use of the edema-free body weight (also known as dry weight) when the patient is between 90% and 115% median standard weight as determined by the National Health and Nutrition Examination Survey (NHANES) II weight tables (Table 4). If the patient's body weight is outside this range, adjusted edema-free body weight should be used (Fig. 1).\n\nTABLE 4\n\nAverage Weights for Men and Women, by Age and Height in the United States\n\n_Source:_ \"Clinical Practice Guidelines for Nutrition\" (1999). NFK-DOQL. National Kidney Foundation, New York. National Kidney Foundation\n\nFIGURE 1 Calculation of adjusted body weight. [Reprinted with permission from K\/DOQN nutrition in chronic renal failure: Adult guidelines (2000). _Am. J. Kidney Dis._ **35** (6), S17\u2013S104.]\n\n#### 3. DIETARY ASSESSMENT\n\nControversy exists on the best method for dietary assessment of this population. Some studies suggest that 7-day diet records are necessary [165], whereas others suggest that a 24-hour recall is just as accurate and may increase the likelihood of successful data collection in this group [166]. Assessment of the patient's typical food intake can identify areas of educational need as well as provide valuable information for individualizing dietary counseling. Nutrition screening is a first step in identifying nutrition-related issues in renal patients. Figure 2 provides an example of an excellent nutrition screening tool.\n\nFIGURE 2 DETERMINE checklist. [Reprinted with permission from Leung, J., and Dwyer, J. (1998). Renal DETERMINE nutrition awareness checklist for health care professionals. _J. Renal Nutr._ **2,** 95\u2013103.]\n\n#### 4. BIOCHEMICAL INDICES\n\nThe results of a variety of biochemical indices and calculations need to be integrated in order to evaluate nutritional status in ESRD patients. Table 5 categorizes a number of these parameters. The most widely used parameters are highlighted below.\n\nTABLE 5\n\nBiochemical Parameters for Assessing the Nutritional Status of the Patient with Renal Disease\n\n_A. Visceral protein stores_\n\nAlbumin\n\nPrealbumin (thyroxin binding prealbumin)\n\nRetinol-binding protein\n\nTransferrin (siderophilin)\n\nSomatomedin c (insulin-like growth factor 1)\n\nAcute-phase proteins (ceruloplasmin, complement components, c-reactive protein, fibrinogen)\n\nFibronectin\n\nPseudocholinesterase\n\nRibonuclease\n\nTotal protein\n\nAlbumin\/globulin ratio\n\n_B. Static (somatic) protein reserves_\n\nUrinary and serum creatinine\n\nCreatinine height index\n\n3-Methylhistidine\n\n_C. Other estimates of protein reserves_\n\nPlasma amino acid profiles\n\nProtein turnover studies\n\nBiochemical analysis of skeletal muscle\n\nNitrogen balance\n\n_D. Immune competence_\n\nTotal lymphocyte counts\n\nDelayed cutaneous hypersensitivity responses: _Candida,_ mumps, _Trichophyton,_ streptokinase-streptodornase (sksd), and purified protein derivative (ppd)\n\nSpecific immunoglobulin levels\n\nComplement proteins\n\n_E. Vitamins, mineral, trace element nutriture_\n\nSerum levels of water-soluble vitamins, fat-soluble vitamins, and specific minerals and trace elements\n\nNutrition physical examination\n\n_F. Fluid, electrolyte, and acid\u2013base status_\n\nSerum chemistries: sodium, potassium, calcium, phosphorus, bicarbonate, chloride, glucose\n\n_G. Indirect indices of renal function and dialysis adequacy_ Serum creatinine\n\nBlood urea nitrogen\n\n_H. Anemia_\n\nHemoglobin\n\nHematocrit\n\nMean cell corpuscular volume\n\nTotal iron-binding capacity\n\nSerum iron\n\nPercent transferrin saturation\n\nFerritin\n\nRed blood cell count\n\nReticulocyte count\n\nWhite blood cell count\n\n_I. Hyperlipidemia_\n\nSerum: cholesterol, triglycerides\n\n_J. Renal osteodystrophy_\n\nSerum: calcium, vitamin D, alkaline phosphatase\n\n_Source:_ Reprinted with permission from Goldstein, D. J. (1998). Assessment of nutritional status in renal disease. _In_ \"Handbook of Nutrition and the Kidney\" (W. E. Mitch and S. Klahr, Eds.), pp. 45\u20136. Lippincott Williams & Wilkins, Philadelphia, PA. Lippincott Williams & Wilkins\n\na. Serum Albumin.: A low serum albumin is strongly correlated with increased morbidity and mortality and may be related to poor nutritional intake [57, 59, 61, 70]. Ideally, serum albumin levels should be maintained greater than 4.0 g\/dL. Of note, hospital admissions have been demonstrated to be shorter with higher serum albumin levels [61]. Although serum albumin may provide information regarding the patient's nutritional status, it is adversely influenced by the coexistence of chronic inflammatory states, liver disease, pancreatic disease, and nephrotic syndrome, among others, and must be interpreted accordingly [70, 96].\n\nb. Serum Prealbumin.: Serum prealbumin has a half-life of only 2 days, making it a potentially more sensitive marker of protein status than albumin, and is another predictor of morbidity and mortality in patients with ESRD. More specifically, a prealbumin <30 mg\/dL may indicate malnutrition [149]. Patients with ESRD initiating dialysis with a prealbumin > 30 mg\/dL were found to have a higher observed and expected survival than those who did not [167].\n\nOf note, serum prealbumin levels are related to the level of renal function and may increase with declining renal function (due to decreased catabolism), regardless of nutritional status [168]. Evaluation of prealbumin levels may be most helpful once a patient's kidney function is in a steady state [169, 170]. Similar to albumin, prealbumin is also adversely influenced by infection, inflammation, and comorbid conditions.\n\nc. Serum Cholesterol.: Low serum cholesterol levels may indicate protein energy malnutrition or be a reflection of other comorbid conditions. A low serum cholesterol, <150 mg\/dL, may be a higher risk factor for mortality than an elevated serum cholesterol level in this patient population [59, 171, 172]. A desirable serum cholesterol level is between 150 and 200 mg\/dL. Importantly, cholesterol levels may be affected by acute infection, malnutrition, and nephrotic syndrome, or elevated by use of glucocorticoids [96]. If serum cholesterol levels are substantially elevated, patients with ESRD may benefit from a fibric acid analogue or statin therapy [71, 173]. However, the long-term safety of lipid-lowering agents in this population has not been established [71].\n\nd. Serum Creatinine and Creatinine Index.: Serum creatinine and creatinine index measures can be useful in evaluation of patients with ESRD who have negligible renal function. Creatinine is formed at a relatively constant rate from muscle tissue and thus may be used in a patient with a stable GFR to assess muscle mass. The creatinine index is defined as the creatinine synthesis rate [152] and is directly related to skeletal muscle mass.\n\nA low serum creatinine or creatinine index may be indicative of decreased muscle mass and\/or low dietary protein intake [174] and is correlated with increased mortality [59, 171, 175]. Specifically, assuming minimal residual renal function, mortality risk increases with serum creatinine levels of <9\u201311 mg\/dL [59, 169, 171, 176] and\/or a creatinine index that is low or declining [177]. In patients with significant residual renal function, serum creatinine may not be as useful in evaluating nutritional status. In this case, serum creatinine will be lower because the kidney will be excreting higher levels of creatinine into the urine.\n\nAnother technique to assess protein status in patients with ESRD is protein catabolic rate. Protein catabolic rate measures net protein degradation and protein intake in stable hemodialysis and peritoneal dialysis patients and is a derivative of urea kinetic modeling. It reflects the amount of protein that a patient is catabolizing per day and is useful in reviewing patient food intake. In a patient that is nutritionally stable and not catabolic, the protein catabolic rate equals the dietary protein intake [178, 179]. Low values may be reflective of poor nutritional intake and are strongly correlated with increased morbidity and mortality [145, 153, 180]. Of note, protein catabolic rate can be falsely elevated if a patient is catabolic and falsely low if a patient is anabolic [152].\n\n### D. Nutrition Recommendations\n\n1. PROTEIN AND ENERGY\n\nIn general, the dietary needs of patients on chronic hemodialysis are high in energy and protein, and restricted in phosphorus, sodium, potassium, and fluid. Nitrogen balance studies on hemodialysis patients have demonstrated a 30% higher nitrogen appearance rate during dialysis versus the interdialytic period [181, 182], indicating that the dialysis process itself increased net protein catabolism. The dialysis process causes loss of amino acids and stimulates the inflammatory process, thus increasing protein breakdown [96]. Based on studies providing protein and energy supplementation to hemodialysis patients, 1.2 g protein\/kg body weight and 35 kcal\/kg body weight are general recommendations [183]. Peritoneal dialysis patients generally have a more liberalized diet, higher in protein, sodium, potassium, and fluid, due to increased losses during the dialysis process, but are restricted in phosphorus. Based on current literature and research, Table 6 lists nutrition recommendations; however, more research is necessary to verify the efficacy of these guidelines. Frequently, actual energy and protein intake for hemodialysis and peritoneal dialysis patients are below these parameters [184]. When estimating a peritoneal dialysis patient's energy intake, the energy available from the dialysate should also be considered.\n\nTABLE 6\n\nDaily Nutrient Recommendations for ESRD Based on Treatment Modality\n\n_b_ For continuous ambulatory peritoneal dialysis (CAPD) and automated or \"cycler\" peritoneal dialysis (APD), include dialysate calories.\n\naTo come.\n\n_Source:_ Modified with permission from \"Manual of Clinical Dietetics,\" 6th ed. \u00a9 2000 American Dietetic Association, Chicago, IL, and Canadian Dietetic Association. American Dietetic Association\n\n#### 2. VITAMIN AND MINERAL SUPPLEMENTATION\n\nSome patients may require supplementation of water-soluble vitamins due to increased losses during the dialysis process coupled with anorexia and poor food intake [92, 96]. Vitamins specially designed for this patient population are available and should be used if vitamin supplementation is prescribed [91, 92]. In general, a vitamin designed for the ESRD population contains B vitamins, folic acid, and vitamin C (Table 7). Fat-soluble vitamins and minerals are intentionally omitted. The majority of patients will require specific and titrated supplementation of iron and vitamin D or vitamin D analogue.\n\nTABLE 7\n\nVitamin Recommendations for ESRD Patients Undergoing Chronic Dialysis\n\nNutrient | ESRD \n---|--- \nVitamin C | 60 mg\/day \nVitamin B1 | 1.5 mg\/day \nVitamin B2 | 1.7 mg\/day \nNiacinamide | 20 mg\/day \nVitamin B6 | 5\u201310 mg\/day \nVitamin B12 | 0.006 mg\/day \nPantothenic acid | 10 mg\/day \nFolic acid | 0.8\u20131.0 mg\/day \nBiotin | 0.3 mg\/day\n\n_Source:_ Adapted from \"Comparison of Vitamin Formulations Prescribed for Renal Patients\" (2000). R&D Laboratories, Marina Del Rey, CA.\n\n## IV. NUTRITIONAL REQUIREMENTS OF THE POST-TRANSPLANT PATIENT\n\nThe overall goal of the post-transplant diet is to normalize electrolyte imbalances, promote blood pressure control, prevent weight gain, maximize bone density, control blood glucose, and promote overall good nutritional status [185\u2013187]. Although renal transplantation restores near-normal renal function, other abnormalities may arise primarily as a consequence of antirejection medications [188]. Specifically, more than 60% of renal transplant patients demonstrate hyperlipidemia. This is primarily due to necessary corticosteroids, and antirejection medications, such as cyclosporin A, as well as antihypertensive medications. Post-transplant patients are also at increased risk of CVD [189\u2013195]. Approximately 60% of renal transplant patients have elevated serum cholesterol levels with about 40% of deaths post-transplant attributable to CVD [196]. To control body weight, serum triglyceride and serum cholesterol levels, a low-fat diet and an exercise plan are generally recommended [187, 197\u2013199]. Many patients will also require lipid-lowering medication. Moreover, glucose intolerance is a frequent occurrence post-transplant, primarily due to corticosteroids and other antirejection medications such as cyclosporin A [200, 201]. Importantly, in addition to the hyperglycemic effects on CVD, post-transplant patients with glucose intolerance have a higher risk for infection and decreased survival rates [202, 203]. Thus, appropriate control of blood glucose is warranted.\n\nExcessive weight gain is a frequent problem that can exacerbate hyperlipidemia and glucose intolerance. Weight control may be problematic in this patient population because many of the antirejection medications may stimulate appetite. Excess weight gain may have adverse effects on heart disease, lipids, blood pressure, diabetes [199], and, although controversial, possibly increase graft rejection [204, 205].\n\nIn addition to increased CVD, glucose intolerance, and increased weight gain, bone disease and altered bone mineral metabolism is a significant problem post-kidney transplant. Hypophosphatemia occurs in as many as 50% of post-transplant patients [206]. In addition, studies show a 10% decrease in bone density by 5 months post-transplant [207].\n\nIn the post-transplant state, during adaptation to the kidney and medication regimen, the patient may present with electrolyte imbalances. Due to the effects of immunosuppressive medications on bone mineral metabolism, post-transplant patients will need to increase their phosphorus and calcium intake and\/or may need supplementation to prevent bone demineralization. Serum electrolytes should be closely monitored with appropriate interventions taken as warranted. Of note, protein and energy needs are increased during the first 4\u20138 weeks post-transplantation. In general, post-kidney transplant patients will require a high-protein, low-fat diet [187]. Nutrition guidelines for post-transplant patients are summarized in Table 8.\n\nTABLE 8\n\nNutrition Guidelines for Adult Kidney Transplant Recipients\n\n_Source:_ Modified with permission from \"Manual of Clinical Dietetics,\" 6th ed. \u00a9 2000 American Dietetic Association, Chicago, IL, and Canadian Dietetic Association.\n\n## V. CONCLUSION\n\nNutritional factors play an important role in the etiology and management of chronic renal disease. During the past several years, important research questions in this area have been addressed in large-scale clinical studies, which have involved the assessment of nutritional factors as predictors of outcome and randomized controlled diet intervention trials. 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Renal Nutr_. 1997;7:176\u2013181.\n\n162. Yates L.A. Anthropometric worksheet for use with hemodialysis patients. _J. Renal Nutr_. 1996;6:162\u2013164.\n\n163. Kopple J.D., Zhu X.F., Lew N.L., Lowrie E.G. Body weight-for-height relationships predict mortality in maintenance hemodialysis patients. _Kidney Int_. 1999;56:1136\u20131148.\n\n164. Fleischmann E., Teal N., Dudley J., May W., Bower J.D., Salahudeen A.K. Influence of excess weight on mortality and hospital stay in 1346 hemodialysis patients. _Kidney Int_. 1999;55:1560\u20131567.\n\n165. Kloppenburg W.D., Stegeman C.A., Hooyschuur M., van der Ven J., de Jong P.E., Huisman R.M. Assessing dialysis adequacy and dietary intake in the individual hemodialysis patient. _Kidney Int_. 1999;55:1961\u20131969.\n\n166. Griffiths A., Russell L., Breslin M., Russell G., Davies S. A comparison of two methods of dietary assessment in peritoneal dialysis patients. _J. Renal Nutr_. 1999;9:26\u201331.\n\n167. Sreedhara R., Avram M.M., Blanco M., Batish R., Avram M.M., Mittman N. Prealbumin is the best nutritional predictor of survival in hemodialysis and peritoneal dialysis. _Am. J. Kidney Dis_. 1996;28:937\u2013942.\n\n168. Adoncecchi L., Marrocco W., Sucraci C., Pecora P., Gallinella B., Porra R., Cavina G. Effect of renal and liver failure on blood levels of vitamin A, its precursor and its carrier proteins, prealbumin and retinol binding protein. _Bollettino-Societa Italiana Biolgia Sperimentale_. 1984;60:881\u2013886.\n\n169. Avram M.M., Goldwasser P., Erroa M., Fein P.A. Predictors of survival in continuous ambulatory peritoneal dialysis patients: The importance of prealbumin and other nutritional and metabolic markers. _Am. J. Kidney Dis_. 1994;23:91\u201398.\n\n170. Goldwasser P., Michel M.A., Collier J., Mittman N., Fein P., Gusik S., Avram M.M. Prealbumin and lipoprotein(a) in hemodialysis: Relationships with patient and vascular access survival. _Am. J. 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Avram M.M., Fein P.A., Bonomini L., Mittman N., Loutoby R., Avram D.K., Chattopadhyay J. Predictors of survival in continuous ambulatory peritoneal dialysis patients: A five year prospective study. _Periton. Dial. Int_. 1996;16:S190\u2013S194.\n\n176. DeLima J., Sesso R., Abensur H., Lopes H.F., Giorgi M.C., Krieger E.M., Pileggi F. Predictors of mortality in long term haemodialysis patients with a low prevalence of comorbid conditions. _Nephrol. Dial. Transplant_. 1995;10:1708\u20131713.\n\n177. Canaud B., Garred L.J., Argiles A., Flavier J.L., Bouloux C., Mion C. Creatinine kinetic modelling: A simple and reliable tool for the assessment of protein nutritional status in haemodialysis patients. _Nephrol. Dial. Transplant_. 1995;10:1405\u20131410.\n\n178. Goldstein D.J., Frederico C.B. The effect of urea kinetic modeling on the nutrition management of hemodialysis patients. _J. Am. Diet. Assoc_. 1987;4:474\u2013479.\n\n179. Gotch F.A. Kinetic modeling in hemodialysis. In: Nissenson A.R., Fine R.N., Gentile D.E., eds. _Clinical Dialysis_. 3rd ed. Bethesda, MD: Appleton and Lange; 1995:156\u2013188.\n\n180. Gotch F.A., Sargent J.A. A mechanistic analysis of the National Cooperative Dialysis Study. _Kidney Int_. 1985;28:526\u2013534.\n\n181. Ward R.A., et al. Protein catabolism during hemodialysis. _Am. J. Clin. Nutr_. 1978;32:243.\n\n182. Farrell P.C., Hone P.W. Dialysis induced catabolism. _Am. J. Clin. Nutr_. 1980;33:1417.\n\n183. Kluthe R., Luttgen F.M., Capetianu T., Heinze V., Katz N., Sudhoff A. Protein requirements in maintenance hemodialysis. _Am. J. Clin. Nutr_. 1998;31:1812.\n\n184. Kopple J.D. Dietary protein and energy requirements in ESRD patients. _Am. J. Kidney Dis_. 1998;32:S97\u2013S104.\n\n185. Perez R. Managing nutrition problems in transplant patients. _Nutr. Clin. Prac_. 1993;8:28\u201332.\n\n186. Weil S.E. Nutrition in the kidney transplant recipient. In: Danovitch G.M., ed. _Handbook of Kidney Transplantation_. 2nd ed. Norwalk, CT: Little Brown and Co.; 1996:321\u2013335.\n\n187. Hines L. Can low-fat\/cholesterol nutrition counseling improve food intake habits and hyperlipidemia of renal transplant patients? _J. Renal Nutr_. 2000;10(1):30\u201335.\n\n188. Jaggers H.J., Allman M.A., Chan M. Changes in clinical profile and dietary considerations after renal transplantation. _J. Renal Nutr_. 1996;6:12\u201320.\n\n189. Ibels L.S., Alfrey A., Weil R. Hyperlipidemia in adult, pediatric and diabetic renal transplant recipients. _Am. J. Med_. 1978;64:634\u2013643.\n\n190. Ponticelli C., Barbi G.L., Cantaluppi A., Donati C., Annoni G., Brancaccio D. Lipid disorders in renal transplant recipients. _Nephron_. 1978;20:189\u2013195.\n\n191. Chan M.K., Varghese Z., Persaud J.W., Fernando O.N., Moorhead J.F. The role of multiple pharmacotherapy in the pathogenesis of hyperlipidemia after renal transplantation. _Clin. Nephrol_. 1981;15:309\u2013313.\n\n192. Jackson J.M., Lee H.A. The role of propanolol therapy and proteinuria in the etiology of post renal transplantation hyperlipidemia. _Clin. Nephrol_. 1982;18:95\u2013100.\n\n193. Shen S.Y., Lukens C.W., Alongi S.V., Sfeir R.E., Dagner F.J., Sadler J.H. Patient profile and effect of dietary therapy on post transplant hyperlipidemia. _Kidney Int_. 1983;24:S147\u2013S152.\n\n194. Kasiske B.L., Umen A.J. Persistent hyperlipidemia in renal transplant patients. _Medicine_. 1987;66:309\u2013316.\n\n195. Raine A.E., Carter R., Mann J.I., Morris P.J. Adverse effects of cyclosporine on plasma cholesterol in renal transplant recipients. _Nephrol. Dial. Transplant_. 1988;3:458\u2013463.\n\n196. Wolfe R.A., et al. Causes of death, U.S. Renal Data System. _Am. J. Kidney Dis_. 1998;32:S85\u2013S86.\n\n197. Devine W. Review of nutritional status on diet in dialysis and transplant patients. _Dial. Transplant._ 1994;23:38\u201341.Devine W. Review of nutritional status on diet in dialysis and transplant patients. _Dial. Transplant_. 1994;23:47\u201348.\n\n198. Sullivan S.S., Anderson E.J., Best S., et al. The effect of diet on hypercholesterolemia in renal transplant recipients. _J. Renal Nutr_. 1996;6:141\u2013151.\n\n199. Patel M.D. The effect of dietary intervention on weight gain after renal transplantation. _J. Renal Nutr_. 1998;8:137\u2013141.\n\n200. Nakai I., Omoni Y., Aikawa I., Yasumura T., Suzuki S., Yoshimura N., Arakawa K., Matsui S., Oka T., Effect of cyclosporine on glucose metabolism in kidney transplant recipients. Transplant Proc. 1988;20:969\u2013978.\n\n201. Roth D., Milgrom N., Esquenazi V., Fuller L., Burke G., Miller J. Post transplant hyperglycemia: Increased incidence in cyclosporine treated allograft recipients. _Transplantation_. 1989;47:278\u2013281.\n\n202. Friedman E.A., Shyh T., Beyer M.M., Maris T., Butt K.M. Post transplant diabetes in kidney transplant recipients. _Am. J. Nephrol_. 1985;5:196\u2013202.\n\n203. Bordreaux J.P., McHugh L., Canfax D.M., Ascher N., Sutherland D.E., Payne W., Simmons R.L., Najarian J.S., Fryd D.S. The impact of cyclosporine and combination immunosuppression on the incidence of posttransplant diabetes in renal allograft recipients. _Transplantation_. 1987;44:371\u2013381.\n\n204. Pirsch J.D., Armbrust M.D., Knechtle S.J., D'Allessandro A.M., Sollinger H.W., Heisey D.M., Belzer F.O. Obesity as a risk factor following renal transplantation. _Transplantation_. 1995;59:631\u2013663.\n\n205. Dimeny E., Fellstrom B. Metabolic abnormalities in renal transplant recipients. Risk factors and predictors of chronic graft dysfunction. _Nephrol. Dial. Transplant_. 1997;12:21\u201324.\n\n206. Massari P.U. Disorders of bone and mineral metabolism after renal transplantation. _Kidney Int_. 1997;52:1412\u20131421.\n\n207. Horber F., Casez J., Steiger U., Czerniak A., Montando A., Jaeger P. Changes in bone mass early after kidney transplantation. _J. Bone Mineral Res_. 1994;9:1\u20139.\nCHAPTER 41\n\nNutritional Management of Parkinson's Disease and Other Conditions Like Alzheimer's Disease\n\nCHRISTY TANGNEY, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois\n\n## I. INTRODUCTION\n\nNeurodegenerative disorders commonly increase in incidence with advancing age. These disorders broadly can be classified into two categories: (1) cognitive decline, such as Alzheimer's disease, vascular dementia, and Parkinson's disease; and (2) physical decline, which may also include these disorders. Alzheimer's disease is the most common form of dementia observed after 60 years of age. From 6% to 8% of those older than 65 have Alzheimer's disease with the prevalence doubling every 5 years after age 60 [1]. Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease, with a prevalence of 2% among persons over the age of 65 years [2]. Parkinson's disease has an unknown etiology, affects more than 1 million people in North America, and has serious health consequences. Mortality is 2\u20135 times as high among affected persons as among age-matched controls [3\u20135]. The average age at diagnosis is 57 years, and the disease is rarely seen in those under 30, becoming more common after age 55 years.\n\nCognitive performance and nutritional status are important indications of general health and functional status in elderly persons. Nutrition and cognition are interrelated. Nutrition surveys of the elderly often indicate increased risk of malnutrition, most often subclinical deficiencies (i.e., folate, vitamin B12), which can impair cognition and mood. Impaired cognition and depression, in turn, decrease appetite.\n\nThis chapter will address the interrelationship of nutrition and cognition. In the broadest sense, the primary objective is to review the evidence for nutritional components as potential risk factors for cognitive decline, Alzheimer's disease, and Parkinson's disease. The literature to be reviewed includes animal studies, pathological comparisons, retrospective case-control studies, cross-sectional studies, and longitudinal studies. Evidence from clinical trials for use of specific nutritional therapies for Alzheimer's disease and Parkinson's disease will be described. Finally, nutritional management issues specific for Alzheimer's disease and Parkinson's disease will be examined.\n\n## II. NUTRITIONAL FACTORS IN RELATION TO COGNITIVE FUNCTION\n\nA. Laboratory Animal Studies\n\nTo define a relationship between a dietary component and cognitive performance, it is important to demonstrate that varying dietary consumption can be reflected in changes in brain tissue, which affect changes in cognitive function. Laboratory animal studies provide a means to illustrate this cause-and-effect relationship. A number of animal studies have examined the influence of specific dietary nutrients at inadequate, adequate, and supranormal levels on tissue concentrations and, specifically, critical regions of the brain, such as the hippocampus, cortex, and striatum. For example, there is ample evidence that tissue levels of alpha-tocopherol are increased with dietary supplementation, but longer feeding periods are required to elevate vitamin E concentration in cerebrum, especially in older animals [6\u201310]. Serum, adipose tissues, and other organs exhibit increases more rapidly than brain, suggesting that serum levels may not be a good indicator of brain tissue content. Moreover, even with vitamin E-adequate diets, older animals were more likely to manifest lipofuscin, an indicator of lipid peroxidative damaged proteins, than younger animals [10]. In humans, studies suggest that age may exert an effect on serum vitamin E concentrations independent of intake of vitamin E [11].\n\nBecause animal brain tissue levels of vitamin E can be elevated with dietary and supplemental vitamin E, what is the evidence that such changes alter cognitive performance? In aged laboratory animals, chronic supranormal feeding of antioxidants may improve cognitive skills. For example, Socci and colleagues [12] found that 24-month-old rats administered intraperitoneal injections of alpha-tocopherol (200 mg\/kg\/day body weight) and of a spin trapping compound at 32 mg\/kg body weight, and vitamin C in their drinking water (600 mg\/week) for 2 months had a greater rate of acquisition and greater memory retention than vehicle-treated animals. Motor activity was not changed [12]. Other groups have also found that vitamin E supplementation in older animals not only elevates brain vitamin E content, but also is associated with improved cognitive performance and less oxidative damage [13\u201316]. Martin and colleagues [14] fed 6-month-old rats diets enriched in vitamin E (500 mg all rac-alpha-tocopherol acetate\/kg diet), spinach, or strawberry extracts for 8 months and observed a marked enhancement in striatal dopamine release in those fed diets enriched in fruits and vegetables but low in vitamin E or those enriched in vitamin E content as compared to controls. Cortex, hippocampus, cerebellum, and striatum vitamin E concentrations were significantly increased in response to supplementation. The risk of neurologic degeneration was reduced with alpha-tocopherol treatment of transgenic mice expressing human variants of amyloid precursor protein, a protein generally considered to reflect brain damage seen in Alzheimer's disease [17].\n\n### B. Case-Control, Cross-Sectional, and Prospective Studies of Dietary Components in Relation to Cognitive Function\n\nA number of cross-sectional and longitudinal studies provide both dietary and biochemical evidence (largely serum, plasma, or erythrocyte concentrations) of possible relationships between nutritional factors and cognitive changes. Diet is a particularly difficult exposure to measure because of its complexity. As outlined in Table 1 [18\u201335], the majority of studies in the literature represent cross-sectional comparisons. For example, when cognitive scores using the Mattis dementia rating in relatively healthy older adults, aged 50\u201375 years, were dichotomized according to age and education, plasma alpha-tocopherol concentrations were found to be highly correlated with cognitive function [29]. Although a number of antioxidant compounds such as carotenoids and vitamin C were quantified in this large sample, many other dietary components were not examined. In one of the largest cross-sectional studies of the relationship between cognitive performance (memory) and serum antioxidant micronutrient concentrations (vitamin E, vitamin C, beta-carotene, vitamin A, and selenium), low vitamin E\/cholesterol ratios were highly predictive of poor memory performance after adjustment for age, education, income, vascular risk factors, and other trace elements (Table 2) [32]. Years of education was a predictor of memory performance [odds ratio (OR) 3.3, _p <_ 0.0001]. As found in the entire sample, a strong direct relationship between blood vitamin E\/cholesterol levels and memory was also observed in Mexican-Americans and non-Hispanic Caucasians [OR = 4.1, 95% confidence intervals (CI), 1.4\u201311.99, _p <_ 0.02 and OR 2.32, CI 1.0\u20135.4, _p_ < 0.05]. A trend was also observed among non-Hispanic African-Americans, but this association was nonsignificant. Only 7% of the entire sample presented with poor memory overall, 5.9% among individuals who had ever smoked versus 8.4% who had never smoked, and in 14% with a history of prior strokes and 6.6% with no previous stroke.\n\nTABLE 1\n\nBiochemical and Dietary Studies of Dietary Components in Relation to Cognitive Performance\n\nTABLE 2\n\nProportion and Odds of Having Poor Memory for Selected Demographic Attributes and Biochemical Measures of Antioxidants in 4809 U.S. Elderly from NHANES III, 1988\u20131994\n\naReference category.\n\n_Source:_ Adapted from Perkins, A. J., Hendrie, H. C., Callahan, C. M., Gao, S., Unverzagt, F. W., Xu, Y., Hall, K. S., and Hui, S. L. (1999). Association of antioxidants with memory in a multiethnic elderly sample using the Third National Health and Nutrition Survey. _Am. J. Epidemiol._ **150,** 37\u201344 [32].\n\nIt has been suggested that estimating the influence of single nutrients rather than dietary patterns is too restrictive. Certainly people eat foods, not nutrients, and the combination of nutrients usually ingested is what may afford protection against cognitive decline. In a cross-sectional study derived from five older male cohorts (aged 70\u201391 years) from the Seven Countries Study, Huijbregts and colleagues [36] looked at overall dietary quality (obtained through diet histories and evaluated using the Healthy Diet Indicator) and found that this indicator was associated with better cognitive function using the Mini Mental Status Examination.\n\nRelatively few studies examined dietary components in relation to future cognitive performance. These longitudinal studies afford greater confidence in possible relationships between biochemical indices or dietary intakes and functioning. In the cross-sectional analyses of elderly Swiss individuals, for example, blood concentrations of vitamin C and beta-carotene, but not those of vitamin E, were found to be correlated with cognitive function in 442 subjects [28]. Again, concurrent status may not necessarily reflect what is available for specific brain regions. Cross-sectional analyses are particularly prone to selection biases and do not account for the latency period between exposures and cognitive decline. In the longitudinal analyses by the same group, however, both beta-carotene and vitamin C status remained significant predictors of cognition.\n\nIn another study of 137 elderly individuals from the New Mexico Aging Project [27], both past (6 years) and concurrent nutrition status (assessed by dietary records and blood indicators) were also related to cognitive performance. These investigators performed an extensive nutrition evaluation that included vitamin C, vitamin E, many B vitamins (i.e., folate, vitamin B12), and several minerals. In many cases, both dietary and biochemical indices indicated potential protective effects against cognitive decline. Surprisingly, no biochemical determinations of vitamin E were reported in this study. Several longitudinal studies in which food frequency questionnaires or queries regarding supplement use appear to confirm the possible role afforded by vitamin E [21, 28, 34, 35] and\/or vitamin C [21, 30, 34, 35]. Finally, fish intake appears to have a protective effect on stroke and dementia [25]. Estimating fish intake on the basis of response to four fish items on the Chicago Health and Aging Project food frequency questionnaire, and cognitive performance data measured at baseline, and at both 6-month and 3-year follow-up revealed less decline in cognitive function among persons with higher fish intake ( _p_ for trend = 0.04 for fish consumption ranging from several times per month to more than 2 times per week). Whether the reduction in cognitive decline is attributable to fish consumption patterns, specific fatty acids (i.e., omega 3 fatty acids), or some unidentified covariates requires further study.\n\nThe inconsistencies between these findings are notable. There are many possible reasons for these differences, which may include the study sample, the type of cognitive tests used, and the ability of such tests to detect specific cognitive deficits. Results are also influenced by the type of food or nutrient exposure and the period of time this index reflects (dietary tool, static biochemical indicator in plasma or erythrocyte, or a functional index such as enzyme activity). Serum vitamin E concentration, when expressed per unit of serum cholesterol, was associated with memory performance of participants in National Health and Nutrition Examination Survey (NHANES) III [32]. Only one other group corrected for cholesterol concentration [28], which provides greater accuracy in assessment.\n\nSeveral vitamins, including folate, vitamin B6, and vitamin B12, are involved in methylation reactions in brain tissue and, indirectly, in the prevention of homocysteinemia, which has been associated with increased risk of vascular occlusive disease, stroke, and thrombosis [37]. Many have proposed that a loss of cognitive function may be related to inadequate status for these vitamins via reduced production of S-adenosylmethionine [38], which serves as one of the universal methyl donors in a variety of reactions including the biosynthesis of neurotransmitters [39]. As briefly summarized in Table 1 and more extensively addressed in an excellent review [40], mostly positive or null associations between status for these vitamins and cognitive function have been reported.\n\n## III. NUTRITIONAL FACTORS IN RELATION TO ALZHEIMER'S DISEASE AND PARKINSON'S DISEASE\n\nA. Pathological Studies in Alzheimer's Disease and Parkinson's Disease\n\nWhen postmortem brain tissues of neurological normal controls and groups of patients with neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease were compared to specific micronutrients, such as vitamin E, the results are far from definitive. Some data suggest higher levels in brains of patients with Alzheimer's disease and Parkinson's disease; others do not [41\u201343]. Vitamin C and beta-carotene concentrations in brain tissue were not quantified in these studies.\n\nIn pathological comparisons of Alzheimer's disease cases and controls, comparisons of postmortem brains of controls and Parkinson's disease cases suggest that basal lipid peroxidation is exaggerated in nigral tissue of Parkinson's disease on the basis of higher levels of malondialdehyde (an intermediate product of lipid peroxidation) concentrations and lower concentrations of polyunsaturated fatty acids in Parkinson's disease cases [43, 44]. Postmortem findings are far from conclusive because tissues are susceptible to complications and artifacts with sample processing. Cerebrospinal fluids may afford the ideal biological matrix because these can be obtained in living patients and controls. Here again, some controversy concerning the site of fluid sampling clouds the picture. An important concentration gradient may exist for vitamin E from ventricular to lumbar fluids. Stepped increases in vitamin E supplements (400\u20134000 IU\/day) did not appear to elevate _ventricular_ cerebrospinal fluid of patients with Parkinson's disease with Ommaya shunts [45], but _lumbar_ cerebrospinal fluids of patients with Parkinson's disease did reflect vitamin E supplementation (2000 IU\/day), with increases being directly related to the duration of supplementation [46]. Lumbar cerebrospinal fluid concentration of alpha-tocopherol were also markedly reduced in patients with Alzheimer's disease when compared to those of controls [47]. Ikeda and coworkers [48] showed lower vitamin B12 concentrations in cerebrospinal fluid, but not plasma of patients with Alzheimer's disease as compared to those of patients with other dementias. Further work in this area may clarify the reasons for these seemingly disparate results.\n\n### B. Observational Studies of Nutritional Factors in Relation to Alzheimer's Disease\n\nWith chronic diseases such as Alzheimer's disease and Parkinson's disease, disease onset is usually a long period of time prior to actual diagnosis. Case-control studies are cost-effective designs for uncommon disease, but as a result are more prone to biased estimates of exposure than are longitudinal studies. A number of case-control studies exist for Alzheimer's disease [49\u201353] with no consistent outcomes that identify nutritional components. Folate and antioxidant nutrient inadequacies such as vitamin E are most often cited as possible risk factors. In a longitudinal study of aging and Alzheimer's disease in a community of nuns, the severity of atrophy of the cortex (via postmortem magnetic resonance imaging) was examined in relation to serum folate levels. The age-adjusted correlation between cortical atrophy and serum folate levels for all 30 deceased participants was \u20130.40, _p =_ 0.03; no other nutrients examined (including vitamin B12, vitamin B6, thiamin, vitamin E, and carotenoids) showed any linear relationship to cortical atrophy [54]. In many studies, serum alpha-tocopherol levels were found to be lower in persons with Alzheimer's disease [50, 55\u201358], but not all studies found this relationship [20].\n\nLongitudinal studies of subjects before the onset of overt symptoms of Alzheimer's disease provide greater confidence in the relationships between nutrient exposures and disease. In a random sample acquired from a prospective study of elderly residents of Boston, the relationship between incident Alzheimer's disease and use of vitamin C and vitamin E supplements was examined [34]. After 4.3 years of follow-up, 91 of the 642 sample participants with vitamin information met the accepted criteria for diagnosis of Alzheimer's disease. None of the 27 vitamin E supplement users had Alzheimer's disease, compared to 3.9 predicted, based on the crude observed incidence among nonusers ( _p_ = 0.04). Alzheimer's disease incidence for supplement users when adjusted for sex, age, years of education, and length of follow-up was not significant ( _p =_ 0.1). In contrast, the adjusted number of Alzheimer's disease cases among vitamin C users were significantly less than among nonusers ( _p_ = 0.04). No relationship was detected for incident Alzheimer's disease with multivitamin use. The relationship for other nutritional supplements could not be examined because too few persons in the cohort reported use of these supplements. A major strength of this study was that the information regarding vitamin supplement use obtained through direct observation of all medications from the entire cohort. Hence, biased estimates of vitamin use are unlikely. The small number of persons taking vitamin supplements raises the possibility that the findings may be due to chance. Results may also be biased due to the selected participant and nonparticipant bias (i.e., 20% refused to participate, 13% died). The absence of an association among multivitamin users may be the result of low statistical power; thus, investigators were unable to detect a small effect of lower doses of these vitamins or other vitamins in multivitamin mixtures. Large-scale randomized trials will likely resolve some of these issues. Equally important is the addition of more longitudinal studies that examine diet and the incidence of Alzheimer's disease that may confirm or refute these findings.\n\nIn preliminary analyses of biracial community participants in the Chicago Health and Aging Project, a significant inverse relationship between total intake (diet and supplements combined) of vitamin E and change in global cognitive score over 3 years (using the average of Z-transformed scores for four cognitive tests) was found [35]. Compared with an average decline of 0.07 standard deviation (SD)\/year for a person with an intake in the lowest quintile, the average decline in the global _Z_ -score in the highest quintile was 0.05 SD\/year (Table 3). There was also a marginally significant reduction in cognitive decline in those with the highest intake of vitamin C. Because there was a significant reduction in cognitive decline in the highest quintile of vitamin E intake (median 386 IU\/day), these data suggest that supplement use probably contributes to this relationship. When intakes of these vitamins from food sources were examined separately, decline in global score was reduced by 0.03 SD\/year per log (IU) increase in vitamin E intake ( _p_ = 0.0005). Intake from dietary vitamin C was not associated with change in cognitive decline [35]. Dietary intake data were based on a food frequency questionnaire, and a preliminary data provide support for the accuracy of this instrument with respect to vitamin supplement reporting behaviors [59]. If food intake is more stable over time than supplement use, the observed inverse relationship suggests that vitamin E intake from foods may be sufficient to protect against cognitive decline.\n\nTABLE 3\n\nAnnual Change in Global Cognitive Score over 3 Years by Quintile of Intake of Vitamins E and C among 2878 Participants Based on Random Effects Models Adjusted for Age, Sex, Race, Education, and Total Energy Intake\n\na _p =_ 0.01.\n\nb _p =_ 0.07.\n\n_Source:_ Adapted from [35].\n\nIn another community study of 3777 elderly subjects aged >65 years living in southwestern France, mean alcohol consumption through use of a structured questionnaire was recorded with the main objective of identifying new Alzheimer's disease cases after 1 and 3 years [60]. After 3 years, the incidence of Alzheimer's disease was lower in those who drank a moderate amount of wine (3\u20134 glasses\/day) compared to nondrinkers (adjusted OR = 0.28, _p <_ 0.05) and for mild drinkers (<1\u20132 glasses per day), the adjusted OR for Alzheimer's disease was 0.55, _p_ <0.05. Further information is needed to provide confirmation of this finding. The antioxidant contribution of wine is consistent with findings for vitamin C and vitamin E, mentioned above.\n\n### C. Observational Studies of Nutritional Factors in Relation to Parkinson's Disease\n\nParkinson's disease is less common than Alzheimer's disease, and there are a greater number of case-control studies addressing potential dietary risk factors and Parkinson's disease (Table 4) [61\u201373]. There are several reasons to examine diet as a possible exposure that may explain Parkinson's disease. First, the rare disease, amyotrophic lateral sclerosis-parkinsonism-dementia, observed in the islands of Guam and Rota, is thought to be caused by a dietary toxin from the cycad plant [74]. Second, a number of neuropathological studies implicate oxidative stress in the pathogenesis of Parkinson's disease [43, 44]. Also, it was discovered that a synthetic heroin analogue, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was selectively toxic to dopaminergic neurons in the substantia nigra. The toxicity attributable to MPTP involves oxidation and the formation of a reactive intermediate that impairs mitochondrial function and results in cell death in the substantia nigra pars compacta and promotes oxidative stress [75]. The appealing attribute of the oxidative stress hypothesis is that cumulative oxidative damage over time could account for the late life onset and the progressive nature of this disorder.\n\nTABLE 4\n\nBiochemical and Dietary Studies of Parkinson's Disease\n\nTwo retrospective case-control studies suggest an inverse association between vitamin E and Parkinson's disease which initiated considerable interest in antioxidant protection and Parkinson's disease [61, 76]. Dietary intake was not quantified directly, but patients with Parkinson's disease rate their food consumption of specific foods different than that of their spouses. Consumption of foods rich in vitamin E and possibly other antioxidant-rich foods (e.g., salad dressing, peanuts, plums) were inversely associated with risk of Parkinson's disease. Tanner and colleagues [62] found that early life use of multivitamins, vitamin E supplements, and cod liver oil was greater among 70 controls as compared to 35 Parkinson's disease subjects. In a secondary analyses of these data, acquired by interviewer-administered food frequency questionnaires using a well-recognized nutrient database (University of Minnesota Nutrient Data System, version 2.4), yielded a null finding, except when females, ages 31\u201350 years, were examined separately, OR = 0.89, _p =_ 0.01 [64]. Another two case-control studies found no inverse relationship for any antioxidant nutrients examined, although the designs were fraught with problems [66, 67]. Finally, in a nestled case-control study of Hawaiian men of Japanese ancestry, an inverse association was shown between Parkinson's disease and consumption of foods containing vitamin E (in particular, legumes) [3]. Intriguingly, three of these case-control studies suggest that consumption of animal fats or meats is associated with increased risk for Parkinson's disease [67, 69, 73]. Thus far, the most compelling data are those from a recent large community-based study in which vitamin E intakes assessed from an interviewer-administered food frequency questionnaire were significantly lower ( _p_ = 0.03) in those with Parkinson's disease ( _n_ = 31) than those without Parkinson's disease ( _n_ = 5311) [70]. Those with higher vitamin E intakes had Parkinson's disease significantly less often than those with lower vitamin E intakes (OR = 0.5, 95% CI, 0.2\u20130.9). Even excluding those with late stage Parkinson's disease and supplement users, the associations did not change. When vitamin E, beta-carotene, vitamin C, and flavonoid intakes were categorized into tertiles, a dose-dependent trend for vitamin E intakes and risk for Parkinson's disease was observed. These findings need to be confirmed in prospective studies in which dietary habits have been measured before the onset of Parkinson's disease. The Rotterdam study may provide such data shortly [70\u201371]. In addition, further investigation of the role of animal fats and meats should be explored in longitudinal studies in which clinical diagnoses of Parkinson's disease can be ascertained.\n\nAt present there are two large prospective studies reporting relationships between diet and Parkinson's disease incidence. As is characteristic of longitudinal studies, subjects were disease free when reporting their usual dietary intakes via dietary history questionnaire or food frequency questionnaire. In the first of these studies, 41,836 women from Iowa Women's Health Study were followed up for an average of 6 years. A significant protective effect was noted for vitamin C and manganese intakes while increased risk was observed for vitamin A intake [65]. Unfortunately, these results are only available as a preliminary report and more information is needed. The Honolulu Heart Program followed Hawaiian men of Japanese and Okinawan ancestry in a nested case-control design [3]. Once again, consumption of foods containing vitamin E 25 years earlier appear to protect against Parkinson's disease. In the longitudinal analyses [68], incident Parkinson's disease and coffee intake were compared in a 30-year follow-up of the 8004 men in the study. Age-adjusted incidence of Parkinson's disease declined consistently with increased coffee intake from 10.4 per 10,000 person years in no coffee drinkers to 1.9 in men who drank more than 28 oz\/day. Similar relationships were observed for total caffeine intake and appeared independent of smoking. Further studies are warranted to confirm or refute these findings.\n\n## IV. SPECIFIC NUTRITIONAL TRIALS IN COGNITIVE, ALZHEIMER'S, AND PARKINSON'S DISEASES\n\nWhile reports of several trials aimed at treating cognitive impairment or dementia are available [77, 78], few can truly be classified as rigorous double-blind, clinical trials with random assignment of treatments. On the other hand, rigorously controlled clinical trials have limitations when studying these diseases. First, if the chronic disease has a long latency period, is it appropriate to test whether short-term nutrient exposure affords protection in individuals with advanced disease? The second important issue is to examine extant observational studies and neuropathological studies in order to test the hypothesis of nutrient exposure and disease risk. More of a nutrient is not always better.\n\nWith respect to alternative therapies with free radical scavenging properties, Gingko biloba extract Egb 761 (containing terpenoids and several organic acids) has been shown to inhibit toxicity of beta amyloid peptide (a marker of Alzheimer's disease on hippocampal neuronal cultures [79]. Egb 761 has been studied extensively in Germany and the United States and approved in the former for the treatment of dementia [80, 81]. In the former trial, in which 200 demented patients received either 40 mg Egb 761, three times\/day, or placebo for 1 year, modest improvements were observed in cognitive performance and social functioning, but these changes were large enough to be discerned by a care-givers' scale. A small but significant improvement on cognitive function has also been noted following a review of more than 30 studies of Egb 761 [82]. A major clinical trial is currently under way to test the efficacy of a daily supplement (240 mg) in 1500 subjects against 1500 subjects assigned placebo over 5 years of treatment.\n\n### A. Alzheimer's Disease Trials\n\nAt this point in time, we are aware of only one completed double-blind clinical trial, the Alzheimer's Disease Cooperative Study of 324 subjects with moderate disease. The main objective of this placebo-controlled, parallel group, factorial design, multicenter trial was to determine whether vitamin E (2000 IU\/day or 1342 alpha-tocopherol equivalents\/day) or selegiline (10 mg\/day) or both could slow functional decline in Alzheimer's disease patients [83]. Selegiline is a monoamine oxidase B inhibitor with antioxidant properties [84]. Time to functional decline was operationalized as the time to reach any one of the following endpoints: institutionalization, loss of basic activities of daily living as measured [85], severe dementia as defined by a clinical rating of 3 [86], or death. Risk of reaching this outcome was significantly reduced by vitamin E ( _p_ < 0.001), selegiline ( _p_ <0.01), and combined treatments ( _p_ <0.05). There was no evidence of additional improvement with both treatments over each treatment alone, however. Although significant benefits of vitamin E supplementation alone as compared to placebo were found for functional assessments, none was shown for cognitive tests. A large proportion of subjects were unable to complete the cognitive testing at the end of 2 years. The lack of cognitive benefit has also been ascribed to the relatively advanced nature of the disease of subjects at study entry. Therefore, Grundman and others [87] have proposed a primary prevention trial with 2000 IU\/day vitamin E or placebo in those at risk for Alzheimer's disease who have minor cognitive deficits that are insufficient to meet clinical criteria for Alzheimer's disease.\n\n### B. Parkinson's Disease Trials\n\nA double-blind clinical trial of the monoamine oxidase B inhibitor, selegiline, and alpha-tocopherol, known as the deprenyl and tocopherol antioxidant therapy of Parkinson's disease or DATATOP, was designed to test whether either treatment or both might retard the progression of Parkinson's disease. Initial analyses at the end of the trial showed that vitamin E at 2000 IU\/day was without effect [88] but selegiline markedly delayed the onset of disability. Follow-up studies showed that the effects were not sustained [89, 90]. One concern with DATATOP and the Alzheimer's Disease Cooperative Study is that the latency period for development of the disease with that afforded by the nutrient exposure is missed. A 2- or 4-year follow-up may be too brief to discern any treatment effect in patients with established disease.\n\nIdentification of subjects at high risk of either disease rather than patients with clearly defined diagnosis and longterm nutrient (or food) exposure may be necessary to truly illustrate neuroprotection. Again, the vitamin E dose selected for aforementioned trials is thought to elevate brain levels by as much as 50%, which is consistent with animal studies suggesting increases in vitamin E levels in brain by 30\u201360% of the elevation seen in plasma [8, 10, 91], as well as lumbar cerebrospinal fluid levels observed in DATATOP participants [8]. On the other hand, data from both ventricular cerebrospinal fluid levels of patients with Parkinson's disease on stepped doses from 400 to 4000 IU\/day of vitamin E [45] and prospective data [23, 70, 71] suggest that even lower doses may be effective.\n\nAnother randomized clinical trial is currently recruiting young and older patients with Parkinson's disease and age-matched controls in order to determine whether glucose aides memory in healthy elderly and young people, and second, whether glucose facilitates working memory in people with Parkinson's disease. The premise for the use of glucose relies on its role as a precursor of acetylcholine synthesis as well as the fact that many drugs used for cognition enhancement produce their effect through enhanced glucose utilization [92]. Moreover, glucose may enhance retention and or retrieval from long-term verbal memory [93]. In a series of experiments, older men were shown to be more susceptible to cognitive impairment during hypoglycemia than were young men [94]. Additionally, a recent review by Benton and Parker [95] shows that breakfast consumption influences cognition, primarily memory, through a variety of mechanisms, one of which is through an elevation of blood and brain glucose levels.\n\n## V. NUTRITIONAL MANAGEMENT ISSUES IN NEUROCOGNITIVE DISORDERS\n\nOne important component of nutritional management of patients with any neurodegenerative disorder is rigorous scrutiny of weight change patterns of patients, because weight loss is so common, particularly in advanced stages. Because cognitive deficiencies lead to orientation disturbances, loss of independence, and altered eating behaviors, the nutritional care of patients with these diseases becomes critical.\n\n### A. Weight Loss in Patients with Alzheimer's Disease\n\nThe National Institute of Neurological and Communicative Disorders and Strokes Task Force included weight loss as a clinical sign of Alzheimer's disease [96]. Alzheimer's disease was a risk factor for weight loss in 467 subjects ages 65 years and older followed for 5 annual exams in the Chicago Health and Aging Project, but weight loss was not associated with disease severity [97]. Conversely, changes in stage of Alzheimer's disease were significantly related to weight change, and losses greater than 5% were highly predictive of mortality [98]. Equally important to evaluate is the perceived burden of the caregivers [99], which has been shown to be highly predictive of weight loss in Alzheimer's disease [100].\n\nIt is unclear why weight loss occurs in Alzheimer's disease. Contrary to earlier studies in which only resting energy expenditure was measured [101, 102], increased total energy expenditures cannot explain observed weight losses, even those with severe cachexia (weight loss >2 kg over the 12-month period). In fact, both lower physical activity and lower resting energy expenditures were observed in Alzheimer's disease [103]. Because both weight gain as well as weight loss are observed in patients with Alzheimer's disease, some aberration in weight regulation may be present. Grundman and colleagues [104] showed that atrophy of the mesial temporal cortex in Alzheimer's disease patients is associated with lower body mass index and lower cognitive function. Because the mesial temporal cortex plays an important role in eating behavior, memory, and emotions, this relationship should be explored further.\n\nDocumentation of food intakes of patients with Alzheimer's disease are sparse in the literature. Refusal of food is a commonly reported problem and may be related to abnormalities in the neuroendocrine system. Neurotransmitters such as neuropeptide Y and norepinephrine are reduced in patients with Alzheimer's disease type dementia [105]. Little research has explored these possibilities, however [106]. One group examined the use of dronabinol in 15 patients with Alzheimer's disease who were refusing food in whom weight gain, but no change in energy intake, was observed. Some of the behaviors associated with food refusal may be due to dysphagia or failure to recognize edible items as food (agnosia) [107]. Refusal to eat may even be a symptom of depression; antidepressant therapy has been shown to improve intakes even in severely demented patients [108].\n\n### B. Weight Loss in Parkinson's Disease\n\nIn patients with Parkinson's disease, impaired cognition and motor disturbances (i.e., dysphagia) may result in marked anorexia. The prevalence of weight loss (as well as a reduction in muscle mass) in Parkinson's disease will vary with the degree of gastrointestinal dysfunction. Dysphagia and constipation are not a sequelae of the disease but often the result of the medications used to treat Parkinson's disease. Weight loss estimates vary from 20 to 50% of patients with Parkinson's disease [109, 110]. In one study, patients with Parkinson's disease were four times more likely than age-matched controls to lose more than 10 pounds [111]. In a doubly labeled water studies of patients with Parkinson's disease and healthy controls, Toth and coworkers [112] have shown that although resting energy expenditures are greater among patients with Parkinson's disease (1655 \u00b1 283 versus 1561 \u00b1 219 kcal\/day), physical activity expenditure is considerably less. Again, as observed for patients with Alzheimer's disease, daily total energy expenditures are nearly 15% lower (2214 \u00b1 460 kcal\/day versus 2590 \u00b1 497 kcal\/day) in patients with Parkinson's disease, when compared to controls.\n\nTanner and colleagues [62] have summarized three general gastrointestinal disorders in Parkinson's disease: motility disorders (including dysphagia and constipation), sialorrhea, and appetite disorders. Aspiration and malnutrition are common outcomes of these disorders. Because swallowing abnormalities and excessive drooling are often related, it is important that swallowing studies be performed to define the nature of the dysphagia and presence or absence of silent aspiration. Soft diets can help most types of dysphagia by making it easier to move foods through the mouth and esophagus. These foods can also minimize aspiration by reducing the need for separate fluid intake [113]. The use of feeding gastrostomies or jejunostomies should be the last alternative.\n\nIn the case of patients with Parkinson's disease, constipation management is important. This consists of dietary changes, exercise, and pharmacotherapy. High-fiber foods should be encouraged along with increased fluids (in particular, water) [114, 115], and exercise should be as vigorous as possible. Use of stool softeners can be added if given with meals. Patients with Parkinson's disease are also at risk for osteoporosis [116, 117]. Patients following any protein redistribution or restriction plan (discussed below) are often prone to inadequate calcium intakes [118, 119].\n\n### C. Protein Modified Diets in Parkinson's Disease\n\nAdvanced Parkinson's disease and prolonged levodopa treatment can cause patients to become more resistant to levodopa therapy [120]. Approximately 50% of patients with Parkinson's disease prescribed levodopa for 3 or more years experience motor-response fluctuations [121], an unpredictable ''on\" and \"off\" of motor function. Periods of drug unresponsiveness in Parkinson's disease may be due to the competition between the large neutral amino acids and levodopa across the blood brain barrier [120, 122\u2013125]. This competition decreases the availability of levodopa for the brain, thereby resulting in reduced ability to control motor functions. Although there is no specific definition, low-protein diets typically limit total daily protein intakes to 0.8 g\/kg body weight, which is lower than the average American intake of 1.6 g\/kg [124, 126\u2013128]. A protein-restricted or redistributed diet is one in which daytime protein intake is usually below 10 g with unrestricted evening intake. The rationale for this approach is that levodopa will be better absorbed and effective during the restrictive protein period when mobility is most desired. Other factors may also play a role in the effectiveness of levodopa treatment, such as time between levodopa administration and the meal, frequency of meals, usual dietary intake of energy, carbohydrates, fat, or fiber. Unfortunately, little systematic study of factors other than protein intake has been addressed by investigators.\n\nLow-protein and protein redistribution studies have been associated with positive effects for some patients suffering from \"on\u2013off\" fluctuations. In a double-blind crossover study, Tsui and coworkers [124] compared the motor performance and blood levels of levodopa in 10 fluctuating patients with Parkinson's disease on high-(80 or 70 g protein\/day for men and women, respectively) and low-protein milkshakes (50 or 40 g protein\/day for men and women, respectively). For 5 days, subjects were given either a high-or low-protein milkshake for breakfast and lunch and ate a regular diet in the evening. After a 2-day rest period, the subjects were given the alternate milkshake for another 5 days. Although plate waste studies were done, no record was made of fat, carbohydrate, or fiber content of the participants' diets other than the amount found in the milkshakes (Table 5). Seven out of 10 patients reported a statistically significant improvement ( _p_ < 0.05) in performance while on a low-protein diet, but blood levodopa levels did not differ. Because the intervention was so short and intake data are incomplete, no firm conclusions can be drawn concerning the efficacy of this dietary manipulation for patients with Parkinson's disease suffering from motor oscillation.\n\nTABLE 5\n\nNutrient Content of Milkshakes Provided in a Crossover Study\n\naValues are per 100-mL milkshake.\n\n_Source:_ Adapted from Tsui, J. K., Ross, S., Poulin, K., Douglas, J., Postnikoff, D., Calne, S., Woodward, W., and Calne, D. B. (1989). The effect of dietary protein on the efficacy of L-dopa: A double blind study. _Neurology_ **39,** 549\u2013552 [124].\n\nCarter and coworkers [127] also looked at the effectiveness of protein restriction and redistribution in five elderly patients. Subjects were prescribed a high-protein diet (1.6 g\/kg\/day) for 1 day and then changed to either a protein-restricted diet (0.8 g\/kg body weight\/day consumed throughout the day) or a protein-redistributed diet (0.8 gm\/kg body weight\/day in which 90% of the protein is consumed at night) for 3 consecutive days. Patients experienced significantly better motor performance and reduced levels of plasma large neutral amino acid concentrations ( _p_ < 0.001) on the protein-restricted diet and protein-redistributed diet than on a high-protein diets. During the day, the patients on the protein-redistributed diet had the most ''on\" time as compared the protein-restricted diet and the high-protein diet (77%, 67%, and 51%, respectively). Although these investigators reported dietary intakes of energy, protein, carbohydrate, and fat, no information regarding fiber intakes or background dietary patterns was provided. Again, these findings suggest that such strategies may provide some additional motor control, but further research that carefully defines the dietary patterns of patients prior to the test diet and careful dietary monitoring with long-term follow-up is warranted.\n\nThe relative amounts of carbohydrate and protein in foods consumed together may also influence how much levodopa enters the brain and, thereby, may modify unpredictable fluctuations [128]. Nine men suffering from unpredictable motor oscillations (mean age of 60) were fed three diets in which the carbohydrate-to-protein ratio was varied: 21:1, 5:1, and 0.3:1; a crossover design was used [128]. Plasma levels of large neutral amino acids were 24% higher and 18% lower on the 0.3:1 and the 21:1 diets, respectively (p<0.001) as compared to those when on the 5:1 plan. Consumption of the 0.3:1 diet caused increased Parkinsonian symptoms while with the 21:1 diet, patients became dyskinetic. A decrease in motor performance, assessed by decreased ability to place pegs in a peg board and a decrease in accuracy in writing, was also observed in patients on the 0.3:1 and the 21:1 diets. The patients on a 5:1 diet maintained stable plasma large neutral amino acids (<3% change), while motor performance on the pegboard and their writing improved. Based on these outcomes, the researchers concluded that the 5:1 diet was associated with improved clinical performance and recommended further study with manipulations. Further work by Feldman [129] and O'Brien [118] confirm that in the short-term, milkshakes with a 5:1 or 7:1 provide enhanced motor function. However, no evidence is available concerning the likelihood of long-term compliance with such dietary plans or whether the observed motor improvements can be sustained. Pare and coworkers [119] examined the impact of protein-restricted diets in patients with Parkinson's disease who were prescribed these dietary plans (maximum of 10 g protein before evening) on nutritional adequacy based on food records completed by the study participants. Calcium, iron, phosphorus, and niacin intakes were marginal (Table 6). We have also found that, in designing a strict 7:1 dietary plan, a multivitamin multimineral supplement is indicated [130].\n\nTABLE 6\n\nMean Energy and Nutrient Intakes in Patients with Parkinson's Disease during Usual Diet and Protein-Restricted Diet Periods Effect of daytime protein restriction on nutrient intakes of free-living Parkinson's disease patients. _Am. J. Clin. Nutr._ **55,** 701\u2013707 [119].\n\naValues represent mean \u00b1 standard deviation.\n\n_Source:_ Adapted from Pare, S., Barr, S. I., and Ross, S. E. (1992).\n\nFinally, data are very limited regarding the value of fiber-rich diets and an improvement in motor skills of patients with Parkinson's disease with unpredictable fluctuations. Nineteen fluctuating patients with Parkinson's disease (13 women and 6 men) suffering from constipation consumed a 28 g fiber\/day diet [131]. A supplement of 18 g dietary fiber (made of wheat, pectin, and dimethylpolyoxylhexane) was mixed with water and administered to all the subjects in addition to 10 g fiber consumed in their diet. Disability scores were measured using the United Parkinson's Disease Rating Scale motor exam before the study began, at 2 weeks into the study, and at the end of 2 months. With significant improvement ( _p_ <0.0001) in frequency of bowel movements came borderline significant improvement in gait ( _p =_ 0.05) and reduction in 3\u20130-methyl dopamine ( _p_ = 0.034). The compound 3-O-methyl dopamine reflects the amount of dopamine metabolized in the periphery. The investigators suggest that levodopa absorption was accelerated by increased gastric motility and shortened gastric emptying, which may improve clinical response [131]. Unfortunately, dietary intakes prior to the intervention as well as those during the intervention were not recorded, so again, this study cannot provide support for a unique role of such diets in motor performance.\n\n## VI. SUMMARY\n\nThere is definitely a need to explore the possible protective and negative associations between specific exposures of both food and nutrients as well as overall usual dietary patterns (quantified by tools such as the Healthy Diet Indicator [36], the Healthy Eating Index [132] or the Dietary Quality Index\u2013Revised [133]) and cognitive performance. Because further information is needed to estimate intake levels associated with change in cognitive performance, several ongoing longitudinal studies provide a unique opportunity to examine these factors in their study populations. We await future reports from many of these studies, including the Rotterdam, Iowa Women's Health, SENECA, and CHAP studies. 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INTRODUCTION\n\nIn 1990 osteoporosis was redefined, for the first time in nearly a century, as a condition of skeletal fragility due to decreased bone mass and to microarchitectural deterioration of bone tissue, with consequent increased risk of fracture [1]. This definition was conceptually important because it both acknowledged and encouraged a shift in thinking about osteoporosis from an anatomic to a dynamic condition. Low bone mass became a risk factor for fracture, rather than, as formerly, the defining characteristic of the disease. This redefinition accompanied a growing recognition that osteoporosis is not a single disorder, but a group of more or less discrete fracture syndromes, multifactorial both in etiology and in pathogenesis.\n\nThe recognition not only of a multiplicity of pathogenetic factors, but of _disease_ heterogeneity adds another dimension of complexity that must be considered when describing and assessing the role of any single factor, whether hormones, exercise, or nutrition (as in this case). Thus, not only is nutrition just one of several interacting factors in any given fracture syndrome, but it may play quite different roles, or none at all, in certain of those syndromes, while being of greater importance in others. This was first suggested in the 1979 report by Matkovic and his colleagues [2] from Croatia, which showed that high calcium intake was associated with strikingly reduced hip fracture risk, but not with altered risk of distal forearm fracture in the same population.\n\n## II. NUTRITION IN THE OSTEOPOROTIC FRACTURE CONTEXT\n\nNutrition affects bone health in two qualitatively distinct ways. Bone tissue deposition, maintenance, and repair are the result of cellular processes, and the cells of bone responsible for these functions are as dependent on nutrition as are the cells of any other tissue. The production of bone matrix, for example, requires the synthesis and post-translational modification of collagen and an array of other proteins. Nutrients involved in such synthesis include protein, the vitamins C, D, and K, and the minerals copper, manganese, and zinc. Phosphorus also is indirectly involved in these cellular activities. Additionally, the skeleton serves as a very large nutrient reserve for two minerals, calcium and phosphorus, and the size of that reserve (i.e., the massiveness of the skeletal structures) will be dependent in part on the daily balance between absorbed intake and excretory loss of these two minerals. Bone mass is also dependent on a variety of non-nutritional factors, such as genetics, mechanical loading, hormonal status, and others. These dependencies complicate the interpretation of low bone mass values because, while low bone mass always means a reduced calcium reserve, simple reduction in bone mass does not necessarily mean that it had a nutritional cause.\n\nFactors involved in osteoporotic fractures can be organized hierarchically to include the injury itself, the strength of the bone, the mass and density of the bone, and the adequacy of nutrition as it affects bone mass. Hip fracture is perhaps the most serious of the fragility fractures, inasmuch as it carries an excess mortality, is expensive, and causes significant deterioration in quality of life for most of its survivors. It is, as well, a good example of the many interacting factors that constitute this fracture domain, and I will use it as such in this preliminary overview. Figure 1 illustrates, schematically, how the various contributing factors interact for hip fracture. It also highlights probable sites in this schema at which nutrition plays a role.\n\nFIGURE 1 Schematic representation of the interplay of principal factors thought to be important in hip fracture. Asterisks denote factors with a recognized nutritional determinant. (Copyright Robert P. Heaney, 1995. Reproduced with permission.)\n\n### A. Frailty and Injury\n\nAlmost all fractures, even those we term _low trauma,_ occur as a result of some injury\u2014the application of more force to the bone than it is able to sustain. Usually this is a result of a fall or the application of bad body mechanics (e.g., bending forward to lift a heavy object). Although fracture incidence patterns differ somewhat from site to site, the risk of virtually all fractures rises with age, and all fractures contribute to the burden of illness, disability, and expense that the elderly (and society) bear.\n\nThe first factor to consider is the fall itself. Normally postural reflexes operate to get the arms into position to break the force of the fall or to swing the body so that it lands on the buttocks (or both). These reflexes are almost always effective in younger individuals, but they commonly fail in the elderly. As a result, young people rarely strike the lateral portion of the trochanteric region of the hip when they fall, whereas the fragile elderly more commonly do so. The force of the impact, when falling from standing height, may well be sufficient to breakeven a healthy femur if that force is concentrated in a small enough impact area [3]. Additionally, hip fracture is a particularly serious problem in undernourished elderly individuals who have less muscle and fat mass around the hip, and therefore less soft tissue through which the force of the impact can be distributed to a larger area of the lateral surface of the trochanter.\n\nNutrition enters into this region of the fracture domain both through its effect on propensity to fall [4] and on maintenance of the soft tissue mass. This latter factor, particularly, is the rationale for the development and successful deployment of hip pads as protection against hip fracture in the elderly [5]. In some cases, nutrition may also influence central nervous system processing time or contribute to the general feebleness that predisposes to falling. The implication here is that we should attempt to improve general nutrition in the elderly or, failing that, we should certainly attend to coexisting nutritional problems at the time of fracture repair.\n\n### B. Intrinsic Bony Strength and Fragility\n\nStrength in bone, as in most engineering structures, is dependent on its mass density, on the three-dimensional arrangement of its material in space, and upon the intrinsic strength of its component material (particularly, in bone, as that strength is influenced over long periods of use by the accumulation of unrepaired fatigue damage). All three factors play some role in most low-trauma fractures, and it is not possible to say which may be the most important in any given case. Nevertheless, most of the investigative effort in this regard in the past 30 years has been devoted to the measurement of bone mass and density, and hence much of what we know about bone strength in living individuals comes from our observation of this facet of the bone strength triad. There is, in fact, a general consensus that decreased bone mass produces a decrease in bone strength.\n\nBut, clearly, other fragility factors exist as well, although there is less of a consensus as to how large a role they play [6]. The data of Ross _et al._ [7] show that prior spine fracture signifies the presence of fragility independent of, and at least as, important as the fragility due to low bone density. Similarly Hui _et al._ [8] showed that the fracture risk gradient for age, holding density constant, was greater than the risk gradient for density itself. These effects, independent of bone mass, may be partly explained by structural and qualitative defects in bone.\n\nFor example, individuals with compression fractures of the vertebrae have been found to have excessive loss of horizontal, cross-bracing trabeculae in their cancellous bone [9, 10], whereas other individuals with the same overall degree of bone loss, but with the bracing trabeculae maintained intact, are less apt to fracture. This may be the basis for the predictive value of prior spine fracture [7]. It appears that women, in particular, are more prone to loss of horizontal trabeculae than are men, and this fact is probably also the explanation for the 6:1 to 8:1 female:male sex differential in vertebral osteoporosis. The data of Eventov _et al._ [11] indicate the probable importance of repair of fatigue damage. Faulkner _et al._ [12] and Gl\u00fcer _et al._ [13] have called attention to a probable role of geometric factors at the hip, specifically to hip axis length, and Gilsanz _et al._ [14] to the importance of vertebral body size.1 In summary, evidence from several quarters makes it clear that bony fragility has bases other than reduced bone density.\n\nNevertheless, fracture risk rises by a factor in the range of 1.5\u20132.5 times for every drop in bone mass\/density of one standard deviation. And whatever may be the role of nonmass factors, it is an inescapable fact that most elderly individuals have bone mass values that are more than two standard deviations below the young adult mean; hence, they all can be said to be at considerably increased risk for fragility fracture. Why some older persons do fracture and others do not appears to be explainable by a combination of random chance, differences in falling patterns, and the structural differences just described.\n\nNutrition enters into this portion of the fracture domain predominantly through its influence on bone mass (or density). Because nonmass factors also influence bone strength, nutritional inadequacies can never explain more than a part of the problem, and nutritional interventions can never completely eliminate fragility fractures. It may also be that trace nutrients such as certain of the vitamins (e.g., C, D, K) or minerals such as manganese, copper, and zinc (see below) directly influence the remodeling process and\/or the character of the remodeled bone, and hence affect bone strength through their impact on the repair of inevitable fatigue damage. However, little is known about these possibilities in the adult skeleton. Hence, in most of what follows, the emphasis will be on the nutritional factors that influence bone _mass._\n\n### C. Bone Mass and Density\n\nBone mass and density are themselves influenced by many factors. Holding body weight constant, the three most important\u2014or at least the three most commonly found to be limiting in industrialized nations\u2014are physical activity, gonadal hormones, and nutrition. In adults of industrialized nations the nutrients most critical for bone health are calcium and vitamin D. Calcium intake, specifically, may be inadequate for the straightforward reason that it is low; however, even when statistically \"normal,\" it may still be inadequate because of subnormal absorption [16] or greater than normal excretory loss [17, 18]. Other nutrients are also essential for building a healthy skeleton, but, except for calcium, their effects are usually seen most clearly during growth. Once built, the skeleton tends to be relatively insulated from many subsequent nutritional deficiencies. In addition, a number of other factors also influence bone mass, such as smoking, alcohol abuse, and various drugs used to treat a variety of medical illnesses, as well as those illnesses themselves.\n\nThe effects of each of these factors are largely independent. In other words, altering any one of them will not substitute for, or compensate for, adverse effects of the others. Thus, a high calcium intake will not prevent the loss of bone that occurs immediately following menopause in women or castration in men. Similarly, physical activity will not compensate for an inadequate calcium intake. Neither will a high calcium intake offset the effects of alcohol abuse or smoking. Much of the apparent confusion in the bone field during the past 25 years could have been avoided if we had better understood that these factors, while interactive, are substantially independent.\n\nFinally, although much of the following discussion will focus on calcium, it is necessary to stress what should perhaps go without saying, that calcium is a nutrient, not a drug, and hence its beneficial effects will be confined to individuals whose intake of calcium is insufficient. Also, calcium is not an isolated nutrient; it occurs in foods along with other nutrients, and it has been shown that diets low in calcium tend also to be inadequate in other respects as well [19, 20]. Thus, while it is necessary to deal with nutrients one at a time in an analysis such as this, the disorders in our patients are likely to be more complex.\n\n## III. PROBLEMS IN THE INVESTIGATION OF NUTRITIONAL EFFECTS ON BONE\n\nSignificant problems arise for both observational and experimental approaches to the elucidation of nutrient effects on the skeleton, and failure to recognize or overcome them has led both to seemingly contradictory results among various studies and to substantial confusion about the role of nutrition in bone health. Some of these problems are nutrition specific; others are inherent in bone biology.\n\n### A. Nutrition-Specific Problems\n\n1. ESTIMATION OF NUTRIENT INTAKE\n\nTwo nutrients with clearly established effects on bone are vitamin D and calcium. For both, there are substantial difficulties in estimating intake [21, 22]. Vitamin D is found naturally in very few foods (mostly fish oils and, to a limited extent, egg yolks).2 For primitive humans, solar exposure would have been the principal source of vitamin D, as is still the case in rural cultures and in the young of even many urbanized societies. Vitamin D is added as a fortificant to fluid and dry milkin the United States and Canada (but not to most other dairy foods).3 Serum 25(OH vitamin D levels have long been recognized as the best available indicator of vitamin D status. Even so, their significance is not fully clear [26]. Furthermore, such levels are affected by season, so no single value in any given individual adequately captures his or her year-round average. Such measurements are also sufficiently costly and invasive so as to be precluded in most epidemiological studies involving large numbers of subjects. Finally, while vitamins D2 and D3 have heretofor been considered equivalent in potency (and both measured and used as a fortificant interchangeably), Vieth [27] has shown that D2 exhibits only 60% the potency of D3 in humans.\n\nCalcium also presents serious difficulties to the investigator who would attempt to estimate its intake. Food calcium content often varies widely from published food table values\u2014sometimes by a factor of 2\u20133\u2014reflecting variations in soil mineral content and plant tissue hydration (among other factors). Even commercial milkexhibits 10\u201320% variability from dairy to dairy or state to state. Charles [28] found, in a chemical analysis of foods consumed in a series of metabolic balance studies, that less than 70% of the actual variability in intake among a group of subjects was reflected in the _calculated_ intakes derived from food table values for the foods consumed, despite the fact that the precise quantities of every food eaten were known with high accuracy. Outside of the metabolic ward environment, and particularly in epidemiological studies, there is the added uncertainty of portion size estimation and food item recall. Another problem is presented by large differences in bioavailability. The calcium of kale or collard greens is highly available [29], while that of spinach is nearly totally unavailable [30]. Thus _actual_ intake and _effective_ intake can differ substantially. Finally, broad daily and seasonal variations are seen in intake patterns. In this regard, Heaney _et al._ [31] showed, in a large series of 7-day consecutive diet records, that any random day elected out of the total record captured only 12.6% of the interday variance, and that the error of the estimate of the 7-day average from one of its days was \u00b1178 mg (which means that the 95% confidence interval covers a range of more than 700 mg!).\n\nThe difficulty of estimating effective calcium intake is compounded by two further problems. First is the use of calcium salts as excipients or \"inert\" ingredients in many medications or as non-nutritive additives to various bulk foods. In both cases their calcium content goes unrecognized and often unacknowledged on the product label. Second is the increased use of explicit calcium supplements since 1982. This should not, of itself, create a problem for estimating calcium intake. However, many tablets in the past exhibited highly variable pharmaceutical formulations [32, 33], and, hence, unpredictable absorbability. Excipient calcium will not often produce major errors in intake estimates unless food source intakes are low (in which case undocumented medication calcium can easily account for half the actual calcium intake); nevertheless Heaney _et al._ [31] reported several cases in which such unrecognized calcium contributed more than 1000 mg\/day to the intake.\n\nIn any event, both causes can lead to serious misclassification of individual intakes in observational or epidemiological studies, therefore biasing toward the null any investigation dependent on intake estimates. An illustration of the effect of this bias is found in a meta-analysis by Heaney [34] of 28 studies in late postmenopausal women published between 1988 and 1992. Twenty-three of these 28 studies reported a positive effect of calcium intake on bone mass, bone loss, or fracture. However, when they were subdivided according to whether the investigators controlled the calcium intake directly or relied on estimates of intake derived from questionnaires and food records, it turned out that all of the 12 studies in which investigators controlled the intake had demonstrated a significant calcium benefit, while all of the inconclusive studies had been those in which intake had been merely estimated. The difference is explainable by errors in intake estimates in the questionnaire-based studies.\n\n#### 2. MAGNITUDE OF NUTRIENT\u2013NUTRIENT INTERACTIONS\n\nIt is a commonplace of nutritional science that nutrients interact, thereby altering one another's requirements.4 It is to be expected, therefore, that the nutrients important for bone health would also exhibit this sort of interaction. What may not have been expected is the very considerable magnitude of those interactions with regard to critical bone nutrients. Coingested nutrients alter both obligatory renal loss and intestinal absorption of calcium and phosphorus. While effects on absorption are comparatively modest, effects on obligatory loss can alter the minimum daily requirement for calcium very substantially. (These effects are covered in more detail later in this chapter.)\n\nFor our purposes here, it is sufficient only to note that other nutrients, ingested within the normal range of human intakes, so alter ability to maintain calcium equilibrium as to produce a fourfold difference between the lowest and the highest values for the minimum requirement. This is a quite extraordinary range and is virtually without parallel among other nutrients. It is for this reason that it is usually misleading to make comparisons among populations that may differ not only in calcium intake, but in intakes of protein and sodium particularly, as well as in the proportion of animal and vegetable food sources in the customary diet. It is likely that much of the seeming differences in the relationship of calcium to bone status across populations [35] can be attributed to differences in minimum requirement related to nutrient\u2013nutrient interactions, and much of the apparent confusion surrounding this topic attributed to failure to give adequate consideration to the influence of these interactions.\n\n### B. Bone-Specific Problems\n\n1. THE BONE REMODELING TRANSIENT\n\nThe bone remodeling transient is dealt with in greater depth elsewhere [36, 37]. It is important to mention it briefly in this context because, whenever bone remodeling is altered by an intervention (nutritional in this context), the changes in calcium balance or bone mass that follow will, for a period of 6\u201312 months, reflect not the effects (if any) of the intervention on _steady-state_ bone balance, but shrinkage or expansion of the bone remodeling space caused by asynchrony of the changes produced in bone formation and resorption.\n\nThis is a particular problem for calcium, because calcium alters endogenous parathyroid hormone production, and parathyroid hormone is the principal determinant of the amount of global skeletal remodeling. But any other nutrient (such as vitamin D or phosphorus), that also alters parathyroid hormone production (whether directly or indirectly) may produce qualitatively similar effects.\n\nThus, the classical stratagem of measuring nutrient balance in individuals on differing intake levels for periods of up to a few weeks, then giving them a short rest period, then trying yet another intake for a few more weeks (and so forth), will not workfor bone or its measurable surrogates. Unfortunately, there are no easy alternatives. Balance for nutrients that are bulkbone constituents can be assessed only under steady-state conditions, and for calcium, that means either studying persons on their habitual intakes, or deferring study for 6\u201312 months after altering intake of a given nutrient. Both options severely limit what the investigator can do to test various hypotheses involving nutrition and bone status.\n\n#### 2. ISOLATION OF BULK BONE FROM CURRENT NUTRITIONAL INFLUENCES\n\nBone is very much a living tissue, with its cells responding both to systemic influences and to strain patterns within the bony structure. Nevertheless, the mechanical properties of bone reside exclusively in the intercellular, nonliving, two-phase composite of fibrous protein and mineral. With the exception of use-related, accumulating fatigue damage, the inherent mechanical properties of this material are largely (though not entirely) determined at the time a unit of bone is formed. The entire skeleton is turned over at a rate of only 8\u201310% per year (and some regions much more slowly). Because only currently forming bone will be affected by current conditions, nutritional stresses have predictably small effects on current bone strength. The bulk of bone is, in effect, isolated from the systemic and environmental influences that can rapidly produce outspoken effects in soft tissues. This is not to say that there are _no_ effects on bone. It is possible that bone cells, damaged by current nutritional problems, may die or otherwise fail in one or another of their monitoring functions. But the effects of that failure may become evident only years in the future, and they are, accordingly, extremely difficult to study.\n\n#### 3. SLOW RESPONSE TIME OF BONE\n\nA corollary of the slow turnover of bone tissue is that bone mass changes relatively slowly in response to nutritional influences, either positive or negative. A gain or loss of bone amounting to at most 1\u20132% per year is typically all that many interventions can produce in adults. Continued over many years, such a rate of change can have profound effects on skeletal strength, but it is a change that is hard to detect by absorptiometric methods in short-term investigations, and essentially impossible to detect reliably in individuals. Although the gain or loss associated with a nutritional intervention may be real enough, its presence is dwarfed by the relatively huge mass of preexisting bone, and its detection tends to be swamped by the inevitable noise of measurement. Balance studies can sensitively detect much smaller changes (since the background bone mass is not reflected in the balance value), but they are subject to the problem of the remodeling transient discussed earlier, and sufficient time must be allowed for the system to come into equilibrium if they are to be useful. Serum and urine biomarkers can sensitively signal qualitative changes in bone remodeling processes, but they are not sufficiently quantitative to tell us the size of any change in bone balance that may have been produced by an intervention.\n\n#### 4. LIFE PHASE SPECIFICITY OF BONY RESPONSE\n\nAs will be developed in more detail below, the skeleton is the body's reserve of the nutrient, calcium. It is the largest reserve of all the nutrients, and one that has acquired an unrelated function in its own right\u2014the mechanical support of our bodies. While bone strength is clearly an inverse function of bone density, and any decrease in bone density must have mechanical consequences, nevertheless reserves, by their very nature, are designed to be called on, and it should not be surprising to find that there may be physiological circumstances in which the reserve will reduce some of its store of mineral, not always because the diet is insufficient to offset excretory and dermal losses, but precisely because the physiological situation demands it or because the body senses that some of the reserve is no longer needed. Lactation may be one such situation and menopause another. In any event, nutritional interventions should be expected to produce qualitatively different effects when they are deployed under such differing physiological circumstances.\n\n#### 5. COMMENT\n\nThis discussion of investigative problems is, of necessity, brief. My purpose has been to highlight the inherent difficulty involved in investigating problems at the interface of nutrition and bone status. Failure of bone biologists to recognize nutritional measurement problems and failure of nutritionists to reckon adequately with the complexities of bone biology will lead (and has led) to badly designed, inconclusive, or misleading investigations. This is a problem not only for investigators, but for those who attempt to make sense of what they report. It is not that easy alternatives are being overlooked. Rather, there are no easy alternatives. But, while the problems are difficult, they are not intractable.\n\n## IV. THE NOTION OF A NUTRIENT REQUIREMENT\n\nNutritional science was born about a century ago with the then-revolutionary recognition that the absence of something could produce disease.5 Once nutritional deficiency was accepted as the cause of disease the notion of a requirement centered on the intake needed to avoid the recognizable deficiency disease concerned. While the science of nutrition has advanced notably since its beginnings, particularly in understanding precisely what various nutrients do in the body, our definitions of a requirement are still often pegged to early 20th-century ability to recognize and characterize disease. There is growing dissatisfaction with this disease-centered approach, and increasingly one reads that the field ought to redefine a requirement as the intake needed to produce optimal health. But the main problem with the traditional approach to a requirement is not that it is negative (i.e., disease centered) but that its definition of disease is primitive. It is centered on disorders that develop rapidly and have distinct clinical expression recognizable with the tools of 70 years ago. However, a deficiency that takes 10 years to develop or to make its presence evident is no less a deficiency than one that develops in 10 days.6\n\nVitamin K deficiency, for example, produces a bleeding disorder, and this is the defining disease associated with the nutrient. Does absence of bleeding mean that vitamin K nutriture is adequate? We now recognize that vitamin K is necessary for gamma-carboxylation of a large number of proteins in addition to the clotting factors, three of them involved in bone matrix (see section below on vitamin K). We also recognize that gamma-carboxylation of these proteins can be very incomplete even when the clotting factors are normally carboxylated, and that physiological vitamin K supplementation completely repairs this deficit. It is not known whether this undercarboxylation expresses itself as disease, but our ignorance in that regard does not guarantee that the absence of clotting disturbance means vitamin K sufficiency.\n\nIn this chapter I define a requirement as the intake that ensures full expression of known functions of the nutrient concerned, and I will presume that any substantial deviation from full physiological expression is harmful until proved safe.\n\n## V. THE NATURAL INTAKE OF CALCIUM AND VITAMIN D\n\nIt has only recently been recognized that both calcium and vitamin D were present in superabundance in the environment in which the human species evolved. It seems likely that, over the millennia of evolution, human physiology developed mechanisms to protect the organism from getting too much of these important nutrients. By contrast, contemporary adult humans, living in industrialized nations at higher latitudes, have intakes of these nutrients that are often only a small fraction of what their primitive ancestors experienced, and our physiologies are, therefore, maladapted to what our environment currently provides.\n\nVitamin D is produced normally in the skin by a photochemical reaction in which ultraviolet light from the sun changes 7-dehydrocholesterol into previtamin D. As the human species evolved in equatorial East Africa, with ample sunlight year-round, two mechanisms coevolved that prevented the accumulation of an excess of vitamin D. One was skin pigmentation, which slowed the photochemical reaction, and the other was the fact that continued solar radiation degrades previtamin D to inert products before it is taken up into the circulating blood. As a result, vitamin D accumulation in the skin plateaus after a few minutes of sun exposure, with the time varying with skin pigmentation. Circulating levels of 25(OH) vitamin D under early conditions can be estimated from values observed in dark-skinned, outdoor laborers at tropical latitudes, which have been reported to be in the range of 150 nmol\/L [27] or \u223c4\u20136 times what is typically measured in city dwellers at midlatitudes.\n\nAs humans moved farther and farther north (away from the equator), and needed all the ultraviolet they could get, skin pigmentation became lighter and lighter. Still, in latitudes such as that of Boston and farther north, the sun is so low in the sky in winter that effectively none of the responsible ultraviolet rays gets through the atmosphere, even on a sunny day [38]. As a result, vitamin D tends to be a scarce nutrient at high latitudes, and without careful attention to maintaining adequacy, varying degrees of vitamin D insufficiency will be common. Just 75 years ago, more than 80% of the children in England showed evidences of rickets [39]. Thanks to nearly universal vitamin D prophylaxis in children, rickets is now a relatively rare disorder.\n\nCalcium, too, was present in abundance in the environment in which the human species evolved. The plant foods eaten by hunter-gatherers provided a calcium intake that, adjusted for differences in body size, would have been in the range of 2000\u20134000 mg\/day for 60\u201370 kg adults [40, 41]. (Contrast that figure with the median value for women 20 and over in the United States in the NHANES III study: in the range of 600 mg\/day [42].) Sources available to our ancestors included a very large number of greens, tubers, roots, nuts, and berries, many of them with very high calcium nutrient densities [41]. Moreover, invertebrate and reptilian sources of animal protein typically have calcium-to-calorie ratios that are six-fold higher than fish or mammalian meats [43]. By contrast, cultivated cereal plants, legumes, and fruits\u2014the plant foods modern humans mainly consume\u2014exhibit augmented levels of carbohydrate and\/or fat without a proportionate increase in minerals and vitamins; thus, they almost always have lower calcium densities than do their wild cousins.\n\nThe agricultural\/pastoral revolution, which occurred from roughly 3000 to 10,000 years ago in various parts of the world, made it possible to feed vastly more people than the hunter-gatherer mode permitted. This was partly because of the increased energy content of polypoid cereal mutants (which occur spontaneously, but which, because of their greater seed weight, need human intervention for their efficient propagation). At the same time the agricultural revolution produced striking changes in micronutrient intake, generally for the worse. We see this reflected in modern times in the nutritional deficiencies that result when hunter-gatherers such as the !Kung San people are forced by restriction of their range to take up farming [44].\n\nThe effect on the calcium density of the diet is depicted in Figure 2. Diets of hunter-gatherers would have been in the range of 70\u201390 mg calcium\/100 kcal (somewhat higher if invertebrate protein sources featured prominently in the diet). Those who then domesticated animals and lived mainly off their milk(as do the Masai, the Ariaal, and, indeed, all pastoralist societies today) would have had a shift in diet to somewhere in the neighborhood of 200 mg calcium\/100 kcal.7 By contrast, those who settled on the land and subsisted mainly on cereal crops and legumes would, at least from these food sources alone, have had diets with calcium densities under 20 mg\/100 kcal. While vegetable greens would have helped when available, calcium intakes based solely on cereals and legumes would probably not have been sufficient to sustain bone health. However, there are numerous, well-attested examples of peoples living in stable equilibrium with their environments who have developed nonfood ways of augmenting the meager calcium intake provided by a diet based on seed foods. The addition of lime to corn meal by indigenous peoples in Central America is one well-known example. Less well known is the practice of pregnant Southeast Asian women of drinking a liquid produced by soaking bones in vinegar [45]. Andean Indians have been reported to add both a particular plant ash and a heat-treated rock powder to their cereal gruel [46]. All of these practices represent a kind of conscious addition of a substance that, _de facto,_ augmented the calcium intake of a cereal-based diet.\n\nFIGURE 2 Changes in calcium concentration of the diet associated with the agricultural\/pastoral revolution. (Copyright Robert P. Heaney, 1995. Reproduced with permission.)\n\nIt seems likely that _unconscious_ additions of the same sort were nearly universal among neolithic farming communities. The archaeological record has preserved numerous examples of stone mortars used for dehulling cereal grains and stone querns for grinding the seeds into flour [47]. In the fertile crescent at least, limestone would have been the most readily available and the most workable stone, and the hours of hand grinding of dehulled cereal grains would inevitably have added substantial calcium (as calcium carbonate) to the resulting flour.8 As technology advanced, and millstones were made of harder and harder rock(usually silicon- and aluminum-based rather than calcium-based minerals), aggregate calcium intakes would have declined toward the lower line depicted in Figure 2. Thus, the low calcium intakes that we take for granted today are relatively quite late arrivals on the human diet scene.\n\nBecause hominid and early human diets were very rich in calcium, the human intestine either failed to develop effective absorptive transport mechanisms or actually developed an absorptive barrier to protect against too much calcium. Nor did mechanisms to conserve absorbed calcium develop. (Presumably, there would be little need to conserve in the face of environmental surfeit.) Humans typically absorb only about 25\u201335% of the calcium in contemporary diets [48] and put about 150 mg\/day back in to the gut in the digestive secretions [49]. Thus, net absorption of a dietary calcium increment is usually in the range of 10\u201315% even during growth when skeletal need is greatest [50]. Additionally, dermal losses are completely unregulated and renal conservation is limited as well. These are precisely the physiological patterns one would expect with an environmentally abundant nutrient.9\n\nThis is the background to why, despite a high standard of living and the potential to nourish ourselves at a level never previously achieved in the history of the race, civilized diets tend to be deficient in precisely these two critical nutrients, calcium and vitamin D.\n\n## VI. CALCIUM\n\nA. The Skeleton as a Nutrient Reserve\n\nThroughout the course of vertebrate evolution, bone developed several times and has served many functions, such as dermal armor and internal stiffening [51]. Evidence from a variety of lines suggests that the most primitive function of the skeleton is actually to buffer the internal milieu for several essential minerals, notably calcium and phosphorus [52]. In some species, phosphorus would have been the critical element; in others, calcium. For both nutrients, the skeleton serves both as a source and as a sink, that is, as a reserve to offset shortages and, to a limited extent, as a place for safely storing surpluses.\n\nWe see this reserve feature of skeletal function expressed in diverse ways. For example, there is the long-established fact that laboratory animals such as cats, rats, and dogs will reduce bone mass as needed to maintain near constancy of calcium levels in the extracellular fluid [53\u201355]. This activity is mediated by parathyroid hormone and involves actual bone destruction, not leaching of calcium from bone. When calcium-deprived animals are parathyroidectomized, bone is spared, but severe hypocalcemia develops [56]. More physiologically, perhaps, deer temporarily increase bone resorption each year to meet the calcium and phosphorus demands of annual antler formation (which exceed the nutrient supply of late winter and spring foliage) [57]. Finally, we see the opposite side of the same function expressed in the now well-established fact that augmented calcium intake will slow or reduce age-related bone loss in humans (see below).10\n\nWhile retaining this primitive, reserve function, bone in the higher, terrestrial vertebrates acquired a second role, namely, internal stiffening and rigidity\u2014what is today the most apparent feature of the skeleton. As such, calcium (or phosphorus) is the only nutrient with a reserve that possesses such a secondary function (with the possible exception of the thermal insulation provided by energy reserves). Figure 3 presents this situation schematically and contrasts calcium with the bulk of other nutrients, depicting what happens for each when intake is curtailed. For typical nutrients, the reserve is first depleted, without detectable impact on the health or functioning of the organism. Then, after the reserve is exhausted and the metabolic pool begins to be depleted, clinical disease expresses itself. For some nutrients (e.g., vitamin A\u2014energy), the reserve can be quite large, and the latent period may last many months. But for others (e.g., thiamin, the reserve may be very small, and detectable dysfunction develops soon after intake drops.\n\nFIGURE 3 Schematic illustration of the relationship between health status and body depletion of a nutrient. (A) Depletion of body stores of a typical nutrient after placing the organism on a deficient intake. (B) In the pattern exhibited by calcium, the reserve is very large relative to the metabolic pool, but health, as reflected in skeletal strength, declines steadily as the reserve itself is depleted. (Copyright Robert P. Heaney, 1995. Reproduced with permission.)\n\nWith calcium, the reserve is vast relative to the cellular and extracellular metabolic pools of calcium. As a result, dietary insufficiency virtually never impairs biochemical functions that are dependent on calcium, at least in ways we can now recognize. However, because bone strength is a function of bone mass, it follows inexorably that any decrease whatsoever in the size of the calcium reserve\u2014any decrease in bone mass\u2014will produce a corresponding decrease in bone strength. We literally walkabout on our calcium reserve. It is this unique relationship that is both the basis for the linkage of calcium nutriture with bone status and the explanation why reduction in the size of the reserve is the sole defining characteristic of the major human calcium deficiency syndrome.\n\n### B. Defining the Requirement for Calcium\n\nUnlike other nutrients, the requirement for calcium relates solely to this secondary function, i.e., to the size of the calcium reserve, in other words, to total skeletal and regional bone mass. However, unlike energy, which can be stored as fat without practical limit, the size of the calcium reserve is limited, even in the face of dietary surfeit, by genetic and mechanical factors (see below). As a result, calcium functions as a threshold nutrient, much as does iron. This means that, below some critical value, the effect (bone mass for calcium, or hemoglobin mass for iron) will be limited by available supplies, while above that value, i.e., the _threshold,_ no further benefit will accrue from additional intake. This biphasic relationship is depicted schematically in Figure 4, in which the intake-effect relationship is depicted schematically in Figure 4A, and then exemplified in Figure 4B by data derived from a growing animal model. In Figure 4B, the effect of the nutrient is expressed directly as the amount of bone calcium an animal is able to accumulate from any given intake.\n\nFIGURE 4 Threshold behavior of calcium intake. (A) Theoretical relationship of bone accumulation to intake. Below a certain value\u2014the threshold\u2014bone accumulation is a linear function of intake (the ascending line); in other words, the amount of bone that can be accumulated is limited by the amount of calcium ingested. Above the threshold (the horizontal line), bone accumulation is limited by other factors and is no longer related to changes in calcium intake. (B) Actual data from two experiments in growing rats, showing how bone accumulation does, in fact, exhibit a threshold pattern. (Redrawn from data in Forbes _et al._ [59]. Copyright Robert P. Heaney, 1992. Reproduced with permission.)\n\nHowever, if _effect_ is broadened to mean ''any change whatsoever,\" then the diagram fits all life stages, even when bone may be undergoing some degree of involution. This generalized form of the threshold diagram is set forth in Figure 5, which shows schematically what the intake\/retention curves look like during growth, maturity, and involution. In brief, the plateau occurs at a positive value during growth, at zero retention in the mature individual, and sometimes at a negative value in the elderly. (Available evidence suggests that there are probably several involutional curves, with the plateau during involution at a negative value in the first 3\u20135 years after menopause, at zero for approximately the next 10 years, and then at increasingly negative values with advancing age.)\n\nFIGURE 5 (A) Schematic calcium intake and retention curves for three life stages. Retention is greater than zero during growth, zero at maturity, and may be negative during involution. Asterisks represent minimum daily requirement. (B) The involution curve only. Point B designates an intake below the maximal calcium retention threshold, whereas point A designates an intake above the threshold. (Copyright Robert P. Heaney, 1998. Reproduced with permission.)\n\nIn Figure 5B, which shows only a composite involutional curve, two points are identified: one below (B) and one above (A) the threshold. At A, calcium retention is negative for reasons intrinsic to the skeleton, whereas at B, involutional effects are compounded by inadequate intake, which makes the balance more negative than it needs to be. Point B (or below) is probably where most older adults in the industrialized nations would be situated today. The goal of calcium nutrition in this life stage is to move them to point A, thereby making certain that insufficient calcium intake is not aggravating any underlying bone loss.\n\nThe functional indicator of nutritional adequacy for such a threshold nutrient is termed _maximal retention_ and can be located in Figures 4A and 5A at the asterisks above the curves. The intake corresponding to this point represents the minimum daily requirement. Calcium retention in this sense is ''maximal\" only in that further intake of calcium will produce no further retention. (This is in contrast to treatment with hormones or drugs, which can sometimes produce further calcium retention.) This approach was used by the Food and Nutrition Board of the National Academy of Sciences for the first time in its development of recommended intakes for calcium in 1997 [60].\n\nMuch uncertainty and confusion have arisen in recent years about what the threshold intake may be for various ages and physiological states. With the 1994 Consensus Development Conference on Optimal Calcium Intake [61] and the report of the Panel on Calcium and Related Nutrients [60], the bulk of that confusion has been resolved. The evidence for the intakes recommended by the consensus panel is summarized both in the conference and panel reports and in recent reviews of the relationship of nutrition and osteoporosis [34, 62], and will be summarized only briefly in ensuing sections of this chapter.\n\nIt is worth noting, however, that the recommendations of the consensus conference, while expressed in quantitative terms, were basically qualitative: Contemporary calcium intakes in North America and Northern Europe, by both men and women, are too low for optimal bone health in Caucasian individuals. The most persuasive of the evidence leading to this conclusion came in the form of several randomized controlled trials showing both reduction in age-related bone loss and reduction in fractures following augmentation of prevailing calcium intakes [63\u201372]. But randomized controlled trials, at least as performed to date, are not well suited to dose ranging (largely because of the problem of the remodeling transient; see above). Hence, while the panel was convinced that prevailing intakes were too low, their recommended levels in several cases involved ranges, and were clearly prudential judgments, centered of necessity on intakes employed in the trials concerned.\n\nIt is instructive, therefore, to review all the recent recommendations, in concert with the evidence from balance studies (to be discussed further below). Table 1 sets forth these various recommendations. As can be seen, while the 1994 NIH recommendations are, for most ages, substantially higher than the 1989 RDAs [73], they are actually quite close both to values derivable from available balance studies and to the 1997 recommendations of the Food and Nutrition Board.\n\nTABLE 1\n\nVarious Estimates of the Calcium Requirement in Women\n\naFrom Ref. 73.\n\nbRecommendations for women as proposed by the Consensus Development Conference on Optimal Calcium Intake [61].\n\ncThe so-called \"adequate intakes\" of the new DRI values, multiplied by a factor of 1.2 to convert them into RDA format [60].\n\ndEstimates derived from published balance studies [50].\n\n### C. The Requirement at Various Life Stages\n\nIn excess of 140 studies have been published relating calcium intake to bone status as summarized and reviewed elsewhere [62]. In more than 40% of this total, the investigators controlled calcium intake, and essentially all of these studies showed that calcium intakes above the then-prevailing RDAs conferred a bone benefit. Even among the observational studies, in which calcium intake was not investigator controlled and could only be estimated, about 80% were positive. There is, thus, an overwhelming mass of evidence establishing the importance both of calcium for bone and of ensuring intakes higher than prevailing levels or former recommendations. What cannot easily be determined from controlled trials (as has already been noted) is the precise location of the intake threshold, i.e., the point where bony retention is maximal and further intake confers no further bony benefit. The following sections focus on estimating this intake level by age and physiological state.\n\n#### 1. GROWTH\n\nThe human skeleton at birth contains approximately 25 g calcium and, in adult women, 1000\u20131200 g. All of this difference must come in by way of the diet. Further, unlike other structural nutrients such as protein, the amount of calcium retained is always substantially less than the amount ingested. This is because, as already noted, absorption efficiency is relatively low even during growth, and because calcium is lost daily through shed skin, nails, hair, and sweat, as well as in urine and nonreabsorbed digestive secretions. The gap between calcium intake and calcium retention is larger than is generally appreciated. In the adult with a modest but repairable skeletal deficiency, only about 4\u20138% of ingested calcium is retained. While retention efficiency is generally higher during growth, even when bone accumulation is most rapid, less than half of the intake is actually retained\u2014ranging from a high of 40% in term infants to 20% in young adults [50]. Even premature infants, with a permeable gut membrane and a relatively huge mineralization demand, exhibit net absorption of less than 60% [74], and dermal and urinary losses mean that they retain even less than that figure. This inefficient retention is not so much because ability to build bone is limited but because, as noted elsewhere, human physiology is optimized to prevent calcium intoxication, not to cope with chronic shortage.\n\nAside from the obvious fact that one cannot store what one does not ingest, how does suboptimal calcium intake limit bone mass accumulation? Except in unusual circumstances, it is not through limiting bone deposition. In most animal experiments as well as human observations, low calcium intake probably does not limit the growth in bone length or breadth. This is because bone-forming sites do not ''see\" the diet. They are exposed only to circulating levels of calcium, phosphorus, and the calciotrophic hormones, and even in the face of frank dietary calcium restriction, blood calcium levels change very little. An inadequate calcium intake does, however, result in a bone with a thinner cortex and fewer, thinner trabeculae. This comes about through modulation of the balance between the normal, ongoing processes of bone formation and bone resorption.\n\nTo understand how dietary intake interacts with the modeling process, it is necessary to recall that bone reshapes itself continuously during growth. In growing long bones, new bone is deposited at the periosteal surface of the mid-shaft, at the endosteal surface of the submetaphyseal shaft, and at the growth plate. At the same time, bone is resorbed at the endosteal-trabecular surface and on the outer surface of the metaphyseal funnel. This produces concentric expansion of both external shaft diameter and medullary cavity diameter. This dual process reshapes bones so that they conform to growth in body size. The difference between the amount deposited and the amount resorbed is equal to the net bone gain (or loss).\n\nWhen ingested calcium is less than optimal, the endosteal-trabecular resorptive process increases, and the balance between formation and resorption, normally positive during growth, falls toward zero. This occurs because parathyroid hormone augments bone resorption at the endosteal-trabecular surface in order to sustain the level of ionized calcium in the extracellular fluid. When the demands of mineralization at the periosteum and growth plates exceed the amount of calcium absorbed from the diet and released from growth-related bone modeling, more parathyroid hormone is secreted and resorption increases still further, until balance becomes zero or even negative. If calcium is the only limiting nutrient, it is usually considered that growth in size continues normally, but that a limited quantity of mineral now has to be redistributed over an ever larger volume.\n\nUsually children's diets in Western nations are not so calcium deprived as to preclude entirely any increase in bone mass, but occasional instances of severe restriction have been reported. Then, high levels of parathyroid hormone drive phosphorus levels in the extracellular fluid so low that mineralization is inhibited and a rachitic type of lesion develops [75, 76], even though vitamin D status may be normal. In such circumstances, bone growth does slow.11 Short of such extreme situations, the principal perceptible effect of inadequate calcium intake during growth in developed countries is a skeleton of low mass\u2014normally shaped and sized, but containing a smaller than normal amount of bone tissue.\n\nHaving said that, it must be noted that at least two studies suggest that augmented calcium intake may influence bone size as well as bone mass [77, 78]. The first [77] is difficult to interpret because the supplementation included extra protein, phosphorus, and other key nutrients as well as calcium. But the second [78], with better matching of other nutrient intakes, also showed a small effect of extra calcium on both bone mass and stature.\n\nMost of the periosteal expansion and growth in length and much of the endosteal expansion during growth are genetically and mechanically determined. Studies in twins have shown that a large fraction of the variability in peak bone mass is accounted for by the genetic program [79]. However, as already noted, endosteal expansion can be increased in the face of insufficient calcium intake beyond what would be dictated by the genetic program. Thus, while an abundant diet will not produce more bone than the genetic program calls for, a deficient diet must restrict what a person is able to accumulate. Optimal peakbone mass for any given individual can be defined as a skeleton in which the balance between the concentric expansions of growth is solely determined by the individual's genetic program and is not reduced by an exogenous shortage of calcium. Correspondingly, optimal calcium intake can be operationally defined as the intake that permits this full expression of the genetic program.\n\nAs just noted, net bone accumulation will be greater as calcium intake increases, but only to the point where endosteal-trabecular resorption is due solely to the genetic program governing growth, and is not being driven by body needs for calcium. Above that level, as seen in Figures 4 and 5, further increases in calcium intake will produce no further bony accumulation. The intake required to achieve the full genetic program, and thus to ensure peakbone mass, is the intake that corresponds to the beginning of the plateau region in Figs. 4 and 5. This value will be different for different stages of growth, in part because growth rates are not constant and also because, as body size increases, obligatory calcium losses through skin and excreta increase as well.\n\nThe best approach to determine this value in humans lies, as with the laboratory animal, in testing various intakes for their influence on calcium retention, i.e., finding the plateau and locating its threshold. (In healthy individuals, calcium retention amounts to the same thing as bone tissue accumulation, because calcium is normally stored in the body only in the form of bone.) Over the past 75 years, many such studies have been performed. When these reports are combined, it is possible to make out the pattern of plateau behavior found in laboratory animals and, from the aggregated data, to estimate the intake values that correspond to the threshold [50]. Figure 6 represents one example of the relationship between intake and retention, combining the results of many published studies of calcium balance. It is derived from a subset of the adolescents whose balances were assembled by Matkovic [80]. More recently, Jackman _et al._ [81] studied a series of adolescent girls (each at two intakes, varying from subject to subject), and reported an intake threshold at very nearly the same level as that found by Matkovic and Heaney in their meta-analysis [50]. Both approaches clearly show the plateau type of behavior that both animal studies and theoretical considerations predict. They also confirm that, at intakes less than the plateau threshold, daily storage is less than optimal, i.e., accumulation of bone is being limited by intake. Any such limiting intake must be considered inadequate.\n\nFIGURE 6 The relationship of calcium intake, on the horizontal axis, to calcium retention (balance), on the vertical axis, for a subset of the adolescents described by Matkovic and Heaney [50]. Note that, despite the \"noisiness\" that is inevitable in measurements of balance in humans, there is clear evidence of an intake plateau, as observed in the animal experiments of Fig. 4. Note also that, for this age, the threshold of the plateau occurs at about 1500 mg calcium\/day. (Copyright Robert P. Heaney, 1992. Reproduced with permission.)\n\nTable 2 and Figure 7 summarize some of the relevant calculations flowing from the type of analysis of aggregated balance data exemplified in Fig. 6 for various stages of growth [50]. First are the threshold intake values, as judged from the assembled balance studies. In some instances these values are slightly higher than both the NIH consensus conference figures and current Recommended Dietary Intakes (RDIs) (Table 1), but, in general, the various recent estimates are quite close to one another. Aside from the usefulness of these threshold values themselves, one especially notable feature of the data in Table 2 is that even after linear growth has ceased (i.e., in young adults), calcium retention still occurs if the intake is high enough to support it. In other words, bony consolidation can continue after growth in stature has ceased. For this reason, calcium intake in young adults needs to be sufficient not only to maintain skeletal equilibrium but to support this continuing augmentation of bone mass.\n\nTABLE 2\n\nCritical Values for Calcium Intake and Retention Efficiency by _Age_ _a_\n\naDerived from analyses of published balance studies during growth [50].\n\nbSlope of the relationship of retention on intake.\n\nFIGURE 7 Regression lines for the subthreshold regions of the intake-balance relationships in infants, children, adolescents, and young adults, from the data of Matkovic and Heaney [50]. (Copyright Robert P. Heaney, 1992. Reproduced with permission.)\n\nFigure 7 shows, for the four age groups delineated in Table 2, the best-fit regression lines for the intake regions below the age-specific thresholds. This analysis of the data reveals a number of interesting features. First, although the slopes are qualitatively similar, there are nevertheless some important quantitative differences among the age groups. The ability to make use of an increment in calcium intake is greater in infancy and adolescence (i.e., the slope is larger) when skeletal growth is most rapid, and lower in childhood and the young adult years, when growth is slower, as would be expected. Perhaps of even greater interest is the rightward displacement of the regression lines in Figure 6 with advancing age. This phenomenon, reflected in the values in Table 2 for the X-axis intercept, reflects the effect of age on obligatory loss. While zero balance is obviously not healthy for a _growing_ organism, these zero-balance intake values are useful in that they reflect how much calcium an individual must ingest just to stay even (i.e., not to _lose_ bone) at the ages concerned. As Figure 7 shows, infants can reduce calcium loss to nearly zero on zero calcium intake. For older children and young adults, larger and larger calcium intakes are required to sustain even zero balance. Most of this effect is accounted for by a rise in urine calcium with age. It is probably body size that is forcing the higher obligatory requirement since, in a multiple regression model of these data, body size continues to have an effect even after controlling for age [50].\n\nAt least 10 randomized controlled trials of calcium supplementation in children and adolescents have been published [66, 67, 78, 81\u201383], together with several longitudinal observational studies in young adults [84]. All of the controlled trials were positive, as were three-fourths of the observational studies. As mentioned above, the bone remodeling transient contributes to the measured difference in these controlled trials. The relative size of its contribution remains uncertain; nevertheless, simulation of the remodeling transient indicates that the gain reported in these studies is greater than can be explained by that mechanism alone. In all studies, supplemental calcium elevated the children's intakes above the 1989 RDAs. The finding of greater bone gain in the supplemented children than in the control group underscores the inadequacy of the earlier RDA values. In other words, they indicate that the RDAs for 1989 and earlier lie on the ascending portion of the threshold curves of Figures 4 and 5, rather than on the plateau. Hence, these studies reinforce the higher requirement values set forth in Table 2.\n\nIn a third trial [80], one group of pubescent girls received approximately the 1989 RDA while the other group was held to a calcium intake of 450 mg\/day (far less than the RDA but unfortunately not uncommon for girls of that age). As predicted, growth in stature was the same in both groups, but bone mass failed to increase in the low intake group, while it did in the high intake group. Matkovic _et al._ [85] had previously shown that intakes as low as 450 mg per day in adolescent girls did not support positive calcium balance, mainly because, despite intense skeletal demand at that life stage, urinary conservation of calcium remained inefficient. While this third study does not specifically address the issue of what the intake requirement ought to be during adolescence, it does clearly document the deleterious effects of intakes well below the RDA.\n\nAll of these intervention studies, as already noted, produce a remodeling transient. None was designed to evaluate steady-state changes, and hence their positive findings cannot be translated directly into a specific intake recommendation. However, the 4-year longitudinal study of young adults by Recker and his colleagues [84] involved no alteration of calcium intake and hence avoided the problem of the transient. This study showed prospectively that bone augmentation continues well into the third decade. Bone mass gains in their subjects ranged from 0.5% per year for the forearm to 1.25% per year for total body bone mineral. The single most important correlate of the rate of bone accumulation in this Caucasian group of women was calcium intake. The rate of accumulation was inversely proportional to age, with the best estimate of the age at which the rate reached zero being approximately 29\u201330 years. This suggests that the window of opportunity to achieve the full genetic program appears to remain at least partly open until about age 30. It is not certain that the gains observed in this study were as great as might have been possible, since subjects were studied on their habitual calcium intakes, which were below the threshold values of Table 2. Nevertheless, these observations of Recker _et al._ [84] provide qualitative confirmation of the analyses for this age group derived from the balance studies (Table 2), which showed that young adults were able to retain calcium if intakes were at or above the threshold. They also illustrate well how the data from observational studies can complement the data from controlled trials.\n\nThe importance of ensuring full realization of the genetic potential for skeletal development lies in the fact that bone mass seems to track throughout life. Newton-John and Morgan [86] first noted this phenomenon more than 30 years ago in cross-sectional data, and Matkovic _et al._ [87] showed very clearly in their study of two Croatian populations that those who had higher mass at age 30 remained higher than the others out to age 75, even though both groups were apparently losing bone with age. The same phenomenon has been seen in shorter term, longitudinal studies [88\u201390], both across puberty and in the postmenopausal years. Dertina _et al._ [88] have gone so far as to suggest that those most at riskfor late life osteoporosis can be detected before puberty.\n\n#### 2. MATURITY\n\nOnce peakbone mass has been achieved, the principal force acting on the skeleton is no longer the impetus of growth, but the mechanical loads imposed in ordinary, everyday usage. Skeletal structures, like all engineering structures, deform slightly under load. The skeleton senses that degree of deformation, and attempts to adjust its mass (by controlling the balance between bone resorption and bone formation) so that this deformation remains on the order of 0.1\u20130.15% in any given dimension. If a bone is loaded so heavily that it consistently bends more than that amount, then the balance between local formation and resorption is adjusted to favor formation, thus making that region stiffer. Conversely, if a bony segment is little used, and its bending is less than that critical amount, the skeleton senses that it has an excess of bone in the region concerned, and adjusts remodeling balance to remove some of the apparent surplus.\n\nThis reference level of bending is one of the fascinating physiological constants of nature. Across the vertebrates, for all species and all bones studied to date, bone mass is regulated such that any given bone deforms by about that critical 0.1\u20130.15% in ordinary use. This reference level of bending is termed a _setpoint,_ and the bone remodeling apparatus operates to minimize local deviations from this critical value. The cellular basis for the setpoint and the precise nature of the apparatus that detects departures from it remain un-known12; however, there is suggestive evidence that localizes this sensing apparatus to the network of osteocytes embedded in bone. For several years, it has seemed likely that one of the principal determinants of the setpoint of this mass-regulating system is the level of gonadal hormones. Circumstantial evidence in support of this connection includes the facts that estrogen receptors in bone are concentrated in osteocytes [91], and that true bone density rises sharply at puberty [92] and declines by about the same amount at menopause (whether natural or artificial) [93]. These life-phase changes are what one would expect if estrogen influences the setpoint.13\n\nWhile adjustments in mass around the setpoint presume an adequate calcium intake, it turns out that prevailing intakes tend to be closer to adequate during the ages 25\u201350 in women, since estrogen improves the efficiency of intestinal calcium absorption [48, 93] and of renal calcium conservation [94, 95]. Thus estrogen not only increases the reference level of bone density, but it helps the body access and retain the mineral necessary to augment bone to that higher level. For this reason, except for the special circumstances of pregnancy and lactation (discussed below), the years from 25 to 50 are a time in life when a woman's skeletal calcium need is at its lowest. She is no longer storing calcium, and her absorption and retention are operating at their adult peak efficiency.\n\nWelten _et al._ [96] in a meta-analysis of 33 studies performed in adults between 18 and 50 years of age, found a positive association between calcium intake and bone mass in this age group, and noted that it seemed prudent to maintain an intake of 1500 mg\/day during this life period. This is a higher figure than either the NIH consensus conference recommendations or the 1997 DRIs of Table 1. Heaney _et al._ [94], using balance methods in estrogen-replete women ingesting their habitual calcium intakes, found a mean intake for zero balance of slightly under 1000 mg\/day, and Nordin _et al._ [97], also using balance methods, arrived at a figure slightly above 800 mg\/day. Recker _et al._ [98], in a small prospective study of bone mass in premenopausal women found no detectable bone loss over a 2-year period on an estimated mean calcium intake of 651 mg. Baran _et al._ [99], studying women in their fifth decade, found bone loss in a control group receiving 892 mg calcium\/day. Loss occurred only from year 2 to year 3, and not during the first 2 years of observation, and it is not clear from that paper whether the apparent loss in year 3 was related to the loss of sampling units that occurred between years 2 and 3, or whether there was actual loss in those who remained in the study.\n\nRequirement estimates based on balance studies make no provision for sweat loss of calcium since, by design, balance methods usually eliminate vigorous exercise. The importance of sweat loss has been highlighted by a study in male college athletes showing sweat calcium losses of over 200 mg in a single vigorous workout session [100]. Moreover, there was a perceptible loss of bone mineral density across a playing season that was preventable by adding calcium supplements to the athletes' already good diets. It is likely that the lost bone would have been regained after the playing season was over (as long as the diet was adequate); that is, that the skeleton was acting in its capacity as a calcium reserve during the playing season. Nevertheless, the study makes clear how large and important sweat losses can be. Undoubtedly such losses contribute to the low bone mass described in women athletes who often have less than generous calcium intakes.\n\nIn conclusion, the bulk of the available evidence suggests that it is important to maintain an intake of 1000\u20131500 mg\/ day during the mature years. Moreover, there are other health reasons for maintaining a high calcium intake during this period [101], even if bone health can be supported adequately by an intake in the range of 800\u20131000 mg\/day.\n\n#### 3. PREGNANCY AND LACTATION\n\nPregnancy and lactation are circumstances in which the mother must provide for maintenance of her own skeleton as well as for construction of her child's. Specifically, during the 9 months of pregnancy, she provides the fetus with 25\u201330 g calcium, and in her milk during the ensuing nine months of lactation, another 50\u201375 g. This aggregate is in the range of 7\u201310% of her own total body calcium and would, presumably, produce a corresponding decrease in bone mass if she were not able to obtain some or all of the required quantities from ingested calcium. It has always seemed intuitively attractive, therefore, to recommend an increased calcium intake during these physiologically demanding life-stages [73] (see also Table 1). Moreover, given the relatively low calcium intakes of modern industrialized societies, one might have expected that a history of multiparity and extended lactation would be associated with lower bone mass and increased risk of osteoporosis. In general, however, epidemiological studies have found, if anything, the contrary. Most studies report a positive association between parity and bone mass density [102\u2013107], although occasional reports of negative associations can be found [108]. Much of the positive association turns out to be due to increased ponderosity, and after correcting for weight, the positive correlations tend to become statistically nonsignificant. Nevertheless, most are still on the positive side, and there is little or no hint in the available evidence that the calcium drain of pregnancy and lactation adversely affects the maternal skeleton.\n\nBone remodeling accelerates in pregnancy [109\u2013114], and maternal intestinal calcium absorption efficiency increases to the highest level since early infancy. Both changes begin well before significant fetal skeletal accumulation of calcium [109, 115]. Both humans and rats show anticipatory storage of skeletal minerals prior to onset of fetal skeletal mineralization [107, 109], and Heaney and Skillman [109] estimated, from balance studies in pregnant women studied on their habitual calcium intakes, that cumulative calcium balance at term exceeded fetal needs, and that the mother, therefore, went into lactation with a skeletal surplus. Brommage and Baxter [110] reported data consistent with a skeletal surplus in rats at delivery, and ultrasound methods suggest that the same occurs in pregnant mares [111]. However, this will not be possible if calcium intake is very low. Barger-Lux _et al._ [116] reported bone loss across pregnancy in young women with dietary calcium to protein ratios averaging 6.6 mg\/g.\n\nDuring lactation, the majority of reports indicate that some degree of bone loss regularly occurs [101, 102, 105\u2013108, 112, 113, 117\u2013120], particularly in presumably reactive bony sites such as the centers of the vertebral bodies and the ultradistal radius [117, 118]. On the other hand, this loss appears to reverse after weaning and may, therefore, represent to some extent a negative remodeling transient like what occurs in deer at the time of antler formation [57]. Immediately following delivery, absorption efficiency falls to or toward nonpregnant, nonlactating levels and remains at this relatively low level throughout lactation, despite the continuing drain of lactational calcium loss [109, 113, 121]; however, urinary calcium falls at the same time and remains low throughout lactation and for several months post-weaning [112], while bone remodeling remains elevated [112, 113]. This is a physiological situation conducive to replacement of lost bone.14\n\nLactating rats lose nearly one-third of their skeleton during milk production [119]. This loss doubles if the animals are fed low- calcium diets, but it does not diminish when the normally high calcium diet of a rat is increased as much as threefold [119]. It is likely that this bone loss represents an anticipatory phenomenon; that is, rather than the calcium being drawn out of bone by the drain of lactation, the bone pumps calcium into the circulation for milk production. This is suggested by the reduced parathyroid hormone levels during lactation [122], by the usually reported failure of increased calcium intake to reduce the loss, and by the high serum phosphorus levels during lactation.15 How this outpouring of skeletal mineral for the benefit of lactation occurs is less clear, although it is certainly plausible that the hypoestrin state of lactation would, like menopause or athletic amenorrhea, shift the bone setpoint, and result in some downward reduction in bone density (thereby, effectively, releasing stored calcium and phosphorus).\n\nWhile Kalkwarf _et al._ [123] found no effect of calcium supplementation, a few reports suggest that even the modest reductions in bone mass normally found during human lactation can be reduced or eliminated by extra calcium [117, 118]. The relatively slow growth of human infants (in comparison, for example, with rats) imposes a lower lactational burden on a human mother, and some of the differences between species may be attributable to quite significant differences in lactational demands for mineral.\n\nGiven the concordance of the balance data and the epidemiological evidence, it seems likely either that adaptive mechanisms are usually sufficient to accommodate the calcium demands of pregnancy and lactation or that postweaning adjustments compensate for whatever bone may have been lost. As it turns out, there is physiological evidence to indicate that both occur.\n\nCompensatory physiological adjustments surrounding pregnancy and lactation are more vigorous than at other lifesta-ges, and the current consensus is that a high calcium intake makes less long-term difference to a woman's skeleton at this lifestage than at most other times in her life. In summarizing the available literature, the panel responsible for the 1997 DRIs noted that there was no evidence on which to base a recommendation for a higher calcium intake during pregnancy and lactation than that considered optimal for other women of the same age. They did add, however, that the situation with adolescent pregnancy was problematic and inadequately studied, and that perhaps some increment above the adolescent recommendation in such individuals might be prudent.\n\n#### 4. MENOPAUSE\n\nIt has been noted already that estrogen seems to adjust the bending setpoint of bone. Accordingly, whenever women lose ovarian hormones, either naturally at menopause or earlier as a result of anorexia nervosa or athletic amenorrhea, the skeleton seems to sense that it has more bone than it needs, and hence allows resorption to carry away more bone than formation replaces. (Precisely the same change occurs when men lose testosterone for any reason.) This is equivalent to raising the bone bending setpoint, as described above. While varying somewhat from site to site across the skeleton, the downward adjustment in bone mass due to lack of gonadal hormone amounts to approximately 10\u201315% of the bone a woman had in the lumbar spine and 6 percent at the total hip prior to menopause [93].\n\nThe importance of this phenomenon in a discussion of nutrient effects is to help distinguish menopausal bone loss from nutrient deficiency loss and to stress that menopausal loss, which is due to absence of gonadal hormones, not to nutrient deficiency, cannot be substantially influenced by diet. Almost all of the published studies of calcium supplementation within 5 years following menopause failed to prevent bone loss [63, 124, 125]. Even Elders _et al.,_ [125] who employed a calcium intake in excess of 3000 mg\/day, succeeded only in slowing menopausal loss, not in preventing it. However, Dutch women tend to be calcium replete, because of high national dairy product consumption, and other studies have shown effects of calcium supplementation in the early menopausal years that are intermediate between placebo and estrogen [72, 124, 125]. It is likely that, in any group of early menopausal women, there are some whose calcium intake is so inadequate that they are losing bone for two reasons (estrogen lackplus calcium insufficiency).\n\nAs important as menopausal bone loss is, it is only a one-time, downward adjustment, and, if nutrition is adequate, the loss continues for only a few years, after which the skeleton comes into a new steady state (although at a 5\u201315% lower bone mass). It is in this context that the importance of a high peak skeletal mass becomes apparent. One standard deviation for lumbar spine bone mineral content in normal women is about 10\u201315% of the young adult mean, and for total body bone mineral, about 12%. Hence a woman at or above one standard deviation above the mean can sustain the 15% menopausal loss and still end up with as much bone as the average woman has before menopause. By contrast, a woman at or under one standard deviation below the young adult mean premenopausally drops to two standard deviations below the mean as she crosses menopause and is, therefore, by the WHO criteria [127], already osteopenic and verging on frankly osteoporotic.\n\nAs noted, the menopausal bone mass adjustment amounts to a loss at the spine of 10\u201315%, and at the hip, \u20136% [128]. Hip bone change, both immediately before and after this menopausal downward adjustment, averages about \u20135% per year, while, except for the menopausal loss, the spine curve is flat. But this is so only as long as calcium intake is adequate. In this regard, it is important to recall the nonskeletal effects of estrogen described above; improvement of intestinal absorption and renal conservation [48, 94, 95]. Because of these effects, an estrogen-deficient woman has a higher calcium requirement, and unless she raises her calcium intake after menopause, she will continue to lose bone after the estrogen-dependent quantum has been lost, even if the same diet would have been adequate to maintain her skeleton before menopause. In other words, early in the menopausal period, her bone loss is mainly (or entirely) because of estrogen withdrawal, while later it is because of inadequate calcium intake.\n\nFigure 8 assembles, schematically, the set of factors contributing to bone loss in the postmenopausal period. The figure shows both the self-limiting character of the loss due to estrogen deficiency and the usually slower, but continuing, loss due to nutritional deficiency, when present. Unlike the estrogen-related loss, which mostly plays itself out in 3\u20136 years, an ongoing calcium deficiency loss will continue to deplete the skeleton indefinitely for the remainder of a woman's life, that is, unless calcium intake is raised to a level sufficient to stop it. Furthermore, since both absorption efficiency [48] and calcium intake [42] decline with age, the degree of calcium shortfall typically worsens with age.\n\nFIGURE 8 Partition of age-related bone loss in a typical postmenopausal woman with an inadequate calcium intake. Based on a model described in detail elsewhere [93]. (Copyright Robert P. Heaney, 1990. Reproduced with permission.)\n\nThus it is important for a woman to increase her calcium intake after menopause, even though, for the first few years, doing so will not prevent estrogen-withdrawal bone loss. Both the 1984 NIH consensus conference on osteoporosis [129], and the 1994 Consensus Conference on Optimal Calcium Intake [61] recommended intakes of 1500 mg\/day for estrogen-deprived postmenopausal women. It may be that the optimal intake is somewhat higher still (see below), but median intake in the United States for women of this age is in the range of 500\u2013600 mg\/day [42], and if the bulk of the diets could be raised even to 1500 mg\/day, the impact on skeletal health would be considerable.\n\n#### 5. SENESCENCE\n\nThere is general agreement that bone is lost with aging. Early cross-sectional data suggested that spine loss began as early as age 30\u201335, but, except for the hip, longitudinal studies have not borne that out for most skeletal regions (e.g., spine, forearm) [98]. Significant loss probably does not begin until sometime in the sixth or seventh decade.16 This age-related bone loss occurs in both sexes, regardless of gonadal hormone levels. However, it is obscured at the commonly measured spine site in the years immediately following menopause in women by the substantially larger effect of estrogen withdrawal (see Figure 8). It probably occurs, however, even in estrogen-treated women, at about the same rate as in men. This rate varies by skeletal region and is generally reported to be on the order of 0.5\u20131.0% per year by the seventh decade, and accelerates with advancing age. Age-related loss involves both cortical and trabecular bone and can be due to several causes. These include disuse atrophy consequent upon reduced physical activity, an entropic kind of loss due to accumulation of random remodeling errors which, of their nature tend to be irreversible17; reduction in androgenic steroid levels, and finally nutritional deficiency loss. These types of bone loss are summarized in Figure 8.\n\nWhile nutrient deficiency is clearly only a part of the problem, nevertheless it is common. Intestinal calcium absorption efficiency declines with age [48], at the same time as nutrient intake itself generally declines [42]; the result is that the diet of aging individuals becomes doubly inadequate. This inadequacy is clearly expressed, for example, in the rate of bone loss reported by Chapuy _et al._ [64] in the untreated control group of their large randomized trial of calcium and vitamin D supplementation. These women, with an average age of 84 and with calcium intakes that averaged 514 mg\/day, were losing bone from the femur at rates of slightly more than 35% per year. That there was a causal connection between intake and bone loss is demonstrated by the fact that the loss was completely obliterated with calcium (and vitamin D) supplementation.\n\nIt is in this age group that the most dramatic and persuasive evidence for the importance of a high calcium intake has been produced in recent years. This is primarily because most fragility fractures rise in frequency with age and, hence, the opportunity to see a fracture benefit (if one exists) is greater then.18 Chapuy _et al._ [64] showed a reduction in hip fracture risk of 43% by 18 months after starting supplementation with calcium and vitamin D, and a 32% reduction in other extremity fractures. Chevalley _et al._ [68], in another study in elderly women, resolved the question left unanswered in the study of Chapuy _et al._ (whether the calcium or the vitamin D was responsible for the effect) by giving vitamin D to both controls and treated subjects, but calcium only to the treated group. They, too, found a reduction in femoral bone loss and in fracture incidence (vertebral in this case) in the calcium-supplemented women. Recker _et al._ [69] in a 4-year, randomized controlled trial in elderly women (mean age 73), showed that a calcium supplement reduced both age-related bone loss and incident vertebral fractures. Their subjects had all received a multivitamin supplement containing 400 IU of vitamin D; hence, most or all of the effect in the calcium-supplemented group can be attributed to the calcium alone.\n\nThe studies of Chevalley _et al._ [68] and Recker _et al._ [69] should not be interpreted to mean that vitamin D is unimportant in this age group. It is likely that intakes of both calcium and vitamin D are commonly inadequate in the elderly (see below), and the high prevalence of combined deficiency has complicated study of the actual requirements of either nutrient in this age group. The importance of these studies lies in the fact that, even after ensuring vitamin D repletion, there was still a calcium benefit and, hence, presumptively a calcium deficiency in this age group. Heikinheimo _et al._ [132] had earlier shown the converse in an elderly Finnish population. Vitamin D supplementation in this population (which tends to be calcium replete) significantly reduced all fractures, both in institutionalized and in free-living individuals.\n\nThe calcium intake achieved in the Chapuy _et al._ [64] study was about 1700 mg\/day, in the Chevalley _et al._ [68] study, 1400 mg\/day, and in the Recker _et al._ [69] study, about 1600 mg\/day. These values are in the range of the intake found by Heaney _et al._ [94, 133] to be the mean requirement for healthy estrogen-deprived older women (1500\u20131700 mg\/day). All of these studies are, therefore, fully consistent with the more recent recommendations in the range of 1500 mg\/ day (Table 1).\n\nAn important feature of these controlled trials in already elderly individuals was that bone mass was low in both treated and control groups at the start of the study, and while a significant difference in fracture rate was produced by calcium supplementation, even the supplemented groups would have to be considered as having an unacceptably high fracture rate. What these studies do not establish is how much lower the fracture rate might have been if a high calcium intake had been provided for the preceding 20\u201330 years of these women's lives. The studies of Matkovic _et al._ [4] and Holbrook _et al._ [134], although not randomized trials, strongly suggest that the effect may be larger than has been found with treatment started in the eighth and ninth decades of life. Both of these observational studies reported a hip fracture rate that was roughly 60% lower in elderly whose habitual calcium intakes had been high. While findings from observational studies such as these had not been considered persuasive in the absence of proof from controlled trials, the trials with fracture endpoints have now met that need.\n\nAdditional reinforcement comes from recent work of McKane _et al._ [135], who studied the effect of a large calcium supplement on parathyroid hormone secretory dynamics in elderly women. In brief, a mean calcium intake of 2413 mg\/day lowered parathyroid hormone levels 40% to the young normal range, and normalized the abnormal parathyroid hormone secretory dynamics typical of the elderly female. They concluded that the combination of declining oral calcium intake, deteriorating vitamin D status, reduced calcium absorption, and impaired renal conservation of calcium in the elderly lead to parathyroid gland hyperactivity and increased bone resorption.\n\nTogether the aggregate of available studies underscores the importance of achieving at least the 1400\u20131500 mg\/day target figure of the new recommendations for the elderly. At the same time it must be stressed, once again, that osteoporosis is a multifactorial condition, and that removing one of these factors (i.e., ensuring an adequate calcium intake) cannot be expected to eradicate all osteoporotic fractures.\n\n### D. Nutritional Factors That Influence the Requirement\n\nSeveral nutritional factors influence or have been proposed to influence the calcium requirement (Table 3). The principal interacting nutrients are sodium, protein, caffeine, and fiber. Fiber and caffeine influence calcium absorption [136\u2013138] and typically exert relatively minor effects, while sodium and protein influence urinary excretion of calcium [136, 137], and can be of much greater significance for the calcium economy. Phosphorus and fat are sometimes mentioned in connection with calcium absorption, but their effect in humans seems minor to nonexistent.\n\nTABLE 3\n\nFood Factors and the Calcium Requirement\n\nThe basis for the importance of nutrients acting on absorpion and excretion is illustrated in Fig. 9, which partitions the variance in calcium balance observed in 560 balances in healthy middle-aged women studied in the author's laboratory. As Fig. 9 shows, only 11% of the variance in balance among these women is explained by differences in their calcium intakes, and absorption efficiency explains only another 15%. By contrast, urinary losses explain slightly more than 50%.19 The dominance in Fig. 9 of renal excretion would be trivial in primary bone-losing syndromes, but it is particularly noteworthy that it appears to be operative in conditions of health, because it means that obligatory losses through the kidney pull calcium out of the skeleton. This is a concept for which we are particularly indebted to the work of Nordin and his associates [139, 140].\n\nFIGURE 9 Partition of variance in calcium balance in normal women among the input-output processes involved in calculation of balance. (Copyright Robert P. Heaney, 1994. Reproduced with permission.)\n\n#### 1. INFLUENCES ON INTESTINAL ABSORPTION OF CALCIUM\n\na. Fiber.: The effect of fiber is variable, and generally small. In acute, single-meal absorption tests, many kinds of fiber have no influence at all on absorption, such as the fiber in green, leafy vegetables [30, 34]. Moreover, fibers of the class termed nondigestible oligosaccharides, rather than interfering with absorption, have been shown in rats to increase both mucosal mass and calcium absorption [141], and there are reports in humans suggesting a similar effect, at least on absorption [142, 143] (which is why fiber is listed in both columns of Table 3). The current theory is that volatile fatty acids produced in fermentation of the nondigestible oligosaccharides by colonic flora evoke gut hormone responses that regulate mucosal mass (thereby serving to match the metabolic cost of replacing the mucosa every 5 days to the level of food intake). The fiber in wheat bran, by contrast, reduces absorption of co-ingested calcium in single-meal tests, although except for extremes of fiber intake [144], the antiabsorptive effect is generally relatively small.\n\nOften lumped together with fiber are associated food constituents such as phytate and oxalate, both of which can reduce the availability of any calcium contained in the same food. For example, for equal ingested loads, the calcium of beans is only about half as available as the calcium of milk [145], while the calcium of spinach and rhubarb is nearly totally unavailable [30, 146]. For spinach and rhubarb, the inhibition is mostly due to oxalate. For common beans, phytate is responsible for about half the interference, and oxalate, the other half. The effects of phytate and oxalate are highly variable. There is a sufficient quantity of both antiabsorbers in beans to complex all the calcium also present, and yet their combined absorptive interference is only half what might have been predicted. With the exception of bran, these interferences generally operate only on calcium contained in the same food. This is because the antiabsorber is usually already fully complexed with calcium in the ingested food. Thus, spinach does not typically interfere with absorption of co-ingested milk calcium.\n\nb. Caffeine.: Often considered to have a deleterious effect on the calcium economy, caffeine actually has the smallest effect of the known interacting nutrients. A single cup of brewed coffee causes deterioration in calcium balance of 3 mg [137, 138, 147], mainly by reducing absorption of calcium [137]. The effect is probably on active transport, although this is not known for certain. This effect is so small as to be more than adequately offset by a tablespoon or two of milk [137, 147]; and caf\u00e9 au lait or caffe latte produce a substantial net calcium gain, despite their caffeine content.\n\nc. Fat.: Fat also has sometimes been presumed to reduce calcium absorption by a similar mechanism, that is formation of calcium soaps with unesterified fatty acids released in the chyme by intestinal lipases. However, in healthy adult humans, no appreciable effect of fat intake on calcium absorption has been found. This is at least partly explained, as with phosphorus (see below), by the fact that the normal small intestine absorbs fat much more avidly than it does calcium. At intakes in the range of recommended levels, the feces contain a considerable stoichiometric excess of calcium relative to fatty acids.\n\n#### 2. INFLUENCES ON RENAL CONSERVATION OF CALCIUM\n\na. Protein and Sodium.: As noted, the effects of protein and of sodium are substantial [17, 18, 138, 148]. Both nutrients increase urinary calcium loss across the full range of their own intakes, from very low to very high\u2014so it is not a question of harmful effects of an excess of these nutrients. Sodium and calcium share the same transport system in the proximal tubule, and every 2300 mg sodium excreted by the kidney pulls 20\u201360 mg of calcium out with it. And every gram of protein metabolized in adults causes an increment in urine calcium loss of about 1 mg.20 This latter effect is probably due to excretion of the sulfate load produced in the metabolism of sulfur-containing amino acids (and is thus a kind of endogenous analog of the acid rain problem). At low sodium and protein intakes, the minimum calcium requirement for skeletal maintenance for an adult female may be as little as 450 mg\/day [139], whereas if her intake of both nutrients is high, she may require as much as 2000 mg\/day to maintain calcium balance. A forceful illustration of the importance of sodium intake is provided by the report of Matkovic _et al._ [85] that urine calcium remains high in adolescent girls on calcium intakes too low to permit bone gain. The principal determinant of urinary calcium in such young women is sodium intake [149], not calcium intake.\n\nDifferences in protein and sodium intake from one national group to another are part of the explanation of why studies in different countries have shown sometimes strikingly different calcium requirements [35]. At the same time, one usually finds a positive correlation between calcium intake and bone mass within the national range of intakes [150]. Hence, while sodium (and protein) intake differences across cultures may obscure the calcium effect, they do not obliterate it.\n\nThe acid\/alkaline ash characteristic of the diet is also important, although the quantitative relationship of this diet feature to the calcium requirement is less completely developed. Nevertheless, it has clearly been shown that substitution of metabolizable anions (e.g., bicarbonate or acetate) for fixed anions (e.g., chloride) in various test diets will lower obligatory urinary calcium loss substantially [151, 152]. This suggests that primarily vegetarian diets create a lower calcium requirement, and provides a further explanation for the seemingly lower requirement in many nonindustrialized populations. However, it is not yet clear whether, within a population, vegetarians have higher bone mass values than omnivores [153], and such limited data as are available suggest, in fact, the contrary [154\u2013156].\n\nb. Phosphorus.: Phosphorus is commonly believed to reduce calcium absorption, but the evidence for that effect is scant to nonexistent, and there is much contrary evidence. Spencer _et al._ [157] showed no effect of even large increments in phosphate intake on overall calcium balance at low, normal, and high intakes of calcium. In adults, calcium to phosphorus ratios ranging from 0.2 to above 2.0 are without effect on calcium balance when studied under metabolic ward conditions and adjustments are made for calcium intake [138]. Still phosphorus intake is not without effect on the calcium economy. It depresses urinary calcium loss and elevates digestive juice secretion of calcium by approximately equal amounts (which is why there is no net effect on balance [49, 148]). While it is true that stoichiometric excesses of phosphate will tend to form complexes with calcium in the chyme, various calcium phosphate salts have been shown to exhibit absorbability similar to other calcium salts [158], and phosphate is, of course, a principal anion of the major food source of calcium (dairy products). In any case, phosphate itself is more readily absorbed than calcium (by a factor of at least 2\u20133 times), and at intakes of both nutrients in the range of their respective DRIs, absorption will leave a stoichiometric excess of calcium in the ileum, not the other way around. This explains the seeming paradox that high calcium intakes can block phosphate absorption (as in management of end-stage renal disease), while achievably high phosphate intakes have little or no effect on calcium absorption.\n\nc. Aluminum.: Although not in any sense a nutrient, aluminum, in the form of aluminum-containing antacids, also exerts significant effects on obligatory calcium loss in the urine [159]. By binding phosphate in the gut, these substances reduce phosphate absorption, lower integrated 24-hour serum phosphate levels, and thereby elevate urinary calcium loss. This is the opposite of the more familiar hypocalciuric effect of oral phosphate supplements. Therapeutic doses of Aluminum-containing antacids can elevate urine calcium by 50 mg\/day or more.\n\n#### 3. ENHANCERS OF CALCIUM ABSORPTION\n\nRelatively little work has been done on enhancers of calcium absorption. Lactose is said to improve absorption, but the effect may be confined to the rat. Human studies using various carbohydrates have generally shown some enhancement [160], but the effect may be confined to intestines damaged by disease or surgery, because it has been hard to find in healthy subjects [161]. Also, the effect of various carbohydrates may be nonspecific, due instead to alteration of the gastric emptying pattern associated with co-ingestion of other food constituents\u2014what we have elsewhere characterized as the ''meal effect\" [162]. (That is the meaning of the entry ''food\" in Table 3.)\n\nNevertheless, given the generally low absorbability of calcium, the prospect of finding substances that might improve calcium bioavailability has enticed many food processors. Various food fractions, such as casein phosphopeptide, derived from milk, have been found to improve calcium absorbability in certain experimental systems [163], although its effect in humans is probably small [164]. Likewise, certain amino acids, notably lysine, have been thought to enhance calcium absorption [165], but human evidence in their regard is sparse and inconsistent. Even fat might theoretically be viewed as an enhancer, since it is known to slow gastric emptying. However, we have been unable to find, using multiple regression methods, any effect of even large variations in fat intake on absorption fraction in our observational study of middle-aged women.\n\n#### 4. INTAKE VS INTERFERENCE\n\nFor diets high in calcium, as would have been the case for our hunter-gatherer ancestors, high protein and possibly high sodium intakes could have been handled by the body without adverse effects. These nutrients create problems for the calcium economy of contemporary adult humans mainly because we typically have calcium intakes that are low relative to those of preagricultural humans. This is because at prevailing low intakes, compensatory adjustment mechanisms are already operating, and for many individuals, capacity for further adaptation (e.g., increased absorption efficiency) is very limited. An increased demand for only 40 mg calcium\/day would require a nearly 40% increase in intestinal absorption at intakes at the bottom quartile for North American and European women today, while the same demand can be met by an increase of only 1\u20132% in absorption efficiency at intakes such as those that prevailed during hominid evolution. The former is not possible, while the latter is easily accomplished. Thus, while there is some emphasis today among nutritionists on regulating intake of interfering nutrients, the real problem is not so much that the intakes of these other nutrients are high, as that calcium intake is too low to allow us to adjust to the inevitable nutrient\u2013nutrient interactions that occur with any diet.\n\n## VII. VITAMIN D\n\nIt has long been recognized that vitamin D is important for absorption of calcium from the diet. Its role in that regard lies in facilitating active transport, probably by inducing the formation of a calcium-binding transport protein in intestinal mucosal cells. This function is particularly important for adaptation to low intakes. There is also, apparently, a second vitamin D-related absorption mechanism, transcaltachia [166], which is nongenomic in expression but nevertheless requires occupancy of the classical vitamin D receptor. Finally, absorption also occurs passively, probably mainly by way of paracellular diffusion. This route is not dependent on vitamin D and is not as well studied. The proportion of absorption by the three mechanisms varies with intake and is not well characterized in humans; at high calcium intakes (above 2000 mg\/day) the absorption fraction approaches that observed in anephric individuals (\u223c10\u201315% of intake). Under these circumstances it is likely that active transport contributes relatively little to the total absorbed load. Nevertheless, it is clear, at prevailing calcium intakes, that vitamin D status influences absorptive performance and that it thereby influences the minimum calcium requirement.\n\nA simple calculation suffices to establish the magnitude of this influence. Assume an intake of 1000 mg calcium\/day. To that is added about 150 mg in the form of digestive secretions and sloughed off mucosa. If passive absorption is at a level of 12.5% of intake, net absorption would amount to 144 mg, leaving the individual in _negative_ balance across the gut of \u20136 mg\/day (and, of course, producing no calcium gain for the body to offset renal and dermal losses). If, however, vitamin D-mediated, active transport is operating, so that, for example, total absorption was 27.5%, net absorption becomes %109 mg. The relationship of active transport to net absorption is shown graphically, for various intakes, in Fig. 10, which makes clear that meeting physiological demands for calcium would require very high calcium intakes in the absence of vitamin D. (That situation is depicted by the bottom line in the figure, which is the net absorption contour for zero active absorption, as well as by the other lines depicting lower levels of active transport, reflecting, in turn, varying degrees of vitamin D insufficiency.)\n\nFIGURE 10 Relationship of vitamin D-mediated, active calcium absorption, calcium intake, and net calcium gain across the gut. Each of the contours represents a different level of active absorption above a baseline passive absorption of 12.5%. (The values along each contour represent the sum total of passive and variable active absorption.) The horizontal, dashed lines indicate zero and 200 mg\/day net absorption, respectively. The former is the value at which the gut switches from a net excretory to a net absorptive mode, and the latter is the value needed to offset typical urinary and dermal losses in mature adults. (Copyright Robert P. Heaney, 1999. Reproduced with permission.)\n\nA principal storage form of the vitamin is 25-hydroxyvitamin D (25OHD), and its plasma level is generally regarded as the best clinical indicator of vitamin D status. Although usually considered to be about three orders of magnitude less potent than calcitriol in promoting active transport in animal receptor assays, there is growing evidence that it may possess physiological functions in its own right [167\u2013172], and in the only human dose\u2013response studies performed to date, 25OHD was found to have a molar potency in the range of 1\/125 to 1\/400 that of 1,25(OH)2D3 [171\u2013173], not the 1\/2000 figure usually considered to reflect relative 25OHD activity.\n\nVitamin D status commonly deteriorates in the elderly, whose plasma 25OHD levels are generally lower than in young adults [174, 175]. These elderly persons, without histological or biochemical evidence of osteomalacia, nevertheless exhibit high parathyroid hormone levels, high serum alkaline phosphatase levels, and low absorptive performance, all of which move to or toward normal with physiological amounts of supplemental vitamin D [174\u2013177]. The rate of age-related loss of bone has been found to be inversely correlated to dietary vitamin D [178]. Low dosage vitamin D supplementation of ostensibly healthy postmenopausal women significantly slows wintertime bone loss and reduces the annual parathyroid-mediated activation of the bone remodeling system that occurs in winter through late spring [175]. These changes all suggest relative vitamin D insufficiency.\n\nLow 25OHD levels in the elderly are partly due to decreased solar exposure, partly to decreased efficiency of skin vitamin D synthesis, and partly to decreased intake of milk, the principal dietary source of the vitamin in North America. Moreover, the elderly exhibit other abnormalities of the vitamin D endocrine system, which may further impair their ability to adapt to reduced calcium intake. These include decreased responsiveness of the renal 1-\u03b1-hydroxylase to parathyroid hormone [179] and possibly, also, decreased mucosal responsiveness to calcitriol [180] (although available data do not permit distinguishing a decrease in mucosal responsiveness from a simple decrease in mucosal mass).\n\nFor all of these reasons, there is a growing body of opinion that the requirement for vitamin D rises with age [27, 176, 181\u2013184], and a body of data that strongly suggests that relative vitamin D deficiency plays a role in several components of the osteoporosis syndrome. Perhaps most persuasive of all is the finding by Heikinheimo _et al._ [132], in a randomized, controlled trial, of substantial reduction in all fractures in an elderly Finnish population given a single injection of 150, 000\u2013300,000 IU vitamin D each fall.21 Lips _et al._ [185], on the other hand, found no benefit from an additional 400 IU of vitamin D in a Dutch population.\n\nThe foregoing studies (as well as others) lead inexorably to the conclusion that vitamin D insufficiency is prevalent in the middle-aged and elderly of Northern Europe and North America. Moreover, in virtually none of the studies showing a benefit of supplemental vitamin D was frank osteomalacia a significant feature of the problem. Hence, as discussed above, this criterion for true vitamin D deficiency may well be much too strict to be nutritionally useful today. How the vitamin D requirement ought to be defined is another matter. Holick [184] has presented data showing that it takes an intake of at least 600 IU\/day, from all sources, to sustain serum 25(OH)D levels, and the doses of vitamin D used in the studies summarized above also suggest that an intake in the range of 500\u2013800 IU\/day is required for full expression of the known effects of vitamin D in adults. Vieth [27] presents evidence that the requirement may be higher still.\n\nWhat is not clear from the above is how much of the effect of vitamin D in studies such as the fracture prevention trial of Heikinheimo _et al._ [132] is due to facilitating gut adaptation to marginal calcium intakes, and how much may represent an extraintestinal effect of the vitamin in its own right, for example, on muscle tone or coordination. Calcitriol receptors are widely distributed in many tissues, and calcitriol enhances parathyroid hormone-mediated bone resorption and exhibits autocrine action in cell differentiation and in the immune response. Furthermore, calcitriol elicits a prompt and sizable increase in osteoblast synthesis of osteocalcin [186]. Additionally, elevating serum 25OHD levels in the elderly improves the often incomplete gamma carboxylation of osteocalcin (see next section). Nevertheless, patients with vitamin D-dependent rickets type II, who lack functional calcitriol receptors, show essentially complete remission of most of their skeletal pathophysiology with intravenous calcium infusions alone [187]. Furthermore, while subtle impairment of immune function can be demonstrated in nutritional vitamin D deficiency, the defects appear to be sufficiently mild to be of little or no clinical consequence in most individuals. Hence the issue of the extraintestinal importance of vitamin D remains unclear.\n\n## VIII. VITAMIN K\n\nThe chemistry and physiology of vitamin K have been extensively reviewed elsewhere [186, 188\u2013191]. In brief, vitamin K is necessary for the gamma-carboxylation of glutamic acid residues in a large number of proteins. Most familiar are those related to coagulation, in which seven vitamin K-dependent proteins are involved in one way or another. The gamma-carboxyglutamic acid residues in the peptide chain bind calcium, either free or on the surface layers of crystals, and have been thought to function in varying ways including catalysis of the coagulation cascade, inhibiting mineralization (as in urine) [192], and serving as osteoclast chemotactic signals [193]. Vitamin K deficiency classically produces bleeding disorders, but the liver, where the clotting factors are produced, is highly efficient in extracting vitamin K from the circulation, and gamma-carboxylation declines substantially in other tissues before the deficiency is severe enough to result in bleeding disorders. It may thus be that the bleeding tendencies that have been the hallmark of vitamin K deficiency are, in fact, the _last_ manifestation of deficiency. If so, what the other clinical expressions of deficiency may be remain uncertain.\n\nThree vitamin K-dependent proteins are found in bone matrix: osteocalcin (bone gla protein), matrix gla-protein, and protein S. Only osteocalcin is unique to bone. There is also a kidney gla protein (nephrocalcin) [194], that may be involved in renal conservation of calcium. Osteocalcin binds avidly to hydroxyapatite (but not to amorphous calcium phosphate) and is chemotactic for bone-resorbing cells. Originally thought to be synthesized and gamma-carboxylated by osteoblasts as they deposit bone matrix, it now seems that osteocalcin is synthesized by osteocytes [195], particularly those newly embedded in forming bone matrix. Roughly 30% of the synthesized osteocalcin is not incorporated into matrix, but is released instead into the circulation, where, like alkaline phosphatase, it can be measured and used as an indicator of new bone formation.\n\nIn vitamin K deficiency, such as would occur with cou-marin anticoagulants, serum osteocalcin levels decline, and the degree of carboxylation of the circulating osteocalcin falls dramatically. Further, binding to hydroxyapatite of the osteocalcin produced under these conditions falls precipitously soon after starting anticoagulant therapy. It would seem, therefore, that vitamin K deficiency would have detectable skeletal effects. The problem is that they have been very hard to find. Rats reared and sustained to adult life under near total suppression of osteocalcin gamma-carboxylation show only minor skeletal defects, mostly related to abnormalities in the growth apparatus [186]. Warfarin anticoagulation therapy in humans has generally not been found to be associated with decreased bone mineral density or increased fractures [196, 197]. However, an osteocalcin knockout mouse exhibits a skeleton significantly more dense than normal [198], a finding compatible with osteocalcin's playing a role in facilitating resorption. In aging humans, the problem of detecting skeletal abnormalities is compounded by the relative isolation of bone from current nutritional stresses, discussed briefly above.\n\nVarious vitamin K-related abnormalities have been described in association with osteoporosis, but their pathogenetic significance remains unclear. Circulating vitamin K and menaquinone levels are low in hip fracture patients [199], but since these levels reflect only recent dietary intake [200, 201], it is uncertain to what extent they reflect prefracture vitamin K status. Osteocalcin is undercarboxylated in osteoporotics, and this defect responds to physiological doses of vitamin K [202]. Finally, urine calcium has been reported to be high in some patients with osteoporosis and to fall in response to physiological doses of vitamin K [203, 204]. In the same subjects, urine hydroxyproline was also found to be high and to fall on vitamin K treatment. The effect was confined to subjects with pretreatment hypercalciuria, and could plausibly be explained as a defect first in a calcium transport protein, with a consequent renal leak of calcium, and a corresponding parathyroid hormone-mediated increase in bone resorption (reflected in the increased hydroxyproline excretion). In a prospective study Feskanich _et al._ [205] found lower hip fracture rates in those with the highest vitamin K intakes, but the cohort was relatively young (mean age: 61) and the relevance of this finding to more typical hip fracture patients is uncertain.\n\nWhether or not vitamin K is important for bone health, serum vitamin K levels are indicators of general nutritional status, and it may simply be that the observation of low vitamin K levels in osteoporotics, especially in those with hip fracture, is a reflection mainly of the often poor nutrition of these individuals [206\u2013208]. Manifestly, much about vitamin K and bone health remains unclear and more work must be done. Until such questions are resolved, it would seem prudent to ensure in the elderly a sufficient vitamin K intake to achieve full expression of the gamma-carboxylation of all vitamin-K dependent proteins.\n\n## IX. OTHER ESSENTIAL NUTRIENTS\n\nA. Magnesium\n\nMagnesium is an essential intracellular cation, a cofactor of many basic cellular processes, particularly those involving energy metabolism. In the face of true magnesium deficiency, there is widespread cellular dysfunction, including the cells and tissues that control the calcium economy and bone remodeling, among others. While slightly more than half the body magnesium is contained in the mineral of bone, it is less certain whether it plays any role there or is, like zinc (see below), present simply accidentally, insofar as it was present in the extracellular fluid bathing the mineralizing site. On the other hand, magnesium alters the surface properties of calcium phosphate crystals, and its concentration in bone is sufficiently high to exert such an effect there. However, the physical-chemical equilibrium between bone crystals and the dissolved minerals in the extracellular fluid is itself poorly understood; hence, any role of magnesium therein is correspondingly uncertain.\n\nMagnesium deficiency clearly occurs in humans of all ages, most often resulting from severe alcoholism or intestinal magnesium leaks, as from sprue or from ileostomy losses. One well-studied manifestation is hypocalcemia, now recognized to be due to refractoriness of the parathyroid glands to the hypocalcemic stimulus itself, coupled with refractoriness of the bone resorption apparatus to parathyroid hormone.\n\nLow bone mass is also a common feature in these situations. But individuals with magnesium deficiency commonly have calcium deficiency as well, and for the same reasons\u2014a varying combination of low intake, renal wastage, and intestinal leakage. One would therefore expect osteoporosis to be very common in such individuals, as is the case. How much of this bony deficit is due to the magnesium deficiency and how much to the calcium deficiency is unclear. (In a clinical sense the question is moot: both deficiencies need repair.) Treating the underlying condition and replacing lost calcium increases bone density in these patients, but Rude and Olerich [209] have shown that even when the underlying condition is controlled and serum magnesium seemingly normal, additional magnesium supplementation will produce a further increase in bone mineral density.\n\nThis latter observation highlights one of the difficulties besetting this field\u2014the assessment of magnesium status. Serum magnesium is recognized not to be a reliable indicator of tissue magnesium repletion. Many investigators favor the magnesium tolerance test [210], that is, measuring percent retention of an intravenous infusion of magnesium. This is, of course, not practical in clinical practice. Nevertheless the observations of Rude and Olerich [209] highlight the fact that serum magnesium values within the ''normal\" reference range may mask a capacity to respond to further magnesium supplementation.\n\nThis is precisely the point at which magnesium intersects the arena of the pathogenesis and treatment of common postmenopausal osteoporosis. Unfortunately no segment of the osteoporosis field is probably more beset with poorly designed, poorly executed, and inadequately powered studies than this one. For example, two small trials, one not randomized, the other with high loss of subjects during the trial, reported bone gain in postmenopausal women given a supplement containing magnesium [211, 212]. Neither study constitutes persuasive evidence of a magnesium effect. The upshot of these and many other even weaker studies is that it is simply not possible to say with any certainty what, if any, role magnesium plays in pathogenesis or treatment of osteoporosis.\n\nOne fact seems certain: in any unselected group of individuals with low bone mass, calcium and\/or vitamin D supplementation results in clear skeletal benefits (see above), _without using extra magnesium._ And despite the fact that magnesium may be necessary for the functioning of such cells as those responsible for synthesizing 1,25(OH)2D [213], there is clear proof that supplemental magnesium does not enhance calcium absorption in ostensibly healthy older adults. Spencer _et al._ [214] more than doubled daily magnesium intake in a group of volunteers and could find no effect on calcium absorption, whether from low or normal calcium intakes. Similarly, the many randomized controlled trials demonstrating efficacy of calcium supplementation in reducing age-related bone loss and fractures all achieved their effect without supplementing with magnesium.\n\nBut absence of proof is not the same as absence of effect. One cannot say, in the routine management of osteoporosis, that the results would not have been even better had extra magnesium been provided as well. Because sprue syndromes can be silent [215], subtle magnesium deficiency could well exist in some individuals with otherwise typical osteoporosis (to mention only one potential cause of magnesium deficiency). Hence, lacking the ability easily to identify individuals with unrecognized magnesium deficiency, it is hard to argue against prudent attention to magnesium supplementation in individuals who have osteoporosis or are at high risk for fragility fractures.\n\n### B. Trace Minerals\n\nSeveral trace minerals, notably zinc, manganese, and copper, are essential metallic cofactors for enzymes involved in synthesis of various bone matrix constituents. Vitamin C (along with zinc) is needed for collagen cross-links. In growing animals, diets deficient in these nutrients produce definite skeletal abnormalities [216, 217]. Additionally, zinc deficiency is well known to produce growth retardation and other abnormalities in humans. But it is not known with certainty whether significant deficiencies of these elements develop in previously healthy adults, or at least, if they do, whether such deficiencies contribute detectably to the osteoporosis problem.\n\n#### 1. COPPER\n\nCopper is of particular interest. The principal sources of copper in the diet are shellfish, nuts, legumes, whole-grain cereals, and organ meats. True dietary copper deficiency is considered to be rare and to be confined to special circumstances, such as with total parenteral nutrition or infants recovering from malnutrition. Recognized manifestations in humans have usually centered on disorders of hemopoiesis, mainly as an iron-refractory, hypochromic anemia and leukopenia. Osteoporosis or fragility fractures have not been generally considered to be a part of the syndrome. However, copper-deficient premature infants have underdeveloped, weak bones that fracture easily and respond to copper supplementation [218], and in one human with copper deficiency due to a copper transport defect, the patient's morbidity included osteoporosis [219].\n\nCopper is a necessary cofactor for lysyl oxidase, one of the principal enzymes involved in collagen cross-linking. These cross-links are important for connective tissue strength, both in tension and in compression, because they prevent the fibrils from sliding along one another's length. Bone formed under conditions of lysyl oxidase inhibition is mechanically weak, independent of mass. Copper deficiency is reported to be associated with osteoporotic lesions in sheep, cattle, and rats [216, 220]. Copper has not been much studied in connection with human osteoporosis, but in one study in which serum copper was measured, levels were negatively correlated with lumbar spine bone mineral density, even after adjusting for body weight and dietary calcium intake [221]. In another [222] postmortem specimens of bone from osteoporotic individuals were reported to contain fewer cross-links than bone from age-matched controls.\n\n#### 2. ZINC\n\nZinc is a known constituent of about 300 enzymes, including alkaline phosphatase, and it plays a role with other proteins, such as the estrogen receptor molecule. Its principal sources in the human diet are red meat, whole-grain cereals, shellfish, and legumes. A 70-kg adult body contains 2\u20133 g zinc, about half in bone. Most of this bony zinc is located on the surfaces of the calcium phosphate crystals and probably has no metabolic significance. (Many cations present in the mineralizing environment adsorb to the oxygen-rich phosphate groups on crystal surfaces and get stuck there as free water is displaced by new mineral deposition.) A fortuitous consequence of this situation is that urine zinc reflects bone resorption. Thus, Herzberg _et al._ [223] have shown that urine zinc rises with age, is higher in patients with osteoporosis, and is reduced when postmenopausal women are given estrogen [224]. While some etiologic connection between zinc and osteoporosis cannot be ruled out, these observations are most easily explained as reflections of the enhanced bone resorption found in many patients with osteoporosis, the elevated resorption of the estrogen-deprived, postmenopausal state, and the well-known antiresorptive effect of estrogen. Urinary zinc excretion probably functions as a marker for bone resorption, rather than as a reflection of the underlying disease mechanisms.\n\nOn the other hand, of known nutrients, zinc is the one most strongly related to serum IGF-1 [225], a growth factor known to be osteotrophic even in adults. In this connection, Sch\u00fcrch _et al._ [226] have shown the importance of IGF-1 in recovery from hip fracture. In an observational study from Sweden, fracture risk was higher in individuals with low zinc intakes [227], and, after adjusting for other nutrients, the risk gradient showed the expected dose\u2013response relationship. New _et al._ [228], in a dietary survey of nearly 1000 British premenopausal women, found high zinc intakes to be associated with higher bone density values at both spine and hip.\n\nSuggestive paleolithic evidence connecting zinc intake with bone status is provided by ancient skeletons discovered in Canary Island cave burials (where contamination by, or leeching of minerals into, groundwater is considered not to have occurred). Bones with normal zinc content per unit ash, concentrated in one region of the islands, tended to be robust, while those with low zinc contents, on another island, were found to be osteoporotic [229]. Zinc content of bone, as suggested above, is determined by the circulating zinc levels when bone is mineralizing, and thus low bone zinc probably reflects low zinc intake throughout life. Whether this exposure played an etiologic role in the low bone mass of these skeletal remains is conjectural.\n\n#### 3. MANGANESE\n\nAlthough manganese is also recognized as an essential nutrient, its precise role in nutrition is much less well characterized than that of copper and zinc. Although manganese deficiency is well recognized in both laboratory and farm animals, there is no generally recognized manganese deficiency syndrome in humans. Manganese is widely distributed in foods and is especially rich in tea.\n\nBone manganese content is, like that of copper and zinc, a reflection mainly of serum levels prevailing at the time bone is formed, and thus a reflection of dietary manganese. Bone manganese probably has no other metabolic significance per se. Manganese is capable of activating many enzymes, but for most the effect is nonspecific. Manganese is, however, believed to be the preferred metal ion for certain glycosylation reactions involved in mucopolysaccharide synthesis. In this connection, manganese deficiency could interfere with both cartilage and bone matrix formation.\n\nAnimals reared on manganese-deficient diets exhibit general growth retardation, but careful measurements indicate that long bone growth is disproportionately affected [230], possibly reflecting a specific problem with endochondral bone formation. There is also indication of delayed skeletal maturation, suggesting a role of manganese in chondrogenesis. Strause _et al._ [231] showed this quite nicely in a rat model in which demineralized bone powder is implanted subcutaneously. In control animals cartilage forms around the powder implant, then osteogenesis occurs. In manganese-deficient animals neither development took place. In further work, Strause _et al._ [232] showed that manganese-deficient rats had both disordered regulation of calcium homeostasis and decreased bone mineral density. Because histology was not performed, it is not possible to say whether this represented impaired mineralization or osteoporosis. Finally, Reginster _et al._ [233] found low serum manganese in a group of 10 women with osteoporosis. What significance any of these findings may have for the bulk of human osteoporosis is uncertain.\n\nIn one four-way, randomized intervention trial, a trace mineral cocktail including copper, zinc, and manganese slowed bone mineral loss in postmenopausal women, when given either with or without supplemental calcium [234]. There appeared to be a small additional benefit from the extra trace minerals; however, the only statistically significant effect in this study was associated with the calcium supplement. This could mean that trace mineral deficiency plays no role in osteoporosis, but it could also mean that not all of the women treated suffered from such deficiency. In fact, because both osteoporotic and age-related bone loss are multifactorial, one would presume that only some of the subjects in such a study would be deficient, since there is no known way to select subjects for inclusion on the basis of presumed trace mineral need. Thus, the suggestive findings of this study have to be considered grounds for further exploration of this issue.\n\n## X. CONCLUSION\n\nMany nutrients and lifestyle interact to determine bone density and risk for osteoporosis. Knowledge of bone acquisition during childhood and adolescents has assumed a key role in predicting age-related bone loss in later years. There are also unique issues of bone metabolism that occur with pregnancy and lactation that help determine nutrient intake recommendations for women during those years. Although dietary adequacy of calcium is primarily associated with bone density, many other vitamins and minerals play important roles in the development and maintenance of normal bone. 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Szulc P., Delmas P.D., Is there a role for vitamin K deficiency in osteoporosis?. Burckhardt P., Heaney R.P., eds. Nutritional Aspects of Osteoporosis. _Challenges in Modern Medicine_. Rome, Italy: AresSerono Publications; 1995;Vol. 7:357\u2013366.\n\n190. Vermeer C., Knapen M.H.J., Jie K.-S.G., Increased vitamin K-intake may retard postmenopausal loss of bone mass. Burckhardt P., Heaney R.P., eds. Nutritional Aspects of Osteoporosis. _Challenges in Modern Medicine_. Rome, Italy: Ares-Serono Publications; 1995;Vol. 7:367\u2013379.\n\n191. Binkley N.C., Suttie J.W. Vitamin K nutrition and osteoporosis. _J. Nutr_. 1995;125:1812\u20131821.\n\n192. Nakagawa Y., Ahmed M.A., Hall S.L., Deganello S., Coe F.L. Isolation from human calcium oxalate renal stones of nephrocalcin, a glycoprotein inhibitor of calcium oxalate crystal growth. _J. Clin. Invest_. 1987;79:1782\u20131787.\n\n193. Hauschka P.V., Lian J.B., Cole D.E.C., Gundberg C.M. Osteocalcin and matrix gla protein: Vitamin K-dependent proteins in bone. _Physiol. Rev_. 1989;69:990\u20131047.\n\n194. Nakagawa Y., Abram V., Kezdy F.J., Kaiser E.T., Coe F.L. Purification and characterization of the principal inhibitor of calcium monohydrate crystal growth in human urines. _J. Biolog. Chem_. 1983;258:12594\u201312600.\n\n195. Kasai R., Bianco P., Robey P.G., Kahn A.J. Production and characterization of an antibody against the human bone GLA protein (BGP\/osteocalcin) propeptide and its use in immunocytochemistry of bone cells. _Bone Miner_. 1994;25:167\u2013182.\n\n196. Jamal S.A., Browner W.S., Bauer D.C., Cummings S.R. Warfarin use and risk for osteoporosis in elderly women. _Ann. Intern. Med_. 1998;128:829\u2013832.\n\n197. Caraballo P.J., Gabriel S.E., Castro M.R., Atkinson E.J., Melton L.J., III. Changes in bone density after exposure to oral anticoagulants: A meta-analysis. _Osteoporos. Int_. 1999;9:441\u2013448.\n\n198. Ducy P., Desbois C., Boyce B., Pinero G., Story B., Dunstan C., Smith E., Bonadio J., Goldstein S., Gundberg C., Bradley A., Karsenty G. Increased bone formation in osteocalcin-deficient mice. _Nature_. 1996;382:448\u2013452.\n\n199. Hodges S.J., Pilkington M.J., Stamp T.C.B., Catterall A., Shearer M.J., Bitensky L., Chayen J. Depressed levels of circulating menaquinones in patients with osteoporotic fractures of the spine and femoral neck. _Bone_. 1991;12:387\u2013389.\n\n200. Gerland G., Sadowski J.A., O'Brien M.D. Dietary induced subclinical vitamin K deficiency in normal human subjects. _J. Clin. Invest_. 1993;91:1761\u20131768.\n\n201. Roberts N.B., Holding J.D., Walsh H.P.J., Klenerman L., Helliwell T., King D., Shearer M. Serial changes in serum vitamin K1, triglyceride, cholesterol, osteocalcin and 25-hydroxyvitamin D3 in patients after hip replacement for fractured neck of femur or osteoarthritis. _Eur. J. Clin. Invest_. 1996;26:24\u201329.\n\n202. Plantalech L.C., Chapuy M.C., Guillaumont M., Chapuy P., Leclerq M., Delmas P.D. Impaired carboxylation of serum osteocalcin in elderly women: Effect of vitamin K1. In: Christiansen C., Overgaard K., eds. _Osteoporosis 1990_. Rome, Italy: Osteopress Ap; 1990:345\u2013347.\n\n203. Knapen M.H.J., Hamulyak K., Vermeer C. The effect of vitamin K supplementation on circulating osteocalcin (bone gla protein) and urinary calcium excretion. _Ann. Intern. Med_. 1989;111:1001\u20131005.\n\n204. Vermeer C., Hamulyak K. Pathophysiology of vitamin K-deficiency and oral anticoagulants. _Thromb. Haemostas_. 1991;66:153\u2013159.\n\n205. Feskanich D., Weber P., Willett W.C., Rockett H., Booth S.L., Colditz G.A. Vitamin K intake and hip fractures in women: A prospective study. _Am. J. Clin. Nutr_. 1999;69:74\u201379.\n\n206. Geinoz G., Rapin C.H., Rizzoli R., Kraemer R., Buchs B., Slosman D., Michel J.P., Bonjour J.P. Relationship between bone mineral density and dietary intakes in elderly. _Osteoporos. Int_. 1993;3:242\u2013248.\n\n207. Rico H., Refilla M., Villa L.F., Hernandez E.R., Fernandez J.P. Crush fracture syndrome in senile osteoporosis: A nutritional consequence. _J. Bone Miner. Res_. 1992;7:317\u2013319.\n\n208. Delmi M., Rapin C.-H., Bengoa J.-M., Delmas P.D., Vasey H., Bonjour J.-P. Dietary supplementation in elderly patients with fractured neck of the femur. _Lancet_. 1990;335:1013\u20131016.\n\n209. Rude R.K., Olerich M. Magnesium deficiency: Possible role in osteoporosis associated with gluten-sensitive enteropathy. _Osteoporos. Int_. 1996;6:453\u2013461.\n\n210. Ryzen E., Elbaum N., Singer F.R., Rude R.K. Parenteral magnesium tolerance testing in the evaluation of magnesium deficiency. _Magnesium_. 1985;4:137\u2013147.\n\n211. Abraham G.E. The importance of magnesium in the management of primary postmenopausal osteoporosis. _J. Nutr. Med_. 1991;2:165\u2013178.\n\n212. Stendig-Lindberg G., Tepper R., Leichter I. Trabecular bone density in a two-year controlled trial of peroral magnesium in osteoporosis. _Magnesium Res_. 1994;6:155\u2013163.\n\n213. Rude R.K., Adams J.S., Ryzen E., Endres D.B., Niimi H., Horst R., Haddad J.G., Jr., Singer F.R. Low serum concentrations of 1,25-dihydroxyvitamin D in human magnesium deficiency. _J. Clin. Endocrinol. Metab_. 1985;61:933\u2013940.\n\n214. Spencer H., Fuller H., Norris C., Williams D. Effect of magnesium on the intestinal absorption of calcium in man. _J. Am. College Nutr_. 1994;13:485\u2013492.\n\n215. Ott S., Tucci J.R., Heaney R.P., Marx S.J. Hypocalciuria and abnormalities in mineral and skeletal homeostatis in patients with celiac sprue without intestinal symptoms. _Endocrinol. Metab_. 1997;4:201\u2013206.\n\n216. Davis G.K., Mertz W., Copper. Mertz W., ed. Trace Elements in Human and Animal Nutrition, 5th ed., Vol. I. Copenhagen: Academic Press, Inc., 1987;301\u2013364.\n\n217. Mertz W., ed. Trace Elements in Human and Animal Nutrition, 5th ed., San Diego, CA: Academic Press, 1987.\n\n218. Schmidt H., Herwig J., Greinacher I. The skeletal changes in premature infants with copper deficiency. Rofo. _Fortschritte aud dem Gebiete der Rontgenstrahlen und der Neuen Bildgebenden Verfahren_. 1991;155:38\u201342.\n\n219. Buchman A.L., Keen C.L., Vinters H.V., Harris E., Chugani H.T., Bateman B., Rodgerson D., Vargas J., Verity A., Ament M. Copper deficiency secondary to a copper transport defect: A new copper metabolic disturbance. _Metabolism_. 1994;43:1462\u20131469.\n\n220. Strain J.J. A reassessment of diet and osteoporosis\u2014Possible role for copper. _Med. Hypotheses_. 1988;27:333\u2013338.\n\n221. Howard G., Andon M., Bracker M., Saltman P., Strause L. Low serum copper, a risk factor additional to low dietary calcium in postmenopausal bone loss. _J. Trace Elements Exp. Med_. 1992;5:23\u201331.\n\n222. Oxlund H., Mosekilde L., Ortoft G. Reduced concentration of collagen reducible cross-links in human trabecular bone with respect to age and osteoporosis. _Bone_. 1996;19:479\u2013484.\n\n223. Herzberg M., Foldes J., Steinberg R., Menczel J. Zinc excretion in osteoporotic women. _J. Bone Miner. Res_. 1990;5:251\u2013257.\n\n224. Herzberg M., Lusky A., Blonder J., Frenkel Y. The effect of estrogen replacement on zinc in serum and urine. _Obstet. Gynecol_. 1996;87:1035\u20131040.\n\n225. Devine A., Rosen C., Mohan S., Baylink D.J., Prince R.L. Effects of zinc and other nutritional factors on IGF-1 and IGF binding proteins in postmenopausal women. _Am. J. Clin. Nutr_. 1998;68:200\u2013206.\n\n226. Sch\u00fcrch M.A., Rizzoli R., Slosman D., Bonjour J.-Ph. Protein supplements increase serum IGF-1 and decrease proximal femur bone loss in patients with a recent hip fracture. In: Papapoulos S.E., Lips P., Pols H.A.P., Johnston C.C., Delmas P.D., eds. _Osteoporosis 1996_. San Diego, CA: Elsevier Science B. V.; 1996:327\u2013329.\n\n227. Elmstahl S., Gullberg B., Janzon L., Johnell O., Elmstahl B. Increased incidence of fractures in middle-aged and elderly men with low intakes of phosphorus and zinc. _Osteoporos. Int_. 1998;8:333\u2013340.\n\n228. New S.A., Bolton-Smith C., Grubb D.A., Reid D.M. Nutritional influences on bone mineral density: A cross-sectional study in premenopausal women. _Am. J. Clin. Nutr_. 1997;65:1831\u20131839.\n\n229. Gonz\u00e1lez-Reimers E., Arnay-de-la-Rosa M. Ancient skeletal remains of the Canary Islands: Bone histology and chemical analysis. _Anthrop. Anz_. 1992;50:201\u2013215.\n\n230. Asling C.W., Hurley L.S. The influence of trace elements on the skeleton. _Clin. Orthop_. 1963;27:213\u2013264.\n\n231. Strause L., Saltman P., Glowacki J. The effect of deficiencies of manganese and copper on osteoinduction and on resorption of bone particles in rats. _Calcif. Tissue Int_. 1987;41:145\u2013150.\n\n232. Strause L.G., Hegenauer J., Saltman P., Cone R., Resnick D. Effects of long-term dietary manganese and copper deficiency on rat skeleton. _J. Nutr_. 1986;116:135\u2013141.\n\n233. Reginster J.Y., Strause L.G., Saltman P., Franchimont P. Trace elements and postmenopausal osteoporosis: A preminary study of decreased serum manganese. _Med. Sci. Res_. 1988;16:337\u2013338.\n\n234. Strause L., Saltman P., Smith K.T., Bracker M., Andon M.B. Spinal bone loss in postmenopausal women supplemented with calcium and trace minerals. _J. Nutr_. 1994;124:1060\u20131064.\n\n* * *\n\n1Other things being equal, a long hip axis increases hip fracture risk, and a small cross-sectional area for vertebral bodies increases spine fracture risk.\n\n2One could argue that vitamin D ought not be considered a nutrient at all, because it is not a constituent of most foods and is naturally synthesized in abundance in our skin, given adequate solar exposure. But it was lumped accidentally with the other vitamins (nutrients in the strict sense) in the early days of development of nutritional science, and that is where we treat it and think about it today. That is not just an historical curiosity. Nutritionists in the past have often held that one can get all the nutrients one needs from a balanced, varied diet, and have been slow to embrace the notions of engineered foods and food fortification. That clearly is a misguided approach to an accidental nutrient such as vitamin D. As the species moved out of equatorial regions, humans did not hesitate to develop warm clothing and shelter as protection from a cold environment. For vitamin D, at least, the same approach would seem to be indicated, now that the biochemistry involved is understood. The industrialized nations of the high latitudes have done that for infants and children for the past 60+ years, and rickets is rare today in those countries, but vitamin D insufficiency is still common among adults\u2014particularly among the elderly (see section below on vitamin D).\n\n3Unfortunately quality control has been poor in the past [23\u201325], i.e., the level of fortification can be highly variable (ranging from near-toxic levels to absent altogether in many skim milk samples\u2014despite what may appear on the label). For both reasons, it has been extremely difficult to assess effective vitamin D intake by any sort of questionnaire.\n\n4Recommended dietary allowances (RDAs) are designed, in theory, to be generous enough to accommodate this food-related variability in requirement.\n\n5The prevailing notion at the time was that all disease was caused by infections or intoxications\u2014i.e., by some noxious influence from outside the organism.\n\n6The first clearly identified deficiency disease, beriberi, typically develops in 30\u201390 days after onset of thiamin deprivation, and it responds to treatment with roughly equal speed. One can speculate whether nutritional science would have developed at all if its disease states had typically had long latency periods.\n\n7The Masai typically have calcium intakes in the range of 6000\u20137000 mg calcium\/day, essentially all from milk.\n\n8The addition of calcium carbonate to bread flour in the United Kingdom during and after World War II and in Japan in the postwar years, as well as the recent fortification of certain breads in the United States with calcium sulfate, are but modern, conscious instances of what must have been an unwitting ancient practice.\n\n9It is instructive to compare the body's handling of calcium with that of sodium, which was an environmentally scarce nutrient during hominid evolution. By contrast with calcium, essentially 100% of dietary sodium is absorbed, and dermal and renal sodium losses can be reduced to near zero.\n\n10That this reduction in bone loss is not simply a pharmacologic effect of calcium, as suggested by Kanis and Passmore [58], is indicated by two facts: (1) The effects are greater in those with low baseline calcium intakes and (2) even the augmented intakes employed in these studies are usually well below what primitive humans would have ingested, i.e., they are in the _nutritional_ range, not the _pharmacological_ range, of calcium intakes.\n\n11This issue is complicated by the fact that diets so severely deficient in calcium are commonly inadequate on other grounds as well, e. g., protein and energy. Consequently, the growth-stunting undoubtedly has multiple causes.\n\n12Bone is not unique in this regard: The molecular basis for the setpoint in most biological feedback systems is not known.\n\n13The same level of bending sensed as tolerable in an estrogen-deprived state lies above the reference level when estrogen is present (and the set-point is lower). The bone remodeling apparatus responds by adding bone to reduce the size of the difference from the reference level of bending.\n\n14The importance of urinary loss for balance is discussed in the section on nutritional factors that influence the requirement.\n\n15In this latter respect, phosphate is as necessary for milk production as is calcium, and high serum phosphorus levels serve that important purpose. The contrary causal flow, that is, lactation pulling calcium out of bone, would work against the lactational need for phosphorus, since parathyroid hormone, mediating the response to all calcium needs, lowers serum phosphorus.\n\n16For certain bony regions, _density_ may begin to decline earlier [130], but in most such instances a countervailing periosteal expansion occurs such that total regional bone mass remains constant and bone strength is, if anything, greater.\n\n17Examples include overlarge Haversian cavities, fenestrated trabecular plates, and severed trabecular spicules that, once disconnected, become unloaded and hence are subject to rapid resorption [131].\n\n18Reduction in bone loss is only presumptively beneficial. Until it can be shown that fracture incidence is reduced, bone mass effects are less persuasive, and despite the abundant theoretical underpinnings of why bone mass should be important, only fracture reduction is ultimately convincing.\n\n19I have already remarked on the importance of urinary calcium loss in the context of the declining retention efficiency with age in growing children, and have noted that the drop in urine calcium during lactation and postweaning helps to compensate for lactational demands.\n\n20This protein effect would be predicted to be less during growth, and particularly when growth is rapid, as in infancy. Then, much of the ingested protein is incorporated into tissue, while in adults, with no net tissue gain, protein catabolism matches protein intake.\n\n21This dose amounts to a daily average exposure of 410\u2013820 IU and can thus be considered a physiological intake.\nCHAPTER 43\n\nEating Disorders: Anorexia Nervosa, Bulimia Nervosa, and Binge Eating Disorder\n\nCHERYL L. ROCK1, 1University of California at San Diego, La Jolla, California\n\nWALTER H. KAYE2, 2University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania\n\n## I. INTRODUCTION\n\nAs a general theme, the eating disorders are characterized by abnormal eating patterns and cognitive distortions related to food and weight, which, in turn, result in adverse effects on nutritional status, medical complications, and impaired health status and function. Diagnostic criteria for the eating disorders and the approaches to management have evolved through various stages during the past several decades, and research efforts continue to increase knowledge of the biological and behavioral aspects of eating pathology.\n\n## II. DEFINITIONS AND DIAGNOSTIC CRITERIA\n\nA. Diagnostic Criteria and Distinguishing Characteristics\n\nClinical diagnosis of these disorders is based on the psychological, behavioral, and physiological characteristics described by the _Diagnostic and Statistical Manual of Mental Disorders,_ fourth edition (DSM-IV), criteria [1]. Current DSM-IV criteria for anorexia nervosa, bulimia nervosa, eating disorder not otherwise specified, and binge eating disorder are listed in Table 1. For anorexia nervosa, the diagnostic criteria specify body weight <85% of that expected based on age and height (with the latter cutpoint typically interpreted as being a body mass index \u2264 17.5 kg\/m2 in adults), intense fear of gaining weight, disturbance in the way in which body size or weight is perceived, and amenorrhea if the patient is a postmenarchal female. With the most recent DSM criteria, subgroups of anorexia nervosa are further defined as restricting type and binge eating\/purging type. Criteria for the diagnosis of bulimia nervosa are recurrent binge eating, recurrent purging behavior or excessive exercise or fasting, excessive concern about body weight or shape, and absence of anorexia nervosa. Patients with bulimia nervosa are also categorized on the basis of the nature of the behavior utilized to prevent weight gain despite recurrent binge eating episodes. A clinically relevant concept that results from these criteria is that a patient cannot be diagnosed with both anorexia nervosa and bulimia nervosa at any given point in time.\n\nTABLE 1\n\nCurrent Diagnostic Criteria for Various Eating Disorders\n\n**Diagnostic citeria for anorexia nervosa**\n\nA Refusal to maintain body weight at or above a minimally normal weight for age and height (e.g., weight loss leading to maintenance of body weight <85% of that expected; or failure to make expected weight gain during period of growth, leading to body weight <85% of that expected).\n\nB Intense fear of gaining weight or becoming fat, even though underweight.\n\nC Disturbance in the way in which one's body weight or shape is experienced, undue influence of body weight or shape on self-evaluation, or denial of the seriousness of the current low body weight.\n\nD In postmenarchal females, amenorrhea, i.e., the absence of at least three consecutive menstrual cycles. (A woman is considered to have amenorrhea if her periods occur only following hormone, e.g., estrogen, administration.)\n\n_Specify type:_\n\nRestricting type: during the current episode of anorexia nervosa, the person has not regularly engaged in binge eating or purging behavior (i.e., self-induced vomiting or the misuse of laxatives, diuretics, or enemas).\n\nBinge eating\/purging type: during the current episode of anorexia nervosa, the person has regularly engaged in binge eating or purging behavior (i.e., self-induced vomiting or the misuse of laxatives, diuretics, or enemas).\n\n**Diagnostic criteria for bulimia nervosa**\n\nA Recurrent episodes of binge eating. An episode of binge eating is characterized by both of the following:\n\n(1) eating, in a discrete period of time (e.g., within any 2-hour period), an amount of food that is definitely larger than most people would eat during a similar period of time and under similar circumstances;\n\n(2) a sense of lack of control over eating during the episode (e.g., a feeling that one cannot stop eating or control what or how much one is eating).\n\nB Recurrent inappropriate compensatory behavior in order to prevent weight gain, such as self-induced vomiting; misuse of laxatives, diuretics, enemas, or other medications; fasting; or excessive exercise.\n\nC The binge eating and inappropriate compensatory behaviors both occur, on average, at least twice a week for 3 months.\n\nD Self-evaluation is unduly influenced by body shape and weight.\n\nE The disturbance does not occur exclusively during episodes of anorexia nervosa.\n\n_Specify type:_\n\nPurging type: during the current episode of bulimia nervosa, the person has regularly engaged in self-induced vomiting or the misuse of laxatives.\n\nNon-purging type: during the current episode of bulimia nervosa, the person has used other inappropriate compensatory behaviors, such as fasting or excessive exercise, but has not regularly engaged in self-induced vomiting or the misuse of laxatives, diuretics, or enemas.\n\n**Diagnostic criteria for eating disorder not otherwise specified**\n\nThis category is for disorders of eating that do not meet the criteria for any specific eating disorder. Examples include:\n\nA All of the criteria for anorexia nervosa are met except the individual has regular menses.\n\nB All of the criteria for anorexia nervosa are met except that, despite substantial weight loss, the individual's current weight is in the normal range.\n\nC All of the criteria for bulimia nervosa are met except binges occur at a frequency of less than twice a week or for a duration of less than 3 months.\n\nD An individual of normal body weight who regularly engages in inappropriate compensatory behavior after eating small amounts of food (e.g., self-induced vomiting after the consumption of two cookies).\n\nE An individual who repeatedly chews and spits out, but does not swallow, large amounts of food.\n\nF Binge eating disorder; recurrent episodes of binge eating in the absence of the regular use of inappropriate compensatory behaviors characteristic of bulimia nervosa.\n\n**Provisional criteria for binge eating disorder**\n\nA Recurrent episodes of binge eating. An episode of binge eating is characterized by both of the following:\n\n(1) eating, in a discrete period of time (e.g., within any 2-hour period), an amount of food that is definitely larger than most people would eat in a similar period of time under similar circumstances;\n\n(2) a sense of lack of control over eating during the episode (e.g., a feeling that one cannot stop eating or control what or how much one is eating).\n\nB The binge eating episodes are associated with three (or more) of the following:\n\n(1) eating much more rapidly than normal;\n\n(2) eating until feeling uncomfortably full;\n\n(3) eating large amounts of food when not feeling physically hungry;\n\n(4) eating alone because of being embarrassed by how much one is eating;\n\n(5) feeling disgusted with oneself, depressed, or very guilty after overeating.\n\nC Marked distress regarding binge eating is present.\n\nD The binge eating occurs, on average, at least 2 days a week for 6 months.\n\nE The binge eating is not associated with the regular use of inappropriate compensatory behaviors (e.g., purging, fasting, excessive exercise) and does not occur exclusively during the course of anorexia nervosa.\n\n_Source:_ Reprinted with permission from _Diagnostic and Statistical Manual of Mental Disorders,_ Fourth Edition. Copyright 1994 Psychiatric Association, Washington, DC. Psychiatric Association\n\nA third major diagnostic category, eating disorder not otherwise specified, is used to describe patients who meet most but not all of the criteria for the better defined disorders, patients who practice other types of behavior to prevent weight gain (such as chewing and spitting out food), and diagnoses for which more research is believed necessary to better define the condition. Binge eating disorder is currently considered as the latter type and is classified within this grouping, although provisional criteria have been developed for binge eating disorder and are being used until further refinements are established. The provisional criteria for binge eating disorder include recurrent episodes of binge eating, associated with at least three behavioral and attitudinal characteristics, such as eating large amounts when not physically hungry or feeling disgusted or guilty after overeating. In binge eating disorder, the absence of compensatory behaviors despite recurrent episodes of overeating typically results in obesity, although the diagnostic criteria for this condition do not specifically define or address body weight.\n\nOver a lifetime, some patients may meet diagnostic criteria for more than one of these conditions, suggesting a continuum of eating disorders [2], and there is substantial overlap in attitudes and behaviors relating to food and weight. However, distinctive patterns of comorbidity and risk factors have been identified for each of these disorders, and the nutritional and medical consequences differ a great deal. The different types of eating disorders and their subtypes are associated with different nutritional problems and management issues, despite attitudinal and behavioral similarities.\n\n### B. Prevalence\n\nCurrent prevalence of the clinical eating disorders in the general population has been estimated to be 0.5\u20131% for anorexia nervosa, 2% for bulimia nervosa, and 2% for binge eating disorder [2]. Overall, the overwhelming majority (>95%) of patients diagnosed with clinical eating disorders are female. An epidemic of eating disorders among high school students and on college campuses has been suggested in some reports, based on variable criteria and approaches to estimate prevalence of the problem, but the figures cited do not necessarily reflect the actual prevalence of clinically diagnosable eating disorders [3\u20137]. Epidemiological studies often report numbers of subjects with mild eating disturbances or bulimic behavior, typically based on self-report survey data, rather than using the DSM criteria [3, 6]. Many young women engage in pathological dieting behaviors without meeting the current diagnostic criteria for anorexia or bulimia nervosa and may be regarded as having subclinical eating disorders. For example, 40\u201360% of high school girls in the United States are reported to be dieting to lose weight, and about 13% induce vomiting or use diet pills, laxatives, or diuretics [7]. In one university survey study, 32% of women reported binge eating at least twice per month, but only 2.7% reported that they feared loss of control over eating during binges, and even fewer (1.2%) met additional criteria for bulimia nervosa [8]. Other studies of college student populations have found that at least 30% of women of reproductive age are practicing some pathological weight control activities, with 15% regularly engaging in bulimic behaviors, including both binge eating and purging [4, 5].\n\nCurrent evidence suggests that the clinical eating disorders are only the most extreme form of pathological eating attitudes and behaviors that are present in many young women. As described by Fairburn and Beglin [3], a broad spectrum of eating psychopathology likely exists in the general population, as a continuum of dieting behavior and weight concerns, especially among women. Knowledge of risk factors, demographic characteristics, and comorbidities of the eating disorders are likely distorted by the nature of the subgroup that seeks treatment, because many individuals with eating disorders do not seek treatment.\n\n## III. ETIOLOGY\n\nEating disorders have many causes, and it is likely that several factors contribute to the development of the disorder in any given case. Table 2 lists various factors associated with increased risk for eating disorders. Sociocultural influences may explain why eating disorders are mainly observed in economically advantaged communities and countries, and a cultural obsession with weight and thinness in women has been linked with increasing incidence during the past two decades [6]. Interpersonal factors and family interactions have also been suggested to contribute to risk, based on interviews and observations of patients and their families [9].\n\nTABLE 2\n\nVarious Factors Associated with Increased Risk for Eating Disorders\n\nFemale gender\n\nFamily history of eating disorders\n\nAffective disorders Substance abuse\n\nCertain character traits (i.e., perfectionism, excessive compliance)\n\nLow self-esteem\n\nFamily dysfunction\n\nProfession or pursuit that stresses maintaining a certain body weight (i.e., dance, modeling, acting, athletics)\n\nDiseases for which management involves emphasis on diet and diet regulations (i.e., type 1 diabetes mellitus, cystic fibrosis)\n\n### A. Nutritional and Dietary Risk Factors\n\nNutritional factors and dieting behavior may contribute to the development and course of eating disorders. In the pathogenesis of anorexia nervosa, dieting or other purposeful changes in food choices contribute enormously to the course of the disease because the physiological and psychological consequences of starvation serve as perpetuating factors. In bulimia nervosa, dieting precedes the development of the pattern of binge eating compensatory behaviors for the majority of patients with this disorder, although a minority (approximately 15%) report that they started binge eating prior to dieting [10]. The onset of bulimia nervosa usually follows a period of dieting to lose weight [10, 11], and a causative link between dietary restraint and bulimia is strengthened by similar behavior among obese patients who binge eat following diet restriction and among normal subjects following a period of food deprivation [12, 13]. Abnormal eating patterns, as well as their physiological consequences, serve to perpetuate the disorder and contribute to its often intractable nature. Higher prevalence rates among specific groups, such as athletes and patients with type 1 diabetes mellitus, support the concept that increased risk occurs with conditions in which dietary restraint and body weight assume great importance. However, only a small proportion of individuals who diet or restrict intake develop an eating disorder.\n\n### B. Genetic and Biologic Factors\n\nResults from recent family and twin studies suggest that eating disorders are highly heritable conditions [14, 15]. This raises the possibility that biological factors may be crucial determinants of vulnerability, with certain individuals more susceptible to developing an eating disorder in response to the environmental factors that increase risk. For example, recent evidence suggests that both anorexia nervosa and bulimia nervosa are familial and that clustering of the disorders in families may arise partly from genetic transmission of risk [16, 17]. Moreover, a more broadly defined eating disorder phenotype that differs from anorexia nervosa or bulimia nervosa in degree of severity occurs far more often in relatives of both anorexia and bulimia nervosa probands compared to relatives of normal controls. Likewise, analysis of data from a large epidemiological sample of twins obtained via the Virginia Twin Registry [18, 19] adds to evidence of a strong association between anorexia nervosa and bulimia nervosa. In short, evidence suggests at least some sharing of familial risk and liability factors between anorexia nervosa and bulimia nervosa. Paralleling these accounts are several reports of greater pairwise concordance rates of eating disorders in monozygotic compared to dyzygotic twin pairs, with several large-scale, community-based studies suggesting that as much as 50\u201380% of the variance in the development of eating disorders is due to genetic factors [15].\n\nVarious general psychiatric symptoms are found commonly in patients with anorexia nervosa or bulimia nervosa. In many cases, they develop secondary to malnutrition and other disabling psychological effects of aberrant eating psychological disability, as noted above; yet, in some, they clearly antedate disordered eating, or arise following recovery from low body weight or binge eating. Whether or not particular psychiatric disorders increase liability to eating disorders or are expressions of a shared underlying diathesis is a question of heuristic and clinical importance. For example, anorexia nervosa and bulimia nervosa often co-occur with major mood disorders, but the two conditions do not seem to express a single, shared transmitted liability [20]. Similarly, despite the high prevalence of substance abuse in bulimia nervosa, these appear to be independently transmitted disorders [21\u201323]. On the other hand, preliminary data suggest a common familial transmission of anorexia nervosa and obsessive compulsive personality disorder [16], thus suggesting the existence of a broad, genetically influenced phenotype with core features of rigid perfectionism and propensity for extreme behavioral constraint.\n\nDetermining whether behavioral symptoms are a consequence or a potential cause of pathological feeding behavior or malnutrition is a major methodological issue in the study of eating disorders. It is impractical to study eating disorders prospectively due to the young age of onset and difficulty in premorbid identification of people who will develop an eating disorder. However, subjects can be studied after longterm recovery from an eating disorder. The assumed absence of confounding nutritional influences in recovered women raises a possibility that persistent psychobiological abnormalities might be trait related and potentially contribute to the pathogenesis of this disorder. Investigators [24\u201327] have found that women who are long-term recovered from anorexia nervosa have a persistence of obsessional behaviors as well as inflexible thinking, restraint in emotional expression, and a high degree of self-and impulse-control. In addition, they have social introversion, overly compliant behavior, and limited social spontaneity as well as greater risk avoidance and harm avoidance. Moreover, long-term recovered anorexics have been found to have continued core eating disorder symptoms, such as ineffectiveness, a drive for thinness, and significant psychopathology related to eating habits. Similarly, people who have recovered from bulimia nervosa continue to be over concerned with body shape and weight, abnormal eating behaviors, and dysphoric mood [28\u201332]. Individuals recovered from anorexia nervosa or bulimia nervosa have increased perfectionism, and their most common obsessional target symptoms are the need for symmetry and ordering\/arranging. Considered together, these residual behaviors can be characterized as over concerns with body image and thinness, obsession with symmetry, exactness, and perfectionism, and dysphoric\/negative affect. In general, pathologic eating behavior and malnutrition appear to exaggerate the magnitude of these concerns. Thus, the intensity of these symptoms is reduced after recovery but the content of these concerns remains unchanged. The persistence of these symptoms after recovery raises the question of whether a disturbance of such behaviors indicates morbid traits contributing to the pathogenesis of anorexia nervosa and bulimia nervosa.\n\nThere has been considerable interest in the role that serotonin may play in anorexia nervosa and bulimia nervosa. That is because brain serotonin pathways have been implicated in behaviors typically found in people with eating disorders, such as disturbance of feeding, mood, impulse regulation, and obsessionality. A substantial number of studies have shown alterations in serotonergic activity in the ill state [33, 34]. While less well studied, serotonergic disturbances appear to persist after recovery in both anorexia nervosa and bulimia nervosa. For example, both recovered anorexics and bulimic women have been found to have elevated levels of cerebrospinal fluid concentrations of 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of serotonin [31, 35]. _Low_ levels of cerebrospinal fluid 5-HIAA are associated with impulsive and nonpremeditated aggressive behaviors, which cut across traditional diagnostic boundaries [36]. Behaviors noted after recovery from anorexia nervosa or bulimia nervosa, such as obsessions with symmetry, exactness, and perfectionism, and negative affect tend to be opposite in character to behaviors displayed by people with low 5-HIAA levels. These data support the hypothesis that increased cerebrospinal fluid 5-HIAA concentrations may be associated with exaggerated anticipatory overconcern with negative consequences (i.e., harm avoidance), while the lack of such concerns may explain impulsive, aggressive acts that are associated with low cerebrospinal fluid 5-HIAA levels.\n\n## IV. ANOREXIA NERVOSA\n\nA. Diagnosis and Nutritional Assessment\n\nAnorexia nervosa occurs much more often in girls and women (compared with males), in adolescence and early adulthood (rather than later in life), and in higher (versus lower) income groups. However, it does occur in males and can be diagnosed at any age and in any socioeconomic or cultural subgroup. By definition, patients with anorexia nervosa present with a very low weight for height, with marked reductions in both adipose tissue stores and lean body mass. In contrast with the patient who is underweight due to a condition such as infection or malabsorption, low body weight in the patient with anorexia nervosa is the result of a purposeful effort to lose weight and maintain low weight by limiting energy intake and\/or increasing energy expenditure, typically over a period of months.\n\n#### 1. PHYSICAL EXAMINATION AND CLINICAL DIAGNOSIS\n\nIn patients with eating disorders, a full physical examination should be performed by a physician familiar with common findings, with particular attention to vital signs; physical and sexual growth and development (including height and weight); the cardiovascular system; and evidence of dehydration, acrocyanosis, lanugo, salivary gland enlargement, and scarring on the dorsum of the hands (Russell's sign). A dental examination should also be performed. It is generally useful to assess growth, sexual development, and general physical development in younger patients. The use of a pediatric growth chart may permit identification of patients who have failed to gain weight and who have growth retardation. A discussion of pertinent laboratory evaluations and physical findings is provided in detail in the _Practice Guidelines for the Treatment of Patients with Eating Disorders_ [37].\n\n#### 2. ENERGY REQUIREMENTS\n\nSome investigators and clinicians have hypothesized that patients with anorexia nervosa may have increased energy requirements due to psychological stress or other metabolic abnormalities, which would theoretically facilitate weight loss. However, current evidence suggests that resting energy expenditure in anorexia nervosa patients who are maintaining a low body weight does not differ from control subjects when expressed as a function of lean body mass [38]. In ambulatory anorexia nervosa patients maintaining low body weights, Casper _et al._ [39] demonstrated that substantially increased levels of physical activity (rather than hypermetabolism per se) explain higher-than-expected total energy expenditure in low-weight patients. During the refeeding process, resting energy expenditure increases dramatically compared to prediction equations derived from observations of normal subjects who are not eating high-energy diets [40], presumably as a result of metabolic effects of the refeeding regimen. In clinical practice, this means that higher-than-expected levels of intake may be necessary to enable progressive weight restoration. Some differences in energy expenditure have been observed across subgroups of patients, with the restricting subtype observed to require more energy to promote weight gain than the binge eating\/purging type during the refeeding and immediate postweight restoration phases [41].\n\n#### 3. CLINICAL LABORATORY FINDINGS AND VITAMIN AND MINERAL STATUS\n\nTable 3 lists clinical and laboratory abnormalities that are common in anorexia nervosa patients. The endocrine abnormalities are a consequence of low body weight and malnutrition and are reversible with weight restoration. Electrolyte disturbances are more likely to occur in the binge eating\/purging type than in the restricting type. Circulating concentrations of secretory proteins that are often used as laboratory markers of overall nutritional status, such as serum albumin, are sometimes (but not always) reduced, depending in part on the state of hydration of the patient when the blood sample was collected and the severity of the weight loss.\n\nTABLE 3\n\nClinical and Laboratory Abnormalities That Are Commonly Observed in Patients with Anorexia Nervosa\n\nClinical abnormalities\n\nHypothermia\n\nDehydration\n\nHypotension, bradycardia, prolonged QT interval, arrhythmia\n\nDecreased heart size\n\nDry skin\n\nAcrocyanosis\n\nLanugo hair growth\n\nDelayed gastric emptying\n\nSevere constipation, bowel obstruction\n\nSwollen salivary glands, dental caries, and erosion of enamel (if self-induced vomiting present)\n\nOsteopenia\n\nLaboratory abnormalities\n\nLeukopenia, neutropenia, anemia, thrombocytopenia\n\nAbnormal liver enzyme concentrations\n\nHypoglycemia\n\nHypercortisolemia, elevated urinary free cortisol\n\nHypercholesterolemia\n\nHypercarotenemia\n\nLow T3 levels, sick euthyroid syndrome\n\nElectolyte disturbances (hypokalemia, hyponatremia, hypomagnesemia, hyperphosphatemia)\n\nLow serum estradiol (testosterone, if male) concentration\n\nIncreased growth hormone\n\nNotably, clinical laboratory data must be interpreted within the context of the neuroendocrine and other physiological abnormalities that are present in starvation [42\u201344]. For example, hypercholesterolemia, if present, is a consequence of thyroid abnormalities in response to dietary restriction. Hypercarotenemia is often present in these patients due to the consumption of high-carotenoid foods (i.e., deeply pigmented vegetables and fruits) by a low-weight individual [45] and is innocuous. Carotenoid absorption is inefficient but unregulated, so peripheral tissue concentrations are largely determined by intake versus relative body mass and tissue capacity (e.g., an individual with a smaller body mass has a higher tissue concentration than an individual with greater body mass, at a given dose of carotenoid intake). The conversion of provitamin A carotenoids to vitamin A is very well regulated, so hypercarotenemia does not cause hypervitaminosis A.\n\nResults from clinical studies and case series reports suggest that vitamin deficiencies occur in up to one-third of anorexia nervosa patients [46\u201349]. Overall, inadequate dietary intake and poor overall nutritional status are major causative factors. Recent evidence suggests that thyroid hormone abnormalities that result from severe dietary restriction may adversely affect riboflavin status by causing an impaired metabolism of the vitamin to the active coenzyme forms [50]. The latter finding is of clinical importance because this suggests that an improvement in energy balance and, thus, overall nutritional status (rather than simply providing the vitamin) may be necessary to correct the problem.\n\nOsteopenia is a serious and possibly irreversible medical complication of anorexia nervosa, with 50% of patients having bone density measurements greater than two standard deviations below age- and gender-matched controls [51]. Results from several studies indicate that some recovery of trabecular bone may be possible with weight restoration and recovery, but compromised bone density and deficits in bone mineral are evident after 11 years of follow-up after weight restoration and recovery [52\u201354]. Several causative factors have been identified, including hypoestrogenemia, elevated cortisol levels, decreased calcium intake, deficiency of insulin-like growth factor-1 (a nonspecific result of compromised nutritional status), and excessive physical activity [51, 55]. Unlike other conditions in which low circulating estrogen concentrations are associated with bone loss (i. e., the perimenopause), providing exogenous estrogen has not been shown to preserve or restore bone mass in the majority of patients with anorexia nervosa [56]. Calcium supplementation alone, even at a level of 1500 mg\/day, has also not been found to promote increased bone density in these patients [51, 56]. Factors most consistently observed to be associated with lower bone mineral density in these patients at diagnosis and follow-up are longer duration of illness, which is often reflected in years of amenorrhea, and lower current and\/or lowest relative body weight [51\u201354, 57, 58].\n\n### B. Medical and Nutritional Management of Anorexia Nervosa\n\nWeight restoration is the most critical component of the treatment of anorexia nervosa [37, 59, 60]. However, weight restoration alone clearly does not indicate recovery, and forcing weight gain without psychological support and counseling is ill advised. The treatment of the patient with anorexia nervosa involves a concerted and rigorous effort to improve nutritional status and restore body weight, concurrent with intensive psychotherapeutic counseling that often involves family therapy if the patient is young or resides at home.\n\n#### 1. NUTRITIONAL MANAGEMENT\n\nWeight restoration is best achieved by ensuring sufficient energy intake to promote weight gain, facilitated by nutritional counseling to provide guidance, reassurance, and assistance with specific food choices. Hospitalization is indicated for refeeding if the patient is severely nutritionally compromised (e.g., <75% of recommended weight for height) [37]. Weight restoration can also be achieved through outpatient medical care and counseling, although a slower rate of weight gain should be expected with that approach. The usual strategy is to set a target weight goal and aim for a weight gain of 2\u20133 pounds\/week for inpatients and 1\u20132 pounds\/week for outpatients during weight restoration.\n\nMost patients with anorexia nervosa are initially very conflicted about their involvement in treatment programs, because maintaining a low body weight and other aspects of the eating disorder are serving as coping mechanisms, and the eating disorder may also be the sole source of identity. Rather than perceiving the patient as being manipulative and treatment resistant, the informed view is to recognize that the behaviors are simply being tenaciously held until belief in the ability to develop another way of life and self-identify, as well as new thinking patterns, can enable the patient to relinquish the eating disorder thinking patterns and behavior. Several treatment team members are involved in the longterm care, and frequent contact and good communications among the members of the treatment team are essential.\n\nThe nutritional counseling process consists of establishing and monitoring dietary and behavioral goals in a stepwise manner, with the application of counseling strategies to help the patient expand his or her diet [60]. Typically, the patient is terrified of weight gain and may be struggling with hunger and urges to binge eat, yet the food choices allowed (by the patient) are too limited to enable sufficient energy intake. Thus, individualized guidance and a meal plan that provides a framework for meals and food choices can be enormously helpful. Specific strategies for effective nutrition intervention and counseling techniques have been described [60, 61]. Cognitive restructuring, which involves challenging deeply held beliefs and thought patterns with more accurate and healthy perceptions and interpretations, also includes nutrition education and the provision of factual information regarding dieting, nutrition, and the relationship between starvation and physical symptoms. Nutrition counseling begins with assessment and the development of shortterm and long-term goals, as rapport and a trusting relationship develop, and extends over some time with counseling sessions scheduled at a frequency that is individualized for the patient and his or her needs.\n\nIn most inpatient treatment programs, certain dietary rules about food choices and goals for energy intake must be agreed on and followed in order for patients to normalize meals and eating patterns. Typically, patients are permitted to select a limited number of foods (i.e., three to five) that they may refuse to eat, and some personal preferences can be accommodated. For example, a lactovegetarian diet can be nutritionally adequate and sufficiently dense in energy to enable weight restoration, while more extreme diets (i.e., one that excludes all animal products) present more problems because of difficulty in achieving adequate energy and nutrient intakes.\n\nWater retention during refeeding should be anticipated, due to shifts in the secretion of aldosterone in response to changes in dietary intake and other hormonal changes. Also, guidance with food choices to promote normal bowel function can be helpful, such as increased intake of grain products that are high in insoluble fiber. Delayed gastric emptying and related gastrointestinal complaints (i.e., bloating) resolve with continued refeeding and weight restoration. Fluid intake and output, electrolytes (especially phosphate and potassium), body weight, and vital signs should be monitored during refeeding.\n\nResults from several clinical studies provide some insight about changes in body composition that may be anticipated to occur in the anorexia nervosa patient during weight restoration. In a recent study of 26 low-weight female anorexia nervosa patients in which dual-energy X-ray absorptiometry and skinfold measurements were used to monitor body composition and fat distribution, increased energy intake to promote weight restoration was associated with significant increases in all major body compartments (e.g., body fat, lean body mass, bone mineral content) [62]. Although fat comprised the largest amount of the weight gained in this study by Orphanidou _et al._ [62], results from the skinfold measurements did not suggest preferential fat deposition in any area. As observed in previous reports [63], even though a greater net gain of body fat typically results from weight restoration in these patients, their body fat usually remains considerably lower than healthy control subjects. A critical concept is that change in weight or body mass index does not necessarily predict change in lean tissue or fat mass, and a meaningful change in fat mass or fat-free mass may occur without a change in body mass index or weight [64]. This is a clinically relevant issue because weight gain has historically been used as evidence of adherence to target eating patterns and, thus, is often an inaccurate basis for expanded privileges and rewards in behavioral therapy-based programs.\n\nIn very rare instances, enteral or parenteral feeding may be necessary. However, risks associated with aggressive nutrition support in these patients are hypophosphatemia, refeeding edema, cardiac failure, seizures, aspiration of enteral formula, and death [37, 65]. Thus, in the rare cases in which enteral or parenteral nutrition support may be necessary, careful and knowledgeable medical monitoring is of paramount importance. Notably, reliance on foods (rather than enteral or parenteral nutrition support) as the primary method of weight restoration contributes a great deal to the longterm recovery. The overall goal is to help the patient normalize eating patterns, and learning that behavior must involve planning and practicing with real food.\n\nIn the initial treatment phases, physical activity almost always must be limited, due to risk for injury and to create an energy balance that permits weight gain. The counseling effort should communicate that in the healthy state, exercise is an activity that should be undertaken for enjoyment and fitness (rather than a strategy to simply expend energy and promote weight loss), and that excessive exercise is often a common component of the eating disorder itself. Supervised, low-weight strength training is less likely to impede weight gain than other forms of activity and can be psychologically helpful for patients.\n\nWeight restoration is the only specific factor or intervention that has been consistently observed to promote recovery of bone mass [51, 52]. However, ensuring adequate vitamin D (400 IU\/day) and calcium (1000\u20131500 mg\/day) intakes is an additional consideration in management [37, 43, 51, 66], because these dietary factors enable bone mineralization with improved endocrine and nutritional status.\n\n#### 2. PHARMACOLOGIC MANAGEMENT AND PSYCHOLOGIC THERAPIES\n\nPatients with anorexia nervosa often do not respond effectively to treatment [67]. Extended hospitalizations can be lifesaving because such treatment can restore weight to emaciated people, which, in turn, reverses medical complications. However, such hospitalizations can be lengthy and expensive, and relapse after hospitalization has been reported to be high [68]. Limited efficacy of pharmacologic and psychologic treatment in anorexia nervosa may be due, in part, to the fact that past treatments have focused mainly on attempts to increase the rate of weight gain of emaciated patients in a hospital setting. Inpatient treatment, consisting of nursing care, behavior modification and supportive psychotherapy, succeeds in restoring the weight of most emaciated anorectics. Thus, the evaluation of the efficacy of medications in augmenting weight gain in anorexia nervosa has been limited, because most trials have been conducted on outpatients or inpatients already participating in behavioral and nutritional eating disorders treatment programs, which are themselves effective in the short run. In these settings, controlled trials have not provided consistent evidence for the efficacy of antidepressant medications in the treatment of anorexia nervosa [69\u201371].\n\nA more recent series of randomized controlled studies have examined the efficacy of various types of psychological therapies in promoting weight gain in acutely ill patients [68, 72]. Overall, the results indicate that substantial improvement in body mass and general psychosocial adjustment can be achieved in some subjects with anorexia nervosa using cognitive-behavioral, psychoeducational, and family therapy techniques (in some studies, coupled with dietary counseling), although treatment gains are not as robust in patients with more chronic, long-standing disability. A few recent studies have focused on preventing relapse in anorexia nervosa and appear to show more promise [68, 72]; for example, some psychotherapies specifically developed to treat anorexia nervosa appear to show reduced relapse at 1\u20132 years follow-up.\n\nRecent studies suggest that fluoxetine may be useful in the prevention of relapse in anorexia nervosa. In separate open and double-blind placebo-controlled studies, fluoxetine was shown to improve outcome and reduce relapse when administered _after_ weight restoration [73, 74], associated with a significant reduction in core eating disorder symptoms, depression, anxiety, and obsessions and compulsions. In a recent double-blind, placebo-controlled study, 10 of 16 (63%) subjects on fluoxetine remained well during 1 year of outpatient follow-up, whereas only 3 of 19 (16%) remained well on placebo ( _P_ = 0.006) [74]. Fluoxetine administration was associated with a significant weight gain and a significant reduction in obsessions and compulsions and, thus, appears to improve outcome in patients with anorexia nervosa by reducing symptoms and helping to maintain a healthy body weight in continuing outpatient treatment.\n\nInterestingly, two studies [75, 76] have found that selective serotonin reuptake inhibitors are specifically not useful when patients with anorexia nervosa are malnourished and underweight. As noted by Tollefson [77], these medications are dependent on neuronal release of serotonin for their action. If the release of serotonin from presynaptic neuronal storage sites is substantially compromised, and net synaptic serotonin concentration is negligible, a clinically meaningful response to a selective serotonin reuptake inhibitor might not occur. In fact, malnourished individuals with anorexia nervosa have reduced cerebrospinal fluid 5-HIAA, the major serotonin metabolite in the brain [78], suggesting that there are reduced levels of synaptic serotonin. This could be due to reduced availability of tryptophan, the essential amino acid precursor to serotonin [79]. This link between dietary intake and efficacy of selective serotonin reuptake inhibitors is supported by data that have consistently shown that dieting in healthy normal-weight and obese women reduces tryptophan availability, thereby limiting potential serotonergic production [80]. Moreover, studies in animals show that food restriction decreases serotonin and its synthesis rate in the brain [81]. In addition, depletion of tryptophan, the precursor of serotonin, reverses the effects of selective serotonin reuptake inhibitors in depressed patients [82] and plasma tryptophan was observed to be inversely related to depression scores. In anorexia nervosa, weight restoration promotes normalized nutritional status, and cerebrospinal fluid 5-HIAA concentrations then become elevated [35]. These changes in nutritional status and serotonergic activity might explain why individuals with anorexia nervosa may become responsive to fluoxetine after weight restoration.\n\n## V. BULIMIA NERVOSA\n\nA. Diagnosis and Nutritional Assessment\n\nPatients with bulimia nervosa are usually within the normal range of weight for height. The diagnostic criteria focus on the binge eating (and purging) behaviors, but another key characteristic aspect of the dietary pattern in bulimia nervosa is dietary restriction. In fact, the patient's efforts to restrict his or her diet according to rules such as good or bad and safe or forbidden (rather than simply calories per se), may actually set the stage for binge eating when the forbidden foods are consumed or the allowed level of intake is exceeded [83, 84]. A small subgroup of patients with bulimia nervosa reports that binge eating preceded dieting, and these patients appear to more closely resemble individuals with binge eating disorder (discussed below), with consistently higher body weights [10]. The majority of patients with bulimia nervosa use purging behaviors to prevent weight gain, and self-induced vomiting is the most common strategy utilized [2, 11, 37]. Purging behaviors do not completely prevent the utilization of energy from episodic binge eating, and an average retention of approximately 1200 kcal from binges of various sizes and energy contents was observed in one study [85].\n\nLaxative abuse is also very common among patients with bulimia nervosa (as well as among patients with binge eating\/purging type anorexia nervosa). Patients often use laxatives to reduce anxiety and feelings of fullness or bloating [86], although laxatives, even at the high doses used by patients with eating disorders, have been shown to have a minimal effect on absorption of energy-producing macronutrients [87].\n\n#### 1. PHYSICAL EXAMINATION AND CLINICAL DIAGNOSIS\n\nBy definition, patients with bulimia nervosa do not present with low body weight, as is characteristic of anorexia nervosa. Clinical evidence of purging behavior, such as dental changes, dehydration, salivary gland enlargement, and scarring on the dorsum of the hands (Russell's sign), are likely to be present. Gastrointestinal complaints, such as bloating, flatulence, and constipation, are common in bulimia nervosa, and the severity of these complaints has been found to correlate with severity of depression [88]. Ipecac, an over-the-counter emetic, is used by some patients with bulimia nervosa (and also by some patients with binge eating\/purging type anorexia nervosa). Chronic ipecac use can result in skeletal myopathy, electrocardiographic changes, and cardiomyopathy resulting in congestive heart failure and arrhythmia [89]. Dental complications caused by self-induced vomiting can be serious and permanent, and the typical appearance of the characteristic dental erosion that results from this behavior allows it to be distinguished from other causes [90].\n\n#### 2. CLINICAL LABORATORY FINDINGS\n\nClinical and laboratory abnormalities observed in the patient with bulimia nervosa are very similar to those observed in anorexia nervosa (see Table 3), with the degree of nutritional compromise and starvation being the most important determinant of risk for these abnormalities. Depending on the nonbinge diet, various degrees of energy imbalance occur in bulimia nervosa, so abnormalities that result from a severe energy deficit (i.e., low serum estradiol, hypercholesterolemia, osteopenia) are often, but not always, observed in these patients. Dehydration and electrolyte abnormalities caused by purging, such as hypokalemia and hypochloremic alkalosis, are common [37].\n\n### B. Medical and Nutritional Management of Bulimia Nervosa\n\nThe majority of patients with bulimia nervosa are managed as outpatients, in private practice settings, or in day treatment programs. Hospitalization is considered necessary only when life-threatening medical problems or risk for suicide is present [37]. Monitoring of electrolytes, vital signs, and body weight are the major components of medical management. Cognitive-behavioral therapy, interpersonal psychotherapy, and pharmacologic treatments have all been shown to be effective in the treatment of bulimia nervosa.\n\n#### 1. NUTRITIONAL MANAGEMENT\n\nThe primary goal of nutritional management is to normalize the patient's eating pattern, which is typically very chaotic and characterized by an overall pattern of food rules, restrictions, or dieting, regularly interspersed with episodes of binge eating and purging. Although many patients with bulimia nervosa desire weight loss (whether or not they are truly overweight), it is important to communicate that the sole and primary goal of intervention is to normalize the eating patterns and discontinue purging behavior.\n\nCognitive-behavioral therapy, which is the most well-established, effective treatment of bulimia nervosa, includes dietary guidance and nutrition education as core components of the therapy [37, 91]. The primary nutritional aspects of cognitive-behavioral therapy include meal planning, encouraging a pattern of regular eating, and discouraging dieting. Didactic nutrition education includes teaching about body weight regulation, energy balance, the psychological and physiological effects of starvation, misconceptions about dieting and weight control, and physical consequences of bulimic behavior. Patients are encouraged to eat three planned meals (plus two to three planned snacks) each day, with a minimum of 1500 kcal\/day to prevent hunger, because hunger has been shown to greatly increase susceptibility to binge eating. Helping the patient expand her diet to begin to include forbidden foods (initially, under controlled circumstances) is usually necessary and helpful. Severe and self-imposed dietary restraint regarding specific food types and choices also promotes perpetuation of the binge eating pattern. Other strategies that are helpful include keeping food records, weighing no more than once per week, and stimulus control (i.e., not shopping while hungry). Nutritional counseling includes developing a food plan and individualized guidance with specific dietary goals that are established and monitored as the diet is expanded, and a regular eating pattern is established. Additional counseling strategies have been summarized [60].\n\nDiscontinuing purging behaviors is an important aspect of intervention for most patients with bulimia nervosa, and this usually results in rebound fluid retention that is very disturbing to the patient due to overconcern with weight. Discussing the nature of this temporary weight gain is helpful, and encouraging a focus on fitness and lean body tissue (rather than body weight per se) can be reassuring. Regular eating, activity, and sleeping patterns, plus consistent daily intakes of carbohydrate, sodium, and energy, will eventually result in normal sodium (and fluid) balance. Severe dietary sodium restriction may actually aggravate the problem of fluid retention in these patients, because counterregulatory mechanisms, such as increased aldosterone secretion, are highly responsive due to periods of dehydration and electrolyte depletion. Education and guidance with the effects of withdrawal from stimulant laxatives is also often necessary, and specific clinical protocols for normalizing bowel function have been reported [37, 86]. Useful dietary strategies include increased intakes of whole grains, wheat bran, and bulk-forming agents, concurrent with adequate fluids to reduce risk for bowel obstruction.\n\nAlthough the goal is to eliminate self-induced vomiting behavior, strategies to minimize loss of dental enamel until that point is reached have been suggested. For example, toothbrushing after vomiting should be discouraged, because the enamel is particularly susceptible to abrasion at that time, but rinses with bicarbonate and fluoride solutions after vomiting, in addition to regular topical fluoride applications, may help to minimize enamel loss [90].\n\n#### 2. PHARMACOLOGIC MANAGEMENT AND PSYCHOLOGICAL THERAPIES\n\nIt is fair to say that progress to date in establishing the efficacy of specific psychological and pharmacological therapies for eating disorders has been more dramatic for bulimia nervosa than for anorexia nervosa [67, 91, 92]. With regard to psychotherapy, although the number of controlled clinical trials is still small, most indicate that cognitive-behavioral therapy is an effective treatment for upwards of 60\u201370% of individuals with bulimia nervosa, with remission of binge eating and purging achieved in 30\u201350% of cases. Direct comparisons of cognitive-behavioral therapy with other psychological treatments suggest that this modality is more effective than psychodynamically oriented supportive expressive psychotherapy in reducing core symptoms of bulimia nervosa, and is more effective than a strictly behavioral treatment in preventing early relapse into dietary restriction, binge eating, and purging. Evidence further suggests that cognitive-behavioral therapy results in improvements in certain core features of the illness, such as body dissatisfaction, pursuit of thinness, and perfectionism.\n\nEvidence for the efficacy of antidepressant pharmacotherapy in bulimia nervosa is impressive; however, the benefits may diminish over time in a significant proportion of individuals with bulimia nervosa who respond initially, and only a minority have complete suppression of their symptoms with antidepressant monotherapy [69\u201371]. The results of most double-blind, placebo-controlled randomized trials reported to date indicate that antidepressants show at least some superiority over placebo in reducing the frequency of binge eating episodes. In addition, some studies show a reduction in intensity of some other symptoms commonly seen in bulimia nervosa, such as preoccupation with food and depression. These findings have been demonstrated with a variety of antidepressive medications, including tricyclic agents, monoamine oxidase inhibitors, and selective serotonin reuptake inhibitors. Patients participating in these trials typically reported from 8\u201310 episodes of binge eating per week at baseline. The average decrease in binge frequency for patients receiving the antidepressant medication has been observed to be about 55%, with wide variation across studies. Placebo responses were similarly variable, but generally the decrease of binge frequency was less than half of the magnitude of the response for the active treatment. However, only a minority of the patients actually achieved full abstinence from binge eating and purging behaviors. Most trials have shown no correlation between improvement in mood and reduction in symptoms of bulimia nervosa. Additionally, antidepressants suppress symptoms of bulimia nervosa also in nondepressed bulimic patients, suggesting a mode of action other than through their antidepressant effects. In some studies, the patients receiving the antidepressive medication demonstrated a reduction in the tendency for stressors to trigger binge eating.\n\nA few studies [93\u201398] have assessed the relative efficacy of combining psychotherapy (in most trials, of the cognitive-behavioral therapy type) and antidepressants for the management of bulimia nervosa, compared with the isolated treatments themselves. Although differing in many respects, these studies suggest that the improvement in symptoms of bulimia nervosa with cognitive-behavioral therapy alone was greater than with the medication alone. Adding medication to the psychotherapy generally did not significantly improve the outcome over psychotherapy alone in terms of eating behaviors, nor did it increase the speed of the therapeutic response. However, one prolonged follow-up evaluation found that combined treatment was more effective for a number of eating variables than cognitive-behavioral therapy alone. Another study showed the superiority of combined therapy in reducing the rates of anxiety and depression.\n\n## VI. EATING DISORDER NOT OTHERWISE SPECIFIED\n\nA. Overview of Eating Disorder Not Otherwise Specified\n\nEating disorder not otherwise specified is a large and heterogeneous diagnostic category that is applicable for individuals who have clinically significant eating disorders but who fail to meet all of the diagnostic criteria for the main eating disorders, anorexia nervosa or bulimia nervosa [99]. Binge eating disorder is included in this category at this time, as evidence accumulates to sufficiently support the specific diagnostic criteria for this disorder [1].\n\nPatients with this diagnosis can have serious eating problems that adversely affect health status, and the nutritional and medical management issues in these patients are similar to those in the patient with anorexia nervosa or bulimia nervosa. For example, some women can menstruate at a very low body weight, possibly due to a biological adaptation to the low weight, although they are otherwise comparable to the patient who meets the full diagnostic criteria for anorexia nervosa (which includes amenorrhea for at least three consecutive menstrual cycles). Another example is an obese individual who has lost a substantial amount of weight due to extreme and unhealthy eating patterns and cognitive distortions related to food and weight, but whose current weight is in the normal range. The eating problems and attitudes of the latter type of patient are characterized by an intense fear of regaining weight. Individuals with unusual food rules or eating patterns to promote weight loss or maintenance of low body weight, although not below a weight that meets the criteria for anorexia nervosa, and associated with psychological distress and abnormal attitudes about food, would also be assigned to this diagnostic category.\n\nBecause this is a heterogeneous group, the medical and nutritional management of a patient with a diagnosis of eating disorder not otherwise specified is determined by the specific eating behaviors and attitudes and clinical problems that are present. In general, the same issues, principles and treatment strategies relevant to the other eating disorders are applicable to these patients.\n\n### B. Binge Eating Disorder\n\n1. DIAGNOSIS AND NUTRITIONAL ASSESSMENT OF BINGE EATING DISORDER\n\nThe diagnostic criteria for binge eating disorder are considered provisional rather than definite at this time, because less evidence has been accumulated to define the disorder and to differentiate it from other eating disorders as a distinct condition [1]. Also, fewer studies that might support the efficacy of various treatments and interventions have been conducted and reported, compared to research in anorexia nervosa and bulimia nervosa [100\u2013102].\n\nPatients with binge eating disorder do not use purging behaviors on a regular basis, yet they binge eat regularly, so they are typically obese [12]. Patients with binge eating disorder are usually identified when they seek treatment for their obesity, rather than presenting for treatment of an eating disorder. In fact, >20% of individuals seeking weight loss treatment from university or commercial programs report binge eating problems and related psychological distress [12]. The characteristics and behaviors of patients with binge eating disorder have been compared with those of non-binge-eating obese patients in several studies. A consistent finding in these studies is that patients with binge eating disorder are typically more obese than non-binge-eating obese patients [103]. Another consistent finding is that patients with binge eating disorder, compared to non-binge-eating obese patients, have an increased frequency of psychiatric symptoms and diagnoses, independent of degree of obesity [101\u2013104]. Major depression accounts for the greatest difference between these two groups.\n\nWhile anorexia nervosa and bulimia nervosa occur predominantly in women, males comprise a comparatively higher percentage of patients with binge eating disorder (suggested to be 40% of patients with this disorder), and similar prevalence rates have been observed in men and women in community and college samples [105, 106]. Another demographic feature that distinguishes binge eating disorder from the other eating disorders is that it appears to occur at similar rates across different racial and ethnic groups [106\u2013108].\n\nAlthough the majority of patients with binge eating disorder report frequent dieting and concern with body weight, a pattern of dieting behavior preceding binge eating is not characteristic of patients with binge eating disorder, in contrast with the temporal sequence that occurs in the majority of patients with bulimia nervosa [102]. Approximately 50% of patients with binge eating disorder report that they started binge eating before they started dieting or trying to lose weight [109, 110]. Because of the concern that dietary restriction increases the susceptibility to binge eating in bulimia nervosa, normalized eating patterns and guidance away from energy restriction is stressed in bulimia nervosa treatment programs. However, this concern may not be as relevant in the treatment of binge eating disorder, in which patients appear to have a more generalized and consistent lack of control over their eating patterns and food choices, even when they are not binge eating.\n\n#### 2. PSYCHOLOGICAL, MEDICAL, AND NUTRITIONAL MANAGEMENT OF BINGE EATING DISORDER\n\nSimilar to bulimia nervosa, cognitive-behavioral therapy, interpersonal psychotherapy, and pharmacologic treatments have all been shown to have utility in the treatment of binge eating disorder [102]. Psychotherapy has been shown to be effective in reducing the frequency of binge eating in patients with binge eating disorder, but cognitive behavior therapy (adapted from protocols used in the treatment of bulimia nervosa) and interpersonal therapy are the approaches most often utilized [12, 101, 102].\n\na. Nutritional Management.: In the adaptation of cognitive-behavioral therapy to the treatment of binge eating disorder, the goal is reduced frequency of binge eating and also a pattern of overall moderation of food intake. Although a framework or meal plan for regular meals may be useful for many patients, strict food rules and rigid diet plans are discouraged. The goal is to promote a healthy overall pattern of eating and exercise, and achieving that goal is accomplished by planning and by identifying and modifying maladaptive thoughts and beliefs that lead to overeating and binge eating. Nutritional guidance emphasizes three planned meals and two to three planned snacks each day, no more than a 3-hour interval between meals or snacks, a varied and balanced diet, and food servings that are of average portion size. Many patients with binge eating disorder benefit from basic nutrition education on the components of a healthy diet and discussion to dispel common myths about diet and nutrition.\n\nAn important issue in the nutritional management of patients with binge eating disorder is whether weight loss should be a goal of the intervention, in addition to normalized eating patterns. For the vast majority of these patients, obesity is the major health risk resulting from the disorder. Efforts that are focused solely on eliminating binge eating have not been shown to result in weight loss in the majority of patients with binge eating disorder 110\u2013114]. Also, studies suggest that guidance toward energy restriction through the application of behavioral weight control strategies does not appear to exacerbate binge eating in these patients [101, 102, 111]. However, the long-term effects on weight management that may be achieved in these patients by behavioral intervention programs that incorporate energy restriction are unknown. Decades of research and long-term follow-up of participants in behavioral treatment programs aimed toward weight reduction suggest that few patients are likely to sustain the energy deficit necessary for continued weight loss and maintenance without considerable motivation and lifestyle modification (see [Chapter 34). Thus, the cognitive-behavioral treatment programs for binge eating currently place the greatest emphasis on the goal of normalized eating patterns, clarifying with patients that simply eliminating the binge eating is unlikely to cause substantial weight loss.\n\nb. Pharmacologic Management.: The use of antidepressants, such as the tricyclic antidepressants, in binge eating disorder has been suggested to have a possible role in the treatment of binge eating disorder, based on results from some studies [101]. However, a role for pharmacotherapy has not been firmly established at this point, and more studies are needed.\n\n## VII. OUTCOME\n\nAs recently reviewed by Pike [115], most studies of longterm outcome of patients with anorexia nervosa suggest that 50\u201375% of patients achieve a good to intermediate outcome and 15\u201325% are chronically symptomatic, although the length of time of follow-up contributes to large variability in these figures. Estimates of overall mortality associated with anorexia nervosa range up to 20%, mainly due to starvation-related medical problems and suicide. In a 10- to 15-year follow-up study conducted by Strober and colleagues [116], nearly 76% of 95 adolescent patients with anorexia nervosa experienced full recovery, defined as being free of all criterion symptoms of anorexia nervosa or bulimia nervosa for not less than 8 consecutive weeks. Nearly 14% failed to achieve even partial recovery at any time during the follow-up period, although no deaths occurred in this case series. Time to recovery ranged from 57 to 79 months, but once recovered, subsyndromal relapse was rare (observed in only 7% of patients who achieved full recovery, and most of these patients returned to full recovery).\n\nIn another long-term follow-up study of 84 female patients with anorexia nervosa reported by Zipfel and colleagues [117], 51% of the patients had achieved full recovery, 10% still met full diagnostic criteria for anorexia nervosa, and 16% had died from causes related to anorexia nervosa, after an average of 21 years following the initial inpatient treatment. Significant predictors of poorer outcome were longer duration of illness before first admission, low body mass index, inadequate weight gain during the first hospitalization, and severe psychological or social problems.\n\nSimilar to long-term follow-up studies of anorexia nervosa, outcome data on bulimia nervosa vary considerably across studies, with recovery rates ranging from 13% to 71% [118]. Factors reported to be predictive of poorer outcome in bulimia nervosa were severity of eating pathology, frequency of vomiting at baseline, extreme weight fluctuations, comorbid disorders, impulsivity, low self-esteem, and suicidal behavior. In a follow-up study of patients with bulimia nervosa who were followed for an average of 11.5 years [119], full or partial remission occurred in nearly 70% of the 173 patients. Of the remainder, 11% still met full criteria for bulimia nervosa, 18.5% met criteria for eating disorder not otherwise specified, and 1 patient died.\n\nAlthough long-term follow-up data on binge eating disorder are scant, good outcome of 68 patients with binge eating disorder over a 6-year course has been reported [120], with the majority having no eating disorder at follow-up. Nearly 6% still met criteria for binge eating disorder, 7.4% met criteria for bulimia nervosa, 7.4% met criteria for eating disorder not otherwise specified, and 1 patient died.\n\n## VIII. PREVENTION OF EATING DISORDERS\n\nFew would argue that prevention or early detection and treatment are the best approaches to reducing the morbidity and mortality associated with the eating disorders. Although current evidence suggests that genetic factors play a crucial role in determining the individual susceptibility to eating disorders, familial, sociocultural, and nutritional factors (i.e., dieting behavior) create the environment in which the susceptibility is more likely to result in a full-blown eating disorder. Chaotic eating patterns and malnutrition adversely affect cognitive function and response to psychotherapy, so early intervention and restoration of good nutritional status can be important in determining the course of the disorder.\n\nAlthough there is general agreement that prevention is a good strategy, the best focus of these efforts is still unclear [121]. Organized prevention efforts, which are typically provided and evaluated in school and university settings [122\u2013124], usually emphasize the value of individuality, promoting increased self-confidence and self-esteem, and encouraging the development of skills to resist social pressure [125, 126]. Attitudes and beliefs that promote dieting are driven by powerful sociocultural attitudes that may be resistant to change. However, health care providers can be sensitive to the attitudes about food, nutrition, and body weight that are communicated to individual patients and community groups, recognizing the potential impact of these communications on dieting behavior and weight concerns in those at risk.\n\n## IX. SUMMARY AND CONCLUSIONS\n\nNutrition is an important aspect of the prevention and treatment of the eating disorders, which appear to develop as a consequence of both genetic and environmental factors in susceptible individuals. 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Attia E., Haiman C., Walsh B.T., Flater S.R. Does fluoxetine augment the inpatient treatment of anorexia nervosa? _Am. J. Psychiatry_. 1998;155:548\u2013551.\n\n76. Ferguson C.P., La Via M.C., Crossan P.J., Kaye W.H. Are SSRI's effective in underweight anorexia nervosa? _Int. J. Eating Disord_. 1999;25:11\u201317.\n\n77. Tollefson G.D. Selective serotonin reuptake inhibitors. In: Schatzberg A.F., Nemeroff C.B., eds. _Textbook of Psychopharmacology_. Washington, DC: American Psychiatric Press; 1995:161\u2013182.\n\n78. Kaye W.H., Ebert M.H., Raleigh M., Lake R. Abnormalities in CNS monoamine metabolism in anorexia nervosa. _Arch. Gen. Psychiatry_. 1984;41:350\u2013355.\n\n79. Schweiger U., Warnhoff M., Pahl J., Pirke K.M. Effects of carbohydrate and protein meals on plasma large neutral amino acids, glucose, and insulin levels of anorexic patients. _Metabolism_. 1986;35:938\u2013943.\n\n80. Anderson I.M., Parry-Billings M., Newsholme E.A., Fairburn C.G., Cowen P.J. Dieting reduces plasma tryptophan and alters brain 5-HT function in women. _Psycholog. Med_. 1990;20:785\u2013791.\n\n81. Haleem D.J., Haider S. Food restriction decreases serotonin and its synthesis rate in the hypothalamus. _Neuro. Report_. 1996;7:1153\u20131155.\n\n82. Delgado P.L., Price L.H., Aghajanian G.K., Landis H., Heninger G.R. Serotonin function and the mechanism of antidepressant action. Reversal of antidepressant-induced remission by rapid depletion of plasma tryptophan. _Arch. Gen. Psychiatry_. 1990;47:411\u2013418.\n\n83. Kales E.F. Macronutrient analysis of binge eating in bulimia. _Physiol. Behav_. 1990;43:837\u2013840.\n\n84. Heatherington M.M., Altemus M., Nelson M.L. Eating behavior in bulimia nervosa: Multiple meal analyses. _Am. J. Clin. Nutr_. 1994;60:864\u2013873.\n\n85. Kaye W.H., Weltzin T.E., Hsu L.K., McConaha C.W., Bolton B. Amount of calories retained after binge eating and vomiting. _Am. J. Psychiatry_. 1993;150:969\u2013971.\n\n86. Colton P., Woodside D.B., Kaplan A.S. Laxative withdrawal in eating disorders: treatment protocol and 3- to 20-month follow-up. _Int. J. Eating Disord_. 1999;25:311\u2013317.\n\n87. Bo-Linn G.W., Santa Ana C.A., Morawski S.G., Fordtran J.S. Purging and calorie absorption in bulimic patients and normal women. _Ann. Intern. Med_. 1983;99:14\u201317.\n\n88. Chami T.N., Anderson A.E., Crowell M.D., Schuster M.M., Whitehead W.E. Gastrointestinal symptoms in bulimia nervosa: Effects of treatment. _Am. J. Gastroenterol_. 1995;90:88\u201392.\n\n89. Palmer E.P., Guay A.T. Reversible myopathy secondary to abuse of ipecac in patients with major eating disorders. _N. Engl. J. Med_. 1985;313:1457\u20131459.\n\n90. Bouquot J.E., Seime R.J. Bulimia nervosa: Dental perspectives. _Prac. Periodont. Aesth. Dentistry_. 1997;9:655\u2013663.\n\n91. Fairburn C.G., Marcus M.D., Wilson G.T. Cognitive-behavioral therapy for binge eating and bulimia nervosa. In: Fairburn C.B., Wilson G.T., eds. _Binge Eating_. Washington, DC: Guilford Press; 1993:361\u2013404.\n\n92. Arnow B. Psychotherapy of anorexia and bulimia. _Balliere's Clin. Psych_. 1997;3:235\u2013257.\n\n93. Agras W.S., Rossiter E.M., Arnow B., Schneider J.A., Telch C.F., Raeburn S.D., Bruc B., Perl M., Koran L.M. Pharmacologic and cognitive-behavioral treatment for bulimia nervosa: A controlled comparison. _Am. J. Psychiatry_. 1992;149:82\u201387.\n\n94. Fichter M.M., Leibl K., Rief W., Brunner E., Schmidt-Auberger S., Engel R.R. Fluoxetine versus placebo: a double-blind study with bulimic inpatients undergoing intensive psychotherapy. _Pharmacopsychiatry_. 1991;24:1\u20137.\n\n95. Goldbloom D.S., Olmsted M., Davis R., Clewes J., Heinmaa M., Rockert W., Shaw B. A randomized controlled trial of fluoxetine and cognitive behavioral therapy for bulimia nervosa: Short-term outcome. _Behav. Res. Ther_. 1997;35:803\u2013811.\n\n96. Leitenberg J., Rosen J.C., Wolf J., Vara L.S., Detzer M.J., Srebnik D. Comparison of cognitive-behavior therapy and desipramine in the treatment of bulimia nervosa. _Behav. Res. Ther_. 1994;32:37\u201345.\n\n97. Mitchell J.E., Pyle R.L., Eckert E.D., Hatsukami D., Pomeroy C., Zimmerman R. A comparison study of antidepressants and structured intensive group psychotherapy in the treatment of bulimia nervosa. _Arch. Gen. Psychiatry_. 1990;47:149\u2013157.\n\n98. Walsh B.T., Wilson G.O., Leob K.L., Devlin M.J., Pike K.M., Roose S.P., Fleiss J., Waternaux C. Medication and psychotherapy in the treatment of bulimia nervosa. _Am. J. Psychiatry_. 1997;154:523\u2013531.\n\n99. Walsh B.T., Garner D.M. Diagnostic issues. In: Garner D.M., Garfinkel P.E., eds. _Handbook of Treatment for Eating Disorders_. 2nd ed. New York: Guilford Press; 1997:25\u201333.\n\n100. Baker C.W., Brownell K.D. Binge eating disorder: Identification and management. _Nutr. Clin. Care_. 1999;2:344\u2013353.\n\n101. Devlin M.J. Assessment and treatment of binge-eating disorder. _Psych. Clin. N. Am_. 1996;19:761\u2013772.\n\n102. Marcus M.D. Adapting treatment for patients with binge-eating disorder. In: Garner D.M., Garfinkel P.E., eds. _Handbook of Treatment for Eating Disorders_. 2nd ed. New York: Guilford Press; 1997:484\u2013493.\n\n103. Telch C.F., Agras W.S., Rossiter E.M. Binge eating increases with increasing adiposity. _Int. J. Eating Disord_. 1988;7:115\u2013119.\n\n104. Marcus M.D., Wing R.R., Ewing L., Kern E., Gooding W., McDermott M. Psychiatric disorders among obese binge eaters. _Int. J. Eating Disord_. 1996;19:45\u201352.\n\n105. Yanovski S.Z., Nelson J.E., Dubbert B.K., Spitzer R.L. Association of binge eating disorder and psychiatric comorbidity in obese subjects. _Am. J. Psychiatry_. 1993;150:1472\u20131479.\n\n106. Spitzer R.L., Yanovski S.Z., Wadden T., Wing R., Marcus M.D., Stunkard A., Devlin M., Mitchell J., Hasin D., Horne R.L. Binge eating disorder: Its further validation in a multisite study. _Int. J. Eating Disord_. 1993;13:137\u2013153.\n\n107. Smith D.E., Marcus M.D., Lewis C., Fitzgibbon M., Schreiner P. Prevalence of binge eating disorder, obesity, and depression in a biracial cohort of young adults. _Ann. Behav. Med_. 1998;20:232\u2013277.\n\n108. Smith D.E. Binge eating in racial minority groups. _Addict. Behav_. 1995;20:695\u2013703.\n\n109. Wilson G.T., Nonas C.A., Rosenblum G.D. Assessment of binge-eating in obese patients. _Int. J. Eating Disord_. 1993;13:25\u201333.\n\n110. Mussell M.P., Mitchell J.E., Weller C.L., Raymond N.C., Crow S.F., Crosby R.D. Onset of binge eating, dieting, obesity, and mood disorders among subjects seeking treatment for binge eating disorder. _Int. J. Eating Disord_. 1995;17:395\u2013410.\n\n111. Marcus M.D., Wing R.R., Fairburn C.G. Cognitive treatment of binge eating versus behavioral weight control in the treatment of binge eating disorder. _Ann. Behav. Med_. 1995;17:S090.\n\n112. Wilfley D.E., Agras W.S., Telch C.F., Rossiter E.M., Schneider J.A., Cole A.G., Sifford L.A., Raeburn S.D. Group cognitive-behavioral therapy and group inter-presonal therapy for the nonpurging bulimic individual: A controlled comparison. _J. Consult. Clin. Psychol_. 1993;61:296\u2013305.\n\n113. Carter J.C., Fairburn C.G. Cognitive-behavioral self-help for binge eating disorder: A controlled effectiveness study. _J. Consult. Clin. Psychol_. 1998;66:616\u2013623.\n\n114. Tech C.F., Agras W.S., Rossiter E.M., Wilfley D., Kenardy J. Group cognitive-behavioral treatment for the nonpurging bulimic: An initial evaluation. _J. Consult. Clin. Psych_. 1990;58:629\u2013635.\n\n115. Pike K.M. Long-term course of anorexia nervosa: Response, relapse, remission, and recovery. _Clin. Psychol. Rev_. 1998;18:447\u2013475.\n\n116. Strober M., Freeman R., Morrell W. The long-term course of severe anorexia nervosa in adolescents: Survival analysis of recovery, relapse, and outcome predictors over 10\u201315 years in a prospective study. _Int. J. Eating Disord_. 1997;22:339\u2013360.\n\n117. Zipfel S., Lowe B., Reas D.L., Deter H.C., Herzog W. Long-term prognosis in anorexia nervosa: lessons from a 21-year follow-up study. _Lancet_. 2000;355:721\u2013722.\n\n118. Herzog D.B., Nussbaum K.M., Marmor A.K. Comorbidity and outcome in eating disorders. _Psych. Clin. N. Am_. 1996;19:843\u2013859.\n\n119. Keel P.K., Mitchell J.E., Miller K.B., Davis T.L., Crow S.J. Long-term outcome of bulimia nervosa. _Arch. Gen. Psychiatry_. 1999;56:63\u201369.\n\n120. Fichter M.M., Quadflieg N., Gnutzmann A. Binge eating disorder: Treatment outcome over a 6-year course. _J. Psychosom. Res_. 1998;44:385\u2013405.\n\n121. Huon G.F., Braganza C., Brown L.B., Richie J.E., Roncolato W.G. Reflections on prevention in dieting-induced disorders. _Int. J. Eating Disord_. 1998;23:455\u2013458.\n\n122. Rosen D.S., Neumark-Sztainer D. Review of options for primary prevention of eating disturbances among adolescents. _J Adolesc. Health_. 1998;23:354\u2013363.\n\n123. Schwitzer A.M., Bergholz K., Dore T., Salimi L. Eating disorders among college women: Prevention, education, and treatment responses. _J. Am. College Health_. 1998;46:199\u2013207.\n\n124. Neumark-Sztainer D. School-based programs for preventing eating disturbances. _J. School Health_. 1996;66:64\u201371.\n\n125. Killen J.D., Taylor B.C., Hammer L.D., Litt I., Wilson D.M., Rich T., Hayward C., Simmonds B., Krawmer H., Varady A. An attempt to modify unhealthful eating attitudes and weight regulation practices of young adolescent girls. _Int. J. Eating Disord_. 1993;13:369\u2013384.\n\n126. Huon G.F. Towards the prevention of dieting-induced disorders: Modifying negative food- and body-related attitudes. _Int. J. Eating Disord_. 1994;16:395\u2013399.\nCHAPTER 44\n\nNutrition and Food Allergy\n\nABBA I. TERR, San Francisco, California\n\n## I. INTRODUCTION\n\nFood is the primary source of nutrients required for maintaining the structure and continued functioning of the body. It is also a potential source of disease. Excessive, insufficient, or improper balance of foods in the diet may cause pathologic conditions such as obesity or malnutrition. Foods and their natural or artificial additives and contaminants can be the source of infection, toxic diseases, or allergy.\n\nFood allergy has been recognized since antiquity. It is based on the fact that foods are chemically foreign to the body. An allergic reaction can range in severity from an annoyance to death. Thus, diagnosing food allergy and identifying the specific food that caused it are essential, so that future reactions can be avoided.\n\nFood allergy is a confusing and often misunderstood subject. It is frequently misdiagnosed. This chapter will focus on the mechanisms of disease and the methods of diagnosis and treatment that are based on current scientific evidence.\n\n## II. DEFINITIONS\n\nConfusion about what constitutes food allergy occurs among medical professionals as well as the public. This can often be traced to a lack of precise terminology and clear understanding of physiologic mechanisms of food-induced disease.\n\n_Adverse reactions_ are any conditions\u2014whether pathologic or only subjective\u2014that occur when a particular food is eaten. _Food allergy_ is one of main adverse reactions and refers to disease in which a component of food, most often a protein, is an antigen that induces an immune response in the patient. The antigen responsible for allergy is called an _allergen._ Not all immune responses to food antigens result in allergy.\n\n_Allergy_ refers to immune responses to environmental allergens that result in inflammation detrimental to the patient. In contrast, _immunity_ refers to immune responses that protect from infectious microorganisms or toxic chemicals.\n\nThere are a number of allergic diseases, reflecting the complexity of the immune response. They are classified by the Gell and Coombs system, which is based on the particular immune pathway involved in the disease (Table 1). This classification system is fully discussed later in the chapter.\n\nTABLE 1\n\nGell and Coombs Classification of Allergic Diseases\n\n_Food intolerance_ is the term used by allergists for adverse reactions that are not allergic. Certain foods contain chemicals that have _pharmacologic_ effects, such as caffeine in coffee causing stimulation, or histamine-like compounds in wines causing flushing and headache (Table 2). Some persons are susceptible to these effects, while others are not. Those who are may misinterpret their symptoms as an allergy. _Toxicity_ is likewise a direct effect of either a naturally occurring chemical in the food, such as mushroom poisoning, or a toxin from a bacterial contaminant, such as the _Staphyloccocus_ or _Escherichia coli._\n\nTABLE 2\n\nSome Foods with Naturally Occurring Chemicals That May Simulate the Effects of Allergy\n\n## III. THE IMMUNE RESPONSE AND ALLERGY\n\nThe immune response is a complicated process by which the immune system generates both antibodies and sensitized lymphocytes that recognize a specific antigen. Antibodies are referred to chemically as _immunoglobulins._ The five different classes of immunoglobulin molecules are IgG, IgA, IgM, IgD, and IgE. Antibodies of the IgG class are the most abundant in the serum and are responsible for long-lasting immunity. IgM antibodies are the ones that are produced first during the immune response. IgE antibodies are responsible for the most common forms of allergic diseases. Because immune responses are normal processes, small amounts of antibodies to foods are normal and do not cause disease or interfere with health.\n\nSpecificity and cross-reactivity are important concepts in allergic disease. _Specificity_ refers to the ability of the immune system to recognize very small differences between \"foreign\" proteins (i.e., those from the environment) and proteins that exist in the person's own body. Each individual's immune responses are determined by his or her genetic makeup. This fact is especially evident when immunologic rejection of a tissue or organ graft occurs.\n\n_Cross-reactivity_ arises when two or more different proteins have the same or closely similar chemical structure so that they are recognized by the same antibody. There are numerous examples, especially among food allergens and among allergens in foods and other environmental substances, such as pollens and natural rubber latex (Table 3).\n\nTABLE 3\n\nCross-Reactivity of Foods and Other Allergens\n\nAny food protein is a potential allergen. Nevertheless, the majority of people tolerate all of the food proteins they eat, even though these protein products are rapidly and efficiently absorbed from the gastrointestinal mucosa and reach the systemic circulation, from where they have access to the immune system. The immune system does, in fact, recognize many food protein products as antigenic and does make specific IgG, IgA, and IgM antibodies and T-cell immune responses to them. However, these are normal and do not cause disease. They may be present in higher amounts in infants who are weaned early.\n\nThe gastrointestinal tract has a unique immune system of its own, called the gastrointestinal associated lymphoid tissue, which is a part of the larger mucosal associated lymphoid tissue of the gastrointestinal tract, respiratory mucosa, and the ductal tissue of the mammary gland during lactation. Gastrointestinal associated lymphoid tissue consists of lymphoid tissue that includes the Peyer's patches of the intestinal mucosa, the lymphoid follicles in the appendix, lymphocytes and plasma cells in the intestinal lamina propria, the intra-epithelial lymphocytes, and the mesenteric lymph nodes.\n\nA unique feature of gastrointestinal associated lymphoid tissue is the production of secretory IgA antibodies (sIgA). These antiodies form molecular complexes with their corresponding food antigens (Ag). The Ag\u2013sIgA complexes are retained in the glycocalyx, slowing absorption of food antigens and promoting digestion by proteolytic enzymes.\n\n_Immunological tolerance_ refers to the failure of the immune system to respond to an antigen. An individual's own protein molecules (and other potential allergens) are normally in a state of immunologic self-tolerance. There are certain \"autoimmune\" diseases, such as systemic lupus erythematosus, in which self-tolerance to some of the body's natural chemicals is lost, so that the immune system produces autoantibodies and autoimmune disease. _Oral tolerance_ can be produced experimentally in animals by the oral administration of antigen. This causes a decreased or absent systemic immune response. It requires a high dose of antigen, and the effect is specific for that antigen only and is not a general immune depression. The significance of oral tolerance as a normal process is not certain. However, eating food exposes the immune system to large oral quantities of potential food antigens, so it is possible that oral immunologic tolerance to foods may be necessary for normal health.\n\nIt has been postulated that a decrease in oral tolerance to foods may enhance production of IgE antibodies and lead to food allergy. Several studies show that exclusive breast feeding increases oral tolerance and helps prevent food allergy. Although this is theoretical, allergists and pediatricians encourage breast feeding, hoping to prevent or delay the development of allergy in infants.\n\n### A. The Allergic Response\n\nAllergy is a two-step process. The first step is _sensitization._ The immune system recognizes a foreign protein (e.g., in a food) as an antigen when it enters the body. It generates an immune response consisting of specific antibodies or specifically sensitized T lymphocytes. The antibodies or lymphocytes are ''specific\" because they carry the information needed to recognize the corresponding antigen. If the antibody is an IgE antibody, it will respond to the presence of the allergen by generating an inflammatory reaction, called _allergy._ Thus, allergy refers to disease and not simply to the existence of antibodies.\n\nThe distinction between sensitization (sensitivity) and allergy is important. An allergy test is not a test of allergic disease. It shows sensitization, i.e., that a person has the specific antibodies or T cells being tested. Sensitization is necessary but not alone sufficient to have an allergic disease. Many people have specific antibodies to one or more food allergens, yet they have no reaction when eating the food. Treatment, including elimination diets, must not be recommended on the basis of a positive allergy test only.\n\n### B. Types of Allergic Diseases\n\nThe classification of allergic diseases is based on the mechanisms of immunopathology and, therefore, the clinical manifestations (Table 1). A description of these mechanisms and their resulting diseases follows.\n\n_Type I reactions_ are mediated by IgE antibodies that react with the allergen on tissue mast cells or circulating basophils. Once the interaction between the allergen and the IgE antibody occurs, these cells rapidly generate and release certain chemical mediators, such as histamine, which results in pathologic effects locally or systemically. These mediators have effects on blood vessels causing vasodilatation, which causes erythema and, if extensive, could lead to hypotension and shock. Cutaneous effects almost invariably result in pruritus and flushing, usually accompanied by urticaria and\/or angioedema because of increased vascular permeability. Airborne allergens cause conjunctival and eyelid itching, rhinitis, and bronchospasm. Orally ingested allergens cause itching and swelling of the lips, tongue, and palate; laryngeal edema; and gastrointestinal smooth muscle spasm resulting in vomiting, diarrhea, and pain. Obviously, not all of these effects necessarily occur with each allergen exposure. If the exposure is by injection or oral ingestion, multiple organs may be involved, and this is referred to as systemic anaphylaxis.\n\n_Type II reactions_ are mediated by IgG or IgM antibodies, which have the property of activating the complement system when combined with antigen to form immune complexes. If the antigen is on a cell, such as a virus or a transfused red blood cell, complement activation causes lysis (destruction) of the cell. This is useful in responding to a viral infection, but undesirable in the case of a mismatched blood transfusion. Type II allergic reactions are caused by drugs that bind to the patient's blood cells, and they have been postulated to occur in very rare instances of milk-induced thrombocytopenia.\n\n_Type III reactions_ also involve IgG or IgM antibodies and complement, but in this case the result is tissue or organ inflammation. This mechanism may be important in celiac disease and dermatitis herpetiformis. It has also been attributed to a form of food allergy in some cases of arthritis, pulmonary hemosiderosis, and intestinal blood loss, but so far without scientifically supportive evidence.\n\n_Type IV reactions_ are cell-mediated forms of hypersensitivity that do not involve antibodies. This is the mechanism of allergic contact dermatitis, but the same mechanism has been postulated as contributing to celiac disease, dermatitis herpetiformis, enterocolitis, and even some cases of malabsorption.\n\n_Atopy_ is the most common manifestation of type I allergy, affecting about 20% of the population who produce IgE antibodies to certain proteins from organic sources in the everyday environment. The allergens include organic airborne particles: pollens of trees, grasses, and weeds; mold spores; the house dust mite; and emanations from animals, such as cats, dogs, and horses. They also include\u2014but to a lesser extent\u2014some of the natural proteins in foods. The atopic diseases are allergic rhinitis, bronchial asthma, and atopic dermatitis. One or more may coexist in the same patient. A number of genes predispose to atopy, so the condition is often familial. In the 80% of the population without the genetic predisposition to atopy, no amount of natural exposure to allergens in pollens, molds, dust, or foods will induce the formation of specific IgE antibodies and atopic disease. Atopic persons frequently have IgE antibodies to food allergens that sensitize without causing clinical allergic disease.\n\n_Anaphylaxis,_ a less common form of type I allergy, is a systemic disease caused by IgE antibodies that is a medical emergency with a rapid appearance of urticaria, angioedema, bronchospasm, shock, and effects on other organs. It may be fatal. The allergens that are most often responsible for anaphylaxis are proteins and nonprotein organic chemicals, especially drugs, foods, and venom from _Hymenoptera_ stinging insects. Exposure is therefore by injection or ingestion. Rarely, in a highly sensitive patient, inhalation or contact of the allergen on a mucous membrane or on the skin may trigger anaphylaxis. _Urticaria\/angioedema_ is a limited form of anaphylaxis involving the skin only, and it is caused by similar allergens. Anaphylaxis and urticaria\/angioedema may affect either atopic or nonatopic persons.\n\n_Serum sickness_ is a disease characterized by skin rash, joint inflammation, and fever. It is an allergic reaction to drugs but has never been clearly associated with foods. It is caused by IgG or IgM antibodies, which activate the complement system, generating systemic inflammation. It resolves when the patient discontinues using the drug that caused the disease.\n\n_Allergic contact dermatitis_ is an acute inflammatory disease of the skin causing redness, swelling, papules, vesicles, and scaling that appears directly on the area of skin in contact with the allergen. It does not involve antibodies, but is caused by a particular immune cell, the effector T lymphocyte, that specifically recognizes the contact allergen. The most common contact allergen is urushiol, a chemical in poison ivy, poison oak, and poison sumac. Allergic contact dermatitis is also frequently caused by allergens in jewelry, perfumes, cosmetics, rubber products, some topical medications, and other items that may contact the skin. Allergic contact dermatitis can also be caused by food allergy, in which a skin eruption appears where the food has contacted the skin, primarily around the mouth in infants and on the hands as an occupational disease of food handlers. Unlike diseases caused by IgE antibodies, which begin within minutes after exposure to the allergen, allergic contact dermatitis caused by T lymphocytes requires many hours, typically 1 to several days, after skin contact with the food before the reaction begins. It is thus called _delayed hypersensitivity,_ and the delayed latency period makes diagnosing the condition more challenging. IgE-mediated allergic diseases, both atopy and anaphylaxis, are often referred to an _immediate hypersensitivity._\n\n## IV. PREVALENCE OF FOOD ALLERGY\n\nFood allergy is much less common than is perceived by the general public. A number of surveys indicate that about 20% of the population consider themselves to be allergic to foods [1, 2]. However, subjective self-diagnosis cannot be confirmed scientifically in a vast majority of these cases. Research by allergy specialists who base a definitive diagnosis on an expert history and examination, proper testing, and confirmation by double-blind placebo-controlled food challenge\u2014the ''gold standard\" for diagnosis\u2014reveals a more accurate estimate that only 1\u20132% of adults have bona fide food allergy [3]. Furthermore, the prevalence of food allergy differs among different population groups. It depends on age (most common prior to age 2), atopy (more common, especially in patients with atopic dermatitis), diet (availability of particular foods, depending on geography and culture), breast feeding practices and the presence of other diseases. Anaphylaxis, the most serious form of food allergy, occurs in 0.004% of the U.S. population (1 in 250,000).\n\nAllergy to food additives is difficult to estimate because different criteria have been used for diagnosis, but most studies report that it affects between 0.01% and 0.20% in the general population [4], although one report from Denmark documented a 1\u20132% prevalence in schoolchildren [5]. These figures may be much higher than the true prevalence as determined by blinded placebo-controlled food challenge.\n\nThere is also a strong public perception that food additives frequently cause allergy [6]. Estimates of prevalence vary at least 100-fold and are related to the type of data with subjective reports being the least reliable. Double-blind challenges are the only reliable methods for accurate diagnosis. Based on challenge data, prevalence in atopic children is 1\u20132%, in atopic adults <0.15% but possibly as low as 0.03%. This includes all adverse reported events, many of which are not allergic, such as subjective headache and behavioral\/mood changes [6].\n\n## V. FOOD ALLERGENS\n\nAny food is potentially allergenic, but certain foods cause allergic reactions more commonly than others. In almost all cases the allergen is a protein, usually about 10,000\u201360,000 (10\u201360 kDa) in molecular weight. Each foodstuff typically contains a number of different proteins, but not all are allergens. The protein allergens in foods, like those in airborne particles such as pollens, often contain a small amount of carbohydrate and are therefore called glycoproteins, but the allergen is in the protein and not the carbohydrate portion of the molecule. The common allergens are called major and those found infrequently are considered minor.\n\nMany common food allergen molecules have been isolated from food and purified. The purified allergens are named using an abbreviated form of the scientific name of their biologic source (Table 4).\n\nTABLE 4\n\nNomenclature of Selected Food Allergens\n\n_Cow's milk_ contains numerous proteins, many of which have been identified as allergens [7]. They are present in both the casein and whey fractions. Pasteurization\u2014flash heating to destroy pathogenic microbes\u2014does not significantly denature the proteins, so the allergens survive this process. Most patients with cow's milk allergy tolerate beef without reactions, even though they may have a positive skin test to beef. On the other hand, a high degree of cross-reactivity is seen with goat and sheep milk, so these may not be suitable substitutes in cases of cow's milk allergy.\n\n_Chicken egg_ allergy is almost always an allergy to the egg white and not the yolk. Egg white contains 23 different glycoproteins, and 4 of these are major allergens, of which ovomucoid is the most frequent [8]. Although most patients who are allergic to chicken egg can tolerate the chicken meat (muscle, skin), a few with exquisite anaphylactic sensitivity may react to the meat also. Cross-reactions with egg allergens from other birds, such as ducks, turkeys, and geese, are common [9].\n\n_Legumes_ are the most common foods causing food allergy in the United States. _Peanut_ allergy is a particularly prominent cause of food-induced anaphylaxis and therefore the subject of considerable research interest. Sixteen allergenic proteins have been identified to date, and 3 of them are major peanut allergens. The allergenicity is not found in peanut oil, but it remains in processing of virtually all peanut products, such as flour. _Soybean_ allergy occurs especially in children and infants, and this is a problem that must be considered in those infants fed on a soy-based formula because of their allergy or intolerance to cow's milk. Soy is extensively used in many food preparations because of its high protein content and low cost. Several allergenic soy proteins have been identified. These are not found in pure soybean oil, but some allergens are present in commercial sources of the oil, in lecithin preparations, and in margarines. There is extensive potential allergic cross-reaction among legumes when tested in the laboratory, but other legumes such as beans, peas, and licorice are far less likely to cause clinical allergic reactions. Most peanut-sensitive patients can tolerate other legumes [10].\n\n_Tree nuts_ cause allergy especially in adults. These include brazil nuts, cashews, filberts (hazelnut), hickory nuts, pecans, pine nuts, pistachios, and walnuts. Cross-reactions among these are common but not always present. There is no known allergenic cross-reactivity between any of these tree nuts and any legumes, including peanut.\n\n_Fish_ is very likely the most common type of food causing allergy in adults [11]. There are hundreds of edible species of fish, and cross-reactivity is extensive.\n\n_Crustaceans and mollusks_ are also important, although the majority of allergy-like reactions, especially urticaria and angioedema, are not immunologic. Allergenic cross-reactivity occurs among different crustaceans and among different mollusks, but not between them.\n\n_Cereal grains_ are a more important cause of allergy in children than in adults. They include wheat, barley, rye, oats, rice, corn, sorghum, and millet, accounting for 70% of the world protein consumption. The allergens vary by disease, e.g., globulin and glutenin in IgE oral allergy, and albumin in baker's asthma. Gliadin is the protein that causes celiac disease. Allergenic cross-reactivity among grains is common. Occupational food allergy is usually manifested as asthma, rhinitis, and\/or dermatitis and is cased by inhalation of airborne flour dust. In baker's asthma, skin tests to cereal grains are positive in 54% of cases, whereas clinical disease occurs in only 20% [12]. Most patients wih baker's asthma have no reaction to the ingestion of the same food causing the asthma.\n\n## VI. CLINICAL MANIFESTATIONS OF FOOD ALLERGY\n\nAllergic diseases from foods are best classified and understood on the basis of their immunologic mechanism (Table 1).\n\n### A. Type I\u2014IgE-Mediated Allergic Diseases\n\nIgE antibodies cause the large majority of well-documented food allergies. They are recognized by (1) the rapid onset of the clinical symptoms and signs following ingestion of the food, (2) objective evidence of allergic inflammation, and (3) evidence that the patient has the specific IgE antibody to the food that initiated the reaction. In some cases, the fact that the disease is caused by IgE antibody is supported by pathologic evidence of eosinophils in the affected tissue or organ and positive therapeutic response to appropriate medications. IgE-mediated reactions to foods, including anaphylaxis and urticaria, can be mimicked by certain chemicals present in some foods that are capable of causing nonallergic reactions.\n\nAnaphylaxis is the most serious form of allergy because of the potential for a rapid fatal outcome. Food allergy is a frequent cause of anaphylaxis [13]. About 100 fatalities occur per year in the United States from food-induced anaphylaxis [14], and an estimated 1000 cases are severe but not fatal [10]. As a general rule, the more quickly the reaction begins after the food is eaten, the more severe the reaction will become. The amount of allergen causing anaphylaxis can be exceedingly small. The likelihood of a fatal outcome is increased in patients with asthma, even if the asthma is not active at the time of the reaction. Although atopic diseases (allergic rhinitis, asthma, atopic dermatitis) and anaphylaxis are both cased by IgE antibodies, atopy is not a prerequisite for anaphylaxis. Nevertheless, most patients with anaphylaxis to a food are in fact atopic. Anaphylaxis to a food affects females twice as often as males. In some infants, the reaction occurs on the first feeding of the food, in which case it is likely that sensitization occured from exposure to the allergen through breast feeding. In most cases, however, the patient reports a history of prior ingestion of the food without a reaction.\n\nIn nonfatal reactions, anaphylaxis can be mild to severe. The reaction may consist of pruritus, urticaria, angioedema, contact urticaria, erythema, laryngeal edema, rhinitis, conjunctivitis, bronchospasm, hypotension or shock, nausea, vomiting, abdominal cramps, diarrhea, and uterine or bladder cramps. Not all of these findings occur in every case, but at least two organ systems are involved. Immediate treatment is essential in order for the reaction to subside without permanent organ damage.\n\nThe allergist evaluating a patient for food anaphylaxis generally does so after the incident has passed and therefore relies on the patient's history. Review of an emergency room record, if available, will provide the necessary information to confirm the presence and severity of the reaction. The most common foods reported to cause anaphylaxis are wheat, shellfish, fruit, milk, celery, and fish. Although peanut and egg white are reported less frequently, they are especially important because minute amounts may be present in some food preparations in a form in which they are likely to be unknowingly ingested by a patient with a known allergy to that food. The allergen is particularly hidden when it is contained in a pastry, salad, sandwich, hors d'ouvres, or candy. Testing to confirm the specific food sensitivity is done by either the skin test method or by the radioallergosorbent test, which detects the specific IgE antibody in the patient's serum. Either test is usually highly reliable to confirm the specific sensitivity, and double-blind, placebo-controlled challenge is almost never used because of the potentially severe nature of this disease.\n\nBy definition, anaphylaxis is a systemic disease. However, the term _gastrointestinal anaphylaxis_ may be used for a reaction that begins within minutes and less than 2 hours after ingestion of a food and results in symptoms of nausea, abdominal pain, cramps, vomiting, and sometimes diarrhea, but without any other symptoms or signs of systemic anaphylaxis. In some children with a mild form of this condition who eat the food frequently, it may cause poor appetite and abdominal pain.\n\n_Food-dependent, exercise-induced anaphylaxis_ is an IgE-mediated food reaction that occurs in a patient who exercises 2\u20133 hours or less either after or before eating a meal containing the food allergen [15\u201317]. In some cases, urticaria or anaphylaxis without other systemic effects may occur. Foods that are especially likely to be implicated in this rare disease are celery, shrimp, oyster, chicken, peach, and wheat. The patient can eat the food without a reaction if there is no physical exercise within 2 hours of ingestion. Exercise in the absence of eating the food during this time interval also does not cause anaphylaxis.\n\nAn _anaphylactoid reaction_ is identical to anaphylaxis, but the patient is not allergic to the agent that causes the reaction. Certain chemicals are capable of directly activating mast cells or basophils without the need for IgE antibodies. This causes the cells to release the same mediators that cause allergic anaphylaxis. Chemicals capable of doing this are present in some foods, especially shellfish or berries, although the precise chemicals that are responsible are unknown. Unlike allergic anaphylaxis, it does not occur each time the food is eaten, and the risk is higher if a large quanity is eaten or if the food is not fresh and possibly contaminated with bacteria or other microorganisms. The distinction between anaphylactic and anaphylactoid reactions should be made in each case by an allergic evaluation and proper testing, because the food causing anaphylaxis must be avoided, whereas avoidance might not be necessary if there is no allergy to the food.\n\n_Cutaneous reactions_ are common. Immediate reactions caused by IgE antibodies produce acute urticaria, angioedema, or both. These occur especially after ingestion of fish, shellfish, tree nuts, and peanuts in adults, and eggs, milk, peanuts, and tree nuts in children. The reaction subsides promptly and does not recur unless the food is eaten again. Contact urticaria results from direct skin contact with raw meats, fish, fruits, and occasionally other foods. The urticarial lesions appear only on the areas of contact with the food. Chronic urticaria\/angioedema is a condition of frequently recurring hives or swelling that persists for weeks or months and is rarely if ever caused by foods. In fact, it is almost always idiopathic, because a search for any causative allergen or condition is usually negative.\n\nUrticaria or angioedema may result from eating a food in the absence of an IgE allergy to the food. This is analogous to an anaphylactoid reaction as discussed above, and the same foods (shellfish and berries) are the usual causes.\n\n_Atopic dermatitis_ is one of the three manifestation of atopy. Patients with this disease may also have asthma and\/or allergic rhinitis in addition to the skin disease. Allergy testing will frequently, but not always, show evidence of IgE sensitivities to inhalant allergens, especially the house dust mite, and foods. Nevertheless, the role of food allergy in causing or exacerbating the skin eruption is controversial and frequently disputed.\n\nIt is unusual for a patient with atopic dermatitis to report a history of a flare of the dermatitis following ingestion of certain foods and improvement on elimination diets. However, when 400 children with atopic dermatitis were challenged with a double-blind, placebo-controlled food challenge in a research setting, one-third demonstrated an immediate allergic reaction [3]. These children had severe atopic dermatitis, high total serum IgE levels, and most of them had respiratory allergy as well. Reactions occurred in minutes and almost always within 2 hours. Seventy-five percent of the food reactions involved the skin, and 30% involved the skin only. The reactions were pruritus, erythema, and morbilliform eruptions, especially at sites of existing eczema. In individual cases, the allergy was limited to only a few foods. Elimination of the food from the diet then resulted in improvement or clearing of the rash in some of the cases, and later challenge with the same food caused urticaria in those whose eczema had cleared. These children usually have positive skin tests or radioallergosorbent tests to some foods. Only 50% of the foods with a positive skin test produced a positive challenge, and foods that gave a negative skin test almost always resulted in a negative challenge. Gastrointestinal symptoms occurred in 51% of the challenges, although there was no history of these symptoms occurring prior to the challenge testing. Upper respiratory symptoms occurred in 45%, with wheezing in 15%. A positive challenge was associated with a significant rise in plasma histamine, consistent with an immediate-phase IgE allergic response. Many of the reactions included a pruritic rash that appeared 4\u20138 hours after the food challenge, accompanied by blood and tissue biopsy evidence consistent with a late-phase IgE allergic response. Long-term elimination diets achieved significant clinical improvement, including elimination of subclinical gastrointestinal malabsorption. Ninety percent of these food reactions to double-blind, placebo-controlled food challenges were to egg, milk, peanut, soy, and wheat. After 1\u20132 years of elimination of the allergenic food from the diet, food hypersensitivity was permanently lost in a third of the patients, especially to soy, but only rarely to peanut. The positive skin test reactions persisted. Based on these and other findings, it is likely that only about one-third of children\u2014and very few adults\u2014with atopic dermatitis have demonstrable food allergy.\n\nThe _oral allergy syndrome_ is a localized ''contact\" allergic reaction of the oropharynx. The symptoms are pruritus and angioedema of the lips, tongue, palate, and throat, beginning about 5 minutes after the food is eaten and resolving promptly thereafter. It results especially from eating uncooked fruits and vegetables, and it occurs in patients with allergy to those food allergens that cross-react with certain plant pollens (Table 3). These patients usually also have symptomatic allergic rhinoconjunctivitis to the cross-reacting pollen [18].\n\n_Allergic eosinophilic gastroenteritis_ is an uncommon condition in some atopic patients who are allergic to both inhalant and food allergens. In adults with this disease, the ingestion of the food allergen causes postprandial nausea and vomiting, abdominal pain, diarrhea, and occasionally steatorrhea and weight loss [19]. Complications include hypoalbuminemia and iron deficiency anemia. In infants it causes failure to thrive, and in some cases a protein-losing enteropathy may result [20]. The food allergen can be identified by skin test or radioallergosorbent test, and when it is eliminated from the diet the disease resolves, although this may take up to 12 weeks of avoidance of the food. Laboratory studies confirm the allergic nature of the disease. Peripheral blood eosinophilia may be as high as 50%, and eosinophils are present in intestinal secretions and in ascites fluid. There is eosinophilic inflammation of the esophagus, stomach, or intestine, involving the mucosa, muscular, or serosal layers, and Charcot-Leyden crystals can be found in stool samples. Compared to normals, there is an increase in the total amount of IgE normally found in duodenal secretions and serum.\n\nSome children and adolescents with this disease do not have specific IgE antibody to the food that causes the disease to flare. A very low protein diet for these patients may cause resolution in 6\u20138 weeks. The condition also responds favorably to systemic corticosteroid therapy if the specific causative food is not identified and eliminated.\n\n_Respiratory food hypersensitivity_ is rare. Patients with asthma sometimes suspect that their attacks are caused by certain foods, but double-blind, placebo-controlled food challenge\u2014the only reliable way to prove food-induced asthma\u2014rarely provokes an asthma attack. In cases where this does happen, it is almost always in an atopic child, and the reaction is accompanied by a skin rash or a gastrointestinal response as well. Interestingly, however, ingestion of food allergens may increase a state of nonspecific bronchial hyperreactivity, as shown by methacholine challenge testing. The clinical significance of this is unknown.\n\nSome patients with exquisite sensitivity to a food may experience an allergic reaction by simply inhaling fumes of the food. This may cause symptoms of allergic rhinitis, conjunctivitis, laryngeal edema, bronchospasm, vomiting, and even on rare occasions anaphylactic shock [21]. The foods most commonly responsible are fish, mollusks, crustaceans, eggs, and peanut.\n\n### B. Type II\u2014IgG- or IgM-Mediated Cytotoxic Allergic Diseases\n\nAllergic reactions to drugs that adhere to blood cells in which the cell is destroyed because of antibody to the drug have been recognized for many years. There have been rare reports of thrombocytopenia apparently caused by milk [22], possibly by this mechanism.\n\n### C. Type III\u2014IgG- or IgM-Mediated Inflammatory Allergic Diseases\n\nRare instances of inflammatory gastrointestinal and respiratory conditions that are caused by a particular food and ameliorated when the food is eliminated from the diet have been suspected to be allergic diseases mediated by IgG antibodies. As discussed earlier, small amounts of IgG antibodies are normal and harmless, but in these cases, an IgG antibody to the food is present in unusually large amounts, and the inflammation is thought to be a consequence of immune complexes that activate complement. The diagnosis of a specific food causing the disease should be made by clinical evidence of improvement when the food is eliminated and exacerbation when the food is reintroduced before an elimination diet is prescribed for treatment.\n\n_Heiner's syndrome_ is a rare disease of infants who have produced high concentrations of IgG antibodies to milk because of frequent aspiration [23]. As a consequence, ingestion of milk triggers a type III immune complex allergic reaction in the lung, causing pulmonary infiltrates. Recurrent attacks may result in anemia and in some cases pulmonary hemosiderosis. The diagnosis can be confirmed by measuring precipitating antibodies to milk in the serum.\n\n### D. Type IV\u2014T-Cell-Mediated Allergic Diseases\n\nThere are two types of contact dermatitis: allergic and irritant. The latter is an irritation where the food contacts the skin, because of a chemical or physical property of the food. It does not involve the immune system [24]. The allergen in _allergic contact dermatitis_ to a food is usually an organic chemical contained naturally within the food or an additive, rather than a food protein. It is first suspected by the history and skin examination and then confirmed by a 48-hour skin patch test.\n\nThe condition is an inflammatory dermatitis, which is pruritic and may be similar in appearance to atopic eczema, but with a tendency to include vesicles and blisters. Numerous foods have been shown to cause the disease. Garlic and onion are the most common, especially among people who handle food often [25]. Citrus fruits, many green vegetables, and some spices should also be suspected.\n\nSome potent contact allergens, especially metals such as nickel, cobalt, and chrome, may occur naturally in certain foods. Patients sensitized by contact from jewelry or from an occupational exposure can experience a flare of the dermatitis after ingesting food that contains the metal allergen [26]. A special diet free of that particular metallic ion may then be necessary. There is also a problem of cross-reactivity between an allergen in a plant and an unrelated food. Urushiol, the allergen in the poison ivy leaf, is antigenically similar to a chemical in the oil of cashew nut and in mango. A person with severe poison ivy sensitivity may experience a rash from contact with those foods or exacerbation of the poison ivy rash from eating the food.\n\n## VII. NONALLERGIC IMMUNOLOGIC DISEASES\n\nThere are a group of conditions that are caused by nonallergic intolerance to certain foods in which the immune system nevertheless plays a role. A common one is gluten-sensitive enteropathy, also known as _celiac disease,_ in which the intestinal mucosa becomes inflamed and atrophic from ingestion of gliadin, a component of gluten in wheat and other grains [27]. Ingestion of gliadin causes diarrhea, steatorrhea, weight loss, malabsorption, and other clinical manifestations. Immunologic involvement includes IgA antibodies to gliadin [28], lymphocyte inflammation of the intestinal mucosa, and some evidence of cell-mediated (T-cell) immunity to gliadin. It is unclear whether these specific immune responses are the cause or the effect of the disease, and there is some feeling that the cause is a toxic effect of gluten [29]. Nevertheless, a lifelong gluten-free diet keeps the condition under control.\n\n_Dermatitis herpetiformis_ is a skin disease that is associated with gluten-sensitive enteropathy [30, 31]. The skin eruption consists of a pruritic symmetric rash that is papulo-vesicular. Like celiac disease, there is a genetic predisposition in persons with certain HLA haplotypes. The gastrointestinal pathology is similar to celiac disease, and IgA is deposited in the skin. It responds to a gluten-free diet.\n\n_Enterocolitis syndrome_ is a disease of infants 1 week to 3 months of age, who have protracted vomiting and diarrhea resulting in dehydration, acidosis, and methemoglobinemia [32]. Cow's milk and soy are the predominant foods eaten at that age, but occasionally other foods may cause a similar disease in older children, and rarely seafoods may do so in adults. The stools contain blood, eosinophils, and neutrophils. Jejunal biopsy reveals flattened villi, edema, lymphocytes, eosinophils, and mast cells. Deliberate diagnostic challenge with the suspected food causes diarrhea and vomiting in 1\u20133 hours. Skin testing is negative to the food that is responsible for the disease. It is not an IgE-mediated allergic disease, and the immunopathogenesis is uncertain at this time. Symptoms resolve in 72 hours when the food is eliminated from the diet.\n\n_Eosinophilic colitis_ occurs during the first few months of life in infants who are fed cow's milk and\/or soy, but it may also occur in breast-fed infants [33]. There is no apparent gastrointestinal discomfort in the infant, but there is gross or occult blood in the stools. Microscopic pathology includes eosinophilia in the epithelium and lamina propria and edema and neutrophils in the distal colon. Elimination of the responsible food results in clearing in 72 hours.\n\n_Enteropathy_ is also a disease of early infancy. There is diarrhea with or without vomiting, failure to thrive, and mal-absorption. Cow's milk is the usual cause, but soy, egg, wheat, rice, chicken, or fish may also be responsible [34]. Biopsy of the intestinal tract shows patchy villous atrophy, milk lymphocytic infiltration, and edema.\n\n## VIII. NONIMMUNOLOGIC REACTIONS TO FOODS\n\n_Gustatory rhinorrhea_ refers to a condition of excessive and annoying nasal secretions that some persons experience after eating. It may occur after eating certain foods, especially spicy ones, or after any meal. The mechanism of rhinorrhea is probably a simple stimulation of muscarinic parasympathetic nerves.\n\n_Lactose intolerance_ results from deficient intestinal lactase so that dietary lactose ferments in the bowel, with excessive production of gas. Primary lactase deficiency varies in prevalence among different ethnic and geographic populations. Secondary lactase deficiency is transient and may last for 2 weeks or more after an acute gastrointestinal infection. More persistent secondary deficiency complicates chronic gastrointestinal diseases. (See Chapter 37 on lactose intolerance).\n\n_Scombroid fish poisoning_ results from contamination by _Klebsiella_ of spoiled tuna, mackerel, bonito, mahi, and blue-fish. The bacterial decarboxylate histadine [35] generates histamine and simulates an allergic reaction. Symptoms are a sharp peppery taste, burning of the mouth, vomiting, diarrhea, nausea, facial flushing, and headache.\n\n## IX. CONDITIONS WITH NO PROVEN RELATIONSHIP TO FOODS\n\nSo many medical and nonmedical conditions have been incorrectly attributed to ''food allergy\" that only a few will be reviewed here.\n\n_Infantile colic_ affects 15\u201320% of infants [36], causing uncontrollable crying and abdominal distension. It starts before age 6 weeks and lasts for 3\u20136 months. Despite numerous theories, the cause remains unknown. Although food allergy is claimed to contribute in up to 10\u201315% of cases [37], there is no scientific proof that colic is an allergic condition, and it occurs regardless of the diet, even with exclusive breastfeeding.\n\n_Behavioral changes_ in children and even in adults have often been attributed to the ingestion of certain foods and food additives. Sugar, dietary salicylates, aspartame and certain other chemicals, food preservatives, and food coloring agents have achieved popular notoriety as the cause of attention deficit hyperactivity disorder, but properly controlled food challenge and elimination protocols fail to substantiate these claims [38].\n\nThe _Chinese restaurant syndrome_ encompasses a variety of symptoms such as headache, sweating, thirst, facial flushing, facial burning, abdominal pain, tearing of eyes, and a crawling sensation of the skin. It begins 15\u201320 minutes after eating and subsides several hours later without treatment. Although widely attributed to monosodium glutamate, controlled deliberate challenge studies frequently fail to reproduce the symptoms [39]. There is no evidence that it is caused by allergy to monosodium glutamate or to Chinese food.\n\nFood allergy has also been claimed to cause sudden infant death syndrome, allergic tension-fatigue syndrome, enuresis, and headache in children, and rheumatoid arthritis, chronic fatigue syndrome, migraine, epilepsy, and inflammatory bowel disease (Crohn's disease or chronic ulcerative colitis) in adults and children. There is no scientific proof for any of these conditions being caused by food allergy.\n\n## X. DIAGNOSIS OF FOOD ALLERGY\n\nThe diagnostic process begins with a thorough history and complete physical examination to establish the nature, extent, and duration of symptoms and their association with the ingestion of a suspected food. Important information includes the timing of the reaction after the food is eaten, the quantity of food required, and consistency of the reaction to the food over time. The history is generally more reliable for acute reactions than for chronic diseases. A hidden ingredient must also be considered. A personal and family history of atopy or other diseases may be important. A complete physical examination is critical to detect objective signs of allergic inflammation and to rule out other possible diseases. In some cases the diagnosis may be obvious, as in a child with recurrent anaphylactic reactions to peanut. In most cases, however, a differential diagnosis and appropriate testing are necessary.\n\n_Diet-symptom diaries_ have the advantage that they provide a prospective ''history\" of reactions and a more accurate documentation than is possible by recall. They are especially useful for disproving a false assumption that a particular food caused reactions. Diaries are especially helpful for patients with intermittent urticaria\/angioedema.\n\n_Diagnostic elimination diets,_ accompanied by a written diary, have long been used by allergists. They are generally based on the results of skin testing and\/or from the history. To be accurate, the diary must include the timing of every item of food eaten, all symptoms or reactions, and any other items or events that may explain the patient's symptoms, such as medications. The purpose of the diet is to eliminate symptoms and\/or objective physical or laboratory findings. When this occurs, an open challenge is performed with one of the suspect foods. The other foods are then challenged one at a time while the diary record is maintained. If a particular food causes a reaction consistent with the previous symptoms, it is again eliminated and rechallenged. In general, three successive positive provocative unblinded tests to a food can be considered probably significant, although the mechanism must then be determined. A reaction to the challenge suggests the cause but not the mechanism. For example, provoking a food reaction in an open challenge could result from allergy, toxicity, an enzyme deficiency, or even a psychological response. If ongoing chronic symptoms fail to improve in 2 weeks, the diet should be abandoned.\n\nTests for a specific immune response must be appropriate to the suspected allergic reaction (Table 5). In most cases, the suspected disease is caused by IgE antibodies, and both the immediate wheal\/erythema skin test and the _in vitro_ serum assay for specific IgE antibodies to foods are reliable. The skin test is more sensitive, but reading and interpreting the result is subjective and requires training and experience. Immediate skin testing to food allergens is done by the prick testing method [40]. A false negative result can occur from an inactive extract, because some food allergens are labile. Skin testing is most useful for suspected food allergy causing anaphylaxis.\n\nTABLE 5\n\nTests of Specific Immune Responses for Each Type of Allergic Reaction, Based on the Gell and Coombs Classification\n\nThe standard _in vitro_ test is the radioallergosorbent test, which measures the quantity of specific IgE antibodies in a sample of the patient's serum. Most commercial medical laboratories offer a limited panel of foods. A negative test to a food by either skin testing or radioallergosorbent test virtually ensures that the patient does not have an IgE-mediated allergic reaction to that food. On the other hand, a positive test in a child indicates only a 50% chance of clinical allergy, while in adults it is only about 3%.\n\n_Patch testing_ to foods is used for suspected allergic contact dermatitis. It is accurate because it simulates the disease ''in miniature.\" The food extract is applied to a small area of skin for 48 hours. A positive test consists of a localized skin inflammation at the patch test site [40].\n\n_Endoscopy_ of the stomach or small bowel during oral challenge with a food allergen to observe for objective changes, with or without biopsy, may be required in some patients with suspected food-induced gastroenteropathy.\n\nThe _double-blind, placebo-controlled food challenge_ is the gold standard for proof of a specific food-induced disease [41], but a positive result does not mean that the disease is necessarily allergic. It is usually appropriate to perform oral food challenge unblinded or single-blind first. If these are negative, there is no need to proceed further. The double-blind, placebo-controlled food challenge is an essential method for many clinical research studies in food allergy, and it has been especially valuable for analyzing food-related complaints without objective evidence of a physical reaction.\n\nThe procedure for double-blind, placebo-controlled food challenge is demanding [42, 43]. The suspect food is eliminated from the diet for 7\u201313 days, or longer if necessary, until the patient is asymptomatic. The challenges are administered while the patient is fasting and free of antihistamines or any other drug that might prevent or mask the reaction. The first challenge is performed with a dose selected as unlikely to cause a reaction. Subsequent challenges are doubled in dose and administered every 15\u201360 minutes until a reaction occurs or until 10 g of the test food is tolerated, at which time the test is considered negative. Food extracts and an equal number of placebo controls are given in random order. The food challenges are administered as lyophilized extracts packed into capsules or dissolved in liquid. Because rare false-negative reactions (2\u20135%) have occurred, a negative result should be repeated with an open challenge. Following the session, the patient should be observed for up to 2 hours if an IgE reaction is suspected, up to 4\u20138 hours for milk enterocolitis, and up to 24\u201348 hours for other gastrointestinal reactions. Wherever appropriate and possible, an objective measure consistent with the disease should be included, such as pulmonary function testing, nasal smear for eosinophils, or histamine or tryptase content of plasma or urine.\n\n## XI. DIFFERENTIAL DIAGNOSIS\n\nThe diagnosis of food allergy rests on the identification of the disease as an allergy, objective evidence of sensitization to a food, and reasonable assurance that the food is responsible for the disease. This requires a suspicion that the patient has food allergy and elimination of other possible diseases and other potential adverse effects from foods. Gastrointestinal anatomic abnormalities, infection, peptic ulcer disease, and gall bladder disease are some conditions that can be misdiagnosed as gastrointestinal food allergy. Nonallergic reactions from foods include poisoning, intolerance because of intestinal enzyme deficiency, and a response to foods from psychosocial factors.\n\n## XII. TREATMENT\n\nThe only sure treatment of food allergy is elimination of the food from the diet. In most cases, allergy is limited to one food, one class of food, or a small number of different foods. Therefore, avoidance therapy is not likely to cause nutritional deficiency. Some infants and a few adults, however, have multiple specific food allergies, requiring diet supplements to maintain good nutritional status.\n\nIn some cases, incomplete elimination accompanied by appropriate drug therapy is possible. Drugs that may offer such protection in IgE-mediated disease include antihistamines, mast cell stabilizers (cromolyn, ketotifen), and corticosteroids. The efficacy of leukotriene inhibitors alone or in combination with other drugs has yet to be studied.\n\nThe acute anaphylactic reaction is treated with epinephrine, intravenous fluid replacement for shock, bronchodilator drugs, antihistamines, and maintenance of the circulation and airways.\n\nImmunotherapy (desensitization) for IgE-mediated food allergy could theoretically eliminate or reduce allergic sensitivity to foods. It has long been established as effective and safe for patients with allergic rhinitis caused by inhalant allergens and for those with anaphylaxis to venom from the stings of _Hymenoptera_ insects. Currently, uncertainty remains about its effectiveness in allergic asthma from inhalant allergens. Clinical trials using extracts of foods in patients with food-induced anaphylaxis to date have been frustrated by excessive numbers of serious and even fatal systemic reactions. Clinical studies of food immunotherapy are continuing, however, and may eventually prove to be useful. If so, it is likely to require some type of modification of the allergen to avoid reactions from the treatment itself.\n\nA small number of atopic infants and small children have severe allergic reactions to multiple food proteins with gastrointestinal and skin involvement. They are unable to tolerate enough variety of foods to permit adequate intake of nutrients. The disease usually begins when foods are first introduced into the diet. These are believed to be true IgE-mediated allergic sensitivities. Various ''elemental\" amino acid-based formula diets have been devised to satisfy all of the infant's nutritional needs for protein to sustain growth.\n\n## XIII. PROGNOSIS OF FOOD ALLERGY\n\nThere is very little reliable information about the prognosis of food allergy. In infants and children, allergy to foods frequently remits after several years [44], with the notable exception of anaphylaxis, especially to nuts and peanut. Children with documented food allergy should be monitored by serial skin testing on an annual basis. They should scrupulously avoid the allergenic food as long as the skin test remains positive. Only when the test reverts to negative should careful administration of a small test dose of the food be given. This is best done in a medical facility where appropriate treatment is available for immediate use.\n\nMost adult-onset food allergy reactions are also transient, although they may persist for years before remitting. Celiac disease, which is an immunologically mediated disease but not an allergic one, is a permanent condition.\n\nMultiple food allergies encompassing almost all ingested proteins in infants persist for 1 to several years with gradual loss of food sensitivities over a period of a number of years, permitting slow and gradual introduction of natural foods into the diet.\n\n## XIV. PREVENTION OF FOOD ALLERGY\n\nAllergists have long been interested in preventing the development of allergic diseases and specific allergies, including foods. Expectant parents who are allergic frequently request strategies to prevent, diminish, or at least delay the onset of allergy in their offspring. A number of dietary measures have been tried empirically, and limited clinical research has been done on this important issue. At this time, there are no firm recommendations for primary prevention (i.e., before the onset of the disease).\n\nIn 12 studies published to date [45], various ''highly allergenic\" foods such as egg, cow milk, peanut, fish, soy, and others were eliminated for varying periods of time from the diet of the mother (during pregnancy, after delivery, or both) and\/or from the infant. Follow-up evaluations were typically done after 18 months, but they ranged from 12 months to 15 years. Ten studies reported some reduction in severity of the infant's atopic disease, especially dermatitis, but no effect was observed in the other two studies. There were significant differences in the protocols, and even the best results suggest that a highly restrictive diet that is difficult to follow for either the pregnant woman or the infant cannot be recommended as a reliable prevention strategy for infants of allergic parents-to-be. Nevertheless, most allergists do recommend breast feeding to delay the introduction of cow's milk for the infant, delayed introduction of solid foods until about 6 months, and elimination of milk, egg, nuts, and fish until 12 months [45]. These particular foods are selected empirically, because there is obviously no way to know what foods should be avoided in a diet for primary prevention before allergies are present.\n\n## XV. FOOD ADDITIVES\n\nApproximately 3000 different substances are intentionally added to the food supply to provide color, flavor, appearance, nutritional value, disease prevention, (e.g., antimicrobials, vitamins, minerals), texture, preservative properties, or enhanced cooking properties. They may or may not be listed as ingredients in the final product. Additives may be foodstuffs (e.g., sugar, cornstarch), minerals, synthetic organic compounds, or natural organic compounds [4, 46\u201347].\n\nUnintentional additives may also appear in the final food product during processing of the food by inadvertent mixture or by contamination with microorganisms.\n\nIt is possible that any one of these food additives can cause an adverse reaction, including allergy, and many people believe they do so frequently. The additives most often claimed to cause allergic or allergic-like reactions are synthetic colorants, sulfites, monosodium glutamate, aspartame, and benzoates. Controlled challenge studies to date indicate that adverse reactions to food additives are actually very rare.\n\n_Sulfites_ in foods cause an allergy-like reaction in some asthmatic patients. The bronchial mucosa in asthma is unusually sensitive to certain irritants and chemicals, including sulfur-containing compounds such as sulfites [48]. Inhalation of sulfur fumes or sulfur dioxide gas results in an acute asthmatic attack and may be fatal. Because sulfites are widely added to certain foods to prevent oxidation, eating such foods may cause acute bronchospasm [49]. This occurs only in persons with asthma. The reaction probably results from inhaling sulfur dioxide fumes generated from the sulfites while the food is being chewed. The reaction is not an allergic one, and no immunologic mechanism is involved. Some asthmatic patients are especially sensitive, because they are deficient in the enzyme sulfite oxidase. Those who have experienced exacerbation of their disease from eating sulfite-containing foods should always avoid such foods, and some allergists recommend avoidance for all persons with asthma. The foods most likely to contain sulfites are listed in Table 6.\n\nTABLE 6\n\nSome Foods Containing Sulfites Likely to Exacerbate Asthma\n\n_Source:_ Adapted from Simon, R. A. (1996). Adverse reactions to food and drug additives. _Immunol. Allergy Clin. N. Am._ **16,** 137.\n\nSome allergists believe that chronic urticaria\/angioedema in certain patients is caused by food additives, especially tartrazine (yellow dye #5). However, the reaction cannot be confirmed in patients who report hives from tartrazine when they are challenged in a double-blind protocol [50].\n\nVegetable gums are natural products that are added to many foods as thickeners and to increase the bulk of the product. They have long been suspected to cause IgE-mediated allergic reactions and are frequently included in the standard skin testing panel used by allergists. Although some atopic patients do react to these skin tests and there is an occasional case report [51], no data are available on the actual prevalence, if any, of clinical reactions to vegetable gums. Their presence in a particular food is not always indicated on the product label.\n\nAntioxidants, especially butylated hydroxyanisol, butylated hydroxytoluene, nitrates, nitrites, citric acid, and benzoates, have all been suspected to cause allergy, especially chronic urticaria. In fact, there is no documentation that any of these cause allergic disease. They may have other adverse health effects in some patients, but these are pharmacologic or toxic, not allergic.\n\n## XVI. GENETICALLY ENGINEERED FOODS\n\nThe science and methods of biotechnology are being increasingly applied to agriculture for selectively producing crops with desirable characteristics, such as enhanced nutritional properties or resistance to herbicides. This is done by inserting DNA with the genetic code for a desired trait into the chromosome of the plant cells in the laboratory, then culturing the cells to produce plantlets that are eventually transferred to soil and propagated. The resulting bioengineered foods in theory could have been produced by the traditional breeding methods that have been used for more than a century, but bioengineering is much more specific and predictable.\n\nAlthough there is concern about the safety of genetically engineered foods, includng the possible emergence of new food allergens, this has yet to occur. In fact, it is theoretically possible to produce crops, such as peanuts, that lack the known major allergens, and attempts along these lines are currently in progress.\n\nReferences\n\n1. Young E., Stoneham M.D., Petruckevitch A., Barton J., Rona R. A population study of food intolerance. _Lancet_. 1994;93:446\u2013456.\n\n2. Altman D.R., Cerement L.T. Public perceptions of food allergy. _J. Allergy Clin. Immunol_. 1996;97:1247\u20131251.\n\n3. Sampson H., Metcalfe D.D. Immediate reactions to foods. In: Metcalfe D.D., Sampson H., Simon R.A., eds. _Food Allergy: Adverse Reactions to Foods and Food Additives_. New York: Blackwell Scientific Publications; 1991:100\u2013112.\n\n4. Chap. 83Bush R.K., Taylor S.L. Adverse reactions to food and drug additives. In: Middleton E., Jr., Reed C.E., Ellis E.F., Atkinson N.F., Jr., Yunginger J.W., Busse W.W., eds. _Allergy: Principles and Practice_. 5th ed. Boston: Mosby; 1989:1183\u20131198.\n\n5. Fuglsang G., Madsen C., Saval P. Prevalence of intolerance to food additives among Danish schoolchildren. _Pediatr. Allergy Immunol_. 1993;4:123\u2013129.\n\n6. Madsen C. Prevalence of food additive intolerance. _Hum. Exp. Toxicol_. 1994;13:393\u2013399.\n\n7. Bleumink E., Young E. Identification of the atopic allergens in cow's milk. _Int. Arch. Allergy Appl. Immunol_. 1968;34:521\u2013543.\n\n8. Hoffman D.R. Immunochemical identification of the allergens in egg white. _J. Allergy Clin. Immunol_. 1983;71:481\u2013486.\n\n9. Langland T. A clinical and immunological study of hen's egg white. VI. Occurrence of proteins cross-reacting with allergens in hen's egg white as studied in egg white from turkey, duck, goose, seagull, and in hen's egg yolk, and hen and chicken sera and flesh. _Allergy_. 1983;38:399\u2013412.\n\n10. Bock S.A., Atkins F.M. The natural history of peanut allergy. _J. Allergy Clin. Immunol_. 1989;83:900\u2013904.\n\n11. Aas K. Studies of hypersensitivity to fish: A clinical study. _Int. Arch. Allergy Appl. Immunol_. 1966;29:346\u2013363.\n\n12. Thiel H., Ulmer W. Baker's asthma: Development and possibility for treatment. _Chest_. 1980;78:400\u2013405.\n\n13. Kemp S.F., Lockey R.F., Wolf B.L., Lieberman P. Anaphylaxis: A review of 266 cases. _Arch. Intern. Med_. 1995;155:1749\u20131754.\n\n14. Yunginger J.W., Sweeney K.G., Sturner W.Q., Giannandrea L.A., Teigland J.D., Bray M., Benson P.A., York J.A., Biedrzycki L., Squillace D.L. Fatal food-induced anaphylaxis. _JAMA_. 1988;260:1450\u20131452.\n\n15. Kidd J.M., III., Cohen S.H., Sosman A.J., Fink J.N. Food-dependent exercise-induced anaphylaxis. _J. Allergy Clin. Immunol_. 1983;71:407\u2013411.\n\n16. Dohi M., Suko M., Sugiyami H., Yamashita N., Tadokoro K., Juji F., Okudaira H., Sano Y., Ito K., Miyamoto T. Food-dependent, exercise-induced anaphylaxis: A study of 11 Japanese cases. _J. Allergy Clin. Immunol_. 1991;87:34\u201340.\n\n17. Horan R., Sheffer A. Food-dependent, exercise-induced anaphylaxis. _Immunol. Allergy Clin. N. Am_. 1991;11:757\u2013766.\n\n18. Anderson L.B., Dreyfuss E.M., Logan J., Johnstone D.E., Glaser J. Melon and banana sensitivity coincident with ragweed pollinosis. _J. Allergy_. 1970;45:310\u2013319.\n\n19. Min K., Metcalfe D. Eosinophilic gastroenteritis. _Immunol. Allergy Clin. N. Am_. 1991;11:799\u2013813.\n\n20. Moon A., Kleinman R. Allergic gastroenteropathy in children. _Ann. Allergy Asthma Immunol_. 1995;74:5\u201312.\n\n21. Crespo J.F., Pascual C., Dominguez C., Ojeda I., Munoz F.M., Esteban M.M. Allergic reactions associated with airborne fish particles in IgE-mediated fish hypersensitive patients. _Allergy_. 1995;50:257\u2013261.\n\n22. Caffrey E.A., Sladen G.E., Isaacs P.E.T., Clark K.G.A. Thrombocytopenia caused by cow's milk. _Lancet_. 1981;2:226\u2013240.\n\n23. Heiner D.C., Sears J.W. Chronic respiratory disease associated with multiple circulating precipitins to cow's milk. _Am. J. Dis. Child_. 1960;100:500\u2013502.\n\n24. Beltrani V.S., Beltrani V.P. Contact dermatitis: A review. _Ann. Allergy_. 1997;78:160\u2013195.\n\n25. Burks J.W. Classic aspects of onion and garlic dermatitis in housewives. _Ann. Allergy_. 1954;12:592\u2013595.\n\n26. Rietschel R.L., Fowler J.F., Jr., eds. Fisher's Contact Dermatitis. 4th ed. St. Louis: Williams & Wilkins; 1996:813\u2013814.\n\n27. Trier J.S. Celiac sprue. (1991). _N. Engl. J. Med_. 1991;325:1709\u20131719.\n\n28. Scott H., Fausa O., Ek J., Brandtzaeg P. Immune response patter in coeliac disease: Serum antibodies to dietary antigens measured by an enzyme-linked immunosorbent assay. _Clin. Exp. Immunol_. 1984;57:25\u201332.\n\n29. Cornell H.J. Amino acid composition of peptides remaining after in vitro digestion of gliadin sub-fraction with duodenal mucosa from patients with coeliac disease. _Clin. Chim. Acta_. 1988;176:279\u2013289.\n\n30. Hall R.P. The pathogenesis of dermatitis herpetiformis: Recent advances. _J. Am. Acad. Dermatol_. 1987;16:1129\u20131144.\n\n31. Katz A.I., Hall R.P., Lawley T.J., Strober W. Dermatitis herpetiformis: The skin and the gut. _Ann. Intern. Med_. 1980;93:857\u2013874.\n\n32. Powell G.K. Milk- and soy-induced enterocolitis of infancy. _J. Pediatr_. 1978;93:553\u2013560.\n\n33. Machida H.M., Catto Smith A.G., Gall D.G., Trevenen C., Scott R.B. Allergic colitis of infancy: Clinical and pathologic aspects. _J. Pediatr. Gastroenterol_. 1994;19:22\u201326.\n\n34. Kuitunen P., Visakorpi J.K., Savilahti E., Pelkonen P. Malabsorption syndrome with cow's milk intolerance: Clinical findings and course in 54 cases. _Arch. Dis. Child_. 1975;50:351\u2013356.\n\n35. Hughes J.M., Merson M.H. Fish and shellfish poisoning. _N. Engl. J. Med_. 1976;2295:1117\u20131120.\n\n36. Hide D.W., Guyer B.M. Prevalence of infantile colic. _Arch. Dis. Child_. 1982;57:559\u2013560.\n\n37. Taubman B. Parental counseling compared with elimination of cow's milk or soy milk protein for the treatment of infant colic syndrome: A randomized trial. _Pediatrics_. 1988;81:756\u2013761.\n\n38. Consensus Conference. Defined diets and childhood hyperactivity. _JAMA_. 1982;248:290\u2013292.\n\n39. Filer L.J., Stegink L.D. A report of the proceedings of an MSG workshop held August 1991. _CRC Crit. Rev. Food. Sci. Nutr_. 1994;34:159\u2013174.\n\n40. Chap. 26Terr A.I. Mechanisms of hypersensitivity. In: Stites D.P., Terr A.I., Parslow T.G., eds. _Human Immunology_. 9th ed. Baltimore, MD: Appleton & Lange; 1997:376\u2013388.\n\n41. Sampson H.A., Metcalfe D.D. Food allergies. _JAMA_. 1992;268:2840\u20132844.\n\n42. Bock S.A., Sampson H.A., Atkins F.M., Zeiger R.S., Lehrer S., Sachs M., Bush R.K., Metcalfe D.D. Double-blind, placebo-controlled food challenge (DBPCFC) as an office procedure: A manual. _J. Allergy Clin. Immunol_. 1988;82:986\u2013997.\n\n43. Metcalfe D., Sampson H. Workshop on experimental methodology for clinical studies of adverse reactions to foods and food additives. _J. Allergy Clin. Immunol_. 1990;86:421\u2013442.\n\n44. Bock S.A. The natural history of food sensitivity. _J. Allergy Clin. Immunol_. 1982;69:173\u2013177.\n\n45. Chap. 21Hill D.J., Hosking C.S. The management and prevention of food allergy. In: Frieri M., Kettelhut B., eds. _Food Hypersensitivity and Adverse Reactions: A Practical Guide for Diagnosis and Management_. Stamford, CT: Marcel Dekker; 1999:423\u2013448.\n\n46. Simon R.A. Adverse reactions to food and drug additives. _Immunol. Allergy Clin. North Am_. 1996;16:137\u2013176.\n\n47. Chap. 6Finegold I. Adverse reactions to food additives. In: Frieri M., Kettelhut B., eds. _Food Hypersensitivity and Adverse Reactions: A Practical Guide for Diagnosis and Management_. New York: Marcel Dekker; 1999:117.\n\n48. Stevenson D.D., Simon R.A. Sensitivity to ingested metabisulfites in asthmatic subjects. _J. Allergy Clin. Immunol_. 1981;68:26\u201332.\n\n49. Delohery J., Simmul R., Castle W.D., Allen D.H. The relationship of inhaled sulfur dioxide reactivity to ingested metabisulfite sensitivity in patients with asthma. _Am. Rev. Resp. Dis_. 1984;130:1027\u20131032.\n\n50. Stevenson D.D., Simon R.A., Lumry W.R., Mathison D.A. Adverse reactions to tartrazine. _J. Allergy Clin. Immunol_. 1986;78:182\u2013191.\n\n52. Danoff D., Lincoln L., Thomson D.M., Gold P. Big Mac attack. _N. Engl. J. Med_. 1978;298:1095\u20131096.\nCHAPTER 45\n\nNutrition and Cystic Fibrosis\n\nPHILIP M. FARRELL and HUI-CHUAN LAI, University of Wisconsin\u2014Madison, Madison, Wisconsin\n\n## I. INTRODUCTION\n\nCystic fibrosis (CF) is the most common, life-threatening autosomal recessive disorder with estimated incidences of 1 in 2900 white live births, 1 in 17,000 black live births, and 1 in 90,000 Asian live births [1, 2]. CF was recognized as a distinct clinical entity in 1938. It is a generalized disease of the exocrine glands characterized by abnormal sodium and chloride transport, leading to elevated electrolyte levels in the sweat glands [3]. Dysfunction of the other exocrine glands occurs, producing viscid secretions of low water content. This results in pancreatic insufficiency, which leads to malabsorption and failure to gain weight, as well as airway obstructions, which leads to increased susceptibility to recurrent bronchial infection, progressive lung damage, and eventual respiratory failure.\n\n### A. Clinical Presentation\n\nThe three categories of major clinical abnormalities in CF are (1) gastrointestinal tract involvement, characterized by pancreatic insufficiency leading to malabsorption and malnutrition; (2) respiratory tract involvement, characterized by chronic obstructive pulmonary disease with recurrent infections; and (3) salt loss in sweat that can lead to severe hyponatremic dehydration. The pancreatic disturbance begins prenatally and can cause intestinal obstruction in newborns with CF(a problem referred to as meconium ileus). It has been estimated that 85\u201390% of CF patients have functional pancreatic insufficiency [4], and 15\u201320% have meconium ileus [5]. Unlike pancreatic insufficiency, pulmonary status of patients with CF often appears normal at birth, however, it inevitably shows obstruction and infection. The onset and rate of progression of CF lung disease are not well understood but appear to vary widely among individuals [6, 7]. Other complications may occur as the disease progresses. For example, glucose intolerance and diabetes mellitus are prevalent [8] and about 15% of adults with CF develop diabetes requiring insulin therapy [9]; up to 5% of CF patients develop overt liver disease in adolescence or adulthood [10]; and infertility in males with CF is virtually universal [11].\n\n### B. Pathogenesis\n\nOn the basis of molecular genetics research [12, 13], CF fundamentally can be attributed to mutations occurring in the long arm of chromosome 7. With cloning of the CF gene, it has been demonstrated that the most common mutation is a 3-base-pair deletion, which results in the loss of a phenylalanine residue at amino acid position 508 of the predicted gene product, namely, the cystic fibrosis transmembrane regulator ( _CFTR_ ) [13, 14]. The 3-base-pair deletion mutant (commonly referred to as the \u0394 _F508_ mutation) occurs in about 70% of the CF chromosomes [14, 15] and more than 85% of CF patients in the United States have at least one \u0394 _F508_ allele [16]. However, more than 800 other DNA mutations in the CF gene have been identified.\n\nThe abnormal CFTR protein is the underlying pathogenic factor in the disease process due to its role in regulating ion transport across the apical membrane of epithelial cells, particularly chloride conductance, which is invariably defective in CF [2, 17]. The most recent research suggests that the CFTR protein is a structural component of the chloride channel and may itself account for the channel core [13].\n\n### C. Diagnosis and Treatment\n\nThe diagnosis of CF is customarily made because of (1) a positive family history, (2) the occurrence of meconium ileus, or (3) symptoms of intestinal malabsorption or pulmonary disease with infection, which occur at variable ages [18, 19]. Once the characteristic signs and symptoms become evident, the diagnosis of CF can be readily established by performing a sweat test using pilocarpine iontophoresis [20]. Although these traditional methods are generally effective in diagnosing CF, there are often delays in diagnosis and referral to a CF center. Accordingly, there has been considerable interest in establishing neonatal screening methods for detection of presymptomatic cases for the purpose of instituting early treatment and preventing ameliorating symptoms. In 1979, Crossley _et al._ [21] first described the use of dry-blood specimen obtained from the newborns to measure immunoreactive trypsinogen level, which was shown to be highly elevated in patients with CF [21, 22]. The discovery of the _CFTR_ gene in 1989 [12] has promoted the development of new screening methods where the immunoreactive trypsinogen test is coupled with detection of the most common mutant allele (\u0394 _F508_ ) or with _CFTR_ mutation analysis [23]. The potential benefits and risks associated with CF neonatal screening programs have been under investigation in various regions of the world [24\u201327] In several recent reports [27\u201332], clear evidence of nutritional benefits attributable to early diagnosis was demonstrated by anthropometric indexes. The most convincing evidence was obtained from a randomized clinical trial in Wisconsin after 10 years of investigation [28, 29]. Although some other states and all of Australia have implemented routine neonatal screening for CF, more evidence of pulmonary benefits and information on cost effectiveness are needed before other regions proceed to implement this method of early diagnosis.\n\nClinical management of CF involves treatment programs with three principal objectives: (1) improve nutritional status, (2) promote clearance of respiratory secretions, and (3) control bronchopulmonary infections. Care programs for CF patients in North America and many European countries are organized in specialized regional centers. These centers have placed particular emphasis on enhancing nutritional status and using aggressive strategies to prevent progressive pulmonary disease [33]. Although CF lung disease cannot be cured, treatment programs have been generally effective, as evidenced by the increasing longevity of CF patients in the United States from less than 20 years to approximately 30 years during the past two decades [9, 34]. The primary causes of deaths in patients with CF are cardiorespiratory complications, accounting for 85% of the deaths from CF that occurred in 1997 [9]. For this reason, most CF Centers in the United States place a great deal of emphasis on respiratory management for patients with CF.\n\n## II. OVERVIEW OF NUTRITIONAL PROBLEMS IN CYSTIC FIBROSIS\n\nCF is associated with an increased risk of protein energy malnutrition, as well as deficiencies in fat-soluble vitamins and other micronutrients. At the mild end of the malnutrition spectrum, CF patients may have depleted stores or low circulating concentrations of a given nutrient, but no associated signs or symptoms. More pronounced nutritional deficiencies lead to metabolic abnormalities, structural changes, functional disturbances, growth failure, developmental delay, and a variety of other characteristics of malnutrition. Malnutrition is most likely to occur during periods of rapid growth when nutritional requirements are high, during pulmonary exacerbations, and with increased severity of lung disease.\n\nHistorically, malnutrition in patients with CF was thought to represent either an inherent consequence of disease process or a physiologic adaptation to advanced pulmonary disease. However, it is now recognized that the causes of malnutrition in CF are multiple and can be attributed to three primary mechanisms [35\u201337]: increased energy and nutrient losses, increased energy expenditure; and decreased energy and nutrient intakes. Table 1 lists the major risk factors for malnutrition associated with CF.\n\nTABLE 1\n\nFactors Contributing to Malnutrition in Cystic Fibrosis\n\n_Disease factors_\n\nPresence of pancreatic insufficiency\n\nSeverity of pancreatic insufficiency (the degree of steatorrhea and azotorrhea)\n\nPartial intestinal resection secondary to bowel obstruction (caused by meconium ileus)\n\nSeverity of respiratory disease\n\nLoss of bile salts associated with steatorrhea\n\nCholestatic liver disease\n\nDiabetes mellitus\n\n_Nutritional factors_\n\nGrowth velocity (of particular concern in young children and adolescents with CF)\n\nMacronutrient intakes (e.g., the quantity and quality of food consumed)\n\nMicronutrient deficiencies (e.g., vitamin E)\n\nEnergy expenditure\n\nEating behaviors\n\n### A. Causes of Malnutrition\n\n1. INCREASED LOSSES\n\nMaldigestion or malabsorption resultant from pancreatic insufficiency is the primary factor contributing to energy and nutrient losses in patients with CF. Approximately 85% of CF patients have pancreatic insufficiency at the time of diagnosis. However, the severity of maldigestion and\/or mal-absorption depends on the residual pancreatic function and varies greatly among individual CF patients. In addition to pancreatic insufficiency, losses of bile salts and bile acids are commonly associated with steatorrhea and can exacerbate maldigestion and malabsorption. Patients presenting with meconium ileus, in particular those who have undergone intestinal resection, have further reduction in intestinal absorptive capabilities. Other factors may also contribute to energy and nutrient losses. For example, diabetes mellitus may increase energy losses due to glycosuria if not adequately controlled.\n\n#### 2. INCREASED REQUIREMENT\n\nEnergy requirements in patients with CF are highly variable. Several studies have reported that patients with CF have increased energy expenditure compared with non-CF patients [38\u201341]. A variety of explanations have been proposed to explain the increased energy expenditure observed in CF patients. These include chronic lung infection, increase in work of breathing, genetic and cellular defects, and changes in body composition.\n\nChronic lung infections, particularly with _Pseudomonas aeruginosa,_ have been shown to be associated with 25\u201380% increase in metabolic rate and energy requirements [42]. The link between CF genotype and energy requirement was reported in a study by Tomezsko _et al._ [41], who demonstrated that energy expenditure was increased by 23% in CF patients with homozygous \u0394 _F508_ mutation as compared with non-CF controls [41]. The hypothesis that a basic cellular defect may increase energy requirement was supported by _in vitro_ studies showing that mitochondria from cultured fibroblasts obtained from CF patients had higher rates of oxygen consumption compared with control tissues [43, 44]. Last, the observation that CF patients often have a lower percentage of body stores of metabolic substrates relative to their muscle mass compared with non-CF individuals [41] may contribute to increased energy expenditure in CF.\n\n#### 3. DECREASED CONSUMPTION\n\nThe appetite or energy intake of CF patients may be limited due to a variety of disease complications. Acute respiratory exacerbations are a common cause of anorexia, and chest infections often give rise to nausea and vomitting, which may further reduce intake [35]. The biochemical causes of anorexia associated with acute infection are unclear, but elevated circulating levels of tumor necrosis factor may play a role [45]. In addition to pulmonary complications, a variety of gastrointestinal complications also contribute to anorexia and inadequate caloric intake [35]. Increased occurrence of gastroesophageal reflux and esophagitis are observed in patients with CF. Distal intestinal obstruction syndrome, a form of subacute or chronic partial bowel obstruction, usually occurs in older patients with pancreatic insufficiency. Large fecal masses, palpable in the abdomen, give rise to intermittent abdominal distention and cramping accompanied with reduced appetite. Constipation in the absence of distal intestinal obstruction syndrome is another cause of anorexia and abdominal discomfort in older patients with CF.\n\n### B. Common Nutritional Deficiencies\n\n1. ENERGY AND MACRONUTRIENTS\n\nAs discussed earlier, patients with CF are at high risk of energy inadequacy due to their increased requirement and decreased consumption. Fat is the highest density source of energy and is needed to provide sufficient essential fatty acids. However, prior to 1980, restriction of fat intake was often recommended in an effort to lessen the symptoms of steatorrhea and malabsorption.\n\nProtein poses less of a nutritional problem than does fat in the CF population. The major risk of protein deficiency in CF patients occurs during the first year of life, when the average requirement is at least three times as great as that in adulthood. Human milk, which is relatively low in protein (7% of energy), and soy-based formula have been particularly associated with hypoproteinemic edema and growth retardation before instituting pancreatic enzyme therapy. It is recommended that protein should provide at least 15% of total daily energy intake for CF patients with pancreatic insufficiency.\n\n#### 2. FAT-SOLUBLE VITAMINS\n\nDeficiencies of fat-soluble vitamins in the CF population have been demonstrated in many studies [46\u201353]. Vitamin A and E are of the greatest concern, particularly in patients with severe malabsorption or liver disease. Deficiencies in Vitamin D or vitamin K are less common, and most likely occur in association with advanced cholestatic liver disease.\n\nVitamin A deficiency was the first micronutrient deficit demonstrated in patients with CF. Clinical symptoms of vitamin A deficiency reported in CF patients include keratinizing metaplasia of the bronchial epithelium, xerophthalmia, and night blindness. Several mechanisms for vitamin A deficiency in CF have been proposed, ranging from a defect in the mobilizing hepatic storage of vitamin A due to liver disease to low levels of retinol binding protein, which is responsible for transporting vitamin A in the circulation [48, 49].\n\nVitamin E deficiency in CF is most commonly evidenced by low plasma levels of \u03b1-tocopherol, which has been shown to be prevalent in infants with CF identified from neonatal screening programs [48, 54]. Vitamin K deficiency has not been routinely demonstrated in patients with CF. However, vitamin K deficiency is likely to develop in patients with CF who also have severe cholestatic liver disease, short-bowel syndrome, and lung disease requiring frequent antibiotic use [53].\n\n#### 3. MINERALS\n\nMacrominerals of concern in CF include sodium, calcium, and phosphorus. Sodium is of concern in CF patients because of its abnomally high content in the sweat. In hot climates, salt depletion can be catastrophic, leading to severe hyponatremic dehydration and shock. Therefore, sodium requirement may be considerably higher for CF patients than that of normal individuals. Nevertheless, routine sodium supplements appear to be unnecessary because the average American diet contains an overabundance of sodium. Sodium supplements are needed only in conditions that may cause prolonged sweat loss.\n\nThere is no clear evidence of deficiency in trace minerals in patients with CF. However, low plasma levels of zinc, calcium, magnesium, and iron in patients with CF have all been reported [55, 56]. In particular, iron-deficiency anemia with low serum ferritin has been shown to be quite frequent in CF patients with advanced pulmonary disease [56, 57].\n\n#### 4. ESSENTIAL FATTY ACIDS\n\nEssential fatty acid (EFA) deficiency has been known to occur in patients with CF [54, 58\u201360]. In infancy, particularly before diagnosis, EFA deficiency can occur with desquamating skin lesions, increased susceptibility to infection, poor wound healing, thrombocytopenia, and growth retardation. In older patients who are adequately treated, clinical evidence of EFA deficiency is rare, although biochemical abnormalities of EFA status remain common [61\u201363]. The major abnormalities in fatty acid profile found in patients with CF is a low level of linoleic acid and elevated levels of palmitoleic, oleic, and eicosatrienoic acid.\n\nMultiple hypotheses have been proposed to explain the underlying mechanisms of abnormal EFA status associated with CF. Fat malabsorption secondary to pancreatic insufficiency is the most common explanation for EFA. However, some investigators have postulated a primary metabolic defect in fatty acid metabolism [61\u201363]. In addition, van Egmond and colleagues [64] found that the growth rates of infants with CF were closely correlated with linoleic acid status.\n\n## III. PREVALENCE OF MALNUTRITION\n\nMalnutrition associated with CF is characterized by its early onset, and is often present at the time of CF diagnosis. Growth impairment, abnormalities in the biochemical markers of nutritional status, and clinical symptoms of malnutrition all have been reported in patients with CF. Because CF is associated with many risk factors of malnutrition, and growth impairment is very common in children with CF, there have been doubts whether normal growth can occur in children with CF [65]. The recent results from the Wisconsin CF neonatal screening study [28, 29] demonstrated unequivocally that young children with CF who are diagnosed early can grow normally with energy intakes averaging 110% of RDA. The prevalence of growth impairment, biochemical abnormalities, and suboptimal intakes is discussed further below.\n\n### A. Growth Impairment\n\nWeight retardation and linear growth failure are the most common observations documented in the CF clinics [66, 67], although its severity and prevalence vary greatly. Accurate estimates on the prevalence of malnutrition in the North American CF populations have been difficult to obtain in the past, due to lack of sufficient data. In recent years, national databases compiled by the U.S. and Canadian Cystic Fibrosis Foundations, known as the CF Foundation Patient Registries, have become available, making it possible to determine population estimates on the prevalence of malnutrition associated with CF. In a comprehensive study by Lai _et al._ [68], the nutritional status of more than 13,000 pediatric patients with CF seen in 144 CF centers in the United States during 1993 was examined using anthropometric criteria. Physical growth of children with CF was found to be substantially below normal at all ages, when plotted against growth curves developed by the National Center for Health Statistics for U.S. children [69], as shown in Fig. 1. The prevalence of stunting, defined as height below the 5th percentile, and underweight, defined as weight below the 5th percentile, was at approximately 25%. In addition, malnutrition was found to be particularly prevalent in infants (47%) and adolescents (34%) as compared with children at other ages (22%), and in patients with newly diagnosed, untreated CF(44%). In another study by Lai _et al._ [70], underweight was also found to be prevalent in adults with CF. Approximately 35% of the 7200 adults with CF documented in the 1992\u20131994 U.S. and Canadian CF Foundation Patient Registries were below the 5th percentile for weight when evaluated based on adult weight standards published from the second National Health and Nutrition Examination Survey [71].\n\nFIGURE 1 Height and weight percentile curves of children with CF(dotted curves) compared with those of the National Center for Health Statistics reference population (solid curves). [Modified from Lai, H. C., Kosorok, M. R., Sondel, S. A., Chen, S. T., FitzSimmons, S. C., Green, C., Shen, G., Walker, S., and Farrell, P. M. (1998). Growth status in children with cystic fibrosis based on National Cystic Fibrosis Patient Registry data: Evaluation of various criteria to identify malnutrition. _J. Pediatr._ **132,** 478\u2013485.]\n\n### B. Biochemical Abnormalities\n\nIn addition to growth impairment, abnormalities in the biochemical indexes of nutritional status also exist in patients with CF. Serum markers of protein status (e.g., albumin, prealbumin, retinol binding protein), fat (e.g., cholesterol, EFA profile), and fat-soluble vitamins (e.g., retinol, \u03b1-tocopherol) are typically found to be low in patients with CF. Abnormalities in biochemical indices are particularly prevalent in newly diagnosed infants with CF. Sokol _et al._ [72] reported that, of the 36 infants identified via CF newborn screening, 36% were hypoalbuminemic, 21% had low serum retinol, 35% had low serum 25-hydoxyvitamin D, and 38% had low serum \u03b1-tocopherol. Similarly, Lai _et al._ [54] reported that, of the 50 infants diagnosed before 3 months of age via neonatal screening, 46% had low serum albumin levels, 40% had low serum retinol levels, 72% had low \u03b1-tocopherol levels, and 50% had low plasma linoleic acid levels. Normalization of the biochemical nutritional indices can occur following comprehensive nutrition therapy. As observed by Marcus _et al._ [73], the prevalence of low serum albumin and retinol reduced substantially after 6 months of treatment. However, plasma \u03b1-tocopherol and linoleic acid remained low in 20\u201330% of patients despite ongoing nutrition therapy. In another recent cross-sectional study by Benabdeslam _et al._ [74], who examined 56 patients with CF aged 4\u201326 years, the prevalence of low plasma levels of albumin, cholesterol, and retinol binding protein remained high at 42, 25, and 12%, respectively, despite regular treatment. Serum concentrations of retinol and \u03b1-tocopherol were also significantly lower in CF patients compared to those of non-CF controls.\n\n### C. Suboptimal Dietary Intakes\n\nThe literature often describes infants and young children with CF as having voracious appetites. However, a number of dietary surveys particularly during the 1970s and early 1980s, when patients with CF were commonly prescribed with fat-resricted diets on the assumption that a reduction in dietary fat intake might improve bowel symptoms, indicate that patients with CF often eat less than normal. [75]. The observation of better growth and survival in patients with CF who received an unrestricted-fat, high-energy diet in combination with pancreatic enzyme supplementation compared with those who received low-fat diet in the early 1980s has changed dietary practices in most CF centers [76]. Energy intakes of 120% or greater than the Recommended Dietary Allowances (RDA), with 40% of energy from fat are now recommended for patients with CF [77]. However, patients with CF often fail to consume such high amounts of energy and\/or fat because of their disease manifestation. In several cross-sectional or short-term studies [78\u201382] and a small prospective 3-year study of 25 patients [83], energy and fat intakes of patients with CF were reported to be much lower than these recommendations. More recently, a longitudinal study [84] evaluating dietary intake patterns in children with CF from the time of diagnosis to age 10 years revealed that mean energy intake was at \u223c110% of RDA with fat consisting of \u223c37% of energy.\n\n## IV. NUTRITIONAL ASSESSMENT AND INTERVENTION\n\nFrequent monitoring of the nutritional status for patients with CF is ential to ensure early detection of any deterioration and prompt initiation of nutrition intervention. Patients with CF are most vulnerable to developing nutrition deficiencies during times of rapid growth, e.g., during infancy and adolescence, and during periods of pulmonary declines. During these periods, close monitoring and intervention are critical to prevent nutritional failure. Guidelines for nutritional assessment and intervention for patients with CF were developed in the early 1990s and reported by the US Cystic Fibrosis Foundation [85, 86]. The following section describes these guidelines. In addition, the Consensus Committee on Nutrition from the Cystic Fibrosis Foundation is in the process of updating the 1990 guidelines.\n\n### A. Guidelines for Nutritional Assessment\n\nAssessment of nutritional status for patients with CF must include anthropometric, biochemical, and dietary assessments. The frequency at which the different indices of nutritional status monitoring should be measured is given in Table 2.\n\nTABLE 2\n\nGuidelines for Assessment of Nutritional Status in Patients with Cystic Fibrosis\n\naIf there is any evidence of iron deficiency, iron status should be measured (i.e., serum iron, iron-binding capacity, and serum ferritin).\n\nbUsually consists of a 24-hour dietary recall with assessment of dietary patterns; should be performed by a dietitian.\n\ncInclude both dietary records to determine energy and fat intakes as well as a determination of stool fat excretion concurrent with evaluation of dietary fat intake to permit calculation of absorption coefficient and assessment of the degree of malabsorption.\n\n_Source:_ 1990 Guidelines: Adapted from Ramsey, B. W., Farrell, P. M., and Pencharz, P., for the Consensus Committee (1992). Nutritional assessment and management in cystic fibrosis: A consensus report. _Am. J. Clin. Nutr._ **55,** 108\u2013116. Proposed Additional Guidelines (2001): Cystic Fibrosis Foundation Consensus Committee on Nutrition, March 2001.\n\n#### 1. ANTHROPOMETRIC ASSESSMENT\n\nAnthropometric assessment, with an emphasis on physical growth, is an important component of nutritional assessment in children with CF. Measurements of height (or length in children aged 0\u20132 years), weight, and head circumference (in children aged 0\u20132 years) should be obtained at each clinic visit. To evaluate if deviation from normal growth occurs, sequential measurements of height and weight should be plotted on standard growth charts and converted to sex- and age-specific percentiles. The growth charts developed by the National Center for Health Statistics can be used for this purpose [69, 87]. The infant growth charts should be used for children aged 0\u20133 years, and the 2- to 18-year charts should be used for older children. For adolescents with CF, it is recommended to supplement National Center for Health Statistics growth charts with the Tanner charts to monitor growth and pubertal development [88].\n\nIn addition to height\/length and weight, it is important to evaluate whether the patient's weight is ideal for his or her height. For this purpose, the Cystic Fibrosis Foundation recommends the use of a weight-for-height index [89], which is referred as the percentage of ideal weight-for-height and can be calculated by the following steps:\n\nStep 1. Plot the patient's height\/length on the growth chart and determine the height percentile.\n\nStep 2. Determine the patient's ''ideal weight,\" which is the weight at the same percentile as that for his or her height. For example, for a 6-year-old girl with a height on the 25th percentile, her ideal weight is the weight that corresponds to the 25th percentile weight at age 6 year.\n\nStep 3. Express actual weight as a percentage of ideal weight, that is, Percentage of ideal weight = (actual weight\/ideal weight) \u00d7 100.\n\nStep 4. Classify nutritional status using the following criteria:\n\nNormal\u201490\u2013100%\n\nUnderweight\u201485\u201389%\n\nMild malnutrition\u201480\u201384%\n\nModerate malnutrition\u201475\u201379%\n\nSevere malnutrition\u2014<75%.\n\nSpecial attention should be given to patients with poor growth. Using various height and weight indices, the following criteria are recommended to identify patients with nutritional failure:\n\nFor patients <5 years of age\u2014a weight-for-height index <85% of ideal weight, weight loss for >2 months, or a plateau in weight gain for 2\u20133 months\n\nFor patients 5\u201318 years of age\u2014weight-for-height index <85% of ideal weight, weight loss for >2 months, or a plateau in weight gain for 6 months\n\nFor patients > 18 years of age\u2014a weight-for-height index <85% of ideal weight, or weight loss >5% for >2 months.\n\nIn addition to the above, body mass index, midarm circumference, and skinfold thickness should be used to further assess the patient's lean body mass and body fatness.\n\n#### 2. BIOCHEMICAL ASSESSMENT\n\nRoutine measurements of the following biochemical indices of nutritional status should be obtained: electrolytes and acid\u2013base status, complete blood count (CBC), serum albumin, retinol, and \u03b1-tocopherol. In addition to these routine measurements, when the results of CBC show evidence of iron deficiency, iron status should be assessed (i.e., serum iron, iron-binding capacity, and serum ferritin).\n\nRoutine assessments of vitamin D (25-hydroxycholecalciferol), vitamin K status, and EFA status are not presently regarded as necessary. However, annual assessments of these nutrients are likely to be recommended in the update guidelines because recent findings indicate increased prevalence of abnormal status of these nutrients among patients with CF [52\u201354, 62].\n\n#### 3. DIETARY ASSESSMENT\n\nAssessments of energy requirement and dietary intakes are important ways of determining whether the patient is in negative energy balance. Energy requirements vary greatly among individual CF patients due to their wide range of the degree of malabsorption and hypermetabolism. Therefore, energy requirements for patients with CF are best determined by estimating the basal metabolic rate, the degree of malabsorption, and the severity of pulmonary disease. For patients who are growing normally and whose steatorrhea is under good control, the estimated energy requirement is consistent with that recommended by the RDA for their gender and age [90]. For patients with increased energy requirement due to malabsorption and\/or pulmonary disease, energy requirement can be estimated by the following method:\n\nStep 1. Estimate basal metabolic rate by using the equations provided by the World Health Organization [91].\n\nStep 2. Estimate daily energy expenditure using the following equation:\n\nDaily energy expenditure = basal metabolic rate \u00d7 (activity coefficient % disease coefficient) where the activity coefficient is 1.3 (confine to bed), 1.5 (sedentary) or 1.7 (active), and the disease coefficient is 0 (normal lung function, i.e., forced expiratory volume in one second (FEV1) >80% predicted), 0.2 (moderate lung disease, i.e., FEV1 40\u201379% predicted), or 0.3 (severe lung disease, i.e., FEV1 <40%).\n\nStep 3. Estimate daily energy requirement, taking into account the degree of fat malabsorption, which can be estimated by the coefficient of fat absorption based on 3-day fat balance studies:\n\na. Pancreatic-sufficient patients (including patients on enzyme with coefficient of fat absorption \u226593%), daily energy requirement = daily energy expenditure.\n\nb. For pancreatic-insufficient patients with a coefficient of fat absorption <93%, daily energy requirement = daily energy expenditure \u00d7 (0.93\/coefficient of fat absorption).\n\nc. For pancreatic-insufficient patients whose coefficient of fat absorption has not been determined, an approximate value of 0.85 may be used to estimate daily energy requirement, i.e., daily energy requirement = daily energy expenditure \u00d7 (0.93\/0.85).\n\nEvaluation of dietary intake is best performed by nutritionists specializing in the care of patients with CF. For patients with good nutritional status, a dietitian may assess dietary habits and the quality of dietary intake using a 24-hour dietary recall. However, for patients with suboptimal nutritional status, a 3-day prospective food record is the best way to obtain quantitative estimates of energy and nutrient intakes. This assessment can then be used as the basis for initiating appropriate nutrition intervention.\n\n### B. Guidelines for Nutrition Management\n\nNutrition management for patients with CF varies and depends on the patient's age and disease severity. Nutrition intervention begins at the time of CF diagnosis, which corresponds to the first 2 years of life in the majority of patients with CF. After the diagnosis of CF, nutrition management is based on the clinical status of patients with CF and can be categorized into five response categories: routine management, anticipatory guidance, supportive intervention, rehabilitative care, and resuscitative and palliative care. The goals of the nutritional management for each response category are summarized in Table 3.\n\nTABLE 3\n\nGuidelines for Nutritional Management in Patients with Cystic Fibrosis\n\nCategory | Target group | Goals \n---|---|--- \nAt diagnosis and routine management | All patients | Nutritional education and dietary counseling; pancreatic enzyme replacement and vitamin supplementation for patients with pancreatic insufficiency \nAnticipatory guidance | Patients at risk of developing energy imbalance (i.e., severe pancreatic insufficiency, frequent pulmonary infections, periods of rapid growth) but maintaining a weight-for-height index of \u226590%> of ideal weight | Further education to prepare patients for increased energy needs; increased monitoring of dietary intakes; increased caloric density in diet as needed; behavioral assessment and counseling \nSupportive intervention | Patients with decreased weight velocity and\/or a weight-for-height index of 85\u201390% of ideal weight | All of the above plus oral supplements as needed \nRehabilitative care | Patients with a weight-for-height index consistently \u226485% of ideal weight | All of the above plus enteral supplementation via nasogastric tube or enterostomy as indicated \nResuscitative and palliative care | Patients with a weight-for-height index consistently \u226475% of ideal weight or with progressive nutritional failure | All of the above plus continuous enteral feeds or total parenteral nutrition\n\n_Source:_ Adapted from Ramsey, B. W., Farrell, P. M., and Pencharz, P., for the Consensus Committee (1992). Nutritional assessment and management in cystic fibrosis: A consensus report. _Am. J. Clin. Nutr._ **55,** 108\u2013116.\n\n#### 1. AT DIAGNOSIS\n\nThe time of diagnosis is a crucial period for beginning nutritional education, dietary counseling, and therapeutic interventions. All nutritional indices should be measured at the time of CF diagnosis. These include anthropometry (height, weight, and\/or head circumference), biochemical nutritional markers (serum or plasma albumin, vitamin A, vitamin E, and EFA), and dietary intake. In addition, assessment of pancreatic function status is essential, and is best determined based on a 3-day fat balance study. If maldigestion and mal-absorption are identified, pancreatic enzyme replacement therapy should be initiated. Nutritional interventions can then be individualized to each patient's needs. Extensive discussion of nutritional management with patients and\/or parents is another important aspect of care during this period of time. By concentrating on nutrition during the family's first few visits to the CF center, care-givers will be able to stress the importance of establishing and maintaining good nutrition.\n\n#### 2. ROUTINE MANAGEMENT\n\nThe primary goal of routine nutrition management is to optimize pancreatic enzyme replacement, as well as nutritional and vitamin supplements. Management should be based on the patient's clinical status and energy requirement. Nutritional status should be assessed at each clinic visit. Presently, the Cystic Fibrosis Foundation recommends that patients with CF be seen on a routine follow-up basis every 3\u20134 months. Thus, at a minimum, growth and nutritional status should be monitored at these intervals. Because nutritional requirements are influenced by age, routine management from infancy to adulthood is further discussed below.\n\na. Infancy to 2 Years.: The first 2 years represent the phase of life with the highest growth rate and energy requirement. Infants diagnosed with CF vary widely with regard to their presenting symptoms. Infants diagnosed as a result of gastrointestinal and\/or pulmonary symptoms often present with growth failure at the time of CF diagnosis. These infants should be evaluated weekly or every other week until normal weight gain has been established.\n\nInfants born with meconium ileus, in particular, those who have bowel resection resultant from intestinal surgery, represent another special group with high risk of nutritional failure. To promote optimal growth, these infants may require enteral or parenteral nutrition support in addition to predigested formula and pancreatic enzyme therapy. Intensive monitoring on growth, biochemical nutritional indices, and dietary intake are critical in maintaining proper growth rate in CF patients with meconium ileus.\n\nUnlike infants with more severe CF as described above, infants identified through a positive family history or through neonatal screening within the first 3 months of life are often asymptomatic and show relatively good growth at the time of CF diagnosis. The goals of nutrition management for this group of infants are to maintain normal growth and to prevent the occurrence of malnutrition.\n\nMilk products serve as the predominant source of nutrients for infants with CF during the first year of life. Breast feeding is encouraged in infants who show appropriate growth. However, breast-fed infants should be closely monitored with regard to their growth velocity, protein status, and electrolyte status. Supplementation with formula should be initiated if growth faltering is observed. If an infant with CF is growing at a normal rate, a change to whole cow's milk may be recommended beginning at 12 months of age, although formula feeding may be continued up to 24 months of age. Introduction of solid food should be made according to the guidelines of the American Academy of Pediatrics, namely, between 4 and 6 months of age or earlier if necessary.\n\nb. Toddlers to Preschool (Ages 2\u20136 years).: Children in this age group have developed self-feeding behaviors and express individual food preferences. Dietary intake and degree of physical activity vary from day to day. For these reasons, close monitoring of dietary habits, energy intake, and growth velocity are important.\n\nc. School Age (Ages 6\u201312 years).: Children in this age group are exposed to significant degrees of peer pressure and are challenged to self-manage their disease. Compliance with prescribed medications such as pancreatic enzymes and fat-soluble vitamins can become a major problem during this period. In addition, acceptance and understanding by teachers and fellow students may be lacking, further stressing a child with CF.\n\nd. Adolescence and Puberty (Ages 12\u201318 years).: This developmental stage represents another vulnerable period of developing malnutrition because of increased nutritional requirement associated with accelerated growth, endocrine development, and high levels of physical activity. In addition, pulmonary disease often becomes more severe in this period, increasing energy requirement. Pubertal delay and growth failure are common and come at a time of extreme psychosocial stresses.\n\nFrequent monitoring of dietary intake, growth velocity, and nutritional status are important; every 3 months is optimal. Nutritional counseling must be directed toward the patient rather than the parents.\n\ne. Adulthood.: Patients with CF who reach adulthood are usually responsible for the entire management of their disease, as well as for the financial burden of a chronic illness. While in college or working, adults with CF are constantly adapting to new schedules and stresses. The goal of nutrition management is to maintain ideal weight-for-height and to prevent weight loss. Nutritional counseling must be practical and pragmatic to help adults adjust to these changes.\n\nf. Pregnancy and Lactation.: Widespread experience in recent years has demonstrated that pregnancy and lactation can be accomplished successfully by some women with CF. Pregnant women with CF should follow the RDA guidelines for nutrient intakes. In addition, special attention should be given to appropriate weight gain, particularly during the last trimester of pregnancy. In addition to the usual multivitamin supplementation for CF, one prenatal vitamin should be consumed daily. During lactation, marked increase in energy intake is necessary to meet the high energy requirement during this period.\n\ng. Vitamin Supplementation.: In CF patients with pancreatic insufficiency, vitamin supplementation is necessary to prevent the occurrence of deficiencies. For infants and children younger than 2 years of age, liquid multivitamin supplementation at a dose equivalent to 1 mL of polyvisol (Mead Johnson Nutritionals, Evansville, IN), has been shown to provide sufficient vitamins A and D. Children aged 2\u20138 years need a standard multivitamin containing 400 IU vitamin D and 5000 IU vitamin A in a dose of 1 tablet\/day. Older children, adolescents and adults need a standard adult multivitamin preparation, 1\u20132 tablets\/day.\n\nIn addition to standard multivitamin supplementation, additional supplementation with vitamin E with the following doses are recommended: ages 0\u20136 months, 25 IU\/day; 6\u201312 months, 50 IU\/day; ages 1\u20134 years, 100 IU\/day; 4\u201310 years, 100-200 IU\/day; and >10 years, 200\u2013400 IU\/day. For the first 2 years of life, vitamin E is given as Aquasol E (Rover Pharmaceuticals, Fort Washington, PA) or liquid-E (Twin Labs, Ronkonkoma, NY). More research is needed to define the optimal supplementation regimen for vitamin K in patients with CF. Until further information is available, recommendations are to prescribe vitamin K supplementation as follows: age 0\u201312 months, 2.5 mg\/week, or 2.5 mg twice weekly if on antibiotics; ages >1 year, 5.0 mg twice weekly when on antibiotics or if cholestatic liver disease is present.\n\n#### 3. GUIDELINES FOR NUTRITIONAL SUPPORT\n\nFor CF patients who are at risk of developing, and those who are experiencing, nutritional failure, nutritional support beyond the level of routine maintenance care is required. Nutritional support is delivered at various levels, beginning with dietary modification, to oral supplementation, to enteral and parenteral supplementation. Guidelines for nutritional support of these patients are divided into four categories based on the severity of malnutrition (Table 3).\n\na. Anticipatory Guidance.: Patients with CF in this category maintain relatively normal nutritional status (i.e., weight-for-height index >90%), but are at high risk of developing energy deficiencies. These include patients who are growing rapidly, e.g., infants and adolescents, and patients who have more severe clinical symptoms, e.g., pancreatic insufficiency, meconium ileus, and frequent pulmonary infections. Nutrition counseling is directed to dietary and behavioral modifications for the purpose of increasing energy intake and minimizing the risk of nutritional decline by introducing the concept of boosted oral intake. This concept considers the patient's usual food preferences and habits and then increases the nutrient density of the diet without dramatically increasing the amount of food consumed. For example, margarine or butter may be added to many foods, and light cream can be used in place of skim milk or water when preparing canned soup. Instead of snacks like fruit juice, nutrient-dense foods such as nuts, cheese and crackers, cold cuts, whole milk, peanut butter sandwiches, and pizza may be provided to patients requiring additional nutrients.\n\nIn addition to the quality of the diets, psychosocial and behavioral problems can have significant impact on the nutritional status of patients with CF. An in-depth assessment of eating behavior, feeding patterns, and family interactions at mealtimes should be performed when a patient shows signs of decline in nutritional status.\n\nb. Supportive Intervention.: This level of nutritional support is initiated when patients with CF show signs of mild malnutrition, as indicated by a weight-for-height index between 85% and 90%. Initial management follows the same guidelines as outlined in ''anticipatory guidance.\" However, additional oral supplements in the form of homemade or proprietary products are strongly encouraged to maximize caloric and nutrient intakes. Homemade supplements are often more palatable and less expensive than commercial products, although the latter may be more convenient. If these treatments are not successful within 3 months or if the patient's weight falls below 85% of ideal, more aggressive nutritional management should be considered, as described below.\n\nc. Rehabilitative Care.: This level of nutritional support is initiated for CF patients who are experiencing nutritional failure (as indicated by a weight below 85% of ideal) and who are unable to improve weight gain with the use of oral supplementation and behavioral modification. In this case, further nutrition intervention with nocturnal enteral feedings can be helpful. Several studies have shown that this intervention improves body composition, increases strength and sense of well-being, increases sense of mastery over body weight, improves body image, encourages normal pubertal development, and reduces weight loss during pulmonary exacerbations.\n\nEnteral feeding can be delivered via nasogastric tubes, gastrostomy tubes, and jejunostomy tubes. The choice of an enterostomy tube and technique for its placement should be based on local factors and the expertise of the CF center involved. Nasogastric tubes are appropriate for short-term nutritional support in highly motivated patients. Patients can learn to pass soft Silastic feeding tubes to receive overnight feeding. High-calorie formula can be infused, allowing patients to receive up to 50% of their energy requirement while asleep.\n\nGastrostomy tubes can be used for patients who need chronic enteral nutritional support. Gastrostomy tubes or buttons can be placed by the percutaneous endoscopic route or surgically. Gastroesophageal reflux may occur with gastrostomy tube feeding. This problem can often be alleviated by adjusting the feeding rate, the patient's positioning during sleep, and administration of prokinetic agents (e.g., domperidone). Jejunostomy feedings potentially increase the problem of nutrient malabsorption, but have been used very successfully in some CF centers. Use of predigested or elemental formula may be needed with jejunostomy feeding.\n\nd. Resuscitative and Palliative Care.: This level of nutritional support is targeted to CF patients who are severely malnourished, with a weight below 75% of the ideal. Aggressive nutritional rehabilitation in terminally ill patients is usually not medically indicated (unless the patients are awaiting organ transplantation). Nevertheless, parenteral nutrition may be used for short-term support when enteral feedings are contraindicated, for example, for patients with short-gut syndrome, pancreatitis, and severe gastroenteritis, and during postoperative period after intestinal surgery.\n\n## V. CONCLUSIONS\n\nThe clear associations between nutritional status and clinical outcomes in CF mandate careful nutritional assessment, management, and monitoring of all patients with CF. In recent years, there has been a shift away from the idea that malnutrition is inevitable for most patients with CF toward the more optimistic view that normal nutrition and growth are possible if early diagnosis and aggressive nutritional monitoring and therapy are made to each individual patient, based on high-quality scientific studies. This task is best accomplished by involving a multidisciplinary team that includes a dietitian in the care and management of CF patients. In this way, the goals of normal growth and prevention of malnutrition can be attained, which will improve the prognosis and quality of life for patients with CF.\n\nReferences\n\n1. 1996Kosorok M.R., Wei W.H., Farrell P.M. The incidence of cystic fibrosis. _Stat Med_. 1996;15:449\u2013462.\n\n2. Boat T.F., Welsh M.J., Beaudet A.L. Cystic Fibrosis. In: Scriver C.R., Beaudet Al L., Sly W.S., Valle D., eds. _The Metabolic Basis of Inherited Disease_. 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Cystic Fibrosis Foundation. _Clinical Practice Guidelines for Cystic Fibrosis_. Cystic Fibrosis Foundation; 1997.\n\n34. FitzSimmons S.C. The changing epidemiology of cystic fibrosis. _J. Pediatr_. 1993;122:1\u20139.\n\n35. Durie P.R., Pencharz P.B. Nutrition in cystic fibrosis. _Br. Med. Bull_. 1992;48:823\u2013847.\n\n36. Pencharz P.B., Durie P.R. Nutritional management of cystic fibrosi. _Annu. Rev. Nutr_. 1993;13:111\u2013136.\n\n37. Stallings V.A. Nutritional deficiencies in cystic fibrosis: Causes and consequences. _New Insights Cyst. Fibros_. 1994;2:1\u20135.\n\n38. Vaisman N., Pencharz P.B., Corey M., Canny G.J., Hahn E. Energy expenditure of patients with cystic fibrosis. _J. Pediatr_. 1987;111:496\u2013500.\n\n39. Buchdahl R.M., Cox M., Fulleylove C., Marchant J.L., Tomkins A.M., Brueton M.J., Warner J.O. Increased resting energy expenditure in cystic fibrosis. _J. Appl. Physiol_. 1988;64:1810\u20131816.\n\n40. Anthony H., Bines J., Phelan P., Paxton S. 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Plasma tumour necrosis factor alpha in cystic fibrosis. _Thorax_. 1991;46:91\u201395.\n\n46. Chap. 9.14.3Farrell P.M. Nutrition in malabsorptive disorders. In: Rudolph A.M., ed. _Rudolph's Pediatrics_. 19th ed. Bethesda, MD: Appleton and Lange; 1991:249\u2013252.\n\n47. Congden P.J., Bruce G., Rothburn M.M., Clarke P.C., Littlewood J.M., Kelleher J., Losowsky M.S. Vitamin status in treated patients with cystic fibrosis. _Arch. Dis. Child_. 1981;81:708\u2013714.\n\n48. Feranchak A.P., Sontag M.K., Wagener J.S., Hammond K.B., Accurso F.J., Sokol R.J. Prospective, long-term study of fat-soluble vitamin status in children with cystic fibrosis identified by newborn screen. _J. Pediatr_. 1999;135:601\u2013610.\n\n49. Ahmed F., Ellis J., Murphy J., Wootton S., Jackson A.A. Excessive faecal losses of vitamin A (retinol) in cystic fibrosis. _Arch. Dis. Child_. 1990;65:589\u2013593.\n\n50. Wilfond B.S., Farrell P.M., Laxova A., Mischler E. Severe hemolytic anemia associated with vitamin E deficiency in infants with cystic fibrosis. Implications for neonatal screening. _Clin. Pediatr_. 1994;33:2\u20137.\n\n51. Lancellotti L., D'Orazio C., Mastella G., Mazzi G., Lippi U. Deficiency of vitamins E and A in cystic fibrosis is independent of pancreatic function and current enzyme and vitamin supplementation. _Eur. J. Pediatr_. 1996;155:281\u2013285.\n\n52. Henderson R.C., Lester G. Vitamin D levels in children with cystic fibrosis. _Southern Med. J_. 1997;90:378\u2013383.\n\n53. Durie P.R. Vitamin K and the management of patients with cystic fibrosis. _Can. Med. Assoc. J_. 1994;15:933\u2013936.\n\n54. Lai H.C., Kosorok M.R., Laxova A., Davis L.A., Fitz-Simmons S., Farrell P.M. Nutritional status of patients with cystic fibrosis with meconium ileus: A comparison with patients without meconium ileus and diagnosed early through neonatal screening. _Pediatrics_. 2000;105:53\u201361.\n\n55. Vormann J., Gunther T., Magdorf K., Wahn U. Mineral metabolism in erythrocytes from patients with cystic fibrosis. _Eur. J. Clin. Chem. Clin. Biochem_. 1992;30:193\u2013196.\n\n56. Pond M.N., Morton A.M., Conway S.P. Functional iron deficiency in adults with cystic fibrosis. _Resp. Med_. 1996;90:409\u2013413.\n\n57. Ater J.L., Herbst J.J., Landaw S.A., O'Brien R.T. Relative anemia and iron deficiency in cystic fibrosis. _Pediatrics_. 1983;71:810\u2013814.\n\n58. Farrell P.M., Mischler E.H., Engle M.J., Brown D.J., Lau S. Fatty acid abnormalities in cystic fibrosis. _Pediatr. Res_. 1985;19:104\u2013109.\n\n59. Christophe A., Robberecht E. Current knowledge on fatty acids in cystic fibrosis. _Prostagland. Leukotr. Essent. Fatty Acids_. 1996;55(3):129\u2013138.\n\n60. Roulet M., Frascarolo P., Rappaz I., Pilet M. Essential fatty acid deficiency in well nourished young cystic fibrosis patients. _Eur. J. Pediatr_. 1997;156:952\u2013956.\n\n61. Ozsoylu S. Clinical importance of essential fatty acid deficiency. _Eur. J. Pediatr_. 1998;157:779.\n\n62. Lloyd-Still J.D., Bibus D.M., Powers C.A., Johnson S.B., Holman R.T. Essential fatty acid deficiency and predisposition to lung disease in cystic fibrosis. _Acta Paediatr_. 1996;85:1426\u20131432.\n\n63. Hubbard V.S., Dunn D.G., di Sant Agnese P.A. Abnormal fatty acid composition of plasma lipids in cystic fibrosis: A primary or secondary effect? _Lancet_. 1977;2:1302\u20131304.\n\n64. van Egmond A.W., Kosorok M.R., Koscik R., Laxova R., Farrell P.M. Effect of linoleic acid intake on growth of infants with cystic fibrosis. _Am. J. Clin. Nutr_. 1996;63:746\u2013752.\n\n65. Davis P.B., Kercsmar C.M. Growth in children with chronic lung disease. _N. Engl. J. Med_. 2000;342:887\u2013888.\n\n66. Kraemer R., Rudeberg A., Hadorn B., Rossi E. Relative underweight in cystic fibrosis and its prognostic value. _Acta Paediatr. Scand_. 1978;67:33\u201337.\n\n67. Soutter V.L., Kristidis P., Gruca M.A., Gaskin K.J. Chronic undernutrition\/growth retardation in cystic fibrosis. _Clin. Gastroenterol_. 1986;15:137\u2013154.\n\n68. Lai H.C., Kosorok M.R., Sondel S.A., Chen S.T., Fitz-Simmons S.C., Green C., Shen G., Walker S., Farrell P.M. Growth status in children with cystic fibrosis based on National Cystic Fibrosis Patient Registry data: Evaluation of various criteria to identify malnutrition. _J. Pediatr_. 1998;132:478\u2013485.\n\n69. Hamill P.V.V., Drizd T.A., Johnson C.L., Reed R.B., Roche A.F., Moore W.M. Physical growth: National Center for Health Statistics percentiles. _Am. J. Clin. Nutr_. 1979;55:108\u2013116.\n\n70. Lai H.C., Corey M., FitzSimmons S.C., Kosorok M.R., Farrell P.M. Comparison of growth status in patients with cystic fibrosis in the United States and Canada. _Am. J. Clin. Nutr_. 1999;69:531\u2013538.\n\n71. data from the national Health Survey Series 11, No. 238, DHHS Publication No. (PHS) 87-1688U.S. Department of Health and Human Services. _Anthropometric Reference Data and Prevalence of Overweight, United States, 1976\u20131980_. San Mateo, CA: NCHS, Hyattsville; 1987.\n\n72. Sokol R.J., Reardon M.C., Accurso F.J., Stall C., Narkewicz M., Abman S.H., Hammond K.B. Fatsoluble vitamin status during the first year of life in infants with cystic fibrosis identified by screening of newborns. _Am. J. Clin. Nutr_. 1989;50:1064\u20131071.\n\n73. Marcus M.S., Sondel S.A., Farrell P.M., Laxova A., Carey P.M., Langhough R., Mischler E.H. Nutritional status of infants with cystic fibrosis associated with early diagnosis and intervention. _Am. J. Clin. Nutr_. 1991;54:578\u2013585.\n\n74. Benabdeslam H., Garcia I., Bellon G., Gilly R., Revol A. Biochemical assessment of the nutritional status of cystic fibrosis patients treated with pancreatic enzyme extracts. _Am. J. Clin. Nutr_. 1998;67:912\u2013918.\n\n75. Dodge J.A., Yassa J.G. Food intake and supplemental feeding programs. In: Sturgess J.M., ed. _Perspectives in Cystic Fibrosis_. MD: Canadian Cystic Fibrosis Foundation; 1980:125\u2013136.\n\n76. Corey M., McLaughlin F.J., Williams M., Levison H. A comparison of survival, growth, and pulmonary function in patients with cystic fibrosis in Boston and Toronto. _J. Clin. Epidemiol_. 1988;41:583\u2013591.\n\n77. Pencharz P.B. Energy intakes and low-fat diets in children with cystic fibrosis. _J. Pediatr. Gastroenterol. Nutr_. 1983;2:400\u2013402.\n\n78. Bell D., Durie P., Forstner G.G. What do children with cystic fibrosis eat? _J. Pediatr. Gastroenterol. Nutr_. 1984;3(Suppl. 1):S137\u2013S146.\n\n79. Buchdahl R.M., Fulleylov C., Matchant J.L., Warner J.O., Brueton M.J. Energy and nutrient intakes in cystic fibrosis. _Arch. Dis. Child_. 1989;64:373\u2013378.\n\n80. Hodges P., Sauriol D., Man S.F., Reichert A., Grace M., Talbot T.W., Brown N., Thomson A.B. Nutrient intake of patients with cystic fibrosis. _J. Am. Diet. Assoc_. 1984;84:664\u2013669.\n\n81. Lloyd-Still J.D., Smith A.E., Wessel H.U. Fat intake is low in cystic fibrosis despite unrestricted dietary practices. _J. Parenteral Enteral Nutr_. 1989;13:296\u2013298.\n\n82. Tomezsko J.L., Stallings V.A., Scanlin T.F. Dietary intake of healthy children with cystic fibrosis compared with normal control children. _Pediatrics_. 1992;90:547\u2013553.\n\n83. Kawchak D.A., Zhao H., Scanlin T.F., Tomezsko J.L., Cnaan A., Stallings V.A. Longitudinal, prospective analysis of dietary intake in children with cystic fibrosis. _J. Pediatr_. 1996;129:119\u2013129.\n\n84. Lai H.C., Kosorok M.R., Laxova A., Davis L.A., Farrell P.M. Long-term dietary intakes in children with cystic fibrosis: Evaluation from diagnosis to age 10 years. _Pediatr. Pulmonol_. 1999(Suppl.):297\n\n85. Ramsey B.W., Farrell P.M., Pencharz P., for the Consensus Committee. Nutritional assessment and management in cystic fibrosis: A consensus report. _Am. J. Clin. Nutr_. 1992;55:108\u2013116.\n\n86. Cystic Fibrosis Foundation. _Clinical Practice Guidelines for Cystic Fibrosis_. Toronto: Cystic Fibrosis Foundation; 1997.\n\n87. advanced data from Vital and Health Statistics, No. 314Kuczmarski R.J., Ogden C.L., Grummer-Strawn L.M., Flegal K.M., Guo S.S., Wei R., Mei Z., Curtin L.R., Roche A.F., Johnson C.L. _CDC Growth Charts: United States_. Bethesda, MD: National Center for Health Statistics; 2000.\n\n88. Tanner J.M., Whitehouse R.H. Clinical longitudinal standards for height, weight, height velocity, weight velocity, and stages of puberty. _Arch. Dis. Child_. 1976;51:170\u2013179.\n\n89. Moore D.J., Durie P.R., Forstner G.G., Pencharz P.B. The assessment of nutritional status in children. _Nutr. Res_. 1985;5:797\u2013799.\n\n90. National Research Council. _Recommended Dietary Allowances_ , 10th ed. Hyattsville, MD: National Academy Press; 1989.\n\n91. World Health Organization. Energy and Protein Requirements. _WHO Tech. Report Series No. 724_. Washington, DC: World Health Organization; 1985.\nCHAPTER 46\n\nOsteomalacia\n\nROBERT MARCUS, Stanford University, Palo Alto, California\n\n## I. INTRODUCTION\n\nMature human bone is a proteinaceous matrix that embeds a highly organized crystalline mineral, hydroxyapatite. The mineral phase of adult bone normally approximates about 40% of the total bone mass. In osteoporotic patients, despite the occasional presence of subtle abnormalities of bone mineralization, the customary ratio of mineral to matrix is not seriously altered. _Osteomalacia_ is the name given a condition in which the total amount of bone may be low, normal, or high, but in which the matrix is notably undermineralized (Fig. 1). The mechanical consequences of undermineralization may be severe. Osteomalacic bone behaves poorly in standardized tests of bone strength, and long bones may deform in response to routine mechanical loads.\n\nFIGURE 1 Relative proportion of bone mineral and matrix in normal, osteoporotic, and osteomalacic bone.\n\nThe term _osteomalacia_ describes a disorder of adult bone. In children, circumstances leading to osteomalacia also produce the specific disorder of growth plates known as rickets. The bones of children with rickets also undermineralize, and are therefore osteomalacic. If untreated, affected children may experience potentially serious consequences of osteomalacia, including reduced pulmonary function due to poor chest expansion and inability to undergo normal childbearing due to pelvic deformity.\n\nThis chapter reviews adult osteomalacia with a primary focus on its antecedents. It describes the clinical settings in which osteomalacia likely occurs and briefly summarizes elements of diagnosis and management of this condition. To begin, it briefly summarizes normal bone mineralization and the disruptions in this process that lead to osteomalacia.\n\n## II. MINERALIZATION\n\nThe concentrations of calcium and phosphate found normally in extracellular fluid substantially exceed their solubility product in aqueous solution. The normal extracellular fluid ionized Ca2% activity in most species approximates 2.5 mEq\/L (1.25 mM), whereas concentrations of inorganic phosphate, which fluctuate considerably with dietary intake, are generally about 1 mM in fasting adult humans. Thus, the degree of body fluid saturation can be expressed as a simple ion product (Ca2+ \u00d7 Pi\u22123) = \u223c1.3 m _M_ 2, which approaches the effective solubility of aqueous calcium-phosphate. In the absence of a catalyzing nucleus on which orderly crystal growth can proceed, crystal precipitation will not occur in stable supersaturated solutions with (Ca2+ \u00d7 PO4\u22123) ion products even as high as 6 m _M_ 2 because of a thermodynamic barrier related to mineral complexity.\n\nIn bone, a number of organic molecules influence the nucleation process and thereby serve to initiate crystal deposition. Matrix components with clustered phosphate groups bind calcium and promote mineralization. Once nucleation occurs, crystal growth proceeds rapidly in proportion to the concentrations of the mineral constituents. One model for this process involves the osteoblast enzyme, alkaline phosphatase, which is thought to initiate nucleation by hydrolyzing organic phosphates, thereby raising the local concentration of inorganic phosphate above supersaturation [1]. The specific phosphate-containing substrates for this process have not been identified, but likely include matrix phosphoproteins and phospholipids.\n\nGiven the fact that extracellular fluids are supersaturated with mineral, why, as with Lot's wife, does mineralization not occur everywhere all the time? To a major degree, the _metastable_ condition of bone reflects the existence of small inorganic, as well as proteinaceous, compounds that act as mineralization inhibitors. The first of these to be identified was pyrophosphate [2]. This ubiquitous by-product of ATP hydrolysis circulates in extracellular fluids at millimolar concentration, which suffices to inhibit mineral deposition. In aqueous solutions of inorganic calcium and phosphorus, pyrophosphate constrains both mineral growth and dissolution in super- and undersaturated conditions, respectively.\n\nAfter osteoblasts have laid down new bone matrix, cleavage of pyrophosphate and possibly other phosphorylated molecules, such as phosphoserine residues in type I collagen, must occur before mineral can be deposited. Pyrophosphate cleavage is accomplished by the skeletal enzyme alkaline phosphatase, which is incorporated into the osteoblast plasma membrane. As osteoblasts move away from their newly secreted osteoid, membrane fragments are torn from the cells, permitting alkaline phosphatase to establish contact with and hydrolyze pyrophosphate and other phosphorus-containing mineralization inhibitors, so that mineral precipitation ensues (Fig. 2). Other mineralization inhibitors are also found in bone, including citrate, magnesium, and the large molecular weight glycosaminoglycans, but their relative importance to normal mineral deposition and crystal growth is not certain.\n\nFIGURE 2 General scheme for bone mineralization.\n\nCollagen fibers in bone are highly ordered and thus direct the nucleation of mineral from metastable solutions. This is termed _heterogeneous nucleation,_ as opposed to the spontaneous precipitation of mineral from supersaturated solutions. Deposition of mineral into collagen is not a random event. Indeed, the structural periodicity of pores and spaces in and around the collagen fibers creates specific sites at which a nidus of mineralization occurs. The initial crystals are formed within the hole zones that result from the quarter-stagger of collagen molecules (Fig. 3) [3, 4]. Thus, mineralization does not occur as a wave throughout collagen fibers, but rather by nucleation at multiple, independent sites in the hole zones. It is likely that other matrix components, especially phosphoproteins, also facilitate nucleation at these sites [5\u20138]. With time, the entire collagenous structure becomes encased in mineral so that the final product, mature calcified bone, is analogous to a freeway tower in which the original interconnected steel poles and girders are encased in cement.\n\nFIGURE 3 Mineral deposition on collagen.\n\nTo succeed, mineral deposition must satisfy multiple conditions, including adequate local concentrations of calcium and phosphorus, normally active alkaline phosphatase, ability of collagen to undergo crystal nidation and foster mineral accumulation, and, finally, absence of nondegradable mineralization inhibitors. Failure to satisfy any of these conditions can jeopardize final mineralization and give rise to osteomalacia (Fig. 4).\n\nFIGURE 4 Disruptors of mineralization.\n\n## III. OSTEOMALACIC BONE\n\nAbnormalities encountered on routine skeletal radiographs of osteomalacic bone reflect the decrease in the amount of mineralized bone along with an increased prevalence of undermineralized bone surfaces (Fig. 5). These include an overall washed out appearance of the skeleton, coarsened trabecular patterns with poorly defined edges, and, in severe cases, bony deformation and pseudofractures. The latter, known also as Looser's zones, represent incomplete fragility fractures that show focal accumulation of osteoid. They tend to be oriented perpendicularly to the long axis of the cortical surface and are frequently bilateral, occurring commonly in the scapulae, pubic rami, proximal femurs, ribs, and long bones [9].\n\nFIGURE 5 Radiographic appearance of osteomalacic bone. [Reprinted with permission from Steiner, E. J., Jergas, M., and Genant, H. K. (1995). Radiology of osteoporosis. _In_ ''Osteoporosis\" (R. Marcus, D. Feldman, and J. Kelsey, Eds.), pp. 1019\u20131954. Academic Press, San Diego.]\n\nOne hallmark of osteomalacic bone is its histologic appearance. Bone remodeling normally occurs on about 10% of bony surfaces, the remainder being quiescent. Sites that have been subjected to recent bone resorption contain surfaces that are actively replacing lost bone with fresh bone matrix. Although this new bone is destined ultimately to be fully calcified, a number of bone surfaces that have not yet mineralized will persist until the process has gone to completion. These partially mineralized surfaces reveal a thin covering of unmineralized matrix, or _osteoid._ Under normal circumstances the total prevalence of osteoid-lined surfaces is low, and the osteoid layers themselves do not exceed 15 micrometers in average thickness (Fig. 6, top; see color plate at the back of the book). By contrast, osteomalacic bone is characterized by the diffuse accumulation of thick osteoid clumps over the majority of, or even the entire, trabecular surface (Fig. 6, bottom) [10].\n\nFIGURE 6 Histologic appearance of normal (top) and osteomalacic (bottom) bone. [Reprinted with permission from Compston, J. (1997). Bone histomorphometry. _In_ ''Vitamin D\" (D. G. Feldman, F. H. Glorieux, and J. W. Pike, Eds.), pp. 573\u2013586. Academic Press, San Diego.]\n\nIt may be difficult at times to distinguish true osteomalacia from a state in which an increased prevalence of osteoid-covered surfaces reflects increased whole-body bone remodeling activity (so-called ''high-turnover\" states), as happens frequently in hyperthyroidism, hyperparathyroidism, and hypervitaminosis A or D. To distinguish these two conditions requires that an iliac crest bone biopsy be obtained in which bone is labeled _in vivo_ prior to taking the biopsy (so-called \"dynamic histomorphometry\"). A typical method to accomplish this is to administer two separate doses of the antibiotic, tetracycline, approximately 10 days apart. Tetracycline binds avidly to bone matrix at sites of ongoing mineral deposition. When examined under ultraviolet light, areas of tetracycline uptake are denoted as bands of yellow fluorescence. When the patient is doubly labeled, two bands appear, and the average distance between them, divided by the number of days between dose administration, gives a value called the mineral apposition rate representing the amount of new bone mineralized per day (Fig. 7; see color plate at the back of the book) [9]. The mineral apposition rate is normally about 70 \u03bcm per day. In states of high bone turnover, surface prevalence of tetracycline uptake is high and the mineral apposition rate is normal or high. By contrast, osteomalacia shows both a reduction in the number of bone surfaces taking up tetracycline and a very low mineral apposition rate.\n\nFIGURE 7 Dynamic histomorphometry of trabecular bone shown by tetracycline double labeling. [Reprinted with permission from Compston, J. (1997). Bone histomorphometry. _In_ ''Vitamin D\" (D. G. Feldman, F. H. Glorieux, and J. W. Pike, Eds.), pp. 573\u2013586. Academic Press, San Diego.]\n\nThis discussion gains particular relevance in light of the nutritional focus of this volume. Several nutritionally based clinical situations lead to increased osteoid prevalence on bone surfaces. These multiple states are associated with increased secretion of parathyroid hormone, such as in response to low calcium intake, or as a toxic manifestation of high-dose vitamins A or D. In these instances, normal mineral apposition rates on tetracycline-labeled biopsies indicate a generalized increase in bone turnover rate but are not compatible with a diagnosis of osteomalacia.\n\n## IV. CAUSES OF OSTEOMALACIA\n\nA. Vitamin D Deficiency\n\n#### 1. VITAMIN D PHYSIOLOGY\n\nVitamin D, whether obtained through the action of ultraviolet light on 7-dehydrocholesterol in the skin or through dietary ingestion, is biologically inert until it is converted to its polar active metabolites by hydroxylation (Fig. 8). The first step in this pathway involves a hepatic cytochrome _P_ -450 enzyme that hydroxylates the parent vitamin in the 25 position. The resulting compound, 25-hydroxyvitamin D (25OHD) constitutes the most abundant circulating form of vitamin D. In the circulation, 25OHD binds highly to a specific vitamin D binding globulin. The metabolic fate of 25OHD is determined by the relative activities of two enzymes located in the renal tubules. The first, the 1-\u03b1 hydroxylase, converts 25OHD into its most active form, 1,25-dihydroxyvitamin D, or _calcitriol._ The alternative step involves a 24-hydroxylase that converts 25OHD into the practically inert product, 24, 25-dihydroxyvitamin D [11].\n\nFIGURE 8 Vitamin D pathway. [Reprinted with permission from Henry, H. (1997). The 25-hydroxyvitamin D1 alpha-hydroxylase. _In_ ''Vitamin D\" (D. G. Feldman, F. H. Glorieux, and J. W. Pike, Eds.), pp. 41\u201368. Academic Press, San Diego.]\n\nThe major regulators of calcitriol synthesis are parathyroid hormone, hypophosphatemia, and hypocalcemia. In the face of dietary calcium deficiency, small reductions in plasma ionized calcium activity lead to compensatory hypersecretion of parathyroid hormone, which in turn stimulates the 1-\u03b1 hydroxylase to promote calcitriol production. Parathyroid hormone also indirectly stimulates this enzyme by causing the kidney tubule to reabsorb less phosphate, thereby creating a state of renal phosphate depletion. Phosphate depletion exerts a powerful stimulatory effect on 1-\u03b1 hydroxylase. Because serum calcium concentrations actually increase (and those of parathyroid hormone decrease) in the presence of hypophosphatemia, the increased hydroxylase activity must be a direct response to low phosphorus content rather than to parathyroid hormone [12]. Increased calcitriol production results in higher circulating concentrations of this metabolite and, therefore, increased vitamin D action on the intestine to enhance calcium absorption efficiency. Thus, the primary physiological role for vitamin D is to increase intestinal calcium absorption efficiency [13].\n\nAlthough considered a ''prehormone\" insofar as it is a substrate for calcitriol synthesis, 25OHD itself possesses intrinsic bioactivity. Indeed, 25OHD has an affinity for the vitamin D receptor that is only 1:1000 that of calcitriol, but as it circulates at 1000-fold molar excess relative to calcitriol (\u223c30 ng\/mL versus 30 pg\/mL),25OHD actually accounts for an important fraction of total vitamin D activity _in vivo._\n\nAn additional feature of vitamin D physiology warranting comment is the suggestion that vitamin D metabolites normally are excreted into the bile, only to be reabsorbed further down the intestine (so-called \"enterohepatic circulation\"). If such a process does truly occur, it would explain the vitamin D deficiency states associated with surgical diversion of biliary contents, as occurs with jejunal or ileal bypass surgery, or use of bile acid sequestering medications, such as cholestyramine. Evidence for an enterohepatic mechanism includes the recovery of radioactivity in the bile following injection of isotopic 25OHD [14]. In a recent review of this topic, however, Mawer _et al._ [15] found little evidence to support the biological significance of those metabolites that are excreted into the bile, and concluded it is ''unlikely that any enterohepatic circulation of vitamin D metabolites that does occur is... of physiologic significance.\"\n\nAs stated, the primary action of vitamin D is to increase the efficiency of intestinal calcium absorption. Calcium is normally absorbed by two distinct processes. The first, which is sensitive to vitamin D, involves an active transport process confined largely to the duodenum. The second, which is independent of vitamin D, involves facilitated diffusion throughout the small intestine (transcaltachia). Although multiple and diverse effects of vitamin D metabolites have been demonstrated in bone and kidney, the increase in intestinal calcium absorption has by far the greatest importance for skeletal homeostasis.\n\n#### 2. VITAMIN D ADEQUACY, INADEQUACY, AND DEFICIENCY\n\nThe definition of vitamin D adequacy remains a controversial topic (see review by Chapuy and Meunier [16]). Clinical laboratories generally publish a ''normal\" range for 25OHD spanning 10\u201350 ng\/mL. Consensus exists that values below this range, particularly below 6 ng\/mL, are highly associated with clinical, biochemical, and histological evidence of osteomalacia. However, evidence suggests that values above 10 ng\/mL are not necessarily adequate for optimal bone balance. A highly significant inverse relationship exists between circulating concentrations of 25OHD and parathyroid hormone. Thus, in the presence of low circulating 25OHD, parathyroid hormone secretion increases, leading to higher rates of bone turnover and loss. However, at circulating 25OHD concentrations in excess of \u223c30 ng\/mL, the negative association with parathyroid hormone loses significance, suggesting that a vitamin D replete state exists. Although extremely low 25OHD concentrations are, at most, uncommon in the general population of Western countries, values associated with increased parathyroid hormone secretion (<\u223c20 ng\/mL), which are very common, might legitimately be considered to reflect a degree of vitamin ''inadequacy.\" At substantial risk for vitamin D inadequacy are people living in the Northern tier of the United States, in Canada, or in Northern or Central Europe, particularly during winter months. Vitamin D inadequacy is also frequently encountered, even in more temperate climates, among shut-ins and others who avoid sunlight exposure, as well as institutionalized patients who do not receive full-spectrum ultraviolet light. Many studies indicate that low vitamin D concentrations are very common in elderly men and women. There is marked seasonal variation in circulating 25OHD, so that the prevalence of vitamin D insufficiency during the winter ranges, depending on country, from 8% to 60% [17\u201319].\n\nFull discussion of vitamin D adequacy exceeds the scope of this chapter, but this issue bears directly on the consideration of osteomalacia. Earlier descriptions of ''osteomalacia\" in association with various medications and pathological states frequently offered no histological corroboration of the diagnosis, and even when that was done, did not apply contemporary diagnostic criteria for osteomalacia. Examination of this literature, particularly with respect to so-called ''anticonvulsant osteomalacia,\" indicates that many reports more likely represented examples of secondary hyperparathyroidism and ''high-turnover\" osteoporosis due to modest or moderate vitamin D inadequacy [20].\n\n#### 3. VITAMIN D DEFICIENCY BASED ON POOR VITAMIN INTAKE OR SUNLIGHT EXPOSURE\n\nFlorid vitamin D deficiency characterized life in Northern Europe during the 18th and 19th centuries, but overt vitamin D deficiency is rare today in industrialized societies. That being said, it is true that certain ethnic groups may undertake dietary and clothing habits that place them at higher risk for deficiency. The primary dietary sources of vitamin D include fish oils and supplemented dairy products. In the United States, milk is supplemented to a nominal concentration of 400 units vitamin D per quart, albeit with great variability in quality control. Milk supplementation is not frequent in other countries. Some [21,22] albeit not all [23] studies indicate that the cutaneous production of vitamin D in response to ultraviolet irradiation declines with age. Older men and women who may not achieve adequate solar exposure, and who drink little milk, are at particular risk for marked vitamin D inadequacy, if not of true deficiency.\n\nInadequate solar exposure has been identified as a cause of florid osteomalacia, particularly in women, when traditional dress and indoor lifestyles keep people from receiving even a modicum of sunlight. Osteomalacia on this basis has been reported even from areas of the world, such as Turkey and the Arabian peninsula, that are drenched in sunlight most of the year [24]. In addition, use of sun block creams reduces the rise in vitamin D concentration following a standard dose of solar exposure, leading to lower circulating 25OHD concentrations [25,26].\n\n#### 4. VITAMIN D DEFICIENCY BASED ON GASTROINTESTINAL PATHOLOGY\n\nIn Western society, true vitamin D deficiency commonly occurs when intestinal function has been disrupted. Affected individuals include those who have undergone gastroduodenal (Billroth I) or gastrojejunal (Billroth II) anastomoses or jejuno- or ileal-bypass surgery, individuals with bowel infarction, or patients with malabsorption syndromes such as those associated with coeliac or inflammatory bowel disease and extensive diverticulosis. Patients with primary biliary disorders or those taking medications, such as bile acid sequestrants (Cholestyramine, Questran\u00ae) or products aimed at inducing fat malabsorption (Orlistat, Xenical\u00ae) may also be at added risk for vitamin D deficiency.\n\nPatients who have undergone gastrojejunal bypass surgery suffer a very high prevalence of osteoporosis and, to a lesser extent, osteomalacia, although when osteomalacia is assessed using iliac crest biopsy criteria, the prevalence may exceed 20% [15,27]. Abnormalities in mineral metabolism in these patients reflect multiple deficits in intestinal function. Vitamin D-sensitive active calcium absorption occurs relatively high in the duodenum, so that bypassing this portion of the gut severely jeopardizes the adaption to low calcium intake. Patients with intestinal bypass surgery experience deficits in both vitamin D and calcium homeostasis resulting in a state of secondary hyperparathyroidism to maintain plasma calcium normalcy. High circulating concentrations of parathyroid hormone in turn promote urinary phosphate excretion and hypophosphatemia, all of which contribute to osteomalacia. As discussed above, the possibility that vitamin D metabolites in bile undergo enteric recirculation has been considered a convenient explanation for the depletion of 25OHD in patients undergoing intestinal bypass surgery. However, the importance of this mechanism has been placed into serious question [15]. Alternatively, it has been shown that hyperparathyroid states are associated with increased clearance of 25OHD, and that clearance normalizes when parathyroid hormone concentrations are reduced by calcium supplementation [28]. Thus, the 25OHD depletion characteristic of intestinal bypass patients may be due to compensatory parathyroid hormone hypersecretion.\n\nUnfortunately, many patients who have undergone disruptive intestinal operations fail to receive adequate medical follow-up once the acute postoperative period has passed. Because of the insidious onset of bone disease, skeletal complaints develop very slowly, generally coming to medical attention only decades later, at which time the patients frequently neglect to mention (or their physicians neglect to ask about or even recognize the significance of) the previous surgical history.\n\nOsteomalacia and osteoporosis, occasionally severe, are recognized to occur in the setting of chronic liver disease. Early studies indicating a high prevalence of osteomalacia were based on relatively nonrigorous histological analyses. Recent studies indicate that true osteomalacia is actually very uncommon in liver disease, although it occurs occasionally in association with severe vitamin D deficiency [29,30].\n\n#### 5. INHERITED FORMS OF VITAMIN D DEFICIENCY OR RESISTANCE\n\nMutations in the 1-\u03b1 hydroxylase enzyme (vitamin D-dependent rickets) lead to a condition of extreme vitamin D resistance and are associated with very low blood concentrations of calcitriol [31,32]. Affected children respond exuberantly to small doses of exogenous calcitriol and, if treated in a timely fashion, experience cure of rickets and do not subsequently develop osteomalacia. By contrast, children with mutations in the vitamin D receptor have profound hypocalcemia, rickets and osteomalacia, and total body alopecia. They show normal to high circulating concentrations of vitamin D and its metabolites, but fail completely to respond to the exogenous administration of these agents [33]. Clinical and biochemical remissions have been observed in these children with long-term administration of calcium infusions. These children tend to undergo a substantial degree of remission when they enter puberty, and adult osteomalacia has not been a major problem.\n\nX-linked hypophosphatemic rickets (or vitamin D-resistant rickets) is an inherited disorder of renal tubular function resulting in wasting of inorganic phosphorus and hypophosphatemia and leading clinically to rickets and osteomalacia. Clinical expression of this disorder is highly variable, ranging from very mild to severe rickets and\/or osteomalacia. In adults with X-linked hypophosphatemic rickets, serum concentrations of 250HD are normal, and those of calcitriol are either normal or only mildly depressed [34]. However, given the degree of hypophosphatemia that occurs in this disorder, much higher concentrations of calcitriol would be predicted. Thus, regulation of the renal 1\u03b1-hydroxylase appears to be abnormal [35]. Recent discovery of the _PHEX_ gene and its gene product, a membrane-bound endopeptidase, has clarified the genetic, if not the physiologic, basis of this disorder. It is speculated that inactivating mutations of _PHEX_ produce X-linked hypophosphatemic rickets because affected people fail to break down phosphatonin, a (still hypothetical) hormone that is predicted to promote hypophosphatemia by impairing renal tubular reabsorption of phosphorus [36]. In addition to X-linked hypophosphatemic rickets, other forms of hereditary rickets\/osteomalacia have been described [35].\n\n### B. Phytate Ingestion\n\nAbout 30 years ago, attention was called to a substantial prevalence of rickets and osteomalacia in Pakistani and Indian immigrants to Great Britain [37,38]. Multiple reasons can be invoked to explain such an occurrence, including changes imposed on these people in diet, dress, and sunlight access. However, one additional aspect of this condition, shared also with Bedouins living in the Middle East, is exposure to unleavened bread made from a high phytate flour (the traditional chapati bread) [39]. Phytate is a 6-carbon cyclic compound in which each carbon atom is linked covalently to a phosphate moiety. These multiple phosphate groups effectively chelate calcium ions, thereby preventing their absorption.\n\n### C. Inadequate Calcium Intake\n\nBecause ionized calcium is critical for proper function of nerves, heart and other muscles, and other vital physiological processes, maintaining its circulating concentration within fairly narrow limits is a matter of high priority. Humans, mammals, and indeed birds, reptiles, and amphibia, possess an elaborate parathyroid-vitamin D axis that compensates for inadequate intake or intestinal absorption of calcium by drawing on skeletal reserves to support the plasma calcium concentration. Hence, although isolated calcium inadequacy, if extremely severe, could theoretically cause osteomalacia, this must be a very rare occurrence. It is likely that a calcium deficiency state of sufficient magnitude and duration to overwhelm the parathyroid compensatory mechanism might actually prove lethal to neurological and cardiac function before clinical osteomalacia could emerge.\n\nDespite the fact that tropical countries have abundant sunlight, nutritional rickets remains prevalent in such countries. The possibility has been raised that dietary calcium deficiency may be the underlying factor in such cases. In a Nigerian study [40],123 children with rickets were observed to have very low calcium intakes averaging 200 mg\/day. Children were treated with vitamin D (600,000 U intramuscularly at enrollment and at 12 weeks), calcium (1000 mg daily), or a combination of both. Treatment produced a smaller increase in the average serum calcium concentration in the vitamin D intervention group (\u223c0.5 mg\/dL at 24 weeks) than in either the calcium (\u223c1.5 mg\/dL, _P_ < 0.001) or combination-therapy groups (\u223c1.4 mg\/dL, _P <_ 0.001). A greater proportion of children in the calcium and combination-therapy groups than in the vitamin D group reached desired clinical outcomes, i.e., suppression of serum alkaline phosphatase activity and radiographic healing of rickets [39]. Thus, the results suggest a contribution of inadequate dietary calcium to the pathogenesis of rickets in these children. A similar suggestion has been made for a cohort of Bangladeshi children with rickets [41].\n\nAn occasional patient with chronic anorexia nervosa has been found to have osteomalacia. However, given the global nature of nutritional deficits in this disorder, it is not possible to assign sole culpability to calcium deficiency in these cases [42].\n\n### D. Phosphorus Depletion\n\nSustained decreases in the plasma phosphorus concentration reliably induce osteomalacia in experimental animals and emerge as a common basis for osteomalacia in humans. Hypophosphatemia (inorganic phosphate concentrations below 1 m _M_ , or \u20133.0 mg\/dL) is encountered in diverse clinical situations. Intestinal causes include impaired phosphorus absorption due to vitamin D deficiency or resistance, and excessive ingestion of phosphorus-binding aluminum-containing antacids or laxatives. Compelling evidence was presented several decades ago that sustained use of phosphate-binding antacids (primarily aluminum hydroxide) produced hypophosphatemia and muscle weakness typical of patients with osteomalacia [43]. During the past decade, these compounds have been virtually abandoned for antacid use and have been replaced by calcium carbonate for the control of serum phosphorus concentrations in renal failure patients. Osteomalacia rarely has been described in patients subjected to chronic laxative abuse.\n\n### E. Tumor-Induced Osteomalacia\n\nBeginning more than 50 years ago, a series of case reports brought attention to an association of hypophosphatemic osteomalacia with the presence of unusual tumors [44,45]. Patients complained typically of progressive weakness, primarily involving large muscle groups of the legs, with marked limitation in the ability to rise from a chair or climb stairs. Hypophosphatemia was severe, with serum phosphate concentrations frequently below 2.0 mg\/dL. Generalized bone pain was also frequent. Symptoms and metabolic abnormalities were characteristically present for years before a tumor was discovered. Some tumors were extremely small, being found as tiny skin nodules or lumps in the webs of toes. In some cases, tumor resection led rapidly to dramatic resolution of biochemical features and symptoms. In many cases, however, tumors were inoperable or surgery was only partly successful. Histologically, these tumors have been highly unusual in their appearance. They have been variably called hemangiopericytomas and ossifying or nonossifying fibromas, among other names.\n\nSome progress has been made in understanding the underlying mechanism of tumor-induced osteomalacia. The syndrome is characterized by paraneoplastic defects in vitamin D metabolism, abnormal proximal renal tubular function, and grossly diminished renal phosphate reabsorption. Serum calcitriol concentrations are vanishingly low, which, in the face of profound hypophosphatemia, constitutes _prima facie_ evidence for defective synthesis or accelerated removal of this hormone. Most investigators consider it likely that these tumors produce humoral factor(s) that alter renal proximal tubular function [35]. Evidence from a single instructive case suggests that the tumor produces a phosphaturic protein or proteins, which could explain the defective phosphorus reabsorption, although it does not suggest a basis for the low calcitriol values [46]. For patients in whom a tumor is discovered, surgical excision may be curative. When the causative tumor cannot be found or completely resected, palliative treatment with calcium, vitamin D, and phosphate replacement is helpful [47].\n\nOsteomalacia has also been noted to occur in patients with a variety of other tumor types, including typical carcinomas, myelomas, and lymphomas. Whether osteomalacia in such patients reflects a mechanism similar to that of tumor-induced osteomalacia, described above, is uncertain because it generally has not been possible to resect the tumors. Alternatively, the bone disorder could be a reflection of severe cachexia associated with systemic malignancy. Osteomalacia also has been reported in association with neuro-fibromatosis, but nothing definitive can be stated about its cause [35].\n\n### F. Renal Tubular Defects\n\nThe Fanconi syndrome is a generalized disorder of proximal renal tubular transport. Its features include wasting of phosphate, glucose, bicarbonate, uric acid, and one or more amino acids [48]. In addition to several hereditary forms, Fanconi syndrome can also be produced by renal tubular toxins, including heavy metals [49, 50], drugs (outdated tetracyclines) and precipitated immunoglobulins or their subunits, as in myeloma, amyloidosis, and paraprotein states [51]. Osteomalacia occurs in Fanconi syndrome as a consequence of profound phosphorus wasting.\n\nSome patients who have undergone ureterosigmoidostomy have developed osteomalacia. The underlying pathophysiology is not entirely clear, but appears to reflect the degree of metabolic acidosis that develops in these patients [52].\n\n### G. Deficits in Osteoblast Function\n\nAs described above, normal initiation of mineralization requires the initial action of skeletal alkaline phosphatase on pyrophosphate. Hypophosphatasia is a genetic disorder characterized by severe deficiency or complete absence of multiple isoforms of alkaline phosphatase [53] and which on iliac crest biopsy shows osteomalacia. Although profound deficits in phosphatase activity have been demonstrated in diverse tissues, it is the absence of this enzyme in bone that underlies the clinically important manifestations of the disease. Severely affected individuals experience bone pain, fractures, and deformity. Rarely, homozygous individuals may die in infancy, the cause of death perhaps related to increased intracranial pressure due to premature craniosynostosis (closure of cranial sutures). Less severely affected children may show rickets, dental hypoplasia, craniosynostosis, ectopic soft-tissue calcifications and growth retardation. Biochemical features include increased plasma concentrations of pyridoxal 5\u2032-phosphate and an increased urinary excretion of phosphatidyl ethanolamine [54]. At present, there is no specific or effective therapy for patients with hypophosphatasia. In this disorder, high doses of vitamin D and phosphate are probably ineffective at best, and potentially dangerous.\n\n### H. Presence of Mineralization Inhibitors\n\nFluoride is the most common naturally occurring inhibitor of mineralization. Indeed, although pitting of dental enamel is the hallmark of mild fluoride toxicity, severe endemic fluorosis leads to profound osteomalacia and bony sclerosis. This condition occurs primarily in areas of the world in which groundwater fluoride content is naturally high. Such areas include portions of Southern Italy, Central Europe, and arid regions of India. Skeletal fluorosis is commonly observed in deer and other animals that graze in those areas [55]. In the United States, groundwater in sections of Colorado and the arid Southwest have very high natural fluoride content, and endemic fluorosis in grazing animals is frequent. Human skeletal fluorosis does not occur in the United States because federal statutes require communities having excessive water fluoride content to adjust the ambient fluoride concentration or to provide all children a supply of freshwater within tolerable fluoride limits. Industrial fluoride exposure may sometimes reach toxic levels in aluminum smelter workers, and there are occasional reports of patients experiencing acute fluoride toxicity in response to a breakdown in hemodialysis quality control.\n\nHuman osteomalacia related to fluoride exposure has been a frequent concern with respect to therapeutic fluoride treatment of osteoporosis. The risk of osteomalacia is particularly high when adequate vitamin D and supplemental calcium are not also prescribed. In addition, the bisphosphonate etidronate (Didronel\u00ae) has been known to cause osteomalacia since its early use at high dose (15\u201320 mg\/kg\/day) for management of Paget's disease of bone. Even at lower doses (5 mg\/kg\/day), continuous use of this agent has been associated with focal osteomalacia. This attribute of etidronate limits its use in the treatment of osteoporosis, because it is necessary to prescribe it on a cyclical basis if osteomalacia is to be avoided. For example, in one standard treatment regimen, etidronate is generally given for 14 days every 3 months with adequate provision of calcium and vitamin D for the remaining 76 days [55a]. Second- and third-generation potent bisphosphonates, such as alendronate (Fosamax\u00ae), pamidronate (Aredia\u00ae), or risedronate (Actonel\u00ae) do not impair mineralization, and therefore do not cause osteomalacia at clinically relevant doses.\n\nAluminum constitutes a special case of mineralization inhibitor. Its role in preventing intestinal phosphorus absorption has been described above. In addition, it has been implicated as the cause of a severe form of osteomalacia in patients with chronic renal failure who have received aluminum-containing antacids for control of plasma phosphorus concentration. Bone biopsy specimens in such patients reveal extensive accumulation of aluminum at mineralizing surfaces and are reported as indicative of a very low turnover (adynamic) state. Treatment of aluminum-related osteomalacia requires its removal with desferroxamine, a chelating agent [56]. Although this therapy is often dramatically successful, the frequency of serious infections in treated individuals has raised concern about the strategy of treating all patients who have been exposed to aluminum. At present, it is recommended that treatment be reserved for patients with symptomatic aluminum-related toxicity. Despite the fact that aluminum antacids have been replaced by calcium salts as the mainstay for managing hyperphosphatemia, adynamic osteomalacia unrelated to aluminum exposure persists and has actually increased in prevalence as an important clinical problem for patients with chronic renal failure. The basis remains obscure.\n\n## V. TREATMENT OF OSTEOMALACIA\n\nSuccessful therapy of osteomalacia requires elimination of its underlying factors in individual cases. For patients who suffer nutritional vitamin D deficiency, restoration of normal circulating concentrations of 25OHD is the desired endpoint. Multiple treatment schedules have been described. Therapy can be initiated with high doses of vitamin D, such as 25,000 units\/day, for 6 months, at which time the dose can be reduced toward maintenance amounts (400\u2013800 units\/day). Adequate supplemental calcium, perhaps 1500 mg\/day, should also be provided. For patients with short-bowel syndromes, intestinal bypass, or other disruptions of normal bowel architecture, vitamin D should be administered by a parenteral route. Intramuscular injection of 50,000\u2013100,000 units\/month is sufficient. The rare child who is completely refractory to the actions of calcitriol generally requires longterm administration of intravenous calcium infusions, at least until the time of puberty.\n\nFor patients with renal phosphorus wasting, restoration of tissue phosphorus content is the primary aim. This generally involves the oral administration of various phosphate salts in amounts sufficient to provide 1\u20132 g\/day of elemental phosphorus. Preparations that are currently available include sodium\/hydrogen salts (e.g., Fleet's PhosphoSoda\u00ae), or mixtures of sodium and potassium phosphate (e.g., K-Phos\u00ae). The latter may be preferable for patients who cannot tolerate large sodium loads. Tolerability of phosphate salts may be limited by diarrhea. Some patients develop hypocalcemia and secondary hyperparathyroidism in response to phosphorus supplementation. When that occurs, additional vitamin D and calcium may be required.\n\nIn patients with primary abnormalities in osteoblastic function, such as those with hypophosphatasia, administration of excess vitamin D and calcium may be counterproductive and actually toxic. These patients should be treated only to be certain that vitamin D deficiency does not confound the underlying disorder. For patients with tumor-related osteomalacia, all attempts should be made to locate and extirpate the tumor.\n\nFinally, where osteomalacia is based on excessive exposure to fluoride, etidronate, or nonabsorbable antacids, the offending exposure must be terminated. Unfortunately, the skeletal half-life of fluoride and bisphosphonate is long, and resolution of osteomalacia may require years of follow-up. 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In: Feldman D.G., Glorieux F.H., Pike J.W., eds. _Vitamin D_. San Diego: Academic Press; 1997:679\u2013693.\n\n17. Stamp T.C.B., Round J.M. Seasonal changes in human plasma levels of 25-hydroxyvitamin D. _Nature (Lond.)_. 1974;247:563\u2013565.\n\n18. Lund B., Sorenson O.H. Measurement of 25-hydroxyvitamin D in serum and its relation to sunshine, age, and vitamin D. _Scand. J. Clin. Lab. Invest_. 1979;39:23\u201330.\n\n19. Gloth F.M., Tobin J.D. Vitamin D deficiency in older people. _J. Am. Geriatr. Soc_. 1995;43:822\u2013828.\n\n20. Weinstein R.S., Bryce G.F., Sappington L.J., King D.W., Gallagher B.B. Decreased serum ionized calcium and normal vitamin D metabolite levels with anticonvulsant drug treatment. _J. Clin. Endocrinol. Metab_. 1984;58:1003\u20131009.\n\n21. MacLaughlin J., Holick M.F. Aging decreases the capacity of human skin to produce vitamin D3. _J. Clin. Invest_. 1985;76:1536\u20131538.\n\n22. Holick M.F., Matsuoka L.Y., Wortsman J. Age, vitamin D and solar ultraviolet. _Lancet_. 1989;2:1104\u20131105.\n\n23. Davie M., Lawson D.E.M. Assessment of plasma 25-hydroxyvitamin D response to ultraviolet irradiation over a controlled area in young and elderly subjects. _Clin. Sci_. 1980;58:235\u2013242.\n\n24. Gullu S., Erodgan M.F., Uysal A.R., Baskal N., Kamel A.N., Erdogan G. A potential risk for osteomalacia due to sociocultural lifestyle in Turkish women. _Endocrine J_. 1998;45:675\u2013678.\n\n25. Matsuoka L.Y., Ide L., Wortsman J., MacLaughlin J.A., Holick M.F. Sunscreens suppress cutaneous vitamin D3 synthesis. _J. Clin. Endocrinol. Metab_. 1987;64:1165\u20131168.\n\n26. Matsuoka L.Y., Wortsman J., Hanifan N., Holick M.F. Chronic sunscreen use decreases circulating concentrations of 25-hydroxyvitamin D: A preliminary study. _Arch. Dermatol_. 1988;124:1802\u20131804.\n\n27. Eddy R.L. Metabolic bone disease after gastrectomy. _Am. J. Med_. 1971;50:442\u2013449.\n\n28. Davies M., Heys S.E., Selby P.S., Berry J.L., Mawer E.B. Increased catabolism of 25-hydroxyvitamin D in patients with partial gastrectomy and elevated 1,25-dihydroxyvitamin D levels. Implications for metabolic bone disease. _J. Clin. Endocrinol. Metab_. 1997;82:209\u2013212.\n\n29. Compston J.E., Crowe J.P., Wells I.P., Horton L.W.L., Hirst D., Merrett A.L., Woodhead J.S., Williams R. Vitamin D prophylaxis and osteomalacia in chronic cholestatic liver disease. _Dig. Dis. Sci_. 1980;25:28\u201332.\n\n30. Parfitt A.M. Osteomalacia and related disorders. In: Avioli S.M., ed. _Metabolic Bone Disease and Clinically Related Disorders_. San Diego: W. B. Saunders; 1990:329\u2013396.\n\n31. Scriver C.R. Vitamin D dependency. _Pediatrics_. 1970;45:361\u2013363.\n\n32. Fraser D., Kooh S.W., Kind H.P., Hollick M.F., Tanaka Y., DeLuca H.F. Pathogenesis of hereditary vitamin D dependent rickets. An inborn error of vitamin D metabolism involving defective conversion of 25-hydroxyvitamin D to 1-alpha, 25-dihydroxyvitamin D. _N. Engl. J. Med_. 1973;289:817\u2013822.\n\n33. Malloy P.J., Pike J.W., Feldman D. Hereditary 1,25-dihydroxyvitamin D resistant rickets. In: Feldman D.G., Glorieux F.H., Pike J.W., eds. _Vitamin D_. Philadelphia, PA: Academic Press; 1997:765\u2013788.\n\n34. Lyles K.W., Clark A.G., Drezner M.K. Serum 1,25-dihydroxyvitamin D levels in subjects with X-linked hypophosphatemic rickets and osteomalacia. _Calcif. Tiss. Intl_. 1982;34:125\u2013130.\n\n35. Drezner M.K. Clinical disorders of phosphate homeostasis. In: Feldman F.H., Pike J.W., eds. _Vitamin D_. San Diego: Academic Press; 1997:733\u2013753.\n\n36. Drezner M.K. PHEX gene and hypophosphatemia. _Kidney Int_. 2000;57:9\u201318.\n\n37. Hodgkin P., Hine P.M., Kay G.H., Lumb G.A., Stanbury S.W. Vitamin D-deficiency in Asians at home and in Britain. _Lancet_. 1973;2:167\u2013171.\n\n38. Holmes A.M., Enoch B.A., Taylor J., Jones M.E. Occult rickets and osteomalacia amongst the Asian immigrant population. _Quart. J. Med_. 1973;42:125\u2013149.\n\n39. Berlyne G.M., Ben-Ari J., Norde E. Bedouin osteomalacia due to calcium deprivation caused by high phytic acid content of unleavened bread. _Am. J. Clin. Nutr_. 1973;26:910\u2013911.\n\n40. Thacher T.D., Fischer P.R., Pettifor J.M., Lawson J.O., Isichei C.O., Reading J.C., Chan G.M. A comparison of calcium, vitamin D, or both for nutritional rickets in Nigerian children. _N. Engl. J. Med_. 1999;341:602\u2013604.\n\n41. Fischer R., Rahman A., Cimma J., Kyaw-Myint T.O., Kabir A.R., Talukder K., Hassan N., Manaster B.J., Staab D.B., Duxbury J.M., Welch R.M., Meisner C.A., Haque S., Combs G.F., Jr. Nutritional rickets without vitamin D deficiency in Bangladesh. _J. Trop. Pediatr_. 1999;45:291\u2013293.\n\n42. Oliveri B., Gomez Acotto C., Mautalen C. Osteomalacia in a patient with severe anorexia nervosa. _Rev. Rhum. (Engl. ed.)_. 1999;66:505\u2013508.\n\n43. Lotz M., Zisman E., Bartter F.C. Evidence for a phosphorus-depletion syndrome in man. _N. Engl. J. Med_. 1968;278:409\u2013415.\n\n44. Salassa R.M., Jowsey J., Arnaud C.D. Hypophosphatemic osteomalacia associated with \"nonendocrine\" tumors. _N. Engl. J. Med_. 1970;283:65\u201370.\n\n45. Evans D.J., Azzopardi J.G. Distinctive tumours of bone and soft tissue causing acquired vitamin-D resistant osteomalacia. _Lancet_. 1972;1:353\u2013354.\n\n46. Cai Q., Hodgson S.F., Kao P.C., Lennon V.A., Klee G.G., Zinsmiester A.R., Kumar R. Brief report: Inhibition of renal phosphate transport by a tumor product in a patient with oncogenic osteomalacia. _N. Engl. J. Med_. 1994;330:1645\u20131649.\n\n47. Yeung S.J., McCutcheon I.E., Schultz P., Gagel R.F. Use of long-term intravenous phosphate infusion in the palliative treatment of tumor-induced osteomalacia. _J. Clin. Endocrinol. Metab_. 2000;85:549\u2013555.\n\n48. Lee D.B., Drinkard J.P., Rosen V.J., Gonick H. The adult Fanconi syndrome: Observations on etiology, morphology, renal function and mineral metabolism in three patients. _Medicine_. 1972;51:107\u2013138.\n\n49. Adams R.G., Harrison J.F., Scott P. The development of cadmium-induced proteinuria, impaired renal function, and osteomalacia in alkaline battery workers. _Quart. J. Med_. 1969;38:425\u2013443.\n\n50. Emmerson B.T. \"Ouch-ouch\" disease: The osteomalacia of cadmium nephropathy. _Ann. Intern. Med_. 1970;73:854\u2013855.\n\n51. Bate K.L., Clouston D., Packham D., Ratnaike S., Ebeling P.R. Lambda light chain induced nephropathy: A rare cause of the Fanconi syndrome and severe osteomalacia. _Am. J. Kidney. Dis_. 1998;32:E3.\n\n52. Donohoe J.F., Freaney R., Muldowney F.P. Osteomalacia in ureterosigmoidostomy. _Irish J. Med. Sci_. 1969;8:523\u2013530.\n\n53. Henthorn P.S., Raducha M., Fedde K.N., Lafferty M.A., Whyte M.P. Different missense mutations at the tissue-nonspecific alkaline phosphatase gene locus in autosomal recessively inherited forms of mild and severe hypophosphatasia. _Proc. Natl. Acad. Sci. USA_. 1992;89:9924\u20139928\n\n54. Whyte M.P. Hypophosphatasia. In: Scriver C.B., Sly W.S., Valle D., eds. _The Metabolic Basis of Inherited Disease_. 6th ed. San Diego: McGraw-Hill; 1989:2843\u20132856.\n\n55. Schultz M., Kierdorf U., Sedlacek F., Kierdorf H. Pathological bone changes in the mandibles of wild red deer. ( _Cervas elaphus L._ ) exposed to high environmental levels of fluoride. _J. Anat_. 1998;193:431\u2013432.\n\n55a. Watts N.B., Harris S.T., Genant H.K., Wasnich R.D., Miller P.D., Jackson R.D., Licata A.A., Ross P., Woodson G.C., III., Yanover M.J., Mysiw J., Kohse L., Rao M.B., Steiger P., Richmond B., Chesnut C.H., III. Intermittent cyclical etidronate treatment of postmenopausal osteoporosis. _N. Engl. J. Med_. 1990;323:73\u201379.\n\n56. Malluche H.H., Smith A.J., Abreo K., Faugere M.C. The use of desferoxamine in the management of aluminum accumulation in bone in patients with renal failure. _N. Engl. J. Med_. 1984;311:140\u2013144.\nCHAPTER 47\n\nNutrition and Immunodeficiency Syndromes\n\nJUL GERRIOR and CHRISTINE WANKE, Tufts University School of Medicine, Boston, Massachusetts\n\n## I. INTRODUCTION\n\nAs we enter the third decade of the acquired immunodeficiency syndrome (AIDS) epidemic, nutrition and metabolic concerns continue to challenge health professionals, medical scientists, and individuals infected with the human immunodeficiency virus (HIV). In the developed world, the rate of death caused by HIV and AIDS has declined largely due to the advent of highly active antiretroviral therapy. However, even in the setting of advanced HIV treatment, complications of infection continue. In countries where there is limited access to medicine, the mortality and infection rate has grown substantially, contributing to abbreviated life expectancy. Weight loss and other nutritional factors have been and continue to be problems for HIV-infected patients. Malnutrition is a common complication of HIV infection and is an independent predictor of morbidity and mortality [1]. Compromised nutritional status continues to exist as well as a newly described metabolic syndrome called lipodystrophy or fat redistribution syndrome.\n\nThis chapter summarizes the available data on nutrition and AIDS including the wasting syndrome, HIV-related weight loss, and the lipodystrophy syndrome. A significant portion of this chapter will be devoted to addressing the clinical importance of assessment as well as relevant data on intervention strategies for HIV-related weight loss. Because the metabolic abnormalities and associated body shape changes are often clinically disturbing to the patient and also the providers, we will discuss the role of assessment and possible intervention strategies for this syndrome.\n\n## II. HIV-ASSOCIATED WEIGHT LOSS\n\nThe wasting syndrome is defined as progressive involuntary weight loss of >10% of baseline body weight in the setting of a chronic infection and\/or chronic diarrhea, and it has distinct features. It is classically associated with advanced and possibly untreated HIV disease [2]. HIV-associated weight and muscle loss was noted early in the AIDS epidemic. In fact, HIV infection or AIDS is known in Africa as \"slim disease\" [3]. In Africa and other developing countries, wasting is due both to HIV infection opportunistic infections, such as gastrointestinal infections, and tuberculosis. Before the antiretroviral therapy era, opportunistic infections also had a clinical impact on weight loss, because fever, anorexia, and malabsorption accelerated the rate of weight loss in the developed world as well. Even in the active antiretroviral therapy era, the etiology of continued weight loss is not so clear, in spite of maximal viral suppression.\n\nIn the past, wasting syndrome, determined by physical examination, presented as loss of body weight and lean body mass. Today, there are validated techniques to measure more accurately body composition of HIV-infected patients. Although the wasting syndrome may appear obvious upon physical examination, further definition of the overall complexity and nature of HIV-associated weight loss is possible.\n\nThe Center for Disease Control (CDC) definition of wasting syndrome as an AIDS-defining illness is: \"Involuntary weight loss of >10% of baseline body weight plus either chronic diarrhea defined as at least one stool per day for >30 days. Or chronic weakness and fever for >30 days in the absence of a condition other than HIV infection that could explain the findings (e.g., tuberculosis, cancer, or microsporidiosis)\" [2].\n\nNahlen and colleagues [4] studied the prevalence of wasting syndrome between 1987 and 1991 for the CDC. They reported a 17.8% wasting syndrome in the 16,773 females and 130,852 males studied [4]. This study examined the prevalence data in a cohort of cases reported to the CDC. Despite issues raised of ethnic or racial bias of case reporting, the highest percentage of wasting syndrome was seen in Hispanics, followed by African Americans, Caucasians, and Asians\/others. A report [5] on a longitudinal study in Puerto Rico, during a similar time period, demonstrated that HIV wasting syndrome accounted for 9.7% of 1520 cases between May 1992 to December 1996. This is comparable to the percentage of Caucasians with AIDS-defining wasting syndrome from the CDC report.\n\nAmong other analyses from the CDC data set include gender differences in AIDS-defining wasting syndrome. These data reported a higher incidence of AIDS wasting in females (10.2%) compared to males (6.7%) [4]. Thus, the conclusion of this report stated AIDS wasting to be significant in both genders, but more common in women.\n\nTo further assess the incidence of wasting syndrome prior to the highly active antiretroviral therapy era, a study was conducted in a cohort of homosexual males. The multicenter AIDS cohort study [6], established to assess the progression of HIV infection to AIDS, also led to an accumulation of data available to determine the incidence of weight loss in this cohort. A longitudinal, prospective study that drew from populations in the Baltimore, Washington (DC), Chicago, Pittsburgh, and Los Angeles areas was included. Weight loss occurred during 6 months before recognized seroconversion and up to 18 months prior to the onset of AIDS. The data analysis determined that weight loss was an early predictor of progression to AIDS in this cohort [6].\n\nWeight loss associated with HIV infection is multifactorial and not completely understood. It is recognized that wasting, particularly of lean muscle mass, is an independent predictor of death [1]. In addition to the devastation of morbidity and mortality due to wasting syndrome, many have described other related consequences of weight loss including increased progression of disease, decline in function and strength, and loss of muscle protein [7\u201311]. Despite the potency of antiretroviral therapy today and a noted decline in secondary infections, HIV-related weight loss continues to occur across all categories [12].\n\nWanke and colleagues [12] assessed the prevalence of wasting in a large cohort of HIV-positive subjects treated with active antiretroviral therapy and found that 33.6% of all participants met the study definition of wasting. They defined the wasting syndrome as loss of >10% body weight since diagnosis, loss of >5% of body weight in the past 6 months, and sustained body mass index of <20 kg\/m2 since the previous 6-month visit. These data suggest that wasting in the era of active antiretroviral therapy remains a problem and cannot be ignored even when patients are on potent therapy.\n\nCriticism of the CDC definition for wasting includes a lack of specification of \"baseline body weight,\" a time frame for weight loss, and lack of criteria for body composition. Defining wasting as a sustained body weight loss of 5% over 6 months serves to represent nutritional risk better than 10% body weight loss from baseline or preinfection body weight.\n\n## III. HIV-ASSOCIATED LIPODYSTROPHY SYNDROME\n\nA newly identified nutritional-related complication has been observed in HIV-infected patients. It is referred to by many pseudonyms such as \"lipodystrophy syndrome,\" \"fat redistribution syndrome,\" \"peripheral lipodystrophy syndrome,\" \"crix-belly,\" and \"protease paunch.\" The etiology of this complication (similar to wasting) is unclear and does not have a generally accepted definition. This syndrome will be referred to as _lipodystrophy_ in the context of this chapter.\n\nLipodystrophy is associated with body shape changes and metabolic abnormalities, but should not to be equated with classic lipodystrophy. The most common change in body shape is enlarged truncal girth resulting from a deposit of adipose tissue in the visceral area. In some cases, patients develop a dorsocervical fat pad or \"buffalo hump\" on the posterior neck. Alternatively, this syndrome may be limited to or include peripheral fat atrophy of the extremities, buttocks, and face. It is common to observe increasing venous markings in the periphery due to the frank loss of fatty tissue. Initially, these changes in body shape were thought to be associated with protease inhibitor therapy. More careful observation noted that these changes occur with or without protease inhibitor therapy [13\u201318].\n\nMetabolic complications have also been documented in the lipodystrophy syndrome. These include, but are not limited to hyperlipidemia, insulin resistance, and, rarely, hyperglycemia [15, 19]. The lipodystrophy syndrome will be discussed in more detail as part of the assessment and intervention strategies of this chapter.\n\n## IV. CAUSES OF WEIGHT LOSS IN HIV\/AIDS\n\nAs suggested above, weight loss due to HIV infection is complex, multifaceted, and not completely understood. The associated causes of weight loss include inadequate intake induced by loss of appetite, malabsorption caused by intestinal dysfunction or diarrheal illness, metabolic abnormalities related to cytokines or the HIV infection itself, or hormonal imbalances. Each of these components may be present independent of one another or in combination.\n\nMalnutrition in HIV infection may be classified as starvation or cachexia. Starvation is simply a lack of food, such as is seen in developing countries or in poverty-stricken areas of developed countries. It may occur either voluntarily or involuntarily and be reversed by providing sufficient energy, electrolytes, and water. Long-term starvation often leads to a physiological response noted by a decrease in energy expenditure, preservation of glucose and protein metabolism, and an increase in lipid oxidation. In comparison, cachexia is defined as a depletion or breakdown of lean body mass. This is commonly associated with an acute illness or an infection, because the body expends protein and glucose substrate in response to an acute insult. This metabolic alteration is significant and life sustaining in the short term, but in the long term will result in nitrogen and protein depletion. Starvation can be corrected by refeeding, but for the cachectic patient, alterations in carbohydrate and protein metabolism have to be closely monitored.\n\n### A. Anorexia and Oral Intake\n\nAnorexia is a common manifestation in HIV infection and AIDS. Two studies have documented that negative energy balance is the primary determinant of HIV-associated weight loss [20, 21]. The causes of inadequate intake include a variety of metabolic, gastrointestinal, and neurologic symptoms. HIV-induced proinflammatory cytokines may contribute to anorexia as does HIV infection itself. The majority of antiviral medications for HIV have anorexigenic effects. Many patients are taking up to 30 or more pills daily in an attempt to control the virus and related infections. Side effects from these agents commonly cause nausea, vomiting, or diarrhea. Other barriers to adequate intake may be due to taste changes, difficulty swallowing, malignancies (Kaposi's sarcoma), opportunistic infections, such as oral thrush, cytomegalovirus, herpes, and apthous ulcers. Dental infections may also contribute to inadequate intake and subsequent weight loss.\n\nPsychological issues as well as mood-altering medications may cause changes in appetite and often contribute to a disinterest in food. \"Battle fatigue\" is a common term associated with HIV-infected individuals who have been fighting this disease for many years. They often complain of overall exhaustion for a variety of reasons. Some may be preparing to give up the fight. This in itself can be both a physical and a psychological reason for appetite loss. Economic, social, and cultural factors may limit adequate nutritional intake in some patients.\n\n### B. Diarrhea and Malabsorption in HIV and AIDS\n\nChronic diarrhea and malabsorption of nutrients is common in these patients. In the United States, 60% of all HIV-infected patients experience diarrhea at some point in the course of their disease. The incidence of diarrhea is 100% of HIV-infected patients in developing countries [22]. A population survey reported chronic diarrhea in 3\u201324% of patients and correlated with reduced quality of life. In the same survey, when patients were further questioned, the prevalence of diarrhea reported increased to 38% [23]. These data suggest that the prevalence of diarrhea is likely to be higher than expected due to interpretation of questionnaires and definitions of diarrhea. Weight loss due to malabsorption of nutrients is more common in patients who experience small bowel diarrhea, where most nutrient absorption occurs, than large bowel diarrhea. Symptoms of small bowel diarrhea include frequent, high-volume stools often accompanied by discomfort in the abdomen, dehydration, and weight loss. Large bowel diarrhea is commonly associated with urgency or tenesmus. Dehydration is common with both types of diarrhea and leads to skin breakdown.\n\nPathogens most likely found in HIV-infected patients include the bacteria _Salmonella, Campylobacter, Shigella, Mycobacterium avium,_ and _Clostridium difficile;_ the protozoa _Cryptosporidia, Isospora belli,_ and _microsporidium;_ and cytomegalovirus, adenovirus, and herpes simplex virus. Entero-aggregative _Escherichia coli_ has been described in HIV infection and may be the cause of chronic diarrhea [24].\n\nAfter a complete workup of potential causes for diarrhea in HIV-infected patients, 10\u201320% of cases have no identifiable pathogen. In the setting of idiopathic diarrhea, other probable causes may include chronic inflammation of the small intestinal mucosa, atrophy of the villi, or pancreatic insufficiency. Primary HIV infection of the enterocytes adhering to the small intestine or invading the gut-associated lymphoid tissue has been postulated to be causal but this remains unclear. Other possible explanations for persistent diarrhea and malabsorption include the presence of unidentified pathogens, motility issues, autonomic dysfunction, colonic inflammation, bacterial overgrowth, medications, dietary habits, and oral nutrition supplements [25, 26].\n\n### C. Metabolic Abnormalities\n\nWeight loss due to metabolic abnormalities or alterations has been described in HIV\/AIDS patients. It has been documented that total energy expenditure and resting energy expenditure increase in HIV infection; however, data do not agree whether weight loss is due to increased energy expenditure or associated with decreased energy intake [20, 21]. Macallan and colleagues [20] assessed resting energy expenditure, total energy expenditure, and energy intake in 27 men with HIV infection. The results indicated no difference in total energy expenditure as compared to HIV-negative controls. However, there was a positive association between the rate of weight loss and decreased total energy expenditure in all subjects. These changes were correlated with a reduction in physical activity and energy intake. Conclusions from this study indicate that total energy expenditure is reduced during episodes of weight loss and the primary determinant is due to a deficit in energy intake and reduced activity level.\n\nGrunfeld and colleagues [21] reported an increase in resting energy expenditure in HIV-positive subjects at varying stages of disease. Resting energy expenditure was increased in 11% of HIV-positive subjects, in 25% with CDC-defined AIDS, and in 29% of AIDS subjects with secondary infections. Kotler and colleagues [27] reported a reduction in resting energy expenditure, predicted by the Harris-Benedict equation, in clinically stable, HIV-infected men compared to controls. It is clear from these studies that energy expenditure appears to be altered by HIV disease and therefore energy requirements need to be carefully determined for medical nutrition therapy.\n\nAn alteration in intermediary metabolism has been described in HIV\/AIDS patients, but the mechanism remains unclear. An increase in plasma triglyceride concentration and acceleration of protein turnover has been documented in AIDS wasting patients with weight loss [28, 29]. Furthermore, carbohydrate oxidation is suppressed and insulin resistance is increased resulting in an increase in fat storage rather than lean tissue [30]. Another explanation derived to understand HIV-related weight loss is the role that cytokine production may play. The production of tumor necrosis factor (TNF), interferon \u03b1 (IFN-\u03b1), interleukin 1 (IL-1), interleukin 6 (IL-6), and possibly other cytokines may be partly responsible for anorexia and wasting as part of an inflammatory response to infection [31]. Elevated plasma triglyceride and cholesterol concentration, insulin resistance, and, in some cases, the development of diabetes have been associated with HIV infection, specifically in the extended life span associated with the active antiretroviral therapy era. These metabolic abnormalities are often associated with the lipodystrophy syndrome. Similar to the derangement in metabolism described in HIV-related weight loss, the phenomenon described in HIV-associated lipodystrophy is poorly understood. Intervention strategies for this myriad of syndromes will be examined later in this chapter.\n\n## V. HORMONAL IMBALANCES\n\nThe most common endocrine abnormality in men with HIV infection is testosterone deficiency, which results in hypogonadism [32]. Symptoms include low sex drive, weight loss, and fatigue. Testosterone, a major anabolic hormone in men, occurs in low levels in women. In men, chronically low testosterone levels may lead to reduced anabolism and poor lean tissue preservation. Low testosterone may be seen in patients who experience an acute insult or acute infection followed by weight loss. Other reported endocrine manifestations associated with HIV infection include the thyroid, pituitary, and adrenal glands. Adrenal insufficiency, growth hormone abnormalities, and low thyroid function have been associated with HIV infection [33]. Because wasting syndrome is poorly understood, so are the endocrine abnormalities.\n\n## VI. NUTRITIONAL ASSESSMENT\n\nAn assessment of nutritional status in HIV\/AIDS is similar to that in other disease states. The information gathered in a nutritional assessment should provide appropriate and relevant data toward the development of intervention and monitoring strategies of individual patients. The goal of this section is to suggest assessment parameters that are critical in the evaluation of nutritional status in HIV infection in both general terms and specific concerns for those experiencing lipodystrophy syndrome. In addition, the American Dietetic Association has prepared a useful clinical guide and a position statement for professionals working in HIV [34, 35]. The guidelines state the minimum nutrition services that should be provided to children, women, and men infected with HIV.\n\nA standard nutritional evaluation of HIV-infected adults should include the following parameters: body weight, ideal and usual body weight, body mass index, and biochemical indices. Clinical research studies are evaluating the use of bioelectrical impedance analysis as a supplement to anthropometry for body composition analysis. Appetite status, dietary intake, and gastrointestinal symptoms are also included. Other relevant clinical data may include duration of HIV infection, age, sex, medication and medication history, medical history, socioeconomic status (including access to food), and psychosocial history.\n\nFor patients with the lipodystrophy syndrome, body shape changes result in an evaluation that differs from a standard nutrition assessment of HIV-infected adults. Assessment for patients with lypodystrophy includes waist and hip circumference, waist:hip ratio, midarm circumference, midthigh circumference, and skinfold thickness over the triceps, suprailiac, subscapular, and thigh. If applicable, photographic evaluation of body habitus, including the dorsocervical fat pad and facial wasting if present, may be used to monitor body shape changes over time and impact of clinical interventions. In research settings, visceral adipose tissue evaluation may be achieved through the use of computed tomography imaging or dual-energy x-ray absorptiometry calibrated for body composition, not bone density. Other assessment parameters to evaluate lipodystrophy may include plasma lipid and glucose concentrations, oral glucose tolerance test, insulin sensitivity studies, and careful clinical examination of arms, legs, and face to identify venous markings due to fat atrophy.\n\n### A. Body Weight Assessment\n\nAlthough body weight is documented in most routine assessments, in patients with HIV\/AIDS, body composition is a better predictor of nutritional and health status than body weight. Although body weight can serve as a convenient and simple predictor of general nutritional status, it is often subject to errors in interpretation due to fluid retention associated with cardiac, renal, and hepatic diseases, or dehydration from diarrhea or chronic illness. Nonetheless, body weight is useful to document so trends can be examined over time. Obtain a weight history prior to HIV infection, during HIV infection, and recently. These questions may be a useful way to characterize the complete weight history of an individual. From the information provided, the clinician can establish baseline data and determine therapeutic goals.\n\n#### 1. IDEAL AND USUAL BODY WEIGHT FOR HEIGHT\n\nEstimating usual and ideal body weight is another key component in the nutrition assessment. This allows determination of percent weight change from actual weight to usual body weight. Estimation of ideal body weight can be done by consulting the weight\u2013height reference chart established from Metropolitan Life Insurance Company actuarial data [36]. The Metropolitan height\u2013weight tables do not offer ideal body weights of individuals from ethnic and low socioeconomic background because the data are derived from healthy men and women who were offered life insurance in the late 1950s. However, this does present a point of reference for an estimated ideal body weight for individuals so is often used in clinical settings. A practical approach to estimate ideal body weight for height involves selecting the midpoint of the medium frame and determine an ideal body weight with a range of \u00b1 5%.\n\nOnce this information is established, develop an understanding of the patient's perception of his or her own ideal body weight. Discrepancy between the provider and the patient may occur if there is not a clear understanding of patient needs.\n\n#### 2. BODY MASS INDEX\n\nBody mass index (BMI) is a ratio of weight to height used to assess degree of fatness or adiposity of an individual [37]. The measurement is obtained from the calculation of weight in kilograms divided by height in meters squared. BMI is an assessment tool used to estimate degree of overweight or obesity. In the general population, a BMI of 30 kg\/m2 or above indicates obesity. A BMI below 19 kg\/m2 indicates a risk of malnutrition. However, BMI is only one component of nutrition assessment and, like body weight, it should not be the only data used to assess nutritional status especially in diseases like AIDS, which can greatly alter body composition.\n\n### B. Body Composition Assessment\n\nBody composition provides information on amounts of various body tissues beyond total weight. In this section, we will discuss the techniques that are useful in the clinical assessment of body composition in HIV\/AIDS and review the relevance of the specific body compartments.\n\n#### 1. BIOELECTRIAL IMPEDANCE ANALYSIS\n\nBioelectrical impedance analysis is a method used to estimate body compartments through a mechanism of resistance and reactance. The compartments measured include body cell mass, fat mass, extracellular tissue, and fat-free mass. The fact that the analyzer is portable and the measurement quick and without discomfort makes bioelectrical impedance analysis popular in the clinical setting. The analyzer detects resistance and reactance and, at high frequencies, total body water is the conducting medium. At low frequencies, the membrane lipid content of the body cell mass compartment acts as a capacitor, which limits the flow to intracellular space only. The calculations estimate different body compartments [38]. Accuracy of the measurements depends on adequate hydration. Estimates of lean body mass are improved when age, sex, and body weight are included in the calculation. In addition to these compartments, estimates of total body water along with extracellular and intra-cellular water are obtained.\n\nThe importance of body composition analysis in HIV\/AIDS is to estimate the amount and percentage of body cell mass present in these patients. As we described earlier, the prevalence of wasting (loss of body cell mass) persists despite improved treatment regimens. Thus, early detection and monitoring of body cell mass is critical, because it is the compartment of body tissue that is most representative of functional status. This compartment accounts for intracellular water, red blood cells, and organ and skeletal tissue, and is the site of all metabolic activity [39]. Ideal body cell mass in men is estimated as 40\u201345% of ideal body weight for height. For women, the ideal body cell mass is estimated at 30\u201335% of ideal body weight for height.\n\nOther compartments of clinical significance include fat mass, extracellular tissue, and fat-free mass. Fat mass is the compartment of tissue responsible for stored energy or fuel and insulation for temperature regulation of organs. Depletion of fat stores is commonly seen during starvation or insufficient energy intake. During starvation, the body becomes efficient at sparing proteins and lean tissue; however, in the setting of HIV infection, the opposite occurs. Fat stores remain as depletion of body cell mass occurs in HIV infection. Ideal fat mass for men is estimated to be between 10\u201320% of actual body weight. Ideally, 16\u201319% is a more suitable fat mass for HIV\/AIDS infection. Ideal fat mass for women is estimated at 20\u201330% of actual body weight.\n\nExtracellular tissue is the transport and structure compartment that includes bone, collagen, cartilage, and other tissues. Assessment of extracellular tissue can provide information pertaining to hydration status. Low extracellular tissue indicates that dehydration may be present. Normal or euhydration status will return extracellular tissue to normal. Edema will often cause extracellular tissue to appear abnormally high. This fluid shift occurs to provide more transport fluid to the \"stressed\" area. When extracellular tissue continues to increase, this indicates poor response to nutritional repletion. This may also identify a candidate for anabolic therapy in the setting of advanced weight loss. Ideal extracellular tissue should be the same as estimated body cell mass requirements, 40\u201345% of ideal body weight for height should contain extracellular tissue in men, and the estimated extracellular tissue for women is 30\u201335% of ideal body weight for height.\n\nFat-free mass is total body weight minus fat mass. This compartment contains extracellular tissue and body cell mass and therefore does not represent ideal functional tissue. Anthropometric assessment using skinfold thickness measurements will yield a two-compartment model containing fat-free mass and fat mass. In a research setting, common skinfold thickness sites measured include fat tissue over the triceps, subscapular, suprailiac, and thigh, performed in triplicate. These measurements are use in calculations of fat-free mass and fat mass percentages [40].\n\n### C. Nutrition Assessment in Lipodystrophy Syndrome\n\nThe lipodystrophy syndrome, as described earlier, is associated with metabolic abnormalities including elevated plasma lipid levels, glucose intolerance, insulin resistance, and changes in body shape. Because clinical focus has shifted from the wasting syndrome to the lipodystrophy syndrome, it is as important to obtain data relevant to body shape and body image as it is to obtain body weight. Nutritional assessment of lipodystrophy requires additional information, compared to the standard nutritional assessment for HIV\/AIDS, but how to characterize these changes is not yet defined.\n\nFat accumulation and fat atrophy are the two body shape changes seen with lipodystrophy syndrome. Although it is important to define usual and ideal body weight in lipodystrophy syndrome, it is especially useful to examine body deposition of fat and\/or areas of fat atrophy. The waist-to-hip measurement ratio provides an important indicator of central obesity. The standard waist measurement should be performed directly under the lowest rib or usually at the narrowest point around the waist. The standard hip measurement is directed at the maximum extension point around the buttocks. Normality is defined in men as waist circumference <88.2 cm and waist-to-hip ratio <0.95, and in women, a waist circumference <75.3 cm and waist-to-hip ratio \u22650.9 for decreased risk of cardiovascular disease [41]. Note that although there are standard methods to measure waist and hip, the distribution of body fat seen in these patients might affect the interpretation of these values.\n\nMeasuring body composition in lipodystrophy syndrome is complicated by the abnormal distribution of body fat. In research settings, magnetic resonance imaging and computed tomography scan can also be used to define the extent of visceral abdominal fat. Dual-energy x-ray absorptiometry (DXA) can be calibrated to determine regional body fat as well. Their high cost and limited accessibility makes these approaches less likely to be used in routine clinical care. Total body magnetic resonance imaging may be the most ideal and accurate method to quantify fat distribution in lipodystrophy. Increased visceral fat on magnetic resonance imaging has been correlated to waist circumference in lipodystrophy; therefore, measuring waist and hip circumferences seems a reliable, valid, and practical method of assessment, although some HIV-infected individuals may also have increased subcutaneous fat [42].\n\nFat accumulation may be associated with insulin resistance, lipid abnormalities, and increased cardiac risk in patients with this syndrome [43]. Computed tomography imaging is a common research method of evaluating this area of prominent fat accumulation seen in this syndrome. Serial measurements of DXA, magnetic resonance imaging, or computed tomography scans will allow for comparison of longitudinal data over time.\n\n### D. Biochemical Indices of Nutrition Assessment\n\nRoutine biochemical parameters in HIV\/AIDS nutrition assessment include the serum albumin concentrations, fasting cholesterol concentrations, fasting triglyceride concentrations, electrolyte concentrations, and complete blood count. Symptoms of fatigue, weight loss, and low sexual drive may be suggestive of hypogonadism. As discussed earlier, the role of testosterone status in HIV-associated weight loss suggests that the evaluation of testosterone levels may be important in HIV\/AIDS and should include total and free testosterone, specifically in the presence of the described symptoms.\n\nChronic diarrhea or loose stools is another common complication in HIV\/AIDS and suggests an evaluation of mal-absorption and micronutrient levels when diarrhea persists in the absence of negative stool cultures or other potential gastrointestinal illnesses. The following tests may be recommended: serum zinc and magnesium concentrations, vitamin B12 concentrations, D-xylose, 24\u201372 hour fecal fat test, and folate concentrations. Evaluation of gastrointestinal symptoms is essential and should be addressed with a gastrointestinal specialist. Recommendations of gastrointestinal assessment parameters include stool number and consistency; severity of abdominal bloating, cramping, and pain; routine stool examination for opportunistic pathogens; and upper and lower endoscopy and biopsy (if necessary).\n\nFor the lipodystrophy syndrome, evaluation of biochemical metabolic parameters may include fasting blood glucose concentration, lipoprotein profile, oral glucose tolerance test, and serum insulin concentrations.\n\nHIV monitoring should involve a specialist in infectious diseases or a clinician with experience in the treatment of HIV infection. Common immune or HIV-related parameters are absolute CD4 count and percentage, absolute CD8 count and percentage, and viral load or the amount of virus present in blood.\n\n### E. Appetite Status and Assessment of Oral Intake\n\nOf primary importance in the nutritional management of HIV\/AIDS is the assessment of appetite status and nutrient intake. Earlier in this chapter many potential causes of anorexia in HIV\/AIDS were described. Medications, opportunistic infections, and depression are among a few of the possible causes of anorexia in HIV infection. The consequences of anorexia often result in low energy intake and subsequent weight loss. In the clinical setting, the three most common methods to evaluate estimated oral intake include 24-hour recall, food frequency questionnaire, and 3-day food diary 44]. Each method has its advantages and limitations. [Chapter 1 of this text discusses these methods in detail.\n\n## VII. NUTRITIONAL RECOMMENDATIONS\n\nMany pharmacological agents are available to enhance or optimize the nutritional health of individuals infected with HIV infection. These include appetite stimulants, anabolic analogues, and anticytokine therapies. A majority of the evidence suggests that these therapies may play a role in the improvement of nutritional status, but a detailed review is beyond the scope of this chapter. The goal of this section is to address the specific nutritional needs of HIV\/AIDS individuals and provide recommendations for intervention.\n\n### A. Recommended Energy Needs\n\nStudies of resting energy expenditure in patients with HIV infection were described above and these data suggest that resting energy expenditure increases 10\u201325% due to the progression of disease or, specifically, an acquired opportunistic infection. [20\u201323]. Therefore, it is reasonable to recommend an increase is estimated energy requirement by 10\u201315%. If an HIV-infected individual is weight stable without weight loss, a 10% increase from the estimated basal energy expenditure needs is recommended. However, if weight loss is evident, estimated energy needs should be adjusted to 15% above the basal energy expenditure requirements. The estimated energy calculation is based on the Harris-Benedict equation for energy needs plus an additional 10\u201315% for HIV infection and an activity factor.\n\nA simple guideline to assist patients with achieving energy needs is to encourage the intake of six small meals or three adequate meals and two snacks daily. If appropriate, an energy-dense, high-protein oral liquid supplement may be needed.\n\n### B. Recommended Protein Needs\n\nMuch attention has been given to protein status and the maintenance of the immune system [45]. Low serum albumin is an indicator of worsening nutritional status and is seen with disease progression. The Recommended Dietary Allowance (RDA) for protein is 0.8\u20131.0 g\/kg body weight for a healthy person [46]. However, based on data in HIV\/AIDS, protein requirements are estimated to be 1.2\u20132.0 g\/kg body weight [47, 48]. For example, in the 70-kg male, the estimated protein needs would be 84\u2013140 g\/day. A general daily approximation of estimated protein needs is 100\u2013150 g\/day for men and 80\u2013100 g\/day for women. To maintain the optimal protein intake, protein supplementation may be considered.\n\n### C. Vitamin and Mineral Recommendations\n\nAltered nutritional status may influence the course of HIV disease progression and survival [49, 50]. Micronutrient deficiencies, widespread among various HIV-infected cohorts, cause disruption in immune function, disease progression, viral expression, and related morbidity [51, 52]. Decreased plasma concentrations of zinc, selenium, and vitamins B6, B12, A, and E are evident and appear to be functionally relevant in maintaining the integrity of immune responses. Baum and colleagues [53] reported a low concentration of vitamins A and E in the plasma of homosexual males with HIV infection. In the same study, 40\u201350% of HIV-infected men and women who abuse drugs also had severely decreased plasma concentrations of vitamins A and E. In a cohort of pediatric patients, perinatally exposed, aged 15\u201318 months, a high proportion had zinc and selenium deficiency [54].\n\nSelenium deficiency has been demonstrated to be a significant predictor of HIV-related mortality, independent of CD4 counts over time or antiretroviral therapy. Others have demonstrated selenium deficiency to be predictive of HIV-related prognosis, immune dysfunction, and decreased survival [55, 56]. This antioxidant at the cellular level is an integral part of the enzyme glutathione peroxidase system, which increases the oxygen consumption of cells and protects membranes against oxidative stress. Selenium status may have an important role in slowing the progression of HIV disease and oxidative stress in HIV disease may be an important mechanistic factor [57]. Recent investigations indicate that supplementation of selenium at doses of 200 mcg\/day may help to increase the enzymatic defense systems in patients with HIV infection [58].\n\nRecent studies have also suggested that vitamin E, another antioxidant nutrient, may be able to increase the CD4-to-CD8 cell ratio and increase lymphocyte counts while reducing levels of suppressor cells [59]. A study evaluating vitamin E status in HIV-infected patients showed that those patients who had the highest plasma concentrations of vitamin E had a 34% decrease in risk progression to AIDS compared to the lowest quartile [60]. In a review of the immune effects of vitamin E, Allard and colleagues [61] examined the effects of vitamin E supplementation (800 IU) in HIV-infected patients. They showed an increase in plasma alpha-tocopherol concentration ( _p_ < 0.0005), a reduction in lipid peroxidation ( _p_ < 0.025), and also a reduction in plasma HIV viral load ( _p_ < 0.1) compared to placebo.\n\nBased on these observations of micronutrient status and possible physiological role in HIV\/AIDS, supplementation with vitamins C, E, beta-carotene, and selenium may be warranted.\n\n### D. Glutamine and Cysteine Status\n\nGlutamine has been noted to have an effect on enhancing lean body mass in HIV infected patients as well as supporting the integrity of the gut mucosa [62\u201368]. It has been shown that HIV-infected individuals may have abnormal levels of the amino acids cysteine and glutathione, complicating the body's ability to maintain lean body mass [63]. Supplementation with N-acetyl-cysteine has been postulated as a modality to enhance glutathione levels in these patients [64]. Glutathione plays an important role in the maintenance of cellular function and immunological reactivity [62]. Recognizing the importance of maintaining cellular and skeletal lean body mass, supplementation with cysteine and glutamine may be recommended.\n\n### E. Nutritional Recommendations for the Lipodystrophy Syndrome\n\nBecause there is no clear definition of the lipodystrophy syndrome, there are also no specific dietary considerations established. For HIV-infected patients with the lipodystrophy syndrome, weight gain is not an issue, rather the distribution of body weight and body fat is the concern. Weight loss is typically not the goal for patients in this situation. A recommendation to promote normal blood lipid levels is a standard clinical approach. Because these patients have insulin resistance, a dyslipidemia develops when low-fat, high-carbohydrate diets are followed. Like patients with type 2 diabetes (see Chapter 29), plasma lipid normalization results from diets containing moderate amounts of unsaturated fatty acids and moderate in total carbohydrate.\n\n## VIII. NUTRITIONAL COUNSELING\n\nData are limited on the effectiveness of nutrition counseling and outcome of enhanced oral intake and body weight. A recent study compared nutritional counseling alone versus nutritional counseling and enteral supplements for 6 weeks in malnourished patients with HIV infection. Half of the patients in each treatment group achieved at least 80% of their target energy intake and the supplement group had larger increases in fat-free mass and grip strength [69]. An earlier study, conducted before the availability of active antiretroviral therapy, concluded that malnourished HIV-infected patients who received oral supplementation and nutritional counseling for 6 weeks achieved a mean weight gain of 1.1 kg [70]. These data are useful and demonstrate that nutrition counseling may assist patients with increasing energy and protein intakes; however, more outcome data are needed.\n\nTo standardize care for HIV\/AIDS patients and foster outcome data collection, a nutrition therapy guideline has been developed [34]. The protocol, designed for HIV-infected adults, children, and adolescents, provides details for screening and nutrition therapy delivered with expected outcomes. Major goals are to optimize nutrition status, prevent the development of nutrient deficiencies, prevent loss of weight and lean body mass, maximize the effectiveness of medical and pharmacological treatments, and minimize health care costs [34].\n\n## IX. SUMMARY\n\nNutrition complications in HIV\/AIDS include HIV-associated weight loss, the wasting syndrome, and the lipodystrophy syndrome. Metabolic abnormalities may include dyslipidemia, insulin resistance, and in some cases type 2 diabetes mellitus. The lipodystrophy syndrome may cause body shape changes including increased truncal fat, a dorsocervical fat pad, thinning of the extremities, and loss of facial fat. Adequate energy and protein intake is necessary to maintain adipose reserve and lean body cell mass. Specific micronutrients of particular importance for HIV\/AIDS patients are antioxidant nutrients because requirements may be increased due to oxidative stress. Nutritional therapy is an important component of the overall health care of patients who live with HIV\/AIDS.\n\nReferences\n\n1. Kotler D.P., Tierney A.R., Wang J., Pierson R.N., Jr. The magnitude of body cell mass depletion determines the timing of death from wasting in AIDS. _Am. J. Clin. 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Association between serum vitamin A and E levels and HIV-1 disease progression. _AIDS_. 1997;11:613\u2013620.\n\n61. Allard J.P., Aghclassi E., Chau J., Tam C., Kovacs C., Salit I., Walmsley S. Effects of vitamin E and C supplementation on oxidative stress and viral load in HIV-infected subjects. _AIDS_. 1998;12:1653\u20131659.\n\n62. Beutler E. Nutritional and metabolic aspects of glutathione. _Annu. Rev. Nutr_. 1989;9:287\u2013302.\n\n63. Droge W., Eck H.P., Haher H., Pekar U., Daniel V. Abnormal amino acid concentrations in the blood of patients with acquired immunodeficiency syndrome (AIDS) may contribute to the immunological defect. _Biol. Chem. Hoppe-Seyler_. 1988;369:143\u2013148.\n\n64. Droge W., Breitkreutz R. N-Acetyl-cysteine in the therapy of HIV-positive patients. _Curr. Opin. Clin. Nutr. Metabol. Care_. 1999;2:493\u2013498.\n\n65. Shabert J.K., Wilmore D.W. Glutamine deficiency as a cause of human immunodeficiency virus wasting. _Med. Hypoth_. 1996;46:252\u2013256.\n\n66. Shabert J., Winslow C., Lacey J., Wilmore D. Glutamine-antioxident supplementation increases body cell mass in AIDS patients with weight loss: A randomized doubleblind controlled trial. _Nutrition_. 1999;15:860\u2013864.\n\n67. Clark R.H., Feleke G., Din M., Yasmin T., Singh G., Khan F., Rathmacher J. Nutritional treatment for acquired immunodeficiency virus-associated wasting using \u03b2-hydroxy, \u03b2-methylbutyrate, glutamine, and arginine: A randomized, double-blind, placebo-controlled study. _J. Parenteral Enteral Nutr_. 2000;24:133\u2013139.\n\n68. Souba W.W., Klimberg V.S., Plumley D.A., Salloum R., Flynn T., Bland K., Copeland E. The role of glutamine in maintaining a healthy gut and supporting the metabolic response to injury and infection. _J. Surg. Res_. 1990;48:383\u2013391.\n\n69. Rabeneck L., Palmer A., Knowles J.B., Seidehamel R.J., Harris C.L., Merkel K.L., Risser J., Akrabawi S. A randomized controlled trial evaluating nutrition counseling with or without oral supplementation in malnourished HIV-infected patients. _J. Amer. Diet. Assoc_. 1998;98:434\u2013438.\n\n70. Stack J.A., Bell S.J., Burke P.A., Forse R.A. Use of supplements in patients with human immunodeficiency virus infection. _J. Amer. Diet. Assoc_. 1996;96:337\u2013341.\nG.\n\nOverall Disease Prevention\nCHAPTER 48\n\nNutrition Guidelines to Maintain Health\n\nSUZANNE P. MURPHY, University of Hawaii, Honolulu, Hawaii\n\n## I. INTRODUCTION\n\nNutrition guidelines for Americans fall into two broad categories: those that focus primarily on nutrient intakes and those that are primarily oriented to food choices. In addition, clinicians and researchers have come to realize that healthy food and nutrient intakes are inevitably linked to healthy activity levels, so that physical activity guidance is often included with dietary guidance.\n\nThe Food and Nutrition Board of the National Academy of Sciences has periodically released recommendations for nutrient intakes by Americans. The last complete set of recommendations was published in 1989 [1], but these Recommended Dietary Allowances are now being replaced by Dietary Reference Intakes (DRIs), most recently in 2000 [2\u20134]. DRI is an umbrella term for four types of nutrient recommendations:\n\n\u2022 Estimated average requirement\n\n\u2022 Recommended Dietary Allowance\n\n\u2022 Adequate intake\n\n\u2022 Tolerable upper intake level.\n\nTo date, DRIs have been set for 17 nutrients. Until the DRI process is complete, health professionals and the public will need to use a combination of the new DRIs and the 1989 RDAs when deciding what nutrient intakes are appropriate (Tables 1\u20133).\n\nTABLE 1\n\nDietary Reference Intakes: Recommended Intakes for Individuals; Food and Nutrition Board, Institute of Medicine, National Academies\n\n_Note:_ This table presents Recommended Dietary Allowances (RDAs) in bold type and Adequate Intakes (AIs) in ordinary type followed by an asterisk (*). RDAs and AIs may both be used as goals for individual intake. RDAs are set to meet the needs of almost all (97 to 98 percent) individuals in a group. For healthy breastfed infants, the AI is the mean intake. The AI for other life-stage and gender groups is believed to cover needs of all individuals in the group, but lack of data or uncertainty in the data prevent being able to specify with confidence the percentage of individuals covered by this intake.\n\naAs cholecalciferol. 1 \u03bcg cholecalciferol = 40 IU vitamin D.\n\nbIn the absence of adequate exposure to sunlight.\n\ncAs niacin equivalents (NE). 1 mg of niacin = 60 mg of tryptophan; 0\u20136 months = preformed niacin (not NE).\n\ndAs dietary folate equivalents (DFE). 1 DEF = 1 \u03bcg food folate = 0.6 \u03bcg of folic acid from fortified food or as a supplement consumed with food = 0.5 \u03bcg of a supplement taken on an empty stomach.\n\neAlthough AIs have been set for choline, there are few data to assess whether a dietary supply of choline is needed at all stages of the life cycle, and it may be that the choline requirement can be met by endogenous synthesis at some of these stages.\n\nfAs \u03b1-tocopherol. \u03b1-Tocopherol includes _RRR_ -\u03b1-tocopherol, the only form of \u03b1-tocopherol that occurs naturally in foods, and the _2R_ -stereoisomeric forms of \u03b1-tocopherol _(RRR-, RSR-, RRS-,_ and _RSS-_ \u03b1-tocopherol) that occur in fortified foods and supplements. It does not include the _2S-_ stereoisomeric forms of \u03b1-tocopherol _(SRR-, SSR-, SRS-,_ and _SSS-_ \u03b1-tocopherol), also found in fortified foods and supplements.\n\ngBecause 10 to 30 percent of older people may malabsorb food-bound B **12,** it is advisable for those older than 50 years to meet their RDA mainly by consuming foods fortified with B **12** or a supplement containing B **12.**\n\nhIn view of evidence linking folate intake with neural tube defects in the fetus, it is recommended that all women capable of becoming pregnant consume 400 \u03bcg from supplements or fortified foods in addition to intake of food folate from a varied diet.\n\niIt is assumed that women will continue consuming 400 \u03bcg from supplements or fortified food until their pregnancy is confirmed and they enter prenatal care, which ordinarily occurs after the end of the periconceptual period\u2014the critical time for formation of the neural tube.\n\n_Source:_ Reprinted with permission from \"Dietary Reference Intakes.\" Copyright 2000 by the National Academy of Sciences. Courtesy of the National Academy Press, Washington, DC.\n\nTABLE 2\n\nRecommended Dietary Allowances _a_, Revised 1989 (Abridged), Designed for the Maintenance of Good Nutrition of Practically All Healthy People in the United States, Food and Nutrition Board, National Academy of Sciences, National Research Council\n\n_Note:_ This table does not include nutrients for which Dietary Reference Intakes have recently been established (see \"Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride\" [1997], \"Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline\" [1998], and \"Dietary Reference Intakes for Vitamin E, Vitamin C, Selenium, and Carotenoids\" [2000]).\n\naThe allowances, expressed as average daily intakes over time, are intended to provide for individual variations among most normal persons as they live in the United States under usual environmental stresses. Diets should be based on a variety of common foods in order to provide other nutrients for which human requirements have been less well defined.\n\nbWeights and heights of Reference Adults are actual medians for the U.S. population of the designated age, as reported by NHANES II. The median weights and heights of those under 19 years of age were taken from Hamill et al. (1979). The use of these figures does not imply that the height-to-weight ratios are ideal.\n\ncRetinol equivalents. 1 retinol equivalent = 1 \u03bcg retinol or 6 \u03bcg \u03b2-carotene.\n\n_Source:_ Reprinted with permission from \"Recommended Dietary Allowances,\" 10th ed. Copyright 1989 by the National Academy of Sciences. Courtesy of the National Academy Press, Washington, DC.\n\nTABLE 3\n\nDietary Reference Intakes: Tolerable Upper Intake Levels (UL _a_); Food and Nutrition Board, Institute of Medicine, National Academies\n\naUL = The maximum level of daily nutrient intake that is likely to pose no risk of adverse effects. Unless otherwise specified, the UL represents total intake from food, water, and supplements. Due to lack of suitable data, ULs could not be established for thiamin, riboflavin, vitamin B12, pantothenic acid, or biotin. In the absence of ULs, extra caution may be warranted in consuming levels above recommended intakes.\n\nbThe ULs for magnesium represent intake from a pharmacological agent only and do not include intake from food and water.\n\ncThe ULs for niacin and folate apply to synthetic forms obtained from supplements, fortified foods, or a combination of the two.\n\ndAs \u03b1-tocopherol; applies to any form of supplemental \u03b1-tocopherol.\n\neND = Not determinable due to lack of data of adverse effects in this age group and concern with regard to lack of ability to handle excess amounts. Source of intake should be from food only to prevent high levels of intake.\n\n_Source:_ Reprinted with permission from \"Dietary Reference Intakes.\" Copyright 2000 by the National Academy of Sciences. Courtesy of the National Academy Press, Washington, DC. Courtesy of the National Academy Press\n\nThree types of guidance have been developed by federal government agencies to help consumers make healthy food and nutrient choices. The Dietary Guidelines for Americans have been jointly issued by USDA and USDHHS every 5 years since 1980. The most recent were released in 2000 [5, 6]. The number of guidelines has been increased from 7 to 10 (Table 4). Two of the three new guidelines result from dividing previous guidelines: recommendations regarding fruit and vegetable intakes have been separated from those regarding grain intake; and the former guideline suggesting balancing food intake with physical activity now is divided into a guideline on healthy weight and a guideline on physical activity. An entirely new guideline on food safety reflects the increasing importance of food safety when selecting and preparing foods.\n\nTABLE 4\n\nDietary Guidelines for Americans\n\n_Source:_ U.S. Department of Agriculture and U.S. Department of Health and Human Services (2000). \"Dietary Guidelines for Americans,\" 5th ed., Home and Garden Bulletin No. 232. U.S. Government Printing Office, Washington, DC. U.S. Government Printing Office\n\nThe Food Guide Pyramid is a tool that is widely used in nutrition education efforts, and has proved to be particularly useful in helping the public make healthy food choices [7]. The Food Guide Pyramid translates the guidance offered by both the RDAs and the dietary guidelines into a graphic format that illustrates the recommended number of servings from each of six food groups (Figs. 1 and 2). Nutrition facts labels [8] and supplement facts labels [9] are now required by law, and provide consumers with useful information at the point of purchase.\n\nFIGURE 1 The Food Guide Pyramid, a guide to daily food choices. [From U.S. Department of Agriculture and U.S. Department of Health and Human Services (2000). ''Dietary Guidelines for Americans,\" 5th ed., Home and Garden Bulletin No. 232. U.S. Government Printing Office, Washington, DC.]\n\nFIGURE 2 How many servings do you need each day? [From U.S. Department of Agriculture and U.S. Department of Health and Human Services (2000). ''Dietary Guidelines for Americans,\" 5th ed., Home and Garden Bulletin No. 232. U.S. Government Printing Office, Washington, DC.]\n\nPhysical activity guidance has been offered by several organizations, but the most widely disseminated recent recommendation is that from the Centers for Disease Control and Prevention and the American College of Sports Medicine: ''Exercise moderately for 30 minutes almost every day of the week\" [10]. The new dietary guideline on physical activity states ''Be physically active every day.\" Physical activity guidance now frequently accompanies dietary guidance because health professionals recognize that maintenance of a healthy body size and sustained cardiopulmonary fitness can only be achieved through an active lifestyle coupled with healthy dietary choices.\n\n## II. GUIDELINES FOR NUTRIENT ADEQUACY AND SAFETY\n\nA. Dietary Reference Intakes for Nutrient Adequacy\n\nThe DRIs offer guidance on the level of nutrient intake that will promote health and reduce the risk of chronic disease. The process for setting the new DRIs was developed by the Food and Nutrition Board after substantial feedback from the nutrition professionals [11]. Several new concepts were incorporated:\n\n\u2022 An increased focus on reduction of chronic disease risk as well as the prevention of nutritional deficiencies.\n\n\u2022 Determination of an estimated average requirement (EAR), which would be the intake that would be adequate for approximately 50% of a healthy population.\n\n\u2022 Calculation of a Recommended Dietary Allowance (RDA) from the EAR, by adding two standard deviations of the requirement distribution. The RDA is thus the level of intake that would be adequate for 97.5% of the population.\n\n\u2022 Use of an adequate intake (AI) when the scientific database is not sufficient to set an EAR (and its associated RDA).\n\n\u2022 Determination of a tolerable upper intake level (UL), which represents the upper level of intake that poses a low-risk of adverse effects. Usual intakes above this level are not recommended.\n\nTo date, DRIs have been set for the bone-related nutrients (calcium, phosphorus, magnesium, vitamin D, and fluoride) [2], for the B vitamins (thiamin, riboflavin, niacin, vitamin B6, vitamin B12, folate, and choline) [3], and for the antioxidant nutrients (vitamin C, vitamin E, and selenium) [4]. Reports are expected by the time of publication on micronutrients (vitamins A and K, iron, zinc, copper, molybdenum, boron, etc.) and on macronutrients (energy, protein, fat, carbohydrates, and dietary fiber). As mentioned earlier, until DRIs have been set for all nutrients, health professionals will need to use a combination of the new DRIs and the former RDAs, as shown in Tables 1 through 3.\n\nBecause there are many new uses of the DRIs, a Subcommittee on Uses and Interpretation of the DRIs was convened, and will publish two reports: one on assessing intakes [12] and the other on planning intakes (expected in mid-2001).\n\nWhen offering guidance to consumers on healthy nutrient intakes, health professionals should suggest the RDA (or the AI if an RDA is not available) as the appropriate target. Because an individual's actual requirement is almost never known, the goal is to reduce to a very low level the risk that an intake is inadequate. By definition, usual intake at the level of the RDA or AI has a low-risk of inadequacy (2\u20133% for the RDA). For example, the appropriate target for magnesium intake for a woman 31\u201350 years of age is the RDA of 320 mg\/day (Table 1). Her target for calcium intake should be the AI of 1000 mg\/day.\n\nTo reflect newer information on bioavailability, the RDAs for two nutrients are in different forms than were used in the past: folate is in micrograms of dietary folate equivalents rather than total micrograms, while vitamin E is in milligrams of \u03b1-tocopherol, rather than in milligrams of \u03b1-tocopherol equivalents. For folate, the new dietary folate equivalent unit reflects an increased availability of fortification and supplemental forms of folate, and thus will tend to increase estimated intakes of this nutrient.\n\nThe situation is reversed for vitamin E. Forms of tocopherol other than \u03b1-tocopherol (such as \u03bb-tocopherol and \u03b2-tocopherol) are not considered active forms of the vitamin due to poor transport from the liver. Furthermore, the activity of the all rac-\u03b1-tocopherol form that is commonly used for fortification and in dietary supplements has a lower activity because only the 2R stereoisomers of \u03b1-tocopherol (which constitute 50% of all rac-\u03b1-tocopherol) are maintained in human plasma and thus serve as vitamin E in tissues. However, the current unit for measuring vitamin E intake is \u03b1-tocopherol equivalents, which includes all forms of tocopherol (although the activity is less for forms other than \u03b1) and all the stereoisomers of \u03b1-tocopherol. Therefore, intakes measured in \u03b1-tocopherol equivalents will overestimate intakes of the 2R stereoisomers of \u03b1-tocopherol.\n\n### B. The Tolerable Upper Intake Level: A New DRI to Reduce the Risk of Adverse Effects\n\nFor the first time, the nutrient intake recommendations from the Food and Nutrition Board also include a level of intake that should not be exceeded. The tolerable upper intake level may be used by health professionals and consumers to ensure that nutrient intakes are not too high. Table 3 shows the ULs that have been set for the first three sets of nutrients. For example, the UL for calcium is 2500 mg\/day for children and adults. It is unlikely that this level of intake could be achieved from unfortified foods alone. However, through the use of heavily fortified foods, calcium supplements and\/or antacids, intake exceeding 2500 mg\/day is possible. Intakes above the UL for calcium carry an increased risk of milk-alkalai syndrome, a potentially serious metabolic disorder.\n\nThe UL for magnesium for adults is 350 mg\/day from pharmacologic forms. Because magnesium salts can cause osmotic diarrhea, intakes above 350 mg\/day are not recommended. Food sources of magnesium do not cause diarrhea, and thus are not included in this UL. Indeed, the UL for magnesium is below the RDA for magnesium for adult men (420 mg\/day), reflecting the different forms of the nutrient in each of these DRIs. Several other nutrient DRIs also use different forms for the RDA and the UL: niacin and folate (for which the UL is from fortification or supplemental forms only), and vitamin E (where the UL is for all forms of \u03b1-tocopherol, whereas the RDA is only for the 2R stereoisomers of \u03b1-tocopherol).\n\nULs are not available for all nutrients, not because intake at any level in considered safe, but because scientific data were not sufficient to set an upper level. ULs should never be considered a target intake; the RDA and AI are the appropriate targets. In some instances, controlled trials, feeding studies, or theraputic prescriptions may utilize nutrient levels above the UL. In these cases, when medical supervision is provided, intakes above the UL may be appropriate.\n\n### C. How Do Current Nutrient Intakes Compare to the RDAs and DRIs?\n\nEvaluating nutrient intakes of either individuals or of groups presents many challenges, as noted by a number of authors [12\u201317]. National nutrition surveys are conducted periodically by both the U.S. Department of Agriculture (the Continuing Survey of Food Intakes by Individuals, CSFII) and the U.S. Department of Health and Human Services (the National Health and Nutrition Examination Survey, NHANES). Table 5 gives some examples of possible ways to evaluate the intake data from these surveys, illustrated using intake data for men and women 31\u201350 years of age.\n\nTABLE 5\n\nHow Well Are American Adults Meeting the RDAs? Selected Nutrients for Men and Women 31 to 50 Years Old\n\naFrom Ref. [4] for vitamin C and vitamin E; Ref. [2] for calcium and magnesium; Ref. [3] for vitamin B6 and folate; and Ref. [1] for iron, zinc, and vitamin A.\n\nbThe percent below the EAR approximates the percent of the population with inadequate nutrient intakes. N\/A indicates that the percent below the EAR cannot be determined because an EAR is not available; no EAR was set for nutrients in 1989, and an AI, rather than an EAR and RDA, was set for calcium. All intake data are adjusted for day-to-day variation in intakes before examining the proportion below the EAR.\n\ncIntake of food and supplements as reported in NHANES III 1988\u20131994. From Ref. [4] for vitamin C and vitamin E; Ref. [3] for vitamin B6; and Ref. [18] for folate. Due to the large variance in intakes, mean nutrient intakes at the RDA do not ensure that there is a low prevalence of dietary inadequacy for a population.\n\ndIntake data for vitamin E are in milligrams of \u03b1-tocopherol equivalents, while the recommendations are in milligrams of \u03b1-tocopherol. Thus actual intake data in milligrams of \u03b1-tocopherol would be lower because forms other than \u03b1 (such as \u03b3 and \u03b2) are not included, and the availability of fortification and supplemental all rac-\u03b1-tocopherol is reduced.\n\neIntake from food only as reported in CSFII 1994\u20131996. From Ref. [3]. Due to the large variance in intakes, mean nutrient intakes at the RDA do not ensure that there is a low prevalence of dietary inadequacy for a population. Mean intakes would increase if supplements were included.\n\nfFolate intake data are in micrograms of dietary folate equivalents, and assumes current levels of fortification of cereal grains with folate. They reflect intake from food plus supplements for men 45\u201369 years of age and women 20\u201349 years of age in NHANES III (no data were given for men 20\u201349 years of age). From Ref. [18].\n\ngIntake from food only as reported in NHANES III 1988\u20131991. Average for ages 30\u201339 and 40\u201349. From Ref. [20]. Due to the large variance in intakes, mean nutrient intakes at the RDA do not ensure that there is a low prevalence of dietary inadequacy for a population. Mean intakes would increase if supplements were included.\n\nBecause a complete evaluation of nutrient intakes at this time requires using a combination of the 2000 DRIs and the 1989 RDAs, the recommended intakes shown in Table 5 are DRI\u2013RDAs for vitamin C, vitamin E, magnesium, vitamin B _6_ , and folate, DRI\u2013AI for calcium, and 1989 RDAs for iron, zinc, and vitamin A.\n\nFood composition tables, and thus intakes estimated from the national surveys, usually are not in dietary folate equivalents. Thus, folate intakes will be underestimated. Furthermore, at the time the surveys were conducted, folate fortification of grain products was not required. Since 1998, however, folate intake will have risen as a result of folate fortification of grains. A recent analysis of the NHANES III data has been published showing folate intakes adjusted for the new dietary folate equivalents and for grain fortification [18]. To illustrate the importance of these intake adjustments, the adjusted total folate intake (from food and supplements) shown in Table 5 can be compared to the unadjusted total intakes that have been published [3]. For example, for men 45\u201369 years of age, mean intake of folate after adjustment was 709 \u03bcg dietary folate equivalents\/day, compared to 429 \u03bcg\/day (for men aged 51\u201370 years) without the adjustment. For women, the differences are also large: 710 \u03bcg dietary folate equivalents\/day if adjusted (women 20\u201349 years of age) versus 407 \u03bcg\/day unadjusted (women 31\u201350 years of age). Although the age groups do not match exactly, it is still possible to conclude that estimated intakes would be much higher if the recommended adjustments are made. The adjustments also have a large impact on the percent of the population below the EAR (an estimate of the prevalence of inadequacy): For men in the above age groups, the prevalence is only 8% with adjusted data, but more than 50% with unadjusted data. For women, the comparable figures are 15% versus more than 50%.\n\nFor vitamin E, the intakes shown in Table 5 are probably underestimates because the intake data are in \u03b1-tocopherol equivalents. An additional consideration is the difficulty of accurately measuring vitamin E intakes, because this vitamin is most concentrated in oils, and these foods are frequently underreported in surveys [19]. Underreporting of fats could lead to underreporting of vitamin E and, in turn, to overestimates of the prevalence of inadequacy. It is difficult to know how the combination of these two effects would change the evaluation of vitamin E intake.\n\nAn important issue to consider when evaluating intakes is whether all sources of a nutrient are measured and included in total intake. In particular, many Americans take dietary supplements, and the contribution of supplements to intakes should be considered before evaluating the adequacy of an individual diet or the prevalence of inadequacy for a group of individuals. NHANES III, conducted in 1988 to 1994, collected and quantified nutrient intakes from supplements, but the 1994\u20131996 CSFII did not. Table 5 shows total intakes for vitamins C and E, and for vitamin B6 and folate. However, total intake data for calcium, magnesium, iron, zinc, and vitamin A have not been published, so the values in Table 5 are for food sources only. Therefore, the mean intakes for these nutrients are lower than they would have been with supplements included, and the percent below the EAR is probably an overestimate of the true prevalence of dietary inadequacy.\n\nWhen evaluating intakes from the national surveys, it is desirable to know the percent of the population below the EAR, because that percentage is an accurate estimate of the prevalence of inadequate intakes for most nutrients (this approach cannot be used if requirements are skewed, however, such as with iron requirements for menstruating women). Of the nutrients shown in Table 5, only five currently have an EAR, so the prevalence of dietary inadequacy can only be estimated for these five nutrients. The findings for vitamin E illustrate the importance of examining the distribution of intakes, not just the means. More than 50% of the population was below the EAR, although mean vitamin E intake was well above the RDA of 15 mg\/day. The intake data are obviously skewed by supplement users, whose high intakes raise the mean, but have little effect on the percent of the population below the EAR.\n\nThus, nutrient intakes appear to be low for several nutrients for adults aged 31\u201350 years. Although comparable analyses have not been summarized here for other age and gender groups, others have reported low intakes of several nutrients for most age groups [2\u20134, 20, 21]\n\n## III. GUIDELINES FOR HEALTHY FOOD CHOICES\n\nA. Dietary Guidelines for Americans\n\nBy law, the secretaries of Agriculture and of Health and Human Services are required to jointly issue dietary guidelines for Americans every five years. The recently released Dietary Guidelines for Americans [5, 6] fulfill that requirement for the year 2000. Changes in both the number and content strengthen the guidelines [22]. Because the number of guidelines increased to 10, the committee advising the federal agencies recommended an overarching theme that would help consumers remember the purpose of the guidelines. Thus, the guidelines were put into three categories with an ABC theme: **A** im for Fitness, **B** uild a Healthy Base, **C** hoose Sensibly. As shown in Table 4, the 10 guidelines fall under one of these three themes.\n\n#### 1. AIM FOR FITNESS\n\nTwo guidelines are included under the Aim for Fitness category: the healthy weight guideline (\"Aim for a healthy weight\") and the physical activity guideline (\"Be physically active every day\"). In the past, there was a single guideline promoting a healthy weight through appropriate dietary choices as well as sufficient physical activity. However, the remarkable increases in the rates of obesity among Americans have added urgency to the need to find better ways to help individuals avoid accumulating body fat [23, 24]. Using the newer range for healthy weight as a body mass index between 19.0 and 25.0, only 41% of all individuals 20 years of age and older were classified as having a healthy weight (39% of men and 44% of women) [25]. Furthermore, the low rates of physical activity among both youth and adults are a cause for concern not only as a contributing factor to the percent of the population that is overweight and obese, but also to a declining level of muscular and cardiopulmonary fitness that is likely to be associated with cardiovascular disease, diabetes, and osteoporosis, especially among older adults [26]. Physical activity is discussed in more detail later (Section IV.A).\n\n#### 2. BUILD A HEALTHY BASE\n\nFor the first time since the initiation of the dietary guidelines, there is not a guideline focusing on dietary variety (\"Eat a variety of foods\" was included with the guidelines from 1980 through 1995). Instead, the 2000 guideline reads ''Let the Pyramid guide your food choices.\" Several advantages are seen for this new wording. First, the primary purposes of the guideline are to communicate the importance of consuming a nutritionally adequate diet and to provide specific information on how to achieve this goal. Although eating a variety of foods indeed helps to promote better nutrient intakes within a population [27], there is no guarantee that variety alone will allow individuals to ensure that their diets meet the nutrient recommendations (such as the RDAs and AIs discussed in the previous section). The Food Guide Pyramid was specifically developed to provide guidance on the type of dietary pattern that can promote nutritional adequacy (see Fig. 1). It has been a very successful nutrition education tool and is widely incorporated into educational messages and programs. The Food Guide Pyramid was the central focus of the previous variety guideline, so it follows that incorporating a reference to the pyramid directly into the guideline text more clearly communicates the essence of a guideline to achieve nutritional adequacy. Secondly, there was some concern that a broad guideline to increase variety might inadvertently promote overconsumption of energy. There is some evidence that a variety of carbohydrate foods, for example, is associated with a higher body fatness [28]. Finally, it was clear that consumers did not always realize that a variety of _healthy_ foods was being suggested\u2014not a variety of snack foods and desserts. More information on the Food Guide Pyramid is given in the next section.\n\nThe concept of variety is still a valid one, however, as long as consumers understand that a variety of nutrient-dense foods are being promoted. Thus, both the grain guideline (\"Eat a variety of grains daily, especially whole grains\") and the fruit and vegetable guideline (\"Eat a variety of fruits and vegetables daily\") specifically mention the desirability of selecting a variety of these foods. The separation of the former fruit\/vegetable\/grain guideline into two guidelines emphasizes the importance these foods play in a healthy diet. They are the base of the Food Guide Pyramid, and form the foundation of a diet that is nutritionally adequate. In addition, high consumption of these foods has been shown to reduce the risk of a variety of chronic diseases, including cardiovascular disease and cancer [29\u201331]. Indeed, the association between fruit, vegetable, and whole-grain intakes and reduced risk of disease is often stronger than the association between intakes of their nutrient components (such as fiber, vitamins and minerals, or carotenoids) and risk. The reasons why this occurs are not well understood, but might include unmeasured food components, interactions among the components within a food, and inaccurate composition data for the components. Furthermore, fruits, vegetables, and whole grains tend to be low in both fat and energy, and thus can be consumed in greater volume than more energy-dense foods without exceeding energy needs. There is evidence that a diet that is based on foods with a low energy density can both promote weight loss and prevent weight gain [32, 33].\n\nA food safety guideline (\"Keep food safe to eat\") was included for the first time in response to increasing concerns about foodborne illness. The U.S. Government Accounting Office has estimated a range of 6.5 to 33 million cases of foodborne illness a year [34]. More recently, the Centers for Disease Control and Prevention estimated that foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5000 deaths in the United States each year [35]. The estimates are difficult to derive because the majority of cases are not reported to health care providers. Even the lower end of the range raises substantial concern about the burden of illness (and the cost of lost productivity) attributable to these illnesses. Although consumers cannot control all sources of food contamination, there is reason to believe that high-risk behaviors by consumers contribute to a substantial portion of foodborne illness [36]. Therefore, an important objective of this guideline is to improve food handling practices. The advice to consumers follows the same four topics that are promoted by the Fight BAC! campaign by the Partnership for Food Safety Education [37]:\n\n\u2022 Clean (wash hand and surfaces often)\n\n\u2022 Separate (separate raw, cooked, and ready-to-eat foods while shopping, preparing, or storing)\n\n\u2022 Cook (cook foods to a safe temperature)\n\n\u2022 Chill (refrigerate perishable foods promptly).\n\n#### 3. CHOOSE SENSIBLY\n\nThe first two of the ''Choose Sensibly\" guidelines focus on fat and sugar intakes. The fat guideline (\"Choose a diet that is low in saturated fat and cholesterol and moderate in total fat\") represents recent research on the role of fat intake on overall health. First, the evidence is strong that intakes of both saturated fat and cholesterol are positively associated with cardiovascular disease 38, 39] (see [Chapter 18). Thus, the guideline continues to promote a maximum of 10% of energy from saturated fat, and a maximum of 300 mg\/day of dietary cholesterol. Table 6 shows that more than 70% of Americans are meeting the cholesterol goal, but only 36% meet the saturated fat goal. The guideline also continues to recommend that less than 30% of energy intake come from fat, as has been suggested by a variety of public and private health organizations. However, the guideline committee recognized that very low fat diets (those well below 30% of energy from fat) may not be ideal for all individuals and, indeed, that diets that are high in carbohydrates may actually contribute to cardiovascular disease for some people 40, 41]. Therefore, the guideline was worded to suggest a ''moderate\" consumption of total fat, to avoid the implication that all individuals should aim for fat intakes below 30% of energy. As explained in an earlier chapter of this book ([Chapter 18), advice regarding the distribution of macronutrients in a diet should be tailored, whenever possible, to an individual's specific risk factors (such as low-density lipoprotein and high-density lipoprotein cholesterol levels).\n\nTABLE 6\n\nHow Well Are Americans Following the Food Guide Pyramid and the Dietary Guidelines for Fat Intake?\n\naFrom Refs. [5] and [7].\n\nbAll individuals aged 2 years and older. From Refs. [21] and [50]. Reflects 2-day averages based on data reported in the 1996 CSFII, except for fat, saturated fat, and cholesterol, where only the first day of data is used in calculating the averages. Excludes breast-fed children.\n\ncBased on servings recommended for a specific caloric intake (for example, 6 servings\/day of grains if 2200 kcal\/day or less; 9 servings\/day if 2200 to 2800 kcal\/day; and 11 servings\/day if greater than 2800 kcal\/day) [7]. Two-day averages are used when determining the percent below the recommendations.\n\ndUses 3 servings\/day as the recommendation for women who were pregnant or lactating, and for individuals aged 11\u201324 years (USDA FGP booklet). This percentage would be likely to decrease if the newer guideline was used: 3 servings\/day for all individuals over age 50 and for those aged 9\u201318 years, based on the increased calcium recommendations for these age categories; 2 servings\/day for younger children, and for persons aged 19\u201350 years; no increased dairy intake is necessary during pregnancy and lactation [5].\n\nThe sugar guideline (\"Choose beverages and foods to moderate your intake of sugars\") reflects concerns that Americans are overconsuming energy in the form of sugar. Although sugars are chemically identical whether they are added to foods or occur naturally, the intent of the guideline was to suggest a reduction in intake of foods that are high in sugar and low in vitamins and minerals, particularly for individuals with low energy intakes or those wishing to reduce their energy intakes. For example, many fruits, vegetables, and dairy products are high in sugars such as fructose and lactose, but these foods are also important sources of many vitamins and minerals. In contrast, foods such as nondiet carbonated beverages or fruit-flavored punches usually supply very few nutrients other than energy. Thus, the guideline urges Americans to avoid foods that are high in added sugars (and the text lists examples of these foods: soft drinks, cakes, cookies, pies, fruitades and fruit punch, ice cream, and candy). Furthermore, dairy product consumption among children and adolescents is decreasing at the same time that soft drink consumption is increasing [42]. Thus, there is concern that high intakes of carbonated beverages may be replacing milk intake among adolescents, for whom bone mineral accretion is especially important if osteoporosis later in life is to be avoided [43]. High intakes of sugars are also associated with an increased incidence of dental caries, providing yet another reason to moderate intake of sugar [44].\n\nThe remaining two guidelines in this section on choosing sensibly address salt intake (\"Choose and prepare foods with less salt\") and alcohol intake (\"If you drink alcoholic beverages, do so in moderation). Although the wording has changed somewhat since the 1995 guidelines, the intent of these two guidelines remains essentially unchanged. The salt guideline refers to the 2400-mg daily value for sodium that is listed on the nutrition facts label on food products, but does not specifically state this should be the upper limit of intake (see Section III.D for more details on daily values). Moderate intake of alcohol is defined as no more than one drink per day for women and two drinks per day for men, where 12 ounces of beer, 5 ounces of wine, or 1.5 ounces of distilled spirits count as a serving. The alcohol guideline also lists individuals who should not drink:\n\n\u2022 Children and adolescents\n\n\u2022 Individuals of any age who cannot restrict their drinking to moderate levels\n\n\u2022 Women who are pregnant or may become pregnant\n\n\u2022 Individuals who plan to drive, operate machinery, or take part in other activities that require attention, skill, or coordination\n\n\u2022 Individuals taking prescription or over-the-counter medications that can interact with alcohol.\n\n### B. The Food Guide Pyramid\n\nThe Food Guide Pyramid was released by the USDA in 1992 [7], and has been widely distributed to both health professionals and consumers. Its appeal is twofold: It is a simple and actionable graphic, and it is based on detailed analyses that demonstrate its scientific accuracy. Both the form and the content of the Food Guide Pyramid underwent extensive testing. Numerous focus groups were conducted to ensure that consumers understood the messages being conveyed, and that the pyramid was a meaningful graphic for offering dietary guidance [45]. In addition, extensive analyses were conducted to determine what guidance would ensure adequacy (provision of recommended levels of nutrients) and moderation (low-fat, low-energy choices from each of the groups) [45]. Finally, typical dietary patterns in the United States were considered, which led to the selection of the food groups, and the number of servings of each, that would provide the recommended levels of nutrients at three daily energy levels (1600, 2200, and 2800 kcal\/day) as shown in Fig. 2. Because the food groups are broadly defined, they can be adapted to meet specific cultural and personal preferences. Thus, the Food Guide Pyramid combines the dietary guidelines and the RDAs\/DRIs into a single tool that is both scientifically based and consumer friendly.\n\nServing sizes for the foods in each Food Guide Pyramid food group, except the tip, are specified [7] as shown in Fig. 3. Serving sizes are smaller than many typical portions. For example, a slice of bread or one-half cup of pasta is a serving of the grains group, although typical portions are substantially larger than these. (Adults typically consume about two slices of bread per eating occasion, and about one cup of pasta [46].)\n\nFIGURE 3 What counts as a serving? [From U.S. Department of Agriculture and U.S. Department of Health and Human Services (2000). ''Dietary Guidelines for Americans,\" 5th ed., Home and Garden Bulletin No. 232. U.S. Government Printing Office, Washington, DC.]\n\nMany variations of the Food Guide Pyramid have been proposed, but most are specific interpretations of the pyramid's more general guidance. For example, a children's pyramid was released by the USDA in 1999 that offers a graphic that includes foods frequently consumed by children [47] (see Fig. 4). It is also a departure from the original (adult) Food Guide Pyramid, in that the importance of physical activity is emphasized with pictures of children engaged in several types of physical activity shown around the pyramid graphic. Although pyramids for various cultural or ethnic groups have been proposed, there is seldom the same level of analytic research for these variations as was conducted for the original, more general, Food Guide Pyramid. Health professionals should be cautious about recommending food guides that have not undergone the rigorous testing of the Food Guide Pyramid.\n\nFIGURE 4 Food Guide Pyramid for young children; a daily guide for 2- to 6-year-olds. [From U.S. Department of Agriculture (1999). ''Tips for Using the Food Guide Pyramid for Young Children 2 to 6 Years Old,\" Program Aid 1647. U.S. Department of Agriculture, Center for Nutrition Policy and Promotion, Washington, DC.]\n\nThe Food Guide Pyramid is based on the 1989 RDAs and on the 1990 dietary guidelines. As explained in Section II of this chapter, the 1989 RDAs are being substantially revised as part of the process of setting the new DRIs. In addition, the dietary guidelines have been modified and expanded twice (in 1995 and 2000) since the pyramid was developed. To date, the only change in either type of guidance that is likely to significantly affect the Food Guide Pyramid is the increased recommendation for calcium. As reflected in the Food Guide Pyramid that is included in the 2000 dietary guidelines [5], three (rather than two) servings of dairy products would be required for adults over the age of 50 in order to meet the new AI for calcium of 1200 mg\/day. Three servings would also be recommended for children and young adults 9\u201318 years of age (rather than those 13\u201324 years of age). It is anticipated that the Food Guide Pyramid will be reevaluated when the process of setting the DRIs is complete.\n\n### C. Food Exchanges to Design Meal Plans\n\nFood exchanges were originally developed by the American Dietetic Association and the American Diabetes Association as a consumer-friendly tool that health professionals could use in meal planning for diabetic patients [48]. Starting in 1989, the exchange lists were adapted for use in weight management as well. These exchanges have been widely used by dietitians to provide simple guidelines for their clients who wished to control macronutrient intake. Although the food exchanges were developed many years before the Food Guide Pyramid was published in 1992, the format of the exchanges is remarkably similar to the food groups used in the pyramid. These are the exchange list food groups:\n\n\u2022 Starch\/bread\n\n\u2022 Meat: lean, medium-fat, and high-fat\n\n\u2022 Vegetable\n\n\u2022 Fruit\n\n\u2022 Milk: skim, low-fat, whole\n\n\u2022 Fat.\n\nEach of the exchanges is assigned an approximate value for energy and for grams of carbohydrate, protein, and fat. A dietitian or diabetes educator can then plan a diet for a patient to include a specified number of exchanges from each group. Once the number of exchanges is determined, the grams of the macronutrients and the energy content of the diet can be easily estimated.\n\nThe patient then uses the booklet to decide what food selections fit within each exchange. For example, if a patient's plan includes six starch\/bread exchanges, he or she may choose among a variety of cereals, grains, pasta, beans, starchy vegetables such as corn or potatoes, bread, and crackers. High-fat starchy foods like muffins or french fried potatoes count as a starch exchange _and_ a fat exchange. Portion sizes for one exchange of each food are specified, such as one (1-ounce) slice of bread or one-half cup of mashed potatoes.\n\nWhen using exchanges, health professionals should be aware that while the exchange food groups and the Food Guide Pyramid food groups are similar, there are some important differences that may lead to confusion if a patient is also familiar with the Food Guide Pyramid. For example, as mentioned above, potatoes count as a starch exchange, not as a vegetable. Dried beans and peas, and corn, are also considered starches. High-fat foods like nuts and seeds, avocados, and bacon count as fat exchanges only. Many vegetables are considered ''free\" foods, rather than vegetables, because they contain very few calories per serving (e.g., lettuce, cabbage, cucumber, celery, and zucchini). Furthermore, there are several differences in the portion sizes for foods in the exchange groups compared to these same foods in the Food Guide Pyramid servings (e.g., meat exchanges are measured in ounces, not in 2- to 3-ounce servings like the pyramid).\n\nA primary appeal of the exchange lists continues to be the flexibility offered to the health professional in planning macronutrient-controlled diets for patients. If combined with the minimum recommended number of servings from the Food Guide Pyramid, the exchange lists provide an attractive alternative for a health professional who wishes to work with a client to design a meal plan that is nutritionally adequate as well as individually tailored for the client's specific macronutrient goals.\n\n### D. The Role of Food and Supplement Labels in Helping Consumers Follow the Dietary Guidelines\n\nThe nutrition facts on food labels provide information that can help consumers follow the dietary guidelines (see Fig. 5). The Nutrition Labeling and Education Act (NLEA) of 1990 requires a nutrition facts panel on most packaged food products [8]. Consumers can use this information to monitor their consumption of energy, fat, saturated fat, sugar, and sodium, as recommended in the body weight, fat, sugar, and sodium guidelines. Persons who wish to increase their intakes, for example of vitamins, minerals, and dietary fiber, also will find the nutrition facts useful. For example, tofu may be a good source of calcium if it is precipitated with a calcium salt, but some tofu products have very little calcium. By examining either the ingredient list (for a calcium salt) or the nutrition facts, it is possible to tell quickly if the product provides substantial amounts of calcium.\n\nFIGURE 5 How to read a nutrition facts label. [From U.S. Department of Agriculture and U.S. Department of Health and Human Services (2000). ''Dietary Guidelines for Americans,\" 5th ed., Home and Garden Bulletin No. 232. U.S. Government Printing Office, Washington, DC.]\n\nFor most nutrients, amounts in weights such as grams, milligrams, or micrograms would not be meaningful to most consumers. As a result, the nutrients are shown as a percent of a daily value (DV). For vitamins and minerals, these DVs are based on RDIs, which in turn are based on the RDAs from 1968 [49]. For macronutrients, cholesterol, fiber, sodium, and potassium, the DVs are based on Daily Reference Values (DRVs). The DRVs parallel the recommendations from the dietary guidelines regarding fat (less than 30% of energy intake) and saturated fat (less than 10% of energy intake) consumption. The DV for dietary fiber intake is 11.5 g\/1000 kcal, for cholesterol is 300 mg\/day, for sodium is 2400 mg\/day, and for potassium is 3500 mg\/day. For example, for a 2000-calorie diet, the DV for fat is 65 g\/day, the DV for saturated fat is 20 g\/day, and the DV for dietary fiber is 25 g. The DVs for two calorie intake levels (2000 and 2500 kcal\/day) are specified on the label for total fat, saturated fat, cholesterol, sodium, total carbohydrate, and dietary fiber. The percent of the 2000 kcal\/day DV that is contained in the specific product is also given in larger type in the center of the panel. For calories, calories from fat, total fat, saturated fat, cholesterol, and sodium, the actual amount per serving is also given. For example, the macaroni and cheese nutrition facts shown in Fig. 5 indicate that one serving of this product (1 cup) contains 18% of the DV for total fat (12 g) and 15% of the DV for saturated fat (3 g). Consumers can readily scan the %DVs to see if this food item is a high or low source of specific nutrients. Nutrition information on unpackaged fruits and vegetables must be posted in the produce department of grocery stores.\n\nBecause they serve different purposes, the serving sizes used for the food label do not always correspond to those used for the Food Guide Pyramid. The servings on the food label are specified by the NLEA and are intended to reflect usual portion sizes that a consumer might typically select. In contrast, the Food Guide Pyramid servings were not necessarily usual portion sizes, but rather were portions that allowed flexibility in providing dietary guidance. In most cases, the pyramid serving size is smaller than the corresponding food label serving size. For example, as noted above, a Food Guide Pyramid serving of pasta is one-half-cup, although a typical portion is closer to 1 cup. Thus, the serving size of pasta on the food label is 1 cup to correspond to this typical portion.\n\nStarting in March 1999, a supplement facts label was required for most dietary supplements [9]. The format of this label is similar to that for the nutrition facts label on foods, but also allows for more flexibility in reporting nonnutrient components. For example, a ginseng supplement would indicate the number of micrograms in the supplement, but no daily value is given.\n\n### E. How Do Current Food Group Intakes Compare to Recommendations?\n\nTable 6 shows the recommended servings from the five major food groups of the Food Guide Pyramid, and the actual intakes reported by a national sample of Americans [50]. Although mean intakes of grains and vegetables exceed the minimum recommended number of servings (six and three, respectively), approximately 60% of the population is not meeting the recommendations. The minimum number of servings applies to persons who are consuming a diet of 1600 kcal (or less), so those with higher energy intakes should also have a higher number of servings from each of the pyramid food groups (up to the maximum number of servings shown in Fig. 2, for a 2800-kcal diet). Intakes of fruit, dairy, and meats or meat substitutes are even lower, with only 22\u201330% of Americans reporting diets that meet these recommendations.\n\nAs can also be seen in Table 6, Americans consume a substantial portion of their energy intake as discretionary fat and added sugar; together these items contribute 41% of the calories in a typical American diet. For the average sedentary woman, low-fat choices from the Food Guide Pyramid would total about 1220 kcal for a 1600-kcal diet, or about 75% of caloric intake [43]. For a sedentary man, appropriate Food Guide Pyramid choices would total about 1650 kcal for a 2200-kcal diet, which is also about 75% of caloric intake. Clearly, individuals who are consuming 41% of their energy from discretionary fat and added sugars are either substituting these items for foods that are more nutrient dense, or are adding these items to their diets at the risk of overconsumption of energy. Although the contribution of fat and sugar to obesity has been difficult to quantify (probably in large part due to underreporting of these foods by survey subjects [19]), if energy consumption exceeds energy expenditure (and, for children and adolescents, the energy requirements for growth), fat storage will occur. Until the role of these foods as a factor in obesity can be better understood, it remains appropriate to encourage a reduction in the use of foods with a low nutrient-to-calorie ratio.\n\nAbout one-third of the population reported diets that met the guidelines for total fat (less than 30% of energy intake) or for saturated fat (less than 10% of energy intake). However, the cholesterol goal (less than 300 mg\/day) was being met by more than 70% of the sample.\n\nOverall, Americans are not choosing diets that follow either the dietary guidelines or the Food Guide Pyramid. The only recommendation that is being met by at least half the population is the one for cholesterol. The Healthy Eating Index provides a tool for evaluating overall conformance with the dietary guidelines [51, 52]. An individual is scored on 10 dietary parameters, most of which are shown in Table 6 (consumption of the correct number of food group servings, and meeting the guidelines for fat and cholesterol; in addition, a diet is scored for the level of sodium intake, and also for a measure of dietary variety based on the number of different food commodities that were consumed). Out of a possible score of 100, the average was 64 in 1994\u20131996 [52]. By this measure as well, there is room for substantial improvement in the pattern of the American diet.\n\n## IV. BEYOND FOOD AND NUTRIENT GUIDELINES: PHYSICAL ACTIVITY GUIDELINES\n\nA. A New Dietary Guideline on Physical Activity\n\nIncreasing concerns about both the fitness level and the obesity rates among Americans have led to a dietary guideline that focuses specifically on physical activity: ''Be physically active every day.\" The text following the guidelines gives specific advice on a minimum level of physical activity: 30 minutes of moderate physical activity most days of the week, preferably daily, for adults, and 60 minutes for children.\n\nChapter 32 examined the role of inactivity in the etiology of obesity. Inactivity not only predisposes an individual to weight gain, but also can reduce the efficacy of weight loss attempts 26]. Obesity in turn is a risk factor for a variety of chronic diseases. Furthermore, an inactive lifestyle is a direct risk factor for several chronic diseases, beyond the indirect role of inactivity in promoting obesity [26]. The roles of both obesity and inactivity as risk factors for chronic disease are discussed elsewhere in this book: osteoporosis ([Chapter 42), colon cancer (Chapter 23), cardiovascular disease (Chapters 18 through Chapter 19 Chapter 20 Chapter 21), and diabetes (Chapter 27).\n\nIn addition, an active lifestyle can promote a healthy diet by increasing energy requirements. With a larger energy ''budget,\" individuals can spare calories for optional foods containing added sugars and discretionary fats, without displacing foods from the healthy foundation of their diets. The ability to choose a larger variety of foods, without gaining weight, also increases the enjoyment of eating, and decreases the guilt associated with occasional choices of foods of low nutrient density.\n\nThe text in the dietary guidelines booklet lists the following benefits of regular physical activity:\n\n\u2022 Increases physical fitness.\n\n\u2022 Helps build and maintain healthy bones, muscles, and joints.\n\n\u2022 Builds endurance and muscular strength.\n\n\u2022 Helps manage weight.\n\n\u2022 Lowers risk factors for cardiovascular disease, colon cancer, and type 2 diabetes.\n\n\u2022 Helps control blood pressure.\n\n\u2022 Promotes psychological well-being and self-esteem.\n\n\u2022 Reduces feelings of depression and anxiety.\n\n### B. Other Recommendations\n\nThe physical activity dietary guideline is a simplified version of the guidance offered in 1995 by the Centers for Disease Control and Prevention and the American College of Sports Medicine: ''Every U.S. adult should accumulate 30 minutes or more of moderate-intensity physical activity on most, preferably all, days of the week\" [10, p. 402]. This message has been widely disseminated, and provides an achievable goal for most Americans. One of the appealing concepts for the public has been the ability to count short periods of exercise toward the daily goal of 30 minutes. Thus, walking up stairs, or walking an extra few blocks to work, can accumulate toward the total, as long as the activity is carried out at a moderate pace. Moderate activity is defined as an activity that requires three to six metabolic equivalents, where a metabolic equivalent is the ratio of the metabolic rate while performing the activity to the resting metabolic rate (measured in kcal\/min). Examples of moderate activity include walking briskly (3\u20134 mph), cycling for pleasure or transportation, and swimming [10]. Numerous activities that can contribute to the 30 minutes of daily exercise are given in the dietary guidelines book [5].\n\nThe guidance for children's physical activity is based primarily on recommendations from the Centers for Disease Control and Prevention [53] suggesting that 60 minutes of moderate activity per day is appropriate. In addition, evidence linking television watching and body weight and level of fatness suggest that television viewing should be limited for children [54].\n\n### C. How Do Current Activity Levels Compare to These Guidelines?\n\nData on activity levels for Americans are given in the 1998\u20131999 Healthy People 2000 Review [55] and are summarized in Table 7. Few American adults (only 16%) engage in even light to moderate activity daily, and only 23% engage in this level of activity at least five times per week. Furthermore, improvements between 1985 and 1995 are almost nonexistent. Clearly, it would take a substantial change in lifestyle to expand daily light to moderate activities to most American adults. A similar number of adults (about 16%) engage in vigorous activity at least three times per week, although this number has increased (from 12%) since 1985. Furthermore, the number of adults with a sedentary lifestyle is essentially unchanged, at almost one-fourth of the population. Data from the 1994 Behavioral Risk Factor Surveillance System indicate that 33% of overweight men and 41% of overweight women 18 years of age and older reported no leisure time physical activity during the previous month [56].\n\nTABLE 7\n\nHow Well Are Americans Following the Physical Activity Guidelines? Changes in Physical Activity Rates and Year 2000 Targets\n\naN\/A = not available.\n\n_Source:_ Adapted from U.S. Department of Health and Human Services (1999). \"Healthy People 2000 Review, 1998\u201399,\" Publication No. (PS) 99\u20131256. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Health Statistics, Hyattsville, MD. National Center for Health Statistics\n\nFor adolescents in grades 9\u201312, physical activity in the schools is declining (Table 7). The percent engaging in daily physical education declined from 42% to only 27% between 1991 and 1997, and those engaging in active physical education time at least 30 minutes per week declined from 24% to 21% over this same time period. No recent data have been compiled for younger children, but in 1984\u20131986, only 36% of children in grades 1\u201312 participated in daily school physical education.\n\nHowever, there is some evidence of an increase in the percent of adolescents who engage in vigorous activity at least three times per week: Although the groups are not exactly comparable, 59% of children 10\u201317 years of age exercised vigorously at least three times per week in 1984, while the comparable figure was 64% for those in grades 9\u201312 in 1997, 13 years later. Because physical activity in schools appears to be declining, these increases in vigorous activity may be attributable to after-school sports or similar non-school activities.\n\nThus, a public health priority is to identify ways to provide safe and enjoyable opportunities to engage in physical activity for both adults and children. Physical education in schools provides an obvious opportunity to increase the activity level of younger children. For older children, team sports may offer more appeal. Community-based activities may be the best way to reach the large majority of sedentary adults. It will take a concerted effort by health professionals if activity levels in America are to increase substantially.\n\n## V. SUMMARY\n\nCurrently a variety of consumer-friendly tools are available to promote the consumption of healthful diets and the need for daily physical activity. In general, these interact smoothly to provide incentives for the selection of nutritious foods and at least moderately strenuous activity levels.\n\nHowever, despite these educational tools, consumers do not appear to be motivated to make the dietary changes recommended by these nutrient and food guidelines, or to select more frequent or more strenuous physical activities. As can be seen from the survey summaries in Tables 5, 6, and 7, many improvements could be made to American food and activity choices. The burden falls on nutrition educators and behavioral scientists to provide approaches that will inspire the public to change their food practices and activity levels in a positive direction. The rewards are many, both at the individual level (in reduced rates of chronic disease) and the societal level (in reduced medical care costs and lost productivity). A recent estimate suggests that 14% of all deaths in the United States in 1990 (300,000 deaths) were due to poor diet and activity patterns [57]. The challenge for health professionals is to provide the motivation and the environment that will facilitate more healthful diet and activity patterns by the American public. Successful and practical intervention programs are greatly needed.\n\nReferences\n\n1. National Research Council, Food and Nutrition Board. _Recommended Dietary Allowances_. Washington, DC: National Academy Press; 1989.\n\n2. Institute of Medicine. _Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride_. Washington, DC: National Academy Press; 1997.\n\n3. Institute of Medicine. _Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline_. Washington, DC: National Academy Press; 1998.\n\n4. Institute of Medicine. _Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids_. Washington, DC: National Academy Press; 2000.\n\n5. U.S. Department of Agriculture, U.S. Department of Health and Human Services. Dietary Guidelines for Americans. 5th ed. Home and Garden Bulletin No. 232. Washington, DC: U.S. Government Printing Office; 2000.\n\n6. U.S. Department of Agriculture. _Report of the Dietary Guidelines Advisory Committee on the Dietary Guidelines for Americans, 2000_. Washington, DC: U.S. Government Printing Office; 2000.\n\n7. U.S. Department of Agriculture. The Food Guide Pyramid. _Home and Garden Bulletin No. 252_. Washington, DC: U.S. Government Printing Office; 1992.\n\n8. Kurtzweil P. Nutrition facts to help consumers eat smart. _FDA Consumer_. 1993;27(4):22\u201327.\n\n9. Kurtzweil P. An FDA guide to dietary supplements. _FDA Consumer_. 1998;32(5):28\u201335.\n\n10. 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Life Sciences Research Office, Federation of American Societies for Experimental Biology. _Guidelines for Use of Dietary Intake Data_. Washington, DC: Life Sciences Research Office; 1986.\n\n15. Thompson F.E., Beyers T. Dietary assessment resource manual. _J. Nutr_. 1994;124:2245S\u20132317S.\n\n16. Buzzard I.M., Willett W.C., eds. Dietary assessment methods. _Am. J. Clin. Nutr_. 1994;59:143S\u2013306S\n\n17. Willett W.C., Sampson L., eds. Dietary assessment methods. _Am. J. Clin. Nutr_. 1997;65:1097S\u20131368S\n\n18. Lewis C.J., Crane N.T., Wilson D.B., Yetley E.A. Estimated folate intakes: Data updated to reflect food fortification, increased bioavailability, and dietary supplement use. _Am. J. Clin. Nutr_. 1999;70:198\u2013207.\n\n19. Briefel R.R., Sempos C.T., McDowell M.A., Chien S., Alaimo K. Dietary methods research in the Third National Health and Nutrition Examination Survey: Underreporting of energy intake. _Am. J. Clin. Nutr_. 1997;65:1203S\u20131209S.\n\n20. 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National Center for Health Statistics, Centers for Disease Control and Prevention. Update: Prevalence of overweight among children, adolescents, and adults\u2014United States 1988\u20131994. _Morbid. Mortal. Wkly Rept_. 1997;46:199\u2013202.\n\n25. U.S. Department of Health and Human Services, Office of Public Health and Science. _Healthy People 2010 Objectives: Draft for Public Comment_. Riverdale, MD: U.S. Department of Health and Human Services; 1998.\n\n26. U.S. Department of Health and Human Services. _Physical Activity and Health: A Report of the Surgeon General_. Washington, DC: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion; 1996.\n\n27. Krebs-Smith S.M., Smiciklas-Wright H., Guthrie H.A., Krebs-Smith J. The effects of variety in food choices on dietary quality. _J. Am. Diet. Assoc_. 1987;87:897\u2013903.\n\n28. McCrory M.A., Fuss P.J., McCallum J.E., Yao M., Vinken A.G., Hays N.P., Roberts S.B. Dietary variety within foodgroups: Association with energy intake and body fatness in men and women. _Am. J. Clin. Nutr_. 1999;69:440\u2013447.\n\n29. Steinmetz K.A., Potter J.D. Vegetables, fruit, and cancer prevention: A review. _J. Am. Diet. Assoc_. 1996;96:1027\u20131039.\n\n30. Ness A.R., Powles J.W. Fruit and vegetables, and cardio-vascular disease: A review. _Int. J. Epidemiol_. 1997;26:1\u201313.\n\n31. Jacobs D.R., Jr., Meyer K.A., Kushi L.H., Folsom A.R. Whole grain intake may reduce the risk of ischemic heart disease death in postmenopausal women: The Iowa Women's Health Study. _Am. J. Clin. Nutr_. 1998;68:248\u2013257.\n\n32. Rolls B.J., Bell E.A., Castellanos V.H., Chow M., Pelkman C.L., Thorwart M.L. Energy density but not fat content of foods affected energy intake in lean and obese women. _Am. J. Clin. Nutr_. 1999;69(5):863\u2013871.\n\n33. Rolls B.J., Castellanos V.H., Halford J.C., Kilara A., Panyam D., Pelkman C.L., Smith G.P., Thorwart M.L. Volume of food consumed affects satiety in men. _Am. J. Clin. Nutr_. 1998;67:1170\u20131177.\n\n34. GAO\/RCED-96-96U.S. General Accounting Office. _Food Safety: Information on Foodborne Illnesses: Report to Congressional Committees_. Atlanta, GA: U.S. General Accounting Office; 1996.\n\n35. Mead P.S., Slutsker L., Dietz V., McCaig L.F., Bressee J.S., Shapiro C., Griffin P.M., Tauxe R.V. Food-related illness and death in the United States. _Emerg. Infect. Dis_. 1999;5:607\u2013625.\n\n36. Yang S., Leff M.G., McTague D., Horvath K.A., Jackson-Thompson J., Murayi T., Boeselager G.K., Melnik T.A., Gildemaster M.C., Ridings D.L., Altekruse S.F., Angulo F.J. Multistate surveillance for food-handling, preparation, and consumption behaviors associated with foodborne diseases: 1995 and 1996 BRFSS food-safety questions. _Morbid. Mortal. Wkly Rept_. 1998;47:33\u201357.\n\n37. Kurtzweil P. A year of food safety accomplishments. _FDA Consumer_. 1998;32(5):8\u20139.\n\n38. Gordon D.J. Cholesterol and mortality: What can meta-analyses tell us? In: Gallo L.L., ed. _Cardiovascular Disease_. Washington, DC: Plenum Press; 1995:333\u2013340.\n\n39. Gordon D.J. Cholesterol lowering and total mortality. In: Rifkind B.M., ed. _Lowering Cholesterol in High Risk Individuals and Populations_. New York: Marcek Dekker; 1995:33\u201348.\n\n40. Grundy S.M. Overview: Second International Conference on Fats and Oil Consumption in Health and Disease: How we can optimize dietary composition to combat metabolic complications and decrease obesity. _Am. J. Clin. Nutr_. 1998;67(3):497S\u2013499S.\n\n41. Krauss R.M. Triglycerides and atherogenic lipoproteins: Rationale for lipid management. _Am. J. Med_. 1998;105(1A):58S\u201362S.\n\n42. Morton J.F., Guthrie J.F. Changes in children's total fat intakes and their food group sources of fat, 1989\u201391 versus 1994\u201395: Implications for diet quality. _Fam. Econ. Nutr. Rev_. 1998;11:44\u201357.\n\n43. Harnack L., Stang J., Story M. Soft drink consumption among U.S. children and adolescents: Nutritional consequences. _J. Am. Diet. Assoc_. 1999;99:436\u2013441.\n\n44. Depaola D.P., Faine M.P., Palmer C.A. Nutrition in relation to dental medicine. In: Shils M.E., Olson J.A., Shike M., Ross A.C., eds. _Modern Nutrition in Health and Disease_. 9th ed. New York: Williams and Wilkins; 1999:1099\u20131124.\n\n45. U.S. Department of Agriculture, Human Nutrition Information Service. USDA's Food Guide: Background and Development. _Misc. Pub. No. 1514. U.S. Department of Agriculture, Hyattsville, MD_. 1993.\n\n46. Krebs-Smith S.M., Guenther P.M., Cook A., Thompson F.E., Cucinelli J., Ulder J. Foods Commonly Consumed Per Eating Occasion and in a Day, 1989\u201391. _NFS Report No. 91-3. U.S. Government Printing Office, Washington, DC_. 1997.\n\n47. Program Aid 1647U.S. Department of Agriculture. _Tips for Using the Food Guide Pyramid for Young Children 2 to 6 Years Old_. Baltimore, MD: U.S. Department of Agriculture, Center for Nutrition Policy and Promotion; 1999.\n\n48. American Dietetic Association and American Diabetes Association. _Exchange Lists for Meal Planning_. Washington, DC: American Dietetic Association; 1995.\n\n49. Kurtzweil P. Daily values encourage healthy diet. _FDA Consumer_. 1993;27(4):28\u201332.\n\n50. U.S. Department of Agriculture, Food Surveys Research Group. _Pyramid Servings Data: Results from USDA's 1995 and 1996 Continuing Survey of Food Intakes by Individuals_. Chicago, IL: U.S. Department of Agriculture; 1997.\n\n51. CNPP-1U.S. Department of Agriculture, Center for Nutrition Policy and Promotion. _The Healthy Eating Index_. Riverdale, MD: U.S. Department of Agriculture, Center for Nutrition Policy and Promotion; 1995.\n\n52. CNPP-5Bowman S.A., Lino M., Gerrior S.A., Basiotis P.P. _The Healthy Eating Index 1994\u201396_. Washington, DC: U.S. Department of Agriculture, Center for Nutrition Policy and Promotion; 1998.\n\n53. Centers for Disease Control and Prevention. Guidelines for school and community health programs to promote lifelong physical activity among young people. _Morbid. Mortal. Wkly Rept_. 1997;46(RR-6):1\u201334.\n\n54. Andersen R.E., Crespo C.J., Bartlett S.J., Cheskin L.J., Pratt M. Relationship of physical activity and television watching with body weight and level of fatness among children: Results from the Third National Health and Nutrition Examination Survey. _JAMA_. 1998;279:938\u2013942.\n\n55. U.S. Department of Health and Human Services. Healthy People 2000 Review, 1998\u201399. _Publication No. (PS) 99-1256. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Health Statistics, Hyattsville, MD_. 1999.\n\n56. Centers for Disease Control and Prevention. Prevalence of physical inactivity during leisure time among overweight persons\u2014Behavioral Risk Factor Surveillance System, 1994. _Morbid. Mortal. Wkly Rept_. 1996;45:185\u2013188.\n\n57. McGinnis J.M., Foege W.H. Actual causes of death in the United States. _JAMA_. 1993;270:2207\u20132212.\nEpilogue\n\nJANET KING, USDA, Western Human Nutrition Research Center, University of California at Davis, Davis, California\n\nThis book, _Nutrition in the Prevention and Treatment of Disease,_ emphasizes the past and current scientific evidence of the role played by nutrition in the prevention and treatment of chronic disease. New emerging areas of study are discussed with an emphasis on the _evolutionary_ process in science and its application. Thus, the book prepares the clinical nutrition investigator or practitioner for a lifelong commitment to learning and change.\n\nAs we start the 21st century, we have entered a new era of nutrition in the prevention and treatment of disease. The definition of clinical nutrition has changed. Today, clinical nutrition involves the integration of diet, genetics, environment, and behavior to promote health and well-being throughout life. This definition requires the capacity to deal with complexity and to utilize multidisciplinary approaches. Now, more than ever before, nutritionists need broad training in a variety of disciplines while being expert in one well-defined aspect.\n\nThe complexity of the relationship between diet, genetics, environment, and behavior is demonstrated by the unfolding story of diet and atherosclerotic heart disease. Early investigators discovered the links between dietary cholesterol, saturated fats, and atherosclerosis. Later, relationships with polyunsaturated fats were explored. This led to studies of monounsaturates, fish oils, and _trans_ fatty acids. More recently, body iron and ferritin levels have been linked to coronary heart disease, as have intakes of antioxidants and folic acid. Also, evidence is emerging that immune function may play a role. The picture becomes much less clear as conflicting data on the role of specific fats, other nutrients, food components, and dietary patterns evolve. This is only part of the picture, however. Epidemiological studies show that gender, perhaps racial and ethnic background, and personal behaviors, such as physical activity, also influence progression of the disorder.\n\nWhen one adds the effect of the interindividual variations in genes on risk factors for heart disease, the situation becomes overwhelming. Studies in the United States show that 50% of the variance in plasma cholesterol concentration is genetically determined, and the response of plasma cholesterol to dietary interventions is heterogeneous. For example, on a low-fat\/high-cholesterol diet, individuals with apoE4\/4 phenotype respond with an increase in serum cholesterol whereas those with apoE2\/2,3\/2, and 3\/4 do not [1]. Genetics and diet also influence low-density lipoprotein peak particle size. Low-fat diets induce an atherogenic lipoprotein phenotype characterized by a predominance of small, dense low-density lipoprotein particles (subclass pattern B) in healthy subjects who originally had large low-density lipoprotein particles (subclass pattern A) [2]. Also, feeding a very low-fat (10% of energy) diet to children of parents with pattern B induces the atherogenic small dense low-density lipoproteins; this did not occur in children of parents with pattern A [3]. Thus, the influence of genetics on response to a dietary intervention is evident in childhood. These data imply that it is not prudent to recommend a low-fat diet for all children and adults until the complex interaction between diet, genetics, and environment is better defined.\n\nDuring the last 25 years, the impact of diet and environment on disease was uncovered; during the next two decades, the impact of genetics will be added into the equation. This integration will not be easy. First, changes in the diet induce many regulatory responses that attenuate the effect of these changes on the organism's metabolism, making the response of individual genes subtle. Also, many genes are involved in the metabolic pathways so that any differences observed are due to groups of genes rather than individual genes. Furthermore, although the human genome has been identified, functions of the genes have only been described for a small portion of the DNA sequences. As we await data on gene function, new information about the phenotypic responses to dietary interventions, such as increases in serum cholesterol and lipoprotein concentrations, will be helpful in deriving appropriate dietary interventions. Data on phenotypic responses to diets can be used to match a dietary intervention to an individual's specific genetic pattern.\n\nEpidemiological studies help describe the link between diet and phenotypic responses. Nutritional epidemiology, or the study of diet and the occurrence of disease in human populations, also emerged within the past 25 years. During the next 25 years, nutritional and molecular epidemiology will merge. This will enable large-scale, population studies of the associations between dietary patterns and phenotypes for chronic disease. To date, most of the studies have linked dietary patterns with the _occurrence_ of disease, i.e., _trans_ fatty acid intake and cardiovascular disease, \u03b2-carotene intake and breast or lung cancer, and dietary fiber and colorectal cancer. Knowledge of the associations between diet and early biomarkers of disease is much more useful. That information will emerge as phenotypes associated with high risk for chronic disease are identified.\n\nIn sum, the tool chest of the clinical nutritionist 25\u201330 years ago included a diet manual, a good dietary history and food composition data table, a tape measure, and a pair of skinfold calipers. Using empirical data generated from those tools, simple dietary prescriptions were derived. Today, in the 21st century, the clinical nutritionist must have a broad understanding of the scientific basis for disease and the influence of diet, genetics, and the environment on the progress of the disorder. The capacity to deal with complex interactions across multiple disciplines is essential. At the same time, the clinical nutritionist or investigator in human nutrition needs a thorough, in-depth understanding of cultural and socioeconomic influences on food behaviors and the competency to integrate that knowledge with phenotypic or functional markers of the disease. Comprehensive textbooks, such as this one, that integrate basic principles and concepts across disciplines while showing how to apply this knowledge in new, creative ways are an essential part of the tool chest needed in this new era of practice.\n\n## References\n\n1. Miettinen T.A., Gylling H., Sanhanen H. Serum cholesterol response to dietary cholesterol and apolipoprotein E phenotype. _Lancet_. 1988;2:1261.\n\n2. Dreon D.M., Fernstrom H.A., Miller B., Krauss R.M. Apolipoprotein E isoform phenotype and LDL subclass response to a reduced-fat diet. _Arterioscler. Thromb. Vasc. Biol_. 1995;15:105\u2013111.\n\n3. Dreon D.M., Fernstrom H.A., Williams P.T., Krauss R.M. Reduced LDL particle size in children consuming a very-low-fat diet is related to parental LDL-subclass patterns. _Am. J. Clin. Nutr_. 2000;71:1611\u20131616.\n\n# Index\n\nA\n\nAcetate salts\n\nin parenteral nutrition, 251\u2013252\n\ntyrosinemia relationship\n\ndescription,\n\nnewborn screening programs, 210\u2013211\n\nnutritional therapy, , ,\n\nAcid reflux disease, description, , 552\u2013553\n\nAcquired immunodeficiency syndrome, _See_ HIV\/AIDS\n\nAcrocyanosis, anorexia nervosa\n\nassociation,\n\nAdditives, allergic reactions,\n\nAdequate intake\n\ndescription, 72\u201374,\n\ndietary reference intake levels, , 753\u2013760\n\nestimated average requirement, 72\u201374, , 759\u2013760\n\nfood guide pyramid, , ,\n\nrecommended dietary allowance, 72\u201374, 753\u2013760\n\nserving equivalencies, , 764\u2013765\n\ntolerable upper intake levels, ,\n\nAdiposity, _See also_ Obesity\n\nbreast cancer association, 342\u2013344\n\ndiabetes relationship, 419\u2013421\n\nlung cancer relationship,\n\nphysical activity role,\n\nsingle-gene adipose tissue atrophy,\n\nAdrenal hyperplasia, newborn screening programs, 210\u2013211\n\nAdvertising, obesity effects, 526\u2013527,\n\nAflatoxin, liver cancer association,\n\nAge, _See also_ Elderly; Pediatric care\n\nlearning relationship,\n\nresting energy expenditure measurement,\n\nAIDS, _See_ HIV\/AIDS\n\nAim for Fitness, health maintenance nutrition guidelines, ,\n\nAlagille syndrome, description,\n\nAlbumin, serum\n\ncystic fibrosis assessment, ,\n\nfood allergy role,\n\nmortality prediction,\n\nrenal disease assessment,\n\nsurgery outcome prediction,\n\nAlcohol consumption\n\nAlzheimer's disease relationship,\n\nbreast cancer risk, ,\n\ncognitive function relationship,\n\ncolon cancer relationship, , , 365\u2013366\n\ncongestive heart failure relationship,\n\nhypertension relationship, 311\u2013312,\n\nliver disease role,\n\nnutrient status effects,\n\nprostate cancer relationship, 375\u2013376\n\nAlcohol dehydrogenase, breast cancer\n\nassociation,\n\nAldosterone\n\ncongestive heart failure role, 325\u2013326\n\neating disorder treatment role, ,\n\nAllergies, _See_ Food allergy\n\nAllylic sulfides, health benefits, 296\u2013297\n\nAloe, health benefits,\n\nAlstrom syndrome, obesity onset,\n\nAlternative medicine, 401\u2013402 _See also_ Botanicals cancer management,\n\nAluminum\n\nbone mineralization inhibition, 737\u2013738\n\nosteomalacia relationship,\n\nosteoporosis relationship,\n\nrenal conservation of calcium,\n\nAlzheimer's disease\n\nenergy requirement effects,\n\nnutritional trials, 644\u2013645\n\nnutrition\u2013cognitive function relationship, 637\u2013641\n\nanimal studies, 637\u2013638\n\ncross-sectional studies, 638\u2013641\n\nlongitudinal studies, 638\u2013641\n\nobservational studies, 641\u2013642\n\noverview, ,\n\npathological studies,\n\ntreatment\n\nginkgo, ,\n\nmedical nutritional therapy, ,\n\nnutritional management, 645\u2013648\n\nweight loss management, 645\u2013646\n\nAmino acids, _See also_ specific amino acids\n\nappetite effects,\n\nenteral feeding formulas, 246\u2013248\n\ngenetic metabolic error treatment, 216\u2013218\n\nhypertension effects,\n\nparenteral feeding formulas, 250\u2013251,\n\nL-Amino acids, inborn errors in metabolism treatment,\n\nAmphetamines, nutrient status effects,\n\nAnaphylaxis, in food allergies, ,\n\nAnemia\n\nanorexia nervosa association,\n\ndrug therapies,\n\ninflammatory bowel disease association,\n\nphysical assessment,\n\nrenal disease association, 622\u2013623\n\nAngioedema, _See_ Food allergy\n\nAngiotensin II, congestive heart failure role, 325\u2013326\n\nAnorexia, _See also_ Binge eating; Bulimia\n\nin cancer patients\n\ncachexia association,\n\nnutrition management strategies,\n\ndiagnostic criteria,\n\netiology, 687\u2013688\n\ngenetic factors, 687\u2013688\n\nHIV\/AIDS relationship,\n\nin inflammatory bowel diseases,\n\nnutritional assessment, 688\u2013690\n\nclinical diagnosis, 689\u2013690\n\nenergy requirements, ,\n\nphysical examination,\n\nnutritional risk factors,\n\noutcome, 695\u2013696\n\noverview, ,\n\nprevalence, 685\u2013687\n\nprevention,\n\npsychogenic vomiting, 558\u2013559\n\ntreatment\n\ndrug therapies, , , , 691\u2013692\n\nnutritional management, , 690\u2013691\n\npsychologic therapies, 691\u2013692\n\nAntacids, gastrointestinal symptom treatment, 551\u2013552\n\nAnthocyanidins, cholesterol synthesis inhibition,\n\nAnthropometric assessment, _See also_ Clinical assessment\n\nbody fat distribution, , 47\u201348, ,\n\nbreast cancer risk, 342\u2013344\n\ncircumference measurement, , 47\u201348, ,\n\ncystic fibrosis assessment, 720\u2013721\n\nheight, 44\u201345\n\nnutrition intervention evaluation,\n\nrenal disease nutrition assessment, 623\u2013624\n\nskinfold thickness measurement, 47\u201348\n\nweight, 45\u201346,\n\nweight for height, 46\u201347\n\nAntibiotics, nutrient status effects,\n\nAnticonvulsants, nutrient status effects,\n\nAntidepressants\n\nanorexia nervosa therapy, ,\n\nbulimia therapy, 693\u2013694\n\nnutrient status effects,\n\nAntioxidants, _See also_ Oxidative stress; _specific antioxidants_\n\nallergic reactions,\n\nbreast cancer risk reduction, 340\u2013341\n\nin cancer treatment, 400\u2013401\n\ncardiovascular disease risk reduction, 295\u2013296\n\ncolon cancer effects, 361\u2013362\n\nApolipoprotein E receptor, soy protein effects,\n\nApolipoproteins\n\nexogenous lipoprotein pathway, 158\u2013164\n\napolipoprotein A-IV, 160\u2013161\n\napolipoprotein B, 158\u2013160\n\napolipoprotein C-III, 163\u2013164\n\napolipoprotein E, 161\u2013163\n\nreverse cholesterol transport role, 167\u2013170\n\napolipoprotein A-I, 167\u2013168\n\nAppetite\n\nenergy appetite,\n\ngenetic relationship, 539\u2013540\n\nin HIV\/AIDS patients, 746\u2013747\n\nobesity effects, 541\u2013542\n\nArachidonic acid, function biomarkers,\n\nArginine\n\nenteral nutrition,\n\nin soy protein,\n\nvasodilator function,\n\nArgininosuccinic aciduria, nutritional therapy,\n\nArtificial sweeteners\n\nbody weight effects, 503\u2013506\n\ngestational diabetes management,\n\nAscorbic acid\n\nbiomarkers of exposure, ,\n\nbreast cancer risk reduction, , ,\n\ncognitive function relationship, 638\u2013642\n\ncolon cancer relationship, ,\n\ndeficiency signs,\n\ndietary reference intakes, , ,\n\nlung cancer relationship, ,\n\noxidative stress reduction,\n\nin parenteral nutrition supplements,\n\nParkinson's disease relationship,\n\nrenal disease therapy, ,\n\nsmoking effects,\n\nAspartame, body weight effects, 503\u2013506\n\nAspiration pneumonia, enteral feeding\n\ncomplications,\n\nAspirin, food interactions,\n\nAssessment methods, _See_ Dietary assessment; Physical assessment; _specific methods_\n\nAstragalus, health benefits,\n\nAtenolol, food interactions,\n\nAtkins diet, description, , ,\n\nAtopic dermatitis, in allergic food reactions, 707\u2013708\n\nATP-binding cassette 1, reverse cholesterol transport role, 169\u2013170\n\nAutoimmune hepatitis, description,\n\nAzathioprine, inflammatory bowel disease treatment,\n\nB\n\nBardet\u2013Biedl syndrome, obesity onset,\n\nBasal energy expenditure, definition,\n\nBasal metabolic rate, definition, 31\u201332\n\nBeano\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nBeans, excessive gas production, 550\u2013551\n\nBehavioral Risk Factor Surveillance System, brief assessment, 7\u20138\n\nBehavior modification, _See also_ Nutrition intervention\n\nbulimia therapy,\n\ngestational diabetes management, 455\u2013456\n\ninfluences\n\nage,\n\nbarriers to action, 90\u201391\n\nbinge eating, , , 694\u2013695\n\nbiobehavioral factors,\n\ncontrol beliefs,\n\ncounseling implications, ,\n\nculture,\n\ndiet programs,\n\nfamily influences, 528\u2013529\n\nfood choice, , 540\u2013541\n\ninfluence levels, 83\u201384\n\nintention versus action,\n\nlearner style, 106\u2013107\n\nliteracy, 105\u2013106\n\nlong-term change, 84\u201390, 219\u2013220\n\nmeal patterns,\n\nmotivation versus intention,\n\npacing,\n\nrecommendation reactions,\n\nrestraint, 527\u2013528\n\nself-efficacy, ,\n\nspecial populations,\n\ntechniques\n\nconsciousness raising, 113\u2013115\n\neducation techniques, 107\u2013110\n\nlearning needs assessment, 107\u2013108\n\nlevels of education,\n\nmethods, 108\u2013109\n\nschool and community-based youth programs, 529\u2013531\n\ntools, 109\u2013110\n\ngoal setting, 117\u2013120\n\ncoping strategies,\n\nproblem-solving skills,\n\nself-management skills training, 118\u2013119\n\nsocial support,\n\nimportance and confidence technique, 115\u2013117\n\nlearning domains, 106\u2013107\n\nmotivational intervention method, 98\u2013102\n\nnot ready to change level, 98\u2013101\n\nready to change level,\n\nunsure about change level,\n\nmotivational interviews,\n\nmotivation models, 95\u201396\n\ndecisional balance,\n\nhealth belief model, 95\u201396\n\nRokeach's value theory,\n\nself-regulation theory, , 118\u2013119\n\noverview, ,\n\nself-reported dietary behavior assessment, , 130\u2013134\n\nstages of change technique, 111\u2013113\n\nteaching\/learning process, 105\u2013107\n\nweight reduction effects,\n\nBenign prostatic hyperplasia, description,\n\nBenzphetamine, obesity intervention,\n\nBeriberi, description,\n\nBeta blockers, congestive heart failure treatment,\n\nBeta-carotene\n\nanorexia nervosa association, 689\u2013690\n\nantioxidant properties,\n\nbiomarker indicators,\n\nbreast cancer risk reduction, , ,\n\ncognitive function relationship, 638\u2013640\n\ncolon cancer relationship, ,\n\ncontent in foods,\n\nlung cancer relationship, 388\u2013390\n\nParkinson's disease relationship,\n\nprostate cancer risk reduction, 379\u2013380\n\ntoxicity signs,\n\nBiliary tract\n\nbile acid sequestrants, nutrient status effects,\n\nbiliary atresia, 606\u2013607\n\ncholestasis\n\nByler's syndrome,\n\ncholedochal cysts,\n\ncholedocholithiasis,\n\ngenetic defects,\n\nneonatal hepatitis association,\n\nnutritional therapy, , 608\u2013609\n\nprimary sclerosing cholangitis,\n\ncolon cancer relationship, 358\u2013359\n\ncystic fibrosis relationship, 716\u2013717\n\nelectrolyte composition,\n\nmetabolism,\n\nprimary biliary cirrhosis,\n\nin short bowel syndrome, 589\u2013591,\n\nBinge eating, _See also_ Bulimia\n\ndiagnostic criteria, 685\u2013686, 694\u2013695\n\nmanagement,\n\nobesity effects,\n\noverview,\n\nBioelectrical impedance analysis, HIV\/AIDS assessment, 745\u2013746\n\nBiomarkers of change\n\nexposure measurement\n\nenergy intake, 142\u2013143\n\nepidemiology exposure,\n\ngeneral dietary indicators,\n\nmicromolecule intake, 140\u2013142\n\nnutrient intake, 139\u2013140\n\nfunctional biomarkers, 143\u2013147\n\nbone health, 146\u2013147\n\ncell turnover,\n\nenzymes, 144\u2013145\n\nimmune function,\n\noxidative stress, 145\u2013146\n\ngenetic susceptibility biomarkers, 147\u2013148\n\nintervention evaluation,\n\noverview, ,\n\nselection, 148\u2013149\n\nBiotin\n\nbiomarkers of exposure,\n\ndeficiency signs, , 210\u2013211\n\ndietary reference intakes,\n\nin parenteral nutrition supplements,\n\nBiotinidase deficiency, newborn screening programs, 210\u2013211\n\nBismuth subsalicylate, gastrointestinal symptom treatment, 551\u2013552\n\nBlack cohosh\n\ncancer management,\n\nhealth benefits,\n\nBlood glucose\n\nappetite effects,\n\ndiabetes management\n\ngestational diabetes, ,\n\ntype 1 diabetes,\n\nBlood pressure, _See_ Hypertension; Hypotension\n\nBody composition\n\nelectrolyte composition, 251\u2013252\n\nHIV\/AIDS nutritional assessment, 745\u2013746\n\nresting energy expenditure measurement,\n\nBody fat, _See also_ Body mass index; Dietary fat; Obesity\n\nbody mass index correlation, , 465\u2013467,\n\ndiabetes relationship, visceral adiposity, 419\u2013421\n\ndistribution\n\ncircumference measurement, , 47\u201348, ,\n\nfat-free mass, ,\n\nhypercortisolism relationship,\n\nBody fluids, _See also_ Liquid dietary supplements\n\nelectrolyte composition, 251\u2013252\n\nfluid intake\n\nanorexia nervosa therapy,\n\nintravenous feeding, 251\u2013252\n\nrenal disease therapy, 619\u2013620, 628\u2013629\n\nrestriction during congestive heart failure,\n\nBody mass index\n\nbody fat assessment, , 465\u2013467,\n\nin cancer management, 399\u2013400\n\ndiabetes risk, 415\u2013417\n\ndietary fiber intake relationship,\n\nHIV\/AIDS assessment,\n\nlimitations,\n\nmortality risk,\n\nweight for height measurement, , 46\u201347\n\nBody weight, _See also_ Behavior modification; Obesity\n\nanthropometric assessment\n\nmethods, 45\u201346,\n\nweight for height, 46\u201347\n\ngain\n\nin cancer patients, 346\u2013347,\n\nin gestational diabetes management, 459\u2013460\n\ngenetic factors, 183\u2013205\n\ncandidate gene association studies, 189\u2013192\n\naltered function causes, 190\u2013192\n\nsingle-gene obesity role,\n\nclinical implications, 192\u2013194\n\ndiagnosis, 192\u2013193\n\ntreatment,\n\nepidemiology, 183\u2013184\n\ngene\u2013environment interactions,\n\ngene map, 184\u2013185\n\noverview, 183\u2013184\n\nphenotype evidence from linkage studies, 188\u2013189\n\nhuman studies,\n\nloci mapping in animals, 188\u2013189\n\nmouse studies,\n\nrare Mendelian syndromes,\n\nsingle-gene adipose tissue atrophy,\n\nsingle-gene obesity, 185\u2013188\n\nleptin deficiency, 185\u2013186,\n\nleptin receptor deficiency, 186\u2013187\n\nmelanocortin-4 receptor gene mutation, 187\u2013188\n\nproopiomelanocortin deficiency,\n\nproprotein convertase subtilisin\/kexin type 1 deficiency,\n\ntaste and appetite relationship, 539\u2013540\n\nhealthy weight guidelines,\n\nhypertension effects, 311\u2013312, ,\n\nloss\n\nin Alzheimer's disease patients, 645\u2013646\n\nin cancer patients, 233\u2013234, , ,\n\ndrug therapies,\n\nin HIV\/AIDS patients\n\nassessment, 744\u2013745\n\ncauses, 742\u2013744, 743\u2013744\n\nmechanisms, 741\u2013742\n\ninsulin sensitivity effects, 421\u2013422\n\nin Parkinson's disease patients,\n\nmacronutrient intake effects, 499\u2013509\n\ncarbohydrates, 502\u2013503\n\ncausal studies, 499\u2013501\n\nclinical studies, 500\u2013501\n\nlaboratory studies,\n\ndietary fats,\n\nenergy density, 501\u2013502\n\nfat density, 501\u2013502\n\nfat substitutes, 503\u2013506\n\noverview, , 508\u2013509\n\nprotein role, 506\u2013508\n\nsugar substitutes, 503\u2013506\n\nresting energy expenditure measurement,\n\nBone density, osteoporosis relationship,\n\nBone disease, _See_ Osteomalacia; Osteoporosis\n\nBone marrow transplantation, nutritional effects, 408\u2013410\n\nBone mineralization\n\ninhibitors, 737\u2013738\n\nmechanisms, 729\u2013730\n\nphysical assessment,\n\nBotanicals, 261\u2013271\n\nadverse effects,\n\nclinical recommendations, 269\u2013271\n\ncost effectiveness,\n\ndietetics professionals, 270\u2013271\n\nlegal issues, 270\u2013271\n\nmedical professionals, 270\u2013271\n\nefficacy, 263\u2013264\n\nhistorical perspectives,\n\noverview, , 271\u2013272\n\npatients,\n\npopularity factors, 262\u2013263\n\nregulation, 268\u2013269\n\nreported use, 261\u2013262\n\nsafety,\n\nsupporting evidence, 264\u2013268\n\nechinacea, 264\u2013265, , ,\n\nfeverfew, ,\n\ngarlic, , , , 296\u2013297\n\nginkgo,\n\nsaw palmetto, 267\u2013268\n\nscientific process,\n\nSt. John's wort, , ,\n\nvalerian,\n\nBowel disease, _See_ Gastrointestinal system\n\nBranched-chain ketoaciduria\n\nnewborn screening programs, 210\u2013212\n\nnutritional therapy, ,\n\nBreast cancer\n\ngenetic factors, 337\u2013338\n\nmammary cell biology, 337\u2013338\n\nnutritional factors, 338\u2013345\n\ndietary fat association, 339\u2013345\n\ndiet\u2013genetic interactions, 204\u2013205, 337\u2013338\n\nprimary risk factors, 338\u2013344\n\nadiposity association, 342\u2013344\n\nanthropometric factors, 342\u2013344\n\ndietary intake, 338\u2013342\n\nphysical activity effects,\n\nskeletal marker association,\n\nprognosis, 344\u2013345\n\nrecurrence risk, 344\u2013345\n\noverview, 337\u2013338, 349\u2013350\n\npost diagnosis issues, 345\u2013349\n\ninitial treatment, 346\u2013348\n\nadjuvant chemotherapy, 346\u2013347\n\nhormone therapy, 347\u2013348\n\nradiation therapy,\n\nsurgery-related nutritional issues,\n\nlong-term perspective, 348\u2013349\n\nscreening, 345\u2013346\n\nstaging methods,\n\nsurgical side effects,\n\nBreath tests, lactose intolerance measurement,\n\nBrief assessment\n\nadvantages and disadvantages,\n\nBehavioral Risk Factor Surveillance System, 7\u20138\n\nfood frequency questionnaires, 7\u20138, 132\u2013133\n\nmethods, 7\u20138\n\nBuild a Healthy Base, Dietary Guidelines for Americans, ,\n\nBulimia, _See also_ Anorexia; Binge eating\n\ndiagnostic criteria,\n\netiology, 687\u2013688\n\ngenetic factors, 687\u2013688\n\nnutritional assessment, 688\u2013690\n\nclinical diagnosis, 689\u2013690\n\nenergy requirements,\n\nphysical examination,\n\nnutritional management, 690\u2013691\n\nnutritional risk factors,\n\noverview, ,\n\npharmacologic management, 691\u2013692\n\nprevalence, 685\u2013687\n\npsychogenic vomiting, 558\u2013559\n\npsychologic therapies, 691\u2013692\n\nBurns, energy requirement effects,\n\nByler's syndrome, description,\n\nC\n\nCachexia\n\ncancer cachexia, nutritional factors, 398\u2013399\n\nanorexia effects,\n\nintake inadequacies,\n\nmetabolic abnormalities, 398\u2013399\n\ncardiac cachexia, mechanisms, 330\u2013331\n\ntreatment\n\nmedical nutritional therapy, 234\u2013235\n\npharmaceutical management,\n\nCadmium, prostate cancer relationship,\n\nCafestol palmitate, colon cancer relationship,\n\nCaffeine\n\nbreast cancer risk,\n\ncalcium absorption effects,\n\ncongestive heart failure effects,\n\nfood allergy role,\n\nobesity intervention, 472\u2013473\n\nParkinson's disease relationship,\n\nCalcium\n\nbiomarkers of exposure,\n\nbone mineralization, 729\u2013730\n\ncolon cancer relationship, ,\n\ndaily requirements, 251\u2013252\n\ndeficiency, in liver disease,\n\ndietary reference intakes, , 756\u2013757,\n\nhypertension effects, 308\u2013309,\n\nlactose intolerance effects,\n\nosteomalacia relationship,\n\nosteoporosis relationship\n\nlife stage requirements, 662\u2013670\n\ngrowth, 663\u2013666\n\nlactation, 667\u2013668\n\nmaturity, 666\u2013667\n\nmenopause, 668\u2013669\n\npregnancy, 667\u2013668\n\nsenescence, 669\u2013670\n\nnatural intake, 658\u2013659\n\nnutritional influences, 670\u2013672\n\nbone density,\n\ncaffeine,\n\ncalcium absorption enhancers,\n\nfat,\n\nfiber,\n\nintake versus interference,\n\nintestinal absorption,\n\nphosphorus,\n\nprotein, 671\u2013672\n\nrenal conservation of calcium, 671\u2013672\n\nsodium, 671\u2013672\n\nnutritional requirement, 660\u2013662, 670\u2013672\n\nskeletal reserves,\n\nparenteral nutrition, 251\u2013252,\n\nParkinson's disease therapy, 647\u2013648\n\nprostate cancer relationship, 376\u2013377\n\nrenal disease therapy, , , 628\u2013629\n\nCalibration studies, dietary assessment methods, , 18\u201319\n\nCalorimetry\n\nresting energy expenditure measurement,\n\ntotal energy expenditure measurement,\n\nCampesterol, health benefits, 296\u2013297\n\nCancer, _See also_ specific types\n\nnutritional factors\n\ncachexia, 398\u2013399\n\nanorexia effects, ,\n\nintake inadequacies,\n\nmetabolic abnormalities, 398\u2013399\n\npharmaceutical management,\n\ndiet\u2013genetic interactions\n\ncarcinogen metabolism,\n\nenzyme induction,\n\nfuture research directions,\n\nmethodologic issues, 202\u2013203\n\none-carbon metabolism, 201\u2013202\n\noverview, 199\u2013200\n\noxidative stress, 200\u2013201\n\nvitamin pathway variation,\n\nissues, 401\u2013410\n\nadvanced cancer,\n\nbone marrow transplantation, 408\u2013410\n\nchemotherapy, , 406\u2013407\n\nenteral nutrition, ,\n\nnutritional caregiving, 403\u2013406,\n\nnutritional screening,\n\noral intake,\n\npalliative care,\n\nparenteral nutrition, ,\n\npediatric patients,\n\nradiation therapy,\n\nrecovery, 407\u2013408\n\nsurgery,\n\nmanagement concepts, 399\u2013401\n\nalternative medicine, 401\u2013402\n\nbody weight management, 399\u2013400\n\ncomplementary medicine, 401\u2013402\n\nenergy intake, 399\u2013400\n\nmicronutrients,\n\nphysical activity role, ,\n\nprotein intake, 399\u2013400\n\nmedical nutritional therapy, , 233\u2013235\n\nnutrition guidelines,\n\noptimal nutrition, 397\u2013398\n\noverview, ,\n\nosteomalacia induction, 736\u2013737\n\nphysical assessment,\n\nweight loss, 233\u2013234, ,\n\nCapsaicin, cardiovascular disease effects,\n\nCarbamyl phosphate synthetase deficiency, nutritional therapy,\n\nCarbohydrates\n\nbody weight effects\n\nconsumption, 502\u2013503\n\nsugar substitutes, 503\u2013506\n\ncardiovascular disease effects, 282\u2013284\n\nfiber, 283\u2013284, 291\u2013293,\n\nfructooligosaccharides,\n\nglycemic index, ,\n\nresistant starch,\n\ndiabetes nutritional management\n\ngestational diabetes\n\ncarbohydrate consistency,\n\ncarbohydrate distribution,\n\ncarbohydrate type,\n\nmorning carbohydrate, 456\u2013458\n\ntotal carbohydrate,\n\ntype 1 diabetes, 431\u2013433\n\ntype 2 diabetes, 444\u2013445\n\nexcessive gas production, 550\u2013551\n\nfiber, _See_ Dietary fiber\n\nhypertension effects, ,\n\ninflammatory bowel disease effects,\n\nlow-fat high-carbohydrate diet, 281\u2013283\n\nmetabolic liver disease,\n\nin Parkinson's disease therapy, 647\u2013648\n\nrenal disease therapy, ,\n\nCarbonated beverages, excessive gas production, 550\u2013551\n\nCardiovascular disease\n\ncardiomyopathy, _See_ Congestive heart failure\n\ncongestive heart failure, 325\u2013331\n\nalcohol effects,\n\ncaffeine effects,\n\ncardiac cachexia, 330\u2013331\n\ndrug interactions,\n\nfluid intake restriction,\n\nnutritional supplements, 329\u2013330\n\nnutrition recommendations, 328\u2013329\n\noverview,\n\npathophysiology, 325\u2013326\n\nprevalence,\n\nright-sided heart failure,\n\nsodium restriction, 326\u2013327\n\nstandard medical care,\n\ntobacco effects, 328\u2013329\n\ndietary effects, 279\u2013286\n\ncarbohydrates, 282\u2013284\n\nfiber, 283\u2013284, 291\u2013293,\n\nfructooligosaccharides,\n\nglycemic index, ,\n\nresistant starch,\n\nfat, 279\u2013282\n\nmonounsaturated fatty acids, 279\u2013282\n\nomega-3 fatty acids,\n\nomega-6 fatty acids,\n\npolyunsaturated fatty acids,\n\nsaturated fatty acids, 279\u2013281,\n\ntotal fat, 279\u2013280,\n\n_trans_ fatty acids, 279\u2013280,\n\nunsaturated fatty acids, 281\u2013282\n\nhomocysteine, 293\u2013294\n\noverview, ,\n\nprotein, 284\u2013286\n\nanimal protein, , 284\u2013285\n\nsoy protein, , 285\u2013286, 294\u2013295\n\ngenetic influences, 157\u2013171\n\nendogenous lipoprotein metabolism, 166\u2013167\n\n3-hydroxy-3-methylglutaryl-coenzyme A reductase,\n\nlow-density lipoprotein receptor, 166\u2013167\n\nexogenous lipoprotein pathway, 158\u2013166\n\napolipoprotein A-IV, 160\u2013161\n\napolipoprotein B, 158\u2013160\n\napolipoprotein C-III, 163\u2013164\n\napolipoprotein E, 161\u2013163\n\nintestinal fatty acid binding protein, 165\u2013166\n\nlipoprotein lipase, 164\u2013165\n\nmicrosomal triglyceride transfer protein,\n\noverview, 157\u2013158, 170\u2013171\n\nreverse cholesterol transport, 167\u2013170\n\napolipoprotein A-I, 167\u2013168\n\nATP-binding cassette , , 169\u2013170\n\ncholesterol 7 alpha-hydroxylase,\n\ncholesteryl ester transfer protein,\n\nhepatic lipase, 168\u2013169\n\nscavenger receptor B type I,\n\nphysical activity role, 493\u2013494\n\nphysical assessment,\n\nrenal disease association,\n\nrisk factors,\n\nstroke therapy,\n\nCarotenoids\n\nanorexia nervosa association, 689\u2013690\n\nantioxidant properties,\n\nbiomarker indicators,\n\nbreast cancer risk reduction, , ,\n\ncognitive function relationship, 638\u2013640\n\ncolon cancer relationship, ,\n\ncontent in foods,\n\nlung cancer relationship, 388\u2013390\n\nParkinson's disease relationship,\n\nprostate cancer risk reduction, 379\u2013380\n\ntoxicity signs,\n\nCarvedilol, food interactions,\n\nCase-control studies\n\ndescription, 10\u201312\n\nnutritional epidemiology study,\n\nCatechins, cardiovascular disease effects,\n\nCatopril, food interactions,\n\nCat's claw, health benefits,\n\nCell growth\n\nbreast cancer biology, 337\u2013338\n\nregulator model in colon cancer, 363\u2013365\n\ncalcium role, ,\n\nvitamin D role, ,\n\nCell turnover, functional biomarkers,\n\nCerebral palsy, energy requirement effects,\n\nCerebral vascular accident, medical nutritional therapy, , 231\u2013232\n\nChamomile, health benefits,\n\nChemotherapy\n\nbreast cancer treatment, nutritional issues, 346\u2013347\n\ncancer treatment, nutritional issues, , 406\u2013407\n\nChildren, _See_ Pediatric care\n\nChinese restaurant syndrome, description,\n\nChloride\n\ndaily requirements, 251\u2013252\n\nin parenteral nutrition, 251\u2013252,\n\nChlorothiazide, food interactions,\n\nCholestasis\n\nByler's syndrome,\n\ncholedochal cysts,\n\ncholedocholithiasis,\n\ngenetic defects,\n\nneonatal hepatitis association,\n\nnutritional therapy, , 608\u2013609\n\nprimary sclerosing cholangitis,\n\nCholesterol\n\ncardiovascular disease risk reduction, , 279\u2013282, 296\u2013298,\n\ncognitive function relationship,\n\nhypercholesterolemia\n\nanimal protein effects, 284\u2013285\n\nanorexia nervosa association,\n\ndiet strategies, , , 283\u2013285\n\nfiber effects, ,\n\nlung cancer relationship,\n\nrenal disease relationship, 624\u2013625\n\nreverse cholesterol transport, 167\u2013170\n\napolipoprotein A-I, 167\u2013168\n\nATP-binding cassette , , 169\u2013170\n\ncholesterol 7 alpha-hydroxylase,\n\ncholesteryl ester transfer protein,\n\nhepatic lipase, 168\u2013169\n\nscavenger receptor B type I,\n\nCholesterol 7 alpha-hydroxylase, reverse cholesterol transport role,\n\nCholesteryl ester transfer protein, reverse cholesterol transport role,\n\nCholine, dietary reference intakes, ,\n\nChromium\n\ndiabetes management,\n\nin parenteral nutrition supplements,\n\nChronic disease, _See_ Nutritional epidemiology; _specific diseases_\n\nChronic obstructive pulmonary disease\n\ncystic fibrosis relationship,\n\nenteral feeding formulas,\n\nmedical nutritional therapy, 229\u2013230, 235\u2013236\n\nphysical assessment methods,\n\nCiprofloxacin, inflammatory bowel disease treatment,\n\nCircumference measurement, body fat assessment, , 47\u201348, ,\n\nCirrhosis\n\nalcohol-induced cirrhosis,\n\nend-stage nutritional therapy,\n\nprimary biliary cirrhosis,\n\nClinical assessment, _See also_ Anthropometric assessment\n\nbotanical recommendations, 269\u2013271\n\ncost effectiveness,\n\ndietetics professionals, 270\u2013271\n\nlegal issues, 270\u2013271\n\nmedical professionals, 270\u2013271\n\ngestational diabetes, ,\n\nlimitations,\n\nnutrition intervention trials, 95\u2013103\n\nDiabetes Control and Complications Trial, , ,\n\nDiet Intervention Study in Children, 96\u201398\n\nModification of Diet and Renal Disease Study, 102\u2013103\n\nmotivational intervention method, 98\u2013102\n\nnot ready to change level, 98\u2013101\n\nready to change level,\n\nunsure about change level,\n\nmotivation models, 95\u201396\n\ndecisional balance,\n\nhealth belief model, 95\u201396\n\nRokeach's value theory,\n\nself-regulation theory, , 118\u2013119\n\noverview, ,\n\nWomen's Health Initiative,\n\nobesity genetics, 192\u2013194\n\ndiagnosis, 192\u2013193\n\ntreatment,\n\nphysical assessment\n\nAIDS,\n\ncancer,\n\ncardiovascular system,\n\ngastrointestinal system, 51\u201352\n\nhair,\n\nhematologic system,\n\ninfectious disease,\n\nmethods, 11\u201312, 43\u201344\n\nmusculoskeletal system,\n\nnails,\n\nneurologic disorders,\n\noral and dental health, 49\u201351\n\npsychiatric disorders,\n\npulmonary system,\n\nrenal system, 52\u201353, 623\u2013627\n\nskin,\n\nscreening, 11\u201312\n\nCoenzyme Q10, congestive heart failure effects, ,\n\nCognitive research\n\ngingko effects, 644\u2013645\n\nlearning domains, 106\u2013107\n\nnutrition\u2013cognitive function relationship, 637\u2013641\n\nanimal studies, 637\u2013638\n\ncross-sectional studies, 638\u2013641\n\nlongitudinal studies, 638\u2013641\n\nnutrition intervention theoretical bases, 85\u201386,\n\nsocial cognitive theory\n\ndietary assessment issues,\n\nnutrition intervention theoretical bases, 85\u201386,\n\nCohort studies\n\ndescription, 11\u201312\n\nnutritional epidemiology study, 61\u201362\n\nColic, description,\n\nCollagen, bone mineralization role, 729\u2013730\n\nColon cancer, _See also_ Gastrointestinal system\n\ncell growth regulator model, 363\u2013365\n\ncalcium role, ,\n\nvitamin D role, ,\n\nDNA methylation model, 358\u2013359, ,\n\nalcohol association, , , 365\u2013366\n\nfolate role, , , 365\u2013366\n\nmethionine role, ,\n\nvitamin B6 role, ,\n\nfood intake relationships\n\ncooked foods model, , ,\n\ndietary interactions, 364\u2013366\n\ndietary patterns,\n\ndiet\u2013genetic interactions, 203\u2013204\n\ngenetic interactions, 364\u2013366\n\nnutrient model, 357\u2013362\n\nantioxidants, 361\u2013362\n\ndietary fat, , ,\n\ndietary fiber, 358\u2013361,\n\nmeat, 358\u2013360,\n\nplant foods,\n\nvegetables, , ,\n\nin inflammatory bowel diseases,\n\ninsulin resistance model, , 362\u2013363\n\nenergy balance, ,\n\nglycemic index, , 362\u2013363,\n\nsugar consumption, 362\u2013363\n\noverview,\n\nprevention,\n\nsurgical side effects,\n\nCompazine\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nComplementary medicine, _See also_ Botanicals\n\ncancer management, 401\u2013402\n\nComputer-based analysis\n\ndata analysis in nutritional epidemiology, 69\u201371\n\nnutrient database choice, , 69\u201370\n\nsoftware selection, 16\u201317\n\nCongenital adrenal hyperplasia, newborn screening programs, 210\u2013211\n\nCongenital hypothyroidism, newborn screening programs, 210\u2013211\n\nCongestive heart failure, 325\u2013331\n\nalcohol effects,\n\ncaffeine effects,\n\ncardiac cachexia, 330\u2013331\n\ndrug interactions,\n\nfluid intake restriction,\n\nnutritional supplements, 329\u2013330\n\nnutrition recommendations, 328\u2013329\n\noverview,\n\npathophysiology, 325\u2013326\n\nprevalence,\n\nright-sided heart failure,\n\nsodium restriction, 326\u2013327\n\nstandard medical care,\n\ntobacco effects, 328\u2013329\n\nConglutin, food allergy role,\n\nConsciousness raising techniques, behavior change, 113\u2013115\n\nConstipation\n\nanorexia nervosa association,\n\ncystic fibrosis association,\n\nfood effects, 556\u2013558,\n\nprevalence,\n\ntherapy, , ,\n\nConsumer information, food choice decisions,\n\nControl beliefs, behavior change influences,\n\nCooked foods\n\ncolon cancer relationship, 358\u2013359, ,\n\nlung cancer relationship, 392\u2013393\n\nprostate cancer relationship, 375\u2013376\n\nCoping strategies, goal setting,\n\nCopper\n\nbiomarkers of exposure,\n\ndeficiency\n\nin liver disease, , ,\n\nWilson's disease, ,\n\nosteoporosis relationship,\n\nin parenteral nutrition supplements,\n\ntoxicity signs,\n\nCoronary heart disease, _See_ Cardiovascular disease\n\nCorrectol\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nCorticosteroids, nutrient status effects,\n\nCounseling, _See_ Nutritional counseling\n\nCranberries, health benefits,\n\nCreatinine, renal disease relationship, 625\u2013627\n\nCrohn's disease\n\ndietary factors, 581\u2013584\n\nfood allergies,\n\nnutritional-related problems, 582\u2013584\n\nspecific foods, 581\u2013582\n\netiology, 581\u2013582\n\nnutrition assessment, 585\u2013586\n\noverview, 577\u2013578,\n\npathology, 580\u2013581\n\nprevalence, 578\u2013580\n\nshort bowel syndrome\n\ncauses,\n\nmedical management, 594\u2013595\n\nnutritional management, 593\u2013594\n\noverview, , ,\n\nsurgery, 594\u2013595\n\nvariants, 589\u2013593\n\ncolon loss, 591\u2013592\n\nileal resections, 590\u2013591\n\nileum loss, 591\u2013592\n\njejunal resections,\n\nmajor intestinal resections, 592\u2013593\n\ntreatment, 584\u2013587\n\ndiet effectiveness,\n\nenteral nutrition, 587\u2013589\n\nmedical management, 584\u2013585\n\nnutritional treatment, 585\u2013587\n\nparenteral nutrition, 587\u2013589\n\nsurgery,\n\nCross-sectional surveys\n\ndescription, 9\u201310,\n\nnutrition\u2013cognitive function relationship, 638\u2013641\n\nCrustaceans, allergic reactions, 705\u2013706\n\nCultural differences\n\ndietary assessment methods, , 13\u201314\n\nfood frequency questionnaires, 5\u20136,\n\nnutritional epidemiology study,\n\nnutritional learning influence,\n\nprostate cancer incidence, 374\u2013375\n\nCyclosporine, inflammatory bowel disease treatment,\n\nCysteine, HIV\/AIDS patient recommendations, 747\u2013748\n\nCystic fibrosis\n\nclinical presentation,\n\ndiagnosis, 715\u2013716\n\nnewborn screening programs, 210\u2013211,\n\nnutritional assessment, 719\u2013722\n\nanthropometric assessment, 720\u2013721\n\nbiochemical assessment,\n\ndietary assessment, 720\u2013722\n\nnutritional problems\n\nbiochemical abnormalities,\n\ndeficiencies, 717\u2013718\n\ngrowth impairment,\n\nmalnutrition\n\ncauses, 716\u2013717\n\nprevalence, 718\u2013719\n\nsuboptimal dietary intakes,\n\noverview, , 715\u2013716, 724\u2013725\n\npathogenesis,\n\ntreatment\n\nclinical fibrosis,\n\nnutrition management, 722\u2013724\n\nat diagnosis,\n\nroutine management, 722\u2013723\n\nCytokines, inflammatory bowel disease association, 580\u2013581\n\nD\n\nDairy products\n\nallergic reactions, _See_ Food allergy\n\nexcessive gas production, 550\u2013551\n\ninflammatory bowel disease association,\n\nlactose intolerance, 563\u2013572\n\ncalcium intake effects, ,\n\ndiagnosis, 564\u2013566\n\ndirect assessment, 564\u2013565\n\nindirect assessment, 565\u2013566\n\nlactase activity loss,\n\nlactose digestion, ,\n\nlactose sources, 563\u2013564\n\nmanagement, 567\u2013572\n\ncolonic fermentation, 570\u2013571\n\ndose response, 567\u2013568\n\ngastrointestinal transit time,\n\ngene therapy, 571\u2013572\n\nlactase supplements,\n\nlactose-reduced milks,\n\nunfermented acidophilus milk, 569\u2013570\n\nyogurt, 568\u2013569\n\nosteoporosis association,\n\noverview, ,\n\nprevalence,\n\nsymptoms, 566\u2013567\n\nprostate cancer relationship, 375\u2013376\n\nDatabase of nutrients, selection, , 69\u201370\n\nDecisional balance model, behavioral motivation,\n\nDehydration\n\neating disorder association, ,\n\ninborn error in metabolism treatment,\n\nintravenous feeding complications,\n\nDelayed-type hypersensitivity skin test, immune function measurement,\n\nDementia\n\nenergy requirement effects,\n\nnutritional trials, 644\u2013645\n\nnutrition\u2013cognitive function relationship, 637\u2013641\n\nanimal studies, 637\u2013638\n\ncross-sectional studies, 638\u2013641\n\nlongitudinal studies, 638\u2013641\n\nobservational studies, 641\u2013642\n\noverview, ,\n\npathological studies,\n\ntreatment\n\nginkgo, ,\n\nmedical nutritional therapy, ,\n\nnutritional management, 645\u2013648\n\nweight loss management, 645\u2013646\n\nDental health, physical assessment, 49\u201351\n\nDepression\n\nanorexia nervosa therapy, ,\n\nbulimia therapy, 693\u2013694\n\nnutrient status effects,\n\nSt. John's wort effects, , ,\n\nDermatitis, in allergic food reactions, 707\u2013708\n\nDesferroxamine, osteomalacia treatment,\n\nDexfenfluramine, obesity intervention,\n\nDextrose, in parenteral feeding formulas, 250\u2013251,\n\nDiabetes\n\nDiabetes Control and Complications Trial, , , ,\n\nnutrition management\n\nenteral feeding formulas,\n\ngestational diabetes, 453\u2013461\n\nartificial sweeteners,\n\nbehavioral outcomes, 455\u2013456\n\nblood glucose monitoring, ,\n\ncarbohydrate consistency,\n\ncarbohydrate distribution,\n\ncarbohydrate type,\n\nclinical outcomes, ,\n\ndiagnosis, 453\u2013454\n\nenergy intake,\n\nfat, 458\u2013459\n\nfetal complications,\n\ninsulin therapy, 459\u2013461\n\nmorning carbohydrate, 456\u2013458\n\nnutrition therapy, 454\u2013459\n\noverview, ,\n\npostpartum recommendations,\n\nprotein,\n\nreferral,\n\nrisk factors,\n\nscreening, 453\u2013454\n\nself-management, 455\u2013456\n\ntotal carbohydrate,\n\nweight gain, 459\u2013460\n\nmedical nutritional therapy, 229\u2013230, 236\u2013237\n\ntype 1 diabetes, 429\u2013438\n\ncarbohydrate intake, 431\u2013433\n\nfat intake, 431\u2013433\n\nguidelines,\n\ninsulin regimens, 433\u2013438\n\nmeal planning approaches, 437\u2013438\n\nmedical nutrition therapy, 430\u2013433\n\noverview, 429\u2013430,\n\nprotein intake,\n\nself-monitoring of blood glucose,\n\ntype 2 diabetes, 441\u2013448\n\nbody weight management, ,\n\ncarbohydrate intake, 444\u2013445\n\nchromium effects,\n\nenergy intake effects,\n\nfat intake, 445\u2013446\n\nmagnesium effects, 446\u2013447\n\noverview, 441\u2013442, 447\u2013448\n\nprotein intake, 442\u2013444\n\nobesity effects\n\ndefinitions, 415\u2013417\n\ndiabetes mellitus, 416\u2013417\n\nobesity, 415\u2013416\n\ndiabetes treatment,\n\nenergy intake restriction effects, 421\u2013422\n\nepidemiology, 417\u2013418\n\ninsulin insensitivity\n\nin nonobese,\n\nin obesity, 420\u2013421\n\ninsulin resistance, 418\u2013419,\n\noverview, , 422\u2013423\n\nprevalence correlations, 418\u2013421\n\npsychological stress, 419\u2013421\n\ntaste effects on food choice, 540\u2013541\n\ntype 2 diabetes\n\nfetal origins,\n\nmetabolic alterations, 419\u2013421\n\nphysical activity role,\n\npredictors,\n\nvisceral adiposity, 419\u2013421\n\nweight loss effects, 421\u2013422\n\nDialysis, renal replacement therapy, 619\u2013621\n\nDiarrhea\n\ncauses, 553\u2013554\n\nenteral feeding complications, 249\u2013251\n\ngastric bypass side effects,\n\nHIV\/AIDS relationship, ,\n\nin inflammatory bowel diseases, , 582\u2013583\n\nnutrition management strategies in cancer patients,\n\nprevalence, 553\u2013554\n\nspecific foods, 554\u2013555\n\ntherapy, , 555\u2013556\n\nDietary Approaches to Stop Hypertension diet\n\ncomputer-based analysis,\n\nhypertension intervention, , , , 313\u2013317\n\nDietary assessment, _See also_ specific diseases\n\ncystic fibrosis, 720\u2013722\n\ndata analysis, 69\u201377\n\ncomputer-based analysis, 69\u201371\n\ninterpretation, 74\u201377\n\nassessment methods,\n\noverreporting,\n\nscientific evidence hierarchy, 74\u201375\n\nunderreporting, 75\u201377\n\nvalidity, 75\u201377\n\npresentation, 72\u201374\n\ntotal diet analysis, 71\u201372\n\nmethods, 3\u201319\n\nbrief assessment methods, 7\u20138, 132\u2013133\n\ndietary records, 3\u20134, , 15\u201316\n\nfood frequency, 5\u20137,\n\n24-hour dietary recall, 4\u20135,\n\nissues, 14\u201319\n\nadministration,\n\ncalibration studies, , 18\u201319\n\ncognitive research,\n\ncomputer software choice, 16\u201317\n\nenergy adjustment,\n\nmultiple day records, 15\u201316\n\nnutrient database choice, , 69\u201370\n\nnutritional supplements assessment, 17\u201318\n\nportion size estimation, 14\u201315\n\noverview,\n\nspecial populations, 12\u201314\n\nchildren, 13\u201314\n\nelderly, , 54\u201355\n\nethnic populations, , 13\u201314\n\nsurrogate reporters, 12\u201314\n\nstudy situations\n\ncase-control studies, 10\u201312\n\ncohort studies, 11\u201312\n\ncross-sectional surveys, 9\u201310,\n\ndietary monitoring,\n\nintervention studies,\n\nprospective studies,\n\nretrospective studies, 10\u201311\n\nrenal disease, 623\u2013627\n\nanthropometrics, 623\u2013624\n\nbiochemical indices, 624\u2013627\n\ndietary assessment,\n\nserum albumin,\n\nserum cholesterol, 624\u2013625\n\nserum creatinine, 625\u2013627\n\nserum prealbumin,\n\nsubjective global assessment,\n\nDietary fat\n\nappetite effects,\n\nbody fat distribution\n\nanthropometric assessment,\n\nfat-free mass, ,\n\nbody weight effects\n\ncausal links, 499\u2013501\n\nconsumption,\n\nfat substitutes, 503\u2013506\n\nbreast cancer association, 339\u2013345\n\ncardiovascular disease effects, 279\u2013282\n\nmonounsaturated fatty acids, 279\u2013282\n\nomega-3 fatty acids,\n\nomega-6 fatty acids,\n\npolyunsaturated fatty acids,\n\nsaturated fatty acids, 279\u2013281\n\ntotal fat, 279\u2013280,\n\n_trans_ fatty acids, 279\u2013280,\n\nunsaturated fatty acids, 281\u2013282,\n\nchoose sensibly guidelines, 761\u2013762\n\ncolon cancer relationship, , ,\n\ndiabetes management\n\ngestational diabetes, 458\u2013459\n\ntype 1 diabetes, 431\u2013433\n\ntype 2 diabetes, 445\u2013446\n\nenteral feeding, 246\u2013247\n\nhypertension effects, 310\u2013311,\n\ninborn metabolic errors, treatment,\n\ninflammatory bowel disease association, 581\u2013582\n\nlow-fat high-carbohydrate diet, 281\u2013283\n\nlung cancer relationship,\n\nobesity relationship, 279\u2013280, , 523\u2013524\n\nparenteral nutrition,\n\nin Parkinson's disease therapy, 647\u2013648\n\nprostate cancer relationship,\n\nprotein content relationship, 506\u2013508\n\nrenal disease effects,\n\nrenal disease therapy, ,\n\nDietary fiber\n\nbiomarkers of exposure,\n\nbody mass index relationship,\n\nbreast cancer risk reduction, 340\u2013343,\n\ncalcium absorption effects,\n\ncardiovascular disease effects, , 291\u2013293,\n\ncolon cancer relationship, 358\u2013361,\n\nconstipation relationship, 556\u2013557,\n\ndiabetes nutritional management, type 2, 444\u2013445\n\nenteral feeding,\n\nhypertension effects, ,\n\nin inflammatory bowel disease treatment, ,\n\nin Parkinson's disease therapy, 647\u2013648\n\nrenal disease therapy,\n\nserum cholesterol reduction, ,\n\nin short bowel syndrome treatment, 594\u2013595\n\nsources, 291\u2013293\n\nDietary intake, _See also_ Supplements; _specific diseases_ ; _specific foods_\n\nadequate intake, 72\u201374\n\ndescription, 72\u201374,\n\nhealth maintenance nutrition guidelines\n\ndietary reference intake levels, , 753\u2013760\n\nestimated average requirement, 72\u201374, , 759\u2013760\n\nfood guide pyramid, , ,\n\nrecommended dietary allowance, 72\u201374, 753\u2013760\n\nserving equivalencies, , 764\u2013765\n\ntolerable upper intake levels, ,\n\nDietary Reference Intake, , 753\u2013760\n\nenergy requirements, 39\u201340,\n\nexposure measurement using biomarkers\n\nenergy intake, 142\u2013143\n\nmicromolecule intake, 140\u2013142\n\nnutrient intake, 139\u2013140\n\nunderreporting,\n\nDietary practices\n\ncolon cancer effects,\n\nhypertension effects, 312\u2013314\n\nobesity effects, 527\u2013529\n\nbinge eating, , , 694\u2013695\n\nfamily influences, 528\u2013529\n\nmeal patterns,\n\npacing,\n\nrestraint, 527\u2013528\n\nDietary recall\n\nadvantages and disadvantages,\n\ndescription, 4\u20135,\n\nfocused recalls,\n\nintervention-associated bias,\n\nDietary records\n\nadvantages and disadvantages,\n\ndescription, 3\u20134\n\nmultiple day records, 15\u201316\n\nDietary Reference Intake, health maintenance, , 753\u2013760\n\nDiet-habits questionnaires, description,\n\nDiet history\n\nadvantages and disadvantages,\n\ndescription, 8\u20139\n\nexercise history,\n\nfood allergy diagnosis, 709\u2013710\n\nDiethylpropion, obesity intervention, 472\u2013473\n\nDiet-induced therogenesis, resting energy expenditure measurement, 33\u201334\n\nDieting, _See_ Behavior modification; Nutrition intervention; _specific diets_\n\nDiet Intervention Study in Children, nutrition intervention trials, 96\u201398\n\nDigestive system, _See_ Gastrointestinal system\n\nDigoxin\n\ncongestive heart failure treatment,\n\nfood interactions,\n\nDimenhydrinate, gastrointestinal symptom treatment, 551\u2013552\n\nDining out, obesity effects,\n\nDisease, _See_ Nutritional epidemiology; _specific diseases_\n\nDiuretics, _See also_ Caffeine\n\ncongestive heart failure treatment,\n\nnutrient status effects,\n\nDNA methylation\n\nalcohol association, , , 365\u2013366\n\ncolon cancer relationship, 358\u2013359, ,\n\nfolate role, , , 365\u2013366\n\nmethionine role, ,\n\nvitamin B6 role, ,\n\nDocosahexanoic acid\n\ncardiovascular disease effects,\n\nrenal disease effects,\n\nDoubly-labeled water method\n\nresting energy expenditure measurement,\n\ntotal energy expenditure measurement, 36\u201338\n\nDramamine\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nDrugs, _See_ Botanicals; Medications; _specific drugs_\n\nDumping syndrome, gastric bypass side effects,\n\nDyes, allergic reactions,\n\nDyslipoproteinemia, physical activity role,\n\nE\n\nEating disorders, _See_ Anorexia; Binge eating; Bulimia\n\nEating disorders not otherwise specified\n\ndiagnostic criteria, 685\u2013686, 694\u2013695\n\nmanagement,\n\noverview,\n\nEating out, obesity effects,\n\nEating practices\n\ncolon cancer effects,\n\nhypertension effects, 312\u2013314\n\nobesity effects, 527\u2013529\n\nbinge eating, , , 694\u2013695\n\nfamily influences, 528\u2013529\n\nmeal patterns,\n\npacing,\n\nrestraint, 527\u2013528\n\nEchinacea\n\nadverse effects, ,\n\ncancer management,\n\nhealth benefits, 264\u2013265, , ,\n\nEcologic studies, nutritional epidemiology study,\n\nEducation\n\ncognitive function relationship,\n\nlearner style, 106\u2013107\n\nlevels of education,\n\nmethods, 108\u2013109\n\nNational Cholesterol and Education Program,\n\nObesity Education Initiative, 481\u2013483\n\nschool and community-based youth programs, 529\u2013531\n\ntechniques\n\nlearning domains, 106\u2013107\n\nneeds assessment, 107\u2013108\n\nteaching\/learning process, 105\u2013107\n\ntools, 109\u2013110\n\nmeal planning, 109\u2013110\n\nskills training,\n\nEggs, allergic reactions, 705\u2013707\n\nEicosapentaenoic acid\n\ncancer nutritional therapy, 234\u2013235\n\ncardiovascular disease effects,\n\nElderly\n\ncalcium requirements, 669\u2013670\n\ncommunity meal programs,\n\ndietary assessment methods, , 54\u201355\n\nmalnutrition, , ,\n\nmenopause, 668\u2013669\n\nvitamin D requirements, 669\u2013670,\n\nElectrolytes, _See also_ specific electrolytes\n\ndaily requirements, 251\u2013252\n\nin parenteral nutrition, 251\u2013252,\n\nin urine, ,\n\nEnergy intake, _See also_ Metabolism\n\nanorexia nervosa relationship,\n\ncancer management, 399\u2013400\n\ncystic fibrosis relationship,\n\ndiabetes management\n\ngestational diabetes,\n\ntype 2 diabetes,\n\nHIV\/AIDS patient recommendations,\n\ninsulin sensitivity effects, 421\u2013422\n\nlifestyle changes, 399\u2013400, , 519\u2013520\n\nlung cancer relationship,\n\nnutrition intervention evaluation, exposure measurement using biomarkers, 142\u2013143\n\nobesity effects, 501\u2013502, 522\u2013523\n\nin Parkinson's disease therapy, 647\u2013648\n\nprostate cancer relationship,\n\nrenal disease therapy, , 627\u2013629\n\nrequirements, 39\u201340,\n\nEnteral nutrition\n\nadministration,\n\ncancer patients, enteral nutrition, ,\n\ncomplications, 249\u2013250\n\ncontraindications,\n\ndisease-specific formulas, 247\u2013248\n\nenteral access, 245\u2013246\n\nformula classification, 247\u2013248\n\nformula composition, 246\u2013247\n\nformula selection, 248\u2013249\n\nindications,\n\ninflammatory bowel disease treatment, 587\u2013589\n\nliver disease treatment,\n\noverview, ,\n\nEnterocolitis syndrome, description, 708\u2013709\n\nEnvironment\n\nintervention evaluation measures, 124\u2013125, 134\u2013135\n\nobesity relationship, gene\u2013environment interactions,\n\nresting energy expenditure study methodology,\n\nEnzymes, _See also_ specific enzymes\n\nfunction biomarkers, 144\u2013145\n\ngastrointestinal symptom treatment, 551\u2013552,\n\ngenetic errors in cardiovascular disease,\n\ninduction in cancer,\n\nEosinophilic gastroenteritis, allergic food reactions,\n\nEphedrine, obesity intervention, 472\u2013473\n\nEpidemiology, _See_ Nutritional epidemiology\n\nEstimated average requirement\n\ndescription, 72\u201374, , 759\u2013760\n\nhealth maintenance guidelines, 759\u2013760\n\nEstrogen\n\nbreast cancer association, 339\u2013340,\n\nhormone replacement therapy, nutritional issues, 347\u2013348\n\nEthnic populations\n\ndietary assessment methods, , 13\u201314\n\nfood frequency questionnaires, 5\u20136,\n\nnutritional epidemiology study,\n\nnutritional learning influence,\n\nprostate cancer incidence, 374\u2013375\n\nEvening primrose, health benefits,\n\nExchanges, nutrition guidelines, 763\u2013764\n\nExercise, _See_ Physical activity\n\nF\n\nFamily, obesity influence, 528\u2013529\n\nFanconi syndrome, osteomalacia relationship,\n\nFast foods, obesity effects, 525\u2013526\n\nFat, _See_ Dietary fat, Obesity\n\nFat-free mass\n\nbody fat distribution,\n\nresting metabolic rate relationship,\n\nFatigue in\n\ninflammatory bowel diseases,\n\nnutrition management strategies in cancer patients,\n\nFat substitutes, body weight effects, 503\u2013506\n\nFatty acids\n\nbiomarker indicators,\n\ncolon cancer relationship, 359\u2013360\n\nessential fatty acids\n\ncystic fibrosis relationship,\n\nenteral feeding,\n\ngenetic influences on cardiovascular disease, 165\u2013166\n\ninflammatory bowel disease association, 581\u2013582, 586\u2013587\n\nintestinal fatty acid binding protein, 165\u2013166\n\noxidation disorders\n\nnutritional therapy, 214\u2013215\n\ntreatment,\n\nshort bowel syndrome treatment, 594\u2013595\n\nFeeding by tube, _See_ Enteral nutrition\n\nFenfluramine, obesity intervention,\n\nFeverfew\n\nadverse effects, ,\n\nhealth benefits, ,\n\nFiber, _See_ Dietary fiber\n\nFinger nails, physical assessment,\n\nFish, allergic reactions, ,\n\nFish oil, hypertriglyceridemia treatment,\n\nFitness, _See_ Physical activity\n\nFlatulence\n\ncauses, 549\u2013550\n\ngas production and composition,\n\nnutrition management strategies in cancer patients,\n\nprevalence, 549\u2013550\n\nspecific foods, 550\u2013551,\n\ntherapy, , 551\u2013552\n\nFlavonoids\n\nbiomarkers of exposure, 140\u2013142\n\nbreast cancer risk reduction, 342\u2013343\n\ncontent in foods,\n\nhealth benefits,\n\nprostate cancer risk reduction, 378\u2013379\n\nin soy protein, 285\u2013286, ,\n\nFlaxseed, cancer management,\n\nFleet's PhosphoSoda\u00ae, osteomalacia treatment,\n\nFluid intake, _See also_ Liquid dietary supplements\n\nanorexia nervosa therapy,\n\nintravenous feeding, 251\u2013252\n\nrenal disease therapy, 619\u2013620, 628\u2013629\n\nrestriction during congestive heart failure,\n\nFluoride\n\nbone mineralization inhibition, 737\u2013738\n\ndietary reference intakes, ,\n\nFluoxetine\n\nanorexia treatment, 691\u2013692\n\nobesity intervention, 472\u2013473\n\nFolate\n\nbreast cancer risk reduction, 340\u2013341,\n\ncognitive function relationship, 638\u2013639,\n\ncolon cancer relationship, , , 365\u2013366\n\ndeficiency signs, ,\n\ndietary reference intakes, , 756\u2013757, 759\u2013760\n\nhyperhomocysteinemia association, 293\u2013294\n\nlung cancer relationship, 391\u2013392\n\nin parenteral nutrition supplements,\n\nrenal disease therapy,\n\nFood additives, allergic reactions,\n\nFood allergy, 701\u2013713\n\nallergens, 704\u2013705\n\nclinical manifestations, 705\u2013708\n\ndefinitions,\n\ndiagnosis, 709\u2013711\n\ndisease types, , 703\u2013704, 706\u2013708\n\nfood additives,\n\ngenetically engineered foods, 712\u2013713\n\nimmune response mechanisms, 701\u2013704\n\nimmunoglobulin-mediated allergic diseases, 706\u2013708\n\nin inflammatory bowel disease,\n\nlactose intolerance, _See_ Lactose intolerance\n\nnonallergic immunologic diseases, 708\u2013709\n\nnonimmunologic food reactions,\n\nnonproven conditions,\n\noverview,\n\nprevalence,\n\nprevention, 711\u2013712\n\nprognosis,\n\nT cell-mediated allergic diseases,\n\ntreatment,\n\nFood dyes, allergic reactions,\n\nFood exchanges, nutrition guidelines, 763\u2013764\n\nFood frequency questionnaires\n\nadvantages and disadvantages, ,\n\nbrief assessment, 7\u20138, 132\u2013133\n\nerror sources, 64\u201366\n\nexposure measurement principles, 60\u201361\n\nmethods, 5\u20137,\n\nreliability assessment, 64\u201366\n\nvalidity assessment, 64\u201366\n\nFood Guide Pyramid, Dietary Guidelines for Americans, , ,\n\nFood history, _See_ Diet history\n\nFood industry\n\nhypertension prevalence,\n\nobesity effects\n\nadvertising, 526\u2013527,\n\neating out,\n\nfast food, 525\u2013526\n\nU.S. food supply, 524\u2013525\n\nFood labels\n\nconsumer information processing,\n\nnutrition guidelines, 764\u2013766\n\nFood records\n\nadvantages and disadvantages,\n\ndescription, 3\u20134\n\nmultiple day records, 15\u201316\n\nFood safety\n\nbotanical use,\n\nkeep food safe to eat program,\n\nFormative evaluations, description,\n\nFosinopril, food interactions,\n\nFried foods\n\ncolon cancer relationship, 358\u2013359, ,\n\nlung cancer relationship, 392\u2013393\n\nprostate cancer relationship, 375\u2013376\n\nFructooligosaccharides, cardiovascular disease effects,\n\nFructose\n\nintolerance\n\nliver disease,\n\nnutritional therapy,\n\nprostate cancer risk reduction,\n\nFruits\n\nallergic reactions, _See_ Food allergy\n\nbreast cancer risk reduction, 340\u2013341, , , 348\u2013349\n\ncardiovascular disease effects, 296\u2013297\n\ncolon cancer relationship, ,\n\nDietary Approaches to Stop\n\nHypertension diet,\n\nexcessive gas production, 550\u2013551\n\nfiber source, 291\u2013292\n\ngrapefruit diet,\n\nhypertension reduction, ,\n\ninflammatory bowel disease effects,\n\njuice therapies,\n\nlung cancer relationship, 387\u2013388\n\nprostate cancer risk reduction, 377\u2013378\n\nFumarylacetoacetate hydrolase deficiency, tyrosinemia\n\ndescription,\n\nnewborn screening programs, 210\u2013211\n\nnutritional therapy, , ,\n\nFunction assessment\n\nbiomarkers of change, 143\u2013147\n\nbone health, 146\u2013147\n\ncell turnover,\n\nenzymes, 144\u2013145\n\nimmune function,\n\noxidative stress, 145\u2013146\n\nobesity candidate gene association studies, 190\u2013192\n\nphysical assessment, 48\u201349\n\nFurosemide, food interactions,\n\nG\n\nGalactosemia\n\ndescription,\n\nnewborn screening programs, 210\u2013212\n\nnutritional therapy, ,\n\npregnancy management, 222\u2013223\n\nGamma-glutamyl allyic cysteines, health benefits,\n\nGarlic\n\nadverse effects, ,\n\nhealth benefits, , , , 296\u2013297\n\nGastric bypass, obesity treatment,\n\nGastrointestinal system\n\nacute complaints\n\nconstipation\n\nanorexia nervosa association,\n\ncystic fibrosis association,\n\nfood effects, 556\u2013558,\n\nprevalence,\n\ntherapy, , ,\n\ndiarrhea\n\ncauses, 553\u2013554\n\nenteral feeding complications, 249\u2013251\n\ngastric bypass side effects,\n\nHIV\/AIDS relationship, ,\n\nin inflammatory bowel diseases, , 582\u2013583\n\nnutrition management strategies in\n\ncancer patients,\n\nprevalence, 553\u2013554\n\nspecific foods, 554\u2013555\n\ntherapy, , 555\u2013556\n\nexcessive gas\n\ncauses, 549\u2013550\n\ngas production and composition,\n\nnutrition management strategies in cancer patients,\n\nprevalence, 549\u2013550\n\nspecific foods, 550\u2013551,\n\ntherapy, , 551\u2013552\n\ngastroesophageal reflux disease, , 552\u2013553\n\nheartburn, , 552\u2013553\n\nnausea\n\ncauses, 557\u2013559\n\ndrug therapies, , , 559\u2013560\n\nfood effects,\n\nginger effects, , , , 559\u2013560\n\nnutrition management strategies in\n\ncancer patients,\n\nprevalence, 557\u2013558\n\noverview, ,\n\nvomiting\n\ncauses, 557\u2013559\n\nfood effects,\n\ngastric bypass side effects,\n\nnutrition management strategies in cancer patients,\n\nprevalence, 557\u2013558\n\ntherapy, , , , 559\u2013560\n\nallergic reactions to foods, _See_ Food allergy\n\ncancer, _See_ Colon cancer; Rectal cancer\n\nenteral nutrition\n\nadministration,\n\ncomplications, 249\u2013250\n\ncontraindications,\n\ndisease-specific formulas, 247\u2013248\n\nenteral access, 245\u2013246\n\nformula classification, 247\u2013248\n\nformula composition, 246\u2013247\n\nformula selection, 248\u2013249\n\nindications,\n\noverview, ,\n\ninflammatory bowel disease\n\ndietary factors, 581\u2013584\n\nfood allergies,\n\nnutritional-related problems, 582\u2013584\n\nspecific foods, 581\u2013582\n\netiology, 581\u2013582\n\nnutrition assessment, 585\u2013586\n\noverview, 577\u2013578,\n\npathology, 580\u2013581\n\nprevalence, 578\u2013580\n\ntreatment, 584\u2013587\n\ndiet effectiveness,\n\nenteral nutrition, 587\u2013589\n\nmedical management, 584\u2013585\n\nnutritional treatment, 585\u2013587\n\nparenteral nutrition, 587\u2013589\n\nsurgery,\n\nlactose intolerance, _See_ Lactose intolerance\n\nparenteral nutrition supplements, _See_ Parenteral nutrition\n\nphysical assessment, 51\u201352\n\nshort bowel syndrome\n\ncauses,\n\nmedical management, 594\u2013595\n\nnutritional management, 593\u2013594\n\noverview, , ,\n\nsurgery, 594\u2013595, ,\n\nvariants, 589\u2013593\n\ncolon loss, 591\u2013592\n\nileal resections, 590\u2013591\n\nileum loss, 591\u2013592\n\njejunal resections,\n\nmajor intestinal resections, 592\u2013593\n\nvitamin D deficiency,\n\nGas X\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nGender\n\ncognitive function relationship,\n\neating disorder risk relationship, 686\u2013687\n\nresting energy expenditure measurement, 32\u201333\n\nGenetically engineered foods, allergic reactions, 712\u2013713\n\nGenetic disease\n\nbiomarkers of susceptibility, 147\u2013148\n\ncancer, diet-genetic interactions, 200\u2013203\n\nbreast cancer, 204\u2013205, 337\u2013338\n\ncarcinogen metabolism,\n\ncolon cancer, 203\u2013204, 364\u2013366\n\nenzyme induction,\n\nfuture research directions,\n\nmethodologic issues, 202\u2013203\n\nnasopharyngeal cancer,\n\none-carbon metabolism, 201\u2013202\n\noral cancer,\n\noverview, 199\u2013200\n\noxidative stress, 200\u2013201\n\nprostate cancer, 204\u2013205,\n\nrectal cancer, 203\u2013204\n\nvitamin pathway variation,\n\ncardiovascular system reactions, 157\u2013171\n\nendogenous lipoprotein metabolism, 166\u2013167\n\n3-hydroxy-3-methylglutaryl-coenzyme A reductase,\n\nlow-density lipoprotein receptor, 166\u2013167\n\nexogenous lipoprotein pathway, 158\u2013166\n\napolipoprotein A-IV, 160\u2013161\n\napolipoprotein B, 158\u2013160\n\napolipoprotein C-III, 163\u2013164\n\napolipoprotein E, 161\u2013163\n\nintestinal fatty acid binding protein, 165\u2013166\n\nlipoprotein lipase, 164\u2013165\n\nmicrosomal triglyceride transfer protein,\n\noverview, 157\u2013158, 170\u2013171\n\nreverse cholesterol transport, 167\u2013170\n\napolipoprotein A-I, 167\u2013168\n\nATP-binding cassette , , 169\u2013170\n\ncholesterol 7 alpha-hydroxylase,\n\ncholesteryl ester transfer protein,\n\nhepatic lipase, 168\u2013169\n\nscavenger receptor B type I,\n\nobesity, 183\u2013205\n\ncandidate gene association studies, 189\u2013192\n\naltered function causes, 190\u2013192\n\nsingle-gene obesity role,\n\nclinical implications, 192\u2013194\n\ndiagnosis, 192\u2013193\n\ntreatment,\n\nepidemiology, 183\u2013184\n\ngene\u2013environment interactions,\n\ngene map, 184\u2013185\n\noverview, 183\u2013184\n\nphenotype evidence from linkage studies, 188\u2013189\n\nhuman studies,\n\nloci mapping in animals, 188\u2013189\n\nmouse studies,\n\nrare Mendelian syndromes,\n\nsingle-gene adipose tissue atrophy,\n\nsingle-gene obesity, 185\u2013188\n\nleptin deficiency, 185\u2013186,\n\nleptin receptor deficiency, 186\u2013187\n\nmelanocortin-4 receptor gene mutation, 187\u2013188\n\nproopiomelanocortin deficiency,\n\nproprotein convertase subtilisin\/ kexin type 1 deficiency,\n\nGerson therapy, cancer management,\n\nGinger\n\ncancer management,\n\nnausea treatment, , , , 559\u2013560\n\nGingko\n\nadverse effects, ,\n\ncognitive function relationship, 644\u2013645\n\nhealth benefits, , ,\n\nGinseng\n\nadverse effects, ,\n\nhealth benefits, ,\n\nGlucose\n\nappetite effects,\n\ndiabetes management\n\ngestational diabetes, ,\n\ntype 1 diabetes,\n\nGlutamine\n\nenteral nutrition,\n\nHIV\/AIDS patient recommendations, 747\u2013748\n\nGlutaric acidemia, nutritional therapy, 212\u2013213\n\nGlutathione S-transferase\n\ncarcinogen metabolism,\n\ncolon cancer relationship,\n\nfunction biomarkers,\n\nGlycemic index\n\ncardiovascular disease effects, ,\n\ncolon cancer relationship, , 362\u2013363,\n\nGlycogen storage diseases\n\ndescription,\n\nnutritional therapy, , ,\n\nGoal setting\n\ncoping strategies,\n\nproblem-solving skills,\n\nself-management skills training, 118\u2013119\n\nsocial support,\n\nweight management,\n\nGotu kola, health benefits,\n\nGrains, _See also_ Carbohydrates\n\nallergic reactions, 705\u2013707\n\ncolon cancer relationship, 358\u2013359,\n\nDietary Approaches to Stop Hypertension diet,\n\nfiber source, 291\u2013292\n\nGrapefruit diet, description,\n\nGrapes, health benefits,\n\nGreen tea\n\nhealth benefits,\n\nprostate cancer risk reduction,\n\nGrowth assessment, 215\u2013216 _See also_ Body weight children with genetic metabolism errors,\n\nGrowth regulators, colon cancer relationship, , 363\u2013365\n\nGustatory rhinorrhea, description,\n\nH\n\nHair, physical assessment,\n\nHandgrip strength test, description,\n\nHarris\u2013Benedict equation, resting energy expenditure measurement,\n\nHawthorne, health benefits,\n\nHeadaches, treatment, feverfew,\n\nHealth belief model\n\nbehavioral motivation, 95\u201396\n\ncontrol beliefs,\n\ndescription,\n\nHealth maintenance nutrition guidelines\n\nadequacy, 753\u2013760\n\ndietary reference intake levels, , 753\u2013760\n\nestimated average requirement, 72\u201374, , 759\u2013760\n\nfood guide pyramid, , ,\n\nrecommended dietary allowance, 72\u201374\n\nserving equivalencies, , 764\u2013765\n\ntolerable upper intake levels, ,\n\nDietary Guidelines for Americans, 760\u2013763\n\nAim for Fitness, ,\n\nBuild a Healthy Base, ,\n\nChoose Sensibly, , 761\u2013763\n\ncurrent intakes compared, 757\u2013760, 766\u2013767\n\ndietary guidelines for fat, 761\u2013762\n\nfood exchanges, 763\u2013764\n\nFood Guide Pyramid, , ,\n\nnutrition labels, 764\u2013766\n\noverview, ,\n\nphysical activity recommendations, _See_ Physical activity\n\nHealth services, _See_ Public health services\n\nHealthy Eating Index, _See also_ Health\n\nmaintenance nutrition guidelines;\n\nRecommended Dietary Allowances\n\ndescription, 72\u201373\n\nHeartburn, food relationship, , 552\u2013553\n\nHeart rate, energy expenditure measurement, 35\u201336\n\nHeight, anthropometric assessment\n\nbreast cancer risk,\n\nmethods, 44\u201345\n\nrenal disease assessment, 623\u2013624\n\nweight for height, 46\u201347\n\nHeiner's syndrome, allergic food reactions,\n\nHematologic system, physical assessment,\n\nHemoglobinopathies, newborn screening programs, 210\u2013211\n\nHeparin, in parenteral nutrition supplements,\n\nHepatic lipase, reverse cholesterol transport role, 168\u2013169\n\nHepatitis\n\nautoimmune hepatitis,\n\nneonatal hepatitis, 604\u2013605\n\nnonalcoholic steatohepatitis,\n\npregnancy-related hepatic disorders,\n\nviral hepatitis, 603\u2013604\n\nHerbs, _See_ Botanicals\n\nHereditary fructose intolerance\n\nliver disease,\n\nnutritional therapy,\n\nHeritability, _See_ Genetic disease\n\nHeterocyclic amines\n\nbreast cancer risk,\n\ncolon cancer relationship, 358\u2013360,\n\nlung cancer relationship, 392\u2013393\n\nprostate cancer relationship, 375\u2013376\n\nHigh blood pressure, _See_ Hypertension\n\nHigh-density lipoprotein\n\ncardiovascular disease risk reduction, , 279\u2013281,\n\ndiabetes management, 445\u2013446\n\nexogenous lipoprotein pathway, 158\u2013166\n\nphysical activity effects,\n\nreverse cholesterol transport, 167\u2013170\n\nHip fractures\n\nfrailty role,\n\nmedical nutritional therapy,\n\nHistamine, food allergy role,\n\nHistory records, _See_ Diet history\n\nHIV\/AIDS\n\nhormone imbalances,\n\nlipodystrophy syndrome association, , ,\n\nmedical nutritional therapy, 229\u2013230,\n\nnutritional assessment, 744\u2013747\n\nappetite status, 746\u2013747\n\nbiochemical indices,\n\nbioelectrical impedance analysis, 745\u2013746\n\nbody composition, 745\u2013746\n\nbody mass index,\n\nbody weight, 744\u2013745\n\nin lipodystrophy syndrome patients,\n\noral intake assessment, 746\u2013747\n\nnutritional recommendations, 747\u2013748\n\ncysteine, 747\u2013748\n\nenergy intake, ,\n\nglutamine, 747\u2013748\n\nnutritional counseling,\n\nprotein,\n\noverview, ,\n\nweight loss association\n\nassessment, 744\u2013745\n\ncauses, 742\u2013744\n\nanorexia,\n\ndiarrhea, ,\n\nmalabsorption, ,\n\nmetabolic abnormalities, 743\u2013744\n\noral intake deficiencies,\n\nmechanisms, 741\u2013742\n\nHome care, medical nutritional therapy,\n\nHome meal replacements\n\nmeal replacement drinks,\n\nobesity effects,\n\nHomocysteine\n\ncardiovascular disease effects, 293\u2013294\n\nrenal disease association, 621\u2013622\n\nvitamin interactions, 293\u2013294\n\nHomocystinuria\n\nnewborn screening programs, 210\u2013212\n\nnutritional therapy, ,\n\nHormone replacement therapy, breast cancer treatment, nutritional issues, 347\u2013348\n\nHormones, _See also_ specific hormones\n\nHIV\/AIDS patient imbalances,\n\nreplacement therapy nutritional issues, 347\u2013348\n\nresting energy expenditure study methodology,\n\nHunger, 541\u2013542 _See also_ Malnutrition obesity effects,\n\nHydralazine, food interactions,\n\nHydration, inborn error in metabolism treatment,\n\nHydrochlorothiazide, food interactions,\n\nHydrogen receptor antagonists, gastrointestinal symptom treatment, 551\u2013553\n\n8-hydroxydeoxyguanosine, oxidative stress biomarker, 145\u2013146\n\n5-Hydroxyindoleacetic acid, eating disorder association, ,\n\n3-Hydroxy-3-methylglutaryl-coenzyme A reductase, endogenous lipoprotein metabolism role,\n\nHypercarotenemia, anorexia nervosa association, 689\u2013690\n\nHypercholesterolemia\n\nanimal protein effects, 284\u2013285\n\nanorexia nervosa association,\n\ndiet strategies, , , 283\u2013285\n\nfiber effects, ,\n\nHypercortisolism\n\nanorexia nervosa association,\n\nbody fat relationship,\n\nHyperglycemia\n\nintravenous feeding complications,\n\nnutrition management strategies in cancer patients,\n\nHyperhomocysteinemia\n\ndescription,\n\nrenal disease association, 621\u2013622\n\nvitamins role, 293\u2013294\n\nHyperlipidemia, intravenous feeding complications,\n\nHypertension\n\nclassification,\n\nlifestyle modification effects\n\nalcohol consumption, 311\u2013312,\n\ndietary patterns, 312\u2013314\n\nintervention diets, 313\u2013314\n\nphysical activity role,\n\nweight reduction, 311\u2013312, ,\n\nnutrient effects\n\ncalcium, 308\u2013309,\n\ncarbohydrates, ,\n\nDietary Approaches to Stop Hypertension diet, , , , 313\u2013317\n\ndietary fat, 310\u2013311,\n\nfiber, ,\n\ngarlic, ,\n\nmacronutrients, 309\u2013311\n\nmagnesium, ,\n\nmicronutrients, 305\u2013309\n\npotassium, ,\n\nprotein, 309\u2013310,\n\nsodium, 305\u2013308,\n\nintervention studies, 306\u2013307\n\nobservational studies, 305\u2013306\n\nsalt sensitivity, 307\u2013308\n\noverview, 303\u2013305, 314\u2013315\n\nlow prevalence populations, 304\u2013305\n\nstudy methodology, 303\u2013304\n\nprevalence\n\nindustrialized populations,\n\nlow prevalence populations, 304\u2013305\n\nHypertriglyceridemia, diet strategies, 281\u2013282\n\nHypoglycemia, anorexia nervosa association,\n\nHypolactasia, _See_ Lactose intolerance\n\nHyponatremia, parenteral nutrition treatment,\n\nHypotension, anorexia nervosa association,\n\nHypothermia, anorexia nervosa association,\n\nHypothyroidism, newborn screening programs, 210\u2013211\nI\n\nImmune function\n\nallergic reactions to food, _See_ Food allergy\n\nautoimmune hepatitis,\n\nbiomarkers of change,\n\nImmunodeficiency syndromes, _See_ HIV\/AIDS\n\nImmunoglobulins, food allergies role, , 703\u2013704, 706\u2013708\n\nImmunotherapy, food allergy treatment,\n\nImodium AD\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nInborn disorders, _See_ Genetic disease\n\nIndinavir, depression treatment,\n\nIndoles, health benefits,\n\nInfantile colic, description,\n\nInfectious disease, _See also_ specific diseases\n\nphysical assessment,\n\nInflammatory bowel disease\n\ndietary factors, 581\u2013584\n\nfood allergies,\n\nnutritional-related problems, 582\u2013584\n\nspecific foods, 581\u2013582\n\netiology, 581\u2013582\n\nnutrition assessment, 585\u2013586\n\noverview, 577\u2013578,\n\npathology, 580\u2013581\n\nprevalence, 578\u2013580\n\ntreatment, 584\u2013587\n\ndiet effectiveness,\n\nenteral nutrition, 587\u2013589\n\nmedical management, 584\u2013585\n\nnutritional treatment, 585\u2013587\n\nparenteral nutrition, 587\u2013589\n\nsurgery,\n\nInfluenza, treatment, echinacea, 264\u2013265, ,\n\nInsomnia, treatment, valerian,\n\nInsulin\n\ndiabetes nutrition management\n\ngestational diabetes, 459\u2013461\n\ntype 1 diabetes, 433\u2013438\n\ninsulin regimens, 433\u2013437\n\nmeal planning approaches, 437\u2013438\n\nself-monitoring of blood glucose,\n\nnutrient status effects,\n\nresistance\n\ncolon cancer relationship, , 362\u2013363\n\nenergy balance, ,\n\nglycemic index, , 362\u2013363,\n\nsugar consumption, 362\u2013363\n\ndiet strategies,\n\nobesity effects on diabetes, 418\u2013419,\n\nIntervention, _See_ Nutrition intervention\n\nInterviews, motivational interviews,\n\nIntestinal disease, _See_ Gastrointestinal system\n\nIntestinal fatty acid binding protein, genetic influences on cardiovascular disease, 165\u2013166\n\nIntravenous feeding, _See_ Parenteral nutrition\n\nIodine\n\nbiomarkers of exposure,\n\ndietary reference intakes,\n\nIron\n\nbiomarkers of exposure,\n\ndeficiency\n\nin liver disease,\n\nsigns, ,\n\ndietary reference intakes, ,\n\nin Parkinson's disease therapy, 647\u2013648\n\nrenal disease therapy, ,\n\nIsoflavones\n\nbiomarkers of exposure, 140\u2013142\n\nbreast cancer risk reduction, 342\u2013343\n\ncontent in foods,\n\nhealth benefits,\n\nprostate cancer risk reduction, 378\u2013379\n\nin soy protein, 285\u2013286, ,\n\nIsoniazid, nutrient status effects,\n\nIsothiocyanates\n\ncontent in foods,\n\nhealth benefits,\n\nIsovaleriac acidemia\n\nnutritional therapy, 212\u2013213\n\ntreatment,\n\nJ\n\nJenny Craig, behavior modification training,\n\nJuice therapies, cancer management,\n\nK\n\nKahweol palmitate, colon cancer relationship,\n\nKaolin, gastrointestinal symptom treatment, 551\u2013552\n\nKaopectate\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nKava\n\nadverse effects, ,\n\nhealth benefits,\n\nKeep food safe to eat, health guidelines,\n\nKelley regime, cancer management,\n\nKetoaciduria\n\nnewborn screening programs, 210\u2013212\n\nnutritional therapy, ,\n\nKidney disease, _See_ Renal disease\n\nK-Phos\u00ae, osteomalacia treatment,\n\nL\n\nLabetalol, food interactions,\n\nLactaid\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\n\u03b1-Lactalbumin, food allergy role,\n\nLactase\n\nactivity loss,\n\nsupplements,\n\nLactation\n\ncalcium requirements, 667\u2013668\n\ncystic fibrosis management, 722\u2013723\n\nenergy requirements,\n\nLactose intolerance, 563\u2013572\n\ncalcium intake effects, ,\n\ndiagnosis, 564\u2013566\n\ndirect assessment, 564\u2013565\n\nindirect assessment, 565\u2013566\n\nlactase activity loss,\n\nlactose digestion, ,\n\nlactose sources, 563\u2013564\n\nmanagement, 567\u2013572\n\ncolonic fermentation, 570\u2013571\n\ndose response, 567\u2013568\n\ngastrointestinal transit time,\n\ngene therapy, 571\u2013572\n\nlactase supplements,\n\nlactose-reduced milks,\n\nunfermented acidophilus milk, 569\u2013570\n\nyogurt, 568\u2013569\n\nosteoporosis association,\n\noverview, ,\n\nprevalence,\n\nsymptoms, 566\u2013567\n\nLaxatives\n\nabuse in eating disorders, 692\u2013693\n\ngastrointestinal symptom treatment, 551\u2013552,\n\nLearning process, behavior change influences\n\nlearner style, 106\u2013107\n\ntechniques\n\nlearning domains, 106\u2013107\n\nneeds assessment, 107\u2013108\n\nteaching\/learning process, 105\u2013107\n\nLegumes, _See also_ specific legumes\n\nallergic reactions, 705\u2013707\n\ncolon cancer relationship,\n\nexcessive gas production, 550\u2013551\n\nfiber source, 291\u2013292\n\nprostate cancer risk reduction,\n\nLeptin, obesity role, 185\u2013186,\n\nLeptin receptor, obesity role, 186\u2013187\n\nLeukopenia, anorexia nervosa association,\n\nLevodopa, in Parkinson's disease therapy, 646\u2013648\n\nLicorice, adverse effects,\n\nLifestyle modification, _See_ _specific behaviors_\n\nLignans\n\nbiomarker indicators,\n\ncontent in foods,\n\nhealth benefits,\n\nLimonoids, health benefits,\n\nLinolenic acids\n\ncholesterol synthesis inhibition,\n\ninflammatory bowel disease association, 586\u2013587\n\nLipids, _See also_ Dietary fat; Lipoproteins\n\nhyperlipidemia, intravenous feeding complications,\n\nsynthesis, liver disease effects, ,\n\nLipodystrophy syndrome, in HIV\/AIDS patients\n\nmechanisms,\n\nnutritional assessment, ,\n\nnutritional recommendations,\n\nLipoprotein lipase, genetic influences on cardiovascular disease, 164\u2013165\n\nLipoproteins\n\nendogenous lipoprotein metabolism, 166\u2013167\n\n3-hydroxy-3-methylglutaryl-coenzyme A reductase,\n\nlow-density lipoprotein receptor, 166\u2013167\n\nexogenous lipoprotein pathway, 158\u2013166\n\napolipoprotein A-IV, 160\u2013161\n\napolipoprotein B, 158\u2013160\n\napolipoprotein C-III, 163\u2013164\n\napolipoprotein E, 161\u2013163\n\nintestinal fatty acid binding protein, 165\u2013166\n\nlipoprotein lipase, 164\u2013165\n\nmicrosomal triglyceride transfer protein,\n\nhigh-density lipoproteins\n\ncardiovascular disease risk reduction, , 279\u2013281,\n\ndiabetes management, 445\u2013446\n\nexogenous lipoprotein pathway, 158\u2013166\n\nphysical activity effects,\n\nreverse cholesterol transport, 167\u2013170\n\nlow-density lipoproteins\n\ncardiovascular disease risk, , 280\u2013285, 294\u2013295\n\nexogenous lipoprotein pathway, 158\u2013166\n\nplant sterol effects, 296\u2013297\n\nreverse cholesterol transport, 167\u2013170\n\noverview, 157\u2013158\n\nrenal disease,\n\nreverse cholesterol transport, apolipoprotein A-I, 167\u2013168\n\nLiquid dietary supplements, 229\u2013238\n\nbarriers,\n\ncommunity programs, 232\u2013233\n\ndisease-specific conditions, 233\u2013238\n\ncancer, 233\u2013235\n\nchronic obstructive pulmonary disease, 229\u2013230, 235\u2013236\n\ndiabetes mellitus, 236\u2013237\n\nHIV\/AIDS,\n\nrenal disease,\n\nFDA definition, 229\u2013231\n\noverview, ,\n\nstandard supplement conditions, 231\u2013233\n\nAlzheimer's disease, ,\n\ncerebral vascular accident, 231\u2013232\n\ngeneral medicine,\n\nhip fracture,\n\nhome care,\n\nlong-term care,\n\nsurgery, ,\n\nLithium, nutrient status effects,\n\nLiver disease\n\nacute liver disease\n\nnutritional management,\n\npathogenesis,\n\nAlagille syndrome,\n\nalcoholic liver disease,\n\nautoimmune hepatitis,\n\nbile acid metabolism,\n\nbiliary atresia, 606\u2013607\n\nByler's syndrome,\n\ncancer, diet\u2013genetic interactions,\n\ncarbohydrate metabolism,\n\ncholedochal cysts,\n\ncholedocholithiasis,\n\nchronic liver disease\n\nnutritional management, 608\u2013609\n\npathogenesis, 601\u2013602\n\ncirrhosis\n\nalcohol-induced cirrhosis,\n\nend-stage nutritional therapy,\n\nprimary biliary cirrhosis,\n\ncystic fibrosis,\n\nenteral feeding formula,\n\ngalactosemia\n\ndescription,\n\nnewborn screening programs, 210\u2013212\n\nnutritional therapy, ,\n\npregnancy management, 222\u2013223\n\nglycogen storage diseases,\n\nhereditary fructose intolerance, ,\n\nlipid synthesis, ,\n\nmetabolic liver disease, 605\u2013606\n\nmineral absorption, 602\u2013603\n\nneonatal hepatitis, 604\u2013605\n\nnonalcoholic steatohepatitis,\n\nnutritional management, 607\u2013609\n\nacute liver failure,\n\nchronic liver diseases, 608\u2013609\n\nend-stage liver disease,\n\nstatus assessment, 607\u2013608\n\noverview, ,\n\npregnancy-related hepatic disorders,\n\nprimary biliary cirrhosis,\n\nprimary sclerosing cholangitis,\n\nprotein effects, ,\n\ntransplant preparation, ,\n\ntyrosinemia\n\ndescription,\n\nnewborn screening programs, 210\u2013211\n\nnutritional therapy, , ,\n\nurea cycle defects, 605\u2013606\n\nviral hepatitis, 603\u2013604\n\nvitamin absorption,\n\nWilson's disease, ,\n\nLomotil\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nLong-term care, medical nutritional therapy,\n\nLoperamide diphenoxylate, gastrointestinal symptom treatment, 551\u2013552\n\nLow blood pressure, anorexia nervosa association,\n\nLow-density lipoprotein\n\ncardiovascular disease risk, , 280\u2013285, 294\u2013295\n\nexogenous lipoprotein pathway, 158\u2013166\n\nplant sterol effects, 296\u2013297\n\nreverse cholesterol transport, 167\u2013170\n\nLow-density lipoprotein receptor,\n\nendogenous lipoprotein metabolism, 166\u2013167\n\nLow-fat high-carbohydrate diet, cardiovascular disease effects, 281\u2013283\n\nLow-protein diet, cardiovascular disease effects,\n\nLung disease\n\ncancer\n\ndietary factors, 387\u2013393\n\nadiposity,\n\ncarotenoids, 388\u2013390\n\ncholesterol,\n\ndietary fat,\n\nenergy balance,\n\nfolate, 391\u2013392\n\nfruits, 387\u2013388\n\nheterocyclic amines, 392\u2013393\n\nprevention,\n\nretinol, 388\u2013390\n\nselenium,\n\nvegetables, 387\u2013388\n\nvitamin C, ,\n\nvitamin E, 390\u2013391\n\noverview, , 393\u2013394\n\nsurgical side effects,\n\nchronic obstructive pulmonary disease\n\ncystic fibrosis relationship,\n\nenteral feeding formulas,\n\nmedical nutritional therapy, 229\u2013230, 235\u2013236\n\nphysical assessment methods,\n\nechinacea effects, 264\u2013265, ,\n\nLutein\n\nbreast cancer risk reduction, 340\u2013341,\n\ncolon cancer relationship,\n\nLycopene\n\ncolon cancer relationship,\n\nhealth benefits,\n\nprostate cancer risk reduction,\n\nLysozyme, food allergy role,\n\nM\n\nMaastrich Index, description,\n\nMacrobiotic diet, cancer management,\n\nMagnesium\n\nbiomarkers of exposure,\n\ndaily requirements, 251\u2013252\n\ndiabetes management, 446\u2013447\n\ndietary reference intakes, , 756\u2013757,\n\nhypertension effects, ,\n\nosteoporosis relationship, 675\u2013676\n\nin parenteral nutrition, 251\u2013252,\n\nrenal disease therapy,\n\nMalnutrition\n\nin HIV\/AIDS patients, 741\u2013746\n\nin inflammatory bowel diseases,\n\nmedical nutritional therapy, , ,\n\nmicronutrients, ,\n\nprevalence,\n\nrenal disease association,\n\nMammary cells, 337\u2013338 _See also_ Lactation breast cancer biology,\n\nManganese\n\nbiomarkers of exposure,\n\nosteoporosis relationship,\n\nin parenteral nutrition supplements,\n\nParkinson's disease relationship,\n\nMaple syrup urine disease\n\nnewborn screening programs, 210\u2013212\n\nnutritional therapy, ,\n\npregnancy management, 222\u2013223\n\nMazindol, obesity intervention,\n\nMeal patterns\n\ncolon cancer effects,\n\nhypertension effects, 312\u2013314\n\nobesity effects, 527\u2013529\n\nbinge eating, , , 694\u2013695\n\nfamily influences, 528\u2013529\n\npacing,\n\nrestraint, 527\u2013528\n\nMeal replacement drinks,\n\nMeals on Wheels, medical nutritional supplements,\n\nMeat\n\nallergic reactions, _See_ Food allergy\n\nbreast cancer risk,\n\ncolon cancer relationship, 358\u2013360,\n\nlung cancer relationship, 392\u2013393\n\nprostate cancer relationship, 375\u2013376\n\nMedical nutritional supplements, 229\u2013238\n\nbarriers,\n\ncommunity programs, 232\u2013233\n\ncongestive heart failure effects,\n\ndisease-specific conditions, 233\u2013238\n\ncancer, 233\u2013235\n\nchronic obstructive pulmonary\n\ndisease, 229\u2013230, 235\u2013236\n\ndiabetes mellitus, 236\u2013237\n\nHIV\/AIDS,\n\nrenal disease,\n\nFDA definition, 229\u2013231\n\noverview, ,\n\nstandard supplement conditions, 231\u2013233\n\nAlzheimer's disease, ,\n\ncerebral vascular accident, 231\u2013232\n\ngeneral medicine,\n\nhip fracture,\n\nhome care,\n\nlong-term care,\n\nsurgery, ,\n\ntype 1 diabetes treatment, 430\u2013433\n\ncarbohydrates, 431\u2013433\n\nfats, 431\u2013433\n\nprotein,\n\nMedication Pass Supplement Program, description,\n\nMedications\n\nanorexia treatment, , , , 691\u2013692\n\ncancer cachexia,\n\ncongestive heart failure, drug\n\ninteractions,\n\nfood interactions,\n\ngastrointestinal symptom treatment, 551\u2013552\n\nnutritional interactions,\n\nobesity intervention, 471\u2013474\n\nbackground, 471\u2013472\n\nefficacy predictors,\n\nlifestyle modification combined,\n\nlong-term medications, 473\u2013474\n\norlistat, , , 473\u2013474\n\nsibutramine,\n\nnonapproved medications, 472\u2013473\n\npatient selection,\n\nrisk\/benefit ratio,\n\nshort-term medications,\n\nvomiting induction,\n\nMedicinal herbs, _See_ Botanicals\n\nMediterranean diet, hypertension effects,\n\nMedium-chain triglyceride, AIDS nutritional therapy,\n\nMelanocortin-4 receptor gene, obesity role, 187\u2013188\n\nMenopause, calcium requirements, 668\u2013669\n\nMenstrual cycle, resting energy expenditure measurement, 32\u201333\n\n6-Mercaptopurine, inflammatory bowel disease treatment,\n\nMetabolic stress, enteral feeding formula,\n\nMetabolism, _See also_ Energy intake; _specific metabolic diseases_\n\ncancer cachexia relationship, 398\u2013399\n\ncolon cancer relationship,\n\nenergy requirements\n\nbiomarkers of exposure, 142\u2013143\n\nrecommended intakes, 39\u201340,\n\nstudy methodology, 31\u201355\n\nage,\n\nbody composition,\n\nbody size,\n\ncomponents, 31\u201336\n\ndeterminants,\n\ndirect calorimetry measurement,\n\ndoubly-labeled water technique, 36\u201338\n\nenergy adjustment,\n\nenvironmental influences,\n\nestimation, , 38\u201339\n\nfitness,\n\ngender, 32\u201333\n\nHarris\u2013Benedict equation,\n\nhormonal status,\n\nindirect calorimetry measurement,\n\noverview,\n\nphysical activity energy\n\nexpenditure, 34\u201336\n\nresting energy expenditure, 31\u201335\n\nspecial populations, 38\u201339\n\nthermic effect of food, 33\u201334,\n\ntotal energy expenditure, 36\u201339\n\nunderreporting, 76\u201377\n\ngenetic errors\n\ncancer association\n\ncarcinogen metabolism,\n\none-carbon metabolism, 201\u2013202\n\ncardiovascular disease association\n\nendogenous lipoprotein metabolism, 166\u2013167\n\n3-hydroxy-3-methylglutaryl-coenzyme A reductase,\n\nlow-density lipoprotein receptor, 166\u2013167\n\ndiagnosis, 210\u2013211\n\nmanagement strategies, 219\u2013220\n\nchildhood illnesses, 216\u2013217\n\nfeeding,\n\nfood-related behaviors, 219\u2013220\n\nhydration,\n\noral health,\n\nparent\u2013child interactions,\n\nnewborn screening programs, 210\u2013211\n\noverview, 209\u2013210,\n\npublic health services, 209\u2013210\n\ntreatment, 211\u2013220\n\nadult treatment, 221\u2013223\n\namino acid requirements, 216\u2013218\n\nbiochemical parameter assessment,\n\nbranched-chain ketoaciduria, ,\n\nbreastfeeding,\n\nenergy requirements,\n\nfats,\n\ngalactosemia, ,\n\nglycogen storage disease, , ,\n\ngrowth assessment, 215\u2013216\n\nhomocystinuria, ,\n\nmanagement strategies, 219\u2013220\n\nmaple syrup urine disease, , , 222\u2013223\n\nmetabolic specialists team,\n\nminerals, 218\u2013219\n\nnutritional assessment, 215\u2013216\n\nnutritionist role,\n\nphenylketonuria, 212\u2013213, 220\u2013221\n\npregnancy, 222\u2013223\n\nprinciples, 212\u2013215\n\nprotein provision,\n\ntyrosinemia, ,\n\nvitamins, , 218\u2013219\n\nHIV\/AIDS patients, 741\u2013742\n\nintravenous feeding complications,\n\nMetamucil\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nMethionine\n\ncolon cancer relationship, ,\n\nmetabolism, B vitamin's role, 293\u2013294\n\nMethotrexate\n\ninflammatory bowel disease treatment,\n\nnutrient status effects,\n\nMethylmalonic aciduria, nutritional therapy,\n\nMethylprednisolone, in parenteral nutrition supplements,\n\nMetoprolol, food interactions,\n\nMetronidazole, inflammatory bowel disease treatment,\n\nMicrosomal triglyceride transfer protein, genetic influences on cardiovascular disease,\n\nMigraines, treatment, feverfew,\n\nMigrant studies, nutritional epidemiology study,\n\nMilk, _See_ Dairy products\n\nMilk thistle\n\ncancer management,\n\nhealth benefits,\n\nMinerals, _See also_ specific minerals\n\nbone mineralization, 729\u2013730\n\nenteral feeding,\n\ninborn metabolic error treatment, 218\u2013219\n\nintake, HIV\/AIDS patient\n\nrecommendations,\n\nliver disease effects on absorption, 602\u2013603\n\nrenal disease therapy,\n\nsupplement assessment,\n\nMini nutritional assessment, _See also_ Brief assessment\n\ndescription, 54\u201355\n\nModification of Diet and Renal Disease Study, description, 102\u2013103\n\nMollusks, allergic reactions,\n\nMolybdenum, biomarkers of exposure,\n\nMonosodium glutamate, food allergy role,\n\nMonoterpenes, health benefits,\n\nMonounsaturated fatty acids\n\ncardiovascular disease effects, 279\u2013282\n\ndiabetes management,\n\nhypertension effects,\n\nMorning sickness, vomiting induction,\n\nMotion sickness\n\ntreatment\n\nDramamine\u00ae, 551\u2013552\n\nginger, , , , 559\u2013560\n\nvomiting induction,\n\nMotivation\n\nmotivation versus intention,\n\ntechniques\n\nmotivational intervention method, 98\u2013102\n\nnot ready to change level, 98\u2013101\n\nready to change level,\n\nunsure about change level,\n\nmotivational interviews,\n\nmotivation models, 95\u201396\n\ndecisional balance,\n\nhealth belief model, 95\u201396\n\nRokeach's value theory,\n\nself-regulation theory, , 118\u2013119\n\nMucositis\n\ndrug therapies,\n\nnutrition management strategies in cancer patients,\n\nMultiattribute utility theory, description, 87\u201388\n\nMycotoxins, biomarkers of exposure,\n\nMylicon\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nN\n\nNails, physical assessment,\n\nNasopharyngeal cancer, diet\u2013genetic interactions,\n\nNational Cholesterol and Education Program,\n\nNausea\n\ncauses, 557\u2013559\n\nfood effects,\n\nnutrition management strategies in cancer patients,\n\nprevalence, 557\u2013558\n\ntreatment\n\ndrug therapies, , , 559\u2013560\n\nginger, , , , 559\u2013560\n\nNeonatal hepatitis, description, 604\u2013605\n\nNeurologic disorders, _See also_ Alzheimer's disease; Parkinson's disease\n\nphysical assessment,\n\ntreatment, ,\n\nNeutropenia\n\nanorexia nervosa association,\n\ndrug therapies,\n\nnutrition management strategies in cancer patients,\n\nNewborn screening programs, 210\u2013211,\n\nNiacin\n\nbiomarkers of exposure,\n\ndeficiency signs,\n\ndietary reference intakes, ,\n\nin parenteral nutrition supplements,\n\nin Parkinson's disease therapy, 647\u2013648\n\nrenal disease therapy,\n\nNicotine, _See_ Smoking\n\nNitrogen, protein conversion,\n\nNonalcoholic steatohepatitis, description,\n\nNonexercise activity thermogenesis, measurement, 34\u201335\n\nNovel diets, _See also_ _specific diets_ description,\n\nNutrient database, selection, , 69\u201370\n\nNutrient intake, _See_ Dietary intake\n\nNutritional counseling, _See also_ Behavior modification\n\nbehavior influence,\n\nHIV\/AIDS patients,\n\nNutritional epidemiology\n\ncause and effect interpretation, 62\u201367\n\nassociation measures, 62\u201364\n\ncausality evidence, 63\u201364\n\nconfounding,\n\nobstacles to association, 64\u201367\n\ndesign limitations, 66\u201367\n\nquestionnaire error sources, 64\u201366\n\nreliability assessment, 64\u201366\n\nvalidity assessment, 64\u201366\n\ncommunity-based research,\n\ndata analysis, 69\u201377\n\ncomputer-based analysis, 69\u201371\n\ninterpretation, 74\u201377\n\nassessment methods,\n\noverreporting,\n\nscientific evidence hierarchy, 74\u201375\n\nunderreporting, 75\u201377\n\nvalidity, 75\u201377\n\npresentation, 72\u201374\n\ntotal diet analysis, 71\u201372\n\ndisease development timeline,\n\nexposure measurement principles, 60\u201361\n\nbiomarker use,\n\nfood frequency questionnaires, 60\u201361\n\nsupplement assessment,\n\nfuture research directions, 67\u201368,\n\nmultifactorial nature of chronic disease,\n\nobesity studies\n\ndiabetes relationship, 417\u2013418\n\ngenetic factors, 183\u2013184\n\noverview, 59\u201360, ,\n\nstudy design, 61\u201362\n\ncase-control studies,\n\ncohort studies, 61\u201362\n\nintervention studies,\n\nmigrant studies,\n\nobservational studies, 61\u201362,\n\nNutritional Risk Index, description,\n\nNutritional screening programs\n\ncancer patients,\n\nNutrition Screening Initiative,\n\nScored Patient-Generated Subjective Global Assessment,\n\nNutritional supplements, _See_ Supplements; _specific supplements_\n\nNutrition education, _See_ Education\n\nNutrition guidelines\n\nadequacy, 753\u2013760\n\ndietary reference intake levels, , 753\u2013760\n\nestimated average requirement, 72\u201374, , 759\u2013760\n\nfood guide pyramid, , ,\n\nrecommended dietary allowance, 72\u201374\n\nserving equivalencies, , 764\u2013765\n\ntolerable upper intake levels, ,\n\nDietary Guidelines for Americans, 760\u2013763\n\nAim for Fitness, ,\n\nBuild a Healthy Base, ,\n\nChoose Sensibly, , 761\u2013763\n\ncurrent intakes compared, 757\u2013760, 766\u2013767\n\ndietary guidelines for fat, 761\u2013762\n\nfood exchanges, 763\u2013764\n\nFood Guide Pyramid, , ,\n\nnutrition labels, 764\u2013766\n\noverview, ,\n\nphysical activity recommendations, _See_ Physical activity\n\nNutrition intervention, _See also_ specific ailments\n\nclinical trials, 95\u2013103\n\nDiabetes Control and Complications Trial, , ,\n\nDiet Intervention Study in Children, 96\u201398\n\nModification of Diet and Renal Disease Study, 102\u2013103\n\nmotivational intervention method, 98\u2013102\n\nnot ready to change level, 98\u2013101\n\nready to change level,\n\nunsure about change level,\n\nmotivation models, 95\u201396\n\ndecisional balance,\n\nhealth belief model, 95\u201396\n\nRokeach's value theory,\n\nself-regulation theory, , 118\u2013119\n\nnutritional epidemiology study,\n\noverview, ,\n\nWomen's Health Initiative,\n\nevaluation, 123\u2013135\n\ncomparison group,\n\ndesign, 125\u2013126\n\neffects analysis, 126\u2013128\n\nconfounding factors, 127\u2013128\n\nmeasurement level, 126\u2013127\n\nsociodemographic effects, 127\u2013128\n\nstatistical power,\n\nunits of analysis, 126\u2013127\n\nexposure measurement using biomarkers\n\nenergy intake, 142\u2013143\n\nepidemiology exposure,\n\ngeneral dietary indicators,\n\nmicromolecule intake, 140\u2013142\n\nnutrient intake, 139\u2013140\n\ninstruments, 130\u2013135\n\nanthropometric measures, 45\u201346,\n\nbiochemical measures, , 624\u2013627\n\ncomplexity,\n\nenvironmental measures, 124\u2013125, 134\u2013135\n\nself-reported dietary behavior, 130\u2013134\n\nsurrogate measures, 134\u2013135\n\nmeasurement issues, 128\u2013130\n\ncosts,\n\ninstrument complexity,\n\nintervention-associated recall bias,\n\nparticipant burden, 129\u2013130\n\nreliability,\n\nresponsiveness,\n\nvalidity, 128\u2013129\n\noutcomes\n\ndescription, 123\u2013124\n\ntypes, 124\u2013125\n\noverview, 11\u201312, 123\u2013124,\n\npostintervention assessments,\n\npreintervention measures,\n\nrandomized assignment,\n\nrepresentative samples,\n\ntypes of evaluation, 123\u2013124\n\nformative evaluation,\n\noutcome evaluation, 123\u2013124\n\nprocess evaluation,\n\nmedical nutritional supplements, 229\u2013238\n\nbarriers,\n\ncommunity programs, 232\u2013233\n\ndisease-specific conditions, 233\u2013238\n\ncancer, 233\u2013235\n\nchronic obstructive pulmonary\n\ndisease, 229\u2013230, 235\u2013236\n\ndiabetes mellitus, 236\u2013237\n\nHIV\/AIDS, 229\u2013230,\n\nrenal disease,\n\nFDA definition, 229\u2013231\n\noverview, ,\n\nstandard supplement conditions, 231\u2013233\n\nAlzheimer's disease, ,\n\ncerebral vascular accident, 231\u2013232\n\ngeneral medicine,\n\nhip fracture,\n\nhome care,\n\nlong-term care,\n\nsurgery, ,\n\ntheoretical bases, 83\u201391\n\napplication findings, 88\u201399\n\nbehavior influences\n\nbarriers to action, 90\u201391\n\nbiobehavioral factors,\n\ncontrol beliefs,\n\ncounseling implications, ,\n\nfood choice,\n\ninfluence levels, 83\u201384\n\nintention versus action,\n\nlong-term change, 84\u201390, 219\u2013220\n\nmotivation versus intention,\n\nrecommendation reactions,\n\nself-efficacy, ,\n\nspecial populations,\n\nchange stages, 86\u201387\n\nchange theories,\n\nconstructs, 89\u201391\n\nconsumer information processing,\n\nexplanatory theories,\n\nhealth belief model,\n\nimplications,\n\ninnovation diffusion,\n\nissues, 89\u201391\n\nmodel selection,\n\nmultiattribute utility theory, 87\u201388\n\nopportunities,\n\noverview,\n\npatient' perceptions,\n\nsocial cognitive theory, 85\u201386,\n\ntheory definition,\n\nNutritionists, inborn error in metabolism treatment,\n\nNutrition labels\n\nconsumer information processing,\n\nnutrition guidelines, 764\u2013766\n\nNuts, allergic reactions,\n\nO\n\nOats, health benefits,\n\nObesity, _See also_ Body weight\n\nacute versus long-term weight loss maintenance, 476\u2013477\n\nassessment methods, 465\u2013468\n\nbody mass index, , 465\u2013467,\n\nclinical assessments, 465\u2013467\n\ngoal setting,\n\nreadiness assessment,\n\nrisk factor assessment,\n\ntreatment option selection,\n\nwaist circumference, , 47\u201348, ,\n\nbehavior modification, _See_ Behavior modification\n\nbreast cancer association, 339\u2013340, 342\u2013345\n\ndiabetes relationship\n\ndefinitions, 415\u2013417\n\ndiabetes mellitus, 416\u2013417\n\nobesity, 415\u2013416\n\ndiabetes treatment,\n\nenergy intake restriction effects, 421\u2013422\n\nepidemiology, 417\u2013418\n\ninsulin insensitivity\n\nin nonobese,\n\nin obesity, 420\u2013421\n\ninsulin resistance, 418\u2013419,\n\noverview, , 422\u2013423\n\nprevalence correlations, 418\u2013421\n\npsychological stress, 419\u2013421\n\ntype 2 diabetes\n\nfetal origins,\n\nmetabolic alterations, 419\u2013421\n\nphysical activity role,\n\nvisceral adiposity, 419\u2013421\n\nweight loss effects, 421\u2013422\n\ndietary factors\n\nappetite effects, 541\u2013543\n\neating behaviors, 527\u2013529\n\nbinge eating, , , 694\u2013695\n\nfamily influences, 528\u2013529\n\nmeal patterns,\n\npacing,\n\nrestraint, 527\u2013528\n\nenergy deficit creation, 468\u2013469\n\nenergy intake, , 522\u2013523, 542\u2013543\n\nenvironmental influences, , 524\u2013527\n\neating out,\n\nfast foods, 525\u2013526\n\nfood advertising, 526\u2013527,\n\ngene\u2013environment interactions,\n\nportion size,\n\nU.S. food supply, 524\u2013525\n\nfat intake, _See_ Dietary fat\n\nfood choices, , 540\u2013541\n\nfuture research directions,\n\nhunger effects, 541\u2013542\n\nmacronutrient composition, 469\u2013470, 522\u2013524\n\nmeal replacement, ,\n\nmedication combined,\n\nnondiet approach, 470\u2013471\n\noverview, 530\u2013532\n\npublic health recommendations, 529\u2013530\n\nsatiety, 541\u2013542\n\nsugar intake, 502\u2013503,\n\ntaste factors, 539\u2013541\n\nunderreporting food intake,\n\ngenetic factors, 183\u2013205\n\ncandidate gene association studies, 189\u2013192\n\naltered function causes, 190\u2013192\n\nsingle-gene obesity role,\n\nclinical implications, 192\u2013194\n\ndiagnosis, 192\u2013193\n\ntreatment,\n\nepidemiology, 183\u2013184\n\ngene\u2013environment interactions,\n\ngene map, 184\u2013185\n\noverview, 183\u2013184\n\nphenotype evidence from linkage studies, 188\u2013189\n\nhuman studies,\n\nloci mapping in animals, 188\u2013189\n\nmouse studies,\n\nrare Mendelian syndromes,\n\nsingle-gene adipose tissue atrophy,\n\nsingle-gene obesity, 185\u2013188\n\nleptin deficiency, 185\u2013186,\n\nleptin receptor deficiency, 186\u2013187\n\nmelanocortin-4 receptor gene mutation, 187\u2013188\n\nproopiomelanocortin deficiency,\n\nproprotein convertase subtilisin\/kexin type 1 deficiency,\n\ntaste and appetite relationship, 539\u2013540\n\nhypertension effects, 311\u2013312, ,\n\nlung cancer relationship,\n\nObesity Education Initiative, 481\u2013483\n\noverview,\n\npediatric treatment, 475\u2013476\n\npharmaceutical intervention, 471\u2013474\n\nbackground, 471\u2013472\n\nefficacy predictors,\n\nlifestyle modification combined,\n\nlong-term medications, 473\u2013474\n\norlistat, , , 473\u2013474\n\nsibutramine,\n\nnonapproved medications, 472\u2013473\n\npatient selection,\n\nrisk\/benefit ratio,\n\nshort-term medications,\n\nphysical activity effects\n\nactivity determinants, 482\u2013483\n\nadherence, 491\u2013493\n\naerobic exercise only, 483\u2013487\n\naerobic exercise plus diet,\n\nbarriers to activity, 521\u2013522\n\naccess,\n\ndiscomfort,\n\nenvironmental factors,\n\ntime, 521\u2013522\n\nbehavior modification,\n\ncomorbidity treatment, 493\u2013494\n\netiology, 482\u2013483\n\nexercise history,\n\nleisure-time activity,\n\nmaintenance programs, 490\u2013491\n\nobesity development, 518\u2013522\n\nobesity prevalence\u2013activity relationship, 481\u2013482\n\noverview, , ,\n\nresistance exercise, 487\u2013490\n\nsedentary behavior, 519\u2013520\n\nself-efficacy, 520\u2013521\n\nsocial support,\n\nweight loss programs, 483\u2013493\n\nprevalence, 481\u2013482,\n\nprostate cancer relationship,\n\nsingle-gene adipose tissue atrophy,\n\nsleep apnea association,\n\nsurgical treatment, 474\u2013475\n\ncomorbid condition reduction,\n\ncomplications and side effects,\n\nweight loss amounts,\n\nvery low calorie\/energy diets,\n\nObservational studies\n\nAlzheimer's disease study, 641\u2013642\n\nhypertension study, 305\u2013306\n\nlimitations, 66\u201367\n\nnutritional epidemiology study design, 61\u201362,\n\nParkinson's disease study, 642\u2013644\n\nOctreotide, in parenteral nutrition supplements,\n\nOdynophagia\n\ndrug therapies,\n\nnutrition management strategies in cancer patients,\n\nOlestra, computer-based analysis, 70\u201371\n\nOmega-3 fatty acids\n\ncancer nutritional therapy,\n\ncardiovascular disease effects,\n\nenteral feeding, 246\u2013248\n\nhypertension effects,\n\ninflammatory bowel disease association, 581\u2013582, 586\u2013587\n\nprostate cancer relationship,\n\nOmega-6 fatty acids\n\ncardiovascular disease effects,\n\nhypertension effects,\n\ninflammatory bowel disease association, 581\u2013582, 586\u2013587\n\nprostate cancer relationship,\n\nOral allergy syndrome, allergic food reactions,\n\nOral and dental health, physical assessment, 49\u201351\n\nOral cancer, diet\u2013genetic interactions,\n\nOral candidiasis, drug therapies,\n\nOrganosulfides, content in foods,\n\nOrlistat\n\ncomputer-based analysis,\n\nnutrient status effects,\n\nobesity intervention, 473\u2013474\n\nOrnithine transcarbamylase deficiency, nutritional therapy,\n\nOsteomalacia\n\ncauses, 732\u2013738\n\ncalcium intake deficiencies,\n\nmineralization inhibitors, 737\u2013738\n\nosteoblast dysfunction,\n\nphosphorus depletion,\n\nphytate ingestion, 735\u2013736\n\nrenal tubular defects,\n\ntumor-induced osteomalacia, 736\u2013737\n\nvitamin D deficiency, 732\u2013735\n\ngastrointestinal pathology,\n\ninadequacy versus deficiency,\n\ninherited forms,\n\nintake deficiencies,\n\nsunlight exposure,\n\nvitamin D physiology, 732\u2013734\n\nmineralization mechanisms, 729\u2013730\n\nosteoporosis compared, 729\u2013730\n\noverview,\n\ntreatment,\n\nOsteopenia, anorexia nervosa association, 689\u2013690\n\nOsteoporosis\n\nbone health biomarkers, 146\u2013147\n\ncalcium effects\n\nlife stage requirements, 662\u2013670\n\ngrowth, 663\u2013666\n\nlactation, 667\u2013668\n\nmaturity, 666\u2013667\n\nmenopause, 668\u2013669\n\npregnancy, 667\u2013668\n\nsenescence, 669\u2013670\n\nnatural intake, 658\u2013659\n\nnutritional influences, 670\u2013672\n\naluminum,\n\nbone density,\n\ncaffeine,\n\ncalcium absorption enhancers,\n\nfat,\n\nfiber,\n\nintake versus interference,\n\nintestinal absorption,\n\nphosphorus,\n\nprotein, 671\u2013672\n\nrenal conservation of calcium, 671\u2013672\n\nsodium, 671\u2013672\n\nnutritional requirement, 660\u2013662, 670\u2013672\n\nskeletal reserves,\n\nhip fractures\n\nfrailty role,\n\nmedical nutritional therapy,\n\nlactose intolerance effects,\n\nmineral effects\n\naluminum,\n\ncopper effects,\n\nmagnesium, 675\u2013676\n\nmanganese,\n\nzinc,\n\nnutritional effects, 653\u2013657\n\nbone density,\n\nbone mass,\n\nfrailty, 654\u2013655\n\ninjury,\n\nintrinsic bony strength, 654\u2013655\n\ninvestigation problems, 655\u2013657\n\nbone remodeling transient, 656\u2013657\n\nbone response time,\n\nlife phase specificity,\n\nnutrient intake estimation, 655\u2013656\n\nnutrient\u2013nutrient interactions,\n\nnutritional requirement\n\ncalcium, 660\u2013662\n\ntheory, 657\u2013658\n\nosteomalacia compared, 729\u2013730\n\noverview, 653\u2013677,\n\nrenal disease association,\n\nvitamin D effects, , 658\u2013659, 672\u2013674\n\nvitamin K effects, 674\u2013675\n\nOvalbumin, food allergy role,\n\nOvereaters Anonymous, behavior modification training,\n\nOxidative stress, _See also_ Antioxidants\n\nbiomarkers of change, 145\u2013146\n\ndiet\u2013genetic interactions in cancer, 200\u2013201\n\nP\n\nPain\n\ndrug therapies,\n\nnutrition management strategies in cancer patients,\n\nPancreatic disease, _See_ Cystic fibrosis\n\nPantothenic acid\n\ndietary reference intakes,\n\nin parenteral nutrition supplements,\n\nrenal disease therapy,\n\nParenteral nutrition\n\nadministration,\n\nanorexia nervosa patients,\n\ncancer patients, ,\n\ncomplications, 253\u2013256\n\nmetabolic complications, 254\u2013256\n\nsepsis,\n\ntechnical complications, 253\u2013254\n\ncontraindications,\n\nelectrolytes, 251\u2013252\n\nfluids, 251\u2013252\n\nin HIV\/AIDS patients,\n\nindications,\n\ninflammatory bowel disease treatment, 587\u2013589\n\nmacronutrients, 250\u2013251\n\nmedications,\n\nmicronutrients, 252\u2013253\n\noverview, ,\n\nparenteral access,\n\nshort bowel syndrome treatment, 593\u2013594\n\nParkinson's disease\n\nnutritional management, 645\u2013648\n\nprotein modified diets, 646\u2013648\n\nweight loss,\n\nnutritional trials, 644\u2013645\n\nnutrition\u2013cognitive function relationship, 637\u2013641\n\nanimal studies, 637\u2013638\n\ncross-sectional studies, 638\u2013641\n\nlongitudinal studies, 638\u2013641\n\nobservational studies, 642\u2013644\n\noverview, ,\n\npathological studies,\n\nParvalbumin-\u03b2, food allergy role,\n\nPeanuts, allergic reactions, 705\u2013707\n\nPectin, gastrointestinal symptom treatment, 551\u2013552\n\nPediatric care\n\nallergic reactions, _See_ Food allergy\n\ncalcium requirements, 663\u2013666\n\ncancer patient nutritional issues,\n\nchronic liver disease,\n\ncystic fibrosis management, 722\u2013723\n\ndietary assessment methods, 13\u201314\n\ninborn metabolic errors, management strategies\n\nchildhood illnesses, 216\u2013217\n\nparent-child interactions,\n\nleisure-time physical activity,\n\nnutrition intervention trials, 96\u201398\n\nobesity treatment, 475\u2013476\n\nD-Penicillamine, Wilson's disease\n\ntreatment, ,\n\nPepsid AC\u00ae, gastrointestinal symptom treatment, 551\u2013553\n\nPepto Bismol\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nPharmaceuticals, _See_ Medications; _specific drugs_\n\nPhenergan\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nPhenolic acids, health benefits,\n\nPhentermine, obesity intervention, 472\u2013473\n\nPhenylalkylketones, health benefits,\n\nPhenylethylamine, food allergy role,\n\nPhenylketonuria\n\nnewborn screening programs, 210\u2013212\n\nnutritional therapy, 212\u2013213, 220\u2013221\n\noutcome analysis,\n\npregnancy management, 222\u2013223\n\nPhenylpropanolamine, obesity intervention, 472\u2013473\n\nPhosphorus\n\nbiomarkers of exposure,\n\nbone mineralization, 729\u2013730\n\ndaily requirements, 251\u2013252\n\ndietary reference intakes, ,\n\nosteomalacia relationship, ,\n\nin parenteral nutrition, 251\u2013252,\n\nin Parkinson's disease therapy, 647\u2013648\n\nrenal conservation of calcium,\n\nrenal disease therapy, , , 628\u2013629\n\nPhthalides, health benefits,\n\nPhysical activity\n\nanorexia nervosa patients,\n\nbreast cancer risk reduction,\n\ncancer risk reduction,\n\ncolon cancer relationship,\n\ngastrointestinal disease reduction,\n\nguidelines, 767\u2013769\n\nhandgrip strength test,\n\nhealth maintenance nutrition guidelines, Aim for Fitness, ,\n\nnonexercise activity thermogenesis, 34\u201335\n\nobesity relationship\n\nactivity determinants, 482\u2013483\n\nadherence, 491\u2013493\n\naerobic exercise only, 483\u2013487\n\naerobic exercise plus diet,\n\nbarriers to activity, 521\u2013522\n\naccess,\n\ndiscomfort,\n\nenvironmental factors,\n\ntime, 521\u2013522\n\nbehavior modification,\n\ncomorbidity treatment, 493\u2013494\n\netiology, 482\u2013483\n\nexercise history,\n\nleisure-time activity,\n\nmaintenance programs, 490\u2013491\n\nobesity development, 518\u2013522\n\nobesity prevalence\u2013activity relationship, 481\u2013482\n\noverview, , ,\n\nresistance exercise, 487\u2013490\n\nsedentary behavior, 519\u2013520\n\nself-efficacy, 520\u2013521\n\nsocial support,\n\nweight loss programs, 483\u2013493\n\nprostate cancer risk reduction, 379\u2013380\n\nresting energy expenditure measurement, 33\u201336\n\ndoubly-labeled water method,\n\nheart rate monitoring, 35\u201336\n\nmovement counters,\n\nobesity relationship, 482\u2013483\n\nquestionnaires,\n\nweight reduction effects,\n\nPhysical assessment\n\nanthropometric assessment\n\nbody fat distribution, , 47\u201348, ,\n\nbreast cancer risk, 342\u2013344\n\ncircumference measurement, , 47\u201348, ,\n\ncystic fibrosis assessment, 720\u2013721\n\nheight, 44\u201345\n\nnutrition intervention evaluation,\n\nrenal disease nutrition assessment, 623\u2013624\n\nskinfold thickness measurement, 47\u201348\n\nweight, 45\u201346,\n\nweight for height, 46\u201347\n\nclinical assessment\n\nAIDS,\n\ncancer,\n\ncardiovascular system,\n\ngastrointestinal system, 51\u201352\n\nhair,\n\nhematologic system,\n\ninfectious disease,\n\nmethods, 11\u201312, 43\u201344\n\nmusculoskeletal system,\n\nnails,\n\nneurologic disorders,\n\noral and dental health, 49\u201351\n\npsychiatric disorders,\n\npulmonary system,\n\nrenal system, 52\u201353\n\nskin,\n\nfunction assessment, _See_ Function assessment\n\noverview, , ,\n\nprognostic indicators, 53\u201355\n\nMini Nutritional Assessment, 54\u201355\n\nNutrition Screening Initiative,\n\nPrognostic Nutritional Index, 53\u201354\n\nSubjective Global Assessment, 53\u201354\n\nPhytates\n\ncontent in foods,\n\nosteomalacia relationship, 735\u2013736\n\nPhytoestrogens\n\nbiomarkers of exposure, 140\u2013142\n\nbreast cancer risk reduction, 342\u2013343\n\ncontent in foods,\n\nhealth benefits,\n\nprostate cancer risk reduction, 378\u2013379\n\nin soy protein, 285\u2013286, ,\n\nPhytosterols, health benefits, 296\u2013297\n\nPneumonia, enteral feeding complications,\n\nPolyacetylenes, health benefits,\n\nPolysaccharides, _See also_ Carbohydrates health benefits,\n\nPolyunsaturated fatty acids, cardiovascular disease effects,\n\nPortion size\n\nestimation, 14\u201315\n\nobesity effects,\n\nPotassium\n\ndaily requirements, 251\u2013252\n\ndeficiency effects, vomiting,\n\nfood interactions,\n\nhypertension effects, ,\n\nosteomalacia treatment,\n\nin parenteral nutrition,\n\nrenal disease therapy, , 628\u2013629\n\nPrediction equations, description,\n\nPregnancy\n\ncalcium requirements, 667\u2013668\n\ncystic fibrosis management, 722\u2013723\n\nenergy requirement effects,\n\ngestational diabetes nutrition management, 453\u2013461\n\nartificial sweeteners,\n\nbehavioral outcomes, 455\u2013456\n\nblood glucose monitoring, ,\n\ncarbohydrate consistency,\n\ncarbohydrate distribution,\n\ncarbohydrate type,\n\nclinical outcomes, ,\n\ndiagnosis, 453\u2013454\n\nenergy intake,\n\nfat, 458\u2013459\n\nfetal complications,\n\ninsulin therapy, 459\u2013461\n\nmorning carbohydrate, 456\u2013458\n\nnutrition therapy, 454\u2013459\n\noverview, ,\n\npostpartum recommendations,\n\nprotein,\n\nreferral,\n\nrisk factors,\n\nscreening, 453\u2013454\n\nself-management, 455\u2013456\n\ntotal carbohydrate,\n\nweight gain, 459\u2013460\n\nmanagement in women with metabolic disorders, 222\u2013223\n\nmorning sickness,\n\npregnancy-related hepatic disorders,\n\nPrimary biliary cirrhosis, description,\n\nPrimary sclerosing cholangitis, pregnancy,\n\nProanthocyanidins, health benefits,\n\nProblem-solving, goal setting,\n\nProcess evaluations, description,\n\nProchlorperazine, gastrointestinal symptom treatment, 551\u2013552\n\nProfilin, food allergy role,\n\nPrognostic indicators, 53\u201355\n\nMini Nutritional Assessment, 54\u201355\n\nNutrition Screening Initiative,\n\nPrognostic Nutritional Index, 53\u201354\n\nSubjective Global Assessment, 53\u201354\n\nPrognostic Nutritional Index, description, 53\u201354\n\nPromethazine, gastrointestinal symptom treatment, 551\u2013552\n\nProopiomelanocortin, obesity role,\n\nPropionic acidemia, nutritional therapy,\n\nPropranolol, food interactions,\n\nProprotein convertase subtilisin\/kexin type 1, obesity role,\n\nProspective studies, _See_ Cohort studies\n\nProstate cancer\n\ndiagnosis,\n\ndietary intervention trials, 380\u2013381\n\ndiet\u2013genetic interactions, 204\u2013205\n\ndiet relationship, 374\u2013380\n\nalcohol consumption, 375\u2013376\n\ncadmium,\n\ncalcium, 376\u2013377\n\ndairy products, 375\u2013376\n\ndiet\u2013prostate cancer hypothesis, 374\u2013375\n\nenergy intake,\n\nfat,\n\nobesity,\n\nred meat, 375\u2013376\n\nrisk reduction, 377\u2013380\n\ncarotenoids, 379\u2013380\n\nfructose,\n\nfruits, 377\u2013378\n\nisoflavones, 378\u2013379\n\nlegumes,\n\nlycopene,\n\nphysical activity, 379\u2013380\n\nselenium, 379\u2013380\n\nsoy protein,\n\ntea consumption,\n\nvegetables,\n\nvitamin D, 377\u2013378\n\nvitamin E, 378\u2013380\n\nzinc,\n\nepidemiology, 373\u2013374\n\nincidence, 373\u2013374\n\nmortality trends, 373\u2013374\n\nrisk factors,\n\ngene\u2013environment interactions,\n\nnormal anatomy and function,\n\noverview, , 380\u2013381\n\npathology,\n\nsurgical side effects,\n\nProstate-specific antigen test, description,\n\nProstatic hypertrophy, treatment, saw palmetto, 267\u2013268\n\nProtein, _See also_ specific proteins\n\nbody weight effects, 506\u2013508\n\nbreast cancer risk, ,\n\ncardiovascular disease effects, 284\u2013286\n\nanimal protein, , 284\u2013285\n\nsoy protein, , 285\u2013286, 294\u2013295\n\ndiabetes medical nutritional therapy\n\ngestational diabetes,\n\ntype 1 diabetes,\n\ntype 2 diabetes, 442\u2013444\n\ndietary reference intakes,\n\nenteral feeding,\n\nfat content relationship, 506\u2013508\n\nhigh protein diets, 506\u2013508\n\nin HIV\/AIDS therapy,\n\nhypertension effects, 309\u2013310,\n\nliver disease effects, ,\n\nin Parkinson's disease therapy, 647\u2013648\n\npostsurgical protein energy malnutrition, medical nutritional therapy, ,\n\nrenal conservation of calcium, 671\u2013672\n\nrenal disease therapy, , , 627\u2013629\n\nrequirements, children with genetic metabolism errors, 216\u2013219\n\nsoy protein, _See_ Soy protein\n\nProton pump inhibitors, nutrient status effects,\n\nPsychiatric disorders, _See also_ specific disorders\n\nphysical assessment,\n\nPsychosocial outcomes, intervention effectiveness assessment, ,\n\nPsyllium, fiber source,\n\nPublic health services\n\ninborn metabolism error services, 209\u2013210\n\nobesity prevention policy, 530\u2013531\n\nschool and community-based youth programs, 529\u2013531\n\nPulmonary disease, _See_ Lung disease\n\nPyridoxine, renal disease therapy,\n\nQ\n\nQuinapril, food interactions,\n\nR\n\nRace, _See_ Cultural differences\n\nRadiation therapy\n\nnutritional issues, ,\n\nside effects,\n\nRamapril, food interactions,\n\nRecommended Dietary Allowances, _See also_ Dietary Approaches to Stop Hypertension diet; Health maintenance nutrition guidelines; Healthy Eating Index\n\ncholesterol levels,\n\ndescription, 72\u201374\n\nenergy requirements, 39\u201340\n\nhealth maintenance guidelines, 753\u2013760\n\nRectal cancer\n\ndiet\u2013genetic interactions, 203\u2013204\n\nin inflammatory bowel diseases,\n\nsurgical side effects,\n\nRed meat, _See_ Meat\n\nRelative risk, nutritional epidemiology association measures, 62\u201364\n\nReliability assessment\n\ncause and effect interpretation, 64\u201366\n\nfood frequency questionnaires, 64\u201366\n\nin nutrition intervention,\n\nRenal disease\n\nchronic renal failure, 617\u2013619\n\nenteral feeding formula, 247\u2013248\n\nFanconi syndrome,\n\nmaple syrup urine disease\n\nnewborn screening programs, 210\u2013212\n\nnutritional therapy, ,\n\npregnancy management, 222\u2013223\n\nmedical nutritional therapy,\n\nModification of Diet and Renal Disease Study, 102\u2013103,\n\nosteomalacia relationship,\n\noverview, ,\n\nphysical assessment, 52\u201353\n\nrenal replacement therapy, 619\u2013628\n\ncomorbid conditions, 621\u2013623\n\nanemia, 622\u2013623\n\nbone disease,\n\ncardiovascular disease,\n\nhyperhomocysteinemia, 621\u2013622\n\nlipoprotein metabolism abnormalities,\n\nmalnutrition,\n\nnutrition assessment, 623\u2013627\n\nanthropometrics, 623\u2013624\n\nbiochemical indices, 624\u2013627\n\ndietary assessment,\n\nserum albumin,\n\nserum cholesterol, 624\u2013625\n\nserum creatinine, 625\u2013627\n\nserum prealbumin,\n\nsubjective global assessment,\n\nnutrition recommendations, 627\u2013628\n\nenergy intake, , 627\u2013629\n\nminerals, 627\u2013628\n\npost-transplant patients, 628\u2013629\n\nprotein, , , 627\u2013629\n\nvitamins, , 627\u2013628\n\nRenal system\n\ncalcium conservation, 671\u2013672\n\nurea cycle\n\nliver defects, 605\u2013606\n\nmaintenance, sodium benzoate role,\n\nurine\n\nelectrolyte composition, ,\n\nlactose intolerance measurement,\n\nsodium excretion, 305\u2013306\n\nRespiratory disease, _See_ Lung disease\n\nRespiratory food hypersensitivity, allergic food reactions, 707\u2013708\n\nResting metabolic rate, study methodology, 31\u201335\n\nage,\n\nbody composition,\n\nbody size,\n\ndeterminants,\n\nenvironmental influences,\n\nestimation,\n\nfat-free mass relationship,\n\nfitness,\n\ngender, 32\u201333\n\nHarris\u2013Benedict equation,\n\nhormonal status,\n\nindirect calorimetry measurement,\n\nphysical activity energy expenditure, 34\u201336, 482\u2013483\n\nthermic effect of food, 33\u201334,\n\nRetinol\n\nbiomarkers of exposure,\n\nbreast cancer risk reduction, ,\n\ncognitive function relationship,\n\ncystic fibrosis relationship, , 720\u2013723\n\ndeficiency signs,\n\ndietary reference intakes, ,\n\nlung cancer relationship, 388\u2013390\n\nin parenteral nutrition supplements,\n\nParkinson's disease relationship,\n\nrenal disease therapy,\n\ntoxicity signs,\n\nRetrospective studies\n\ndescription, 10\u201312\n\nnutritional epidemiology study,\n\nRhinorrhea, description,\n\nRiboflavin\n\nbiomarkers of exposure,\n\ndeficiency signs,\n\ndietary reference intakes,\n\nhomocysteine association,\n\nin parenteral nutrition supplements,\n\nin Parkinson's disease therapy, 647\u2013648\n\nrenal disease therapy,\n\nRickets\n\ncauses\n\ncalcium intake deficiencies,\n\ninherited vitamin D deficiency,\n\nphytate ingestion, 735\u2013736\n\nosteomalacia compared,\n\nRight-sided heart failure, description,\n\nRokeach's value theory, behavioral motivation,\n\nRolaids\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\nRoux-en-Y gastric bypass, obesity treatment,\n\nRussell's sign, bulimia diagnosis,\n\nS\n\nSaccarine, body weight effects, 503\u2013506\n\nSaliva, electrolyte composition,\n\nSalt, _See_ Sodium\n\nSalt sensitivity, hypertension association, 307\u2013308\n\nSatiety\n\nenergy dense foods, 542\u2013543\n\nfats, 541\u2013543\n\nmacronutrients,\n\nobesity effects, 541\u2013542\n\nproteins, 507\u2013508,\n\nSaturated fatty acids, cardiovascular disease effects, 279\u2013281\n\nSaw palmetto\n\ncancer management,\n\nhealth benefits, 266\u2013268\n\nScavenger receptor B type I, reverse\n\ncholesterol transport role,\n\nSchisandra, health benefits,\n\nScombroid fish poisoning, description,\n\nScored Patient-Generated Subjective Global Assessment\n\ncancer patient screening,\n\ndescription, 53\u201354\n\nScreening programs\n\ncancer patients, 345\u2013346,\n\nclinical assessment, 11\u201312\n\nnewborns, inborn metabolism error services, 210\u2013211,\n\nNutrition Screening Initiative,\n\nScored Patient-Generated Subjective Global Assessment,\n\nSedentary lifestyle, obesity effects, 519\u2013520\n\nSelegiline, neurologic degeneration treatment,\n\nSelenium\n\nbiomarkers of exposure,\n\ncognitive function relationship, ,\n\ndeficiency, in liver disease,\n\ndietary reference intakes, ,\n\nlung cancer relationship,\n\nin parenteral nutrition supplements,\n\nprostate cancer risk reduction, 379\u2013380\n\ntoxicity signs,\n\nSelf-efficacy\n\nbehavior change influences, ,\n\nphysical activity regimes, 520\u2013521\n\nSelf-regulation\n\nbehavioral motivation, , 118\u2013119\n\ngoal setting training, 118\u2013119\n\nSelf-reported dietary behavior, _See also_ specific measures\n\nintervention effectiveness assessment, , 130\u2013134\n\nSenescence, _See_ Elderly\n\nSepsis, intravenous feeding complications,\n\nSerotonin, food allergy role,\n\nSerotonin reuptake inhibitors\n\nanorexia nervosa association, ,\n\nnutrient status effects,\n\nSerum albumin\n\ncystic fibrosis assessment,\n\nfood allergy role,\n\nmortality prediction,\n\nrenal disease assessment,\n\nsurgery outcome prediction,\n\nSerum sickness,\n\nServices, _See_ Public health services\n\nServing equivalencies, nutrition guidelines, , 764\u2013765\n\nShell fish, allergic reactions, 705\u2013706\n\nShort bowel syndrome\n\ncauses,\n\nmedical management, 594\u2013595\n\nnutritional management, 593\u2013594\n\noverview, , ,\n\nsurgery, 594\u2013595, ,\n\nvariants, 589\u2013593\n\ncolon loss, 591\u2013592\n\nileal resections, 590\u2013591\n\nileum loss, 591\u2013592\n\njejunal resections,\n\nmajor intestinal resections, 592\u2013593\n\nShrimp, allergic reactions,\n\nSibutramine, obesity intervention,\n\nSimethicone, gastrointestinal symptom treatment, 551\u2013552\n\nSitosterol, health benefits, 296\u2013297\n\nSkeletal markers, breast cancer risk association,\n\nSkeletal system, physical assessment,\n\nSkin\n\ndelayed-type hypersensitivity immune function measurement,\n\nphysical assessment,\n\nskinfold thickness measurement, 47\u201348\n\nSleep apnea, obesity association,\n\nSmoking\n\ncongestive heart failure effects, 328\u2013329\n\nlung cancer risk, 387\u2013389, 393\u2013394\n\nParkinson's disease relationship,\n\nSocial cognitive theory\n\ndietary assessment issues,\n\nnutrition intervention theoretical bases, 85\u201386,\n\nSocial support\n\nfamily influence on obesity, 528\u2013529\n\ngoal setting,\n\nfor physical activity,\n\nSocioeconomic status, cognitive function relationship,\n\nSodium\n\ncongestive heart failure role, 326\u2013327\n\ncystic fibrosis relationship, ,\n\ndaily requirements, 251\u2013252\n\ndeficiency effects, vomiting,\n\nhidden salt ingredients, ,\n\nhigh-salt foods,\n\nhypertension role, 305\u2013308,\n\nintervention studies, 306\u2013307\n\nobservational studies, 305\u2013306\n\nsalt sensitivity, 307\u2013308\n\nliver disease therapy, 608\u2013609\n\nin medications,\n\nmonosodium glutamate allergy,\n\nosteomalacia treatment,\n\nin parenteral nutrition, 251\u2013252,\n\nrenal conservation of calcium, 671\u2013672\n\nrenal disease therapy, , 628\u2013629\n\nSodium benzoate, urea cycle maintenance,\n\nSoft drinks\n\nexcessive gas production, 550\u2013551\n\nobesity effects, 524\u2013526\n\nSoftware, dietary assessment, selection, 16\u201317\n\nSotolol, food interactions,\n\nSoy protein\n\nallergic reactions, 705\u2013707\n\nbreast cancer risk reduction, 342\u2013343\n\ncancer management,\n\ncardiovascular disease effects, , 285\u2013286, 294\u2013297\n\nin enteral feeding formulas,\n\nhypertension effects, 309\u2013310\n\nin parenteral feeding formulas,\n\nprostate cancer risk reduction,\n\nSpinal cord injury, energy requirement effects,\n\nSpironolactone, food interactions,\n\nSt. John's wort\n\nadverse effects, ,\n\nhealth benefits, , ,\n\nStages of change technique, behavior change, 111\u2013113\n\nStarch, _See_ Carbohydrates\n\nStigmasterol, health benefits, 296\u2013297\n\nStinging nettle, health benefits,\n\nStomatitis, nutrition management strategies in cancer patients,\n\nStress, _See also_ Oxidative stress diabetes relationship,\n\nStroke, medical nutritional therapy,\n\nSubjective Global Assessment\n\ncancer patient screening,\n\ndescription, 53\u201354\n\nSugars, _See also_ Carbohydrates\n\ncolon cancer relationship, , 362\u2013363\n\ndiabetes nutritional management, type 2 diabetes, 444\u2013445\n\ndietary guidelines,\n\ninflammatory bowel disease effects,\n\nobesity effects, 502\u2013503,\n\ntaste effects on food choice, 540\u2013541\n\nSugar substitutes\n\nbody weight effects, 503\u2013506\n\ngestational diabetes management,\n\nSulfasalazine, nutrient status effects,\n\nSulfides\n\nallylic sulfides, 296\u2013297\n\norganosulfides,\n\nSulfites, allergic reactions,\n\nSulfonylureas, nutrient status effects,\n\nSulforaphane, colon cancer relationship,\n\nSulfur, plant sources,\n\nSunlight exposure, vitamin D deficiency,\n\nSupplements\n\nassessment methods, 17\u201318\n\nbotanicals, 261\u2013271\n\nadverse effects,\n\nclinical recommendations, 269\u2013271\n\ncost effectiveness,\n\ndietetics professionals, 270\u2013271\n\nlegal issues, 270\u2013271\n\nmedical professionals, 270\u2013271\n\nefficacy, 263\u2013264\n\nhistorical perspectives,\n\noverview, , 271\u2013272\n\npatients,\n\npopularity factors, 262\u2013263\n\nregulation, 268\u2013269\n\nreported use, 261\u2013262\n\nsafety,\n\nsupporting evidence, 264\u2013268\n\nechinacea, 264\u2013265, , ,\n\nfeverfew, ,\n\ngarlic, , , , 296\u2013297\n\ngingko, , , , 644\u2013645\n\nsaw palmetto, 267\u2013268\n\nscientific process,\n\nSt. John's wort, , ,\n\nvalerian,\n\ncongestive heart failure effects,\n\nmedical nutritional supplements, 229\u2013238\n\nbarriers,\n\ncommunity programs, 232\u2013233\n\ndisease-specific conditions, 233\u2013238\n\ncancer, 233\u2013235\n\nchronic obstructive pulmonary disease, 229\u2013230, 235\u2013236\n\ndiabetes mellitus, 236\u2013237\n\nHIV\/AIDS, 229\u2013230,\n\nrenal disease,\n\nFDA definition, 229\u2013231\n\noverview, ,\n\nstandard supplement conditions, 231\u2013233\n\nAlzheimer's disease, ,\n\ncerebral vascular accident, 231\u2013232\n\ngeneral medicine,\n\nhip fracture,\n\nhome care,\n\nlong-term care,\n\nsurgery, ,\n\nSurgery\n\nbone marrow transplantation, 408\u2013410\n\nbreast cancer treatment, nutritional issues,\n\nin cancer patients, common side effects,\n\ninflammatory bowel disease treatment,\n\nliver transplant preparation, ,\n\nmedical nutritional therapy, ,\n\nnutrition management strategies in cancer patients,\n\nobesity treatment, 474\u2013475\n\ncomorbid condition reduction,\n\ncomplications and side effects,\n\nweight loss amounts,\n\nshort bowel syndrome treatment, 594\u2013595, ,\n\nulcerative colitis treatment,\n\nSurrogate reporters\n\ndietary assessment methods, 12\u201314\n\nnutrition intervention, 134\u2013135\n\nSurveillance methods\n\nBehavioral Risk Factor Surveillance System, 7\u20138\n\ndescription,\n\nSweat, electrolyte composition,\n\nT\n\nTagamet\u00ae, gastrointestinal symptom treatment, 551\u2013553\n\nTake Off Pounds Sensibly, behavior modification training,\n\nTangier disease, HDL deficiency,\n\nTaste\n\ngenetic factors, 539\u2013540\n\nmedical nutritional supplement barrier,\n\nnutrition management strategies in cancer patients,\n\nobesity regulation effects, 539\u2013541\n\nTaurine, congestive heart failure effects, ,\n\nTeas\n\ncancer management,\n\ngreen tea\n\nhealth benefits,\n\nprostate cancer risk reduction,\n\nTelevision watching, obesity effects, 519\u2013520, 526\u2013527\n\nTerpenes\n\ncontent in foods,\n\nhealth benefits,\n\nTheobromine, food allergy role,\n\nThermic effect of food, resting energy expenditure measurement, 33\u201334,\n\nTherogenesis, resting energy expenditure measurement, 33\u201334\n\nThiamin\n\nbiomarkers of exposure,\n\ncognitive function relationship, 638\u2013639\n\ndeficiency, in liver disease,\n\ndeficiency signs,\n\ndietary reference intakes,\n\nin parenteral nutrition supplements,\n\nrenal disease therapy,\n\nThrombocytopenia\n\nanorexia nervosa association,\n\ndrug therapies,\n\nThyme, health benefits,\n\nTobacco, _See_ Smoking\n\n\u03b1-Tocopherol\n\nAlzheimer's disease relationship,\n\nbioavailability,\n\nbiomarkers of exposure,\n\nbreast cancer risk reduction, ,\n\ncognitive function relationship, 637\u2013642\n\ncolon cancer relationship, , 361\u2013362\n\ncystic fibrosis relationship, , 720\u2013723\n\ndeficiency signs, ,\n\ndietary reference intakes, , 756\u2013757, 759\u2013760\n\nlung cancer relationship, 390\u2013391\n\nneurologic degeneration treatment, ,\n\noxidative stress reduction,\n\nin parenteral nutrition supplements,\n\nParkinson's disease relationship, 644\u2013645\n\nprostate cancer risk reduction, 378\u2013380\n\nrenal disease therapy,\n\nsmoking effects,\n\nTolerable upper intake levels, health maintenance nutrition guidelines, ,\n\nTomatoes, health benefits,\n\nTotal energy expenditure, measurement, 36\u201339\n\ndirect calorimetry,\n\ndoubly-labeled water method, 36\u201338\n\nestimation, 38\u201339\n\nspecial populations, 38\u201339\n\nTotal nutrient admixture, _See_ Parenteral nutrition\n\nTrandolapril, food interactions,\n\n_Trans_ fatty acids\n\ncardiovascular disease effects, 279\u2013280,\n\ninflammatory bowel disease association, 586\u2013587\n\nTransplants\n\nbone marrow transplantation, 408\u2013410\n\nliver transplant preparation, ,\n\nrenal replacement therapy in post-transplant patients, 628\u2013629\n\nTreophylline, nutrient status effects,\n\nTricyclic antidepressants\n\nbulimia therapy, 693\u2013694\n\nnutrient status effects,\n\nTriglycerides\n\ncardiovascular disease risk, , 280\u2013281\n\nhypertriglyceridemia treatment,\n\nTriterpenoids, health benefits,\n\nTropomysin, food allergy role,\n\nTube feeding, _See_ Enteral nutrition; Parenteral nutrition\n\nTumeric, health benefits,\n\nTumors, _See_ Cancer\n\nTumor suppressor genes, breast cancer association,\n\nTums\u00ae, gastrointestinal symptom treatment, 551\u2013552\n\n24-Hour recall, _See_ Dietary recall\n\nTyramine, food allergy role,\n\nTyrosinemia\n\ndescription,\n\nnewborn screening programs, 210\u2013211\n\nnutritional therapy, ,\n\nU\n\nUbiquinone, congestive heart failure effects, ,\n\nUlcerative colitis\n\ndietary factors, 581\u2013584\n\nfood allergies,\n\nnutritional-related problems, 582\u2013584\n\nspecific foods, 581\u2013582\n\netiology, 581\u2013582\n\nnutrition assessment, 585\u2013586\n\noverview, 577\u2013578,\n\npathology, 580\u2013581\n\nprevalence, 578\u2013580\n\ntreatment, 584\u2013587\n\ndiet effectiveness,\n\nenteral nutrition, 587\u2013589\n\nmedical management, 584\u2013585\n\nnutritional treatment, 585\u2013587\n\nparenteral nutrition, 587\u2013589\n\nsurgery,\n\nUnsaturated fatty acids\n\ncardiovascular disease effects, 279\u2013282\n\ndiabetes management,\n\nhypertension effects,\n\nUrea cycle\n\nliver defects, 605\u2013606\n\nmaintenance, sodium benzoate role,\n\nUrine\n\nelectrolyte composition, ,\n\nlactose intolerance measurement,\n\nmaple syrup urine disease\n\nnewborn screening programs, 210\u2013212\n\nnutritional therapy, ,\n\npregnancy management, 222\u2013223\n\nsodium excretion, 305\u2013306\n\nUrsodeoxycholic acid, cholestasis treatment,\n\nUrticaria, _See_ Food allergy\n\nUtility theory, description, 87\u201388\n\nV\n\nValerian, health benefits, 266\u2013267\n\nValidation studies, dietary assessment methods, 18\u201319\n\nValidity assessment\n\ncause and effect interpretation, 64\u201366\n\nin nutritional epidemiology, 75\u201377\n\nin nutrition intervention, 128\u2013129\n\nVasopressin, congestive heart failure role, 325\u2013326\n\nVegetable gums, allergic reactions,\n\nVegetables\n\nallergic reactions, _See_ Food allergy\n\nbreast cancer risk reduction, 340\u2013341, , , 348\u2013349\n\ncardiovascular disease effects, 296\u2013297\n\ncolon cancer relationship, , ,\n\nDietary Approaches to Stop Hypertension diet,\n\nexcessive gas production, 550\u2013551\n\nfiber source, 291\u2013292\n\nhypertension reduction, , , , 313\u2013317\n\ninflammatory bowel disease effects,\n\njuice therapies,\n\nlung cancer relationship, 387\u2013388\n\nprostate cancer risk reduction,\n\nVery low calorie diets, lifestyle modification,\n\nVicilin, food allergy role,\n\nViral hepatitis, description, 603\u2013604\n\nVitamins\n\nin cancer treatment, 400\u2013401\n\ndeficiency signs, , 52\u201353\n\nenteral feeding,\n\nHIV\/AIDS patient recommendations,\n\ninborn metabolic error treatment, , 218\u2013219\n\ninflammatory bowel disease effects,\n\nliver disease effects on absorption, ,\n\nrenal disease therapy, , 627\u2013628\n\nsupplement assessment,\n\nVitamin A\n\nbiomarkers of exposure,\n\nbreast cancer risk reduction, ,\n\ncognitive function relationship,\n\ncystic fibrosis relationship, , 720\u2013723\n\ndeficiency signs,\n\ndietary reference intakes, ,\n\nlung cancer relationship, 388\u2013390\n\nin parenteral nutrition supplements,\n\nParkinson's disease relationship,\n\nrenal disease therapy,\n\ntoxicity signs,\n\nVitamin B2, cognitive function relationship, 638\u2013639\n\nVitamin B6\n\nbiomarkers of exposure,\n\ncognitive function relationship, 638\u2013639,\n\ncolon cancer relationship, ,\n\ndeficiency signs,\n\ndietary reference intakes, , ,\n\nhyperhomocysteinemia association, 293\u2013294\n\nmalabsorption in short bowel syndrome,\n\nin parenteral nutrition supplements,\n\nVitamin B12\n\nbiomarkers of exposure,\n\ncognitive function relationship, 638\u2013639,\n\ndeficiency signs, ,\n\ndietary reference intakes,\n\nhyperhomocysteinemia association, 293\u2013294\n\nin parenteral nutrition supplements,\n\nrenal disease therapy, ,\n\nVitamin C\n\nbiomarkers of exposure, ,\n\nbreast cancer risk reduction, , ,\n\ncognitive function relationship, 638\u2013642\n\ncolon cancer relationship, ,\n\ndeficiency signs,\n\ndietary reference intakes, , ,\n\nlung cancer relationship, ,\n\noxidative stress reduction,\n\nin parenteral nutrition supplements,\n\nParkinson's disease relationship,\n\nrenal disease therapy, ,\n\nsmoking effects,\n\nVitamin D\n\nanorexia nervosa therapy,\n\nbiomarkers of exposure,\n\ncolon cancer relationship, ,\n\ncystic fibrosis relationship, 722\u2013723\n\ndeficiency signs, , 52\u201353, ,\n\ndietary reference intakes, ,\n\nosteomalacia role, 732\u2013735\n\ngastrointestinal pathology,\n\ninadequacy versus deficiency,\n\ninherited forms,\n\nintake deficiencies,\n\nsunlight exposure,\n\ntreatment,\n\nvitamin D physiology, 732\u2013734\n\nosteoporosis relationship, , 658\u2013659, 672\u2013674\n\nin parenteral nutrition supplements,\n\nprostate cancer association, , 377\u2013378\n\nrenal disease therapy, ,\n\nVitamin E\n\nAlzheimer's disease relationship,\n\nbioavailability,\n\nbiomarkers of exposure,\n\nbreast cancer risk reduction, ,\n\ncognitive function relationship, 637\u2013642\n\ncolon cancer relationship, , 361\u2013362\n\ncystic fibrosis assessment, 720\u2013721\n\ncystic fibrosis relationship, , 722\u2013723\n\ndeficiency signs, ,\n\ndietary reference intakes, , 756\u2013757, 759\u2013760\n\nlung cancer relationship, 390\u2013391\n\nneurologic degeneration treatment, ,\n\noxidative stress reduction,\n\nin parenteral nutrition supplements,\n\nParkinson's disease relationship, 644\u2013645\n\nprostate cancer risk reduction, 378\u2013380\n\nrenal disease therapy,\n\nsmoking effects,\n\nVitamin K\n\nbiomarkers of exposure,\n\ndeficiency signs, ,\n\ndietary reference intakes,\n\nenteral feeding,\n\nliver disease therapy,\n\nosteoporosis relationship, 674\u2013675\n\nin parenteral nutrition supplements,\n\nrenal disease therapy,\n\nVomiting, _See also_ Binge eating; Bulimia\n\ncauses, 557\u2013559\n\nfood effects,\n\ngastric bypass side effects,\n\nnutrition management strategies in cancer patients,\n\nprevalence, 557\u2013558\n\ntherapy, , 559\u2013560\n\nW\n\nWaist circumference, body fat assessment, , 47\u201348, ,\n\nWasting syndrome, in HIV\/AIDS patients, 741\u2013742\n\nWeight, _See_ Body weight; Obesity\n\nWeight Watchers, behavior modification training,\n\nWilson's disease, description, ,\n\nWine, health benefits,\n\nWomen's Health Initiative, nutrition intervention trials,\n\nX\n\nXerostomia\n\ndrug therapies,\n\nnutrition management strategies in cancer patients,\n\nY\n\nYogurt, lactose intolerance management, 568\u2013569\n\nZ\n\nZantac\u00ae, gastrointestinal symptom treatment, 551\u2013553\n\nZeaxanthin, breast cancer risk reduction, ,\n\nZinc\n\nbiomarkers of exposure,\n\ncognitive function relationship, 638\u2013639\n\ndietary reference intakes, ,\n\nosteoporosis relationship,\n\nin parenteral nutrition supplements,\n\nprostate cancer relationship,\n\nrenal disease therapy,\n\nZone diet, description, , \n","meta":{"redpajama_set_name":"RedPajamaBook"}} +{"text":"\n\nSochoolhouse Mystery\n\nGERTRUDE CHANDLER WARNER \nIllustrated by David Cunningham\n\nALBERT WHITMAN & Company, Chicago, Illinois\nContents\n\nCHAPTER\n\n1 Benny's Plan\n\n2 Being Watched\n\n3 Wanted: A Schoolhouse\n\n4 A Woman of Few Words\n\n5 The Money Man\n\n6 Mystery Painting\n\n7 Grandfather Makes a Call\n\n8 Caught by the Tide\n\n9 The Disappearing Stranger\n\n10 A Discovery\n\n11 Who Is the Englishman?\n\n12 A Sockful of Money\n\n13 Surprise for Violet\n\n14 Caught!\n\n15 The Last Song\n\nAbout the Author\n\nCHAPTER 1\n\nBenny's Plan\n\nThe whole Alden family sat on the front porch reading. It was one of those hot vacation days in June. Supper was over and the sun had not yet set.\n\n\"Plenty of light to read by,\" said Benny as he took his favorite book to his favorite seat in the corner. Jessie and Violet, his sisters, were already sitting in the porch swing. Henry, the oldest of the Aldens, was just home from college. He sat in one easy chair, and Grandfather sat in another.\n\nSuddenly Grandfather looked up. Benny was not reading any more. He was looking straight ahead. But he was not looking at anything.\n\n\"What's the matter, Ben?\" asked Mr. Alden.\n\n\"I'm thinking,\" said Benny. He did not move.\n\n\"What are you thinking about, old man?\" asked Henry. \"It seems to be important.\"\n\n\"No, it isn't important,\" Benny said, but he did not go back to his book.\n\n\"You might as well tell us,\" said his older sister Jessie. \"It must be interesting.\"\n\nViolet added, \"Please, Benny.\"\n\n\"Well,\" said Benny, \"it's something Max said.\"\n\n\"Oh, your friend Max?\" said Henry. \"What did Max say?\"\n\n\"Well, it wasn't much,\" said Benny, \"but it got me thinking. He said that we Aldens always seem to have an exciting time on vacation no matter where we go. Always some adventure.\"\n\n\"Max was right,\" said Violet.\n\n\"Yes,\" said Benny, looking at Violet. \"I told Max he was dead right. But then he said he'd like to see us have any exciting adventures if we went to his father's favorite fishing town up on the northern coast. He said it was a tiny village with nothing there. He is sure we couldn't go there and have any adventures. He said even an Alden couldn't find anything exciting in that place.\"\n\nMr. Alden was quick to read Benny's mind. He laughed and said, \"So I suppose you want to go to this fishing village and try it?\"\n\nBenny turned and looked at his grandfather. \"Well,\" he said, \"you see it sounded pretty interesting. I mean I can't imagine being dull anywhere, can you?\"\n\n\"No, Benny, I can't,\" said Mr. Alden. \"This is not a family to have a dull time. It never was. How would you like to go there for a short time? There would still be half the summer left to go somewhere else.\"\n\n\"Oh, I remember!\" said Jessie. \"You had something all planned for this summer, Grandfather.\"\n\n\"It can wait,\" Mr. Alden said, smiling to himself.\n\n\"It certainly would be fun to visit a dull town,\" said Jessie. \"Is it right on the sea?\"\n\n\"Yes,\" said Benny. \"It's an island at high tide with water on all sides. But at low tide the ocean goes out and leaves a roadway made of rocks and gravel. You can drive a car across or walk across. But the people don't go off the island very often, Max says. Just the summer visitors.\"\n\n\"And I guess there are not many of them,\" said Henry.\n\n\"Where do the visitors stay?\" asked Violet.\n\n\"There's only one place,\" Benny answered. \"It's something like an old country hotel with six rooms for summer fishermen like Max's father. The village is tiny. There's a schoolhouse and a store. There isn't even a post office. There are houses for the people who live there, and a sardine factory where they work. They use the schoolhouse for town meetings. And that's all.\"\n\nGrandfather looked around at the family. \"If you all want to go, I'm ready. But every one of you must want to go.\"\n\n\"Of course we do, Grandfather,\" said Henry. \"It will be fun to prove old Max is wrong. We have exciting times just by ourselves.\"\n\n\"We'll show Max!\" said Benny.\n\nHenry added, \"It would be interesting to study a village where people are so cut off from everyone else. I might even write a college paper on it.\"\n\n\"What's the name of this village?\" asked Grandfather. \"Maybe I know it from my old fishing trips.\"\n\n\"Maybe you do,\" said Benny. \"You know a lot of things. It is called Port Elizabeth.\"\n\nMr. Alden shook his head. \"No, I don't know that name. We can look for it on a map. It can't be too far away.\"\n\nViolet ran into the house and soon came back with a book of maps. \"You look it up,\" said Grandfather.\n\n\"Here it is,\" cried Violet. \"It must be very small, the name is in such fine print. And here's the island. The only town near it is Northport.\"\n\nHenry looked at the map. \"It looks as if Northport is about thirty miles away,\" he said. \"It must be a very small town, too.\"\n\n\"It's bigger than Port Elizabeth, though,\" Jessie said. \"We can probably buy things there.\"\n\n\"We can take some things with us, too,\" said Grandfather.\n\nBenny began to laugh. \"I never thought you would want to go to Max's village,\" he said. \"I just can't help thinking about the surprise we'll have for Max.\"\n\n\"Neither can I,\" said Henry. \"We'll let Benny tell Max when we get home what a dull time we had.\"\n\n\"You seem to be very sure you will have an adventure,\" said Grandfather, his eyes twinkling. \"What happens if it is dull, just as Max said?\"\n\n\"Let's try it anyway, Grandfather,\" said Jessie. \"If it is dull, we won't mind.\"\n\nViolet said, \"I'll take my watercolors. I love to paint the sea.\"\n\nHenry winked at Benny and said, \"We'll find something to do. We can always fish.\"\n\nJust a week later Henry drove the station wagon into the fishing village of Port Elizabeth. The tide was out. It was exactly as Max had said. There was the old hotel with six rooms, the schoolhouse, and a small store. They knew that the storekeeper, Mr. Fenton, owned the hotel. He had rented them three rooms by telephone.\n\n\"Shall we go to the store first?\" asked Henry.\n\n\"Yes, I'm sure Mr. Fenton will have the keys. We can let him know we have arrived,\" said Grandfather. \"I hope the beds are comfortable.\"\n\nEveryone was surprised when Mr. Fenton opened the door of the first room. They had not expected anything so pleasant. There were two comfortable beds. The floor was bare except for two handmade rugs near the beds.\n\n\"Very good, very good,\" said Mr. Alden nodding his head at the storekeeper. \"I can see that we are going to enjoy this.\"\n\n\"It's lovely,\" said Violet. \"It's just right for us.\"\n\n\"Now,\" said Mr. Fenton, \"I'm going to show you something I don't show all my guests. It's a kitchen.\"\n\nHe opened another door at the end of the hall, and, sure enough, there was a small kitchen. There was a large table in the middle of the room. The sink and stove were old fashioned, but the refrigerator looked new. There were cupboards full of dishes.\n\n\"It will be a pleasure to let you use it,\" Mr. Fenton said. \"There's no place to eat in Port Elizabeth, and you'd have to go to Northport for your meals\u2014and that's thirty miles away.\"\n\n\"Oh, aren't you kind!\" cried Jessie, \"A kitchen like this is just what we need.\"\n\n\"Can you cook?\" asked Mr. Fenton.\n\n\"They all can cook, even Benny,\" said Mr. Alden, laughing. \"I tell them they can make something out of nothing.\"\n\n\"Good,\" said Mr. Fenton. \"I thought you people looked as if you could take care of yourselves. I'm pretty good at guessing what people are like. Don't often make a mistake. That's why I showed you the kitchen.\"\n\n\"Today we brought our own supper,\" said Benny. \"We didn't know about the kitchen.\"\n\n\"That's fine,\" said Mr. Fenton. \"You must be tired. You have had a long drive. Maybe you'll go to bed early and start up again tomorrow.\"\n\n\"We'll just drive around and see the town first,\" said Mr. Alden.\n\n\"It won't take you long,\" said Mr. Fenton with a laugh. \"It's a very small place.\"\n\nHenry drove slowly along the sandy road by the ocean. Two large wharves stood out in the water. The sardine factory could be seen in the distance, but it was closed for the day. Some boats were tied up there. Beyond the factory was a high cliff, and on top was a very large and beautiful old house.\n\n\"I wonder who lives in that house,\" said Benny. \"Isn't it huge? Must be a big family. I guess there are twenty rooms.\"\n\n\"It looks to me like an old family mansion,\" said Grandfather. \"It is certainly not new. The house seems to be closed, too.\"\n\nIt was not, but the Aldens did not know that until later. The road curved around past the cliff, past the store and a row of fishermen's houses, and back to the hotel. That was all there was to see, except for the schoolhouse. A great many bushes and trees stood between the schoolhouse and their hotel.\n\nThe schoolhouse had once been painted white, but much of the paint had worn off. On top there was a belfry with a bell in it.\n\n\"Listen!\" said Benny. \"Is that the school bell ringing?\"\n\n\"Oh, Benny!\" Jessie said. \"What an imagination!\"\n\nViolet looked hard at the building. She said, \"I think it's a pretty little school. That big chimney is a queer shape, isn't it?\"\n\nAnd so it proved to be.\n\n\"We have done the town,\" said Grandfather. \"That is all there is. Maybe Max is right, Benny, and it is dull.\"\n\n\"Well, maybe,\" said Benny. \"We haven't been here very long. Anyway I'm hungry.\"\n\n\"You shouldn't be hungry,\" said Jessie. She looked at her watch. Then she looked up in surprise. \"It's half past five!\" she exclaimed. \"I thought it was about three o'clock.\"\n\n\"It's a long time since we stopped for lunch,\" said Henry. \"Let's get unpacked and have supper.\"\n\nThe girls were delighted. They put the straw basket on the kitchen table and opened it. They took out chicken sandwiches, pickles, cheese, doughnuts, and potato chips. Benny ran over to the store to get cold milk.\n\nGrandfather had hardly had time to unpack when he heard Violet call, \"Supper!\"\n\nThe children sat on long benches, but Grandfather had a chair at the head of the table.\n\n\"This reminds me of Surprise Island,\" said Henry. \"Only there we didn't have a real table. We used two barrels with a board across them.\"\n\n\"One thing is the same,\" said Benny. \"I'm starved. Let's begin!\"\n\nEveryone else must have been hungry, too. The food was soon gone. Then the boys went to their room to unpack, and the girls to another. Grandfather had a big room alone.\n\nThe hotel was so near the ocean that the splash of the waves kept them awake for a while. But they loved the sound of waves and quickly fell asleep.\n\nThe Aldens did not know that soon they would meet a wonderful friend and find some people who were not friendly at all.\nCHAPTER 2\n\nBeing Watched\n\nThe next day the Aldens began in earnest to learn more about Port Elizabeth.\n\nFirst they walked over to the little store. They found that they could buy almost any kind of food there. The girls bought enough food to last for a few days. Then Benny asked Mr. Fenton, \"Who lives in the big mansion on the cliff?\"\n\n\"Well,\" said Mr. Fenton, \"Miss Gray lives there. Miss Elizabeth Gray. She is the last of her family.\"\n\n\"She lives alone in that great house?\" cried Benny.\n\n\"No, not really alone. She has a woman to get her meals and a man to mow the grass and shovel snow in the winter. But she has no family. Her grandfather built that house, and twelve children grew up there. But some of them died and some left. She hasn't anyone close to her. People say she writes books.\"\n\nJessie said, \"I should think she would be very lonesome. Does she ever go out?\"\n\n\"No,\" said Mr. Fenton. He shook his head. \"Almost never. Nobody calls there, either. She is shy, and people don't feel comfortable with her. She has plenty of money, and she has done a lot for the town. Her grandfather built the schoolhouse many years ago. He called it the Elizabeth Gray School for his wife, Miss Gray's grandmother. Miss Gray is the one who has the schoolhouse cleaned in the fall, and she keeps the keys. But she stays by herself.\"\n\n\"I'm sorry for anyone like that,\" said Benny. \"She must miss a lot of fun.\"\n\nAs the Alden family talked with the storekeeper, a red-haired boy and girl came in together. They looked alike and were certainly twins.\n\nThe island children did not look at the Aldens. In fact, they looked the other way. But Benny thought, \"They must have looked us over before they came in.\"\n\n\"We want some flour,\" said the boy. \"And Ma wants some more tea.\" His voice was as rough as his looks. His sister looked rough, too. Her hair hung in wisps around her face.\n\nThe girl counted out the money carefully, but she did it slowly. It seemed hard for her, so at last Mr. Fenton helped her. When they had gone, Mr. Fenton said, \"To tell you the truth, Mr. Alden, I'm sorry for the children who are brought up here. Living is hard on this island, and the people have no education. The children never have a chance to learn any other way to live. They don't even have TV. They don't see magazines, and they never go off this island.\"\n\n\"Oh, dear,\" said Jessie, \"I shouldn't think they would know anything about the world.\"\n\n\"They don't,\" said Mr. Fenton. \"As soon as they're old enough, they work in the sardine factory. Those two are the Moss twins. They work in the factory whenever it is open.\"\n\n\"Then they earn some money,\" said Benny.\n\nMr. Fenton smiled. \"I can see you don't know what the island people are like. The children don't ever see that money. The fathers take it and keep it. They don't spend much. All these people save all the money they can, but they never put it in the bank. They put it in an old stocking.\"\n\n\"That's a queer thing to do,\" said Benny. \"They might lose it that way.\"\n\nMr. Fenton gave a short laugh. \"Yes, they certainly might,\" he said. \"And then again, they might not! Here's something funny. A strange man came here last summer and he gave Mr. Moss ten dollars for an old quarter. He gave me three dollars for an old penny, but I didn't trust him.\"\n\nSuddenly Grandfather was interested. \"What was this man's name?\" he asked.\n\n\"Mr. Fred Willet,\" said Mr. Fenton. \"He said he would come back this summer.\"\n\n\"Well, if he does,\" said Mr. Alden, \"I hope to see him.\"\n\n\"You will,\" said Mr. Fenton. \"Everyone will see Freddy Willet if he's around. He's very friendly. I'll say that much for him.\"\n\nThen Jessie paid for the groceries and thanked Mr. Fenton for telling them about the island. Henry and Benny raced home to the hotel to put the food away.\n\nViolet said, \"Imagine how it would be to live on this island and not know anything else.\"\n\n\"I can't imagine,\" said Jessie. \"I don't blame that girl if she can't count very well.\"\n\nWhen the boys came back, they and the girls went down to the beach. Mr. Alden did not go this time. He had to write some letters.\n\nAs the young people walked along, Violet said, \"I have a funny feeling that people are staring at us.\"\n\n\"Me, too,\" said Benny. \"And how is it we don't see any men around? I see kids and some women, but not a single man. I thought a lot of fishermen lived here. But where are they?\"\n\nHenry looked all around. \"You're right, Ben. I hadn't really noticed. But I think I know the answer. If the men are fishermen, they must be out in their boats.\"\n\n\"Let's go down to the wharf and see what is going on,\" said Benny.\n\nThere were people on the beach. There were many children of all ages and some mothers. Some of the older girls and boys were sitting on the sand working on nets. They tied knot after knot. They all worked fast. They did not look up when the Aldens went by.\n\nLittle children were playing in the water. They all swam like fishes. Some boys dived off the wharf into deep water.\n\n\"Aren't you afraid to be under water?\" Benny asked a small boy.\n\n\"No, I like it. I can always come up,\" the boy said. He seemed surprised that anyone would ask him such a question.\n\n\"I can see you are a fine swimmer,\" Benny told him. \"Probably you've been swimming all your life.\"\n\nHenry laughed. He said, \"Probably all these children can swim as soon as they can walk.\"\n\nJessie smiled and said hello to a woman who was knitting. The woman answered gruffly, but she quickly looked the other way.\n\nWhen the Aldens went home for lunch, Jessie said to her grandfather, \"These people aren't very friendly, are they?\"\n\n\"No,\" said Mr. Alden. \"They don't trust strangers. I've seen people like this before.\"\n\nBenny frowned. \"What have we done wrong?\" he asked.\n\n\"Nothing,\" said Mr. Alden. \"You'll just have to get used to the idea that these people have different ways.\"\n\nBenny said, \"Well, even if they do, I should think they could smile.\"\n\n\"Do you want to go home?\" Grandfather asked.\n\n\"Oh, no, not yet!\" said all four Aldens together. Then they laughed, for they had all agreed to keep trying.\n\nGrandfather smiled. He liked to see his grandchildren stick to something that was not easy.\n\nIt was lucky that the Aldens could not hear what people were saying about them. The people had watched them quietly ever since they had come.\n\nOne girl said, \"They're stuck-up rich kids. Look at their clothes! I bet those girls never had to work.\"\n\nSome of the older people said, \"Summer people! We've seen plenty of them. I wish we had half the money they spend.\"\n\n\"That car is air-conditioned,\" said a big boy. \"Showing off, they are.\"\n\nBut his mother spoke up. \"I don't think they're stuck-up. And I don't think they're showing off. They all smile and act friendly. You are just jealous.\"\n\nIt was true. The island children wished they were like the Aldens with nothing to do. They watched them every minute. They could hear the four young people laughing and talking together as they went around the town.\n\nBut as time passed, things began to change. People began to like the Aldens. They were such pleasant visitors. The island children really wanted to be friends, but not one of them knew how to say so.\nCHAPTER 3\n\nWanted: A Schoolhouse\n\nIt was a few days later, and the girls were getting lunch. Grandfather came in from the store. He said, \"While you were at the beach Mr. Fenton told me that the fishermen get up at three o'clock in the morning to take their boats out. That's why we never see any men in the morning.\"\n\nHenry said, \"Well, we know they come back at four or five in the afternoon. We've noticed that every day.\"\n\n\"They looAnd thankk fine coming in one by one,\" said Benny. \"I'm going down to the beach to watch those boats. I don't care if nobody speaks to me. But I'm going early. I don't want to miss anything.\"\n\nLong before three o'clock the four Aldens went down to the sandy beach. Violet took her watercolors, brushes, and paper.\n\nA few boats were coming in early. The fishermen started to shovel fish into boxes. Some of the fish were spread out in nets to dry. Others were packed in ice to go to Northport. Gulls were flying all around the wharf, trying to get leftover fish. They made a great noise.\n\n\"Fish smells awful, doesn't it?\" said Benny.\n\n\"Well, there is so much of it the whole town smells of fish,\" said Henry. \"I suppose it is the fish drying that smells.\"\n\nJust then the red-haired girl and her brother came slowly out of their house next to the store. They passed the Aldens.\n\n\"Oh, hello there!\" said Jessie.\n\n\"Hello,\" said the girl, but she did not stop. In fact she seemed to walk faster.\n\nJessie said sadly, \"Nobody wants to be friends.\"\n\n\"Maybe Max is right,\" said Benny. \"It will be dull if nobody is friendly.\"\n\n\"Come on, Ben, don't give up so easily,\" Henry said. \"If anyone can make friends, you can.\"\n\nAfter Violet had watched the boats for a while, she climbed up on the rocks. She could see the harbor better from there. The other Aldens stayed on the sand below.\n\n\"Oh, my!\" Violet called down. \"There are a lot of small pools of seawater all over the top of this rock. I'm going to use salt water for my watercolors!\" So she washed her brushes in a pool of seawater.\n\nShe had just painted a blue band for the sea and some yellow sand, when she heard someone climbing up the rock behind her. She knew her family was sitting on the sand below her because she could see them. Who could this be? She turned her head to look. It was the red-haired girl!\n\n\"Oh, can I watch you? Do you mind?\" asked the girl.\n\nViolet was so surprised she could hardly answer. But she said, \"Of course I don't mind! I'm not much of a painter, though.\"\n\nThe girl climbed the last rock and sat down beside Violet. \"I just couldn't keep away when I saw you painting!\" she said.\n\nThen Violet saw that her twin brother was right behind her.\n\n\"Are you twins?\" she asked.\n\n\"Yes,\" said the girl. \"My name is Marie Moss, and Hal and I are just crazy about painting! There was a man up here when we were kids. He came here to paint the view. Ever since then, Hal and I have wanted to learn to paint.\"\n\n\"Did the man show you how?\" asked Violet. She looked from one to the other.\n\n\"Him? Oh, no! He chased us away,\" said Hal with a frown. \"He didn't like to have us watch him.\"\n\nMarie said, \"He didn't stay here long. He painted just two pictures. One was of the harbor and one was our house. They were beautiful!\"\n\n\"Then I guess you did watch him,\" said Violet.\n\n\"Yes, we did,\" said Hal. \"The man never guessed. We knew the rocks better than he did. So we got behind a rock only a few feet away. We saw everything he did. We saw just what colors he used. He had oil paints.\"\n\n\"And you remember all this time!\" said Violet.\n\n\"Oh, yes!\" said Marie. \"He mixed a lovely blue with green. It looked just exactly like the water. Then he put purple in it! See? Under the rocks, it's purple.\"\n\n\"You know,\" said Violet slowly, \"you see things the way an artist would. Did you ever have any paints?\"\n\n\"No,\" said Hal. \"We tried to make pictures with old crayons. But if we only had some paints\u2014!\"\n\nThe other Aldens heard what was going on. They climbed up the rocks and sat down.\n\n\"Are you going to stay very long?\" asked Marie.\n\n\"Well, I think so,\" said Jessie, smiling. \"We like it here.\"\n\n\"You see,\" began the boy and stopped. He seemed to be having trouble with his words.\n\n\"Did you want something?\" asked Benny.\n\nThe two strangers laughed a little. The girl said, \"I guess we are scared.\"\n\nBenny said, \"Say! You can't be scared of us, can you?\"\n\nThe twins looked at each other, and Marie said, \"We aren't supposed to talk to summer people.\"\n\nHenry said, \"Come on! Don't call us summer people. We just came up here to visit Port Elizabeth and see what it is like. A fishing village is new to us, you know.\"\n\n\"We've been wanting to make friends with someone,\" said Benny, \"but it's been very hard.\"\n\nThen Marie spoke very fast. \"Everybody said you were stuck-up, but I said you weren't. You aren't stuck-up at all! I was right! I know we haven't been very nice to you on this island, but Hal and I wondered. . . .\"\n\n\"What did you wonder?\" asked Benny. \"I'd like to know.\"\n\nThe twins were quiet for a minute.\n\nThen Marie said, \"You see, we go to school in the winter. But the teachers don't like it here. They don't stay long. We had five teachers last year.\"\n\n\"Imagine having five teachers in one year!\" said Benny.\n\n\"It's awful,\" said Hal. \"Every new teacher thinks we have forgotten everything. So they all start back at the beginning.\"\n\n\"You don't get very far that way,\" said Henry.\n\n\"No,\" said Marie. \"The last teacher told us to study this summer, but we don't know how. And we would love to learn to paint.\"\n\nJessie said, \"Violet learned to paint in school. She loves it, too.\"\n\n\"Oh, would you teach us?\" cried the twins.\n\n\"Of course,\" said Violet. \"I'll do my best.\"\n\n\"Oh, thank you,\" said Marie, her eyes bright.\n\nHal said slowly, \"We'd like to learn other things, too, but we don't know how.\"\n\nThen Jessie woke up. Violet woke up. Henry woke up. As for Benny, he was already wide awake. They all saw what the twins really wanted.\n\nThen the whole story came out. The Moss twins talked faster than ever.\n\nMarie said, \"The little children can't read, and they love stories. All the children in this village ought to go to school. Even the little ones would love it if you taught it. And all our mothers would be so glad.\"\n\n\"OK,\" said Benny. \"So you want to go to summer school. Henry, let's teach school! Even I could teach. Just give me a few small kids, and I'll teach 'em to read. And I could teach singing. Now how about that old schoolhouse? That's empty, doing nothing.\"\n\n\"That belongs to Miss Elizabeth Gray,\" said Marie. \"She has the key.\"\n\n\"Is she cross?\" asked Benny. \"Would she let us use the schoolhouse?\"\n\n\"She might. She isn't cross, anyway. You could ask her. I wouldn't dare,\" said Marie. \"It would be grand if we could use the schoolhouse, wouldn't it?\"\n\nHenry began to climb down the rock. He said, \"Never mind your painting this time, Violet. Let's go!\"\n\n\"Oh, do you dare?\" asked Hal.\n\n\"Why not?\" said Benny. \"She doesn't bite, does she?\"\n\n\"No,\" said Hal, laughing a little. \"But we won't go. You go.\"\n\nHenry smiled. He said, \"Yes, I guess four of us will be enough. We'll tell you later what she says.\"\n\nJust then a loud whistle sounded. It whistled and whistled.\n\n\"That's for us!\" cried Marie. \"It's the sardine factory! A school of sardines has come in. Sardines have to be canned quick. We have to go. Please don't forget our school.\"\n\n\"No, we won't!\" called Benny.\n\nAs they climbed down, Henry said to Jessie, \"Remember my friend Larry in Adams College? He is going to live in a city this summer and help boys who live in a poor neighborhood catch up in school. Then they won't be drop-outs. And here we are on an island, doing about the same thing.\"\n\n\"I think it's an exciting idea,\" said Violet. \"We didn't think we would be schoolteachers this summer, did we?\"\n\n\"That's the last thing I thought of,\" said Henry. \"Just look at everybody going to work.\"\n\nIndeed somebody came out of every house. Sometimes there were three or four people from one house. They all hurried down to the factory.\n\n\"Well, well,\" said Jessie. \"We have made friends at last.\"\n\n\"We made two, anyway,\" said Benny.\n\nBy that time Marie and Hal were almost out of sight.\nCHAPTER 4\n\nA Woman of Few Words\n\nHere we go, up to a strange mansion to see a strange lady,\" said Benny.\n\n\"Right!\" said Henry. \"I'm sure it's all right to ring a doorbell, even if we don't get in.\"\n\nThe four Aldens started along the beach, past the factory, and up the high cliff walk. They could see the mansion above them. It was a large square house, painted white. There was a square room on top that looked out to sea.\n\nThe Aldens climbed the steps to the front door and rang the bell. They could hear it ringing inside.\n\nAfter a few minutes they heard someone coming very slowly. The door opened to show a tall, thin woman with straight gray hair. She did not smile. She just stood there.\n\nJessie began, \"Miss Gray, I hope you will forgive us for coming to see you. But we are staying here for a while, and we want to ask you a favor.\"\n\nElizabeth Gray's eyes went sharply from one to another. Still she did not smile. Then she said shortly, \"Come in.\" She stood aside for them to pass. \"Go straight ahead,\" she said.\n\nBenny thought, \"Not a very warm welcome! Maybe we won't get that schoolhouse after all.\"\n\nNone of the Aldens knew that they were the first real callers Miss Gray had had for many years.\n\nThey all sat down. Miss Gray did not say a word. Henry found it hard to begin, but he knew he must say something. He began, \"This is a funny question, Miss Gray. You see, we had no idea we would do this. But some of the children in Port Elizabeth want to go to school this summer to learn more. They asked us to help them. Maybe you think that is a queer idea.\"\n\n\"No,\" said Miss Gray, \"I don't.\"\n\nThat was all she said. Still no smile.\n\nBenny could not stand this. He said, \"We came to ask you if we could use your schoolhouse. It's a wonderful schoolhouse! It has a big bell and everything. I'd be the one to ring the bell. Could we use it, do you think?\"\n\n\"What would you do with it?\"\n\nJessie answered this. She said, \"We'd have a real school every morning, but we wouldn't use the schoolhouse at all in the afternoon.\"\n\nViolet added, \"We'd be very careful of it. We'd keep it clean, too.\"\n\n\"We'd always remember to lock the door,\" said Benny.\n\n\"No reason why you can't have it,\" said Miss Gray. \"Certainly these children have never learned much.\"\n\n\"You are very kind,\" said Jessie. \"We are strangers, and we have asked for a lot. I know that you are interested in the children. Everyone says so.\"\n\n\"Humph!\" said Miss Gray.\n\nJessie went on, \"We haven't told our grandfather yet. But I'm sure he will think this school is a good idea.\"\n\nBenny laughed. He said, \"Grandfather will think it is funny, though. _Me_ teaching school!\"\n\nThe sharp voice said, \"What are you going to teach, boy?\"\n\n\"Well, I don't know,\" said Benny. \"Maybe I could teach them about the moon.\"\n\nMiss Gray looked at Henry and said, \"I'm sure they don't know much about the moon.\"\n\nBenny said, \"We didn't expect to teach school. We came up here to find a mystery.\"\n\n\"What? A mystery?\" said Miss Gray. \"Why?\"\n\n\"Because my friend Max said we couldn't,\" said Benny. \"He said this island was dull, and we couldn't have any adventure here.\"\n\nMiss Gray said nothing. The Aldens had never met anyone before who talked so little and never smiled.\n\n\"Maybe we'd better go now,\" said Violet softly.\n\nMiss Gray went stiffly over to a desk and took out two keys. \"Back door, front door,\" she said. \"They are marked.\" She gave the keys to Henry.\n\n\"We won't lose them,\" said Jessie. \"We'll give them back to you when we go home.\"\n\n\"Give me your names,\" said Miss Gray. She picked up a small notebook from the table. Then the Aldens saw that a new book was lying on the table. The name of the book was _The Woman Who Talked Too Much,_ by E. Gray.\n\nThey all thought E. Gray is Elizabeth Gray! She wrote that book. But she certainly doesn't talk much herself! But nobody dared to ask her any questions.\n\nJessie gave her the names.\n\n\"Your grandfather's name?\"\n\nHenry said, \"He is James Henry Alden, and I'm Henry James Alden. We live in Greenfield.\"\n\nMiss Gray had heard of the Alden Library in one town and the Alden Museum in another. But she did not say so.\n\nBenny said, \"Oh, Grandfather's wonderful! He's the best man you ever saw. We'll go and tell him right now that you are letting us use your schoolhouse.\"\n\nThe others wanted to go, so they were glad Benny had started toward the door. When they stood on the step they all said, \"Goodbye! And thank you!\"\n\nMiss Gray did not say goodbye. Instead she called suddenly, \"Did you ever see a blond-haired man anywhere who smiles and shows his teeth all the time?\"\n\n\"No, I'm sorry,\" began Henry.\n\n\"Don't be sorry,\" said Miss Gray sharply and shut the door.\n\n\"Well, what do you know!\" said Benny, in a very low voice. \"What does she mean by that?\"\n\n\"I don't know,\" said Henry. \"But keep your eyes open, Ben. Miss Gray doesn't ask questions for nothing.\"\n\nThe Aldens said no more until they were on the beach.\n\nThen Benny said, \"I don't think Miss Gray is really polite, but I suppose that's her way.\"\n\n\"We knew she was different,\" said Jessie. \"That's why she lives all alone and never goes out. Maybe she doesn't like people.\"\n\nHenry said, \"We know she's a writer. Maybe she has to be alone. In a way I thought she liked the idea of the school, even if she didn't say so.\"\n\n\"Anyway, she gave us the schoolhouse,\" said Benny. \"And that's why we went up there.\"\n\nThen they walked as fast as they could to find Mr. Alden.\n\n\"Oh, Grandfather!\" cried Benny when he saw a figure sitting in the sun by the hotel. \"Please let us go to Northport this afternoon and buy paints and school things. We're going to teach school!\"\n\n\"Teach school!\" said Grandfather.\n\nThe young people tried not to talk all at once.\n\n\"We went up to ask Miss Gray\u2014\" began Benny.\n\n\"Oh, you did? I thought she didn't like visitors.\"\n\n\"Well, that's right, she doesn't. That's sure! But she gave us everything we asked for.\"\n\n\"Bless my soul! What did you ask for? From a perfect stranger!\"\n\nHenry looked straight at his grandfather and said, \"It turned out very well, Grandfather. You see the red-haired twins asked us if we would help them with schoolwork. Just to catch up, you know. They said every child on the island would come. So that's why we wanted the schoolhouse.\"\n\n\"And she gave it to you?\" asked Mr. Alden, laughing.\n\n\"Well, yes,\" said Jessie. \"She doesn't talk much. But she must have liked the idea, really. She gave us the keys.\"\n\n\"She gave us a rough time, too,\" said Benny.\n\n\"Hard to talk to?\" asked Mr. Alden.\n\n\"Very!\" said Henry.\n\nBenny said suddenly, \"I'd like to forget Miss Gray. She is no fun at all. I'd rather think about our school. I'm going to ring that bell. And I'll ring it for recess, too.\"\n\nGrandfather nodded. \"Yes,\" he said. \"I know how you feel, Benny. I never saw a bell rope myself without wanting to pull it.\" But he could see that his grandchildren had not received a very warm welcome from Miss Gray. He decided to talk with Henry later, when they were alone.\n\nViolet said, \"Grandfather, we think Marie and Hal Moss can paint. Wouldn't it be wonderful if they really could paint well?\"\n\n\"Yes, my dear, it is a great thing to discover an artist. This school idea is a surprise to me, but it's fine. I never know what you will think of next!\"\n\nHenry said, \"We thought we could go over to Northport and buy some paints and school things.\"\n\nMr. Alden laughed. \"Yes,\" he said, \"you can take the car. And maybe you will take me, too.\"\n\nEveryone laughed at this. They always had a better time when Mr. Alden came along.\n\nThirty miles seemed a long drive because they wanted to get there and buy the things. They went first to a store to buy paints.\n\n\"Better get enough to go around,\" said Mr. Alden. \"Give them a bit of fun. It isn't much fun with two sharing a paintbox.\"\n\nIn the end they bought ten boxes of paints. The older children could each have a box. They bought colored paper, small scissors, gold stars for Benny, and a stapler.\n\nJessie said, \"Marie told us that there is plenty of chalk in the schoolroom closet. And there are pencils and paper left from last year, and some school books.\"\n\n\"Well,\" said Grandfather, \"if you forget anything, we can come again. Let's eat supper at this little restaurant. It seems to be called the Sea Shell.\"\n\nThe Aldens noticed a bright red sports car parked in the yard. The top was down. \"That car looks strange up here in such a quiet town,\" said Henry.\n\n\"It looks as if it goes fast,\" said Benny.\n\nAs they went into the Sea Shell a man with blond hair came out. He smiled at the Aldens and showed all his teeth. He said, \"Hi, there!\" although he was a perfect stranger. They watched him as he started the red car. Off he went like the wind.\n\n\"I told you that car could go fast,\" said Benny. \"And wait! That man had blond hair, all right, and he smiled and showed all his teeth. There can't be two men like _him!_ Just what Miss Gray said.\"\nCHAPTER 5\n\nThe Money Man\n\nWhen the Aldens drove back to the island they saw Hal and Marie waving to them from the beach. Marie shouted, \"Please come over!\"\n\n\"You go on,\" said Grandfather. \"You don't need me.\"\n\nThe four Aldens ran down to the beach.\n\nHal said, \"Guess who is coming to the island! The Money Man!\"\n\nBenny said, \"The Money Man? Now who is that?\"\n\nMarie said, \"Oh, he's a wonderful man who buys money. Pa had a quarter, and the Money Man gave him ten dollars for it.\"\n\n\"Whew, that's a lot of money!\" said Benny. \"How do you know he is coming again?\"\n\n\"Somebody saw him in Northport and told Mr. Fenton,\" said Hal.\n\n\"Does he have blond hair?\" asked Benny.\n\n\"Yes, he does, and white teeth,\" said Hal. \"He has a big smile. He smiles at everybody.\"\n\n\"There you are, Jessie,\" said Henry with a nod.\n\nJessie said, \"Yes, Mr. Fenton was right. You couldn't miss that man.\"\n\nMarie said, \"My pa says we ought to help you clean up the school. After all, it's for us. The big boys can help carry water, and there are more children to help if you want them.\"\n\n\"Good!\" said Henry. \"Let me see. We do need help, but it's too late to do anything now. Let's clean the school tomorrow morning and begin school the next day. That'll be Thursday.\"\n\n\"Funny day to begin school,\" said Benny. \"But what do we care? We bought some paints and things in Northport.\"\n\n\"Let's meet at eight o'clock tomorrow morning at the school,\" said Jessie. \"Eight of us can do a lot of work.\"\n\nAs it turned out, there were more than eight. Several other children were waiting with Hal and Marie when the Aldens arrived the next morning.\n\nHenry unlocked the front door, and they all went in and looked around.\n\n\"The room is dusty,\" said Marie.\n\nIt was an old-fashioned schoolroom. The desks and seats were fastened to the floor.\n\n\"I'd like my children in the front row,\" said Benny. \"Your big children can sit in the back seats.\"\n\nThis was really the only way, because the front seats were small and the back seats were large.\n\nHenry set everyone to work. The children took the books off the shelves and washed the shelves. Every book was dusted and set back. The children were delighted with their own work. The biggest boys washed the seats and desks. The teacher's desk was washed, and a new pink blotter put in the middle.\n\n\"Will somebody bring flowers for this desk tomorrow?\" asked Jessie.\n\n\"Oh, I will!\" cried a child named Isabelle. \"We have a big vine of pink roses all over our roof.\"\n\nThere was a wood stove in the back of the room and a woodbox. Hal wiped off the stove. Then he said, \"How about the woodbox? It looks all right to me.\"\n\nHenry agreed. \"Just brush off the top. We certainly won't have a fire. Leave the wood for winter.\"\n\nA little later Benny was standing still, looking at a big picture on the wall. It hung in the front of the room where the big chimney was. He said, \"Look at this picture of George Washington. White wig and hair ribbon and all! What can we do about that? It looks dark and dirty.\"\n\nThe painting showed the first President in his old-fashioned costume, standing beside a table.\n\n\"You can't clean that, Benny,\" said Violet. \"It would spoil it to wash it. And it's too high, anyway.\"\n\n\"Just let it alone, huh?\" said Benny. \"I'm glad we don't have to wear fancy clothes like that with all those buttons down the coat.\"\n\n\"So am I,\" said Jimmy. \"I've got to go home now, Mr. Benny.\"\n\n\"Don't call me Mr. Benny. I'm not really a teacher. Why do you have to go home?\"\n\n\"I've got to find some old bottles out in the fish house,\" said Jimmy. \"The Money Man wants 'em. I've got to have them all ready.\"\n\n\"Yes,\" agreed his sister. \"He even wants an old olive bottle! I've found that already. It says Queen Olives, 1875, on it. Ma says her ma must have bought it. Isn't he a funny man? To want an old dusty bottle almost a hundred years old?\" Then she added quickly, \"But he's wonderful just the same.\"\n\nAll the children were listening now. They nodded their heads. An older boy named Jeffrey Frost said, \"My pa and ma have been waiting for the Money Man to come. He told them to find all the funny old things they could. He likes 'em! Ma found an old glass pitcher and a whale's tooth with pictures on it.\"\n\nHenry said, \"Jeffrey, do you know where that whale's tooth came from?\"\n\n\"Oh, yes, my great uncle made the pictures when he sailed on an old whaling ship. That tooth is so heavy you wouldn't believe it!\"\n\nJessie said, \"Well, we've finished our work here, anyway. We'll all go home now and start school tomorrow.\"\n\n\"What time does school begin?\" asked Jeffrey.\n\n\"Nine o'clock,\" said Henry. \"You'll hear the bell.\"\n\n\"I'm going to ring the bell,\" added Benny.\n\n\"That's lots of fun,\" said Jimmy.\n\n\"Maybe you can be the bell ringer later,\" said Benny. \"Tell all the other children, won't you?\"\n\n\"Tell them?\" said Marie, laughing. \"Most of the children are here now. The rest will know just as soon as we get home.\"\n\n\"No school in the afternoon?\" asked Jeffrey.\n\n\"No, I'm sorry,\" said Henry. \"You ought to help at home.\"\n\n\"I suppose so,\" said Marie. Then her face lighted up and she said, \"I'm going to clean up my room just like this schoolroom. My sister will help me. She sleeps there, too.\"\n\nThe schoolroom was indeed clean. Henry locked the door, and the children ran off in all directions.\n\nThe Aldens found Grandfather waiting for lunch.\n\nWhen they were sitting down at the table, Henry said, \"We are worried, Grandfather, about the Money Man. I'm afraid he is cheating everyone on the island.\"\n\n\"I'm sure he is,\" said Jessie. \"You see, he paid ten dollars for an old quarter. That made them all trust him. But who knows? That quarter may be worth a hundred dollars.\"\n\n\"Or a thousand!\" said Benny. \"You see, Grandfather, there really is a mystery here. I don't think the Money Man is as wonderful as he seems.\"\n\nGrandfather smiled. \"Well, Benny,\" he said, \"I'm sure you will keep your eyes open. If this man goes too far, we'll try to stop him. But I agree with you. He isn't wonderful at all!\"\nCHAPTER 6\n\nMystery Painting\n\nIt was a wonderful moment when Benny rang the school bell. Dingdong! Dingdong! Everyone on the island heard it and smiled. Benny did not really need to ring the bell at all\u2014because every child was already in the school yard.\n\nThe girls and boys rushed in. They could hardly wait to see what was going to happen. They had no idea what their four young teachers would do.\n\n\"Here's my seat,\" cried Isabelle. \"I sat here last year. And here are the pink roses.\"\n\nAll of Benny's class rushed up to the small front seats. The big boys and girls sat down in the back seats. And the middle-sized ones sat in the middle. There were twenty children when Benny counted them.\n\nHenry began to talk. The children were suddenly very quiet. He said, \"Today we will have four classes. First you tell us what grade you were in last year. Now, who was in Isabelle's class? You stand up, and Benny will see how many there are.\"\n\n\"Don't we call him Mr. Benny?\" asked Isabelle.\n\n\"No,\" said Benny, \"just call me Benny. And don't ever call me Teacher.\"\n\n\"I always call my teachers Teacher,\" said Isabelle.\n\nBenny laughed. \"I guess you are going to be my talker. There's always a talker in every class.\"\n\nSoon Jessie said, \"We will have a spelling match today and a singing school and a new game at recess.\"\n\n\"Ah!\" whispered the children. They were all smiles. School had never been like this.\n\nThere were five children in Benny's class and five in Violet's, six in Jessie's, and four in Henry's. Henry said, \"Sometimes we will change teachers. Now we will write down your names and get started.\"\n\nBenny sat down with his class and took all their names. Then he said, \"Isabelle, you let somebody else tell me what book you can read.\"\n\nTommy Spoon said, \"We can't read any book. The teacher took the books with her.\"\n\nBenny was all ready for this. He had not found any easy books on the shelf.\n\n\"I'll write on the board,\" he said. He printed in big letters, \"I have a dog.\"\n\n\"I have a dog,\" said Tommy Spoon. \"That's what it says.\"\n\n\"Right!\" said Benny. \"You read it, Eddie.\" Eddie was the smallest child.\n\n\"No,\" said Eddie, shaking his head, \"no dog.\"\n\n\"You mean you haven't any dog?\" asked Benny. \"Well, can't you read it, even if you haven't any dog?\"\n\n\"No,\" said Eddie crossly. \"No dog!\"\n\n\"Well,\" said Benny, laughing, \"have you a cat?\"\n\nYes, Eddie had a cat. So Benny printed, \"I have a cat.\" Eddie read it very well. \"I know _cat_ is the last word. My cat's name is Fish.\"\n\n\"Fish!\" said Benny. \"Why?\"\n\n\"Because he always comes when we call 'Fish, Fish.'\"\n\nBenny printed the whole story. They all read it together.\n\nI have a cat.\n\nHis name is Fish.\n\nI call, \"Fish, Fish.\"\n\nFish comes.\n\n\"Oh, I like to read about myself!\" said Eddie.\n\nThen Benny saw that he would have to make his own reading books. While the children were busy making figure 2's, Benny printed the story about Eddie's cat five times on paper. Every child could read it. Benny said they could take the story home.\n\n\"Write about me next time,\" begged Isabelle. \"I've got a boat.\"\n\n\"So have I,\" said every child.\n\n\"We'll all read about Isabelle's boat tomorrow,\" said Benny.\n\nThe older children had books. They even had spelling books. After reading and a spelling match, Henry said, \"Now we will have an art lesson all together. Let's study that painting of George Washington.\"\n\nAs Henry pointed out the different things in the picture, Benny was interested.\n\nHenry went on, \"You may think it is strange that a strong man would wear ruffles. But every well-dressed gentleman wore short knee pants and long white stockings and shoes with buckles. And now look at the hair. What color is it?\"\n\n\"White!\" shouted every child.\n\n\"Yes, it is white. But it is a wig. This is a very good painting, you see. That wig looks exactly like hair.\"\n\nJessie said, \"Now notice the _eyes._ George Washington seems to be looking right at you. It takes a fine artist to do that.\"\n\nEveryone looked at the eyes. All but Benny. He happened to look at the coat buttons. There was a long row of buttons down the right side of Washington's velvet coat.\n\nBenny thought, \"One of those buttons looks awfully strange to me. It looks like a round hole instead of a button.\"\n\nHe couldn't very well tell Henry, so he wisely said nothing. After the art lesson, Henry said, \"Now ring the bell for recess, Ben.\"\n\n\"Recess!\" said Jimmy. \"It can't be time for recess.\"\n\nBut it was. Time had gone fast for all the children.\n\n\"Come outdoors, and we'll teach you a new game,\" said Jessie.\n\nThe children made a circle and had already played the game once when they heard a car. They all looked, and a bright red sports car came bumping over the rocky road to the island. It was low tide. The game stopped.\n\nThe children called out, \"Oh, oh! Look, there's the Money Man!\"\n\nA man put his head out of the car window and waved and smiled. He called \"Hello! Hello, kids!\" But he did not stop.\n\nWhen he had gone, Benny said, \"Tell me about this Money Man, Isabelle.\"\n\n\"Oh, he gives us things. He came down here last year. You ought to see the big doll he gave me!\"\n\n\"Why did he give you a doll?\" asked Henry.\n\n\"He traded it for my old one,\" said Isabelle. \"I had a teeny little wooden doll only this long.\" She showed with her small hands. \"My grandfather made it with his knife. And the man said if I'd trade, he'd give me a beautiful doll with a pink silk dress. And he did.\"\n\n\"Money Man isn't his name,\" said Hal. \"We just call him that. His name is Mr. Fred Willet.\"\n\nMarie showed Jessie a chain of bright red beads she had around her neck. \"The man gave me this, too,\" she said.\n\n\"And what did you trade it for?\" asked Violet.\n\n\"Oh, you'd laugh! It was a string of old buttons. Ma said I could string the buttons and wear them for a necklace. They were old things anyway. Ma said most of them were on her grandmother's dresses. You can see they were old.\"\n\nHenry looked at Jessie. Then he said, \"Why do you think he is here again? To trade?\"\n\n\"Oh, I hope so,\" said Eddie. \"I've got an old iron bank. You put in a penny and a little man comes out and goes back in. The Money Man said I could have a real watch for it or a new cap. I couldn't decide then. But I know now. I want the watch.\"\n\nThen the game went on. Benny knew that something was wrong. He saw Henry whisper to Jessie, \"I wish we could get word to Grandfather that the Money Man has arrived.\"\n\nViolet heard this, too. She whispered to Henry, \"Don't worry. Grandfather's eyes are wide open. And he doesn't miss much.\"\n\nThen it was time to go in for the singing school.\n\nJessie taught the children a new song. It was a round with three parts.\n\nRight after the song, the sardine factory whistle blew. Everybody except the Aldens ran.\n\nBenny went up to look at the picture of George Washington again. He said softly to Henry, \"Don't go yet. Wait till the children are out of sight.\"\n\nWhen the last one had shouted goodbye, Benny took his family up to the front of the room to look at Washington. \"Look at button number five,\" he said. \"There's a hole in the picture. The fifth button _is_ a hole.\"\n\n\"Why, so it is!\" cried Jessie. \"I wonder why.\"\n\n\"I wonder what is behind it,\" said Violet.\n\n\"The back hall,\" said Henry.\n\nThey all went into the tiny back hall. The back door was there with a window beside it. The huge chimney made one wall of the room. There was a big pile of wood.\n\n\"We haven't time to look around any more,\" said Jessie. \"I'm worried about that Mr. Willet. He's here now. I wish Grandfather could stop him before he cheats any more people.\"\n\nJessie did not need to worry so much. The Money Man had gone from the schoolhouse straight to Mr. Fenton's store. When he went in, there was Grandfather leaning on the counter, drinking a cup of coffee.\nCHAPTER 7\n\nGrandfather Makes a Call\n\nThe Money Man was not at all shy when he saw Mr. Alden. He said brightly, \"I'm Freddy Willet, at your service. Introduce me, Fenton, to your friend. I'm a coin dealer. A few antiques, too. Call me Freddy.\"\n\nMr. Alden nodded and said, \"How do you do, Mr. Willet. So you're a coin dealer?\"\n\n\"Yes, sir! And let me tell you, the people on this island save everything. Never throw anything away. It's a great place.\"\n\n\"Really?\" said Mr. Alden. \"Will these people sell you their things?\"\n\n\"Sure. They have a lot of old money tucked away. It came from their great-grandfathers, I guess. They trust me because I gave one of them ten dollars for an old quarter. Who wouldn't take ten dollars for a quarter?\"\n\nMr. Alden wanted to say \"I wouldn't,\" but he said nothing. He went on drinking coffee.\n\nMr. Willet looked at Mr. Alden's watch chain. He said, \"Do you mind if I look? That big old penny on your chain\u2014do you know what that's worth?\"\n\n\"Well,\" said Grandfather, \"not much, I guess.\"\n\n\"You're dead right,\" said Mr. Willet. \"It's got that hole in it, so it isn't worth anything at all. But if some idiot hadn't made that hole it would be worth two hundred and fifty dollars. Yes, sir, no fooling! It's very rare.\"\n\nMr. Alden said, \"Yes, I know. That's why I wear it. I was the idiot who made that hole.\"\n\n\"Sorry!\" said Mr. Willet with a wink. \"Didn't mean to hurt your feelings. By the way, have you any coins on you? I just take a quick look and I know.\"\n\nMr. Alden emptied his pockets on the counter. Mr. Willet quickly turned each piece of money over to see the date.\n\n\"No.\" He shook his head. \"Nothing there.\"\n\n\"How about this?\" Mr. Alden took an old dime from his vest pocket.\n\nFreddy took one look and said, \"Ah!\" Then he took a magnifying glass from his pocket and held it over the dime.\n\nAt last he looked up and said slowly, \"Mr. A., you have a rare dime here. I'll be glad to buy it for a hundred dollars. And _cash.\"_\n\nMr. Alden shook his head again. \"No, I ought to have told you it is not for sale. I just wanted to know how much it was worth.\"\n\n\"Well, now you know,\" said Freddy. \"Sure you don't want to sell it?\"\n\n\"Sure,\" said Mr. Alden.\n\nThis did not stop Mr. Willet. He went on, \"I sell coins to fine places. To colleges and museums. Other things, too. I know people who would be delighted to get that dime.\"\n\nBut Mr. Alden still said no. He looked at his watch.\n\nMr. Willet went closer to Mr. Alden. He said in a low voice, \"Listen, Mr. A., you seem to be a nice sort of chap. That watch of yours is very old. I collect things like that. I'll give you a beautiful modern watch for that watch. I bet it doesn't keep very good time.\"\n\n\"It keeps excellent time,\" said Mr. Alden. \"I don't want to sell it or trade it.\"\n\n\"Well,\" said Mr. Willet brightly, \"maybe you'll change your mind. I'll be around.\" He winked and went out of the store.\n\nGrandfather watched Freddy from the door. \"Now I do hope he won't go up and try to buy anything from Miss Elizabeth Gray,\" he said.\n\n\"Oh, he won't!\" said Mr. Fenton. \"He'll never get in there. The maid Eva sees to that. She doesn't let anyone in.\"\n\n\"I see,\" said Grandfather. \"By the way, what is the matter with Miss Gray? Why does she keep herself shut up in that house?\"\n\n\"I don't really know,\" said Mr. Fenton. \"She's always been that way. She is almost a hermit. All I know is that she hardly ever comes out of her house.\"\n\n\"She must care about the children of the island if she lets them use the schoolhouse this summer.\"\n\n\"Yes, I suppose so,\" said Mr. Fenton. \"Here's another thing. Have you seen that little white building on one side of my store? Well, that's the Gray Library. Miss Gray's grandfather was a book collector. Miss Gray built that library and gave a lot of her grandfather's books to the island people. But nobody ever goes there.\"\n\n\"Why not?\"\n\n\"Well, the books aren't interesting,\" said Mr. Fenton. \"They are all old fashioned and in fine print. Nobody can read 'em.\"\n\n\"How do you get in?\" asked Mr. Alden.\n\n\"Walk in!\" said Mr. Fenton, laughing. \"It's never locked. A young girl used to stay there on Saturdays, but she just sat there. Nobody came. So she doesn't come any more, and the library's always empty.\"\n\nMr. Alden was thinking it very strange that the island people didn't know who Miss Gray was. They did not know that she was a famous author. Long before the Aldens had come to the island, Grandfather had read all her books. He knew that she was well known for her help to many schools all over the country.\n\n\"That's too bad,\" said Mr. Alden. \"Probably they are all good books.\" But then he changed the subject, asking, \"What do you think of Freddy?\"\n\n\"I don't like him,\" said Mr. Fenton at once. \"He may be a coin dealer, but he's something else, too. I don't trust him.\"\n\n\"I wonder why these island people trust him?\" said Mr. Alden.\n\n\"Oh, that's easy. That ten dollar bill he gave to Moss for an old quarter, that did it! Nobody had ever heard of such a thing. I tried to tell them, but they wouldn't listen.\"\n\nGrandfather said, \"I think I'll go up to Miss Gray's and see if I get in.\"\n\n\"You will,\" said Mr. Fenton. \"Eva will know you should be let in.\"\n\nMr. Alden nodded. \"But first I think I had better call up a friend of mine. Could I use your telephone?\"\n\nThere was a telephone on the wall near the door.\n\n\"Go right ahead,\" Mr. Fenton said. \"I have to step out just now.\"\n\nMr. Alden's call was short, but he seemed pleased. He walked up to the mansion. He took off his hat and rang the bell. Eva knew who he was the minute she saw him through the curtain.\n\nShe opened the door and said, \"You are Mr. Alden. Miss Gray will see you.\"\n\nMr. Alden followed Eva into the parlor. \"It's Mr. Alden,\" said Eva.\n\nMiss Gray was sitting at her desk with her back to the door. She turned around and said, \"Sit down, please.\"\n\nGrandfather drew a chair nearer to Miss Gray and said, \"You have been very kind to my grandchildren. I want to thank you. I can't stay long because they will miss me and wonder where I am. But before I go, I want to tell you that a queer-acting coin dealer is loose on this island.\"\n\nMiss Gray rapped on her desk with a pencil. Then she said sharply, \"Freddy Willet!\"\n\n\"I see you know his name. I just wanted you to know what was going on. I think he plans to cheat these island people this time. Then they'll never see him again.\"\n\nThere was a pause, then Miss Gray said, \"Thank you very much.\"\nCHAPTER 8\n\nCaught by the Tide\n\nWhen Mr. Alden met his grandchildren at noon they were all bursting with news. Mr. Alden certainly had news, too, but he did not tell it then. Nobody thought of lunch. They were too busy talking.\n\nHenry was the one to tell his grandfather about the toy bank and the old wooden doll and the string of buttons. He said, \"This man is cheating the people in two ways. First he doesn't pay enough for the old coins, and then he trades their treasures for cheap things. The new things are bright colored, so the children are delighted. They haven't any idea that old buttons are worth anything.\"\n\nBenny added, \"Of course my little Isabelle thinks her new doll is lots better than an old wooden doll. It really is prettier.\"\n\nGrandfather nodded and said, \"This Mr. Willet is certainly not honest. I'm sure of that. Those old iron banks are worth much more than a knife.\"\n\nHenry said, \"Freddy is clever. So far he hasn't done a thing that is against the law. The people here think he pays enough. They are delighted to trade.\"\n\n\"So he hasn't really stolen a thing,\" finished Benny.\n\n\"Exactly right! You just wait,\" said Jessie. \"He will go from one house to another. But even if he should steal things, what can we do, Grandfather?\"\n\nMr. Alden said, \"You children can't arrest him, certainly.\" They all laughed at the idea.\n\nThen they began to tell Mr. Alden about school. He watched Jessie and Violet. They looked tired to him. He said, \"Let's have another quick lunch today. Then we can eat an early supper at the Sea Shell. You girls look tired.\"\n\n\"Oh, no, we're not tired!\" Jessie said. \"We are just thinking hard.\"\n\nBut both girls were willing to set out the peanut butter and jelly for sandwiches and have doughnuts and milk for dessert. Then the four teachers began to study their lessons for the next day.\n\n\"It's a funny feeling,\" said Mr. Alden, \"to see you all sitting around doing schoolwork in vacation.\"\n\n\"This doesn't seem like work,\" said Violet. \"It's just something very interesting mixed up with a thief and a poor rich lady living all alone.\"\n\n\"And lots of cute children,\" said Benny. \"Anyway, I can tell Max that something happened on his old island\u2014but I'm not quite sure what it is yet.\"\n\nThe Aldens sat out in the yard by their hotel with their work. From there they could see the store and the beach and the houses. They could see everything in the village except the mansion. But nobody saw Freddy Willet. The afternoon passed quickly.\n\nThen about five o'clock they saw the red car go bumping over the rocky road to Northport.\n\nMr. Alden got up and took his hat. \"I have an idea,\" he said. \"Let's go right now to the Sea Shell and have supper.\"\n\n\"Right!\" said Henry. He went at once to get the car. They all thought that Grandfather was following Freddy, but they did not say so.\n\nWhen Henry drove into the parking place of the Sea Shell, there was the red sports car!\n\n\"I wonder why the Money Man comes over here to eat,\" said Benny.\n\n\"He has to,\" said Henry. \"There's no place to eat on the island.\"\n\nSure enough, there was the Money Man sitting at a table with two other people, a man and a woman. He said to the Aldens \"Hello, there,\" and went right on talking to his guests.\n\nSuddenly Henry whispered to Benny, \"Change seats with me, Ben, will you?\" Benny got up at once without asking why. He knew that Henry would tell him later. Now Henry sat with his back to the other table. Grandfather himself wondered why.\n\nWhen their waitress had left them, Henry said in a low voice, \"I know those people with Mr. Willet. The man is Dr. James English. He runs the museum at Adams College, and the woman is Miss Cox, the librarian. I hope they won't see me.\"\n\n\"I bet Freddy is trying to sell them something,\" whispered Benny.\n\nGrandfather said, \"That's just what he's doing. I'm sure of it.\"\n\n\"How do you know?\" whispered Benny.\n\n\"I've met him,\" said Grandfather. \"I met him this morning in the store. He tried to buy my watch and the big penny on my watch chain.\"\n\n\"Oh, you'd never sell those! Mr. Willet picked the wrong man that time,\" Benny said. \"Nobody could cheat you, Grandfather.\"\n\nMr. Alden laughed quietly. He was watching Mr. Willet out of the corner of his eye. But he didn't see him give anything to the strangers. Suddenly he said, \"Let's get out of here before Freddy gets through with his dinner. We don't want his friends to see Henry.\"\n\nThis was easy. Mr. Willet and his friends had just started on their dinner, and they were eating very slowly. The Aldens just went without dessert. When they left the table, they all stood behind Henry to cover him up. Henry went out at once to the car on the other side of the parking lot, and Mr. Alden waited to pay the bill.\n\nFreddy Willet looked up and saw Mr. Alden.\n\nThe Aldens sat in the car for a few minutes. \"What do you think Freddy is going to do, Grandfather?\" asked Benny.\n\n\"That's what I wanted to find out. He saw me, but he didn't speak,\" said Mr. Alden. \"I think he's making a deal with Adams College. I hope he won't fool the librarian.\"\n\nHenry said, \"She's pretty smart, and she would know what coins are worth.\"\n\nHe started the car and turned into the road.\n\nThe drive of thirty miles seemed longer than ever. As they came near the island, Violet was looking ahead. \"Oh, look, Henry! The tide!\"\n\n\"Oh, dear,\" said Jessie. \"The tide has turned! Why didn't we think of that? It's getting dark, too.\"\n\nHenry stopped the car and they all looked ahead. There was no road to the island. It was covered with seawater, which was getting deeper every minute.\n\n\"I don't dare try it, Grandfather,\" said Henry, frowning.\n\n\"Right, my boy! If we got stuck half way over, it would be no fun, I can tell you.\"\n\nHenry said, \"I thought it seemed awfully wet when we came over. But I knew you must be following Freddy, and I don't think he gets caught like this very often.\"\n\n\"By the way,\" cried Benny, \"I wonder what Freddy will do? He'll be caught, too. It will be six hours before it's low tide again.\"\n\n\"It will be nearer twelve hours, Ben,\" said Henry. \"You see the tide hasn't been coming in long.\"\n\n\"Let's see,\" said Jessie. \"It is about eight o'clock now. The tide will be high at midnight, and low again at six o'clock in the morning!\"\n\n\"We're not in any danger, though, are we, Grandfather?\" said Violet.\n\n\"No, my dear. We will just have to find a place to sleep.\"\n\n\"Oh, boy!\" shouted Benny. \"We could sleep in the car! That's what this station wagon is made for. We could do it, Grandfather!\"\n\n\"Five people?\" asked Grandfather, smiling.\n\nBenny said, \"I could take the car blanket and sleep on the top of the car. Oh, please!\"\n\nGrandfather laughed. He said, \"I don't think we would get much sleep. But I suppose you young people don't mind that.\"\n\n\"I don't,\" said Jessie. \"I'd like to wait and see what Freddy will do.\"\n\n\"Very well,\" said Grandfather. \"Find a place off the road, Henry, and we'll try it.\"\nCHAPTER 9\n\nThe Disappearing Stranger\n\nHenry backed the car into the woods a little way. Nobody could see it from the road. The Aldens all got out and took out every car rug that they could find for bedclothes. It was so warm that they didn't really need any covers.\n\nHenry and Benny let down the seats that made beds for four people. By nine o'clock all was ready. Benny climbed up on top of the car.\n\n\"I won't fall down,\" he called. \"There's a railing all around the edge. I'm going to put this blanket under me instead of over me.\" He folded the car blanket and lay down. \"A very soft bed,\" he said.\n\n\"I should think Freddy would be along soon\u2014if he's coming,\" said Jessie. \"I wonder what he will do.\"\n\nIn about an hour they heard a car coming. It did not make much noise, but everyone was awake. Benny sat up and looked through the trees. \"Yes, it's a car,\" he whispered.\n\nBut the car stopped. Everyone was watching it now. Mr. Freddy must have seen that the tide was in, because he quietly turned his car around and went back.\n\n\"Now what?\" Benny whispered down. \"I don't think he is going to give up like that. He would just love to be on the island when we are away. He knows we are watching him, I bet.\"\n\nThey all lay down again. But nobody went to sleep. It was not too long before they heard another very soft sound. It sounded like water, and it was ahead of them. They all looked hard through the darkness.\n\n\"He's got a boat!\" whispered Henry. They all watched. Then they all saw it. It was a rowboat with Mr. Willet's head showing against the dark sky.\n\n\"Come down now, Benny,\" whispered Grandfather. \"We'll get a boat, too!\"\n\n\"Oh, can we really?\" whispered Benny. \"Where can we get a boat?\"\n\n\"Where Mr. Willet got his, I think,\" said Grandfather.\n\nThey all sat up in the car, and Henry drove back over the road very carefully.\n\n\"There's a light!\" said Violet. \"It's in a fisherman's hut.\"\n\nHenry stopped the car in front of the house.\n\n\"Somebody is awake,\" said Jessie. \"And do I see the end of that red car sticking out behind the house?\"\n\n\"You do,\" said Mr. Alden. \"I do, anyway. I think this is the place where Freddy got his boat. You do the talking, Henry.\"\n\nHenry went to the door and knocked. At once a fisherman came to the door. He wore an old coat.\n\n\"I'm sorry to come so late,\" began Henry, \"but we need a boat. It's high tide, and we're stuck.\"\n\n\"Yep, I know that,\" said the man. \"Seems as if everybody is stuck tonight on the wrong side.\"\n\n\"Can we rent a boat from you?\" asked Henry.\n\n\"Sure! I've got three. I just rented two.\"\n\n\"Two!\" said Henry in surprise.\n\n\"Yep, two. One man came, and then another man came. You can have the last one.\"\n\n\"Where is it?\" asked Henry.\n\n\"It's tied down near the road, when there is a road. You'll see it on the bank on the left, tied to a post. Can't miss it.\"\n\n\"How much will it be?\" asked Henry. He couldn't help wondering about two men. He had been thinking only of Freddy Willet.\n\n\"A dollar,\" said the man. \"Be sure and tie her up on the island side. My boys can row 'em back tomorrow. It ain't far.\"\n\nHenry quickly gave the man a dollar and thanked him. Then he went back to the family. He said, \"Do you suppose five of us can get into one boat?\"\n\n\"We'll see,\" said Grandfather. \"I think so.\"\n\nBenny had heard about the two men. He could hardly wait to speak. \"Who do you suppose that other man was? A mystery, for sure.\"\n\nMr. Alden was thinking, too. \"I wonder,\" he said.\n\nHenry drove back to the water. Sure enough, a boat was tied up on the bank.\n\n\"Good! It's a big one,\" said Grandfather. \"I wonder why we didn't see it. Back the car off the road again, Henry. I guess this night's sleep is over.\"\n\nEverybody got out of the car and went carefully down to the boat. When Henry came, they all pushed the boat into the water. Henry helped Violet and Jessie into the front of the boat, and Benny stepped into the other end. When Grandfather was sitting down on the middle seat, Henry pushed off. He and his grandfather began to row.\n\n\"We'll tie up just below the schoolhouse,\" said Henry. \"I suppose we will see the other two boats there.\"\n\nIt was not very far to the island, but it was dark, and the tide was very high. They could see a little through the darkness. But there were no boats to be seen!\n\n\"What do you know!\" said Benny. \"Where would Freddy go? Do you think he has a partner, Grandfather?\"\n\n\"Well,\" said Mr. Alden, \"I don't know. If the man is a partner of Willet's, why would he come by himself?\"\n\n\"That's so,\" Jessie said.\n\nBenny added, \"But I'm almost sure the first man must have crossed over to the island before we came. We were watching and listening all the time.\"\n\nHenry ran the boat up on the beach. He tied it to a tree.\n\nSuddenly Violet looked around and asked, \"Where do you suppose Freddy has gone?\" She shivered a little.\n\n\"Not to the hotel,\" Jessie said. \"And I don't think he would stay with any of the island people.\"\n\n\"I wonder,\" said Henry. \"I just wonder\u2014\"\n\n\"I know,\" said Benny. \"The schoolhouse!\"\n\nBenny would have run to peek in the windows, but Grandfather said, \"How do you know that Freddy isn't going back to the other side after getting something? The best thing we can do now is to get some sleep.\"\n\n\"And there's school tomorrow,\" said Violet.\nCHAPTER 10\n\nA Discovery\n\nAt breakfast they talked about the two boats and the two men.\n\n\"It's time to ring the bell,\" said Benny. \"I have to go.\"\n\nBut Benny really had something else in mind. He wanted to look around the schoolhouse. He wanted to see where the hole went in George Washington's coat button.\n\nBenny unlocked the schoolroom door and went at once into the back hall. The wood for the winter was there, piled high. There was the back door, a window, and the big chimney. He rattled the window. It was unlocked. The lock was very old and looked as if it hadn't been locked for a long time. Benny tried it. The lock was broken. It certainly had been broken for years.\n\nBenny looked at the floor under the window and saw some flakes of dry white paint just exactly like the dry paint on the windowsill.\n\n\"I bet someone came through this window last night,\" he thought. \"But why? I like to do things by myself, but this time I wish I could tell John Carter who works for Grandfather. After all, he used to be an F.B.I. man.\"\n\nBenny looked around again. Only the big chimney was left, and the broom closet next to it. He opened the small door. Just a broom closet. There was a broom in it.\n\nAt recess Benny got word to his family to wait after the children had gone home at noon. When they were alone in the school yard, Jessie said, \"Well, Benny, what's all this?\"\n\n\"The back hall,\" said Benny. \"Come and see.\"\n\nThe four Aldens all went into the tiny back hall.\n\n\"See that old dry paint on the floor?\" said Benny.\n\n\"Yes, old man,\" said Henry. He bent over to look. \"You are clever! This hasn't been here long. Somebody must have come in through the window. Maybe this is where Freddy slept last night.\"\n\n\"This broom closet is just a broom closet,\" said Benny. He opened the door beside the huge chimney. \"See, there's a broom in it.\"\n\n\"Wait!\" said Henry. \"See those wood planks this closet is made of? If we could only pry them off\u2014\"\n\nHenry opened his knife and pried away at one board. It was loose and came off easily. And there was a little brick room in the chimney!\n\n\"I thought this chimney was awfully big,\" said Henry. \"And it has a queer shape. This is the reason, of course. Somebody wanted to make a place to hide in.\"\n\n\"Who?\" asked Benny.\n\n\"Well, I don't know. It was made that way when the schoolhouse was built, and that was a long time ago. I do know that a lot of New England people built their houses with a hiding place in the chimney. I'm sure this is one of them. A perfect place!\"\n\n\"It is a perfect place for us, too,\" said Benny, \"if we want to watch what is going on in the schoolroom.\"\n\n\"What's the idea, Benny?\" asked Violet. \"Why would we ever want to watch the children?\"\n\n\"Not the children, Violet!\" said Benny. \"Not in the daytime, either. At night! We can watch Freddy Willet if he ever comes in here, and I think he does.\"\n\n\"I think so, too, Ben,\" said Henry. \"He must have some place to hide his things.\"\n\nThey put the planks back, locked the front door, and went home to lunch. They told Mr. Alden all about the room in the chimney. After lunch, Grandfather said, \"I know something you don't know!\"\n\nThe children laughed. \"What is it?\" asked Violet.\n\nThen Mr. Alden told them about the Gray Library and the old books. When Benny heard it, he pushed back his bench. \"That's where I'm going,\" he said. \"Maybe I can find some pictures for my class.\"\n\n\"I don't think you will, Benny,\" said Mr. Alden. \"Mr. Fenton said the books were all old.\"\n\n\"Well, maybe there's an old picture of George Washington,\" said Benny. \"Anyway I want to see the library.\"\n\nBenny took a notebook, and off he went. Sure enough, the door of the library was not locked. Benny gave it a push and went right in. He found himself in a room just the size of the building. There were bookcases all around the room and one tall one down the middle.\n\n\"Well, here goes!\" said Benny out loud. He began to whistle. He went to the first bookcase. He looked at the dusty books on the top shelves. Then he dropped to his knees to look at the books near the floor. All at once he had a queer feeling that someone was in the room. He listened, but he didn't hear a sound.\n\n\"This is nonsense,\" he thought. \"Mr. Fenton says nobody ever comes here.\"\n\nThen he heard a very, very soft noise. \"What is that?\" he thought. \"Somebody is certainly in here!\" But he did not get up or turn around. He listened. Then he heard the noise again. It was behind the middle bookcase. Benny knew that he couldn't see over it if he did turn around. Then there was a soft thud and then another. Benny rushed around the bookcase just in time to see the fingers of a hand disappear from the sill of the open window.\n\n\"Well, well, Mr. Willet!\" said Benny out loud. He rushed to the window and looked out. Nobody was in sight.\n\n\"Freddy is hiding, that's sure,\" said Benny to himself. \"He hasn't had time to get anywhere. But I know what I'm going to do.\" He ran up the cliff walk to the mansion!\n\nBenny smiled as he rang the bell because he saw Eva peeking at him. In a minute Miss Gray herself opened the door.\n\n\"Sorry, Miss Gray,\" said Benny, very fast, \"I think someone just stole some books out of the library. I thought I ought to tell you first. Maybe those books are valuable.\"\n\nEva's eyes grew big and round as Miss Gray said, \"Thank you, Benny. I'll look myself. Nobody else knows the books. You come, too, Eva.\"\n\nThe three people went quickly down the cliff. Miss Gray went into the library and straight over to the last rows of books. There was the empty space exactly where she thought it would be. She knew which were the most valuable books, and so did Mr. Willet.\n\n\"There are four books gone here,\" she said. \"They were a set. I never had an idea they would be stolen. And over here! Yes, some more are missing. Mr. Willet must have been here before.\"\n\nShe went around the room and found empty spaces everywhere. \"Oh, dear!\" said Miss Gray. \"He has taken the very best books! He can sell them for a lot of money.\"\n\n\"But look here, Miss Gray,\" said Benny. \"See this sign? It says you can borrow anything you want and sign your name. And here is Freddy Willet's name!\"\n\n\"I don't think he will bring them back,\" said Miss Gray. \"How foolish I was not to lock the door!\"\n\n\"I don't think so,\" said Benny. \"Freddy would have climbed in the window. Windows and doors don't stop our Freddy. That's why I think he is a thief. Don't worry too much, Miss Gray. My grandfather has Freddy on his mind. And my grandfather doesn't slip up very often.\"\nCHAPTER 11\n\nWho Is the Englishman?\n\nBenny went slowly back to the hotel. He looked on every side as he walked, but Mr. Willet had disappeared.\n\nBenny did some hard thinking on that walk. He had been sure that Freddy Willet was going to do more trading. But as far as Benny could tell, the Aldens and Miss Gray were the only people who knew Freddy was on the island today. After all, he hadn't come in his red sports car. He had come secretly in the middle of the night. Why?\n\nOne idea came to Benny. Freddy had planned to get the library books on this day while the Aldens were still busy with their school. It was just bad luck for Freddy that Benny had gone to the library to hunt for pictures.\n\nWhen he reached home, Benny told the family about his adventures. Henry said, \"Ben, you certainly have made a friend of Miss Gray. I thought you could. Why, you have even made her come out of her house.\"\n\nJessie said, \"We've been here in the yard, but we haven't seen Mr. Willet or anyone new.\"\n\nMr. Alden said, \"It's a mystery how two men can keep themselves out of sight. This is such a small place.\"\n\nNothing else happened that day. Everything seemed peaceful\u2014too peaceful, the Aldens thought.\n\nThe next day was Saturday. The first thing the Aldens saw in the morning was children fishing. The island children sat on the edge of the wharf with long fishlines. They did not need poles.\n\nGrandfather said, \"Let's go fishing!\"\n\n\"Fine,\" said Jessie. \"We can watch the whole island from the wharf.\"\n\nIn a short time the whole family was sitting on the edge of the wharf with the children. Mr. Alden gave Hal a dollar for five fishlines. Eddie said, \"I'll show you how to fish, Mr. Alden. You let the line way down. Then pull it up just a little and let it down again. Keep the hook wiggling.\"\n\nThe Aldens did as Eddie said. The island children were catching fish after fish. But the Aldens caught nothing. Suddenly Mr. Alden had a bite.\n\n\"Pull her in, mister!\" cried Eddie. \"Don't rush it. Hand over hand! But keep it even-like.\"\n\nMr. Alden pulled as well as he could through thirty feet of water, and landed a beautiful big fish on the wharf.\n\nBenny said, \"Aren't you going to fish any more, Grandfather?\"\n\n\"No. I'm afraid I'll catch another.\"\n\n\"Well, I'm not,\" said Benny. \"I wish I could catch one. I would eat that whole fish myself.\"\n\nEveryone was quiet. The island children went on pulling in the fish. But the Aldens still caught nothing.\n\n_All_ at once Grandfather said slowly, \"I believe I see our strange man at last!\" Something in Grandfather's voice made the Aldens look up quickly at the cliff walk. The island children paid no attention.\n\n\"Who in the world is that?\" said Jessie. \"He is dressed like an Englishman.\"\n\nThe stranger wore a cap that shaded his eyes. He had on dark blue shorts and a soft shirt with a scarf tucked in at the neck. His socks were red and blue and came up to his knees. He carried a walking stick.\n\n\"Maybe he's a friend of Miss Gray's,\" said Henry. \"He is coming from that way.\"\n\nThen Violet said in a very low voice, \"Grandfather! Do look at him! It's Mr. Carter!\"\n\n\"It is,\" said Henry softly. \"Look at his walk! It's John Carter's walk. Now why is he here?\"\n\nMr. Alden said, \"I telephoned him about Willet, but I didn't send for him. Maybe he began to look up Willet's business and thinks we need help.\"\n\nHenry whispered, \"He's coming this way. Act as if you don't know him.\"\n\nThe man in shorts came down to the wharf. Benny wanted to smile, but he stopped in time.\n\n\"Hello, everybody! Fishing?\" said the man, as if he had never seen the Aldens before.\n\n\"Well, sir, these children are fishing,\" said Mr. Alden. \"But we have caught only one.\"\n\n\"I say, that's a big one!\" It was exactly as if an Englishman were speaking. Not one of the island children turned around. But they heard every word.\n\n\"You're a stranger here, sir,\" said Grandfather. \"Are you staying long?\"\n\n\"A week or so, don't you know? My name is Wilder-Smith. Guest of Miss Gray's. My mother went to college with Miss Gray. Great old friends.\"\n\n\"We are the Aldens,\" said Grandfather. \"Henry, Jessie, Violet, and Benny.\"\n\nMr. Wilder-Smith bowed. He knew that the island children were listening. He went on, \"There's another stranger here, Mr. Willet. Today he is going to visit the good people and look over some dusty old things. He likes old things. Bit odd, don't you know?\"\n\nIsabelle spoke up loudly. \"He isn't odd. He's wonderful!\"\n\n\"There, now,\" said Mr. Wilder-Smith, \"you see! He's wonderful. I may go with Mr. Willet on his calls.\"\n\n\"Go with him?\" asked Benny, surprised.\n\n\"The man invited me. Of course I don't know anything about old things. By the way, did you know there _is_ no post office on this island?\"\n\n\"Yes, the mail has to come from Northport,\" said Henry.\n\nMr. Wilder-Smith shook his head. \"I'll have to find some other way. I have to get my letters by fast post.\"\n\n\"Maybe we can think of a way to help you,\" said Benny. \"We've got a car.\"\n\n\"Right,\" said Mr. Wilder-Smith. \"I have important letters. Maybe you can help. But I must toddle along. Cheerio! Top-hole to meet you.\"\n\nMr. Wilder-Smith walked back along the wharf. Not a child spoke. They went right on fishing.\n\n\"Let's go!\" said Henry. \"Thanks, Eddie, for the fishing lessons.\"\n\n\"OK,\" said Eddie.\n\nWhen the Aldens reached the sand, Jessie said, \"Why do you suppose Freddy ever asked Mr. Carter to come with him?\"\n\n\"Because he's a smart man,\" said Grandfather. \"Freddy wants us to think he is honest. But he'll find that John Carter is smarter than he is.\"\n\nNobody on the wharf could hear what the Aldens said now. Benny said in a low voice, \"Mr. Carter wanted to find some way to send us messages, didn't he\u2014talking about the post office?\"\n\n\"That's what I thought,\" said Jessie.\n\n\"I know a way,\" said Benny. \"You know that old log on the beach that Grandfather sits on? There's a deep hole in one end. We can put a small piece of paper in the hole any time. And so can he.\"\n\n\"Good!\" said Jessie. \"Let's send him our message right away. The people know that we often sit on that log. They won't notice.\"\n\nHenry wrote it. He made it as short as he could. \"Light in schoolhouse at night. Back window has no lock. Broom closet goes into secret room in chimney. Look out of Washington's coat button.\"\n\n\"Now how shall we put it in the log?\" asked Violet.\n\n\"I know!\" said Jessie. \"We'll make some sandwiches and have a picnic on the beach. While we are eating we can hide the paper.\"\n\nThe girls quickly made some cheese sandwiches. They packed the basket with many other things. Mr. Alden sat on the log. \"Why don't you hide the paper yourself, Gramps?\" said Benny. \"You know where the hole is.\"\n\n\"Don't watch me,\" said Mr. Alden. Then in a minute he said, \"It's all done.\" Not a sign of paper showed.\n\n\"I wonder how Mr. Carter will get it out?\" said Benny.\n\n\"Well, that's his lookout,\" said Mr. Alden. \"I wouldn't worry about him.\"\nCHAPTER 12\n\nA Sockful of Money\n\nThe picnic was over. The Aldens sat looking out over the blue water when the factory whistle blew.\n\n\"Oh, dear,\" said Benny. \"Now everyone will go to work and nobody will be at home when Freddy comes.\"\n\n\"They don't all go to work,\" said Jessie. \"By the way, I need some stuffing for the fish tonight. I'll go to the store while you take the picnic things home.\"\n\n\"I'll go for you, Jessie,\" said Benny. \"I'd like to.\"\n\n\"Good boy,\" said Henry.\n\nBenny walked up to the store, whistling. Mr. Fenton had the fish stuffing, and the two began to talk. The door opened, and who should come in but Freddy Willet!\n\n\"Hello, kid,\" said Freddy. \"I saw you come in. You one of the youngsters that teaches the funny school?\"\n\n\"It isn't a funny school,\" said Benny with good nature. \"And I do teach the smallest ones. They're pretty keen kids, too.\"\n\n\"Your name?\" asked Mr. Willet.\n\n\"Benny.\"\n\n\"Well, Benny, how would you like to learn something yourself? Come with me to the Easton family and watch me buy their old money. I bet you don't know money when you see it. You'd never think a common penny was worth a dollar, would you?\"\n\n\"No,\" said Benny. He kept wondering why Freddy had invited him and Mr. Wilder-Smith to watch while he cheated the people. Benny took his box of stuffing and the two walked over to the house where the Eastons lived.\n\nMrs. Easton was waiting at the door with a smile. At that moment Mr. Wilder-Smith came along.\n\n\"Well, here we all are,\" said Freddy. \"Now you can watch me work. Mrs. Easton, we are all happy to be here. Let's get started.\"\n\nBenny could hardly believe his eyes when Mrs. Easton put an old stocking on the table and poured out a pile of money. Some of the coins were very old, and a few had hardly been used at all.\n\nMrs. Easton said, \"Some of that money was saved by my grandfather.\"\n\nThey sat down at the table. Mr. Willet moved like lightning. He picked out one coin after another and dropped each in an empty box. He checked some coins by using a magnifying glass. Mr. Wilder-Smith just looked on. He did not seem to know a thing about coins.\n\n\"Is this an old penny?\" asked Benny. It was almost as big as a half dollar, but it was made of copper.\n\n\"Yes, that's an old penny. Let's see. The date is 1864. I can pay Mrs. Easton ten dollars for it.\" He put a ten-dollar bill on the table and the penny in the box.\n\nMrs. Easton smiled and said, \"Mr. Willet works fast. He knows his business.\"\n\n\"You can say that again!\" said Freddy. He showed his teeth in a wide smile. He went on, \"This is a nice job, Mr. Wilder-Smith. I have already bought five-hundred dollars worth of coins and things from these fishermen. They are very grateful to me.\"\n\n\"No wonder, old boy,\" said Mr. Wilder-Smith. \"What in the world do you want them for?\"\n\n\"Oh, I'm crazy about old things,\" said Freddy. \"I like 'em all. But I like coins best.\"\n\nBenny said, \"I don't see how you remember the prices without a book.\"\n\n\"Easy!\" said Freddy. \"I've been at this business for many years. I know a coin the minute I see it.\"\n\n\"I say,\" said Mr. Wilder-Smith, \"you might make a mistake.\"\n\n\"Not me,\" said Freddy. \"I know these coins like old friends. I never make a mistake. There! This is the best one I've found.\"\n\nHe pushed a dime over to the two visitors. \"Here is a new coin in perfect condition. But look at the date. 1901S. I'll pay Mrs. Easton plenty for that. She'll never forget Freddy Willet, you bet.\"\n\n\"I certainly won't,\" said Mrs. Easton.\n\n\"I'm sure of that,\" said Mr. Wilder-Smith. \"That dime looks as if it had never been used.\"\n\n\"That's right. It never has been used. That dime has sat here for more than sixty years.\"\n\n\"By Jove, I thought a worn-out coin was worth more,\" said the Englishman.\n\n\"No, not if it's worn smooth. Then it's worth nothing. See this big copper penny? Smooth as silk. I can't read the date. That penny is old enough, but it's not worth a cent!\" He laughed loudly at his own joke.\n\n\"That's all,\" said Freddy. \"The rest are no good. I'll just settle up with Mrs. Easton.\"\n\n\"Thanks very much,\" said Mr. Wilder-Smith. \"It was a jolly good show. I learned a lot from you.\"\n\n\"I guess you learned something today, too, young man,\" said Freddy.\n\n\"Yes, I did,\" said Benny. He said goodbye to Mrs. Easton, and he and Mr. Wilder-Smith went out together.\n\nBenny whispered quickly, \"Hole in the log.\" And Mr. Wilder-Smith whispered, \"Look three times a day.\"\n\nThen Benny went one way and Mr. Wilder-Smith another. They did not seem to know each other, for the Englishman took off his hat to the boy.\n\nBenny had a great deal to tell his family. He told it in a whisper.\n\nJessie said, \"It's funny how we all whisper. It seems as if somebody is listening all the time.\"\n\n\"Maybe someone is,\" said Violet.\n\nThey were noisy enough when Jessie put the baked stuffed fish on the table. They had baked potatoes, too, and onions, and apple pie and cheese.\n\n\"A big dinner at last,\" said Benny. \"I'm hungry.\"\n\nWhen it grew dark, the Aldens sat out in the yard in front of the hotel. Henry was on one end. He turned suddenly as he heard a noise coming from the bushes behind the house. It was a whisper.\n\n\"Benny! Come with me to the schoolhouse. Bring the back door key. Follow me through the bushes.\" It was John Carter's voice.\n\nThey all heard it. Henry gave the key to his brother. Benny went without a sound.\n\n\"Why did he want Benny?\" Jessie wondered.\n\n\"Maybe his small size is better than my big size,\" guessed Henry, speaking low. Then he began to talk about other things in his own tone of voice.\n\nAt ten o'clock Benny came back. He was very much excited.\n\n\"Whisper, Ben,\" said Henry. \"Who knows who is listening?\"\n\nSo Benny said in a whisper, \"We tried the room in the chimney. We could both get in, and we could see right through the coat button into the schoolroom. Mr. Carter thinks Freddy is hiding things in the schoolroom. But we couldn't find them. He is going to hunt again. He thinks Freddy will stay a while longer because he has a lot of families left to visit.\"\n\n\"Well, well,\" said Mr. Alden. \"You will have something to tell Max. It won't be dull, either.\"\n\n\"It's funny,\" said Benny. \"We didn't do anything to make this happen.\"\n\n\"No, Benny,\" said his grandfather. \"But you all kept your eyes open. You cared enough about your new friends to worry about them. And you listened when they told you about the man who paid so much for a quarter. You guessed that he was cheating in every trade.\"\n\n\"But we can't prove a thing,\" said Henry.\n\n\"No,\" said Grandfather. \"That's why I am so glad to see John Carter.\"\nCHAPTER 13\n\nSurprise for Violet\n\nThe Aldens could hardly teach school on Monday with Mr. Willet making calls on every family in the village. All the children knew about it. They couldn't help talking about it. At recess they did not want to play. They wanted to talk.\n\n\"Oh, my ma's got an old gold pin with a lion's head on it,\" said one girl. \"The Money Man's going to pay her real money for it. And she's got an old garnet ring, too.\"\n\n\"And my grandma's got a big box of old valentines,\" said her brother. \"The Money Man is going to buy those.\"\n\n\"My pa has got an old tin dish they used to cut meat on,\" said Tommy Spoon.\n\n\"Could that be pewter?\" asked Violet.\n\n\"Pewter? No, I never heard of that.\"\n\n\"And we've got an old hourglass they used in church over in Northport,\" said Eddie.\n\n\"We've got a glass paperweight full of flowers. The Money Man is just crazy over it,\" said another child.\n\nThen suddenly Hal got up on a rock and began to talk. \"You kids, listen here!\" he began. \"You got all the afternoon to talk about the Money Man. And this morning you got a good chance to learn something. We don't ever get any teachers like these Aldens, teaching us interesting things. And they won't be here much longer. Let's stop talking and learn. That's all I've got to say.\"\n\nSome of the children clapped for Hal's speech, and soon everyone was busy. The morning flew by. Then Violet said, \"Now let's paint. We can go down to the beach.\"\n\nMarie and Hal were more excited than the other children. Marie begged, \"Please teach me to paint the harbor the way the artist did.\"\n\n\"I'll help you as much as I can,\" Violet promised.\n\nThe girls and boys took their painting things down to the water. The Aldens helped them get started. Violet had Marie and Hal draw the harbor first with pencil. She showed them only one thing. She said, \"Notice that the lines of the wharf look as if they get closer together as they stretch into the water.\"\n\nAs soon as Violet had shown the children how to hold their brushes she said, \"Now paint away, just as you feel.\"\n\nSoon Jessie and Henry came over by Violet to watch the Moss twins.\n\nBoth Hal and Marie dashed on blue and green paint that was just the right color for the sea. The boats that they made looked like real boats, but they were drawn with only a few lines. It was hard to believe that this was the first time the twins had had watercolors.\n\nAll at once Violet knew the truth. Here were two natural artists, much better than she would ever be. There was something different about their painting\u2014something all their own.\n\nMarie cried, \"Oh, here's that purple place,\" and she splashed purple and black under the wharf. She was not careful at all. Her picture made Jessie exclaim, \"Beautiful!\"\n\nHal's picture was just as good, but it was different.\n\n\"May I take your paintings to show our grandfather?\" Violet asked the twins.\n\n\"I could paint another!\" cried Hal. \"I know a different way to paint the ocean, the way it looks in a storm.\"\n\nJust then Mr. Alden came walking along the beach to see what was going on. Violet ran to show Mr. Alden the two paintings.\n\n\"Well, well!\" he said. \"What have we here? Watercolors? These are really good! These artists ought to go to art school. Not now, but later.\"\n\nSchool was over for the morning. The bell was ringing. Henry knew that Benny would soon lock the schoolroom door and come back to the beach.\n\n\"Old Ben wants his lunch,\" he said, laughing.\n\n\"So do I,\" said Mr. Alden. \"I wish Benny would come.\"\n\nAs they were eating, Violet said slowly, \"I think Miss Gray ought to see these paintings.\"\n\n\"Oh, so do I,\" said Benny. \"I'm sure she'd like them.\"\n\nAfter lunch the whole family set out with the two pictures. They climbed the cliff walk and rang Miss Gray's bell.\n\nEva opened the door. She laughed. \"I don't have to ask any more when it's you,\" she said. \"Come right in.\"\n\nWhen Miss Gray saw the Aldens she really smiled. It was the first time the Aldens had seen her smile like this.\n\n\"How is Freddy?\" she asked.\n\n\"Well, Freddy is getting into trouble,\" said Grandfather. \"He is too brave. He is going a little too far.\"\n\n\"How about the books?\"\n\nMr. Alden had to say, \"We don't know yet about the books.\"\n\n\"I'm sure you will get them back,\" said Benny. \"You see\u2014\"\n\nHenry looked at his brother. Benny stopped.\n\nMiss Gray said, \"Yes? What were you going to say?\"\n\n\"Well, I guess it wasn't important,\" said Benny. \"We're watching Freddy most of the time.\" Benny was not sure what Miss Gray knew about her English guest.\n\nMr. Alden said, \"We really came to show you two watercolors. We'd like to know what you think of them.\" He passed them to Miss Gray.\n\n\"The harbor,\" said Miss Gray. \"Very good. Were they done by one artist or two?\"\n\n\"Two,\" said Violet with bright eyes. \"The twins, Hal and Marie Moss.\"\n\n\"Hal and Marie!\" said Miss Gray. \"They never had a lesson!\"\n\n\"No,\" said Grandfather, \"they were born that way.\"\n\nJessie said, \"They have seen only three paintings in their lives. An artist came here long ago and painted their house and the harbor. And there's George Washington in the schoolroom.\"\n\n\"That's why they have a style of their own,\" said Miss Gray with a nod.\n\nViolet said, \"We have to go home the first of August. Somebody ought to look after those twins.\"\n\nMiss Gray looked straight at Violet. \"I will,\" she said. Then she seemed to be having a hard time with her words. At last she said, \"When you go home, I'm going to teach your school myself.\"\n\n\"You?\" cried Jessie. \"Are you strong enough?\"\n\n\"I'm strong enough,\" said Miss Gray. \"Those twins live in my own town, and here I sit writing books. Nobody ever knew those children could paint. If you hadn't come along, I don't believe anyone would ever have found them.\"\n\nThe Aldens started to go. Miss Gray went to the door with them. As Grandfather bowed to her, she said, \"Mr. Alden, Violet found the two artists, and your Benny found _me.\"_\nCHAPTER 14\n\nCaught!\n\nThe next day everyone saw Freddy Willet coming and going about the island in his red sports car. But no one ever saw him open the trunk, and there were no boxes in the front of the car.\n\n\"He must be hiding the library books in the schoolhouse,\" said Benny. \"There's no other place here where he could hide the bigger things he has traded. But Mr. Carter can't find a thing.\"\n\nHenry looked serious. \"Time is getting short,\" he said. \"In another day or two Freddy will have everything of value that can be found on this island.\"\n\nAfter supper the Aldens found a long note hidden in the hollow log on the beach. In it John Carter said, \"Fred Willet is Harold K. Frederic. He has two other names he also uses. Canadian police want him for smuggling. All we have to do is get him to the border.\"\n\nThat evening the Aldens sat outside their hotel as they always did. When it was quite dark, they grew more and more excited. They were sure something was going to happen. They were not at all surprised to hear a whisper: \"Benny, follow me.\"\n\nWhile the others talked about the weather, Benny slipped after John Carter.\n\nAt first Benny thought that the schoolhouse was dark. Then he saw that something had been hung over a window. It was a car blanket. A dim light showed through another window.\n\nWithout a word, John Carter and Benny crept in at the back door. They went into the tiny chimney room. Mr. Carter looked through the hole in Washington's coat button. He let Benny look.\n\nThe room was faintly lighted by a square flashlight on one of the desks in the back of the room. At first Benny could not tell what Freddy Willet was doing. Then he saw. The man was taking the wood out of the woodbox!\n\n\"His hiding place!\" whispered Mr. Carter. \"Now let me look.\"\n\nWhen all the wood was out of the woodbox, Mr. Willet turned the box upside down very carefully and poured coin after coin on the back desk. He swept them quickly into a strong bag.\n\n\"The books?\" whispered Benny.\n\nMr. Carter shook his head. Then he was really surprised. Mr. Willet went to the bookcase that held the school books and took them all out. He took out a board in the back of the bookcase. Then he began to take out the old books from the library.\n\nMr. Carter stepped aside to let Benny look through the hole. Book after book came out of the space behind the shelf. Then came the doll, the buttons, the gold pins, the iron bank\u2014everything.\n\nBenny was thinking hard. \"Mr. Willet must have been in the schoolhouse many times before. It took a long time to fix that hole behind the school books. That's what he was doing when we couldn't find him. My, what a lot of books! What will he do with them?\"\n\nHe soon found out. Freddy took six books and began to tie them up with string. He set them on another desk. Soon he had sixty books. Then he put the board back, and fixed the school books just as they had been.\n\nMr. Carter took Benny's place at the peephole. He was not a minute too soon, for Freddy took some books in one hand and was reaching to put out the flashlight when he heard Mr. Carter's voice.\n\n\"Hold it, Willet! Don't move! The front door is locked now!\"\n\nMr. Carter and Benny dashed down the back hall and into the schoolroom. Mr. Willet did the only thing he could do. He put out his light.\n\nBut this did him no good, for when his light went out, Mr. Carter's went on.\n\n\"What's the matter with you?\" shouted Freddy. \"I haven't done anything!\"\n\n\"Oh, yes, you have!\" said John Carter. \"Just put those books down. What about all the little things you traded? What about the coins?\"\n\n\"They were fair trades!\" shouted Freddy. \"Everyone was satisfied. They were glad to trade.\"\n\n\"That's because they did not know the things were antiques,\" said Mr. Carter. \"And what about these books? A trade?\"\n\n\"I borrowed those books. You just sign your name and take what you want. And I signed mine!\"\n\n\"Which name did you sign, Freddy?\" asked Mr. Carter softly.\n\nThen Freddy knew he was in trouble. He had three or four names, and John Carter knew every one of them. Mr. Carter went on. \"The Canadian police want you, Freddy. They will be very glad to see you. You have been smuggling for years. You made a great mistake to try it again. No, leave all the things right where they are!\"\n\nMr. Willet made one last try. \"You aren't a policeman. You can't arrest me.\" He was very angry.\n\n\"You're wrong,\" said John Carter. \"I was made a special policeman a week ago in Northport. But you'll be glad to know that the chief of police from Northport is sitting in your red car out in the bushes.\"\n\nThat stopped Freddy for a minute. Then he said, \"Now, listen! I've paid these people a lot of money for old coins.\"\n\n\"Not enough,\" said John Carter. \"You were going to sell the coins to the Adams College Museum. We can do that for you, and thanks very much for picking out the best ones!\"\n\n\"You'll have to pay me back what I paid,\" said Freddy.\n\n\"We will, in time,\" said Mr. Carter. \"Nobody is going to cheat you, Freddy. But money won't do you much good in prison. Ah, hello, Anderson! Benny and I are glad to see you.\"\n\nA tall policeman from Northport had come in the back door.\n\nFreddy growled, \"The Alden kids did this!\"\n\n\"Yes,\" agreed Mr. Carter, \"the Alden kids did this. They did their duty as American citizens. They just did what was right.\"\n\nMr. Anderson said to Benny, \"We all thank you, and so will Canada. We have tried for years to catch Mr. North.\"\n\n\"Mr. North!\"\n\n\"Yes, and Mr. Frederic and Mr. Benson. They are all Freddy Willet. We will go in his own car.\"\n\nFreddy Willet was smart. He knew when he was caught. He went with the policeman without another word. It was low tide, just as Freddy had planned, and the road to Canada was straight before the two men.\n\nStanding outside the hotel, Grandfather, Henry, Jessie, and Violet saw the car disappear over the stones and gravel to the mainland.\nCHAPTER 15\n\nThe Last Song\n\nWhat excitement there was in Port Elizabeth when the people heard about Freddy! It was hard for them to believe that the Money Man had cheated them. But when Mr. Carter told them that just one of the old valentines was worth many dollars, they changed their minds.\n\n\"Oh, it seems wonderful to talk out loud again,\" said Benny. \"I don't like to have people hiding and listening and whispering. Now I can yell if I want to.\"\n\n\"It's good to talk to you again, Mr. Carter,\" said Henry, \"and no Mr. Wilder-Smith.\"\n\n\"What did you find out at Adams College, John?\" asked Grandfather.\n\n\"They want to buy many of the coins,\" said Mr. Carter. \"And I found two collectors who want the rest. I am going to call on every family and pay them the right price for their coins.\"\n\n\"What a job!\" said Henry.\n\n\"All in a day's work,\" Mr. Carter said with a smile.\n\n\"What about the little things?\" asked Jessie.\n\n\"I will give them all back,\" said Mr. Carter. \"An honest antique dealer will come up here a little later. The people can sell them or keep them, whatever they wish.\"\n\n\"Mr. Willet did give Marie a red necklace,\" said Benny slowly.\n\nMr. Carter smiled. \"That necklace cost him twenty-five cents,\" he said. \"I guess you don't need to worry about that.\"\n\nViolet said suddenly, \"Maybe Miss Gray would like to sell her old books to an honest man. Nobody here reads them.\"\n\n\"Right, Violet,\" said Mr. Carter. \"That's exactly what she told me. But she said a lot more. She thought she was foolish to think anybody would read them. She plans to buy a whole library of colored picture books, stories, and other new books. The books will be for both children and grown-ups.\"\n\n\"Good,\" said Jessie. \"She has changed, and now she's really wonderful.\"\n\nThe Aldens had planned to have a picnic for the school children on the last day. But no\u2014the children wanted to go to school!\n\n\"What children!\" said Violet. \"I never saw any children like them anywhere.\"\n\nAs the children were singing a last song, someone came in the back door. The children could hardly believe their eyes. They clapped without knowing it. They had seen Miss Gray only from a distance, but they knew who she was.\n\nHenry said, \"Miss Gray is going to be your teacher until a new one comes. And she will choose somebody to ring the bell.\"\n\n\"We won't say goodbye,\" said Benny. \"We hate to say goodbye. We never do, we just go.\"\n\nIt was hard, but everyone did just go. Mr. Alden sat outside in the station wagon. Miss Gray's gardener took her home. The Aldens piled into the station wagon, and the school children waved and waved. Then the car rattled over the rocks and stones, and the Aldens were soon on their way home.\n\nWhen they reached home, Benny called up his friend Max.\n\n\"Hello, Max! It's me!\"\n\n\"Oh, hi, Ben! How about adventures? Did you catch a thief or find hidden treasure?\"\n\n\"Both!\" said Benny. \"Come over on your bike and we'll tell you all about the thief and the money hidden in old socks.\"\n\n\"Don't try to fool me, Ben,\" said Max. \"You couldn't find all that on such a dull island.\"\n\n\"Well, we did,\" said Benny. \"It was so exciting that we could hardly pay attention to our schoolteaching.\"\n\n\"Schoolteaching! Are you crazy?\"\n\n\"No,\" said Benny. \"We did more than that. We met a famous author and we discovered two artists.\"\n\n\"I don't believe it,\" said Max. But he always believed Benny Alden.\n\nIt was not long before Max leaned his bike against the front steps. The Aldens took turns telling him about the schoolhouse mystery.\n\n\"It's too much,\" said Max at last. \"I've been up there two summers. And not a thing happened except that we ate our meals and went fishing. Not a thing! But, Mr. Alden, I thought you were going to take the family somewhere else this summer?\"\n\n\"Well, so I am, Max,\" said Grandfather. He winked.\n\n\"Not much time left, sir.\"\n\n\"There's enough. There's all of August and part of September,\" said Mr. Alden.\n\n\"Almost time to go to the moon,\" said Benny.\n**About the Author**\n\nGERTRUDE CHANDLER WARNER discovered when she was teaching that many readers who like an exciting story could find no books that were both easy and fun to read. She decided to try to meet this need, and her first book, _The Boxcar Children,_ quickly proved she had succeeded.\n\nMiss Warner drew on her own experiences to write the mystery. As a child she spent hours watching trains go by on the tracks opposite her family home. She often dreamed about what it would be like to set up housekeeping in a caboose or freight car\u2014the situation the Alden children find themselves in.\n\nWhen Miss Warner received requests for more adventures involving Henry, Jessie, Violet, and Benny Alden, she began additional stories. In each, she chose a special setting and introduced unusual or eccentric characters who liked the unpredictable.\n\nWhile the mystery element is central to each of Miss Warner's books, she never thought of them as strictly juvenile mysteries. She liked to stress the Aldens' independence and resourcefulness and their solid New England devotion to using up and making do. The Aldens go about most of their adventures with as little adult supervision as possible\u2014something else that delights young readers.\n\nMiss Warner lived in Putnam, Connecticut, until her death in 1979. During her lifetime, she received hundreds of letters from girls and boys telling her how much they liked her book. And so she continued the Aldens' adventures, writing a total of nineteen books in the Boxcar Children series.\n_The Boxcar Children Mysteries_\n\nTHE BOXCAR CHILDREN\n\nSURPRISE ISLAND\n\nTHE YELLOW HOUSE MYSTERY\n\nMYSTERY RANCH\n\nMIKE'S MYSTERY\n\nBLUE BAY MYSTERY\n\nTHE WOODSHED MYSTERY\n\nTHE LIGHTHOUSE MYSTERY\n\nMOUNTAIN TOP MYSTERY\n\nSCHOOLHOUSE MYSTERY\n\nCABOOSE MYSTERY\n\nHOUSEBOAT MYSTERY\n\nSNOWBOUND MYSTERY\n\nTREE HOUSE MYSTERY\n\nBICYCLE MYSTERY\n\nMYSTERY IN THE SAND\n\nMYSTERY BEHIND THE WALL\n\nBUS STATION MYSTERY\n\nBENNY UNCOVERS A MYSTERY\n\nTHE HAUNTED CABIN MYSTERY\n\nTHE DESERTED LIBRARY MYSTERY\n\nTHE ANIMAL SHELTER MYSTERY\n\nTHE OLD MOTEL MYSTERY\n\nTHE MYSTERY OF THE HIDDEN \nPAINTING\n\nTHE AMUSEMENT PARK MYSTERY\n\nTHE MYSTERY OF THE MIXED-UP ZOO\n\nTHE CAMP-OUT MYSTERY\n\nTHE MYSTERY GIRL\n\nTHE MYSTERY CRUISE\n\nTHE DISAPPEARING FRIEND MYSTERY\n\nTHE MYSTERY OF THE SINGING GHOST\n\nMYSTERY IN THE SNOW\n\nTHE PIZZA MYSTERY\n\nTHE MYSTERY HORSE\n\nTHE MYSTERY AT THE DOG SHOW\n\nTHE CASTLE MYSTERY\n\nTHE MYSTERY OF THE LOST VILLAGE\n\nTHE MYSTERY ON THE ICE\n\nTHE MYSTERY OF THE PURPLE POOL\n\nTHE GHOST SHIP MYSTERY\n\nTHE MYSTERY IN WASHINGTON, DC\n\nTHE CANOE TRIP MYSTERY\n\nTHE MYSTERY OF THE HIDDEN BEACH\n\nTHE MYSTERY OF THE MISSING CAT\n\nTHE MYSTERY AT SNOWFLAKE INN\n\nTHE MYSTERY ON STAGE\n\nTHE DINOSAUR MYSTERY\n\nTHE MYSTERY OF THE STOLEN MUSIC\n\nTHE MYSTERY AT THE BALL PARK\n\nTHE CHOCOLATE SUNDAE MYSTERY\n\nTHE MYSTERY OF THE HOT \nAIR BALLOON\n\nTHE MYSTERY BOOKSTORE\n\nTHE PILGRIM VILLAGE MYSTERY\n\nTHE MYSTERY OF THE STOLEN \nBOXCAR\n\nTHE MYSTERY IN THE CAVE\n\nTHE MYSTERY ON THE TRAIN\n\nTHE MYSTERY AT THE FAIR\n\nTHE MYSTERY OF THE LOST MINE\n\nTHE GUIDE DOG MYSTERY\n\nTHE HURRICANE MYSTERY\n\nTHE PET SHOP MYSTERY\n\nTHE MYSTERY OF THE SECRET MESSAGE\n\nTHE FIREHOUSE MYSTERY\n\nTHE MYSTERY IN SAN FRANCISCO\n\nTHE NIAGARA FALLS MYSTERY\n\nTHE MYSTERY AT THE ALAMO\n\nTHE OUTER SPACE MYSTERY\n\nTHE SOCCER MYSTERY\n\nTHE MYSTERY IN THE OLD ATTIC\n\nTHE GROWLING BEAR MYSTERY\n\nTHE MYSTERY OF THE LAKE MONSTER\n\nTHE MYSTERY AT PEACOCK HALL\n\nTHE WINDY CITY MYSTERY\n\nTHE BLACK PEARL MYSTERY\n\nTHE CEREAL BOX MYSTERY\n\nTHE PANTHER MYSTERY\n\nTHE MYSTERY OF THE QUEEN'S JEWELS\n\nTHE STOLEN SWORD MYSTERY\n\nTHE BASKETBALL MYSTERY\n\nTHE MOVIE STAR MYSTERY\n\nTHE MYSTERY OF THE PIRATE'S MAP\n\nTHE GHOST TOWN MYSTERY\n\nTHE MYSTERY OF THE BLACK RAVEN\n\nTHE MYSTERY IN THE MALL\n\nTHE MYSTERY IN NEW YORK\n\nTHE GYMNASTICS MYSTERY\n\nTHE POISON FROG MYSTERY\n\nTHE MYSTERY OF THE EMPTY SAFE\n\nTHE HOME RUN MYSTERY\n\nTHE GREAT BICYCLE RACE MYSTERY\n\nTHE MYSTERY OF THE WILD PONIES\n\nTHE MYSTERY IN THE COMPUTER \nGAME\n\nTHE MYSTERY AT THE CROOKED \nHOUSE\n\nTHE HOCKEY MYSTERY\n\nTHE MYSTERY OF THE MIDNIGHT DOG\n\nTHE MYSTERY OF THE SCREECH OWL\n\nTHE SUMMER CAMP MYSTERY\n\nTHE COPYCAT MYSTERY\n\nTHE HAUNTED CLOCK TOWER \nMYSTERY\n\nTHE MYSTERY OF THE TIGER'S EYE\n\nTHE DISAPPEARING STAIRCASE \nMYSTERY\n\nTHE MYSTERY ON BLIZZARD \nMOUNTAIN\n\nTHE MYSTERY OF THE SPIDER'S CLUE\n\nTHE CANDY FACTORY MYSTERY\n\nTHE MYSTERY OF THE MUMMY'S \nCURSE\n\nTHE MYSTERY OF THE STAR RUBY\n\nTHE STUFFED BEAR MYSTERY\n\nTHE MYSTERY OF ALLIGATOR SWAMP\n\nTHE MYSTERY AT SKELETON POINT\n\nTHE TATTLETALE MYSTERY\n\nTHE COMIC BOOK MYSTERY\n\nTHE GREAT SHARK MYSTERY\n\nTHE ICE CREAM MYSTERY\n\nTHE MIDNIGHT MYSTERY\n\nTHE MYSTERY IN THE FORTUNE \nCOOKIE\n\nTHE BLACK WIDOW SPIDER MYSTERY\n\nTHE RADIO MYSTERY\n\nTHE MYSTERY OF THE RUNAWAY \nGHOST\n\nTHE FINDERS KEEPERS MYSTERY\n\nTHE MYSTERY OF THE HAUNTED \nBOXCAR\n\nTHE CLUE IN THE CORN MAZE\n\nTHE GHOST OF THE CHATTERING \nBONES\n\nTHE SWORD OF THE SILVER KNIGHT\n\nTHE GAME STORE MYSTERY\n\nTHE MYSTERY OF THE ORPHAN TRAIN\n\nTHE VANISHING PASSENGER\n\nTHE GIANT YO-YO MYSTERY\n\nTHE CREATURE IN OGOPOGO LAKE\n\nTHE ROCK 'N' ROLL MYSTERY\n\nTHE SECRET OF THE MASK\n\nTHE SEATTLE PUZZLE\n\nTHE GHOST IN THE FIRST ROW\n\nTHE BOX THAT WATCH FOUND\n\nA HORSE NAMED DRAGON\n\nTHE GREAT DETECTIVE RACE\n\nTHE GHOST AT THE DRIVE-IN MOVIE\n\nTHE MYSTERY OF THE TRAVELING \nTOMATOES\n\nAll rights reserved under International and Pan-American Copyright Conventions. By payment of the required fees, you have been granted the non-exclusive, non-transferable right to access and read the text of this ebook onscreen. No part of this text may be reproduced, transmitted, downloaded, decompiled, reverse engineered, or stored in or introduced into any information storage and retrieval system, in any form or by any means, whether electronic or mechanical, now known or hereinafter invented, without the express written permission of the publisher.\n\nThis is a work of fiction. Names, characters, places, and incidents either are the product of the author's imagination or are used fictitiously. Any resemblance to actual persons, living or dead, businesses, companies, events, or locales is entirely coincidental.\n\ncopyright \u00a9 1965 by Albert Whitman & Company\n\nISBN: 978-1-4532-0794-9\n\nThis 2010 edition distributed by Open Road Integrated Media \n180 Varick Street \nNew York, NY 10014 \nwww.openroadmedia.com\n\n","meta":{"redpajama_set_name":"RedPajamaBook"}}