Handbook of Vitamins, Fourth Edition

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Handbook of

VITAMINS

F O U RT H

E D I T I O N

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CRC Press is an imprint of the

Taylor & Francis Group, an informa business Boca Raton London New York

Handbook of

VITAMINS

F O U RT H E D I T I O N

Edited by

Janos Zempleni Robert B. Rucker Donald B. McCormick

John W. Suttie

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Library of Congress Cataloging-in-Publication Data Handbook of vitamins / editors, Robert B. Rucker ... [et al.]. -- 4th ed.

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1. Vitamins. 2. Vitamins in human nutrition. I. Rucker, Robert B.

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Preface

In keeping with the tradition of previous editions, the fourth edition of the Handbook of Vitaminswas assembled to update and provide contemporary perspectives on dietary acces- sory factors commonly classified as vitamins. One of the challenges in assembling this volume was to maintain the clinical focus of previous editions, while addressing important concepts that have evolved in recent years owing to the advances in molecular and cellular biology as well as those in analytical chemistry and nanotechnology. The reader will find comprehensive summaries that focus on chemical, physiological, and nutritional relationships and highlights of newly described and identified functions for all the recognized vitamins. Our goal was to assemble the best currently available reference text on vitamins for an audience ranging from basic scientists to clinicians to advanced students and educators with a commitment to better understanding vitamin function.

As examples, apparent vitamin-dependent modifications that are important to epigenetic events and genomic stability are described, as well as new information on the role and importance of maintaining optimal vitamin status for antioxidant and anti-inflammatory defense. Important analytical advances in vitamin analysis and assessment are discussed in a chapter dealing with accelerated mass spectrometry (AMS) applications. Recent AMS applications have provided the basis for studies of vitamin metabolism and turnover in humans at levels corresponding to physiological concentrations and fluxes. It is also important to underscore that much of the interest in vitamins stems from an appreciation that there remains regrettably sizable populations at risk for vitamin deficiencies. In this regard, classic examples are included along with examples of vitamin-related polymorphisms and genetic factors that influence the relative needs for given vitamins.

This volume is written by a group of authors who have made major contributions to our understanding of vitamins. Over half of the authors are new to this series; each chapter is written by individuals who have made clearly important contributions in their respective areas of research as judged by the scientific impact of their work. In addition, Dr. Janos Zempleni joins the group of editors who assembled the third edition. Dr. Zempleni adds a molecular biology perspective to complement the biochemical and physiological expertise of the other editors. We also wish to note that we miss the input of Dr. Lawrence Machlin, a renowned researcher on vitamin E, who was sole editor of the first two editions in this series and who died shortly after the release of the third edition. We know that he would be pleased with the progress and advances in vitamin research summarized in the fourth edition.

This volume comes at an important time and represents a new treatment of this topic.

With the possible exception of the earlier days of vitamin discovery, this period of vitamin research is particularly exciting because of the newly identified roles of vitamins in cellular and organismal regulation and their obvious and continuing importance in health and disease.

Janos Zempleni Robert B. Rucker Donald B. McCormick John W. Suttie

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Editors

Janos Zemplenireceived his undergraduate and graduate training in nutrition at the Univer- sity of Giessen in Germany. He received postdoctoral training in nutrition, biochemistry, and molecular biology at the University of Innsbruck (Austria), Emory University, and Arkansas Children’s Hospital Research Institute. Janos Zempleni is currently an assistant professor of molecular nutrition at the University of Nebraska at Lincoln. He has published more than 100 manuscripts and books and is the recipient of the 2006 Mead Johnson Award by the American Society for Nutrition. Zempleni’s research focuses on roles of the vitamin biotin in chromatin structure.

Robert B. Rucker received his PhD in biochemistry from Purdue University in 1968 and worked for two years as a postdoctoral fellow at the University of Missouri, before joining the faculty of nutrition at the University of California (UC), Davis, in 1970. He currently holds the title of distinguished professor. He serves as vice chair of the department of nutrition in the College of Agriculture and Environmental Sciences and holds an appointment in endo- crinology, nutrition, and vascular medicine, department of internal medicine, UC Davis, School of Medicine.

Dr. Rucker’s research focuses on cofactor function. His current research addresses problems associated with extracellular matrix assembly, the role of copper in early growth and development, and the physiological roles of quinone cofactors derived from tyrosine, such as pyrroloquinoline quinone.

Honors and activities include serving as a past president, American Society for Nutrition;

appointment as a fellow in the American Association for the Advancement of Science and the American Society for Nutrition; service as chair or cochairperson for FASEB Summer and Gordon Conferences; service on Program and Executive Committees for American Society for Nutrition and FASEB, as well as service on committees of the Society for Experimental Biology and Medicine. He also currently serves as senior associate editor,American Journal of Clinical Nutrition and is a past editorial board member of theJournal of Nutrition, Experi- mental Biology and Medicine, Nutrition Research, and theAnnual Review of Nutrition. He is a past recipient of UC Davis and the American Society for Nutrition Research awards.

Donald B. McCormickearned his bachelor’s degree (chemistry and math, 1953) and doctorate (biochemistry, 1958) at Vanderbilt University in Nashville, Tennessee. His dissertation was on pentose and pentitol metabolism. He was then an NIH postdoctoral fellow (1958–1960) at the University of California-Berkeley, where his research was on enzymes that convert vitamin B6 to the coenzyme pyridoxal phosphate. He has had sabbatics in the chemistry departments in Basel University (Switzerland) and in the University of Arizona, and in the biochemistry department in Wageningen (Netherlands).

Dr. McCormick’s academic appointments have been at Cornell University (1960–1978) in Ithaca, New York, where he became the Liberty Hyde Bailey Professor of nutritional biochemistry and at Emory University (1979–present) in Atlanta, Georgia, where he served as the Fuller E. Callaway professor and chairman of the department of biochemistry and the executive associate dean for basic sciences in the school of medicine. His research has been on

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cofactors with emphases on chemistry, biochemistry, and nutrition of vitamins (especially B6, riboflavin, biotin, and lipoate), coenzymes, and metal ions.

Dr. McCormick has been a consultant and served on numerous committees that include service for NIH, NCI, FASEB, IOM=NAS, NASA, FAO=WHO, and several organizing groups for international symposia on cofactors. He has been on the editorial boards of several journals of biochemistry and nutrition, and he has served as editor of volumes on vitamins and coenzymes in theMethods in Enzymologyseries,Vitamins and Hormones, theHandbook of Vitamins, and theAnnual Review of Nutrition.

Dr. McCormick is a member of numerous scientific societies, including those in biochemistry and nutrition and has received such honors as a Westinghouse Science Scholarship, Guggenheim Fellowship, Wellcome Visiting Professorships (University of Florida, Medical College of Pennsylvania), and named visiting professorships (University of California-Davis, University of Missouri). He has received awards from the American Institute of Nutrition (Mead Johnson, Osborne and Mendel) and Bristol-Myers Squibb=Mead Johnson Award for Distinguished Achievement in Nutrition Research. He is a fellow of AAAS and a fellow of the American Society of Nutritional Sciences.

John W. Suttie is the Katherine Berns Van Donk Steenbock professor emeritus in the department of biochemistry and former chair of the department of nutritional sciences at the University of Wisconsin-Madison. He has broad expertise in biochemistry and human nutrition. Dr. Suttie received his BS, MS, and PhD degrees from the University of Wisconsin-Madison. He was an NIH postdoctoral fellow at the National Institute for Medical Research, Mill Hill, England, before joining the University of Wisconsin faculty.

His research activities are directed toward the metabolism, mechanism of action, and nutritional significance of vitamin K. Dr. Suttie has served as president of the American Society for Nutritional Sciences (ASNS) and recently resigned his position as editor-in-chief of The Journal of Nutrition. He has received the Mead Johnson Award, the Osborne and Mendel Award, and the Conrad Elvehjem Award of the ASNS, the ARS Atwater Lectureship, and the Bristol Myers-Squibb Award for Distinguished Achievement in Nutrition Research. In 1996 Dr. Suttie was elected to the National Academy of Sciences. He has served as chairman of the Board of Experimental Biology, as president of the Federation of American Societies for Experimental Biology (FASEB), and as a member of the NRC’s Board on Agriculture and Natural Resources, the FDA Blood Products Advisory Committee, and the American Heart Association Nutrition Committee. He presently serves on the Public Policy Committees of the ASNS and the American Society for Biochemistry and Molecular Biology (ASBMB), the USDA=NAREEE Advisory Board, the ILSI Food, Nutrition and Safety Committee, and the Food and Nutrition Board of the Institute of Medicine.

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Contributors

Lynn B. Bailey

Department of Food Science and Human Nutrition

University of Florida Gainesville, Florida Susan I. Barr

Department of Food, Nutrition, and Health

University of British Columbia Vancouver, British Columbia, Canada

Chris J. Bates

MRC Human Nutrition Research Elsie Widdowson Laboratory Cambridge, United Kingdom

Kathryn Bauerly

Department of Nutrition University of California Davis, California

Linda K. Buckles

Department of Biochemistry and Molecular Biology

University of Nebraska Medical Center Omaha, Nebraska

Betty Jane Burri

Department of Nutrition University of California Davis, California Judith K. Christman

Department of Biochemistry and Molecular Biology and UNMC=Eppley

Cancer Center

University of Nebraska Medical Center Omaha, Nebraska

Andrew J. Clifford Department of Nutrition University of California Davis, California

Krishnamurti Dakshinamurti

Department of Biochemistry and Medical Genetics

University of Manitoba Winnipeg, Manitoba, Canada

Shyamala Dakshinamurti

Department of Pediatrics and Physiology University of Manitoba

Winnipeg, Manitoba, Canada

Timothy A. Garrow

Department of Food Science and Human Nutrition

University of Illinois at Urbana-Champaign Urbana, Illinois

Ralph Green

Department of Pathology and Laboratory Medicine University of California Davis, California

Earl H. Harrison

Department of Human Nutrition The Ohio State University Columbus, Ohio

Helen L. Henry

Department of Biochemistry University of California Riverside, California

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Carol S. Johnston Department of Nutrition Arizona State University Mesa, Arizona

James B. Kirkland

Department of Human Health and Nutritional Sciences

University of Guelph Guelph, Ontario, Canada Donald B. McCormick Department of Biochemistry Emory University

Atlanta, Georgia Joshua W. Miller

Department of Pathology and Laboratory Medicine University of California Davis, California Donald M. Mock

Department of Biochemistry and Molecular Biology

University of Arkansas for Medical Sciences Little Rock, Arkansas

Fabiana Fonseca de Moura Department of Nutrition University of California Davis, California Suzanne P. Murphy

Cancer Research Center of Hawaii University of Hawaii

Honolulu, Hawaii Anthony W. Norman

Department of Biochemistry University of California Riverside, California

Richard S. Rivlin Department of Medicine Weill Medical College

of Cornell University New York, New York

A. Catharine Ross

Department of Nutritional Sciences Pennsylvania State University University Park, Pennsylvania

Robert B. Rucker Department of Nutrition University of California Davis, California

Francene M. Steinberg Department of Nutrition University of California Davis, California John W. Suttie

Department of Biochemistry University of Wisconsin Madison, Wisconsin Maret G. Traber

Department of Nutrition and Exercise Sciences, Linus Pauling Institute Oregon State University

Corvallis, Oregon

Janos Zempleni

Department of Nutrition and Health Sciences

University of Nebraska Lincoln, Nebraska

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1 Vitamin A: Nutritional Aspects of Retinoids and Carotenoids

A. Catharine Ross and Earl H. Harrison

CONTENTS

Introduction ... 2

Nutritional Aspects of Vitamin A and Carotenoids... 2

Historical ... 2

Definitions of Vitamin A, Retinoids, and Carotenoids ... 3

Properties, Nutritional Equivalency, and Recommended Intakes ... 3

Properties of Nutritionally Important Retinoids ... 3

Properties of Nutritionally Important Carotenoids ... 7

Nutritional Equivalency... 8

Transport and Metabolism... 10

Transport and Binding Proteins ... 10

Retinol-Binding Protein ... 12

Albumin ... 13

Lipoproteins... 13

Intracellular Retinoid-Binding Proteins ... 13

Nuclear Retinoid Receptors... 14

Intestinal Metabolism ... 15

Conversion of Provitamin A Carotenoids to Retinoids... 15

Intestinal Absorption of Vitamin A... 17

Reesterification, Incorporation into Chylomicrons, and Lymphatic Secretion ... 18

Hepatic Uptake, Storage, and Release of Vitamin A ... 18

Hepatic Uptake ... 18

Extrahepatic Uptake ... 19

Storage ... 19

Release ... 20

Plasma Transport ... 20

Plasma Retinol ... 20

Conditions in Which Plasma Retinol May Be Low... 22

Other Retinoids in Plasma ... 23

Plasma Carotenoids ... 23

Plasma Retinol Kinetics and Recycling ... 23

Intracellular Retinoid Metabolism ... 23

Hydrolysis ... 23

Oxidation–Reduction and Irreversible Oxidation Reactions ... 24

Formation of More Polar Retinoids ... 24

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Conjugation ... 25

Isomerization ... 25

Vitamin A and Public Health... 25

Prevention of Xerophthalmia ... 25

Actions of Vitamin A in the Eye... 26

Morbidity and Mortality ... 27

Subclinical Deficiency ... 27

Immune System Changes ... 27

Medical Uses of Retinoids... 28

Dermatology ... 28

Acute Promyelocytic Leukemia ... 29

Prevention of Hypervitaminosis A of Nutritional Origin ... 29

Excessive Consumption ofb-Carotene ... 29

References ... 30

INTRODUCTION

Vitamin A (retinol) is an essential micronutrient for all vertebrates. It is required for normal vision, reproduction, embryonic development, cell and tissue differentiation, and immune function. Many aspects of the transport and metabolism of vitamin A, as well as its functions, are well conserved among species. Dietary vitamin A is ingested in two main forms—preformed vitamin A (retinyl esters and retinol) and provitamin A carotenoids (b-carotene, a-carotene, and b-cryptoxanthin)—although the proportion of vitamin A obtained from each of these form varies considerably among animal species and among individual human diets. These precursors serve as substrates for the biosynthesis of two essential metabolites of vitamin A: 11-cis-retinal, required for vision, and all-trans-retinoic acid, required for cell differentiation and the regulation of gene transcription in nearly all tissues.

Research on vitamin A now spans nine decades. Over 34,000 citations to vitamin A, 7,000 tob-carotene, and 20,000 to retinoic acid can be found in the National Library of Medicine’s PubMed database [1], covering topics related to nutrition, biochemistry, molecular and cell biology, physiology, toxicology, public health, and medical therapy. Besides the naturally occurring forms of vitamin A indicated earlier, numerous structural analogs have been synthesized. Some retinoids have become widely used as therapeutic agents, particularly in the treatment of dermatological diseases and certain cancers.

In this chapter, we focus first on vitamin A from a nutritional perspective, addressing its chemical forms and properties, the nutritional equivalency of compounds that provide vitamin A activity, and current dietary recommendations. We then cover the metabolism of carotenoids and vitamin A. Finally, we provide a brief discussion of the key uses of vitamin A and retinoids in public health and medicine, referring to their benefits as well as some of the adverse effects caused by ingesting excessive amounts of this highly potent group of compounds.

NUTRITIONAL ASPECTS OF VITAMIN A AND CAROTENOIDS HISTORICAL

Vitamin A was discovered in the early 1900s by McCollum and colleagues at the University of Wisconsin and independently by Osborne and Mendel at Yale. Both groups were studying the effects of diets made from purified protein and carbohydrate sources, such as casein and rice flour, on the growth and survival of young rats. They observed that growth ceased and the animals died unless the diet was supplemented with butter, fish oils, or a quantitatively

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minor ether-soluble fraction extracted from these substances, from milk, or from meats. The unknown substance was then called ‘‘fat-soluble A.’’ Not long thereafter, it was recognized that the yellow carotenes present in plant extracts had similar nutritional properties, and it was postulated that this carotenoid fraction could give rise through metabolism to the bioactive form of fat-soluble A, now called vitamin A, in animal tissues. This was shown to be correct afterb-carotene and retinol were isolated and characterized, and it was shown that dietary b-carotene gives rise to retinol in animal tissues. Within the first few decades of vitamin A research, vitamin A deficiency was shown to cause several specific disease conditions, including xerophthalmia; squamous metaplasia of epithelial and mucosal tissues;

increased susceptibility to infections; and abnormalities of reproduction. Each of these seminal discoveries paved the way for many subsequent investigations that have greatly expanded our knowledge about vitamin A. Although the discoveries made in the early 1900s may now seem long ago, it is interesting to note, as reviewed by Wolf [2], that physicians in ancient Egypt, around 1500BC, were already using the liver of ox, a very rich source of vitamin A, to cure what is now referred to as night blindness.

DEFINITIONS OFVITAMINA, RETINOIDS,ANDCAROTENOIDS

Vitamin A is a generic term that refers to compounds with the biological activity of retinol.

These include the provitamin A carotenoids, principally b-carotene, a-carotene, and b-cryptoxanthin, which are provided in the diet by green and yellow or orange vegetables and some fruits and preformed vitamin A, namely retinyl esters and retinol itself, present in foods of animal origin, mainly in organ meats such as liver, other meats, eggs, and dairy products.

The term retinoid was coined to describe synthetically produced structural analogs of the naturally occurring vitamin A family, but the term is now used for natural as well as synthetic compounds [3]. Retinoids and carotenoids are defined based on molecular structure. According to the Joint Commission on Biochemical Nomenclature of the International Union of Pure and Applied Chemistry and International Union of Biochemistry and Molecular Biology (IUPAC–IUB), retinoids are ‘‘a class of compounds consisting of four isoprenoid units joined in a head-to-tail manner’’ [4]. All-trans-retinol is the parent molecule of this family.

The retinoid molecule can be divided into three parts: a trimethylated cyclohexene ring, a conjugated tetraene side chain, and a polar carbon–oxygen functional group. Additional examples of key retinoids and structural subgroups, a history of the naming of these compounds, and current nomenclature of retinoids are available online [4].

The IUPAC–IUB defines carotenoids [5] as ‘‘a class of hydrocarbons (carotenes) and their oxygenated derivatives (xanthophylls) consisting of eight isoprenoid units joined in such a manner that the arrangement of isoprenoid units is reversed at the center of the molecule.’’ All carotenoids may be formally derived from the acyclic C40H56structure that has a long central chain of conjugated double bonds, by (i) hydrogenation, (ii) dehydrogenation, (iii) cyclization, or (iv) oxidation, or any combination of these processes.

PROPERTIES, NUTRITIONALEQUIVALENCY,ANDRECOMMENDEDINTAKES

Properties of Nutritionally Important Retinoids

Nutritionally important retinoids and some of their metabolites are illustrated in Figure 1.1.

The conventional numbering of carbon atoms in the retinoid molecule is shown in the structure of all-trans-retinol in Figure 1.1a. Due to the conjugated double-bond structure of retinoids and carotenoids, these molecules possess very characteristic UV or visible light absorption spectra that are useful in their identification and quantification [6,7].

Vitamin A: Nutritional Aspects of Retinoids and Carotenoids 3

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Furr and colleagues have summarized the light absorption properties of over 50 retinoids [8] and nutritionally active carotenoids [9]. Some of the properties of several retinoids related to dietary vitamin A are summarized in Table 1.1.

Retinoids tend to be most stable in the all-transconfiguration. Retinol is most often present in tissues in esterified form, where the fatty acyl group is usually palmitate with lesser amounts of stearate and oleate esters. Esterification protects the hydroxyl group from oxidation and significantly alters the molecule’s physical properties (Table 1.1). Retinyl esters in tissues are usually admixed with triglycerides and other neutral lipids, including the antioxidant a-tocopherol. Retinyl esters are the major form of vitamin A in the body as a whole and the predominant form (often more than 95%) in chylomicrons, cellular lipid droplets, and milk fat globules. Thus, they are also the major form in foods of animal origin. Retinol contained in nutritional supplements and fortified foods is usually produced synthetically and is stabil- ized by formation of the acetate, propionate, or palmitate ester. Minor forms of vitamin A may be present in the diet, such as vitamin A₂(3,4-didehydroretinol) (Figure 1.1b), which is present in the oils of fresh-water fish and serves as a visual pigment in these species [10].

Several retinoids that are crucial for function are either absent or insignificant in the diet, but are generated metabolically from dietary precursors. Due to the potential for the double bonds of the molecules in the vitamin A family to exist in either thetrans- orcis-isomeric form, a large number of retinoid isomers are possible. The terminal functional group can be in one of several oxidation states, varying from hydrocarbon, as in anhydroretinol, to alcohol, aldehyde, and carboxylic acid. Many of these forms may be further modified through the addition of substituents to the ring, side chain, or end group. These changes in molecular structure significantly alter the physical properties of the molecules in the vitamin A family and may markedly affect their biological activity. While dozens of natural retinoids

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20 19

18 17

16 15

14 13 12 11 10 9 8 7 6 45 3

2 CH₂OH

All-trans-retinol (a)

CHO 11-cis-Retinal

(d)

COOH

(e) All-trans-retinoic acid

9-cis-Retinoic acid

COOH (f)

OH OH

OH

O O

Retinoyl-β-glucuronide

COOH O

(g) CH₂OH

3,4-Didehydroretinol (b)

CHO

(c)

All-trans-retinal

FIGURE 1.1 Nutritionally important retinoids and major metabolites. The conventional numbering system for retinoids is shown for all-trans-retinol, the parent molecule of the retinoid family.

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TABLE 1.1

Properties of Vitamin A Compounds and Their Metabolites

Compound

Formula and

Molecular Mass Solvents in Which Soluble Physical State

Wavelength of Maximum Absorption,l_max

Molar Extinction Coefficient,«, in Indicated Solvent All-trans-retinol

(vitamin A₁)

C₂₀H₃₀O 286.44

Absolute alcohol, methanol, chloroform, petroleum ether, fats, and oils

Crystalline solid 324–325 52,770 in ethanol

51,770 in hexane 3,4-Didehydroretinol

(vitamin A2)

C₂₀H₂₈O 284.44

Alcohols, ether Crystalline solid 350 41,320 in ethanol

Retinyl palmitate C₃₆H₆₀O₂ 528

Hexane, ether, dimethylsulfoxide Viscous oil 325 49,260 in ethanol

All-trans-retinal C₂₀H₂₈O;

284.44

Ethanol, chloroform, cyclohexane, petroleum ether, oils

Crystalline solid 383

368

42,880 in ethanol 48,000 in hexane

11-cis-Retinal C₂₀H₂₈O;

284.44

Ethanol, chloroform, cyclohexane, petroleum ether, oils

Crystalline solid 380

365

24,935 in ethanol 26,360 in hexane All-trans-retinoic acid C₂₀H₂₈O₂;

300.4

Ethanol, methanol, isopropanol, dimethyl sulfoxide

Crystalline solid 350 45,300 in ethanol

9-cis-Retinoic acid C₂₀H₂₈O₂; 300.4

13-cis-Retinoic acid C₂₀H₂₈O₂; 300.4

Retinoyl-b-glucuronide C₂₆H₃₆O₈ Aqueous methanol Crystalline solid 360 50,700 in methanol

476.1 4-Oxo-all-trans-retinoic acid C₂₀H₂₆O₃;

314.4

Ethanol, methanol, dimethyl sulfoxide

Note: For additional absorption spectrum data, see Furr et al. [8,9].

VitaminA:NutritionalAspectsofRetinoidsandCarotenoids5

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have been isolated, the molecules illustrated in Figure 1.1 and Figure 1.2 are the principal retinoids and carotenoids, respectively, of nutritional importance, and thus are the main focus of this chapter. Nevertheless, it is important to recognize that numerous minor metabolites can be formed at several branch points as retinol and the provitamin A carotenoids are metabolized.

All-trans-retinal (Figure 1.1c) is the immediate product of the central cleavage of b-carotene as well as an intermediate in the oxidative metabolism of retinol to all-trans- retinoic acid. The 11-cisisomer of retinal (Figure 1.1d) is formed in the retina and most of it is covalently bound to one of the visual pigments, rhodopsin in rods or iodopsin in cones. The aldehyde functional group of 11-cis-retinal combines with specific lysine residues in these proteins as a Schiff’s base.

All-trans-retinoic acid (Figure 1.1e) is the most bioactive form of vitamin A. When fed to vitamin A-deficient animals, retinoic acid restores growth and tissue differentiation and prevents mortality, indicating that this form alone, or metabolites made from it, is able to support nearly all of the functions attributed to vitamin A. A notable exception is vision, which is not restored by retinoic acid because retinoic acid cannot be reduced to retinal in vivo. Retinoic acid is also the most potent natural ligand of the retinoid receptors, RAR and RXR (described later), as demonstrated in transactivation assays. Severalcisisomers of retinoic acid have been studied rather extensively, but they are still somewhat enigmatic as to origin and function. 9-cis-Retinoic acid (Figure 1.1f ) is capable of binding to the nuclear receptors and may be a principal ligand of the RXR. 13-cis-Retinoic acid is present in plasma, often at a concentration similar to all-trans-retinoic acid, and its therapeutic effects are well demonstrated (see the section Dermatology), but it is not known to be a high-affinity ligand for the nuclear retinoid receptors. It is possible that 13-cis-retinoic acid acts as a relatively stable precursor or prodrug that can be metabolized to all-trans-retinoic acid or perhaps

Lycopene (a)

(b)

(c)

(d)

(e)

Lutein HO

OH

HO

All-trans-β-carotene

β-Cryptoxanthin α-Carotene

BCO BCO-2

15 15⬘

10⬘ 9⬘

FIGURE 1.2 Nutritionally important carotenoids. (a) Lycopene, a nonprovitamin A carotene; (b) all- trans-bb⁰-carotene; arrows indicate sites of cleavage byb-carotene monooxygenase, BCO, and BCO-2;

(c) all-trans(a,b⁰) carotene; (d) lutein, a nonprovitamin A xanthophyll; (e)b-cryptoxanthin.

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another bioactive metabolite. Di-cis isomers of retinoic acid also have been detected in plasma, further illustrating the complex mix of retinoids in biological systems.

Retinoids that are more polar than retinol or retinoic acid are formed through oxidative metabolism of the ionone ring and side chain. These include 4-hydroxy, 4-oxo, 18-hydroxy, and 5,6-epoxy derivatives of retinoic acid, and similar modifications of other retinoids.

Conjugation of the lipophilic retinoids with very polar molecules such as glucuronic acid renders them water-soluble. As an example, retinoyl-b-glucuronide (Figure 1.1g) is present as a significant metabolite in the plasma and bile. Although some of these polar retinoids are active in some assays, most of the more polar and water-soluble retinoids appear to result from phase I and phase II metabolic or detoxification reactions. They may, however, be deconjugated to some extent and recycled as the free compound.

Many retinoids have been chemically synthesized. A large number of structural analogs have been synthesized and tested for their potential as drugs that may be able to induce cell differentiation. In the field of dermatology, 13-cis-retinoic acid (isotretinoin) and the 1,2,4-trimethyl-3-methoxyphenyl analog of retinoic acid (acetretin) are prominent differentiation-promoting and keratinolytic compounds. Other retinoids have been developed as agents able to selectively bind to and activate only a subset of retinoid receptors. Some synthetic retinoids show none of the biological activities of vitamin A, but still are related in terms of structure. Retinoids that show selectivity in binding to the RXR receptors rather than RAR, sometimes referred to as rexinoids, also have been synthesized [11,12].

As analytical methods have improved, additional retinoids have been discovered. Retinol metabolites have been identified in which the terminal group is dehydrated (anhydroretinol);

the 13,14 position is saturated or hydroxylated; or the double bonds of the retinoid side chain are flipped back into a form known as a retro retinoid [4]. These retinoids tend to be quantitatively minor or limited in their distribution, and their significance is still uncertain.

Properties of Nutritionally Important Carotenoids

Carotenoids are synthesized by photosynthetic plants and some algae and bacteria, but not by animal tissues. The initial stage of biosynthesis results in the formation of the basic poly- isoprenoid structure of the hydrocarbon lycopene (Figure 1.2a), a 40-carbon linear structure with an extended system of 13 conjugated double bonds. Further biosynthetic reactions result in the cyclization of the ends of this linear molecule to form eithera- orb-ionone rings. The carotene group of carotenoids comprises hydrocarbon carotenoids in which the ionone rings bear no other substituents. The addition of oxygen to the carotene structure results in the formation of the xanthophyll group of carotenoids. The double bonds in most carotenoids are present in the more stable all-transconfiguration, althoughcisisomers can exist. Carotenoids are widespread in nature and are responsible for the yellow, orange, red, and purple colors of many fruits, flowers, birds, insects, and marine animals. In photosynthetic plants, carotenoids improve the efficiency of photosynthesis, while they are important to insects, birds, animals, and humans for their colorful and attractive sensory properties.

Although some 600 carotenoids have been isolated from natural sources, only about one-tenth of them are present in human diets [13], and only about 20 have been detected in blood and tissues. b-carotene (Figure 1.2b), a-carotene (Figure 1.2c), lycopene, lutein (Figure 1.2d), and b-cryptoxanthin (Figure 1.2e) are the five most prominent carotenoids in the human body. However, only b-carotene, a-carotene, and b-cryptoxanthin possess significant vitamin A activity. To be active as vitamin A, a carotenoid must have an unsubstituted b-ionone ring and an unsaturated hydrocarbon chain. The bioactivity of all-trans-b-carotene, with two symmetrical halves, is about twice that of an equal amount ofa-carotene andb-cryptoxanthin, in which only one unsubstitutedb-ionone ring is present.

Even though lycopene, lutein, and zeaxanthin can be relatively abundant in the diet and

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humans can absorb them across the intestine into plasma, they lack vitamin A activity because of the absence of a closed unsubstituted ring. In plants, provitamin A carotenoids are embedded in complex cellular structures such as the cellulose-containing matrix of chloroplasts or pigment-containing chromoplasts. Their association with these matrices of plants is a significant factor affecting the efficiency of their digestion, release, and bioavailability [14,15].

Nutritional Equivalency Units of Activity

Different forms of vitamin A differ in their biological activity per unit of mass. For this reason, the bioactivity of vitamin A in the diet is expressed in equivalents (with respect to all-trans- retinol) rather than in mass units. Several different units have been adopted over time and most of them are still used in some capacity. In 1967, the World Health Organization (WHO)=FAO recommended replacing the international unit (IU), a bioactivity unit, with the retinol equivalent (RE); 1 RE was defined as 1mg of all-trans-retinol or 6mg ofb-carotene in foods [16]. In 2001, the U.S. Institute of Medicine recommended replacing the RE with the retinol activity equivalent (RAE) and redefining the average equivalency values for carotenoids in foods in comparison with retinol [15]. These sequential changes in units were in large part a response to better knowledge of the efficiency of utilization of carotenoids [15,16]; 1mg RAE is defined as 1mg of all-trans-retinol, and therefore is the same as 1mg RE. Both are equal to 3.3 IU of retinol. The equivalency of provitamin A carotenoids and retinol in the RAE system is illustrated in Figure 1.3. These currently adopted conversion factors are necessarily approx- imations. Because the RAE terminology is not yet fully used, the vitamin A values in some food tables, food labels, and supplements are still expressed in RE or IU.

Another term, daily value (% DV), is used in food labeling. It is not a true unit of activity, but provides an indication of the percentage of the recommended dietary allowance (RDA)*

present in one serving of a given food.

Form consumed Equivalency

after bioconversion Dietary or supplemental

vitamin A (1µg) Retinol (1µg)

Supplemental β-carotene (pure, in oily solution) (2 µg)

Retinol (1µg)

Dietary β-carotene (in food matrix) (12 µg)

Retinol (1µg)

Dietary α-carotene or β-cryptoxanthin (in food matrix) (24 µg)

Retinol (1µg)

FIGURE 1.3 Approximate nutritional equivalency of dietary provitamin A carotenoids and retinol, as revised in 2001. The values shown are used to convert the contents of carotenoids in supplements and foods to equivalent amounts of dietary retinol. (From Institute of Medicine,Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc,National Academy Press, Washington, 2002, pp. 8–9.)

* Based on the percentage of the RDA of a nutrient, for a person consuming a 2000 kcal diet.

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Recommended Intakes

The conceptual framework and values for dietary reference intakes (DRI) are discussed by Murphy and Barr in Chapter 18. DRI values for vitamin A, established in 2001 [15], are summarized in Table 1.2. A tolerable upper intake level (UL, see Chapter 18) for vitamin A was defined at this time [15]; similarly, a safe upper level for vitamin A andb-carotene has been defined in the United Kingdom [17]. It is important to note that the UL applies only to chronic intakes of preformed vitamin A (not carotenoids, which do not cause adverse effects).

For several life stage groups, the UL values are less than three times higher than the RDA.

Dietary Sources

Detailed tables of the vitamin A contents of foods can be found in several reference sources and online resources. A database for carotenoids in foods is available online [18]. It should be noted that nutrient databases provide only approximate values. The contents of vitamin A and carotenoids in foods can vary substantially with crop variety or cultivar, the environment in which it is grown, and with processing and storage conditions [19,20].

Foods in the U.S. diet with the highest concentrations of preformed vitamin A are liver (4–20 mg retinol=100 g) and fortified foods such as powdered breakfast drinks (3–6 mg=100 g), ready-to-eat cereals (0.7–1.5 mg=100 g), and margarines (~0.8 mg=100 g) [18]. The highest

TABLE 1.2

Recommended Dietary Allowances (RDA) and Upper Level (UL) Values for Vitamin A by Life Stage Group

Life Stage Group RDA (mg=day)^a UL (mg=day)^b Infants

0–12 months 400^c 600

Children

1–3 years 300 600

4–8 years 400 900

Adolescent and adult males

9–13 years 600 1700

14–18 years 900 2800

19 to70 years 900 3000

Adolescent and adult females

9–13 years 600 1700

14–18 years 700 2800

19 to70 years 700 3000

Pregnancy

<18 years 750 2800

19–50 years 770 3000

Lactation

<18 years 1200 2800

19–50 years 1300 3000

Source: From Institute of Medicine inDietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc, National Academy Press, Washington, 2002, pp. 8–9.

a As retinol activity equivalents (RAEs).

b Asmg preformed vitamin A (retinol).

c Adequate intake (RAEs).

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levels of provitamin A carotenoids are found in carrots, sweet potatoes, pumpkin, kale, spinach, collards, and squash (roughly 5–10 mg RAE=100 g) [18].

Data from NHANES 2001–2002 for food consumption in the United States showed that the major contributors to the intake of preformed vitamin A are milk, margarine, eggs, beef liver, and ready-to-eat cereals, whereas the major sources of provitamin A carotenoids are carrots, cantaloupes, sweet potatoes, and spinach [21]. These data, compiled for both genders and all age groups, showed that the mean intake of vitamin A is ~600mg RAE=day from food and that 70% –75% of this is as preformed vitamin A (retinol). The provitamin A carotenoids b-carotene, a-carotene, and b-cryptoxanthin were ingested in amounts of ~1750, 350, and 150mg=day, respectively. By comparison, the intakes of the nonprovitamin A carotene lycopene and the xanthophylls (zeaxanthin and lutein) were ~6000 and 1300mg=day, respectively. The Institute of Medicine’s Micronutrients Report [15] includes sample menus to illustrate that an adequate intake of vitamin A can be obtained even if a vegetarian diet containing only provitamin A carotenoids is consumed.

TRANSPORT AND METABOLISM

Taken as a whole, the processes of vitamin A metabolism can be viewed as supporting two main biological functions: providing appropriate retinoids to tissues throughout the body for the local production of retinoic acid, which is required to maintain normal gene expression and tissue differentiation, and providing retinol to the retina for adequate production of 11-cis-retinal. Major interconversions and metabolic reactions are diagrammed in Figure 1.4.

TRANSPORT ANDBINDINGPROTEINS

Carotenoids and retinyl esters are transported by lipoproteins and stored within the fat fraction of tissues, whereas retinol, retinal, and retinoic acid are mostly found in plasma and cells in association with specific retinoid-binding proteins. The associations of carotenoids and retinoids with proteins greatly influence their distribution, metabolism, and physiological functions. Amphiphilic retinoids—principally retinol, retinal, and retinoic acid—bind to retinoid-binding proteins, which confer aqueous solubility on these otherwise insoluble molecules. The concentration of free retinoid is very low. Binding proteins thus reduce the potential for retinoids to cause membrane damage [22]. Different retinoid-binding proteins function in plasma, interstitial fluid, and the cytosolic compartment of cells as chaperones that direct the bound retinoids to enzymes that then carry out their metabolism. Table 1.3 summarizes

RA Storage

Retinal

Further oxidation and conjugation reactions Retinol

Conjugation, oxidation, and

excretion

Carotenoids Retinyl

esters Polar metabolites

Cleavage Dietary precursors

11-cis-Retinoids,ocular processes

Oxidative metabolism

Mobilization Bioactivation

FIGURE 1.4 Principal metabolic reactions of vitamin A. RA, retinoic acid.

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TABLE 1.3

Major Extracellular and Intracellular Retinoid-Binding Proteins

Protein Gene Family=Type Size

Ligand (All-transIsomer

Unless Indicated) Function

Retinol-binding protein, RBP Lipocalin 21 kDa Retinol Transport of retinol between

liver and extrahepatic tissues Cellular retinol-binding proteins,

CRBP-I, CRBP-II, CRBP-III, CRBP-IV

Fatty acid-binding protein=cellular retinoid-binding protein

~14.6 kDa CRBP-I: retinol

CRBP-II: retinol and retinal CRBP-III:trans- andcis-retinol CRBP-IV: retinol

Binding of retinol and

chaperone function to enzymes of metabolism

Cellular retinoic acid-binding proteins, CRABP-I, CRABP-II

Fatty acid-binding protein=cellular retinoid-binding protein

~14.8 kDa CRABP-I: retinoic acid CRABP-II: retinoic acid

Binding of retinoic acid and chaperone function to enzymes of metabolism;

possible coreceptor function for CRABP-II

Cellular retinal-binding protein, CRALBP

CRAL=TRIO ~36 kDa All-trans-retinol and 11-cis-retinol;

11-cis-retinal

Binding of retinol, retinal;

chaperone function to enzymes of retinoid cycle that regenerate visual pigments in the retina

Interstitial retinoid-binding protein, IRBP

Partial homology to enoyl-CoA isomerase=hydratases

136 kDa All-transand 11-cis-retinal; other lipophilic substances

Improving the efficiency of translocation of retinoids between the RPE and photoreceptor cells

Transthyretin, TTR Transthyretin 55 kDa Thyroxin homotetramer Cotransport protein for holo-RBP;

prevents rapid loss by renal filtration

Albumin Albumin 67 kDa Retinoic acid; other acidic

retinoids; fatty acids

General carrier for acidic lipids

VitaminA:NutritionalAspectsofRetinoidsandCarotenoids11

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some of the characteristics of the retinoid-binding proteins involved in these absorptive, transport, and metabolic processes.

Retinol-Binding Protein

Plasma retinol is transported by a retinol-binding protein (RBP) [23]. One molecule of retinol is bound noncovalently within the beta-barrel pocket of the RBP protein. Although most RBP is produced in liver parenchymal cells, the kidney, adipose tissue, lacrimal gland, and some other extrahepatic organs also contain RBP mRNA, generally at levels less than 10% of that in hepatocytes, and may synthesize RBP [24]. The maintenance of a normal rate of RBP synthesis depends on an adequate intake of protein, calories, and some micronutrients (Table 1.4); conversely, a deficiency of any of these can reduce the plasma concentrations of retinol–RBP. RBP is synthesized in the endoplasmic reticulum of hepatocytes, transported through the Golgi apparatus where apo-RBP combines with a molecule of retinol to form holo-RBP [25], and then released into plasma. Nearly all circulating holo-RBP is bound noncovalently to another hepatically synthesized protein, transthyretin (TTR), which also binds thyroxine [26,27]. RBP protects retinol from oxidation, while TTR stabilizes the retinol–

RBP interaction [28]. Although retinol is the natural ligand of RBP, other retinoids such as 4-hydroxyphenylretinamide (4HPR) can compete for binding to RBP in vitro [29], destabilize the RBP–TTR complex [30], and result in reduced levels of plasma retinol [31].

TABLE 1.4

Causes of a Low Level of Plasma Retinol

Etiology Mechanisms

Nutritional

Inadequate vitamin A in liver Reduced secretion of holo-RBP Inadequate protein or energy

Inadequate micronutrients (zinc, iron) Reduced RBP synthesis, release Disease-related

Infection or inflammation Reduced production and section of RBP, and TTR (retinol not limiting)

Liver diseases Reduced synthesis of hepatic proteins,

including RBP, TTR

Renal diseases Reduced reabsorption

Treatment-related

Retinoid treatment (4HPR, RA) Displacement of retinol from RBP;

possibly altered synthesis is RBP Genetic

Hereditary disorders Rare natural mutations that affect the production or the stability of RBP and TTR proteins

Toxicologic

Alcohol-related Impaired vitamin A storage; generally

poor nutritional or health status Environmental toxin-related Altered retinol kinetics (e.g., dioxin or

TCDD exposure)

Note: 4HPR, 4-hydroxphenylretinamide; RA, retinoic acid; RBP, retinol-binding protein; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TTR, transthyretin.

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Albumin

Retinoic acid circulates in plasma bound to albumin [32], and it is likely that other retinoids with a carboxylic acid function also bind to albumin.

Lipoproteins

Retinyl esters, when present in plasma, are bound to lipoproteins. Retinyl esters are a normal constituent of chylomicrons and chylomicron remnants in the postprandial phase, but they are not present in appreciable amounts in the fasting state in humans, except in the patho- physiological state of hypervitaminosis A [33]. Plasma retinyl esters may also indicate delayed metabolism of retinyl ester-containing chylomicrons or remnants [34]. As noted later, some species (carnivores) carry most vitamin A as lipoprotein-bound retinyl esters.

Humans and a few other species absorb a fraction of intestinally absorbed carotenoids without cleaving them; thus, carotenoids are present transiently in chylomicrons and remnants, as well as low-density (LDL) and high-density lipoproteins (HDL) [35], which also carry carotenoids in the fasting state. The nonpolar carotenes and lycopene are associated mostly with LDL whereas the relatively more polar xanthophylls are equally distributed between LDL and HDL [36].

Intracellular Retinoid-Binding Proteins CRBP Family

Cellular retinoid-binding proteins belonging to the fatty acid-binding or cellular retinoid- binding protein gene family are present in the cytoplasm of many types of cells [37]. The structural motif of this family has been described as a beta clam in which a single molecule of ligand fits into the binding pocket with the functional group (hydroxyl group of retinol) oriented inward. Various cellular retinoid-binding proteins bind ligands selectively (see Table 1.3). Four cellular RBPs (CRBP-I, CRBP-II, CRBP-III, and CRBP-IV) and two cellular retinoic acid-binding proteins (CRABP-I and CRABP-II), which may have arisen through gene duplication, are expressed at different levels in cell type-specific and tissue-specific patterns (reviewed by O’Bryne and Blaner [38] and Ross [39]). These proteins confer aqueous solubility on otherwise insoluble retinoids; protect them from degradation; protect membranes from accumulating retinoids; and escort retinoids to enzymes that metabolize them [39,40].

CRBP-I is widely distributed in retinol-metabolizing tissues. In liver, its principal endogenous ligand, all-trans-retinol, is a substrate for lecithin:retinol acyltransferase (LRAT) [41] and a retinol dehydrogenase [42]. It may also function in the uptake of retinol by effectively removing retinol from solution and providing a driving force for its continued uptake [43]. In the small intestine, CRBP-II is abundant and CRBP-II-bound retinol is the substrate for LRAT [44]. Apo-CRBP, when present, increases retinyl ester hydrolysis [42].

Mice lacking these proteins do not exhibit a significant phenotype so long as they are fed a diet high in vitamin A [45,46]. However, retinol is rapidly lost when the diet is low in vitamin A.

Therefore, the CRBPs apparently serve as efficiency factors that help to conserve vitamin A, and therefore they may be of significant advantage when dietary vitamin A is scarce.

The sequences of CRBP-III and CRBP-IV are ~50%–60% homologous to CRBP-I and CRBP-II. CRBP-III is distributed mainly in liver, kidney, mammary tissue, and heart and binds, in addition to all-trans-retinol, several other retinoids as well as fatty acids [47,48].

Another member of the CRBP family, CRBP-IV, is more similar to CRBP-II than CRBP-I and CRBP-III [30]. CRBP-IV has a higher affinity for retinol and exhibits a somewhat different absorption spectrum, suggesting it binds retinol somewhat differently as compared with the binding of retinol by the other CRBPs. The mRNA for CRBP-IV is most abundant in kidney, heart, and colon, but is relatively widely expressed.

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CRABP-I and CRABP-II bind all-trans-retinoic acid. CRABP-I is expressed, albeit at low concentrations, in numerous tissues whereas CRABP-II has a more limited distribution range but is inducible by retinoic acid [49]. These proteins have been studied most extensively for their roles in embryonic development and tissue differentiation [50–52]. CRABP-I has been implicated in the oxidation of retinoic acid [42], whereas CRABP-II may aid in the distribution of retinoic acid within cells and as a cotranscription factor in the nucleus [53,54].

Nevertheless, mice lacking either CRABP-I or CRABP-II, and even double-mutant mice lacking both proteins, appear essentially normal [55].

Specialized Intracellular and Extracellular Retinoid-Binding Proteins

Two proteins unrelated to CRBP and CRABP are highly expressed in the retina: an intracellular retinal-binding protein, CRALBP belonging to the CRAL-TRIO family [56], and interstitial retinoid-binding protein, IRBP, a large multiligand-binding protein that is abundant in the extracellular space of the retina, known as the interphotoreceptor matrix [57].

CRALBP binds 11-cis-retinoids, retinol, and retinal and functions in the retinal pigment epithelium (RPE) in the visual cycle. Disruption of this gene resulted in reduced dark adaptation after exposure to light [58]. IRBP is implicated in the transport, distribution, and protection of retinol and 11-cis-retinal between the RPE and photoreceptor cells [57].

Another protein, RPE65, which is also highly expressed in the retina, can bind retinyl esters and may facilitate the storage of vitamin A in the RPE [59].

Nuclear Retinoid Receptors

Nuclear retinoid receptor proteins may be considered a subset of retinoid-binding proteins, as ligand binding is crucial for their function. The nuclear retinoid receptors, RAR and RXR, are transcription factors that bind as dimers to specific DNA sequences present in retinoid responsive genes (RAREs and RXREs, respectively) and thereby induce or repress gene transcription. The retinoid receptor gene family, a subset of the superfamily of steroid hormone receptors [60], is composed of six genes, encoding RARa,b, andg, and RXRa,b, andg. Each protein contains several domains that are similarly organized and well conserved within the RAR and RXR subfamilies, including a ligand-binding domain (LBD) that binds the preferred retinoid ligand with high affinity and a DNA-binding domain (DBD) that binds to the retinoic acid receptor response elements (RAREs). These are usually located within the promoter region upstream of the transcription start site of target genes [61].

All-trans-retinoic acid binds with high affinity to the RAR, while 9-cis-retinoic acid binds with high affinity to RXR and RAR proteins in vitro, but is believed to mainly activate members of the RXR family in vivo [62]. Each nuclear receptor protein also includes a dimerization domain and a transactivation domain through which the RAR–RXR heterodimer interacts with coreceptor molecules [63]. In the absence of ligand, RAR–RXR heterodimers associate with a multiprotein complex containing transcriptional repressor proteins (e.g., N-CoR and SMRT), which induce histone deacetylation, chromatin condensation, maintaining transcription in a repressed state. Crystallographic studies have shown that the binding of all-trans-retinoic acid to the RAR causes a change in receptor conformation [61], which induces the receptors to dissociate from their corepressors and associate with coactivators that have histone acetyltransferase activity and induce the local opening up of the condensed chromosome. In turn, these changes allow the binding of the RNA polymerase II complex, thus enabling the activation of the target gene and the transcription of DNA to RNA [63,64].

There are still many open questions about ligand-activated transcription involving the RAR and RXR receptors, including the significance of subtle differences between the three RARs,a,b, andg[61,65]; the specificity of RARE to which these receptors bind; and how receptor levels are regulated. Regarding RARE, certain canonical elements are well defined,

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such as the direct repeat (DR)-2 and DR-5 nucleotide sequences of Pu=GGTCA-N(2 or 5)-Pu=

GGTCA to which the RAR–RXR heterodimer binds well, but widely spaced or noncanonical response elements have also been discovered [66]. Regarding receptor levels, the expression RAR-bis regulated at least in part by retinoids through a DR-5 RARE in its promoter [67].

This receptor is frequently downregulated in cancers, possibly related to increased methylation of its gene or other changes in chromatin structure [68,69]. Other important but unresolved questions concern the role of posttranscriptional or posttranslational modifications in modulating retinoid receptor activity [64] and the ways in which coactivator and corepressor proteins are recruited and, in turn, regulate the outcome of gene expression by retinoids [63,70]. The ligand specificity of the RXR in vivo is still a subject of speculation. Although 9-cis-retinoic acid can bind to the RXR in vitro and is an excellent RXR ligand in transfection and gene promoter assays, it has been suggested that other ligands such as phytanic acid [71]

and docosahexaenoic acid [72] might also be RXR ligands. Overall, the nature and the production of the endogenous ligands for the RXR are less clear than those for the RAR.

Besides forming heterodimers with RAR, the RXR also bind with several other ligand-activated nuclear receptors: the vitamin D receptor (VDR), thyroid hormone receptor (TR), and the PPAR, LXR, FXR, and CAR proteins.

It is interesting that mice do well in the absence of one and sometimes more of these binding proteins and nuclear receptors. However, mice do not survive a nutritional deficiency of vitamin A, which effectively knocks out all of these ligand-dependent functions.

INTESTINALMETABOLISM

Conversion of Provitamin A Carotenoids to Retinoids

Humans are apparently relatively unusual in their ability to absorb an appreciable proportion of dietary b-carotene across the intestine in intact form. In contrast, most species cleave nearly all of the absorbedb-carotene during digestion and absorption [73]. Most of what is known about human carotenoid absorption has been derived directly from metabolic studies in humans and in vitro cell culture models. Ferrets, preruminant calves, and gerbils [74–76]

have been used as models although none of these completely represents human carotenoid metabolism [77].

In human studies, the intestinal absorption of carotenoids has been estimated through the intake–excretion balance approach, and by assessing the total plasma carotenoid response to carotenoid ingestion, which provides only a rough estimate of intestinal absorption. The bioavailability of a single dose of purified oil-dissolved b-carotene appears to be relatively low: 9%–17% using the lymph-cannulation approach [78], 11% using the carotenoid and retinyl ester response in the triglyceride-rich lipoprotein plasma fraction [79], and 3%–22%

using isotopic tracer approaches [80,81]. Even though quantitative data are few and numerous aspects of the absorption process require further study, the framework for the intestinal absorption of carotenoids is reasonably well known. The process can be divided into several steps: (1) release of carotenoids from the food matrix, (2) solubilization of carotenoids into mixed lipid micelles in the lumen, (3) cellular uptake of carotenoids by intestinal mucosal cells, (4) intracellular metabolism, and (5) incorporation of carotenoids into chylomicrons and their secretion into lymph.

Release of Carotenoids

The type of carotenoid and its physical form affect the efficiency of carotene absorption. Pure b-carotene in an oily solution or supplements is absorbed more efficiently than an equivalent amount of b-carotene in foods. Carotenoids in foods are often bound within plant matrices of indigestible polysaccharides, fibers, and phenolic compounds, which reduces the