Crilical Reviews in Food Science and NUlrilion, 31 (liZ): 103-163 (1992)

Carotenoids and in Human Nutrition

E-Siong Tee Division of Human Nutrition, Institute for Medical Research, 50588 Kuala Lumpur, Malaysia

Referee: C. Y. Lee, Ph.D., Department of Food Science and Technology, Cornell University, Geneva, New York 14456

ABSTRACT: Since the discovery of as a fat-soluble growth factor in thl~ early part of the century. research into and retinoids has attracted the attention of many scientists. These two groups of compounds are still being actively studied all over the world since many gaps in knowledge exist and new frontiers are being pursued. Recent developments in studies into the possible roles of carotl~noids and retinoids beyond their classical functions in vision have created a great deal of excitement in the biomedical community. This review covers a wide range of topics pertaining to these two closely related compounds. Particular emphasis is given to the functions of these compounds and their roles in human nutrition. V2,rious aspects of vitamin A deficiency and ,tudies on carotenoids and retmoids in cancer development and prevention are reviewed in some detail.

KEY WORDS: carotenoids. retinoids, human nutrition, vitamin A deficiency, cancer development and prevention.

I. INTRODUCTION chemical synthesis are all more recent achieve­ ments, accomplished during the 20th century. Some 75 years ago, vitamin A was discov-­ ered as a fat-soluble growth factor present in Vitamin A, or , is an essential nutrient and recognized as an essential biochemical for for man and all mammalian species since it can­ normal vision in man and animals. It is, in fact.. not be synthesized within the body. Deficiency the first vitamin to be discovered. Today, after of the vitamin results in adverse effects on growth, several decades, very little is known about the reproduction, and resistance to infections. Var­ vitamin's biochemical mechanisms of action in ious mild to severe biochemical and clinical signs the different functions except for the process of develop, depending on the degree of severity of vision. 1 Vitamin A deficiency remains one of the the insufficiency. The most important manifes­ major public health nutritional problems in many tation of severe vitamin A deficiency is xeroph­ developing countries and is an important cause thalmia, and irreversible blindness may eventu­ of preventable blindness. Afflicting large num .. ally occur in one or both eyes. bers of pre-school children in southeast Asia, Xerophthalmia is not a condition of recent vitamin A deficiency is often associated with pro.. recognition. The ancient Papyrus Ebers, an tein-energy malnutrition, parasitic infestation, and Egyptian medical treatise of about 1500 B. C. , diarrheal disease. The interaction between vita .. and the Chinese at about the same time, had min A and infection has recently been given a already documented the occurrence of night great deal of attention. The synergistic effect be· blindness. 1.2 For the treatment of this early stage tween vitamin A deficiency and infection may of xerophthalmia, liver or liver extract was rec­ be responsible for excessi ve childhood morbidity ommended. However, the identification, isola­ and mortality in many developing regions of the tion, synthesis, and production of vitamin A by world. Recent studies on vitamin A supp1emen­

1040-8398/92/$ .50 © 1992 by CRC Press, Inc. tation and child survival and mortality have cre­ ular attention to their importance to human nu­ ated a great deal of interest and debate in the trition. A detailed discussiOll of analytical meth­ international health and nutrition community. ods for retinoids and carotenoids is treated in a The major source of vitamin A in the diet of separate review by the author." most communities in developing countries is ca­ rotenoids. A review of vitamin A in human nu­ trition would thus be incomplete without due con­ II. HISTORICAL PERSPECTIVE sideration to these pigments of provitamin A activity. Carotenoids and retinoids have had a long Carotenoids are synthesized exclusively by and interesting history. Biologists, organic chem­ photosynthetic microorganisms and by members ists, and biochemists were fascinated by the of the kingdom where they play a funda­ brightly colored pigments more than mental role in metabolism. Aside from providing a century ago. Studies on these pigments have aesthetic qualities as colorants in the plant and been traced to the beginning of the 19th century, the animal kingdoms, these pigments also take when the crystalline yellow pigment was part in the photosynthetic process. Most impor­ first isolated in 1831 and the naming in 1837 of tantly, the carotenoids serve the animal kingdom the yellow pigments of autumn as xan­ as sources of vitamin A activity. thophylls.7-9 In the early part of the 20th century, A large number of pigments in living organ­ it was clearly shown that there existed a whole isms - carotenoids, chlorophylls, anthocyanins, family of carotenoids, and that these were iso­ and porphyrins - provide the rich variety of prenoid derivatives. color in nature. The carotenoids, believed to have In 1913 McCollum and Davis 10 reported the derived their name from the fact that they con­ existence in certain foods of an essential Iipid­ stitute the major pigment in the root, Dau­ soluble substance capable of promoting growth cus carata, are undoubtedly among the most in rats. They called this substance "fat-soluble widespread and important. This group of pig­ A" to distinguish it from essential water-soluble ments is found throughout the plant kingdom (al­ nutrients, which they called' . water-soluble B,', It though the~ir presence is often masked by chlo­ During the subsequent decade, further informa­ rophyll) and in insects, fish, birds, and other tion about the existence and properties of this animals. These pigments provide a whole range soluble essential nutrient was obtained from the of light yellow to dark red colorings, and when work of a number of investigators, including comp1exed with proteins, green and blue color­ McCollum and associates Osborne and Mendel, ations are achieved. Thus, a wide variety offoods Stepp, Steenbock, Moore, and others. It was and feeds - yellow vegetables, tomatoes, apri­ shown that • 'fat-soluble A" (now known as vi­ cots, oranges, egg yolk, chicken, , shrimp, tamin A) not only maintained growth in rats but lobsters, salmon, trout, yellow corn, etc. owe was capable of preventing night blindness and their color principally to carotenoids, as do cer­ xerophthalmia. Subsequently, the relationship tain food color extracts from natural sources such between vitamin A in animals and the provitamin as palm oil, paprika, annatto, and saffron. 3-5 carotene in was also clarified, particularly This review discusses these two large groups after Karrer and associat,~sI2 determined the of interrelated compounds, which are still being chemical structure of ~-carotene in 1930 and of actively studied all over the world. Many gaps retinol in 1931 using a highly purified vitamin A in knowledge exist, and new frontiers are being extract that they had obtained from shark liver pursued. Recent developments in studies into the oil. 13.14 Using such retinol preparations, the first possible roles of carotenoids and retinoids be­ oily retinol esters (e,g., retinyl acetate) were yond their classical functions have created a great prepared. 13 deal of excitement in the biomedical community. The structure of ~-carotene had been deter­ This is one of the few reviews on the subj(~ct that mined as early as 1930. 12 These two results made deals with the carotenoids and retinoids together. it possible to see from a chemical standpoint why A wide range of topics is discussed, with partic­ ~-car()tene is a natural precursor of vitamin A

104 compounds. This relationship was discovered be­ noic acid, but nevertheless retain many of the tween 1928 and 1930 in a large number of ammal biological activities of these substances. Ad­ experiments. 15.16 The elucidation of the vtamin vances in tracer chemistry now permit the syn­ A precursor function of these pigments has proved thesis of almost any desired in radioac­ to have great scientific and economic importance. tively labeled form.30 Remarkable progress has In 1937. Holmes and Corbet 17 were a.bIe to been made in the synthesis of carotl.~noids, par­ crystallize pure retinol from fish liver. Various ticularly to meet the needs of a wide range of crystalline esters of retinol were obtained from commercial uses. 31 fish liver oils by Baxter and Robeson. 18.19 These Major advances have occurred in understand­ pure compounds made possible the accurate deter­ ing the intermediary metabolism as well as the mination of a number of physicochemical c:harac­ transport systems of rctinoids, both extracellular teristics of carotenoids. Several years later. Arens and intracellular. The development of cell culture and Van Dorp20 and Isler and associates:~l suc­ techniques has facilitated study of the effects of ceeded in achieving the chemical synthesis of retinoids on cell differentiation and proliferation, pure and retinol. Shortly thereafter. one of the most basic problems of biology ..12 Fun­ the total synthesis of f3-carotene was also damental to these new studies is the realization reported. 22 that retinoids are highly potent agents for control Many studies were conducted in the first half of these processes_ In this regard, the retinoids of this century dealing with various aspects of have had a particularly striking role in helping to the physiology and metabolism of vitamin A. illuminate the problem of rnalignam cell differ­ Particularly noteworthy was the identification by entiation. There has thus been a thrust in the Wald,23 Morton,24 and Morton and Good"",in25 of development of new retinoids for prevention or the chromophore of the visual pigment a~, retin­ treatment of disease, particularly in the areas of aldehyde. These various studies provided con­ oncology and dermatology 33-<5 Th~: domain of siderable information about the role of vitamin retinoid research is said to have moved well be­ A in vision, and about the pathology and patho­ yond its classical rools in the study of nutrition physiology of vitamin A deficiency. and vision. In the later half of the century, major ad­ Similarly, in addition to their role as vitamin vances in carotenoid and retinoid research have A precursors, the carotenoids are now thought to taken place. Since the middle of the century, play specific roles in mammalian tissues related rapid development in spectroscopic techniques, to their function in piantsY36 Carotenoids, with particularly nuclear magnetic resonance (NMR) their highly reactive conjugated double bonds, and mass spectrometry (MS). have revolution­ act as free radical traps or antioxidants. Based ized structural studies of these compounds. In the on the ability of carotenoids to protect plants and area of separation and analysis, various new bacteria against photosensitivity, trials have been methods have been developed.6.26-28 The devel­ carried out in the use of these pigments for the opment of high-performance liquid chromatog­ treatment oferythropoietic porphyria, a condition raphy (HPLC) is said to have revolutionized the in which patients suffer from an extreme degree problem of isolation and characterization of ca­ of photosensitivity. 37 These led to studies in the rotenoids and retinoids, and their metabolites. A use of f3-carotene and other carotenoids in the whole array of new in ~'itro bioassay systems for treatment of various skin tumors. retinoids have been developed, some of which are said to be even more sensitive than the chem­ ical detection methods. 29 III. NOMENCLATURE AND STRUCTURE Rapid progress has taken place in the area of chemical synthesis, particularly for retinoids. A. Nomenclature Advances in synthetic organic chemistry have resulted in the synthesis of well over a thousand The International Union of Pure and Applied new retinoids. Some of these compounds super­ Chemistry (IUPAC) Commission on Nomencla­ ficially bear little resemblance to retinol or reti­ ture of Organic Chemistry and the International

105 Union of Biochemistry (IUB) Commission on eight five-carbon isoprenoid units (ip), joined in Biochemical Nomenclature jointly recommended the nonnal head-to·tail manner, except at the cen­ revised rules for the nomenclature of carot­ ter of the molecule where the order is reversed J8 enoids. Known as the IUPAC-IUB 1974 Rec­ so that the C40 skeleton, viewed as a whole. is ommendations, these rules are adhered to in this symmetrical. Thus, the two central methyl groups review. are in a 1,6 position relative to each other while Relatively recent reviews on the nomencla­ the remaining nonterminal methyl groups are in ture and structure of carotenoids include those of a 1,5 relatiollship,8,38 as shown in Structure 1: 39 Weedon,4 Isler, Davies. and Goodwin. 0 These, and the 1UPAC-IUB document,38 have been par­ I -C=CH-CH=CH -C=CH-CH== I ==CH -CH=C-CH=CH -CH=C­ ticularly useful in the preparation of this section. I I I I I The term ., carotenoids" embraces a large CH3 CH3 Centre of CH3 CH3 number of naturally occurring fat-soluble com­ I I molecule I I 111------(5) I I 1(1) ------(5) pounds that are colored red and yellow. The char­ 111 ------.. ------••••- (6) acteristic chromophore is due to the presence of a series of conjugated double bonds. This large family of pigments has been grouped as hydro­ Using the abbreviation of ip for the isoprenoid carbons ( and 0'.-, 13-, and 'V-) residue, the C40 carotenoids can be represented and their oxygenated derivatives (oxycarotenoids as: ipipipip-pipipipi. or ). The acyclic C4oH56 conjugated polyenelyco­ With regard to retinoids, the IUPAC-IUB pene (II) can be taken as the prototype of the Joint Commission on Biochemical Nomencla­ carotenoids. ture40 has defined them as: a class of compounds consisting of four isoprenoid uniTS joined in a head-to-tail manner. All retinoids may be for­ mally derived from a monocyclic parent compound con­ (lI) taining five carbon-carbon double bonds and a lUlietional group at fhe terminus of the acyclic portion. To avoid con­ fusion with previously used names in this field 110 parent The shorthand form of the carotenoid formula is hydrocarbon is named. also often used (l1a), the broken lines indicating formal division into isoprenoid units: In recent years, the term, "retinoid" has been taken to include both the naturally occurring com­ pounds with vitamin A activity and synthetic an­ alogs, with or without biological activity, of re­ tinol. Carotenoids are not included in thIS group of compounds. 41 The Commission further recommended that In the case of retinoids, the basic hydrocarbon the term vitamin A should be used as the generic (III), also known as axerophthene, has been des­ descriptor for retinoids that exhibit qualitatively ignated as deoxyretinol: the biological activities of retinol. This term should be used in derived terms such as vitamin A activity, vitamin A deficiency, and vitamin A antagonist. 40

B. Structure

1. Basic Structure

The basic structure of carotenoids consists of Deaxyretlnol Inn

106 2. Modifications to the Parent of these end groups, and their corresponding Compound Greek letter prefixes, have been compiled from Goodwin8 and IUPAC-IUB38 and are given m Based on the basic structure of the carot­ Table 1. enoids (I and II above), other carotenoids can then be considered as related to it by means of R structural changes in one or both halves of the , 6 J -0 "a'·.4 S 18 molecule. These changes may be brought about by reactions involving (I) hydrogenation, (2) de­ (VlI)e hydrogenation, (3) cyclization, (4) insertion of oxygen in various forms, (5) double bond mi­ 16 gration, (6) methyl migration, (7) chain e:lon­ 8 gation, and (8) chain shortening. Retinol (vita­ "a'62 6 R min A) and related C20 compounds are not ;3 {) 18 included in this class of compounds. 4 Though the number of basic structural, mod­ (VIII) 1 ifications are comparatively few, their occur­ rence in different combinations accounts for the 19 20 remarkabk variety of natural carotenoids. About R 400 are now known and the number is increasing rapidly. The number of hydrocarbons (carotenes) is relatively small, most being oxygenated ca­ Examples of carotenes are rotenoids (xanthophylls).8

Similarly, for retinoids, changes to the basic 18' ,",' 17 16 y' ~ ~3' hydrocarbon give rise to the various reti,noids. .r 7 :9 \1 13 '!> '" 12 10' "l~;1 ~ ~~ A 2 These include changes to the functional group at 1 16: 8 10 12 14 15' 1'13' j, " 3 ~I,'11' l~ 1&' ~7 5 20' 19 the 15 position of the basic hydrocarbon (III), Lt­• '8 the hydrogenation level, and chain length. ~-Carotene I~,~-carotenel (XlII:

a. Carotenoid Hydrocarbons 17 16 :/' :3 t /' 7 '919 11~ '3 i5 ,.' 12' 10' 8YQ'8'5'" 6' ,~~~~/~ , B '0 " 14 '5' ;" " ,. .3 6 ~"P' 1 : 15' if The carotenoid hydrocarbons are known as l~. 5 ,. 20' 1!J' carotenes. Specific names of compounds in this group are based on the stem name "carotene". ,,-Carotene (p.•-carotene) (XlIII: The structure and numbering of the parent car­ otene is given below (IV).

19 17 16 ~ '6~~'4' 3' , 7 ~ '3 15 ,4' '2' 10' s' 6' C l"Carotene 1~.,,;;aroteneJ (XI'I),

'0 '2 ,4 5 • l' 2' "2~6 ..J ~ ~~~15' 1'3' 11' 1 1' ji;6 17' 5 2a 19' • 18 In the above examples, as well as others dis­

(IV) cussed in this section, their systematIC names are given in brackets behind their trivial names. The The semi-circles at the two terminations. rep­ first Greek-letter prefix in the systematic name resent two double-bond equivalents. The system­ is separated from the second by a comma, and atic name of a specific carotenoid hydrocarbon the second is connected to the stem name by a is constructed by adding two Greek letters as hyphen. These prefixes are cited in alphabetical prefixes to the stem name carotene, depending order ([3 [beta], E [epsilon], K [kappa], [phi], on the two Cg end groups which it contains. Some X [chi], \~ [psi]).

107 TABLE 1 End Group Designation of Carotenes

Type Prefix Formula Struc~ture

Acyclic w(psi) C9 H'5 V Cyclohexane 13, E (beta, epsilon) C9 H'5 VI, VII Methylene-cyclohexane 'Y (gamma) C9 H'5 VIII Cyclopentene K (kappa) C.H" IX Aryl q" x (phi, chi) C.Hl1 X, XI

Adapted from References 8 and 38,

In numbering a carotenoid formula which is However, almost all naturally occurring acetyl­ asymmetrical (the two end groups are disslmilar), enic and allenic carotenoids contain oxygen and unprimed numbers are allotted to the end of the are considered in the next section, molecule associated with the Greek letter prefix cited first in the systematic name, as indicated in the numbering of

Zeta·Carotene [7,8, 7,8'·tetrahydrolycopenel (XV]

(ii) methoxy, as in spirilJoxanthin [1.1' -dime­ ['1 thoxy-3 ,4,3'4'-tetrahydro-l ,2, 1'2'-tetrahydro­ tV, w-carotene] (XX): 3,4'Dldehydro''V,'V'carotenc (XI!!)

CH,o

7,8·Didehydro,<,<·carotene (XVII) (iii) glycosyloxy, as in myxoxanthophyll [2' -(13­ L-rhamno-pyranosyloxy)-3' ,4'-didehydro-I' ,2' dihydro-13 ,E-carotene-3, I'-diol] (XXI):

6,7·Didehydco,[,[·carotene [XVIII)

108 (iv) carboxy, as in torularhodin [3',4'-clidehy­ d. Seco-carotenoids dro-[3,tjJ-carotene-16' -oic acid] (XXII): Seco-carotenoids is a carote~noid which undergoes fission of the bond between two ad­ jacent carbon atoms (other than carbon atoms 1 and 6) of a cyclic end group, witt retention of

the C40-carbon skeleton. e,g.,

(v) oxo, as in [[3 , [3-caroten-4-one ] (XXIII): ~~~ 2,3·Seco·,,£-<:arotene IXXVlIJ o e. Apo-carotenofds

A carotenoid in which the carbon skeleton (vi) aldehyde, as in torularhodin aldehyde [3' ,4'­ has been shortened by the formal removal of frag­ didehydro-[3, tjJ-carotene··16' -all (XXIV): ments from one or both ends is named apo-car­ otenoid, e.g.,

(vii) epoxy, as in [5,6-epoxy-5,6­ dihydro-[3,[3-carotene,3,3' -diol] (XXV): For purposes of nomenclature, the C,o carot­ enoids are treated as apo-carotenoids. e.g.,

4,4'-Dlaponeurosporene (XXIX) c. Retro-carotenoids f. Nor-carotenoids Retro-carotenoids are those in which all sin­ gle and double bonds of the conjugated polyene In nor-carotenoids, carbon atoms have been system have shifted by one position. The carbon formally removed from the carotenoid by pro­ atoms delineating the conjugated systems are in­ cesses other than cleavage of carbon-carbon dou­ dicated in the systematic numbering, The prefix ble bonds. Examples of these are retro is preceded by the pair of locants, e.g.,

3-Hydroxy-S',7- 'relTo'-p,E-carotene-S'.one!XXVI1 2,2'-Dinor-~,~-carolene (XXX)

10S1 16.17.16'. JT-Tetranor-<.<-carotene (XXXf1

Retinol (XXXIII) g. Higher Carotenoids (ii) retinaldehyde, also known as , vita­ Until recently, no natural carotenoid was min AJ aldehyde, retinene, or retinene,. The name known in which the carbon skeleton contained retinal, however, is to be avoided so as not to more than 40 carbon atoms_ Over the last few be confused with the adjective retinal (pertaining years, however, studies on the pigments of var­ to the retina). ious nonphotosynthetic bacteria have revealed the occurrence of a number of C4S - and Cso"carot­ enoids, the higher carotenoids. 4 These are a class of hydrocarbons and their oxygenated derivatives consisting of more than eight isoprenoid units joined in a manner similar to that of the C40­ carotenoids. They are named as mono- or di­ substituted C4o-carotenoids. Retlnaldehyde OOOOV)

(iii) retinoic acid, also known as vitamin A acid, h. Retinoids vitamin Al add, or ,

For numbering the carbon atoms of the re­ tinoids, the Commission has recommended that the system described for carotenoids38 is to be adopted. The suggested representation of the skeletal formula is as given in (XXXII): 19 20 Retino1c acid (XXXV) 1:cc167~~~ On the basis of this numbering scheme, geo­ I 6 8 10 12 14 metric isomers or substituted compounds can be 3 named unambiguously, e.g., 13-cis-retinoic acid 5 18 or (XXXVI), a::1d 3-hydroxy-retinol 4 (XXXVII).

Skeletal formula of retinoid (XXXII).

Structures of the three naturally occurring reti­ noids are

(i) retinol" also known as vitamin A, vitamin A alcohol, vitamin AI, vitamin Al alcohol, axer­ ophthol, or axerol. 13-cfs-Retlno1c acid (XXXVI)

110 pounds substituted by the bivalent retinylidene ~~"OH moiety, e.g., 3 I HO ft 3-Hydraxyretinol (XXXVIij

To name the retinoids systematically, however, a different numbering scheme has to be used. 4ll.42 The carbon atom bonded to the functional group RetJnaIdehyde oxime (}ooo(J is given the number I. The systematic name for retinol is thus a11­ trans-3, 7 -dimethy 1-9-(2,6, 6-trimethylcyclo-hex­ l-en-I-yl)-nona-2,4,6,8-tetraen-I-ol.

19 20 N-Rettnyl1dene··l-aminopropan-2-o1 (XXXXI) 17((:167 19 11 113 15 2 ~~'OH I 6 8 10 12 14 Similarly, derivatives of retinoic acids are named 3 as carboxylic acid derivatives, e.g., 5 18 4

Retinol (XXXIllA)

Functional substitution at the C-IS position (see [XXXII] above) of the basic hydrocarbon is denoted by the use of the group names retinyl (R Ethyl retinoate or retinolc acid ethyl ester (XXXXIn is CH2 -) or retinylidene (R is CH=), with reten­ tion of the original numbering of the basic hy­ drocarbon. Examples of these are

N -Ethyllretinamlde or rettno1c ail:ld ethylamlde (XXXXIm

Retinyl acetate (XXXVIm i. "Hydro" and "Oehydro" RetinOids

Retinoids that differ in hydrogenation level from ilhe con-esponding parent compound are named by use of the prefixes "hydro" and"de­ hydro" together with locants specifying the car­ bon atoms at which hydrogen atoms have been Retinylamine (XXXIX) added or removed. These prefixes are nonde­ tachable and, if both occur in one name, are cited Compounds derived from retinaldehyde are in the order dehydro before hydro. Example of named either as aldehyde derivatives or as com­ these are

111 3,4-DidehydroretJnol (also known as dehydroretJnol or v1tamin A?J (JOOOIJV)

4,5-Didebydro-5,6-dihydroretJnol, also known as a-vitamin A (XXXXV) j. Other Retinoids monly used method of stereomutation is to ex­ pose a solution of the carotenoid, containing Other modifications covered by the IUPAC­ catalytic amounts of iodine, to light (iodine-cat­ IUB report40 include the substituted retinoids, al yzed photo isomerization ). seco-retinoids, nor-retinoids, and retro-reti­ The number of theoretllcally possible isomers noids. These are generally similar to those de­ of a carotenoid is determined by the number of scribed for carotenoids. sterically effective double bonds, and by the sym­ metry of the molecule (more isomers being pos­ sible in the asymmenical molecule). For a con­ C. Cis and Trans Isomerization jugated system with n nOl1cyclic double bonds. the number of stereoisomers N is given by the It is now known that most, if not all, carot­ expressions:43 enoids can be converted into mixtures of geo­ metrical isomers in vitro under appropriate con­ N = 2n for unsymmetrical systems ditions. The principal methods are refluxmg in N = 2(n- 1)/2. (2(n- 1),2 + 1) for symmetrical sys­ an organic solvent, photochemical, contact with tems, n odd active surfaces, and irradiation in the presence ru2 N = 2(nl2)-1 • (2 + 1) for symmetrical systems, 39 of iodine. .43 n even A corollary to these methods is that inad­ vertent isomerization may be induced by pro­ Thus, ~-carotene (9 sterically effective dou­ longed heating of carotenoid solutions (e.g., re­ ble bonds, symmetrical) has 272 possible iso­ fluxing in light petroleum, hexane, or benzene mers, and lycopene (11 effective double bonds, for 30 min can isomerize up to 40% of a caro­ symmetrical) should be capable of existing in tenoid), or by exposure of carotenoid solutions 1056 isomers. However, because of steric hin­ at room temperature to sunlight or artificial light drance between lateral methyl groups and olefinic (50% of all-trans zeaxanthin can be lost in this protons, some of the double bonds are "hind­ way from a benzene solution in 15 min) ..\9 Isom­ ered", leaving four "unhindered" (methyl-sub­ erization can also result from prolonged contact stituted) bonds in the case of ~-carotene and six with active surfaces, for example, when the car­ in the case of lycopene. Hence, the numbers of otenoid is adsorbed on alumina. The most com­ sterically unhindered isomers are much fewer than

112 those theoretically possible, 20 being expected etc. in order of their decreasing polarity. while for fj-carotene and 76 for lycopene.'Y those more polar than the all-trans form are named The stereomutation products of all-trans ca­ neo U, neo V, etc. in order of their increasing rotenoids exhibit certain characteristic features.:19 adsorption affinities. 39 Cis carotenoids are generally less stable than their For the retinoids, the polyene chain in the aU-trans isomers. They are also more soluble parent compound has the trans configuration about than the corresponding all-trans compounds. The all the double bonds, unless the contrary is 1n­ melting points of cis isomers are lower (by 20 to dicated:m The stereochemical prefixes E or Z 120°C) than those of their all-trans countf~rparts. may be used, and should always be used where Because of stereomutation on fusion, some cis cis or trans might be ambiguous. For example, isomers exhibit double melting points. The most 13-cis-retinol (XXXXVI). also known as neo­ important of these features. from the analytical vitamin A, is (7E,9E, 11 E, 13Z)-retinol. point of view, is the relationship between cis­ trans isomerism and UV-vis light absorption and the different adsorption affinities. Cis carotenoids exhibit light absorption of lower intensity than their all-trans isomers and, as a general rule, the principal light absorption maxima are shifted to shorter wavelengths (ap­ proximately 2 to 5 nm for one and 10 nm for two sterically "unhindered" cis double bonds). One of the most noticeable features in the spectra of mono- "unhindered" -cis carotenoids is the ap­ pearance of a subsidiary peak at about 340 nm, Simil,arly. 13-cis-retinoic acid, also known as in the near-UV region. The wavelength differ­ isotretinoin. IS designated as OE,9E, lIE, 13Z)­ ence between this cis peak and the longest ,vave­ retinoic acid (see [XXXVI] above) length maximum of the all-trans compound is The six known isomers of retinol are a11­ said to be rather constant, 142 2 nm for C40­ trans, II-cis (neo b), 13-cis (neo a), 9, 13-cis (iso carotenoids possessing 10 or 11 conjugated dou­ b), 9-cis (iso a), and 11, l3··cis (neo c). The older ble bonds. When compared on a molar basis, cis designations for these isomers are given in brack­ carotenoids show less intense absorption than their ets. Both the biological activity (Table 7, Section all-trans isomers.43 These changes are illustrated VI. B) and physical propeJ1ies of retinol, retin­ in Figure 1. Other spectroscopic changes (in­ aldehyde, and retinoic acid are highly dependent frared, proton magnetic resonance) in relation to on their isomeric forms. 44 cis-trans isomerization have also been described The isomerization of all-trans retinol is stim­ by Weedon. 43 ulated by temperature, light, and iodine. The The geometrical configuration of a caroten­ isomerization of retinaldehyde is pmticularly well oid has a considerable effect on its adsorption studied due to its involvement as a visual pig­ affinity. Thus, mixtures of carotenoid )somers ment. Details of this are given in Frickel42 and can be resolved by the various techniques of ad­ Olson.44 sorption chromatography. The isomerization of an all-trans carotenoid may either increase or decrease the adsorption affinity with the result IV. PHYSICAL AND CHEMICAL that, in a column chromatogram, some CIS zones PROPERTIIES appear above the all-trans carotenoid while oth­ ers run below. In the absence of a precise: con­ Before 1950, a main tool for the analysis of figurational assignment, cis isomers may be chromatographic fractions was various color re­ named according to their positions on such a actions, the most important being the Carr-Price chromatogram. Those isomers that are less polar reaction. In this reaction, a polyene dissolved in than the all-trans carotenoid are neo A, neo B, chloroform reacts with antimony trichloride and

113 0.4

A W'~\. J \. I •••• ~.. . 0.3 J ~ • ...... fl·· . /: •·· , • Q) u •· c · rn j • -e 0.2 · o · .c(/J / ~ ~ : · .- ·· · .. l 0.1 ,,- /:i ,...... ~ . • " I" , ~ .. -- .. - I ... I J ~ '-~ ~,. " .:-;: ~."''"':.~ 0.0 ,....,."'.. 280 320 360 400 440 480 520 560 600 Wavelength (nm)

FIGURE 1. Ught absorption spectra of all-trans ~-carotene, and cis isomers after treat· ment with 30 ILg (- -- -) and 50 ILg (....) iodine per 100 ILg carotene. the transient deep blue coloration measured at for the synthesis of polyenes and elucidate new 620 nm. This reaction is still used for vitamin A structures of minor or complicated carotenoids. 45 determination. Besides these reactions, C and H analysis were the only methods readily available for characterizing new structures. The availabil­ A. Physical Characteristics ity of a number of physical methods has greatly influenced carotenoid research and the pace of Being a group of very diverse compounds, progress has quickened. With the ever-increasing the carotenoids can be expected to have very number of synthetic and naturally occurring ma­ different physical characteristics. However, some terials available for comparison, the physical generalizations in these properties have been given methods led in tum to valuable data and empirical by Borenstein and Bunnell.' Carotenoids are said rules. It was possible to develop new methods to crystallize in a variety of forms, the color of

114 the crystals varying from deep red through violet tures, e.g., the substituents and cis-trans ISO­ to almost black. Their melting points are usually merism, and by the solvent,45 fairly high and tend to increase with increasing First, with regard to an increase in a chain molecular weight and functional groups. For ex­ doubJ.e bond, it has been shown that this results ample, [3-apo-8' -carotenal, [3-carotene, and can­ in an increase of the absorption maximum vary­ thaxanthin have melting points of 136 to 140°C, ing from 7 to 35 nm.45.4 8 An example of this 15 170 to 182°C, and 208 to 210°C respectively. seen in going from trans- (7,8,11,12. The conjugated double bond system of the ca­ 7' ,8' ,11', IY·octahydrolycopene) (XXXXVII) to rotenoids renders the crystalline materials very trans- (7,8,11,12,7' ,8' -hexahydro­ sensitive to oxidative decomposition when ex­ lycopene) (XXXXVlIl), which involves the ex­ posed to air. The carotenoids are insoluble in tension of the chromo ph ore by two double bonds, water, slightly soluble in vegetable oils, mod­ The increase in absorption maximum is 61 nm, erately soluble in aliphatic and aromatic hydro­ Le., from 286 to 347 nm (Table 3). carbons, and very soluble in chlorinated hydro­ carbons (e.g., chloroform). Some physicochemical data of vitamin A and esters are summarized in Table 2. Physicochem­ ical characteristics of several cis-isomers of the tran&-phytoene (7,8,11.12.7'.8'.11', 12'·octahydrolycopene, C'iXXXVlD vitamin have been tabulated by Schwkter and Isler.46

trans-pl:>ytofluene (7.8.11,12. 7'.S·-hemhydrolycopene, (XXl(l{>1m B. Absorption Spectroscopy When a tit (psi)-end group of lycopene cy­ This technique has been the cornerstone for clizes to a [3 (beta)-end group, as in 'V-carotene, the characterization of carotenoids for more than there is a hypsochromic shift (shift to higher fre­ 50 years. It is still the diagnostic tool most easily quencies, i.e., lower wavelength), a loss of fine available to many laboratories. structure, and a reduced intensity (hypochromic The characteristic absorption spectrum of a effect). These effects are even more pronounced carotenoid is determined by the number of double upon formation of a second ring as in [3-carotene. bonds and of various additional structural fea- These changes are illustrated in Figure 2.

TABLE 2A Physicochemical Data of Some Vitamin A Com~,ounds

Physical Vitamin A Vitamin A Vitamin A characteristics alcohol acetate palmitate

Appearance Pale yellow Yellow prismatic Yellowamor­ prism crystals crystals phous or crystalline Melting point (0C) 62-64 57-60 27-29

Molecular formula C2oH30O C22H3202 C3sHSOO2 Molecular weight 286.44 328.5 524.8 Solubility (g/100 ml) Soluble in most organic solvEmts; soluble in fats and oils; insoluble in water, glyc'9rol Spectrophotometric data

Amax :325 nm (in 326 nm (in 325-328 nm (in ethanol) ethanol) ethanol)

% E'1 em 1835 1550 940-Sl75

Adapted from Reference 47,

1H' TABLE 2B Physicochemical Data of Some Vitamin A Compounds

Physical Vitamin A Vitamin A Vitamin A2 characteristics aldehyde acid (dehydroretinol)

Appearance crystals Crystals Yellow crystals or oil Melting point (0C) 61-134 180-182 17-'19

Molecular formula CZO H28O C2oH2BOZ C2oH2BO Molecular weight 284.4~~ 300.44 284.42 Solubility (g 1100 ml) Soluble in most organic solvents: solution in fats and oils; practically insoluble in water Spectrophotometric data Amax 373 nm 350 nm 288,352 nm (cyclohexane) (ethanol) (ethanol) E~c/~m 1548 1510 820; 1450

Adapted from Reference 47.

TABLE 3 Visible Light Absorption Properties of Some Isoprenoid Polyene Hydrocarbonsa

Number of conjugated Principal IiQlht absorption Polyene double bonds maxirna (nm)

Phytoene 3 298 286 276 Phytofluene 5 366 347 331 ,-Carotene 7 425 401 380 a-Carotene 8 449 421 398 9 470 440 416 E-Carotene 9 471 440 418 o-Carotene 10 487 456 431 Lycopene 11 504 472 443 1,2-Dihydro-3,4-dehydrolycopene 12 518 483 457 3,4-Dehydrolycopene 13 535 !;OO 468 3,4,3',4' -bisDehydrolycopene 15 540 S10 480

a Determined on dilute solutions of the all-trans isomers in light petroleum or hElxane. From Moss, G. P. and Weedon, B. C. L., in Chemistry and Biochemistry of Plant Pigments. Vol. 1, 2nd ed., Goodwin, T. w., Ed., Academic Press, London, 1976. chap. 3. With permission.

The effect of trans .....,.. cis isomerization on the position of the maxima and on the mo­ (stereomutation) is the appearance of a subsidiary lecular absorbance of the compound. These var­ cis peak about 140 nm below the absorption max­ iations, with ~-carotene as an example, are given imum of the longest wavelength of the corre­ in Table 4. Figure 3 shows the absorption spectra sponding all-trans-carotene. There is also a lower of ~-carotene in several solvents. A more com­ extinction coefficient. These effects were dis­ plete tabulation of the absorption maxima of some cussed in greater detail in Section III. C. well-known carotenoids in various solvents has The solvent in which the absorption spectrum been given in Davies. 39 of a caroH:noid is measured has a marked effect From the beginning of research into reti­

116 0.6

~-carotene ,..

"'><:'0::.....: 1 ...... 1...... A>. A>...... ~ ~ ~ ~ ~ ~ ~ ~ ~ ~I.,. ~,~~~~-~----,~~,1\ 0.5 - I I I • ~ :. ____ y-carotene ::I. :• 1: .':' .:f • ~ .l~-.~J.....__~~~ . : ...... lycopene 11 i : 0.4 , :' :: ~:~, :. ,. " , : 1 1'.' : •• , : , ',! (J) 1 : (.) I' c ,! •• !,i I.:', ::" ..4"· ~ :, : .ccc:s !:f;1\. \~: i ! '- 0.3 0 , : II \ I. (/) ,:11 I! .c« !: /\ l : IY,/ \ \ ! 0.2 I/f , :

~\ '~. If. !, I .;~ I : \ 1 0.1 l t\ ~ ,'J ! Q

0.0 240 280 320 :360 400 440 480 520 560 Wavelength (nm)

FIGURE 2. Ultraviolet and visible light absorption spectra of l3-carotene (-), "I-carotene (- " -), and Iycopene (.... ) in hexane. noids, absorption spectroscopy has been em­ green fluorescence, with excitation at 327 nm and ployed as an aid in the characterization of struc­ emission at 510 nm. ture. 42 VV absorption characteristics of some vitamin A compounds have been included in Ta­ ble 2. Similar data for the cis-forms of retinoL C. Infrared Spectroscopy retinaldehyde, and retinoic acid have been tab­ ulated by Morton. 50 This technique has not been used routinely Retinol and its esters fluoresce in VV light. for identifying carotenoids, mainly because the This fluorescence property of retinol has been conjugated polyene system gives rise to only very extensively studied and has been used in the assay weak bands. With the advances in other spectro­ of the vitamin. Retinol emits a strong yeUow­ scopic techniques, JR spectroscopy has become

117 TABLE 4 thetic derivatives have been published. This data Effect of Solvent on Absorption Maxima and bank makes it possible in most cases to determine Absorbance of J.l-Carotene the configuration of a novel retinoid or carotenoid structure rapidly and reliably. Moreover, the ef­ Position of ficiency of commercial instruments has been im­ main absorption Solvent maximum (nm) El%cm proved greatly in the past few years, so that very small amounts of the material are required to Hexane 453 2592 obtain the NMR spectra. In combination with a Cyclohexane 457 2505 chromatographic method such as HPLC, it is pos­ Benzene 465 2337 sible to achieve a reliable structure assignment. Chloroform 465 2396 even in the case of very small amounts of a re­ Methylene chloride 463 2448 Carbon disulfide 484 2008 tinoid as obtained, for example, when a retinoid 42 Dioxan 461 2471 is isolated from biological material. Frickel has Ethanol 453 2620 given a detailed account of the use of NMR spec­ troscopy in structural studies of retinoids. Adapted from Reference 49. even less widely used. It has, however, proved to be of value for detecting certain special struc­ E. Mass Spectrometry tural features such as acetylenic, allenk, hy­ droxy, and unreactive keto groups, e.g., in fu­ MS is said to have had tremendous impact coxanthin and capsanthin. Vetter et al. 45 and Moss on the elucidation of carotenoid structures, par­ and Weedon48 have tabulated some infrared ab­ ticularly when frequently not more than 100 J.Lg sorption bands of carotenoids. of a pigment are required for analysis. Three Similarly, neither the retinol nor the retin­ different kinds of information useful for struc­ aldehyde series show characteristic differences tural analysis can be obtained from a mass 45 for the various isomers in IR spectroscopy. 46 spectrum: Frickel42 made only a brief mention of IR ab­ sorption spectra in his comprehensive account of 1. The molecular weight and, if measured to the physical properties of retinoids. an accuracy of a few parts per million, the elementary composition of a compound. 2. Certain structural features may be deducible D. Nuclear Magnetic Resonance from the fragmentation pattern, provided Spectroscopy that spectra of a number of structurally re­ lated compounds are available for NMR spectroscopy, particularly proton mag­ compaJison. netic resonance (PMR), has been described as a 3. Proof of the identity of the different sam­ very important and effective tool for the eluci­ ples; mass spectra usually contain numerous dation of carotenoid structures ..~5 New develop­ peaks and are easily reproducible, so thal ments in 13C-magnetic resonance spectroscopy they are generally considered to be excellenl are said to suggest that this technique will prove for this purpose. to be an even more powerful method for struc­ 48 tural studies. Detailed discussions on the use Vetter et al. 45 have also discussed some prob­ of NMR spectroscopy in the study of carotenoids lems in using MS for investigating carotenoid have been given by Vetter et al. 45 and Moss and structure. Because of the partial decompositior:. Weedon.48 of samples during vaporization for MS, repro· NMR spectroscopy has also proved useful in ducibility of the mass spectra of carotenoids is studies of retinoids. A large number of IH-NMR said to be poor. This poor reproducibility does and 13C-NMR spectra of geometric isomers of not, however, normally interfere with the use of the naturally occurring retinoids and of their syn­ MS for molecular weight determinations. It was

118 0.6

Ii I, 0.5 ~---+---+--I----I----+4--+--/\ -+----II-I I \, , :..... \1 \ 0.4 , : ~ I : ~ ,

, / · jI ~/\l~ \• ~ c: i i~;l '-.,~ • l ~ ~ 0.3 ~~----+-----~---'---+-----+-~~~I ---~__--~----~-I o 'll I .8 Ifl) l\\ « r+------r------+----~------+~/.r~f/~!I (11 f I I I' I:! i II 0.2 ~~----+-----~-----+-----r~:Ij~/~~!r-----+~~~.'~~----~~

j II i I' I rl. " I:V \i 0.1 J~' \.n

0.0 240 280 320 360 400 440 480 520 560 Wavelength (nm)

FIGURE 3. Absorption spectra of g-carotene in chloroform and dichloromethane tetra­ hydrofuran (- - -), ethyl acetate (.... 1, and n-hexane (- -- -). nevertheless felt that MS remains a very useful the chemical properties of carotenoids refer to tool for the elucidation of carotenoid structure. the use of these reactions in assisting the struc· A detailed discussion of the use of MS in tural elucidation of these compounds. Carefully carotenoid study has been given by Vetter et al. 45 chosen chemical tests are said to be useful in and Moss and Weedon.48 Both publications also confirming suggestions of structure arising from include a compilation of the mass spectra data of spectroscopic evidence and are often crucial in various carotenoids which should serve as good the recognition and differentiation of the number reference materials. and type of the functional groups. Comprehen .. sive reviews on the subject include those 0:' Goodwin,S Davies,39 moss and Weedon,48 and F. Chemical Properties Liaaen-JensenY Some of these reactions are dis· cussed below. 1. Reactions of Carotenoids Oxidative degradation of carotenoids had been carried out classically by ozonolysis. Oxidative Most of the discussions in the literature on cleavage of carbon-carbon double bonds could

11H be achieved, and from the carboxylic acids pro­ isomerization to the unstable II-cis and other duced, conclusions were drawn concerning the more stable isomers. In light of higher intensity, end groups of carotenoids. In the early structural other more drastic photochemical reactions take studies on carotenoids, much valuable informa­ place, leading to dimerization to form kitol and tion was obtained by partial degradation using other polymers. I chromic acid and permanganate. Stepwise deg­ Acidic reagents give transient - mainly blue radation of carotenoids allowed early workers to - color reactions with vitamin A. The brilliant isolate large degradation products such as apo­ blue color obtained with the Carr-Price reagent carotenals and ketonic products, which permitted (antimony trichloride in chloroform), with an ab­ conclusions to be drawn concerning the structure sorption maximum at 620 nm, has become a of the natural carotenoids. 51 widely used color reaction for vitamin A deter­ Most carotenoids are stable to alkalis, hence mination. Other reagents that have been used in­ the common use of saponification as an early step clude trifl uoroacetic acid,52 trichloroacetic in the purification process. 8 acid, and, 1 ,3-dichloropropan-2-01 55 The Treatment of a ca!'Otenoid containing a con­ fading of the color in these tests is rapid and jugated carbonyl group with sodium borohydride makes quantitative measurement difficult. They in ethanol or tetrahydrofuran will result in a dra­ are, however, especially useful for qualitative or matic change in absorption spectrum. 8 There is comparative measurements, The use of the re­ a hyposochromic shift of 20 to 30 nm and an action for the biochemical assessment of nutri­ enhancement of the fine structure of the spectrum. tional status is discussed in Section IX.B.2.b. Chemically, dehydroretinol or vitamin A2 is much more sensitive to the effects of oxygen than 2. Reactions of Retinoids retinol and decomposition occurs rapidly. In other aspects, it resembles r,etinol in many of its chem­ In the absence of antioxidants, retinol and its ical properties. There is some evidence that, in derivatives are very unstable toward oxygen. In addition to the all-trans isomer. the I3-cis isomer practical terms, oxidation is the most important occurs naturally. It is however relatively unim­ cause of destruction. Traces of metallic ions, no­ portant in mammals and has been shown to have tably copper and iron, catalyze the oxidation. 1 only 30 to 40% of the biological activity of The degradation process can be monitored by the retinol. 2 rapid decrease of the extinction at 328 nm. The oxidation products are said to be ill defined, usu­ ally exhibiting a rather broad band at 270 to 280 V. METABOLISM nm.46 Other chemical oxidizing agents are also a A. Biosynthesis of Carotenoids threat to unprotected vitamin A structures. Se­ lective mild oxidizing agents, such as manganese Comprehensive accounts of the biosynthesis dioxide, can transform retinol into retinal. On the of carotenoids are given in Goodwin56 and Brit­ other hand, reducing agents such as lithium al­ ton. 57 The following summary has been con­ uminium hydride or borohydrides can reduce re­ densed from the above, and particularly from 58 tinoic acid or retinal to retinol. I Goodman and Blaner and Simpson and Vitamin A is extremely sensitive toward Chichester. 59 acids, which can cause rearrangement of the dou­ Although C30 , C.~5' and Cso carotenoids are ble bonds and dehydration, followed eventually produced by some nonphotosynthetic bacteria, by the addition of the solvent and by cis-trans most naturally occurring carotenoids (in partic­ 46 isomerization. Ethanolic hydrochloric acid and ular those of higher plants) are C40 tetraterpenes, hydrobromic acid are particularly reactive in this biosynthesized by the well-established respect. 2 In the absence of oxygen, retinol and pathway. The basic feature of these compounds its derivatives are stable toward alkali. is the repeating isoprenoid units. This isoprenoid­ Light. together with iodine, catalyzes bond like carbon skeleton is also found in such diverse

120 compounds as , bile acids, ,. sex more unsaturated compounds, leading to the for­ hormones, ubiquinones, , essential mation of lycopene. Lycopene is then cyclized oils, phytol (in chlorophyll), and the side chains to the monocyclic o-and -y-carotenes, which in of vitamins E and K. turn are further cyclized to yield a- and ~-car­ The ubiquitous acetyl coenzyme A is the basic otene, respectively. The introduction of oxygen carbon source in the synthesis of carotenoids, results in the formation of the xanthophylls. three molecules of which result in the formation of 3-hydroxy-3-methyl-glutaryl coenzyme A (Figure 4). This is then converted to mevalonic B. Conversion of Carotenoids to acid, a pivotal compound in the synthesis path­ Vitamin A way. Phosphorylation and decarboxylation of this give rise to isopentenyl pyrophosphate, which is After the existence of fat-soluble vitamin A also a key compound, serving as the building was well recognized and accepted, considerable block for successive C-5 units. Condensation of confm.ion arose during the 1920s regarding two two molecules of isopcntenyl pyrophosphate (after quite different types of substances showing vi­ isomerization of one of them to dimethylallyl tamin A activity in animals. 60 One group was the pyrophosphate) yields the IO-carbon compound highly colored hydrocarbon compounds found geranyl pyrophosphate. Subsequent addition of extensively in the plant kingdom and collectively 5-carbon isoprene units (via isopentenyl pyro­ called carotenoids. The other compound is almost phosphate) leads to the formation of the 5-car­ colorless and is concentrated in the liver. This bon and 20-carbon intermediates, namely, far­ confusion was. ultimately resolved following two nesyl pyrophosphate and geranylgeranyl important studies in the early 1930:-.. Yloore l6 pyrophosphate, respectively. In the pathway of demonstrated that rats fed massive amounts of sterol biosynthesis, two molecules offarnesyl py­ carotenoids extracted from deposited large rophosphate condense to form one molecule of amounts of vitamin A in their . At about squalene, which is subsequently cyclized to the the same time, the classica:. studies of Karrer et sterol ring structure. In carotenoid biosynthesis, a1. 12.13 decisively established the chemical rela­ in contrast, two molecules of geranylgeranyl tionship between ~-carotene and VItamin A. It pyrophosphate condense to form the first com­ became clear that carotene acts as a precursor for mon carotenoid precursor, the 40-C hydrocarbon vitamin A and is converted to the vitamin in the phytoene. This is then converted to progressively animal body. The conversion of ~-carotene to retinol is an important biological process since, throughout the course of evolution, most, if not all, of the +0, vitamin A utilized by animals was ultimately de­ rived from this process. Although retinol has been

I a-corotene 15. 15'-oxYgenOse synthesized chemically by several procedures in f (intestine. liver) recent years, human requirements of vitamin A depend largl~ly on dietary sources of the provitamins.

retinoldehyde After absorption, the carotenoid is cleaved in the mucosal cell of the intestine to form vi­ retinaJdehyde reductase l' (+ NAO~) ~ alcohol dehydr0genose tamin A. The enzymatic cleavage of !3-carotene (intestine. liver, eye) into retinaldehyde with soluble enzyme prepa­ 2~OH rations from rat intestinal mucosa and liver was 61 retlno! reported by Goodman and Huang. Subsequent studies have shown the mechanism of the reaction FIGURE 4. Biosynthetic formation of carotenoids. to be that of a dioxygenase reaction, in which (From Simpson, K. L. and Chichester, C. 0., Annu. molecular oxygen reacts with the central two car­ Rev. Nutr., 1, 351, 1981. With permission.) bon atoms of !3-carotene, followed by the cleav­

121 age of the central double bond of carotene to yield C. Absorption and Utilization 62 two molecules of retinaldehyde ,63 (Figure 5), The enzyme has been designated as J3-carotene 1. Absorption and Factors Affecting ] 5,] 5' -dioxygenase, Partially purified forms of the enzyme have been obtained from raL hog, After the ingestion of food, the provitamin and rabbit intestines. However, because of ]a­ carotenoids of vegetables and other plant sources. bility during purification. the pure enzyme has and pre-formed vitamin A of animal tissues are not yet been isolated. 58 released from associated proteins by the proteo­ lytic action of pepsin in the stomach, and of chy­ motrypsin and trypsin in the small intestine. The Acetyl CcA liberated carotenoids and vitamin A are dissolved in fatty globules which then pass into the lumen 2 Acetyl CoA --Acetoacetyl GoA J-. 6-0H- ~-methyl-9IutO)l1 CcA of the duodenum. Here, the globules encounter ? 7 7H5 OH-?-~-C-CH,-CH20H NADPH the bile salts and the pancreatic enzymes, which H OH release various products of lipid digestion, Vi­ Mevolonic ocid tamin A esters are also hydrolyzed at this stage. j ATP These products of dig,estion then interact with bile ?H5 yH3 CH 2;C-CH,-CH2-OPP CH 3-C;CH-CHr OPP salts and cholesterol to form soapy aggregates, Isopen~tenYIpyrophosphate DimethylOlr pyrophosphate termed mixed micelles, which solubilize the vi­ j tamin A and carotenes, These micelles diffuse Ceronyl pyrophsophote C- 10 into the glycoprotein layer surrounding the mi­ crovilli or brushborder of the mucosal cells, where Sterols -Squolene -{Fornesyl pyrophosphote C-15 they come into contact with the cell membranes. · t The components of the micelles, except the bile cerreronYI pyrophosphate C-20 salts, then individually penetrate the lipid phase of the mucosal cell membranes to reach the

Phyloene C40 cytoplasm.

14(-2H) The above summary outlines the processes I involving the absorption of vitamin A and car­ .·-carotene. B-coralene. Iyeopen. (elc.) otenes. Further details, especially the experi­ 1(0) mental evidence for the various processes, are . given in Wolf, I Goodman and Blaner,58 and FIGURE 5. Conversion of p-carotene to retinol. (From Moore. 66 A discussion on the factors affecting Frolik, C. A., in The Retinoids, Vol. 2, Sporn, M. B., the absorption of the vitamin and its prewrsor is Roberts, A. B., and Goodman, D. S" Eds., Academic given below, as these have an important bearing Press, Orlando, FL. 1984, chap. 11. With permission.) on human nutrition. The efficiency of dispersion of vitamin A and carotenes is known to be affected by the presence According to Ganguly and Sastry,60 the bi­ or absence of other components in the diet, as ological conversion of carotene into vitamin A well as by the general nutritional status of the may not be as straightforward as by a cleavage subject. First, soluble protein and the peptides of the centra] double bond, He pointed out that, derived therefrom are able to assist in the dis­ aside from this central cleavage theory. another persion of the vitamin. Increased protein level in theory has proposed a random cleavage: of the the diet is also known to aid in the intracellular carotene molecule. This discussion will not delve formation of retinaldehyde from carotene,67 dis­ into these two different theories, which have been cussed below. dealt with in some detail in the reviews of Gan­ Second, fat in the diet provides the vehicle guly and Sastry60 and Glover. 65 for transporting the vitamin A and carotenoids

122 from the stomach into the intestinal lumen and as reduced secretion of pancreatic juice or bile, is the source of some of the digestion products or impaired cell activity. which take part in micelle formation, as outlined After absorption, the carotenoid is cleaved earlier. In addition, some dietary lipids, sllch as in the mucosal cell of the intestine to form vi­ seed oils, may also contain a-tocopherol, which tamin A in the form of retinaldehyde. Current has a protective antioxidant effect on vitamin A. 67 understanding of this conversion process was out­ Studies by Hollande~8 show that the presence of lined previously in Section V. B. The retinalde­ fatty acids of varying chain lengths and the de­ hyde so formed is reduced promptly to retinol. gree of saturation also affect absorption rates. During its passage through the intestinal epithe­ Third, from the summary of the absorption lium, up to 75% of the retinol, irrespective of of vitamin A and carotenoid above, it is clear source, becomes esterified with long-chain fatty that an ample supply of bile salts is necessary for acids. These esters, mainly in the form of the efficient absorption. palmitate, are then incorporated together wi.th Carotenes in foods are usually less wdl ab­ other lipids, and with apolipoproteins, into chy­ sorbed than pre-formed vitamin A from the in­ lomicron particles that are secreted from the cell testine. Under normal conditions, over 90% of into the lymph. 58 ,67 ingested vitamin A is absorbed, and the effi­ Some unconverted carotenoids are directly ciency of absorption decreases only slowly as the absorbed and pass into the blood where their dose increases. On the other hand. about 70% of composition reflects the diet. They may be de­ the ingested l3-carotene is absorbed, and the ef­ posited in the liver or elsewhere, such as the fat ficiency of absorption decreases rapidly as the depots and and various organs. 59 dose increases. 69 A wide range of absorption rates has been reported, depending on the vegetable sources. In some vegetables, as low as 5% of the 2. Uptake and Storage in Liver carotene may be absorbed. It has been estimated that carotene in green leafy vegetables is two or After leaving the intestinal cells, retinyl es­ three times better absorbed than that from red or ters in low density lipoproteins of the plasma are yellow root vegetables such as camot. 2 Zha070 probably hydrolyzed by esterases on liver cell reported absorption rates ranging from 20 to 45% membranes. lntracellularly, unesterified retinol for various leafy vegetables, roots, and tubers. is bound by a specific cellular retinol-binding Children and infants are also known to absorb protein (cRB P) and transported to the endo­ carotene inefficient! y. 2 The absorption rate for plasmic reticulum, where it is again esterified, carotenoids is lower probably because the solu­ largely to the palmitate ester. This is then trans­ bility of carotene and other carotenoids in fat is ferred and stored in a soluble complex macro­ limited, whereas vitamin A is more or less mis­ molecule made up of various lipids, various poly­ cible with fats. In most rich sources, with the peptide chains, and carbohydrates linked notable exception of red palm oil, carotene is covalently to the protein. 67 present in the tissues of the vegetable in much Vitamin A is remarkable for its preference greater concentration than can be dissolved in the of the liver as its site of storage and for its stability accompanying fat. 66 Being less polar than vita­ when stored. 66 Hepatic vitamin A (95% as long­ min A, l3-carotene is also solubilized less well chain retinylesters, mostly retinyl palmitate) nor­ by surface-active agents, including the bile salts. 69 mally represents over 90% of the total body re­ Thus, vitamin A deficiency in human sub­ serves of vitamin A. 58 In any individual, the mag­ jects can still arise when the diet supplies caro­ nitude of the store will depend not only upon the tenes, instead of pre-formed vitamin A, but in­ dietary intake of vitamin A and its provitamins, sufficient fat to facilitate the extraction of the but also on the efficiency of absorption of the provitamin and its carriage into the intestinal vitamin and its rate of expenditure. These factors wall. 71 Absorption of carotenoids is also reduced may, in tum, be influenced by sex, rate of growth, in conditions that upset intestinal function, such state of health, etc. 1.66

123 3. Transport in Plasma carotene. each with one 6- ring, are bio­ logically active at approximately half the l3-car­ As outlined above, the liver rapidly immo­ otene value. 65 bilizes and stores vitamin A which has reached Because of the importance of carotenes (par­ the bloodstream after absorption from the diet. ticularly j3-carotene) and other carotenoids as a In the reverse direction, the liver can rekase vi­ main source of vitamin A supply in human nu­ tamin A into the bloodstream to maintain a con­ trition, many efforts have been made to evaluate stant level in the blood even when the diet is the exact vitamin A potencies of individual car­ deficient in the vitamin. 66 otenes and carotenoids (reviewed in Reference Whereas absorbed dietary retinol is carried 78). The activity of the carotenoid vitamin A to the liver mainly as its ester in complex with precursors can be illustrated in a number of ways: lipoproteins, delivery of the vitamin to the target (I) by feeding the carotenoid to an intact animal tissues is in the alcohol form. This is achieved under prescribed bioassay conditions and noting by the hydrolytic action of retinyl ester hydrolase, tissue changes, blood, livl';r. etc., or effect on which is also present in the storage complex. growth; and (2) by using a purified or partially Upon hydrolysis, retinol is directly transferred purified carotenoid cleavage enzyme in contact from its binding site in the storage complex onto with the carotenoid under in vitro conditions and a protein, a retinol-binding protein (RBP) for measuring retinaldehyde formation. 67 transport to other tissues. The RBP is the pri­ Bauernfeind et 76 tabulated the vitamin A mary carrier for retinol in the plasma, and can activities of some pure carotenoids measured in exist as holo-RBP, i.e., a 1: I molar complex with biological trials under well-defined laboratory retinol, and as apo-RBP, which is free of retinol. conditions. WH078 has used the relationship of Various detailed reviews of RBP, including its I J-Lg of f3-carotene as equivalent to 0.167 J-Lg of chemical structure, metabolism, and synthesis, retinol or 0.56 IU of vitamin A. This has gained have been given by Wolf. I Glover, 72 Chytil and acceptance (see References 77 and 79) and is also Ong,7J and Goodman. 74 used in the Malaysian Food Composition Table. so The earlier publication of Borenstein and BunnelP estimated that only 6 or 7 of the more VI. BIOLOGICAL ACTIVITY than 100 carotenoids could have provitamin A activity. More recently, Simpson and ChichesterW A. Vitamin A Activity of Carotenoids estimated that 50 to 60 carotenoids and apocar­ otenoid compounds could have provitamin A ac­ Over 60 years ago, Steenbock75 first con­ tivity. However, only a few of the identified ca­ ceived that yellow color might be associated with rotenoids have both vitamin A activity and occur biological activity; and in 1930, Moore showed in significant amounts in natural foods commonly by biological criteria that l3-carotene is converted eaten by vertebrate animals. 5 to vitamin A. There is now considerable literature Table 5, summarized from a more compre­ on the vitamin A activity of carotenoids. and hensive tabulation from Bauernfeind,5 tabulates recent reviews include those of Bauernfeind, vitamin A activity of some carotenoids present Bauernfeind et al.. 76 and Underwood. in foods, expressed as a percentage of the 13­ For vitamin A activity in mammals, it is now carotene activity, the latter taken as 100%. 11 known that a carotenoid provitamin A compound includes various carotenoid hydrocarbons. Some must have at least one unsubstituted j3(beta)-io­ compounds, such as 5,6-monoepoxide and 5,8­ none ring with an attached polyene side. The monofuranoxide, are believed to possess biolog­ other end of the molecule may vary in cyclic or ical activity despite the alteration of the l3-ionone acylic structure and can be lengthened but not ring. This is presumably due to in vivo conversion shortened to less than an II-carbon po]ye:ne frag­ back to the unaltered ring structure. 5 Oxygenated. ment. j3-Carotene possesses two j3-ionone rings, carotenoids are also biologically active and in­ one at either end of a long polyene chain, and is clude cryptoxanthin. isocryptoxanthin, 5,6-di, a provitamin A with high activity. 0'.- and "Y­ hydroxy-f3-carotene. . and torula·

124 TABLE 5 Provitamin A Activity of Some Common Carotenoids

Activity (%)

f3-Carotene 100 a-Carotene 50-54 3,4-Dehydro-f3-carotEme 75 'Y-Carotene 42-45 7' ,8' -Dihydro-'Y-carotene (f3­ 20-40 zeacarotene) f3-Carotene 5' ,6' -monoepoxide 21 f3-Carotene 5' ,8' -monofuranoxide 50 (mutatochrome, citroxanthin, flavacin) 3-Hydroxy-f3-carotene (cryptoxanthin) 50-60 4-Hydroxy-f3-carotene (isocryptoxanthin) 48 5.6-Dihydroxy-J)-carotene ActivH f3-Apo-2' -carotenal Active f3-Apo-8' -carotenal 72 I3-Apo-1 0'-carotenal Active I3-Apo-12' -carotenal 120 I3-Apo-8' -carotenoic acid Active Torularhodin Active <50 Citranaxanthin 44

Adapted from Reference 5. rhodin. Cryptoxanthin, present in maize, B. Activity of Vitamin A Congeners contributes significantly to vitamin A intake when high levels of this grain are present in animal The biological activity of the vitamin A mol­ feed. ecule may be affected by (I) cis-trans isomerism: As discussed in Section IILC, a great number (2) other changes in the general structure of the of possible geometrical isomers can exist in the molecule: (3) the substitution of the terminal hy­ case of carotenoids. An all-trans configuration droxyl radical by other radicals. Many laborious in the side chain provides higher biological ac­ biological tests, mainly with rats and chicks. have tivity than any of the cis isomers. However. be­ been made to compare the activities of vitamin cause of the relative ease of trans-cis isomeriza­ A and its numerous congeners. In generaL minor tion in solution, it is sometimes difficult to decide changes from the structure of all-trans vitamin whether a cis isomer occurs in nature or whether A leads to a reduction in biological activity. Gross it is formed during its isolation. One view of the changes, such as saturation of double bonds, usu­ biological activity ofthe cis isomers is that during ally cause complete loss of activity,66 digestion and/or absorption they are rotated, at Esterification of the hydroxyl group ha5, no least partially, on their asymmetric C atoms to fundamental effect on activity, although minor the linear all-trans form.5 It was, however. also differences may result if the efficiency of ab­ pointed out that it is not known for certain whether sorption is affected. Oxidation of retinol to the such transformation must first take place before aldehyde also has little effect on activity. Cis­ it can be converted to vitamin A. isomers of retinol have reduced activity, ranging The mechanism of cleavage of carotenoids from 23% for II-cis and 'I-cis isomers to about to form vitamin A was discussed in Section V. B. 75% for the 13-cis isomer. 50

125 The vitamin A2 analogs of all the above sub­ there was considerable confusion concerning the stances, which differ only in having an additional exact activity of the vitamin since few pure prep­ double bond between C-3 and C-4 in their aro­ arations of either vitamin A or of the provitamin matic rings, are generally considerably less active A were available. Various criteria have been used than the corresponding vitamin A congeners. 66 for the determination of vitamin A activity in rat All-trans dehydroretinol (compound number assays, such as increase in body weight occur­ XXXXIV in Section III.B.2.i), for example, pos­ rence of xerophthalmia, and changes in vaginal sesses approximately half the activity of all-trans epithelium determined by vaginal smears. H:> Bio­ retinol. 50 logical tests to detemline the activity of the .i­ Retinoic acid is not converted to retinol, but tamins were tedious and did not produce repro­ lies on the pathway of the metabolism of retinol, ducible results when comparing data obtained in possibly to an active metabolite. 1 It has high ac­ different laboratories employing different types tivity in the general system, but not in the visual of animals, cages, temperature conditions. and 47 66 system. . Its activity appears to depend greatly even basic diets. on the method of dosing. According to van Dorp In order to arrive at some degree of uniform­ and Arens, R 1 it has 10% of the activity of retinol ity in comparing results, early workers then de­ if given orally dissolved in arachis oiL 50% if cided on reference standards. These were single given as the sodium salt by injection. and 100% preparations of a compound either isolated from if given as the sodium salt orally. natural sources or synthesized in the laboratory, The interconversion of the various congeners and maintained and distributed by an interna­ of vitamin A after digestion by animals is a rather tional agency such as the Health Organization of complicated problem, and many areas appear to the League of Nations. These international stan­ be unclear. Conversions appear to occur readily dards were of varying degrees of purity and were between vitamin A and its esters, and between modified from time to time as purity improved retinol and retinaldehyde. Some degree of cis­ through technique or synthesis. These original trans isomerism presumably occurs since the ret­ standard preparations are no longer in existence, ina seems to be able to obtain from dietary sup­ as each of the individual vitamins is now readily plies of the all-trans retinol the particular isomer available in high purity. 8' of retinaldehyde which it requires for rhodopsin Because of the variation in the food sources formation. On the whole, however, chatiges in of vitamin A activity in diets. i.e., pre-formed the animal body between cis and trans isomers and precursor, a common mode of expressing are much Jess extensive than might be expected. their vitamin A value was needed. Vitamin A There seems to be no clear evidence of COllver­ activity of foods was expressed in IU where I sions between vitamin AI and vitamin A2 IU of vitamin A is equivalent to 0.3 IJ,g of retinol, derivatives. 66 0.344 IJ,g of retinyl acetate, or 0.55 IJ,g of retinyl Wolfl has summarized the requirements for palmitate; and I IU of provitamin A is equivalent biological activity as: provided the substance to 0.6 Il-g of f1-carotene or 1.2 Il-g of provitamin reaches its target tissue (e.g., the tracheal epi­ A carotenoids other than ~~-carotene. 6978.84 Be­ thelium), a hydrocarbon ring (five- or six-mem­ cause of the considerably poorer utilization of bered or aromatic) linked to a hydrocarbon chain dietary provitamins as compared with retinoL the of four conjugated double bonds with two methyl expression of the total vitamin A activity of a groups attached in the mode of two isoprene units, diet has to be qualified by indicating the per­ and ending with almost any functional group (po­ centages of the activity coming from retinol and lar terminal group), can have vitamin A activity from provitamins. 69 in maintaining epithelial differentiation. The use onus caused some confusion among scientists in estimating dietary vitamin A activ­ ity.85 In the 1967 WHO report, it was recom­ C. Units and Equivalency mended that the practice of expressing vitamin A values in terms of IU be discontinued in view In the early years of the study of vitamin A, of the availability of crystalline retinol as a ref­

126 erence standard. It was suggested that units of Subsequently. the United States National weight should be used instead, , micrograms Academy of Sciences (NAS)7\I described the use of retinol. The report also discussed the relative of the term "retinol equivalent": biological activities of the various vitamin (and provitamin) A compounds, and the calculation I retinol equivalent of vitamin A value of diets. These are outlined I jJ.g of retinol in the following paragraphs. 6 j-Lg of l3-carotene The major source of vitamin A in the diet of 12 j-Lg of other provitamin A carotenoids most communities is fj-carotene. As has been 3.33 IU of vitamin activity from retinol discussed in the previous section on some aspects 10 IU of vitamin A activity from 13­ of metabolism, fj-carotene is not utilized as ef­ carotene ficiently as retinol by the human body. Based on the structure of fj-carotene and retinol, one mol­ The total vitamin A value, in micrograms. ecule of the provitamin can be expected to be of a food or composite diet would thus be cal­ enzymatically converted to two molecules of vi­ culated as the amount of retinol plus the equiv­ tamin A. However, the overall biological effi­ alent amount of retinol that can be derived from ciency of conversion, when absorbed by the in­ the provitamins:~3 testine, is at best only about 50%, and occurs at lower levels of carotene intake, declining as in­ j-Lg l3-carotem: take ri ses. 77 .78 j-Lg retinol + 6 The variable efficiency of conversion is said + f..lg provitami~:. to be more a function of the efficiency of ab­ 12 sorption into mucosal cells, rather than in the efficiency of conversion to retinol from absorbed designated as micrograms of retinol equivalents fj-carotene. Increasing levels of carotenoid in­ (RE). take are inversely related to the efficiency of ab­ The formula may also be rewritten: 77 sorption, and this, in tum, is influenced by var­ ious factors, including the character of the mixed RE = lILg retinol] diet, e.g., the amount of fat, the antioxidant con­ + [0.167 x j-Lg of fj-carotene] tent, and the digestibility of the carrier food. The + [0.083 x f..lg of other WH078 report gave an extensive tabulation of provitamin carotenoidsJ (1) studies of human carotene absorption, which showed wide variations. It was suggested that, 7 for practical purposes. the best approximation for The NAS 'J report has given some examples absorbability of fj-carotene, in man, should be of calc:ulating the retinol equivalents In a diet or assumed to be one third (33%) of the amount foodstuff. based on the above·mentioned ingested. 78 relationships. Hence, on a weight basis, one sixth of the Bieri and McKenna~3 have emphasized that l3-carotene found in foods is estimated to be con­ the term' 'retinol equivalent" is a dietary concept verted to vitamin A in the body. Since dietary for approximating the vitamin A activity in foods. retinol is assumed to be 100% utilized by hu­ It is not an equivalency in the usual chemical mans, 1 j-Lg of l3-carotene in the diet is take:n to sense. It was further noted that the vitamin A have the same biological activity as 0.167 f,I,g of value for many foods will be a rough approxi­ retinol. 78 Since the activity of other carotenoids mation. When the form of vitamin A in the food varies considerably. some having no activity and is predominantly retinol, the analysis for retinol others about 50% of that of l3-carotene, this Com­ will be a reasonably accurate estimation of the mittee of the WHO also accepted that the activity retinol equivalents. On the other hand, when the of other total mixed carotenoids (with vitamin A provitamins are the predominant or only form of activity) be taken as one half of that of 13­ vitamin A, then the calculation of RE may con­ carotene. 78 tain a considerable error because of the approx­

127 imations made regarding the activity of the for Preventive Medicine, 95 carotenes are tabu­ carotenoids. lated for plant foods, in micrograms, whereas vitamin A values in IU are given for foods of animal origin. D. Units Used in Reported Values The Malaysian Food Composition Table 80 has also tabulated the retinol and carotene values sep­ The food composition tables commonly used arately on a weight basis (micrograms). Total in the U.S. have expressed vitamin A ve.lues in vitamin A activity, expressed as RE, has been IU. or both as IU and as RE.77 The equivalency calculated based on the formula discussed in the values of the IU and RE were discussed in Section previous section, namely: Vl.c. These values have been used for calculat­ ing the vitamin A values reported in food tables. . I p,g J3-carotene I-Lg retmo + Since analytical methods used for determination 6 of many food tables are not able to providf: values As discussed above, the limitation of vitamin for individual provitamin A carotenoids, the con­ A value so calculated is that it has been assumed version has usually been made based on the as­ that the carotenoids determined are J3-carotene. sumption that most of the carotenoids are ~)-car­ otene. For foods in which the provitamin A Such an assumption is, of course, not necessarily valid and depends a great deal on the foodstuff. carotenoid consists wholly or almost completely of J3-carotene, e.g., green leaves, the values for More accurate vitamin A values can become available only when the various carotenoids can vitamin A activity obtained from such calcula­ be separately determined. tions are good. On the other hand, the values are poor if other partially active carotenoids are pres­ ent in significant quantities. 86 Food composition tables all over the:! world VII. OCCURRENCIE have adopted different formats for reporting vi­ tamin A values of foods. Beecher and KhachikK7 A. Occurrence of Carotenoids in Plant have also highlighted this lack of uniformity in Materials the presentation of these data. Food tables for use in EnglandRH have provided separate values The ability to produce carotenoids seems to on a weight basis (microgram) for retinol and have been developed at an early stage in evolu­ carotene. Total vitamin A values were not tab­ tion. Photosynthetic bacteria, the algae, spore­ ulated. The authors pointed out that carotene val­ bearing vascular plants, and the higher plants ues in most foods are for J3-carotene; thus, the preserve this capability. Animals, certainly the total vitamin A activity calculated from these val­ higher orders, are not capable of de novo caro­ ues could be slightly overestimated. tenogenesis and are dependent for their carot­ 89 A recent edition of the Japanese food tables enoids on those present in their diet. 4 This dis­ have also tabulated carotene and retinol. values cussion is concernec. mainly with those separately in micrograms. However, total vita­ carotenoids present :tn higher plants, which are min A activity was expressed as "retinol po­ the more important food sources. tency" and given in IUs. Between 1933 and .I 948, the number of The FAO food tables for use in East Asia90 known naturally occurring carotenoids increased have tabulated, in micrograms, the retinol con­ from 15 to about 80, and in the early 1970s,7 tent and the "J3-carotene equivalent" of the foods. rose sharply to about 300. A decade and a half No total vitamin A values were given. Other ta­ later, Goodwin96 reported that over 500 were bles using this format are the Australian'll and known. Most of these, however, are xanthophylls the PhilippinesY2 food tables. which, as discussed previously, are oxygenated The food tables for Taiwan91 and Thailand94 carotenoids. The common name and the semi­ have tabulated all foods in IUs of vitamin A. In systematic equivalent, together with the structure the food tables compiled by China National Centre of about 300 natural carotet1oids, have been listed

128 in the 800-reference publication of Straub. 97 In prior to 1970) using column chromawgraphy and another publication 5 years later, Straub98 up­ thin-layer chromatography, as well as those ob­ dated the list to 400 compounds in a 1600-ref­ tained more recently (after the 1970s) using erence monograph. Bauernfeind' has tabulated HPLC. HPLC procedures have become more the occurrence of some 40 COmmon carotenoids widely used for the analysis of carotenoids in in various foodstuffs. foods, mainly because of the ability of the tech­ It has been estimated that nature produces nique to effect rapid separation, its nondestruc­ about 100 million tons of carotenoid pigments tivencss, and (more importantly) the better res­ per year. 99 most of this output is in the form of olution that is achieved. 6 Since older analytlcal four major carotenoids,4 viz., , the methods have been known to be msufficiently characteristic pigment of many marine algae and specific for the quantitation of individual carot­ undoubtedly the most abundant natural caroten­ enoids, it is probable that some of the data quoted oid, and the three main carotenoids in green using these methods could be inaccurate. leaves, i.e., , , and neoxan­ thin. All other carotenoids are produced in rel­ atively small amounts. However, some, like 13­ carotene and zeaxanthin, occur very widely; and 1. Vegetables others, such as Iycopene, capsanthin, . and spirilloxanthin, constitute the principal pigments Various studies (summarized by Goodwin 1(1) in a particular organism. have shown that leaves of higher plants usually How, and in which chemical and physical contain the same carotenoids: l3-carotene, lutein. form, carotenoids occur in nature is of impor­ violaxanthin, and . Pigments which tance, particularly to the food technologist in­ appear frequently, but not constantly, and in volved in the processing and development of new smaller amounts are a-carotene, mutatochrome food products. These aspects are also important (l3-carotene-5,8-epoxide), l3-cryptoxanthin, lu­ to the nutritionist because carotenoids are sources tein 5,6-epoxide, eloxanthin, taraxanthin, zeax­ of vitamin A. The overall carotenoid patte:rn may anthin, and antheraxanthin. The colorless po­ vary from relatively simple mixtures to extn:mely lyenes, phytoene and phytofluene, are known to complex ones. The simplest mixtures may be be rather widely distributed, but only at levels found in foods of animal origin due to the limited about one two-hundredth that of l3-carotene. Cis ability of the animal to absorb, modify, and de­ isomers of l3-carotene were also reported earlier, posit carotenoids. The other extreme is ':he for­ but it is now thought that they ra:~ely, if ever, midable array of carotenoids encountered in, for occur naturally, and are more likely to be artifacts example, citrus products, dehydrated alfalfa meal, of isolation. 5 or paprika. 3, The constancy of the qualitative distribution The concentrations in which carotenoids oc­ in leaves of high plants is remarkable and is said cur vary enormously from one source to another. to be applicable to the most diverse plants from The highest concentration of carotenes has been varying habitats. 101 In 35 green leafy and nOI1­ found in the red fringe of the corona of the pheas­ leafy tropical vegetables studied by Tee and Limg6 ant's-eye narcissus, Narcissus majalis. Here 13­ using HPLC, the composition of the carotenoids carotene can constitute up to 16% of the dry was strikingly similar, with lutein and l3-carolene weight. Furthermore, the daily rate of l3-carotene invariably present as the major carotenoids. The formation reaches 70 IJ-g/mg dry weight, which root and fruit vegetables (generally colored) stud­ is over 10,000 times the rate observed in carrot ied showed slightly greater variations in caroten­ roots. 100 oid composition. There are, however. consider­ The following is a summary of the distri­ able quantitative variations of these: carotenoids, bution of carotenoids in various plant materials, I3-Carotene in the vegetables was found to vary emphasizing particularly those products that are from 20 to 85% of the total carotenoids. Simi­ of importance to human nutrition. Data presented larly. total carotenoid content of the se vegetables include those reported some years ago (generally varies widely, ranging from <0.2 mg/IOO g edi­

129 ble portion for lettuce to mg!lOO g for sev­ values of the sweet corn cultivars were calculated eral of these vegetables. 86 to range from 5.0 to 7.7 RE. Green soya (Soja glycine), cow pea (Vigna sinensis). Lima bean (Phaseo/us lunatus). 2. Seeds pea (Pisum sativum val'. Thomas Laxton), and French (Phaseolus vulgaris) have been In mature cereal seeds used in human nutri­ found to contain 200 to 700 f.Lg/100 g (fresh tion, the carotenoid level is about 100 f.Lgi 100 g weight) of carotene. 102 The qualitative distribu­ fresh weight, of which 10% is carotene, le2 f)­ tion in peas and beans is said to be probably very Carotene is usually the only carotene present, but similar to that in leav(~,s. a-carotene is said to be the major pigment in millet. Total carotenoid in varieties of sorghum with yellow endosperm falls within the range 8,4 3. Fruits to 118 f.Lg per 100 g. and the percentage of f)­ carotene varies from "trace" to 26%. The xan­ There are relatively more data on the occur­ thophyll lTIixture in cereal seeds is complex and rence of carotenoids in fruit. In ripening fruit. generally silTlilar to that in green leaves, with the decrease in chlorophylls is frequently accom­ lutein as the major component. panied by an increasing concentration of carot­ Maize (yellow com) is widely used as food enoids and by an increase in the ratio of carotenes in the fresh, frozen, and canned forms, and is to xanthophylls.3 Goodwin 102 has tabulated the also processed in the form of com flour, meal, qualitative distribution of camtenoids in 150 types and grits. The pigments of maize have been stud­ of fruit. The quantitative distribution of total ca­ ied extensively by various investigators. Indeed, rotenoids and f)-carotene :in 50 fruits are also zeaxanthin and f)-cryptoxanthin were first iso­ tabulated in this publication. The changes in the lated from this source. 102 distribution of carotenoids during ripening of fruits Total carotenoid content in maize has been were also discussed. A discussion of the distri­ reported to range from 1 to 5.5 mglloo g." There bution of carotenoids in several individual fruits are, however, considerable qualitative and quan­ is given in Borenstein and Bunnell.' titative variations in the carotenoids of maize va­ Unlike the leafy vegetables, fruits tend to rieties and hybrids grown in different countries. show greater variations in carotenoid composi­ Quackenbush et al. 103 studied 125 strains of maize tion. f)-Carotene was invariably present, but its and reported the presence of 11 pigments. The proportion varied considerably, ranging from major pigments are phytoene, lutein, and zeax­ 100% in a variety of Thai to less than anthin. Others are phytofluene, f)-carotene, f)­ 10% for watermelon. f)-Cryptoxanthin, known zeacarotene, ,-carotene, a-cryptoxanthin. f)­ to possess provitamin A activity, was found in cryptoxanthin, zeaxanthin esters, and polyhy­ significant proportions in most of the fruits stud­ droxy pigments. ied. Vitamin A activity of the fruits was much Sweet com is another commonly consumed lower than that of the leafy vegetables. 86 food item. Lee et aI., 104 in one of the few recent A fruit that has particular significance in car­ studies available that detem1ines individual ca­ otenoid content is oil palm (Elaeis guineensis). rotenoids in several cultivars of processed sweet The pericarp of the fr~it yields the deep-red col­ com, reported monohydroxy carotenoids as the ored palm oil, which has been well known for major carotenoids present, followed by hydro­ its unusually high content of carotenoids. Un­ carbon and polyhydroxy carotenoids. The first refined palm oil is one of the richest known nat­ group consisted mainly of zeinoxanthin and f)­ ural sources of f)-carotene.!05 The food compo­ cryptoxanthin. Zeinoxanthin was, in fact, the ma­ sition table for use in Africa has tabulated the f)­ jor carotenoid in all four cultivars studied. Hy­ carotene equivalent content of palm oil as ranging drocarbon carotenoids consisted mainly of a-, from 37,300 to 128,700 f.Lg/100 g.106 A later study f)-, and 'V-carotenes, and f)-zeacarotene. Based of 30 samples of palm oil bought from market on the deltail composition available, vitamin A places in Nsukka, Nigeria, Mudambi, and

130 Rajagopal 107 reported f)-carotene equivalent con­ of palm oil, including effects on development of tents ranging from 85,920 to 165,200 f.lg/100 g, coronary heart disease and cancer. with a mean value of 137,835 f.LgIlOO g. The Malaysian food table has given an entry of 57, 100 f.lg/IOO g. 80 4. Rc)ots Early studies on the fractionation of carot­ en aids in palm oil have been reported by Hunter The most important plants from the point of l1O and co-workers. 108.109 Recently, Tan et al. re­ view of root carotenoids are the carrot and the ported the detailed separation and identification . In fact, the term carotenoids and! of carotenoids in several Malaysian palm oil pro­ or carotene is believed to have been derived from cessed fractions. Seven previously unreported the fact that they constitute the major pigment in hydrocarbon carotenoids were identified, namely, the carrot root, D. carota. 5 The major pigments phytoene, phytofluene, ~-carotene, o.-zeacaro­ are Q,-, f)-, and 'Y-carotenes; and traces of Iyco­ tene, f)-zeacarotene, neurosporene, and a-caro­ pene, a-carotene, ~-carotene, neurosporene, phy­ tene. In addition, the presence of a 0.-, f) .. , and toene:, and phytofluene have been reported. The 'Y-carotenes and Jycopene was confirmed. Crude relative amounts of o.-carotene to f)-carotene may palm oil, crude palm olein, and filtered palm vary from 5-I 0 to 51 % depending on the strain; olein were found to have a total carotenoid con­ deep orange-red strains usually have relatively centration ranging from 70,000 to 80,000 f.lg/ less o.-carotene than f)-carotene. The usual values 100 g, and most of this was f)-carotene. On the for the carotene content of carrots are 6 to 12 other hand, refined, filtered, and deodorized palm mg!IOO g fresh weight. 102 The xanthophyll frac­ olein was devoid of hydrocarbon carotenoids due tIon of commercial carrots is only 5 to 10% of to the rigorous refining process the fraction had the total pigments, but the percentage rises to 75 undergone. The carotenoid profile of palm kernel to 93% in yellow carrots, and to at least 95% in oil was also rather different, containing only 30 wild carrots. 102 f.lg/IOO g of o.-zcacarotene. Carrot has been studied by various investi­ Crude palm oil, with its high carotene con­ gators in recent years using HPLC. 6 U sing a com­ tent, is of particular nutritional importance. It has bination of HPLC and thin-layer chromatogra­ been consumed for years in Africa. LathamIII phy, Simon and Wolff!l3 studied in detail the has urged the wider cultivation and consumption carotenoid composition of several genetically di­ of palm oil in Africa for the prevention of vitamin verse collections of carrots. Total carotenoid was A deficiency. Locally, the importance of palm rep0l1ed to range from 6.3 to 54.8 mgllOO g fresh oil in the treatment of vitamin A deficiency has weight. Six carotenes were identified, namely, long been emphasized. 112 However, the available 0.-, ~1-, 'Y-, ~-carotene; f)-zeacarotene; Iycopene. palm oil currently marketed for cooking has been The major carotenoids were, however, 0.- and f)­ refined to the extent that carotene content has carotene, together contributing over 90% of the been reduced to a low level. Although a red­ total carotenoids. Of the two carotenes, the latter colored crude palm oil may not be acceptable to was found in higher concentrations, accounting consumers, some effort could be made to accus­ for 44 to 79% of all carotenoids, Similarly, tom them to a semi-refined oil. Such an oil would Heinonen l14 also reported 0.- and f)-carotene as retain some of its f)-carotene content as well as the major carotenes in a number of carrot culti­ other carotenoids which, although not important vars studied, Lutein was reported as a minor car­ as vitamin A precursors, may provide beneficial otenoid in the samples studied. health effects. In addition, a good proportion of The carotenoids of sweet potatoes (Ipomea the tocopherols, which are present in high con­ batalas) have received considerable attention, centrations in palm oil, could be retained. and are of particular importance to this region Chong 104 has reviewed the beneficial properties since it is an important item in the diet. f)-Car­

13"1 otene is also the main pigment present in sweet et a1. 120 for a number of Finnish foods. Retinol potatoes and only traces of xanthophylls are found. and several carotenoids ([3-carotene, lutein, and Singh and Bradbury 11S provided more recent data lycopene) in the foods were determined by two on carotenoids in several South Pacific root crops. HPLC procedures. Vitamin A value of the crops studied were. in In addition to the more commonly found form decreasing order. > sweet potato> Colo­ (vitamin AI)' a second molecular variation known casia taro> Xanthosoma taro> giant taro. as A2 (3'-dehydroretinol) exists in the tissues of fresh water fish.77 The latter has approximately a third of the biological potency of the former. B. Distribution of Retinoids The contribution of this form to human vitamin A intake is significant only in areas where fresh­ Vitamin A is found exclusively in animals, water fish is eaten. Small amounts of vitamin AI where it occurs as the free alcohol and also in aldehyde may be derived from fish roes and from esterified form. It is widely found in mammals, hen's eggs, but their practical importance is prob­ birds, amphibia, reptiles, and marine fish. Lower ably negligible. 66 Other substances with vitamin marine invertebrates usually do not contain pre­ A activity are also said to have been found in formed vitamin A. Crustacea usually contain only fish oils. These are, however, usually of consid­ carotenoids, commonly in crabs, erably lower biological activity than retinol. Ex­ lobsters, and shrimps. 116 amples of these are 13-clS-retinol, kitol, anhy­ 2 llo Livers of the vertebrate classes mentioned are droretinol, and rehydroretinol. . With regard the richest food sources of pre-formed vitamin A to retinoic acid, Moore06 pointed out that there because of the special role of the liver in the is no evidence that it contributes significantly to uptake, storage, and controlled release of dietary the vitamin A content of human or animal diets, pre-formed and biosynthesized vitamin A. 77 Be­ and that there is, indeed, little or no evidence fore the availability of chemically synthesized that it is present at all in natural foods. vitamin A, the principal source of vitamin A con­ centrates was, in fact, the liver and/or body oils of marine fish.1 The vitamin is believed to be VIII. FUNCTIONS AND USES synthesized by fishes from the large amounts of carotenoids they ingest with the plankton, algae, A. Carotenoids as Precursors of and smaller crustaceans that form their diet. 116 Vitamin A Animal milk and eggs contain pre-formed vita­ min A in variable amounts, depending on the diet The varied functions of carotenoids include of the animals from which they are obtained. provitamin A activity. light yellow to dark red Among free-living herbivores, the level is said food colorants, absorbers of light energy, oxygen to fluctuate with the carotenoid level in the diet. 77 transporters, and probably other functions un­ Recent data on retinol and carotenoid content known at this time. Although it is incorrect to of foods using HPLC have been provided by var­ consider vitamin A activity as a general function ious investigators. Retinol and carotenoids in var­ of carotenoids, the provitamin A activity of the ious foods in the Netherlands were determined carotenoids is nevertheless given emphasis in this separately by two HPLC procedures, and the re­ discussion because of the importance of vitamin sults used for calculation of vitamin A value of A in human nutrition. total diets. 118 Using a novel approach, Tee and Animals are not capable of de novo synthesis Lim l19 reported the simultaneous determination of vitamin A-active substances, neither pre­ of retinol and the content of major carotenoids formed retinol and its derivatives nor the caro­ of 40 foods of animal origin. [3-Carotene content tenoid precursor forms. 7~ Thus, apart from the in the foods studied was generally low, and the pre-formed vitamin A contained in foods such as contribution of other provitamin A carotenoids milk, eggs, liver, and fish liver oils, and their is probably insignificant. One of the few studies derivatives (and of course synthetic vitamin A), on ready-to-eat foods was reported by Heinonen the main sources of supply of this vitamin for

132 man are the carotenes and related carotenoids. that absorbed by chlorophyll, they extend the These pigments possess vitamin A activity and wavelength of light that can be used in are the so-called provitamin A. Indeed. ~-caro­ . 37 tene is the most important vitamin A precursor It has been observed that carotenoid pigments in human nutrition since its concentration in food in all photosynthetic organisms, bacleria. algae. and feed ingredients. particularly of origin. and higher plants, play an important role in pro­ greatly exceeds that of the other vitamin A active tecting these organisms against the seriously compounds. 76 damaging effects of photooxidation by their own The diets of population groups in the tropical endogenous photosensitizer, chlorophyll. Exper­ world rarely contain milk, eggs, or liver, which iments made in photosynthetic organisms lacking are the rich sources of pre-formed vitamin A. colored carotenoid pigments have supported these Thus, there has been a great deal of emphaslls on observations. Many workers have confirmed these carotenoids, particularly from leafy vegetables, findings and have extended the observations to as the sources of vitamin A to these communities. nonphotosynthetic bacteria. plants, and ani­ Vitamin A deficiency is prevalent, and the tragic mals. 127 Extending these data to disease treatment irony is that there is an abundance of vegetation in humans, carotenoids have been used for the in these countries. 121 treatment of patients with photosensitivity dis­ In the U. S .. the average diet of the usually ease. In this disease, known as light-sensitive available food is estimated to provide about 7500 porphyria, the porphyrins produced resemble the IV (2250 RE) of vitamin A per day. About 3500 porphyrin ring of chlorophyll and act as photo­ IV (1050 RE) is derived from vegetables and sensitizers in the patient. Mathews-Roth37m re­ fruits, 2000 IV (600 RE) from fats and oils. and ported increased tolerance to sun exposure by dairy products, and 200 IV (600 RE) from meat. erythropoietic protoporphyria patients after ~­ fish, and eggs. 5 Various estimates of percentage carotene administration. The use of carotene in contribution of provitamin A to the human diet the treatment of other photosensitive disorders place the figure at around 50%.79.112.123 has been reported. 129 As discussed earlier. provitamin A activity may be derived from various carotenoids. The above estimates of contributions of carotenoids C. Antioxidant Functions of Carotenoids to total vitamin A activity of diets must be as­ sumed as tentative, until food tables become more Various proposals have been put forth to ex­ complete in listing both l3-carotene and other plain the protective function of carotenoids against provitamins A. harmful photosensitized oxidations discussed in the previous section. Krinsky 127 discussed the major mechanisms whereby these pigments exert B. Carotenoids in Photosynthetic this function: (1) quenching of triplet sensitizers;

Tissues (2) quenching of singlet oxygen CO 2 ); (3) inhi­ bition of free radical reactions. The photochem­ The other established functions of carot­ ical reactions that can induce photodamage were enoids in plants are related to their ability to reviewed, and the possible mechanism of action absorb visible light. In the case of photosynthetic of carotenoids on the reactive chemical species tissues, they appear to have two well-defined produced was discussed. In photochemically in­ functions: (1) in photosynthesis itself, and (2) in duced oxidations, carotenoid pigments have been protection of the photosynthetic tissue against shown to have the capacity to quench the first photosensitized oxidation. Comprehensive re­ potentially harmful intermediate, the triplet sen­ views of these functions are given by Krinsky, 124 sitizer, at a significant rate. The remaining triplet Burnett,125 and Goodwin. 126 sensitizer species could then continue to initiate Carotenoids could act as accessory light-ab­ a series of reactions, depending on the availa­ sorbing pigments in the photosynthesis process. bility of oxygen and the nature of other poten­ By their absorption at wavelengths lower than tially reactive species in the environment, with

133 the production of singlet oxygen and free radi­ continually being formed in human tissues and cals. These three mechanisms of carotenoid pro­ their safe sequestration IS an important part of tection against oxidation are summarized in antioxidant defense. Thus, the proteetive effeets ure 6. In an updated review of the 5,ubject, of carotenoids against the harmful effects of ox­ Krinsky!3O described in some detail these biolog­ idation would be expected to have a protective ical actions of carotenoids. effect against cancer. The role of carotenoids and retinoids in cancer is discussed in Section IX. D.

~:/SH' -+­ R'~RH C J O. Carotenoids as Food Colors S ~"S CAR GAR' (1) "o~ There is much to the saying: "Man eats with 3 ~ ' .... 8 .... '0 CAR CAR ~2 his eyes as well." Thus, color of food is a sig­ nificant factor in determining its acceptabihty. ~ "CAR CAR Man associates a particular food with its specific "natural" color. He becomes cautious when a FIGURE 6. Mechanisms of carotenoid protection food shows an unexpected color, interpreting it against oxidations: (1) quench triplet sensitizers; (2) as a possible sign of spoilage, poor processing, quench singlet oxygen; (3) quench free radical inter­ or adulteration. An important use of carotenoids mediates. hv = light; 8 = sensitizer molecuh~; '8 (especially carotene) is in . singlet excited species; 38 = triplet sensitizer; '0 2 Natural extracts containing carotenoids have singlet oxygen; 302 = triplet oxygen; R· = freE! radical species; CAR = carotenoidies; 3CAR = triplet state of been used for coloring food for centuries: annatto carotenoid. (From Krinsky, N. I., Pure Appl. Chf1m., 51, with bixin as the main coloring component, saf­ 649, 1979. With permission.) fron with derivatives of and other ca­ rotenoids, paprika containing the two pigments capsanthin and capsorubin, xanthophyll extracts The ability of carotenoids to deactivate re­ from leaves, carrot extracts of varying purity, and active chemical species such as singlet oxygen, red palm oil. 3,76 triplet photochemical sensitizers, and free radi­ Several synthetic carotenoids are presently cals has been actively studied in recent years, available, making it possible for them to be used with the main focus on fj-carotene. Some insight widely in coloring processed and fabricated into the antioxidant activities of other naturally foods. 5 fj-Carotene was the first synthetic caro­ occurring carotenoids has also been reported. tenoid to be marketed in 195476 and is now prob­ Studying the antioxidant activity of l3-carotene ably the most widely used carotenoid for coloring and other related carotenoids on the free radical foods. With the different commercial forms of oxidation of methyl linoleate, Terao l31 reported this carotene, it is technically and economically that canthanxanthin and astaxanthin were more feasible to color a wide variety of fat- or water­ effective antioxidants. It was postulated [hat the based foods including butter, margarine, cheese, introduction of oxo groups at 4 and 4' positions ice cream, wheat products, vegetable oils, cake into these two carotenoids enhanced the antiox­ mixes, candy, soups, desserts, fruit juices, and idant activity of these compounds. Di Mascio et beverages. a1. 132 studied the singlet oxygen quenching ca­ Another commercially available carotenoid, pability of several carotenoids and reported that l3-apo-8' -carotenal, is used where an orange to lycopene was the most efficient in this action. It reddish-orange shade is desired, such as in top­ would appear that opening of the l3-ionon(: ring pings, frostings, candies, confections, pastry fill­ to an open chain, as in lycopene, increases the ings, cheese sauces, cheese spreads, cake mixes, quenching ability. soups, salad dressings. etc. has an It has been suggested that reactive oxygen unusually high tinctorial potency and is a useful species and free radicah may play an important food colorant in the red range, It is used, for role in cancer development. These species are example, in products, soups, barbecue

134 sauces, fruit drinks, simulated meat, and shrimp two kinds of light receptors: rods, for vision in and lobster products. Hathcock et al.133 have cau­ dim light, and cones, for vision in bright light tioned against the excessive use of cantbanxan­ and color vision. Each of these organs contains thin. Persons consuming large number of tanning a photosensitive pigment which bleaches on ex­ tablets (to produce a skin color similar to suntan) posure to light. Some aspects of this process lead containing mainly canthaxanthin were found to to a r,ervous excitation, which, transmitted from have crystalline golden deposits on their retinas. one neuron to another along the optic pathways The use of ~-apo-8' -carotenal in combination with to the brain, ends in exciting visual sensations. 135 ~-carotene, or either one in combination with Four such visual pigments are widely dis­ canthaxanthin, expands the color range of the tributed among vertebrate eyes: rhodopsin and individual compounds. 5 A more comprehensive porphyropsin in rods, and iodopsin and cyanop­ survey of the application of carotenoids in col­ sin in cones. Each is composed of a polyene oring foods and pharmaceuticals is given in Bor­ chromophore united with a specific type of pro­ enstein and BunnelP and Bauernfeind et a1. 76 tein, called an opsin. The visual pigments and Bauernfeind5 surveyed the worldwide legal their opsins are insoluble structural components status of ~-carotene, ~-apo-8' -carotenal, can­ of the receptor organelles, the photosensitive outer thaxanthin, and ~-apo-8' -carotenoic acid ethyl segments of the rods and cones. ester as food colors. ~-Carotene is permitted in Two chromophores are known to be in­ some 40 countries, whereas over 20 countries volved, retinaldehyde and 3-dehydroretinalde­ allow the use of the other three carotenoids. The hyde. together with two main types of opsin, one Food Regulations currently enforced in Malaysia found in rod outer segments and the other in cone also permit the usage of these four pigments in outer segments. The two retinaldehydes, both in foods.!34 the cis form, combine with the two kinds of op­ sins 10 yield the four main classes of visual pig­ ments. The need for retinol to supply the chro­ E. Role of Vitamin A in Vision mophores of the visual pigments is the reason why, in vitamin A deficiency, the first symptom The different categories of function of vita­ is a fall in sensitivity of both rod and cone vision, min A in mammals can be broadly grouped under a condition known as "night blindness" or five headings: (l) vision, (2) bone growth, (3) "nyctalopia" ,135 reproduction, (4) maintenance of epithelia, and (5) overall growth.! The following discussion concentrates on two of these functions: its role in vision and the maintenance of epithelia. F. Vitamin A in Maintenance of Epithelia It has become evident from the work of Wald and Hubbard1J5 that the active retinoid in the vis­ The most striking and extensive lesions caused ual process is ll-cis-retinaldehyde, but the bio­ by vi:tamin A deficiency are those affecting ep­ chemical mechanisms of the other biological ithelial growth and differentiation. They arc all functions of retinoids, for example, maintenance defects of the outer and inner linings of the body of epithelial tissues (discussed below), and other which, consequently, invite invasion by micro­ functions listed above remain unclear. 1,64 organisms. There is generally an increase in the The only established function of retinol in proportion of squamous keratinizing cells (cor­ the retina is to serve as the precursor of ll-cis­ nification), accompanied by a decrease in the retinaldehyde, the chromophore of all known vis­ proportion of columnar, mucus-secreting cells. ual pigments. An outline of the involvement of It is, however, known that different epithelia are the vitamin in the photosensitive system is given affected differently. In epithelial tissues such as below. Comprehensive reviews of the subject have intestinal mucosa, where there are normally no been given by Wald and Hubbard DS and the more keratinizing cells, there is simply a decline in recent publications of Wolf! and Bridges. 136 mucus-secreting cells. In other tissues such as The retinas of most vertebrate eyes possess the cornea and epidermis, where there are nor­

135; mally no mucus-secreting cells, hyperkeratosis have also been estimated on the basis of data results from the deficiency. I .67 obtained from control1ed animal experiments. In the eye, apart from the defects in the retina As with other nutrients, recommended levels caused by disappearance of rhodopsin, many ep­ of intake of vitamin A vary from country to coun­ ithelial lesions occur in vitamin A deficiency. try. This is clearly shown in a study of the rec­ These lesions are manifest in both the conjunctiva ommended dietary allowances (RDA) of 17 and cornea as a result of lack of vitamin A for countries for 9 nutrients by the Committee on maintaining the normal differentiation of epithe­ Recommended Dietary Allowances of the Inter­ lial tissue. The earliest change is in the conjunc­ national Union of Nutritional Sciences. 138 For vi­ tiva tissue, resulting in one or more patches of tamin A, the recommended level for adult men dry, nonwettable conjunctiva, a condition termed and women ranged from 600 to J500 f.Lg RE, conjunctival xerosis. This is sometimes accom­ with a mean value of 910 f.Lg RE. The addition panied by the appearance of small plagues of a amount for pregnancy ranged from 0 to 1000 I-lg silvery grey hue, usually with a foamy surface, RE, and for lactation, from 200 to 1920 f.Lg RE. called bito!' s spots. In more severe vitamin A The safe level of vitamin A intake recom­ deficiency, the structural changes go on further mended by FAO/WHOD9 is tabulated in Table to involve the cornea. In the beginning, corneal 6. These levels are considerably different from xerosis takes place, giving the cornea a hazy ap­ the 1967 recommendations. 7H The recent rec­ pearance. This progresses to corneal ulceration, ommendations are appreciably lower for adults frequently referred to as keratomalacia, and may and older children, but higher for young children. eventually end in impairment of vision in varying The recommendations by the U.S. NAS 79 for degree. These structural changes are described in vitamin A are generally higher than the above­ Section IX.B.2.a. mentioned FAO/WHO recommendations. The recommended allowance for adult men is 1000 RE per day, and for women, 800 RE. For preg­ IX. VITAMIN A IN HUMAN NUTRITION nancy, an additional 200 RE is recommended,

A. Recommended Levels of Vitamin A TABLE 6 Intake Safe Level of Vitamin A Intake

Present knowledge of human vitamin A re­ Age RE per day quirements has been obtained from field surveys Group (yealrs) <....9) and controlled dietary studies. Rodriguez and Irwin!37 have given a thorough review of the stud­ Infants and chil­ 0-1 350 1-6 400 ies carried out to determine these requirements. dre·n (both sexes) 6-10 400 Field surveys provide a general idea of the total 10-12 500 vitamin A intake of popUlation groups, with and 12 15 600 without overt clinical and biochemical vitamin A deficiency symptoms. They are, however, oflim­ ited value in quantitating human vitamin A re­ Boys 15-18 600 Girls 15-18 500 quirements. Most of the controlled studies have Men 8+ 600 been based on dark-adaptation tests. The role of Women li8.,.. 500 vitamin A in the visual cycle is the only metabolic Pregnant women 600 function for which the mechanism has been elu­ Lactating women 850 cidated. Serum vitamin A concentration has been From FAO/WHO, Requirements of Vitamin A, Iron, the second most commonly used criterion. Some Fa/ate and Vitamin B,2• Report of ajoint FAO;WHO investigators have also measured growth, sus­ Expert Consultation, FAO Food and Nutrition Se­ ceptibility to infections, and changes in the ep­ ries No. 23. Food and Agriculture Organization, ithelial tissue. Human vitamin A requirements Rome, 1988. With permission.

136 and for lactation, an extra 400 RE. For children 1. Epidemiology of Vitamin A Deficiency 1 to 3 years of age, however, the recommended allowance of 400 RE per day is the same as the The basic underlying cause of vitamin A de­ FAO/WHO recommended safe intake. ficiency is a chronic inadequate dietary intake. The epidemiology of the problem is, however. rather complex. The factors involved may be con­ B. Vitamin A Deficiency sidered in terms of a host-environment-agent in­ terrelationship, as in other diseases or disorders. Vitamin A malnutrition, as in other nutrient malnutrition, can be of two kinds, namely, ov­ ernutrition and undernutrition. In the fornler, there a. Host Factors is acute or chronic . At the other extreme is hypovitaminosis A or vitamin Age is considered predominant among all of A deficiency. Hypervitaminosis A is relatively the host factors involved. 69 Young children con­ rare and could be caused by self-prescription of stitute the most vulnerable age group, and the large pharmacological doses of vitamin A. In most serious eye lesions commonly occur in them. contrast, vitamin A deficiency is fairly common, This is related to their relatively high vitamin A particularly among poor children in developing requirements for growth, increased needs due to countries. It has been described as among the the frequent occurrence of infections. low intake most widespread and serious nutritional disorders from milk of undernourished mothers. and failure to afflict mankind. 140 The problem has been said to supplement carotene-poor staples with dark

to have remained largely unchecked and contin­ green leaves and other rich sources. 141 ued to be the cause of a high toll in bl indness There appears to be evidence that males are and death among young children. more susceptible to xerophthalmia. This is said The term "xerophthalmia" literally means to be true for all stages of ocular lesions, all ages. , 'dry eye", and in a restricted sense is a term and in many countries. 69 However, reasons for used by ophthalmologists to describe the changes this sex difference are unclear. It is felt that the in the eye that occur when the secretions of the reasons could be more cultural than biologic. 141 paraocular glands or of the goblet cells of the conjunctiva dry up, leading to discontinuity of the fluid films usually present over the surface b. Agent Factors: The Diet of the conjunctiva and cornea. However, in a broader sense, and in a public health context, the Vitamin A deficiency !IS highly prevalent in term has been applied to the syndrome of severe communities where the dietary staple is rice, with vitamin A deficiency. 69 The term has been taken little or no consumption of animal foods, dark­ to cover all the ocular manifestations of vitamin green leafy vegetables, or of yellow/orange fruits. A deficiency (including night blindness), and This is also true for communities dependent on would thus be taken to denote an advanced degree cassava, white potato, or other carbohydrate­ of vitamin A deficiency with a potential threat to dense foods that are virtually devoid of vitamin

sight. 140 Particular emphasis has been given to A and carotenoids. Vitamin A deficiency is es­ the term because of its direct relation to the most sentially a condition of a poor socioeconomic tragic sequel of the deficiency - blindness. environment. 142 In these communities where foods "Vitamin A deficiency" refers to any state from animal sources are too expensive, carotenes in which the vitamin A status is subnOlwal. It from plant sources are of paramount importance. can be presumed to occur when the habitual in­ However, due to ignorance and/or neglect, even take of the total vitamin A is markedly below the these cheaper sources of vitamin A are very often recommended dietary intake. 69 It therefore in­ not given to the children. Thus, it is common to cludes xerophthalmia. find "poverty in the midst of plenty", and de­

137 struction of eyes by xerophthalmia in envlron­ the occurrence of infectious diseases and xeroph­ ments where carotene-rich green leaves are abun­ thalmia.69.77.I44 Any serious illness, whether acute dant. 69 It is therefore ironic that vitamin A or chronic, depresses the appetite, impairs ab­ deficiency should be prevalent in southeast Asia, sorption, increases vitamin A requirements, and amidst plenty of greens and a variety of colored has other effects on general metabolism that may fruits. lower the vitamin status, thus precipitating the 61 As discussed in Section V. C. 1, fats are im­ occurrence of the disease. '.14J Hence, there is a portant in the absorption and metabolism of ca­ close association between xerophthalmia and res­ rotenoids and vitamin A. Where diets are unusu­ piratory tract infections, gastrointestinal dis­ ally low in fat, inefficient absorption of dietary eases, and intestinal parasitic infestations, es­ carotenoids has been implicated as contributing pecially ascariasis. Measles appear to occupy a to development of xerophthalmia. Roels et al. rather special position, and many cases of xe­ reported improved absorption of carotenoids fol­ rophthalmia reported from parts of Africa have lowing improved fat intakes in Ruanda? 1 and been associated with measles. 141 It takes a severe Indonesia. 143 form in the undernourished child, with marked Unsatisfactory early childhood feeding prac­ impairment of the cell-mediated immune re­ tices have an important bearing on the develop­ sponse, and affects the cornea even in well-nour­ ment of vitamin A deficiency. Xerophthalmia is ished children. rarely reported among breast-fed infants and sel­ The interactions betwel.;:n vitamin A and in­ dom reported in children who continue to be fection have recently been given a great deal of breast-fed in the second year. 77 However, this is attention. Various pieces of evidence. including not to be so in many communities since the moth­ laboratory experiments, clinical and epidemio­ ers themselves tend to be undernourished, with logic clues, have shown that vitamin A deficiency a very low vitamin A status, and consequently plays an important role in resistance to infection, the milk produced has a low concentration of the most apparent for respiratory infection. The syn­ vitamin. Early weaning from the breast would ergistic effect between vitamin A deficiency and worsen the situation. This is further aggravated infection may be responsible for excessive child­ when the child is weaned to an inadequate diet hood morbidity and mortality in many developing of rice and other cereals or tubers, somewhat regions of the world. 145 In a group of West Java devoid of vitamin A. Indonesian preschool-age mral children, Sommer et a1. 146 reported that the mortality rate among children with mild xerophthalmia (night blind­ c. Environmental Factors ness and/or Bitor's spots) was on the average 4 times the rate, and in some age groups 8 to 12 Where xerophthalmia endemicity is ex­ times the rate, among children without xeroph­ tremely high, as in parts of Indonesia, the disease thalmia. These investigators further reported that tends to occur all year round, with less seasonal children with mild xerophthalmia were more likely variations. On the other hand, where the inci­ to dt~veJop respiratory disease and diarrhea than dence rate drops, vitamin A deficiency appears non-xerophthalmia children, and that this in­ highly seasonal. 69 The deficiency is associated creased risk was more closely associated with with, among other things, particular seasons in their vitamin A status than with their general which precipitating factors occur, such as dry nutritional status. 147 Further studies carried out periods when the supply of fresh fruits and veg­ by Sommer et al. 14H in Sumatra showed that vi­ etables are scarce. tamin A supplementation (200.000 IU vitamin A twicl~ at six-month intervals) was able to reduce mortality among the preschool children by as d. Vitamin A Deficiency, Infection, and much as 34%. More recently, Rahmathullah et Child Survival al. 149 also reported a drastic reduction (on average by 54%) in mortality among children in southern There is an indisputable relationship between India supplemented with a small (8333 IV) weekly

138 dose of vitamin A. To avoid criticism of the study the population. Before any intervention program design, a randomized, placebo-controlled, mask.ed is launched, a careful assessment of the situation clinical trial among a large number of children should be made. Such surveys should be aimed (over 15,000) was carried out. at establishing: These studies on child survival and mortality created a great deal of interest and debate in the I. Whether vitamin A deficiency exists in the international health and nutrition community. vulnerable age group These findings also had wide policy implica­ 2. The nature, magnitude, severity, and geo­ tions. 150 Questions have been raised regarding the graphical distribution of this deficiency validity of the findings, including those pertain­ 3. Whether this deficiency constitutes a prob­ ing to experimental designs and measurements, lem of public health magnitude and data analysis and interpretation. 15 J.I S2 4. A baseline for evaluating the effectiveness Keusch,153 however, felt that the evidence avail­ of future intervention able support the implementation of vitamin A programs in specific cases - wherever there is Assessment of vitamin A status should include. evidence that a population has a vitamin A de­ whenever possible, clinical examination, bio­ ficiency, wherever protein-energy malnutrition is chemical determinations. and dietary assessment. common, and wherever there is an excess of mea­ Sommer et al.,15M WHO,140 and, more recently, sles deaths. It was, however, pointed out that Underwood l59 have provided some general vitamin A supplementation should not be a sub­ guidelines for the conduct and analysis of such stitute for immunization, primary health care. and surveys. The following subsections outline the improved sanitation and water supplies. The con­ important aspects of each of the assessment meth­ troversy continues, and the Subcommittee on Vi­ odologies mentioned. tamin A Deficiency Prevention and Control of the U.S. NAS has considered studies into vitamin A and child mortality of significant research a. Clinical Assessment priority. 154 In the meantime, the International Vi­ tamin A Consultative Group (IV ACG) has issued As discussed in Section IX. B, vitamin A de­ an interim statement that evidence is accumulat­ ficiency is a systemic disease affecting epithelial ing that vitamin A does reduce mortality by a structures in a variety of organs. the eye being mechanism(s) which is still unclear. 155 The state­ the most obvious and dramatic example. Clinical ment also made it clear that the impact of im­ signs and symptoms OCCUlTing in the eye are spe­ proved vitamin A nutrition will vary with the cific to vitamin A deficiency. Thus. these have severity of vitamin A deficiency and the contri­ been widely used in c1inica.l assessment of vita­ butions of other ecological factors. min A status. Studies into the mechanisms of the interac­ The ocular signs of vitamin A deficiency have tion between vitamin A and infection have been been grouped under the term xerophthalmia, undertaken for many years. Recent findings in­ which literally means "dry eye". The major xe­ dicate that vitamin A deficiency could adversely rophthalmia signs have been classified at the affect epithelial integrity and function, lymphoid WHO/USAlD meeting on Vitamin A Deficiency mass, specific immunity (i.e., cell-mediated and and Xerophthalmia. 69 This was subsequently humoral), and nonspecific mechanisms of host modified in another WHO meeting. 140 The clas­ resistance. 144.145.156.157 sification is reproduced in Table 7. Following this, a brief description of the signs is givi~n. Details have been published in various mono­ 2. Assessment of Vitamin A Status graphs and reviews.o\>,77,I-iO,'4LI60,162 A field guide for the detection and control of xerophthalmia Regional or nationwide surveys should be was also published by WHO. 163 Most of these carried out to determine the frequency (preva­ publications include colored photographs of the lence) and severity of vitamin A deficiency in ocular signs.

139 TABLE 7 Xerophthalmia Classification by Ocular Signs

Night blindness XN Conjunctival xerosis X 1A Bitors spot X1 B Corneal xerosis X2 Corneal ulceration/keratomalacia <113 corneal surface X3A Corneal ulceration/keratomalacia ~'1/3 corneal surface X3B Corneal scar XS XerophthalmiC fundus XF

Reproduced from Control of Vitamin A Deficiency and Xerophthalmia, Report of a Joint WHOiUNICEF/USAIDi Helen Keller International/lVACG Meeting, World Health Organization, Geneva, 1982. With permission.

With the exception of corneal scar (XS) and with a foamy surface, consisting of keratinized, xerophthalmic fundus (XF), the ocular signs in desquamated conjunctival epithelial cells. XIB Table 7 are arranged in increasing order of se­ usually appears on the temporal quadrant of the verity of active vitamin A deficiency. However, conjunctival surface and may occur on one or this does not mean that all the earlier stages have both eyes .. The concurrent appearance of XIA necessarily occurred when a later stage is de­ and Xl B is usually consistent with hypovitamin­ tected. The earliest ocular symptom is that of osis A. night blindness (X~), or impaired dark adapta­ The more serious, blinding effects of severe tion, and is the result oftoo little available retinol vitamin A deficiency involving the cornea consist for the rapid regeneration of rhodopsin in the eye. in the early stages of xerosis, loss of transpar­ The symptom is said to be quite specific for vi­ ency, and nonwettability, as in the conjunctiva, tamin A deficiency among young children, but giving the cornea a hazy appearance (X2 in Table 77 4 is less so in older children and adults. WHOl 0 7). Subsequent to X2, tt.ere is loss of continuity considered it a useful screening tool in that it ofthe epithelium with forrnation of inflammatory correlated closely with other evidence of vitamin "ulcer". Further progression of this corneal ul­ A deficiency, such as serum retinol levels. How­ ceration, frequently referred to as keratomalacia ever, its use requires a careful detailed history (X3A and X3B in Table 7), may eventually end taking and is particularly useful in communities in impairment of vision to varying degrees. where the phenomenon of night blindness is rec­ XS may be indicative of past severe vitamin ognized by the community. A deficiency, but they may be due to other etiol­ The earliest structural change in xerophthal­ ogies as well. When observed, a careful history mia is conjunctival xerosis (XIA in Table 7) con­ must be taken for evidence of trauma rather than sisting of one or more patches of dry. nonwet­ xerophthalmia causative. 77 XF is a rare sign, usu­ table conjunctiva, with loss of transparency. X I A ally accompanied by XN and/or XIA and XIB. may be associated with various degrees of con­ Patients with prolonged and severe vitamin junctival thickening. wrinkling, and pigmenta­ A deficiency may have, in addition to the eye tion. It has, however, been pointed out that these lesions, a widespread dryness, wrinkling, slate­ latter signs are poorly reproducible, highly vari­ gray discoloration, and hyperkeratosis of the skin. able, and nonspecific, and should not be used in It is, however, difficult to separate the role of 141 isolation in making a diagnosis of XIA. ),:64 vitamin A deficiency in ,::ausing these signs from The xerosis process in X I A extends to the the part played by other nutrient deficiencies. formation of Bitot's spots (XIB in Table 7), These Even more controversial is the etiology of fol­ are small plaques of a silvery gray hue, usually licular hyperkeratosis or phrynoderma, ascribed

140 by some workers to be due to vitamin A with older children and adults. Based on analysis deficiency.14o.IM of the HANES data, these investigators found that younger children (aged 4 to 5 years) have lower serum vitamin A levels than the older chil­ b. Biochemical Examination dren (aged 9 to I I years). Plasma carotene concentration is generally Measurement of the serum or plasma vitamin considered not to be a reliable indicator of vi­ A level remains the most practical, available bio­ tamin A status because it retlects the level of chemical means of assessing the status of nutri­ immediate dietary intake. 69 Furthermore, de­ ture in spite of limitations in sensitivity and spec­ pending on the dietary compositlon, a consid­ ificity.77 Since vitamin A is stored in high erablle proportion of the plasma carotenoids may concentrations in the body. almost entirely in the be non-vitamin A compounds. However, when liver. the plasma level does not closely rellect considered with plasma retinol levels, carotenoid the level in the body as a whole. The plasma analysis can be useful. 771Sg The ICNND 16s used concentration reflects the body status only under the levels given in Table 8 for the interpretation two very different circumstances:69 when the body of serum "carotene" leve Is. It would be more stores have been critically depleted, and when appropriate to refer to these levels as . 'total car­ the liver has become saturated with vitamin A. otenoid" since the colorimetric method used is The U.S. Interdepartmental Committee on Nu­ not able to determine individual ,·arotenes. (, trition for National Defense (lCNND)16' recom­ A common method for the analysis of serum mended the interpretation of plasma vitamin A retinol is based on the transient blue-colored com­ levels summarized in Table 8. plex which retinol and its esters form under an­ hydrous conditions with antimony trichloride­ chlorofoml reagent (Carr-Price reaction). After TABLE 8 precipitation of protein with ethanol. vitamin A Interpretation of Plasma Vitamin A and and carotene in serum are extracted into petro­ Carotene Levels leum ether. Carotene in solution can be quanti­ tated by reading at 450 nm. The petroleum ether Vitamin Al100 ml "Carotene"/100 ml is evaporated off and the residue is reacted with Level (l-1g) (l-1g) antimony trichloride-chloroform reagent. The blue High >50 >100 color formed is read at 620 nm. Details of the Acceptable 20-49 40--99 procedure are described in various publications Low 10-19 20--39 (see, for example, Underwood and StekeP67'1. A Deficient <10 <20 common problem encount,;red is that the reagent develops turbidity in the presence of trace amounts Adapted from Reference 164. of moisture. Another difficulty encountered is that the characteristic blue color formed by the Of the categories thus outlined, only the' 'de­ reagent with vitamin A is subject 1:0 rapid fading. ficiency" state, i.e., <10 f-lg/lOO ml, is recog­ In view of these difficulties, various modi­ nized to be universally associated with both low fications have been made to the method. These liver reserves of vitamin A and an increased prev­ include the substitution of trifluoroacetic or tri­ alence of clinical signs of deficiency. The so­ chloroacetic acid for antimony trichloride re­ called "low" category (10 to 20 f-lgIlOO m]) agent, and the development of a micro-method should be interpreted with caution since low for small volume of blood. loX Other methods that plasma values may not be associated with vitamin have been in use include the spectrophotometric l A deficiency, but instead with other conditions method based on CV inactivation "" (also de­ such as inadequate protein intake, parasitic in­ scribed in detail in Underwood and Stekel festation, add liver diseases.69 Lewis et a1. 166 and a fluorometric method.]7O Further examples pointed out the need for different cutoff levels of of all other methods have been described in detail serum vitamin A for young children as compared in the compilation by Arroyave et a1. 171 of se­

141 lee ted methods for the analysis of vitamin A and based qualitalive data to determine the frequency carotenoids in nutrition surveys. More recently, with which different foods are eaten. A compar­ several HPLC methods have also been pro­ ison of three dietary methods for estimating vi­ posed.m.177 A more detailed discussion on de­ tamin A intake was recently reported by Russell­ velopments in the analysis of retinol in serum is Briefel et al. 17:, given in the review by Tee and Lim.n The IVACG recently introduced guidelines for the development of a simplified dietary a~­ sessment to identify groups '" at risk" for inad­ c. Dietary Assessment equate intake of vitamin A.179 The semiquanti­ tative method is particularly suitable for The difficulty of obtaining food consumption communities where almost all the vitamin A in­ data is well documented. Many factors are dif­ take is from carotenoids contained in a limited ficult to control, making the results obtainable number of food groups. Based on food compo­ by even the most conscientious workers approx­ sition tables, these foods are classified into groups imations only. Dedicated workers are defmitely of high, moderate, or low levels of vitamin A a must. activity. It has been emphasized that the simpli­ For the calculation of nutrient intake from fied method will not provide precise quantitative the food consumption data collected, a good food information on levels of intake and, alone, will composition data base is required. It would be not be adequate for a quantitative assessment of quite pointless in taking great pains to carry out vitamin A status of either communities or the data collection, but lack good data on nutrient individuals. composition of foods for calculations. For vita­ min A, obtaining good nutrient intake data is particularly difficult. This is related to the non­ d. Other Assessment Procedures availability of good food composition data bases with accurate vitamin A and carotene values. As Functional biochemical measures have been has been previously pointed out in Section VI.D, proposed as more dynamic means of identifying vitamin A activity of foods in many food tables marginal vitamin A status, either among individ­ may be unreliable due to the lack of precision of uals or population groups 159 Two such functional methodologies for the analysis of vitamin A ,md tests recently advocated are the relative dose­ carotenoid content of foods. In recent years, there response (RDR) test and conjunctival impression has been particular emphasis on obtaining more cytology (CIC). accurate data on the types and concentrations of In the RDR test, two representative serum various carotenoids and retinoids in foods. b levels obtained before and after supplementation Various approaches have been used to assess with vitamin A form the basis of the test. After dietary intake of nutrients. Each has strengths and an initial fasting blood sample is obtained, a liq­ weaknesses, and the method of choice would have uid solution containing about 450 to 1000 IJ.g to depend on the objectives of the study. Dietary retinyl palmitate is given orally. A small meal history to quantitate the exact amounts of foods containing foods with some fat but minimal vi­ eaten, e.g., using the 3 days 24-h recall method, tamin A is given after the dosing. After 5 h, a can provide fairly good data. The method is, second blood sample is taken. Vitamin A in the however, time-consuming, especially when used blood samples is determined and RDR calcu­ for large population groups. In addition, memory lated. An RDR > 20% is considered to be po­ recalls are difficult to administer to young chil­ sitive: and indicative of inadequate hepatic stores dren. Household food consumption data can be of vitamin A. 159 It is clear that the test would be obtained using the frequency of food consump­ very difficult to administer under field conditions. tion and purchase method. Such data, however, CIC has been proposed to be a sensitive and cannot be relied upon to provide an indication of specific indicator for the detection of marginal the intake of individual members of the house­ vitamin A deficiency among young children. hold. A compromise would be to collect family- Specimens of conjunctival epithelium are ob­

142 tained atraumatically by applying strips of cel­ preva]'ence survey in populous Indonesia. The lulose acetate paper to the lower. outer (temporal) incidence (appearance of new cases) of active portion of the conjunctiva for 3 to 5 s. then gently corneal disease in Indonesia was estimated to be peeled away. The paper with the adherent epi­ around 2.7 per 1000 preschool children per thelial cells is then transferred to a fixative so­ year. 162 Given an incidence of this order. the lution, stained, and examined under a micro­ Administrative Committee on Coordination-Sub­ scope. The principle underlying the technique is committee on Nutrition of the United Nations that, since vitamin A is needed for maintenance (ACC-SCN)18t> has estimated thaI 400.000 to of nonnal mucous epithelium, deficiency states 500,000 preschool children in Bangladesh, India, brings about changes to the morphological char­ Indonesia, and the Philippines combined will de­ acteristics of the epithelial cells. The number of velop active corneal lesions resulting in partial goblet cells decreases and may even be absent in or total blindness. It has been further pointed out vitamin A deficiency. At the same time, meta­ that these are conversative estimates since the plasia of the epithelial cells occurs. 180 It is clear assumption that identical incidence rates prevail that some training and experience is required to for all four countries is not correct: higher rates obtain a good impression of cells from the con­ are known to occur in Bangladesh and India. The junctiva. The criteria for normal and abnonnal incidence of non-corneal xerophthalmia (mild. cytology should be clearly defined. The proce­ and generally reversible forms of the disease) in dure has been tried out by various investigators these countries has been estimated to be in the (e.g.. Natadisastra et aL I81 Kjolhede ct al. .. l~2 order of 5 million preschool children per year. and Reddy et al. 183). Reports on the validity of Applying these estimates from the four Asian the procedure have not been consistent and m:ed countries mentioned previously, a worldwide confinnation. Natadisastra et al. 181 reported ele projection has been made of 700.000 cases an­ to be highly sensitive and specific for the detec­ nually of new active corneal lesions among pre­ tion of physiologically significant vitamin A de­ school children, The total incidence of non-cor­ ficiency. On the other hand, when compared with neal xerophthalmia among preschool children has fasting serum vitamin A levels and RDR, Ga­ been similarly estimated to be 6 to 7 million new domski et al. IM reported low sensitivity and po­ cases per year, and 20 to 40 million suffering sitive predictive value of Cle. from at least mild deficiency at anyone time. of which nearly half are in India. 187 WH069 had suggested that. with respect to 3. Prevalence of Vitamin A Deficiency prevalence of vitamin A ddiciency. the world may be divided into three regions: In most reviews on the prevalence of vitamin A deficiency, the global survey by Oomen et 1. Technologically developed countries where al. 185 has been quoted. Drs. Oomen. McLaren. there is no xerophthalmia problem but some and Escapini visited 36 countries and compiled hypovitaminosis A may exist data based on hospital records. government s.ta­ 2. Some rice-dependent developing countries tistics, questionnaire studies, and personal ob­ of Asia where there is a problem of public servations. Based on this WHO-sponsored global health magnitude survey and a detailed notification system In Jor­ 3, The rest of the world, including Africa. Latin dan, the annual incidence of xerophthalmia in the America, and the Middle East, where the world prior to 1970 was estimated to be problem is not ex.tensive but is intermit­ 100,000. 140 The areas found to be most affected tently intensive and highly sensitive to were the overpopulated, rice-dependent countries changing social and economic conditions of Asia. Other regions that were affected to a lesser extent include Africa, Latin America. and Some detailed descriptions of the problem as en­ the Middle East. countered in each of these three regions are given Subsequently, surveys have been conducted in several rev iews (e, g .. see Reference 69, 77, in several countries, most notably the nationwide 140, and 188). A map showing the world geo­

143 graphical distribution of vitamin A deficiency and 4. Prevention and Control of Vitamin A xerophthalmia in 1984 has been provided by re­ Deficiency ports of the ACC-SCN. Igo-187 A summary of the vitamin A deficiency problem of over 30 coun­ Vitamin A deficiency has been considered tries is also given in the 1985 report. one of the "big four" nutritional deficiencies in A review of the vitamin A deficiency prob­ developing countries; the other three are protein­ lem in Malaysia was earried out, based mainly energy malnutrition, iron deficiency anemia, and on the annotated bibliography of nutrition re­ endemic goiter due to iodine deficiency. Vitamin search in the country by Tee l89 for the period A deficiency is the most important cause of pre­ 1900 to 1979 and Tee 190 for the 1980 to 1984 ventable childhood blindness in these countries. period, and various recently published reports. In recent years, there has been an increasing ap­ Various studies carried out in different parts of preciation by governments and international the country have shown vitamin A deficiency to agencies of the magnitude of the problem and of be an important sight-threatening disorder, af­ the means available for dealing with xerophthal­ fecting mainly young children on imbalanced mia. 1'l1 Various intervention strategies have been diets. It was found to be the major single cause implemented to combat the problem, and vol­ of blindness. The disease was most prevalent umes have been written on these pro­ among the lower socioeconomic segments of the grams. 69.140. 192· 195 Intervention programs to pre­ population. Vitamin A intake was generally low, vent vitamin A deficiency are in operation with little or no retinol, and most of it from the nationally in at least 8 of the 34 countries with provitamins. Even then, the amount of vegetables known vitamin A deficiency. 187 Coverage of the and fruits consumed was generally low. population at risk in some of these countries has No exact estimates of the magnitude of the reached sufficiently higb levels, while in others problem were available However, as seen from coverage is still unsatisfactory. The Administra­ reports in the literature, the problem app,eared to tive Committee on Coordination of the United be confined to certain groups, mainly in the rural Nations has emphasized that, if intervention pro­ areas, and did not pose a major health hazard grams are energetically implemented, vitamin A nationwide. The problem appeared to have less­ deficiency could be reduced to a level tbat it no ened over the years, judging from reports up to longer poses a public health problem. 187 the late 1970s and the early 1980s. There are The objectives of an intervention program probably very few cases of children with eye must necessarily depcnd on many factors, such signs past XIA, and serum vitamin A <10 f,Lgl as the severity of the problem. Inadequate vita­ m1. It is, however, recognized that there are many min A status varies from a marginal condition of remote areas in the country where the vitamin A inadequate body reserves of the vitamin without status is not known, including parts of peninsular clinical signs, through the presence of early and Malaysia. Furthermore, the problem among ur­ reversible clinical signs, to a severely depleted ban squatters is little studied. Most of the studies statle mainly characterized by advanced corneal relied on the less precise clinical signs and dietary changes and the high probability of blindness. 14U inquires due to the lack of laboratory facilities to Control programs should therefore be directed carry out biochemical investigations. Even for toward the particular problem at hand. the latter, investigators have been faced with the Control of vitamin A deficiency may be ap­ difficulty of obtaining sufficient blood from sub· proached through one of three major intervention jects. A reliable micro-method would have to be strategies: established for local use. Extensive mapping of the vitamin A status of children in the country I . Improvement of dietary vitamin A intake remains an important task. 2. Vitamin A fortification of foods

144 3. Periodic administration of a massive dose for vitamin A fortification. it should have the of vitamin A following characteristics:

I. The food should be technically fortifiable. a. Improvement of Dietary Vitamin A Intake 2. It should be widely or universally consumed by the popu lation of the target area, in quan­ Dietary improvement has been recognized as tities that will make a significant contri­ the major long-term solution to controlling vi­ bution to the diet. tamin A deficiency in a community. Through a 3. There should be little variation in per capita combination of horticultural and related activities daily consumption of the food. and educational and socioeconomic inputs the 4. The food item should show no appreciable habitual intake of food sources of vitamm A is changes in its organoleptic characteristics, gradually increased and maintained at a level so that its acceptability is not affected after which minimizes the risk of developing vitamin addition of vitamin A. A deficiency among the vulnerable segments of 5. It should be economically feasible to fortify a population. 194 Even when other intervention the food on an industrial scale. strategies have been implemented. thesl~ mea­ sures should be carried out as on-going, long­ Other important factors in nutrient fortifi­ term strategies. cation of foods include: In those parts of the world where vitamin A deficiency is prevalent, vegetable products are 1 . The addition of the nutrient should not cre­ the main source of dietary vitamin A in the form ate an imbalance of essential nutrients. of carotenes. Thus. horticultural and related ac­ 2. The nutrient added should be stable under tivities to increase the availability of carotene­ proper conditions of storage and use. rich fruits and vegetables have been empha­ 3. The nutrient should be physiologically sized. 140 These would include not only matters available from the food. related to production (varieties for promotion, 4. There should be n~asonable assurance pest and disease contro!), but also problems of against excessive intake to a level of nu­ transport, marketing, storage, and preservation. trient toxicity. These activities should be coordinated with the aspects of nutrition education programs since Examples of fortification of vitamin A in availability does not necessarily mean consump­ sugar in Central America, monosodium gluta­ tion of these foods by the vulnerable segments mate in the Philippines, and dried skim milk in of the population, especially young children. The various food aid programs have been described reasons why these foods are not consumed in by WH 0. 140 The fortification of milk powder and significant amounts, even in areas where they are margarine with vitamin A in this country has been abundant, should be understood. practiced for some years. Bauernfeindll7 has dis­ cussed in detail the fortification of various foods and the technology of the process. b. Vitamin A Fortification of Foods Depending upon local factors and program costs, fortification can offer both short- and long­ Fortification or nutrification of a widely con­ term solutions in a chronlcally vitamin A-defi­ sumed food with vitamin A offers a major tech­ cient population. 194 Since the amounts of vitamin nically feasible intervention which can be rela­ A added are adjusted to permit the recipient to 19 tively inexpensive and effective. 1.l94 Various receive the approximate RDA for vitamin A, there considerations would have to be given before is no risk of potential hypervitaminosis A. To fortification can be considered a viable interven­ date, there are no indications clinically or bio­ tion. First, with respect to a suitable food vehicle chemically that hypervitaminosis A has resulted

145 from any of the nutrification projects cUlTently has the advantage of immediate implementation. in operation. 193 but suffers from the disadvantage that it applies only to the isolated nutrient vitamin A, and re­ quires repetitive administration. In addition. c. Periodic Administration of a Massive complete coverage is seldom achieved. Of the Dose of Vitamin A total cost, the major percentage is in the mech­ anism for delivery and not of the nutrient per se. On the basis of available evidence from nearly Education in health and nutrition should be an 2 decades of experience, large-dose vitamin A integral part of the periodic dosing program. distribution can generally be regarded as a safe Underwood '97 has emphasized that, although these and potentially effective intervention to prevent programs have proven to be beneficial, the focus 195 xerophthaimia. ,196 The underlying objective is should be on approaches that foster practical so­ to maximize liver stores from a single dose with lutions attainable through better utilization of little, if any, risk of acute toxicity. A 200.000 available food and other resources. Although these IU dose of vitamin A (l10 mg retinyl palmitate preventive measures are more difficult to imple­ or 66 mg retinol acetate). with 40 IU vitamin E ment and take longer to bring about the needed in an oil solution for oral administration, given behavioral changes, they are more permanent. every 3 to 6 months is used in most prevention More importantly, these measures address health programs described in the literature. 69, 117, 1~O.194.195 and nutrition issues that commonly coexist with The program had been instituted in the early and vitamin A deficiency. mid-1970s in several countries with a severe xe­ Three models of delivery system have been 140 l rophthalmia problem. These included India, suggested: 4(j medical, universal, and targeted. Bangladesh, Indonesia, Haiti, Sri Lanka, and the The medical (or therapeutic) system is appropri­ Philippines. The successful implementation of a ate as an initial measure in any endemic area, massive vitamin A supplementation program and provides vitamin A prophylactically (as part covering 229 villages in Sumatra has b(:en re­ of their treatment) to all preschool-age children cently reported by Sommer et al. 148 who are ill or with deficiency signs and come to A large number of studies have been carried hospitals, clinics, and health centers. The tar­ out on various aspects of the massive dosing of geted system focuses on specific high-risk groups, vitamin A. The IVACG monograph 194 and a re­ defined usually by age and/or location. In the cent WHO'96 booklet provide details of various universal (or prophylactic) system. all preschool­ aspects of the subject, including program imple­ age children within a designated area receive a mentation and effecti veness, estimates of the pro­ periodic dose of vitamin A at established intervals. tective period, and efficiency of absorption and retention of the administered dose. Bauernfeind '93 has discussed in detail aspects related to safety d. Evaluation of Vitamin A Deficiency of use of vitamin A in the program. Intervention Programs Although the approach appears conceptually simple, the adequacy and efficiency of programs A great deal of attention has been given to pose major challenges and determine their suc­ the development of strategies for the control and cess. Since the intervention involves a deJiverer­ eradication of vitamin A deficiency all over the recipient individual contact at specified times, a world. Many of these intervention programs have delivery system with a high requirement of per­ been carried out without appropriate evaluation. sonnel is needed to execute the program, How­ As with other intervention measures, lack of ever, the personnel involved need not have a high proper evaluation has resulted in the inability to level of technical expertise. Distribution systems determine whether the programs have produced should be designed and implemented with em­ the expected results. Even if changes are ob­ phasis on primary level conditions, existing in­ served, it is not possible to estimate the extent frastructures, and ongoing health activities, with to which those changes may be attributed to the the involvement of local leadership. This strategy programs. Recognizing thi~,. the IV ACG has put

146 forth a useful manual for monitoring and eval­ a detailed review of the animal studies carried uating vitamin A deficiency intervention pro­ out on toxicity and overdose of vitamin A. grams. 198 Details for evaluating the intervention Bauernfeind, 193 who has given a detailed ac­ programs discussed herein are given. Methodol­ count of the subject, estimated that worldwide ogies described include indicators for evaluation several hundred instances of hypervitaminosis A and sampling design. probably occur annually, 90% of which cause fleeting side effects which are self-correcting within hours or a day after a single high level C. Hypervitaminosis A dosage. The remaining cases may cause suffi­ ciently severe symptoms to require weeks or The intake of vitamin A above that \\/hich an months to reverse. No deaths have been attributed individual can metabolize, either in a single ex­ solely to hypervitaminosis A. Vitamin A toxicity cessively high intake (acute) or very high intakes therefore remains a very minor clinical or nutri­ for prolonged periods (chronic), causes hyper­ tional problem. When hypervitaminosis A is sus­ vitaminosis A. It is essentially a result of abuse pected, a history of vitamin A consumption should of this essential nutrient. Acute hypervitaminosis be taken from the patient and serum retinol1evel A has been reported to occur with ingestion of a determined. If overdosage is ascertained, the single massive dose of 100,000 f,Lg vitamin A in simple remedy is to stop the excessive vitamin infants and young children, and 600,000 f,Lg in A intake. adults. Chronic hypervitaminosis A may occur However, there is growing concern, espe­ in infants after 4 months of receiving 10,000 f,Lg cially in devdoped countries, that prevalence of daily, and in children and adults taking between chronic hypervitaminosis A may increase in fu­ 20,000 and 50,000 f,Lg daily for several years. 194 ture years. 77 ,201 There is fear that excessive intake Reviewing the evidence, Hathcock et al. 33 con­ could result from misuse or overconsumption. cluded that exposure to high doses of vitamin A either out of ignorance or carelessness or out of (2:30,000 f,Lg/day) for relatively short periods the misconception among food faddists that con­ (days or a few weeks) or lower doses (7500 to tinued excessively high intakes will provide some 15,000 f,Lg/day) for periods of several months or unusual health benefits. Vitamin A preparations more can produce multiple adverse effects. Sev­ are available commercially without prescription eral predisposing conditions, such as viral hep­ in concentrations of up to 7500 RE, and there atitis, cirrhosis, and other liver diseases, may arc those who advocate routine megavitamin greatly increase susceptibility and thus lower the ingestion. Furthermore, for young children who amount of vitamin A necessary to produce ad­ are given concentrated vitamin supplements as verse effects. 133 Blood levels of vitamin A be­ well as fortified foods, they could be on the verge come very high in hypervitaminosis A, well in of toxicity. Such fears are real, and even devel­ excess of 100 f,Lg/dl. 77 Various clinical symptoms oping countries should be wary of the situation. 7 have been recorded, including nausea, head­ Underwood ! had also pointed out that in view aches, vomiting, diarrhea, irritability, drowsi­ of the linkage of retinoids in animal studies to ness, fatigue, and abnormal responses of the cancer prevention, and the epidemiologic asso­ blood, skin, hair, and bone.2.143.2(XJ.201 These ad­ ciations in humans of dietary and serum levels verse symptoms are expected to vary with the of vitamin A to cancer risk, there is the danger dose and the duration of exposure, as well as that this will lead the uninformed public to in­ with the age of the individual exposed. A rapid creased consumption of megadoses. The Com­ recovery usually results when excessive intakes mittee on Dietary Allowances of the U.S. Na­ are discontinued, and no permanent health effects tional Research Council has recommended that ZllO are known to occur. 77 Bendich and Langseth regular ingestion of supplements of retinol ex­ and 0lson201 have, however, emphasized the dan­ ceeding 3000 RE by infants and children be un­ ger of possible teratogenic and embryotoxic ef­ dertaken only under the direction of a physician, fects of excess vitamin A intake in women of and that for adults, regular ingestion of more than child-bearing age. Hathcock et al. m have given 7500 RE daily is not prudeat. 79 Hathcock et al. 1J,'

147 have emphasized that educational efforts s,hould a permanent change to a few cells of the affected point out clearly that there are no well-established tissue. In the preneoplastic or latent stage, the therapeutic or prophylactic benefits to the inges­ affected cells gradually develop into cancer cells. tion of intakes above the usual RDA, and that A characteristic feature of this phase is that the toxicity is possible from excessive intakes. preneoplastic cells can sometimes remain trans­ Excessive consumption of carotenoids does formed but not multiply for a long time. These not generate a correspondingly high vitamin A cells can sometimes revert to normal or be pre­ tissue content and does not cause hypervitami­ vented in their progression toward the tumor nosis A, although there is usually a rise in serum phase. Finally, in the third stage, the transformed retinol level. 12Y This has been attributed to the cells are permitted or encouraged to multiply rap­ sharp decline in efficiency of intestinal absorp­ idly, transforming the tissue into a tumor. It is tion and rate of conversion of carotenoids to vi­ now known that cancer incidence is influenced tamin A in the body. I,}} Individuals who consume by various environmental factors, aside from ge­ high levels of carotenoids in pharmacoliogical netic factors. The environmental factors include dosage forms, or as large quantities of carotene­ the air, water, and food. 204 The food we eat daily rich fruits, will develop a yellow or orange pig­ can participate in tumor development in that cer­ mentation of skin, especially the palms of the tain food components can act as initiators (car­ hands and soles of the feet. Hypercarotcmemia cinogens) and/or promoters (cocarcinogens), results, and serum levels of carotenoids may be thereby increasing the incidence or speed of de­ in excess of 300 f.lg/dl. 71 These conditions have velopment of particular tumors. no effect on health and will slowly disappear On the other hand, the foods we eat may following ,elimination of the excessive dietary ca­ contain components (anticarcinogens) that can act rotenoids. Reviewing the evidence, Bendich202 to block initiation. to enhance the immune sys­ and Hathcock et al. In concluded that dai:ly sup­ tem's ability to identify and destroy transfonned plementation with high doses of !3-carotene for cells, or to inhibit development to promotion extended periods of time is not associated with stage, thereby lowering incidence or slowing tu­ other side effects. mor development. Epidemiologic studies, sup­ Hypercarotenemia, also known by other tenns ported by experimental observations in labora­ suc h as "carotenemia", "hyperli pochromia ' , , tory animals have suggested that dietary practices .,xanthemia". and' 'carotenosis cutis" . may also are a promising area to explore in the search for be associated with various conditions other than prevtmtive measures against cancer. 205 As Peto 129 excessive dietary intake. These include dia­ et aJ. 206 explained, it would be more attractive to betes mellitus, , hypothalamic discover anticancer substances in certain foods amenorrhea, , liver disease, and that can be prescribed rather than carcinogens that certain inborn errors of metabolism. Although the must be proscribed since people are more willing mechanisms of hypercarotenemia in these con­ to accept prescription rather than proscription. ditions have not been fully understood, it is known Thus, the role of diet in cancer development that the decreased rate of conversion of carotene has been actively studied in recent years. Various to vitamin A is an important factor. epidemiologic and laboratory studies have been carried out. In the past 20 years or so, attention has been focused on carotenoids and pre-fomled D. Carotenoids, Retinoids, and Cancer vitamin A. The possibility that carotenoids and retinoids may playa role in cancer development Cancer is a condition of unrestrained cellular has added a whole new, exciting dimension to growth, commonly associated with poorly dif­ the studies of these compounds in human ferentiated cells. The condition arises from a nutrition. myriad of causes and may affect cells from es­ Several lines of evidence suggest that im­ sentially all tissues. 203 There appears to be three portant relationships exist between carotenoids, distinct stages in tumor development. I The first retinoids, and cancer. 2(J7~2Ifl These are based on is the initiation stage, when a carcinogen causes experimental studies of retinoids and carotenoids

148 04 2 in cancer, epidemiologic evidence suggesting as­ self.2 . 1 1 Since cancer is essentially a process sociations between reduced risk of cance:r and of loss of cellular differentiation, while retinoids vitamin A status, and findings from clinical trials are involved in the induction or enhancement of and studies in the field of oncology. Some recent cellular differentiation, the possibility that reti­ findings from each of these three areas are high­ noids could arrest or reverse the carcinogenesis lighted herein. process seemed logical and worthy of exploration. 21 Since the late 1970s. there have been nu­ 1. Experimental Studies merous studies into the ability of retinoids to modify the processes of cell differentiation and Little is known, except in the visual process, proliferation. Studies have shown that retinoids about the mechanisms of vitamin A action at the can act directly on cells in culture to suppress molecular level. However. it has become increas­ the process of malignant transformation. whether ingly clear that the "action" of vitamin A is caused by chemical carcinogens, radiation. or shown most dramatically in its ability to control viral transforming factors.:~ 12 They have been and direct differentiation of epithelial tissues. In shown to be able to suppress the process of car­ all species studied thus far, including humans, cinogenesis in vivo in experimental animals. There vitamin A has been shown to exert a profound is now extensive literature on the ability of re­ effect on differentiation. 33 There are numerous tinoids to suppress the development of the ma­ epithelial tissues throughout the body that are lignant phenotype in vitro. More recently. it has totally dependent on retinoids for their proper been shown that retinoids can exert effects on differentiation and growth. In some epithelia, such certain fully transformed, invasive. neoplastic as those of the trachea and bronchi. a potentially cells. leading in certain instances to a suppression premalignant lesion occurs in the absence of re­ of proliferation, and in other instances to terminal tinoids. Sporn et a!. 2ll observed a loss of normal differentiation of these cells, resulting in a more columnar ciliated and mucus cells in tracheal ep­ benign. nonneoplastic pher:Dtype. ,,13 There are, ithelial organ cultures. as well as development however, only a limited number of instances in of lesions of squamous metaplasia with heavy which such profound effects of retinoids on dif­ keratinization. Addition of retinoids to the cul­ ferentiation and proliferation of tumor cells have tures after development of such lesions caused been shown. Several detailed reviews of studles reversal of the process of keratinization and re­ of effects of retinoids and possible mechanisms placement of the abnormal squamous cells by of action have been published.32.213214 columnar ciliated and mucus cells. According to With the encouraging results obtained with these investigators, epithelial tissues that depend retinoids, it was not unexpected that attention on retinoids for normal cellular differentiation soon focused on carotenoic1s as well. However, and growth account for well over half of the total compared with the retinoids, there are fewer ex­ primary cancer in both men and women. Besides perimental data on the effects of carotene or other bronchi and trachea. the affected organ and tissue carotenoids on cancer risks.206 In the recent re­ sites include stomach. intestine uterus. kidney view by Go()dwin.~6 it was pointed out that two and bladder, testis. prostate, pancreatic ducts, carotenoids, I=J.-carotene and canthaxanthin, were and skin. able Ito delay growth of tumors in mice induced From the fact that vitamin A deficiency pro­ by UV-A, UV-B. benzpyrene (DP), BP/UV-A. duces conditions at least morphologically similar and 8-methoxypsoralen, irn~spective of their in­ to those found during the pre neoplastic stages of herent ability or otherwise to act as precursors of carcinogenesis (e.g., squamous metaplasia in tra­ vitamin A. In addition, I=J.-carotene was found to cheal epithelium), it has been predicted that the have a positive effect against 9.1O-dimethyl-l vitamin may prevent the carcinogenic process benzanthracene (DMBAHnduced tumors. It was from proceeding to the final stage by reversal or thought that, since camhaxanthin was not effec­ repair of epithelial, or by enhancement of the tive in this case, prior conversion into vitamin A intrinsic capacity of the tissue to repair it­ may be required for activity. Phytocne was found

149 to be much less effective than the other two ca­ take, can be estimated. The advantage of such rotenoids in protecting against tumors induced by studies is that they yield results faster, without DMBA/croton oil or DMBA/UV-B. Temple and the need to wait for years to see who develops Basu 215 had also reported that (3-carotene has a the disease. Furthermore, they involve much less strong inhibitory action against 1,2-dimethyl-hy­ work. drazine (OMH)-induced colon tumors in mice. An often-quoted investigation into the rela­ The level of carotene supplementation in the study tionship between serum retinol level and risk of was within the nutritionally relevant range for cancer is the study of Wald et a1. In a pro­ humans. spective study of about 16,000 men. serum sam­ Recently. Schwartz and Shklar216 repOlted the ples were collected and frozen. Retinol levels effectiveness of (3-carotene and canthaxanthin in were later measured in the stored samples from bringing about regression of DMBA-induced car­ the 86 men who were subsequently notified as cinomas of hamster buccal pouch. l3-Cls-Reti­ having developed cancer and from 172 controls noic acid was said to have no effect in the study. who did not develop cancer. It was found that In a further communication, these investigators low retinol levels were associated with an in­ reported that algae extract rich in carotenoids, creased risk of cancer, pal1icularly lung cancer when administered by mouth, prevented tumor and gastrointestinal tract cancer. Wald et al. 219 formation in the hamster buccal pouch experi­ reported findings of a similar study a few years mental model. later. Plasma samples from 5004 women were collected and stored. The levels of retinol. 13­ carotene, and vitamin E in 39 women who sub­ 2. Vitamin A Status and Carcinogenesis: sequently developed brea:,t cancer were mea­ Epidemiological Studies sured and compared with levels from 78 controls who did not develop cancer. In this study, how­ As has been discussed previously, studies in ever, it was found that plasma retinol levels were experimental animals have shown that retinol de­ not related to the risk of breast cancer. (3-Caro­ ficiency enhances susceptibility to chemical car­ tene levels showed a tendency to be lower in cinogenesis. Turning now to epidemiologic stud­ women who developed cancer than in controls. ies, several have reported that vitamin A status but the difference was not statistically significant, in humans may be related to cancer development. On the other hand, low plasma vitamin E was With respect to vitamin A status, two groups of found to be associated with a significantly higher studies may be differentiated. The first deals with risk of cancer. serum retinol or carotene levels and their asso­ A similar study was reported a few years later ciation with cancer risk. Investigations into the by Willett et aL 220 Blood specimens were col­ relationship between dietary retinol or carotene lected from 4000 subjects who were free of can­ intake and cancer risk are in the second group. cer at the time. These subjects were enrolled for For both groups of studies, two approaches the Hypertension Detection and Follow-up pro­ to the investigation have been used. The first is gram. During a 5-year follow-up period, I I I sub­ the prospective cohort study. where a group of jects were diagnosed as having cancer. Blood subjects is followed for a sufficient period of from these patients were analyzed for retinol. time. The second approach is the retrm,pective retinol-binding protein, vitamin E, and total ca­ case-control study. More investigations based on rotenoids. These measurements were compared retrospective serum studies or dietary question­ with those in 210 controls 'who were matched for naires have been carried out. In the case of dietary age, sex, race, and time of blood collection, and studies, these involve asking people with cancer who remained free of cancer. Levels of these cases and people without cancer controls their parameters were found to be similar for cases and dietary intake of carotene and pre-formed retinol controls. Thus, the study was not able to confirm in previous years. From these data, the relative other findings that low levels of vitamin A are risk of cancer in people with low vitamin A in­ related to cancer incidence. Similarly, using the take, as opposed to that in people with high in­ same approach, Friedman et al. 221 also reported

150 no difference in retinol and retinol-binding pro­ One of the early prospe,;:tive epidemiological tein between lSI cases who developed cancer investigations into dietary vitamin A intake and after a 7-year follow-up, and 302 controls. It was cancer risk was that of Bjelke.m A total of 8278 concluded that retinol and retinol-binding protein men were followed-up for 5 years through postal levels were not useful in predicting the subse­ questionnaires regarding their smoking and di­ quent development of lung cancer. etary habits. "Vitamin A index" was computed In another recent case-control study of 99 for the respondents and cOlTelated to occulTence patients with lung cancer and 196 matched con­ of lung cancer within the group. It was observed trols in Washington County, Maryland, the re­ that the index for vitamin A intake was inversely lation of serum retinol, ~-carotene, vitamin E, associated with lung cancer incidence at all levels and selenium to cancer risk was reported. 222 A of cigarette smoking, and most clearly expressed strong inverse association between serum ~1-car­ among those aged 45 to 64 years. otene and the risk of squamous-cell carcinoma Results from another prospective epidemio­ of the lung was observed. An association between logical study of 1954 middle-aged men employed low levels of serum vitamin E and the risk of any at the Western Electric Company's Hawthorne type of lung cancer was also reported. Works in the Chicago Metropolitan area was re­ Other investigators had used the approach of ported by Shekelle et al. 224 The study commenced retrospective case-control studies to study the as­ at the end of 1957. and the men were followed sociation. Peto et al. 106 reviewed the findings of up for 19 years. Dietary intake of retinol and nine such studies carried out in India, Pakistan, carotene were assessed. It was found that only East Africa, the U.K., and the U.S .. with most dietary carotene was inversely related to the in­ of them reported in the late 1970s. Blood retinol cidence of lung cancer. Intake of pre-formed vi­ levels in recently diagnosed cancer patients were tamin A and of other nutrients was not signifi­ compared with levels of control patients. i\lI these cantly related. In addition. it was observed that studies reported lower retinol levels in cases than neither carotene nor retinol intake was signifi­ in controls. In six of these studies, blood ~-car­ cantly related to the risk of other carcinomas otene levels were also studied. In the Indian, grouped together. Pakistani" and East African studies, blood car­ Positive results were reported by Colditz et otene levels were found to be significantly lower al. 225 in a prospective cohort study of 1271 Mas­ in cases, compared with the controls. The review, sachusetts elderly residents (66 years and above). however, pointed out that these studies have not In this 5-year follow-up study, dietary informa­ sufficiently shown the inverse association be­ tion was obtained by food frequency question­ tween either retinol or carotene with cancer. It naire. After controlling for age and smoking be­ was felt that these were relatively small studies, havior. it was found that those in the highest and in some of them, the controls might not have quintile of intake of carotene-rich vegetables had been appropriately chosen. a lower risk of cancer mortality than that of those As mentioned earlier, another group of epi­ in the lowest quintile. The trend of decreased demiologic studies investigated the relationship cancer risk with increasicg intake of carotene­ between dietary intake of retinol and carotene and containing vegetables was statistically signifi­ cancer risks. According to Peto et at., 206 many cant. The investigators emphasized that even in of these studies have investigated this relation­ old age, higher intakes of green and yellow veg­ ship almost by accident because dietary ques­ etables are still associated with lowered risks of tionnaires designed for more general purposes cancer death. However, they pointed out that, happened to ask about the main local carotene from such observed associations, it is still not source. It was realized only later that the lower known whether the protective relationship with cancer risks found among users of a particular such vegetables is truly one of cause and effect. foodstuff might be due to its carotene content. and still less is known which components of such Findings from some of these epidemiologic stud­ vegetables are chiefly iEvolved. ies are summarized below to illustrate the design A more recent prospective study was reported of the studies and the findings obtained. by Paganini-Hill et al. 226 of a cohort of 10,473

151 residents in California who were initially free of cases showed a significantly higher proportion cancer and followed from 1981 to 1986. A hea.lth with lowered total vitamin A intake levels. survey questionnaire was completed by all mem­ Peto et aI.206 analyzed findings from 5 pro­ bers of the cohort. and information obtained In­ spective and 15 retrospective studies on the re­ cluded usual frequencies of consumption of cer­ lationship between dietary vitamin A intake and tain food items including fruits and vegetables, cancer risk carried out in various countries. Most dairy products, liver, and cereal, as well as vi­ of these dealt with intake of carotene-rich veg­ tamin supplements taken. Cancer was con finned etables. Varied relativf~ risks (lower:higher in­ in 643 subjects and included occurrence in var­ take) were reported. with many around].5 to 2. ious sites. Findings of the study showed little Many of the epidemiological studies carried indication that increased intake of vitamin A or out have indicated a slightly lower-than-average ~-carotene from the diet or supplements de­ incidence of cancer among people with an above­ creased the risk of development of cancer. average vitamin A status. The studies. howev(~r. The study of Ziegler et al. 227 is an example have not produced consistent results and suffi­ of a retrospective case-control dietary investi­ cient proof. IY9.206.227.230 The negative results from gation of the association. It was a study of lung some of these studies could be due to the limited cancer in white males. conducted between 1980 number of high-risk individuals in the study pop­ to ]981 in six high-risk areas of New Jersey. ulations. Willett et al.~20 felt that, if indeed an Dietary intake of carotene, pre-formed retinol, inverse association between serum retinol and and total vitamin A were determined by inter­ overall cancer incidence exists, it is probably viewing 763 cases and 900 controls or their next complex and limited to certain population of kin. Subjects were asked about their usual subgroups. Such subgroups may be defined by frequency of consumption, several years earlier, extreme values of serum retinol, certain levels of of 44 food items, which were thought to provide other nutrients, or specific types of cancers. 83% of the vitamin A in the American diet. and There appears to be greater emphasis on the about their use of vitamin supplements. A sta­ possible protective effects of carotene. Inverse tistically significant association between in­ associations were noted more frequently with in­ creased risk of lung cancer and decreased caro­ dices of carotene intake or carotene-containing tene intake was obtained. No inverse asso·:::iation foods than with indices of pre-fonned retinol or between retinol intake or total vitamin A intake retinol-containing foods. 203.207 .209.2::7 Carotenoids with risk of lung cancer was observed. as a whole, appear to be more promising as pos­ Another case-control study was conducted sible protective agents.36.203.231 It appears that in among the multiethnic population of Hawaii be­ the nonnal dietary range the intake of pre-fomled tween 1979 and 1982 by Hinds et al. m Detailed vitamin A in individuals with an adequate vitamin dietary interviews were carried out for 364 pri­ A status, such as in developed countries, does mary lung cancer patients and 627 general pop­ not seem to be associated with protection against ulation matched controls. It was found that total cancer. In other words, although some protection vitamin A intake (food sources plus supple­ may be seen by increasing vitamin A intake in ments), vitamin A intake from food sources only, the lower consumption levels, intakes of retinol and carotene intake were each inversely associ­ above RDAs would exhibit a threshold effect and ated with lung cancer risk in males, but not in would not reduce cancer risk further. In contrast. females. Dietary vitamin C intake was also in­ when total vitamin A for a population is marginal vestigated and shown to be not significantly as­ and carotenoids are the predominant source of sociated with lung cancer risk. vitamin A activity, such as in developing coun­ A smaller, similar study was reported by Stehr tries, both retinol and carotenoids, alone or in et al. 229 for III cases and a pair-matched control combination, would appear to reduce cancer group in southwestern Pennsylvania, during the risk. 36 years 1978 and 1979. Interviews and/or mail cor­ Although many observational studies of di­ respondence included questions on food fre­ etary factors and cancer incidence have already quency history. It was found that stomach cancer indicated a slightly lower than average incidence

152 of cancer among people with an above average studies) do not have entries specifically for 13­ intake of I)-carotene. the association is probably carotene. It would be more accurate to speak in rather complex. There are several possible inter­ terms of carotenoids. Even for serum levels. if pretations for the inverse association ob­ a more specific method was not used. such as served: 2()3.::04.2oo (1) it may be just an artifact due using HPLC, it would not be correct to assume merely to association of l3-carotene ingestion with that l3-carotene levels had been determined. 1n some truly protective dietary habits or compo­ fact, very few reports of serum levels of other nents: (2) it may be due to association of 1)­ carottenoids exist in the literature. It is entirely carotene ingestion with avoidance of some truly possible that carotenoids other than l3-carotene, harmful dietary habits or components: (3) it may which do not possess provitamin A activity. are be due to a genuine protective effect of ~I-caro­ the active agents in the associations observed. tene against the onset of cancer; and (4) it may With further advancement in analytical methods. be factors characteristic of a given life style, which more aecurate data on carotenoid lypes in foods by chance are related to dietary (carotene) pattern. as well as blood would become available. Ifcarotenes do indeed offer some prottection, it is thought that their anticarcinogenicity effect does not require their prior conversion to retinol. 3. Cancer Treatment ,md Prevention It is perhaps the !3-carotene that is absorbed un­ Trials changed from the intestine which is chicfly re­ sponsible for this protective effect rather than any A considerable amount of literature has ac­ retinol or retinaldehyde generated in the intesline cumulated on the use of relinoids in the chemo­ from l3-carotene. 206.23 I prevention of experimentally induced cancer in Peto et al. 206 have suggested various mech­ animals. "Chemoprevention". according to anisms by which carotenoids may reduce cancer Sporn et aI., 111 is a different approach from che­ risks. These include (l) a direct retinoid-like motherapy, which is used for trcatment of in­ fect of some carotenoids on cellular differentia­ vasive, malignant cells. Chemotherapy is a cy­ tion in the target tissues: (2) conversion in the totoxic approach to invasive cancer in which a target tissue of some carotenoids into molecules deliberate attempt is made to kill cancer cells by with some such retinoid-like effects: and (3) pro­ blocking key metabolic pathways. In contrast, tection by carotenoids of the target tissues via chemoprevention is a new pharmacological ap­ mechanisms not related to control of cellular dif­ proach to arrest or reverse premalignant cells dur­ ferentiation. In the last-named group, these ing their progression to invasive malignancy, i.e., mechanisms may include enhancing some im­ during the preneoplasia period, using physiolog­ munological functions, or by quenching singlet ical mechanisms that are not cytotoxic. It makes oxygen, a highly reactive excited molecular spe­ use of defined chemicals such as provitamins 1.1)­ cies which may be generated as a toxic byproduct carotene), vitamins (A, C, and E). synthetic an­ of many normal metabolic processes in both an­ alogs, or other substances (e:.g .. the trace element imals and plants. According to these authors, 13­ selenium) for reducing cancer incidence. Chemo­ carotene is the most efficient quencher of singlet prevention is being emphasized by the National oxygen thus far discovered. In plants, the chief Cancer Institute (NCI) as having the potential for functions of carotenoids may be to quench the making an important impact on nutrition-related singlet oxygen produced by photosynthesis (Sec­ cancer incidence. 20:; tion VIILB). It is believed that synthetic rctinoids would Almost all the studies encountered have men­ be more appropriate for the chemoprevention of tioned the relationship of carotene or !3-carotene cancer. 211.232 The naturally occurring forms of the to cancer risk. In the case of dietary studies, most vitamin A molecule have limited tissue distri­ of the investigators had also used these terms. bution and am too ineffective. Furthermore, they This usage of these terms would not be quite are too toxic to be used practically for the pre­ correct since it is known that most food tables vention of cancer in man. Since the 1970s. many (used for calculating carotene intake in dietary new synthetic analogs of vitamin A have been

153 prepared for this purpose. In the Roche labora­ cies, joints, and bones, as well as changes in tories alone, about 1000 retinoids were synthe­ serum lipids and hepatic function and teratogen­ sized and tested biologically. m Molecular mod­ icity.235 Thus.. retinoids for the use of the pre­ ifications were made to all the three components vention and therapy of cancer conditions are still of the vitamin A molecule: the hydrocarbon ring, undergoing modifications to arrive at compounds hydrocarbon side chain, and polar terminal satisfactory for clinical use. group.233 These synthetic retinoids have an en­ Peck}; has described the use of synthetic re­ tirely different pattern of specific biological ac­ tinoids, particularly isotretinoin and . in tivity, tissue distribution, and toxicity compared the treatment of several dermatologic disorders. with natural forms of vitamin A.2Il Studies into Extending from this success, current interest is the toxicity of retinol and the three synthetic re­ to examine the use of retinoids in cancer pre­ tinoids tretinoin, isotretinoin, and etretinate in vention and therapy for cutaneous tumors. Re­ animals and man have been reviewed in detail sults are still preliminary, requiring further in­ by Kamm et al.234 vestigation and clarification. Several detailed reviews of the use of reti­ The clinical use of [3-carotene and other ca­ noids in the chemoprevention of cancer in ex­ rotenoids in the treatment of patients with a pho­ perimental animals have been published. Moon tosensitivity disease, known as light-sensitive and ItrP4 have described the induction of various porphyria, has been described by Mathews­ cancers and the successful use of retinoids in the Roth37.128 (St:ction VIII.B). Work in this area chemoprevention of these cancers. They include soon led to trials of f3-carotene for a variety of cancers of the skin, lung, mammary glands, uri­ other conditions related to photosensitivity, with nary bladder, and the digestive tract and asso­ positive results in some.J6.128 ciated organs. Ong and ChytiP' have tabulated Since supplemental [3-carotene can be in­ various studies carried out in this field. gested for long periods virtually without risk of Following successes obtained with labora­ toxicity, it was thought that long-term random­ tory animals, retinoids have been used in the ized cancer intervention studies with this com­ treatment of various human carcinomas. Ong and pound may be carried out. Stich et aJ.236 reported Chytip3 tabulated results from some 15 reports the supplementation of both f3-carotene and re­ on the use of retinoids in man, which showed tinol to a group of 40 rural Filipino betel chewers. limited success. Moon and Itri/~ in reviewing Chewing of betel nut, also a common practice of the use of several retinoids, felt that early clinical rural Malaysians and other Asians, has been trials were largely un interpretable for efficacy known to be an important cause of oral cancers. because of the small number of patients involved, These investigators studied the effect of these the lack of control groups, variability in n~sponse supplements on the frequency of micronuclei in definitions, and imprecise reporting of data. cells scraped from inside the cheeks of the sub­ Nevertheless, evidence of activity is said to be jects. Micronuclei formation is a measure of apparent in several malignant conditions. Many chromosome breakage in earlier cell divisions, of the studies have employed l3-cis-retinoic acid, and it is known to be increased by carcinogenic etretinate, tretinoin, and isotretinoin. In some of stimuli. It was found that after 3 months of sup­ the studies, a retinoid has been used with other plementation, there was a threefold decrease in agents in combination chemotherapy. Moon and the mean proportion of cells with micronuclei in ItrF4 also described the use of retinoids in cancer 37 of the 40 subjects studied. In 11 unsupple­ prevention, one of the most exciting potential mented betel chewers in a nearby cluster of uses for these compounds. Some of these studies houses, there was no change in the mean pro­ had reported reversal or regression of the: malig­ portion of micronuclei. nant condition or the prevention of reCUITcnt le­ Munoz et al. m reported that a randomized sions in the patients. However, the promising double-blind intervention trial was carried out in results achieved with certain premalignant and Huixian, Henan Province, People's Republic of malignant changes were accompanied by a series China, to determine whether combined treatment of side effects in skin. mucus membranes, mus­ with retinol, riboflavine, and zinc and could lower

154 the prevalence of precancerous lesions of the of carotenoids. There should indeed be no vita­ esophagus. A group of 610 individuals. aged 35 min A deficiency problem. This would be cor­ to 64 years, with equal numbers of each sex, was rect, provided young children do consume veg­ randomly allocated to weekly vitamins plus zinc etables; the vegetables and fruits consumed treatment or to placebo. Examination conducted contain those carotenoids possessing vitamin A at the end of the treatment, about 14 months later, activity; the carotenes ingested are efficiently ab­ showed that the prevalence of esophageal lesions sorbed and utilized in the body. These are sup­ among the vitamins plus zinc treatment group positions that do not hold true for these most was not different from the placebo group. vulnerable segments of the population. It is known Several other long-term follow-up studies that vegetable consumption among young chil­ have been initiated. results of which are still dren is poor. Whatever small amounts of carot­ 34 36 awaited. . In a large cooperative study spon­ enoids ingested together with a diet poor in fat sored jointly by NCI and the National Heart, and protein makes these precursors of vitamin A Lung and Blood Institute, approximately 20,000 poorly absorbed. At the same time, it is also not male physicians over 40 years of age are being clear which of the fruits and vegetables are richest followed for at least 5 years to evaluate the po­ in l3-carotene. These are obviously important areas tential of l3-carotene in lowering cancer inci­ for studies and intervention. dence. NCI sponsored another trial to assess the A new frontier in vitamin A research has been protective effects of orally administered l3-car­ the examination of a possible association between otene in combination with canthaxanthin in a carotenoids and retinoids and the development population of albino Africans in Tanzania. This and prevention of cancer. Research in this area study is in line with the suggestion that carotenes can be likened to a huge jigsaw puzzle. with the are able to prevent the development of skin can­ pieces slowly being put into their places. Evi­ cer induced by UV light in this high-risk popu­ dence has been accumulating from various sec­ lation. NCI is in fact sponsoring some 22 cancer tors to support this association. Publications in chemoprevention human trials, and most of these this field have been increasing at a very rapid involve the use of l3-carotene and/or retinoL and rate. Nevertheless, it appears that it will take a few using trans-retinoic acid, or l3-cis-retinoic some time before a clear picture can be seen. It acid. 205 is still too early for recommendations to be made for routine retinol or carotenoid supplementation to prevent cancer. X. CONCLUDING REMARKS Since the discovery of vitamin A as a fat­ soluble growth factor in the early part of this Vitamin A deficiency remains a serious prob­ century, research into carotenoids and retinoids lem in many developing countries of the world, has attracted the attention of many scienti:.ts. affecting mainly young children. An estimate is Volumes have been written about them, and many that at anyone time, 20 to 40 million preschool more are certain to follow since many questions children suffer from at least mild deficiency of remain to be answered. Even the biochemical the vitamin. Such numbers are mind-boggling mechanisms of action of the vitamin and pre­ and beyond the imagination of people living in cursors remain largely unknown. There is thus a countries relatively free of the problem. A large great venue for studies, for scientists looking for proportion of these affected children an~ from excitement in research. countries j,n south and east Asia. The main source of vitamin A in the diet of the rural communities in developing countries is ACKNOWLEDGMENTS carotenoids, the precursors of vitamin A, from vegetables and fruits. Pre-formed vitamin A in I thank Dr. M. Jegathesam, Director of the meat, liver, and eggs is frequently out or reach Institute for Medical Research Malaysia, for of the economically deprived. Many these granting permission to publish this review. I am countries have abundant vegetation. rich sources grateful to Dr. Chin-Lam Lim. Associate Pro­

155 fessor in the Food Technology Programme, 12. Karrer, P., Helfenstein. A., Wehrli, H .• and School of Industrial Technology. Universiti Sains Wettstein, A., Plant pigments. XXV. Lycopene and carotene. Hell'. Chim. Ana. 13. 1084. 1930. Malaysia, Penang, and Dr. Yoon-Hin Chong, 13. Karrer, P., Morf, R., and Schopp, K., Vitamin A Nutrition Consultant to the Palm Oil Research in fish oils. Hell'. Chim. Acta. 14. 1431. 1931. Institute of Malaysia. for their valuable com­ 14. von Euler, H. and Karrer, P., Highly concentrated ments on the manuscript. This study was funded preparation of vitamin A. Helv. Chim. Acta. 14. 1040, by the Intensification of Research Priorit) Areas 1931. 15. von Euler, R., von Euler, H., and Karrer, P., (IRPA) Programme of the Ministry of Science, Biochemi~try of carotenoids. Helv. Chim. Acta. l2. Technology and Environment Malaysia (Project 278. 1929. No. IMR 53). 16. Moore, T., V. The absence of the liver oil vitamin A from carotene. VI. The convers!On of carotene to vitamin A in vim. Biochem. 1.. 24, 692. 1930. 17. Holmes, H. N. and Corbet, R. E., The isolation of crystalline vitamin A, 1. Am. Chern. Soc .. 59. 2042. 1937. REFERENCES 18. Baxter, J. G. and Robeson, C. I).. Crystalline al­ iphatic esters of vitamin A. 1. Am. Chem. Soc .. 64. 1. Wolf, G., Vitamin A. in Human NUlrition A Com­ 2407. 1942. prehensive Treallse, Vol. 3B. Nutrition and the Adult: 19. Baxter, J. G. and Robeson, C. I).. Crystalline vi­ Micronutrients. Alfin-Slater. R. B. and Kritchevsky. tamin A, 1. Am. Chem. Soc .. 64. 2411. 1942. D .. Eds .. Plenum Press. New York. 1980. :hap. 3. 20 Arens, J. F. and Van Dorp, D. A .• Synthesis of 2. Barker, B. M., Vitamin A. in Vitamins in Medicine, some compounds possessing vitamin A activity. Na­ Vol. 2. 4th ed .. Barker. B. M. and Bender. D. A .. ture, 157. 190. 1946. Eds .. William Heinemann. London. 1982. chap. 7. 21. Isler, 0 .., Huber, W., Ronco, A .. and Kofler, M., 3. Borenstein, B. and Bunnell, R. H., Carotenoids: Synthesis of vitamin A. Helv. Chim. Acta. 30. 1911. properties. occurrence. and utilization in food, Ad\,. 1947. Food Res., 15. 195. 1966. 22. Karrer, P. and Eugster, C. H., Synthesis of ca­ 4. Weedon, B. C. L., Carotenoids: occurrence. in Ca­ rotenoids. II. Total synthesis of [3-carotene. Helv. rotenoidy. Isler. 0 .. Gutmann, H., and Solms. U .. Chim. Acta. 33. 1172. 1950. Eds .. Birkhauser. Basel. 1971. chap. 2. 23. Wald, G., Nature. 134.65, 1934. cited in Sporn, 5. Bauernfeind, J. C.. Carotenoid vitanlln A precur­ M. B., Roberts, A. B., and Goodman, D. S.. in sors and analogs in foods and feeds. 1. A/-:.rlc Food The Retinoids. Vol. 1. Sporn. M. B .. Roberts. A. B. Chem" 20.456. 1972. and Goodman. D. S.• Eds .. ACddemic Press. Or­ 6. Tee, E. S. and Lim, C. L., The analYSIS of carot­ lando. FL. 1984. chap. 1. enoids and retinoids: a review. Food Chem., 41. 147. 24. Morton, R. A., ChemIcal a~pects of the visual pro­ 1991. cess, NaLUre. 153.69. 1944. 7. Isler, 0., Carotenoids: introduction. in Carotenoid:;. 25. Morton, R. A. and Goodwin, T. W., Preparation Isler. 0., Gutmann. H .. and Solrns. U .. Ed" .. Birk­ of retinene in vitro. Nature. 153.405. 1944. hauser. Basel. 1971. chap. I. 26. Taylor, R. F., Chromatography of carotenoids and 8. Goodwin, T. W., Nature and properties. il'. The Bio­ retinoids, Adv. ChromCilogr., 22.157, 1983. chemistn' of the Carorenoids. VoL I. Plants. 2nd 27. Frolik, C. A. and Olson, .J. A., Extraction. sepa­ ed .. Chapman and Hall. New York. 1980. chap. I. ration and chemical analysis of rerinoids. in The Re­ 9. Karnaukhov, V. N .• Carotenoids: recent progress. tinoid.l, Vol. I, Sporn. M. B .. Roberts. A. B .. and problems and prospects. Compo Biochem. Physiol .. Goodman. D. S.• Eds , Academic Press. Orlando. 958. 1. 1990. FL. 1984. chap. 4. 10. McCollum, E. V. and Davis, M., I BioI. Chem .. 28. Thompson, J. N., Problems of official methods and 15. 167. 1913. cited in Sporn, M. B., Roberts, new techniques for analysis of foods and feeds for A. B., and Goodman, D. S., in The Retinoid.I·, Vol. vitamin A. f. A:;soc. Off'. Anal. Chem., 69. 727. I, Spom, M. B.. Roberts, A. B .. and Goodman, 1986. D. S. Eds .. Academic Press. Orlando, FL. 1984. 29. Sporn, M. B. and Roberts, A. B., Biological meth­ chap. 1. ods for analysis and assay of retinoids - relatiollship 11. McCollum, E. V. and Davies, M., The nature of between <;tructure and activity. in The Retinoid:;. Vol. the dietary deficiencies of rice. f. Bioi. Chem .. 23. I, Sporn. M. B .. Roberts, A. B .. and Goodman. 181. 1915. cited in Wolf, G., in Human Vutrition D. S" Eds .. Academic Press, Orlando, FL. 1984. -A Comprehensive Treatise. Alfin-Slater. R. B. and chap. 5. Kritchevsky. D .. Eds .. Plenum Press, New York. 301. Kaegi, H. H., Synthesis of retinoids labelled with 1980. chap. 3. radioisotopes. in The Retinoids, Vol. I. Sporn. M. B ..

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