Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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combination of methanol (or acetonitrile) and water,

and in some cases may be entirely nonaqueous.

0027 Many foods, including poorly characterized or un-

known samples, will usually be assayed subsequent to

saponification, which offers the option of employing

either chromatographic mode, and permits analytical

simplification through conversion of multiple esters

to a single retinol form. Milks, dairy products, and

fortified cereals, may, alternatively, be tested by direct

injection of a total lipid extract. Figure 3 illustrates a

typical chromatogram obtained for a milk-based

infant formula by this procedure. While retinyl acet-

ate is the additive, the long-chain endogenous esters

are visible as a composite peak, which are resolvable

by independent nonaqueous, reversed-phase chroma-

tography, as illustrated in the insert.

0028In fats and oil products, retinyl esters can

be assayed by direct dissolution in hydrocarbon,

followed by clarification and injection into the

chromatographic system. Analytical success with

these strategies relies on the selective nature of spec-

tral detection modes, the narrow elution window of

the various esters, and their extinction equivalence.

Retinyl

acetate

cis-isomers

Trans-

retinyl

acetate

Long-chain esters

10 min

Time (min)

10 15

cis-isomers

fig0003 Figure 3 The lipids from 2 g of milk-based infant formula

(22% fat) were extracted without saponification into 5 ml of

organic solvent: 100 ml was injected directly into high-perform-

ance liquid chromatography (HPLC) equipped with a silica

column and a fluorescence detector (325 nm excitation, 470 nm

emission). Mobile-phase composition was hexane:2-propanol

(99.92:0.08 v/v). Insert: The endogenous esters were collected

and separated by nonaqueous reversed-phase HPLC on a C

column with an isocratic mobile phase of acetonitrile:dichloro-

methane (80:20). The major identified long-chain esters are

retinyl oleate (O), retinyl palmitate (P), and retinyl stearate (S).

Time (min)

3⬘

1⬘

fig0004Figure 4 Retinyl acetate was extracted from a multivitamin

formulation without saponification, using dimethyl sulfoxide

(DMSO). High-performance liquid chromatographic (HPLC)

analysis was achieved on a Waters C

Radial-PAK column with

a methanol (100%) mobile phase flowing at 2 ml min

1

. Detection

was achieved by ultraviolet at 280 nm, 0.3 aufs. Vitamins D, E, and

K can be assayed concurrently. Peak identification: 1, retinyl

acetate (vitamin A); 2, cholecalciferol (vitamin D

); 3, tocopheryl

acetate (vitamin E); 4, phylloquinone (vitamin K

). The elution

positions of retinol (1

) and tocopherols (3

) are also indicated.

4956 RETINOL/Properties and Determination

Limitations of these approaches include the require-

ment for normal-phase conditions in order to avoid

triglyceride column fouling, and anhydrous or iso-

hydric eluents to obtain reproducible retention. In

addition, any nonesterified retinol present in the

sample will not be concurrently viewed owing to its

tenacious retention on silica relative to the esters.

0029 High-potency pharmaceutical preparations and

vitamin premix formulations can also be successfully

assayed without the need for saponification. Thus,

providing the carrier materials are free from signifi-

cant fat, the retinyl esters can be extracted into a

suitable solvent (e.g., dimethylsulfoxide), insuring

release from encapsulation, diluted into ethanol,

clarified, and injected on to a reversed-phase column.

Such products usually contain vitamins D, E, and K,

all of which can be concurrently assayed using both

wavelength and sensitivity attenuation. However,

under these conditions, it is difficult to analyze for

retinyl acetate and palmitate concurrently, as the

latter is highly retained by nonpolar columns. This

problem can be circumvented by performing the

analyses subsequent to saponification and thereby

recovering total vitamin A as retinol. Figure 4 illus-

trates a typical chromatogram of retinyl acetate in a

pharmaceutical product. The elution position of

retinol is also indicated.

See also: Antioxidants: Natural Antioxidants;

Carotenoids: Occurrence, Properties, and

Determination; Physiology; Chromatography: Thin-layer

Chromatography; High-performance Liquid

Chromatography; Gas Chromatography; Emulsifiers:

Uses in Processed Foods; Food Fortification;

Spectroscopy: Fluorescence

Further Reading

Ball GFM (1988) Fat-Soluble Vitamin Assays in Food

Analysis. A Comprehensive Review. London: Elsevier

Applied Science.

Ball GFM (1998) Bioavailability and Analysis of Vitamins

in Food, pp. 115–161. London: Chapman and Hall.

Bates CJ (2000) Vitamins: fat and water soluble: analysis.

In: Meyers RA (ed.) Encyclopedia of Analytical

Chemistry, pp. 1–35. Chichester: John Wiley.

De Leenheer AP, Lambert WE and Boxdaer R (2000) Vita-

min A: retinol, carotenoids and related compounds.

Modern Chromatographic Analysis of the Vitamins,

Chromatographic Series, vol. 84, pp. 1–74. New York:

Marcel Dekker.

Eitenmillar RR and Larder WO (1999) Vitamin Analysis

for the Helath and Food Sciences, pp. 3–75. Boca Raton,

USA: CRC Press.

Friedrich W (1988) Vitamins, pp. 63–140. Berlin: de

Gruyter.

Klaui HM, Hausheer W and Huske G (1970) Technological

aspects of the use of fat-soluble vitamins and carotenoids

and of the development of stabilised marketable forms.

In: Morton RA (ed.) Fat Soluble Vitamins; International

Encyclopaedia of Food and Nutrition, vol. 9, pp. 113–

159. Oxford: Pergamon Press.

McLaren DS and Frigg M (2001) Sight and Life. Guide-

book on Vitamin A in Health and Disease. Basel, Switz-

erland: Roche.

Noll GN and Becker C (2000) HPLC of non-polar retinoid

isomers. Journal of Chromatography A 881: 183–188.

Olson JA (1991) Vitamin A. In: Machlin LJ (ed.) Handbook

of Vitamins, pp. 1–57. New York: Marcel Dekker.

Pitt GAJ (1985) Vitamin A. In: Diplock AT (ed.) Fat-Soluble

Vitamins. Their Biochemistry and Applications, pp.

1–75. London: Heinemann.

Thiex N, Smallidge R and Beine R (1996) Sources of error

in vitamin A analysis. Journal of the Association of

Official Analytical Chemists 79: 1269–1275.

Thompson JN (1986) Review: official methods for meas-

urement of vitamin A. Journal of the Association of

Official Analytical Chemists 69(5): 727–738.

United States Department of Agriculture (2002) USDA

National Nutrient Database for Standard Reference,

Release 15. Beltsville, MD, USA: Agricultural Research

Services.

Wagner AF and Folkers K (1975) Vitamins and Coenzymes,

pp. 280–307. New York: Robert E. Krieger.

Wyss R (1990) Chromatography of retinoids. Journal of

Chromatography 531: 481–505.

Physiology

H C Furr, Craft Technologies, Wilson, NC, USA

M M McGrane, University of Connecticut, Storrs,

CT, USA

Absorption, Bioavailability, Transport, and

Distribution

0001‘Vitamin A’ is the collective term for compounds that

show the biological properties of retinol, including

maintenance of epithelial tissue and visual function.

This classification includes retinol, retinyl esters, and

retinal. Retinoic acid is included, even though it does

not sustain visual function, because it has been found

to be the active metabolite that carries out many of

the functions of vitamin A. These are isoprenoid

compounds, having in common an 11-carbon poly-

ene chain attached to a trimethyl substituted cyclo-

hexenyl ring (Figure 1). The term ‘retinoids’ refers to

all compounds, natural or synthetic, that show some

biological activity typical of vitamin A, such as pro-

moting differentation of cells in culture; not all reti-

noids can support all the functions of vitamin A, such

RETINOL/Physiology 4957

as vision. Vitamin A compounds are not found as

such in plant tissues, but rather are characteristic of

the animal kingdom; the notable exception is 13-cis

retinal, which serves as a chromophore in the purple

membrane of certain halobacteria.

0002 Dietary vitamin A is provided by two sources: pre-

formed vitamin A and provitamin A carotenoids.

Preformed vitamin A (mostly as esters of retinol

with long-chain fatty acids) comes from animal prod-

ucts or from dietary supplements; retinyl esters, e.g.,

retinyl acetate and retinyl palmitate, are more stable

chemically than is free retinol. Provitamin A carote-

noids arise mostly from plant products: b-carotene is

the most active and is widely distributed in plants,

but other carotenoids (such as a-carotene and

b-cryptoxanthin and b-apo-carotenals) can be im-

portant sources of vitamin A from particular foods

(See Carotenoids: Physiology). The relative import-

ance of these sources of vitamin A is very dependent

on diet. Other carotenoids, such as lycopene and

xanthophyls (including lutein and zeaxanthin), are

major carotenoids in some foods and may have

other important physiological functions, but they

have no provitamin A activity in higher animals.

0003Typical estimates of dietary vitamin A absorption

efficiency are 70–85%. Estimates of carotenoid ab-

sorption are usually much lower, but are confounded

by slow intestinal absorption and rapid metabolism;

there is considerable species variability in absorption

efficiency and in metabolism of carotenoids. Animal-

feeding studies show that the biological matrix of

food carotenoids has profound effects on their bio-

availability.

0004Since both vitamin A and carotenoids are lipids,

intestinal micelle formation with bile acids is essential

for their absorption. Human subjects with impaired

bile acid formation or flow (e.g., with biliary atresia)

may require intramuscular supplementation with

vitamin A and the other fat-soluble vitamins. Within

the intestinal lumen, vitamin A esters (retinyl esters)

all-trans Retinol

(vitamin A alcohol)

OOC

all-trans Retinyl Palmitate

all-trans Retinal

COH

all-trans Retinoic acid

(Tretinoin)

13-cis Retinoic acid

(Isotretinoin, Accutane)

COH

COOH

Retinoyl β-glucuronide

4-oxo Retinoic acid

3,4-didehydroretinol (vitamin A

alcohol)

COH

11-cis Retinal

4-hydroxyphenylretinamide (4-HPR, Fenretinide)

COC

Acitretin (Etretinate, Ro 10-9359)

TTNPB (an arotinoid)

β-Carotene

COOH

fig0001 Figure 1 Chemical structures of some representative retinoids.

4958 RETINOL/Physiology

are hydrolyzed to free retinol and are absorbed as

such; this free retinol is promptly reesterified within

the intestinal cells. (Figure 2). Provitamin A carote-

noids, such as b-carotene, are often cleaved within

intestinal cells; this metabolism is predominantly by

central cleavage (mediated by the enzyme carotene

15,15

-dioxygenase), although asymmetric cleavage

followed by chain shortening may play a role. Retinal

(also called retinaldehyde or vitamin A aldehyde), the

final product of carotenoid cleavage, is enzymatically

reduced to retinol and is then esterified. The physio-

logical ligand for this esterification process seems to

be retinol bound to an intracellular retinol-binding

protein (CRBP II, molecular weight (MW) approxi-

mately 14 600 Da, one of several small intracellular

retinoid-binding proteins); the primary retinyl ester

synthesizing activity transfers fatty acid from phos-

phatidyl choline (lecithin:retinol acyltransferase,

LRAT), although an acyl-coenzyme A-dependent

esterifying activity (acylCoA:retinol acyltransferase,

ARAT) is also present.

0005 Regardless of their dietary source, the retinyl esters

are incorporated in the core of chylomicra and trans-

ported in the lymph. After removal of triacylglycerols

by lipoprotein lipase as the lipoprotein particle

circulates through peripheral tissues, the chylomicron

remnants are rapidly taken up by the liver, and the

vitamin A esters are hydrolyzed by retinyl ester

hydrolase there. The resulting retinol is then either

reesterified (primarily by LRAT, although ARAT ac-

tivity may be important at high retinol concentra-

tions) and stored in the liver, or released into the

plasma as a complex with plasma retinol-binding

protein (RBP). The two cell types involved in liver

vitamin A metabolism are the hepatocytes (parenchy-

mal cells) and the lipocytes (also called stellate cells,

Ito cells, or fat-storing cells). The hepatocytes are the

major site of RBP synthesis and retinol-RBP release;

some retinyl esters are also found here. The lipocytes

store retinyl esters in cytoplasmic lipid droplets,

which also contain triacylglycerols and some choles-

teryl esters within a phospholipid coat. It has been

suggested that RBP transfers vitamin A, as retinol,

between lipocytes and hepatocytes.

0006Because most forms of vitamin A are hydrophobic,

the transport, metabolism, and function of vitamin A

are dependent on a series of binding proteins, each

specific for its ligand and tissue. Plasma RBP typically

has molecular weight of approximately 21 000 Da

in mammalian species; the complete amino acid com-

position has been determined for several species, and

the gene from some species has been cloned. RBP

binds retinol with 1:1 stoichiometry. The hydropho-

bic retinol molecule fits into a ‘barrel’ within the

protein, shielded from interactions with the aqueous

environment. Holo-RBP, in turn, binds transthyretin

in plasma (TTR; formerly called prealbumin); a TTR

tetramer can bind up to four molecules of RBP. Usual

concentrations of human plasma RBP are 1.9–2.4 mM

(40–50 mgml

1

); typically, total circulating RBP is

80–90% saturated with retinol ligand. Although ret-

inol, retinal, and retinoic acid bind to RBP with simi-

lar affinity, retinol is present in highest concentrations

and is the predominant ligand. Retinyl esters have

much less affinity for RBP and are transported by

serum lipoproteins instead.

0007The release of holo-RBP from liver hepatocytes is

carefully controlled to maintain levels of circulating

Dietary Vitamin A,

Carotenoids

Eye (visual cycle)

(11-cis Retinal <==> all-trans Retinal)

Intestines

(carotenoid cleavage)

Chylomicrons

(retinyl esters)

Liver

(retinol <===> retinyl esters)

Metabolism, excretion

(including retinoid glucuronides)

Serum

(retinol-RBP-TTR)

Extrahepatic tissues

(retinol, retinyl esters,

retinoic acid)

Metabolism,

excretion

fig0002 Figure 2 Overview of vitamin A metabolism.

RETINOL/Physiology 4959

retinol within narrow limits, but the mechanism of

this control is not yet clear. In the absence of adequate

vitamin A, apo-RBP accumulates in the liver, ready to

be released as soon as vitamin A is available (this is

the basis of the Relative Dose Response assay for

vitamin A deficiency, as discussed below). Clearly,

this mechanism has developed because of the dichot-

omy of vitamin A action: vitamin A is essential, in

small amounts, for proper differentiation and main-

tenance of epithelial cells, but excesses of vitamin A

are toxic and must be avoided by the organism.

Metabolism, Storage, and Excretion

0008 Vitamin A in excess of immediate requirements is

stored in the liver as esters of long-chain fatty acids

(Figure 2); the ratio of liver retinol to retinyl esters

decreases as total liver vitamin A increases, but typic-

ally, 95% of total liver vitamin A is presentas the esters.

Retinyl palmitate is the primary ester in the liver of the

human, rat, cow, sheep, rabbit, cat, frog, trout and

polar bear, althoughsignificant amounts ofother esters

are found (especially oleate and stearate), and the liver

retinyl ester composition can be affected by dietary

fatty acid composition. (Remarkably, the predominant

vitamin A ester in rat adrenal cortex is retinyl stearate.)

Liver frequently contains as much as 90% of total body

vitamin A, although other organs, such as kidney,

testes, and adrenal glands, also contain detectable

retinyl esters. The efficiency of liver storage of vita-

min A is particularly noteworthy in the polar bear,

where concentrations as much as 36 mmol (10 380 mg)

per gram of liver have been reported. Typical values

for human liver vitamin A (autopsy specimens) are

0.44–0.74 mmol g

1

(126–211 mgg

1

) in the USA.

0009 Kinetic studies carried out in rats have shown that

there is extensive cycling of liver vitamin A stores, and

extensive recycling of vitamin A between liver and

other tissues; this assures an ample supply of retinoid

to tissues in response to immediate needs. It is certain

that retinol-RBP is the major form of transport from

liver to peripheral tissues; although the transport of

vitamin A from other tissues back to the liver may be

via retinyl esters transported by lipoproteins (normally

present at about 5–10% the serum concentration of

retinol-RBP), transport via retinol on RBP synthesized

in the outlying tissues may be an important pathway

(mRNA for plasma RBP has been detected in a number

of other tissues in addition to liver). In vitamin A-

deficient rats, the recycling is even more extensive,

and catabolism of vitamin A is markedly diminished.

0010 The rat has served as a valuable model for vitamin A

metabolism in the human, particularly for aspects that

can not be studied readily in humans. In the rat, catab-

olism of vitamin A occurs by oxidation of the cyclo-

hexenyl ring (particularly at the 4-position), epoxide

formation at the 5,6-positions, hydroxylation of the

ring methyl groups, and chain shortening. The

resulting metabolites are generally inactive, although

some may have a little biological activity. These more

polar retinoids are excreted in the urine and in the bile.

0011Retinol can be reversibly oxidized to retinal (vita-

min A aldehyde); retinal can be oxidized to retinoic

acid, but retinoic acid cannot be converted back to

the other forms. Thus, retinol, retinyl esters, and

retinal have equal biological activity because they

are freely interconverted; retinoic acid fills some

(but not all) of the functions of retinol, but it and

its derivatives are not stored. Typical human serum

concentrations of retinoic acid are 5–10 nM (1.5–

3ngml

1

), compared with typical retinol concentra-

tions of 1–2 mM. Retinoyl b-glucuronide and retinyl

b-glucuronide (formed in the liver from retinoic acid

and retinol, respectively) are secreted into the bile but

can be hydrolyzed and reabsorbed in the intestine

(enterohepatic circulation); both these compounds

have vitamin A activity in a variety of tests, are

found in human blood, and may have regulatory

roles in vitamin A function.

Roles in the Body

Vision

0012The major roles of vitamin A are in vision and in

control of gene expression (including differentiation

of epithelial tissues, and in the immune system, as well

as a host of other systems). Metabolism of vitamin A

in the retina of the eye is unique, in keeping with the

unusual role of vitamin A in that tissue. Vitamin A is

stored in the retinal pigment epithelium as retinyl

esters. All-trans retinyl esters are simultaneously

hydrolyzed and isomerized to 11-cis retinol by an

isomerohydrolase enzyme that uses the free energy of

hydrolysis of the retinyl esters to drive the isomeriza-

tion reaction. 11-cis Retinol, a compound unique to

the eye, is then oxidized to 11-cis retinal by an alcohol

dehydrogenase. 11-cis Retinal is transferred from the

pigment epithelium cells to the rod cells by interstitial

retinoid binding protein (IRBP), a distinct binding

protein (MW 140 000). In the rod cells, 11-cis retinal

binds (as a Schiff base with the e-amino group of a

specific lysine residue) to the protein opsin to form the

visual pigment, rhodopsin. When a photon of light is

absorbed by a rhodopsin complex, the 11-cis retinal is

isomerized to all-trans retinal and released from the

protein complex; the resulting conformation change

of the protein initiates a cascade of reactions, resulting

in a neural signal to the brain. The protein opsin is

then available to bind another molecule of 11-cis

4960 RETINOL/Physiology

retinal for another round of the visual cycle. The all-

trans retinal that was released from the protein com-

plex is transferred back to the pigment epithelium via

IRBP, enzymatically reduced to all-trans retinol, and

esterified again. In contrast to the high turnover rates

of vitamin A in other tissues, vitamin A in the eye is

highly conserved, with little leakage back to the liver.

Prolonged vitamin A deficiency, however, leads to re-

duced sensitivity to light, usually first noted as

impaired vision at night (night blindness). These

effects of vitamin A deficiency are generally reversible

by subsequent vitamin A supplementation.

0013 In a very different role of vitamin A in the eye, the

cornea depends on vitamin A for proper cell differen-

tiation and for secretion of protective glycoproteins.

In vitamin A deficiency, these tissues are susceptible

to attack by opportunistic bacteria; such attack may

result in permanent scarring and permanent vision

loss. These effects of vitamin A deficiency, unlike

those in the retina, may not be reversible by subse-

quent vitamin A supplementation. Such vitamin A-

dependent corneal degeneration (given the general

name ‘xerophthalmia’) accounts for an estimated

500 000 new cases of blindness in preschool children

in the world each year.

Control of Gene Expression

0014 Overview of control of gene expression Vitamin A

and its metabolites are required for embryonic

development, cellular differentiation, growth, repro-

duction, adult tissue homeostasis, vision, and main-

tenance of the immune system. Over the past several

years, it has become clear that retinoids mediate

many of their pleiotropic effects by regulating the

activation or inhibition of gene transcription. It is

well documented that retinoids regulate the expres-

sion of many genes involved in their own transport

and metabolism. The genes for different retinoid re-

ceptor subtypes, the cellular retinol binding proteins

(CRBP) I and II, and the cellular retinoic acid binding

protein II (CRABP II) are regulated by either all-trans

retinoic acid (RA) or 9-cis RA (Figure 3). These vita-

min A metabolites exhibit hormonal action by bind-

ing to their nuclear receptors that interact with DNA

responseelementswithintargetgenestoaffectgenetran-

scription. The retinoid receptors are members of the

steroid/thyroid/retinoid superfamily of nuclear recep-

tors. There are two distinct families of nuclear

retinoid receptors: the retinoic acid receptors

(RARs) and the retinoid X receptors (RXRs). It has

been suggested that the two classes of receptors have

a distinct functional activity, the RARs being import-

ant in cell development and differentiation, and the

RXRs being important in metabolic regulation. Nu-

clear receptors of the steroid/thyroid/retinoid super-

family can act as either inhibitors or activators of

transcription. The mechanism whereby these regula-

tory properties are propagated is via the recruitment

of accessory proteins to the nuclear receptor. In the

absence of ligand, such as all-trans RA, corepressor

proteins are bound to retinoid receptors and cause

inhibition of transcription of the target gene.

Conversely, binding of ligand causes a conforma-

tional change in the receptor such that corepressor

proteins are released, and coactivator proteins are

recruited to the retinoid receptor. The association of

coactivators with the nuclear retinoid receptor causes

induction of transcription of the target gene, initiated

by retinoic acid binding to its specific nuclear receptor.

0015Nuclear retinoid receptors As indicated previously,

there are two distinct classes of nuclear retinoid re-

ceptors, the RARs and RXRs. Both RARs and RXRs

bind the naturally occurring metabolites of vitamin A

with a similar order of affinity (all-trans RA > reti-

nal > retinyl acetate > retinol), but RXRs require from

10- to 40-fold more ligand for comparable tran-

scriptional activation. A downstream metabolite of

ROH

RCHO

ROH RE

all-trans RA

9-cis RA

RAR

RXR RXR

RXRE

Nucleus

Cytoplasm

RXR

RARE

fig0003 Figure 3 Retinoid action in the nucleus.

RETINOL/Physiology 4961

all-trans RA, 9-cis RA, has been shown to be the

naturally occurring, high-affinity, ligand for the

RXR. The binding of ligand to nuclear retinoid recep-

tors induces dimer formation and initiates transcrip-

tional activation of a target gene via the DNA

response element(s) in the promoter/regulatory region

of the gene. Retinoid receptors act in a dimeric con-

formation and bind to specific RA response elements

(RARE) within the 5

promoter/regulatory regions of

target genes. The DNA response elements are usually

a direct repeat (DR) of six nucleotides in a specific

sequence separated by a defined number of nucleotides

that vary depending on the type of DNA response

element. RAR/RXR heterodimers can activate or re-

press gene transcription, depending on the type of

RARE to which they bind in the promoter region of

a target gene. If the RAR/RXR heterodimer binds to a

DR-5 RARE, the RAR/RXR complex activates tran-

scription in response to the RAR ligand all-trans RA.

In contrast, RAR/RXR heterodimers bound to a

DR-1 RARE exhibit no response to RAR-specific

ligands and repress the response to RXR-specific

ligands such as 9-cis RA. Therefore, the RAR/RXR

heterodimer can be stimulatory, neutral, or inhibi-

tory, depending on ligand, all-trans RA or 9-cis RA,

and the DNA RARE to which it is bound. This is

partially explained by the fact that there is a specific

polarity to the binding of RAR and RXR hetero-

dimeric partners, determined by the DNA itself.

RAR/RXR heterodimers bind with the RXR occupy-

ing the upstream half-site of a DR-5 element, but this

orientation is reversed on a DR-1 element. Further

investigation of this polarity of RAR/RXR heterodi-

mer binding has shown that transactivation by RAR

bound to a DR-5 RARE involves the ligand-depend-

ent dissociation of a bound corepressor (discussed

below), but RAR bound to a DR-1 RARE does not

respond to ligand by dissociation of a bound core-

pressor. Therefore, transcription is not stimulated in

the later response. In this regard, the DNA itself

is considered an allosteric regulator of receptor–

accessory protein interaction.

0016 All of the RAR and RXR subtypes, a, b,andg, are

encoded by separate genes at distinct chromosomal

loci. However, the protein products encoded by these

genes have certain common modular structures, par-

ticularly in the DNA-binding and ligand-binding

domains, which define, in part, their membership

within the steroid/thyroid/retinoid superfamily. The

amino acid sequence of the RARs can be divided into

six regions (A–F). The amino acid sequence of

the RXR genes has a similar domain structure

to the RARs. Among the retinoid receptors, the

DNA-binding domain (C) is a highly conserved

region of approximately 70 amino acids that contains

two zinc fingers; the zinc-finger configuration is re-

sponsible for specific DNA response element recogni-

tion. According to Evans and colleagues, a sequence

of three amino acids within the first zinc finger (the P

box) determines recognition of the half-site sequence

of a DNA response element, and five amino acids in

the second zinc finger (the D box) are critical

for recognition of the spacing between half-sites.

Nuclear magnetic resonance spectroscopy of the

DNA-binding domain indicates a secondary structure

comprising three a-helices; the first helix is postulated

to fit across the major groove of the DNA response

element. In this orientation, the third helix interacts

with the minor groove of the DNA, such that Lys and

Arg residues make contact with the phosphate back-

bone and stabilize protein–DNA interactions. This

third helix in the DNA-binding domain is also

thought to mediate protein–protein interaction re-

quired for dimer formation. The ligand-binding

domain (E) is also highly conserved among the RAR

and RXR genes and mediates the ligand-dependent

transcriptional activation function. The amino-

terminal region (A) is less well conserved and is sub-

type-specific. Both regions A and B are responsible for

ligand-independent transcriptional activation. The

amino-terminal part of domain D may function as a

nuclear localization site.

0017In addition to subtype differences in RARs and

RXRs, there are also different mRNA isoforms for

each gene. These isoforms are generated by different

transcription start sites and alternative splicing,

which affects the 5

untranslated region (UTR) and

the A domain such that either a deleted or altered A

region is combined with a common B–F region.

0018The biological activity of vitamin A metabolites is

further extended by the interaction of the retinoid

receptors with other members of the receptor super-

family. The RXRs serve as heterodimeric partners

with RARs, thyroid hormone receptors (TR), and

the vitamin D receptors (VDR) and have been

shown to increase the affinity of RAR, VDR, and

TR, for their respective response elements (favored

DR motifs DR-2/DR-5, DR-3, and DR-4, respect-

ively), and to activate transcription in the presence

of the appropriate ligand. This indicates a central role

for the RXR as a heterodimeric partner with the other

nuclear receptors of this superfamily. Interestingly,

only one ligand is required to stimulate the hetero-

dimeric complex and affect transcriptional acti-

vation. However, the RXR is unique in that it can

function as a homodimer. In this capacity, it can

stimulate gene transcription favoring the DR-1 hor-

mone response element and is dependent upon the

9-cis RA ligand. The RXR also forms a heterodimeric

complex with the peroxisome proliferator-activated

4962 RETINOL/Physiology

receptor (PPAR) and, for example, mediated by a DR-

1 motif in the promoter of the acyl-CoA oxidase gene,

stimulates transcription in response to both 9-cis RA

or clofibric acid (a peroxisomal proliferator). Re-

cently, it has been shown that dietary polyunsaturated

fatty acids (PUFA) activate PPAR, and via PPAR–

RXR heterodimer formation, dietary PUFA regulate

both lipid and glucose metabolism. Clarke refers to

dietary PUFA as ‘fuel partitioners’ that act, via PPAR

activation, to increase glycogen storage and fatty acid

oxidation while decreasing triglyceride synthesis.

0019 The multiplicity of nuclear retinoid receptors re-

flects developmental and tissue-specific differences in

their expression. In the mouse embryo, RARa is ubi-

quitously expressed, whereas RARb and RARg are

more restricted, and nonoverlapping, in their expres-

sion. RXRb genes are widely expressed in the mouse

embryo, whereas RXRa is found in liver, kidney,

intestine, and skin, and RXRg is more restricted

still. In the adult, both RARa and RXRb are found

in most tissues, whereas RARb,RARg, RXRa, and

RXRg are more limited. RXRa is highly expressed in

liver, kidney, spleen, and visceral tissues, indicating

a possible role for this RXR in retinoid, lipid,

and carbohydrate metabolism. The demonstrated

RXR responsiveness of numerous genes including

the CRBP II, apolipoprotein (apoAl), acyl-CoA oxi-

dase and the phosphoenolpyruvate carboxykinase

(PEPCK) genes further supports this hypothesis.

0020 Nuclear coactivators and corepressors associated

with nuclear retinoid receptors Recently, several

coactivators and corepressors that associate with nu-

clear retinoid receptors and affect their ability to

regulate target genes have been cloned and character-

ized. It has been established that retinoid binding

induces conformational changes in nuclear retinoid

receptors and promotes their association with a

diverse group of nuclear proteins that function as

coactivators of transcription. Alternatively, unbound

receptors recruit nuclear proteins that repress tran-

scription of target genes. It has been demonstrated

that all-trans RA binding to RAR, with subsequent

activation of transcription of a target gene, requires

dissociation of corepressor proteins and recruitment

of coactivator proteins. It is thought that corepressors

contribute to the formation of condensed chromatin

structure, prohibiting activation of target genes. Con-

versely, most coactivators cause a loosening of chro-

matin conformation, allowing nuclear receptors to

activate transcription of target genes.

0021Regulation of the PEPCK gene by retinoids The

multifaceted action of vitamin A is indicated by its

role in regulation of the gene encoding PEPCK, the

rate-determining enzyme of gluconeogenesis in liver

(Figure 4). All-trans RA and 9-cis RA, in equal con-

centrations, induce the PEPCK promoter by approxi-

mately sevenfold in hepatoma cells in culture. Studies

in mice in vivo have shown that vitamin A deficiency

causes a decrease in PEPCK gene expression. Specif-

ically, with food deprivation, hepatic PEPCK mRNA

levels are not induced in vitamin A-deficient mice.

Induction of the PEPCK gene can be reestablished

with retinoid treatment of vitamin A deficiency. Reti-

noid regulation of the PEPCK gene has been tested

in vivo in two lines of transgenic mice. Results from

these experiments indicate that expression driven by

both the 460/þ73 and 355/þ73 PEPCK DNA

sequences is decreased with vitamin A deprivation.

Subsequent treatment of vitamin A deficiency with

all-trans RA increases expression driven by the

460/þ73 promoter or the minimal 355/þ73 pro-

moter. The second retinoid isomer, 9-cis RA, however,

9-cis RA

DR-1 RARE:

Required for 9-cis RA induction

Inhibitory for all-trans RA

DR-5 RARE:

Mediates all-trans RA induction

No response to 9-cis RA

DR-1

RARE

TGACCT

TTGGCCG

−451 −439

DR-5

RARE TATA

AGCCCT

GTCCTTGACCC

−337 −321

RXR RXR

all-trans RA

RXR RXR

fig0004 Figure 4 Retinoic acid response elements involved in regulation of the PEPCK gene.

RETINOL/Physiology 4963

increases expression driven by the 460/þ73 pro-

moter, but not the 355/þ73 promoter. Therefore,

the two active retinoid isomers in liver have different

molecular signaling mechanisms and activate the

PEPCK gene at different DNA response elements.

0022 Additionally, it has been shown that appropriate

developmental regulation of the PEPCK gene during

fetal development and the perinatal period is depend-

ent on adequate vitamin A nutriture. Vitamin A defi-

ciency causes a decrease in hepatic PEPCK mRNA

during late gestation, a stage in development when

the PEPCK gene is first expressed in liver. At birth,

with adequate vitamin A nutriture, the PEPCK gene

is rapidly induced. This is consistent with the need of

the newborn to conduct gluconeogenesis because of

removal from the maternal glucose supply. The rapid

induction of PEPCK gene expression at birth is sig-

nificantly decreased with vitamin A depletion in the

newborn. Overall, adequate vitamin A is required

for the appropriate developmental expression of the

PEPCK gene in late gestation and at birth. The fact

that vitamin A is an active regulator of expression of

the PEPCK gene indicates a significant role for this

nutrient in the regulation of carbohydrate metabol-

ism overall. This is a new area of investigation of

vitamin A regulation of gene expression as it expands

the known role of vitamin A beyond the regulation of

genes involved in vitamin A metabolism itself.

Human Health

Infection and Disease

0023 Early in the study of vitamin A requirements, it was

appreciated that vitamin A-deficient animals are

more susceptible to disease. Attempts to extend

these observations to human health long gave incon-

sistent results, however. Recently, it has been shown

that supplementation of children with vitamin A re-

duces the severity of measles and diarrhea (although

vitamin A supplementation may not reduce the inci-

dence and duration of diarrhea). Although vitamin A

prophylaxis may not reduce measles severity, sup-

plementation of measles patients with vitamin A

(200 000 international units (IU), i.e., 60 000 mgof

RE, 209 mmol) during infection does reduce hospital

stay and mortality rate and is now recommended by

WHO in communities where vitamin A deficiency is

prevalent. Currently, it is not believed that vitamin A

supplementation can provide any benefit for other

respiratory diseases (nonmeasles pneumonia). The

possible role of vitamin A status in tuberculosis, mal-

arial infection, and HIV infection is not clear. How-

ever, vitamin A public health programs can have a

tremendous impact on child morbidity and mortality

in developing countries.

0024The mechanism(s) by which retinoids affect the

immune system have not yet been delineated, but it

seems safe to assume that control of gene expression is

involved. In at least some experiments, the function of

vitamin A can be filled by all-trans or 13-cis retinoic

acid, consistent with current knowledge of retinoids

and gene expression. Both antibody-mediated and

cell-mediated immune responses are reduced in vita-

min A deficiency. Interpretation of human studies of

the relationship between vitamin A status and immune

response is complicated by the fact that plasma retinol

concentrations (often used as a convenient measure of

vitamin A status) fall during infection even in vitamin

A-replete subjects, and chronic infection reduces vita-

min A absorption and mobilization.

0025Dermatology One of the first pharmaceutical appli-

cations of retinoids was the use of 13-cis retinoic acid

(isotretinoin) to combat cystic acne, ichthyosis, and

follicular keratosis; 13-cis RA inhibits sebaceous

gland size and activity by promoting CRABP II

mRNA expression. The process of keratinization

(differentiation and maturation) of skin cells is

affected both by deficiency and by excess of retinoids.

Dehydroretinol and its esters (vitamin A

) are found

in human skin (more so in epidermis than in dermis)

and may have specific roles there. Retinoids are now

used clinically to treat psoriasis, ichthyosis, Darier’s

disease, and other disorders of keratinization, acne,

and photoaged skin. Synthetic retinoids, such as etre-

tinate and the ethyl ester of the arotinoid TTNPB

(Figure 1), as well as the naturally occurring retinoyl

b-glucuronide, have been developed in attempts to

reduce the toxicity of the naturally occurring retinoic

acids without losing efficacy.

0026Cancer prevention and treatment Retinoids can in-

hibit cell transformation and tumor development in a

variety of cell and animal models, but the effects are

not uniform with all malignancies. In general, it

seems that retinoids may not prevent initiation of

tumors, but more likely interfere with the promotion

process. They exert these effects by modulating

growth, differentiation, and apoptosis of cells; most,

if not all, of these effects may be explained by the role

of retinoids in control of gene expression.

0027Retinoids have been tested against a variety of

cancers in human and animal studies. N-(4-Hydroxy-

phenyl)-retinamide (4-HPR, Fenretinide) has been

tested clinically for prevention of recurrence of

human breast cancer; although results are promising,

some toxicity (owing to interference with normal

retinol metabolism) has been observed. Incidence of

skin tumors associated with xeroderma pigmentosum

is reduced by treatment with 13-cis retinoic acid.

4964 RETINOL/Physiology

Perhaps the most dramatic application is the use of

retinoic acid (tretinoin) against promyelocytic leuke-

mia; this disease results from a chromosomal trans-

location producing the fusion of the RARa gene with

the PML gene to produce an oncogene and responds

well to high doses of all-trans retinoic acid.

Requirements and Recommended Intakes

0028 Because of confusion among the various units for

presenting vitamin A values, the concept retinol

equivalent (RE) has been proposed. One retinol

equivalent equals 1 mg of all-trans retinol, either free

or as the retinyl component of a retinyl ester; 1 RE ¼

3.33 IU of vitamin A, or 3.5 nmol of retinol or retinyl

ester. Although the exact vitamin A value of caro-

tenoids depends on several factors (See Carotenoids:

Occurrence, Properties, and Determination), the ret-

inol equivalent was defined as 6 mg of all-trans b-

carotene or 12 mg of other provitamin A carotenoids

(10 IU of provitamin A carotenoid). To account for

updated findings on provitamin A value of carote-

noids, the retinol activity equivalent (RAE) has been

defined more recently as 1 mg of all-trans retinol,

12 mg of all-trans b-carotene, or 24 mg of other pro-

vitamin A carotenoids.

0029 Liver concentrations provide the best appraisal of

vitamin A status. Human liver specimens can be diffi-

cult to obtain, but analysis of autopsy specimens can

be useful in evaluating the vitamin A status of a

population. As indicated above, serum retinol levels

are maintained nearly constant and are not generally

useful in assessing vitamin A status, except when liver

vitamin A reserves fall well below 0.07 mmol g

1

(20 mg per gram of liver). Serum (or plasma) retinol

levels are normally 1–2 mM (290–570 ng ml

1

) across

a wide range of mammalian species.

0030 Because liver concentrations of vitamin A are not

readily measured, and serum retinol values are not an

adequate indicator of an individual’s vitamin A

status, several indirect methods have been developed.

Conjunctival impression cytology (CIC) evaluates the

development of squamous metaplasia (enlarged epi-

thelial cells) and loss of goblet cells from the conjunc-

tiva of the eye by histological examination of cells

transferred from the cornea to filter paper. The rela-

tive dose response (RDR) depends on the release of

retinol-RBP from liver into serum after a large oral

dose of vitamin A (typically, 450 mg of retinyl acetate

in human studies); as described above, apo-RBP ac-

cumulates in the liver in vitamin A depletion. The

RDR is calculated as:

RDR ¼

 A

 100%,

where A

represents the serum retinol concentration

at 5 h after the oral dose, and A

represents the fasting

serum retinol concentration at the time of the dose.

Studies in both humans and rats indicate that an RDR

value greater than 20% indicates liver vitamin A

reserves less than 0.07 mmol g

1

(< 20 mg per gram of

liver), i.e., inadequate vitamin A status. The RDR

assay requires, of course, that the oral dose be nor-

mally absorbed (an intramuscular injection has been

used in human subjects with biliary atresia), and that

protein metabolism is normal: protein deficiency or

zinc deficiency or liver cirrhosis impairs the dose

response. An alternate approach, the modified rela-

tive dose response (MRDR), uses the vitamin A

analog 3,4-didehydroretinol (vitamin A

, a form

found in some freshwater fish and found in very low

levels in human skin). The ratio of vitamin A

vitamin A

in serum retinol at 5 h after an oral dose

is used as a measure of vitamin A status, with high

values (ratio > 0.03 after an oral dose of 100 mg per

kilogram of body weight) indicating poor vitamin A

status. Chlorinated vitamin A analogs have been

used in a similar fashion. The RDR and MRDR, as

well as CIC, have been used successfully in human

population studies. Isotope dilution of tracer-labeled

vitamin A (radioactive or stable-isotope labeling) has

been used to estimate human vitamin A status, but

technical difficulties have so far prevented general use

of the technique.

0031Nutritional requirements for vitamin A have not

been well defined because of the diversity of vitamin

A functions. In animal studies, intakes of 3–8 RE per

day per kilogram of body weight (10–28 nmol per day

per kilogram of body weight) cure deficiency symp-

toms and intakes of 30–60 RE per day per kilogram of

body weight (100–210 nmol per day per kilogram

of body weight) produce optimal growth. Kinetic

studies of vitamin A metabolism in rats show that

irreversible loss of vitamin A is decreased on low

vitamin A intakes. Studies on vitamin A requirements

have not yet addressed functional criteria such as

immune function and possible anticancer effects.

0032To provide an adequate and safe human intake,

current WHO/FAO dietary recommendations (1988)

and suggested recommended dietary intakes (RDIs)

for adults are based on intakes of 9.3 RE per day per

kilogram of body weight (33 nmol of vitamin per day

per kilogram of body weight). The European Union

Scientific Committee for Food has recommended

population reference intakes of 700 RE per day

(men) and 600 RE per day (women), with estimated

average requirements of 500 and 400 RE, respect-

ively, and lowest threshold intakes of 300 and

250 RE, respectively. In contrast, the recommended

dietary allowances (RDAs) of the US National

RETINOL/Physiology 4965