Chapter 7. Riboflavin (Vitamin B2)

The classical glossitis, angular stomatitis, and dermatitis observed in advanced cases are not specific to riboflavin deficiency and may be due to other vitamin deficiencies as well.. An

7 Riboflavin (Vitamin B 2 )

Richard S Rivlin

CONTENTS

Introduction 233

History 233

Chemistry 234

Riboflavin Deficiency and Food-Related Issues 236

Riboflavin Deficiency 236

Food-Related Issues 238

Physiology 240

Absorption, Transport, Storage, Turnover, and Excretion 240

Specific Functions 241

Antioxidant Activity 241

Riboflavin and Malaria 242

Riboflavin and Homocysteine 243

Inborn Errors of Metabolism 243

Pharmacology, Toxicology, and Carcinogenesis 244

Requirements and Assessment 245

Acknowledgments 246

References 247

INTRODUCTION

Within the last few years, much has been learned about the role of riboflavin in intermediary metabolism and in several categories of disease The relationship of riboflavin to other

B vitamins has undergone further clarification Like many of the B vitamins, its metabolically active forms are as coenzyme derivatives, the formation of which is regulated by the nutri-tional state, hormones, drugs, and other stimuli There are now new approaches to riboflavin supplementation for specific purposes

Several recent reviews have emphasized the role of riboflavin in health (1), the regulation

of riboflavin metabolism (2), and the inborn errors of riboflavin metabolism with neuro-logical sequelae (3)

HISTORY

Perhaps the earliest scientific studies showing prevention of a deficiency state by riboflavin and other factors were those of McCollum and Kennedy (4), who observed its efficacy against a pellagra-like condition In later studies, a heat-labile and heat-stable fraction were identified The heat-stable fraction contained a yellow growth factor that was able to fluoresce After purification, the factor was named riboflavin (5) This heat-stable fraction

con tained a numb er of other essent ial nutri ents, includi ng niacin (variably call ed vita min B3) and vita min B6

The physiolo gical role of the yell ow grow th facto r was shown later by Warburg and Christ ian (6) who describe d the fact or as ‘‘old yell ow en zyme,’’ co mposed of an ap oenzyme and a yell ow cofact or as co enzyme The coenzyme was fou nd to have an isoalloxazi ne ring (7) and a phos phate-co ntaining side chain (8)

Ribof lavin was synthes ized by Kuh n et al (9) and Karr er et al (10) The structure of the first coen zyme formed sequenti ally from ribof lavin , ribof lavi n-50 -phosphat e, also called flavin mono nucleot ide (FMN), was establ ished by Theor ell (11) The struc ture of the secon d coen zyme form ed, flavin adenine dinucleot ide (FAD), was establis hed by Warbu rg an d Christ ian (12) Thi s co enzyme was synthes ized from its coenzyme precurs or, FMN

CHEMISTRY

FMN and FAD serve as co enzymes for enzymes in a wid e variety of react ions in intermed iary meta bolism There are also tissue form s of FAD, whi ch are covalent ly linked from the 8-al pha pos ition of the isoalloxazi ne por tion of the flav in via N(1) or N(3) of histidyl or the S of cysteinyl resi dues within specif ic enzymes that have a number of signi ficant roles in meta bolism (13) Thos e mamm alian enzymes with co valently bound flavins include sarcos ine deh ydrogenas e, succini c deh ydrogenas e, mono amine oxidase , and L -gulonola ctone oxidase (14) L-gulonol actone oxidase syn thesizes ascorbic acid from its precurs ors an d is not present

as a functional holoenzyme in human tissues

The planar isoalloxazine ring forms the basic structure for riboflavin, FMN, and FAD, as shown in Figure 7.1 The sequ ence of events in the syn thesis of the flavin coen zymes from ribof lavin and its c ontrol by thyroid hormones are shown in Figure 7.2 Thyroid hormones regulate the activities of the flavin biosynthetic enzymes (15), the synthesis of the flavopro-teins apoenzymes, and the formation of covalently bound flavins (16) The first biosynthetic enzyme, flavokinase, catalyzes the initial phosphorylation of riboflavin from ATP to form FMN A fraction of FMN is directly used in this form as a coenzyme The largest fraction of FMN, however, combines with a second molecule of ATP to form FAD, the predominant tissue flavin, in a reaction catalyzed by FAD synthetase, also called FAD pyrophosphorylase The covalent attachment of flavins to specific tissue proteins occurs after FAD has been synthesized A sequence of phosphatases returns FAD to FMN, and FMN, in turn, to riboflavin (15) Most flavoproteins use FAD rather than FMN as coenzyme for a wide variety of metabolic reactions

Microsomal NADPH-cytochrome P450 reductase is the first mammalian enzyme shown

to contain both FMN and FAD as coenzymes and in equimolar ratios Human novel reductase 1, like other diflavin reductases, also contains both FMN and FAD as prosthetic groups (17,18) In addition, nitric oxide synthase (19) and methionine synthase (20) also contain both FMN and FAD as coenzymes

Riboflavin is yellow in color and has a high degree of natural fluorescence when excited by

UV light, a property that can be used conveniently in its assay There are a number of variations

in structure in the naturally occurring flavins Riboflavin and its coenzymes are sensitive to alkali and to acid, particularly in the presence of UV light Under alkaline conditions, ribofla-vin is photodegraded to yield lumiflaribofla-vin (7,8,10-trimethylisoalloxazine), which is inactive biologically Riboflavin is photodegraded under acidic conditions to lumichrome (7,8-dimethylalloxazine), a product that is also biologically inactive Thus, an important physical property of riboflavin and its derivatives is their sensitivity to UV light, resulting in rapid inactivation Therefore, phototherapy of neonatal jaundice and of certain skin disorders has the potential to promote systemic riboflavin deficiency The structure–function relationships

of the various biologically active flavins have been comprehensively reviewed (21)

CH4 (CHOH)3 CH

2 OH N

NH N

N

CH3

(CHOH)2 CH2O

O O P OH

O

O

OH CH HOCH

CH N

N

N

N CH

NH3

C HOCH

CH

C CO

O

NH N

N

CH3

C CO

O

CH2 H O H

H O H H

OH O OH

CH3OP C

O H N

NH N

CH3

CH2

C CO

O Riboflavin-5 ⬘-phosphate (Flavin mononucleotide)

Flavin adenine dinucleotide (FAD)

Riboflavin

FIGURE 7.1 Structural formulas of riboflavin and the two coenzymes derived from riboflavin, FMN and FAD FMN is formed from riboflavin by the addition in the 50position of a phosphate group derived from adenosine triphosphate FAD is formed from FMN after combination with a second molecule of adenosine triphosphate

Thyroid

Hormone

Riboflavin

Flavokinase FMN Phosphatase

Flavin mononucleotide (FMN)

Unstable flavoprotein apoenzymes FAD Pyrophosphorylase Pyrophosphatase Stable flavoprotein holoenzymes

Flavin adenine dinucleotide (FAD)

FIGURE 7.2 Metabolic pathway of conversion of riboflavin into FMN, FAD, and covalently bound flavin, together with its control by thyroid hormones (From Rivlin, R.S., N Engl J Med., 283, 463, 1970.)

RIBOFLAVIN DEFICIENCY AND FOOD-RELATED ISSUES

RIBOFLAVINDEFICIENCY

Isolated clinical deficiency of riboflavin is not recognizable at the bedside by any unique or characteristic physical feature The classical glossitis, angular stomatitis, and dermatitis observed in advanced cases are not specific to riboflavin deficiency and may be due to other vitamin deficiencies as well In fact, when deficiency of riboflavin does occur, it is almost invariably in association with multiple nutrient deficits (22)

With the onset of riboflavin deficiency, one of the adaptations that occurs is a fall in the hepatic free riboflavin pool to nearly undetectable levels, with relative sparing of the pools of FMN and FAD that are needed to fulfill critical metabolic functions (23) Another adapta-tion to riboflavin deficiency in its early stages is an increase in the de novo synthesis of reduced glutathione (GSH) from its amino acid precursors, in response to the diminished conversion of oxidized glutathione back to GSH (24) This may represent a compensatory reaction resulting from depressed activity of glutathione reductase, a key FAD-requiring enzyme, as shown in Figure 7.3

Dietary inadequacy is not the only cause of riboflavin deficiency Certain endocrine abnormalities, such as adrenal and thyroid hormone insufficiency, specific drugs, and diseases may interfere significantly with vitamin utilization (24,25) Psychotropic agents, such

as chlorpromazine; antidepressants, including imipramine and amitriptyline (26); cancer

FAD

Oxidized glutathione (G55G)

FAD Glutathione reductase

Glutathione synthetase

y-Glutamylcysteine

glycine glycine

y-Glutamylcysteine synthetase

Olvtamate + Cysteine

Reduced glutathione (GSM)

FIGURE 7.3 Regeneration of reduced glutathione (GSH) under normal and riboflavin-deficient condi-tions The diagram represents two major pathways for the formation of GSH in erythrocytes, that is, reduction of oxidized glutathione (GSSG) via the glutathione reductase pathway and de novo biosyn-thesis via glutamylcysteine synthetase and glutathione synthetase Bold arrows are used to emphasize the predominant pathways, thin arrows represent pathways that are operating below maximal levels, and the dotted arrow indicates diminished enzymatic activity

chemotherapeutic drugs, for example, adriamycin; and some antimalarial agents, for example, quinacrine (27), impair riboflavin utilization by inhibiting the conversion of this vitamin into its active coenzyme derivatives Figure 7.4 shows the structural similarities among riboflavin, imipramine, chlorpromazine, and amitriptyline There is evidence that alcohol causes riboflavin deficiency by inhibiting both its digestion from dietary sources and its intestinal absorption (28)

In approaching riboflavin deficiency, as well as other nutrient deficiencies, it may be useful

to think in terms of risk factors That is to say, the consequences of a poor diet may be intensified if the patient is also abusing alcohol, using certain drugs for prolonged periods, is elderly, or has malabsorption or other underlying illnesses affecting vitamin metabolism (24)

In experimental animals, hepatic architecture is markedly disrupted in riboflavin defi-ciency Mitochondria in riboflavin-deficient mice increase greatly in size, and cristae increase

in both number and size (29) These structural abnormalities may disturb energy metabolism

by interfering with the electron transport chain and metabolism of fatty acids Villi decrease

in number in the rat small intestine; villus length increases, as does the rate of transit of developing enterocytes along the villus (30) These structural abnormalities, together with the accelerated rate of intestinal cell turnover (31), may help to explain why dietary riboflavin deficiency leads to both decreased iron absorption and increased iron loss from the intestine There are many other effects of riboflavin deficiency on intermediary metabolism, par-ticularly in lipid, protein, and vitamin metabolism Of particular relevance is the impaired conversion of vitamin B6to its coenzyme derivative, pyridoxal-50-phosphate (32) Riboflavin deficiency has been studied in many animal species and has several vital effects, foremost of which is failure to grow Other effects include loss of hair, skin disturbances, degenerative changes in the nervous system, and impaired reproduction Congenital malformations occur

in the offspring of female rats that are riboflavin-deficient The conjunctivae become inflamed, and the cornea is vascularized and eventually opaque with cataract formation (33) Changes in the skin consist of scaliness and incrustation of red–brown material consistent with changes in lipid metabolism Alopecia may develop, lips become red and swollen, and filiform papillae on the tongue deteriorate During late deficiency, anemia develops Fatty degeneration of the liver occurs Important metabolic changes occur, so that deficient rats require 15% to 20% more energy than control animals to maintain the same body weight

CH−(CH 2 )2−N(CH 3 )2

S

CI N

CH2−(CHOH) 3 −CH 2 OH N

N

NH CO

O

CH3

CH3

CH2−(CH 2 )2−N(CH 3 )2

N

CH2−(CH2)2−N(CH3)2

FIGURE 7.4 Structural formulas of riboflavin, chlorpromazine, imipramine, and amitriptyline showing their similarities

(34, 35) Thus , in a ll specie s studied , ribof lavin deficiency ca uses profound struc tural and functi onal changes in an ord ered sequence Early chan ges are v ery read ily revers ible Later anatom ical changes, such as form ation of cataract, are largely irre versible despit e treatment with ribof lavin

In huma ns, as noted earlier, the clini cal featu res of human ribof lavin de ficiency do not have absolut e specif icity Early sympt oms may include weakne ss, fatigue, mo uth pain and tendernes s, burn ing and itching of the eyes, an d possibl e person ality chan ges More ad vanced defic iency may give rise to cheilo sis, angu lar stomati tis, dermat itis, corneal vascul ariza tion, anemi a, an d brain dysfunct ion Thus , the syn drome of dietary ribof lavin deficiency in human s has many sim ilarities to that in anima ls, wi th one notab le exception The spectr um

of congen ital malforma tions obs erved in rodents (33) with mate rnal ribof lavin de ficiency has not been clear ly identi fied in humans

The role of ribof lavin in catar act ha s be en the su bject of recent ren ewed inter est For some time, higher intake of riboflavi n has bee n associ ated with reduced cataract form ation (36) The use of riboflavi n for a 5 year period in the Nurses Health Stud y was associated with a decreas ing rate of de velopm ent of lens opacif ication (37) In ano ther study of patie nts who alrea dy had keratoc onus, ribof lavin admini stered in eye drops delayed its progres sion (38) Treatm ent of keratoc onus with ribof lavin and UV light increa ses the sti ffness of the cornea, increa ses the cross-lin king of c ollagen, and in this manner may inhibit pro gression of the disorde r UV A light reduces the acti vity of gluta thione redu ctase in the lens because of the light sensitiv ity of its FAD coe nzyme (39)

F OOD-RELATED ISSUES

The mo st signifi cant dieta ry sources of ribof lavin in the Unit ed States today are meat and meat produ cts, includi ng poultr y and fis h, as well as milk and dairy products , such as eggs and ch eese In developi ng countri es, plant sources co ntribute most of the dieta ry riboflavi n intake Green vegeta bles, su ch as broccoli , collard greens, an d turni p greens, are reasonabl y goo d sources of ribof lavi n Natur al grain pro ducts tend to be relat ively low in ribof lavin, but fort ification and enrichm ent of grains and cereal s has led to a consider able increa se in ribof lavin intake from these food items

The food sou rces of ribof lavi n are simila r to those of other B vitamins Therefor e, it is not surpri sing that if a given individu al’s diet has inadequat e amoun ts of ribof lavin, it is very likely to be inadequat e in other vita mins as wel l A prima ry de ficiency of dieta ry riboflavi n has wide impl ications for other vitamins , as flav in coen zymes are involv ed in the meta bolism

of folic acid, pyridoxi ne, vitamin K, niaci n, and vita min D (22)

Se veral facto rs in food preparat ion and process ing may influence the amoun t of riboflavi n that is actual ly bioavai lable from dieta ry sources In view of the light sensitiv ity of ribof la-vin noted earlier, it is not surprising that appreci able amounts of ribof lala-vin may be lost with expo sure to UV light, parti cularly during co oking and pro cessing Pro longed stora ge of milk

in c lear bottles or contai ners may resul t in flavin deg radation (40) For tunate ly, most milk is

no longer sold in clear bottles There has been some con troversy a s to wheth er opaqu e plastic containers provide greater protection than do cartons, particularly when milk is stored on a grocery shelf exposed to continuous fluorescent lighting Milk must be perfectly protected against light; otherwise significant amounts of riboflavin and vitamin A will be lost and the flavor will deteriorate (41)

It is highly likely that large amounts of riboflavin are lost during the sun-drying of fruits and vegetables The precise magnitude of the loss is not known but varies with the duration

of exposure The practice of adding sodium bicarbonate as baking soda to green vegetables

to make them appear fresh can result in accelerated photodegradation of riboflavin The riboflavi n content of common food items with the highest amo unts is shown in Tabl e 7.1

TABLE 7.1

Top Sources of Riboflavin and Their Caloric Content

Top Food Sources

Riboflavin (mg=100 g)

Energy (kcal=100 g) Top Food Sources

Riboflavin (mg=100 g)

Energy (kcal=100 g) Yeast baker’s dry (active) 5.41 282 Cheese, pasteurized, process American 3.53 375 Liver, lamb, broiled 5.11 261 Liver, chicken, simmered 1.75 165 Yeast, torula 5.06 277 Corn flakes, with added nutrients 1.40 380 Kidneys, beef, braised 4.58 252 Almonds, shelled 0.93 598 Liver, hog, fried in margarine 4.36 241 Cheese, natural, Roquefort 0.59 369 Yeast, brewer’s, debittered 4.28 283 Eggs, chicken, fried 0.54 210 Liver, beef or calf, fried 4.18 242 Beef, tenderloin steak, broiled 0.46 224 Brewer’s yeast, tablet form 4.04 — Mushrooms, raw 0.46 28 Cheese, pasteurized, process American 3.53 375 Cheese, natural Swiss (American) 0.40 372 Turkey, giblets, cooked (some gizzard fat), simmered 2.72 233 Wheat flour, all-purpose, enriched 0.40 365 Kidneys, lamb, raw 2.42 105 Turnip greens, raw 0.39 28 Kidneys, calf, raw 2.40 113 Cheese, natural Cheddar 0.38 402 Eggs, chicken, dried, white powder 2.32 372 Wheat bran 0.35 353 Whey, sweet, dry 2.21 354 Soybean flour 0.35 333 Eggs, chicken, dried, white flakes 2.16 351 Bacon, cured, cooked, drained, sliced medium 0.34 575 Liver, turkey, simmered 2.09 174 Pork, loin, lean, broiled 0.33 391 Whey, acid dry 2.06 339 Lamb, leg, good or choice, separable lean roasted 0.30 186 Heart, hog, braised 1.89 195 Corn meal, degermed, enriched 0.26 362 Milk, cow’s dry, skim, solids, instant 1.78 353 Chicken, dark meat without skin, fried 0.25 220 Liver, chicken, simmered 1.75 165 Bread, white, enriched 0.24 270 Liver, beef or calf, fried 4.18 242 Milk, cow’s, whole, 3.7% 0.17 66 Source: From Ensminger, A.M., Ensminger, M.E., Konlande, J.E., and Robson, J.R.K in Food and Nutrition Encyclopedia, CRC Press, Boca Raton, FL, p 1927, 1994 Figures are given in terms of the riboflavin and calorie amounts in 100 g (approximately 3.5 oz) of the items as usually consumed Portion size and moisture content differ among food items.

ABSORPTION, TRANSPORT, STORAGE, TURNOVER,ANDEXCRETION

Since dietary sources of riboflavin are largely in the form of their coenzyme derivatives, these molecules must be hydrolyzed before absorption Very little dietary riboflavin is found as free riboflavin from sources in nature Under ordinary circumstances, the main sources of free riboflavin are commercial multivitamin preparations, which are consumed increasingly by the general public

The absorptive process for flavins occurs in the upper gastrointestinal tract by specialized transport involving a dephosphorylation–rephosphorylation mechanism, rather than by pas-sive diffusion This process is sodium-dependent and involves an ATPase-active transport system that can be saturated (35) It has been estimated that under normal conditions the upper limit of intestinal absorption of riboflavin at any one time is approximately 25 mg (34) This amount represents approximately 15 times the recommended dietary allowance (RDA) Therefore, the common practice of some megavitamin enthusiasts to consume massive doses of multivitamins has little benefit with respect to riboflavin, as the additional amounts would be passed in the stool Dietary covalent-bound flavins are largely inaccessible

as nutritional sources In experimental animals, the uptake of riboflavin from the intestine is increased in dietary riboflavin deficiency (35), which likely represents an adaptive mechanism

to vitamin deficiency

A number of physiological factors influence the rate of intestinal absorption of riboflavin (24) Diets high in psyllium gum decrease the rate of riboflavin absorption, whereas wheat bran has no detectable effect The time from oral administration to peak urinary excretion of riboflavin is prolonged by the antacids, aluminum hydroxide, and magnesium hydroxide Total urinary excretion is unchanged by these drugs, however, and their major effects appear

to be delaying the rate of intestinal absorption rather than inhibiting net absorption As noted earlier, alcohol interferes with both the digestion of food flavins into riboflavin and the direct intestinal absorption of the vitamin (28) This observation suggests that the initial rehabili-tation of malnourished alcoholic patients may be accomplished more rapidly and efficiently with vitamin supplements containing riboflavin rather than with food sources comprising predominantly phosphorylated flavin derivates This hypothesis needs to be tested directly There is evidence that the magnitude of intestinal absorption of riboflavin is increased by the presence of food This effect of food may be due to decreasing the rates of gastric emptying and intestinal transit, thereby permitting more prolonged contact of dietary ribo-flavin with the absorptive surface of the intestinal mucosal cells In general, delaying the rate

of gastric emptying tends to increase the intestinal absorption of riboflavin Bile salts also increase the rate of intestinal absorption of riboflavin (24)

A number of metals and drugs form chelates or complexes with riboflavin and

riboflavin-50-phosphate that may affect their bioavailability (42) Among the agents in this category are the metals, copper, zinc, and iron; the drugs, caffeine, theophylline, and saccharin; and the vitamins, nicotinamide and ascorbic acid; as well as tryptophan and urea The clinical significance of this complex formation is not known with certainty in most instances and deserves further study

In human blood, the transport of flavins involves loose binding to albumin and tight binding to a number of globulins The major binding of riboflavin and its phosphorylated derivatives in serum is to several classes of immunoglobulins, that is, IgA, IgG, and IgM (42)

In human erythrocytes, there is very little free riboflavin, compared with the much larger amounts of FMN and FAD (43) Following supplementation of human subjects with 1.6 mg=day of riboflavin, the concentrations of riboflavin in serum and FMN in erythrocytes are increased more than 80% compared with levels in placebo-fed controls (43)

Pregnancy induces the formation of flavin-specific binding proteins initially found in birds (44) Riboflavin-binding proteins have also been found in sera from pregnant cows, monkeys, and humans A comprehensive review of riboflavin-binding proteins covers the nature of the binding proteins in various species and provides evidence that, as in birds, these proteins are crucial for successful mammalian reproduction (13) Pregnancy-specific binding proteins may help transport riboflavin to the fetus

Serum riboflavin-binding proteins appear to influence placental transfer and fetal or maternal distribution of riboflavin There are differential rates of uptake of riboflavin at the maternal and fetal surfaces of the human placenta (45) Riboflavin-binding proteins regulate the activity of flavokinase, the first biosynthetic enzyme in the riboflavin-to-FAD pathway (21)

Urinary excretion of flavins occurs predominantly in the form of riboflavin; FMN and FAD are not found in urine McCormick (13) has identified and described a large number of flavins and their derivatives in human urine Besides the 60% to 70% of urinary flavins contributed by riboflavin itself, other major derivatives include 7-hydroxymethylriboflavin (10% to 15%), 8a-sulfonylriboflavin (5% to 10%), 8-hydroxymethylriboflavin (4% to 7%), riboflavinyl peptide ester (5%), and 10-hydroxyethylflavin (1% to 3%), representing largely metabolites from covalently bound flavoproteins and intestinal riboflavin degradation by microorganisms Traces of lumiflavin and other derivatives have also been found

Accidental ingestion of boric acid greatly increases urinary excretion of riboflavin (24) This agent when consumed forms a complex with the side chain of riboflavin and other molecules that have polyhydroxyl groups, such as glucose and ascorbic acid In rodents, riboflavin treatment greatly ameliorates the toxicity of administered boric acid This treat-ment should also be effective in humans with accidental exposure of boric acid, although in practice it may be difficult to provide adequate amounts of riboflavin because of its low solubility and limited absorptive capacity from the intestinal tract

Urinary excretion of riboflavin in rats is also greatly increased by chlorpromazine (46) Levels are twice those of age- and sex-matched pair-fed control rats In addition, chlorpromazine accelerates urinary excretion of riboflavin during dietary deficiency Urinary concentrations of riboflavin are increased within 6 h of treatment with this drug

SPECIFIC FUNCTIONS

The major function of riboflavin, as noted earlier, is to serve as the precursor of the flavin coenzymes, FMN and FAD, and of covalently bound flavins These coenzymes are widely distributed in intermediary metabolism and catalyze numerous oxidation–reduction reac-tions As FAD is part of the respiratory chain, riboflavin is central to energy production Other major functions of riboflavin include drug and steroid metabolism, in conjunction with cytochrome P450 enzymes, and lipid metabolism The redox functions of flavin coenzymes include both one-electron transfers and two-electron transfers from the substrate to the flavin coenzymes (13)

Flavoproteins catalyze dehydrogenation reactions as well as hydroxylations, oxidative decarboxylations, dioxygenations, and reductions of oxygen to hydrogen peroxide Thus, many different kinds of oxidative and reductive reactions are catalyzed by flavoproteins

ANTIOXIDANTACTIVITY

In the wake of contemporary interest in dietary antioxidants, one vitamin that is often not appreciated sufficiently as a member of this category is riboflavin Riboflavin has little, if any, significant antioxidant action per se, but powerful antioxidant activity is derived from its role

as a precursor to FMN and FAD A major protective role against lipid peroxides is provided

by the glutathione redox cycle (47) Glutathione peroxidase breaks down reactive lipid peroxides This enzyme requires GSH, which in turn is regenerated from its oxidized form (GSSG) by the FAD-containing enzyme glutathione reductase Thus, riboflavin nutrition may be critical in regulating the rate of inactivation of lipid peroxides Diminished glu-tathione reductase activity would be expected to lead to diminished concentrations of GSH that serve as substrate for glutathione peroxidase and glutathione S-transferase, and therefore would limit the rate of degradation of lipid peroxides and xenobiotic substances (48) Furthermore, the reducing equivalents provided by NADPH, the other substrate required

by glutathione reductase, are primarily generated by an enzyme of the pentose monopho-sphate shunt, glucose-6-phomonopho-sphate dehydrogenase Taniguchi and Hara (49), as well as Dutta

et al (50), have found that the activity of glucose-6-phosphate dehydrogenase is significantly diminished during riboflavin deficiency This observation provides an additional mechanism

to explain the diminished glutathione reductase activity in vivo during riboflavin deficiency and the eventual decrease in antioxidant activity

There have been reports (51,52) indicating that riboflavin deficiency is associated with compromised oxidant defense and furthermore that supplementation of riboflavin and its active analogs improves oxidant status Riboflavin deficiency is associated with increased hepatic lipid peroxidation and riboflavin supplementation limits this process (49–52) In our laboratory, we have shown that feeding a riboflavin-deficient diet to rats increases basal as well as stimulated lipid peroxidation (48)

RIBOFLAVIN ANDMALARIA

There is increasing evidence that riboflavin deficiency may be protective against malaria both

in experimental animals and in humans (53,54) With dietary riboflavin deficiency, parasite-mia is decreased dramatically, and symptomatology of infection may be diminished In a study with human infants suffering from malaria, normal riboflavin nutritional status was associated with high levels of parasitemia In similar fashion, supplementation with iron and vitamins that included riboflavin resulted in increased malaria parasitemia (55,56)

Further evidence for a beneficial role of riboflavin deficiency in malaria is provided by studies using specific antagonists of riboflavin, for example, galactoflavin and 10-(40 -chlorophenyl)-3-methylflavin (57,58) These flavin analogs as well as newer isoalloxazines derivatives are glutathione reductase inhibitors and possess clear antimalarial efficacy The exact mechanism

by which riboflavin deficiency appears to inhibit malarial parasitemia is not yet established One possibility relates to effects on the redox status of erythrocytes, which is an important determinant of growth of malaria parasites Protection from malaria is afforded by several oxidant drugs, vitamin E deficiency, and specific genetic abnormalities in which oxidative defense

is compromised (47)

It is well known that malaria parasites (Plasmodium berghei) are highly susceptible to activated oxygen species Parasites are relatively more susceptible than erythrocytes to the damaging effects of lipid peroxidation (47) We have hypothesized that the requirement of parasites for riboflavin should be higher than that of the host cells and therefore that marginal riboflavin deficiency should be selectively detrimental to parasites Support for this hypoth-esis comes from the finding that the uptake of riboflavin and its conversion to FMN and FAD are significantly higher in parasitized than in unparasitized erythrocytes and further-more that the rate of uptake of riboflavin is proportional to the degree of parasitemia (59) These results strongly suggest that parasites have a higher requirement for riboflavin than do host erythrocytes

In a recent report of malaria patients in Gabon (60), plasma levels of FAD, FMN, and riboflavin were normal, but the authors point out that because of the high degree of