Photo Addition Reaction Of Riboflavin

7-carboxyriboflavin O

8-carboxylumichrome O

photolysis or bacterial metabolism h3c.

lumichrome o




Figure 7.2. Products of riboflavin metabolism. Relative molecular masses (Mr): riboflavin, 376.4; lumiflavin, 256.3; 8- and 7-carboxylumichrome, 296.2; and lumichrome, 242.2.

Table 7.2 Urinary Excretion of

Riboflavin Metabolites

% of Total



7-Hydroxymethyl riboflavin


8-a-Sulfonyl riboflavin


8-Hydroxymethyl riboflavin


Riboflavin peptide esters


10-Hydroxyethyl riboflavin


flavokinase. There is some evidence that 8-hydroxymethylriboflavin may have biological activity; it is not known whether or not it is involved in the formation of 8-a-amino acid covalent links in proteins. Significant amounts of both of these hydroxylated derivatives and their onward oxidation products (7- and 8-carboxyriboflavin) are excreted in the urine (see Figure 7.2 and Table 7.2).

Intestinal bacterial cleavage of the ribityl side chain results in the formation of 10-hydroxyethylflavin (an oxidation product of lumiflavin), lumichrome, and 7- and 8-carboxy-lumichromes, which are also excreted in the urine. Some of the lumichromes detected in urine may result from photolysis of riboflavin in the circulation.

7.2.6 Biosynthesis of Riboflavin

A number of fungi have a failure of the normal regulation of riboflavin synthesis and are overproducers of the vitamin. Mutants of Ashbya gossypii may accumulate up to 150 ^mol of riboflavin per gram of protein, compared with a normal content of 0.25 ^mol per gram of protein. They can produce and excrete so much that riboflavin crystallizes in the culture medium. Such fungi are used for the commercial production of riboflavin by fermentation, as an alternative to chemical synthesis.

The precursors for riboflavin biosynthesis in plants and microorganisms are guanosine triphosphate and ribulose 5-phosphate. As shown in Figure 7.3, the first step is hydrolytic opening of the imidazole ring of GTP, with release of carbon-8 as formate, and concomitant release of pyrophosphate. This is the same as the first reaction in the synthesis ofpterins (Section 10.2.4), but utilizes a different isoenzyme of GTP cyclohydrolase (Bacher et al., 2000, 2001).

In yeasts and fungi, opening of the imidazole ring is followed by reduction of the ribose side chain to ribitol, deamination, and dephosphorylation to yield amino-ribitylamino-pyrimidinedione; in bacteria, deamination occurs before

Riboflavin Methylene Reductase Mechanism
Figure 7.3. Biosynthesis of riboflavin in fungi; in bacteria, deamination precedes reduction of sugar. GTP cyclohydrolase, EC; and riboflavin synthase, EC

reduction of the sugar. Amino-ribitylamino-pyrimidinedione condenses with dihydroxybutanone 4-phosphate, which is formed from ribulose 5-phosphate by an unusual reaction involving loss of carbon-4 via an intramolecular rearrangement. The product of the reaction catalyzed by lumazine synthase is dimethyllumazine.

The final stepis a dismutation reaction between two molecules of dimethyl-lumazine, catalyzed by riboflavin synthase, yielding riboflavin and amino-ribitylamino-pyrimidinedione. This latter product can undergo reaction with dihydroxybutanone 4-phosphate to yield dimethyl-ribityllumazine.


The metabolic function of the flavin coenzymes is as electron carriers in a wide variety of oxidation and reduction reactions central to all metabolic processes, including the mitochondrial electron transport chain. Unlike the nicotinamide nucleotide coenzymes (Section 8.4.1), which act as cosubstrates, leaving the catalytic site of the enzyme at the end of the reaction, the flavin coenzymes remain bound to the enzyme throughout the catalytic cycle.

FAD is the prosthetic group of the bacterial photolyase that reduces cyclobutane thymine dimers formed in DNA as a result of ultraviolet (UV) irradiation; closely homologous proteins in the human eye (the cryptochromes) are the blue-sensitive pigments that are responsible for day-length sensitivity and the setting of circadian rhythms.

7.3.1 The Flavin Coenzymes: FAD and Riboflavin Phosphate

The majority of flavoproteins have FAD as the prosthetic group rather than riboflavin phosphate. Some have both flavin coenzymes, and some have other prosthetic groups.

As shown in Figure 7.4, flavins can undergo a one-electron reduction to the semiquinone radical or a two-electron reduction to dihydroflavin. This means that flavins can act as intermediates between obligatory two-electron redox reactions involving nicotinamide nucleotides (Section 8.4.1) and obligatory one-electron reactions involving cytochromes, iron-sulfur proteins, and ubiquinone (Section 14.6).

In solution, the flavin semiquinone radical is highly unstable, undergoing rapid equilibration to a mixture of the oxidized and fully reduced flavins. It is stabilized by protein binding in enzymes.

The neutral flavin radical has an absorption maximum at 580 nm and hence a blue color; it is sometimes referred to as the blue radical. It can undergo either protonation at N-1 to yield a cation radical or deprotonation at N-5 to yield an anion radical, if the enzyme has appropriate proton donating or withdrawing amino acid residues at the catalytic site. Both protonation and deprotonation result in the same spectral shift to give an absorption maximum at 470 nm and hence a red color. Both the blue and red radicals are seen as intermediates in enzyme reactions, suggesting that some enzymes form the neutral radical, whereas others form one of the charged radicals.

Riboflavin Lumiflavin Reactions
Figure 7.4. One- and two-electron redox reactions of riboflavin.

Dihydroflavin can be oxidized by reaction with a substrate, NAD(P)+ or cytochromes in a variety of dehydrogenases, or can react with molecular oxygen in oxygenases and mixed-function oxidases.

7.3.2 Single-Electron-Transferring Flavoproteins

Flavoproteins catalyzing single-electron transfer provide the link between substrate oxidation catalyzed by dehydrogenases and the mitochondrial electron transport chain.

The simplest such single-electron-transferring flavoproteins are the flavo-doxins of obligate anaerobic bacteria, which catalyze a single-electron-transfer reaction, cycling between dihydroflavin and the semiquinone radical. In all other organisms, the electron transport iron-sulfur flavoprotein undergoes a two-electron reduction at the expense of NADH; this may result in either the formation of dihydroflavin or reduction to the semiquinone radical of two molecules of flavin in the enzyme. The reduced enzyme then transfers electrons singly to cytochrome b.

The reduction of cytochrome P450 by NADPH involves a single enzyme, NADPH-cytochrome P450 reductase, which contains both FAD and riboflavin phosphate. The FAD undergoes a two-electron reduction at the expense of NADPH, then transfers electrons singly to the riboflavin phosphate, which in turn reduces cytochrome P450. The semiquinone radicals of both FAD and riboflavin phosphate are intermediates in this reaction.

A distinct electron transfer flavoprotein (ETF) is the single-electron acceptor for a variety of flavoprotein dehydrogenases, including acyl CoA, glutaryl CoA, sarcosine, and dimethylglycine dehydrogenases. It then transfers the electrons to ETF-ubiquinone reductase, the iron-sulfur flavoprotein that reduces ubiquinone in the mitochondrial electron transport chain.

7.3.3 Two-Electron-Transferring Flavoprotein Dehydrogenases

The initial step of the two-electron-transferring reactions is the removal of a proton from the substrate, followed by the intermediate formation of an adduct between the substrate and prosthetic group at N-5 of the flavin. This undergoes cleavage to yield dihydroflavin and the oxidized product, which is commonly a carbon-carbon double bond. The reduced flavin is then reoxidized by reaction with an electron-transferring flavoprotein, as discussed above, or in some cases by reaction with nicotinamide nucleotide coenzymes.

The nicotinamide nucleotide independent flavoprotein dehydrogenases include the following:

1. Succinate dehydrogenase in the tricarboxylic acid cycle, which reacts directly with ubiquinone in the mitochondrial electron transport chain.

2. Acyl CoA dehydrogenases in fatty acid f-oxidation. These enzymes are especially sensitive to riboflavin depletion, and riboflavin deficiency is characterized by impaired fatty acid oxidation and organic aciduria (Section 7.4.1). These are also the enzymes affected in riboflavin-responsive organic acidurias.

3. Dimethylglycine and sarcosine dehydrogenases in the catabolism of choline (Section 14.2.1). In these reactions, a methyl group in the substrate is oxidized by FAD, then the intermediate adduct undergoes hydrolysis to release formaldehyde, which reacts with tetrahydrofolate to form 5,10-methylene tetrahydrofolate.

7.3.4 Nicotinamide Nucleotide Disulfide Oxidoreductases

Glutathione reductase, thioredoxin reductase, and lipoamide dehydrogenase are members of a group of flavoproteins that contain an active site disulfide as well as FAD. They catalyze the NAD(P)H dependent reduction of a disulfide

Figure 7.5. Reaction of glutathione peroxidase (EC and glutathione reductase (EC Relative molecular masses (Mr): glutathione, 307.3; and oxidized glutathione, 612.6.

substrate to its dithiol form. The initial step in the reaction is reduction of the disulfide to yield a sulfhydryl group and a flavin-cysteine adduct, followed by release of the oxidized flavin to leave a second sulfhydryl group at the active site. It is these two disulfide groups that catalyze the reduction of the disulfide substrate. The reaction of glutathione reductase is shown in Figure 7.5, and that of lipoamide dehydrogenase in Figure 6.2.

7.3.5 Flavin Oxidases

Flavin oxidases include d- and L-amino acid oxidases, and some amine oxidases, although others are quinoproteins (Section 9.8.3). In these enzymes, the flavin is reduced by dehydrogenation of the substrate, byway of an intermediate substrate-flavin adduct, as occurs in the dehydrogenases (Section 7.3.3).

Alter the oxidized product has left the enzyme, the reduced flavin reacts with oxygen to form, initially, the flavin semiquinone radical and superoxide. These undergo the sequence of rapid reactions shown in Table 7.3, ultimately resulting in reoxidation of the flavin and formation of hydrogen peroxide.

Table 7.3 Reoxidation of Reduced Flavins in Flavoprotein Oxidases

The overall reaction is:

X-H2 + flavin ^ X + flavin-H2, flavin-H2 + O2 ^ flavin + H2O2 Fully reduced flavin-H2 reacts with oxygen to form the flavin semiquinone radical and superoxide flavin-H2 + O2 ^ flavin-H' + 'O2-Flavin semiquinone and superoxide react to form flavin hydroperoxide flavin-H' + 'O2- ^ flavin-HOOH Flavin hydroperoxide slowly breaks down to yield flavin semiquinone and perhydroxyl flavin-HOOH ^ flavin-H' + 'O2H Perhydroxyl decays to superoxide plus a proton

In the presence of H+, flavin semiquinone and superoxide yield peroxide and oxidized flavin flavin-H' + H+ + 'O2- ^ flavin + H2O2

By their production of superoxide and perhydroxyl radicals and hydrogen peroxide, flavin oxidases make a significant contribution to the so-called oxidant stress of the body. Overall, some 3% to 5% of the daily consumption of about 30 mol of oxygen by an adult human being is converted to singlet oxygen, hydrogen peroxide, and the superoxide, perhydroxyl and hydroxyl radicals, rather than undergoing complete reduction to water in the electron transport chain. There is thus a total production of 1.5 mol of reactive oxygen species daily, potentially capable of causing damage to membrane lipids, proteins, and nucleic acids.

Paradoxically, although the oxidation of reduced flavins contributes significantly to oxidant stress, it may also have a protective role. There is a significant amount of reduced riboflavin bound to protein inhepatocyte (and presumably other cell) membranes. This undergoes oxidation when the cells are exposed to oxygen. The superoxide so produced may have a protective role in trapping the considerably more reactive and damaging hydroxyl radicals produced in other reactions (Nokubo et al., 1989). Nevertheless, the fact that reduced flavins react nonenzymically with oxygen to yield superoxide and perhydroxy radicals (the autoxidation of flavins) suggests that they are potentially toxic in excess. This may explain not only the limitation of the absorption of riboflavin from the intestinal tract, but also the active efflux of free riboflavin from the central nervous system and the active secretion of the vitamin in the renal tubule.

7.3.6 NADPH Oxidase, the Respiratory Burst Oxidase

NADPH oxidase was originally described in activated macrophages, whose function is to generate reactive oxygen species and halogen radicals as part of the cytotoxic action against phagocytosed microorganisms. It catalyzes transfer of electrons from NADPH onto cytochrome b558, which then reduces oxygen to yield 2 mol of superoxide and two protons. Activation of the oxidase requires increased formation of NADPH and hence increased oxidation of glucose through the pentose phosphate pathway (see Figure 6.4), the so-called respiratory burst.

The oxidase is a cell membrane-multienzyme complex. It has a cell surface receptor linked to a G-protein that activates a phosphatidyl inositol cascade leading to assembly and activation of the oxidase complex. The receptor is activated by the following:

1. Complement fragment C5a, which arises from the antibody-antigen reaction.

2. Peptides containing the sequence N-formyl-Met-Leu-Phe. These may be bacterial peptides or peptides arising from mitochondria of damaged tissue.

3. A variety of endogenous responses to infection and mediators of inflammatory reactions, including platelet activating factor, leukotriene p 4, and interleukin-8.

The NADPH binding site is on the cytosolic side of the membrane, whereas the superoxide release site is either extracellular or on the luminal side of the phagocytic vesicle. The enzyme acts as an ion pump, because it releases superoxide without an accompanying cation; protons remain inside the cell, resulting in considerable membrane depolarization (Babior, 1992; Chanock etal., 1994).

NADPH oxidase activity has also been demonstrated in a wide variety of cells other than macrophages, where it is not involved in cytotoxic action against engulfed microorganisms. It seems likely that reactive oxygen species produced by NADPH oxidase have a role in cell signaling, possibly as part of the mechanism of apoptosis.

7.3.7 Molybdenum-Containing Flavoprotein Hydroxylases

Xanthine oxidoreductase and aldehyde oxidase represent a distinct class of flavin-dependent oxidases. They both dehydrogenate andhydroxylate the substrate. However, unlike the mixed-function oxidases (Section 7.3.8), the oxygen introduced into the substrate by these enzymes is derived from water, and the role of molecular oxygen is in the reoxidation of the reduced flavin. Among other reactions, aldehyde oxidase is important in the oxidation of N1 -methyl nicotinamide to methyl pyridone carboxamide (Section 8.2.4) and pyridoxal to 4-pyridoxic acid (Section 9.2).

The initial step in the reaction is dehydrogenation of the substrate at the expense of the molybdenum, which is reduced from MoVI to MoIV. This is followed by attack by the persulfide group on the carbon atom of the substrate at which the hydroxyl group will be introduced. Hydrolysis of the persulfidecarbon bond then introduces a hydroxyl group. The reduced molybdopt-erin is reoxidized by the iron-sulfur groups, which in turn reduce the FAD. The reduced flavin then reacts with oxygen, eventually forming hydrogen peroxide.

Xanthine oxidoreductase contains two molecules of molybdenum as molybdopterin (Section 10.5), two molecules of FAD, eight non-heme iron-sulfide groups, and two persulfide (—S—S—) groups. It exists as two interconvertible forms:

1. The xanthine dehydrogenase form catalyzes the oxidation of xanthine to hypoxanthine at the expense of NAD+.

2. The xanthine oxidase form cannot utilize NAD+, but reduces oxygen to hydrogen peroxide.

The dehydrogenase form of the enzyme is converted to the oxidase form by reversible oxidation of cysteine to form a disulfide bridge. The redox potential of the dehydrogenase form of the enzyme is considerably lower than that of the oxidase form, because the protein confers greater stability on the neutral flavin semiquinone radical (Rajagopalan and Johnson, 1992; Kiskeretal., 1997; Nishino and Okamoto, 2000).

7.3.8 Flavin Mixed-Function Oxidases (Hydroxylases)

The flavin-dependent mixed-function oxidases include amine N-oxidases and a variety of S-oxidases. They provide an alternative to cytochrome P450-dependent enzymes in the metabolism of xenobiotics.

In most of these enzymes, the flavin is reduced to dihydroflavin by NADPH, although some also act as dehydrogenases, both oxidizing and hydroxylating the substrate so that it is the substrate that is the source of hydrogen to form the dihydroflavin. This then forms a hydroperoxide by reaction with oxygen. Rather than decaying to the flavin and perhydroxyl radicals as in the oxidases discussed in Section 7.3.5, the hydroperoxide is stabilized by the enzyme protein and is cleaved by the substrate, resulting in transfer of a hydroxyl group and leaving the flavin C-4 hydroxide, that breaks down to yield water and the oxidized flavin, via the sequence of reactions shown in Table 7.4, again adding to the overall radical burden in the body.

Table 7.4 Reoxidation of Reduced Flavins in Flavin Mixed-Function Oxidases

The overall reaction is:

X-H2 + O2 ^ X-OH + H2O or X + NADPH + O2 ^ X-OH + NADP+ + H2O Flavin is reduced by reaction with either substrate-H2 or NADPH. Fully reduced flavin-H2 reacts with oxygen to form the flavin semiquinone radical and superoxide flavin-H2 + O2 ^ flavin-H' + 'O2-Flavin semiquinone and superoxide react to form flavin hydroperoxide flavin-H' + 'O2- ^ flavin-HOOH Flavin hydroperoxide reacts with substrate flavin-HOOH + X ^ flavin-HOOH-X Intermediate complex breaks down to hydroxylated product + flavin hydroxide flavin-HOOH-X ^ flavin-OH + X-OH Flavin hydroxide breaks down to regenerate fully oxidized flavin + H2O flavin-OH ^ flavin + H2O

7.3.9 The Role of Riboflavin in the Cryptochromes

One of the mechanisms of DNA repair in bacteria, acting to reduce cyclobutane dipyrimidines and pyrimidine-pyrimidone dimers formed by exposure to UV light, is the blue light-activated photolyase. The primary light-trapping pigment is 5,10-methylene tetrahydrofolate (Section 10.1), which then transfers the excitation energy of the trapped photon to FADH, which reduces the substrate.

Cryptochromes in the human eye have a considerable sequence and structure homology with the photolyases, binding both methylene tetrahydrofolate and FAD. They have the same DNA binding pocket as photolyase, although they do not catalyze the reduction of DNA pyrimidine dimers. They are found in the nucleus of cells of the inner layer of the retina, behind the rods and cones involved in vision (Section 2.3.1), and absorb blue light, with maximum absorbance at 420 nm.

The function of cryptochromes is in setting the circadian clock in response to day-length. In response to excitation by light, there are changes in the expression in the retinal cells of genes known to regulate the circadian cycle, possibly as a result of increased ubiquitination and hence catabolism of the TIM protein (the product of the timeless gene). In addition, a nerve impulse is generated along fibers of the optic nerve that innervate the suprachiasmatic nucleus in the anterior hypothalamus, rather than the visual cortex. It is not known how this nerve impulse is initiated in response to photoexcitation of cryptochrome (Sancar, 2000; Lin et al., 2001).


Riboflavin deficiency is relatively common, yet there is no clear deficiency disease and the condition never seems to be fatal. This presumably reflects the high degree of conservation of riboflavin in tissues (Section 7.2.3). There is only a relatively small difference between the concentration of flavins at which tissues are saturated and the lowest levels in prolonged depletion of experimental animals. In deficiency, most of the flavin coenzymes released by the catabolism of enzymes are reutilized.

Clinically, riboflavin deficiency is characterized by lesions of the margin of the lips (cheilosis) and corners of the mouth (angular stomatitis), a painful desquamation of the tongue, so that it is red, dry, and atrophic (so-called magenta tongue) and a sebhorroic dermatitis, with filiform excrescences, affecting especially the naso-labial folds, eyelids, and ears, with abnormalities of the skin around the vulva and anus and at the free border of the prepuce. The lesions of the mouth may respond to either riboflavin or vitamin B6 in apparently riboflavin-deficient subjects (Lakshmiand Bamji, 1974).

There may also be conjunctivitis with vascularization of the cornea and opacity of the lens. This is the only lesion for which we know a possible biochemical basis - glutathione is important in maintaining the normal clarity of crystallin in the lens, and glutathione reductase is a flavoprotein that is particularly sensitive to riboflavin depletion.

7.4.1 Impairment of Lipid Metabolism in Riboflavin Deficiency

Riboflavin-deficient animals have a lower metabolic rate than controls, and require a 15% to 20% higher food intake to maintain body weight. There is increased accumulation of triglycerides in the liver, with an increase in liver weight as a proportion of body weight. There is no impairment of electron transport in the liver, although in brown adipose tissue both electron transport and the thermogenic response to adrenergic stimulation are impaired (Duerden and Bates, 1985).

The main effect of riboflavin deficiency is on lipid metabolism. In experimental animals on a riboflavin-free diet, feeding a high-fat diet leads to more marked impairment of growth, and a higher requirement for riboflavin to restore growth. There are also changes in the patterns of long-chain polyunsat-urated fatty acids in membrane phospholipids.

Within a day of initiating a riboflavin-free diet in weanling rats, there is a 35% decrease in the oxidation of palmitoyl CoA. All three mitochondrial acyl CoA dehydrogenases are affected, although it is the short-chain acyl CoA

dehydrogenase that is most severely impaired and that becomes the rate-limiting step of fatty acid oxidation. The accumulating short-chain fatty acyl CoA derivatives may undergo microsomal «-oxidation and possibly peroxisomal p -oxidation of the resultant dicarboxylic acids. Although mitochondrial P-oxidation is impaired in riboflavin deficiency, the peroxisomes appear to be protected. As a result, a number of dicarboxylic acids (including adipic, suberic, sebacic, octenedioic, hexenedioic, and decendioic acids) are excreted in the urine. In addition, a number of conjugates of the substrates of impaired acyl CoA dehydrogenases are excreted, including butyryl-, isovaleryl-, 2-methylbutyl-, and isobutyl-glycine conjugates. There are a number of ribo-flavin responsive organic acidurias caused by impairment of one or another of the acyl CoA dehydrogenases (Goodman, 1981; Veitch et al., 1988).

In animals, the production of 14CO2 from [14C]palmitate or octanoate is not consistently affected by riboflavin deficiency, possibly as a result of increased activity of carnitine palmitoyl transferase, which is more a response to food deprivation than to riboflavin deficiency. However, the production of 14CO2 from [14C]adipic acid is significantly reduced, and responds rapidly (with some overshoot) to repletion with the vitamin. It has been suggested that the ability to metabolize a test dose of [13C]adipic acid may provide a sensitive means of investigating riboflavin nutritional status in human beings (Bates, 1989, 1990).

7.4.2 Resistance to Malaria in Riboflavin Deficiency

A number of studies have noted that, in areas where malaria is endemic, riboflavin-deficient subjects are relatively resistant and have a lower parasite burden than adequately nourished subjects. Dietary deficiency of riboflavin, hypothyroidism, which induces functional riboflavin deficiency by lowering the synthesis of flavokinase (Section 7.2.4), or the administration of chlor-promazine, which inhibits flavokinase and can cause functional riboflavin deficiency (Section 7.4.4), all inhibit the growth of malarial parasites in experimental animals. However, although parasitemia is less in riboflavin deficiency, the course of the disease may be more severe (Dutta et al., 1985; Dutta, 1991; Akompong et al., 2000a, 2000b; Shankar, 2000).

The biochemical basis of this resistance to malaria in riboflavin deficiency is not known, but a number of mechanisms have been proposed, including the following:

1. The malarial parasites may have a particularly high requirement for riboflavin. A number of flavin analogs have antimalarial action.

2. The impairment of glutathione reductase activity may result in lower availability of glutathione in erythrocytes and hence a more oxidizing environment, which is hostile to the parasites.

3. As a result of impaired antioxidant activity in erythrocytes, there may be increased fragility of erythrocyte membranes or reduced membrane fluidity. As in sickle cell trait, which also protects against malaria, this may result in:

(a) Exposure of the parasites to the host's immune system at a vulnerable stage in their development, resulting in the production of protective antibodies.

(b) Release into the circulation of immature forms of the parasite that are not capable of either surviving outside the erythrocyte or infecting new cells.

In vitro, high concentrations of riboflavin also impair parasite growth, apparently as a result of increased reduction of methemoglobin. The parasites can only hydrolyze methemoglobin, not native hemoglobin.

7.4.3 Secondary Nutrient Deficiencies in Riboflavin Deficiency

Riboflavin deficiency is associated with hypochromic anemia as a result of secondary iron deficiency. The absorption of iron is impaired in riboflavin-deficient animals, with a greater proportion of a test dose retained in the intestinal mucosal cells bound to ferritin, and hence lost in the feces, rather than being absorbed. The mobilization of iron bound to ferritin, in either intestinal mucosal cells or the liver, for transfer to transferrin, requires oxidation of Fe2+ to Fe3+, a reaction catalyzed by NAD-riboflavin phosphate oxidoreductase (Powers et al., 1991; Powers, 1995; Williams et al., 1995).

At least part of the impairment of iron absorption in riboflavin deficiency is a result of morphological changes in the intestinal mucosa, with hyperpro-liferation, an increased rate of enterocyte transit along the villi and a reduced number of (longer) villi and deeper crypts (Williams et al., 1996).

In addition to the role of flavoproteins in iron metabolism, it is possible that the anemia associated with riboflavin deficiency is a consequence of the impairment of vitamin B6 metabolism in riboflavin deficiency. Pyridoxine oxidase is a flavoprotein and, like glutathione reductase, is very sensitive to riboflavin depletion (McCormick, 1989). Vitamin B6 deficiency can result in hypochromic anemia as a result of impaired porphyrin synthesis. Although riboflavin depletion decreases the oxidation of dietary vitamin B6 to pyridoxal (Section 9.2), it is not clear to what extent there is secondary vitamin B6 deficiency in riboflavin deficiency. This is partly because vitamin B6 nutritional status is commonly assessed by the metabolism of a test dose of tryptophan (Section 9.5.4), and kynurenine hydroxylase in the tryptophan oxidative pathway is a flavopro-tein (Section; riboflavin deficiency can therefore disturb tryptophan metabolism quite separately from its effects on vitamin B6 nutritional status. In riboflavin-deficient animals, despite a decrease inpyridoxine oxidase to 15% of the control activity, and an increase in the concentration of pyridoxine in tissues, there is no significant decrease in the tissue concentration of pyridoxal phosphate (Lakshmi and Bamji, 1974).

The disturbance of tryptophan metabolism in riboflavin deficiency, caused by impairment of kynurenine hydroxylase, can also result in reduced synthesis of NAD from tryptophan. This may therefore be a factor in the etiology of pellagra (Section

In species for which ascorbate is not a vitamin, riboflavin deficiency can also lead to considerably reduced synthesis and low tissue concentrations of ascorbate, since gulonolactone oxidase, the key enzyme in ascorbate synthesis (Section 13.2), is a flavoprotein.

7.4.4 Iatrogenic Riboflavin Deficiency

The phenothiazines, such as chlorpromazine, used in the treatment of schizophrenia, the tricyclic antidepressant drugs such as imipramine and amitryp-tiline, antimalarials such as quinacrine, and the anticancer agent adriamycin are structural analogs of riboflavin (see Figure 7.6) and inhibit flavokinase. In experimental animals, administration of these drugs at doses equivalent to those used clinically results in an increase in the EGR activation coefficient (Section 7.5.2) and increased urinary excretion of riboflavin, with reduced tissue concentrations of riboflavin phosphate and FAD, despite feeding diets providing more riboflavin than is needed to meet requirements (Pinto et al., 1981).

Although there is no evidence that patients treated with these drugs for a prolonged period develop clinical signs of riboflavin deficiency, long-term use of chlorpromazine is associated with a reduction in metabolic rate.

Neonatal hyperbilirubinemia is normally treated by phototherapy. The peak wavelength for photolysis of bilirubin is 450 nm, the same as that for photolysis of riboflavin (Section 7.1). Infants undergoing phototherapy show biochemical evidence of riboflavin depletion, with a significant increase in the EGR activation coefficient. Provision of additional riboflavin to maintain plasma concentrations enhances the photolysis of bilirubin, apparently as a result of reactive oxygen radicals generated by the products of photolysis of riboflavin.

Figure 7.6. Drugs that are structural analogs of riboflavin and may cause deficiency. Relative molecular masses (Mr): riboflavin, 376.4; quinacrine, 472.9 (dihydrochloride); chlorpromazine, 318.9; imipramine, 280.4; amitryptyline, 277.4; and adriamycin (doxorubicin), 543.5.

However, even relatively low concentrations of riboflavin can cause damage to DNA under conditions of photolysis, with damage to deoxy-guanosine in isolated DNA, and activation of DNA repair mechanisms in cells in culture. It is therefore not common practice to use riboflavin supplements as an adjunct

Table 7.5 Indices of Riboflavin Nutritional Status




Urine riboflavin

|xg/g creatinine




mol/mol creatinine




lg/24 h




nmol/24 h




mg over 4 h after




5 mg dose

| mol over 4 h after




5 mg dose

Erythrocyte riboflavin

| g/g hemoglobin


nmol/g hemoglobin


Glutathione reductase

Activation coefficient




Sources: From data reported by Sauberlich et al., 1974; Bates, 1993.

Sources: From data reported by Sauberlich et al., 1974; Bates, 1993.

to phototherapy of neonatal hyperbilirubinemia (Speck et al., 1975; Gromisch et al., 1977).


Two methods of assessing riboflavin status are generally used: urinary excretion of the vitamin and its metabolites, and activation of EGR. Criteria of riboflavin adequacy are shown in Table 7.5.

7.5.1 Urinary Excretion of Riboflavin

Clinical signs of riboflavin deficiency are seen at intakes below about 1 mg per day. At intakes below about 1.1 mg per day, there is very little urinary excretion of riboflavin; thereafter, as intake increases, there is a sharp increase in excretion. Up to about 2.5 mg per day, there is a linear relationship between intake and excretion. At higher levels of intake, excretion increases sharply, reflecting active renal secretion of excessive vitamin (Section 7.2.5).

Riboflavin excretion is only correlated with intake in subjects who are maintaining nitrogen balance. In subjects in negative nitrogen balance, there may be more urinary excretion than would be expected, largely as a result of the catabolism of tissue flavoproteins and loss of their prosthetic groups. Higher intakes of protein than are required to maintain nitrogen balance do not affect the requirement for riboflavin or indices of riboflavin nutritional status, although, as might be expected, more riboflavin is retained in subjects in positive nitrogen balance, as a result of increased net synthesis of flavoproteins.

7.5.2 Erythrocyte Glutathione Reductase (EGR) Activation Coefficient

Glutathione reductase is especially sensitive to riboflavin depletion. In deficient animals, the activity of glutathione reductase responds earlier and more markedly than any other index of riboflavin status apart from liver concentrations of flavin coenzymes and the activity of hepatic flavokinase (Prentice and Bates, 1981a, 1981b). The activity of the enzyme in erythrocytes can therefore be used as an index of riboflavin status.

Interpretation of the results can be complicated by anemia, and it is more usual to use the activation of EGR by FAD added in vitro. An activation coefficient of 1.0 to 1.3 reflects adequate nutritional status; >1.7 indicates deficiency. EGR activation coefficient between 1.3 to 1.7 represents a marginal status, with no clinical signs of deficiency.

Like glutathione reductase, pyridoxine oxidase is sensitive to riboflavin depletion. In normal subjects and in experimental animals, the EGR and pyridox-ine oxidase activation coefficients are correlated, and both reflect riboflavin nutritional status. In subjects with glucose 6-phosphate dehydrogenase deficiency, there is an apparent protection of EGR, so that even in riboflavin deficiency it does not lose its cofactor, and the EGR activation coefficient remains within the normal range. The mechanism of this protection is unknown. In such subjects, the erythrocyte pyridoxine oxidase activation coefficient gives a response that mirrors riboflavin nutritional status (Clements and Anderson, 1980).


On the basis of depletion/repletion studies, the minimum adult requirement for riboflavin is 0.5 to 0.8 mg per day. In population studies, values of the EGR activation coefficient <1.3 are seen in subjects whose habitual intake of riboflavin is 1.2 to 1.5 mg per day. At intakes between 1.1 to 1.6 mg per day, urinary excretion rises sharply, suggesting that tissue reserves are saturated. On the basis of such studies, reference intakes (see Table 7.6) are in the range of 1.2 to 1.6 mg per day (Bates, 1987a, 1987b).

Because of the central role of flavin coenzymes in energy-yielding metabolism, reference intakes are sometimes calculated on the basis of energy intake: 0.6 to 0.8 mg per 1,000 kcal (0.14 to 0.19 mg per MJ). However, in view of the wide range of riboflavin-dependent reactions, in addition to energy-yielding metabolism, it is difficult to justify this basis for the calculation of requirements.

Table 7.6 Reference Intakes of Riboflavin (mg/day)

Table 7.6 Reference Intakes of Riboflavin (mg/day)






0-6 m




7-12 m





1-3 y





4-6 y





7-8 y






9-10 y





11-13 y





> 14 y






9-10 y





11-13 y





>14 y















EU, European Union; FAO, Food and Agriculture Organization; WHO, World Health Organization. Sources: Department of Health, 1991; Scientific Committee for Food, 1993; Institute of Medicine, 1998; FAO/WHO, 2001.

EU, European Union; FAO, Food and Agriculture Organization; WHO, World Health Organization. Sources: Department of Health, 1991; Scientific Committee for Food, 1993; Institute of Medicine, 1998; FAO/WHO, 2001.


Because of its intense yellow color and low toxicity, riboflavin is widely used as a food color (E-101). It is also used in relatively high doses in the treatment of recessive familial methemoglobinemia and some organic acidurias.

Recessive familial methemoglobinemia is from lack of NADH-dependent cytochrome b5 methemoglobin reductase, which is the major enzyme involved in reduction of methemoglobin. (The more common methemoglobinemias are caused by mutations in the hemoglobin gene and are commonly dominant conditions.) Reduced flavins will reduce methemoglobin nonenzymically, and doses of the vitamin of 20 to 40 mg per day result in a significant accumulation of reduced flavins in erythrocytes, as a result of the activity of NADH-flavin reductase (Yubisui et al., 1977).

Some congenital organic acidurias resulting from apparent deficiency of acyl CoA dehydrogenases are riboflavin responsive. The defects seem to result from impaired coenzyme binding to either the electron-transferring flavopro-tein that transfers electrons from a variety of acyl CoA dehydrogenases into the electron transport chain, or electron-transferring flavoprotein-ubiquinone reductase. Administration of 100 mg of riboflavin per day or more seems to permit accumulation of sufficient flavin coenzymes to give useful activity of the affected enzyme, despite the limitation of riboflavin absorption (Christensen et al., 1984; Gregersen et al., 1986).

There is some evidence that riboflavin status affects the stability of the ther-molabile variant of methylene tetrahydrofolate reductase (Section, and that supplements of riboflavin may lower plasma homocysteine (Section in people who are homozygous for the variant enzyme (McNulty et al., 2002).

Because of its low solubility and limited absorption from the gastrointestinal tract, riboflavin has no significant or measurable toxicity by mouth. At extremely high parenteral doses (300 to 400 mg per kg of body weight), there may be crystallization of riboflavin in the kidney because of its low solubility.


Adiga PR (1994) Riboflavin carrier protein in reproduction. In Vitamin Receptors: Vitamins as Ligands in Cell Communication, K Dakshinamurti (ed.), pp. 137-76. Cambridge: Cambridge University Press. Bates CJ (1987) Human requirements for riboflavin. American Journal of Clinical Nutrition 46, 122-3.

Bates CJ (1987) Human riboflavin requirements, and metabolic consequences of deficiency in man and animals. World Review of Nutrition and Dietetics 50, 215-65. Ghisla S and Massey V (1989) Mechanisms of flavoprotein-catalyzed reactions. European Journal of Biochemistry 181, 1-17. Massey V (2000) The chemical and biological versatility of riboflavin. Biochemical Society

Transactions 28, 283-96. McCormick DB (1989) Two interconnected B vitamins: riboflavin and pyridoxine. Physiological Reviews 69, 1170-98. Shankar AH (2000) Nutritional modulation of malaria morbidity and mortality. Journal of Infectious Diseases 182(Suppl. 1), S37-S53.

References cited in the text are listed in the Bibliography.

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