Riboflavin has a central role as a redox coenzyme in energy-yielding metabolism and a more recently discovered role as the prosthetic group of the cryptochromes in the eye - the blue-sensitive pigments that are responsible for day-length sensitivity and the setting of circadian rhythms.
Dietary deficiency is relatively widespread, yet is apparently never fatal; there is not even a clearly characteristic riboflavin deficiency disease. In addition to intestinal bacterial synthesis of the vitamin, there is very efficient conservation and reutilization of riboflavin in tissues. Flavin coenzymes are tightly enzyme bound, in some cases covalently, and control of tissue flavins is largely at the level of synthesis and catabolism of flavin-dependent enzymes.
Reoxidation of reduced flavin coenzymes is the major source of oxygen radicals in the body, and riboflavin is also capable of generating reactive oxygen species nonenzymically. As protection against this, there is very strict control over the body content of riboflavin. Absorption is limited, and any in excess of requirements is rapidly excreted.
In bacteria, flavin adenine dinucleotide (FAD) is the prosthetic group of the photolyases that catalyze reductive repair of light-induced pyrimidine dimers in DNA. Riboflavin is the light-emitting molecule in some bioluminescent fungi and bacteria, and is the precursor for synthesis of the dimethylbenzimidazole ring of vitamin B12 (Section 10.7.3).
As shown in Figure 7.1, riboflavin consists of a tricyclic dimethyl-isoalloxazine ring conjugated to the sugar alcohol ribitol. The metabolically active coenzymes are riboflavin 5 -phosphate and flavin adenine dinucleotide (FAD). In some enzymes the prosthetic group is riboflavin, bound covalently at the catalytic site.
The ribityl moiety is not linked to the isoalloxazine ring by a glycosidic linkage, and it is not strictly correct to call FAD a dinucleotide. Nevertheless, this trivial name is accepted, as indeed is the even less correct term flavin mononucleotide for riboflavin phosphate.
Riboflavin phosphate and FAD may be either covalently or noncovalently bound at the catalytic sites of enzymes. Even in those enzymes in which the binding is not covalent, the flavin is tightly bound; in many cases, the flavin has a role in maintaining or determining the conformation of the enzyme protein. In some cases, the flavin is incorporated into the nascent polypeptide chain, while it is still attached to the ribosome. However, in others a flavin-free apoenzyme is synthesized and accumulates in riboflavin deficiency (Section 7.5.2).
Covalent binding of the flavin coenzymes is normally through the 8-a-methyl group. 8-Hydroxymethyl-riboflavin is formed by microsomal mixed-function oxidases (Section 7.2.5), but it is not known whether or not this is a precursor of covalently bound flavin coenzymes. A variety of amino acid residues may be involved in covalent binding of flavin coenzymes to enzymes, including the following:
1. flavin 8-a-carbon linkage to imidazole N-3 of a histidine residue (e.g., in succinate, sarcosine, and dimethylglycine dehydrogenases in mammals and bacterial 6-hydroxynicotine oxidase);
2. flavin 8-a-carbon linkage to imidazole N-1 of a histidine residue (e.g., in bacterial thiamin dehydrogenase and mammalian gulonolactone oxidase in those species for which ascorbate is not a vitamin) (Section 13.3.4);
3. flavin 8-a-carbon thioether linkage to a cysteine residue (e.g., in monoamine oxidase) - the 8-ethyl analog of riboflavin is incorporated into monoamine oxidase, although the resultant holo-enzyme analog is cat-alytically inactive;
4. flavin 8-a-carbon thio-hemiacetal linkage to a cysteine residue (e.g., in bacterial cytochrome c552);
5. flavin 8-a-carbon O-tyrosyl ether linkage (e.g., in bacterial p-cresol methyl hydroxylase); and
6. linkage from carbon-6 of the flavin to a cysteine residue (e.g., in bacterial trimethylamine dehydrogenase).
Although the ribitol moiety is not involved in the redox function of the flavin coenzymes, both the stereochemistry and nature of the sugar alcohol are important. Although some riboflavin analogs have partial vitamin action, most are inactive or have antivitamin activity, although they may be active in microbiological assays. The galactitol (dulcitol) analog, galactoflavin, has been widely used as a means of inducing riboflavin deficiency in animal and human studies.
Photolysis of riboflavin leads to the formation of lumiflavin in alkaline solution and lumichrome in acidic or neutral solution (see Figure 7.2). Because lumiflavin is chloroform extractable, photolysis in alkaline solution, followed by chloroform extraction and fluorimetric determination, is the basis of commonly used chemical methods of assaying riboflavin. The photolysis proceeds byway of intermediate formation of cytotoxic riboflavin radicals, and the addition of riboflavin and exposure to light has been suggested as a means of inactivating viruses and bacteria in blood products (Goodrich, 2000).
Exposure of milk in clear glass bottles to sunlight or fluorescent light (with a peak wavelength of400 to 550 nm) can result in the loss of significant amounts of riboflavin as a result of photolysis. This is potentially nutritionally important, because on average, in Western diets, 25% to 30% of riboflavin comes from milk. The resultant lumiflavin and lumichrome catalyze the oxidation of vitamin C, so that even relatively brief exposure to light, causing little loss of riboflavin, can lead to a considerable loss of vitamin C. This is nutritionally unimportant, because milk is normally an insignificant source of vitamin C. Lumiflavin and lumichrome also catalyze oxidation of lipids (to lipid peroxides) and methionine (to methional), resulting in the development of an unpleasant flavor - the so-called sunlight flavor.
Photolysis of riboflavin occurs in vivo during phototherapy for neonatal hyperbilirubinemia (Section 7.4.4). There is no evidence that normal exposure to sunlight results in significant photolysis of riboflavin, although it is possible that some of the lumichromes found in urine may arise in this way.
7.2 THE METABOLISM OF RIBOFLAVIN
7.2.1 Absorption, Tissue Uptake, and Coenzyme Synthesis
Apart from milk and eggs, which contain relatively large amounts of free riboflavin bound to specific binding proteins, most of the vitamin in foods is as flavin coenzymes bound to enzymes, with about 60% to 90% as FAD.
FAD and riboflavin phosphate in foods are hydrolyzed in the intestinal lumen by nucleotide diphosphatase and a variety of nonspecific phosphatases to yield free riboflavin, which is absorbed in the upper small intestines by a sodium-dependent saturable mechanism; the peak plasma concentration is related to the dose only up to about 15 to 20 mg (40 to 50 ^mol). Thereafter,
Table 7.1 Tissue Flavins in the Rat
% Present as
Total ^mol/kg Riboflavin Riboflavin P FAD
7 23 41 12
28 74 55 85
Liver 58.0 Kidney 63.2 Muscle 4.1
there is little or no absorption of higher single doses of riboflavin (Zempleni et al., 1996).
Intestinal bacteria synthesize riboflavin, and fecal losses of the vitamin may be five- to six-fold higher than intake. It is possible that bacterial synthesis makes a significant contribution to riboflavin intake, because there is carrier-mediated uptake of riboflavin into colonocytes in culture. The activity of the carrier is increased in riboflavin deficiency and decreased when the cells are cultured in the presence of high concentrations of riboflavin. The same carrier mechanism seems to be involved in tissue uptake of riboflavin (Said et al., 2000).
Much of the absorbed riboflavin is phosphorylated in the intestinal mucosa by flavokinase and enters the bloodstream as riboflavin phosphate; this metabolic trapping is essential for concentrative uptake of riboflavin into en-terocytes (Gastaldi et al., 2000). Parenterally administered free riboflavin is also largely phosphorylated in the intestinal mucosa. It is not clear whether this is the result of enterohepatic recycling of the vitamin or simply uptake of free riboflavin into the intestinal mucosa from the bloodstream.
About 7% of dietary riboflavin is covalently bound to proteins (mainly as riboflavin-8-a-histidine or riboflavin-8-a-cysteine). The riboflavin-amino acid complexes released by proteolysis are not biologically available; although they are absorbed from the gastrointestinal tract, they are excreted in the urine (Chia et al., 1978).
As shown in Table 7.1, the total riboflavin concentration in plasma is very much lower than in most tissues. About 50% of plasma riboflavin is free riboflavin, which is the main transport form, with 44% as FAD and the remainder as riboflavin phosphate. The vitamin is largely protein bound in plasma; free riboflavin binds to both albumin and a- and ^-globulins, and both riboflavin and the coenzymes also bind to immunoglobulins. The products of photolysis of riboflavin bind to albumin with considerably higher affinity than riboflavin itself; this albumin binding may represent a mechanism to prevent tissue uptake of these potential antimetabolites, or it may be an artifact of the exposure of samples to light during analysis.
Most tissues contain very little free riboflavin and, except in the kidneys, where 30% is as riboflavin phosphate, more than 80% is FAD, almost all bound to enzymes. Isolated hepatocytes (and presumably other tissues) show saturable concentrative uptake of riboflavin. The Km of the uptake process is the same as that of flavokinase, and uptake is inhibited by inhibitors of flavokinase, suggesting that tissue uptake is the result of carrier-mediated diffusion, fol-lowedbymetabolic trapping as riboflavin phosphate, then onward metabolism to FAD, catalyzed by FAD pyrophosphorylase. FAD is a potent inhibitor of the pyrophosphorylase and acts to limit its own synthesis. FAD, which is not protein bound is rapidly hydrolyzed to riboflavin phosphate by nucleotide pyrophosphatase; unbound riboflavin phosphate is similarly rapidly hydrolyzed to riboflavin by nonspecific phosphatases (Awetal., 1983; Yamadaetal., 1990).
FAD is cleaved by an FAD-adenosine monophosphate (AMP) lyase in liver to yield AMP and riboflavin 4 ,5'-cyclic phosphate; it is not known whether this has any coenzyme or cell signaling function, but it is a substrate for phosphodiesterase and has also been identified in small amounts in yeast (Fraiz et al., 1998; Cabezas et al., 2001).
There is a specific plasma riboflavin binding protein that is induced by estrogens; in female animals, its concentration varies through the estrous cycle. The same protein is also synthesized in the testes and is found on the acrosomal surface of spermatozoa. In females, it acts to transport the vitamin across the placenta, which is impermeable to free riboflavin or the coenzymes. The protein is essential for fetal uptake of riboflavin, and immunoneutralization of the protein causes a considerable decrease in the uptake of riboflavin by the fetus, leading to death of the fetus and termination of the pregnancy, with no apparent effect on maternal riboflavin metabolism. It has been suggested that active or passive immunization against the binding protein may provide both male and female contraception (Krishnamurthy et al., 1984; Adiga, 1994; Adiga et al., 1997).
In pregnant women, there is a progressive increase in the erythrocyte glutathione reductase activation coefficient (an index of functional riboflavin nutritional status; Section 7.5.2), which resolves on parturition despite the daily secretion of 200 to 400 fig (0.5 to 1 f mol) of riboflavin into milk. This suggests that the estrogen-induced riboflavin binding protein can sequester the vitamin for fetal uptake at the expense of causing functional deficiency in the mother.
In laying hens, induction of this riboflavin protein results in a 100-fold increase in plasma riboflavin, compared with males or nonlaying females. In mutant chickens lacking the protein, the adult has massive urinary loss of riboflavin. The embryo develops normally for about 10 days, then develops severe hypoglycemia associated with a reduction in medium-chain acyl coenzyme A (CoA) dehydrogenase to 20% of normal activity and the accumulation of intermediates of fatty acid oxidation (White, 1996).
The riboflavin binding protein that occurs in eggs has been exploited for the radio-ligand binding assay of riboflavin. Because binding to the protein quenches the native fluorescence of riboflavin, it can be exploited for a direct titrimetric fluorescence assay of the vitamin in urine and other biological samples (Kodentsova et al., 1995).
There is no evidence of any significant storage of riboflavin; in addition to the limited absorption, any surplus intake is excreted rapidly; thus, once metabolic requirements have been met, urinary excretion of riboflavin and its metabolites reflects intake until intestinal absorption is saturated. In depleted animals, the maximum growth response is achieved with intakes that give about 75% saturation of tissues, and the intake to achieve tissue saturation is that at which there is quantitative urinary excretion of the vitamin.
Equally, there is very efficient conservation of tissue riboflavin in deficiency. There is only a four-fold difference between the minimum concentration of flavins in the liver in deficiency and the level at which saturation occurs. In the central nervous system, there is only a 35% difference between deficiency and saturation.
Control over tissue concentrations of riboflavin coenzymes seems to be largely by control of the activity of flavokinase, and the synthesis and cata-bolism of flavin-dependent enzymes. Almost all the vitamin in tissues is enzyme bound, and free riboflavin phosphate and FAD are rapidly hydrolyzed to riboflavin. If this is not rephosphorylated, it rapidly diffuses out of tissues and is excreted.
In deficiency, virtually the only loss of riboflavin from tissues will be the small amount that is covalently bound to enzymes. The 8a-linkage is not cleaved by mammalian enzymes and 8a-derivatives of riboflavin are not substrates for flavokinase and cannot be reutilized.
7.2.4 The Effect of Thyroid Hormones on Riboflavin Metabolism
The activities of a variety of flavin-dependent enzymes are depressed in hy-pothyroidism. They are increased by the administration of thyroxine or triiodothyronine, as a result of increased synthesis of riboflavin phosphate and
FAD, leading to increased saturation of enzyme proteins with coenzymes. This increases the stability of the enzymes against proteolysis and increases their activity in tissues (Rivlin and Langdon, 1966).
Tissue concentrations of flavin coenzymes in hypothyroid animals may be as low as in those fed a riboflavin-deficient diet. In hypothyroid patients, erythrocyte glutathione reductase (EGR) activity may be as low, and its activation by FAD added in vitro (Section 7.5.2) as high, as in riboflavin-deficient subjects. Tissue concentrations of flavin coenzymes and EGR are normalized by the administration of thyroid hormones, with no increase in riboflavin intake (Cimino et al., 1987).
The administration of thyroid hormones to hypothyroid animals results in a rapid increase in flavokinase activity as a result of the activation of an inactive precursor protein; as flavokinase activity increases, there is a parallel decrease in the tissue content of an apparently inactive riboflavin binding protein (Lee and McCormick, 1985).
Hyperthyroidism is not associated with elevated tissue concentrations of flavin coenzymes, despite increased activity of flavokinase. Again, this demonstrates the importance of the enzyme binding of flavin coenzymes and the rapid hydrolysis of unbound FAD and riboflavin phosphate in the regulation of tissue concentrations of the vitamin.
Riboflavin may also be involved in the metabolism of thyroid hormones. In the presence of oxygen, riboflavin phosphate catalyzes a photolytic deiod-ination of thyroxine. The lower tissue concentration of riboflavin phosphate in hypothyroidism may thus serve to protect such thyroid hormone as is available against catabolism and prolong its action.
Riboflavin and riboflavin phosphate that are not bound to plasma proteins are filtered at the glomerulus; the phosphate is generally dephosphorylated in the bladder. Renal tubular reabsorption of riboflavin is saturated at normal plasma concentrations, and there is also active tubular secretion of the vitamin, so that urinary clearance of riboflavin can be two- to three-fold greater than the glomerular filtration rate.
Under normal conditions, about 25% of the urinary excretion of riboflavin is as the unchanged vitamin, with a small amount as a variety of glycosides of riboflavin and its metabolites. Riboflavin-8-a-histidine and riboflavin-8-a-cysteine arising from the catabolism of enzymes in which the coenzyme is covalently bound are excreted unchanged.
Liver cytochrome P450-linked mixed-function oxidases result in the production of 7- and 8-hydroxymethylriboflavin, both of which are substrates for h3c. h3c-
lumiflavin photolysis ch3
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