Riboflavin requirement

Some vitamins undetfio a rather unique transformation prior to becoming functional. They are covalently attached to specific enzymes. Biotin, for example, is covalently bound to the biotin-requiring enzymes. Pantothenic acid, in a modified form, is covalently bound to fatty acid synthase. Riboflavin, following conversion to thecofaclor form, is bound to succinate dehydrogenase, as well as to a few other enzymes requiring riboflavin-based cofactors. 

A newly isolated soil bacterium grows without oxygen but requires ferric ion in the growth medium. Succinate suffices as a carbon source, but neither hexoses nor pyruvate can be utilized. The bacteria require riboflavin as a growth supplement. Neither niacin nor thiamin is required, and neither substance nor compounds derived from them can be found in the cells. The electron carriers found in the bacteria are cytochrome b, cytochrome c, FAD, and coenzyme Q. 

Supplement vitamin B2 deficiency states, impaired absorption, increased requirements Riboflavin 5-phosphate sodium, riboflavin Tablets, ampules 10-30 mg 0.4-1.4 mg 1.2-1.6 mg... 

In addition to thiamine, all mammalian species, so far as studied, require riboflavin, nicotinic acid, and vitamin A in their food. We conclude that the supply of food available to an earlier ancestor provided an adequate supply of these vitamins, enough to make it advantageous to discard the mechanism for synthesizing them. There is little doubt that this food was plant food, probably not greatly different from present day plant food. 

As mentioned earlier, nearly all tissues require riboflavin. The free vitamin is trapped as one of its phosphorylated coenzyme forms, which then become specifically associated (and in a few cases covalently linked) to the protein chains of catalytic flavoenzymes. If not already covalently linked, the flavin coenzyme can often be liberated by extremes of pH or by other nonphysiological maneuvers. In a few biological locations, such as the mature red cell, flavoenzymes such as glutathione reductase (NADPH oxidized glutathione oxidoreductase EC 1.6.4.2) may exist partly in their apoenzyme form, i.e., without the flavin coenzyme and therefore without enzyme activity. An increased supply of riboflavin will permit the depleted coenzyme (in this case FAD) to be synthesized so that enzyme activity can be restored. 

The specificity of riboflavin in bacterial growth has been examined by Snell and Strong (362) using Lactobacillus casei and B. lactis acidi (both of which require riboflavin) and using a number of synthetic flavins, including —... 

The microbial assay is based on the growth of l ctobacillus casei in the natural (72) or modified form. The lactic acid formed is titrated or, preferably, the turbidity measured photometrically. In a more sensitive assay, l euconostoc mesenteroides is employed as the assay organism (73). It is 50 times more sensitive than T. casei for assaying riboflavin and its analogues (0.1 ng/mL vs 20 ng/mL for T. casei). A very useful method for measuring total riboflavin in body fluids and tissues is based on the riboflavin requirement of the proto2oan cHate Tetrahjmenapyriformis which is sensitive and specific for riboflavin. 

Flavin mononucleotide was first isolated from the yellow en2yme in yeast by Warburg and Christian in 1932 (4). The yellow en2yme was spHt into the protein and the yellow prosthetic group (coen2yme) by dialysis under acidic conditions. Flavin mononucleotide was isolated as its crystalline calcium salt and shown to be riboflavin-5Lphosphate its stmeture was confirmed by chemical synthesis by Kuhn and Rudy (94). It is commercially available as the monosodium salt dihydrate [6184-17 /, with a water solubiUty of more than 200 times that of riboflavin. It has wide appHcation in multivitamin and B-complex solutions, where it does not require the solubili2ers needed for riboflavin. 

Biotransformations are carried out by either whole cells (microbial, plant, or animal) or by isolated enzymes. Both methods have advantages and disadvantages. In general, multistep transformations, such as hydroxylations of steroids, or the synthesis of amino acids, riboflavin, vitamins, and alkaloids that require the presence of several enzymes and cofactors are carried out by whole cells. Simple one- or two-step transformations, on the other hand, are usually carried out by isolated enzymes. Compared to fermentations, enzymatic reactions have a number of advantages including simple instmmentation reduced side reactions, easy control, and product isolation. 

Riboflavin was first isolated from whey in 1879 by Blyth, and the structure was determined by Kuhn and coworkers in 1933. For the structure determination, this group isolated 30 mg of pure riboflavin from the whites of about 10,000 eggs. The discovery of the actions of riboflavin in biological systems arose from the work of Otto Warburg in Germany and Hugo Theorell in Sweden, both of whom identified yellow substances bound to a yeast enzyme involved in the oxidation of pyridine nucleotides. Theorell showed that riboflavin 5 -phosphate was the source of the yellow color in this old yellow enzyme. By 1938, Warburg had identified FAD, the second common form of riboflavin, as the coenzyme in D-amino acid oxidase, another yellow protein. Riboflavin deficiencies are not at all common. Humans require only about 2 mg per day, and the vitamin is prevalent in many foods. This vitamin... 

In terms of amino acids bacterial protein is similar to fish protein. The yeast s protein is almost identical to soya protein fungal protein is lower than yeast protein. In addition, SCP is deficient in amino acids with a sulphur bridge, such as cystine, cysteine and methionine. SCP as a food may require supplements of cysteine and methionine whereas they have high levels of lysine vitamins and other amino acids. The vitamins of microorganisms are primarily of the B type. Vitamin B12 occurs mostly hi bacteria, whereas algae are usually rich in vitamin A. The most common vitamins in SCP are thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, choline, folic acid, inositol, biotin, B12 and P-aminobenzoic acid. Table 14.4 shows the essential amino acid analysis of SCP compared with several sources of protein. 

The human lens is rich in ascorbate, which is required for normal collagen synthesis and acts as a water-soluble antioxidant, reacting rapidly with superoxide, hydroxyl and peroxyl radicals. However, ascorbic acid can undergo auto-oxidation and, at certain concentrations, can form hydroxyl radicals with hydrogen peroxide in the presence of light and riboflavin as described above (Delaye and Tardieu, 1983 Ueno et al., 1987). 

Water-soluble vitamins removed by hemodialysis (HD) contribute to malnutrition and vitamin deficiency syndromes. Patients receiving HD often require replacement of water-soluble vitamins to prevent adverse effects. The vitamins that may require replacement are ascorbic acid, thiamine, biotin, folic acid, riboflavin, and pyridoxine. Patients receiving HD should receive a multivitamin B complex with vitamin C supplement, but should not take supplements that include fat-soluble vitamins, such as vitamins A, E, or K, which can accumulate in patients with renal failure. 

Since many essential nutrients (e.g., monosaccharides, amino acids, and vitamins) are water-soluble, they have low oil/water partition coefficients, which would suggest poor absorption from the GIT. However, to ensure adequate uptake of these materials from food, the intestine has developed specialized absorption mechanisms that depend on membrane participation and require the compound to have a specific chemical structure. Since these processes are discussed in Chapter 4, we will not dwell on them here. This carrier transport mechanism is illustrated in Fig. 9C. Absorption by a specialized carrier mechanism (from the rat intestine) has been shown to exist for several agents used in cancer chemotherapy (5-fluorouracil and 5-bromouracil) [37,38], which may be considered false nutrients in that their chemical structures are very similar to essential nutrients for which the intestine has a specialized transport mechanism. It would be instructive to examine some studies concerned with riboflavin and ascorbic acid absorption in humans, as these illustrate how one may treat urine data to explore the mechanism of absorption. If a compound is... 

FAO/WHO. 1967. Requirements of Vitamin A, Thiamine, Riboflavine and Niacin. WHO Technical Report Series No. 362. Geneva World Health Organization. 

Group-transfer reactions often involve vitamins3, which humans need to have in then-diet, since we are incapable of realizing their synthesis. These include nicotinamide (derived from the vitamin nicotinic acid) and riboflavin (vitamin B2) derivatives, required for electron transfer reactions, biotin for the transfer of C02, pantothenate for acyl group transfer, thiamine (vitamin as thiamine pyrophosphate) for transfer of aldehyde groups and folic acid (as tetrahydrofolate) for exchange of one-carbon fragments. Lipoic acid (not a vitamin) is both an acyl and an electron carrier. In addition, vitamins such as pyridoxine (vitamin B6, as pyridoxal phosphate), vitamin B12 and vitamin C (ascorbic acid) participate as cofactors in an important number of metabolic reactions. 

The movement of cutaneous interstitial fluids varies from a 3- to 4-fold range in normal individuals as determined by injecting riboflavin intradermally and recording the time required for one-half its fluorescence to disappear.31 When repeated tests were made on three individuals it was found that one gave relatively highly variable results. (S. D. 48.4), and the others gave relatively constant results (S.D. 9.5 and 5.8). 

Riboflavin needs have been found to vary with different strains of white Leghorn chickens,56, 57 and there appears to be a sex difference in that females require less and thus show greater resistance to riboflavin deficiency. 

Note that this overall reaction requires three coenzymes that we encountered as metabolites of vitamins in chapter 15 NAD+, derived from lucotiiuc acid or nicotinamide FAD, derived from riboflavin and coenzyme A(CoASH), derived from pantothenic acid. In the overall process, acetyl-SCoA is oxidized to two molecules of carbon dioxide with the release of CoASH. Both NAD+ and FAD are reduced to, respectively, NADH and FADH2. Note that one molecule of guanosine triphosphate, GTP, functionally equivalent to ATP, is generated in the process. 

In the form in which they are consumed, many vitamins are not biologically active. For several water-soluble vitamins such as thiamine, riboflavin, nicotinic acid, pyridoxine, activation includes phosphorylation or, as is the case with riboflavin and nicotinic acid, coupling to purine or pyridine nucleotides is required. In their major known actions, water-soluble vitamins participate as cofactors for specific enzymes, whereas at least two fat-soluble... 

---