Riboflavine phosphate

Infusion solutions of pyridoxine (274) hydrochloride were unaffected by ward lighting, but when riboflavine phosphate sodium was added the pyridoxine was completely decomposed in about 3 h. The photosensitized oxidation gave... 

Riboflavine Phosphate Sodium 0.1 g Water/Acetate Buffer 444 323... 

The procedure to phosphorylate riboflavin derivatives on a preparative scale has recently been improved . These preparations, and also commercial FMN, contain a considerable amount of riboflavin phosphate isomers, which are difficult to separate by column chromatography. This problem is emphasized in the chemical synthesis of FAD where the yield is rather low (20-25 %). In this context, it is surprising that a modification of the synthesis of FAD from FMN published by Cramer and Neuhoeffer has not been noticed by workers in the flavin field. According to Cramer and Neuhoeffer, the yield of the chemical synthesis of FAD is drastically improved ( 70 % pure FAD). The procedure was successfully applied in the author s own laboratory (yield 60-70%). It is expected that the improved procedure of the FAD synthesis will stimulate the active-site directed studies on flavoproteins because the problem of separating FMN or FAD from their synthetic by-products has already been solved by use of FMN- or FAD-specific affinity column... 

Why are there four major hydrogen transfer coenzymes, NAD+, NADP+, FAD, and riboflavin phosphate (FMN), instead of just one Part of the answer is that the reduced pyridine nucleotides NADPH and NADH are more powerful reducing agents than are reduced flavins (Table 6-7). Conversely, flavin coenzymes are more powerful oxidizing agents than are... 

Some metalloflavoproteins contain heme groups. The previously mentioned flavocytochrome b2 of yeast is a 230-kDa tetramer, one domain of which carries riboflavin phosphate and another heme. A flavocytochrome from the photosynthetic sulfur bacterium Chromatium (cytochrome c-552)279 is a complex of a 21-kDa cytochrome c and a 46-kDa flavoprotein containing 8a-(S-cysteinyl)-FAD. The 670-kDa sulfite reductase of E. coli has an a8P4 subunit structure. The eight a chains bind four molecules of FAD and four of riboflavin phosphate, while the P chains bind three or four molecules of siroheme (Fig. 16-6) and also contain Fe4S4 clusters.280 281 Many nitrate and some nitrite reductases are flavoproteins which also contain Mo or... 

Due to the high loss of riboflavin phosphate sodium it should be substituted by riboflavin. 

P Nielsen, P Rauschenbach, A Bacher. Preparation, properties and separation by high-performance liquid chromatography of riboflavin phosphates. Methods Enzymol 122G 209-220, 1986. 

FMN is the standard biochemical abbreviation for flavin mononucleotide (or riboflavin phosphate). The sodium salt (95-97% pure) of FMN is used. This grade 1s inexpensive and is available from Sigma Chemical Company. Its purpose is to effect recycling of the catalytic amount used of the much more costly NAD. A larger than stoichiometric amount of FMN is employed in order to ensure rapid recycling of the NAO. 

Figure 9-15 Structural Formula of Riboflavin. Riboflavin R = OH Riboflavin phosphate R = POjNaOH.

Figure 7.1. Riboflavin, the flavin coenzymes and covalently bound flavins in proteins. Relative molecular masses (Mr) riboflavin, 376.4 riboflavin phosphate, 456.6 and FAD, 785.6.

The ribityl moiety is not linked to the isoalloxazine ring by a glycosidic linkage, and it is not strictly correct to caU 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 noncovalenfly 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 stUl attached to the ribosome. However, in others a flavin-free apoenzyme is synthesized and accumulates in riboflavin deficiency (Section 7.5.2). 

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 /xmol). Thereafter,... 

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. 

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 /3-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... 

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 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 (Aw et al., 1983 Yamada et al., 1990). 

Control over tissue concentrations of riboflavin coenzymes seems to be largely by control of the activity of flavokinase, and the synthesis and catabolism 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. 

The activities of a variety of flavin-dependent enzymes are depressed in hypothyroidism. They are increased by the administration of thyroxine or triiodothyronine, as a result of increased synthesis of riboflavin phosphate and... 

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. 

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. 

The reduction of cytochrome P450 hy 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. 

The phenothiazines, such as chlorpromazine, used in the treatment of schizophrenia, the tricyclic antidepressant drugs such as imipramine and amitryp-tUine, 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). 

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