Cyanocobalamin activity

The lUPAC-IUB Commission on Biochemical Nomenclature (13) recommends that the term vitamin B 2 be used as the genetic descriptor for aU. cottiaoids exhibiting quaUtatively the biological activity of cyanocobalamin. However, because of its commercial importance, cyaaocobalamin is used iaterchangeably with vitamin B 2 heteia. 

Cobalt is one of twenty-seven known elements essential to humans (28) (see Mineral NUTRIENTS). It is an integral part of the cyanocobalamin [68-19-9] molecule, ie, vitamin B 2> only documented biochemically active cobalt component in humans (29,30) (see Vitamins, VITAMIN Vitamin B 2 is not synthesized by animals or higher plants, rather the primary source is bacterial flora in the digestive system of sheep and cattle (8). Except for humans, nonmminants do not appear to requite cobalt. Humans have between 2 and 5 mg of vitamin B22, and deficiency results in the development of pernicious anemia. The wasting disease in sheep and cattle is known as bush sickness in New Zealand, salt sickness in Florida, pine sickness in Scotland, and coast disease in AustraUa. These are essentially the same symptomatically, and are caused by cobalt deficiency. Symptoms include initial lack of appetite followed by scaliness of skin, lack of coordination, loss of flesh, pale mucous membranes, and retarded growth. The total laboratory synthesis of vitamin B 2 was completed in 65—70 steps over a period of eleven years (31). The complex stmcture was reported by Dorothy Crowfoot-Hodgkin in 1961 (32) for which she was awarded a Nobel prize in 1964. 

Conversion of inactive vitamin Bj2 to active 5 -deoxyadenosylcobal-amin is thought to involve three steps (see figure). Two flavopro-tein reductases sequentially convert Co in cyanocobalamin to the Co state and then to the Co state. Co is an extremely powerful nucleophile. It attacks the C-5 carbon of ATP as shown, expelling the triphosphate anion to form 5 -deoxyadenosyl-... 

Pernicious anaemia was a fatal disease first reported in 1880. It was not until 1926 that it was discovered that eating raw liver effected a remission. The active principle was later isolated and called vitamin B or cyanocobalamin. It was initially obtained... 

Both the active enzyme, the heat-inactivated enzyme from Sulfurospirillum (Dehalos-pirillum) multivorans, and cyanocobalamin are capable of dehalogenating haloacetates (Nenmann et al. 2002), and the rate of abiotic dehalogenation depends on the catalyst that is nsed. 

The fibroblasts do not convert cyanocobalamin or hydroxocobalamin to methylcobalamin or adenosyl-cobalamin, resulting in diminished activity of both N5-methyltetrahydrofolate homocysteine methyltransferase and methylmalonyl-CoA mutase. Supplementation with hydroxocobalamin rectifies the aberrant biochemistry. The precise nature of the underlying defect remains obscure. Diagnosis should be suspected in a child with homocystinuria, methylmalonic aciduria, megaloblastic anemia, hypomethioninemia and normal blood levels of folate and vitamin B12. A definitive diagnosis requires demonstration of these abnormalities in fibroblasts. Prenatal diagnosis is possible. 

Methylcobalamin is the coenzyme form of vitamin It is neurologically active, most bioavailable and best utilized. Unlike cyanocobalamin, it does not require any conversion after absorption by the body and is better retained by the liver and other tissues. It has exhibited beneficial effects against brain aging, irregular sleep patterns. It supports immune function and promote normal cell growth. It represents one of the best values in nutritional products, given its comparably low cost and its wide range of potential benefits. 

Vitamin B12 consists of a porphyrin-like ring with a central cobalt atom attached to a nucleotide. Various organic groups may be covalently bound to the cobalt atom, forming different cobalamins. Deoxyadenosylcobalamin and methylcobalamin are the active forms of the vitamin in humans. Cyanocobalamin and hydroxocobalamin (both available for therapeutic use) and other cobalamins found in food sources are converted to the active forms. The ultimate source of vitamin Bi2 is from microbial synthesis the vitamin is not synthesized by animals or plants. The chief dietary source of vitamin Bi2 is microbially derived vitamin B12 in meat (especially liver), eggs, and dairy products. Vitamin Bi2 is sometimes called extrinsic factor to differentiate it from intrinsic factor, a protein normally secreted by the stomach that is required for gastrointestinal uptake of dietary vitamin B12. 

Treatment of cyanide poisoning includes rapid administration of activated charcoal (although charcoal binds cyanide poorly, it can reduce absorption) and general supportive care. The conventional antidote kit available in the USA includes two forms of nitrite (amyl nitrite and sodium nitrite) and sodium thiosulfate. The nitrites induce methemoglobinemia, which binds to free CI T creating the less toxic cyanomethemoglobin thiosulfate is a cofactor in the enzymatic conversion of CN to the much less toxic thiocyanate (SCINT). Recently, the FDA approved a concentrated form of hydroxocobalamin, which is now available as the Cyanokit (EMD Pharmaceuticals, Durham, North Carolina). Hydroxocobalamin (one form of vitamin B12) combines rapidly with CN to form cyanocobalamin (another form of vitamin B12). 

Cyanocobalamin appears to be the most stable of the B12 vitamers (167,168). It can be autoclaved at 120°C in aqueous solution at pH 4-7. It is susceptible to degradation and loss of vitamin activity under alkaline conditions. Short exposure to UV or visible light causes conversion to HOCbl prolonged exposure results in irreversible decomposition. CNCbl is soluble in water, short-chain alcohols, and phenol, but it is insoluble in acetone, chloroform, and ether. 

The vitamin is stable in aqueous solution at room temperature. It may be heated to 120°C with little loss of activity provided the pH is kept between 4 and 6. However, it breaks down rapidly when exposed to a pH below 2 or above 9. Crystals of cyanocobalamin are dark red, needle-like, and contain about 12% water. 

Two forms of vitamin B12 are available for treatment, cyanocobalamin and hydroxocobalamin, both showing similar activity in man. Hydroxocobalamin is retained in the body more efficiently than cyanocobalamin. It disperses more slowly from the injection site (K8) and therefore a larger percentage of the dose is retained. 

Homocystinuria may result from one or several abnormalities in the mechanism whereby homocysteine is methylated to form methionine. About half of the patients respond to treatment with pyridoxine and it is thought that the vitamin overcomes a block at the homocysteine/cystathionine level by mass action (C23). However, Schuh et al. (S22) have recently described a patient who responded to vitamin B12. The infant presented with severe developmental delay, homocystinuria, and a megaloblastic anemia. Treatment with cyanocobalamin was without effect but treatment with hydroxocobalamin resulted in a rapid clinical improvement, and the homocystinuria disappeared. Methionine synthetase activity in cell extracts was normal, while cultured fibroblasts showed an absolute growth requirement for methionine. The defect appeared to be limited to methyleobalamin accumulation and an inability to transfer the methyl group from 5-methyltetrahydrofolate to homocysteine. 

In 1926 Minot and Murphy (4) announced that whole liver was effective in the treatment of pernicious anemia. The initial assay methods, which were clinical (5), coupled with what we now know are the exceptionally small amounts of Bi2 (even in a relatively rich source such as liver) required that two more decades pass before Folkers (6) and Smith (7) in 1948 simultaneously isolated crystalline vitamin Bi2 (1, R = CN). A further decade passed before it was realized that the so-called vitamin (cyanocobalamin) was an artifact of the isolation procedure and that the enzymatically active species is the vitamin Bi2 coenzyme (5 -deoxyadenosylcobalamin, 1, R = 5 -deoxyadenosyl). This initial observation arose during Barkers study on the conversion... 

Interaction with Adenosylcobalamin. It has been considered generally that adenosylcobalamin or its analogs binds to the apoprotein of diol dehydrase or other adenosylcobalamin-dependent enzymes almost irreversibly (4). However, we found that the holo-enzyme of diol dehydrase was resolved completely into intact apoen-zyme and adenosylcobalamin when subjected to gel filtration on a Sephadex G-25 column in the absence of K+ (9, 10). Among the inactive complexes of diol dehydrase with irreversible cobalamin inhibitors, those with cyanocobalamin and methylcobalamin also were resolved upon gel filtration on Sephadex G-25 in the absence of both K+ and substrate, yielding the apoenzyme, which was reconstitutable into the active holoenzyme (II). The enzyme-hydroxocobalamin complex, however, was not resolvable under the same conditions. The enzyme-cobalamin complexes were not resolved at all by gel filtration in the presence of both K+ and substrate. When gel filtration of the holoenzyme was carried out in the presence of K+ only, the holoen-... 

Both Components F and S were required for irreversible cleavage of the C-Co bond of adenosylcobalamin by oxygen upon aerobic incubation with the coenzyme in the absence of substrate. This suggests that activation of the C-Co bond of the coenzyme is dependent on both components. Sephadex G-25 filtration experiments showed that neither adenosylcobalamin nor cyanocobalamin was bound by the individual components, F or S. Both of them were necessary for the cobalamin binding (8). 

Various vitamin B12 derivatives are shown in Figure 6.2, where the R group is usually taken as CN-. This form of vitamin B12 is cyanocobalamin. In the active coenzyme, the CN" is replaced by a 5 -deoxyriboadenosyl residue or by -CH3. Vitamin B12 seems to be the only mammalian substance that contains cobalt. It also has a unique corrin ring structure, which is very similar to that of heme. Metabolic reactions requiring vitamin B12 are discussed in Chapters 19 and 20. 

The ligand attached to the cobalt atom determines the activity of vitamin B12 in human enzymatic reactions. The two active coenzyme forms are methyl-cobalamin and 5 -adenosylcobalamin, the primary form of vitamin B12 in tissues. Cyanocobalamin, the therapeutic form of vitamin B12 contained in vitamin supplements, is produced by the cleavage of the unstable fink... 

Vitamin B12, cyanocobalamin, is an important biologically active compound. It serves as a hematopoietic factor in mammals and as a growth factor for many microbial and animal species. Its markets are divided into pharmaceutical (96-98% pure) and animal feed (80% pure) applications. All vitamin B 2 is now made commercially by fermentation.50... 

Cyanocobalamin (la) is a relatively inert complex and, apart from being involved in the detoxification of small amounts of hydrogen cyanide [5], does not appear to serve any major biological function [10]. The only difference between this species and the metabolically active forms of B12 (methylcobalamin (MeCbl Ib) and 5 -deoxyadenosylcobalamin (AdoCbl Ic)) is the ligand that occupies the... 

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