Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Vitamins
Water-Soluble Vitamins
Vitamin B12

Vitamin B₁₂ (cobalamin, or the anti-anemic vitamin) was isolated from Liver in crystalline form in 1948. Long before that, it was known that animal liver contains a specific substance that regulates hematopoiesis and has a therapeutic effect on pernicious (malignant) anemia in humans. However, it was not until 1955 that D. Hodgkin* deciphered its Structure, including its three-dimensional spatial configuration, primarily using physical Research Methods (X-ray crystallography). Based on these data and the results of chemical composition studies, the following structure was proposed for vitamin B₁₂:

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* Dorothy Hodgkin was awarded the Nobel Prize (1964) for deciphering The structure of vitamin B₁₂ by X-Ray Diffraction Analysis. She was also elected a foreign member of the USSR Academy of Sciences.

In the vitamin B₁₂ molecule, the central cobalt atom is bonded to the nitrogen atoms of four reduced pyrrole rings, which form a porphyrin-like corrin ring, and to the nitrogen atom of 5,6-dimethylbenzimidazole*. The cobalt-containing portion of the molecule forms a planar structure; perpendicular to it lies the nucleotide Ligand, which, In addition to 5,6-dimethylbenzimidazole, contains ribose and a phosphate residue attached to the 3rd carbon atom. This entire structure was named "cobalamin." Various derivatives of vitamin B₁₂ have been obtained containing an OH group (hydroxycobalamin), chlorine (chlorocobalamin), H₂O (aquacobalamin), and nitrous acid (nitritocobalamin). In addition, B₁₂ analogs have been isolated from natural sources in which 5,6-dimethylbenzimidazole is replaced by 5-hydroxybenzimidazole, adenine, 2-methyladenine, hypoxanthine, or methylhypoxanthine. All of these exhibit lower biological activity than cobalamin. Typically, vitamin B₁₂ is isolated from microbial biomass or animal Tissues using solutions containing cyanide ions, which serve as the 6th ligand of cobalt. However, cyanocobalamin is metabolically inactive. In B₁₂ Coenzymes, the CN group is replaced by a 5-deoxyadenosine residue or a methyl group.

* This is the only vitamin that contains a metal atom in its molecule.

In humans and animals, a deficiency of vitamin B₁₂ leads to The Development of malignant macrocytic, megaloblastic anemia. Besides hematopoietic disorders, B₁₂ avitaminosis is also characterized by specific dysfunctions of The Nervous system and a sharp drop in gastric juice acidity. It was found that for the active absorption of vitamin B₁₂ in the Small Intestine, the presence of a specific gastric protein—gastromucoprotein, known as Castle's intrinsic factor—is a mandatory condition. This factor specifically binds vitamin B₁₂ into a unique complex. The exact role of this factor in B₁₂ absorption remains unclear. It is hypothesized that vitamin B₁₂ enters the mucosal Cells of the ileum bound to this factor, then slowly passes into the portal Blood system, while the intrinsic factor undergoes Hydrolysis (breakdown). It should be noted that B₁₂ enters the portal Circulation not in a free state, but complexed with two Proteins called transcobalamins I and II; one of these (I) acts as a B₁₂ depot because it binds more tightly to the vitamin. Therefore, impaired Synthesis of the intrinsic factor in the gastric mucosa leads to B₁₂ deficiency even when Dietary intake of cobalamin is adequate. In such cases, the vitamin is usually administered therapeutically via parenteral routes or orally combined with neutralized gastric juice, which contains the intrinsic factor. This Treatment method is effective for pernicious anemia. This indicates a clear link between the development of human malignant anemia and impaired gastric function. It can likely be stated that while pernicious anemia is a consequence of vitamin B₁₂ deficiency, it develops against the Background of organic gastric lesions that disrupt the synthesis of Castle's intrinsic factor in gastric mucosal cells, or following a total surgical gastrectomy.

Vitamin B₁₂ is used clinically to treat not only pernicious anemia, but also other forms—such as megaloblastic anemias with neurological complications that typically do not respond to other Vitamins, particularly Folic acid.

Biological role. Enzyme systems have been identified in which the prosthetic group is not free vitamin B₁₂, but rather the so-called B₁₂ coenzymes, or cobamide coenzymes. The latter differ in that they contain Two Types of ligands: a methyl group and a 5'-deoxyadenosine group. Accordingly, a distinction is made between methylcobalamin (CH₃-B₁₂) and deoxyadenosylcobalamin. The conversion of free vitamin B₁₂ into B₁₂ coenzymes occurs in several stages within the body, mediated by specific Enzymes in the presence of FAD, reduced NAD, ATP, and Glutathione as Cofactors. Notably, during The formation of the 5-deoxycobalamin coenzyme, ATP undergoes an unusual Cleavage involving the release of a triphosphate residue, similarly to the unique synthesis of 5-adenosylmethionine from Methionine and ATP (see Chapter 12). B₁₂ coenzymes were first isolated by H. Barker et al. in 1958 from microorganisms, and their existence in animal tissues was later proven.

Chemical Reactions in which vitamin B₁₂ Functions as a coenzyme are conventionally divided into two groups according to its chemical nature. The first group includes Transmethylation reactions, in which methylcobalamin acts as an intermediate carrier of the methyl group (the synthesis reactions of methionine and acetate).

The synthesis of methionine requires, in addition to homocysteine, N⁵-methyl-THF and reduced FAD, and proceeds According to the equation:

The enzyme catalyzing this reaction has been discovered in the liver of humans and several animals, as well as in microorganisms. Evidence has been obtained showing that the reaction mechanism involves The transfer of the methyl group from N⁵-CH₃-THF to the Active Site of the enzyme, forming a methyl-B₁₂-enzyme complex, followed by the transfer of this group to homocysteine. The blocking of this reaction, observed in B₁₂ deficiency, leads to the accumulation of N⁵-CH₃-THF and, consequently, puts another vital coenzyme out of action.

The second group of reactions involving B₁₂ coenzymes entails intramolecular hydrogen transfer in isomerization reactions. The Mechanism of these reactions follows the scheme:

It can be seen that the hydrogen proton moves (migrates) between two adjacent carbon atoms and does not exchange with Water protons. It is suggested that hydrogen is first transferred from the substrate to 5-deoxycobalamin and then back to the substrate at a new position. Examples include the glutamate mutase reaction (the interconversion of glutamic and β-methylaspartic acids), the methylmalonyl-CoA mutase reaction (the reversible conversion of methylmalonyl-CoA to succinyl-CoA), glycerol and diol dehydratase reactions, enzymatic reduction of ribonucleotides to deoxyribonucleotides, and others. Among these processes, only the isomerization of methylmalonyl-CoA to succinyl-CoA has been discovered in The Human Body.

It should be emphasized that the methylmalonyl-CoA isomerization reaction requires 5'-deoxyadenosylcobalamin as a coenzyme, whereas the methylation reaction (see above) requires methylcobalamin. These circumstances can explain certain biochemical symptoms of vitamin B₁₂ deficiency, particularly methylmalonic aciduria and homocystinuria. Furthermore, disorders caused by inherited defects in the synthesis of either deoxyadenosylcobalamin alone or both B₁₂ coenzymes have been described; in such cases, even a 1000-fold dose of vitamin B₁₂ yields no therapeutic effect. Currently, it is hypothesized that B₁₂ coenzymes participate more broadly in enzymatic transmethylation and deamination reactions (such as the ethanolamine deaminase reaction), among others. However, considerable efforts are still required to elucidate the molecular mechanisms by which vitamin B₁₂ affects hematopoiesis. The positive therapeutic effect of semi-raw liver in treating pernicious anemia is due, as is now known, to the presence of vitamin B₁₂, although greater therapeutic efficacy can be achieved by the simultaneous administration of gastric mucosal intrinsic factor.

Occurrence in nature and daily requirement. Vitamin B₁₂ is the only vitamin synthesized exclusively by microorganisms; neither plants nor animal tissues possess this capability. The main dietary sources of vitamin B₁₂ for humans are meat, beef liver, Kidneys, fish, milk, and eggs. The primary storage site for vitamin B₁₂ in the human body is the liver, which can contain up to several milligrams of the vitamin. It reaches the liver via animal-derived foods, particularly meat, or is synthesized by the intestinal microflora provided that dietary cobalt is available. The recommended daily allowance of vitamin B₁₂ for an adult is approximately 3 μg (0.003 mg).