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COBAIAMINS (VITAMIN B12)

    More than 150 years ago, Combe and Addison described an anemia associated with gastric and neurologic disorders, with death usually occurring two to five years after onset. Successful treatment of perni­cious anemia was not achieved until 1926, when Minot and Murphy showed the effectiveness of feeding whole liver (Nobel Prize, 1934). Shortly thereafter, Castle demonstrated the need for both an extrinsic, dietary factor and an intrinsic, gastric factor. The structure of isolated, crystallized vitamin B12 was determined by Dorothy Hodgkins using x-ray diffraction (Nobel Prize, 1964).
    Chemistry. The stable pharmaceutical form of vi­tamin B12, and the form first isolated, is cyanocoba­lamin. It is not the natural form of the vitamin, however. The molecule consists of two major parta, a corrin nucleus similar to heme and a nucleotide, 5,6-dimethylbenzimidazole (Fig. 10). At the center of the corrin nucleus is an atom of cobalt. The major parts of the molec~ile are linked by a bridge consisting of D-1-aniino-2-propanol and a bond between cobalt and one of the nitrogens of the nucleotide. Also attached to the cobalt atom is one of several anionic CR—) groups that distinguish the various congeners. Cobalamin is the term used to describe the molecule in the absence of an R — group. In its presence, the name of the partic­ular R — group is prefixed to cobalamin.

    Distribution and Bioavailability The sole source of vitamin B12 in nature is synthesis by microorga­nisms. Plants are totally devoid of the vitamin unless contaminated by microorganisms. The usual dietary sources are the organs of domestic animals, which absorb the vitamin produced by microorganisms in the gastrointestinal tract. Vitamin B12 is also synthesized in the colon of humans, but it is not absorbed. Prime dietary sources include beef liver, kidney, whole milk, eggs, oysters, fresh shrimp, pork, and chicken. The average American diet supplies 7 to 30 ug of the vitamin per day.
 Absorption. Vitamin B12 is absorbed by two mech­anisms. Pharmacologically, 1 per cent of any dose of the free vitamin is absorbed by diffusion along the entire small intestine. Physiologically, a maximum of 1.5 to 3 ug of the vitamin is absorbed. The vitamin in food is released from its polypeptide linkage by gastric and intestinal enzymes and combines with the gastric intrinsic factor. Dimerization occurs, and a complex is formed consisting of two molecules of intrinsic factor and two molecules of vitamin B12. The complex subse­quently attaches to the brush border of the ileum. In the presence of calcium ions and a pH greater than 6.0, the complex enters the mucosa cell and B12 is released. It then enters the bloodstream, where it is bound by vitamin B12—binding proteins.
    Transport and Storage. The normal plasma con­centration of vitamin B12 is 200 to 900 pg/ml. The vitamin is bound by three proteins in plasma, trans­cobalamin (TC) I, II, and III. Most of the B12 in plasma is bound to TC I, but it appears to be nonfunctional. Transcobalamins I and Ill are glycoproteins synthe­sized primarily by granulocytes and appear to serve a storage function. They are referred to collectively as cobalophilins. Unlike the cobalophilins, transcoba­lamin II is the vitamin B12 transport protein. It is a beta-globulin synthesized by the liver, and it delivers B12 to liver, bone marrow, lymphoblasts, fibroblasts, and tumor cells. It has a molecular weight of 38,000 compared with 60,000 for the cobalophilins, and it is not a glycoprotein.
The body stores of B12 range from 1 to 10 mg, 90 per cent of which is stored in the liver. Virtually the only
loss is through excretion into bile.  B12 is excreted into the bile and is reabsorbed in the ileum with an entero­hepatic circulation of 3 to 8 ug/day. The stored form of B12 is deoxyadenosylcobalamin, and the plasma form is methylcobalamin. Because of the efficiency of the enterohepatic circulation, B12 stores are not totally depleted until five to six years after cessation of intake or absorption.
    Transformation and Function. In the body, vita­min B12 is reduced and converted to two active coen­zyme forms, deoxyadenosylcobalamin and methylco­balamin. Only two metabolic pathways are known to require this cofactor.

    1. 5-Deoxyadenosylcobalamin is required for hydro­gen transfer and isomerization of methylmalonyl CoA to succinyl CoA. This reaction is involved in both fat and carbohydrate metabolism and may be related 

to the abnormality of the lipid portion of the myelin sheath seen in B12 deficiency.
    2. Methylcobalamin acts as coenzyme in synthesis of methionine from homocysteine (Fig. 1). This reaction also regenerates tetrahydrofolate. In the absence of B12, the body becomes functionally folate deficient be­cause folate is trapped as methyl THF.

    Excretion. Vitamin B12 is not further metabolized in the body, and the major route of excretion is into bile. Losses at this step depend on the efficiency of reabsorption in the ileum. The vitamin is not excreated into urine until the renal tubular reabsorptive capacity has been exceeded.
    Requirement. Only small amounts of vitamin B12 are required by the body. The MDR is 1 ug/day, and 1 to 1.5 ug is absorbed from the 3 ug/day RDA. With the exception of vegetarian diets without supplements, most deficiencies do not result from inadequate intake but from a defect in absorption. Absorption can be impaired in two ways: (1) failure to produce the gly­copirotein gastric intrinsic factor and (2) interference with the absorption of the intrinsic factor—vitamin B12 complex, as in loss of the terminal ileum or intralum­inal catabolism of B12 by overgrowth of bacteria in intestinal stasis.
    Deficiency State. Deficiency is clinically manifest both hematopoietically and in the nervous system. Hematopoietic damage is caused by an inadequate amount of the vitamin to promote demethylation of N­methyltetrahydrofolate to THF, which is required for the synthesis of thymidine (and therefore of DNA). The sensitivity of the red blood cell to vitamin B12 deficiency is due to its rapid turnover rate. Vitamin B12 deficiency is suggested by large red blood cells (mean corpuscular volume> 95 fi) and is highly likely if the mean erythrocyte volume is> 110 fi and abnor­mally large numbers of segments (> 6) exist in nuclei of neutrophils. Deficiency in the nervous system can cause irreparable damage. Demyelination, cell death, and swelling of myelinated neurons are often seen.
    Deficiency may be determined in several ways: (1) determination of plasma concentration of B12 (2) gas­tric function tests; (3) excretion of methylmalonate; and (4) demonstration of reticulocytosis after a therapeutic dose of vitamin B12. The simplest screening test is the serum B12 level, usually by a radioimmunoassay which unfortunately has a decreasing accuracy at low levels of serum B12.
    The Schillings test is more sensitive for conditions that prevent proper cyanocobalamin absorption. It has four parts, of which only the first two are usually necessary: (1) The subject is given a known amount of radiolabeled vitamin B12 by mouth and a parenteral dose of 1 mg nonlabeled B12. This saturates vitamin B12—binding proteins and facilitates maximal urinary excretion of absorbed radioactive B12. More than 7 per cent of the ingested radioactive cobalt should be re­covered in the urine if normal absorption occurs. (2) The Schilling test is similarly repeated but with the ingested radiolabeled vitamin B12 attached to intrinsic factor. A normal result this time, after an abnormal result from the first part, confirms the presence of a gastric disorder causing insufficient production of in­trinsic factor.
    Parts three and four of the Schilling test involve treatment with antibiotics and pancreatic enzymes, respectively, and can be used with patients in whom small bowel bacterial overgrowth or pancreatic insuf­ficiency is a possible cause of vitamin B12 malabsorp­tion.
    Methylmalonicaciduria is a chemical sign of vitamin B12 deficiency because of the vitamin’s role as cofactor in the conversion of methylmalonate to succinate. Homocystinuria is also present, since the demethyla­tion of methyl THF, which normally converts homocysteine to methionine, is impaired.
    Therapeutic trials of B12 can be misleading, since large doses of B12 can partially correct a folate deficiency, thereby masking the true cause of the macrocytic anemia. Parenteral administration of’ 1 to 10 big/day of B12 should be followed by reticulocytosis in three to five days.
    Toxicity. Vitamin B12 is nontoxic in humans even at 10,000 times the minimum daily requirement. Excess amounts are excreted into the urine.