The biosynthesis of vitamin B12.

The use of 13C-Fourier transform nuclear magnetic resonance (F.t.-n.m.r.) has led to the observation that while 8 molecules of [2-13C]ALA are incorporated into vitamin B12 in P. shermanii, [5-13C]ALA labels only seven of the carbon atoms of cyanocobalamin, i.e. one of the amino methyl groups of ALA is "lost" in the process. It has also been confirmed that seven of the methyl groups of B12 are derived from 13CH3-enriched methionine and further that the chirality of the gemdimethyl grouping at C12 labelled with [13CH3]methionine is R. A soluble enzyme mixture from the 37000 or 100000 g supernatant of disrupted cells of P. shermanii converts both 14 C-labelled ALA and [14C]uro'gen III to cobyrinic acid, the simplest corrinoid material on the pathway to vitamin B12 and the coenzyme, in presence of NADPH, Co2+, Mg2+, S-adenosyl-methionine and glutathione. Multiply-labelled uro'gens (13C, 14C and 3H) have been used to show that incorporation takes place without randomization. A sequence for corrin synthesis from uro'gen III is presented.     

Malabsorption of protein bound vitamin B12.

Patients with subnormal serum vitamin B12 concentrations were tested for absorption of protein bound vitamin B12 and compared with controls. Absorption of the protein bound vitamin appeared to decrease with increasing age in healthy subjects. Differences between the result of this test and the result of the Schilling test in patients who had undergone gastric surgery were confirmed; such differences were also seen in some patients who had iron deficiency anaemia, an excessive alcohol intake, or folate deficiency. Defective absorption was also found in six patients with an adequate dietary intake of vitamin B12, normal Schilling test results, low serum vitamin concentrations, and tissue changes responding to treatment with vitamin B12. Malabsorption of the vitamin from protein bound sources, which is not detected by the Schilling test, may produce vitamin B12 deficiency of clinical importance.     

Biosynthesis of vitamin B12

Radioactivity from [1-¹⁴C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B₁₂ in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum.As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-¹⁴C]glycine and [2-¹⁴C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of l-[methyl-¹⁴C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven extra methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor.In Clostridium thermoaceticum, forming the vitamin B₁₂ analog 5-methoxybenzimidazolylcobamide, [1-¹⁴C]glycine and [2-¹⁴C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring.In E. limosum l-[U-¹⁴C]glutamate led to the labeling of the corrin ring of vitamin B₁₂, but not of its base moiety.There results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B₁₂ only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.     

Relationships between biotin and vitamin B12. Effects of biotin and vitamin B12 on folic acid metabolism

1. The effects of dietary biotin compared with vitamin B₁₂ on the total content and on the distribution of the various folate derivatives in the liver of rats given a biotin-free diet have been studied. The effect of both vitamins on the conversion in vitro of folic acid into citrovorum factor in the same experimental conditions was also examined. 2. In biotin-treated rats as well as in vitamin B₁₂-treated rats the total content of folic acid-active substances measured microbiologically by Pediococcus cerevisiae, Streptococcus faecalis and Lactobacillus casei is significantly higher than that in biotin-deficient rats. The liver distribution of various folate derivatives in the three groups of animals is also markedly modified. 3. The amount of citrovorum factor formed in systems with liver homogenate of rats receiving biotin or vitamin B₁₂ is higher than that with liver homogenates of deficient rats. 4. The results obtained demonstrate the influence of biotin in the metabolism of folic acid, and the similar actions at this level of both biotin and vitamin B₁₂. These results are discussed in relation to the participation of the two vitamins in the metabolism of C₁ units, as a biochemical interpretation of the relationships between vitamin B₁₂ and biotin.     

Physician's management of suspected vitamin B12 deficiency.

A retrospective study was undertaken to audit physician''s management of patients with a low serum level of vitamin B12 who were admitted to a university-affiliated teaching hospital during 1 year. Among the 34 patients 13 were proved to have pernicious anemia or vitamin B12 malabsorption, but for 12 of them there were unnecessary delays (several days or weeks) before initiation of investigation and therapy. An additional six patients, who had low serum levels of vitamin B12 and macrocytosis, most likely had true vitamin B12 deficiency, but proper investigation was not done and they did not receive any vitamin B12 or folic acid therapy. In another nine cases unexplained low serum levels of vitamin B12 were not properly investigated, and the patients either did not receive any vitamin B12 therapy or received it without proper documentation of a deficiency. Suggestions for facilitating early detection, investigation and treatment of megaloblastic anemia or vitamin B12 deficiency are given.     

A spectroscopic analysis of vitamin B12 derivatives.

1. Several vitamin B12 derivatives including descobalt B12 show unusual inversion of their CD signs upon rapid cooling to liquid N2 temperature, although the room temperature CD signs are conserved by a slower cooling to the same temperature. As a possible explanation for this puzzling observation, a micro-environmental birefringence around the chromophore imbedded in an organic glass is proposed. 2. Absorption, fluorescence, phosphorescence, and polarization spectra of descobalt B12 can be correlated with those of porphyrin free bases, as these two molecular systems share many similarites in their electronic structure. 3. Molecular orbital calculations of polarization directions further support the analoby between the spectroscopic characteristics of corrins and porphyrins, and are generally in good agreement with the fluorescence polarization data.     

Effect of bile on vitamin B12 absorption.

The standard double-isotope Schilling test was used to study vitamin B12 absorption in seven patients with obstructive jaundice and 10 with T-tube bile duct drainage after cholecystectomy and bile duct exploration. In three and five of these patients respectively absorption was impaired. In the second group six patients were restudied after removal of the T tube, and in each case absorption was improved. Similar results were obtained after bile duct ligation in rats. Bile exclusion produced a 50-60% reduction in renal and hepatic uptake of vitamin B12 from the intestinal lumen. The malabsorption was corrected by replacing bile. These studies suggest that bile plays a part in the normal absorption of vitamin B12.