Microbiological assays cobalamins

Spectrophotometric deterrnination at 550 nm is relatively insensitive and is useful for the deterrnination of vitamin B 2 in high potency products such as premixes. Thin-layer chromatography and open-column chromatography have been appHed to both the direct assay of cobalamins and to the fractionation and removal of interfering substances from sample extracts prior to microbiological or radioassay. Atomic absorption spectrophotometry of cobalt has been proposed for the deterrnination of vitamin B 2 in dry feeds. Chemical methods based on the estimation of cyanide or the presence of 5,6-dimethylben2irnida2ole in the vitamin B 2 molecule have not been widely used. 

Major vitamin Bi2-dependent metabolic processes include the formation of methionine from homocysteine, and the formation of succinyl coenzyme A from methylmalonyl coenzyme A. Thus, apart from directly determining vitamin B12 concentration in serum, elevated levels of both methylmalonic acid and homocysteine may indicate a vitamin B12 deficiency. Serum cobalamine concentration is often determined by automated immunoassays using an intrinsic factor as binding agent. These assays have mainly replaced the microbiological methods. Literature data about vitamin B12 concentration in serum varies. Values <110-150pmoll are considered to reflect deficiency, whereas values >150-200pmoll represents an adequate status. 

Measurement of serum cobalamin includes release of cobalamin from its binding proteins transcobalamin and haptocorrin, conversion of the various forms of cobalamin into one form of the vitamin, and quantification employing microbiological or protein binding assays. 

Other ions and neutral molecules can replace the cyano group in cyanocobalamin. Compounds such as hydroxocobalamin (vitamin Biaa), chloro-cobalamin, cyanatocobalamin, thiocyanatocobalamin, and nitrocobalamin, are known. The microbiological activity of these compounds by the L. lactis and L. leichmannii assays is from 30-100% of the activity of cyanocobalamin, depending upon the assay conditions employed. 

In certain mixtures with other vitamins, or in natural products such as liver extracts, feeding stuffs, etc., the only reliable assay may be a microbiological one (see page 813). In the first case the preparation of the sample for assay presents no difficulty and simple dilution to the required level is sufficient, but with the natural products the vitamin may be present in complex-bound forms or as hydroxocobalamin, which is relatively unstable to heat. Both problems can be resolved by treating the sample with cyanide, thus assisting the extraction by breaking the complexes and converting all of the cobalamin to the more stable cyano compound. ... 

---