This chapter discusses the measurement of tissue vitamin B12 by radioisotopic competitive inhibition assay and quantitation of tissue cobalamin fractions. In this method, the measurement of tissue vitamin B12 by the competitive inhibition approach requires an initial complete separation of all endogenous B12 from the tissue binders and the removal or inactivation of these binders. Because B12 in tissue exists as a group of coenzyme forms with differing affinities for tissue binding proteins and subcellular moieties, extraction of the tissue B12 from these binders is accomplished by vigorous boiling of the homogenate of tissue in an acetate–cyanide buffer. This also results in the conversion of all the B12 forms into that of cyanocobalamin. Prior to this extraction, addition of radiolabeled vitamin B12 ([57Co]B12) permits the determination of the recoverable B12 originally present. Analysis is accomplished by the subsequent incubation of these extracts with a predetermined amount of material with unsaturated B12 binding sites. The percentage of [57Co]B12 that is protein bound is inversely proportional to the total B12 concentration. The bound B12 can be removed by batch adsorption onto diethylaminoethyl (DEAE) cellulose and subsequent centrifugation or by adsorption onto coated charcoal. The percentage binding of any unknown can then be determined from previously prepared standard curves.
ASJC Scopus subject areas
- Molecular Biology