Determination of cobalamins in biological material. II. The cobalamins in human plasma and erythrocytes after desalting on nonpolar adsorbent material, and separation by one-dimensional thin-layer chromatography

An improved method for determination of cobalamins in biological materials is described. The cobalamins are extracted with ethanol after preincubation with cadmium acetate, which inhibits nonspecific adsorption of hydroxocobalamin to proteins. The extracts are desalted on Amberlite XAD-2 columns, which at least doubles the capacity of the analysis, as compared to the previous phenol procedure. The cobalamins are separated by one-dimensional thin-layer chromatography. Bioautography with Escherichia coli strain 113-3 is performed after the light-sensitive cobalamins and hydroxocobalamin have been converted to sulfitocobalamin. This is done in order to ensure comparable growth responses to equal amounts of the cobalamins. Reference intervals of plasma and erythrocytes of healthy blood donors for methyl-, 5'-deoxyadenosyl-, cyano-, hydroxo/aquo-, and sulfitocobalamin are presented. The differences between the present and previous results are discussed.