Improved distribution analysis of cobalamins and cobalamin analogues in human plasma in which the use of thiol-blocking agents is a prerequisite.

In this paper the quantitative analysis of cobalamins and cobalamin analogues in human plasma by means of high performance liquid chromatography (HPLC) and radioisotope dilution assay (RIDA) is described. Current methods for the extraction of cobalamins from plasma proved impracticable due to the selective loss of hydroxo- and sulphitocobalamin, caused by concomitant reduction to the Co2+ form and tight binding to thiol groups of denatured plasma proteins. Although this process can be prevented by exchange of the hydroxyl group with sulphite, azide or nitrite ions, further separation of the respective cobalamin forms from the other endogenous cobalamins by HPLC proved to be impossible. Efficient extraction of hydroxo- and sulphitocobalamin has been obtained in the presence of the thiol-blocking agent N-ethylmaleimide, which does not interfere with the subsequent chromatographic separation of any of the plasma cobalamin forms. The use of R-binder free Intrinsic Factor and salivary R-binder separately as binding substances in RIDA made it possible to analyse the distribution of biologically active cobalamins as well as cobalamin analogues over the various HPLC fractions. Analyses are reported of 15 normal human plasma samples, in which the major components were found to be methylcobalamin (46.9 +/- 4.5%, mean +/- SD) and hydroxocobalamin (40.4 +/- 7.1%, mean +/- SD). Analyses of plasma extracts in the absence of N-ethylmaleimide showed a gross relative overestimation of the amount of methylcobalamin, due to loss of hydroxocobalamin during the extraction procedure. Cobalamin analogues appeared to be evenly distributed over the cobalamin fractions in a pattern almost similar to that of the biologically active forms.