Determination of cobalamins in biological material. I. Improvement in the unequal recovery of cobalamins by preincubation with cadmium acetate.

Ethanol extraction of 28 specimens of human plasma in the dark showed that the total cobalamin value is increased up to 2 1/2 times by adding 400 micrometers of potassium cyanide. The addition of cyanide significantly changed the substance concentration (fractional content times substance concentration of total cobalamins) for all the cobalamin forms except 5'-deoxyadenosylcobalamin. The ethanol extraction was also studied with 57Co-labeled cobalamin forms. The recovery of [57Co]hydroxo- and [57Co]sulfitocobalamin from both plasma and hemolysate was inadequate. The recovery of [57Co]5'-deoxyadenosylcobalamin was only studied from liver tissue; the extraction recovery of this form was equal to that of 57Co]cyano- and [57Co]methylcobalamin. Preincubation with 0.25 M of cadmium acetate or extraction with 400 micrometers of potassium cyanide almost equalized the recovery of all five cobalamins. It is concluded that (i) the substance concentrations of the individual cobalamins presented hitherto are erroneous, owing to inadequate ethanol extraction of hydroxo- and sulfitocobalamin; (ii) about one-third of the cobalamins in plasma consists of hydroxocobalamin, which is not extractable by the methods used hitherto; (iii) studies of the cobalamin forms in body fluids and tissue cells need to be repeated by methods ensuring equal extraction of all the cobalamins; and (iv) the extraction of the cobalamins must be uniform for determination of the substance concentration of total cobalamins as well as the fractional amounts of all cobalamins.