Measurement of Serum Vitamin B12 Level Using Radioisotope Dilution and Coated Charcoal.

I N PREVIOUS REPORTS’3 it was shown that uncoated charcoal adsorbs both free vitamin B12 and B12 bound to proteins, but charcoal precoated by mixing with saturating quantities of albumin adsorbs only free B12. This finding has been confirmed.4 The charcoal particles may be considered as solid microsponges and the albumin coat as a molecular sieve surrounding each sponge. Small molecules such as B12 pass through the coat and continue to be adsorbed, but large molecules such as proteins are excluded from adsorption. This provides a system akin to, but much more rapid than dialysis for the separation of free from bound B12. This property of coated charcoal has been used in assays for: ( 1 ) intrinsic factor ( IF ) ; ( 2) unsaturated B12 binding capacity of gastric juice; ( 3 ) antibody to IF; ( 4 ) unsaturated B12 binding capacity of serum.3 These assays suggested the use of coated charcoal for measuring serum B12 levels. The principle of radioisotope dilution is also applied, by using the unknown quantity of nonradioactive B12 released from serum to dilute the specific activity of a known quantity of Co57B12. A solution of intrinsic factor concentrate ( IFC ) with a B12 binding capacity less than the quantity of added Co57B12 is used to bind a portion of the mixture of radioactive and nonradioactive B12-i.e., to “biopsy” the pool of B12. The B12 not bound to IFC is removed by addition of coated charcoal. This series of events is depicted in figure 1. The quantity of radioactive B12 bound to IFC is compared with the IFC control, and the B12 level of the unknown serum is then obtained from a simple equation.