An Improved Radioisotope Dilution Assay for Serum Vitamin B₁₂ Using Hemoglobin-coated Charcoal

A radioisotope dilution assay for vitamin B₁₂, using hemoglobin-coated charcoal, was modified in several respects, the most prominent of which was the use of cyanide extraction of sera. With these modifications, the B₁₂ levels were significantly higher in sera from normal subjects and patients with folate deficiency, whereas sera from patients with pernicious anemia and other B₁₂-deficient states were only minimally affected. The use of cyanide extraction thus resulted in a clearer differentiation of B₁₂-deficient sera from other sera; all 23 patients with pernicious anemia had B₁₂ levels below 140 pg/ml, whereas all of 85 normal and 218 folate-deficient subjects had B₁₂ levels above 156 pg/ml. The microbiologic assay with Euglena gracilis was similarly affected by cyanide extraction of sera. Thus, it appears that the use of cyanide results in more complete extraction of B₁₂ from serum proteins.