Vitamin B12 is normally present in plasma mainly bound to a specific binding protein. Addition of exogenous vitamin ultimately results in saturation of protein binding sites and excess vitamin remains in free form. The ratio of free to bound fractions thus quantitatively depends upon the concentration of exogenous compound. This observation has been utilized to determine amounts of vitamin B12 extracted from the blood of normal subjects and of patients with certain pathologic conditions.

The method is simple and reproducible. The sensitivity of the method is such that vitamin levels down to roughly 20 µµg./ml. may be evaluated using labeled vitamin B12 of a specific activity of about 1 µc./µg. Repeated assays on identical specimens of normal plasma have shown a reproducibility of about 5-6 per cent. Results on 39 normal subjects gave a range of 330-1070 µµg./ml. with an average of 611 ± 167. Values observed in plasma taken from patients suffering from pernicious anemia were around 100 µµg./ml. or less. Results on subjects with other pathologic conditions are also presented and the limitations of the method are discussed.

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