A sensitive competitive protein binding assay for vitamin D in plasma

Abstract

A sensitive protein binding assay for vitamin D is described. The vitamin D 3 was extracted from plasma with diethyl ether and methylene chloride. The lipid extract was purified on Sephadex LH-20 followed by Lipidex 5000 and finally by high pressure liquid chromatography on a Zorbax Sil column (0.79 × 25 cm) developed in 0.25:99.75 isopropanol: methylene chloride. The vitamin D fraction was collected and quantitated by competitive protein binding assay with a 1/50,000 dilution of sheep plasma in 0.05 M potassium phosphate buffer (pH 7.5) containing 0.01% gelatin. [ 3H]-25-Hydroxyvitamin D 3 was used as a radioactive tracer in the assay. We found that under these conditions, sheep plasma had equal affinity for vitamin D 2 and vitamin D 3 and could detect as little as 0.1 ng of vitamin D. When rat, cow, or human plasma was substituted for the sheep plasma, the decline in sensitivity to vitamin D 2 was fivefold to tenfold. With this assay, we found excellent agreement (r = 0.98) between the results obtained by competitive protein binding analysis and direct U.V. absorbance analysis by high pressure liquid chromatography.