An improved competitive protein binding assay for 1,25-dihydroxyvitamin D

Abstract A practical procedure for the routine analysis of blood levels of 1,25-dihydroxyvitamin D has been developed. 1,25-Dihydroxyvitamin D was extracted from serum with methylene chloride:methanol. The concentration of acidic lipids in the extract was reduced with an alkaline wash. The serum extract was chromatographed on Sephadex LH-20, which was extracted with methanol prior to use in the columns. The chromatography was aided by a device which simultaneously collected and evaporated the eluates. High pressure liquid chromatography to further purify 1,25-dihydroxyvitamin D was found to be unnecessary. These modifications resulted in negligible 1,25-dihydroxyvitamin D values ( 3 standard curve capable of measuring 1.5 pg of 1,25-dihydroxyvitamin D 3 was achieved with the use of high specific activity 3 H-labeled 1,25-dihydroxyvitamin D 3 (92 Ci/mmol). The sensitivity of the standard curve, combined with changes in the resuspension of the assay sample, reduced the required amount of serum for an assay from 5 to 2 ml. A simplified centrifugation procedure for the separation of bound from free 1,25-dihydroxyvitamin D was attained by the inclusion of bovine γ-globulin in the polyethylene glycol precipitation.