Bioassay of 1,25-dihydroxyvitamin D in human plasma purified by partition, alkaline extraction, and high-pressure chromatography

A method is described which permits the assay of 1,25-dihydroxyvitamin D in 1 ml of normal human plasma. Samples are extracted with benzene or isopropyl ether. The organic phase is then back-extracted with 0.1 m Na₂HPO₄, pH 10.5. The final purification step involves high-pressure liquid chromatography on μPorosil with a solvent system of 13% isopropanol in hexane. Recovery through the entire purification procedure averages 76.8%, the purified extract is assayed for 1,25-dihydroxyvitamin D by determining its effect on the resorption of fetal rat bones in vitro. 1,25-Dihydroxyvitamin D_a standards in the 4- to 25 pg/ml range gave responses which fitted a logarithmic dose-response curve with an average r² of 0.986. Repeat assays with normal human plasma gave a coefficient of variation ( SD x) between experiments of 16.1 ± 0.3%. Normal human serum contains 33.4 ± 6.4 pg/ml (x ± SD) 1,25-dihydroxyvitamin D by this assay. Values averaging less than normal are found in plasmas of patients with renal failure and in patients with pseudohypoparathyroidism. Average values greater than normal were found in patients with hyperparathyroidism. 1,25-Dihydroxyvitamin D was not detectable in plasma of anephric patients.