Analysis for 1,25-dihydroxyvitamin D in human plasma, after a liquid-chromatographic purification procedure, with a modified competitive protein-binding assay.

Abstract

1,25-Dihydroxyvitamin D in plasma is measured by competitive protein-binding assay after isolation from plasma. The present method is improved, as compared with those hitherto described, with regard to receptor preparation, isolation of vitamin D metabolites from plasma, and procedure for the competitive protein binding. Receptor preparation from healthy rather than rachitic chicks, together with a fast isolation procedure, results in a high yield of active receptor protein: 12 animals provide receptor for about 4000 incubations. No loss of binding activity was observed during one year. 1,25-Dihydroxyvitamin D is isolated from plasma by a single extraction and a one-step chromatographic purification. Analytical recovery for the entire procedure averaged 78.1% (SD 4.7%). Other vitamin D metabolites (25-hydroxyvitamin D and 24,25-dihydroxyvitamin D) can also be separated with this procedure. The main features of the modified binding assay are the use of a stabilized cytosol receptor and dextran-coated charcoal instead of polyethylene glycol. The smallest detectable amount in the binding assay is 1-2 pg (2.4-4.8 fmol). Intra- and interassay CVs are 7.0% and 4.8%, respectively. 1,25-Dihydroxyvitamin D concentrations in plasma of 20 healthy subjects averaged 51.7 (SD 10.8) ng/L [124 (SD 26) pmol/L]. Three anephric patients showed values of 3, 6, and 7 ng/L (7, 14, and 17 pmol/L).