Estimation of 1,25 dihydroxyvitamin D by cytoreceptor and competitive protein binding assays without high pressure liquid chromatography.

Using two different cultured rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8) we have investigated the recently described cytoreceptor assay for 1,25-dihydroxyvitamin D (1,25-(OH)2D). The assay method is relatively simple and sensitive to 2.4 fmole per tube. Using either cell line, assay of serum samples, whose only preparation consisted of extraction and purification on a disposable diatomaceous earth column, produced variable values for serum 1,25-(OH)2D. Additional purification, using a disposable silicic acid minicolumn to remove other vitamin D metabolites resulted in consistent values and additional HPLC resulted in no further decrease in the values obtained. Our results show that a single two stage non-HPLC column can purify serum samples for assay in the cytoreceptor assay. The method is also applicable to the competitive protein binding assay employing calf thymus cytosol and the correlation between values obtained by both methods is highly significant. It is a sensitive, simple, and accurate method with technical advantages which allow greater sample throughput than other 1,25-(OH)2D assays.