[63] A Sensitive Radioreceptor Assay for 1α,25-Dihydroxyvitamin D in Biological Fluid

This chapter discusses a sensitive radioreceptor assay for lα,25-dihydroxyvitamin D in biological fluids. Vitamin D has been recognized as a principal regulator of calcium and phosphate homeostasis in a variety of animal species, including humans. Some studies describe 1α,25-dihydroxyvitamin D [lα,25-(OH)₂D] as the hormonal metabolite responsible for mediating intestinal transport of these minerals and hence necessitate development of a precise method for quantitating this sterol in biological fluids, particularly serum. It has been demonstrated that chick intestinal mucosa contained a cytosolic protein that rapidly and specifically bound lα,25-(OH)₂D₃ both in vivo and in vitro with high affinity. Furthermore, the sterol–protein complex was rapidly translocated to the intestinal cell nucleus, suggesting a classic steroid hormone action. The ligand affinity and specificity of this cytosolic receptor has been exploited to develop a sensitive competitive protein binding assay for lα,25-(OH)₂D. This radioreceptor assay has been instrumental in the phosphate-related disease states, as well as in characterizing the physiological regulation of lα,25-(OH)₂D. Specific components for the radioreceptor assay are described in the chapter. Procedures for hormone extraction, purification, preparation, and reconstitution of cytosol-chromatin, etc, are also discussed in detail.