Filter assay for 1α, 25-dihydroxyvitamin D₃. Utilization of the hormone's target tissue chromatin receptor

A radioreceptor assay for 1α, 25-dihydroxyvitamin D₃ (1α, 25-(OH)₂-D₃) has been developed by utilizing competitive binding to the receptor system from chick intestinal mucosa. This target tissue binding system consists of a high affinity cytosol receptor protein and acceptor sites on nuclear chromatin. Reconstituted cytosol-chromatin is incubated with radioactive 1α, 25-(OH)₂-D₃, and the bound and free sterol are separated by trapping the hormone-receptor-chromatin complex on glass fiber filters. Washing away of free or nonspecifically bound sterol is carried out with 1% Triton X-100. The amount of specifically bound sterol is linearly related to the quantity of cytosol-chromatin in the incubation and the receptor system is saturated at 5 nM la, 25-(OH)2-[3H]D3. An isotope-dilution standard curve obtained with increasing amounts of authentic nonradioactive 1α, 25-(OH)₂-D₃ indicates that 17 pg of hormone can be detected by this assay. After extraction and extensive purification of the suspected 1α, 25-(OH)₂-D₃ fraction from normal chick and human plasma, the circulating levels of the hormone were determined to be 8.0 and 4.0 ng/100 ml, respectively. These values were verified by demonstrating that the assayable material elutes with tracer 1α, 25- (OH)₂-[³H]D₃ on Celite liquid-liquid partition columns. By similar analysis, rachitic chick plasma contained <0.05 ng of 1α, 25-(OH)₂-D₃/100 ml. The lower limit of sensitivity of the assay for human plasma samples (20 ml) is 1.0 ng/100 ml of 1α, 25-(OH)₂-D₃ and the coefficient of variation is 12%. Patients with chronic renal failure and anephric subjects have undetectable circulating levels of the kidney-produced la, 25- (OH)2-D3 hormone as determined by the assay. These data implicate a lack of 1α, 25-(OH)₂-D₃ in the etiology of renal osteodystrophy and point to the kidney as the unique site of synthesis of 1α, 25-(OH)₂-D₃ in humans. This assay should not only be of great utility in diagnosing other defects in vitamin D metabolism, but the concept of employing the two-step receptor binding system for the sterol may find application in the assay of steroid hormones.