Application of the rat thymus receptor in a specific, sensitive and simplified assay of 1,25-dihydroxyvitamin D in blood serum

Abstract A simple method has been employed to prepare crude nuclear extract from rat thymus, using hypertonic buffer after previous treatment with hypotonic buffer. The preparation is free from serum vitamin D-binding protein and contains a 3.7 S receptor molecule, which specifically binds 1,25-dihydroxyvitamin D-3 (1,25-(OH) 2 D 3 ). The receptor is of high affinity K D = 0.85·10 −11 M at 0°C) and low capacity (260–460 fmol/g tissue). The Scatchard analysis of ligand binding results in a concave downward curve. The Hill analysis of the same data gives good linear fitting ( r = +0.971) with the Hill coefficient n H = 1.63. These facts indicate positive cooperativity between two ligand binding sites of the rat thymus 1,25-(OH) 2 D 3 receptor. The preparation was used in a competitive protein binding assay of 1,25-(OH) 2 D in serum extracts, purified on Sep-Pak C 18 followed by silica Sep-Pak cartridges. The method was sensitive to 0.5 pg/tube (2.0 ng/l) when 1 ml of serum was extracted. Intra- and interassay coefficients of variation were 9% and 14%, respectively. The serum 1,25-(OH) 2 D concentration estimated in 33 children (mean age 6.5 ± 3 years) was 46.6 ± 18.4 ng/l (mean ± S.D.).