Studies on the Mode of Action of Calciferol. XIII. Development of a Radioimmunoassay for Vitamin DDependent Chick Intestinal Calcium-Binding Protein and Tissue Distribution*

Abstract

A RIA for chick intestinal calcium-binding protein (CaBP) has been developed with a sensitivity of 1 ng. The antiserum was generated in rabbits injected with highly purified vitamin D-dependent chick intestinal CaBP. The assay employs the double antibody technique, and 125-labeled CaBP was prepared using chloramine T. Low molecular weight peptide hormones and normal rabbit, rat, and human serum proteins show no cross-reactivity in the assay. Measurements of chick intestinal and kidney CaBP by RIA showed a good correlation with measurements of CaBP by the radial immunodiffusion method. The assay is reproducible (interassay variability, 16.3%) and precise (intraassay variability, 4.0%). The concentration of immunoreactive CaBP (iCaBP) in chick serum (2.7 ng/ml serum) can now be measured as early as 8 h after the administration of 6.5 nmol 1,25-dihydroxyvitamin D3; a maximum of 11 ng/ml is reached at 20 h. The level of CaBP in chick serum was found to be dependent on the dose of vitamin D3 or 1,25-dihydroxyvitamin D3 administered to the animal. The concentration of iCaBP in various tissues of the vitamin D-replete as well as the rachitic chick was significantly higher than serum levels of iCaBP. The values for iCaBP in the rachitic chick ranged from a high value for the kidney (480 ng/mg protein) and hypothalamus (275 ng/ mg protein) to a barely detectable level in the myocardium of 1.1 ng/mg protein. After administering 1.3 nmol vitamin D3 daily for 2 weeks, the level of iCaBP was highest in the duodenum (25μg/mg protein), jejunum (32 μg/mg), ileum (10 ng/mg), kidney (3.1 μg/mg), pancreas (141 ng/mg), and bone (110 ng/mg). Lower concentrations of iCaBP were detected in several other tissues. In all tissues except liver and skeletal muscle, the protein crossreacting in the RIA was immunochemically similar (as determined by parallel immunodilution curves) to highly purified intestinal CaBP. iCaBP in parathyroid glands responded to vitamin D and its metabolites 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D3 in a qualitatively similar pattern to intestinal CaBP. Levels of iCaBP in bone responded inversely to changes in dietary calcium and phosphorus, as did the levels of CaBP in the intestine. By employing the RIA, we can now quantitate minute levels of chick CaBP thus enabling us to probe more effectively the vitamin D endocrine system.