Vitamin D3 and its biologically active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] are shown to induce in the chick intestine and kidney the biosynthesis of a calcium binding protein (CaBP). In vitamin D3-replete chickens raised under adequate dietary calcium (Ca) and phosphorus (P) conditions, the steady-state level of intestinal CaBP (30–50 g/mg protein) is 5- to 20-fold greater than that of renal CaBP. Whereas dietary phosphorus restriction is known to elevate both intestinal and renal CaBP levels, dietary calcium restriction elevates only intestinal CaBP. The present study reports the rates of biosynthesis in vivo and in vitro, and of biodegradation in vivo, of both intestinal and renal CaBP after administration of vitamin D3 or 1,25(OH)2D3 to rachitic chicks. The apparent rate constant of degradation for intestinal CaBP was 0.024 h−1 (t12 = 29 h) and that for renal CaBP was 0.019 h−1 (t12 = 36 h) while total cellular soluble protein in the intestine and kidney had half-lives of 43 and 70 h, respectively. The time course of induction of the synthesis of CaBP was determined in intestine and kidney after administration of a physiological dose of 1,25(OH)2D3 to rachitic chicks. Intestinal CaBP synthesis was detectable by 3 hours, reached a maximal rate by 10 hours, and sharply decayed by 16–20 hours. The time course of induction of renal CaBP synthesis was very similar, although the rate of renal CaBP synthesis was readily detectable at the initial time of administration of 1,25(OH)2D3. The relative rates of synthesis of CaBP in the intestine and kidney under a variety of dietary Ca and P conditions in the vitamin D3-replete chick exactly paralleled the steady-state level of CaBP in these two tissues. These results are consistent with a model in which the steady-state levels of intestinal and renal CaBP are solely determined by their respective rates of biosynthesis; the CaBP biosynthetic capability, in turn, is regulated by the availability of 1,25(OH)2D3 to each target organ.

This chapter describes the purification of chick intestinal calcium-binding protein. Intestinal calcium-binding proteins (CaBP) have been identified and purified from several species of animals. The method described in this chapter enables isolation of chick intestinal CaBP in an electrophoretically homogeneous state. The procedure employed is a modification of the method of Wasserman; it utilizes an extra (NH4)2SO4 fractionation step and substitutes two anion-exchange steps for the originally reported preparative acrylamide disc gel electrophoresis procedure. In first step, duodenum obtained from vitamin D-repleted chickens was immediately cooled to 4°, slit open, and rinsed with cold 0.12 M NaCl. The mucosal tissue was scraped from the underlying muscle layers with a glass slide and then homogenized in a chilled buffer mixture (20% w/v) with a Potter-Elvehjem homogenizer utilizing a Teflon pestle. The composition of the buffer utilized was 13.7 mM Tris, 0.12 M NaC1 with 0.01% B-mercaptoethanol; this solution was adjusted to pH 7.4 with HCl. Other steps in this method that include (NH4)2SO4 precipitation, gel filtration, and anion exchange are also discussed in detail.

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.

The biological activity of 1α,24R,25-trihydroxyvitamin D3 [1α,24R,25(OH)3D3] was elevated in comparison to the hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], in the rachitic chick in terms of its ability to (a) stimulate intestinal calcium absorption, (b) mobilize bone calcium, (c) induce intestinal calcium binding protein, (d) modulate the level of enzyme activity of the renal 25-OH-D3-1-hydroxylase system, and (e) interact with the intestinal cystosol-chromatin receptor system for the 1α,25(OH)2D3 receptor system. In each of these assays, the relative ratio of activity of 1α,24R,25(OH)3D3 to 1α,25(OH)2D3was (a) 25–50, (b) ca. 20, (c) 10, (d) 50, and (e) 36%, respectively.

Calcium and phosphorus homeostasis is exquisitely regulated by the three hormones, calcitonin, parathyroid hormone, and cholecalciferol (vitamin D3). The most notable advance in our understanding of the mechanism of action of vitamin D has been the elucidation of the complex metabolic pathway which has evolved to produce the biologically active form, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Coupled with these developments concerning our understanding of the metabolic pathway of conversion of vitamin D into its active form, has been the realization that the mechanism of action of the fat soluble vitamin D is in reality similar to that of many of the classical steroid hormones, e.g. aldosterone, testosterone, estrogen, hydrocortisone, and ecdysterone. It should be noted that chemically vitamin D is in reality a steroid, in particular a seco steroid. Seco steroids are those in which one of the rings has undergone fission; in the instance of calciferol, this is ring B.

In recent years, there have been intensive efforts by several laboratories to elucidate various aspects of the parameters involved in the regulation of calcium and phosphorus metabolism. Three of the most important of these biological regulators are the seco steroid, calciferol (vitamin D), parathyroid hormone, and calcitonin. With each of these regulators, outstanding developments and advances have been made in recent years. It is the purpose of this article to briefly outline some of the developments that have occurred specifically with regard to our understanding of the shape or chemical conformation of vitamin D, its subsequent metabolism by the kidney in a regulated fashion, the interaction of the product steroid hormone (1,25-dihydroxycholecalciferol) with both the intestinal mucosal system where it mediates calcium absorption, and with the parathyroid gland. We have reviewed this subject in depth previously (2, 6).