A new radioreceptor assay was used to quantify changes in serum concentration of 1alpha,25-dihydroxyvitamin D3 in rats with low calcium or low phosphate diets. Low availability of either ion elicits a fivefold increase in the circulating concentration of 1alpha,25-dihydroxyvitamin D3. The enhancement of 1alpha,25-dihydroxyvitamin D3 concentration in response to calcium deficiency is dependent on the presence of the parathyroid or thyroid glands (or both), suggesting that this effect is mediated by parathyroid hormone. In contrast, the response of phosphate deficiency is independent of these glands and may result from an action of low serum phosphate concentration or some factor associated with phosphate depletion on the renal synthesis of the 1alpha,25-dihydroxyvitamin D3 hormone.

A competitive protein binding assay with a sensitivity of 80 picograms has been developed for 1alpha,25-dihydroxyvitamin D(3), the hormonal form of vitamin D(3). lalpha,25-Dihydroxyvitamin D(3) displaced tritiated hormone from a cytosol-chromatin receptor preparation isolated from chick small intestine, providing a simple assay for the hormone. The concentration of lalpha, 25-dihydroxyvitamin D(3) in human plasma, as determined by this assay, is approximately 6 nanograms per 100 milliliters; in patients with renal disease the concentration of this kidney-produced hormone is significantly lower.

A sensitive radioreceptor assay for 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) is utilized to quantitate the circulating concentration of this sterol in experimental animals and humans. When weanling rats are grown for 2 weeks on low calcium or low phosphate diets, limited availability of either ion elicits a five-fold increase in the plasma level of 1alpha,25-(OH)2D3. The enhancement of 1alpha,25-(OH)2D3 in calcium deficiency is dependent upon the presence of the parathyroid and/or thyroid glands, which is consistent with parathyroid hormone (PTH) mediation of this effect. In contrast, the response to phosphate deficiency is independent of these glands and may result from a direct action of low phosphate on the renal synthesis of 1alpha,25-(OH)2D3. Studies in humans indicate that the normal level of 1alpha,25-(OH)2D is 2.1--4.5 ng/100 ml plasma. Patients with chronic renal failure have markedly lower circulating 1alpha,25-(OH)2D and this kidney hormone is undetectable in anephric subjects, but returns to normal within 1 day after successful renal transplantation. Hypoparathyroidism and pseudohypoparathyroidism are associated with reduced plasma 1alpha,25-(OH)2D while patients with primary hyperparathyroidism have significantly elevated sterol hormone levels. Thus, from measurements in rats and humans, it appears that circulating 1alpha,25-(OH)2D3 is regulated by PTH and/or phosphate and that abnormal plasma 1alpha,25-(OH)2D3 is a part of the pathophysiology of renal osteodystrophy and parathyroid disorders.

A radioreceptor assay for 1 a,25-dihydroxyvitamin D3 (la,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

Specific binding of la,25-dihydroxyvitamin D3 [la,25(OH) (1) they sediment at 6 S in 0.3 M KCI-sucrose gradients, (2) they are observed in all tissues examined, (3) they have a higher affinity for 25-(OH)D3 than la,25-(0H)2D3, and (4) they are not found in the nucleus of the parathyroid glands, in vitro. The discovery of unique la,25.OH)2D3-binding components in the parathyroid glands is consistent with the sterol hormone's action at this endocrine site and possible involvement in the regulation of parathyroid hormone synthesis and secretion. Vitamin D3 action to mobilize calcium and phosphate at intestine and bone is thought to be mediated by the metabolite la,25-dihydroxyvitamin D3 [la,25-(OH)2D3] (1-4). Its production from 25-hydroxyvitamin D3 [25-(OH)D3] by the renal la-hydroxylase appears to be regulated by calcium (5), phosphate (6, 7), parathyroid hormone (PTH) (7,8), and the vitamin D status of the animal (9). Evidence suggests that hypocalcemia stimulates PTH secretion which in turn enhances the production of la,25-(OH)2D3 at the kidney (7, 8). Thus PTH, rather than calcium, may be the dominant modulator of the renal la-hydroxylase (10) and the finding of abnormal circulating 1a,25-(OH)2D3 in humans with parathyroid disease is consistent with this concept (11,12). DeLuca has proposed that PTH and phosphate deficiency may be functioning through a common intracellular mechanism to enhance the la-hydroxylase by lowering the phosphate level in the renal cell (10). Moreover, MacIntyre and associates (13) have suggested that la,25-(OH)2D3 might control its own biosynthesis directly at the kidney by a negative feedback mechanism involving the de novo synthesis of the la-hydroxylase enzyme. Clearly, the fashion in which la,25-(OH)2D3, PTH, and phosphate deficiency interact to control the formation of 1a,25-(OH)2D3 at its kidney endocrine site must be completely understood before the exact role of 1a,25-(OH)2D3 in the homeostatic regulation of calcium and phosphate can be elucidated.Henry and Norman (14) Homogenates were centrifuged at 1200 X g for 10 min. Nuclear pellets were removed, and the resulting supernatant was centrifuged at 100,000 X g for 1 hr at 0°to yield a final supernatant fraction (cytosol). The cytosol (0.2-1.0 ml) was incubated with sterol (in 20,l ethanol) for 1 hr at 00 and then analyzed for sterol binding components.Purified nuclear extracts (chromatin) were prepared from nuclear pellets by a modification (7) of the method of Haussler et al. (1) and were resuspended in 0.01 M Tris-HCl, pH 7.5, and centrifuged for 20. min at 48,000 X g. The pellet from 300 mg of tissue was reconstituted with cytosol (2.5 ml)Abbreviations: 25-(OH)D3, 25-hydroxyvitamin D3; la-(OH)D3, la-hydroxyvitamin D3; la,25-(OH)2D3, la,25-dihydroxyvitamin D3; PTH, parathyroid hormone. 4871