Radioreceptor assay for 1 alpha,25-dihydroxyvitamin D3.

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.

Regulation of serum 1alpha,25-dihydroxyvitamin D3 by calcium and phosphate in the rat.

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.

Specific binding of 1alpha,25-dihydroxycholecalciferol to nuclear components of chick intestine.

Specific binding of 1alpha,25-dihydroxycholecalciferol to macromolecular components of small intestinal mucosa nuclei is demonstrated in vitamin D-deficient chicks. The nuclear 1alpha,25-dihydroxycholecalciferol-macromolecule complex was isolated on sucrose density gradients and sediments at 3.7 S in the presence of 0.3 M KCl. Agarose gel filtration of the nuclear component indicated an apparent molecular weight of 47,000. The nuclear receptor complexes could not be distinguished from previously described cytoplasmic 1alpha,25-dihydroxycholecalciferol-binding components by the ultracentrifugation and chromatographic procedures employed. The association of the 3-H-sterol with the nuclear component is thermolabile and is destroyed by treatment with pronase, but not by nucleases; the receptor component is therefore presumed to be a protein. The macromolecular-1alpha,25-dihydroxycholecalciferol complex formed in vivo or in vitro at 25 degrees can be extracted from intestinal nuclei by 0.3 M KCl, but not by low salt buffers. Smaller amounts of the 3.7 S binding component can be detected in isolated purified chromatin or after incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin at 0 degrees. Following incubation of the labeled hormone with reconstituted cytosol-chromatin at 0 degrees, 1alpha,25-dihydroxy[3-H]cholecalciferol is primarily associated with the cytoplasmic receptor, After shifting the incubation temperature to 25 degrees, a progressive increase in the concentration of the nuclear receptor complex and a concomitant decrease in the concentration of the cytoplasmic binding component occur. Thus the 1alpha,25-dihydroxycholecalciferol binding molecules appear to exist primarily in the cytoplasm, where they presumably function to transport the hormone into the nucleus. Experiments employing incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin from nontarget tissues indicate a requirement for both intestinal cytosol and chromatin for maximal formation of the nuclear hormone-receptor complex. These results suggest that the nuclear-binding component arises from hormone-dependent transfer of the cytoplasmic 1alpha,25-dihydroxycholecalciferol receptor to intestinal chromatin acceptor sites.

1 alpha, 25-Dihydroxyvitamin D3 a metabolite of vitamin D that promotes bone repair.

1 alpha, 25-dihydroxyvitamin D3, the hormonal form of vitamin D3 that mediates calcium translocation in intestine and bone, was tested for its ability to promote fracture repair. Chicks were raised on a vitamin D-deficient diet supplemented with 1 alpha, 25-dihydroxyvitamin D3 for 3 weeks. Following fracture of the humerus, those chicks that did not receive continued 1 alpha, 25-dihydroxyvitamin D3 supplementation showed prolonged fracture healing, abnormal enchondral bone formation delayed remodeling of woven bone and osseous union, but normal formation of callus. Fracture repair in chicks receiving 1 alpha, 25-dihydroxyvitamin D3 was normal. These data indicate that 1 alpha, 25-dihydroxyvitamin D3 promotes bone repair in the absence of vitamin D3, 25-hydroxyvitamin D3, and 24,25-dihydroxyvitamin D3.

Cytoplasmic and nuclear binding components for 1alpha25-dihydroxyvitamin D3 in chick parathyroid glands.

Specific binding of 1 alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] to macromolecular components in the cytoplasm and nucleus is demonstrated in parathyroid glands of vitamin-D-deficient chicks. The interaction of 1alpha,25-(OH)2D3 with the cytoplasmic binding component is of high affinity (Kd = 3.2 X 10(-10) M) and high specificity [1alpha,25-(OH)2D3 greater than 25-hydroxyvitamin D3 greater than 1alpha-hydroxyvitamin D3 greater than vitamin D3 in competing with radioactive 1alpha,25-(OH)2D3]. Both cytoplasmic and nuclear hormone-macromolecular complexes sediment at 3.1 S in 0.3 M KC1-sucrose gradients, and agarose gel filtration of the components indicates an apparent molecular weight of 58,000. The 3.1S binding molecules are not observed in adrenal gland, testes, liver, or kidney, but similar receptors for 1alpha,25-(OH)2D3 have been found previously in intestine. Macromolecular species with a high affinity and preference for 25-hydroxyvitamin D3 [25-(OH)D3] are also identified in parathyroid cytosol and differ from the parathyroid 1alpha,25-(OH)2D3-binding component in that: (1) they sediment at 6 S in 0.3 M KC1-sucrose gradients, (2) they are observed in all tissues examined, (3) they have a higher affinity for 25-(OH)D3 than 1alpha,25-(OH)2d3, and (4) they are not found in the nucleus of the parathyroid glands, in vitro. The discovery of unique 1alpha,25-(OH)2D3-binding components in the parathyroid glands is consistent with the sterol hormone's action at this endocrin site and possible involvement in the regulation of parathyroid hormone synthesis and secretion.

A cellulose acetate electrophoretic procedure evaluated for quantitation of high-density lipoprotein cholesterol.

We evaluated the performance of a commercially available cellulose acetate electrophoretic method for quantitating high-density lipoprotein cholesterol (I) in serum by comparing it to a method involving precipitation with dextran sulfate-500/Mg2+. In both methods, enzymic reagents are used for cholesterol measurement. For electrophoretic measurement of I the mean intramembrane CV ws 4.1% (at 220 to 360 mg/L) and the intermembrane CV ranged from 12.2 to 21.0% (at 220 to 880 mg/L). Interassay precision was significantly better for the precipitation method (CV = 3.9% at 390 mg/L). The electrophoretic procedure demonstrated significant measurement bias, both at high and low I concentrations. However, low-density lipoprotein cholesterol, measured electrophoretically correlated well with its calculated concentrations obtained by the precipitation method. Measurement of I by this electrophoretic procedure did not achieve the accuracy and reproducibility that have been demonstrated for precipitation methods and that are necessary for reliable clinical interpretation of results for I.

Physiological importance of vitamin D metabolism.

1,25(OH)2D3 and 24,25(OH)2D3 are biosynthesized from 25(OH)D3 in kidney. A cytosol receptor for 1,25(OH)2D3, but not 24,25(OH)2D3, is detected in kidney as well as bone and gut. The function of 1,25(OH)2D3 in kidney is probably to enhance calcium-binding protein and mineral reabsorption and to induce vitamin D-24-OHase enzyme. Receptors for 1,25(OH)2D3 are present also in endocrine sites such as parathyroid, pancreas, pituitary, and placenta, possibly to mediate central control of mineral homeostasis. Finally, 1,25(OH)2D3 elicits normal bone mineralization and fracture healing, contrary to other reports that 24,25(OH)2D3 is uniquely required for such physiologic vitamin D actions.

The assay of 1alpha,25-dihydroxyvitamin D3: physiologic and pathologic modulation of circulating hormone levels.

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.