Synthesis and biological activity of (22E,24R)- and (22E,24S)-1 alpha,24-dihydroxy-22-dehydrovitamin D3.
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Animals↗
Biomedical subjects
H F DeLuca
Publications and source records attributed to H F DeLuca.
Effects of 1 alpha OHD2 on bone tissue. Studies of 1 alpha OHD2 and 1 alpha OHD3 in normal rats and in rats treated with prednisolone.
Three months old male rats were fed a diet containing 0.3% calcium and 0.5% phosphate. Prednisolone was added to the diet at a dose of 1.5 mg a day and the effect of oral treatment with 0.5 micrograms 1 alpha OHD2 or 0.5 micrograms 1 alpha OHD3 was studied. After 6 weeks rats given prednisolone had a lower bone mass and bone density. The serum phosphate concentration was also lower. Rats given 1 alpha OHD3 had an increased serum calcium concentration even when simultaneously treated with prednisolone. 1 alpha OHD2 did not cause hypercalcaemia. The decrease in bone density caused by prednisolone was prevented by the administration of 1 alpha OHD2. Rats receiving 1 alpha OHD3 together with prednisolone still exhibited a lower bone density. Furthermore, 1 alpha OHD3 decreased bone mass in normal rats and caused a higher mean value of osteoclasts/area whereas 1 alpha OHD2 reduced the number of osteoclasts/area. The study indicates that 1 alpha OHD2 is an effective vitamin D analogue with a lower tendency to cause bone resorption.
Animals↗
24,25-Dihydroxyvitamin D3 administration increases incidence of parturient paresis.
Fifty percent (7/14) of aged cows treated with 4 mg 24,25-dihydroxyvitamin D3 intramuscularly precalving developed parturient paresis shortly after calving compared with 7% (1/14) of controls. Injection of 24,25-dihydroxyvitamin D3 increased concentrations in blood plasma 15 times that in control cows. Blood plasma 1,25-dihydroxyvitamin D concentrations were elevated in all groups on day of calving but were not different. Injection of 24,25-dihydroxyvitamin D3 did not alter the typical plasma profile for calcium, phosphorus, or 1,25-dihydroxyvitamin D of paretic or nonparetic cows around parturition. Although injection of 24,25-dihydroxyvitamin D3 was associated with an increased incidence of parturient paresis, the mechanism remains unknown.
24,25-Dihydroxyvitamin D 3↗
Fluoride substitution of vitamin D analogs at C-26 and C-27: enhancement of activity of 25-hydroxyvitamin D but not of 1,25-dihydroxyvitamin D on bone and intestine in vitro.
A series of analogs of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] with multiple fluorine substitutions in positions 26 and 27 have been tested for their activities 1) in competing with 1,25-(OH)2D3 for binding to partially purified chick intestinal cytosol, 2) in stimulating resorption of fetal rat limb bones in vitro and 3) in competing with 25-hydroxyvitamin D3 for binding sites in rat plasma. The relative potencies of 26,26,26,27,27,27-hexafluoro-25-hydroxyvitamin D3, 26,26,26-trifluoro-25-hydroxyvitamin D3, 27-nor-26,26,26-trifluoro-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 in competing for intestinal cytosolic binding were 17:11:1:1. The relative potencies of the same series of compounds on stimulating resorption of fetal rat bones was 25:21:9:1. The relative ability of these four compounds to compete for plasma binding sites was 0.3:0.3:0.4:1.0. The results indicate that multiple fluorine substitution in the vicinity of the 25-hydroxyl group can markedly enhance the direct effects of 25-hydroxyvitamin D3 on its target tissues. This is postulated to result from the electronegativity of the fluorines which increases the acidity of the 25-hydroxyl group and enhances its affinity for the receptor. In contrast to the effects seen with the 25-hydroxyvitamin D3 analogs, multiple fluorine substitution in positions 26 and 27 did not enhance the activity of 1,25-(OH)2D3 on either cytosolic binding or bone resorption. Presumably, this is because biological activity, expressed in terms of affinity for the receptor, is already optimal in the 1,25-(OH)2D3 structure. The relative activities of the 26,27-hexafluoro derivative and 1,25-(OH)2D3 in competition for the plasma binding sites was 0.4:1.0.(ABSTRACT TRUNCATED AT 250 WORDS)
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Renal-nonresponsive, bone-responsive pseudohypoparathyroidism. A case with normal vitamin D metabolite levels and clinical features of rickets.
Clinical signs of rickets developed in a previously healthy 13-year-old girl with normal features. She had hypocalcemia, hyperphosphatemia, elevated alkaline phosphatase and parathyroid hormone levels, and normal vitamin D metabolite levels, with osteitis fibrosa cystica on bone biopsy specimen. Her renal function was normal. Treatment with 1 microgram of calcitriol each day resulted in symptomatic and clinical relief and improvement of the serum chemical values. This patient probably has pseudohypoparathyroidism type 1, with renal nonresponsiveness and bone responsiveness. This disorder has the clinical features of rickets, but represents hyperparathyroid bone disease.
Adolescent↗
Induction of macrophage differentiation of human normal and leukemic myeloid stem cells by 1,25-dihydroxyvitamin D3 and its fluorinated analogues.
This study shows for the first time that 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] and two fluorinated analogues of 1,25-(OH)2D3 [24,24-F2-1,25-(OH)2D3 and 26, 26, 26, 27, 27, 27-F6-1,25-(OH)2D3] induce macrophage differentiation of human normal and leukemic myeloid stem cells. The addition of either 1,25-(OH)2D3 or one of the two fluorinated analogues of 1,25-(OH)2D3 at concentrations as low as 10(-9) M to culture plates containing normal human marrow cells stimulated myeloid stem cells to preferentially differentiate to colonies of monocytes and macrophages. Over 80% of the normal human myeloid colonies were composed of only monocytes and macrophages in culture plates containing 10(-7) M 1,25-(OH)2D3 or one of the fluorinated analogues. In contrast, control plates not containing 1,25-(OH)2D3 had less than 35% macrophage colonies. Likewise, 1,25-(OH)2D3 and the two vitamin D analogues induced macrophage differentiation of leukemic colony-forming cells taken from patients. In plates containing 10(-7) M 1,25-(OH)2D3 or one of the analogues at 10(-8) M, 80% of chronic myelogenous leukemia and approximately 50% of acute nonlymphocytic leukemia colony-forming cells differentiated to macrophage-like cells. In contrast, control plates had about 30 and 20% macrophage colonies in cultures from chronic myelogenous leukemia and acute nonlymphocytic leukemia patients, respectively. Our findings suggest that 1,25-(OH)2D3 may play a role in hematopoiesis and that the compound or a related analogue may possibly have a therapeutic role in some leukemias.
Bone Marrow↗
Vitamin D and parathyroid hormone status in children with the nephrotic syndrome and chronic mild glomerulonephritis.
The serum concentrations of 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (1,25(OH)2D), calcium, creatinine, albumin and immunoreactive parathyroid hormone (iPTH) were measured in eight children with chronic glomerulonephritis not treated with prednisone (group I), in nine non-edematous children with the nephrotic syndrome treated with prednisone for more than 18 months (group II) and in five children with overt edema also treated with prednisone (group III). Serum creatinine was under 1.2 mg/dl in all 22 patients. Reductions in serum calcium, albumin and 25(OH)D were found in group III patients only, whereas both group II and group III patients showed reduced values of 1,25(OH)2D (p less than 0.001 vs. group I or controls). Chronic glucocorticoid administration in children with glomerulonephritis and minimally impaired renal function (group II) is associated with a reduction in the circulating level of 1,25(OH)2D, since children with comparable type and degree of renal disease but non glucocorticoid treatment (group I) have normal 1,25 (OH)2D values. Children with nephrotic edema (group III) have greater reduction of 1,25(OH)2D values, as well as lower 25(OH)D values and serum calcium values, presumably related to a urinary loss of vitamin D-binding protein. No changes in iPTH were evident in either glucocorticoid-treated or edematous patients, suggesting that the acute elevation in iPTH seen after prednisone treatment is an acute phenomenon. Additional short-term studies are needed to more clearly define the effect of glucocorticoids on vitamin D metabolism.
Calcium↗
Support of embryonic chick survival by vitamin D metabolites.
The provision of 1,25-dihydroxyvitamin D3 as the only source of dietary vitamin D3 to laying hens failed to support normal embryonic development in their fertile eggs. Significant (P less than .001) improvement in embryonic survival to hatching in these eggs resulted from injections of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or 24,24-difluoro-25-hydroxyvitamin D3 prior to incubation. Maximum embryonic survival with lowest embryonic mortality was observed when 0.20 micrograms/egg of 1,25-dihydroxyvitamin D3 or 0.60 micrograms/egg 25-hydroxyvitamin D3 was injected. These results indicate that several forms of vitamin D, two of which cannot be converted to 24,25-dihydroxyvitamin D3, can provide this activity; and of the vitamin D compounds tested, 1,25-dihydroxyvitamin D3 may be the most active in supporting embryonic survival in the chick when delivered directly by injection.
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Stimulation of 1,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in the hypocalcaemic rat.
Serum 1,25-dihydroxyvitamin D3 concentration and renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity were measured in rats fed various levels of calcium, phosphorus and vitamin D3. Both calcium deprivation and phosphorus deprivation greatly increased circulating levels of 1,25-dihydroxyvitamin D3. The circulating level of 1,25-dihydroxyvitamin D3 in rats on a low-calcium diet increased with increasing doses of vitamin D3, whereas it did not change in rats on a low-phosphorus diet given increasing doses of vitamin D3. In concert with these results, the 25-hydroxyvitamin D 1 alpha-hydroxylase activity was markedly increased by vitamin D3 administration to rats on a low-calcium diet, whereas the same treatment of rats on a low-phosphorus diet had no effect and actually suppressed the 1 alpha-hydroxylase in rats fed an adequate-calcium/adequate-phosphorus diet. The administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats on a low-calcium diet also increased the renal 25-hydroxy-vitamin D 1 alpha-hydroxylase activity. These results demonstrate that the regulatory action of 1,25-dihydroxyvitamin D3 on the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is complex and not simply a suppressant of this system.
25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
Biliary metabolites of all-trans-retinoic acid in the rat.
Biliary metabolites from physiological doses of all-trans-[10-3H]retinoic acid were examined in normal and vitamin A-deficient rats. The bile from normal and vitamin A-deficient rats contained approximately 60% of the administered dose following a 24-h collection period. However, vitamin A-deficient rats show a 6-h delay in the excretion of radioactivity compared to normal rats. Retinoyl-beta-glucuronide excretion was particularly sensitive to the vitamin A status of the rats. In normal rats, retinoyl-beta-glucuronide reached a maximum concentration of 235 pmol/ml of bile 2 h following the dose and then rapidly declined. Vitamin A-deficient rats show a relatively constant concentration of this metabolite (100-150 pmol/ml of bile) over a 10-h collection period. Retinoic acid excretion was low in both normal and deficient rats. The concentration of retinotaurine, a recently identified biliary metabolite, was approximately equal to retinoyl-beta-glucuronide in normal rats and appeared in the bile 2 h later than the glucuronide.
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Purification of the receptor for 1 alpha,25-dihydroxyvitamin D3 from chicken intestine.
Methods were investigated for use in the purification of the chicken intestinal receptor for 1 alpha,25-dihydroxyvitamin D3. The techniques investigated include column isoelectric focusing, gel exclusion, polyacrylamide gel electrophoresis, and DNA-cellulose, DEAE-cellulose, and hydroxylapatite chromatography. For the starting receptor preparation, a nuclear extract of chicken intestinal mucosa was found to be enriched above cytosol preparations and a plentiful source of receptor. A five-step purification scheme that resulted in the purification of the receptor protein by 5800-fold with 8% yield has been described. Analysis of the purified proteins on polyacrylamide gel electrophoresis containing sodium dodecyl sulfate suggests homogeneity. Analysis using two-dimensional polyacrylamide electrophoresis characterized the purified protein as having a molecular weight of approximately 63 000 and a pI of 6.0-6.2. Furthermore, assessment of protein purity by 125I iodination followed by sucrose gradient analysis revealed that approximately 90% of the iodinated macromolecules have the same sedimentation coefficient as the titrated 1 alpha,25-dihydroxyvitamin D3 receptor complex. The final purified receptor that bound tritiated 1 alpha,25-dihydroxyvitamin D3 retained affinity for DNA-cellulose and possesses a 3.7S sedimentation coefficient. The receptor has an estimated Stokes radius of 37 A.
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Studies on the site of 1,25-dihydroxyvitamin D3 synthesis in vivo.
Anephric, vitamin D-deficient male rats were injected with a physiologic dose of 25-hydroxy[26,27-3H]vitamin D3 (specific activity of 160 Ci/mmol), and 18-20 h later, intestine, bone, and serum were analyzed by high performance liquid chromatography for 1,25-dihydroxy-[26,27-3H]vitamin D3. Identical studies were carried out using sham-operated rats and rats with ligated ureters. No 1,25-dihydroxy[26,27-3H]vitamin D3 was detected in the tissues from anephric rats, while large amounts were detected in sham-operated and ureteric ligated controls. This result demonstrates that in the nonpregnant rat, 1,25-dihydroxyvitamin D3 is either not synthesized or is synthesized in vanishingly small amounts in bone and intestine in vivo, casting considerable doubt of the physiological importance of reports of in vitro synthesis of 1,25-dihydroxyvitamin D3 by cells in culture derived from bone and elsewhere.
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In vitro inhibitor studies of vitamin D 25-hydroxylase in rat liver microsomes.
The ability of four vitamin D analogs to inhibit the liver microsomal vitamin D-25-hydroxylase was determined. 19-Hydroxy-10(S),19-dihydrovitamin D3,25-fluorovitamin D3, 3 beta-hydroxy-9,10-seco-5,7,10(19)-choletrien-24-oic acid dimethylamide and 25-aza-vitamin D3 were competitive inhibitors with apparent KI values of 44, 137, and 870 nM, and 6.4 microM, respectively. The values for the 19-hydroxy-10(S), 19-dihydrovitamin D3, 25-fluorovitamin D3, and 25-aza-vitamin D3 correspond well to other literature reports with respect to their relative in vivo inhibitory properties. 24-Oxovitamin D3 oxime also proved to be a potent inhibitor but a detailed analysis was prohibited by the lack of material. The 3 beta-hydroxy-9,10-seco-5,7,10(19)-choletrien-24-oic acid dimethylamide was also tested in vivo but had no antagonistic activity when provided at a 2000-fold excess over vitamin D3.
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The biological activity of rat intestinal retinoic acid metabolites in the vaginal smear assay.
Vitamin A-deficient rats were given a single intrajugular injection of 1 mg all-trans-[11-3H]retinoic acid and 3 h later the rats were killed. The small intestines were extracted and chromatographed by high-performance liquid chromatography to yield distinct metabolites. These were quantitated using the assumption that the specific activity of the metabolite is equal to that of the parent [3H]retinoic acid. The biological activity of all discernible metabolites was determined in the vitamin A-deficient female rat by vaginal smear assay. Retinoic acid and retinoyl-beta-glucuronide from the preparation had equal activity while no activity was found for any of the other metabolite fractions. Thus, no evidence for an unknown metabolite having potent epithelial differentiating activity could be found in this target tissue of vitamin A action.
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Induction of calcium-binding protein before 1,25-dihydroxyvitamin D3 stimulation of duodenal calcium uptake.
Protein synthesis occurring before the onset of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) stimulation of calcium uptake was examined in embryonic chick duodena by double label autoradiography. Duodena from 19-day-old embryos were cultured for 24 h. Duodena exposed to a saturating concentration of 1,25-(OH)2D3 during the last 2, 4, or 6 h were either cultured with [3H]leucine for the final 2 h or used for calcium uptake assays. Duodena not exposed to the hormone were either cultured with [14C]leucine for the final 2 h or used for calcium uptake assays. Calcium uptake by nonradiolabeled duodena was increased (p less than 0.05) only in tissues exposed to the hormone for 6 h. Tissue uptakes of [3H]leucine and [14C]leucine were identical and linear with time. Soluble proteins were extracted from 3H- and 14C-labeled tissues, mixed, and simultaneously resolved by two-dimensional polyacrylamide gel electrophoresis. Analysis of fluorographs and autoradiographs prepared from the double-labeled gels revealed a protein induced by 1,25-(OH)2D3 within 2 h of exposure to 1,25-(OH)2D3 and at lest 2 h before the calcium uptake response. The induced protein co-migrated with purified chick calcium-binding protein during two-dimensional electrophoresis. These results demonstrate that 1) this protein is the calcium-binding protein and 2) calcium-binding protein is clearly synthesized in the embryonic duodenum before the calcium uptake response to 1,25-(OH)2D3 occurs.
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Oral 1,25(OH)2D3: an effective prophylactic treatment for glucocorticoid osteopenia in rats.
Eighty-eight adult male Sprague-Dawley rats were given a diet with either (a) 0.5% Ca and 0.6% P or (b) 0.01% Ca and 0.6% P. Osteopenia was created by adding prednisolone to the diet. The prophylactic effect of oral 1,25(OH)2D3 on the osteopenia was studied. It was found that prednisolone osteopenia in the rat was associated with defective Ca absorption. By giving an oral dose of 1,25(OH)2D3, it was possible to maintain normal Ca absorption during prednisolone treatment and to prevent the bone loss. No significant hypercalcemia or any kidney calcifications were seen. These results are in contrast to earlier findings, in which subcutaneous administration of 1,25(OH)2D3 failed to prevent prednisolone osteopenia because of its tendency to increase bone resorption.
Animals↗
Autoradiographic localization of target cells for 1 alpha, 25-dihydroxyvitamin D3 in bones from fetal rats.
Thaw-mount autoradiographic studies after injection of 3H-1,25-D3 were conducted on 18- and 20-day-old rat fetuses. In maxillary bones, ribs, and tibia, nuclear concentration of radioactivity was found in osteoprogenitor cells and osteoblasts. Osteocytes and chondrocytes in epiphyseal plates were either unlabeled or weakly labeled. In competition experiments, nuclear concentration of radioactivity was blocked by the injection of a high dose of nonradioactive 1,25-D3 prior to the administration of the labeled hormone, but not by a similar dose of nonradioactive 25-D3. The results are interpreted as indicating that osteoprogenitor cells and osteoblasts are target cells for the direct action of 1,25-D3 on fetal bone.
Animals↗
Studies on the role of vitamin D in early skeletal development, mineralization, and growth in rats.
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