Effects of the administration of phosphate on nuclear 1,25-dihydroxyvitamin D3 uptake by duodenal mucosal cells of Hyp mice

Effects of the administration of phosphate on nuclear 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] uptake by duodenal mucosal cells of Hyp mice were investigated. In Hyp mice fed a high phosphate diet (1.1% Ca and 2.0% phosphate) for 2 weeks, maximal nuclear 1,25-(OH)2D3 binding by duodenal mucosal cells is significantly increased from 5.01 +/- 0.49 x 10(3) to 8.23 +/- 1.10 x 10(3) sites/cell (P less than 0.05). No significant change was observed in normal mice fed the same diet. The serum phosphate concentration of Hyp mice increased significantly (P less than 0.01), whereas no significant change was found in normal mice. On this regimen, serum calcium, urinary cAMP to creatinine ratio, and cytosolic 1,25-(OH)2D3 receptor number in Hyp mice were not changed significantly. On the basis of these data, we speculate that the recovery of serum phosphate in Hyp mice fed a high phosphate diet affects the recovery of nuclear 1,25-(OH)2D3 uptake by duodenal mucosal cells. The mechanism for this recovery is not related to either the secondary hyperparathyroidism or the change in cytosolic 1,25-(OH)2D3 receptor content but, rather, to increased binding of 1,25-(OH)2D3-receptor complex to nuclei. Hypophosphatemia, therefore, appears to play a role in the vitamin D resistance in Hyp mice.     

Administration of pharmacological amounts of 25(s),26-dihydroxyvitamin D3 reduces serum 1,25-dihydroxyvitamin D3 levels in rats

Pharmacological amounts of 25(s),26-dihydroxyvitamin D3 were administered to normal, vitamin D-replete rats in order to assess its pharmacological activity. Treatment with 25(s),26-dihydroxyvitamin D3 (20 micrograms/day for 1 week) caused a marked and significant fall in the circulating concentration of 1,25-dihydroxyvitamin D (16 +/- 5 SEM vs. 28 +/- 4 pg/ml, P = 0.02). This reduction of 1,25-dihydroxyvitamin D was dependent on the dose of 25,26-dihydroxyvitamin D3 administered since a 5 micrograms/day dosing regimen failed to alter serum 1,25-dihydroxyvitamin D levels. Despite the 25-66% reduction in circulating 1,25-dihydroxyvitamin D concentration produced by 25,26-dihydroxyvitamin D3 therapy, serum calcium and intestinal calcium absorption remained normal. These results suggested that 25,26-dihydroxyvitamin D has a weak agonist action or that a further metabolite that stimulates bone calcium resorption and/or intestinal calcium absorption is formed. Rats predosed with 25,26-dihydroxyvitamin D3 (20 micrograms/day) for 4 days and subsequently dosed with both 1,25-dihydroxyvitamin D3 (0.15 micrograms/day) and 25,26-dihydroxyvitamin D3 for an additional 3 days, demonstrated serum 1,25-dihydroxyvitamin D levels significantly higher than that found for control rats (47 +/- 5 vs. 25 +/- 4 pg/ml, P less than 0.001) but significantly reduced from the value observed for rats receiving only 1,25-dihydroxyvitamin D3 (47 +/- 5 vs. 187 +/- 38 pg/ml, P less than 0.001). These results suggest that 25,26-dihydroxyvitamin D3 has a previously unrecognized action in affecting the metabolism of 1,25-dihydroxyvitamin D3.     

24- and 26-homo-1,25-dihydroxyvitamin D3: preferential activity in inducing differentiation of human leukemia cells HL-60 in vitro.

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D3, promotes the differentiation of HL-60 human promyelocytic leukemia cells into monocytes. Differentiation changes include the induction of phagocytosis, the initiation of nitroblue tetrazolium-reducing activity, and the appearance of nonspecific acid esterase. We have found that the 24-homo- and 26-homo-1,25-dihydroxyvitamin D3 and their delta 22 analogues are 10-fold more potent than 1,25-dihydroxyvitamin D3 in inducing differentiation of HL-60 cells in vitro. In vivo, these analogues show activity similar to 1,25-dihydroxyvitamin D3 in stimulating intestinal calcium transport in vitamin D-deficient rats. The 24-homoanalogues are significantly less active, whereas the 26-homo derivatives are more active than the natural hormone in mobilizing calcium from bone. This unusual activity pattern cannot be explained on the basis of the affinity of these analogues for the 1,25-dihydroxyvitamin D3 intracellular receptor: both 24-homo- and 26-homo-1,25-dihydroxyvitamin D3 have the same effectiveness as 1,25-dihydroxyvitamin D3 in displacing the tritiated hormone from its receptor in rat intestine or HL-60 cells. These analogues of 1,25-dihydroxyvitamin D3 may be of some interest as possible therapeutic substances, or as tools in understanding the action of 1,25-dihydroxyvitamin D3 in inducing differentiation.     

Action of 1,25-dihydroxyvitamin D3 on phospholipid metabolism of human bone cells in culture

It has recently been proposed that the action of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on bone metabolism may be mediated by changes in phospholipid metabolism. The effects of vitamin D metabolites on the incorporation of radiolabelled precursors into corresponding phospholipid classes were investigated using cells arising from cultured explants of normal human bone with osteoblast-like characteristics. Treatment with 1,25-(OH)2D3 increased the incorporation of serine, measured as the ratio of [3H]serine in phosphatidylserine (PS) to [14C]ethanolamine in phosphatidylethanolamine (PE), in a time- and dose-dependent manner. The maximum effect on PS/PE of 141.6 +/- 5.9% over control (P = 0.022) was observed at a dose of 0.1 nmol 1,25-(OH)2D3/l, maintained for 24 h. Incubations with 25-hydroxyvitamin D3 (0.1 mumol/l) and 24,25-dihydroxyvitamin D3 (10 nmol/l) had no effect. Supraphysiological doses (0.1 mumol/l) of 1,24,25- and 1,25,26-trihydroxyvitamin D3 showed similar effects to those of 1,25-(OH)2D3, emphasizing the importance of 1 alpha-hydroxylation. Incorporation of [14C]choline into phosphatidylcholine, calculated as a ratio to PE, was not affected by treatment with vitamin D metabolites. However, [3H]inositol uptake into phosphatidylinositol was almost doubled when compared with control uptake within 2 h of treatment with 1,25-(OH)2D3 (0.1 mumol/l). This may be of relevance, considering the importance of phosphoinositide metabolism in influencing the intracellular calcium concentration. These results support a role for 1,25-(OH)2D3 in the modulation of phospholipid metabolism in human bone cells, which in turn may be involved in the action of 1,25-(OH)2D3 in bone mineralization.     

Bone biochemistry and physiology from the perspectives of the vitamin D endocrine system.

Vitamin D plays an indispensable role in the dual processes of bone formation (mediated by osteoblasts) and bone resorption (mediated by osteoclasts). More recently, researchers have confirmed the existence of a vitamin D endocrine system, which is responsible for describing the "sphere of biological influence" of vitamin D3. In that system, the kidney serves as the endocrine gland that produces 1,25-dihydroxyvitamin D3. This hormonally active form of vitamin D3 generates many, if not all, of the biologic responses attributed to the parent vitamin D3, including its role in bone formation and bone resorption. In addition, 1,25-dihydroxyvitamin D3 is able to generate biologic responses via both genomic and nongenomic pathways. The classic nuclear receptor for 1,25-dihydroxyvitamin D3 is present in more than 30 target tissues. This paper reviews evidence for the critical role of 1,25-dihydroxyvitamin D3 in cell differentiation, particularly of hematopoietic cells, as well as in the generation of the bone resorptive cell--the osteoclast. In the past year, much evidence has been accumulated supporting the claim that 1,25-dihydroxyvitamin D3 tightly regulates differentiation of osteoclast progenitors into osteoclasts. Osteoclast progenitors are believed to be derived from the monocyte-macrophage lineage. However, the generation of new osteoclasts is modulated by osteoblastic stromal cells, which are one of the target cells for the nuclear actions of 1,25-dihydroxyvitamin D3.     

Rapid changes in skeletal muscle calcium uptake induced in vitro by 1,25-dihydroxyvitamin D3 are suppressed by calcium channel blockers

Previous investigations have shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] stimulates muscle Ca uptake through a nuclear mechanism. The possibility that 1,25-(OH)2D3 would induce rapid changes in muscle Ca fluxes independent of de novo protein synthesis was investigated in the present work. In vitro preparations of soleus muscles obtained from vitamin D-deficient chicks were used. A significant increase in 45Ca labeling of the tissue was already observed after 3-min treatment with 2.4 X 10(-10) M 1,25-(OH)2D3. This early stimulation in muscle Ca uptake became maximal at 10-15 min. Cycloheximide (50 microM) did not block the effect of the metabolite at 15 and 30 min. However, the antibiotic effectively blocked the increase in Ca uptake induced by 1,25-(OH)2D3 after 1-h treatment. The rapid 1,25-(OH)2D3-dependent stimulation of 45Ca labeling of soleus muscle was not associated to changes in lipid synthesis as assessed by measurements of 3H-glycerol incorporation into the tissue lipids. However, the calcium antagonists verapamil and nifedipine (50 microM) abolished the stimulation in Ca uptake produced by 1,25-(OH)2D3 in 5 min. These results suggest that 1,25-(OH)2D3 can act directly at the muscle membrane level affecting Ca fluxes through Ca channels.     

The mobilization of bone mineral by 1,25-dihydroxyvitamin D3 in hypophosphatemic rats.

Administration of 1,25-dihydroxyvitamin D3 to thyroparathyroidectomized, hypophosphatemic rats on a low-phosphorus diet increases serum inorganic phosphorus and calcium concentrations. Experiments utilizing such rats treated with 45Ca and 32P ten days before use revealed that the rise in serum phosphorus in response in 1,25-dihydroxy-vitamin D3 is derived at least in part from bone mineral. This suggests that under conditions of hypophosphatemia, 1,25-dihydroxyvitamin D3 can mobilize bone in the absence of parathyroid hormone.     

1,25-dihydroxyvitamin D3 stimulates vesicular transport within 5 s in polarized intestinal epithelial cells

Controversy remains regarding whether the seco-steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances calcium and phosphate movement across the intestinal epithelial cell by facilitated diffusion or a vesicular transport mechanism. In this study we investigated whether membrane trafficking, as judged by confocal microscopy, was sufficiently rapid in comparison to hormone-stimulated uptake of phosphate (32P). Primary cultures of chick intestinal cells were established overnight either in Petri dishes (uptake studies) or chambered coverslips (confocal microscopy). Addition of 130 pM 1,25(OH)2D3 resulted in an apparent increase in 32P uptake within 1 min, relative to controls, that was statistically significant from 3-10 min of incubation. Using the endocytic marker dye, FM1-43, confocal microscopy revealed a profound decrease in membrane-associated fluorescence (apical> basal) within 10 s of hormone treatment, a return of fluorescence at 15-65 s, followed by another round of decreasing and increasing fluorescence. Between 3-9 min of incubation, fluorescence intensity increased 50% (apical region) and 20% (basal region) over control conditions. An antibody (Ab 099) directed against a putative membrane receptor for 1,25(OH)2D3 (1,25D3-MARRS) inhibited both 32P uptake, and changes in fluorescence. In addition, the protein kinase C (PKC) inhibitor, calphostin C, inhibited both 32P uptake and the observed 1,25(OH)2D3-mediated changes in fluorescence. At the microscopic level, calphostin C pretreatment abolished the very rapid redistribution of the endocytic marker dye, although a slight increase in fluorescence was still observed. We conclude that 1,25(OH)2D3-stimulated vesicular trafficking is mediated by the 1,25D3-MARRS protein, implicates a PKC signaling mechanism, and occurs in a time frame that is commensurate with a role in ion transport.     

Decreased intestinal calcium absorption in vivo and normal brush border membrane vesicle calcium uptake in cortisol-treated chickens: evidence for dissociation of calcium absorption from brush border vesicle uptake.

The influence of cortisol on intestinal calcium transport was studied in isolated duodenal loops and brush border membrane (BBM) vesicles of vitamin D-deficient or replete chickens. Four- to five-week-old vitamin D-deficient cockerels were dosed intraperitoneally with 1 microgram of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] alone 15 hr before sacrifice or in combination with 1, 3, or 5 mg of cortisol 24 and 48 hr before sacrifice. After a 1-microgram dose of 1,25-)OH)2D3 the in situ intestinal ligated loop technique revealed a 60% increase in calcium absorption compared to control birds (P less than or equal to 0.001). However, the administration of cortisol in various doses (3 and 5 mg) to chickens given 1,25-(OH)2D3 resulted in significant decreases in intestinal calcium transport in vivo (P less than or equal to 0.05; P less than or equal to 0.05). When intestinal BBM vesicles were prepared from birds treated in a manner identical with that described above, there was no observable difference between calcium uptake in BBM vesicles of the 1,25-(OH)2D3-treated birds and that of the cortisol plus 1,25-(OH)2D3-treated birds. 1,25-(OH)2D3-treated and 1,25-(OH)2D3 plus cortisol-treated chicks had intestinal BBM vesicle uptakes that were significantly greater than those of vitamin D-deficient controls (P less than or equal to 0.02; P less than or equal to 0.025). These data show that in vivo intestinal calcium transport may be markedly reduced in the presence of normal intestinal BBM vesicle calcium uptake. This suggest that factors other than BBM calcium uptake (e.g., protein synthesis or contraluminal membrane events) play an important role in the movement of calcium from the intestinal lumen into the bloodstream and extracellular fluid of the organism.     

Decreased nuclear uptake of 1,25-dihydroxyvitamin D3 by duodenal mucosal cells in the X-linked hypophosphatemic mouse

We have shown that there is a significant decrease in the nuclear uptake of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by duodenal mucosal cells in the X-linked hypophosphatemic (Hyp) mouse. Duodenal mucosal cells prepared from control and Hyp mice were incubated with 1,25(OH)2[26,27-methyl-3H]D3 ([3H]-1,25(OH)2D3) for 30 min. to evaluate the time-course and perform saturation analysis. The results of time-course studies showed that saturation was attained in 30 min., reaching an average nuclear uptake of 10.4 fmol/tube in the control mice and 6.1 fmol/tube in the Hyp mice. The results of Scatchard analyses were as follows: dissociation constant (Kd) 5.71 X 10(-10) M and maximal binding sites 7.31 X 10(4) sites/cell in the control mice, and Kd 2.92 X 10(-10) M and maximal binding sites 4.88 X 10(4) sites/cell in the Hyp mice, the maximal binding sites of the latter showed a significant decrease (P less than 0.05) by Student's t test. In addition, there was no significant difference in the binding of [3H]-1,25(OH)2D3 to its residual cytosol receptors between the control and Hyp mice. On the basis of these data, we speculate that the reported resistance of Hyp mice to vitamin D may be due to decreased nuclear uptake of 1,25(OH)2D3 by their duodenal mucosal cells.     

The effects of chronic prednisone administration on intestinal receptors for 1,25-dihydroxyvitamin D3 in the dog

Glucocorticoid inhibits intestinal calcium absorption. To further explore the mechanism of this inhibition, we studied dogs during the administration of oral prednisone (1.2-1.5 mg/kg X day) for 20 to 28 weeks in comparison to untreated dogs. Prednisone administration had no effect on serum 25-hydroxyvitamin D concentrations, but was accompanied by a fall in serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations from 87 +/- 20 pM (control) to 62 +/- 28 pM (prednisone-treated; P less than 0.01). Cytosol prepared from the duodenal, jejunal, and ileal mucosa of control dogs was found to contain a specific 3.2S [3H]1,25-(OH)2D3 binder analogous to the binder that has been observed in the intestine of other species and in other tissues. The apparent concentration of this binder decreased progressively from duodenum to ileum. Prednisone administration increased the apparent duodenal concentration of the binder from 170 +/- 91 (control) to 363 +/- 124 fmol/mg protein (prednisone-treated; P less than 0.025). The intestinal content of calcium-binding protein also declined progressively from the duodenum to the ileum, but was not affected by prednisone administration. These data suggest that events other than alterations in intestinal 1,25-(OH)2D3 receptors must mediate the inhibition of intestinal calcium absorption during chronic glucocorticoid administration.     

Cloning of a functional vitamin D receptor from the lamprey (Petromyzon marinus), an ancient vertebrate lacking a calcified skeleton and teeth

The nuclear vitamin D receptor (VDR) mediates the actions of its 1,25-dihydroxyvitamin D(3) ligand to control gene expression in terrestrial vertebrates. Prominent functions of VDR-regulated genes are to promote intestinal absorption of calcium and phosphate for bone mineralization and to potentiate the hair cycle in mammals. We report the cloning of VDR from Petromyzon marinus, an unexpected finding because lampreys lack mineralized tissues and hair. Lamprey VDR (lampVDR) clones were obtained via RT-PCR from larval protospleen tissue and skin and mouth of juveniles. LampVDR expressed in transfected mammalian COS-7 cells bound 1,25-dihydroxyvitamin D(3) with high affinity, and transactivated a reporter gene linked to a vitamin D-responsive element from the human CYP3A4 gene, which encodes a P450 enzyme involved in xenobiotic detoxification. In tests with other vitamin D responsive elements, such as that from the rat osteocalcin gene, lampVDR showed little or no activity. Phylogenetic comparisons with nuclear receptors from other vertebrates revealed that lampVDR is a basal member of the VDR grouping, also closely related to the pregnane X receptors and constitutive androstane receptors. We propose that, in this evolutionarily ancient vertebrate, VDR may function in part, like pregnane X receptors and constitutive androstane receptors, to induce P450 enzymes for xenobiotic detoxification.     

The role of 1,25-dihydroxyvitamin D in the inhibition of bone formation induced by skeletal unloading

Skeletal unloading results in osteopenia. To examine the involvement of vitamin D in this process, the rear limbs of growing rats were unloaded, and alterations in bone calcium and bone histology were related to changes in serum calcium (Ca), inorganic phosphorus, 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D [24,25-(OH)2D], and 1,25-dihydroxyvitamin D [1,25-(OH)2D]. Acute skeletal unloading induced a transitory inhibition of Ca accumulation in unloaded bones. This was accompanied by a transitory rise in serum Ca, a 21% decrease in longitudinal bone growth (P less than 0.01), a 32% decrease in bone surface lined with osteoblasts (P less than 0.05), no change in bone surface lined with osteoclasts, and a decrease in circulating 1,25-(OH)2D from 130 +/- 10 to 53 +/- 11 pg/ml. No significant changes in the serum concentrations of inorganic phosphorus, 25-hydroxyvitamin D, or 24,25-(OH)2D were observed. After 2 weeks of unloading, bone Ca stabilized at approximately 70% of control values, and serum Ca and 1,25-(OH)2D returned to control values. Maintenance of a constant serum 1,25-(OH)2D concentration by chronic infusion of 1,25-(OH)2D (Alza osmotic minipump) throughout the study period did not prevent the bone changes induced by acute unloading. These results suggest that acute skeletal unloading in the growing rat produces a transitory inhibition of bone formation, which, in turn, produces a transitory hypercalcemia, leading to a temporary decrease in serum 1,25-(OH)2D. No evidence could be found for a direct involvement of 1,25-(OH)2D in the bone changes induced by skeletal unloading.     

23(S),25(R)-1,25-dihydroxyvitamin D3-26,23-lactone stimulates murine bone formation in vivo

23(S),25(R)-1,25-Dihydroxyvitamin D3-26,23-lactone (1,25-lactone) has been shown to have unique actions different from those of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. In contrast to 1,25-(OH)2D3, 1,25-lactone causes a significant reduction in the serum Ca2+ level, stimulates collagen production in an osteoblastic cell line, and inhibits bone resorption induced by 1,25-(OH)2D3. A possible effect of 1,25-lactone on bone formation was examined in experiments on ectopic bone formation using a bone-inducing factor derived from Dunn osteosarcomas. 1,25-Lactone, a metabolite of 1,25-(OH)2D3, increased [3H]proline uptake at the stage of chondrogenesis and 85Sr uptake during bone formation. Significantly enlarged bone was also induced by this compound 3 weeks after implantation. These results suggest that the 1,25-lactone may be able to stimulate bone formation under in vivo conditions.     

Immunocytochemical localization of the 1,25-dihydroxyvitamin D3 receptor in target cells

We have used a monoclonal antibody (9A7) against the purified avian 1,25-dihydroxyvitamin D3 receptor to develop an immunocytochemical technique for visualization of the protein in fixed tissues and cultured cells. In Bouin's-fixed, chick intestine, 1,25-dihydroxyvitamin D3 receptor-like immunoreactivity was localized mainly in nuclei of epithelial cells and was more abundant in the crypt than in the villar cells. Receptor staining was low or undetectable in liver hepatocytes but was present in nuclei of cells lining the hepatic sinusoids. In rat brain, receptor-like immunoreactivity was abundant and widely distributed, but did not always coincide with the presence of vitamin D-dependent calcium-binding protein; 1,25-dihydroxyvitamin D3 receptor was absent from cerebellar Purkinje cells that contained abundant calcium-binding protein. In disaggregated rat bone cells, receptor immunoreactivity was present in mononuclear cells including osteoblasts and fibroblasts but was absent from osteoclasts. Two separate clones of osteoblast-like, rat osteosarcoma cells, shown in previous studies to be either receptor positive (17/2.8) or negative (24.1), demonstrated nuclear immunoreactivity in exact concordance with receptor levels as determined by ligand binding. The phenomenon of hormone-induced up-regulation of receptor was visualized in receptor-positive 3T6 fibroblasts by demonstration of markedly enhanced nuclear reactivity in cells treated with 10(-7) M 1,25-dihydroxyvitamin D3 for 48 h. Our studies demonstrate the feasibility of the immunocytochemical approach to visualize the 1,25-dihydroxyvitamin D3 receptor in target tissues and show that it is predominantly a nuclear protein in the relatively unoccupied and fully activated states. Moreover, the vitamin D-dependent calcium binding is not a universal marker for 1,25-dihydroxyvitamin D3 action. Rather, our observations suggest that the expression of the 1,25-dihydroxyvitamin D3 receptor may be connected with the state of cellular differentiation.     

Characterization of a receptor-like protein for 1,25-dihydroxyvitamin D3 in rat skin.

Isolated rat epidermis possesses a cytosolic 3.5 S receptor-like protein for 1,25-dihydroxyvitamin D3. This 3.5S binder has a high affinity (Kd = 1.4 X 10(-10) M) for 1,25-dihydroxyvitamin D3 and is present in low concentrations (31 fmol of binding sites per mg of cytosol protein). Analog competition for receptor binding revealed the following potency order: 1,25-dihydroxyvitamin D3 > 25-hydroxyvitamin D3 > 1 alpha-hydroxyvitamin D3 > 24 (R),25-dihydroxyvitamin D3 > vitamin D3. The receptor has a molecular weight of 60,000, has affinity for DNA-cellulose, and aggregates in the presence of low potassium concentrations. The 1,25-dihydroxyvitamin D3 binder is stabilized by sodium molybdate (10 mM). Addition of phenylmethylsulfonyl fluoride (3 mM) was found to yield more reproducible receptor preparations. The presence of a 1,25-dihydroxyvitamin D3 binding macromolecule with properties similar to those of the bone and intestinal receptor suggests that skin is an additional target organ for this hormone.     

Female sex hormone replacement therapy increases serum free 1,25-dihydroxyvitamin D3: a 1-year prospective study

OBJECTIVE: Currently, hormone replacement therapy is applied successfully to reduce post-menopausal bone resorption. However, the exact mechanism by which oestrogen exerts its effect has not yet been fully elucidated. In order to determine whether changes in the biologically active 1,25-dihydroxyvitamin D3 may be of importance in this process, the concentrations of both total and free 1,25-dihydroxyvitamin D3 in serum were assessed. DESIGN: In 36 post-menopausal women the effect of hormone replacement therapy, with a combination of 17 beta-oestradiol and norethisterone acetate, on the serum levels of total and free 1,25-dihydroxyvitamin D3 was studied after 0, 3, 6 and 12 cycles. MEASUREMENTS: The total concentration of 1,25-dihydroxyvitamin D3 in serum was assessed using a radioreceptor assay after diethylether extraction of the samples followed by paper chromatography. The free fraction of 1,25-dihydroxyvitamin D3 was measured using symmetric dialysis. The free 1,25 dihydroxyvitamin D3 concentration was calculated by multiplying the total concentration by the free fraction. RESULTS: During therapy, mean serum total 1,25-dihydroxyvitamin D3 concentrations (+/- SD) were 106.4 pmol/l (+/- 27.5), 155.0 pmol/l (+/- 49.5), 176.7 pmol/l (+/- 70.0) and 161.1 pmol/l (+/- 55.3) at 0, 3, 6 and 12 cycles, respectively. Serum free 1,25-dihydroxyvitamin D3 concentrations were 68 fmol/l (+/- 22), 107 fmol/l (+/- 35), 120 fmol/l (+/- 43) and 108 fmol/l (+/- 37), respectively. Baseline values of both total and free 1,25-dihydroxyvitamin D3 were significantly lower than those during therapy at all time (P < or = 0.001). CONCLUSION: Both the serum total 1,25-dihydroxyvitamin D3 and the serum free 1,25-dihydroxyvitamin D3 concentrations are increased during combined 17 beta-oestradiol and norethisterone acetate therapy for a year. Assuming that the free concentration 1,25-dihydroxyvitamin D3 reflects the biologically active fraction, this rise may in part explain the preventive effect of hormone replacement therapy on osteoporosis.     

Effect of oral 1,25-dihydroxyvitamin D and calcium on glucocorticoid-induced osteopenia in patients with rheumatic diseases

Twenty-three rheumatic disease patients with glucocorticoid-induced osteopenia (defined by measurement of forearm bone mass) completed an 18-month double-blind, randomized study to assess the effect of oral calcium and 1,25-dihydroxyvitamin D (1,25-OH2D) or calcium and placebo on bone and mineral metabolism. Intestinal 47Ca absorption was increased (P less than 0.05) and serum parathyroid hormone levels were suppressed (P less than 0.01) by 1,25-OH2D (mean dose 0.4 micrograms/day); however, no significant gain in forearm bone mass occurred, and bone fractures were frequent in both groups. In the 1,25-OH2D group, histomorphometric analysis of iliac crest biopsy specimens demonstrated a decrease in osteoclasts/mm2 of trabecular bone (P less than 0.05) and parameters of osteoblastic activity (P less than 0.05), indicating that 1,25-OH2D reduced both bone resorption and formation. We conclude that 1,25-OH2D should not be used for treatment of glucocorticoid-induced osteopenia. Since patients receiving calcium and placebo did not exhibit a loss of forearm bone mass, elemental calcium supplementation of 500 mg daily might be useful to maintain skeletal mass in patients receiving long-term glucocorticord therapy.     

A bioassay capable of measuring 1 picogram of 1,25-dihydroxyvitamin D3.

Fetal rat bones in organ culture constitute a sensitive system for assay of the vitamin D metabolite, 1 alpha,25-dihydroxyvitamin D3. Significant bone resorption is obtained with as little as 2 pg 1,25-dihydroxyvitamin D3 after 48 h of culture and with 1 pg after 64 h of culture. In the current study, organ cultures of fetal rat bone are used as a bioassay for 1,25-dihydroxyvitamin D i normal human plasma, which was prepared for assay by extraction with dichloromethane, chromatography on Sephadex LH-20, and purification on silicic acid by high pressure liquid chromatography. The concentration of 1 alpha,25-dihydroxyvitamin D3 in normal adult human plasma was 24.8 +/- 2.0 pg/ml (n = 19) by this assay.