Role of 1,25-Dihydroxyvitamin D3 in Maintaining Serum Phosphorus and Curing Rickets

The intravenous injection of a single dose of 650 pmoles of 1,25-dihydroxyvitamin D(3) to rats fed a vitamin D-deficient, low-phosphorus diet caused an elevation of serum phosphorus within 5 hours which reached a maximum in about 10-12 hours. This elevated serum phosphorus returned to deficiency levels 2-3 days later. On the other hand, a single injection of 650 pmoles of 25-hydroxyvitamin D(3) produced a significant rise at 12 hours, reached a maximum in 24-36 hours, and was maintained for at least 7 days. The single dose of 1,25-dihydroxyvitamin D(3) supported little calcification of bone, whereas the 25-hydroxyvitamin D(3) produced marked calcification. Six-hundred and fifty pmoles of 24,25-dihydroxyvitamin D(3) increased serum phosphorus only slightly and induced no calcification. When 1,25-dihydroxyvitamin D(3) was given each day, a sustained increase in serum phosphorus and marked bone calcification resulted. In contrast to the serum phosphorus responses, intestinal calcium transport remained high 5 days after administration of a single dose of 1,25-dihydroxyvitamin D(3). Serum calcium was not changed appreciably by any of the metabolites.Thyroparathyroidectomized rats or rats fed a diet extremely deficient in phosphate still exhibited a marked elevation of serum phosphorus in response to 1,25-dihydroxyvitamin D(3). The effect of 1,25-dihydroxyvitamin D(3) on serum phosphorus was greatly reduced in nephrectomized rats, suggesting that the serum phosphorus response to 1,25-dihydroxyvitamin D(3) may arise from an enhancement of phosphate reabsorption in the renal tubules.It is suggested that 1,25-dihydroxyvitamin D(3) cures rickets in rats by increasing the concentration of serum phosphorus rather than by increasing serum calcium concentration and calcium absorption.     

Serum calcium and vitamin D regulate 1,25-dihydroxyvitamin D3 receptor concentration in rat kidney in vivo.

The effects of vitamin D status, serum calcium, and serum phosphorus levels on 1,25-dihydroxyvitamin D receptor levels in kidney were investigated. Weanling rats were fed for 4 weeks on a diet with various levels of calcium and phosphorus with or without vitamin D. The 1,25-dihydroxyvitamin D3 receptor concentration in kidney was determined by an immunoradiometric assay. In the absence of vitamin D, total receptor concentration is increased 2-fold by an increase in serum calcium concentration. At normal serum calcium levels, the administration of vitamin D resulted in a 5-fold increase in receptor concentration. In hypocalcemic animals, however, vitamin D did not change receptor levels. Serum phosphorus levels could not be linked to any changes in 1,25-dihydroxyvitamin D3 receptor concentration. This study demonstrates that serum calcium levels and vitamin D regulate 1,25-dihydroxyvitamin D3 receptor concentration in vivo in kidney. On the other hand, vitamin D is unable to exert control of receptor levels in kidney under hypocalcemic conditions.     

Do tissues other than the kidney produce 1,25-dihydroxyvitamin D3 in vivo? A reexamination.

Recent experiments have shown that 1,25-dihydroxyvitamin D3-like material is produced in cultured nonrenal cells and may be present in the sera of anephric patients. We reexamined the question of whether 1,25-dihydroxyvitamin D3 can be synthesized extrarenally in the rat in vivo. To intact, sham-operated, ureter-ligated, or acutely nephrectomized vitamin D-deficient rats raised on a diet normal in calcium and phosphorus, we gave a physiologic dose of high-specific-activity 25-hydroxy-[3H]vitamin D3 (3.6-3.8 microCi; approximately equal to 25 pmol per rat). Twenty-four hours later we examined their tissues and plasma for the presence of radiolabeled 1,25-dihydroxyvitamin D3. Large amounts of radioactivity that behaved chromatographically as identical with authentic 1,25-dihydroxyvitamin D3 were present in the plasma, bone, and intestine of the intact, sham-operated, or ureter-ligated rats. However, no radioactivity eluting in a manner similar to 1,25-dihydroxyvitamin D3 was found in plasma, bone, or intestine of acutely nephrectomized rats. We conclude that, in the acutely nephrectomized living rat, 1,25-dihydroxyvitamin D3 is not present in plasma, bone, or intestine in quantities detectable by the sensitive techniques we have used. No conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 was observed during a 24-hr period after nephrectomy of vitamin D-deprived rats. This fact casts doubt upon the significance of the in vitro production of 1,25-dihydroxyvitamin D3 by nonrenal cells as an in vivo phenomenon.     

Vitamin D endocrinology.

Current status of our understanding of the metabolism of vitamin D and its implications in metabolic bone disease is reviewed. The details of metabolism of vitamin D3 to 25-hydroxyvitamin D3 in the liver and its further conversion in the kidneys to either 1,25-dihydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 are presented. The latter conversions are regulated by the vitamin D status, serum calcium through the parathyroid gland system, and serum inorganic phosphorus concentration. The 1,25-dihydroxyvitamin D3 can now be regarded as a calcium- and a phosphate-mobilizing hormone and must be considered as one of the most important serum calcium-regulating hormones. Disruption of the vitamin D metabolic sequence or the signal system for 1,25-dihydroxyvitamin D3 results in several bone and calcium metabolism disorders such as renal osteodystrophy, hypoparathyroidism, pseudohypoparathyroidism, and vitamin D-dependency rickets. The use of the synthetic analogs of 1,25-dihydroxyvitamin D3 as well as 1,25-dihydroxyvitamin D3 itself in the management of these disease states is discussed.     

Tumor-induced osteomalacia. Kinetics of calcium, phosphorus, and vitamin D metabolism and characteristics of bone histomorphometry

A patient with a mesenchymal tumor and hypophosphatemic osteomalacia was studied before and after tumor excision. Initial laboratory values included normal serum calcium, decreased serum phosphorus and tubular reabsorption of phosphate, undetectable 1,25-dihydroxyvitamin D, and normal parathyroid hormone. Histomorphometry of a bone biopsy specimen showed evidence of increased osteoclastic bone resorption. By 16 hours after tumor removal, 1,25-dihydroxyvitamin D level had normalized, but serum phosphorus level was unchanged; at 28 hours, both serum phosphorus value and tubular reabsorption of phosphate were within normal limits. It is concluded that tumor removal is associated with rapid correction both of 1,25-dihydroxyvitamin D production and of renal phosphate wasting. Increased bone resorption suggests the production of an osteoclast activator by the tumor and may explain the typically normal serum calcium value in this disorder.     

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.     

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.     

Dietary phosphorus deprivation induces 25-hydroxyvitamin D(3) 1alpha-hydroxylase gene expression

Dietary phosphorus deprivation causes hypophosphatemia and an increase in serum 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] concentrations. To determine the molecular mechanisms of this regulation, the effects of dietary phosphorus deprivation and hypophysectomy on 25-hydroxyvitamin D(3) 1alpha-hydroxylase (1alpha-hydroxylase) protein and messenger RNA (mRNA) expression were examined in rats. A low phosphorus diet (LPD) for 4 days resulted in hypophosphatemia and an increase in serum 1,25-(OH)(2)D(3) levels. This increase was caused by the induction of 1alpha-hydroxylase protein and mRNA expression (4- and 10-fold increases, respectively). Administration of the LPD or normal phosphorus diet to hypophysectomized (HPX) rats resulted in hypophosphatemia and suppression of 1alpha-hydroxylase gene expression, indicating that hypophosphatemia itself is not sufficient to induce 1alpha-hydroxylase mRNA expression. Administration of GH to HPX rats fed LPD could partially restore 1alpha-hydroxylase mRNA expression, whereas supplementation with insulin-like growth factor I, T(3), estrogen, or corticosterone had no effect. We also examined Phex gene expression in the bone, because the clinical features of X-linked hypophosphatemia resemble those of HPX rats. Phex mRNA expression, however, was not altered in HPX rats. In conclusion, we demonstrated that the increase in serum 1,25-(OH)(2)D(3) levels caused by dietary phosphorus deprivation is due to the induction of 1alpha-hydroxylase mRNA expression, and this increase is mediated in part by a GH-dependent mechanism.     

Skeletal resistance to 1,25-dihydroxyvitamin D3 in osteopetrotic rats

The osteopetrotic (op/op) rat mutation is a lethal mutation in which decreased osteoclast function (bone resorption) coexists with markedly elevated serum levels of 1 ,25-dihydroxyvitamin D3[1,25(OH)2D3]. Increased circulating levels of 1,25(OH)2D3 have been reported in other osteopetrotic animal mutations and in some osteopetrotic children. This study examined the effects of 1,25(OH)2D3 infusions on serum and skeletal parameters in normal and mutant rats of op stock. We also examined vitamin D receptor expression and binding in bone cells from op normal and mutant animals. Four-week-old normal and mutant rats were infused either with propylene glycol (used as controls) or with 12.5-125 ng of 1,25(OH)2D3/d using osmotic minipumps implanted subcutaneously for 1 wk. Sera were analyzed for calcium, phosphorus, and 1,25(OH)2D3 levels. Histomorphometric analyses of proximal tibiae from treated normal (50 ng/d) and op mutant (125 ng/d) rats and their vehicle-infused controls were performed. Normal animals infused with 1,25(OH)2D3 exhibited a dose-dependent increase in serum calcium levels. Histomorphometric analyses of metaphyseal bone within the primary spongiosae region showed that 1,25(OH)2D3 increased osteoclast number with a reduction in osteoblast surface associated with a decrease in growth plate cartilage thickness. However, similar analyses on secondary spongiosae showed a decrease in osteoclast number and surface associated with an anabolic response. Op mutants infused with 1,25(OH)2D3 did not exhibit any change in serum calcium levels or histomorphometric parameters related to growth plate cartilage and metaphyseal bone compared with mutant controls. Vitamin D mRNA and protein levels were increased twoto threefold in op mutants compared to age-matched normal rats. However, binding affinity of 1,25(OH)2D3 to its receptor was similar between op mutant and normal animals. High dose calcitriol therapy, under the conditions and period of treatment used in this study, failed to stimulate bone turnover in op rats, suggesting that they are resistant to the skeletal effects of 1,25(OH)2D3. The failure of osteoclast activation in response to 1,25(OH)2D3 treatment may be associated with osteoblast incompetence in this mutation.     

Dietary phosphorus and 1,25-dihydroxyvitamin D metabolism: influence of insulin-like growth factor I

Hypophysectomy abolishes the increase in serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] induced by restriction of dietary phosphorus. Administration of GH increases circulating insulin-like growth factor I levels (IGF-I) and restores, in part, the responsiveness of serum 1,25-(OH)2D to restriction of dietary phosphorus. To determine whether the GH-dependent increase in serum 1,25-(OH)2D induced by restriction of dietary phosphorus is mediated by IGF-I, we measured the serum concentration of 1,25-(OH)2D in hypophysectomized rats treated with either GH (100 micrograms/day) or recombinant human IGF-I (150 micrograms/day) and fed either a normal or low phosphorus diet for 6 days. Restriction of dietary phosphorus in sham-hypophysectomized rats increased serum 1,25-(OH)2D from 97 +/- 13 to 251 +/- 36 pg/ml, or 159%, but had no effect on serum 1,25-(OH)2D in hypophysectomized rats. Restriction of dietary phosphorus in rats receiving GH increased, (P less than 0.001) serum 1,25-(OH)2D from 52 +/- 8 to 133 +/- 18 pg/ml, or 156%. Restriction of dietary phosphorus in rats receiving IGF-I increased (P less than 0.001) serum 1,25-(OH)2D from 33 +/- 5 to 94 +/- 11 pg/ml, or 185%, an increase equivalent to that observed in animals receiving GH. For a given diet, no significant differences were seen between the serum concentrations of 1,25-(OH)2D in animals receiving GH or IGF-I. These data indicate that IGF-I can restore the increase in serum 1,25-(OH)2D induced by restriction of dietary phosphorus to the same degree as GH. This strongly suggests that the GH-dependent increase in serum 1,25-(OH)2D induced by restriction of dietary phosphorus is mediated by IGF-I.     

Role of insulin in the increase in serum 1,25-dihydroxyvitamin D concentrations in response to phosphorus deprivation in streptozotocin-induced diabetic rats

To evaluate the role of insulin in the regulation of circulating 1,25-dihydroxyvitamin D [1,25(OH)2D] levels, serum 1,25(OH)2D concentrations in response to phosphorus (P) deprivation were examined in control, streptozotocin-diabetic and insulin-treated diabetic rats. Dietary P deprivation for 1 week caused a marked increase in serum 1,25(OH)2D level from 75 +/- 4 pg/ml to 274 +/- 16 pg/ml in control rats. In contrast, serum 1,25(OH)2D level was significantly lower in diabetic rats on a normal P diet (20 +/- 2 pg/ml) compared to that in control rats and increased only slightly by P deprivation (33 +/- 4 pg/ml). Treatment of the diabetic rats on normal P diet with insulin caused an increase in serum 1,25(OH)2D concentration to a level (82 +/- 10 pg/ml) similar to that in control rats and restored the increase in serum 1,25(OH)2D concentration in response to P deprivation (315 +/- 38 pg/ml). Although there was a marked decrease in serum phosphate level by P deprivation in all groups of animals, the rise in serum calcium level by P deprivation seen in control rats was abolished in diabetic rats. In addition, while bone mineral contents decreased significantly in response to P deprivation in control rats, no significant changes in either bone calcium or P contents were observed after P deprivation in diabetic rats. Insulin treatment of the diabetic rats recovered the responsiveness to P deprivation in both serum calcium level and bone mineral contents. P deprivation did not affect plasma glucose or serum creatinine level in any group of rats. These results suggest that insulin, either directly or indirectly, is required for the increase in circulating 1,25(OH)2D concentrations in response to P deprivation, and that the rise in serum 1,25(OH)2D level may play a role in the hypercalcemic response to P deprivation.     

Evidence that somatomedins mediate the effect of hypophosphatemia to increase serum 1,25-dihydroxyvitamin D3 levels in rats

The present studies were undertaken in an effort to determine whether somatomedins (SMs) play a role in the elevation of serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels during dietary phosphate deprivation. Serum 1,25-(OH)2D3,SM-C, and phosphate levels were measured in rats fed diets containing adequate or very low levels of dietary phosphorus under circumstances known to affect SM levels, including hypophysectomy with and without GH replacement, normal protein vs. low protein diets, and streptozotocin-induced diabetes with and without insulin replacement. In all circumstances, serum 1,25-(OH)2D3 concentrations were directly related to serum SM-C levels. However, the slope for the relationship was increased 2- to 10-fold in animals fed the low phosphorus diets. As observed previously, serum 1,25-(OH)2D3 levels were inversely related to serum phosphate levels, but the slope for this relationship was deceased in the presence of low SM levels and absent in animals with very low SM levels. These results suggest that SM are required for elevation of serum 1,25-(OH)2D3 levels in response to phosphate deprivation.     

Production of 1,25-dihydroxyvitamin D3 and formation of medullary bone in the egg-laying hen.

Renal 25-hydroxyvitamin D-1-hydroxylase, the percentage of medullary bone in the femur, plasma calcium, and plasma phosphorus were measured in female chickens reaching maturity. These parameters and plasma 1,25-dihydroxyvitamin D levels were also measured during the daily egg-laying cycle in mature hens. The renal 25-hydroxyvitamin D3-l-hydroxylase becomes elevated in maturing hens before and at the time of ovulation. This elevation in the 1-hydroxylase correlates with the elevation in total plasma calcium concentration but lags and at the time of ovulation and is followed by a further elevation of plasma 1,25-dihydroxyvitamin D levels. The plasma 1,25-dihydroxyvitamin D level remains high until 12 h postovulation. At this time, it falls to the preovulation level. No relationship could be found between the plasma 1,25-dihydroxyvitamin D levels and the changes in medullary bone found during the egg-laying cycle. However, plasma 1,25-dihydroxyvitamin D levels are highest immediately before and during the egg shell calcification phase of the egg-laying cycle.     

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.     

Effects of high doses of vitamin D3 and 1,25-dihydroxyvitamin D3 in lactating rats on milk composition and calcium homeostasis of the suckling pups

Changes in serum Ca and phosphorus and in kidney Ca were determined in lactating rats and their suckling pups after the mothers received high doses of vitamin D3 or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. High dietary vitamin D3 intake (300 IU/g diet) or daily oral doses of vitamin D3 (1 microgram/g BW) to vitamin I)-replete (+D) lactating rats for 8 or 12 days caused significant increases in serum Ca in the mothers (1-2 mg/dl) and in their suckling pups (1.5 mg/dl). Daily oral doses of 1,25-(OH)2D3 (2 ng/g BW) to +D lactating rats caused a similar increase in serum Ca in the mothers, but did not affect the serum Ca of the pups. The administration of a high dose of 1,25-(OH)2D3 to vitamin D-deficient lactating rats or high doses of vitamin D3 to +D rats, caused no change in milk Ca, Mg, or phosphorus. Milk from +D rats given high doses of [3H]vitamin D3 (1 microgram/g BW) contained mostly [3H]vitamin D3 (85%) and a small amount of [3H]25-hydroxyvitamin D3 (6%). The results indicate that high doses of vitamin D3, but not 1,25-(OH)2D3, given to +D lactating rats can cause hypercalcemia in the suckling pups. The hypercalcemic effect on the pups observed after vitamin D3 treatment of the mother is probably a result of transport of toxic amounts of primarily vitamin D3 into the milk and is not due to altered mineral composition of the milk.     

The effects of short-term, high-dose treatment with prednisone on the nuclear uptake of 1,25-dihydroxyvitamin D3 in monocytes from normal human subjects

Animal and cell culture studies indicate glucocorticoid regulation of 1,25-dihydroxyvitamin D3 receptors and interference with cellular effects of vitamin D. These investigations prompted us to examine the effects of prednisone on the nuclear uptake of 1,25-dihydroxyvitamin D3 in freshly isolated human monocytes. Eighteen normal subjects were studied in a randomized, double-blind, placebo-controlled study. Analysis of receptor kinetics revealed that the maximal nuclear uptake (Bmax) of (3H)-1,25-dihydroxyvitamin D3 in monocytes decreased 40% (P less than .001) after prednisone treatment. In the group treated with prednisone (40 mg/d for 5 days) s-1,25-dihydroxyvitamin D increased 46% (P less than .01). S-25-Hydroxyvitamin D, S-phosphat, S-calcium, and S-iPTH remained unchanged. Osteoblastic production of the matrix protein, bone gla protein (BGP) is stimulated by 1,25-dihydroxyvitamin D3. However, despite increased serum levels of 1,25-dihydroxyvitamin D3, the prednisone-treated group revealed a 75% reduction in s-BGP. The present data indicate that corticosteroids decrease the nuclear uptake of 1,25-dihydroxyvitamin D3 in human monocytes. Further investigations are necessary, however, to elucidate the biologic mechanism for this observation and whether the mechanism is operative in other human tissues including bone.     

Vitamin D deficiency causes a selective reduction in deposition of transforming growth factor beta in rat bone: possible mechanism for impaired osteoinduction.

We demonstrated previously that implants of bone matrix prepared from vitamin D-deficient (-D) rats were less osteoinductive and contained less extractable mitogenic activity compared with control implants prepared from vitamin D-replete (+D) rats and proposed that bone from -D rats is deficient in one or more specific growth factors. To test this hypothesis, bones from rats that were fed either +D or -D diets and kept in the dark for 8 wk were extracted and assayed for insulin-like growth factors I and II (IGF-I and IGF-II) and transforming growth factor beta (TGF-beta), the three most abundant growth factors in rat bone, and osteocalcin. Serum calcium, 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were determined at sacrifice. In -D rats, there were significant reductions in serum calcium, 25-hydroxyvitamin D3, and 1,25(OH)2D3 and skeletal TGF-beta but no differences in extractable skeletal protein, IGF-I, IGF-II, or osteocalcin compared with +D rats. To determine whether 1,25(OH)2D3 increased TGF-beta production by bone cells, we treated mouse calvaria for 6 days and mouse osteoblasts for 2 days with 10 nM 1,25(OH)2D3. Production of TGF-beta was increased almost 100% by 1,25(OH)2D3. We conclude that vitamin D deficiency reduces deposition of TGF-beta in rat bone and that diminished skeletal TGF-beta could contribute to the previously observed decrease in osteoinduction in implants from -D rat bone. The findings support the possibility that vitamin D and bone-derived TGF-beta are required for normal repair of the skeleton.     

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.     

Serum levels of vitamin D metabolites and bone remodelling in hyperthyroidism

Serum levels of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D were measured in 25 untreated hyperthyroid patients in whom histomorphometric evaluations of iliac crest bone biopsies were performed after in vivo tetracycline doublelabeling. The serum concentration of 25-hydroxyvitamin D was normal. The serum concentration of 1,25-dihydroxyvitamin D was reduced (p < 0.02) compared to normal whereas the serum concentration of 24,25-dihydroxyvitamin D was increased (p < 0.02). The bone changes were characterized by an enhanced turn-over in trabecular and cortical bone leading to an increased porosity of cortical bone and mobilisation of bone mineral. The observed changes in vitamin D metabolism could be explained by a reduced renal 1-alpha-hydroxylase activity secondary to hypercalcaemia with suppressed parathyroid secretion and hyperphosphataemia. The bone changes were unrelated to the serum levels of vitamin D metabolites. In trabecular bone the appositional rate and mineralization rate of osteoid were increased and the mineralization lag time was shortened showing that the mineralization and formation of osteoid in the hyperthyroid state can progress with an enhanced rate in spite of a reduced mean serum level of the active vitamin D metabolite, 1,25-dihydroxyvitamin D.     

Effects of parathyroidectomy on circulating levels of 1 alpha,25-dihydroxyvitamin D and bone Gla protein in dialyzed patients

The effects of decreasing serum levels of PTH after parathyroidectomy on circulating levels of 1 alpha,25-dihydroxyvitamin D [1,25-(OH)2D] and bone Gla protein (BGP) were studied in seven patients treated with chronic maintenance dialysis. Before surgery, all patients had extremely elevated levels of PTH, low normal or low levels of 1,25-(OH)2D, high levels of BGP, and histological signs of excess PTH action on bone. The fall in PTH levels after surgery resulted in a further decline in 1,25-(OH)2D concentrations and a reduction in circulating BGP levels. This fall in serum 1,25-(OH)2D and BGP levels was not related to serum calcium or phosphorus. Administration of 1,25-(OH)2D3 from 4-6 months after surgery did not significantly affect serum levels of BGP or PTH. These data indicate that 1,25-(OH)2D is still under regulatory control of PTH in patients without excretory kidney function. This might reflect some remaining endocrine activity of the kidneys in these dialyzed patients or extrarenal production of 1,25-(OH)2D. In addition, the data show that serum BGP levels in renal failure are elevated due not only to impaired clearance but also to PTH-mediated acceleration in bone turnover. Therapy with 1,25-(OH)2D3 in these patients resulted in supraphysiological serum 1,25-(OH)2D levels which did not stimulate BGP production.