The effect of hemodialysis, vitamin D metabolites and renal transplantation on the skeletal demineralization associated with renal osteodystrophy: a computerized histomorphometric analysis

Twenty-three patients with end-stage renal failure treated by hemodialysis or transplantation were followed for up to 10 years. Sequential full thickness iliac crest bone biopsies were obtained to assess the effects on bone disease of hemodialysis, treatment with 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25-(OH)2D3] and renal transplantation. The biopsies were analyzed by a computerized histomorphometric technique which allowed accurate measurements of calcified bone and osteoid areas. Serum aluminum and parathyroid hormone concentrations were also monitored. Hemodialysis was associated with a loss of calcified bone and an increase in osteoid areas. The progressive bone loss was arrested but not reversed following treatment with either 1,25-(OH)2D3 or 24,25-(OH)2D3. Osteoid area was unchanged or reduced following treatment with 1,25-(OH)2D3 in all but three patients who had serum aluminum concentrations in excess of 5 mumol/l. 24,25-(OH)2D3 was not effective in reducing osteoid area, and combined treatment with 1,25 and 24,25-(OH)2D3 had no effect beyond that expected with 1,25-(OH)2D3 alone. Bone biopsies showed loss of calcified bone and an increase in osteoid areas one year and more after successful renal transplantation in five patients. Nineteen of the 23 patients developed serum aluminum concentrations greater than 3 mumol/l, probably because of the use of oral aluminum hydroxide as a phosphate binding agent. In these patients serum parathyroid hormone concentrations greater than 600 pg/ml appeared to prevent the development of osteopenia.     

Histogenesis of hyperosteoidosis in 1,25(OH)2D3-treated rats fed high levels of dietary calcium

The purpose of this investigation was to determine by sequential quantitative morphometry the histogenesis of metaphyseal changes induced in rats fed high levels of dietary calcium and treated with pharmacologic doses of 1,25(OH)2D3. Young adult female rats were placed on a diet containing 2.5% calcium and 0.3% phosphorus and administered either ethanol or 135 ng (5 units) 1,25(OH)2D3 in ethanol IP daily for 10 days. Rats were terminated at Days 1, 2, 3, 4, 6, 8, and 10. At Day 1 the proximal tibias from rats treated with 1,25(OH)2D3 had a dramatic increase in osteoclasts/mm total trabecular surface perimeter compared with placebo-treated rats. Osteoclast numbers decreased in 1,25(OH)2D3-treated rats to the levels in placebo-treated rats by Days 3 and 4 and decreased significantly below placebo-treated levels at Days 6, 8, and 10. Active resorbing surface was significantly increased at Days 1 and 2 and decreased at Days 8 and 10 in 1,25(OH)2D3-treated rats compared with placebo-treated rats. From Day 4 through Day 10 in 1,25(OH)2D3-treated rats, there was a progressive increase in osteoblasts/mm total trabecular surface perimeter, osteoid surface, active osteoid surface, and metaphyseal osteoid. Metaphyseal osteoid increased markedly at Days 8 and 10 in 1,25(OH)2D3-treated rats and caused a significant increase in the amount of osseous tissue in the metaphysis. Metaphyseal mineralized bone, however, was not consistently affected by 1,25(OH)2D3 treatment. Serum calcium and phosphorus were elevated in 1,25(OH)2D3-treated rats at more time periods. In rats fed high levels of dietary calcium, repeated supraphysiologic doses of 1,25(OH)2D3 result in a net increase in metaphyseal osseous tissue, predominantly osteoid.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of uremia and dietary phosphorus on the bone of rats

In order to evaluate the effects of uremia and low levels of dietary phosphorus on bone, male Sprague-Dawley rats weighing 320 +/- 20 g (12 weeks old) were subjected to either a two-step, subtotal nephrectomy or sham-operation (SO), and then fed a custom diet with either normal calcium (0.5%) and normal phosphorus (0.3%) (NCNP), or normal calcium and low phosphorus (0.03%). When compared to the NCNP SO group after seven days, only uremic rats fed low phosphorus diets developed osteomalacia characterized by an increase in the osteoid thickness, surface and volume, a prolonged osteoid maturation time, and a decreased bone formation rate. No other groups developed these changes. This osteomalacia was also associated with hypophosphatemia, a reduced serum PTH and an elevation in the serum 1,25(OH)2D3. It was concluded that while neither this degree of uremia nor the low phosphorus diets alone had any significant effect, the combination of uremia and low dietary phosphorus resulted in the initiation of osteomalacia. This animal model should prove useful in investigations dealing with the influence of uremia on the mineralization process.     

Bone response to phosphate and vitamin D metabolites in the hypophosphatemic male mouse

The hypophosphatemic male mouse (Hyp/y), the proposed model for human vitamin D-resistant rickets (VDRR), is characterized by chronic hypophosphatemia, dwarfism, and rachitic and osteomalacic bone lesions. We have reported that treatment of Hyp/y mice with phosphate salts (Pi) heals rickets but does not correct the defective endosteal bone mineralization. In an attempt to cure osteomalacia, mutant male animals were treated with Pi combined with 25-hydroxyvitamin D3 (25OHD3, 1 microgram/kg/day), 24,25-dihydroxyvitamin D3 [24,25(OH)2D3, 0.5 microgram/kg/day], or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3, 0.05--0.25 microgram/kg/day] infused constantly for 3 weeks. The biochemical and skeletal effects of treatment were assessed by analytical methods and bone histomorphometry. The results show that only 1,25(OH)2D3 produced a dose-dependent elevation of serum calcium and phosphorus, and greatly improved bone mineralization at doses high enough to increase serum calcium and phosphorus concentrations within or above the normal range. Better improvement of bone mineralization was obtained when Pi was combined to 1,25(OH)2D3. In conjunction with the correction of hypocalcemia, Pi + 1,25(OH)2D3 suppressed the stimulation of bone turnover induced by Pi supplementation. The results show that, as in VDRR children, 1,25(OH)2D3 produces beneficial effects on bone lesions in Hyp/y mice, mainly through enhancement of mineral availability. However, the persistence of osteomalacia despite correction of serum mineral concentrations suggests that there is a specific bone cell resistance to mineral and/or hormonal influences in Hyp/y mice.     

Extrarenal metabolism of 25-hydroxycholecalciferol in the rat: regulation by 1,25-dihydroxycholecalciferol

To determine the role of the kidney in regulation of 25-hydroxycholecalciferol (25OHD3, metabolism, the effects of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] on 3H-25OHD3 were compared in intact and nephrectomized vitamin D-deficient rats. Sixteen hours after the intravenous administration of 3H-25OHD3, extracts of serum and pooled small intestinal mucosa were fractionated by Sephadex LH-20 column chromatography followed by high performance liquid chromatography. In intact rats, 1,25(OH)2D3 (50 ng/day i.p. for 7 days) increased mean serum 3H-24,25-dihydroxycholecalciferol [3H-24,25(OH)2D3] from 2 +/- 2-210 +/- 80 fmol/ml (mean +/- 1 SD), increased mean serum 3H-25,26-dihydroxycholecalciferol [3H-25,26(OH)2D3] from 2 +/- 2-12 +/- 6 fmol/ml and lowered mean serum 3H-1,25(OH)2D3 from 210 +/- 40-4 +/- 4 fmol/ml. Similarly, in nephrectomized animals, 1,25(OH)2D3 increased mean serum 3H-24,25-(OH)2D3 from 6 +/- 11-115 +/- 30 fmol/ml and increased mean serum 3H-25,26(OH)2D3 from 3 +/- 3-26 +/- 10 fmol/ml. Nephrectomy increased serum 3H-25(OH)D3 in untreated (from 1450 +/- 225-2675 +/- 225 fmol/ml serum) and 1,25(OH)2D3 treated rats (from 1600 +/- 175-3075 +/- 100 fmol/ml). 3H-1,25(OH)2D3 averaged 74 +/- 16% of total radioactivity in intestinal mucosa of untreated intact rats and was not detected in either the serum or intestinal mucosa of nephrectomized animals. The results suggest that in intact animals, extrarenal synthesis can account for substantial 24,25(OH)2D3 production and for most 25,26(OH)2D3 production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of 1 alpha,25- and 24R,25-dihydroxyvitamin D3 on aluminum-induced rickets in growing uremic rats

Rats were subjected to a two-stage subtotal nephrectomy or sham operation, and treated with aluminum (Al) or both aluminum and vitamin D3 metabolites for 5 weeks with a cumulative dose of 13.6 mg aluminum. Animals were injected with 3H-thymidine and 3H-proline. The following analyses were performed: quantitative histology of tibial metaphyses and cytomorphometric electron microscopy of osteoclasts, quantitative (ICP-spectroscopy) and qualitative determination (histochemical staining) of aluminum within organs, and serum biochemistry (Ca, P, Mg, vitamin D3 metabolites, alkaline phosphatase, urea). The following new facts of the aluminum-related bone disease became evident: (a) Application of aluminum to growing uremic rats induced rickets, whose major epiphyseal growth plate changes were 1 alpha,25(OH)2D3-dependent. Addition of 1 alpha,25(OH)2D3 prevented the formation of rachitic metaphysis, but failed to prevent osteoid accumulation on epiphyseal and metaphyseal trabecular surfaces. Moreover, calcitriol produced hyperosteoidosis and osteosclerosis in the same rats. Aluminum did not alter the function of osteoblasts, while osteoclasts seemed inactivated. (b) The development of rickets was associated with suppressed serum levels of 1,25(OH)2D3, reduced phosphorus level and the high content of aluminum in the bone, kidney, and liver. The addition of 24R,25(OH)2D3 markedly exaggerated the reduction of serum levels of calcitriol. We suggested that aluminum induces rickets in growing uremic rats, which consists of two components: vitamin D refractory osteomalacia and 1 alpha,25(OH)2D3-dependent epiphyseal growth plate changes.     

Serum calcium regulating hormones in the perinatal period

To clarify perinatal vitamin D metabolism, we measured 25-hydroxyvitamin D (25OHD), 24,25-dihydroxyvitamin D [24,25(OH)2D], 1,25-dihydroxyvitamin D [1,25(OH)2D], calcium (Ca), phosphorus (P), parathyroid hormone (PTH), and human calcitonin (CT) in paired maternal, cord, and infant serum. Cord serum 25OHD was significantly lower than the maternal level, and cord serum 24,25(OH)2D was also significantly below the maternal concentrations. Maternal, cord, and infant serum 1,25(OH)2D, on the other hand, was significantly higher than the normal adult level. The serum PTH was low, but the CT concentration was high in the cord. Cord serum Ca and P levels were significantly higher than maternal. The reason for the elevated circulating 1,25(OH)2D level in the perinatal period is uncertain, and we speculate that the possible factors are gonadal steroids, placental lactogen, prolactin, and CT. In addition, serum 24,25(OH)2D and 1,25(OH)2D concentrations are under some control by the fetus.     

Ultrastructural development of hyperosteoidosis in 1,25(OH)2D3-treated rats fed high levels of dietary calcium

To evaluate the sequential ultrastructural pathogenesis of the increase in osseous tissue and hyperosteoidosis previously demonstrated in rats administered supraphysiologic doses of 1,25(OH)2D3 and fed high levels of dietary calcium, young adult female rats were placed on a 2.5% calcium and 0.3% phosphorus diet, administered ethanol or 135 ng (5 units) 1,25(OH)2D3 IP daily, and killed after 2, 4, 6, 8, and 10 days. Metaphyseal trabeculae from 1,25(OH)2D3 and placebo-treated rats were examined. Osteoblast hypertrophy characterized by increased cytoplasmic area, abundant rough endoplasmic reticulum, and prominent Golgi apparatus was evident in 1,25(OH)2D3-treated rats at Day 4. These osteoblasts were interpreted to be active in matrix synthesis. Widened osteoid seams were present at Day 6. Osteoblast hypertrophy and widened osteoid seams persisted through Day 10 in 1,25(OH)2D3-treated rats. The unmineralized bone matrix in 1,25(OH)2D3-treated rats contained more numerous cytoplasmic processes from adjacent osteoblasts than did control animals and loosely arranged collagen fibrils, which failed to aggregate in regions adjacent to the osteoid-mineralized bone interface as in placebo-treated rats. Osteoid seams in 1,25(OH)2D3-treated rats contained irregular electron-dense foci, which were often concentrated around embedded cytoplasmic processes. Osteocytic hypertrophy characterized by increased cytoplasmic area, developed rough endoplasmic reticulum, and increased numbers of mitochondria was evident at Day 2 and was sustained through Day 10 in 1,25(OH)2D3-treated rats. Variable-sized aggregates of electron-dense deposits similar to those concentrated around osteoblast cytoplasmic processes were observed in the pericellular space and on and immediately adjacent to the plasma membranes of osteocytes and embedding osteoblasts in 1,25(OH)2D3-treated rats as early as Day 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in vitamin D metabolites and bone histology in rats during recovery from rickets

Summary The relative roles of 25-hydroxyvitamin D (25-OHD), 1,25-dihydroxyvitamin D (1,25-(OH)₂D) and 24,25-dihydroxyvitamin D (24,25-(OH)₂D) in bone mineralization are largely unknown. Young vitamin D depleted rats were fed increasing amounts of vitamin D and grouped radiologically in accordance with the rat line test. They ranged from severely rachitic to normal. Radiology was correlated with serum levels of 25-OHD, 1,25-(OH)₂D, 24,25-(OH)₂D, ionized calcium, magnesium, and phosphate, with bone histology, and with the total mineral content of the animals. Serum 1,25-(OH)₂D rose in a linear fashion to supranormal values during bone healing and correlated with the radiological degree of rickets. Serum 25-OHD was below detection limit in the most rachitic and low in the radiologicall normal rats, whereas 24,25-(OH)₂D was low in all groups. These two metabolites showed no correlation with the radiologic, histologic or biochemical parameters. In rachitic rats, 1,25-(OH)₂D appears to play a major role in bone healing and possibly exerts a direct effect on bone cells. It cannot be ruled out, however, that the effect is mediated through a rise in serum levels of calcium and phosphorus, although signs of bone healing were seen in the presence of a subnormal calcium x phosphorus product. Initiation of mineralization can take place with unmeasurable 25-OHD, and 24,25-(OH)₂D seems to be without importance.     

24,25-dihydroxyvitamin D3 in combination with 1,25-dihydroxyvitamin D3 ameliorates renal osteodystrophy in rats with chronic renal failure

AIMS: This study compares the effects of 1,25(OH)2D3 and 24,25(OH)2D3 alone or in combination on renal osteodystrophy in rats with chronic renal failure (CRF). MATERIAL AND METHOD: One month subsequent to 5/6 nephrectomy animals were divided into four groups and treated for one or four weeks with either vehicle, 1,25(OH)2D3, 24,25(OH)2D3 or 1,25(OH)2D3 + 24,25(OH)2D3. A sham-operated group with normal renal function matched for age and weight was used as control. At the termination of the study blood chemistry, parathyroid hormone (PTH) level and bone histomorphometry were analyzed. RESULTS: The main findings were: amelioration of 1,25(OH)2D3-induced hypercalcemia by 24,25(OH)2D3, and similar suppression of PTH by the two metabolites of vitamin D when administered alone or in combination. Bone histomorphometry showed that 1,25(OH)2D3 alone exerts a potent proliferative effect on the osteoblasts but severely depresses their mineralizing capacity in a dose- and time-dependent manner. By contrast, 24,25(OH)2D3 improved the mineralizing activity with only a limited effect on osteoblast proliferation. Addition of 24,25(OH)2D3 potentiated the beneficial effect of 1,25(OH)2D3 on bone-resorbing parameters and corrected the mineralization failure. CONCLUSIONS: Based on the above observations we suggest that the combined treatment with 1,25(OH)2D3 and 24,25(OH)2D3 markedly improves the morphologic and metabolic abnormalities of renal osteodystrophy.     

Action of vitamin D metabolites on PTH secretion in man

We have examined the effects of metabolites of vitamin D [25OHD3, 1,25(OH)2D3, 24,25(OH)2D3, and 25,26(OH)2D3] on serum calcium and iPTH in human deficient-D osteomalacia. The four metabolites decreased iPTH, but only for 1,25(OH)2D3 was a significant correlation between increase of serum calcium and decrease of iPTH observed. The 24,25(OH)2D3 and 25,26(OH)2D3 decreased iPTH despite a decrease of serum calcium at the beginning of treatment. The 25OHD decreased iPTH before increased serum calcium. These results could be interpreted as a direct effect of metabolites of vitamin D on PTH secretion. However, the conversion of other metabolites and the calcium concentration in parathyroid cells must be determined before this hypothesis can be accepted.     

Relative effectiveness of vitamin D metabolites in increasing bone mineral solubility

Weanling rats were given a vitamin D-deficient diet containing 1.4% calcium and 1.0% phosphorus. After 4 weeks these deficient animals were injected for 7 days with selected doses of one of the following vitamin D metabolites: 25(OH)D3, 1,25(OH)2D3, 24,25(OH)2D3, 25,26(OH)2D3 or the ethanol vehicle. A vitamin D-replete group was placed on the same diet but injected with 50 IU of vitamin D3 once a week for the entire 5-week period. By the use of a modified Ussing chamber [1], the measurements of calcium fluxes into and from the rat calvaria were possible. These data enabled the apparent mineral solubilities to be derived. After 5 weeks on this diet the vitamin D-deficient rats had low levels of serum calcium (1.41 mM) and decreased mineral solubility when compared to the vitamin D-replete group. The apparent solubility of the bone mineral increased toward the vitamin D-replete level in calvaria from vitamin D metabolite-treated rats. However, these changes did not directly reflect the alterations in the level of serum calcium. At any given dose level, 1,25(OH)2D3 was the most effective metabolite in increasing serum calcium. In fact, the high dose (250 pmoles/day) was hypercalcemic. Next in effectiveness was 25(OH)D3. These two metabolites were equally effective in increasing mineral solubility. At a 10 times higher dose, the 24,25(OH)2D3 metabolite was able to normalize serum calcium and improve but not normalize mineral solubility. At the high dose (260 pmoles/day), the 25,26(OH)2D3 metabolite caused no effect on mineral solubility and minimal increases in serum calcium.     

Absence of effect of 24,25-dihydroxycholecalciferol on serum immunoreactive PTH in patients with persistent hyperparathyroidism after renal transplantation

Three hypercalcemic renal transplant recipients with stable, excellent renal function (creatinine clearance 74 +/- 11.8 ml/min) were treated with 60 micrograms 24,25(OH)2D3 by mouth daily for three months. Immunoreactive c-terminal PTH, intact PTH, 1,25(OH)2D3, 25(OH)D3, 24,25(OH)2D3, and serum and 24 h urine calcium, phosphate, magnesium and creatinine were obtained before, at one week, one month and three months of treatment, and at six weeks post-treatment. Significant elevations in serum levels of 24,25(OH)2D3 were induced by therapy (1.32 +/- .16 ng/ml to 30.06 +/- 5.18 ng/ml at one month). Moderate elevations of c-terminal PTH and normal levels of intact PTH remained unchanged throughout the study. Serum calcium remained elevated, serum phosphate and magnesium remained depressed and creatinine clearance and urinary excretion of calcium, phosphate, and magnesium remained unchanged. Furthermore, 1,25(OH)2D3 and 25(OH)D3 remained in the normal range throughout the study. We conclude that 24,25(OH)2D3 did not have a suppressant effect on levels of iPTH in the clinical setting of persistent hyperparathyroidism after successful renal transplantation.     

Histological observations on the failure of rachitic rat bones to respond to 1,25/OH)2D3

Rachitic rats, maintained on diets with low or normal P contents, were given daily intraperitoneal doses of 1,25(OH)2D3 or 25OHD3 at levels of 100 or 200 ng. Plasma chemistry was measured and the ash content and histological appearance of the bones investigated. Using labeled material it was shown that the dosing levels of 1,25(OH)2D3 employed ensured a higher than normal plasma concentration of that metabolite over the period between doses. 1,25(OH)2D3 was not effective as 25OHD3 in raising bone ash or reducing the amount of osteoid. The difference between the effects of the metabolites was evident at both dietary P levels, but more marked at the higher P level. In contrast, the metabolites reduced the width of the epiphyseal plate to an approximately similar degree, and this is possibly the reason why there are discrepancies between previous reports of the effectiveness of 1,25(OH)2D3 compared with 25OHD3 or vitamin D3. Dosing with 1,25(OH)2D3 failed to maintain a constant plasma Pi value over the period between doses in animals fed the low P diet.     

Effects of vitamin D metabolites on healing of low phosphate, vitamin D-deficient induced rickets in rats

A model of low-phosphate, vitamin D-deficient rachitic rats was used to compare the effects of 1 alpha(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3 on cartilage and bone. The rats were maintained for 3 weeks on a high-calcium, low-phosphate, vitamin D-deficient diet, during which period they developed severe rickets. The rachitic rats were injected for 2 or 3 consecutive days with a physiologic dose of either metabolite. Other littermates were given a single dose of 50,000 IU of cholecalciferol in combination with a normal diet. Samples of cartilage fluid (Cfl) and of blood were removed prior to sacrifice for biochemical studies of some parameters of calcification. These parameters were correlated with the results of light and electron microscopic studies of the growth plate cartilage and bone. Treatment with 1 alpha (OH)D3 or with 1,25(OH)2D3, in spite of increasing Ca and P levels in the Cfl, induced only partial healing of the rickets. In contrast, 24,25(OH)2D3 or vitamin D with a normal diet resulted in complete morphologic and biochemical healing of the rickets. Transmission electron microscopic (TEM) studies have shown partial mineralization of the wide hypertrophic zone of the growth plate following treatment with 1 alpha(OH)D3 or with 1,25(OH)2D3. Mineralization was more complete with 24,25(OH)2D3 treatment. The results of this study emphasize the importance of 24,25(OH)2D3 for normal endochondral bone formation and mineralization.     

Serum calcium regulating hormones in the perinatal period

Summary To clarify perinatal vitamin D metabolism, we measured 25-hydroxyvitamin D (25OHD), 24,25-dihydroxyvitamin D [24,25(OH)₂D], 1,25-dihydroxyvitamin D [1,25(OH)₂D], calcium (Ca), phosphorus (P), parathyroid hormone (PTH), and human calcitonin (CT) in paired maternal, cord, and infant serum. Cord serum 25OHD was significantly lower than the maternal level, and cord serum 24,25(OH)₂D was also significantly below the maternal concentrations. Maternal, cord, and infant serum 1,25(OH)₂D, on the other hand, was significantly higher than the normal adult level. The serum PTH was low, but the CT concentration was high in the cord. Cord serum Ca and P levels were significantly higher than maternal. The reason for the elevated circulating 1,25(OH)₂D level in the perinatal period is uncertain, and we speculate that the possible factors are gonadal steroids, placental lactogen, prolactin, and CT. In addition, serum 24,25(OH)₂D and 1,25(OH)₂D concentrations are under some control by the fetus.     

Differential effects of 1,25-dihydroxy-vitamin D3 and 19-nor-1,25-dihydroxy-vitamin D2 on calcium and phosphorus resorption in bone

1,25-Dihydroxy-vitamin D3 [1,25-(OH)2D3] suppresses the secretion and synthesis of parathyroid hormone (PTH) and has been used in the treatment of secondary hyperparathyroidism. However, 1,25-(OH)2D3 can induce hypercalcemia, which often precludes its use. Therefore, an analog of 1,25-(OH)2D3 that would retain its therapeutic effects but produce minor effects on calcium and phosphorus metabolism could be an ideal tool for the treatment of secondary hyperparathyroidism. It has been shown that 19-nor-1,25-dihydroxy-vitamin D2 [19-nor-1,25-(OH)2D2], an analog of 1,25-(OH)2D3, can suppress PTH levels in uremic rats at doses that do not affect plasma ionized calcium levels. The experiments presented here, using parathyroidectomized rats fed diets deficient in either calcium (0.02%) or phosphorus (0.02%), were performed to compare the effects of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 on calcium and phosphorus resorption in bone. Parathyroidectomized rats received daily intraperitoneal injections of vehicle, 1,25-(OH)2D3 (100 ng), or 19-nor-1,25-(OH)2D2 (100 or 1000 ng) for 9 d. Plasma calcium and phosphorus levels were monitored during the study, and ionized calcium levels were determined at the end of the study. By 9 d, 1,25-(OH)2D3 (100 ng/d) increased total calcium levels to 12.4+/-0.26 mg/dl, compared with 6.32+/-0.25 mg/dl (P<0.001) in control animals. The same dose of 19-nor-1,25-(OH)2D2 (100 ng/d) was much less potent (9.45+/-0.28 mg/dl, P<0.001). Similar results were seen with ionized calcium levels [19-nor-1,25-(OH)2D2, 3.61+/-0.12 mg/dl; 1,25-(OH)2D3, 5.03+/-0.16 mg/dl; P<0.001]. Ionized calcium levels were also lower in rats receiving the higher dose (1000 ng) of 19-nor-1,25-(OH)2D2 (4.59+/-0.09 mg/dl, P<0.05). Similar results were seen in rats fed the phosphorus-deficient diet. 1,25-(OH)2D3 (100 ng) increased plasma phosphorus levels from 4.30+/-0.39 mg/dl in vehicle-treated rats to 7.43+/-0.26 mg/dl (P<0.001). The same dose of 19-nor-1,25-(OH)2D2 had no effect (5.19+/-0.32 mg/dl), whereas the high dose (1000 ng) increased plasma phosphorus levels (7.31+/-0.24 mg/dl) in a manner similar to that of 1,25-(OH)2D3 (100 ng). Therefore, 19-nor-1,25-(OH)2D2 is approximately 10 times less effective in mobilizing calcium and phosphorus from the skeleton, compared with 1,25-(OH)2D3. With its ability to suppress PTH at noncalcemic doses, 19-nor-1,25-(OH)2D2 is a potential therapeutic tool for the treatment of secondary hyperparathyroidism in chronic renal failure.     

Presystemic 24-hydroxylation of oral 25-hydroxyvitamin D3 in rats

The metabolism of 25-hydroxyvitamin D3 (25-OHD3) was compared following its intracardial or gastric administration. The rats were deprived of calcium and vitamin D. A mixture of radiolabeled (0.3 microCi) and stable (2 micrograms) 25-OHD3 was given as a single dose. After 24 h the rats given the dose by gastric tube had significantly lower serum concentrations of 25-OHD3 and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] than those injected intracardially. In contrast, serum 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] was much higher in the rats given the 25-OHD3 dose by gastric tube (6.2 nmol/liter +/- 1.3 SD, n = 7) compared to the intracardial group (0.9 nmol/liter +/- 0.5, p less than 0.001). The preceding results were based on specific radioactivity of metabolites. The same findings were obtained by reanalyzing the samples using conventional competitive binding assays for 25-OHD3, 1,25-(OH)2D3, and 24,25-(OH)2D3. The results show that orally administered 25-OHD3 is partly metabolized to 24,25-(OH)2D3 presystemically.     

Regulation of 25-hydroxyvitamin D3 metabolism in cultures of osteoblastic cells

This study was designed to investigate the mechanisms involved in the regulation of the conversion of 25-hydroxyvitamin D3 (25-OHD3) to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] in primary cultures of osteoblastlike cells from neonatal mouse calvariae. These cells, when incubated with tritiated 25-OHD3 ([3H]25-OHD3), spontaneously synthesized [3H]24,25-(OH)2D3 20-50 times more efficiently than [3H]1,25-(OH)2D3 at a rate of conversion that was substrate dependent and linear from 1 to 36 h. Gas chromatography-mass spectrometry verified the identity of the dihydroxylated metabolites. The calcium ionophore A23187 (5 microM) consistently stimulated the synthesis of 1,25-(OH)2D3 while suppressing the production of 24,25-(OH)2D3. This effect was sustained for 36 h and was dose dependent for concentrations from 0.05 to 10 microM. Furthermore, A23187 stimulated cAMP production and indomethacin (50 ng/ml) blocked the A23187-induced production of cAMP and 1,25-(OH)2D3 but had no effect on the suppression of 24,25-(OH)2D3 by A23187. This led to other experiments to find out whether the stimulative effect of A23187 on 1,25-(OH)2D3 synthesis is mediated by prostaglandins or cAMP, or both. PGE2 (10(-8)-10(-6) M) increased the production of 1,25-(OH)2D3 and of 24,25-(OH)2D3. Forskolin (0.01-10 microM) and dibutyryl cAMP (0.1-10 mM) increased the production of both metabolites but to a lesser degree than PGE2. These data suggest that osteoblastlike cells are stimulated by A23187 to increase the synthesis of 1,25-(OH)2D3 through mechanisms involving prostaglandins and cAMP. The synthesis of 24,25-(OH)2D3 is suppressed by A23187 through different mechanisms.     

Vitamin D deficiency in guinea pigs: exacerbation of bone phenotype during pregnancy and disturbed fetal mineralization,with recovery by 1,25(OH)2D3 infusion or dietary calcium-phosphate supplementation

Vitamin D (D) deficiency during human pregnancy appears to disturb fetal growth and mineralization, but fetal development is normal in D-deficient rats and vitamin D receptor gene-ablated mice. We used the guinea pig model to investigate maternal and fetal effects of D deficiency. Pregnant (Pr) and nonpregnant (NPr) animals were fed a D-replete (+D) or D-deficient diet (-D) for 8 weeks. We further studied whether the effects of a -D diet are reversed by continuous 1,25(OH)2D3 infusion (-D+1,25) and/or by a lactose-, Ca- and P-enriched D-deficient diet (-D+Ca/P). Bone analyses included histomorphometry of the proximal tibiae, dual-energy X-ray absorptiometry (DXA), and quantitative computed tomography (QCT) of the femora. Depletion of 25(OH)D3 and 1,25(OH)2D3 levels and the D-deficiency syndrome were more severe in pregnant animals. Indeed, Pr/-D but not NPr/-D guinea pigs were hypophosphatemic, and showed robust increases in growth plate width and osteoid surface and thickness; in addition, bone mineral density on DXA was lower in Pr/-D animals only, which was exclusively in cortical bone on QCT. Bone phenotype was partly normalized in Pr/-D+1,25 and Pr/-D+Ca/P animals. Compared with +D fetuses, -D fetuses had very low or undetectable 25(OH)D3 and 1,25(OH)2D3, were hypercalcemic and hypophosphatemic, and had lower osteocalcin levels. In addition, body weight and total body bone mineral content were 10-15% lower; histomorphometry showed hypertrophic chondrocyte zone expansion and hyperosteoidosis. 1,25(OH)2D3 levels were restored in -D+1,25 fetuses, and the phenotype was partially corrected. Similarly, the fetal +D phenotype was rescued in large part in -D+Ca/P fetuses, despite undetectable circulating 25(OH)D3 and 1,25(OH)2D3. We conclude that pregnancy markedly exacerbates D deficiency, and that augmenting Ca and P intake overrides the deleterious effects of D deficiency on fetal development.