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We previously demonstrated in normal subjects that 1,25-dihydroxyvitamin D3 (1,25(OH)2D) can prevent the increase in serum 25-hydroxyvitamin D (25-OHD) which occurs in response to vitamin D. An investigation was carried out in eight normal subjects, therefore, to determine whether increases in calcium intake would alter the response of serum 25-OHD to challenge with vitamin D. In control studies, vitamin D, 100,000 U/d for 4 d, significantly increased mean serum 25-OHD from 18 +/- 3 to 42 +/- 5 ng/ml (p less than 0.001), an increment of 24 ng/ml (133%). Mean serum calcium, ionized calcium, phosphorus, creatinine, and 1,25(OH)2D did not change. In contrast, the same dose of vitamin D and calcium, 2,000 mg/d for 4 d, administered to the same eight subjects produced an increase in mean serum 25-OHD from 19 +/- 3 to 31 +/- 4 ng/ml (p less than 0.001), an increment of only 12 ng/ml (63%) and significantly less than the control (p less than 0.02). Mean serum calcium (8.8 +/- 0.1 vs. 9.2 +/- 0.1 mg/dl, p less than 0.01) and ionized calcium (4.79 +/- 0.07 vs. 4.85 +/- 0.08 mg/dl, p less than 0.05) increased significantly in response to vitamin D and calcium, mean serum phosphorus and creatinine did not change, and mean serum 1,25(OH)2D decreased significantly (37 +/- 2 vs. 31 +/- 4 pg/ml, p less than 0.02). In a postcontrol study in six of the normal subjects, vitamin D again significantly increased mean serum 25-OHD from 17 +/- 3 to 39 +/- 9 ng/ml (p less than 0.02), an increment of 22 ng/ml (129%).(ABSTRACT TRUNCATED AT 250 WORDS)

To determine whether immobilization acts directly on bone by alteration of mechanical loading or systemically, studies of the effects of immobilization were carried out on histomorphometry of diaphyses of tibiae and on subcutaneous implants of demineralized allogenic bone matrix of rats. The right hind leg of growing rats was denervated by severing the tibial nerve. A sham operation on the right hind leg was performed in control animals. Bone formation at the endosteal and periosteal surfaces was significantly lower in tibiae from limbs with severed nerves as compared to tibiae from the intact limbs of nerve-sectioned rats and from both limbs of sham-operated control rats. Bone formation was decreased at both 3 and 7 weeks after immobilization. The decreased formation resulted in significant reductions in cross-sectional area. At 3 weeks post denervation, the periosteal bone formation rate was lower in tibiae of intact limbs from denervated rats as compared to tibiae from intact limbs of sham-operated animals. This finding was attributed to reduced physical activity of the denervated rats. In the implants, nerve section did not alter the amount of implant matrix resorbed, the amount of bone matrix synthesized, or the amount of calcium in the implant. These findings support the hypothesis that inhibition of bone formation at the tibial diaphysis in response to immobilization resulted from altered mechanical loading and not from the production of substances acting systemically. Whereas the mean medullary area of tibiae was not altered by nerve section, it was decreased in tibiae of all groups compared to the values of basal controls, indicating that bone formation was greater than bone resorption. The findings are interpreted to mean that, in the rat, reducing the mechanical loading of an extremity inhibits bone formation at the tibial diaphysis of the affected limb and does not influence bone resorption at that site.

Gallium nitrate lowers the serum calcium in patients with hypercalcemia caused by malignancy and is available for clinical use. The mechanism for the hypocalcemic action is unknown, however. The present studies were undertaken to determine the effects of gallium on bone metabolism. Normal male rats were implanted subcutaneously with mineralized allogeneic bone matrix. Histomorphometry of the implants and of tibiae was determined after three doses of tetracycline administered at intervals of 1 week. Gallium as nitrate was administered daily by intraperitoneal injection at doses of 0.9, 1.8, and 3.6 mg elemental gallium per kg body weight for 21 days in one study and at 3.5 mg/kg for 33 days in a second study. All the gallium-treated rats gained weight. Rats given gallium at doses of 3.5 mg/kg or more grew at a lower rate than untreated controls (-7 and -10% at doses of 3.5 and 3.6 mg/kg, respectively; p < 0.05). At a dose of 0.9 mg/kg, gallium did not inhibit bone resorption or lower serum calcium but inhibited bone formation by 32% and bone apposition by 36% at the endosteal surface of the tibia. At a dose of 1.8 mg/kg, gallium produced modest hypocalcemia, prevented a rise in circulating 1,25-dihydroxyvitamin D [1,25-(OH)2D], inhibited bone resorption in implants, and inhibited bone formation by 19% and bone apposition by 18%. At a dose of 3.5 mg/kg, gallium lowered the serum calcium and serum 1,25-(OH)2D, inhibited growth, and accentuated the antiresorptive and antiformative effects seen at the two lower doses. The results indicate that gallium inhibits bone resorption, produces hypocalcemia, alters vitamin D metabolism, and inhibits bone formation at doses below those that inhibit bone resorption. The mechanism for hypocalcemia may be by direct action on bone and could involve an indirect effect on calcium metabolism mediated by changes in the metabolism of vitamin D.

A 35-year-old white male with rheumatoid arthritis who had developed hypercalcemia, hypercalciuria, and nephrolithiasis was found to be abnormally sensitive to vitamin D as a result of lack of regulation of circulating 1,25-dihydroxyvitamin D (1,25-(OH)2D). An increase in daily intake of vitamin D from 10 micrograms (400 units) per day to 50 micrograms (2000 units) per day produced an abnormal elevation in serum 1,25-(OH)2D, hypercalcemia, and hypercalciuria which were corrected by prednisone. Serum 25-hydroxyvitamin D initially was abnormally low, and increased with vitamin D to values which were in the low normal range. There were significant positive correlations between serum 1,25-(OH)2D (p less than .05) and serum calcium and between serum 1,25-(OH)2D and urinary calcium (p less than .05). Serum immunoreactive parathyroid hormone, initially in the lower range of normal, decreased further during hypercalcemia. A radiograph of the chest, gallium scan, and serum angiotensin-converting enzyme activity were normal. No granulomas or evidence of lymphoma were found in biopsies of the liver and of several lymph nodes. It is concluded that the abnormal calcium metabolism in this patient resulted from increased circulating 1,25-(OH)2D and that the defect in vitamin D metabolism was not related to sarcoidosis, other granulomatous disease, Hodgkin's disease, or lymphoma. The relationship, if any, of the abnormal metabolism of vitamin D and calcium to rheumatoid arthritis remains to be established.

To determine whether the nonsteroidal antiestrogen tamoxifen behaves as either an agonist or antagonist of estrogen on bone, the effects of ovariectomy, 17 beta-estradiol, and tamoxifen were compared on radial growth at the tibial diaphysis in young adult female rats. Ovariectomy and 17 beta-estradiol did not alter serum calcium, phosphate, or 25-hydroxyvitamin D. Ovariectomy increased serum 1,25-dihydroxyvitamin D in one experiment but not in the other. Tamoxifen increased the serum calcium and phosphate by itself and did not change serum 1,25-dihydroxyvitamin D in ovariectomized rats. Ovariectomy produced significant increases in medullary area, periosteal bone formation rate, and periosteal bone apposition rate compared to values in sham-operated animals and did not change endosteal bone formation rate. The increase in medullary area resulted from an increase in osteoclast number and resorbing surface length. Although endosteal forming surface length decreased, this was compensated for by an increase in the apposition rate. 17 beta-estradiol and tamoxifen each prevented the increases in bone formation rate and medullary area in ovariectomized rats. Tamoxifen reduced the length of the resorbing surface and osteoclast number to values observed in sham-operated animals. The findings demonstrate that in the rat, tamoxifen acts as an estrogen agonist by preventing the skeletal alterations that result from ovarian hormone deficiency.

The effects of castration on bone histomorphometry and mineral homeostasis were compared in male and female rats. Measurements were performed 4 weeks after sham operation or gonadectomy. Orchiectomy produced increases in serum calcium and decreases in serum testosterone and androstenedione, whereas ovariectomy produced decreases in serum estradiol and testosterone. Orchiectomy did not alter static bone histomorphometric measurements of the tibial diaphysis, whereas ovariectomy increased cross-sectional and medullary areas, lowered endosteal tetracycline-labeled surface length, and markedly increased endosteal nonlabeled surface length. Orchiectomy decreased mean periosteal bone formation rate and mean periosteal bone apposition rate, whereas ovariectomy increased both measurements. Orchiectomy and ovariectomy markedly diminished trabecular area and trabecular surface length at the tibial metaphysis. Orchiectomy did not alter the number of osteoclasts per mm trabecular surface or the percentage of trabecular surface covered by osteoclasts, whereas ovariectomy increased both measurements. These findings indicate that gonadal hormones produce separate and distinct effects on bone metabolism as determined by histomorphometry in male and female rats.

The results of slow-release sodium fluoride (SR-NaF) treatment in two nonrandomized trials involving 65 patients with postmenopausal osteoporosis from the primary site and 121 patients from collaborative sites were compared with those obtained from 54 treated patients and 56 patients taking placebo from a randomized controlled trial. Spinal fracture data were analyzed separately in mild to moderate bone loss of lumbar spine (baseline L2-L4 bone density [BD] ≥ 65% young normal) and in severe bone loss (BD < 65%). Since demographic and fracture data were similar among fluoride-treated patients from the three trials at each stratum of bone loss, their data were combined. In mild to moderate bone loss, SR-NaF treatment in the combined group virtually eliminated new spinal fractures with 96.6% of patients remaining fracture-free. The Fluoride group had a markedly lower individual vertebral fracture rate (0.025 vs. 0.188/patient year, p = 0.0001) and group vertebral fracture rate (0.029 vs. 0.175/patient year, relative risk [RR] 0.12, p = 0.0001) than the Placebo group. In severe bone loss, the combined treated group had a significantly lower new spinal fracture rate than the Placebo group, although the differences were not as marked (group vertebral fracture rate of 0.150 vs. 0.276/patient year, RR 0.54, p = 0.03). In the combined fluoride-treated group, the L2-L4 bone mass rose by 4-6%/year for 4 years, and the femoral neck BD increased by 1-2%/year during first 2 years. The radial shaft BD did not change. The Placebo group did not show a change in bone mass at any site. The prevalence (percentage) of patients with related gastrointestinal side effects and nonvertebral fracture rates did not differ significantly between the combined SR-NaF group and the Placebo group (hip fracture rate of 0.0045/patient year in SR-NaF and 0.0053/patient year in Placebo; appendicular fracture other than hip (see text) rate of 0.0193/patient year in SR-NaF and 0.0159/patient year in Placebo). A subgroup analysis showed a low baseline L2-L4 BD, high prevalent spinal fractures, and reduced body weight to be important determinants of the development of spinal fracture during SR-NaF treatment. Concomitant medications (estrogen, vitamin D, thiazide and thyroid hormone) were not independent predictors of the spinal fracture risk. Only 17% of fluoride-treated patients were nonresponders (new spinal fractures or a fall/no change in L2-L4 bone mass). Thus, the effects of SR-NaF treatment on the spinal fracture rate from nonrandomized trials were similar to those of the treated group of the randomized trial but different from those of the Placebo group. The similarity of response of nonrandomized trials with that of the randomized controlled trial and the resultant combined analysis further validate the efficacy and safety of SR-NaF in the treatment of postmenopausal osteoporosis.

Exercise and muscle strength are important determinants of bone mass. Studies were carried out in normal young adult white males to determine the effects of exercise on vitamin D and mineral metabolism. Fourteen men who had engaged in regular muscle-building exercises for at least 1 year and 14 age-matched controls (age range, 19-36 year) were hospitalized on a metabolic ward and were given a constant daily diet estimated to contain 400 mg of calcium, 900 mg of phosphorus, 110 mEq of sodium, 65 mEq of potassium, and 18 mEq of magnesium. Body weight averaged 78 +/- 3 kg in the exercisers and 72 +/- 2 kg in the controls (NS). Serum calcium, ionized calcium, phosphate, magnesium, somatomedin-C, and immunoreactive parathyroid hormone (PTH) were not different in the two groups, whereas serum Gla-protein (39 +/- 5 vs. 24 +/- 2 ng/ml, p less than 0.01), 25-hydroxyvitamin D (23 +/- 2 vs. 16 +/- 2, p less than 0.05) and 1,25-dihydroxyvitamin D [1,25(OH)2D] (40 +/- 2 vs. 29 +/- 2 pg/ml, p less than 0.01) were higher in the exercisers than in the controls. Urinary calcium, phosphorus, sodium, potassium, creatinine clearance, and norepinephrine were not different in the two groups, whereas urinary magnesium (12.6 +/- 1.0 vs. 9.4 +/- 0.5 mEq/d, p less than 0.01) and urinary cyclic adenosine 3',5'-monophosphate (cyclic AMP) (2.52 +/- 0.19 vs. 1.72 +/- 0.20 nM/dl glomerular filtrate, p less than 0.01) were higher in the exercisers than in the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Neither the efficiency of the 25-hydroxylation of vitamin D nor the steady state relation between vitamin D 3 and 25-hydroxyvitamin D [25(OH)D] has been studied in humans. Objective: We aimed to examine the relation between serum vitamin D 3 and 25(OH)D in normal subjects after either oral administration of vitamin D 3 or ultraviolet-B radiation across a broad range of inputs. Design: Values for serum vitamin D 3 and (OH)D 3 were aggregated from 6 studies¯1 acute and 5 near-steady state-at various vitamin D 3 inputs. In 3 of the steady state studies, vitamin D 3 had been administered for 18-26 wk in doses of 0 to 11 000 IU/d; in 2 studies, subjects had received solar or ultraviolet-B irradiation. Results: In the acute study, subjects receiving a single 100 000-IU dose of vitamin D 3 had a rise in serum cholecalciferol to a mean of 521 nmol/L at 1 d and then a fall to near-baseline values by 7-14 d. Serum 25(OH)D peaked at 103 nmol/L on day 7 and fell slowly to baseline by day 112. In the 5 steady state studies, the relation of serum 25(OH)D to serum vitamin D 3 was biphasic and was well described by a combined exponential and linear function: Y = 0.433X + 87.81[1-exp (-0.468X)], with R 2 = 0.448. Conclusions: At physiologic inputs, there is rapid conversion of precursor to product at low vitamin D 3 concentrations and a much slower rate of conversion at higher concentrations. These data suggest that, at typical vitamin D 3 inputs and serum concentrations, there is very little native cholecalciferol in the body, and 25(OH)D constitutes the bulk of vitamin D reserves. However, at supraphysiologic inputs, large quantities of vitamin D 3 are stored as the native compound, presumably in body fat, and are slowly released to be converted to 25(OH)D.

Studies were performed to further characterize the male-specific hepatic recombinant microsomal vitamin D 25-hydroxlase CYP2C11, expressed in baculovirus-infected insect cells, and determine whether it is also a vitamin D 24-hydroxylase. 25- and 24-hydroxylase activities were compared with those of 10 other recombinant hepatic microsomal cytochrome P-450 enzymes expressed in baculovirus-infected insect cells. Each of them 25-hydroxylated vitamin D2, vitamin D3, 1α-hydroxyvitamin D2(1αOHD2), and 1α-hydroxyvitamin D3(1αOHD3). CYP2C11 had the greatest activity with these substrates, except vitamin D3, which had the same activity as four of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1αOHD3> 1αOHD2> vitamin D2> vitamin D3. Each of the recombinant cytochrome P-450 enzymes 24-hydroxylated 1αOHD2. CYP2C11 had the greatest activity. 24-Hydroxylation of 1αOHD3was very low, and there was none with vitamin D3. Only CYP2C11 24-hydroxylated vitamin D2. Structures of vitamin D metabolites, including 24-hydroxyvitamin D2, 1,24( S)-dihydroxyvitamin D2, and 1,24-dihydroxyvitamin D3, were confirmed by HPLC and gas chromatography retention times and characteristic mass spectrometric fragmentation patterns. In male rats, hypophysectomy significantly reduced body weight, liver weight, hepatic CYP2C11 mRNA expression, and 24- and 25-hydroxylation of 1αOHD2. Expression of CYP2J3 and CYP2R1 mRNA did not change. In male rat hepatocytes, CYP2C11 mRNA expression and 24- and 25-hydroxylation were significantly reduced after culture for 24 h compared with uncultured cells. Expression of CYP2J3 and CYP2R1 either increased or did not change. It is concluded that CYP2C11 is a male-specific hepatic microsomal vitamin D 25-hydroxylase that hydroxylates vitamin D2, vitamin D3, 1αOHD2, and 1αOHD3. CYP2C11 is also a vitamin D 24-hydroxylase.

Studies were performed to identify the microsomal enzyme that 24-hydroxylates vitamin D, whether 25-hydroxylation occurs, and structure function of the enzyme. Sixteen hepatic recombinant microsomal cytochrome P450 enzymes expressed in baculovirus-infected insect cells were screened for 24-hydroxylase activity. CYP3A4, a vitamin D-25-hydroxylase, and CYP1A1 had the highest 24-hydroxylase activity with 1 alpha-hydroxyvitamin D(2) (1 alpha OHD(2)) as substrate. The ratio of rates of 24-hydroxylation of 1 alpha-hydroxyvitamin D(3) (1 alpha OHD(3)), 1 alpha OHD(2), and vitamin D(2) by CYP3A4 was 3.6/2.8/1.0. Structures of 24-hydroxyvitamin D(2), 1,24(S)-dihydroxyvitamin D(2), and 1,24-dihydroxyvitamin D(3) were confirmed by HPLC and gas chromatography retention time and mass spectroscopy. In characterized human liver microsomes, 24-hydroxylation of 1 alpha OHD(2) by CYP3A4 correlated significantly with 6 beta-hydroxylation of testosterone, a marker of CYP3A4 activity. 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dose-dependent inhibition by ketoconazole, troleandomycin, alpha-naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24- and 25-hydroxylation of 1 alpha OHD(2) and 1 alpha OHD(3) were determined in recombinant wild-type CYP3A4 and site-directed mutants and naturally occurring variants expressed in Escherichia coli. Substitution of residues showed the most prominent alterations of function at residues 119, 120, 301, 305, and 479. Thus, CYP3A4 is both a 24- and 25-hydroxylase for vitamin D(2), 1 alpha OHD(2), and 1 alpha OHD(3).

The synthesis of bioactive vitamin D requires hydroxylation at the 1α and 25 positions by cytochrome P450 enzymes in the kidney and liver, respectively. The mitochondrial enzyme CYP27B1 catalyzes 1α-hydroxylation in the kidney but the identity of the hepatic 25-hydroxylase has remained unclear for >30 years. We previously identified the microsomal CYP2R1 protein as a potential candidate for the liver vitamin D 25-hydroxylase based on the enzyme's biochemical properties, conservation, and expression pattern. Here, we report a molecular analysis of a patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency. This individual was found to be homozygous for a transition mutation in exon 2 of the CYP2R1 gene on chromosome 11p15.2. The inherited mutation caused the substitution of a proline for an evolutionarily conserved leucine at amino acid 99 in the CYP2R1 protein and eliminated vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency.

The human hepatic microsomal vitamin D 25-hydroxylase protein and gene have not been identified with certainty. Sixteen hepatic recombinant microsomal enzymes were screened for 25-hydroxylase activity; 11 had some 25-hydroxylase activity, but CYP3A4 had the highest activity. In characterized liver microsomes, 25-hydroxylase activity correlated significantly with CYP3A4 testosterone 6β-hydroxylase activity. Activity in pooled liver microsomes was inhibited by known inhibitors of CYP3A4 and by an antibody to CYP3A2. Thus, CYP3A4 is a hepatic microsomal vitamin D 25-hydroxylase. Introduction: Studies were performed to identify human microsomal vitamin D-25 hydroxylase. Materials and Methods: Sixteen major hepatic microsomal recombinant enzymes derived from cytochrome P450 cDNAs expressed in baculovirus-infected insect cells were screened for 25-hydroxylase activity with 1α-hydroxyvitamin D2 [1α(OH)D2], 1α-hydroxyvitamin D3 [1α(OH)D3], vitamin D2, and vitamin D3 as substrates. Activity was correlated with known biological activities of enzymes in a panel of 12 characterized human liver microsomes. The effects of known inhibitors and specific antibodies on activity also were determined. Results: CYP3A4, the most abundant cytochrome P450 enzyme in human liver and intestine, had 7-fold greater activity than that of any of the other enzymes with 1α(OH)D2 as substrate. CYP3A4 25-hydroxylase activity was four times higher with 1α(OH)D2 than with 1α(OH)D3 as substrate, was much less with vitamin D2, and was not detected with vitamin D3. 1α(OH)D2 was the substrate in subsequent experiments. In a panel of characterized human liver microsomes, 25-hydroxylase activity correlated with CYP3A4 testosterone 6β-hydroxylase activity (r = 0.93, p < 0.001) and CYP2C91 diclofenac 4′-hydroxylase activity (r = 0.65, p < 0.05), but not with activity of any of the other enzymes. Activity in recombinant CYP3A4 and pooled liver microsomes was dose-dependently inhibited by ketoconazole, troleandomycin, isoniazid, and α-naphthoflavone, known inhibitors of CYP3A4. Activity in pooled liver microsomes was inhibited by antibodies to CYP3A2 that are known to inhibit CYP3A4 activity. Conclusion: CYP3A4 is a vitamin D 25-hydroxylase for vitamin D2 in human hepatic microsomes and hydroxylates both 1α(OH)D2 and 1α(OH)D3.

Previous studies indicated that aminobisphosphonate alendronate sodium, a potent inhibitor of bone resorption, increases bone mineral density (BMD) at the hip and spine, reduces markers of bone turnover, and reduces the risk of fractures in Caucasian postmenopausal women. The purpose of the present study was to investigate whether alendronate increases BMD and reduces markers of bone turnover in African-American postmenopausal women. In a multicenter, randomized, double-blind, placebo-controlled study, 65 African-American women, aged 45 to 88 yr, were randomly assigned to either placebo (n = 33) or alendronate 10 mg daily (n = 32) for 2 yr. Mean BMD T scores of the lumbar spine at baseline were -3.18 in the placebo-treated group and -3.09 in the alendronate-treated group. All women took 500 mg elemental calcium daily in the form of calcium carbonate and 500 IU vitamin D. Alendronate significantly increased BMD and reduced markers of bone formation and resorption, compared with placebo. At 2 yr, mean changes +/- SE in BMD were 6.5% +/- 0.7% for the lumbar spine (P < 0.001), 4.5% +/- 1.0% for the femoral neck (P < 0.001), 6.4% +/- 0.6% for the femoral trochanter (P < 0.001), 4.1% +/- 0.7% for the total hip (P < 0.001), 0.7% +/- 0.5% for the one third forearm (NS), and 2.0% +/- 0.4% for the total body (P < 0.001) in women treated with alendronate, compared with 0.9% +/- 0.6% (NS), 0.5% +/- 1.1% (NS), -0.2 +/- 0.8 (NS), -1.1 +/- 0.7% (NS), -0.8% +/- 0.6% (NS), and -1.2% +/- 0.6% (P < 0.05) for the lumbar spine, femoral neck, trochanter, total hip, one third forearm, and total body, respectively, in women treated with placebo. At 2 yr, mean serum bone-specific alkaline phosphatase had declined by 46.3% with alendronate (P < 0.001) and 13.6% with placebo (P < 0.01), and mean urinary N-telopeptide of type I collagen/creatinine ratio had declined by 70.5% with alendronate (P < 0.001) and 6.7% with placebo (NS). The incidence of adverse experiences was not different between the two groups. We conclude that in postmenopausal African-American women with osteoporosis, alendronate, 10 mg daily for 2 yr, increases BMD at the lumbar spine, hip, and total body and reduces markers of bone remodeling and is well tolerated.

Racial differences in bone mineral density (BMD) appear to account in part for racial differences in the incidence of osteoporosis and fractures. We previously reported that the greater BMD in adult blacks compared with whites is associated with a higher serum 17 beta-estradiol and greater secretion of growth hormone (GH) in men but not women. To determine whether these racial differences occur in prepubertal boys, we measured spontaneous overnight GH secretion, serum testosterone, 17 beta-estradiol, IGF-I, and IGFBP3, IGF-I/ IGFBP3 ratio, BMD of the total body, forearm, lumbar spine, trochanter, and femoral neck, and lean body mass and body fat in 14 healthy black and 16 white boys ages 6-7 years. Measurements of GH were obtained at 20-minute intervals for 12 hours. Results were analyzed by deconvolution and are expressed as mean +/- SE. Whereas BMD of the hip (0.755 +/- 0.020 vs 0.663 +/- 0.021 g/cm(2), P = 0.0037), trochanter (0.617 +/- 0.014 vs 0.552 +/- 0.018 g/cm(2), P = 0.0102) and femoral neck (0.710+/-0.018 vs 0.6381 +/- 0.021 g/cm(2), P = 0.0157) were significantly greater in black compared with white boys, BMD of the total body (0.768 +/- 0.010 vs 0.741 +/- 0.012 g/cm(2), NS), forearm (0.405 +/- 0.010 vs 0.380 +/- 0.008 g/cm(2), NS), and lumbar spine (0.612 +/- 0.013 vs 0.609 +/- 0.021 g/cm(2), NS) was not different in the two groups. Stepwise regression analysis showed significant correlations between BMD and race at each skeletal site except the lumbar spine and trochanter. Deconvolution analysis revealed no racial difference in any of the GH measurements. Whereas serum testosterone, serum 17 beta-estradiol, and serum IGF-I were not different, serum IGFBP-3 was higher and the molar ratio of serum IGF-l/IGFBP-3 was lower in white than in black males. In summary, prepubertal BMD is higher in black than in white males at the hip, trochanter, and femoral neck, and the racial difference does not result from differences in secretion of GH.

Drugs used to treat osteoporosis act either by inhibiting bone resorption or stimulating bone formation. Osteoclast formation and bone resorption require cell-to-cell contact between osteoblasts and osteoclast precursors and osteoclasts in the marrow. Interaction between the receptor for activation of nuclear factor kappa B (RANK) on the surface of preosteoclasts and osteoclasts and RANK ligand on the surface of osteoblasts is required to stimulate osteoclast formation and activation. Binding of the RANK ligand to its receptor and osteoclastogenesis are prevented by osteoprotegerin (OPG), a decoy receptor produced by osteoblasts and marrow stromal cells. Thus, interference in binding of the RANK ligand to RANK by OPG determines the rate of bone resorption. Antiresorptive drugs such as estrogen, raloxifene, bisphosphonates, salmon calcitonin, and osteoprotegerin increase bone mass by inhibiting osteoclast function and bone resorption. Osteoprotegerin is more potent since it also inhibits osteoclast formation. Raloxifene, a selective estrogen receptor modulator (SERM), is a member of a class of compounds that act through estrogen receptors and are agonists for bone, antagonists for breast and uterine tissue and may be cardioprotective. The drug was shown to prevent vertebral fractures. Alendronate and bisphosphonates are the only antiresorptive drugs that have been shown to decrease fracture rates for the hip in addition to spine and other sites. Bone morphogenetic proteins stimulate bone formation at local sites and are being developed to stimulate fracture healing. Parathyroid hormone (1-34) stimulates osteoblastic bone formation, markedly increases bone mass, prevents vertebral fractures and is under development to treat osteoporosis.

The bisphosphonate alendronate and conjugated equine estrogens are both widely used for the treatment of postmenopausal osteoporosis. Acting by different mechanisms, these two agents decrease bone resorption and thereby increase or preserve bone mineral density (BMD). The comparative and combined effects of these medications have not been rigorously studied. This prospective, double blind, placebo-controlled, randomized clinical trial examined the effects of oral alendronate and conjugated estrogen, in combination and separately, on BMD, biochemical markers of bone turnover, safety, and tolerability in 425 hysterectomized postmenopausal women with low bone mass. In addition, bone biopsy with histomorphometry was performed in a subset of subjects. Treatment included placebo, alendronate (10 mg daily), conjugated equine estrogen (CEE; 0.625 mg daily), or alendronate (10 mg daily) plus CEE (0.625 mg daily) for 2 yr. All of the women received a supplement of 500 mg calcium daily. At 2 yr, placebo-treated patients showed a mean 0.6% loss in lumbar spine BMD, compared with mean increases in women receiving alendronate, CEE, and alendronate plus CEE of 6.0% (P < 0.001 vs. placebo), 6.0% (P < 0.001 vs. placebo), and 8.3% (P < 0.001 vs. placebo and CEE; P = 0.022 vs. alendronate), respectively. The corresponding changes in total proximal femur bone mineral density were +4.0%, +3.4%, +4.7%, and +0.3% for the alendronate, estrogen, alendronate plus estrogen, and placebo groups, respectively. Both alendronate and CEE significantly decreased biochemical markers of bone turnover, specifically urinary N-telopeptide of type I collagen and serum bone-specific alkaline phosphatase. The alendronate plus CEE combination produced slightly greater decreases in these markers than either treatment alone, but the mean absolute values remained within the normal premenopausal range. Alendronate, alone or in combination with CEE, was well tolerated. In the subset of patients who underwent bone biopsies, histomorphometry showed normal bone histology with the expected decrease in bone turnover, which was somewhat more pronounced in the combination group. Thus, alendronate and estrogen produced favorable effects on BMD. Combined use of alendronate and estrogen produced somewhat larger increases in BMD than either agent alone and was well tolerated.

The bisphosphonate alendronate and conjugated equine estrogens are both widely used for the treatment of postmenopausal osteoporosis. Acting by different mechanisms, these two agents decrease bone resorption and thereby increase or preserve bone mineral density (BMD). The comparative and combined effects of these medications have not been rigorously studied. This prospective, double blind, placebo-controlled, randomized clinical trial examined the effects of oral alendronate and conjugated estrogen, in combination and separately, on BMD, biochemical markers of bone turnover, safety, and tolerability in 425 hysterectomized postmenopausal women with low bone mass. In addition, bone biopsy with histomorphometry was performed in a subset of subjects. Treatment included placebo, alendronate (10 mg daily), conjugated equine estrogen (CEE; 0.625 mg daily), or alendronate (10 mg daily) plus CEE (0.625 mg daily) for 2 yr. All of the women received a supplement of 500 mg calcium daily. At 2 yr, placebo-treated pati...

Biochemical markers of bone resorption are useful for evaluating metabolic bone diseases. A three-center study was performed in 253 men, 21–86 yr of age, to determine the normal range of urinary N-telopeptide of type I collagen (NTX/creatinine) in a nonfasting, second void, morning specimen, to define the biological variability and to examine the relationship between NTX/creatinine and age. Men with disorders or taking medications known to alter bone turnover, or with a serum creatinine level greater than 2 mg/dL were excluded. Results are expressed as nanomoles of bone collagen equivalents (BCE) per mmol creatinine. In a subset of individuals over age 30 yr, additional second void morning urine specimens were obtained at 2, 3, and 4 days (short term study) and at 2, 3, and 4 months (long term study) after the first specimen. After collection, samples were shipped to one laboratory for analysis. Multiple samples from the same subject were analyzed in separate assays. It was found that urinary NTX/creatinine was significantly higher in 45 men, aged 21–30 yr, than in 206 men, aged 31–86 yr (48 ± 22 vs. 33 ± 15 nmol/L BCE/mmol/L creatinine; P < 0.00001). Values did not otherwise change with age. The range of values in men aged 21–30 yr was 4–92 nmol/L BCE/mmol/L creatinine. The range for men over age 30 yr was 3–63 nmol/L BCE/mmol/L creatinine, essentially the same as that previously reported for premenopausal women. The coefficient of variation was determined in each individual for the short term (n= 36) and long term studies (n = 35) and averaged 18% and 19%, respectively. There was no correlation between short term and long term coefficient of variations. In summary, urinary NTX/creatinine is higher in men aged 21–30 yr than in men over age 30 yr and may reflect continued skeletal maturation. Intrasubject variability of urinary NTX/creatinine in short term and long term studies has been defined for clinical purposes.

Asian Indians who immigrate to northern Europe have lower serum 25-hydroxyvitamin D [25(OH)D] than Caucasians, and they develop vitamin D deficiency, rickets, and osteomalacia. We investigated vitamin D metabolism, the effects of 25(OH)D3 on vitamin D metabolism and activity of 25(OH) D-24-hydroxylase, the rate-limiting enzyme for degradation of 25(OH)D, from cultured skin fibroblasts of Asian Indians and compared them with cultured skin fibroblasts of Caucasians in the southern United States. Normal subjects, ages 20-40 yr, were admitted to a metabolic ward for 2.5 days and given a daily diet containing 400 mg calcium and 900 mg phosphorus. Serum vitamin D, serum 25(OH)D, urinary calcium, and urinary phosphorus were significantly lower, whereas serum immunoreactive intact parathyroid hormone (PTH) and serum 1,25-dihydroxyvitamin D [1,25(OH)2D] were significantly higher in Asian Indians than in Caucasians. Administration of 25(OH)D3 increased serum 25(OH)D and urinary calcium but did not change serum PTH or serum 1,25(OH)2D in Asian Indians. In cultured skin fibroblasts, Emax and Vmax of 25(OH)D-24-hydroxylase activity were significantly higher in Asian Indians. In summary, in Asian Indians serum vitamin D and 25(OH)D are markedly reduced, altered vitamin D metabolism is only partially reversed by 25(OH)D3, and 25(OH)D-24-hydroxylase activity in cultured skin fibroblasts is markedly increased. Thus, Asian Indians residing in the U.S. are at risk for developing vitamin D deficiency, rickets, and osteomalacia.

PURPOSE: Previously we have reported a significant increase in bone mineral density (BMD) of the spine and the hip and reductions in biochemical indices of bone turnover in postmenopausal women with osteoporosis treated with alendronate at various doses over 1 to 2 years. We have followed BMD and biochemical parameters in these patients for 1 or 2 years after discontinuation of alendronate to determine resolution of alendronate effects. PATIENTS AND METHODS: Participants received daily oral doses of placebo, 5 or 10 mg of alendronate for 2 years, or 20 or 40 mg of alendronate for 1 year followed by 1 year of placebo. No treatment was given in the third year of study. RESULTS: Lumbar spine BMD changes in the 5- and 10-mg groups (−1.4 and −0.4%) were similar to those in the placebo group (−1.2%) 1 year after discontinuation of drug and lumbar spine BMD changes in the 20- and 40-mg groups (−1.2% and 0.8%) were similar to those in the placebo group (−0.9%) 2 years after discontinuation of drug. BMD of the total hip followed the same pattern of resolution. The difference in BMD between alendronate and placebo groups at the end of alendronate treatment was maintained up to 2 years. Residual reductions in the bone resorption markers urinary deoxypyridinoline (d-Pyr) and collagen type 1 cross-linked N telopeptides and the bone formation markers serum bone-specific alkaline phosphatase and osteocalcin remained for 1 year after discontinuation of 5 and 10 mg of alendronate and for 2 years after discontinuation of 20 and 40 mg of alendronate, other than return of D-Pyr to baseline 1 year after cessation of treatment with the 5- and 10-mg doses. CONCLUSIONS: A residual decrease in bone turnover may be found up to 2 years after discontinuation of alendronate. Accelerated bone loss is not observed when treatment is discontinued. However, continuous therapy with alendronate is required to achieve a continuous gain in BMD.

To assess the ability of the urinary N-telopeptide of type I collagen (NTx) to monitor and predict therapeutic effects of hormone replacement therapy (HRT) in postmenopausal women.To assess the relationship between baseline or change in NTx (predictive variable), and change in lumbar and hip bone mineral density (BMD; outcome variable), we conducted a 2-year randomized controlled study at academic university and private practice medical centers in 236 healthy women 1 to 3 years postmenopausal; 227 women completed the study. Women received estrogen plus progesterone plus calcium (treated group) or calcium alone (control group).In the treated group NTx significantly (P0.0001) decreased, and spine and hip BMD significantly (P0.00001 and P0.005, respectively) increased; in the control group NTx did not change but BMD decreased significantly (P0.01). Subjects in the highest quartiles for baseline NTx (67 to 188 units) or decreasing NTx (-66% to -87%) through 6 months demonstrated the greatest gain in BMD in response to HRT (P0.05 and P0.005). For every increase of 30 units in baseline NTx the odds of gain in BMD in response to HRT increased by a factor of 5.0 (95% confidence interval [CI] 1.9 to 13.3); for every 30% decrease in NTx through 6 months, the odds of gaining BMD in response to HRT increased by a factor of 2.6 (95% CI 1.6 to 4.4). In the control group an increase of 30 units in mean NTx across the study indicated a higher odds of losing BMD by a factor of 3.2 (95% CI 1.6 to 6.5). A high baseline NTx (67 units) indicated a 17.3 times higher risk of BMD loss if not treated with HRT.These data support the clinical utility of NTx to monitor the antiresorptive effect of HRT in recently postmenopausal women, and to predict changes in BMD in response to HRT.

Background Postmenopausal osteoporosis is a serious health problem, and additional treatments are needed. Methods We studied the effects of oral alendronate, an aminobisphosphonate, on bone mineral density and the incidence of fractures and height loss in 994 women with postmenopausal osteoporosis. The women were treated with placebo or alendronate (5 or 10 mg daily for three years, or 20 mg for two years followed by 5 mg for one year); all the women received 500 mg of calcium daily. Bone mineral density was measured by dual-energy x-ray absorptiometry. The occurrence of new vertebral fractures and the progression of vertebral deformities were determined by an analysis of digitized radiographs, and loss of height was determined by sequential height measurements. Results The women receiving alendronate had significant, progressive increases in bone mineral density at all skeletal sites, whereas those receiving placebo had decreases in bone mineral density. At three years, the mean (±SE) differences in bone...

BACKGROUND The effects of the aminobisphosphonate alendronate sodium on bone mass and markers of bone remodeling were investigated. PATIENTS AND METHODS In a multicenter, randomized, double-blind, placebo-controlled, 2-year study, 188 postmenopausal women, aged 42 to 75 years, with low bone mineral density (BMD) of the lumbar spine were randomly assigned to 1 of 6 daily treatment groups: placebo for 2 years, alendronate 5 or 10 mg for 2 years, alendronate 20 or 40 mg for 1 year followed by placebo for 1 year, or alendronate 40 mg for 3 months followed by 2.5 mg for 21 months. All subjects were given 500 mg/d of elemental calcium as calcium carbonate. RESULTS At each dose, alendronate produced significant reductions in markers of bone resorption and formation, and significantly increased bone mass at the lumbar spine, hip, and total body, as compared with decreases (significant at lumbar spine) in subjects receiving placebo. In the 10-mg group, mean urinary deoxypyridinoline/creatinine had declined by 47% at 3 months, and mean serum osteocalcin by 53% at 6 months. Mean changes in BMD over 24 months with 10 mg alendronate were +7.21% +/- 0.49% for the lumbar spine, +5.27% +/- 0.70% for total hip, and +2.53% +/- 0.68% for total body (each P < 0.01) compared to changes of -1.35% +/- 0.61%, -1.20% +/- 0.64% and -0.31% +/- 0.44% at these sites, respectively, with placebo treatment. Progressive increases in BMD of both lumbar spine and total hip were observed in the second year of treatment with 10 mg alendronate (both P < 0.05). CONCLUSION Alendronate, a potent inhibitor of bone resorption, reduces markers of bone remodeling and significantly increases BMD at the lumbar spine, hip, and total body, and is well tolerated at therapeutic doses (5 or 10 mg daily) in the treatment of osteoporosis in postmenopausal women.

To determine why blacks have a higher bone mineral density (BMD) and lower incidence of osteoporosis and fractures than whites, we investigated whether the secretion of GH is higher in black than in white men. Measurements of GH were obtained at 20-min intervals over 24 h and analyzed by deconvolution. BMD was determined by dual energy x-ray absorptiometry in 16 normal black and 17 normal white men, aged 20-40 yr. The 24-h integrated GH concentration 942 +/- 174 vs. 602 +/- 104 micrograms/L; P = 0.0495) and GH secretory burst amplitude (0.499 +/- 0.163 vs. 0.169 +/- 0.027 micrograms/L.min; P = 0.0482) were higher in black than in white men. GH burst frequency, half-duration, mass, and half-life were not different in the 2 groups. The serum 17 beta-estradiol level (162 +/- 12 vs. 108 +/- 11 pmol/L; P = 0.0011) was higher, and the serum insulin-like growth factor-binding protein 3 level (2.2 +/- 0.1 vs. 2.8 +/- 0.1 microgram/mL; P = 0.0001) was lower in black than in white men. BMD values for total body (1.22 +/- 0.02 vs. 1.14 +/- 0.02 g/cm2; P = 0.0041), forearm (0.69 +/- 0.01 vs. 0.66 +/- 0.01 g/cm2; P = 0.0211), trochanter (0.91 +/- 0.03 vs. 0.77 +/- 0.03 g/cm2; P = 0.0003), and femoral neck (1.08 +/- 0.03 vs. 0.93 +/- 0.03 g/cm2; P = 0.0007) were higher in black than in white men. Thus, serum 17 beta-estradiol level, GH secretion, and BMD values for the total body, forearm, trochanter, and femoral neck are greater in black than in white men. As estrogen is known to increase GH secretion and GH to increase bone mass, increases in circulating 17 beta-estradiol may contribute to the higher GH secretion and bone mass in black men.

Prednisone inhibits bone formation and causes bone loss. To investigate possible mechanisms and sites, the effects of sham operation, ovariectomy, and prednisone were determined on bone and mineral metabolism in 7-week-old growing female rats. Forty animals were divided into groups of 10 each. Sham operation and ovariectomy were performed. One week later, pellets containing 5 mg prednisone or drug free were implanted S.C. at the back of the neck. Four weeks later, animals were sacrificed and tibiae were removed for histomorphometric analysis of the middiaphysis and proximal metaphysis. In both sham-operated and ovariectomized rats, prednisone (1) reduced weight gain (P

Alterations in vitamin D metabolism normally occur after the menopause and with aging. Increases in skeletal remodeling as a consequence of estrogen deficiency lead to increased bone resorption, suppression of serum 1,25-dihydroxy-vitamin D and intestinal absorption of calcium and increases in urinary calcium. Age-related changes in vitamin D metabolism include diminished dermal production of 7-dehydrocholesterol, the precursor of previtamin D3, vitamin D deficiency as a consequence of inadequate intake or exposure to sunshine (individuals may be institutionalized or housebound), a decline in intestinal vitamin D receptors and diminished intestinal absorption of calcium and secondary hyperparathyroidism which leads to further bone loss. These changes also have been implicated in the pathogenesis of senile osteoporosis. Treatment with vitamin D and its analogues such as 1,25-dihydroxyvitamin D3 is sometimes of value in preventing bone loss and fractures in patients with postmenopausal osteoporosis and senile osteoporosis. However, the drugs have not been approved for treatment and prevention of osteoporosis.

It has long been known that estrogen deficiency in animal models and postmenopausal women is associated with increased osteoclastic bone resorption and bone loss (1–5). In the past decade, several important discoveries of some of the key factors involved in osteoclast formation, survival, function, and regulation by estrogen have been made. A number of hormones and cytokines modulate osteoclastogenesis by enhancing osteoclast differentiation, activation, lifespan, and function. These include parathyroid hormone (PTH), calcitriol, PTH-related protein, prostaglandin E2, thyroxine, and IL-11 (3–5). The formation of active osteoclasts requires M-CSF (1, 3–6) and involves cell-to-cell contact between precursors of the monocyte/macrophage lineage and osteoblasts, marrow stromal cells, and T and B cells. These cells express the receptor activator of NF-κB ligand (RANKL), a member of the TNF ligand family, which is essential for this process. RANKL attaches to RANK, a receptor on the cell surface of osteoclasts and osteoclast precursors, to stimulate proliferation and differentiation of cells to form the osteoclast phenotype and inhibit apoptosis. Osteoprotegerin (OPG), a soluble decoy receptor produced by osteoblasts, marrow stromal cells, and other cells, profoundly modifies the effects of RANKL by inhibiting RANKL/RANK interaction (1, 3–5). In laboratory animals, estrogen deficiency leads to increased osteoclastogenesis and bone loss, and inhibition of osteoclastogenesis is the major means by which estrogen prevents the loss of bone. This is accomplished by diminishing production of IL-1, IL-6, and TNF-α — cytokines that enhance production of M-CSF and RANKL (1, 3–5) — and downregulating NF-κB and RANKL-induced activation of JNK1 and osteoclastogenic activator protein–1 transcription factors c-Fos and c-Jun (7). Estrogen upregulates OPG (8) and TGF-β (9), and TGF-β increases OPG expression by osteoblasts and stromal cells (10) and inhibits bone resorption by increasing apoptosis of osteoclasts (11) (Figure ​(Figure11). Figure 1 Regulation of osteoclast formation, function, and apoptosis by cytokines produced by bone marrow stromal cells, osteoblasts, monocytes, T cells, and B cells. Stimulatory factors are shown in orange and inhibitory factors in green. The effects of E to ...

background: The results of experimental studies with animals indicated that prostaglandins stimulate bone resorption, that skeletal production of prostaglandin E 2 is enhanced by ovariectomy and is diminished by 17 β -estradiol, and that the nonsteroidal anti-inflammatory drug (NSAID) naproxyn prevents bone loss after ovariectomy. Studies were carried out to investigate the effects of the NSAID diclofenac sodium on bone and mineral metabolism in premenopausal women and to compare the effects of diclofenac sodium and conjugated estrogens on bone and mineral metabolism in postmenopausal women. patients and methods: Ten healthy premenopausal women and 17 healthy postmenopausal women were studied while not being treated and again after 4 weeks of treatment with diclofenac sodium, 150 mg per day in divided doses (both groups), and conjugated estrogens, 0.625 mg per day (postmenopausal women). Cross-linked N-telopeptides of type 1 collagen were measured in the urine as an index of bone resorption. The postmenopausal women were separated into two groups, responders and nonresponders, based on their response to conjugated estrogens as assessed by linear discriminant analysis for groups. Conjugated estrogens lowered urinary N-telopeptides of type I collagen in responders, but not in nonresponders. results: Urinary cross-linked N-telopeptides were higher in the eight postmenopausal women responders than in the nine postmenopausal nonresponders or in the premenopausal women, and were not altered by diclofenac sodium in premenopausal women. In the eight postmenopausal women with higher rates of bone resorption, diclofenac sodium and conjugated estrogens significantly lowered both urinary calcium concentration and urinary cross-linked N-telopeptides. The effects of the two drugs were comparable. conclusion: The preliminary results demonstrate that, at the dose used, diclofenac sodium is almost as effective as conjugated estrogens for decreasing bone loss in postmenopausal women. Further studies will be needed to determine whether diclofenac sodium can prevent postmenopausal bone loss.

Sarcoidosis is a multisystem disorder of unknown etiology that frequently involves the lymph nodes, lungs, eyes, and skin. The disease can involve any organ system, and noncaseating granulomas are characteristically present. Synthesis of 1,25-dihydroxyvitamin D, the most biologically active form of vitamin D, occurs in granulomatous tissue and may give rise to increases in its concentration in the peripheral circulation and to hypercalcemia and hypercalciuria. Infiltration of endocrine organs also occurs. Involvement of the hypothalamus and pituitary can cause primary polydipsia and disordered regulation of thirst; diabetes insipidus, impaired secretion of anterior pituitary hormones (with clinically apparent hypothyroidism, hypogonadism, hypoadrenalism, or impaired growth), and increases in serum prolactin may also result. Galactorrhea, however, seldom occurs. Involvement of the thyroid and adrenal glands rarely leads to hypofunction. Involvement of the pancreas rarely occurs but does not produce diabetes mellitus. Involvement of the male reproductive system results in epididymitis and hypogonadism, and involvement of the uterus causes abnormalities in menstrual function.

Studies were performed to investigate vitamin D metabolism in Mexican-Americans. Groups of 15 whites and 16 Mexican-Americans ranging in age from 18 to 41 years were evaluated. All of them were within 25% of their ideal body weight. Each of them was admitted to the Clinical Research Center of the University of Texas Health Science Center and placed on a daily diet estimated to contain 400 mg calcium and 900 mg phosphate. It was found that whereas serum vitamin D (1.8 ± 0.5 versus 7.6 ± 1.3 ng/ml, P < 0.001) and serum 25-hydroxyvitamin D (9 ± 1 versus 17 ± 2 ng/ml, P < 0.01) were significantly lower and serum 1,25-dihydroxyvitamin D (37 ± 2 versus 28 ± 2 pg/ml, P < 0.001) was significantly higher in the Mexican-Americans than in the whites, serum calcium (9.1 ± 0.1 versus 9.2 ± 0.1 mg/dl), magnesium (1.84 ± 0.07 versus 1.80 ± 0.07 mEq/liter), and Gla protein (19 ± 3 versus 21 ± 2 ng/ml) were not different in the two groups. Urinary calcium (144 ± 14 versus 124 ± 16 mg/day), phosphate (672 ± 51 versus 683 ± 44 mg/ day), sodium (110 Ω 8 versus 99 ± 9 mEq/day), potassium (43 ± 4 versus 49 ± 3 mEq/day), and magnesium (7.3 ± 0.7 versus 7.7 ± 0.5 mEq/day) and creatinine clearance (173 ± 14 versus 154 ± 7 liters/day) were not different in the two groups. Serum immunoreactive PTH (390 ± 25 versus 299 ± 21 pg/ml, P < 0.02) was significantly higher in the Mexican-Americans than in the whites and urinary cyclic AMP (2.64 Ω 0.27 versus 2.59 ± 0.39 nM/dl GF) was not different in the two groups. The results indicate that Mexican-Americans are depleted of vitamin D and, as a result, have low serum 25-hydroxyvitamin D and compensatory increases in serum immunoreactive PTH and 1, 25-dihydroxyvitamin D. The depletion of vitamin D in Mexican-Americans is attributed to increased skin pigment

The CYP27A1 gene encodes a mitochondrial enzyme that modulates the acidic biosynthetic pathway for bile acids beginning with the 27-hydroxylation of cholesterol. CYP27A1 also 25-hydroxylates vitamin D(3). Gene mutations cause cerebrotendinous xanthomatosis (CTX), an autosomal recessive disorder, and may cause 25-hydroxyvitamin D deficiency and early-onset osteoporosis and fractures in affected patients. To examine the effects of mutations of CYP27A1 on vitamin D and cholesterol hydroxylating activity, recombinant CYP27A1 and mutant complementary DNAs produced by site-directed mutagenesis were stably expressed in either Escherichia coli or COS-1 cells. Activities of wild-type and mutant enzymes were determined with cholesterol, vitamin D(3), and 1alpha-hydroxyvitamin D(3) (1alphaOHD(3)) as substrates. Of the 15 mutants tested, 11 expressed protein and 4 expressed little or no protein. Functional heme activity, estimated by reduced CO difference spectra at 450 nm, was absent in 12 mutants. When expressed in E. coli, 3 mutants, K226R, D321G, and P408S, each known to cause clinically CTX, showed modest decreases in reduced CO spectra peak and either no change or decreases of less than 50% in hydroxylation of cholesterol, vitamin D(3), and 1alphaOHD(3) compared with wild type. When expressed transiently in COS-1 cells, each of these mutants showed 25-hydroxylation activity for 1alphaOHD(3) as well as wild type. Thus, 3 mutants, K226R, D321G, and P408S, known to occur clinically with nonfunctioning mutants, hydroxylated cholesterol, vitamin D(3), and 1alphaOHD(3). How they contribute to the pathogenesis of CTX despite being biologically active in vitro remains to be determined.

To assess the effects of growth hormone (GH) on serum 1,25-dihydroxyvitamin D [1,25(OH)2D], we performed the following prospective crossover study in six healthy, young, adult, white men. During each of two admissions for 2½ days to a general clinical research center, subjects were placed on a daily dietary calcium intake of 400 mg. Serum calcium, phosphorus, 1,25(OH)2D, immunoreactive intact parathyroid hormone (PTH), insulin-like growth factor I (IGF-I), IGF binding protein 3 (IGFBP3), tubular reabsorption of phosphate (TRP), and maximum tubular reabsorption of phosphate (TMP/GFR) were measured. Recombinant human GH (rhGH, Humatrope) (25 μg/kg/day subcutaneously for 1 week) was administered prior to and during one of the admissions. Results are expressed as mean ± SEM. Whereas serum 1,25(OH)2D (58.9 ± 7.7 versus 51.6 ± 7.4 pg/ml, P < 0.01), serum phosphorus (4.5 ± 0.1 versus 3.7 ± 0.1 mg/dl, P < 0.01), TRP (92.0 ± 0.5 versus 87.8 ± 0.7 mg/dl, P < 0.005), TMP/GFR (4.6 ± 0.1 versus 3.5 ± 0.2, P < 0.005), and urinary calcium (602 ± 49 versus 346 ± 25 mg/day, P < 0.001) increased significantly, serum PTH decreased significantly (19.9 ± 1.9 versus 26.8 ± 4.0 pg/ml, P < 0.05) and serum calcium did not change when subjects received rhGH. These findings indicate that in humans, GH affects serum 1,25(OH)2D independently of circulating PTH and that this effect is mediated by IGF-I. We propose, therefore, that one potential mechanism by which GH stimulates increases in bone mass is via modest increases in serum 1,25(OH)2D.

The incidence of osteoporosis and fractures of the hip and spine is lower in black than in white subjects. To determine whether bone mass is increased in black men and to assess the influence of body weight and age, bone mineral density (BMD) of the lumbar spine, trochanter, and femoral neck was measured by dual-photon absorptiometry in 59 normal white men and 40 black men between the ages of 20 and 50 years. Body weight and age were not different from each other in the two groups. BMD of the midradius was measured by single-photon absorptiometry. Multivariate regression was used for independent analysis of each group and for analysis of the two groups together. After adjusting for body weight, age was inversely related to BMD of the femoral neck in both blacks and whites and of the trochanter in blacks. When body weight was analyzed independently of age, it was a positive predictor for BMD of the midradius of black men and of the femoral neck in white men. Despite the racial differences in age and weight on BMD, there were no significant interactions between race and age or race and weight when the data from black and white men were combined. Race had a highly significant effect on BMD of the lumbar spine, trochanter, and femoral neck midradius, and BMD was higher in blacks than in whites at these sites. There were significant declines in BMD with age at the midradius and femoral neck and significant increases in BMD with body weight at the trochanter and femoral neck. Thus, bone mass is higher in black than in white men and the difference in bone mass may contribute to the lower incidence of osteoporosis and fractures in blacks.

Previous studies showed differences in bone and mineral metabolism in African-Americans and Caucasians: reductions in serum 25-hydroxyvitamin D [25(OH)D], urinary calcium and skeletal remodeling and moderate secondary hyperparathyroidism. Diurnal studies were carried out in 7 African-American and 7 white normal premenopausal women matched for age, weight and height to further characterize these racial differences in calcium homeostasis. Serum 25(OH)D was significantly lower and serum intact parathyroid hormone (PTH) was significantly higher in the African-American compared with the white women, whereas serum total calcium, Ca2+, phosphorus and 1,25-dihydroxyvitamin D [1,25(OH)2D] were not different in the two groups. Serum intact PTH increased significantly at night in the white women and did not change in the African-American women. Urinary calcium was 47% lower in the African-American than in the white women during the day but was not different at night. Urinary calcium declined at night by 53% in the white women and by 40% in the African-American women. Stepwise multivariate analysis showed that determinants of urinary calcium were mean 24 h serum intact PTH and serum Ca2+ in the two groups together, mean 24 h serum intact PTH, body mass index (BMI) and serum 25(OH)D in the white women, and BMI in the African-American women. Urinary N-telopeptide of type I collagen, a marker of bone resorption, increased by over 60% at night in both groups and was 25% lower in African-American compared with white women, but the difference was not statistically different. Urinary free deoxypyridinoline also increased at night in both groups and was not racially different. Thus, African-American women show higher serum intact PTH and greater conservation of calcium than white women throughout the day. In both groups, maintenance of serum calcium at night is achieved by increased bone resorption and renal conservation of calcium.

A number of previous investigations showed significant associations between polymorphisms of the vitamin D receptor (VDR) gene and bone mineral density (BMD). BMD is influenced by hormones and the rate of skeletal remodeling. A study was performed to investigate the possible relationship between Apa I, Bsm I, Taq I, and Fok I polymorphisms of the VDR gene and serum 1,25-dihydroxyvitamin D (1,25[OH]2D), osteocalcin, and propeptide of type I collagen (PICP)-markers of bone turnover, total body calcium, and BMD of the total body, radius, lumbar spine, trochanter, and femoral neck-in 39 young adult black men of 20 to 40 years of age and 44 age-, height-, and weight-matched white men. The distribution of each of the four alleles of the VDR genotypes was similar in the two racial groups. The Apa I VDR genotype was associated with serum PICP (P =.0494) but not with serum 1,25(OH)2D or serum osteocalcin. A significant association between the Apa I VDR genotype and BMD of the lumbar spine (P =.0291) was also observed. However, the Bsm I, Taq I, and Fok I genotypes were not significantly associated with BMD or serum osteocalcin, PICP, or 1,25(OH)2D. Multivariate stepwise analysis indicated that (1) the Apa I VDR genotype was associated with BMD of the lumbar spine in the two groups together; with total body calcium and BMD of the total body, radius, trochanter, and femoral neck in the black men; and with BMD of the radius in the white men; analysis also indicated that (2) race was significantly associated with total body calcium and BMD of the total body, lumbar spine, and femoral neck. In summary, the Apa I VDR genotype is associated with serum PICP and BMD at a number of sites but does not contribute to or account for racial differences in BMD in young adult men.

For steroid hormone function to occur, nuclear receptors interact with a series of coactivators including steroid receptor coactivator-1 (SRC-1). The SRC-1 binds the vitamin D receptor (VDR) in the presence of ligand in an activation function 2 (AF-2)-dependent manner. In order to understand the role of this interaction in 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]-mediated gene expression, the level of SRC-1 expression was altered in MG-63 cells. Previous studies had demonstrated that MG-63 cells express the VDR and that 1,25(OH)(2)D(3) regulates expression of alkaline phosphatase (ALP). Analysis of MG-63 cells demonstrated that SRC-1 is expressed. A full-length cDNA coding for SRC-1 was inserted in antisense orientation into an expression vector (anti-SRC-1). The MG-63 cells were transfected with anti-SRC-1 or mock vector and stable transformants were selected. Western blot analysis showed a 95% reduction in SRC-1 protein as compared with mock cells. We determined the effect of normal and reduced SRC-1 expression in MG-63 cells on 1,25(OH)(2)D(3)-mediated stimulation of ALP. Whereas 10(-8) M 1,25(OH)(2)D(3) produced a 3.6-fold stimulation in ALP in mock cells expressing normal levels of SRC-1, it did not alter ALP in cells expressing reduced levels of SRC-1. Thus, SRC-1 is required for 1,25(OH)(2)D(3)-mediated gene expression of ALP by human MG-63 cells.

Race is a major determinant of peak bone mass, bone density, and fracture. Available evidence indicates that Caucasian women and men have a lower bone mineral density and a higher risk of hip fracture than African American women and men. While Asian women have a bone mineral density similar to that of Caucasian women, their risk of fracture is less. African American, Hispanic, and Asian women have at least a 40% lower incidence of hip fracture and 50% lower incidence of fractures at other sites than Caucasian women. The difference in fracture rates appears to be related to a variety of hormonal, biochemical, and life-style differences between Caucasian and other racial groups. The fact that differences in bone mineral density, especially between African Americans and Caucasians, can be traced to early childhood provides evidence that bone mass may be determined by genetic as well as by environmental factors. A number of factors, some of which are controversial, have been proposed that would contribute to or be responsible for the racial difference in bone density. These include differences in metabolism of vitamin D and calcium; skeletal resistance to parathyroid hormone; lower rate of skeletal remodeling; differences in distribution of alleles of the vitamin D receptor; higher serum leptin and earlier puberty in girls; and higher serum testosterone, 17/3- estradiol, and rate of secretion of growth hormone in men.

Studies were performed to determine if the nonsteroidal anti-inflammatory drug ibuprofen alters bone and mineral metabolism in female rats. In experiment 1, four groups of growing rats underwent either sham operation or ovariectomy (OVX). One week later, controlled-release pellets with ibuprofen or placebo were implanted subcutaneously at the back of the neck. Following 3 weeks of treatment, rats were sacrificed and blood and bone samples were removed for serum assays and histomorphometric analysis. Body growth rate and the static cortical bone measurements made at the tibial diaphysis did not change in response to OVX. OVX, however, did increase radial bone growth, lowered serum 17beta-estradiol, reduced uterine weight, and decreased the cancellous bone area of the tibial metaphysis in the rats. Ibuprofen did not alter serum 17beta-estradiol or uterine weight but reduced radial bone growth as well as cancellous bone area of the tibial metaphysis in both sham-operated and OVX animals. In experiments 2 and 3, we tested the influence of ibuprofen on the effects of the tissue-selective estrogen agonist tamoxifen and of exogenous 17beta-estradiol in the OVX rat. Ibuprofen completely blocked the effects of tamoxifen and partially blocked the effects of 17beta-estradiol to prevent cancellous osteopenia. In contrast, ibuprofen did not influence the effects of tamoxifen and 17beta-estradiol to reduce radial bone growth. Besides the skeletal effects, ibuprofen suppressed estrogen-induced uterine growth. Our data suggest that ibuprofen blocks selective estrogen receptor-mediated activities in the rat.