Effect of a long-term treatment with 1,25-dihydroxyvitamin D3 on osteocalcin in postmenopausal osteoporosis

Summary Serum bone Gla-protein (BGP or osteocalcin) was measured in 25 women with histologically confirmed postmenopausal osteoporosis before and during long-term treatment with 1 μg/day of 1,25-dihydroxyvitamin D₃(1,25(OH)₂D₃). Basal serum BGP was significantly lower in osteoporotic women (3.8±1.4 ng/ml) than in agematched controls (6.8±2.0 ng/ml). During 1,25(OH)₂D₃ therapy serum BGP increased so that the mean of the values observed on treatment (4.8±1.5) was significantly higher than the mean basal value. It is known that BGP synthesis is stimulated by 1,25 (OH)₂D₃ and that serum BGP is a specific marker of bone formation; therefore, it is possible that the low basal levels of osteocalcin we observed were related to the low serum 1,25(OH)₂D concentrations reported in osteoporotic women and that the increase in BGP levels observed under 1,25(OH)₂D₃ treatment was a consequence of osteoblast stimulation.     

Alteration in osteoblast activity and nutritional vitamin-D deficiency in non-hypercalcemic malignancy

Summary The biochemical parameters of bone mineral metabolism in patients with nonhypercalcemic malignancy have not been extensively investigated. Therefore, a group of 29 such patients with different types of malignancy was studied. Ten patients received corticosteroids. In the entire group, serum ionized calcium (Ca²⁺), bone gla protein (BGP), 25-hydroxyvitamin D (25OHD), and 1,25-dihydroxyvitamin D (1,25(OH)₂D) were all lower than in age-matched controls, and carboxy-terminal parathyroid hormone (CPTH) was higher. Although both corticosteroid- and noncorticosteroid-treated patients had decreased BGP values, the corticosteroid-treated patients had lower BGP levels than those not on steroids (4.24±0.70 SE vs. 11.50±2.20 ng/ml;P<0.005). Patients on corticosteroids had lower 1,25(OH)₂D values than controls (18.81 ±2.71 vs. 27.83±1.17 pg/ml;P<0.01), whereas those not on corticosteroids had normal 1,25(OH)₂D values. These results suggest that patients with nonhydpercalcemic malignancy have nutritional vitamin-D deficiency and secondary hyperparathyroidism with perhaps corticosteroid-induced suppression of serum 1,25(OH)₂D and BGP. The decreased levels of serum BGP in the nonsteroid-treated patients suggest, in addition, a defect in osteoblast function.     

Comparison of metabolism of vitamins D2 and D3 in children with nutritional rickets

Children with calcium‐deficiency rickets may have increased vitamin D requirements and respond differently to vitamin D₂ and vitamin D₃. Our objective was to compare the metabolism of vitamins D₂ and D₃ in rachitic and control children. We administered an oral single dose of vitamin D₂ or D₃ of 1.25 mg to 49 Nigerian children—28 with active rickets and 21 healthy controls. The primary outcome measure was the incremental change in vitamin D metabolites. Baseline serum 25‐hydroxyvitamin D [25(OH)D] concentrations ranged from 7 to 24 and 15 to 34 ng/mL in rachitic and control children, respectively (p < .001), whereas baseline 1,25‐dihydroxyvitamin D [1,25(OH)₂D] values (mean ± SD) were 224 ± 72 and 121 ± 34 pg/mL, respectively (p < .001), and baseline 24,25‐dihydroxyvitamin D [24,25(OH)₂D] values were 1.13 ± 0.59 and 4.03 ± 1.33 ng/mL, respectively (p < .001). The peak increment in 25(OH)D was on day 3 and was similar with vitamins D₂ and D₃ in children with rickets (29 ± 17 and 25 ± 11 ng/mL, respectively) and in control children (33 ± 13 and 31 ± 16 ng/mL, respectively). 1,25(OH)₂D rose significantly (p < .001) and similarly (p = .18) on day 3 by 166 ± 80 and 209 ± 83 pg/mL after vitamin D₂ and D₃ administration, respectively, in children with rickets. By contrast, control children had no significant increase in 1,25(OH)₂D (19 ± 28 and 16 ± 38 pg/mL after vitamin D₂ and D₃ administration, respectively). We conclude that in the short term, vitamins D₂ and D₃ similarly increase serum 25(OH)D concentrations in rachitic and healthy children. A marked increase in 1,25(OH)₂D in response to vitamin D distinguishes children with putative dietary calcium‐deficiency rickets from healthy children, consistent with increased vitamin D requirements in children with calcium‐deficiency rickets. © 2010 American Society for Bone and Mineral Research     

Serum bone gla-protein in osteoporosis treated with 1α-hydroxyvitamin D3 for more than five years

It has been reported that 1,25-dihydroxyvitamin D₃ increases serum bone Gla-protein (BGP) in a short period in osteoporotic patients as well as in normal subjects. There have been, however, no reports on serum BGP in osteoporotic patients under long term treatment with 1α-hydroxyvitamin D₃. We measured serum BGP in 11 osteoporotic women treated with 1α-hydroxyvitamin D₃ and calcium for 5–12 years, 8.4 years on average. Bone mineral density of distal radius was assessed by single photon absorptiometry. Other biochemical parameters such as serum alkaline phosphatase, fasting urinary hydroxyproline/creatinine and calcium/creatinine were also measured. Serum BGP levels were 6.25±0.36ng/ml (mean±S.E.), being all within the normal range (6.2±3.86ng/ml). We found no significant correlation between serum BGP and other biochemical parameters. Significant correlation was found neither between serum BGP and period of treatment nor between serum BGP and bone mineral density. Our result that serum BGP is within the normal range in osteoporotic patients whose bone mineral density has been maintained by long-term treatment suggests the normal bone turnover in these patients.     

Vitamin D metabolism in pregnant and pseudopregnant rats: Identification of 25-hydroxycholecalciferol-1-hydroxylase in decidual tissue

Summary Elevated levels of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are found in late pregnancy but the factors responsible for this are not known. To determine if the maternal-fetal calcium flux or the presence of a previously described extrarenal 25-hydroxycholecalciferol-1-hydroxylase (25(OH)-D₃-1-hydroxylase) play a role, serum calcium and 1,25(OH)₂D₃ were measured in pregnant, nonpregnant, and decidua-bearing pseudopregnant rats. Serum calcium was 8.74± 0.26 mg/dl (mean±SEM) in nonpregnant rats. In pregnant rats, serum calcium was not significantly different from nonpregnant controls on day 12 and only slightly higher on day 15. Pseudopregnant rats were significantly hypercalcemic on days 12 (11.93±0.19 mg/dl) and 15 (11.45±0.23 mg/dl) compared with nonpregnant rats (P<0.001). In nonpregnant controls the serum level of 1,25(OH)₂D₃ was 44.6±6.3 pg/ml. Levels in pregnant rats were not significantly different on days 12 or 15 but tended to be higher by day 15 (75.2±19.7 pg/ml). Pseudopregnant rats had levels of 72.6±13.5 pg/ml on day 12 and 102.8±10.9 pg/ml on day 15, the latter of which was significantly higher than nonpregnant values (P<0.05). 25(OH)D₃-1-hydroxylase activity was determined in whole tissue homogenates of placenta and decidua. Placenta from pregnant rats and decidua from pregnant and pseudopregnant rats both formed putative 1,25(OH)₂D₃ in short-term incubation with 25(OH)D₃ as identified by comigration with authentic 1,25(OH)₂D₃ on high pressure liquid chromatography (HPLC). The results suggest that hypercalcemia during pseudopregnancy is due to unregulated production of 1,25(OH)₂D₃ by decidual tissue. Pregnant rats may not become hypercalcemic because the fetus acts as a calcium sink or the 25(OH)D₃-1-hydroxylase is under regulation in the pregnant state.     

Idiopathic juvenile osteoporosis: Evidence of normal osteoblast function by 1,25-dihydroxyvitamin D3 stimulation test

Summary Idiopathic juvenile osteoporosis (IJO) is a rare form of bone demineralization that occurs during childhood. The mechanism of bone loss is unclear. Some bone hystomorphometric studies have found osteoblast failure and decreased bone formation in the affected patients whereas others have reported increased bone resorption. To elucidate this issue, we studied osteoblast function in six patients with IJO (five males, one female; aged 2.3–14.6 years) and five healthy sex- and age-matched subjects (four males, one female; aged 2.0–15.1 years) measuring serum values of osteocalcin under basal condition and during an osteoblast stimulation test performed by oral 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] administration (1.8 g/1.73 m²/daily). After a baseline day (day 0), all the subjects (patients and controls) received 1,25(OH)₂D₃ in four divided doses for 6 days (days 1–6). Fasting blood samples were obtained every morning (0800 h) for the determination of serum osteocalcin. Baseline osteocalcin levels were not significantly different between IJO and controls (13.58±6.05 ng/ml versus 16.04±5.09 ng/ml, respectively) even if two patients had low osteocalcin values. During 1,25(OH)₂D₃ administration, serum osteocalcin values significantly increased (P<0.001) from baseline in both children with IJO and controls, reaching peak values not significantly different in the two groups. Our results do not support the hypothesis that defective osteoblast function is the primary factor of bone demineralization in IJO.     

Low Bone Density with Normal Bone Turnover in Ovariectomized and Streptozotocin-Induced Diabetic Rats

Diabetes and estrogen deficit are known causes of osteopenia, diabetes being associated with a low bone turnover and estrogen deficit with a high bone turnover. In the present work, we studied the effect of combined ovariectomy and diabetes on bone mineral content (BMC) and bone mineral density (BMD) and several bone markers in the rat. Four groups of rats were studied: control (C), ovariectomized (O), diabetic (D), and ovariectomized and diabetic (DO). Twelve weeks after starting the experiments, BMC and BMD of the first six lumbar vertebrae were measured; a bone formation marker (BGP) and a bone resorption marker (free collagen cross-links, PYD) were also analyzed. Diabetic rats showed diminished gain in bone mass, BMC (D: 0.417 ± 0.028 g, DO: 0.422 ± 0.020 g) and BMDs (D: 0.171 ± 0.006 g/cm², DO: 0.174 ± 0.006 g/cm²) both being significantly (P < 0.001) lower than those of control (C: BMC 0.727 ± 0.024 g and BMD 0.258 ± 0.004 g/cm²) and ovariectomized (O: BMC 0.640 ± 0.044 g and BMD 0.240 ± 0.009 g/cm²) groups. Moreover, the BMC and BMD of the C group were significantly (P < 0.05) higher than that of the O group. BGP and PYD levels were significantly (P < 0.01) higher in the O group (BGP: 138.2 ± 16.8 ng/ml, PYD: 270.2 ± 17.8 nM/mM) than those found in the control rats (BGP: 44.7 ± 4.8 ng/ml, PYD: 165.6 ± 12.5 nM/mM); the D group showed significantly (P < 0.01) lower values (BGP: 27.4 ± 14.6 ng/ml, PYD: 55.0 ± 7.4 nM/mM) than those of the control group. The DO group showed similar levels (BGP: 43.4 ± 5.1 ng/ml, PYD: 146.7 ± 14.6 nM/mM) to those found in the C group. Although bone marker levels in the O and D groups were in accordance with those expected in these situations, in the DO group the corresponding levels are apparently ``normal.' Also, the decrease of gain in bone mass observed after combining estrogen deficit and diabetes (DO group) did not seem to be more marked than that caused by diabetes alone. Received: 7 January 1997 / Accepted: 7 August 1997     

Effects of 1,25(OH)2D3 on bone tissue in the rabbit: Studies on fracture healing,disuse osteoporosis,and prednisone osteoporosis

Summary A closed tibial fracture, which was controlled by an intramedullary stainless steel pin, was created in 16 rabbits. Eight rabbits were treated with 75 ng of 1,25(OH)₂D₃ daily as subcutaneous (s.c.) injections. After three weeks, the fractured tibia resisted a force of 101,7±21.0 Newtons in the control group and 57.3±8.0 Newtons in animals given 1,25(OH)₂D₃ (m±SE,P<0.05). In another group of eight rabbits, the left hindleg was immobilized in a plastic splint. Four rabbits were given 75 ng of 1,25(OH)₂D₃/day s.c. and the effect of immobilization was studied on the calcaneus. Bone ash/cm³ of the calcaneus on the immobilized side was decreased by 11±2% in control rabbits and by 20±2% in the group treated with 1,25(OH)₂D₃ indicating a more advanced immobilization osteoporosis (m±SE,P<0.05), which was also demonstrated by studies of bone density. Eighteen rabbits were used in a study of the effects of 1,25(OH)₂D₃ on the development of prednisolone osteoporosis. The dose of prednisolone was 2.5 mg per day, given by the oral route. After four months, the density of the femur was 1.53±0.02 g/cm² in control rabbits and 1.42±0.01 in prednisolonetreated animals (P<0.01). In rabbits additionally given 1,25(OH)₂D₃, the mean value for bone density was further lowered (n.s.). It appears that 1,25(OH)₂D₃ exaggerates disuse osteoporosis and prednisolone osteoporosis and impairs fracture healing in rabbits. These results differ from what has been shown earlier with 1,25(OH)₂D₃ treatment in the rat.     

Enhanced suppression of 1,25(OH)2D3 and intact parathyroid hormone in Graves' disease as compared to toxic nodular goiter

Summary 1,25(OH)₂D₃, 25OHD₃, and intact parathyroid hormone, as well as various parameters of calcium-phosphorus metabolism were measured in 38 patients with Graves' disease (GD) and in 24 patients with toxic nodular goiter (TNG). Plasma 1,25(OH)₂D₃ levels were lower in GD patients (82 ±29 pmol/liter) than in those with TNG (155±32 pmol/liter) (P<0.0005). The mean value of 1,25(OH)₂D₃ in 45 controls was intermediate between the two groups of patients (140±41) and the difference was statistically significant. GD patients before and after treatment had higher alkaline phosphatase (P<0.05), lower intact parathyroid hormone (PTH) (P<0.05), and lower 1,25(OH)₂D₃ levels (P<0.0005 in the hyperthyroid andP<0.01 in the euthyroid state) than TNG patients. We conclude that increased skeletal calcium resorption is due to elevated levels of T₃ causing suppression of 1,25(OH)₂D₃ production and of PTH levels in both groups of patients albeit of different degrees. Furthermore, we postulate that the profound suppression of 1,25(OH)₂D₃ in GD is secondary to an immune-mediated phenomenon.     

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

Summary To determine the role of the kidney in regulation of 25-hydroxycholecalciferol (25OHD₃, metabolism, the effects of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] on³H-25OHD₃ were compared in intact and nephrectomized vitamin D-deficient rats. Sixteen hours after the intravenous administration of³H-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 serum³H-24,25-dihydroxycholecalciferol [³H-24,25(OH)₂D₃] from 2±2–210±80 fmol/ml (mean±1 SD), increased mean serum³H-25,26-dihydroxycholecalciferol [³H-25,26(OH)₂D₃] from 2±2–12±6 fmol/ml and lowered mean serum³H-1,25(OH)₂D₃ from 210±40–4±4 fmol/ml. Similarly, in nephrectomized animals, 1,25(OH)₂D₃ increased mean serum³H-24,25-(OH)₂D₃ from 6±11–115±30 fmol/ml and increased mean serum³H-25,26(OH)₂D₃ from 3±3–26 ± 10 fmol/ml. Nephrectomy increased serum³H-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. Further, the observed stimulation of production of 24,25(OH)₂D₃ and 25,26(OH)₂D₃ by 1,25(OH)₂D₃ in anephric — D rats providesin vivo evidence for regulation of extrarenal 25OHD₃: 24- and 26-hydroxylases.     

1,25 dihydroxyvitamin D3 modifies cyclosporine-induced bone loss

Summary We have previously shown that cyclosporin A (CsA) produces high bone remodeling with resorption exceeding formation and loss of bone volume in the rat. This may have important clinical implications where CsA is widely used in organ transplantation. 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) is a bone mineralizing hormone which also has immune modifying properties. Consequently, we studied the effect of combined CsA and 1,25(OH)₂D₃ administration over 28 days in four groups of rats. Group A received vehicle (n=10), group B CsA (15 mg/kg) (n=10) alone, group C 1,25(OH)₂D₃ plus CsA (n=15), and group D 1,25(OH)₂D₃ alone (20 ng/100 g) (n=15). Rats were bled periodically at day 0, 7, 14, and 28 and Ca, parathyroid hormone (PTH), 1,25(OH)₂D, osteocalcin (bone Gla-protein, BGP), BUN, and creatinine were measured. Rats were sacrificed on day 28 and bones were examined histomorphometrically. Compared to controls, CsA resulted in significant elevation of BGP and a transient increase in 1,25(OH)₂D with excess bone remodeling and loss of bone volume. 1,25(OH)₂D₃ administration produced hypercalcemia, a significant rise in BGP, with suppression of PTH and increased osteoid volume. Combined therapy prevented the loss of bone volume probably due to increased osteoid tissue and enhanced osteoblast activity. Renal dysfunction, a side-affect of CsA, was not a factor. In conclusion, 1,25(OH)₂D₃ combined with CsA restores bone volume which is accompanied by increases in serum calcium and BGP.     

Calcitonin and bisphosphonates treatment in bone loss after liver transplantation

Osteopenia is a major complication of orthotopic liver transplantation (OLT). However, no effective therapy for bone disease has been defined. We have studied vertebral bone mineral density (VMD) and fasting serum markers of bone formation [bone gla protein (BGP), procollagen I carboxyterminal peptide (PICP)] and metabolism (serum Ca, P, intact parathyroid hormone (iPTH), 25OHD₃ and 1,25(OH)₂D₃) in 120 patents after OLT. VMD was measured by dual-energy X-ray absorptiometry (DXA) using a Hologic QDR 1000 densitometer on two occasions, 12 months apart. Patients with OLT had a VBD significantly lower compared with age- and sexed-matched Spanish controls (P<0.05). Prevalence of osteoporosis (Z score below-2 SD) was 35.8%. Serum BGP (8.6±0.7 ng/ml) and PICP (222.9±81.9 ng/dl) were higher than those of controls. However, serum calcium, phosphorus, iPTH, 25OHD₃, and 1,25(OH)₂D₃ were within normal range. Patients with osteoporosis were randomly treated with 40 IU/day of calcitonin i.m. (Diatin, Ferrer Int. Laboratories) (n=17) or 400 mg p.o., 15 days every 3 months, of sodium ethiodronate (Difosfen, Rubio Laboratories) (n=23). All patients received 500 mg/12 hours of elemental calcium p.o. After 12 months of treatment, a significant increment of vertebral mineral density (VMD) was observed (6.4% and 8.2%, respectively). Serum BGP and PICP values remained elevated without a difference between the two drugs. Our results indicate that antiresorptive drugs may be of benefit in the high turnover osteoporosis of OLT recipients.     

Effects of hPTH(1‐34) Infusion on Circulating Serum Phosphate, 1,25‐Dihydroxyvitamin D,and FGF23 Levels in Healthy Men

Fibroblast growth factor 23 (FGF23) promotes phosphaturia and suppresses 1,25‐dihydroxyvitamin D [1,25(OH)₂D] production. PTH also promotes phosphaturia, but, in contrast, stimulates 1,25(OH)₂D production. The relationship between FGF23 and PTH is unclear, and the acute effect of pharmacologically dosed PTH on FGF23 secretion is unknown. Twenty healthy men were infused with human PTH(1‐34) [hPTH(1‐34)] at 44 ng/kg/h for 24 h. Compared with baseline, FGF23, 1,25(OH)₂D, ionized calcium (iCa), and serum N‐telopeptide (NTX) increased significantly over the 18‐h hPTH(1‐34) infusion (p < 0.0001), whereas serum phosphate (PO₄) transiently increased and then returned to baseline. FGF23 increased from 35 ± 10 pg/ml at baseline to 53 ± 20 pg/ml at 18 h (p = 0.0002); 1,25(OH)₂D increased from 36 ± 16 pg/ml at baseline to 80 ± 33 pg/ml at 18 h (p < 0.0001); iCa increased from 1.23 ± 0.03 mM at baseline to 1.46 ± 0.05 mM at hour 18 (p < 0.0001); and NTX increased from 17 ± 4 nM BCE at baseline to 28 ± 8 nM BCE at peak (p < 0.0001). PO₄ was 3.3 ± 0.6 mg/dl at baseline, transiently rose to 3.7 ± 0.4 mg/dl at hour 6 (p = 0.016), and then returned to 3.4 ± 0.5 mg/dl at hour 12 (p = 0.651). hPTH(1‐34) infusion increases endogenous 1,25(OH)₂D and FGF23 within 18 h in healthy men. Whereas it is possible that the rise in PO₄ contributed to the observed increase in FGF23, the increase in 1,25(OH)₂D was more substantial and longer sustained than the change in serum phosphate. Given prior data that suggest that neither PTH nor calcium stimulate FGF23 secretion, these data support the assertion that 1,25(OH)₂D is a potent physiologic stimulator of FGF23 secretion.     

Regulation of Murine Osteoblast Macrophage Colony-Stimulating Factor Production by 1,25(OH)2D3

Macrophage colony stimulating factor (MCSF) is important for formation of osteoclasts. We investigated the ability of 1,25(OH)₂D₃ to regulate osteoblast production of MCSF. Mouse calvarial osteoblasts were cultured for 2 days ± 1,25(OH)₂D₃. Since 1,25(OH)₂D₃ decreased osteoblast proliferation by 17.6 ± 1% at 10 nM and 11 ± 4% at 1 nM, the effect of growth rate on MCSF secretion was examined. Limiting cell proliferation by serum did not affect MCSF production. 1,25(OH)₂D₃ (1 nM) increased MCSF production (U/10⁵ cells) maximally by 68 ± 33% (n = 3) with an ED₅₀ for 1,25(OH)₂D₃ of 5 × 10⁻¹¹ M. To investigate effects of 1,25(OH)₂D₃ on MCSF gene regulation, RT-PCR primers were designed to identify the mRNA coding for the membrane-bound isoform of MCSF. Simultaneous RT-PCR of glyceraldehyde-phosphate dehydrogenase (GAP) allowed semiquantitative assessment of MCSF mRNA between treatment groups expressed as the MCSF/GAP RT-PCR product ratio; both MCSF and GAP (+) primers were labeled with ³²P-ATP for phosphorimage quantitation. The membrane-bound MCSF/GAP PCR product ratio was not affected by proliferative rate when growth was limited by [serum]. The MCSF/GAP RT-PCR product ratio was dose dependently increased by 1,25(OH)₂D₃, maximally at 1 nM at 2.2 ± 0.2 = fold (n = 10). 1,25 (OH)₂D₃ also increased the expression of an RT-PCR MCSF/GAP product ratio which represented the secreted isoform of MCSF. The ability of 1,25(OH)₂D₃ to pretranslationally regulate expression of membrane-bound osteoblast MCSF may be important in osteoblast:osteoclast interactions. Received: 12 August 1995 / Accepted: 25 March 1996     

Human parathyroid hormone (1–34) and salmon calcitonin do not reverse impaired mineralization produced by high doses of 1,25 dihydroxyvitamin D3

Summary We have reported recently that pharmacologic doses of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) stimulated bone matrix formation but impaired mineralization. The objective of this study was to determine if parathyroid hormone (hPTH 1-34) or calcitonin (sCT) would mineralize the osteoid induced by 1,25(OH)₂D₃ in rat long bones. In one experiment, male Sprague-Dawley rats were given daily subcutaneous injections of vehicle: 8 μg hPTH(1-34); 125 ng 1,25(OH)₂D₃; or both 8 μg hPTH and 125 ng 1,25(OH)₂D₃ per 100 g body weight for 12 days. In a second experiment, rats received daily injections of vehicle: 2 U sCT; 125 ng 1,25(OH)₂D₃; or both 2 U sCT and 125 ng 1,25(OH)₂D₃ per 100 g body weight for 18 days. Calcium (Ca), hydroxyproline (Hyp), and dry weight (DW) of the distal femur and serum calcium, phosphate, and serum bone Gla protein (BGP) were measured. In rats given both 1,25(OH)₂D₃ and hPTH, total bone DW and Hyp increased (P<.01) without a corresponding increase in bone Ca so that Ca/Hyp decreased 47% (P<.01) from control and remained comparable to values for rats treated with 1,25(OH)₂D₃ alone. In rats treated with both 1,25(OH)₂D₃ and sCT, total bone DW and Hyp increased while Ca decreased so that Ca/Hyp decreased 38% from control (P<.05), and remained comparable to values for rats treated with 1,25(OH)₂D₃ alone. These results indicate that hPTH or sCT, given by intermittent injection to rats for 12 or 18 days respectively, failed to mineralize the osteoid induced by high doses of 1,25(OH)₂D₃.     

Persistent hypercalciuria and elevated 25-hydroxyvitamin D3 in children with infantile hypercalcaemia

The aim of the study was to characterize abnormalities of calcium-phosphate and vitamin D₃ metabolism in children with a past history of “mild” Lightwood-type idiopathic infantile hypercalcaemia. Seventeen seemingly healthy children aged 2 – 12 years, with long-term idiopathic hypercalcaemic syndrome since infancy were studied. Two reference groups were also included (vitamin D₃ intoxication/healthy and Williams groups). Despite a long-term milk-restricted diet and a restricted vitamin D₃ intake, urinary calcium excretion in the study group was 0.117±0.07 mmol/kg per 24 h. Compared with the reference groups (0.047±0.029 and 0.067±0.06 mmol/kg per 24 h, P<0.05), there was significant hypercalciuria in the children with idiopathic hypercalcaemia since infancy. Serum concentrations of 25-hydroxyvitamin D₃ in the study group were also elevated compared with the reference groups (57.4±15.5 vs. 34.6±9.3 and 22.7±10.5 ng/ml). 1,25-Dihydroxyvitamin D₃ levels were at the upper limit of normal (45.9±13.1 vs. 35.0±8.1 and 30.0±13.7 pg/ml). Non-progressive, clinically silent nephrocalcinosis was visible on ultrasound examinations. The disturbances of vitamin D₃ and calcium-phosphate metabolism persistent in the normocalcaemic phase of idiopathic infantile hypercalcaemia may be a primary metabolic defect of the condition. The mechanisms leading to elevation of metabolites of 1,25-dihydroxy- and 25-hydroxyvitamin D₃ and the relationship between this and persistent hypercalciuria and nephrocalcinosis need pathophysiological explanation. Received September 22, 1995; received in revised form May 3, 1996; accepted May 7, 1996     

The dichotomy in the effects of 1,25 dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 on bone γ-carboxyglutamic acid-containing protein in serum and bone in vitamin D-deficient rats

Summary Vitamin D-deficient, second generation, rachitic rats showed significant decrease in bone Gla protein (BGP) levels in circulation and in the skeleton. 1,25 dehydroxyvitamin D₃ (1,25 (OH)₂D₃) exhibited the most potent influence on serum BGP levels in a dose-dependent manner. At a dose 25 ng/100 g body weight 1,25 (OH)₂D₃ showed a cumulative effect, i.e., the longer the treatment, the more circulating BGP was detected 24,25 dehydroxyvitamin D₃ (24,25(OH)₂D₃) at the same doses did not show similar effect on the serum BGP levels, regardless of the serum calcium levels. Bone BGP levels assayed at various sites representing endochondral and intramenbranous ossification demonstrated an opposite pattern. 1,25(OH)₂D₃ administration was not sufficient to restore bone BGP levels to normalcy, whereas in animals treated with 24,25(OH)₂D₃ bone BGP and calcium levels were significantly higher than control (Vitamin D₃-repleted) levels. The present results can be explained by the dual action of 1,25 (OH)₂D₃ on both synthesis and release of BGP by bone turnover, whereas 24,25 (OH)₂D₃ stimulates synthesis and accumulation of BGP in bone. These observations imply that caution is required in the interpretation of clinical data based solely on serum BGP determination.     

Serum bone GLA protein in streak gonad syndrome

Summary Osteoporosis is one of the most common complications of streak gonad syndrome (SGS), however its pathogenesis is still unclear. Bone Gla protein (BGP) has been found to be a serum marker of bone turnover in various metabolic disease states. In the present study serum BGP and alkaline phosphatase (AP) were measured in 13 osteoporotic patients with SGS and in 56 healthy women. Mean (±SD) serum BGP levels were normal (7.5±2.0 ng/ml) in seven patients who had been on estrogen-progestin replacement therapy and became significantly elevated (P<0.001) 2 and 3 months after discontinuation of the treatment (15.3±2.3 and 13.2±1.0 ng/ml, respectively). Mean (±SD) serum AP (207±65 U/l) showed significant increases (P<0.05) 2 months after withdrawal of hormonal substitution (287±74 U/l). Mean (±SD) serum BGP (15.4±3.5) and AP (287±49) levels were significantly higher (P<0.001 and <0.05, respectively) in six patients with SGS who had not been on hormonal substitution. These findings are consistent with those obtained in postmenopausal women suffering from “high remodelling osteoporosis” and suggest that bone turnover in osteoporotic patients with SGS is increased and the skeletal loss is a consequence of accelerated bone loss rather than decreased bone formation.     

Modulation by epidermal growth factor of the basal 1,25(OH)2D3 receptor level and the heterologous up-regulation of the 1,25(OH)2D3 receptor in clonal osteoblast-like cells

Summary The effects of epidermal growth factor (EGF) on basal 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) receptor level and on parathyroid hormone (PTH)-induced 1,25-(OH)₂D₃ (OH)₂D₃ receptor up-regulation were studied in the phenotypically osteoblastic cell line UMR 106. EGF in concentrations exceeding 0.1 ng/ml reduced the number of 1,25(OH)₂D₃ binding sites without changing the binding affinity. Maximal reduction was 30% at about 1 ng/ml. This reduction was independent of a change in cAMP content. EGF dose-dependently attenuated both PTH-induced 1,25(OH)₂D₃ receptor up-regulation and PTH-stimulated cAMP production without and effect on the ED₅₀ of the PTH effects. For both PTH responses the IC₅₀ and the maximal effective dose were similar, 0.1 ng/ml an 1 ng/ml EGF, respectively. Reduction was first seen at 0.01 ng/ml EGF. At this concentration. EGF reduced PTH-stimulated 1,25-(OH)₂D₃ receptor binding without an inhibition of the cAMP response. Time-course studies with 1 ng/ml EGF revealed that at 2 h preincubation EGF reduced the heterologous up regulation by PTH, and maximal inhibition was seen after 4 h. In contrast, PTH-stimulated cAMP production was just significantly inhibited only after 6 h, with 60% inhibition after 24 h preincubation. The effects of prostaglandin E₂ and forskolin on both 1,25(OH)₂D₃ binding and cAMP production were inhibited in a similar fashion. On the other hand, dibutyryl cAMP- and 3-isobutyl-1-methylxanthinestimulated 1,25(OH)₂D₃ binding were not affected by EGF. Taken together, our results demonstrate that EGF reduces both the basal number of 1,25(OH)₂D₃ binding sites and the heterologous up-regulation of the 1,25(OH)₂D₃ receptor. The current data suggest that EGF reduces heterologous upregulation of the 1,25(OH)₂D₃ receptor independent of as well as dependent on the cAMP messenger system. The EGF effect is not primarily located at the PTH receptor, at cAMP phosphodiesterase, or at protein kinase A level.