Bone resorption induced by 1 alpha,25 dihydroxyvitamin D(3) in vivo is not altered by inactivation of the plasminogen activator inhibitor 1

One of the proteolytic systems produced by bone cells is the plasminogen activator/plasmin pathway, which involves the two plasminogen activators and the type 1 plasminogen activator inhibitor (PAI-1) and results in plasmin generation. We have recently demonstrated that this pathway plays a specific role in the degradation of the nonmineralized matrix of bone in vitro. To evaluate whether PAI-1 is required during bone resorption in vivo, we studied the effects of PAI-1 inactivation on bone metabolism using systemic administration of 1alpha,25 dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)] as model. PAI-1-deficient (PAI-1-/-) and wild-type (WT) mice were injected intraperitoneally with 1,25(OH)(2)D(3) (2 microg/kg) or vehicle every other day during 4 weeks and analyzed using biochemical parameters of bone turnover, histomorphometric analysis of the proximal tibial metaphysis, and pQCT analysis of the distal femoral metaphysis. PAI-1 inactivation did not affect bone metabolism in vehicle-treated mice. Treatment with 1,25(OH)(2)D(3) induced bone resorption similarly in PAI-1-/- and WT mice, as assessed by the increase in the urinary excretion of calcium (2. 2-fold and 2.3-fold, respectively) and of pyridinoline crosslinks (by 24% and 22%, respectively). In addition, a comparable reduction in bone mass was observed in PAI-1-/- and WT mice after treatment with 1,25(OH)(2)D(3), as evidenced by the decrease in the femoral calcium content (by 25% and 32%, respectively), in the trabecular bone volume (by 50% and 40%, respectively), in the trabecular mineral content (by 17% and 15%, respectively), and in the cortical mineral content (by 45% and 52%, respectively). The parameters of bone turnover also increased after 1,25(OH)(2)D(3) treatment. Serum osteocalcin was, respectively, 25% and 28% higher in PAI-1-/- and WT mice treated with 1,25(OH)(2)D(3) compared with the mice injected with vehicle. Similarly, the osteoid surface increased in 1, 25(OH)(2)D(3)-treated PAI-1-/- and WT mice by 40% and 51%, respectively, the mineral apposition rate increased by 15% and 8%, respectively, and the bone formation rate by 54% and 48%, respectively. These data indicate that PAI-1 is not critical during bone resorption induced by 1,25(OH)(2)D(3) in vivo.     

Bone metabolism in cholestatic children before and after living-related liver transplantation--a long-term prospective study

Bone disorders are common in children with end-stage liver diseases, especially those associated with cholestasis. Abnormal hepatocyte function, disordered vitamin D metabolism and calcium-phosphorous homeostasis, malnutrition, and immunosuppressive treatment are potential risk factors of bone tissue pathology before and after transplantation. The aim of the study was to analyze the long-term effect of successful living-related liver transplantation (LRLTx) on skeletal status and bone metabolism in cholestatic children. Eighteen cholestatic children (1.4±0.5yr old; 12 females [F]/6 males [M]) qualified for LRLTx were analyzed; 16 (5F/11M) of them participated in long-term observation (V4). Serum levels of osteocalcin (OC), procollagen type 1 N-terminal propeptide (P1NP), cross-linked telopeptide of type 1 collagen (CTx), insulin-like growth factor I (IGF-I), IGF-I binding protein 3 (IGFBP-3), parathyroid hormone (PTH), 25-hydroxyvitamin D (25(OH)D), and 1,25-dihydroxyvitamin D (1,25(OH)(2)D) were assayed before (V0) and 6mo (V1), 12mo (V2), 18mo (V3), and 4.4yr (V4) after LRLTx. Total body bone mineral content (TBBMC) and total body bone mineral density (TBBMD) were measured by dual-energy X-ray absorptiometry (DXA) at the same pattern. Before LRLTx, the OC, P1NP, CTx, IGF-I, and IGFBP-3 levels as well as TBBMC and TBBMD were decreased compared with age-matched control group. The mean serum levels of 25(OH)D and 1,25(OH)(2)D were within reference ranges from V0 to V4. After LRLTx, the OC, P1NP, CTx, IGF-I, and IGFBP-3 as well as TBBMC and TBBMD reached the age-matched reference values. At V4, the level of P1NP decreased below and the PTH increased above the reference range that coincided with reduced Z-scores of both TBBMC (-1.11±1.24) and TBBMD (-1.00±1.19). P1NP and CTx, both measured at V3, correlated with IGF-I at V2 (R=0.86, p=0.014 and R=0.78, p=0.021, respectively) and PTH at V3 for P1NP and V1 for CTx (R=0.64, p=0.048 and R=0.54, p=0.038, respectively). The TBBMC changes between V0 and V4 correlated with IGF-I (R=0.68, p=0.015) and 1,25(OH)(2)D (R=0.54, p=0.025), both assayed at V1. The change of TBBMC Z-scores between V0 and V4 correlated with P1NP at V1 (R=0.69, p=0.002). The TBBMD changes between V0 and V4 correlated with CTx at V1 (R=0.54, p=0.027) and P1NP change between V0 and V1 (R=0.51, p=0.038). In short-term observation, successful LRLTx led to bone metabolism normalization triggered by probable anabolic action of IGF-I and PTH and manifested by TBBMC and TBBMD increases. In long-term horizon, moderately impaired DXA assessed bone status coincided with disturbances in bone metabolism. Bone metabolism markers, especially P1NP and CTx, appeared to be good predictors of changes in bone status evaluated by DXA.     

1,25-Dihydroxy-19-nor-vitamin D(2), a vitamin D analog with reduced bone resorbing activity in vitro

1,25-Dihydroxy-19-nor-vitamin D(2) (19-norD(2)), a new analog of 1,25(OH)(2)D(3), suppresses parathyroid hormone in renal failure patients and in uremic rats but has less calcemic activity than 1,25(OH)(2)D(3). Although 19-norD(2) has high affinity for the vitamin D receptor and similar pharmacokinetics to those of 1,25(OH)(2)D(3), it has much less bone resorbing activity in vivo. The intrinsic activity of 19-norD(2) on osteoclastogenesis and activation of bone resorption in mouse bone marrow cultures was examined to determine the mechanism involved. 19-norD(2) and 1,25(OH)(2)D(3) (10 nM) were equivalent in stimulating the formation and maintenance of large multinucleated, tartrate-resistant acid phosphatase-positive cells. However, the amount of bone resorbed by osteoclasts stimulated by 10 nM 19-norD(2), as measured by pit-forming assays, was reduced 62% compared with 10 nM 1,25(OH)(2)D(3)-stimulated osteoclasts (P < 0. 05). This difference could not be attributed to enhanced catabolism or to downregulated vitamin D receptor. The rate of degradation of 19-norD(2) in cultures was approximately 20% greater than 1, 25(OH)(2)D(3), not enough to account for the different effects on bone resorption. The VDR levels were identical in cultures that were treated with 19-norD(2) and 1,25(OH)(2)D(3). In summary, 19-norD(2) is less effective than 1,25(OH)(2)D(3) in stimulating mouse marrow osteoclasts to resorb bone. The reason for this difference is not clear but seems to involve the late maturation and/or activation of osteoclasts as the number of pits produced by each tartrate-resistant acid phosphatase-positive cell is reduced under stimulation by 19-norD(2) compared with 1,25(OH)(2)D(3).     

Combined treatment with cyclosporin A and cortisone acetate minimizes the adverse bone effects of either agent alone

Although cyclosporin A (CsA) and cortisone acetate (CRT) adversely affect bone, their combined effect on bone is unknown. Sprague Dawley rats were therefore administered either vehicle or CsA (7.5 mg/kg/day) by gavage and saline or CRT (2 mg/100 mg/day) by s.c. injection for 28 days. Group A received vehicle plus saline, group B CsA plus saline, group C vehicle plus CRT, and group D CsA/CRT. Serial bloods were sampled over a 28-day period for ionized calcium (Ca), PTH, 1,25 dihydroxyvitamin D (1,25(OH)2D), and bone gla protein (BGP osteocalcin) and tibia were examined on day 28 for histomorphometry. Results were compared with group A. Ca and PTH levels in groups B, C, and D were similar to those in group A during the study period. Group B had lower body weights, elevated levels of BGP, and an increase in 1,25(OH)2D. Group C developed weight loss and a decrease in BGP and 1,25(OH)2D. Group D had weight loss, BGP levels between those of group A and group C, and 1,25(OH)2D values similar to group A. Bone histomorphometry revealed high turnover osteopenia in group B and hyperostosis in group C with a decrease in bone formation and osteoclastlike cells. Combination therapy returned these to control values. In conclusion, the adverse effects of either CsA or CRT on bone in rats are minimized by combined therapy.     

Regulation of bone turnover and prevention of bone atrophy in ovariectomized beagle dogs by the administration of 24R, 25(OH)2D3

Summary In order to determine whether the administration of 24R,25(OH)₂D₃ had any beneficial effect on the regulation of bone turnover and the prevention of bone atrophy, we examined beagles for 31 months after ovariectomy (OVX). Fourteen beagle dogs (8.54±1.22 kg body wt-b.w.) were divided into four groups. Group 1 (n=3) was the sham, and Group 2 (n=3) served as the OVX control. In Group 3 (n=4) and Group 4 (n=4), 24,25-dihydroxyvitamin D₃(24R,25(OH)₂D₃) was given daily at dose levels of 2 and 10 mcg/kg B.W., respectively. In Group 4, the dose level was increased to 100 mcg/kg by 17 months. During the experiments, urinary hydroxyproline (U-HPr), serum chemistry, serum bone gla-protein (BGP), and vitamin D metabolite levels were monitored. At the end of the experiment, bone mineral content (BMC) in the 6th and 7th lumbar vertebrae and right femur was determined by single photon absorptiometry. The left iliac bone sample was obtained after tetracycline labeling, and undecalcified sections were observed. In Group 2, excretion of U-HPr increased after OVX and had reached a level of approximately twice the baseline values by 10 months; then it gradually came down to the original level. In Group 3, however, U-HPr excretion remained at the same level as the baseline value, as it did in Group 1. In Group 4, it was remarkably reduced down to 50–60% of the baseline values. Serum BGP level was markedly reduced in Group 4. Serum 24,25(OH)₂D levels were markedly increased in Groups 3 and 4. BMC levels of both vertebrae and epi-metaphyseal regions in the femur showed a significant reduction of approximately 25% in Group 2. In Groups 3 and 4, however, they remained at the same level as in Group 1. Histomorphometrical data showed a reduction in the parameters of osteoblast functions in Group 2. In Group 3, both kinetic and static parameters maintained the same level as in Group 1. In Group 4, eroded surface and osteoclast number decreased significantly, but mineral appositional rate and wall thickness maintained the same level as in Group 1. From these findings, it was concluded that, in beagle dogs, the administration of 24R,25(OH)₂D₃ inhibited the increase of bone turnover and prevented the reduction of cancellous bone mass after a long time postovariectomy.     

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

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

Continuous infusion of 1,25-dihydroxyvitamin D3 stimulates bone turnover in the normal young mouse

The effects of continuous administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on mineral and bone metabolism have been examined in the normal mouse. Four doses (0.05-0.25 micrograms/kg/day) of 1,25(OH)2D3 were infused continuously for 4 weeks in 21-day-old intact animals. Mineral and skeletal changes were evaluated by analytical methods and by histomorphometric analysis of endosteal bone formation and resorption parameters. All doses of 1,25(OH)2D3 increased the fractional osteoclastic surface and the osteoclast number in conjunction with increased hydroxyproline excretion. 1,25(OH)2D3 induced a dose-dependent elevation of the calcification rate, reduction of the mean osteoid seam thickness, and shortening of the mineralization lag time. In addition, there was a dose-related increase in the extent of tetracycline double-labeled osteoid surface and a concurrent rise in the fractional osteoblastic surface associated with elevated serum alkaline phosphatase levels. Increased bone formation appeared to have been balanced by increased bone resorption since the trabecular bone volume remained unchanged. Except at the highest dose given, serum calcium and phosphate concentrations remained normal in spite of increased bone mobilization and presumably enhanced intestinal absorption of minerals. Urinary cAMP and TmP/GFR remained normal, suggesting that parathormone secretion was not altered. The results show that continuous 1,25(OH)2D3 infusion in the young mouse produces a dose-dependent stimulation of bone mineralization rate in response to increased osteoclastic bone resorption. The data indicate that 1,25(OH)2D3 can regulate bone turnover as well as mineral homeostasis in the young mouse.     

Calcemic activity of 19-Nor-1,25(OH)(2)D(2) decreases with duration of treatment

19-Nor-1,25(OH)(2)D(2) (19-norD(2)) has been shown to suppress parathyroid hormone effectively, but with lower calcemic activity than 1,25(OH)(2)D(3). The present study investigated potential mechanisms to explain the reduced calcemic response to 19-norD(2). Tissue localization of [(3)H]19-norD(2) or[(3)H]1,25(OH)(2)D(3) after a single injection was not different. Intestinal calcium absorption and bone mobilization, measured in vitamin D-deficient rats 24 h after single injections of 60 or 600 pmol of 19-norD(2) or 1,25(OH)(2)D(3), were enhanced to a similar degree by the two compounds. However, when normal rats were treated every other day with 240 pmol of 19-norD(2) or 1,25(OH)(2)D(3), increases in serum calcium were identical 24 h after the first injection but diverged thereafter with significantly lower serum calcium in the 19-norD(2)-treated rats by 5 d. Intestinal calcium absorption and bone calcium mobilization were reassessed in vitamin D-deficient rats after seven daily injections of 600 pmol of 19-norD(2) or 1, 25(OH)(2)D(3), and both parameters were significantly lower in the 19-norD(2)-treated rats. Pharmacokinetic analysis after seven daily injections of 600 pmol of 19-norD(2) or 1,25(OH)(2)D(3) showed similar localization to the intestine and bone. In addition, intestinal vitamin D receptor levels were not different after 1 wk of treatment with 19-norD(2) or 1,25(OH)(2)D(3). In conclusion, the low calcemic activity of 19-norD(2) seems to be due to an acquired, postreceptor resistance of the intestine and bone to chronic treatment with the analog.     

Vitamin D binding protein, bone status and body composition in community-dwelling elderly men

The vitamin D binding protein (DBP) is the major carrier protein for vitamin D metabolites in plasma. Polymorphisms in DBP have been described to be associated with an increased bone fracture risk and diabetes. The present study investigates the influence of both phenotypic and (TAAA)(n)-Alu repeat DBP-polymorphism and DBP-concentration on bone mineral density, body composition, bone turnover- and metabolic markers in a cohort of ambulatory elderly men. We included 211 men (>70 years) in this study. Bone mineral density (BMD) was determined by dual energy X-ray absorptiometry. Bone turnover was assessed by measurement of serum osteocalcin, serum and urinary C-terminal telopeptides of type I collagen and urinary deoxypyridinoline, together with 25(OH)-vitamin D and 1,25(OH)(2)-vitamin D concentrations. DBP-phenotypes were determined electrophoretically and the (TAAA)(n)-Alu repeat polymorphism was determined by polymerase chain reaction. Body composition was estimated using bioelectrical impedance analysis, together with handgrip and arm strength, fasting serum glucose and leptin concentrations. No differences in BMD or bone turnover markers among DBP-phenotypes or (TAAA)(n)-genotypes were observed in this study. Serum 25(OH)-vitamin D was comparable among DBP-variants and did not relate to DBP-concentrations, whereas 1,25(OH)(2)-vitamin D was different among DBP-phenotypes and was correlated positively with DBP-concentrations. DBP-concentrations related positively to body mass index, fat mass, leptin and glucose concentration. The correlation with leptin remained significant after correction for fat mass. Fasting glucose concentrations were different among DBP-phenotypes, whereas no difference was observed between (TAAA)(n)-genotypes. In conclusion, serum 1,25(OH)(2)-vitamin D concentrations are codetermined by DBP-phenotypes and DBP-concentrations. No major effect of DBP-polymorphism was demonstrated on BMD, bone turnover markers or body composition.     

Relative potencies of 1,25-(OH)(2)D(3) and 19-Nor-1,25-(OH)(2)D(2) on inducing differentiation and markers of bone formation in MG-63 cells.

19-Nor-1,25-(OH)(2)D(2), an analog of 1,25-(OH)(2)D(3), is used to treat secondary hyperparathyroidism because it suppresses parathyroid hormone synthesis and secretion with lower calcemic and phosphatemic activities. 19-Nor-1,25-(OH)(2)D(2) is approximately 10 times less active than 1,25-(OH)(2)D(3) in promoting bone resorption, which accounts in part for the low potency of this analog in increasing serum calcium and phosphorus. Concern that 19-nor-1,25-(OH)(2)D(2) also could be less potent than 1,25-(OH)(2)D(3) on bone formation led to a comparison of the potency of both compounds on osteoblasts. In the human osteoblast-like cell line MG-63, 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) had a similar potency in upregulating vitamin D receptor content and suppressing proliferation. Both sterols caused a similar reduction in DNA content and proliferating cell nuclear antigen protein expression. Time-course and dose-response studies on 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) induction of the marker of bone formation, osteocalcin, showed overlapping curves. The effects on alkaline phosphatase (ALP) activity also were studied in MG-63 cells that had been co-treated with either sterol and transforming growth factor-beta, an enhancer of 1,25-(OH)(2)D(3)-induced ALP activity in this cell line. Transforming growth factor-beta alone had no effect, whereas 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) increased ALP activity similarly. These studies demonstrate that 19-nor-1,25-(OH)(2)D(2) has the same potency as 1,25-(OH)(2)D(3) not only in inducing vitamin D receptor content, osteocalcin levels, and ALP activity but also in controlling osteoblastic growth. Therefore, it is unlikely that 19-nor-1,25-(OH)(2)D(2) would have deleterious effects on bone remodeling.     

Pharmacokinetics of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) in normal and uraemic men.

BACKGROUND: The therapeutic equivalence of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) on the suppression of PTH synthesis and secretion has not clearly been established. The aim of the present study was to evaluate the pharmacokinetics of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) after oral and i.v. administration in healthy volunteers and uraemic patients. METHODS: Six healthy volunteers and 12 uraemic patients were included in the study. With an interval of 2 weeks, 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, 4 microg of 1alpha(OH)D(3) i.v. and 4 microg of 1alpha(OH)D(3) orally were administered. Blood samples for analysis of plasma-Ca(2+), plasma-1,25(OH)(2)D(3), and plasma-PTH were drawn at time 0, 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, 48, and 72 h. The healthy volunteers were studied in all four protocols and the uraemic patients in either the 1alpha(OH)D(3) (n=6) or the 1,25(OH)(2)D(3) (n=6) protocol. RESULTS: After oral administration of 1,25(OH)(2)D(3) the bioavailability of 1,25(OH)(2)D(3) was 70.6+/-5.8/72.2+/-4.8% in healthy volunteers/uraemic patients (n.s.). After i.v. administration the volume of distribution of 1,25(OH)(2)D(3) was similar, 0.49+/-0.14 vs 0.27+/-0.06 l/kg in healthy volunteers vs uraemic patients (n.s.), while the metabolic clearance rate of 1,25(OH)(2)D(3) was 57% lower in the uraemic patients, 23.5+/-4.34 vs 10.1+/-1.35 ml/min in healthy volunteers vs uraemic patients, respectively (P<0.03). The bioavailability of 1,25(OH)(2)D(3) after i.v. administration of 1alpha(OH)D(3) was 42.4+/-11.0/42.0+/-2.0% in healthy volunteers/uraemic patients (n.s.); and after oral administration of 1alpha(OH)D(3) 42.0+/-2.0/29.8+/-3.1% in healthy volunteers/uraemic patients (n.s.). A small, but significant increase in plasma-Ca(2+) was seen after administration of 1,25(OH)(2)D(3) to the uraemic patients, while no increase was seen after administration of 1alpha(OH)D(3). PTH levels were significantly suppressed in the healthy volunteers 24 h after administration of 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, and 4 microg of 1alpha(OH)D(3) orally by 35+/-7, 30+/-8, and 35+/-4%, respectively (all P<0.03). In the uraemic patients, PTH levels were significantly suppressed after administration of 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, and 4 microg of 1alpha(OH)D(3) i.v. by 30+/-10, 45+/-7, and 40+/-7%, respectively (all P<0.04). The effect was transitory in the healthy volunteers and lasted for at least 72 h in the uraemic patients. CONCLUSION: The present study found a 57% lower metabolic clearance rate of 1,25(OH)(2)D(3) in uraemic patients, as compared with that of healthy volunteers (P<0.03). The bioavailability of 1,25(OH)(2)D(3) following administration of 1alpha(OH)D(3) i.v. and orally in both healthy volunteers and uraemic patients was markedly lower than after administration of oral 1,25(OH)(2)D(3) (P<0.03). In spite of lower plasma-1,25(OH)(2)D(3) levels after administration of 1alpha(OH)D(3), no significant difference was observed on the suppressive effect of 4 microg i.v. of either 1,25(OH)(2)D(3) or 1alpha(OH)D(3) on the plasma-PTH levels in the uraemic patients. This might suggest the existence of an effect of 1alpha(OH)D(3) on the parathyroid glands which is independent of the plasma-1,25(OH)(2)D(3) levels, that are achieved after oral or i.v. administration of 1alpha(OH)D(3).     

New insights into mineral and skeletal regulation by active forms of vitamin D

Recent studies in mice using genetic approaches have shed new light on the physiological effects of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) and the vitamin D receptor (VDR) in skeletal and mineral homeostasis, and on their interaction with calcium. These studies in mice with targeted deletion of the 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha(OH)ase), and of the VDR or of double mutants, have shown the discrete effects of calcium in inhibiting parathyroid hormone secretion and in enhancing bone mineralization, but overlapping effects of calcium and 1,25(OH)(2)D on inhibiting parathyroid growth and on normal development of the cartilaginous growth plate. The 1,25(OH)(2)D/VDR system is essential, however, in enhancing intestinal calcium absorption and in optimally increasing osteoclastic activation. In addition, the 1,25(OH)(2)D/VDR system has important anabolic effects on bone, thus defining a dual role for this system in bone turnover. These studies are revealing functions of the vitamin D/VDR system which have relevance for new concepts of the pathophysiology of renal bone disease and, in particular, of the adynamic bone disorder, and for the development of new analogs of the active form of vitamin D, which have less calcemic activity and greater skeletal anabolic effects.     

Reductions in bone turnover, mineral, and structure associated with mechanical properties of lumbar vertebra and femur in glucocorticoid-treated growing minipigs

The present study was designed to determine the effects of glucocorticoid (GC) on bone turnover, minerals, structure, and bone mechanical properties in minipigs. Six 8-month-old G?ttingen minipigs were subcutaneously injected with prednisolone (PN, 0.5 mg/kg body wt (BW)/day, 5 days/week for 26 weeks (Group GC)), 6 were treated with vehicle alone (Group VC), and 4 were sacrificed at start of the study for baseline controls (Group BC). The increase in BW was similar in all groups. PN significantly reduced serum osteocalcin and urinary type-1 collagen N-telopeptide levels at 13 weeks and thereafter, compared with baseline and control, and also reduced serum bone specific alkaline phosphatase levels relative to baseline. At 26 weeks, the longitudinal axis of the lumbar bone and length of femur were smaller in Group GC than Group VC. The total cross-sectional area of femur, but not the lumbar bone, in Group GC was significantly different from Group VC. BMD of the femur, but not L2, measured by DXA, was lower in Group GC than in Groups BC and VC. The cortical shell structure measured by 2D-micro-CT deteriorated and age-dependent increases in trabecular bone structure 3D micro-CT were reduced by PN. PN also caused deterioration of the cortical structure of the mid-femur. In L2 and femur, PN significantly reduced the ultimate load and maximum absorption energy of the femur and L2 compared with Group VC. The structural modulus in Group GC was lower than in Group BC. Regression analyses revealed that bone minerals, bone structure, and chemical markers correlated with mechanical properties of L2 and mid-femur. Our results indicate that PN reduced systemic bone formation and resorption and suppressed the age-dependent increases in bone minerals, structure, and mechanical properties of L2 and mid-femur. Reduced bone turnover seemed to be associated with a reduction in mechanical properties. The growing minipig could be a suitable model of GCs-induced osteoporosis in humans.     

Melatonin and vitamin D3 increase TGF-beta1 release and induce growth inhibition in breast cancer cell cultures

BACKGROUND: Evidence has accumulated that 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] is involved in the regulation of the proliferation of breast tumor cells. For complete tumor suppression high hypercalcemic doses of 1,25-(OH)(2)D(3) are needed. The aim of this study was to assess the effect of combined treatment of 1,25-(OH)(2)D(3) at low doses and melatonin (MEL) on the proliferation of estrogen-responsive rat breast cancer cell line RM4. MATERIALS AND METHODS: RM4 cell proliferation was assessed by [3H]thymidine uptake. The presence of TGF-beta(1) in serum-free conditioned medium was determined by inhibition antibody binding assay. RESULTS: In 17-betaE cultured RM4 cells both MEL and 1,25-(OH)(2)D(3) alone and in combination significantly reduced [3H]thymidine incorporation in a dose-related fashion. MEL by itself was ineffective in inhibiting the FCS-cultured RM4 cells, while 1,25-(OH)(2)D(3) strongly inhibited [3H]thymidine incorporation. Meanwhile, MEL increased the sensitivity of the FCS-cultured RM4 cells to 1,25-(OH)(2)D(3) in the combined regimen, from 20- to 100-fold. MEL significantly enhanced the TGF-beta(1) secretion from RM4 cells and vitamin D(3) increased the TGF-beta(1) secretion in a dose-dependent manner, from 2- to 7-fold. Moreover, a further enhancement of the TGF-beta(1) release was obtained with the combined treatment, but only for low 1,25-(OH)(2)D(3) concentrations. The addition of monoclonal anti-TGF-beta(1) antibody to the medium of RM4 cells exposed to vitamin D(3) alone or in combination with MEL increased the [3H]thymidine uptake compared to the correspondent cells cultured without antibody. CONCLUSIONS: Our data point to a potential benefit of combination therapy with 1,25-(OH)(2)D(3) and MEL in the treatment of breast cancer and suggest that the growth inhibition could be related, at least in part, to the enhanced TGF-beta(1) secretion.     

Osteoporosis in hypogonadal men: role of decreased plasma 1,25-dihydroxyvitamin D, calcium malabsorption, and low bone formation

To investigate the pathogenesis of osteoporosis in male hypogonadism we have investigated a heterogeneous group of 13 men with hypogonadism: 7 men (median age 60, range 31-79) with two or more vertebral crush fractures and 6 men (median age 61.5, range 28-76) without vertebral fractures. The group with crush fractures had trabecular and cortical osteoporosis as assessed by Singh grade, iliac crest trabecular bone volume, and metacarpal cortical area/total area. This was accompanied by an altered trabecular architecture with a reduction in number of trabeculae but no change in trabecular width, which contrasts with age-related bone loss in men where there is no reduction in trabecular number but thinning of trabeculae. The fracture group had significantly lower plasma 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations than the nonfracture group, and this was associated with malabsorption of calcium. Irrespective of the presence or absence of osteoporosis, treatment with testosterone led to a significant increase in total and free plasma 1,25(OH)2D and an improvement in calcium absorption measured with radiocalcium and by balance techniques. In addition, urine biochemistry, metabolic balance studies, and bone biopsy suggest that skeletal retention of calcium and bone formation are increased by testosterone treatment. We conclude that male hypogonadism causes both cortical and trabecular osteoporosis and altered trabecular architecture. A major risk factor for the development of osteoporosis is reduction in plasma 1,25(OH)2D, leading to malabsorption of calcium and reduced bone formation.     

Vector-averaged gravity-induced changes in cell signaling and vitamin D receptor activity in MG-63 cells are reversed by a 1,25-(OH)2D3 analog,EB1089

Skeletal unloading in an animal hindlimb suspension model and microgravity experienced by astronauts or as a result of prolonged bed rest causes site-specific losses in bone mineral density of 1%-2% per month. This is accompanied by reductions in circulating levels of 1,25-(OH)(2)D(3), the active metabolite of vitamin D. 1,25-(OH)(2)D(3), the ligand for the vitamin D receptor (VDR), is important for calcium absorption and plays a role in differentiation of osteoblasts and osteoclasts. To examine the responses of cells to activators of the VDR in a simulated microgravity environment, we used slow-turning lateral vessels (STLVs) in a rotating cell culture system. We found that, similar to cells grown in microgravity, MG-63 cells grown in the STLVs produce less osteocalcin, alkaline phosphatase, and collagen Ialpha1 mRNA and are less responsive to 1,25-(OH)(2)D(3). In addition, expression of VDR was reduced. Moreover, growth in the STLV caused activation of the stress-activated protein kinase pathway (SAPK), a kinase that inhibits VDR activity. In contrast, the 1,25-(OH)(2)D(3) analog, EB1089, was able to compensate for some of the STLV-associated responses by reducing SAPK activity, elevating VDR levels, and increasing expression of osteocalcin and alkaline phosphatase. These studies suggest that, not only does simulated microgravity reduce differentiation of MG-63 cells, but the activity of the VDR, an important regulator of bone metabolism, is reduced. Use of potent, less calcemic analogs of 1,25-(OH)(2)D(3) may aid in overcoming this defect.     

Investigating the mechanism for maintaining eucalcemia despite immobility and anuria in the hibernating American black bear (Ursus americanus)

Ursine hibernation uniquely combines prolonged skeletal unloading, anuria, pregnancy, lactation, protein recycling, and lipolysis. This study presents a radiographic and biochemical picture of bone metabolism in free-ranging, female American black bears (Ursus americanus) that were active (spring bears and autumn bears) or hibernating (hibernating bears). Hibernating bears included lactating and non-lactating individuals. We measured serum calcium, albumin, inorganic phosphate, creatinine, bone specific alkaline phosphatase (BSALP), CTX, parathyroid hormone, insulin-like growth factor-I (IGF-l), leptin, 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)(2)D] and sclerostin from 35 to 50 tranquilized hibernating bears and 14 to 35 tranquilized spring bears. We compared metacarpal cortical indices (MCI), measured by digital X-ray radiogrammetry, from 60 hunter-killed autumn bears and 79 tranquilized, hibernating bears. MCI was greater in autumn than winter in younger bears, but showed no seasonal difference in older bears. During hibernation eucalcemia was maintained, BSALP was suppressed, and CTX was in the range expected for anuria. During hibernation 1,25(OH)(2)D was produced despite anuria. 1,25(OH)(2)D and IGF-I were less in hibernating than spring bears. In a quarter of hibernating bears, sclerostin was elevated. Leptin was greater in hibernating than spring bears. In hibernating bears, leptin correlated positively with BSALP in non-lactating bears and with CTX in lactating bears. Taken together the biochemical and radiographic findings indicate that during hibernation, bone turnover was persistent, balanced, and suppressed; bone resorption was lower than expected for an unloaded skeleton; and there was no unloading-induced bone loss. The skeleton appears to perceive that it was loaded when it was actually unloaded during hibernation. However, at the level of sclerostin, the skeleton recognized that it was unloaded. During hibernation leptin appeared anabolic in non-lactating bears and catabolic in lactating bears. We hypothesize that ursine hibernation may represent a natural model in which suppression of the sympathetic nervous system prevents unloading-induced bone loss by influencing leptin's skeletal effects and preventing transmission of loading information.     

Effect of 2-methylene-19-nor-(20S)-1 alpha-hydroxy-bishomopregnacalciferol (2MbisP), an analog of vitamin D, on secondary hyperparathyroidism.

Vitamin D analogs are being developed that retain therapeutic effects but are less calcemic and phosphatemic, a concern in CKD patients who are prone to vascular calcification. We tested a new analog of vitamin D, 2MbisP, and found that it suppresses PTH at doses that do not affect serum Ca or P. INTRODUCTION: Calcitriol is used for the treatment of secondary hyperparathyroidism. However, its use is often limited by the development of hypercalcemia and hyperphosphatemia, an important consideration in patients with chronic kidney disease (CKD) because they are prone to vascular calcification. To minimize this toxicity, structural modifications in the vitamin D molecule have led to the development of calcitriol analogs with selective actions. MATERIALS AND METHODS: In this study, we compared the effects of 1,25(OH)(2)D(3) and a new analog, 2-methylene-19-nor-(20S)-1 alpha-hydroxy-bishomopregnacalciferol (2MbisP), on the development of secondary hyperparathyroidism and established secondary hyperparathyroidism in uremic rats and on mobilization of calcium and phosphorus from bone in parathyroidectomized rats. The clearance from circulation, half-life, and binding affinities to the vitamin D-binding protein and vitamin D receptor of this compound were also evaluated. RESULTS: Uremia produced a marked rise in plasma PTH, but treatment every other day for 2 wk with either 1,25(OH)(2)D(3) (4 ng) or 2MbisP (250, 750, 1500, or 3000 ng) suppressed this increase by >50%. The suppression by 1,25(OH)(2)D(3), however, was accompanied by increases in ionized calcium, phosphorus, and the calcium x phosphorus product, whereas these three parameters were unchanged by 2MbisP. The binding affinity of 2MbisP was 10-20 times less for the vitamin D receptor and 1000 times less for the serum vitamin D-binding protein compared with 1,25(OH)(2)D(3). Also, 2MbisP was cleared more rapidly from the circulation (t1/2 = 10 min) than 1,25-(OH)(2)D(3) (t1/2=7-9 h). In parathyroidectomized rats fed calcium-or phosphorus-deficient diets, daily injections of 2MbisP (1500 or 3000 ng), unlike 1,25(OH)(2)D(3) (50 ng), had no effect on calcium or phosphorus mobilization from bone. CONCLUSIONS: In uremic rats, 2MbisP can suppress PTH at doses that do not affect plasma calcium, phosphorus, and calcium x phosphorus product. This new vitamin D analog may represent an important tool in the treatment of secondary hyperparathyroidism in patients with CKD.     

Pharmacokinetics of doxercalciferol, a new vitamin D analogue that lowers parathyroid hormone.

BACKGROUND: This is the first detailed pharmacokinetic report published on the administration of doxercalciferol [1alpha(OH)D(2)] recently introduced to treat secondary hyperparathyroidism. METHODS: 1alpha(OH)D(2) was administered in a range of single and multiple doses to volunteers with and without normal renal and/or hepatic function. Subsequent serial blood samples were assayed by HPLC/radioimmunoassay for the metabolite 1,25-dihydroxyvitamin D(2) [1,25(OH)(2)D(2)], the major active species. RESULTS: Bioavailability of 1,25(OH)(2)D(2) from a single 5 micro g 1alpha(OH)D(2) oral-capsule dose was estimated to be normally approximately 42% of that from a 5 micro g intravenous injection. Steady-state serum concentrations of 1,25(OH)(2)D(2) were attainable within 8 day, and fluctuated approximately 2.5-fold from peak to trough when oral 1alpha(OH)D(2) doses were taken every second day, and the terminal half-life was 34+/-14 h. Mean steady-state serum concentrations rose less than proportionally (from 20 to 45 pg/ml) on increasing oral 1alpha(OH)D(2) doses from 5 to 15 micro g every 48 h. Renal patients showed 39+/-37% increase in serum 1,25(OH)(2)D(2) concentration during 3-4 h haemodialysis sessions, but no other difference in steady-state pharmacokinetics was found between these or hepatically impaired patients and normal subjects. CONCLUSIONS: Given the sensitivity limits of current assays, the pharmacokinetics of this and other vitamin-D compounds is best elucidated from steady-state studies. The pharmacokinetics of 1,25(OH)(2)D(2) from 1alpha(OH)D(2) doses appears to be similar to that of 1,25(OH)(2)D(3) from 1alpha(OH)D(3) doses, albeit D(3) data have to date largely derived from single-dose studies. Deviation of 1,25(OH)(2)D(2) pharmacokinetics from linearity appears to be marginal enough to be clinically manageable with adequate precaution.