Parathyroid Hormone Modulates the Response of Osteoblast-Like Cells to Mechanical Stimulation

Mechanical loading stimulates many responses in bone and osteoblasts associated with osteogenesis. Since loading and parathyroid hormone (PTH) activate similar signaling pathways in osteoblasts, we postulate that PTH can potentiate the effects of mechanical stimulation. Using an in vitro four-point bending device, we found that expression of COX-2, the inducible isoform of cyclooxygenase, was dependent on fluid forces generated across the culture plate, but not physiologic levels of strain in MC3T3-E1 osteoblast-like cells. Addition of 50 nM PTH during loading increased COX-2 expression at both subthreshold and threshold levels of fluid forces compared with either stimuli alone. We also demonstrated that application of fluid shear to MC3T3-E1 cells induced a rapid increase in [Ca²⁺]_i. Although PTH did not significantly change [Ca²⁺]_i levels, flow and PTH did produce a significantly greater [Ca²⁺]_i response and increased the number of responding cells than is found in fluid shear alone. The [Ca²⁺]_i response to these stimuli was significantly decreased when the mechanosensitive channel inhibitor, gadolinium, was present. These studies indicate that PTH increases the cellular responses of osteoblasts to mechanical loading. Furthermore, this response may be mediated by alterations in [Ca²⁺]_i by modulating the mechanosensitive channel. Received: 5 October 1999 / Accepted: 15 February 2000     

Effects of Lead, Mercury, and Methyl Mercury on Gap Junctions and [Ca2+]i in Bone Cells

Heavy metals such as lead (Pb), mercury (Hg), and methyl mercury (MeHg) impair cell functions. For bone it is known that Pb changes bone formation rates, which depend on intracellular free calcium concentration ([Ca²⁺]_i). Since heavy metals compete with Ca²⁺ at multiple sites and increased [Ca²⁺]_i reduces gap junctional coupling between bone cells, we analyzed the effects of extracellular (e) and intracellular (i) application of Pb, Hg, and MeHg on these channels. Using primary cultures of osteoblast-like cells, relative changes of [Ca²⁺]_i were studied in Fura-2/AM loaded cells. Parallel intracellular recordings of neighboring cells were obtained using a conventional and a patch electrode. Pb_(e) (5 μmol/liter; n = 3) and Hg_(e) (5 μmol/liter; n = 3) as well as Pb_(i) (25 μmol/liter; n = 7) did not change the coupling (ΔMP₂/ΔMP₁). In contrast, MeHg_(e) (1–10 μmol/liter; n = 6) and Hg_(i) (≥5 μmol/liter; n = 8) reduced the coupling to 79.5 ± 19.3% and 62.4 ± 15.3%, respectively, within 15–20 minutes. The reduction of coupling followed individual time courses, and in no case was a steady state of decoupling reached within 20 minutes. Extracellular application of Pb_(e) (5 μmol/liter, n = 74) for 20 minutes, linearly elevated the Fura emission ratio reflecting transmembrane Pb permeation rather than [Ca²⁺]_i increase. Hg_(e) (n = 48) slightly increased [Ca²⁺]_i from 100 to ≤200 nmol/liter, whereas MeHg_(e) (5 μmol/liter, n = 52) released Ca²⁺ from internal stores, thus increasing [Ca²⁺]_i up to 2 μmol/liter. In conclusion, Pb_(e), Pb_(i) and Hg_(e) do not affect gap junctional coupling per se. Since MeHg_(e) and Hg_(i) deplete calcium stores, the decrease of the electric coupling is attributable to increased [Ca²⁺]_i, which affects gap junction channels. Received: 6 May 1997 / Accepted: 15 October 1997     

Effects of Calcium on Gap Junctions Between Osteoblast-Like Cells in Culture

The present study investigated the effects of elevated cytoplasmic free calcium concentrations ([Ca²⁺]_i) on the permeability of gap junctions between cultured osteoblast-like (OB) cells derived from calvarial and periosteal fragments of newborn rats. This was studied using the double whole cell patch clamp technique and intracellular dye injections. To increase [Ca²⁺]_i, patch pipette solutions contained 100 μmol/liter Ca²⁺. About 1–2 minutes after whole cell modes had been attained, the total number of gap junction channels was reduced from an average of 400 in normal Ca²⁺ to 20 in high Ca²⁺. Thereafter, remaining gap junction channels were active for up to 8 minutes. In normal rat kidney (NRK) cells, gap junction channels were closed by high Ca²⁺ within 1 minute, pointing to a similar sensitivity of Connexin43 gap junction channels in OB and NRK cells. To study the effects of elevated [Ca²⁺]_i on the dye permeability of gap junctions between extended OB cells, the spread of Lucifer Yellow to neighboring cells was evaluated. [Ca²⁺]_i was gradually increased from 1.5- to 14-fold the normal value by application of either ouabain, Na⁺-free/ouabain, or A23187. Reduced dye spread correlated with the increase of [Ca²⁺]_i measured by analyzing the fluorescence of fura-2. These data show that gap junctions in OB cells are sensitive to Ca²⁺. Received: 15 August 1995 / Accepted: 12 February 1996     

Increased Serum 1,25-Dihydroxyvitamin D after Growth Hormone Administration is not Parathyroid Hormone-Mediated

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 2? 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. Received: 2 May 1996 / Accepted: 18 October 1996     

Net Fluxes Over Working Thigh of Hormones,Growth Factors and Biomarkers of Bone Metabolism During Short Lasting Dynamic Exercise

The purpose of this study was to evaluate the responses of hormones, growth factors, and biomarkers involved in bone and muscle metabolism during exercise and in recovery. One leg knee-extension exercise and concomitant sampling from the artery and vein were performed. In 12 healthy individuals (6 men and 6 women; age 21–36 years) blood was drawn from the femoral artery and vein at rest, after 10 minutes warm-up, after 15 minutes work at 61% of peak one leg VO₂, and after 5 minutes work at peak one leg VO₂, as well as 5, 30, and 60 minutes in recovery. Blood flow in the femoral vein was measured using the thermodilution technique. Arteriovenous differences were measured over working thigh for growth hormone (GH), insulin-like growth factor I (IGF-I), insulin-like growth factor binding protein 3 (IGF BP3), parathyroid hormone (PTH) and bone biomarkers, i.e., the carboxyterminal propeptide of type I procollagen (PICP), the carboxyterminal cross-linked telopeptide of type I collagen (ICTP), osteocalcin, and bone-specific alkaline phosphatase (b-ALP). There was an uptake of GH (3.1 ± 1.2 mU · min⁻¹, P < 0.001; mean ± SE) over thigh during exercise and a release of IGF-I at the end of exercise (60 ± 36 μg · min⁻¹; P < 0.01). PICP was also released after the maximal exercise (23 ± 12 μg · min⁻¹; P < 0.01) as well as ICTP (0.5 ± 0.3 μg · min⁻¹; P < 0.05) and b-ALP (0.2 ± 0.1 μkat · min⁻¹; P < 0.05). Osteocalcin, IGF BP3, and PTH revealed no clearcut pattern. In the present study, exercise induces endocrine changes which point to anabolic effects on muscle and bone tissue. Received: 12 February 1996 / Accepted: 6 June 1996     

A Calcium Release Activated Calcium Influx in Primary Cultures of Rat Osteoblast-like Cells

Osteoblast-like (OBL) cells in primary culture were tested for their ability to generate a calcium release activated calcium flux (CRAC). Influx of Ca²⁺ was optically detected by fura-2. Intracellular calcium stores (ICS) were emptied in the absence of extracellular calcium ([Ca²⁺]_e) by 5 μM thapsigargin (TG) or 2 μM A23187. Readdition of 1.8 mM [Ca²⁺]_e increased the free intracellular Ca²⁺ ([Ca²⁺]_i) after a delay of 30–60 seconds at a rate of 2.3 nM/s due to CRAC. This rate depended on [Ca²⁺]_e and was substantially lowered if readdition of 1.8 mM [Ca²⁺]_e was preceded by, e.g., 0.72 mM [Ca²⁺]_e. CRAC-induced [Ca²⁺]_i peaks were correlated (r= 0.543) with [Ca²⁺]_i peaks during the complete depletion of ICS with A23187. Ca²⁺ influx due to CRAC could be blocked by flufenamic acid (100 μM) but not verapamil (20 μM). Ni²⁺ (1 mM), although reversibly blocking CRAC, accelerated the initial [Ca²⁺]_i influx rate. Induction of CRAC enhanced the influx of Mn²⁺ 4.3-fold, as measured by quenching of fura-2 fluorescence. In summary, OBL cells exhibit a CRAC which allows for the permeation of ions other than Ca²⁺. This Ca²⁺ flux may be activated by transmembraneous gradients of Ca²⁺ and Ni²⁺. Received: 8 September 1997 / Accepted: 27 January 1998     

The Effect of Intermittent Slow-Release Sodium Fluoride and Continuous Calcium Citrate Therapy on Calcitropic Hormones, Biochemical Markers of Bone Metabolism, and Blood Chemistry in Postmenopausal Osteoporosis

A detailed examination of calcitropic hormones and biochemical markers of bone turnover, serum chemistry, and blood hematology was performed in 75 postmenopausal women allocated to two groups: placebo plus calcium citrate (400 mg Ca B.I.D.) (n = 36) or intermittent slow-release sodium fluoride (SRNaF, 25 mg B.I.D.) plus calcium citrate (n = 39). After 2 years of therapy, a significant reduction in serum immunoreactive parathyroid hormone (PTH) was seen for both groups (43 ± 18 SD–30 ± 11 ng/liter, in placebo and 46 ± 24–36 ± 10, in SRNaF P < 0.0001 for both groups). Serum 1,25(OH)₂D significantly fell in placebo-treated patients (91 ± 31–75 ± 34 pmol/liter, P= 0.001) but did not change for SRNaF-treated patients. This difference in response between placebo and SRNaF-treated groups was significant, P= 0.005. Urinary hydroxyproline significantly declined during treatment in both groups (130 ± 61–76 ± 38 μmol/day, for placebo and 138 ± 84–84 ± 38 for SRNaF, P= 0.001). Similar decreases in urinary N-telopeptide of type I collagen were also observed for both groups (305 ± 192–252 ± 197 nmoles BCE/day for placebo and 356 ± 230–220 ± 197, P= 0.0001 for SRNaF). Serum carboxyterminal propeptide of type I collagen (PICP) declined significantly in both the placebo and SRNaF groups (118 ± 38–101 ± 36 μg/liter, and 116 ± 47–105 ± 39, P= 0.0027). Serum osteocalcin did not change significantly for either group, but bone-specific alkaline phosphatase (BS-ALPase), another marker of bone formation, demonstrated a significant fall in the placebo group at 2 years of therapy (16.2 ± 6.7 U/liter–12.1 ± 3.5, P= 0.009) and a small increase in the SRNaF-treated patients (13.0 ± 4.1–15.0 ± 4.5). The observed difference in response of BS-ALPase between the placebo and treated groups was significant (P= 0.007). There were no significant changes within or between treatment groups for blood hematology or serum chemistries. Mean values for all parameters remained within established normal ranges. These findings suggest that administration of calcium citrate inhibited PTH secretion and thereby reduced bone resorption in both groups, indicated by a decline in serum PTH, urinary hydroxyproline, and N-telopeptide. A low turnover state of bone may have been produced in the placebo group taking calcium citrate alone, since serum PICP, BS-ALPase, and 1,25(OH)₂D also decreased. The addition of SRNaF prevented serum 1,25(OH)₂D from falling by an unknown mechanism. However, its anabolic action on the skeleton was best reflected by changes in BS-ALPase. Moreover, SRNaF appeared to exert no deleterious effects on blood chemistries or hematology during 2 years of administration. Received: 28 January 1996 / Accepted: 25 April 1997     

The PTH-Calcium Relationship During a Range of Infused PTH Doses in the Parathyroidectomized Rat

To establish the PTH dosage that maintains normal mineral homeostasis in the PTX rat, a series of doses of rat 1-34 PTH were infused via a subcutaneously implanted miniosmotic pump. The doses were 0, 0.011, 0.022, 0.044, and 0.11 μg/100 g/hour. After 48 hours, serum calcium ranged from 5.56 ± 0.02 to 16.29 ± 0.25 mg/dl, ANOVA P < 0.001, and serum phosphorus from 12.49 ± 0.03 to 5.33 ± 0.34 mg/dl, ANOVA P < 0.001. By post hoc test, the serum calcium level was different (P < 0.05) at every PTH dose; the serum phosphorus level was different (P < 0.05) at every PTH dose except between the two highest doses. The PTH dosage that produced a normal serum calcium (10.09 ± 0.10 mg/dl) and phosphorus (6.90 ± 0.18 mg/dl) was 0.022 μg/100 g/hour. The relationship between increasing doses of PTH and both serum calcium and phosphorus was curvilinear and the calcium-phosphorus product was remarkably constant from a serum calcium of 7–13 mg/dl. The increase in serum calcium and the decrease in serum phosphorus were more rapid at lower than at higher PTH doses so that for both, an asymptote was reached. At the highest serum calcium values, the calcium-phosphorus product increased and in individual rats, an increase in serum phosphorus was associated with a decrease in serum calcium. In summary, this study shows that (1) for rat 1-34 PTH, the normal replacement dose in the PTX rat with normal renal function on a normal diet is 0.022 μg/100 g/hour; (2) the relationship between PTH and both serum calcium and phosphorus is curvilinear, and an asymptote is reached for both; and (3) the calcium-phosphorus product is remarkably constant as the serum calcium increases from 7 to 13 mg/dl and only increased during marked hypercalcemia when serum phosphorus did not decrease further or even tended to increase. Received: 30 May 1997 / Accepted: 15 October 1997     

Parathyroid hormone decreases in vivo insulin effect on glucose utilization

Hyperparathyroidism is associated with impaired glucose tolerance, and parathyroidectomy may improve carbohydrate homeostasis. It has been suggested that parathyroid hormone (PTH) suppresses insulin secretion but it is unclear whether it also interferes with the peripheral action of insulin. To evaluate in vivo effects of PTH on insulinmediated glucose utilization, 15 male Sprague Dawley rats were continuously infused with rat PTH (1–34) using an Alzet miniosmotic pump at a rate of 0.03 nm/hour. Controls were infused with the vehicle alone. Following 5 days of PTH infusion, plasma calcium (Ca) levels were higher in the PTH-infused rats (12.3±0.2 versus 9.9±0.1 mg/dl, P<0.01). On the 5th day, glucose (700 mg/kg) and insulin (0.175 U/kg) were given as a bolus infusion through the left femoral vein, blood samples were obtained from the right femoral vein, and plasma glucose and insulin were measured at basal (0 minutes) and at 2, 5, 10, and 20 minutes postinfusion. Basal, nonfasting glucose levels were higher (166±4 versus 155±4 mg/dL, P<0.04) in the PTH-infused rats but their insulin levels were similar to those of controls (6.5±0.6 versus 5.6 ±0.5 ng/ml). Postinfusions and maximal (2 minutes) glucose and insulin levels were similar in both groups. However, although insulin levels were similar in both groups at all measured time points, glucose levels at 20 minutes were higher in the PTH-treated rats (205±13 versus 173±9; P<0.03). Also, calculated glucose disappearance rates (Kg) were decreased in the PTH-infused rats (4.05±0.3 versus 4.63±0.8; P=0.054), suggesting an impaired peripheral effect of insulin on glucose utilization. To gain insight into the potential contribution of the hypercalcemia or the PTH to these abnormalities, correlation evaluations were performed. Only in PTH-infused rats did plasma Ca correlate with plasma glucose at 0 and 20 minutes (r=0.6, P=0.02; r=0.7, P=0.01) and with the area under the glucose curve (r=0.6, P=0.03) during the glucose-insulin infusion. Also only in PTH-infused rats did PTH correlate with 0 (P=0.07) and 20-minute (P=0.02) plasma glucose levels. There was no correlation between either Ca or PTH and basal insulin levels or the area under the insulin curve in either group. Consequently, we suggest that in the rat, PTH infusion associated with hypercalcemia impairs insulin effect on glucose utilization in vivo and this defect may be induced by the Ca, PTH, or both.This study was presented in part at the 76th Annual Meeting of the Endocrine Society, Anaheim, CA, USA, June 1994.     

Calcium Mobilization is Required for Spreading in Human Osteoblasts

Adhesion-induced changes in intracellular calcium concentration ([Ca²⁺]_i) were measured in populations of human osteoblasts spreading on bone matrix proteins. In cells spreading on collagen type I, fibronectin, or laminin, average values for [Ca²⁺]_i were found to increase approximately 2× over baseline and then decline. The speed with which [Ca²⁺]_i increased and declined was dependent upon the matrix protein on which the cells were plated but was generally complete within 1 hour from the time of plating. Calcium mobilization was found to be due to influx of calcium across the osteoblast plasma membrane and was integrin dependent. Carboxyamido triazole (CAI), a specific inhibitor of nonvoltage-dependent calcium channels, or BAPTA-AM, a chelator of intracellular calcium, inhibited osteoblast adhesion and spreading on collagen type I, fibronectin and laminin in a dose-dependent manner. In conclusion, these results demonstrate that calcium mobilization is induced upon integrin-ligand contact and that calcium influx is required for cell adhesion and spreading. Received: 10 September 1999 / Accepted: 18 January 2000     

Effects of moderate endurance exercise on calcium, parathyroid hormone, and markers of bone metabolism in young women

We investigated the short-term (1 hour–3 days) effects of a 45 minute run on calcium, parathyroid hormone, the carboxyterminal propeptide of type I procollagen (PICP), and the immunoactive carboxyterminal telopeptide of type I collagen in serum (ICTP) in young females. Fourteen healthy young women, aged 25.2 ± 0.6 years (mean ± SEM) with regular menstruations, participated. The test was outdoor jogging for 45 minutes at an intensity of 50% of VO_{2 max}. Blood samples were collected 15 minutes before the test and 1, 24, and 72 hours after the test. The measured values were adjusted for changes in plasma volume. A significant decrease of ionized calcium was observed at 1 hour (P < 0.001) and 72 hours (P < 0.05) and a significant increase of parathyroid hormone (PTH) was noted 24 (P < 0.01) and 72 hours (P < 0.05) after the test. A significant decrease of PICP at 1 hour (P < 0.05) was followed by an increase after 24 (P < 0.01) and 72 hours (P < 0.001) and a significant increase in ICTP was noted at 24 and 72 hours (P < 0.05). A strong positive correlation was found between serum levels of PICP and ICTP (r = 0.55–0.84; P < 0.05) throughout the experiment. In conclusion, young females showed biochemical signs of increased bone collagen turnover and altered homeostasis of calcium and PTH after a single bout of moderate endurance exercise. Received: 19 October 1995 / Accepted: 14 June 1996     

Age dependence of tolerance to anoxia and changes in cytosolic calcium in rabbit renal proximal tubules

Calcium(Ca²⁺)-dependent processes mediate, in part, anoxic cell injury. These may account for the difference in sensitivity to anoxia between certain immature and mature renal cells. To address this question, we studied the effects of anoxia on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), cell integrity, and transport functions in microdissected proximal convoluted tubules (PCT) of <3-week-old (newborn) and >12-week-old (adult) rabbits. Tubules were loaded with 10 μM fura-2 AM by incubation for 60 min at 37°C, and then superfused with isosmotic saline solution gassed with either 95%O₂-5%CO₂ (control group) or 95%N₂-5%CO₂ (anoxia group) for 30 min. [Ca²⁺]_i was measured ratiometrically; cell damage was assessed by nuclear binding of propidium iodide (PI). Anoxia resulted in a fourfold increase in [Ca²⁺]_i in adult tubules (from resting values of 245±10 to 975±100 nM, P <0.001), whereas in newborn tubules the rise was significantly less (from resting values of 137±5 to 165±5 nM, P <0.001 between anoxic groups). Transient exposure to 100 mM potassium chloride, which depolarizes the PCT cells, induced increases in [Ca²⁺]_i from baseline, to 920±90 nM in tubules from adult and to 396±16 nM in those from newborn rabbits (P <0.001 between age groups). After exposure to ligands such as parathyroid hormone (PTH) and ATP, [Ca²⁺]_i increased in both newborn and adult tubules, but to lower levels in newborn tubules. The response to PTH and ATP was transient in both age groups, [Ca²⁺]_i returning to baseline levels after 2 min. Following anoxia, tubules from adult animals exhibited staining of all cell nuclei by 1 min exposure to PI, indicative of gross permeabilization of the cells. Nuclei of anoxic immatures tubules did not stain with PI. The sodium-dependent uptakes of a glucose analogue (¹⁴C-α-methyl-glucopyranoside) and phosphate (³²Pi) were preserved in agarose-filled tubules of newborns after anoxia, whereas in those of adults recovery from anoxia was associated with drastic reduction in the uptake of these solutes. Overall, our results suggest that: (1) during anoxia, cell Ca²⁺ rises to critical levels in PCTs of adults compared with those of <3-week-old animals, (2) Ca²⁺ influx occurs via a pathway activated by exposure to high [K⁺]_o, presumably voltage-sensitive Ca²⁺ channels or reversal of Na⁺-Ca²⁺ exchange, (3) these pathways are either less active or less abundant in proximal tubules of newborn compared with adult rabbits, and (4) secondary active transport activity and cellular integrity are well preserved after anoxia in PCT cells of newborn but not of adult rabbits. Received June 27, 1995; received in revised form December 1, 1995; accepted December 8, 1995     

Peripheral Bone Mass is Not Affected by Winter Vitamin D Deficiency in Children and Young Adults from Ushuaia

Low vitamin D levels in elderly people are associated with reduced bone mass, secondary hyperparathyroidism, and increased fracture risk. Its effect on the growing skeleton is not well known. The aim of this study was to evaluate the possible influence of chronic winter vitamin D deficiency and higher winter parathyroid hormone (PTH) levels on bone mass in prepubertal children and young adults. The study was carried out in male and female Caucasian subjects. A total of 163 prepubertal children (X age ± 1 SD: 8.9 ± 0.7 years) and 234 young adults (22.9 ± 3.6 years) who had never received vitamin D supplementation were recruited from two areas in Argentina: (1)Ushuaia (55° South latitude), where the population is known to have low winter 25OHD levels and higher levels of PTH in winter than in summer, and (2)Buenos Aires (34°S), where ultraviolet (UV) radiation and vitamin D nutritional status in the population are adequate all year round. Bone mineral content (BMC) and bone mineral density (BMD) of the ultradistal and distal radius were measured in the young adults. Only distal radius measurements were taken in the children. Similar results were obtained in age-sex matched groups from both areas. The only results showing significant difference corresponded to comparison among the Ushuaian women: those whose calcium (Ca) intake was below 800 mg/day presented lower BMD and BMC values than those whose Ca intake was above that level (0.469 ± 0.046 versus 0.498 ± 0.041 g/cm², P < 0.02; 3.131 ± 0.367 versus 3.339 ± 0.386 g, P < 0.05, respectively). In conclusion, peripheral BMD and BMC were similar in children and young adults from Ushuaia and Buenos Aires in spite of the previously documented difference between both areas regarding UV radiation and winter vitamin D status. BMD of axial skeletal areas as well the concomitant effect of a low Ca diet and vitamin D deficiency on the growing skeleton should be studied further. Received: 7 June 1999 / Accepted: 22 March 2000     

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     

Abnormal cell calcium concentrations in cultured bone cells obtained from femurs of obese and noninsulin-dependent diabetic rats

Summary Cytoplasmic free calcium concentration [Ca²⁺]i was quantified in cultured bone cells with osteoblastic characteristics. The cells were obtained from femurs of obese (fa/fa) Wistar-Kyoto rats, from nonobese, noninsulin-dependent diabetic (NIDD) Sprague Dawley rats, and from their appropriate controls. [Ca²⁺]i was also determined in bone cells obtained fromin vivo insulin-treated NIDD rats. Obese (Wistar Kyoto) rats had increased body weight (313±13 vs. 249±4 g;P<0.01), decreased femur weights (0.68±0.05 vs. 0.89±0.05 g;P<0.05), similar glucose levels (148±5 vs. 139±3 mg/dl), and higher plasma insulin levels (6.0±0.5 vs. 0.7±0.1 ng/ml;P<0.01) when compared with their nonobese [(fa/+); (+/+)] littermates. Nonobese, NIDD rats, compared with their appropriate controls (nondiabetic Sprague Dawley rats) had higher plasma glucose levels (235±32 vs. 145±3 mg/dl;P<0.01) but their plasma insulins, body weights, and femur weights were similar to controls (0.7±0.1 vs 0.6±0.1 ng/ml; 302±4 vs. 318±14 g; 0.97±0.4 vs. 0.98±0.04 g, respectively). Long-term (4 weeks) daily insulin treatment (2 u/100 g) of the NIDD rats increased their plasma insulin (1.9 ng/ml;P<0.05) and body weight (369±13 g;P<0.05) but did not change their plasma glucose levels (225±5 mg/dl), or femur weights (0.98±0.4 g). [Ca²⁺]i in bone cells derived from the femurs of obese animals was higher than in cells derived from their nonobese littermates (156±22 vs. 71±13 nM;P<0.01). In bone cells from NIDD rats, [Ca²⁺]i was lower compared with controls (146±22 vs. 229±19 nM;P<0.001). Insulin treatment of the diabetic animals augmented the decrease in [Ca²⁺]i in their bone cells (68±11 nM;P<0.005 compared with nontreated rats). These data reveal that [Ca²⁺]i in cultured bone cells from obese nondiabetic and nonobese NIDD rats differs from that of their controls. Compared with their controls, the changes in [Ca²⁺]i in bone cells from the NIDD and obese animals were in opposite directions. Whether the underlying mechanisms for the changes in cell [Ca²⁺]i in nondiabetic obese and nonobese NIDD animals differ, and whether the changes in [Ca²⁺]i observed in the cultured cells reflect thein vivo condition in bone cells of these animals are questions that await further investigations.     

Calcium-Induced Natriuresis: Physiologic and Clinical Implications

Assessment of the tubular reabsorption of calcium (Ca) by infusion is complicated by suppression of parathyroid hormone (PTH) secretion, and by activation of the serpentine Ca sensing receptor in the renal tubule, which inhibits Ca and sodium reabsorption, but little is known about the magnitude of the natriuretic effect of Ca in human subjects. Accordingly, we reanalyzed the relationship between serum Ca and urine Ca and sodium excretion expressed per unit of creatinine clearance (CaE and NaE), and per unit of time (UCa and UNa), during a standard Ca infusion, in 14 healthy volunteers and in 8 primary hyperparathyroid patients. In healthy subjects we observed a large effect of Ca infusion on NaE, which rose as high as 8 mmol/liter GFR. In patients with primary hyperparathyroidism both CaE and NaE during Ca infusion were significantly lower overall than in healthy subjects for comparable values of serum Ca (P < 0.05 by covariance analysis), due mainly to a decline or reversal of the slopes at the highest serum Ca levels. In both controls and primary hyperparathyroid subjects the variance of CaE as dependent variable was explained by both serum Ca and by NaE as independent variables (P < 0.001). We conclude that (1) The natriuretic effect of hypercalcemia was unexpected large and if maintained would lead to substantial depletion of extracellular fluid. (2) Patients with chronic hypercalcemia, including primary hyperparathyroidism, probably have mild sodium depletion, and are more susceptible to volume depletion. (3) Calcium reabsorption during Ca infusion is reduced by suppression of PTH secretion and increased by volume contraction due to sodium depletion. Discrimination between different basal levels of parathyroid function is successful because these effects usually cancel out. (4) The increase in tubular reabsorption of Ca due to volume contraction can initiate a vicious circle, of importance to the pathogenesis and treatment of severe hypercalcemia. Received: 11 May 1999 / Accepted: 13 January 2000     

Elevated Collagen Turnover in Nigerian Children with Calcium-Deficiency Rickets

Calcium deficiency is a major etiological determinant of rickets in Nigerian children and is accompanied by undermineralization of the developing bone matrix which is composed largely of type I collagen. We have assessed types I and III collagen metabolism by measuring the circulating concentrations of the N- and C-terminal propeptides (intact PINP and PICP) and the C-terminal telopeptide (ICTP) of type I collagen, and the N-terminal propeptide (PIIINP) of type III collagen in 94 healthy Nigerian children and in 44 children aged 1–5 years with active calcium-deficiency rickets. In active rickets the mean levels of the four collagen metabolites were approximately twofold higher than in the healthy children, despite a wide variation of individual values. Mean intact PINP was 812 ± 279 versus 403 ± 189 μg/liter; PICP was 573 ± 265 versus 348 ± 229 μg/liter; PIIINP was 16.8 ± 8.6 versus 10.8 ± 3.6 μg/liter, and ICTP was 28.4 ± 17.2 versus 11.9 ± 4.1 μg/liter (all P < 0.001), in rachitic and healthy children, respectively. Healthy children younger than 3 years had higher levels of all the collagen metabolites than those between 3 and 5 years (all P < 0.05). Alkaline phosphatase was greater in rickets than in the healthy group (P < 0.001) whereas mean osteocalcin levels were slightly lower (P= 0.09). 1,25(OH)₂D correlated with all the collagen propeptides, but not with ICTP in the healthy children. No such correlations were found in rickets, where there was a poor inverse correlation between 1,25(OH)₂D and ICTP. These data suggest that collagen turnover is elevated in cases of calcium-deficiency rickets, where vitamin D status is adequate, possibly indicating increased turnover of undermineralized osteoid. Received: 15 October 1996 / Accepted: 5 March 1997     

Hypovitaminosis D Myopathy Without Biochemical Signs of Osteomalacic Bone Involvement

The aims of this study were to investigate myopathy in relation to vitamin D status, and to study the muscular effects of vitamin D treatment on vitamin D-deficient individuals. Further, hypovitaminosis D myopathy was investigated in relation to alkaline phosphatase (ALP), the most commonly used marker for hypovitaminosis D osteopathy. Eight patients with osteomalacia had an isokinetic dynamometer test of all major muscle groups before and after 3 months of vitamin D treatment. The most pronounced improvements in muscle power were seen in the weight-bearing antigravity muscles of the lower limbs. A cross-sectional study was performed among 55 vitamin D-deficient veiled Arab women living in Denmark and 22 Danish controls. An isometric dynamometer model was used for determination of quadriceps muscle power. Both maximal voluntary contraction (MVC) and electrically stimulated values (single twitch, maximal production rate (MPR), and maximal relaxation rate (MRR)) were determined. The women underwent high-dose vitamin D treatment and were retested after 3 and 6 months. Prior to vitamin D treatment all parameters of muscle function in the group of vitamin D-deficient Arab women were significantly reduced compared with Danish controls. MVC: 259.4 ± 11.0 N (Newton) versus 392.6 ± 11.4 N (P < 10⁻⁶), single twitch: 47.0 ± 1.8 N versus 74.6 ± 2.2 N (P < 10⁻⁵), MPR 8.9 ± 0.3 N/10 ms versus 14.3 ± 0.4 N/10 ms (P < 10⁻⁶), MRR 4.5 ± 0.2 N/10 ms versus 6.2 ± 0.2 N/10 ms (P < 10⁻⁶). Muscle function was affected to a similar degree in women with and without bone involvement (as indicated by elevated ALP). After 3 months of vitamin D treatment all muscle-related parameters improved significantly. After 6 months only MVC was reduced compared with Danish controls (320.7 ± 14.3 N (P < 0.02)), whereas all other measurements were normalized. Hypovitaminosis D myopathy is a prominent symptom of vitamin D deficiency, and severely impaired muscle function may be present even before biochemical signs of bone disease develop. Full normalization of hypovitaminosis D myopathy demands high-dose vitamin D treatment for 6 months or more. Our findings indicate that serum levels of ALP cannot be used in the screening for hypovitaminosis D myopathy. Assessment of s-25OHD is the only reliable test. Received: 10 September 1999 / Accepted: 11 January 2000     

Prednisolone Induces an Increase in Serum Calcium Concentration: Possible Involvement of the Kidney, the Bone, and the Intestine

To evaluate the early effect of glucocorticoids on calcium metabolism, 15 subjects aged 22–58 years (5 males, 10 females) with chronic glomerulonephritis were orally treated with 40 mg daily of prednisolone. Five of these subjects were diagnosed with nephrotic syndrome and none had a serum creatinine concentration of more than 1.4 mg/dl. Serum specimens and 24-hour urine specimens were obtained just before and 24 hours after a single oral dose of prednisolone. Serum calcium, ionized calcium, phosphate, intact parathyroid hormone (PTH), intact osteocalcin and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and urinary excretion of calcium, phosphate, and deoxypyridinoline were measured. Both serum calcium and ionized calcium concentrations were significantly increased from 4.39 ± 0.10 to 4.47 ± 0.09 mEq/liter (P= 0.037) and from 2.48 ± 0.04 to 2.55 ± 0.04 mEq/liter (P= 0.002), respectively, 24 hours following a single oral dose of prednisolone. Serum intact PTH concentration slightly decreased, but the difference was not significant by statistical analysis. Serum intact osteocalcin concentration was markedly suppressed. In contrast, no significant changes were observed in urinary excretion of deoxypyridinoline. Serum 1,25(OH)₂D₃ concentration measured in five patients was significantly increased. No significant changes in urinary excretion of calcium was observed in the face of these findings. It thus follows that a single oral dose of prednisolone administration increases serum calcium and ionized calcium concentrations, possibly mediated by suppressed bone formation, increased intestinal absorption of calcium, and impaired urinary excretion of calcium. Received: 19 February 1998 / Accepted: 12 March 1999     

Calcium Supplementation Suppresses Bone Resorption in Early Postmenopausal Women

In order to establish whether calcium supplementation suppresses bone resorption in early postmenopausal women and whether any response is related to calcium absorption status, we studied 22 healthy women (median age 52 years) all within 5 years of the menopause. Urine was collected between 9.00 p.m. and 9.00 a.m., and 9.00 a.m. and 9.00 p.m., (2 days) and a fasting blood and spot urine sample was obtained at 9 a.m. On the first day, 5 μCi of ⁴⁵Ca in 250 ml water with 20 mg calcium carrier as the chloride was given at 9.00 a.m. and a further blood sample was obtained at 10.00 a.m. to measure calcium absorption. A 1 g calcium load was given at 9.00 p.m., immediately before the second 24-hour urine collection. There was a rise in plasma ionized calcium (1.18 ± 0.010 mmol/liter versus 1.21 ± 0.011 mmol/liter, P < 0.01) and a fall in plasma PTH (4.2 ± 0.34 pmol/liter versus 3.5 ± 0.31 pmol/liter, P < 0.01) from baseline after the calcium load, and a trend for the magnitude of the change in PTH to be inversely related to calcium absorption (r =−0.33, P= 0.13). In the fasting spot urine samples, there were falls in hydroxyproline (OHPr/Cr; 14.6 ± 0.71 versus 12.6 ± 0.83, P < 0.001), pyridinoline (Pyr/Cr; 75 ± 2.8 versus 70 ± 3.5, P < 0.05), and deoxypyridinoline (Dpd/Cr; 22.7 ± 1.2 versus 19.5 ± 1.1, P < 0.005) after the calcium load. The calcium load suppressed urinary Dpd/Cr between 9.00 p.m. and 9.00 a.m. (P < 0.005), but not between 9.00 a.m. and 9.00 p.m. We conclude that acute administration of a 1 g calcium load suppresses bone resorption in early postmenopausal women, probably by decreasing PTH secretion. Received: 2 December 1996 / Accepted: 21 May 1997