Plasma Osteocalcin Concentrations Research Articles

Dose-dependent bone-sparing effects of dietary isoflavones in the ovariectomised rat.

The dose-dependent bone-sparing effects of dietary isoflavones (IF) were investigated in adult (7-month-old) Wistar rats. Forty animals were ovariectomised, allocated into four groups of ten rats each, and immediately treated orally with IF at 0 (OVX), 20 (IF20), 40 (IF40) or 80 (IF80) microg/g body weight per d for 91 d; ten sham-operated (SH) controls received the same diet without added IF. Animals were killed on day 91. Both femoral failure load and total femoral, diaphyseal or metaphyseal bone mineral densities (BMD) were lower in OVX animals than in SH animals. Urinary deoxypyridinoline (DPD) excretion, a marker of bone resorption, and plasma osteocalcin (OC) levels, a marker of osteoblast activity, were higher in OVX animals than in SH animals. Total femoral and diaphyseal BMD and femoral failure load were similar in IF-treated rats and SH rats. Although metaphyseal BMD in IF40 or IF80 rats was similar to that in SH rats, its value was lower in IF20 rats than in controls. The day 91 urinary DPD excretion in IF40 and IF80 rats, but not in IF20 rats, was similar to that in SH rats. Day 91 plasma OC concentrations in IF-treated rats were similar to day 45 values, but were decreased in OVX and SH rats. Thus, daily IF consumption prevented ovariectomy-induced bone loss, both by depressing bone resorption and stimulating osteoblast activity. Moreover, as only the highest IF level induced a weak uterotrophic activity, the optimal IF dose which preserves both cancellous and cortical bone, but exhibits no oestrogen-like effects on the uterus, was 40 microg/g body weight per d.

Rutin inhibits ovariectomy-induced osteopenia in rats.

Several studies suggest that polyphenols might exert a protective effect against osteopenia. The present experiment was conducted to observe the effects of rutin (quercetin-3-O-glucose rhamnose) on bone metabolism in ovariectomized (OVX) rats. Thirty 3-month-old Wistar rats were used. Twenty were OVX while the 10 controls were sham-operated (SH). Among the 20 OVX, for 90 days after surgery 10 were fed the same synthetic diet as the SH or OVX ones, but 0. 25% rutin (OVX + R) was added. At necropsy, the decrease in uterine weight was not different in OVX and OVX + R rats. Ovariectomy also induced a significant decrease in both total and distal metaphyseal femoral mineral density, which was prevented by rutin consumption. Moreover, femoral failure load, which was not different in OVX and SH rats, was even higher in OVX + R rats than in OVX or SH rats. In the same way, on day 90, both urinary deoxypyridinoline (DPD) excretion (a marker for bone resorption) and calciuria were higher in OVX rats than in OVX + R or SH rats. Simultaneously, plasma osteocalcin (OC) concentration (a marker for osteoblastic activity) was higher in OVX + R rats than in SH rats. High-performance liquid chromatography (HPLC) profiles of plasma samples from OVX + R rats revealed that mean plasma concentration of active metabolites (quercetin and isorhamnetin) from rutin was 9.46+/-1 microM, whereas it was undetectable in SH and OVX rats. These results indicate that rutin (and/or its metabolites), which appeared devoid of any uterotrophic activity, inhibits ovariectomy-induced trabecular bone loss in rats, both by slowing down resorption and increasing osteoblastic activity.

Daidzein Is More Efficient than Genistein in Preventing Ovariectomy-Induced Bone Loss in Rats

We investigated the ability of genistein and daidzein, two soybean isoflavones, compared with that of 17 alpha-ethinylestradiol, to prevent bone loss in ovariectomized rats, a model for postmenopausal osteoporosis. Female Wistar rats (n = 65; 12 mo old) were either sham-operated (SH; n = 13) or ovariectomized (OVX; n = 52). On d 0, OVX rats were randomly assigned to groups as follows: 13 received genistein [G; 10 mcg/(g body weight. d)], 13 were treated with daidzein [D; 10 mcg/(g body weight. d)], 13 received 17 alpha-ethinylestradiol [E(2); 30 mcg/kg body weight. d)] and 13 were untreated (OVX). Compounds were mixed with a soy protein-free powdered semipurified diet and given orally for 3 mo. On d 90, the bone mineral density (BMD) in lumbar vertebrae, femur and its metaphyseal and diaphyseal zones (rich in cancellous and cortical bone, respectively) was lower in OVX than in SH (P < 0.01). In D or E(2), the four BMD were not different from SH, whereas in G, only the diaphyseal BMD was not different from SH. Image analysis performed in the distal femur metaphysis revealed that the cancellous bone area was lower in OVX than in SH (P < 0.01). Only the area in D was not different from that in SH. Finally, the bone turnover, which was higher in OVX than in SH (P < 0.005 and P < 0.05 for plasma osteocalcin concentration and urinary deoxypyridinoline excretion, respectively), was not different in G, D or E(2) compared with SH. Therefore, consumption of 17 alpha-ethinylestradiol or daidzein was more efficient than genistein in preventing ovariectomy-induced bone loss in rats.

Amylin inhibits ovariectomy-induced bone loss in rats.

Amylin (AMY), a peptide co-secreted with insulin by pancreatic beta-cells, inhibits bone resorption and stimulates osteoblastic activity. The ovariectomized (OVX) rat is an established animal model for human osteoporosis. Thus, the present experiment was performed to study the effects of AMY on estrogen deficiency-induced bone loss in rats. Thirty-one 6-month-old Wistar rats were randomized by body weight (BW) into two groups. The first underwent surgical OVX (n=21). The second was sham-operated (SH; n=10). Sixty days after surgery, 11 OVX rats were s.c. injected with rat AMY (3 microg/100 g BW/day, for 30 days; OVX+AMY), and 10 with solvent alone in the same way (0.15 ml/100 g BW; OVX). Each rat, housed in an individual cage, was fed daily the mean quantity of diet consumed the day before by SH rats. This diet contained 0.24% calcium and 0. 16% phosphorus. The 31 animals were killed on day 90. No difference in daily weight gain and BW was observed between groups. Neither AMY treatment nor OVX had any significant effect upon femoral morphology, femoral failure load, diaphyseal femoral density (representative of cortical bone) and total femoral calcium content. Nevertheless, both distal metaphyseal (representative of cancellous bone) and total femoral bone densities were higher in SH and OVX+AMY than in OVX rats. The highest plasma osteocalcin concentration was measured in OVX+AMY rats. Simultaneously, urinary deoxypyridinoline excretion was lower in OVX+AMY than in OVX rats. These results indicate that in OVX rats, AMY treatment inhibited trabecular bone loss both by inhibiting resorption and by stimulating osteoblastic activity.

Effect of calcium supplementation on bone mineral accretion in Gambian children accustomed to a low-calcium diet

Rural Gambian children have poor growth, delayed puberty, a low bone mineral content, and a low calcium intake. We investigated the effect of a calcium supplement on bone mineral accretion in rural Gambian children. A randomized, double-blind, placebo-controlled study was conducted in 160 children (80 boys, 80 girls) aged 8.3-11.9 y. Bone mineral content (BMC), bone mineral density (BMD), and BMC adjusted for bone width, body weight, and height (size-adjusted BMC) were measured at the midshaft and distal radius. Each child received either 1000 mg Ca/d (as calcium carbonate) or a placebo 5 d/wk for 12 mo. Supplementation increased calcium intake from 342 to 1056 mg/d (8.6 to 26.4 mmol/d). Calcium supplementation resulted in a higher BMC, BMD, and size-adjusted BMC (&xmacr; difference +/- SE): midshaft radius-BMC (3.0 +/- 1.4%; P = 0.034), BMD (4.5 +/- 0.9%; P </= 0.0001), and size-adjusted BMC (4.6 +/- 0.9%; P </= 0.0001); distal radius-BMC (8. 4 +/- 3.2%; P = 0.009), BMD (7.0 +/- 2.7%; P = 0.011), and size-adjusted BMC (5.5 +/- 2.7%; P = 0.042). Supplementation had no significant effect on height, weight, or bone width at the midshaft radius or distal radius. At the end of the study, the calcium group had a significantly lower mean plasma osteocalcin concentration than the placebo group after adjustment for baseline concentration, sex, and pubertal status (-21.9 +/- 6.5%; P = 0.001). Increased calcium intake resulted in increased bone mineral status, possibly in association with a decreased bone remodeling space. Further studies are needed to determine whether an increased calcium intake has long-term benefits in Gambian children.

Bone Mineral Density in Patients with Apolipoprotein E Type 2/2 and 4/4 Genotype

The peak bone mass and the rate of bone loss are in part genetically determined. It has been suggested that bone mineral density (BMD) may be related to allelic variation in the apolipoprotein E (ApoE) gene locus. ApoE is important in the receptor-mediated clearance of chylomicron particles from the plasma, Apo E4 having the highest and Apo E2 the lowest receptor affinity. Chylomicrons are the main carrier of vitamin K in the plasma; vitamin K plays an important role in the carboxylation of osteocalcin. We have tested the hypothesis that persons with E4 variant would have lower BMD and increased bone turnover than those with E2 variant. A total of 18 ApoE 2/2 and ApoE 4/4 homozygotes were selected from 873 patients who were examined for the ApoE genotype. BMD in lumbar vertebral, femoral neck and distal forearm was measured and plasma concentrations of osteocalcin and C-terminal fragments of collagen (CTx) were determined. BMD values (expressed as T-score) at the three specified sites were –0.12± 1.72, –0.52± 1.32 and –0.52± 0.81 in ApoE 2/2 group and –0.24± 1.22, 0.00± 0.84 and –0.17± 1.07 in the ApoE 4/4 group. Plasma osteocalcin and CTx were within normal limits in both groups. In conclusion, we did not observe any association of ApoE genotype with BMD and biochemical markers of bone metabolism in ApoE 2/2 and ApoE 4/4 homozygotes.

The New Selective Estrogen Receptor Modulator MDL 103,323 Increases Bone Mineral Density and Bone Strength in Adult Ovariectomized Rats

Selective estrogen receptor modulators (SERMs) can prevent the bone loss induced by ovariectomy (OVX), but it is not established whether they can increase bone mass and strength in a curative protocol in ovariectomized osteopenic animals. We investigated the influence of a SERM of the new generation, MDL 103,323, on areal bone mineral density (BMD), as measured by dual-energy X-ray absorptiometry, bone strength and remodeling in OVX osteopenic rats. Nine weeks after OVX, 8-month-old rats were divided into six groups of 10 animals. MDL 103,323 was given by gavage at doses of 0.01, 0.1 or 0.6 mg/kg body weight, 5 days a week. The effect of MDL 103,323 was compared with that of the bisphosphonate pamidronate (APD), which was injected subcutaneously at a dose of 1.6 mmol/kg body weight for 5 days every 4 weeks. Lumbar spine (LS), femoral neck (FN), proximal tibia (PT) and midshaft tibia (MT) BMD, bone strength, and proximal tibia histomorphometry, serum osteocalcin, urinary total deoxypyridinoline and serum insulin-like growth factor I (IGF-I) were measured. After 16 weeks of treatment, BMD changes (means +/- SEM) were -11.4 +/- 2. 2, +4.0 +/- 2.1 and +6.4 +/- 1.0% respectively in OVX controls, in rats treated with 0.1 mg/kg MDL 103,323 (p<0.05) and in APD-treated rats (p<0.02) at the level of LS; -0.4 +/- 1.1, +6.7 +/- 1.4, +7.2 +/- 1.8% (p<0.01 and NS) at the level of FN; and -2.6 +/- 1.2%, +5.8 +/- 1.2, +6.9 +/- 1.4% (p<0.03 and 0.01) at the level of PT. MDL 103, 323-treated animals had a higher trabecular bone volume, a higher number of trabeculae and smaller intertrabecular spaces compared with OVX controls. Vertebral body ultimate strength was 186 +/- 13, 292 +/- 16, 249 +/- 23 N (p<0.05) in OVX controls, MDL 103, 323-treated rats and APD-treated rats, respectively. The administration of 0.6 mg/kg of MDL 103,323 did not further increase BMD or bone strength, indicating a bell-shaped dose-response curve. MDL 103,323 lowered plasma osteocalcin concentration and urinary deoxypyridinoline excretion. In rats treated with 0.1 mg/kg MDL 103, 323, plasma IGF-I was increased as compared with OVX controls (664 +/- 36 ng/ml vs 527 +/- 39 ng/ml, p<0.05). In conclusion, these results indicate that this new SERM positively influences BMD and lumbar spine bone strength in estrogen-deficient rats.

Etomidate and the osteocalcin response to gynaecological surgery

Circulating osteocalcin is a good marker of osteoblastic activity and decreases significantly after stressful physiological states such as major surgery. Glucocorticoids are known to inhibit osteoblastic activity and result in a decline in circulating osteocalcin. We used etomidate to inhibit the cortisol response to routine gynaecological surgery to determine if this would prevent the postoperative decline in osteocalcin. Twenty-four patients were allocated randomly to receive either thiopental or etomidate for induction of anaesthesia; all other aspects of anaesthesia and perioperative management were standardized. In the thiopental group, circulating cortisol increased significantly at 2 and 6 h after the start of surgery and plasma osteocalcin concentrations decreased significantly to almost 50% of baseline values at 48 h. Etomidate abolished the cortisol response to surgery, and circulating osteocalcin concentrations did not change after operation. There was a significant difference in osteocalcin concentration between the groups at 48 h. We conclude that the cortisol response to surgery is associated with a postoperative decrease in circulating osteocalcin.

Treadmill running starting 3 months after orchidectomy restores femoral bone mass in rats.

The present study was designed to provide data on the effects on femoral bone of endurance training starting only 3 months after orchidectomy in rats. A total of 70 Wistar male rats were used at 8 weeks of age. On day 0 of the experiment, 10 rats were killed by cervical dislocation to be used as first controls. Among the 60 other animals, half was surgically castrated (CX) or sham operated (SH). On day 90, 10 CX and 10 SH were killed and used as intermediary controls (ICX and ISH). Among the other 20 CX and 20 SH, 10 within each group (CXE, SHE) were selected for treadmill running (60% maximal oxygen uptake, 1 h x day(-1), 5 days x week(-1) for 12 weeks). The 20 other rats were used as sedentary controls (CXR, SHR) and killed (as runners) on day 180. On day 90 femoral bone density (BMD) and mineral content (BMC) were lower in ICX than in ISH. On day 180 total femoral BMD was lower in CXR than in CXE. Simultaneously metaphyseal femoral BMD was lower in CXR than in CXE, SHR or SHE. Furthermore, at that time, no significant difference concerning BMD and BMC was observed between SHR and CXE. This would indicate that treadmill running starting only 3 months after orchidectomy is able to restore BMD and BMC to control values, mainly by inhibiting bone resorption (as shown by decreased urinary deoxypyridinoline excretion in CXE) without decreasing osteoblastic activity (evaluated by plasma osteocalcin concentration).

Influence of Aging on Cortical and Trabecular Bone Response to Estradiol Treatment in Ovariectomized Rats

Three groups (n = 15/group) of 6-, 12- and 30-month-old (mature, old and senescent animals, respectively) female Wistar rats on a diet (6 g/100 g BW/day) containing 0.8% calcium and 0.8% inorganic phosphorus were studied. Within each group, 10 rats were ovariectomized surgically and 5 injected s.c. with 17β-estradiol (E rats, 10 µg/kg BW/48 h) and 5 with solvent alone (OVX rats) from day 2 until day 60 after ovariectomy. Five other rats were sham-operated (SH rats) and received solvent only. All rats were killed by exsanguination 60 days after ovariectomy. Neither ovariectomy nor estradiol treatment had a significant effect upon tibial mechanical properties in 6-, 12- and 30-month-old animals. Bone mineral density (BMD) and bone mineral content (BMC) of the distal femur and BMC of the whole femur were decreased by ovariectomy in 6- and 12-month-old rats, but were not different in the SH and E groups. In senescent animals, in which the lowest BMD and BMC were measured, estradiol treatment was more effective in increasing these parameters than in adult and old rats. Image analysis of the distal femoral diaphysis showed that estradiol treatment prevented trabecular bone loss induced by senescence and/or ovariectomy. In each group, urinary deoxypyridinoline excretion and plasma osteocalcin concentration were higher in the OVX animals than in the controls, consistent with increased bone turnover in the estrogen-deficient state. Both biochemical turnover markers were reduced in the estrogen-treated groups. These results indicate that 17β-estradiol is particularly effective at preventing high-turnover-induced osteopenia in 30-month-old animals.

Decreased osteoblast activity in spontaneously diabetic rats. In vivo studies on the pathogenesis.

Diabetes in both humans and rats is accompanied by low bone formation, which is presumably caused by serum-borne factors. To explore its pathogenesis, we carried out experiments in diabetic and nondiabetic BB rats, using plasma osteocalcin concentrations (OC) as a marker for osteoblast activity. In nondiabetic rats, the i.v. infusion of glucose (30%, 4 d) did not change OC; s.c. insulin infusion (4 U/d, 14 d) reduced OC by 27% (p < 0.01). In diabetic rats, OC were decreased from the first day of glycosuria (71 +/- 5% of paired controls), declining exponentially to 24 +/- 3% after 5 wk. Insulin infusion (1, 2, and 3 U/d, 14 d) produced gradual restoration of OC. OC were better correlated with insulin-like growth factor-I (IGF-I) than with insulin levels in these experiments. OC were dramatically increased 4 d after adrenalectomy (ADX) in all diabetic rats (73 +/- 8 vs 22 +/- 4 micrograms/L before ADX; p < 0.001), but not if corticosterone was administered. Ligand blotting of IGF binding proteins showed a marked decrease in two bands (44-49 and 32-35 kDa) 10-14 d after diabetes onset; the density of these bands was increased, but not normalized after ADX. Thus, decreased osteoblast activity is present from the onset of diabetes, is dependent on endogenous corticosterone, and cannot be reproduced by hyperglycemia in nondiabetic rats.

Influence of treadmill running on femoral bone in young orchidectomized rats.

Forty 6-wk-old male Wistar rats weighing 308 +/- 24 g were divided into two groups. On day 0, the 20 animals in one group were surgically castrated and the other group was sham operated. Within each group, 10 rats were selected for treadmill running (60% maximal O2 consumption, 1 h/day, 6 days/wk for 15 wk). The 20 sedentary rats were used as controls. At the time the rats were killed (day 105), running had no significant effect on femoral mechanical properties either in castrated or in sham-operated rats. Femoral bone density was lower in orchidectomized than in sham-operated rats. Nevertheless, it was higher in exercised than in sedentary rats. Femoral Ca content paralleled changes in bone density. Treadmill running had no significant effect on plasma osteocalcin concentration but inhibited the increase in urinary deoxypyridinoline excretion observed in castrated rats. Image analysis (measured at the distal femoral diaphysis) revealed that these effects mainly resulted from decreased trabecular bone resorption in castrated exercised rats.

Influence of Ovariectomy and Estradiol Treatment on Calcium Homeostasis During Aging in Rats

Study was carried out on Wistar female rats to evaluate the consequences of ovariectomy and 17β-estradiol substitutive treatment during aging on bone, Ca metabolism and calciotropic hormones. Three groups of fifteen rats, mature, old and senescent (4–, 10–, and 28 month-old) female were fed a diet (6 g/100 g BW/day) containing 0.9% Ca and 0.8% Pi. Within each group, 10 rats were surgically ovariectomized (OVX). From day 1 until day 60 after OVX, they were subcutaneously injected with either 17β-estradiol (E: 10 µg/kg BW/48 h; n = 5) or with solvent alone (OVX; n = 5). Five other rats were sham operated (SH) and received solvent alone. Animals were put in balance 1 day per week to determine Ca and Pi intestinal apparent absorption and urinary pyridinium cross-links excretion was measured by HPLC. All rats were killed by exsanguination 60 days after OVX. Plasma was collected for measurement of intact parathyroid hormone (PTH), calcitonin (CT), insulin-like growth factor-1 (IGF-1), Ca and Pi. The success of OVX was confirmed at necropsy by observation of marked atrophy of the uterine horns. The right femur was collected, cleaned from adjacent tissue and used for mineral analysis. Despite correct matching for feeding, BW was significantly larger in 6 and 12 month-old OVX rats. OVX and 17β-estradiol had no significant effect upon plasma Ca, Pi and CT concentrations. Aging is associated with increased circulating PTH levels (pg/ml) (SH - 6 months: 50.8 ± 12.6; 12 months: 219.1 ± 34.9; 30 months: 158.7 ± 23.5; P < 0.05). Ur inary and fecal Ca and Pi excretion in senescent animals were higher than in adult or old rats, thus resulting in a drastic fall in both intestinal apparent absorption and retention of Ca and Pi in 30 month-old animals. In each group, urinary pyridinium cross-links excretion and plasma osteocalcin concentration were higher in the OVX animals than in the controls, consistent with increased bone turnover in the estrogen deficient state. Both biochemical turnover markers were reduced in the estrogen-treated groups. In the same way, OVX increased and estrogen decreased the plasma IGF-1 levels. We conclude that 17β-estradiol prevents high turnoverinduced osteopenia even in 30 month-old rats.

Effect of diets varying in nitrogen or phosphorus content on indicators of bone growth in lambs.

Growing lambs were fed diets low in nitrogen and phosphorus (LNLP), low in nitrogen and high in phosphorus (LNHP), high in nitrogen and low in phosphorus (HNLP) or high in nitrogen and phosphorus (HNHP) and the effects on bone growth and on blood and urinary bone marker levels or excretion rates were monitored. Plasma calcium concentrations were higher, and phosphorus concentrations lower, in lambs fed the low phosphorus diets but there were no differences in plasma 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) concentrations. Lambs fed both low phosphorus diets (LNLP and HNLP) had lower plasma osteocalcin and higher bone-specific alkaline phosphatase concentrations than those fed the high phosphorus diets. Urinary pyridinoline and deoxypyridinoline excretion were also affected by treatment, with their rates of excretion being highest in lambs fed the diet low in both nitrogen and phosphorus (LNLP). Lambs fed the low phosphorus diets were lighter in weight at slaughter and had lighter bones that were less well mineralized than those fed the high phosphorus diets. Reducing the nitrogen content of the diet appeared to have little effect on bone composition. These results suggest that bone markers that have proved useful in the diagnosis and treatment of bone disease are sensitive to variation in nutrient supply and may prove useful in early detection of nutrient deficiencies that affect bone growth.

Plasma osteocalcin in healthy Nigerian children and in children with calcium-deficiency rickets.

Osteocalcin is an osteoblast-specific protein believed to be associated with events occurring during bone mineralization, which has been widely used clinically as an indicator of osteoblast function. Plasma osteocalcin concentrations (pOC) were studied in 94 (59 male, 35 female) healthy and 44 (21 male, 23 female) rachitic Nigerian children, all one to five years of age. The study was aimed at establishing a reference range for healthy Nigerian children determining any changes in plasma osteocalcin levels occurring in children with calcium-deficiency rickets. In the controls, pOC levels ranged from 3-89 ng/ml, with a mean value of 23 +/- 19 ng/ml. The values were higher in girls (29 +/- 21 ng/ml) than in boys (21 +/- 18 ng/ml), though not significantly. The controls had values consistent with other published pediatric ranges from Europe and North America. In the younger rachitic children (under 3 years) the mean pOC was lower than in the controls (P = 0.04) despite the much elevated plasma levels of 1,25(OH)2D. In the controls, pOC correlated with 1,25(OH)2D (r = 0.59, P = 0.003), alkaline phosphatase (r = 0. 22, P = 0.03), and inorganic phosphate (r = 0.27, P = 0.01). These correlations were lost in the rickets group. The findings in the controls confirm the known association between plasma 1,25(OH)2D and circulating osteocalcin levels, whereas the findings in the rickets group suggest that the stimulatory effects of 1,25(OH)2D on osteocalcin may depend on other permissive factors, such as normal circulating levels of calcium and phosphate.

Influence of ovariectomy on bone metabolism in very old rats.

Twenty-five 30-month-old Lou rats fed a diet (6 g/100 g BW/day) containing 0.9% Ca and 0.8% Pi were divided into five groups. Four groups were surgically ovariectomized. From day 2 until day 29 after ovariectomy, they were S.C. injected either with 17 beta estradiol (E2; 10 micrograms/kg BW/48 hours) or progesterone (P; 140 micrograms/kg BW/48 hours), or 17 beta estradiol + progesterone (E2P) at the same doses, or solvent alone (OVX). The fifth group was sham operated (SH) and injected with solvent. Urine was collected in metabolic cages from day 24 to 29 after ovx, and urinary pyridinoline (PYD) and deoxypyridinoline (DPD) excretion (markers of bone resorption) was measured by HPLC. All animals were killed 30 days after ovariectomy. Serum was then collected for measurement of osteocalcin (OC), alkaline phosphatase (ALP), parathyroid hormone (PTH), and calcitonin (CT). At necropsy, the success of ovariectomy was checked by marked atrophy of the uterine horns. Left and right femur were harvested for densitometric and mineral analysis, respectively. Ovariectomy had no significant effect upon plasma calcium and PTH concentrations. E2 or E2P treatment significantly increased plasma PTH and calcitonin concentrations. Plasma OC concentrations and ALP were not different in any of the groups. In contrast, urinary excretion of PYD and DPD was higher in OVX than in SH rats. Bone mineral density (BMD) of the distal femur was decreased by OVX, but was not different in the E2P and SH groups. A similar pattern was observed for the mineral or Ca content of whole femur. Thus, OVX decreased BMD and bone mineral content (BMC) in very old female rats. Plasma OC concentration and ALP activity failed to demonstrate any significant effect of OVX, whereas PYD and DPD were elevated. These results suggest that bone resorption is increased in OVX rats, even when supplemented with E2 or P alone. However, no significant difference was observed between SH and OVX rats treated with supplementation of both E2 and P. Thus, in very old rats, a combination of E2 and P is much more effective than E2 or P alone to prevent bone loss following ovariectomy.

Metacarpal growth and systemic markers of bone metabolism in the ovine fetus.

Bone metabolism was assessed in 56 Ile de Francex(RomanovxLimousine) fetal lambs killed between Day 80 and Day 145 of gestation. In each fetus, the length, width and weight, as well as the calcium and phosphorus content of the left diaphyseal metacarpal bone were measured. Plasma alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities, bone ALP (B-ALP), osteocalcin (OC) and insulin-like growth factor-I (IGF-I) were assayed in these fetuses and in six newborn lambs from birth until one month after birth. Fetal growth is characterized by an increase in bodyweight, bone size and bone mineral content from Day 80 to Day 132 of gestation. These parameters did not significantly vary until birth. Plasma concentrations of IGF-1, OC and B-ALP increased from Day 80 to Day 132. Between Day 132 and birth, plasma IGF-I and B-ALP concentrations did not significantly vary, whereas plasma OC concentration decreased, confirming the usefulness of OC as a marker of osteoblastic activity and bone formation in the ovine species. The increase in plasma IGF-I, B-ALP and OC concentrations observed during the first two weeks of postnatal life indicate an intense skeletal growth in these animals, which was confirmed by the bodyweight growth curve.

Circadian rhythm in type I collagen formation in postmenopausal women with and without osteopenia.

A circadian rhythm in the serum concentration of the procollagen type I carboxyl-terminal propeptide (sPICP) has previously been demonstrated in premenopausal women. This study was performed to investigate the circadian rhythm in sPICP in healthy and osteopenic postmenopausal women. Blood samples were taken every third hour for 27 h from three groups of women: 12 early postmenopausal women (aged 55 +/- 2 years; mean +/- SD); 12 late postmenopausal women (aged 73 +/- 1 years); and 12 osteopenic but otherwise healthy late postmenopausal women (aged 73 +/- 1 years). A circadian rhythm in sPICP was found in all three groups, as shown by cosinor analysis (p = 0.000003-0.03). The circadian rhythm in sPICP was significantly different between the osteopenic group and the age-matched healthy group (p < 0.008). The amplitude of the circadian rhythm in sPICP was about twice as high in the osteopenic group, and the time of the maximum tended to be about 3 h later, as compared with the age-matched healthy group. The plasma concentration of osteocalcin, as measured by a recently developed two-site enzyme-linked immunosorbent assay, also showed a circadian rhythm in all three groups (p = 0.0001-0.05), with no significant differences between groups. In conclusion, we have found a significant circadian rhythm in sPICP in both early and late postmenopausal women. In osteopenic women the nightly peak in sPICP is larger and persists later into the night as compared with non-osteopenic women.

Regulation of plasma osteocalcin by corticosterone and norepinephrine during restraint stress

Osteocalcin (OC), an extracellular calcium-binding protein of bone origin, is synthesized by osteoblasts and binds with high specificity to bone mineral crystals. A small, but relatively consistent portion of newly synthesized OC which is released to circulation has been well correlated with histological indices of osteoblastic activity. Synthesis of OC is regulated by numerous hormones including glucocorticoids. We previously reported that mild mental stressors such as cage change or cold exposure decreased rat plasma OC by up to 40% within 1 h. A similar response was induced in a time- and dose-related manner by injection of physiological levels of corticosterone (CS), the active glucocorticoid in rats. Prone immobilization by foot restraint of conscious rats for up to 2 h (IMMO) is a well-characterized model of classic "fight-or-flight" response. This model induces an immediate and prolonged elevation of CS, as well as the catecholamines epinephrine (E) and norepinephrine (NE). In marked contrast to milder stressors, immobilization induced an immediate increase of plasma OC, greater than 50% within 5-20 min, which returned toward normal after 2 h of restraint. Selective ablation of the hormones by adrenal medulectomy, adrenalectomy, or blockade of sympathetic ganglia did not abolish the initial rapid rise of plasma OC. Even before IMMO, plasma OC was increased by about 50% in the absence of sympathetic neural function or adrenal CS production. The presence of both CS and NE, but not E, was required to return plasma OC concentrations to basal levels. This strongly suggests interaction of CS and NE to regulate plasma OC and its release from bone. As expected, prior cold exposure lowered plasma OC, but did not abolish a subsequent increase in response to IMMO, nor did IMMO repeated daily for 7 days. The stimulus for the initial rapid elevation of OC is unknown, but likely to be of importance in the role OC plays in response to stress. Further investigation of the OC under mental stress should help to understand the function of this abundant and highly conserved bone protein.

Oral and inhaled corticosteroids reduce bone formation as shown by plasma osteocalcin levels.

Osteoporosis is a well-known, serious complication of long-term high-dose corticosteroid therapy. This study was performed to determine the effects of commonly used doses of oral and inhaled steroids on biochemical indices of bone formation. Initially we examined the long-term effects of oral steroids. Thirty-four outpatients with symptomatic asthma or chronic obstructive airways disease (COAD) receiving long-term oral prednisolone (mean 10.1 mg daily) were compared with 34 control subjects with asthma or COAD matched individually for age, sex, and menopausal status who were not taking oral steroids. Plasma osteocalcin concentrations were significantly lower (patients 6.3 +/- 0.1 ng/ml; control subjects 8.6 +/- 0.5 ng/ml, mean +/- SEM; p < 0.01) in patients on steroids with no difference in alkaline phosphatase. To examine the short-term effects of oral and inhaled corticosteroids, healthy male volunteers were given a 7-d course of either 15 mg oral prednisolone daily (n = 10) or 500 micrograms inhaled beclomethasone twice daily (n = 20). After 1 wk of oral prednisolone, mean plasma osteocalcin decreased from 11.8 +/- 1.1 ng/ml to 6.9 +/- 0.8 ng/ml (p < 0.001). With inhaled beclomethasone mean plasma osteocalcin decreased from 11.6 +/- 0.6 ng/ml to 9.6 +/- 0.6 ng/ml (p < 0.001) with no change in alkaline phosphatase. In doses routinely prescribed for the prophylaxis and treatment of asthma, oral and inhaled steroids suppress osteocalcin levels and may therefore inhibit bone formation. This effect is seen with short courses of steroids and also with chronic administration.(ABSTRACT TRUNCATED AT 250 WORDS)