Effects of Promethazine on Bone Mass and on Bone Remodeling in Ovariectomized Rats: A Morphometric, Densitometric, and Histomorphometric Experimental Study

The effect of promethazine on bone is debated. We studied the effect of promethazine on bone and the mechanism of action involved by densitometric and histomorphometric measurements in female Wistar rats (100 days old, mean weight 25 ± 20 g). A control group of 15 rats was not manipulated. An experimental group of 15 rats were ovariectomized (OVX) at 100 days of life and fed a diet supplemented with 4.8 mg/kg promethazine hydrochloride (OVX + Prom). The group that underwent OVX and a group of 15 rats that underwent sham ovariectomy (Sham-OVX) were not treated with promethazine. After 30 days, all the rats were killed. Their femur and 5th lumbar vertebra were dissected and cleaned of soft tissue. Femoral length and vertebral height were measured with a caliper and bones were weighed on a precision balance. The bone mineral content (BMC) and bone mineral density (BMD) of the whole right femurs and 5th lumbar vertebras were measured by dual-energy X-ray absorptiometry (DXA). Trabecular bone volume (Cn-BV-TV%), trabecular number (Tb-N mm⁻¹), trabecular thickness (Tb-Th μm), and trabecular separation (Tb-Sp μm) were measured in the femurs by histomorphometric study of nondecalcified bone. Our results showed that promethazine significantly inhibited postovariectomy loss of bone mass (P < 0.0001) by significantly reducing bone resorption, as shown by the smaller trabecular spaces observed in the treated OVX rats (P < 0.0001). Received: 1 June 1998 / Accepted: 17 February 1999     

Effect of silicon supplement on osteopenia induced by ovariectomy in rats

The effect of silicon (Si) supplement on preventing bone mass loss induced by ovariectomy (OVX) in rats was investigated. Three groups of 15, 100-day-old female Wistar rats each, with a mean initial weight of ∼260 g per animal, were selected for the present study. One of the experimental group consisting of 15 OVX rats was fed a diet supplemented with 500 mg of Si per kg of feed (Si + OVX). The other two groups consisting of 15 OVX and 15 sham-OVX rats did not receive these supplements. Morphometric (weight and length) and densitometric studies with dual-energy X-ray absorptiometry were performed on the whole femur and 5th lumbar vertebra of each animal 30 days after the experiment. The Si + OVX rats did not show a loss of bone mass induced by OVX at axial level (5th lumbar vertebra) or periphery (femur). Nonetheless, a significant increase (ANOVA with Bonferroni/Dunn post hocs test) of longitudinal development of the femur (P < 0.0001) was patent. These results, obtained through the measurements of axial and peripheral bones, warrant closer scrutiny in connection with the Si inhibitory effect on bone mass loss as well as the stimulatory effect on bone formation. Both actions, namely, inhibition of resorption and stimulation of formation, infer that Si may have a potential therapeutic application in the treatment of involutive osteoporosis. Received: 15 February 1999 / Accepted: 25 June 1999     

Restoration of ovariectomy-induced osteopenia by nitroglycerin

Nitric oxide (NO) is known to inhibit osteoclastic bone resorption. Previously, we demonstrated that the NO donor nitroglycerin (NG) prevented ovariectomy (OVX)-induced bone loss. The current study shows that NG restores ovariectomy-induced osteopenia. Twenty-four female Sprague-Dawley rats, 36 weeks of age, underwent OVX, and a further six rats were sham-operated. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometric (DXA) scanning prior to OVX, at 6 weeks postsurgery, and at 6 weeks posttreatment. OVX rats were then assigned to four groups and treated with either (1) vehicle, (2) 17-β-estradiol, (3) NG (0.2 mg/kg/day), or (4) a combination of estrogen and NG (n = 6/group). During the first 6-week post-OVX period, there was a significant decrease in the BMD in all ovariectomized (OVXed) rats (−11.0%, P < 0.001). There were no significant changes in BMD during the entire 12-week period in sham-operated rats. During the second 6-week period (after developing bone loss), there was no further significant loss of BMD in OVXed controls. BMD loss and loss of femur weight produced by OVXed were restored by treatment with estrogen, NG, or the two agents together during the second 6-week period (P < 0.01). The effects of estrogen and NG together, however, were not additive. The BMD of rats treated with NG alone, at 12 weeks, was similar to that of animals treated with estrogen alone or with estrogen and NG, and was comparable to that of sham-operated rats. The increased urinary excretion of deoxypyridinolines caused by OVX was negated by estrogen, NG, and estrogen together with NG (P < 0.01). In contrast to estrogen, NG did not decrease the post-OVX-induced increase of serum osteocalcin levels, suggesting that NG may also have a positive effect on bone formation. In summary, the results suggest that the NO donor, NG, reverses the OVX-induced bone loss in rats, and these effects are likely due to decreased bone resorption and, perhaps, increased bone formation. Received: 24 December 1998 / Accepted: 1 July 1999     

Ovariectomy-Induced Bone Loss Can be Affected by Different Intensities of Treadmill Running Exercise in Rats

Fifty-six Sprague-Dawley rats were either ovariectomized (OVX, n= 24), sham-operated (Sham, n= 24), or sacrificed (n= 8) at the beginning of the experiment to serve as a baseline group. The OVX and Sham groups were further randomly divided into control (CTRL), slow running (R10), and faster running (R18) groups. R10 and R18 groups ran for 2 × 30 min/day for 8 weeks at speeds of 10 m/min and 18 m/min, respectively. Exercise did not affect the mechanical or histomorphometric parameters of bone in the sham-operated rats. There was no effect of exercise on body weight gain in the OVX-R10 group, but in OVX-R18 it decreased the gain of body weight. In the OVX–CTRL group the maximal load and energy absorption of the femoral neck were 16.7% (P < 0.001) and 30.0% (P < 0.001) lower than in the Sham–CTRL group, respectively. In OVX animals, slow running had a positive effect on the maximal load of the femoral neck (86.5 N) when compared with OVX–CTRL rats (77.1 N, P < 0.07). 51.7% of the trabecular bone was lost in the distal femur as a result of OVX and exercise reduced this loss to 30.2% (R10) and 39.9% (R18). Ovariectomy increased the bone formation rate (BFR) and the mineral apposition rate (MAR) on the periosteum of the femoral shaft. Exercise decreased the periosteal BFR and MAR in OVX rats, but increased it at the endosteum. Osteoclast numbers in the femoral metaphysis were increased after OVX and running exercise inhibited this effect significantly. The maximal bending load of the humerus increased after OVX by 12.1% (P < 0.05). Exercise enhanced this effect, the slow running being more effective. These results suggest that bone in OVX rats is either more sensitive to exercise than in sham-operated rats or that the higher body weight with slow running induces optimal loading and strengthens the bones. Received: 2 May 1996 / Accepted: 15 October 1996     

The synthetic phytoestrogen, ipriflavone, and estrogen prevent bone loss by different mechanisms

Ipriflavone (IP), a synthetic isoflavone has been reported to prevent bone loss in both postmenopausal women and ovariectomized (ovx) rats. The purpose of this study was to compare and contrast some of the bone protective mechanisms of IP to those of 17β-estradiol (E₂) in ovarian hormone deficiency. Forty-eight 95-day-old Sprague-Dawley rats were assigned to four groups: sham, ovx, ovx+IP, and ovx+E₂. The doses of IP and E₂ were 100 mg and 10 μg/kg body weight per day, respectively. Rats were fed a diet that contained 0.4% calcium, 0.3% phosphorus, and 0.195 nmol vitamin D₃/g diet. After sacrifice, left femoral bone densities were measured and bone histomorphometry was performed on the proximal tibial metaphysis. Ipriflavone as well as E₂ treatment completely prevented the ovx-induced femoral bone density loss. However, in contrast to E₂, IP did not lower the ovx-induced rise in serum alkaline phosphatase (ALP) activity or insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3 concentrations. On histomorphometry analysis, the ovariectomy-induced increase (P < 0.09) in bone formation rate (BFR) was significantly (P < 0.05) suppressed by E₂ treatment, whereas this higher BFR was maintained in IP-treated animals. These findings indicate that IP is effective in preventing the ovx-associated bone loss. The bone protective mechanisms of IP in ovarian hormone deficiency may be different from those of E₂ and may involve increased rates of bone formation. Received: 21 October 1998 / Acccepted: 26 July 1999     

The Effect of Tamoxifen on Bone Metabolism and Skeletal Growth is Different in Ovariectomized and Intact Rats

The effects of tamoxifen (TAM) treatment on bone metabolism and skeletal growth were studied in sexually mature intact or ovariectomized (OVX) rats. Experiment 1 was designed to observe the effects of TAM on bone metabolism and skeletal growth in intact rats and included two groups: (1) intact plus vehicle and (2) intact plus TAM. Experiment 2 was designed to investigate the effects of TAM on OVX rats and included the other two groups: (3) OVX plus vehicle and (4) OVX plus TAM. Serum calcium osteocalcin and urinary pyridinoline (Pyr) and deoxypyridinoline (Dpyr) were measured serially before and after TAM treatment for 6 weeks in order to monitor bone turnover. Bone mineral density (BMD) and bone mineral content (BMC) of excised right femora and lumbar vertebrae were determined by dual energy X-ray absorptiometry (DXA). To examine the effect of TAM on skeletal growth, the conventional parameters of femora and the histology of right tibiae were also measured. TAM did not induce significant change in the biochemical markers in intact rats during the 6-week experiment. Bone mass and skeletal growth were not changed by TAM treatment in intact rats. However, TAM treatment reduced the increase in serum osteocalcin and urinary pyridinium cross-links from 1 week to 6 weeks postovariectomy in the OVX rats. TAM inhibited the skeletal growth in OVX rats, because TAM treatment shortened femoral length and decreased the cell number in the growth plate in OVX rats in this study. Our findings indicate that TAM exerts an effect of estrogen agonist on bone metabolism and skeletal growth in OVX rats, however, it does not affect them in intact rats. Received: 1 September 1995 / Accepted: 20 February 1996     

Vitamin K Status and Bone Mass in Women With and Without Aortic Atherosclerosis: A Population-Based Study

Gammacarboxyglutamate (Gla) is an uncommon amino acid formed by vitamin K action. Increasing evidence indicates that Gla-proteins are involved in the regulation of calcification processes in both bone tissue and atherosclerotic vessel wall. In a population-based study we have previously shown that in a group of 113 postmenopausal women the presence of abdominal aortic calcifications is associated with a reduced vitamin K status. In the present study we investigated whether this reduced vitamin K status was also associated with differences in bone mass or circulating calciotropic hormone levels. Serum immunoreactive osteocalcin with low affinity for hydroxyapatite (irOC_free) was used as a marker for vitamin K status. After correction for age it was found that women with atherosclerotic calcifications had a 7% lower bone mass as measured by metacarpal radiogrammetry (mean difference: 3.2 mm², 95% CI: −0.2–6.5, P= 0.06). No differences between both groups of women were observed for serum intact parathyroid hormone (PTH) and serum 25-hydroxyvitamin D levels. In the atherosclerotic women (n = 34), markers for vitamin K status were inversely associated with bone mass (r =−0.47, P= 0.013), whereas no such association was found in the nonatherosclerotic women (n = 79). It is concluded that the atherosclerotic women in this study may be at higher risk for osteoporotic fractures as evidenced by their lower bone mass and higher serum irOC_free levels. The finding that in atherosclerotic women vitamin K status is associated with bone mass supports our hypothesis that vitamin K status affects the mineralization processes in both bone and in atherosclerotic plaques. Received: 15 January 1996 / Accepted: 3 May 1996     

The Phytoestrogen Genistein Reduces Bone Loss in Short-Term Ovariectomized Rats

The incidence of fractures and of osteoporosis differs between Oriental and Western Caucasian women. This may depend, at least in part, on nutritional factors, including dissimilarities in dietary intake of phytoestrogens. To investigate this possibility, 2-month-old female rats were ovariectomized (OVX) or sham-operated (SHAM), fed a casein-based diet, injected daily with subcutaneous genistein (GEN), the most abundant and best characterized phytoestrogen, or vehicle (Veh) and killed 21 days after surgery. As expected, ovariectomy resulted in loss of bone mineral density (BMD) and in uterine atrophy. However, administration of 5 mg GEN per gram body weight (b.w.) ameliorated the ovariectomy-induced loss of BMD (189 ± 2 mg/cm² in OVX and 192 ± 2 in OVX with 5 mg GEN/g b.w. per day; p<0.05). One microgram GEN per gram body weight did not affect the BMD loss and the effect of the 5 mg and 25 mg GEN per gram body weight were statistically not different. A trend toward reduced uterine atrophy (21% reduction) was noted with the 25 mg GEN dose, but not with the 1 mg and 5 mg doses. A separate experiment with 2 x 2 factorial design was conducted to elucidate the mechanism by which GEN ameliorates ovariectomy-induced bone loss. In this experiment, histomorphometry demonstrated a dramatic reduction in trabecular bone volume after ovariectomy (7.6 ± 0.7% of total bone volume in SHAM-Veh vs 3.3 ± 0.2% in OVX-Veh; p<0.01) and less bone loss in OVX rats injected with 5 mg GEN per gram per day (3.3 ± 0.2% of total bone volume in OVX-Veh vs 5.2 ± 0.4% in OVX-GEN; p<0.01). Administration of GEN was associated with higher bone formation rate per tissue volume and with a trend toward a higher number of osteoblasts per bone perimeter. The parameters of bone resorption were not affected by GEN. The concentration of serum osteocalcin and the urinary excretion of deoxypyridinoline provided corroborating results. Since production of proinflammatory cytokines is intimately involved in the pathogenesis of postmenopausal osteoporosis, the effect of GEN on lipopolysaccharide-induced in vitro production of Tumor necrosis factor-alpha (TNFa) was tested in monocytic cells from the same four rat groups. Production of TNFa was markedly elevated in OVX-Veh as compared with the SHAM-Veh rats, but this was blocked by GEN in the OVX rats. This study shows that GEN reduces both trabecular and compact bone loss after ovariectomy and that this protective effect differs from that of estrogen, since it depends on stimulation of bone formation rather than on suppression of bone resorption. Lack of action of GEN on uterine atrophy supports the possibility that this GEN dose affects target tissues via non-estrogenic mechanisms. Modulation of cytokine production may be involved in the effect of GEN on bone. Received: 27 June 1997 / Accepted: 27 October 1997     

Effects of Postmenopausal Hormone Replacement Therapy With and Without Vitamin D3 on Circulating Levels of 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D

The effects of postmenopausal hormone replacement therapy (HRT) and vitamin D₃ on vitamin D metabolites (25OHD and 1,25(OH)₂D) were studied in a population-based prospective 1-year study. The serum concentrations of intact parathyroid hormone (PTH), calcium, and phosphate were also studied. A total of 72 women were randomized into four treatment groups: HRT group (sequential combination of 2 mg estradiol valerate and 1 mg cyproterone acetate), Vit D₃ group (vitamin D₃ 300 IU/day + calcium lactate 500 mg/day), HRT + Vit D₃ group (both above) and placebo group (calcium lactate 500 mg/day). Serum samples were taken in March–April, when vitamin D formation from sunlight in Finland is minimal after the dark winter. Serum concentrations of 25OHD increased in the Vit D₃ group (33.5%, P < 0.001) and in the HRT + Vit D₃ group (38.2%, P < 0.001) but had not changed significantly in the HRT and placebo groups at the 1-year follow-up examination. Serum concentrations of calcitriol (1,25(OH)₂D) increased, however, only in the HRT group (23.7%, P < 0.05), and remained unchanged in other groups. Serum concentrations of PTH decreased by 23.2% (P < 0.05) in the placebo group, but did not change significantly in the other three groups. The concentrations of serum calcium increased in the nonhormone groups (P < 0.001), whereas serum phosphate concentrations decreased in the hormone groups (P < 0.05 and 0.001). Our results confirm the positive effect of 1 year of HRT on serum calcitriol. Vitamin D₃ supplementation increased 25OHD concentrations, but did not affect calcitriol concentrations even though the initial levels were low. Interestingly, the combination of HRT and vitamin D₃ did not increase serum calcitriol concentrations as much as HRT alone. Received: 14 June 1996 / Accepted: 17 June 1997     

Treatment of Glucocorticoid-Induced Osteoporosis with Alfacalcidol/Calcium Versus Vitamin D/Calcium

Vitamin D/calcium substitution is generally regarded as an effective first step treatment for glucocorticoid-induced osteoporosis (GIOP). The aim of our study was to evaluate the efficacy of the active vitamin D metabolite alfacalcidol (1α) compared with the native vitamin D₃ in patients with established GIOP with or without vertebral fractures. Patients on long-term corticoid therapy were given either 1 μg alfacalcidol plus 500 mg calcium per day (group A, n = 43) or 1000 IU vitamin D₃ plus 500 mg calcium (group B, n = 42). The two groups were alike in age range, sex ratio, percentages of underlying diseases, average initial bone density values (lumbar spine: mean T-score −3.28 and −3.25, respectively), and rates of vertebral and nonvertebral fractures. During the 3-year study we found a small but significant increase of lumbar spine density in group 1α (+2.0%, P < 0.0001) and no significant changes at the femoral neck. In the D₃ group, there were no significant changes at both sites. At the end of the study, 12 new vertebral fractures had occurred in 10 patients of the group 1α and 21 in 17 patients of the D₃ group. In accordance with the observed fracture rates, the alfacalcidol group showed a significant decrease in back pain (P < 0.0001) whereas no change was seen in the vitamin D group. We conclude that with the doses used in this trial, alfacalcidol is superior to vitamin D in the treatment of established GIOP.     

Specific Effects of γ-Linolenic,Eicosapentaenoic, and Docosahexaenoic Ethyl Esters on Bone Post-ovariectomy in Rats

Long chain polyunsaturated fatty acids (LCPUFAs) are involved in the regulation of bone metabolism. Increased dietary consumption of n-3, and possibly some n-6, LCPUFAs may limit postmenopausal bone loss. The aim of this study was to determine the effects on bone of specific fatty acids within the n-3 and n-6 LCPUFA families in ovariectomized (OVX) rats. Rats were OVX or sham-operated and fed either a control diet (OVX and sham) or a diet supplemented with 0.5 g/kg body weight/day of γ-linolenic (GLA), eicosapentaenoic (EPA), docosahexaenoic (DHA) ethyl esters or a mixture of all three (MIX) for 16 weeks. Bone mineral content (BMC), area, and density and plasma concentrations of insulin-like growth factor-I, vitamin D, selected biochemical markers of bone metabolism, and parathyroid hormone (PTH) were determined. The OVX-induced decrease in lumbar spine BMC was significantly attenuated by DHA but not by EPA or GLA supplementation or supplementation with a mixture of all three LCPUFAs. Endosteal circumferences of tibiae were significantly greater in DHA and EPA compared to OVX. Plasma C-terminal telopeptide of type I collagen and osteocalcin concentrations were not significantly different in the DHA group compared to OVX. Femur BMC decreased by a significantly greater amount in GLA than OVX, and final plasma PTH concentrations were significantly higher in GLA compared to all other groups. In conclusion, DHA ameliorated OVX-induced bone mineral loss. GLA exacerbated post-OVX bone mineral loss, possibly as a result of PTH-induced bone catabolism.     

1α-Hydroxyvitamin D2 Partially Dissociates Between Preservation of Cancellous Bone Mass and Effects on Calcium Homeostasis in Ovariectomized Rats

Vitamin D metabolites can prevent estrogen depletion-induced bone loss in ovariectomized (OVX) rats. Our aim was to compare the bone-protective effects of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 1α,25-dihydroxyvitamin D₂ (1,25(OH)₂D₂), 1α-hydroxyvitamin D₃ (1α(OH)D₃), and 1α-hydroxyvitamin D₂ (1α(OH)D₂) in OVX rats. 1α(OH)D₃ and 1α(OH)D₂ are thought to be activated in the liver to form 1,25(OH)₂D₃ and 1,25(OH)₂D₂, respectively. Forty-four 12-week-old female Fischer-344 rats were either OVX or sham-operated (SHAM). Groups of OVX rats (n = 7 each) received vehicle alone, 1,25(OH)₂D₃, 1,25(OH)₂D₂, 1α(OH)D₃, or 1α(OH)D₂, starting 2 weeks after surgery. All vitamin D metabolites were administered orally at a dose of 15 ng/day/rat. Urine and blood samples were collected 6, 9, 12, and 16 weeks after surgery. Serum samples were analyzed for total calcium and phosphate. Calcium, phosphate, creatinine, and free collagen cross-links (ELISA) were determined in urine. After tetracycline double labeling, the rats were sacrificed 16 weeks postsurgery, and the proximal tibiae and the first lumbar vertebrae were processed undecalcified for static and dynamic bone histomorphometry. 1,25(OH)₂D₃ and, to a slightly lesser extent, 1,25(OH)₂D₂ elevated vertebral cancellous bone mass in OVX rats to a level beyond that observed in SHAM animals, and both compounds increased serum calcium and urinary calcium excretion to similar extents. 1α(OH)D₃ and 1α(OH)D₂ resulted in a 64% and 84%, respectively, inhibition of ovariectomy-induced vertebral cancellous bone loss. In the proximal tibial metaphysis, all vitamin D metabolites tested could only partially prevent post-OVX trabecular bone loss, with a tendency for 1α(OH)D₃ to be the least active compound. The effects of 1α(OH)D₃ and 1α(OH)D₂ on calcium homeostasis differed markedly, however. The mean increase in urinary calcium excretion over the whole experiment was fivefold for 1α(OH)D₃, whereas the corresponding increase for 1α(OH)D₂ was only twofold. We conclude that, compared with 1α(OH)D₃, 1α(OH)D₂ combined at least equal or higher bone-protective activity in OVX rats with distinctly less pronounced effects on calcium homeostasis. This effect was not due to a differential action of the corresponding main activation products, 1,25(OH)₂D₃ and 1,25(OH)₂D₂. Received: 2 May 1996 / Accepted: 18 October 1996     

Differences in Bone Turnover and Skeletal Response to Thyroid Hormone Treatment Between Estrogen-Depleted and Repleted Rats

This study was undertaken to compare the effect of supraphysiological doses of thyroxine (T4) on bone metabolism in SHAM and OVX young adult rats. Female Sprague Dawley rats (220 ± 2 g, approx. 5 months of age) were divided into four groups of eight animals each. The animals were intraperitoneally injected 6 days per week with vehicle (Vh): 0.001 N NaOH/0.9% NaCl (SHAM+Vh and OVX+Vh) or 250 μg of thyroxine/kg/day (SHAM+T4 and OVX+T4) during a 5-week period. Serum T4 and osteocalcin (BGP), urinary pyridinolines (Pyr), and creatinine (creat) were determined. At the beginning and at end of the experiment, skeletal bone mineral content (BMC), bone mineral density (BMD), and area (A) of the total skeleton, femur, spine, and whole tibia, as well as proximal, middle, and distal areas of the tibia were assessed by dual X-ray absorptiometry (DXA) in an ultra-high-resolution mode. T4 treatment of the SHAM rats did not induce significant changes in BGP level or Pyr/creat excretion compared with the SHAM+Vh control group. However, these two biochemical bone markers significantly increased due to T4 treatment in OVX rats compared with both OVX+Vh and SHAM+T4 groups (P < 0.05 and P < 0.001, respectively). The OVX+T4 group had a significantly lower ΔBMD than SHAM+T4 rats in all studied regions (P < 0.05) except for the middle tibia region. OVX+T4 groups presented a significantly lower ΔBMC and ΔA compared with SHAM+T4 animals (P < 0.001). OVX+T4 rats significantly impaired the ΔBMD in the femur (P < 0.01), spine (P < 0.05), whole (P < 0.05) and middle (P < 0.05) tibia whereas T4 treatment of SHAM rats only affected, significantly, the whole (P < 0.05) and the proximal tibia region (P < 0.01). T4 treatment affects bone growth in young adult rats. The effect is significantly greater in the estrogen-depleted than in the estrogen-repleted state. The bone site most adversely affected by T4 treatment depends on the estrogen status. The proximal tibia (principally trabecular bone) was the most affected area in estrogen-repleted rats. Conversely, in OVX rats, the middle tibia (principally cortical bone) presented the greatest decrease in bone density. Received: 20 May 1999 / Accepted: 4 February 2000     

Local bone turnover in the metaphysis of the proximal tibia and the lumbar vertebra during the early periods after ovariectomy in rats

To clarify the local changes in bone formation and resorption during the early period after ovariectomy (OVX), 200 SD rats, 4 months of age, underwent OVX or sham surgeries and seven to nine rats from each group were terminated at 1, 3, 7, 11, 15, 19, 23, 28, 35, 63, and 91 days postsurgery after tetracycline labeling. Serum intact osteocalcin levels were measured. Undecalcified sections of the 5th lumbar body (L5) and the right proximal tibia were measured for trabecular bone area, the labeled perimeters and the interlabeling distances after Villanueva's staining. On the 4th lumbar body (L4) and the left proximal tibia, undecalcified sections were measured for the trabecular osteoclast by tartrate-resistant acid phosphatase staining. The uterine horns were atrophied on the 3rd postovariectomy day (day 3). Serum osteocalcin levels increased on day 7 and reached the highest value on day 23. In either L5 or the metaphysis of the proximal tibia, trabecular bone volume (BV/TV) significantly decreased on day 15. The trabecular bone loss on day 28 was approximately 50% in the tibia and 15% in the L5. In either the lumbar or the tibia, osteoclast numbers significantly increased at day 3, and peaked between days 15 and 23. In the tibia, however, the bone formation rates (BFR/BS) were significantly reduced on the 3rd and 7th postsurgical days compared with the start value for both the OVX and sham groups. The BFR/BS values in L5 did not decrease during the first 7 days in either group. The BFR/BS values were then increased for both L5 and the tibia after day 7. These data clearly demonstrated that the local bone turnover 7 days post-OVX was identical in the proximal tibia and the lumbar vertebra. In the proximal tibia, however, it may be suggested that the increased bone resorption and reduced formation within 7 days after OVX due to the combined effects of both an estrogen deficiency and the surgical intervention would possibly play a critical role in the greater magnitude of the trabecular bone loss. Received: 29 April 1995 / Accepted: 18 August 1995     

Partial Prevention of Long-Term Femoral Bone Loss in Aged Ovariectomized Rats Supplemented with Choline-Stabilized Orthosilicic Acid

Silicon (Si) deficiency in animals results in bone defects. Choline-stabilized orthosilicic acid (ch-OSA) was found to have a high bioavailability compared to other Si supplements. The effect of ch-OSA supplementation was investigated on bone loss in aged ovariectomized (OVX) rats. Female Wistar rats (n = 58, age 9 months) were randomized in three groups. One group was sham-operated (sham, n = 21), and bilateral OVX was performed in the other two groups. OVX rats were supplemented orally with ch-OSA over 30 weeks (OVX1, n = 20; 1 mg Si/kg body weight daily) or used as controls (OVX0, n = 17). The serum Si concentration and the 24-hour urinary Si excretion of supplemented OVX rats was significantly higher compared to sham and OVX controls. Supplementation with ch-OSA significantly but partially reversed the decrease in Ca excretion, which was observed after OVX. The increase in bone turnover in OVX rats tended to be reduced by ch-OSA supplementation. ch-OSA supplementation increased significantly the femoral bone mineral content (BMC) in the distal region and total femoral BMC in OVX rats, whereas lumbar BMC was marginally increased. Femoral BMD was significantly increased at two sites in the distal region in OVX rats supplemented with ch-OSA compared to OVX controls. Total lumbar bone mineral density was marginally increased by ch-OSA supplementation. In conclusion, ch-OSA supplementation partially prevents femoral bone loss in the aged OVX rat model.     

Effect of Salmon Calcitonin on Femoral Bone Quality in Adult Ovariectomized Ewes

The aim of this study was to evaluate the effect of intermittent calcitonin on femoral bone quality in adult ewes from the time of ovariectomy. Six months after the start of the experiment, bone density measurements and mechanical testing (torsion and resonant frequency analysis of the diaphysis and compression of an excised trabecular bone cylinder from the femoral neck) were performed in sham-control and ovariectomized (OVX) ewes treated with placebo or salmon calcitonin (50 or 100 units, 3 times/week). Crystallinity of bone was evaluated by measuring X-ray diffraction line broadening. After OVX, a nonsignificant bone loss was found at all measured sites in the femur (−3 to −9%) together with a decreased biomechanical competence in the trabecular bone (compressive strain −28%, P < 0.05). Treatment with salmon calcitonin, 50 or 100 IU subcutaneously three times a week from the time of ovariectomy, resulted in a significant dose-dependent preservation of bone strength in the trabecular bone of the femoral neck compared with OVX. No adverse effects of calcitonin were observed on bone crystal composition as assessed by diffractiometry. We conclude that in adult ewes intermittent calcitonin treatment from the time of OVX was associated with a significant preservation of cancellous bone strength and strain in trabecular bone of the femoral neck, without affecting crystalline properties of bone. Received: 20 October 1995 / Accepted: 19 February 1996     

Effects of Risedronate Treatment on Bone Density and Vertebral Fracture in Patients on Corticosteroid Therapy

Men and women (n = 518) receiving moderate-to-high doses of corticosteroids were enrolled in two studies with similar protocols and randomly assigned to receive either placebo or risedronate (2.5 or 5 mg) for 1 year. All patients received daily calcium supplementation (500–1000 mg), and most also received supplemental vitamin D (400 IU). The primary endpoint was the difference between the placebo and active groups in lumbar spine bone mineral density (BMD) at 1 year; changes in BMD at other sites, biochemical markers of bone turnover, and the incidence of vertebral fractures were also assessed. In the overall population, the mean (SE) lumbar spine BMD increased 1.9 ± 0.38% from baseline in the risedronate 5 mg group (P < 0.001) and decreased 1.0 ± 0.4% in the placebo group (P= 0.005). BMD at the femoral neck, trochanter, and distal radius increased or was maintained with risedronate 5 mg treatment, but decreased in the placebo group. Midshaft radius BMD did not change significantly in either treatment group. The difference in BMD between the risedronate 5 mg and placebo groups was significant at all skeletal sites (P < 0.05) except the midshaft radius at 1 year. The 2.5 mg dose also had a positive effect on BMD, although of a lesser magnitude than that seen with risedronate 5 mg. A significant reduction of 70% in vertebral fracture risk was observed in the risedronate 5 mg group compared with the placebo group (P= 0.01). Risedronate was efficacious in both men and women, irrespective of underlying disease and duration of corticosteroid therapy, and had a favorable safety profile, with a similar incidence of upper gastrointestinal adverse events in the placebo and active treatment groups. Daily treatment with risedronate 5 mg significantly increases BMD and decreases vertebral fracture risk in patients receiving moderate-to-high doses of corticosteroid therapy. Received: 11 October 1999 / Accepted: 1 May 2000 / Online publication: 27 July 2000     

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     

Clodronate Prevents Bone Loss in Aged Ovariectomized Rats

The purpose of this study was to investigate the ability of clodronate to prevent ovariectomy (OVX)-induced osteopenia in aged rats. Fourteen-month-old female Sprague-Dawley rats (n = 166) were randomized into six groups. One group was sacrificed at the start of the study, four groups were ovariectomized, and one group was sham-operated (Sham). The OVX rats were given subcutaneously either vehicle (veh) or clodronate at doses of 3, 7, or 25 mg/kg once a week for 3 months, and the Sham rats were given the vehicle. At all dose levels clodronate inhibited trabecular bone loss in the distal femur and in the fourth lumbar vertebral body (L4), and decreased bone resorption as evidenced by urinary deoxypyridinoline excretion. The lowest dose of clodronate preserved serum osteocalcin and endosteal bone formation of secondary spongiosa in L4 at the level of the Sham/veh group. The OVX-induced increase in periosteal bone formation of femoral diaphysis was unaffected by two smaller doses of clodronate, but was decreased to the level of Sham rats after the highest dose. After 3 mg/kg clodronate, the percentage of femoral cortical bone area and the mean relative cortical thickness were higher compared with the OVX/veh group. There was a good positive correlation between the maximum load in three-point bending of the tibia and tibial ash weight. Normal lamellar pattern of newly formed cancellous and cortical bone was found after clodronate treatment. No signs of adverse accumulation of osteoid or any deleterious effect on mechanical strength of long bones and lumbar vertebrae were found. Received: 28 August 1996 / Accepted: 5 March 1997     

Short-Term Effect of Vitamin K Administration on Prednisolone-Induced Loss of Bone Mineral Density in Patients with Chronic Glomerulonephritis

Glucocorticoid-induced osteoporosis has been reported to be caused by enhanced bone resorption and suppressed bone formation. To clarify whether administration of vitamin K, which enhances bone formation, prevents prednisolone-induced loss of bone mineral density (BMD), a randomized, prospective, controlled study was conducted on 20 patients with chronic glomerulonephritis scheduled for treatment with prednisolone. All patients were initially treated with 0.8 mg/kg body weight/day of prednisolone (maximum of 40 mg) for 4 weeks, tapering to 20 mg/day over approximately 6 weeks. Ten patients received prednisolone alone (Group 1), and the other 10 patients received prednisolone plus 15 mg of menatetrenone, vitamin K, three times per day (Group 2). BMD of the lumbar spine measured by dual-energy X-ray absorptiometry (DXA) and biochemical markers of bone metabolism in blood and urine were evaluated before and 10 weeks after administration of prednisolone alone or with menatetrenone. In Group 1, treatment with prednisolone significantly reduced BMD of the lumbar spine from 1.14 ± 0.12 to 1.10 ± 0.11 g/cm² (P= 0.0029). Serum intact osteocalcin and procollagen type I C-peptide (PICP) concentrations, biochemical markers of bone formation, were markedly reduced. A biochemical marker of bone resorption, urinary excretion of deoxypyridinoline, was significantly reduced. In Group 2, prednisolone-induced reduction of BMD was prevented by menatetrenone administration (1.09 ± 0.09 to 1.07 ± 0.07 g/cm², P= 0.153). Menatetrenone prevented reduction of PICP concentration by prednisolone but not in serum intact osteocalcin concentration and urinary excretion of deoxypyridinoline. Thus, treatment with prednisolone resulted in loss of BMD of the lumbar spine associated with suppression of both bone formation and bone resorption. Menatetrenone is a useful agent in preventing prednisolone-induced loss of BMD. Received: 7 July 1998 / Accepted: 13 August 1999