Dietary Xylitol Retards the Ovariectomy-Induced Increase of Bone Turnover in Rats

The effects of 10% dietary xylitol supplementation in ovariectomized rats were studied on the degradation of bone organic and inorganic structures. The osseal concentrations of hydroxyproline, pyridinoline, and deoxypyridinoline were analyzed by high-performance liquid chromatography. Bone resorption was measured in [³H]tetracycline-prelabeled rats by urinary excretion of ³H, and by the amount of ³H preserved in bone. Bone trabeculation was measured by a computer image analyzer from sections stained by the method of von Kossa. The amount of collagen in bone organic fraction was lower in ovariectomized rats as compared with the sham-operated controls. This most likely is partly a consequence of an increased resorption, and partly a consequence of a higher proportion of immature periosteal bone in the ovariectomized animals, leading to a higher ratio of noncollagenous protein to collagen. The number of pyridinium crosslinks was lower in proportion, indicating no selective changes in the structure of collagen. Dietary xylitol significantly retarded the ovariectomy-associated decrease in the relative amount of collagen and the number of its mature crosslinks. Ovariectomy doubled the excretion of ³H and caused a significant decrease in the amount of ³H preserved in bone; both these changes were significantly retarded by the 10% dietary xylitol supplementation. Ovariectomy significantly decreased the volume of bone trabeculae, but this effect was also significantly inhibited by the xylitol supplementation in the diet. In conclusion, these findings suggest a dietary xylitol-induced normalizing effect on the rate of bone turnover in ovariectomized rats. Received: 12 February 1996 / Accepted: 20 August 1996     

Biochemical Markers of Bone Turnover and Bone Loss at the Lumbar Spine and Femoral Neck: The Taiji Study

The purpose of this study was to ascertain whether biochemical markers of bone turnover predict bone loss. The survey was carried out in Taiji, Wakayama Prefecture, Japan. From a list of inhabitants aged 40–79 years, 400 participants (50 men and 50 women in each of four age groups) were selected randomly. Bone mineral density (BMD) was measured, and blood and urine samples of all participants were examined to obtain values for eight biochemical markers: alkaline phosphatase (ALP), bone Gla protein (BGP), type I procollagen (carboxyterminal peptide of type I procollagen; PICP), cross-linked carboxyterminal telopeptide region of type I collagen (ICTP), and urinary excretion of calcium (Ca), phosphate (P), pyridinoline (Pyr), and deoxypyridinoline (D-Pyr). Each marker was evaluated as a predictor of the rate of bone change in lumbar spine and femoral neck BMD over a 3-year period. The value of Pyr was significantly related to the change of lumbar spine BMD in men (P= 0.009), and that of BGP was found to be significant in women (P= 0.045). By contrast, none of the bone markers significantly correlated with bone loss at the femoral neck. The coefficient of determination at the lumbar spine was 5% and 7% at the femoral neck only. We conclude that biochemical markers of bone turnover cannot predict bone loss rates in middle-aged or elderly Japanese men and women over a 3-year period with sufficient accuracy for use in clinical decision making. Received: 26 January 1998 / Accepted: 9 July 1998     

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     

Effect of Estrogen Deficiency on Cancellous and Cortical Bone Structure and Strength of the Femoral Neck in Rats

Eighty mature Sprague-Dawley rats were weight matched before ovariectomy (Ovx) or Sham surgery (Sham). Sham rats had free access to food and water throughout the experiment, whereas Ovx rats were kept on the pair-fed diet. Rats were euthanized at 4, 8, and 12 weeks after surgery, and had received fluorochrome bone markers at 9 and 2 days prior to euthanasia. In addition 10 rats were euthanized at the time of surgery serving as baseline controls. All rats were also scanned for body composition and bone mineral parameters by DEXA before surgery and euthanasia. Left proximal femurs (femoral necks) were used for bone histomorphometry, whereas right femurs were used for in vitro DEXA measurements and mechanical testing. Despite pair-feeding, ovariectomized rats had increased body weights and fat body mass, whereas the percent lean body mass steadily declined throughout the experiment. Mineral density of the whole femur and femoral neck was significantly higher in the Sham rats relative to Ovx animals. Ovariectomy reduced trabecular number and thickness, and increased trabecular separation and bone marrow space at the femoral midneck location. The structure of the remaining trabeculae was dramatically changed toward simpler struts as revealed by nodal analyses. Cortical thickness in Ovx rats was reduced because of the high endocortical resorption, which, in addition to cancellous bone resorption, resulted in fewer endocortico-trabecular connections. Femoral necks obtained from ovariectomized rats had reduced strength and were less stiff relative to controls. Because of the enormous clinical significance of the proximal femur for osteoporosis in humans, and the opportunity for studying bone BMD, mass, structure, and strength at the same skeletal location, the femoral neck appears superior to other skeletal sites routinely used for bone histomorphometry or mechanical testing in the Ovx rat model. Received: 25 September 1996 / Accepted: 24 March 1997     

Bone Density, Vitamin D Status, and Disordered Bone Remodeling in End-Stage Chronic Liver Disease

Hepatic osteodystrophy occurs in up to 50% of patients with chronic liver disease (CLD). The aim of this study was to determine the relative contribution of increased resorption and decreased formation to hepatic osteodystrophy by measuring biochemical markers. Twenty-seven patients with advanced CLD (14 female, 13 male) were enrolled. Bone mineral density (BMD), measured at the lumbar spine, and femoral neck, were measured by dual energy X-ray absorptiometry (DXA); bone turnover was assessed using biochemical markers of bone formation and resorption. Based on WHO criteria, osteoporosis and osteopenia were present in 41% and 18% of patients, respectively. All three markers of bone resorption (free deoxypyridinoline, pyridinoline, and hydroxyproline) were increased significantly in patients with CLD. There was a less marked change in the markers of bone formation (osteocalcin, procollagen type 1 peptide, and bone alkaline phosphatase), resulting in a negative uncoupling index in 23/27 (85%) of the patients. Only two (7%) patients had biochemical changes consistent with osteomalacia. The results suggest that increased bone resorption is the predominant cause of hepatic osteodystrophy and therapeutic strategies should be designed to suppress bone resorption, especially in preparation for liver transplantation. Bone biomarkers may be useful alternatives to bone biopsy in evaluating hepatic osteodystrophy. Received: 11 September 1997 / Accepted: 22 September 1998     

Age-Associated Decline in Human Femoral Neck Cortical and Trabecular Content of Insulin-Like Growth Factor I: Potential Implications for Age-Related (Type II) Osteoporotic Fracture Occurrence

Recent evidence suggests that regulatory peptides such as insulin-like growth factor-I (IGF-I) are released locally from bone during resorption, and may then act in a sequential manner to regulate the cellular events required for the coupling of bone formation to resorption. Among other factors, a decrease in bone-associated IGF-I levels could therefore result in remodeling imbalance and contribute to the gradual loss of bone that occurs with age. As the femoral neck region is of primary concern for the clinical manifestations of osteoporosis, the current study was intended to assess the IGF-I contents in femoral neck cortical and trabecular bone from aging individuals. Bone samples from the neck region were obtained at postmortem from 39 females and 35 males, aged 23–92 years. Concentrations of IGF-I and osteocalcin were measured by radioimmunoassay in the supernatants obtained after EDTA and guanidine hydrochloride extraction. The total amount of protein present in the extracts was determined by spectrophotometry. IGF-I levels were significantly lower in trabecular compared with cortical bone. Though femoral neck total protein did not vary with donor age, both IGF-I and osteocalcin were found to decline markedly. Between the ages of 23 and 92 years, average yearly rates of loss of 0.30 and 0.21 ng IGF-I/mg protein were observed in cortical and trabecular bone, respectively, corresponding with net losses of nearly 35% of the cortical skeletal content of IGF-I and 41% of the trabecular skeletal content of IGF-I. These changes in bone-associated IGF-I paralleled those of osteocalcin, consistent with an overall decrease in osteoblast function with aging. In women, the rate of decline was significantly faster for trabecular than for cortical IGF-I, however in men, age-dependent changes in cortical and trabecular IGF-I were similar. These findings support the hypothesis that changes in the local IGF regulatory system over time could be a pathophysiologic component of the age-related (type II) femoral neck osteoporotic syndrome. Received: 12 December 1996 / Accepted: 23 April 1997     

Alteration in the Extent of Collagen I Hydroxylation,Isolated from Femoral Heads of Women with a Femoral Neck Fracture Caused by Osteoporosis

The aim of this study was to investigate the extent of lysyl and prolyl hydroxylation of collagen I in osteoporosis and compare it with collagen I from ``bone healthy' individuals. Collagen I was isolated from femoral heads of osteoporotic women, from women suffering from osteoarthrosis of the hip, and from healthy women 60–85 years of age. The femoral heads were dissected into compact and trabecular bone of the neck region and from trabecular bone of the head region, and collagen I was extracted by limited pepsin digestion. The amino acid analysis of individual α-chains showed a remarkably higher degree of hydroxylation of lysine residues both in the α1(I)- and in the α2(I)-chains in osteoporotic bone compared with osteoarthrotic and ``normal' bone, whereas the prolyl hydroxylation was nearly unchanged. The lysyl overhydroxylation was observed in the compact as well as in the trabecular bone of osteoporotic femoral heads. These biochemical alterations may play a crucial role in the pathogenesis of osteoporosis. Received: 2 May 1996 / Accepted: 31 December 1996     

Peripheral Quantitative Computed Tomography of the Femoral Neck in 60 Japanese Women

Peripheral quantitative computed tomography (pQCT) is able to evaluate trabecular and cortical bone separately, and to determine geometric properties from cross-sectional images for noninvasive assessments of mechanical strength. In order to assess the diagnostic value of pQCT of the femoral neck, 60 healthy women were examined with a new pQCT machine, XCT-3000 (Norland-Stratec, Germany), which is suitable for direct measurement of the hip. The region of interest chosen was the center of the femoral neck. pQCT of the distal radius and dual energy X-ray absorptiometry (DXA) of the lumbar spine and femoral neck were also performed. The study demonstrated that total bone mineral density (BMD) (femoral MD) and trabecular BMD (femoral-TBD) decreased with advancing age. Percent cortical area showed a small but significant decrease with advancing age and % trabecular area increased slightly. Both the endosteal perimeter and the periosteal perimeter were relatively constant with aging. Bone strength index (BSI) and stress-strain index (SSI), which reflect the mechanical strength of bone, declined with advancing age, especially after menopause. Femoral TBD correlated strongly with femoral neck BMD by DXA and L2-L4 BMD by DXA but femoral-CBD did not correlate with femoral neck BMD by DXA. Volumetric BMD of the femoral neck and distal radius were closely correlated. It is concluded that (1) cortical thinning occurs with aging by endocortical resorption and loss of femoral-TBD; (2) loss of femoral-CBD occurred at a slower rate than radial CBD, perhaps due to the weight-bearing effect; (3) biomechanical parameters such as the BSI and SSI may reflect increasing fragility of the femoral neck in pre- and postmenopausal women; (4) pQCT of the femoral neck had diagnostic value at least equivalent to that of DXA or pQCT of the distal radius. Received: 23 June 1998 / Accepted: 1 July 1999     

Bone Biomechanical Property Deterioration Due to Tobacco Smoke Exposure

Tobacco smoking has been implicated in the development of osteoporosis and early onset of menopause in women smokers. We measured various biomechanical properties of femurs and tibiae obtained from smoke-exposed and control mice to determine cigarette smoke influences on bone mass, structure, and strength. Growing female C57BL mice were exposed to sidestream cigarette smoke in a whole-body exposure chamber, set at 30 ± 2 mg smoke particulates/m³ for 4 hours/day and 5 days/week for 12 consecutive weeks. Elevated levels of urinary cotinine and pulmonary ethoxyresorufin deethylase activity in smoke-exposed mice confirmed their effective exposure to cigarette smoke. There were no differences in body weight and physical size (length, medial-lateral and anterior-posterior widths, midshaft cortical area and thickness) of femurs and tibiae between smoke-exposed and control mice. The femoral mid-shaft yield load, stiffness, yield stress, and modulus were, respectively 8%, 13%, 10%, and 14% lower (P < 0.05) in smoke-exposed compared to control mice. The ultimate load and stress in mid-shaft femurs showed decreasing trends (P < 0.1) in smoke-exposed mice. In the femoral neck, the ultimate load and stiffness were 9% and 12% lower (P < 0.05) in smoke-exposed mice, respectively. Further, the ash-to-dry bone weight ratio was smaller (∼6%, P < 0.05), and micro-computed tomographic scanning of distal femoral bone volume/total volume (%) and trabecular thickness showed decreasing trends in smoke-exposed mice compared to the control group. We conclude that exposure to tobacco smoke deteriorates some of the biomechanical properties of bone in growing female mice.     

Bone Mass and Markers of Bone and Calcium Metabolism in Postmenopausal Women Treated with 1,25-Dihydroxyvitamin D (Calcitriol) for Four Years

To evaluate the long-term effect of calcitriol treatment on bone mineral density (BMD) of the femoral neck and lumbar spine and the parameters of calcium and bone metabolism in elderly women, 55 healthy, postmenopausal women, all aged 66 years, were enrolled in the study. Eighteen started a 4-year supplementation with 0.5 μg of calcitriol daily and 37 served as controls. Calcium intake of all the subjects was adjusted to 800 mg daily. In 4 years femoral neck BMD increased by 3.0% in the calcitriol group, but decreased by 1.6% in the control group (P= 0.009). The respective changes in lumbar spine BMD were +2.3% and +0.9% (P= 0.067). Two years' treatment with calcitriol increased the intestinal absorption of strontium by 57% (P < 0.001), doubled the urinary excretion of calcium (P < 0.001), and decreased the mean parathyroid hormone (PTH) level by 32% (P < 0.01). In the calcitriol group the marker of bone formation, serum osteocalcin, decreased by 27% (P < 0.01), and the marker of bone resorption, serum C-telopeptide of type I collagen (CTx), by 33% (P= 0.05) after 2 years. In two subjects the calcitriol dose had to be reduced because of hypercalciuria. We conclude that calcitriol treatment increases bone mass at the femoral neck and lumbar spine, the increases being maintained for up to 4 years. The gain in bone mass results from reduced bone turnover which is partly a consequence of the enhanced intestinal absorption of calcium and suppressed serum PTH levels. Received: 8 January 1999 / Accepted: 29 February 2000     

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     

Excretion of pyridinium cross-links of collagen in ovariectomized rats as urinary markers for increased bone resorption

Summary Groups of 19-day-old rats were ovariectomized or were given sham operations. Measurements in urine of the pyridinium cross-links of collagen, pyridinoline and deoxypyridinoline, 7 weeks after surgery showed significantly higher amounts of cross-links relative to creatinine in the ovariectomized groups compared with the controls. Analyses before and after acid hydrolysis of the urine revealed that the increased excretion was only as free cross-link with no change in the concentrations of the bound forms. The loss of trabecular bone in the ovariectomized group was confirmed by immunocytochemical staining with antibodies to type I collagen. There were no differences between the ovariectomized and control groups in the concentrations of cross-links in the tibial bone or the articular cartilage. Measurements of free pyridinoline and deoxypyridinoline in urine therefore appear to provide a good index of the increased bone resorption induced by estrogen deficiency.     

Biochemical Markers of Bone Turnover Reflect Femoral Bone Loss in Elderly Women

Although over 90% of hip fractures occur in patients over age 70, few data are available on femoral bone loss in this age group. To examine the relationship between biochemical markers of bone turnover and femoral bone loss in the elderly, 36 female and 17 male, healthy, community-dwelling elderly over age 65 (mean ± SD age: women 71 ± 4 years, men 75 ± 5 years) were followed for 3 years. Annual bone mineral density measurements of the hip and lumbar spine by dual-energy x-ray absorptiometry (DXA) were obtained and biochemical markers of bone resorption (urinary N-telopeptide crosslinks, free pyridinoline, total pyridinoline, total deoxypyridinoline, and hydroxyproline) and bone formation (serum osteocalcin, bone-specific alkaline phosphatase) were obtained at the end of year 3. In elderly women, longitudinal bone loss at the total hip was negatively correlated with markers of bone resorption (r =−0.39 to −0.52, P < 0.05), bone formation (r =−0.38, P < 0.05), and age (r =−0.39, P < 0.05). Markers of bone resorption were correlated with markers of bone formation (r = 0.63 to 0.74, P < 0.01). In multiple regression analysis, urinary N-telopeptide crosslinks (marker of resorption), serum osteocalcin (marker of formation), and serum parathyroid hormone explained 43% of the variability of bone loss at the total hip in women. These parameters were not related to bone loss in men. We conclude that femoral bone loss increases with age in women over 65. Measurements of specific biochemical markers of bone turnover are correlated with longitudinal bone loss in elderly women. These markers may help identify women at greatest risk for bone loss who would benefit most from therapeutic interventions. Received: 28 January 1996 / Accepted: 3 May 1996     

Age-Related Changes in the Biochemical Properties of Human Cancellous Bone Collagen: Relationship to Bone Strength

The metabolism of bone collagen has received little attention in relation to age-related loss of bone mass and strength. The aim of the present study was to analyze bone collagen content and metabolism in human bone with respect to age. The material consisted of iliac crest bone biopsies from 94 individuals: 46 women (ages 18–96, mean age 60.8 years) and 48 men (ages 23–92, mean age 59.5 years). Excluded from the study were all individuals with known osteoporotic lumbar vertebral fractures and renal, hepatic, or malignant diseases. Prior to collagen analysis the biopsies were scanned in a pQCT scanner for density assessment and then tested biomechanically. The results showed a decline in apparent bone density with age (P < 0.0001), a decline in maximum stress, Young's modulus, and energy absorption with age (P < 0.001). Concomittantly, there was an age-related decline in the intrinsic collagen content with age (P < 0.001). However, there were no biochemical modifications of the bone collagen during aging. There were no significant differences between women and men in the slopes of the regressions-curves. When multiple regression analyses were performed, only apparent bone density came out as a significant contributor in the correlation to biomechanical properties. Nevertheless, the decrease in bone collagen content with age might indicate an increase in the mineralization degree (probably due to decreased bone turnover) and thereby a change in material properties of bone. In conclusion, the present study has shown that loss of bone mass plays the major role in loss of bone strength. However, there is also a change in bone composition during normal aging, leading to a decrease in collagen content and an increase in the degree of mineralization. At this skeletal site, in a normal population there was no change in the biochemical properties of bone collagen. Received: 3 November 1998 / Accepted: 12 March 1999     

Cyclical Etidronate Therapy for Prevention of Postmenopausal Bone Loss: A 1-Year Open-Label Follow-Up Study

The objective of this study was to evaluate whether the pharmacological activity of cyclical etidronate therapy is sustained beyond the dosing period. A group of 121 postmenopausal women who had completed a 2-year, double-blind, placebo-controlled parallel study with etidronate or placebo (400 mg/day for 14 days every 3 months) and calcium agreed to participate in a 1-year open-label follow-up study to evaluate the effect of discontinuing etidronate treatment. Fifty-nine subjects in the former etidronate group and 62 in the placebo group received 500 mg/day of elemental calcium; 54/59 and 58/62 subjects, respectively, completed the study. Outcomes of the study were bone mineral density (BMD), measured by dual energy X-ray absorptiometry (DXA), and biochemical markers of bone turnover (urinary deoxypyridinoline/creatinine and serum osteocalcin). To determine whether there was a residual effect of previous therapy we compared mean percentage changes from baseline (year 0) to year 3 for both spinal and femoral neck BMD and markers of bone turnover in the former cyclical etidronate and placebo groups. To evaluate the carryover effect of treatment we compared the percent change from year 2 to year 3 for the same variables. Mean percentage change (SEM) from year 2 to year 3 for spinal BMD in the former cyclical etidronate group was −2.87% (0.48%) versus −0.99% (0.36%) in the placebo group (P= 0.0022). In the femoral neck, the BMD changes were −0.86% (0.42%) versus −1.01% (0.41%) (NS). Biochemical markers increased within 6 months toward baseline levels. Mean percentage changes from baseline (year 0) in both spinal and femoral neck BMD were significantly different between groups 1 year after treatment discontinuation. No differences between groups were maintained in deoxypyridinoline and osteocalcin. It is concluded that following withdrawal of cyclical etidronate therapy bone loss resumes at a normal and moderately accelerated rate in the proximal femur and lumbar spine, respectively. A positive effect on BMD at both cortical and trabecular sites is maintained for 1 year after treatment withdrawal. Received: 8 May 1999 / Accepted: 10 December 1999     

Ultrasound Characterization of Bone Demineralization

Quantitative ultrasound (QUS) assessment of bone may permit an assessment of bone properties currently not available by bone densitometry techniques. To explore the effects of the quantity of bone mineral on acoustic parameters, we carried out an in vitro study of the impact of demineralization on attenuation of ultrasound in trabecular bone. Ten fresh cubes of trabecular bone obtained from bovine distal femurs were progressively demineralized using formic acid solution. The progression of demineralization was controlled by monitoring the specimen bone mineral density (BMD) using dual x-ray absorptiometry (DXA). At five stages of demineralization—0% (baseline), 25%, 50%, 75%, and 100% (all mineral removed)—the US properties of the specimens were assessed (Walker Sonix UBA 575+). The US parameters investigated were broadband ultrasound attenuation (BUA) and ultrasound attenuation in bone (UAB). Both DXA and QUS measurements were made along the three orthogonal axes of each cube. Our results demonstrated significant variability in both BUA and UAB along the three principle axes of the cubes whereas BMD did not differ in the different directions. A strong but nonlinear correlation was found between BMD and US attenuation. A reduction in BMD to 50% of the baseline values resulted in BUA (UAB) reduction to 25% and 19%, respectively. A random effect model analysis supported a multiplicative relationship between BMD and the US parameters. US attenuation is a sensitive indicator of bone mineral changes with nonlinear dependence on bone mineral loss. Bone collagen structure reinforced by hydroxyapatite crystal accounts for fundamental US characteristics. Ultrasound attenuation associated with trabecular orientation is basically dominated by the mineral spread in a collagen framework. Received: 3 February 1997 / Accepted: 2 July 1997     

Does Leptin Have an Effect on Bone in Adult Women?

Recent studies have implicated leptin in the modulation of bone mass during skeletal development. Whether leptin also exerts an influence on bone after growth has stopped is unknown at present. In this cross-sectional study on 94 women (60 premenopausal, 34 postmenopausal) aged 40–60 years, we analyzed the relationship between serum leptin and bone density and bone cortex geometry and bone metabolism. Total and trabecular bone density as well as total and cortical bone area were determined by quantitative computed tomography (QCT) at the distal radius. Bone metabolism was assessed by measuring bone-specific alkaline phosphatase, osteocalcin, procollagen type I C-terminal propeptide (PICP) and collagen type I C-terminal telopeptide in serum, and deoxypyridinoline in urine samples. None of the indices of bone density or geometry was significantly related to leptin serum concentrations (P > 0.05) before or after adjustment for body mass index (BMI). PICP was associated with serum leptin in the postmenopausal group only (r =−0.40 after adjustment for BMI; P= 0.009). Yet, as none of the other markers of bone metabolism exhibited a significant correlation with serum leptin in any of the menopausal groups, this association is likely to be due to the influence of extraskeletal factors on PICP serum levels. Thus, it appears that leptin has less influence on the mature than on the growing skeleton. Received: 5 January 1998 / Accepted: 12 May 1998     

Influence of Electromagnetic Fields on Bone Mass and Growth in Developing Rats: A Morphometric,Densitometric, and Histomorphometric Study

The effect of electromagnetic fields on bone is debated. In an experimental study of this effect, we compared two lots of growing female rates (both lots n = 15, age 3 weeks, average weight 23.2 ± 3.3 g), one of which was exposed to a 3-mT, 100-Hz, Helmholtz-type electromagnetic field for 24 hours a day for 30 days, and the other of which served as the control. Bone development and bone mass were evaluated by morphometry, densitometry, and histomorphometry. The rats were killed at 30 days and weighed. The right femurs were dissected, measured, and weighed; bone densitometry was used to determine femoral bone mineral content (BMC) and density (BMD), and histomorphometry of the nondecalcified bone was used to determine trabecular bone volume (Cn-BV-TV%), number (Tb-N mm) and thickness (Tb-Th μm), intertrabecular space (Tb-Sp μm) and growth cartilage thickness (Gc-Th μm). In the rats exposed to the electromagnetic field, BMC and BMD (P= 0.019 and P= 0.002, respectively) and Cn-BV-TV, Tb-N, Tb-Th (P= 0.005, P= 0.036, and P= 0.027, respectively) all were decreased, whereas Tb-Sp was increased (P= 0.002). There were no significant differences in initial and final body weight, or in final femur weight, femur length, and GC-Th. These findings indicate that electromagnetic fields of the type used here reduced bone formation and increased bone resorption without affecting bone development in rats. Received: 12 February 1996 / Accepted: 2 December 1996     

Effects of Clodronate on Cortical and Trabecular Bone in Ovariectomized Rats on a Low Calcium Diet

The aim of this study was to evaluate the contribution of a low calcium diet to the cortical and trabecular osteoporosis seen in ovariectomized rats after 7 weeks on a low calcium diet and to investigate the effects of the bisphosphonate clodronate on this development of osteoporosis. Thirty-six mature, female Wistar rats were randomized into four groups: Ovx−B (bisphosphonate) and Ovx−C (control) were ovariectomized, and Sham−Ca (low calcium) and Sham+Ca (normal calcium) were sham operated. The first three groups were fed a low calcium diet (0.01%) and Sham+Ca normal rat chow (Ca 1.1%). The Ovx−B received 10 mg/kg s.c. clodronate daily for nine weeks, and Ovx−C, Sham−Ca, and Sham+Ca received the same volumes of saline. Bone mineral turnover measured as ⁸⁵Sr-uptake was increased in all low calcium groups compared to Sham+Ca. The Sham+Ca femora had higher dry weight and ash weight than the other groups, and Ovx−C had higher dry weight compared with Ovx−B and Sham−Ca. Calcium content was lower in both Ovx groups compared to both Sham groups. Magnesium was lower in all groups compared to Sham+Ca and higher in Ovx−B compared with Ovx−C. In the femoral shaft, Sham+Ca had significantly higher ultimate bending moment, energy absorption, and deflection compared to the other three groups. Ultimate bending moment was higher in Sham−Ca than in Ovx−C. Stiffness was increased in both Sham+Ca and Ovx−B compared to Ovx−C. The maximum stress in the femoral midshaft was higher in Sham+Ca than in the other groups, and higher in Ovx−B than in Ovx−C. Histomorphometry showed increased medullary area in all low calcium groups compared to Sham+Ca and larger cortical area in Sham+Ca and Ovx−B compared to Ovx−C. Compared to Sham+Ca the trabecular bone volume was decreased to 30% in Sham−Ca and to 9% in Ovx−C, but was unchanged in Ovx−B. The low calcium diet generally increased bone mineral turnover and reduced the tibial bone volume. Femoral changes led to a reduction of cortical fracture strength and maximal stress. Ovariectomy in addition to a low calcium diet reduced femoral strength even more. Daily injections of clodronate to ovariectomized rats on a low calcium diet increased femoral shaft stiffness and maximum stress, and clodronate preserved both trabecular and cortical tibial bone volume completely. Received: 11 June 1996 / Accepted: 5 March 1997     

The Effect of Modifying Dietary Calcium Intake Pattern on the Circadian Rhythm of Bone Resorption

In order to assess day-to-day variations of the circadian rhythm of biochemical bone resorption markers, urinary morning (6–8 a.m.) and evening (7–10 p.m.) samples from 35 individuals were monitored during 3 subsequent days. The bone-specific deoxypyridinoline (DPD) crosslinks of type I collagen followed a circadian rhythm in all individuals. In contrast, no such pattern was observed in the urinary hydroxyproline/creatinine and calcium/creatinine measurements. The DPD crosslink measurements showed a much larger difference between the morning and evening samples collected within 1 day compared with the variation between the samples collected in the morning or evening on subsequent days, indicating the importance of adequate timing of urine sampling for clinical trials aiming to monitor effects on bone resorption. The analysis of DPD crosslinks was then used to evaluate the effects of different patterns of dietary calcium intake on the circadian rhythm of bone resorption in osteoporotic patients. No significant effect on the circadian rhythm of the DPD crosslinks was found after concentrating the normal daily calcium intake to the evening (6–10 p.m.) during 8 days (n = 7). Ingestion of a dietary calcium supplement (600 mg) at 10 p.m. during 8 days (n = 7) resulted in an increased urinary calcium excretion in the morning, and a flattening of the circadian peak and nadir concentrations of urinary DPD/creatinine. The absolute levels of DPD/creatinine in the morning and evening urine samples, respectively, were not significantly altered compared with the control day. We conclude that dietary calcium supplementation in the evening only marginally affects the circadian rhythm of urinary DPD crosslinks in established osteoporosis patients. Received: 23 December 1996 / Accepted: 1 November 1998