James K. Yeh

Calcium Supplementation with and without Hormone Replacement Therapy To Prevent Postmenopausal Bone Loss

Postmenopausal bone loss is a major factor in the increasing prevalence of osteoporotic fractures. Evidence is abundant that hormonal replacement therapy prevents the bone loss that follows natural or surgical menopause and reduces the prevalence of osteoporotic fractures in later life [1-4]. However, only about 10% of American women elect to receive replacement therapy because of attitudes of physicians and patients, the undesirability of menstrual bleeding, and unresolved questions about the relation of the use of estrogen to breast cancer [5]. Moreover, the duration of hormonal therapy may need to be prolonged because bone loss recurs when therapy is discontinued, yet the incidence of some adverse effects increases with the duration of estrogen use. Safer alternatives to estrogen use have been sought. Epidemiologic and cross-sectional studies have suggested that increasing calcium intake might prevent postmenopausal bone loss, and prospective studies have yielded conflicting results [6-17]. Moreover, some investigators have suggested that effects differ on the various skeletal sites used to determine the rate of bone loss [18]. We compared the efficacy of calcium augmentation in early postmenopause with calcium augmentation plus hormonal replacement therapy and with placebo. The study had a three-arm, randomized, parallel design. The patients receiving hormonal replacement therapy were obviously not blinded nor were their physicians, whereas the placebo and calcium groups were double blinded. Methods Healthy, white women between 6 months and 6 years after a natural menopause were recruited to participate in the study. The protocol was approved by the Human Investigation Review Committees of Winthrop-University Hospital and Brookhaven National Laboratory; written informed consent was obtained from each participant. Participants were recruited by announcements in the local press and in hospital and university publications and through a direct mail campaign. All participants had a history and physical examination. Exclusion characteristics included any disorder known to affect bone metabolism such as glucocorticoid use, gastrointestinal disease, or any chronic illness. Previous or current malignancy was an exclusion characteristic as were absolute contraindications to estrogen replacement or calcium supplements. Absolute contraindications to estrogen replacement therapy included estrogen-dependent neoplasm (breast or uterus), undiagnosed vaginal bleeding, thrombophlebitis or thromboembolism, and acute liver disease. Women with the following problems considered by some investigators to be relative contraindications to estrogen therapy were also excluded: gallbladder disease, history of liver disease, first-degree relatives with breast cancer, and hypertension. Calcium urolithiasis was also an exclusion factor. Women with known osteoporosis or with a vertebral compression fracture were not eligible for the study. One hundred eighteen women entered the study. The women were randomly assigned to three groups: 1) hormonal replacement [estrogen-progesterone-calcium carbonate], 2) calcium carbonate, or 3) placebo. Assignment to the groups was based on computer-generated random numbers provided by the statistician, with stratification for years postmenopause. The women in the hormonal replacement group took conjugated equine estrogens (Premarin, Wyeth-Ayerst Laboratories, Inc.; Philadelphia, Pennsylvania), 0.625 mg daily for 25 days of the month along with medroxyprogesterone (Provera, Upjohn; Kalamazoo, Michigan), 10 mg from days 16 to 25. All women received 400 IU of vitamin D daily in the form of a multivitamin, and calcium supplementation (as Caltrate, Lederle; Clifton, New Jersey) was provided to the two treatment groups. The duration of the study was 2.9 1.1 years (mean SD). A 7-day dietary history was reviewed with a nutritionist every 2 months; calcium was provided as calcium carbonate, 600 mg (Caltrate), and used to supplement the diet to approximate a total daily intake of 1700 mg of elemental calcium (the mean + 2 SD found by Heaney and colleagues [7] to result in zero calcium balance in estrogen-deprived women). The calcium supplements were taken with meals in divided doses. The placebo appeared identical to the calcium carbonate tablets. No patients took antacids or histamine-2 blockers. All women had a baseline mammogram. Measurements Routine laboratory studies included a complete blood count, urinalysis, and serum fasting calcium, phosphorus, urea nitrogen, creatinine, alkaline phosphatase, cholesterol, and aminotransferase measurements [19, 20]. In addition, follicle-stimulating hormone, estradiol, parathyroid hormone, osteocalcin, free thyroxine, and bone alkaline phosphatase were measured, and a urine specimen was collected after an overnight fast for hydroxyproline, calcium, and creatinine determinations, following a 3-day low-hydroxyproline diet [21-23]. Total body calcium was measured annually in the participants, using the delayed neutron activation method at Brookhaven National Laboratory [24, 25]. This method uses a whole-body counter to measure the characteristic rays emitted from the neutron capture of Calcium-48 (natural abundance of 0.187%) in the body. The Brookhaven National Laboratory whole-body counter was upgraded in 1987 to use 32 NaI (T1) detectors of 10 cm 10 cm 46 cm positioned symmetrically above and below the patient [25]. The activated isotope, Calcium-49, decays with a half-life of 8.72 minutes, emitting a 3.08 MeV characteristic line. More than 99.5% of the body calcium is contained in the bone [26]. The method provides total body calcium with a coefficient of variation of about 1.5% when no substantial change in the body weight occurs during the period of repeated studies. The measurements were made annually. The bone mineral density of the distal radius site was measured using a Lunar Radiation (Madison, Wisconsin) single-photon absorptiometer (SP2). Bone mineral density of the spine (L2-L4) and femur (neck, trochanter, and Ward triangle) was measured using a Lunar Radiation DP4 dual-photon absorptiometer. The software version used for the analysis of scans was DP4 Lunar Corporation Version 1.1. All scans were analyzed using the same software version, which corrects for source decay. Instruments were calibrated daily, and the radioactive source was changed annually. Each measurement was done every 6 months. The coefficient of variation of these measurements was 2%, except for the Ward triangle (2.5%). Activity was measured using activity monitors (large-scale integrated monitors), which were worn about the waist [27]. The average of 2 weekdays and 1 weekend day was used as an activity score. Activity was measured at baseline and at one other point during the study to ensure that differences among the groups were not due to varied levels of exercise. Statistical Analysis Total body calcium was selected as the primary criterion for efficacy for the following reasons: It measures mass rather than density per unit area; it measures calcium balance precisely and accurately in the free living state and may be better related to previous studies using the balance technique; it is more precise than the other measurements; and it avoids sampling error by measuring the entire skeleton rather than a specific region of the appendicular or axial skeleton. The rate of change in bone mineral was calculated for each woman at each of the sites used in the study. Standard linear regression procedures were used to estimate the rate of bone mineral change for each woman, and the regression intercept was used as the best estimate of the baseline value. Because some women terminated their participation in the study before others, the rate-of-change data were weighted by the inverse variance to reflect the fit of the regression line for each woman [28]. Analyses of covariance were done using body mass index, activity scores, cigarette smoking, calcium intake, age, and years postmenopause as covariates. The data reported in this article are based on all women who provided at least three observations for a particular skeletal site. We considered other criteria, such as using data only from women who had participated in the study for at least 2 years, and all data analyses were done for this subgroup as well. The results of these analyses were invariably similar to those reported here and therefore are not presented separately. The mean rates of change in bone mineral for each condition at each site were characterized in terms of both raw units and percentages; separate analyses were carried out for each. The two indices were similar. Evidence from recent research is substantial that estrogen replacement therapy is effective, whereas the efficacy of calcium supplements is questionable. Our expectation was that our data would confirm the efficacy of estrogen-progesterone-calcium therapy, and the critical question was whether or not a beneficial effect of calcium supplements given alone could be shown. A separate one-way analysis of covariance was done for each of the bone mineral measurements to compare the mean rates of change in bone mineral for each of the three conditions. We used two a priori contrasts: the first contrasting women taking estrogen with those receiving calcium and the second comparing women receiving calcium supplements with those on placebo. All P values reported are two-tailed. Results Baseline data for historical data and bone mineral measurements and chemical studies are given in Table 1. Analysis of variance showed no significant differences in the baseline variables. The initial and final activity scores did not differ significantly. Table 1. Baseline Values for Patient Characteristics, Bone Mineral Measurements, and Chemical Variables The range of initial daily calcium intake in the overall study group was 150 to 1263 mg; in the calcium augmentation group, it was 222 to 806

Differential effect of treadmill exercise on three cancellous bone sites in the young growing rat

The aim of the present study was to examine cancellous bone changes induced by exercise on three different skeletal sites, the lumbar vertebra, the proximal, and the distal tibia, in the young growing rat. Forty 4-week-old female Sprague-Dawley rats were randomized into 4 groups of 10 animals each; 8 weeks exercise (8EX), 8 weeks sedentary control (8CON), 12 weeks exercise (12EX), and 12 weeks sedentary control (12CON). The exercise regimen consisted of treadmill running at 24 m/min 1 hr per day 5 days a week. After each period of exercise, the proximal and distal tibial metaphyses (PTM and DTM, respectively) and the fifth lumbar (L5) vertebral body were processed for histomorphometry of the cancellous bone (secondary spongiosa) and cortical periosteum. Eight and twelve weeks of exercise significantly increased the mineral apposition rate and bone formation rate in the PTM and DTM, and 12 weeks of exercise significantly increased the labeled perimeter in the DTM, compared with the age-matched controls. Eight and twelve weeks of exercise significantly increased cancellous bone volume in the PTM (mean +/- standard deviation, 8EX; 19.1 +/- 2.9% vs 8CON; 14.3 +/- 3.1%, P < 0.05 and 12EX; 18.8 +/- 3.5% vs 12CON; 15.2 +/- 3.3%, P < 0.05), and 12 weeks exercise significantly increased cancellous bone volume in the DTM, compared with age-matched control (12EX; 32.5 +/- 7.7%, 12CON; 22.2 +/- 4.8%, P < 0.05). The increase in cancellous bone volume by 12 weeks exercise was higher in the DTM than that in the PTM (43.4% and 24.0%, respectively). On the other hand, the exercise did not significantly affect cancellous bone volume and bone formation in the L5 vertebral body, although the cortical periosteal bone formation rate and the L5 vertebral bone mass were increased. These findings suggest that cancellous bone adaptation to treadmill exercise is site specific, and the effect may be influenced by factors such as mechanical loading and metaphyseal bone architecture in the young growing rat.

Treatment of osteoporosis with calcitonin, with and without growth hormone

A 24 month randomized parallel study of the treatment of postmenopausal osteoporosis with calcitonin alone v calcitonin alternating with growth hormone (combined treatment) was conducted. Each group received 1000 mg daily of oral calcium supplements. The rate of change in total body calcium for the combined and calcitonin groups was + 1.68%/yr and + 1.33/yr, respectively (P less than .05). However, the difference in the two groups was not statistically significant. Further, the total body calcium level did not increase after 12 to 18 months of treatment. There was significant difference in the rates of change of bone mineral content (BMC) of the radius for the two groups, with a loss of BMC in the combined treatment group (F = 4.80, P less than .05). Calcitonin treatment is effective in producing an increment in bone mass. The addition of growth hormone to this regimen appears to have a deleterious effect on cortical bone mass.

Effects of Growth Hormone and Testosterone on Cortical Bone Formation and Bone Density in Aged Orchiectomized Rats

Osteoporosis in men is a disease that is increasing in incidence, and with an increasing elderly population it poses a serious health problem. Since both testosterone (T) and growth hormone (GH) have an anabolic effect on bone and both decrease with aging, we were prompted to test whether the administration of these hormones in combination would increase bone mass in orchiectomized (orx) senile rats more than administration of either agent alone. Twenty-month-old male Wistar rats were divided into five groups with seven animals each: (a) age-matched intact control, (b) orx, (c) orx+GH (2.5 mg/kg/day), (d) orx+T [10 mg/kg, subcutaneous (s.c.), injection given twice a week], and (e) orx+GH+T. Testosterone and GH were given subcutaneously for 4 weeks. Bone histomorphometry of the tibial shaft showed that the orx group had lower cortical bone area than the intact control group. The decrease in cortical bone area was due to increased intracortical porosis as well as decreased periosteal bone formation rate (BFR). Administration of T to the orx animals prevented the development of the porosis and the decrease in periosteal BFR. The bone mineral content (BMC) and bone mineral density (BMD) of the femur as tested by dual-energy X-ray absorptiometry were significantly higher in the orx+T than in the orx group and were not significantly different from that of the intact control group. Administration of GH to the orx rats increased periosteal BFR significantly; however, the BMC and BMD measured were not increased significantly in comparison to the orx group. When GH and T were combined in treatment, the cortical bone area, periosteal BFR, and femoral BMD were all significantly higher than that of the orx and even higher than the intact control rats. Two-way analysis of variance shows that the individual effect of GH and T treatment on the periosteal BFR and cortical bone area was significant. The effect of T, but not GH, on femoral BMC and BMD was also significant; however, there is no synergistic interaction between the two treatments. Four weeks of orx with or without GH or T administration had no significant effect on tibial metaphyseal cancellous bone volume. In conclusion, this short-term study suggests that the combined intervention of GH and T in androgen-deficient aged male rats may have an independent effect in preventing osteopenia. The significant effect of GH+T may be attributed to the prevention of intracortical porosis, and an increase in periosteal bone formation and cortical bone mass.

Effect of Deconditioning on Cortical and Cancellous Bone Growth in the Exercise Trained Young Rats

Exercise enhances bone growth and increases peak bone mass. The aim of this study was to determine whether or not 4 weeks of deconditioning after 8 weeks of exercise in growing rats would result in a decrease in bone gain or reverse the benefits of exercise. Fifty 4‐week‐old female Sprague‐Dawley rats were randomized by a stratified weight method into 5 groups with 10 rats in each group: 8 weeks exercise (8EX), 8 weeks sedentary control (8S), 12 weeks exercise (12EX), 8 weeks exercise followed by 4 weeks sedentary (8EX4S), and 12 weeks sedentary control (12S). The exercise consisted of running on a treadmill with a 5° slope at 24 m/minute for 1 h/day and 5 days/week. After each period of exercise, cancellous and cortical bone histomorphometry were performed on double fluorescent labeled 5‐μm‐thick sections of the proximal tibia and 40‐μm‐thick sections of the tibial shaft, respectively. Eight and 12 weeks of exercise resulted in a significant increase in the body weight and gastrocnemius muscle weight by two‐way analysis of variance (ANOVA). The femoral wet weight (mg; mean ± SD; 8EX, 781 ± 45.1 vs. 8S, 713 ± 40.5; p < 0.05; 12EX, 892 ± 41.6 vs. 12S, 807 ± 19.8; p < 0.05) was significantly higher in the exercise group than that in the respective control groups. The femoral wet weight and bone volume (BV) of the 8EX4S group (818 ± 46.2 mg and 531 ± 31.2 μl, respectively) were significantly lower than those of the 12EX group (p < 0.05) and did not differ significantly from those of the 12S groups. The cancellous BV was significantly higher in the 8EX and 12EX groups than that in the respective sedentary groups (p < 0.05). The cortical bone area of the tibial shaft was also significantly higher in the 12EX than that in the 12S group (p < 0.05). The increase in the cancellous BV or cortical bone area was caused by an increase in the mineral apposition rate (MAR), without a significant effect in the labeled perimeter. The bone formation rate (BFR; μm3/μm2 per day) in the cancellous bone (12EX, 27.9 ± 7.74 vs. 12S, 15.4 ± 4.56; p < 0.05) or periosteal surface (12EX, 127.6 ± 27.7 vs. 12S, 79.5 ± 18.6; p < 0.05) was significantly higher in the exercised groups than that in the respective control group (p < 0.05). Again, deconditioning resulted in a decrease in the cancellous BFR, BV, periosteal BFR, and cortical bone area to levels not significantly different from the 12S group. In conclusion, our findings showed that exercised growing rats, when deconditioned, lost the benefits gained through exercise and their bone parameters were reduced to levels not different from the sedentary control. Thus, continued exercise is required to maintain high bone mass.

Effect of Treadmill Exercise on Tibial Cortical Bone in Aged Female Rats: A Histomorphometry and Dual . Energy X-Ray Absorptiometry Study

The aim of this study was to increase our understanding of the effect of exercise on cortical bone mass and turnover in aged female rats. Female Sprague-Dawley rats, 14 months of age, were divided into four groups: 8 controls and 10 exercised for the 9-week study, and 8 controls and 9 exercised for the 16-week study. Exercise consisted of treadmill running at 17 m/min for one h/day and 5 days/week for 9 and 16 weeks. All animals received double fluorochrome labeling of bone prior to sacrifice. Histomorphometric analysis was performed on 30-microns-thick Villanueva-stained, undecalcified cross-sections of the tibial shaft. Tibial diaphyseal mineral density of each rat in the 16-week study was measured by dual energy x-ray absorptiometry in vivo at 0, 9, and 16 weeks. The diaphyseal mineral density of the exercised group was significantly greater than that of the control group (p < 0.05 by two-way ANOVA) and the individual slopes of the density vs. time was found to be higher in the exercised than in the control animals (mean +/- SE of exercised 0.56 +/- 0.13 vs. control 0.19 +/- 0.07 mg/cm2/week, p < 0.05) by the end of the experiment. The results of the histomorphometric analysis after 9 weeks of exercise showed that the periosteal labeled surface, mineral apposition rate, and bone formation rate were profoundly increased by 192% (p < 0.001), 35%, and 206% (p < 0.01), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

REFERENCE RANGE FOR SERUM PARATHYROID HORMONE

OBJECTIVEnTo determine whether the reference range for parathyroid hormone (PTH) should be lowered (from 65 pg/mL to a proposed value of 46 pg/mL) with use of the Allegro radioimmunometric assay.nnnMETHODSnWe examined the reference range for PTH, adjusted for serum 25-hydroxyvitamin D (25-OHD), in 503 healthy African American and white women, who were 20 to 80 years old. We also analyzed other factors that are thought to influence PTH levels.nnnRESULTSnUnivariate predictors of PTH were identified, and a multivariate model was developed with use of the variables and PTH. Serum PTH was significantly higher in black study subjects than in white study subjects (P<0.02). Increasing PTH was also significantly correlated with increasing body mass index, age, and serum creatinine and with decreasing dietary calcium intake and serum 25-OHD levels. A stepwise multiple linear regression analysis yielded the following predictors of PTH: body mass index (R2=9.4%), age (R2=1.0%), and serum 25-OHD (R2=0.8%). In our study population, many PTH values were above the proposed new upper limit of 46 pg/mL.nnnCONCLUSIONnThe upper limit of the reference range for serum PTH should not be changed. Factors to be considered in analysis of serum PTH values in the upper reference range in patients with normocalcemia include obesity, race, 25-OHD levels, advanced age, serum creatinine, and dietary calcium intake.

Vitamin D and Serum Cytokines in a Randomized Clinical Trial

Background. The role of vitamin D in the bodys ability to fight influenza and URIs may be dependent on regulation of specific cytokines that participate in the host inflammatory response. The aim of this study was to test the hypothesis that vitamin D can influence intracellular signaling to regulate the production of cytokines. Subjects and Methods. This study was a 3-month prospective placebo-controlled trial of vitamin D3 supplementation in ambulatory adults [Li-Ng et al., 2009]. 162 volunteers were randomized to receive either 50u2009μg/d (2000 IU) of vitamin D3 or matching placebo. 25(OH)D and the levels of 10 different cytokines (IL-2, 4, 5, 6, 8, 10, 13, GM-CSF, IFN-γ, TNF-α) were measured in the serum of participants at baseline and the final visit. There were 6 drop-outs from the active vitamin D group and 8 from the placebo group. Results. In the active vitamin D group, we found a significant median percent decline in levels of GM-CSF (−62.9%, P < .0001), IFN-γ (−38.9%, P < .0001), IL-4 (−50.8%, P = .001), IL-8 (−48.4%, P < .0001), and IL-10 (−70.4%, P < .0001). In the placebo group, there were significant declines for GM-CSF (−53.2%, P = .0007) and IFN-γ (−34.4%, P = .0011). For each cytokine, there was no significant difference in the rate of decline between the two groups. 25(OH)D levels increased in the active vitamin D group from a mean of 64.3 ± 25.4u2009nmol/L to 88.5 ± 23.2u2009nmol/L. Conclusions. The present study did not show that vitamin D3 supplementation changed circulating cytokine levels among healthy adults.

Ovariectomy-induced high turnover in cortical bone is dependent on pituitary hormone in rats

The aim of this study is to examine the interrelationship of pituitary and ovarian hormone deficiency on the regulation of bone growth and bone formation rate. 48 female rats, at 3 months of age, were divided into age-matched intact control, hypophysectomized (HX), ovariectomized (OV), and HX + OV groups. Ten rats were killed at 3 months of age as baseline controls, and the rest of the animals were killed 5 weeks after surgery. Serum levels of osteocalcin and dynamic histomorphometry on the periosteal surface of the tibial shaft and fifth lumbar vertebrae were measured to evaluate systemic and local bone turnover. Tibial and fourth lumbar vertebral bone area, bone mineral content, and bone density were measured by dual-energy X-ray absorptiometry (DXA). Our results confirmed that OV increased and HX suppressed systemic and periosteal bone formation parameters in both bone sites, OV increased and HX suppressed the gain in bone size and bone mass. When OV rats were HX, the serum levels of osteocalcin and periosteal bone formation parameters of the tibial shaft and the fifth lumbar vertebrae were, however, depressed and did not differ from that of the HX alone. DXA results show that the effect of OV on bone size and bone mass is also abolished by HX. In conclusion, we have demonstrated that OV increases tibial and lumbar vertebral bone formation and bone growth and this effect is pituitary hormone dependent.

Effects of 17β-estradiol replacement and treadmill exercise on vertebral and femoral bones of the ovariectomized rat

To evaluate the effect of 17 beta-estradiol replacement (10 micrograms, twice a week) (E2) and treadmill exercise (18 m/min, 45 min/day) (EX) on long bone and vertebral bone mass and density, 10-month-old rats were ovariectomized (OV) and divided into four groups: OV, OV + E2, OV + EX, OV + EX + E2 2 months after surgery. After 7 weeks intervention, the calcium content and the density of lumbar-5 were higher in both OV + E2 and OV + EX + E2 groups than in the OV group, but, only the OV + EX + E2 group had a significantly higher femoral bone weight and density than the OV group. After 16 weeks intervention, the bone-conserving effects of E2 and EX were significant on lumbar-5 and femoral dry weight and density. The effect of E2 on both two sides of bones was due to the suppression of the bone turnover rate, while EX suppressed bone turnover rate primarily on the femur. We conclude that the effect of the two interventions on lumbar-5 and femoral bone mass were additive and independent.