IN WOMEN, BONE loss associated with an increase in

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1 X/03/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 88(11): Printed in U.S.A. Copyright 2003 by The Endocrine Society doi: /jc Prediction of Bone Mass Density Variation by Bone Remodeling Markers in Postmenopausal Women with Vitamin D Insufficiency Treated with Calcium and Vitamin D Supplementation FRANCK GRADOS, MICHEL BRAZIER, SAÏD KAMEL, MARC MATHIEU, NATHALIE HURTEBIZE, MOHAMED MAAMER, MICHÈLE GARABÉDIAN, JEAN-LUC SEBERT, AND PATRICE FARDELLONE Departments of Rheumatology and Biochemistry (F.G., M.B., S.K., J.-L.S., P.F.), and Laboratory of Clinical Pharmacy, Faculty of Pharmacy (M.B., S.K.), CHU, Amiens 80054, France; Laboratoires Innothera (M.Mat., N.H., M.Maa.), Arcueil 94111, France; and UPR 1524, Hôpital Saint Vincent de Paul (M.G.), Paris 75010, France The aim of this study was to determine whether early changes in bone markers could predict long-term response in bone mineral density (BMD) after calcium (500 mg) and vitamin D (400 IU) supplementation twice daily in ambulatory elderly women with vitamin D insufficiency (25-hydroxyvitamin D, <12 ng/ml). One hundred and ninety-two women (mean age, 75 7 yr) were randomized to receive either the supplementation (n 95) or a placebo (n 97) in a double-blind, controlled clinical trial for 1 yr. In comparison with the placebo group, supplementation significantly increased BMD, normalized 25-hydroxyvitamin D and significantly decreased intact PTH and bone remodeling markers. The initial values of Abbreviations: b-ap, Bone alkaline phosphatase; BMD, bone mineral density; Ca, calcium; Ca-Vit D, calcium and vitamin D group; Cr, creatinine; CTX, C-terminal telopeptide of type I collagen; f-dpd, free deoxypyridinoline; i-pth, intact PTH; 25(OH)D, 25-hydroxyvitamin D; NTX, N-terminal telopeptide of type I collagen; P, placebo group; s-, serum; u-, urinary. telopeptide cross-links were correlated with improvement in total body BMD [urinary N-telopeptides (NTX), r 0.38; C- telopeptides (CTX), r 0.32; serum CTX, r 0.28], and the 3-month changes in the same markers were correlated with improvement in total body (urinary N-telopeptides, r 0.29; serum CTX, r 0.26) and vertebral BMD (CTX, r 0.26; all P < 0.05). We concluded that short-term changes in bone resorption markers can predict long-term variations in BMD in elderly women with vitamin D insufficiency receiving calcium and vitamin D supplementation. (J Clin Endocrinol Metab 88: , 2003) IN WOMEN, BONE loss associated with an increase in bone remodeling is observed at the time of menopause, mainly due to estrogen deficiency, and persists thereafter when other determinant factors of bone loss are encountered, such as calcium and vitamin D insufficiencies. In fact, the prevalence of insufficiency in calcium and vitamin D is high and well documented in elderly subjects (1, 2). This insufficiency is responsible for increased PTH levels and subsequent accelerated bone remodeling (3, 4), leading to a further decrease in bone mass and an increase in fracture risk (5, 6). In this case, the calcium and vitamin D supplementation is able to normalize the secondary hyperparathyroidism and consequently to reduce bone remodeling and stop bone loss (7, 8). Furthermore, several studies have shown the ability of this supplementation to reduce the incidence of peripheral fractures (8, 9). These observations, well known in elderly women, also contribute to bone loss in younger populations, particularly in women around 65 yr of age (10). However, the effects on bone mass and bone remodeling of vitamin D and calcium supplementation in women with vitamin D insufficiency are poorly documented; moreover, no data are available on the link existing between bone turnover and bone mass density during a lengthy course of treatment with calcium and vitamin D supplementation. The purpose of the study was to assess the effect of long-term treatment with calcium and vitamin D in comparison with a placebo on bone mineral density (BMD) and the ability of bone markers to afford an early prediction of this effect on bone in a population of postmenopausal women over 65 yr of age with vitamin D insufficiency. Subjects Subjects and Methods In 10 centers in France, we studied ambulatory women, aged 65 yr and over, from the usual practice of the physicians participating in the study. The women were selected because the physicians considered them at high risk for vitamin D insufficiency, and they were enrolled when the 25-hydroxyvitamin D [25(OH)D] serum level was less than 12 ng/ml. The exclusion criteria were hypercalcemia or primary hyperparathyroidism, renal or hepatic insufficiency, and treatment with a bisphosphonate, calcitonin, vitamin D and its metabolites, estrogen, raloxifene, fluoride, anticonvulsants, or other treatments, such as glucocorticoid, that are active on bone metabolism in the previous 6 months. All women were living at home, were ambulatory, and gave written informed consent. A power analysis was performed before the experiment began. Once the statistical hypothesis were stated (type 1 error: 5%, by one-sided test; type two error, 15%), we obtained a number of 49 subjects in both arms (i.e. 98 patients in total). Taking into account the predictable risk of drop-out of about 30% (in the light of previous studies in the same field), we increased the number of patients included to reach 80 patients in each arm (i.e. 160 patients). The protocol was approved by the local ethics committee, and the trial was conducted in accordance with good clinical practices and guidelines proposed in the Declaration of Helsinki. 5175

2 5176 J Clin Endocrinol Metab, November 2003, 88(11): Grados et al. Calcium and Vitamin D Supplementation in Postmenopausal Women Study design In the 12-month, double-blind, placebo-controlled trial, the subjects were randomly assigned to either the placebo (P) group or the calcium and vitamin D (Ca-Vit D) group. Dietary calcium and vitamin D intakes were assessed at admission. At baseline and during the course of treatment (3, 6, 9, and 12 months), a complete clinical examination (height, weight, blood pressure, and resting pulse rate) was performed. We also assessed biological parameters of calcium homeostasis and of bone remodeling, and BMD was evaluated at baseline and after 12 months of treatment. The women received either calcium carbonate (500 mg elemental calcium) and cholecalciferol (400 IU) as tablets (n 95; Ideos Laboratoires, Innothera, France) or matching placebo tablets (n 97) twice a day. Status of subjects and compliance Three hundred and sixty women were selected; among them 192 fulfilled the inclusion criteria and were included. During the trial 61 subjects discontinued treatment, without a statistical difference between P and Ca-Vit D groups. The mean sd rate of compliance with treatment, assessed on the basis of tablet counts, was 88 14% for the Ca-Vit D group and 87 18% for the P group. Measurements The daily calcium intake was assessed using a validated food frequency questionnaire (11); at the same time, we developed a vitamin D dietary questionnaire to evaluate the daily vitamin D intake. BMD in the hip, spine, and total body was measured by dual energy x-ray absorptiometry using the device available in each region (Hologic, Inc., Bedford, MA; Lunar Corp., Madison, WI; Norland Medical Systems, Inc., Fort Atkinson, WI). Intrascanner precision ranged from % (12). The results of osteodensitometric evaluations were established by central analysis. The BMD values were standardized by the universal method proposed by Genant (13). Specimen collection Blood and urine samples were collected at baseline, then after 3, 6, 9, and 12 months of treatment. Fasting blood samples were taken, and serum was removed, then frozen at 80 C. The 24-h urine samples of the previous day were stored at 80 C. Biochemical measurements In serum. Measurements of 25-(OH)D were performed using a competitive protein binding assay after ethanol extraction, followed by chromatographic purification (14). Intact PTH (i-pth) was measured by an immunoluminometric assay (Magic LITE intact PTH, Ciba-Corning, Cergy-Pontoise, France), bone alkaline phosphatase (b-ap) by immunoenzymatic kits (Alkphase-B, Metra-Biosystems Inc., Palo Alto, CA), serum C-telopeptide derivatives (s-ctx) by ELISA (serum Crosslaps one-step, Osteometer A/S, Ballrup, Denmark). Serum calcium was measured by colorimetric methods using an EKTACHEM 500 autoanalyzer (Kodak-Pathe, Paris, France). Interassay reproducibility, expressed as the coefficient of variation, was 10.8% for 25-(OH)D, 3.4% for i-pth, 7.8% for b-ap, 6.3% for s-ctx, and 1.4% for calcium. In urine. Measurements were performed on the 24-h urine samples and expressed as the ratio to creatinine. Urinary (u-) N- and C-telopeptides (NTX and CTX) excretion was measured using ELISA with the Osteomark kit (u-ntx; Ostex, Inc., Seattle, WA) and the Crosslaps kit (u-ctx; Osteometer A/S), respectively. Free deoxypyridinoline (f-dpd) was assayed using an immunological kit (Pyrilinks-D ACS, Chiron, Cergy- Pontoise, France). With these assays, the interassay coefficients of variation were less than 10%. u-ca and creatinine (u-cr) were determined by colorimetric methods using the automated system (EKTACHEM 500). u-ca was expressed as the ratio to creatinine ( 24h uca/cr). The interassay reproducibility of both u-ca and u-cr, expressed as coefficients of variation, were, respectively, 2.6% and 5.3%. Statistical analysis The results of the full analysis set were provided. The statistical analysis was carried out using SAS software version 6.12 (SAS Institute, Inc., Cary, NC). The data were analyzed using the signed-rank test for intragroup comparison and Wilcoxon test to compare the difference in the changes between the two treatments. Two-sided tests were performed with a type I error of 5%. The relationship between variations in BMD at the end point and initial or changes in biological parameters were tested using the Spearman correlation coefficient. Results The baseline clinical characteristics of the 192 women enrolled in this trial are shown in Table 1. The 2 groups did not differ according to age, body mass index, dietary calcium, and vitamin D intakes. There were no statistically significant differences in BMD (Table 2) or in biochemical markers of calcium homeostasis and bone turnover values (Table 3). Several baseline biological parameters showed a variety of weak correlations, particularly dietary calcium intake that negatively correlated with urinary NTX (r 0.12; P 0.05) and CTX (r 0.11; P 0.05), when dietary vitamin D intake was correlated, positively with 25(OH)D (r 0.15; P 0.01) and 24H uca/cr (r 0.13; P 0.05) and negatively with s-ctx (r 0.19; P 0.05). The 25(OH)D levels showed a negative correlation with i-pth (r 0.24; P 0.01), u-ntx (r 0.12; P 0.05), and f-dpd (r 0.18; P 0.001). Finally, i-pth and b-ap values were correlated with all bone resorption markers, which, in turn, correlated among themselves. After 1-yr treatment, Ca-Vit D supplementation had a significant effect on BMD at every site except for the trochanter. Compared with the placebo, the supplementation showed a positive and significant effect on the change in BMD over 1 yr, measured at every site (Table 2). The women who had received the Ca-Vit D supplementation showed a marked increase in 25(OH)D associated with enhanced excretion of urinary calcium and a dramatic decrease (median [Q 1 ;Q 3 ]) in i-pth levels ( 34% [ 54; 15]; p ), but without a significant change in calcemia (Table 3). Simultaneously, we observed a reduction of bone turnover markers, except for f-dpd. When expressed as a percentage of baseline, the changes (median [Q 1 ;Q 3 ]) in bone resorption markers were: u-ntx, 41% [ 57; 11]; u-ctx, 46% [ 66; 16]; s-ctx, 26% [ 46; 13] (all P ). The changes in bone formation markers were: b-ap, 14% [ 34; 13] (P ; Table 3). The decrease in bone remodeling markers was observed as early as the third month of treatment, particularly for s-ctx, and reached a maximum level after 6 months in the case of u-ntx and u-ctx (Fig. 1). In the P group a slight, but significant, increase in 25(OH)D associated with a moderate and significant decrease in i-pth was TABLE 1. Means ( SD) of baseline clinical characteristics of the 192 subjects in the two treated groups Characteristics Groups Ca-Vit D (n 95) Placebo (n 97) Age (yr) Body mass index (kg/m 2 ) Dietary calcium intake (mg/d) Dietary vitamin D intake (IU/d)

3 Grados et al. Calcium and Vitamin D Supplementation in Postmenopausal Women J Clin Endocrinol Metab, November 2003, 88(11): TABLE 2. Means ( SD) of initial values and changes in BMDs (milligrams per square centimeter) at 12 months (intention to treat) in the two treated groups Ca-Vit D Placebo n Initial value Change P value a n Initial value Change P value a P value b Lumbar spine (L2 L4) Neck Trochanter Total body a P values from the signed rank test (intragroup comparison). b P values from Wilcoxon test with normal approximation of the difference between the two treatments for the change. TABLE 3. Means ( SD) of initial laboratory values and changes at 12 months (intention to treat) of calcium homeostasis parameters and bone remodeling markers in the two treated groups Ca-Vit D Placebo Initial value Changes P value a Initial value Changes P value a P value b 25(OH)D (ng/ml) c-ca (mmol/liter) ipth (pg/ml) hUCa/Cr (mmol/mmol) b-ap ( g/liter) s-ctx (pmol/liter) NTX/Cr (nmol BCE/mmol) CTX/Cr ( g/mmol) f-dpd/cr (nmol/mmol) c-ca, Corrected value of calcium (mmol/liter) Ca (mmol/liter) [40-albuminemia (g/liter)]/40; BCE, bone collagen equivalent. a P values from the signed rank test (intragroup comparison). b P values from Wilcoxon test with normal approximation of the difference between the two treatments for the change. FIG. 1. Mean ( SD) decrease in bone remodeling markers, expressed as a percentage of their levels calculated from baseline in the Ca-Vit D-treated group of postmenopausal women at the different times of the study: 3 (m 3 ),6(m 6 ),9(m 9 ), and 12 (m 12 ) months. A signed-rank test for intragroup comparisons was performed to assess the statistical differences between baseline (m 0 ) and each time point. *, P 0.05; **, P 0.01; ***, P observed. Similarly, a significant variation in bone remodeling markers was seen at 1 yr only for u-ntx and u-ctx. When we compared the two groups, a significant positive effect of the supplementation was obtained simultaneously for the biological parameters of calcium homeostasis, such as 25 OH-D, i-pth, and 24H uca/cr, and for the bone remodeling markers, with the exception of u-ctx and f-dpd (Table 3). In the Ca-Vit D group, there were few significant correlations between baseline or 3-month changes in biochemical markers and 1-yr changes in BMD. The change in total body BMD correlated positively with the baseline levels of u-ntx, u-ctx, and s-ctx. Moreover, the decreases at 3 months in u-ntx and s-ctx also correlated with the total body BMD change (Table 4). Finally, using the Spearman correlation coefficient, the decreases in u- CTX at 3 and 6 month correlated with the change at the lumbar spine (Fig. 2).

4 5178 J Clin Endocrinol Metab, November 2003, 88(11): Grados et al. Calcium and Vitamin D Supplementation in Postmenopausal Women Discussion TABLE 4. Correlation of BMD changes at 1 yr by biochemical parameters (intention to treat) Site 1 yr BMD increase r value P value Initial value NTX/Cr (nmol BCE/mmol) Total body CTX/Cr (nmol/mmol) Total body s-ctx (pmol/liter) Total body month decrease NTX/Cr (nmol/mmol) Total body s-ctx (pmol/liter) Total body BCE, Bone collagen equivalent. FIG. 2. Scatterplots showing the percent change in lumbar spine BMD at 1 yr according to the percent change in urinary CTX bone resorption markers at 3 and 6 months. In the Ca-Vit D-treated group (heavy solid line), Spearman s correlation coefficient demonstrated a significant negative correlation at each time (r 0.26; P 0.05 and r 0.31; P 0.02, respectively), whereas no statistical relationship was established in the P group (light solid line). In this study we investigated the effects on BMD and bone remodeling of Ca-Vit D supplementation in ambulatory women with vitamin D insufficiency. We found positive effects on BMD and bone turnover simultaneously, and we demonstrated the ability of bone resorption markers to predict changes in BMD after supplementation for 1 yr. Among our population, the prevalence of vitamin D insufficiency and the levels of dietary calcium intake were very closely related to prior results (10), confirming the high prevalence of these insufficiencies in French postmenopausal women. For that reason high baseline i-pth levels were observed, close to those usually reported in studies performed during the winter when 25(OH)D levels are at their lowest (9, 15, 16); however, the increase in i-pth levels was less dramatic because of the ambulatory status of our population (17). Moreover, as i-pth concentrations were obtained using different assays, it is difficult to compare the data from various studies. We confirmed the relationship between negative calcium balance and high bone turnover (4), the low BMD status in elderly patients with vitamin D and calcium insufficiency (18), and the ability of vitamin D and calcium supplementation to increase BMD, particularly at sites at major risk of fracture in elderly. After 1 yr, the BMD changes at all sites in our population were higher than those reported by Dawson-Hughes et al. (8), showing a significant effect only in the spine and total body, but not in femoral neck. In the Dawson-Hughes study changes were obtained giving 500 mg calcium and 700 IU vitamin D/d, whereas we used a similar dosage of vitamin D (800 IU), but twice the amount of calcium (1000 mg). Obviously the vitamin D insufficiency characterizing our population plays a partial role in the dramatic effect on BMD; unfortunately, we were unable to assess the effect of such supplementation over a longer period as Dawson-Hughes did. The gain in BMD was approximately equivalent to that in the Chapuy study, in which the same dosages of vitamin D and calcium were provided to an elderly, dramatically vitamin D-depleted, institutionalized population (9). However, we noted that BMD changes after such supplementation for 1 yr remain at only half the level achieved with bisphosphonates such as alendronate (19) and risedronate (20). Here the positive effects of vitamin D and calcium on BMD were accompanied by a reduction in bone turnover as previously reported, first by Chapuy et al. (9) in an elderly institutionalized population, then by Dawson-Hughes (8) in an ambulatory population of men and women. The supplementation strongly increased 25(OH)D levels and moderately increase u-ca excretion without modification of calcemia. We observed a normalization of i-pth values associated with a dramatic decrease in bone turnover markers, and the difference between treated and placebo groups was highly significant. In the P group, a slight decrease in some bone resorption markers without any change in BMD was probably due to a seasonal effect on 25(OH)D levels. Indeed, the vitamin D insufficiency displayed by our subjects, who were mainly recruited during the winter, was partially corrected by sun exposure during the course of spring and summer, resulting in a significant decrease in i-pth levels and simultaneously in the more sensitive markers of bone resorption (21). Using the new sensitive assays of bone resorption such as CTX or NTX of type I collagen derivatives, we demonstrated that supplementation resulted in dramatic changes in bone resorption as early as 3 months. Moreover, we did not observe significant changes in f-dpd as previously reported by Garnero (22), confirming that f-dpd is not a relevant bone marker for monitoring antiresorptive treatment. For the first time we demonstrated in a population of postmenopausal women with vitamin D insufficiency treated with vitamin D and calcium supplementation at doses known to prevent hip fracture in elderly people (8, 9) that various bone resorption markers could predict changes in BMD. High baseline values of bone resorption markers or their changes at 3 or 6 months were correlated with variations in BMD after 1 yr. Furthermore, the reduction in bone resorption markers is in line with the cut-off proposed by recent recommendations for the use of biological indexes to monitor osteoporosis (23). These properties have been ob-

5 Grados et al. Calcium and Vitamin D Supplementation in Postmenopausal Women J Clin Endocrinol Metab, November 2003, 88(11): served with powerful antiresorptive agents such as bisphosphonates (24, 25) or hormone replacement therapy (26). This is clinically relevant because no short-term evaluation is currently available by another method, such as densitometry, which takes at least 2 yr to show any significant change in a given patient. However in our study the relationship between BMD variations and bone resorption markers was tenuous, probably because the supplementation is a less potent antiresorptive agent than bisphosphonates or hormone replacement therapy, but also because the duration of the study was shorter than in previous studies by Greenspan (24, 25) or Rosen (26). In conclusion, we confirmed in women with vitamin D insufficiency the ability of a large amount of vitamin D and calcium to enhance bone mass and reduce bone turnover. Moreover, we showed that bone resorption markers, such as derivatives of CTX and NTX of type I collagen, could be useful to the practitioner as a mean of monitoring the effects of such treatment. Acknowledgments We extend grateful thanks to Mrs. Fabienne Hervy and the association PREDOS, and to Alain Morel and Claude Diot for their technical assistance. Received December 13, Accepted July 15, Address all correspondence and requests for reprints to: Prof. P. Fardellone, Service de Rhumatologie, Hôpital Nord, Amiens Cedex 1, France. fardellone.patrice@chu-amiens.fr. This work was supported by the Laboratoires Innothera. References 1. Omdahl JL, Garry PJ, Hunsaker LA, Hunt WC, Goodwin JS 1982 Nutritional status in a healthy elderly population: vitamin D. Am J Clin Nutr 36: Gloth FM III, Tobin JD 1995 Vitamin D deficiency in older people. 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