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Early Changes in Serum N-Telopeptide and C-Telopeptide Cross-Linked Collagen Type 1 Predict Long-Term Response to Alendronate Therapy in Elderly Women¹

Divisions of Endocrinology and Metabolism and Geriatrics, Department of Medicine, University of Pittsburgh Medical Center (S.L.G.), Pittsburgh, Pennsylvania 15213; and Charles A. Dana Research Institute, Harvard-Thorndike General Clinical Research Center (S.L.G., H.N.R., R.A.P.); Divisions of Bone and Mineral Metabolism and Gerontology, Department of Medicine (H.N.R.); and Biometrics Center (R.A.P.), Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Susan L. Greenspan, M.D., University of Pittsburgh Medical Center, Osteoporosis Prevention and Treatment Center, 1110 Kaufmann Building, 3471 Fifth Avenue, Pittsburgh, Pennsylvania 15213. E-mail: greenspans{at}msx

	Abstract

Top Abstract Introduction Experimental Subjects Materials and Methods Results Discussion References

 
The aim of this study was to determine whether early changes in serum markers of bone resorption could predict long-term responses in bone mineral density (BMD) after alendronate therapy in elderly women. One hundred and twenty women (mean age, 70 yr) were randomized to alendronate or placebo in this double blind, placebo-controlled clinical trial for 2.5 yr. Outcome measures were hip and spine BMD and biochemical markers of bone resorption, including serum N-telopeptide and C-telopeptide cross-linked collagen type I (NTx and CTx, respectively). Serum NTx and CTx were highly correlated at baseline (r = 0.73; P < 0.001) and remained so throughout the study (range, r = 0.36–0.56; all P < 0.05). After treatment with alendronate, serum NTx decreased 30.4 ± 16.0% at 6 months, reaching a nadir of -36.7 ± 18.0% by 24 months (P < 0.001). Serum CTx decreased 43.5 ± 67.0% at 6 months and continued to decrease to 67.3 ± 19.3% at 2.5 yr (P < 0.001). Moreover, decreases in serum NTx and CTx at 6 months were correlated with long-term improvements in vertebral BMD at 2.5 yr in patients receiving alendronate therapy (NTx: r = -0.42; CTx: r = -0.31; both P < 0.05). We conclude that early changes in serum NTx and CTx, markers of bone resorption, predict long-term changes in vertebral BMD in elderly women receiving alendronate therapy and provide a useful tool to assess skeletal health.

	Introduction

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BIOCHEMICAL markers of bone turnover provide valuable information to clinicians regarding the efficacy of antiresorptive therapy (1, 2, 3, 4, 5). Although fracture reduction is still considered the gold standard of therapeutic outcomes, bone mineral densitometry often provides supportive information to the clinician, because larger gains in bone mass are associated with greater fracture reduction with alendronate (6). However, even follow-up bone mass measurements may be misleading with regard to the ultimate response to therapy (7). Biochemical markers complement bone mineral density (BMD) measurements in current practice (8). Previous studies have shown that the rate of bone loss is associated with levels of biochemical markers (9, 10, 11). Decreases in biochemical markers are often observed 3 months after antiresorptive therapy and before BMD changes are evident (2). Furthermore, the rate of bone turnover may provide independent information regarding fracture risk (12).

Early changes in urinary N-telopeptide cross-linked collagen type 1 (NTx) after hormone replacement therapy have been shown to be predictive of BMD response in postmenopausal women (3). We have previously shown that urinary NTx is also associated with long-term efficacy after alendronate therapy in elderly women (13). However, patients and clinicians often prefer the ease and simplicity of a single serum test vs. a morning urine collection. The aim of the present study was to determine whether serum markers of bone resorption, serum NTx and C-telopeptide cross-linked collagen type I (CTx), could also predict the response to alendronate therapy.

	Experimental Subjects

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One hundred twenty community-dwelling elderly women, aged 65 yr and older, were enrolled as previously described (13). Subjects with illnesses or medications known to affect bone mineral metabolism were excluded. The protocol was approved by the committee on clinical investigations, new devices, and new forms of therapy at the Beth Israel Deaconess Medical Center (Boston, MA). All subjects were advised of the nature of the study, and written informed consent was obtained before enrollment.

	Materials and Methods

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Study design

Subjects were randomized to receive alendronate (5 mg daily) or placebo for this 2.5-yr, double-blind, placebo-controlled trial. After 1.5 yr, the dose of alendronate was increased to 10 mg for the final year of the protocol. Subjects received supplementary calcium if necessary to ensure that their daily intake was greater than 1000 mg/day. The calcium carbonate used in this study also contained vitamin D (125 IU vitamin D/250 mg elemental calcium).

Outcome variables

BMD. BMD of the hip (femoral neck, greater trochanter, intertrochanter, and total hip), lumbar spine [posterior anterior (PA) and lateral], total body, and forearm were determined at baseline and every 6 months thereafter.

Biochemical markers. Serum was drawn after an overnight fast and stored at -80 C. Serum NTx [nanomoles per L of bone collagen equivalents (BCE)] was measured by a chemiluminescence assay in competitive inhibition format, as previously described (14) [Ostex International, Inc., Seattle, WA; interassay coefficient of variation (CV), <12%]. Serum CTx was measured with an enzyme-linked immunosorbent assay (with monoclonal antibodies), as previously described (15) (Serum CrossLapsJ One Step ELISA, Osteometer Biotech, Herlev, Denmark; interassay CV, 5.4–7.9%). Urinary NTx (nanomoles per L of BCE/creatinine) was obtained as the second void urine (0600–0800 h) after an overnight fast and was frozen at -20 C. Urinary NTx was measured with an enzyme-linked immunosorbent assay (Osteomark7, Ostex International, Inc.; interassay CV, 5–19%). All assays were simultaneously run at the end of the study by the same technician.

Statistical methods

For analysis of baseline characteristics, we included data from all subjects. For analyses involving changes over time, we analyzed data separately by treatment group. Descriptive statistics are presented as the mean ± SD unless otherwise noted. Comparisons over time within subjects were assessed using the Wilcoxon signed rank test. Comparisons between treatment groups used the Wilcoxon rank-sum test. The Kruskal-Wallis test (a nonparametric equivalent of ANOVA) was used for comparison between tertiles of the marker changes. Spearman rank correlation was used for the assessment of correlations between variables. If a BMD measurement was missing at 30 months, we carried the last value forward and used standard linear regression to predict the change in BMD measurement at 2.5 yr for a given change at 6 months in a marker of bone turnover.

	Results

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Baseline characteristics were previously described (13). Mean age was 69.9 ± 4.6 yr. At baseline, serum NTx averaged 16.3 ± 4.4 nmol/L BCE, serum CTx averaged 2881 ± 1980 pmol/L, and urinary NTx averaged 67.2 ± 35.1 nmol/L BCE/creatinine. Similar values for all variables were found in both groups (treatment vs. placebo, all P > 0.50).

In patients who were treated with alendronate therapy, serum NTx decreased significantly by 30 ± 16% (mean ± SD) at 6 months, reaching a nadir of -37 ± 18% by 24 months (both P < 0.001; Fig. 1). Serum CTx decreased to -43 ± 67% at 6 months and continued to decrease to -67 ± 19% at 30 months (both P < 0.001). Baseline serum NTx was correlated with baseline serum CTx (r = 0.73; P < 0.001) and urinary NTx (r = 0.52; P < 0.001); serum CTx was correlated with urinary NTx (r = 0.56; P < 0.001). From months 6–24, the correlation coefficients of these three measurements ranged from 0.42–0.83 (all P < 0.01) in the treatment group; the correlation was poorer in month 30, but was still significant (P < 0.05), except for serum NTx and urinary NTx (r = 0.28; P = 0.06).

View larger version (14K): [in this window] [in a new window]   Figure 1. Mean change from baseline in markers of bone resorption (a, serum NTx; b, serum CTx) in women receiving alendronate or placebo for 30 months. Results are the mean ± SEM. *, P < 0.05; , P < 0.001 (vs. baseline). , P < 0.001 (alendronate vs. placebo).

After 2.5 yr of alendronate therapy, BMD significantly increased in the PA spine (7.8%), lateral spine (10.6%), total hip (4.0%), and femoral neck (3.1%), as previously described (13). Decreases in serum NTx at 6 months were correlated with long-term improvements in BMD at the PA spine (r = -0.42; P < 0.01), with a trend for improvements at the total hip (r = -0.26; P = 0.06) and femoral neck (r = -0.27; P = 0.06). A 15% decrease in serum NTx at 6 months predicted BMD increases of 3.4% at the total hip, 5.0% at the greater trochanter, 3.3% at the intertrochanteric region, 5.6% at the PA spine, and 6.6% at the lateral spine at 2.5 yr (all P < 0.05). Short-term decreases in serum CTx were also associated with long-term improvements at the PA spine (r = -0.31; P < 0.05). A 20% decrease in serum CTx at 6 months predicted an increase of 4.2% at the total hip, 5.7% at the greater trochanter, 4.0% at the intertrochanter, 8.0% BMD at the spine, and 10.1% at the lateral spine (all P < 0.05). The early decreases in urinary NTx were associated with long-term changes at the hip (r = -0.35; P < 0.01), femoral neck (r = -0.28; P < 0.05), trochanter (r = -0.36; P < 0.01), PA spine (r = -0.41; P < 0.01), and whole body (r = -0.34; P < 0.05) as previously reported (13). In patients in the placebo group, short-term changes in markers were not associated with long-term changes in bone density, except for one association thought to be a chance finding.

When patients receiving alendronate were separated into tertiles, those with the greatest decreases in serum or urinary NTx at 6 months were found to have the greatest gains in spine BMD at 2.5 yr (P < 0.05; Fig. 2); this association was not statistically significant for serum CTx.

View larger version (33K): [in this window] [in a new window]   Figure 2. Changes in vertebral BMD after 2.5 yr of alendronate therapy in groups by tertiles of the percent decrease in serum NTx (a) and serum CTx (b) at 6 months. Results are the mean ± SEM. P < 0.05 for serum NTx.

	Discussion

Top Abstract Introduction Experimental Subjects Materials and Methods Results Discussion References

There are currently several urinary biochemical markers available to assess bone turnover (16, 17). Although urinary NTx is very responsive in patients treated with antiresorptive therapy (1, 3, 4, 13), urinary NTx has a diurnal variation, resulting in significant variability in the measurement (18). Both serum NTx and CTx appear as responsive as urinary NTx, but serum NTx is less variable than serum CTx (see error bars in Fig. 1). Furthermore, while short-term changes in urinary and serum NTx and CTx were associated with long-term changes in spinal BMD after alendronate therapy, urinary NTx was associated with similar findings at the hip; serum NTx and CTx revealed only a trend.

The strengths of this study include the fact that both serum and urine were simultaneously assessed in a population of elderly, community-dwelling women with both osteopenia and osteoporosis. All subjects were assessed at one center with the same densitometer by one technician. However, there are several potential weaknesses. First, our data show associations with group trends; serum NTx and CTx may not prove as useful in assessing individual patients (19). In addition, these data were drawn from an elderly cohort, and therefore may not be applicable to perimenopausal women or men. Moreover, although serum NTx reached a nadir by 6 months with little further decrease after 1.5 yr, serum CTx had a continual decrease through the study, reaching a nadir at 2.5 yr. This suggests that the dose increase in alendronate from 5 to 10 mg at 1.5 yr may have impacted the outcome of serum CTx, but not serum NTx. Finally, the serum and urine collections were obtained while subjects were in a fasting state. It is not clear whether similar results would have been obtained if patients were not in a fasting state (19).

Although other urinary markers of bone resorption, such as deoxypyridinoline, have been associated with rates of bone loss (9, 11), therapeutic response (1, 3, 4, 13), and hip fracture prediction (12), serum products reflective of bone resorption have not been especially useful. For example, the collagen type 1 cross-linked C-telopeptide assay, a serum assay directed at the C-terminal telopeptide fragment of type 1 collagen, has demonstrated a poor response to hormone replacement therapy and bisphosphonates (20, 21, 22). However, serum CTx, an assay derived from the carboxyl-terminal telopeptide region of type 1 collagen -chain, is responsive to hormone replacement therapy (23). This serum resorption marker decreased approximately 75% in postmenopausal women treated with hormone replacement therapy, returning to a level seen in premenopausal women (16). Serum CTx remained stable in the placebo group.

These and other studies examine group trends and predictions (1, 3, 4, 13). However, clinicians use markers for information for an individual patient. We previously examined the minimum significant change in a biochemical marker of bone turnover necessary to determine whether a real change in the marker had occurred after therapy (19). Unfortunately, because of the variability coupled with the diurnal variation, markers may have limited clinical utility and require changes of 20–54% to be certain of therapeutic efficacy (19, 24). Similar assessment will be necessary for serum NTx and CTx to examine clinical utility for management in an individual patient.

In summary, serum NTx and CTx were highly correlated with urinary NTx in elderly postmenopausal women. Changes for baseline to 6 month levels of these variables were associated with long-term changes in spinal BMD in women treated with alendronate. Future studies are needed to determine whether changes in serum NTx and CTx after therapy are associated with fracture prediction and long-term bone density outcomes for individual patients.

	Acknowledgments

 
We are indebted to the nursing staff of the General Clinical Research Center at Beth Israel Deaconess Medical Center, and to Dawn Griffiths for excellent preparation of the manuscript. We also acknowledge Ostex International, Inc., Osteometer Biotech, and Merck Research Laboratories for their support of this study.

	Footnotes

 
¹ This work was supported by NIH Grant RR-01032 (to Harvard-Thorndike General Clinical Research Center, Beth Israel Deaconess Medical Center, Boston, MA) and Centers for Disease Control (Atlanta, GA) Grant CC102550.

Received April 19, 2000.

Revised June 23, 2000.

Accepted June 30, 2000.

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Top Abstract Introduction Experimental Subjects Materials and Methods Results Discussion References

 

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