Clinical Chemistry 47:6 10831088 (2001)

Endocrinology and Metabolism

Biochemical Markers of Bone Turnover and Response of Bone Mineral Density to Intervention in Early Postmenopausal Women: An Experience in a Clinical Laboratory
La-or Chailurkit,* Boonsong Ongphiphadhanakul, Noppawan Piaseu, Sunee Saetung, and Rajata Rajatanavin

Background: Markers of bone formation and resorption may be useful as early indicators of response to therapy. Our aim in this study was to investigate the use of bone markers for monitoring of intervention for bone loss in early postmenopausal women and to assess the relationships between these markers and changes in bone mineral density (BMD). Methods: Subjects were randomly assigned to the following groups: a control group; a group receiving calcium alone; groups receiving calcium plus low or conventional doses of conjugated equine estrogen; and groups receiving calcium plus low or conventional doses of calcitriol. At baseline and at 1 and 3 months after intervention, we measured serum intact osteocalcin, serum N-terminal midfragment osteocalcin, serum C-terminal telopeptide of type I collagen (CTx), urinary deoxypyridinoline cross-links, and urinary CTx. The BMD of the lumbar spine and the femoral neck was measured at baseline and after 1 and 2 years of intervention. Results: No marker changed significantly in the control group except urinary CTx, which increased at 3 months. Serum CTx decreased in all regimens at 1 or 3 months of intervention. In addition, the changes of all markers at 3 months were inversely associated with the change in the BMD of the lumbar spine at 1 or 2 years (r 0.144 to 0.314), whereas only the changes of bone resorption markers at 3 months were inversely correlated with the

changes in femoral BMD at 1 or 2 years (r 0.143 to 0.366). Conclusions: Biochemical markers of bone turnover appear to be of use in assessing early response to therapy. Bone resorption markers, especially serum CTx, are better indicators than bone formation markers for estimating the response to intervention in early postmenopausal women. However, the early changes in bone markers were weakly related to the later changes in BMD.
2001 American Association for Clinical Chemistry

To reduce accelerated bone loss in early postmenopause (15 ) and to minimize the risk of fracture, a variety of antiresorptive treatments (e.g., calcium, calcitonin, estrogens, and bisphosphonates) have been used. A positive response to therapy can be assessed by the measurement of bone mineral density (BMD),1 but a statistically significant change usually requires 1 or more years because of the imprecision of the BMD measurement (6, 7 ). This limitation may be avoided by use of biochemical markers of bone turnover in the serum or urine, which can show the effects of intervention sooner. In addition, bone turnover markers provide a more representative indicator of overall skeletal bone loss than the results obtained by measuring rates of change in BMD at a single skeletal site. The ability to determine a positive response to therapy in advance of BMD facilitates the clinical decision to alter a patients regimen or dosage of medication to achieve the desired antiresorptive effect. Assays for several biochem-

Division of Endocrinology and Metabolism, Department of Medicine, Ramathibodi Hopital, Mahidol University, Rama VI Road, Bangkok 10400, Thailand. *Author for correspondence. Fax 66-2-201-1715; e-mail ralcl@mahidol.ac.th. Received November 20, 2000; accepted April 5, 2001.

1 Nonstandard abbreviations: BMD, bone mineral density; OC, osteocalcin; CTx, C-terminal telopeptide of type I collagen; DPD, deoxypyridinoline cross-links; N-mid OC, N-terminal midfragment osteocalcin; and Cr, creatinine.

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ical markers of bone turnover are commercially available, but their relative sensitivities are unclear. In this study, we investigated, in a clinical laboratory setting, biochemical markers of bone turnover for monitoring of several types of intervention for early postmenopausal bone loss and evaluated whether the responsiveness of biochemical markers is related to the response in BMD.

3.6%, 5.2%, 4.3%, and 3.4%, respectively, and the betweenassay CVs were 11%, 12%, 7.8%, 4.7%, and 4.1%.

bmd measurements
BMD was measured by dual-energy x-ray absorptiometry (Lunar DPX-L; Lunar Corp.). Daily calibration and quality control were performed regularly according to the manufacturers instructions. The BMD of the anteroposterior lumbar spine (L2L4) and the femoral neck was measured for all subjects at baseline and at 1 and 3 months of intervention. In vivo CVs for these sites were 1.2% and 1.6%, respectively.

Materials and Methods subjects

Subjects consisted of 214 healthy postmenopausal women (time after menopause 6 years). None was taking any medications that might interfere with bone metabolism. None had diseases known to affect skeletal turnover. The participants were randomly divided into six groups. Group 1 (n 46) included untreated controls. Group 2 (n 36) received 750 mg of calcium (calcium carbonate capsule) supplementation daily. Group 3 (n 34) received 750 mg of calcium plus 0.3 mg of conjugated equine estrogen (Wyeth-Ayerst) daily together with 5 mg of medrogestrone (Wyeth-Ayerst) 12 days per month. Group 4 (n 33) received the same treatment as group 3, except that the estrogen dose was increased to 0.625 mg daily. Group 5 (n 33) received 750 mg of calcium plus 0.25 g of calcitriol (Roche) supplement daily. Group 6 (n 32) received 750 mg of calcium plus 0.5 g of calcitriol supplement daily. The study was approved by the Ethical Clearance Committee on Human Rights related to research involving human subjects of the Faculty of Medicine, Ramathibodi Hospital, Mahidol University. Informed consent was obtained from all participants before a course of treatment was initiated.

statistical analyses
We analyzed the actual values and the percentages of change from baseline of the bone markers and BMD. Descriptive results are presented as the mean SE. The normality of results was assessed by the Kolmogorov Smirnov test. Differences between baseline and aftertreatment values were analyzed by repeated-measures ANOVA for parametric and the Friedman test for nonparametric variables. The Student paired t-test or the Wilcoxon rank-sum test was used to test the significance within a group if there was a time-related change within the group by repeated-measures ANOVA or the Friedman test. To correct for multiple testing, P was adjusted according to the Bonferroni correction. Associations between markers were identified by Spearman correlations. All analyses were performed using SPSS/PC (Release 9.0).

Results
In the untreated (control) postmenopausal women in this study, the mean values for all markers of bone remodeling at 1 or 3 months of treatment were not significantly different from baseline (Table 1). However, the mean value of urinary CTx was significantly increased at 3 months. In group 2, who received only calcium supplementation, the mean values of serum N-mid OC and CTx were significantly decreased at 3 months and at 1 and 3 months of treatment, respectively. In groups 3 and 4, who received low and traditional doses of estrogen, the mean values of all bone markers decreased consistently in response to treatment. Significant decreases were seen at 1 month for serum intact OC, urinary CTx, and serum CTx in group 3 and for urinary DPD, urinary CTx, and serum CTx in group 4. After 3 months of treatment, all markers in both groups except urinary DPD in group 3 were significantly decreased from baseline values. In groups 5 and 6, who received low and traditional doses of calcitriol, only urinary CTx and serum CTx were significantly decreased after 1 and 3 months of intervention. In addition, in group 6, a significant decrease was found at 3 months for serum N-mid OC. The mean BMD of either the lumbar spine or the femoral neck of the untreated (control) and the calciumtreated postmenopausal (group 2) groups was slightly

laboratory assays
Blood was collected between 0700 and 0930 after an overnight fast. Urine was collected over a 24-h period. Blood and urine samples were collected from all subjects before and 1 and 3 months after initial intervention. Serum and urine samples were frozen at 80 and 20 C, respectively, until analysis. Serum intact osteocalcin (OC; NovoCalcin; Metra Biosystem), urinary deoxypyridinoline cross-links (DPD; Pyrilinks-D; Metra Biosystem), and urinary C-terminal telopeptide fragment of type I collagen (CTx; -CrossLaps; Roche Diagnostics, Mannheim, Germany) were measured by competitive enzyme immunoassay. Serum N-terminal midfragment OC (Nmid OC; N-MIDTM Osteocalcin; Roche Diagnostics) was measured by sandwich enzyme immunoassay. Serum CTx (-CrossLaps; Roche Diagnostics) was determined by sandwich electrochemiluminescence immunoassay. All results for urinary markers were expressed relative to urinary creatinine (Cr). Blood and urine samples collected before and after intervention were analyzed in the same assay. The within-assay CVs for serum intact OC (mean concentration, 6.5 g/L), serum N-mid OC (16.0 g/L), urinary DPD (4.0 mol/mol of Cr), urinary CTx (5.5 mol/mol of Cr), and serum CTx (0.41 g/L) were 13%,

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Table 1. Responses of biochemical markers of bone turnover to treatments.

After treatmenta Baselinea 1 month 3 months

Controls Serum intact OC, g/L Serum N-mid OC, g/L Urinary DPD, mol/mol Cr Urinary CTx, mol/mol Cr Serum CTx, g/L Group 2 Serum intact OC, g/L Serum N-mid OC, g/L Urinary DPD, mol/mol Cr Urinary CTx, mol/mol Cr Serum CTx, g/L Group 3 Serum intact OC, g/L Serum N-mid OC, g/L Urinary DPD, mol/mol Cr Urinary CTx, mol/mol Cr Serum CTx, g/L Group 4 Serum intact OC, g/L Serum N-mid OC, g/L Urinary DPD, mol/mol Cr Urinary CTx, mol/mol Cr Serum CTx, g/L Group 5 Serum intact OC, g/L Serum N-mid OC, g/L Urinary DPD, mol/mol Cr Urinary CTx, mol/mol Cr Serum CTx, g/L Group 6 Serum intact OC, g/L Serum N-mid OC, g/L Urinary DPD, mol/mol Cr Urinary CTx, mol/mol Cr Serum CTx, g/L
a b

10.47 0.65 20.73 1.42 4.79 0.24 5.20 0.35 0.42 0.02 9.15 0.73 17.55 1.17 4.76 0.28 5.37 0.50 0.46 0.04 9.98 0.66 16.27 1.40 4.21 0.24 4.21 0.24 0.43 0.04 8.70 0.73 16.92 1.54 5.27 0.46 5.30 0.65 0.44 0.04 9.41 0.83 13.97 0.92 4.50 0.32 4.73 0.53 0.37 0.04 9.47 0.64 17.81 1.63 4.22 0.29 4.75 0.41 0.39 0.03

10.36 0.58 20.94 1.46 4.74 0.33 5.43 0.43 0.43 0.03 8.45 0.63 17.47 1.31 4.02 0.23 4.70 0.59 0.37 0.03b 9.01 0.71b 14.87 1.27 3.72 0.30 3.72 0.30b 0.24 0.03b 7.18 0.82 16.04 1.48 3.71 0.29b 2.61 0.27b 0.24 0.03b 9.23 0.53 14.52 0.84 3.77 0.30 3.47 0.39b 0.31 0.04b 9.13 0.54 16.59 1.46 4.16 0.30 3.5 0.32b 0.34 0.04b

10.82 0.61 20.79 1.49 5.40 0.31 6.51 0.60b 0.45 0.04 9.18 0.61 15.71 1.05b 4.47 0.35 4.39 0.40 0.36 0.03b 7.66 0.60b 11.80 0.97b 3.83 0.32 3.83 0.32b 0.18 0.02b 6.47 0.53b 11.68 1.04b 4.16 0.37b 2.53 0.37b 0.21 0.03b 9.34 0.73 12.99 0.88 4.27 0.41 3.11 0.33b 0.31 0.04b 9.22 0.57 14.66 1.30b 4.54 0.28 3.84 0.40b 0.32 0.04b

Values are the mean SE. Significantly different from baseline: P 0.0167.

decreased at 1 or 2 years compared with baseline values (Table 2). In contrast, in the estrogen-treated postmenopausal groups (groups 3 and 4), the mean BMD of the both the lumbar spine and the femoral neck was increased after 1 or 2 years of intervention, but a significant increase in the mean BMD from baseline was observed only at the lumbar spine after 1 year of intervention for group 3 and after 2 years of intervention for group 4. In the calcitrioltreated groups (group 5 and 6), there were no significant changes of BMD in either the lumbar spine or the femoral neck after intervention compared with baseline. Associations between the percentages of change in bone markers and the percentages of change in BMD after intervention are shown in Table 3. At 1 month, there was no correlation between serum intact OC, N-mid OC, or

urinary DPD and the long-term BMD change in both the anteroposterior lumbar spine and the femoral neck. The change in urinary CTx at 1 month was inversely correlated only with the change in femoral BMD at 1 year, and the change in serum CTx at 1 month was inversely correlated with the lumbar spine BMD at 1 or 2 years and femoral BMD at 1 year. At 3 months, for the changes of bone formation markers, serum intact OC and N-mid OC were inversely correlated with the changes in the BMD of the lumbar spine at 1 or 2 years. Only the change in N-mid OC was related to the change in femoral BMD only at 1 year. With regard to bone resorption markers, the changes of urinary DPD, urinary CTx, and serum CTx at 3 months were inversely correlated with the BMD of the lumbar spine or femoral neck at both 1 and 2 years.

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Table 2. Responses of BMD (g/cm2) to treatments.

After treatmenta Baselinea 1 year 2 years

Control Anteroposterior Femoral neck Group 2 Anteroposterior Femoral neck Group 3 Anteroposterior Femoral neck Group 4 Anteroposterior Femoral neck Group 5 Anteroposterior Femoral neck Group 6 Anteroposterior Femoral neck
a b

spine

0.9908 0.027 0.8361 0.023 1.0579 0.043 0.8316 0.032 1.0020 0.041 0.8331 0.031 1.0416 0.028 0.8754 0.024 1.0231 0.044 0.8371 0.033 1.0518 0.030 0.8339 0.022

0.9639 0.037 0.8040 0.025b 1.0553 0.042 0.8186 0.032 1.0665 0.028b 0.8689 0.018 1.0377 0.057 0.8120 0.044 1.0470 0.044 0.8347 0.033 1.0670 0.031 0.8381 0.022

0.9429 0.037 0.8047 0.026b 1.0432 0.049b 0.8212 0.035 1.0399 0.032 0.8627 0.021 1.0947 0.035b 0.8807 0.026 1.0248 0.044 0.8361 0.032 1.0535 0.027 0.8356 0.022

spine

spine

spine

spine

spine

Values are the mean SE. Significantly different from baseline: P 0.0167.

Discussion
In this study, we measured five different commercially available biochemical markers of bone turnover. Two of the markers were bone formation markers (serum intact OC and serum N-mid OC), and the others were bone resorption markers (urinary DPD, and urinary and serum CTx). Our results showed that the bone resorption markers were more sensitive than the bone formation markers in response to different modalities of intervention. This might be attributable to a more rapid decrease in the bone resorption markers than in the formation markers as a result of bone remodeling during the early postmenopausal period (8 10 ). Among the bone resorption markers, serum CTx was

the most sensitive; it showed a positive response to all regimens at 1 or 3 months of intervention. Urinary CTx was comparable to serum CTx but slightly less sensitive, whereas urinary DPD was the least sensitive marker. The lower sensitivities of urinary DPD and urinary CTx might be attributable to an inherent biologic variability (8, 11 ). A much lower variability and greater changes with antiresorptive therapy were seen in the serum measurements (12, 13 ). In addition, serum CTx has been reported to be more specific for bone resorption than other measurements (12 ). Fall et al. (14 ) also found that the responses of urinary and serum N-terminal telopeptides of type I collagen in postmenopausal women receiving hormone replacement therapy were comparable to those of serum

Table 3. Correlation between biochemical markers of bone turnover and BMD.

% change from baseline of biochemical marker of bone turnover Marker Month 1 year % change from baseline of BMD Anteroposterior spine 2 years 1 year Femoral neck 2 years

Intact OC N-mid OC DPD Urinary CTx Serum CTx

a b

1 3 1 3 1 3 1 3 1 3

r 0.025 r 0.178a r 0.006 r 0.198b r 0.015 r 0.205b r 0.134 r 0.254b r 0.294b r 0.314b

r 0.044 r 0.145a r 0.035 r 0.184a r 0.003 r 0.144a r 0.080 r 0.281b r 0.212b r 0.306b

r 0.076 r 0.109 r 0.042 r 0.167a r 0.062 r 0.247b r 0.151a r 0.366b r 0.157a r 0.191b

r 0.010 r 0.131 r 0.031 r 0.066 r 0.003 r 0.162a r 0.106 r 0.264b r 0.110 r 0.143a

P 0.05 (two-tailed). P 0.01 (two-tailed).

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and urinary CTx. Therefore, bone resorption markers might be preferable for follow-up of antiresorptive interventions. Regarding markers of bone formation, the performance of both intact OC and N-mid OC (intact OC and Nterminal fragments) in response to intervention in this study was similar. However, previous data have shown differences in the performance of different assays used in different laboratories. This might be attributable to OC consisting of multiple fragments and the presence of immunologic heterogeneity (1517 ). In addition, at room temperature a fraction of serum intact OC is rapidly cleaved into smaller fragments. The large N-terminal midfragment is the main breakdown product (18 ). Measuring both the intact molecule and the N-terminal midfragment in a single assay has been reported to give more robust and sensitive results (19, 20 ). Therefore, this marker requires accurate control of both temperature and length of storage of samples. In the present study, our serum samples were stored at 80 C immediately after separation of the whole blood and had never been thawed until assayed. This may explain why our two different OC assays showed quite similar responses. Among the five different regimens for prevention of early postmenopausal bone loss, estrogen replacement was shown to be the most effective modality. Almost all biochemical markers decreased after 1 or 3 months of estrogen replacement. Moreover, BMD in the estrogentreated postmenopausal groups was slightly increased after 1 and 2 years of intervention. Estrogen is known to be a potent inhibitor of osteoclastic activity (21 ) and to reduce the rate of bone turnover (22, 23 ). Additionally, the lower dose of estrogen appeared to have the same effect as the traditional dose in decreasing bone remodeling. This finding was in accordance with other studies demonstrating that in early postmenopausal women, a lower dose of estrogen was sufficient to maintain bone mass compared with the traditional dose after 1 or 2 years of treatment (24 26 ). It is also noteworthy that the antiresorptive effect of treatment, as assessed by the changes in bone turnover markers after 3 months of therapy, could be used to predict the subsequent change in BMD (6, 12, 27 ). Our study indicated that the early changes in the bone markers in this study were weakly related to the later changes in BMD. However, bone resorption markers were better indicators of BMD change than bone formation markers. Because antiresorptive agents have a direct inhibitory effect on bone resorption and a resulting indirect effect on bone formation, the change in bone formation markers might lag behind the change in bone resorption markers in predicting the change in BMD. Moreover, bone resorption markers probably reflect the overall contribution of bone turnover throughout the whole skeleton because the changes of bone resorption markers at 3 months could predict skeletal responsiveness in both the spine and femur in the first year of treatment. In addition, serum

CTx was found to be a better indicator than other markers, providing an earlier indication of response than other markers. The change in serum CTx at 1 month could predict the long-term change in bone density after intervention. The changes in bone formation markers, however, were insufficient to demonstrate a significant response in hip BMD. This result was in accordance with the result reported previously by Cosman et al. (28 ), who hypothesized that this lack of a significant response in hip BMD might be attributable to the higher cancellous bone content in the spine than in the hip. A greater proportion of cellular product (OC, alkaline phosphatase, and tartrate-resistant acid phosphatase) from the total active cell population might be liberated into the circulation from the spine than from the hip, which could account for the better relationships between these variables and spinal bone turnover vs hip bone turnover. In conclusion, biochemical markers of bone turnover appear to be of use in assessing early response to therapy. Bone resorption markers, especially serum CTx, are better indicators than bone formation markers for estimating the response to intervention in early postmenopausal women. Moreover, in this clinical laboratory setting, changes in serum CTx were a better indicator of the long-term changes in BMD after therapeutic intervention than were other markers.

This work was supported by the Thailand Research Fund. We thank Roche Diagnostics for kindly providing the serum N-mid OC and urinary and serum CTx immunoassays for this study.

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