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Technical Briefs |
1 Division of Endocrinology and Metabolism,2 General Clinical Research Center, and3 Department of Geriatrics, University of Connecticut Health Center, Farmington, CT;4 New Britain General Hospital, New Britain, CT
aauthor for correspondence: e-mail taxel{at}nso.uchc.edu
Bone is a dynamic tissue that is constantly undergoing remodeling or turnover, a requirement for maintenance of bone health. Our group and others have shown the usefulness of biochemical markers of bone turnover, specifically those that reflect bone resorption, in the evaluation of metabolic bone disease and the determination of response to treatment in the clinical as well as research setting (1)(2)(3).
Measurement of the degradation products of bone reflect the rate of bone turnover (2). Type 1 collagen, a triple helical molecule composed of two 
1 chains and one 2 chain with cross-linking at the N- and C-telopeptides, comprises 90% of the organic matrix of bone (3). Degradation products of this molecule have been the most useful markers of bone resorption. During resorption of bone, type 1 collagen is cleaved by proteinases in both the helical and telopeptide regions. Initially markers were based on measurement of breakdown products of the cross-linked telopeptide region. Recently, a peptide consisting of residues 620–633 derived from the helical region of the 1 chain of type 1 collagen was isolated from the urine of patients with Paget disease of bone, and a competitive enzyme immunoassay for this peptide was developed (4)(5). We compared the changes in this new marker with changes in existing urinary markers, including N-telopeptide collagen cross-links (NTx), C-telopeptide collagen cross-links (CTx), and free deoxypyridinoline (DPD), from three different intervention studies in older men and women with osteoporosis.
We studied participants in three separate osteoporosis studies, each approved by the Institutional Review Board. Informed consent was obtained from each volunteer. Exclusion criteria were diseases known to affect bone metabolism; use of estrogen, androgens, corticosteroids, heparin, anticonvulsants, vitamin D (other than multivitamin), or calcitonin currently or in the past year; past or present use of bisphosphonates or sodium fluoride; chronic medical conditions (kidney, gastrointestinal, or liver disease); and significant coronary disease or thromboembolic disorders.
Study 1 was a 9-week randomized, double-blind placebo-controlled trial of 1 mg/day micronized 17ß-estradiol (E2) vs placebo, in which 27 community-living men >60 years receiving treatment with luteinizing hormone-releasing hormone agonist therapy for prostate cancer (or neoadjuvant treatment before radiation or seed implantation) were enrolled. Men with bone metastases or prostate-specific antigen >20 µg/L were excluded. Twenty-five men completed the trial.
Study 2 was an open label study for which 45 community-dwelling women >70 years were recruited. Women with a history of breast or endometrial cancer, undiagnosed vaginal bleeding, or baseline endometrial thickness >5 mm were ineligible for the study. Thirty-one women were randomized to one of two treatment groups: (a) 12 weeks of 1500 mg/day elemental calcium (carbonate) with 800 IU/day vitamin D followed by 12 weeks of calcium plus vitamin D plus 0.5 mg/day micronized E2 (Estrace®; Bristol-Myers-Squibb) or (b) 12 weeks of E2 followed by 12 weeks of E2 plus calcium plus vitamin D. Fourteen additional women served as a control group. Baseline and 12-week values for helical peptide (HelP) were compared with other markers.
Study 3 was an open label study of community-dwelling postmenopausal women and men referred to an osteoporosis clinic. Participants were evaluated as part of the usual care for osteoporotic patients, and treatment was individualized for each patient with either 10 mg/day alendronate or 60 mg/day raloxifene. Participants collected three samples of fasting second-void morning urine for measurement of the three biochemical markers of bone resorption. The samples were collected at baseline and after 12 weeks of treatment.
Markers of bone resorption included NTx, CTx, DPD, and HelP. All markers were measured on fasting, second-void morning urine samples because this is the standard practice for the resorption markers NTx, CTx, and DPD (1) as well as for HelP (4)(5)(6).
NTx was measured in urine by a competitive inhibition ELISA (Ostex International). Assay values were corrected for creatinine. Intraassay imprecision (CV) reported by the manufacturer on 8 urine specimens (40 replicates) ranging from 26 to 2640 nmol/L bone-collagen equivalents (BCE) was 7.6%. The detection limit (reported by the manufacturer) was 20 nmol/L BCE. CTx was measured in the urine by ELISA (Osteometer A/S). The mean intraassay imprecision (CV) reported by the manufacturer on 25 replicates of 4 samples (240, 480, 1340, and 3790 µg/L) was 5.7%, 2.9%, 3.5%, and 5.4%, respectively, and the detection limit was reported to be 50 µg/L as determined from the mean minus 2 SD of the zero calibrator (n = 21). Neither assay requires the cross-link for antibody recognition. Urinary free DPD was measured by a competitive enzyme monoclonal immunoassay (Quidel Corp). Results are reported as the ratio to the urinary concentration of creatinine. Intraassay imprecision (CV) determined by the manufacturer on 21 replicates of 3 urine samples (10.7, 30, and 175 nmol/L) was 8.4%, 4.3%, and 5.5%, respectively. The detection limit, calculated by the manufacturer as the mean minus three SD for the zero calibrator, was 1.1 nmol/L.
HelP was measured by a competitive enzyme immunoassay (Quidel Corp) using a monoclonal anti-helical antibody coated on the strip to capture HelP. HelP in the sample competes with conjugated HelP–alkaline phosphatase for the antibody; bound labeled antigen is detected with p-nitrophenyl phosphate. Results are expressed as a ratio to the concentration of creatinine. The detection limit was 8 µg/L. The day-to-day variability for creatinine-corrected HelP has been measured as 28%, compared with 17% for DPD, 23% for NTx, and 24% for CTx (7).
Repeated-measures ANOVA models were constructed to examine changes in bone markers over time, by group, and time-by-group interactions. All of the participants in all three studies were assessed at baseline (time 1) and again after treatment (time 2 was 9 weeks for study 1 and 12 weeks for studies 2 and 3). Both the absolute change and the percentage change from baseline for each marker were examined. Pearson product-moment correlations were calculated to examine the associations between bone markers at baseline and posttreatment time points.
The mean (SE) intraassay CV for HelP obtained from duplicate analyses (n = 228) was 4.0 (0.3)%. The interassay CV of a low control (60 µg/L), obtained by use of duplicate samples in six different assays, was 7.7%, and the CV of a high control (311 µg/L) was 7.6%. Analysis of samples from individuals in the alendronate and raloxifene groups (total n = 12) on 3 consecutive days at both baseline and posttreatment time points gave mean (SD) interassay CV of 9.0 (1.4)% for NTx, 15 (2.1)% for CTx, 7.4 (1.0)% for DPD, and 12 (1.6)% for HelP.
Shown in Table 1
are the mean (SD) baseline values for the bone resorption markers for each of the groups in the three studies and the values for posttreatment markers, as well as the percentage changes from baseline in HelP, NTx, CTx, and free DPD for individuals on antiresorptive therapy, including E2, calcium and vitamin D, alendronate, and raloxifene. There were no significant differences in baseline values for resorption markers within the groups. The changes in posttreatment markers were significant on repeated-measures ANOVA in each of the studies by time, group, or time-by-group interactions, as shown on Table 1
. The percentage change from baseline in markers (shown in brackets in the posttreatment column) was significant for NTx, CTx, and HelP in the E2 group (study 1); for all markers in the calcium plus vitamin D group (study 2); for DPD, CTx, and HelP in the E2 group (study 2); and for NTx, CTx, and HelP in alendronate group (study 3).
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At baseline, there were significant correlations (P <0.01) between HelP and all three markers of resorption (NTx, CTx, and DPD); similar results were obtained at the posttreatment time points and for percentage changes (Table 1
in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue4/). Fig. 1
shows the absolute values of HelP vs the three markers of bone turnover for all time points in the combined three studies.
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This study suggests the potential usefulness of a novel urinary marker of bone resorption, HelP, a degradation product of the helicoidal region of type 1 collagen. The intra- and interassay CV (<4% and 8%, respectively) compare favorably to previous data on CTx, NTx, and free DPD (8)(9)(10). The assay was highly correlated with the established resorption markers urinary CTx and NTx and was equally sensitive in the detection of response to antiresorptive treatment in both men and women. We also found no difference in the relationship between markers for treated and control individuals.
The HelP has previously been shown to be a reliable index of bone resorption in mice during a rapid growth phase as well as in response to a bisphosphonate (11). Garnero and Delmas (6) have recently reported that HelP was highly correlated with urinary CTx in a cross-sectional analysis of 89 healthy and 59 postmenopausal women treated with either alendronate or estradiol. Hannon et al. (12) reported a decrease in HelP in response to risedronate that was similar to the change in urinary NTx, consistent with our own data. We also measured NTx and free DPD, as well as the percentage change with treatment in both men and women and found significant correlations.
The high correlations of HelP with CTx and NTx as well as the magnitude of the response to antiresorptive therapy were somewhat surprising because the HelP portion of the collagen molecule is identical in bone and nonbone collagen. Hence, degradation of skin and other collagens should affect HelP more than NTx and CTx. Our results suggest that nonbone collagen makes a relatively small contribution in individuals with high bone turnover; however, the importance of other sources must be tested in diseases affecting other connective tissues. Nevertheless, HelP could be a useful marker of bone turnover in patients with primary osteoporosis.
Acknowledgments
This study was supported by the General Clinical Research Center (NIH Grant M01 RRO6192) and by Donaghue Foundation (West Hartford, CT) Award DF99-072.
References
-1 chain. [Abstract]. J Bone Miner Res 1997;12(Suppl 1):S178.
-1 620–633 derived form type 1 collagen -1 chain [Abstract]. Bone 1998;23(Suppl):S629.[CrossRef]
1 helicoidal peptide 620–633, a new marker of bone resorption in osteoporosis. Bone 2003;32:20-26.[Medline]
[Order article via Infotrieve]
1 helical peptide as a marker of bone resorption in mice. J Bone Miner Res 2002;17:S315.
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represent untreated patients (placebo). The solid lines represent the linear regressions of the control groups, and the dashed lines represent the linear regressions of the treated groups. CR, creatinine.