Elsevier

Bone

Volume 26, Issue 5, May 2000, Pages 505-511
Bone

Original Articles
Serum CrossLaps for monitoring the response in individuals undergoing antiresorptive therapy

https://doi.org/10.1016/S8756-3282(00)00248-9Get rights and content

Abstract

The Serum CrossLaps (CTx) enzyme-linked immunosorbent assay (ELISA) is specific for a cross-linked, β-aspartate-isomerized form of the epitope EKAHDGGR derived from the carboxyterminal telopeptide region of type I collagen α1 chain. Collagen type I fragments reactive in the CTx assay are released during osteoclastic bone resorption and can be used as a measure of bone resorption activity. Our objectives were to assess the intraindividual variation of serum CTx concentration as well as the clinical value of the serum CTx assay for monitoring antiresorptive therapy in individual patients. The influence of the sampling time and fasting on the serum CTx measurements was studied with the aim of determining an optimal sampling protocol. Studies of circadian variation in serum CTx concentration in 15 postmenopausal women showed that fasting significantly reduced the average circadian variation of the marker from 36% to 8.7%. This was further supported by assessing short-term (2 weeks) intraindividual variation in ten postmenopausal women who were sampled in the morning, either fasting or nonfasting. The average short-term intraindividual coefficient of variation (CV) was 7.9% in the samples obtained from fasting women, and 14.3% in the samples obtained from nonfasting women. The long-term intraindividual biological variation was 13.4% in 44 postmenopausal women sampled every 6 months (fasting morning samples) over a 1 year period. The ability of the serum CTx assay to monitor individual responses to antiresorptive therapy was assessed in studies of the effects of hormone replacement therapy (HRT) and bisphosphonate (alendronate). Serum samples (morning fasting) were obtained from postmenopausal women treated with either bisphosphonate or HRT at baseline and then after various timepoints of therapy. Spine bone mineral density (BMD) measurements were carried out and the annual percentage change in spine BMD (αBMD) was calculated. Sixteen of 17 (94%) of the HRT-treated and 12 of 13 (92%) of the bisphosphonate-treated women showed a decrease in serum CTx after 6 months that was greater than the calculated least significant change (LSC) of the marker (LSCCTx). In contrast, only 59% of the HRT-treated and 64% of the bisphosphonate-treated women showed a response in spine BMD greater than the LSCBMD after 1 year follow-up. When the two study populations were combined, the decrease in serum CTx after 6 months showed an analytical sensitivity of 83.8% and specificity of 100% to differentiate women with a gain in spine BMD (αBMD > 0%) from women with a loss in spine BMD (αBMD < 0%). In conclusion, the serum CTx showed high specificity and sensitivity for monitoring individual responses to antiresorptive therapy. More than 92% of the treated women showed significant responses in serum CTx measurements after 6 months.

Introduction

Biochemical markers of bone resorption have shown considerable promise for prediction of the risk of osteoporosis and accurate assessment of the clinical progression of the disease.3, 4, 9, 16, 19 The Serum CrossLaps (CTx) assay measures collagen type I fragments derived from the C-terminal telopeptide region of the protein.25 The assay uses monoclonal antibodies specific for an isomerized form of the sequence EKAHD-β-GGR from the α1 chain of human collagen type I.10 Postmenopausal women or patients with Paget’s disease who are treated with antiresorptive therapy show a pronounced decrease in serum CTx concentration, indicating that the assay provides a clinically useful index of bone resorption.7, 22

An important issue for the use of biochemical markers of any physiological process is the intraindividual variation. This has been reported to be relatively high for specific markers of bone resorption,13, 14, 20 and has hampered their routine use for monitoring antiresorptive therapies. However, the intraindividual variation in marker level has to be considered in parallel with the magnitude of the changes observed in marker concentration as a result of antiresorptive therapy.4, 16 Thus, specific biochemical markers of bone resorption, such as the CTx assay, have, in several studies, shown a rapid and pronounced decrease in concentration in patients undergoing antiresorptive therapy of a magnitude larger than the intraindividual and analytical variation of the marker.7, 8, 22

In contrast to biochemical markers of bone resorption, bone mineral density (BMD) measurements are subject to low intraindividual variation. However, only small annual responses in BMD are seen when using antiresorptive therapy. In fact, the BMD response of individual patients over a 1 year treatment period is typically of the same magnitude as the precision error for BMD measurements: 1%–5%.12, 21, 23 Thus, follow-up periods of >1 year are necessary to monitor a statistically significant effect of antiresorptive therapy with BMD measurements.18 This represents a problem in relation to the average adherence to antiresorptive therapy in postmenopausal women. These women are generally asymptomatic, and they only reluctantly accept side effects or other concerns with regard to long-term medical intervention.5 Hence, methods for rapid evaluation of the response to antiresorptive therapy, as well as assessment of prognosis, will be of considerable value for its clinical use.16

In this study, we assess the intraindividual biological variation in serum CTx concentration in relation to response to antiresorptive therapy, which can be monitored in individual patients. First, we examine circadian variation and the optimal sampling protocol for obtaining serum samples for serum CTx measurements. Using this sampling protocol, the long-term intraindividual variation of the serum CTx level is determined and the least significant change (LSC) in marker level as well as for spine BMD measurements is calculated. Finally, we evaluate the individual responses in postmenopausal women treated with either HRT or bisphosphonate with the serum CTx enzyme-linked immunosorbent assay (ELISA), as well as with spine BMD measurements.

Section snippets

Study populations and trial design

The clinical trials were performed according to the Helsinki Declaration II and the European standard for good clinical practice after approval from the county research ethics committee of Copenhagen. All subjects provided written informed consent to participate.

Study of circadian variation was performed by sampling 15 postmenopausal women at 8:00, 9:00, 10:00, and 11:00 a.m. and then every 3 h throughout 24 h. Prior to the study, the women fasted overnight for 10 h. The women had an average

Circadian variation and influence of sampling time on biological variation in serum CTx concentration

Circadian variation was measured in 15 postmenopausal women subjected to a randomized cross-over study. In one part of the study, the women fasted overnight (10:00 p.m. to 8:00 a.m.), prior to initiation of the study, but were then allowed to drink and eat snacks and regular meals during the 24 h study period. In the other part of the study, the women fasted from 10 h before the study and then during 24 h throughout the study. Serum samples were obtained at 8:00, 9:00, 10:00, and 11:00 a.m. and

Discussion

A major impediment to the widespread use of biochemical markers of bone resorption has been the reported intraindividual variation in markers, which has raised questions about their applicability for monitoring the individual patient.13, 14 These studies were carried out with urinary markers of bone resorption such as d-pyridine and urine CTx. Recently, a newly developed assay for measurement of type I collagen degradation products in serum (Serum CTx ELISA) was introduced,7, 26 and recent

Acknowledgements

The authors thank Dr. Pernille Ravn from the Centre of Clinical and Basic Research for providing access to the clinical samples in the bisphosphonate study. The expert technical assistance of Marianne Helsberg, Marianne Littau, and Bodil Nielsen is gratefully acknowledged. S.C., O.B.J., E.G.H., P.Q., and D.B.H. are employees of Osteometer BioTech.

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