The amino- and carboxyterminal cross-linked telopeptides of collagen type I, NTX-I and CTX-I: A comparative review

Clinica Chimica Acta 393 (2008) 57–75

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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

Invited critical review

The amino- and carboxyterminal cross-linked telopeptides of collagen type I, NTX-I and CTX-I: A comparative review Markus Herrmann, Markus Seibel ⁎ ANZAC Research Institute, University of Sydney, Sydney NSW, Australia

A R T I C L E

I N F O

Article history: Received 30 January 2008 Received in revised form 13 March 2008 Accepted 18 March 2008 Available online 27 March 2008 Keywords: CTX-I NTX-I Bone resorption markers

A B S T R A C T Bone diseases such as osteoporosis or bone metastases are a continuously growing problem in the ageing populations across the world. In recent years, great efforts have been made to develop specific and sensitive biochemical markers of bone turnover that could help in the assessment and monitoring of bone turnover. The amino- and carboxyterminal cross-linked telopeptides of type I collagen (NTX-I and CTX-I, respectively) are two widely used bone resorption markers that attracted great attention due to their relatively high sensitivity and specificity for the degradation of type I collagen, and their rapid adaptation to automated analyzers. However, the clinical performance of both markers differs significantly depending on the clinical situation. These differences have caused considerable confusion and uncertainty. If used correctly, both markers have great potential to improve the management of many bone diseases. We here review the biochemistry, analytical background and clinical performance of NTX-I and CTX-I, as documented in the accessible literature until March 2008. © 2008 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemistry of aminoterminal cross-linked telopeptides of type I collagen (NTX-1) collagen (CTX-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analytical background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Cross-linked N-terminal telopeptide of type I collagen (NTX-1) in urine . . . 3.2. Cross-linked N-terminal telopeptide of type I collagen (NTX-I) in serum . . . . 3.3. Cross-linked C-terminal telopeptide of type I collagen (CTX-I) in urine . . . 3.4. C-terminal telopeptide of type I collagen (CTX-I) in serum . . . . . . . . . 3.5. Comparison of CTX-I and NTX-I in urine and serum . . . . . . . . . . . . Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Technical sources of variability . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Thermodegradation . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Diurnal variation . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4. Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Biological sources of variability. . . . . . . . . . . . . . . . . . . . . . 4.2.1. Effects of age and changes in sex hormone levels . . . . . . . . . 4.2.2. Intraindividual variation. . . . . . . . . . . . . . . . . . . . . 4.2.3. Seasonal variation . . . . . . . . . . . . . . . . . . . . . . . 4.2.4. Other biological sources of variability . . . . . . . . . . . . . . 4.3. How to deal with variability? . . . . . . . . . . . . . . . . . . . . . . 4.4. Reference values in serum and urine . . . . . . . . . . . . . . . . . . . Clinical use of CTX-I and NTX-I . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Menopause . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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. . . . . . . . carboxyterminal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . cross-linked . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . telopeptides of type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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58 58 58 58 59 61 61 61 61 61 61 62 62 62 62 63 63 63 63 64 65 65

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⁎ Corresponding author. ANZAC Research Institute, The University of Sydney, Concord Campus, Concord, NSW 2139, Australia. Tel.: +61 2 9767 5000; fax: +61 2 9767 7472. E-mail address: [email protected] (M. Seibel). Abbreviations: BMD, bone mineral density; CTX-I, carboxyterminal cross-linked telopeptides of type I collagen; HRT, hormone replacement therapy; ICTP, carboxyterminal crosslinked telopeptides of type I collagen; NTX-I, aminoterminal cross-linked telopeptides of type I collagen. 0009-8981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2008.03.020

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5.2. Osteoporosis. . . . . . . . . . . . . . . . . . . . . . 5.3. Bone turnover and bone loss . . . . . . . . . . . . . . 5.4. Bone turnover and fracture risk. . . . . . . . . . . . . 5.5. Pre-treatment bone turnover and therapeutic effect . . . 5.6. Bone turnover markers and therapeutic monitoring . . . 5.7. Monitoring patient compliance using bone markers . . . 5.8. Comparison of CTX-I and NTX-I in hemodialysis patients . 5.9. Paget’s disease. . . . . . . . . . . . . . . . . . . . . 5.10. Cancer and bone metastasis . . . . . . . . . . . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Bone diseases such as osteoporosis or bone metastases are a continuously growing problem in the ageing populations across the world. For example, osteoporosis represents one of the most common agerelated diseases worldwide, affecting about 75 million people in Europe, the USA and Japan alone [1,2]. At present, the diagnosis of osteoporosis is mainly based upon a history of a previous fragility fracture and the measurement of bone mineral density (BMD) [3,4]. However, the onset of most bone diseases precedes measurable changes in BMD, or the occurrence of fractures by years if not decades. Beside BMD, other factors, such as bone remodelling, are major determinants of bone strength [5–10]. In recent years, great efforts have been made to develop specific and sensitive biochemical markers of bone turnover that could help in the assessment and monitoring of bone turnover. At present, there are more than 10 different bone turnover markers commercially available. The three major macro-molecular products of collagen degradation, namely the aminoterminal (NTX) and the carboxyterminal (CTX-I, ICTP) cross-linked telopeptides of type I collagen can be measured by specific immunoassays, some of which have been adapted to automated analyzers. While the assay for ICTP in serum was the first of the telopeptide markers to be developed, the assays for NTX-I and CTX-I have become the most commonly used methods to measure bone resorption rates. This review will therefore concentrate on NTX-I and CTX-I. While both these markers may be measured in serum and urine, their clinical performance differs significantly depending on the clinical situation [11–13]. These differences have caused considerable confusion and uncertainty. However, if used correctly, both markers have great potential to improve the management of many bone diseases. We here review the biochemistry, analytical background and clinical performance of NTX-I and CTX-I, as documented in the accessible literature until November 2007. 2. Biochemistry of aminoterminal cross-linked telopeptides of type I collagen (NTX-1) and carboxyterminal cross-linked telopeptides of type I collagen (CTX-1) NTX-I and CTX-I are degradation products of type I collagen (Fig. 1). Collagen type I is the major organic component of the extracellular matrix and is present as a triple helix. The cross-links covalently link individual collagen molecules within the triple helix. The main molecular sites involved in collagen cross-linking are the short non-helical peptides at both ends of the collagen molecule, termed amino- (N) and carboxy- (C) terminal telopeptides. In normal collagen, these telopeptides are each linked via pyridinium or pyrrole compounds to the helical portion of neighbouring collagen molecules (Fig. 1) [14–17]. During collagen breakdown, N- and C-terminal telopeptide fragments of various sizes, still attached to the helical portions of a nearby molecule by a pyridinium or pyrrole cross-link, are released into the circulation. NTX-I and CTX-I are small enough to be readily cleared by the kidneys. Hence, they can be measured in both serum and urine. Of note, NTX-I and CTX-I are also present in tissues other than bone and non-

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skeletal processes may therefore influence their circulating or urinary levels [18–23]. Consequently, both markers are not disease specific but reflect, as an integral measure, alterations in bone metabolism independently of the underlying cause. Hence, results of CTX-I and NTX-I measurement should always be interpreted against the background of their basic science and the clinical picture. 3. Analytical background 3.1. Cross-linked N-terminal telopeptide of type I collagen (NTX-1) in urine The NTX-I urine assay is based on a monoclonal antibody that specifically recognizes an epitope embedded in the α2-chain of the Ntelopeptide fragment. The peptide has the sequence QYDGKGVG, where K is involved in a trivalent cross-linking site. The compound still contains the pyridinium cross-link, but the antibody does not recognize the pyridinoline or deoxypyridinoline per se [24]. Collagen must be broken down to small cross-linked peptides that contain the exact sequence before antibody binding occurs with the NTX-I antigen. The antibody recognizes peptides in culture medium conditioned by osteoclasts resorbing human bone particles [25,26]. These data suggest that the NTX-I peptide is a direct product of osteoclastic proteolysis and appears not to be metabolized further [24,25]. However, cross-reaction of the antibody with peptides derived from skin has also been reported [15,18]. The NTX-I assay is calibrated using standard amounts of human bone collagen digested with bacterial collagenases, or a synthetic sequence of the NTX-I epitope fs™, Ostex International, Seattle, WA) [27]. Generally, measurement is performed in second morning spot urine. Results are reported as bone collagen equivalents (BCE), in nM, corrected for creatinine excretion to compensate for differences in urine dilution. For information regarding sensitivity, intra- and interassay coefficients of variation see Table 1. As the α2-chain of the N-terminal telopeptide of the collagen I molecule contains an Asp-Gly bond, isomerization of the α2-chain may occur [28,29]. Preliminary data indicate that the α-NTX-I represents native or newly formed type I collagen, while the isomerized form, βNTX-I, appears to correspond to older collagen molecules. A lower β to α-peptide ratio was observed in the urine of growing children, indicative of a higher rate of bone metabolism allowing less time for the isomerization to occur [28]. No significant differences were found between postmenopausal healthy and osteoporotic women. However, the currently available NTX-I assays do not account for isomerization and its clinical relevance needs further exploration. 3.2. Cross-linked N-terminal telopeptide of type I collagen (NTX-I) in serum An assay for the measurement of NTX-I in serum had been developed in the late 1990s [30]. Experimental and clinical data demonstrated this assay to provide a useful index of bone resorption, however, mostly for technical reasons, such as assay stability and variability, the serum assay has not become as widely used as the assay for NTX-I in urine [12,27,31–38].

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59

Fig. 1. Molecular basis of currently used markers of type I collagen-related degradation. For more details, see text and Table 1 [29].

3.3. Cross-linked C-terminal telopeptide of type I collagen (CTX-I) in urine There are several assays currently available for the measurement of CTX-I in urine (Table 1). Most of the antibodies used in these assays bind to a region of the C-terminal α1-chain telopeptide, where a lysine

and an aspartyl are present. While the lysine participates in intermolecular cross-linking, the aspartyl is subject to isomerization and racemazation. It can be found in four different forms: αL-, βL-, βD- and αD-aspartyl (Fig. 2). In newly formed bone the αL-isomer is predominant. In a recent in vitro study, Garnero et al. showed that bone

Table 1 Characteristics of NTX-I and CTX-I assays Analyte

Product name

Manufacturer

Material

Isoform

Automation

Detection limit

Interassay-CV (%)

Intraassay-CV (%)

Type of assay

CTX-I

β-CrossLaps Serum CrossLaps Urine CrossLaps Urine Beta CrossLaps ALPHA CTX ELISA ALPHA CTX RIA Osteomark NTX ELISA serum Osteomark NTX ELISA urin Osteomark NTX POC

Roche Diagnostics Nordic Bioscience Nordic Bioscience Nordic Bioscience Nordic Bioscience No longer available Ostex Inc. Ostex Inc. No longer available

Serum Serum Urine Urine Urine Urine Serum Urine Urine

ββ ββ ββ and βα ββ αα αα and αβ ? ? ?

Yes No No No No No No No No

2.7 pg/ml 0.02 ng/ml 50 ng/ml 0.80 ng/ml 0.80 ng/ml 0.04 mg/L 3.2 nM BCE 20 nM BCE 30 nM BCE

2.6 10.9 5.7 6.9 9.4 b5 4.6 4.6 10.9

4.1 3 9.4 3.9 2.3 7 6.9 6.9 ?

CLIA ELISA ELISA ELISA ELISA RIA ELISA ELISA CLIA

NTX

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Fig. 2. β-isomerization and racemization of the C-terminal telopeptide of collagen type I [29].

M. Herrmann, M. Seibel / Clinica Chimica Acta 393 (2008) 57–75

ageing is associated with increased isomerization of type I collagen and reduced biomechanical properties [39]. Measurement of the relative amounts of newly synthesized αL-CTX-I and age-modified β-CTX-I has been suggested to provide information about the age of resorbed bone and the activity (frequency) of bone turnover [40–44]. The first α-CTX-I and β-CTX-I assays only required one CTX-I chain to react, however the lysine residue within the CTX-I epitope can participate in inter- and intra-molecular covalent cross-links joining two epitopes [40]. Hence, the α-CTX-I assay measured αα- and αβ-CTX-I and the β-CTX-I assay detected ββ-CTX-I and βα-CTX-I fragments [45]. Modern assays are able to measure exclusively αα- and ββ-CTX-I (Table 1). It is important to make a clear distinction between the bone turnover rate as measured by α- or β-CTX-I and collagen maturation as assessed by the α/β-CTX-ratio. The differential use of these assays has been suggested to provide additional information on the agedependent changes of collagen in health and disease [18], although the clinical relevance of this approach has yet to be shown. 3.4. C-terminal telopeptide of type I collagen (CTX-I) in serum An initial competitive ELISA for the measurement of β-CTX-I in serum had been developed about a decade ago [46]. The polyclonal antibody employed in this assay binds to the β-isomerized C-terminal octapeptide EKAHD-β-GGR. The newer version of the serum CTX-I assay uses two monoclonal antibodies which recognize di-peptides containing a cross-link and two β-isomerized peptides with the same sequence as shown for the urinary assay (ββ-CTX-I). Assays analyzing other isoforms of CTX-I in serum are not available at the moment. The manufacturers' claim that in certain clinical settings, such as the prediction of fracture risk or the diagnosis of bone metastases, measurement of both α and β-CTX-I in urine, and hence the resulting α/β-CTX-I ratio performs better than a single measurement of serum ββ-CTX-I. At the moment no head-to-head comparisons between serum ββ-CTX-I and the urinary α/β-CTX-I ratio are available. However, a comparison of different longitudinal studies where either serum ββ-CTX-I or the α/ β-CTX-I ratio was used to assess osteoporotic fracture risk yields rather comparable predictive values [47,48]. Hence, based on the existing data, there is no evidence that the ratio of urinary α/β-CTX-I performs differently from serum ββ-CTX-I. 3.5. Comparison of CTX-I and NTX-I in urine and serum Any urine measurement has the disadvantage that results have to be corrected for creatinine excretion to adjust for the effect of urine concentration or dilution. This implies the measurement of a second analyte with all its analytical and physiological shortcomings. Therefore, measurement of CTX-I or NTX-I in serum may improve the practicability, variability and, possibly, the sensitivity of these markers. For example, a head-to-head comparison of serum vs. urine CTX-I or NTX-I in postmenopausal women showed a reduction in the marker-specific variability by 20–30%; this effect was independent of the use of hormone replacement therapy (HRT) [12]. However, in men of comparable age, the variabilities of NTX-I and CTX-I did not differ between urinary and serum analyses. Compared to serum NTX-I, the variability of serum CTX-I was approximately 50% higher in postmenopausal women (independently of HRT use) but equivalent in men. The correlation between urinary and serum measurements of bone markers is an important and partly unresolved issue. In the head-tohead comparison by Fall et al., serum CTX-I and NTX-I correlated well with the analyses in urine (Table 2) [12]. However, considering other studies the correlation coefficient differs over a wide range (r = 0.5–0.9) [49–51]. It should be noted that a marker with a lower variability is not necessarily “better” or “worse” than a marker with a higher variability. What matters in the clinical setting is the signal-to-noise ratio, i.e. the ratio between the true change (“signal”) and the non-specific variation

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Table 2 Correlation coefficients between serum and urine telopeptide markers [12]

uCTX sNTX uNTX

sCTX

uCTX

sNTX

0.87 0.80 0.66

– 0.77 0.65

– 0.63

Log-transformed data were used for analysis. All r values are significant.

(“noise”) of a given analyte (see below; variability). Hence, a marker with a higher variability may be clinically as useful as a marker with a lower variability if the signal-to-noise ratios of both markers are similar. For example, in a study of untreated postmenopausal women (mean age: 63 years), urinary levels of CTX-I and NTX-I correlated slightly better with BMD than the respective concentrations in serum (Table 3); correlation coefficients between CTX-I and NTX-I were similar [12]. Of note, no significant correlations between BMD and serum NTX-I or serum CTX-I were found in healthy men of the same age. This head-to-head comparison of NTX-I and CTX-I therefore found no relevant differences between the two markers, independent of whether they were measured in serum or urine. 4. Variability Both CTX-I and NTX-I exhibit significant within-subject variability. Therefore, knowledge of the sources of variability and the strategies used to cope with “background noise” are essential for the meaningful interpretation of bone markers. 4.1. Technical sources of variability In addition to parameters of assay performance, factors such as the choice of sample (i.e. serum vs. urine), mode of sample collection (e.g. 24-hour collection vs. first or second morning void), the appropriate preparation of the patient (e.g. pre-sampling diet/fasting/exercise before phlebotomy), the correct handling, processing and storage of specimens are all important as these technical sources of variability are controllable and hence modifiable. 4.1.1. Thermodegradation Serum β-CTX-I levels have been shown to decrease by up to 30% if kept at room temperature for more than 1 day. At 4 °C, serum β-CTX-I levels are stable for up to 5 days [52,53]. No comparable studies are available for serum NTX-I. Urinary levels of CTX-I and NTX-I are stable at room temperature for at least 3 days [54]. At −20 °C, the epitope quantified by the NTX-I assay denatures significantly within 4 months. However, this change is usually masked by the simultaneous decrease in creatinine [55]. However, several freeze–thaw cycles of urine samples appeared to have no effect on the concentrations of urinary CTX-I and NTX-I [55]. 4.1.2. Diurnal variation It is long known that both serum and urine levels of CTX-I and NTX-I are subject to significant diurnal variation, with highest values in the early morning hours and lowest values during the afternoon and evening [56]. Most studies report daily amplitudes of 20–30% (Table 4) [57–61], although the most pronounced diurnal changes (up to 90%) have been communicated for CTX-I [62]. It should be borne in mind that the slope of diurnal changes is steepest during the morning hours, which is usually the time at which urine or blood samples are collected (Fig. 3). The etiology of diurnal variations is unknown. Several hormones, such as parathyroid hormone, growth hormone, or cortisol show diurnal changes and may therefore be involved [63,64]. Measurement of CTX-I and NTX-I in urine is usually performed either in first or second morning voids, or in 2 h collections. In each

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Table 3 Correlation coefficients between NTX-I/CTX-I and BMD in postmenopausal women without HRT [12]

BMD lumbar spine BMD femoral neck BMD total body

sCTX

uCTX

sNTX

uNTX

−0.27 −0.23 −0.29*

− 0.36* − 0.30* − 0.34*

−0.23 −0.26 −0.34*

−0.30* −0.27 −0.38*

*p b 0.05.

case, values need to be corrected for urinary creatinine, which introduces additional pre-analytical and analytical variability. Creatinine output has been reported to be fairly constant with time (variations within 10%) but to correlate with lean body mass [65]. However, other reports suggest that the correction for creatinine in a urine spot sample could be misleading because the creatinine excretion rate has been found to show a considerable diurnal variation [66]. Serum markers usually show less pronounced changes during the day than urine based indices and therefore can avoid most of these limitations. In any case, controlling the timing of sample collection is a “bare necessity” for all types of markers. 4.1.3. Nutrition A single meal within 60–120 min before blood collection can reduce serum CTX-I levels by up to 50% (Fig. 4) [67–69]. The postprandial reduction of bone resorption is probably mediated by the intestinal release of glucagon-like peptide 2 [68,69]. No comparable studies have been published for urinary or serum NTX-I levels. In addition to the above, the composition of the diet over a longer period of time also affects bone resorption [70–75]. Diet changes that increase net acid excretion by the kidneys are associated with increases in urinary NTX-I [76,77]. Conversely, milk basic protein has been shown to reduce urinary NTX-I levels [70,71]. Interestingly, the intake of omega-3 fatty acids also appears to affect serum NTX-I concentrations [73]. Consequently, blood and urine sampling for CTX-I and NTX-I quantification should be done in a fasting state and, ideally, the patient's diet should remain stable in the weeks before sample collection. 4.1.4. Exercise Urinary NTX-I levels have been found to be reduced after a single session of whole-body vibration exercise [78]. In contrast, serum CTX-I does not seem to change consistently in response to acute exercise [79–81]. 4.2. Biological sources of variability Biological causes of variability are much harder to control then the technical aspects of variability. While many biological factors cannot be modified at all (e.g. age, gender, ethnicity etc.), every effort should be made to account for these factors when interpreting the results of bone marker measurements. 4.2.1. Effects of age and changes in sex hormone levels During early childhood and then again during the pubertal growth spurt, CTX-I and NTX-I levels in urine and serum are significantly higher than during adulthood [82–84]. After birth, concentrations of

Fig. 3. Circadian rhythm of urine NTX-I and serum CTX-I [236].

both markers increase attaining their peak at approximately 3 months. Throughout the childhood, urinary CTX-I and NTX-I levels remain constant or decrease. Values are similar in age matched pre-pubertal boys and girls. During the growth spurt, both CTX-I and NTX-I concentrations increase (Fig. 5). In girls, peak levels are observed two years earlier than in boys. In men between 20 and 30 years of age, values of most bone turnover markers are usually higher than in women of comparable age. After the age of 50, most bone turnover markers tend to increase with further ageing (Fig. 6), but less in men than in women. In the latter, the age-related increase in bone turnover is more pronounced due to the menopause [85,86]. Menopause is associated with a substantial acceleration in bone turnover, which is mirrored by a 50–100% increase in NTX-I and CTX-I levels (Table 5) [51,57,87–96]. A prospective study covering the perimenopausal transition in healthy women suggested that changes in bone turnover commence during late premenopause with a decrease in bone formation, which only later is followed by a rise in bone resorption [97]. Serum and urine NTX-I and CTX-I concentrations continue to be increased during late menopause [98]. In men, the pattern of age-dependent change in bone markers is quite different from that observed in women. Both NTX-I and CTX-I

Table 4 Variability of NTX-I and CTX-I in urine and serum Premenopausal women

Postmenopausal women

Men

Parameter

Intra-day

Day-to-day (%)

Seasonal

Intra-day (%)

Day-to-day (%)

Seasonal

Intra-day

Day-to-day

Seasonal

uNTX-I uCTX-I sCTX-I sNTX-I

23 24–54% 60–80% –

20 26 13 11

0–50% – No change –

31 24 22–90 25

15–25 24–48 8–13 6–12

0–44% – 21% –

24–40% 23% 75% –

15–19% 23% 15% –

0–50% – No change –

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In conclusion, day-to-day variability adds considerably to the total variation of both serum and urine NTX-I and CTX-I levels. Unlike diurnal variation, day-to-day variability cannot be controlled easily. 4.2.3. Seasonal variation In temperate climates, bone turnover is also subject to circannual (seasonal) variation [109,110]. Seasonal changes have been described for serum CTX-I levels, with a 20–30% lower turnover rate in summer than in winter [111,112]. The increase in bone turnover during the winter period may be due, at least in part, to variations in serum vitamin D and PTH concentrations. Seasonal variation of NTX-I has only been investigated in urine and the existing results are conflicting (Table 4) [103,113,114]. 4.2.4. Other biological sources of variability A number of other conditions and disorders have been shown to strongly affect bone turnover markers. For example, renal disease may affect CTX-I and NTX-I levels through both impaired clearance and disease-specific alterations in bone metabolism. Thus, even moderate impairment of renal function (GFR 50 ml/min) has been shown to have significant effects on the serum levels of NTX-I and CTX-I [115]. Another study has shown that immobility, disease severity and balance all affect bone turnover markers in an independent fashion [116]. Finally, eating disorders such as bulimia or anorexia have significant and lasting effects on bone turnover markers that are only partly explained through the actual bone disease [117]. In summary, a great number of sources of variability need to be taken into account when measuring NTX-I and CTX-I. To minimize some of the limitations linked to pre-analytical and analytical variability, standardized sampling and sample handling are mandatory. Controllable factors such as the mode of sample collection, sample handling and storage, diurnal and menstrual rhythms, pre-sampling exercise and pre-sampling diet should be taken care of wherever possible. 4.3. How to deal with variability? Fig. 4. Effects of glucose, normal diet, and fasting on bone turnover. Intervention with normal diet (squares), OGTT (diamonds), IVGTT (triangles), and fasting (open circles) on s-CTx (A) and u-CTx (B) in healthy, postmenopausal women [69].

levels are high in men aged 20 to 30 years, which corresponds to the late phase of peak bone mass accrual. Thereafter, both markers decrease reaching their lowest levels between 50 and 60 years [99–101]. Data on bone turnover rates in men over the age of 60 years are discordant. Some studies evaluating age-related changes of NTX-I and CTX-I levels observed an increase in serum and urinary indices [100,102]. However, this was not confirmed in other studies showing no change or a decrease in NTX-I values with age [99,103,104]. As NTX-I and CTX-I are cleared by the kidney, the age-associated decrease in glomerular filtration rate may affect urinary and, to a larger extent, serum marker concentrations. 4.2.2. Intraindividual variation The serum and urine concentrations of NTX-I and CTX-I not only vary within a single day but also between consecutive days (Table 4) [105,106]. This day-to-day variability is due to genuine variations in marker levels and not to analytical imprecision. In general, serum NTX-I and CTX-I levels show less day-to-day variability than their counterparts in urine [107]. Another factor contributing to the biological variability of most bone markers is the menstrual cycle. Bone resorption is highest during the mid-follicular, late-follicular and early luteal phase, resulting in an overall amplitude of variation of 10–20% [108]. Hence, menstrual variability should be taken into account when appropriate, with the best timing for sampling being the first 3–7 days of the menstrual cycle.

The pronounced variability of NTX-I and CTX-I levels makes it difficult to determine precise thresholds or cut-offs for practical use in individual patients. In particular serial measurements are compromised by the variability of both markers. The concept of least significant change is an elegant approach to overcome some of the problems linked to variability: A true (“significant”) change in a given marker can only be assumed when the signal believed to be a “specific response” is at least greater than the imprecision of the measurement. This minimal required change has been termed “least significant change” (LSC) and defines certain cut-off levels that a change in a marker must exceed to be considered “clinically significant”. Therefore, the LSC is an overall measure of variability within a pre-specified confidence interval and is used to distinguish between non-specific (noise) and specific (signal) changes in bone turnover markers. The LSC determines the signal to noise ratio of any biological surrogate marker. For most bone resorption markers, including NTX-I and CTX-I, the LSC is around 60–80%. Not surprisingly, the LSC is lower for markers measured in serum than for those determined in urine. Another widely used method to assess therapy-induced changes is to compare the actual marker level to a predefined range, such as the ‘reference’ or ‘normal’ range. This approach is based upon the assumption that patients with accelerated bone turnover are likely to benefit from a treatment if their bone markers return to the respective ‘normal’, i.e. premenopausal range. Such a “normalisation” would be considered a valid response. The problem with this method is that the reference ranges for most markers have not been well defined. Also, a reduction into the ‘normal’ range can only be achieved if the pretreatment values are abnormally high, which is the case in less than 50% of patients with osteoporosis.

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Fig. 5. Reference curves for serum CTX-I and NTX-I in boys (left plot) and girls (right plot). Curves represent the 50th centile (straight lines) and 3rd/97th centile (dotted lines) [118,237].

4.4. Reference values in serum and urine Apart from the reference values provided by the assay manufacturers, independently published reference values add useful information. Rauchenzauner et al. analyzed serum CTX-I in 572 children and adolescents (300 boys) aged 2 months to 18 yr [118]. In children aged 0–10 years, serum CTX-I values ranged from 500 to 3000 ng/L, while in adolescents values between 500–4000 ng/ml were found (Fig. 5).

Crofton et al. investigated 346 individuals between 0–19 years and reported significantly lower reference intervals, suggesting that reference values vary between study populations [119]. Two studies from Poland and China have established reference values for serum CTX-I and urinary NTX-I in pre- and postmenopausal women, respectively (Fig. 6) [120,121]. The manufacturer of the β-CTXI assay (Roche Diagnostics) provides comparable values derived from the OFELY study. The reference range for men may be deduced from

Fig. 6. Scatter plots and cubic regression curves of age-related changes in serum and urinary CTX-I in Chinese women [121].

M. Herrmann, M. Seibel / Clinica Chimica Acta 393 (2008) 57–75 Table 5 The effect of menopause on CTX-I concentrations in urine and serum [51] Premenopause

Postmenopause

Age-matched Premenopause

n Mean age ± SD (years) s-CTX u-αCTX u-βCTX a b c

Postmenopause

36 45.0 ± 4.1

35 55.3 ± 3.7a

10 49.3 ± 2.9

10 50.9 ± 3.6

1345 ± 764 299 ± 193 258 ± 159

2527 ± 1505a 361 ± 219 418 ± 241c

905 ± 615 184 ± 93 173 ± 105

2297 ± 818a 349 ± 172b 393 ± 131a

p b 0.05 vs. premenopause. p b 0.01 vs. premenopause. p b 0.001 vs. premenopause.

65

increases in NTX-I levels of around 15–50% [127,133], but one should keep in mind that NTX-I measurements generally show lower background noise than those of CTX-I. A prospective study covering the perimenopausal transition in healthy women suggests that changes in bone turnover (as measured by urinary NTX-I) occur during the late premenopause [131]. Studies employing serum CTX-I and NTX-I measurements indicate that the circulating levels continue to be increased (and to be associated with bone loss) during late menopause. In early postmenopausal women, increased bone turnover can be attenuated by oral calcium supplementation [134–138]. Long-term treatment of women with oestrogen was shown to reduce both NTX-I and CTX-I values to premenopausal levels [137,139–141;141–143]. 5.2. Osteoporosis

recent results of the MINOS study (Table 6) [122]. In elderly individuals the following median levels (interquartile range) of serum CTX-I can be expected: men: 0.24 (0.16–0.39), women: 0.31 (0.2–0.47) [116]. These values have been confirmed by two other studies [123,124]. Reference intervals for serum NTX-I in children have been published by van der Sluis et al. [125]. An adult reference range has only been established for urinary NTX-I. For serum NTX-I, the only reference interval available is the one provided in the package insert of the OSTEOMARK® assay. The mean ± 2 standard deviations range was 6.2– 19 nmol BCE/L for men and 5.4–24.2 nmol BCE/L for premenopausal women. No reference values are available for postmenopausal women. Additional information may be obtained from various clinical studies where a control group of healthy and untreated individuals has been included. Thus, Clemens et al. measured serum and urinary NTX-I in 32 pre- and 21 postmenopausal women and found a mean concentration of 4.7 ± 2.2 nmol BCE/L in premenopausal women and 7.3 ± 2.8 nmol BCE/L in postmenopausal women [30]. Gertz et al. observed comparable values for serum NTX-I [126]. In contrast, Scariano et al. reported significantly higher NTX concentrations of 12.2 (premenopausal) and 17.9 (postmenopausal) nmol BCE/L [127]. In a large population of untreated postmenopausal women, Eastell et al. found urinary NTX-I levels of 56.4 ± 26.9 nM BCE/mM creatinine and serum NTX-I values of 17.8 ± 4.9 nmol/BCE/L [37]. For middle-aged and elderly men reference values can be obtained from the Rancho Bernardo Study [128]. In 276 elderly men (mean age 73 ± 9 years) the median (interquartile range) for urinary NTX was 29.0 (22.0–40.0) nmol/BCE/L. In middle-aged healthy men (54 ± 1 years) mean urinary NTX-I was 30 nmol/BCE/L. In young Finnish men median urinary NTX-I values have been reported to be 85 (29-363) nmol/BCE/L [129]. Similar levels have been described by Kristensen et al. [77]. 5. Clinical use of CTX-I and NTX-I In clinical practice, measurements of either serum or urine CTX-I and NTX-I are used in the diagnosis and management of a range of metabolic and malignant bone diseases. However, for each bone disorder and treatment it is important to consider which marker, measured with what assay, will provide the most relevant clinical information. In addition, knowledge of the relationship between the level of a certain marker and clinical outcome creates the option of using this as a surrogate marker of outcome, similar to bone mineral density in osteoporosis. 5.1. Menopause Menopause is associated with a substantial acceleration in bone loss and bone turnover, mirrored by a 50–100% increase in serum and urinary CTX-I levels (Fig. 6, Table 5) [51,121,130]. Urinary NTX-I concentrations have also been found significantly elevated in postmenopausal women, as compared to premenopausal subjects [97,131,132]. Some studies have described smaller menopause-induced

Since osteoporosis is a heterogeneous disease rates of bone turnover tend to vary over a wide range. Although most cross-sectional studies show accelerated bone turnover in a certain proportion of postmenopausal osteoporotic women, there is usually broad overlap between diseased and healthy populations [88,144–149]. In a population-based study, measurement of urinary NTX-1 levels could discriminate between older individuals with normal hip bone density, osteopenia and osteoporosis [150]. However, this association did not hold true for men at the level of the spine. Comparable data exist for serum CTX-I [121,151,152]. In a study measuring serum CTX-I and urinary NTX-I in over 800 elderly women, serum CTX-I levels were the strongest discriminator between normal, osteopenic and osteoporotic subjects [151]. In this study, the α/β-CTX-I ratio was elevated in post- compared with premenopausal women, but there was no difference in the ratio among normal, osteopenic or osteoporotic postmenopausal women. Serum CTX-I concentrations have also been found to predict bone loss in postmenopausal women. Results are less homogeneous for elderly men. Chandani et al. investigated 78 elderly men and found a significant correlation between serum NTX-I levels and BMD at the femoral neck (r = −0.26) [36]. In osteoporotic men, serum NTX-I levels correlated with BMD at the lumbar spine (r = −0.66). In contrast, similar analyses using the Dubbo study data showed no differences in serum β-CTX-I levels between osteoporotic and non-osteoporotic elderly men [153] (whereas ICTP did). Of note, in a study of osteoporotic men, Drake et al. found no significant correlation between urinary NTX-I levels and BMD [154]. 5.3. Bone turnover and bone loss Bone mass, rates of bone loss, and the risk of osteoporotic fractures are interrelated, and both low bone mass and rapid bone loss have been shown to be independent predictors of future fracture risk [155]. The rate of bone loss is determined by a number of factors, one of which appears to be the rate of bone turnover. Most longitudinal studies support the notion that individuals with high rates of bone turnover loose bone at a faster rate than subjects with normal or low bone turnover [137,139,156–162]. Markers of bone resorption, such as NTX-I and CTX-I, seem to be stronger predictors of future bone loss than markers of bone formation, and correlations are stronger in elderly than in younger women [163].

Table 6 Serum and urine CTX-I reference values in men [122] Age group (years)

19–30

n 56 Body weight (kg) 71 ± 10 urine β-CTX-I (μg/mM 186 ± 75 creatinine) serum β-CTX-I (mmol/L) 5.0 ± 2.0

31–40

41–50

51–60

61–70

88 79 ± 11 134 ± 46

105 82 ± 14 122 ± 59

352 153 81 ± 12 77 ± 10 120 ± 60 122 ± 69

3.0 ± 1.3

2.6 ± 1.3

179 83 ± 14 118 ± 571 2.4 ± 1.1

2.4 ± 1.1

71–85

2.4 ± 1.2

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In a prospective study 1283 randomly selected Japanese women aged 15–79 years at baseline were followed for 6 years [163]. Premenopausal women aged 45 years or older with elevated levels of CTXI showed significantly greater bone loss at most skeletal sites during the follow-up period than those with lower levels. However, early postmenopausal women showed an association that was limited mostly to the distal radius, and other postmenopausal groups had virtually no association. In the prospective OFELY study women with abnormally high CTX-I levels lost bone at a 2- to 6-fold higher rate than women with low turnover, according to bone turnover markers [164]. In the logistic regression model, the odds ratio of fast bone loss, defined as the rate of bone loss in the upper tertile of the population, was increased by 1.8- to 3.2-fold for levels of CTX-I in the high turnover group compared with levels within the premenopausal range. For urinary NTX-I, comparable predictive values have been shown. In addition to healthy peri- and postmenopausal women, serum CTX-I and NTX-I levels have also been found to predict bone loss in hemodialysis and chronic renal failure patients [165,166]. Taken together, there is evidence that high NTX-I and/or CTX-I levels are associated with accelerated bone loss. However, the strength of this association seems to depend on a number of factors, such as menopausal age, skeletal site and gender. However, both markers are no substitute for individual bone mass measurements, or for a careful assessment of the patient's personal and family history. 5.4. Bone turnover and fracture risk Since bone turnover is an independent predictor of fracture risk and fractures are the major complication of osteoporosis it seems reasonable to ask whether bone turnover markers, such as NTX-I or CTX-I, are related to fractures and fracture risk. In the prospective OFELY study, baseline levels of serum CTX-I and urinary NTX-I of 55 women who subsequently had a fracture (20 vertebral and 35 peripheral fractures) were compared with those of 380 women who were not fractured during a mean follow-up of 5 years [167]. Women with levels in the highest quartile of serum CTX-I and urinary NTX-I had about a 2-fold increased risk of fractures compared with women with levels in the three lowest quartiles. In addition, an increased urinary α/β-ratio of CTX-I, reflecting a decreased degree of type I collagen racemization/ isomerization, was associated with increased fracture risk independently of BMD and partly of bone turnover rate [48]. In contrast, the EPIDOS Study showed no significant predictive value of serum CTX-I for future fractures in 212 hip fracture cases and 642 controls [168]. However, when the analysis was restricted to samples taken in the early afternoon, serum CTX-I was significantly predictive with a relative hazard of 1.86 for values above the premenopausal range (mean + 2 SD). Similar findings have been reported for urinary CTX-I but not for urinary NTX-I [169]. In contrast, a longitudinal trial from the Netherlands demonstrated a significant predictive value for urinary NTX-I in 348 healthy postmenopausal women [170]. Garnero et al. reported that combining both BMD and bone resorption markers improves the prediction of hip fractures: Women with both a femoral BMD value of 2.5 SD or more below the mean of young adults (T-score) and high CTX-I levels were at greater risk of hip fracture (odds ratio: 4.8) than those with only low BMD or high levels of the bone resorption marker [169]. In a recent large-scale study from Sweden, Gerdhem et al. confirmed a predictive value of serum CTX-I for fractures of any type, including vertebral fractures [47]. In addition, prospective data from the Australian FREE study of 1112 frail elderly men and women indicate that high serum CTX-I levels are also independent predictors of all cause mortality [123]. In contrast to the studies discussed above, serum CTX-I was not associated with fractures in a mixed population of frail elderly subjects with vitamin D deficiency and high falls risk [124]. Moreover, prospective data from the Dubbo study, investigating a subset of 50 men with incident low-trauma fractures and comparing

these to 101 men without fracture, did not reveal any relationship between baseline serum CTX-I and fractures [153]. Unfortunately, there is no study analyzing serum NTX-I and its value in assessing fracture risk. In summary, there are only a limited number of publications analyzing the relation between NTX-I and CTX-I levels on the one side, and fracture risk on the other side. While in elderly women, serum CTX-I and urinary NTX-I seem to have a significant predictive value for future vertebral and non-vertebral fractures, little is known about this relationship in older men. 5.5. Pre-treatment bone turnover and therapeutic effect From both a theoretical and clinical point of view, it is conceivable that intervention strategies may differ between patients with accelerated, normal or even abnormally low bone turnover at the time of diagnosis. This theory raises the question if pre-treatment bone marker measurements are helpful in guiding the selection of therapy for individual patients. There is some evidence that urinary NTX-I as well as urinary and serum CTX-I concentrations prior to intervention can predict the improvement of BMD under treatment. In a one year intervention trial 192 vitamin D insufficient elderly women were treated with either calcium (500 mg) and vitamin D (400 IU) supplementation twice daily or placebo [171]. The initial values of urinary NTX-I (r = 0.38), urinary CTX-I (r = 0.32) and serum CTX-I (r = 0.28) correlated significantly with the changes in BMD after one year of treatment. Similar results were reported by Rosen et al. in 239 postmenopausal women, who were treated with HRT or calcium [137]. When baseline urinary NTX-I was stratified by quartile, there was a significantly greater increase in BMD among those with the highest NTX-I levels compared to those with the lowest NTX-I concentrations. Chesnut et al. also found a significant predictive value of urinary NTX-I for the gain of BMD in postmenopausal women on HRT [139]. A post-hoc analysis of the Fracture Intervention Trial (FIT), examining the influence of pre-treatment bone turnover on the anti-fracture efficacy of daily alendronate in postmenopausal women found that serum CTX-I was significantly associated with changes of BMD at the hip but not at the spine. In addition, CTX-I was not associated with fracture outcomes at any site [172]. In contrast, in a longitudinal study from Marcus et al. serum CTX-I and NTX-I levels offered little useful information for predicting BMD changes for untreated or HRT-treated postmenopausal women [173]. Moreover, Kyd et al. [174] and Delmas et al. [175] failed to find a significant relation between baseline serum CTX-I or urinary NTX-I and the change of BMD in alendronate or teriparatide treated individuals. Taken together, it remains unclear whether there is a clinically relevant relationship between bone turnover at baseline and the response to anti-resorptive treatment. Another interesting approach which may be helpful to individualize anti-osteoporotic therapy might be the monitoring of changes of bone turnover markers during therapy. It can be speculated that the magnitude of these changes is related to future fracture risk and changes in BMD. Data from a Danish study treating 67 women with alendronate demonstrated a highly significant correlation between changes of serum as well as urinary NTX-I and CTX-I from baseline at months 3–12 and the 2 year response in spine BMD (r = −0.45 to r = −0.78, p b 0.001) [176]. Sensitivity and specificity were used to assess the accuracy of a 50% decrease from baseline at month 6 in the biochemical markers for predicting prevention of bone loss in the spine over 2 years. Sensitivity levels were 78% (serum CTX-I and NTXI), and 89% (urinary CTX-I). Specificities were 100% (serum CTX-I), 71% (urinary CTX-I), and 84% (NTX-I). Positive predictive values were 100% (serum CTX-I), 87% (urinary CTX-I), and 90% (NTX-I). In comparison, the predictive capacities of change from baseline at year 2 in hip BMD in predicting prevention of bone loss at the spine were similar [177].

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These results have been confirmed in a larger cohort from the same study (n = 306) [177]. Similar results have been published by Okabe et al. [178], Chen et al. [179] and Delmas et al. [180]. Sornay-Rendu et al. reported that early changes in serum CTX-I levels also correlate with the loss of BMD after cessation of HRT [181]. While for serum CTX-I the association between early changes of circulating levels and the change in BMD is unequivocal the situation for urinary and serum NTX-I is not. Rosen et al. found only serum CTX-I but not urinary or serum NTXI predictive for the gain of BMD in estrogen treated postmenopausal women [138]. In another study early changes of urinary NTX-I did not correlate with structural changes of cancellous bone as assessed by histomorphometry and μ-CT [182]. In conclusion, early changes of serum CTX-I seem to have a predictive value for long term changes of BMD. For NTX-I the situation is equivocal.

In summary, treatment induced changes of CTX-I and NTX-I may have a predictive value for future fracture risk. However, existing publications are rare and not sufficient for a final conclusion.

5.6. Bone turnover markers and therapeutic monitoring

5.7. Monitoring patient compliance using bone markers

Bisphosphonates improve bone mineral density (BMD) and reduce fracture risk to varying degrees. However, the reduction in fracture risk is usually much greater than predicted from improvements in BMD [183–185]. Hence, it has been estimated that changes in BMD explain only 4% to 28% of the reduction in vertebral fracture risk attributed to anti-resorptive treatments [186]. It is therefore likely that changes in other determinants of bone strength, including the rate of bone turnover may be better predictors of anti-fracture efficacy (Fig. 7). In fact, several studies confirmed that short-term reductions in bone turnover were associated with a reduction in vertebral and/or non-vertebral fracture risk in women treated with HRT [186], raloxifene [187–189], risedronate [190], alendronate [35,191] and ibandronate [192]. Two studies have investigated the associations between change in CTX-I and NTX-I levels and fracture risk in bisphosphonate-treated postmenopausal women [190,191]. Post-hoc analyses of data from the VERT studies including postmenopausal women with at least one vertebral fracture demonstrated that reductions in urinary CTX-I (by 60%) and NTX-I (by 51%) at 3–6 months of risedronate treatment were significantly associated with the reduction in vertebral and nonvertebral fracture risk after 3 years [190]. The change in bone resorption markers explained 50–60% of the risedronate-related fracture risk reduction for both, vertebral and non-vertebral fractures. Bauer et al. reported that in alendronate-treated women, greater reductions in bone turnover were associated with fewer osteoporotic spine fractures (Table 7) [191]. Another study investigated the predictive value of early changes in urinary CTX-I under treatment with raloxifene in 2622 women from the MORE trial [189]. Change in urinary CTX-I after 6 and 12 months was significantly related to future risk of vertebral fracture.

Long-term compliance with treatment for osteoporosis is usually poor [193]. Several studies reported that up to 50% of postmenopausal women were not adherent to their treatment after one to 5 years of HRT [194–196]. Hence, monitoring patients on anti-resorptive medication is an eminent part of patient management. As bone resorption markers decrease rapidly after initiation of treatment within 3–6 months, they might represent useful surrogate markers for monitoring patient compliance. Only few data, however, are available to support this theoretical approach. Chapurlat et al. observed that monitoring osteoporotic women with measurements of bone markers early during the treatment course may increase effectiveness of treatment with greater quality adjusted life years than no follow-up [197]. In another study of 75 postmenopausal women treated with raloxifene, Clowes et al. examined whether monitoring (nursemonitoring or marker-monitoring) enhances adherence and persistence with anti-resorptive therapy, and whether presenting information on the biochemical response to therapy provided additional benefit [198]. In the group being monitored, cumulative adherence to therapy increased by 57% compared with no monitoring. Adherence at 1 yr was correlated with percent change in hip BMD (r = 0.28; p = 0.01) and in uNTX-I (r = −0.36; p = 0.002). However, presentation of results of effects on NTX-I levels did not improve compliance to therapy compared with nurse-monitoring alone. Nevertheless, results from the IMPACT study in postmenopausal women on risedronate have shown that a reinforcement message based on a verbal feedback on the change of urinary NTX-I improved the one-year persistence compared to non-reinforced subjects [199].

Fig. 7. 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 b 0.05 for serum NTX [35].

Table 7 Fracture risk per SD of 1-year decrease in serum CTX-I among alendronate-treated women [191] Variable

SD of change over 1 year (%)

Spine fracture OR (95% CI)

Non-spine fracture RH (95% CI)

Hip fracture RH (95% CI)

Serum β-CTX-I1.31)

33.1

Serum β-CTX-I (fasting at baseline)

35.5

0.83 (0.73–0.95) 0.77 (0.58–1.03)

0.94 (0.84–1.06) 1.02 (0.75–1.37)

0.89 (0.61–1.31) –

5.8. Comparison of CTX-I and NTX-I in hemodialysis patients Renal osteodystrophy (ROD) is one of the major complications in patients with severe renal failure. Untreated ROD is usually associated with a substantial increase in fracture risk as well as increased morbidity and mortality [200,201]. The gold standard for the diagnosis of renal osteodystrophy is bone biopsy and histology. Since this is an invasive and time-consuming method the determination of serum intact parathyroid hormone has been used for many years as an indirect predictor of bone disease in ROD [165,166,202]. The introduction of modern biomarkers of bone metabolism offers the possibility of a direct assessment of bone metabolism. However, most of the bone resorption markers including CTX-I and NTX-I are cleared by the kidney. Therefore, in patients with renal failure, these markers may not accurately reflect bone turnover. Moreover, hemodialysis (HD) may have additional effects on the circulating levels of bone turnover markers. Several well-performed studies have unequivocally shown that serum CTX-I and NTX-I concentrations are significantly elevated in HD patients and correlate well with both serum creatinine levels and BMD

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Table 8 Sensitivity, specificity, Youden index, positive predictive value (PPV), and negative predictive value (NPV) of fourth quartile CTX-I levels in identification of male hemodialysis patients with BMD loss at 1/3 distal radius of more than 1.59% per year (highest tertile of rate of loss) Marker

Sensitivitya (%)

Specificityb (%)

Youden index

β-CTX-I

41

83

0.24

c

PPVd (%)

NPVc (%)

55

73

PPV positive predictive value; NPV negative predictive value [165]. a Sensitivity: proportion of male HD patients with rapid bone loss (N1.59% per year at distal radius) with marker levels above the fourth quartile of bone marker. b Specificity: proportion of male HD patients with slow bone loss (b 1.59% per year at distal radius) with marker levels below the fourth quartile of bone marker. c Youden Index: sensitivity + specificity − 1. d NPV: incidence of slow bone loss among male HD patients with marker levels below the fourth quartile of bone marker.

measurements [165,166]. Maeno et al. reported that CTX-I and NTX-I levels significantly correlate with BMD and annual bone loss in HD patients [166]. In addition, serum CTX-I and NTX-I concentrations were highly correlated with each other (r = 0.929, p b 0.0001). Sensitivity and specificity for predicting annual bone loss were comparable for both markers. Similar results have been published by Okuno et al. (Table 8) [165]. However, in another study including 137 male HD patients, serum CTX-I levels were not significantly different between patients exhibiting a significant loss of BMD compared to those without loss of BMD at the distal radius [203]. In chronic renal failure patients urinary NTX-I levels have been shown to correlate well with histomorphometric results from bone biopsies [204]. However, in HD patients this correlation was not present. The missing correlation between histomorphometric results and NTX-I in HD patients is probably due to the fact that HD decreases NTX-I concentrations. A longitudinal study by Alvarez et al. demonstrated a 30% decrease of CTX-I within 4 h after HD [202]. Since HD reduces CTX-I and NTX-I but does not improve the secondary hyperparathyroidism, HD may mask the relation between bone resorption markers and bone loss. Taken together, CTX-I and NTX-I levels can be massively elevated in patients with advanced renal disease, correlating with both creatinine and BMD. However, the utility of bone turnover markers in HD patients is questionable, as the latter lowers the concentrations of both markers and attenuates the correlation between bone turnover markers and histomorphometric parameters and/or BMD measurements, 5.9. Paget’s disease Biochemical markers of bone turnover play a clear-cut role in the diagnosis and management of Paget’s disease of bone. In active disease, most bone formation and resorption markers are elevated [205,206]. Alterations in the rate of bone turnover in this disease are so pronounced that bone marker measurements require relatively

Fig. 8. Serum CTX-I in breast cancer patients with and without bone metastasis [216].

little sensitivity and specificity, and in most cases, measurement of total alkaline phosphatase and/or urinary hydroxyproline will be adequate for diagnosis [205,206]. Serum CTX-I and urinary NTX-I seem to perform comparably well in diagnosis and monitoring treatment in Paget’s disease patients [207–210]. In addition, several recent studies indicate that the non-isomerized αα-CTX-I exhibits the highest sensitivity and the best prognostic value [206,209,211]. In general, these more sensitive and specific bone turnover markers, provide minimal additional value [205,206]. While the initial response to anti-resorptive treatment may be reliably monitored with a bone resorption marker, biochemical remission and relapse are often based on changes in a bone formation marker [205,206]. 5.10. Cancer and bone metastasis Bone metastases profoundly perturb normal bone remodelling [212]. Biochemical markers of bone turnover have been shown to reflect these tumour-induced changes in bone remodelling and may therefore be useful in the diagnosis, follow-up and prognosis of patients with malignant (bone) disease [213,214]. Most markers of bone turnover, particularly those of bone resorption, are elevated in patients with established bone metastases [215]. Numerous studies investigated the value of urinary NTX-I and urinary as well as serum CTX-I for the diagnosis of bone metastasis. Both markers have been found to be significantly elevated in breast and prostate cancer patients with bone metastasis (Fig. 8) [216–221]. Initially it was believed that CTX-I would not be a sensitive marker of bone metastasis since its epitope is generated by cathepsin K digestion and cathepsin K is secreted by mature osteoclasts. However, in a metastatic environment, matrix metalloproteinases are mainly involved in bone resorption and release predominantly epitopes associated with ICTP rather than CTX-I [222]. Of note, the NTX molecule is not subject to such differential mechanisms of release. In contrast to this hypothesis, which was based on in vitro data, clinical studies clearly demonstrate that ICTP, CTX-I as well as NTX-I are all useful tools in the diagnosis of bone metastasis. Assays for the various isoforms of CTX-I have been shown to perform differently in patients affected by bone metastases. The αα-CTX-I isoform appeared to provide the best differentiation of patients affected by breast cancerinduced bone metastases [43]. Moreover, recent evidence from a study including 90 breast cancer patients showed an accumulation αα-CTX-I in the proximity of bone metastasis [223]. The amount of αα-CTX-I was associated with the urinary excretion of αα-CTX-I in these patients. The estimated relative increases in αα-CTX-I associated with the presence of one, two, or three metastases were 38%, 57%, and 81%, respectively. The measurement of CTX-I and NTX-I has also been found to contribute to the management of patients with monoclonal gammopathy [224,225]. However, due to the interindividual variability of CTX-I and NTX-I they cannot substitute conven-

Fig. 9. Percentage change of serum CTX-I in cancer patients with metastatic bone disease treated with oral clodronate at various daily doses [231].

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tional diagnostic tools such as bone scintigraphy [218]. In addition, CTX-I and NTX-I levels are also significantly elevated in postmenopausal women (see above), which limits their utility in these individuals. Other markers, such as serum ICTP are less variable and are probably more suitable for the biochemical diagnosis and management of bone metastases [218,226–228]. Another potential use of bone markers in cancer patients is the monitoring and management of treatment in cancer patients with bone metastasis. Serum and urinary levels of both CTX-I and NTX-I respond promptly and profoundly to bisphosphonate therapy, and this response appears to be associated with a favourable clinical outcome [229,230]. Chen et al. reported that zoledronic acid produced significant declines from baseline in serum and/or creatininecorrected urine CTX-I (by 74%) and NTX-I (by 69%) [229]. There was no relationship of the magnitude and duration of these changes with zoledronic acid dose. The anti-resorptive effects were evident within 1 day postdose and were maintained over 28 days across all dose levels, supporting monthly dosing with 4 mg zoledronic acid. Brown et al., in a recent study of 125 cancer patients with metastatic bone disease, demonstrated a dose-dependent decrease of urinary NTX-I and serum CTX-I following treatment with clodronate at daily oral doses of 800, 1,600, 2,400 or 3,200 mg (Fig. 9). In breast cancer patients a dose of 1600 mg daily was most appropriate while in prostate cancer patients 2400 mg were more effective [231]. Longitudinal measurements of CTX-I have been shown to predict progression of bone metastasis in bisphosphonate-treated prostate cancer patients [232]. A current retrospective analysis of three large longitudinal intervention trials treating 1824 cancer patients with bisphosphonates demonstrated that urinary NTX-I levels above 50 nmol/mmol creatinine have a 2-fold increased risk for skeletal complications and disease progression compared with patients with low NTX-I levels [233]. It should be mentioned that other therapeutic regimens, such as androgen ablation or estrogen substitution, do not change or increase CTX-I and NTX-I [234,235]. However, the pathophysiology behind the unchanged or increasing CTX-I and NTX-I levels in androgen ablated or estrogen substituted patients is not understood at present. Based on the available data, both CTX-I and NTX-I may play a role in the diagnosis and management of cancer patients with bone metastases. However, it remains unknown whether the use of bone markers has any defined beneficial effects on overall outcome in cancer patients. In particular, no study has addressed the question whether patients with bone metastases should be treated according to their rate of bone turnover, and what the treatment goals are in this respect. 6. Conclusion Although the above-mentioned studies represent only a selection of the published literature, they all demonstrate that the bone resorption markers CTX-I and NTX-I are helpful tools in evaluating the physiology and pathophysiology of bone metabolism, and in elucidating the pathogenesis of bone disease. With regards to their clinical use, it appears that measurement of both serum and urinary CTX-I is limited by its marked variability, a problem which appears less pronounced with measurements of NTX-I. While the utility of serum NTX-I and CTX-I in relevant clinical situations is rather well documented, the use of NTX-I is compromised by the insufficient documentation of reference ranges. Finally, it remains unclear whether CTX-I or NTX-I perform better than other bone resorption markers such as pyridinoline cross-links or hydroxyproline excretion. Acknowledgment The authors dedicate this review to the memory of the late Professor Heinrich Schmidt-Gayk, who's measured guidance and

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