Serum levels of C-Terminal Telopeptide (CTX) are Associated with Muscle Function in Community-Dwelling Older Adults

Abstract

Background

Markers of bone metabolism have been associated with muscle mass and function. Whether serum cross-linked C-terminal telopeptides of type I collagen (CTX) is also associated with these measures in older adults remains unknown.

Methods

In community-dwelling older adults at high risk of falls and fractures, serum CTX (biochemical immunoassays) was used as the exposure, while appendicular lean mass (dual-energy x-ray absorptiometry) and muscle function (grip strength [hydraulic dynamometer], short physical performance battery [SPPB], gait speed, sit-to-stand, balance, Timed Up and Go [TUG]) were used as outcomes. Potential covariates including demographic, lifestyle, and clinical factors were considered in statistical models. Areas under the receiver operating characteristic (ROC) curves were calculated for significant outcomes.

Results

Two hundred and ninety-nine older adults (median age: 79 years, interquartile range: 73, 84; 75.6% women) were included. In multivariable models, CTX was negatively associated with SPPB (β = 0.95, 95% confidence interval [CI]: 0.92, 0.98) and balance (β = 0.92, 0.86, 0.99) scores, and positively associated with sit-to-stand (β = 1.02, 95% CI: 1.00, 1.05) and TUG (β = 1.03, 95% CI: 1.00, 1.05). Trend line for gait speed (β = 0.99, 95% CI: 0.98, 1.01) was in the hypothesized direction but did not reach significance. Area under the ROC curves showed low diagnostic power (<0.7) of CTX in identifying poor muscle function (SPPB: 0.63; sit-to-stand: 0.64; TUG: 0.61).

Conclusions

In older adults, higher CTX levels were associated with poorer lower-limb muscle function (but showed poor diagnostic power for these measures). These clinical data build on the biomedical link between bone and muscle.

As the world population rises, the prevalence of musculoskeletal diseases characterized by low bone density/structure (osteopenia/osteoporosis) and low muscle mass and strength/physical function (sarcopenia) will inevitably increase the risk of adverse clinical outcomes such as disability, falls, and fractures (1–3).

In previous decades, bone and muscle loss have largely been studied in isolation. However, more recent work from preclinical models has shown that bone and muscle interact biomechanically and biochemically, with the latter mediated by bone and muscle cells which secrete anabolic and catabolic molecules in a bidirectional manner, and with fat as a third player that intervenes in this cross talk via the release of adipokines and fatty acids from adipocytes (4). In human studies, markers of bone metabolism such as osteocalcin (a marker of bone formation) and sclerostin (a regulator of bone formation) have been connected with muscle mass and function (5,6). Other clinical works in older men and women have also shown that osteopenia/osteoporosis and sarcopenia are associated with one another (7,8). However, it remained to be seen if a causal biological link exists between bone and muscle loss.

Since then, a notable advancement in the bone–muscle cross-talking field has emerged. Osteoclast differentiation is regulated by receptor activator of nuclear factor-κB (RANK), its ligand (RANKL), and a decoy receptor for RANKL, osteoprotegerin (OPG) (9). The RANK/RANKL/OPG system is key in orchestrating bone resorption/turnover (9). Indeed, the role of RANKL is not exclusive to bone loss alone. Compelling work by Bonnet et al. (10) demonstrated that RANKL is also expressed in the muscles of osteoporotic mice with sarcopenia. In the same trial, treatment with denosumab, a well-known inhibitor for RANKL, preserved lean (muscle) and bone mass and increased grip strength in postmenopausal women with osteoporosis over a 3-year period (10). Follow-up data from an observational study also found improvements in some measures of muscle function in older adults treated with denosumab for 6 months (11). Taken together, these data suggest that markers of bone resorption may be associated with muscle mass and function in older adults.

At present, several serum biomarkers are used in clinical practice to monitor the efficacy of osteoporosis treatments. Of these, serum cross-linked C-terminal telopeptides of type I collagen (CTX) is a marker of bone resorption, and higher levels are adversely associated with bone loss (12), as well as weight-bearing and non-weight-bearing fractures (13). Moreover, data from the Avon Longitudinal Study of Parents and Children (14) and the European Male Aging Study (15) found that CTX is positively associated with multiple genetic variants in the RANK/RANKL/OPG signaling pathway. Considering this, in addition to the notion that RANKL is expressed in the muscles of sarcopenic mice (10), CTX may be associated with lean mass and function in community-dwelling older adults at high risk of falls, fractures, and mobility limitations. However, to our knowledge, no previous study has explored this.

Here, we investigated the associations between CTX and lean mass and function in community-dwelling older adults referred to an outpatient high-risk falls and fractures clinic. In addition, we investigated the diagnostic power of this bone resorption biomarker in identifying poor muscle function. We hypothesized that higher levels of CTX would be associated with lower lean mass and poorer muscle function after adjusting for relevant covariates.

Materials and Methods

Study Population

The population consisted of community-dwelling men and women (aged ≥65 years) attending the Falls and Fractures Outpatients Clinic at the Australian Institute for Musculoskeletal Science (Western Health, Melbourne, Australia). As previously reported (16), for referral to this clinic, participants must be fully ambulant, free of major cognitive deficits, and present with at least 1 risk factor for falls or fractures. Patients are only permitted to attend this clinic at least 3 months postfracture. Ethical approval for the Falls and Fractures Clinic Databank was granted by the Western Health Low Risk Ethics Panel at Sunshine Hospital (ID: DB2017.13), and this project was approved by the same ethical panel (ID: QA2018.106_44499). Informed consent was waived as data were collected as part of standard care. Exclusion criteria were based on any known factors affecting the exposure (CTX status) or the outcomes (lean mass or muscle function) (17). These included individuals with a medical condition affecting CTX or bone metabolism (ie, multiple myeloma, confirmed kidney disease, primary hyperparathyroidism, Paget’s disease, active rheumatoid arthritis), conditions affecting lean mass or muscle function (ie, cancers, heart disease, carpal tunnel syndrome, De Quervain’s tenosynovitis, Parkinson’s disease, cerebellar ataxia, spinocerebellar degeneration, or other neurological disorders), participants on any osteoporosis treatments (denosumab, risedronate, zoledronate, alendronate, or teriparatide) which can affect CTX levels, or medications affecting bone/muscle metabolism (ie, estradiol, anastrozole, raloxifene, clonazepam, sodium valproate, phenytoin, gabapentin, carbamazepine, prednisolone). For instances where pathology reports (ie, estimated glomerular filtration rate [eGFR], calcium or parathyroid hormone [PTH] levels) were outside normative values, but no associated disease was listed on medical history, we did not exclude these participants as we were able to adjust for the potential confounders along with a comorbidity index.

Biochemical Assessments

Fasting venous blood samples were collected by a certified phlebotomist and processed by an accredited pathology laboratory service. The instrument, assay, and interassay coefficient of variation (COV) for the biochemical tests were as follows: CTX (Roche Cobas e411; Electrochemiluminescence Immunoassay; COV: 2.8%–8.4%), 25 hydroxyvitamin D (DiaSorin LiasonXL; Chemiluminescent Immunoassay; COV: 6.0%–9.8%), calcium (Beckman Coulter Au5800; Photometric Color; COV: 0.68%–1.34%), PTH (Beckman Coulter DXI 800; Paramagnetic Particle Chemiluminescent Immunoassay; COV: 2.5%–3.4%), and creatinine (Beckman Coulter Au5800; Kinetic Jaffe Method; COV: 1%–1.5%). eGFR was calculated using the Modification of Diet in Renal Disease Study equation: eGFR (mL/min/1.73 m²) = 175 × (S_cr)^−1.154 × (Age)^−0.203 × (0.742 if female) × (1.212 if African American) (18). Age- and sex-specific reference ranges for CTX were reported using data from an Australian population (19). Cut points for vitamin D deficiency (≤50 nmol/L), high PTH (>6.8 pmol/L), bone biomarkers, and abnormal eGFR (<60 mL/min/1.73 m²) were reported following recommended criteria (18,20).

Anthropometry, Lean Mass, and Muscle Function

Height (nearest 0.1 m; SECA Stadiometer) and weight (nearest 0.1 kg; TANITA) were measured using standard laboratory procedures, and body mass index (BMI) was calculated. Whole-body and regional lean mass, fat mass, and bone density were assessed by an imaging specialist using dual-energy x-ray absorptiometry (DXA; Hologic Inc., Bedford, MA), with appendicular lean mass adjusted for BMI (ALM/BMI), whole-body fat mass and femoral neck bone density used in the analysis. The neck of femur was used to identify osteopenia (T-score between −1 and −2.5) and osteoporosis (T-score ≤ −2.5) according to recommended criteria (21). For handgrip strength, participants were seated upright in an armless chair (46–49 cm in height) with elbow flexed at 90° and instructed to apply maximal pressure to a handheld Jamar hydraulic dynamometer (Sammons Preston Inc., Bolingbrook, Illinois) (16). Three trials were performed on each arm with the best used for analysis.

For physical function tests, the short physical performance battery (SPPB) (22) was used to assess 3 timed components; 5 times sit-to-stand, standing balance (tandem, semi-tandem, full tandem), and 4-m gait speed. The 4-m gait speed was measured using the GAIT Rite (CIRSystems Inc., Havertown, PA) system or by a standardized marked course (including 1-m acceleration and deceleration zones) and a stopwatch. Participants were asked to walk at normal speed with the fastest of 2× trials used for analysis. The Timed Up and Go (TUG) (23) was assessed at normal speed using a stopwatch and a 3-m marked track whereby the participant was instructed to rise from the chair, walk around a cone, and then return to the seated position. The best of 2 trials was used for analysis. If required, a gait aid was permitted during gait speed and TUG tests. Poor muscle function was defined either as low SPPB (≤8 points), slow sit-to-stand time (>15 seconds or unable to perform), or slow TUG (≥20 seconds) following validated cut points (24). A musculoskeletal imaging expert and an accredited exercise physiologist performed all imaging and muscle function tests, respectively, as previously reported (16).

Demographic, Lifestyle, and Clinical Assessments

All participants answered demographics, lifestyle, and clinical questionnaires led by either the fracture liaison nurse or the exercise physiologist. These included details on age (date of birth), sex, alcohol/smoking habits (current, previous, never), medications/medical history, physical activity levels (current, previous, never), and the number of falls and fractures occurring in the past 1 and 5 years, respectively, as previously reported (16). The Geriatric Depression Scale (25), Charlson Comorbidity Index (26), and the Mini Nutritional Assessment (27) were used to evaluate depression, comorbidities, and nutritional status, respectively. Wherever possible, demographic, lifestyle, and clinical information were cross-referenced with reports on medical records.

Statistical Analyses

Statistical analysis was performed using StataCorp LLC 16 (College Station, TX). Data are presented as frequency (percentage [%]) for categorical variables and mean (standard deviation) or median (interquartile range 25–75th [IQR]) for continuous variables, as appropriate. Scatter plots were used to visualize the relationship between the exposure, potential confounders, and outcomes of interest. Several variables, including the exposure (CTX), required transformation using natural logarithm to meet assumptions of a linear regression. Univariable linear regression was then performed to examine the associations between the exposure (CTX), potential confounders, and outcomes of interest. Multivariable linear regression was performed for each outcome, adjusted for confounders that had p < .100 on univariable regression. Beta (β) coefficients with 95% confidence interval (CI) present the fold change in CTX when variable increases for 1 unit. In cases where the variable was transformed, the coefficient represents the fold change in CTX with 10% increase in the variable. The diagnostic power of CTX was examined by calculating the area under the receiver operating characteristic (ROC) curve for the muscle function measures that were significant in multivariable analysis. Area under the ROC curve (AUC) was considered acceptable (0.7–0.8), excellent (0.8–0.9), or outstanding (>0.9), as previously described (28). p < .05 was considered statistically significant.

Results

Excluded Participants

After removing participants with excluded medications (n = 104) and medical conditions (n = 47), as well as missing CTX (n = 60) and implausible CTX (n = 4, <15 ng/L) values, a total of 299 community-dwelling older adults were included in the final analytical sample (Supplementary Figure 1).

Study Population

As seen in Table 1, median age of participants was 79 years (IQR: 73, 84), and over two-thirds (75.6%) were women. Median concentrations of CTX were 284 ng/L (IQR: 175, 440). According to normative values for CTX in Australia, 236 (79.0%) had normal values, 33 (11.0%) had low values, and 30 (10.0%) had high values (Table 2). Regarding demographic and clinical factors, 110 (36.8%) were previous smokers, 91 (30.4%) were alcohol consumers, 42 (14.0%) were at risk of malnutrition, and 104 (34.8%) performed physical activity at least once per week. Regarding biochemical status, 64 (21.7%) presented with vitamin D deficiency, while 147 (49.5%) had elevated PTH and 94 (32.6%) had declining eGFR. The prevalence of osteopenia and osteoporosis was 144 (52.6%) and 102 (37.2%), respectively, while 188 (66%) of participants had poor muscle function on the SPPB. A respective 126 (42.1%) and 179 (59.9%) of participants had experienced at least 1 fall or fracture in the past 1 and 5 years.

Univariable Associations Between CTX and Demographic, Lifestyle, and Clinical Factors

In univariable analyses, CTX was positively associated with men, previous smoking, malnutrition risk, comorbidities, depression, and serum PTH, and negatively associated with eGFR (p < .05). No association was observed between CTX and falls/fracture history or other possible confounders (Table 1).

Univariable- and Multivariable-Associations Between CTX and Outcome Measures

In univariable analyses, CTX was positively associated with ALM/BMI, sit-to-stand, and TUG, and negatively associated with handgrip strength, SPPB score, and standing balance (p < .05; Table 3). After adjusting for age, sex, smoking, malnutrition risk, comorbidities, depression, PTH, and eGFR, associations between CTX and lower-limb muscle function (SPPB, sit-to-stand, standing balance, and TUG) were slightly attenuated but remained significant in the fully adjusted model (p < .05; Table 2 and Figure 1). Associations between CTX and ALM/BMI and handgrip strength were no longer significant in multivariable analyses, nor were there any associations detected between CTX and gait speed, although the slope was in the hypothesized direction. In a subgroup analysis of the population, those with mobility limitations (low SPPB scores [≤8]) were positively associated with CTX in univariable and multivariable models (p < .05; Supplementary Table 1).

Diagnostic Power of CTX in Identifying Poor Muscle Function

As seen in Figure 2, the area under the ROC curves showed low diagnostic power (<0.70) of CTX in identifying poor performance on the SPPB (AUC: 0.63, 95% CI: 0.56, 0.70), chair rise (AUC: 0.64, 95% CI: 0.56, 0.72), and TUG (AUC: 0.61, 95% CI: 0.54, 0.68).

Discussion

We investigated the association between CTX and lean mass and function in community-dwelling older adults at high risk of falls, fractures, and mobility limitations, and found higher concentrations of CTX were associated with poorer lower-limb muscle function (but showed low diagnostic power for these measures) after controlling for covariates in multivariable models.

In multiple studies, CTX correlates with poorer bone microarchitecture (29,30), increased fracture risk (31), as well as being used as an efficacious biomarker to monitor the effect of antiresorptive treatments in older adults (32). Here, for the first time, higher levels of CTX demonstrated clinically relevant associations with lower-limb muscle function. Indeed, older adults with elevated concentrations of CTX were negatively associated with lower SPPB and standing balance scores, and positively associated with slower sit-to-stand and TUG times (all of which are predictors of adverse outcomes in older persons (33–35)). These findings add to the clinical data showing the associations between markers of bone metabolism (such as osteocalcin and sclerostin) and muscle mass and function (5,6), thus suggesting that bone and muscle are biologically linked through systemic and/or localized factors (4).

One possible explanation for the association between CTX and lower-limb muscle function may be its close biological links with the catabolic cytokine RANKL. Serum CTX has previously been shown to strongly correlate with multiple single-nucleotide polymorphisms in the OPG/RANKL/RANK signaling pathway in 1 130 adolescents (14) and 2 653 middle–older aged men (15). High levels of RANKL expression in mice (10) not only induces bone loss but also activates the nuclear factor-κB pathway and inhibits myogenic differentiation (36,37), leading to skeletal muscle dysfunction and alterations in glucose homeostasis. Thus, associations between higher levels of CTX and poorer lower-limb muscle function, as observed here, may be related to an upregulation of the RANK/RANKL/OPG signaling pathway in a population of older adults at high risk of falls, fractures, and mobility limitations (66% scored ≤ 8 points on the SPPB). Although as RANLK expression was not measured in serum or muscle, our suggested mechanism is yet to be confirmed.

We also calculated area under the ROC curves for the outcomes measures that were significant in multivariable analysis. CTX showed low diagnostic power in discriminating poor lower-limb muscle function. These findings were in line with our hypothesis, given that type 1 collagen is the most abundant protein found in the extracellular matrix of bone, and collagen cross-links (peptide fragments) are released into the serum during bone resorption (13,17,38). In contrast, the extracellular matrix surrounding skeletal muscle fibers is primarily composed of type IV collagen along with other proteins such as fibronectin, which make up this protective scaffolding (39).

CTX was not associated with bone density in our study but, given a substantial proportion (~90%) of participants were either osteopenic or osteoporotic, this finding was not surprising. It was interesting to note that CTX was not consistently associated with all outcome measures. The trend line for gait speed was in the hypothesized direction (ie, CTX negatively associated with gait speed) but did not reach statistical significance and grip strength showed no association. The sample size may explain the lack of statistical associations with these measures. Effect sizes of exercise interventions on grip strength/gait speed are small and sometimes negligible compared to other muscle function tests (40–44). This is likely due to the inherent nature of these tests. For instance, additional factors such as joint stiffness, range of motion, and spinal neural drive play an equal or greater contribution to gait speed than muscle size (45,46), and the upper-limb muscles required for grip strength are less affected by aging compared to the lower-limb muscles (47) required for sit-to-stand and TUG tests (24). Hence, larger sample sizes were likely needed to observe any possible associations between CTX and gait speed or grip strength. The lack of relationship between CTX and DXA-derived lean mass was unsurprising when considering this measure is precise but not accurate at measuring muscle mass due to the confounding inclusion of protein, water, and other connective tissues (48). Epidemiological studies including Osteoporotic Fractures in Men Study (2,49) and the UK Biobank (50) have shown that DXA-lean mass, when compared to accurate measures of muscle mass/size such as high-resolution peripheral quantitative computed tomography, magnetic resonance imaging, or D3-creatine dilution, produces conflicting associations with sarcopenia-related outcomes.

There are several strengths of our study, including careful exclusion of medications and medical conditions known to affect CTX status or lean mass/function, and our ability to control for multiple covariates (demographic, lifestyle, and clinical factors) in a population of community-dwelling older adults at high risk of osteoporosis, sarcopenia, and falls. However, our findings should also be taken in the context of the study limitations. First, no cause-and-effect can be derived from cross-sectional data, and our suggested mechanism cannot be confirmed as we did not measure RANKL in serum or muscle. Second, as these data were sought from a cross-sectional study of older adults attending a high-risk falls and fracture clinic, we cannot rule out the possibility of residual confounding due to the fracture date not being accounted for. In saying this, we do not believe this significantly affected our findings as there was no correlation between CTX and fractures (or falls). As described in the methodology, eligibility criteria for referral to our clinic also require a patient to be fully ambulant, and attendance is only permitted at least 3 months postfracture (CTX levels peak 4 weeks postfracture).

To conclude, higher CTX levels were associated with poorer lower-limb muscle function (but showed low diagnostic power for these measures) in community-dwelling older adults at high risk of falls, fractures, and mobility limitations. These clinical data build on the biomedical link between bone and muscle. Future longitudinal studies should now investigate the associations between CTX and muscle mass and function in older populations.

Acknowledgments

The authors would like to thank the Australian Institute for Musculoskeletal Science (AIMSS) for supporting the authors of this study.

Funding

None declared.

Conflict of Interest

None declared.

Author Contributions

Conception and study design: B.K., N.L., and G.D. Study conduct and data collection: B.K., N.L., M.S., and G.D. Data interpretation and analysis: B.K., N.L., M.S., S.V., and G.D. Drafting of the manuscript: B.K., N.L., S.V., M.S., J.A.P., and G.D. All authors reviewed the manuscript and approved the final version. B.K., N.L., S.V., and G.D. take responsibility for the integrity of the data analysis.

References

Author notes