Advanced glycation end products (AGE) in bone tissue are associated with impaired biomechanical properties and increased fracture risk. Here we examine whether serum levels of the AGE carboxy-methyl-lysine (CML) are associated with risk of hip fracture. We followed 3373 participants from the Cardiovascular Health Study (age 78 years; range, 68–102 years; 39.8% male) for a median of 9.22 years (range, 0.01–12.07 years). Rates of incident hip fracture were calculated by quartiles of baseline CML levels, and hazard ratios were adjusted for covariates associated with hip fracture risk. A subcohort of 1315 participants had bone mineral density (BMD) measurement. There were 348 hip fractures during follow-up, with incidence rates of hip fracture by CML quartiles of 0.94, 1.34, 1.18, and 1.69 per 100 participant-years. The unadjusted hazard ratio of hip fracture increased with each 1 SD increase (189 ng/mL) of CML level (hazard ratio, 1.27; 95% confidence interval [CI], 1.16–1.40]; p < 0.001). Sequential adjustment for age, gender, race/ethnicity, body mass index (BMI), smoking, alcohol consumption, prevalent coronary heart disease (CHD), energy expenditure, and estimated glomerular filtration rate (based on cystatin C), moderately attenuated the hazard ratio for fracture (1.17; 95% CI, 1.05–1.31; p = 0.006). In the cohort with BMD testing, total hip BMD was not significantly associated with CML levels. We conclude that increasing levels of CML are associated with hip fracture risk in older adults, independent of hip BMD. These results implicate AGE in the pathogenesis of hip fractures. © 2014 American Society for Bone and Mineral Research.
A characteristic of biological aging is the non-enzymatic glycation of long-lasting proteins, such as collagen. Approximately 90% of bone matrix protein consists of collagen. In contrast to enzymatic cross-linking of collagen fibrils, which orients collagen fibrils and contributes to tensile strength, non-enzymatic glycation of collagen weakens collagen's biomechanical properties. This occurs because of three factors. (1) Glycation is a marker of ambient oxidative and carbonyl stress that increases with “biological” aging. Although more extreme in diabetes (DM), glycation increases with age in all people. (2) Glycation products—termed advanced glycation end products (AGEs)—are inducers of oxidative stress and inflammation acting through the receptor for AGE (RAGE) to activate NF-kB and its downstream inflammatory proteins. (3) Accumulation of AGEs in bone leads to increased bone matrix stiffening and fragility. In vitro cell culture studies show that non-enzymatic glycation of collagen makes it resistant to osteoclastic bone resorption and decreases osteoblast differentiation and proliferation. Excess amounts of “old” bone result from these processes.
Carboxy-methyl-lysine (CML) is the major AGE epitope recognized by antibodies prepared against AGE proteins. It forms when oxoaldehyde glyoxal reacts with lysine. It reflects the combination of oxidation and glycation of proteins. CML accumulation has been shown to parallel AGE formation. In addition, CML appears to be the dominant component of AGEs; on average, 30% of lysine residues present on a protein are converted to CML after glycation.
Few clinical investigations have studied AGEs and bone fracture risk. Several that have examined AGEs and fracture risk have relied upon cadaveric bone specimens and tissue AGE levels. These types of study are time-consuming, expensive, and too invasive for clinical use. Other studies have used urine levels of the AGE pentosidine. The results have been inconsistent and primarily focused upon vertebral fractures. One study showed no independent association, others showed positive associations,[7, 8] another showed a positive effect in women but not men, and yet another, an association in people with DM but not in people without DM. The relationship of circulating AGEs with fracture risk has not been evaluated, although one study examined the association of serum CML levels with the degree of osteoporosis. In the present study, we examine serum CML levels in a cohort of older adults to determine whether they are prospectively associated with hip fracture risk. The cohort is drawn from the Cardiovascular Health Study (CHS), an ongoing population-based study of individuals with a mean age of 78 years at the time of these measurements.
Subjects and Methods
The CHS is a prospective, observational population-based cohort study of 5,888 Medicare-eligible adults ≥65 years old in four U.S. communities. Two cohorts were recruited. In the original cohort, 5201 eligible men and women were enrolled during 1989–1990. In the second recruitment, during 1992–1993, an additional 687 predominantly black men and women were enrolled. Clinic examinations were performed at study baseline and at annual visits through 1998–1999, and again in 2005–2006. Participants were contacted by telephone annually between exams, and twice per year during 2000–2004 and in 2007, when no clinic examinations occurred. All participants signed informed consent upon study entry.
The cohort for this analysis was taken from the 1996–1997 examination. At that point, 1180 of the original 5888 participants had died, and 296 were lost to follow-up or refused further visits. Of the remaining 4412 participants, 3373 had an adequate blood sample available from the 1996–1997 visit.
Laboratory tests from the 1996–1997 visit were done using methods previously reported. As a measure of renal function, cystatin C levels (mg/L) were measured from stored samples using a BNII nephelometer (Dade Behring Inc., Deerfield, IL, USA) that used a particle enhanced immunonephelometric assay (N Latex Cystatin-C). Estimated glomerular filtration rate (eGFR) was calculated based on cystatin C levels. Information regarding smoking history, medication use, history of falling in the preceding year, amount of energy (kcal) expended per week based on the Minnesota Leisure Time Activity Questionnaire, and alcohol use were obtained at the time of the visit. Technicians directly measured weight, blood pressure, waist circumference, height, grip strength, and the time needed to walk 15 feet (in seconds). Frailty was defined using a phenotype that requires at least three of the following criteria to be present: unintended weight loss >10 lbs in the prior year; self-reported exhaustion most of the time; physical activity in the lowest 20% of CHS cohort (<383 kcal/week in men; <270 kcal/week in women); weakness as measured by grip strength (lowest 20% of CHS cohort: ≤23 kg/m2 in men, <17 kg/m2 in women); and slowness of walking (lowest 20% in each sex, adjusted for height). Those with one or two criteria were considered “pre-frail,” an intermediate syndrome with increased risk for the development of frailty. Random urine specimens were used to measure urinary albumin to creatinine ratio; albuminuria testing was available for 2972 participants.
Measurement of hip fracture
Data on hip fracture was obtained through patient report and confirmed from hospital discharge codes. Incident hip fracture was identified using International Classification of Diseases, Ninth Revision (ICD-9) codes from hospitalization records from the time of the 1996–1997 visit through June 30, 2008. CHS prospectively gathers all hospitalization data, including discharge summaries, from participants every 6 months. To ensure completeness of hospitalization records, data were checked against Medicare claims data to identify any hospitalizations that were not reported by the participant. Hip fracture was defined as an ICD-9 code of 820.xx. Admissions for pathologic fractures (ICD-9 code 773.1x) and motor vehicle accidents (E810.xx–E825.xx) were excluded.
Measurement of CML
Fasting samples were stored at the University of Vermont (Burlington, VT, USA) at –80°C until assays for CML were measured using a competitive enzyme-linked immunosorbent assay (ELISA) (AGE-CML ELISA; Microcoat, Penzberg, Germany). This assay has been validated, is specific, and shows no cross-reactivity with other compounds. The minimum level of detectability of the assay is 5 ng/mL, below the concentration found in human studies. Both the intraassay and interassay coefficients of variation were <5%. Further details of the assay are described elsewhere.
Bone mineral density measurement
A subset of the cohort from two field centers (n = 1315) underwent bone mineral density (BMD) scanning 1 to 2 years before CML levels were obtained. BMD was measured by dual-energy X-ray absorptiometry (DXA) (QDR 2000 or 2000 ± ; Hologic, Inc, Bedford, MA, USA). All scans were completed using the array beam mode. Scans were read blindly at the University of California, San Francisco reading center with Hologic software version 7.10. The coefficient of variation for total hip BMD was <0.75%.
We graphically examined the distribution of CML levels. Because the distribution of CML is highly skewed at large values, we Winsorized the top 1% (ie, we set values above the 99th percentile to that value; Supplementary Fig. 1). This approach resulted in replacing CML values for 33 individuals with the 99th CML percentile of 1435 ng/mL. For descriptive purposes, participants were categorized by quartiles of the distribution of Winsorized CML levels. We show means ± SDs for continuous variables and counts (%) for categorical variables. Linear trend tests across quartiles of CML were used for continuous variables, and χ2 tests for categorical variables.
Kaplan-Meier curves were generated to describe the association between CML concentrations and time to incident hip fracture. Time to event was calculated as the interval in years from the baseline visit in 1996–1997 to the earliest date of first incident hip fracture, death, loss to follow-up, or end of follow-up on June 30, 2008. Cox proportional hazards models were used to estimate the hazard ratio (HR) of incident fracture associated with a 1 SD increase in CML level (189 ng/mL) after confirming that the association was linear using generalized additive models. Adjustments were made in nested fashion as follows: unadjusted; Model 1 (demographic adjusted) adjusted for age, gender, race/ethnicity, and clinic site; Model 2 (osteoporosis adjustment) was adjusted for factors in Model 1 and for factors known to be associated with risk of osteoporosis: prevalent coronary heart disease, smoking, body mass index (BMI), alcohol use, level of physical activity, and baseline eGFR. Model 3 additionally included a history of falls, which could be a consequence of CML levels. We also tested additional adjustment for log2-transformed albuminuria among individuals with available measurements. Age categories (<75, 75–84, and 85+ years) were used as strata to improve the fit of the model. We evaluated the validity of the proportional hazards assumption graphically and numerically using Schoenfeld residuals and found no meaningful departures.
We examined the association of CML and fracture separately in men and women, and in those with and without DM, and tested for interaction with cross-product terms.
In the subcohort with BMD measurements, we calculated the correlation between CML levels Winsorized at the upper 1st percentile with total hip BMD. We also examined the association of CML levels on total hip BMD adjusted for factors related to BMD using models similar to Models 1 to 3 (with frailty status added to Model 3). Cox regression models were used to gauge whether CML levels were associated with hip fracture risk after adjusting for Model 2 covariates and hip BMD. We also repeated our analyses of CML and hip fracture excluding individuals who reported use of calcium and/or vitamin D supplements; only one individual reported use of a bisphosphonate.
Analyses were conducted using R (R Development Core Team).
The median value of CML was 584 ng/mL (interquartile range [IQR]: 498, 703 ng/mL; Supplementary Fig. 1). Baseline characteristics of participants by quartiles of CML levels are shown in Table 1.
|Quartile I (n = 844)||Quartile II (n = 843)||Quartile III (n = 843)||Quartile IV (n = 843)||p|
|CML level (ng/mL)a||439.8 ± 47.7||546.7 ± 35.3||636.3 ± 34.2||876.4 ± 182.7|
|Black race (%)||16.8||15.1||17.1||15.1||<0.001|
|Age (years)||77.0 ± 4.3||77.9 ± 4.8||78.5 ± 4.8||78.9 ± 5.2||<0.001|
|BMI (kg/m2)||28.4 ± 4.7||27.1 ± 4.5||26.4 ± 4.5||25.8 ± 4.5||<0.001|
|SBP (mmHg)||136.2 ± 19.2||136.2 ± 20.2||136.7 ± 21.6||138.2 ± 21.6||0.04|
|DBP (mmHg)||69.9 ± 11.2||70.4 ± 10.2||70.2 ± 11.8||69.3 ± 11.8||0.25|
|Time to walk 15 feet (seconds)||5.6 ± 2.2||5.8 ± 3.2||5.8 ± 2.7||6.2 ± 4.3||<0.001|
|Fell in last year (%)||15.7||18.5||18.7||21.9||0.015|
|Energy expenditure/week (mean, kcal)||1319 ± 1787||1297 ± 1607||1284 ± 1638||1160 ± 1612||0.06|
|Education >12 years (%)||44.5||47.3||49.5||49.6||0.12|
|C-reactive protein (nmol/L)||50 ± 84||50 ± 88||40 ± 56||42 ± 74||0.01|
|eGFR cystatin (mL/min)||73.9 ± 17.3||71.8 ± 18.9||70.2 ± 18.3||65.6 ± 22.2||<0.001|
|Fasting glucose (mmol/L)||5.9 ± 1.8||5.7 ± 1.8||5.7 ± 1.9||5.5 ± 1.6||<0.001|
Over a median follow-up period of 9.22 years (IQR: 5.12, 11.42 years), there were 348 hip fractures during 27,409 person-years of follow-up (Table 2). Survival free of hip fracture was highest among individuals with the lowest CML levels, similar among those in the intermediate quartiles, and lowest among those with the highest levels (log-rank p < 0.001; Fig. 1).
|Total||Quartile 1||Quartile 2||Quartile 3||Quartile 4|
|Hip fracture events||348||69||94||81||104|
|Person-years of follow up||27409||7371||7029||6860||6149|
|Incidence rate per 100 person-years (95% CI)||1.27 (1.14–1.41)||0.94 (0.74–1.19)||1.34 (1.09–1.64)||1.18 (0.95–1.47)||1.69 (1.40–2.05)|
The unadjusted HR for hip fracture risk per 1-SD (189 ng/mL) increment in CML levels, Winsorized at the upper 1% of the distribution, was 1.27 (95% confidence interval [CI], 1.15–1.40) (Table 3). Adjustment for demographic factors had little impact on the HR. Additional adjustment for factors associated with osteoporosis moderately attenuated the HR of hip fracture with CML levels. Further adjustment for falling—in the causal pathway for fractures—did not attenuate the association of CML level change with hip fracture risk, nor did additional adjustment for frailty status or albuminuria (HR 1.17; 95% CI, 1.05–1.31; p = 0.006). Similarly, the association differed little even after exclusion of individuals taking calcium supplements or a bisphosphonate (HR adjusted for Model 2 covariates 1.19; 95% CI, 1.04–1.35; p = 0.009).
|Hazard ratio||95% CI||p|
When the cohort was stratified by gender, men and women had similar HR for hip fracture in association with CML in the fully adjusted model (Supplementary Table 1). There were also no differences in HR between participants with or without diabetes.
CML levels and total hip bone density
Among those undergoing DXA scanning the mean total hip BMD was 0.83 ± 0.18 mg calcium/cm2 (median 0.82; IQR: 0.70, 0.95) and the mean T-score was –1.4 ± 1.42. Osteoporosis (T-score < –2.5) was present in 23.3% of individuals.
The correlation between the hip bone density and CML levels was small (r = –0.073; p = 0.01). In adjusted linear regression models, the association of CML with hip BMD was modest and not statistically significant (decrease in BMD per SD increase –0.007; SEM 0.004; p = 0.07). In Cox regression models, a 1-SD increase of Winsorized CML levels remained significantly associated with hip fracture risk (HR, 1.25; 95% CI, 1.02–1.52; p = 0.03) even after adjustment for total hip BMD and Model 2 covariates.
To the best of our knowledge, this is the first study to examine circulating levels of CML as a biomarker of hip fracture risk. The HR of hip fracture risk increased with increasing CML levels, even after adjustment for osteoporosis risk factors. This association was independent of eGFR, albuminuria, and hip BMD.
There have been few clinical investigations of the contribution of bone, serum, or urine AGEs to fracture risk. Most reports have examined pentosidine, an AGE whose levels can be measured in bone through its fluorescent properties. Cadaveric models show a correlation between increased bone AGEs and bone fracture. In hip fracture specimens,[20, 21] pentosidine levels were elevated in cortical and cancellous bone compared to age-matched controls. In observational studies, urine or serum pentosidine levels predicted vertebral fractures in postmenopausal women and older adults with DM.[7, 9, 10] These studies, like our own, showed that AGE appear to be associated with fracture risk independent of hip BMD. Of note, pentosidine leads to collagen cross-linking in bone, whereas CML is a chemical adduct on proteins, and hence the biological effects of these two AGEs may differ.
We did not find a significant relationship between hip BMD and circulating CML levels in regression models. Further, there remained a significant association of CML levels with risk of hip fracture, even adjusting for hip BMD. These findings are consistent with the hypothesis that fracture risk in association with CML levels is through impaired bone quality, not through bone quantity. Whether pharmacological treatment of osteoporosis can modify this apparent effect is uncertain, but in vitro studies suggest opposing effects of bisphosphonates and AGEs on osteoblasts. Similarly, AGE crosslink breakers exist, although their effects on bone quality remain to be tested.
Advantages of this study include a well-characterized cohort with long follow-up, information on multiple covariates, the examination of men and women, and rigorous ascertainment of study outcomes. The number of hip fractures was large and more than in any other study that has examined AGEs and the risk of fracture. The mean eGFR (based on cystatin C levels) was >60 mL/min/1.73 m2 in all quartiles of CML, mitigating the potential confounding effects of chronic kidney disease. A limitation of our study is that CHS is limited in its characterization of bone health. In particular, markers of bone turnover and resorption were not performed. Also, CML and BMD were each only measured once, and biological variability may have led us to underestimate the association of repeated measures of CML with hip fracture over time. Finally, glycosylated hemoglobin (HbA1c) and pentosidine levels were not measured, thereby preventing us from comparing our findings to those of other studies.
In conclusion, we observed a statistically significant prospective association of CML levels with incident hip fracture risk. The mechanisms of this association are uncertain, but the association was independent of BMD. Our findings suggest that AGEs may represent a promising marker for categorizing and perhaps even ameliorating future hip fracture risk.
All authors state that they have no conflicts of interest.
This research was supported by contracts HHSN268201200036C, HHSN268200800007C, N01 HC55222, N01HC85079, N01HC85080, N01HC85081, N01HC85082, N01HC85083, N01HC85086, and grants HL094555 and HL080295 from the National Heart, Lung, and Blood Institute (NHLBI), with additional contribution from the National Institute of Neurological Disorders and Stroke (NINDS). Additional support was provided by AG023629 from the National Institute on Aging (NIA). A full list of principal CHS investigators and institutions can be found at CHS-NHLBI.org.
Authors' roles: JIB wrote the paper, helped with analysis, and contributed to the intellectual content of the paper. PB performed statistical analyses and contributed to the intellectual content of the paper. SJZ obtained funding for the project and contributed to the intellectual content of the paper. JRK obtained funding for the project and contributed to the intellectual content of the paper. LD obtained funding for the project and contributed to the intellectual content of the paper. JHI obtained funding for the project and contributed to the intellectual content of the paper. RPT performed laboratory assays and contributed to the intellectual content of the paper. DSS oversees the diabetes arm of the CHS extension project, is a member of the CHS steering committee, and contributed to the intellectual content of the paper. JAC contributed to the intellectual content of the paper. KJM obtained funding for the project, oversaw this project, contributed to the intellectual content of the paper and its writing, and takes responsibility for data integrity.