PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Eur J Appl Physiol. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2809783
NIHMSID: NIHMS156729

Relationship of an advanced glycation end product, plasma carboxymethyl-lysine, with slow walking speed in older adults: the InCHIANTI study

Abstract

Advanced glycation end products (AGEs) are bioactive molecules found in foods and generated endogenously in the body. AGEs induce cross-linking of collagen and increase the stiffness of skeletal muscle and cartilage. We characterized the relationship between a plasma AGE, carboxymethyl-lysine (CML), and slow walking speed (lowest quintile of walking speed) in older adults. Walking speed over a 4 m course was assessed in 944 adults, aged ≥65 years, in the InCHIANTI study, a population-based study of aging and mobility disability conducted in two towns in Tuscany, Italy. Participants in the highest quartile of plasma CML were at higher risk of slow walking speed (Odds Ratio [O.R.] 1.56, 95% Confidence Interval [C.I.] 1.02-2.38, P = 0.04) compared to those in the lower three quartiles of plasma CML in a logistic regression models adjusting for age, education, cognitive function, smoking, and chronic diseases. After exclusion of participants with diabetes, participants in the highest quartile of plasma CML were at higher risk of slow walking speed (O.R. 1.87, 95% C.I. 1.15-3.04, P = 0.01) adjusting for the same covariates. In older community-dwelling adults, elevated plasma CML is independently associated with slow walking speed.

Keywords: Advanced glycation end products, aging, carboxymethyl-lysine, physical performance, walking speed

Introduction

Sarcopenia, or loss of muscle strength and muscle mass, is an important factor underlying mobility difficulties such as slow walking speed in older adults (Lauretani et al. 2003). Advanced glycation end products have been hypothesized to play a role in the pathogenesis of sarcopenia through AGE-mediated increases in inflammation and endothelial dysfunction in the microcirculation of skeletal muscle (Payne 2006) and through cross-linking of collagen in skeletal muscle (Haus et al. 2007). The connective tissue scaffold that surrounds individual muscle fibers, muscle bundles, the whole muscle plays an important role in the transfer of force from contractile units of muscle to tendon and bone. AGE concentrations in skeletal muscle of older adults is more than two-fold higher than that found in younger adults, suggesting that glycated-related cross-linking of intramuscular connective tissue may contribute to the decline in muscle function with aging (Haus et al. 2007). In addition, AGE-induced cross-linking of collagen has been shown to increase the stiffness of human articular cartilage (Verzijl et al. 2000; Verzijl et al. 2002).

Recently, it was shown that older, disabled, community-dwelling women with elevated serum carboxymethyl-lysine (CML), a dominant AGE in serum and tissues, were at higher risk of poor grip strength (Dalal et al. in press). It is not known whether older adults with high serum AGE concentrations are at higher risk of impaired physical performance and greater disability. We hypothesized that older community-dwelling adults with elevated plasma CML were at greater risk of having a slow walking speed. To address this hypothesis, we characterized the relationship between plasma CML and walking speed in a population-based study of older adults.

Subjects and Methods

The study participants consisted of men and women, aged 65 and older, who participated in the Invecchiare in Chianti, “Aging in the Chianti Area” (InCHIANTI) study, conducted in two small towns in Tuscany, Italy. The rationale, design, and data collection have been described elsewhere, and the main outcome of this longitudinal study is mobility disability (Ferrucci et al. 1999). Briefly, in August 1998, 1270 people aged 65 years and older were randomly selected from the population registry of Greve in Chianti (population 11,709) and Bagno a Ripoli (pop. 4,704), and of 1256 eligible subjects, 1155 (90.1%) agreed to participate. Participants received an extensive description of the study and participated after written, informed consent. The study protocol complied with the Declaration of Helsinki and was approved by the Italian National Institute of Research and Care on Aging Ethical Committee. The plan for additional laboratory and data analyses were approved by the Institutional Review Board of the Johns Hopkins University School of Medicine.

Of the 1155 participants ≥65 years seen at enrollment, 1055 (91.3%) participated in the blood drawing. There were 944 (81.7%) participants at enrollment with both plasma carboxymethyl-lysine and 4 m walking speed available for this analysis. The subjects who did not participate in the blood drawing were generally older and had greater comorbidity than the subjects who participated in the blood drawing, as reported elsewhere (Schrager et al. 2007). None of the participants were taking dietary supplements.

Data collection consisted of a home interview and a medical evaluation at the study clinic, which took place within 21 days after the home interview. Demographic information was collected, including sociodemographic variables (age, sex, and years of education), smoking history, and use of medications. All participants were examined by a trained geriatrician, and diseases were ascertained according to standard, pre-established criteria and algorithms based upon those used in the Women’s Health and Aging Study for coronary heart disease, congestive heart failure, peripheral artery diseases, diabetes mellitus, chronic obstructive pulmonary disease, and osteoarthritis (Guralnik et al. 1995). Weight was measured using a high-precision mechanical scale. Standing height was measured to the nearest 0.1 cm. Body mass index (BMI) was calculated as weight/height2 (kg/m2). BMI was categorized as underweight (<18.5 kg/m2), normal range (18.5-24.9 kg/m2), overweight (≥25-29.9 kg/m2) and obese (≥30 kg/m2) according to World Health Organization criteria (James et al. 2001). Mini-Mental Status Examination (MMSE) was administered at enrollment, and an MMSE score <24 was considered consistent with cognitive impairment (Folstein et al. 1975). Renal insufficiency was defined as estimated glomerular filtration rate of <60 mL/min/1.73 m2 using the four-variable Modification of Diet in Renal Disease Study equation of Levey and colleagues (Levey et al. 1999)

In the 4-meter walking test, the participants were instructed to walk at their normal pace over a 4 meter distance, were repeated twice and the average time was used (Bandinelli et al. 2006). Participants were categorized as having slow walking speed if they were in the slowest quintile of walking speed, which in this population was <0.79 m/sec.

Blood samples were collected in the morning after a 12-h fast. Aliquots of serum and plasma were immediately obtained and stored at -80° C. The measure of plasma AGEs in this study was plasma carboxymethyl-lysine (CML). CML is a dominant circulating AGE, one of the best characterized of all the AGEs, and a dominant AGE in tissue proteins (Reddy et al. 1995). CML was measured using a competitive enzyme-linked immunosorbent assay (ELISA) (AGE-CML ELISA, Microcoat, Penzberg, Germany) (Boehm et al. 2004). This assay has been validated (Zhang et al. 2005), is specific, and shows no cross-reactivity with other compounds (Boehm et al. 2004). The minimum level of detectability of the assay is 5 ng/mL (Boehm et al. 2004), which is below the concentration that has been found in human studies. The intra-assay and inter-assay coefficients of variation were both <5%.

Variables are reported as medians (25th, 75th percentiles) or as percentages. Plasma CML was divided into quartiles, and the cut-off at the highest quartile was 424 ng/mL. Age and BMI were used as categorical variables because the relationship between age and BMI, respectively, with slow walking speed was not linear. Logistic regression models were used to examine the relationship between plasma CML and other risk factors with slow walking speed. Covariates that were significant in univariate analyses were included in the final multivariate models. All analyses were performed using SAS (v. 9.1.3, SAS Institute, Inc., Cary, NC) with a statistical significance level set at P <0.05.

Results

The demographic, anthropometric, and disease characteristics of participants with and without slow walking speed are shown in Table 1. Participants with slow walking speed were older and more likely to be female, obese, not currently smoking, with MMSE score <24, and with depression, hypertension, congestive heart failure, peripheral artery disease, stroke, diabetes, and renal insufficiency compared with participants without slow walking speed. Plasma CML concentrations were higher in participants with slow walking speed compared to those without slow walking speed. There were no significant differences in the proportion of angina or cancer among participants with and without slow walking speed.

Table 1
Demographic and Health Characteristics of Adults, Aged ≥65 Years, in the InCHIANTI Study in Lowest versus Upper Four Quintiles of Walking Speed

Participants in the highest quartile of plasma CML had greater odds of slow walking speed in separate multivariate logistic regression models, adjusted for age and sex, and additionally for education, smoking, MMSE, and for chronic diseases, respectively. The association between plasma CML and slow walking speed remained significant in similar models after excluding participants with diabetes.

Discussion

The present study shows that elevated plasma CML is independently associated with slow walking speed in older community-dwelling adults. To our knowledge, this is the first study to show an association between elevated AGEs and impaired physical performance in older, community-dwelling men and women. This observation is consistent with the hypothesis that AGEs play a role in sarcopenia (Payne 2006). Increased AGEs may contribute to increased stiffness in muscle tissue and reduced viscoelastic properties of muscle and thus impair muscle function (Haus et al. 2007). In rats, AGEs accumulate in skeletal muscle with aging (Snow et al. 2008). In a rabbit model, cross-linking of collagen by nonenzymatic glycation increased the structural stiffness of Achilles tendon (Reddy 2004). Elevated AGEs are associated with increased arterial stiffness (Schram et al. 2005; Semba et al. 2008) and bone rigidity (Vashisth et al. 2001; Hernandez et al. 2005; Tang et al. 2007) which is thought to occur through cross-linking of collagen. Increased levels of CML have been described in skeletal muscle of healthy middle-aged men who reported weight increase compared to men who maintained stable weight (de la Maza et al 2008). In addition, immunostaining of skeletal muscle for CML was co-localized with immunostaining for markers of oxidative injury (8-hydroxy-deoxyguanosine and 4-hydroxy-2-nonenal) and inflammation (tumor necrosis factor-α).

AGEs are a potentially modifiable risk factor, since the diet is a major source of exogenous AGEs (Goldberg et al. 2004). The Western diet is rich in AGES, especially in foods that are processed at high temperatures. Serum CML concentrations show a moderate correlation with dietary intake of AGEs in older adults (Uribarri et al. 2007), and serum CML concentrations vary with dietary intake of AGEs (Negrean et al. 2007). Elevated serum CML concentrations have been associated with low grip strength (Dalal et al, 2009).

A limitation of this study is that causality cannot be strongly inferred from this cross-sectional study. It is possible that older adults with slow walking speed were less physically able to have access to a more healthy diet, i.e., greater intake of fruits and vegetables and lower intake of foods processed at very high temperatures. The relationship between serum AGEs and changes in walking speed needs to be examined in the future in large, prospective studies to determine whether elevated serum AGEs predict a decline in walking speed. An important consideration in longitudinal studies is the competing risk of mortality, since older adults with elevated serum AGEs are at an increased risk of all-cause and cardiovascular disease mortality (Semba et al. 2009).

In the present study, the relationship between elevated CML and slow walking speed remained strong even after exclusion of participants with diabetes. The relationship between AGEs and adverse clinical outcomes has mostly been studied in patients with diabetes and/or end-stage renal disease (Basta et al. 2004; Bohlender et al. 2005). Recent investigations show that elevated AGEs are associated with arterial stiffness (Semba et al. 2008) and cardiovascular mortality (Semba et al, 2009) in persons without diabetes. These recent findings together suggest that elevated AGEs are an important independent risk factor for adverse aging-related outcomes in older, community-dwelling adults.

In conclusion, older adults with elevated plasma CML, an advanced glycation end product, had greater risk of slow walking speed. Further studies are needed to determine whether elevated circulating AGEs are predictive of a decline in walking speed in older adults and to corroborate these findings in other cohort studies of aging.

Table 2
Multivariate logistic regression models of the relation of plasma CML with slow walking speed1 in adults, aged ≥65 years, in the InCHIANTI Study

Acknowledgments

This work was supported by National Institute on Aging Grant R01 AG027012, R56 AG027012, R01 AG029148 and the Intramural Research Program, National Institute on Aging, NIH.

Contributor Information

Richard D. Semba, Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Stefania Bandinelli, Azienda Sanitaria Firenze, Florence, Italy.

Kai Sun, Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Jack M. Guralnik, Laboratory of Epidemiology, Demography, and Biometry, National Institute on Aging, Bethesda, Maryland.

Luigi Ferrucci, Longitudinal Studies Section, Clinical Research Branch, National Institute on Aging, Baltimore, Maryland.

References

  • Bandinelli S, Pozzi M, Lauretani F, Phillips C, Shumway-Cook A, Guralnik JM, Ferrucci L. Adding challenge to performance-based tests of walking: The Walking InCHIANTI Toolkit (WIT) Am J Phys Med Rehabil. 2006;85:986–991. [PMC free article] [PubMed]
  • Basta G, Schmidt AM, de Caterina R. Advanced glycation end products and vascular inflammation: implications for accelerated atherosclerosis in diabetes. Cardiovasc Res. 2004;63:582–592. [PubMed]
  • Boehm BO, Schilling S, Rosinger S, Lang GE, Lang GK, Kietsch-Engel R, Stahl P. Elevated serum levels of Nε-carboxymethyl-lysine, an advanced glycation end product, are associated with proliferative diabetic retinopathy and macular oedema. Diabetologia. 2004;47:1376–1379. [PubMed]
  • Bohlender JM, Franke S, Stein G, Wolf G. Advanced glycation end products and the kidney. Am J Renal Physiol. 2005;289:F645–F659. [PubMed]
  • Dalal M, Ferrucci L, Sun K, Beck J, Fried LP, Semba RD. Elevated serum advanced glycation end products and poor grip strength in older community-dwelling women. J Gerontol A Biol Sci Med Sci. 2009;64:132–137. [PMC free article] [PubMed]
  • De la Maza MP, Uribarri J, Olivares D, Hirsch S, Leiva L, Barrera G, Bunout D. Weight increase is associated with skeletal muscle immunostaining for advanced glycation end products, receptor for advanced glycation end products, and oxidation injury. Rejuvenation Res. 2008;11:1041–1048. [PubMed]
  • Folstein MF, Folstein SE, McHugh PR. “Mini-Mental State” A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975;12:189–198. [PubMed]
  • Goldberg T, Cai W, Peppa M, Dardaine V, Baliga BW, Uribarri J, Vlassara H. Advanced glycoxidation end products in commonly consumed foods. J Am Diet Assoc. 2004;104:1287–1291. [PubMed]
  • Guralnik JM, Fried LP, Simonsick EM, Kasper D, Lafferty ME. The Women’s Health and Aging Study: Health and Social Characteristics of Older Women with Disability. Bethesda, MD: National Institute on Aging; 1995. NIH Publication No 95-4009.
  • Haus JM, Carrithers JA, Trappe SW, Trappe TA. Collagen, cross-linking and advanced glycation endproducts in aging human skeletal muscle. J Appl Physiol. 2007;103:2068–2076. [PubMed]
  • Hernandez CJ, Tang SY, Baumbach BM, Hwu PB, Sakkee AN, van der Ham F, DeGroot J, Bank RA, Keaveny TM. Trabecular microfracture and the influence of pyridinium and non-enzymatic glycation-mediated collagen cross-links. Bone. 2005;37:825–832. [PMC free article] [PubMed]
  • James PT, Leach R, Kalamara E, Shayeghi M. The worldwide obesity epidemic. Obes Res. 2001;9(suppl 4):228S–233S. [PubMed]
  • Lauretani F, Russo CR, Bandinelli S, Bartali B, Cavazzini C, Di Iorio A, Corsi AM, Rantanen T, Guralnik JM, Ferrucci L. Age-associated changes in skeletal muscles and their effect on mobility: an operational diagnosis of sarcopenia. J Appl Physiol. 2003;95:1851–1860. [PubMed]
  • Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Ann Intern Med. 1999;130:461–470. [PubMed]
  • Negrean M, Stirban A, Stratmann B, Gawlowski T, Horstmann T, Götting C, Kleesiek K, Mueller-Roesel M, Koschinsky T, Uribarri J, Vlassara H, Tschoepe D. Effects of low-and high-advanced glycation endproduct meals on macro-and microvascular endothelial function and oxidative stress in patients with type 2 diabetes mellitus. Am J Clin Nutr. 2007;85:1236–1243. [PubMed]
  • Payne GW. Effect of inflammation on the aging microcirculation: impact on skeletal muscle blood flow control. Microcirculation. 2006;13:343–352. [PubMed]
  • Reddy GK. Cross-linking in collagen by nonenzymatic glycation increases the matrix stiffness in rabbit Achilles tendon. Exp Diab Res. 2004;5:143–153. [PMC free article] [PubMed]
  • Reddy S, Bichler J, Wells-Knecht KJ, Thorpe SR, Baynes JW. N epsilon-(carboxymethyl)lysine is a dominant advanced glycation end product (AGE) antigen in tissue proteins. Biochemistry. 1995;34:10872–10878. [PubMed]
  • Schrager MA, Metter EJ, Simonsick E, Ble A, Bandinelli S, Lauretani F, Ferrucci L. Sarcopenic obesity and inflammation in the InCHIANTI study. J Appl Physiol. 2007;102:919–925. [PMC free article] [PubMed]
  • Schram MT, Schalkwijk CG, Bootsma AH, Fuller JH, Chaturvedi N, Stehouwer CDA. Advanced glycation end products are associated with pulse pressure in type 1 diabetes. The EURODIAB prospective complications study Hypertension. 2005;46:232–237. [PubMed]
  • Semba RD, Najjar SS, Sun K, Lakatta EG, Ferrucci L. Serum carboxymethyl-lysine, an advanced glycation end product, is associated with increased aortic pulse wave velocity in adults. Am J Hypertens Nov. 2008;20 Epub ahead of print. [PMC free article] [PubMed]
  • Semba RD, Ferrucci L, Sun K, Beck J, Dalal M, Varadhan R, Walston J, Guralnik JM, Fried LP. Advanced glycation end products and their circulating receptors predict cardiovascular disease mortality in older community-dwelling women. Aging Clin Exp Res. 2009;21:183–190. [PMC free article] [PubMed]
  • Snow LM, Fugere NA, Thompson LV. Advanced glycation end-product accumulation and associated protein modification in type II skeletal muscle with aging. J Gerontol A Biol Sci Med Sci. 2007;62:1204–1210. [PubMed]
  • Tang SY, Zeenath U, Vashishth D. Effects of non-enzymatic glycation on cancellous bone fragility. Bone. 2007;40:1144–1151. [PubMed]
  • Uribarri J, Cai W, Peppa M, Goodman S, Ferrucci L, Striker G, Vlassara H. Circulating glycotoxins and dietary advanced glyation endproducts: two links to inflammatory response, oxidative stress, and aging. J Gerontol A Biol Sci Med Sci. 2007;72:427–433. [PMC free article] [PubMed]
  • Vashishth D, Gibson GJ, Khoury JI, Schaffler MB, Kimura J, Fyhrie DP. Influence of nonenzymatic glycation on biomechanical properties of cortical bone. Bone. 2001;28:195–201. [PubMed]
  • Verzijl N, deGroot J, Oldehinkel E, Bank RA, Thorpe SR, Baynes JW, Bayliss MT, Bijlsma JWJ, Lafeber FPJG, teKoppele JM. Age-related accumulation of Maillard reaction products in human articular cartilage collagen. Biochem J. 2000;350:381–387. [PubMed]
  • Verzijl N, deGroot J, Ben Zaken C, Braun-Benjamin O, Maroudas A, Bank RA, Mizrahi J, Schalkwijk CG, Thorpe SR, Baynes JW, Bijlsma WJW, Lafeber FPJG, teKoppele JM. Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: a possible mechanism through which age is a risk factor for osteoarthritis. Arthritis Rheum. 2002;46:114–123. [PubMed]
  • Zhang X, Frischmann M, Kientsch-Engel R, Steinmann K, Stopper H, Niwa T, Pischetsrieder M. Two immunochemical assays to measure advanced glycation end-products in serum from dialysis patients. Clin Chem Lab Med. 2005;43:503–511. [PubMed]