Semba RD, Cotch MF, Gudnason V, Eiríksdottir G, Harris TB, Sun K, Klein R, Jonasson F, Ferrucci L, Schaumberg DA. Serum Carboxymethyllysine, an Advanced Glycation End Product, and Age-Related Macular DegenerationThe Age, Gene/Environment Susceptibility–Reykjavik Study. JAMA Ophthalmol. 2014;132(4):464-470. doi:10.1001/jamaophthalmol.2013.7664
Advanced glycation end products have been implicated in the pathogenesis of age-related macular degeneration (AMD).
To investigate the relationship between serum carboxymethyllysine (CML), a major circulating advanced glycation end product, and AMD in older adults.
Design, Setting, and Participants
Cross-sectional study of a population-based sample of 4907 older adults (aged ≥66 years) in the Age, Gene/Environment Susceptibility–Reykjavik Study in Iceland.
Serum CML and risk factors for AMD.
Main Outcomes and Measures
Early or late AMD, assessed through fundus images taken through dilated pupils using a 45° digital camera and grading for drusen size, type, area, increased retinal pigment, retinal pigment epithelial depigmentation, neovascular lesions, and geographic atrophy using the modified Wisconsin Age-Related Maculopathy Grading System.
Of the 4907 participants, 1025 (20.9%) had early AMD and 276 (5.6%) had late AMD. Mean (SD) serum CML concentrations among adults with no AMD, early AMD, and late AMD (exudative AMD and pure geographic atrophy) were 618.8 (195.5), 634.2 (206.4), and 638.4 (192.0) ng/mL, respectively (to convert to micromoles per liter, multiply by 0.00489; P = .07). Log serum CML (per 1-SD increase) was not associated with any AMD (early and late AMD) (odds ratio = 0.97; 95% CI, 0.90-1.04; P = .44) or with late AMD (odds ratio = 0.94; 95% CI, 0.82-1.08; P = .36) in respective multivariable logistic regression models adjusting for age, sex, body mass index, smoking, and renal function.
Conclusions and Relevance
Higher serum CML concentration had no significant cross-sectional association with prevalent AMD in this large population-based cohort of older adults in Iceland.
Age-related macular degeneration (AMD) is the leading cause of vision loss among adults aged 65 years and older in developed countries.1 With the growing population of older adults, the prevalence of advanced AMD is projected to increase by 50% to nearly 3 million in 2020 in the United States alone.2 The global cost of visual impairment due to AMD alone was an estimated $343 billion in 2010, including $255 billion in direct health care costs.3 Lifestyle and dietary modifications, intravitreal antiangiogenic therapy, and antioxidant supplementation are among the current strategies to reduce the morbidity of AMD.1 Despite advances in treatment and prevention, AMD has no effective cure and remains the primary cause of irreversible blindness in older adults.
The pathogenesis of AMD has been linked to mechanisms involving inflammation or innate immune dysregulation as well as oxidative stress. Age is a strong risk factor for AMD, with the prevalence of advanced AMD increasing from about 0.2% in ages 55 to 64 years to 13% in those older than 85 years.4 Smoking,5- 7 obesity, white race,8 and low intake of dietary antioxidants9 and ω-3 fatty acids10 are associated with an increased risk of AMD. There is a strong genetic susceptibility to AMD as shown in twin studies,11 familial aggregation analyses,12 and a large and growing body of association studies that have identified several common AMD-associated variants, for example, in and around complement factor H and the ARMS2/HTRA1 region.13- 17 Other studies have implicated lipid metabolism genes such as apolipoprotein E, hepatic lipase, cholesterol ester transfer protein, lipoprotein lipase, and very low-density lipoprotein receptor as well as extracellular matrix genes such as hemicentin 1 and fibulin 5 in AMD risk.16,17 Although variants within identified major susceptibility genes to AMD play a role in more than half of AMD cases, many individuals carrying AMD risk genotypes never develop the disease, and only a fraction diagnosed as having it progress to advanced AMD with vision loss.
Advanced glycation end products (AGEs) are a heterogeneous group of bioactive molecules formed by the nonenzymatic glycation of proteins, lipids, and nucleic acids. They are implicated in a wide number of adverse age-related outcomes, including cardiovascular disease, diabetes mellitus, chronic kidney disease, osteoporosis, and sarcopenia.18 They alter the structural integrity of tissues by cross-linking collagen and are thought to upregulate inflammation through binding with the receptor for AGEs (RAGE).18 The AGEs are implicated in the pathogenesis of AMD through various lines of evidence. Immunohistochemical studies have shown accumulation of AGEs such as pentosidine in the Bruch membrane with increasing age,19 carboxymethyllysine (CML) in drusen of eyes with AMD,20 and AGEs and RAGE in photoreceptors and retinal pigment epithelium of eyes with AMD.21 Basal laminar deposits, which develop between the retinal pigment epithelial cells and the basement membrane and are specific for AMD, show greatly increased expression of RAGE.22 A key factor in the pathogenesis of neovascular AMD is the expression of vascular endothelial growth factor. The activation of RAGE leads to the increased expression of vascular endothelial growth factor via the activation of NF-κB.23 One study suggested that plasma CML and pentosidine concentrations were higher in 58 patients with AMD compared with 32 control participants,24 but further corroboration is needed.
We hypothesized that an elevated level of circulating CML is independently associated with AMD in older adults. To address this hypothesis, we measured serum CML concentration and assessed its relationship with AMD in a large, population-based cohort of older adults in Iceland.
The Age, Gene/Environment Susceptibility (AGES)–Reykjavik Study is a population-based study aimed to investigate genetic and environmental factors contributing to health, disability, and disease (including systemic disease as well as eye disease) in older people. The study design and assessment of the cohort have been described elsewhere.25 In 2002, when the AGES-Reykjavik Study began, 11 549 previously examined cohort members of the Icelandic Heart Association’s Reykjavik cohort (1967-1996) were still alive according to the Icelandic Census Database, and a random sample of 5764 individuals were examined for the AGES-Reykjavik Study in 2002 to 2006.25 The comprehensive AGES-Reykjavik Study protocol required each participant to complete 3 visits to the Icelandic Heart Association Research Center within 3 to 6 months. The ocular component was included as part of the third visit in which 5330 persons participated. As part of the assessments at the Icelandic Heart Association Research Center, a questionnaire was administered, visual acuity was assessed, and images were acquired from the retina.26 Fundus images were available from 5272 individuals for the determination of AMD status. Written informed consent was obtained from all participants. The AGES-Reykjavik Study was approved by the Icelandic National Bioethics Committee, which acts as the institutional review board for the Icelandic Heart Association, and by the institutional review board for the US National Institute on Aging, National Institutes of Health. The Johns Hopkins School of Medicine institutional review board approved the ancillary study protocol for measurement of serum CML concentration.
A standardized protocol was used for fundus photography and is described in detail elsewhere.27 In brief, after pharmacologic dilation of the pupils, photography was performed in each eye using a 45° 6.3-megapixel digital nonmydriatic camera (Canon). Two photographic fields were taken of each eye, with the first centered on the optic disc and the second centered on the fovea. Software was used for image acquisition and archiving (Eye QSL; Digital Healthcare Inc). Retinal images were evaluated by the University of Wisconsin Ocular Epidemiology Reading Center for assessment of AMD in a semiquantitative fashion by a grader using EyeQ Lite (an image-processing database for storage, retrieval, and manipulation of digital images) and a standard AMD grading protocol,28 including the modified Wisconsin Age-Related Maculopathy Grading System used in the Multi-Ethnic Study of Atherosclerosis.29 Early AMD required one of the following: (1) the presence of any soft drusen and pigmentary abnormalities; (2) the presence of a large soft drusen 125 μm or larger in diameter with a large drusen area (>500-μm-diameter circle); or (3) large (≥125 μm in diameter) soft indistinct drusen and no signs of late AMD. Late AMD was defined by the presence of geographic atrophy or exudative AMD. A participant’s AMD status was based on the eye with the more severe disease classification or the eye with gradable signs if only 1 eye was graded.
Diagnoses of chronic diseases were made as described elsewhere.25 The definition of diabetes was based on self-reported diabetes in the questionnaire, use of diabetes medication, or a hemoglobin A1c level of 6.5% or greater.
Fasting venous blood samples were obtained by brachial venipuncture during the first visit of the 2002 to 2006 study round. Aliquots of serum were obtained and stored at −80°C. Serum was available for 4709 of the 5272 participants who had fundus images. The measure of circulating AGEs in this study was serum CML, one of the best-characterized AGEs found in the circulation and in tissue.18,30 The CML was measured in a masked fashion using a competitive enzyme-linked immunosorbent assay (AGE-CML enzyme-linked immunosorbent assay; Microcoat). This assay has been validated, is specific, and shows no cross-reactivity with other compounds.31,32 The interassay coefficient of variation for serum CML was 6%. Serum creatinine was measured using the Jaffe method. The estimated glomerular filtration rate (eGFR) in milliliters per minute per 1.73 m2 was calculated from the serum creatinine concentration using the Chronic Kidney Disease Epidemiology Collaboration equation of Levey et al.33
Continuous and categorical variables were compared across quartiles of serum CML concentration using Kruskal-Wallis tests and χ2 tests, respectively. Univariable and multivariable logistic regression models were used to compare the relationship of serum CML level with AMD. Covariates with established associations with AMD such as age, smoking, and body mass index (BMI; calculated as weight in kilograms divided by height in meters squared) were included in the multivariable models. The eGFR was included in the final multivariable models because of its known association with circulating CML. All analyses were performed using SAS version 9.1.3 statistical software (SAS Institute, Inc) with a type I error of 0.05 to determine statistical significance.
The mean (SD) age was 76.4 (5.5) years for the 4907 participants in the study. The mean (SD) serum CML concentration was 623.1 (197.7) ng/mL (to convert to micromoles per liter, multiply by 0.00489). Of the 4907 participants, 1025 (20.9%) had early AMD and 276 (5.6%) had late AMD. The characteristics of the participants are shown by AMD status in Table 1. Participants with early or late AMD were significantly older, had a lower BMI, were current smokers, had higher high-density lipoprotein cholesterol and C-reactive protein levels, and had lower triglycerides levels compared with participants without AMD. Participants with early or late AMD were also significantly more likely to have a history of myocardial infarction and chronic kidney disease. There were no significant differences between participants with and without AMD by sex, alcohol consumption, total cholesterol level, or low-density lipoprotein cholesterol level. The prevalences of hypertension, angina, and diabetes were not associated with AMD status. Higher plasma CML concentrations were weakly associated with AMD (P = .07).
The characteristics of the participants by quartile of serum CML concentration are shown in Table 2. Being older, being male, not smoking, having a lower BMI, and having a higher HDL cholesterol level were associated with higher quartiles of serum CML concentration. Higher triglycerides level, C-reactive protein level, and eGFR were associated with lower quartiles of CML concentration. The prevalence of diabetes was lower and the prevalence of chronic kidney disease was higher among those with higher serum CML concentration. Higher quartiles of CML concentration showed a trend toward a higher prevalence of hypertension (P = .06) and a lower prevalence of angina (P = .07). The prevalence of AMD was highest in the top quartile of serum CML concentration. There were no significant associations of total cholesterol level, low-density lipoprotein cholesterol level, or myocardial infarction with quartiles of serum CML concentration.
Multivariable logistic regression models were used to examine the relationship between serum CML and any AMD (early or late AMD) or late AMD only after controlling for potential confounding (Table 3). In models for the outcome of any AMD (early or late AMD), we observed no significant relationship with log CML (per 1-SD increase) after adjusting for age and sex (model 1), additionally adjusting for BMI and smoking (model 2), with the addition of eGFR (model 3), and finally with addition of diabetes, alcohol consumption, total cholesterol level, and HDL cholesterol level (model 4). There was also no significant relationship between the highest vs lowest quartiles of CML concentration in association with any AMD in multivariable models adjusting for the same covariates as described earlier. For late AMD, we observed a suggestion of an association between log CML (per 1-SD increase) when adjusting for age and sex (model 1, P = .10), but the relationship diminished after adjusting for BMI and smoking (model 2, P = .21), additionally for eGFR (model 3, P = .36), and finally with the addition of diabetes, alcohol consumption, total cholesterol level, and HDL cholesterol level (model 4, P = .14). In similar multivariable models comparing the highest quartile of CML concentration vs the lowest quartile, there was no significant relationship between serum CML concentration and late AMD. Alternative multivariable logistic regression models were explored in which either neovascular AMD or geographic atrophy was the dependent variable; serum CML concentration was not significantly associated with either form of late AMD (data not shown).
This study shows that the distribution of circulating CML levels is comparable to that described in other populations; it is positively associated with age and inversely associated with kidney function and BMI, as is already shown in the literature.18 Contrary to our original hypothesis, the study shows that circulating CML is not associated with prevalent AMD in community-dwelling older adults. To our knowledge, this is the first population-based study to examine the relationship between a circulating AGE and AMD. The findings from this study do not corroborate a previous clinic-based study in which mean plasma CML concentrations were more than 50% higher in 58 cases with AMD (27 with early AMD and 31 with late AMD) compared with 32 control participants.24 In our study, although mean serum CML concentrations were higher in participants with AMD compared with those without AMD, the difference in circulating CML concentration was minor (approximately 3%) and not statistically significant.
The strengths of our study are that it involved a large sample with more than 1300 cases of AMD, the participants were a population-based sample of community-dwelling adults, AMD was carefully documented using standardized fundus photography and AMD grading at the University of Wisconsin Ocular Epidemiology Reading Center, and serum CML was measured using a well-characterized assay with low coefficients of variability. Serum CML is the best-characterized circulating AGE in epidemiological studies.18 The limitations of the study are its cross-sectional design, single measurement of CML, and measurement of only a single type of circulating AGE. Specifically, other AGEs such as pentosidine and hydroimidazolone were not measured; however, previous studies show that circulating CML and pentosidine are moderately correlated.24 The findings of this study in a white population cannot necessarily be extrapolated to other study populations. The relationship between other circulating AGEs and AMD could be explored in future studies.
Elevated concentration of circulating CML has been associated with other adverse age-related outcomes such as cardiovascular and all-cause mortality, arterial stiffness, decline in skeletal muscle strength, and chronic kidney disease.18 The factors that regulate circulating CML are not clear. Cigarette smoke is a source of AGEs, but the prevalence of current smoking was actually lower among those with higher serum CML levels. A large population-based study from Finland also found no significant association between higher serum CML levels and smoking.34 Activation of the AGE-RAGE pathway is thought to increase inflammation,18 but in our study, serum CML and C-reactive protein levels were inversely related. This association was not adjusted for age, sex, or other possible confounders. The lack of an association between serum CML and C-reactive protein levels is consistent with the Finnish study.34 In our study, although current smoking was higher among those with late AMD, this association was not statistically significant. A review of smoking and AMD indicated that 13 of 17 studies showed a statistically significant association between smoking and AMD.35
Although it is thought that diabetes contributes to the increased formation of AGEs, in our study we found no association between plasma CML level and diabetes. These findings are consistent with a previous study of glucose metabolism in the Baltimore Longitudinal Study of Aging,36 a study of patients with type 1 diabetes,37 and a population-based study of more than 800 adults with type 2 diabetes in Finland.34 In addition, no significant relationship has been found between serum CML and hemoglobin A1c levels34,36- 38 or between levels of hemoglobin A1c and low-molecular-weight AGEs39 or serum hydroimidazolone.40
It has been hypothesized that AGEs contained in food contribute substantially to circulating AGEs.18 This hypothesis is attractive because it would suggest that dietary modification may reduce circulating CML levels. The relationship between dietary AGEs and circulating AGEs has not been rigorously studied using stable isotopes. However, recent studies suggest that dietary intake of AGEs does not correlate with either plasma CML concentrations or plasma pentosidine concentrations.41 Another study of 261 adults showed that both serum and urinary CML concentrations were not associated with dietary intake of AGEs, as rigorously assessed by 6 separate 24-hour dietary recalls.42 Intake of AGE-rich foods was not significantly correlated with serum or urinary CML concentration. Urinary CML concentration was negatively correlated with intake of fast foods.42
Although other studies suggest that AGEs in the retina and retinal pigment epithelium play a role in the pathology of AMD, it remains possible that local production and action of AGEs in the eye may participate in the development of AMD. Our study suggests that systemic levels of circulating AGEs are not associated with AMD. Because this study was cross-sectional, it does not necessarily exclude a role for AGEs in the development of AMD. It is possible that elevated concentrations of circulating AGEs increase the long-term risk of AMD over time for a subgroup of individuals. Future longitudinal studies are needed to determine whether elevated levels of circulating AGEs are associated with the development or progression of AMD.
Corresponding Author: Richard D. Semba, MD, Wilmer Eye Institute, Johns Hopkins University School of Medicine, 400 N Broadway, Smith Bldg, M015, Baltimore, MD 21287 (firstname.lastname@example.org).
Submitted for Publication: May 16, 2013; final revision received August 8, 2013; accepted August 19, 2013.
Published Online: January 30, 2014. doi:10.1001/jamaophthalmol.2013.7664.
Author Contributions: Mr Sun had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Semba, Gudnason, Eiríksdottir, Jonasson, Ferrucci, Schaumberg.
Acquisition of data: Semba, Gudnason, Harris, Klein, Schaumberg.
Analysis and interpretation of data: Semba, Cotch, Sun, Jonasson, Schaumberg.
Drafting of the manuscript: Semba.
Critical revision of the manuscript for important intellectual content: All authors.
Statistical analysis: Sun.
Obtained funding: Cotch, Gudnason, Eiríksdottir, Harris, Klein, Ferrucci.
Administrative, technical, and material support: Semba, Cotch, Gudnason, Eiríksdottir, Jonasson, Schaumberg.
Study supervision: Gudnason, Jonasson, Schaumberg.
Conflict of Interest Disclosures: None reported.
Funding/Support: This work was supported by grants R01 AG027012 and R01 EY017362 from the National Institutes of Health, grant ZIAEY00401 from the Intramural Research Program of the National Eye Institute, grant N01-AG-1-2100 from the Intramural Research Program of the National Institute on Aging, the Icelandic Heart Association, the Icelandic Parliament, the University of Iceland Research Fund, and a Lew Wasserman Merit Award to Dr Semba from Research to Prevent Blindness.
Role of the Sponsor: The National Eye Institute was involved in the design and conduct of the study in regard to collection of fundus photographs but had no role in the collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. The other funding organizations had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.