To compare plasma levels of oxidative stress biomarkers in patients with age-related macular degeneration (AMD) and controls and to evaluate a potential relationship between biochemical markers of oxidative stress and AMD susceptibility genotypes.
Prospective case-control study.
Plasma levels of oxidative stress biomarkers were determined in 77 AMD patients and 75 controls recruited from a clinical practice. Cysteine, cystine (CySS), glutathione, isoprostane, and isofuran were measured, and participants were genotyped for polymorphisms in the complement factor H ( CFH ) and age-related maculopathy susceptibility 2 ( ARMS2 ) genes.
CySS was elevated in cases compared with controls ( P = .013). After adjustment for age, sex, and smoking, this association was not significant. In all participants, CySS levels were associated with the CFH polymorphism rs3753394 ( P = .028) as well as an 8-allele CFH haplotype ( P = .029) after correction for age, gender, and smoking. None of the other plasma markers was related to AMD status in our cohort.
Our investigation of the gene–environment interaction involved in AMD revealed a relationship between a plasma biomarker of oxidative stress, CySS, and CFH genotype. These data suggest a potential association between inflammatory regulators and redox status in AMD pathogenesis.
Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in older individuals in the Western world. Approximately 1.75 million people in the United States older than 40 years have the sight-threatening advanced stages of the disease, and this number is projected to approach 3 million by 2020.
Oxidative stress seems to play a role in the pathogenesis of AMD. Several demographic and environmental risk factors for AMD, such as aging, smoking, and light exposure, have been linked to increased production of reactive oxygen species, and thus cumulative cellular oxidative injury. Supplementation with antioxidants (vitamin C, vitamin E, and β-carotene) and zinc was shown to slow AMD progression in the Age-Related Eye Disease Study (AREDS), a multicenter, randomized clinical trial, and high dietary intake of antioxidants (particularly carotenoids) has been correlated with lower AMD prevalence and incidence. Additionally, plasma biomarkers of oxidative stress, such as the lipid peroxidation product carboxyethylpyrrole and homocysteine, have been associated with AMD.
Quantification of oxidative stress by the measurement of cysteine thiol/disulfide couples and lipid peroxidation products in plasma may be an effective tool for identifying patients at risk of developing AMD, progressing to later stages of the disease, or both. The thiolated amino acid cysteine (Cys) and the Cys-derived antioxidant glutathione (GSH) are oxidized to their respective disulfides, cystine (CySS) and glutathione disulfide (GSSG). The redox potentials of Cys/CySS and GSH/GSSG couples reflect the body’s overall redox status. In our previous studies, the redox potentials of Cys/CySS and GSH/GSSG showed increased oxidation in association with age and smoking, both risk factors for AMD. Altered plasma redox status of these thiol metabolites also has been demonstrated in a range of human diseases, including Alzheimer disease, cystic fibrosis, diabetes mellitus, and cardiovascular disease. Levels of these thiol metabolites in plasma have not yet been compared in AMD patients versus controls.
Increased plasma levels of the lipid peroxidation metabolites F 2 -isoprostanes (F 2 -IsoPs) and isofurans (IsoFs), produced by the nonenzymatic free radical-catalyzed peroxidation of arachidonic acid, indicate impaired clearance of lipid peroxidation products throughout the body. Abnormal lipid metabolism may be detrimental to the retina–retinal pigment epithelium (RPE) complex, which is particularly susceptible to oxidative damage given the high rate of phagocytosis in RPE cells and the high prevalence of readily oxidized polyunsaturated fatty acids in photoreceptor outer segments. Increased plasma levels of IsoPs have been associated with disease risk factors such as smoking as well as multiple systemic diseases. Because an environment high in oxygen favors the production of IsoFs over IsoPs, the combined measurement of these 2 metabolites offers a comprehensive and reliable approach to evaluating oxidative stress status in vivo. Small-scale studies have linked increased IsoFs, but not F 2 -IsoPs, with diseases involving mitochondrial dysfunction, abnormal oxygen levels, or both: Parkinson disease, hypoxic lung injury, and bronchopulmonary dysplasia. The potential relationship of IsoPs and IsoFs with age-related eye diseases has not been investigated fully.
Recent studies have established genetic variation as an important factor in assessing AMD risk. Polymorphisms in the complement factor H gene ( CFH ) and the age-related maculopathy susceptibility 2 ( ARMS2 ) / high temperature requirement factor A-1 ( HTRA1 ) locus consistently have been linked with AMD. Most AMD-associated genes are related to the complement cascade, and current evidence suggests that the interaction between oxidative stress and inflammation may play a key role in AMD pathophysiology.
The purpose of the present study was to determine identifiable differences between AMD patients and controls using 2 independent measures of oxidative stress: thiol redox metabolites and lipid peroxidation products. We also aimed to evaluate a potential relationship between these biochemical markers of oxidative stress and AMD susceptibility genotypes.
For this prospective case-control study, blood was drawn from 152 individuals (77 AMD patients and 75 non-AMD controls) for measurement of plasma biomarkers of oxidative stress and genotyping. Individuals older than 55 years were recruited from the Retina Division at the Vanderbilt Eye Institute. Cases were diagnosed with intermediate or advanced AMD (AREDS categories 3 or 4) and were required to have at least intermediate drusen in both eyes. Controls had no clinical signs of AMD. Exclusion criteria included the presence of any retinopathy other than AMD, active uveitis, or ocular infection and any ocular surgery within the 60 days before enrollment. Patients with diabetes mellitus were excluded, given the potential role of reactive oxygen species in the pathogenesis of diabetic complications. Disease status was confirmed by high-resolution fundus photography. Fifty-degree fundus images were examined by a masked retina specialist (P.S.) for the presence or absence of the following AMD-related findings: drusen; RPE changes; neurosensory retinal detachment; pigment epithelial detachment; subretinal or intraretinal exudation (hemorrhage, lipid, or both), or both; choroidal neovascularization; and fibrovascular tissue. AMD patients then were classified by disease stage using the AREDS criteria. For phenotypic analysis, patients with intermediate AMD had intermediate drusen (AREDS category 3) in both eyes, and patients with advanced AMD had either geographic atrophy or neovascular AMD (AREDS category 4) in at least 1 eye. Patients with neovascular AMD in one or both eyes constituted the neovascular AMD group. Smoking history was obtained from all participants.
Blood Sample Collection and Plasma Biomarker Measurement
At the time of study enrollment, blood was collected from each participant using a 23-gauge butterfly needle. For the measurement of plasma thiol metabolites, 1.5 mL blood immediately was transferred to a microcentrifuge tube containing 0.5 mL serine-borate preservation solution, which has been demonstrated to protect against auto-oxidation. After centrifugation to remove blood cells, 200 μL supernatant was transferred to another microcentrifuge tube containing 200 μL 10% perchloric acid, 0.2 M boric acid, and 10 μM γ-glutamyl-glutamate (internal standard). Samples were frozen at −80 C for no more than 6 to 8 weeks until derivatization with dansyl chloride. Plasma Cys, CySS, and GSH were measured by high-performance liquid chromatography. Levels of GSSG were below the detection limit for most specimens. All patients with available biomarker measurements were included in data analyses.
For the measurement of lipid peroxidation metabolites, 8 mL blood immediately was transferred to two 4-mL blood collection tubes containing 7.2 mg K 2 ethylenediamine-tetraacetic acid each. These tubes were centrifuged at 4 C to remove blood cells, and 2 mL supernatant from each tube was transferred to one of two 15-mL conical tubes. Plasma was frozen immediately at −80 C and not thawed before analysis, which took place within 4 to 6 months. Samples were analyzed for F 2 -IsoP and IsoF concentration by the Vanderbilt University Eicosanoid Core Laboratory using gas chromatography/negative-ion chemical ionization mass spectrometry, as described below.
First, 1.0 ng of the internal standard [ 2 H 4 ]-15-F 2t -IsoP (8-iso-PGF 2α ) was added to each plasma sample. Samples were applied to a C 18 Sep-Pak cartridge (Waters, Milford, Massachusetts, USA) and eluted with ethyl acetate:heptane (50:50, vol/vol). The eluate was applied to a silica Sep-Pak cartridge (Waters) and eluted with ethyl acetate:methanol (50:50, vol/vol). The resulting eluate was dried under nitrogen, converted to pentafluorobenzyl esters by the addition of pentafluorobenzyl bromide and diisopropyl ethanolamine in acetonitrile, and incubated at 37 C for 30 minutes. Products were dried under nitrogen and reconstituted in 30 μL chloroform and 20 μL methanol. The initial separation of IsoPs, PGF 2α , and IsoFs from other lipid metabolites was achieved by thin-layer chromatography in a solvent system of chloroform:methanol (93:7, vol/vol). The methyl ester of PGF 2α was used as a standard on a separate lane and was visualized by staining with 10% phosphomolybdic acid in ethanol followed by heating. The R f of PGF 2α methyl ester in this solvent system is 0.15. Compounds migrating in the region ± 1 cm of the PGF 2α standard were collected from the thin-layer chromatography plate, extracted with 1 mL ethyl acetate, and dried under nitrogen.
After thin-layer chromatography purification, compounds were converted to trimethylsilyl ether derivatives by addition of 20 μL N,O-bis(trimethylsilyl)trifluoroacetamide and 10 μL dimethylformamide and were dried under nitrogen. The residue was dissolved for gas chromatography/mass spectrometry analysis in 20 μL undecane that had been stored over a bed of calcium hydride. Gas chromatography/negative-ion chemical ionization mass spectrometry was carried out on an Agilent 5973 Inert Mass Selective Detector coupled with an Agilent 6890n Network gas chromatography system (Agilent Labs, Torrance, California, USA) and interfaced with an Agilent computer. Gas chromatography was performed using a 15 m × 0.25-μm (film thickness) DB-1701-fused silica capillary column (J and W Scientific, Folsom, California, USA). The column temperature was programmed to increase from 190 C to 300 C at 20 C per minute. The major ion generated in the NICI mass spectrum of the PFB ester trimethylsilyl ether derivative of F 2 -IsoPs was the m/z 569 carboxylate anion (M-181 [M-CH 2 C 6 F 5 ]), and the major ion for IsoFs was the m/z 585 carboxylate anion ([M-181 [M-CH 2 C 6 F 5 ]). The corresponding ion generated by the deuterated internal standard [ 2 H 4 ]-15-F 2t -IsoP was m/z 573. Levels of endogenous F 2 -IsoPs and IsoFs in each biological sample were calculated from the ratio of intensities of the ions m/z 569 (IsoPs) or m/z 585 (IsoFs) to m/z 573. Using this assay, the lower limit of detection of F 2 -IsoPs and IsoFs is in the range of 4 pg, using an internal standard with a blank of 3 parts per thousand. Validation of this assay has shown precision of ± 6% and accuracy of 94% in biological fluids.
Genomic DNA was isolated from whole blood using the PureGene system (Gentra Systems, Minneapolis, Minnesota, USA), and all white participants were genotyped for AMD-associated single nucleotide polymorphisms (SNPs) in CFH and ARMS2. Genotyping was conducted by the DNA Resources Core in the Center for Human Genetics Research at Vanderbilt University using the Sequenom MassARRAY platform. All patients with available genotypes were included in data analyses.
Descriptive statistics for all demographic and clinical variables were calculated, and comparisons between cases and controls were made using the 2-sample t test for continuous data (eg, age) and the chi-square test for categorical data (eg, gender, race, and smoking). Comparisons of the biomarker levels between cases and controls were made using the t test and Wilcoxon rank sum test. Logistic regression models were fitted to evaluate the effect of plasma biomarkers on AMD risk while adjusting for the effects of age, gender, and smoking. Stratified analysis was performed for multiple AMD severity levels, including intermediate, neovascular, and advanced. The Hardy-Weinberg equilibrium test was carried out for all SNPs in the control group. The influence of individual CFH and ARMS2 genotypes on CySS levels was evaluated by linear regression. Haplotype analysis was used to test the association between plasma CySS and each CFH haplotype. When appropriate, adjustments were made for the potential confounders age, gender, and smoking. All analyses were performed with R ( www.r-project.org ). The haplotype analysis was performed through the R package haplo.stats, in which the haplo.score program yielded both global and haplotype-specific scores and P values while allowing for covariate adjustment. For all statistical analyses, P < .05 was considered to be significant.
Plasma levels of thiol metabolites (Cys, CySS, and GSH) and lipid peroxidation products (IsoP and IsoF) were measured in 152 participants, 77 AMD patients and 75 controls. The demographics of all participants are presented in Table 1 . Age and gender were significantly different between cases and controls.
|Variable||Patients (n = 77)||Controls (n = 75)||P Value|
|Mean age (SD), y||75.9 (6.4)||71.6 (7.8)||<.001|
|Gender, n (% female)||50 (65%)||34 (45%)||.023|
|Race, n (% white)||75 (97%)||73 (97%)||1.000|
|Current or past smokers, n (%)||69 (91%)||66 (88%)||.770|
In our study population, the mean plasma level of CySS was 9.1% higher in AMD patients than controls ( P = .013). After adjusting for age, gender, and smoking, the association was no longer significant ( P = .108). None of the other oxidative stress markers was associated with AMD status. Mean levels of IsoFs were 47% higher in AMD patients (0.22 ng/mL) than in controls (0.15 ng/mL), but this difference did not reach significance in our study population ( P = .087). Levels for all measured biomarkers in AMD patients and controls, with both unadjusted and adjusted P values, are reported in Table 2 .
|Biomarker||No. a||Mean ± SD||Unadjusted P Value||Adjusted P Value b|
|Patients||Controls||Patients||Controls||t Test||Wilcoxon Rank-Sum Test|
|Cys (μM)||69||68||4.18 ± 1.45||4.11 ± 1.39||.770||.835||.632|
|CySS (μM)||69||68||55.27 ± 11.21||50.66 ± 10.27||.013||.025||.108|
|GSH (μM)||69||67||1.77 ± 0.65||1.89 ± 0.97||.396||.492||.167|
|IsoP (ng/mL)||72||67||0.051 ± 0.034||0.059 ± 0.064||.372||.850||.346|
|IsoF (ng/mL)||72||67||0.22 ± 0.38||0.15 ± 0.18||.115||.087||.194|
To determine if the severity of disease affected the association of CySS levels with AMD, plasma biomarker levels were compared in patients with varying stages of AMD versus controls. Of the AMD patients, 26 had intermediate AMD (AREDS category 3) in both eyes, 9 had geographic atrophy in one or both eyes and no neovascular AMD, and 37 had neovascular AMD in one or both eyes. The 46 patients with either geographic atrophy or neovascular AMD (AREDS category 4) in at least 1 eye were considered advanced AMD patients. Plasma CySS levels were significantly different between neovascular AMD patients and controls ( P = .048), as well as between advanced AMD patients and controls ( P = .016). These differences were no longer significant after adjustment for age, gender, and smoking. In contrast, plasma CySS levels were not significantly different between intermediate AMD patients and controls ( Table 3 ). As in the study population at large, no differences between any of the AMD severity stages and controls were found for Cys, GSH, IsoP, or IsoF.
|AMD Stage||No. a||Mean Level (μM)||Unadjusted P Value||Adjusted P Value b|
|Patients||Controls||Patients||Controls||t Test||Wilcoxon Rank Sum Test|
|Intermediate c||24||68||55.03 ± 14.21||50.66 ± 10.27||.176||.264||.205|
|Neovascular d||34||68||54.68 ± 9.11||50.66 ± 10.27||.048||.063||.163|
|Advanced e||41||68||55.40 ± 9.39||50.66 ± 10.27||.016||.022||.121|
All white participants (75 cases, 73 controls) were genotyped for AMD-associated SNPs in CFH and ARMS2 to identify any potential genotype–biomarker relationships. No significant departure from Hardy-Weinberg equilibrium was detected for any of the tested SNPs. CySS levels in all subjects were found to be associated with CFH SNP rs3753394 ( P = .028) and an 8-allele CFH haplotype ( P = .029) after correction for age, gender, and smoking. SNP rs3753394 also was associated with CySS levels in AMD patients ( P = .024) and controls ( P = .040) when considered independently. Results for all tested SNPs are given in Table 4 , and haplotype data are presented in Table 5 .
|Gene||SNP||No. a||Unadjusted P Value||Adjusted P Value b|