Study Participants

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.

Genotyping

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.

Data Analysis

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.