Study Participants

For this pilot interventional study, individuals over the age of 55 were recruited from the Retina Division at the Vanderbilt Eye Institute using the inclusion/exclusion criteria of the ARED Study. 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 showed no clinical signs of AMD. Priority was given to spouses, as these individuals would tend to have similar dietary and environmental exposures. Exclusion criteria included the presence of any retinopathy other than AMD, active uveitis or ocular infection, any ocular surgery within the 60 days prior to enrollment, and current or previous participation in a clinical trial using an investigational drug or treatment within 30 days prior to the study start date. Patients with diabetes mellitus were excluded given the potential role of oxidative stress in the pathogenesis of diabetic complications. All participants had a dilated eye examination performed by an experienced retina specialist (P.S.) during the study week. 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, retinal pigment epithelium (RPE) changes, neurosensory retinal detachment, pigment epithelial detachment, subretinal and/or intraretinal exudation (hemorrhage and/or lipid), choroidal neovascularization, and fibrovascular tissue. AMD patients were then classified by disease stage using the AREDS criteria. Smoking history was obtained from all participants. The risks and benefits of supplementation, including the association between long-term β-carotene intake and lung cancer, were carefully explained to all patients who wished to participate.

Controlled Diet

Six meal plans with varying caloric loads were designed to accommodate study participants with different caloric needs. Daily kilocalorie (kcal) intake ranged from 1600 to 2600 kcal per day, increasing in 200-kcal increments. All meal plans were controlled for dietary antioxidant intake, including ±10% of the recommended daily amount (RDA) of vitamins A, C, and E for women aged 51 to 70 years: 700 μg/day vitamin A, 75 mg/day vitamin C, 15 mg/day vitamin E. The macronutrient distribution was 27% to 30% fat, 55% to 60% carbohydrates, and 15% to 20% protein. No juice was allowed in the study diet. A sample diet plan is included as an Appendix (Supplemental Material available at AJO.com ).

Prior to admission day, the resting energy expenditure (REE) was calculated for each patient using the Mifflin-St. Jeor equation. The REE was multiplied by an appropriate activity factor to estimate caloric needs, and the appropriate calorie level was assigned.

Stay at the General Clinical Research Center

The 6 AMD patients who were taking AREDS supplements prior to study enrollment were instructed to discontinue all antioxidant and zinc supplements 2 weeks prior to admission to the Vanderbilt General Clinical Research Center (GCRC). Throughout the duration of their 7-day stay at the GCRC, patients received a controlled diet, as described in detail above. Patients were allowed to leave the facility but were required to eat all meals at the GCRC during the study week. The nutritionist confirmed that the patients were receiving the appropriate amount of food every day. Three patients requested an increase in calorie level during the study week. Each patient’s progress was monitored daily by a study physician.

The first 48 hours on a regulated diet served to normalize baseline parameters. Dietary supplementation with the AREDS formula of 500 mg vitamin C, 400 IU vitamin E, 15 mg β-carotene, 80 mg zinc oxide, and 2 mg cupric oxide per day was instituted on study day 2. Blood (19 mL) was drawn at 4:00 PM with a 23-gauge butterfly needle on days 2 and 7 for measurement of plasma biomarkers of oxidative stress.

Plasma Biomarker Measurement

For the measurement of plasma thiol metabolites, 1.5 mL blood was immediately transferred to a microcentrifuge tube containing 0.5 mL of serine-borate preservation solution, which has been demonstrated to protect against auto-oxidation. Following centrifugation to remove blood cells, 200 μL of supernatant was transferred to another microcentrifuge tube containing 200 μL of 10% perchloric acid, 0.2 M boric acid, and 10 μM γ-glutamyl-glutamate (internal standard). Samples were frozen at −80° C until derivatization with dansyl chloride. Plasma Cys, CySS, and GSH were measured by high-performance liquid chromatography (HPLC). Levels of GSSG were below the detection limit for the majority of specimens. All patients with available thiol biomarker measurements at both time points (11 AMD patients and 7 controls) were included in data analyses.

For the measurement of lipid peroxidation metabolites, 2 4-mL blood collection tubes containing 7.2 mg K ₂ ethylenediamine-tetraacetic acid each were centrifuged at 4° C to remove blood cells, and 2 mL supernatant from each tube was transferred to 1 of 2 15-mL conical tubes, which were immediately frozen at −80° C and not thawed prior to analysis. 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 (GC/NICI-MS) as described previously.

After the addition of the internal standard [ ² H ₄ ]-15-F _2t -IsoP (8-iso-PGF _2α ), plasma samples were applied to a C18 Sep-Pak cartridge (Waters, Milford, Massachusetts, USA) followed by a silica Sep-Pak cartridge (Waters). Initial separation of IsoPs, PGF _2α , and IsoFs from other lipid metabolites was achieved by thin layer chromatography (TLC) in a solvent system of chloroform:methanol (93:7, v/v), and compounds migrating in the region ±1 cm of the PGF _2α standard were collected from the TLC plate.

Following TLC purification, GC/NICI-MS was carried out on an Agilent 5973 inert mass selective detector coupled with an Agilent 6890n Network GC system (Agilent Labs, Torrance, California, USA) and interfaced with an Agilent computer. GC 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. 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. Validation of this assay has shown precision of ±6% and accuracy of 94% in biological fluids. Levels of IsoPs and IsoFs were available at both time points for 14 participants (10 AMD patients and 4 controls).

Data Analysis

Descriptive statistics for all demographic and clinical variables were calculated. Biomarker levels before (day 2) and after (day 7) 5 days of AREDS supplementation were compared in AMD patients only, controls only, and all participants using 2-tailed paired t tests. Linear regression models adjusting for age, sex, and AMD status were fitted to each biomarker independently. All analyses were performed with Microsoft Excel and R ( www.r-project.org ). For all statistical analyses, P < .05 was considered to be significant.