Forty-eight apparently healthy young men and women (18 to 30 years old, 18 men, 30 women) volunteered as participants for this study. Participants were recruited by flyers, newspaper, institutional Internet sites, and clinic advertisements from the central Virginia region. All participants had to meet the following criteria before enrollment in the study: no participation in regular physical activity (vigorous exercise two times or more per week); no chronic health problems or current smoking; no history of cardiovascular, metabolic, or respiratory disease; no consumption of AOX supplements within the past 6 months; and no current usage of any form of hormonal forms of contraceptives. All participants read and signed a written informed consent statement consistent with university policy on protection of human subjects. The protocol of the study was approved by the Institutional Review Board for Studies Involving Human Subjects at the University of Virginia (UVA) and conforms to the guidelines involving the use of human participants as outlined by the American College of Sports Medicine.
Participants and Study Groups
After the investigators had determined BMI values [normal weight (BMI < 25 kg/m2) and overweight and obese (BMI ≥ 25 kg/m2)], participants were stratified into normal-weight or overweight groups. Within each group, participants were randomized by the UVA investigational pharmacist to receive either AOX treatment or placebo (PL). Hereafter, the participant stratifications will be referred to as normal-weight, AOX-treated (N-AOX); normal-weight, PL (N-PL); overweight, AOX-treated (O-AOX); and overweight, PL (O-PL). The first character refers to the adiposity status (normalweight, overweight), and the second characters refer to the AOX treatment or PL (AOX, PL). A total of 65 participants were enrolled in the study; among all normal-weight and overweight participants, those who withdrew were 11 participants who had overcommitted themselves and requested to withdraw. Three were withdrawn by the investigators for failure to comply with the study visits. Two participants completed two visits and stopped responding to investigator contact. One had child care issues and could not complete the study.
Groups assigned to the AOX treatment were prescribed a mixture of vitamin E (800 IU/d), vitamin C (500 mg/d), and β-carotene (10 mg/d) by the physician investigator. The supplements or PL were administered by the UVA investigational pharmacist. Supplements were provided in opaque bottles. These dosages of AOXs were chosen based on previous studies that showed reductions in lipid peroxidation with this range of combined supplements (24). PLs were provided in identical opaque bottles with color-matched, odorless capsules similar to those of the AOX. Treatment was administered for 8 weeks, a time by which plasma concentrations of vitamin E stabilize with supplementation (25).
All participants visited the testing area five times (three pre-intervention, two post-intervention) and were acclimated to the UVA Exercise Physiology Laboratory at the General Clinical Research Center (GCRC). During Visit 1, body composition and vital signs were measured, and 3-day dietary record forms were provided to participants. During Visit 2, aerobic fitness levels [peak oxygen consumption (Vo2peak)] were estimated using a load-incremented cycle ergometer protocol. During Visit 3, a constant-load cycle ergometer test was administered to each participant with blood draws before and after the constant-load test. All visits were completed within a 14-day period. Visits 4 and 5 were repeats of Visits 2 and 3 that occurred after the supplementation period.
Height and weight were measured using a standard medical grade scale. For classification of obesity, waist and hip girths were measured using a soft cloth measuring tape at anatomical landmarks described by the American College of Sports Medicine (26). BMI values were determined by the following: BMI = weight (kilograms)/height (meters)2. Body volume was estimated using air displacement plethysmography in a BodPod device (Bod-Pod; Life Measurement Instruments, Concord, CA) corrected for thoracic gas volume; body density was calculated and used to predict body fat using the Siri equation (27).
To determine whether nutritional intake was different between groups or over time, 3-day dietary record forms were provided to each participant with standard instructions on how to complete the record before Visit 3. Participants were instructed to estimate servings of foods using household measurements (volume) as described in national dietary guidance documents as previously described (28). Each participant received individual training sessions with the same investigator with regard to measuring technique and volume estimation. Picture books of portion sizes were also provided after the dietary estimation training session (28). Diet records were assessed by the same investigator using Nutritionist Pro Software (version 2.1.13; First DataBank, San Bruno, CA) and were analyzed for macronutrient, AOX, and caloric intake. To ensure the stability of the habitual diet of the subjects, all subjects completed a second 3-day dietary record 8 weeks later (28).
Vo2peak/Lactate Threshold (LT) Test
To reduce the potential effects of the weight-bearing effect on lipid hydroperoxide (PEROX) responses, a cycle ergometry mode of exercise was selected for this study. After a 12-hour overnight fast, participants arrived at the GCRC. A venous catheter was inserted into a forearm vein. Participants completed a Vo2peak/LT test on an electronically braked cycle ergometer (Ergo Metrics 800S; Sensor Medics, Yorba Linda, CA). The initial power output (PO) was set at 20 W, and the PO was increased 15 W every 3 minutes until volitional fatigue. Blood samples were taken at rest and during the last 15 seconds of each exercise stage for the measurement of blood lactate concentration (model 2700; YSI Instruments, Yellow Springs, OH). Heart rates (HRs), blood pressures, and ratings of perceived exertion were collected at every exercise stage (26).
Constant-Load Cycle Ergometer Test
To standardize the duration and relative intensity of the exercise challenge among all subjects, a 30-minute constant-load exercise test was performed in a fasting state. The PO for the 30-minute constant-load PO (CLPO) aerobic exercise sessions (see below) was calculated as follows: CLPO = PO at LT + 0.50 (PO at Vo2peak − PO at LT) (1.5 LT). Each individual self-selected his/her seat height and the front console option during this exercise test. Each participant pedaled at ∼50% of the CLPO during Minute 1 and at ∼75% of the CLPO during Minute 2, and CLPO was attained by Minute 3. Subjects were required to pedal between 60 and 100 rpm. If pedaling cadence dropped to <60 rpm, the PO was reduced, but the flywheel resistance was increased by the ergometer to keep the work consistent at all times.
Metabolic, Hemodynamic, and Perceptual Responses
Metabolic data were collected during both the Vo2peak/LT protocol and the CLPO tests, using standard open-circuit spirometric techniques (Vmax 229; Sensor Medics). Ratings of perceived exertion were assessed at the end of each stage during the Vo2peak/LT protocol and every 10 minutes during the CLPO protocol using the Borg Scale. HR was determined electrocardiographically (Marquette Max-1 electrocardiograph, Marquette, WI). HR, blood pressures, and lactate were assessed every 10 minutes during the CLPO test.
Blood samples were collected from the participants’ venous catheter into heparinized Vacutainer tubes before and immediately post-CLPO test. Blood samples were analyzed for PEROX, TAS, cholesterol, and lipid subfractions. Other portions of whole blood were analyzed for glucose and hemoglobin A1c (HbA1c) levels, and inflammatory cytokines and adiponectin. A portion of the blood was immediately centrifuged at 1500g for 5 minutes to separate plasma from red blood cell pellets. Plasma samples were immediately frozen and stored at −70 °C until analysis.
Cholesterol, Glucose, and HbA1c
To document levels of lipid substrates in the blood available for oxidation, plasma cholesterol subfractions [total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), triglycerides] were analyzed by the UVA Health System Clinical and Toxicology Laboratories using standard automated spectrophotometric laboratory procedures (Olympus AU640, Olympus calibrator catalog no. DR0040, Olympus, Tokyo, Japan; and Genzyme HDL-C calibrator catalog no. 80-4529-00, Genzyme, Cambridge, MA). Low-density lipoprotein-cholesterol (LDL-C) was estimated from the following equation: LDL-C = TC − HDL-C − (triglycerides/5). Blood glucose was assessed using an Olympus AU640 procedure, in which glucose was phosphorylated by hexokinase in the presence of ATP and magnesium. The resultant glucose-6-phosphonate dehydrogenase oxidized glucose-6-phosphonate to 6-phosphogluconate and reduced nicotinamide adenine dinucleotide to NADH. The change in absorbance at 340/380 was proportional to the amount of glucose in the sample (Olympus Glucose Reagent, Calibrator catalog no. DR0040). HbA1c was analyzed using automated high-performance liquid chromatography (Tosoh G7 Automated HPLC Analyzer, using TSKgel G and HSi elution columns). All samples were performed in duplicate.
As an estimate of the total AOX capacity of the plasma, a colorimetric commercial kit was used (Randox Laboratories Ltd., Antrim, UK; TAS catalog no. NX2332). In brief, 2,2-azion-di-[3-ethylbenzenthiazoline sulfonate] (ABTS) was incubated with a peroxidase and hydrogen peroxide to produce a radical cation ABTS. The suppression of the ABTS radical in vitro was proportional to the AOX level in the plasma samples. Samples were read in a single batch at 600 nm on a spectrophotometer. Samples were expressed in AOX capacity in millimolar plasma. All samples were performed in duplicate. The coefficient of variation for this assay was 4%.
Inflammatory cytokines IL-6 and CRP were measured as the major adipokines in this study. Cytokines were measured in the GCRC Core Laboratory using the following techniques: an enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN) for measurement of IL-6 and an Immunolite 2000 ELISA for high-sensitivity (hs)-CRP. All samples were performed in duplicate. Measurement of adiponectin was made using an ELISA technique (LINCO Research Inc., St. Charles, MO). Because adiponectin levels are not acutely changed with exercise (29), only resting adiponectin levels at baseline and 8 weeks were collected.
Lipid Peroxidation Measurements
PEROX was quantified using the colorimetric ferrous oxidation/xylenol orange spectrophotometric technique previously described, where cumene hydroperoxide was used as the standard for this assay, and samples were read at 580 nm (30). All samples were performed in triplicate in a single batch analysis. The coefficient of variation for this assay was 4%. The PEROX values were adjusted by Vo2 values as previously described (7) to account for possible differences in the oxygen exposure during exercise.
All data are expressed as mean ± standard error. Data were analyzed using the SPSS/PC statistical program (version 12.0 for Windows; SPSS, Inc., Chicago, IL). Descriptive variables were analyzed using a two-way ANOVA. If differences did not exist between groups at baseline, repeated measures ANOVAs were performed for the change scores (Δ values from pre- to post-CLPO exercise at baseline and 8 weeks) for the inflammatory cytokines, blood lipids, PEROX, and TAS. The between-group factors were adiposity status (non-obese, overweight) and treatment (AOXs, PL), and the within-group factor was time (pre- and post-exercise, baseline, and 8 weeks). When baseline differences existed for blood measures, two-way analyses of covariance were performed using the baseline value as the covariate. The between-group factors were adiposity status (non-obese, overweight) and treatment (AOXs, PL).
Lastly, a hierarchical regression analysis was performed on variables postulated to contribute to exercise induced oxidative stress in obesity to determine which variables contribute most to the ΔPEROX/ΔVo2 (the change in adjusted lipid peroxidation during exercise from baseline to 8 weeks). The level of significance was set at 0.05 for all statistical tests.