Eccentric exercise was reported in several investigations to damage skeletal muscle in a fiber specific manner (9,31). It has been purported that reactive oxygen species (ROS) play a role in both the initiation and the progression of muscle fiber injury after the initial mechanical insult. A single bout of exercise can result in activation of several distinct systems of radical generation and may be separated into both primary as well as secondary sources (15). A significant portion of ROS production from aerobic exercise is thought to result due to an increased flux in electron transport leading to an increased leakage of superoxide radicals (25). The generation of ROS during and after anaerobic exercise has been attributed to the xanthine and NADPH oxidase production, ischemia reperfusion, prostanoid metabolism, phagocytic respiratory bursts, disruption of iron-containing proteins, and excessive calcium accumulation, often resulting from the performance of high-force eccentric exercise, which commonly produces muscle injury (23). Several studies have reported increases in oxidative stress markers in blood after eccentric exercise (7,8,17,26). Childs et al. (7) reported an increase in blood MDA after eccentric exercise, whereas Lee et al. (17) noted an increase in protein carbonyls (PC) in blood after eccentric resistance exercise. Additionally, downhill running resulted in MDA, and F2α-isoprostane increases from serum (26).
Antioxidant vitamins, in particular the water-soluble vitamin C and the lipid soluble vitamin E have been used in an attempt to protect against ROS mediated oxidative stress in response to exercise. Whereas several investigations have used aerobic exercise as the physical stressor, few have examined high-intensity anaerobic work (5,7,22). In addition, few studies have used resistance exercise with an eccentric bias as the chosen exercise mode, and these studies have reported mixed findings within the blood (7,17,22). Vitamin E supplementation before eccentric exercise-induced muscle damage was reported to show little protection within the muscle (2,31). A combination antioxidant treatment was shown to attenuate DOMS and CK increases with minimal changes in muscle force and range of motion (3).
With the exception of Petersen et al. (24), who used downhill running as the exercise stressor, no investigation has studied the role of combination antioxidant (vitamin E and C) therapy in relation to eccentric exercise-induced oxidative stress and reported on oxidative-stress markers. These vitamins are known to act together in redox reactions in order to maintain each other in the reduced and active form (11). Additionally, selenium is an important mineral for the enzyme glutathione peroxidase (GPX). GPX in the presence of reduced glutathione catalyzes the conversion of hydrogen peroxide to water and oxidized glutathione and as such is important in reducing the amount of hydrogen peroxide in the system. Therefore, the purpose of this study was to determine the effects of a combination antioxidant therapy (vitamins E and C and selenium) on blood biomarkers of oxidative stress after eccentric exercise. It was hypothesized that this antioxidant therapy would attenuate blood biomarkers of oxidative stress as represented by the rise in protein, lipid, and glutathione oxidation after eccentric exercise.
METHODS
Subjects
Eighteen healthy women (aged 19–31 yr) volunteered to participate as subjects after explanation of all experimental procedures and signed the written informed consent. All subjects completed a medical history, diet and supplementation history, and physical activity questionnaire in order to determine eligibility. No subject was a smoker, used oral contraceptives, antiinflammatory drugs, or dietary supplements (i.e., antioxidants). Subjects were nonresistance trained (i.e., had not performed resistance training within the past 12 months). The University IRB Board for Human Subjects Committee approved all experimental procedures.
Antioxidant Therapy
Subjects were randomly assigned to either an antioxidant supplement (S) (N = 9) or a lactose placebo (P) (N = 9) in a double-blind manner. Subjects in the antioxidant group consumed a total of 400 IU of vitamin E (100% d-α-tocopherol), 1 g of vitamin C (ascorbic acid), and 90 μg of selenium per day, delivered in three capsules per day for a total of 14 d before the eccentric protocol and for 2 d postexercise. This should have allowed adequate time for incorporation of vitamin C and vitamin E into plasma and cells before the eccentric exercise bout (4,19,29). Subjects in the placebo group consumed three capsules per day for the same duration before and after exercise. Both the P and S capsules were identical in appearance. All subjects were instructed to maintain their normal diet during the study period and completed daily food records for the 3 d before and 2 d after the eccentric protocol to allow for nutrient intake comparisons between the two groups (Diet Analysis Plus, ESHA Research, Salem, OR). Capsules were counted upon return of the capsule bottles to assess compliance with the treatment.
Baseline Measurements
Three to four weeks before the eccentric protocol, measurements of height, weight, and percent body fat by three skinfold sites (16) were obtained by the same trained technician. Subjects were familiarized and fitted to the Biodex isokinetic dynamometer (Biodex Medical Systems Inc., Ronkonkoma, NY), and all settings were recorded for future reference during the eccentric protocol. Subjects were asked to perform three maximal isometric contractions with their nondominant arm elbow flexors, each lasting 3 s, with 60 s of rest between each effort. These efforts served as a familiarization to the Biodex and also to obtain maximum isometric force for all subjects. Subjects were then provided their bottle containing the capsules, a detailed instruction sheet pertaining to the study design, including dates when to begin and cease taking their capsules, diet record sheets and a scheduled to return for the eccentric protocol.
Experimental Procedures
All subjects reported back to the laboratory during the first few days (follicular phase) of their menstrual cycle in an attempt to avoid any variance in estrogen levels, in a 10-h postabsorptive state. Subjects were instructed not to perform any strenuous physical activity for the entire study duration. Subjects were seated in a chair, and after at least a 10-min rest period 7 mL of blood were drawn by Vacutainer from an antecubital vein by a trained phlebotomist. Subjects were then seated on the Biodex and began the eccentric protocol. A total of four sets of 12 repetitions were performed by all subjects using their nondominant arm elbow flexors, at an angular velocity of 20°·s−1. The range of motion (ROM) was set from full extension to 100° of flexion so that the muscle would be exercised over that entire length and result in greater muscle damage than would be achieved using a shorter-range muscle length ROM (30). Subjects were instructed to complete the eccentric action required in 5 s. After each eccentric action, the investigator returned the dynamometer lever arm to the start (fully flexed) position over the course of 5 s and the subject resumed the repetitions. Each set consisted of 12 repetitions, with the subjects rested for 60 s before performing another set, until all four sets were completed. The lever arm was programmed to move against the subjects’ resistance so long as the subjects produce at least 10 N·m of torque. The total amount of work performed during each of four sets was recorded for all subjects, and the percent decrease in work from set 1 to set 4 was calculated. Blood samples were obtained immediately 0, 2, 6, 24, and 48 h after the eccentric protocol. During the 0 h after blood draw, attempts were made to take blood from the nondominant arm. However, no preference was given regarding which arm was used for subsequent blood draws. All blood samples were analyzed for plasma protein, plasma protein carbonyls (PC), plasma malondialdehyde (MDA), and whole blood total (TGSH) and oxidized (GSSG) glutathione.
Blood handling and analysis.
Three milliliters of whole blood were immediately removed, treated, and stored at −80°C (Revco, Asheville, NC) until analyzed for glutathione status. The remaining blood was immediately centrifuged at 3000 rpm for 15 min at 4°C in a Beckman (J2–21) centrifuge (Fullerton, CA) to obtain plasma and stored at −80°C until analyzed for the dependent variables. All assay procedures described below were performed in duplicate.
Plasma protein.
Plasma protein was determined by the method of Lowry et al. (21), comparing values to known standards.
Protein carbonyls.
After adjustment to 4 mg·mL−1 protein using phosphate buffer, protein carbonyls were determined from plasma using the 2,4-dinitrophenolhydrazine (DNPH) spectrophotometric method as described by Levine et al. (20). Briefly, samples containing either 2N HCL or DNPH were passed through columns containing Sephadex G 10 and rinsed with 2N HCl. The effluent was collected, mixed with guanidine HCL, and the absorbance determined at 360 nm on a Shimadzu UV-1601 spectrophotometer (Shimadzu, Columbia, MD). The change in absorbance with and without DNPH was calculated for all samples. Values are expressed as molar quantities using the extinction coefficient 22,000 M−1·cm−1 (nM·mg−1 protein).
Malondialdehyde.
Total malondialdehyde was determined as described by Gerard-Monnier et al. (10). Samples were mixed with 500 mM BHT in acetonitrile and 12 N HCl, and allowed to incubate at 60°C for 80 min. Samples were then cooled to room temperature and working reagent (i.e., one volume of 100% methanol with three volumes of 10.3 mM N-methyl-2-phenylindole in acetonitrile) was added to each sample tube, mixed, and centrifuged (Eppendorf 5415C, Brinkman Instruments, Westbury, NY) at 13,000 rpm for 5 min. The clear supernatants were transferred to microcentrifuge tubes, 12 N HCl added, and tubes centrifuged at 13,000 rpm for 5 min. The supernatants were transferred to cuvettes and the absorbance measured at 586 nm on a Shimadzu UV-1601 spectrophotometer and compared with known standards. All values are expressed in picomoles per milligram of protein.
Glutathione status.
Whole blood was used for determination of glutathione status, using the method described by Anderson (1). Blood mixed with EDTA was immediately treated with ice-cold 10% 5-suflosalicylic acid containing 1 mM bathophenantrolinedisulfonic acid (BPDS). This mixture was centrifuged at 10,000 rpm for 15 min at 4°C in a Beckman (J2–21) centrifuge (Fullerton, CA). The supernatants were used for determination of TGSH and GSSG and compared with known standards. Both TGSH and GSSG were analyzed using 5,5′-diothiobis-2 nitrobenzoic acid (DTNB) to combine with GSH to form 5-thio-2-nitrobenzoic acid (TNB). Before analysis of GSSG samples, the supernatants were neutralized with NaOH (pH 7.0–7.5), and 2 μL of 2-vinylpyridine was added per 100 μL of supernatant and vigorously mixed for 5 min to derivatize GSH. The resultant supernatants were then analyzed in the same manner as those for TGSH. GSSG was reduced back to GSH by glutathione reductase in the presence of NADPH. The rate of TNB formation at 412 nm using a Shimadzu UV-1601 spectrophotometer (Shimadzu) was used. GSH was computed by subtracting two times the value obtained for GSSG from TGSH [GSH = TGSH − 2(GSSG)].
Statistical Analyses
The data obtained for PC, MDA, TGSH, GSSG, GSH, and GSSG:TGSH were analyzed using a 2 × 6 repeated measures ANOVA. Significant interactions and main effects were further analyzed using Tukey’s post hoc tests. Subject characteristics, dietary variables, total work, and percent decrease in work during the eccentric protocol were compared between groups using a one-way ANOVA. All analyses were performed using JMP statistical software (SAS Institute, Cary, NC). Statistical significance was set at P < 0.05. The data are presented as mean ± SEM.
RESULTS
All subjects successfully completed testing with 100% compliance. Capsule consumption, assessed via counting the capsules provided upon delivery and return of bottles, was near 100% for both groups (99.3% and 98.1% for supplement (S and P, respectively). There were no statistical differences between groups for age (22 ± 1.8 and 23 ± 1.4 yr), height (163 ± 1.4 and 161 ± 2.5 cm), weight (64 ± 5 and 60 ± 4.3 kg), or percent body fat (28 ± 2.1 and 25 ± 1.8) for S and P, respectively. Dietary intake assessed over a 5-d period showed no significant differences in any measured variable (Table 1). No differences were noted between groups for total work during the eccentric protocol (1137 ± 126 and 1117 ± 130 N·m), or for the percent decrease in work during the eccentric protocol (37 ± 5.3 and 38 ± 8.2) for S and P, respectively.
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TABLE 1:
Mean dietary intake during the 3 d before and 2 d after eccentric exercise for subjects provided antioxidant supplement or placebo.
Protein carbonyls.
Plasma PC data before and during the 48 h after eccentric exercise are presented in Figure 1. There was no condition by time interaction for plasma PC. There was a significant time main effect, with PC higher at 24 and 48 h postexercise compared with preexercise. Additionally, there was a condition main effect, with PC in the supplement group lower than the placebo group. The PC increase in the control group to the eccentric exercise was about 200%, whereas the increase in the supplemented group was only 50%.
Malondialdehyde.
Plasma MDA data before and during the 48 h after eccentric exercise are presented in Figure 2. There was no condition by time interaction for plasma MDA. There was a significant time main effect, with MDA higher at 48 h postexercise compared with preexercise. Additionally, there was a condition main effect, with MDA in the supplement group significantly lower than the placebo group.
Glutathione status.
There was no significant condition by time interactions or condition main effects for TGSH, GSSG, GSH, or GSSG:TGSH. However, significant time main effects were evident for all of these variables. TGSH and GSH were lower at 0 and 2 h postexercise compared with preexercise, whereas GSSG was elevated above preexercise at these times. As a result the GSSG:TGSH was statistically elevated above preexercise at 0 and 2 h postexercise independent of group (Fig. 3).
DISCUSSION
The major findings of this study indicate that: 1) eccentric exercise can increase biomarkers of oxidative stress in nonresistance trained females, with the greatest increase in protein oxidation; and 2) the antioxidant combination therapy used in this study can significantly attenuate the rise in blood protein oxidation after eccentric exercise, whereas having a modest impact on blood MDA and no impact on blood glutathione status. This is the first investigation that we are aware of that assessed oxidative stress to eccentric exercise in women supplemented with antioxidants.
Few published reports related to PC after anaerobic exercise currently exist, and only one with the inclusion of antioxidant supplements (5). Lee et al. (17) demonstrated a statistically significant increase in PC at 24 and 48 h after 60 maximal eccentric muscle actions performed by the elbow flexors. The results in the present study report similar findings in the placebo group. Bryer and Goldfarb (5) also reported an attenuated PC response with subjects supplemented with 3 g of vitamin C per day for 2 wk prior and the days after a similar eccentric protocol that used a similar protocol with elbow flexors as reported by Lee et al. (17).
The rise in PC after eccentric exercise, in which muscle damage is often produced, may be attributed to invasion of phagocytic cells into the damaged tissue, which typically occurs several hours postexercise and can generate a substantial amount of ROS. Additionally, the disruption of iron containing proteins such as erythrocytes can lead to an increase in free iron, which is known to catalyze radical reactions (15). Therefore, exercise that creates a significant degree of trauma (e.g., high-force eccentric muscle actions) can lead to destruction of these heme proteins, potentially increasing free iron to aid in the production of ROS. An imbalance in calcium homeostasis, which is believed to be a major contributor to muscle injury resulting from resistance type exercise (9), has also been associated with ROS generation through activation of phospholipase and proteolytic enzymes (15). Furthermore, the enzyme xanthine oxidase has been noted to be a radical species generator (15) and may have been produced during conditions of ischemia/ reperfusion to the muscles.
The increase in ROS production resulting from any of the above described sources, in addition to the increase in oxygen uptake during exercise, could lead to oxidation of amino acid side chains and fragmentation of polypeptides, as all amino acids are susceptible to metal-catalyzed oxidation (25). These factors could help to explain the elevation in protein oxidation observed after eccentric exercise. The data from the present study, taken together with those of Lee et al. (17) and Bryer and Goldfarb (5), suggest that high-force eccentric exercise can increase blood protein oxidation. Additionally, the prophylactic antioxidant combination therapy as prescribed in the present investigation attenuated this increase, as evidenced by the marked reduction in PC in subjects pretreated with this combined antioxidant treatment.
The lipid peroxidation data from the present study corroborates the findings of others suggesting modest changes in MDA after eccentric exercise in either blood (6,18,26) or skeletal muscle (13,27). However, Childs et al. (7) reported elevated lipid peroxidation after eccentric exercise as measured by lipid hydroperoxides. Additionally, McBride et al. (22) reported an increase in MDA during the 48 h after full-body isotonic resistance training independent of vitamin E supplementation with 1200 IU·d−1 for 2 wk before exercise or those taking a placebo.
Taken together, these findings suggest that lipid peroxidation as assessed by MDA may be modestly affected by eccentric exercise using small muscle groups. It is possible that a combination of concentric/eccentric work as used in the McBride et al. (22) investigation may promote greater lipid peroxidation, possibly due to the greater oxygen cost of including the concentric muscle action and/or that a larger muscle mass could increase plasma MDA levels. Elevations in MDA due to ROS production may be more dependent on increased oxygen uptake compared with the factors associated with muscle damage/injury. In the present study, MDA was only elevated at 48 h postexercise in the placebo group. Subjects supplemented with antioxidants did not demonstrate any change at this time. Although the difference was small and the physiological importance not established, MDA was significantly lower in the supplemented group compared with the placebo group. It is possible that larger differences between treatments might have occurred if more muscle mass was recruited because McBride et al. (22) reported whole-body exercise increased MDA. Additionally, it is possible that MDA might have continued to increase if the time course was extended. Close et al. (8) noted an increase in MDA at 72 h after downhill running; however, Lee et al. (17) reported that PC had returned to normal levels at this time with a similar eccentric protocol as used in the present investigation. It is probable that MDA has a different time course than PC. However, based on the existing research, it appears as though resistance eccentric exercise by itself does not stimulate large MDA increases. Additional research is needed to more fully elucidate the impact of eccentric exercise and antioxidant therapy on altering this biomarker because results are limited and inconsistent on MDA changes.
Glutathione status did not differ between treatments in the present study, with TGSH and GSH lower during the initial 6 h postexercise compared with preexercise, and GSSG elevated at this time. Therefore, the ratio of oxidized to total glutathione GSSG:TGSH was significantly elevated above preexercise at 0 and 2 h postexercise. These results are in agreement with previously reported studies indicating a transient decrease in GSH after isometric (28) and sprint exercise (12,14).
Only two investigations to date have studied glutathione status after eccentric exercise (5,17), and both demonstrated minimal change. Furthermore, Bryer and Goldfarb (5) failed to observe group differences in glutathione oxidation in subjects consuming a placebo or 3 g of vitamin C per day for 14 d before exercise. However, both these studies did not measure glutathione values 2 h after their eccentric exercise protocols. In the present study, GSSG was elevated and GSH suppressed during the 6-h period after eccentric exercise. In a similar manner as MDA, it appears as though glutathione oxidation was not as dramatically affected by ROS pathways associated with muscle soreness (3), as was PC. Additionally, the glutathione status in the blood was not affected by the antioxidant treatment. Such short-lived changes in glutathione status are likely due to the rapid conversion of GSSG back to GSH, through the activity of glutathione reductase. The inability of the antioxidant treatment to influence glutathione status suggests that there may have been adequate protection within the blood to the eccentric stress. In contrast, the PC increase most likely arose from damaged muscle. Future investigations should examine whether antioxidant pretreatment within muscle can protect oxidation of PC, glutathione, and lipid peroxidation.
In summary, eccentric exercise can increase blood biomarkers of oxidative stress in nonresistance trained females, with the greatest increase in protein carbonyls. The antioxidant therapy that included vitamin C, vitamin E, and selenium attenuated the rise in PC, and MDA at 48 h after the eccentric exercise with no effect on blood glutathione status. Future work using combined antioxidants, in varying combinations and dosages after eccentric exercise, and examining the blood and muscle influences may provide further insight into the role of these micronutrients in attenuating exercise-induced oxidative stress.
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