Physical exercise of sufficient intensity or duration enhances, through several pathways, the generation of reactive oxygen species (ROS), which can oxidative damage proteins, nucleic acids, and lipids, leading to impaired cellular function (7,14,19,20). ROS-mediated sarcolemmal phospholipids peroxidation may play a role in the etiology of exercise-induced muscle damage (21,35). A correlation was found between myocellular creatine kinase (CK) efflux, an indicator of muscle damage, and thiobarbituric reactive acid substances (TBARS), a biomarker of lipid peroxidation (22).
Muscle damage and/or ROS increase after exercise promote an acute phase local inflammatory response characterized by the release of inflammatory cytokines (such as interleukin-6, IL-6) (12) from various cell types (36), with the aim of stimulating the recruitment of neutrophils and monocytes to inflammation areas to repair damaged tissue (20). The mobilization and activation of immune cells during exercise seem to be mediated by stress hormones, such as cortisol (26). Infiltrated phagocytes produce additional superoxide (O2•−) that may amplify muscle injury (8), which may be the cause of the delayed-onset muscle soreness. There is also evidence that ROS produced during exhaustive exercise are responsible for protein oxidation, contributing to the development of muscle fatigue (27). This ROS-mediated disturbance in cellular homeostasis might result in muscular injury, soreness, and fatigue, and, consequently, decrements in physical performance (3).
To minimize exercise-related oxidative harmful effects, the body contains intracellular and extracellular defense equipment composed of both enzymatic and nonenzymatic antioxidants (AOX) (27). Trained individuals seem to have an enhanced endogenous AOX system because of regular training (6,7,19). However, intensive and sustained exercise can increase ROS to a level that outpaces tissues' AOX capacity (7). Therefore, the high training load of elite athletes could impose a cellular oxidative stress even in highly adapted muscles (7).
As physiologic adaptations induced by training may not completely counterbalance the increased oxidant insult associated with exhaustive exercise, athletes' dietary AOX requirements may be increased, particularly during periods of intensive training and competition (19). Strenuous physical training can diminish plasma AOX vitamins to a suboptimal level even if dietary intakes respect the recommendations (32). Athletes may have a greater likelihood of AOX vitamin deficiency. Moreover, sportsmen's AOX dietary intake is frequently inadequate (19). Thus, a normal diet may not be always sufficient for athletes and supplementing them with AOX would probably have beneficial effects against exercise-induced oxidative stress (19,32).
The use of AOX supplements in an attempt to minimize the extent of muscle injury or oxidative stress in response to exercise has attracted the interest of researchers and is a common practice among the athletic community (20). However, the results of works that have evaluated its effectiveness are not unequivocal and conclusive (6,7,20). Furthermore, the majority of these studies focused on exercise-unaccustomed subjects and/or used exercise protocols unrealistic for athletes (6). The extrapolation of the findings of laboratory-based intervention studies in untrained individuals to the athletic population is questionable.
The proper functioning of the AOX system depends on the concerted action of AOX, as each one of them play a specific role and functionally complement the others. It has been suggested that the combination of several AOX may be more effective than a single-compound supplementation (19). The association of AOX may maximize the biological AOX effect by allowing the scavenging of ROS generated in different environments, either lipid (such as in muscle cell lipid membranes and in plasma lipoproteins) or aqueous (such as in cytosol and in extracellular fluid) (7,9,19).
The objective of this work was to evaluate the effects of combined AOX supplementation in attenuating the oxidative stress, lipid peroxidation, muscle damage, and inflammation induced by a well-known exercise protocol for elite kayakers in a field situation while following their habitual dietary pattern and training and competition program.
Twenty volunteer athletes (14 men and 6 women) recruited from the Portuguese National Kayaking and Canoeing Senior and Junior Teams participated in this 4-wk double-blind study. During most of the time frame (3 wk), these elite athletes were in the National Team Sport Centre engaged in a controlled competitive period of training to the European Championship, consisting of resistance, endurance, and kayaking skills training sessions. Although we have not laboratorially demonstrated their high fitness level (e.g., V˙O2max), all kayakers were international representatives, regularly training and competing since their official affiliation on the National Federation (AOX group: 8.5 ± 4.4 yr; placebo (PLA) group: 8.0 ± 4.6 yr; P = 0.807). Athletes, or their parents, gave written informed consent after having been explained verbally and in writing the purpose, demands, and possible risks associated with the study, in accordance with the Helsinki Declaration. The protocol for this study was approved by the Scientific Council of the Faculty of Nutrition and Food Sciences at the University of Porto. All sportsmen were healthy, nonsmokers, reported no use of nonsteroidal anti-inflammatory drugs or oral contraceptives, and did not take AOX supplements for at least 3 months (20). Subjects were instructed to refrain from making any drastic changes in the diet, to abstain from anti-inflammatory or analgesic drugs throughout the study, and to not consume caffeine or alcoholic beverages for 24 h before the blood draws.
Subjects were randomly assigned in a double-blind fashion to two groups taking either an antioxidant complex supplement (AOX, n = 10) or a PLA (n = 10) during the 4 wk. Each AOX capsule contained 136 mg of α-tocopherol, 200 mg of vitamin C, 15 mg of β-carotene, 1 mg of lutein, 200 μg of selenium, 15 mg of zinc, and 300 mg of magnesium. AOX and PLA (lactose) capsules were generously donated by a pharmaceutical company. Kayakers were told before the beginning of the study to comply carefully with the treatment and to take two capsules daily, one before lunch and the other before dinner. Supplementation was started after the completion of the first exercise bout, continued for 28 d and ended in the night before the second kayak trial. Capsules were counted upon return of the capsule bottles to assess compliance with the treatment. The average compliance was 90.1% with no differences between treatments groups (P = 0.873).
Subjects were asked to complete in the least time possible a 1000-m maximal flat-water kayaking trial on two occasions at an interval of 4 wk. The exercises were carried out under the same equipment conditions and in the same place, time of the day (between 8:30 and 9:00 a.m.), day of the week and month to avoid circadian variations and menstrual cycle variations in women (37). Before each test, all canoeists did a standardized warming up for 15 min by jogging, stretching, and kayaking slowly. The training program in the day before both exercise trials was a short low-intensity skills session (50 min). Athletes were asked to avoid additional intense physical activity afterward. Each subject served as self-control to eliminate any biological variability in the response to AOX supplementation. This design was chosen instead of a randomized crossover design because of the storage kinetics of liposoluble AOX vitamins (36).
Since 2 d before the first trial, athletes ate identical meals prepared and provided in the same local in almost all days throughout the study. To estimate average energy and nutritional intake, athletes recorded their dietary intake during seven consecutive days, starting 2 d before the first 1000-m bout. A trained nutritionist gave detailed oral and written instructions about proper nutritional recording, including estimating portion sizes. Athletes were asked to describe the foods and fluids consumed and estimate the amount ingested by using standardized household measures or record the weight/volume and the commercial name of packaged food. Dietary records' information was transformed into energy and nutrients using ESHA Food Processor 8.0 for Windows (Salem, OR), which does not allow the quantification of α-carotene, lycopene, lutein, and zeaxanthin intakes. The adequacy of nutritional intake was assessed by reference to the Food and Nutrition Board Dietary Reference Intakes (13).
Physical activity and estimated energy expenditure.
To estimate energy expenditure, athletes recorded their daily activities in physical activity records at 15-min intervals during a 7-d period, comprising the same days of their similar dietary counterpart (2 d before and 5 d after the first 1000-m trial). The technical staff provided the detailed information about the training program during the 7 d that consisted of 11 sessions (∼560 min), comprising on-water (n = 7, 74 km, 395 min), weight training (n = 2, 115 min), and running (n = 2, 10 km, 50 min) sessions. The estimate of the mean energy expenditure for each recorded activity was calculated according to its duration and corresponding physical activity level (2) that enables assessment of kayakers' daily energy expenditure. The basal metabolic rate was calculated using the Cunningham equation.
Body composition and anthropometry.
Weight (lightly dressed and barefooted) and height were measured with a stadiometer and electronic scale (Model 701; SECA, United Kingdom), with a precision of 0.5 cm and 100 g, respectively, according to the international recommended methodology. Skinfold thicknesses were measured at four sites (bicep, tricep, subscapula, and iliac crest) using a Harpenden caliper (John Bull, United Kingdom), and body fat percentage was calculated using the formula of Siri (33).
Blood collection and handling.
Antecubital venous blood samples were collected at the onset of the study (baseline) and after 4 wk of supplementation (compliance) in preexercise and postexercise conditions. The time of day for basal blood test was standardized to within 10 min for each subject, and all samples were taken between 8:00 and 9:30 a.m. after an overnight fast (>12 h). The preexercise samples were drawn after subjects had been seated at rest for at least 15 min and immediately before warming up. The postexercise blood samples were obtained 15 min after the 1000-m canoeing trial has ended, which allows time for subjects to get from the finish line to our research site. Although more blood collections in the postexercise period would have been useful to provide additional information about the oxidative status response, the peak changes in oxidative stress biomarkers after a relatively short-duration aerobic exercise seem to occur at this time point (7). Blood samples were drawn into ethylenediaminetetraacetic acid (EDTA)-treated vacutainer tubes and nonadditive serum vacutainer tubes and immediately placed on ice in the dark until centrifugation. An aliquot of whole blood was separated to measure hematocrit and hemoglobin. Whole blood in serum tubes was allowed to clot for 30 min at room temperature and then centrifuged at 2000 rpm for 10 min for serum separation. To obtain the plasma fraction, the remaining whole blood in EDTA-containing tubes was immediately centrifuged. Erythrocytes were washed and centrifuged three times with a 0.9% sodium chloride solution and lysed with ice-cold distilled deionized water. Serum, plasma, and washed erythrocytes were separated into several aliquots and frozen at −80°C for later biochemical analysis. In evaluating the results, all plasma or serum postexercise values were adjusted by the equation suggested by Dill and Costill (11) to correct for plasma volume shift, with the exception of superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities that were expressed relative to hemoglobin concentration. The preexercise analytical values were presented without a correction for plasma volume changes. All assay procedures were performed in duplicate.
Hemoglobin and hematocrit were assessed from EDTA-treated blood using an automated analyzer (Horiba ABX Micros 60; ABX Diagnostic, Montpellier, France). The concentrations of cholesterol and triglycerides in plasma and lipoprotein subfractions were determined by enzymatic colorimetric assays (kit 07 3680 5 for triglycerides and 07 3664 3 for cholesterol; Hoffman-La Roche, Basel, Switzerland) in an autoanalyzer (Cobas Mira; Hoffman-La Roche). LDL cholesterol (LDL-C) was calculated using the Friedewald equation. Uric acid was determined by an enzymatic method at 550 nm using a commercial kit (Horiba ABX A11A01670; ABX Diagnostic), according to the manufacturer's specifications. Serum total AOX status (TAS) was measured spectrophotometrically using a commercial kit (Randox NX2332; Randox, Crumlin, UK). In brief, the assay is based on the reduction of free radicals (2,2′-azino-di-(3-ethylbenzothiazoline-6-sulfonate-ABTSΡ+), measuring the decreased of absorbance at 600 nm. The radical cation ABTSΡ+ is formed by the interaction of ABTS with ferrylmyoglobin radical species generated by the activation of metmyoglobin with hydrogen peroxide (H2O2). The suppression of the absorbance of the radical cation ABTSΡ+ by plasma AOX was compared with that from Trolox. Samples were expressed in AOX capacity in millimoles per liter of Trolox equivalents. Enzyme activities were analyzed according to the standard spectrophotometric-colorimetric procedures provided with the commercial kits in a Cobas Mira Plus analyzer (Roche Diagnostic Systems) at 37°C. Whole-blood GPx activity was determined spectrophotometrically using cumene hydroperoxide as the oxidant of glutathione (GSH; Ransel RS 505; Randox). The oxidized glutathione (GSSG) is immediately reduced to GSH by glutathione reductase (Gr), with a concomitant oxidation of NADPH to NADP+. GPx activity was measured by the decrease in absorbance at 340 nm and expressed in units per gram of mercury. The plasmatic activity of glutathione reductase (Gr) was measured by monitoring the oxidation of NADPH to NADP+ during the reduction of GSSG (Ransel GR 2368; Randox) and expressed in units per liter. A value of 10 U·L−1 was considered as the detection limit. Cu/Zn-SOD activity was measured in washed erythrocytes after lysis by using a commercial kit (Ransod SD 125; Randox). For this purpose, xanthine and xanthine oxidase were used to generate superoxide anion, which reacts with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazan dye. SOD activity was calculated by assessing the extent of inhibition of the reaction on the basis of the change in the absorbance for 3 min at 505 nm and data from the standard curve generated from purified SOD obtained from the manufacturer. SOD activity was expressed in units per gram of mercury. Plasma-liposoluble vitamins were extracted with n-hexane, after protein precipitation with ethanol, using β-apo-carotenal and tocol as internal standards. The chromatographic analyses were performed by High Performance Liquid Chromatography (Jasco, Tokyo, Japan) on a Chromolith performance RP-18 column (100 × 3 mm; Merck, Darmstadt, Germany), using methanol and acetonitrile (50:50) as mobile phase. Lutein (coeluting with zeaxanthin), α-carotene, β-carotene, and the carotenoids' internal standard (β-apo-8′-carotenal) were quantified at 450 nm, retinol at 325 nm, and lycopene at 425 nm, using a diode-array detector. α-Tocopherol quantification was based on the fluorescence readings (excitation: 290 nm; emission: 330 nm) using tocol as the internal standard. After deproteinization with metaphosphoric acid (0.75 M), serum vitamin C was determined by atomic absorption spectrometry at 520 nm using a Lambda 2 UV-visible spectrophotometer (Perkin Elmer Ltd, Norwalk, CT). Serum creatine kinase (CK) and uric acid were determined at 37°C using commercially available methods (Roche Products, United Kingdom) and an automated system (Cobas Mira Plus; Roche Diagnostic Systems). The determination of TBARS in serum was performed using a commercial kit (Oxi-tek TBARS assay kit; Zeptometrix Corporation, Buffalo, NY) according to the manufacturer's instructions. Briefly, serum (100 μL) was mixed with an equal volume of 8.1% sodium dodecyl sulfate and 2.5 mL of 5% thiobarbituric acid-acetic acid reagent. Sample was incubated at 95°C in capped tubes for 60 min and, thereafter, cooled to room temperature in an ice bath for 10 min before being centrifuged at 3000 rpm for 15 min. The supernatant was removed, and its absorbance was read at 532 nm. The results are expressed as malondialdehyde (MDA) equivalents by interpolation from an MDA standard curve (0-100 nmol·mL−1). Plasma IL-6 was measured with a commercially available solid-phase high-sensitivity enzyme-linked immunosorbent assay (ELISA) kit (Human Quantikine IL-6 Immunoassay D6050; R&D Systems, Minneapolis, MN). The limit of detection of IL-6 was <0.70 pg·mL−1. Serum cortisol was measured with an enzyme-linked fluorescent assay (ELFA; Vidas Biomerieux, Marcy I'Étoile, France) technique using a commercial kit (cat. no. 30417; Biomerieux, Marcy I'Étoile, France).
Data and statistical analysis
Statistical analyses were performed with the software SPSS 13.0 (Chicago, IL). Group data were expressed as mean ± SD. By convention, the a priori level of significance was set at α < 0.05. All data were assessed for normality (one-sample Kolmogorov-Smirnov test). Subjects' baseline characteristics, nutritional parameters, and plasma AOX levels between treatment groups were compared using independent-sample t-tests. Significant changes in preexercise plasma AOX levels before and after supplementation were analyzed using paired-sample t-tests. The other biochemical data were analyzed using mixed-model repeated-measures general linear model, with treatment (AOX vs PLA) as the between-subject factor and time (presupplementation vs postsupplementation) and exercise (preexercise vs postexercise) as the two within-subject factors. The effect of each factor and of the combined factors (interaction) on each parameter was analyzed. Because no significant gender by exercise, treatment, or time interaction effects were found for muscle injury and oxidative stress parameters (P > 0.05), data of both sexes were pooled to increase the power of analysis.
Subject's descriptive characteristics are presented in Table 1 for the AOX (n = 10, 7 males and 3 females) and PLA (n = 10, 7 males and 3 females) groups. There were no significant differences between treatment groups, also within each gender, with respect to these characteristics at baseline.
The dietary data are listed in Table 2. The supplemented and PLA groups did not differ in the estimated average energy expenditure and energetic and nutritional intakes (P > 0.05).
Before the supplementation, plasma AOX levels were similar in both groups (Table 3). In response to 4 wk of supplementation, plasma α-tocopherol and β-carotene augmented in the AOX supplement group (P = 0.003 and P = 0.007, respectively) became higher than those in the PLA group (P = 0.041 and P = 0.007, respectively), whereas the levels in the PLA group were unchanged. The levels of the other vitamins and the activities of AOX enzymes were similar in both groups, either in pre- and postsupplementation conditions, and were not modified significantly during the study and/or by supplementation. We observed a nonsignificant decrease in GPx after supplementation in the PLA group. There were no statistically significant differences in race performance between groups either before or after intervention.
A significant interaction effect among supplementation, time, and exercise on plasma TAS was found (P = 0.034), with elevated values above preexercise after the kayak race in AOX-supplemented athletes after the 4-wk period, whereas a decrease in response to exercise was observed in all the other conditions (Table 4). Uric acid and IL-6 increased significantly 15 min after exercise (P = 0.032 and P = 0.039, respectively) and did not seem to be affected by time or treatment because no statistically significant treatment and time main or interaction effects were noted (P > 0.05). Muscle protein leakage enhanced in response to the kayak trial (exercise main effect, P < 0.001) and decreased from week 0 to week 4 (time main effect, P = 0.001) more markedly in the AOX group (supplementation × time interaction effect, P = 0.049). Serum concentrations of TBARS rose significantly after the kayak bout (exercise main effect, P < 0.001) and showed a tendency to augment during the study (time main effect, P = 0.069) in all participants, although the difference was not statistically significant. ANOVA revealed an almost significant supplementation × time × exercise interaction effect (P = 0.085), with a lower TBARS increase in response to exercise in the AOX-supplemented athletes after intervention. With regard to cortisol, participants responded differently to the treatment during the study (treatment × time interaction effect, P = 0.002), with the PLA group experiencing a greater increase from pre- to postsupplementation.
As expected from other works' findings (1), we observed a significant elevation of α-tocopherol and β-carotene concentrations after the administration of the AOX supplement, whereas the levels of those receiving the PLA remained unchanged. As previously found (32), retinol levels did not augment with carotenoids supplementation. In accordance to our data, lutein (1 mg) administration for 24 d was also unable to induce an increase in its plasma levels (30). The nonsignificant trend (P = 0.149) we found for decreased lutein concentrations in the supplemented group after supplementation might be explained by its competition for intestinal absorption with β-carotene. Actually, β-carotene supplementation demonstrated significantly lower plasma lutein status (18). The supplementation led to a small increase in serum vitamin C concentrations according to another study using the same amount (400 mg) (35). However, we did not observe a statistically significant increase as they did, in accordance with other authors' findings (31,32), which may reflect the hydrosoluble nature of this vitamin and the existence of homeostatic control of its plasma levels. Schröder et al. supplemented professional basketball players with 1000 mg of vitamin C for 32 d (32) and 35 d (31), and their plasma levels also did not increment, although they have avoided the drop seen in the PLA group (32).
We observed an increase in TAS with exercise in the postsupplementation period in the athletes that have received AOX. Despite AOX supplements did not have ameliorated plasmatic AOX capacity at rest, in agreement with other works (32), they seemed to help to counterbalance the exercise oxidative insult. In line with this, a supplementation with selenium, retinol, vitamin C, and α-tocopherol has been shown to significantly augment the exercise-induced TAS increase, whereas a nonsignificant reduction in TAS in resting conditions was observed (19). The TAS response to the kayak trial may have been influenced, at least partially, by the significant augmented uric acid synthesis (exercise main effect, P = 0.032) consecutive to an enhanced activation of xanthine oxidase. Consistent with other studies' data (20,35), the postkayaking increase above preexercise levels of this aqueous AOX did not differ significantly between treatment groups.
The higher TBARS levels at baseline we observed reflect the high physical stress sportsmen are exposed to during training (32) and the tendency to increase from pre- to postsupplementation period (time main effect, P = 0.069) may be due to the cumulative augment through the season (29). Although several studies indicate that AOX supplementation attenuates oxidative damage to lipids caused by exercise (9,10,12,20,40), there are, likewise, published literature that suggests their ineffectiveness (3,24,35) or that even report a prooxidative effect (8,25). The discrepancy in the research outcomes do not seem to be significantly influenced by the type, form (natural vs synthetic), and dose of the AOX, the exercise protocol, and the training level of subjects (38). We noticed a trend of supplementation to lower the increase in TBARS after kayaking (supplementation × time × exercise interaction effect, P = 0.085). A limitation of this study is that we only have a single blood sampling time (15 min after exercise), and because TBARS may follow specific time courses, it is unknown whether differences may have occurred latter (6). Zembron-Lacny et al. (40) found lowered erythrocytes TBARS levels in the supplemented group only 30 min after the exercise ended that were not statistically evident immediately after. Although TBARS is the most widely used biomarker of lipid peroxidation because it is inexpensive and easy to assay, there is some concern about its specificity and sensitivity. It has been demonstrated that AOX supplements significantly influenced lipid hydroperoxide and F2-isoprostane levels in response to exercise, whereas they did not alter TBARS (8).
Oxidative stress and inflammation have been proposed to be involved in muscle soreness and impaired recovery after damaging exercise (26,35). AOX supplements can potentially attenuate inflammatory responses to exercise by neutralizing ROS, which are able to activate redox-sensitive signal transduction pathways that control cytokine production (12,26). Nieman et al. (25) found that the triathletes with the highest plasma F2-isoprostanes levels also presented the highest plasma concentrations of IL-6. However, very few studies have studied the effects of AOX on both exercise-induced oxidative stress and inflammation. In our work, AOX were ineffective in blunting TBARS increase with exercise and, consequently, did not influence IL-6 levels, in accordance with previous results (24). By contrast, vitamins C and E supplementation demonstrated to be efficient in repressing exercise-induced lipid peroxidation as well as in attenuating cytokine (IL-6) translocation from contracting skeletal muscle into the circulation (12). However, AOX supplementation did not downregulate the cytokine response to an ultramarathon, despite having blunted the exercise-induced rise in F2-isoprostane levels seen in the PLA group (20). Actually, the findings of studies involving AOX supplementation on alterations in markers of inflammation in response to exercise are equivocal. It has been suggested (10,26) that differences in the subjects' training status, the type, dose, and timing of supplementation, and/or the mode of exercise may explain the discrepant responsiveness to AOX supplementation. Nevertheless, conflicting data have been described either after supplementation with a combination of (10,12) or single (25,35) AOX in athletic (20,25) or nonathletic populations (8,12,35) and in response to concentric (10,12), high-intensity intermittent running (35), or ultraendurance (20,25) exercise protocols. Our findings do not support that AOX supplements are useful in limiting the increase in cytokine levels in response to exercise in highly trained athletes. The exercise studied was what kayakers' actually do in competition, and the inflammation it elicited was relatively small in comparison with the earlier investigation. The response pattern of IL-6 to exercise is influenced by the type, duration, and intensity of the exercise; the muscle mass recruited and damaged; the subjects' training status; and the blood sampling time. It is possible that the exercise used in the present investigation was not strenuous enough to induce significant inflammation, potentially disallowing for any effect to be noted. However, the magnitude of increment of cytokines did not seem to be crucial for an effect of AOX supplements (26). Carbohydrate intake may modulate the cytokine release with exercise (20) but is not expected to have influenced our findings because their intakes were not different between treatment groups.
Resting cortisol concentrations were similar to those of the Polish Olympic kayaking team during training (39) and did not significantly increase after the kayak trial. Plasma cortisol responds to exercise in an intensity- and duration-dependent manner (31). Interestingly, augmented cortisol levels were described after repeated short anaerobic cycling bouts but not after a single 1-min all-out test on a cycling ergometer (23). The lack of effect might be related to the very modest increase observed in IL-6 in response to exercise because this cytokine is, recognizably, a stimulus for cortisol secretion (34). In the present study, a significant supplementation × time interaction (P = 0.002) was observed, with the PLA group experiencing an augment in cortisol levels from the pre- to the postsupplementation period. The effects of AOX supplements on cortisol responses to exercise are variable, with some (10,12), but not all (25,31,35), studies demonstrating an attenuation. The mechanism could involve a direct action on the hypothalamic-pituitary-adrenal axis (10) or indirectly by modulating IL-6 (12). However, even when AOX supplements significantly blunt cortisol, it has not always evidenced the desirable effect on the modulation of postexercise neutrophilia and neutrophil function (10).
An extensive lipid peroxidation of membrane unsaturated fatty acids could lead to augmented permeability eliciting a CK efflux from the muscle cells. The similar increases in serum activity of CK with exercise in both groups indicate that AOX had no effect on sarcolemmal integrity, which is supported by the similar TBARS response to exercise with or without supplementation. The exercise used in the present investigation produced similar muscle damage (CK: AOX ↑ 16%, PLA ↑ 25%) and lipid peroxidation (TBARS: AOX ↑ 30%, PLA ↑ 59%) that rowers exhibited after performing a laboratory 2000-m rowing trial that lasted ∼407 s (CK: AOX ↑ 19%, PLA ↑ 32%; TBARS: AOX ↑ 70%, PLA ↑ 71%). Our observation that supplementation had no effect on exercise-related increases in CK is in agreement with the literature (21,35,40), and this outcome does not seem to depend on the amount, duration, and type of AOX supplemented, as well as the subjects' fitness status and the mode of exercise investigated. This calls into question the role of ROS in the etiology of muscle damage that may instead be a by-product of muscle damage (26). ROS play a dual mission regarding to the regulation of muscle contraction. Although the low ROS levels continuously generated by skeletal muscle under basal conditions are involved in force production, higher concentrations, as those produced during strenuous exercise, can contribute to the development of muscle fatigue by inhibiting calcium sensitivity (28). Thus, AOX interventions have the potential to delay muscle fatigue. However, research generally does not support that nutritional AOX supplementation has ergogenic effects, with very few exceptions (1). AOX supplementation does not seem to enhance exercise performance in nondeficient athletes (27). However, Aguiló et al. (1) showed that administration of vitamin E and β-carotene for 3 months and vitamin C for 15 d induces a lower maximal blood lactate concentration after a maximal exercise and might improve the efficiency of aerobic metabolism. We must considerer that ROS-mediated decline in contractile function can be a protective mechanism to limit further muscle injury and that the use of AOX supplements can override that protection, causing greater exercise-induced damage and increasing recovery times (28). We theorize that the minor decrease in the resting CK levels after supplementation that we observed in the AOX-supplemented athletes (supplementation × time interaction effect, P = 0.049) can be illustrative of this phenomenon. Interestingly, significantly (3) and nonsignificantly (4,6,8) elevated plasma CK activities in the supplement versus PLA groups have already been encountered in the days (1to 10) after the exercise, even when these had been initially minor (4). Our data biochemically support the previously reported observations of a delayed recovery in muscle function (9) and an attenuated muscle torque at specific velocities after vitamin C supplementation (35). These undesirable outcomes were observed in studies with diverse designs, concerning individuals' training status (sedentary  or physically active [3,6,9,35]) and type (vitamin C [9,35], vitamin E [3,4], vitamins C and E , or vitamin C and N-acetylcysteine ), dosage (e.g., 400  or 1000 mg  of vitamin C), length (from 7  to 30 d ), and timing (before [3,4,6,35] and after  or after  exercise) of supplement administration. It is noteworthy that most of these studies (3,4,6,8,9) used exercise protocols involving eccentric muscle contractions, which induce a delayed-onset damage caused by activated phagocytes attracted into the inflammation area to repair injured tissue (36). ROS generated by phagocytic cells may have an important physiological role in muscle regeneration, and attempts to prevent their postexercise production through AOX intervention can be detrimental to the recovery process by hampering the removal of degraded tissue proteins (9) and posterior muscle fiber regeneration (4). An AOX supplementation after muscle injury caused by eccentric exercise has been shown to increase further tissue damage in the following days (8). Furthermore, ROS are recognized modulators of cell signaling and gene expression and might, in this way, be involved in the adaptive responses to exercise (14). Actually, there is evidence that decreasing the exercise-induced ROS formation suppresses activation of important cellular signals implicated in cellular adaptations to training (16). Furthermore, vitamin C administration mitigated, albeit nonsignificantly, the training-induced increases in V˙O2max (15). Moreover, the International Olympic Committee warned that as many as one in four supplements may have a positive test result (17). Not to mention that a recent article associates β-carotene, vitamin A, and vitamin E supplementation with increased mortality (5). Thus, the recommendation that athletes should take AOX supplements needs to be seriously reconsidered either for health- or for performance-related issues. Actually, exercise itself may be already an AOX (14).
The results of the present research indicate that the supplementation with a combination of AOX does not afford protection against lipid peroxidation, muscle damage, and inflammation elicited by a 1000-m kayaking bout in highly trained athletes. Our findings do not recommend the use of the AOX supplements for attenuating exercise-induced oxidative stress and muscle injury in already well-trained individuals. Further experiments are needed to explore the hypothesis that acute AOX administration before damaging exercise can alleviate lipid peroxidation and muscle injury, whereas chronic supplementation may delay muscle recovery.
The authors thank the Portuguese Canoeing Federation and respective kayakers, coaches, and physician who participated in this study. This study was partially funded by a pharmaceutical company (PRISFAR SA) that also provided the AOX and PLA capsules and had no other source of funding, including from National Institutes of Health, Wellcome Trust, or Howard Hughes Medical Institute or others. The results of the present study do not constitute endorsement of any products by the authors or by ACSM.
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