More than 50% of all elite endurance athletes and approximately 40% of the nonelite athletes in the United States consume vitamin supplements daily (36) and in much higher doses than the recommended daily allowance (RDA) (33). Consumption of vitamin supplements is not limited to athletes, however. Among individuals participating in a regular exercise program within the United States, 25% of the women and 16% of the men reportedly ingest supplements on a daily basis (36).
The reason for this interest in vitamin supplements is primarily because of the observation that enhanced production of reactive oxygen and nitrogen species (RONS) in response to exercise may lead to modifications of lipids, proteins, nucleic acid, and other cellular compounds (2). The damaging effects of RONS may consequently induce decreased muscular functionality, histological changes, and muscular soreness (22,37) and thus may attenuate exercise performance. This observation has led to research into whether antioxidant supplementation could prevent the damaging effects of RONS and thereby enhance performance.
From the initial view that RONS were, in general, harmful and that preventing their actions would be beneficial (26), it now seems possible that these substances also play fundamental roles in the regulation of gene transcription and protein synthesis (16,18). Several transcription factors seem to be regulated by the intracellular redox state (17). Two of the most well-documented redox-sensitive transcription factors are nuclear factor κB, which regulates the transcription of immune and inflammatory genes, and activator protein, which regulates genes involved in growth and differentiation (34). In addition, RONS have been shown to act as important signals for the induction of endogenous antioxidant enzymes during endurance training (14,27).
According to the theory of the hormesis curve (31), RONS production is necessary during regular exercise for the initiation of adaptive processes. These adaptive processes include up-regulation of antioxidant and damage-repair enzymes, lowering of the basal levels of RONS, and reduction in oxidative damage during exercise (30). Thus, RONS production during regular exercise, which is placed on the top of the hormesis curve, provides beneficial and protective effects concerning health.
The suggested roles of RONS as inducers of adaptive responses to muscle contraction seem to contradict the use of antioxidants. The latter could potentially suppress beneficial adaptive responses and thereby diminish or even abolish the effect of regular training on exercise performance. Recent data by Gomez-Cabrera et al. (12) are in accordance with the view that the well-known beneficial effects of exercise are attributable to the capability of exercise to produce increased levels of RONS. The latter study demonstrated that a high daily dosage of vitamin C during endurance training attenuates normal training-induced mitochondrial biogenesis and endurance capacity in rodents (12).
Therefore, we hypothesized that vitamin C and E supplementation during endurance training would attenuate the expected increases in exercise performance in individuals with normal levels of vitamins C and E at entry. We measured physiological parameters such as maximal oxygen consumption, maximal power output, and workload at lactate threshold (LT) that are used as parameters of aerobic performance. We also measured metabolic parameters such as skeletal muscle glycogen content and mitochondrial enzymes-citrate synthase (CS) and β-hydroxyacyl-CoA dehydrogenase (β-HAD)-which represent the oxidative capacity of skeletal muscle and are increased with endurance training (11,29). To obtain a marked improvement in cardiorespiratory fitness, we subjected the participants to a training protocol with high training intensity and a large volume of training.
SUBJECTS AND METHODS
Twenty-one young, healthy, physically active men (aged 18-40 yr old) participated in the study. Participants' characteristics are listed in Table 1. Before inclusion in the study, a medical examination with blood test screening, a test for maximal power output (Pmax) assessment, and an oral glucose tolerance test were performed. Exclusion criteria included physical exercise more than two times per week, body mass index (BMI) > 30 kg·m−2, smoking, impaired glucose tolerance, use of medication, and supplementation with antioxidants.
A double-blinded placebo-controlled design with minimization was used for the allocation of the participants into two groups: antioxidant (AO; n = 11) and placebo (PL; n = 10). The participants were allocated according to age, BMI, and Pmax. Participants in the AO group received oral supplementation (tablets) with vitamin C (ascorbic acid, 500 mg daily) and vitamin E (RRR-α-tocopherol, 400 IU daily) for 16 wk, whereas the PL group received placebo tablets. The active compound for the vitamin E tablets was d-α-tocopherylsuccinate, and the active compounds for vitamin C tablets were ascorbic acid 200 mg per tablet and sodium ascorbate 300 mg per tablet. The placebo tablets for vitamin E consisted mainly of microcrystalline cellulose, calcium hydrogen phosphate anhydrous, and starch 1500 PT. The placebo tablets for vitamin C consisted mainly of citric acid, mannitol, and sorbitol.
The supplement dosage for vitamin C was >5 times higher than the recommended dietary allowance, whereas the dosage for vitamin E was >15 times higher than the recommended dietary allowance. Of note, this combination has previously been shown to attenuate oxidative stress after acute exercise (9). All participants were instructed to take the supplementation once a day with breakfast. The supplementation started 4 wk before onset of the training and continued throughout the training period.
The study was approved by the local ethical committee of Copenhagen and Frederiksberg and was performed in accordance with the Declaration of Helsinki. The purpose of the study and its possible risks and discomforts were explained to the participants before their written consent was obtained.
The mode of exercise selected for training was cycling, and the training frequency was five times per week for 12 wk. A Pmax test was performed at the beginning of each training week to determine the intensity of the training for the following days of the week. The Pmax test was performed the same way as the V˙O2max test. On Tuesdays, the training consisted of ten 3-min intervals at 85% Pmax interspersed by 3-min intervals at 40% Pmax. The next day, the training was continuous at 60% Pmax, and the duration was 60 min. On Thursdays, the training consisted of five 8-min intervals at 75% Pmax with 4-min rest at 40% Pmax in between. On Fridays, the training was continuous for 120 min at 55% Pmax. For the first 6 wk, the duration of each training session was increased by 5%·wk−1, while within the last 6 wk, the duration remained stable and the intensity was increased by 1%·wk−1. The participants were allowed to miss only 5% of the total amount of the training, which was equal to three training days. If they had to refrain from training for more than 3 d, training was performed during the weekend.
Maximal oxygen consumption test (V˙O2max) and determination of LT.
Maximal oxygen consumption (V˙O2max) was measured on an electrically braked cadence-independent cycle ergometer (Monark 839E; Monark Ltd., Varberg, Sweden) using an indirect calorimetry system (Quark b2; CosMed, Rome, Italy) during an incremental exercise test to volitional fatigue. Participants were asked to abstain from strenuous exercise at least 2 d before the test. After a 5-min warm-up at 40% of V˙O2max, the workload increased every minute by 0.1 of 60% of expected V˙O2max (7). The expected V˙O2max was calculated on the basis of the Pmax obtained from the screening test. The test was terminated when the participant was unable to maintain a cadence of 60 rpm for more than 15 s despite verbal encouragement. Expired oxygen and carbon dioxide were recorded in real time (Quark b2; CosMed). HR was recorded during the whole duration of the test. Blood samples for lactate determination were collected from a flexible catheter (Becton Dickinson, Helsingborg, Sweden) placed in the antecubital vein, at the start of every minute during the incremental phase, and at 1, 3, 5, and 20 min after the end of the incremental test while the participant remained seated on the cycle ergometer. Blood was drawn into preheparinized (80 IU of dry electrolyte-balanced heparin; Pico 50; Radiometer, Copenhagen, Denmark) syringes, which were kept on ice until analyzed for lactate concentration. The blood samples were analyzed immediately after the cessation of the test on an ABL 700 series (Radiometer).
The workload at which the concentration of blood lactate reached 4 mmol·L−1 was selected to determine the LT. The exercise intensity for a lactate concentration [La] of 4 mmol·L−1 is considered the intensity at which the blood [La] begins to increase exponentially. The onset of blood lactate accumulation has been used as an indicator of endurance performance (13).
Muscle biopsies were obtained at rest, before, and after the training period from the vastus lateralis using the percutaneous needle method with suction (4) under local anesthesia, using 3-5 mL of 20 mg·mL−1 lidocaine (SAD, Copenhagen, Denmark). Muscle tissue was immediately frozen in liquid nitrogen and stored at −80°C until further analysis. Muscle biopsies were obtained between 72 and 96 h after the last exercise session.
Blood samples were drawn before supplementation and before and after the training period. The blood samples were drawn from a venous catheter into EDTA-containing glass tubes, and they were immediately centrifuged at 3500g for 15 min at 4°C. Plasma samples for vitamin C measurements were treated as described below and, subsequently, stored at −20°C until analysis.
The participants were instructed to maintain their habitual diet. Mean vitamin C and E daily consumption was determined by registration of dietary food and activity in 3 d (including a weekend day) in the beginning, after 6 wk of training, and at the end of the training period. Data were analyzed by a software (Dankost Sport; Dansk Catering Center A/S, Copenhagen, Denmark). Complete dietary food registration was, however, provided from only five participants of each group.
Glycogen concentration and enzymatic activity.
About 5-10 and 10-20 mg of muscle tissue were used for glycogen concentration and for enzymatic activity measurements, respectively. The tissue was freeze-dried and dissected-free of visible blood and connective tissue. Glycogen concentration was determined by boiling the dried muscle tissue in 1 M HCl for 2 h at 100°C and by analyzing the supernatants for glucose concentration (Roche UniKit, Paris, France) on an automatic analyzer (Cobas Fara; Roche, Basel, Switzerland). The maximal activities of CS and β-HAD were determined using enzymatic fluorometric assays at 30°C and 37°C, respectively (28).
Analysis of vitamin C and α-tocopherol.
Plasma samples for vitamin C measurements were mixed with an equal amount of 10% meta-phosphoric acid containing 2 mM disodium EDTA and were frozen immediately until analysis. The concentration of ascorbate was measured by reverse-phase HPLC with colorimetric detection (25). The concentration of α-tocopherol in plasma samples was measured by HPLC with amperometric detection (8).
Twenty-five milligrams of skeletal muscle samples was used for protein extraction. Muscle lysates were then prepared adding 500 μL of ice-cold homogenization buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM PMSF, 1 mM dithiothreitol, 0.5% (v/v) protease inhibitor cocktail, and 1% (v/v) Nonidet P-40) to the muscle tissue. The muscle tissue was then homogenized using cooled racks in a TissueLyser (Qiagen, Valencia, CA) for 1 min at 20 Hz followed by 15 min of incubation on ice. The procedure was repeated twice and, if tissue particles were still visible, also a third time. Homogenates were then rotated end over end for 1 h at 4°C and centrifuged at 13,000g at 4°C for 15 min. The supernatant was collected and stored at −80°C. An aliquot of 10 μL of each lysate was collected and diluted for protein concentration determination before storage.
The protein concentration of tissue extracts was determined in triplicate using the bicinchoninic acid (BCA) method using bovine serum albumin standards (Pierce, Rockford, IL) and BCA assay reagents (Pierce). A maximal coefficient of variance of 5% was accepted between replicates.
Equal amounts of denatured proteins (23) from the tissue homogenates were separated by gel electrophoresis using a NuPage 8% Bis-Tris gel (Invitrogen, Taastrup, Denmark), followed by immunoblotting to PVDF membranes (hybond-P; GE Healthcare, Amersham Place, UK). Membranes were then incubated in blocking buffer (5% skimmed milk) for 1 h at room temperature and washed three times for 5 min in wash buffer (Tris-buffered saline with 0.1% Tween-20). Subsequently, the membranes were incubated overnight at 4°C in a blocking buffer containing a primary antibody against manganese superoxide dismutase (MnSOD, catalog No. 06-984; Upstate-Millipore, Billerica, MA). The membranes were then washed three times in wash buffer and incubated for 1 h at room temperature with a secondary antibody (Dako, Glostrup, Denmark). Protein bands were detected using Supersignal West Femto (Pierce) and were quantified using a CCD image sensor (ChemiDocXRS; Bio-Rad, Hercules, CA) and software (Quantity One; Bio-Rad). Preliminary experiments demonstrated that the amounts of protein loaded were within the dynamic range for the conditions used and the results obtained (data not shown), and immunoreactive bands migrated at expected relative mobilities. Reactive brown protein stain (39) was used as a loading control.
All data were tested for normality of distribution before further analysis using histograms and probability plots. Plasma ascorbic acid concentration, V˙O2max, Pmax, and protein content of MnSOD were normally distributed. Plasma concentrations of α-tocopherol, glycogen, CS and β-HAD activity as well as workload at which LT occurred, and dietary data were normally distributed after log transformation. Of note, statistical analysis of log-transformed data evaluates relative rather than absolute differences.
A general linear mixed model was used to analyze the effect of time and supplementation. An interaction between the two was also tested. A random subject-specific component was introduced to adjust for the interindividual variations. The fit of the model was evaluated by testing the residuals for normal distribution and variance homogeneity. In the post hoc analysis, possible effects of time were tested by Student's paired t-tests. The level of significance was set at P < 0.05. Statistical analysis was performed using SAS statistical software (version 9.1; SAS Institute Inc., Cary, NC).
Results are presented as mean ± SEM or geometric mean ± 95% confidence interval (CI) for the log-transformed data. The geometric mean was calculated from the arithmetic mean of the log-transformed data (back-transformation) to present data in a meaningful way. Similarly, the 95% CI was calculated from the 95% CI of the log-transformed data. Compared with the mean of nontransformed data, the geometric mean is less sensitive to high values; thus, it is often lower than the corresponding arithmetic mean.
The two groups did not differ at baseline regarding Pmax, anthropometrical measurements, and vitamin plasma concentration (Table 1).
Vitamin C and E concentration in plasma.
Plasma ascorbic acid concentration (Fig. 1A) increased (P < 0.05) in the AO group after 1 month of supplementation and remained elevated (P < 0.05) during the whole training period. Plasma vitamin E (α-tocopherol) concentration (Fig. 1B) also increased after 1 month of supplementation (P < 0.001) in the AO group and remained elevated until the end of the training period (P < 0.005). There was no difference in the plasma vitamin levels over time in the PL group (P > 0.05).
Mean vitamin C and E daily consumption did not change during the training period for any of the groups. Data are shown in Table 2.
In response to training, all physiological parameters measured increased in both groups. V˙O2max (Fig. 2A) increased by approximately 17% (P < 0.0001) in the AO group and 20% in the PL (P < 0.0001). Pmax (Fig. 2B) increased approximately 24% the AO (P < 0.0001) and 20% in the PL (P < 0.0001) group. The workload at the LT (Fig. 2C) also increased in both groups in response to training. The increase was approximately 25% (P < 0.0001) in the AO group and 23% (P < 0.005) in the PL group. There were no differences between the two groups concerning any of the variables mentioned above.
Effects on skeletal muscle.
Skeletal muscle glycogen concentration (Fig. 3A) was 45% (P < 0.001) higher in the AO group and 42% (P < 0.01) higher in the PL group after the 12 wk of training. Activity of the metabolic enzymes in the skeletal muscle also increased significantly. CS activity (Fig. 4A) increased approximately 62% (P < 0.0005) in the AO group and 60% (P < 0.005) in the PL group. β-HAD activity (Fig. 4B) was 28% (P < 0.005) higher in the AO group and 31% (P < 0.05) higher in the PL group. Finally, the protein content of MnSOD (Fig. 5) was 40% higher (P < 0.001) in the AO group and 30% higher (P < 0.0005) in the PL group after the 12 wk of training. There were no differences between the two groups concerning any of the variables mentioned above.
The main aim of the present study was to test if antioxidant supplementation to healthy individuals, having normal vitamin C and E levels at entry, would influence training adaptation with focus on performance. In contrast to our hypothesis, the present study showed that combined supplementation with vitamins C and E before and during 12 wk of supervised, strenuous bicycle exercise training of a frequency of 5 d·wk−1 had no effect on maximal oxygen consumption, maximal power output, workload at LT, glycogen content, and CS and β-HAD activities in muscle.
The RDA for vitamin C is 90 mg for men and 75 mg for women to maintain a normal plasma concentration of around 50 μmol·L−1 (6). For vitamin E, the RDA is 10-30 IU to maintain a normal plasma concentration of 8-28 μmol·L−1 (36). Before the beginning of supplementation, the plasma concentration was within the reference range for both vitamins C and E in both groups. The participants managed to maintain a steady diet during the whole training period (Table 2), and with the additional supplementation, the plasma concentration for both vitamins increased up to saturation levels (Fig. 1) in the AO group.
We evaluated a training protocol that aimed at inducing maximally improved performance in only 12 wk. In addition, each training session was supervised to ensure that all training was performed and to obtain a very clear training effect. Using a combination of interval and continuous endurance training, healthy, moderately trained, young men improved their V˙O2max by approximately 17%. Mitochondrial enzyme activity and muscle glycogen content were also significantly increased in response to training. These findings are in accordance with previous studies (11,29), which show that endurance training increases the oxidative capacity of the skeletal muscle.
There are few data on the effect of vitamin C or E or the combined vitamin C and E supplementation on training adaptation in response to endurance training, and the results are controversial. Early studies from the 1970s and 1980s have tested the ergogenic effect of vitamin C during training. Some of them have shown no beneficial effect of vitamin C on endurance (10) or on aerobic and anaerobic capacity (20) in response to endurance training, although in one study, 1000 mg of vitamin C supplementation daily for 2 wk significantly reduced the HR during submaximal work (15). In a recent study, Gomez-Cabrera et al. (12) found that the same supplementation had no effect on V˙O2max after 8 wk of endurance training in sedentary men, whereas vitamin C supplementation in rodents attenuated training-induced mitochondrial biogenesis and endurance capacity. The dosage of vitamin C used was fourfold higher compared with the dosage given in humans in the same study, and furthermore, it was eightfold higher than the dosage used in our study.
Training studies with vitamin E supplementation alone have shown no effect on endurance capacity (24) or on cardiorespiratory efficiency and motor fitness (35) in young, competitive swimmers. However, a recent rodent study indicated a beneficial effect of vitamin E supplementation on endurance capacity in aging rats (3).
The combined supplementation of vitamins C and E had no effect on performance in soccer players during the precompetitive period (40) but led to a reduction in maximal blood lactate concentration during an incremental test in endurance athletes (1) and increased aerobic power in nonathletes (19). Jourkesh et al. (19) found an increase in aerobic power after vitamin C and E supplementation, and Aguilo et al. (1) found a decrease in lactate concentration after an incremental test after 90 d of antioxidant supplementation, indicating a positive effect of the supplementation.
Recent studies suggest that antioxidants may have detrimental effects when applied in patients with hypertension. Thus, when a 6-wk aerobic exercise training program was applied in patients with hypertension, supplementation of antioxidants (vitamins C and E and α-lipoic acid) led to an enhancement of blood pressure and an inhibition of exercise-induced flow-mediated vasodilatation (38). Another recent study showed that antioxidant supplementation with vitamins C and E inhibited health beneficial effects of 4 wk of exercise on insulin sensitivity and other markers of metabolic training adaptation (32).
Recent data have also shown potentially negative effects of high doses of antioxidants on the adaptive responses of endogenous antioxidant enzymes and stress proteins (21), which are considered important defense mechanisms against free radicals. In the present study, we found a significant increase in protein content of MnSOD in skeletal muscle in response to training in both the AO and the PL groups. A possible explanation for the discrepancies may be that the high fitness level of the participants in the present study prevents large changes in the redox status.
Taken together, there is little consistency in the literature about the effects of training adaptation and antioxidants. However, it seems that when regular moderate exercise is initiated in a relatively untrained group of people, antioxidants may tend to blunt training adaptation. In our study, the participants had a relatively high fitness level at entry and underwent a training protocol with a larger amount of training and much higher intensity than that in previous studies. Most studies differ concerning the dose and nature of antioxidant supplementation. However, it is not possible to ascribe differences in study outcomes to differences in supplementation.
The hormesis theory suggests that biological systems respond to exposure to RONS with a bell-shaped curve. According to this theory, the beneficial effects of regular moderate exercise are based on the RONS-generating capability of exercise, which would explain the somewhat detrimental effects of antioxidants in some studies (32,38). Our participants were subjected to a very high training load in order for exercise-induced adaptation to occur. The volunteers in our study obtained maximal improvements in performance and adaptation. According to the hormesis theory, this indicates that whole body and skeletal muscle maximal physiological functionality and resistance to stress were achieved (31).
Therefore, we believe that any possible, biologically relevant effect of the vitamin supplementation would have been detected. We suggest that with highly intense endurance exercise, the beneficial effects of RONS may be counteracted by RONS-induced cell damage effects, explaining why we were unable to find any effects of the antioxidant supplementation.
The clear finding in the present study that antioxidants did not influence training adaptation does not exclude the possibility that antioxidant supplementation will exert different effects when applied in combination with less intense training protocols or if given to individuals, who are older, less trained, metabolically impaired, or vitamin deficient at entry.
Considering that the health-conscious part of the population generally consumes a balanced diet, rich in fruits and vegetables, and low in fat (5), our data suggest that this particular population will not experience any effect-positive or negative-from moderate daily vitamin supplements on training adaptation in response to strenuous endurance training. In conclusion, healthy people who just exercise regularly to improve or maintain a certain fitness level should be more critical toward antioxidant supplements.
Ruth Rousing, Hanne Villumsen, Jytte Nielsen, and Annie B. Kristensen are acknowledged for their excellent technical assistance. The Center of Inflammation and Metabolism is supported by a grant from the Danish National Research Foundation (No. 02-512-55). This study was further supported by the Danish Medical Research Council, the Commission of the European Communities (contract no. LSHM-CT-2004-005272 EXGENESIS), and by grants from the Greek State Scholarships Foundation and the Danish Ministry of Culture committee on sports research.
The results of the present study do not constitute endorsement by American College of Sports Medicine.
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