Athletic competitions of long duration, such as marathon and ultramarathon, are demanding in terms of physical conditioning, thus challenging for the participants, whether they are competitive or recreational athletes. Although regular physical activity induces beneficial adaptations to the human body (46), very long-duration (>4 hours) ultraendurance exercise is associated with musculoskeletal injuries and gastrointestinal complaints (40).
Ultraendurance exercise-induced oxidative damage is an issue of a growing interest (20). Regular training may enhance the antioxidant defense mechanisms (13), yet acute exercise, particularly of long duration and/or high intensity, has been found to affect redox status in favor of the oxidants (3,11). In this case, the overwhelming production of free radicals cannot be compensated by the antioxidant defense mechanisms, thus resulting in oxidative stress (41), a state allegedly implicated in several pathophysiological conditions (8). From the relatively few studies conducted to investigate the effects of ultraendurance exercise on redox status (reviewed in ), it appears that ultraendurance efforts may induce oxidative stress, yet there are also studies reporting no change in oxidative stress in response to ultraendurance exercise (20). To explain the contradictory findings among studies, several factors have been put forward including the intensity and duration of exercise, the type of exercise, the training level of the participants and the markers examined (20).
The majority of studies investigating the effects of ultraendurance exercise on oxidative stress employed a variety of exercise stimulus including running, cycling, and ironman triathlon (1,12,18). To our knowledge, there is no evidence in the literature about the effects of marathon swimming on redox balance. Even though swimming is a popular sport and official competitions in open water up to a distance of 25 km are organized, only few oxidative stress studies implementing short duration swimming protocols were found in the literature (10,16,36,45). These studies have provided some useful data indicating a disturbance of the redox balance after acute short duration swimming; however, the effects of long-duration swimming on redox status are yet to be addressed.
In addition, it has been suggested that the intensity rather than the duration of exercise is the critical component for inducing oxidative damage (22,37). In this regard, marathon swimming is a useful model to study oxidative stress because it combines the extremely long exercise duration with low intensity and dissociates the production of free radicals from muscle damage induced by impact forces. Therefore, a potential increase in oxidative stress markers during ultramarathon swimming could be more confidently attributed to the metabolic rather than the mechanical component of exercise.
Thus, the objective of the present study was to investigate selective oxidative stress markers in response to ultramarathon swimming in well-trained male swimmers. In particular, thiobarbituric acid-reactive substances (TBARS), as an index of lipid peroxidation, protein carbonyls, as an index of protein oxidation, total antioxidant capacity (TAC), and full-blood count analysis pre and post ultramarathon swimming were examined. We hypothesized that ultramarathon swimming will not induce oxidative stress in well-trained rowers as critical factors associated with the increased free-radical generation, namely, the high exercise intensity and the vertical impact forces inducing muscle damage are not involved in this physical activity. Therefore, we expected that the low exercise intensity and the nonimpact nature of the effort would prevent any increase in blood oxidative stress markers.
Experimental Approach to the Problem
This work attempts to address a rather fundamental issue related to exercise-induced oxidative stress research, that is, whether oxidative stress is induced by physical activity of long duration or the increased production of reactive species is rather associated with exercise intensity. The experimental approach to this issue is simple, yet it is straightforward to its nature, because it implements an ultralong duration, very low-intensity, non-muscle-damaging physical activity. In these terms, ultramarathon swimming is a suitable model to study oxidative stress because it is characterized by very long duration and low intensity; furthermore, it dissociates the potential generation of free radicals from muscle injury induced by impact forces. To investigate oxidative stress in response to ultralong duration, low-intensity, non-muscle-damaging exercise, blood oxidative stress indices (dependent variables) before and immediately after ultramarathon swimming (independent variable) in well-trained male swimmers were assessed using a paired Student's t-test. In particular, the concentration of TBARS and protein carbonyls, as markers of lipid peroxidation and protein oxidation, respectively, TAC and full blood count analysis were evaluated. It was expected that postmarathon swimming blood oxidative stress markers will not be significantly altered from baseline. This was probably because of the low exercise intensity and the minimal mechanically induced muscle injury associated with swimming. The findings of the present study would be of interest to the scientists for conducting research with competitive or novice athletes and the coaches and trainers alike for designing training sessions of minimal oxidative stress.
Five adult male swimmers who were well trained volunteered to participate in the study. The age of the subjects was 28.8 (6.0) years, their height was 178 (8.4) cm, and their weight was 79.4 (15.6) kg. For eligibility, all participants had to be adults and provide medical affirmation signed by a pathologist and cardiologist that the subjects were healthy to participate in such exhaustive physical activity. In addition, the participants had to be experienced swimmers who have taken part previously in at least 1 swimming contest of 10 km minimum length or a triathlon race. A written informed consent was provided by the athletes after they were fully informed of the nature, potential risks, discomforts, and benefits involved in this investigation. All experimental procedures were performed in accordance with the policy statement of the American College of Sports Medicine on research with human subjects as published in Medicine and Science in Sports and Exercise and were approved by the Institutional Review Board.
Athletes were instructed to abstain from consuming alcohol and caffeine-containing beverages for 24 hours before the race and to avoid taking any vitamin supplements or changing their regular diet the last week before testing. On the day of the contest, swimmers were instructed to have a light lunch 3-4 hours before the race.
The contest was set to start at 17:00. The event took place in an outdoor 50-m length swimming pool. Each subject swam independently in the lane he was assigned to. The duration of the race was set at 24 hours of continuous swimming. However, the athletes had the right to exit the pool for 10 minutes every 60 minutes of swimming. When the swimmers were in the pool, swimming time was counted, independently of whether they were actually swimming or buoying. Subjects swam at their own pace that was constrainedly slow, for the athletes being able to complete the marathon. Judges located proximal to the pool recorded both the total duration and distance swum by the swimmers. Members of the research and support teams including a physician were present in the testing area throughout the ultramarathon race. During the race, the participants were allowed to consume snacks that consisted of potato chips, wheat crackers, and bananas and were rehydrated by drinking water and liquid isotonic solutions ad libitum.
Blood Sample Collection and Handling
Two days before exercise and within 10 minutes of the completion of exercise, a venous blood sample was drawn from the antecubital vein of the reclining subjects using standard phlebotomy procedures. Blood was collected into tubes containing dipotassium ethylenediamine tetraacetic acid as the anticoagulant. A portion of blood collected was used for full blood count. Another portion of blood was centrifuged at 1,370 g for 10 minutes at 4°C; plasma was collected and used for the determination of TBARS, protein carbonyls, and TAC.
Hematocrit (Hct), hemoglobin (Hb), erythrocyte count, leukocyte count, and platelet count were measured in a Coulter Microdiff autoanalyzer (Miami, FL, USA). Postexercise plasma volume changes were calculated on the basis of Hct and Hb using the method employed by Dill and Costill (7).
Oxidative Stress Markers Assays
Plasma protein carbonyls, an index of protein oxidation, were determined based on the method of Patsoukis et al. (38). Fifty microliters of 20% trichloroacetic acid (TCA) was added to 50 μL of plasma, and this mixture was incubated in an ice bath for 15 minutes and centrifuged at 15,000 g for 5 minutes at 4°C. The supernatant was discarded, and 500 μL of 10 mM 2,4-dinitrophenylhydrazine (in 2.5 N HCl) for the sample, or 500 μL of 2.5 N HCl for the blank, was added to the pellet. The samples were incubated in the dark at room temperature for 1 hour, with intermittent vortexing every 15 minutes, and were centrifuged at 15,000 g for 5 minutes at 4°C. The supernatant was discarded, and 1 mL of 10% TCA was added, vortexed, and centrifuged at 15,000 g for 5 minutes at 4°C. The supernatant was discarded and 1 mL of ethanol-ethyl acetate (1:1 v/v) was added, vortexed, and centrifuged at 15,000 g for 5 minutes at 4°C. This washing step was repeated twice. The supernatant was discarded, and 1 mL of 5 M urea (pH 2.3) was added, vortexed, and incubated at 37°C for 15 minutes. The samples were centrifuged at 15,000 g for 3 minutes at 4°C, and the absorbance was read at 375 nm. Total plasma protein was assayed using a Bradford reagent from Sigma-Aldrich.
Thiobarbituric acid-Reactive Substances
For plasma TBARS, a marker of lipid peroxidation, a slightly modified assay of Keles et al. (19) was used. One hundred microliters of plasma was mixed with 500 μL of 35% TCA and 500 μl of Tris-HCl (200 mM, pH 7.4) and incubated for 10 minutes at room temperature. One milliliter of 2 M Na2SO4 and 55 mM thiobarbituric acid solution was added, and the samples were incubated at 95°C for 45 minutes. The samples were cooled on ice for 5 minutes and were vortexed after adding 1 mL of 70% TCA. The samples were centrifuged at 15,000 g for 3 minutes at 25°C and the absorbance of the supernatant was read at 530 nm. A baseline shift in absorbance was taken into account by running a blank along with all samples during the measurement.
Total Antioxidant Capacity
The determination of TAC was based on the method of Janaszewska and Bartosz (17). Four hundred and eighty microliters of 10 mM sodium potassium phosphate (pH 7.4) and 500 μL of 0.1 mM 2,2-diphenyl-1-picrylhydrazyl free radicals were added to 20 μl of plasma, and the samples were incubated in the dark for 60 minutes at room temperature. The samples were centrifuged for 3 minutes at 20,000 g, and the absorbance was read at 520 nm.
Each assay was performed in duplicate. The intra and interassay coefficients of variation for each measurement were 5.2 and 7.1% for protein carbonyls, 4.5 and 5.7% for TBARS, and 3.5 and 5.6% for TAC, respectively. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Data were analyzed using SPSS, version 14 (SPSS, Chicago, IL, USA) and presented as mean ± SD or mean ± SEM. The distribution of all dependent variables was examined by the Shapiro-Wilk test and was found not to differ significantly from normal values. To evaluate any differences in the dependent variables between pre and postexercise, a paired Student's t-test was applied. The level of statistical significance was set at α = 0.05. To determine the meaningfulness of the effect of exercise on oxidative stress markers, effect sizes were calculated as the difference between post and pre-exercise mean concentrations divided by the SD of the pre-exercise concentration. Effect sizes of 0.2, 0.6, 1.2, 2.0, and 4.0 were considered to be small, moderate, large, very large, and nearly perfect, respectively, according to a modified Cohen's scale (1998) (http://newstats.org). For comparison, the values of the original Cohen's scale are 0.2 for small, 0.5 for moderate, and 0.8 for large effects (5).
A post hoc power analysis to determine the sample size (n) that would be needed for a significant increase in oxidative stress markers was performed. The acceptable statistical power was set at 0.70. The power calculations were based on the effect sizes computed between the pre and postexercise value for each dependent variable. The SDs of the pre and postexercise values were used for these calculations, whereas the α level was set at 0.05. The statistical power for each dependent parameter was performed using the software G*Power 3 (9). For TAC, the difference between pre and postexercise values was indistinguishable, and therefore, it was not meaningful to continue with further analysis. With respect to the other dependent variables, for the magnitude of differences detected between pre and postexercise values, the sample size required for a statistical power of 0.70 would be n = 18 for PC and n = 11 for TBARS.
The mean swimming time and distance covered were 1,165 (203) minutes and 50.5 (15.0) km, respectively. The results of the hematologic parameters are shown in Table 1.
There was a significant increase (p < 0.05) in Hct values postexercise (46.6 ± 2.3%) vs. pre-exercise (43.9 ± 0.8%). Hemoglobin also increased postexercise (15.3 ± 0.8 vs. 14.7 ± 0.5 g·dl−1), yet this increase was not significant.
Based on the method of Dill and Costill (7), the calculated postexercise plasma volume relatively to pre-exercise value was 0.919 ± 0.07. However, no alteration in the output of the statistical analyses was detected regardless of whether postexercise values were treated as raw data or were corrected for plasma volume change. Therefore, only the raw data are presented in the Results section.
Leukocytosis occurred after swimming, as evidenced by the increase in leukocyte count that was mainly the result of the neutrophil and monocyte count increases (p < 0.05). Oxidative stress markers data are presented in Table 2. No significant differences between the pre- and postexercise values were found. The effect size of exercise on protein carbonyls, TBARS, and TAC was +0.53, +0.70, and +0.20, respectively.
To our knowledge, this is the first attempt to examine redox status in response to ultramarathon swimming. The data of the present study did not demonstrate significant increases in blood oxidative stress markers of well-trained swimmers after ultramarathon swimming. Plasma protein carbonyls, TBARS, and TAC concentration remained unchanged compared to prerace values.
The findings of previous studies regarding oxidative stress in response to ultraendurance exercise are equivocal suggesting that a consensus is yet to be reached. There are studies reporting increased oxidative damage after ultraendurance cycling, ultraendurance running, and triathlon (1,4,18,21,27,28,32-34), whereas other relevant studies have found no alteration in oxidative stress markers after these types of activities (12,25,39). Our findings are in agreement with those of the latter group of studies indicating no significant change in oxidative stress parameters. This inconsistency in the literature could be possibly attributed to the different exercise types and intensities used in the relevant studies, the different training level and age of the participants, and the variety of markers and methods used to assess oxidative stress.
In the present study, the fact that there was no significant increase in oxidative stress markers may be explained on the basis of the relatively low intensity of marathon swimming. It appears that despite the very long exercise duration, oxidative stress is not initiated when the stimulus is of low intensity. It has been suggested that high exercise intensity and low adaptation to the particular physical activity are main factors to induce oxidative damage (2,11). According to this view, it appears that there is an intensity threshold below which oxidative stress does not occur.
Most studies reporting increased oxidative stress at exercise intensities greater than 60-70% of o2max (2). In our study, it was practically difficult to measure directly o2 during marathon swimming. However, there was an indirect estimation of the average exercise intensity via heart rate (HR), because it is well established that HR is linearly related to o2 during exercise (6). When athletes exited the pool for their short break, HR was randomly measured by the means of carotid artery palpation. On average, the athletes' HR during swimming represented about 70-75% of the subjects' predicted maximal HR, which in turn corresponds to about 50-60% of their predicted maximal oxygen consumption (29,44).
With regard to adaptation, it has been recently shown that high-volume ultraendurance activity produces a significant decrease in resting malondialdehyde concentration suggesting protection against oxidative damage (21). Trained subjects exhibited greater resistance to oxidative damage after acute exercise (11).
We hypothesize that during the long exercise duration of marathon swimming, the human body managed to adapt to the stress of exercise and regulate the redox homeostasis. In 2 recent studies, serum reactive oxygen species (14) and urinary DNA oxidative damage marker of 8-hydroxy-2′-deoxyguanosine (30) increased after the first day of the race of a 2-day ultramarathon running and returned to baseline levels after the second day of the race, indicating that an antioxidant defense was developed to confront the exercise-induced oxidative stress. In another study, Martarelli and Pompei (26) found elevated levels of reactive oxygen metabolites and a concomitant increase in biological antioxidant potential after a 24-hour mountain bike competition. This possibly indicates a regulatory response of the body for re-establishing the balance between oxidant production and antioxidant defense mechanisms that was disturbed by exercise. In our study, the fact that neither the oxidative damage markers of protein carbonyls and TBARS nor the TAC was affected by marathon swimming may indicate that a redox balance was attained during exercise.
Another noteworthy finding of the present study was the leukocytosis observed after marathon swimming mainly resulted from the increased neutrophil and monocyte count, indicating postexercise inflammatory response. Inflammatory neutrophilia is an important source of reactive oxygen species after endurance exercise and may lead to oxidative damage (15). However, it has been suggested that the ability of neutrophils to generate reactive oxygen species was reduced in trained individuals (42,43).
In addition, leucocytosis and increased oxidative stress have been mainly associated with exhaustive and unaccustomed exercise inducing muscle damage (23,24,35). Swimming is considered a non-muscle-damaging type of exercise because it involves mainly non-weight-bearing activity and concentric contraction causing no or minor muscle damage (31). Therefore, had the swimming affected oxidative stress markers, it would unlikely be attributed to muscle damage.
A limitation of the present study was the small sample examined because of the nature of the study. The ultraendurance effort undertaken by the athletes is considered physically and mentally intense and required stamina and persistence. Nevertheless, to our knowledge, this is the first time that data of oxidative stress parameters after an ultramarathon swimming are reported. Another limitation of this study was the fact that we have not detected additional sensitive markers of oxidative damage. For example F2-isoprostanes are considered a sensitive marker of lipid peroxidation. Had we measured isoprostanes, there was a possibility that slightly different findings may have been noted.
Furthermore, it would have been helpful if we had assessed the activity of the endogenous antioxidant enzymes, namely, superoxide dismutase, catalase, and glutathione peroxidase pre and postexercise. This information would have shown whether exercise induced any alterations in blood redox status compared to the baseline. Alternatively, when the resting TAC values (reflecting the concentration of nonenzymatic antioxidant molecules) of our subjects were compared to those of untrained subjects of similar characteristics from our laboratory database, it appears that TAC values were 20% higher in the trained swimmers compared to the untrained individuals.
The fact that our subjects were allowed to consume food throughout the event is unlikely that it affected oxidative stress status, because no antioxidant supplements were provided. Rather the food consisted of snacks and isotonic sport drinks aimed at providing athletes with energy and maintained them in an adequately rehydrated state. Previous ultramarathon studies in which subjects were also consuming food throughout the race reported either increase (27) or no change (39) in oxidative stress.
In conclusion, no alteration was observed in protein carbonyls, TBARS, and TAC, as markers of oxidative stress, after an ultramarathon swimming. The well-trained and well-adapted swimmers seemed to be able to regulate a redox homeostasis during swimming of ultralong duration, likely because of the relatively low intensity of marathon swimming. Furthermore, the antioxidant defense mechanism of our well-trained swimmers may have been amplified to meet the demands of such exercise conditions. Additional research is needed to further clarify the effect of ultraendurance exercise on oxidative stress, assessing more parameters associated with oxidative stress and controlling those constrain variables and factors that may interfere with the oxidative damage.
The data of the present investigation support the findings of previous studies reporting that the critical factors associated with oxidative stress in physical activity include the intensity of exercise, the training level of the athlete, the type of exercise, and whether the athlete is accustomed to the type of exercise implemented. Marathon swimming performed by well-trained swimmers did not induce oxidative stress because these well-adapted athletes were able to modulate a redox balance during exercise. It appears that swimming, even though of ultralong duration, is unlikely to induce oxidative stress probably because of the low-intensity and the nonimpact force, thus the non-muscle-damaging nature of this exercise modality.
The findings of the present study would be of interest to the scientists conducting research with competitive athletes trained with high-intensity or novice individuals engaged in recreational physical activities of long duration and low intensity. Designing an experimental protocol involving oxidative stress, research scientists could use the findings of this paper in terms of whether duration alone in exercise regimen can be an adequate stimulus to induce oxidative stress and cite the data of this study to support the experimental approach they may follow. Equally importantly, coaches of competitive sports when design long-term and short-term training plans should take into consideration that the implementation of low-intensity long-duration exercise protocols are unlikely to associate with oxidative damage; rather this type of exercise regimen may facilitate recovery. In this regard, coaches can more flexibly schedule the content of the training sessions that precede and follow a low-intensity long-duration training session.
Furthermore, trainers of recreational activities can design training sessions inducing minimal oxidative stress. For recreational athletes, swimming may be a more suitable exercise modality than other types of physical activity because it is associated with less oxidative stress compared to high impact sports like running and jumping.
In any case, it appears that the long duration and low-to-moderate intensity exercise is health oriented because it dissociates from oxidative damage. In a related issue, recreational athletes who participate in these types of activities may not take any antioxidant supplements because the necessity for boosting the endogenous antioxidant system is questionable.
No external financial support for this project was received.
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