Effect of Different Running Exercise Modalities on Post-Exercise Oxidative Stress Markers in Trained Athletes

aim of this study was to examine the e ect of running exercise modality on oxidative stress. Thirteen endurance athletes (age: 21.46 0.66 years) performed three di erent running exercise modalities (Continuous running exercise (CR): continuous running exercise at 75% of VO2maxfor 25 min; intermittent running exercise #1 (15/15): intermittent running protocol, 15 s running at 75% of VO2max, 15 s passive recovery, performed for 50 min; intermittent running exercise #2 (30/30): intermittent running protocol, 30 s running at 75% of VO2max, 30 s passive recovery, performed for 50 min) in a randomized order. Blood samples were drawn at rest and immediately after each running exercise and assessed for malondialdehyde (MDA), advanced oxidation protein products (AOPP), superoxide dismutase(SOD), and glutathione peroxidase (GPX) activities. MDA increased by 55% following 30/30 exercise (p<0.01), while it remained unchanged with CR and15/15 exercise. SOD increased after CR (+13.9%,p<0.05), and also remained unchanged after 15/15 (p>0.05) and decreased after 30/30 ( 19.7%p<0.05). GPX and AOPP did not change after exercise in all experimental sessions (p>0.05). In conclusion, 30/30

protein products (AOPP), superoxide dismutase(SOD), and glutathione peroxidase (GPX) activities. MDA increased by 55% following 30/30 exercise (p<0.01), while it remained unchanged with CR and15/15 exercise. SOD increased after CR (+13.9%,p<0.05), and also remained unchanged after 15/15 (p>0.05) and decreased after 30/30 ( 19.7%p<0.05). GPX and AOPP did not change after exercise in all experimental sessions (p>0.05). In conclusion, 30/30 intermittent running induced higher lipid damages than the 15/15 and CR exercise. 15/15 intermittent exercise promoted a better balance between free radicals production and antioxidant defense compared to continuous exercise and intermittent 30/30 exercise. Keywords:oxidative stress; exercise; athletes; free radical damages; antioxidant defenses 1. Introduction The practice of regular physical activity [1] and running [2] are recognized as essential factors for maintaining good health (e.g., ghting against cardiovascular diseases, osteoarthritis, diabetes, and osteoporosis) [1]. However, the practice of long and/or intense physical exercise can expose athletes to muscle injuries and chronic fatigue, which can be directly linked to the toxic e ects of free radicals (FR) [3]. Int. J. Environ. Res. Public Health2020,17, 3729; doi:10.3390 /ijerph17103729 /journal/ijerph

Int. J. Environ. Res. Public Health2020,17, 3729 2 of 12 Oxidative stress is an imbalance between the biochemical processes of FR production and antioxidant defenses [4]. Classically, elevated levels of oxidative damage markers, likemalondialdehyde (MDA) and advanced oxidation levels of the protein products (AOPP) are associated with increased oxidative stress, while elevated levels of antioxidants, such as superoxide dismutase (SOD) and glutathione peroxidase (GXP) are associated with decreased oxidative stress. When the redox equilibrium or homeostasis is disrupted, cells become vulnerable to free radical attack, resulting in oxidative damage to cellular components [5]. FR are widely recognized for their dual roles as being both deleterious and bene cial, since they can be either harmful or bene cial to living systems, particularly by playing a physiological role in intracellular signaling and regulation as secondary messengers of the expression of antioxidant enzymes, such as SOD, and improves oxidation resistance [6]. However, under certain conditions, FR produced during exercise may exceed the body's antioxidant capacity and contribute to muscle fatigue [7], in ammation, and tissue damage caused by the oxidation of macromolecules [8]. Physical exercise a ects FR production and the antioxidant capacity that could contribute to a disruption of the balance between these two entities. Many studies focused on sports, involving aerobic metabolism, like running or swimming [9–11], showed an increase in FR production of, as well as an increase in the activity of antioxidant enzymes, such as SOD, GPX, and catalase (CAT). In several studies, changes in oxidant/antioxidant balance could be explained by an increase in oxygen consumption during exercise [12–14]. Indeed, the increase in oxygen consumption during exercise promotes a large leakage of FR in the mitochondria, and then results in an antioxidant reaction. In endurance training sessions, coaches and athletes adopt two types of exercise: either continuous exercise or intermittent exercise. Although these two types of exercise do promote the development of the aerobic capacity, their solicitation of energy metabolism is not the same. Indeed, Combes et al. [15] reported higher oxygen consumption during continuous exercise compared to intermittent exercise, consisting of 30 s of running, interspersed with 30

athletes adopt two types of exercise: either continuous exercise or intermittent exercise. Although these two types of exercise do promote the development of the aerobic capacity, their solicitation of energy metabolism is not the same. Indeed, Combes et al. [15] reported higher oxygen consumption during continuous exercise compared to intermittent exercise, consisting of 30 s of running, interspersed with 30 s passive recovery. The same authors in another study compared di erent types of intermittent exercise (i.e., 30/30 s, 60/60 s, and 120/120 s) and showed that oxygen consumption increases proportionally with the duration of intermittent exercise [16]. As a result, this variation in oxygen consumption could potentially have di erent e ects on the production of FR and FR damage in athletes. The impact of the exercise modality (continuous vs. intermittent) on oxidative stress is an ongoing debate. Numerous studies compared the exercise modalities with the same energy expenditure and di erent exercise durations, and reported that both modality and duration of the exercise could impact the oxygen consumptiondi erently [17–19]. As a result, the comparison of variable exercise load (intermittent) vs. constant exercise load (continuous) with the same overall energy expenditure and the same exercise duration would clearly identify the e ect of the exercise modality on oxidative stress. Therefore, the present study aims to determine the type of exercise (continuous vs. intermittent) allowing a minimal level of oxidative stress. The results will allow a better understanding of oxidative stress responses to di erent modalities of aerobic running exercise. It would improve the prescription of endurance running training by targeting training modalities favoring less radical damage, in order to preserve the athlete's health and to optimize the process of recovery after training. Therefore, the aim of this study wasto investigate the e ects of di erent running exercise modalities (continuous, intermittent 30/30 s, and intermittent 15/15 s) on antioxidant defenses and markers of radical damage in male athletes. We hypothesized that continuous exercise could lead to moreradical damage compared to intermittent exercise, and that intermittent exercise 15/15 could lead to higher antioxidant defenses.

wasto investigate the e ects of di erent running exercise modalities (continuous, intermittent 30/30 s, and intermittent 15/15 s) on antioxidant defenses and markers of radical damage in male athletes. We hypothesized that continuous exercise could lead to moreradical damage compared to intermittent exercise, and that intermittent exercise 15/15 could lead to higher antioxidant defenses.

Int. J. Environ. Res. Public Health2020,17, 3729 3 of 12 2. Materials and Methods 2.1. Participants Thirteen male athletes (Mean standard deviation: age: 21.46 0.66 years, weight: 76.62 7.53 kg, height: 1.79 0.08 m) voluntarily participated in this study. The inclusion criteria were: (i) training a minimum of 8 h per week; (ii) healthy; (iii) does not consume alcohol and ca eine. After receiving a full description of the study protocol and the possible risks and bene ts associated with the study, each participant signed a written informed consent form prior to participation. The study was conducted in accordance with the Declaration of Helsinki [20]. Additionally, the protocol was fully approved by the Research Ethics Committee of the High Institute of Sport and Physical Education of Sfax, University of Sfax, Tunisia before the commencement of the assessments. 2.2. Experimental Protocol All participants completed an initial session to ensure familiarization with all measures and procedures. Athletes performed a time-to-exhaustion test (VAMEVAL) 72 h following the familiarization session in order to determine their maximal aerobic running speed (MAS). VAMEVAL is a triangular test in which participants exercise to exhaustion. The test starts at 8.5 km h 1 , and the increment of speed was 0.5 km h 1 every 60 s. Thereafter, and in counterbalanced design, participants performed the three di erent running exercise modalities in three di erent experimental sessions, separated by three days of recovery for all participants. Onesessionconsists of a continuous running exercise (CR) at 75% of MAS for 25 min. In another session, athletes performed an intermittent running exercise #1 (15/15) consisting of 15 s of running at 75% of MAS, interspersed by 15 s of passive recovery, performed for 50 min. The third session consisted of an intermittent running exercise #2 (30/30) in which participants performed an intermittent running protocol, consisting of 30 s of running at 75% of MAS interspersed by 30 s of passive recovery, performed for 50 min. The three running protocols were performed outdoors on a PVC running surface. Pacing was controlled using time emitted for each 50 m running distance. In order

intermittent running exercise #2 (30/30) in which participants performed an intermittent running protocol, consisting of 30 s of running at 75% of MAS interspersed by 30 s of passive recovery, performed for 50 min. The three running protocols were performed outdoors on a PVC running surface. Pacing was controlled using time emitted for each 50 m running distance. In order to limit the in uence of exogenous factors on oxidative stress parameters and on running performance, written indications were given to each participant before the commencement of the experiment—three days prior to each experimental session, no training sessions were allowed and participants were requested to refrain from any recovery treatments (e.g., massage, compression garments, cold water immersion). The participants were asked to refrain from consuming any alcoholic or ca einated beverages 24 h prior to each experimental session and to maintain their normal dietary habits for the duration of the study. Finally, heart rate (HR) was recorded for each experimental session (Polar Team2 Pro, Polar, Finland). The experimental protocol is represented in Figure. Each testing session was performed at the same time of day to minimize the diurnal variation e ect on performance (between 4:30 p.m. and 6:30 p.m.).Int. J. Environ. Res. Public Health 2020, 17, x 4 of 14 Figure 1.protocol design. BS: blood sample; MAS: maximal aerobic speed 2.3. VAMEVAL Test For assessment of maximum aerobic speed (MAS), participants performed the VAMEVAL field test [21]. It is an incremental running test performed on a 400 m outdoor running track. Participants were required to run between markers set 20 m apart at varying speeds dictated by an audio signal. The test started with 8.5 km/h −1 , and the running speed increased by 0.5 km/h −1 every minute until voluntary exhaustion of the participant. The test ended when participants were not able to maintain the required running speed dictated by the audio signal at two consecutive occasions. MAS was considered as the highest running velocity maintained throughout a complete stage during the VAMEVAL test. 2.4. Dietary Records During the period, the three different running exercise modalities were performed, and

voluntary exhaustion of the participant. The test ended when participants were not able to maintain the required running speed dictated by the audio signal at two consecutive occasions. MAS was considered as the highest running velocity maintained throughout a complete stage during the VAMEVAL test. 2.4. Dietary Records During the period, the three different running exercise modalities were performed, and participants recorded food intake. At the first visit tothe laboratory, a standardized individual information session was performed to instruct subjects to record their daily food intake. Food quantities were estimated, specifying the number of units and a code corresponding to the size of the portion, using a reference portion guideline book. Data collected from each participant was analyzed using the Bilnut 4 software package (SCDA Nutrisoft, Cerelles, France) and the food composition tables published by the Tunisian National Institute of Statistics in 1978 [22]. 2.5. Blood Sampling and Analysis Blood samples (5 mL of blood) were taken from an antecubital vein at rest and immediately after each running exercise. After the blood samples were drawn, samples were immediately Figure 1.Protocol design. BS: blood sample; MAS: maximal aerobic speed.

Int. J. Environ. Res. Public Health2020,17, 3729 4 of 12 2.3. VAMEVAL Test For assessment of maximum aerobic speed (MAS), participants performed the VAMEVAL eld test [21]. It is an incremental running test performed on a 400 m outdoor running track. Participants were required to run between markers set 20 m apart at varying speeds dictated by an audio signal. The test started with 8.5 km/h 1 , and the running speed increased by 0.5 km/h 1 every minute until voluntary exhaustion of the participant. The test ended when participants were not able to maintain the required running speed dictated by the audio signal at two consecutive occasions. MAS was considered as the highest running velocity maintained throughout a complete stage during the VAMEVAL test. 2.4. Dietary Records During the period, the three di erent running exercise modalities were performed, and participants recorded food intake. At the rst visit tothe laboratory, a standardized individual information session was performed to instruct subjects to record their daily food intake. Food quantities were estimated, specifying the number of units and a code corresponding to the size of the portion, using a reference portion guideline book. Data collected from each participant was analyzed using the Bilnut 4 software package (SCDA Nutrisoft, Cerelles, France) and the food composition tables published by the Tunisian National Institute of Statistics in 1978 [22]. 2.5. Blood Sampling and Analysis Blood samples (5 mL of blood) were taken from an antecubital vein at rest and immediately after each running exercise. After the blood samples were drawn, samples were immediately centrifuged at 3000 rpm at a temperature of 4 C for 10 min. Then, the plasma obtained was divided into 8 tubes and frozen at 80 C. Subsequently to defreezing, the blood samples were analyzed for MDA, AOPP, SOD, and GPX. Blood was collected in EDTA, cut o from oxygen and light. Hematocrit and hemoglobin were measured as part of a complete blood count using an automated cell counter (Coulter LH 750; Beckman Coulter, Brea, CA, USA). Finally, plasma volume was then corrected using the guidelines provided by Dill and Costill

blood samples were analyzed for MDA, AOPP, SOD, and GPX. Blood was collected in EDTA, cut o from oxygen and light. Hematocrit and hemoglobin were measured as part of a complete blood count using an automated cell counter (Coulter LH 750; Beckman Coulter, Brea, CA, USA). Finally, plasma volume was then corrected using the guidelines provided by Dill and Costill [23]. 2.6. Protein Rate Determination Total protein concentration was determined by the Bradford method [24], calibrated with bovine serum albumin. 2.7. Antioxidants Measurement (SOD) According to Beauchamp and Fridovich [25], SOD activity was evaluated for its ability to inhibit photochemical reduction of nitrobluetetrazolium (NBT). The reaction mixture contained 0.1 M potassium phosphate bu er (pH 7.4), 0.26 mM ribo avin, 2.69 mM methionine, and 2.64 mM NBT, with a plasma suitably diluted in a total volume of 1.5 mL. The assay mixture was illuminated for 20 min with a 20 W uorescent lamp in an aluminum lined container. Reduction of NBT by the blue-colored formazan superoxide radicals was monitored at 580 nm. SOD activity was expressed in U/mg protein. 2.8. GPX GPX activity was quanti ed by the procedure of Floh²and Günzler [26]. The plasma was added to a reaction mixture containing 0.1 M potassium phosphate bu er (pH 7.4) and 4 mM GSH. H2O2 (5 mM) was added, then mixed after 10 min of incubation at 37 C. TCA (5%) stopped the reaction. After centrifugation at 3000 rpm for 10 min at 4 C, the supernatant was combined with phosphate bu er and 10 mM DTNB and the absorbance read at 412 nm. GPx activity was expressed in nmol GSH consumed/min/mg protein.

Int. J. Environ. Res. Public Health2020,17, 3729 5 of 12 2.9. Markers of Radical Damages (MDA) MDA is the principal and most studied product of polyunsaturated fatty acid peroxidation. It is also one of the most popular markers used to evaluate oxidative stress damage on lipids in the literature in view of the facility and accessibility of its detection in comparison with other markers of lipid peroxidation, like F2-isoprostanes and lipid hydroperoxyde. MDA was assessed by thiobarbituric acid (TBA) reactive substances by measuring plasma levels of MDA, using the method described by Buege and Aust [27]. Samples were mixed with TBA solution (15% trichloroacetic acid [TCA], 0.8% TBA, 0.25 N HCl), then incubated at 95 C for 15 min. The mixture was then centrifuged at 3000 rpm for 10 min and cooled in ice for 5 min. Absorption of supernatants was read at a wavelength of 532 nm. Concentrations were reported as nmol MDA/mg protein. 2.10. AOPP The method of Kayali et al. [28] was used to determine the advanced oxidation levels of the protein products (AOPP). Plasma was treated with phosphate bu er (0.1 M, pH 7.4). After 2 min of incubation, 1.16 Mpotassiumiodide and 10% of TCA were added to the mixture. The AOPP concentration for each sample was calculated based on an extinction coe cient of 261 cm-1mM-1 at 340 nm and expressed in nmol/mg protein. 2.11. Statistical Analysis All data are presented as means standard deviation (SD), and were analyzed using STATISTICA for Windows software (version 6.0, StatSoft, Inc, Tulsa, OK, USA). The normality of every dependent variable and homogeneity of the variances of the distributions (equal variance) were con rmed using the Shapiro–Wilk test and the Levene test, respectively. For all physical and biochemical data, a two-way ANOVA with repeated measures [exercise (continuous, 15/15, 30/30) time (pre-exercise vs. post-exercise)] was used. When appropriate, post hoc comparisons were made with the Bonferroni test. Statistical signi cance was accepted atp<0.05. 3. Results 3.1. Physiological Parameters Heart rate values were signi cantly higher in 15/15 compared to CR (168 11 bpm and 146 12 bpm respectively) (p<0.05). However,

a two-way ANOVA with repeated measures [exercise (continuous, 15/15, 30/30) time (pre-exercise vs. post-exercise)] was used. When appropriate, post hoc comparisons were made with the Bonferroni test. Statistical signi cance was accepted atp<0.05. 3. Results 3.1. Physiological Parameters Heart rate values were signi cantly higher in 15/15 compared to CR (168 11 bpm and 146 12 bpm respectively) (p<0.05). However, no signi cant di erences were revealed between heart rate values during 30/30 (155 8 bpm) and the two other running exercise protocols (p>0.05). 3.2. Dietary Intake No signi cant di erences in dietary intake were reported between the three experimental sessions (Table). 3.3. MDA Level Statistical analysis revealed a signi cant interaction modality time (F=9.69;p<0.01; ïp 2 =0.51). The post hoc test showed a signi cant increase of MDA concentration immediately after 30/30 intermittent running, compared to the resting values (+55.05 % 8.5) (p<0.01). In addition, post-exercise MDA levels in 30/30 intermittent running were signi cantly higher compared to CR and 15/15 intermittent running (p<0.05) (Figure).

This study analyzes the effects of different running modalities on oxidative stress in trained athletes.