Effects of Aerobic-, Anaerobic- and Combined-Based Exercises on Plasma Oxidative Stress Biomarkers in Healthy Untrained Young Adults

tly, it is well accepted that physical exercise-induced oxidative stress may damage biological structures and impair cellular functions. However, it is still unclear which type of exercise results in the greatest oxidative stress responses among a healthy untrained population. The aim of the present study was to compare the acute oxidative stress response (i.e., 0 to 20 min) following di erent types of exercise (anaerobic, aerobic, and combined). Ten healthy, untrained males (19.5 1.7 years ) performed three randomized exercise bouts: anaerobic (30 s Wingate test), aerobic (30 min at 60% maximal aerobic power (MAP))

a healthy untrained population. The aim of the present study was to compare the acute oxidative stress response (i.e., 0 to 20 min) following di erent types of exercise (anaerobic, aerobic, and combined). Ten healthy, untrained males (19.5 1.7 years ) performed three randomized exercise bouts: anaerobic (30 s Wingate test), aerobic (30 min at 60% maximal aerobic power (MAP)) or combined (anaerobic and aerobic). Venous blood samples were collected before, as well as at 0 (P0), 5 (P5), 10 (P10), and 20 (P20) min after each session. Rates of malondialdehyde (MDA) and antioxidant activities (i.e., glutathione peroxidase (GPX), superoxide dismutase (SOD), glutathione reductase (GR), -tocopherol, and total antioxidant status (TAS)) were assessed. Independent of exercise type, plasma MDA, GPX, SOD, and GR contents increased above baseline, whereas plasma -tocopherol decreased under baseline after the test sessions (p<0.05). Aerobic and anaerobic exercises generated faster responses (at P0) when compared to the combined exercise (P5 to P10) for the majority of the tested parameters. Plasma TAS content only increased following the aerobic exercise at P10 (p=0.03). Five to twenty-minutes post exercise, the highest MDA response was registered in the aerobic condition, and the highest GPX and SOD responses were recorded in the anaerobic (at P5) and aerobic (at P20) conditions (p<0.05). In conclusion, aerobic, anaerobic, or combined exercises have the potential to acutely increase oxidative stress and Int. J. Environ. Res. Public Health2020,17, 2601; doi:10.3390 /ijerph17072601 /journal/ijerph

Int. J. Environ. Res. Public Health2020,17, 2601 2 of 12 antioxidant activities, but with di erent responses magnitude. These ndings con rm that oxidative stress response seems to be dependent on the intensity and the duration of the physical exercise and may help in understanding how varying exercise bouts in uence the degree of oxidative stress among healthy untrained young adults. Keywords: redox status; physical exercise; untrained healthy young adults; physiological responses; anaerobic; antioxidant 1. Introduction Oxidative stress is characterized by the imbalance between pro-oxidant and antioxidant status, with the former outweighing the latter [1]. This imbalance can lead to physiopathological e ects by increasing cells and cellular components' (i.e., membranes, lipids, proteins, deoxyribonucleic acid (DNA), and lipoproteins, among the others) vulnerability to reactive oxygen species (ROS) attacks [1,2]. If not strictly controlled and counteracted, oxidative stress can cause acute pathologies (i.e., trauma and stroke) and be responsible for the insurgence of several chronic and degenerative diseases [3]. In order to properly protect the cells against the harmful e ects of free radicals, the human body is able to mount a cascade of defense mechanisms, including preventative, repairing, and scavenging ones, as well as through the amelioration of antioxidant activities [4,5]. It is well accepted that physical exercise is an activity that increases ROS production via increased phospholipase A2 (PLA2), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and xanthine oxidase (XO) activities, which, when combined, lead to oxidative stress [5–8]. During strength-based exercises [5,6,9], short-term maximal sprints [10,11], and exercise performed near the anaerobic threshold [12], the level of lipid peroxidation signi cantly increases immediately and up to 48 h following the e ort. The mitochondrial electron transport chain complex, the phenomenon of ischemia-reperfusion injury, and local in ammation have all been identi ed as major sources of free radical production and induced oxidative stress during exercise [13]. These acute changes in oxidative stress-related biomarkers following exercise are also accompanied by an increase in antioxidant responses. Indeed, immediate increases in the content of uric acid (UA), catalase (CAT), and glutathione peroxidase (GPX) are registered following intensive strength, sprint, and Wingate e

been identi ed as major sources of free radical production and induced oxidative stress during exercise [13]. These acute changes in oxidative stress-related biomarkers following exercise are also accompanied by an increase in antioxidant responses. Indeed, immediate increases in the content of uric acid (UA), catalase (CAT), and glutathione peroxidase (GPX) are registered following intensive strength, sprint, and Wingate e orts [14,15], with a return to baseline occurring from 10 min [16,17] to 4–8 h [5,6]. The aforementioned studies clearly describe intensive physical exercise as a situation resulting in oxidative stress, characterized by acute and delayed redox imbalance (i.e., between pro-oxidants and antioxidants). However, it is still unclear which type (aerobic, anaerobic, or combined anaerobic plus aerobic) of exercise results in the greatest oxidative stress responses [18,19]. For instance, limited investigations have explored the e ects of exercises' type on redox balance [20–25]. These studies have been limited only on aerobic- and anaerobic-based exercises, with no rm conclusion. Indeed, Bloomer et al. [20–22] and Parker et al. [23,24] suggested aerobic exercise induces a greater increase in pro-oxidant status when compared to anaerobic exercise, and Inal et al. [25] and Marzatico et al. [26] demonstrated the activities of enzymatic antioxidant defense increase similarly following aerobic and anaerobic exercises, while Parker et al. [23,24] showed that increasing exercise intensity resulted in greater endogenous antioxidant defenses. Discrepancies between ndings may be attributable to participant training level. In this sense, it is well described that longitudinal steady state exercise [25,26], as well as an e ective resistance training program [1,5–8,27,28], could prevent or suppress increases in malondialdehyde (MDA) levels after physical e orts and reinforce the body's defense against other oxidative attacks via activation of the redox sensitive transcription factors that nely regulate gene and protein expression within skeletal muscle and increased production of endogenous antioxidants (GPX, CAT, superoxide dismutase (SOD), and glutathione (GSH)) [19].

Int. J. Environ. Res. Public Health2020,17, 2601 3 of 12 To the authors' knowledge, there are only one study focused on the e ect of three types of exercise (aerobic-based exercise, anaerobic based exercise, and combined exercise) on oxidative stress response [29]. This recent study was conducted by our research team and reported that redox-related biomarkers exhibited divergent response dynamics at 20 min following exercises with aerobic-based exercise generates greater MDA response, while anaerobic-based exercise generates lower antioxidant responses (e.g., GPX, SOD, glutathione reductase (GR), and total antioxidant status (TAS)) compared to the two other type of exercises. However, given that this study investigated only well-trained athletes and given that training level was shown to be a disruptive factor in previous studies, it was suggested that the practical application of these preliminary ndings is limited to a trained population, and more research is needed to corroborate it in a non-athletic population. Therefore, the aim of the present study was to compare levels of lipid peroxidation and antioxidant biomarkers immediately and up to 20 min following anaerobic, aerobic, or combined (anaerobic and aerobic) exercise performed by healthy untrained young adult males. Resolving which exercise type elicits the greatest oxidative stress response in healthy untrained subjects is important as this will help with understanding of redox homeostasis and preventing the harmful e ect of exercise-induced oxidative stress among this population. 2. Materials and Methods 2.1. Participants Selection: Inclusion and Exclusion Criteria Ten healthy untrained males (19.5 1.7 years, 71.8 2.1 kg, 1.76 0.17 m (mean SD)) volunteered to participate in this study. The participants were recruited on the basis that they had not participated in any type of regular physical training for at least one year before the experiment, as measured using the “International Physical Activity Questionnaire” and that they were not su ering from any kind of acute or chronic diseases and any kind of injury within three months of the start of the experiment. To avoid any possible bias related to nutrition-derived assets of antioxidants (e.g., di erent nutrients' protection levels), participants were instructed to avoid the consumption of any

the “International Physical Activity Questionnaire” and that they were not su ering from any kind of acute or chronic diseases and any kind of injury within three months of the start of the experiment. To avoid any possible bias related to nutrition-derived assets of antioxidants (e.g., di erent nutrients' protection levels), participants were instructed to avoid the consumption of any medications (e.g., antioxidant or anti-in ammatory drugs) or dietary supplements (e.g., creatine, foods rich in antioxidants or polyphenols, such as blueberries, co ee, green tea, grapes, cherries, curcuma, red wine, and dark chocolate) during the experimental period and for at least 8 weeks before the commencement of the study. 2.2. Ethical Clearance Participants were informed of all procedures, potential risks, and bene ts associated with the study and they provided written informed consent to take part in the experiment. The study was conducted according to the declaration of Helsinki, and the protocol was fully approved (identi cation code: 8/16) by the university institutional review board before the commencement of the assessment. 2.3. Experimental Design One week before the start of the experimental period, VO2 peakand maximal aerobic power (MAP) output was determined for each participant from an incremental laboratory cycling test [29,30]. After a 10 min warm-up at 100 W, the test began at an initial power output of 200 W. Subsequently, power output was increased by 30 W every 4 min until respiratory exchange ratio (RER) 1. Thereafter, power output was increased by 10 W/min until exhaustion. During the test, VO2was measured breath by breath using an indirect calorimetry system (Quark PFT, Cosmed, Rome, Italy) [29,30]. MAP was calculated using the equation proposed by Kuipers et al. [31]. The VO2 peakwas determined from the mean VO2over the last 30 s of the test [29]. As part of a repeated-measures, cross-over experimental design, participants performed three randomized test sessions, with a recovery period of 72 h in between. To avoid any chronobiological e ects [5,7,32–34], all test sessions were performed at the same time of day (around 08.00 hours).

s of the test [29]. As part of a repeated-measures, cross-over experimental design, participants performed three randomized test sessions, with a recovery period of 72 h in between. To avoid any chronobiological e ects [5,7,32–34], all test sessions were performed at the same time of day (around 08.00 hours).

Int. J. Environ. Res. Public Health2020,17, 2601 4 of 12 The test sessions consisted of either anaerobic-based (i.e., 30 s standard Wingate test), aerobic-based (i.e., 30 min low-intensity pedaling exercise), or combined (aerobic and anaerobic) exercise. Upon arrival for their rst test session, each participant's body mass (Tanita, Tokyo, Japan) and height were recorded. Before completing the experimental testing sessions, a standardized 5 min cycling warm-up was completed at 75 W. The anaerobic-based protocol comprised a single standard 30 s Wingate test on an electronically-braked cycle ergometer (Excalibur Sport, Lode B.V, Medical Technology, Groningen, Netherlands) connected to a computer with diagnostic software (Ergocard ® , Medisoft, Dinant, Belgium). Following the warm-up, participants were instructed to pedal as fast as possible during a 6 s acceleration phase to attain peak cadence [29]. Immediately following the acceleration phase, the load was electronically applied to the ywheel and subjects pedaled “all-out” for the entirety of 30 s [29]. The aerobic-based protocol consisted of pedaling on the same cycle ergometer at an intensity equal to 60% of MAP output for a duration of 30 min at a cadence of 60 rpm [29]. The combined protocol involved the completion of the anaerobic-based protocol followed by the aerobic-based protocol with 3 min passive recovery between these protocols. Before and after (i.e., at 0 min (P0), 5 min (P5), 10 min (P10), and 20 min (P20)) each training session, blood samples were collected from a forearm vein (dominant arm) through an intravenous cannula. 2.4. Dietary Records To assess the adequacy and consistency of nutrient intake, a daily dietary record was completed over seven days. All participants received detailed verbal and written instructions on the process of recording their diet. Participants were asked to continue with their usual dietary habits during the period of dietary recording and to be as accurate as possible in recording the amounts and types of food and uid consumed. A list of common household measures (e.g., tablespoons, cups) and speci c information about the quantity in each measurement (grams, etc.) was given to each participant. The individual's diet was evaluated using the Bilnu

dietary habits during the period of dietary recording and to be as accurate as possible in recording the amounts and types of food and uid consumed. A list of common household measures (e.g., tablespoons, cups) and speci c information about the quantity in each measurement (grams, etc.) was given to each participant. The individual's diet was evaluated using the Bilnu 4 software (SCDA Nutrisoft, Cerelles, France), and the food composition tables published by the Tunisian National Institute of Statistics in 1978. Estimated nutrient intakes were compared to reference dietary intakes for physically active people, and the daily nutriment data showed that total calorie, macronutrient, and micronutrient intakes were within the reference dietary intakes for healthy Tunisian adults, with no signi cant di erences between the three test sessions (e.g., test session 1: 2875 365 kcal/day; test session 2: 2798 402 kcal/day; 2906 438 kcal/day). 2.5. Blood Analysis To eliminate inter-assay variance, all samples were analyzed in the same assay run. All assays were performed in duplicate in the same laboratory, with simultaneous use of commercially assay kits from Randox (Randox Laboratories Limited, 55 Diamond Road, Crumlin, County Antrim, BT29 4QY, United Kingdom). SOD, GPX, glutathione reductase (GR), and total antioxidant status (TAS) were measured using standard colorimetric assays (Randox Laboratories Limited, 55 Diamond Road, Crumlin, County Antrim, BT29 4QY, United Kingdom) as described by El Abed et al. [16,29]. Intra- and inter-assay coe cient of variation for the SOD were 0.8% and 0.9%; intra- and inter-assay coe cient of variation for the GPX were 0.9% and 1.0%; intra- and inter-assay coe cient of variation for the GR were 0.7% and 0.8%; and intra- and inter-assay coe cient of variation for the TAS were 0.6% and 0.7%. -tocopherol was extracted with hexane from human plasma and then measured via high performance liquid chromatography (HPLC). For specimen preparation, 100 Lof internal standard was mixed with100 L of plasma in a 1.5 mL Eppendorf tube and vortexed for 5 s. Subsequently, 200 L of ethanol was added and vortexed for 30 s, followed by 500 L of Hexane and a further 1 min

hexane from human plasma and then measured via high performance liquid chromatography (HPLC). For specimen preparation, 100 Lof internal standard was mixed with100 L of plasma in a 1.5 mL Eppendorf tube and vortexed for 5 s. Subsequently, 200 L of ethanol was added and vortexed for 30 s, followed by 500 L of Hexane and a further 1 min vortex. The mixture was centrifuged at 4000 rpm and 4 C for 8 min with 450 L of the supernatant removed and evaporated to dryness under a stream of nitrogen at room temperature. Solids were extracted via the addition of 250 L of methanol, followed by a 30 s vortex and the same centrifugation protocol described above, before being analyzed using the HPLC method

Int. J. Environ. Res. Public Health2020,17, 2601 5 of 12 described by Siluk et al. [35]. Intra- and inter-assay coe cient of variation for the a-tocopherol were 1.1 and 1.2% MDA was assayed as a marker of lipid peroxidation using a colorimetric reaction, which uses 1-methyl-2-phenylindole as chromogen. Condensation of one molecule of MDA with 2 molecules of 1-methyl-2-phenylindole (MPI) under acidic conditions results in the formation of a chromophore with an absorbance maximum at 586 nm. A 7.6 mM solution of MPI was prepared immediately prior to use in 33% methanol in acetonitrile. A 650 L aliquot of MPI was placed in each test tube, to which was added a solution of 200 L of plasma. The tubes were well mixed, and 150 L of 10 M HCl was added. After mixing once more, the tubes were sealed, and incubated for 60 min at 45 C. After incubation, the tubes were chilled in an ice bath and spun at 10,000 gfor 5 min, in order to fully remove debris. The absorbance at 586 nm was measured and subtracted from the blank value, obtained by replacing plasma with water. A calibration graph was prepared using 4 mol/L, 8 mol/L, 16 mol/L, and 20 mol/L of 1,1,3,3-tetramethoxypropane in 20 mM Tris-HCl, bu er, pH 7.4. Intra- and inter-assay coe cient of variation for the MDA were 1.6 and 1.7%. 2.6. Statistical Analysis All statistical analyses were performed using the commercial statistical software STATISTICA (StatSoft, Paris, France, version 10.0). Normality of the data distribution was con rmed using the Shapiro-Wilks-W-test. Values were computed and reported as mean SEM (standard error of the mean). The data obtained for all antioxidants and oxidative stress markers were analyzed using a two-way ANOVA (3 levels (exercise type (anaerobic, aerobic, and combined—aerobic and anaerobic)) 5 levels (samples-time (before, P0, P5, P10, and P20))) with repeated measure. Fisher's least signi cant di erence (LSD)post-hoctests were conducted when a statistically signi cant main e ect was found. E ect sizes were calculated as partial eta-squared ( p 2) to assess the potential practical signi cance of the ndings. For

type (anaerobic, aerobic, and combined—aerobic and anaerobic)) 5 levels (samples-time (before, P0, P5, P10, and P20))) with repeated measure. Fisher's least signi cant di erence (LSD)post-hoctests were conducted when a statistically signi cant main e ect was found. E ect sizes were calculated as partial eta-squared ( p 2) to assess the potential practical signi cance of the ndings. For all analyses, statistical signi cance was set atp<0.05. 3. Results The levels of lipid peroxidation at pre-test and at P0, P5, P10, and P20 following aerobic, anaerobic, and combined (anaerobic and aerobic) exercise are presented in Figure.Int. J. Environ. Res. Public Health 2020, 17, x 5 of 12 Solids were extracted via the addition of 250 µL of methanol, followed by a 30 s vortex and the same centrifugation protocol described above, before being analyzed using the HPLC method described by Siluk et al. [35]. Intra- and inter-assay coefficient of variation for the a-tocopherol were 1.1 and 1.2% MDA was assayed as a marker of lipid peroxidation using a colorimetric reaction, which uses 1- methyl-2-phenylindole as chromogen. Condensation of one molecule of MDA with 2 molecules of 1-methyl-2-phenylindole (MPI) under acidic conditions results in the formation of a chromophore with an absorbance maximum at 586 nm. A 7.6 mM solution of MPI was prepared immediately prior to use in 33% methanol in acetonitrile. A 650 µl aliquot of MPI was placed in each test tube, to which was added a solution of 200 µl of plasma. The tubes were well mixed, and 150 µl of 10 M HCl was added. After mixing once more, the tubes were sealed, and incubated for 60 min at 45 °C. After incubation, the tubes were chilled in an ice bath and spun at 10,000 x g for 5 min, in order to fully remove debris. The absorbance at 586 nm was measured and subtracted from the blank value, obtained by replacing plasma with water. A calibration graph was prepared using 4 µmol/L, 8 µmol/L, 16 µmol/L, and 20 µmol/L of 1,1,3,3- tetramethoxypropane in 20 mM Tris-HCl, buffer, pH 7.4. Intra- and inter-assay coefficient of

The study investigates how various exercises affect oxidative stress biomarkers.