Reactive oxygen species (ROS) are continuously produced during cellular oxygen metabolism and they have been closely linked to the pathophysiology of a vast number of chronic diseases and aging (12). Although ROS are important signalling molecules in various cellular pathways, marked changes in redox status caused by elevated ROS generation may be detrimental to cellular homeostasis and function because they induce oxidative modifications to macromolecules such as lipids, proteins, and nucleic acids. Because ROS represent normal physiological by-products, there is a wide range of antioxidants that minimize oxidative damage by neutralizing ROS while still allowing for the maintenance of redox status, cellular function, and intracellular signalling (14). Oxidative stress refers to an imbalance in the pro- and antioxidant status in favor of the former. Oxidative stress following intense aerobic/anaerobic exercise results from the production of excess ROS such as superoxide, hydrogen peroxide (H2O2), hydroxyl radical, and organic hydroperoxide, mainly resulting from phagocytic respiratory burst activity (37).
Anaerobic exercise is a major component of multiple-sprint sports, such as soccer, which are characterized by periods of high-intensity activity (sprinting, running, kicking, jumping, and tackling) interspersed with lower-intensity actions (jogging, walking) and/or active or passive recovery (5,23). Repetitive intense eccentric muscle contractions generated during a soccer game (i.e., running, kicking, jumping, tackling) have been associated with muscle damage that is clinically presented as muscular pain developed several days post-exercise (11). Muscle damage is mainly induced by mechanical stress and calcium homeostasis disturbances while a sensation of discomfort within the muscle may be experienced (8). Exercise-induced muscle damage is associated with an acute-phase inflammatory response characterized by phagocyte infiltration into muscle, free radical production, and elevation of cytokines and other inflammatory molecules (2). ROS can assist in repairing damaged tissue via phagocytosis and respiratory burst activity (14). However, large amounts of ROS may damage vital cellular structures and oxidative damage can result. Elite soccer players must fully recover and be ready to compete for a full 90 minutes plus stoppage time in the next game within 3 to 6 days. Oxidation end-products accumulation may be elevated for 24 to 96 hours following strenuous exercise as a result of skeletal muscle injury causing macromolecule oxidation (20) and recovery retardation because ROS production promotes muscle fatigue (29). ROS-mediated macromolecule oxidation may promote muscle protein breakdown of certain proteolytic systems (10). Previous investigations suggest that ROS may act as second messengers in intracellular signaling pathways that mediate proteolysis and cell death via apoptosis (28). This function of ROS may be linked to their level and the overall redox status in the cell (i.e., lower ROS concentrations lead to cell adaptation and survival, whereas higher concentrations activate signalling pathways that lead to proteolysis and cell death) (28).
Although exercise-induced oxidative stress is a well-known phenomenon described by hundreds of research papers, limited information exists regarding oxidative stress and antioxidant status responses following multiple-sprint, intermittent-type sports such as soccer. Knowledge of oxidative stress manifestations during recovery from intense activities such as soccer would inform sport practitioners and scientists regarding the time-course of post-game inflammatory response because redox status alterations represent an integral part of post-exercise inflammatory response and proteolytic activity in skeletal muscle. The time-course of inflammatory and oxidative stress responses may be sport-specific because game intensity, duration and activity pattern differ considerably between sports and various types of exercise. Indeed, soccer appears to induce a marked inflammatory response during recovery lasting 48 to 96 hours (11). To date, only 2 studies have described soccer-related oxidative stress responses as a part of exercise-induced inflammation (3,11). Ascencão et al. (3) reported that a soccer game upregulates oxidative stress and muscle damage responses for as long as 72 hours. However, in that study researchers examined only a lipid peroxidation biomarker (malondialdehyde [MDA]) and a disulphide linkage marker (plasma sulphydryl residues, a glutathione metabolism marker), whereas antioxidant status was monitored by measuring only total antioxidant status but not antioxidant enzymes. Ispirlidis et al. (11) described the inflammatory response during an entire microcycle of training that follows a soccer competition including 2 oxidative stress markers (thiobarbituric acid reactive substances [TBARS] and protein carbonyls) but no antioxidant status markers. Therefore, the purpose of the present investigation was to test the hypothesis that a single soccer game alters oxidative stress and antioxidant status indices during the recovery period.
Experimental Approach to the Problem
To determine the time-course of changes in oxidative stress and antioxidant status markers, a 2-group (including a control group to account for the day-to-day variation of the physiological and biochemical assays), repeated-measures design was used. Twenty soccer players (experimental group; data collected from the goalkeepers of each team were not included in the study) were assigned to 2 different teams that competed against each other in a regular soccer game (2 × 45 minutes). Ten other players served as controls (rested) by participating only in the measurement sessions. Subjects abstained from any strenuous physical activity for at least 7 days before the game and 3 days after the game with the exception of performance tests. Athletes were instructed to maintain their normal eating pattern for 2 weeks prior to data collection. Subjects were not taking any medication or dietary supplements with antioxidant action for 6 months prior to the study. To examine whether dietary changes influenced oxidative stress variables and antioxidant status outcomes, 5-day diet recalls were completed prior to the game. A trained dietician taught the subjects how to complete diet recall questionnaires and determine food serving and sizes. Diet records were analyzed using the computerized nutritional analysis system Science Fit Diet 200A (Science Technologies, Athens, Greece) (20). During the game, participants were allowed to drink only water ad libitum. Subjects had their weight, height, body composition, maximal oxygen uptake, and maximal heart rate measured 1 week prior to the match.
On the game day (between 8:00 am and 12:00 pm), participants gave a resting blood sample after an overnight fast. Following blood sampling, athletes consumed a light standardized meal (1.5-1.8 g/kg of white bread and 0.3 g/kg of low-fat spread), drank 1 glass of orange juice (containing approximately 100 calories, 91% carbohydrates, 4% fat, 5% protein, 25 mg calcium, 25 mg magnesium, 40 mg phosphorus, and 500 mg potassium, but no iron, zinc, copper, selenium, manganese, fluoride), and rested (2 hours). Pre-game meal consumption was utilized to control for carbohydrate, protein, fat, vitamins, and selenium intake (which influence oxidative stress responses). Following the pre-game meal and before the start of the game (2 hours post-meal), participants had their performance (speed and vertical jumping ability) measured. Heart rate (Polar Electro, Kempele, Finland; measured in 5-second intervals during the game) was used to monitor the game's intensity. Blood sampling was repeated 30 minutes post-match and 24, 48, and 72 hours after the match at the same time following an overnight fast and resting for 30 minutes. After blood sampling, players had their performance measured at 24, 48, and 72 hours of recovery.
Thirty injury-free, male soccer players (age 20.3 ± 0.3 years, weight 75.4 ± 3.1 kg, height 177 ± 1.3 cm, percent body fat 7.9 ± 0.7 %, O2max 59.7 ± 3.1 mL/kg/min) who had participated for at least 8 years in elite youth soccer competition participated in the present study. Participants competed in under-21 year's division 1 soccer league in Greece for at least 3 years and attended an average of 6 practices per week during their pre- and in-season period. After receiving a detailed explanation of the study's benefits and risks, each subject signed an informed consent document that was approved by the local ethics committee.
Body mass was measured to the nearest 0.5 kg (Beam Balance 710, Seca, Birmingham, England, United Kingdom) with subjects wearing their underclothes and barefooted. Standing height was measured to the nearest 0.5 cm (Stadiometer 208, Seca). Percentage body fat was calculated from 7 skinfold measures (average of 2 measurements of each site) using a Harpenden caliper (John Bull, British Indicators, St Albans, United Kingdom).
O2max was determined during a graded exercise test on a treadmill until voluntary exhaustion. A 12-lead electrocardiogram, heart rate, brachial artery cuff pressure, and ratings of perceived exertion (6-20 Borg scale) were monitored continuously during the test and for 30 minutes during recovery (19). O2 was measured continuously by open-circuit spirometry and averaged every 30 seconds with the use of an automated online pulmonary gas exchange system via breath-by-breath analysis (Oxycon Champion IEC 601-1, Erich Jaeger, Würzburg, Germany). To ascertain that O2max had been attained, standard criteria had to be met (7).
Time for speed testing during a 20-m sprint was recorded by infrared light sensors with a precision of 0.01 seconds (Newtest, Finland) (11). Vertical jump height (VJ) was measured in 3 maximal efforts (the best jump was recorded) on an Ergojump contact platform (Newtest, Oulu, Finland) (11). Subjects started from a standing position, allowed a preparatory countermovement motion, and had their hands on their waist throughout the jump. Flight times were measured by means of a digital timer connected to the platform and was used to calculate jump height. The coefficient of variation (CV) for test-retest trials were 3.1% and 3.4% for sprinting and VJ, respectively.
Muscle Damage Markers
DOMS was determined by palpation of the muscle belly and the distal region of relaxed vastus medialis, vastus lateralis, and rectus femoris using a visual analog scale (VAS), with ‘‘no pain’’ at 1 end of a 100-mm line and ‘‘extremely sore’’ at the other (24). During palpation, pressure was applied with the tips of 3 fingers (II, III, and IV) for approximately 3 seconds with the participant standing (24). Palpation for DOMS assessment was performed by the same examiner so that applied pressure on muscle was standardized. The test-retest reliability of DOMS measurement by palpation was 0.95.
Blood Sampling and Assays
Blood samples were collected from an antecubital arm vein into evacuated tubes containing ethylenediaminetetraacetic acid (EDTA), heparin, or SST-Gel and clot activator. Plasma and serum were separated by centrifugation (1,500 g, 4°C, 15 minutes). A blood aliquot (1-mL) was immediately mixed with EDTA to prevent clotting for hematology. A small quantity of blood (200 uL) was immediately added to 400 uL of 5% trichloroacetic acid (TCA) and centrifuged (2,500g, 15 minutes). Complete blood count and uric acid (UA) were determined within 24 hours via matching duplicate counts using an automated hematology analyzer (Sysmex K-1000, TOA Electronics, Hyogo, Japan). Multiple aliquots of each sample were stored at −80oC (heparinized whole-blood samples were stored at −20oC). Blood samples thawed only once before analysis and assays were performed in duplicate. Results were corrected for match-induced plasma volume changes based on hematocrit and hemoglobin changes. Blood samples were protected from light and auto-oxidation.
Blood lactate and glucose were determined spectrophotometrically by an enzymatic method with reagents purchased from Sigma Chemicals (St. Louis, Missouri, USA). CK was determined using a commercially available kit (Spinreact, Sant Esteve, Spain). Total protein in serum was assayed using a Bradford reagent. MDA levels were measured by reverse-phase high-performance liquid chromatography (rp-HPLC) with fluorimetric detection (excitation 532 nm and emission 550 nm) as described (18). Protein carbonyls (PC) were assayed spectrophotometrically at 375 nm as described (26). Reduced (GSH) and oxidized (GSSG) glutathione were analyzed spectrophotometrically (at 412 nm) in TCA-treated blood as described (30,36). Serum catalase activity was assayed spectrophotometrically at 240 nm (1). Whole-blood glutathione peroxidase (GPX) activity was measured spectrophotometrically at 37°C using cumene hydroperoxide as the oxidant of glutathione (Ransel RS 505, Randox, Crumlin, United Kingdom). Total antioxidant capacity (TAC) in serum was assayed spectrophotometrically at 520 nm (13). The interassay and intraassay coefficients of variation in all assays performed were 2.4 to 7.3 and 3.5 to 8.6, respectively.
Data are presented as means ± SD. Data normality was verified with the 1-sample Kolmogorov-Smirnoff test; therefore, a nonparametric test was not necessary. Data were analyzed through 2-way (group × time) repeated-measures analysis of variance (ANOVA) with planned contrasts on different time points. When a significant effect was found, post hoc was performed through the Bonferroni test. Significance was accepted at p < 0.05.
Table 1 summarizes participants' physiological responses to the soccer games. Mean heart rate during the games was 168.6 ± 8.2 beats/min, whereas peak heart rate reached 194.6 ± 12.1 beats/min. Glucose levels increased (4.1 ± 0.4 mM vs. 4.8 ± 1.2 mM, p < 0.05), and blood lactate concentration reached 4.9 ± 1.6 mM (p < 0.05) post-game.
Although sprinting ability (%CV: 11.1-13.9% for the experimental group and 15.7-18% for the control group) declined by approximately 2% (p < 0.05) throughout recovery, VJ (%CV: 0.9-1.1% for the experimental group and 0.7-0.9% for the control group) decreased by 10% only for 24 hours (Figure 1). Leukocyte count (%CV: 7.9-8.9% for the experimental group and 7.8-9.5% for the control group) increased (p < 0.05) immediately post-match (26%), remained elevated (14%) for 24-hours, and normalized thereafter (Figure 2). Uric acid (%CV: 8.7-10.5% for the experimental group and 6.2-10.3% for the control group) concentration increased (34%, p < 0.05) after 24 hours, peaked at 48 hours of recovery (47%), and returned to baseline thereafter (Figure 2). DOMS (%CV: 11.5-39% for the experimental group) increased immediately post-game (7-fold, p < 0.05), peaked at 24 hours (8.6-fold, p < 0.05), remained elevated at 48 hours (5-fold, p < 0.05), and returned to baseline thereafter (Figure 2). CK activity (Figure 2) increased (%CV: 15-25% for the experimental group and 14-23% for the control group) immediately post-match (3-fold, p < 0.05) and at 24 hours (4.5-fold, p < 0.05), peaked at 48 hours (7-fold, p < 0.05), and remained elevated throughout recovery (6-fold).
MDA (%CV: 18-24% for the experimental group and 13-25% for the control group) increased post-game (24%, p < 0.05), peaked at 24 hours (48%, p < 0.05), remained elevated at 48 hours (32%, p < 0.05), and normalized at 72 hours (Figure 3). PC (%CV: 10-28% for the experimental group and 12-33% for the control group) increased post-game (19%, p < 0.05), peaked at 24 hours and 48 hours (65%, p < 0.05), and remained elevated at 72 hours (41%, p < 0.05) (Figure 3). GSH concentration (%CV: 10-21% for the experimental group and 10-17% for the control group) declined only at 24 hours (33%, p < 0.05) and returned to normal thereafter (Figure 4). In contrast, GSSG concentration (%CV: 35-81% for the experimental group and 31-61% for the control group) increased (p < 0.05) at 24 hours and peaked at 48 hours by 1.2-fold and 3-fold, respectively, and normalized thereafter (Figure 4). The GSH/GSSG ratio (%CV: 33-94% for the experimental group and 39-70% for the control group) declined post-game (13%, p < 0.05), reached its lowest values at 24 hours and 48 hours (55%, p < 0.05), and returned to baseline at 72 hours (Figure 4).
TAC (%CV: 14-22% for the experimental group and 12-28% for the control group) increased at 24 hours (26%, p < 0.05), peaked at 48 hours (29%, p < 0.05), and normalized thereafter (Figure 5). Serum catalase activity (%CV: 21-30% for the experimental group and 23-41% for the control group) increased (45%, p < 0.05) immediately post-game and returned to baseline thereafter (Figure 5). GPX activity (%CV: 6-8% for the experimental group and 8-11% for the control group) increased at 24 hours (20%, p < 0.05), peaked at 48 hours (23%, p < 0.05), and normalized thereafter (Figure 5).
The sport of soccer is associated with physiological adaptations (i.e., eccentric work, leukocytosis, catecholamine increase) that predispose athletes to muscle damage and inflammation. Currently, there is limited information regarding oxidative stress responses following a soccer game. The present investigation suggests that a competitive (participants achieved maximal heart rates) soccer event induces time-dependent changes in various circulating oxidative stress markers, a phenomenon likely related to exercise-induced inflammation and a decline of lower-limb functional status. It is worth noting that despite some wide SD in some markers (such as the leukocyte count, uric acid concentration, GPX activity, and catalase activity), the subjects' response following the soccer game was homogeneous (more than 85% of the participants followed the mean response for all markers). Although overload data during the game (such as time-motion analysis data) are lacking, an effort was made so that all participants competed at a relatively high intensity level. This is verified by the homogeneous response in heart rate and lactate values recorded during and following the game.
In the present investigation, a soccer game was performed by Greek under-21 years first division players. Therefore, a question may arise as to whether that match resembled elite soccer competition. Players' physiological overload during the match approximated the values observed during elite soccer competition (5). Mean and peak heart rate reached values corresponding to 80% and 94% of their maximal value, whereas blood lactate accumulation reached 4.8 mM, a value that has previously been reported for elite soccer competition (5). Furthermore, blood glucose levels were elevated at the end of the game as it has been consistently described previously for elite soccer competition (15,16). Glucose increase at the end of the game results from increased hepatic glycogenolysis following an increase in catecholamine concentration and a reduction in insulin levels (5,15). This coincides with the higher glucose utilization during the game as it is reflected by the elevated lactate concentration in blood and muscle (15). This glucose increase probably compensates for the progressive reduction of muscle glycogen seen during a soccer game (15).
In agreement with previous reports (3,11), speed and VJ declined by almost 8% during recovery, suggesting that players may not be able to perform intense anaerobic activities at maximal levels for at least 48 hours following a game. This response has been attributed mainly to elevated cortisol; reduced protein synthesis; and the loss of contractile proteins, neurotransmitters, and muscular force resulting in strength reductions (9). Because elite athletes cannot abstain from training for more than 24 hours during the in-season period, performance deterioration may be greater than reported in this study, suggesting that additional recovery may be needed for full recuperation of the athletes.
The large DOMS (8-fold) and CK elevation (7-fold) provide indirect support for muscle microtrauma following the game. Peak CK activity, 48 hours after the game, was 800 U/L, a value 4 times greater than clinical ranges and almost as high as after a rugby match or a marathon race (34). This CK protein efflux from muscle may be attributed to the increased permeability of plasma membrane and/or intramuscular vasculature (8). However, CK values were lower than those usually seen following eccentric exercise-induced muscle damage (6). This may be attributed to soccer's intermittent nature and players' high conditioning level (27). In agreement with previous studies, DOMS increased within the first 24 hours post-game and peaked at 48 hours of recovery (3,25). Exercise-induced inflammation was also verified by post-game leukocytosis, indicating a systemic acute-phase inflammatory response with leukocyte infiltration in the damaged tissue (19,27). Neutrophils demonstrate a transient increase immediately post-exercise followed by a delayed increase several hours later-a response that also occurred in the present investigation (11).
DOMS, CK, and leukocyte elevations reveal a muscle damage response following a soccer game. Muscle microtrauma seen following a soccer game may be attributed, at least partially, to intermittent repetitions of intense eccentric actions such as running, jumping, and rapid acceleration and deceleration movements (e.g., hamstrings act eccentrically to decelerate hip flexion and knee extension during running's landing phase) that represent an integral part of this sport. Eccentric contractions induce higher tension per cross-sectional area of active muscle mass compared to concentric actions, resulting in significant structural muscle damage (6). Similar findings have been shown for other field sports that involve prolonged high-intensity shuttle running (rugby and hockey) (35).
ROS may be involved in the regulation of exercise-induced inflammation that accompanies muscle microtrauma by promoting the nuclear translocation of redox-sensitive transcription factors and molecules mediating inflammation (i.e., chemokines, cytokines, adhesion molecules) (2). Exercise-induced ROS production may be related to DOMS through its association with phagocyte infiltration and the leukocytosis observed for 24 hours. Leukocytes generate ROS that promote post-exercise inflammation, removal of traumatized tissue, and healing (8). Superoxide produced by neutrophils and macrophages is converted to H2O2, which then reacts with superoxide in the presence of a transition metal to form the toxic hydroxyl radical that oxidizes lipids and proteins (4). In agreement with previous studies, MDA, a lipid peroxidation marker, remained elevated for 48 hours, peaking 24 hours post-game (3,20). This MDA increase in blood is probably caused by peroxidation of lipoprotein phospholipids and nonesterified fatty acids and oxygen-mediated injury of muscle cell membranes (31). Increased circulating levels of CK and MDA levels may indicate a relationship between the exercise-induced ROS insult and the protein leakage into plasma. A novel finding of the present study is that soccer elevated protein oxidation in the circulation, as previously shown with damaging exercise (20). PC, a protein oxidation biomarker, levels remained elevated throughout recovery probably as a result of increased oxidation of albumin and other serum proteins (22).
Exhaustive exercise decreases liver and muscle GSH concentration probably because acute exercise decreases cystine in the liver, which is the rate-limiting precursor for glutathione synthesis (32). Exercise-induced decreases in GSH and cystine may account for the increased MDA and PC seen in the present study. MDA and PC are negatively correlated with GSH and cystine following acute exercise, respectively (17). In contrast, GSSG increased post-game as it has been previously been shown for high-intensity exercise (20). Consequently, the GSH/GSSG ratio declined during recovery, indicating the onset of oxidative stress.
TAC demonstrated a modest increase during recovery, suggesting that soccer activity stimulated the body's antioxidant defenses in serum to counteract elevated ROS production and confirming previously published data (3). Uric acid's elevation paralleled TAC's increase during the same timeframe. Uric acid elevation has been estimated to account for nearly one-third of TAC increase (38). High-intensity exercise-associated muscle ischemia upregulates purine nucleotide metabolism leading to adenosine elimination of adenosine monophosphate (AMP) and accumulation of hypoxanthine in the muscle compartment and plasma (4,15). Although hypoxanthine may be converted back to AMP at rest and during mild exercise, it is converted to uric acid and oxygen radicals (4). Uric acid results in the present study are consistent with these observations. The involvement of purine nucleotide metabolism in soccer has been reported recently, demonstrating a marked adenosine triphosphate (ATP) reduction and inosine monophosphate (IMP) increase in muscle following soccer activity (15). Furthermore, soccer has been shown to increase ammonia, uric acid, and hypoxanthine levels in the circulation (5).
It is questionable whether this TAC increase is related to antioxidant enzyme increase. Mobilization of tissue antioxidant stores into the plasma is a widely accepted phenomenon that would help maintain antioxidant status in plasma in times of need (4). Soccer effects on antioxidant enzyme activity have not been studied so far. Increased GPX activity seen after a soccer game in the present study may be dependent on scavenger cell migration into damaged muscle tissue. Macrophage migration in damaged muscle fibers has been observed 24 to 72 hours after exercise (37). Exhaustive exercise increased GPX activity by 87% in lymphocytes, indicating blood cell damage (33). However, these responses were seen only 3 hours post-exercise and with an entirely different training mode and limiting the inference that can be made to the present study. Soccer engages large muscle groups during intermittent types of activity at varying intensities and types of muscular contractions. Catalase increased only immediately post-game in agreement with previous findings (21). However, catalase has no apparent function in serum because it is an intracellular enzyme. Therefore, its increased activity after exercise probably indicates increased damage of erythrocyte membranes, which results in its increased leakage into the circulation.
In summary, our results suggest that oxidative stress is markedly upregulated by a soccer game, probably as a part of the exercise-induced inflammatory response, and is accompanied by a marked deterioration of anaerobic performance for as long as 72 hours.
Results of the present study not only verify previously published results suggesting that a soccer game may elevate lipid and protein oxidation for as long as 72 hours post-game, but also extend these findings by reporting a substantial increase in the antioxidant system activity and a significant performance decline during the same timeframe. Monitoring of these oxidative stress markers helps sport scientists and practitioners not only to understand the physiological mechanisms involved in soccer-induced inflammatory reaction, but also to describe possible factors underpinning improved recovery in the team-sport setting. Soccer appears to induce a 48 to 72-hour increase of the inflammatory response during recovery. Based on these findings, practitioners should design their daily training program so that inflammation is allowed to subside while at the same time performance is recovered. Knowing that muscle power activities such as sprinting and jumping return to pre-game levels after 24 to 48 hours, trainers should modify training intensity and volume to accommodate these adaptations. Use of maximal intensity may be contraindicated earlier than 48 hours during the post-game period. Because of the intense daily training and the ongoing increase of game rates in elite soccer, possible anti-inflammatory or antioxidant interventions should be tested with well-designed field trials to determine whether it is possible to reduce the time needed for full recovery following a soccer game.
The authors wish to thank all the subjects for their participation and commitment to the study. This study was supported by departmental funding.
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