Biochemical Impact of a Soccer Match

Available online at www.sciencedirect.com

(2008) 841–851
Clinical Biochemistry 41
Biochemical impact of a soccer match — analysis of oxidative stress andmuscle damage markers throughout recovery

António Ascensão a,b,⁎,1, António Rebelo c,1, Eduardo Oliveira b, Franklim Marques d,e,Laura Pereira d, José Magalhães a,b,1

a Research Centre in Physical Activity, Health and Leisure, University of Porto, Portugalb Department of Sport Biology, Faculty of Sport Sciences, University of Porto, Portugal

c Department of Soccer, Faculty of Sport Sciences, University of Porto, Portugald Department of Clinical Analysis, Faculty of Pharmacy, University of Porto, Portugal

e Institute for Molecular and Cell Biology, University of Porto, Portugal

Received 11 December 2007; received in revised form 3 April 2008; accepted 8 April 2008Available online 23 April 2008

Abstract

Background: Exercise is a prone condition to enhanced oxidative stress and damage and the specific activity pattern of a soccer match mayfavour additional pro-oxidant redox alterations. To date, no studies have reported the impact of a soccer match on oxidative stress and muscledamage markers.

Aim: To analyse the effect of a competitive soccer match on plasma levels of oxidative stress and muscle damage markers, and to relate thesefindings with lower limb functional data.

Methods: Blood samples, leg muscle strength, sprint ability and delayed-onset muscle soreness (DOMS) were obtained in 16 soccerplayers before, at 30 min, 24, 48 and 72 h after a soccer match. Plasma creatine kinase (CK), myoglobin (Mb), malondialdehyde (MDA),sulfhydryl (–SH) groups, total antioxidant status (TAS), uric acid (UA) and blood leukocyte counts were determined.

Results: A soccer match elevated plasma Mb following 30 min and CK levels throughout the 72 h-recovery period. MDA increasedthroughout the recovery period and –SH decreased until 48 h post-match. TAS increased at 30 min and UA increased throughout the 72 hrecovery. Blood neutrophils increased at 30 min whereas lymphocytes decreased and returned to baseline from 24 to 72 h. DOMS was higher thanbaseline until 72 h. Lower limb strength and sprint ability were lower than baseline until 72 h recovery.

Conclusion: The present data suggest that a soccer match increases the levels of oxidative stress and muscle damage throughout the 72 h-recovery period. The extent to which the redox alterations are associated with the recovery of muscle function should be further analysed.© 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Keywords: Football game; Antioxidants; Strength; Sprint

Introduction

Reactive oxygen and nitrogen species (RONS) have thepotential to react with a variety of chemical compounds, being

⁎ Corresponding author. Research Centre in Physical Activity, Health andLeisure, Faculty of Sport Sciences, University of Porto, Portugal, Rua Dr.Plácido Costa, 91, 4200-450 Porto, Portugal. Fax: +351 225500689.

E-mail address: [email protected] (A. Ascensão).1 Contributed equally to this manuscript.

0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemistsdoi:10.1016/j.clinbiochem.2008.04.008

closely related to the physiopathology of a wide range of modernwestern country diseases [1]. The imbalance between enhancedRONS production and the ability of antioxidant systems torender these inactive, lead to cellular loss of redox homeostasisand to prone conditions of oxidative damage to cellular lipids,proteins and DNA. In fact, despite RONS having a fundamentalrole as signalling molecules in several determinant cellularpathways, redox changes induced by increased RONS produc-tion during exercise are negatively related to cellular home-ostasis and might compromise cellular function. Additionally,the emerging role of free radicals in the delayed-onset muscle

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http://dx.doi.org/10.1016/j.clinbiochem.2008.04.008

842 A. Ascensão et al. / Clinical Biochemistry 41 (2008) 841–851

soreness and contraction-induced muscle injury has been re-cently reported [for refs see [2]].

The activity of soccer players during the competitive seasonentails 1 week cycles of training, taper, competition and recovery.At the top level, this cycle is altered by several irregularities in thecompetitive fixture list, being match day not necessarily the samefrom 1 week to another. Moreover, players from top level teamsmay be involved in additional commitments such as nationalcups and other knock-out matches, or representing their coun-tries in international championships. These competitive demandsmay impose strains to various physiological systems, includingmusculoskeletal, nervous, immune and metabolic, to a pointwhere recovery strategies post-exercise became influential inpreparing for the next match [3].

The high absolute levels of mitochondrial oxygen consump-tion, the increased circulating catecholamine, the elevated par-ticipation of eccentric muscle contraction-induced damageand inflammatory response, the intermittent and repeated sprintactions—causing temporary ischemia–reperfusion events inskeletal muscle are plausible factors that may influence RONSproduction during and after a soccer effort. Thus, and despitechronic exercise training having a protective effect throughimprovement of antioxidant capacity [4,5], it is likely thattraining sessions as well as the competitive matches exposeparticipants to oxidative stress and damage with consequentmuscle damage, both during, immediately post-exercise andthroughout the recovery.

Although scarce data have been published regarding theeffects of oxidative stress on exercise performance, there is apossibility that prior oxidative damage caused by intensivetraining periods and/or oxidative modifications while exercisingmight compromise the healthy status of the players as well asexercise performance [6].

Several reports focused on some stress biomarkers, in-cluding those of oxidative damage, as well as on the anti-oxidant status of soccer players under regular training periodsthat have been provided [4,5,7]. However, despite post-exercise vitamin C supplementation failing to attenuate legmuscle dysfunction, plasma malondialdehyde, interleukin-6,creatine kinase and myoglobin increase during the 72 h-re-covery period after 90 min of a shuttle running designed tocorrespond to the average exercise intensity of playing soccer[8], no data have been provided regarding the impact of asoccer match on oxidative stress and damage markers so far.Moreover, the impact of a soccer match on muscle damagemarkers throughout the post-game recovery period has alsobeen scarcely studied.

The understanding of the redox-based alterations imposed bya soccer match can contribute with additional physiologicalknowledge on the effects of a soccer game on players andparticularly, to improve possible recovery strategies based onpossible antioxidant supplementation. In this regard, the purposeof the present study was to determine the impact of a soccermatch on the recovery of plasma markers of oxidative stress andmuscle damage in the 72 h-post-match. Leg muscle functionaldata, sprint ability, muscle damage as well as leukocyte countswere also determined throughout the same period.

Methods

Subjects

Sixteen male soccer players from secondary divisionsparticipated in this study after being informed about the aims,experimental protocol, procedures and after delivering writtenconsents. The experimental protocol was approved by theEthical Committee of the Faculty of Sport Sciences, Universityof Porto, Portugal, and followed the Declaration of Helsinki ofthe World Medical Association for research with humans.

Experimental design and procedures

For 2 weeks prior to data collection and during the protocolperiod, soccer players were instructed not to change theirnormal eating habits and to refrain from additional vitamin orantioxidant dietary supplementation. Subjects were alsoinstructed to abstain from exhaustive exercise during the 72-hpre- and post-match, with exception of functional tests. Subjectsvisited the lab 5 days prior the match to determine maximaloxygen uptake and maximal heart rate.

Blood samples and functional data (quadriceps and ham-strings muscle strength and 20 m sprint ability) were assessedpre-match and 30 min, 24, 48 and 72 h of the recovery period inresponse to a competitive (2×45 min) soccer match. On the dayof the game, players arrived at the laboratory after an overnightfast of between 10 and 12 h. A resting blood sample was takenafter subjects had been standing for at least 15 min, after whichsubjects consumed a light standardized meal and drink andrested for 2 h. The meal consisted of 1.7 g white bread and 0.3 gof low-fat spread; both values are per kilogram of body mass[8]. Pre-match muscle strength and sprint ability were assessedduring the 2 h period between the consumption of the pre-exercise meal and the start of the soccer match.

For 3 days after the match, subjects returned to the laboratoryafter an overnight fast and at approximately the same time of themorning (within 1 h). A blood sample was taken from theforearm vein after the subjects had been at complete rest for atleast 15 min. Subsequently, the players performed the strengthand speed tests as outlined below.

Maximal oxygen uptake and heart rate

Five days prior the match, the subjects performed an in-cremental treadmill (Quasar-Med, Nussdorf, Germany) testuntil voluntary exhaustion to determine maximal oxygen uptake(VO2max) and maximal heart rate (HRmax). Expired respira-tory gas fractions were measured using an open circuit breath-by-breath automated gas-analysis system (Cortex, Metalyzer,3 B, Leipzig, Germany). HR was measured using a HR monitor(Vantage NV, Polar Electro, Kempele, Finland).

Intensity of the match

Heart rate was measured during the match and recordedevery 5 s using a HRmonitor (POLAR TEAM SYSTEMTM, Polar

843A. Ascensão et al. / Clinical Biochemistry 41 (2008) 841–851

Electro, Kempele, Finland). For time-motion analysis eachplayer was video-filmed close up during the entire match. TheVHS-format movie cameras (NV-M50, Panasonic, Germany)were positioned at the side of the pitch at the level of thehalfway line, at a height of about 15 m and at an approximatedistance of 30–40 m of the touching line. The videotapes werelater replayed for computerized time-motion analyses accord-ing to the procedures described by Mohr et al. [9]. The usedmotor pattern categories included standing (0 km.h− 1), walk-ing (6 km.h− 1), jogging (8 km.h− 1), low-speed running(12 km.h− 1), moderate-speed running (15 km.h− 1), high-speed running (18 km.h−1), sprinting (30 km.h− 1), sideways,and backwards (10 km.h− 1) running. The match activitieswere later analysed considering standing, walking, jogging,cruising, sprinting, backwards running and sideways running.

Delayed onset muscle soreness (DOMS)

Prior to taking blood samples, each subject was asked tocomplete a muscle soreness questionnaire, in which they ratedtheir perceived muscle soreness on a scale from 0 (normalabsence of soreness) to 10 (very intense sore) [10]. They wereinstructed to indicate the general soreness of the entire musclearea when moving or using it. The areas included on the ques-tionnaire were the quadriceps, hamstrings, gastrocnemius andtibialis muscles.

Strength assessment

In order to evaluate muscle function, maximal gravity cor-rected concentric peak torque of quadriceps and hamstrings weremeasured during isokinetic knee joint movement of thedominant leg at an angular velocity of 90°.s−1 (1.57 rad.s−1)according with [11] using an isokinetic dynamometer (BiodexSystem 2, NY, USA). After individual self-report, the dominantleg was determined by a routine visual inspection in a simpletarget kicking test requiring accuracy according to the pro-cedures described elsewhere [12].

Briefly, the subjects were familiarized with the muscle func-tion test on at least two occasions during preliminary visits to thelaboratory. They were seated on the dynamometer chair at 85°inclination (external angle from the horizontal) with stabilizationstraps at the trunk, abdomen and thigh to prevent inaccuratejoint movements. The knee to be tested was positioned at 90° offlexion (0°=fully extended knee) and the axis of the dyna-mometer lever armwas aligned with the distal point of the lateralfemoral condyle. Before the anatomical alignments andprocedures, all the subjects were instructed to kick and also tobend the tested leg as hard and fast as they could through acomplete range of motion (from 90° to 0°). Subjects were alsoinstructed to hold their arms comfortably across their chest tofurther isolate knee joint flexion and extension movements.

Prior to muscle function measurements, subjects were taken toa standardized warm-up consisting of 5 min of gentle running andstretching. All subjects performed a specific sub-maximal warm-up protocol on the isokinetic device. Three maximal repetitions atangular velocity 90°.s−1 (1.57 rad.s−1) were therefore carried out.

20 m sprint ability

Sprint ability measurements were carried out using telemetricphotoelectric cells placed at 0 and 20m (Brower Timing System,IRD-T175, Utah, USA). The players stood 1 m behind thestarting line, started on a verbal signal being time activated whenplayers cross the first pair of photocells, and then ran as fast asthey could to complete the 20m distance. Players completed tworuns and the best time at each distance was registered.

Blood sampling and preparations

All the venous blood samples were taken by conventionalclinical procedures using EDTA as anticoagulant. Nevertheless,no tourniquet constriction was used in order to minimize po-tentially enhanced oxidative stress induced by an ischemia–reperfusion maneuver.

An aliquot of the whole blood was used to perform leukocytecounts. The remaining freshly withdrawn blood was immedi-ately centrifuged at 3000 rpm during 10 min for careful removalof the plasma. Plasma was separated into several aliquotsand rapidly frozen at −80 °C for later biochemical analysis ofmyoglobin (Mb), creatine kinase (CK), total antioxidant status(TAS), uric acid (UA), malondialdehyde (MDA) and proteinsulfhydryl groups (SH).

Biochemical assays

Plasma creatine kinase (CK) activity was determined spectro-photometrically using a commercial test kit (ABX A11A01632,Mompelier, FR). Plasma myoglobin concentration was assessedusing a commercial test kit (myoglobin bioMerieux 30446,Carnaxide, PT).

PlasmaMDAwas assayed according to the method describedby Rohn et al. [13] with somemodifications andmeasured by theformation of thiobarbituric acid reactive substances at 535 nm.Briefly, plasma was incubated, at 25 °C, in 500 μL of a mediumconsisting of 175 mM KCl, 10 mM Tris, pH 7.4. Samples of50 μL were taken and mixed with 450 μL of a TBARS reagent(1% thiobarbituric acid, 0.6 N HCl, 0.0056% butylatedhydroxytoluene). The mixture was heated at 80–90 °C during15 min, and re-cooled in ice for 10 min before centrifugation inEppendorf centrifuge (1500 g, 5 min). The amount of TBARSformed was calculated using a molar extinction coefficient of1.56×105 M− 1 cm−1 and expressed as nanomoles of MDA permilligram of protein [14].

Oxidative modification of protein SH groups in plasmawas quantified by spectrophotometric measurement at 414 nmaccording to the method proposed by Hu [15]. Briefly, thecolorimetric assay was performed after the reaction of 50 μLaliquot of plasma with 10 μL of 5,5′-dithio-bis (2-nitrobenzoicacid) (10 mM) in a medium containing 150 μL of Tris (0.25 M)and 790 μL methanol, at 414 nm against a blank test. SHcontent was expressed in nmol/mg of plasma protein (ɛ414=13.6 mM−1 cm−1).

TAS was measured spectrophotometrically using a commer-cial kit (Randox NX2332 Crumlin, UK). The assay is based on

Table 1Anthropometric and physiological characteristics of the subjects

Variables Mean±SD

Age (yr) 21.3±1.1Mass (kg) 70.7±6.3Height (cm) 175.0±6.0% Body fat 8.3±1.9VO2max (mL.Kg−1.min−1) 55.1±5.1Hrmax (bpm) 196.0±7.0

VO2max — maximal oxygen uptake, HR — heart rate.


the reduction of free radicals (ABTSU+–2,2′-azinobis (3ehyl-

benzothiazoline-6-sulfonate)) measured as a decrease ofabsorbance at 600 nm at 3 min by antioxidants. The ABTS

U+radical cation is formed by the interaction of ABTS with ferryl-myoglobin radical species, generated by the activation ofmetmyoglobin with hydrogen peroxide. The suppression of theabsorbance of the ABTSU+ radical cation by plasma antioxidantswas compared with that from a Trolox (6-hydroxy-2,5,7,8-te-tramethylchroman-2-carboxylic acid). The results are expressedas mmol/L of Trolox equivalents.

Uric acid was determined by an enzymatic method at 550 nmusing a commercial kit (ABX A11A01670, Montpellier, FR)according to the specifications of the manufacturer.

Protein content was spectrophotometrically assayed usingbovine serum albumin as standard according to Lowry et al.[16].

Samples were analysed in duplicate and the mean of the twovalues was used for statistical analysis.

Leukocyte count were assessed by an automatic cell counter(Horiba ABX Micros 60; ABX Diagnostics, Montpellier,France) calibrated with an ABX Minocal (ABX Diagnostics);the intra-assay coefficient of variation (CV) determined on fivereplicates of each leukocyte measurement was b1%. Wholeblood smears on glass slides (VBS 655/A Microscope —Biosigma) were used for white blood cell differential analysis.Smears were stained using Wright coloring (Merck) and air-dried. Cell differentials were performed using an Olympusmicroscope equipped with 1000× oil immersion lens. Specifi-cally, the leukocyte counts including neutrophils and lympho-cytes were recorded.

Statistics

Mean, standard deviation (SD) and standard errormean (SEM)were calculated for all variables in each of the experimentalgroups. The ANOVA with repeated measures was used to es-tablish whether any of the subsequent test results were sig-nificantly different from baseline results. Pearson's correlation

Table 2Frequency, mean duration and percent of match time spent on the considered motor

Standing Walking Jogging C

Frequency (n) 114±44.8 408.6±93.8 441.9±96.2 6Mean duration (min) 7.0±2.5 39.5±3.6 31.8±7.4% total time 7.8±3.4 43.8±7.9 35.3±5.6

Values are mean±SD.

coefficient was used to analyse the intercorrelations betweenbiochemical variables and lower limb functional data. The Sta-tistical Package for the Social Sciences (SPSS Inc., version 14.0)was used for all analysis. The significance level was set at 5%.

Results

Physiological and anthropometric characteristics of thesoccer players are presented in Table 1.

The mean heart rate during the match play was 173.0±8.8 bpm, and the peak heart rate was 195.6±6.0 bpm, whichcorrespond to 87.1±3.2% and 99.7±7.0%, respectively, of themaximal heart rate previously determined.

As can be seen in Table 2, time-motion analysis showed thatthe players were around 80 min of the total match time involvedin lower intensity activities including standing, walking, joggingand cruising, and 8 min in high intensity activities includingsprinting, backwards and sideways running, corresponding to~92 and 8% of the total match time.

Sprint ability as well as quadriceps and hamstrings peaktorque levels decreased significantly throughout the 72 h-re-covery period after the soccer match (Figs. 1 and 2). Meanquadriceps peak torque decreased by approximately 10% until48 h of recovery and remained ~5% lower than pre-match valuesat 72 h. The soccer match induced a ~15% decrease in ham-strings peak torque until 24 h. Hamstrings strength remained~10% lower than pre-match values at 48 and 72 h. The soccermatch also increased 20 m running time by ~7% at 30 min afterthe end of the match. Sprint ability remained lower than pre-game values by ~5% until 72 h recovery.

As shown in Fig. 3, delayed-onset muscle soreness increasedsignificantly until 48 h after the game.

The soccer match induced a significant increase in plasmamyoglobin content only at 30 min recovery (pb0.05). Plasmacreatine kinase activity during the 72 h-recovery period wassignificantly increased when compared to pre-match values(pb0.05).

Plasma levels of malondialdehyde (MDA) significantlyincreased until 72 h post-soccer match when compared tobaseline (pb0.05) and the content of protein sulfhydryl groups(–SH) during the analysed recovery period where lower thanbefore the match (pb0.05) with the exception of 72 h.

Plasma total antioxidant status (TAS) increased significantlyonly 30 min after the game (pb0.05). Uric acid (UA) con-centration in plasma increased significantly during the 72 h-recovery period after the game compared to pre-match values(pb0.05).

As can be depicted fromFig. 6, blood leukocyte and neutrophilcounts significantly increased 30min after the match and returned

categories

ruising Sprinting Backwards running Sideways running

9.6±10.4 41.7±18.0 122.1±26.6 64.9±4.82.9±1.5 2.2±1.5 4.3±1.3 1.4±0.45.8±2.3 2.5±1.3 4.8±1.9 1.6±0.6

Fig. 1. Sprint ability evaluated as running time under 20 m before and throughout the 72 h-post-match recovery period. Values are means±SEM. ⁎ vs. Pre (pb0.05).

Fig. 2. Dominant leg quadriceps (2A) and hamstrings (2B) peak torques evaluated before and throughout the 72 h-post-match recovery period. Values are means±SEM.⁎ vs. Pre (pb0.05).


Fig. 3. Delayed onset muscle soreness before and 30 min, 24, 48 and 72 h after a soccer match. Values are means±SEM, ⁎ vs. Pre (pb0.05).


to pre-match levels at 24, 48 and 72 h post-exercise (pb0.05). Asignificant decrease in lymphocyte count was observed 30 minafter the game, which returned to baseline levels during theremaining recovery period (pb0.05).

Bivariate correlations were performed in an effort to studypossible relationships between the functional measurementsand the considered biochemical variables. In fact, significantcorrelations were only found between sprint time and Mb(r=0.65; pb0.01), CK (r=0.55; pb0.01) and DOMS (r=0.48;pb0.01). No significant correlations were found between anyoxidative stress and damage markers analysed and the takenlower limb functional measurements. Significant correlationwasfound between plasma TAS and UA contents (r=0.48, pb0.01).

Discussion

A soccer match effort implies several acute physiologicalchanges such as increased cardiac output and blood flow, aug-mented catecholamine release, high contractile eccentric de-mands, mobilization of blood leukocytes and importantly relieson aerobic metabolism. Given that these are predisposing con-ditions for pro-oxidant redox changes in human body, we testedthe hypothesis that a standard 2×45 min soccer match inducesalterations in plasma markers of oxidative damage, antioxidantcapacity and muscle damage. Our data demonstrated for the firsttime that a soccer match resulted in changes in the expression ofmuscle damage and oxidative damage markers in plasma. Also,significant impairments in lower limb muscle function hadoccurred during the considered recovery period after the game.

The match examined in the present study was a friendly gameplayed by Portuguese secondary division players, and it shouldbe thus considered how close it is from games played at an elitelevel. The mean heart rate measured was 173.0±8.8 bpm andcorresponded to 87.1±3.2% of the maximal heart rate. Theabsolute heart rate values were similar to values recently re-ported for Danish soccer players from a similar level [17].Additionally, time-motion analysis from the examined players(Table 2) showed that the frequency and the percentage of time

both at low and high intensity activities were also similar to thatdescribed for players of the same level, and below to thoseobserved in elite players [9]. These observations may suggestthat the analysed match intensity was somewhat similar to othernon-elite games, and probably lower than the intensityperformed by elite soccer players.

As an estimated 1–5% of the total VO2 results in the for-mation of O2

U− [18] and given the high level of VO2 ac-companying soccer, it is not surprising that the biomarkers ofoxidative stress and damage had increased. In addition, otherconcurrent factors can influence cellular and blood antioxidantstatus. For example, stress hormones undergoing autoxidation[19] and circulating neutrophil-induced oxidative burst [20,21]can contribute to the observed blood oxidative stress anddamage. The influence of eccentric exercise-mediating musculardamage-like events on the formation of RONS has also beenreported [22]. Considering the specific physiological demandsimposed by a soccer match, none of these potential RONSsources should be ruled out in the current study. However, it isimportant to note that under the technical constrains of thepresent study we cannot conclusively demonstrate a casual linkbetween any of those potential sources and the increased plasmaoxidative stress and damage found.

The increased oxidative damage induced by the soccermatch can be observed by the additional accumulation of lipidperoxidation by-products, measured as plasma MDA (Fig. 7).Accordingly, the game also induced a significant decrease inplasma sulphydryl residues (Fig. 7), indicating increaseddisulphide linkages (–S–S–) from both proteins and reducedglutathione (GSH). It is important to highlight that the increasein the levels of oxidative damage after the match occurreddespite the possible up-regulation of antioxidant systemspreviously observed under rest conditions in players regularlyinvolved in training and competition when compared withsedentary controls [4,5]. Moreover, as soccer players undergoingregular training showed higher plasma oxidative stress anddamage levels than sedentary controls [4,5] and consideringthe present data assessed in regularly trained subjects, it would


be expected that pro-oxidant changes after a soccer matchwould be further increased in a non-athlete population.

Surprisingly, we found that plasma TAS significantly in-creased after the match, which may indicate compensationin response to intense exercise (Fig. 5A). Previous studies haveshown that half-marathon in trained male runners [23] andtreadmill running until exhaustion [24] also induced an increasein total antioxidant capacity. Considering that TAS assay onlymeasures the antioxidant capacity of the aqueous blood com-partment, which relies mostly on protein (10–28%), UA (7–58%) and ascorbic acid (3–27%) [25], the increase in TASobserved immediately after exercise seems to reflect and/or beinfluenced, at least partially, by the significant increase observedin UA (Fig. 5B), as suggested by the significant correlationfound between TAS and UA. In fact, although being an endproduct of the purine nucleotide system, UA scavenge OH

U−radicals as well, and there is evidence that it may be an importantbiological scavenger against free radicals in human plasma andin skeletal muscle during and after acute hard exercise [26]. Thiswell-known free radical quenching action of UA might have

Fig. 4. Plasma Mb (4A) and CK (4B) levels before and 30 min, 24, 48 and

contributed in this particular case to an attenuation of the rise inplasma oxidative damage.

During high intensity exercise and muscle ischemic condi-tions, the purine nucleotide system is extremely active and theelimination of adenosine monophosphate (AMP) causes a build-up of hypoxanthine in skeletal muscle and in plasma. Despitethat some may be converted back to AMP during rest and atlower exercise intensities, hypoxanthine is also converted to UAgenerating O2

U−. The observation that plasma UA levelsincreased in response to the match is consistent with the findingsfrom other studies using exercise [26]. Confirming theinvolvement of purine nucleotide metabolism in soccer, recentdata from Krustrup et al. [17] showed a significant decrease inmuscle ATP levels after an intense exercise period in the secondhalf and after the entire soccer match as well as significantincrease in muscle IMP content after an intense exercise periodin the second half. Moreover, increased venous blood ammonia,venous plasma UA and hypoxanthine contents were earlierreported [27]. Therefore, it is likely that the observed increasedoxidative stress and damage during the intense exercise periods

72 h after a soccer match. Values are means±SEM, ⁎ vs. Pre (pb0.05).

Fig. 5. Plasma TAS (A) and UA (B) levels before and 30min, 24, 48 and 72h after soccer match. Values are means±SEM, ⁎ vs. Pre (b0.05).


comprised during a soccer match might have the contribution,at least partially, of a xanthine oxidase free radical generatingsystem.

As previously reported in other types of exercise [21,28–30]the present data showed that a soccer match induced a leu-kocytosis dependent on neutrophilia (Fig. 6), which can beascribed to the mobilization of blood cells from marginal poolsby hemodynamic redistribution and augmentation that re-sulted from exercise-related metabolic conditions, such as en-hanced catecholamine secretion imposed by game conditions[27]. Regardless of some controversy on the involvement ofneutrophils in exercise-induced oxidative stress [31], previousstudies using chemiluminescence techniques had shown thatintense exercise was able to increase the capacity of neutro-phils for RONS generation [20,21]. Nevertheless, the casual linkbetween the increased oxidative damage and neutrophiliaobserved in the present study should be cautiously established,as we did not measure the levels of neutrophil activation.

In accordance with others, data from the present study re-ported a marked lymphocytopenia during the subsequent periodafter the end of exercise [32,33]. Although Steensberg et al [33]

observed that, even in a study in which high levels of apoptosis-inducing factors were generated, such as cortisol and isopros-tanes, lymphocyte apoptosis did not contribute to post-exerciselymphocytopenia, others suggested that apoptosis may partiallyaccount for the transient loss of lymphocytes after intense ex-ercise with consequent immunosuppression [32]. Moreover,hormonal changes such as catecholamine over production dur-ing exercise have been described to be responsible for inducingapoptosis [34].

As indirect evidence of delayed-onset muscle damageinduced by the soccer match we considered the levels of musclesoreness (Fig. 3), the lower limb muscle strength (Fig. 2), theappearance of the muscle proteins creatine kinase andmyoglobin (Fig. 4) in plasma and the counts of blood in-flammatory cells over a 72 h period after the match. In thepresent study, the magnitude of changes induced by the soccermatch in these parameters was rather lower than those ob-served following other specific models of exercise-inducedmuscle damage, such as repeated maximal eccentric contrac-tions, which were reported to be severely affected [22]. Ex-pectedly, the significant alterations observed after the soccer

Fig. 6. Changes in blood leukotype, neutrophil and lymphocyte counts before and throughout the 72 h-post match recovery period. Values are means±SEM, ⁎ vs.Pre (b0.05).

Fig. 7. Plasma MDA (A) and -SH (B) levels before and 30min, 24, 48 and 72h after soccer match. Values are means±SEM, ⁎ vs. Pre (b0.05).



match were somewhat close to some reported after an inter-mittent exercise protocol designed to simulate a soccer matchplay [8,35–37].

Several reports [38,39] about the mechanisms related todelayed-onset muscle damage have demonstrated that RONSnot only directly causes damage by oxidation of macromolecularcellular components such as lipids, proteins and DNA, but alsoacts as a regulator of inflammation. Increased RONS productionpromotes the translocation to the nucleus of certain redox-sen-sitive transcription factors, regulating inflammatory mediatorssuch as cytokines, chemokines and adhesion molecules. There-fore, increased RONS production due to exercise is possiblyinvolved in delayed-onset muscle damage associated withphagocyte infiltration secondary to the increased expression ofinflammatory mediators. However, given that the soccer matchdid not represent a sufficient severe muscular stimulus to causeleukocyte infiltration, as shown by the maintenance of bloodleukocyte counts from 24 to 72 h-recovery period when com-pared to baseline (Fig. 6), the possible effects of neutrophil-related oxidative burst on muscle damage induced by soccershould probably be ruled out. This lack of variation in bloodleukocyte counts, with the significant increase in the plasmalevels of oxidative damage that had occurred in the present studywas initially not expected. It is likely that the contribution ofother RONS sources during the post-exercise periods might beconsidered, such as monocyte and macrophage oxidative burst[40]. It is possible that a delayed and continuous monocytemobilization from bone marrow, thus compensating possibleinfiltration of these cells into muscle tissue after damagingexercise [40,41] had occurred in the present study maskingleukocyte count changes in blood. This delayed inflammatoryresponse may be responsible for amplifying and/or repairingskeletal muscle injury [41].

Actually, other adaptive responses induced by contractileactivity in general, and by soccer muscular effort in this par-ticular case, appear to be directly mediated by RONS as sig-naling molecules in the activity and/or expression of severaltranscription factors such as HSF1, NFkB and AP-1 [42,43],which are potentially involved in the up-regulation of cellulardefense against deleterious stress conditions, including muscledamage. Animal studies demonstrated that the overexpression ofskeletal muscle heat shock proteins (HSPs) is associated withan increased superoxide production during non-damaging con-traction and a subsequent transient and reversible oxidation ofprotein thiol groups within the muscle [44], a signal for theactivation of HSP-mediated adaptive response. These data weresupported by evidence that the increased HSP content thatoccurs following an acute period of non-damaging exercise inhumanswas attenuated by prior vitamin C supplementation [45].Moreover, inhibition of free radical production after damagingexercise (downhill running) by ascorbic acid supplementationdid not affect muscle soreness and delayed the recovery ofmuscle function [46]. Extrapolating this hypothesis to a situationwhere exercise-induced muscle damage occurred such as asoccer match, cellular pro-oxidant redox changes, particularly inmild levels, might hypothetically have little direct effect onthe scale of muscle dysfunction, but may stimulate signaling-

mediated adaptive response through the recovery period fol-lowing exercise [2].

Acknowledgments

We would like to thank the soccer players involved inthe study for their committed participation. The excellent tech-nical and practical assistance and skilful involvement ofSergio Ribeiro, João Renato, Ricardo Ladeira, Bárbara Duarte,Henrique Reguengo, and camera operators is appreciated.

The authors are grateful to the City Council of Maia forproviding the pitch where a soccer match was carried out.

António Ascensão is supported by a grant from the PortugueseFoundation for Science and Technology (SFRH/BPD/42525/2007).

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Biochemical Impact of a Soccer Match