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Clinical Biochemistry 41 (2008) 841 851

Biochemical impact of a soccer match analysis of oxidative stress and


muscle damage markers throughout recovery
Antnio Ascenso a,b,,1 , Antnio Rebelo c,1 , Eduardo Oliveira b , Franklim Marques d,e ,
Laura Pereira d , Jos Magalhes a,b,1
a

Research Centre in Physical Activity, Health and Leisure, University of Porto, Portugal
Department of Sport Biology, Faculty of Sport Sciences, University of Porto, Portugal
c
Department of Soccer, Faculty of Sport Sciences, University of Porto, Portugal
d
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 2008
Available 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 may
favour additional pro-oxidant redox alterations. To date, no studies have reported the impact of a soccer match on oxidative stress and muscle
damage markers.
Aim: To analyse the effect of a competitive soccer match on plasma levels of oxidative stress and muscle damage markers, and to relate these
findings with lower limb functional data.
Methods: Blood samples, leg muscle strength, sprint ability and delayed-onset muscle soreness (DOMS) were obtained in 16 soccer
players 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 increased
throughout the recovery period and SH decreased until 48 h post-match. TAS increased at 30 min and UA increased throughout the 72 h
recovery. Blood neutrophils increased at 30 min whereas lymphocytes decreased and returned to baseline from 24 to 72 h. DOMS was higher than
baseline 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 hrecovery 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 the
potential to react with a variety of chemical compounds, being

Corresponding author. Research Centre in Physical Activity, Health and


Leisure, Faculty of Sport Sciences, University of Porto, Portugal, Rua Dr.
Plcido Costa, 91, 4200-450 Porto, Portugal. Fax: +351 225500689.
E-mail address: aascensao@fcdef.up.pt (A. Ascenso).
1
Contributed equally to this manuscript.

closely related to the physiopathology of a wide range of modern


western country diseases [1]. The imbalance between enhanced
RONS production and the ability of antioxidant systems to
render these inactive, lead to cellular loss of redox homeostasis
and to prone conditions of oxidative damage to cellular lipids,
proteins and DNA. In fact, despite RONS having a fundamental
role as signalling molecules in several determinant cellular
pathways, redox changes induced by increased RONS production during exercise are negatively related to cellular homeostasis and might compromise cellular function. Additionally,
the emerging role of free radicals in the delayed-onset muscle

0009-9120/$ - see front matter 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.clinbiochem.2008.04.008

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A. Ascenso et al. / Clinical Biochemistry 41 (2008) 841851

soreness and contraction-induced muscle injury has been recently reported [for refs see [2]].
The activity of soccer players during the competitive season
entails 1 week cycles of training, taper, competition and recovery.
At the top level, this cycle is altered by several irregularities in the
competitive fixture list, being match day not necessarily the same
from 1 week to another. Moreover, players from top level teams
may be involved in additional commitments such as national
cups and other knock-out matches, or representing their countries in international championships. These competitive demands
may impose strains to various physiological systems, including
musculoskeletal, nervous, immune and metabolic, to a point
where recovery strategies post-exercise became influential in
preparing for the next match [3].
The high absolute levels of mitochondrial oxygen consumption, the increased circulating catecholamine, the elevated participation of eccentric muscle contraction-induced damage
and inflammatory response, the intermittent and repeated sprint
actionscausing temporary ischemiareperfusion events in
skeletal muscle are plausible factors that may influence RONS
production during and after a soccer effort. Thus, and despite
chronic exercise training having a protective effect through
improvement of antioxidant capacity [4,5], it is likely that
training sessions as well as the competitive matches expose
participants to oxidative stress and damage with consequent
muscle damage, both during, immediately post-exercise and
throughout the recovery.
Although scarce data have been published regarding the
effects of oxidative stress on exercise performance, there is a
possibility that prior oxidative damage caused by intensive
training periods and/or oxidative modifications while exercising
might compromise the healthy status of the players as well as
exercise performance [6].
Several reports focused on some stress biomarkers, including those of oxidative damage, as well as on the antioxidant status of soccer players under regular training periods
that have been provided [4,5,7]. However, despite postexercise vitamin C supplementation failing to attenuate leg
muscle dysfunction, plasma malondialdehyde, interleukin-6,
creatine kinase and myoglobin increase during the 72 h-recovery period after 90 min of a shuttle running designed to
correspond to the average exercise intensity of playing soccer
[8], no data have been provided regarding the impact of a
soccer match on oxidative stress and damage markers so far.
Moreover, the impact of a soccer match on muscle damage
markers throughout the post-game recovery period has also
been scarcely studied.
The understanding of the redox-based alterations imposed by
a soccer match can contribute with additional physiological
knowledge on the effects of a soccer game on players and
particularly, to improve possible recovery strategies based on
possible antioxidant supplementation. In this regard, the purpose
of the present study was to determine the impact of a soccer
match on the recovery of plasma markers of oxidative stress and
muscle damage in the 72 h-post-match. Leg muscle functional
data, sprint ability, muscle damage as well as leukocyte counts
were also determined throughout the same period.

Methods
Subjects
Sixteen male soccer players from secondary divisions
participated in this study after being informed about the aims,
experimental protocol, procedures and after delivering written
consents. The experimental protocol was approved by the
Ethical Committee of the Faculty of Sport Sciences, University
of Porto, Portugal, and followed the Declaration of Helsinki of
the World Medical Association for research with humans.
Experimental design and procedures
For 2 weeks prior to data collection and during the protocol
period, soccer players were instructed not to change their
normal eating habits and to refrain from additional vitamin or
antioxidant dietary supplementation. Subjects were also
instructed to abstain from exhaustive exercise during the 72-h
pre- and post-match, with exception of functional tests. Subjects
visited the lab 5 days prior the match to determine maximal
oxygen uptake and maximal heart rate.
Blood samples and functional data (quadriceps and hamstrings muscle strength and 20 m sprint ability) were assessed
pre-match and 30 min, 24, 48 and 72 h of the recovery period in
response to a competitive (2 45 min) soccer match. On the day
of the game, players arrived at the laboratory after an overnight
fast of between 10 and 12 h. A resting blood sample was taken
after subjects had been standing for at least 15 min, after which
subjects consumed a light standardized meal and drink and
rested for 2 h. The meal consisted of 1.7 g white bread and 0.3 g
of low-fat spread; both values are per kilogram of body mass
[8]. Pre-match muscle strength and sprint ability were assessed
during the 2 h period between the consumption of the preexercise meal and the start of the soccer match.
For 3 days after the match, subjects returned to the laboratory
after an overnight fast and at approximately the same time of the
morning (within 1 h). A blood sample was taken from the
forearm vein after the subjects had been at complete rest for at
least 15 min. Subsequently, the players performed the strength
and speed tests as outlined below.
Maximal oxygen uptake and heart rate
Five days prior the match, the subjects performed an incremental treadmill (Quasar-Med, Nussdorf, Germany) test
until voluntary exhaustion to determine maximal oxygen uptake
(VO2max) and maximal heart rate (HRmax). Expired respiratory gas fractions were measured using an open circuit breathby-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 recorded
every 5 s using a HR monitor (POLAR TEAM SYSTEMTM, Polar

A. Ascenso et al. / Clinical Biochemistry 41 (2008) 841851

Electro, Kempele, Finland). For time-motion analysis each


player was video-filmed close up during the entire match. The
VHS-format movie cameras (NV-M50, Panasonic, Germany)
were positioned at the side of the pitch at the level of the
halfway line, at a height of about 15 m and at an approximate
distance of 3040 m of the touching line. The videotapes were
later replayed for computerized time-motion analyses according to the procedures described by Mohr et al. [9]. The used
motor pattern categories included standing (0 km.h 1 ), walking (6 km.h 1 ), jogging (8 km.h 1 ), low-speed running
(12 km.h 1 ), moderate-speed running (15 km.h 1 ), highspeed running (18 km.h 1 ), sprinting (30 km.h 1 ), sideways,
and backwards (10 km.h 1 ) running. The match activities
were 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 to
complete a muscle soreness questionnaire, in which they rated
their perceived muscle soreness on a scale from 0 (normal
absence of soreness) to 10 (very intense sore) [10]. They were
instructed to indicate the general soreness of the entire muscle
area when moving or using it. The areas included on the questionnaire were the quadriceps, hamstrings, gastrocnemius and
tibialis muscles.

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20 m sprint ability
Sprint ability measurements were carried out using telemetric
photoelectric cells placed at 0 and 20 m (Brower Timing System,
IRD-T175, Utah, USA). The players stood 1 m behind the
starting line, started on a verbal signal being time activated when
players cross the first pair of photocells, and then ran as fast as
they could to complete the 20 m distance. Players completed two
runs and the best time at each distance was registered.
Blood sampling and preparations
All the venous blood samples were taken by conventional
clinical procedures using EDTA as anticoagulant. Nevertheless,
no tourniquet constriction was used in order to minimize potentially enhanced oxidative stress induced by an ischemia
reperfusion maneuver.
An aliquot of the whole blood was used to perform leukocyte
counts. The remaining freshly withdrawn blood was immediately centrifuged at 3000 rpm during 10 min for careful removal
of the plasma. Plasma was separated into several aliquots
and rapidly frozen at 80 C for later biochemical analysis of
myoglobin (Mb), creatine kinase (CK), total antioxidant status
(TAS), uric acid (UA), malondialdehyde (MDA) and protein
sulfhydryl groups (SH).
Biochemical assays

Strength assessment
In order to evaluate muscle function, maximal gravity corrected concentric peak torque of quadriceps and hamstrings were
measured during isokinetic knee joint movement of the
dominant leg at an angular velocity of 90.s 1 (1.57 rad.s 1)
according with [11] using an isokinetic dynamometer (Biodex
System 2, NY, USA). After individual self-report, the dominant
leg was determined by a routine visual inspection in a simple
target kicking test requiring accuracy according to the procedures described elsewhere [12].
Briefly, the subjects were familiarized with the muscle function test on at least two occasions during preliminary visits to the
laboratory. They were seated on the dynamometer chair at 85
inclination (external angle from the horizontal) with stabilization
straps at the trunk, abdomen and thigh to prevent inaccurate
joint movements. The knee to be tested was positioned at 90 of
flexion (0 = fully extended knee) and the axis of the dynamometer lever arm was aligned with the distal point of the lateral
femoral condyle. Before the anatomical alignments and
procedures, all the subjects were instructed to kick and also to
bend the tested leg as hard and fast as they could through a
complete range of motion (from 90 to 0). Subjects were also
instructed to hold their arms comfortably across their chest to
further isolate knee joint flexion and extension movements.
Prior to muscle function measurements, subjects were taken to
a standardized warm-up consisting of 5 min of gentle running and
stretching. All subjects performed a specific sub-maximal warmup protocol on the isokinetic device. Three maximal repetitions at
angular velocity 90.s 1 (1.57 rad.s 1) were therefore carried out.

Plasma creatine kinase (CK) activity was determined spectrophotometrically using a commercial test kit (ABX A11A01632,
Mompelier, FR). Plasma myoglobin concentration was assessed
using a commercial test kit (myoglobin bioMerieux 30446,
Carnaxide, PT).
Plasma MDA was assayed according to the method described
by Rohn et al. [13] with some modifications and measured by the
formation of thiobarbituric acid reactive substances at 535 nm.
Briefly, plasma was incubated, at 25 C, in 500 L of a medium
consisting of 175 mM KCl, 10 mM Tris, pH 7.4. Samples of
50 L were taken and mixed with 450 L of a TBARS reagent
(1% thiobarbituric acid, 0.6 N HCl, 0.0056% butylated
hydroxytoluene). The mixture was heated at 8090 C during
15 min, and re-cooled in ice for 10 min before centrifugation in
Eppendorf centrifuge (1500 g, 5 min). The amount of TBARS
formed was calculated using a molar extinction coefficient of
1.56 105 M 1 cm 1 and expressed as nanomoles of MDA per
milligram of protein [14].
Oxidative modification of protein SH groups in plasma
was quantified by spectrophotometric measurement at 414 nm
according to the method proposed by Hu [15]. Briefly, the
colorimetric assay was performed after the reaction of 50 L
aliquot of plasma with 10 L of 5,5-dithio-bis (2-nitrobenzoic
acid) (10 mM) in a medium containing 150 L of Tris (0.25 M)
and 790 L methanol, at 414 nm against a blank test. SH
content was expressed in nmol/mg of plasma protein (414 =
13.6 mM 1 cm 1 ).
TAS was measured spectrophotometrically using a commercial kit (Randox NX2332 Crumlin, UK). The assay is based on

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A. Ascenso et al. / Clinical Biochemistry 41 (2008) 841851

Table 1
Anthropometric and physiological characteristics of the subjects
Variables

Mean SD

Age (yr)
Mass (kg)
Height (cm)
% Body fat
VO2max (mL.Kg 1.min 1)
Hrmax (bpm)

21.3 1.1
70.7 6.3
175.0 6.0
8.3 1.9
55.1 5.1
196.0 7.0

VO2max maximal oxygen uptake, HR heart rate.

the reduction of free radicals (ABTS +2,2-azinobis (3ehylbenzothiazoline-6-sulfonate)) measured as a decrease of


U
absorbance at 600 nm at 3 min by antioxidants. The ABTS +
radical cation is formed by the interaction of ABTS with ferrylmyoglobin radical species, generated by the activation of
metmyoglobin with hydrogen peroxide. The suppression of the
U
absorbance of the ABTS + radical cation by plasma antioxidants
was compared with that from a Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). The results are expressed
as mmol/L of Trolox equivalents.
Uric acid was determined by an enzymatic method at 550 nm
using a commercial kit (ABX A11A01670, Montpellier, FR)
according to the specifications of the manufacturer.
Protein content was spectrophotometrically assayed using
bovine serum albumin as standard according to Lowry et al.
[16].
Samples were analysed in duplicate and the mean of the two
values 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 five
replicates of each leukocyte measurement was b 1%. Whole
blood 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 airdried. Cell differentials were performed using an Olympus
microscope equipped with 1000 oil immersion lens. Specifically, the leukocyte counts including neutrophils and lymphocytes were recorded.
Statistics
Mean, standard deviation (SD) and standard error mean (SEM)
were calculated for all variables in each of the experimental
groups. The ANOVA with repeated measures was used to establish whether any of the subsequent test results were significantly different from baseline results. Pearson's correlation

coefficient was used to analyse the intercorrelations between


biochemical variables and lower limb functional data. The Statistical 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 the
soccer 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, which
correspond to 87.1 3.2% and 99.7 7.0%, respectively, of the
maximal heart rate previously determined.
As can be seen in Table 2, time-motion analysis showed that
the players were around 80 min of the total match time involved
in lower intensity activities including standing, walking, jogging
and cruising, and 8 min in high intensity activities including
sprinting, backwards and sideways running, corresponding to
~ 92 and 8% of the total match time.
Sprint ability as well as quadriceps and hamstrings peak
torque levels decreased significantly throughout the 72 h-recovery period after the soccer match (Figs. 1 and 2). Mean
quadriceps peak torque decreased by approximately 10% until
48 h of recovery and remained ~ 5% lower than pre-match values
at 72 h. The soccer match induced a ~ 15% decrease in hamstrings peak torque until 24 h. Hamstrings strength remained
~ 10% lower than pre-match values at 48 and 72 h. The soccer
match also increased 20 m running time by ~ 7% at 30 min after
the end of the match. Sprint ability remained lower than pregame values by ~ 5% until 72 h recovery.
As shown in Fig. 3, delayed-onset muscle soreness increased
significantly until 48 h after the game.
The soccer match induced a significant increase in plasma
myoglobin content only at 30 min recovery (p b 0.05). Plasma
creatine kinase activity during the 72 h-recovery period was
significantly increased when compared to pre-match values
(p b 0.05).
Plasma levels of malondialdehyde (MDA) significantly
increased until 72 h post-soccer match when compared to
baseline (p b 0.05) and the content of protein sulfhydryl groups
(SH) during the analysed recovery period where lower than
before the match (p b 0.05) with the exception of 72 h.
Plasma total antioxidant status (TAS) increased significantly
only 30 min after the game (p b 0.05). Uric acid (UA) concentration in plasma increased significantly during the 72 hrecovery period after the game compared to pre-match values
(p b 0.05).
As can be depicted from Fig. 6, blood leukocyte and neutrophil
counts significantly increased 30 min after the match and returned

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

Frequency (n)
Mean duration (min)
% total time
Values are mean SD.

Standing

Walking

Jogging

Cruising

Sprinting

Backwards running

Sideways running

114 44.8
7.0 2.5
7.8 3.4

408.6 93.8
39.5 3.6
43.8 7.9

441.9 96.2
31.8 7.4
35.3 5.6

69.6 10.4
2.9 1.5
5.8 2.3

41.7 18.0
2.2 1.5
2.5 1.3

122.1 26.6
4.3 1.3
4.8 1.9

64.9 4.8
1.4 0.4
1.6 0.6

A. Ascenso et al. / Clinical Biochemistry 41 (2008) 841851

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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 (p b 0.05).

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 (p b 0.05).

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A. Ascenso et al. / Clinical Biochemistry 41 (2008) 841851

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 (p b 0.05).

to pre-match levels at 24, 48 and 72 h post-exercise (p b 0.05). A


significant decrease in lymphocyte count was observed 30 min
after the game, which returned to baseline levels during the
remaining recovery period (p b 0.05).
Bivariate correlations were performed in an effort to study
possible relationships between the functional measurements
and the considered biochemical variables. In fact, significant
correlations were only found between sprint time and Mb
(r = 0.65; p b 0.01), CK (r = 0.55; p b 0.01) and DOMS (r = 0.48;
p b 0.01). No significant correlations were found between any
oxidative stress and damage markers analysed and the taken
lower limb functional measurements. Significant correlation was
found between plasma TAS and UA contents (r = 0.48, p b 0.01).
Discussion
A soccer match effort implies several acute physiological
changes such as increased cardiac output and blood flow, augmented catecholamine release, high contractile eccentric demands, mobilization of blood leukocytes and importantly relies
on aerobic metabolism. Given that these are predisposing conditions for pro-oxidant redox changes in human body, we tested
the hypothesis that a standard 2 45 min soccer match induces
alterations in plasma markers of oxidative damage, antioxidant
capacity and muscle damage. Our data demonstrated for the first
time that a soccer match resulted in changes in the expression of
muscle damage and oxidative damage markers in plasma. Also,
significant impairments in lower limb muscle function had
occurred during the considered recovery period after the game.
The match examined in the present study was a friendly game
played by Portuguese secondary division players, and it should
be thus considered how close it is from games played at an elite
level. The mean heart rate measured was 173.0 8.8 bpm and
corresponded to 87.1 3.2% of the maximal heart rate. The
absolute heart rate values were similar to values recently reported 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 that
described for players of the same level, and below to those
observed in elite players [9]. These observations may suggest
that the analysed match intensity was somewhat similar to other
non-elite games, and probably lower than the intensity
performed by elite soccer players.
As an estimated 15% of the total VO2 results in the forU
mation of O2 [18] and given the high level of VO2 accompanying soccer, it is not surprising that the biomarkers of
oxidative stress and damage had increased. In addition, other
concurrent factors can influence cellular and blood antioxidant
status. For example, stress hormones undergoing autoxidation
[19] and circulating neutrophil-induced oxidative burst [20,21]
can contribute to the observed blood oxidative stress and
damage. The influence of eccentric exercise-mediating muscular
damage-like events on the formation of RONS has also been
reported [22]. Considering the specific physiological demands
imposed by a soccer match, none of these potential RONS
sources should be ruled out in the current study. However, it is
important to note that under the technical constrains of the
present study we cannot conclusively demonstrate a casual link
between any of those potential sources and the increased plasma
oxidative stress and damage found.
The increased oxidative damage induced by the soccer
match can be observed by the additional accumulation of lipid
peroxidation by-products, measured as plasma MDA (Fig. 7).
Accordingly, the game also induced a significant decrease in
plasma sulphydryl residues (Fig. 7), indicating increased
disulphide linkages (SS) from both proteins and reduced
glutathione (GSH). It is important to highlight that the increase
in the levels of oxidative damage after the match occurred
despite the possible up-regulation of antioxidant systems
previously observed under rest conditions in players regularly
involved in training and competition when compared with
sedentary controls [4,5]. Moreover, as soccer players undergoing
regular training showed higher plasma oxidative stress and
damage levels than sedentary controls [4,5] and considering
the present data assessed in regularly trained subjects, it would

A. Ascenso et al. / Clinical Biochemistry 41 (2008) 841851

be expected that pro-oxidant changes after a soccer match


would be further increased in a non-athlete population.
Surprisingly, we found that plasma TAS significantly increased after the match, which may indicate compensation
in response to intense exercise (Fig. 5A). Previous studies have
shown that half-marathon in trained male runners [23] and
treadmill running until exhaustion [24] also induced an increase
in total antioxidant capacity. Considering that TAS assay only
measures the antioxidant capacity of the aqueous blood compartment, which relies mostly on protein (1028%), UA (7
58%) and ascorbic acid (327%) [25], the increase in TAS
observed immediately after exercise seems to reflect and/or be
influenced, at least partially, by the significant increase observed
in UA (Fig. 5B), as suggested by the significant correlation
found between TAS and UA. In fact, although being an end
U
product of the purine nucleotide system, UA scavenge OH
radicals as well, and there is evidence that it may be an important
biological scavenger against free radicals in human plasma and
in skeletal muscle during and after acute hard exercise [26]. This
well-known free radical quenching action of UA might have

847

contributed in this particular case to an attenuation of the rise in


plasma oxidative damage.
During high intensity exercise and muscle ischemic conditions, the purine nucleotide system is extremely active and the
elimination of adenosine monophosphate (AMP) causes a buildup of hypoxanthine in skeletal muscle and in plasma. Despite
that some may be converted back to AMP during rest and at
lower exercise intensities, hypoxanthine is also converted to UA
U
generating O2 . The observation that plasma UA levels
increased in response to the match is consistent with the findings
from other studies using exercise [26]. Confirming the
involvement of purine nucleotide metabolism in soccer, recent
data from Krustrup et al. [17] showed a significant decrease in
muscle ATP levels after an intense exercise period in the second
half and after the entire soccer match as well as significant
increase in muscle IMP content after an intense exercise period
in the second half. Moreover, increased venous blood ammonia,
venous plasma UA and hypoxanthine contents were earlier
reported [27]. Therefore, it is likely that the observed increased
oxidative stress and damage during the intense exercise periods

Fig. 4. Plasma Mb (4A) and CK (4B) levels before and 30 min, 24, 48 and 72 h after a soccer match. Values are means SEM, vs. Pre (p b 0.05).

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A. Ascenso et al. / Clinical Biochemistry 41 (2008) 841851

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 (b 0.05).

comprised during a soccer match might have the contribution,


at least partially, of a xanthine oxidase free radical generating
system.
As previously reported in other types of exercise [21,2830]
the present data showed that a soccer match induced a leukocytosis dependent on neutrophilia (Fig. 6), which can be
ascribed to the mobilization of blood cells from marginal pools
by hemodynamic redistribution and augmentation that resulted from exercise-related metabolic conditions, such as enhanced catecholamine secretion imposed by game conditions
[27]. Regardless of some controversy on the involvement of
neutrophils in exercise-induced oxidative stress [31], previous
studies using chemiluminescence techniques had shown that
intense exercise was able to increase the capacity of neutrophils for RONS generation [20,21]. Nevertheless, the casual link
between the increased oxidative damage and neutrophilia
observed 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 reported a marked lymphocytopenia during the subsequent period
after the end of exercise [32,33]. Although Steensberg et al [33]

observed that, even in a study in which high levels of apoptosisinducing factors were generated, such as cortisol and isoprostanes, lymphocyte apoptosis did not contribute to post-exercise
lymphocytopenia, others suggested that apoptosis may partially
account for the transient loss of lymphocytes after intense exercise with consequent immunosuppression [32]. Moreover,
hormonal changes such as catecholamine over production during exercise have been described to be responsible for inducing
apoptosis [34].
As indirect evidence of delayed-onset muscle damage
induced by the soccer match we considered the levels of muscle
soreness (Fig. 3), the lower limb muscle strength (Fig. 2), the
appearance of the muscle proteins creatine kinase and
myoglobin (Fig. 4) in plasma and the counts of blood inflammatory cells over a 72 h period after the match. In the
present study, the magnitude of changes induced by the soccer
match in these parameters was rather lower than those observed following other specific models of exercise-induced
muscle damage, such as repeated maximal eccentric contractions, which were reported to be severely affected [22]. Expectedly, the significant alterations observed after the soccer

A. Ascenso et al. / Clinical Biochemistry 41 (2008) 841851

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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 (b 0.05).

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 (b 0.05).

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A. Ascenso et al. / Clinical Biochemistry 41 (2008) 841851

match were somewhat close to some reported after an intermittent exercise protocol designed to simulate a soccer match
play [8,3537].
Several reports [38,39] about the mechanisms related to
delayed-onset muscle damage have demonstrated that RONS
not only directly causes damage by oxidation of macromolecular
cellular components such as lipids, proteins and DNA, but also
acts as a regulator of inflammation. Increased RONS production
promotes the translocation to the nucleus of certain redox-sensitive transcription factors, regulating inflammatory mediators
such as cytokines, chemokines and adhesion molecules. Therefore, increased RONS production due to exercise is possibly
involved in delayed-onset muscle damage associated with
phagocyte infiltration secondary to the increased expression of
inflammatory mediators. However, given that the soccer match
did not represent a sufficient severe muscular stimulus to cause
leukocyte infiltration, as shown by the maintenance of blood
leukocyte counts from 24 to 72 h-recovery period when compared to baseline (Fig. 6), the possible effects of neutrophilrelated oxidative burst on muscle damage induced by soccer
should probably be ruled out. This lack of variation in blood
leukocyte counts, with the significant increase in the plasma
levels of oxidative damage that had occurred in the present study
was initially not expected. It is likely that the contribution of
other RONS sources during the post-exercise periods might be
considered, such as monocyte and macrophage oxidative burst
[40]. It is possible that a delayed and continuous monocyte
mobilization from bone marrow, thus compensating possible
infiltration of these cells into muscle tissue after damaging
exercise [40,41] had occurred in the present study masking
leukocyte count changes in blood. This delayed inflammatory
response may be responsible for amplifying and/or repairing
skeletal muscle injury [41].
Actually, other adaptive responses induced by contractile
activity in general, and by soccer muscular effort in this particular case, appear to be directly mediated by RONS as signaling molecules in the activity and/or expression of several
transcription factors such as HSF1, NFkB and AP-1 [42,43],
which are potentially involved in the up-regulation of cellular
defense against deleterious stress conditions, including muscle
damage. Animal studies demonstrated that the overexpression of
skeletal muscle heat shock proteins (HSPs) is associated with
an increased superoxide production during non-damaging contraction and a subsequent transient and reversible oxidation of
protein thiol groups within the muscle [44], a signal for the
activation of HSP-mediated adaptive response. These data were
supported by evidence that the increased HSP content that
occurs following an acute period of non-damaging exercise in
humans was attenuated by prior vitamin C supplementation [45].
Moreover, inhibition of free radical production after damaging
exercise (downhill running) by ascorbic acid supplementation
did not affect muscle soreness and delayed the recovery of
muscle function [46]. Extrapolating this hypothesis to a situation
where exercise-induced muscle damage occurred such as a
soccer match, cellular pro-oxidant redox changes, particularly in
mild levels, might hypothetically have little direct effect on
the scale of muscle dysfunction, but may stimulate signaling-

mediated adaptive response through the recovery period following exercise [2].
Acknowledgments
We would like to thank the soccer players involved in
the study for their committed participation. The excellent technical and practical assistance and skilful involvement of
Sergio Ribeiro, Joo Renato, Ricardo Ladeira, Brbara Duarte,
Henrique Reguengo, and camera operators is appreciated.
The authors are grateful to the City Council of Maia for
providing the pitch where a soccer match was carried out.
Antnio Ascenso is supported by a grant from the Portuguese
Foundation for Science and Technology (SFRH/BPD/42525/
2007).
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