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Module 1 British Journal of Sports Medicine 14/6/08 07:42:56
Original article
Effects of exercise intensity on lymphocyte H2O2
production and antioxidant defences in soccer
players
;
A Sureda,1 M D Ferrer,1 P Tauler,1 D Romaguera,1 F Drobnic,2 P Pujol,3 J A Tur,1 A Pons1
1
Laboratori de Ciències de
l’Activitat Fı́sica, Departament
de Biologia Fonamental i
Ciències de la Salut, Universitat
de les Illes Balears, Spain;
2
Medical Services, F.C.
Barcelona, Spain; 3 Sports
Physiology Department, Centre
d’Alt Rendiment de Barcelona,
Barcelona, Spain
Correspondence to:
Dr Antoni Pons, Laboratori de
Ciències de l’Activitat Fı́sica,
Universitat de les Illes Balears,
Crtra. Valldemossa, km 7,5, E07122 - Palma de Mallorca,
Balears, Spain; antonipons@uib.
es
Accepted 20 November 2007
Published Online First
10 December 2007
ABSTRACT
Objective: Physical exercise is capable of enhancing or
suppressing the immune response depending on the
intensity and duration of exercise. This study investigated
how exercise intensity influences the lymphocyte
antioxidant response and the induction of cellular
oxidative damage.
Design: Eighteen voluntary male pre-professional soccer
players participated in this study. Sportsmen played a
60 min training match, and were divided into three groups
depending on the intensity degree during the match: low,
medium and high intensities.
Measurements: Malondialdehyde (MDA), vitamins C
and E and haem oxygenase-1 (HO-1) gene expression
were measured in lymphocytes. Reactive oxygen species
(ROS) production was determined in lymphocytes and
neutrophils.
Results: Lymphocyte MDA levels and H2O2 production
were significantly increased in the group which performed
the most intense exercise. Neutrophil counts and ROS
production increased progressively with the exercise
intensity. Vitamin C significantly decreased after exercise
in the highest-intensity group in comparison with initial
values, whereas vitamin E levels significantly increased in
the medium and high-intensity groups. HO-1 gene
expression significantly increased in the medium and highintensity groups.
Conclusions: Exercise intensity affects the lymphocyte
and neutrophil oxidant/antioxidant balance, but only
exercise of high intensity induces lymphocyte oxidative
damage.
The physiological response to physical exercise
involves a number of changes in the oxidative
balance and in the metabolism of some important
biological molecules.1 Physical exercise is characterised by an increase in oxygen consumption by
the whole body and by an increase in reactive
oxygen species (ROS) production.2 The main
sources of ROS during exercise are the mitochondrial respiratory chain, xanthine oxidase-catalysed
reaction and neutrophil activation.3 ROS are
known to cause oxidative modifications of lipids,
proteins and nucleic acids leading to cell and tissue
damage.4–7 Under physiological conditions, these
deleterious species are mostly removed by the
cellular antioxidant systems. Exercise-induced ROS
are also thought to modulate acute-phase inflammatory responses8 and to have a role in cell
signalling inducing specific cellular adaptations to
exercise.9 10
Exhaustive exercise elicits a stress response
similar to the acute phase immune response.8
Br J Sports Med 2008;000:1–6. doi:10.1136/bjsm.2007.043943
Exercise affects lymphocytes as reflected in total
blood cell counts and lymphocyte proliferative
response.11 12 It is widely accepted that athletes
undergoing intensive training and competition
schedules are at increased risk of developing upper
respiratory tract infections.13 Strenuous physical
exercise, characterised by a remarkable increase in
oxygen consumption with concomitant ROS
production, could lead to an oxidative stress
situation and cell damage.3 It has been evidenced
that athletes undergoing regular and adequate
training show improved intracellular antioxidant
status14 15 and increased resistance to upper respiratory tract infections.16
Soccer is one of the most popular sports worldwide, but only a few studies have focused on the
effect of soccer practice on the antioxidant status
of players. Competitive soccer engages many of the
body’s systems to a major extent, to a point where
oxidative-derived damage can appear.17 Some studies have compared the oxidant and antioxidant
status of soccer players and sedentary controls.
Total antioxidant capacity, superoxide dismutase
activity, uric acid, ascorbic acid and tocopherol
plasma levels were all higher in soccer players,
while MDA levels were lower.14 18 The levels of
autoantibodies against oxidised LDL in professional
soccer players are increased as result of intensive
training-induced oxidative stress.19 Despite these
studies, there are few data concerning the effects of
a soccer match on the antioxidant status of
players.
The aim of this study was to determine the
differential effects of the exercise intensity on the
antioxidant response and on the cellular oxidative
damage in lymphocytes induced by a training
soccer match. Neutrophil capability to produce
ROS was also determined for comparison with
lymphocytes.
MATERIALS AND METHODS
Subjects and protocol
Eighteen voluntary male pre-professional soccer
players participated in this study after giving their
written consent to participate. The protocol was in
accordance with the Declaration of Helsinki for
research on human subjects and was approved by
the Ethical Committee of Clinical Investigation of
CAR-Sant Cugat (Barcelona). All sportsmen participating in the study had a controlled diet and
expended similar periods of training and competition.
The sportsmen played a training soccer match
for 60 minutes. The exercise intensity was deter1
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Module 1 British Journal of Sports Medicine 14/6/08 07:42:57
Original article
=
reproducibility and the interference by several agents of the
classical determination by the TBARs method.23
mined by a pulsometer, and sportsmen were divided into three
groups (n = 6) depending on intensity degree. As the cardiac
heart rate increases linearly with oxygen consumption,20 we can
indirectly evaluate the work done during maximal and
intervallic exercise through the heart rate.21 We categorise the
subjects according to the perspective of the work performed
during the training sessions and the competition in relation to
the reference values of a progressive and maximal exercise test.
Five metabolic zones are usually considered. From zone one (Z1)
to zone five (Z5), the relation to maximal oxygen consumption
is respectively ,70%, 70–80%, 80–90%, 90–100% and 100% or
higher. Groups were simplified to three (table 1), the lowintensity (more than 50% of match into zones Z1 plus Z2) the
medium-intensity (more than 50% of match into zone Z3) and
the high-intensity (more than 50% of the match into zones Z4
plus Z5).
The three groups had similar anthropometric values (table 2).
Venous blood samples were obtained from the antecubital
vein with EDTA as anticoagulant. Samples were obtained in
basal conditions and immediately after exercise finished. Blood
samples were used to purify lymphocytes and neutrophils, and
to obtain plasma. Lymphocyte and neutrophil counts were
quantified in an automatic flow cytometer analyser Techicon
H2 (Bayer) VCS system.
Lymphocyte vitamin C and vitamin E determination
Samples for vitamin C were deproteinised with 5% metaphosphoric acid and centrifuged for 5 min at 15 0006g at 4uC, and
the supernatants recovered. The mobile phase consisted of
0.05 mol/l sodium phosphate, 0.05 mol/l sodium acetate,
189 mmol/l
dodecyltrimethylammonium
chloride
and
36.6 mmol/l tetraoctylammonium bromide in 25/75 methanol/water (v/v), pH 4.8. The HPLC system was a Shimadzu
with a Waters Inc. electrochemical detector and a Nova Pak,
C18, 3.96150 mm column. The potential of the chromatographic detection was set at 0.7 V versus an Ag/AgCl reference
electrode. Vitamin C was quantified by using a standard curve
of known concentration.
Vitamin E was extracted from lymphocyte lysates using nhexane after deproteinisation with ethanol. Vitamin E concentration was determined by HPLC in the n-hexane extract after
samples had been dried under nitrogen and dissolved in
methanol. The mobile phase consisted of 550:370:80
acetonitrile:tetrahydrofuran:H2O, and the column was Nova
Pak, C18, 3.96150 mm. a-tocopherol isoform was determined
at 290 nm and was quantified by comparison to a standard
curve of known concentration.
Vitamin concentrations were calculated by taking into
account the lymphocyte volume of 2161026 ml/lymphocyte.24
Lymphocyte, neutrophil and plasma purification
>
Blood cells were immediately purified from whole blood
following an adaptation of the method of Boyum.22 Blood was
introduced onto Ficoll and centrifuged at 9006g at 4uC for
30 min. The lymphocyte layer was carefully removed and
washed twice with PBS and centrifuged for 10 min at 10006g at
4uC. This method ensures that 95% (SD 5%) of cells in fraction
are mononucleocytes with 95% (SD 5%) viability. The cellular
precipitate of lymphocytes was lysed with distilled water. The
precipitate obtained after centrifugation with Ficoll, containing
erythrocytes and neutrophils, was incubated at 4uC with
ammonium chloride 0.15 mol/l to haemolyse erythrocytes.
The suspension was centrifuged at 7506g at 4uC for 15 min
and the supernatant was discarded. The neutrophil phase at the
bottom was washed first with ammonium chloride and then
with PBS. Neutrophils were resuspended in Hank’s balanced salt
solution (HBSS) for chemiluminescence assays.
Plasma was obtained after centrifugation for 15 min at
10006g at 4uC of another blood sample and was stored at
280uC until use.
Lymphocyte hydrogen peroxide production
H2O2 production in lymphocytes was measured before and after
stimulation with phorbol myristate acetate (PMA) using 2,7dichlorofluorescin-diacetate (DCFH-DA) as indicator. DCFHDA (30 mg/ml) in PBS was added to a 96-well microplate
containing lymphocyte suspension. PMA (3 mmol/l) prepared in
HBSS or HBSS alone was added to the wells and the
fluorescence (Ex 480 nm; Em 530 nm) was recorded at 37uC
for 1 h in a FLx800 Microplate Fluorescence Reader (Bio-tek
Instruments, Inc., USA).
Neutrophil chemiluminescence assay
Opsonised zymosan (OZ) was used as neutrophil stimulant.
Zymosan A (Sigma) was suspended in HBSS at a concentration
of 1 mg/ml and incubated with 10% human serum at 37uC for
30 min, followed by centrifugation at 7506g for 10 min at 4uC.
The precipitate was washed twice in HBSS and finally
resuspended in HBSS at 1 mg/ml. OZ suspension (100 ml) was
added to a 96-well microplate containing 50 ml neutrophil
suspension and 50 ml luminol solution (2 mmol/l in PBS,
pH 7.4). Chemiluminescence was measured at 37uC for
90 min in a FLx800 Microplate Fluorescence Reader.
Lymphocyte MDA concentration
MDA as a marker of lipid peroxidation was analysed in
lymphocytes using a colorimetric assay kit (Calbiochem, San
Diego, CA, USA) by following the manufacturer’s instructions.
This assay kit is specific for MDA, avoiding the poor
Real-time reverse transcriptase–polymerase chain reaction
Table 1 Distribution of groups
Low intensity (%)
Medium intensity (%)
High intensity (%)
Z1–Z2
Z3
Z4–Z5
61.2 (5.7)
26.0 (3.7)
2.70 (0.63)
36.5 (5.2)
62.8 (3.8)
21.7 (4.3)
2.32 (1.16)
11.1 (3.2)
75.6 (4.8)
Percentage of time during which the players performed at low, medium or high
exercise intensity. From zone one (Z1) to zone five (Z5), the relation to maximal
oxygen consumption was respectively ,70%,70–80%, 80–90%,90–100% and 100% or
higher. Groups were simplified to three: the low-intensity (Z1 plus Z2 more than 50%
of the total time), the medium-intensity (Z3 more than 50% of the total time) and the
high-intensity (Z4 plus Z5 more than 50% of the total time).
Results expressed as mean (SEM).
2
<
Lymphocyte mRNA was isolated by phenol–chloroform extraction. cDNA was synthesised from 1 mg total RNA using reverse
transcriptase with oligo-dT primers. Quantitative PCR was
performed using the LightCycler instrument (Roche
Diagnostics) with DNA-master SYBR Green I. The primers
used
were:
HO-1,
forward:
59-CCAGCG
GGCCAGCAACAAAGTGC-39,
reverse:
59AAGCCTTCAGTGCCCACGGTAAGG-39 and 18S, forward:
59-ATGTGAAGTCACTGTGCCAG-39,
reverse:
59GTGTAATCCGTCTCCACAGA-39. The PCR conditions were
as follows: 95uC for 10 min, followed by 40 cycles of
Br J Sports Med 2008;000:1–6. doi:10.1136/bjsm.2007.043943
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Table 2 Anthropometric and physiological parameters
Age (years)
Height (cm)
Weight (kg)
BMI
VO2max (ml/kg/min)
Low intensity
Medium intensity High intensity
20.2
179 (2)
76.1
23.6
56.6
19.8
176 (2)
72.0
23.0
58.4
(0.4)
(1.8)
(0.5)
(0.8)
(0.3)
(1.9)
(0.7)
(2.4)
19.7
175 (3)
75.5
24.8
56.2
(0.4)
(2.4)
(0.9)
(1.5)
One-way ANOVA; no significant differences were evidenced between groups. Results
are expressed as mean (SEM), n = 6.
amplification at 95uC for 0 s, 60uC for 5 s and 72uC for 10 s for
HO-1 and for ribosomal 18S 40 cycles at 95uC for 10 s, 60uC for
7 s and 72uC for 12 s. The relative quantification was performed
by standard calculations considering 2(-DDCt). Basal mRNA levels
of the low-intensity group were arbitrarily referred to as 1. The
expression of target gene was normalised with respect to
ribosomal 18S.
after exercise. The after-match values in the most intense group
were significantly higher than the ones obtained in the lowintensity group after the match. There were no significant
differences in the basal values of MDA concentration between
groups. MDA levels were significantly increased in the highintensity group after the match (42%), whereas the other two
groups maintained initial values. The increase in MDA levels in
the high-intensity group after the match was not significantly
different from the results obtained in the medium and lowintensity groups.
HO-1 expression was similar in the three groups in the basal
point (Figure 2). After the training match, HO-1 expression
significantly increased in the groups which performed the
medium and high exercise intensities (57% and 86% respectively). However, the post-exercise values in the medium and
high intensity groups were not significantly different respect
the post-exercise values measured in the low intensity group.
DISCUSSION
Statistical analysis
Statistical analysis was carried out using a statistical package
(SPSS 12.0 for Windows). Results are expressed as mean (SEM)
and p,0.05 was considered statistically significant. The
statistical significance of the data was assessed by two-way
analysis of variance (ANOVA). The statistical factors analysed
were the exercise intensity (Fc) and the soccer match (E). When
significant effects were found, a one-way ANOVA was used to
determine the differences between the groups involved.
RESULTS
Lymphocyte number and haematocrit were similar in the three
groups in basal conditions and did not change during the
exercise in any of the three studied groups (data not shown).
No significant differences were observed between groups in
the lymphocytes’ basal ROS production (fig 1). ROS production
in non-activated lymphocytes significantly increased only in the
most intense group after the match (24%). This increase is
significantly different when compared with the post-exercise
values obtained in the low-intensity group. In PMA-activated
lymphocytes, ROS production maintained initial values in the
groups which performed the lower and medium-intensity
exercise. ROS production significantly increased in the highintensity group after exercise (33%), the final values in this
group being significantly higher than the final values measured
in the low and medium-intensity groups.
Neutrophil number and ROS production were similar in the
three groups in basal conditions (table 3).
Exercise induced an increase in the number of circulating
neutrophils, and the increase degree depends on the exercise
intensity. In the low-intensity group circulating neutrophils
increased 36%, in the medium group 61% and in the high group
83%. ROS production in zymosan-stimulated neutrophils only
increased significantly in the high-intensity group with respect
to pre-exercise values. This increase is significantly different
compared with post-exercise values of the low-intensity group.
Both exercise and exercise intensity influenced lymphocyte
vitamin C and E levels (table 4).
Vitamin C significantly decreased after exercise in the
highest-intensity group with respect to initial values (34%),
whereas basal values were maintained in the other groups. This
vitamin C decrease was significantly different when compared
with the vitamin C levels obtained in the lower-intensity group,
measured after the match. Vitamin E levels significantly
increased in the medium (28%) and high (34%)-intensity groups
Br J Sports Med 2008;000:1–6. doi:10.1136/bjsm.2007.043943
Moderate exercise is a healthy practice; however, exhaustive
exercise induces oxidative stress. Moderate exercise attenuates
lymphocyte apoptosis induced by oxidative stress, possibly by
improving intracellular antioxidative capacity.15 In previous
studies, we reported that a mountain cycling stage induced a
significant lymphopenia and high levels of oxidative stress in
lymphocytes,25 whereas a cycling stage without mountainous
terrain maintained lymphocyte counts.26 These results suggest
that the exercise intensity could be responsible for the stressinduced changes. In fact, in the present study, MDA concentration increased only in the group that performed the most
intense exercise. We also reported an inverse correlation
between lymphocyte protein carbonyl derivatives and lymphocyte number.11
We studied the lymphocytes’ capability to produce ROS by
using DCFH-DA before and after stimulation with PMA as a
possible source of oxidative stress. However, DCFH can suffer
an auto-oxidation that appears to form trace amounts of H2O2,
but the rate of auto-oxidation should be of equal importance in
each sample.27 The progressive increase in the capabilities of
ROS production in the groups which performed the moderate
(with no significant differences) and high-intensity exercise
after the match indicates a relationship between exercise
intensity and the appearance of oxidative stress. Exercise
probably induces an increase in mitochondrial oxidant production as result of the increased oxygen availability. However,
recent publications on the production of ROS show that a small
increase in H2O2 is necessary for the activation of some
intracellular signalling pathways, responsible for the development of an adaptive response to exercise-induced oxidative
damage.28 29
Exercise induces an increase in the number of circulating
neutrophils related to the intensity of the physical activity. In
previous studies, we evidenced that an exhaustive exercise such
as a duathlon competition or a cycling mountain stage increases
the neutrophil counts about fourfold,30 31 while in another
study, following a flat cycling stage, neutrophil number only
increased twofold.32 Circulating neutrophils in the high-intensity group, but not in the low and medium-intensity groups,
seem primed for an oxidative burst after exercise, as is evidenced
by the progressive increase in the maximum luminol chemiluminescence. This response is similar to that obtained for
lymphocytes. The increased preactivation state of neutrophils
from the most intensely exercised group could contribute, at
least in part, to the instauration of oxidative stress.
3
@
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Table 3
Neutrophil counts and ROS production
Low intensity
Medium intensity
High intensity
6
Neutrophils (10 /ml)
Before
After
RLU/min/106 cells
Before
After
3.26 (0.21)
4.41 (0.44)*
1887 (120)
1765 (103)
3.16 (0.23)
5.06 (0.43)* {
1908 (116)
2208 (292)
3.37 (0.22)
6.17 (0.51)* {
1912 (126)
2680 (182)* {
Two-way ANOVA, p,0.05.
No significant differences were evidenced between basal values. *before vs after;
{differences with respect to the low post-exercise group; {differences with respect to
the medium post-exercise group.
Results expressed as mean (SEM), n = 6.
In conclusion, exercise affects the lymphocyte antioxidant
system response and induces cellular oxidative damage depending on its intensity. Intense exercise enhances lymphocyte and
neutrophil ROS production and produces lipid peroxidative
damage and lymphocyte ascorbate consumption, activating the
antioxidative response as evidenced by the increase in lymphocyte HO-1 expression. Intense exercise-induced oxidative stress
is probably related to a disturbance in redox homeostasis. Thus,
physical exercise provides an excellent model to study the
relationship between pro-oxidants and the antioxidant defenses
in healthy subjects.
Figure 1 Lymphocyte ROS production. Lymphocyte ROS production
without stimulation (A) and stimulated with PMA (B).Two-way ANOVA,
p,0.05. No significant differences were evidenced between basal
values. * before vs after; { differences with respect to low and medium
post-exercise groups; { differences with respect to the low post-exercise
group.Results expressed as mean (SEM), n = 6.
Deficiency of antioxidant nutrients appears to hamper
antioxidant systems and augment exercise-induced oxidative
stress and tissue damage.3 33 Vitamin C prevents initiation of
lipid peroxidation and spares other critical antioxidants including a-tocopherol and urate.34 Vitamin C is also an essential
metabolite for cell and tissue metabolism, especially for collagen
synthesis and for regulation of HIF-1a.35 36 Although vitamin C
may interact with ‘free’ active metal ions contributing to
oxidative damage,37 its relevance in vivo has been a matter of
controversy. Lymphocyte vitamin C levels decreased significantly only in the group which performed the most intense
exercise, probably because this group produced more ROS and
consequently more vitamin C was consumed. Vitamin E levels
were unchanged in the low-intensity group, but significantly
increased in the other two groups. These results are in
agreement with the increase observed in lymphocyte vitamin
E just after a half-marathon or after a mountain cycling
stage.28 38 It seems that lymphocyte vitamin E uptake is
activated by oxidative stress in order to protect the cell from
the action of ROS, according to the levels of oxidative stress.
When HO-1 gene expression was analysed, a significant
increase was evidenced in the medium and higher-intensity
groups. HO-1 is an antioxidant stress protein induced by
stressful and inflammatory stimuli.39 HO-1 is particularly
sensitive to acute exercise, being activated by the NF-kB
pathway. Several authors have evidenced an increase in HO-1
after a half-marathon39 or after a treadmill test until exhaustion,40 but not after a short run or eccentric exercise.39 Our
results suggest that HO-1 is activated depending on the degree
of ROS production.
4
Acknowledgements: This work was supported by a grant from the Spanish Ministry
of Science and Education (Projects DEP2005-00238-CO4-01 and DEP2005-00238-CO402/EQUI) and the FEDER funding.
Competing interests: None.
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Before
After
Vitamin E (mmol/l)
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After
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Br J Sports Med 2008;000:1–6. doi:10.1136/bjsm.2007.043943
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Module 1 British Journal of Sports Medicine 14/6/08 07:43:02
Original article
What is already known on this topic
Data studying the effects of soccer practice on the antioxidant
status of players are scarce. Several studies evidenced the
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exercise resulting in oxidative stress. However, these reactive
species also have a role in cell signalling, being important in the
adaptive response to exercise.
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What this study adds
The present manuscript shows that soccer induces oxidative
stress. This study evidences the importance of intensity and
duration on exercise-induced oxidative stress. The predominance
of ROS’ deleterious effects over their signalling role depends on
the intensity degree of exercise. Only very intense exercise
results in evident signals of oxidative damage and induces an
antioxidant response in lymphocytes.
Br J Sports Med 2008;000:1–6. doi:10.1136/bjsm.2007.043943
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sm43943
Topics:
Module 1 British Journal of Sports Medicine 14/6/08 07:43:03
Original article
Authors Queries
Journal: British Journal of Sports Medicine
Paper: sm43943
Title: Effects of exercise intensity on lymphocyte H2O2 production and antioxidant defences in soccer players
Dear Author
During the preparation of your manuscript for publication, the questions listed below have arisen. Please attend to these matters
and return this form with your proof. Many thanks for your assistance
6
Query
Reference
Query
Remarks
1
You have opted not to pay a fee to
make your article free online
(Unlocked). If you wish to change
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dtl
2
(Are results in table 1 expressed as
means ¡ SEM?
3
Technicon?
4
Are the figures for cell fraction and
viability mean ¡ SD?
5
Could lymphocyte volume
expressed as 2.161025?
6
I am not sure of the meaning of
‘‘instauration’’ here; could it be
replaced by, for example, ‘‘induction’’?
7
Should ‘‘diseades’’ be changed to
‘‘diseased’’ in ref. 4?
be
Br J Sports Med 2008;000:1–6. doi:10.1136/bjsm.2007.043943