Exercise-Induced Changes on Lipid
Peroxides and Antioxidant Enzymes
Levels Changes in Plasma of Show
Jumping and Dressage Horses
Barbara Muñoz-Escassi, DVM1
Gonzalo Marañón, DVM, PhD1
William Manley, DVM, PhD1
Mercedes Sánchez de la Muela, DVM, PhD2
Cristina Riber, DVM, PhD3
Patricia Cayado, DVM1
Rosa León, DVM1
Cruz Garcia, PharmD, PhD4
Maria Suárez, DVM2
Elena Vara, BD, PhD4
Horsepital SL
Madrid, Spain
2
Department of Animal Medicine and Surgery
School of Veterinary
Complutense University
Madrid, Spain
3
Department of Animal Medicine and Surgery
School of Veterinary
Cordoba, Spain
4
Department of Biochemistry and Molecular Biology
School of Medicine
Complutense University
Madrid, Spain
1
KEY WORDS: oxidative stress, exercise,
horse
ABSTRACT
The objective of this study was to evaluate
changes in oxidant and antioxidant parameters in plasma of horses competing in both
jumping and dressage contests. In this
study, 57 horses regularly involved in competition were included. Blood samples were
taken at 3 time points: baseline at rest, upon
reaching the schooling area but before exercise, and post-performance over a jump or
dressage course. Fourteen healthy horses
274
were considered as controls. Exercise
induced an increase in lipid hydroperoxide
(LPO) concentration in the 2 levels of horse
jumping-show. We also observed an exercise-induced increase of LPO in dressage
horse, although the effect was less apparent.
By contrary, exercise decreased glutathione,
glutathione peroxidase, and glutathione
transferase levels. In conclusion, exerciseinduced oxidative stress is linked with
increased lipid peroxidation.
INTRODUCTION
Exercise is known to exert numerous physiologIntern J Appl Res Vet Med • Vol. 4, No. 3, 2006.
ical changes in vital organ system of the body.
Among those changes, the most important is the
enhanced respiration and utilization of oxygen
in the body. Recently, much evidence has accumulating indicating that exhaustive exercise
generates free radicals.1-3 Abnormal production
of free radicals leads to damage of some macromolecules, including proteins, lipids, and nucleic
acids,2,4-6 and this is believed to be involved in
the etiology of many diseases.1,7 Under normal
conditions, excessive formation of free radicals
and concomitant damage at cellular and tissue
concentrations is controlled by cellular defense
systems. These preventive defense systems can
be accomplished by enzymatic or non-enzymatic mechanisms, including vitamin E, vitamin C,
and glutathione. The antioxidant enzymes such
as glutathione peroxidase (GPx) and glutathione-S-transferase (GST) may also have an
important function in mitigating the toxic effects
of reactive oxygen species.8
Tissue glutathione plays a central role in
antioxidant defense.9,10 Reduced glutathione
detoxifies reactive oxygen species, such as
hydrogen peroxide, and lipid peroxides
directly or in a GPx-catalyzed mechanism.
Glutathione also regenerates the major
aqueous and lipid phase antioxidants, ascorbate and α-tocopherol. Glutathione-S-transferase catalyzes the reaction between the
-SH group and potential alkylating agents,
rendering them more water soluble and
suitable for transport out of the cell.
Glutathione transferase can also use peroxides as a substrate.11
Although the exact mechanism for the
exercise-induced cell and tissue damage is
still elusive, there is increasing evidence
that during strenuous exercise, the oxidation
rate increases about 30-fold in horses. Thus,
the exercising horse is a likely model to
observe the balance between pro-oxidant
assault and antioxidant defense.
In this study, we evaluated changes in
oxidant and antioxidant parameters in plasma of horses competing in both jumping
and dressage contests.
MATERIALS AND METHODS
Fifty seven horses between the ages of 5 to
12 years were sampled from 3 levels of
horse show experience: moderate experience, intermediate experience: and most
experimented. Fourteen healthy horses not
involved in competition were used as control group.
Blood samples were collected from the
jugular vein of each horse into EDTA tubes
at 3 time points: baseline at rest, upon reaching the schooling but before exercise, and
post-performance over a jump or dressage
course. After sampling, plasma was separated and stored at -80ºC until determinations
were performed. Lactate concentration and
lactate dehydrogenase (LDH) activity were
measured spectrophotometrically.
Activities of GPx and GST, and lipid
hydroperoxide (LPO) levels were determined using commercially available kits
(Cayman Chemical Company, Ann Arbor,
Michigan, USA).
Reproducibility within the assays was
evaluated in 3 independent experiments.
Each assay was carried out with 3 replicates. The overall intra-assay coefficient of
variation has been calculated to be <5%.
Assay-to-assay reproducibility was evaluated in 3 independent experiments. The overall inter-assay coefficient of variation has
been calculated to be <6%.
Intern J Appl Res Vet Med • Vol. 4, No. 3, 2006.
275
Statistical Analysis
Results are expressed as the mean ±
SEM. Mean comparison was done by the
Kuskal-Wallis test followed by a Mann
Whitney test; a confidence level of 95%
(P < 0.05) was considered significant.
RESULTS
Baseline LPO (Figure 1A) and GPx plasma
activity (Figure 1B) from both jumping and
dressage horses did not differ from control
horses, while both jumping and dressage
horses show lower GST plasma levels than
the control group (Figure 1C).
Competition induced a significant
decrease in GPx levels in plasma at the 3
Figure 1. Baseline LPO (A) and GPx plasma
activities (B) from both jumping and dressage
horses.
20
Figure 2. Effect of competition on GPx activity in plasma at the 3 levels of jumping horses
studied: moderate, intermediate, and most
experienced horses.
control (n=14)
jumping (n=36)
dressage (n=21)
Moderate experience (n=12)
basal
prior comp.
after comp.
50
GPx (Ui/L)
40
10
30
p<0.001
20
5
GPx (Ui/L)
LPO (nmol/ml)
LPO (nmol/ml)
15
0
10
0
50
control
jumping (n=36)
dressage (n=21)
Intermediate experience (n=12)
40
GPx (Ui/L)
30
20
GPx (Ui/L)
GPx (Ui/L)
GPx (Ui/L)
basal
prior comp.
after comp.
50
40
10
30
p<0.001
20
10
0
0
500
Most experienced (n=12)
control
jumping (n=36)
dressage (n=21)
basal
prior comp.
after comp.
50
40
300
100
GPx (Ui/L)
GST (Ui/L)
200
GPx (Ui/L)
GST (Ui/L)
400
30
p<0.001
20
10
0
0
levels of horse jumping-show studied: moderate, intermediate, and most experienced
horses (Figure 2). The exercise-induced
effect was more apparent in dressage horses
(Figure 3).
Plasma GST activity followed a pattern
similar to that of GPx. After competition,
GST activity was reduced in sport horses
compared with baseline and before competition levels, both in jumping (Figure 4) and
dressage (Figure 5) horses.
As shown in Figure 4, competition
induced a dramatically decrease of GST levels in jumping horses with moderate experience. Although less marked, the
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Intern J Appl Res Vet Med • Vol. 4, No. 3, 2006.
Figure 3. Effect of competition on GPx activity in plasma at the 3 levels of dressage horses
studied: moderate, intermediate, and most
experienced horses.
Figure 4. Effect of competition on GST activity
in plasma at the 3 levels of jumping horses
studied: moderate, intermediate, and most
experienced horses.
Moderate experience (n=12)
Moderate experience (n=7)
basal
prior comp.
after comp.
50
basal
prior comp.
after comp.
300
250
GST (Ui/L)
GPx (Ui/L)
40
30
20
200
p<0.05
150
100
GST (Ui/L)
GPx (Ui/L)
p<0.001
10
50
p<0.001
0
0
Intermediate experience (n=12)
Intermediate experience (n=7)
basal
prior comp.
after comp.
50
250
GST (Ui/L)
40
GPx (Ui/L)
basal
prior comp.
after comp.
300
30
GST(Ui/L)
GPx (Ui/L)
20
p<0.001
10
0
200
150
p<0.001
100
50
0
Most experienced (n=7)
Most experienced (n=12)
basal
prior comp.
after comp.
50
basal
prior comp.
after comp.
300
250
40
p<0.001
10
0
GST (Ui/L)
20
GST(Ui/L)
GPx (Ui/L)
GPx (Ui/L)
200
30
p<0.001
150
100
50
0
exercise-induced GST decrease was also
observed in horses with intermediate experience, and most experienced horses. In dressage horses, this competition effect was
even more apparent (Figure 5)
The decrease in antioxidant enzymes
was accompanied by a competition-induced
significant increase in LPO concentration in
plasma at the 3 levels of horse jumpingshow studied: moderate, intermediate, and
most experienced horses (Figure 6). We also
observed a competition-induced increase of
LPO in dressage horses, although the effect
was less apparent (Figure 7).
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277
Figure 5. Effect of competition on GST activity
in plasma at the 3 levels of dressage horses
studied: moderate, intermediate, and most
experienced horses.
Figure 6. Effect of competition on LPO plasma concentration activity at the 3 levels of
jumping horses studied: moderate, intermediate, and most experienced horses.
Moderate experience (n=12)
Moderate experience (n=7)
basal
prior comp.
after comp.
300
basal
prior comp.
after comp.
20
p<0.001
LPO (nmol/ml)
GST (Ui/L)
250
200
150
LPO (nmol/ml)
100
GST (Ui/L)
50
p<0.001
15
10
5
0
0
Intermediate experience (n=12)
Intermediate experience (n=7)
basal
prior comp.
after comp.
300
p<0.001
20
basal
prior comp.
after comp.
LPO (nmol/ml)
GST (Ui/L)
250
200
150
LPO (nmol/ml)
GST(Ui/L)
100
p<0.001
50
15
10
5
0
0
Most experienced (n=7)
Most experienced (n=12)
basal
prior comp.
after comp.
300
20
p<0.001
basal
prior comp.
after comp.
250
LPO (nmol/ml)
15
150
100
50
p<0.001
0
Competition also increases plasma concentrations of total protein, lactate, and
LDH activity (data not shown).
LPO (nmol/ml)
GST(Ui/L)
GST (Ui/L)
200
10
5
0
DISCUSSION
There is growing evidence to support the
theory that increased oxygen consumption
during exercise increases free radical generation creating oxidative stress.2,7,12-16 Free
radicals may play important roles within
the body. They can act as chemical messengers and are also produced by phagocytes
during the respiratory burst. Free radical
formation can also increase under condi-
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tions of altered metabolism, ischemia-reperfusion, and exercise.
Free radicals and oxidative stress have
been implicated in the pathogenesis of different diseases, including exercise-induced
cells and tissue damage.17 The role of reactive oxygen species (ROS) in the exercise
process is supported by many studies.2,12,18-20
Production of ROS has been found to
increase with exercise, thus augmenting the
amount of oxidative damage induced to
lipids, proteins, and DNA.21 This in turn
may lead to dysfunction and inflammation.
In horses, oxidative stress has been
shown to occur in some horses as a result of
endurance exercise, in racehorses over several months of training, and as a result of
exercise in several stressful environmental
conditions. In the present study, we investigated the effect of jumping and dressage
competition on free radical generation in
sport horses.
Lipid peroxidation may be due to the
oxidation of molecular oxygen to produce
superoxide radicals. This reaction is also the
source of H2O2, initiating the peroxidation
of unsaturated fatty acids in the membrane.
Both H2O2 and O2- produced highly reactive
hydroxyl radical with Haber-Weiss reaction.
The hydroxyl radical can initiate lipid peroxidation, which is a free radical chain leading to loss of membrane structure and
function.22,23
In this study, the rate of lipid peroxidation, measured in the form of lipid peroxides, increased in the plasma after
competition, suggesting exercise-induced
oxidant stress. This is also supported by the
simultaneous decrease in antioxidant
enzymes, GPx and GST, which is in accordance with earlier reports of other authors.7
Some works, however, have shown either
an increase or no change in antioxidant
enzymes activity after exercise.17
The aim of this study was to examine
the effect of competition, and the control
horses involved in the study were welltrained horses not participating in competitions.
In healthy individuals, there is balance
between free radical generation and removal
by antioxidants. The antioxidant defense is
made up of both enzymatic and non-enzymatic parts, and changes in antioxidant
enzyme activities in erythrocytes have been
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279
Figure 7. Effect of competition on LPO plasma concentration activity at the 3 levels of
dressage horses studied: moderate, intermediate, and most experienced horses
Moderate experience (n=7)
basal
prior comp.
after comp.
20
LPO (nmol/ml)
15
p<0.001
10
LPO (nmol/ml)
5
0
Intermediate experience (n=7)
basal
prior comp.
after comp.
LPO (nmol/ml)
20
15
p<0.001
10
LPO (nmol/ml)
5
0
Most experienced (n=7)
basal
prior comp.
after comp.
20
LPO (nmol/ml)
15
p<0.001
10
LPO (nmol/ml)
5
0
used to document oxidative stress; between
them, GPx and GST have been widely used.
An increase in oxygen consumption during exercise activates the enzyme GPx to
remove hydrogen peroxide and organic
hydroperoxides from the cell.24 Glutathione
peroxidase is located in both the mitochondria and the cytosol where it serves as an
important cellular protectant against free
radical-induced damage to membrane lipids,
proteins, and nucleic acids.25 During normal
function of the antioxidant defense system,
reduced glutathione is used by GPx to
detoxify hydrogen peroxide. The tendency
for GPx activity to decrease after competition may denote the effect of competition
inducing oxidative stress on horses.
Furthermore, as has been previously indicated, the observed increase of LPO plasma
levels in sport horses after competition is an
indicator of competition-induced lipid peroxidation.
As has been previously mentioned, free
radicals have been implicated as mediators
of exercise-induced cellular dysfunction. On
the other hand, it is accepted that mitochondrial production of oxygen-derived radicals
could be increased during exercise and this
fact could be a major mechanism for the
exercise-related increase in oxidative damage. Our finding of increased plasma LPO
levels after competition support the hypothesis of augmented oxidative stress and damage with exercise, and it is in accordance
with previous studies from other groups.19
In conclusion, competition produced
evidence of lipid peroxidation and changes
in antioxidant enzymes. Competitioninduced oxidative stress is linked with
increased lipid peroxidation. These observations provide evidence that competition
increases oxygen consumption and causes a
disturbance of intracellular pro-oxidantantioxidant homeostasis.
ACKNOWLEDGEMENTS
We would like to thank the Real Federación
Hípica Española for its interest and support.
We wish to thank the Spanish riders for
280
their kind cooperation. Special thanks to
Rutherford Latham, Andrea Rueda, Alfonso
Arango, Asis Arango, and Susana GarcíaCereceda for their continued interest and
cooperation in our work. The skilful technical assistance of Ana García-Quiralte is
gratefully acknowledged.
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