Exercise-induced Quadriceps Oxidative Stress
and Peripheral Muscle Dysfunction in Patients
with Chronic Obstructive Pulmonary Disease
Annabelle Couillard, Francois Maltais, Didier Saey, Richard Debigare, Annie Michaud, Christelle Koechlin,
Pierre LeBlanc, and Christian Prefaut
UPRES-EA 701, Laboratory of Physiologie des Interactions, Service Central de Physiologie Clinique, Ho
Montpellier, France; and Centre de Recherche, Ho pital Laval, Institut Universitaire de Cardiologie et de Pneumologie de lUniversite Laval,
Sainte-Foy, Quebec, Canada
Exercise-induced muscle oxidative stress may be involved in the
myopathy associated with chronic obstructive pulmonary disease
(COPD). This study was designed to look at whether local exercise
induces muscle oxidative stress and whether this oxidative stress
may be associated with the reduced muscle endurance in patients
with COPD. Quadriceps endurance was measured in 12 patients
with COPD (FEV1 0.96 0.14 SEM) and 10 healthy sedentary
subjects by repeated knee extensions of the dominant leg. Biopsies
of the vastus lateralis muscle were obtained before and 48 hours
after exercise. Muscle oxidative stress was measured by lipid peroxi-
dation and oxidized proteins. Muscle antioxidant was evaluated by
peroxidase glutathion activity. Quadriceps endurance was signifi-
cantly reduced in patients with COPD when compared with the
healthy control subjects (p 0.01). Forty-eight hours postexercise,
only patients with COPD had a significant increase in muscle lipid
peroxidation (p 0.05) and oxidized proteins (p 0.05), whereas
increased peroxidase glutathion activity was only observed in con-
trol subjects (p 0.05). Both increases in muscle lipid peroxidation
and oxidized proteins were significantly and inversely correlated
with quadriceps endurance capacity in COPD (p 0.05). In sum-
mary, local exercise induced muscle oxidative stress in patients
with COPD, whereas it failed to raise antioxidant activity. In these
individuals, muscle oxidative stress was associated with a reduced
quadriceps endurance.
Keywords: malondialdehyde; skeletal muscle; glutathion peroxidase;
myopathy; chronic obstructive pulmonary disease
The peripheral muscle dysfunction observed in patients with
chronic obstructive pulmonary disease (COPD) is, in part,
characterized by a marked reduction in quadriceps endurance
(1, 2). Because this peripheral myopathy is associated with
reduced exercise capacity (3) and quality of life (4), the docu-
mentation of its underlying mechanisms is likely to be clini-
cally relevant.
Deconditioning related to progressive reduction of daily
(Received in original form September 11, 2002; accepted in final form March 25, 2003)
Supported in part by Canadian Institutes of Health Research grant MOP-53135.
A.C. was supported by a traveling grant from la Cooperation Franco-Quebecoise.
F.M. is a research scholar of the Fonds de la Recherche en Sante du Quebec. R.D.
and D.S. are recipients of a Ph.D. training award of Fonds de la Recherche en
Sante du Quebec.
Correspondence and requests for reprints should be addressed to Annabelle Couil-
lard, Laboratory of Physiologie des Interactions, Service Central de Physiologie
Clinique, Ho
pital Arnaud de Villeneuve, 34295 Montpellier cedex 5, France. E-mail:
annabelle_couillard@yahoo.fr
This article has an online supplement, which is accessible from this issues table
of contents online at www.atsjournals.org
Am J Respir Crit Care Med Vol 167. pp 16641669, 2003
Originally Published in Press as DOI: 10.1164/rccm.200209-1028OC on April 2, 2003
Internet address: www.atsjournals.org
pital Arnaud de Villeneuve,
activities is often quoted as being one of the main reasons why
patients with COPD have peripheral myopathy (5). Recent
studies, however, have suggested that other factors such as
exposure to systemic corticosteroids (6), malnutrition (7),
hypoxia (8), and apoptosis (9) may also contribute to the
alteration in peripheral muscle function. Oxidative stress,
resulting from an inability of the antioxidant systems to cope
with elevated oxidant production, is also believed to play
an important role in altering peripheral muscle function in
patients with COPD (10, 11). Indeed, our group has recently
documented evidences of lipid peroxidation, a marker of
oxidative stress, in the plasma of patients with COPD but
not in healthy subjects after local quadriceps exercise per-
formed to exhaustion (2). Although we hypothesized that
the contracting quadriceps was the source of this oxidative
stress, it was not confirmed because no muscle biopsies were
done. This is an important question because muscle oxidative
stress causes noticeable myocyte damage (12, 13) and may
potentially be detrimental to muscle function, thus contribut-
ing to muscle fatigue (14) and reduced endurance (13). On
the basis of this observation, we hypothesized that muscle
oxidative stress could be associated with reduced peripheral
muscle endurance in patients with COPD.
The objectives of the present study were therefore to deter-
mine (1) whether local muscle exercise performed to exhaus-
tion induces oxidative stress within the quadriceps itself and
(2 ) whether this oxidative stress is associated with reduced
quadriceps endurance capacity in patients with COPD.
Some of the results of these studies have been reported
previously in the form of an abstract (15).
METHODS
Study Population
This study involved 22 male subjects, with 12 subjects having COPD
as determined by moderate to severe irreversible airflow obstruction
(16) (FEV1 60% predicted and FEV1/FVC 70% and 10%
improvement in FEV1 after 2-agonist inhalation). These 12 patients
were all ex-smokers, had no resting hypoxemia, and were clinically stable
at the time of the evaluation. They had neither experienced respiratory
tract infection nor exacerbation of their disease for at least 4 weeks
before being studied. All were receiving inhaled anticholinergic and/
or 2 agonists, and some also received inhaled steroids. None was
treated with oral corticosteroids. To avoid potentially confounding fac-
tors, we excluded from the study subjects with known muscle disorders,
cardiac failure, diabetes mellitus, or alcoholism, as well as those with any
other comorbidity that could have impaired their capacity to exercise.
Ten age-matched nonsmoker healthy males with only sedentary activities
were recruited as control subjects through newspaper advertisements.
All participants had a body mass index smaller than 35 kg/m2, and all
were questioned on their dietary habits so that those taking antioxidants
or vitamin supplements could be excluded. None of the subjects involved
in a previous study on systemic oxidative stress and local muscle exercise
in COPD (2) participated in the present investigation. The study was
Couillard, Maltais, Saey, et al.: Muscle Oxidative Stress in COPD
approved by the institutional ethics committee, and written consent
was obtained after subjects had received a complete explanation of the
objectives of the study protocol.
Study Protocol
Pulmonary function test. All subjects underwent spirometry including
measurements of FVC and FEV1. The FEV1/FVC ratio was also calcu-
lated. The results of pulmonary function testing were related to the
normal values of Knudson and coworkers (17)
Physical activity. Levels of physical activity were assessed through
a physical activity questionnaire adapted for older retired adults (18).
This questionnaire provides a reliable and valid method for classifying
the activity level of older subjects as high, medium, or low with a score
of 9 or more indicating a low physical activity level, thus classifying the
subjects as being sedentary. Additional details on this measurement
are provided in the online supplement.
Midthigh muscle cross-sectional area measurement. A computed to-
mography of the dominant (the stronger one) thigh halfway between
the pubic symphisis and the inferior condyle of the femur was performed
using a fourth-generation Toshiba Scanner 900S (Toshiba Inc., Tokyo,
Japan) (19). Additional details on this measurement are provided in
the online supplement.
Quadriceps strength measurement. The quadriceps maximal volun-
tary strength was measured while subjects performed dynamic knee
extensions against a hydraulic resistance (HF STAR, Hydrafitness Total
Power; Henley Health Care, Belton, TX) that could be adjusted to six
different levels of resistance. Starting with the lowest level of resistance,
the subjects were asked to perform three sets of movements at 5-minute
intervals until they reached a level of resistance at which they could
no longer complete the full range of movement. Under these conditions,
the generated strength reached a plateau, defined as maximal voluntary
contraction. Additional details on this measurement are provided in
the online supplement.
Quadriceps endurance measurement. To assess the quadriceps en-
durance of the dominant leg, we used an exercise bench and followed
the method described by Serres and coworkers (1). Subjects were asked
to perform repeated knee extensions of the leg against weights corre-
sponding to 30% of their maximal voluntary capacity at a pace of six
movements per minute imposed by audio signals (metronome) until
exhaustion. The dynamic knee extension was performed for 3 seconds,
immediately followed by active leg return (eccentric flexion) against
resistance for 3 seconds, and by rest before the next extension. The
test was concluded when subjects could no longer perform maximal
extension or if they could not sustain, despite verbal encouragement,
the required frequency two consecutive times. The duration of the test
was then recorded as the quadriceps endurance time. The dyspnea
score was measured at rest and immediately after exercise on an ana-
logic visual scale ranging from 0 to 10.
Twitch quadriceps force assessment. To determine whether local
exercise induced contractile muscle fatigue, we quantified the force of
the dominant quadriceps during maximal magnetic stimulation of the
femoral nerve before and 10 minutes after exercise. This was done
using a commercially available magnetic stimulator (Magstim 200; Mag-
stim Co Ltd, Whitland, Wales, UK) equipped with a figure-of-eight
42-mm coil according to the method of Mador and coworkers (20).
Additional details on this method are available in the online supple-
ment.
Muscle biopsies. Percutaneous biopsy specimens of the vastus later-
alis muscle of the nondominant leg at baseline and of the dominant leg
48 hours after exercise were taken at midthigh (15 cm above the patella)
as described by Bergstrom (21). The biopsy specimens were first dis-
sected free of visible connective tissue and fat and the remaining muscle
tissue was immediately frozen in isopentane cooled to freezing point
with liquid nitrogen and stored at 80C until analysis.
Skeletal Muscle Data Analysis
Fiber typing determination. All muscle specimens were coded and ana-
lyzed without knowledge of the clinical data. Muscle samples were cut
into 10-m thick transverse sections in a cryostat at 20C. One of
these sections was stained for myofibrillar adenosine triphosphatase
activity according to the single-step ethanol-modified technique (22).
Depending on the staining intensity, fibers were labeled as being type
1665
I (nonstained), type IIa (lightly stained), or type IIx (darkly stained).
A small proportion of fibers stained intermediate between IIa and IIx
and they were classified as IIab, but because of their relative scarcity
they were not included in the analysis. Muscle sections were magnified
and transmitted to an image analysis software (Photoshop L5) for fiber
counting and classification. For each subject, the fiber-type composition
was calculated as the total number of fibers of a given type divided by
the total number of fibers.
Determination of markers of oxidative stress. Muscle thiobarbituric
acid reactive substances (TBARs) were used as markers of muscle lipid
peroxidation and were determined fluorimetrically using the method
described by Ohkawa and coworkers (23). The final results were ex-
pressed in nmol/g wet weight. The reproducibility of TBARs, calculated
as coefficients of variation, was less than 10%. In subjects in whom
sufficient amount of muscle tissue was still available (COPD, n 8;
control subjects, n 7), we also measured protein oxidation by evaluat-
ing the levels of protein carbonyls using immunoblotting (Oxyblot kit;
Serologicals Corporation, Norcross, GA). Muscle protein carbonyl con-
tents were calculated by adding the integrated density of individual
protein bands (Alpha Innotech Corporation, San Leandro, CA) ob-
tained by Western blot analysis (24). Additional details on this method
are available in the online supplement.
Determination of muscle antioxidant activity. Muscle activity of glu-
tathion peroxidase (GPX) was quantified spectrophotometrically ac-
cording to the method of Nakamura and coworkers (25). The reproduc-
ibility of GPX activity, calculated as coefficients of variation, was less
than 10%.
Venous Blood Analysis
Determination of muscle damage. Blood samples were drawn in heparin-
ized tubes, and plasma was obtained by centrifugation (2,500 rpm for
10 minutes at 4C) and stored at 80C until analysis. The plasma
activity of creatine kinase (CK) was determined at rest as well as 6 and
48 hours after exercise using biochemical assay kits.
Study Design
Subjects were instructed to abstain from strenuous physical activity 4
days before and 2 days after being studied. On Day 1, the subjects
underwent spirometry and a computed tomography of the thigh, and
they had to answer the physical activity questionnaire. After the first
venous blood sample was obtained at rest, strength was evaluated in
each leg and a baseline muscle biopsy was performed on the nondomi-
nant leg. On Day 2, the subjects were familiarized with the endurance
test procedures by performing five consecutive dynamic knee extensions
of the dominant leg. They then performed the local muscle endurance
exercise test. A second venous blood sample was obtained 6 hours after
the end of exercise. On Day 4 (48 hours after local exercise), a third
venous blood sample and a biopsy of the dominant quadriceps were
obtained. This second biopsy was taken 48 hours postexercise because
previous investigations have shown that peak increases in TBARs con-
tent and enzymatic antioxidant activity in muscle samples occur in this
time frame (26, 27).
Statistical Analysis
Results are expressed as mean SEM. According to the homogeneity
and normality of the data, comparisons between groups before and
after local exercise were performed using the Students test or the
MannWhitney test. Comparisons within groups were performed with
the Students paired t test or the Wilcoxon test. Possible correlations
between variables were evaluated using Spearman correlation coeffi-
cients. The results were considered statistically significant with p values
less than or equal to 0.05.
RESULTS
Subjects Characteristics
The anthropometric data did not show any significant differences
between patients with COPD and control subjects (Table 1).
Spirometric values showed that patients with COPD had on
average severe airflow obstruction (Table 1). All subjects had a
low level of physical activity (Table 1).
1666 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 167 2003

TABLE 1. CHARACTERISTICS OF THE STUDY POPULATION

Control Figure 1. Local quadriceps
COPD Subjects endurance exercise induced a
(n 12) (n 10) p Value significant increase in the mus-
Age, yr 68 2 64 2 NS cle levels of thiobarbituric acid
Height, cm 166 2 173 1 NS reactive substances (TBARs)
Weight, kg 74 4 82 4 NS in patients with chronic ob-
BMI, kg/m2 26.6 1.2 27.2 1.0 NS structive pulmonary disease
FEV1, L 0.96 0.13 3.20 0.18 0.001 (COPD) (black bars; p 0.05)
FEV1, % predicted 33 3 104 4 0.001 but not in the healthy subjects
FEV1/FVC, % 42 2 76 2 0.001
(white bars). *p value less than
Physical activity score 6 1 8 2 NS
0.05 compared with baseline
Definition of abbreviations: BMI body mass index; COPD chronic obstructive values.
pulmonary disease; NS nonsignificant difference between groups.
Values are expressed as mean SEM.

COPD before and after exercise are shown in Figure 2. Four to

Morphologic and Functional Muscle Characteristics
five bands whose molecular weight varied from 27 to 68 kD
In patients with COPD, there were fewer type I fibers, but a larg- were detected in patients with COPD and in control subjects.
er proportion of type II fibers than in control subjects (Table 2). The migration pattern of these bands was similar between the
Midthigh muscle cross-sectional area was considerably lesser in two groups. After quadriceps exercise, these bands became more
patients with COPD (Table 2). Peripheral muscle performance intense in patients with COPD, but no new bands were detected.
as determined by quadriceps strength also showed significant Quadriceps exercise induced a significant increase in muscle
intergroup difference (Table 2). A significant positive correlation TBARs (Figure 1) and in muscle protein carbonyl contents (Fig-
was found between midthigh muscle cross-sectional area and ure 2) in patients with COPD (p 0.05) but not in control
quadriceps strength (r2 0.88; p 0.05), but the strength/mid- subjects. The difference between postexercise and baseline mus-
thigh muscle cross-sectional area ratio did not significantly differ cle TBARs (Figure 4) and between postexercise and baseline
between patients with COPD and control subjects. Quadriceps muscle protein carbonyls significantly and inversely correlat-
endurance time was twofold lower in the patients with COPD ed with the quadriceps endurance time in patients with COPD
compared with control subjects (Table 2). Resting and post- (r 0.66, p 0.05 and r 0.70, p 0.05, respectively). The
exercise dyspnea scores varied from 0 to 2.6 0.1 and from 0 quadriceps endurance time did not significantly correlate with
to 2.7 0.2 in patients with COPD and healthy subjects, re- FEV1 or the physical activity score (r 0.27 and r 0.32,
spectively. Resting twitch quadriceps force/midthigh muscle cross- respectively, p 0.05). There was no significant correlation
sectional area ratio was not significantly different between both between the proportion of type II fibers and the increase in
groups. When compared with the baseline values, there was a muscle TBARs or muscle protein carbonyl contents.
25 7% and 27 8% decrease in twitch quadriceps force after
local muscle exercise in patients with COPD and healthy subjects,
respectively.
Figure 2. Representative im-
Oxidative Stress and Muscle Damages at Rest and after munoblots of muscle protein
Local Muscle Exercise carbonyl groups in a control
No difference between patients and control subjects was found subject and a patient with
in the resting levels of muscle lipid peroxidation (TBARs) COPD before (pre) and after
(Figure 1), protein carbonyl contents (Figure 2), or GPX activity (post) quadriceps exercise (A ).
(Figure 3). Representative immunoblots of muscle protein car- Four to five bands whose mo-
bonyl groups obtained in a control subject and in a patient with lecular weight varied from 27
to 68 kD were detected in both
subjects. Compared with the
preexercise levels, these bands
TABLE 2. HISTOLOGIC AND FUNCTIONAL CHARACTERISTICS became more intense after
OF THE QUADRICEPS quadriceps exercise in the pa-
tient with COPD, but no new
COPD (n 12) Control Subjects (n 10 ) bands were detected. Negative
% Fiber IIx 35 5 29 3
controls (underivatized protein)
% Fiber IIa 38 6 28 4 are also shown. As indicated by
% Fiber I 27 4* 43 5 the group mean data (B ), local
Fibers counted 329 33 318 64 quadriceps endurance exercise
MTCSA, mm2 7247 526 9425 408 induced a significant increase
MVC, kg 32 2 44 2 in the muscle protein carbonyl
Quadriceps endurance contents in patients with
time, s 378 72 843 177 COPD (black bars; n 8) but
Definition of abbreviations: COPD chronic obstructive pulmonary disease;
not in the healthy subjects
MTCSA midthigh cross-sectional area; MVC maximal quadriceps strength. (white bars; n 7). * p value
Values are expressed as mean SEM. less than 0.05 compared with
* p 0.05 compared with control subjects. baseline values. ID inte-

p 0.01 compared with control subjects. grated density.
Couillard, Maltais, Saey, et al.: Muscle Oxidative Stress in COPD
Exercise induced a significant increase in muscle GPX activity
only in the healthy subjects (p 0.05) in such a way that muscle
GPX activity was significantly different between both groups 48
hours after exercise (Figure 3).
Plasma CK levels were significantly increased 6 hours after
local exercise in both groups (from 133 36 IU/L to 169 34
in COPD, p 0.01 and from 126 20 to 181 28 in healthy
subjects, p 0.01). However, the increase in CK from baseline to
6 hours postexercise ( CK) only correlated with the quadriceps
endurance time in healthy subjects (r 0.83; p 0.01).
DISCUSSION
On the basis of previous studies that have shown increased
systemic oxidized glutathion and lipid peroxidation after exercise
in patients with COPD, several investigators have suggested that
this oxidative stress may originate, at least in part, within the
contracting muscles (2, 11, 28). With muscle biopsy, the current
study extends these results by demonstrating that local quadri-
ceps exercise of sufficient intensity to cause fatigue can induce
oxidative stress within the contracting quadriceps in patients
with COPD but not in healthy subjects. Furthermore, local exer-
cise failed to induce the expected physiologic increase in antioxi-
dant defenses, as assessed by GPX activity, in patients with
COPD, a possible explanation for the greater susceptibility to
muscle oxidative stress in these individuals. Another important
finding of the present study was the relationship between exer-
cise-induced oxidative stress and quadriceps endurance, support-
ing the concept that muscle oxidative stress may have important
functional consequences in patients with COPD.
Oxidative Stress and Antioxidant Defenses at Rest and after
Local Quadriceps Exercise
Like Rabinovich and coworkers (29), we were unable to docu-
ment elevated muscle lipid peroxidation in resting patients with
COPD. Those findings, however, contrast with those of a recent
study that showed greater accumulation of lipofuscin, a marker
of lipid peroxidation, in the vastus lateralis muscle of patients
with COPD when compared with healthy subjects (30). This
discrepancy may be explained by the different methods that were
used to assess lipid peroxidation; whereas TBARs are markers of
acute oxidative stress, lipofuscin, which accumulates in the cells,
is a reflector of cumulative stress. One may thus speculate that
TBARs muscle content returns to normal levels between re-
peated short courses of oxidative stress, provided the recupera-
tion period is adequate. On the other hand, repeated bursts of
oxidative stress are likely to generate long-term accumulation
of lipofuscin.
At rest, GPX activity was similar in both groups despite
Figure 3. Local quadriceps
endurance exercise induced
a significant increase in glu-
tathion peroxidase (GPX)
activity in the healthy sub-
jects but not in patients with
COPD. *p value less than
0.05 compared with base-
line values, p value less than
0.05 compared with values
of patients with COPD 48
hours after exercise.
1667
Figure 4. There was an inverse and significant correlation between quad-
riceps endurance time and exercise-induced increased muscle TBARs
in patients with COPD (r 0.66; p 0.05).
muscle histologic changes in favor of type II fiber in patients
with COPD. This result is somewhat surprising because type II
muscle fibers have lower levels of GPX activity when compared
with type I fiber (31). Thus, normal resting GPX activity in
COPD may reflect an adaptive mechanism in COPD to repeated
bursts of oxidant production (27). However, this possible adap-
tive mechanism seems to be insufficient as indicated by the lack
of increase in GPX activity after exercise.
The present study confirms the hypothesis (2) that local mus-
cle exercise performed until exhaustion can produce oxidative
stress within the contracting muscles. Conversely, Rabinovich
and colleagues (29) were unable to document such evidence
after whole body exercise in patients with COPD. This may be
explained by differences in patient characteristics and exercise
intensity, which in the Rabinovich study was probably insuffi-
cient to induce muscle oxidative stress. Another significant dif-
ference between the two studies is that our local exercise protocol
was purposely designed to minimize other possible sources of
oxidative stress (lungs, liver, etc.). When analyzed together, the
results of these two studies indicate that intensity and type of
exercise are important factors in the genesis of muscle oxidative
stress in patients with COPD.
Potential Mechanisms of Exercise-induced Oxidative Stress
Because the results of this study suggest that there is a role for
oxidative stress as a mediator of peripheral muscle dysfunction
in patients with COPD, it would be clinically relevant to try
to determine its underlying mechanisms. Exercise can lead to
oxidative stress either by increasing prooxidant activity or be-
cause of an inadequate antioxidant activity. Xanthine oxidase,
within the muscle or its capillary endothelium, can also be an
important source of oxidant production during exercise in pa-
tients with COPD in the presence of O2 and of adenosine triphos-
phate breakdown products, including inosine monophosphate.
In support of this hypothesis, Heunks and coworkers (11) were
recently able to demonstrate that exercise-induced increase in
lipid peroxides observed during whole body exercise in patients
with COPD can be prevented by pretreating the patients with
allopurinol, a potent xanthine oxidase inhibitor. The potential
role of this enzyme in generating oxidative stress has been further
substantiated by Pouw and coworkers (32) who reported, in
patients with COPD, elevated muscle inosine monophosphate
levels, as a precursor of hypoxanthine that serves as a substrate
for xanthine oxidase activity.
An inefficient mitochondrial handling of oxygen could also
contribute to an increase in oxidant production by the respiratory
chain. On the basis of studies that have shown, in the quadriceps
of patients with COPD, a low citric acid cycle and fatty acid
1668 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 167 2003
-oxidation enzyme activities (33) and also on the basis of other
studies that have demonstrated increased cytochrome oxidase
activity (8), an enzyme of the mitochondrial electron transport
chain, it can be speculated that mitochondrial uncoupling may
also enhance oxidant production during exercise in COPD.
The possibility that increased muscle lipid peroxidation and
protein oxidation observed 48 hours after exercise in patients
with COPD could be related to exercise-induced inflammatory
response cannot be excluded. Muscle trauma during exercise
can cause injury, which may lead to a prolonged inflammation
response, and accumulation of neutrophils and macrophages
within the capillary bed of damaged muscle tissues (34). It is
therefore possible that those inflammatory cells, which are capa-
ble of producing free oxygen radicals, may contribute to the
increased muscle lipid peroxidation observed after exercise in
patients with COPD (35). We, however, believe that postexercise
muscle inflammation is unlikely to be the main mechanism of
oxidative stress based on the modest rise in CK increase and on
the fact that no patient reported muscle pain 48 hours postexer-
cise, indicating only mild muscle damage. In previous experi-
ments done in our laboratory, we were also unable to show any
evidence muscle inflammatory cell infiltration after local quad-
riceps exercise in patients with COPD (Maltais and Debigare,
unpublished observation).
There is increasing evidence that oxidant production may be
used by cells to signal an adaptive enzymatic antioxidant re-
sponse. Indeed, a recent report (27) suggested that exercise-
induced oxidant production serves to raise enzymatic antioxidant
responses. In the current study, the finding of a significant postex-
ercise increase in GPX activity in healthy subjects supports these
observations. In contrast, muscle GPX activity did not increase
appropriately in response to the increase in oxidant production in
patients with COPD. A depletion in muscle glutathion, a substrate
required for GPX catalyzed reactions, has been reported pre-
viously in patients with COPD (36) and is a possible explanation
for this observation. Other important antioxidant systems such as
vitamin E blood levels may also be deficient in patients with
COPD (2). In summary, the available information suggests that
the increased susceptibility to muscle oxidative stress in COPD is
due to an imbalance between oxidant production and antioxidant
defenses in favor of the former.
Peripheral Muscle Dysfunction in Patients with COPD
Because subjects in both groups had similar levels of physical
activity, our results suggest that in COPD, factors other than
inactivity may be involved in the development of peripheral mus-
cle dysfunction. In this context, there is increasing evidence that
suggests a role for oxidative stress as a mediator of peripheral
muscle dysfunction in patients with COPD. In the present study,
inverse and significant relationships were found between mark-
ers of exercise-induced muscle oxidative stress and quadriceps
endurance. These findings support the role of cytotoxic oxidant
and oxidative stress in the development of muscle fatigue and
altered endurance capacity in patients with COPD. Indeed, mus-
cle oxidative stress causes noticeable damage to myocytes organ-
elles such as DNA, proteins, and lipids, resulting in excessive
rise in intracellular free calcium, mitochondrial dysfunction, and
bioenergetic enzymes downregulation (12, 13). Additional inves-
tigations evaluating, for example, the effects of antioxidants on
peripheral muscle function are required to further demonstrate
the specific role of muscle oxidative stress in mediating periph-
eral myopathy in patients with COPD.
The release of CK in the extracellular environment is influ-
enced by various factors including magnitude of mechanical cell
injury, increase in body temperature, content of high-energy
substrate within the cells, and duration of exercise (37). Hence,
the positive and significant relationship observed between exer-
cise-induced CK release and quadriceps endurance time in the
control subjects was not unexpected. However, we were surprised
to find similar exercise-induced CK increases in both groups, de-
spite a much shorter duration of exercise in patients with COPD.
This probably indicates that other mechanisms may have initiated
the rise in CK in patients with COPD. Given that products of
peroxidation decrease membrane fluidity, increase membrane fra-
gility and, therefore, susceptibility to rupture (38), the increase in
peroxidative damage observed after local exercise could have
contributed to the CK release.
Methodologic Considerations
Physical activity. A specific physical activity questionnaire adapted
for older retired adults able to carry on usual daily activities was
selected for this study. Although this questionnaire does not
provide a direct quantification of the number and intensity of
daily activities, it has been validated against other activity mea-
sures such as 24-hour activity self-reporting and pedometer
scores (18). We are therefore confident that both groups had
similar low levels of physical activity.
Quadriceps endurance. To study local muscle endurance, we
used a protocol similar to the one described by Andersen and
coworkers (39). This protocol minimizes the ventilatory response
and provides highly reproducible values for muscle endurance
(40), whereas electromyographic recordings confirm that the
quadriceps is the main active muscle during the local exercise.
The increase in dyspnea and presumably in ventilation after local
exercise was of small amplitude in this study, and we, therefore,
assume that quadriceps exhaustion was the main factor limiting
exercise. The 25% decrease in quadriceps twitch force 10 minutes
postexercise also supports this contention. Interestingly, the reduc-
tions in quadriceps twitch force observed after local exercise were
of greater magnitude than those reported by Mador and coworkers
(20) after whole body cycling exercise, suggesting that a greater
degree of muscle fatigue may be reached after local exercise than
after whole body exercise.
Muscle Markers of Oxidative Stress
Tissue lipid peroxidation measurement (TBARs), which is an
indicator of reactive oxygen species molecular reactions, was
used to assess global oxidative stress. One potential limitation
of TBARs is that under oxidative stress conditions, malondialde-
hyde, hydroperoxides, and some carbohydrates and amino acids
may yield products that are able to react with thiobarbituric
acid (41). To circumvent this limitation, the oxidation of muscle
protein was also evaluated. The fact that a similar conclusion
about oxidative stress was reached with both methods is reassur-
ing in terms of the validity of our findings.
In summary, this study shows that exhaustive local exercise
may induce oxidative stress in the quadriceps of patients with
COPD and that the expected increase in antioxidant defenses
after exercise is compromised in these individuals. From a clinical
perspective, this oxidant/antioxidant imbalance may contribute
to the reduction of muscle endurance. Further studies are needed
to determine more precisely the mechanisms involved in the
exercise-induced muscle oxidative stress in COPD and to confirm
its implication in the myopathy of these patients.
Acknowledgment : The authors thank Marthe Belanger, Marie-Josee Breton,
Brigitte Jean, and Catherine Scott-Carmeni for valuable technical assistance and
Dr. Jean Deslauriers for editing the manuscript.
References
1. Serres I, Gauthier V, Varray A, Prefaut C. Impaired skeletal muscle
endurance related to physical inactivity and altered lung function in
COPD patients. Chest 1998;113:900905.
Couillard, Maltais, Saey, et al.: Muscle Oxidative Stress in COPD
2. Couillard A, Koechlin C, Cristol JP, Varray A, Prefaut C. Evidence of
local exercise-induced systemic oxidative stress in chronic obstructive
pulmonary disease patients. Eur Respir J 2002;20:11231129.
3. Gosselink R, Troosters T, Decramer M. Peripheral muscle weakness
contributes to exercise limitation in COPD. Am J Respir Crit Care
Med 1996;153:976980.
4. Oga T, Nishimura K, Tsukino M, Hajiro T, Ikeda A, Mishima M. Rela-
tionship between different indices of exercise capacity and clinical
measures in patients with chronic obstructive pulmonary disease. Heart
Lung 2002;31:374381.
5. Young A. Rehabilitation of patients with pulmonary disease. Ann Acad
Med Singapore 1983;12:410416.
6. Decramer M, Lacquet LM, Fagard R, Rogiers P. Corticosteroids contrib-
ute to muscle weakness in chronic airflow obstruction. Am Rev Respir
Dis 1994;150:1116.
7. Engelen MPKJ, Schols AMWJ, Baken WC, Wesseling GJ, Wouters EFM.
Nutritional depletion in relation to respiratory and peripheral skeletal
muscle function in out-patients with COPD. Eur Respir J 1994;7:1793
1797.
8. Sauleda J, Garca-Palmer F, Wiesner RJ, Tarraga S, Harting I, Tomas
P, Gomez C, Saus C, Palou A, Agust AGN. Cytochrome oxidase
activity and mitochondrial gene expression in skeletal muscle of pa-
tients with chronic obstructive pulmonary disease. Am J Respir Crit
Care Med 1998;157:14131417.
9. Agusti AG, Sauleda J, Miralles C, Gomez C, Togores B, Sala E, Batle
S, Busquets X. Skeletal muscle apoptosis and weight loss in chronic
obstructive pulmonary disease. Am J Respir Crit Care Med 2002;166:
485489.
10. Reid M. COPD as a muscle disease. Am J Respir Crit Care Med 2001;164:
11011102.
11. Heunks LM, Vina J, van Herwaarden CLA, Folgering HTM, Gimeno
A, Dekhuijzen PNR. Xanthine oxidase is involved in exercise-induced
oxidative stress in chronic obstructive pulmonary disease. Am J Physiol
1999;277:R1697R1704.
12. Davies K, Quintanihla A, Brooks G, Packer L. Free radicals and tissue
damage produced by exercise. Biochem Biophys Res Commun 1982;
107:11981205.
13. Travaline JM, Sudarshan S, Roy BG, Cordova F, Leyenson V, Criner
GJ. Effects of N-acetylcystein on human diaphragm strength and fati-
guability. Am J Respir Crit Care Med 1997;156:15671571.
14. Reid MB, Stokic DS, Koch SM, Khawli FA, Leis AA. N-acetylcysteine
inhibits muscle fatigue in humans. J Clin Invest 1994;94:24682474.
15. Couillard A, Debigare R, Saey D, Michaud A, LeBlanc P, Prefaut C,
Maltais F. Increased susceptibility of the quadriceps to oxidative stress
in patients with COPD [abstract]. Am J Respir Crit Care Med 2002;165:
A505.
16. American Thoracic Society. Standards for the diagnosis and care of
patients with chronic obstructive pulmonary disease. Am J Respir Crit
Care Med 1995;152:S77S120.
17. Knudson RJ, Slatin RC, Lebowitz MD, Burrows B. The maximal expir-
atory flow-volume curve: normal standards, variability and effects of
age. Am Rev Respir Dis 1976;113:587600.
18. Voorips LE, Ravelli ACJ, Dongelmans PCA. A physical activity ques-
tionnaire for the elderly. Med Sci Sports Exerc 1991;8:974979.
19. Bernard S, Leblanc P, Whittom F, Carrier G, Jobin J, Belleau R, Maltais
F. Peripheral muscle weakness in patients with chronic obstructive
pulmonary disease. Am J Respir Crit Care Med 1998;158:629634.
20. Mador MJ, Kufel TJ, Pineda L. Quadriceps fatigue following cycle exer-
cise in patients with COPD. Am J Respir Crit Care Med 2000;161:447
453.
21. Bergstrom J. Muscle electrolytes in man: determination by neutron acti-
vation analysis on needle biopsy specimens: a study on normal subjects,
kidney patients and patients with chronic diarrhoea. Scand J Clin Lab
Invest 1962;14:1110.
1669
22. Mabuchi K, Sreter FA. Actomyosin ATPase: quantitative measurement
of activity in cryostat sections. Muscle Nerve 1980;3:227232.
23. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animals tissues
by thiobarbituric acid reaction. Anal Biochem 1979;95:351358.
24. Barreiro E, Comtois A, Mohammed S, Lands L, Hussain S. Role of heme
oxygenase in sepsis-induced diaphragmatic contractile dysfunction and
oxidative stress. Am J Physiol Lung Cell Mol Physiol 2002;283:L476
L484.
25. Nakamura W, Hosoda S, Hayashi K. Purification and properties of rat
liver glutathione peroxidase. Biochim Biophys Acta 1974;358:251261.
26. Salminen A, Vihko V. Lipid peroxidation in exercise myopathy. Exp
Mol Pathol 1983;38:380388.
27. Khassaf M, Child RB, McArdle A, Brodie DA, Esanu C, Jackson MJ.
Time course of responses of human skeletal muscle to oxidative stress
induced by nondamaging exercise. J Appl Physiol 2001;90:10311035.
28. Vina J, Servera E, Asensi M, Sastre J, Pallardo FV, Ferrero JA, Garca-
De-La-Asuncion J, Anton V, Marn J. Exercise causes blood glutathi-
one oxidation in chronic obstructive pulmonary disease: prevention
by O2 therapy. J Appl Physiol 1996;81:21992202.
29. Rabinovich RA, Ardite E, Troosters T, Carbo N, Alonso J, Gonzalez
de Suso JM, Vilaro J, Barbera JA, Polo MF, Argiles JM, et al. Reduced
muscle redox capacity after endurance training in patients with chronic
obstructive pulmonary disease. Am J Respir Crit Care Med 2001;164:
11141118.
30. Allaire J, Maltais F, Leblanc P, Simard PM, Whittom F, Doyon JF,
Simard C, Jobin J. Lipofuscin accumulation in the vastus lateralis
muscle in patients with chronic obstructive pulmonary disease. Muscle
Nerve 2002;25:383389.
31. Ji LL, Fu R, Mitchell EW. Glutathione and antioxidant enzymes in
skeletal muscle: effects of fiber type and exercise intensity. J Appl
Physiol 1992;73:18541859.
32. Pouw EM, Schols AMWJ, van der Vusse GJ, Wouters EFM. Elevated
inosine monophosphate levels in resting muscle of patients with stable
chronic obstructive pulmonary disease. Am J Respir Crit Care Med
1998;157:453457.
33. Maltais F, Simard AA, Simard C, Jobin J, Desgagnes P, Leblanc P.
Oxidative capacity of the skeletal muscle and lactic acid kinetics during
exercise in normal subjects and in patients with COPD. Am J Respir
Crit Care Med 1996;153:288293.
34. Jones D, Newham D, Round J, Tolfree S. Experimental human muscle
damage: morphological changes in relation to other indices of damage.
J Physiol 1986;375:435448.
35. Smith J, Grisham M, Granger D, Korthuis R. Free radical defense mecha-
nism and neutrophil infiltration in postischemic skeletal muscle. Am
J Physiol 1989;25:789793.
36. Engelen MPKJ, Schols AMWJ, Does JD, Deutz NEP, Wouters EFM.
Altered glutamate metabolism is associated with reduced muscle gluta-
thione levels in patients with emphysema. Am J Respir Crit Care Med
2000;161:98103.
37. Kanter MM, Lesmes GR, Kaminsky LA, Ham-Saeger J, Nequin ND.
Serum creatine kinase and lactate dehydrogenase changes following
an eighty kilometer race: relationship to lipid peroxidation. Eur J Appl
Physiol Occup Physiol 1988;57:6063.
38. Rajguru S, Yeargans G, Seidler N. Exercise causes oxidative damage to
rat skeletal muscle microsomes while increasing cellular sulphydryls.
Life Sci 1991;80:611618.
39. Andersen P, Adams RP, Sjogaard G, Thorboe A, Saltin B. Dynamic
knee extension as model for study of isolated exercising muscle in
humans. J Appl Physiol 1985;59:16471653.
40. Serres I, Varray A, Vallet G, Micallef JP, Prefaut C. Improved skeletal
muscle performance after individualized exercise training in patients
with chronic obstructive pulmonary disease. J Cardiopulm Rehabil 1997;
17:232238.
41. Halliwell B, Chirico S. Lipid peroxidation: its mechanism, measurement,
and significance. Am J Clin Nutr 1993;57:715724.