Antioxidant status and lipid peroxidation after short-term

maximal exercise in trained and untrained humans

NIELS IZIRTENBLAD,l KLAVS MADSEN,’ AND MOGENS S. DJURHUUS2
‘Department of Phy sical Education, Odense University, 5230 Odense M; and
2Department of Clinical Chemistry, Odense University Hospital, 5000 Odense C, Denmark

Q)rtenblad, Niels, Klavs Madsen, and Mogens S. tathione (GSH) and vitamins] as well as enzymatic
Djurhuus. Antioxidant status and lipid peroxidation after defense systems [e.g., superoxide dismutase (SOD),
short-term maximal exercise in trained and untrained hu- catalase (Cat), glutathione peroxidase (GPX), and gluta-
mans. Am. J. Physiol. 272 (Regulatory Integrative Comp. thione reductase (GR)]. It is believed that the antioxida-
Physiol. 41): Rl258-Rl263,1997.-The purpose of this study tive defense system is in a dynamic equilibrium, so that
was to measure resting muscle and blood antioxidant status a decrease in one of the factors will to a certain extent
in untrained (n = 8) and jump-trained (n = 8) humans and to
evaluate free radical-mediated muscle damage after a strenu- be compensated through increased capacity of other
ous jump test consisting of six bouts of 30-s continuous antioxidants. This implies that evaluating the capacity
jumping separated by 2 min of rest. Resting muscle antioxi- of the antioxidative defense system requires determina-
dant activities [superoxide dismutase (SOD), glutathione tion of the whole antioxidant status at the time.
peroxidase (GPX), glutathione reductase (GR), and manga- Skeletal muscle soreness and increased membrane
nese SOD] were significantly higher in jump-trained com- permeability after strenuous exercise are known to be
pared with untrained subjects. Blood antioxidant enzyme reduced by training (3). Training increases the antioxi-
activities and muscle catalase, however, were not different dant activity, thereby protecting skeletal muscle from
between the two groups. Creatine kinase activities increased radical-induced damage after exhaustive exercise (1,2,
significantly (P < 0.0001) after the jump test in untrained 15). Most studies have been done using long-term
individuals, but remained unchanged in the jump trained. submaximal exercise, but it is possible that supermaxi-
Plasma and muscle malonaldehyde (MDA) after the jump test ma1 exercise has a larger radical-producing and damag-
were not significantly different from rest. These data suggest
ing potential. This is supported by observations of
that jump training is associated with elevated activities of
Alessio and Goldfarb (1) and Salminen and Vihko (25),
SOD and the coupled enzymes GPX and GR in muscle tissue,
but other antioxidants remain unchanged. High-intensity who have noted a larger increase in thiobarbituric acid
jump exercise induces muscle enzyme leakage in untrained (TBA)-reactive substances (TBARS) after high-inten-
humans, but muscle lipid peroxidation, measured as changes sity compared with moderate-intensity running.
in MDA, was not different in the two groups despite the The purpose of the present study was to compare
varied muscle antioxidant enzyme levels. blood and muscle antioxidant enzyme activities in elite
volleyball players and untrained subjects. In addition,
antioxidant activity; oxygen free radicals; delayed onset
muscle damage; malonaldehyde we evaluated indexes of muscle damage after high-
intensity exercise in these subjects, measured by plasma
creatine kinase (CK), and the possible role of oxygen
free radical-mediated muscle damage, measured as
UNACCUSTOMED EXERCISE or extremely strenuous exer- plasma and skeletal muscle malonaldehyde (MDA).
cise, particularly eccentric contractions, can be accom-
METHODS
panied by ihjury to fibers in the active skeletal muscles
(3, 19). The biochemical mechanisms behind this have Subjects. Eight male, jump-trained, Danish elite volleyball
remained unclear. However, recent evidence suggests players, age 23 -+ 5 yr, body mass 84 -+ 8 kg, and height 191 -+
that generation of free radicals, leading to lipid peroxi- 6 cm (mean -t SD), and eight nontrained males, age 23 * 2 yr,
dation of cell membranes and thereby loss of fluid body mass 75 ? 10 kg, and height 183 2 6 cm, volunteered as
property, could be important in eliciting muscle damage the two experimental groups. The volleyball players trained
five to eight times per week, with an average weekly training
(1, 3, 27). Free oxygen radicals rapidly react with
of 12 t 5 h, including a considerable amount ofjumping. None
polyunsaturated fatty acids in the cell membranes, of the subjects used antioxidant supplements, except for three
proteins, and other cellular components. Lipid peroxida- trained and three untrained subjects, who had been taken a
tion of cell membranes causes loss of fluid property and standard multivitamin for >l mo. However, there was not a
protein flexibility due to alteration in the fluid-mosaic higher vitamin content in the subjects consuming vitamins at
bilayer, which perturbs cell integrity. Studies in hu- rest or immediately and 24 h after the exercise. All subjects were
mans on the influence of exercise on lipid peroxidation fully informed of the risks and stresses associated with the
marker levels are, however, limited, and the findings project, which was approved by the local Ethics Committee.
are contradictory. Exercise protocol. The subjects were instructed to fast for 4
h before samples were taken and refrain from strenuous
During metabolic stress, free radical production in-
physical exercise 24 h before control samples were taken on
creases, and the subsequent removal depends on the the day preceding and the day after the exercise test. After a
capacity of the well-developed scavenger and antioxi- standard warm-up period of 15 min, the subjects performed a
dant systems. The antioxidative capacity might be modified Bosco test (5). During this test, they performed a
important in protection against muscle damage and series of six bouts of 30 s of continuous jumping. Maximal
includes nonenzymatic antioxidants [e.g., reduced glu- effort was exerted at a standardized knee angular displace-

R1258 0363-6119/97 $5.00 Copyright o 1997 the American Physiological Society

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 ANTIOXIDANT STATUS AND LIPID PEROXIDATION AFTER EXERCISE R1259

ment at 90” during the contact phase, with 2-min rest period and manganese SOD (MnSOD), the supernatant fraction was
between each bout. The jumping was performed on a Digitest assayed both directly and after incubation in 6 mM KCN for
Jumping Mat (Newtest 2,000 testing system). Flight time 30 min. Incubation for 30 min with 6 mM KCN completely
was recorded, and the average mechanical power output in inhibits the cytosol isoform (CuZnSOD), whereas the mito-
watts per kilogram of body weight was calculated from the chondrial matrix isoform (MnSOD) is not inhibited nor is the
following formula: (g2 Tf= T,)l[4n(30
l - Tf)] , where g is gravity, assay affected. Hemolysate was diluted to a final concentra-
Tf is total flight time, Y’t is total contact time, and n is the tion of 1:200 before assaying. Cat (EC 1.11.1.6) was assayed
number ofjumps. The subjects performed a standard stretch according to Rodrigues et al. (23) and modified as follows:
program for 15 min after performance and rested in the muscle homogenate supernatant fractions were diluted 60
laboratory while blood samples were obtained. times in 50 mM phosphate buffer and incubated for 30 min at
Muscle biopsy preparation. At 1 wk before and 24 h after 0°C with 0.01 vol of 96% ethanol and 0.01 vol of 1% Triton-X
the jump test, muscle biopsies were taken while the subjects before the assay. Hemolysate was diluted 1,000 times before
rested on a couch. With local anesthesia (lidocaine) of the the assay. One unit is defined as the amount of Cat that
skin, subcutaneous tissue, and fascia, a biopsy of the vastus decomposes 1 mmol HZ02/min. GPX (EC 1.11.1.9) was as-
lateralis (quadriceps femoris) was taken 15 cm above the sayed with FLAIR GPX assay (28) and modified as follows:
knee joint by use of a Bergstrom needle (4). This muscle was the enzyme stabilization step, adding DTT, was omitted
preferred because it is highly active in the present exercise because the standard buffer contained DTT. One unit is
(30). The biopsies were taken from different legs (random defined as micromoles of p-NADPH oxidized per minute. GR
order) before and after exercise to avoid any interfering (EC 1.6.4.2.) activity was measured according to Goldberg
damage from the first biopsy. Each muscle biopsy, yielding and Spooner (lo), where one unit is defined as micromoles of
120-200 mg tissue wet wt, was divided into two specimens. P-NADPH oxidized per minute.
One sample (lo-20 mg) was placed in a mounting medium Muscle and plasma lipid peroxidation was determined by
(OCT compound), rapidly frozen in isopentane cooled over measuring the TBA-MDA complex, fractionated on high-
liquid nitrogen, and stored at -80°C for later analysis of fiber- performance liquid chromatography (HPLC) according to
type composition. The second specimen was homogenized in Wong et al. (31), and modified as follows: a clear aliquot of the
ice-cold standard buffer (1:lO wt/vol) with 20 strokes on a muscle homogenate was made according to Draper and
Potter-Elvehjem homogenizer. The standard buffer consisting Hadley (9) with slight modifications; 150 ul of 1:lO (wt/vol>
of 100 mM KHZP04 (pH 7.4), 1 mM dithiothreiol (DTT), and 2 homogenate, 37.5 ul of 30% trichloroacetic acid (TCA), and
mM EDTA was used in all preparations. A 200~ul portion of 37.5 ul of butylated hydroxytoluene (BHT, 0.5 g/l) were
the homogenate was used for MDA determination, and the heated for 30 min and precipitated. Supernatant (150 ul), 200
remaining homogenate was immediately frozen in liquid ul of saturated TBA solution, and 650 ul of 1% H3P04 were
nitrogen in 150-ul portions and stored at -80°C until ana- heated for further 30 min, neutralized with methanol-NaOH,
lyzed. Before analysis for protein content and antioxidant precipitated, and injected onto the HPLC column to separate
enzyme activity at rest, a clear supernatant was obtained by the MDA-TBA2 adduct from interfering chromogens. One-
centrifuging homogenates at 13,000 g for 15 min. tenth volume of 20 mM ethanol-BHT was added to plasma
Blood sample preparation. Heparinized blood samples were and placed at room temperature for 10 min (12), after which a
obtained from a forearm vein at rest, immediately after 200~ul aliquot of 1,400 ul saturated solution of TBA reagent
exercise, and 1, 2, and 24 h after cessation of exercise. Two and 400 ul of 1% H3P04 was boiled for 1 h, then placed on
portions of 500 ul plasma were withdrawn and stored at ice-neutralized with methanol-NaOH, precipitated, and in-
-80°C until analyzed for MDA, vitamins, and CK. Erythro- jected onto the HPLC column.
cytes were washed three times in 0.9% NaCl. Washed erythro- Plasma concentrations of the vitamins cx-tocopherol, reti-
cytes (500 ul) were lysed in 2 ml distilled water, further nol, and P-carotene were determined with HPLC method
diluted in the same standard buffer as the muscle preparation according to Lee et al. (18).
to obtain a final 1:lO lysate, and stored at -80°C until Plasma CK activity was measured on Kodak Ektachem DT
analysis. 60 (Eastman Kodak, Rochester), where 10 ul of plasma
dispense onto a multilayered film slide, which is then read by
Analyses. Protein content, hemoglobin (Hb) concentration,
and SOD, GPX, and GR activity were analyzed with a Cobas reflectance spectrometry.
Serial transverse sections (IO urn thick) of muscle samples
Mira AutoAnalyser, and Cat activity was determined on a
were cut with a microtome at -20°C. The distribution of type
conventional spectrophotometer. To evaluate the effect of
I and II fibers was determined from sections stained for
freezing on the activity of SOD, Cat, GPX, and GR, separate
myosin adenosinetriphosphatase activity at pH 4.37 and 10.3
aliquots of muscle homogenate and blood were stored for up to
(6). The stained sections were magnified (X 120) with a
2 mo at -80°C. The activity of the enzymes was not affected
microscope and photographed, and an area with 100-200
by storage of up to 2 mo in the freezer. The blood and muscle
fibers was classified and counted.
homogenate samples were obtained within 3 days, and the
Statistical comparisons were made for mean differences by
assays were carried out within 1 mo from the sample collec-
use of the Student’s t-test for paired observations. Parametric
tion. Protein content in supernatant from homogenates centri-
data were analyzed with a two-way repeated-measures analy-
fuged at 13,000 g for 15 min was determined by the Biuret
sis of variance. Significance was established at the 0.05 level.
method (standard assay, Randox Laboratory). Hb concentra-
Values are given as means t SD.
tion was measured by the cyanmethemoglobin method (Ran-
dox Laboratory). SOD (EC 1.15.1.1) was assayed with a SOD
assay (Ransod Laboratories). This method uses xanthine and RESULTS
xanthine oxidase to generate superoxide radicals, which react
with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium Performance. Data for mean power output during the
chloride to form a red formazan dye. In the presence of SOD, six jump bouts are presented in Fig. 1. Trained individu-
dye formation will be inhibited, and 1 SOD activity unit is als performed a higher (P < 0.01) mean total power
defined as the amount of SOD that inhibits dye formation output of 25% of the untrained volleyball players in
50%. To distinguish between copper-zinc SOD (CuZnSOD) each bout (21 t 2 and 17 t 2 W/kg, respectively, in 1st

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 ANTIOXIDANT STATUS AND LIPID PEROXIDATION AFTER EXERCISE

G 7000
2 6500 J T
-2 3000
‘3 2500 x-x-
i?i
k4 2000
0
aE: 1500 >2
2 1000
z
500
0 jI : ? ?:i
Pre 0 1 2 24
Time ( hours after jumping)

Fig. 2. Plasma creatine kinase (CK) activity in jump-trained (0) and

untrained (0) male subjects at rest and after 6 bouts of 30-s maximal
jumping. Values are means + SD; y2 = 8/group. *Significantly
different from trained, P < 0.0001.

between rest and 24 h after exercise. However, there

was a tendency for increased plasma MDA (from 0.84 t
0.13 to 0.98 t 0.31 uM, mean of both groups) and
30 120 SE?C
skeletal muscle MDA (from 91.5 t 33.6 to 109.8 t 36.1
Fig. 1. Mechanical power output (in W/kg body wt) in jump-trained nmol/g protein, mean of both groups) in response to
(filled bars) and untrained (open bars) male subjects during 6 bouts of
30-s continuous jumping with a 2-min rest period between each bout. exercise.
Values are means + SD; n = 8/group. Vitamins. Plasma levels of a-tocopherol did not change
significantly from resting values directly after exercise
bout). Both groups had a similar and linear decrease in or 24 h after the exercise (Fig. 3). A paired comparison
mean power output of 23% between bouts 1 and 6. showed a significantly lower p-carotene value after 24 h
Antioxidant enzymes. Antioxidant enzyme activities (P < 0.02) when all subjects where compared, but the
are presented in Table 1. We found significantly higher differences were not significant for the untrained and
(P < 0.01) act ivi t ies of total SOD (23%), GPX (13%), and trained groups (Fig. 3). In addition, there were no
GR (25%) in muscle of high-intensity-trained volleyball significant differences between the two groups. The
players compared with untrained individuals. In addi- large variation in the p-carotene values in the un-
tion, the volleyball players had significantly higher trained group, both at rest and after 24 h, are due to
(P < 0.02) mean muscle mitochondrial MnSOD activity extremely high values of two subjects.
than untrained subjects (31%). The fraction of MnSOD Fiber type composition. A slight dominance of type I
to total SOD was 18% and not significantly different (slow-twitch) fibers was observed in both trained (56 t
between the two groups. No group specific differences 11%) and untrained (54 t 11%) individuals. For the
were seen in any of the blood antioxidant enzyme entire group of subjects, the abundance of type I fibers
activities and muscle Cat activity. varied from 37 to 70%. No differences in fiber type
Plasma CK. Twenty-four hours after high-intensity distribution were found between the two groups and
exercise, we found a significant increase (P < 0.0001) in there was no correlation between fiber type distribution
plasma CK activity in untrained individuals, from a and skeletal muscle antioxidant enzyme activity.
mean value of 104 2 29 IU/l at rest to 2,241 2 4,628 DISCUSSION
IU/l at 24 h after exercise (Fig. 2), indicating muscle
damage. However, the plasma CK activity of the volley- Antioxidant enzymes. An important finding in the
ball players remained at resting level (228 t 181 IU/l present investigation is that the activities of the antioxi-
before exercise and 342 IT 201 IU/l24 h after exercise; dant enzymes MnSOD, total SOD, GPX, and GR in
NS). There were no significant differences in plasma skeletal muscle were markedly elevated in highly
CK activity in the two groups at rest, immediately after trained jumpers, whereas muscle Cat as well as any of
exercise, and 1 and 2 h after the cessation of exercise. the blood antioxidant enzymes were at the untrained
Plasma and muscle MIA. No significant differences levels. To our knowledge, this is the first study so far
in plasma and muscle MDA levels were observed concerning antioxidant enzyme adaptations to high-

Table 1. Blood and muscle antioxidant enzyme activity at rest

Total SOD Mn SOD Cat, X10” GPX GR

Blood
Trained 672 + 58 169 t 17 79 2 5 8+-l
Untrained 655 2 46 161 t 13 78+8 822
Muscle
Trained 1,210 2 175* 231 t 5O”i 22+7 5655”” 112 1*
Untrained 985 t 112 176 t 24 25+6 5023 8+1

Values are means + SD; n = 8. Blood values are given in U/g hemoglobin and muscle values in U/g protein. SOD, superoxide dismutase; Cat,
catalase; GPX, glutathione peroxidase; GR, glutathione reductase. Significantly different from untrained: ‘I:P < 0.01; TP < 0.02.

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Copyright © 1997 American Physiological Society. All rights reserved.
 ANTIOXIDANT STATUS
14 A 1.2- B which we found 13% higher GPX and 25% higher GR
12- 1.0.
8 enzymes in the GSH-GSSG cycle, keeping the GSH/
z . 3 0.6.
26 -2
0.4 -
4-
0.2 -
2-
O- - 0.0.
rest Oh 24 h rest
Fig. 3. Plasma vitamins a-tocopherol (A) and p-carotene
trained (filled bars) and untrained (open bars) male subjects
sured at rest and immediately and 24 h after exercise. Values
means + SD; II = Wgroup.
intensity exercise such as jumping. Our findings sug-
gest that high-intensity training, i.e., jumping, in-
creases the antioxidant enzyme activity and thereby
enhances the defense against radical-induced skeletal
muscle damage. This supports an increasing number of
studies reporting elevated antioxidant enzyme activi-
ties in endurance-trained skeletal muscle (1,13,15,16).
However, the studies are not consistent with respect to
the relative importance of the various antioxidant
enzymes and their relationship to training, and obser-
vations, especially for SOD and Cat, are contradictory.
Alessio and Goldfarb (1) and Ji et al. (13) have reported
elevated Cat but unchanged SOD activity in response
to endurance training, suggesting that SOD is not a
limiting factor in muscle defense against free oxygen
radicals. In contrast, our finding of a significant higher
activity of SOD in the high-intensity trained compared
with untrained subjects indicates that SOD is an
important antioxidant enzyme in respect to exercise.
Furthermore, Krotkiewski et al. (17) showed that
supplementation of a pollen extract containing SOD
lowered MDA in muscle immediately after exercise.
Some of the conflicting results reported could be due to
biological differences between humans and experimen-
tal animals and differences in experimental conditions,
e.g., type and duration of training and differences in
exercise protocol.
The MnSOD isoform located in the mitochondria is
reported to account for 8-16s of total SOD, whereas
the CuZnSOD isoform is found in the cytosol (for
review, see Ref. 27). This is in agreement with our
findings of 18% MnSOD in both groups. It has been
estimated that 80% of the superoxide radicals formed
in the mitochondria are reduced by the mitochondrial
MnSOD (21), suggesting that MnSOD would be the
most susceptible isoform to exercise. However, we
found that the training-induced elevation of total SOD
was due to equal elevation in activity of both isoforms.
Observations with regard to GPX and GR are more
consistent, showing that endurance training elevates
the activity (14-16, 22), as in the present study, in
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Copyright © 1997 American Physiological Society. All rights reserved.
AND LIPID PEROXIDATION AFTER EXERCISE Rl261
skeletal muscle activity in high-intensity-trained than
in untrained subjects. This agrees with the general
observations that the reduced-to-oxidized glutathione
ratio (GSH/GSSGj decreases after exercise and that the
coupled enzymes GPX and GR are the most important
GSSG level high. It has been reported that endurance
training elevates the antioxidant enzyme activities in
blood (16). A similar response was not observed in
jump-trained subjects in the present study.
A number of studies have found higher antioxidant
enzyme activities in type I fibers (2, 13), suggesting
that type II fibers, recruited during high-intensity
24 h exercise, are more susceptible to exercise-induced oxida-
(B) in
tive stress. However, no group specific differences in
mea- fiber type composition could explain the differences in
are enzyme levels.
Plasma CK. An evidence of muscular damage is the
release of muscle proteins, e.g., CK, which are normally
unable to cross the cell membrane. This enzyme efflux
is considered to reflect a change in the normal mem-
brane structure, making it permeable to large mol-
ecules such as proteins. In the present investigation,
we found a highly significant increase (P < 0.0001) in
plasma CK in the untrained subjects 24 h after high-
intensity exercise, indicating some kind of muscle
damage. In trained individuals, however, CK remained
at resting levels. We are aware of the problems regard-
ing interpretation of the CK values, since the explana-
tion for the increased blood CK values is inadequate.
Furthermore, there seems to be a preadaptation in the
CK response to eccentric exercise, so that one bout of
eccentric exercise generates an adaptation so that less
damage is produced when the same exercise is per-
formed up to several months later (7). This phenom-
enon could to some extent explain the differences in the
CK pattern between trained and untrained subjects.
Nevertheless, the CK values illustrate a clear differ-
ence in exercise response in the two groups. In addition,
the general observations of delayed onset elevation of
muscle protein levels in blood show that the mecha-
nism behind leakage is not caused by direct mechanical
disruption of the cell, which would probably give an
immediate response in plasma protein levels.
Lipid peroxidation after exercise. There were no
significant changes in the level of plasma or muscle
TBA-MDA complexes after high-intensity exercise. Sax-
ton et al. (26) reported a similar pattern of unaltered
skeletal muscle TBARS but significantly elevated
plasma CK after serial repetitions of maximal volun-
tary eccentric and concentric exercise. This is also in
agreement with the observations by Sahlin et al. (24),
who found unchanged plasma levels of MDA after
prolonged exhaustive cycling. These results indicate
that elevated plasma CK levels are not induced by free
radical-induced lipid peroxidation. Other exercise stud-
ies measuring lipid peroxidation marker levels are few,
and the findings are contradictory. Elevated levels of
plasma and serum TBARS have been observed after
strenuous exercise (8, 19), but Viinikka et al. (29)
R1262 ANTIOXIDANT STATUS AND LIPID PEROXIDATION AFTER EXERCISE

reported that neither long-term physical training nor peroxidation, measured as changes in MDA, was not
acute exercise affected the TBARS level. Moreover, significantly different in the two groups despite the
most measurements in human studies have been on varied muscle antioxidant enzyme levels.
plasma and not on skeletal muscle, which would pro-
This study was supported by grants from Team Denmark.
vide a more accurate assessment of exercise-induced Address for reprint requests: N. 0rtenblad, Dept. of Physical Educa-
lipid peroxidation. Studies of sedentary rats have shown tion, Odense University, Campusvej 555230 Odense M, Denmark.
a slight increase in skeletal muscle TBARS after exhaus- Received 17 November 1995; accepted in final form 8 October 1996.
tive exercise (2, 8). Ji et al. (14) and Salminen and
Vihko (25) reported no changes in muscle MDAlevels in REFERENCES
response to acute exercise. These results clearly illus-
1. Alessio, H. M., and A. H. Goldfarb. Lipid peroxidation and
trate that further research is required to evaluate the scavenger enzymes during exercise: adaptive response to train-
role of lipid peroxidation on exercise-induced muscle ing. J. Appl. Physiol. 64: 1333-1336, 1988.
damage. Part of the reason for the contradictory find- 2. Alessio, H. M., A. H. Goldfarb, and R. G. Cutler. MDAcontent
ings could be the use of different types and intensities of increases in fast- and slow-twitch skeletal muscle with intensity
of exercise in a rat. Am. J. Physiol. 255 (Cell Physiol. 24):
exercise and different times of sampling after exercise. C874-C877,1988.
In a study of ultrastructural changes of the cytoskel- 3. Armstrong, R. B., G. L. Warren, and J. A. Warren. Mecha-
eton after a bout of eccentric exercise, Newham et al. nisms of exercise-induced muscle fibre injury. Sports Med. 12:
(20) reported a greater number of disrupted fibers 24 184-207,199l.
4. Bergstrom, J. Muscle electrolytes in man determined by neu-
and 48 h after the exercise compared with immediately
tron activation analyses on needle biopsy specimens. A study on
after exercise. On the basis of this observation, we normal subjects, kidney patients and patients with chronic
decided to obtain the biopsies for MDA measurements diarrhoea. Stand. J. Clin. Lab. Invest. 14, Suppl. 68: l-110,1962.
24 h after the test to compare muscle damage with 5. BOSCO, C., P. Luhtanen, and P. V Komi. A simple method for
MDA levels. The fact that we only measured muscle measurement of mechanical mower in jumping. Eur. J. Appl.
Physiol. Occup. Physiol. 50: 273-282, 1983.
and blood MDA 24 h postexercise may explain why we 6. Brooke, M., and K. Kaiser. Three “myosin ATPase” systems.
did not find any exercise-induced MDA. Another reason The nature of their pH lability and sulfhydryl dependence. J.
for the contradictory findings could be that lipid peroxi- Histochem. Cytochem. 18: 670-672,197O.
dation measured by the TBA-MDA assay implies some 7. Clarkson, P. M., K. Nosaka, and B. Braun. Muscle function
after exercise-induced muscle damage and rapid adaptation.
problems as outlined by Halliwell and Chirico (12). Med. Sci. Sports Exercise 24: 512-20, 1992.
Most of the problems can to some extent be averted or 8. Davies, K. J. A., A. T. Quintanilha, G. A. Brooks, and L.
overcome by the use of HPLC and the methods de- Packer. Free radicals and tissue damage produced by exercise.
scribed (12). With HPLC, MDA can be determined Biochem. Biophys. Res. Commun. 107: 1198-1205,1982.
9. Draper, H. H., and M. Hadley. Malonaldehyde determination
unambiguously as the TBA-MDA complex contrary to
as index of lipid peroxidation. Methods Enzymol. 186: 421-431,
TBARS (12). It must also be kept in mind that -98% of 1990.
the MDA reacting in the TBA test is not “free” MDA 10. Goldberg, D. M., and R. J. Spooner. In: Methods ofEnzymatic
from the sample assayed but is formed by decomposi- Analysis, edited by H. U. Bergemeyer. Deerfield Beach, FL:
tion of lipid peroxides, an early stage of the lipid Verlag Chemie, 1983, p. 258-268.
11. Gutterridge, J. M. C. Aspects to consider when detecting and
peroxidation, during the acid heating stage of TBA measurering lipid peroxidation. Free Radical Res. Commun. 1:
assay (11). 173-184,1986.
Vitamins. Plasma resting levels of the vitamins cw-to- 12. Halliwell, B., and S. Chirico. Lipid peroxidation: its mecha-
copherol and p-carotene were unchanged immediately nism, measurement, and significance. Am. J. Clin. Nuts. 57:
715S-725S, 1993.
after and 24 h after high-intensity exercise. This is in 13. Ji, L. L., R. Fu, and E. W. Mitchell. Glutathione and antioxi-
agreement with the observation by Krotkiewski et al. dant enzymes in skeletal muscle: effects of fiber type and exercise
(17), who found unaltered plasma vitamin E concentra- intensity. J. Appl. Physiol. 73: 1854-1859, 1992.
tions 5 min and 24 and 48 h after acute exercise. 14. Ji, L. L., F. W. Stratman, and H.A. Lardy. Antioxidant enzyme
systems in rat liver and skeletal muscle; influences of selenium
Endurance training may require a greater need for
deficiency, chronic training and acute exercise. Arch. Biochem.
vitamin E. Decreased vitamin E levels have been Biophys. 263: 150-160,1988.
reported in rat skeletal muscle after training (22); 15. Ji, L. L., E. Wu, and D. Thomas. Effect of exercise training on
moreover, vitamin E-depleted rats have decreased en- antioxidant and metabolic functions in senescent rat skeletal
durance capacity (8). As for MDA, a determination of muscle. Gerontology 37: 317-325, 1991.
16. Kanter, M. M., R. L. Hamlin, D. V. Unverferth, H. W. Davis,
muscle vitamin levels in contrast to plasma may more and A. J. Merola. Effect of exercise training on antioxidant
accurately assess the role of cx-tocopherol in preventing enzymes and cardiotoxicity of doxorubicin. J. Appl. Physiol. 59:
oxidative damage to skeletal muscle. 1298-1303,1985.
Conclusions. Our results indicate that the antioxi- 17. Krotkiewski, M., Z. Brzezinska, B. Liv, G. Grimby, and S.
Palm. Prevention of muscle soreness by pretreatment with
dant enzyme system of skeletal muscle adapts to
antioxidants. Stand. J. Med. Sci. Sport 4: 191-199, 1994.
high-intensity training. The adaptation is reflected in a 18. Lee, B. L., S. C. Chua, H. Y. Ong, and C. N. Ong. High-
significant elevated activity of muscle GPX, GR, and performance liquid chromatographic method for routine determi-
both isoforms of SOD. There were no significantly nation of vitamins A and E and p-carotene in plasma. J.
differences in blood antioxidant enzyme activities or in Chromatogr. 581: 41-47,1992.
19. Maughan, R. J., A. E. Donnelly, M. Gleeson, P. H. Whiting,
muscle Cat activity between trained jumpers and un- K. A. Walker, and P. J. Clough. Delayed-onset muscle damage
trained. High-intensity exercise induces muscle en- and lipid peroxidation in man after a downhill run. Muscle Nerve
zyme leakage in untrained humans; however, lipid 12: 332-336,1989.

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 ANTIOXIDANT STATUS AND LIPID PEROXIDATION AFTER EXERCISE R1263

20. Newham, D. J., G. McPhail, K. R. Mills, R. H. Edwards. 27. Sjiidin, B., Y. H. Westing, and F. S. Apple. Biochemical
Ultrastructural changes after concentric and eccentric contrac- mechanisms for oxygen free radical formation during exercise.
tions of human muscle. J. Neural. Sci. 61: 109-22, 1983. SportsMed. 10:236-254,199O.
21. Nohl, H., and D. Hegner. Do mitochondria produce oxygen 28. Van den Berg, H., H. Heseker, M. Lamand, B. Sandstrom,
radicals in vivo? Eur. J. Biochem. 82: 563-567, 1978. and D. Thurnham. FLAIR concerted action no. 10 status
22. Quintanilha, A. T. Effects of physical exercise and/or vitamin E papers. Introduction, conclusions and recommendations. Int. J.
on tissue oxidation metabolism. Biochem. Sot. Dans. 12: 403-4, Vitamin Nuts. Res. 63: 247-251, 1993.
1984. 29. Viinikka, L., J. Vuori, and 0. Ylikorkala. Lipid peroxides,
23. Rodrigues, R. E., M. Misra, and K. S. Kasparzak. Effects of
protacyclin, and thromboxane AZ in runners during acute exer-
nickel on catalase activity in vitro and in vivo. Toxicology 63: cise. Med. Sci. Sport 16: 275-277, 1984.
45-52,199O.
24. Sahlin, K., K. Ekberg, and S. Cizinsky. Changes in plasma 30. Voigt, M., E. B. Simonsen, P. Dyhre-Pot&en, and K. Klau-
sen. Mechanical and muscular factors influencing the perfor-
hypoxanthine and free radical markers during exercise in man.
Acta Physiol. Stand. 142: 275-281, 1991. mance in maximal vertical jumping after different prestretch
25. Salminen, A., and V; Vihko. Lipid peroxidation in exercise loads. J. Biomech. 28: 293-307, 1995.
myopathy. Exp. Mol. Pathol. 38: 380-388, 1983. 31. Wong, S. H. Y., J. A. Knight, S. M. Hopfer, 0. Zaharia, C. N.
26. Saxton, J. M., A. E. Donnelly, and H. P. Roper. Indices of free Leach, Jr., and F. W. Sunderman, Jr. Lipoperoxides in plasma
radical-mediated damage following maximum voluntary eccen- as measured by liquid-chromatographic separation of malondial-
tric and concentric muscular work. J. Appl. Physiol. 68: 189-193, dehyde-thiobarbituric acid adduct. Clin. Chem. 33: 214-220,
1994. 1987.

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