Effects of acute swimming exercise on muscle and erythrocyte malondialdehyde, serum myoglobin, and plasma ascorbic acid concentrations MITAT KOZ AND
DENIZE R B A ~ ~
Department of PBzysdo60gyJ Medical FacukfyJ Gazi Uns'versiry, 06510 Begevker, Ankara, Turkey AND
A Y ~ BIHA~IHAN E AND AYSELAMCI&L&T Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 12/29/14 For personal use only.
Depamnent of Bischernis~~, Medical Facukq): Gazi Universiry, 06510 Begevker, Ankara, Turkey
Received March 18, 1992
Kcsz, M., EWBA~, Ds, BILGIHAN, A., and ARICIOCLU, A. 1992. Effects of acute swimming exercise on muscle and erythrocyte malondialdehyde, semm wsyoglobin, and plasma ascorbic acid concentrations. Can. J. Physiol. Pharmacol. 70: 1392- 1395. Acute exercise may induce free-radical production in mitochondria during basal metabolism of aerobic cells. Ascorbic acid is a strong antioxidant agent, whereas myoglobin is known to act as an oxygen reservoir. The purpose of this study was to investigate the effects of acute exercise on malondialdehyde Bevels in gastrocnemius, vastus medialis, and triceps brachi muscles and erythrocytes. In addition, we investigated the ascorbic acid levels and semm myoglobin concentrations in rats, following acute swimming exercise. We found that the levels of muscle rnalondiaidehyde and serum rnyoglobin increased and the levels of plasma ascorbic acid decreased, in proportion to the duration of exercise; however, the levels of erythrocyte malondialdehyde did not change. Key words: lipid peroxidation, malondialdehyde, myoglobin, ascorbic acid, acute swimming exercise.
KOZ,M., ERBA~, B., BILGIHAW, A.. et ARICIOELU, A. 1992. Effects of acute swimming exercise on muscle and erythrocyte malondialdehyde, semm myoglobin, and plasma ascorbic acid concentrations. Can. J . PhysioH. Pharmacol. 70 : 1392- 1395. Un exercice intense peut engendrer la production de radicaux Bibres dans les mitochondries durant le rnCbbolisme basal des cellules akrobies. E'acide ascorbique est aan puissant antisxydant, aHors que la rnysglobine est connue pour stocker I'oxygkne. Le but de cette Ctude a CtC d'exarniner les effets d'un exercice aigu sur les taux de malondialdkhyde dans Hes muscles gastrocnCmien, vaste interne du membre infCrieur et triceps brachial, ainsi que dans les erythrocytes. Be plus, nous avons examine Hes taux d'acide ascorbique et les concentrations de myoglobine skrique chez des rats apr23s un exercice aigu en priscine. Dans notre expkrience, nous avons eonstat6 une augmentation des taux de malondialdkhyde musculaire et de myoglobine skrique, et une diminution des taux d'acide ascorbique plasmatique propsrtionnelles a la duree de l'exercice; toutefois, les taux de malondialdkhyde krytgrthrocytaire n'ont pas varik. Mots cO& peroxydation lipidique, rnalondialdkhyde, myoglobine, acide ascorbique, exercice aigu en priscine. [Traduit par la rCdaction]
Introduction During mitochsndrial oxidative phosphorylation, superoxide radicals (CIS2-) are produced when single electrons bind to molecular oxygen (Deneke and Fanburg 1980). During basal metabolism, some Om2-originating in mitochondria may leak into the cytosol (Fridovich 1978), where iron or copper catalysis can promote the formation of highly reactive hydroxyl radicals (OH' -) (Halliwell 1987). Oxygen radicals, especially hydroxyl radicals, can extract hydrogen from a wide range of biomolecules including the polyunsaturated fatty acids of cell membranes and may initiate lipid peroxidation, thus causing cell injury (Del Maestro 1980). Within the erythrocytes, the constant conversion of oxyhaemoglobin to rnethaernoglobin (3 % of total haemcsglobin mass per day) results in a concsmitant production of superoxide anions (Clemens and WaHler 1987). Being exposed to high oxygen tension and rich in polyunsaturated lipids and iron, a potent catalyst for free radical reactions, erythrocytes are perforce in an environment in which they are constantly exposed to both extracellular and intracellular sources of free radicals (Chiu el al. 2982; Chiu and Claster 1988). During physical exercise the aerobic metabolic rate may increase up to 10-fold and the oxygen uptake of skeletal 'Author for correspondence. Printed in Canada / ImpnmC au Canada
muscles may increase up to 100-200 times (Guyton 1991), enhancing leakage of O e 2 - from the mitochondria to the cytosol (Davies ef a / . 1982). This rise in oxygen free radical concentration could exceed the protective capacity of cell antioxidant defence systems, and strenuous exercise promotes free-radical formation and lipid peroxidation in skeletal muscle. liver, and blood (Duehie ef a!. 1998; Davies et a / . 1982; Salminen and Vihko 1983). Ascorbic acid (AA) is a strong antioxidant agent (Hershko 1989), which also increases performance (Brouns m d Saris 1989). Vitamins E and C play an important role in antioxidant defence mechanisms. Vitamin E radicals produced by the oxidation of vitamin E can be reduced back to vitamin E by ascorbic acid (Clemens and Waller 1987). Myoglobin is known to facilitate oxygen transport f r ~ mthe muscle cell membrane to the mitochondria and also acts as an oxygen store (Svedenhag er a / . 1983). Muscle proteins such as myoglobin can be released during and after prolonged exercise as a result of muscle damage (Roxin et al. 1986; Lbandin et ale 1986). The purpose of this study was to investigate the effects of acute exercise on malondialdehyde (MDA) levels of skeletal muscle and erythrocytes, In addition we also investigated the plasma AA and serum myoglobin concentrations of rats which developed leakage from damaged muscles following acute swimming exercise.
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KQZ ET AL.
TABLEI. Lipid peroxidation in muscle (nmol MBA/g tissue, X
+ SD)
Group
n
Gastrocnemius
Vastus medialis
Triceps brachi
(1) Control (2) 60 min exercise (3)90rninexercise (4) 12Ominexercise
7 6 5 6
29.54f 3.1 36.0f4.9" 43.0f10.6** 48.8f3.9
32.5k3.3 37.5 f 4.4 45.4+4** 48.2k 3.9**
29.6k3.9 41.6+5.7** 43.4+5.8** 61.4f 8.9**
TABLE2. Lipid peroxidation in erythrocyte (nrnsl ME)A/g haemoglobin, X f SD) Group (1) (2) (3) (4)
Control 60 min exercise 90 min exercise 120 min exercise
TABLE3. Plasma AA concentrations (mg AA/ 100 mL, X f SD)
n 7 7 4 7
Group B782+212 1727f 154 I797f 177 1766 k 2 3 1
Materials and methods In this study 28 male Wistar rats (288-250 g) were used. Animals were divided into four groups: group 1 was the control and groups 2, 3, and 4 were exposed to exercise for 60. 90, and 120 min, respectively. Rats were exercised by swimming in steel barrels filled with water maintained at 35°C. They each swam with a weight equivalent to approximately 2% of body weight affixed to the base of the tail. After the exercise, exercised and control animals were immediately killed by decapitation after ether anesthesia. Their blood samples were collected in standard heparinized glass tubes. Gastrocnemius (muscle I), vastus medialis (muscle 21, and triceps brachi (muscle 3) were rapidly removed, washed in 0.9% NaC1, and kept in ice. Muscle MBA levels were estimated by the method of Uchiyarna and Mihara (1978). To 0.5 mL homogenate, 3 mL 1% phosphoric acid and 1 mL 8.6% thiobarbituric acid (TBA) were added, and the mixture was kept in a water bath at 95°C for 45 min. The colored reaction product was extracted with 4 mL n-butanol, and the difference of the absorbance at 535 and 520 nm was recorded. The breakdown product of 1,1,3,3-tetraethoxypropamae was used as standard, and the results were expressed as mnornoles MDA per gram muscle tissue. Erythrocyte susceptibility to lipid peroxidation was determined by MDA formation, using the addition technique of Stocks and Bormandy (1971) in which erythrocyte suspensions were incubated with hydrogen peroxide at 37'C for 2 h. The results were expressed as wanomoles MDA per gram haerrasglobin. Plasma AA concentrations were determined using a modified 2,4-dinitrophenylhydrazin method by which AA levels in 0.01 mL blood can be determined (Roe 196'7). The results were expressed as milligrams AA/dL plasma. Serum rnyoglobin concentrations were determined by an RIA kit. The results were expressed as nanograms per millilitre. Differences between groups were detected using Duncan's multiple range test. The significance of the differences was assessed by analyses of variance.
Results The muscle (gastrocnemius, vastus medialis, and triceps brachi) and erythrocyte MDA levels are given in Tables I and 2, respectively. When compared with controls, MDA levels of muscles I and 3 were found to be significantly increased in group 2 (g < 0.05 and g < 0.01, respectively), group 3 ( p < 0.01 andp < 8.01, respectively), and group 4 ( p < 0.01 and p < 0.05, respectively) afier exercise. Increased MDA levels
(1) (2) (3) (4)
Control 689 min exercise 90 min exercise 120 min exercise
n
6 6 6 6
1.37 f0.2 1.23 1 0 . 2 8.86+0.2** 0.55+89.2**
of muscle 2 in group 2 were not statistically significant ( p > 0.05), but in groups 3 and 4 the increases were significant ( p < 0.81 and p < 0.01, respectively). Erythrocyte MDA levels did not change in any of the exercised groups when compared with controls. Plasma AA concentrations (Table 3) decreased in all exercised groups. Decreased AA concentrations in group 2 were not statistically significant ( p > O.05), but in groups 3 and 4 were significant ( p < 0.01 and p < 0.01, respectively). Serum myoglobin concentrations, haernoglobin, and hematocrit values are given in Table 4. Increases of serum myoglobin concentrations in group 4 and increases of hernatocrit values in groups 3 and 4 were statistically significant ( p < 0.05, p < 0.01, and g < 0.05, respectively). Increases of serum mysglobin concentrations, haemoglobin, and hematocrit values in the other groups were not significant ( g > 0.05).
Results of this study indicate that acute swimming exercise increased lipid peroxidation in muscles. The detection of MDA using its reaction with TBA has been the most widely used indicator of lipid peroxidation (Chiu el al. 1989; Saltman 1989). Alessio et al. (1988) observed increasing MDA levels of fast- and slow-twitch skeletal muscle with intensity sf exercise in rats. Davies el al. (1982) observed an 869% increase in MDA content of gastrocnemius, soleus, and plantaris muscles after maximal running exercise. Brady et al. (1979) observed increased lipid peroxidation levels in liver and muscle, follswing swimming exercise in rats. Salminen and Vihko (1983) observed a significant increase in MDA content of muscle after treadmill exercise, and the peroxidation rate was slightly higher in white muscles than in red muscles. The fiber type of muscles used in our experiment has been reported as, for gastrscnemius, 27 % fast oxidative glycolytic, 60 % fast glycolytic (white muscle), 13% slow oxidative (red muscle) ; for vastus medialis; 12% fast oxidative glycolytic, 86 % fast glycalytic (white muscle), 2% slow oxidative (red muscle); and for triceps brachi, fast glycolytic fibers are higher than others (Armstrong and Phelps 1984). Although the same muscle fiber type (white muscle) forms the majority of all the muscles,
CAN. J. PHYSIOL. PHARMACOL. VOL. 70, 1992
TABLE4. Serum mysglobin (amg/mL). haemoglobin (g/dL), and hematocrit (76) values (T k SD)
Group
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(1) (2) (3) (4)
Control 60 min exercise 90 min exercise 120 min exercise
n
My oglobin
Haemoglobin
Hematocrit
7 6 6 6
10.9k 1.0 11.6+_0.9 H2.3f 1.3 14.0f 1.0"
20.6f 1. H 1H.1+0.6 12.5f0.9 l 1.6f 0.5
42.3k4.8 45.3k1.5 49.6+0.6** 47.2 1 1 . 5 "
increases of MDA levels differed in each muscle. This can be explained by variation in degree of activity of muscIes during swimming exercise in rats; it is possible that upper extremity muscles. for example, triceps brachi, are more active than lower extremities during prolonged swimming, in accordance with MDA levels. Our results and several reports (Alessio et al. 1988; Davies et a%.1982; Brady et al. 1979; Sumida et a&. 1989; Duthie et a&.1990) strongly suggest that acute exercise of sufficient intensity can increase active oxygen radical species, which accelerates lipid peroxidation and related reactions. We suggest that the degree of lipid peroxidation in exercised muscles depends on the intensity and duration of exercise. In our experiment, a proportional increase in the levels of MDA in each group supports this view. Increased oxygen utilization during exercise causes increased mitochondrial Ow2-production and haemoglobin autoxidation (Davies et a&. 19821, and any resulting enhanced leakage of CIS2- could promote lipid peroxidation and tissue damage (Duthie et al. 1990). In addition, ischemia occurs in some tissues during acute heavy exercise. Thus Kellog and Fridovich (1975) have indicated that reperfusion of hypoxic tissues may cause free-radical production by the xanthine oxidase system. Sumida et al. (1989) hypothesized that generation of free radicals during acute heavy exercise may occur not only via the mitochondrial respiratory chain but also through activation of the xanthine oxidase system. According to Stanley et al. (B985), the more intense the exercise the more lactate is produced. Since appearance rate of lactate exceeds the disappearance rate at maximal work loads, this indicates a possible drop in the concentrations of cytoplasmic NADH and (or) NABPH (Lowlin et al. 1987). The oxidized glutathione is reduced to glutathione. This reaction is catalysed by glutathione reductase enzyme. Only NADPH is effective as a substrate for glutathione reductase in vivo. The known mechanism for the recovery of NADPH from NADH depends on the oxidation of glucose via the hexose monophosphate shunt (Clemens and Waller 1987). Consequently, at maximal exercise, the activity of free radical scavenging enzymes such as superoxide disrnutase, catalase, glutathione reductase, and glutathione peroxidase may be compromised, and substrates that generate free radicals would accumulate (Lowlin et al. 1987). Any stress on the system, such as hypoxia in tissues, which results in depletion of glycolytic substrates may cause a decrease in the generation of NADH and NADPH (Lowlin et aE. 1987). Erythrocyte MDA levels did not change immediately in groups 2, 3, and 4, because erythrocytes are endowed with a variety of extremely efficient protective antioxidant mechanisms that can block autoxiidation in vivo (Clemens and Waller 1987). These include enzyme systems such as superoxide dis-
mutase, catalase, glutathione reductase, and glutathione peroxidase and antioxidant substrates such as vitamin E, vitamin C, cemloplasmin, tramsferrin, apotransferrin, and haptoglobin (Clemens and Waller 1987; Chiu et al. 1989). Duthie et al. (1990) found that the susceptibility of erythrocytes to in vitro peroxidation did not change immediately up to 24 h postexercise in runners, but increased after 24 h. Results of our study are similar to those of Duthie et al. Immediately afier exercise there were decreased AA concentrations in all exercised groups, which were related to exercise durations. It has been suggested that, especially as a result of intensive endurance activity, the body may become vitamin C depleted, and studies have shown that vitamin C is released from the liver and adrenal glands in some animal species after exercise (Brouns and Saris 1989). Immediately after exercise, hematocrit levels significantly increased in groups 3 and 4. These increases in hematocrit can be explained with hemsconcentration, where some fluid leaks out of the vessels, which results in increases in haemoglobin. erythrocyte, and plasma protein levels in the blood and increases in the volume of interstitial fluid (Ganong 1989). Increased haemoglobin levels were not significant in any of the exercise groups. Repeated muscle contraction during strenuous exercise can reduce the plasma volume by as much as 10 to 20% in three ways: it increases pressure in capilleries by compressing venules in muscles; it generates metabolites such as lactic acid, which increase osmotic pressure in the muscle tissue surrounding the capilleries, and these forces drive plasma water into the tissues; and much of the fluid lost in sweat comes from plasma (Eichner 1991). Although an increase of 17.3% hematocrit exists in group 3, increases of group 4 are less than increases of group 3 (1 1.5%). In addition to hematocrit values, semm myoglobin concentration significantly increased in group 4. Increased semm myoglobin levels were regularly found after performance of long-term exercise. Exercise-induced leakage of myoglobin from muscle cells may reduce the muscle oxygen store and the metabolic capacity of the muscle cell. Moreover, increases in plasma creatine kinase activity may indicate that muscle damage occurs by a mechanism that influences exercise-induced damage (Duthie et al. % 990). Although acute exercise leads to increased lipid peroxidation in skeletal muscle, liver, and semm, chronic aerobic training has been reported to help reduce lipid peroxidation by increasing defence capabilities (Alessio and Goldfarb 1988; Salminen and Vihko 1983; Higuchi et a&.1985; Ghandwaney and Ji 1992; Kanter et a&. 1985). Our results indicated that acute swimming exercise increased muscle lipid peroxidation, serum myoglobin concentration, haemoglobin, and hematocrit values and decreased plasma AA concentrations but did not affect erythrocyte lipid peroxidation.
KOZ BT AL.
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These findings support the view that acute exercise may damage tissues via free radical and lipid peroxidation, whereas chronic and regular exercise may be useful in terms s f increasing defence capabilities of tissues against lipid peroxidation. Alessio, M. M., and Goldfarb, A. HI. 1988. Lipid peroxidation and scavenger enzymes during exercise: adaptive response to training. J. Appl. Physiol. 64(4): 1333- 1336. Alessio, H. M . , Coldfarb, A. H., and Cutler, R. G. 1988. MDA content increases in fast- and slow-twitch skeletal muscle with intensity of exercise in a rat. Am. J. Physiol. 255: C874-C877. Armstrong, R. B., and Phelps, R. D. 1984. Muscle fiber type composition of the rat hindlimb. Am. J. Anat. 171: 259 -272. Brady, P. S., Brady, L. J., and Ullrey, D. E. 1979. Selenium, vitamin E and the response to swimming stress in the rat. J. Nutr. 109: 1103- 1109. Brouns, F., and Saris, W. 1989. How vitamins affect performance. J . Sport Med. Phys. Fitness, 29(4): 400-404. Chandwaney, 8. H., and Ji, L. L. 1992. Exercise training attenuates muscle mitochondria1 damage by oxygen free radicals. Med. Sci. Sports Exercise Suppl. 24(5): 17. (Abstr.) Chiu, D. T. Y., and Claster, S. 1988. Measurement of red cell membrane oxidation and the generation of oxidative intermediates. In The cell membrane. Edited by S. B . Shotet and W. Mohandes. Churchill Livingston, New York. pp. 2683 -234. Chiu, D., Lubin, B., and Shotet, S. B. 1982. Peroxidative reactions in red cell biology. I n Free radicals in biology. Vol. 5. Edited by W. Pryor. Academic Press. San Diego. pp. 115- 160. Chiu, D. L., Kuypers, F., and Lubin, B. 1989. Lipid peroxidation in human red cells. Semin. Hematol. 26(4): 257 -276. Clemens, M. R., and Waller, H. D. 1987. Lipid peroxidation in erythrocytes. Chem. Phys. Lipids, 45: 251 -268. Davies, K. J. A., Quintanilha, A. T., Brooks, G. A., and Packer, L. 1982. Free radicals and tissue damage produced by exercise. Biochem. Biophys. Res. Commun. 107(4): 1198- 1205. Bel Maestro, 8. F. 1980. An approach 6 0 free radicals in medicine and biology. Acta Physiol. Scand. Suppl. 492: 153 - 168. Deneke. S. M., and Fanburg, B. L. 1980. Normobaric oxygen toxicity of the lung. N.Eng1. J . Med. 303(2): 76-85. Duthie, G . G., Robertson, J. D.. Maughan, W. J., and Morrice, P. C. 1990. Antioxidant status and erythrocyte lipid peroxidation following distance running. Arch. Biwhem. Biophys. 282: 7883. Eichner, E. R. 1991. Hematologic problems. %nClinical sports medicine. Edited by A. Grana and A. Ka'lenak. W. B. Saunders Company, Philadelphia, Pa. pp . 209 -222. Fridovich, 1. 1978. The biology of oxygen radicals. Science (Washington, D.C.), 201: 875 - 879. Ganong, W. F. 1989. Dynamics of blood and Hymph flow. I n Review of medical physiology. 14th ed. Appleton and Lange, Los Altos, Calif. pp. 477 -492.
1395
Guyton, A. C. 1991. Sports physiology. I n Textbook of medical physiology. 8th ed. W. B. Saunders Company, Philadelphia, Pa. pp. 939-951. Malliwell, B. 1987. Oxidants and human disease: some new concepts. FASEB J. 1: 358-364. Hershko, C. 1989. Mechanism of iron toxicity and its possible role in red cell membrane damage. Semin. Hernatol. 26(4): 227-288. Higuchi, M., Cartier, L. J., Chen, M., and Holloszy, J. 0. 1985. Superoxide dismutase and catdase in skeletal muscle: adaptive response to exercise. J. Gerontol. 40(3): 281 -286. Kanter, M. M., Hamlin, R. L., Unverferth, D. V., Davis, H. N., and Merola, A. J . 1985. Effect of exercise training on antioxidant enzymes and cardiotoxicity of doxorubicin. J. Appl. Physiol. 59(4): 1298- 1303. Kellog, E. W., and Fridovich, I. 1975. Superoxide, hydrogen peroxide and singlet oxygen in lipid peroxidation by a xanthine oxidase system. J. Biol. Chem. 250: 8812-8817. Lowlin, R., Cottle, W., Pyke, I., Kavarnagh, M., and Belcastro, A. N. 1987. Are indices of free radical damage related to exercise intensity? Eur. J. Appl. Physiol. 56: 3 13-3 16. Laandin, L., Hallgren, R., Lidell, C., Roxin, L. E., and Venge, P. 1986. Ethanol reduces myoglobin release during isokinetic muscle exercise. Acta Med. Scand. 219: 415-419. Roe, W. R. 1967. Ascorbic acid. I n The vitamins. Vol. 7. Edited by B. Gyorgy and W. N. Pearson. Academic Press, New York and London. Woxin, L. E., Hedin, T., and Venge, P. 1986. Muscle cell leakage of rnyoglobin after long-term exercise and relation to the individual performances. Hnt. J. Sports Med. 7: 259 -263. Salminen, A., and Vihko, V. 1983. Endurance training reduces the susceptibility of mouse skeletal muscle lipid peroxidation in vitro. Acta Physiol. Scand. 117: 1689 - 113. Saltman, P. 1989. Oxidative stress: a radical view. Semin. Hernatol. 26: 249-256. Stanley, W. C., G e m , E. W., Wisneski, J. A., Morris, D. L., Neese, R. A., and Brooks, G. A. 1985. Systemic lactate kinetics during graded exercise in man. Am. 9. Physiol. 249: E595 -E607. Stocks, J . , and Dormandy, T. L. 1971. The autsxidation of human red ccll lipids induced by hydrogen peroxide. Br. J. Haematol. 20: 95 - 109. Sumida, S., Tanaka, K., Kitao, H., and Nakadomo, F. 1989. Exercise-induced lipid peroxidation and leakage of enzymes before and after vitamin E supplementation. Hnt. J. Biochem. 28: 835 838. Svedenhag, J., Henriksson, J., and Sylven, C. 1983. Dissociation of training effects on skeletal muscle mitochondria1 enzymes and myoglobin in man. Acta Physiol. Scand. 117: 213 -218. Uchiyarna, M., and Mihara, M. 1978. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Ann. Biochem. 86: 271 -278.
DENIZE R B A ~ ~
Department of PBzysdo60gyJ Medical FacukfyJ Gazi Uns'versiry, 06510 Begevker, Ankara, Turkey AND
A Y ~ BIHA~IHAN E AND AYSELAMCI&L&T Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 12/29/14 For personal use only.
Depamnent of Bischernis~~, Medical Facukq): Gazi Universiry, 06510 Begevker, Ankara, Turkey
Received March 18, 1992
Kcsz, M., EWBA~, Ds, BILGIHAN, A., and ARICIOCLU, A. 1992. Effects of acute swimming exercise on muscle and erythrocyte malondialdehyde, semm wsyoglobin, and plasma ascorbic acid concentrations. Can. J. Physiol. Pharmacol. 70: 1392- 1395. Acute exercise may induce free-radical production in mitochondria during basal metabolism of aerobic cells. Ascorbic acid is a strong antioxidant agent, whereas myoglobin is known to act as an oxygen reservoir. The purpose of this study was to investigate the effects of acute exercise on malondialdehyde Bevels in gastrocnemius, vastus medialis, and triceps brachi muscles and erythrocytes. In addition, we investigated the ascorbic acid levels and semm myoglobin concentrations in rats, following acute swimming exercise. We found that the levels of muscle rnalondiaidehyde and serum rnyoglobin increased and the levels of plasma ascorbic acid decreased, in proportion to the duration of exercise; however, the levels of erythrocyte malondialdehyde did not change. Key words: lipid peroxidation, malondialdehyde, myoglobin, ascorbic acid, acute swimming exercise.
KOZ,M., ERBA~, B., BILGIHAW, A.. et ARICIOELU, A. 1992. Effects of acute swimming exercise on muscle and erythrocyte malondialdehyde, semm myoglobin, and plasma ascorbic acid concentrations. Can. J . PhysioH. Pharmacol. 70 : 1392- 1395. Un exercice intense peut engendrer la production de radicaux Bibres dans les mitochondries durant le rnCbbolisme basal des cellules akrobies. E'acide ascorbique est aan puissant antisxydant, aHors que la rnysglobine est connue pour stocker I'oxygkne. Le but de cette Ctude a CtC d'exarniner les effets d'un exercice aigu sur les taux de malondialdkhyde dans Hes muscles gastrocnCmien, vaste interne du membre infCrieur et triceps brachial, ainsi que dans les erythrocytes. Be plus, nous avons examine Hes taux d'acide ascorbique et les concentrations de myoglobine skrique chez des rats apr23s un exercice aigu en priscine. Dans notre expkrience, nous avons eonstat6 une augmentation des taux de malondialdkhyde musculaire et de myoglobine skrique, et une diminution des taux d'acide ascorbique plasmatique propsrtionnelles a la duree de l'exercice; toutefois, les taux de malondialdkhyde krytgrthrocytaire n'ont pas varik. Mots cO& peroxydation lipidique, rnalondialdkhyde, myoglobine, acide ascorbique, exercice aigu en priscine. [Traduit par la rCdaction]
Introduction During mitochsndrial oxidative phosphorylation, superoxide radicals (CIS2-) are produced when single electrons bind to molecular oxygen (Deneke and Fanburg 1980). During basal metabolism, some Om2-originating in mitochondria may leak into the cytosol (Fridovich 1978), where iron or copper catalysis can promote the formation of highly reactive hydroxyl radicals (OH' -) (Halliwell 1987). Oxygen radicals, especially hydroxyl radicals, can extract hydrogen from a wide range of biomolecules including the polyunsaturated fatty acids of cell membranes and may initiate lipid peroxidation, thus causing cell injury (Del Maestro 1980). Within the erythrocytes, the constant conversion of oxyhaemoglobin to rnethaernoglobin (3 % of total haemcsglobin mass per day) results in a concsmitant production of superoxide anions (Clemens and WaHler 1987). Being exposed to high oxygen tension and rich in polyunsaturated lipids and iron, a potent catalyst for free radical reactions, erythrocytes are perforce in an environment in which they are constantly exposed to both extracellular and intracellular sources of free radicals (Chiu el al. 2982; Chiu and Claster 1988). During physical exercise the aerobic metabolic rate may increase up to 10-fold and the oxygen uptake of skeletal 'Author for correspondence. Printed in Canada / ImpnmC au Canada
muscles may increase up to 100-200 times (Guyton 1991), enhancing leakage of O e 2 - from the mitochondria to the cytosol (Davies ef a / . 1982). This rise in oxygen free radical concentration could exceed the protective capacity of cell antioxidant defence systems, and strenuous exercise promotes free-radical formation and lipid peroxidation in skeletal muscle. liver, and blood (Duehie ef a!. 1998; Davies et a / . 1982; Salminen and Vihko 1983). Ascorbic acid (AA) is a strong antioxidant agent (Hershko 1989), which also increases performance (Brouns m d Saris 1989). Vitamins E and C play an important role in antioxidant defence mechanisms. Vitamin E radicals produced by the oxidation of vitamin E can be reduced back to vitamin E by ascorbic acid (Clemens and Waller 1987). Myoglobin is known to facilitate oxygen transport f r ~ mthe muscle cell membrane to the mitochondria and also acts as an oxygen store (Svedenhag er a / . 1983). Muscle proteins such as myoglobin can be released during and after prolonged exercise as a result of muscle damage (Roxin et al. 1986; Lbandin et ale 1986). The purpose of this study was to investigate the effects of acute exercise on malondialdehyde (MDA) levels of skeletal muscle and erythrocytes, In addition we also investigated the plasma AA and serum myoglobin concentrations of rats which developed leakage from damaged muscles following acute swimming exercise.
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 12/29/14 For personal use only.
KQZ ET AL.
TABLEI. Lipid peroxidation in muscle (nmol MBA/g tissue, X
+ SD)
Group
n
Gastrocnemius
Vastus medialis
Triceps brachi
(1) Control (2) 60 min exercise (3)90rninexercise (4) 12Ominexercise
7 6 5 6
29.54f 3.1 36.0f4.9" 43.0f10.6** 48.8f3.9
32.5k3.3 37.5 f 4.4 45.4+4** 48.2k 3.9**
29.6k3.9 41.6+5.7** 43.4+5.8** 61.4f 8.9**
TABLE2. Lipid peroxidation in erythrocyte (nrnsl ME)A/g haemoglobin, X f SD) Group (1) (2) (3) (4)
Control 60 min exercise 90 min exercise 120 min exercise
TABLE3. Plasma AA concentrations (mg AA/ 100 mL, X f SD)
n 7 7 4 7
Group B782+212 1727f 154 I797f 177 1766 k 2 3 1
Materials and methods In this study 28 male Wistar rats (288-250 g) were used. Animals were divided into four groups: group 1 was the control and groups 2, 3, and 4 were exposed to exercise for 60. 90, and 120 min, respectively. Rats were exercised by swimming in steel barrels filled with water maintained at 35°C. They each swam with a weight equivalent to approximately 2% of body weight affixed to the base of the tail. After the exercise, exercised and control animals were immediately killed by decapitation after ether anesthesia. Their blood samples were collected in standard heparinized glass tubes. Gastrocnemius (muscle I), vastus medialis (muscle 21, and triceps brachi (muscle 3) were rapidly removed, washed in 0.9% NaC1, and kept in ice. Muscle MBA levels were estimated by the method of Uchiyarna and Mihara (1978). To 0.5 mL homogenate, 3 mL 1% phosphoric acid and 1 mL 8.6% thiobarbituric acid (TBA) were added, and the mixture was kept in a water bath at 95°C for 45 min. The colored reaction product was extracted with 4 mL n-butanol, and the difference of the absorbance at 535 and 520 nm was recorded. The breakdown product of 1,1,3,3-tetraethoxypropamae was used as standard, and the results were expressed as mnornoles MDA per gram muscle tissue. Erythrocyte susceptibility to lipid peroxidation was determined by MDA formation, using the addition technique of Stocks and Bormandy (1971) in which erythrocyte suspensions were incubated with hydrogen peroxide at 37'C for 2 h. The results were expressed as wanomoles MDA per gram haerrasglobin. Plasma AA concentrations were determined using a modified 2,4-dinitrophenylhydrazin method by which AA levels in 0.01 mL blood can be determined (Roe 196'7). The results were expressed as milligrams AA/dL plasma. Serum rnyoglobin concentrations were determined by an RIA kit. The results were expressed as nanograms per millilitre. Differences between groups were detected using Duncan's multiple range test. The significance of the differences was assessed by analyses of variance.
Results The muscle (gastrocnemius, vastus medialis, and triceps brachi) and erythrocyte MDA levels are given in Tables I and 2, respectively. When compared with controls, MDA levels of muscles I and 3 were found to be significantly increased in group 2 (g < 0.05 and g < 0.01, respectively), group 3 ( p < 0.01 andp < 8.01, respectively), and group 4 ( p < 0.01 and p < 0.05, respectively) afier exercise. Increased MDA levels
(1) (2) (3) (4)
Control 689 min exercise 90 min exercise 120 min exercise
n
6 6 6 6
1.37 f0.2 1.23 1 0 . 2 8.86+0.2** 0.55+89.2**
of muscle 2 in group 2 were not statistically significant ( p > 0.05), but in groups 3 and 4 the increases were significant ( p < 0.81 and p < 0.01, respectively). Erythrocyte MDA levels did not change in any of the exercised groups when compared with controls. Plasma AA concentrations (Table 3) decreased in all exercised groups. Decreased AA concentrations in group 2 were not statistically significant ( p > O.05), but in groups 3 and 4 were significant ( p < 0.01 and p < 0.01, respectively). Serum myoglobin concentrations, haernoglobin, and hematocrit values are given in Table 4. Increases of serum myoglobin concentrations in group 4 and increases of hernatocrit values in groups 3 and 4 were statistically significant ( p < 0.05, p < 0.01, and g < 0.05, respectively). Increases of serum mysglobin concentrations, haemoglobin, and hematocrit values in the other groups were not significant ( g > 0.05).
Results of this study indicate that acute swimming exercise increased lipid peroxidation in muscles. The detection of MDA using its reaction with TBA has been the most widely used indicator of lipid peroxidation (Chiu el al. 1989; Saltman 1989). Alessio et al. (1988) observed increasing MDA levels of fast- and slow-twitch skeletal muscle with intensity sf exercise in rats. Davies el al. (1982) observed an 869% increase in MDA content of gastrocnemius, soleus, and plantaris muscles after maximal running exercise. Brady et al. (1979) observed increased lipid peroxidation levels in liver and muscle, follswing swimming exercise in rats. Salminen and Vihko (1983) observed a significant increase in MDA content of muscle after treadmill exercise, and the peroxidation rate was slightly higher in white muscles than in red muscles. The fiber type of muscles used in our experiment has been reported as, for gastrscnemius, 27 % fast oxidative glycolytic, 60 % fast glycolytic (white muscle), 13% slow oxidative (red muscle) ; for vastus medialis; 12% fast oxidative glycolytic, 86 % fast glycalytic (white muscle), 2% slow oxidative (red muscle); and for triceps brachi, fast glycolytic fibers are higher than others (Armstrong and Phelps 1984). Although the same muscle fiber type (white muscle) forms the majority of all the muscles,
CAN. J. PHYSIOL. PHARMACOL. VOL. 70, 1992
TABLE4. Serum mysglobin (amg/mL). haemoglobin (g/dL), and hematocrit (76) values (T k SD)
Group
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(1) (2) (3) (4)
Control 60 min exercise 90 min exercise 120 min exercise
n
My oglobin
Haemoglobin
Hematocrit
7 6 6 6
10.9k 1.0 11.6+_0.9 H2.3f 1.3 14.0f 1.0"
20.6f 1. H 1H.1+0.6 12.5f0.9 l 1.6f 0.5
42.3k4.8 45.3k1.5 49.6+0.6** 47.2 1 1 . 5 "
increases of MDA levels differed in each muscle. This can be explained by variation in degree of activity of muscIes during swimming exercise in rats; it is possible that upper extremity muscles. for example, triceps brachi, are more active than lower extremities during prolonged swimming, in accordance with MDA levels. Our results and several reports (Alessio et al. 1988; Davies et a%.1982; Brady et al. 1979; Sumida et a&. 1989; Duthie et a&.1990) strongly suggest that acute exercise of sufficient intensity can increase active oxygen radical species, which accelerates lipid peroxidation and related reactions. We suggest that the degree of lipid peroxidation in exercised muscles depends on the intensity and duration of exercise. In our experiment, a proportional increase in the levels of MDA in each group supports this view. Increased oxygen utilization during exercise causes increased mitochondrial Ow2-production and haemoglobin autoxidation (Davies et a&. 19821, and any resulting enhanced leakage of CIS2- could promote lipid peroxidation and tissue damage (Duthie et al. 1990). In addition, ischemia occurs in some tissues during acute heavy exercise. Thus Kellog and Fridovich (1975) have indicated that reperfusion of hypoxic tissues may cause free-radical production by the xanthine oxidase system. Sumida et al. (1989) hypothesized that generation of free radicals during acute heavy exercise may occur not only via the mitochondrial respiratory chain but also through activation of the xanthine oxidase system. According to Stanley et al. (B985), the more intense the exercise the more lactate is produced. Since appearance rate of lactate exceeds the disappearance rate at maximal work loads, this indicates a possible drop in the concentrations of cytoplasmic NADH and (or) NABPH (Lowlin et al. 1987). The oxidized glutathione is reduced to glutathione. This reaction is catalysed by glutathione reductase enzyme. Only NADPH is effective as a substrate for glutathione reductase in vivo. The known mechanism for the recovery of NADPH from NADH depends on the oxidation of glucose via the hexose monophosphate shunt (Clemens and Waller 1987). Consequently, at maximal exercise, the activity of free radical scavenging enzymes such as superoxide disrnutase, catalase, glutathione reductase, and glutathione peroxidase may be compromised, and substrates that generate free radicals would accumulate (Lowlin et al. 1987). Any stress on the system, such as hypoxia in tissues, which results in depletion of glycolytic substrates may cause a decrease in the generation of NADH and NADPH (Lowlin et aE. 1987). Erythrocyte MDA levels did not change immediately in groups 2, 3, and 4, because erythrocytes are endowed with a variety of extremely efficient protective antioxidant mechanisms that can block autoxiidation in vivo (Clemens and Waller 1987). These include enzyme systems such as superoxide dis-
mutase, catalase, glutathione reductase, and glutathione peroxidase and antioxidant substrates such as vitamin E, vitamin C, cemloplasmin, tramsferrin, apotransferrin, and haptoglobin (Clemens and Waller 1987; Chiu et al. 1989). Duthie et al. (1990) found that the susceptibility of erythrocytes to in vitro peroxidation did not change immediately up to 24 h postexercise in runners, but increased after 24 h. Results of our study are similar to those of Duthie et al. Immediately afier exercise there were decreased AA concentrations in all exercised groups, which were related to exercise durations. It has been suggested that, especially as a result of intensive endurance activity, the body may become vitamin C depleted, and studies have shown that vitamin C is released from the liver and adrenal glands in some animal species after exercise (Brouns and Saris 1989). Immediately after exercise, hematocrit levels significantly increased in groups 3 and 4. These increases in hematocrit can be explained with hemsconcentration, where some fluid leaks out of the vessels, which results in increases in haemoglobin. erythrocyte, and plasma protein levels in the blood and increases in the volume of interstitial fluid (Ganong 1989). Increased haemoglobin levels were not significant in any of the exercise groups. Repeated muscle contraction during strenuous exercise can reduce the plasma volume by as much as 10 to 20% in three ways: it increases pressure in capilleries by compressing venules in muscles; it generates metabolites such as lactic acid, which increase osmotic pressure in the muscle tissue surrounding the capilleries, and these forces drive plasma water into the tissues; and much of the fluid lost in sweat comes from plasma (Eichner 1991). Although an increase of 17.3% hematocrit exists in group 3, increases of group 4 are less than increases of group 3 (1 1.5%). In addition to hematocrit values, semm myoglobin concentration significantly increased in group 4. Increased semm myoglobin levels were regularly found after performance of long-term exercise. Exercise-induced leakage of myoglobin from muscle cells may reduce the muscle oxygen store and the metabolic capacity of the muscle cell. Moreover, increases in plasma creatine kinase activity may indicate that muscle damage occurs by a mechanism that influences exercise-induced damage (Duthie et al. % 990). Although acute exercise leads to increased lipid peroxidation in skeletal muscle, liver, and semm, chronic aerobic training has been reported to help reduce lipid peroxidation by increasing defence capabilities (Alessio and Goldfarb 1988; Salminen and Vihko 1983; Higuchi et a&.1985; Ghandwaney and Ji 1992; Kanter et a&. 1985). Our results indicated that acute swimming exercise increased muscle lipid peroxidation, serum myoglobin concentration, haemoglobin, and hematocrit values and decreased plasma AA concentrations but did not affect erythrocyte lipid peroxidation.
KOZ BT AL.
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