- Email: [email protected]
Physical exercise, oxidative stress and muscle damage in racehorses
Comparative Biochemistry and Physiology Part B 119 (1998) 833 – 836
Physical exercise, oxidative stress and muscle damage in racehorses E. Chiaradia a, L. Avellini a, F. Rueca b, A. Spaterna b, F. Porciello b, M.T. Antonioni b, A. Gaiti a,* a
Istituto di Biochimica e Chimica Medica, Uni6ersity of Perugia, Via del Giochetto, 06122 Perugia, Italy b Istituto Patologia Speciale e Clinica Medica Veterinaria, Uni6ersity of Perugia, Perugia, Italy Received 8 August 1997; received in revised form 17 February 1998; accepted 26 February 1998
Abstract Since it has been suggested that lipid peroxidation following free radical overproduction may be one of the causes of physical exercise-induced myopathies and hemolysis in horses, we looked for the possible relationships between these phenomena and muscle fiber damage. We use a homogeneous group of Maremmana stallions which, after a 3-month training period, underwent a series of physical exercises of increasing intensity. We determined the contents of malondialdehyde (MDA), one of the main lipid peroxidation end-products, and glutathione the substrate of one of the most important free radical scavenger enzymes. We also measured creatine phosphokinase and serum lactate dehydrogenase isoenzyme activities whose modification may be indicative of muscle fiber damage. The results obtained indicated that the physical exercise we adopted was able to modify both MDA and glutathione contents in blood. However, its effect on some LDH isoenzyme activities suggested possible damage to tissues other than muscle. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Creatine kinase; Glutathione; Lactate dehydrogenase; Malondialdehyde; Muscle damage; Peroxidative stress; Physical exercise; Racehorse
1. Introduction The causes of oxidative stress (i.e. generation of an excess of reactive oxygen species) are many. Among others, physical exercise can cause oxidative stress, and it has been suggested that the pathogenesis of exerciseinduced myopathies and hemolysis in horses may be related to changes in lipid peroxidation caused by free radicals generated during either acute or chronic physical exercise [6,8,16,19]. Enzymatic (superoxide dismutase, glutathione peroxidase, etc.) and non-enzymatic (e.g. vitamin E) antioxidants usually protect tissues from excessive oxidative damage [2,5,14]. Depletion of any of the antioxidant systems increases the vulnerability of tissues and cellular components to reactive * Corresponding author. Fax: + 39 75 5853428; e-mail: [email protected] 0305-0491/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)10001-9
oxygen species, while tissues seem to increase their antioxidant defences under chronic activation. In this regard, controlled training may be one of the ways of increasing antioxidant defences in the tissues of subjects undergoing physical exercise. The aim of this study was to look for possible relationships between physical exercise, lipid peroxidation and muscle fibre damage in trained horses. To this end, we report the results obtained while studying the effect of a physical exercise test on a group of 3-yearold Maremmana stallions after a 3-month training period. In particular, we determined both malondialdehyde (MDA) and glutathione levels in the blood of these animals after a series of physical exercises of increasing intensity and tried to correlate them with plasma creatine phosphokinase (CK) and lactate dehydrogenase (LDH) isoenzyme activities whose modification may be indicative of muscle fiber damage [17].
834
E. Chiaradia et al. / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 833–836
Fig. 1. MDA (a) and glutathione (b) contents in the plasma. T0, before exercise; T1, immediately after limbering up; T2, after the ride; T3, 18 h after exercise. The values are presented as mean 9 S.D. Significant differences were computed by ANOVA.
2. Materials and methods
2.1. Animals Ten 3-year-old Maremmana stallions (Equus caballus) were selected on the basis of genetic screening and clinical check-up to determine their health and their suitability to be used as stallions. The animals were fed with a diet that was considered adequate for light work from the caloric point of view (about seven fodder units per day per horse [13]), which also ensured at least 12 mg of selenium and 1000 IU of vitamin E per day. The horses underwent physical training for 3 months, 30 min a day, 6 days a week, and the relative intensity of exercise was gradually increased. The period of training was followed by a series of tests especially established to evaluate the suitability of the horses for show-jumping. In particular, this test consists of a series of physical exercises of increasing intensity: after a session of 8 min of limbering up (a series of six jumps without rider on the riding track), the horses were forced to gallop twice (10 min between one run and other) with rider over a distance of 200 m. In the first trial, the average speed was 7229 104 m min − 1; in the second trial, the speed was significantly increased by 10% (P B0.0004). Blood samples were collected by jugular venipuncture before the exercise (T0), immediately after the limbering up (T1), after the ride (T2) and 18 h after exercise (T3).
2.2. Chemical and enzymatic analyses The serum enzymes CK and LDH were assayed with an automatic analyzer (Super Z818; SCLAVO, Italy) and the SCLAVO kits CK-NAC and LDH-P [1]. The isoenzymes of CK and LDH activities were separated electrophoretically on agarose gel using kits from SEBIA (Issy les Moulineaux, France)
After collection, the plasma was immediately separated from the blood by centrifugation. The plasma was mixed with either butylated hydroxytoluene (BHT) or 5,5%-dithio-bis-(2-nitrobenzoic acid) (Ellman’s reagent) and than stored at − 30°C until the MDA or the glutathione assay, respectively. Total glutathione, which included both reduced glutathione and glutathione disulphide, content of the plasma was measured using the glutathione reductase– Ellman’s reagent recirculating assay [7,18]. Due to the very low amount of glutathione present in the plasma, the above enzymatic cycling was used as it continuously reduces glutathione–Ellman’s adduct using NADPH. The MDA content in the blood, as a measure of lipid peroxidation end-products, was assayed by separating the MDA–thiobarbituric acid adduct by high-performance liquid chromatoraphy HPLC and using fluorescence detection [3].
2.3. Statistical analyses The values are represented as mean9 S.D. Significant differences between pre- and post-exercise values on individual and consecutive days were computed by analysis of variance (ANOVA) with Fischer (significance at 95%) post hoc test.
3. Results
3.1. MDA and glutathione content in the plasma In Fig. 1 we report the results obtained by testing the MDA (Fig. 1a) and glutathione (Fig. 1b) contents in the plasma (ANOVA: PB0.0001). The MDA content in plasma significantly increases only after the gallop. The pattern of the glutathione content in the plasma is similar at T0 and T1, increases at T2 and decreases to pre-exercise level at T3.
E. Chiaradia et al. / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 833–836
835
Fig. 2. LDH-3 and LDH-5 isoenzyme activities in horse serum. T0, before exercise; T1, immediately after limbering up; T2, after the ride; T3, 18 h after exercise.
3.2. LDH isoenzymes in the serum
4. Discussion
Fig. 2 shows the slopes of the activity of LDH-3 and LDH-5 isoenzymes in the serum. The LDH-3 activity content shows a dramatic increase after the physical exercise, while the LDH-5 increase is low. The LDH-4 values found at the different times were very low (B 15 U l − 1) and not statistically significant (data not shown). In Fig. 3, we report the slope of the LDH-1/LDH-2 ratio. The ratio increases about five times after the gallop and rapidly decreases within the 18 h of rest.
Lipid peroxidation is a complex phenomenon [9] involving the generation of many products. However, the content of malondialdehyde (MDA), one of most important end-products of lipid peroxidation, in the tissues is usually accepted as an index of lipid peroxidation intensity. The MDA content in the plasma (Fig. 1a) confirms that: (1) physical exercise is able to generate free radicals which, in turn, cause lipid peroxidation; (2) the effect is related to the intensity of the exercise (T0 compared to T1 and T2); (3) the elimination of lipid peroxidation products is a slow process (T2 compared to T3). Glutathione is generally considered to be a multifunctional antioxidant which links the different scavenger systems (lipophilic/hydrophilic, intracellular/ extracellular). As a consequence, glutathione content in the blood may be used as one of the indices of the total antioxidant capacity of the body [4,8]. The glutathione content (Fig. 1b) in the plasma confirms the above because it shows a trend similar to that of MDA except for T3. After an oxidative stress, glutathione is released in the blood; the oxidized form (GSSG) is transferred from the cells to the liver to be reduced and the reduced form (GSH) is then released by the liver to support increased requirement of cells for this substrate which is necessary for the activity of glutathione peroxidase [4,8]. Lipid peroxidation caused by the physical exercise in our conditions is unable to cause significant cell damage to skeletal muscle as demonstrated by the absence of changes in the CK-MM activity in the blood as well as LDH isoenzyme 4 (data not shown), which is considered characteristic of skeletal muscle. The increased content of isoenzyme LDH-5 is also considered to be a marker of muscle damage; however, the increase in LDH-5 activity found (Fig. 2b) is statistically signifi-
3.3. CK-MM isoenzyme in the serum No statistically significant modification in CK-MM isoenzyme was found (U l − 1): T0, 75.7597.96; T1, 76.599 6.68; T2, 80.5193.52; T3, 78.6094.37.
Fig. 3. LDH-1/LDH-2 ratio. T0, before exercise; T1, immediately after limbering up; T2, after the ride; T3, 18 h after exercise. The data were transformed in arcsin before statistical evaluation.
836
E. Chiaradia et al. / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 833–836
cant, but too low to be considered of any clinical meaning, also because this isoenzyme might be released by the liver [15]. In contrast, the LDH-1/LDH-2 ratio [15] seems to indicate that the first session of challenging physical exercise is able to cause moderate but significant injury to the heart cells even if, on the basis of our present knowledge, we could not discard other hypotheses. In consideration of the reduced cardiac mass/body mass ratio of Maremmana stallions compared with that of other racehorse breeds [10,11] (personal observations), this damage might be caused by short-term hypoxemia during the above session. On the other hand, we believe we can exclude that LDH-1 increase might be caused by hemolysis because the content in the serum of the other isoenzyme activities present in the erythrocytes (i.e. LDH-4 and LDH-5) was respectively low and unchanged or moderately modified by physical exercise. Finally, the impressive increase in the LDH-3 content in the blood is more difficult to explain. Indeed, this isoenzyme is mainly localized in the lungs, kidney and spleen [12,17], but these organs are not usually considered the first target of free radical attack. Nevertheless, we may hypothesize that the first session of challenging physical exercise in our horses was also able to induce cell damage in the above organs. Such damage may have been induced by a variety of causes such as over increased respiratory and blood flow rates due to the smaller heart size of these animals as compared with other breeds of racehorses. In conclusion, regarding our working hypothesis, our results seem to indicate that in trained horses: (1) acute exercise (able to increase lactate content in the serum over 4 mmol l − 1; data not shown) generates free radicals and, as a consequence, lipid peroxidation, as demonstrated by the increase in both MDA and glutathione contents in the plasma (Fig. 1a,b; T2 compared to T0); (2) 18 h of rest are insufficient to remove all the lipid peroxidation products from the body (Fig. 1a; T3 compared to T0 and T2) and, without an adequate rest period, this may cause an accumulation of dangerous substances which, in turn, may ultimately damage the body structure; (3) the lipid peroxidation following exercise of the intensity and duration examined is insufficient to cause a significant lysis of skeletal muscle cells; (4) light exercise does not cause oxidative stress as demonstrated by the lack of both MDA and glutathione increase in the plasma (T0 compared to T1; Fig. 1a,b). This last result may be of some importance in evaluating the level of training by correlating the content of glutathione and MDA in the blood with the degree of physical exercise, although more evidence is needed to do this.
Acknowledgements We are grateful to the Racehorse Study Centre, University of Perugia, for technical support. This work was supported in part by a grant from the National Research Council, Rome (CNR 95/0277CT04), and MURST.
References [1] Bergmeyer HU. Methods of Enzymatic Analysis, vol. 1. New York: Academic Press, 1974. [2] Chow CK. Vitamin E and oxidative stress. Free Radic Biol Med 1991;11:215 – 32. [3] Draper HH, Squire EJ, Mahmoody H, Wu J, Agarwal S, Hadley MA. Comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radic Biol Med 1993;15:353 – 63. [4] Gohil K, Viguie C, Stanley WC, Brooks GA, Packer L. Blood glutathione during human exercise. J Appl Physiol 1988;64:115– 9. [5] Halliwell B, Gutteridge JMC. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990;280:1 – 8. [6] Hodgson DR. Exercise-associated myopathy: is calcium the culprit? Equine Vet J 1993;25:1 – 3. [7] Hughes H, Hartmut J, Mitchell JR. Measurement of oxidant stress in vivo. Methods Enzymol 1990;186:681 – 5. [8] Ji LL. Oxidative stress during exercise: implication of antioxidant nutrients. Free Radic Biol Med 1995;18:1079 – 86. [9] Kagan VE. Lipid Peroxidation in Biomembranes. Boca Raton, FL: CRC Press, 1988. [10] Kubo K, Senta T, Sugimoto O. Relationship between training and heart in the thoroughbred racehorse. Exp Rep Equine Health Lab 1974;11:87 – 93. [11] Kuramoto K, Shiraishi A, Nakanishi Y, Kai M, Ueno Y, Ueda Y. Application of echocardiography for assessing left ventricular function of thoroughbred horses at resting stage. Bull Equine Res Inst 1989;26:23 – 30. [12] Littlejohn A, Blackmore DJ. Blood and tissue content of the iso-enzymes of lactate dehydrogenase in the thoroughbred. Res Vet Sci 1978;25:118 – 9. [13] Martin-Rosset W. L’Alimentation des Chevaux. Paris: INRA Press, 1990. [14] Michiels C, Raes M, Toussiant O, Remacle J. Importance of Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress. Free Radic Biol Med 1994;17:235 – 48. [15] Moss DW. Multiple forms of enzymes in diagnostic enzymology. In: Moss DW, editor. Isoenzymes. London: Chapman & Hall, 1982:159 – 184. [16] Ono K, Inui K, Hasegawa T, Matsuki N, Watanabe H, Takagi S, Hasegawa A. The change of antioxidative enzyme activities in equine erythrocytes following exercise. Jpn J Vet Sci 1990;52:759 – 65. [17] Thornton JR, Lohni MD. Tissue and plasma activity of lactate dehydrogenase and creatine kinase in the horse. Equine Vet J 1979;11:235 – 8. [18] Tieze F. Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969;27:502 – 22. [19] Valberg S, Ha¨ggendal J, Lindholm A. Blood chemistry and skeletal muscle metabolic responses to exercise in horses with recurrent exertional rhabdomyolysis. Equine Vet J 1993;25:17– 22.
Physical exercise, oxidative stress and muscle damage in racehorses E. Chiaradia a, L. Avellini a, F. Rueca b, A. Spaterna b, F. Porciello b, M.T. Antonioni b, A. Gaiti a,* a
Istituto di Biochimica e Chimica Medica, Uni6ersity of Perugia, Via del Giochetto, 06122 Perugia, Italy b Istituto Patologia Speciale e Clinica Medica Veterinaria, Uni6ersity of Perugia, Perugia, Italy Received 8 August 1997; received in revised form 17 February 1998; accepted 26 February 1998
Abstract Since it has been suggested that lipid peroxidation following free radical overproduction may be one of the causes of physical exercise-induced myopathies and hemolysis in horses, we looked for the possible relationships between these phenomena and muscle fiber damage. We use a homogeneous group of Maremmana stallions which, after a 3-month training period, underwent a series of physical exercises of increasing intensity. We determined the contents of malondialdehyde (MDA), one of the main lipid peroxidation end-products, and glutathione the substrate of one of the most important free radical scavenger enzymes. We also measured creatine phosphokinase and serum lactate dehydrogenase isoenzyme activities whose modification may be indicative of muscle fiber damage. The results obtained indicated that the physical exercise we adopted was able to modify both MDA and glutathione contents in blood. However, its effect on some LDH isoenzyme activities suggested possible damage to tissues other than muscle. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Creatine kinase; Glutathione; Lactate dehydrogenase; Malondialdehyde; Muscle damage; Peroxidative stress; Physical exercise; Racehorse
1. Introduction The causes of oxidative stress (i.e. generation of an excess of reactive oxygen species) are many. Among others, physical exercise can cause oxidative stress, and it has been suggested that the pathogenesis of exerciseinduced myopathies and hemolysis in horses may be related to changes in lipid peroxidation caused by free radicals generated during either acute or chronic physical exercise [6,8,16,19]. Enzymatic (superoxide dismutase, glutathione peroxidase, etc.) and non-enzymatic (e.g. vitamin E) antioxidants usually protect tissues from excessive oxidative damage [2,5,14]. Depletion of any of the antioxidant systems increases the vulnerability of tissues and cellular components to reactive * Corresponding author. Fax: + 39 75 5853428; e-mail: [email protected] 0305-0491/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)10001-9
oxygen species, while tissues seem to increase their antioxidant defences under chronic activation. In this regard, controlled training may be one of the ways of increasing antioxidant defences in the tissues of subjects undergoing physical exercise. The aim of this study was to look for possible relationships between physical exercise, lipid peroxidation and muscle fibre damage in trained horses. To this end, we report the results obtained while studying the effect of a physical exercise test on a group of 3-yearold Maremmana stallions after a 3-month training period. In particular, we determined both malondialdehyde (MDA) and glutathione levels in the blood of these animals after a series of physical exercises of increasing intensity and tried to correlate them with plasma creatine phosphokinase (CK) and lactate dehydrogenase (LDH) isoenzyme activities whose modification may be indicative of muscle fiber damage [17].
834
E. Chiaradia et al. / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 833–836
Fig. 1. MDA (a) and glutathione (b) contents in the plasma. T0, before exercise; T1, immediately after limbering up; T2, after the ride; T3, 18 h after exercise. The values are presented as mean 9 S.D. Significant differences were computed by ANOVA.
2. Materials and methods
2.1. Animals Ten 3-year-old Maremmana stallions (Equus caballus) were selected on the basis of genetic screening and clinical check-up to determine their health and their suitability to be used as stallions. The animals were fed with a diet that was considered adequate for light work from the caloric point of view (about seven fodder units per day per horse [13]), which also ensured at least 12 mg of selenium and 1000 IU of vitamin E per day. The horses underwent physical training for 3 months, 30 min a day, 6 days a week, and the relative intensity of exercise was gradually increased. The period of training was followed by a series of tests especially established to evaluate the suitability of the horses for show-jumping. In particular, this test consists of a series of physical exercises of increasing intensity: after a session of 8 min of limbering up (a series of six jumps without rider on the riding track), the horses were forced to gallop twice (10 min between one run and other) with rider over a distance of 200 m. In the first trial, the average speed was 7229 104 m min − 1; in the second trial, the speed was significantly increased by 10% (P B0.0004). Blood samples were collected by jugular venipuncture before the exercise (T0), immediately after the limbering up (T1), after the ride (T2) and 18 h after exercise (T3).
2.2. Chemical and enzymatic analyses The serum enzymes CK and LDH were assayed with an automatic analyzer (Super Z818; SCLAVO, Italy) and the SCLAVO kits CK-NAC and LDH-P [1]. The isoenzymes of CK and LDH activities were separated electrophoretically on agarose gel using kits from SEBIA (Issy les Moulineaux, France)
After collection, the plasma was immediately separated from the blood by centrifugation. The plasma was mixed with either butylated hydroxytoluene (BHT) or 5,5%-dithio-bis-(2-nitrobenzoic acid) (Ellman’s reagent) and than stored at − 30°C until the MDA or the glutathione assay, respectively. Total glutathione, which included both reduced glutathione and glutathione disulphide, content of the plasma was measured using the glutathione reductase– Ellman’s reagent recirculating assay [7,18]. Due to the very low amount of glutathione present in the plasma, the above enzymatic cycling was used as it continuously reduces glutathione–Ellman’s adduct using NADPH. The MDA content in the blood, as a measure of lipid peroxidation end-products, was assayed by separating the MDA–thiobarbituric acid adduct by high-performance liquid chromatoraphy HPLC and using fluorescence detection [3].
2.3. Statistical analyses The values are represented as mean9 S.D. Significant differences between pre- and post-exercise values on individual and consecutive days were computed by analysis of variance (ANOVA) with Fischer (significance at 95%) post hoc test.
3. Results
3.1. MDA and glutathione content in the plasma In Fig. 1 we report the results obtained by testing the MDA (Fig. 1a) and glutathione (Fig. 1b) contents in the plasma (ANOVA: PB0.0001). The MDA content in plasma significantly increases only after the gallop. The pattern of the glutathione content in the plasma is similar at T0 and T1, increases at T2 and decreases to pre-exercise level at T3.
E. Chiaradia et al. / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 833–836
835
Fig. 2. LDH-3 and LDH-5 isoenzyme activities in horse serum. T0, before exercise; T1, immediately after limbering up; T2, after the ride; T3, 18 h after exercise.
3.2. LDH isoenzymes in the serum
4. Discussion
Fig. 2 shows the slopes of the activity of LDH-3 and LDH-5 isoenzymes in the serum. The LDH-3 activity content shows a dramatic increase after the physical exercise, while the LDH-5 increase is low. The LDH-4 values found at the different times were very low (B 15 U l − 1) and not statistically significant (data not shown). In Fig. 3, we report the slope of the LDH-1/LDH-2 ratio. The ratio increases about five times after the gallop and rapidly decreases within the 18 h of rest.
Lipid peroxidation is a complex phenomenon [9] involving the generation of many products. However, the content of malondialdehyde (MDA), one of most important end-products of lipid peroxidation, in the tissues is usually accepted as an index of lipid peroxidation intensity. The MDA content in the plasma (Fig. 1a) confirms that: (1) physical exercise is able to generate free radicals which, in turn, cause lipid peroxidation; (2) the effect is related to the intensity of the exercise (T0 compared to T1 and T2); (3) the elimination of lipid peroxidation products is a slow process (T2 compared to T3). Glutathione is generally considered to be a multifunctional antioxidant which links the different scavenger systems (lipophilic/hydrophilic, intracellular/ extracellular). As a consequence, glutathione content in the blood may be used as one of the indices of the total antioxidant capacity of the body [4,8]. The glutathione content (Fig. 1b) in the plasma confirms the above because it shows a trend similar to that of MDA except for T3. After an oxidative stress, glutathione is released in the blood; the oxidized form (GSSG) is transferred from the cells to the liver to be reduced and the reduced form (GSH) is then released by the liver to support increased requirement of cells for this substrate which is necessary for the activity of glutathione peroxidase [4,8]. Lipid peroxidation caused by the physical exercise in our conditions is unable to cause significant cell damage to skeletal muscle as demonstrated by the absence of changes in the CK-MM activity in the blood as well as LDH isoenzyme 4 (data not shown), which is considered characteristic of skeletal muscle. The increased content of isoenzyme LDH-5 is also considered to be a marker of muscle damage; however, the increase in LDH-5 activity found (Fig. 2b) is statistically signifi-
3.3. CK-MM isoenzyme in the serum No statistically significant modification in CK-MM isoenzyme was found (U l − 1): T0, 75.7597.96; T1, 76.599 6.68; T2, 80.5193.52; T3, 78.6094.37.
Fig. 3. LDH-1/LDH-2 ratio. T0, before exercise; T1, immediately after limbering up; T2, after the ride; T3, 18 h after exercise. The data were transformed in arcsin before statistical evaluation.
836
E. Chiaradia et al. / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 833–836
cant, but too low to be considered of any clinical meaning, also because this isoenzyme might be released by the liver [15]. In contrast, the LDH-1/LDH-2 ratio [15] seems to indicate that the first session of challenging physical exercise is able to cause moderate but significant injury to the heart cells even if, on the basis of our present knowledge, we could not discard other hypotheses. In consideration of the reduced cardiac mass/body mass ratio of Maremmana stallions compared with that of other racehorse breeds [10,11] (personal observations), this damage might be caused by short-term hypoxemia during the above session. On the other hand, we believe we can exclude that LDH-1 increase might be caused by hemolysis because the content in the serum of the other isoenzyme activities present in the erythrocytes (i.e. LDH-4 and LDH-5) was respectively low and unchanged or moderately modified by physical exercise. Finally, the impressive increase in the LDH-3 content in the blood is more difficult to explain. Indeed, this isoenzyme is mainly localized in the lungs, kidney and spleen [12,17], but these organs are not usually considered the first target of free radical attack. Nevertheless, we may hypothesize that the first session of challenging physical exercise in our horses was also able to induce cell damage in the above organs. Such damage may have been induced by a variety of causes such as over increased respiratory and blood flow rates due to the smaller heart size of these animals as compared with other breeds of racehorses. In conclusion, regarding our working hypothesis, our results seem to indicate that in trained horses: (1) acute exercise (able to increase lactate content in the serum over 4 mmol l − 1; data not shown) generates free radicals and, as a consequence, lipid peroxidation, as demonstrated by the increase in both MDA and glutathione contents in the plasma (Fig. 1a,b; T2 compared to T0); (2) 18 h of rest are insufficient to remove all the lipid peroxidation products from the body (Fig. 1a; T3 compared to T0 and T2) and, without an adequate rest period, this may cause an accumulation of dangerous substances which, in turn, may ultimately damage the body structure; (3) the lipid peroxidation following exercise of the intensity and duration examined is insufficient to cause a significant lysis of skeletal muscle cells; (4) light exercise does not cause oxidative stress as demonstrated by the lack of both MDA and glutathione increase in the plasma (T0 compared to T1; Fig. 1a,b). This last result may be of some importance in evaluating the level of training by correlating the content of glutathione and MDA in the blood with the degree of physical exercise, although more evidence is needed to do this.
Acknowledgements We are grateful to the Racehorse Study Centre, University of Perugia, for technical support. This work was supported in part by a grant from the National Research Council, Rome (CNR 95/0277CT04), and MURST.
References [1] Bergmeyer HU. Methods of Enzymatic Analysis, vol. 1. New York: Academic Press, 1974. [2] Chow CK. Vitamin E and oxidative stress. Free Radic Biol Med 1991;11:215 – 32. [3] Draper HH, Squire EJ, Mahmoody H, Wu J, Agarwal S, Hadley MA. Comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radic Biol Med 1993;15:353 – 63. [4] Gohil K, Viguie C, Stanley WC, Brooks GA, Packer L. Blood glutathione during human exercise. J Appl Physiol 1988;64:115– 9. [5] Halliwell B, Gutteridge JMC. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990;280:1 – 8. [6] Hodgson DR. Exercise-associated myopathy: is calcium the culprit? Equine Vet J 1993;25:1 – 3. [7] Hughes H, Hartmut J, Mitchell JR. Measurement of oxidant stress in vivo. Methods Enzymol 1990;186:681 – 5. [8] Ji LL. Oxidative stress during exercise: implication of antioxidant nutrients. Free Radic Biol Med 1995;18:1079 – 86. [9] Kagan VE. Lipid Peroxidation in Biomembranes. Boca Raton, FL: CRC Press, 1988. [10] Kubo K, Senta T, Sugimoto O. Relationship between training and heart in the thoroughbred racehorse. Exp Rep Equine Health Lab 1974;11:87 – 93. [11] Kuramoto K, Shiraishi A, Nakanishi Y, Kai M, Ueno Y, Ueda Y. Application of echocardiography for assessing left ventricular function of thoroughbred horses at resting stage. Bull Equine Res Inst 1989;26:23 – 30. [12] Littlejohn A, Blackmore DJ. Blood and tissue content of the iso-enzymes of lactate dehydrogenase in the thoroughbred. Res Vet Sci 1978;25:118 – 9. [13] Martin-Rosset W. L’Alimentation des Chevaux. Paris: INRA Press, 1990. [14] Michiels C, Raes M, Toussiant O, Remacle J. Importance of Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress. Free Radic Biol Med 1994;17:235 – 48. [15] Moss DW. Multiple forms of enzymes in diagnostic enzymology. In: Moss DW, editor. Isoenzymes. London: Chapman & Hall, 1982:159 – 184. [16] Ono K, Inui K, Hasegawa T, Matsuki N, Watanabe H, Takagi S, Hasegawa A. The change of antioxidative enzyme activities in equine erythrocytes following exercise. Jpn J Vet Sci 1990;52:759 – 65. [17] Thornton JR, Lohni MD. Tissue and plasma activity of lactate dehydrogenase and creatine kinase in the horse. Equine Vet J 1979;11:235 – 8. [18] Tieze F. Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969;27:502 – 22. [19] Valberg S, Ha¨ggendal J, Lindholm A. Blood chemistry and skeletal muscle metabolic responses to exercise in horses with recurrent exertional rhabdomyolysis. Equine Vet J 1993;25:17– 22.