Tohoku
J. Exp.
Med., 1992,
167, 301-303
Short Effects
of Brief
Concentrations Dismutase
Physical
Exercise
on
of Immunoreactive Isoenzymes
Report
the
Superoxide
in Human
Plasma
HIDEKIOHNO,HITOSHIYAMASHITA, T0M0MIOOKAWARA, DAIZOSAITOH*, KAZUOMIMURA* and NAOYUKI TANIGUCHIt Departments of Hygiene and *Emergency,National Defense Medical College, Tokorozawa 359, and 'Department of Biochemistry,Osaka University Medical School,Suita 565
OHNO, H., YAMASHITA, H., OOKAWARA, T., SAITOH,D., MIMURA,K. and TANIGUCHI,N. Effects of Brief Physical Exercise on the Concentrations of Immunoreactive Superoxide Dismutase Isoenzymes in Human Plasma. Tohoku J. Exp. Med., 1992, 167 (4), 301-303 Effects of cycle ergometer exercise (- 75% Vo2max for 15 min) on the concentrations of immunoreactive Mn- and CuZnsuperoxide dismutases (SOD) in plasma were studied on 10 male students. During the experimental period, Mn-SOD concentration did not vary substantially. On the other hand, CuZn-SOD concentration decreased markedly at 15 min and 24 hr after the exercise ; that is, CuZn-SOD appeared to differ virtually from Mn-SOD in recovery pattern, physical exercise ; Mn-superoxide dismutase ; CuZnsuperoxide dismutase ; ELISA Superoxide dismutases (SOD) catalyze the dismutation of superoxide anion to molecular oxygen and hydrogen peroxide, and it is likely that they protect cells against the potential toxicity of free radical produced from oxygen. In mammalian tissues, two main forms of SOD are found : CuZn-SOD is predominantly located in the cytosol, whereas Mn-SOD appears to be localized mostly in the mitochondrial matrix (Fridovich 1975). The oxygen uptake of skeletal muscles may increase up to 100-200 times during strenuous exercise. The possibility that this could cause oxidative tissue damage including skeletal muscles is upheld by much experimental work (Clark et al. 1985 ; Jenkins 1988). Dillard et al. (1978), for example, were the first to show that pentane (which might be formed as a lipid peroxidation product) was increased in the expired gas of human subjects who had exercised at 50% Vo2max. However, although the effect of an acute bout of exercise in animals has uniformly resulted in an increase in tissue lipid peroxidation, the data on lipid peroxidation in humans is sparse and not at all in agreement (Jenkins 1988). Likewise, there is little available information concerning the effect of physical exercise on SOD in humans (Jenkins 1988). In specialty, our recent study seems to be the only published work to date on Mn-SOD in humans, in which it was demonstrated that athletes had a significantly higher plasma level of immunoreactive Mn-SOD as compared to sedentary individuals (Ohno et al. 1992). The present study was undertaken to determine the effect Received
August
10, 1992;
revision
Address for reprints : Hideki Ohno, Medical College, 3-2 Namiki, Tokorozawa
accepted
for publication
M.D., Department 359, Japan. 301
August
of Hygiene,
27, 1992. National
Defense
H. Ohno
302 TABLE 1.
Variations
in plasma
enzyme
et al.
values
and hematocrit
level at
75% Vo2max
on a cycle ergometer
of acute physical exercise on both immunoreactive SOD isoenzymes in human plasma, since activity might not reflect the true amount of the enzyme protein, due to the presence or absence of cofactors, activators and inhibitors. Ten healthy male students (volleyball players), aged 19-22 years, volunteered for this study after informed consent according to the Declaration of Helsinki. The exercise protocols, performed in the morning after an overnight fast, consisted of 15 min work periods on a Monark bicycle ergometer at 75% Vo2max. On the day preceding the experiment they were requested not to take exercise in order to minimize the residual effect of the last exercise. Heparinized blood samples were collected by venepuncture at the start and end of the exercise session and then 15 min and 24 hr later, respectively. Plasma was carefully aspirated off, leaving the buffy coat and packed erythrocytes and then stored at -80°C for 2 weeks until theptime of enzyme analysis. Measurements of immunoreactive Mn-SOD and CuZn-SOD were made by an ELISA with the use of a monoclonal antibody against each isoenzyme purified from human tissues, originally developed in our group (Oka et al. 1989 ; Kawaguchi et al. 1990). Mitochondrial aspartate aminotransferase (AST-m) was also immunologically separated from plasma with the tests "Eiken" (Eiken Chemical Company, Tokyo). The activities of AST-m and creatine kinase (CK) in plasma were determined on a kinetic enzyme analyzer using optimized AST and CK substrate reagents. Scheffe's test was applied to the data. In all statistical analyses, the 0.05 level of significance was used. The results are shown in Table 1. As was expected and previously reported (Forssell et al. 1975; Ohno et al. 1978), there were significant increases in plasma CK and AST-m activities as well as hematocrit level immediately after the exercise, suggesting some effect of acute physical exercise on skeletal muscles. Mn-SOD concentration did not vary substantially at any time. AST-m is localized in the mitochondrial matrix like Mn-SOD. It seems likely, therefore, that Mn-SOD and AST-m have different release mechanisms from tissues, probably from skeletal muscles, into the bloodstream during exercise. On the other hand, although CuZn-SOD concentration did not change significantly immediately after exercise, surprisingly, it was markedly decreased throughout the recovery period compared to the initial value ; CuZn-SOD appeared to differ virtually from Mn-SOD in the recovery pattern. The reduced plasma concentration of CuZu-SOD, the molecular weight of which 33,000 is smaller than that of Mn-SOD (88,000), could be the result of an increased glomerular permeability of which the mechanism remained obscure. Actually, significant increases in CuZn-SOD concentration were noted in urine collected from 10 healthy male high-school students immediately after 2-hr soccer training and at 30-min rest after it (data not shown) ;
during changes
the
experiment,
in plasma
total
Effects
of E
no
Mn-SOD
on Sup eroxi de Dismutases
xercise
SOD activity
exercise, however, seem to be required. line to clarify such points.
was
detected
and the renal
in urine. clearance
We are at present
303
Further
investigations
of CuZn-SOD continuing
after
the work
into physical
along
the
Acknowledgments We wish technical
to thank
Dr. T. Kawaguchi
(Ube Industries
Ltd.,
Ube) for his excellent
assistance. References
1) Clark, IA., Cowden, W.B. & Hunt, N.H. (1985) Free radical-induced pathology. Med. Res. Rev., 5, 297-332. 2) Dillard, C.J., Litov, RE., Savin, W.M., Dumelin, E.E. & Tappel, AL. (1978) Effects of exercise, vitamin E, and ozone on pulmonary function and lipid peroxidation. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol., 45, 927-932. 3) Fridovich, I. (1975) Superoxide dismutases. Ann. Rev. Biochem., 44, 147-159. 4) Forssell, G., Nordlander, R., Nyquist, 0., Qrinius, E. & Styrelius, I. (1975) Creatine phosphokinase after submaximal physical exercise in untrained individuals. Acta Med. &and., 197, 503-505. 5) Jenkins, R.R. (1988) Free radical chemistry. Relationship to exercise. Sports Med., 5, 156-170. 6) Kawaguchi, T., Suzuki, K., Matsuda, Y., Nishiura, T., Uda, T., Ono, M., Sekiya, C., Ishikawa, M., lino, S., Endo, Y. & Taniguchi, N. (1990) Serum-manganesesuperoxide dismutase : Normal values and increased levels in patients with acute myocardial infarction and several malignant diseases determined by an enzyme-linked immunosorbent assay using a monoclonal antibody. J. Immunol. Methods, 127, 249254. 7) Ohno, H., Watanabe, H., Kishihara, C., Taniguchi, N. & Takakuwa, E. (1978) Effect of physical exercise on the activity of GOT isozyme in human plasma. Tohoku J. Exp. Med., 126, 371-376. 8) Ohno, H., Yamashita, H., Ookawara, T., Saitoh, D., Wakabayashi, K. & Taniguchi, N. (1992) Training effects on concentrations of immunoreactive superoxide dismutase isoenzymes in human plasma. Acta Physiol. Scand., 146, 291-292. 9) Oka, S., Ogino, K., Matsuura, S., Yamamoto, K., Okazaki, Y., Takemoto, T., Kato, N. & Uda, T. (1989) Human serum immunoreactive copper, zinc-superoxide dismutase assayed with an enzyme monoclonal immunosorbent in patients with digestive cancer. Olin. Chim. Acta, 182, 209-220.
J. Exp.
Med., 1992,
167, 301-303
Short Effects
of Brief
Concentrations Dismutase
Physical
Exercise
on
of Immunoreactive Isoenzymes
Report
the
Superoxide
in Human
Plasma
HIDEKIOHNO,HITOSHIYAMASHITA, T0M0MIOOKAWARA, DAIZOSAITOH*, KAZUOMIMURA* and NAOYUKI TANIGUCHIt Departments of Hygiene and *Emergency,National Defense Medical College, Tokorozawa 359, and 'Department of Biochemistry,Osaka University Medical School,Suita 565
OHNO, H., YAMASHITA, H., OOKAWARA, T., SAITOH,D., MIMURA,K. and TANIGUCHI,N. Effects of Brief Physical Exercise on the Concentrations of Immunoreactive Superoxide Dismutase Isoenzymes in Human Plasma. Tohoku J. Exp. Med., 1992, 167 (4), 301-303 Effects of cycle ergometer exercise (- 75% Vo2max for 15 min) on the concentrations of immunoreactive Mn- and CuZnsuperoxide dismutases (SOD) in plasma were studied on 10 male students. During the experimental period, Mn-SOD concentration did not vary substantially. On the other hand, CuZn-SOD concentration decreased markedly at 15 min and 24 hr after the exercise ; that is, CuZn-SOD appeared to differ virtually from Mn-SOD in recovery pattern, physical exercise ; Mn-superoxide dismutase ; CuZnsuperoxide dismutase ; ELISA Superoxide dismutases (SOD) catalyze the dismutation of superoxide anion to molecular oxygen and hydrogen peroxide, and it is likely that they protect cells against the potential toxicity of free radical produced from oxygen. In mammalian tissues, two main forms of SOD are found : CuZn-SOD is predominantly located in the cytosol, whereas Mn-SOD appears to be localized mostly in the mitochondrial matrix (Fridovich 1975). The oxygen uptake of skeletal muscles may increase up to 100-200 times during strenuous exercise. The possibility that this could cause oxidative tissue damage including skeletal muscles is upheld by much experimental work (Clark et al. 1985 ; Jenkins 1988). Dillard et al. (1978), for example, were the first to show that pentane (which might be formed as a lipid peroxidation product) was increased in the expired gas of human subjects who had exercised at 50% Vo2max. However, although the effect of an acute bout of exercise in animals has uniformly resulted in an increase in tissue lipid peroxidation, the data on lipid peroxidation in humans is sparse and not at all in agreement (Jenkins 1988). Likewise, there is little available information concerning the effect of physical exercise on SOD in humans (Jenkins 1988). In specialty, our recent study seems to be the only published work to date on Mn-SOD in humans, in which it was demonstrated that athletes had a significantly higher plasma level of immunoreactive Mn-SOD as compared to sedentary individuals (Ohno et al. 1992). The present study was undertaken to determine the effect Received
August
10, 1992;
revision
Address for reprints : Hideki Ohno, Medical College, 3-2 Namiki, Tokorozawa
accepted
for publication
M.D., Department 359, Japan. 301
August
of Hygiene,
27, 1992. National
Defense
H. Ohno
302 TABLE 1.
Variations
in plasma
enzyme
et al.
values
and hematocrit
level at
75% Vo2max
on a cycle ergometer
of acute physical exercise on both immunoreactive SOD isoenzymes in human plasma, since activity might not reflect the true amount of the enzyme protein, due to the presence or absence of cofactors, activators and inhibitors. Ten healthy male students (volleyball players), aged 19-22 years, volunteered for this study after informed consent according to the Declaration of Helsinki. The exercise protocols, performed in the morning after an overnight fast, consisted of 15 min work periods on a Monark bicycle ergometer at 75% Vo2max. On the day preceding the experiment they were requested not to take exercise in order to minimize the residual effect of the last exercise. Heparinized blood samples were collected by venepuncture at the start and end of the exercise session and then 15 min and 24 hr later, respectively. Plasma was carefully aspirated off, leaving the buffy coat and packed erythrocytes and then stored at -80°C for 2 weeks until theptime of enzyme analysis. Measurements of immunoreactive Mn-SOD and CuZn-SOD were made by an ELISA with the use of a monoclonal antibody against each isoenzyme purified from human tissues, originally developed in our group (Oka et al. 1989 ; Kawaguchi et al. 1990). Mitochondrial aspartate aminotransferase (AST-m) was also immunologically separated from plasma with the tests "Eiken" (Eiken Chemical Company, Tokyo). The activities of AST-m and creatine kinase (CK) in plasma were determined on a kinetic enzyme analyzer using optimized AST and CK substrate reagents. Scheffe's test was applied to the data. In all statistical analyses, the 0.05 level of significance was used. The results are shown in Table 1. As was expected and previously reported (Forssell et al. 1975; Ohno et al. 1978), there were significant increases in plasma CK and AST-m activities as well as hematocrit level immediately after the exercise, suggesting some effect of acute physical exercise on skeletal muscles. Mn-SOD concentration did not vary substantially at any time. AST-m is localized in the mitochondrial matrix like Mn-SOD. It seems likely, therefore, that Mn-SOD and AST-m have different release mechanisms from tissues, probably from skeletal muscles, into the bloodstream during exercise. On the other hand, although CuZn-SOD concentration did not change significantly immediately after exercise, surprisingly, it was markedly decreased throughout the recovery period compared to the initial value ; CuZn-SOD appeared to differ virtually from Mn-SOD in the recovery pattern. The reduced plasma concentration of CuZu-SOD, the molecular weight of which 33,000 is smaller than that of Mn-SOD (88,000), could be the result of an increased glomerular permeability of which the mechanism remained obscure. Actually, significant increases in CuZn-SOD concentration were noted in urine collected from 10 healthy male high-school students immediately after 2-hr soccer training and at 30-min rest after it (data not shown) ;
during changes
the
experiment,
in plasma
total
Effects
of E
no
Mn-SOD
on Sup eroxi de Dismutases
xercise
SOD activity
exercise, however, seem to be required. line to clarify such points.
was
detected
and the renal
in urine. clearance
We are at present
303
Further
investigations
of CuZn-SOD continuing
after
the work
into physical
along
the
Acknowledgments We wish technical
to thank
Dr. T. Kawaguchi
(Ube Industries
Ltd.,
Ube) for his excellent
assistance. References
1) Clark, IA., Cowden, W.B. & Hunt, N.H. (1985) Free radical-induced pathology. Med. Res. Rev., 5, 297-332. 2) Dillard, C.J., Litov, RE., Savin, W.M., Dumelin, E.E. & Tappel, AL. (1978) Effects of exercise, vitamin E, and ozone on pulmonary function and lipid peroxidation. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol., 45, 927-932. 3) Fridovich, I. (1975) Superoxide dismutases. Ann. Rev. Biochem., 44, 147-159. 4) Forssell, G., Nordlander, R., Nyquist, 0., Qrinius, E. & Styrelius, I. (1975) Creatine phosphokinase after submaximal physical exercise in untrained individuals. Acta Med. &and., 197, 503-505. 5) Jenkins, R.R. (1988) Free radical chemistry. Relationship to exercise. Sports Med., 5, 156-170. 6) Kawaguchi, T., Suzuki, K., Matsuda, Y., Nishiura, T., Uda, T., Ono, M., Sekiya, C., Ishikawa, M., lino, S., Endo, Y. & Taniguchi, N. (1990) Serum-manganesesuperoxide dismutase : Normal values and increased levels in patients with acute myocardial infarction and several malignant diseases determined by an enzyme-linked immunosorbent assay using a monoclonal antibody. J. Immunol. Methods, 127, 249254. 7) Ohno, H., Watanabe, H., Kishihara, C., Taniguchi, N. & Takakuwa, E. (1978) Effect of physical exercise on the activity of GOT isozyme in human plasma. Tohoku J. Exp. Med., 126, 371-376. 8) Ohno, H., Yamashita, H., Ookawara, T., Saitoh, D., Wakabayashi, K. & Taniguchi, N. (1992) Training effects on concentrations of immunoreactive superoxide dismutase isoenzymes in human plasma. Acta Physiol. Scand., 146, 291-292. 9) Oka, S., Ogino, K., Matsuura, S., Yamamoto, K., Okazaki, Y., Takemoto, T., Kato, N. & Uda, T. (1989) Human serum immunoreactive copper, zinc-superoxide dismutase assayed with an enzyme monoclonal immunosorbent in patients with digestive cancer. Olin. Chim. Acta, 182, 209-220.
