Journal of Applied Physiology
Vol. 81, No. 4, pp. 1484-1487, October 1996
METABOLISM

Effect of sprint cycle training on activities of antioxidant enzymes in human skeletal muscle

Ylva Hellsten, Fred S. Apple, and Bertil Sjödin

Department of Physiology III, Karolinska Institute, S-11486 Stockholm, Sweden; Copenhagen Muscle Research Centre, August Krogh Institute, DK-2200 Copenhagen N, Denmark; and Department of Laboratory Medicine and Pathology, Hennepin County Medical Center, University of Minnesota Medical School, Minneapolis, Minnesota 55455

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Hellsten, Ylva, Fred S. Apple, and Bertil Sjödin. Effect of sprint cycle training on activities of antioxidant enzymes in human skeletal muscle. J. Appl. Physiol. 81(4): 1484-1487, 1996.The effect of intermittent sprint cycle training on the level of muscle antioxidant enzyme protection was investigated. Resting muscle biopsies, obtained before and after 6 wk of training and 3, 24, and 72 h after the final session of an additional 1 wk of more frequent training, were analyzed for activities of the antioxidant enzymes glutathione peroxidase (GPX), glutathione reductase (GR), and superoxide dismutase (SOD). Activities of several muscle metabolic enzymes were determined to assess the effectiveness of the training. After the first 6-wk training period, no change in GPX, GR, or SOD was observed, but after the 7th week of training there was an increase in GPX from 120 ± 12 (SE) to 164 ± 24 µmol ^· min^{1 ·} g dry wt¹ (P < 0.05) and in GR from 10.8 ± 0.8 to 16.8 ± 2.4 µmol ^· min^{1 ·} g dry wt¹ (P < 0.05). There was no significant change in SOD. Sprint cycle training induced a significant (P < 0.05) elevation in the activity of phosphofructokinase and creatine kinase, implying an enhanced anaerobic capacity in the trained muscle. The present study demonstrates that intermittent sprint cycle training that induces an enhanced capacity for anaerobic energy generation also improves the level of antioxidant protection in the muscle.

free radicals; scavengers

INTRODUCTION

OXYGEN RADICALS may be derived from various biological processes within the body, and the magnitude of generation may be elevated in a tissue during energy metabolic stress. Exercise has been reported to lead to generation of free radicals in animal muscle, as evidenced by direct measurements of free radicals with the electron paramagnetic resonance technique (4, 12) and by indirect determinations of products of free radical reactions (2, 20). Indications of exercise-induced oxygen radical generation in skeletal muscle may also be obtained by studying the levels of antioxidant enzymes with training. Antioxidant enzymes act directly or indirectly to remove reactive oxygen species, and thus an elevation of these enzymes with training suggests an increased need for protection against free radicals. One of the most important physiological antioxidant systems is the glutathione system in which glutathione peroxidase (GPX) utilizes reduced glutathione as a hydrogen donor for the removal of peroxides. The produced oxidized form of glutathione may in the presence of NADPH be reduced to glutathione via glutathione reductase (GR). Superoxide dismutase (SOD) is another important antioxidant enzyme that converts superoxide radicals to hydrogen peroxide, which then can be further converted to water via GPX or catalase. Endurance training in rats has been found to result in an elevated activity of GPX, GR, and SOD (1, 11, 19), but often a training effect has only been observed for some of the measured antioxidant enzymes (14, 15). The discrepancies in findings among studies suggest that the type, frequency, and intensity of exercise used in training may affect the response in the antioxidant systems. In a recent study we have found strong indications that high-intensity exercise leads to free radical formation in human muscle (20); however, no data exist on the effect of sprint training on the levels of antioxidant enzyme activities in human skeletal muscle.

In the present study, we tested the hypothesis that intermittent sprint cycle training could lead to an enhanced activity of GPX, GR, and SOD in human skeletal muscle. To examine whether sprint cycle training improved the muscle anaerobic capacity and whether this capacity was related to the activity level of SOD, GPX, and GR, the activities of some anaerobic enzymes were also determined.

MATERIALS AND METHODS

Eleven men, with a mean age of 23.6 ± 5.9 (SE) yr and a mean body mass of 79.2 ± 7.7 kg, participated in the study. Training consisted of maximal cycling sprints of 10-s duration, repeated 15 times with 50 s of rest between each sprint. Resistance was set at 7% of body mass. Training was performed three times per week for 6 wk followed by training two times per day for 7 consecutive days. Resting muscle biopsies were obtained from the vastus lateralis muscle before all testing; 24 h after the termination of the 6-wk training period; and 3, 24, and 72 h after the additional 1 wk of more frequent training. Multiple resting muscle biopsies were obtained after the final week of training to have more than one time point for assessment. Samples were freeze-dried before analysis. Activities of muscle GPX, GR, and SOD were analyzed according to methods described elsewhere (3). Muscle activities of phosphofructokinase (PFK), lactate dehydrogenase (LDH), and creatine kinase (CK) were assessed as indications of anaerobic capacity; citrate synthase (CS) and -hydroxyacyl-CoA dehydrogenase (HAD) were determined as indicators of oxidative capacity. All metabolic enzyme activities were analyzed according to methods by Lowry and Passonneau (17).

Statistical analysis. Values are presented as means ± SE. One-way analysis of variance with repeated measures was used for comparison of dependent variables. Scheffé's test was used to determine differences. Regression analysis was performed on values from biopsies obtained before training and on differences between pre- and posttraining values.

RESULTS

There were no significant changes in antioxidant enzyme activities after the first 6 wk of training, whereas after the additional week of more frequent training there was an increase in the activity of GPX (37%) and GR (56%; Fig. 1, A and B). For both GPX and GR, the activity levels reached significance 24 h after training (P < 0.05). No change in SOD activity was observed with the training (Fig. 1C). After the first 6 wk of training, there was a significantly higher (P < 0.05) muscle activity of PFK from a pretraining value of 191 ± 8.4 to 222 ± 6.9 µmol ^· min^{1 ·} g dry wt¹), and after the seventh week (24 h posttraining), the activity was 226 ± 10.6 µmol ^· min^{1 ·} g dry wt¹. The muscle CK activity increased (P < 0.05) from 6,147 ± 275 µmol ^· min^{1 ·} g dry wt¹ pretraining to 6,919 ± 370 µmol ^· min^{1 ·} g dry wt¹ after the first 6-wk period, and the activity was 6,725 ± 455 µmol ^· min^{1 ·} g dry wt¹ after the seventh week (24 h). There was no difference in the activity of LDH [1,534 ± 91 vs. 1,614 ± 112 and 1,652 ± 107 µmol ^· min^{1 ·} g dry wt¹ after 6 and 7 wk (24 h), respectively], CS [100 ± 10.9 vs. 101 ± 4.4 and 117 ± 8.4 µmol ^· min^{1 ·} g dry wt¹ after 6 and 7 wk (24 h), respectively], or HAD [36.9 ± 2.5 vs. 41.3 ± 2.3 and 42.1 ± 2.4 µmol ^· min^{1 ·} g dry wt¹ after 6 and 7 wk (24 h), respectively]. No significant relationship was observed between the activities of GPX, GR, or SOD and activities of the metabolic enzymes (P > 0.05). Nor was there a relationship between the training-induced change (seventh week, 24-h post- vs. pretraining value) in GPX or GR and the change in metabolic enzymes (seventh week, 24-h post- vs. pretraining value) (P > 0.05).

DISCUSSION

A novel finding in the present study was that intermittent sprint training induced an enhanced activity of GPX and GR in human skeletal muscle. The exercise used in the present study consisted of repeated 10-s sprints, performed at intensities corresponding to ~300-400% of an intensity eliciting maximal oxygen uptake. This type of exercise requires a maximal rate of energy production via dephosphorylation of adenosine triphosphate and creatine phosphate and via glycolysis, whereas the oxidative energy systems mainly are required in the resting phases between sprints for recovery of the energy stores. The high rate of energy turnover that is required in the active muscle during intermittent sprint exercise may well induce a marked level of oxidative stress. Short-term high-intensity exercise has been shown to result in nonenzymatic oxidation of urate in human muscle, strongly suggesting free radical generation (20). In animal models, the extent of free radical generation in muscle has also been found to increase with increasing exercise intensity (2, 18). The present experimental design was such that after 6 wk of training three times per week, the training dose was increased to two sessions per day for 1 wk, a fivefold increase in training frequency, to examine the effect of frequent training sessions. Because the antioxidant enzyme levels were significantly higher than pretraining levels after the seventh week of training but not after the sixth week of training, it appears that the more frequently performed sprint training imposed a greater level of oxidative stress on the muscle.

The actual sources of free radicals in muscle during exercise can only be speculated upon as no evidence exists from experiments performed in vivo and during exercise. In the mitochondria a relatively high flux through the electron transport chain, combined with a possible loss of cytochrome oxidase (7) during intermittent sprint exercise, may lead to an increased use of coenzyme Q as an electron acceptor and the consequent formation of semiquinones. Semiquinones readily reduce oxygen to form superoxide radicals (16). Another proposed source of reactive oxygen species during intense exercise is the superoxide-generating enzyme xanthine oxidase, located in the blood vessel walls of most tissues including skeletal muscle (9). Xanthine oxidase has been demonstrated to generate free radicals during other forms of metabolic stress to tissue, such as ischemia-reperfusion, but direct evidence for xanthine oxidase as a radical generator in muscle during exercise is lacking (see Ref. 8).

To focus on the role of metabolic factors in oxidative stress during exercise, cycling was used as an exercise model to avoid eccentric contractions that may cause inflammatory responses. Absence of muscle damage was indicated by unaltered plasma CK activity throughout and after the seventh week of training, during which the training frequency was severalfold that of the prior 6-wk period (see Ref. 10, subject group A). Because the muscle activities of GPX and GR were significantly elevated after the seventh week only, it does not appear that inflammation could have been the main determinant for the elevated antioxidant protection level. In addition, at no time during the 7 wk of training did the subjects experience subjective muscle soreness or show a decline in training performance, factors often coinciding with muscular damage (see Ref. 5). Although inflammatory events cannot be completely ruled out, we propose that the enhancement in muscle antioxidant protection, as indicated by increased levels of GPX and GR, was mainly the result of an elevation in metabolically induced oxygen radical formation.

It has been proposed that the level of antioxidant enzyme protection in muscle is related to tissue oxygen consumption (13, 15). A relationship between muscle antioxidant enzyme activities and oxidative capacity, as determined by succinate dehydrogenase activity and fiber type composition of rat muscle, has been reported (15), and a rank-order relationship between the level of oxygen consumption at rest of various rat tissues and the activity of SOD has been described (13). In the present study no relationship was observed between pretraining levels of GPX, GR, or SOD and the mitochondrial enzymes CS and HAD. The lack of increase in CS and HAD with the present training, furthermore, suggests that the antioxidant capacity in muscle may be improved without a simultaneous enhancement in the oxidative capacity.

We also examined whether human muscle antioxidant enzyme activities could be related to indicators of anaerobic capacity. In addition to the assessment of anaerobic enzymes, the fiber type distribution was determined in the current subjects and it was found that the fiber type distribution was altered toward a more fast-twitch oxidative muscle: type IIa fibers increased from 40 ± 9 to 56 ± 11% and the proportion of type IIb fibers decreased from 14 ± 11 to 1 ± 2% over the 7-wk period (6). There was no significant relationship among the activities of GPX, GR, or SOD measured before training and the activities of PFK, LDH, CK, or percent type II fibers before training. Neither was there a relationship between the training-induced change in antioxidant enzymes and the change in anaerobic enzyme activities or fiber type distribution. In conclusion, intermittent sprint training, which leads to an enhancement in the muscle capacity for anaerobic energy production without an alteration in the oxidative capacity, can also result in enhanced muscle antioxidant protection. No apparent relationship between anaerobic capacity and the levels of scavenger enzymes was found.

ACKNOWLEDGEMENTS

The study was funded by grants from the Swedish Work Environment Fund, the Swedish Sports Research Council, the Karolinska Institute, Stockholm, Sweden, and the American Heart Association, Minnesota Affiliate.

FOOTNOTES

Address for reprint requests: Y. Hellsten, Copenhagen Muscle Research Centre, August Krogh Institute, Universitetsparken 13, DK-2200 Copenhagen N, Denmark.

Received 2 January 1996; accepted in final form 2 April 1996.

REFERENCES