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Martial Art Training Enhances the Glutathione Antioxidant System in Middle-Aged Adults

Douris, Peter C1; Elokda, Ahmed S1; Handrakis, John P1; Principal, Suze1; Rondo, Eleni1; Bovell, Juan1; Coughlin, William P1; Mastroianni, Charles N1; Wong, Michael J1; Zimmerman, Thomas2

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Journal of Strength and Conditioning Research: August 2009 - Volume 23 - Issue 5 - p 1518-1523
doi: 10.1519/JSC.0b013e3181b339ac
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Abstract

Introduction

Free radicals are endogenously formed on a continual basis during normal metabolic processes (1,4). Antioxidants act as free radical scavengers. Under normal circumstances, the antioxidants are sufficient at preventing major cellular damage (1,4,15). When free radical production exceeds antioxidant production, the disparity will produce the state of oxidative stress in which cell damage can occur (1,4,15). Free radicals destroy macromolecules in cells, limiting their physiologic effectiveness while leading to cell death by necrosis or apoptosis. The free radical theory of aging proposes that there is an accumulation of oxidative damage with aging. This damage is the principal cause of age-related declines in cellular function (15). Free radical damage has been implicated in cardiovascular disease (13,14,18), cancer (1), Alzheimer's disease (9,21), Parkinson disease (15,21), diabetes, and macular degeneration (20).

Maher (15) states that glutathione (GSH), the body's most highly concentrated antioxidant, is the central component of the antioxidant system and plays an essential role in protecting tissues against oxidative stress. Free radical oxidation will lead to the transformation of GSH to glutathione disulfide (GSSG). GSSG is the oxidized form of GSH (5,6,15). Under normal conditions, the body maintains the resting intracellular levels of GSH and GSSG by reducing GSSG to GSH (5,6,15). Strenuous exercise creates a situation in which free radical formation causes GSSG synthesis to exceed GSH supply and thus leads to free radical accumulation (5,6). Oxidative stress is associated with a decrease in the GSH ratio (GSH:GSSG), the maintenance of which is essential to a variety of cell functions (5,6,15).

Aging is associated with increased levels of oxidative stress according to the free radical theory of aging. A number of studies have investigated levels of GSH during aging (7,10,16,20,23). Erden-Inal et al. (7) reported that GSH levels were highest from age 12 to 24 years of age and declined steadily after 40 years. After 40 years of age, GSSG levels were highest and the GSH:GSSG ratio was lowest, indicating oxidative stress increased after age 40. Jones et al. (10) reported that the capacity of the GSH antioxidant system is maintained until 45 years of age and then declines rapidly thereafter. There was no significant difference in decline of GSH with age between men and women according to Yang et al. (23).

Exercise increases free radical production (2). There is a paradox between acute exercise increasing oxidative stress and regular exercise having a protective and beneficial effect on health (2). Subudhi et al. (22) reported that a single bout of exhaustive treadmill exercise induced a state of oxidative stress in untrained young adults but not in the trained group. Elokda and Nielsen (5) demonstrated that a 6-week exercise program in sedentary young adults consisting of aerobic exercise training, circuit weight training, or a combination of the two were all effective in improving the GSH antioxidant system when compared with a control group. The antioxidant system can improve in young adults with regular exercise to reduce oxidative stress (5,22). Habitual physical activity has been shown to favorably affect the antioxidant defense system in older adults. Physically active older adults possessed significantly higher GSH levels than sedentary older adults; GSH was positively correlated with the level of activity (11). Fatouros et al. (8) studied the effect of an endurance-training program on oxidative stress in previously inactive older men. They reported that total antioxidant capacity improved over a 16-week period and that training cessation 4 months later reversed those adaptations. These studies provide evidence that, although the antioxidant defense system declines with age, it is a dynamic system that can improve with training.

Soo Bahk Do is traditional Korean martial art. Soo Bahk Do training has been shown to increase the overall physical fitness and health of middle-aged practitioners (3). According to Douris et al. (3), the Soo Bahk Do practitioners displayed greater aerobic capacity, balance, flexibility, muscle strength, and endurance as compared with sedentary controls. Elokda and Nielsen (5) has confirmed that exercise training that improves aerobic capacity, muscle strength, and endurance can also promote positive changes in the GSH antioxidant system. Antioxidant defense declines and oxidative damage begins to increase during middle age. Is martial art training an effective exercise intervention that can alter the age-related declines in GSH and the antioxidant defense system? The purpose of this study was to compare the antioxidant capacity of physically active middle-aged martial artists with age-matched sedentary controls.

Methods

Experimental Approach to the Problem

This study used a matched-pairs design to investigate the difference in antioxidant capacities at rest and as a result of a graded exercise test (GXT) between actively practicing middle-aged martial artists and age- and sex-matched sedentary controls. GSH is the body's most highly concentrated antioxidant, is the central component of the antioxidant system, and plays an essential role in protecting tissues against oxidative stress. Free radical oxidation leads to the transformation of GSH to GSSG. Venous blood samples for GSH and GSSG were collected before and immediately after the GXT. Khan et al. (12) have suggested that body mass index is associated with a significant decrease in GSH. The Soo Bahk Do and sedentary subjects in this study differed in body mass index by 29.56 ± 4.25 for the sedentary group vs. 25.8 ± 1.14 for the Soo Bahk Do group. The authors investigated this difference by using body mass index as a covariate, and body mass index was found not to be a significant factor. The analysis of covariance model is appropriate only if there is a linear relationship between the covariate and the dependent variable; it is most effective when r > 0.60, according to Portney and Watkins (17). The correlation between body mass index and GSH for this study was r = −0.44, and the use of body mass index as a covariate did not change the results. Consequently, we performed the analyses without body mass index as a covariate. Repeated measures analysis of variance (ANOVA) were performed on the resting baseline values and immediate post-GXT values of GSH, GSSG, and GSH:GSSG to compare the antioxidant capacities of both groups.

Subjects

The institutional review board at New York Institute of Technology approved the design of this study. Written informed consent was obtained from each subject before the start of the study. Subject characteristics are presented in Table 1 for the 4 women and 14 men who took part in the study. Ages ranged from 41 to 58 years. The two groups were matched by age and sex. Subjects were recruited from the New York Institute of Technology community and a local Soo Bahk Do martial art school. The means and SD for the physiologic characteristics for each group are presented in Table 1.

Table 1
Table 1:
Physiologic characteristics of subjects (mean ±SD).

The inclusion criteria were (a) subjects to be 40 to 60 years of age; (b) subjects to present written medical clearance by their physician who judged them to be physically able to tolerate a GXT; (c) Soo Bahk Do practitioners to have been practicing for at least 4 years at a minimum of twice a week for 1 hour per day; (d) Soo Bahk Do practitioners to participate in Soo Bahk Do only and no other formal exercise; (e) sedentary/physically inactive subjects not to participate in any formal program of physical exercise or physically active occupation. The exclusion criteria were (a) subjects who do not fit the inclusion criteria; (b) subjects unable to physically perform a GXT; (c) cigarette smokers, and (d) a history of taking any antioxidants such as vitamin E during the last year before this study.

Procedures

The researchers were responsible for taking the medical history of each prospective subject to evaluate for the presence of any of the exclusion criteria. To minimize the effects of diurnal variations, all testing was performed in the morning hours in a fasting state. Resting blood pressure (BP), heart rate (HR), height, and body mass were measured and recorded. Resting blood samples were drawn before and immediately after a GXT using a modified Bruce protocol to induce oxidative stress. Previous research has demonstrated that a GXT is an effective technique for inducing oxidative stress (5,6,21). Venous blood samples (3 mL) were taken from the antecubital vein using a standard butterfly catheter. Venous blood samples were cooled (placed in ice bath) then frozen at -70° C and stored until final analysis.

Graded Exercise Test

Each subject was oriented to the treadmill-testing protocol and equipment (Q4500 Stress Test Monitor, Bothell, WA). The Borg 6 to 20 category scale for rating of perceived exertion (RPE) was explained to the subjects, and they were told that they would be asked to rate the difficulty of the test at various stages with this RPE scale. Subjects were monitored continuously with a 12-lead electrocardiogram (ECG). BP was measured at the brachial artery while subjects were asked to indicate their RPE 15 seconds before the end of each stage. Subjects were monitored closely for signs and symptoms of exercise intolerance throughout the test. The subjects underwent the various stages of the modified Bruce protocol until they attained one of the following endpoints for terminating the test. Criteria for termination of the test consisted of (a) subject request, (b) abnormal ECG changes suggestive of ischemia or significant arrhythmia, (c) a drop in systolic BP (≥20 mm Hg) or an abnormal rise in diastolic BP (≥20 mm Hg over baseline), (d) signs or symptoms suggestive of subject not tolerating the workload, (e) attainment of 90% of the subject's age-predicted maximal HR, and (f) a subjective rating of 19 or 20 on the RPE scale. The post-GXT blood samples were drawn immediately upon completion of the test. The subjects then sat quietly for 5 to 10 minutes while recovery HR and BP were monitored.

Venous Collection and Blood Processing Procedures

Determination of both GSH and GSSG in the blood has been considered an essential index of whole-body GSH status because blood GSH concentrations may reflect GSH status in other less accessible tissues (19). These levels are reliable and valid biomarkers of oxidative stress and of disease risk in humans (19). A relatively new assay analysis technique that uses a commercially available assay kit (5,6,22) (GSH:GSSG-412 assay: Colorimetric Determination of Reduced and Oxidized Glutathione, Oxis Research, Portland, OR) was used for erythrocyte total glutathione (GSHt) and GSSG determinations. The assay uses the scavenger reagent 1-methyle-2-vinylpyridinium trifloromethane sulfonate at a level that rapidly (1 min) scavenges 99% of GSHt but does not interfere with GSH reductase. The original technique was slower and involved up to 60 minutes with only 70% removal of GSHt from the blood. During this extended time period, uncontrolled oxidation of GSH could occur, resulting in significant overestimation of the GSSG concentration. The new assay procedure uses an enzymatic method for quantitative determination of GSHt and GSSG. The method uses Ellman's reagent DTNB, which reacts with GSHt to form a spectrophotometrically detectable product at 412 nm. GSSG was determined by reduction of GSSG to GSHt by nicotinamide adenine dinucleotide phosphate-oxidase and the reaction of formed GSHt with DTNB. For clarification, the assay analysis and the spectrophotometric measurements allowed for the direct determination of GSHt and GSSG. GSH and the GSH:GSSG values were indirectly calculated from the following equations:

Spectrophotometric readings were performed at a wavelength of 412 nm. A computer software program automatically computed the necessary standard referenced based calibration curves (absorbance vs. time and net reaction rate vs. concentrations) as well as the blank and the unknown blood sample analyte absorbance vs. time regression equations and the final calculations of GSH, GSSG, and GSH:GSSG ratios. To enhance measurement reliability, triplicate assay analysis was performed on each blood sample. To insure blinding of the sampling procedure, each collection tube was assigned a random number, which was generated from a computer-indexing program. The same index number was used for all subsequent sample tubes with additional identification according to the specific assay procedures.

Statistical Analyses

Statistical analyses were performed using SPSS for Windows (version 15.0, Chicago, IL) using a repeated measures design. Descriptive statistics were calculated for age, height, body mass, and resting HR and BP for both groups. The independent variables are the matched Soo Bahk Do group and sedentary group. The dependent variables are the GSH, GSSG levels, and the ratio of GSG:GSSG. Repeated measures ANOVA were performed on the resting and immediately post-GXT values of GSH, GSSG, and GSG:GSSG to compare both groups. When a significant F ratio was identified, Tukey's post hoc analysis was used to locate pairwise differences between the means. A priori sample size calculations revealed that 9 subjects were required in each group to detect observed differences at a power of 80%. Statistical significance for this study was set at the p < 0.05.

Results

The means and SD of GSH, GSSG, and the ratio of GSH:GSSG from the following conditions are presented in Table 2: condition 1: pre-GXT blood test for the Soo Bahk Do group (PreSBD); condition 2: post-GXT blood test for the Soo Bahk Do group (PostSBD); condition 3: pre-GXT blood test for the sedentary group (PreSED); condition 4: post-GXT blood test for the sedentary group (PostSED).

Table 2
Table 2:
Mean ±SD of GSH, GSSG, and GSH:GSSH.

GSH

The repeated-measures ANOVA for GSH revealed a significant F statistic (p < 0.001) for the 4 measurement conditions. Post hoc pairwise comparisons using Tukey's honestly significantly different (HSD) test identified that a comparison of 1 vs. 3 was significant (p = 0.002) for a difference at rest between the PreSBD and the PreSED. A comparison of 1 vs. 2 was not significant (p = 0.646), demonstrating that that the Soo Bahk Do GSH levels did not change as a result of the GXT. The comparison of 3 vs. 4 was significant (p < 0.001) for GXT significantly lowering GSH levels in the sedentary group.

GSSG

The repeated-measures ANOVA for GSSG revealed a significant F statistic (p < 0.001) for the 4 measurement conditions. Post hoc pairwise comparisons using Tukey's HSD identified that a comparison of 1 vs. 3 was significant (p < 0.001) for a difference between PreSBD and PreSed. A comparison of 1 vs. 2 was not significant (p = 0.145) for the Soo Bahk Do GSSG levels changing as a result of the GXT. The comparison of 3 vs. 4 was significant (p < 0.001) for the GXT significantly raising the GSSG levels in the sedentary group.

GSH:GSSG

The repeated-measures ANOVA for the GSH:GSSG ratio revealed a significant F statistic (p < 0.001) for the 4 measurement conditions. Post hoc pairwise comparisons using Tukey's HSD identified that a comparison of 1 vs. 3 was significant (p < 0.001) for a difference between PreSBD and PreSED. A comparison of 1 vs. 2 was not significant (p = 0.122) for the Soo Bahk Do ratio of GSH:GSSG changing as a result of the GXT. The comparison of 3 vs. 4 was significant (p < 0.001) for the GXT significantly lowering the ratio of GSH:GSSG in the sedentary group.

Discussion

This study has provided evidence that practicing martial arts, specifically Soo Bahk Do, may be an effective intervention for promoting health because it protects against the harmful effects of excessive free radical formation. Normal aging has shown to decrease the levels of GSH and increase the levels of GSSG, especially after the age of 40, predisposing these individuals to oxidative damage (7,10,16,20,23). The results of this study has demonstrated that Soo Bahk Do training can limit this decline and allow for maintenance of higher levels of GSH, enabling the body to effectively limit free radical formation and cellular damage. From a clinical perspective, resting values of GSH, GSSG, and GSH:GSSG ratios may serve as general indicators of a person's oxidative stress status as well as overall resistance to oxidative stress. Immediate postmaximal exercise GSH, GSSG, and GSH:GSSG values may be considered indicators of the body's ability to cope with acute oxidative stress.

Soo Bahk Do is a traditional Korean martial art similar to karate. A typical Soo Bahk Do class is 1 hour in length. The class usually includes a 15-minute warm-up of flexibility exercises, calisthenics, punching, blocking, and kicking drills. The core of the class may include an additional 10 minutes of basic kicking, blocking, and punching drills, 20 minutes of hyung (set forms in pre-established sequences of defensive and offensive movements), 10 minutes of sparring against an opponent, and self-defense techniques. The class typically ends with a 5-minute cool down of calisthenics and flexibility exercises (3). The martial art practitioners in the present study were all black belts with an average experience of approximately 10 years in Soo Bahk Do.

The decline in GSH levels has been shown to contribute to the deterioration of function and the presence of disease states associated with aging (1). Elokda et al. (6) reported an average GSH level of 1025.8 μM for a group of sedentary subjects with an average age of 32.5 years. The sedentary subjects in the current study, which used similar procedures as Elokda et al.'s study (6), presented with a mean GSH level of 613.1 μM at an average age of 52.9 years, a 40.2% decline over a 20-year period. The Soo Bahk Do group's GSH levels were significantly higher than the sedentary group at 710.9 μM, representing a 30.7% decline. Soo Bahk Do training appears to limit the age-related decline of GSH levels. A treadmill GXT is an effective stimulus for inducing oxidative stress in subjects according to Elokda et al. (5,6) and Subudhi et al. (22). As a result of our GXT, the Soo Bahk Do group demonstrated a nonsignificant decline of 4.9% in GSH, whereas the sedentary group declined a significant 17.0%. It should be noted that the Soo Bahk Do group achieved higher workrates and spent more time (mean 13:46 vs.12:03 min) on the treadmill before attaining at least 90% of their target HR.

GSSG is the product of the oxidation of GSH by GSH peroxidase. GSSG is released from cells as a consequence of oxidative stress (7,10,20,22). An increase in GSSG is toxic to the cells according to Maher (15). Several researchers have demonstrated that GSSG levels begin to significantly increase after the age of 40 years (7,10,16,20,23). Elokda et al. (6) reported GSSG levels at rest were 2.24 μM and increased significantly to 3.15 μM as a result of a GXT in their sedentary subjects. The sedentary subjects in this study displayed resting values of 3.10 μM, which increased significantly to 3.82 μM after the GXT. However, the Soo Bahk Do group at rest displayed GSSG resting values of 2.53 μM with a nonsignificant increase to 2.65 μM after the GXT. Our findings are in agreement with the findings of several other studies, that resting GSSG levels begin to increase after 40 years of age (7,10,20,23), given that our Soo Bahk Do group presented with significantly lower GSSG levels both at rest and after the GXT. Soo Bahk Do training appears to limit the increase of GSSG levels seen with aging as compared with age- and sex-matched sedentary controls. It appears that the Soo Bahk Do practitioners produce less free radicals at rest than their age-matched sedentary counterparts, theoretically limiting the damage of free radical accumulation.

The GSH:GSSG ratio can reflect the imbalance that occurs when GSSG formation exceeds GSH supply and uptake that can occur during strenuous exercise. Decreases in this ratio after exercise have been interpreted as a sign of acute oxidative stress (5,6,22). Oxidative stress increases with age as shown by the decrease in the GSH:GSSG ratio, leading to the development of age-related toxicities and pathologies (7,10,20). The sedentary subjects in Elokda et al.'s (6) study displayed a ratio of 462.1 that dropped significantly to 276.4 after the GXT. The older sedentary adults in this study exhibited 197.8 at rest and a significant decline to 133.2 after the GXT. The Soo Bahk Do group's ratio was 277.6, significantly higher than the sedentary group at rest, and they only declined to a nonsignificant 255.7 after the GXT. Therefore, the Soo Bahk Do subjects experienced less acute oxidative stress than their sedentary counterparts as a result of the GXT. Our results compare favorably with a similar study using young trained and untrained subjects. Subudhi et al. (22) reported that the ratio significantly declined in untrained young adults compared with trained subjects in response to exhaustive treadmill exercise. This study demonstrates that, although the ratio will decline with aging, Soo Bahk Do training can attenuate that decline and protect against oxidative stress.

Previous research has shown antioxidant defense system declines with age. However, it is a dynamic system that can improve with training in young adults (5,22) and older adults (8,11). Soo Bahk Do practitioners in this study had higher resting levels of GSH, lower levels of GSSG, higher GSH:GSSG ratios, and responded more effectively to acute oxidative stress than age-matched sedentary controls. This study provides evidence that practicing Soo Bahk Do or a similar martial art may be a beneficial intervention for improving health because it protects against the dangers of excessive free radical formation that are detailed in the free radical theory of aging.

Practical Applications

Health professionals should be aware of alternative methods of training, conditioning, and exercise that can improve the general adaptation response to oxidative stress. Oxidative damage has been shown to increase with aging. This damage has been implicated as a contributing factor in a variety of age-related diseases and pathologies. Millions practice martial arts worldwide, and it can be considered one of the oldest forms of exercise known to humans. The conditioning practices of the martial arts have been passed down from generation to generation for thousand of years, and it is only recently that scientific investigation has begun to validate its health and exercise benefits. Soo Bahk Do or a similar martial art appears to enhance the antioxidant defense system and thus may retard the aging process and protect against the development of age-related disease and pathologies. Soo Bahk Do training is not only applicable as self-defense against an attacker but can be considered self-defense against aging.

Acknowledgments

This work was supported by an ISRC grant from New York Institute of Technology for faculty research.

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Keywords:

oxidative stress; free radicals; graded exercise testing; karate

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