Physical activity exhibits beneficial health effects for adolescents and adults, such as improved bone health, cardiorespiratory, cardiovascular, and metabolic adaptations that play a pivotal role in the prevention of different chronic diseases (37).
However, exercise increases the endogenous production of reactive oxygen species (ROS), and excessive generation of ROS is discussed to be associated with cardiovascular diseases, diabetes mellitus, rheumatoid arthritis, and cancer. During exercise, mitochondrial superoxide generation, xanthine oxidase-catalyzed AMP breakdown, increased activity of muscular oxidoreductases, and activation of immune responsive cells are considered as the main sources of ROS generation (23,32). Disruptions of the redox homeostasis with a shift toward a pro-oxidant milieu may occur when ROS production is elevated and/or antioxidant activity is decreased (24,35).
Intensive or prolonged exercise might exceed the individual ROS detoxifying capacity and result in oxidative stress. As reported in a review article by Reid (26), increased ROS production in athletes may be associated with muscular fatigue and reduction in force production as a result of structural and functional modifications of lipids or proteins. ROS seems to be involved in contraction-induced muscle cell damage, inflammation, and delayed onset of muscle soreness (3,6). Markers of oxidative stress, such as an increase of lipid peroxidation products as well as oxidative protein modifications, were shown after different types of exercise (32,39). Thus, supplemental antioxidant intake is often discussed to prevent exercise-induced oxidative damage in athletes (16,17).
However, functional consequences of exercise-induced ROS formation include an upregulation of antioxidant defense mechanisms, such as the regulation of gene expression or activity of antioxidant enzymes (24). Chronic exercise may elevate resting levels of antioxidant enzymes, such as erythrocyte glutathione peroxidase and superoxide dismutase in muscle cells (20,21). Subsequently, trained individuals seem to be less susceptible to exercise-induced oxidative damage compared with subjects not regularly exposed to exercise (21,32). In adults, chronic exercise may increase blood antioxidant capacity and can result in efficient protection and improved tolerance against potentially damaging effects of later periods of exercise (15,23).
In adolescent athletes, the oxidative metabolism and the exercise-induced ROS release may be different compared with adult athletes (7). However, little is known about the effect of chronic exercise on the antioxidant capacity and oxidative modifications in the juvenile organism. Gougoura et al. (13) reported about increased oxidative stress markers in adolescent swimmers, suggesting that young athletes might not be sufficiently protected against oxidative damage. Besides endogenous antioxidant enzymes, exogenous dietary substances may contribute to the antioxidant defense system. Although the amount of dietary and plasma antioxidants, such as vitamins C and E and carotenoids, has been well documented in adult athletes (31,40), little is known about habitual antioxidant intake in adolescent athletes.
Whether regular moderate or vigorous exercise modifies the antioxidant capacity and subsequently lowers the susceptibility toward oxidative protein modifications in adolescent athletes is still unclear. Therefore, the purpose of this study was, first, to measure habitual antioxidant intake and, second, to evaluate the impact of regular training with different chronic workloads on the antioxidant capacity, substantial plasma antioxidants, and plasma protein modifications in adolescent athletes and moderately active controls.
A total of 253 adolescent subjects volunteered to participate in the study; 108 of them were finally included. Reasons for exclusion from the study were not handing in activity reports (n = 37), not meeting quality criteria for the dietary reports (n = 8), or both (n = 100). Participants were classified as competitive athletes in case of exercising for competitions (n = 90, 16.3 ± 2.1 yr; range = 12.5-19.7 yr) or moderately active controls in case of recreational or no physical exercise training (n = 18, 16.3 ± 2.3 yr; range = 13.3-19.6 yr). Athletes participated in different sports, mostly in track and field (n = 38), soccer or handball (n = 15), and rowing or canoeing (n = 12). Triathlon, swimming, judo, or modern pentathlon were represented only with small groups of athletes. Mean energy expenditure during exercise was 4.8 ± 3.1 MJ·d−1 for male athletes (n = 35), 2.5 ± 1.3 MJ·d−1 for male controls (n = 7), 3.7 ± 2.2 MJ·d−1 for female athletes (n = 55), and 1.2 ± 0.7 MJ·d−1 for female controls (n = 11).
Data collection consisted of a short questionnaire ahead of laboratory measurements, physical activity reports, dietary protocols to obtain antioxidant intake, anthropometric examination, and fasting blood withdrawal to examine plasma levels of antioxidants, antioxidant capacity, and protein modifications. All data were obtained in a cross-sectional study design within 6 wk to avoid differences between athletes and controls in either antioxidant intake or antioxidant plasma levels due to seasonal variations. The questionnaire (physical activity, medical history, recent medication, dietary habits, and use of nutritional supplements) was completed at home and brought into the laboratory, where subjects underwent anthropometric examination and fasting blood sampling before the morning training session. Anthropometrical measurements (height, weight, and percent body fat) were conducted in a fasting state using bioelectrical impedance analysis for the assessment of body fat (Multifrequence Body-Composition Analyzer 2000-M; Data-Input, Frankfurt/Main, Germany).
All subjects and their parents in cases where participants were underage gave written informed consent after the study was approved by the local ethics committee.
Dietary and Activity Protocols
Dietary intake was recorded using a 4-d dietary protocol, and nutritional antioxidant intake was calculated on the basis of the German Food Code and Nutrition database BLS II.3. A 7-d activity protocol validated against doubly labeled water (18) was used to determine total energy expenditure and energy spent for exercise (EEtrain). EEtrain was calculated on the basis of the MET intensities according to Ainsworth et al. (1) and served to express metabolic costs for exercise as an integral parameter of frequency, duration, and intensity of trainings session. Both athletes and moderately active controls were clustered into four groups of different physical activity levels according to gender-specific quartiles of calculated EEtrain (Table 1). These activity groups were characterized as I (EEtrain below 1.7 MJ·d−1 for women and below 2.2 MJ·d−1 for men), II (EEtrain between 1.7 and 2.6 MJ·d−1 for women and 2.2 and 4.0 MJ·d−1 for men), III (EEtrain between 2.6 and 4.2 MJ·d−1 for women and 4.0 and 5.5 MJ·d−1 for men), and IV (EEtrain higher than 4.2 MJ·d−1 for women and 5.5 MJ·d−1 for men).
Blood withdrawal was performed after an overnight fasting period using prechilled test tubes with ethylenediaminetetraacetic acid as an anticoagulant (Kabevette E301; Kabe, Nümbrecht-Elsenroth, Germany). Plasma was obtained by centrifugation for 15 min at 2500g, until analysis aliquots were stored at −80°C. Plasma levels of carotenoids and α-tocopherol were analyzed within a month, and further analysis was performed within 1 yr after blood sample collection.
Plasma antioxidant status.
To evaluate plasma antioxidant status, we measured the concentration of fat and water-soluble components of antioxidant capacity and antioxidant capacity itself. As recommended by different authors (25,33), two different methods for assessing plasma antioxidant capacity were applied, Trolox equivalent antioxidant capacity (TEAC) and electron spin resonance (ESR) spectrometry.
Small-molecular contributers to antioxidant capacity.
According to the literature, uric acid, α-tocopherol, and carotenoids as determinants of antioxidant capacity (5,8) were analyzed. For quantification of α-tocopherol and carotenoids, a modified reverse-phased high-performance liquid chromatography (HPLC) system was used as previously described (34). Briefly, deproteinized diluted plasma samples were extracted with n-hexane, and supernatants were evaporated under nitrogen, reconstituted in isopropanol, and injected into the HPLC system (Waters, Eschborn, Germany). Absorption was measured at 450 and 290 nm and quantified by comparison with external standards (Sigma-Aldrich, Deisenhofen, Germany). Uric acid quantification was carried out on Olympus AU 600 using the uricase-PAP test (Olympus Europe GmbH, Hamburg, Germany).
Plasma antioxidant activity was evaluated using ESR by monitoring the ability of the samples to donate a hydrogen atom or a free electron using a synthetic stabilized radical (Fremy's salt (FS), i.e., potassium nitrosodisulfonate) as previously described (29). Briefly, a 10-fold dilution of plasma aliquots was added to the same volume of FS solution (1 mM of phosphate-buffered saline, pH 7.4). The low-field resonance spectrum was recorded 20 min after mixing the samples with the radical solution (Miniscope MS 100; Magnettech GmbH, Berlin, Germany). Signal intensity was obtained by double integration, and the concentration of FS was calculated by comparison with a control reaction (phosphate buffer). Plasma antioxidant activity was expressed as the percentage of FS radicals reduced by the plasma samples (millimolar of FS per liter of plasma):
Trolox equivalent antioxidant capacity.
Equation (Uncited)Image Tools
Measurement of TEAC is based on the scavenging of long-living 2,2-azinobis-3-ethylbenzthiazolin-6-sulfonic acid radical anions by antioxidants contained in the sample. The radicals are generated by potassium persulfate and were detected at 734 nm (Spekord 40 and WinAspect 184.108.40.206; Analytic Jena, Jena, Germany). A modified TEAC assay with Trolox (Sigma-Aldrich) as reference substance was used as described in previous studies (30). The antioxidant activity of the samples is quantified by constructing a calibration curve and is given in Trolox equivalents (TE; millimolar of Trolox per liter).
Analysis of Transthyretin Microheterogeneity
Posttranslational protein modifications were assessed by analyzing structural changes of transthyretin (TTR), which contains cysteine residues that are susceptible to oxidation. Therefore, transthyretin is an eligible model for analyzing oxidative modifications using proteomic approaches (19). Microheterogeneity was quantified using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Plasma samples of 10 athletes and 10 moderately active controls each with five men and women were analyzed for transthyretin modifications. As described in previous studies (9), serum aliquots were mixed with half the volume of polyclonal rabbit antihuman prealbumin antibody (DAKO, Hamburg, Germany). After incubation for 2 h at 37°C, samples were centrifuged, the resulting supernatant removed, and the pellet was washed twice with phosphate-buffered saline, once with HEPES and then resuspended in HPLC-grade water. Saturated sinapinic acid solution served as the matrix, and MALDI mass spectra of immunoprecipitated TTR were detected using a Reflex II MALDI time-of-flight mass spectrometer (Bruker-Daltonik, Bremen, Germany) and external calibration standards.
Data are presented as means ± SD, and the 95% confidence intervals are shown in Figures 1-5. To test for differences in parameters of the antioxidant status between the groups, we calculated a two-factor ANOVA (factor 1: quartiles of energy expenditure during training, EEtrain; factor 2: age group). For post hoc comparisons between groups, the Tukey-Kramer test was used. To test for correlation between energy intake and dietary supply with antioxidants, we conducted a bivariate linear regression analysis. P < 0.05 for the α error was considered significant for all tests. Finally, a multiple regression analysis was conducted to test for effects of dietary antioxidant intake, plasma antioxidants, age, and exercise load on plasma antioxidant capacity. All statistical calculations were carried out on the JMP 5.0 statistical software (SAS, Cary, NC).
Mean intake of vitamin C (188 ± 120 mg·d−1), total vitamin E (18.7 ± 10 mg·d−1), and β-carotene (4.2 ± 2 mg·d−1) were in accordance with the recommended daily amounts (10). Because the supply of vitamin E (P < 0.001, r = 0.34) and vitamin C (P < 0.001, r = 0.25) by habitual diet was weakly correlated with energy intake (Fig. 1), antioxidant intake was slightly but not significantly higher in men than that in women (vitamin E: 20.9 ± 11 mg·d−1 for boys and 17.3 ± 10 mg·d−1 for girls, P = 0.08; vitamin C: 210 ± 136 mg·d−1 for boys and 174 ± 107 mg·d−1 for girls, P = 0.12). The groups of different amounts of physical activity (1st-4th quartile of EEtrain) did not differ in their habitual antioxidant intake (Table 2), and there were no differences depending on age (Table 3).
Plasma levels of antioxidants
The mean plasma level of α-tocopherol (19.7 ± 5.2 μmol·L−1, range = 9.68-42.8 μmol·L−1) was above the cutoff point for deficiency (12 μmol·L−1) (10). Concentrations of plasma β-carotene (0.72 ± 0.5 μmol·L−1, range = 0.1-2.5 μmol·L−1) and total carotenoids (1.57 ± 0.8 μmol·L−1, range = 0.3-3.7 μmol·L−1) were within the values measured in European adults (2). Mean serum levels of uric acid (262 ± 69 μmol·L−1, range = 122-458 μmol·L−1) were within reference limits. There were no differences in the plasma concentrations of α-tocopherol (P = 0.61), β-carotene (P = 0.96), total carotenoids (P = 0.89), or uric acid (P = 0.99) considering differing exercise loads (Table 2). In addition, no differences were observed for α-tocopherol (P = 0.24), β-carotene (P = 0.49), total carotenoids (P = 0.23), or uric acid (P = 0.50; Table 3) in the different age groups.
The mean of ESR spectrometry to assess plasma antioxidant activity was 5.7 ± 0.3 mmol·L−1 of FS (range = 4.9-6.6 mmol·L−1 of FS). There were no differences between the groups of different exercise loads (P = 0.74; Table 2). A trend toward higher ESR values in the plasma of older subjects with 5.5 ± 0.3 mmol·L−1 of FS in the 12- to 14.5-yr-olds and 5.8 ± 0.4 mmol·L−1 of FS in subjects older than 18 yr was observed (P = 0.06, Table 3).
For TEAC, there were significant differences according to both exercise load (P = 0.007) and age (P = 0.0001; Figs. 2 and 3). Mean antioxidant capacity was 1.41 ± 0.2 mmol·L−1 of TE, with a range of 1.11-1.90 mmol·L−1 of TE. Subjects with the highest daily exercise load exhibited significantly elevated TEAC levels compared with those with the lowest amount of daily exercise (1.55 ± 0.2 and 1.53 ± 0.1 mmol·L−1 of TE for men and women, respectively, in the 4th quartile of EEtrain vs 1.37 ± 0.2 and 1.27 ± 0.2 mmol·L−1 of TE for men and women, respectively, in the 1st quartile of EEtrain). When conducting multiple regression analysis with dietary intake and plasma levels of all analyzed antioxidants, age, and daily exercise loads, only age (r = 0.18, P < 0.001) and exercise load (r = 0.32, P < 0.001) turned out to significantly effect the TEAC value. In our model, exercise load was the most important yet weak predictor of TEAC value.
Protein modifications were assessed by calculating the relative amount of cysteinylated transthyretin (% cysTTR) compared with native TTR. The mean rate of modified transthyretin was 110% ± 39% cysTTR, with no differences between men and women (P = 0.61, 105% ± 45% and 114% ± 36% cysTTR for men and women, respectively). Athletes exhibited a higher rate of protein modifications compared with moderately active controls (P = 0.002, 135% ± 41% and 86% ± 15% cysTTR; Table 4). The rate of modified transthyretin was similar across the age groups (P = 0.13; Fig. 4). Comparison between the groups of different exercise loads showed significant differences (P = 0.006), with a higher percentage of modified transthyretin in the moderately active subjects (2nd quartile of EEtrain) compared with the less active individuals (1st quartile of EEtrain, Fig. 5).
The purpose of this study was to evaluate antioxidant intake and antioxidant plasma status in adolescent individuals considering their age and physical activity. Antioxidant intake, as measured for the vitamins C and E and β-carotene, did not differ between competitive athletes and moderately active controls. We found that the majority of adolescents were able to meet recommended daily amounts of antioxidants with the habitual diet. However, in 24% of the subjects, vitamin C intake was below the recommendations, and 26% of them did not meet recommended daily amounts for vitamin E. Insufficient supply with vitamins C and E was more prevalent in women (28% and 31%, respectively) than that in men (17% for both vitamins C and E). Although a considerable number of adolescents would benefit from nutritional guidance, the prevalence of insufficient dietary antioxidant intake in adolescent athletes observed in this study is much lower compared with adult athletes reported by others. Rousseau et al. (31) observed that in 60% of elite athletes, vitamin C intake was less than recommended, and 81% of athletes documented vitamin E intakes below the recommendations. Admittedly, these authors used the French recommended daily amounts for athletes, which suggest higher antioxidant requirements for athletes compared with less physically active individuals. No such sports-specific validated recommendation is available for Europe. Because dietary antioxidant supply (β-carotene and vitamins C and E) was weakly but significantly correlated with energy intake in the present study, it can be suggested that a balanced, isocaloric diet may be sufficient to meet the recommended daily amounts and individual requirements of dietary antioxidants.
This hypothesis is underlined when considering the plasma levels of the measured antioxidants. Only two subjects exhibited plasma α-tocopherol concentrations below the cutoff point for deficiency (12 μmol·L−1 of α-tocopherol), one male rower with calculated isocaloric diet (9.7 μmol·L−1 of α-tocopherol) and one female soccer player with a calculated negative energy balance (−0.8 MJ·d−1; 10.8 μmol·L−1 of α-tocopherol). For carotenoids and α-tocopherol, mean plasma values of athletes and moderately active controls were in accordance to results presented by others. Irwig et al. (14) studied healthy Costa Ricans aged 12-20 yr and observed concentrations of 16.9 ± 3 μmol·L−1 of α-tocopherol and 0.47 ± 0.3 μmol·L−1 of β-carotene for men and 17.1 ± 3 μmol·L−1 of α-tocopherol and 0.60 ± 0.5 μmol·L−1 of β-carotene for women. In 12- to 17-yr-old Americans, β-carotene concentration was even lower with 0.22 ± 0.03 μmol·L−1 for men and 0.24 ± 0.03 μmol·L−1 for women, but α-tocopherol levels (16.9 ± 4 and 17.3 ± 4 μmol·L−1 for men and women, respectively) were comparable to the results of the present study (22). Considering that plasma levels of β-carotene and total carotenoids were within the reference values measured in 3089 individuals of nine European countries (2) and that α-tocopherol concentrations were slightly above the levels reported by others (14,22), we conclude that the diets of both athletes and controls are sufficient to meet the subject's requirements.
Plasma levels of α-tocopherol and carotenoids were not correlated with blood antioxidant capacity, which might be due to the fact that antioxidant intake and plasma levels varied within a small range. Correlations with antioxidant capacity might have been observed if some subjects had antioxidant plasma levels below the cutoff points for deficiency. Otherwise, our data suggest that additional antioxidant intake above the recommended daily amounts does not promote plasma antioxidant capacity. Indeed, there is growing evidence that large doses of supplemental antioxidants may have adverse effects on health and/or training adaptation. Very recently, Ristow et al. (27) studied the effect of 4 wk of endurance training on different markers of glucose metabolism and reported that increased ROS formation because of exercise efficiently counteracts insulin resistance. However, these health benefits of exercise were significantly attenuated in participants who were supplemented with antioxidants (1 g of vitamin C and 400 IU of vitamin E per day) compared with placebo (27). Regarding performance, Gomez-Cabrera et al. (11) observed a significantly impaired improvement in V˙O2max in supplemented subjects (1 g·d−1 vitamin C) compared with the placebo group after 8 wk of endurance training. It appears that supplementation with antioxidants may prevent useful exercise-induced adaptations of the endogenous antioxidant system, such as the activation of mitogen-activated protein kinase or nuclear factor kappa B pathways with subsequent upregulation of superoxide dismutase and/or glutathione peroxidase after exercise (23,32).
In our study, adolescent's plasma antioxidant capacity seems to increase with age and daily exercise load. These findings are in accordance with the recent literature, which shows that regular exercise improves antioxidant defense mechanisms. In 1991, Robertson et al. (28) reported significant positive correlations between resting activities of erythrocyte antioxidant enzymes and weekly training distance in runners. In addition, Miyazaki et al. (21) studied antioxidant enzyme activities after a training intervention in previously untrained men and found enhanced resting antioxidant enzyme activities after 12 wk of endurance training. Jackson (15) suggested that these kinds of response to regular exercise may result in significant protection of the muscle cells against possible damaging effects of later periods of exercise. Thus, the observed changes in antioxidant capacity in athletes with moderate to high daily exercise loads might be due to induction of antioxidant enzymes because both uric acid and nutritional antioxidants were not correlated with the TEAC value.
Considering age, little is known about antioxidant enzymes and blood redox status in athletes during growth and maturation. Gougoura et al. (13) compared the blood redox status of adolescent competitive swimmers with age-matched controls and found a 43% lower GSH/GSSG ratio and 28% lower total antioxidant capacity in the athletes. In contrast, Kabasakalis et al. (16a) observed an increase in the GSH/GSSG ratio and no change of antioxidant capacity in child swimmers during a training period of 23 wk. To our knowledge, there is only one study that compared the response of oxidative stress markers between adolescents and adults after the same exercise load. In this study, Timmons and Raha (38) did not find any difference in plasma carbonyl content between 9- and 10-yr-old boys and 20- and 25-yr-old men after 1 h of cycling at 70% of individual aerobic power.
Regarding protein modifications in this study, athletes in general exhibited a higher rate of modified transthyretin compared with less active controls (Table 4). However, post hoc comparisons revealed that only the moderate active subjects with a mean exercise energy expenditure of 2.7 MJ·d−1 (2nd quartile of EEtrain) showed significantly elevated rates of transthyretin cysteinylation compared with the less active subjects with an average of 1.3 MJ·d−1 spent for exercise (1st quartile of EEtrain). Considering that both groups of high exercise loads (3rd and 4th quartile of EEtrain) show significantly elevated blood antioxidant capacity suggesting antioxidant adaptations because of regular training, it could be hypothesized that subjects who are not well conditioned to exercise (2nd quartile of EEtrain) suffer the most from oxidative stress. Having observed less muscle damage after intensive exercise in trained animals compared with untrained, Gomez-Cabrera et al. (12) suggested moderate physical activity to be an antioxidant because physical activity was associated with an up-regulation in antioxidant enzyme activities in the trained animals. In mice, 8 wk of treadmill running resulted in reduced ROS generation and decreased oxidative stress in skeletal muscles (4). In addition, Simar et al. (36) found significantly elevated plasma antioxidant capacity (TEAC) and a 50% reduction of oxidative stress markers in physically active old men compared with age-matched controls with low physical activity. However, because of the little available data dealing with the redox status in exercising children, extrapolating data from animal or adult literature to adolescents needs to be done with caution (38).
In conclusion, habitual diets of adolescent athletes seem to provide enough antioxidants to meet recommended daily amounts and the individual's requirements. Data suggest that antioxidant intake increases with energy intake, and therefore adolescent athletes should be counseled to consume a balanced diet naturally rich in antioxidants instead of consuming supplements. However, special attention may be needed in cases where athletes restrict their energy intake. Currently, there is no evidence that adolescent athletes who consume a diet naturally rich in antioxidants experience adverse effects on health or redox status because of competitive exercise training. Indeed, regular exercise seems to improve antioxidant capacity in young athletes. Thus, beneficial effects of exercise training on antioxidant status rather than oxidative stress may be anticipated.
No funding was received for this work from any of the following organizations or others: National Institutes of Health, Wellcome Trust, and Howard Hughes Medical Institute.
This study has no professional relationships with companies or manufacturers who will benefit from the results of the study.
The results of this study do not constitute endorsement of the product by the authors or the American College of Sports Medicine.
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