There is convincing evidence showing that daily supplementation with a mixture of antioxidants for 4-8 wk can significantly reduce the cortisol and interleukin-6 (IL-6) responses to prolonged exercise (10,29). Fischer et al. (10) have demonstrated that 28 d of supplementation with antioxidants (500 mg of vitamin C) and 400 IU RRR-alpha-tocopherol, per day) completely blunted the cortisol response to exercise. Fischer et al. (10) also observed a significantly lower exercise-induced release of IL-6 from the contracting skeletal muscle in their supplemented subjects, and they suggest that this may contribute to attenuation of the cortisol response. A reduced IL-6 response to exercise with antioxidant supplementation has also been shown by Vassilakopoulos et al. (29). They observed a postexercise increase in plasma IL-6 concentration that was significantly reduced when subjects were supplemented daily with an antioxidant mixture of vitamin A (50,000 IU), vitamin C (1000 mg), and vitamin E (200 mg) for 60 d, allopurinol (600 mg) for 15 d, and N-acetylcysteine (2000 mg) for 3 d before (and 800 mg immediately before) exercise (45 min of cycling at 70% V˙O2max). Taken together, these studies provide substantial evidence for the notion that supplementation with antioxidants can reduce the cortisol and IL-6 responses to exercise; however, it remains unclear whether such effects are also associated with a reduction in the magnitude of immunodepression.
A reduced infection incidence after ultramarathon races has been observed when large doses of vitamin C were consumed for at least 7 d before the event (21,22). It is possible that increased levels of cortisol and some cytokines may have direct inhibitory effects on neutrophil function or an indirect effect because they contribute to exercise-induced leukocytosis (5,33). Increased leukocytosis (and consequently neutrophilia) leads to immature neutrophils constituting a greater proportion of total blood neutrophils. Neutrophils that are immature or that have been released prematurely from the bone marrow have a lower capacity to respond to stimulation (2). Therefore, a lower cortisol, IL-6, and leukocytosis response to exercise may be associated with a smaller depression in neutrophil function. Furthermore, antioxidant supplementation may result in improved antioxidant defense. This may modulate the postexercise decrease in neutrophil function by protecting these cells from oxidative damage (24). However, Nieman et al. (15) found greater IL-6 and oxidative stress responses (and no effect on cortisol) after a competitive triathlon event in athletes who had consumed vitamin E, compared with placebo, supplements daily for 2 months before the event. Furthermore, Nieman et al. (16,17) also have suggested that oxidative stress has little influence on immunoendocrine changes during and after prolonged exercise. Therefore, the aim of the present study was to examine the effects of a 4-wk period of oral supplementation with antioxidant vitamins (vitamin C, 1000 mg·d−1 and vitamin E, 400 IU·d−1) before a single bout of prolonged exercise, on plasma markers of oxidative stress, hormones of the hypothalamic-pituitary-adrenal (HPA) axis (plasma cortisol and adrenocorticotrophic hormone (ACTH) concentration), plasma IL-6 concentration, and neutrophil functional capacity.
Ethics approval was obtained from the local research ethics committee. Subjects were informed of the experimental procedures (verbally and in writing), and subjects gave written informed consent. Subjects also completed a medical questionnaire before participating in each test. The data of Davison et al. (7) were used to estimate that a sample size of 10 (per group) gives a 92.2% power to detect differences in the postexercise plasma cortisol concentration using a post hoc independent t-test.
Twenty healthy recreationally active men (age 23 ± 3 yr, body mass 71.7 ± 9.3 kg, V˙O2max 55.3 ± 10.4 mL·min−1·kg−1, power output at V˙O2max 294 ± 50 W; means ± standard deviation) participated in this study. Subjects were randomly assigned to one of two groups, either placebo (PLA) supplementation (N = 10) or antioxidant (AO) supplementation (N = 10). Subject characteristics for the two groups independently are listed in Table 1. Subjects in the AO group received oral supplementation with vitamin C (as L-ascorbic acid, 1000 mg·d−1) and vitamin E (as RRR-alpha-tocopherol, 400 IU·d−1), whereas the PLA group received a placebo containing calcium carbonate (267 mg·d−1) for 28 d. The supplement or placebo was taken in two equal doses per day, one with breakfast and one with the evening meal.
All subjects completed two exercise bouts: a preliminary trial (V˙O2max determination) and a main trial. For V˙O2max determination, subjects performed a continuous incremental exercise test to volitional exhaustion on an electrically braked cycle ergometer (Lode Excalibur, Holland) as previously described (7). Heart rate and rating of perceived exertion (RPE) were recorded during this period using a telemetric device (Polar, Kempele, Finland) and the Borg scale (3), respectively.
Subjects were instructed to consume 500 mL of water 2 h before arrival at the laboratory at 9:30 a.m. on the morning of the main trial, after an overnight fast of at least 10 h. Subjects sat quietly in the laboratory before beginning exercise at 10:00 a.m. for 2.5 h at an intensity of about 60% V˙O2max. Expired gas was collected into Douglas bags during the 15th, 30th, 45th, 60th, 90th, 120th, and 150th minutes of exercise, for analysis of V˙O2 and V˙CO2. Heart rate and RPE were recorded every 15 min during exercise. Subjects were given 2.5 mL·kg−1 body mass of a beverage (a low-calorie, lemon-flavored squash with artificial sweetener, providing 21 kJ of energy, 0.1 g of protein, 1.1 g of carbohydrate, trace amounts of fat, and trace amounts of sodium per liter of solution) every 15 min during exercise. This was equivalent to approximately 1.97 L of beverage throughout the course of the 2.5-h trial.
Subjects completed a weighed food record diary for the 48-h period before the main trial. The dietary records were analyzed using computer dietary-analysis software (CompEat, Nutrition Systems, London, UK). Subjects were all nonsmokers and were required to abstain from alcohol, caffeine, and strenuous activity for 48 h before the main trial. It was also stipulated that subjects should not take any mineral or vitamin supplement (other than those provided) or any other antioxidant supplements for the 4 wk before and during the study.
Blood samples were obtained by venepuncture, with minimal stasis, from an antecubital forearm vein and were collected into vacutainer tubes (9 mL in K3EDTA-treated tubes, and 7 mL into a heparin-treated tube). An initial baseline sample was taken after an overnight fast, before beginning the supplementation period. Further venous blood samples were taken immediately before beginning the 2.5-h exercise bout (Pre-Ex), within a few minutes of completing the exercise bout (Post-Ex), and after 1 h of recovery (1 h Post-Ex), during which subjects remained in the laboratory and were allowed to undertake restful activities such as reading or using a computer. All blood samples were obtained while subjects were in the seated position. Subjects were asked to sit without changing posture and with minimal movement for 15 min before all blood samples were drawn, except the Post-Ex sample, which was drawn as soon as possible after completing the exercise (within 3 min).
Hematological analysis was performed on the blood samples collected into one of the K3EDTA tubes using an automated hematology analyzer (ACT 5 diff, Beckman Coulter). Blood hemoglobin concentration, hematocrit, and total and differential leukocyte counts were measured. A 1-mL aliquot of blood from the heparin tube was used for measurement of neutrophil degranulation (see below). The remaining blood (K3EDTA and heparin tubes) was centrifuged at 1500g for 10 min at 4°C, and aliquots of plasma were stored at −80°C for later analysis. All samples were thawed only once before analysis. Estimates of postexercise plasma volume change (9) were small and similar for both groups, so it was not deemed necessary to correct any plasma variables for changes in plasma volume.
In vitro bacteria-stimulated neutrophil degranulation.
The neutrophil degranulation response to bacterial stimulant (840-15, Sigma, Poole, UK) was determined as previously described (25). Neutrophil degranulation was expressed as the amount of stimulated elastase release per neutrophil. Elastase concentration was determined using a commercially available ELISA kit (Merck Calibiochem, Darmstadt, Germany).
Plasma antioxidant capacity and vitamin C concentration.
Aliquots of heparin plasma were used for the determination of plasma antioxidant capacity (PAC) using a commercially available chemiluminescence (CL) test (ABEL-21M, Knight Scientific) with the use of a microplate luminometer equipped with an automated reagent dispenser (Anthos Lucy 1, Salzburg, Austria) as previously described (6). The capacity of plasma to scavenge the superoxide radical was expressed in ascorbate equivalent antioxidant units (μM). Aliquots of heparin plasma were used for the determination of plasma vitamin C concentration, according to Liu et al. (11), using a specific enzymatic (ascorbate oxidase, E 220.127.116.11) spectrophotometric assay.
Plasma cortisol, ACTH, and IL-6 concentrations.
Aliquots of K3EDTA plasma were analyzed to determine the concentrations of cortisol, ACTH, and IL-6 using commercially available ELISA kits (DRG, Biomercia, and R&D Systems, respectively).
Plasma free F2-isoprostane concentration.
To prevent the ex vivo formation of isoprostanes before analysis, samples were centrifuged immediately after collection, and K3EDTA-treated plasma was stored immediately at −80°C in the presence of 0.005% 3,5-Di-tert-4butylhydroxytoluene. Plasma free F2-isoprostane concentration was determined using a commercially available ELISA kit (Cayman Chemical Co.).
Plasma thiobarbituric acid reactive substances concentration.
A specific colorimetric method based on that used by Satoh (28) was used for determination of plasma thiobarbituric acid reactive substances (TBARS) concentration. Briefly, 20% trichloroacetic acid (1.25 mL) was added to plasma samples (250 μL) and left at room temperature for 10 min. The mixture was then centrifuged at 3000g for 10 min, and the supernatant was decanted. A weak solution (0.05 M) of H2SO4 (1 mL) was added to the precipitate, which was mixed thoroughly and centrifuged again, and then the supernatant was decanted. Both liberation of TBARS and color reaction were then performed simultaneously by heating (30 min in a boiling water bath) the precipitate with 1.25 mL of H2SO4 and 1.5 mL of the sodium sulphate/thiobarbituric acid (Na2SO4/TBA) reagent mixture (the concentration of TBA was 0.2% and Na2SO4 was 2 M; the solution also contained 20 μM Fe2+). Resulting chromogen was extracted with butyl alcohol, the absorbance of which was read immediately at 535 nm against butyl alcohol.
Statistical analysis was carried out using the statistical computer software package SPSS (version 12.00; SPSS Inc., Chicago, IL). Demographic information relating to subjects and results from the dietary analysis were normally distributed. Subjects were compared between groups with independent-samples t-tests (Tables 1 and 2). Total leukocyte count, neutrophil count, elastase release, plasma vitamin C concentration, and PAC data were normally distributed. The other variables were not normally distributed but were normalized with log transformation (cortisol, F2-isoprostane, IL-6, TBARS) or square-root transformation (ACTH) before analysis. To compare the two independent groups' responses to exercise, a two-way between-groups ANOVA (group × time) was used. Post hoc independent t-tests with the Holm-Bonferroni correction were used, where appropriate, to compare time point-specific differences between groups. When there was evidence of an interaction, the effect of time was analyzed in each group independently with one-way repeated-measures ANOVA. The Greenhouse-Geisser correction was applied to all ANOVA P values. All results are presented as mean ± standard deviation.
Physiologic Responses, Markers of Oxidative Stress, and Plasma Antioxidant Capacity
There was no difference between groups' demographic data or dietary composition during the 48 h before the main trial or relative and absolute exercise intensity during the main trial (Tables 1 and 2). Plasma free F2-isoprostane concentration (Table 3) was significantly increased at Post-Ex (P = 0.002) and 1 h Post-Ex (P = 0.002), but there was no difference between the PLA and AO groups (interaction P = 0.740). A significant group × time interaction was observed (P = 0.023) for plasma TBARS concentration, and there was a significant main effect of group (P = 0.008). Plasma TBARS concentration (Table 3) was significantly lower at Post-Ex (P = 0.003) and 1 h Post-Ex (P = 0.024) in the AO group compared with the PLA group. Furthermore, one-way ANOVA on each group independently showed that there was no change in plasma TBARS concentration across all time points in the PLA group (P = 0.088), whereas there was a significant decrease below Pre-Ex values in the AO group at Post-Ex (P = 0.014) but not at 1 h Post-Ex (P = 0.086).
There were significant main effects of group (P = 0.002) and time (P = 0.012) for plasma vitamin C concentration (Fig. 1) and a trend for a group × time interaction (P = 0.056). The two-way ANOVA showed that plasma vitamin C concentration was significantly higher than baseline at Post-Ex (P = 0.032) and 1 h Post-Ex (P = 0.006). However, one-way ANOVA on each group individually revealed no effect of time in the PLA group (P = 0.407), whereas in the AO group, there was a significant increase above baseline after the 4-wk supplementation period (P = 0.018), and a further increase above Pre-Ex values at Post-Ex (P = 0.034) and 1 h Post-Ex (P = 0.042). PAC (Fig. 2) was not significantly affected by the supplementation (interaction P = 0.771, main effect of group P = 0.232) and did not change significantly over time (main effect of time P = 0.506).
Plasma ACTH, Cortisol, and IL-6 Concentrations
Plasma ACTH concentration (Table 4) was significantly increased at Post-Ex (P < 0.001) and returned to near baseline by 1 h Post-Ex (P = 0.765); there was a trend for the temporal pattern to be different between the PLA and AO groups (interaction P = 0.077). There was a significant group × time interaction effect for plasma cortisol concentration (Fig. 3) (P = 0.008), with the increase above baseline significantly greater in the PLA group compared with the AO group at Post-Ex (P = 0.042) and 1 h Post-Ex (P = 0.038). Furthermore, one-way ANOVA analysis on each group independently revealed an effect of time (P < 0.001) for the PLA group, with the Post-Ex and 1 h Post-Ex values significantly increased above baseline (P < 0.001 and P = 0.028, respectively), whereas the main effect of time for the AO group was not quite significant (P = 0.052). Plasma IL-6 concentration (Table 4) was significantly increased at Post-Ex (P < 0.001) and 1 h Post-Ex (P < 0.001), and the temporal pattern was not different between the PLA and AO groups (interaction P = 0.167).
Circulating Leukocyte Number and Neutrophil Function
Circulating leukocyte count (Table 4) was significantly increased Post-Ex (P < 0.001) and 1 h Post-Ex (P < 0.001) and the temporal pattern was not different between the PLA and AO groups (interaction P = 0.441). Similarly, circulating neutrophil count (Table 4) was significantly increased Post-Ex (P < 0.001) and 1 h Post-Ex (P < 0.001) and the temporal pattern was not different between the PLA and AO groups (interaction P = 0.459). There was a significant decrease in the amount of elastase released per neutrophil in response to in vitro bacteria stimulation after exercise (Fig. 4) in both groups (P < 0.001). However, there was no difference between groups in this response (interaction P = 0.423).
The main findings of this study are that 4 wk of antioxidant vitamin supplementation may blunt the cortisol response to a single bout of prolonged exercise independently of changes in oxidative stress or plasma IL-6 concentration. However, there were no significant effects on total leukocyte or neutrophil trafficking, and it was not effective at modulating the postexercise reduction of neutrophil function. The findings from the present study are in line with the suggestion of Peake (18) that adrenal cortisol and vitamin C release do not occur together (corelease) in response to oxidative stress. For example, there was no change from pre- to postexercise in plasma vitamin C concentration in the PLA group, despite a significant increase of plasma cortisol concentration. Furthermore, it has been shown previously that vitamin C supplementation can influence the temporal response of plasma cortisol concentration, without any effect on markers of oxidative stress (6). It is possible that there are some direct effects of vitamin C on the HPA axis, and it has been suggested that this occurs at the level of the adrenal glands (6,18-20). However, the trend for lower ACTH responses in the AO compared with the PLA group in the present study suggests that there also may be some effects elsewhere on the HPA axis, possibly at the level of the hypothalamus or the anterior pituitary gland.
The results from the present study agree with the findings of Peters et al. (19,20) and Fischer et al. (10) with regard to the plasma cortisol response, but they are not in line with the studies of Vassilakopoulos et al. (29) or Fischer et al. (10) with regard to the systemic IL-6 response. Fischer et al. (10) have demonstrated that IL-6 release from contracting skeletal muscle is reduced with antioxidant supplementation, probably by reducing its translocation from active skeletal muscle tissue into the circulation. We hypothesized that this may result from vitamin E exerting an effect in the lipid milieu and somehow interfering with cellular membrane transport mechanisms and reducing translocation out of the muscle fibers (6). The present results do not support this idea, because there was no effect on plasma IL-6 concentration despite the inclusion of vitamin E in the AO supplement. It is possible that greater IL-6 responses are required (as observed with the exercise protocol used by Fischer et al. (10)) for AO supplementation to modulate IL-6 (and immune function) changes with exercise. The cycle ergometer exercise employed in the present study was, perhaps, more comparable with what athletes would actually do in training. Therefore, methodological differences and, perhaps, fitness and training status differences may account for some of the different observations between studies. It should also be noted that in a study by Nieman et al. (15), 2 months of vitamin E supplementation resulted in a greater IL-6 response to prolonged exercise (with no effect on the cortisol response). However, the exercise undertaken in that study was extreme by comparison (a competitive event of approximately 12-h duration), with mean postexercise plasma IL-6 concentrations more than 10-fold higher than in the present study.
It is difficult to compare the plasma free F2-isoprostane results of the present study with previous exercise and antioxidant supplementation studies. That is because many previous studies (12,13,26) report plasma total (free plus esterified) F2-isoprostane concentration. Moreover, there is a great deal of variation in the values reported in the literature; for example, Mastaloudis et al. have reported baseline values between 70 and 90 pg·mL−1 (13) and between 25 and 30 pg·mL−1 (12), and Sacheck et al. (26) have reported baseline values between 2 and 3 ng·mL−1. Therefore, it is probably more appropriate to just consider the temporal patterns within a study. However, the baseline values observed in the present study are comparable with those of Fischer et al. (10), who observed baseline levels of approximately 30 pM (~10.6 pg·mL−1). Fischer et al. (10) also observed that the significant increase in plasma F2-isoprostane concentration, which peaked immediately after exercise, was blunted with antioxidant supplementation. It may be that differences are more likely to be observed at the time of peak plasma F2-isoprostane concentration, which may have been missed in the present study (Table 3). This is one possible explanation for the lack of effect in the present study. Alternatively, it may be attributable, at least in part, to methodological differences in terms of type and duration of exercise. It is possible that antioxidant supplementation is more likely to blunt the plasma F2-isoprostane response to longer-duration exercise. For example, in the study by Sacheck et al. (26), antioxidant supplementation (1000 IU of RRR-alpha-tocopherol per day for 12 wk) did not influence the peak plasma F2-isoprostane response, which occurred at 72 h after exercise, after 45 min of downhill treadmill running at 75% V˙O2max in both young (mean age 26 yr) and older (mean age 71 yr) men. However, the exercise undertaken in the study by Fischer et al. (10) was of 3-h duration, and in the study by Mastaloudis et al. (12), antioxidant supplementation (300 mg of RRR-alpha-tocopherol and 1000 mg of ascorbic acid per day, for 6 wk) was associated with significantly lower plasma F2-isoprostane concentrations immediately after an ultramarathon event that had lasted approximately 7 h. The significant main effects of group and group × time interaction observed for plasma TBARS concentration (Table 3) are in line with the findings of Watson et al. (32), who showed that plasma F2-isoprostane concentration was decreased after exercise in subjects who had consumed a high-antioxidant diet for 2 wk. However, the TBARS results of the present study should not be considered alone as evidence that the supplementation was effective at blunting oxidative stress. Indeed, the TBARS results are not in line with the plasma free F2-isoprostane results in the present study. This highlights the need for a number of measures of oxidative stress and demonstrates that the TBARS assay, which has been criticized for a lack of specificity (30), should not be used as a sole measure of oxidative stress. However, it is known that plasma MDA and F2-isoprostane responses to exercise differ in time course (26). There was a significant difference in the plasma TBARS response, but not the plasma free F2-isoprostane response, between groups in the present study. This suggests that, in future studies, measurements should be made more frequently after exercise and for a longer postexercise period (up to 72 h) to determine whether peak plasma levels are observed at a time point not measured in the present study, and also to determine whether differences would be apparent at such times.
The AO supplementation in the present study was not effective at modulating prolonged exercise-induced neutrophilia. This is not in line with a previous study from our laboratory (6) in which 2 wk of vitamin C supplementation was associated with a reduced cortisol response and significantly reduced postexercise leukocytosis and neutrophilia responses. However, in the present study there was a trend for higher plasma cortisol at Pre-Ex in the AO compared with PLA group, and it is not clear whether this was a direct consequence of the supplementation or was attributable to some unforeseen difference(s) between the groups. Therefore, although the antioxidant supplementation seemed to blunt the increase in cortisol from pre- to postexercise, the higher levels in the antioxidant group before exercise may partly account for the observation of a similar neutrophil count after exercise. This may help explain the observation of no difference between groups in the magnitude of the postexercise decrease in neutrophil degranulation. For example, although cortisol does not have a direct inhibitory effect on neutrophil degranulation (31), the associated delayed neutrophilia may have an indirect effect, because immature neutrophils have a lower capacity to respond to stimulation (2). It is not surprising, therefore, that there is no difference between groups in postexercise neutrophil function.
Davison and Gleeson (8) and Robson et al. (24) have demonstrated that neutrophil function (measured by in vitro stimulated oxidative burst capacity) is protected after prolonged exercise when antioxidants are supplemented for a short period before and/or on the day of exercise. The results of these two studies (8,24) may be related to antioxidant protection against (auto)oxidative damage directly to neutrophils. Alessio et al. (1) have demonstrated that vitamin C supplementation (1000 mg·d−1) for 1 d is more effective than 2 wk of supplementation at modulating exercise-induced oxidative stress. Therefore, it may be that a relatively short period of supplementation is more beneficial because it protects neutrophils from (auto)oxidative damage or modification. The same benefit may not occur with longer-duration supplementation periods (as employed in the present study), because it is likely that the natural endogenous antioxidant defenses are downregulated in response and/or adaptation to the higher intake of exogenous antioxidant compounds so that total antioxidant capacity is kept fairly constant (4,23). The plasma vitamin C and PAC results of the present study support this contention and are in line with the findings of McAnulty et al. (14), who observed that 2 months of vitamin E supplementation significantly increased plasma alpha-tocopherol concentration without significantly affecting plasma ferric reducing antioxidant power, a marker of plasma total antioxidant capacity. However, the antioxidant supplements were not taken on the day of exercise in the present study. It is possible, therefore, that there would have been higher plasma antioxidant defense (PAC) if that had been the case, considering that the supplement also contained vitamin C, which can result in an acute peak in plasma ascorbate concentration within 2-3 h of ingestion (27).
In conclusion, supplementation with high doses of the antioxidant vitamins C and E for 4 wk before a single 2.5-h bout of prolonged cycling modulates the cortisol response but does not affect the IL-6 response, oxidative stress, or perturbations in neutrophil function, which typically follow. It is possible that greater IL-6 responses are required for antioxidant supplementation to modulate IL-6 and immune function changes with exercise, but this requires further investigation. The present findings indicate that daily high-dose antioxidant vitamin supplementation is unlikely to be of real practical benefit.
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