Physical inactivity is a recognized risk factor in the development of obesity, diabetes, and cardiovascular disease (CVD), and the health benefits associated with regular moderate-intensity exercise are incontrovertible. Physical training decreases the incidence of hypertension (2) and results in favorable changes in the blood lipid profile (5). However, aerobic exercise increases the rate of oxygen consumption, which, in turn, increases the production of reactive oxygen species (ROS) (1). In some circumstances, ROS can assist in repairing damaged tissue and destroying invading microorganisms via phagocytosis and respiratory burst activity (12). However, large amounts of ROS, like those identified to occur from ultraendurance exercise, can damage vital cellular structures, and oxidative damage can result. Cellular damage and oxidative stress have been associated with a number of pathophysiological conditions such as atherosclerosis, malignancies, and neurologic diseases.
There are some epidemiological data that implicate an increased risk of disease in individuals who regularly undertake large volumes of exercise (11,18,19,24). In the Harvard Alumni Health Study, individuals in the group with the highest energy expenditure had an increased relative risk of death compared with two groups who completed less exercise (18). Ten years later, in the same cohort, there was an increase in the age-standardized mortality rate in subjects reporting physical activity energy expenditure greater than 14,700 kJ·wk−1 (11). In addition, if the activity was vigorous, the increase in mortality occurred when energy expenditure exceeded 12,600 kJ·wk−1. This finding is consistent with that reported in the British Regional Heart Study, where vigorously active men had higher rates of heart attacks than men performing moderate or moderately vigorous activity (24). Finally, Quinn et al. (19) examined relationships between caloric expenditure and mortality in their Michigan State University Longevity Study (19). The authors reported that the most active subjects had the same risk of CVD as those who were least active (19). A plausible rationale linking high-volume physical activity and increased disease incidence may include oxidative stress in disease etiology (11,19). Furthermore, antioxidant fortifications may be an important mitigating factor in combating the oxidative stress thought to contribute to disease.
Although a few studies have investigated antioxidant status and oxidative stress with athletes involved in ultraendurance exercise, the findings have been inconsistent (7,9,13,15,17,22). A potential reason for the discrepancies is the different exercise durations reported by the authors. The primary aim of the present study was to examine the dose-response relationship of erythrocyte antioxidant enzyme activity and plasma oxidative stress in ultraendurance athletes training for and competing in half and full Ironman triathlons. Furthermore, recent findings of increased oxidative stress in ultraendurance athletes supplementing with vitamin E (17) prompted the final aim: to determine whether oxidative stress and antioxidant status are different in individuals who supplement with antioxidants.
MATERIALS AND METHODS
Thirteen male and three female ultraendurance triathletes who were training to compete in a half Ironman triathlon were recruited into the study 4 wk before the event. Subjects were (mean ± SD) 29.8 ± 7 yr of age, had a cycling V˙O2peak of 65.4 ± 5.8 mL·kg−1·min−1, and were undertaking medium- to high-intensity training 14.5 ± 3.4 h·wk−1. They had been training for and competing in ultraendurance events for 4.7 ± 2.4 yr. Sixteen control subjects (13 males and 3 females) were matched to the athletes on age, sex, height, and weight. None of the control subjects were active for more than 180 min·wk−1. Twenty-nine ultraendurance triathletes (23 males and 6 females) and 29 matched control subjects (who were different controls from those involved in the half Ironman study, because of the timing of each race) volunteered to participate in the full Ironman study. The full Ironman athletes were 36.2 ± 7.8 yr of age (mean ± SD), had been training for and competing in ultraendurance events for 6.9 ± 6.4 yr, and were training 17.19 ± 3.4 h·wk−1. These athletes were following a periodized program that focused on high-volume, low-intensity exercise.
Three athletes (two females and one male) who competed in the half Ironman also competed in the full Ironman. The 29 control subjects were matched to athletes on sex, weight, age, and height, and none were active for more than 180 min·wk−1. Each subject provided written informed consent, and the investigation was approved by the medical research ethics committee of The University of Queensland.
On entry into the study, all participants completed medical history, exercise, and lifestyle questionnaires to assess their current levels of physical activity, antioxidant supplementation, and menstrual status. Athletes reported for initial testing between 2 and 10 d before each race; the control subjects had resting blood sampled 2 wk before or 2 wk after the triathlon races. All participants had fasted overnight and had refrained from exercise for at least 36 h before being tested. On arriving at the laboratory (~21°C, 40-60% relative humidity, 760-770 mm Hg), participants had their weight and height measured, and a 10-mL sample of blood was drawn from an antecubital vein.
Within 15 min of completing the half or full Ironman triathlons, 10 mL of blood was again taken from the athletes via the antecubital vein. Blood was collected into lithium heparin vacutainers and centrifuged (International Equipment Company, Centra MP4R) at 1500 rpm for 10 min at 4°C. Aliquots of washed/lysed red blood cells and plasma samples were stored at −80°C until biochemical assays were performed. A mobile testing laboratory was used at the races to ensure appropriate collection, separation, and storage of samples.
Concentrations of total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, and triglycerides (TG) were measured on a Kodak Ektachem analyzer (DT60, Eastman Kodak Company, Rochester, NY).
Plasma malondialdehyde (MDA) concentration, a by-product of the peroxidation of ω-3 and ω-6 polyunsaturated fatty acids, was determined using an assay described by Sim et al. (25) using high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) with ultraviolet detection. The coefficient of variation for the measurement of MDA was 3.7%.
The methods used to measure hematocrit and hemoglobin followed those used by Dill and Costill (4). Hematocrit was determined in triplicate by spinning blood in a microcapillary centrifuge for 10 min at 11,800 rpm (Jouan, Saint Herblan, France; serial no. 25990013). Hemoglobin concentration was measured in triplicate using a Unicam UV/VIS Spectrometer 5625 series Cambridge, UK), and values were averaged for analysis.
Blood was analyzed for erythrocyte GPX and CAT activities using modified versions of assays described by Wheeler et al. (27) and Slaughter and O'Brien (26), respectively, using an automated spectrophotometer (Cobas Mira, Roche, Switzerland). SOD activity was measured using a modified version of the assay described by Madesh and Balasubramanian (14) using a microplate reader (Titertek, Helsinki, Finland). The within-run coefficients of variation obtained for 10 aliquots of the same sample were 1.6, 1.8, and 4.6% for GPX, CAT, and SOD activities, respectively, and were expressed relative to hemoglobin concentration.
The half Ironman triathlon race was held in Coomera, Australia, and athletes were required to complete a 1.9-km swim, a 90.1-km cycle, and a 21.1-km run. The race started at 6:00 a.m., when the air temperature was 27.0°C and relative humidity was 80%. By 10:30 a.m., the temperature and relative humidity were 33.3°C and 66%; by finishing time (median time for participants approximately 11:14 a.m.), the temperature and relative humidity were 34.6°C and 64%, respectively.
The Ironman triathlon was held in Forster, Australia and comprised a 3.8-km swim, a 180-km cycle, and a 42.2-km run. The race started at 6:15 a.m., when the air temperature and relative humidity were 14.6°C and 86%, respectively. By midday, the air temperature and relative humidity were 22.6°C and 56%, and by finishing time (median time for participants approximately 5:35 p.m.), temperature and relative humidity were 19.0°C and 74%, respectively.
Data were tested for normality using the Kolmogorov-Smirnov test, with all data sets found to be normally distributed. Independent t-tests were then used to assess differences in descriptive variables and lipid concentrations between athletes and control subjects. One-way analysis of variance (ANOVA) was used to compare resting activities of GPX, SOD, and CAT and MDA concentration between control subjects, all athletes, antioxidant-supplementing athletes, and nonsupplementing athletes. Post hoc analysis was performed using Tukey's test. Paired t-tests were used to investigate changes in lipid concentrations from pre- to postrace, and one-way repeated-measures ANOVA was used to determine whether there were differences from pre- to postrace between the four groups. Pearson's product-moment correlation coefficients were used to examine potential relationships between training volume, supplementation dosages, and time spent training. Significance was set at the 0.05 level of confidence, and statistical analyses were performed on SPSS (version 12) software.
Half Ironman versus Ironman athletes.
Table 1 shows that, compared with control subjects, the half Ironman athletes had significantly (P < 0.001) greater resting erythrocyte GPX activity (19%). As a result of the half Ironman triathlon, there was a significant (P < 0.05) increase in serum triglycerides (67%) and decreases in the activities of erythrocyte GPX (6.7%), SOD (16.4%), and CAT (15.4%, P < 0.001) (Table 1). Plasma MDA significantly (P < 0.05) increased (25.1%) after the half Ironman triathlon.
Compared with the control subjects, those athletes training for and competing in the full Ironman triathlon also had a significantly (P < 0.05) greater resting activity of GPX (7%) (Table 1). In addition, these athletes had significantly (P < 0.001) greater CAT activity (29%)and a lower concentration of plasma MDA (13%, P < 0.05). Table 1 shows that after the Ironman triathlon, athletes had significant increases in serum triglycerides (55%, P < 0.001) and HDL (7%, P < 0.05) and significant (P < 0.001) decreases in the activities of GPX (12.1%), CAT (31.1%), and SOD (22.1%). Plasma MDA was also significantly (P < 0.05) increased (17.4%) after the races.
Ten of the 16 half Ironman athletes were regularly taking antioxidant supplements (1095 ± 447 mg·d−1 of vitamin C for 4.9 ± 4.7 yr and 314 ± 128 mg·d−1 of vitamin E for 5.6 ± 5.2 yr). A comparison of resting values showed no differences between the two groups (supplementers and nonsupplementers) in erythrocyte antioxidant enzyme activities or plasma MDA (Table 2 and Fig. 1). Differences between the supplementers and nonsupplementers emerged after the half Ironman race. Supplementers had significant (P < 0.05) decreases in the concentrations of the antioxidant enzymes SOD (10.5%) and GPX (19.3%) and an increase in plasma MDA (31.2%) in response to the triathlon race. In the nonsupplementation group, there was a significant decrease (P < 0.05) in CAT (25%) after the races compared with before races. There were no significant (P > 0.05) correlations between the amount of supplemented vitamin C or vitamin E and any of the resting or exercise-induced changes in antioxidant enzyme activities or MDA concentration.
Eight of the 29 Ironman athletes were regularly taking antioxidant supplements (558 ± 350 mg·d−1 vitamin C for 0.8 ± 0.6 yr and 702 ± 756 mg·d−1 of vitamin E for 1.6 ± 0.8 yr), but there were no differences between the two groups in any of the resting antioxidant enzyme activities or MDA. After the Ironman race, those athletes who were supplementing had a significant decrease in the activities of GPX (15%, P < 0.05) and CAT (31%, P < 0.001) (Table 2 and Fig. 2). Athletes who were not taking antioxidant supplements also showed significant decreases (P < 0.001) in the activities of GPX (11%) and CAT (31%), but they also had a significant decrease (23%, P < 0.001) in SOD activity that was not observed in the supplementation group. Only those athletes who were supplementing with antioxidants showed a significant (P < 0.05) increase in plasma MDA (30%) after the race. There were no significant (P > 0.05) correlations between the amount of supplemented vitamin C or vitamin E and any of the resting or exercise-induced changes in antioxidant enzyme activities or MDA concentration.
There were significant relationships for the full Ironman athletes between time spent training and resting plasma MDA concentration (r = 0.37; P < 0.05) and erythrocyte SOD activity (r = 0.57; P < 0.001).
The main findings from the present study were that training for a half Ironman triathlon resulted in an increase in erythrocyte GPX activity, whereas training for a full Ironman triathlon resulted in increases in the activities of erythrocyte CAT and GPX. Furthermore, resting concentrations of plasma MDA concentration were significantly lower in the full Ironman athletes compared with controls. Both races resulted in significant increases in plasma MDA concentration and decreases in the activities of erythrocyte antioxidant enzymes. Of further interest was the finding that athletes taking antioxidant supplements had a significantly greater increase in the plasma concentrations of MDA after each race compared with nonsupplementers.
Half Ironman versus Ironman athletes: effects of ultraendurance training.
The present data suggest a dose effect of half versus full Ironman training on resting erythrocyte antioxidant enzyme activity and plasma MDA concentration. The full Ironman athletes trained an extra 2.7 h·wk−1 compared with the half Ironman participants, and their resting levels of both CAT and GPX were significantly higher than the controls; for the half Ironman athletes, only GPX was increased. It is proposed that the extra training and subsequent increased production of ROS upregulated CAT. The increase in CAT activity in the full Ironman athletes may explain why plasma MDA was significantly lower in this group compared with controls. CAT is mainly located in peroxisomes and is responsible for reducing organic hydroperoxides such as hydrogen peroxide, which has the capability of causing lipid peroxidation and increasing MDA. Furthermore, the significant positive correlation between time spent training and resting MDA concentration in the full Ironman triathletes lends support to a dose-response relationship.
Compared with control values, resting GPX erythrocyte activity was significantly higher in both half Ironman and Ironman athletes. This is consistent with the findings from previous studies (6) and supports the notion that this enzyme may be the most responsive to exercise-induced oxidative stress (3). It has been suggested that higher-intensity activity generates hydrogen peroxide, which, in turn, is capable of increasing the activity of GPX (3).
Although there was no significant difference in SOD activity between the Ironman athletes and control subjects, the time spent training by the athletes was positively related to resting SOD activity. It is possible that SOD activity is less sensitive to training than the activities of GPX and CAT. Indeed, this was the case in the investigation by Elosua et al. (6), in which it was reported that GPX activity significantly increased in response to a 16-wk training program, whereas SOD activity remained unchanged.
Acute effects of ultraendurance exercise.
Plasma concentrations of MDA significantly increased in response to the half and full Ironman triathlons. Previous investigations on this topic using a range of single exercise bouts from a marathon to Ironman triathlons have yielded equivocal results (7,9,13,15,22). Kanter et al. (9), Sanchez-Quesada et al. (22), and Liu et al. (13) all reported significant increases in oxidative stress in response to marathon and ultramarathon distance running races. In contrast, Ginsburg et al. (7) reported a decrease in the oxidizability of LDL, and Margaritis et al. (15) found no change in thiobarbituric reactive substances (TBARS) or in the susceptibility of lipids to peroxidation in response to an ultraendurance triathlon race. The discrepancy in the results may be related to the distance and intensity of the bouts of exercise, coupled with the analytic methods and assays used to detect oxidative stress. The use of TBARS and conjugated dienes to detect oxidative stress has been criticized for their lack of accuracy and validity (8,16). In contrast, measurement of plasma MDA concentration using HPLC is considered both sensitive and reproducible (25) in the assessment of oxidative stress (10) and is arguably more reliable and sensitive than the measurement of TBARS and conjugated dienes that have been used by others to estimate oxidative stress (8,16).
Our finding of decreases in antioxidant enzyme activities after the races is somewhat unexpected and seems to be in contrast to the majority of studies, which report either an increase (9,13,22) or no change (15) in activity after exercise. The decrease in antioxidant enzyme activity observed may reflect allosteric downregulation of the enzymes in addition to enzyme inactivation attributable to overwhelming oxidative stress. Alternatively, Elosua et al. (6) have shown that there is a transient decrease (at 30 min postexercise) in erythrocyte SOD, whole-blood GPX, and glutathione reductase after a 30-min exercise bout. The authors postulate that exercise-induced ROS production leads to the consumption of enzyme activity with a subsequent rebound recovery. In the present study, the approximate time between finishing the race and obtaining the blood sample was 15 min. During this time, exhausted athletes sought fluids, nourishment, and the accolades of supporters. These findings may illustrate the importance of the timing of blood samples after exercise.
Resting MDA concentrations and erythrocyte antioxidant activities were not significantly different between the supplementation and nonsupplementation groups of half and full Ironman athletes. Interestingly, only the athletes taking antioxidant supplements showed a significant increase in MDA concentration from before to after both races. Despite the obvious limitations of interpreting these observational data, we feel this finding is worth reporting because it is consistent with recent research with Ironman athletes that found antioxidant supplementation (vitamin E) had a prooxidant effect (17). Indeed, it has been proposed that a high dose of vitamin E in the presence of oxidative stress creates free radicals capable of initiating lipid peroxidation (20).
There were no relationships found between any of the dependent variables and antioxidant supplementation in the half or full Ironman triathletes. Previous research examining the effects of antioxidant supplementation on markers of oxidative stress in response to endurance running has, again, yielded equivocal results (21,23). Rokitzki et al. (21) have reported no difference in TBARS between 24 runners supplemented for 4.5 wk with vitamin C and vitamin E (and who completed a marathon) and a placebo group. However, Sanchez-Quesada et al. (23) have shown that 1 g of ascorbic acid consumed before a 4-h running race reduced LDL susceptibility to oxidation in seven well-trained marathon runners compared with seven controls. The inconsistency in these findings may again be explained, at least in part, by the analytic methods used to assess oxidative stress and by the differences in intensity and duration of exercise. Future studies that approach the problem of oxidative stress in ultraendurance exercise may be advanced through the use of Trolox-equivalent antioxidant capacity and ferric reducing antioxidant power assays, to better resolve discrepancies regarding erythrocytes and plasma oxidative stress variables.
In summary, the present investigation has shown that high-volume ultraendurance activity produces a significant decrease in resting MDA concentration (with athletes involved in full Ironman triathlon training) and increases in the resting activities of GPX (Ironman and half Ironman triathlon training) and CAT (Ironman triathlon training). Both the half and full Ironman triathlons resulted in increases in plasma MDA, coupled with decreases in the activities of GPX, SOD, and CAT. Athletes taking antioxidant supplements had significantly greater increases in plasma MDA after the Ironman triathlon. Combined, these data indicate that there is a dose-response relationship between adaptations of antioxidant enzymes and responses to ultraendurance exercise. It seems that the volumes and intensities of activity associated with ultraendurance training confer protection against increases in free radical damage, resulting in improved oxidative balance.
The authors wish to thank Jim Sharman, Paul Laursen, Gary Wilson, Sue Marsh, Shannon Ahern, and Warrick Dalziel for their contributions to the study, and the participants for their valuable time and effort.
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Keywords:©2007The American College of Sports Medicine
ACUTE ULTRAENDURANCE EXERCISE; MALONDIALDEHYDE; GLUTATHIONE PEROXIDASE; SUPEROXIDE DISMUTASE AND CATALASE