Despite the many health-related benefits of endurance exercise, there is also evidence suggesting that the 10- to 15-fold rise from rest in oxygen consumption occurring during strenuous exercise promotes formation of free radicals (1). Exercise-induced increase in oxidative stress can result in lipid peroxidation, which has been implicated in a number of destructive biological processes including DNA modification, aging, membrane ion transport damage, cancer, and atherosclerosis (6,16,20).
The effect of decreased estrogen levels, specifically estradiol, in physically active women on oxidative stress is of particular interest especially because a protective role has been demonstrated for estrogen as a free radical scavenger (4,10,11,17,19,22). Estrogens have been shown to inhibit low density lipoprotein (LDL) peroxidation in vitro (10,22) and in vivo (19), and it has been suggested that this effect is involved in the protection against cardiovascular diseases in postmenopausal female subjects (3,5,14,26). The purpose of the present study is to characterize the effect of an endurance run (22.1 km) on lipid peroxidation as measured by the formation of conjugated dienes in trained eumenorrheic runners during a low estrogen state of the menstrual cycle.
Seven eumenorrheic females (mean age ± yr) who ran an average of 5.5 d·wk−1 for 50 min·d−1 provided voluntary informed consent in accordance with the university’s institutional review board. Each subject completed a medical questionnaire, which included a detailed exercise and menstrual history. Subjects were nontobacco users and reported no history of coronary artery disease, hypertension, diabetes, or hyperlipidemia. Subjects did not consume estrogen supplements, oral contraceptives, or antioxidant supplements (vitamin C, E, and beta-carotene) within the past 3 months. Descriptive data for the subject’s is presented in Table 1.
To determine dietary antioxidant intake, all subjects completed a diet history, which included a 7-d dietary record for the week preceding the run. Diets were analyzed by the Nutritionist IV computer program (First Data Bank, San Bruno, CA).
Skin-fold measurements, made with Lange calipers (Cambridge Scientific Industries, Cambridge, MD) were taken from three sites: the triceps, supra-iliac, and abdomen. The average of three repeated trial values was used as the representative score for a given site for use in the equation of Pollock et al. (15) for estimation of body fat.
The race was a 22.1-km (half-marathon) run. Fasting blood samples were drawn 2 h before and 5-min post run. Subjects were either postovulatory (day 21–25 on a 28-d cycle) or currently menstruating (day 2–5). Pre- and post-exercise blood samples were collected in EDTA (1 mg·mL−1) for LDL isolation (10 mL). Plasma was separated by centrifugation at 1,500 ×g for 14 min at 4°C. The samples were immediately subjected to density gradient ultracentrifugation for isolation of lipoproteins based on the procedures of Havel et al. (8). In brief, LDL were isolated with solid potassium bromide at density 1.109–1.063 (42,000 rpm for 24 h) with an L3–50 ultracentrifuge and Ti 50.3 rotor (Beckman Instruments, Palo Alto, CA). The lipoproteins were flushed with N2 and dialyzed (Dulbecco’s phosphate-buffered saline (PBS)) without EDTA for 24 h with two changes of dialzying solution. The samples were then flushed with N2 again and kept at 4°C until ready for LDL oxidation assay. LDL oxidation was determined by the formation of conjugated dienes as described by Esterbauer et al. (6). LDL (100 μg·mL−1) in PBS was incubated with 2,2′=azobisdihydrocholoride (2 μM) and absorption 234 nM was monitored every 5 min for 150 min. The specimens were present in cuvettes and maintained in a water bath (Scientific Product/Temp-Blok Module Heater) at 37°C during the reaction process/between measurements. The measurements were taken in a UV/Visible Spectrophotometer (Pharmacia Biotech, Piscataway, NJ/Ultraospec 2000). The initial absorbance was set at zero. The delay absorption at 234 nM was divided into phases: lag, propagation, and decomposition. The lag time was determined from the intercept of the lines drawn through the linear portion of the propagation phase and the lag phase.
Enzymatic methods were used for plasma total cholesterol, high density lipoprotein cholesterol, and triglyceride levels (25) and levels of 17β-estradiol was measured by radioimmunoassay (21).
Statistica for Windows (Tulsa, OK) was used to perform the data analysis. Mean and standard errors were obtained for the descriptive data. To evaluate the pre- and post-values of LDL peroxidation lag time, a paired t-test was performed. To determine the correlation between estradiol levels and lag time, the Pearson product correlation was used. Level of significance was set at P < 0.05.
Results of biochemical characteristics are presented in Table 2. Total cholesterol, triglyceride, and lipoprotein values were within normal limits and representative of endurance-trained women.
Estradiol levels ranged from 18 to 78 pg·mL−1. LDL oxidation showed an increase in lag phase time of conjugated diene formation in all samples (Table 3). The increase (25.7 ± 18.2%) in lag time when comparing pre- and post-race values was significant (P < 0.05).
In this study, we examined the issue of exercise induced oxidative stress in female endurance athletes during a low estrogen phase of the menstrual cycle. We observed that prolonged exercise was associated with a decrease in susceptibility to lipid peroxidation despite subject’s relatively low estrogen levels. To date, the issue of exercise induced oxidative stress after prolonged exercise has been studied primarily on male athletes with varying results. Endurance athletes (26 male, 13 female) participating in the Hawaii Ironman Triathlon demonstrated a decrease in susceptibility of lipids to peroxidation as determined by the formation of hydroxyl radicals (7). This effect was not related to subject’s antioxidant use or levels of vitamins A, C, or E. The authors suggested that iron may play a role in mediating lipid peroxidation during prolonged ultraendurance exercise due to the associated 45% decline in serum iron, an effect that was weakly correlated with changes in lipid peroxidation. Similarly, Vasankari et al. (23) demonstrated a slight increase in serum diene concentration in male endurance athletes after a 31-km run. Conversely, Sanchez-Quesada et al. (20) studied six male athletes and reported an increase of LDL susceptibility to oxidation occurring after intense, long duration exercise (4 h run @ 11.95 km·h−1). Viinikka and colleagues (24) studied 10 trained long-distance runners (9 male, 1 female) and demonstrated that acute exhaustive exercise did not affect lipid peroxidation as measured by thiobarbituric acid reactive species. Discrepancy in these studies may be a result of varying training protocols, varying training levels of the athletes and differences in methods used in the detection of lipid peroxidation.
Although only a few studies have specifically addressed the issue of exercise induced oxidative stress in female athletes with varying estradiol levels (2,9), a definite increase in lipid peroxidation parameters in athletes with low estradiol levels has not been demonstrated. Kanaley and Ji (9) evaluated lipid peroxidation in amenorrhiec and eumenorrheic (estradiol: 43.2 ± 5.7 pg·mL−1 and 64.3 ± 6.5 pg·mL−1, respectively) runners after prolonged exercise (90 min at 60% V̇O2max). They noted a nonsignificant trend toward an increase in plasma malonadelyde (MDA) levels in amenorrheic athletes. The athletes in the present study had a mean plasma estradiol level comprabable to Kanaley and Ji’s amenorrheic athletes, but the range was much larger (42.17 ± 21.65pg·mL−1 vs 43.2 ± 5.7 pg·mL−1, respectively). Also, the methodology for measurement of lipid peroxidation differed (conjugated diene vs MDA) as did exercise intensity and duration.
Recently, Ayres et al. (2) reported a significant decrease in lag time of formation of conjugated dienes in both amenorrheic and eumenorrheic (22.4 ± 2.0 pg·mL−1 and 66.0 ± 14.8 pg·mL−1, respectively), after 15 min of maximal treadmill exercise (V̇O2max test). The magnitude of difference in lag time was significantly greater (P < 0.05) in the amenorrheic group, and the authors also reported modest correlations between estradiol and conjugated dienes. The amenorrheic runners studied by Ayers et al. had a mean estradiol level lower than the subjects in the present study and they participated in a shorter but more intense exercise bout. Exercise intensity has been shown to be a factor in lipid peroxidation in some (13,24) but not all (12) studies.
We did not observe a relationship between estrogen level and of lipid peroxidaiton. In fact, the subject with the lowest estradiol level (18 pg·mL−1) had a similar short lipid peroxidation lag phase time compared to the subject with the highest estradiol level (78 pg·mL−1) (Table 3).
Vitamin E (alpha-tocopherol) and vitamin C are nutrient antioxidants that can scavenge or reduce free radicals. They may also be influenced by exercise. Robertson et al. (18) studied male subjects of various training levels and found that erythrocyte alpha-tocopherol levels were significantly higher in the runners versus sedentary males. Measurement of serum antioxidants was not determined in this study. Nutrient analysis revealed a mean alpha tocopherol dietary intake of 3.5 ± 0.7 mg, which is 44% of the RDA of 8 mg·d−1 for women 24–50 yr. Vitamin C intake was 166% subject’s RDA.
The results of this study have shown an increase in lag time of conjugated diene formation after a 22.1-km running event in female athletes during a low estrogen phase of the menstrual cycle. This suggests that aerobically trained women who report normal menses do not seem to be at increased risk for lipid peroxidation in response to a single competitive endurance running event during a low estrogen phase of their menstrual cycle.
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