Long-duration and ultramarathon running have been shown to increase oxidative stress (17,21). Ultramarathoners are known to consume nonsteroidal antiinflammatory drugs (NSAID) such as ibuprofen in an attempt to prevent muscle damage and soreness and, thereby, increase race performance (22). However, NSAID use along with strenuous exercise during ultramarathons may increase gut permeability and enhance endotoxin leakage from the gut into the blood, resulting in inflammation and oxidative stress, but these events have not yet been verified using appropriate research designs (1,3,6). In support of the concept of increased inflammation, we recently reported that inflammatory plasma cytokines were two to three times greater in runners reporting use of NSAID after a 160-km race event compared with nonusers (22).
Specifically, NSAID such as ibuprofen may contribute to oxidative stress, because nonspecific COX inhibitors are known to induce nephrotoxicity (10) and to induce liver function abnormalities (4). It is known that ibuprofen is capable of inducing renal damage, including functional acute renal failure, water and electrolyte disorders, and interstitial nephritis, but nephropathy is not a routinely documented consequence of ibuprofen therapy. Renal side-effects of ibuprofen seem to be dose dependent and are rarely reported at the recommended dosage as an over-the-counter drug (0.2-0.8 g·d−1). Even at antiinflammatory doses (> 1.6 g·d−1), renal side-effects are almost exclusively encountered only in individuals predisposed to renal problems (15). Despite this, the consequences of NSAID use on oxidative stress during extreme exercise are not well known.
In a previous study demonstrating a linkage between ibuprofen use and elevated plasma cytokines, the ibuprofen dose was not controlled or reported (22). As a follow-up to this study, we designed a controlled study in which athletes used 600 and 1200 mg of ibuprofen the day before and during the race, respectively. Ultramarathon race events allow the relationships between NSAID use and oxidative stress to be tested in an extreme exercise environment of long-lasting duration. Changes in oxidative stress indicators should occur within the first few hours of the race and should then be maintained or become pronounced 20-30 h later at the end of the race.
PGE2 is a major cyclooxygenase (COX) product that is important in human physiology and pathophysiology. Increased PGE2 production is associated with malignancy, vascular tone, kidney function, and proliferation of carcinoma cells. Quantification of systemic PGE2 production in humans can be assessed by measuring urinary metabolites. The major urinary metabolite of PGE2 is 11-alpha-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M) (20). PGE2 concentrations are reduced by ibuprofen and, as such, can serve as an end point of ibuprofen activity (11).
Therefore, given the potential detrimental effects of ibuprofen, the purpose of this study was to examine the effect of ibuprofen use with extreme exercise on oxidative stress and PGE2. Changes in plasma and urinary F2-isoprostane formation and plasma nitrite and urate were used as indicators of oxidative stress. Urinary PGE-M was used as an end point to assess ibuprofen use during the race. We hypothesized that ibuprofen use during the 160-km race would augment endotoxemia and associated drug-action mechanisms, which would lead to increased oxidative stress and decreased PGE-M (18).
MATERIALS AND METHODS
Sixty-three experienced male and female ultramarathoners from the 2005 Western States 100-Mile Endurance Run were recruited and provided prerace blood and urine samples. Athletes were placed into ibuprofen (N = 33) and control groups (N = 30) on the basis of their historical use during training and competition and their willingness to use or avoid ibuprofen before and during the race. Permission for a randomized, placebo-controlled research design was not granted by the race medical board because of ethical concerns regarding compliance in athletes suffering from pain during the latter stages of the race. Fifty-four subjects (N = 29 in the ibuprofen group, N = 25 for controls) completed the race and provided postrace blood and urine samples. Written informed consent was obtained from each subject, and the study's experimental procedures were approved by the institutional review board of Appalachian State University and in conformance with the policies of the American College of Sports Medicine. To enter the study, subjects had to have completed a 160-km race and have qualified for the 2004 160-km Western States Endurance Run. To qualify for the Western States Endurance Run, runners must have completed a 160-km race in less than 24 h, or a 100-km race in 12-13 h, depending on age. The 160-km Western States Endurance Run is a point-to-point trail run in the Sierra Nevada of northern California. It is regarded as one of the most arduous organized running events in the United States.
Subjects provided urine and blood samples during registration (held the morning before the race) and 5-15 min after the race. Blood samples were drawn from an antecubital vein with subjects in the seated position. Urine and plasma samples were transported on dry ice and then were stored at −80°C until analysis. Prerace body mass and percent body fat (via three-site skinfolds) were measured, and subjects filled in a questionnaire on basic demographics and training history. Subjects in the ibuprofen group ingested 600 mg (three 200-mg tablets) during the afternoon before the race, and 1200 mg on race day (six 200-mg tablets, with one taken before the race and one approximately every 4 h thereafter). Subjects in the control group avoided use of all medications including ibuprofen, and subjects in the ibuprofen group avoided all other medication use. On race day, body mass was measured at the 90-km aid station (Michigan Bluff, 1220 m) and within 5-15 min after the race at Auburn. Subjects completed a postrace questionnaire indicating adherence to the research design. Subjects consumed food and beverages ad libitum during the race. Plasma volume changes were estimated using the method of Dill and Costill (9).
Oxidative Stress Indicators
Urate was measured in a clinical laboratory using a LX-20 clinical analyzer (Beckman, Brea, CA).
F2-isoprostanes and PGE-M.
Plasma and urinary F2-isoprostanes were determined using gas chromatography mass spectrometry (GC-MS) according to the methodology of Morrow and Roberts (19). Briefly, free F2-isoprostanes were extracted from 1 mL of plasma. One to five picomoles of deuterated [2H4] PGF2α internal standard was added, and the mixture was vortexed. This mixture was then added to a C18 Sep Pak column, followed by silica solid-phase extractions. F2-isoprostanes were converted into pentafluorobenzyl esters, subjected to thin-layer chromatography, and then converted to trimethylsilyl ether derivatives. Samples were then analyzed by a negative ion chemical ionization GC-MS using a Nermag R10-10C mass spectrometer interfaced with an Agilent computer system. PGE-M is the major urinary metabolite of prostaglandin E2 and was extracted from urine and analyzed by GC-MS according to the methodology of Murphey et al. (20).
Total plasma nitrate and nitrite were determined by spectrophotometric analysis using a nitric oxide nonenzymatic assay (Oxis International, Portland, OR). This assay is based on reaction of nitrite with Greiss reagent. Samples were prereacted with cadmium to convert any nitrate into nitrite. Plasma samples were diluted 3.8 times and read on a non-protein-binding microtiter plate at 540 nm. Concentration was determined by linear regression of a standard curve (0-100 μM).
Data are expressed as means ± SEM and analyzed using a 2 × 2 repeated-measures ANOVA. If significant treatment × time interaction was detected, changes from prerace to postrace values were calculated and compared between ibuprofen and control groups using Student's t-tests, with significance set at P ≤ 0.025 after Bonferonni correction. Comparisons between genders were conducted using Student's t-tests. Pearson product-moment correlations were used to test the relationship between changes in measured outcomes. All statistical analyses were done using Instat version 1.01 (San Diego, CA) and SPSS version 13.0 (Chicago, IL).
Fifty-four of 63 subjects completed the 160-km race event. In some cases, numbers do not represent this because of sample loss during analysis. Subject characteristics for the ibuprofen and control groups are compared in Table 1 and indicate no significant differences in age, body composition, training history, and race time. Male (N = 43) and female (N = 11) runners did not differ significantly in race time (25.4 ± 0.6 vs 27.2 ± 0.76 h, respectively, P = 0.141) or any of the other variables measured in this study, except for those related to body mass and composition (data not shown). Thus, male and female runners were combined for this data analysis. Plasma volume did not change appreciably and did not differ significantly between groups (−1.6 ± 0.4 and −1.2 ± 0.3%, respectively; P = 0.381) (data not shown), and body mass was maintained near prerace levels for both groups (Table 1).
There was a significant time effect (P ≤ 0.001) of exercise and a strong trend toward treatment × time interaction (P = 0.066) in plasma F2-isoprostanes. For this reason, pre- to postrace differences were examined. Postrace plasma F2-isoprostanes increased 37% in ibuprofen users (P ≤ 0.001) compared with 20% in nonusers (Fig. 1). Exercise significantly increased urinary F2-isoprostanes over time (P = 0.032), and there was significant interaction (treatment × time effect, P = 0.040) as well. Urinary postrace F2-isoprostanes increased 138% in ibuprofen users (P ≤ 0.01) and were essentially unchanged in nonusers (Fig. 2).
Pre- to postrace increases in plasma nitrite and plasma urate did not differ significantly between the ibuprofen and control groups (Table 2). PGE-M concentration was approximately double in nonusers compared with ibuprofen users, and the treatment effect was significant (P = 0.016) (Fig. 3).
In agreement with our hypothesis, oxidative stress (F2-isoprostanes) and PGE-M were significantly affected by ibuprofen use during the race. Although we have previously observed significant increases in F2-isoprostanes as a result of ultraendurance races (16,21), we are unaware of other published studies indicating changes in oxidative stress in NSAID users compared with nonusers after ultramarathons. The oxidative stress associated with ibuprofen use was manifest through significant increases from pre- to postrace in both urinary and plasma F2-isoprostanes. Interestingly, urinary F2-isoprostanes were more affected (significant treatment × time interaction) than plasma F2-isoprostanes, but the two measures were not correlated. Because urinary F2-isoprostanes represent clearance through constant kidney filtration, this is likely more a cumulative reflection of oxidative stress over time versus plasma. Because we were unable to collect data on the nine subjects who failed to finish the race, we did not conduct analysis of prerace data on these subjects. This was a limitation in that it would have been interesting to examine whether any prerace differences predisposed these subjects to not finish.
It was predicted that one of the likely causes of the increased oxidative stress associated with ibuprofen use would be the potential for endotoxemia to occur. Endotoxemia is caused by increases of bacterial LPS infiltrating into the blood from the gut because of increased gastric permeability from NSAID use. In support of these concepts, Ryan et al. (24) found that aspirin intake during running significantly increased gastric permeability compared with placebo. Endotoxemia has also been found to occur solely as a result of exercise (1,6), and LPS has been used in animal models to induce oxidative stress (18). Interestingly, at least prerace endotoxemia did not occur in our athletes, because plasma and urinary F2-isoprostanes were similar in ibuprofen users and nonusers at this point. However, it cannot be stated that endotoxemia did not occur during the race, when ibuprofen use was accompanied by exercise.
Ibuprofen use is not likely related to direct effects on oxidative stress products via the cyclooxygenase enzyme (COX). It is known that in man, 8-epi-prostaglandin F2 alpha, a major F2-isoprostane, is produced in vivo predominantly by free radical-dependent peroxidation of lipid-esterified arachidonic acid, although both cyclooxygenase isoforms (COX-1 and COX-2) may form free 8-epi-prostaglandin F2 alpha as a minor product. It has been recently seen in human volunteers that the overall formation of 8-epi-prostaglandin F2 alpha in vivo is mostly COX independent, and F2-isoprostanes are, therefore, an accurate marker of oxidative stress in vivo (2). Given that F2-isoprostanes are bioactive compounds that may mediate oxidative stress with detrimental effects, an increase of these compounds during exercise may be of some concern (18).
Although not directly assessed in this study and somewhat speculative, a possible effect on F2-isoprostane formation could have resulted from an ibuprofen-associated effect on liver and kidney function. NSAID have been associated with direct hepatotoxicity in susceptible individuals, but the molecular mechanisms underlying this toxicity have not yet been fully elucidated. However, experimental evidence suggests that these mechanisms include formation of reactive metabolites that covalently modify proteins and produce oxidative stress, and mitochondrial injury (4,5). Exercise-imposed ischemia from redirected blood flow might augment the pathways leading to hepatic toxicity or impede the protective and detoxifying pathways. Additionally, NSAID use may influence changes in the cellular redox states and cytochrome P-450 activity (7).
Kidney cells may be damaged and altered during an ultramarathon as a result of NSAID use. One method is NSAID-induced nephrotoxicity, which may involve production of reactive oxygen species, leading to oxidative stress and DNA fragmentation. Free radical-mediated events may ultimately translate into apoptotic cell death of kidney cells in vivo (13). Secondly, NSAID-induced nonspecific inhibition of cyclooxygenase enzymes results in a decrease in the production of prostaglandins. Normally, kidney function is unchanged by ultraduration exercise (14), but ibuprofen use in our study might have contributed to reduced kidney function, likely through reduction of vasodilatory prostaglandins such as PGE2, which we found to be significantly lower in ibuprofen users (8). Inhibition of prostaglandin production has been suggested to decrease renal perfusion, redistribute blood flow to the cortex, and lead to acute renal vasoconstriction, medullary ischemia, and, possibly, acute renal failure (10). Interestingly, oxidative stress may act synergistically with NSAID use and, ultimately, lead to further damage in renal tissue. Fukunaga et al. (12) found an increase in the potent vasoconstrictive peptide endothelin-1 in a dose-dependent response to F2-isoprostane.
In contrast to the F2-isoprostanes, we did not find plasma nitrate or nitrite to be affected by ibuprofen use, although there was a time effect of the exercise. Because nitrate and nitrite are degradation products of nitric oxide, we interpret this as being an exercise-induced increase in turnover rate, or an increase in production, or both. Exercise studies are extremely limited, but Vassalle et al. (25) found that athletes had significantly higher baseline values of plasma nitrate and nitrite compared with controls. Nitrite has been found to cause nitrosative stress, leading to nitration of aromatic compounds (23). We were also interested in changes in plasma urate because it provides most of the antioxidant potential of plasma (26), but even though it increased significantly after exercise, there were no differences in the patterns of change between groups.
The major urinary metabolite of PGE2 is 11-alpha-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M). Concentrations of the metabolite in urine from healthy humans are nearly twofold greater in men than in women (10.4 ± 1.5 vs 6.0 ± 0.7 ng·mg−1 creatinine) (20). Interestingly, we observed resting and postexercise values of PGE-M approximately threefold higher than this in ibuprofen nonusers. Exercise did not exert any effects on PGE-M concentration. Ibuprofen users had significantly reduced PGE-M compared with nonusers pre- and postrace, indicating an end point of ibuprofen use during the race. Levels of PGE-M in healthy humans are suppressed significantly, not only by the nonselective COX inhibitor ibuprofen but also by selective COX-2 inhibitors (20). Farquhar and Kenney (11) found a similar suppression of PGE2 associated with ibuprofen use during exercise.
In conclusion, our results indicate that ibuprofen use during extreme endurance exercise increases oxidative stress. The physiological ramifications of the exercise-associated increase in F2-isoprostanes are not currently known, but research is planned by our group to further examine this oxidative stress component. Certainly, in the interest of sports medicine, the relationship between oxidative stress and ibuprofen use should be examined more closely in future studies. On the basis of our findings, we recommend caution in using ibuprofen and other NSAID during ultradistance exercise events.
Supported by the Gatorade Sports Science Institute, the 160-km Western States Endurance Run medical board, and National Institutes of Health grants (to J.D. Morrow) DK-48831, GM-15431, CA-77839, and RR00095. J. D. Morrow is the recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.
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