Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used by athletes to reduce existing or prevent anticipated musculoskeletal pain related to physical exercise, especially in the competitive season (1,15). Athletes have reported the use of NSAIDs both during training and competition (16). It is generally assumed that NSAIDs improve athletic performance by enabling more frequent and more intensive training sessions, but no clear evidence exists. The reported prevalence of NSAID use among athletes differs widely between various sport types and ranges from 12% in cyclists to more than 90% in professional soccer players (16,29,32).
A major adverse effect of NSAIDs is their known tendency to cause gastrointestinal (GI) complications such as mucosal ulceration, bleeding, perforation, and the formation of diaphragm-like strictures (2,35). Several mechanisms of action have been suggested to play a role in the development of these NSAID-induced complications: inhibition of cyclooxygenase (COX)-1 and reduction of local nitric oxide production via regulation of the nuclear factor kappa B pathway may impair perfusion of the upper GI tract (20), whereas COX-2 inhibition compromises immunomodulation, potentially resulting in the onset of an inflammatory response (4).
We previously demonstrated that 1 h of exhaustive physical activity leads to small intestinal injury and short-term loss of gut barrier function in otherwise healthy individuals (33). In the light of such exercise-induced GI compromise, it seems that the hazardous effects of NSAIDs on the GI mucosa may put athletes at risk for more severe abdominal distress. We hypothesize that the combination of short-term NSAID consumption with the physiological exercise-induced splanchnic hypoperfusion may lead to aggravated intestinal injury in athletes. The current study shows that the use of NSAIDs intensifies exercise-induced small intestinal injury and loss of intestinal barrier integrity in healthy athletes.
METHODS AND MATERIALS
Power calculation performed before the start of the study revealed that a sample size of nine subjects was required to achieve 80% statistical power and detect statistically significant differences in the current experimental setting. Therefore, nine healthy male cyclists or triathletes (age: 27 ± 0.9 yr; body mass index: 20.6 ± 0.6 kg·m−2) were selected to participate in the present study. The maximal workload capacity (Wmax) was 5.5 ± 0.2 W·kg−1, with maximum heart rate at 192 ± 2 bpm. All volunteers spent 3 to 10 h·wk−1 performing endurance sports as part of their normal lifestyle. The volunteers had no abdominal complaints during daily activities, had not taken any medication for at least 1 month before participation, had no history of GI disease, and had no history of abdominal surgery. Volunteers were informed about the nature and risks of the experiments. Written consent was obtained at least 5 d before the experiments. This study was approved by the medical ethics committee of Maastricht University Medical Center+ and conducted in accordance with the Declaration of Helsinki (revised version, October 2008, Seoul).
Wmax was assessed before the experiments on a stationary cycle ergometer (Lode Excalibur, Groningen, The Netherlands) (18). Electrocardiographic monitoring (MAC 5500; GE Medical Systems, Freiburg, Germany) was performed to exclude cardiologic abnormalities such as exercise-induced cardiac ischemia or arrhythmia. An experienced cardiologic resident interpreted the results and found no abnormalities. Participants maintained normal activities of daily living but refrained from strenuous physical activity for the 2 d before the test days and were not allowed to consume alcohol and artificial sweeteners the day before the experiments. Ibuprofen (400 mg, iso-butyl-propanoic-phenolic acid; GlaxoSmithKline, Brentford, Middlesex, United Kingdom) was ingested by the participants on the evening before the ibuprofen test days. An additional 400 mg of ibuprofen was consumed in the morning of these test days, at 7:00 a.m., being 60 min before start of the experiments to mimic the normal use of ibuprofen by athletes.
Study design and sampling
All nine subjects were tested consecutively in random order in four different situations: 1) during and after cycling after intake of ibuprofen, 2) during and after cycling without ibuprofen, 3) rest with prior intake of ibuprofen, and 4) rest without prior ibuprofen intake. Time between test days was at least 7 d. All experiments were performed after an overnight fast. Upon arrival at the sports laboratory at 8:00 a.m., a catheter (20-gauge; Braun, Melsungen, Germany) was placed in the participant’s forearm vein to obtain blood samples, which were put into prechilled ethylenediaminetetraacetic acid tubes (Vacucontainer; Becton Dickinson, Helsingborg, Sweden) and kept on ice.
On test days that participants were assigned to cycling, participants started cycling at a workload of 150 W after collection of baseline plasma and urine samples. After 3 min, workload was increased to 70% of the individual’s preassessed Wmax (70% of Wmax was 268 ± 9 W (mean ± SEM of the nine participants)). Subjects maintained pedal rates of at least 60 rpm, and workload was decreased by 25 W if participants were unable to maintain 60 rpm. Participants consumed tap water ad libitum, with a minimum of 50 mL and a maximum of 150 mL every 10 min. After 30 min of cycling, the test subjects ingested a 150-mL multisugar drink to enable whole gut permeability analysis. A second urine sample was collected by the participants at 90 min after exercise. Resting test days were similar in setup, with the exception that the participants were tested during and after 1 h of rest in supine position instead of during and after cycling. On all test days, blood and urine samples were centrifuged within 1 h of collection at 4°C at 2300 g for 15 min and stored at −80°C until analysis. Analyses were performed when sample collection was complete for all participants to perform analyses in consecutive runs. Any GI complains of the subjects during the test days were registered by the researcher, and subjects were contacted 2 d after the test day to assess the occurrence of GI complaints on the day after each test day.
Assessment of small intestinal injury
To evaluate the presence and the extent of small intestinal injury in the above-described situations, plasma concentrations of human intestinal fatty acid binding protein (I-FABP) were determined by an analyst who was blinded for the specific test conditions. I-FABP is a 15-kDa cytosolic protein present especially in the mature enterocytes of the small intestine that rapidly diffuses through the interstitial space into the circulation upon enterocyte injury, making it an early and sensitive marker of small intestinal injury (19,25). I-FABP was measured by an in-house developed enzyme-linked immunosorbent assay (ELISA). In short, ELISA plates were coated with anti–I-FABP immunoglobulin G overnight at 4°C; free sites were blocked with 1% bovine serum albumin in phosphate-buffered saline. Plasma samples and human recombinant I-FABP for standard calibration curves were incubated at room temperature, after which biotinylated anti–I-FABP immunoglobulin G was added. After washing, horseradish peroxidase–streptavidin conjugate (Zymed Laboraties Inc., San Francisco, CA) in 0.1% bovine serum albumin–phosphate-buffered saline and 3,3,5,5-tetramethylbenzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD) were added. The reaction was stopped, and color intensity was measured with an ELISA reader at 450 nm. The detection window of the I-FABP assay was 12.5 to 800 pg·mL−1.
Assessment of GI permeability
GI permeability was determined as a measure of GI barrier integrity using a multisugar test drink as described previously (34). The food grade sugar probes included in the test drink were 1 g lactulose (Centrafarm, Etten-Leur, The Netherlands), 1 g sucralose (Brenntag, Sittard, The Netherlands), 1 g erythritol (Danisco, Copenhagen, Denmark), 1 g sucrose (Van Gilse, Dinteloord, The Netherlands), and 0.5 g L-rhamnose (Danisco) dissolved in 150 mL of tap water. GI permeability was assessed by determination of the 0–2 h urinary excretion of these orally ingested sugar probes using a combined high-performance liquid chromatography (model PU-1980 pump; Jasco Benelux, Maarssen, The Netherlands) and mass spectrometry (model LTQ-XL; Thermo Electron, Breda, The Netherlands) approach (34). Gastroduodenal permeability was assessed by calculation of the 0–2 h urinary ratio of sucrose (342 D) and L-rhamnose (164 D), whereas the urinary 0–2 h ratio lactulose (342 D) and L-rhamnose, the lactulose/rhamnose (L/R) ratio, was computed to assess small intestinal permeability.
Statistical analysis was performed using GraphPad Prism (version 5.00; GraphPad Software for Windows, San Diego, CA). Normality of all data was verified by the Kolmogorov–Smirnov test. All normally distributed data are presented as mean ± SEM and not normally distributed data as median and range. Continuous data were analyzed using two-way ANOVA with Bonferroni post hoc test for multiple comparisons. Correlations were determined by calculating the Spearman correlation coefficient (rS). Linear regression was used to visualize the correlation. Correlations between small intestinal injury and small intestinal permeability were computed using individually normalized I-FABP levels, and P < 0.05 was deemed statistically significant.
Ibuprofen aggravates exercise-induced intestinal injury
Plasma I-FABP levels were determined to assess loss of cellular integrity within the small intestine. In line with our previous study (33), plasma I-FABP levels gradually increased during cycling at 70% of Wmax from baseline 295 ± 46 pg·mL−1 to mean peak levels of 474 ± 74 pg·mL−1 immediately postexercise (P < 0.05, Fig. 1A). Interestingly, cycling with ibuprofen resulted in even higher levels of circulating I-FABP (P < 0.0001, Fig. 1A). In the latter situation, peak I-FABP levels of 875 ± 137 pg·mL−1 were observed immediately postexercise, being a significant increase from baseline (328 ± 32 pg·mL−1, P < 0.05; Fig. 1A). These peak I-FABP levels were significantly higher than I-FABP levels after cycling without ibuprofen (875 ± 137 vs 474 ± 74 pg·mL−1, P < 0.05; Fig. 1A, C).
Ibuprofen consumption also increased levels of small intestinal injury at rest (P = 0.0003, Fig. 1B, C). Two of nine participants reported minor abdominal complaints during the study, including epigastric pain, flatulence, and belching after ibuprofen administration. One individual (individual A) reported these symptoms only after ibuprofen administration at rest, and his symptoms were not accompanied by high plasma I-FABP levels (259 vs 273 pg·mL−1 with and without ibuprofen at rest, respectively). The other, individual B, reported symptoms on both test days after ibuprofen administration and had elevated plasma I-FABP levels (579 vs 495 pg·mL−1 with and without ibuprofen at rest and postexercise levels of 1493 vs 865 pg·mL−1 for cycling with and without ibuprofen, respectively). In addition, the I-FABP levels of participant B were considerably higher than the mean I-FABP levels of the total group depicted in Figure 1A, C.
Ibuprofen before exercise increases gastroduodenal and small intestinal permeability
Upper GI permeability increased after cycling with ibuprofen (0–2 h sucrose/rhamnose (S/R) ratio, 0.041 (0.000–1.000) versus 0.000 (0.000–0.020) compared with rest, P = 0.06; Fig. 2A). Urinary S/R ratios showed a weak but statistically significant correlation with peak plasma I-FABP levels (RS = 0.49, P < 0.005).
Consistent with gastroduodenal permeability, small intestinal permeability increased after cycling with ibuprofen: the 0–2 h urinary L/R ratio was 0.08 (0.04–0.56) compared with 0.03 (0.00–0.20) for cycling without ibuprofen. In addition, the 0–2 h urinary L/R ratio increased at rest with ibuprofen use to 0.05 (0.01–0.07) compared with 0.01 (0.01–0.04) without ibuprofen. Interestingly, levels of intestinal injury correlated significantly with small intestinal permeability. Peak plasma I-FABP levels of each test day correlated with 0–2 h urinary L/R ratios. In line, total shedding of I-FABP from injured enterocytes, reflected by the area under the curve of plasma I-FABP levels, correlated with the 0–2 h urinary L/R ratios. Gastroduodenal and small intestinal permeability outcomes were not significantly different between symptomatic and asymptomatic participants, but individual A did show relatively high permeability ratios after ibuprofen consumption. In rest, the 0–2 h S/R ratio of individual A was 0.475 and 0.398 after cycling. The 0–2 h L/R ratio of this individual was 0.058 in rest and 0.564 after cycling.
In summary, both gastroduodenal and small intestinal permeability increased after cycling after ibuprofen intake. In addition, a clear relation was found between exercise-induced intestinal injury and small intestinal permeability after exercise.
NSAID consumption is common among athletes. Alarmingly, its prevalence among athletes has been reported to reach 90% in specific sports (16,29,32). Especially, endurance athletes are well acquainted with the occurrence of GI problems during or after exercise (26,31). However, these athletes have limited awareness of the potentially hazardous effects of NSAIDs on the GI tract and their potential to induce GI discomfort (16). This study demonstrates that the use of ibuprofen before exhaustive exercise exacerbates exercise-induced small intestinal injury resulting in the loss of gut barrier function in healthy men.
Small intestinal injury as assessed by plasma I-FABP levels developed within 1 h of cycling at 70% of Wmax and gradually returned to baseline levels during 60 min of subsequent postexercise recovery. These data corroborated the findings of our previous study, in which we demonstrated that the exercise-induced intestinal compromise is at least partly caused by the rapid onset of splanchnic hypoperfusion (33). The latter is a result of the redistribution of splanchnic blood flow that occurs to secure adequate perfusion of muscles, skin, heart, and lungs after exercise (24,27). Hypoperfusion decreases the supply of oxygen and nutrients to the gut, leading to injury of the enterocytes lining the small intestinal villi (5,6), as reflected by leakage of I-FABP from the affected enterocytes into the circulation (10). In line, injury of the GI tract after exercise has also been established using endoscopy, showing mucosal inflammation and erosive mucosal lesions in athletes after endurance running (8,23).
The exact mechanisms by which NSAID consumption leads to GI damage and increased risk of upper GI complications such as mucosal lesions and perforation (11,12,14) have not yet been fully elucidated. One of the major pathways considered to be involved is the inhibition of COX isotypes 1 and 2, resulting in local inflammation and vascular dysregulation, ultimately reducing perfusion and promoting mucosal integrity loss within the splanchnic area (9,12,30). In addition, interference of NSAIDs with the production of nitric oxide due to prostaglandin-mediated microvascular dysfunction may further reduce mucosal blood flow (20). This NSAID-induced reduction of the splanchnic blood flow may deteriorate the exercise-induced state of splanchnic hypoperfusion, putting athletes at risk for serious GI compromise. Endurance athletes have been observed to have significant GI injury after endurance running without using NSAIDs (8,23), and the combination of exercise and NSAIDs may aggravate this intestinal injury. Moreover, the negative effects of NSAIDs may not be restricted to the GI tract. Recent studies have demonstrated an increased risk of cardiovascular adverse events associated with the use of NSAIDs (7,13,17,22).
In the present study, high levels of plasma I-FABP were observed in athletes after cycling, strongly suggesting intestinal injury. This intestinal damage was significantly more pronounced after administration of two (400 mg) oral doses of the over-the-counter drug ibuprofen than after cycling without ibuprofen. Furthermore, ibuprofen intake induced abdominal discomfort in two of the nine athletes. The recommended maximum daily dose for oral ibuprofen intake varies from 1.2 g for nonprescription use to 2.4 g for prescribed oral administration (28). Our results clearly demonstrate that a considerably lower dose of ibuprofen before strenuous exercise aggravates exercise-induced small intestinal injury, leading to intestinal compromise. Because endurance athletes often use higher doses of NSAIDs and exercise for more than 1 h, it may be expected that the intestinal compromise is more pronounced in daily practice. Although in the current study, the NSAID-induced small intestinal injury is reversible within 2 h, long-term use of NSAIDs may prolong GI compromise, increasing the risk of GI complications and potentially affecting performance and recovery.
Another key issue in the pathophysiology of NSAID-induced intestinal compromise is the development of barrier integrity loss and subsequent translocation of bacteria and permeation of harmful digestive enzymes (4,21). These processes contribute to the local inflammatory response via activation of Toll-like receptor 4 and MyD88 (36). In the current study, loss of gut barrier function was demonstrated by an increase in urinary sugar permeability ratios. The compounding effect of NSAID consumption and heavy physical exercise led to increased gastroduodenal and small intestinal permeability, reflected by elevated urinary S/R and L/R ratios. The strong correlation that was found between cellular injury and the loss of barrier function in the small intestine emphasizes the extent of the intestinal compromise in the current study. In addition, these data lead to the question whether the exercise-induced intestinal injury may also negatively affect the digestive and absorptive function of the GI system, thereby impeding postexercise recovery. In case of extensive intestinal damage, both the intestinal barrier function and the absorptive capacity of the GI tract may be compromised, yet future studies are warranted to confirm the hypothesis.
A limitation of the current study may be that we did not perform measurements to assess hydration status or renal function that may potentially affect plasma and urinary markers of intestinal compromise. GI permeability analysis in the current study was based on the urinary excretion of large and small sugar permeability probes, but because these probes are equally affected by factors as intestinal transit and renal function (3), alterations in renal function could not have influenced permeability analysis. Furthermore, all 15 athletes participating in our previous study had normal hydration status and no detectable kidney damage after completion of the same exercise protocol as applied in the current study (33). These data strongly suggest normal renal function in healthy athletes after 1 h of cycling in our experimental setting. Endurance athletes exercising in a hot environment are more prone to become dehydrated, thereby potentially compromising renal function and clearance of NSAIDs.
In conclusion, the current study shows that in healthy endurance athletes, NSAID consumption can aggravate exercise-induced small intestinal injury and induces loss of gut barrier function. Although the ergogenic properties of NSAIDs remain questionable, evidence is provided to show that NSAIDs consumption is not harmless and may cause abdominal distress (11,12,14). We consider it of utmost importance to increase the awareness of athletes and trainers toward the potential negative effects of NSAIDs and recommend that the use of NSAIDs in the absence of a clear medical indication should be discouraged.
The authors gratefully acknowledge T. van Stipdonk, MD, for his thorough interpretation of the ECGs.
The authors have no funding sources to disclose and report that no conflicts of interest have occurred that are associated with the current study.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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