Gastrointestinal (GI) symptoms are common during strenuous physical exercise and range from mild nausea to “angine abdominale” and hemorrhagic stool (24). Incidence rates of such symptoms vary from 25% to 70%, depending on exercise intensity and duration (24). Although the etiology of exercise-related GI symptoms is thought to be multifactorial, decreased splanchnic perfusion has been postulated as a key mechanism. During strenuous physical exercise, blood is redistributed away from the splanchnic area toward active muscles, the cardiopulmonary system, and the skin, thereby strongly reducing splanchnic blood flow (27). We have recently demonstrated that splanchnic hypoperfusion is associated with small intestinal injury and loss of gut barrier function in healthy men (33). Intestinal injury and barrier integrity loss are undesirable phenomena especially for athletes because they may lead to abdominal distress and impaired nutrient absorption, possibly interfering with early recovery and hampering athletic performance (32). Disruption of the intestinal barrier may lead to further GI injury because it exposes the submucosal tissue to microbiota, potentially harmful pancreatic juices, and digestive products (2).
The splanchnic microcirculatory blood flow is a promising target to prevent exercise-induced splanchnic compromise. Increasing the local availability of nitric oxide (NO) is a potential means to enhance intestinal microcirculation (20). NO is produced via oxidation of L-arginine upon activation of endothelial NO synthase (eNOS) in endothelial cells and inducible NOS during ischemia-induced intestinal damage (21). NO inhalation has been applied as a therapeutic agent in patients with pulmonary arterial hypertension to induce local vasodilatation (12) and was found to attenuate lung ischemia-reperfusion injury (10). However, because NO is a short-lived gas, it cannot easily be applied locally to organs other than the lungs. Therefore, many NO donors have been studied to improve mucosal oxygenation. L-arginine was one of the first agents studied in this respect, and although its administration did increase plasma arginine availability, the results were not unequivocally positive (3,25,26). In addition, negative side effects such as diarrhea, GI discomfort, and nausea were frequently observed after arginine supplementation and limit its practical application (15). Oral supplementation of citrulline, a nonprotein amino acid and important precursor of arginine, has been shown to increase L-arginine availability for NO production more than L-arginine supplementation itself (6,28). The latter is explained by a combination of factors. First, L-citrulline is transported more efficiently from the gut lumen into the circulation than L-arginine (6). Second, a balanced, near-null flux across the liver exists for L-citrulline, which means that hepatic uptake is counterbalanced by L-citrulline release, whereas L-arginine is known to induce ureagenesis in the liver, limiting its systemic availability (6,30). Third, L-citrulline is converted into L-arginine in the kidneys via argininosuccinate synthetase and argininosuccinate lyase (6). Furthermore, L-citrulline is actively converted to L-arginine within endothelial cells, readily available for NO production (39).
Ten grams of oral L-citrulline supplementation in healthy subjects has been shown to increase plasma citrulline and arginine levels and to improve muscle protein synthesis (22) without negative side effects as seen after L-arginine supplementation (15). Therefore, L-citrulline seems to be a promising substrate to enhance intestinal L-arginine availability (22,38) to restore intestinal NO production in endothelial cells and increase intestinal microcirculatory perfusion (38). We hypothesized that oral supplementation of 10-g L-citrulline before exercise increases arginine availability in healthy athletes, thereby improving splanchnic perfusion and reducing exercise-induced GI injury and GI barrier loss. This was tested in a randomized, double-blind, placebo-controlled crossover study (registered as NCT01239303 at clinicaltrials.gov).
METHODS AND MATERIALS
Ten healthy recreationally active men (age, 24.9 ± 1.0 yr; body mass index, 21.1 ± 0.4 kg·m−2) were selected to participate in the present study. Before the experiments, maximal workload capacity (W max), assessed in an incremental exercise test to volitional exhaustion on a stationary cycle ergometer (Lode Excalibur, Groningen, The Netherlands) (19), was 5.0 ± 0.1 W·kg−1, with HRmax of 194 ± 2 bpm. The selected volunteers all spent 3–15 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, had no history of abdominal surgery, and were nonsmokers. Selected volunteers were informed about the nature and risks of the experiments, after which, a written consent was obtained at least 5 d before the experiments. This study was approved by the medical ethics committee of Maastricht University Medical Centre+ (MEC 10-3-064) and conducted in accordance with the Declaration of Helsinki (revised version, October 2008, Seoul, South Korea).
Participants maintained normal activities of daily living but refrained from strenuous physical activity for 2 d before each test day. Participants were not allowed to consume alcohol and artificial sweeteners the day before the experiments. Ranitidine (150 mg; GlaxoSmithKline, Zeist, The Netherlands) was ingested by the participants on the evening before each test day to inhibit gastric acid production and secretion. The latter is necessary to enable gastric tonometry measurements because the presence of acid in the stomach during tonometry can buffer carbon dioxide molecules, thereby interfering with the outcome of the tonometry measurements (18). An additional 150 mg of ranitidine was consumed on the morning of the test days at 07:00, 60 min before the start of the experiments.
Study design and nutritional intervention
In this double-blind, randomized, controlled crossover study, the participants were randomized for two different interventions, i.e., oral L-citrulline supplementation and placebo supplementation. All subjects performed the two test days in an order randomly assigned by computer: 1) after oral intake of a 10-g bolus of L-citrulline dissolved in 125 mL of tap water and 2) after intake of the 125-mL placebo drink containing 20 g of L-alanine in tap water. Both L-citrulline and the placebo were tested safe for human oral consumption by Basic Pharma Group (Geleen, The Netherlands) and supplied as powders labeled as “A” and “B”, which were dissolved 30 min before intake by the participants. The test drinks were identical in appearance and smell. No additives or sweeteners were used to keep drinks highly pure. As a consequence, a difference in taste was unavoidable. However, the researchers in the sports laboratory, the researchers performing data analysis, and the participants were unaware of this taste difference, and no questions were asked regarding the taste of the test drinks. The time between test days was at least 7 d.
Experiments and sampling
All experiments were performed after an overnight fast. Upon arrival at the sports laboratory at 08:00 a.m., participants were instructed to collect a urinary sample. To enable collection of arterialized blood for analysis of arterial pCO2 levels, a catheter (22 gauge; Braun, Melsungen, Germany) was inserted in a dorsal hand vein of the participant, and the hand was placed in a hot box set at 60°C (17). To measure gastric pCO2, an 8 French tonometrics catheter (Datex-Ohmeda Oy, Helsinki, Finland) was introduced via the nose into the stomach of the participant and fixed to the nasal flares. Gastric pCO2 was measured at 10-min intervals before, during, and after cycling using an automated capnograph (Tonocap TC-200; Datex-Ohmeda Oy, Helsinki, Finland). Arterialized blood samples were collected simultaneously with the tonometry measurements in heparin tubes, and gastric-arterialized pCO2 (gapg-apCO2) was calculated. Tonometry data were obtained from nine participants because of inability to introduce the nasogastric catheter into the stomach of one of the participants. At each time point, a second blood sample was collected into prechilled ethylenediaminetetraacetic acid tubes (Vacutainer; Becton Dickinson, Helsingborg, Sweden) for analysis of plasma parameters. After calibration of the tonometry device, sidestream dark field (SDF) sublingual imaging was performed for baseline assessment of the microcirculation, as described in the succeeding section.
After baseline measurements, participants ingested the test drink they were assigned to for that day. Thirty minutes after intake of the test drink, participants started cycling at a workload of 150 W. After 3 min, workload was increased to 70% of the individual’s preassessed Wmax (70% of Wmax was 248.2 ± 7.6 W). Subjects maintained pedal rates of at least 60 rpm, and workload was decreased by 25 W if participants were unable to maintain 60 rpm (this information is used to estimate exercise performance). 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 while cycling to complete the 60-min exercise bout. Immediately postexercise, SDF imaging was performed to measure sublingual microcirculation. Additional SDF imaging was performed at 30 min and 1 h after cycling, whereas a second urinary sample was collected 90 min after cycling. All collected blood and urine samples were centrifuged as soon as possible after collection at 4°C at 2300g for 15 min and stored at −80°C until analysis. Any GI complaints of the subjects during the test days were registered by the researchers, and subjects were contacted 2 d after the test day to assess the occurrence of GI complaints on the day after each test day.
To evaluate whether L-citrulline supplementation resulted in an enhanced arginine availability, plasma amino acid concentrations were measured as described before (31) and the arginine availability index (AAI) was calculated as arginine/(ornithine + lysine) (23). This index is based on the concentrations of arginine, ornithine, and lysine, reflecting the cellular uptake of these amino acids by the same transport system, the y+ transporter.
To evaluate the extent of small intestinal injury during and after cycling in both situations, plasma concentrations of human intestinal fatty acid binding protein (I-FABP) were determined by a researcher blinded for the specific test conditions. I-FABP is a small, 15-kD cytosolic protein that is present especially in mature enterocytes of the small intestine. Upon enterocyte injury, the protein rapidly diffuses through the interstitial space into the circulation, enabling its detection in plasma samples. Plasma I-FABP levels were measured by an in-house developed enzyme-linked immunosorbent assay (34). The detection window of the I-FABP assay is 12.5–800 pg·mL−1. The intra-assay and interassay coefficient of variation of this assay are 4.1% and 6.2%, respectively. The assay is specific for the detection of the I-FABP isoform.
Assessment of microcirculation
To evaluate intestinal microcirculation during exercise, sublingual microcirculation was measured with an SDF imager (16,29). SDF imaging was used to discriminate between small vessels (diameter, <10 µm) and large vessels (10 µm or larger in diameter) to assess the proportion of perfused sublingual vessels. The SDF imager uses 530-nm light absorbed by the hemoglobin in red blood cells, which allows observation of these cells in the microcirculation (4). All imaging measurements were done by an experienced investigator. Using a camera magnification of 5×, sharp real-time images of the sublingual microcirculation were obtained in a field of 1000 × 750 µm. In total, 20-s continuous image sequences per time point, each consisting of 200 images, were recorded at five sublingual sites according to the consensus of a round table conference (4,8,37). Videos were analyzed by two independent researchers according to De Backer et al. (8) using the Automated Vascular Analysis software 3.0 (Microscan, Amsterdam, The Netherlands). The total number of perfused small (smaller than 10 µm) vessels and the total number of perfused vessels were measured.
Assessment of GI permeability
Permeability analysis was performed as a measure of GI barrier integrity using a multisugar test drink, as described previously (35,36). 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 urinary concentrations of these orally ingested sugar probes as described (35). Small intestinal permeability was reflected by the 0- to 2-h urinary excretion ratio between lactulose (342 D) and L-rhamnose (164 D), the L/R ratio.
Statistical analysis was performed using GraphPad Prism (version 5.00; GraphPad, San Diego, CA). Normality of all data was verified by the Kolmogorov–Smirnov test. Data are presented as mean ± SEM. Continuous data were analyzed using two-way repeated-measures ANOVA (time and treatment were the factors) with Bonferroni post hoc test for multiple comparisons to identify the difference between treatments (citrulline or placebo) at respective time points. For within-group comparisons, either one-way ANOVA with Bonferroni post hoc test or Friedman with Dunn post hoc test was used depending on data normality. P < 0.05 was considered statistically significant.
Oral L-citrulline supplementation before exercise enhances the AAI during exercise compared with placebo
As expected, oral L-citrulline supplementation led to increased plasma citrulline concentrations (peak, 1840 ± 142 µM) compared with that in baseline (34 ± 3 µM) and compared with that in placebo L-alanine (peak, 46 ± 5 µM; P < 0.0001) (Fig. 1A). Plasma citrulline concentrations, although slightly increased, remained low throughout the studied period after placebo supplementation (Fig. 1A), whereas L-alanine levels increased profoundly after placebo administration (peak, 1639 ± 112 µM, vs peak, 435 ± 26 µM, after L-citrulline; P < 0.0001) (Fig. 1B).
The increased citrulline concentrations after L-citrulline supplementation were accompanied by a significant increase in plasma arginine concentrations during exercise (239 ± 9 µM directly after exercise compared with 82 ± 3 µM at baseline, P < 0.0001) (Fig. 1C), whereas supplementation with placebo only slightly increased plasma arginine concentrations (102 ± 6 µM vs 78 ± 3 µM, P < 0.0001) (Fig. 1C). The increase in arginine concentrations after oral L-citrulline supplementation significantly enhanced the AAI (0.76 ± 0.02 vs baseline 0.38 ± 0.02, P < 0.0001) (Fig. 1D), whereas placebo supplementation did not influence the AAI (0.38 ± 0.02 to 0.42 ± 0.03) (Fig. 1D). Interestingly, both the plasma L-arginine levels and the AAI remained elevated after oral L-citrulline supplementation during 1 h of exercise and at least until the end of our experiments, 1 h postexercise (Fig. 1C and D). These observations indicate a prolonged effect of a single 10-g bolus of L-citrulline before exercise.
Oral citrulline supplementation before exercise maintains adequate splanchnic perfusion during exercise
Gapg-apCO2 gap levels, measured by gastric tonometry, reflect splanchnic perfusion, with increased levels indicating a reduction in perfusion. Strenuous exercise quickly resulted in a significant increase in gapg-apCO2 levels during exercise after placebo supplementation (−1.67 ± 0.32 to a peak of −0.55 ± 0.46 kPa at 30 min of exercise, P < 0.01), whereas no significant change from baseline was observed during exercise after oral L-citrulline supplementation (−1.70 ± 0.19 to −1.45 ± 0.63 kPa) (Fig. 2A), pointing to preserved perfusion after L-citrulline intake. The inter-individual differences in gapg-apCO2 levels were relatively high during exercise, reflected by relatively large SEM. Nonetheless, area under the curve (AUC) calculations of gapg-apCO2 during exercise revealed a tendency (P = 0.10) toward a difference between L-citrulline and L-alanine supplementation (−3.3 ± 20.1 vs 40.1 ± 20.6 kPa, respectively) (Fig. 2B).
Citrulline maintains sublingual vessel perfusion after exercise
In line with the tonometry data, analysis of the sublingual microcirculation revealed that citrulline preserves the perfusion of microvessels. After L-citrulline supplementation, the number of perfused small vessels under the tongue was higher immediately postexercise and 30 min postexercise than that after L-alanine supplementation (Fig. 3A), indicating a trend (P = 0.06) toward a positive change in perfusion of the small sublingual vessels in favor of L-citrulline (7.8 ± 6.0 for L-citrulline vs −2.0 ± 2.4 after placebo) (Fig. 3B). In addition, the total number of perfused vessels significantly increased in the same time span with L-citrulline supplementation before exercise compared with a decrease in sublingual perfusion in case of placebo administration (12.0 ± 6.1 vs −7.4 ± 1.7, P < 0.01) (Fig. 3D). In line, if L-citrulline was ingested, the total number of perfused vessels immediately postexercise was 50.7 ± 2.9, compared with 38.0 ± 1.7 when L-alanine was ingested (P < 0.001) (Fig. 3C).
Citrulline reduces exercise-induced small intestinal injury during exercise
To assess the effect of oral L-citrulline supplementation on small intestinal enterocyte damage, plasma I-FABP levels were determined every 10 min before, during, and after exercise. Baseline I-FABP levels (before exercise) were not significantly different between the two interventions, being 244 ± 27 and 213 ± 19 pg·mL−1 with citrulline and alanine supplementation, respectively (data of one individual were excluded on both occasions because of six times higher plasma I-FABP levels). The increase in plasma I-FABP levels, depicted as percentage from baseline, reached significance after 1 h of strenuous exercise in case of oral placebo supplementation before exercise (P < 0.01), whereas the increase in plasma I-FABP levels was less pronounced after L-citrulline administration before exercise (Fig. 4A). By reporting changes in I-FABP levels as a percentage of baseline, data of all participants could be included here. Postexercise levels of I-FABP increased by approximately 72% above baseline after both citrulline and alanine, possibly reflecting reperfusion damage, and gradually declined toward baseline levels in the postexercise recovery period (Fig. 4A). A marked difference of the effect of citrulline and alanine supplementations was observed during exercise. AUC calculations of the I-FABP levels (as percentage from baseline) during exercise revealed a significant difference between L-citrulline and L-alanine supplementation (−185 ± 506 vs 1318 ± 553, respectively, P < 0.01) (Fig. 4B). Hence, oral L-citrulline supplementation was able to prevent a net increase in plasma I-FABP levels during exercise, compatible with diminished exercise-induced intestinal injury.
Exercise-induced GI permeability changes
To analyze whether oral L-citrulline supplementation before exercise could maintain GI barrier function during exercise, permeability L/R ratios were determined in urine collected in the 0- to 2-h postexercise recovery period. Urinary L/R ratio was 0.027 ± 0.003 after oral administration of L-alanine before exercise compared with 0.024 ± 0.002 after administration of L-citrulline (Fig. 4C). These data indicate no significant differences between the two nutritional interventions with respect to small intestinal permeability.
GI complaints and exercise performance
No GI complaints were reported by the individuals when specifically asked for any GI disturbances experienced on the test days or the day after each test day. No differences in exercise performance, exhaustion, or HR were observed between the two supplementation strategies (data not shown).
In this randomized, double-blind, controlled crossover study, the effect of oral citrulline supplementation before strenuous exercise on the splanchnic (micro)circulation, small intestinal injury, and gut barrier function was studied. Oral citrulline intake significantly increased plasma levels of citrulline and arginine in healthy athletes compared with those in placebo and, importantly, without causing GI discomfort, as is observed for arginine (15). The AAI increased as a result of elevated plasma arginine levels, reflecting an increased availability of the NO donor L-arginine, which persisted at least until 1 h postexercise. The increased arginine availability after oral L-citrulline supplementation was associated with a tendency to preserve splanchnic perfusion during exercise compared with that in placebo and significantly reduced enterocyte damage. Intracellular arginine generation from citrulline by argininosuccinate synthetase and argininosuccinate lyase might play a role in the protective effect of citrulline observed in the current study. This conversion is found to be essential for endothelial NO synthase-mediated NO production in endothelial cells (13), which may result in enhanced local blood flow through NO-mediated local vasodilatation (6).
Previously, we demonstrated that 1 h of cycling at 70% W max rapidly increased gapg-apCO2 levels by more than 1 kPa, reflecting the development of profound splanchnic hypoperfusion during exercise in the fasted state (33). In the current study, subjects received amino acid drinks before exercise on both test days. This might explain the smaller rise in gapg-apCO2 observed here, as meal ingestion and small intestinal nutrient supply in general have the ability to increase the superior mesenteric artery blood flow and, hence, splanchnic perfusion (5,11). Importantly, however, our study reveals that a single pre-exercise dose of L-citrulline is able to prevent the rise in gapg-apCO2 level during exercise, which is clearly observed after alanine supplementation, indicating that the extent to which GI blood flow depends on the type of nutrient.
Although we could not measure local splanchnic arginine availability in the study participants, experimental data indicated that intravenous L-citrulline supplementation is able to increase both plasma and intestinal arginine availability and the intracellular NO production in the intestine (38). In line, clinical studies indicated that oral supplementation of L-citrulline was able to increase plasma arginine concentrations and tissue NO production without causing side effects (9). Previously, we demonstrated that the small intestinal microcirculation was maintained during endotoxin-induced inflammation in mice if L-citrulline was applied intravenously (38). In accordance, the current study revealed that oral supplementation of L-citrulline before a trigger for splanchnic hypoperfusion, in this case strenuous exercise, improves sublingual microcirculation, strongly suggesting improved intestinal microcirculation (37).
Splanchnic hypoperfusion during strenuous cycling was reported to correlate with small intestinal injury in healthy athletes (33). Hypoperfusion-induced intestinal compromise may hamper athletic performance and can jeopardize early postexercise recovery (32). We hypothesized that oral supplementation of L-citrulline improved splanchnic circulation during exercise, thereby reducing exercise-induced enterocyte injury. The present study revealed significantly lower plasma I-FABP levels during exercise with previous L-citrulline intake compared with those in placebo, indicating that L-citrulline intake helps reduce small intestinal cellular injury. In line with these data, L-citrulline was recently shown to reduce intestinal injury after experimentally induced small bowel obstruction (1) and gastric mucosal injury as a consequence of ischemia-reperfusion (14).
The fact that, in the present study, splanchnic hypoperfusion during exercise is less pronounced than previously reported in the fasted state (33) suggests that oral supply of nutrients (citrulline or alanine) acts protectively on barrier function. This potentially explains the absence of a significant effect between L-citrulline and placebo supplementation with respect to GI permeability during exercise. A differential effect of citrulline compared with that of alanine on GI barrier loss is expected to become detectable during prolonged or more extensive reductions in splanchnic perfusion; however, this needs to be confirmed in additional studies.
The results of this study may be useful for athletes with ischemia-related abdominal symptoms during strenuous exercise, a group in which citrulline supplementation should be tested first. Next, it may also be interesting to extrapolate the results to asymptomatic athletes and to find out whether oral L-citrulline supplementation also has advantages such as improved nutrient uptake and improved early recovery. We recently revealed a negative correlation between plasma I-FABP levels and protein digestion and absorption (34). Reducing enterocyte damage by L-citrulline could therefore result in improved uptake of nutrients directly postexercise. In addition, it was previously reported that oral L-citrulline supplementation improves muscle protein synthesis, comparable to the stimulating effect of leucine (7). Both amino acids seem to exert this effect via the mammalian target of rapamycin (mTOR) signaling pathway. Interestingly, orally administered L-citrulline in combination with a low-protein diet in healthy volunteers was found to increase muscle protein synthesis compared with that in an isonitrogenous diet (7).
In conclusion, the current study demonstrates that a single oral dose of L-citrulline before exercise preserves splanchnic perfusion and reduces intestinal injury during exercise. The mechanism probably entails increased arginine availability for NO-mediated vasodilatation. These data suggest oral L-citrulline supplementation to be a promising strategy to improve GI blood flow and prevent intestinal injury in athletes without adverse GI effects as observed for arginine.
The authors are grateful for the excellent work of Dirk Schellekens and M’hamed Hadfoune on the I-FABP assay validation and gratefully acknowledge the volunteers for participating in this study. The authors have no funding sources to disclose.
The following are the authors’ contributions: K. V. W. and K. W. conception and design, study execution, collection of data, data analysis and interpretation, and manuscript writing; D. M., study execution and collection of data; B. B., collection of data and data analysis; L. V. L., design, provision of study material, and critical revision of manuscript; W. B., conception and design, data interpretation, and critical revision of manuscript; C. D., design, data interpretation, and critical revision of manuscript; K. L., conception and design, data analysis and interpretation, and manuscript writing; M. P., conception and design, data interpretation, and critical revision of manuscript. All authors approved the final version of the manuscript.
The authors report that no conflicts of interest associated with the current study have occurred.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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