Changes of tryptophan metabolism in Japanese runners during an ultra-marathon race
Under the assumption that tryptophan (TRP) metabolism may be modulated by the intensity, duration, and total exercise/energy expenditure of the ultra-marathon, we evaluated the changes in two major TRP metabolic pathway substances [serotonin (5-HT) and kynurenine (KYN)] during a two-day ultra-marathon race.
Blood was sampled at three different time points: before the race (baseline), after running 45 km on the first day, and after running 135 km on the second day.
Serum TRP concentrations decreased in proportion to the distance covered, and the levels after running 135 km were significantly lower than at baseline and after running 45 km. Serum serotonin (5-HT) concentrations increased significantly after running 45 km but reduced towards baseline levels after running 135 km. Serum kynurenine (KYN) concentrations hardly changed after running 45 km but increased significantly after running 135 km compared with after running 45 km values. Serum FFAs levels increased significantly after running 45 km compared with baseline values, and they elevated even further after running 135 km. Serum albumin concentrations reduced significantly after running 45 km but remained at almost the same level after running 135 km. Serum 5-HT levels tended to be consistently correlated to the completion times on the first and second days.
Serum 5-HT concentrations are known to be associated with central fatigue, and may predict exercise performance. KYN levels appeared to reflect the intensity of physical exercise, and its pathway may play a role in reactive oxygen species scavenging systems during a long-duration exercise.
KeywordsExercise intensity Exercise performance Kynurenine Serotonin Tryptophan Ultra-marathon
Tryptophan (TRP) is an essential amino acid that is metabolized into various physiologically active substances via two major pathways. It is converted to 5-hydroxytryptophan (5-HT: serotonin), which is associated with fatigue during prolonged exercise because of its effects on sleep, feelings of lethargy and drowsiness, and loss of motivation [1, 2].
In an alternative reaction series, TRP is catabolized by tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase (IDO) to kynurenine (KYN), which is a precursor of physiologically active chemicals, including nicotinic acid, nicotinamide adenine dinucleotide (NAD), quinolic acid (QUIN), 3-hydroxykynurenine (3-OH-KYN), and kynurenic acid (KYNA) . KYN derivatives, including 3-OH-KYN and QUIN, have toxic effects on neuronal function, but KYNA has been reported to have neuroprotective effects. However, a recent study found it has an ability to promote central fatigue, and TRP and KYNA levels elevated in some brain regions in central fatigue [4, 5].
Exercise generally increases 5-HT levels in the brains of animals [4, 6, 7], and enhances serum KYN concentrations in humans and rats [8, 9, 10]. Long-duration exercise also generated excess reactive oxygen species (ROS) . 5-HT synthesized from TRP is rate limited by tryptophan hydroxylase (THP), whose activity is decreased by ROS [12, 13], but ROS also activates IDO and TDO [14, 15].
In the present study, we examined the effects of long-duration exercise on serum TRP metabolism along with serum free fatty acids (FFAs) and albumin concentrations, and assessed possible associations of TRP metabolite concentrations with completion times in 35 male Japanese non-professional runners participating in a two-day ultra-marathon race.
We invited runners to participate in the present study prior to the race via post mail, and 44 men and 6 women agreed to join the study. We excluded women because they were small in number and 9 men having outlier measurements from the mean evaluated by Smirnov–Grubbs test (p < 0.05), and adopted 35 men as study subjects. For the sake of the subjects’ safety, no specific restrictions, including meals and beverages, were given before and during the race. Anthropometric measurements and sampling of venous blood were carried out at three time points: (1) before the race (baseline), (2) immediately after running 45 km on the first day, and (3) immediately after running 135 km on the second day. The blood samples were stored at −80 °C until assayed.
A non-competitive ultra-marathon race (Yashagaike Legendary Maranic 2003) described elsewhere  was held in Gifu Prefecture, Japan, on July 26 and 27, 2003. In brief, the temperature was 29 °C, and relative humidity was approximately 49 % at noon on both days. The race covered a 135 km distance involving running and mountaineering over the 2 days. On the first day at 11:00 a.m., the participants started an approximately 45 km race to complete the distance within 7 h. On the second day at 3:30 a.m., they resumed the race, covering approximately 90 km, including climbing a mountain approximately 1100 m high, then returning to the starting point within 15 h and 30 min.
Serum TRP and KYN concentrations were measured using a high-performance liquid chromatography (HPLC) method . The HPLC pump was a Shimadzu LC-6A, and the detector was a Shimadzu SPD-6AV for UV detection and Shimadzu RF-550 for fluorescence detection. The analytical column was 150 × 4.6 mm i.d. and packed with Platinum EPS C18 100Å. The mobile phase was a 5 mmol/L aqueous zinc acetate solution: acetone = 92:8(v/v), pH = 4.9. The flow rate was 1.0 mL/min, and the volume per injection was 20 μL. The wavelength of the UV detector was set at 365 nm, and the fluorescence condition was excitation at 220 nm with detection at 354 nm. 6-Methyltryptophan was used as an internal standard. Serum 5-HT levels were determined using an EIA kit (Immunotech S.A., Praha, Czech Republic), and albumin concentrations by kit (Kyowamedex Co., Ltd., Tokyo, Japan). FFAs levels were measured at the SRL laboratory in Japan.
Normal distribution of variables was assessed using the Kolmogorov–Smirnov test. A one-way repeated measures analysis of variance (ANOVA) along with Bonferroni post hoc test was adopted for comparison of the mean levels of each parameter at the three time points. Correlations were calculated using Pearson’s product-moment correlations. A p < 0.05 was considered to be statistically significant.
Average age of the 35 runners was 49.9 ± 9.5 years (mean ± SD) (ranging from 26 to 72 years), and body mass index (BMI: kg/m2) was 22.5 ± 2.1 (from 17.0 to 27.5). Average completion times were 5.2 ± 0.7 h (ranging from 3.6 to 6.3 h) for the first day, 12.8 ± 1.7 h (from 8.9 to 15.3 h) for the second day, and 18.0 ± 2.4 h (from 12.6 to 21.4 h) for the two-day race. Dehydration was not evident because no changes were detected in red blood cell counts, hemoglobin concentrations, or hematocrit figures (%) during the race period (data not shown). All measurement values by age and BMI showed similar patterns for all sampling points and completion times.
Matrices of Pearson correlation coefficients across serum tryptophan metabolite concentrations and completion times
We examined the association of long-duration exercise with serum TRP metabolism along with serum FFAs, and albumin concentrations in 35 male Japanese non-professional runners participating in a 2-day ultra-marathon race. We noted typical changes in serum TRP, 5-HT, and KYN concentrations together with variations in serum FFAs and albumin levels during the ultra-marathon race. Serum TRP levels decreased in accordance with the running distances, and levels reduced significantly at the end of the race. On the other hand, serum 5-HT concentrations increased significantly after running 45 km but declined towards baseline levels after the end of the race. Serum KYN concentrations hardly changed after running 45 km but increased significantly after the race compared with values after running 45 km. Serum FFAs levels increased steeply after running 45 km and even more at the end of the race. Serum albumin concentrations reduced markedly after running 45 km, but remained almost the same up to the end of the race. Serum TRP levels were consistently negatively associated with the completion times, whereas 5-HT concentrations were universally positively related to the times.
Baseline serum TRP levels were above the upper limit of the Japanese reference range (37–75 μmol/L) . The maximum value of TRP was 114 μmol/L; namely, 1.5 times higher than the upper limit, being comparable with the observations in the similar race settings in 2004 . We may consider that the participants regularly consumed TRP-rich food, including animal protein, and had training regularly, as seen from the fact that their average running was greater than 250 km/month .
TRP is conjugated with albumin in serum. Serum albumin concentrations decreased during the race, whereas FFAs levels increased steeply. A rise in FFAs may have displaced TRP from albumin and released free TRP in the peripheral blood because the bond between albumin and FFAs is stronger than that of TRP [2, 20]. Peripheral free TRP increased, then brain TRP uptake and concentrations will rise , and TRP may have been converted into several metabolites, including 5-HT and KYN, in the peripheral tissues and the brain or used for energy expenditure in physical exercise. Eventually, TRP declined as the race distance increased. Yamamoto et al. reported that analbuminemic rats were shorted the treadmill run time to exhaustion and had higher extracellular TRP concentrations in the brain than normal rats when exercise induced fatigue . In the present study, after second day running, serum albumin concentrations were reduced, in contrast, serum FFAs levels increased, suggesting that serum free TRP levels increased and entered the brain.
Serotonergic neurons are considered to be provoked by repetitive motor activities [21, 22]. Physical exercise increased not only brain TRP levels but also 5-HT synthesis in rats . Plasma 5-HT concentrations are reported to be correlated with those of the cerebrospinal fluid (CSF) . Hence, concentrations of serum 5-HT may be a marker for those of CSF. 5-HT concentrations may have been enhanced not only in circulating blood but also in the CSF after running 45 km in this race. 5-HT spillover onto the axon initial segment of motoneurons was reported to induce central fatigue , with 5-HT also inhibiting midbrain dopamine (DA) neurons , and long-duration exercise elevated brain 5-HT levels, reduced brain glycogen levels, and yielded central fatigue [6, 7]. On the other hand, other researchers suggested that 5-HT do not cause strong effects on central fatigue [4, 5].
5-HT in the periphery has multiple functions, promoting hepatic gluconeogenesis, lipolysis in white adipocytes, and 6-phosphofructo-1-kinase activity, and thus it augments skeletal muscle and hepatic glycolysis [27, 28, 29, 30]. Serum 5-HT concentrations increased significantly after running 45 km but declined towards baseline levels after the end of the race, and the above metabolic systems may be elevated up to the 45 km point. If the glycolytic system was activated in skeletal muscle, it may relate to blood lactate concentration elevation. We noted elevation of serum lactate concentrations after running 40 km in similar race conditions in 2002 [before race: 1.39 ± 0.05, after 40 km running: 1.78 ± 0.06, after 130 km running: 1.82 ± 0.07 (mg/dL) (n = 106)] (unpublished). Blood lactate does not influence muscle fatigue directly, but it does reduce blood flow to the brain, which may be a signal related to load intensity during muscle fatigue for the brain [31, 32]. THP, a rate-limiting enzyme in the synthesis of 5-HT from TRP, shows decreased activity in response to ROS [12, 13]. We reported previously that serum ROS levels elevated after running 40 km . Therefore, serum ROS level elevation may be a candidate for 5-HT decline after the end of the race. We considered that the decline in 5-HT after the race ended was due to multiple factors such as the serum TRP level decrease, inhibition of THP activity by ROS, and increased KYN formation from TPR. Serum 5-HT levels tended to be correlated with the race completion times on the first and second day, when the running performance of participants with high blood 5-HT levels may be low. From integrating previous studies and the results of this study, one can conclude that the serum 5-HT may be connected with fatigue.
Serum KYN concentrations hardly changed after running 45 km but increased significantly towards the end of the race, as corroborated by the previous observations in humans and rats [8, 9, 10]. Serum KYN concentrations were associated with the score for vigor after running 20 km, indicating close correlations between serum KYN levels and exercise performance or mood . KYN derivatives, including 3-OH-KYN and QUIN, have toxic effects on neuronal function, and KYNA can induce central fatigue [3, 4, 5, 33]. KYNA was also reported to cause a reduction in striatal DA, and too much or too little DA in the brain yields the onset of fatigue [34, 35]; KYNA may, therefore, relate to central fatigue. KYN is transported through the blood–brain barrier [33, 36], and the precursor of KYN, TRP is also transported into the brain [2, 4, 20]. Very recently, Yamamoto et al. proposed TRP-KYNA and KYNA-QUIN hypotheses, suggesting that central fatigue can be induced by elevated TRP concentrations in the brain and subsequently synthesized KYNA and QUIN, which displayed synergy effects for progress of central fatigue, and 5-HT does not play a key role in central fatigue [4, 5]. Hence, the long-distance running may enhance the concentrations of KYN and its metabolites not only in circulating blood but also in the CSF, and can induce exercise fatigue; however, in-depth studies are warranted on this issue.
KYN is metabolized from TRP by TDO or IDO , and physical exercise elevated IDO activity in rats . IDO and TDO are both activated by ROS [14, 15]. IDO is also potentiated by several pro-inflammatory cytokines such as interferon-gamma (INF-γ), tumor necrosis factor-alpha (TNF-α), and interleukins (Il-1β, IL-2, IL-6, IL-12) [36, 37]. Accordingly, IDO was enhanced by long-duration running and scavenged ROS to catalyze the oxidative cleavage of TRP to KYN [10, 38]. Some ROS may have been scavenged by these routes after running 135 km in the present study. The levels of urinary 8-OHdG, a marker of oxidative DNA damage, and serum ROS levels were elevated after running 40 km but returned to baseline levels after running 130 km in similar settings in Japanese non-professional runners [11, 39]. Therefore, the KYN pathway may be one of the ROS scavenge systems activated during long-distance running.
There exist certain limitations in the present study. The sample size was small, and sub-categorization analyses with sufficient statistical power were unavailable, including analyses adjusting/classifying with age and their fitness/performance level because there were differences in those figures even though the study subjects were amateur runners. The study was conducted during the actual ultra-marathon race, not in a metabolic laboratory room, runners consumed food and beverages at lib before and during the race, and we could not control consumption of TRP from meals. In addition, we did not collate changes and trends of biomarkers with subjective feeling using questionnaires inquiring the level of physical fatigue and mental distress. Thus, further studies managing intake of TRP are warranted for in-depth examinations and discussion.
Serum TRP concentrations decreased with increasing running distance in a long-distance race. Production of 5-HT from TRP was enhanced after running 45 km on the first day but decreased after running 135 km on the second day. KYN formation from TRP, however, gradually predominated during the second day running. Serum 5-HT levels tended to be correlated with the race completion times on the first and second days. Serum KYN levels may demonstrate the severity of physical exercise. 5-HT concentrations seemed to predict exercise performance, and the KYN pathway was thought to be one of the ROS scavenging systems in long-duration exercise. These observations appeared useful for non-professional athletes, including people in public, to know about an optimal physical activity level, duration, and total energy expenditure for monitoring physical/mental conditioning as well as for prevention of overuse and physical injuries.
We thank the volunteer runners for their generous participation in our study together with the chairman and organizing committee of the Maranic race. We also thank Ms. Fujii, T., Ms. Kubo, Y., Ms. Nakanishi, N., Ms. Ito, Y., Ms. Higuchi, K., and Ms. Watanabe, M. for technical assistance.
Compliance with ethical standards
The protocol was approved by the Institutional Review Board of the Nagoya City University Graduate School of Medical Sciences and by the chairman and organizing committee of the race.
Conflict of interest
The authors declare no conflicts of interest that are directly relevant to the content of this article.
All procedures performed in studies involving human participants were in accordance with study approval by the Institutional Review Board of the Nagoya City University Graduate School of Medical Sciences and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent in writing was obtained from each subject enrolled in the study.
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