THE EFFECT OF BCAA ON ISOMETRIC FORCE FOLLOWING ENDURANCE EXERCISE IN A HOT ENVIRONMENT

ABSTRACT Introduction: Fatigue due to endurance exercise results from both peripheral and central changes, and may influence subsequent performance during a strength task. The increase in serotonin concentration is one of the central factors associated with endurance exercise-induced fatigue, particularly in hot environments. A nutritional strategy employed to reduce serotonergic activation is supplementation with branched-chain amino acids (BCAA). Objective: To investigate whether BCAA supplementation attenuates the reduction in isometric force caused by prior endurance exercise in a hot environment. Methods: Nine volunteers (aged 25.4 ± 1.2 years) performed a 2-min maximal voluntary isometric contraction (MVCISO) of upper limb muscles before and after an endurance exercise on a cycle ergometer at 40% of the maximal aerobic power. The volunteers underwent three experimental trials: 1) endurance exercise in a temperate environment (23°C and 60% RH); exercise in a hot environment (35°C and 60% RH) with the ingestion of: 2) a placebo solution or 3) a solution containing BCAA 30 mg.kg−1. During the MVCISO test, the isometric force of flexor muscles of the right elbow, core body temperature (TCORE) and heart rate (HR) were measured. Results: Isometric force decreased following endurance exercise in the hot environment, and BCAA administration did not attenuate this reduction. Greater TCORE and HR values were observed following endurance exercise in the heat, compared to pre-exercise values, and supplementation did not interfere with these physiological responses. Conclusion: The reduction in isometric force, caused by previous endurance exercise in a hot environment, was not diminished by supplementation with BCAA. Level of evidence I; Type of study: Therapeutic studies - Investigation of treatment outcomes.


INTRODUCTION
3][4] One of the acute influences is the increase in core body temperature (T CORE ).Nybo and Nielsen (2001) 5 showed that exercising on a cycle ergometer at ~60% of the maximal oxygen consumption (VO 2max ) in a hot environment promoted greater reduction in force during a subsequent 2-min maximal isometric voluntary contraction (MVC ISO ) of upper-limb muscles, relative to the trial performed in a temperate environment.This exacerbated reduction in force was accompanied by greater reduction in central activation and, therefore, attributed to central mechanisms of fatigue dependent on a rise in T CORE.

6,7
Increased serotonin (5-HT) concentration in the central nervous system (CNS) is a factor associated with the T CORE increase and fatigue during endurance exercise and, therefore, is a possible explanation for the subsequent reduction in strength performance.According to the central fatigue hypothesis, 8,9 increased 5-HT activity during endurance exercise can cause fatigue by inducing lethargy, increasing the rating of perceived exertion and reducing motivation, tolerance to pain/discomfort and central drive to the active musculature. 10,11he 5-HT synthesis in the CNS depends on the passage of its precursor, free tryptophan (TRP free ; not bound to albumin), through the blood-brain barrier, which is mediated by the L-carrier system.During endurance exercise, there is an increase in 5-HT concentration in the CNS resulting from: (1) the release of free fatty acids (FFA) that have a greater affinity for albumin binding than TRP, thereby increasing the concentration of TRP free ; and the oxidation of branched-chain amino acids (BCAA: leucine, isoleucine and valine), which compete with TRP free for the L-carrier system. 9In a hot environment, the 5-HT increase during endurance exercise is exacerbated by the greater stimulus for lipolysis and the greater sympathetic activation, both of which result in increased FFA and TRP free . 12Thus, the 5-HT concentration is directly correlated with T CORE under these conditions. 9,13,14CAA supplementation is a nutritional strategy to reduce the entry of TRP into the CNS to delay fatigue, 11,12 since these amino acids compete with TRP for the same transport system.Considering that nutrition generally provides a small but potentially valuable contribution to performance in elite athletes, and that the use of supplements is widely diffused at all levels of sport, 15 the aim of this study was to evaluate whether an acute supplementation with BCAA attenuates the reduction in isometric force caused by prior endurance exercise performed in a hot environment.Based on the central fatigue hypothesis, we suggested that supplementation would reduce 5-HT brain synthesis and, therefore, would attenuate the reduction in force commonly observed following endurance exercise in a hot environment.

METHOD Participants and ethical care
Nine physically active, non-smoking, male volunteers agreed to participate in the study and signed an informed consent form.The experimental procedures were approved by the Research Ethics Committee of the Universidade Federal de Minas Gerais (protocol 144/05) and respected the norms established by the National Health Council (Resolution 466/2012) and the Declaration of Helsinki (1965) regarding ethical principles involving human research.(Table 1)

Guidance provided to volunteers
Each volunteer was instructed to: 1) not use medications or supplements while participating in this research; 2) refrain from drinking alcohol, caffeine, and from exercising 48 h prior to any data collection; 3) register their food intake during the 48 h prior to the first experimental trial, and to reproduce the same intake before the other trials.

Experimental design
The volunteers completed four visits to the laboratory.During the first visit, which took place 2-8 days before the beginning of the experimental trials, subjects were characterized and familiarized with the MVC ISO task, and an incremental test was performed.On the experimental days, the volunteers performed MVC ISO before and after endurance exercise on a cycloergometer (40% peak power at 50 rpm) until voluntary interruption of effort (~2 h and 45 min in the temperate environment and 2 h during the two trials in the hot environment).Endurance exercise was performed inside an environmental chamber (Russells Inc.® WMF-1150-5, USA) under the following conditions: 1) control trial in a temperate environment (23°C and 60% RH) (TEMP-C) with ingestion of a placebo solution, 2) a hot environment (35°C and 60% RH) with ingestion of a placebo solution (HOT-PLA) or (3) a hot environment with ingestion of a solution containing BCAA (HOT-BCAA).The experiments always began between 7:00 and 8:00 AM.

Incremental test to determine peak oxygen consumption (VO 2peak )
The incremental cycle ergometer test (Monark, Ergomedic 824-E, Sweden) was initiated at a power output of 50 W at 50 rpm, with 25-W increments every 2 min.The criteria for interrupting the test were: an inability to maintain stipulated cadence, a score of 20 on the perceived exertion scale, 16 a request made to stop exercise or the presence of pallor, cyanosis, or nausea.VO 2PEAK was measured directly through a metabolic gas analyzer (BIOPAC Systems ® , GasSys2, USA, accuracy of ± 1% for O 2 and ± 3% for CO 2 ).

Force Task -isometric maximum voluntary contraction (MVC ISO )
The MVC ISO test was performed to evaluate muscle strength.This test allows for monitoring of fatigue development during a sustained task of force production, avoiding compensatory movements, as the volunteer is kept in a standardized position.
Each volunteer performed 2-min MVC ISO with the flexor muscles of the right elbow.While seated in a chair, adjusted to his height with feet resting on the ground, the volunteer had his position stabilized through straps wrapped around his trunk and thighs.The volunteer rested his arm on a metal plate, with a 90° angle between the arm and the trunk, and a relative elbow angle of 90°.To minimize changes in position during the test, there was a 3-cm high metal bulkhead where the elbow was throughout the test.The wrist, between 0° and 30° of extension, was stabilized through an orthosis.A band, wrapped in the volunteer's hand, was attached to a strain-sensitive force cell, against which the force was made.Each volunteer was instructed to perform at maximum force and received verbal stimuli, visual feedback of the force curve (without access to values) and elapsed time throughout the test.The data acquisition was performed using software (Dasylab ® 5.0) and an analog-digital converter (PCMCIA card) NIDAQ-700 at 1000 Hz (National Instruments ® , Austin, USA).

Supplementation protocol
The ingestion of the placebo (PLA) or BCAA solution, in a volume of 4.0 mL.kg -1 body weight, 17 occurred in a double-blind, crossed-over and randomized manner.The beverage was administered 120 and 60 min before, immediately before and every 30 min during the endurance exercise.

Experimental protocol
The volunteers arrived at the laboratory after overnight fasting, ingested a dose of the solution corresponding to the experimental trial (i.e., PLA or BCAA) and a standardized breakfast (301 kcal; 78% carbohydrates, 9% protein and 13% fat).After 30 min, the volunteers performed the pre-exercise MVC ISO test, the endurance exercise, and the post-exercise MVC ISO test in sequence.The MVC ISO tests were performed at room temperature between 22 and 24ºC.(Figure 1) During the MVC ISO test, T CORE and heart rate (HR) were recorded.The T CORE was obtained every 30 s through a disposable rectal probe (Yellow Springs ® , 4491-E, USA) inserted 10 cm beyond the anal sphincter, connected to a telethermometer (Yellow Springs ® , YSI 4600 Series, USA).HR was measured by an HR monitor (Polar, S120 ® , Finland) every 15 s.

Statistical analyses
Data were checked for normality using the Shapiro-Wilk test and, for this purpose, the data at the pre-exercise moment from each group were evaluated in isolation; all evaluated parameters presented a normal distribution.The homoscedasticity of the data was evaluated through Levene's tests, which confirmed the homogeneity of variances.
The data comparisons across time-points and between the different periods in the same experimental trial (Figure 2) or between the three trials (Figures 3 and 4) were performed using two-way ANOVAs with repeated measures and, when applicable (i.e., significant F value), a post-hoc test was chosen according to the coefficient of variation (CV): Tukey (T CORE and HR, CV ≤ 15%) or Student Newman-Keuls (other variables, CV > 15%).The Pearson correlation coefficient was used to assess the degree of association between the changes in T CORE and force.The significance level adopted was set at 0.05.Data were expressed as mean ± standard error of the mean.The Cohen's d effect size (ES) was calculated to assess the magnitude of differences in the data between experimental trials.The ES was calculated by subtracting the mean of a group (lower value) from the mean of a second group (higher value); the result was then divided by the pooled standard deviation of the data.As all volunteers were subjected to all experimental trials, ES was corrected by the Pearson coefficient obtained from the correlation between the values that were being compared.The ES values were classified as trivial (ES < 0.2), small (ES = 0.2 -0.6), moderate (ES = 0.6 -1.2) or large (ES ≥ 1.2) 18 .

RESULTS
A decreased force of elbow flexors was observed, when compared to before the endurance exercise values, during the initial 15 s of the isometric test in TEMP-C (Figure 2A), over the 120 s in HOT-PLA (Figure 2B) and at 15, 30, and 60 to 120 s in HOT-BCAA (Figure 2C).Before the endurance exercise, the force was lower from the 30 th to the 60 th s in the HOT-BCAA than in the HOT-PLA trial (Figure 2D).
The T CORE and HR values were not different between trials before the exercise.For all trials, the T CORE following the endurance exercise was higher when compared to values recorded before the exercise (TEMP-C: +0.48°C, P = 0.005, ES = 1.18;HOT-PLA: +1.46°C, P < 0.001, ES = 3.14; HOT-BCAA: +1.35°C, P < 0.001, ES = 4.32) (Figure 4).Following exercise, the T CORE values in the hot environment were higher than those found in the temperate environment (P < 0.001) (Figure 4), and supplementation with BCAA did not influence the increase in T CORE (P = 0.57; ES = 0.31).The change in T CORE correlated negatively with the change in force production (r = -0.47;P = 0.01) (Figure 5); that is, the increase in T CORE , induced by endurance exercise, was associated with reduced force production during the subsequent MVC ISO test.
There was no difference in HR between MVC ISO tests at pre-and post-endurance exercise in the TEMP-C trial (pre-exercise: 110 ± 4 bpm vs. post-exercise: 113 ± 3 bpm; P = 0.30; ES = 0.22).The HR was higher during the MVC ISO test at post-than at pre-endurance exercise for both trials performed in the hot environment (HOT-PLA: +18 bpm, P < 0.001, ES = 1.80;HOT-BCAA: +12 bpm, P < 0.001, ES = 1.24) (Table 2) and as well higher at post-endurance exercise in the hot trials than in the TEMP-C trial (P < 0.01) (Table 2).However, when comparing the values between the two trials in the hot environment, supplementation with BCAA did not alter the HR (P = 0.60; ES = 0.21).

DISCUSSION
BCAA supplementation did not attenuate the reduction in isometric force caused by prior endurance exercise in a hot environment, thus not justifying the use of BCAA as an ergogenic aid during a concurrent training session, in which fatigue during the strength task is accentuated by body hyperthermia.The reduced post-endurance exercise force in the hot environment was accompanied by higher T CORE and HR values when compared to those recorded in the temperate environment.In addition, BCAA supplementation resulted in reduced force production during evaluation performed prior to endurance exercise.
1][22][23] This assumption may also explain the reduction in force during the pre-exercise MVC ISO in subjects supplemented with BCAA.Dong-Hee et al. (2013) 22 showed a higher ammonia concentration at 40 min after the ingestion of a single dose of BCAA (80 mg.kg -1 ).MacLean, Graham and Saltin (1996) 24 showed that high (308 mg.kg -1 ) and moderate (77 mg.kg -1 ) doses of BCAA, respectively, increased ammonia concentrations during 90 min and 60 min of a dynamic exercise involving knee extensors.However, hyperammonemia was not observed in other studies in which smaller amounts of BCAA were provided, 17,20,25 including the study of Mittleman et al. (1998), 17 with a BCAA dose similar to the one used in the present study.Therefore, the relationship between the dose of BCAA and the reduction in isometric force, possibly due to hyperammonemia, should be better addressed in future studies.The present study reinforces the notion that a central component induces fatigue in concurrent exercises, [5][6][7] as the lower force produced after the endurance exercise in the hot environment cannot be attributed to local factors derived from previous exercise performed with the same muscle group.Indeed, the muscle group tested during the MVC ISO was different from that which was exercised during the endurance exercise.Moreover, the reduction in force was not observed after the endurance exercise under temperate conditions.Thus, the results of this study contradict the position adopted by Raddi et al. (2008), 26 who, after not observing the influence of a running protocol (45 min at 70% VO 2max ) on force production, concluded that interference observed in the concurrent exercise is dependent on prior recruitment of the same muscle group.
The increase in T CORE determined a 22% reduction in isometric force at post-endurance exercise in the hot environment, as shown by the   correlation between these two variables, which corroborates the existence of an association between a rise in T CORE and a reduction in neuromuscular function, 5 regardless of changes in muscle or skin temperature. 6,7Thus, it is likely that in the study of Raddi et al. (2008), 26 the increase in T CORE was not great enough to reduce force -as is supported by our results obtained in the temperate environment.On the other hand, the study by Nybo and Nielsen (2001) 5 reported an 18% force reduction in post-endurance exercise performed in a temperate environment; however, the 1.1°C temperature increase registered by the latter authors (from 36.9 to 38.0°C) was higher than that observed in the present study (0.46°C) and reinforces the relationship between the magnitude of the T CORE rise and the reduction in force.Thus, future experiments evaluating the isometric force during sustained tasks should consider the influence of the magnitude of the T CORE rise in reducing force production.Other factors may have also contributed to the lower strength performance associated with endurance exercise in a warm environment, such as central hypoglycemia, reduced cerebral blood flow caused by hyperventilation, endotoxemia and an increase in circulating pro-inflammatory cytokines. 27R was higher during the post-exercise MVC ISO in a hot environment, despite the reduction in force production.As this increase was not observed during the post-exercise MVC ISO in the temperate environment, HR elevation appears to be a cardiovascular response driven by thermoregulatory stimuli to facilitate cutaneous heat dissipation while exercising in a hot environment.
The results of the present study, performed in a systematized and controlled manner, do not justify the use of a single dose of BCAA as an ergogenic aid for increasing muscle strength when individuals are hyperthermic.In addition, it is important to emphasize that, when prescribing high-intensity training, coaches should consider performing force exercises before endurance exercise, particularly when the latter exercise markedly elevates T CORE .Finally, additional studies are still needed to evaluate the effects of BCAA on force production during specific tests that reproduce sports skills.

CONCLUSION
The reduction in isometric force caused by endurance exercise in a hot environment was not diminished by acute BCAA supplementation.

Figure 2 .
Figure 2. Comparison of the force produced along time-points during the 2-min isometric maximum voluntary contraction (MVC ISO ) performed before and after a cycling exercise in each of the three experimental trials.TEMP-C: control in the temperate environment; HOT-PLA: placebo in the hot environment; HOT-BCAA: branched-chain amino acids in the hot environment.Data are expressed as mean ± SEM. *Difference between experimental trials.n = 9.

Figure 3 . 9 Figure 4 .
Figure 3. Mean values of the force produced during the 2-min isometric maximum voluntary contraction (MVC ISO ) performed before and after a cycling exercise in each of the three experimental trials.TEMP-C: control in the temperate environment; HOT--PLA: placebo in the hot environment; HOT-BCAA: branched-chain amino acids in the hot environment.Data are expressed as mean ± SEM. *Different from the pre-exercise moment within the same experimental trial.#Different from the TEMP-C trial at the same moment.§Different from the pre-exercise moment in the HOT-PLA trial.n = 9

Figure 5 .
Figure 5. Correlation between the changes in core body temperature (T CORE ) and the changes in mean force for the three experimental trials (27 points).TEMP-C: control in the temperate environment; HOT-PLA: placebo in the hot environment; HOT-BCAA: branched-chain amino acids in the hot environment.

THE EFFECT OF BCAA ON ISOMETRIC FORCE FOLLOWING ENDURANCE EXERCISE IN A HOT ENVIRONMENT EFEITO
DOS AACR NA FORÇA ISOMÉTRICA APÓS EXERCÍCIO DE ENDURANCE EM UM AMBIENTE QUENTE EFECTO DE LOS AACR EN LA FUERZA ISOMÉTRICA DESPUÉS DE EJERCICIO DE ENDURANCE EN UN AMBIENTE CÁLIDO

Table 1 .
Physical characteristics of volunteers.