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Basic Sciences: Original Investigations

Pre-exercise branched-chain amino acid administration increases endurance performance in rats

CALDERS, PATRICK; PANNIER, JEAN-LOUIS; MATTHYS, DIRK M.; LACROIX, ERIC M.

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Medicine & Science in Sports & Exercise: September 1997 - Volume 29 - Issue 9 - p 1182-1186
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Abstract

It is well established that the oxidation of branched-chain amino acids (BCAA) increases during prolonged endurance exercise(11,12). This may play a role in the onset of fatigue by different mechanisms. First, it may lead to an excessive formation of ammonia (10). An accumulation of ammonia in skeletal muscle may lead to a rise in the plasma ammonia concentration, leading to an increase of ammonia in the brain with resulting central nervous dysfunction(central fatigue) (15). Second, increased oxidation of BCAA may lead to a decrease in the plasma BCAA concentration, which favors the entry of free-tryptophan (free-TRP) into the brain. This could cause an increase in cerebral serotonin (5-HT) synthesis which may be involved in central fatigue during exercise (16).

Based on the discussion above, the supplementation of BCAA could influence both performance and metabolic responses during exercise. It has been suggested that BCAA supplementation improves endurance performance by reducing the transport of free-TRP across the blood-brain barrier, thereby suppressing the synthesis of serotonin (3,4). In contrast, some investigators have reported that BCAA supplementation has no effect(6,17-19) or even a detrimental effect on physical performance (20). Further, several investigators have observed that compared with placebo, the supplementation of BCAA results in a higher blood concentration of ammonia during exercise(13,19,20). Clearly, additional research is needed to clarify the effects of BCAA supplementation on exercise tolerance. Therefore, this study was designed to investigate the effects of pre-exercise BCAA administration on the magnitude of the “exercise induced” hyperammonemia and on time to exhaustion during treadmill exercise in rats.

MATERIALS AND METHODS

Animals and Exercise Protocols

This study was conducted in conformity with the policies and guiding principles in the care and use of animals of the American College of Sports Medicine. Female Wistar rats (body mass = 150-180 g at the start of the experiment) were provided water and food ad libitum, and were trained to run on a motor driven treadmill. The rats were randomly divided into appropriate experimental groups. Three sets of experiments were performed with and without BCAA supplementation: 1) control experiments, 2) 30 min of submaximal exercise, and 3) exercise to exhaustion. The animals were fasted during 24 h before the beginning of experimentation to ensure identical basal metabolic conditions across treatments.

Control experiments. To determine the acute effects of BCAA supplementation on plasma levels of BCAA and other metabolites, preliminary experiments were performed. Control values were determined using resting rats sacrificed without any treatment (N = 6) and resting rats sacrificed 5 min after i.p. injection of placebo (N = 6) or BCAA (N = 6). Placebo treatment consisted of 1 mL of 0.9% NaCl, while BCAA treatment consisted of 10 mg of each leucine, isoleucine, and valine (30 mg BCAA) dissolved in 1 mL 0.9% NaCl.

30-min submaximal exercise. Thirty-six rats were trained to run on the treadmill 5 d·wk-1 during 3 wk at 1.2 km·h-1 and 0% grade for 15 min. In the fourth week the rats ran the first four days at 1.2 km·h-1 and 0% grade for 30 min, and the experiment was performed on the fifth day. In these experiments, 18 rats received the placebo, and the other 18 rats were injected with BCAA. Five minutes after the injection, half of the placebo (N = 9) and half of the BCAA group (N = 9) started to run for 30 min at 1.2 km·h-1 and 2.5% grade while the remaining rats (placebo(N = 9) and BCAA group (N = 9)) were not exercised. All animals were sacrificed 35 min after the injection.

Run to exhaustion. Twenty-four rats were trained 5 d·wk-1 during 5 wk at 1.2 km·h-1 and 0% grade. The first week they ran for 15 min with the daily run time being increased by 10 min each week to reach 55 min·d-1 during the last week. After these 5 wk the animals were trained during another 4 consecutive weeks. During this period the rats ran the first 4 d of each week for 30 min at 1.2 km·h-1 and 0% grade. Each fifth day, 12 rats ran until exhaustion at 1.2 km·h-1 and 8% grade while the other 12 rats were allowed to rest. For the runs to exhaustion, six rats ran on four separate occasions after receiving the placebo, while the six other rats made the four runs alternately with placebo and with BCAA. On the fifth day of the last week, rats were sacrificed after the last experimental session. For the resting control rats injected either with placebo (N = 6) or with BCAA (N = 6), the same time interval was allowed between injection and sacrifice as observed in their counterparts which had run to exhaustion. Exhaustion was defined as the point in time animals were unable to keep pace with the treadmill despite constant physical prodding of the tail for up to 1 min.

Measurements

After sacrifice blood was collected for the measurement of glucose, nonesterified fatty acid (NEFA), lactic acid, BCAA, ammonia and free-tryptophan (free-TRP). Ammonia was measured immediately with the Ammonia Test kit II in combination with the Ammonia Checker II (Menarini, Italy). An enzymatic method was used for the determination of lactate (LACT, Boehringer Mannheim, Germany), glucose (D-glucose, Boehringer Mannheim, Germany) and NEFA(NEFA PAP, Biomérieux, France). For the determination of BCAA and free-tryptophan the samples were prepared by addition of succinate buffer to reach a pH of 7.4 and centrifuged at 7,000 rpm for 90 min at room temperature. The remaining protein was removed by addition of sulfosalicylic acid, and after neutralization to pH 2.2, amino acids were analyzed by ion exchange column chromatography (LKB-4151 Alpha Plus Amino Acid Analyser, LKB Biochrom). BCAA concentration was calculated by summing the individual concentrations of leucine, isoleucine, and valine.

Data Analysis

Data are expressed as means ± SD. Statistical analysis was performed using ANOVA 1 followed by a Duncan Multiple Range Test post hoc when appropriate. Significance was established at P < 0.05.

RESULTS

Exercise Without Previous Administration of BCAA

Submaximal 30-min exercise. Thirty min of submaximal exercise resulted in a small but not significant decrease of the plasma glucose concentration, a significant increase in the NEFA concentration and a significant large increase in the lactate levels (Table 1). Moreover, while the plasma concentrations of the BCAA were not modified(Fig. 1), the concentrations of free-TRP(Fig. 2A) and of blood ammonia (Fig. 3) were significantly augmented at the end of the exercise, the former rising from 22 ± 8 to 37 ± 6 μmol·L-1(P < 0.01) and the latter from 33 ± 15 to 89 ± 16μmol·L-1 (P < 0.01). Also the ratio of free-TRP to BCAA increased markedly from 0.040 ± 0.004 to 0.088 ± 0.005(P < 0.01) (Fig. 2B).

Exercise until exhaustion. In general, exercise until exhaustion resulted in qualitatively the same changes in the plasma metabolite concentrations as in the submaximal exercise bout (Table 1). The only quantitative difference between the exhaustive exercise bout and the submaximal 30-min run was a slightly larger increase in the NEFA concentration (Table 1) and a more pronounced increase in the blood ammonia levels (Fig. 3), which rose from control levels of 33 ± 15 to 123 ± 19 μmol·L-1(P < 0.01).

Exercise With Previous Administration of BCAA

At rest. At rest administration of 30 mg BCAA i.p. had no influence on the plasma levels of glucose, NEFA, and lactic acid(Table 1). The blood ammonia concentration was not modified 35 min after the BCAA administration, but increased from 37 ± 17 to 83 ± 11 μmol·L-1 (P < 0.01) after 104 min (Fig. 3). Five min after its administration the BCAA concentration had risen from 522 ± 32 (control animals) to 1497± 62 μmol·L-1 (P < 0.01), and then decreased to 1189 ± 131 after 35 min and to 820 ± 60μmol·L-1 104 min after the injection (significant decrease compared with peak value) (P < 0.01) (Fig. 1). The ratio of free-TRP to BCAA was lower after the administration of BCAA (0.016 ± 0.004) compared with placebo (0.040 ± 0.004), and increased progressively to 0.020 ± 0.002 after 35 min and to 0.031± 0.004 after 104 min (Fig. 2B).

Submaximal 30-min exercise. Submaximal 30-min exercise, which began 5 min after the administration of the BCAA, resulted in the same changes in the plasma parameters as observed in the exercising animals that received the placebo injection (Table 1), except for the blood ammonia concentration (Fig. 3) which increased to a higher level, i.e., to 113 ± 25 instead of 89 ± 16μmol·L-1 (P < 0.05). Moreover, compared with the resting rats, the plasma levels of BCAA (Fig. 1) had decreased to a lower level, i.e., to 872 ± 107 instead of 1189 ± 131 μmol·L-1 (P < 0.05).

Exercise until exhaustion. After administration of BCAA the average time to exhaustion increased by approximately 23 min, from 76 ± 4 min without BCAA to 99 ± 9 min after BCAA (P < 0.01)(Fig. 4). The values for the plasma levels of glucose, NEFA, lactic acid (Table 1) and free-TRP(Fig. 2A) at the time of exhaustion were not statistically different from those observed at exhaustion in the rats which did not receive BCAA. However, compared with placebo, BCAA administration resulted in higher (P < 0.05) blood ammonia levels at the time of exhaustion (186 ± 44 vs 123 ± 19 μmol·L-1)(Fig. 3). Moreover, compared with those in resting rats, the plasma levels of BCAA (Fig. 1) had decreased to a lower level reaching 512 ± 37 instead of 820 ± 60μmol·L-1 104 min after the injection of BCAA (P< 0.01). The ratio of free-TRP to BCAA (Fig. 2B) was similar to the value obtained in the run to exhaustion without BCAA.

DISCUSSION

The purpose of the present study was to investigate the effect of pre-exercise BCAA administration on exercise tolerance. The main findings were that compared with placebo, the administration of BCAA to rats before exercise results in higher blood ammonia concentrations during exercise, a lower plasma free-TRP/BCAA ratio, and an increase in running time to exhaustion.

During the placebo run we observed a slight (statistically not significant) decrease in plasma glucose, an increase in plasma NEFA and lactic acid concentrations, and a progressive increase of the blood ammonia levels. The free-TRP levels increased with no change in the BCAA concentration so that the ratio free-TRP/BCAA nearly doubled. These changes are in agreement with the results of previous investigators(2-5,7,13,15,19). The effect of exercise on the plasma BCAA levels is controversial. BCAA levels have been reported to be either decreased (2,8), increased (1,9), or unchanged during exercise compared with resting values (1,9). During endurance exercise there is an uptake of BCAA by leg tissue, and as exercise progresses, a significant output of BCAA from the liver(1,9), so that the plasma pool of BCAA may vary according to the balance between these two processes.

We used BCAA supplementation as a tool to increase the plasma levels of BCAA before the start of exercise. This resulted in a threefold increase of the BCAA levels within 5 min which is in agreement with the results of others(13,14). The more rapid decrease in plasma BCAA levels during exercise as compared with that at rest (Fig. 1) could be explained by an increased oxidation of BCAA during exercise(11,12).

Our findings confirm previous reports(13,14,19,20) that administration of BCAA before exercise results in significantly higher blood ammonia concentrations during exercise compared with placebo supplementation(Fig. 3). The high plasma BCAA concentration also led to a marked reduction in free-TRP/BCAA ratio at rest and during the submaximal 30-min exercise test compared with placebo. When exercise was continued this ratio progressively increased owing to a rise in the levels of free-TRP and to a decrease in the plasma levels of BCAA to reach the same values as in the placebo experiments at the time of exhaustion (Fig. 2).

The marked increase in time to exhaustion after pre-exercise BCAA administration is the most salient observation of the present experiments(Fig. 4). The effect of BCAA supplementation on physical performance is controversial. Oral administration of BCAA before and during physical activity has been reported to shorten run time in marathon and improve mental performance in a 30-km cross-country run(3,4). Confusion exists concerning the effects of BCAA supplementation on exercise tolerance in patients with McArdle's disease. Wagenmakers et al. (20) reported a detrimental effect of BCAA administration on performance during incremental cycle ergometer exercise in McArdle's patients, but Coackley et al. (6) found no consistent change. Also, evidence exists that BCAA supplementation does not improve exercise performance in healthy “glycogen depleted” men(17,19) cycling to exhaustion. Finally, Verger et al. (18) found that BCAA supplementation did not significantly alter exercise performance in running rats.

We can only postulate as to the mechanisms responsible for our finding that pre-exercise BCAA administration prolonged time to exhaustion in the present experiments. The improved endurance performance notwithstanding the much larger increase in blood ammonia seems to indicate that the ammonemia is not a decisive factor in the genesis of fatigue in these circumstances. According to the hypothesis proposed by Newsholme et al. (16), BCAA supplementation could delay fatigue during exercise by preventing the increase in the free-TRP/BCAA ratio which occurs during exhausting endurance exercise. In the present experiments, the high plasma BCAA concentration did not only prevent a rise in the ratio, but even led to a marked reduction of the free-TRP/BCAA ratio at rest and during the submaximal 30-min exercise test compared with placebo. This ratio progressively increased as exercise progressed, but, starting from a lower level at rest, more time was needed before the high values were reached, possibly explaining the longer time to exhaustion (Fig. 2).

In conclusion, the present experiments demonstrate that, compared with placebo, pre-exercise administration of BCAA in fasted rats results in: 1) a significantly higher blood ammonia concentration during exercise, 2) a reduction in the free-TRP/BCAA concentration ratio, and 3) increased running time to exhaustion. Further, our results suggest that the exercise-induced hyperammonemia was not the immediate cause of fatigue as the BCAA-treated rats ran some 23 min longer notwithstanding much higher blood ammonia levels. The improved performance of the BCAA-treated rats may result from a central effect by reducing the uptake of free-TRP in the brain and thereby reducing the formation of serotonin (16).

T1-10
F1-10
Figure 1-Plasma BCAA concentration at rest (----) or during treadmill exercise (―), before (0 min, 6 rats) or after the administration of placebo (▪) or BCAA (•). Mean values ± SD. 5 min: 2 groups of 6 rats; 35 min: 4 groups of 9 rats; at exhaustion: 4 groups of 6 rats.[uArr ], injection of 0.9% NaCl (placebo) or 30 mg BCAA; ↑, start of exercise; X error bars, SD of times-to-exhaustion **:
P < 0.01; * P < 0.05, significantly different from rest.
F2-10
Figure 2-Plasma free tryptophan concentration (3A) and ratio of free tryptophan to BCAA (3B) at rest (----) or during treadmill exercise (―), after the administration of placebo (▪) or BCAA (•). Mean values± SD. 5 min: 2 groups of 6 rats; 35 min: 4 groups of 9 rats; at exhaustion: 4 groups of 6 rats. [uArr ], injection of 0.9% NaCl (placebo) or 30 mg BCAA; ↑, start of exercise; X error bars, SD of times-to-exhaustion.**:
P < 0.01, significantly different from rest (Fig. 3A) ; **: P < 0.01, significantly different from BCAA (Fig. 3B) .
F3-10
Figure 3-Blood ammonia concentration at rest (----) or during treadmill exercise (―), after the administration of placebo (▪) or BCAA (•). Mean values ± SD. 5 min: 2 groups of 6 rats; 35 min: 4 groups of 9 rats; at exhaustion: 4 groups of 6 rats. [uArr ], injection of 0.9% NaCl (placebo) or 30 mg BCAA; ↑, start of exercise; X error bars, SD of times-to-exhaustion. **:
P < 0.01 and * P < 0.05, significantly different from placebo.
F4-10
Figure 4-Time to exhaustion during treadmill exercise after administration of placebo (□) or BCAA (▨). Mean values ± SD. Two groups of 6 rats, which ran 4 times until exhaustion. **:
P < 0.01, significantly different from placebo.

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Keywords:

AMMONIA; EXERCISE; BRANCHED-CHAIN AMINO ACIDS; TIME TO EXHAUSTION

©1997The American College of Sports Medicine