Beruflich Dokumente
Kultur Dokumente
DOI 10.1007/s00421-005-0036-1
O R I GI N A L A R T IC L E
Abstract Branched chain amino acids (BCAA), particularly leucine, have been suggested to be ergogenic for
both endurance and strength/power performance. This
study investigated the eects of dietary leucine supplementation on the exercise performance of outrigger
canoeists. Thirteen (ten female, three male) competitive
outrigger canoeists [aged 31.6 (2.2) year, VO2max 47.1
(2.0) ml kg 1 min 1] underwent testing before and after
6-week supplementation with either capsulated L-leucine
(45 mg kg 1 d 1; n=6) or placebo (cornour; n=7).
Testing included anthropometry, 10 s upper body power
and work and a row to exhaustion at 7075% maximal
aerobic power where perceived exertion (RPE), heart
rate (HR) and plasma BCAA and tryptophan concentrations were assessed. Leucine supplementation resulted
in signicant increases in plasma leucine and total
BCAA concentrations. Upper body power and work
signicantly increased in both groups after supplementation but power was signicantly greater after leucine
supplementation compared to the placebo [6.7 (0.7) v.
6.0 (0.7) W kg 1]. Rowing time signicantly increased
[77.6 (6.3)88.3 (7.3) min] and average RPE signicantly
decreased [14.5 (1.5)12.9 (1.4)] with leucine supplementation while these variables were unchanged with the
placebo. Leucine supplementation had no eect on the
plasma tryptophan to BCAA ratio, HR or anthropometric variables. Six weeks dietary leucine supplemenParts of this work have previously been presented in abstract form:
Crowe MJ, Weatherson JN (2002) The eects of dietary L-leucine
supplementation on exercise performance. Sports Medicine and
Science at the Extremes. Australian Conference of Science and
Medicine in Sport. 1216 October, Melbourne, Australia
M. J. Crowe (&) J. N. Weatherson
Institute of Sport and Exercise Science, James Cook University,
Townsville, QLD 4811, Australia
E-mail: Melissa.Crowe@jcu.edu.au
Tel.: +61-7-47815610
Fax: +61-7-47816688
B. F. Bowden
School of Pharmacy and Molecular Sciences,
James Cook University, Townsville, QLD 4811, Australia
Introduction
Research has shown that branched chain amino acids
(BCAA), particularly leucine, undergo increased oxidation during prolonged endurance exercise (Henderson
et al. 1985) and promote skeletal muscle protein synthesis (Layman 2002). For these reasons, there has been
interest in the potential of BCAA supplementation to
enhance both endurance and strength/power exercise
performance (Blomstrand et al. 1991, 1997; Davis et al.
1999; Mittleman et al. 1998; Pitkanen et al. 2003).
Research interest in BCAA has focussed on the
potential of the BCAA to prevent central fatigue
(Blomstrand et al. 1991, 1997) by competing with freetryptophan (f-TRP) for uptake from the plasma into the
brain. Increased brain f-TRP concentrations lead to increased serotonin (5HT) synthesis which is associated
with lethargy, fatigue and decreased exercise performance (see Davis et al. 2000). BCAA may limit central
fatigue as they compete with f-TRP for uptake into the
brain via the same transport mechanism (Pardridge and
Oldendorf 1975). Therefore, the ratio of plasma f-TRP
and BCAA may be important in preventing increases in
brain 5-HT and fatigue during exercise. Human and
animal studies support the central fatigue hypothesis
(see Davis et al. 2000). However, evidence that BCAA
supplementation can reduce central fatigue and improve
performance is less convincing. BCAA supplementation
studies have reported either decreased perceived exertion
and increased endurance performance (Blomstrand
et al. 1991, 1997; Mittleman et al. 1998) or no ergogenic
eects (Davis et al. 1999; Verger et al. 1994). The
665
Methods
Subjects
Female (n=10) and male (n=3; one male participant
withdrew after injury unrelated to the study) outrigger
canoeists gave their written informed consent to participate in this study. The participant characteristics are
presented in Table 1. All the participants all had a
Males
3
27.3 (3.9)
96.3 (6.2)
185.9 (1.4)
min 1) 55.3 (1.4)
Females
All participants
10
32.9 (3.0)
67.5 (3.3)
166.8 (1.8)
44.6 (2.0)
13
31.6 (2.2)
74.2 (4.5)
171.2 (2.7)
47.1 (2.0)
666
power test and row to exhaustion at pre-supplementation, the anthropometry followed by the VO2max test
were performed on one day with the upper body power
test and row to exhaustion performed on a separate day,
17 days later. As the VO2max test was not repeated at
post-supplementation, the anthropometry was performed followed by the upper body power test and row
to exhaustion on the same day. At least 30-min rest was
provided between the 10 s upper body power test and
commencement of the row to exhaustion at both the preand post-supplementation sessions. Details of these
assessments are provided below.
VO2max assessment
Maximal oxygen consumption was determined using an
incremental rowing protocol modied from that of
Yoshiga and Higuchi (2003) on a rowing ergometer
(ConceptII, Morrisville, VT, USA). Female participants
commenced rowing at 100 W and increased the workload by 15 W every 2 min until volitional exhaustion.
Male participants underwent the same protocol with the
exception of commencing on 150 W with 50 W increments. All participants received verbal encouragement
throughout the duration of the test. Heart rate was recorded telemetrically (Polar Electro OY, Kempele,
Finland) every 30 s. The participants breathed through a
two-way valve (Hans Rudolph Inc.,Kansas City, MO,
USA) with gas analysis via a metabolic cart (Quinton
Instrument Company, Seattle, WA, USA) which was
calibrated with a 3 l syringe (Hans Rudolph Inc.,) and
standard alpha gas mixture (BOC Gases, Sydney, Australia). Oxygen consumption, CO2 production, respiratory exchange ratio (RER) and minute ventilation were
continuously recorded and averaged every 15 s of the
test. Verication of VO2max was established by the
achievement of three of the following four criteria: a
decrease or plateau in VO2 with an increase in the
workload; an RER greater than 1.15; HRmax > 85%;
and failure to maintain the specied workload (American College of Sports Medicine 2000).
Anthropometric characteristics
Anthropometric characteristics were determined in line
with the standards endorsed by the International Society
for the Advancement of Kinanthropometry (Norton and
Olds 1996) and performed by the same investigator
(technical error of measurement <5%). Height was assessed using a wall-mounted stadiometer and body mass
assessed using digital weighing scales (50 g; Precision
Health Scale VC-300, A & D Company Ltd, Japan).
Percentage body fat was calculated using LifeSize software (Version 1.0 Human Kinetics, Champaign, IL,
USA) from nine skinfold (bicep, tricep, subscapular,
iliac crest, supraspinale, abdominal, mid axilla, thigh
and calf), and ve girth (arm relaxed and exed, waist,
667
Blood testing
Blood samples were analysed using high performance
liquid chromatography (HPLC) for plasma concentrations of leucine, isoleucine, valine and tryptophan.
Within 4 h of collection, the blood samples were centrifuged (3,000 rpm for 25 min at 4C) and the plasma
frozen at 20C for later analysis. Following thawing,
plasma preparation was carried out in accordance with
the methods of Gratseld-Hueesgen (1998) to precipitate
protein and extract lipophilic materials. To precipitate
proteinaceous material, 2 ml of methanol was added to
the samples with 4 ml of 1:1 diethyl ether and hexane to
extract lipophilic materials. The sample was then mixed
in a vortex for 60 s and centrifuged (3,000 rpm for
10 min at 4C). After centrifugation, the upper hexane/
diethyl ether phase was removed and discarded. A further 4 ml of the diethyl ether/hexane mixture with 1 ml
of methanol was added and the sample vortexed and
centrifuged (3,000 rpm for 15 min at 4C). The supernatant was removed and retained. The pellet was washed
twice more with 1 and 0.5 ml methanol and then discarded. The sample was divided into two 2 ml aliquots
that were snap-frozen using liquid nitrogen and placed
on a freeze drier overnight.
Amino acid derivatisation and HPLC analysis were
performed with modications to the methods of Carroll
et al. (1996). Following freeze-drying, the aliquots were
treated with 500 ll distilled water. A 200 ll aliquot was
then derivatised with 2.5 mg of 1-uoro-24-dinitophen5-yl-L-alanine amide (FDAA) in 250 ll of acetone and
1N sodium bicarbonate (30 ll) at 50C for 4 h. Samples
were acidied with 125 ll hydrochloric acid and ltered
through 0.22 ll lters (Millipore Corporation, Bedford,
MA, USA) to remove all the remaining proteins and
transferred into 2 ml glass ampoules. High performance
liquid chromatography analysis was performed on a
Reverse Phase Luna phenyl-hexyl column (4.6250 mm;
Phenomonex, Auckland, New Zealand) with an autosampler injector (ICI Instruments, Hubbartson, MA,
USA). The best conditions for providing separation
were found to be a mobile phase of 72:28% triethylammonium phosphate (pH 3.0):acetonitrile, at a ow
rate of 2.0 ml min 1 and UV detection at 340 nm.
Each peak in the chromatographic trace was identied
by comparing the retention time with that of standards of
L-leucine, L-isoleucine, L-valine and L-tryptophan (British
Drug Houses Ltd., Poole, UK). The standards were
prepared by serial dilution and derivatised, acidied and
run on the column in the same manner as the plasma
samples. The concentrations of each amino acid in the
plasma were determined by comparing the integral of the
peak with that of the standard.
Results
VO2max
There was no signicant dierence in the VO2max of the
two groups prior to supplementation (P=0.681) with
the leucine and placebo groups averaging 46.1 (3.3) and
47.9 (2.7) mL kg 1 min 1, respectively.
Dietary analysis
The dietary protein intake of the leucine and placebo
groups did not dier signicantly during the supplementation period [leucine group 0.85 (0.06) g kg 1 d 1;
placebo group 0.85 (0.05) g kg 1 d 1; P=0.775]. Furthermore, dietary intake of protein did not change from
the week preceding pre-supplementation testing to the
supplementation period for either group (P=0.570).
Anthropometric characteristics
There was no signicant dierence between the leucine
and placebo groups in body mass at pre-supplementation [leucine 73.0 (5.1) kg, placebo 75.2 (7.5) kg] or postsupplementation [leucine 73.1 (5.0) kg, placebo 75.1
(7.5) kg; P>0.05]. Similarly, percentage body fat was
not signicantly dierent between the leucine and placebo groups at pre-supplementation [leucine 25.1
(3.9)%, placebo 26.4 (1.6)%] or post-supplementation
[leucine 25.7 (3.6)%, placebo 26.6 (1.6)%; P>0.05].
Statistical analysis
668
groups (P<0.001; power=1.00), however, the postsupplementation peak power achieved by the leucine
group was signicantly higher than that achieved by the
placebo group (P=0.045; power=0.54; Fig. 1a). Total
work achieved during the 10-s-test also increased signicantly for both groups from pre- to post-supplementation (P=0.001; power=0.99). However, there was
no signicant dierence between the leucine and placebo
groups at post-supplementation (P=0.277; Fig. 1b).
Row to exhaustion
A main eect for time showed that regardless of supplementation group, there was an overall improvement
in the rowing time to exhaustion from 72.2 (4.4) min at
pre-supplementation to 76.3 (5.8) min at post-supplementation (P=0.036; power=0.59). The interaction effect (P=0.008; power=0.84) showed a signicant
increase in time to exhaustion for the leucine group
whereas the placebo group showed no improvement
(Fig. 2).
Exercise time to exhaustion varied between 55 and
110 min. Therefore, to analyse a complete data set for
all participants, only the rst 55 min of RPE and HR
data were analysed. There was a signicant main eect
for time on RPE with a signicant increase in RPE
every 10 min throughout the row to exhaustion test
(P<0.001; power=1.00). The average RPE was not
signicantly dierent between the leucine and placebo
groups at pre-supplementation [14.5 (1.5) and 14.6
(1.2), respectively]. However, the average RPE for the
leucine group was signicantly lower than that of the
placebo group during the post-supplementation row to
Fig. 1 Upper body a peak power and b total work for the leucine
and placebo groups at pre- and post-supplementation. Data are
mean (SE). *P<0.05 signicantly dierent from leucine at presupplementation and placebo at pre- and post-supplementation
Fig. 2 Row time to exhaustion for the leucine and placebo groups
at pre- and post-supplementation. Data are mean (SE). *P<0.05
signicantly dierent from leucine at pre-supplementation and
placebo at pre- and post-supplementation
669
Table 2 Mean (SE) plasma amino acid concentrations for all participants (n=13) prior to and following the row to exhaustion
Amino acid (lmol)
Pre-exercise
Post-exercise
Leucine
Isoleucine
Valine
f-tryptophan
Total BCAA
f-TRP:BCAA
145.1 (4.0)
76.8 (2.3)
262.0 (4.6)
13.4 (0.8)
483.9 (10.4)
0.0280 (0.0020)
143.2 (4.0)*
75.0 (2.2)*
250.0 (4.5)*
14.0 (0.7)*
478.2 (10.3)*
0.0294 (0.0017)*
isoleucine concentrations between the leucine and placebo groups pre- or post-exercise following the supplementation period (Table 3).
Plasma valine concentrations did not change signicantly from pre- to post-supplementation in either the
leucine or placebo groups (P=0.632; Table 3). Similarly, plasma f-tryptophan concentrations were unaffected by leucine supplementation with no signicant
dierences from pre- to post-supplementation in the
leucine or placebo groups (P=0.671; Table 3).
The total BCAA concentrations showed a signicant
main eect for time with a signicant increase from preto post-supplementation [476.5 (11.5) lmol v. 485.5
(9.2) lmol; P=0.014; power=0.75]. A condition by
time interaction showed that BCAA were signicantly
increased from pre- to post-supplementation in the leucine group [479.3 (16.9) v. 497.4 (13.5) lmol, respectively] and that the post-supplementation levels in the
leucine group were signicantly greater than those of the
placebo group at both pre- and post-supplementation
[473.7 (15.7) v. 473.6 (12.5) lmol, respectively;
P=0.014; power=0.76].
The ratio of f-tryptophan to BCAA was unaected
by leucine supplementation with no signicant dierence
between the leucine and placebo groups at pre- or postsupplementation before or after exercise (P=0.438;
Table 3).
Side eects
None of the participants reported any side eects as a
result of dietary supplementation.
Discussion
The results of this study supported the hypotheses that
leucine supplementation would increase plasma leucine
670
Table 3 Mean (SE) plasma concentrations of isoleucine, valine, free-tryptophan (f-TRP) and the ratio of f-TRP to BCAA for the leucine
and placebo groups
Pre-supplementation
Leucine (Pre-exercise)
Leucine (Post-exercise)
Placebo (Pre-exercise)
Placebo (Post-exercise)
Post-supplementation
Leucine (Pre-exercise)
Leucine (Post-exercise)
Placebo (Pre-exercise)
Placebo (Post-exercise)
Isoleucine (lmol)
Valine (lmol)
f-TRP (lmol)
f-TRP:BCAA
76.7
74.5
77.4
76.1
(3.4)
(3.5)*
(3.1)
(3.2)
263.7
262.2
260.4
257.6
(6.7)
(6.8)
(7.4)
(7.0)
13.5
14.3
14.1
14.4
(1.5)
(1.3)
(1.5)
(0.9)
0.028
0.030
0.029
0.031
(0.029)
(0.030)
(0.040)
(0.032)
76.3
75.0
76.9
74.3
(3.3)
(3.2)
(3.1)
(2.9)*
263.0
261.3
260.7
258.9
(5.8)
(5.7)
(6.1)
(8.0)
13.0
13.2
13.4
13.9
(0.9)
(0.7)
(0.9)
(0.6)
0.026
0.027
0.029
0.030
(0.022)
(0.018)
(0.025)
(0.017)
studiesparticularly with reference to the exercise protocol, BCAA dose and carbohydrate availabilitymake
comparisons dicult. Past BCAA supplementation
studies have reported an increase in marathon performance in slower but not quicker runners (Blomstrand
et al. 1991) and an increase in exercise time to exhaustion in the heat (Mittleman et al. 1998). The latter study
reported a decrease in concentrations of f-TRP and the
ratio of f-TRP to BCAA in response to BCAA supplementation (Mittleman et al. 1998). In contrast, glycogen-depleted subjects who exercised to exhaustion in the
heat did not benet from BCAA supplementation
(Watson et al. 2004). Plasma ammonia concentrations
were also elevated following exercise with BCAA in this
study (Watson et al. 2004). The dose of BCAA has been
implicated in the outcome of BCAA supplementation
studies with the suggestion that higher doses increase
plasma ammonia concentrations and therefore result in
fatigue (Blomstrand 2001). Consistent with this suggestion, the current study utilised a dose of approximately
3.3 g d 1 and other studies reporting positive eects of
BCAA supplementation have utilised doses of approximately 68 g (Blomstrand et al. 1997) and 9 and 16 g
(Mittleman et al. 1998). In contrast, those studies
reporting no eect of BCAA supplementation on performance utilised approximately 1730 g BCAA (Madsen et al. 1996; Varnier et al. 1994; Watson et al. 2004).
However, one of these studies (Varnier et al. 1994) did
not detect a dierence in plasma ammonia concentrations between the BCAA and placebo treatments. A
dose-response study of BCAA supplementation and
subsequent alterations in plasma BCAA, the ratio of
f-TRP to BCAA and ammonia may assist in clarifying
the exercise response to BCAA supplementation.
Supplementation of BCAA with carbohydrate has
previously been reported to provide no additional performance benets compared to carbohydrate supplementation alone (Davis et al. 1999; Madsen et al. 1996).
The current study provided a pre-exercise carbohydraterich meal in order to simulate competition conditions. It
was therefore interesting that after 6-week leucine supplementation, endurance performance was improved to
a greater extent than after the placebo where the same
671
672
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