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Basic nutritional investigation
Effects of oral supplementation with glutamine and alanyl-glutamine
on glutamine, glutamate, and glutathione status in trained rats and
subjected to long-duration exercise
Vinicius Fernandes Cruzat, M.Sc., and Julio Tirapegui, Ph.D.*
Department of Food Science and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
Manuscript received May 30, 2008; accepted September 21, 2008.
Abstract Objective: We investigated the effect of supplementation with the dipeptide L-alanyl-L-glutamine
(DIP) and a solution containing L-glutamine and L-alanine, both in the free form, on the plasma and
tissue concentrations of glutamine, glutamate, and glutathione (GSH) in rats subjected to long-
duration exercise.
Methods: Rats were subjected to sessions of swim training. Twenty-one days before sacrifice, the
animals were supplemented with DIP (1.5 g/kg, n⫽6), a solution of free L-glutamine (1 g/kg) and
free L-alanine (0.61 g/kg; GLN ⫹ALA, n⫽6), or water (CON, n⫽6). Animals were sacrificed
before (TR, n⫽6) or after (LD, n⫽6) long-duration exercise. Plasma concentrations of glutamine,
glutamate, glucose, and ammonia and liver and muscle concentrations of glutamine, glutamate, and
reduced and oxidized (GSSG) GSH were measured.
Results: Higher concentrations of plasma glutamine were found in the DIP-TR and GLN ⫹
ALA-TR groups. The CON-LD group showed hyperammonemia, whereas the DIP-LD and GLN ⫹
ALA-LD groups exhibited lower concentrations of ammonia. Higher concentrations of glutamine,
glutamate, and GSH/GSSG in the soleus muscle and GSH and GSH/GSSG in the liver were
observed in the DIP-TR and GLN ⫹ALA-TR groups. The DIP-LD and GLN ⫹ALA-LD groups
exhibited higher concentrations of GSH and GSH/GSSG in the soleus muscle and liver compared
with the CON-LD group.
Conclusion: Chronic oral administration of DIP and free GLN ⫹ALA before long-duration
exercise represents an effective source of glutamine and glutamate, which may increase muscle and
liver stores of GSH and improve the redox state of the cell. © 2009 Published by Elsevier Inc.
Keywords: Glutamine; Alanine; L-alanyl-L-glutamine; Glutathione; Long-duration exercise
Introduction
The use of glutamine as a nutritional supplement is well
known within the field of clinical nutrition. However, inter-
est in sports-related glutamine supplementation appears to
have decreased, probably because studies with substantial
amounts of exogenous glutamine in athletes did not appear
to enhance aspects of immune function or performance [1].
Glutamine is classified as a non-essential amino acid, be-
cause it can be synthesized by the body [2]. Further, glu-
tamine is the most abundant free amino acid in the human
body [3] and plays an essential role in promoting and
maintaining the functions of various organs and cells, in-
cluding cellular proliferation, acid–base balance, transport
of ammonia between tissues, and antioxidant synthesis
[2,4,5]. However, studies have demonstrated that in some
catabolic situations, such as prolonged starvation, sepsis,
and long-duration physical exercise, glutamine deficiency
can occur [6–9].
A decreased availability of glutamine may depress the
synthesis of several key molecules, such as the tripeptide
␥
-L-glutamyl-L-cysteinylglycine (GSH), which is involved
This work was supported by the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior and the Fundação de Amparo à Pesquisa do
Estado de São Paulo (process 05/59003-2).
* Corresponding author. Tel.: ⫹55-11-3091-3309; fax: ⫹55-11-3815-
4410.
E-mail address: tirapegu@usp.br (J. Tirapegui).
Nutrition 25 (2009) 428–435
www.nutritionjrnl.com
0899-9007/09/$ – see front matter © 2009 Published by Elsevier Inc.
doi:10.1016/j.nut.2008.09.014
in cellular resistance to lesions, oxidative stress, apoptotic
processes, and detoxification of xenobiotics [3,10,11]. More-
over, GSH is the most abundant antioxidant inside the cell, and
the most important non-enzymatic antioxidant in the body,
reacting directly with the reactive oxygen species as an elec-
tron donator for peroxide reduction [3,12]. Experimental evi-
dence has shown that high reactive oxygen species synthesis
may promote lipid peroxidation, DNA lesions, and oxidation
of essential proteins [12,13].
Supplementation with glutamine may serve as an al-
ternative way to increase bodily stores of GSH and at-
tenuate the oxidative stress that occurs in catabolic situ-
ations [14,15]. Administration of oral free L-glutamine to
humans or animal models has shown minimal effects in
the maintenance of physiologic levels of plasma glu-
tamine [16–21]. The use of glutamine dipeptides such as
L-alanyl-L-glutamine (DIP) has provided an alternative
non-invasive way to increase the concentration of glu-
tamine [20–22]. The greater efficiency of supplementa-
tion rendered by DIP is a result of a more hydrolytically
stable molecular structure and mitigated use by entero-
cytes [23]. Our primary hypothesis was that L-glutamine
can attenuate the depression in GSH concentration and
this may reduce the oxidative stress induced by long-
duration exercise. However, most studies have not tested
solutions containing the same amino acids in the same
quantities as DIP. Thus, this study investigated the effects
of supplementation with DIP and a free L-glutamine/L-alanine
(GLN ⫹ALA) solution on plasma and tissue glutamine and
glutamate levels and the tissue status of reduced and oxi-
dized GSH (GSSG).
Materials and methods
Animals and diet
Thirty-six adult male Wistar rats with average weight
(229 ⫾16 g) were provided by the animal house of the
University of São Paulo for use in this study. The animals
were housed in individual cages in a controlled environment
at 22 ⫾2°C and relative air humidity of 60 ⫾10% under
a 12-h light/12-h dark cycle (lights off from 0700 to 1900 h)
for a period of 6 wk. All animals were allowed to adapt to
the experimental conditions for 1 wk before the beginning
of the experimental protocol. Throughout the experiment,
animals had free access to water and an ad libitum diet,
prepared according to the 1993 recommendations of the
American Institute of Nutrition for adult rats (AIN-93) [24].
All animal procedures were approved by the ethics com-
mittee on animal experimentation of the University of São
Paulo. Weight and food intake were monitored three times
per week, and the final weight was determined immediately
before sacrifice in all groups. The method of sacrifice was
decapitation.
Supplementation
Animals were supplemented with DIP (1.5 g/kg of body
weight per day), manufactured by Cláris, Pharmaceutical Prod-
ucts of Brazil Ltd. (São Paulo, Brazil) and distributed by
Fórmula Medicinal Ltd. (São Paulo, Brazil), or free L-glu-
tamine (1 g/kg of body weight per day) and free L-alanine
(0.67 g/kg of body weight per day; GLN ⫹ALA). Both free
amino acids were supplied by Ajinomoto Interamerican Indus-
try and Commerce Ltd. (São Paulo, Brazil). The animals re-
ceived supplementations through gavage for a period of 21 d
before sacrifice. The amount of DIP was calculated such that
the total amount of glutamine was the same as that of glu-
tamine administered in its free form (1 g of glutamine/kg of
body weight per day). The amount of glutamine was chosen
because studies have found effects with this dosage on plasma
and tissue glutamine concentration [20,21]. Control (CON)
animals received water at the same volume by gavage. Ani-
mals were divided into groups of similar mean body weight 1 d
before the initiation of nutritional intervention.
Training protocol and long-duration exercise
The training protocol has been described by Rogero et al.
[20]. Two days after the last session of training, the animals
were subjected to2hofexercise, twice as long as the maxi-
mum time of the training protocol. The tail weight in the
long-duration exercise was the same as that of the last exercise
session. All the sessions and long-duration exercise were in the
dark cycle. Animals were subdivided into groups TR and LD
of similar mean body weight 1 d before the initiation of
nutritional intervention. The sacrifice of each animal subjected
to long-duration exercise (LD) was concomitant to the sacrifice
of an animal of its respective group not subjected to long-
duration exercise (TR). To exclude the influence of the food
variables, all animals of the TR groups were fasted. All ani-
mals were sacrificed between 1040 and 1440 h.
Biochemical analysis
After sacrifice by decapitation, blood was collected and
centrifuged for plasma and serum separation, which were
stored at ⫺80°C for subsequent determination of glutamine,
glutamate, ammonia, and glucose concentrations. Immediately
after death, the liver and gastrocnemius and soleus muscles
were removed and frozen in liquid nitrogen for subsequent
determination of protein, glutamine, glutamate, GSH, and
GSSG concentrations.
Plasma glutamine and glutamate concentrations were deter-
mined with a commercial kit (Sigma-Aldrich Diagnostics Inc.,
Saint Louis, MO, USA) as described by Lund [25], and plasma
ammonia concentration was measured with a commercial kit
(Raichem Diagnostics, San Diego, CA, USA) as described by
Neeley and Phillipson [26]. Plasma glucose was measured
using a commercial kit obtained from Labtest (São Paulo,
Brazil), as described by Bergmeyer [27].
429V. F. Cruzat and J. Tirapegui / Nutrition 25 (2009) 428 –435
Tissue glutamine and glutamate was extracted as described by
Sahlin et al. [28] and was determined with a commercial kit
(Sigma-Aldrich Diagnostics Inc.) as described by Lund [25].
Mean values are reported as micromoles of glutamine per gram of
fresh tissue and as nanomoles of glutamine per milligram of
protein. Muscle and liver protein concentrations were determined
according to the method of Lowry et al. [29].
Muscle and liver GSH and GSSG concentrations were deter-
mined according to the method of Nogueira et al. [30]. A standard
Shimadzu high-performance liquid chromatographic system (Shi-
madzu Corp.,Toquio, Japan) equipped with a Shim-Pack CLC-
NH
2
(6.0 ⫻150 mm) column (Shimadzu Corp.) was used for
analysis, and retention times were determined according to stan-
dard curves of GSH and GSSG.
Statistical analyses
Results were subjected to multivariate analysis of variance to
track type I errors through an array of univariate tests. Compari-
sons of nutritional treatment and measuring time were performed
in series. One-way analysis of variance was conducted at each of
the two time points, with the nutritional treatment as an indepen-
dent variable. Whenever the analysis of variance resulted in a P
value less than 0.05, statistically significant differences were iden-
tified by the multiple comparison of Tukey’s procedure (honestly
significant difference). The comparisons between the time points
for each nutritional treatment were conducted with ttests. All tests
were processed by SAS 9.1.3 (SAS Institute, Cary, NC, USA).
Results
Food intake (CON-TR 20.9 ⫾1.5 g/d, DIP-TR 20.9 ⫾0.9
g/d, GLN ⫹ALA-TR 20.5 ⫾1.5 g/d, CON-LD 20.1 ⫾0.8
g/d, DIP-LD 20.6 ⫾1.0 g/d, GLN ⫹ALA-LD 21.0 ⫾0.7 g/d)
and body weight at the end of the experiment (CON-TR 317.1 ⫾
16.2 g, DIP-TR 305.0 ⫾14.5 g, GLN ⫹ALA-TR 307.1 ⫾
16.3 g, CON-LD 307.5 ⫾14.6 g, DIP-LD 311.1 ⫾19.3 g,
GLN ⫹ALA-LD 308.2 ⫾11.1 g) did not differ across groups.
The tail weight across groups subjected to the long-duration
exercise session was the same (6% of body weight).
Plasma parameters
Plasma glutamine concentrations were higher in the DIP-TR
and GLN ⫹ALA-TR groups compared with the CON-TR group
(Table 1). After long-duration exercise, plasma ammonia levels
were higher in the CON-LD and DIP-LD groups compared with
the CON-TR and DIP-TR groups, respectively (Table 1). The
DIP-LD and GLN ⫹ALA-LD groups had lower plasma ammo-
nia concentrations compared with the CON-LD group. There was
no significant difference in plasma glutamate and glucose concen-
trations among groups due to nutritional intervention or long-
duration exercise.
Tissue glutamine and glutamate
In the soleus muscle higher glutamine concentration and
glutamine/protein were found in the supplemented groups,
DIP-TR and GLN ⫹ALA-TR, compared with the CON-TR
group (Table 2). In addition, soleus muscle glutamate concen-
trations were higher in the DIP-TR and GLN ⫹ALA-TR
groups compared with the CON-TR. The DIP-TR group had
higher glutamine and glutamate concentrations in the gastroc-
nemius muscle than the CON-TR group (Table 2). In the liver,
glutamate concentrations were higher in the DIP-TR group
than in the CON-TR group. Supplementation with DIP in the
Table 1
Concentrations of plasma ammonia, glutamine, glutamate, and glucose*
CON DIP GLN ⫹ALA P
Plasma ammonia (
mol/mL)
TR 5.79 ⫾1.47
a
4.87 ⫾0.78
a
6.26 ⫾2.09
a
0.314
LD 11.76 ⫾0.59
a†
6.94 ⫾0.60
b†
6.22 ⫾0.66
b
0.001
% 103.09 42.28 ⫺0.64
Plasma glutamine (mmol/L)
TR 0.41 ⫾0.10
a
0.61 ⫾0.11
b
0.64 ⫾0.16
b
0.040
LD 0.69 ⫾0.38
a
0.75 ⫾0.33
a
0.60 ⫾0.32
a
0.743
% 66.81 22.95 ⫺6.79
Plasma glutamate (mmol/L)
TR 0.29 ⫾0.04
a
0.26 ⫾0.05
a
0.33 ⫾0.09
a
0.193
LD 0.31 ⫾0.08
a
0.28 ⫾0.08
a
0.23 ⫾0.06
a†
0.184
% 7.86 6.00 ⫺32.56
Plasma glucose (mg/dL)
TR 96.06 ⫾7.61
a
90.58 ⫾8.96
a
94.66 ⫾8.11
a
0.505
LD 99.87 ⫾4.45
a
92.52 ⫾9.25
a
89.14 ⫾6.59
a
0.011
% 3.97 2.14 ⫺5.83
CON, control; DIP, dipeptide; GLN ⫹ALA, free L-glutamine and L-alanine; LD, long-duration exercise; TR, trained; %, difference between TR rats
and LD rats ([LD – TR]/TR)] ⫻100
* Results presented as mean ⫾SD (n⫽6 per group). Values in the same row with different letters are significantly different (Tukey’s honestly significant difference).
†
Significantly different between TR and LD groups (P⬍0.05, ttest).
430 V. F. Cruzat and J. Tirapegui / Nutrition 25 (2009) 428 –435
DIP-TR group increased the concentration of glutamate in the
gastrocnemius and liver compared with the GLN ⫹ALA-TR
group (Table 2).
After long-duration exercise, gastrocnemius and soleus muscle
glutamine and glutamate concentrations were higher in the
DIP-LD and GLN ⫹ALA-LD groups than in the CON-LD
group (Table 2). Higher concentrations of glutamine/protein in the
gastrocnemius and soleus muscles were found in the DIP-LD and
GLN ⫹ALA-LD groups than in the CON-LD group. Lower
glutamate concentrations in the gastrocnemius and soleus muscles
were found in the CON-LD group compared with the CON-TR
group. Long-duration exercise resulted in lower concentrations of
glutamate in the gastrocnemius muscle of the CON-LD and
DIP-LD groups compared with the CON-TR and DIP-TR,
groups, respectively. Glutamine in the liver (milligrams of fresh
tissue and nanomoles of glutamine/protein) was lower in the
CON-LD and GLN ⫹ALA-LD groups than in the CON-TR and
GLN ⫹ALA-TR groups, respectively (Table 2). The DIP-LD
group had a higher glutamine concentration in the liver than the
CON-LD and GLN ⫹ALA-LD groups. All groups subjected to
long-duration exercise (LD) had lower glutamate concentrations
in the liver compared with their respective groups maintained at
rest (TR, P⬍0.05; Table 2).
Tissues GSH status
As presented in Table 3, soleus muscle GSH concentrations
and GSH/GSSG values were higher in the DIP-TR group
compared with the CON-TR group. Soleus muscle GSH/
GSSG values were also higher in the GLN ⫹ALA-TR group
compared with the CON-TR group. Gastrocnemius muscle
GSH concentrations and GSH/GSSG were higher in the DIP-
Table 2
Skeletal muscle and liver concentrations of glutamine and glutamate*
CON DIP GLN ⫹ALA P
Soleus muscle glutamine (
mol/g fresh tissue)
TR 4.63 ⫾0.62
a
6.37 ⫾0.73
b
8.68 ⫾0.77
c
0.001
LD 4.27 ⫾0.48
a
7.95 ⫾0.26
b†
8.23 ⫾0.88
b
0.001
%⫺7.88 29.24 ⫺8.47
Soleus muscle glutamine (nmol/mg protein)
TR 28.8 ⫾3.8
a
40.3 ⫾1.9
b
52.1 ⫾6.9
c
0.001
LD 28.4 ⫾5.9
a
46.5 ⫾6.5
b
45.1 ⫾6.4
b
0.001
%⫺1.6 15.3 ⫺13.5
Soleus muscle glutamate (
mol/g fresh tissue)
TR 1.37 ⫾0.06
a
2.71 ⫾0.27
b
2.57 ⫾0.05
b
0.001
LD 0.95 ⫾0.17
a†
2.38 ⫾0.22
b
2.42 ⫾0.45
b
0.001
%⫺31.12 ⫺7.45 ⫺10.62
Gastrocnemius muscle glutamine (
mol/g fresh tissue)
TR 2.29 ⫾0.38
a
3.16 ⫾0.31
b
2.67 ⫾0.63
ab
0.018
LD 2.48 ⫾0.65
a
3.34 ⫾0.56
b
3.01 ⫾0.44
b
0.050
% 8.50 5.91 12.53
Gastrocnemius muscle glutamine (nmol/mg protein)
TR 16.4 ⫾2.8
a
17.6 ⫾7.3
a
17.1 ⫾5.0
a
0.919
LD 16.9 ⫾4.8
a
20.9 ⫾7.3
b
22.3 ⫾4.4
b
0.050
% 3.6 18.5 30.1
Gastrocnemius muscle glutamate (
mol/g fresh tissue)
TR 0.53 ⫾0.02
a
0.74 ⫾0.03
b
0.49 ⫾0.03
a
0.001
LD 0.38 ⫾0.04
a†
0.65 ⫾0.06
b†
0.59 ⫾0.01
b†
0.001
%⫺28.64 ⫺20.67 34.76
Liver glutamine (
mol/g fresh tissue)
TR 2.84 ⫾0.48
a
3.18 ⫾0.56
a
3.05 ⫾0.46
a
0.519
LD 2.16 ⫾0.44
a†
3.11 ⫾0.44
b
2.17 ⫾0.46
a†
0.003
%⫺23.81 ⫺2.25 ⫺28.67
Liver glutamine (nmol/mg protein)
TR 17.0 ⫾2.9
a
19.6 ⫾4.9
a
18.7 ⫾3.5
a
0.521
LD 12.3 ⫾2.1
a†
15.6 ⫾3.1
a
12.5 ⫾2.1
a†
0.069
%⫺27.5 ⫺20.4 ⫺33.4
Liver glutamate (
mol/g fresh tissue)
TR 1.37 ⫾0.13
a
2.18 ⫾0.05
b
1.56 ⫾0.18
a
0.001
LD 1.73 ⫾0.18
a†
1.92 ⫾0.11
a†
1.88 ⫾0.08
a†
0.053
%⫺26.47 ⫺11.76 20.55
CON, control; DIP, dipeptide; GLN ⫹ALA, free L-glutamine and L-alanine; LD, long-duration exercise; TR, trained; %, difference between TR rats
and LD rats ([LD ⫺TR]/TR)] ⫻100
* Results presented as mean ⫾SD (n⫽6 per group). Values in the same row with different letters are significantly different (Tukey’s honestly significant
difference).
†
Significantly different between TR and LD groups (P⬍0.05, ttest).
431V. F. Cruzat and J. Tirapegui / Nutrition 25 (2009) 428 –435
supplemented group compared with the CON-TR and GLN ⫹
ALA-TR groups. In the liver, GSH concentrations and GSH/
GSSG were higher in the DIP-TR and GLN ⫹ALA-TR
groups compared with the CON-TR group.
Table 3 also demonstrates that after long-duration exer-
cise, the soleus muscle of the DIP-LD and GLN-ALA-LD
groups had higher GSH concentrations and GSH/GSSG
values than the CON-LD group. In the gastrocnemius mus-
cle, higher GSH concentrations were observed in the DIP-LD
group than the CON-LD group. Long-duration exercise re-
duced the concentration of GSH in the CON-LD group com-
pared with the CON-TR group. In the liver, the GSH concen-
trations and the GSH/GSSG were higher in the DIP-LD and
GLN ⫹ALA-LD groups compared with the CON-LD group.
Liver GSH concentrations were lower in the CON-LD group
than in the CON-TR group. Furthermore, livers of the GLN ⫹
ALA-LD group had higher GSH/GSSG values than livers of
the GLN ⫹ALA-TR group (Table 3).
Discussion
This research demonstrates that chronic oral supplemen-
tation with free L-glutamine or DIP administered before
long-duration exercise is an effective method to provide
glutamine to rats.
The measured effect on plasma and muscle glutamine or glu-
tamate for groups that received free L-glutamine may be attributed
to the addition of free L-alanine to the solution. In similar studies,
despite administering glutamine amounts equivalent to those in
the present study, solutions containing the same amino acids in the
same quantities among groups were not tested [20–22]. The
addition of free L-alanine to the solution likely caused a rapid
increase in the plasma concentration, because its transport
through the intestinal epithelium cell occurs preferentially by a
transporter [31]. Studies evaluating the transport of L-alanine
in intestinal epithelial cells have demonstrated that its absorp-
tion can be reduced in the presence of other neutral amino
acids. However, L-glutamine was not included among these
amino acids [32]. The exact method of oral administration of
L-glutamine is an important factor in determining glutamine
bioavailability. In addition, our results suggest that L-alanine
may also have contributed to the effect of the DIP on glutamine
availability found in our and other studies [20–22].
The chronic oral supplementation of L-glutamine in the
L-alanyl-glutamine form administered before long-duration
exercise represents an efficient means to provide glutamine
to rats. This has been previously demonstrated in our labo-
ratory in studies conducted in sedentary rats subjected to
intense exercise [20,21]. It has also been shown by other
researchers in studies done in humans under conditions of
high-protein catabolism [22,33]. The effects of the utiliza-
Table 3
Skeletal muscle and liver concentrations of GSH and rate of GSH/GSSG*
CON DIP GLN ⫹ALA P
Soleus muscle GSH (
mol/g fresh tissue)
TR 0.27 ⫾0.05
a
0.45 ⫾0.08
b
0.32 ⫾0.06
a
0.001
LD 0.29 ⫾0.10
a
0.55 ⫾0.08
b
0.52 ⫾0.08
b†
0.001
% 6.57 20.59 63.22
Soleus muscle GSH/GSSG
TR 6.35 ⫾0.42
a
10.52 ⫾0.89
b
9.75 ⫾0.38
b
0.001
LD 6.67 ⫾0.50
a
9.50 ⫾1.09
b
10.29 ⫾1.03
b
0.001
% 5.06 ⫺2.24 ⫺2.55
Gastrocnemius muscle GSH (
mol/g fresh tissue)
TR 0.24 ⫾0.01
a
0.39 ⫾0.07
b
0.28 ⫾0.05
a
0.008
LD 0.18 ⫾0.04
a†
0.31 ⫾0.05
b
0.22 ⫾0.02
a
0.005
%⫺24.33 ⫺20.15 ⫺20.21
Gastrocnemius muscle GSH/GSSG
TR 6.18 ⫾0.40
a
8.65 ⫾1.78
b
5.08 ⫾1.29
a
0.009
LD 5.57 ⫾1.26
a
6.44 ⫾1.65
a
6.20 ⫾1.65
a
0.699
%⫺9.81 ⫺25.62 22.15
Liver GSH (
mol/g fresh tissue)
TR 0.65 ⫾0.05
a
0.89 ⫾0.08
b
0.90 ⫾0.07
b
0.001
LD 0.54 ⫾0.09
a†
0.94 ⫾0.06
b
0.88 ⫾0.02
b
0.001
%⫺17.96 6.06 ⫺2.17
Liver GSH/GSSG
TR 6.43 ⫾0.57
a
9.33 ⫾0.66
b
7.50 ⫾0.14
c
0.001
LD 6.59 ⫾0.90
a
9.19 ⫾0.70
b
9.01 ⫾1.39
b†
0.001
% 2.54 ⫺1.48 20.13
CON, control; DIP, dipeptide; GSH, reduced glutathione; GLN ⫹ALA, free L-glutamine and L-alanine; GSSG, oxidized glutathione; LD, long-duration
exercise; TR, trained; %, difference between TR rats and LD rats ([LD ⫺TR]/TR)] ⫻100
* Results presented as mean ⫾SD (n⫽6 per group). Values in the same row with different letters are significantly different (Tukey’s honestly significant
difference).
†
Significantly different between TR and LD groups (P⬍0.05, ttest).
432 V. F. Cruzat and J. Tirapegui / Nutrition 25 (2009) 428 –435
tion of L-glutamine in the form of DIP on glutamine avail-
ability has been attributed to the fact that enterocytes have
a more efficient transport mechanism for the absorption of
dipeptides and tripeptides than for the absorption of free
amino acids [34,35]. The glycopeptides transport protein
(Pept-1), which is located exclusively in the luminal mem-
brane, has broad substrate specificity and actively transports
dipeptides and tripeptides in the intestines of humans and
animals [23,36]. Research utilizing radioactively labeled
glutamine dipeptides has shown that nearly 90% of the
radioactivity accumulates intact in the cytosol [33,37].In
this manner, glutamine can avoid intracellular hydrolysis
and subsequent metabolism by enterocytes, proceeding di-
rectly to systemic circulation [23,20].
The groups not subjected to long-duration exercise and
supplemented with DIP or GLN ⫹ALA had higher plasma
glutamine concentrations compared with the CON-TR
group. This effect may be attributed to the higher concen-
tration of glutamine found in the soleus muscle of the
DIP-TR and GLN ⫹ALA-TR groups and the gastrocne-
mius of the DIP-TR group. Similar results, using DIP, were
observed by Rogero et al. [20] in rats subjected to intense
exercise. The skeletal muscle is the primary site involved in
the synthesis, storage, and delivery of glutamine. Because
skeletal muscle stores more than 60% of total free glutamine
in rats, its intracellular metabolism can influence glutamine
concentrations in the plasma [2]. The concentration of glu-
tamine in the plasma, however, does not correlate with the
concentration in tissues, even under elevated muscular cata-
bolic conditions [38]. In our study, the DIP-TR and GLN ⫹
ALA-TR groups exhibited an increase in concentrations of
glutamine and glutamate in the soleus muscle, without a con-
comitant increase in plasma concentrations.
Among the stress-related factors promoted by prolonged
training is the production of plasma ammonia, combined
with the deamination of purines, and catabolism of amino
acids inside myofibrils [39]. The animals in the CON-LD
group displayed hyperammonemia in comparison with the
concentration in the CON-TR group. The same effect was
found in the DIP-TR group, an observation that may suggest
that no beneficial effect resulted from the administration of
DIP in the presence of high levels of ammonia induced by
long-duration exercise. However, comparisons between the
CON and supplemented groups showed that GLN ⫹ALA
or DIP supplementations reduced the levels of ammonia in
the DIP-LD and GLN ⫹ALA-LD groups compared with
the CON-LD group. A similar effect of supplementation
with L-glutamine on the reduction of plasma ammonia lev-
els was found in human athletes [40]. Supplementation with
glutamine in the free form or in DIP at levels equivalent to
1 g of glutamine per kilogram per day is not regarded as low
dose [41,42]. Although higher levels of supplementation of
amino acids can cause effects such as toxic hyperammone-
mia, these effects were not detected in the present study.
Before and after long-duration exercise, higher concen-
trations of glutamine and glutamate in the soleus muscle
were observed in both supplemented groups. The effects of
the supplementation were also found in the concentration of
glutamine and glutamate of gastrocnemius muscle after
long-duration exercise. These results suggest that the am-
monia produced by the long-duration exercise served as a
substrate for the synthesis of glutamine by the action of the
glutamine synthetase enzyme [2]. In this manner, the effect
of both supplementations not only decreased the production
of ammonia that is induced by long-duration exercise but
also increased the availability of muscular glutamine. In-
tense and prolonged physical exercise and exhaustive or
high frequency training promote a reduction in the synthesis
and storage of glutamine, which decreases the availability of
the amino acid [6,7,17,43].
The effect of chronic oral supplementation with L-glutamine in
the free form or as DIP on the synthesis of GSH, the main
non-enzymatic antioxidant of the organism, was also evalu-
ated. In our study, the DIP-TR and DIP-LD groups had higher
GSH concentrations in the soleus and gastrocnemius muscles
and higher GSH/GSSG in the soleus muscle than the CON-TR
and CON-LD groups. This may be due, in part, to an increase
in tissue glutamine and glutamate concentrations. Experimen-
tal evidence has indicated that higher glutamine availability, by
means of parenteral supplementation in humans subjected to
metabolic stress events (abdominal region surgeries), decreases
muscular depletion of GSH, which improves patient recovery
[14]. After trauma or catabolic situations, muscle concentra-
tions of glutamine and glutamate are reduced, whereas the
other amino acids that comprise GSH (i.e., cystine and glycine)
remain at relatively constant levels [5,10,15].
The higher glutamate concentrations provide sufficient sub-
strate for the enzyme
␥
-glutamylcysteine synthetase, which is
the first regulated step in the synthesis of GSH. However,
although glutamate in high concentrations is considered neu-
rotoxic [13], the supplementation with glutamine, from the use
of DIP or a solution containing GLN ⫹ALA, is an efficient
means to increase the availability of glutamate for the synthesis
of GSH.
In the liver, the concentration of GSH and the cellular
redox state (GSH/GSSG) of the supplemented groups, sub-
jected or not to long-duration exercise, were higher than
those in the control groups of the study. This effect is
particularly important, because the liver is the primary or-
gan for the de novo synthesis of GSH and is responsible for
supplying 90% of circulating GSH in the human body
[12,15]. During prolonged physical exercise, the liver ex-
ports GSH to the plasma under the influence of several hormones
stimulated by cyclic adenosine monophosphate, including gluca-
gon, vasopressin, and catecholamines [13,44], whereas the skele-
tal muscle tissue is responsible for the elevated concentrations of
plasma GSH [13,45].
However, only the DIP-TR and DIP-LD groups showed
a relation between liver GSH concentration and glutamine
and glutamate availability in the same tissue, which indi-
cates that several mechanisms take part in the synthesis of
GSH. It is hypothesized that changes in the cell volume,
433V. F. Cruzat and J. Tirapegui / Nutrition 25 (2009) 428 –435
induced by an increased influx of sodium ions caused by an
increase in the transport of glutamine to the intracellular
medium, can favorably influence protein turnover by in-
creasing or maintaining the availability of substrates for the
synthesis of compounds such as GSH [46]. Other hypotheses
are related to an increase in liver concentrations of adenosine
triphosphate induced by glutamine [45]. Decreased hepatic
adenosine triphosphate levels after stresses have been corre-
lated with intracellular damage, which can lead to cell injury
and death [47].
Conclusion
The present results indicate that when administered be-
fore long-duration exercise, oral supplementation with L-
glutamine in the dipeptide form (DIP) or in the free form
associated with L-alanine represents an effective supple-
mentation to provide glutamine and glutamate to rats, which
increases muscle and liver stores of GSH and improves the
redox state of the cell.
Acknowledgments
The authors thank Ajinomoto do Brasil for the donation
of purified amino acids L-alanine and L-glutamine, Formula
Medicinal for the donation of DIP, and José Maria de
Souza, João Ezequiel de Oliveira, Suzana da Rocha Heller
and Carlos Roberto Jorge Soares (Centro de Biologia Mo-
lecular, do Instituto de Pesquisas Energéticas e Nucleares—
IPEN) and Margareth Braga and Érica Welise (Genese
Produtos Farmacêuticos).
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