The effect of BCAA supplementation upon immune response of triathletes

Abstract

Intense long-duration exercise could lead to immune suppression through a decrease in the circulating level of plasma glutamine. The decrease in plasma glutamine concentration as a consequence of intense long-duration exercise was reversed, in some cases, by supplementing the diet of the athletes with branched-chain amino acids (BCAA). To better address this question, we have evaluated some blood parameters (lymphocyte proliferation, the level of plasma cytokines, plasma glutamine concentration, and in vitro production of cytokines by peripheral blood lymphocytes) before and after the São Paulo International Triathlon, as well as the incidence of symptoms of infections between the groups.
Twelve elite male triathletes of mean age 25.5 +/- 3.2 yr (ranging from 21.4 to 30.1 yr), weighing 74.16 +/- 3.9 kg, swam 1.5 km, cycled 40 km, and ran 10 km (Olympic triathlon) in the São Paulo International Triathlon held in April 1997 and April 1998. In both events, six athletes received BCAA and the others, placebo. RESULTs: Athletes from the BCAA group (BG) presented the same levels of plasma glutamine, before and after the trial, whereas those from the placebo group showed a reduction of 22.8% in plasma glutamine concentration after the competition. Changes in the proliferative response of peripheral blood lymphocytes were accompanied by a reduction in IL-1 production after exercise (22.2%), which was reversed by BCAA supplementation (20.3%), without changes in IL-2 production.
The data obtained show that BCAA supplementation can reverse the reduction in serum glutamine concentration observed after prolonged intense exercise such as an Olympic triathlon. The decrease in plasma glutamine concentration is paralleled by an increased incidence of symptoms of infections that results in augmented proliferative response of lymphocytes cultivated in the absence of mitogens. The prevention of the lowering of plasma glutamine concentration allows an increased response of lymphocytes to ConA and LPS, as well as an increased production of IL-1 and 2, TNF-alpha, and IFN-gamma, possibly linked to the lower incidence of symptoms of infection (33.84%) reported by the supplemented athletes.

Full text

BASIC SCIENCES
Original Investigations
The effect of BCAA supplementation upon
the immune response of triathletes
REINALDO A. BASSIT, LETICIA A. SAWADA, REURY FRANK P. BACURAU, FRANCISCO NAVARRO, and
LUI´S FERNANDO B. P. COSTA ROSA
Department of Physiology and Biophysics and Department of Histology and Embryology, Institute of Biomedical Sciences,
University of Sa˜o Paulo, BRAZIL; Department of Biodynamic of the Movement of the Human Body, School of Sport and
Physical Education, University of Sa˜o Paulo, BRAZIL; and Laboratory of Human Nutrition for Athletes - CEPEUSP,
University of Sa˜o Paulo, BRAZIL
ABSTRACT
BASSIT, R. A., L. A. SAWADA, R. F. P. BACURAU, F. NAVARRO, and L. F. B. P. COSTA ROSA. The effect of BCAA
supplementation upon the immune response of triathletes. Med. Sci. Sports Exerc., Vol. 32, No. 7, pp. 1214–1219, 2000. Introduction:
Intense long-duration exercise could lead to immune suppression through a decrease in the circulating level of plasma glutamine. The
decrease in plasma glutamine concentration as a consequence of intense long-duration exercise was reversed, in some cases, by
supplementing the diet of the athletes with branched-chain amino acids (BCAA). To better address this question, we have evaluated
some blood parameters (lymphocyte proliferation, the level of plasma cytokines, plasma glutamine concentration, and in vitro
production of cytokines by peripheral blood lymphocytes) before and after the Sa˜o Paulo International Triathlon, as well as the
incidence of symptoms of infections between the groups. Methods: Twelve elite male triathletes of mean age 25.5 ⫾3.2 yr (ranging
from 21.4 to 30.1 yr), weighing 74.16 ⫾3.9 kg, swam 1.5 km, cycled 40 km, and ran 10 km (Olympic triathlon) in the Sa˜o Paulo
International Triathlon held in April 1997 and April 1998. In both events, six athletes received BCAA and the others, placebo. Results:
Athletes from the BCAA group (BG) presented the same levels of plasma glutamine, before and after the trial, whereas those from the
placebo group showed a reduction of 22.8% in plasma glutamine concentration after the competition. Changes in the proliferative
response of peripheral blood lymphocytes were accompanied by a reduction in IL-1 production after exercise (22.2%), which was
reversed by BCAA supplementation (20.3%), without changes in IL-2 production. Discussion: The data obtained show that BCAA
supplementation can reverse the reduction in serum glutamine concentration observed after prolonged intense exercise such as an
Olympic triathlon. The decrease in plasma glutamine concentration is paralleled by an increased incidence of symptoms of infections
that results in augmented proliferative response of lymphocytes cultivated in the absence of mitogens. The prevention of the lowering
of plasma glutamine concentration allows an increased response of lymphocytes to ConA and LPS, as well as an increased production
of IL-1 and 2, TNF-
␣
, and IFN-
␥
, possibly linked to the lower incidence of symptoms of infection (33.84%) reported by the
supplemented athletes. Key Words: TRIATHLON, IMMUNE SYSTEM, GLUTAMINE, IMMUNOSUPPRESSION, CYTOKINES,
LYMPHOCYTE PROLIFERATION
The triathlon, which comprises sequential swimming,
cycling, and running, has retained the attention of
many researchers involved in an attempt to charac-
terize the sport and its general features (6,15,25). The grow-
ing interest in triathlon is based on the original structure of
the sport itself and in the wide variety of distances offered
to the participants, ranging from sprint to Ironman distance,
lasting about 1 h and 10 h, respectively.
Cardiorespiratory and metabolic responses during Olym-
pic (1.5 km swimming, 40 km cycling, and 10 km running)
and long triathlon events have been extensively studied in
laboratory and field conditions (7,8,14,15,24,30,32), as well
as the changes in the plasma amino acid concentration
(10,19,20). The effect of this type of exercise upon the
immune system has not, however, been vastly investigated.
Rohde and colleagues (31) reported a fall in natural killer-
cell and lymphokine activated killer-cell cytotoxic activity
and a decrease in serum glutamine concentrations 2 h after
the end of a triathlon, but failed to show a correlation
between plasma glutamine concentration and lymphocyte
proliferative response, as previously proposed by Parry-
Billings and coworkers (27,28).
0195-9131/00/3207-1214/0
MEDICINE & SCIENCE IN SPORTS & EXERCISE
®
Copyright © 2000 by the American College of Sports Medicine
Submitted for publication November 1998.
Accepted for publication October 1999.
1214

The modulatory effect of exercise upon the immune sys-
tem is well known. The mechanisms involved, however, are
not fully understood, mainly at the level of high top perfor-
mance (39). Different hypotheses involve the participation
of cytokines and/or hormones as mediators of the effects,
whereas others suggest that plasma glutamine concentration
could be one of the major factors modulating the immune
response to exercise. Several studies have demonstrated that
glutamine is an important substrate for the cells of the
immune system, which utilize this amino acid as a source of
energy and precursors for the synthesis of nucleic acids
(1,2,27). Based on this hypothesis, Keast and colleagues
(17) showed that intense, long-duration exercise could lead
to immune suppression through a decrease in the level of
plasma glutamine.
The decrease of glutamine concentration in plasma as a
consequence of intense long-duration exercise was reversed,
in some cases, by supplementing the diet of the athletes with
branched-chain amino acids (BCAA) (30). However, this
reversal is not fully accepted as a means of restoring im-
mune function in athletes. Rohde and colleagues (30) ques-
tion the statement that maintenance of plasma glutamine
concentration in vivo is able to prevent postexercise immu-
nosuppression. To better address this question, we have
evaluated the effect of a chronic BCAA supplementation
upon the immune system of triathletes after an Olympic
triathlon. We have evaluated some blood parameters (lym-
phocyte proliferation, plasmatic levels of cytokines, plasma
glutamine concentration, and in vitro production of cyto-
kines by peripheral blood lymphocytes) before and after the
Sa˜o Paulo International Triathlon. As a mean of evaluating
the practical relevance of BCAA supplementation upon
athletes health, we addressed, by a questionnaire, the inci-
dence of symptoms of upper respiratory tract between the
groups.
MATERIALS AND METHODS
Subjects and protocol. The experimental protocol
was approved by the local ethics committee, and after sign-
ing an informed consent term, 12 elite male triathletes of
mean age 25.5 ⫾3.2 yr (range 21.4–30.1 yr) swam 1.5 km,
cycled 40 km, and ran 10 km (Olympic triathlon) in the Sa˜o
Paulo International Triathlon held in April 1997 and April
1998.
The athletes were allowed to drink and eat normally but
received BCAA or placebo for 30 d before the competition
and 1 wk after the event. BCAA was given twice a day, after
each training session (6.0 g: 60% L-leucine, 20% L-valine,
and 20% L-isoleucine) during the first 30 d, and a single
dose of 3.0 g 30 min before the triathlon, as well as a single
dose (3.0 g) daily, in the morning, in the first week after the
test were administered. On the day of the competition, blood
samples were collected (20 mL) from the antecubital vein 45
min before the event and 15 min after the race. During the
37-d period, the athletes answered a questionnaire reporting
their health conditions (Table 1).
Incorporation of [2-
14
C]-thymidine into periph-
eral blood lymphocytes. Peripheral blood lymphocytes
were cultured in RPMI-1640 medium for 24 h at 37°C in an
artificially humidified atmosphere of 5% CO
2
in air, under
sterile conditions. The cells were cultured in a LAB-LINE
Microprocessor CO
2
incubator (Lab Line, Melrose Park, IL)
in 96 well plates (Corning, NY), 1 ⫻10
5
cells per well (total
Figure 1—Number of marks in the questionnaires filled by the ath-
letes for 1 month before the competition and 1 wk after. The results are
expressed as percentile and represent mean ⴞSEM of 8 athletes in the
GP and 11 in the GS. *P<0.05 for comparison with the placebo group
(GP, placebo group; GS, supplemented group).
Figure 2—Plasma glutamine level in athletes before and after an
Olympic triathlon. The results are expressed as nmol䡠mL
ⴚ1
and rep-
resent mean ⴞSEM of 8 athletes in the GP and 11 in the GS. *P<0.05
for comparison between the values obtained before and after the trial.
a
P<0.05 for comparison between GP and GS after the trial (GP,
placebo group; GS, supplemented group).
TABLE 1. Table of symptoms to be filled by the athletes during 37 d, 30 d before
and 1 wk after an Olympic triathlon; each mark corresponds to one point.
Symptoms Days
Fever (°C)
Persistent muscle soreness or tenderness (⬎than 8 h)
Pain in the next exercise session
Painful throat
Catarrh in the throat
Itching or sensation of ardour in the throat
Cough
Sneeze
Cephalitis
Coryza
Cold
Grippe
Herpes
Glossitis
Aphtha
Conjunctivitis
Otitis
Mycosis
Infection by Candida
Tendinitis
Articular pain
Abrupt change of humour
Insomnia (how many hour without sleep)
Weakness
Anorexia
BCAA SUPPLEMENTATION AND IMMUNE RESPONSE Medicine & Science in Sports & Exercise姞
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volume, 200
␮
L). After 24 h in culture, more than 98% of
lymphocytes were viable, as measured by Tripan blue ex-
clusion test.
The cells were pulsed with 20
␮
L of 0.02
␮
Ci [2-
14
C]-
thymidine (sp. Act. 56.0 mCi nM-1) diluted in sterile phos-
phate-buffered saline (PBS), yielding a final concentration
of 1
␮
g䡠mL
⫺1
. Cells were then maintained under these
conditions for an additional 15 h and harvested automati-
cally by a multiple cell harvester onto a filter paper (cat. no.
11731 Skatron Combi, Suffolk, U.K.). The paper disks
containing the labeled cells were added to vials containing
in 5 mL of Bray’s scintillation cocktail (60 g䡠L
⫺1
naphtha-
lene, 4 g䡠L
⫺1
2,5-diphenyloxazole (PPO), 20 mg䡠L
⫺1
1,4-
di-[2-(5-phenyloxazolyl)]-benzene - POPOP, 10% methanol
(by vol.) and 2% ethylene glycol (by vol.)) in p-dioxane
(chromatographic grade) and counted in a Beckman-LS
5000TD liquid scintillator ion counter (Beckman Instru-
ments, Fullerton, CA). All the reagents used in the prepa-
ration of the Brays solution were obtained from Sigma (St.
Louis, MO) or Merck (Darmstadt, Germany).
Measurement of plasmatic glutamine concentra-
tion. Plasmatic glutamine concentration was measured en-
zymatically as described by Windmueller and Spaeth (40).
Determination of cytokines concentration. Each
5-mL blood sample was transferred to a glass tube contain-
ing 5
␮
L of heparin (500 IU䡠mL
⫺1
). The tubes were kept on
ice until centrifuging at 2500 rpm for 8 min. The plasma was
stored at ⫺80°C. The concentration of cytokines in plasma
was measured using commercially available ELISA kits
(Amersham Life Science, Clearbrook, IL): interleukin-1
(IL-1), interleukin-2 (IL-2),
␥
-interferon (IFN), and tumor
necrosis factor-
␣
(TNF).
Cytokines produced by cultivated peripheral blood lym-
phocytes were also measured. Lymphocytes were prepared
by centrifuging the blood in the presence of Hystopaque
(1.007) for 15 min at 2500 rpm. The mononuclear cells (⫾
97% lymphocytes) were plated (1.0 ⫻10
6
cells䡠mL
⫺1
) onto
a plastic Petri dish in the presence of phytohemagglutinin
(PHA) 10
␮
g䡠mL
⫺1
to stimulate IL-2, INF, and TNF pro-
duction or lipopolysaccharide (LPS) 10
␮
g䡠mL
⫺1
to stimu-
late IL-1 production. After 48 h, the concentration of the
cytokines was measured in the supernatant.
Statistical analysis. The data obtained in the two
events were compared using paired t-test, and the level of
significance of P⬍0.05 was chosen for all statistical
comparisons. The data are presented as mean ⫾SEM.
RESULTS
Athletes participating in the Olympic triathlon answered
a questionnaire concerning the incidence of infection symp-
toms before (1 month) and after (1 wk) the competition (18;
Table 1). Supplementation with BCAA induced a decrease
in the symptoms of infection reported (33.84%) when the
answers were compared with those of the group receiving
placebo (PG) (Fig. 1).
Athletes from the BCAA group (BG) presented the same
plasma glutamine level, before and after the trial (Fig. 2),
whereas those from the placebo group showed a reduction of
22.8% in plasma glutamine concentration after the compe-
tition (Fig. 2). This change in glutamine concentration was
accompanied by increased lymphocyte proliferation ob-
tained from resting subjects (Table 2). In such athletes, we
observed a systematic decrease in lymphocyte proliferation
that was, however, not significant (Table 2). Lymphocytes
obtained from subjects of the BG, on the other hand, pre-
sented a smaller index of proliferation before the test (re-
duction of 36.2% as compared with PG), which was slightly
increased by 12.6% after the competition (Table 2). The
effects of BCAA supplementation appeared when the cells
were stimulated by mitogens. In PG lymphocytes the pres-
ence of concanavalin A (ConA) or lipopolysaccharide (LPS)
in the culture medium induced an increase in the prolifera-
tive response (79.9% and 41.1%, respectively) that was
amplified in the BG (3.34-fold and 2.35-fold, respectively,
Table 2). We have also observed in the BG triathletes a
Figure 3—Production of interleukin-1 (IL-1) by peripheral blood
mononuclear cells cultivated for 48 h in the presence of phytohemag-
glutinin 10
␮
g (mL), before and after the trial. The results are ex-
pressed as pg䡠mL
ⴚ1
and represent the mean ⴞSEM of 8 samples in the
placebo group (GP) and 11 in the supplemented group (GS). *P<0.05
for comparison with the placebo group.
TABLE 2. Proliferative response of peripheral blood lymphocytes obtained from athletes of the placebo (GP) and supplemented (GP) groups before and after a triathlon; the
results are expressed in DPM (decay per minute) as mean ⫾of 8 athletes in the GP and 11 in the GS.
Control GP Control GS ConA GP ConA GS LPS GP LPS GS
Average 1373.03 878.53* 2470.12 2936.9* 1938.00 2072.70*
Before
SEM 108.82 59.34 56.09 100.16 49.81 48.59
Average 1200.46 989.88* 2556.82 3253.24* 2056.60 2411.18*
After
SEM 57.16 105.06 73.31 197.81 75.04 65.34
*
P
⬍0.05 for comparison with the supplemented group (GS)
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greater lymphocyte proliferative response after the test
when compared with the changes observed in the placebo
group after the triathlon (27.4% and 17.2% for ConA and
LPS, respectively).
These changes in the proliferative response of peripheral
blood lymphocytes were accompanied by changes in the
production of IL-1 and IL-2, TNF-
␣
, and INF-
␥
by cells
cultivated for 48 h in the presence of LPS (IL-1) or PHA
(IL-2, TNF and INF). The athletes from PG presented a
reduction in IL-1 production after exercise (22.2%), which
was reversed by BCAA supplementation (20.3%, Fig. 3).
There were no changes in IL-2 production induced by the
exercise per se (Fig. 4), but we could notice an increase in
the production of this cytokine in the supplemented group
before (47.9%) and after the trial (84.8%, Fig. 4). The
production of TNF and INF presented the same pattern of
changes as that of IL-1. Cells from PG showed, when
harvested after exercise, a decreased production of these
cytokines (35.1% for IFN and 16.9% for TNF, Figs. 5 and
6, respectively). In the cells obtained from BG this decrease
in cytokines production after exercise was abolished (Figs.
5 and 6).
DISCUSSION
Participation in triathlon is continuously growing. This
sport, which began in the mid-1970s, includes three sequen-
tially performed endurance events: swimming, cycling, and
running. The distance of each segment varies substantially,
so that the total time of competition ranges from 30 min to
several hours (25). The physiological effects of a triathlon
have been extensively studied (5,7,8,12,14,19,24,26,35,41).
It is interesting to note that the physiological and energetic
demands of such a sequence of exercise are unique and
require the triathletes to develop a blend of characteristics
seen in endurance swimming, cycling, and running (33). As
a consequence of these elevated demands, triathletes pre-
sented with some side-effects such as glomerular damage
(41) and the presence of oxidized DNA bases in urine (12)
as well as cellular dehydration and a decrease in serum
amino acids, reflecting a catabolic state (19).
The imbalance in plasma amino acids concentration could
be related to the increased risk of upper respiratory tract
infections that follows intense long-duration exercise
(13,23,29) by inducing, in the host, a suppression of natural
killer and lymphokine activated cells activity and lympho-
cyte proliferation (36,37). Glutamine decrease seems to be
the main factor causing immunosuppression, because this
amino acid is essential for lymphocyte and macrophage
metabolism (2,27,30) and its concentration is reduced in the
plasma of athletes after long-term, strenuous exercise
(17,28). Rohde and colleagues (30) showed that after long-
term intense exercise (a triathlon consisting of 2.5 km swim-
ming, 81 km cycling, and 19 km running), there was a
reduction in NK and LAK cell activities paralleled by a
decrease in serum glutamine concentrations.
In this study, we have evaluated the effect of BCAA
supplementation (BG) upon peripheral blood lymphocyte
proliferation, serum glutamine concentration, and the pro-
duction of IL-1, IL-2, TNF-
␣
, and INF-
␥
, as well as by a
questionnaire, in the incidence of infections.
As previously reported (30), the athletes presented reduced
serum glutamine concentration (22.8%) after the triathlon,
which is very similar to the observed by Parry-Billings and
colleagues (28) after a marathon (a reduction of 16%) and by
Rhode and colleagues (30) after another triathlon: 2.5 km
swimming, 81 km cycling, and 19 km running (reduction of
32%). The supplementation of the athletes with BCAA re-
stored serum glutamine concentration to values similar to those
found before the competition. In fact, it is known that supple-
mentation with BCAA is able to increase the circulating levels
of these amino acids and their metabolization to glutamine in
the skeletal muscle (21) leading to a greater muscle NH
3
production (21). NH
3
, in such model, is derived from the
transamination with 2-oxoglutarate, which forms glutamate
Figure 4—Production of interleukin-2 (IL-2) by peripheral blood
mononuclear cells cultivated for 48 h in the presence of phytohemag-
glutinin 10
␮
g (mL), before and after the trial. The results are ex-
pressed as pg䡠mL
ⴚ1
and represent the mean ⴞSEM of 8 samples in the
placebo group (GP) and 11 in the supplemented group (GS). *P<0.05
for comparison with the placebo group.
Figure 5—Production of INF by peripheral blood mononuclear cells
cultivated for 48 h in the presence of phytohemagglutinin 10
␮
g (mL),
before and after the trial. The results are expressed as pg䡠mL
ⴚ1
and
represent the mean ⴞSEM of 8 samples in the placebo group (GP) and
11 in the supplemented group (GS). *P<0.05 for comparison with the
placebo group.
BCAA SUPPLEMENTATION AND IMMUNE RESPONSE Medicine & Science in Sports & Exercise姞
1217

and branched-chain oxo acids, a reaction catalyzed by BCAA
aminotransferase. Glutamate can then be oxidatively deami-
nated by glutamate dehydrogenase, releasing the NH
3
and
reforming 2-oxoglutarate (22). The NH
3
produced in such
pathway, during exercise, is released in the form of glutamine
(21). This mechanism is reinforced by the fact that the rate
limiting step in BCAAs catabolism involves the nonreversible
decarboxylation of the BCOAs by branched-chain oxo acid
dehydrogenase, which is activated during exercise (11,16,38)
and is responsive to an increase in the intracellular concentra-
tion of BCOAs (21).
It is interesting to note that in the athletes of the PG, who
showed a reduction in serum glutamine concentration after the
triathlon, an increased proliferative response of peripheral
blood lymphocytes, harvested before and after the exercise
session, cultivated without mitogens was also observed. These
cells, however, showed a reduced response to ConA and LPS,
mitogens for T and B cells, respectively, when compared with
those obtained from BG athletes, who presented a normal
serum glutamine concentration after the triathlon.
An important aspect of immune response concerns the
production of cytokines by immune cells. This group of
intercellular signaling proteins regulates local and systemic
immune and inflammatory responses, as well as other bio-
logic processes. Their effect often overlap considerably and
one cytokine may induce the secretion of others, producing
a cascade of biological effects. We have evaluated the effect
of BCAA supplementation upon cultivated blood mononu-
clear cells capacity to produce IL-1 and IL-2, TNF, and INF.
IL-1 is expressed during infections, particularly with Gram-
negative bacteria, and activates B and T cells besides stim-
ulating hemopoiesis (3). IL-1 and TNF share most of their
activities, except for the toxic effect of TNF against tumor
cells (4,9). INF is synthesized by T and NK cells in response
to mitogens, antigens, and IL-2 and enhances macrophage
tumoricidal, antimicrobial, and antigen presenting activities
as well as presents antiviral properties (4,34). IL-2 is pro-
duced by activated T lymphocytes and is essential for clonal
T cell proliferation and for NK cytolytic activity. It also
induces the production of IFN, TNF, IL-3, -4, -5, and -6 by
activated lymphocytes.
BCAA supplementation increased IL-1, IL-2, TNF and
INF production in cells cultivated for 48 h in the presence of
LPS or PHA. Cells obtained from the PG showed a reduc-
tion in cytokine production after the competition, except for
IL-2, whose synthesis was not changed after exercise.
BCAA supplementation increased the production of all cy-
tokines before the competition, but only that of IL-1 and
IL-2 after the test (20% and 41%, respectively). The pro-
duction of IFN and TNF by the BG athletes was the same
before and after the triathlon. Considering the importance of
these cytokines in the establishment and control of immune
response, the supplementation of the athletes with BCAA
seems to be important to keep this signaling and effector
mechanism working properly, leading to a reduced inci-
dence of infections in those athletes.
The data show that BCAA supplementation can reverse
the reduction in serum glutamine concentration observed
after prolonged intense exercise such as an Olympic triath-
lon. This change in plasma glutamine concentration is par-
alleled by an increased incidence of infections that results in
augmented proliferative response of lymphocytes cultivated
in the absence of mitogens. The prevention of the lowering
of plasma glutamine concentration allows an increased re-
sponse of lymphocytes to ConA and LPS, as well as an
increased production of IL-1 and 2, TNF, and IFN, possibly
linked to the lower incidence of infection reported by the
supplemented athletes.
We gratefully acknowledge Twinlab
®
for kindly provide the amino
acids for this project. The work was supported by FAPESP 98/
07141–7.
Address for correspondence: Dr. Luı´s F. B. P. Costa Rosa, Depar-
tamento de Histologia e Embriologia, Instituto de Cieˆ ncias Biome´ dicas
I, Universidade de Sa˜ o Paulo, Av. Lineu Prestes, 1524, 05508 –900,
Butanta˜, Sa˜ o Paulo, SP, Brasil; E-mail: ggrosa@icb.usp.br.
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