Acute amino acids supplementation enhances pituitary responsiveness in athletes

Original Investigations

Acute amino acids supplementation enhances pituitary responsiveness in athletes LUIGI DI LUIGI, LAURA GUIDETTI, FABIO PIGOZZI, CARlO BALDARI, ALESSANDRO CASINI, MAURIZIO NORDIO, and FRANCESCO ROMANELLI Endocrinology Unit , Endocrine Research Laboratory, Sports Medicine Unil, University Inslilure of MOlOr Sciences, Rome, iTALY; and Division of Andro!ogy, University of Rome "La Sapienz.a, " Rome, ITALY

ABSTRACT

OJ LUIGI , L., L. GUIDEITI, F. PIGOZZI, C. BALDARI, A. CASINI, M. NORDIO, and F. ROMANELLI. Acute amino acid s supplementation enhances pituitary responsiveness in athletes. Med. Sci. Spons Exere., Vol. 31, No. 12, pp. 1748-1754, 1999. Purpose: The purpose of this sllldy was to determine the effect of a mixture of amino acids on pituitary responsiveness to a stimulation test (GnRH

+ CRH) in athletes. Methods: In a double blinded counterbalanced experimental protocol, 10 moderately trained male

attdetes performed the pituitary stimulation test 60 min after a single oral administration of a placebo (PI-AS) or an amino acid mi xture sollllion (AS) (L-arginine hydrocloride 100 mg'kg- I + L-orni[hine hydrocloride 80 mg'kg- I + L-branched chain amino acids 140 I11g'kg- l : 50% L-Ieucine, 25% L-isoleucine, 25% L-valine) on two different occasions. Plasma ACTH, LH, FSH, GH, and cortisol were evaluated before (-60, -30,0 min) nnd after (+ 15, +30, +45, +60. +90 min) [he stimulation test. Results: The ACTH, LH and FSH response to CRH + GnRH was significantly higher in AS group both as absolute values and area under CUrve (AUC) values than ill PI-AS group. Pre-test and post-test cortisol AUC levels were significantly higher in PI-AS group although a higher percent increase in post-test corti sol was found in AS group. The roral GH-AUC was higher in AS group and, as expected, the absolute GH concentrations at different time points were not influenced by CRH + GnRH admini stration. Conclusion: The amino acid mixture used enhanced the ACTH. LH, and FSH response to CRH

+ GnRH. Key Words: ACTH, CRH, CORTISOL, GnRH, GH , FSH, LH, BCAA,

ARGININE, ORNITHINE

he effects of amino acid supplementations in athletes have been evaluated in different experimental proto­ cols mainly with respect to possible influences on muscle strength and athletic performance (36). It has been posmlated that the beneficial effects of amino acid supple­ mentation on strength and endurance performance result from, respectively, increased protein synthesis and use of amino acids for energy production (3,5,23,24). Since muscle amino acid utilization increases during physical activity, it is thought that amino acid supplemen­ tations might be useful in sport activities. In fact, the use of such supplementations is widely increasing all over the world for competitive and also noncompetitive athletes, such as body builders, and also for nonathletes. Unfortu­ nately, amino acids are usually supplied and administered without medical supervision. Users frequently decide their

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OJ 95-9131 /99/3112-1748/0

MEDlClNE & SCIENCE IN SPORTS & EXERClSE@

Copyrigh[ © 1999 by [he American College of Spons Medicine

Submi[[ed fOJ" publication December 1997.

Accepted for publication October 1998.

own dosages and combinations without consideration of efficacy, nutritional requirements, and, most importantly, short- and long-term health effects. In our opinion, the observed amino acid-linked "positi ve effects" on muscle strength and athletic performance are probably carried out principally through nonclassical meta­ bolic pathways. In particular, we emphasize the influence of several amino acids on hormonal secretion via neuroendo­ crine pathways. For example, the administration of branched chain amino acids (BCAA), i.e ., leucine, isoleu­ cine, and valine, or other amino acids including arginine, lysine, ornithine, histydine, phenylalanine, methionine, in specific dosages and combinations stimulates growth hor­ mone (GB) secretion (27,42). BCAA supplementation also stimulates basal insulin synthesis and secretion and in­ creases insulin-sensitivity without modifying the insulin re­ sponse to acute physical exercise (9,20,25). It has also been found that a single administration of BCAA increases tes­ tosterone and cortisol response to acute physical exercise (9), while glutamic acid can stimulate ACTB-mediated cor­ tisol secretion (41).

1748

Furthermore, we showed in a previous experiment (14) that the chronic administration of an amino acid mixture (BCAA, lysine, arginine, ornithine) in athletes during a training period induced significant increases in basal corti­ sol and testosterone plasma concentrations, and in 24-h urinary cortisol when compared with placebo-treated ath­ letes. However, the relationship between acute and chronic amino acid supplementation and the observed hormonal modifications is not completely clear. We have no reliable information whether the observed amino acid-linked en­ hanced cortisol and testosterone secretions in athletes are influenced by amino acids directly at the adrenal and go­ nadal level and/or indirectly throughout an amino acid­ dependent pituitary "hyper-activity." We focused our attention on the hypothesis that amino acid administration could amplify pituitary responses to specific hypothalamic hormonal stimuli. To evaluate whether amino acids could enhance the pituitary responsiveness to specific releasing hormones in moderately trained athletes, we admin­ istrated a single oral bolus of an amino acid mixture before a pituitary stimulation test featuring the administration of exog­ enous corticotropic (CRR) and gonadotropic (GnRH) releasing hormones in a controlled experimental protocol.

MATERIALS AND METHODS Subjects. A homogeneous group (N = 10) of moder­ ately trained male volunteers (age 27.1 :::':: 1.9 yr; weight 83.2 :::':: 14.3 kg; height 179 :::':: 10 cm; body mass index 25.68 :::':: 2.1; V0 2mux 55.2 :::':: 3.6 mL·kg-'·min- l ) was selected for the study. All subjects participated only in educational and recreational noncompetitive athletic activ­ ity. They were recruited from the University Institute of Motor Sciences in Rome. The experimental protocol was approved by the local scientific and ethical committee. The nature of the study was explained to each subject in detail and written informed consent was obtained. A preliminary screening assessment was designed to de­ tect risk factors that may have contraindicated participation in the study. All subjects were in good health and were taking no medications or other supplementations including anabolic steroids. The volunteers presented normal physical and sexual development. All subjects were counseled by a nutritionist and had the same diet regimen (about 40 kcal per kg of body weight per day: 55% carbohydrates, 30% lipids, 15% proteins) starting 2 wk before and throughout the experimental period. The subjects were counseled weekly, and their food records reviewed to maintain the correct diet regimen throughout the period of the study. The calculated dietary nutrients consumed were considered sufficient for each individual's needs according to recommended dietary allowances for the Italian population (28). Protocols. In the first experimental session each subject performed a pituitary stimulation test (GnRH + CRH) (16) after a single oral administration of a placebo solution or after an amino acid mixture in a double-blinded, counter­ balanced protocol. Then, after a washout period of 1 wk, AMINO ACIDS AND PITUITARY RESPONSIVENESS

each subject performed a second identical stimulation test following consumption of the mixture opposite of that taken for the first test. Each subject was therefore his own control, receiving both amino acids and placebo. Solution composition. The solution composition of the placebo (PI-AS) was 200-mL orange flavored water. The composition of the amino acids solution (AS) was: 200 mL orange flavored water + L~arginine hydrocloride (100 mg'kg- I ) + L-ornithine hydrocloride (80 mg'kg- I ) + L-BCAA (140 mg'kg- I : 50% L-Leucine, 25% L-Isoleucine, 25% L-Valine). To reduce the possible bitter taste, glucose (10 g) was added to both the solutions. Sampling and blood extraction. During each of 2 days that pituitary stimulation testing occurred, blood sample collection started at about 9 a.m., 2 h after breakfast (white milk, 150 mL + toasted bread, 50 g). A stopcock catheter was introduced into a forearm vein 60 min before starting the stimulation test and maintained in situ through­ out the experiment. Blood collections were performed 60 min before (- 60), 30 min before (- 30), immediately before (0), and after (+ 15, +30, +45, +60, +90 min) an iv single bolus of GnRH (l00 J.Lg; Relisorm, Ares-Serono, Milano, Italy) + CRH (l J.Lg·kg -I; hCRH, Calbiochem, Inalco, Milano, Italy) was administered. Immediately after the first blood collection (- 60), subjects drank the solution contain­ ing either placebo or amino acids. After each blood sample was taken, the catheter was flushed with physiological saline to avoid blood clotting. Following blood sample collection, the plasma was sepa­ rated via centrifugation, and stored at -70°C until hormonal assays for LH, FSH, ACTH, GH, and cortisol. Hormonal assays. ACTH was measured in unex­ tracted plasma by radioimmunoassay kits (CIS Bio Interna­ tional, Gif-Sur-Yvette Cedex, France). The sensitivity of the assay was 2 pg·mL- l . The inter- and intra-assay coefficients of variation (%) were 4.8 and 2.9, respectively (N = 10). LH was measured in unextracted plasma by radioimmu­ noassay kits (Ares-Serono Diagnostici, Milano, Italy). The sensitivity of the assay was 0.3 mIU'mL - I . The inter- and intra-assay coefficients of variation (%) were 4.3 and 1.6, respectively (N = 10). FSH was measured in unextracted plasma by radioimmu­ noassay kits (Ares-Serono Diagnostici, Milano, Italy). The sensitivity of the assay was 0.3 mIU'mL -I. The inter- and intra-assay coefficients of variation (%) were 4.9 and 2.2, respectively (N = 10). GH was measured in unextracted plasma by radioimmu­ noassay kits (CIS Bio International, Gif-Sur-Yvette Cedex, France). The sensitivity of the assay was 0.04 ng·mL - I . The inter- and intra-assay coefficients of variation (%) were 4.4 and 2.8, respectively (N = 10). Cortisol was measured in unextracted plasma by radio­ immunoassay kits (Diagnostic System Laboratory, Webster, TX). The sensitivity of the assay was 0.5 J.Lg·dL- I. The inter- and intra-assay coefficients of variation (%) were 8.9 and 5.3, respectively (N = 10). All assays were performed in duplicate. Medicine & Science in Sports & Exercise®

1749

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Figure 1-ACTH AUC and plasma ACTH (mean ::t SD) response to stimulation test (GnRH + CRR) in moderately trained male subjects (N 10) after placebo (PL-AS) or amino acid solution (AS) administration. • P < 0.05 post-test AUC value vs pre-testj "'" P < 0.05 AS vs PL-ASj * P < 0.05 vs basal (0).

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Statistical analysis. The hormonal data are expressed in SI units (means ± SD) either as absolute values or areas under curves (AUC) calculated by trapezoidal integration. After testing the normal distribution of data, to analyze the difference between AS and placebo treatments for each protocol time, the paired Student's I-test was performed. To analyze the response to test stimulation within each AS and placebo treatment, the repeated measures ANOV A with posl-hoc comparison as appropriate was used. P values ::::; 0.05 were considered to be significant.

RESULTS All subjects completed the experimental protocols. No disturbances, adverse reactions, or side effects were ob­ served at any time during the experimental period. All hormonal results are reported in Figures 1-5. In both AS and PI-AS treated subjects the basal prestimulation test hor­ monal values (areas and absolute values from -60 to 0) were in the normal range, and with the exception of cortisol , no significant differences were found either within groups or between treatments. ACTH. The plasma ACTH-AUC and absolute ACTH plasma levels (means ± SD) are shown in Figure I. There was a significant increase of the ACTH-AUC means after the CRH + GnRH test only in AS group (+ 106.8%), whereas in the PI -AS group there was a small, nonsignifi­ cant rise (+ 9.8 %). The mean absolute ACTH values after the CRH test increased significantly in the AS group from + IS to +45 min after the test (maximum +206% at + IS), whereas in the PI-AS group there was a lower but significant increase at + IS and +30 min (maximum +91.9% at + IS). The ACTH response to the stimulation test was significantly higher in the AS group versus the PI-AS group both in terms of total ACTH-AUC (0, +90) (589.2 ± 146.3 pmol'L-I'h- I vs 292.3 ± 91.9 pmol'L- I'h -I, respectively ; P < 0 .05) and in terms of absolute ACTH values at + IS, + 30, +45 and +60 min after CRH+GnRH (P < 0.05 at each time).

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Figure 2-LH AUC and plasma LH (mean ::t SD) response to stimulation test (GnRH + CRH) in moderately trained male subjects (N = 10) after placebo (PL-AS) or amino acid solution (AS) administration. P < 0.05 post· test AUC value vs pre-testj "'" P < 0.05 AS vs PL-ASj " P < 0.05 vs basal (0).

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LH. The plasma LH-AUC and absolute LH plasma levels (means ± SD) are shown in Figure 2. The mean LH-AUC increased significantly after the stimulation test in both the AS and PI-AS groups (+629% and +418.1 %, respectively). The mean absolute LH values increased significantly in both the AS and PI-AS groups (maximum + 790% and + 579% at +30, respectively).The LH response to the stimulation test was significantly higher in the AS group versus the PI-AS group both in terms of total LH-AUC (0,+90) (2197.5 ± 150.3 IU·L-I·h- I vs 1107.5 ± 181 IU·L- I·90 min-I, re­ spectively; P < 0.05) and in terms of absolute LH values at all times from + 15 to + 90 min after CRH + GnRH (P < 0.05 at each time). FSH. The plasma FSH-AUC and absolute FSH plasma levels (means ± SD) are shown in Figure 3. The mean FSH-AUC increased significantly (P < 0.05) after the stim­ ulation test in both AS and PI-AS groups (+ 99 .2 % and +59%, respectively). The mean absolute FSH values in­ creased significantly in both AS and PI-AS groups from +15 to +90 min (maximum +128.8% and + 65.4% at + 45, respectively; P < 0 .05 at all times). The total FSH-AUC (0, +90) after the test was significantly higher in the AS group than in the PI-AS group (1042.9 ± 209.2 IUL -1'90 min-I vs 838.3 ± 147.2 IUL - 1'90 min-I, respectively; P < 0.05); significant differences were found in the abso­ lute FSH values between treatments at + IS and + 45 min (P < 0.05). GH. The total plasma GH-AUC and absolute GH plasma levels (means ± SD) are shown in Figure 4. A significant difference in the total GH-AUC (from -60 to +90 min) between AS and PI-AS groups was observed (GH-AUC [-60, +90]: 165.8 ± 112.3 f.Lg·L -1'150 min-I and 64.6 ± 33.5 f.Lg·L-I·150 min-I, respectively ; P < 0.05) although no significant differences in the absolute GH levels were observed between treatments. As expected, nonsignificant differences were found in partial GH-AUC and absolute GH values before and after the stimulation test in both groups. The evaluation of partial

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The results of the present study indicate that the respon­ siveness of [he pituitary corticotropin and gonadotropin secretory cells to their specific releasing hormones (CRH and GnRH) is enhanced in moderately trained athletes by the ingestion of a supplied mixture of amino acids (L­ arginine + L-ornithine + L-BCAA). In fact the ACTH, LH, and FSH response to CRH + GnRH after the AS ingestion showed significantly higher levels than after PI-AS admin­ istration. Furthermore, in the PI-AS group, the significant AeTH, LH, and FSH increase after the CRH + GnRH stimulati on test was the normal response to this test in endocrine clinical practice (16). The amino acid-dependent enhancement of pituitary responsiveness could be responsi­ ble for the cortisol and testosterone plasma level increases 0bserved after amino acid supplementation in athletes 300

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(9,14). However, further studies are required to evaluate whether amino acid supplementations could also have a direct action at testicular and adrenal levels in addition to the action we found at the pituitary level. The amino acid mixture used in this study was cho sen on the basis of a previous study in which we observed an increase of cortisol and testosterone plasma levels in ath­ letes using a similar amino acid mixture supplementation over 6 wk (14). The amino acid doses we used were also chosen to have an acute amino acid plasma level modifica­ tion to reduce the risk of acute side effects and to diminish compliance and tolerance problems (11). In the present study we were more interested in the effect of an amino acid mixture than in evaluating the effects of a single amino acid because the use of mixtures is an habitual practice among amino acid users. Amino acids and growth hormone. In this experi­ ment we did not evaluate the influence of the amino acid mixture on the response of GH to its specific releasing hormone (GHRH). We only tested the plasma GH concen­ trations at different times to evaluate a possible direct stim­ ulatory effect of amino acids on GH secretion. Interestingly, we found a significantly higher value of total OH-AUC (- 60, +90) in AS treated subjects versus the PI-AS treated group, without any significant increase of absolute OH mean values at different times after amino acids adminis­ tration. The failure to detect significant increases in absolute GH may be related to high inter-indi vidual variability both in the absolute plasma GH values and in the OH response [0 the amino acids administration. In fact, the GH secretion after amino acid administration may be affected by the type of amino acid administered, on its dosage, and on the specific combination of amino acids (27,42). For example, different single oral dosages of arginine are associated with a plasma GH increase (1.2 g6fL-ARG) or with no plasma modifications of such hormone (2.4 g of L-ARG). It has been demonstrated that the combination of Jysine with arginine improves the GH response (2,27). On the

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basis of existing evidence, both iv and oral arginine adminis­ tration per se stimulates GH secretion (31) and amplifies the GH response to the GHRH at all ages (2,21), probably inhib­ iting somatostatin secretion at hypothalamic level (1,22). How­ ever, an inhibitory effect of arginine on endogenous GHRH secretion has also been reported (30). Amino acids and hypothalamic-pituitary-adrenal axis. The stimulation test was able to increase sign.ificantly the absolute ACTH plasma levels in both groups but with significantly higher values in the AS group. The absence of a significant increase of the ACTH-AUC, after the stimulation test, in placebo-treated subjects might be dependent on the presence of high ACTH basal levels at -60 min observed in both groups that were probably related to mental stress. Inter­ estingly, the ACTH response to the stimulation test was asso­ ciated with higher absolute cortisol levels in the placebo­ treated subjects, but AS subjects displayed higher relative (%) plasma cortisol elevation following the stimulation test. Therefore, we observed an amplification of the ACTH response to the stimulation test in AS-treated subjects, but we do not know whether this effect is mediated at pituitary level and/or the hypothalamic level through neuroendocrine pathways which modulate the activity of corticotropin cells. In fact, the intluence of the amino acids on the hypotha­ lamic-pituitary-adrenal axis has never been extensively evaluated. An intravenous infusion of L-ornithine hydro­ chloride, a constituent of the supplement used here, infused over 30 min in 54 fasting children (4-14 yr with constitu­ tional short stature) was able to increase plasma GH, ACTH, and cortisol concentrations (7). Conflicting data regarding the influence of amino acids on CRH and ACTH secretion were described following administration of excitatory amino acids (EAA: L-glutamic acid and L-aspartic acid) (10,12,13,34) or L-tryptophan (26,32,43), an action that at hypothalamus-pituitary level is also mediated by its metab­ olite serotonin (6,40,46). Amino acids and hypothalamic-pituitary-gonadal axis. As with ACTH secretion, gonadotropin secretion was also higher after the CRH + GnRH stimulation test in AS group, indicating an increased responsiveness of pituitary gonadotropin cells in this group. The observed LH and FSH increase in the PI-AS group represented the physiological response to GnRH administration. Unlike cortisol, in the present experimental protocol we wouldn't expect any significant early modification of testos­ terone plasma level. In fact, after the human chorionic gonad­ otropin (hCG) stimulation test, which is a more direct stimulus for testosterone than GnRH, testosterone response is usually evaluated after 48 -96 h from hCG adrnin.istration (19). 1752

Official Journal of the American College of Sports Medicine

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Very little information exists on the influences of amino acids on the hypothalamic-pituitary-gonadal axis. Experi­ mental evidence, in animals and in vitro, showing a GnRH­ mediated EAA stimulating action on gonadotropin secretion has been reported (8,13,29,35). The amino acids we used (BCAA, arginine, and orni­ thine) might have influenced the pituitary secretion in dif­ ferent ways: they might have acted directly at the hypotha­ lamic-pituitary level or indirectly through a modification of the concentration of neuroendocrine amino acid compo­ nents. Such modification would influence the neuroendo­ crine pathways that regulate pituitary secretion. Central catecholarn.inergic and serotonergic systems influ­ ence the secretion of many pituitary hormones. The rate of synthesis of these neurotransmitters depends on various factors including the availability and rate of uptake of precursors (tyrosine, phenylalanine, and tryptophan) from the circulation (18,44). The uptake of these precursors across the blood brain barrier occurs via a membrane-bound transport system, known as the large neutral arn.ino acids transporter (LNAA). The uptake of these precursors is affected by the plasma levels of other arn.ino acids (isoleucine, leucine, methionine, valine) competing for uptake into the brain via LNAA mediated trans­ port. Thus, in our experiment, the acute administration of BCAA might have decreased the rate of uptake of tyrosine, phenylalanine, and tryptophan in the brain, thereby decreasing the pool sizes of the corresponding neurotransmitters with consequent changes in the neuroendocrine modulation of pi­ tuitary hormone secretion. It has been demonstrated that BCAA administration in rats decreased the brain concentration of several amino acids (tyrosine, GABA, methionine, phenyl­ alanine, glycine, histidine, threonine, etc.) and increased glu­ tamic acid, a metabolite in BCAA catabolism (4) . These results indicate preferential transport of BCAA across the blood-brain barrier by LNAA. Furthermore, a BCAA-dependent reduction of a free-tryptophan in brain, with the consequent ceduction in serotonin production in the central nervous system, may be responsible for the reduced fatigue in athletes consuming BCAA supplementation (5). BCAA are also precursors of other amino acids, and in CNS, where there is a low concentration of branched-chain keto-acid dehydrogenase, BCAA catabolism may provide glutamine (39), which is a precursor for the neurotransmit­ ters glutamate, and GABA, which is essential for detoxifi­ cation of brain ammonia (II). The action of arginine and ornithine on pituitary function might be dependent on: I) the influence of arginine on brain concentration and metabolism of citrulline and ornithine and vice versa; 2) the competition of arginine and ornithine with http://www.msse.org

the amino acid lysine and histidine (Ly carrier system); 3) arginine's function as a specific endogenous modulator of cerebral mitochondrial glutamate transport (15); 4) argin­ ine' s stimulating action on the secretion of hormones (i.e., prolactin, catecholamines, etc .) that may influence the se­ cretion of other pituitary hormones; and 5) the fact that arginine is the main source for production of nitric oxide (NO) in the brain (although it has yet to be determined whether oral intake of L-arginine can affect NO synthesis). NO is formed by the conversion of arginine to citrulline by the enzyme NO synthase (NOS) and has multiple regulatory effects in a variety of tissues and systems. For example, the pulsatile release of LHRH induced by norepinephrine is brought about by an increase in hypothalamic NO, resulting in LHRH secretion into the portal vessels (37). Endogenous NO also modulates the hypothalamic-pituitary-adrenal axis activity via CRH/A CTH secretion and their response to arginine-vasopressin, prostaglandins, the adrenergic system, etc. (38). Finally, rat steroidogenesis is also influenced, at testicular and adrenal levels, by NO (7) and thus indirectly by ,uginine.

eral different pathways involving the neuroendocrine control and modulation of pituitary secretory cells. In our opinion, further experiments are necessary to clarify whether the ob­ served neuroendocrine modifications are a consequence of a dose-dependent effect of a single amino acid andior whether they are linked to the particular mixture we used. Moreover, it is important to point out that, because of the significant effect on pituitary respon siveness, amino acids cannot be considered simply "plastic protein-building substances" used by athletes solely for .muscle protein synthesis. We may also speculate on the possibility that particular combinations andior the prevalence of some amino acids in the diet might influence the characteristics of basal, general­ stress, or even exercise-related hormonal responses. In an ethical light, because of the effects of amino