Athletes have increasingly used caffeine as an ergogenic supplement because it increases the blood epinephrine concentrations, which leads to an increase in the metabolism and promotes a greater power output during high-intensity exercise. This effect is associated with increased glucose levels both at rest and during exercise (2). Caffeine also changes the metabolism through the mobilization of free fatty acids and the sparing of glycogen (12). Although several studies have attempted to elucidate the amino acids metabolism during exercise, the combined effects of caffeine and exercise on the amino acid metabolism remain obscure (6,20,36,37).
In the human body, ammonia (NH3) is mostly present in its ionized form ammonium (NH4+); hereafter, “ammonia” refers to the combination of NH3 and NH4+. At rest, most of the body’s ammonia is produced by enteric microbiota and, to a smaller extent, by either amino acids or the deamination of purine derivatives, mostly AMP (1). The ATP/ADP ratio reflects the energy state of the cell and is effectively regulated by the rate of enzymatic ATP synthesis. During exercise, a decrease in the stored ATP and a transient increase in the ADP levels activate myokinase, which leads to the formation of AMP (3). Both AMP deamination and, to a lesser amount, branched-chain amino acid (BCAA) deamination lead to an increase in the intracellular ammonia, which requires active transport to leave the cytoplasm. The ammonia leaves the cells, enters the blood for further detoxification, and is excreted mostly as urea. Ammonemia increases during exercise in an intensity-dependent manner (5,7). A hyperammonemic state changes the function of the blood–brain barrier and is postulated to cause different cerebral dysfunctions (8) and central fatigue during exercise through the engagement of the metabotropic glutamate receptors (34,38).
We previously proposed that exercise can be used as a tool to study metabolic stress. Also, intermittent exercise, in addition to a continuous exercise protocol, provides invaluable data that can be used to understand metabolic changes (5,6,11,32). We recently proposed a sportomics approach to mimic both the real challenges and the conditions that are faced during sports situations (10,17,33). Sportomics combines the use of “-omics” sciences with classic clinical laboratory analyses to understand sport-induced modifications.
In this article, we describe the changes in the concentrations of amino acids and other metabolites in the blood of elite soccer players during caffeine supplementation using a sportomics-designed study. On the basis of our previous findings, we hypothesize that caffeine might affect both ammonia genesis and metabolism during exercise, and thus, we evaluated the effect of this supplement during exercise.
This double-blind randomized study was conducted according to the guidelines dictated by the Declaration of Helsinki. All of the procedures involving human subjects were approved by the Ethics Committee for Human Research at the Federal University of the State of Rio de Janeiro (117/2007, renewed in 2011) and met the requirements regulating research on human subjects (Health National Council, Brazil, 1996). The nature of the study and the procedures involved were described to all of the subjects, and written informed consent was obtained from all of the subjects. The experiment was conducted as described in a previous study (6). Briefly, professional soccer players (n = 19) from a major league team affiliated with the Confederação Brasileira de Futebol (CBF, Brazilian Soccer Confederation) voluntarily participated in the study. To maximize the control over the large number of experimental variables that can potentially influence the results, we conducted the experiment during the preparation phase training for the Brazilian Soccer Championship to ensure that all of the athletes had similar diets, training regimes, and resting and sleeping conditions.
None of the subjects had a medical history of health problems or were using ergogenic substances or any other drugs. In addition, the athletes were subjected to clinical examinations, anthropometric measurements, and laboratory tests to ensure homogeneity among the groups. The initial laboratory tests included hematological and biochemical analyses, which allowed the diagnosis of any metabolic anomalies that could affect the results or impair their interpretation. As previously described, the 85 biochemical analyses that were conducted allowed us to select individuals with the following conditions: a similar carbohydrate, lipid, and protein metabolism; a similar oxygen transport efficiency; similar macronutrient anabolism and catabolism indicators; a normal water and electrolyte metabolism; no infection or parasitic infestation; a well-balanced body water content; and undisturbed hepatic and renal function (6). All of the subjects exhibited normal metabolic functions, and no differences between the subjects were detected before the experiment (data not shown). In addition, the athletes exhibiting biochemical outliers in the results were removed from the experimental procedure (data not shown). Furthermore, we did not find evidence of hemoconcentration (i.e., < 2%) during the exercise protocol (data not shown).
Caffeine, xanthine, or any other substance that could mask the results were not ingested by the athletes for 72 h before the blood collection. On the day of the experiment, blood was collected from fasting soccer players, i.e., before breakfast (PRE). The players were randomly divided into two groups and provided with a specific breakfast supplemented with either caffeine or lactose.
After receiving breakfast and the supplements, the subjects were driven to the testing location, which took 15 min. After 20 min of warm-up (exercises for articular movement and elongation), the subjects performed the intermittent exercise test protocol under the heart rate accompaniment (Fig. 1).
All of the subjects had previously performed the test twice during the general preparation phase training to ensure their familiarity with the testing procedure. On the day of the test, each subject performed an intermittent exercise protocol. Briefly, this protocol included two sessions of a 45-min variable distance run protocol (VDR) with a 15-min interval in a 50 × 50-m court marked with a 5 × 5-m grid. After finishing the VDR (5 min), the athletes executed the Yo-Yo Intermittent Recovery Test level 2 (Yo-Yo IR2) (4,27) until exhaustion, finishing 190–200 min after the beginning of the exercise protocol; the test duration depended on the athlete. The athletes received a drink containing electrolytes and glucose (Gatorade®) ad libitum throughout the study. Each athlete completed the exercise protocol at different times. The exercise test was immediately followed by the collection of blood, which was then used for the laboratory analyses (POST).
As previously described, anthropometric, hormonal, hematological, and biochemical evaluations were performed to guarantee homogeneity among the participants enrolled in the study (6). We randomly divided the soccer players into two groups and measured the urinary amino acids (Ile, Leu, Met, Phe, Tyr, and Val), which were normalized to the creatinine clearance rate. The different supplements were provided in indistinguishable capsules to ensure that the subjects were not aware of their group assignment. Caffeine (Purifarma, China) was orally administered at a dosage of 5 mg·kg−1, whereas the control group received lactose (Via Farma, Brazil). The lactose content (1 g total) in the capsules was not sufficient to provide a significant amount of metabolizable energy (<4.2 kJ) that could influence the study results.
The venous blood samples were collected from an antecubital vein. Immediately after collection, the blood samples were centrifuged to separate the serum, which was quickly frozen and stored at −70°C. Because of the nature of the study, we used four phlebotomists to ensure that the blood collection occurred within 2 min of the completion of the exercise protocol. As previously described, distinct biochemical and hematological analyses (biomarkers of hepatic and renal function; evaluations of carbohydrate, lipid, and protein metabolism; indicators of oxygen transport efficiency, macronutrient anabolism and catabolism, and water and electrolyte metabolism) were performed to identify any variables that could affect the metabolic interpretation of the results (data not shown) (6).
Serum caffeine measurement.
Caffeine was identified and measured in the serum through gas chromatography/mass spectrometry (GC/MS) using β-naphthoic as an internal control, as previously described (25). The caffeine extraction was performed at a basic pH, and the samples were centrifuged and subjected to solid-phase extraction. After elution with chloroform, the solvent was evaporated and the residue was resuspended in dichloromethane. The samples were then dried with nitrogen and resuspended in ethyl acetate. The alkaline extraction (1 μL) was then analyzed by GC using a mass selective detector with a 5% phenylmethylsiloxane column, VB-5 cross-linking, and a 0.25-mm-thick, 30-m-long film. Because of the nature of the investigation, we used the World Anti-Doping Agency guidelines for the analysis: SCAN mode, injector temperature of 280°C, detector temperature of 295°C, and initial column temperature of 60°C. The column temperature was increased at a rate of 22°C·min−1 to 200°C, then at a rate of 10°C·min−1 to 270°C, then at a rate of 30°C·min−1 to 305°C, and maintained at 305°C for 6 min.
Caffeine absorption and group assignment.
To evaluate the available caffeine concentration during the experiment, we measured the serum levels of caffeine. No differences were detected in the serum caffeine level among any of the groups before the experiment. In addition, the levels of serum caffeine in the nonsupplemented group before and after the test (LEx, n = 8) were compared, and no differences were found. The observed increases in the serum caffeine after supplementation followed two significantly different patterns, which were evaluated as described under the statistical analyses section. Therefore, we divided the supplemented athletes into two different groups: CEx (<900% increase, n = 5) and SCEx (>10,000% increase, n = 6) (Fig. 2).
Serum amino acid determination.
The concentrations of the amino acids were measured through high-performance liquid chromatography, as previously described (23).
The data were analyzed by ANOVA using the test condition and the time as the repeated-measure variables, which were confirmed using Tukey post hoc test. Differences with P < 0.05 were considered to be statistically significant. Data are expressed as means ± SE.
Effect of caffeine effect on performance.
The VDR and Yo-Yo IR2 tests were used to simulate an exercise state similar to that obtained in a soccer match and to induce exhaustion, respectively. Our assumption that these tests generated a similar exercise intensity in the three studied groups was confirmed through the measured cardiac frequency and the blood lactate concentration, which can be used as an indicator of consistent glucose metabolism. All of the athletes in the three groups showed similar results at the end of the Yo-Yo IR2 test. In addition, we were not able to measure any significant caffeine-induced difference in the athletes’ performances (Tables 1 and 2).
Effects of caffeine on metabolism.
Exercise increased the serum concentrations of glucose in the LEx group by 26%. In addition, the experimental protocol significantly increased glycemia by 43% in the CEx group and by 53% in the SCEx group. The insulin concentration followed the rise in glycemia: 54% increase in the control group and 72% and 84% increases in the caffeine-supplemented groups (CEx and SCEx, respectively). In our protocol, the caffeine supplementation did not affect the exercise-induced increase in the glucose, insulin and lactate levels (Table 2).
Exercise produced similar increases in the serum levels of ammonia (five- to sixfold) in all three groups. No significant difference was measured in the exercise-induced increase in ammonemia between the groups. However, a significant dose-dependent effect was observed with uremia. The amount of urea rose by 10% in the LEx and CEx groups because of exercise, whereas the subjects with the highest serum caffeine concentrations (SCEx) did not exhibit an exercise-induced increase in uremia. The creatinine concentration in the blood increased because of exercise in all three groups; however, caffeine supplementation was not found to have a significant difference in the creatinine concentration (Table 3).
Urate is an important metabolite that indirectly measures the level of ammoniagenesis because of AMP deamination. In all three groups, the urate concentration in the blood remained unaffected by either exercise or caffeine supplementation (Table 3).
Effects of caffeine on the serum amino acids.
Caffeine and exercise demonstrated different effects on the serum amino acid concentrations.
The exercise-induced increases in the serum concentrations of Gly (LEx 26%, CEx 28%), Ser (LEx 27%, CEx 27%) and His (LEx 34%, CEx 36%) were inhibited by the high serum caffeine concentration measured in the SCEx group. The serum Pro concentration increased because of exercise, as measured in the LEx group (38%). This increase was inhibited by caffeine because the two groups that received caffeine did not exhibit a significant increase in the concentration of this amino acid (CEx 26%, SCEx 25%). The serum concentrations of Lys, Thr, Tau, Asn, Asp, and Met were not affected by either exercise or caffeine (Table 4).
Exercise increased the total serum concentration of the BCAA by 28%. This increase was inhibited by caffeine, as demonstrated with both caffeine-supplemented groups (CEx 16%, SCEx 4%). The analysis of the serum Val levels showed that this amino acid was the only BCAA that was significantly affected by caffeine. The control group showed an exercise-induced increase in Val of 29%, which was dose-dependently repressed by caffeine supplementation (CEx 16%, SCEx 0%). No significant differences were measured in the concentrations of either Leu (LEx 29%, CEx 18%, SCEx 19%) or Ile (LEx 27%, CEx 12%, SCEx 0%) in response to exercise, and no differences in these amino acids were detected among the groups.
The serum concentration of Trp was not affected by either exercise or caffeine. The Tyr concentration increased in the three groups (LEx 78%, CEx 77%, SCEx 35%) in response to exercise, and caffeine supplementation was not found to have a significant effect. In contrast, the exercise-induced increase in the level of Phe was significantly inhibited by high serum concentrations of caffeine (LEx 53%, CEx 36%, SCEx 11%). Gln and Ala are the major amino acid sources for neoglucogenesis. Higher serum caffeine levels abolished the exercise-induced increase in Gln (LEx 27%, CEx 24%, SCEx 0%) but did not significantly affect the exercise-induced increase in the levels of Ala (LEx 118%, CEx 160%, SCEx 110%) and Glu (LEx 61%, CEx 42%, SCEx 51%) (Table 4).
Caffeine affected the serum concentrations of the three urea cycle intermediates: Arg, Cit, and Orn. The SCEx group showed a significant attenuation of the exercise-induced increases in the serum levels of Cit (LEx 37%, CEx 48%, SCEx 16%) and Orn (LEx 53%, CEx 47%, SCEx 0%). The serum Arg concentrations were also affected by caffeine (LEx 26%, CEx 7%, SCEx 15%) (Table 4).
The analyses of the exercise-induced increases in the serum amino acids revealed two effects of caffeine. The serum Ile, Trp, Val, and BCAA concentrations were affected in both caffeine-supplemented groups. However, the serum Orn, Gln, Gly, His, Phe, Ser, and Tyr levels were not strongly affected by the lower caffeine concentration that was used (Table 4).
In the present study, we used a previously published protocol (6) that combines intermittent and exhaustion exercise to determine the effects of caffeine on the serum amino acid levels and the ammonia metabolism of elite soccer players. The interest in our study lies in the fact that we analyze a small portion of the population (those who have achieved a world-class sports level as professional soccer players from a major league team affiliated with the Brazilian Soccer Confederation, i.e., the top athletes in Brazilian soccer) and follow the metabolic changes in response to modifications in both training and diet using an in-field sportomics protocol. To the best of our knowledge, this study is the first investigation to examine the effects of caffeine on human amino acid metabolism during exercise. It is important to reinforce that further studies and observations are required to extrapolate our interpretations because of the number of subjects who were analyzed in this study.
On the basis of previous data on the pharmacokinetics and dose dependence of caffeine during rest and physical exercise (6,14,19), a 5-mg·kg−1 dose of caffeine was used in the present study. Our assumption that a similar exercise intensity was achieved by the athletes in the three studied groups was confirmed through the lactate concentration changes, which were not different under caffeine supplementation (15), and the measured cardiac frequency. Although athletic performance was not the focus of the study, we did not find a significant difference in performance because of caffeine supplementation. We believe that either the small number of subjects in the study or the different soccer positions that are played by the athletes can explain this result. In addition, the biochemical analyses that we used have a greater sensitivity than the performance measurement tools, which might also account for the lack of the difference in performance found.
Our protocol was designed on the basis of the previously described caffeine absorption curve, which led to the assumption that the serum caffeine level peaks approximately 1 h after oral supplementation (18). Using the gold standard GC/MS technique to measure caffeinemia after caffeine supplementation, we showed that the serum caffeine level increased in two different patterns. The statistical analysis, which was performed using ANOVA followed by Tukey post hoc test, showed a ∼25-fold significant difference (P < 0.001) in the blood caffeine concentration between the SCEx and CEx groups (41.19 ± 2.72 vs 1.69 ± 0.02, median ± SEM). On the basis of the two caffeine levels in the blood and the significant difference in the caffeinemia of the subjects, we divided the caffeine-supplemented subjects into two groups according to their serum caffeine levels. This division allowed us to detect distinct amino acid metabolism profiles in response to the different serum caffeine concentrations, which would not have been observed in the absence of the serum caffeine analysis. Further studies should take into account that caffeinemia may differ even between subjects receiving the same doses of caffeine.
In the present study, we subjected the subjects to a high-intensity exercise session after an intermittent exercise period. Caffeine has been shown to decrease the exercise-induced reduction in glycogen and increase the amount of exercise-induced gluconeogenesis (21). In addition, during recovery, caffeine increases both insulin-independent glucose transport and glycogen resynthesis (31). In our study, the serum concentrations of both glucose and Leu increased in all groups in response to exercise; this finding can be explained by the breakfast, access to a glucose drink ad libitum throughout the training the exercise-induced glycogenolysis and gluconeogenesis, or the combination of these factors (13,16). Also, caffeine ingestion has been shown to impair insulin-mediated glucose disposal, which can be attributed to either β-adrenergic stimulation or adenosine receptor-antagonized elevations of insulin and glucose (35). Therefore, because of the increased concentration of Leu that was observed, we hypothesize that the rise in insulin can be explained by an additive effect of the increases in amino acid levels and glycemia because insulin secretion is primarily stimulated by glucose and amino acids (26).
BCAAs can be oxidized and used as an alternative source of energy by muscles or to provide tricarboxylic acid cycle intermediates (22). Increases in serum BCAAs during or after exercise may indicate the mobilization of BCAAs from either the liver or muscle (28). The changes observed in our study indicated that the intensity and duration of the intermittent exercise induced a significant utilization of BCAAs for energy generation. In our protocol, the total BCAA concentration increase because of exercise was diminished by caffeine supplementation. The serum levels of BCAAs (Ile, Leu, and Val) exhibited no significant differences between the two caffeine-treated groups (SCEx and CEx). The serum caffeine levels inhibited the Val concentration increase in a dose-dependent manner. A similar effect was observed with Leu and Ile, although this effect appears to be hidden because of subject variations. Moreover, the serum concentrations of the amino acids related to the urea cycle (Arg and Orn) were unaffected by exercise in the SCEx and CEx groups. Furthermore, the serum concentration of citrulline did not change significantly in the SCEx group. The serum concentrations of Cit, Arg, and Orn may also be dependent on urea and ammonia metabolism. The other amino acids evaluated in our study (Asp, Asn, Lys, Met, Tau, and Thr) did not show any changes because of either exercise or caffeine supplementation. Our data suggest that, in addition to the effects on performance enhancement, caffeine modifies the concentrations of blood amino acids during exercise. However, the translation of these findings to the athletic performance requires further investigation.
The level of ammonemia increased in all three tested groups in response to exercise. However, even with the approximately 600% increase in ammonemia observed in the SCEx group, no increase in uremia was detected. Muscle and other tissues are widely known to respond to increased levels of ammonia by synthesizing and releasing Gln into the serum. We thus measured increases in the serum concentrations of Gln, Ala, and Glu in response to exercise. These amino acids are metabolized in muscle, thereby providing the amino groups and, most likely, the ammonia required for the synthesis of glutamine and alanine (37). In addition, previous studies have shown that Ala, Gln, and Glu increase the levels of tricarboxylic acid cycle intermediates during exercise and contribute to the increase in glycemia (9). In our study, higher concentrations of serum caffeine prevented the exercise-induced increase in the serum Gln level but did not affect the exercise-induced increases in Ala and Glu. Given that Gln is the major ammonia transporter to the liver, caffeine may decrease urea synthesis via a decrease in the blood Gln concentration.
The effects of the physiological responses to caffeine supplementation on the urea cycle intermediates in humans are not well understood. High caffeine concentrations (4 mmol·L−1) do not cause changes in urea synthesis or in the levels of its intermediates (ornithine, citrulline, and arginine) in murine hepatocytes (24). Previous studies have shown an inhibitory effect of caffeine on urea synthesis; this effect is demonstrated through the in vitro inhibition of several urea cycle enzymes, such as arginase (29,30). During this experiment, the CEx group exhibited serum urea levels that were 10% higher than those of the LEx group. However, the concentration of urea in the serum of the SCEx group was reduced by 16% compared with the nonsupplemented group.
In this study, we measured a caffeine-induced decrease in the blood concentrations of both urea and the urea cycle intermediates (Cit, Orn, and Arg) in response to exercise. Our data suggest that that caffeine might decrease systemic urea by decreasing the concentration of glutamine in the serum, which, in turn, decreases the amount of ammonia transported to the liver and subsequently decreases urea synthesis.
The authors thank Ricardo Freitas-Dias for his help during the first steps of the study. The authors declare that they have no financial competing interests and that no funding agencies supported this project.
The results of this study do not constitute an endorsement by the American College of Sports Medicine.
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