Carnosine (β-alanyl-L-histidine) and anserine (β-alanyl-1methyl-L-histidine) are histidine-containing dipeptides that are present in the brain, heart, skin, liver, kidney, and skeletal muscle of a variety of animals (7,9); however, the physiological role of these dipeptides has not yet been fully clarified. It is suggested that because the pKa values of carnosine and anserine are 6.83 and 7.03, respectively (4,25), their proton sequestering capacity is high; therefore, they are important buffers in the nonbicarbonate buffering system (1,15,19).
Buffering capacity (β) is determined by the concentrations and the proton sequestering capacity of the buffers that are capable of accepting the protons that are released during metabolism (16). It is possible to reduce acidosis during high-intensity exercise, during which a large amount of hydrogen ions are produced, by the oral ingestion of carnosine and anserine. It has also been shown that most of the protons that are produced during exercise and circulated in the blood are buffered by the bicarbonate (5). Because carnosine and anserine supplementation increases the nonbicarbonate buffering action, the contribution of the bicarbonate buffering action during and after exercise may be reduced by oral supplementation. However, it is very difficult to prepare a large amount of pure carnosine and anserine; therefore, to our knowledge, no study has thus far investigated the influence of carnosine and anserine supplementation on the contribution of bicarbonate buffering action and performance during high-intensity exercise. The purpose of this study was to investigate the influence of oral supplementation with chicken breast extract (CBEX™), which is a rich source of carnosine and anserine, on acid-base balance and performance during intense intermittent exercise.
Eight healthy and active, but not well-trained males volunteered as subjects. The mean (±SE) values for age, height, and body mass were 19.8 ± 0.5 yr, 171.8 ± 2.3 cm, and 64.7 ± 2.7 kg, respectively. The objective, methods, and risks of this study were explained in detail, and consent was obtained from all the subjects before the study was conducted. This study was approved by the ethical committee for human studies in Nippon Meat Packers, Inc.
Preparation of CBEX and placebo soups.
CBEX contains large amounts of carnosine and anserine. It has a strong, peculiar flavor and odor, and is thus difficult to ingest directly. Therefore, a consommé soup containing approximately 40 g of CBEX per can (net weight, 190 g) was prepared (CBEX soup). In addition, a placebo soup having the same taste as the CBEX soup was prepared. The nutritional value of the experimental soup is presented in Table 1. The CBEX soup contained 1.5 g of carnosine and anserine. Other than the chicken extract, no animal extracts were used to prepare either the CBEX soup or the placebo soup.
Figure 1 shows the experimental protocol. The subjects were instructed not to eat anything for a period of 3 h before the start of the study. On entering the experimental room, they were asked to rest until the warm-up began. While they rested, an indwelling needle (22 g) was inserted into the radial artery in preparation for the continuous collection of the blood, which was to be investigated for carnosine and anserine concentrations and blood-gas dynamics during exercise. The needle was inserted under local anesthesia to minimize pain. Blood was collected using a heparin treated syringe through an extension tube connected to the indwelling needle and a three-way cock. Blood at rest was also collected after the preparatory process.
After obtaining the sample of blood at rest, all the subjects consumed a can (net weight, 190 g) of either the CBEX or placebo soup, and, subsequently, the warm-up was started. The standardized warm-up consisting of two bouts of 5 min of pedaling at 60 W, with two bouts of maximal pedaling against a resistance of 7.5% of body mass for 5 s between them. The exercise test was started 30 min after the consumption of the experimental soup and 10 min after completion of the warm-up. In a previous study with rats, carnosine and anserine concentrations were shown to reach a peak 30 min after CBEX supplementation (13). Further, in our pilot study on humans, carnosine, anserine, and their related compounds were detected, and their concentrations reached their peak in 30 min. Therefore, the subjects in this study were instructed to consume the soup 30 min before the commencement of exercise. This study was a double-blinded, placebo-controlled, randomized crossover study with a washout period of at least 3 d between the CBEX and placebo supplementations.
The interval test consisted of 10 sets of 5-s maximal cycle sprints, with a 25-s recovery period between sprints. This test was conducted to estimate intermittent exercise performance, and the mean power in each set was measured. Using an electronically braked cycle ergometer, the subjects exercised against a resistance of 7.5% of body mass (TKK1254a, Takei Scientific Instruments Co., Ltd., Tokyo, Japan) after both CBEX and placebo supplementations. The subjects were instructed to remain seated during all the sprints, and their feet were fixed with straps. All the subjects were highly motivated and were instructed to cycle maximally from the beginning of the test; they were given verbal encouragement during each 5-s sprint. This test was a modification of the one used by Gaitanos et al. (11).
Blood was collected at rest (Rest), 1 min before the start of the test (Pre), after each set of the test (1-9), and immediately after the test (Post) to measure blood-gas parameters, blood lactate concentration([La−]), and the concentrations of carnosine, anserine, and their constitutive amino acids.
The blood samples that were used to measure the concentrations of carnosine, anserine, and their constitutive amino acids were flash-frozen with liquid nitrogen immediately after blood collection and stored at −80°C until the assay. One milliliter of thawed blood was mixed with twofold its volume of water to homogenize it. Following this process, the sample was combined with one third its volume of 20% trichloroacetic acid and kept on ice for 10 min. Subsequently, the sample was centrifuged (3000 × g for 10 min) to obtain the supernatant. The residual sediment was suspended in 6% trichloroacetic acid and was centrifuged again (3000 × g for 10 min) to obtain the supernatant; this process was repeated once. The obtained supernatant was mixed with fivefold its volume of diethyl ether and kept for 10 min. Subsequently, the upper layer (ether layer) was removed, and this procedure was repeated twice. The lower layer (water layer) was collected, freeze-dried for complete desiccation, and dissolved in 100 μL of lithiumcitrate buffer solution (the first buffer for the amino acid analyzer; JEOL, Ltd., Tokyo, Japan). The concentrations of carnosine, anserine, and their constitutive amino acids β-alanine, histidine, and 1-methylhistidine were then measured using an amino acid analyzer (JLC-500; JEOL, Ltd., Tokyo, Japan).
The blood samples that were collected in a syringe in order to measure blood-gas parameters and [La−] were immediately cooled on ice; the samples were warmed at 37°C before the blood-gas parameters were measured. The blood-gas parameters, including the hydrogen ion concentration (pH), carbon dioxide partial pressure (pCO2), and bicarbonate ion concentration ([HCO3−]), were measured using an automatic blood-gas analyzer (ABL520, Radiometer Co., Ltd., Copenhagen, Denmark). An automatic lactate analyzer (1500SPORT,YSI Ltd., Ohio, USA) was used to analyze [La−]. These analyses were completed within 15 min after blood collection.
All measured values are expressed as mean ± SE. Paired t-tests were used to investigate the differences between the mean values of CBEX and placebo supplemented groups. One-way analysis of variance (ANOVA) was used to compare the concentrations of carnosine, anserine, and their constitutive amino acids at rest (Rest) and immediately before (Pre) and after (Post) the interval test. When F values were significant, a multiple comparison was performed using Fisher's PLSD method. P values less than 0.05 were considered to be statistically significant.
In the interval test, there were no significant differences were observed between the CBEX and placebo supplemented groups with regard to the mean power in each set. Furthermore, there were no significant differences between the CBEX and placebo supplemented groups withregard to the mean power of all 10 sets (9.35 ± 0.18 W·kg−1 and 9.50 ± 0.19 W·kg−1), the first 5 sets (9.81 ± 0.19 W·kg−1 and 9.98 ± 0.21 W·kg−1), or the second 5 sets (8.90 ± 0.25 W·kg−1 and 8.98 ± 0.21 W·kg−1).
Figure 2 shows the changes in [La−], pCO2, pH, and [HCO3−]. Table 2 shows each value at Rest, Pre (30 min after CBEX and Placebo supplementation), and Post (40 min after CBEX and Placebo supplementation), during and after the interval test. After both the CBEX and placebo supplementations, the [La−] increased throughout the period of exercise, pCO2 transiently increased after the commencement of exercise and decreased afterwards, and the pH and [HCO3−] decreased during the period of exercise. After CBEX supplementation, the pH at Post and [HCO3−] from the end of the fifth set till the completion of the interval test were significantly higher.
Table 3 shows the concentrations of carnosine, anserine, and their constitutive amino acids at Rest, Pre, and Post. Because the samples from two out of eight subjects were not analyzed, samples from the remaining six subjects were used to obtain these data. Carnosine was not detected at Rest (not more than 0.5 μmol·L−1), and the carnosine concentration at Pre or Post was low and did not significantly differ from the concentration at Rest. Anserine was also not detected at Rest; however, the anserine concentrations at Pre and Post were significantly higher than the concentration at Rest (P < 0.05). The concentrations of β-alanine and 1-methyl-histidine at Pre and Post were significantly higher than the concentration at Rest (P < 0.01). However, no significant differences were observed in the histidine concentrations at Rest, Pre, and Post.
This study is the first to examine the effect of supplementation with a nonbicarbonate buffer on acid-base balance and high-intensity exercise performance. The most important finding of the present study is that supplementation with CBEX containing 1.5 g of carnosine and anserine delayed the decrease in [HCO3−] during intense intermittent exercise; however, performance was not observed to improve significantly.
It has been reported that most of the protons that are produced during exercise and released in blood are buffered by the bicarbonate buffering system (5). However, whereas the pKa of the bicarbonate buffering system is 6.1, the pKa values of carnosine and anserine contained in CBEX were 6.83 and 7.03, respectively (4,25); this suggests that the pKa of carnosine and anserine are closer to the physiological pH as compared with the bicarbonate buffering system. The protons that are released during metabolism are considered to be immediately buffered by the nonbicarbonate buffering system, which has a pKa that is closer to the physiological pH, and subsequently buffered by the bicarbonate buffering system (15). This indicates that compared with [HCO3−], carnosine, and anserine can exert a high buffering action and quickly accept protons during high-intensity exercise. Consequently, supplementation with CBEX containing carnosine and anserine may increase the contribution of the nonbicarbonate buffering action in blood, and appears to spare the [HCO3−] during and after interval test.
Because CBEX supplementation decreased the contribution of bicarbonate buffering action during exercise, it is possible that the acceptance of protons released during exercise-induced metabolism increased with the increase in the contribution of the nonbicarbonate buffering action. If this is the case, CBEX supplementation may promote the efflux of protons from the muscle to blood and inhibit the decrease of intramuscular pH, which could improve performance during the latter half of exercise. However, there were no significant differences with regard to the mean power in each set between the CBEX and placebo supplementated groups. The lack of improvement in intense intermittent exercise performance may be attributed to the limited buffering potential of the CBEX supplementation. This may be explained by the fact that the pretest blood [HCO3−] and pH were not different in the two conditions. To use a similar protocol (5 × 6-s all-out sprints every 30 s) as the one used in this study, Bishop et al. (6) reported that NaHCO3 ingestion before exercise increased resting blood [HCO3−] and pH, which resulted in a significant increase in total work, and the latter half of work and power output. The increase in [HCO3−] does not increase the skeletal muscle buffering capacity (6,17); rather, it increases the pH gradient through the cell membrane, facilitates the efflux of protons from the muscle to blood, reduces the decrease of intramuscular pH caused by exercise, and improves single and repeated bouts of exhaustive exercise performance (6,8,10,12). This suggested that in order to improve high-intensity exercise performance, it was necessary to elevate resting blood [HCO3−] and pH.
Although we did not measure the intramuscular pH, it is likely that the intramuscular pH during the interval test did not differ between the two conditions. Because the decrease in intramuscular pH causes muscle fatigue via several mechanisms such as decreasing skeletal muscle tension and relaxation (23) and the inhibition of phosphofructokinase activity (26), this decrease appears to be important in preventing the decrease of intramuscular pH during exercise to achieve high performance levels. It was suggested that proton efflux from the muscle cell does not change regardless of the increasing contribution of the nonbicarbonate buffering system because the contribution of bicarbonate buffering system was decreased.
Carnosine and anserine are present, in particular, in skeletal muscle, and are reported to play a role in the nonbicarbonate buffering action within the cells (1,19,25). Anserine does not exist in the human skeletal muscle (18). Carnosine concentration in human skeletal muscle is greater in Type II fibers than in Type I fibers (14). Moreover, sprinters have higher carnosine concentrations and demonstrate longer endurance time in anaerobic speed tests than marathoners or untrained subjects (20). It has been recently reported that there exists a positive correlation between the carnosine concentration in the skeletal muscle and the mean power of 30-s maximum pedaling (24). These results indicate that whereas carnosine in the skeletal muscle is an important substance that is involved in short-duration, high-intensity exercise performance, blood carnosine is not believed to be involved. Carnosine is rarely found in blood (22) because the carnosine-degrading enzyme, carnosinase, is present in the serum of healthy human adults (2,3,27). In the present study, the carnosine concentration 30 min after CBEX supplementation (Pre) did not significantly differ from that at Rest. With regard to anserine, its degradation speed was found to be slow as compared to that of carnosine, the former being 52% when the assumed degradation speed of carnosine is 100% (21). In addition, anserine content of the CBEX was approximately double (2.5 times) that of carnosine; therefore, anserine seems to peak 30 min after CBEX supplementation.
In conclusion, although oral supplementation with CBEX that is rich in carnosine and anserine increased the contribution of the nonbicarbonate buffering system and decreased bicarbonate buffering action in blood, intense intermittent exercise performance was not observed to significantly improve.
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