Carnosine or β-alanyl-l-histidine is a naturally occurring histidine dipeptide, which occurs in high concentrations in skeletal muscle tissue, particularly in Type II fibers (9,10). Carnosine has been implicated in a variety of cellular functions, including regulation of calcium sensitivity (19), inhibition of protein glycosylation and protein cross-linking (14,15), and free radical scavenging (20) as well as regulation of enzyme activity (16). Still, the only physiological role of carnosine, which is corroborated by observations in humans, is intramyocellular pH buffering (3). The imidazole ring of carnosine has a pKa (6.83) close to intracellular pH (6.5), which turns carnosine into a potent proton buffering molecule. It is currently believed that carnosine accounts for approximately 10% of total buffer capacity in skeletal muscle cells (21).
Although the precise cellular mechanisms involved in the development of muscle fatigue are still debated (1,18) it is believed that intramyocellular accumulation of protons is implicated in fatigue during high-intensity muscle contractions (6-8). Therefore, any intervention that can reduce intramyocellular pH drop during high-intensity exercise, either by facilitating the export of H+ from muscle cells or by directly buffering intracellular H+, conceivably can delay fatigue and thus enhance muscle force and power output. Carnosine, relative to bicarbonate, is believed to play a secondary role in endogenous buffer capacity (23). Nonetheless, recent data seem to indicate that oral β-alanine (βALA) intake to increase muscle carnosine content is at least as effective to enhance performance in 1-2 min maximal exercise bouts than supplementary bicarbonate intake aimed to increase alkaline reserve. Recent studies in humans have consistently shown that supplementary intake of βALA (4-6 g·d−1) over a period of 4-10 wk can substantially increase (>50%) carnosine content in both Type I and Type II muscle fibers (4,11,13). Interestingly, both in less trained volunteers (13) and in elite sprinters (4), increased muscle carnosine content due to βALA intake was associated with enhanced short (1-2 min) maximal exercise performance (4,13).
Success in endurance competitions often depends on the ability to intermittently increase power output or sprint. For instance in cycling, victory is often decided in a final sprint. Furthermore, in most cycling competitions, episodes of submaximal exercise are alternated by short bouts of maximal exercise. Thus, enhanced anaerobic capacity, by virtue of elevated intramyocellular carnosine content and buffering capacity, may conceivably enhance performance in cycling. However, the effect of βALA supplementation on sprint performance in the context of an endurance exercise is unknown. Therefore, in this study, we investigated the effect of short-term βALA administration in a simulated cycling race. We hypothesized that supplementary βALA intake enhances sprint performance at the end of an endurance cycling bout.
METHODS
Subjects
Twenty-one healthy young males (moderately to well-trained cyclists) volunteered to participate in the study after having been informed in detail on the procedures contained in the study protocol. However, four subjects dropped out due to various reasons, which were unrelated to the study protocol. Thus, 17 subjects completed the full study protocol. Their age and body weight and the rate of maximal oxygen uptake obtained from an incremental exercise test (see below) were 24.9 yr (range = 18-30), 72.5 kg (range = 56.6-95.1), and 60.3 mL·min−1·kg−1 (range = 45-72), respectively. Subjects were diagnosed to be healthy using a medical questionnaire. They were nonsmokers and had not been taking dietary supplements for a period of at least 2 months before the start of the study. Subjects were instructed not to change their dietary and training habits throughout the duration of the study. Furthermore, subjects were asked not to participate in any strenuous exercise from 2 d before any experimental session. The study protocol was approved by the local ethics committee (K.U. Leuven), and the subjects signed an informed consent.
Study protocol
A double-blind, placebo-controlled study was performed, which consisted of two experimental test sessions (pretesting vs posttesting) interspersed by an 8-wk β-alanine (βALA) or placebo (PL) supplementation period. The pretesting and the posttesting consisted of an incremental exercise test and a simulated road race on a bicycle ergometer. One week before the start of the study, the subjects reported to the air-conditioned laboratory (18°C; 60% relative humidity) for a familiarization trial to get habituated with the exercise tests.
For the incremental exercise, test subjects reported to the laboratory in the morning, and their personal race bicycle was mounted on an ergometer (Avantronic® Cyclus 2, Leipzig, Germany) with gear fixed at either 53 × 14 or 52 × 14 depending on the bicycle configuration. The ergometer was put in power control mode, which means that the preset power output is automatically regulated independent of cadence or gear selection, by continuous adjustment of the degree of electromagnetic braking. Initial workload was set at 100 W and was further increased by 40 W per 8-min stages until volitional exhaustion was reached. Heart rate was continuously measured (Polar, Kempele, Finland), and V˙O2 and V˙CO2 were measured during the final stage of the test (Cortex Metalyzer II, Leipzig, Germany). Furthermore, capillary blood was sampled from a hyperemic earlobe at 4-min intervals for determination of lactate concentration and estimation of the maximal lactate steady state (MLSS; 31).
One week later, the subjects participated in the simulated road race. The evening before the test, they received a standardized dinner (1280 kcal, 70% En carbohydrates, 20% En fat, 10% En protein). They were instructed thereafter to remain fasted, but water was allowed ad libitum. Next morning they reported to the laboratory 2 h before the start of the exercise test (between 7:00 and 9:00 a.m.) and received a standardized breakfast (1200 kcal; 80% En carbohydrates, 10% En fat, 10% En protein). Their personal race bicycle was mounted on the ergometer (Avantronic® Cyclus 2) with gear fixed at either 52 × 14 or 53 × 14, depending on the bicycle configuration, and automated control of power output independent of cadence or gear selection, and the simulated road race was started (Fig. 1). The exercise protocol started with a 110-min intermittent endurance exercise bout during which exercise intensity was varied between 50% and 90% (10-min stages) of their previously estimated MLSS. Subjects throughout the exercise received a 10% carbohydrate-electrolyte solution at a rate of 10 mL·kg−1 body weight per hour. Immediately at the end of this intermittent exercise bout, a 10-min time trial was started. Initial workload was set at 100% of MLSS, which corresponded with 89 ± 1% of V˙O2max. Subjects could voluntarily increase or decrease the workload at 1-min intervals according to their perception of fatigue, but measured power outputs were blinded to the subject during the test. Before the trial, the subjects were instructed to consistently produce as much power output (W) as possible over the 10-min trial. A recent and preliminary test/retest study in our laboratory in a similar population (n = 7) yielded an intraclass correlation coefficient of 0.97, which proves the high-degree reliability of the time trial procedure used (24). Power output and heart rate (Polar) were continuously recorded. Furthermore, at the start and at the end of the time trial, capillary blood samples were obtained from a hyperemic earlobe for blood lactate and pH determination. At the end of the time trial, subjects were allowed to recover for 5 min at a workload corresponding to 50% of their MLSS. Finally, they performed a 30-s all-out sprint. For this purpose, the ergometer (Avantronic® Cyclus 2) was set in the isokinetic mode, which means that the preset cadence is automatically regulated independent of power output or gear selection by continuous adjustment of the degree of electromagnetic braking. Cadence was fixed at 100 rpm, which is a normal sprint cadence in elite road cyclists. Throughout the sprint, power output was continuously measured. Blood was sampled from a hyperemic earlobe after 3 min of recovery for determination of peak lactate concentration (Analox PLM5, London, UK) and pH (Radiometer ABL510, Copenhagen, Denmark). For both the time trial and the final sprint, subjects were instructed to remain seated in the saddle at all times, and no verbal encouragement was given at any time.
;)
FIGURE 1: Schematic presentation of the exercise protocol used in the pretest and the posttest. Exercise intensity is indicated as a percentage of the estimated MLSS, except during the time trial (10 min) and the sprint (30 s) where maximal performance was requested. Timing of capillary blood sampling is indicated by an arrow.
After the pretesting, subjects were allocated to one of two treatment groups (βALA or PL). On the basis of the results of the pretest, subjects were pair matched for age, body weight, and V˙O2max and total work outputs (Kj) during both the time trials and the final sprint. Thereafter, the pairs were randomly allocated to either the βALA or the PL group, and the 8-wk supplementation period was started. βALA (Aminolabs, Hasselt, Belgium) was administered in 500-mg capsules to be ingested at regular intervals throughout the day. Dosage was gradually increased from 2 g·d−1 (weeks 1 and 2) to 3 g·d−1 (weeks 3 and 4) and eventually 4 g daily from week 5 to the end of the study. PL capsules contained maltodextrin and were identical in appearance to the βALA capsules. Halfway and at the end of the supplementation period, subjects received a standard questionnaire to evaluate the occurrence of possible side effects. At the end of the supplementation period, subjects participated in the posttesting, which was identical to the pretesting. Pre- and posttesting was performed on the same day of the week and on the same time of the day. During the pretesting, each subject could select a convenient cadence in both the incremental exercise tests, the 110-min simulated road race and the time trial. Still, subjects were instructed to maintain their cadence constant within each of the three exercise bouts. Cadence for each bout was registered, and subjects were instructed to reproduce a similar cadence during the posttesting.
Data analysis
For the time trial, mean power output per minute (W) over the entire 10-min bout was calculated. For the 30-s sprint bout, peak power output (W), mean power output (W), and final power output (average power output during the final 2-3 s of the sprint, W) were determined. Furthermore, percent fatigue was calculated as the percent power drop from peak power to final power output. Data are expressed as mean ± SEM, and 95% confidence intervals (95% CI) were calculated as mean ± SEM × 2.365 or 2.306 (for n = 8 and 9, respectively) (24). Differences between the experimental groups were evaluated using two-way analysis of variance with one repeated-measure factor. Paired t-tests were used to evaluate treatment effects within groups (Statistica 7.1 Statsoft, Tulsa, OK). A P value lower than 0.05 was considered to be statistically significant.
RESULTS
Incremental exercise tests
Subjects performed a maximal incremental exercise test in both the pretesting and the posttesting. In the pretesting, time to exhaustion was 48.4 ± 2.5 min in PL versus 49.7 ± 3.6 min in βALA (not significant, NS) and was increased by approximately 10% (95% CI = 2.3-4.0 to 16.1-16.3%; P = 0.02) in either group in the posttesting (PL = 53.3 ± 2.5 min; βALA = 54.2 ± 2.6 min; NS). Rate of peak oxygen uptake (V˙O2peak) in the pretesting was 60.8 ± 2.90 mL·min−1·kg−1 in PL and was similar in βALA (59.9 ± 2.3 mL·min−1·kg−1). Corresponding peak heart rates and blood lactate concentrations were 193 ± 3 beats·min−1 and 6.75 mmol·L−1 in PL versus 190 ± 3 beats·min−1 and 7.9 ± 0.3 mol·L−1 in βALA (NS). V˙O2peak, heart rate, and blood lactate values at exhaustion were similar between the pretesting and the posttesting in either group (NS).
Body weight, side effects, and treatment blinding
Body weight (kg) was similar between groups in the pretesting (PL = 71.5 ± 2.5; βALA = 73.4 ± 2.2; NS) as well as in the posttesting (PL = 70.9 ± 2.9; βALA = 73.1 ± 3.0; NS). None of the subjects reported any significant side effect during the period of the study. At the end of the study, the subjects were asked as to whether they could identify the treatment received. Both in PL and βALA, two subjects correctly identified their treatment, whereas the others were either unsure or guessed the wrong treatment. Thus, treatment identification was not different between the two experimental groups.
Time trial performance
Initial workload was 281 ± 15 W in the PL group versus 289 ± 19 W in the βALA group (NS) in the pretesting and was set identical in the posttesting. Workloads increased by approximately 5-10% from the start to the end of the time trials (Table 1, Fig. 2). Compared with the pretesting, both βALA and PL subjects on average produced slightly higher power outputs in the posttesting (+3.1%; 95% CI = +0.9 to +5.3%; P = 0.01), but there was no difference between the groups at any time. Thus, mean power output was similar between groups during either the pretesting or the posttesting. From the start to the end of the time trial, blood lactate concentration increased from ∼1-2 to ∼6-7 mmol·L−1 (P = 0.0002), whereas pH dropped from ∼7.40 to ∼7.30 (P = 0.0003). However, lactate and pH values were identical between PL and βALA, either in the pretesting or in the posttesting.
TABLE 1: Effect of βALA supplementation on 10-min time trial performance.
FIGURE 2: Effect of βALA intake on 10-min time trial performance. Mean values ± SEM for PL (n = 8) and βALA (n = 9) in the pretest (○) and the posttest (•) are given.
Sprint performance
In the pretesting, peak, mean, and final power outputs as well as percent fatigue were similar between PL and βALA (Table 2). However, in the posttesting and compared with PL, both peak (+11.4%; 95% CI = +7.8 to +14.9%; P = 0.0001) and mean power output (+5.0%; 95% CI = +2.0 to +8.1%; P = 0.005) were higher in βALA. Figure 3 clearly shows that βALA slightly increased power production in every single subject, whereas in PL power output slightly decreased from the pretesting to the posttesting in the majority of the subjects. In both the pretesting and the posttesting, the sprint bout in either group increased blood lactate concentration to a final value of approximately 7 mmol·L−1, whereas blood pH on average was approximately 7.25.
TABLE 2: Effect of βALA supplementation on 30-s sprint performance.
FIGURE 3: Effect of βALA intake on 30-s sprint performance. Individual values and mean ± SEM for PL (○, n = 8) and βALA (•, n = 9) in the pretest and the posttest are given.
DISCUSSION
Performance in endurance competitions such as long-distance running and cycling is largely determined by the potential for oxidative energy provision in active muscles. However, for a similar degree of aerobic performance, victory often depends on the potential to develop high anaerobic power outputs during decisive stages of the race, for instance the final sprint. Therefore, the present study aimed to evaluate the effect of βALA supplementation on sprint performance during the final stage of a simulated endurance cycling event. We did not measure muscle carnosine content. However, based on recent observations by others (4,11,13,17), it is reasonable to assume that the 8-wk supplementation protocol used caused a greater than 50% increment of carnosine content in both Type I and Type II muscle fibers and thus inevitably increased the intramyocellular buffer capacity. We observed here for the first time that such βALA administration regimen enhances sprint power output at the end of a simulated endurance race and therefore could be an effective strategy to improve sprint performance in a real-life competition.
To date, only a small number of studies have looked at the effect of βALA supplementation on performance in short maximal exercise and have yielded equivocal results. Hill et al. (13) first reported that 10 wk of βALA supplementation enhanced total work output during an approximately 150-s cycle capacity test by approximately 16% compared with PL. Moreover, the increase in total work output during the cycle capacity test was well correlated with the increase in muscle carnosine content at the end of the supplementation period (11). More recently, however, Derave et al. (4) found 4 wk of βALA administration in well-trained sprinters not to be ergogenic in either a well-controlled 400-m run on an indoor track or an approximately 3 min of sustained isometric contraction of the knee extensor muscles (45% MVC) to exhaustion or in a single bout of 30 maximal voluntary knee extensions on an isokinetic dynamometer. These differential findings conceivably are at least partly explained by the smaller increase in muscle carnosine content (<50%) in the study by Derave et al. (4) than that in the earlier study by Hill et al. (13) (+80%) as well as by the different exercise protocols used. Interestingly, however, Derave et al. (4) also demonstrated that βALA significantly enhanced torque production during an intermittent exercise bout involving five series of 30 maximal knee extensions, interspersed by 1-min rest intervals. However, the ergogenic effect only emerged during the last two series of muscle contractions. In keeping with such observation, here we demonstrate 8 wk of supplementary βALA intake to consistently enhance sprint power output (+11%; 95% CI = +7.8 to +14.9%; see Fig. 3) at the end of a 2-h intermittent endurance exercise bout with a high glycolytic contribution. The anaerobic nature of the sprint exercise is evidenced by the high blood lactate concentrations (∼7 mmol·L−1) at the end of the 10-min time trial preceding the final sprint. However, time trial performance per se was not beneficially affected by βALA.
On the basis of the current and the previous observations by Derave et al. (4), we postulate that muscle carnosine pool during high-intensity intermittent exercise may act as a recyclable intramyocellular first-line buffer and by this action alleviates exercise-induced depletion of the endogenous bicarbonate pool (∼alkaline reserve). Hence, total buffer capacity in the later stages of high-intensity intermittent exercise is increased. The pKa value of the imidazole ring of carnosine is 6.83, which means that in the range of pH values existing in muscle cells during intermittent high-intensity muscle contractions, carnosine acts as an immediate H+ buffer during episodes of excess H+ production (∼high-intensity contractions). Thereafter, during recovery episodes, protons are progressively released from the imidazole ring as normal intramyocellular pH is restored by an enhanced activity of pyruvate dehydrogenase, increasing the rate of pyruvate oxidation and hence mitochondrial H+ utilization (22) on the one hand, and by H+ export from muscle cells on the other hand. Support for carnosine to act as a first-line buffer pool in muscle fibers also comes from recent data by Stout et al. (25), showing the ventilatory threshold to shift to a higher exercise load during incremental exercise after βALA intake. Such shift of the ventilatory threshold could at least partly result from delayed CO2 production from bicarbonate buffering.
Increased intramyocellular buffer capacity by definition should result in the potential to increase glycolytic energy production during the final stage of a maximal exercise bout and thus result in higher peak lactate concentrations in active muscles and blood. However, we could not demonstrate increased blood lactate level due to βALA intake. Our unpaired study design may have been insufficiently powerful to detect a small increase in blood lactate concentration. Furthermore, blood lactate concentration conceivably may be a too raw measure to detect a small increase in intramyocellular lactate concentration in these motor units accounting for the bulk of power production during the final stage of an exhaustive sprint. Alternatively, however, it must also be considered that increased muscle carnosine content might also be ergogenic via other physiological mechanisms than solely an increment in intramuscular buffer capacity. In fact, the direct role of H+ accumulation in muscle fatigue is still debated, and it has been proposed that Pi and adenosine 5′-diphosphate resulting from net adenosine 5′-triphosphate breakdown, by inhibiting sarcoplasmic reticulum calcium release as well as reducing myofibrillar Ca2+ sensitivity, are more important triggers of muscle fatigue than acidosis. In vitro studies indicate that carnosine does not affect sarcoplasmic reticulum calcium release (5) but can increases myofibrillar Ca2+ sensitivity particularly in glycolytic muscle fibers (5,19). However, whether these observations in skinned muscle fibers exposed to physiological carnosine concentrations also apply to intact human myofibers during high-intensity contractions, indeed, remains to be established.
Research data concerning the physiological and health effects of βALA supplementation in humans to date are very scarce, and therefore caution certainly is warranted. Still, it appears from available studies that supplementary oral βALA intake according to dosage regimens used in the present and previous studies (4,11,13,17), notably ≤5 g·d−1 and ingested as repeated small doses (<1 g) with 1- to 2-h intervals throughout the day for a period up to 10 wk, is probably safe and does not yield harmful side effects. However, symptoms of flushing (∼prickly sensation at the skin) may occur (11). In the current study, subjects ingested 500-mg capsules (∼7 mg·kg−1 body weight) eight times per day, and none of them complained of flushing. However, Harris et al. (11) have previously shown that ingestions in the range of 10 mg·kg−1 body weight may induce mild symptoms of flushing in some individuals (∼25%), whereas doses higher than 20 mg·kg−1 body weight rather consistently induce severe symptoms. Stout et al. (25) recently postulated that a minimum effective dose of 65-86 mg·kg−1 body weight per day is needed for performance enhancement in young males or females. Our current observations contradict this presumption by showing that a daily dose of approximately 55 mg·kg−1 body weight consistently enhances sprint performance in moderately to well-trained young healthy volunteers. Calculated CI indicate that in experimental conditions similar to the present study, such βALA dose in 95% of the studied population may be expected to increase peak power output during a 30-s sprint at the end of an exhaustive endurance event between 8% and 15% whereas mean power output increases by 2-8%.
Creatine is a nutritional supplement that has a well-established ergogenic action during sprint and repeated-sprint exercise (12,26,28-30). We have previously demonstrated that creatine can also be ergogenic in sprint performance at the end of an endurance cycling event (27), which is comparable with the beneficial effect of βALA in the current study. In the latter study, creatine loading for 5 d (20 g·d−1) increased power output during five 10-s intermittent sprints at the end of a 2-h exhaustive endurance exercise bout by approximately 5%. Because creatine and βALA act via different cellular mechanisms, one could prematurely assume that the effects of both ergogenic agents may be additive. However, it has been clearly documented that oral creatine supplementation significantly increases body weight, and in some individuals this increase may be substantial (>2 kg). Hence, creatine supplementation in road cyclists is unpopular because decreased power output (W) per kilogram of body weight by definition impairs uphill riding performance. In this regard, Kendrick et al. (17) have recently demonstrated that βALA supplementation in conjunction with resistance training increased neither body weight nor fat-free mass. Accordingly, both in the present and previous studies (11,13,25), body mass was unchanged by 2-8 wk of βALA supplementation. Therefore, βALA is probably recommended over creatine to enhance final sprint performance in endurance athletes competing in sports where an elevated body weight might negatively impact on performance.
In conclusion, the current study provides novel evidence that supplementary βALA intake is an effective strategy to enhance power output during the final sprint at the end of an endurance cycling competition.
Ruud Van Thienen and Karen Van Proeyen contributed equally to this work.
There is no conflict of interest to be reported. The results of the present study do not constitute endorsement by ACSM.
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