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00005768-201301000-0000800005768_2013_45_43_everaert_supplementation_1miscellaneous-article< 119_0_23_6 >Medicine & Science in Sports & Exercise©2013The American College of Sports MedicineVolume 45(1)January 2013p 43–51Effect of Beta-Alanine and Carnosine Supplementation on Muscle Contractility in Mice[BASIC SCIENCES]EVERAERT, INGE1; STEGEN, SANNE1; VANHEEL, BERT2; TAES, YOURI3; DERAVE, WIM11Department of Movement and Sport Sciences, Ghent University, Ghent, BELGIUM; and 2Department of Basic Medical Sciences, Division of Physiology, Ghent University, Ghent, BELGIUM, and 3Department of Endocrinology, Ghent University Hospital, Ghent, BELGIUMAddress for correspondence: Wim Derave, Ph.D., Watersportlaan 2, B-9000 Ghent, Belgium; E-mail: for publication February 2012.Accepted for publication July 2012.ABSTRACTPurpose: Enhanced carnosine levels have been shown to be ergogenic for high-intensity exercise performances, although the role of carnosine in the control of muscle function is poorly understood. Therefore, the aim of this study was to investigate the effect of long-term supplementation with increasing doses of carnosine and beta-alanine on muscle carnosine, anserine, and taurine levels and on in vitro contractility and fatigue in mice.Methods: Male Naval Medical Research Institute mice (n = 66) were control fed or supplemented with either carnosine (0.1%, 0.5%, or 1.8%) or beta-alanine (0.6 or 1.2%) in their drinking water for 8–12 wk. Soleus and extensor digitorum longus (EDL) were tested for in vitro contractile properties, and carnosine, anserine, and taurine content were measured in EDL and tibialis anterior by high-performance liquid chromatography.Results: Only supplementation with 1.8% carnosine and 1.2% beta-alanine resulted in markedly higher carnosine (up to +160%) and anserine levels (up to +46%) compared with control mice. Beta-alanine supplementation (1.2%) resulted in increased fatigue resistance in the beginning of the fatigue protocol in soleus (+2%–4%) and a marked leftward shift of the force–frequency relation in EDL (10%–31% higher relative forces).Conclusion: Comparable with humans, beta-alanine availability seems to be the rate-limiting step for synthesis of muscle histidine-containing dipeptides in mice. Moreover, muscle histidine-containing dipeptides loading in mice moderately and muscle dependently affects excitation–contraction coupling and fatigue.Mammalian skeletal muscles contain high concentrations of histidine-containing dipeptides (HCD), in the form of carnosine (β-alanyl-L-histidine), anserine (β-alanyl-1-methylhistidine), or ophidine (β-alanyl-3-methylhistidine). The muscular content of carnosine, the only HCD found in humans, has been related to high-intensity exercise performance, as reviewed by Sale et al. (28) and Derave et al. (10). Harris et al. (19,20) were the first to show that the long-term supplementation (4–10 wk, 4–6 g·d−1) with beta-alanine, the rate-limiting amino acid for muscle carnosine synthesis, leads to markedly enhanced human muscle carnosine levels and subsequently improved high-intensity performance. As summarized in a recent meta-analysis by Hobson et al. (21), chronic beta-alanine supplementation has been shown to be ergogenic for cycling tests until exhaustion at 110% of maximal power (approximately 2.5 min [20,29]) or with increasing load (start, 40 W; every 3 min, +20 W [34]), for the sprint at the end of 2-h simulated cycling race (38), for the time to complete a simulated 2000-m rowing race (approximately 6 min [2]), but not for a 400-m run (12) or for repeated sprint performance (10 × 5 s [35]). Despite the increasing evidence for the ergogenic effect of carnosine during high-intensity exercise that lasts several minutes, the role of carnosine in the control of muscle contractile function is poorly understood.To explore the function of carnosine during contraction in rodents, the development of an optimal HCD loading protocol in skeletal muscles is essential. To our knowledge, only two studies have reported enhanced muscle carnosine levels in rats as a result of carnosine supplementation (24,36). However, the researchers did not study the resulting effects on muscle function. Carnosine supplementation for 2 wk with 1.8% and 5% of the diet has been shown to increase the gastrocnemius carnosine content by 2- and 2.2-fold, respectively, (36) and Maynard et al. (24) reported a fivefold increase of soleus, but not of gastrocnemius, carnosine content as a result of 8 wk of carnosine supplementation (1.8% w/w). Although not investigated in these two studies, one would expect that muscle anserine content will be affected by long-term carnosine supplementation. The effect of beta-alanine supplementation on rodent skeletal muscle HCD content has never been reported, despite the clear increases in muscle carnosine of both humans (19) and horses (14) as a result of beta-alanine supplementation. An optimal HCD loading protocol in rodent skeletal muscle can lead to a better understanding of the function of carnosine on intact skeletal muscle or even during in vivo or in situ muscle contraction in rodents. However, caution is warranted, because treatment with 3% beta-alanine for 1 month diminished rat muscle taurine content by 50% (8) and taurine has been shown to be involved in muscle contractility (18). Therefore, this study was undertaken to investigate the effect of increasing doses of carnosine and beta-alanine for several weeks on carnosine, anserine, and taurine muscle content in mice. The effect of carnosine and beta-alanine supplementation on twitch and tetanus characteristics, force–frequency relation, and fatigability during isolated muscle contractions was also examined to gain more insight in muscle contractile function in HCD-enhanced muscles.A reduced fatigability is expected, mainly in muscles with a high proportion of fast-twitch muscle fibers, because the attenuation of intracellular acidosis is the most widely proposed ergogenic mechanism of carnosine due to its pKa of 6.83 (37). Baguet et al. (4) demonstrated the role of carnosine as a pH buffer when the decline in blood pH was attenuated during a 6-min high-intensity cycling exercise bout after 4–5 wk of beta-alanine supplementation. Muscle carnosine content is also positively correlated with total proton buffering capacity (23), and Hill et al. (20) showed that the contribution of carnosine to the nonbicarbonate buffering capacity can be increased from 7% at baseline to 17.3% after 10 wk of beta-alanine supplementation. As already mentioned by Sale et al. (28), the determination of muscle buffering capacity by this titration method is only an assumption and is probably an underestimation of the contribution of carnosine to the nonbicarbonate buffering capacity, meaning that the current estimations of the contribution of muscle carnosine are approximate minimum values.Other than the potential of pH buffering of carnosine, an improved calcium handling during muscle contraction has also been proposed as another mechanism behind the ergogenic potential of carnosine. Russian studies have suggested that the Severin phenomenon (reduced contractile fatigue when frog muscles are exposed to increased extracellular carnosine) is explained by an increase in sarcoplasmic reticulum (SR) Ca2+ release channel activity by carnosine (6,27). In this study, a leftward shift of the force–frequency relation as a result of muscle carnosine loading is expected, because this was shown in chemically skinned fiber preparation of frogs (22) as well as in mechanically skinned rat muscle fibers (15) and, very recently, also in human muscle fibers (both Type I and Type II) (16), suggesting an improved Ca2+ handling in the presence of elevated carnosine levels. Furthermore, the proton buffering action of carnosine can also indirectly improve calcium handling as H+ can compete with calcium ions at the troponin-binding site (17).In this study, Naval Medical Research Institute (NMRI) mice were supplemented with either carnosine (0.1%, 0.5%, and 1.8%) or beta-alanine (0.6% and 1.2%) for 8–12 wk in their drinking water. It was hypothesized that enhanced carnosine levels will contribute to reduced fatigability, mainly in extensor digitorum longus (EDL), and a leftward shift of the force–frequency relation was expected.MATERIALS AND METHODSAnimalsAll animals were allowed free access to food and water at room temperature and were exposed to a light cycle of 12 h·d−1. A total of 66 NMRI mice (45.9 ± 5.9 g body weight (BW); range, 36–62 g BW) were used in this study, divided into six groups. All animals received a standard chow (not containing carnosine or derivatives) but a different drink (carnosine or beta-alanine dissolved in water) according to the experimental groups (Table 1). Eighteen control animals received water. Because the optimal muscle HCD loading protocol in mice was not yet established, two low doses of carnosine were first tested on a small group of eight animals being either 0.1% carnosine (n = 4 (30) and 0.5% carnosine (n = 4). Because these doses did not result in elevated HCD muscle levels, the supplementation dose was increased to 1.8% carnosine (n = 22, [24,36]). Beta-alanine has frequently been shown to elevate muscle carnosine content both in humans (19) and horses (14); therefore, the effect of beta-alanine supplementation with two different doses was tested on muscle HCD levels in mice: 0.6% beta-alanine (n = 9) or 1.2% beta-alanine (n = 9). Caution was warranted as supplementation with 3% beta-alanine for several weeks has frequently been used as a strategy to deplete taurine content of several organs, including skeletal muscle by 50% (8).TABLE 1 Characteristics of control mice, carnosine-supplemented, and beta-alanine–supplemented mice.The intervention period lasted 8 wk, except for the lower carnosine doses (0.1% and 0.5%), which lasted 12 wk. Bottles were refreshed two times a week, and BW and drinking volume per cage (three to four animals) were recorded. One mouse of the 1.8% carnosine-supplemented group died during the supplementation period. After supplementation, mice were anaesthetized by an intraperitoneal infusion of 80% Rompun–20% Ketalar (5 μL·g−1 BW). After careful dissection of soleus, tibialis anterior, and EDL, mice were killed by cervical dislocation. The experimental protocol was approved by the Ethics Committee for Animal Research at University Hospital Ghent, and all procedures adhered to the American College of Sports Medicine animal care standards.Muscle contractile propertiesAfter dissection of the soleus and EDL muscles, wires were attached to the tendons and muscles were mounted vertically in an incubation bath with one tendon attached to a force transducer (PowerLab, ADInstruments, Spechbach, Germany) and stimulated with capacitor discharges (pulse duration, 500 μs, 100 mA) between platinum electrodes. The incubation medium (10 mL) was a Krebs–Henseleit solution (117 mM NaCl, 25 mM NaHCO3, 5 mM KCl, 1 mM MgSO4, 1 mM KH2PO4, 2.5 mM CaCl2, 8 mM mannitol, and 2 mM pyruvate), which was continuously gassed with a mixture of 95% O2 and 5% CO2 and maintained at 30°C. After mounting, a 15-min stabilization period was allowed and optimal muscle length (L0) was determined by tetanic contractions (350-ms duration, with 50- and 100-Hz stimulation frequency and 1- or 2-min interval for soleus and EDL, respectively). Force (mN), specific force (force corrected for cross-sectional area (N·cm−2)), and contraction (maximum slope (N·s−1)) and relaxation speed (minimum slope; N·s−1) were determined on the mean of three twitches and on a tetanic contraction (350 ms, 50 Hz for soleus and 125 Hz for EDL). Cross-sectional area was determined by dividing the wet muscle mass (g) by the product of optimal muscle length (L0 (cm)) and 1.06 g·cm−3, the density of mammalian skeletal muscle. Next, the force–frequency relation was determined by stimulating at 10, 20, 35, 50, 75, and 100 Hz with 1-min rest interval for soleus and stimulating at 25 (1-min rest), 40 (1-min rest), 55 (1-min rest), 70, 100, 125, 150, and 175 Hz with 2-min rest interval, unless otherwise stated, for EDL. Furthermore, fatigability was evaluated as the percentage decrease in tetanic force (relative to initial force) during 8 min of repeated tetanic contractions (train duration, 350 ms; soleus, 50 Hz every 5 s; EDL, 100 Hz every 10 s). Stimulation frequencies and rest intervals were adapted to the differences in muscle fiber type composition between soleus (slow) and EDL (fast) to evoke a similar profile in force–frequency relation and force decline during fatigue.Muscle content of carnosine, anserine, and taurineAny visible connective or fat tissue was removed from tibialis anterior, EDL, and soleus muscle from the contralateral leg, immediately frozen in liquid nitrogen and stored at −80°C. The muscles were freeze dried and subsequently dissolved in phosphate-buffered saline solution (100 μL per 1 mg muscle) for homogenization. Muscle homogenates were deproteinized using 35% sulfosalicylic acid and centrifuged (5 min, 14,000g). Deproteinized supernatant (100 μL) was dried under vacuum (40°C). Dried residues were resolved with 40 μL of coupling reagent: methanol/triethylamine/H2O/phenylisothiocyanate (7:1:1:1) and allowed to react for 20 min at room temperature. The samples were dried again and resolved in 100 μL of sodium acetate buffer (10 mM, pH 6.4). The same method was applied to the combined standard solutions of carnosine (Flamma, Italy), anserine (Sigma), and taurine (Sigma). The derivatized samples (20 μL) were applied to a Waters high-performance liquid chromatography (HPLC) system with a Hypersilica column (4.6 × 150 mm, 5 μm) and ultraviolet detector (wavelength, 210 nm). The column was equilibrated with buffer A (10 mM sodium acetate adjusted to pH 6.4 with 6% acetic acid) and buffer B (60% acetonitrile–40% buffer A) at a flow rate of 0.8 mL·min−1 at room temperature. Limit of detection and quantification of muscle homogenates were 3 and 10 μM, respectively.StatisticsData are presented as mean ± SD, and significance level was set at P ≤ 0.05. The effects of the different supplementation protocols on muscle metabolites, drinking behavior, and BW were analyzed with a one-way ANOVA, and post hoc Tukey tests were performed when a significant group effect was shown. Correlations between carnosine and anserine in one muscle and across the muscles were obtained with Pearson correlations. Each individual stimulation frequency in the force–frequency analysis and every sixth peak in soleus and third peak in EDL during the fatigue protocol were analyzed with independent sample t-tests (supplementation vs control group).RESULTSBW and Drinking BehaviorThe treatment with beta-alanine or carnosine influenced the total drinking volume of mice (P < 0.001, Table 1). Control mice drank significantly more water compared with the 1.8% carnosine (−12.7% vs control, P = 0.003), 0.6% beta-alanine (−28% vs control, P = 0.001), and 1.2% beta-alanine group (−35% vs control, P < 0.001). The dietary intervention had no effect on BW gain (P = 0.132, Table 1) over the supplementation period. The mean BW at the end of the supplementation period was for both the control group and the three carnosine groups approximately 47 g and was slightly lower in the beta-alanine–supplemented groups (0.6% beta-alanine: 43 g, P vs control group = 0.231; 1.2% beta-alanine: 41 g, P vs control group = 0.066).Muscle Content of Carnosine, Anserine and TaurineSoleus samples displayed carnosine and anserine levels, which were too low to be reliably quantifiable <1 mmol/kg dw. In the other muscles, carnosine and anserine content were positively correlated to each other (EDL: n = 52, r = 0.85, R2 = 0.71, P < 0.001; tibialis anterior: n = 64, r = 0.74, R2 = 0.49, P < 0.001), and there was a positive correlation between the carnosine content of EDL and tibialis anterior (n = 51, r = 0.56, R2 = 0.25, P < 0.001), whereas this was less strong for anserine (n = 51, r = 0.23, R2 = 0.10, P = 0.023).Carnosine supplementationIn control mice, carnosine levels were higher in EDL compared with tibialis anterior (4.20 ± 1.34 mmol·kg−1 dry weight (DW) vs 2.48 ± 0.9 mmol·kg−1 DW, respectively, P = 0.002), although anserine levels were higher in tibialis anterior (8.57 ± 2.82 mmol·kg−1 DW vs EDL: 7.03 ± 1.47 mmol·kg−1 DW, P = 0.009). Supplementation for 12 wk with low doses of carnosine (0.1% and 0.5%) in the drinking water did not affect muscle HCD content (Table 2A). Ingestion of 1.8% carnosine in drinking water for 8 wk, however, enhanced carnosine (+57%, P = 0.032) and anserine (+30%, P = 0.01) content in EDL but not in tibialis anterior. Muscle taurine levels were not lowered as a result of carnosine supplementation and were even higher in EDL of 0.5% supplemented mice compared with control mice (P = 0.049, Table 2A).TABLE 2 Effect of increasing doses of carnosine (A) and beta-alanine (B) supplementation on mean ± SD content of carnosine, anserine, and taurine (mmol·kg−1 DW) measured in tibialis anterior and EDL muscles.Beta-alanine supplementationSupplementation with a low dose of beta-alanine (0.6%) did not result in higher muscle HCD levels (Table 2B). Mice supplemented with 1.2% beta-alanine, however, had significantly higher muscle carnosine levels (EDL: +156%, P < 0.001; tibialis anterior: +160%, P < 0.001) compared with control mice (Table 2B). Concerning anserine, 1.2% beta-alanine supplementation resulted in higher EDL (+46%, P < 0.001) and trend to higher tibialis anterior (+32%, P = 0.08) levels compared with control (Table 2B). Muscle taurine content was lower in tibialis anterior of 1.2% beta-alanine–supplemented mice (−18% vs control mice, Table 2B).A significant positive correlation was observed between accumulated ingested dose of beta-alanine (or equivalent of beta-alanine) and carnosine content in EDL (P = 0.001, r = 0.492, Fig. 1) and tibialis anterior (P = 0.024, r = 0.333).FIGURE 1. Relation between total amount of beta-alanine or beta-alanine equivalent (g·kg−1 BW) ingested during supplementation protocol and (A) individual muscle carnosine content (percentage compared with control) for mouse EDL (P = 0.001, r = 0.492) and (B) mean muscle carnosine content (percentage to control) for mouse EDL and tibialis anterior (present study) compared with human data.Effect of Supplementation on Twitch and Tetanus Contractile PropertiesThe following results concerning contractile properties are only displayed for the groups with enhanced HCD levels, namely, 1.8% carnosine and 1.2% beta-alanine compared with controls. Force, specific force, and maximum and minimum slope of twitch and tetanus contractions were not affected by carnosine or beta-alanine treatment (data not shown). The twitch and tetanus (mean ± SD) characteristics for control group were as follows: force, 16.21 ± 6.05 and 139.44 ± 43.48 mN; specific force, 2.78 ± 1.64 and 22.88 ± 10.56 N·cm−2; maximum slope, 2.19 ± 0.75 and 2.05 ± 0.66 N·s−1; minimum slope, −0.68 ± 0.23 and −3.91 ± 1.49 N·s−1, respectively.Effect of Supplementation on Force–Frequency RelationAs depicted in Figure 2A and B, the relation between stimulation frequency and force (percentage of maximum force) was affected by beta-alanine supplementation. The force output at 20 Hz in soleus tended to be higher in 1.2% beta-alanine group compared with control (+32%, P = 0.058) but was not influenced by 1.8% carnosine supplementation (Fig. 2A). In EDL, the amount of relative force produced in the muscle of mice supplemented with 1.2% beta-alanine was increased (+10%–31%; P < 0.05) compared with that of control mice over the range of 25–125 Hz, except for 40 Hz (Fig. 2B). Thus, the largest increase in muscle carnosine content (i.e., by 1.2% beta-alanine supplementation) induced a leftward shift of the force–frequency relation.FIGURE 2. The relation between relative force (percentage of maximum force) and stimulation frequency in soleus (A) and EDL (B) separated by supplementation group (SD was removed for clarity). A, In soleus, a trend for a higher force was observed in the 1.2% beta-alanine–treated group at20 Hz (P = 0.058), whereas supplementation with 1.8% carnosine had no effect on force–frequency relation. B, Beta-alanine supplementation (1.2%), but not carnosine (1.8%), shifted the force–frequency relation to the left (*P < 0.05 vs control) in EDL.Effect of Supplementation on FatigueTo evaluate the effect of carnosine and beta-alanine supplementation on fatigue, repeated tetanic contractions were performed during 8 min. In soleus, the decline in force in the beginning of the fatigue protocol was attenuated as a result of 1.2% beta-alanine supplementation (Fig. 3A). During the first minute, the relative forces were 2%–4% higher as a result of 1.2% beta-alanine supplementation (30 and 60 s: P < 0.05; 90, 120, and 150 s: P < 0.1, Fig. 3B). However, the decline in force during repeated tetani was not attenuated in EDL, neither by 1.2% beta-alanine nor 1.8% carnosine supplementation. The percentage of initial force at 30 s was significantly lower in 1.8% carnosine-supplemented mice (93.4% vs 97.0% of control mice, P = 0.032, Fig. 3B).FIGURE 3. The effect of beta-alanine (1.2%) and carnosine (1.8%) supplementation for 8 wk on EDL and soleus fatigue. The decline in force (percentage of initial force) in soleus (A) was attenuated in the beginning of the fatigue protocol as a result of 1.2% beta-alanine supplementation (*P<0.05 and $P < 0.1 vs control), but not by 1.8% carnosine supplementation nor in EDL (B, *P < 0.05 for 1.8% carnosine vs control). In soleus, theslowing of relaxation during repeated tetani (C) was less pronounced in beta-alanine–supplemented mice (1.2%) versus control mice (*P < 0.05 and$P<0.1: 1.2% beta-alanine vs control) and the slowing of force development was attenuated only in EDL as a result of 1.8% carnosine supplementation at the end of fatigue protocol (D, *P < 0.05 and $P < 0.1: 1.8% carnosine vs control).The slowing of relaxation rate during fatigue (% of initial minimum tetanic slope) was attenuated (P < 0.05: 60–180 s; P < 0.1: 210–270 s; Fig. 3C) during the first 3 min in soleus of 1.2% beta-alanine–supplemented mice but not in EDL (data not shown). The maximum rate of tetanic force development altered during fatigue, with the highest maximum slope at 1 min and diminished to approximately 50% of its maximum velocity after 8 min of repeated tetani. The EDL maximum tetanic slope was less reduced in 1.8% carnosine-supplemented mice compared with control at the end of fatigue protocol (P < 0.05: 270–480 s, except 420 and 450 s; P < 0.1: 210 s; Fig. 3D).DISCUSSIONThis study shows that the long-term supplementation with high doses of both carnosine (1.8%) and beta-alanine (1.2%) can increase HCD content (up to +160%) in skeletal muscle of NMRI mice. Furthermore, these enhanced HCD levels in the beta-alanine treated group (1.2%) are accompanied with a leftward shift of the force–frequency relation in EDL. Beta-alanine supplementation (1.2%) resulted in a reduced fatigability in soleus during isolated muscle contractions.Muscle carnosine, anserine, and taurine levelsLong-term beta-alanine supplementation (3%) has frequently been used in rodents as taurine-depleting agent, although muscle carnosine and anserine content have never been determined. This study shows for the first time that long-term supplementation with 1.2% beta-alanine results in markedly elevated carnosine levels in both EDL (+156%) and tibialis anterior (+160%) and only a minimal decrease in taurine content in one muscle (−18%, tibialis anterior). Carnosine and taurine have some common properties in skeletal muscle (antioxidant and calcium regulation), and it is therefore important to pay attention to the possible interaction between those two metabolites. An increase in muscle carnosine content could maybe explain why there is no deleterious effect of beta-alanine supplementation (that was used to deplete muscle taurine levels) on, for example, exercise-induced muscle injury (TBARS (8)) or on lipid peroxidation in the liver (26) in studies with rodents.The markedly enhanced carnosine content in mouse skeletal muscle as a result of beta-alanine supplementation suggests that beta-alanine is the rate-limiting precursor for carnosine synthesis in mice. Figure 1 shows that the increase in mouse muscle carnosine levels is, like in humans (20,33), dependent on the total amount of ingested beta-alanine or equivalent of beta-alanine, although the process seems to be much slower. Because mice were placed with three to four per cage, we did not control for drinking volume, and therefore no group with isomolar dose of carnosine was included in the study.There are large interindividual differences in response to carnosine/beta-alanine supplementation in mice (Fig. 1). Some mice were nonresponders (mainly with accumulated dose of beta-alanine <60 g·kg−1 BW), and some had very pronounced increases of >300%. The mean 2.6-fold increase in this study as a result of beta-alanine supplementation, together with the apparent absence of a ceiling effect of human carnosine loading, suggests that the increases in human muscle carnosine content can be higher than so far reported (+80% in 10 wk (20) and +85% in 12 wk (9)). However, one should keep the possible side effects of long-term beta-alanine supplementation in mind and take the possible changes in muscle taurine content into account.The enhancement of muscle carnosine content (EDL, +60%; tibialis anterior, +30%, P) as a result of the same carnosine supplementation (1.8%) is less compared with the five- and twofold increases previously reported in soleus (24) and gastrocnemius (36) carnosine content in rats, respectively. Species differences in carnosine metabolism between rat and mouse could underlie these findings, because the mean total HCD content is approximately four times lower compared with rat (unpublished data measured with the same HPLC method) and also three times lower compared with human (3). In contrast to humans who are lacking anserine, the anserine content is markedly higher than carnosine content in both mice (anserine/carnosine ratio: 1.67–3.46) and rats (anserine/carnosine ratio: 1.59–3.79, unpublished data). Nevertheless, the increase in muscle anserine (+30%–50%) was always less compared with carnosine (+60%–160%), across the supplementation protocols, consistent with the study of Derave et al. (11) in which anserine (+40%) and carnosine (+88%) levels were increased after creatine supplementation in senescence-accelerated mice. To date, it is not fully understood whether anserine is synthesized as a result of the condensation between beta-alanine and 1-methyl-L-histidine (13) or by the direct methylation of carnosine by the carnosine-N-methyltransferase enzyme (Fig. 4), as suggested by several in vitro studies (7,25). If the condensation between beta-alanine and 1-methyl-L-histidine was to be the major pathway in anserine biosynthesis, we would expect the loading of carnosine and anserine after beta-alanine supplementation to be similar in this study. Because the differences (both absolute and relative) between mice from the control and 1.2% beta-alanine–supplemented group are higher for carnosine (tibialis anterior: +3.99 mmol·kg−1 DW or +160%; EDL: +6.57 mmol·kg−1 DW or +156%, respectively) than for anserine (tibialis anterior: +2.81 mmol·kg−1 DW or +32% (P > 0.05); EDL: 3.25 mmol·kg−1 DW or +46%), it is suggested that anserine is mainly synthesized by the methylation of carnosine (Fig. 4). Thus, both in vivo experiments with beta-alanine supplementation in mice (current study) and in vitro experiments with beta-alanine–incubated cultures of myoblasts (chicken, [7]) confirm that anserine is formed as a secondary product and therefore suggest that the methylation of carnosine is the major pathway to synthesize anserine.FIGURE 4. Possible pathways for anserine synthesis after beta-alanine and carnosine supplementation in mice. A, Part of the carnosine that enters the muscle is methylated to anserine by carnosine-N-methyl transferase (CMT). B, Elevated beta-alanine availability leads to enhanced carnosine levels through carnosine synthase (CS). Anserine is presumably formed by methylation of carnosine (CMT) rather than direct synthesis of beta-alanine with 1-methyl-histidine through CS (indicated by open arrow).Contractile functionThe loading of carnosine and anserine levels in EDL resulted in a marked leftward shift of the force–frequency relation. This shift was already shown in chemically skinned single muscle fibers of frog Sartorius muscle (22) and confirmed in single muscle fibers of rats and humans by Dutka and Lamb (15) and Dutka et al. (16). This study shows, for the first time, a leftward shift of the force–frequency relation and thus indirectly an improved calcium handling by carnosine loading in the whole and intact EDL muscle and through a nutritional intervention rather than direct exposure in vitro. However, our study is limited by the fact that we cannot differentiate whether this is the result of enhanced calcium release from the SR (6,27) or whether this is the result of improved calcium sensitivity (15). Furthermore, the lack of a shift in the force–frequency relation in soleus or after carnosine supplementation in EDL can be related to muscle-specific differences in contraction mechanism or to higher HCD levels after beta-alanine versus carnosine supplementation.Beta-alanine supplementation (1.2%) for 8 wk also resulted in reduced fatigability in isolated soleus, but not in EDL, which agrees with the study of Derave et al. (11) in which it was shown that an increase in HCD levels resulted in diminished fatigue in soleus, but not in EDL, during repeated tetanic contractions. We must acknowledge that our study is limited by the fact that the HCD levels of soleus were below the limit of detection of our currently used HPLC method. However, based on the facts that muscle carnosine loading in humans, as a result of beta-alanine supplementation, is comparable between all investigated muscle fiber types (5,20) and that carnosine supplementation in rats has been shown to elevate rat soleus carnosine content (24), one can assume that mice soleus HCD levels will be elevated in the current study and that the enhanced fatigue resistance is likely the result of enhanced HCD levels.Because carnosine has been proposed as an intracellular pH buffer, we hypothesized that the attenuation of fatigue would mainly occur in EDL, because it is the muscle with the most anaerobic metabolism. Surprisingly, fatigue evoked by repeated tetanic contractions was only attenuated in slow soleus muscle but not in the fast EDL after beta-alanine supplementation. The bath solution in which the contractions were performed was continuously gassed with 95% O2 and 5% CO2 to mimic the in vivo physiological buffer conditions of the surrounding blood. Therefore, it is possible that the evoked fatigue in these experiments was less dependent on the removal of protons compared with in vivo situations. Therefore, the improved fatigue resistance as a result of beta-alanine supplementation may have resulted from other mechanisms than pH buffering. However, because no direct measurements of H+, ROS, and/or Ca2+ were made, we can only speculate about the muscle-dependent mechanism of enhanced fatigue resistance.One possible mechanism could be the protection against exercise-induced production of reactive oxygen species in the muscle. However, the antioxidative function of carnosine is well documented by in vitro studies; the evidence in vivo is scarce. One animal experiment showed that 3% beta-alanine supplementation for 4 wk could result in enhanced muscle HCD levels (extrapolated from the results of present study) and can diminish the production of TBARS (lipid peroxidation products) during 90-min downhill running in rats although the performance was unaffected (8). In contrast, Smith et al. (32) did not find a significant decrease of exercise-induced lipid peroxidation in healthy female subjects. In the present study, we were unfortunately unable to measure the amount of contraction-induced TBARS production or other ROS markers because of the small muscle size. It remains to be elucidated whether carnosine could decrease exercise-induced oxidative stress.A second possible mechanism to explain the reduced fatigability could be related to the calcium handling (release, sensitivity, and/or reuptake) because there is an attenuated decrease in relaxation and contraction speed during the fatigue protocol. An interaction between the role of carnosine as pH buffer, antioxidant, and calcium regulator is not excluded because 1) ROS formation has been shown to be dependent on pH (31) and 2) both pH decline and ROS production have been associated with a modification of calcium handling during repeated contractions (for review, see Ref. (1)). Thus, further in vivo and in vitro research is needed to elucidate the potential of carnosine to enhance the interaction between H+, ROS, and Ca2+ during muscle contraction and subsequently exercise performance.To conclude, this study is the first to provide an optimal loading protocol for muscle HCD levels in mice, namely, beta-alanine supplementation with 1.2% of drinking water (or preferably somewhat less to avoid a decrease in muscle taurine levels) during 8 wk. This enables further research on contractile properties of carnosine on intact skeletal muscle or even during in vivo or in situ muscle contractions in rodents. Furthermore, beta-alanine supplementation resulted in a leftward shift of the force–frequency relation, predominantly in EDL, and attenuated fatigue during repeated tetani only in soleus. The exact ergogenic mechanism of muscle carnosine loading remains to be elucidated.This study was financially supported by grants from the Research Foundation—Flanders (FWO and G.0046.09). We thank Flamma (Italy) for generously providing carnosine.The practical contribution of Anneke Volkaert and Anouk Vanhoutte is greatly acknowledged. We thank Jean Lebacq for the inspiring discussion.The authors declare that they have no conflict of interest.The results of the present study do not constitute endorsement by the American College of Sports Medicine.REFERENCES1. Allen DG, Lamb GD, Westerblad H. Skeletal muscle fatigue: cellular mechanisms. Physiol Rev. 2008; 88: 287–332. [CrossRef] [Medline Link] [Context Link]2. Baguet A, Bourgois J, Vanhee L, Achten E, Derave W. Important role of muscle carnosine in rowing performance. J Appl Physiol. 2010; 109: 1096–101. [CrossRef] [Medline Link] [Context Link]3. Baguet A, Everaert I, De Naeyer H, et al.. 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