Carnosine (β-alanyl-l-histidine) is a multifunctional histidine-containing dipeptide (HCD) occurring naturally in high concentrations in mammalian skeletal muscle (1). It is also present in relatively large quantities in the central nervous system, particularly the olfactory epithelium and bulb (2), and in smaller amounts in the kidney (3) and myocardium (4). A large body of research shows that elevating skeletal muscle carnosine (m-carn) contents can improve anaerobic exercise capacity and performance (5). Although its ergogenic effects are clear, the actions of carnosine are complex. Putative physiological roles in human skeletal muscle include acting as an intracellular pH buffer (pHi) (6), direct modulation of energy metabolism (7), regulation of Ca2+ handling and myofilament sensitivity (8), quenching of reactive species, and detoxification of reactive aldehydes (9). Several recent publications provide new insights into these roles, which warrant a detailed discussion. In this review, we examine the research relating to each physiological role attributed to carnosine, and its rate-limiting precursor β-alanine, in exercising human skeletal muscle.
Carnosine and β-Alanine Metabolism
β-alanine availability is influenced by diet and, to a lesser extent, endogenous production from uracil degradation in the liver (10). A typical omnivorous diet provides ~300 to 550 mg·d−1, whereas a vegetarian diet provides virtually no β-alanine (11,12). Figure 1 depicts carnosine and β-alanine absorption and transport. After ingestion, carnosine reaches the small intestine where it enters enterocytes via peptide transporter 1. Tissue carnosinase (CN2), mainly present in the jejunal mucosa, hydrolyses carnosine into its constituent amino acids β-alanine and l-histidine (13). Small amounts of carnosine remain intact and enter the bloodstream via a basolateral peptide transporter, whereas β-alanine enters via a basolateral amino acid transporter. Highly active serum carnosinase (CN1) rapidly hydrolyses much of the remaining carnosine (13). Small amounts (≤14%) can be detected in urine during the 5 h after ingestion (14), meaning that a portion of the ingested carnosine circulates intact for a short period. Park et al. (15) confirmed this by showing a small increase in plasma carnosine, which peaked 3.5 h after consumption of cooked beef. Individuals with homozygosity for the (CTG)5 allele display lower CN1 activity which is associated with higher circulating plasma carnosine levels (16) and a reduced risk of developing diabetic nephropathy (17).
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FIGURE 1: A schematic depicting carnosine and β-alanine absorption and metabolism. The presence of carnosine in the blood is dependent on the ingested dose and CN1 activity. Specific transporters on the basolateral membrane have not been fully characterized. β-alanine metabolism in the liver represents a pathway observed in rodents, putative in humans, but requires validation. β-alanine transport is sodium and chloride-dependent. Carnosine transport into and out of skeletal muscle remains to be demonstrated in humans. See the corresponding text for the accompanying references. MSA, malonate semialdehyde; PAT1, proton-assisted amino acid transporter 1; PEP1/2, peptide transporter 1/2; PHT1, peptide-histidine transporter 1; TauT, taurine transporter; TCA, tricarboxylic acid.
Everaert et al. (18) and Saunders et al. (19) have respectively identified mRNA transcripts of peptide-histidine transporter 1 and peptide transporter 2 (PEPT2) in human skeletal muscle; however, direct uptake or release of carnosine has not yet been shown. Uptake of β-alanine into skeletal muscle occurs via proton-assisted amino acid transporter 1 and taurine transporter (18). Subsequently m-carn is synthesized in situ, catalyzed by carnosine synthase (20). Drozak et al. (21) identified the encoding gene as ATP-grasp-domain-containing protein 1 (ATPGD1), which shows a greater affinity for L-histidine (Km ~0.37 mM) than for β-alanine (Km ~0.09 mM). Due to low substrate affinity and availability, endogenous synthesis of carnosine is rate-limited by β-alanine availability (10); and high-dose l-histidine supplementation alone does not augment m-carn contents in humans (22).
Humans exhibit m-carn concentrations in the millimolar range (Fig. 2), similar to levels of adenosine triphosphate, phosphorylcreatine, and taurine (27). Several factors determine m-carn contents, with diet being the easiest to modify. The absence of dietary carnosine or β-alanine does not appear to influence m-carn contents in the short-term (≤6 month vegetarian diet) (28). However, long-term (≥1 yr and ≥8 yr) vegetarians display ~17% to 26% and 35% lower m-carn compared with omnivores, respectively (23,29). Harris et al. (10) first showed, in humans, that β-alanine supplementation (4 wk, 6.4 g·d−1) results in a substantial (~60%) increase in m-carn content. A response since shown to be reproducible, with an initial linear increase in response to the total β-alanine dose supplemented (Fig. 3). Saunders et al. (19) supplemented 1075 g over 24 wk, representing the largest total β-alanine dose in the literature, which led to a 119% ± 42% increase in m-carn from baseline and a plateau from 20 to 24 wk. The average time to reach maximal m-carn content was 18 ± 6 wk, which may indicate a putative saturation point. Some participants continued to exhibit higher m-carn contents between weeks 20 and 24. Therefore, a longer duration or higher dose of β-alanine is likely required to maximize m-carn contents in certain individuals.
FIGURE 2: Representative skeletal m-carn concentrations, specific to fiber-type, across different demographics. All samples obtained using the muscle biopsy technique from
m. vastus lateralis. ● young, healthy males and mixed gender samples; ■ young, healthy females; □ young, healthy vegetarian males (pre- and post-HIIT training intervention); ▲ elderly (~70 yr) males and females. TI; type I muscle fibers, TII; type II muscle fibers (based on data from references [
10,23–26]).
Relative increase in skeletal m-carn contents after β-alanine supplementation. Values obtained using the muscle biopsy technique or proton magnetic resonance spectroscopy (
1H-MRS). Data presented are changes in mixed-muscle fiber-types from 13 independent studies (total 54 samples: references
7,10,11,19,22,24,30–36).
Once augmented, total m-carn has a recorded washout time of approximately 2% to 4% per week (11,30). It remains unclear whether this stability is due to a low or high turnover rate. Although CN2 is present in the skeletal muscle, carnosine degradation is believed to be minimal due to low enzyme activity at intramuscular pH (~7.1) (37). Studies utilizing the microdialysis technique show a large elevation of carnosine in skeletal muscle interstitium after trauma to the sarcolemma from the insertion probe (38). This suggests an efflux of m-carn secondary to muscle damage; however, there is no measurable loss of m-carn immediately after high-intensity interval training (HIIT) (9). At present, the rate of m-carn decay appears to depend on the muscle location, fiber-type, and dietary β-alanine intake, but further investigations into m-carn turnover are warranted.
In humans, the type I:II ratio for m-carn in fibers from m. vastus lateralis is 1.3:2 (24,25,31). Absolute increases in m-carn from β-alanine supplementation are comparable for both fiber-types, which can lead to a doubling of m-carn in type I fibers and ~50% increase in type II fibers (24). A possible explanation for lower type I contents is greater rate of degradation, efflux from the muscle cell, or a lower synthetic rate. Conversely, higher type II m-carn contents may be an adaptive response to anaerobic demands and perturbations in local pH. The latter is supported by cross-sectional data showing higher m-carn in sprinters compared with aerobic athletes and healthy controls (6). Recently a longitudinal study in vegetarians observed a 25% increase in m-carn after 12 wk of HIIT (23). Similar studies, reporting null findings, are all short-term (≤6 wk) with a lower weekly training volume and fail to control for training-induced changes in muscle fiber distribution (39) or dietary intake of β-alanine (31). The precise mechanism of the training-induced increase in m-carn remains unknown, but recent evidence shows an increase in ATPGD1 mRNA by ~28% in response to a 6-wk HIIT regime (40). Furthermore, Hoetker et al. (41) showed m-carn contents and ATPDG1 gene expression concomitantly fluctuate throughout different phases of exercising training, suggesting that the amount of carnosine synthesis is an important regulator of m-carn homeostasis. Changes in hepatic β-alanine synthesis and β-alanine transport into muscle fibers are also likely important to provide sufficient substrate for carnosine synthesis to occur.
Less is known regarding the physiological roles of β-alanine, independent of its role in synthesizing m-carn. Ingested β-alanine is rapidly absorbed and plasma concentrations peak 30 to 60 min postsupplementation (10,42), with small losses occurring via urinary excretion (~1%–3%) and through incorporation into m-carn (~3%–6%) (10,32). Supplementing with a sustained-release β-alanine formulation results in greater retention and higher m-carn contents (43), this presents a more efficient supplementation strategy for use in future studies. The large proportion of remaining β-alanine is thought to be rapidly deaminated and oxidized.
In an early rodent model, Pihl and Fritzson (44) described the metabolic fate of C14-labeled β-alanine after intraperitoneal injection. The cumulative excretion of C14 in expired CO2 over 5 h was 93%, 60%, and 77% of the dose administered, respective to the carbon atom labeled. In humans, β-alanine transamination results in the formation of the keto-acid malonate semialdehyde (45). A reaction, according to rodent data, catalyzed by two mitochondrial enzymes, β-alanine-2-oxoglutarate transaminase and alanine-glyoxylate transaminase (4). Human hepatocytes express both enzymes and β-alanine-2-oxoglutarate transaminase is additionally present in kidney and brain tissue, which are putative sites of β-alanine metabolism. Malonate semialdehyde undergoes oxidative decarboxylation to acetyl-CoA, which provides a substrate for the tricarboxylic acid cycle (Fig. 1). Skeletal muscle β-alanine contents may increase up to ~98% in response to supplementation (33). However, β-alanine does not appear to undergo transamination in skeletal muscle and instead primarily contributes to carnosine synthesis (20). The role of the excess β-alanine is muscle unclear, but it may contribute to physiological function within the cell (e.g., molecular signaling) or be transported to another organ where it is metabolized.
Skeletal Muscle Metabolism
Direct effects on glycolysis and aerobic metabolism
Early research showed carnosine regulates enzyme activity and chelates heavy metal glycolytic inhibitors in isolated skeletal muscle (46), leading to an increase in glycolytic flux (47). Despite showing an ability to exert a direct influence on energy metabolism in vitro, data in human skeletal muscle are equivocal.
An increase in glycolytic flux or capacity, independent of oxidative capacity, is quantifiable by higher lactate accumulation during and after exercise. Some β-alanine supplementation studies have demonstrated this, with higher postexercise plasma lactate values after the special judo fitness test (12) and 4 × 30 s upper-body Wingate tests (48). However, in these studies, total mechanical work was not matched between presupplementation and postsupplementation trials. Direct effects on glycolysis cannot be separated from indirect effects, which are an increase in total work performed (e.g., due to increased buffering capacity). We also note that many studies have failed to show a difference in delta lactate, despite not matching mechanical work (34,35,49).
To our knowledge, only two studies have quantified the effects of β-alanine supplementation on energy system contribution with matched total mechanical work (7,50). da Silva et al. (50) showed no change in oxidative, glycolytic, or ATP-PCr contribution during 4 × 60 s cycling bouts performed with a constant cadence at 110% maximal aerobic power output. In stark opposition to the glycolytic activation hypothesis, Gross et al. (7) observed a decrease in postexercise muscle lactate and oxygen deficit after a 90-s fixed-power cycling test. Activity of the rate-limiting glycolytic enzyme, phosphofructokinase, was also reduced. This occurred alongside a small (~1.3%), but significant, increase in the estimated aerobic energy contribution, although aerobic enzyme activities were unchanged. β-alanine supplementation may also delay the onset of blood lactate accumulation during treadmill running (51), indicative of an improvement in oxidative capacity. Despite this, increasing m-carn contents does not appear to alter ventilatory threshold or V˙O2max/peak (52).
In a cell model, treatment of C2C12 murine skeletal myotubes with 800 μM of β-alanine led to induction of several markers of mitochondrial biogenesis (53). Although interesting, several limitations preclude acceptance of these findings. Intracellular carnosine was not quantified so it is unclear whether the effects are due to β-alanine or carnosine. Furthermore, evidence that β-alanine supplementation evokes favorable oxidative and mitochondrial adaptations is not supported by longitudinal human studies (7,39). The proposed small benefit to aerobic metabolism, shown by Gross et al. (7), is difficult to reconcile with evidence that exercise capacity and performance exceeding 10 min does not typically improve after β-alanine supplementation (5). Despite showing an ability to interact with metabolic pathways in vitro, the influence of carnosine as a direct modulator of energy metabolism in whole skeletal muscle appears less pronounced.
Intramyocellular pH buffering
The early works of Bate Smith (54) and Deutsch and Eggleton (55) first proposed the role of carnosine as a pHi buffer. The addition of β-alanine to l-histidine raises the pKa of the histidine imidazole ring from 6.1 (free histidine) to 6.83 (carnosine), causing it to act as a buffer over the exercise-induced pHi transit range (pH ~7.1 to 6.5) (10). This feature is consistent across species, whereby the highest HCD concentrations are found in animals with the greatest anaerobic energy demands, for example, due to prolonged sprinting (locomotion) or hypoxia (diving) (56). Furthermore, species with a highly oxidative phenotype and contractile properties (i.e., hummingbirds) possess low HCD contents (57). This suggests that HCD are nonessential to aerobic metabolism and muscle contractility, and instead, supports the primary physiological role as a pHi buffer. Due to these functions, there is widespread interest in β-alanine supplementation and m-carn in situations of exercise-induced acidosis.
The role of acidosis in peripheral fatigue during short-duration, high-intensity exercise has been debated. Recent data show elevated levels of H+ (pH ~6.2) and Pi (~30 mM) act synergistically to depress cross-bridge function by inhibiting isometric force, shortening velocity, peak power, and the low to high-force transition of the cross-bridge cycle (58). Regardless of the specific mechanism, increasing m-carn via β-alanine supplementation improves exercise capacity and performance in exercise durations of 30 s to 10 min (5). This outcome is consistent with acting as a pHi buffer, as H+ accumulation is at its highest and more likely contributor to fatigue than with shorter or longer exercise durations. Assessments of exercise capacity (e.g., time to exhaustion) show the greatest benefit from increasing m-carn contents (5). Whereas performance-based tests (e.g., time trials) show a smaller benefit, likely due to being highly influenced by pacing strategy. As such, the physiological milieu at the end of a performance-based task may not represent volitional fatigue or severe acidosis.
Several studies have used the Henderson–Hasselbalch equation (Fig. 4) to estimate the effect of m-carn on nonbicarbonate total muscle buffering capacity (βm) in muscle homogenates. Mannion et al. (59) first estimated that m-carn contributes ~7% to βm. This likely underestimates the in vivo buffering contribution of m-carn (βm-carn) due to methodological limitations, discussed herein. Measurements are recorded in a metabolic composition close to that of rigor mortis and, upon homogenization, there is a complete loss of adenosine triphosphate and phosphorylcreatine (60). As a result, estimates of βm do not include dynamic buffering via rephosphorylation of adenosine diphosphate by phosphorylcreatine. Instead, it encompasses histidine residues of proteins and dipeptides, inorganic phosphate, and hexose monophosphates (60)–βm is quantified without measuring the concentrations of these non-HCD buffers. Lastly, carnosine is a mobile buffer, freely dissolved in the cytoplasm, whereas proteins are fixed buffers. Such mobility allows carnosine to contribute to the prevention of local pHi gradients (61), which likely encompass greater effects than estimated from its proportion of βm alone.
FIGURE 4: During a pH change from 7.1 to 6.5, m-carn concentrations of 10.5 mmol·kg
−1·
dm −1 and 49.7 mmol·kg
−1·
dm −1 provide the capacity to sequester ~3.5 mmol·H
+.kg
−1·
dm −1 and 16.5 mmol·H
+.kg
−1·
dm −1 (■), respectively. Corresponding to a
βm-carn contribution to total
βm of 4.5% and 18.2% (▲). Data show values in omnivore TI muscle fibers at baseline (
27) and TII muscle fibers after 28-d β-alanine supplementation (
10). Nonbicarbonate
βm was determined by titrating muscle homogenates against HCl at 37°C and expressed as the
μmol H
+ required to change the pH of 1 g free-dried muscle from pH 7.1 to 6.5. This encompasses the contribution made by the static buffers (proteins, dipeptides and phosphates) to
in vivo buffering. A derivation of the Henderson–Hasselbalch equation was used to estimate the contribution of m-carn to total buffering (
βm-carn = {[m-carn]/(1 + 10
(6.5 –pKa))} – {[m-carn]/(1 + 10
(7.1 –pKa))}). The linear relationship shown may not reflect pH changes
in vivo, where reductions can be nonlinear.
Exercise models that induce fatigue under solely anaerobic conditions may provide a better estimation of βm-carn in vivo. Several studies show β-alanine supplementation increases isometric knee extension time to fatigue (+11.1%–17.2%) when performed at 45% maximal voluntary isometric contraction (MVIC) (62–64). To our knowledge, when performing identical methods, only one study has yielded null results (65). The test is estimated to cause fatigue in ~78 s, a duration and intensity accompanied by the largest increase in pyruvate and lactate (66). Moreover, an intensity of 45% MVIC raises intramuscular pressure sufficient to prevent muscle reoxygenation (67). This creates a local hypoxic environment and greater reliance on intracellular buffers (e.g., carnosine). These differences partly explain why the studies did not reproduce the earlier findings of Derave et al. (34). Participants recorded an isometric knee extension time to fatigue of ~173 s and ~201 s, substantially longer than the subsequent times of ~75 s (64) and ~55 s (62). To sustain a longer contraction the intensity was likely <45% MVIC (66). The results are further confounded by differences in the knee extension measurement angle of 45° (34) and 90° (62,64). Despite both positions producing comparable MVIC values, the time to fatigue is ~62% longer at a shorter quadriceps muscle length (50° vs 90°) (68). The lower relative force results in less intramuscular pressure and only a partial restriction of tissue oxygenation (67). Therefore, some circulation would have been maintained to enable H+ efflux from the working muscles, reducing the demand on intracellular buffers.
Hill et al. (24) investigated 4- and 10-wk β-alanine supplementation on cycling to exhaustion at 110% V˙O2peak (CCT110%), a test designed to cause fatigue in ~150 s. M-carn contents increased by ~59% and ~80%, leading to improvements in exercise capacity of 13% and 16.2%, respectively. Sale et al. (49) replicated these methods and reported a 12.1% improvement in CCT110% after 4 wk supplementation. The magnitude of improvement is remarkably consistent across the isometric knee extension hold and CCT110% protocols and shows a dose response to β-alanine supplementation (24). Both protocols induce severe acidosis; however, it is challenging to isolate these effects from concomitant changes in Ca2+ handling that may also be causative in fatigue (see following section).
A limitation to the pHi buffer perspective is the absence of evidence showing a carnosine-mediated attenuation of the exercise-induced decline in muscle pH. Gross et al. (7) showed no effect of β-alanine supplementation on postexercise muscle pH after a 90-s fixed-power cycling test. To contrast using a similar model, the findings did not replicate the mild (~0.1 pH units) alkalizing effect of elevated plasma bicarbonate on muscle acid–base balance during exercise (69). One explanation is that, because H+ buffered by m-carn remains within the muscle, on extraction, the protonated carnosine dissociates from its H+ with the cation returning to the muscle homogenate. Furthermore, it is possible the increase in m-carn was insufficient to improve βm by a detectable amount. The low supplementation dose (38 d, 3.2 g·d−1) induced a 24% elevation in m. vastus lateralis carnosine content (7), whereas a more typical β-alanine dose (e.g., 4–6 wk, 6.4 g.d−1) can lead to two- to threefold higher increase (Fig. 2).
The shortcomings in our understanding are likely to persist until reliable methods are available to quantify real-time changes in muscle pH during exercise. However, by triangulating findings from controlled human trials, in vitro research, and comparative physiology, there is robust evidence to support a key physiological role of carnosine as a pHi buffer in skeletal muscle.
Ca2+ Handling and Muscle Contractility
Ca2+ release, reuptake, and myofilament Ca2+ sensitivity
Several studies show that carnosine influences Ca2+ handling, which could partly explain the ergogenic effects of m-carn. Decreases in Ca2+ release from the sarcoplasmic reticulum (SR), myofilament Ca2+ sensitivity, and Ca2+ reuptake into the SR occur during fatiguing contractions (70). These factors act synergistically with the accumulation of metabolic by-products (namely H+ and Pi) to cause loss in muscle function during intense contractile activity.
Early studies in chemically skinned skeletal muscle fibers proposed a role for carnosine, and other HCD, in facilitating Ca2+ release from the SR and increasing myofilament Ca2+ sensitivity (71,72). The chemically skinned muscle fiber model has since been criticized as it results in disruption to normal coupling between Ca2+ release channels and voltage-sensor proteins (dihydropyridine receptors) (73). Furthermore, the positive results were from experiments performed in the presence of subphysiological concentrations of cytoplasmic Mg2+, a potent inhibitor of Ca2+ release.
Dutka and Lamb (73) showed no effect of carnosine on Ca2+ release from the SR when performed in mechanically skinned muscle fibers under conditions that corrected for the previous shortcomings. However, they did confirm that adding carnosine to muscle preparations lowers the amount of Ca2+ ions required to produce half-maximum tension, with minimal change in the maximum Ca2+-activated force. Dutka et al. (8) replicated these findings in human skeletal muscle samples. In a concentration-dependent manner, 8 and 16 mM carnosine increased the pCa50 (−log (10) of Ca2+ concentration at half-maximal force) by ~0.07 and 0.12 pCa units in type I fibers, and by ~0.06 and 0.1 pCa units in type II fibers, respectively. This equates to a leftward shift of the force-pCa relationship (the in vitro analogue of the force-frequency relationship), whereby a fiber producing ~40% of maximal force in the absence of carnosine would produce ~60% in the presence of 16 mM carnosine (Fig. 5) (8). In the studies discussed, experiments involve heavily buffered preparations that maintain pH at ~7.1 and therefore do not assess carnosine on Ca2+ handling throughout the range of exercise-induced acidosis (i.e., pH ~7.1 to ~6.5). This is important as muscle pH and Ca2+ handling are inextricably linked. For example, the sarco/endoplasmic reticulum-ATPase (SERCA) pump rate, responsible for Ca2+ uptake, declines approximately twofold over a pH drop from 7.1 to 6.6 (74). The ability of m-carn to buffer pHi may indirectly improve Ca2+ handling in the muscle cell.
FIGURE 5: Schematic representation of the force–Ca2+ relationship in skeletal muscle. A leftwards shirt indicates an increase in myofilament Ca2+ sensitivity (red line). The result is a large increase in submaximal force (dashed line) with little effect on maximal force.
Human β-alanine supplementation studies show varied responses to in vivo measures of Ca2+ handling. Gross et al. (75) reported a ~7% improvement in maximum and average power during a countermovement jump, despite no change in maximal jump height. The authors attributed this to an increase in contraction velocity secondary to enhanced myofilament Ca2+ sensitivity. Although interesting, subsequent human studies disagree. Hannah et al. (76) and Jones et al. (65) showed no effect of β-alanine supplementation on peak force, time to peak tension, and maximum or explosive force production in voluntary and electrically evoked contractions in fresh and fatigued muscle, refuting earlier in vitro data (8) and the observation of a leftwards shift in the force–frequency curve in mice (77). In whole, contracting human skeletal muscle, carnosine may be less important in sensitizing the myofilaments to Ca2+ than detected in in vitro and rodent models. This is possibly due to differences in relative muscle excitability between species (for a review, see reference 78). Furthermore, β-alanine supplementation does not typically enhance maximal force production in vitro (8,73) or in human studies (36,65,75,76).
Interestingly, both Hannah et al. (76) and Jones et al. (65) showed a significant decrease in knee extensor half-relaxation time, highlighting a potential interaction between m-carn and Ca2+ handling. Resting and potentiated twitches were recorded before and after three sets of MVIC, whereas supramaximal octets (eight impulses at 300 Hz) assessed explosive performance of the musculotendinous unit. β-alanine supplementation decreased half-relaxation time in resting (−12%) and potentiated (−7%) twitches in fresh muscle (76); in resting (−19%) and potentiated (−2%) twitches in fatigued muscle, and supramaximal octets in fresh (−20%) and fatigued (−11%) muscle (65). Everaert et al. (77) reported an attenuation of the slowing in relaxation rate, in predominantly slow-twitch muscle (soleus), during the first 3 min of a fatiguing protocol in mice. This presents a contrast from human data, where the reduction in relaxation time occurred in resting twitch and explosive contractions (65), implying the response is similar in both muscle fiber types. The reason for these differences between studies is unclear, but it may be due to the abundance of anserine in rodents. In human skeletal muscle, anserine is either absent or accounts for only a minor (~2%) portion of the total HCD content (10,41), whereas anserine content is higher than carnosine in mice (ratio ~1:2.1) and rats (ratio ~1:2.4), and increases further with β-alanine supplementation (77). Anserine and carnosine show differences in their Ca2+ handling properties (71); therefore, the changes in muscle function in rodents are due to the total increase in HCD content and not carnosine alone.
The slowing of relaxation can limit performance in exercise where rapidly alternating movements are performed (70). However, repeated resisted muscle contractions to fatigue (e.g., strength endurance exercise) show mixed responses to β-alanine supplementation. Derave et al. (34) showed an attenuation of fatigue in repeated isokinetic knee extensions performed at 180°·s−1 (5 × 30 repetitions). Raising m-carn by ~37% to 47% led to greater average peak torque in sets 3 to 5 compared with the control group. More recently, Bassinello et al. (62) could not reproduce these results, despite using the same experimental methods. There was also no change in total repetitions performed during high volume (eight sets, 70%1RM) smith-machine bench press and 45° leg press exercise. This is consistent with the data that show no improvement in fatigue resistance during exhaustive arm curl exercise (~20–40 repetitions), even with a ~59% increase in m-carn (36).
There is inconsistency between results from in vitro, animal, and human studies. The ability to influence Ca2+ handling and myofilament Ca2+ sensitivity may be important chemical properties of carnosine. However, if these were primary physiological roles in skeletal muscle we might expect improvements in exercise over a wider range of modes and durations. The findings of a decrease in half-relaxation time in humans are interesting and warrant further investigation. Lastly, our discussion is specific to skeletal muscle and the role of carnosine–Ca2+ interactions in other tissues (e.g., cardiomyocytes) may differ.
Cytoplasmic Ca2+–H+ exchanger
Emerging evidence suggests that carnosine may function as a diffusible cytoplasmic Ca2+–H+ exchanger in cardiomyocytes (61). This combines elements of the two previously discussed roles: Ca2+ handling and pHi buffering. The interrelationship between H+ and Ca2+ is important in exercising skeletal muscle. H+ can compete with Ca2+ at the troponin-binding site, thereby limiting the ability of the muscle contractile machinery to operate effectively (79). Both Ca2+ and H+ competitively bind to carnosine, which can cause unloading of Ca2+ in areas of high H+ production (e.g., local glycolytic metabolism) and unloading H+ in areas of high Ca2+ production (e.g., efflux from the RyR1 channels) (61). Through these actions, carnosine is able to regulate highly compartmentalized ionic microdomains and potentially improve contractile function through increasing myofilament Ca2+ sensitivity.
The evidence that an increase in m-carn can reduce half-relaxation time could explain a role of the Ca2+–H+ exchanger in human skeletal muscle (65,76). Relaxation time is influenced by the rate of dissociation of Ca2+ from troponin; translocation of Ca2+ to a site close to the SR; and reuptake of Ca2+ into the SR by SERCA pumps (74). Only 1 in 100 Ca2+ ions is free to diffuse, and the diffusivity of the remaining Ca2+ depends on the mobility of the Ca2+-buffer complex (61). During Ca2+ uptake by SERCA, H+ are countertransported from the SR lumen to the cytosol, simultaneously, H+ are transported from the cytosol to the lumen during Ca2+ release (80), leading to H+ and Ca2+ nonuniformity. As a mobile buffer, carnosine may translocate Ca2+ closer to the SERCA pump for Ca2+ reuptake (Fig. 6). Although the cytoplasmic Ca2+–H+ exchanger is an alluring concept, the model proposed by Swietach et al. (61) was demonstrated in rat ventricular cardiomyocytes, where spatiotemporal responses in Ca2+ sparks differ to skeletal muscle (81). It is conceivable that the role also occurs in human skeletal muscle, but validation is required before drawing strong conclusions.
FIGURE 6: Schematic depicts the proposed role of Ca2+-H+ exchanger in skeletal muscle. Ca2+ release and reuptake from the SR, combined with H+ production from glycolysis, results in ionic microdomains and local pHi gradients. Carnosine is able to bind, transport, and deposit H+/Ca2+ in the cytoplasm to regulate local pHi. Subsequently, H+ appearing in the blood are buffered by bicarbonate (HCO3 −) and expired as CO2. Primary pathways involve the 1) sodium–hydrogen exchanger, 2) sodium bicarbonate cotransporter, and 3) monocarboxylate transporter (MCT) 1 and 4. Key membrane transporters and organelles involved in H+ and Ca2+ homeostasis are depicted: dihydropyridine receptors (DHPR), monocarboxylate transporters 1/4 (MCT1/4), sodium-bicarbonate cotransporter (NBC), Na+–Ca2+ exchanger (NCX), Na+–H+ exchanger (NHE), plasmalemmal Ca2+–H+–ATPase pumps (PMCA), ryanodine receptor 1 (RyR1), sarcoplasmic reticulum (SR), sarco-endoplasmic reticulum Ca2+–H+–ATPase pumps (SERCA).
Redox Activity
Through its diverse chemical properties, carnosine has the ability to scavenge reactive species, form adducts with reactive aldehydes, and chelate metal ions (82). These actions may confer a benefit against oxidative stress and deleterious modifications to biomolecules, including proteins, lipids, and DNA. Here, consistent with the theme of our review, we focus on these roles within the context of exercising skeletal muscle. For a clinical perspective, we direct the reader to a recent review by Artioli et al. (83).
Scavenger of reactive species
The production of reactive oxygen species (ROS) increases in skeletal muscle during exercise (84). Despite endogenous defenses, high rates of ROS production can exceed the antioxidant capacity of muscle fibers, leading to oxidative stress. Carnosine, and other HCD, can quench superoxide anions, hydroxyl radicals, and peroxyl radicals (82) thereby reducing intracellular oxidative stress. It is unknown, however, whether carnosine contributes to redox homeostasis in whole human skeletal muscle. Further complicating the issue is evidence that ROS exert a beneficial or detrimental effect on contractile function depending upon the magnitude and duration of increase, localization of accumulation and the type of ROS produced (84).
In two similar human studies, Smith et al. (85) and Smith-Ryan et al. (86) supplemented recreationally trained participants with β-alanine (4 wk, 4.8 g.d−1) and recorded plasma markers of oxidative stress in response to a 40-min treadmill run. Superoxide dismutase (SOD) activity, total antioxidant capacity, reduced glutathione, and 8-isoprostane were all unaffected by supplementation at baseline and postexercise. The additional interpretation of confidence intervals suggested a likely beneficial effect of supplementation in reducing postexercise 8-isoprostane (85). However, assessing the activity of redox enzymes in plasma is strongly discouraged and the use of nonspecific assays, several of which are inherently flawed (for a review, see reference [87]), means that few conclusions can be drawn from these data.
In a novel comparative physiology model, Dolan et al. (57) examined m. pectoralis samples from two avian species with distinct metabolic phenotypes: hummingbirds (highly oxidative) and chickens (highly glycolytic). Due to their oxidative and contractile demands, hummingbirds have a well-developed primary antioxidant system to neutralize the by-products of aerobic metabolism. Interestingly, total skeletal muscle HCD content was negatively correlated (R2 = 0.79) to SOD activity and positively correlated (R2 = 0.87) to βm capacity. A finding that suggests HCD are nonessential to mitigate oxidative stress in skeletal muscle. Although, HCD exhibit chemical properties of an antioxidant, the importance of this in vivo appears limited due to well-developed primary antioxidant defenses. It is therefore possible that carnosine is in fact more effective at binding and removing secondary redox products, namely saturated and unsaturated aldehydes.
Formation of adducts with reactive aldehydes
Carnosine and other HCD contain reactive nucleophilic amines that can form stable conjugates with highly toxic lipid peroxidation products (e.g., malondialdehyde, 4-hydroxynonenal (HNE), and acrolein) in skeletal muscle (88). Lipid peroxidation products accumulate after intense exercise, which may amplify and prolong tissue damage under conditions of oxidative stress. To minimize protein modification reactions, most tissues metabolize aldehydes via enzymatic pathways catalyzed by aldehyde dehydrogenases and aldo-keto reductases. Emerging evidence shows that m-carn plays an important role in nonenzymatic detoxification of reactive aldehydes, an effect that β-alanine supplementation potentiates.
Following a ~50% increase in m-carn contents, Carvalho et al. (9) detected a twofold greater formation of carnosine-acrolein adducts in muscle biopsy samples taken immediately after a HIIT session (4 × 30 s Wingate tests with 3 min recovery between efforts). There was no effect for exercise- or supplementation-alone, indicating that m-carn conjugated the acrolein generated during exercise. Other markers of lipid peroxidation, carnosine-HHE (4-hydroxy-hexanal) and carnosine-HNE, were either undetectable or did not change with supplementation or exercise. Using similar outcomes, Hoetker et al. (41) put participants through a 9-wk periodized exercise block that included multiple modes of testing, endurance cycling training, and a 6-wk HIIT regime. At the end of the training block, individuals receiving β-alanine supplementation had greater formation of carnosine-aldehyde adducts in postexercise skeletal muscle samples. Compared with the placebo group, formation of carnosine-HNE and reduced carnosine-acrolein conjugates: carnosine-propanal and carnosine-propanol (89) increased by ~58%, ~119%, and ~86%. Interestingly, and in contrast to Carvalho et al. (9), there were no changes in carnosine-adducts after the first session of HIIT, at which point m-carn contents were elevated by ~51% compared with an elevation of ~127% at the end of the 6-wk HIIT program (41).
The reason for the differences between studies is unclear, but both studies found acrolein-based adducts in higher quantities than with other reactive aldehydes. One explanation is that the bimolecular rate constant for carnosine with acrolein is approximately eightfold higher than for carnosine with HNE (90), hence its propensity to form favorably with acrolein or its derivatives. The carnosine-aldehyde adducts are subsequently eliminated from the body via urinary excretion (89). Given that lipid peroxidation products in skeletal muscle are lowest in endurance-trained and highest in sedentary, obese individuals (91), the role of m-carn as a detoxifying agent may be particularly important in clinical populations. Consistent with this hypothesis, carnosine scavenging of damaging reactive aldehyde species has recently been shown to enhance glucose uptake into myotubes, thereby protecting these cells against cellular dysfunction driven by oxidative stress (92).
Perspectives
The enigmatic nature of carnosine makes it challenging to draw a unified conclusion on its physiological roles in human skeletal muscle. However, we have discussed key research in the field, using a combination of in vitro, animal models, comparative physiology, and human intervention studies to show the best available evidence for each role. In the context of exercise physiology, the most robust evidence shows m-carn functions as a pHi buffer; however, this does not exclude the potential of other functions that may be additive to, or in place of, its buffering actions. The possibility that m-carn acts as a cytoplasmic Ca2+–H+ exchanger is an exciting new paradigm. This coupled with emerging evidence that m-carn detoxifies reactive aldehydes, shows that there is still much to learn regarding the physiological roles of carnosine in skeletal muscle.
It is important to note that, outside of skeletal muscle, the actions of carnosine may markedly differ to those we have discussed. Indeed, roles that are less important in skeletal muscle may be of primary importance in other organs and vice-versa. Major pathways of β-alanine metabolism and its turnover require further investigation in humans. It is possible the elevation in skeletal muscle β-alanine, as occurs with high-dose supplementation, plays a functionally relevant role within the muscle cell or in its transport to another organ. Further knowledge from animal and cell models is likely to come from approaches that involve knocking-out or overexpressing genes involved in carnosine metabolism. Whether carnosine can influence gene expression or epigenetics is of interest and will undoubtedly be an area of future research.
Regrettably, due to journal space constraints and reference limitations, we were unable to cite all of the excellent research that has contributed to this field of study.
Disclosure of Funding: No funding was received for writing this article. G. G. A. has been supported financially by Fundação de Amparo à Pesquisa do Estado de São Paulo (FASESP; grant number: 2014/11948-8). M. D. T. has received a British Council award to support a studentship focused on research into carnosine (grant number: 209524711).
Conflict of Interest: J. J. M., G. G. A., and M. D. T. collectively declare that they have no competing interests. C. S. has received β-alanine supplements free of charge from Natural Alternatives International (NAI) for use in experimental investigations; NAI have also supported open access page charges for some manuscripts. The review is presented honestly, and without fabrication, falsification, or inappropriate data manipulation. The viewpoints expressed in the review do not constitute endorsement by the American College of Sports Medicine.
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