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00005768-201407000-0001900005768_2014_46_1426_stegen_maintaining_7miscellaneous-article< 100_0_16_6 >Medicine & Science in Sports & Exercise© 2014 American College of Sports MedicineVolume 46(7)July 2014p 1426–1432β-Alanine Dose for Maintaining Moderately Elevated Muscle Carnosine Levels[APPLIED SCIENCES]STEGEN, SANNE1; BEX, TINE1; VERVAET, CHRIS2; VANHEE, LANDER1; ACHTEN, ERIC3; DERAVE, WIM11Department of Movement and Sports Sciences, Ghent University, Ghent, BELGIUM; 2Laboratory of Pharmaceutical Technology, Ghent University, Ghent, BELGIUM; and 3Department of Radiology, Ghent Institute for Functional and Metabolic Imaging, Ghent University, Ghent, BELGIUMAddress for correspondence: Wim Derave, Ph.D., Ghent University, Watersportlaan 2, B-9000 Ghent, Belgium; E-mail: .Submitted for publication July 2013.Accepted for publication December 2013.ABSTRACTIntroduction: Chronic β-alanine (BA) supplementation is an increasingly popular nutritional strategy, because it can elevate muscle carnosine content and thereby enhance high-intensity exercise performance. The current study investigated 1) whether sex and body mass are determinants of BA-induced muscle carnosine loading and 2) the optimal maintenance dose for ensuring constantly elevated muscle carnosine stores.Methods: During the loading phase, 34 participants (men and women) were supplemented with 3.2 g (4 × 800 mg) BA per day for 46 d (slightly different loading strategies were applied concerning the effect of meal timing and supplementation form). Thereafter, 19 participants (men and women) continued taking free-powder BA for six more weeks (maintenance phase). The participants were matched and redivided into three groups receiving 0.4, 0.8, and 1.2 g·d−1 BA, respectively. Muscle carnosine content was measured in the soleus and gastrocnemius muscles using proton magnetic resonance spectroscopy.Results: Body mass and sex had only minimal effect on the absolute increase in muscle carnosine. Given the lower baseline values in women, the relative increase for women was higher, indicating that women required less BA for the same relative increase. In addition, a significant negative correlation was observed between body mass and the relative increase in muscle carnosine (r = −0.45, P = 0.007). A maintenance dose of ∼1.2 g·d−1 BA was the most effective in keeping muscle carnosine content elevated at the postsupplementation level.Conclusions: Sex and body mass did not markedly affect the absolute increase during muscle carnosine loading, although they are determinants for the relative increase. In addition, we established for the first time an effective maintenance dose of ∼1.2 g·d−1 BA to keep muscle carnosine content elevated at 30%–50% above baseline for a prolonged period.β-Alanine is an increasingly popular nutritional supplement. It is the rate-limiting precursor for carnosine synthesis, a cytoplasmic dipeptide (β-alanyl-L-histidine) in human skeletal muscle (17). Increasing muscle carnosine is associated with performance enhancement in high-intensity exercise (2,8,9,18). The ergogenic mechanism of carnosine presumably relates to its characteristics as a calcium sensitizer (11,14) and proton buffer (4,23). However, various other research fields have investigated this molecule for other biological qualities: scavenger of reactive oxygen species, reactive nitrogen species, and deleterious aldehydes (malondialdehyde, methylglyoxal, hydroxynonenal, etc.); chelator of zinc and copper ions; and antiglycating and anti–cross-linking activities (1,6,7). These properties suggest a therapeutic potential of muscle carnosine loading in clinical populations, a research topic beginning to emerge (8,15,25).An efficient regimen to augment human muscle carnosine content was initially established by Harris et al. (17). The latter and subsequent studies have shown that chronic oral supplementation of 1.6–6.4 g·d−1 of β-alanine (BA) will generally lead to increases in muscle carnosine content of ∼15%–85% in 4–12 wk (5,8,19,28). Although we may not yet have attained the true maximal or optimal loading protocol, supplementation strategies aiming for moderately elevated muscle carnosine levels are frequently used in sports and have proven ergogenic effects (2,9,19). In contrast, not a single study has so far established a suitable maintenance dose. This gap hampers long-term studies on effects of more chronically elevated carnosine levels, which is more relevant in clinical situations and aging.The current study focused on establishing a suitable maintenance dose, although for a specific loading protocol (46 d of supplementation with 3.2 g·d−1 of BA).It is known that on discontinuation, muscle carnosine levels will gradually return to baseline (presupplementation) levels in 6–20 wk (5,28). A first attempt to establish such maintenance dose for BA supplementation was published by Stellingwerff et al. (28). They demonstrated that, after an initial increase achieved by a 4-wk loading protocol of 3.2 g·d−1 BA, a further ∼30% increase in muscle carnosine occurred after 4 wk of taking 1.6 g·d−1 BA. We can conclude that the optimal dose for maintaining elevated muscle carnosine stores must be between 0 and 1.6 g·d−1 BA. Therefore, we will investigate three intermediate doses: 0.4, 0.8, and 1.2 g·d−1.Little is known about the influence of sex, body mass, and fat-free mass (FFM) on muscle carnosine loading via BA supplementation in the current literature. Previously published reports on the effects of BA supplementation on human muscle carnosine content included exclusively male participants (2,5,8,9,17,19,28). Only one study (8) included both elderly men and women, but they did not diversify between sexes. Some studies have reported positive effects of BA supplementation on exercise performance in women (29,30), yet without measurement of muscle carnosine content. In addition, it has been shown that carnosine metabolism becomes sex-specific in humans on sexual maturation (3,13), with muscle carnosine content being lower and serum carnosinase activity being higher in adult women than in men. Several reasons for this sexual dimorphism have been postulated, such as muscle fiber-type distribution or diet, but animal experiments have shown that testosterone is certainly involved (12,24). Up until now, it was not clear whether the factors inducing differences in baseline muscle carnosine content would also result in differences in carnosine loading between men and women. Therefore, we now aim to establish whether women respond equally well, better, or worse to BA supplementation.Since women are, in general, lighter and have less FFM, we could assume a larger muscle carnosine loading because they receive a higher dose per kilogram body mass or FFM. In comparison with other nutritional supplements having ergogenic effects during high-intensity exercise, the recommended dosing protocol is either a fixed amount irrespective of body mass (creatine [16,21]) or body mass corrected (sodium bicarbonate and sodium citrate [31]). However, it remains to be established whether BA should be dosed in relation to body mass.In summary, this study was designed: to define the optimal maintenance dose for ensuring constantly elevated muscle carnosine stores of 30%–50% above baseline, to clarify whether sex and body mass are influencing factors during muscle carnosine loading, and to provide an explanatory model of loading, maintenance, and washout of elevated muscle carnosine stores.METHODSParticipantsThirty-four participants (men and women) volunteered to participate in this study. All participants were healthy, nonvegetarian, and physically active but were not involved in regular training. The study (loading and maintenance phase) was approved by the local ethics committee (Ghent University Hospital, Ghent, Belgium); all participants gave their written informed consent to take part in the study and were aware that they were free to withdraw from the experiment at any point.Study DesignDuring the loading phase, 34 participants (16 men: age = 19.4 ± 1.1 yr, body mass = 73.9 ± 8.2 kg; 18 women: age = 19.3 ± 0.9 yr, body mass = 60.2 ± 6.0 kg) were supplemented with 3.2 g (4 × 800 mg) of BA per day for 6 wk (46 d). These data were collected from a previous study with another research question (26), where all participants were randomized into three sex- and body mass–balanced groups (group 1 received free-powder BA between the meals, group 2 received free-powder BA in between the meals, and group 3 received slow-release [SR] BA). Although the three groups had slightly different loads, they allowed us to use the data set to study the effect of body mass (BW), FFM, and sex on muscle carnosine loading because the participants with different body mass and sex were equally dispersed among the three groups (group 1: BW = 55–84 kg, FFM = 43–72 kg; group 2: BW = 54–85 kg, FFM = 45–81 kg; and group 3: BW = 49–88 kg, FFM = 37–61 kg). Groups 1 and 2 received CarnoSyn powder in capsules and group 3 received CarnoSyn SR tablets, both delivered by Natural Alternatives International and analyzed using the documented HFL screening method by HFL Sport Science. Muscle carnosine content was measured before and after the loading phase. Therefore, the percentage of ingested BA accumulated in skeletal muscles could be calculated (vide infra). Anthropometrical variables (length, body mass, and FFM) and dietary BA ingestion were also assessed at the start of the supplementation period.During the maintenance phase, 19 participants volunteered to continue taking free-powder BA for 6 wk to determine a maintenance dose. The participants (12 men: age = 19.8 ± 1.1 yr, body mass = 74.5 ± 8.1 kg; and 7 women: age = 19.7 ± 1.0 yr, body mass = 60.1 ± 4.3 kg) were redivided into three groups receiving 0.4, 0.8, and 1.2 g·d−1 of free-powder BA, respectively (CarnoSyn powder; one, two, or three capsules, respectively, dispersed among the day [before the meal]). After loading, and because of the high dropout, the participants were restratified for 1) loading history (carnosine content after loading and absolute and relative increase in muscle carnosine during the loading phase), 2) body mass, and 3) sex to obtain three new and matched groups for the maintenance phase. Muscle carnosine was measured again at the end of the 6-wk maintenance dose.Determination of Muscle Carnosine ContentCarnosine content of all the participants was measured by proton magnetic resonance spectroscopy (1H-MRS) in soleus and gastrocnemius medialis muscles, as described by Baguet et al. (2). When both muscles responded similarly, the average of the two muscles was taken and referred to as “muscle carnosine.” Otherwise, the soleus or the gastrocnemius muscle was mentioned separately. The participants were lying in the supine position on their back, and the lower leg was fixed in a holder with the angle of the ankle at 20° plantarflexion. All the MRS measurements were performed on a 3-T whole-body magnetic resonance imaging scanner (Siemens Trio, Erlangen) equipped with a spherical knee coil. Single-voxel point-resolved spectroscopy sequence with the following parameters was used: repetition time = 2000 ms, echo time = 30 ms, number of excitations = 128, data points = 1024, spectral bandwidth = 1200 Hz, and total acquisition time = 4.24 min. The average voxel size for the soleus and gastrocnemius muscle was 40 × 10 × 28 mm and 40 × 11 × 30 mm, respectively. The line width of the water signal for the soleus and gastrocnemius muscles was, on average, 24.6 and 27.6 Hz, respectively, after shimming procedures. The absolute carnosine content (mM) was calculated as described before by Baguet et al. (2), and they reported a relatively low coefficient of variation of 9.8% in the soleus muscle and 14.2% in the gastrocnemius muscle (5).Daily Dietary BA Intake and Percent BA Accumulated in Skeletal MusclesAn estimation of the daily dietary BA ingestion was achieved by a 2-wk record of their meat and fish consumption, as described by Baguet et al. (5). During the loading phase, the percentage of the total ingested amount of BA (147 g) that is accumulated into the skeletal muscles is estimated for every subject separately by assuming that 46% of body mass is muscle mass for 19-yr-old men and 33% for 19-yr-old women (22) and by taking into account their personal average increase in muscle carnosine.AnthropometryHeight and body mass were determined using a digital balance scale and a stadiometer. FFM was assessed by bioimpedance (Bodystat 1500MDD; Bodystat Ltd., Douglas, Isle of Man, UK). For the latter, only a subgroup (n = 23; group 1 = 2/3, group 2 = 4/4, group 3 = 3/5 [men/women]) was measured because the equipment (Bodystat 1500MDD) was not always available. Patients were in supine position for at least 5 min. Surface electrodes were attached to the right hand (red lead: behind the knuckle of the middle finger; black lead: on the wrist to the ulnar head) and foot (red lead: behind the second toe next to the big toe; black lead: on the ankle at the level of and between the medial and lateral malleoli). FFM was derived from the impedance and the deducted total body water. The intraclass correlation coefficient for FFM measurements with the Bodystat 1500MDD is 0.987 (27).StatisticsAll correlations were evaluated by Pearson correlations. An independent two-sample t-test was used to study sex differences during loading. To examine whether our three maintenance dose groups evolved differently in time, we performed a repeated-measures ANOVA (general linear model) with “group” (0.4, 0.8, and 1.2 g·d−1) as the between-participants factor and “time” (after loading and after maintenance) as the within-participants factor. In case of significant interactions and to determine whether a specific maintenance dose results in a decrease/increase compared to post loading, a paired-sample t-test was performed for each group separately. All analyses were done with SPSS statistical software (SPSS version 20, Chicago, IL), and statistical significance was set at P < 0.05.RESULTSEffects of Sex, Body Mass, and FFM on the Loading PhaseThe absolute increase in muscle carnosine, after taking 3.2 g·d−1 BA for 6 wk, was approximately 1.5–2 mM and was equal irrespective of the muscle type and sex (Fig. 1A). Logically, since we confirm that the soleus muscle has lower baseline values compared to the gastrocnemius muscle (SOL: 3.47 ± 0.87 mM, GAS: 4.51 ± 1.36 mM, P < 0.05), the relative increase in the soleus muscle is higher (SOL: +53.4% ± 30.1%, GAS: +41.8% ± 26.8%). Similarly, women have lower baseline carnosine values compared to men (women: 3.28 ± 0.58 mM, men: 4.79 ± 0.85 mM, P < 0.05); therefore, the relative increase in women is significantly higher in gastrocnemius compared to men (women: +50.8% ± 30.7%, men: +31.6% ± 17.5%), however, not for soleus (Fig. 1B). The percentage of the ingested BA that is accumulated in the skeletal muscles is higher in men (3.6% ± 1.1%) compared to women (1.9% ± 0.8%, P < 0.001); however, this is explained by the higher body mass and muscle mass in men compared to that in women (body mass: men 73.9 ± 8.2 kg vs. women 60.2 ± 6.0 kg, P < 0.001; muscle mass: men 34.0 ± 3.7 kg vs. women: 19.6 ± 2.0 kg, P < 0.001).FIGURE 1. Loading phase: Comparison between sexes for absolute (A) and relative (B) increase in muscle carnosine after a supplementation period of 3.2 g·d−1 BA during 46 d. Data are means ± SD. *Significant for P < 0.05 (women vs. men).To assess the effects of body mass and FFM on muscle carnosine loading, correlations were made with the absolute and relative increase in muscle carnosine, separately for sex and muscle type (Fig. 2). Heavier men have a lower absolute increase in soleus carnosine compared to lighter men (r = −0.51, P = 0.044; Fig. 2A). Nevertheless, this finding could not be confirmed for women and gastrocnemius muscle (Figs. 2A and B). In addition, correlations between body mass and the absolute increase in muscle carnosine are not stronger when taking FFM into account. For the relative increase in muscle carnosine, a stronger relationship with body mass is present (r = −0.45, P = 0.007 for muscles and sexes pooled), which remains predominantly present for men (SOL: r = −0.53, P = 0.036; GAS: r = −0.47, P = 0.065) and not for women (SOL: r = −0.10, P = 0.699; GAS: r = −0.44, P = 0.069) (Figs. 2C and D).FIGURE 2. Loading phase: Correlation between body mass and the absolute (soleus [A] and gastrocnemius [B]) and relative (soleus [C] and gastrocnemius [D]) increases in muscle carnosine for a supplementation protocol of 3.2 g·d−1 BA for 46 d. White and black circles represent women and men, respectively. Dashed and solid lines represent significant correlations for men only and the entire group, respectively. Correlations are significant when P < 0.05.Finally, baseline carnosine content did not influence the absolute increase in muscle carnosine (men: r = −0.23, P = 0.222; women: r = −0.28, P = 0.264). As a consequence, men and women with higher baseline values have a lower relative increase compared to those with lower baseline values (men: r = −0.74, P = 0.001; women: r = −0.56, P = 0.015).Maintenance DoseNo significant differences were present between groups before the start of the maintenance phase concerning subject characteristics and loading history of muscle carnosine (Table 1). The group that took 1.2 g·d−1 BA remained at the postloaded level, whereas the other two groups (0.4 and 0.8 g·d−1 BA) showed a decrease relative to the initial increase in muscle carnosine with −46% and −32%, respectively (Fig. 3A). In addition, a strong linear relationship is present (r = 0.71, P = 0.001) between the maintenance dose, corrected for body mass (mg·kg−1 BW per day) and the maintenance in muscle carnosine (with 100% indicating perfect maintenance and 0% indicating complete return to baseline). From this relationship, the optimal maintenance dose is calculated at ∼18 mg·kg−1 BW per day.TABLE 1 Maintenance phase: subject characteristics, muscle carnosine loading history, and postmaintenance carnosine concentration for the three groups (0.4, 0.8, and 1.2 g·d−1 BA as maintenance dose)FIGURE 3. Maintenance phase: Five different maintenance doses on a time scale are shown: before loading (0%), after 6 wk of loading with 3.2 g·d−1 BA (100%), and after 6 wk receiving a maintenance dose of1.2 g·d−1 (n = 7), 0.8 g·d−1 (n = 6), or 0.4 g·d−1 (n = 6). The postmaintenance doses are relative to the initial increase during loading. Data of 0 and 1.6 g·d−1 are derived from Baguet et al. (5) and Stellingwerff et al. (28), respectively. *P < 0.05 different from postloading.DISCUSSIONThe current study further examined possible determinants of BA-induced muscle carnosine loading and newly established an efficient maintenance dose following a 46-d supplementation protocol of 3.2 g·d−1 BA.This study showed that sex and body mass only have a minimal effect on the absolute increase during muscle carnosine loading. It has already been shown that soleus and gastrocnemius muscles respond equally well to BA supplementation in men (5), irrespective of the higher absolute carnosine baseline value of the gastrocnemius. This study confirms similar absolute increases in carnosine in the gastrocnemius and soleus muscles in females, suggesting that there are no muscle type or sex differences in the response to BA supplementation. However, it remains a subject of debate whether the higher relative increases in women (Fig. 1B) or lighter people (Figs. 2C and D) are relevant from a practical point of view. It has already been reported that there is a correlation between the relative change in carnosine and performance enhancement (8,19). In addition, Baguet et al. (2) (data from our own laboratory) published a correlation between the absolute increase in muscle carnosine and the absolute improvement on a 2000-m rowing test in highly trained rowers. Their manuscript reported only absolute values, but the correlation remained present when values were expressed relatively (data not published: P = 0.024, r = −0.543). Nevertheless, it remains questionable whether this is an argument for establishing a model where gain in performance is primarily related to the relative change in carnosine. In Figure 4, we have graphically demonstrated the relevance of body mass and sex for increasing muscle carnosine content by 50%. Although this increase in muscle carnosine is probably not the most optimal increase for the most optimal improvement in performance, it is considered effective for ergogenic and function-enhancing purposes.FIGURE 4. Loading phase: The relationship between the daily BA dose (3.2 g·d−1 for the entire group) and the relative increase in muscle carnosine for lighter (<60 kg) and heavier (>60 kg) women (diamonds) and for lighter (<75 kg) and heavier (>75 kg) men (circles). Dashed lines are the extrapolations to reach a 50% increase in muscle carnosine.A nicely performed meta-analysis (20), covering 18 studies, about the effect of BA supplementation on exercise performance clearly demonstrated an ergogenic effect in exercise lasting 60–240 s. However, there are only four studies that measured muscle carnosine as well. These studies reported increases in muscle carnosine, with +37% (2), +42% (9), +59% (19), and +85% (8), averaging ∼50%. Hence, in Figure 4, we have extrapolated (or intrapolated) the daily dose that would be required to achieve a 50% increase over a 6-wk loading phase in men and women, each divided in a lighter and heavier half, with a cutoff value of 60 kg for women and 75 kg for men. This post hoc calculation indicates that the female participants weighing less than 60 kg, with an average of 56 kg, should ingest 2.5 g·d−1, whereas heavier women (∼64 kg) and lighter men (∼69 kg) should ingest 3.4 and 3.6 g·d−1, respectively, and heavier men (∼84 kg) should ingest 5.5 g·d−1 (Fig. 4). If faster loading is aimed, BA dose can be increased up to 6.4 g·d−1, with serving doses of 800 mg per intake (or 1600 mg in the case of SR BA). Higher doses may not be advised at present because the effectiveness or the side effects have not been studied beyond this dose.The present study aimed to determine the effective maintenance dose. Previous studies (5,28) have shown that after muscle carnosine loading, a slow washout takes place, ultimately leading to a return to baseline values. This indicates that there is some kind of an elimination process. However, the nature of the elimination process is still unclear at the moment: it could be 1) an active release through outward trans-sarcolemmal transporters (e.g., through PHT1 or PHT2 [12]), 2) a passive loss of carnosine through sarcolemmal disruption (10), 3) an enzymatic degradation (hydrolysis into BA and L-histidine) through carnosinase (CN2 [12]), or 4) a combination of these. Carnosine elimination rate is most likely positively related to the muscle carnosine content itself, that is, the more you have, the more you can lose or degrade. Net muscle carnosine turnover is a balance between carnosine synthesis and elimination rate, and an effective maintenance dose would be one where synthesis equals elimination. The carnosine synthesis rate in muscle is mainly determined by the sarcoplasmic BA availability because BA is the rate-limiting precursor. Sarcoplasmic BA availability is largely determined by dietary BA intake (17) and the enzymatic synthesis and degradation pathways of BA.In Figure 5, we have graphically displayed a hypothesis for the alterations in net carnosine turnover rate in different situations (baseline, loading, washout, and maintenance) as a result of changes in carnosine synthesis and/or elimination rate. At baseline, there is no net change in muscle carnosine because synthesis and elimination are in equilibrium and both are at a low level. During BA supplementation (loading), carnosine synthesis will markedly increase because of increased precursor availability, and net turnover will be strongly positive. As supplementation continues, with long duration and high doses, the elimination rate will gradually increase as muscle carnosine content, and thus elimination, becomes higher. As a result, the net positive turnover rate will become smaller, as has been demonstrated by Hill et al. (19), where carnosine loading efficiency was much smaller in the second half (+20%) compared to the first half (+60%) of supplementation. Upon discontinuation of supplementation, elimination rate is still elevated; whereas synthesis rate falls back to baseline values, resulting in a net negative balance, causing washout (5,28). Finally, keeping muscle carnosine at a constantly elevated level (maintenance) requires that the carnosine synthesis rate is increased by a low dose of BA supplementation, to compensate for the increased carnosine degradation rate in carnosine-loaded muscles.FIGURE 5. Graphical representation of the hypothesis on muscle carnosine turnover, which could explain the currently observed effects of the different phases of BA supplementation on muscle carnosine content.We now determined that an average dose of 1.2 g·d−1 is considered optimal to keep muscle carnosine content elevated at 30%–50% above baseline. Similar with the loading phase, it seems that heavier participants need a higher maintenance dose to keep muscle carnosine levels at 100% loaded compared to lighter participants because there is a strong relationship between maintenance dose, corrected for body mass, and maintenance in muscle carnosine. Therefore, we suggest taking ∼18 mg·kg−1 BW per day to keep elevated muscle carnosine levels. However, because the carnosine degradation rate is positively related to the muscle carnosine content, it can be hypothesized that the required maintenance dose may be higher to keep muscle carnosine elevated at supramaximal levels (>100% or even higher above baseline). Future studies will have to investigate this issue.In conclusion, sex and body mass are not major determinants for the absolute increase in muscle carnosine during loading, although they can partly explain the variability in the individual response in muscle carnosine loading when expressed as percentage increase versus baseline. 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Link]|00005768-201407000-00019#xpointer(id(R31-19))|11065213||ovftdb|00005768-200407000-00023SL00005768200436123911065213P91[CrossRef]|00005768-201407000-00019#xpointer(id(R31-19))|11065404||ovftdb|00005768-200407000-00023SL00005768200436123911065404P91[Full Text]|00005768-201407000-00019#xpointer(id(R31-19))|11065405||ovftdb|00005768-200407000-00023SL00005768200436123911065405P91[Medline Link]15235332β-Alanine Dose for Maintaining Moderately Elevated Muscle Carnosine LevelsSTEGEN, SANNE; BEX, TINE; VERVAET, CHRIS; VANHEE, LANDER; ACHTEN, ERIC; DERAVE, WIMApplied Sciences746