Human skeletal muscles contain carnosine (β-alanyl-L-histidine), a cytoplasmic dipeptide, in relatively high concentrations (∼5 mmol·kg−1 wet weight) (19). Chronic supplementation of beta-alanine (BA), the rate-limiting precursor for carnosine synthesis, can significantly increase muscle carnosine content by 40%–80% when ingested in doses of ∼1.6–6.4 g·d−1 for ≥4 wk (2,10,21,23,31). BA supplementation has recently become an intensive research topic, not only in the exercise and performance-enhancement field but also in clinical populations with chronic glycoxidative and inflammatory damage. An increase in muscle carnosine is related to an increase in performance for high-intensity exercise (1,8,22). Moreover, low muscle carnosine levels are possibly found in certain clinical populations such as diabetes type 2 and elderly (8,17), which may suggest that carnosine loading could be beneficial. These findings are assisted by in vitro and preclinical research, suggesting a multitude of physiological roles for carnosine, such as a regulator of muscle excitation–contraction coupling via increasing calcium sensitivity, a proton buffer, an antioxidant, and an inhibitor of protein glycation, as extensively reviewed by Boldyrev (4) and Derave et al. (9).
To understand the mechanism of muscle carnosine loading, it is necessary to gain insight into the metabolic fate of chronically ingested BA in humans. Decombaz et al. (7) acutely supplemented pure BA (PBA) and slow-release BA (SRBA) and showed a very high whole body retention for both forms, 96.3% and 98.9%, respectively. SRBA was developed to avoid high peak concentrations of BA in the blood, which is associated with side effects such as paresthesia; however, its efficiency for muscle carnosine loading has never been directly compared with PBA. When calculating the percentage of the total ingested BA dose (PBA or SRBA ingested over a prolonged period) that is actually incorporated into muscle carnosine, this is less than 6% (2,23,31). Therefore, we expect that the retention of BA, albeit nearly maximal upon a first oral challenge (7), will decrease during chronic supplementation. This would be similar to two other popular nutritional supplements that enhance performance through muscle loading, namely, creatine and carnitine, which are similar small nitrogen-containing molecules with high natural abundance in skeletal muscle. Although the biochemical mode of action with respect to involvement in energetic pathways leading to exercise performance enhancement is different between creatine, carnitine, and carnosine, the mechanism of accumulation in muscle is strikingly similar: the muscular loading is mainly limited by the trans-sarcolemmal transport system, and the transport is dependent on and driven by sodium ion cotransport. The retention of creatine and carnitine is high in a first ingestion but subsequently decreases during chronic supplementation. After 3 d (20 g·d−1), creatine retention decreases from 53% to 41% (14); and after 14 d (3 g·d−1), carnitine retention decreases from 98% to 90% (33).
Interestingly, the urinary loss of creatine and carnitine seems to be inversely related to the muscle loading efficiency, or in other words, when the muscle pools get saturated and reach maximal levels, the compounds (creatine and carnitine) start to appear more abundantly in the urine (13,33). According to this rationale, the search for new interventions to improve the muscle loading efficiency can start with looking at interventions that decrease urinary excretion, as a proxy of muscle accumulation. Indeed, when large amounts of carbohydrates are coingested with creatine or carnitine, its urinary excretion decreases (14,33). The follow-up research then demonstrated that more creatine and carnitine are accumulated in the skeletal muscle under the influence of carbohydrate- and protein-mediated insulin release (13,29,35). Insulin stimulates the sodium-dependent creatine (CreaT) and carnitine (OCTN2) transporter, secondary to its action of increasing sarcolemmal Na+/K+-ATPase pump activity and therefore transmembrane Na+ flux (5,34). Because it has been shown that the most important BA transporter (TauT) is strongly dependent on sodium and chloride cotransport (3), it is equally expected that muscular BA uptake, and hence carnosine loading is enhanced by insulin action.
Therefore, this study was designed to investigate the distribution of chronically ingested BA, by looking at urinary BA excretion and muscle carnosine loading. Second, does the coingestion of carbohydrates and proteins have the potential to alter this distribution of BA in the body? And finally, is SRBA more efficient concerning muscle carnosine loading compared with PBA?
MATERIALS AND METHODS
Forty-five healthy nonvegetarian men and women participated in the present studies (study A and study B), which were approved by the local ethical committee (Ghent University Hospital, Ghent, Belgium). All gave their written informed consent to take part in the studies and were aware that they were free to withdraw from the experiment at any point.
In study A (represented in Fig. 1), seven men (mean ± SD; age = 22.1 ± 1.3 yr, body weight = 80.7 ± 11.8 kg) were supplemented for 5 wk with 4.8 g SRBA (three times a day, two tablets of 800 mg PowerBar). Retention was measured weekly, by the ingestion of 1.6 g SRBA in fasted state and collection of urine up to 6 h postingestion. Total urinary volume was determined, and urine aliquots were collected in EDTA-coated tubes and stored at −20°C for further HPLC analysis of BA. During the 6-h period, the subjects were withdrawn from food but had access to water ad libitum. From week 2, subjects performed the retention twice on consecutive days: in the first condition, subjects took 1.6 g of SRBA in fasted state (FASTED); in the second condition, subjects took 1.6 g SRBA together with the intake of two carbohydrate-rich energy bars of PowerBar (CHO) (PowerBar Europe GmbH, München, Germany). To sustain elevated levels of plasma insulin, the first carbohydrate-rich bar (Energize C2 max: 39.1 g CHO + 5.8 g protein) was taken 10 min after BA intake, and the second protein-rich bar (Protein Plus: 22.2 g CHO + 16.6 g protein) was taken 55 min after BA intake. According to Steenge et al. (30), we can assume that the ingestion of 61.3 g CHO + 22.4 g protein would elevate plasma insulin above 70 mU·L−1.
In study B (represented in Fig. 1), to determine the effect of meal-induced insulin release on muscle carnosine loading, we first aimed to design a protocol in which the area under the curve (AUC) for plasma insulin and BA would either overlap with each other or would be completely separated from each other. For this purpose, we subjected four participants to a small acute pretest (age = 22.5 yr, body weight = 76.6 kg, two men and two women). For a period of 8 h, subjects ingested two meals, starting with a breakfast at 8:00 a.m. (850 kcal: 74% CHO, 14% P, and 12% F) and a lunch at 12:00 p.m. (950 kcal: 66% CHO, 13% P, and 21% F). Both meals were standardized and contained no meat. Breakfast consisted of four slices of white bread with chocolate-hazelnut paste, 200 mL semi-skimmed milk, 1 banana, and 125 g fruit yogurt. Lunch contained a 150-g baguette topped with 40 g young cheese, 30 g mayonnaise and vegetables with a 33-cL orange juice. Subjects all drank 1.5 L of water during the 8 h. Two subjects (one man and one woman) ingested PBA (2 × 800 mg) interspersed in between meals (at 10:30 a.m. and 2:30 p.m.: PBA), and two other subjects ingested PBA at the start of the meals (8:00 a.m. and 12:00 a.m.: PBA + meal). Every 30–45 min, venous blood samples (EDTA-coated tubes and serum tubes for, respectively, BA and insulin determination) were taken from a catheter and placed in an antecubital vein. We defined the AUC where elevated plasma BA values (>30 μM) coexisted with moments of elevated plasma insulin levels (>25 μU·mL−1) induced by meals.
We subsequently adopted this protocol of the pretest, that is, the comparison of ingesting PBA either during or interspersed between meals, into a full study lasting 46 d. In addition, a third experiment group was included that chronically ingested SRBA in the same dose. Thirty-four subjects (age = 19.4 ± 1.0 yr, body weight = 66.6 ± 9.9 kg), both men (n = 16) and women (n = 18), were randomized into three sex-balanced groups. All groups received 3.2 g BA daily, divided over four intakes of 800 mg each, with at least 3 h between intakes. The first group, PBA, received PBA between the meals/snacks (CarnoSyn BA powder in 400 mg gellules; Natural Alternatives International, San Marcos, CA). Subjects were not allowed to eat 150 min before and 90 min after each intake (four times daily) of BA throughout the 46 d. The second group, PBA + meal, received PBA at the start of each meal (three times a day), and a fourth pill had to be taken with a carbohydrate-rich snack. The last group, SRBA + meal, took SRBA (CarnoSyn SR tablets; Natural Alternatives International) 30 min before a meal or snack. Before and after the supplementation period, muscle carnosine content was measured; and during supplementation, subjects had to fill in a diary for the registration of every BA and meal/snack consumption. Finally, all subjects completed a questionnaire of their meat and fish consumption during the first 2 wk of the study to quantify daily dietary BA ingestion, as described by Baguet et al. (2).
Determinations in plasma and urine samples.
The concentration of BA in plasma and urine was determined by high-performance liquid chromatography (HPLC). One hundred microliters of EDTA-containing plasma and urine was taken and added with 11.1 μL of S-sulfosalicylic acid to deproteinize the samples. These deproteinized samples were dried under vacuum (40°C) for 45 min. 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. Samples were dried again and resolved in 100 μL sodium acetate buffer (10 mM, pH 6.7). The same method was applied for the standard solutions of BA (Sigma, Diegem, Belgium; dissolved in deionized distilled water). Derivatized samples (20 μL) were chromatographed on a Waters HPLC system with a Spherisorb C18/ODS2 column (4.6 × 150 mm, 5 μm), ODS2 guard column (80 Å, 5 μm, 4.6 × 10 mm), and UV detector (wavelength = 210 nm). The columns were equilibrated with buffer A (10 mM sodium acetate adjusted to pH 6.8 with 6% acetic acid), buffer B (60% acetonitrile–40% buffer A), and buffer C (100% acetonitrile) at a flow rate of 0.8 mL·min−1 at 25°C. The limit of quantification was 7 μM. Plasma insulin was determined by electrochemiluminescent assay on a Cobas E411 (Roche Diagnostics, Mannheim, Germany).
Determination of muscle carnosine content.
The carnosine content of all the subjects was measured by proton magnetic resonance spectroscopy (1H-MRS) in soleus and gastrocnemius medialis muscles, as described by Baguet et al. (1). The subjects were lying in 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 MRI scanner (Siemens Trio, Erlangen, Germany) equipped with a spherical knee coil. Single voxel point-resolved spectroscopy (PRESS) sequence with the following parameters was used: repetition time = 2.000 ms, echo time = 30 ms, number of excitations = 128, 1.024 = data points, spectral bandwidth = 1.200 Hz, and total acquisition time = 4.24 min. The average voxel size for the soleus and gastrocnemius muscle was, respectively, 40 × 10 × 28 mm and 40 × 11 × 30 mm. The line width of the water signal for the soleus and gastrocnemius muscle was, on average, 24.6 Hz and 27.6 Hz, respectively, following shimming procedures. The absolute carnosine content (mM) was calculated as described before by Baguet et al. (1).
A general linear model repeated-measures ANOVA was used to evaluate a decrease in BA retention over time. To look for differences between PLA and CHO a paired sample t-test was performed at each time point. To examine the absolute and relative difference in muscle carnosine between PBA and PBA + meal, a 2 × 2 general linear model ANOVA was performed, with absolute/relative increase in muscle carnosine as the dependent variable and muscle group and intervention group as the between-subject factors. A post hoc independent two-sample t-test was performed to study the difference for each muscle separately. To investigate the efficiency of PBA + meal versus SRBA + meal, the same statistics were performed as above. All analyses were performed using the Statistical Package for the Social Sciences (version 20; SPSS Inc., Chicago, IL), and statistical significance was set at P < 0.05.
Whole body BA retention (study A).
Urinary BA excretion was found to be very low and unaltered throughout the study in all subjects. Of the ingested 1600 mg SRBA, only ∼26 mg (range = 1–75 mg) was excreted in the urine. Whole body BA retention was 97.98% during a first oral challenge with SRBA. Long-term supplementation did not affect the retention (weeks 1–5: 97.98% ± 1.45%, 98.45% ± 1.20%, 98.05% ± 0.85%, 98.53% ± 0.66%, and 98.79% ± 0.59%, respectively; P > 0.05). Logically, the coingestion of SRBA with carbohydrates and proteins did not enhance BA retention (week 2: 98.37% ± 0.92%; week 3: 98.71% ± 0.83%; week 4: 98.54% ± 0.98%; week 5: 97.56% ± 1.74%; P > 0.05) (Fig. 2).
Plasma insulin and BA (study B).
Figure 3 represents the profile of plasma insulin and BA during 8 h when PBA ingestion is interspersed in between (Fig. 3A) or coincides (Fig. 3B) with meals. In Figure 3A, the moments of insulinemia (>25 μU·mL−1) and beta-alaninemia (>30 μM) are clearly separated; whereas in Figure 3B, the elevations in plasma insulin and BA clearly overlap. This is represented by, respectively, 0% and 82% of the total AUC of plasma BA showing elevated BA levels at the moment of hyperinsulinemia.
Muscle carnosine content (study B).
There were no differences between the groups concerning age, height, weight, and total dietary BA intake (Table 1) nor concerning baseline carnosine concentration in soleus and gastrocnemius (Table 2). Baseline soleus carnosine concentration (3.47 ± 0.87 mM) was lower compared with gastrocnemius muscle (4.51 ± 1.36 mM; P < 0.001). In soleus, the absolute (1.98 ± 0.96 mM) and relative (63.6% ± 41.6%) increase in carnosine was higher when PBA (4 × 800 mg) was taken at the start of each meal/snack (PBA + meal) compared with the group that ingested PBA in between the meals (PBA; 1.40 ± 0.44 mM and 40.7% ± 14.2%, respectively; Table 2 and Fig. 4) (main effect group: P = 0.08; independent two-sample t-test for soleus separately: P = 0.049). In the gastrocnemius muscle, the effect of meal timing was not present (independent two-sample t-test for gastrocnemius separately: P = 0.531). The muscle carnosine loading in the group SRBA + meal was not statistically different from PBA + meal.
Whole body BA retention.
The main finding of study A is that of the total amount of chronically ingested SRBA (168 g during the 5-wk period) only very little (∼2.7 g or 1.6%) is excreted in the urine (Fig. 2). This is surprising because it could be calculated from previous studies (2,21,31) that muscular uptake and the incorporation efficiency of exogenous BA into carnosine are very low as well. In the present study, the amount of BA converted into muscle carnosine was calculated to be ∼4.1 g (2.8%), when assuming that 40% of body mass is muscle mass. Thus, it seems that most (∼160 g or 95%–96%) of the ingested BA is neither going into muscle carnosine nor into the urine. Therefore, the alternative metabolic fate is currently unknown and merits further investigation. These new findings for BA are in sharp contrast with creatine, another popular ergogenic nutritional supplement. Like BA, creatine is a small nitrogen-containing, nonproteinogenic molecule with high abundance in skeletal muscle and a similar trans-sarcolemmal transport system. It has been repeatedly demonstrated, already upon the first publication in 1992 by Harris et al. (20), that the major portion of the ingested creatine is either excreted in the urine or accumulated in the muscle, with a reciprocal relationship between the two options (13,14,29,30). It is clear that for BA, urinary excretion cannot be used to make an indirect estimation of muscular accumulation of carnosine.
This finding brings to mind an important question: where does all the BA go? One possibility is oxidation and energy provision. The contribution of alpha-amino acids to energy delivery in muscle and other tissues is usually quite low as compared with carbohydrates and fat. However, when certain amino acids are available in excess, for example, branched chain α-amino acid supplementation, then they will be oxidized and their contribution to total energy delivery can amount to up to 10% (27). Indications exist that not only α-amino acids but also beta-amino acids can serve as an energy source when available in excess, which would certainly be the case with the ingestion of BA in such high daily doses (3.2–6.4 g·d−1 [2,21,23,31]). To enter the citric acid cycle, the amino group of BA has to be removed through a transamination reaction, catalyzed by BA transaminase (EC:184.108.40.206, also known as GABA transaminase [6,16]). Interestingly, we recently demonstrated its expression in rodent skeletal muscle and showed that its transcription is up-regulated upon BA supplementation (11). Therefore, we suggest that BA oxidation could be an important metabolic fate of chronically ingested BA, possibly in skeletal muscle, but likely in other organs such as liver, kidney, and brain (6,24). However, it is not excluded that other metabolic pathways are involved as well. Yet it seems unlikely that the conversion of BA into carnosine in other (nonmuscle) tissues is of any quantitative significance because the presence of carnosine (12,25) and carnosine synthase (18) in nonmuscle tissues is several orders of magnitude lower than that in muscle.
The effect of macronutrients on BA retention.
We observed that the coingestion of carbohydrates and proteins (61 g CHO + 22 g P) with SRBA did not alter urinary BA excretion and hence BA retention (Fig. 2). However, as mentioned above, urinary BA excretion is not a good inverse estimate of muscle carnosine accumulation. Thus, this observation does not exclude a shift toward more muscle carnosine loading, if this depends on the insulin sensitivity of the sodium-dependent BA transporter. To resolve this question, we needed to look at muscle carnosine loading in a long-term protocol in which BA was ingested either in the presence or absence of hyperinsulinemia.
The effect of meal-timing on muscle carnosine loading.
In study B, we could confirm in a 1-d pretest that subjects taking PBA in between the meals and subjects taking PBA at start of the meals had different (respectively out-of-phase versus in-phase) blood profiles concerning plasma insulin and BA (Fig. 3). Respectively 82% and 0% of the total AUC of plasma BA showed elevated BA levels (>30 μM) at the moment of insulinemia (>25μU·mL−1). In the follow-up chronic supplementation study, we were able to demonstrate that carnosine loading in the insulin-sensitive soleus muscle increased by 64% when PBA (4 × 800 mg) is taken at the start of each meal/snack compared with 41% when PBA was ingested in between the meals (Fig. 4). This finding may have practical applications, that is, regarding the advice on how and when to ingest BA for optimal muscle carnosine loading results. However, it may also provide further insight into the physiological mechanism and limitations to muscle BA uptake and carnosine synthesis.
Most researchers postulate that very high physiological insulin levels (approaching ∼100 μU·mL−1) are necessary to stimulate the sodium-dependent transporters of creatine (CreaT) and carnitine (OCTN2) and hence to alter their retention and muscle concentration (13,14,29,30,32). However, Greenwood et al. (15) suggested that creatine retention can be increased even with relatively small amounts (18 g) of simultaneous carbohydrate ingestion. We could demonstrate that meal timing alone can beneficially influence muscle carnosine loading. In Figure 3, average meal-induced insulin plasma peaks are shown, but individual peaks vary between 53 and 102 μU·mL−1. Clausen et al. (5) showed that Na+/K+ pumps in skeletal muscle are stimulated by insulin over a range of concentrations down to low physiological levels. Therefore, it is possible that meal-induced elevations in serum insulin are capable of triggering the sodium-dependent transporter TauT. In our study, a meal-induced effect could only be confirmed in soleus muscle. Considering the fact that this is a more insulin-sensitive muscle (26), it is not unlikely that the soleus responds better on insulin-induced BA uptake. In addition, Lavoie et al. (28) demonstrated that insulin-induced translocation of Na+/K+ ATPase subunits to the plasma membrane, one of the two possibilities to increase Na+/K+ ATPase activity, is restricted to oxidative fiber-type skeletal muscles that are predominantly present in soleus.
SRBA versus PBA supplementation.
To our knowledge, this is the first study that directly compares the loading efficiency of a similar dose of PBA and SRBA in a chronic supplementation study. It was initially hypothesized that SRBA would be more efficient than PBA because of a better BA retention (98.9% and 96.3%, respectively) upon acute ingestion (7). We now know that whole BA retention is not a good indicator of muscle carnosine loading efficiency. In the current study, we could show that there was no significant difference between the long-term supplementation of SRBA (SRBA + meal) and PBA (PBA + meal) (Fig. 4B). If we believe that the percentage of the AUC of plasma BA that occurs during hyperinsulinemia is of importance for enhancing BA uptake in skeletal muscle, we may expect that SRBA+ meal is more responsive than PBA, but less than PBA + meal. This expected order is observed in the soleus, but not in the gastrocnemius. Possibly, this is merely an epiphenomenon and also other aspects of the kinetics, such as the peak plasma BA concentration and the total AUC for BA may play a role in the effectiveness of BA on muscle carnosine loading.
In this article, we show that despite only a small portion of chronically ingested BA being incorporated into muscle carnosine, there is equally only a very small portion lost through urinary excretion. We therefore hypothesize that most ingested BA is metabolized in noncarnosine directed pathways. Second, we show that BA supplementation is more effective when coingested with a meal, suggesting that insulin could play a role in muscle carnosine loading. Finally, we conclude that SRBA is equally effective as PBA in muscle carnosine loading.
The contribution of Anneke Volkaert, Coussens Brendan, and D’Hondt Anthony is greatly acknowledged.
The authors thank Zibi Szlufcik and PowerBar Europe for generously providing the energy bars and Roger Harris and Natural Alternatives International for generously providing the BA supplements.
This study was financially supported by the Research Foundation–Flanders (grant nos. FWO 1.5.149.08 and G.0046.09).
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
The authors declare no conflict of interest.
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