The Effect of Beta-Alanine versus Alkaline Agent Supplementation Combined with Branched-Chain Amino Acids and Creatine Malate in Highly-Trained Sprinters and Endurance Athletes: A Randomized Double-Blind Crossover Study

study aimed to verify the e ect of intra- (beta-alanine—BA) versus extra- (alkaline agents—ALK) cellular bu ering agent supplementation, combined with customarily used branched-chain amino acids (BCAAs) and creatine malate (TCM) treatment in natural training conditions. Thirty-one elite athletes (11 sprinters and 20 endurance athletes) participated in the study. Eight-week randomized double-blind, crossover, combined supplementation with BA-ALKplaBCAA&TCMand ALK-BAplaBCAA&TCMwas implemented. In the course of the experiment, body composition, aerobic capacity, and selected blood markers were assayed. After BA-ALKplaBCAA&TCMsupplementation, total fat-free mass increased in sprinters (p=0.009). No other di erences were found in body composition, respiratory parameters, aerobic capacity, blood lactate concentration, and hematological indices after BA-ALKplaBCAA&TCM/ALK-BAplaBCAA&TCM supplementation. The maximum post-exercise blood ammonia (NH3) concentration decreased in both groups after BA-ALKplaBCAA&TCMsupplementation (endurance,p=0.002; sprint,p<0.0001). Also, lower NH3concentrations were observed in endurance athletes in the post-exercise recovery period. The results of our study indicate that combined BCAA, TCM, and BA supplementation is more e ective than combined BCAA, TCM and ALK supplementation for an increase in fat-free mass and exercise adaptation, but not for aerobic capacity improvement. Besides, it seems that speci c exercise stimuli and the training status are key factors a ecting exercise performance, even in athletes using e cient supplementation. Keywords: supplements; training support; sport; physical capacity; body composition; biochemical markers 1. Introduction In sports practice, it is often di cult to cover the increased athlete's need for energy and nutrients through only a standard diet. For this reason,

c exercise stimuli and the training status are key factors a ecting exercise performance, even in athletes using e cient supplementation. Keywords: supplements; training support; sport; physical capacity; body composition; biochemical markers 1. Introduction In sports practice, it is often di cult to cover the increased athlete's need for energy and nutrients through only a standard diet. For this reason, adequate supplementation is an important factor supporting the training process. From the multitude of supplements, athletes mostly use preparations such as branched-chain amino acids (BCAAs), creatine (Cr), -alanine ( A) and sodium bicarbonate (SB). These supplements are associated with several mechanisms that a ect the metabolism and adaptation of the human body to physical exercise. Nutrients2019,11, 1961; doi:10.3390 /nu11091961 /journal/nutrients

Nutrients2019,11, 1961 2 of 19 BCAAs can signi cantly a ect the stimulation of the mammalian target of the rapamycin (mTOR) kinase pathway and thus stimulate the synthesis of muscle proteins, as well as have anti-catabolic activity, related to inhibition of muscle protein proteolysis [1–3]. The above mechanisms may explain the observed e ect of BCAA supply on the exercise-induced muscle damage, pain, fatigue or injury reduction, and the acceleration of fat-free mass or strength recovery [1,2,4,5]. Some additional bene ts may also be observed for endurance (aerobic) exercise because BCAAs have ergogenic potential and may a ect energy sources metabolism [6]. Some papers indicated the possible relationship between BCAAs and the reduction of central fatigue, which is associated with the synthesis of serotonin and facilitated by reduced BCAA/tryptophan ratio [7,8]. Such data show the importance of BCAA supplementation during long-term training periods. Ultimately, however, the e ectiveness of BCAAs in athletes may also depend on the availability of other essential amino acids [5,9]. Cr is one of the most commonly used supplements to stimulate phosphocreatine (PCr) synthesis, increase PCr concentration, and enhance the e ectiveness of adenosine triphosphate (ATP) resynthesis in muscle tissue [5,10,11]. Cr may also support high-intensity exercise and upregulate anabolic signals provided by exercise stimuli [5]. These mechanisms allow Cr to a ect the development of muscle strength and power, exercise capacity in single and repeated sprints, and support lean body mass (LBM) increase, which is particularly relevant to strength and speed–strength sport disciplines [5,10,11]. The Cr supplementation is therefore bene cial to the competitors of various sports, although its supply in endurance disciplines does not seem justi ed [12]. However, some observations suggested a Cr-related increase in the e ectiveness of muscle glycogen resynthesis, aerobic metabolism (via improvement of ATP shuttling from mitochondria), anaerobic threshold, and exercise tolerance, accompanied by a decrease in blood lactate accumulation [10,11,13]. This indicates some bene ts of Cr supplementation also in endurance athletes. Cr may also have the potential to counteract injury and support treatment and rehabilitation [5,10,11]. A serious problem is muscle acidi cation, closely related to intense

metabolism (via improvement of ATP shuttling from mitochondria), anaerobic threshold, and exercise tolerance, accompanied by a decrease in blood lactate accumulation [10,11,13]. This indicates some bene ts of Cr supplementation also in endurance athletes. Cr may also have the potential to counteract injury and support treatment and rehabilitation [5,10,11]. A serious problem is muscle acidi cation, closely related to intense training and competition. Acidi cation leads to muscle fatigue due to the competition of H + with calcium ions for the troponin binding site, suppression of phosphocreatine resynthesis and oxidative phosphorylation, inhibition of key enzymes of the glycolytic pathway (such as glycogen phosphorylase and phosphofructokinase), and decrease in the mitochondrial energy production in muscle cells (due to reduced mitochondrial matrix-cell cytoplasm proton gradient) [14–16]. Given the above-mentioned exercise-induced homeostasis disturbances, A supplementation may be an e ective counteraction [5,17–20]. Carnosine, synthesized from A, is the main intracellular factor that bu ers the excess of H + ions (generated in the process of glycolysis in muscle bers) and thus suppresses the muscle acidi cation [5,17–20]. Furthermore, the potential impact of A on muscle sensitivity and e ciency of Ca 2+ release, neuromuscular fatigue suppression, antioxidant and anti-glycation e ects, and detoxi cation of exercise-induced metabolites can also be bene cial in athletes [17,20,21]. It seems that the A supply is particularly e cient in e orts lasting 60–240 s, e.g., in speed–strength disciplines in which the development of speed, power, and endurance strength is most important [5,15,17,19,21–23]. However, further assessment of this compound requires further research in disciplines in which exercise lasting<1 min (e.g., sprint) or>7 min (e.g., endurance disciplines) is dominant, especially in the case of longer supplementation periods under real training conditions. A growing interest in ergogenic support is also seen in the case of alkalizing agents (ALK) such as SB, sodium citrate, and sodium/calcium lactate. Their impact on blood alkalosis and increase in extracellular bu er capacity are described in the literature [5,15,24]. The supply of alkalizing compounds counteracts the exercise-induced muscle acidi cation by H + binding and greater e ux of H + and lactate from

also seen in the case of alkalizing agents (ALK) such as SB, sodium citrate, and sodium/calcium lactate. Their impact on blood alkalosis and increase in extracellular bu er capacity are described in the literature [5,15,24]. The supply of alkalizing compounds counteracts the exercise-induced muscle acidi cation by H + binding and greater e ux of H + and lactate from muscle, thus ensuring sustained muscle contractility during exercise [5,15,25,26]. The bene ts of the extracellular metabolic alkalosis can also be associated with membrane depolarization, mitochondrial adaptations, and acceleration of glycogenolysis, which may enhance exercise performance [27–29]. The above mechanisms seem to explain the e ectiveness of ALK observed in the studies on e orts lasting ~1–4 min, but the results obtained for the exercise of longer duration are inconclusive [5,15,30]. It was

Nutrients2019,11, 1961 3 of 19 observed that SB supplementation increased performance, speed, and muscle power. It also reduced the time to reach peak power, increased total mechanical work, endurance strength, and improved sport-speci c exercise abilities in speed–strength disciplines and multiple bouts of exercise [5,15,31–35]. Our study aimed to assess the e ect of the supplementation of intra- (BA) versus extra- (ALK) cellular bu ering agents, combined with customarily ingested BCAAs and Cr in real training conditions in highly-trained athletes. The above aspects of the most popular supplements often did not re ect the chronic supplementation in highly-trained athletes who use dietary supplements much more frequently than their non-elite counterparts and often concurrently use many preparations [26,36–38]. Moreover, evidence-based supplementation protocols are often isolated from other ergogenic aids or habitual training. These issues include the additive, interactive, or counteractive e ect of combined use of di erent supplements and the repeated use of a supplement during one event or individual responsiveness [26,38]. Also, although there is no evidence of a direct e ect of BA and ALK supplementation on body composition and aerobic capacity, the reduction of exercise-induced muscle acidi cation may a ect muscle e ciency during training. This could a ect training-induced aerobic adaptation and body composition. According to our practical observations, we hypothesize that chronic BA and ALK treatment will support the training process in terms of the favorable e ect on exercise adaptation and body composition. 2. Materials and Methods 2.1. Participants Fifty-two participants (17 sprinters, 22 triathletes, and 13 long-distance runners) were initially enrolled in this study. Eventually, 31 subjects completed the entire study protocol and were included in the analysis: 11 sprinters (10 men, 1 woman) and 20 endurance athletes (12 triathletes (9 men, 3 women) and 8 long-distance runners (7 men, 1 woman)) (Figure, Table). The participants were professionally trained athletes and members of the Polish National Team. The inclusion criteria were age from 18 to 35 years, good health, a valid medical certi cate con rming the athlete's ability to practice competitive sport, and at least 5 years of training experience. The

3 women) and 8 long-distance runners (7 men, 1 woman)) (Figure, Table). The participants were professionally trained athletes and members of the Polish National Team. The inclusion criteria were age from 18 to 35 years, good health, a valid medical certi cate con rming the athlete's ability to practice competitive sport, and at least 5 years of training experience. The gender-related impact of the study was assumed negligible because the crossover design of our study allowed the participants to be their own control group. Furthermore, under real training conditions, the studied female athletes consumed the same supplements as male athletes. Also, there is a lack of evidence in the literature that the applied supplementation protocol was dependent on the sex of the subjects. Moreover, the assumption was also not to change athletes' nutrition and eating habits during the course of the study. Exclusion criteria were less than 6 training sessions per week, cigarette smoking, use of banned stimulants, alcohol consumption more than 1–2 drinks per week, and dietary supplements use beyond the recommendation of the authors of this study. For females, additional exclusion criteria were being pregnant or planning to become pregnant during the study. Before the study, the athletes customarily used preparations such as carbohydrates, BCAA, and Cr. However, they did not use any intra- (BA) or extra- (ALK) cellular bu ering agents. Moreover, during the study period, all athletes declared that they did not introduce any changes in their lifestyles, especially nutrition, and that they did not use any medications and supplements with potential ergogenic e ects, other than those supplied by the authors of this study. The dietary and workout records were collected during the run-in period of the study (Table, Table S1). Furthermore, every second week, the consultations with a dietitian and a member of the research team were held, which ensured that the athletes did not change their dietary habits and training mode during the whole supplementation period. The primary recruitment strategy was to contact the national team coaches. They enabled the identi cation and con rmation of required inclusion criteria declared by the participants

the consultations with a dietitian and a member of the research team were held, which ensured that the athletes did not change their dietary habits and training mode during the whole supplementation period. The primary recruitment strategy was to contact the national team coaches. They enabled the identi cation and con rmation of required inclusion criteria declared by the participants (such as training experience and the number of training sessions per week). They also supported the compliance of supplementation with the study design.

Nutrients2019,11, 1961 4 of 19Nutrients 2019, 11, x; FOR PEER REVIEW 4 of 20 Declaration of Helsinki. Each subject was informed of the testing procedure, purpose, and risks of the study and submitted her/his written consent to participate. The study was conducted from December 2015 to December 2016. The study complies with the CONSORT Statement for randomized trials, as shown in Figure 1. Figure 1. The flow chart of the study design. Abbreviations: ALK—Alkaline agents, BA—Beta- Alanine Carno Rush, BCAA—branched-chain amino acids, DXA—Dual X-ray Absorptiometry, LD— long-distance runners, pla—placebo, TCM—creatine malate, and TRI—triathletes. Figure 1. The ow chart of the study design. Abbreviations: ALK—Alkaline agents, BA—Beta-Alanine Carno Rush, BCAA—branched-chain amino acids, DXA—Dual X-ray Absorptiometry, LD—long-distance runners, pla—placebo, TCM—creatine malate, and TRI—triathletes. The project was approved by the Ethics Committee at the Poznan University of Medical Sciences (143/15 of 5 February 2015) and was performed according to the ethical standards laid down in the Declaration of Helsinki. Each subject was informed of the testing procedure, purpose, and risks of the study and submitted her/his written consent to participate. The study was conducted from December 2015 to December 2016. The study complies with the CONSORT Statement for randomized trials, as shown in Figure.

Nutrients2019,11, 1961 5 of 19 Table 1.Baseline anthropometric, training, and diet characteristics of the studied athletes. Variable SPRINTERS ENDURANCE Sprinters vs. Endurance * Mean SD Mean SD p-Value n 11 20 Age (years) 23.5 3.4 22.3 4.4 0.13 Body height (cm) 184 6 178 7 0.04 Body mass (kg) 77.8 7.8 70.4 11.3 0.01 Body mass index (BMI) (kg m 2 ) 23.0 1.1 22.1 2.3 0.29 Maximal oxygen uptake (VO 2max) (L min 1 ) 4.15 0.53 4.46 0.82 0.27 Training experience (years) 8.6 2.5 8.6 1.8 0.82 Energy intake (kcal day 1 ) 2922 335 3179 491 0.13 (kcal kg 1 ) 37.9 5.3 46.6 8.3 <0.01 Protein intake (g day 1 ) 129 29 138 34 0.48 (g kg 1 ) 1.7 0.3 2.0 0.5 0.052 (% of energy) 17.8 4.0 17.3 3.3 0.68 Fat intake (g day 1 ) 102 18 108 28 0.50 (g kg 1 ) 1.3 0.3 1.6 0.5 0.09 (% of energy) 31.4 4.0 30.4 4.6 0.55 Carbohydrate intake (g day 1 ) 378 57 437 63 0.02 (g kg 1 ) 4.9 0.9 6.4 1.1 <0.001 (% of energy) 51.8 4.4 55.3 5.4 0.07 Values are expressed as the means standard deviation (SD). Abbreviations: BMI—body mass index and VO2max—maximal oxygen uptake. *t-tests or Mann–Whitney U tests for independent samples, depending on data distribution. 2.2. Experimental Design 2.2.1. Supplementation Characteristics The e ect of supplementation was assessed in a randomized, crossover double-blind trial (Figure). The use of combined supplementation was intended to re ect the real conditions in which athletes use these preparations and to assess the synergistic e ect of supplements on the exercise capacity of athletes. Upon being quali ed to the experiment, the athletes were subjected to a randomization procedure and assigned either to (1) the group receiving an BA-ALKplaBCAA&TCM(Beta-Alanine Carno Rush Mega Tabs ® , BCAA Mega Caps ® , TCM Mega Caps ® , and placebo (PLA) instead of Alkagen—) or (2) to the group receiving ALK-BAplaBCAA&TCM(Alkagen—, BCAA Mega Caps ® , TCM Mega Caps ® , and PLA instead of Beta-Alanine Carno Rush Mega Tabs ®

procedure and assigned either to (1) the group receiving an BA-ALKplaBCAA&TCM(Beta-Alanine Carno Rush Mega Tabs ® , BCAA Mega Caps ® , TCM Mega Caps ® , and placebo (PLA) instead of Alkagen—) or (2) to the group receiving ALK-BAplaBCAA&TCM(Alkagen—, BCAA Mega Caps ® , TCM Mega Caps ® , and PLA instead of Beta-Alanine Carno Rush Mega Tabs ® ) preparations. The random allocation and assigning participants to the supplementation with a speci c set of preparations was performed by an impartial scientist who was not a member of the research team. The experimental procedure included 8 weeks of BA-ALKplaBCAA&TCMand ALK-BAplaBCAA&TCMsupplementation. After this period, a 6-week washout period was introduced [20,22]. The next step, after the washout period, was the crossover exchange of the preparations between the groups. During each period of supplementation, the athletes were given the equivalent quantitative doses of 0.2 g kgFFM 1 branched-chain amino acids (BCAA Mega Caps ® ; 1100 mg cap 1 ) and 0.05 g kgFFM 1 creatine malate (TCM Mega Caps ® ; 1100 mg cap 1 ). Depending on the period of study, the following preparations were also administered in the BA-ALKplaBCAA&TCMgroup: 5 g day 1 beta-alanine (Beta-Alanine Carno Rush Mega Tabs ® , containing A (1000 mg cap 1 ), sodium citrate (150 mg cap 1 ), 1-histidine HCl (500 mg cap 1 ), and vitamin B6(0.35 mg cap 1 )) and PLA (maltodextrin) imitating Alkagen ® alkalizing formulation. Concurrently, the following preparations were administers in the ALK-BAplaBCAA&TCMgroup: 0.2 g kgFFM 1 Alkagen ® alkalizing preparation [containing SB

Nutrients2019,11, 1961 6 of 19 (375 mg cap 1 ), potassium bicarbonate (375 mg cap 1 ), calcium phosphate (150 mg cap 1 ), potassium citrate (125 mg cap 1 ), magnesium citrate (125 mg cap 1 ), calcium citrate (90 mg cap 1 ), magnesium oxide (30 mg cap 1 ), and zinc (0.375 mg cap 1 )] and a PLA (maltodextrin instead of a beta-alanine). The preparations were administered in four split doses: upon waking, 45 min before a training session, immediately after a training session, and before sleep. All products and PLA preparations were prepared by Olimp Laboratories (D ebica, Poland), who have experience in manufacturing sports supplements according to high-quality production standards. Preparations were labeled using special codes, making it impossible to identify them and assign the same preparation twice to the same subject. 2.2.2. Study Visits The athletes visited the laboratory four times (T1–4, Figure). At each visit, body mass and composition were measured, and an exercise test was performed. All tests were conducted in the Human Movement Laboratory “LABTHLETICS” in the Department of Athletics, Strength and Conditioning at the Poznan University of Physical Education. The subjects were instructed not to participate in any high-intensity or long-duration training session at least 24 h before testing. The tests were performed in the morning, 3 h after a light breakfast (no co ee or tea). Before the exercise test, subjects underwent body composition analysis. Afterward, an incremental treadmill exercise test until volitional exhaustion was performed. During all examinations, room temperature remained at 20–21 C. The athletes were familiar with the incremental exercise test until volitional exhaustion because they participated in some previous studies and routine examinations. Anthropometrics and Body Composition. Body mass (kg) and height (cm) were measured using a digital stadiometer (SECA 285, Hamburg, Germany). Body mass index (BMI) was calculated by dividing body mass by height squared. The Dual X-ray Absorptiometry (DXA) method, utilizing the Lunar Prodigy Pro device (GE Healthcare, Madison, WI, USA) and enCORE v. 16 SP1 software, were used for body composition analysis. During the DXA examination, subjects only wore their undergarments,

This study investigates the effects of different supplementation strategies on athletic performance.