The effect of multi-ingredient intra- versus extra-cellular buffering supplementation combined with branched-chain amino acids and creatine on exercise-induced ammonia blood concentration and aerobic capacity in taekwondo athletes | Journal of the International Society of Sports Nutrition

Participants

Twelve taekwondo athletes were initially enrolled in this study. Eventually, 9 participants (5 male and 4 female) completed the entire study protocol and were included in analyses. The participants were members of the Polish national team meeting specific criteria, i.e. highest level of training status, physical capacity, performance, and technical skills. All of the participants were black belt holders and medalists in ranked competitions (international open tournaments, Universiade, European, and world championships). One athlete took part in the Olympic Games. The inclusion criteria were age from 18 to 35 years, good health, a valid medical certificate confirming the ability to compete and practice sports, and at least 5 years of professional taekwondo training experience. The gender-related impact on the cross-over design of the study was considered negligible. Exclusion criteria were: current smoking or illicit drug use, alcohol consumption greater than 1–2 drinks/week, and dietary supplements beyond those recommended within this study. For females, additional exclusion criteria were being pregnant or planning to become pregnant during the study. Basic training characteristics were also monitored before and during the whole study protocol (initial period, first treatment period, washout, and second treatment period; Table S1). All athletes maintained a high-intensity exercise regime characteristic of taekwondo training and combat during the whole treatment and washout periods. Importantly, each treatment period was preceded by a similar standard training period. Furthermore, 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 effects other than those supplied by the authors of this study. Dietary records were being performed for two days every second week (current quotation) to ensure that the athletes had not changed their dietary habits during the whole supplementation period. The team member responsible for the diet analysis randomly informed the subjects about the need to start recording their two-day menus. Nutritional data was collected using notes on mobile phones and taking pictures of the meals consumed, which were then sent to the person analyzing the data. Dietary assessment was carried out using the Dietetyk-2 (Jumar, Poznań, Poland) software package following the previously described procedures used in our studies, both including supplementation and nutritional interventions, and weight management [8, 27,28,29]. The national team coaches enabled the confirmation of the required inclusion criteria declared by the participants. They also supported the control of training and supplementation compliance by monitoring the empty supplement containers, athletes’ diaries, and periodic personal observations of supplementation use by athletes. The project was approved by the Ethics Committee at the Poznan University of Medical Sciences (decision No. 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 and submitted her/his written consent to participate. The study had been conducted for 1 year, from December 2015 to December 2016. The study complies with the CONSORT Statement for randomized trials as shown in Fig. 1.

Experimental Design

Supplementation

The effect of supplementation was assessed in a randomized crossover double-blind trial. The primary outcomes were changes in total blood ammonia concentration (NH₃), a specific marker of training adaptation, and aerobic capacity as assessed during an incremental treadmill test until exhaustion. In our previous similar study [8], we assessed the same effects in two extreme cases of exercise performance in highly-trained sprinters vs. endurance athletes. In this study, it was particularly essential to assess the effect of similar supplementation protocol in disciplines with mixed energetics. Of twelve participants, 1 male and 2 females did not complete the study either due to their refusal to participate or resignation without explanation (Fig. 1). After qualifying for the experiment, the athletes were subjected to a randomization procedure and assigned either to the group receiving (i) BA (Beta-Alanine Carno Rush Mega Tabs®, BCAA Mega Caps®, TCM Mega Caps®, and placebo (maltodextrin, PLA) instead of Alkagen™) or (ii) ALK (Alkagen™, BCAA Mega Caps®, TCM Mega Caps®, and PLA instead of Beta-Alanine Carno Rush Mega Tabs®) preparations. The random allocation sequence and assigning participants to supplementation with preparations with specific codes was performed by an impartial scientist who was not a member of the research team. The experimental procedure included an 8-week BA and ALK supplementation. After this period, an 8-week washout period was introduced. The next step was a crossover exchange of the preparations. The 8-week washout and 8-week supplementation periods were established, similar to other studies [30, 31].

Detailed supplementation characteristics are presented in Table 1. During each series of supplementation, all athletes were given the equivalent quantitative doses of about 0.2 g·kg_FFM^− 1 BCAA (11.0 ± 2.2 g·day^− 1 BCAA; BCAA Mega Caps®; 1100 mg·cap^− 1) and about 0.05 g·kg_FFM^− 1 creatine malate (2.8 ± 0.6 g·day^− 1 TCM; TCM Mega Caps®; 1100 mg·cap^− 1). In the BA group, the following preparations were also administered depending on the study phase: 5 capsules of Beta-Alanine Carno Rush Mega Tabs® (containing βA (1000 mg·cap^− 1), sodium citrate (150 mg·cap^− 1), L-histidine HCl (500 mg·cap^− 1), and vitamin B₆ (0.35 mg·cap^− 1)), and PLA (maltodextrin) imitating Alkagen™ alkalizing formulation; the specified doses indicated in Table 1. Concurrently, in the ALK group, the following preparations were administered: about 0.2 g·kg_FFM^− 1 Alkagen™ (11.0 ± 2.2 g·day^− 1 ALK) alkalizing preparation (containing SB (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), zinc (0.375 mg·cap^− 1)), and a PLA (maltodextrin instead of a Beta-Alanine Carno Rush Mega Tabs); the specified doses indicated in Table 1. The number of capsules (Alkagen, BCAA, TCM) was adjusted to match the prescribed dose in g·kg_FFM^− 1 as close as possible. The preparations were administered in 4 split doses. If one training session a day was foreseen, the split doses were administered at the following times: upon awakening, 60 min before the training session, immediately after the training session, and before sleep. If two training sessions a day were scheduled the times were as follows: upon awakening, 60 min before each training session, and before sleep.

All products, including PLA preparations, were prepared by one manufacturer (Olimp Laboratories, Dębica, Poland). Preparations were coded, making it impossible to identify and assign the same preparation twice to the same subject.

Laboratory Visits

The study protocol included four visits to the Human Movement Laboratory “_LA_BTHLETICS” of the Department of Athletics, Strength and Conditioning at the Poznan University of Physical Education, Poznań, Poland (Fig. 1). Subjects were instructed not to participate in any high-intensity or long-duration training sessions at least 24 h before testing. All measurements were performed in the morning, 3 h after a light breakfast (no coffee or tea). At the start, subjects underwent body composition analysis. Afterward, an incremental treadmill test until volitional exhaustion was performed. During all measurements, the ambient temperature remained at 20‒21^oC. The subjects were familiar with the tests and procedures used as they had participated in some previous research projects.

Anthropometry 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, Wisconsin, USA) and enCORE v. 16 SP1 software, was used for body composition assessment. During the examination, subjects only wore their undergarments, without jewelry and metal objects to minimize measurement error. The measurements were carried out according to the standardized scanning protocol as recommended by Nana et al. [32].

Cardiorespiratory Test

An exercise test on the Pulsar mechanical treadmill (H/P Cosmos Sports & Medical GmbH, Nussdorf-Traunstein, Germany) was performed to determine maximal oxygen uptake (V̇O_2max). The initial speed was set at 4 km·h⁻¹ and increased after 3 min to 8 km·h⁻¹. After that point, treadmill speed progressively increased by 2 km·h⁻¹ every 3 min until volitional exhaustion. After the speed of 10 km·h⁻¹ was reached, venous blood samples were drawn at the end of each 3-min stage during pauses of up to 20 s. Minute ventilation (V̇E), oxygen uptake (V̇O₂), carbon dioxide production, respiratory exchange ratio (RER), and other respiratory parameters were measured (breath by breath) by the Metamax 3B R2 ergospirometer and analyzed using MetasoftStudio v. 5.1.0 Software (Cortex Biophysik, Leipzig, Germany). Heart rate (HR) was monitored using the Bluetooth Smart H6 heart rate monitor (Polar Electro Oy, Kempele, Finland). The V̇O₂max and corresponding exercise variables were considered to be reached when at least three of the following criteria were met: (1) a plateau in V̇O₂ despite an increase in speed and minute ventilation; (2) blood lactate concentration at exhaustion ≥ 9 mmol∙L⁻¹, (3) respiratory exchange ratio ≥ 1.10, and (4) heart rate ≥ 95 % of maximum (based on previous measurements) [33]. Also, V̇O₂, V̇E, HR, and RER at the ventilatory threshold were determined based on the ventilatory equivalents and partial pressures of oxygen and carbon dioxide.

Blood Sampling

A catheter (BD Venflon Pro 1.3 × 32 mm, Helsingborg, Sweden) patent with isotonic saline (0.9 % NaCl) was placed in the antecubital vein. Blood samples were taken into two monovettes (S-Monovette 2.7 ml KE, Sarstedt, Nümbrecht, Germany), one with a lithium anticoagulant (heparin) for lactate and NH₃ assay and another containing the EDTA anticoagulant for hematological measurements.

Lactate and Ammonia

To determine lactate concentration (LA), 20 µl of whole blood was placed into a capillary and placed in the Biosen C-line device (EKF diagnostic GmbH, Barleben, Germany). To determine total blood ammonia concentration, the PocketChem BA PA-4140 device (Arkray, Kyoto, Japan) was used with measuring range and accuracy (CV) of 8‒285 µmol·L⁻¹ and 2.3 %, respectively. Immediately after drawing blood, a sample of 20 µl was placed on the test strip (Ammonia Test Kit II, Arkay, Kyoto, Japan) using a pipette. Ammonium ions in the sample were converted into the gaseous form that reacted with an indicator layer to change its color. The light of a wavelength of 635 nm, reflected by the indicator layer, was used to determine the level of color indicative of ammonia concentration.

Hematological Measurements

Ten µl of blood were analyzed in the automated Mythic®18 analyzer (Orphée, Geneva, Switzerland) to determine concentrations of white blood cells (WBC), lymphocytes (Lym), monocytes (Mon), granulocyte (Gra), red blood cells (RBC), hemoglobin (Hb), and hematocrit (Ht).

Statistical analysis

Stratified randomization was performed with fat-free mass (FFM) being a prognostic variable as described previously [8, 30, 34, 35]. The results are presented as means ± standard deviation (and 95 % confidence intervals). The normality of data was tested using the Shapiro-Wilk test. If the distribution was not normal, the Box-Cox transformation was applied. Data were analyzed using repeated measures within-between interaction analysis of variance (ANOVA) with the inclusion of experimental supplementation order (BA first or ALK first), which allowed for the elimination of the potential carryover effect. The analysis also included factors independent of time: treatment (BA/ALK) x period (Before/After). Post hoc analysis was done using Bonferroni correction. If the sphericity assumption was violated, the Greenhouse-Geisser and the Huynh-Feldt corrections were performed. The sample size was estimated a priori, assuming that the effect size of supplementation type on ammonia blood concentration was 0.42 (partial eta-squared) as shown in our previous study [8]. Using an α-level of 0.05, a power (1 – β) of 0.80, it was calculated that at least 8 participants in each supplementation group would be needed to detect a significant change or differences in the variables analyzed (G*Power; Heinrich-Heine-Universität, Düsseldorf, Germany). To compare the anthropometric traits, training history, and diet characteristics, independent samples t-tests or Mann–Whitney U tests were performed, depending on data distribution (parametric or nonparametric, respectively). Statistical significance was set at p < 0.05 and data were analyzed using the Statistica 12 software package (StatSoft Inc., Tulsa, OK, USA).