Fifteen national-level male soccer athletes from China Football College volunteered to participate in this study. Exclusion criteria included: 1) hypertension, diabetes, cardiovascular risk factors and any other diagnosed metabolic disorders (e.g., acid-base imbalance); 2) smoking, ingested alcohol, caffeine and any other ergogenic supplements (e.g., sodium bicarbonate and creatine). Inclusion criteria: 1) age 18-24 years; 2) BMI ranging from 18.5 to 23.9 kg/m2; and 3) at least 5 years of regular soccer training experience with maximal oxygen uptake over 50 ml/kg/min. The study protocol was approved by the Internal Review Board of Beijing Sport University (2020057H). All subjects were informed of the study aims and were asked to sign an informed consent form. One participant dropped out after the graded cycling exercise test. Fourteen athletes completed all the sessions and were included in the data analyses (Table 1).
Randomized, double-blind, crossover trials were used in this research. There were five laboratory visits, each separated by 7 days. The first visit involved personal information collection, body composition measurement, familiarization with the exercise test protocol, and performed a graded cycling exercise test (GXT). In the second visit, participants took sodium pyruvate (PYR) or maltodextrin (Placebo, PLA) (0.1 g/kg/d) for 7 days parallelly and randomly in PYR (n=7) and PLA (n=7), respectively. During the third visit, 45 min after the last supplementation, the subjects took a 15-min resting oxygen uptake test (15-RO2), performed high-intensity interval exercise (HIIE) test to induce lactic acidosis, and collected a 6-min oxygen uptake immediately after HIIE (6-VO2). An RSE test was performed 10 min after HIIE. In the fourth visit, participants who were supplied with pyruvate/maltodextrin in the second visit were switched to maltodextrin/pyruvate, parallelly and randomly in PYR (n=7) and PLA (n=7). After a 1-week, they returned to the laboratory and repeated the tests they had done during the third visit. Each subject performed the tests at the same time of day and in the same laboratory environment.
Participants arrived in a resting and thoroughly hydrated condition following ≥ 2 h postprandial, avoiding any strenuous exercise and consumption with carbonate, alcohol and caffeine 24 h before each exercise test. A 3-day dietary recall was used to measure calorie intake and macronutrient consumption (2 weekdays and 1 weekend). Subjects used a 24-h recall to measure dietary consumption the day before the first trial and were asked to replicate the same diet as accurately as possible in subsequent studies. To ensure compliance, participants were expected to ingest the supplement under supervision. Fig. 1 presents a timeline for this study.
Body composition measurements
A calibrated electronic scale was used to calculate height to the nearest 0.1 cm (GMCS-SGJ3, Jianmin, Beijing, China). Body composition was assessed 2 h after a meal by multifrequency bioelectrical impedance measurement device (Inbody 230, Biospace, Seoul, Korea).
Graded cycling exercise test (GXT)
Each subject underwent GXT on an electromagnetically braked cycle ergometer (EC 3000e, CUSTO Med, Ottobrunn, Germany) to determine peak power (Wmax) and maximum oxygen uptake (VO2max). Participants adjusted the seat and warmed up for 3 min at 50 W. Then, the pedal frequency was kept between 75-80 rpm and the load was increased at 30 W/min. The subjects’ standards for exhaustion included the following: 1) Failed to keep 75 rpm for 5 s; 2) Increased the power load with the oxygen uptake (VO2) rise ≤ 150 ml/kg/min; 3) Respiratory exchange rate (VCO2/VO2) ≥ 1.10 and 4) Heart rate reached 220-age. VO2max is the average oxygen consumption 30 s before reaching exhaustion 26. Wmax is determined as the power of the last completed stage plus the fraction of time spent in the final uncompleted stage multiplied by 30 W 27. During the test, the Borg scale (6-20) 28 was used to determine the rate of perceived exertion (RPE) and Polar V800 (Polar Electro Oy, Oulu, Finland) was used to measure heart rate.
Sodium pyruvate in high quality (Lianlu industrial Co., Ltd., Shanghai, China) and maltodextrin were randomly packed in capsules A or B; both were similar in appearance, size and weight. Each participant took 0.1 g/kg/d 23 pyruvate or maltodextrin for 7 days (ingested 2 capsules just after a meal and 1 capsule before sleep on the first 6 days; ingested all the remaining capsules 60 min prior to the exercise test on the 7th day 23). Subjects took the capsule A were switched to the capsule B and vice versa during the test timeline.
Oxygen uptake test
VO2 was measured with breath-by-breath at GXT, 15-RO2, HIIE test and 6-VO2, using a portable gas analyzer (Cortex Metamax 3B, CORTEX Biophysik, Leipzig, Germany). Participants took a break in a sitting position to measure 15-RO2 and 6-VO2. The RO2 was determined by using the average of the last 10 min of data collected 29. The gas analyzer was calibrated before each test in accordance with the manufacturer’s instructions.
HIIE was conducted in an electromagnetically braked cycle (Ergoline Ergoselect 100K, Ergoline, Bitz, Germany). During the test, subjects took a 5-min warm-up at 60 W. HIIE consisted of 4 x 1 min high-intensity cycling at 110% Wmax and separated by 1 min rest. Cadence was constant (90-100 rpm) during high-intensity bout. Subjects remained seated on the ergometer, taking a 10-min break after finishing the last bout 30.
RSE consisting of 6 x 6 s of all out maximal cycling was performed on a mechanically braked cycle ergometer (894E, Monark, Vansbro, Sweden). Exercise load equaled weight (kg) × 0.087 kp/kg (weight) 31. During the test, participants adjusted the seat and began to fully ride following the staff countdown "3, 2, 1, start". Once the cadence reached 110 rpm, the load of inertial ergometers was adjusted to the predetermined load. Subjects kept fully pedaling to complete a 6 s cycling exercise and encouragement was provided to enable subjects to exert maximum ability. Each sprint had a 24 s rest interval 25.
Relative peak power (RPP): the highest power output (PP) relative to body mass observed in each 6 s; Relative average power (RAP): the average power output (AP) relative to body mass maintained in each 6 s; Power drop (PD%): the percentage of mechanical power decay over the period of the test relative to the peak power, according to the following equation: PD% = [(PP − AP)/PP × 100] 25. All the parameters were calculated via Monark Anaerobic Testing software (Version: 220.127.116.11, developed in cooperation with HUR Labs).
Blood collection and analyses
Capillary blood samples with 10 µl of fingerstick were collected (wiped away the first drop of blood) in a Biosen capillary tube (EKF Diagnostics, Barleben, Germany) at baseline, the end of each bout of HIIE, and 3, 5, 7 and 10 min after HIIE. The samples were used to measure blood lactate concentrations with a lactate analyzer (Biosen C-Line, EKF Diagnostics, Barleben, Germany).
Blood samples of 1.0 ml were obtained from the ulnar vein at baseline, pre-HIIE, post-HIIE, pre-RSE and post-RSE. The samples were collected in sodium heparin tubes (YA1430, Solarbio, Beijing, China) and immediately assessed for blood pH, HCO3-, BE and oxygen partial pressure (pO2) by a blood gas analyzer (Radiometer ABL80, FLEX CO-OX, Willich, Germany). Additionally, the blood HCO3- was calculated from the partial pressure of carbon dioxide and pH values according to the Henderson-Hasselbalch equation and the blood BE was calculated from HCO3- and hemoglobin (Hb) determined by gas analyzer according to the following equation 32:
Estimation of energy contribution
The aerobic energy contribution was estimated by subtracting the resting oxygen consumption from the oxygen consumption obtained during each 110% Wmax bout. The consumed oxygen was transferred to energy. One liter O2 is converted into 20.92 kJ energy equivalent 33. The lactate difference before and after each 110% Wmax bout and the lactate accumulated during HIIE were assumed to calculate the glycolytic energy contribution of each bout and total HIIE, respectively. The accumulation of 1.0 mmol/L lactate corresponds to 3.0 ml O2/kg of body weight 33. The phosphagen energy system contains two sections. First, resting oxygen uptake was subtracted from the oxygen consumption obtained during each 1-min interval. Second, fast component of excess post-exercise oxygen uptake (EPOC fast) accessed during 6-VO2 was adjusted by a biexponential model. The EPOC fast is assumed to be the product of the amplitude and time constant of the first exponential model (OriginPro 8.0, OriginLab, Microcal, Massachusetts, USA) 34.
A power analysis (Power 1-β = 0.9 and α = 0.05) was performed with a priori in G*Power software version 18.104.22.168 (Universitat Kiel, Germany), and at least 12 participants were needed for the present study. Data analyses were carried out by using SPSS, version 22.0 (SPSS Inc. Chicago, IL, USA). The Shapiro-Wilk test was used to verify the assumptions of normality of the data. A non-parametric Kruskal-Wallis test was used to analyze the power drop (%) of each sprint and Dunn-Bonferroni test as a post-hoc test. Two-way repeated-measures ANOVA was performed to assess the interaction between time (baseline, each bout of HIIE and each sprint of RSE) and the cohort (PYR and PLA). Fisher's least square significant difference (LSD) post-hoc analyses were performed when significant interactions were observed. Independent samples t-tests were used to measure relative energy contribution during HIIE and average RPP, RAP and PD% during RSE. Values were expressed as the mean ± SD or median (P25, P75). The statistical significance level was set at p < 0.05.