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Molecular Hydrogen as an Antioxidant in exercise

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Molecular Hydrogen as an Antioxidant in exercise ( molecular-hydrogen-as-an-antioxidant-exercise )

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Nutrients 2021, 13, 459 3 of 14 for the placebo. The amount of hydrogen in the HCP was 0.636 μg/capsule. The subjects were instructed to consume the supplements in one dose of four capsules at 9:00 p.m. (±1 h) for the three consecutive days prior to the experiment day, amounting to the total H2 amount of 2.544 μg/day. 2.3. Experimental Overview The subjects were familiarized with all of the measurement techniques, including the cycling exercise with a face mask. A familiarization test was conducted 1–2 weeks prior to the start of the experiments, and the experiments were separated by a six- to seven-day washout/rest period. On the day prior to testing, the consumption of alcohol and caffeine was prohibited, and when the exercise or training was carried out, the intensity, the timing, and duration were matched for both experiments. The subjects were also instructed to record and replicate their dietary intake for dinner the previous night and for breakfast before the testing. The subjects consumed breakfast at home ≥6 h prior to the start of the experiment (and were instructed to replicate it from their dietary intake report). At 3 h before the experiment, the subjects ate a small meal consisting of the Calorie Mate (four blocks, Otsuka Pharmaceutical, Tokyo) and one bottle of caffeine-free barely tea (Healthy Mineral Barley Tea, 600 mL, ITO EN, Tokyo) that was standardized for all subjects, in order to avoid hunger and minimize fluctuations in significant blood metabolic parameters (specifically blood glucose) among/within subjects. All studies were conducted in a custom-made environmental chamber (LP-2.5PH-SS, NKsystem, Osaka, Japan) maintained at a temperature of 25 ◦C with 50% relative humidity with minimal external stimuli. The subjects performed an incremental exercise test using a cycle ergometer (75XL-III; Konami, Tokyo). The exercise started with 2 min at the workload of 20 W, after which the workload was increased at 20 W/2 min until the subject’s exhaustion or 300 W was reached, and the subjects were instructed to maintain their pedal frequency at 60 rpm . throughout the exercise. When given criteria were met (e.g., a plateau or a drop in VO2, a heart rate (HR) > 95% of the age-predicted maximum [19], or a respiratory exchange . ratio > 1.1), the highest average value of 1-min VO2 was regarded as the individual’s peak oxygen uptake [20]. 2.4. Analyses of Blood Metabolites A peripheral venous catheter was placed in the subject’s forearm to allow free move- ment of his elbow and hands during the experiment, and a 100-μL blood sample was collected at four time points: pre-exercise and at 120, 200, and 240 W during the incremen- tal exercise. The blood samples were analyzed for blood gas, electrolytes, and the metabolic profile with a portable blood analysis system (epoc®, Siemens Healthcare, Tokyo) for the determination of blood gases, acid status of pH, bicarbonate (HCO3−), and blood proper- ties of hemoglobin (Hgb) and hematocrit (Hct). HCO3− was calculated from the partial pressures of CO2 (PCO2) and pH values according to the Henderson–Hasselbalch equation, and the base excess (BEecf) was calculated according to the following equation [21]: BE = (1 − 0.014 × [Hb]) × ([HCO3−] − 24.8 + (1.43 × [Hgb] +7.7) × (pH − 7.4)) The metabolic status of lactate (Lac), glucose (Glu), and creatinine (Crea), the elec- trolytes of the serum sodium (Na+), potassium (K+), chloride (Cl−) concentrations, • and the Aniongap (AGap) and Aniongap potassium (AGapK) were calculated by elec- trolyte parameters. 2.5. Measurements Each subject’s pulmonary gas exchange was measured breath by breath throughout all tests as described [13,22,23]. The breath-by-breath gas exchange system (AE-310s; Minato

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