Abstract
Initially, molecular hydrogen was considered a physiologically inert and non-functional gas. However, experimental and clinical studies have shown that molecular hydrogen has anti-inflammatory, anti-apoptotic, and strong selective antioxidant effects. This study aimed to evaluate the effects of 60 minutes of molecular hydrogen inhalation on respiratory gas analysis parameters using a randomized, double-blind, placebo-controlled, crossover design. The study was conducted at Faculty of Physical Culture, Palacký University Olomouc from September 2022 to March 2023. Twenty, physically active female participants aged 22.1 ± 1.6 years who inhaled either molecular hydrogen or ambient air through a nasal cannula (300 mL/min) for 60 minutes while resting were included in this study. Metabolic response was measured using indirect calorimetry. Breath-by-breath data were averaged over four 15-minute intervals. Compared with placebo (ambient air), molecular hydrogen inhalation significantly decreased respiratory exchange ratio and ventilation across all intervals. Furthermore, the change in respiratory exchange ratio was negatively correlated with body fat percentage from 30 minutes onwards. In conclusion, 60 minutes of resting molecular hydrogen inhalation significantly increased resting fat oxidation, as evidenced by decreased respiratory exchange ratio, particularly in individuals with higher body fat percentages.
Keywords: body fat, fat oxidation, metabolic flexibility, mitochondria respiration, respiratory exchange ratio
Introduction
Experimental and clinical studies have shown anti-inflammatory, antioxidant, and signaling properties of molecular hydrogen.1,2,3 Molecular hydrogen can be delivered into a body by drinking hydrogen-rich water,4 hydrogen bath,5 or inhalation.6 Inhalation of molecular hydrogen through a nasal cannula, even at low flow rates (250 mL/min), is an effective method of transporting molecular hydrogen into the blood.7 Once in the circulation molecular hydrogen is transported throughout the body by advection-diffusion and dynamically metabolized.7 An experimental study showed that a 30-minute inhalation of 3–4% molecular hydrogen concentration induced a plateau in molecular hydrogen level at approximately 10–20 μM in arterial and venous blood within 20 minutes.8 Molecular hydrogen administration has been shown to be safe, with no adverse effects reported in human9 and animal10 studies. A large volume of studies have demonstrated beneficial health effects of molecular hydrogen administration.11,12 Molecular hydrogen administration has also been shown to alter metabolism, improving both lipid and carbohydrate metabolism in patients with type two diabetes mellitus or impaired glucose tolerance,13 increasing lipid metabolism in vitro,14,15 animal models16,17 and humans.18,19,20 Acute administration of molecular hydrogen resulted in decreased blood lactate levels during and post-exercise.21,22,23,24 This reduction in blood lactate in response to molecular hydrogen administration has potentially beneficial implications, because blood lactate accumulation during exercise in individuals with metabolic syndrome, has been understood as a sign of lower metabolic flexibility due to impairment in mitochondrial oxidative function.25 Positive metabolic outcomes of molecular hydrogen link with the evidence showing significantly increased mitochondrial respiratory efficiency through enhanced mitochondrial Q-cycle,26 mitochondrial oxygen consumption (V̇O2), and adenosine triphosphate (ATP) production27 in in vitro experiments.
Investigation of resting metabolic rate using indirect calorimetry is well-established.28 Measurements of V̇O2 and carbon dioxide production (V̇CO2), are used to calculate the respiratory exchange ratio (RER), the rate of fat oxidation (FATox) and the rate of carbohydrate oxidation.25 Specifically, RER values ranging from 0.71 to 1.0, are used to indirectly determine the relative contribution of carbohydrate and lipids to overall energy expenditure. Importantly, a decrease in RER from 0.85 to 0.80 shows a 16% shift from carbohydrates to lipid utilization.29 Recently it was shown that RER variability is also important to measure as it provides an indication of metabolic flexibility.30 Higher RER variability in response to routine daily tasks such as sitting, standing, and sit-stand-sit transition was positively correlated with lower overall central obesity, fat-free mass, and negatively correlated with body fat mass in a young, non-obese population.31 In addition, San-Millán and Brooks25 associated poor metabolic flexibility with mitochondrial respiratory limitations manifesting in low fat and carbohydrate oxidation and poor lactate clearance capacity. Shook et al.32 further showed that young adults with a high resting RER (0.841 ± 0.032) experienced a greater increase in fat mass compared with those with a low or moderate RER (0.766 ± 0.025) over 12 months.
Murakami et al.27 showed that molecular hydrogen can improve mitochondrial respiration capacity within 1 hour, which implies that measures of whole-body oxidative metabolism may be altered. Until now, the effect of molecular hydrogen on metabolic flexibility in females has never been investigated. Therefore, based on this rationale, we conducted this randomized controlled crossover study with the hypothesis that 60 minutes of resting molecular hydrogen inhalation would influence metabolic response. The primary objective was to compare RER values in healthy females at rest with inhalation of molecular hydrogen versus placebo (ambient air). The secondary objective was to analyze potential moderators of changes in RER between molecular hydrogen and placebo.
Participants and Methods
Participants
The study was conducted at Faculty of Physical Culture, Palacký University Olomouc from September 2022 to March 2023 and recruited 24 female sports science students. Inclusion criteria were age 18–26 years, physical activity (at least three times per week 20 minutes of moderate to vigorous intensity33), and a female student in a sports faculty. Exclusion criteria were the use of drugs and dietary supplements that could affect metabolism, 1 month before the first experimental session,34,35 menstruation at the time of experimental measurement, and any known (self-reported) cardiovascular, pulmonary, or metabolic diseases. Three participants were excluded due to technical failure to record ventilation (VE) or heart rate. One participant was lost due to illness during the washout period. Twenty participants successfully completed the study (Table 1). They followed instructions to avoid using dietary supplements (including sports drinks and beverages containing caffeine) and maintain the same individually prescribed training load from 1 week before the first session of the experiment until completion of the second session, including during the washout period. Furthermore, 24 hours before testing, they should not perform any vigorous physical activity36 and not consume any alcoholic beverages. The research was conducted according to the Declaration of Helsinki and was approved by the Ethics Committee of the Faculty of Physical Culture, Palacký University Olomouc on December 27, 2021 (reference number 107/2021). Participation in this research was voluntary and all participants signed informed consent. The manuscript was written and revised in accordance with CONsolidated Standards Of Reporting Trials (CONSORT) (Additional file 1).37
Table 1.
Characteristics of the participants (n = 20)
| Variable | Data | P |
|---|---|---|
| Age (yr) | 22.1±1.6 | 0.61 |
| Body mass (kg) | 64.1±7.0 | 0.55 |
| Body height (cm) | 167.2±6.8 | 0.37 |
| Body mass index (kg/m2) | 22.9±2.1 | 0.63 |
| Body fat (%) | 23.1±4.5 | 0.81 |
Data are expressed as mean ± SD. P indicates significance of Shapiro-Wilk test.
CONSORT 2010 checklist of information to include when reporting a randomised trial*
| Section/Topic | Item No | Checklist item | Reported on page No |
|---|---|---|---|
| Title and abstract | |||
| 1a | Identification as a randomised trial in the title | 1 | |
| 1b | Structured summary of trial design, methods, results, and conclusions (for specific guidance see CONSORT for abstracts) | 1 | |
| Introduction | |||
| Background and objectives | 2a | Scientific background and explanation of rationale | 1, 2 |
| 2b | Specific objectives or hypotheses | 2 | |
| Methods | |||
| Trial design | 3a | Description of trial design (such as parallel, factorial) including allocation ratio | 2 |
| 3b | Important changes to methods after trial commencement (such as eligibility criteria), with reasons | No change | |
| Participants | 4a | Eligibility criteria for participants | 2 |
| 4b | Settings and locations where the data were collected | 2 | |
| Interventions | 5 | The interventions for each group with sufficient details to allow replication, including how and when they were actually administered | 3 |
| Outcomes | 6a | Completely defined pre-specified primary and secondary outcome measures, including how and when they were assessed | 3 |
| 6b | Any changes to trial outcomes after the trial commenced, with reasons | No change | |
| Sample size | 7a | How sample size was determined | 4 |
| 7b | When applicable, explanation of any interim analyses and stopping guidelines | Not applicable | |
| Randomisation: | |||
| Sequence generation | 8a | Method used to generate the random allocation sequence | 2 |
| 8b | Type of randomisation; details of any restriction (such as blocking and block size) | 2 | |
| Allocation concealment mechanism | 9 | Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned | 2 |
| Implementation | 10 | Who generated the random allocation sequence, who enrolled participants, and who assigned participants to interventions | No reported |
| Blinding | 11a | If done, who was blinded after assignment to interventions (for example, participants, care providers, those assessing outcomes) and how | 2 |
| 11b | If relevant, description of the similarity of interventions | 3 | |
| Statistical methods | 12a | Statistical methods used to compare groups for primary and secondary outcomes | 3, 4 |
| 12b | Methods for additional analyses, such as subgroup analyses and adjusted analyses | 4 | |
| Results | |||
| Participant flow (a diagram is strongly recommended) | 13a | For each group, the numbers of participants who were randomly assigned, received intended treatment, and were analysed for the primary outcome | 2 |
| 13b | For each group, losses and exclusions after randomisation, together with reasons | 2 | |
| Recruitment | 14a | Dates defining the periods of recruitment and follow-up | 2 |
| 14b | Why the trial ended or was stopped | 2 | |
| Baseline data | 15 | A table showing baseline demographic and clinical characteristics for each group | 2 |
| Numbers analysed | 16 | For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups | 2 |
| Outcomes and estimation | 17a | For each primary and secondary outcome, results for each group, and the estimated effect size and its precision (such as 95% confidence interval) | 4, 5 |
| 17b | For binary outcomes, presentation of both absolute and relative effect sizes is recommended | Not applicable | |
| Ancillary analyses | 18 | Results of any other analyses performed, including subgroup analyses and adjusted analyses, distinguishing pre-specified from exploratory | 6 |
| Harms | 19 | All important harms or unintended effects in each group (for specific guidance see CONSORT for harms) | No harms |
| Discussion | |||
| Limitations | 20 | Trial limitations, addressing sources of potential bias, imprecision, and, if relevant, multiplicity of analyses | 7 |
| Generalisability | 21 | Generalisability (external validity, applicability) of the trial findings | 7 |
| Interpretation | 22 | Interpretation consistent with results, balancing benefits and harms, and considering other relevant evidence | 6, 7 |
| Other information | |||
| Registration | 23 | Registration number and name of trial registry | Not available |
| Protocol | 24 | Where the full trial protocol can be accessed, if available | Not available |
| Funding | 25 | Sources of funding and other support (such as supply of drugs), role of funders | 1 |
Citation: Schulz KF, Altman DG, Moher D, for the CONSORT Group. CONSORT 2010 Statement: updated guidelines for reporting parallel group randomised trials. BMC Medicine. 2010;8:18. © 2010 Schulz et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
*We strongly recommend reading this statement in conjunction with the CONSORT 2010 Explanation and Elaboration for important clarifications on all the items. If relevant, we also recommend reading CONSORT extensions for cluster randomised trials, non-inferiority and equivalence trials, non-pharmacological treatments, herbal interventions, and pragmatic trials. Additional extensions are forthcoming: for those and for up-to-date references relevant to this checklist, see www.consort-statement.org.
Experimental protocol
The research was conducted as a randomized, double-blind, placebo-controlled crossover study. Figure 1 shows the course of the experiment. At the beginning of the experiment, the participants were familiarized with all aspects of the research (such as measuring equipment and procedures). After signing the informed consent document, they completed an initial examination, which included body composition analysis. The first experimental session started after 7 days. The participants were divided into two experimental groups (molecular hydrogen and placebo) using a randomization table. The table was generated using a random number generator (randperm function available in MATLAB R2020a, MathWorks, Natick, MA, USA) based on block randomization with a block size of four.38 Both groups inhaled molecular hydrogen (99.8%) or a placebo (ambient air) for 60 minutes. Following a 7-day washout period, similarly to previous studies,39,40 a second experimental session was conducted where molecular hydrogen and placebo inhalation were reversed. Measurements took place in the morning (8:00–12:00), with each participant being measured at the same time of day.
Figure 1.
Experimental design.
After arriving at the laboratory, participants were seated and a two-lead electrocardiogram built into a chest strap (DiANS PF8, Dimea Group, Olomouc, Czech Republic) was placed between the breasts and the lower edge of the sternum. They were then fitted with a nasal cannula supplying molecular hydrogen or placebo, with a mask then placed over the cannula, mouth and nose for respiratory analysis (Figure 2). Subsequently, heart rate, gas exchange, and VE parameters were monitored for 60 minutes in an inactive seated position. Any electronic devices, reading, or listening to music were not allowed during the experiment. The laboratory was quiet and dimly lit, with a temperature of 20–22°C and humidity of 30–45%.
Figure 2.
An inserted nasal cannula delivering molecular hydrogen or a placebo covered with a respiratory analysis mask.
(A) Molecular hydrogen or placebo supply; (B) face mask; (C) nasal canula under the face mask.
Anthropometric measurement
Body mass and body fat were measured using bioimpedance analysis (Tanita MC-980 MA, Tanita, Tokyo, Japan). Body height was measured to the nearest 1 cm using a stadiometer SOEHNLE 7307 (Leifheit, Nassau, Germany).
Indirect calorimetry measurement
An Ergostik device (Geratherm Respiratory, Bad Kissingen, Germany) was used to measure breath-by-breath gas exchange and ventilatory characteristics during 60 minutes of molecular hydrogen or placebo inhalation. Recording began immediately after starting the molecular hydrogen/placebo generator. Before each measurement, volume calibration (3 L syringe) and gas calibration were performed according to the manufacturer’s instructions. Breakfast before each experiment was standardized and repeated at the same time for everyone in both parts of the experiment.
Gas exchange, VE, and heart rate recordings were averaged over four 15-minute intervals as follows: 0–15, 15–30, 30–45, and 45–60 minutes. Molecular hydrogen or placebo were delivered through the nasal cannula from underneath the mask and therefore, this gas flow could not be recorded by the flow sensor. In the case of the placebo, a certain amount of oxygen was delivered to the nose without being registered by the Ergostik device. Consequently, it was necessary to make a computational correction for the V̇O2. The amount of bypass oxygen flow was calculated as the flow rate of the placebo generator (300 mL/min) multiplied by the volume concentration of oxygen in the air (20.9%). Thus, an oxygen flow rate of 300 × 0.209 = 62.7 mL/min was added to each value of V̇O2. In the case of the molecular hydrogen, the gas produced contains almost no oxygen (0.055%), and therefore no such correction was necessary. The amount of bypass carbon dioxide was small (molecular hydrogen generator: < 0.002%, placebo: 0.04%) compared with the concentration in exhaled air (4–5%), and therefore no correction was made to V̇CO2. It would be correct to add the bypass flow of 300 mL/min to the VE. However, the correction adds the same value to VE in both molecular hydrogen and placebo groups, resulting in the same difference between the groups whether or not the correction is made. Thus, for simplicity, this correction was omitted. The V̇O2 and V̇CO2 in L/min were calculated relatively to body mass and expressed in mL/(kg·min). The RER was calculated as a ratio of V̇CO2/V̇O2.
The energy released per liter of oxygen consumed was calculated using the values on Kenney et al.29: 4.69, 4.74, 4.80, 4.86, 4.92, 4.99, and 5.05 kcal/L, corresponding to RER values of 0.71, 0.75, 0.80, 0.85, 0.90, 0.95, and 1.00, respectively. Energy expenditure in kJ/min was calculated by multiplying energy per liter of oxygen by V̇O2 and converted to kJ (1 kcal = 4.185 kJ). Finally, energy expenditure was expressed in kJ/(kg·min) by dividing by body mass. To calculate the relative amount of energy derived from FATox, the values were used: 100%, 84%, 67%, 49%, 32%, 16%, and 0%, which correspond to the RER values mentioned above. Calculations of energy expenditure and FATox were performed for each subject and each interval, with intermediate values obtained by linear interpolation, then the values were statistically analyzed.
Molecular hydrogen administration
Molecular hydrogen was produced by a molecular hydrogen generator i300 (Molecular Hydrogen Medical Technologies, Ostrava, Czech Republic). According to the operation manual, molecular hydrogen was produced via electrolysis of purified water using a membrane electrode assembly/proton exchange membrane. The generator produces 300 mL of molecular hydrogen per minute. The composition of the gas (volume concentration) produced by the molecular hydrogen generator was analyzed by gas chromatography in an external commercial laboratory (LABTECH, Paskov, Czech Republic) as follows: 99.8% molecular hydrogen, 0.12% nitrogen, 0.055% oxygen, < 0.002% carbon dioxide. Placebo was generated using a professionally modified generator (Molecular Hydrogen Medical Technologies, Ostrava, Czech Republic) that pumps ambient air free of molecular hydrogen at a flow rate of 300 mL per minute. The air composition in the laboratory was verified using air quality meter AQ-9901SD (Lutron Electronics, Taipei, China) as follows: 0.0% molecular hydrogen, 78% nitrogen, 20.9% oxygen, and 0.05% carbon dioxide. Inhalation was taken place through a nasal cannula while sitting for 60 minutes. Inhalation of molecular hydrogen or placebo could not be distinguished because molecular hydrogen is colorless, odorless, and tasteless.41 For safety reasons, a molecular hydrogen gas detector (Gasman-FL-MPS, Crowcon Detection Instruments, Oxfordshire, UK) was used during the experiment according to the manufacturer’s instructions.
Statistical analysis
Data are presented as arithmetic mean and standard deviation. Normality and sphericity were assessed using the Shapiro-Wilk test and the Mauchly test, respectively. Changes in dependent variables were analyzed using two-way analysis of variance for repeated measures with inhalation factor (molecular hydrogen and placebo), time factor (0–15, 15–30, 30–45, 45–60 minutes), and interaction. If any factor or interaction was significant, pairwise comparisons were performed using Fisher’s least significant difference post hoc tests. The effect size was evaluated as Cohen’s d, where the standard deviation was calculated as the pooled value of the standard deviations of the four intervals when the placebo was administered. The following thresholds were used to interpret the magnitude of d42: trivial 0.00–0.19, small 0.20–0.49, medium 0.50–0.79, and large ≥ 0.80. The association between potential moderators (body fat, body mass index) and changes in dependent variables were analyzed using Pearson’s correlation coefficient (r). The following thresholds were used to interpret the magnitude of r42: trivial 0.00–0.09, small 0.10–0.29, medium 0.30–0.49, and large ≥ 0.50. For all tests, P < 0.05 was considered statistically significant. Statistical analyses were performed using MATLAB R2024a with Statistics Toolbox. Sensitivity type of power analysis was performed using G*Power version 3.1.9.7 (Heinrich-Heine-Universität, Düsseldorf, Germany). The level of statistical significance was set at α = 0.05 and the power was set at 1 – β = 0.80. The calculations were performed for paired t-test and Pearson’s correlation coefficient. With a sample size of 20, the required effect sizes resulted in d = 0.66 and r = 0.55.
Results
The raw data are available in the Additional Tables 1 and 2. Participant characteristics are presented in Table 1. The normal distribution for the variables body mass index (P = 0.63) and body fat (P = 0.81) used in the correlation analysis was not rejected (Table 1). The normality tests of the residuals from the analysis of variance model are shown in Table 2. For breathing frequency (P = 0.019) normality was rejected, therefore, a normal probability plot was visually inspected for this variable. After inspection, the deviation from normality was assessed as acceptable, and parametric statistical methods were used as they are considered robust for such deviations from normality.43 Sphericity was rejected for all variables studied (all P < 0.001; Table 2), which was resolved using the Greenhouse-Geisser adjustment.
Additional Table 1.
Raw data of participants
| Participant | Session 1 | Age (yr) | Body mass (kg) | Body height (cm) | Body fat (%) | FFM (%) |
|---|---|---|---|---|---|---|
| Participant 1 | H2 | 23 | 64.6 | 173 | 21 | 74.9 |
| Participant 2 | Pla | 23 | 60.7 | 161 | 25.2 | 71 |
| Participant 3 | H2 | 20 | 67.5 | 171 | 22 | 74.1 |
| Participant 4 | Pla | 24 | 61.3 | 169 | 17.9 | 78.3 |
| Participant 5 | Pla | 24 | 75.9 | 172 | 25.7 | 70.6 |
| Participant 6 | Pla | 22 | 52.1 | 169 | 15.5 | 80.2 |
| Participant 7 | H2 | 22 | 65.4 | 172 | 21.1 | 74.9 |
| Participant 8 | H2 | 19 | 61.4 | 173 | 23 | 73.1 |
| Participant 9 | Pla | 23 | 54.9 | 164 | 13.5 | 82.1 |
| Participant 10 | Pla | 21 | 62.4 | 166 | 26.1 | 70.2 |
| Participant 11 | H2 | 22 | 64.2 | 170 | 21.7 | 74.5 |
| Participant 12 | Pla | 23 | 68.9 | 163 | 26.4 | 69.8 |
| Participant 13 | Pla | 24 | 67.5 | 163 | 28.4 | 67.9 |
| Participant 14 | H2 | 25 | 71.5 | 176 | 19.9 | 76.1 |
| Participant 15 | Pla | 21 | 75 | 173 | 27.7 | 68.7 |
| Participant 16 | Pla | 22 | 70.2 | 162 | 32.1 | 64.5 |
| Participant 17 | H2 | 21 | 61.7 | 162 | 25.8 | 70.5 |
| Participant 18 | H2 | 20 | 70.2 | 178 | 26.5 | 69.8 |
| Participant 19 | H2 | 21 | 53.9 | 155 | 20.8 | 75.1 |
| Participant 20 | Pla | 21 | 53.4 | 152 | 21.2 | 74.9 |
H2: Hydrogen; Pla: placebo.
Additional Table 2.
Raw data of experiments
| Participant | Session | Interval (min) | RR (ms) | BF (breaths/min) | VE (L/min) | VO2 (L/min) | VCO2 (L/min) |
|---|---|---|---|---|---|---|---|
| Participant 1 | Session 1 | 0-15 | 796.22 | 11.46 | 8.31 | 0.2594 | 0.2277 |
| 15-30 | 784.24 | 13.81 | 8.88 | 0.2588 | 0.2214 | ||
| 30-45 | 812.87 | 13.99 | 8.41 | 0.247 | 0.2085 | ||
| 45-60 | 817.84 | 12.54 | 8 | 0.2381 | 0.2014 | ||
| Session 2 | 0-15 | 796.34 | 14.35 | 9.81 | 0.1843 | 0.2225 | |
| 15-30 | 797.65 | 15.12 | 9.76 | 0.1716 | 0.2133 | ||
| 30-45 | 826.95 | 11.28 | 8.48 | 0.1655 | 0.2026 | ||
| 45-60 | 849.61 | 10.83 | 8.22 | 0.1596 | 0.1941 | ||
| Participant 2 | Session 1 | 0-15 | 870.3 | 14.99 | 8.95 | 0.1539 | 0.1917 |
| 15-30 | 892.12 | 14.45 | 8.95 | 0.1548 | 0.1912 | ||
| 30-45 | 897.7 | 12.89 | 8.76 | 0.1577 | 0.1914 | ||
| 45-60 | 909.28 | 13.27 | 8.79 | 0.1676 | 0.2045 | ||
| Session 2 | 0-15 | 866.11 | 20.18 | 10.14 | 0.2617 | 0.2193 | |
| 15-30 | 830.88 | 19.49 | 9.28 | 0.2479 | 0.1981 | ||
| 30-45 | 827.18 | 17.75 | 9.57 | 0.254 | 0.2161 | ||
| 45-60 | 910.05 | 18.58 | 9.3 | 0.2431 | 0.2087 | ||
| Participant 3 | Session 1 | 0-15 | 636.23 | 11.02 | 6.64 | 0.211 | 0.1729 |
| 15-30 | 629.92 | 11.29 | 6.98 | 0.2123 | 0.1819 | ||
| 30-45 | 668.91 | 11.83 | 6.99 | 0.2153 | 0.1797 | ||
| 45-60 | 691.25 | 11.76 | 7.07 | 0.2162 | 0.1821 | ||
| Session 2 | 0-15 | 674.1 | 12.93 | 9.87 | 0.1815 | 0.2395 | |
| 15-30 | 664.47 | 12.03 | 9.82 | 0.182 | 0.2418 | ||
| 30-45 | 662.22 | 12.39 | 9.82 | 0.1768 | 0.2331 | ||
| 45-60 | 671.06 | 12.37 | 9.87 | 0.1776 | 0.2358 | ||
| Participant 4 | Session 1 | 0-15 | 1032.4 | 16.15 | 8.47 | 0.1821 | 0.2136 |
| 15-30 | 1153.64 | 15.46 | 8.1 | 0.1751 | 0.2092 | ||
| 30-45 | 1183.08 | 14.39 | 7.78 | 0.1571 | 0.186 | ||
| 45-60 | 1136.36 | 15.26 | 7.95 | 0.1577 | 0.1933 | ||
| Session 2 | 0-15 | 834.02 | 15.22 | 7.9 | 0.2554 | 0.2082 | |
| 15-30 | 876.71 | 15.46 | 7.9 | 0.2503 | 0.2058 | ||
| 30-45 | 907.31 | 15.74 | 7.93 | 0.251 | 0.2101 | ||
| 45-60 | 957.26 | 16.02 | 7.72 | 0.2439 | 0.2045 | ||
| Participant 5 | Session 1 | 0-15 | 993.66 | 13.62 | 7.78 | 0.1739 | 0.1809 |
| 15-30 | 1005.55 | 13.27 | 7.61 | 0.1702 | 0.1807 | ||
| 30-45 | 1029.3 | 13.06 | 7.54 | 0.1671 | 0.1818 | ||
| 45-60 | 1005.87 | 13.65 | 8.11 | 0.1678 | 0.1923 | ||
| Session 2 | 0-15 | 887.35 | 15.1 | 7.98 | 0.2244 | 0.1869 | |
| 15-30 | 913.25 | 16.02 | 8.14 | 0.2213 | 0.1828 | ||
| 30-45 | 930.74 | 15.84 | 7.96 | 0.2185 | 0.1786 | ||
| 45-60 | 911.05 | 16.75 | 7.95 | 0.2154 | 0.1709 | ||
| Participant 6 | Session 1 | 0-15 | 763.81 | 15.67 | 8.26 | 0.1407 | 0.1766 |
| 15-30 | 774.44 | 15.18 | 7.65 | 0.13 | 0.1662 | ||
| 30-45 | 856.82 | 16.81 | 7.84 | 0.1231 | 0.1582 | ||
| 45-60 | 770.22 | 14.67 | 7.98 | 0.1295 | 0.1739 | ||
| Session 2 | 0-15 | 841 | 17.18 | 7.14 | 0.1999 | 0.1696 | |
| 15-30 | 940.08 | 17.13 | 6.78 | 0.189 | 0.1537 | ||
| 30-45 | 903.54 | 16.66 | 6.71 | 0.1849 | 0.1563 | ||
| 45-60 | 807.46 | 15.8 | 7.06 | 0.1943 | 0.1738 | ||
| Participant 7 | Session 1 | 0-15 | 876.15 | 11.28 | 7.56 | 0.2366 | 0.2166 |
| 15-30 | 882.37 | 10.66 | 6.88 | 0.2154 | 0.189 | ||
| 30-45 | 854.46 | 12.06 | 7.31 | 0.2195 | 0.1947 | ||
| 45-60 | 862.87 | 10.39 | 7.22 | 0.2258 | 0.1976 | ||
| Session 2 | 0-15 | 821.83 | 12.53 | 8.53 | 0.1669 | 0.2101 | |
| 15-30 | 870.98 | 11.24 | 7.8 | 0.1525 | 0.1857 | ||
| 30-45 | 832.98 | 12.25 | 8.07 | 0.1531 | 0.1806 | ||
| 45-60 | 863.46 | 11.03 | 7.73 | 0.1512 | 0.1769 | ||
| Participant 8 | Session 1 | 0-15 | 958.17 | 17.3 | 5.86 | 0.1539 | 0.1392 |
| 15-30 | 1042.46 | 15.73 | 6.75 | 0.183 | 0.1522 | ||
| 30-45 | 1083.12 | 14.67 | 6.75 | 0.1796 | 0.1453 | ||
| 45-60 | 1065.04 | 14.56 | 7.26 | 0.1936 | 0.1609 | ||
| Session 2 | 0-15 | 786.78 | 13.01 | 8.24 | 0.1665 | 0.1825 | |
| 15-30 | 865 | 13.82 | 7.99 | 0.1577 | 0.178 | ||
| 30-45 | 972.69 | 15.26 | 7.94 | 0.1476 | 0.1747 | ||
| 45-60 | 936.09 | 13.03 | 7.75 | 0.1431 | 0.1742 | ||
| Participant 9 | Session 1 | 0-15 | 929.45 | 11.06 | 6.05 | 0.1739 | 0.1772 |
| 15-30 | 934.81 | 10.77 | 6.47 | 0.1601 | 0.1773 | ||
| 30-45 | 923.98 | 10.2 | 6.01 | 0.1435 | 0.1636 | ||
| 45-60 | 938.55 | 10.42 | 5.91 | 0.1381 | 0.158 | ||
| Session 2 | 0-15 | 988.43 | 10.77 | 5.36 | 0.1992 | 0.1595 | |
| 15-30 | 973.17 | 10.9 | 5.69 | 0.1904 | 0.1665 | ||
| 30-45 | 962.48 | 10.42 | 5.41 | 0.1858 | 0.162 | ||
| 45-60 | 963.38 | 9.87 | 5.15 | 0.1814 | 0.1618 | ||
| Participant 10 | Session 1 | 0-15 | 767.92 | 19.26 | 8.26 | 0.1397 | 0.1739 |
| 15-30 | 850.19 | 17.91 | 7.48 | 0.1284 | 0.1596 | ||
| 30-45 | 935.35 | 16.71 | 6.33 | 0.1125 | 0.1393 | ||
| 45-60 | 937.23 | 17.08 | 7.4 | 0.133 | 0.1635 | ||
| Session 2 | 0-15 | 730.41 | 19.59 | 7.88 | 0.2182 | 0.1811 | |
| 15-30 | 803.25 | 17.97 | 7.4 | 0.2372 | 0.1983 | ||
| 30-45 | 815.56 | 17.37 | 7.46 | 0.2414 | 0.2042 | ||
| 45-60 | 780.43 | 18.72 | 8.01 | 0.246 | 0.2142 | ||
| Participant 11 | Session 1 | 0-15 | 820.63 | 12.88 | 5.41 | 0.1428 | 0.1163 |
| 15-30 | 857.13 | 12.88 | 4.44 | 0.1131 | 0.0922 | ||
| 30-45 | 888.1 | 12.24 | 4.56 | 0.1196 | 0.0975 | ||
| 45-60 | 945.29 | 12.31 | 4.24 | 0.1131 | 0.0902 | ||
| Session 2 | 0-15 | 988.75 | 11.62 | 7.52 | 0.1478 | 0.1554 | |
| 15-30 | 1052.18 | 12.32 | 7.14 | 0.1354 | 0.1501 | ||
| 30-45 | 963.54 | 12.18 | 7.98 | 0.14 | 0.1603 | ||
| 45-60 | 1095.84 | 12.72 | 7.5 | 0.1392 | 0.1536 | ||
| Participant 12 | Session 1 | 0-15 | 840.9 | 15.15 | 8.68 | 0.1742 | 0.2144 |
| 15-30 | 854.96 | 13.8 | 8.48 | 0.1751 | 0.2115 | ||
| 30-45 | 864.98 | 15.66 | 8.5 | 0.1758 | 0.2064 | ||
| 45-60 | 859.86 | 13.66 | 8.35 | 0.1852 | 0.2134 | ||
| Session 2 | 0-15 | 736.54 | 15.83 | 7.31 | 0.2291 | 0.1886 | |
| 15-30 | 744.36 | 15.18 | 7 | 0.2258 | 0.1819 | ||
| 30-45 | 766.86 | 15.39 | 7.03 | 0.2284 | 0.1825 | ||
| 45-60 | 803.77 | 15.26 | 7 | 0.2329 | 0.1828 | ||
| Participant 13 | Session 1 | 0-15 | 1106.83 | 10.43 | 7.41 | 0.1825 | 0.2139 |
| 15-30 | 1111.28 | 11.19 | 7.18 | 0.1726 | 0.2024 | ||
| 30-45 | 1103.27 | 10.94 | 7.26 | 0.172 | 0.2037 | ||
| 45-60 | 1125.6 | 12.2 | 6.93 | 0.1584 | 0.1872 | ||
| Session 2 | 0-15 | 927.11 | 12.53 | 7.41 | 0.2574 | 0.206 | |
| 15-30 | 995.89 | 13.38 | 7.01 | 0.2486 | 0.1922 | ||
| 30-45 | 1000.46 | 13.66 | 7.24 | 0.2486 | 0.1996 | ||
| 45-60 | 980.79 | 12.77 | 6.89 | 0.2424 | 0.1867 | ||
| Participant 14 | Session 1 | 0-15 | 1054.42 | 6.45 | 6.56 | 0.2459 | 0.2238 |
| 15-30 | 1087.94 | 7.74 | 6.27 | 0.2485 | 0.2183 | ||
| 30-45 | 1190.16 | 8.23 | 6.01 | 0.2349 | 0.2014 | ||
| 45-60 | 1173.27 | 7.7 | 5.99 | 0.2238 | 0.1977 | ||
| Session 2 | 0-15 | 1167.05 | 10.96 | 7.41 | 0.1847 | 0.2359 | |
| 15-30 | 1187.23 | 13.75 | 7.53 | 0.1632 | 0.1964 | ||
| 30-45 | 1136.11 | 13.55 | 7.76 | 0.1704 | 0.2103 | ||
| 45-60 | 1110.81 | 11.93 | 7.39 | 0.1617 | 0.196 | ||
| Participant 15 | Session 1 | 0-15 | 991.98 | 9.94 | 7.07 | 0.1774 | 0.2223 |
| 15-30 | 1017.36 | 11.54 | 7.17 | 0.1704 | 0.2123 | ||
| 30-45 | 980.35 | 10.52 | 7.42 | 0.1681 | 0.2156 | ||
| 45-60 | 973.75 | 11.59 | 7.55 | 0.1642 | 0.2109 | ||
| Session 2 | 0-15 | 973.7 | 9.28 | 6.23 | 0.246 | 0.2031 | |
| 15-30 | 1017.05 | 12.05 | 6.6 | 0.2377 | 0.1966 | ||
| 30-45 | 1050.5 | 12.02 | 6.47 | 0.2358 | 0.1996 | ||
| 45-60 | 986.63 | 9.18 | 6.03 | 0.2387 | 0.2041 | ||
| Participant 16 | Session 1 | 0-15 | 662.8 | 8.77 | 7.65 | 0.17 | 0.1981 |
| 15-30 | 679.41 | 12.53 | 8.18 | 0.1688 | 0.1994 | ||
| 30-45 | 674.17 | 8.3 | 9.35 | 0.174 | 0.2198 | ||
| 45-60 | 669.79 | 6.82 | 8.98 | 0.1862 | 0.2214 | ||
| Session 2 | 0-15 | 730.8 | 9.77 | 9.89 | 0.2446 | 0.2349 | |
| 15-30 | 804.28 | 14.38 | 8.24 | 0.2463 | 0.206 | ||
| 30-45 | 798.25 | 15.38 | 7.48 | 0.2368 | 0.1916 | ||
| 45-60 | 752.58 | 12.09 | 7.74 | 0.2306 | 0.1996 | ||
| Participant 17 | Session 1 | 0-15 | 832.19 | 14.38 | 6.34 | 0.2022 | 0.1704 |
| 15-30 | 873.6 | 13.68 | 5.48 | 0.1723 | 0.1355 | ||
| 30-45 | 878.4 | 12.68 | 5.17 | 0.1684 | 0.1297 | ||
| 45-60 | 894.26 | 12.65 | 4.78 | 0.156 | 0.1176 | ||
| Session 2 | 0-15 | 739.7 | 15.52 | 8.35 | 0.1792 | 0.211 | |
| 15-30 | 791.41 | 16.15 | 8.3 | 0.1665 | 0.2019 | ||
| 30-45 | 790.27 | 16.2 | 8.36 | 0.1616 | 0.1957 | ||
| 45-60 | 778.31 | 15.73 | 8.03 | 0.1495 | 0.1777 | ||
| Participant 18 | Session 1 | 0-15 | 930.03 | 16.18 | 6.98 | 0.1982 | 0.1597 |
| 15-30 | 928.73 | 15.49 | 6.8 | 0.1982 | 0.1514 | ||
| 30-45 | 932.33 | 15.6 | 6.97 | 0.2053 | 0.161 | ||
| 45-60 | 929.58 | 15.11 | 7.08 | 0.204 | 0.1597 | ||
| Session 2 | 0-15 | 784.48 | 16.71 | 9.79 | 0.1998 | 0.2263 | |
| 15-30 | 799.85 | 15.66 | 8.51 | 0.1776 | 0.2005 | ||
| 30-45 | 804.46 | 15.36 | 8.91 | 0.1759 | 0.2047 | ||
| 45-60 | 812.63 | 14.84 | 8.79 | 0.1736 | 0.2033 | ||
| Participant 19 | Session 1 | 0-15 | 694.79 | 13.36 | 8.19 | 0.2399 | 0.1973 |
| 15-30 | 714.03 | 12.85 | 7.69 | 0.2284 | 0.1825 | ||
| 30-45 | 707.35 | 12.03 | 7.64 | 0.2287 | 0.1897 | ||
| 45-60 | 712.87 | 11.96 | 7.69 | 0.2281 | 0.1912 | ||
| Session 2 | 0-15 | 643.06 | 13.17 | 7.67 | 0.1686 | 0.2151 | |
| 15-30 | 672.34 | 12.93 | 7.36 | 0.1647 | 0.2063 | ||
| 30-45 | 645.02 | 12.61 | 7.2 | 0.1604 | 0.197 | ||
| 45-60 | 658.54 | 11.65 | 6.81 | 0.1537 | 0.1821 | ||
| Participant 20 | Session 1 | 0-15 | 864.45 | 20.99 | 6.77 | 0.1503 | 0.167 |
| 15-30 | 857.8 | 20.61 | 6.72 | 0.1504 | 0.1646 | ||
| 30-45 | 880.78 | 21.49 | 6.83 | 0.15 | 0.1639 | ||
| 45-60 | 888.8 | 24.14 | 7.2 | 0.1541 | 0.1695 | ||
| Session 2 | 0-15 | 822.21 | 21.41 | 6.27 | 0.1943 | 0.1493 | |
| 15-30 | 862.31 | 20.06 | 6.07 | 0.1958 | 0.1438 | ||
| 30-45 | 849.88 | 20.93 | 6.46 | 0.2025 | 0.153 | ||
| 45-60 | 826.46 | 20.4 | 6.59 | 0.2042 | 0.1549 |
Table 2.
Results from two-way repeated measures analysis of variance and tests of assumptions
| Variable | P Inhalation | P Time | P Interaction | P Mauchly | P Shapiro-Wilk |
|---|---|---|---|---|---|
| HR | 0.90 | < 0.001 | 0.89 | < 0.001 | 0.32 |
| BF | 0.43 | 0.13 | 0.68 | < 0.001 | 0.019 |
| VE | 0.002 | 0.002 | 0.86 | < 0.001 | 0.76 |
| ⟇O2 | 0.25 | 0.001 | 0.25 | < 0.001 | 0.12 |
| ⟇CO2 | 0.042 | 0.001 | 0.43 | < 0.001 | 0.62 |
| RER | 0.034 | 0.084 | 0.47 | < 0.001 | 0.97 |
| FATox | 0.034 | 0.091 | 0.47 | < 0.001 | 0.97 |
| EE | 0.17 | 0.001 | 0.29 | < 0.001 | 0.16 |
BF: Breathing frequency; EE: energy expenditure; FATox: fat oxidation rate; HR: heart rate; P Inhalation: significance of the inhalation factor; P Interaction: significance of interaction; P Mauchiy: significance of Mauchly's test of sphericity; P shapiro-wilk: significance of Shapiro-Wilk normality test; P Time: significance of the time factor; RER: respiratory exchange ratio; ⟇CO2: carbon dioxide production; VE: ventilation; ⟇O2: oxygen consumption.
Effect of hydrogen gas inhalation on indirect calorimetry variables
The effect of hydrogen gas inhalation compared with placebo inhalation expressed as analysis of variance factor is shown in Table 2. A significant effect of hydrogen gas was found for VE (P = 0.002), V̇CO2 (P = 0.042), RER (P = 0.034), and FATox (P = 0.034). For the remaining variables studied, the effect was not significant (all P ≥ 0.17). No significant interaction was found (all P ≥ 0.25). A detailed analysis of the effect of hydrogen gas is shown in Table 3 and Figure 3. Compared with placebo, inhalation of hydrogen gas decreased VE at all intervals (all P < 0.001, d ranging from –1.07 to –0.99), increased V̇CO2 at all intervals (all P ≤ 0.001, d ranging from –0.74 to –0.50), decreased RER at all intervals (all P ≤ 0.012, d ranging from –0.66 to –0.38) and increased FATox at all intervals (all P ≤ 0.013, d ranging from 0.38 to 0.67).
Table 3.
Effect of 60 minutes of hydrogen gas inhalation on heart rate, respiratory and metabolic variables compared to placebo inhalation
| Variable | Time (min) | Placebo | Hydrogen | P | d | Effect |
|---|---|---|---|---|---|---|
| HR (beat/min) | 0–15 | 71.6 ± 11.9 | 72.0 ± 9.5 | 0.04 | Trivial | |
| 15–30 | 69.2 ± 11.8 | 69.5 ± 9.7 | 0.03 | Trivial | ||
| 30–45 | 68.6 ± 11.9 | 68.6 ± 9.5 | –0.01 | Trivial | ||
| 45–60 | 68.5 ± 11.8 | 68.7 ± 9.1 | 0.01 | Trivial | ||
| BF (breath/min) | 0–15 | 13.8 ± 3.1 | 14.1 ± 3.9 | 0.08 | Trivial | |
| 15–30 | 14.0 ± 2.4 | 14.3 ± 3.1 | 0.11 | Trivial | ||
| 30–45 | 13.6 ± 3.0 | 14.2 ± 2.9 | 0.22 | Small | ||
| 45–60 | 13.3 ± 3.4 | 13.7 ± 3.4 | 0.13 | Trivial | ||
| VE (L/min) | 0–15 | 8.1 ± 1.0 | 7.3 ± 1.3 | < 0.001 | –0.99 | Large |
| 15–30 | 7.9 ± 0.9 | 7.0 ± 1.2 | < 0.001 | –1.03 | Large | |
| 30–45 | 7.9 ± 0.9 | 7.0 ± 1.1 | < 0.001 | –1.07 | Large | |
| 45–60 | 7.9 ± 0.9 | 6.9 ± 1.2 | < 0.001 | –1.07 | Large | |
| ⟇O2 (mL/(kg-min)) | 0–15 | 3.66 ± 0.35 | 3.47 ± 0.57 | –0.61 | Medium | |
| 15–30 | 3.53 ± 0.34 | 3.39 ± 0.58 | –0.44 | Small | ||
| 30–45 | 3.46 ± 0.31 | 3.38 ± 0.57 | –0.26 | Small | ||
| 45–60 | 3.46 ± 0.30 | 3.36 ± 0.58 | –0.34 | Small | ||
| ⟇CO2 (mL/(kg-min)) | 0–15 | 3.16 ± 0.37 | 2.92 ± 0.50 | < 0.001 | –0.73 | Medium |
| 15–30 | 3.02 ± 0.37 | 2.78 ± 0.49 | < 0.001 | –0.74 | Medium | |
| 30–45 | 2.96 ± 0.33 | 2.80 ± 0.51 | < 0.001 | –0.53 | Medium | |
| 45–60 | 2.96 ± 0.30 | 2.80 ± 0.54 | 0.001 | –0.50 | Medium | |
| RER | 0–15 | 0.863 ± 0.067 | 0.843 ± 0.047 | 0.012 | –0.38 | Small |
| 15–30 | 0.856 ± 0.056 | 0.821 ± 0.038 | < 0.001 | –0.66 | Medium | |
| 30–45 | 0.856 ± 0.053 | 0.826 ± 0.033 | < 0.001 | –0.57 | Medium | |
| 45–60 | 0.856 ± 0.050 | 0.832 ± 0.045 | 0.004 | –0.45 | Small | |
| FATox (%) | 0–15 | 45 ± 23 | 52 ± 16 | 0.013 | 0.38 | Small |
| 15–30 | 47 ± 19 | 60 ± 14 | < 0.001 | 0.67 | Medium | |
| 30–45 | 47 ± 18 | 58 ± 12 | < 0.001 | 0.57 | Medium | |
| 45–60 | 47 ± 17 | 56 ± 16 | 0.004 | 0.45 | Small | |
| EE (kJ/(kg-min)) | 0–15 | 0.075 ± 0.007 | 0.070 ± 0.012 | –0.66 | Medium | |
| 15–30 | 0.072 ± 0.007 | 0.068 ± 0.012 | –0.53 | Medium | ||
| 30–45 | 0.071 ± 0.006 | 0.068 ± 0.012 | –0.34 | Small | ||
| 45–60 | 0.071 ± 0.006 | 0.068 ± 0.012 | –0.40 | Small |
Data are expressed as mean ± SD (n = 20). BF: Breathing frequency; d: Cohen's d; EE: energy expenditure; FATox: fat oxidation rate; HR: heart rate; P: significance of Fisher's least significant difference test; RER: respiratory exchange ratio; ⟇CO2: carbon dioxide production; VE: ventilation; ⟇O2: oxygen consumption.
Figure 3.
Effect of 60 minutes of hydrogen gas inhalation on heart rate, respiratory and metabolic variables compared to placebo inhalation.
Solid and open circles indicate hydrogen gas inhalation and placebo inhalation, respectively. Data are expressed as mean ± SD. *P < 0.05, hydrogen gas versus placebo (Fisher’s least significant difference test). BF: Breathing frequency; EE: energy expenditure; FATox: fat oxidation rate; HR: heart rate; RER: respiratory exchange ratio; V̇CO2: carbon dioxide production; VE: ventilation; V̇O2: oxygen consumption.
Correlation analysis
Correlation analysis revealed that within 0–15 minutes and 15–30 minutes, none of the variables studied were significantly correlated with body fat (all P ≥ 0.19, Table 4). However, negative significant correlations between body fat and change in RER between hydrogen gas and placebo were found at 30–45 minutes (r = –0.52, P = 0.018) and at 45–60 minutes (r = –0.51, P = 0.022, Figure 4). In addition, there were positive significant correlations between body fat and change in FATox at 30–45 minutes (r = 0.53, P = 0.017) and at 45–60 minutes (r = 0.52, P = 0.020, Figure 4). In comparison to body fat, body mass index was somewhat less corelated with changes in RER and FATox (Table 5), specifically RER at 30–45 minutes (r = –0.46, P = 0.044), FATox at 30–45 minutes (r = 0.46, P = 0.044), and FATox at 45–60 minutes (r = 0.45, P = 0.049). The correlation between body mass index and change in RER at 45–60 minutes was not significant (r = –0.44, P = 0.052). Finally, there was a positive significant correlation between body fat and change in breathing frequency at 30–45 minutes (r = 0.44, P = 0.049).
Table 4.
Correlation analysis between body fat and changes in heart rate, respiratory and metabolic variables after hydrogen gas inhalation
| Variable | Inhalation time (min) |
|||
|---|---|---|---|---|
| 0–15 | 15–30 | 30–45 | 45–60 | |
| ΔHR | –0.02 (0.92) | –0.07 (0.76) | –0.15 (0.53) | –0.08 (0.75) |
| ΔBF | 0.25 (0.28) | 0.28 (0.23) | 0.44 (0.049) | 0.33 (0.15) |
| ΔVE | 0.27 (0.25) | 0.12 (0.62) | –0.06 (0.79) | –0.07 (0.77) |
| Δ⟇O2 | 0.17 (0.47) | 0.17 (0.48) | 0.08 (0.74) | 0.00 (0.99) |
| Δ⟇CO2 | 0.18 (0.46) | 0.03 (0.90) | –0.14 (0.55) | –0.20 (0.39) |
| ΔRER | 0.01 (0.97) | –0.30 (0.20) | –0.52 (0.018) | –0.51 (0.022) |
| ΔFATox | 0.00 (0.99) | 0.30 (0.19) | 0.53 (0.017) | 0.52 (0.020) |
| ΔEE | 0.18 (0.44) | 0.14 (0.55) | 0.03 (0.91) | –0.04 (0.85) |
Values are shown as Pearson's correlation coefficient (significance of the correlation coefficient). BF: Breathing frequency; EE: energy expenditure; FATox: fat oxidation rate; HR: heart rate; RER: respiratory exchange ratio; ⟇CO2: carbon dioxide production; VE: ventilation; ⟇O2: oxygen consumption; Δ: change between hydrogen gas inhalation and placebo inhalation.
Figure 4.
Correlation analysis between body fat and changes in respiration exchange ratio (RER) or fat oxidation (FATox).
Dashed curves indicate 95% confidence area. Δ: Change between hydrogen gas inhalation and placebo inhalation; r: Pearson’s correlation coefficient; P: significance of the correlation coefficient.
Table 5.
Correlation analysis between body mass index and changes in heart rate, respiratory and metabolic variables after hydrogen gas inhalation
| Variable | Inhalation time (min) |
|||
|---|---|---|---|---|
| 0–15 | 15–30 | 30–45 | 45–60 | |
| ΔHR | 0.32 (0.16) | 0.25 (0.29) | 0.05 (0.82) | 0.10 (0.67) |
| ΔBF | 0.03 (0.89) | 0.14 (0.55) | 0.37 (0.11) | 0.17 (0.47) |
| ΔVE | 0.41 (0.070) | 0.19 (0.41) | –0.02 (0.94) | –0.07 (0.76) |
| Δ⟇O2 | 0.30 (0.20) | 0.19 (0.43) | 0.07 (0.77) | –0.02 (0.92) |
| Δ⟇CO2 | 0.26 (0.27) | 0.08 (0.74) | –0.12 (0.60) | –0.21 (0.38) |
| ΔRER | –0.09 (0.72) | –0.22 (0.34) | –0.46 (0.044) | –0.44 (0.052) |
| ΔFATox | 0.09 (0.69) | 0.23 (0.34) | 0.46 (0.044) | 0.45 (0.049) |
| ΔEE | 0.30 (0.19) | 0.17 (0.48) | 0.03 (0.92) | –0.07 (0.78) |
Values are shown as Pearson's correlation coefficient (significance of the correlation coefficient). BF: Breathing frequency; EE: energy expenditure; FATox: fat oxidation rate; HR: heart rate; RER: respiratory exchange ratio; ⟇CO2: carbon dioxide production; VE: ventilation; ⟇O2: oxygen consumption; Δ: change between hydrogen gas inhalation and placebo inhalation.
Discussion
This study is the first to provide new insights into the effects of a 60-minute inhalation of molecular hydrogen under resting conditions on respiratory and metabolic parameters measured by breath gas analysis in healthy females. The research demonstrated that molecular hydrogen administration significantly decreased RER and VE values compared with placebo. Additionally, it was found that the RER reduction positively correlated with body fat percentage between 30 and 60 minutes after the commencement of molecular hydrogen inhalation. The RER is commonly used as an indicator of metabolic function.28 The results of this study indicate that molecular hydrogen inhalation led to a significant decrease in RER compared to placebo from the onset of molecular hydrogen inhalation.
The observed decrease in RER may suggest an influence of molecular hydrogen on metabolic processes towards an increased preference for ATP production through oxidative metabolism using lipids at the expense of carbohydrates. This change in RER corresponded to the calculated increase in FATox percentage, from 47 ± 19% to 56 ± 14%. The rapid onset of the molecular hydrogen metabolic effect in the body is likely due to its easy permeability through the cell membrane into individual cellular compartments,1 and to its low molecular weight44 and electrical neutrality.45 Based on gas chromatography of molecular hydrogen concentration in arterial and venous blood, it was found that after 3% hydrogen gas inhalation, the concentration of molecular hydrogen in the blood reaches a plateau after 20 minutes, but a steep increase occurs around the 5-minute mark.8 However, as shown in a recent study, the maximum saturation levels of hydrogen were reached fastest in the brain (6.3 minutes), liver (7.8 minutes), and kidneys (8.2 minutes) when inhaling the same concentration of molecular hydrogen.46 During transportation through the bloodstream, molecular hydrogen diffuses into and is utilized by tissues.47 The pharmacokinetics of molecular hydrogen in the body may be related to the significant decrease in RER observed in our subjects as early as 5–10 minutes after the start of inhalation. Our results also indicate that it took 30 minutes, after the start of molecular hydrogen inhalation, for there to be a significant difference between hydrogen and placebo relating to the correlation (r = –0.52, P = 0.018 at 30–45 minutes; r = –0.51, P = 0.022 at 45–60 minutes, after molecular hydrogen gas inhalation) between RER and body fat percentage (range from 13.5% to 32.1%). From the results, it can be inferred that molecular hydrogen inhalation under resting conditions must last at least 30 minutes for a stimulatory effect on lipid metabolism to be greater in females with a higher body fat percentage.
A higher positive effect of hydrogen on uphill running performance (4.2 km, 8% incline) and current running performance was previously described by Botek et al.40 who found that the ergogenic effect of molecular hydrogen on performance was negatively influenced by the achieved current running performance. Less fit runners benefited more from short-term molecular hydrogen supplementation compared with well-trained uphill runners. The current study findings of maximum stimulation of oxidative metabolism by molecular hydrogen administration 30 minutes after the start of inhalation. This delay may be related to the pharmacokinetics of molecular hydrogen in muscle tissue, with maximum molecular hydrogen saturation after inhalation of 3% hydrogen has been shown to be reached after 20 minutes,46 likely due to the larger muscle mass volume compared to the size of other organs in the body. Muscles are generally considered to be very energetically active tissues.48 They contain a high percentage of high-oxidative, low glycolytic type I fibers, and higher capillary and mitochondrial density in women,49 which may enhance the conditions for lipid metabolism. It is known that the respiratory activity of mitochondria plays a key role in the level of oxidative metabolism.48 An in vitro study showed that a 60-minute molecular hydrogen treatment slightly activated mitochondria, accompanied by weak oxidative stress, triggering an adaptive antioxidative response against oxidative stress.27 More recently, an increase in mitochondrial respiration rate and ATP production was demonstrated in isolated mitochondria in response to hydrogen administration.26 Although the mechanism underlying the biological actions of molecular hydrogen in mitochondria remains unclear, increasing evidence suggests that the hydrogen-induced mitochondrial response varies depending on mitochondrial functional status50 with molecular hydrogen proposed as both a radical scavenger and a mitohormetic agent against oxidative stress in cells.27 Improvements in mitochondrial ATP production and enhanced oxygen consumption rates induced by molecular hydrogen could also be explained by the increased membrane potential difference, indicating activation of oxidative phosphorylation, which may be regulated by molecular hydrogen-modified calcium signaling to oxidative phosphorylation.27,51 Another mechanism for the action of molecular hydrogen is proposed by Gvozdjáková et al.26 who suggested that enhanced mitochondrial function is driven by molecular hydrogen as a donor of both electrons and protons for the Q-cycle. Lower values of exercise and post-exercise lactate have been reported to reflect indirect evidence of improved mitochondrial activity after acute molecular hydrogen administration.21,22,52,53 Lactate, as a product of anaerobic glycolysis, can be oxidatively metabolized as a preferred energy intermediate in mitochondria.54 In our study, molecular hydrogen inhalation resulted in a significant decrease in VE, which can be attributed to reduced V̇CO2 production and consequently lower stimulation of the medulla oblongata to increase minute VE. Molecular hydrogen ingestion was also shown to reduced minute VE during exercise which was attributed to more efficient breathing and aerobic metabolism.22
Inhalation of molecular hydrogen, generated by electrolysis of water and administered through a nasal cannula, represents a safe and convenient method suitable for outpatient healthcare facilities (e.g. spas) or even for home use. Compared to a pressure cylinder containing a molecular hydrogen gas mixture, the advantage is that generated hydrogen is quickly consumed for inhalation, so that only a very small volume of hydrogen accumulates, and in addition, the electrolysis can be shut down immediately by means of a switch, reducing the risk of explosion. Therefore, the methodology used in this study can be considered ecologically valid for such type of inhalation. On the other hand, the bypass flow of the inhalation gas underneath the mask caused a systematic error in gas exchange measurement, which had to be dealt with using computational correction. The accuracy of the correction used can be questioned and should be verified in a future study using the gold standard, Douglas bags. Our findings suggest that molecular hydrogen inhalation could offer a new strategy for supporting body mass reduction and improving metabolic health. However, further research is needed to thoroughly understand the mechanisms by which molecular hydrogen affects human metabolism.
This study has several limitations. Firstly, participants were not measured at the same phase of their menstrual cycle; they were only requested to avoid participation if menstruating. Secondly, while participants were advised to maintain consistent hydration, sleep, and physical activity routines, these factors were not quantified. Finally, the upper limit of body mass index (26.7 kg/m2) in this study does not allow extrapolation of the results to overweight and obese females. Future study on overweight and obese females is needed to verify the interesting results of this study.
In our study, 60 minutes of molecular hydrogen inhalation significantly decreased RER and VE compared to placebo. The decrease in RER was positively correlated with the percentage of body fat in our cohort of healthy females. Therefore, molecular hydrogen inhalation could be a promising strategy for improving metabolic health, especially in females with higher percentages of body fat.
Additional files:
Additional file 1: CONSORT checklist.
Additional Table 1: Raw data of participants.
Additional Table 2: Raw data of experiments.
Acknowledgments:
The authors would like to thank Rebecca Tanner at the University of Canberra, Australia, for her help in reviewing and editing the manuscript.
Funding Statement
Funding: This study was supported by Palacký University Olomouc under grant IGA FTK 2022_013.
Footnotes
Conflicts of interest: The author MB is the external research consultant of H2 Global Group (Ostrava, Czech Republic). The other authors disclose no conflicts of interest related to this project.
Data availability statement:
All data relevant to the study are included in the article or uploaded as Additional files.
References
- 1.Ohsawa I, Ishikawa M, Takahashi K, et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med. 2007;13:688–694. doi: 10.1038/nm1577. [DOI] [PubMed] [Google Scholar]
- 2.Slezak J, Kura B. Springer Cham; 2024. Molecular hydrogen in health and disease. [Google Scholar]
- 3.Jin Z, Zhao P, Gong W, Ding W, He Q. Fe-porphyrin: A redox-related biosensor of hydrogen molecule. Nano Res. 2023;16:2020–2025. [Google Scholar]
- 4.Ohta S. Molecular hydrogen may activate the transcription factor Nrf2 to alleviate oxidative stress through the hydrogen-targeted porphyrin. Aging Pathobiol Ther. 2023;5:25–32. [Google Scholar]
- 5.Kawamura T, Suzuki K, Takahashi M, et al. Involvement of neutrophil dynamics and function in exercise-induced muscle damage and delayed-onset muscle soreness: effect of hydrogen bath. Antioxidants (Basel) 2018;7:127. doi: 10.3390/antiox7100127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Fukuda K, Asoh S, Ishikawa M, Yamamoto Y, Ohsawa I, Ohta S. Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress. Biochem Biophys Res Commun. 2007;361:670–674. doi: 10.1016/j.bbrc.2007.07.088. [DOI] [PubMed] [Google Scholar]
- 7.Sano M, Shirakawa K, Katsumata Y, Ichihara G, Kobayashi E. Low-flow nasal cannula hydrogen therapy. J Clin Med Res. 2020;12:674–680. doi: 10.14740/jocmr4323. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Ono H, Nishijima Y, Adachi N, et al. A basic study on molecular hydrogen (H2) inhalation in acute cerebral ischemia patients for safety check with physiological parameters and measurement of blood H2 level. Med Gas Res. 2012;2:21. doi: 10.1186/2045-9912-2-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Cole AR, Sperotto F, DiNardo JA, et al. Safety of prolonged inhalation of hydrogen gas in air in healthy adults. Crit Care Explor. 2021;3:e543. doi: 10.1097/CCE.0000000000000543. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Salomez-Ihl C, Tanguy S, Boucher F, et al. Development of a preclinical tool for measuring percutaneous transfer of dihydrogen, with a view to optimizing medical devices adapted to focal therapies in dermatology. In: Slezak J, Kura B, editors. Molecular hydrogen in health and disease. Cham: Springer Nature Switzerland; 2024. pp. 401–416. [Google Scholar]
- 11.Ohta S. Molecular hydrogen as a preventive and therapeutic medical gas: initiation, development and potential of hydrogen medicine. Pharmacol Ther. 2014;144:1–11. doi: 10.1016/j.pharmthera.2014.04.006. [DOI] [PubMed] [Google Scholar]
- 12.Botek M, Krejčí J, Valenta M, et al. Molecular hydrogen positively affects physical and respiratory function in acute post-COVID-19 patients: a new perspective in rehabilitation. Int J Environ Res Public Health. 2022;19:1992. doi: 10.3390/ijerph19041992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Kajiyama S, Hasegawa G, Asano M, et al. Supplementation of hydrogen-rich water improves lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance. Nutr Res. 2008;28:137–143. doi: 10.1016/j.nutres.2008.01.008. [DOI] [PubMed] [Google Scholar]
- 14.Iuchi K, Nishimaki K, Kamimura N, Ohta S. Molecular hydrogen suppresses free-radical-induced cell death by mitigating fatty acid peroxidation and mitochondrial dysfunction. Can J Physiol Pharmacol. 2019;97:999–1005. doi: 10.1139/cjpp-2018-0741. [DOI] [PubMed] [Google Scholar]
- 15.Kamimura N, Ichimiya H, Iuchi K, Ohta S. Molecular hydrogen stimulates the gene expression of transcriptional coactivator PGC-1α to enhance fatty acid metabolism. NPJ Aging Mech Dis. 2016;2:16008. doi: 10.1038/npjamd.2016.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Karapehlivan M, Başer ÖF, Dolanbay T, Alwaze D. Effects of hydrogen-enriched water on lipid profile and some biochemical markers. Rats. 2023;1:47–50. [Google Scholar]
- 17.Kamimura N, Nishimaki K, Ohsawa I, Ohta S. Molecular hydrogen improves obesity and diabetes by inducing hepatic FGF21 and stimulating energy metabolism in db/db mice. Obesity (Silver Spring) 2011;19:1396–1403. doi: 10.1038/oby.2011.6. [DOI] [PubMed] [Google Scholar]
- 18.Korovljev D, Stajer V, Ostojic J, LeBaron TW, Ostojic SM. Hydrogen-rich water reduces liver fat accumulation and improves liver enzyme profiles in patients with non-alcoholic fatty liver disease: a randomized controlled pilot trial. Clin Res Hepatol Gastroenterol. 2019;43:688–693. doi: 10.1016/j.clinre.2019.03.008. [DOI] [PubMed] [Google Scholar]
- 19.Kura B, Szantova M, LeBaron TW, et al. Biological effects of hydrogen water on subjects with NAFLD: a randomized, placebo-controlled trial. Antioxidants (Basel) 2022;11:1935. doi: 10.3390/antiox11101935. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Todorovic N, Fernández-Landa J, Santibañez A, et al. The effects of hydrogen-rich water on blood lipid profiles in clinical populations: a systematic review and meta-analysis. Pharmaceuticals (Basel) 2023;16:142. doi: 10.3390/ph16020142. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Aoki K, Nakao A, Adachi T, Matsui Y, Miyakawa S. Pilot study: effects of drinking hydrogen-rich water on muscle fatigue caused by acute exercise in elite athletes. Med Gas Res. 2012;2:12. doi: 10.1186/2045-9912-2-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Botek M, Krejčí J, McKune AJ, Sládečková B, Naumovski N. Hydrogen rich water improved ventilatory, perceptual and lactate responses to exercise. Int J Sports Med. 2019;40:879–885. doi: 10.1055/a-0991-0268. [DOI] [PubMed] [Google Scholar]
- 23.Botek M, Krejčí J, McKune A, Valenta M, Sládečková B. Hydrogen rich water consumption positively affects muscle performance, lactate response, and alleviates delayed onset of muscle soreness after resistance training. J Strength Cond Res. 2022;36:2792–2799. doi: 10.1519/JSC.0000000000003979. [DOI] [PubMed] [Google Scholar]
- 24.Timón R, Olcina G, González-Custodio A, Camacho-Cardenosa M, Camacho-Cardenosa A, Martínez Guardado I. Effects of 7-day intake of hydrogen-rich water on physical performance of trained and untrained subjects. Biol Sport. 2021;38:269–275. doi: 10.5114/biolsport.2020.98625. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.San-Millán I, Brooks GA. Assessment of metabolic flexibility by means of measuring blood lactate, fat, and carbohydrate oxidation responses to exercise in professional endurance athletes and less-fit individuals. Sports Med. 2018;48:467–479. doi: 10.1007/s40279-017-0751-x. [DOI] [PubMed] [Google Scholar]
- 26.Gvozdjáková A, Kucharská J, Kura B, et al. A new insight into the molecular hydrogen effect on coenzyme Q and mitochondrial function of rats. Can J Physiol Pharmacol. 2020;98:29–34. doi: 10.1139/cjpp-2019-0281. [DOI] [PubMed] [Google Scholar]
- 27.Murakami Y, Ito M, Ohsawa I. Molecular hydrogen protects against oxidative stress-induced SH-SY5Y neuroblastoma cell death through the process of mitohormesis. PLoS One. 2017;12:e0176992. doi: 10.1371/journal.pone.0176992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Brooks GA, Fahey TD, White TP, Baldwin KM. 3rd. Mayfield: McGraw-Hill Education; 2000. Exercise physiology: human bioenergetics and its applications. [Google Scholar]
- 29.Kenney WL, Wilmore JH, Costill DL. Physiology of sport and exercise. Human Kinetics. 2015 [Google Scholar]
- 30.Zhang S, Tanaka Y, Ishihara A, et al. Metabolic flexibility during sleep. Sci Rep. 2021;11:17849. doi: 10.1038/s41598-021-97301-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Júdice PB, Sardinha LB, Silva AM. Variance in respiratory quotient among daily activities and its association with obesity status. Int J Obes (Lond) 2021;45:217–224. doi: 10.1038/s41366-020-0591-x. [DOI] [PubMed] [Google Scholar]
- 32.Shook RP, Hand GA, Paluch AE, et al. High respiratory quotient is associated with increases in body weight and fat mass in young adults. Eur J Clin Nutr. 2016;70:1197–1202. doi: 10.1038/ejcn.2015.198. [DOI] [PubMed] [Google Scholar]
- 33.Cale L, Harris J. Exercise recommendations for young people: an update. Health Educ. 2001;101:126–138. [Google Scholar]
- 34.Kremer JM, Jubiz W, Michalek A, et al. Fish-oil fatty acid supplementation in active rheumatoid arthritis. A double-blinded, controlled, crossover study. Ann Intern Med. 1987;106:497–503. doi: 10.7326/0003-4819-106-4-497. [DOI] [PubMed] [Google Scholar]
- 35.Rawson ES, Persky AM, Price TB, Clarkson PM. Effects of repeated creatine supplementation on muscle, plasma, and urine creatine levels. J Strength Cond Res. 2004;18:162–167. doi: 10.1519/1533-4287(2004)018<0162:eorcso>2.0.co;2. [DOI] [PubMed] [Google Scholar]
- 36.Norton K, Norton L, Sadgrove D. Position statement on physical activity and exercise intensity terminology. J Sci Med Sport. 2010;13:496–502. doi: 10.1016/j.jsams.2009.09.008. [DOI] [PubMed] [Google Scholar]
- 37.Schulz KF, Altman DG, Moher D, CONSORT Group CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. BMJ. 2010;340:c332. doi: 10.1136/bmj.c332. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Kang M, Ragan BG, Park JH. Issues in outcomes research: an overview of randomization techniques for clinical trials. J Athl Train. 2008;43:215–221. doi: 10.4085/1062-6050-43.2.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Botek M, Sládečková B, Krejčí J, Pluháček F, Najmanová EJAG. Acute hydrogen-rich water ingestion stimulates cardiac autonomic activity in healthy females. Acta Gymnica. 2021;51:e2021.009. [Google Scholar]
- 40.Botek M, Krejčí J, McKune AJ, Sládečková B. Hydrogen-rich water supplementation and up-hill running performance: effect of athlete performance level. Int J Sports Physiol Perform. 2020;15:1193–1196. doi: 10.1123/ijspp.2019-0507. [DOI] [PubMed] [Google Scholar]
- 41.Huang CS, Kawamura T, Toyoda Y, Nakao A. Recent advances in hydrogen research as a therapeutic medical gas. Free Radic Res. 2010;44:971–982. doi: 10.3109/10715762.2010.500328. [DOI] [PubMed] [Google Scholar]
- 42.Cohen J. 2nd. Lawerence Erlbaum Associates; 1988. Statistical power analysis for the behavioral sciences. [Google Scholar]
- 43.Ghasemi A, Zahediasl S. Normality tests for statistical analysis: a guide for non-statisticians. Int J Endocrinol Metab. 2012;10:486–489. doi: 10.5812/ijem.3505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Martusevich A, Surovegina A, Popovicheva A, Didenko N, Artamonov M, Nazarov V. Some beneficial effects of inert gases on blood oxidative metabolism: in vivo study. Biomed Res Int. 2022;2022:5857979. doi: 10.1155/2022/5857979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.LeBaron TW, Sharpe R, Pyatakovich FA, Artamonov MY. Hydrogen: from stars to fuel to medicine. In: Slezak J, Kura B, editors. Molecular hydrogen in health and disease. Cham: Springer Nature Switzerland; 2024. pp. 1–20. [Google Scholar]
- 46.Yamamoto R, Homma K, Suzuki S, Sano M, Sasaki J. Hydrogen gas distribution in organs after inhalation: Real-time monitoring of tissue hydrogen concentration in rat. Sci Rep. 2019;9:1255. doi: 10.1038/s41598-018-38180-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Sano M, Ichihara G, Katsumata Y, et al. Pharmacokinetics of a single inhalation of hydrogen gas in pigs. PLoS One. 2020;15:e0234626. doi: 10.1371/journal.pone.0234626. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Hall JE, Guyton AC. Philadelphia: Saunders/Elsevier; 2011. Guyton and Hall textbook of medical physiology. [Google Scholar]
- 49.Roepstorff C, Thiele M, Hillig T, et al. Higher skeletal muscle alpha2AMPK activation and lower energy charge and fat oxidation in men than in women during submaximal exercise. J Physiol. 2006;574:125–138. doi: 10.1113/jphysiol.2006.108720. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Zhang X, Xie F, Ma S, et al. Mitochondria: one of the vital hubs for molecular hydrogen’s biological functions. Front Cell Dev Biol. 2023;11:1283820. doi: 10.3389/fcell.2023.1283820. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Iuchi K, Imoto A, Kamimura N, et al. Molecular hydrogen regulates gene expression by modifying the free radical chain reaction-dependent generation of oxidized phospholipid mediators. Sci Rep. 2016;6:18971. doi: 10.1038/srep18971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Drid P, Trivic T, Casals C, Trivic S, Stojanovic M, Ostojic SM. Is molecular hydrogen beneficial to enhance post-exercise recovery in female athletes? Sci Sports. 2016;31:207–213. [Google Scholar]
- 53.Botek M, Khanna D, Krejčí J, et al. Molecular hydrogen mitigates performance decrement during repeated sprints in professional soccer players. Nutrients. 2022;14:508. doi: 10.3390/nu14030508. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Brooks GA. Lactate as a fulcrum of metabolism. Redox Biol. 2020;35:101454. doi: 10.1016/j.redox.2020.101454. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All data relevant to the study are included in the article or uploaded as Additional files.