Effects of Hydrogen Gas Inhalation on Community-Dwelling Adults of Various Ages: A Single-Arm, Open-Label, Prospective Clinical Trial

Molecular hydrogen (H2) is a versatile therapeutic agent. H2 gas inhalation is reportedly safe and has a positive impact on a range of illnesses, including Alzheimer’s disease (AD). Herein, we investigated the effects of 4 weeks of H2 gas inhalation on community-dwelling adults of various ages. Fifty-four participants, including those who dropped out (5%), were screened and enrolled. The selected participants were treated as a single group without randomization. We evaluated the association between total and differential white blood cell (WBC) counts and AD risk at individual levels after 4 weeks of H2 gas inhalation treatment. The total and differential WBC counts were not adversely affected after H2 gas inhalation, indicating that it was safe and well tolerated. Investigation of oxidative stress markers such as reactive oxygen species and nitric oxide showed that their levels decreased post-treatment. Furthermore, evaluation of dementia-related biomarkers, such as beta-site APP cleaving enzyme 1 (BACE-1), amyloid beta (Aβ), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor A (VEGF-A), T-tau, monocyte chemotactic protein-1 (MCP-1), and inflammatory cytokines (interleukin-6), showed that their cognitive condition significantly improved after treatment, in most cases. Collectively, our results indicate that H2 gas inhalation may be a good candidate for improving AD with cognitive dysfunction in community-dwelling adults of different ages.


Introduction
A crucial component of clinical trial inclusion criteria is the use of biomarkers that reliably identify the pathology of Alzheimer's disease (AD) over the course of an individual's lifetime. The prevalence of AD is 3% at the age of 65 years, which doubles every 5-6 years [1]. By 2025, South Korea is expected to have a super-aged society, only 8 years after becoming an elderly society. By the year 2045, the proportion of elderly persons is anticipated to increase by 37% [2]. Globally, approximately 50 million people suffer from dementia, with nearly 10 million new cases diagnosed every year [1]. The prevalence of dementia among individuals over 65 years of age in Korea was predicted to be 10.25% in 2020, or approximately 830,000 individuals. However, it is predicted to increase by about 15.91% by 2050, with an additional 3.02 million dementia sufferers [2]. Thus, effective therapeutic and preventive strategies are required to overcome the challenges of this global issue. The most common form of dementia-related neuropathological brain changes can reportedly begin as early as 20 years before symptoms appear; these can be further evaluated with potential biomarkers to determine treatment measures [3]. Normal Antioxidants 2023, 12, 1241 3 of 14 from researchers. The study was approved by Yonsei University Wonju Severance Christian Hospital's Research Review Board (No: CR323002) and registered with Clinical Trials.gov under the identifier NCT05891938. Patients were included only if a signed consent was obtained. Participants were free to withdraw from the experiment at any point. The questionnaires in the consent form were not critical of the participants. All the findings of this investigation were kept private and confidential.
Following approval from Yonsei University Wonju Severance Christian Hospital's IRB, subjects signed a permission form. Male and female participants who met the predetermined criteria were selected. The willing participants were assigned screening numbers. The physical examination data (height and weight), demographic data (sex and age), medical/medication history, and vital signs (temperature, blood pressure, and pulse) of each patient were recorded. Finally, 51 participants who met the inclusion criteria were selected for this study design, as shown in Figure 1. The basic characteristics, vital signs, accompanying symptoms, psychological problems, and recent lifestyle changes of the included participants were recorded. H 2 gas 2-3% was produced from an H 2 -generating device designed and provided by the company (GOOTZ Co., Ltd., Yangju-si, Gyeonggi-do, Republic of Korea). Therefore, 2% hydrogen + 98% filtering air inhalation (30 min/day) with a nasal cannula was provided for 4 weeks. The inclusion and exclusion criteria are listed below in Table 1. In addition, gender, education, systolic blood pressure, and diastolic blood pressure were recorded in this study to obtain baseline data, as shown in Table 2. Those who have participated in other clinical trials within 6 months of participating in clinical trials.
Persons who can comply with concomitantly permitted drugs, prohibited drugs, and relief drugs.
Patients who have another autoimmune disease.
Persons who can maintain the same exercise and activity during the clinical trial period.
Patients with tumors other than neurodegenerative diseases.
Fully understand the purpose and procedure of this clinical trial. Pregnant and lactating women. vice designed and provided by the company (GOOTZ Co., Ltd., Gyeonggi-do, Suwon, Republic of Korea). Therefore, 2% hydrogen + 98% filtering air inhalation (30 min/day) with a nasal cannula was provided for 4 weeks. The inclusion and exclusion criteria are listed below in Table 1. In addition, gender, education, systolic blood pressure, and diastolic blood pressure were recorded in this study to obtain baseline data, as shown in Table  2.

Inclusion Criteria Exclusion Criteria
Patients aged 40 to 70 years can walk voluntarily.
Those who have a history of AD disease.

Differential White Blood Cell Count Analysis
Retro-orbital plexus blood was collected and placed in tubes coated with ethylenediaminetetraacetic acid (EDTA). The total and differential white blood cell (WBC) counts, including those of lymphocytes, monocytes, neutrophils, eosinophils, and basophils, were measured using an automatic blood analyzer (HEMAVET HV950 FS; Drew Scientific Inc., Dallas, TX, USA).

Total ROS Estimation
Using the oxidation of 2-4-dichlorodihydrofluorescein diacetate (DCFH-DA) (Abcam, Cambridge, MA, USA), the total ROS production in serum was assessed. A total of 50 µL of serum samples was placed in a 96-well plate. After adding 100 µL of 10µM DCFH-DA to the wells, the plate was incubated in the dark for 30 min. The plate was then read at 488 nm excitation/525 nm emission using a DTX-880 multimode microplate reader (Beckman Counter Inc., Fullerton, CA, USA).

Nitrous Oxide Level Estimation
Griess reagent (Promega Corp., Madison, WI, USA) was used to measure NO generation in blood; the test was performed according to the manufacturer's instructions. Briefly, standards were prepared, and nitrite measurement was performed by adding 50 µL of serum samples into the wells. In each well, 50 µL of sulfanilamide solution was added and incubated for 10 min in the dark at room temperature. Then, in each well, 50 µL of N-(1-Naphthyl) ethylenediamine (NED) solution was added and incubated for 15 min in the dark at room temperature. A SpectraMax ® ABS Plus (Molecular Devices, San Jose, CA, USA) was used to measure the OD at 520 nm.

BDNF Level Measurement
Serum levels of total BDNF were measured using a human BDNF ELISA Kit (catalog number: EH42RB). The test was performed in accordance with the manufacturer's instructions. Serum samples (20 µL) were added to constructed standards and incubated for 2.5 h at room temperature. The samples were washed three times. Subsequently, 100 µL of biotin conjugate was added and incubated for 1 h. The samples were washed three times and 100 µL of Streptavidin-HRP solution was added in each well. These were incubated again for 45 min. Thereafter, 100 µL of this solution was added to the TMB substrate in each well and incubated for 30 min at room temperature. SpectraMax ® ABS Plus (Molecular Devices, San Jose, CA, USA) was used to measure the OD at 450 nm.

BACE-1 Level Measurement
Serum levels of total BACE-1 were measured using a human BACE-1 ELISA Kit (catalog number: ab267637). The test was performed in accordance with the manufacturer's instructions, such as that carried out for BACE-1 level estimation. Serum samples (20 µL) were added to constructed standards and incubated for 2.5 h at room temperature. The samples were washed three times. Subsequently, 100 µL of biotin conjugate was added and incubated for 1 h. The samples were washed three times and 100 µL of Streptavidin-HRP solution was added in each well. These were incubated again for 45 min. Thereafter, 100 µL of this solution was added to the TMB substrate in each well and incubated for 30 min at room temperature. SpectraMax ® ABS Plus (Molecular Devices, San Jose, CA, USA) was used to measure the OD at 450 nm.

Detection of Inflammatory Cytokines, Aβ, and Tau Protein by Multiplex Assay
The previously described approach was utilized to test the blood levels of inflammatory cytokines, including IL-6, MCP-1, Aβ, t-tau, p-tau, and VEGF-A using a Bead Array Suspension Multiplex Kit (Bio-Rad, San Diego, CA, USA). Each standard concentration was resuspended in standard diluents to create serial dilutions of the standard. The bead combination was mixed with the typical serum sample. After being washed, the plate was incubated for a further 18 h at 4 • C. The plate was then incubated for an hour at room temperature after the addition of the detecting antibody. The plate was then coated with streptavidin-phycoerythrin solution and left to sit at room temperature for 30 min. The plate was then examined using the (Millipore Corporation, Billerica, MA, USA) after the washing process; an assay buffer was then applied.

Data Management and Statistical Analysis
Statistical analysis was carried out using Graph Pad Prism (version 8.0; GraphPad Software, La Jolla, CA, USA) using unpaired t-tests. Data are expressed as mean ± standard deviation (SD). Differences were considered statistically significant at p < 0.05.

Effects of H 2 Gas Inhalation on Total and Differential WBC Counts in Community-Dwelling Adults of Different Ages
WBCs are important for both the innate and adaptive immune responses in the body. We did not find any significant changes in the total and differential WBC counts between the post-and pre-treatment values (Figure 2A-F).

Effects of H 2 Gas Inhalation on ROS and NO in Community-Dwelling Adults of Different Ages
Oxidative stress is involved in the progression of aging and AD [25]; it is one of the main mechanisms underlying cognitive aging and neurodegenerative diseases. We estimated the total ROS and NO serum levels to examine the impact of H 2 gas inhalation on community-dwelling adults of various ages. The serum levels of total intracellular ROS (p < 0.05) ( Figure 3A) and NO (p < 0.01) ( Figure 3B) significantly decreased after treatment. Both OS indicators, ROS and NO, indicate that H 2 gas is useful in treating cognitive impairment community-dwelling adults of various ages after 4 weeks of treatment.

Effects of H2 Gas Inhalation on ROS and NO in Community-Dwelling Adults of Different Ages
Oxidative stress is involved in the progression of aging and AD [25]; it is one of the main mechanisms underlying cognitive aging and neurodegenerative diseases. We estimated the total ROS and NO serum levels to examine the impact of H2 gas inhalation on community-dwelling adults of various ages. The serum levels of total intracellular ROS (p < 0.05) ( Figure 3A) and NO (p < 0.01) ( Figure 3B) significantly decreased after treatment. Both OS indicators, ROS and NO, indicate that H2 gas is useful in treating cognitive impairment community-dwelling adults of various ages after 4 weeks of treatment.

Effects of H2 Gas Inhalation on ROS and NO in Community-Dwelling Adults of Different Ages
Oxidative stress is involved in the progression of aging and AD [25]; it is one of the main mechanisms underlying cognitive aging and neurodegenerative diseases. We estimated the total ROS and NO serum levels to examine the impact of H2 gas inhalation on community-dwelling adults of various ages. The serum levels of total intracellular ROS (p < 0.05) ( Figure 3A) and NO (p < 0.01) ( Figure 3B) significantly decreased after treatment. Both OS indicators, ROS and NO, indicate that H2 gas is useful in treating cognitive impairment community-dwelling adults of various ages after 4 weeks of treatment.

Effects of H 2 Gas Inhalation on Serum BACE-1 Levels in Community-Dwelling Adults of Various Ages
BACE-1 levels activity is isolated from post-mortem human brains since AD neuropathology primarily develops in the cortex and hippocampus. We assessed the effect of possible confounding variables on the relationship between BACE-1 serum activity and cognitive diagnosis on community-dwelling adults of various ages. The total intracellular BACE-1 levels (p < 0.01) significantly decreased after treatment ( Figure 4). Additionally, BACE-1 levels are shown in different ages (50-59 years, 60-69 years, and 70-79 years) separately in Supplementary Figure S1. Our findings demonstrated that the dementia marker BACE-1 was effective in reducing dementia on community-dwelling individuals of various ages after 4 weeks of treatment; (50-59 years; * p < 0.05), (60-69 years; * p < 0.05), and (70-79 years; * p < 0.05) are shown in Figure S1. BACE-1 levels (p < 0.01) significantly decreased after treatment ( Figure 4). Additionally, BACE-1 levels are shown in different ages (50-59 years, 60-69 years, and 70-79 years) separately in Supplementary Figure S1. Our findings demonstrated that the dementia marker BACE-1 was effective in reducing dementia on community-dwelling individuals of various ages after 4 weeks of treatment; (50-59 years; *p < 0.05), (60-69 years; *p < 0.05), and (70-79 years; *p < 0.05) are shown in Figure S1.

Effects of H2 Gas Inhalation on the Serum BDNF Levels in Community-Dwelling Adults of Various Ages
The investigation of peripheral BDNF levels in clinical research involving patients is controversial. While several studies have shown that patients with AD have greater peripheral BDNF levels than healthy controls, other studies have found the opposite. Earlier studies examining the levels of serum BDNF in patients with AD and MCI have reported conflicting results [26]. In the present study, we investigated the effects of H2 gas inhalation on serum BDNF levels on community-dwelling adults of various ages. The serum BDNF levels were significantly increased (p < 0.001) after treatment on community-dwelling adults of various ages as compared to before treatment; the results are shown in Figure  5. Our results confirmed that the dementia marker BDNF was effective in reducing cognitive impairment on community-dwelling individuals of various ages after 4 weeks of treatment; (50-59 years; ***p < 0.001), (60-69 years; ***p < 0.001), and (70-79 years; ***p < 0.001) are shown Figure S2.

Effects of H 2 Gas Inhalation on the Serum BDNF Levels in Community-Dwelling Adults of Various Ages
The investigation of peripheral BDNF levels in clinical research involving patients is controversial. While several studies have shown that patients with AD have greater peripheral BDNF levels than healthy controls, other studies have found the opposite. Earlier studies examining the levels of serum BDNF in patients with AD and MCI have reported conflicting results [26]. In the present study, we investigated the effects of H 2 gas inhalation on serum BDNF levels on community-dwelling adults of various ages. The serum BDNF levels were significantly increased (p < 0.001) after treatment on community-dwelling adults of various ages as compared to before treatment; the results are shown in Figure 5. Our results confirmed that the dementia marker BDNF was effective in reducing cognitive impairment on community-dwelling individuals of various ages after 4 weeks of treatment; (50-59 years; *** p < 0.001), (60-69 years; *** p < 0.001), and (70-79 years; *** p < 0.001) are shown Figure S2.

Effects of H2 Gas Inhalation on Serum Levels of Aβ-40, Aβ-42, t-Tau and p-Tau in Community-Dwelling Adults of Various Ages
Neurofibrillary amyloid plaques, abnormal protein deposits made up of Aβ peptides (Aβ1-40 and Aβ1-42), and build-up of intracellular insoluble hyperphosphorylated tau proteins (p-Tau) that are the hallmarks of AD brain pathology. Herein, we evaluated how exposure to H2 gas affects the serum levels of Aβ peptides (Aβ1-40, Aβ1-42), t-tau, and p-Tau on community-dwelling adults of various ages. There were significant differences found in all biomarkers, such as Aβ1-40 (p < 0.01), Aβ1-42 (p < 0.05), t-tau (p < 0.001), and p-Tau (p < 0.05), after treatment compared to before treatment ( Figure 6). Our results Figure 5. Impact of H 2 gas inhalation on serum BDNF levels before and after treatment. Data are presented as mean ± SD. *** p < 0.001.

Discussion
This study was designed to investigate the effectiveness of H2 gas inhalation in adu of different ages. The results of the present clinical trial support the effectiveness of H2 g inhalation in improving cognitive impairment and related symptoms in patients with d mentia. Moreover, no adverse effects were recorded after four weeks of treatment with gas inhalation, and therefore, it can be considered a safe and well-tolerated healthy su plement for the treatment of AD in community-dwelling adults of various ages. Current no blood-based biomarkers can detect AD in its preclinical state. Aβ plaque and neuro brillary tangles develop as a result of inflammation and immunological dysfunction. stimulating microglial cells, the immune response initially lowers the plaque burd against neurodegeneration [27]. To stop the buildup of plaque, more inflammatory cy kines are released, and macrophage numbers increase [28]. During this stage, there is increase in the number of peripheral blood cells, including lymphocytes, neutrophi monocytes, platelets, and lymphocyte subsets, which are involved in inflammation a the immune response [29]. Numerous investigations have demonstrated that patien with AD and MCI had drastically increased peripheral neutrophil counts or decreas peripheral lymphocyte numbers [29]. Our results demonstrated that H2 gas inhalati treatment did not cause any adverse effects, indicating that it was safe. To date, no reliab conclusion has been made regarding the alterations in peripheral blood cell profiles. Se sitivity and other technological constraints have slowed the development of blood-bas biomarkers. Age-related diseases, their developmental stages, and cell signaling pathwa can all be linked to ROS generation [30,31]. Neuronal cells are more susceptible to oxid tive stress than other normal body tissues owing to their high oxygen consumption, hi lipid content, and lack of antioxidant enzymes [32]. One of the major factors causing A

Discussion
This study was designed to investigate the effectiveness of H 2 gas inhalation in adults of different ages. The results of the present clinical trial support the effectiveness of H 2 gas inhalation in improving cognitive impairment and related symptoms in patients with dementia. Moreover, no adverse effects were recorded after four weeks of treatment with H 2 gas inhalation, and therefore, it can be considered a safe and well-tolerated healthy supplement for the treatment of AD in community-dwelling adults of various ages. Currently, no blood-based biomarkers can detect AD in its preclinical state. Aβ plaque and neurofibrillary tangles develop as a result of inflammation and immunological dysfunction. By stimulating microglial cells, the immune response initially lowers the plaque burden against neurodegeneration [27]. To stop the buildup of plaque, more inflammatory cytokines are released, and macrophage numbers increase [28]. During this stage, there is an increase in the number of peripheral blood cells, including lymphocytes, neutrophils, monocytes, platelets, and lymphocyte subsets, which are involved in inflammation and the immune response [29]. Numerous investigations have demonstrated that patients with AD and MCI had drastically increased peripheral neutrophil counts or decreased peripheral lymphocyte numbers [29]. Our results demonstrated that H 2 gas inhalation treatment did not cause any adverse effects, indicating that it was safe. To date, no reliable conclusion has been made regarding the alterations in peripheral blood cell profiles. Sensitivity and other technological constraints have slowed the development of blood-based biomarkers. Agerelated diseases, their developmental stages, and cell signaling pathways can all be linked to ROS generation [30,31]. Neuronal cells are more susceptible to oxidative stress than other normal body tissues owing to their high oxygen consumption, high lipid content, and lack of antioxidant enzymes [32]. One of the major factors causing AD pathogenesis is the brain's vulnerability to ROS. Oxidative stress irreversibly damages cellular biomolecules and disrupts neuronal activity [32]. Our results suggest that H 2 gas inhalation improves cognitive function in patients with dementia by decreasing ROS levels. Due to its crucial physiological role, NO is involved in a variety of neurological disorders, including AD and other neurodegenerative dementias, according to a growing body of research [32]. According to numerous studies, the number of neurons in the hippocampus and BDNF concentrations in the cranial cortex were both found to increase following ischemic brain injury. An asymptomatic inflammatory process is the best description of the chain of events caused by chronic diseases and aging [33]. These findings highlight the neuroprotective function of BDNF in ischemic brain damage. Postmortem examination of the AD brain revealed a higher BDNF concentration which is shown in Figures 4 and S2. This study also advances the theory that a compensatory mechanism boosts the concentration of BDNF [34]. BACE-1 is a novel type 1 transmembrane aspartic acid protease that shares 501 amino acids with pepsin and retroviral aspartic proteases [35]. In the amyloidogenic pathway, BACE1 (β-secretase) breaks down the amyloid precursor protein (APP) to produce Aβ. As a result, the therapeutic inhibition of β-secretase would result in a reduction in the synthesis of all Aβ forms, including the pathogenic Aβ-42 [36]. Therefore, it is crucial to understand the biochemistry of BACE-1 to identify possible therapeutic targets in the etiology of AD. Our study demonstrated that after four weeks, H 2 gas inhalation treatment significantly decreased BACE-1 levels compared to the pre-treatment group. Our results indicate that H 2 gas inhalation may be a good candidate for improving cognitive impairment in patients with dementia, as demonstrated in Figures 3 and S1.
The development of tau neurofibrillary tangles and amyloid plaques inside brain tissues are both associated with AD. Brain damage from neurotoxic peptides, such as soluble or fibrillar Aβ, may be more likely to occur as a result of age-related accumulation of oxidative stress metabolites. Additionally, the buildup of Aβ can lead to an increase in ROS generation; however, it is still unknown whether AD is primary or secondary to excess oxidative stress. As Aβ can easily penetrate the BBB and is generally acknowledged as the oldest AD species, it is a promising choice as a blood biomarker. However, it has not been approved as a reliable signal for blood analysis, likely due to contradictory research findings. One study revealed [33] that the Aβ deposited in the brain is composed of 42 amino acids (Aβ-42) [37]. Many variants of Aβ are present in patients with AD, but the levels of Aβ-40 and Aβ-42 in CSF are the most reliable indicators of the disease. Although this amyloidogenic protein is present in the human body, it has been observed more than once that Aβ-42 concentrations in the CSF of patients correlate with Aβ levels in the brain [38]. Aβ-40, the most prevalent isoform of Aβ that is found in CSF, is another derivative of Aβ, which may function as a possible biomarker for AD. Many studies looking at the levels of A in those at risk for AD have found that Aβ-40 or Aβ-42 [39] levels are raised. In addition, Aβ-40 or Aβ-42 concentrations are decreased in people who are at risk for AD, or Aβ-40, and Aβ-42 levels have no bearing on a patient's likelihood of developing dementia [40,41]. Our research demonstrates that H 2 gas inhalation for four weeks following therapy decreased the level of the Aβ-40/Aβ-42 ratio in post-treatment groups compared to pre-treatment groups and improved cognitive impairments in AD patients, which is shown in Figures 5 and S3.
High levels of CSF t-tau have also been described, in earlier studies, as a sign of more serious cognitive impairment [42], fast progression to AD [43], and conversion to moderate dementia [44]. Based on these findings, it can be concluded that significantly [45] elevated CSF t-tau levels, indicating greater neuronal loss, are associated with a higher propensity for extensive cortical changes. The negative correlation between CSF p-tau levels and neuronal loss outside the medial temporal areas in individuals with high tau levels suggests the possibility of more extensive neuronal loss [46]. Our results suggested that t-Tau and p-Tau levels were significantly reduced after 4 weeks of H 2 gas inhalation treatment, which was effective for improved neurodegenerative diseases, such as AD, which is shown in Figures 5 and S4.
Immune dysregulation promotes neurodegeneration and is a role in cognitive decline, including synapse loss and neuronal death. It is characterized by persistent and increasing glial polarization [47]. Age-related cognitive decline is associated with plasma MCP-1 levels and functional and anatomical changes in the brain [48]. This result supports the newly developed theory that intrathecal inflammation occurs before the clinical onset of AD [48]. The fact that MCP-1 levels are elevated in the serum of patients with MCI and mild-to-moderate AD, whereas they decline throughout AD development, lends credence to this finding. Our results suggest that H 2 gas inhalation improves cognitive function in patients with dementia by decreasing MCP-1 levels, indicating that H 2 might be a good candidate for improving AD with cognitive dysfunction, as shown in Figures 6 and S5. It was reported that H 2 -rich water improved cognitive impairment [49]. Clinical testing revealed that 72 h of exposure to 2.4% H 2 gas had no negative impacts on any physiological measures, indicating that H 2 may not have any negative effects [50].
VEGF-A is a risk factor for chronic diseases, including AD. Although, the VEGF family is crucial for regulating angiogenic activity, neurogenesis, and neuronal survival [48]. Although the results have not been completely consistent, VEGF has been investigated as a possible biomarker of AD. In one study, the intrathecal levels of VEGF in the CSF of individuals with AD and vascular dementia were higher than those in healthy controls (i.e., no neurological disease or deficit) [51]. Another study discovered that there was no difference in CSF VEGF levels between patients with AD and cognitively healthy controls [52]. Similarly, VEGF promoter polymorphisms increased the risk of AD, although there was no change in serum VEGF levels between patients with AD and control [53]. Additionally, the build-up of VEGF with Aβ plaques in the brains of patients with AD may sequester VEGF, leading to a shortage [54]. Our findings leave us uncertain of whether the correlation between low blood VEGF levels and AD is due to the primary protective role of VEGF against AD or a secondary effect of AD pathology, such as Aβ plaques, on serum VEGF levels, which is shown in Figures 6 and S5. Our results also reveal that H 2 gas inhalation significantly decreased dementia biomarkers by reducing oxidative stress and enhancing antioxidant activity.

Conclusions
In summary, H 2 gas inhalation in the elderly is a good candidate for improving the risk of developing a neurodegenerative disease (dementia). However, further mechanistic studies are needed to fully elucidate the effects of H 2 gas inhalation in community-dwelling adults of different ages.

Institutional Review Board Statement:
This study was conducted after obtaining approval from the Institutional Review Board (IRB) of Wonju Severance Christian Hospital, Republic of Korea (IRB number: CR323002). Written informed consent was obtained from all participants included in this study.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data are contained within the article and supplementary materials.