Peripheral endothelial function can be improved by daily consumption of water containing over 7 ppm of dissolved hydrogen: A randomized controlled trial

Toru Ishibashi; Kosuke Kawamoto; Kasumi Matsuno; Genki Ishihara; Takamichi Baba; Nobuaki Komori

doi:10.1371/journal.pone.0233484

. 2020 May 29;15(5):e0233484. doi: 10.1371/journal.pone.0233484

Peripheral endothelial function can be improved by daily consumption of water containing over 7 ppm of dissolved hydrogen: A randomized controlled trial

Toru Ishibashi ^1,^2,^3,^*, Kosuke Kawamoto ¹, Kasumi Matsuno ¹, Genki Ishihara ¹, Takamichi Baba ², Nobuaki Komori ¹

Editor: Giuseppe Andò⁴

PMCID: PMC7259729 PMID: 32470022

Abstract

Background

Measurement of the reactive hyperemia index (RHI) using peripheral arterial tonometry (PAT) has shown benefits in the evaluation of vascular endothelial function and prediction of cardiovascular disease prognosis. Thus, it is important to examine the factors that promote the RHI. In this study, we aimed to investigate the effect of molecular hydrogen (H₂) on reactive hyperemia-PAT of the small arteries of fingers in healthy people.

Methods

To determine the efficacy of H₂ for improving peripheral vascular endothelial function, water containing high H₂ concentrations was administered to participants, and the Ln_RHI was measured in the finger vasculature. Sixty-eight volunteers were randomly divided into two groups: a placebo group (n = 34) that drank molecular nitrogen (N₂)-containing water and a high H₂ group (n = 34) that drank high H₂ water (containing 7 ppm of H₂: 3.5 mg H₂ in 500-mL water). The Ln_RHI was measured before ingesting the placebo or high H₂ water, 1 h and 24 h after the first ingestion, and 14 days after daily ingestion of high H₂ water or the placebo. The mixed effects model for repeated measures was used in data analysis.

Results

The high H₂ group had a significantly greater improvement in Ln_RHI than the placebo group. Ln_RHI improved by 22.2% (p<0.05) at 24 h after the first ingestion of high H₂ water and by 25.4% (p<0.05) after the daily consumption of high H₂ water for 2 weeks.

Conclusions

Daily consumption of high H₂ water improved the endothelial function of the arteries or arterioles assessed by the PAT test. The results suggest that the continuous consumption of high H₂ water contributes to improved cardiovascular health.

Introduction

Vasomotor tone and appropriate blood pressure are maintained by the vascular endothelium, which responds to shear stress generated by increased blood flow and stimulates vasodilation [1]. Vascular damage is caused and intensified by endothelial dysfunction, which induces chronic inflammation of the vasculature, followed by the development of atherosclerosis and cardiovascular disease [2–6]. The endothelium is composed of a monolayer of endothelial cells that function as an endocrine organ and a 0.2-2-μm-thick, negatively charged surface layer called the glycocalyx, which is a primary-functional barrier for endothelial cell protection [7, 8]. The endothelium senses the shear stress and transduces it into chemical or electrical signals, including nitric oxide (NO) and endothelium-derived hyperpolarization factor (EDHF), leading to the relaxation of smooth muscle cells. In smaller diameter arteries, such as peripheral arteries, EDHF predominantly controls the vasomotor function, while in larger diameter vessels, such as conduit arteries, NO plays a pivotal role in the vascular response to blood flow. These factors do not work alone but work in conjunction with each other to maintain a healthy circulation. The method to monitor the response of a conduit artery endothelium is flow-mediated dilation (FMD) [9], and reactive hyperemia-peripheral arterial tonometry (RH-PAT) has been developed to assess endothelial function in the small arteries of the fingers [10]. These methods are thought to significantly predict the risks of cardiovascular and cerebrovascular events, including angina, myocardial infarction, ischemic stroke, cerebral infarction, acute coronary syndrome, and heart failure [2–5]. Recently, RH-PAT and FMD have been compared, and it was suggested that these two methodologies are not associated and reflect different aspects of cardiovascular risks. [10–13]. It is important to not only estimate the function of the endothelium of both conduit arteries and peripheral arteries in healthy people using these non-invasive methodologies, but to also identify the factors or methodologies that can improve endothelial function and prevent endothelial dysfunction in healthy people without administering pharmaceutical drugs.

Recently, we showed that consumption of over 3.5 mg of molecular hydrogen (H₂) dissolved in water (at a concentration of 7 ppm H₂ in 500 ml of water: high H₂ water) can rapidly improve FMD of the conduit brachial artery [14]. In this study, we aimed to investigate the effect of H₂ on RH-PAT of the small arteries of fingers in healthy people. In some interventional studies, attempts to improve the reactive hyperemia index (RHI) without using a pharmaceutical approach, but instead focusing on exercise therapy and/or lifestyle improvement, have been reported in patients suffering from heart failure and metabolic syndrome [15–18]. Improvements in RHI were also reported when postprandial hyperglycemia was treated pharmaceutically [19]. Besides these disease conditions, it is important to identify safe and conventional ways for achieving relatively high (healthy) RHIs in the small vessels of healthy individuals. We demonstrated here whether daily consumption of high H₂ water is beneficial for the endothelial function of which improvement can prevent the development of atherosclerosis and cardiovascular disease.

Methods

Subjects and the measurements of RHI

This study was designed to assess the possibility of H₂ acting as an agonist for NO and/or EDHF, which was suggested previously using FMD (S1 File). The research protocol was registered with the UMIN clinical trial registry (number: UMIN000032510; dated 08/05/2018). Our study was approved by the Huis Ten Bosch Satellite H2 Clinic Ethics Committee (July 3, 2015; approval number: hshc1502). The study was performed in accordance with the Declaration of Helsinki. Seventy-one volunteers were recruited between July 2015 and May 2018 and informed individually about the significance of the study and the instruments of intended measurement. These volunteers enrolled in this study after providing their written informed consent. This clinical trial was performed according to the S1 Checklist, and the CONSORT diagram is shown in Fig 1. All study subjects took no medications or dietary supplements or received any medical treatments for more than 6 months before this study was performed. All subjects were asked to fast and avoid drinking caffeinated drinks or ingesting sugar as well as breakfast for 6 h before the test. The subjects were randomly divided into two groups, and three of them did not complete the protocol. Randomization was performed by blinded independent physicians’ assistants. Sixty-eight subjects completed the study protocol, and the average (±standard deviation [SD]) age of the 34 subjects (men: 13; women: 21) in the high H₂ group was 36.3±10.4 years and that of the 34 subjects (men: 11; women: 23) in the placebo group was 38.2±11.2 years. Using the EndPad2000 System (Itamar Medical Inc.), we measured the RHI using endothelium-dependent digital pulse amplitude testing (EndoPAT) according to the reports in previous studies [2,18]. In brief, disposable RH-PAT probes were placed individually on both index fingers, and a blood pressure cuff was placed on the subject’s ipsilateral upper arm region in a quiet and dimmed room. After resting for more than 20 min, a baseline pulse amplitude recording was started, and RH-PAT was induced via reperfusion of blood flow after a 5-min cuff occlusion of the brachial artery at 60 mmHg above the measured systolic pressure. Next, the subjects drank 500 ml of the placebo water or the high H₂ water within 10 min, and after resting for 1 h, the RHI was measured again. On the second day, 24 h after the first ingestion of placebo or high H₂ water and before the second ingestion, the RHI was measured. After the measurements were obtained, a second drink was administered, and the day after, they drank the placebo or the high H₂ water once each day until the day before the last measurements at 14 days. In total, they drank the high H₂ water or placebo 14 times. No adverse events were recorded as a result of the clinical study. The RHI was calculated as follows: RHI = Ln{[RH-PAT ratio]×[0.226×Ln (baseline)–0.2]} [2, 3, 20], and we used the natural logarithmic scaled Ln_RHI.

Preparation of high H₂ water and placebo water

The high H₂ water (Aquela Global Co., Ltd., London, UK) was prepared according to previously described methods [21]. Briefly, H₂ gas was produced in an elastic polyethylene terephthalate (PET) bottle (manufactured by TOMIKAWA Chemical Industry Co., Ltd., or other bottles were used if they were prepared for use with carbonated drinks and had a high elasticity to withstand the high pressures of the dissolved gas). The bottle was filled with 530 ml of water by mixing 75% of metal aluminium grains with 25% of calcium hydroxide (by weight) and 0.5 ml water in an acrylic resin tube placed into the bottle. During the reaction, the H₂ gas pressed against the surface of the water in the standing bottle, which was gradually hardened by the increasing pressure of the emerging hydrogen gas. After the reaction was terminated, the H₂ gas was dissolved in the water for 12 h, and the bottle was shaken for about 30 s just before drinking. The placebo water was prepared by filling up a PET bottle containing water with molecular nitrogen (N₂) gas under 0.8 MPa to make the placebo bottles as firm as the bottles with the high H₂ water. An acrylic resin tube with a placebo non-woven fabric (used to produce H₂ gas in a tube but lacking the reactive compound) was also placed in the placebo bottles so that the volunteers could not distinguish between the two types of water. On the first and second days of the EndoPAT testing, participants were given either a placebo or high H₂ PET bottle of water, which was indistinguishable due to the lack of taste and identical appearance. After the second day, each PET bottle of water was shipped to the participants daily, with each bottle received a day before the administration. It has been clarified and agreed in this field that H₂ is transferred into the human body and circulated from ingested water containing H₂. In cases where rats were administered 15 mL/kg of 0.8 mM H₂ water by a catheter, or mice drank approximately 200 mL/kg of 0.2–0.4 mM H₂ water per day, the concentration of H₂ in their blood was 5–6 μM and about 2 μM, respectively [22, 23]. In a clinical report about the concentration of H₂ in blood 30 minutes after ingesting water containing H₂, we also clarified that the presence of hydrogen gas during exhalation from the lung of the person who ingested the high H₂ water can be explained by the rapid and passive diffusion of H₂ in the aqueous human body including circulation [21].

Statistical analysis

The efficacy of the high H₂ water was estimated by analyzing the change in Ln_RHI at each time point based on the value of Ln_RHI before drinking, which reflected the change in Ln_RHI from baseline. We used the mixed effects model for repeated measures (MMRM) for the analysis. MMRM is a model used to analyze repeated measures continuous outcomes [24]. Using all data obtained at 1 h, 24 h, and 2 weeks, the MMRM included the treatment group, time points, and the interaction between the treatment group and the time points as fixed effects, with the Ln_RHI at baseline as a covariate. In the MMRM analysis, the correlation structure between time points needs to be specified and unstructured (i.e., no specific correlation structure), which is why we used it. Furthermore, we assumed a Gaussian error term in the model. We performed a log-likelihood ratio test and residual analysis in order to check the fit of the model. Under the MMRM, the adjusted mean change from the baseline, adjusted mean difference between the treatment groups, and their 95% confidence intervals (CIs) and p-values were calculated using the software SAS version 9.2 (SAS Institute, Inc. Cary, NC, USA).

Results

The high H₂ water was prepared more than 12 h before the subjects drank it, and we confirmed that the concentration was over 7 ppm, as described before [21]. The participants drank about 500 ml of the water containing 7-ppm H₂ immediately after opening the PET bottle so that they could consume approximately 3.5 mg H₂.

The baseline data of the subjects are presented in Table 1. All of the subjects had normal mean biometric parameters and had no previous diagnosis of, or took medications related to metabolic syndrome, hypertension, chronic inflammatory diseases, or cardiovascular diseases. There were no significant differences in age, sex, height, weight, body mass index (BMI), blood pressure, or heart rate between the two groups. Additionally, there was no significant difference in the RHI value of subjects between the groups (placebo group: 0.61±0.26, high H₂ group: 0.63±0.29). We considered that the subjects in these two groups had physiologically similar characteristics in terms of endothelial function.

Table 1. Characteristics of the enrolled volunteers.

	All cases (N = 68)	High H₂ group (n = 34)	Placebo group (n = 34)
Age	37.3±10.7	36.3±10.4	38.2±11.2
Men, n (%)	24 (35.3)	13 (38.2)	11 (32.3)
Women, n (%)	44 (64.7)	21 (61.8)	23 (67.6)
Height (m)	1.61±0.09	1.62±0.08	1.60±0.09
Weight (kg)	56.4±11.0	57.4±11.1	55.3±10.7
BMI (kg/m²)	21.6±3.34	21.7±3.84	21.5±2.81
SBP (mmHg)	114±14.7	113±15.4	115±14.2
DBP (mmHg)	67.5±10.9	66.9±12.1	68.0±9.76
HR (beats/minute)	71.0±8.75	70.7±8.13	71.4±9.44

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BMI: body mass index, SBP: systolic blood pressure, DBP: diastolic blood pressure, HR: heart rate, H₂: hydrogen. The data are presented as mean±standard deviation.

The concentration of H₂ in the bloodstream is thought to reach its peak within 10 min [21]. The influence of H₂ was observed with FMD of the brachial artery at 30 min after H₂ consumption in a previous study [18]. Thus, we obtained the first measurement of Ln_RHI at 1 h after the subjects drank the high H₂ water so that the endothelium of the peripheral artery had enough time to respond to the H₂ mechanism, whether or not it was NO dependent, as observed in the study using FMD. The Ln_RHI data “before drinking” was collected 30 min before the first drink of the high H₂ water. The interval between the first PAT measurement (before drinking) and the second measurement was 1.5–2.0 h. It has been reported that repetitive PAT measurements over a 1-hour or 2-hour interval do not carry the effects forward [25]. The second measurement was set 24 h after the first drink, but before the second drink on the second day. All of the tests were performed in the afternoon and the subjects drank the placebo or the high H₂ water once a day at roughly the same time in the afternoon for 14 days. The third measurement was performed on the 15^th day after the subjects finished the daily drinking of the placebo or the high H₂ water for 14 days.

As shown in Fig 2, 24 h after the first consumption of 3.5 mg H₂ in water, the Ln_RHI improved significantly compared to the placebo water. By continuous consumption of the high H₂ water for 2 weeks, Ln_RHI was ameliorated compared to the placebo group. The statistical analysis comparing all of the subjects in the two groups is summarized in Table 2. From the result of log-likelihood ratio test (p = 0.0483) and the analysis of residuals (data not shown), we confirmed the appropriateness of the model to the data. The improvement in RH from its level before ingestion was also observed with the H₂ group. At 1 h after the first consumption of the high H₂ water, the Ln_RHI tended to increase from the value observed before consumption (p = 0.0597). At both 24 h and 2 weeks after ingestion, significant improvement was observed in the Ln_RHI, with p-values of 0.0003 and 0.0066 for 24 h and 2 weeks, respectively. By contrast, in the placebo group, no improvement of Ln_RHI was observed throughout the study. The p-values are presented in Table 2.

Table 2. Comparison between the two groups for all of the subjects.

Time Point	Treatment Group	Change from the Ln_RHI Before Drinking			H₂ Group Versus Placebo Group
Time Point	Treatment Group	LS Mean (Standard Error)	95% Confidence Interval	p-value	Difference of Adjusted Mean [95% Confidence Interval]	p-value
1 h	Placebo	0.05 (0.04)	-0.03, 0.13	0.1849
	H₂	0.07 (0.04)	0.00, 0.15	0.0597	0.02 [-0.09, 0.13]	0.6851
24 h	Placebo	0.02 (0.04)	-0.06, 0.09	0.6456
	H₂	0.14 (0.04)	0.07, 0.22	0.0003	0.13 [0.02, 0.23]	0.0193
2 weeks	Placebo	-0.02 (0.04)	-0.11, 0.07	0.6395
	H₂	0.12 (0.04)	0.03, 0.21	0.0066	0.14 [0.02, 0.26]	0.0237

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MMRM analysis: fixed effects are for the treatment groups, time points, and interaction effects.

The covariate was set as the Ln_RHI value before drinking. The covariance structure was unstructured.

RHI: reactive hyperemia index, LS: least square mean, H₂: hydrogen.

p-value of log-likelihood test: 0.0483.

In order to identify the influence of the Ln_RHI value before ingestion on the data at each time point, we divided the subjects into two groups with the cut-off value of 0.71 (see Discussion section), a low Ln_RHI group (<0.71) and a high Ln_RHI group (≥0.71). Nineteen subjects in the high H₂ group and 22 subjects in the placebo group were sorted in the low Ln_RHI group. We also applied MMRM analysis to the low Ln_RHI group. The p-value of the log-likelihood ratio test was 0.3597. It was relatively large because of the small sample size. From the result of the analysis of residuals (data not shown), we confirmed the appropriateness of the model to the data. Comparing the two groups, in the low Ln_RHI group, significant improvement was observed only after daily consumption of the high H₂ water for 2 weeks, as shown in Fig 3. When the Ln_RHI at each time point was compared to the Ln_RHI before ingestion in the high H₂ group, a significant improvement was observed for all of the 3 time points. Significant improvement of Ln_RHI at 1 h (p = 0.0057) and 24 h (p = 0.0205) compared to the values before ingestion was also seen in the placebo group. The statistical analysis is summarized in Table 3. On the other hand, in the high Ln_RHI group, there was no significant difference between the placebo and the high H₂ group, and no significant improvements were observed when the Ln_RHI was compared to the Ln_RHI value before ingestion in either the placebo or the high H₂ group (data not shown).

Fig 3 — Results are presented as the height of the columns at each time point. P: the placebo group, H: the high H₂ group. Error bars are indicated on each column, and p-values are indicated above the compared columns between the two groups. RHI: reactive hyperemia index, H₂: hydrogen.

Table 3. Comparison between the two groups with the low Ln_RHI (Ln_RHI <0.71).

Time Point	Treatment Group	Change from Baseline			H₂ Group Versus Placebo Group
Time Point	Treatment Group	LS Mean (Standard Error)	95% Confidence Interval	p-value	Difference of Adjusted Mean [95% Confidence Interval]	p-value
1 h	Placebo	0.13 (0.05)	0.04, 0.22	0.0057
	H₂	0.11 (0.05)	0.01, 0.20	0.0349	-0.03 [-0.16, 0.11]	0.7011
24 h	Placebo	0.09 (0.04)	0.01, 0.17	0.0205
	H₂	0.17 (0.04)	0.09, 0.25	0.0002	0.08 [-0.04, 0.19]	0.1788
2 weeks	Placebo	0.06 (0.05)	-0.03, 0.16	0.1625
	H₂	0.28 (0.05)	0.18, 0.38	< .0001	0.22 [0.08, 0.35]	0.0024

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MMRM analysis: fixed effects are for the treatment group, time points, and interaction effects.

The covariate is set as the Ln_RHI value before drinking. The covariance structure is unstructured.

RHI: reactive hyperemia index, LS: least square mean, H₂: hydrogen.

p-value of log-likelihood test: 0.3597.

Discussion

In this study, consumption of 3.5 mg of H₂ dissolved in water improved Ln_RHI after daily consumption for 2 weeks (Fig 2). A single drink of the high H₂ water also increased the Ln_RHI in the all of the subjects in the high H₂ group at 24 h after the drink. It was suggested that daily consumption of high H₂ water is important to improve the endothelial function of the peripheral artery. Furthermore, to investigate how the value of Ln_RHI before drinking affects the H₂ mode of action, we divided the subjects into two groups, with high risk and with low risk based on Ln_RHI values. Although not intended for healthy subjects, there are several investigations on the prognostic value of Ln_RHI for predicting cardiovascular risks. The Ln_RHI values reported were 0.531 in 577 patients at high risk for cardiovascular events by Matsuzawa et al [2] and 0.4 in 270 low-risk outpatients with chest pain by Rubinshtein et al [3]. It has been also reported that the risk of arrhythmia recurrence could be estimated by an Ln_RHI value of 0.618 in 92 participants with atrial fibrillation undergoing catheter ablation [26]. Orthopedic patients who underwent total hip or knee arthroplasty and avoided postoperative deep vein thrombosis (DVT) had preoperative ln-RHI values of 0.71±0.25 [27]. Among these reports, the border value of 0.71 for the orthopedic patients avoiding DVT seems to be close to that of the healthy masses. To predict future endothelial health in healthy subjects in this study, it seemed to be appropriate to set the threshold for RHI above the values for patients with cardiovascular risks. We set the Ln_RHI value at 0.71 in order to divide the subjects into two groups.

In the low Ln_RHI group, the continuous consumption of H₂ for 2 weeks significantly improved the Ln_RHI value compared to placebo. An unexpected result that we found was that there was no improvement at 24 h after the first drink. To account for this result, both the time at 1 h and at 24 h after the single drink, showed significant improvement from the values before ingestion of the drink were also observed within the placebo group (Table 3). Because there was no improvement at 2 weeks within the placebo group, the placebo effects are thought to be transient, although it is currently unclear whether it is merely the placebo effect or there is another factor that causes the improvement of Ln_RHI within 1 h after the placebo group drinks the water containing N₂. On the other hand, there was no influence of H₂ intake on RH-PAT in the high Ln_RHI group. The mechanism in which H₂ acts on the peripheral endothelium seems to be mediated by the factors that are lacking or are impaired in the low Ln_RHI group with future risk for endothelial dysfunction. In a recent report, we demonstrated the preliminary potential of H₂ and its agonistic effects on NO-related mechanisms, as well as the functional roles of H₂ on the endothelium of conduit arteries by the FMD test [14]. Similar to the FMD test, NO-mediated vasodilation mechanisms are certainly involved in PAT testing. It was clearly demonstrated that the PAT test evaluates NO production, using N^G-nitro-L-arginine methyl ester (L-NAME) as an inhibitor of nitric oxide synthase [28]. However, when the reactive increase in reperfusion flow in the conduit brachial artery at the site of occlusion in the upper arm is inhibited by L-NAME, the increase in reactive hyperemia and reperfusion flow to the finger vessels seems to decrease. Therefore, it is necessary to consider whether reperfusion flow without the reactive increase is enough to cause the release of EDHF. A prompt release of EDHF may be required in addition to the release of NO to obtain a high enough RHI to maintain the health of the peripheral microcirculation, although there are few studies on EDHF and the RH-PAT test. Furthermore, NO and EDHF are thought to cause vasodilation via mitochondrial function, and endothelial dysfunction is mediated by the overproduction of superoxide and H₂O₂ [29].

The accumulated molecular mechanisms of endothelial function regarding the vasodilation of peripheral described in previous reports are summarized and presented in Fig 4 to help considering the mechanistic insights of H₂ on PAT data and EDHF [29–36].

As illustrated in the left part of Fig 4, as long as the endothelium functions well with high RHI values (it is above 0.7, here), the peripheral vasculature can metabolize and generate appropriate amounts of energy with relevant O₂ consumption. The by-products of mitochondrial electron transfer (H₂O₂ induced by flow-mediated shear stress via the function of superoxide dismutase in respiratory conditions) are moderate and play important roles as signals of vasodilation, as evidenced in the coronary arteries [30,31]. The subsequent activation of large conductance Ca²⁺-activated potassium channels in smooth muscle cells induces EDHF and relaxation of the vessels. The secured flow by EDHF maintains the microcirculation, which supports the appropriate metabolism of the endothelial mitochondria. Then, it creates the positive feedback loop where the higher value of RHI is provided.

By contrast, the hypoxic microcirculation which causes an accumulation of NADH or FADH₂ and an increase in electron leakage from the electron transport chain, with excess production of superoxide and H₂O₂ resulting in the formation of deleterious peroxynitrite (ONOO^-) by exhausting NO as well as the formation of hydroxyl radicals [29]. Once the vicious circle presented in the right part of Fig 4 is established, RH in the peripheral arteries including coronary arterioles could be impaired, as in the low Ln_RHI group.

It should be noted that measuring RH of the peripheral microcirculation in which occlusion of the brachial artery following the release of blood flow is known as the model of ischemia reperfusion (I/R) injury, although the function of the vasodilation of the microvasculature is not fully understood [32]. The mechanisms of the cellular damage by I/R injury are believed to be caused by the superoxide burst in the mitochondrial respiratory chain. Particularly, the reverse electron transport accompanied by the elevated mitochondrial membrane potential is the major factor of electron leakage in mitochondrial complex I [33]. Recently, we have reported that H₂ reduces the mitochondrial membrane potential in the living cultured cells [34]. In the study, the overproduction of superoxide with the high ratio of NADH/NAD⁺, which reflects the highly reduced state of mitochondria during hypoxia following I/R injury [35], was suppressed by H₂. This situation corresponds to the right circle in Fig 4. On the basis of these most recent mechanistic insights of H₂ [34, 36], we suppose that H₂ may improve endothelial function via the mitochondrial mechanisms in healthy subjects. H₂ may shift the circle in Fig 4 from right to left by reducing the elevated mitochondrial membrane potential as well as the overproduction of superoxide under the condition with hypoxia presented in the right circle.

Thus far, the limitations to explain the mechanisms of the H₂ effect on a wide range of diseases or pathological states based on the scavenging property of H₂ with respect to highly reactive radicals have been discussed [37, 38]. In blood flow, the availability of H₂ and/or the frequency with which it reacts with target radicals seems insufficient to explain the distinct biological effects of H₂ in the peripheral vasculature. In the present study, the continuous scavenging of deleterious radicals by H₂ is excluded by the single intake of high H₂ water in a day. Therefore, we consider that the effects of H₂ herein are occur via the mitochondrial mechanisms described in Fig 4 [33–36].

Study limitations

As the aim of this study was to investigate the effect of H₂ on RH-PAT intended for healthy subjects to assess the significance of daily consumption of H₂ to prevent the endothelial dysfunction of small arteries, there are no data for an atherosclerotic group with a low RHI value. It is important to examine the effect of H₂ on the RH-PAT of people with a high risk of hypertension, cardiovascular disease, diabetes mellitus and dyslipidemia due to atherosclerosis. The next study should include participants with these characteristics. As for glucose metabolism and lipid profiles, which are confounding factors for endothelial function, we did not collect blood samples, and our study protocol did not include the blood test. In addition, the number of the subjects in this study was small and the follow-up data for the long-term consumption of high H₂ water was lacking.

Conclusions

The daily consumption of water containing a high concentration of H₂ (over 7 ppm or 3.5 mg in 500 mL of water) could ameliorate the endothelial function of the arteries and arterioles assessed by the PAT test. It was suggested that the high H₂ water is useful for improving the function of the vasculature and decreasing the risk of illness. Based on the agonistic function of H₂ against the endothelium as suggested herein, a controlled study with a larger number of subjects and a long-term observation should be done in the near future. High H₂ water has been available commercially, and now many people have been drinking it in Japan and Europe. A cohort study of this population should also be performed in the future.

Supporting information

S1 Checklist. CONSORT 2010 checklist of information to include when reporting a randomised trial*.

(DOC)

Click here for additional data file.^{(218.5KB, doc)}

S1 File. Trial protocol.

(DOCX)

Click here for additional data file.^{(28.8KB, docx)}

S2 File

(DOCX)

Click here for additional data file.^{(26.2KB, docx)}

S1 Data. Set of the present study.

(XLSX)

Click here for additional data file.^{(27.2KB, xlsx)}

Acknowledgments

We thank T. Nagao, H. Tagomori, K. Kiyota, R. Nawata, K. Fukuoka, and M. Takeda for the technical support and advice. We would like to thank Editage for English language proofreading.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The authors received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0233484.r001

Decision Letter 0

Giuseppe Andò

28 Nov 2019

PONE-D-19-26280

Peripheral endothelial function can be improved by daily consumption of water containing over 7ppm of dissolved hydrogen: A randomized controlled trial

PLOS ONE

Dear Dr. Ishibashi,

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Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: No

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Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, Ishibashi et al. describe the effects of hydrogen water for 2 weeks on endothelial function assessed by reactive hyperemic index (RHI) in 68 Japanese volunteers. The authors observed that hydrogen water significantly increased RHI in these subjects. The topic that the authors investigated is clinically important. However, there are critical points that the authors should address.

Comments

1. The specific purpose of the study is vaguely stated. In addition, conclusion is also vaguely stated. The present study lacks a real atherosclerotic group with endothelial function (e.g., hypertensive patients, diabetes mellitus, dyslipidemia, and cardiovascular disease).

2. A number of studies in vitro reported that hydrogen directly works several types of cells through the various mechanisms. However, it is doubt that dissolved hydrogen in hydrogen water is taken in the vasculature and the cell after drinking hydrogen water in humans.

3. Subjects enrolled in this study were relatively young and healthy subjects. However, values of RHI were markedly small (e.g., 0.61 in the placebo group and 0.63 in the hydrogen water group). In addition, the authors stated that the border value of 0.71 seems to be close to that of the healthy masses (page 25, lines 6 and 7). This interpretation is clearly wrong. Previous studies including guidelines for management of endothelial function have clearly shown that normal range of RHI (healthy endothelial function) is ≥2.10, borderline is 1.67 to 2.10, and abnormal is <1.67.

4. Unfortunately, there are no mechanisms by which hydrogen water significantly increased RHI in healthy subjects. As the authors stated in the Discussion section, measurements of oxidative stress and inflammatory markers would draw more specific conclusions concerning the role of dissolved hydrogen in hydrogen water on vascular function.

5. It is well known that blood pressure, heart rate, glucose metabolism, and lipid profiles) other than BMI also are confounding factors for endothelial function. These parameters should be presented in Tables.

6. Introduction section and the Discussion section are too long. They should be significantly shortened as they contain a number of issues not directly related to the message of this study.

7. Figure 4 is too speculative and is not needed.

Reviewer #2: The manuscript addresses an interesting topic. The data are original and rich of information. The statistical methods employed are sound. Results are of potential interest. Some comments follow.

1. The data are not fully available. This is not in line with the journal's guidelines. More importantly, this does not allow the reviewer to check for the correctness and appropriatness of the methods. Please, add the dataset as a supplementary file.

2. The use of mixed effects models is sound. Linear mixed model could be an interesting option, but several aspects deserve more care. The model must fulfill some assumptions to ensure the reliability of the results. Those assumptions are completely overlooked. A complete analysis of the residuals must be included and tests (e.g log-likelihood ratio) should be included to check for the appropriateness of the random effects model. Moreover, some info on the random terms must be included (e.g. the variance of the random effects) and, as I am guessing that Gaussian distribution is taken from granted, if the assumptions on the random terms are also fulfilled.

3. Please, provide inference on the baseline characteristics as well, bearing in mind that any parametric statistical test is based on some assumptions to be fulfilled.

4. The way results are presented are somehow confusing to me. I would expect to see regression parameters, in which the placebo is taken as reference category. I do not really get how bot Placebo and Treatment can be included in a single model (as p-values are reported for both). Moreover, the term LS is not explained in the text. Please, report the results in a more common way, focusing on regression parameters and heterogeneity due to the longitudinal stracture in the data. Something should be said on the interaction effect as well.

Reviewer #3: The paper entitled “Peripheral endothelial function can be improved by daily consumption of water containing over 7ppm of dissolved hydrogen: A randomized controlled trial” describes the effect of oral intake of H2 improved the endothelial function assessed by reactive hyperemia peripheral arterial tonometry (RH-PAT).

In this study, subjects had an administration of 500mL of water every day. The subjects were divided into 2 groups by the property of water: high H2 water, and control. RH-PAT was performed at baseline, 1hour, 24 hours, and 14 days after the first intake. And the reactive hyperemia index (RHI), an index for a vascular endothelial function was compared between 2 groups. The authors found that the high H2 water group showed an increase in RHI in the later phase. And this effect was observed in subjects with lower RHI at baseline (lnRHI < 0.71). The authors have already reported this topic using flow mediated dilation (FMD), another index for endothelial dysfunction, and confirmed similar findings using another endothelial function test.

The paper suggests that oral intake of H2 might improve the endothelial function in healthy subjects having slightly reduced RHI. However, it is unclear if this treatment has a favorable effect on patients with impaired endothelial function.

Major comment

In general, the manuscript and too long and rambling. In Results, the same data were repeatedly shown in several figures and tables. This manuscript should be restyled with a more concise and clear way.

Minor comments

Introduction

- The introduction is too long, and describes too much for the comparison between FMD and EndoPAT.

- In the introduction (line 85), the authors stated: “the nature of the rosy faces often observed…..”. Do the authors have a reference for this?

Methods

- In line 121, “both 2d fingers” should read “both index fingers”.

- In this paper, RH-PAT was performed in the afternoon. The authors should describe the breakfast and other oral intakes like caffeine.

- The authors should describe the statistical analysis more precisely.

Results

- The authors divided subjects into low and high RHI groups with a cut-off of 0.71. They should explain how they conduced 0.71 as a cut-off.

Discussion

- In line 295-297, the authors stated: “The RH-PAT in the small vessels may be influenced by volume loading…”. Please show some references.

- In line 296-297, the authors said that loading of volume may strain the sympathetic nerve and affect PAT data. One of the advantages of RH-PAT is that RH-PAT might cancel the effect of systemic autonomic nervous tone by normalizing with contralateral PAT.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2020 May 29;15(5):e0233484. doi: 10.1371/journal.pone.0233484.r002

Author response to Decision Letter 0

1 Apr 2020

Responses to the Reviewers’ Comments

5. Review Comments to the Author

Comments

Comment 1: The specific purpose of the study is vaguely stated. In addition, conclusion is also vaguely stated. The present study lacks a real atherosclerotic group with endothelial function (e.g., hypertensive patients, diabetes mellitus, dyslipidemia, and cardiovascular disease).

Response: Thank you very much for your suggestions.

The purposes of the present study were to assess the effects of H2 on endothelial function before morbidity and to provide the primary data of healthy people before applying H2 to a group with diseases related to endothelial dysfunction including atherosclerosis. Our study aims have been clearly stated in the Abstract section (page 2, lines 23-24) and Introduction section (page 5, lines 63-67; page 6, lines 79-83). The EndPad examination itself is the method used to prevent atherosclerosis before the subjects are involved in the group that already developed the diseases.

Therefore, the aims of the study were to investigate the effects of H2 on the endothelium of healthy populations and to determine whether H2 has beneficial effects in improving endothelial function before atherosclerosis and cardiovascular diseases develop. This is a basic study focusing on the primary effects of H2 on endothelial function of a healthy group, not people with morbidities, as noted in the text.

Page 2, lines 23-24_

“In this study, we aimed to investigate the effect of H2 on RH-PAT of the small arteries of fingers in healthy people.”

Page 5, lines 63-67

“It is important to not only estimate the function of the endothelium of both conduit arteries and peripheral arteries in healthy people using these non-invasive methodologies, but to also identify the factors or methodologies that can improve endothelial function and prevent endothelial dysfunction in healthy people without administering pharmaceutical drugs.”

Page 6, lines 76-80

“Besides these disease-conditions, it is important to identify safe and conventional ways for achieving relatively high (healthy) RHIs in the small vessels of healthy individuals.”

Additionally, one of the factors that made the Discussion section of our manuscript too long is the limitation of the insight where the effects of H2 is originated only for the scavenger property against hydroxyl radical. Many researchers and clinicians in this field are pointing to the limitations of the scavenger theory. Our study’s result cannot be explained by the diminishment of hydroxyl radicals and/or peroxynitrite by H2.

Recently, we proposed new mechanistic insights of H2 aside from the scavenger theory, and the first evidence to explain the insights was accepted at the end of 2019 after we submitted this manuscript and published in February 2020 [35]. We have shortened the Discussion and restructured it to make it concise and clear by adding explanations to the mechanisms by which H2 could improve the endothelial functions aside from the scavenger theory (page 22, line 311 to page 23, line 334).

Comment 2: A number of studies in vitro reported that hydrogen directly works several types of cells through the various mechanisms. However, it is doubt that dissolved hydrogen in hydrogen water is taken in the vasculature and the cell after drinking hydrogen water in humans.

Response: It has been clarified and agreed in this field that H2 is transferred into the human body and circulated from the ingested water containing H2. In cases where H2 was dissolved in water (15 ml/kg of 0.8-mM H2 water was administered to rats), H2 effectively alleviated nephrotoxicity caused by cisplatin at a concentration of 5-6 �M of H2 in blood [22]. In a clinical report about the concentration of H2 in blood 30 minutes after ingesting water containing H2, we also clarified that presence of hydrogen gas during exhalation from the lung of the person who digested the high H2 water can be explained by the rapid and passive diffusion of H2 in the aqueous human body including circulation [21].

We have added this explanation to the Methods section (page 9, line 140 to page 10, line 148).

[22] Nakashima-Kamimura N, Mori T, Ohsawa I, Asoh S, Ohta S. Molecular hydrogen alleviates nephrotoxicity induced by an anti-cancer drug cisplatin without compromising anti-tumor activity in mice. Cancer Chemother Pharmacol. 2009;64: 753-761.

Comment 3: Subjects enrolled in this study were relatively young and healthy subjects. However, values of RHI were markedly small (e.g., 0.61 in the placebo group and 0.63 in the hydrogen water group). In addition, the authors stated that the border value of 0.71 seems to be close to that of the healthy masses (page 25, lines 6 and 7). This interpretation is clearly wrong. Previous studies including guidelines for management of endothelial function have clearly shown that normal range of RHI (healthy endothelial function) is ≥2.10, borderline is 1.67 to 2.10, and abnormal is <1.67.

Response: The value you noted is not the natural logarithmic scaled RHI (Ln_RHI). Because RHI values are not normally distributed, RHI here is Ln_RHI, as described in the Methods section, [RHI=Ln{[RH-PAT ratio]×[0.226×Ln (baseline)–0.2]}[2,3, 20]. Therefore, most of the clinical trials using RHI including the large-scale clinicals trials usually adopt Ln_RHI of RHI, and some of them express the Ln_RHI simply as RHI.

We have added “and we used the natural logarithmic scaled Ln_RHI” in the Methods (page 8, lines 115-117) and changed “RHI” to “Ln_RHI” in the Abstract (page 2, lines 27, 30, 34, 35), Methods (page 10, lines 151, 152, 153, 157), Results (page 13, lines 182, 185, 195, 196; page 14, lines 199, 201, 203, 206; page 15, lines 211, 213; page 16, lines 215, 217, 219, 221, 223, 225, 227; page 17, lines 229, 231; page 18, line 234), and Discussion sections (page 18, lines 241, 243, 245, 247, 248, 249; page 19, lines 252, 258, 260, 261; page 20, lines 267, 269, 271; page 22, line 310)

Comment 4: Unfortunately, there are no mechanisms by which hydrogen water significantly increased RHI in healthy subjects. As the authors stated in the Discussion section, measurements of oxidative stress and inflammatory markers would draw more specific conclusions concerning the role of dissolved hydrogen in hydrogen water on vascular function.

Response: Recently, we proposed the new mechanistic insights of H2 aside from the scavenger theory, and the first evidence to explain the insights were accepted at the end of 2019 after we submitted this manuscript. We have shortened the Discussion and restructured it to make it concise and clear by adding the explanations for the mechanisms by which H2 can improve endothelial functions aside from the scavenger theory, as follows (page 22, line 311 to page 23, line 334).

“It should be noted that measuring RH of the peripheral microcirculation in which occlusion of the brachial artery following the release of blood flow is known as the model of ischemia reperfusion (I/R) injury, although the function of the vasodilation of the microvasculature is not fully understood [31]. The mechanisms of the cellular damage by I/R injury are believed to be caused by the superoxide burst in the mitochondrial respiratory chain. Particularly, the reverse electron transport accompanied by the elevated mitochondrial membrane potential is the major factor of electron leakage in mitochondrial complex I [32]. Recently, we have reported that H2 reduces the mitochondrial membrane potential in the living cultured cells [33]. In the study, the overproduction of superoxide with the high ratio of NADH/NAD+, which reflects the highly reduced state of mitochondria during hypoxia following I/R injury [34], was suppressed by H2. This situation corresponds to the right circle in Figure 4. On the basis of these most recent mechanistic insights of H2 [33, 35], we suppose that H2 may improve endothelial function via the mitochondrial mechanisms in healthy subjects. H2 may shift the circle in Figure 4 from right to left by reducing the elevated mitochondrial membrane potential as well as the overproduction of superoxide under the condition with hypoxia presented in the right circle.

Thus far, the limitations to explain the mechanisms of the H2 effect on a wide range of diseases or pathological states based on the scavenging property of H2 with respect to highly reactive radicals have been discussed [36, 37]. In blood flow, the availability of H2 and/or the frequency with which it reacts with target radicals seems insufficient to explain the distinct biological effects of H2 in the peripheral vasculature. In the present study, the continuous scavenging of deleterious radicals by H2 is excluded by the single intake of high H2 water in a day. Therefore, we consider that the effects of H2 herein are occur via the mitochondrial mechanisms described in Figure 4 [32-35].”

Comment 5: It is well known that blood pressure, heart rate, glucose metabolism, and lipid profiles) other than BMI also are confounding factors for endothelial function. These parameters should be presented in Tables.

Response: The alternations of blood pressure and heart rates have been added to Table 1.

We have added the comment below as a study limitation at the end of the Discussion section (page 24, lines 336-340).

As for glucose metabolism and lipid profiles, which are confounding factors for endothelial function, we did not collect blood samples, and our study protocol did not include the blood test.

Comment 6: Introduction section and the Discussion section are too long. They should be significantly shortened as they contain a number of issues not directly related to the message of this study.

Response: Thank you for your suggestion. We have deleted unnecessary sentences and restructured the Introduction section.

Introduction, page 5, line 71 to page 6, line 78

“In some interventional studies, attempts to improve the reactive hyperemia index (RHI) without using a pharmaceutical approach, but instead focusing on exercise therapy and/or lifestyle improvement, have been reported in patients suffering from heart failure and metabolic syndrome [15-18]. Improvements in RHI were also reported when postprandial hyperglycemia was treated pharmaceutically [19]. Besides these disease conditions, it is important to identify safe and conventional ways for achieving relatively high (healthy) RHIs in the small vessels of healthy individuals.”

Deleted sentences

“Although the evidence regarding the use of RH-PAT is lacking, the results obtained and reported after using this relatively new method are similar to those obtained for FMD, and the convenience and/or reproducibility of the methodologies are more universal in RH-PAT compared to that in FMD”

“This type of evaluation can elucidate the prognostic value of a new therapeutic modality, as well as determine the strategies for preventing any deleterious events associated with the use of this method.”

“So far, we have discussed the potential that H2 can react and dissociate to donate electrons to the highly active radical molecules such as �OH and ONOO-. According to guidelines on the safety of ingesting H2, therapeutic applications of H2 including clinical approaches have been emerging in the past decade.”

“Apart from the redox-related signalling including NO and the surrounding radical molecules such as H2O2, EDHF is attributed to electrochemical reactions by hyperpolarization of the endothelial cells first. The changes in the membrane potential of the endothelial cells can reach -85 〜 -80 mv, which is 10 〜 30 mv below the basal membrane potential of arteries and arterioles. This hyperpolarization is dependent on the activation of both endothelial Ca2+-sensitive K+-channels, the small KCa2.3 and KCa3.1. The efflux of K+ affecting the contiguous smooth muscle cells connected to the gap junctions is the primary factor in the mechanism of vasodilation of relatively small arteries. Because H2 is a stable molecule requiring 436 kJ/mol to cleave the covalent bond, and this property simultaneously permits the safety of H2 in our body, it is unlikely that the chemical reactions of H2 are directly involved in the mechanisms of EDHF.”

“In this study, healthy meant that no medications were required and/or not likely to become ill. In this study, however, quite a few subjects had RHI values below 0.71 despite their thinking that they were healthy (22 subjects in the placebo group and 19 subjects in the high H2 group).”

“…probably because of the single overload of the volume on the peripheral vasculature in the condition of low RH. The effects of the intake of 500 ml of water within 10 min is thought to have caused the increase in the volume of blood flow and affected the microcirculation independent of the presence of H2 or N2 in the water for individuals not accustomed to drinking such types of water. The RH-PAT in the small vessels may be influenced by volume loading more than by the diameter of the brachial artery as measured in FMD test. Furthermore, such loading of volume during the test may strain the sympathetic nerve and affect the PAT data. Within the high H2 group, an improvement was seen at 1 h after the drink in addition to at 24 h and 2 weeks after ingestion of the drink. This is consistent with the results we previously reported using FMD, where an H2-specific improvement was observed within 1 h after the consumption of 3.5 mg of H2. Currently, however, we could not assure the immediate efficacy of H2 on the peripheral artery, because of the presence of the placebo effect at 1 h in the low RHI group. Regarding the effect of H2 on RHI at 24 h, there was significant improvement from the RHI before consumption only within the H2 groups (Table 4), whereas in the low RHI groups, the difference from the value before ingestion was improved in the both of the placebo and H2 group. Further investigations about the immediate effects of a single ingestion of H2 within 24 h are necessary.”

Comment 7: Figure 4 is too speculative and is not needed.

Response: According to your suggestion, we have revised Figure 4 and deleting the schema below the circles. The remaining circles summarize the previous publications that are necessary for understanding our data here with the mitochondrial membrane potential, which is affected by H2, particularly for the researchers working on H2 medicine outside of the field of vasculature. We have also revised and shortened the Discussion section with regard to Figure 4, as described above (page 21, line 286 to page 23 line 334).

Comment 1: The data are not fully available. This is not in line with the journal's guidelines. More importantly, this does not allow the reviewer to check for the correctness and appropriatness of the methods. Please, add the dataset as a supplementary file.

Response: Thank you very much for your suggestions. We have added the dataset as Supplementary file S3.

Comment 2: The use of mixed effects models is sound. Linear mixed model could be an interesting option, but several aspects deserve more care. The model must fulfill some assumptions to ensure the reliability of the results. Those assumptions are completely overlooked. A complete analysis of the residuals must be included and tests (e.g log-likelihood ratio) should be included to check for the appropriateness of the random effects model. Moreover, some info on the random terms must be included (e.g. the variance of the random effects) and, as I am guessing that Gaussian distribution is taken from granted, if the assumptions on the random terms are also fulfilled.

Response: In the mixed effect models for repeated measures (MMRM), only correlation structure between outcome variables between time points is needed to be modeled explicitly [23]. Accordingly, the required assumption in this analysis is whether the correlation structure is correct. In this case, our analysis was unstructured; that is, we did not assume any structural assumptions for the correlation structure. MMRM is widely used for continuous outcome in longitudinal trials. We have described MMRM in the statistical analysis section (page 10, lines 153-155, 157-159).

[23] Mallinckrodt CH, Clark WS, David SR. Accounting for dropout bias using mixed-effects models. J Biopharm Stat. 2001;11: 9-21.

Comment 3: Please, provide inference on the baseline characteristics as well, bearing in mind that any parametric statistical test is based on some assumptions to be fulfilled.

Response: Statistical tests for baseline characteristics are not meaningful because these statistical strategies are not of interest in this trial, and a significance difference will occur with a probability of 0.05 [ref]. Therefore, we should interpret the difference based on a clinical viewpoint. In this study, there was no clinically meaningful difference in baseline characteristics between groups. In order to provide clarification, we have changed “The RHI value of the subjects was 0.61 ± 0.26 in the placebo group and 0.63 ± 0.29 in the high H2 group, and there was no significant difference.” to “Additionally, there was no significant difference in the RHI value of subjects between the groups (placebo group: 0.61±0.26, high H2 group: 0.63±0.29). We considered that the subjects in these two groups had physiologically similar characteristics in terms of endothelial function.” (page 11, lines 173-176).

[ref] Pocock SJ, Assmann SE, Enos LE, Kasten LE. Subgroup analysis, covariate adjustment and baseline comparisons in clinical trial reporting: current practice and problems. Stat Med. 2002;21: 2917-2130.

Comment 4: The way results are presented are somehow confusing to me. I would expect to see regression parameters, in which the placebo is taken as reference category. I do not really get how bot Placebo and Treatment can be included in a single model (as p-values are reported for both). Moreover, the term LS is not explained in the text. Please, report the results in a more common way, focusing on regression parameters and heterogeneity due to the longitudinal stracture in the data. Something should be said on the interaction effect as well.

Response: We have revised Tables 3-6 and summarized the data in Tables 2 and 3 for simplicity. The MMRM included the treatment group, time points, and interaction between the treatment group and the time points as fixed effects, with the RHI at baseline as a covariate. Therefore, we were able to estimate the covariate-adjusted mean and mean difference by estimated correlation coefficients. Additionally, the main purpose of this trial was to compare the adjusted mean between the groups, so we did not show many estimated parameters. LS is the covariate-adjusted mean, and we have revised the following sentence in the Results section to provide clarification (page 11, lines 173-176).

“Additionally, there was no significant difference in the RHI value of subjects between the groups (placebo group: 0.61±0.26, high H2 group: 0.63±0.29). We considered that the subjects in these two groups had physiologically similar characteristics in terms of endothelial function.”

Major comment

Comment 1: In general, the manuscript and too long and rambling. In Results, the same data were repeatedly shown in several figures and tables. This manuscript should be restyled with a more concise and clear way.

Response: Thank you very much for your suggestions. One of the factors that made the Discussion section of our manuscript too long is the limitation of the insight where the effects of H2 are originated from only the scavenger property against hydroxyl radical. Many researchers and clinicians in this field are pointing out the limitations of the scavenger theory. Our study’s results cannot be explained by the diminishment of hydroxyl radicals and/or peroxynitrite by H2.

Recently, we proposed new mechanistic insights of H2 aside from the scavenger theory, and the first evidence to explain the insights were accepted at the end of 2019 after we submitted this manuscript and published in February 2020 [35]. We have shortened the Discussion and restructured it to make it concise and clear by adding explanations for the mechanisms by which H2 can improve endothelial functions aside from the scavenger theory.

Further, we have deleted unnecessary sentences and revised text in the Introduction section.

Introduction: page 5, line 71 to page 6, line 78

“In some interventional studies, attempts to improve the reactive hyperemia index (RHI) without using a pharmaceutical approach, but instead focusing on exercise therapy and/or lifestyle improvement, have been reported in patients suffering from heart failure and metabolic syndrome. Improvements in RHI were also reported when postprandial hyperglycemia was treated pharmaceutically. Besides these disease conditions, it is important to identify safe and conventional ways for achieving relatively high (healthy) RHIs in the small vessels of healthy individuals.”

Deleted sentences

We have also restructured the Results section to shorten it and focus on the RHI values. We have revised Table 1 to clearly present the basic data of the subjects by combining the baseline data in Table 2, and we renumbered the tables. Data in Tables 2-5 include the same data of Figures 2 and 3, as you pointed out. We deleted the column for “Observed Value” from Tables 2-5. Although we considered whether Table 2-5 were essential, we considered that they are necessary for the statistical considerations that reviewer #2 requested. We have combined the data in Tables 2 and 3 into Table 2, and combined the data in Tables 4 and 5 into Table 3 in order to simplify the presentation for the statistical data.

We have deleted the following sentences from the Discussion section in order to shorten and restructure it.

“Apart from the redox-related signaling including NO and the surrounding radical molecules such as H2O2, EDHF is attributed to electrochemical reactions by hyperpolarization of the endothelial cells first. The changes in the membrane potential of the endothelial cells can reach -85 ~ -80 mv, which is 10 ~ 30 mv below the basal membrane potential of arteries and arterioles. This hyperpolarization is dependent on the activation of both endothelial Ca2+-sensitive K+-channels, the small KCa2.3 and KCa3.1. The efflux of K+ affecting the contiguous smooth muscle cells connected to the gap junctions is the primary factor in the mechanism of vasodilation of relatively small arteries. Because H2 is a stable molecule requiring 436 kJ/mol to cleave the covalent bond, and this property simultaneously permits the safety of H2 in our body, it is unlikely that the chemical reactions of H2 are directly involved in the mechanisms of EDHF.”

“The RH-PAT in the small vessels may be influenced by volume loading more than by the diameter of the brachial artery as measured in FMD test. Furthermore, such loading of volume during the test may strain the sympathetic nerve and affect the PAT data. Within the high H2 group, an improvement was seen at 1 h after the drink in addition to at 24 h and 2 weeks after ingestion of the drink. This is consistent with the results we previously reported using FMD, where an H2-specific improvement was observed within 1 h after the consumption of 3.5 mg of H2. Currently, however, we could not assure the immediate efficacy of H2 on the peripheral artery, because of the presence of the placebo effect at 1 h in the low RHI group. Regarding the effect of H2 on RHI at 24 h, there was significant improvement from the RHI before consumption only within the H2 groups (Table 4), whereas in the low RHI groups, the difference from the value before ingestion was improved in the both of the placebo and H2 group. Further investigations about the immediate effects of a single ingestion of H2 within 24 h are necessary.”

Minor comments

Introduction

Comment 2: The introduction is too long, and describes too much for the comparison between FMD and EndoPAT.

Response: We have deleted sentences from the Introduction section to shorten and restructure it, as noted above in our response to your comment 1.

We have added the following sentences in the Discussion section (page 22, line 311 to page 23, line 334), according to the recent finding about the new properties of H2.

“It should be noted that measuring RH of the peripheral microcirculation in which occlusion of the brachial artery following the release of blood flow is known as the model of ischemia reperfusion (I/R) injury, although the function of the vasodilation of the microvasculature is not fully understood [29]. The mechanisms of the cellular damage by I/R injury are believed to be caused by the superoxide burst in the mitochondrial respiratory chain. Particularly, the reverse electron transport accompanied by the elevated mitochondrial membrane potential is the major factor of electron leakage in mitochondrial complex I [30]. Recently, we have reported that H2 reduces the mitochondrial membrane potential in the living cultured cells [31]. In the study, the overproduction of superoxide with the high ratio of NADH/NAD+, which reflects the highly reduced state of mitochondria during hypoxia following I/R injury [32], was suppressed by H2. This situation corresponds to the right circle in Figure 4. On the basis of these most recent mechanistic insights of H2 [31, 33], we suppose that H2 may improve endothelial function via the mitochondrial mechanisms in healthy subjects. H2 may shift the circle in Figure 4 from right to left by reducing the elevated mitochondrial membrane potential as well as the overproduction of superoxide under the condition with hypoxia presented in the right circle.

Thus far, the limitations to explain the mechanisms of the H2 effect on a wide range of diseases or pathological states based on the scavenging property of H2 with respect to highly reactive radicals have been discussed [34, 35]. In blood flow, the availability of H2 and/or the frequency with which it reacts with target radicals seems insufficient to explain the distinct biological effects of H2 in the peripheral vasculature. In the present study, the continuous scavenging of deleterious radicals by H2 is excluded by the single intake of high H2 water in a day. Therefore, we consider that the effects of H2 herein are occur via the mitochondrial mechanisms described in Figure 4 [30-33].”

Comment 3: In the introduction (line 85), the authors stated: “the nature of the rosy faces often observed….”. Do the authors have a reference for this?

Response: No, it is just a consensus among the clinicians treating H2; we do not have a reference to support this statement. We have deleted it.

Methods

Comment 4: In line 121, “both 2d fingers” should read “both index fingers”.

Response: Thank you very much for your advice. We have revised the text accordingly (page 7, line 105).

Comment 5: In this paper, RH-PAT was performed in the afternoon. The authors should describe the breakfast and other oral intakes like caffeine.

Response: We have described the 6 h fasting as follows (page 7, lines 95-96): “All subjects were asked to fast and avoid drinking caffeinated drinks or ingesting sugar for 6 h before the test.” We have also described the breakfast (page 7, line 96): “All subjects were asked to fast and avoid drinking caffeinated drinks or ingesting sugar as well as breakfast for 6 h before the test.”

Comment 6: The authors should describe the statistical analysis more precisely.

Response: We responded to the queries and requests of reviewer #2 about the statistical analysis, and we described MMRM (page 10, lines 153-155, 157-159).

Results

Comment 7: The authors divided subjects into low and high RHI groups with a cut-off of 0.71. They should explain how they conduced 0.71 as a cut-off.

Response: We explained the cut-off in the Discussion section, and we have revised the sentence and added a comment in the Results section.

Results: page 16, lines 215-217

“In order to identify the influence of the RHI value before ingestion on the data at each time point, we divided the subjects into two groups with the cut-off value of 0.71 (see Discussion section).”

Discussion: page 18, line 247 to page 19, line 259

“Although not intended for healthy subjects, there are several investigations on the prognostic value of RHI for predicting cardiovascular risks. The RHI values reported were 0.531 in 577 patients at high risk for cardiovascular events by Matsuzawa et al [6] and 0.4 in 270 low-risk outpatients with chest pain by Rubinshtein et al [7]. It has been also reported that the risk of arrhythmia recurrence could be estimated by an RHI value of 0.618 in 92 participants with atrial fibrillation undergoing catheter ablation [23]. Orthopedic patients who underwent total hip or knee arthroplasty and avoided postoperative deep vein thrombosis (DVT) had preoperative ln-RHI values of 0.71 ± 0.25 [24]. Among these reports, the border value of 0.71 for the orthopedic patients avoiding DVT seems to be close to that of the healthy masses. To predict future endothelial health in healthy subjects in this study, it seemed to be appropriate to set the threshold for RHI above the values for patients with cardiovascular risks. We set the RHI value at 0.71 in order to divide the subjects into two groups.”

Discussion

Comment 8: In line 295-297, the authors stated: “The RH-PAT in the small vessels may be influenced by volume loading…”. Please show some references.

Response: Thank you for pointing out this reference. Because the comments concerning the placebo effects were based on our speculative opinion, we searched for appropriate references. Unfortunately, we could not find a reference that clearly explained the relationship between hyperemia of peripheral circulation and intake of 500 ml of water within 1 h. Therefore, we deleted the explanation of the placebo effect attributing to overload of the volume on the peripheral vasculature in the condition of low RH. Instead, we stated (page 19, line 266 to page 20, line 268), “…although it is currently unclear whether it is merely the placebo effect or there is another factor that causes the improvement of RHI within 1 h after the placebo group drinks the water containing N2.”

Comment 9: In line 296-297, the authors said that loading of volume may strain the sympathetic nerve and affect PAT data. One of the advantages of RH-PAT is that RH-PAT might cancel the effect of systemic autonomic nervous tone by normalizing with contralateral PAT.

Response: Thank you very much for pointing this out. This comment is in contradiction with the theory of the PAT test so we deleted it. We also deleted the unnecessary sentence explaining the effects at 1 h compared with the results of the FMD test.

________________________________________

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PLoS One. doi: 10.1371/journal.pone.0233484.r003

Decision Letter 1

Giuseppe Andò

17 Apr 2020

PONE-D-19-26280R1

Peripheral endothelial function can be improved by daily consumption of water containing over 7 ppm of dissolved hydrogen: A randomized controlled trial

PLOS ONE

Dear Dr. Ishibashi,

Statistical reviewer still asks to verify assumption of the modelling while another reviewer still underscores the lack of a control group.

We would appreciate receiving your revised manuscript by Jun 01 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Giuseppe Andò, M.D., Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: The present study lacks a real atherosclerotic group with endothelial function (e.g., hypertensive patients, diabetes mellitus, dyslipidemia, and cardiovascular disease).

A number of studies in vitro reported that hydrogen directly works several types of cells through the various mechanisms. However, it is doubt that dissolved hydrogen in hydrogen water is taken in the vasculature and the cell after drinking hydrogen water in humans.

Reviewer #2: I appreciate most the reply given by the authors to my comments.

There are still some points deserving a better clarification.

Previously, I commented that "A complete analysis of the residuals must be included and

tests (e.g log-likelihood ratio) should be included to check for the appropriateness of

the random effects model. Moreover, some info on the random terms must be included

(e.g. the variance of the random effects) and, as I am guessing that Gaussian

distribution is taken from granted, if the assumptions on the random terms are also

fulfilled.". The authors reply is not satisfactory. The unstructured specification mentioned in their reply refers to the variance-covariance matrix of the random effects. This is quite a flexible assumption and I am fine with it.

The issue is that the random effects follow some distribution. Again I guess it is the Gaussian one, but this must be checked somehow, as departure from the Gaussian assumption may lead to biased estimates. Moreover, as the author replied, random effects modelling is widely used for continuous outcome in longitudinal trials. This is absolutely true. But continuous outcomes may be distributed according to hundreds of different distributions. Again, the most used one is the Gaussian, i.e. the error term in the linear predictor is assumed to follow a zero-mean Gaussian distribution. This is a crucial assumption of the modelling. If heavy tails are evident, i.e. if the error term follows a t distribution instead, regression parameters and all the statistical inference are biased. This is the reason why the analysis of residuals must be provided, because the linear mixed model is based on some assumptions. If those assumptions are not fulfilled, the results are unreliable. Citing a paper in the J. Bioph. Stat is not a good choice, as dozens of statistical books exist (just to mention one https://www.routledge.com/Longitudinal-Data-Analysis/Fitzmaurice-Davidian-Verbeke-Molenberghs/p/book/9781584886587) and discuss longitudinal data analysis, mentioning how to perform a proper data analysis.

Reviewer #3: The authors correctly addressed all the points raised by reviewers, and the revised manuscript is much improved.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

PLoS One. 2020 May 29;15(5):e0233484. doi: 10.1371/journal.pone.0233484.r004

Author response to Decision Letter 1

4 May 2020

Peripheral endothelial function can be improved by daily consumption of water containing over 7 ppm of dissolved hydrogen: A randomized controlled trial (Manuscript: PONE-D-19-26280R1)

Response to Reviewer #1

Thank you for reviewing our manuscript and for your insightful comments. We have responded to each of your comments below, and have revised the manuscript accordingly.

Comment 1: The present study lacks a real atherosclerotic group with endothelial function (e.g., hypertensive patients, diabetes mellitus, dyslipidemia, and cardiovascular disease).

Response: Thank you for your further suggestions.

In the former round of revision, we emphasized the aim and goal of the present study according to your previous suggestion in the Abstract section:

“In this study, we aimed to investigate the effect of H2 on RH-PAT of the small arteries of fingers in healthy people.”

…and in the Introduction section:

“Besides these disease-conditions, it is important to identify safe and conventional ways for achieving relatively high (healthy) RHIs in the small vessels of healthy individuals.”

Furthermore, we added the comments in the Study limitations as follows (page 27, lines 342-347):

“As the aim of this study was to investigate the effect of H2 on RH-PAT intended for healthy subjects to assess the significance of daily consumption of H2 to prevent the endothelial dysfunction of small arteries, data for an atherosclerotic group with a low RHI value are lacking. It is important to examine the effect of H2 on RH-PAT of people having a high risk of hypertension, cardiovascular diseases, diabetes mellitus and dyslipidemia due to atherosclerosis. The next study should include participants with these characteristics.”

Response: As we explained in the Methods section (page 9, lines 138-140), it has been clarified and agreed in this field that H2 is transferred into the human body and the hydrogen is absorbed in vasculature and circulated from ingested water containing H2. (reference [22]).

Moreover, we added reference [23] and revised the sentence as follows (pages 9-10, lines 140-142):

“In cases where rats were administered 15 mL/kg of 0.8 mM H2 water by a catheter or mice drank approximately 200mL/kg of 0.2–0.4 mM H2 water per day, the concentration of H2 in their blood was 5–6 �M and about 2 �M, respectively [22, 23].”

Response to Reviewer #2

Thank you for reviewing our manuscript and for your insightful comments. We have responded to each of your comments below, and have revised the manuscript accordingly.

Comment: I appreciate most the reply given by the authors to my comments.

There are still some points deserving a better clarification.

Previously, I commented that "A complete analysis of the residuals must be included and tests (e.g log-likelihood ratio) should be included to check for the appropriateness of the random effects model. Moreover, some info on the random terms must be included (e.g. the variance of the random effects) and, as I am guessing that Gaussian distribution is taken from granted, if the assumptions on the random terms are also fulfilled.". The authors reply is not satisfactory. The unstructured specification mentioned in their reply refers to the variance-covariance matrix of the random effects. This is quite a flexible assumption and I am fine with it. The issue is that the random effects follow some distribution. Again I guess it is the Gaussian one, but this must be checked somehow, as departure from the Gaussian assumption may lead to biased estimates. Moreover, as the author replied, random effects modelling is widely used for continuous outcome in longitudinal trials. This is absolutely true. But continuous outcomes may be distributed according to hundreds of different distributions. Again, the most used one is the Gaussian, i.e. the error term in the linear predictor is assumed to follow a zero-mean Gaussian distribution. This is a crucial assumption of the modelling. If heavy tails are evident, i.e. if the error term follows a t distribution instead, regression parameters and all the statistical inference are biased. This is the reason why the analysis of residuals must be provided, because the linear mixed model is based on some assumptions. If those assumptions are not fulfilled, the results are unreliable. Citing a paper in the J. Bioph. Stat is not a good choice, as dozens of statistical books exist (just to mention one https://www.routledge.com/Longitudinal-Data-Analysis/Fitzmaurice-Davidian-Verbeke-Molenberghs/p/book/9781584886587) and discuss longitudinal data analysis, mentioning how to perform a proper data analysis.

Response: Thank you very much for your detailed advice. As you guessed, we assumed a Gaussian distribution for the error term in MMRM. I checked distribution of the residuals in the MMRM model for Table 2 and Table 3. Please see attached figure below. The distribution appears to be normal/ Gaussian (upper right) and the points approximately fall on the line in QQ-plot (lower left). In addition, we performed a log-likelihood ratio test in order to check the fit of the MMRM model to the data and its p-values were 0.0483 and 0.3597 for Table 2 and Table 3, respectively. The p-value of Table 3 (0.3597) is relatively large because of the small sample size. We think that the MMRM model is appropriate for the data. We have added a related sentence to the Statistical analysis section (pages 10-11, lines 157-159, and footnotes to Table 2 (page 17, line 215) and Table 3 (pages 20, line 237) as follows:

(Page 10-11, lines 157-159)

“Furthermore, we assumed a Gaussian error term in the model. We performed a log-likelihood ratio test and residual analysis in order to check the fit of the model.”

(Page 17, line 215)

“p-value of log-likelihood test: 0.0483”

(Page 20, line 237)

“p-value of log-likelihood test: 0.3597”

Furthermore, thank you for providing us with an appropriate reference. We replaced reference [24] according to your suggestion as follows:

[24] Garrett Fitzmaurice, Marie D, Geert V, Geert M. (2008). Longitudinal data analysis. Chapman and Hall/CRC.

Residual analysis for Table 1

Residual analysis for Table 2 (Figure in uploaded file)

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(78.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0233484.r005

Decision Letter 2

Giuseppe Andò

7 May 2020

Peripheral endothelial function can be improved by daily consumption of water containing over 7 ppm of dissolved hydrogen: A randomized controlled trial

PONE-D-19-26280R2

Dear Dr. Ishibashi,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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Giuseppe Andò, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0233484.r006

Acceptance letter

Giuseppe Andò

20 May 2020

PONE-D-19-26280R2

Peripheral endothelial function can be improved by daily consumption of water containing over 7 ppm of dissolved hydrogen: A randomized controlled trial

Dear Dr. Ishibashi:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Checklist. CONSORT 2010 checklist of information to include when reporting a randomised trial*.

(DOC)

Click here for additional data file.^{(218.5KB, doc)}

S1 File. Trial protocol.

(DOCX)

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S2 File

(DOCX)

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S1 Data. Set of the present study.

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Data Availability Statement

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PERMALINK

Peripheral endothelial function can be improved by daily consumption of water containing over 7 ppm of dissolved hydrogen: A randomized controlled trial

Toru Ishibashi

Kosuke Kawamoto

Kasumi Matsuno

Genki Ishihara

Takamichi Baba

Nobuaki Komori

Roles

Abstract

Background

Methods

Results

Conclusions

Introduction

Methods

Subjects and the measurements of RHI

Fig 1. CONSORT flow diagram.

Preparation of high H2 water and placebo water

Statistical analysis

Results

Table 1. Characteristics of the enrolled volunteers.

Fig 2. Ln_RHI values for all of the subjects.

Table 2. Comparison between the two groups for all of the subjects.

Fig 3. Ln_RHI values for the low Ln_RHI group (Ln_RHI <0.7).

Table 3. Comparison between the two groups with the low Ln_RHI (Ln_RHI <0.71).

Discussion

Fig 4. Schematic representation of the possible action of H2 for the improvement of RHI.

Study limitations

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Giuseppe Andò

Roles

Author response to Decision Letter 0

Decision Letter 1

Giuseppe Andò

Roles

Author response to Decision Letter 1

Decision Letter 2

Giuseppe Andò

Roles

Acceptance letter

Giuseppe Andò

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Preparation of high H₂ water and placebo water

Fig 4. Schematic representation of the possible action of H₂ for the improvement of RHI.