# Austin Air purifiers – clinical trials

The Austin Air Bedroom Machine® air purifier was used in clinical trials at the University of John Hopkins to reduce Nitrogen Dioxide levels in the homes of local residents. Nitrogen Dioxide (NO2) is a gas often present in homes where there are gas stoves. High levels of NO2 are particularly dangerous for people with COPD. The gas can dramatically increase symptoms of coughing, wheezing and chest tightness.
The results were dramatics. NO2 dropped in the kitchen by 27% and continued to stay low for the length of the study. Within a week NO2 levels in the bedroom dropped by 23%.

In a second study at John Hopkins, the Austin Air Bedroom Machine® air purifier was placed in the homes of women, living with a smoker, who were either pregnant or with very young babies. Again the results were impressive. It was clinically proven that running an Austin Air Bedroom Machine® air purifier will effectively remove indoor pollutants from the home, including nicotine.

The Austin Air Allergy Machine® was selected for use in a clinical trial at the Cincinnati Children’s Hospital Medical Centre. The team monitored a group of children with asthma who were regularly exposed to second hand smoke. The aim of the study was to determine how the use of our air cleaners could help to reduce the number of times the children went to hospital. The results were clear, running one of our air purifiers in the children’s bedrooms while they slept, reduced their hospital visits by nearly 20%.

Read the clinical trials using Austin Air purifiers  outcomes:

Johns Hopkins Austin HEPA AIR purifier Second hand smoke 2018

Johns Hopkins HEPA Austin air filter NO2 study.docx

HEPA Austin air cleaners pedriatic study- asthma symptoms children exposed to second hand smoking summary

Johns Hopkins Inhale #1 In Home Particle Levels and Asthma Morbidity

## Order the only Clinically Proven, Medical Grade Air Purifiers-made in USA.

###### In 2002 following the Battelle Labs tests, Austin Air is selected by the U. S. government for the largest deployment of air purifiers in the world in order to protect residents of Anniston, Alabama during the elimination of chemical weapons

Nearly 3 decades ago, Richard Taylor founded the company Austin Air Systems. Introducing medical grade HEPA air purification to residential customers.

He noticed asthma attacks were more frequent at night as hormonal changes take place and lying down causes the airways to narrow. Together these factors increase the chance of a night time asthma attack. Back then, the only advice doctors had to offer was to avoid asthma triggers like bedding and carpeting or take medication. He knew there had to be a better solution.

Clinical studies(see below) had shown that HEPA air filtration, used in hospitals at that time, was effective at removing contaminants from the environment. So he developed the first HEPA air purifier, designed for domestic use.

To this day, it is still the MOST EFFECTIVE AIR PURIFIER ON THE MARKET

Built for family first.

The Austin Air Healthmate was born out of necessity as President, Richard Taylor, undertook the task of improving what many doctors and specialists couldn’t; his wife’s quality of life.

Since his wife, Joyce, was a child, she had difficulty breathing. An endless round of doctors and specialists didn’t make any difference; neither did medications or dietary changes. She suffered from this debilitating condition for years, until Richard realized the obvious. The air Joyce was breathing was contaminated.

Drawing upon technology used in medical facilities, Richard reproduced the only environment where Joyce had relief of her symptoms, “the hospital clean room”. Using True Medical HEPA, surrounded with Activated Carbon he created a filter that addressed all the issues related to environmental particulate contamination and chemical toxicity. Within a week Joyce was finally sleeping undisturbed through the night.

Today, at 480,000 square feet Austin has the largest air cleaner manufacturing facility in the world, producing everything in house from filter, to metal forming, to the final paint.

Austin air is extremely proud that the Healthmate is the air cleaner of choice by leading doctors throughout the United States and many other parts of the world. As a matter of fact, there is hardly a country in the world today, where an Austin Air cleaner is not hard at work cleaning the air in someone’s home.

We’re proud to be on the cutting edge of the air purification industry. Here are a few key dates in our history:

In 1990 Austin Air Systems, Limited is formed in Buffalo, New York. The flagship air purifier, the Austin HealthMate®, is released. Using True Medical Grade HEPA and granular activated carbon for air filter, it is the first domestic air purifier to remove a broad range of contaminants including chemicals, gases and Volatile Organic Compounds (VOC’s).

In 1992 Austin Air is featured in medical and health newsletters including Dr. Whitaker’s Health & Healing. Proven to remove 99.97% of particles larger than 0.3 microns and 95% of particles larger than 0.1 microns.

In 2000 the Austin HealthMate® undergoes extensive testing along with over 100 other air purifiers at Battelle Laboratories, Aberdeen,MD.It is selected as the TOP AIR PURIFIER for chemical and VOC air filtration.

In 2001  After the 911 attack, Austin Air products are recommended by the Red Cross, FEMA and the U.S. Department of Housing and Urban Development (HUD) to combat the negative health effects of ash, chemicals, VOCs and other miscellaneous airborne particulates released into the air after the attack.

In 2002 following the Battelle Labs tests, Austin Air is selected by the U. S. government for the largest deployment of air purifiers in the world in order to protect residents of Anniston, Alabama during the elimination of chemical weapons.

In 2010 Austin Air receives the California Environmental Protection Agency’s Air Resources Board certification.This confirms Austin Air purifiers are well under the standards for safe ozone emissions.

In 2016 The Austin Air HealthMate Plus® is deployed to many thousands of residents of Porter Ranch, California and the surrounding Aliso Canyon area to help protect against the effects of the Southern California natural gas leak.

Year after year, Austin Air purifiers are chosen to filter the contaminants released into the air during the wildfire season. 2017 was a particularly brutal year for residents of California and surrounding states – the wildfire season started very early and continued through the end of the year.

Of course, there are many other important dates and product innovations that we’re very proud of.  We have been developing and manufacturing air cleaners for nearly 30 years. It’s all we do. We understand the science. We understand what our customers need.

Sold in over 100 countries, we have created a line of robust, reliable, American made products that you can trust.

Read the clinical trials using Austin Air purifiers  outcomes

Integrity in Construction

The Air purifier Body
Austin Air produces all of its products(air purifiers and filters) with solid steel construction and non-toxic powder coated paint for strength, reliability and longevity.

The Air Filter
Austin Air incorporates the only trusted air filtering technology used in hospitals and operating rooms. For an air filter to be effective, gases and sub-micron particles must be removed from the air. That’s why you’ll find True Medical Grade HEPA and Activated Carbon in every Austin Air filter.

True HEPA air filter
The True HEPA air filter was developed by the Atomic Energy Commission specifically to protect the Human Respiratory System (nose, mouth, throat, lungs) from radioactive dust particles. Austin Air takes it a step further, incorporating True Medical Grade HEPA with 99.97% efficiency down to 0.3 microns and 95% efficiency down to 0.1 microns. True Medical Grade HEPA is the most efficient particulate air filtering media on the market.

Activated Carbon in the air filter
Activated Carbon is used to remove Volatile Organic Compounds (VOC’s); noxious gases and chemicals, including smoke & smoke odor. Austin Air filters can use up to 15 lbs of Activated carbon in one filter wich is UNmatched on the market

Powder Coated – Not Painted

Austin Air cleaners are powder coated not painted. This gives the products an unsurpassed finish without the harmful off gassing that painted products can produce. In fact you will find that the control knob, wheels and fan are the only plastic components we use. This is great news for the chemically sensitive. We feel that am air purifier that offers relief to allergy and asthma sufferers shouldn’t contribute to the problem.

Designed from the inside out.

The engineers at Austin Air concentrated on developing the most critical component of the air cleaner first; the air filter.

Austin Air’s 360-degree intake system draws air into all sides of the air cleaner, maximizing efficiency and delivering more clean air faster.

Every minute, 250 cubic feet of air is processed by a 4-stage air filter that progressively removes contaminants out of the air.

This process enables Austin Air cleaners to:

• Achieve the highest levels of performance.
• Attain superior air flow rates.
• Sustain a longer air filter life (5-years under normal residential use).
• Increase the life expectantly of the Medical Grade HEPA, the most critical media in the air filtration process.

5 year Warranty
Austin Air has taken the risk out of the buying and stands by their products (air purifiers and filters) with a 5-year warranty on the material and craftsmanship and 5-year pro-rated warranty on the air filter. Customers always have peace of mind when purchasing an Austin Air cleaner

Austin Air has made a conscious decision to keep Americans employed by keeping their manufacturing facility in the United States. We feel that every company has an obligation to carefully examine the social, economic and moral costs of sending jobs offshore. At Austin Air we believe that American workers still build the best products, that corporations can still make a healthy profit manufacturing in the United States and that the benefits of staying in America far outweigh the costs.

When a company chooses to go offshore to manufacture a product, they are motivated by one thing…greed. As a result, our economy suffers through lost jobs and money, and the American consumer is short changed. The benefits of these corporate cost savings are rarely passed on to the consumer and the product is now of a lesser quality. If this were not true, your running shoes would last 5 times longer and would never cost over \$50 a pair. The next time you buy a product made in China, ask yourself, “Why is this costing as much as it did when it was made in the USA and how is this country going to prosper when our own corporations have no loyalty to us?”

# Daily ingestion of alkaline electrolyzed water containing hydrogen influences human health, including gastrointestinal symptoms

## Abstract

In Japan, alkaline electrolyzed water (AEW) apparatus have been approved as a medical device. And for the patients with gastrointestinal symptoms, drinking AEW has been found to be effective in relieving gastrointestinal symptoms. But some users of AEW apparatus do not have abdominal indefinite complaint. Little attention has been given to the benefit for the users which have no abdominal indefinite complaint. The object of this study is to evaluate the effect on health, including gastrointestinal symptoms, when a person without abdominal indefinite complaint, etc., drinks AEW on a daily basis. A double-blind, randomized controlled trial has been designed. Four-week period of everyday water drinking, PW drinking group: drink purified tap water as a placebo, AEW drinking group: drink alkaline electrolyzed water which made by electrolysis of purified tap water. Before the experiment and after the 4-week period of water drinking, Blood tests, physical fitness evaluations, and questionnaire evaluations is conducted. In this study, we did not specifically select patients with gastrointestinal symptoms. Sufficiently clear effect could not be confirmed. But the stools were more normal, and, as shown in the previous report, that drinking AEW is considered to contribute to intestinal normalization. In addition, when drinking AEW, a high proportion of the respondents said that they felt they were able to sleep soundly, and the proportion of subjects who answered that they felt good when awakening increased. The effect of reducing oxidative stress, thus allowing for improved sleep, was exhibited by drinking AEW containing hydrogen, which is considered to be an antioxidant substance. This research were approved by the Ethics Committee of the Osaka City University Graduate School of Medicine (No. 837) and were registered in the University Hospital Medical Information Network (UMIN) Clinical Trials Registry (UMIN ID: UMIN000031800) on March 22, 2018.

Keywords: alkaline electrolyzed water, gastrointestinal symptoms, hydrogen-dissolved water, physical fitness evaluations, questionnaire evaluations, functional beverage

## Introduction

In Japan, water which is obtained on the cathode side by the electrolysis of tap water is called alkaline electrolyzed water (AEW) or reduced hydrogen water. Improvement of gastrointestinal symptoms by ingesting AEW has been confirmed by Japanese researchers. For example, Naito et al. reported the inhibitory effect of AEW ingestion on gastric mucosal disorder caused by aspirin, and Hayakawa et al.reported the inhibitory effect of AEW ingestion on abnormal intestinal fermentation. Tashiro et al.examined the effect of ingesting AEW or purified tap water (PW; as a placebo) at a rate of at 500 mL per day for 4 weeks in patients who had abdominal pain such as heartburn, stomach discomfort, abdominal bloating, diarrhea, constipation, etc., and reported that the results of the AEW group were superior to those of the placebo group., From these results, apparatus that produce AEW have been approved as medical devices by the Japanese Ministry of Health, Labour and Welfare. AEW is thought to be effective for functional gastrointestinal disorders.

Since AEW is produced by electrolyzing water, hydroxide ions, which are alkaline in nature, are generated. Hydrogen molecules are also generated on the electrode surface and dissolved in water. Therefore, AEW is alkaline water containing hydrogen. In conventional efficacy studies, evaluations with respect to ingesting AEW have typically been conducted focusing on the alkalinity of the water.,,, In recent years, however, the assumed effectiveness of the antioxidant effect of dissolved hydrogen on various diseases has been reported.,,,,,,,, Nevertheless, some users of AEW apparatus do not have any definite abdominal symptoms. In many cases, they are drinking AEW on a daily basis to improve their health, and many users also feel health benefits such as improvement in exercise capacity. These may be thought to be due to the action of dissolved hydrogen. There have been no researched studies of these in detail. The object of this study is to evaluate the effect of daily ingestion of AEW on health, including gastrointestinal symptoms, in subjects without any definite abdominal symptoms.

## Participants and Methods

### Participants

Healthy men and women (20–60 years) who use the Osaka City Citizen Health Development Consultation Center were selected as test subjects to determine the health effect of daily AEW ingestion. It was aimed to clarify whether general subjects without gastrointestinal symptoms have another good effect besides gastrointestinal symptoms by drinking AEW which is good for gastrointestinal symptoms. We explained this purpose to the subjects and asked for research participation. Written informed consent was obtained from all subjects. All procedures used in this research were approved by the Ethics Committee of the Osaka City University Graduate School of Medicine (No. 837) and were registered in the University Hospital Medical Information Network (UMIN) Clinical Trials Registry (UMIN ID: UMIN000031800) on March 22, 2018. This study follows the Consolidated Standards of Reporting Trials (CONSORT) guidelines. A double-blind, randomized controlled trial has been designed, and the research design is shown in Figure 1.

Research design.

Note: PW: Purified tap water; AEW: alkaline electrolyzed water.

Subjects were randomly divided into two groups, with an AEW group (n = 30) and a PW group (n = 30). Blood tests, physical fitness evaluations, and questionnaire evaluations were conducted before the experiment was initiated. Subjects were provided with AEW apparatus that had been modified to produce only AEW or PW. They ingested 500 mL or more of freshly produced AEW or PW per day (they were required to ingest 200 mL immediately after awakening, and 300 mL or more during the rest of the day). After the end of the four-week period, blood tests, physical fitness evaluations, and questionnaire evaluations were conducted again to check whether the ingestion of AEW for four weeks had beneficial effects on the health of the subjects.

### Blood sample/urinalysis

General blood test: Red blood cell count, white blood cell count, hemoglobin, hematocrit, and platelet count.

Blood biochemical examination: Total protein, albumin, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), γ-GTP, total cholesterol, high-density lipoprotein (HDL), cholesterol, low-density lipoprotein (LDL) cholesterol, neutral fat, uric acid, creatinine, and blood sugar.

Urinalysis: Urine sugar, urine protein, urine occult blood, and urine pH.

### Physical measurements

Right/left grip strength, right/left leg muscle strength, vertical jump, whole body reaction time, standing time on one leg with eyes closed, sit-up, seated forward bend, and resting blood pressure.

### Questionnaire variables

Gastrointestinal symptoms (stomachache, heartburn, heavy stomach, lower abdominal pain, bloated stomach), urinary frequency, condition of the stools (fecal properties and bowel movement), and physical condition (sleep quality and upon awakening).

### Statistical analysis

In the blood data, the urinalysis and physical measurement values, the statistical significance of the average difference (before and after AEW, PW drinking) was analysed using a paired t-test (Statcel 4 Software [OMS Publishing, Saitama, Japan). The questionnaire data (before and after AEW, PW drinking) was analysed by the Wilcoxon signed-rank test using the same Statcel 4 software. Differences for which Pvalues of < 0.05 and < 0.01 were inferred as significant.

## Results

### Conditions of subjects and water quality

Subjects with abdominal symptoms such as heartburn, stomach discomfort, abdominal bloating, diarrhea, and constipation were used in the study previously performed., For the current study, subjects aged 20 to 69 years were randomly selected among medical checkup examinees who visited the Osaka City Citizen Health Development Consultation Center, and then divided into two groups. One group ingested PW while the other ingested AEW. Neither the subjects nor the experimenters knew which group the subjects belonged to. Figure 2 shows that no significant differences were found in dispersion of mean values and distribution values.

Age distribution of subjects.

Note: PW: Purified tap water; AEW: alkaline electrolyzed water.

Each subject was provided with an AEW apparatus that had been modified to either produce or not produce AEW, and asked to install it at their home. In order to verify the quality of the drinking water, the water produced by the apparatus was taken into aluminum containers and collected when the subjects came in for measurement. Figure 3 shows the water quality distribution of each drinking water.

Water quality distribution of two types of drinking water.

Note: PW: Purified tap water; AEW: alkaline electrolyzed water.

Because we selected subjects who live in or around Osaka City, the tap water from either the same or a nearby water source was used for the evaluation. For this reason, the tests have been conducted using water of equivalent quality and which shows little bias in the distribution of ions.

Regarding the water before and after the electrolysis, the pH was 7.6 ± 0.2 for the PW group, and 9.2 ± 0.2 for the AEW group. Dissolved hydrogen concentration was not measurable at the subjects’ houses because hydrogen easily escapes water. However, for non-electrolyzed and electrolyzed tap water from the same water source and using the same water apparatus, the hydrogen concentration was confirmed as 0 mg/L in the PW group and 0.2 mg/L for the AEW group for the characteristics of the device.

### Comparison of hematological values

The hematological data of subjects in the PW group and the AEW group were compared before and after the four-week period, but no significant differences were observed in both groups. This is consistent with the contents of the previous report. However, the HDL cholesterol level, a newly measured value this time, of the AEW group showed a tendency to increase with P = 0.097, as shown in Figure 4.

Change in HDL cholesterol before and after drinking.

Note: (A) alkaline electrolyzed water (AEW) drinking group, (B) purified tap water (PW) drinking group. HDL: High-density lipoprotein.

### Comparison of data related to physical abilities

For the seated forward bend, vertical jump, right/left grip strength, and sit-up, there was no significant difference before and after the 4-week period for both the PW group and the AEW group.

Regarding the whole body reaction time, no significant differences were observed before and after the 4-week period in the case of the PW group, as seen in Figure 5B. However, a significant difference (decrease) (P < 0.05) was observed in the AEW group, as seen in Figure 5A. As for standing time on one leg with eyes closed, longer times were observed in the AEW group (P = 0.09), as seen in Figure 6A.

Change in whole body reaction time before and after drinking.

Note: (A) Alkaline electrolyzed water (AEW) drinking group; (B) purified tap water (PW) drinking group.

Changes in the standing time on one leg with eyes closed before and after drinking.

Note: (A) alkaline electrolyzed water (AEW) drinking group, (B) purified tap water (PW) drinking group.

### Questionnaire to subjects

As for the questionnaire items, we asked the subjects to provide answers in 3 to 5 points about gastrointestinal symptoms (Table 1), defecation and urination (Table 2), and physical condition (Table 3).

### Table 1

Gastrointestinal symptoms

Item 1 2 3 4
Stomachache Not at all Slightly Quite a lot Very much
Heartburn Not at all Slightly Quite a lot Very much
Heavy stomach Not at all Slightly Quite a lot Very much
Lower abdominal pain Not at all Slightly Quite a lot Very much
Bloated stomach Not at all Slightly Quite a lot Very much

Note: Scoring 1 to 4, where: Not at all = 1, and Very much = 4.

### Table 2

Defecation and urination

Item 1 2 3 4 5
Uninary frequency Very often Often Sometimes Occasionally Rarely
Fecal properties Hard Slightly hard Normal Slightly soft Soft

### Table 3

Physical conditionn

Item 1 2 3
Sleep quality Good Neither nor Bad
Waking up Good Neither nor Bad

Note: Scoring 1 to 3, where: Good = 1, and Bad = 3.

First, as seen in Figures 77 to to11,11, as for gastrointestinal symptoms, sufficiently clear effect could not be confirmed in this study. Next, as seen in Figure 12, the urinary frequency significantly increased in both groups, likely due to an increase in urine volume resulting from water ingestion. Regarding bowel movement, the stools slightly changed from slightly soft to normal or slightly hard, or from soft to normal (P < 0.05) in the AEW group, as can be seen in Figure 13A. There was no difference between subjects of the two groups who had answered that they were in “good” or “bad” physical condition.

Change in stomach ache before and after drinking.

Note: Left side: alkaline electrolyzed water (AEW), and right side: purified tap water (PW).

Change in bloated stomach before and after drinking.

Note: Left side: alkaline electrolyzed water (AEW), and right side: purified tap water (PW).

Change in urinary frequency before and after drinking.

Note: (A) Alkaline electrolyzed water (AEW) drinking group, and (B) purified tap water (PW) drinking group.

Changes in the condition of stools before and after drinking.

Note: (A) Alkaline electrolyzed water (AEW) drinking group, and (B) purified tap water (PW) drinking group.

Change in heartburn before and after drinking.

Note: Left side: alkaline electrolyzed water (AEW), and right side: purified tap water (PW).

Change in heavy stomach before and after drinking.

Note: Left side: alkaline electrolyzed water (AEW), and right side: purified tap water (PW).

Change in lower abdominal pain before and after drinking.

Note: Left side: alkaline electrolyzed water (AEW), and right side: purified tap water (PW).

Regarding sleep quality, there was a significant increase (P < 0.01) in the number of AEW group subjects who responded that they were able to sleep well, as shown in Figure 14A, and there was a significant increase (P < 0.05) in the number of subjects from the same group who said that they felt good upon awakening, as seen in Figure 15A.

Change in sleep quality before and after drinking.

Note: (A) Alkaline electrolyzed water (AEW) drinking group, and (B) purified tap water (PW) drinking group.

Changes in the state of getting up before and after drinking.

Note: (A) Alkaline electrolyzed water (AEW) drinking group, and (B) purified tap water (PW) drinking group.

## Discussion

In Japan, AEW apparatus have been approved as medical devices, and ingesting AEW has been found to be effective in relieving gastrointestinal symptoms. A clinical evaluation of this effect was conducted with patients with gastrointestinal symptoms (heartburn, stomach discomfort, and abdominal symptoms such as abdominal bloating, diarrhea, and constipation).

Antioxidant action by hydrogen and gastric acid neutralization by alkaline pH have been considered. In addition, recent studies have shown that the intestinal bacterial flora distribution changes. It seems that these are involved in the normalization of the gastrointestinal activity. However, for this study, patients with gastrointestinal symptoms were not specifically selected. As for these as well as the previous results, in general, there was no difference in the hematological values between the PW group and the AEW group. However, the newly measured HDL cholesterol value showed a tendency to increase with P = 0.097. The increase in HDL cholesterol by ingesting water containing hydrogen is reported by Gadek and colleagues. The effect of hydrogen can be considered to have had an effect in the AEW group this time as well.

As for gastrointestinal symptoms—which showed a significant difference during the previous study (significant improvement of abdominal symptoms and improvement of abnormal bowel movement),—sufficiently clear effect could not be confirmed by this study because the subjects did not show gastrointestinal symptoms, and very few of them responded that they had abnormal abdominal symptoms and bowel movement before participating in this study. Therefore, we believe this is the reason the answers of the subjects were the same before and after their participation in the study.

However, with respect to bowel movement, the stools slightly changed from soft to normal or slightly hard, or from loose to normal in the AEW group. This reflects that the stools are more normal, and, as shown in the previous report, that ingesting AEW is considered to contribute to intestinal normalization.,,Regarding items other than the gastrointestinal tract, a high proportion of the respondents said that they felt they were able to sleep well, and the proportion of subjects who answered that they felt good when awakening increased. Various studies on the relationship between the ingestion of antioxidant substances and the condition of sleep have been undertaken, and the effect of reducing oxidative stress, thus allowing for improved sleep quality, is exhibited by ingesting AEW containing hydrogen, which is considered an antioxidant substance.

Regarding sports performance, various reports on the effects of sleep on sports performance have concluded that willingly sleeping longer can lead to faster running, shortened reaction time, and improved motivation during practice and games. Improved sleep quality by ingesting AEW is, therefore, believed to help reduce fatigue, ensure appropriate endurance recovery, and improve overall sports performance.

The findings of this study indicate that ingesting AEW on a daily basis improves health and exercise capacity, even in healthy people who do not have gastrointestinal symptoms.

## Footnotes

Funding: The study was supported by a grant from Matsushita Electric Works Co., Ltd. Home Appliances R&D Center (to HN).

Conflicts of interest

The corresponding author (YT) is a salaried employee of the Panasonic Corporation. One of the authors (SY) was a salaried employee of the Panasonic Corporation. This study does not alter our adherence to Medical Gas Research policies on sharing data and materials. Another authors (KI and HN) report no conflict of interest related to this manuscript.

Financial support

The study was supported by a grant from Matsushita Electric Works Co., Ltd. Home Appliances R&D Center (to HW).

Institutional review board statement

All procedures used in this research were approved by the Ethics Committee of the Osaka City University Graduate School of Medicine (No. 837) and were registered in the University Hospital Medical Information Network (UMIN) Clinical Trials Registry (UMIN ID: UMIN000031800) on March 22, 2018.

Declaration of participant consent

The authors certify that they have obtained participant consent forms. In the form, participant have given their consent for their images andother clinical information to be reported in the journal. The patients understand that their names and initials not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Reporting statement

This study follows the Consolidated Standards of Reporting Trials (CONSORT) guidelines.

Biostatistics statement

The statistical methods of this study were reviewed by the biostatistician of the Osaka City University, Osaka, Japan.

The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement

Individual participant data that underlie the results reported in this article, after deidentification (text, tables, figures, and appendices). Study protocol and informed consent form will be available immediately following publication, without end date. Results will be disseminated through presentations at scientific meetings and/or by publication in a peer-reviewed journal. Anonymized trial data will be available indefinitely at www.figshare.com.

Plagiarism check

Checked twice by iThenticate.

Peer review

Externally peer reviewed.

. 2018 Oct-Dec; 8(4): 160–166.
Published online 2019 Jan 9.
PMCID: PMC6352572
PMID: 30713669

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# role of molecular hydrogen H2 water in the regression of hypercholesterolemia and atherosclerosis

### Abstract

#### CONTEXT:

We have found that hydrogen (dihydrogen [H2] (water)) decreases plasma low-density lipoprotein (LDL) cholesterol levels and improves high-density lipoprotein (HDL) function in patients with potential metabolic syndrome in a before-after self-controlled study.

#### OBJECTIVE:

The purpose of this study was to further characterize the effects of H2-rich water (0.9 L/day) on the content, composition, and biological activities of plasma lipoproteins on patients with hypercholesterolemia and their underlying mechanisms in a double-blinded, randomized, and placebo-controlled trial.

#### DESIGN:

This was a case-control study.

#### SETTING:

The setting was the Zhoudian community, Tai’an, China.

#### PATIENTS:

A total of 68 patients with untreated isolated hypercholesterolemia were randomly allocated to either drinking H2-rich water (n = 34) or placebo water (n = 34) for 10 weeks.

#### RESULTS:

HDL isolated from the H2 group showed an increased ability to promote the ATP-binding cassette transporter A1-mediated cholesterol efflux ex vivo. Plasma pre-β-HDL levels were up-regulated although there were no changes in plasma HDL-cholesterol levels. Moreover, other HDL functions, assessed in protection against LDL oxidation, inhibition of oxidized-LDL-induced inflammation, and protection of endothelial cells from oxidized-LDL-induced apoptosis, were all significantly improved by H2 treatment. In addition, molecular hydrogen water H2 treatment increased the effective rate in down-regulating plasma levels of total cholesterol (47.06% vs 17.65%) and LDL cholesterol (47.06% vs 23.53%). Western blot analysis revealed a marked decrease in apolipoprotein B100 and an increase in apolipoprotein M in plasma of the molecular hydrogen water H2 group. Finally molecular hydrogen water H2 treatment resulted in a significant reduction in the levels of several inflammatory and oxidative stress indicators in whole plasma and HDL particles.

#### CONCLUSIONS:

H2 activates ATP-binding cassette transporter A1-dependent efflux, enhances HDL antiatherosclerotic functions, and has beneficial lipid-lowering effects. The present findings highlight the potential role of H2 in the regression of hypercholesterolemia and atherosclerosis.

PMID:25978109
DOI: 10.1210/jc.2015-1321
2015 Jul;100(7):2724-33. doi: 10.1210/jc.2015-1321. Epub 2015 May 15.
###### Hydrogen Activates ATP-Binding Cassette Transporter A1-Dependent Efflux Ex Vivo and Improves High-Density Lipoprotein Function in Patients With Hypercholesterolemia: A Double-Blinded, Randomized, and Placebo-Controlled Trial.
Song G1Lin Q1Zhao H1Liu M1Ye F1Sun Y1Yu Y1Guo S1Jiao P1Wu Y1Ding G1Xiao Q1Qin S1.

### Author information

1
Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis (G.S., Q.L., H.Z., Y.Y., S.G., P.J., S.Q.), TaiShan Medical University, Tai’an, China 271000; Heart Center of TaiShan Medical University (G.S., Q.L., Y.W., Q.X., S.Q.), Tai’an, China 271000; Zhoudian Community (M.L., Y.S.), Daiyue District, Tai’an, China 271021; Tai’an He Ren Tang Hospital (F.Y.), Tai’an, China 271021; Department of Cardiology (Y.W., Q.X., S.Q.), Affiliated Hospital of Taishan Medical University, Tai’an, China 271000; and Institute of Public Health (G.D.), TaiShan Medical University, Tai’an, China 271000.

# Molecular hydrogen affects body composition, metabolic profiles, and mitochondrial function in middle-aged overweight women

### Abstract

#### BACKGROUND:

Molecular hydrogen (H2) effectively treats obesity-related disorders in animal models, yet no studies have investigated the effectiveness and safety of H2 for improving biomarkers of obesity in humans.

#### AIM:

In this double blind, placebo-controlled, crossover pilot trial, we evaluated the effects of H2 intervention on body composition, hormonal status, and mitochondrial function in ten (n = 10) middle-aged overweight women.

#### METHODS:

Volunteers received either hydrogen-generating minerals (supplying ~6 ppm of H2 per day) or placebo by oral administration of caplets for 4 weeks. The primary end-point of treatment efficacy was the change in the body fat percentage from baseline to 4 weeks. In addition, assessment of other body composition indices, screening laboratory studies, and evaluation of side effects were performed before and at follow-up. Clinical trial registration www.clinicaltrials.gov , ID number NCT02832219.

#### RESULTS:

No significant differences were observed between treatment groups for changes in weight, body mass index, and body circumferences at 4-week follow-up (P > 0.05). H2 treatment significantly reduced body fat percentage (3.2 vs. 0.9%, P = 0.05) and arm fat index (9.7 vs. 6.0%, P = 0.01) compared to placebo administration, respectively. This was accompanied by a significant drop in serum triglycerides after H2 intervention comparing to placebo (21.3 vs. 6.5%; P = 0.04), while other blood lipids remained stable during the study (P > 0.05). Fasting serum insulin levels dropped by 5.4% after H2 administration, while placebo intervention augmented insulin response by 29.3% (P = 0.01).

#### CONCLUSIONS:

It appears that orally administered H2 as a blend of hydrogen-generating minerals might be a beneficial agent in the management of body composition and insulin resistance in obesity.

2018 Feb;187(1):85-89. doi: 10.1007/s11845-017-1638-4. Epub 2017 May 30.

### Author information

1
Faculty of Sport and Physical Education, University of Novi Sad, Novi Sad, Serbia.
2
Faculty of Sport and Physical Education, University of Novi Sad, Novi Sad, Serbia. sergej.ostojic@chess.edu.rs.
3
4
Applied Bioenergetics Laboratory, Faculty of Sport and PE, University of Novi Sad, Lovcenska 16, Novi Sad, 21000, Serbia. sergej.ostojic@chess.edu.rs.

# Novel haemodialysis (HD) treatment employing molecular hydrogen (H2)-enriched dialysis solution improves prognosis of chronic dialysis patients: A prospective observational study

## Abstract

Recent studies have revealed unique biological characteristics of molecular hydrogen (H2) as an anti-inflammatory agent. We developed a novel haemodialysis (E-HD) system delivering an H2 (30–80 ppb)-enriched dialysis solution by water electrolysis, and conducted a non-randomized, non-blinded, prospective observational study exploring its clinical impact. Prevalent chronic HD patients were allocated to either the E-HD (n = 161) group or the conventional HD (C-HD: n = 148) group, and received the respective HD treatments during the study. The primary endpoint was a composite of all-cause mortality and development of non-lethal cardio-cerebrovascular events (cardiac disease, apoplexy, and leg amputation due to peripheral artery disease). During the 3.28-year mean observation period, there were no differences in dialysis parameters between the two groups; however, post-dialysis hypertension was ameliorated with significant reductions in antihypertensive agents in the E-HD patients. There were 91 events (50 in the C-HD group and 41 in the E-HD group). Multivariate analysis of the Cox proportional hazards model revealed E-HD as an independent significant factor for the primary endpoint (hazard ratio 0.59; [95% confidence interval: 0.38–0.92]) after adjusting for confounding factors (age, cardiovascular disease history, serum albumin, and C-reactive protein). HD applying an H2-dissolved HD solution could improve the prognosis of chronic HD patients.

## Introduction

The combination of enhanced oxidative stress and inflammation in patients on chronic haemodialysis (HD) treatment plays a crucial role in the occurrence of excessive cardiovascular events and death,. The bio-incompatibility of the HD procedure is supposed to be involved with this pathology. HD may exaggerate leukocyte activation and injury, which enhance oxidative stress and inflammation. Therefore, we hypothesized that ameliorating the stress to leukocytes during HD may have a beneficial effect on patient outcomes.

Molecular hydrogen (H2) is an inert gas with no known side effects. Recent studies have shown that H2acts as an antioxidant and an anti-inflammatory agent, and ameliorates cellular and organ damage,. We therefore developed a novel HD system using highly dissolved H2 water rendered by the water electrolysis technique. Previous pilot studies, including ours, have reported that suppression of interleukin-6, high-sensitivity C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-1)/chemokine (C-C motif) ligand 2 (CCL2), and myeloperoxidase (MPO), decrease oxidative injury of lymphocytes, improve the redox status of serum albumin, and ameliorate hypertension. In reference to these findings, we conducted a non-randomized, non-blinded, prospective observational study to compare the outcomes between patients receiving haemodialysis using an H2-enriched dialysis solution (E-HD group) and patients receiving conventional haemodialysis (C-HD group).

## Results

### Patient registration and characteristics

Patients were recruited during April 2011 and October 2012. Of the 327 prevalent chronic HD patients who were pre-registered, 18 were excluded because of the lack of data and withdrawal. Ultimately, 148 patients were allocated to the C-HD group and 161 patients were allocated to the E-HD group (Fig. 1). The patients’ characteristics in the two groups at baseline are shown in Table 1. All subjects were treated by the standard HD schedule (three sessions/week, 4–5 h/session), using high-performance biocompatible dialyzers with fixed blood flow rate (QB) (200 mL/min) and dialysate flow rate (QD) (500 mL/min). Patients who had been treated by a vitamin-E coated dialyzer were excluded from this study. At baseline, there was no statistical difference between the groups in the blood urea nitrogen (BUN) reduction rate by HD (69.7 ± 6.9% in the C-HD group and 70.3 ± 8.4% in the E-HD group; p = 0.485).

Flow chart from pre-registration to the end of observation. Abbreviations: C-HD, conventional haemodialysis; E-HD, electrolyzed water haemodialysis; KH, Kashima Hospital; GJC, Gumyoji Jin Clinic; TJC, Tateishi Jin Clinic; NH, Noboribetsu Hospital; NMH, Nikko Memorial Hospital; HMC, Higashi Muroran Clinic; HHC, Higashi Horai Clinic.

### Table 1

Patient characteristics.

Characteristic C-HD E-HD P Value
N 148 161
Age (y) 67.4 ± 11.8 64.0 ± 11.9 <0.05
Gender, male (%) 92 (62.2) 85 (52.8) NS
Dialysis vintage (months) 60 (3, 263) 80 (2, 478) <0.01
Cause of renal failure (DM, (%)) 62 (41.9) 55 (34.2) NS
Patients with CVD history (%)) 36 (24.3) 53 (32.9) NS
with multiple CVDs (%) 5 (3.4) 10 (6.2) NS
with cardiac disease (%) 25 (16.9) 31 (19.3) NS
with apoplexy (%) 11 (7.4) 29 (18.0) <0.01
with PAD (%) 5 (3.4%) 3 (1.9%) NS
Body weight (pre HD, kg) 59.3 ± 12.0 58.9 ± 11.2 NS
Body weight (post HD, kg) 57.0 ± 11.7 56.3 ± 10.9 NS
CTR (%) 48.7 ± 6.0 48.7 ± 5.5 NS
Pre-dialysis SBP (mmHg) 154 ± 27 154 ± 25 NS
Pre-dialysis DBP (mmHg) 79 ± 15 80 ± 16 NS
Post-dialysis SBP (mmHg) 142 ± 24 135 ± 24 <0.05
Post-dialysis DBP (mmHg) 75 ± 14 73 ± 14 NS
Patients on Anti-hypertensive agents (%) 108 (73.0) 107 (66.5) NS
Patients with ESA (%) 124 (83.8) 140 (87.0) NS
Fatigue Grade 2.9 ± 1.0 2.9 ± 1.1 NS
Pruritis Intensity Grade 3.4 ± 0.9 3.2 ± 0.9 <0.05
Puriritis Frequency Grade 3.2 ± 1.0 3.0 ± 1.1 NS

C-HD, conventional haemodialysis; E-HD, electrolyzed water haemodialysis.

CVD, cardio-cerebrovascular disease; HD, haemodialysis; PAD, peripheral arterial disease; SBP, systolic blood pressure; DBP, diastolic blood pressure; ESA, erythropoiesis stimulating agents.

### Changes in laboratory and subjective/objective parameters during the study

HD-related laboratory parameters at the time of the first HD session of the respective weeks are shown in Table 2. No differences were noted between the two groups during the study period. Regarding subjective symptoms, there was a significant difference in the grade of pruritus between the two groups at baseline (with more severe symptoms in the E-HD group); however, no differences were found during the course of the study. Small but significant differences were noted between the two groups in the fatigue grade (fewer symptoms in the E-HD group) at 48 weeks. No differences were observed in the time-course pre-dialysis blood pressures (BPs); however, post-dialysis BPs differed between the two groups. In sub-analysis of the post-dialysis systolic BP (SBP) levels at baseline, there were significant differences in post-dialysis SBP (6 months) and Defined Daily Dose of antihypertensive medication (6, 12, 18 months) in patients with post-dialysis SBP ≥ 140 mmH at baseline, while no statistical differences were found in those parameters in patients with post-dialysis SBP < 140 mmHg (Fig. 2).

### Table 2

Dialysis-related and subjective/objective parameters in the two groups.

Months 0 m 6 m 12 m 18 m 24 m 30 m 36 m 42 m 48 m
WBC count (/µL) C-HD 5504 ± 1653 5597 ± 1840 5461 ± 1669 5321 ± 1778 5251 ± 1996 5404 ± 2093 5701 ± 2014 5543 ± 1840 5541 ± 1985
(n) 148 136 128 126 117 109 104 84 80
E-HD 5852 ± 1803 5865 ± 2091 5734 ± 2083 5648 ± 1851 5779 ± 1823 5584 ± 1751 5620 ± 1684 5637 ± 1759 5642 ± 1793
(n) 161 160 152 145 131 123 121 112 105
Hemoglobin (g/dL) C-HD 10.6 ± 1.1 10.6 ± 1.2 10.4 ± 1.3 10.7 ± 1.4 10.4 ± 1.3 10.5 ± 1.3 10.4 ± 1.3 10.6 ± 1.3 10.7 ± 1.3
(n) 148 136 128 126 117 109 104 83 80
E-HD 11.1 ± 1.2 11.0 ± 1.0 10.7 ± 1.2 10.9 ± 1.2 10.4 ± 1.3 11.1 ± 1.1 10.8 ± 1.1 10.9 ± 1.1 11.1 ± 1.3
(n) 161 159 152 145 131 123 121 112 105
BUN (mg/dL) C-HD 66.8 ± 15.1 63.7 ± 15.0 65.3 ± 13.9 56.1 ± 14.5 58.8 ± 14.3 56.3 ± 14.0 61.3 ± 13.1 57.0 ± 14.0 61.1 ± 13.7
(n) 148 136 128 126 117 109 103 84 80
E-HD 69.0 ± 15.8 67.5 ± 16.5 65.2 ± 15.5 62.9 ± 15.8 64.3 ± 14.5 61.0 ± 13.2 62.5 ± 15.1 63.0 ± 14.8 61.4 ± 13.4
(n) 161 160 152 145 131 123 121 112 105
creatinine (mg/dL) C-HD 10.8 ± 2.6 11.1 ± 2.5 10.9 ± 2.5 10.0 ± 2.3 10.3 ± 2.3 10.4 ± 2.5 10.9 ± 2.5 11.0 ± 2.4 10.8 ± 2.5
(n) 148 136 128 126 117 110 104 84 80
E-HD 10.6 ± 3.0 10.4 ± 2.8 10.7 ± 2.8 10.3 ± 2.8 10.6 ± 2.6 10.7 ± 2.6 10.4 ± 2.3 10.8 ± 2.2 10.6 ± 2.4
(n) 161 159 152 145 131 123 121 112 105
Ca (mg/dL) C-HD 8.8 ± 0.7 8.8 ± 0.8 8.8 ± 0.8 8.8 ± 0.6 8.8 ± 0.7 8.8 ± 0.7 8.8 ± 0.7 8.9 ± 0.8 8.6 ± 0.8
(n) 148 136 128 126 117 110 104 84 79
E-HD 8.8 ± 0.7 8.8 ± 0.6 8.7 ± 0.7 8.8 ± 0.6 8.7 ± 0.7 8.8 ± 0.6 8.8 ± 0.7 8.8 ± 0.6 8.8 ± 0.6
(n) 160 159 152 145 131 123 121 112 105
Pi (mg/dL) C-HD 5.5 ± 1.3 5.5 ± 1.4 5.6 ± 1.4 5.5 ± 1.3 5.6 ± 1.3 5.3 ± 1.3 5.7 ± 1.4 5.5 ± 1.6 5.8 ± 1.4
(n) 148 136 128 126 117 109 104 84 80
E-HD 5.6 ± 1.4 5.6 ± 1.5 5.4 ± 1.3 5.4 ± 1.3 5.4 ± 1.4 5.4 ± 1.1 5.4 ± 1.1 5.3 ± 1.3 5.2 ± 1.1
(n) 161 161 154 147 133 125 123 114 107
B2-microglobulin (mg/L) C-HD 27.7 ± 7.0 28.2 ± 6.6 27.5 ± 6.4 26.9 ± 5.8 26.6 ± 6.0 27.5 ± 5.3 29.9 ± 5.8 29.8 ± 5.7 29.1 ± 6.0
(n) 148 131 126 126 116 108 102 80 78
E-HD 26.9 ± 6.5 27.0 ± 6.9 27.6 ± 6.5 26.0 ± 5.9 26.9 ± 6.3 27.3 ± 5.6 28.4 ± 5.6 28.2 ± 5.7 28.6 ± 5.3
(n) 161 159 149 142 131 122 120 110 104
CRP (mg/dL) C-HD 0.32 ± 0.57 0.23 ± 0.34 0.41 ± 0.93 0.53 ± 2.24 0.26 ± 0.44 0.40 ± 0.95 0.45 ± 0.97 0.99 ± 5.12 0.82 ± 2.10
(n) 148 133 128 126 115 109 101 81 78
E-HD 0.39 ± 0.73 0.45 ± 1.03 0.66 ± 1.52 0.56 ± 1.87 0.57 ± 1.17 0.38 ± 0.88 0.41 ± 0.71 0.35 ± 0.67 0.62 ± 1.91
(n) 161 160 152 145 131 123 121 112 105
albumin (g/dL) C-HD 3.5 ± 0.3 3.6 ± 0.3 3.6 ± 0.4 3.5 ± 0.3 3.5 ± 0.3 3.5 ± 0.4 3.5 ± 0.3 3.5 ± 0.3 3.4 ± 0.3
(n) 148 136 126 124 116 109 103 83 79
E-HD 3.7 ± 0.3 3.6 ± 0.3 3.7 ± 0.4 3.5 ± 0.4 3.5 ± 0.3 3.6 ± 0.3 3.5 ± 0.3 3.6 ± 0.3 3.6 ± 0.3
(n) 161 159 152 145 131 123 121 112 107
Dry weight (kg) C-HD 56.6 ± 11.8 57.0 ± 11.6 57.6 ± 12.3 57.0 ± 11.6 56.9 ± 11.4 56.8 ± 11.1 56.6 ± 11.5 56.4 ± 12.6 56.4 ± 12.3
147 140 133 129 119 114 106 87 82
E-HD 56.4 ± 10.9 56.5 ± 11.0 56.5 ± 11.4 56.3 ± 11.5 56.9 ± 11.8 56.4 ± 11.3 56.5 ± 11.3 56.5 ± 11.6 58.3 ± 12.2
(n) 161 160 152 146 131 125 120 113 107
CTR (%) C-HD 48.7 ± 6.0 49.1 ± 4.2 49.0 ± 4.2 49.0 ± 4.4 49.9 ± 5.3 49.6 ± 5.2 49.7 ± 5.2 49.5 ± 5.8 49.1 ± 6.2
(n) 148 134 131 115 117 112 104 84 79
E-HD 48.7 ± 5.5 49.0 ± 5.4 49.3 ± 5.6 49.4 ± 5.4 49.2 ± 5.3 49.3 ± 5.4 49.5 ± 5.6 48.7 ± 5.4 49.0 ± 5.1
(n) 161 155 148 133 129 123 119 108 101
pre-dialysis MBP (mmHg) C-HD 104 ± 17 97 ± 16 104 ± 15 100 ± 14 100 ± 16 101 ± 17 104 ± 15 101 ± 18 101 ± 18
(n) 148 137 121 112 101 88 78 66 62
E-HD 103 ± 22 94 ± 19 103 ± 18 102 ± 19 103 ± 19 105 ± 15* 105 ± 15 104 ± 16 106 ± 18
(n) 161 163 152 146 131 125 120 115 105
post-dialysis MBP (mmHg) C-HD 97 ± 13 93 ± 18 96 ± 13 96 ± 15 96 ± 13 98 ± 14 98 ± 12 100 ± 12 95 ± 12
(n) 148 137 121 112 101 88 78 66 62
E-HD 93 ± 20 90 ± 18 94 ± 16 92 ± 16* 92 ± 15** 95 ± 16 95 ± 14* 96 ± 16 95 ± 13
(n) 161 162 152 146 131 125 120 115 105
DDD C-HD 1.04 1.03 1.00 1.00 1.22 1.36 1.34 1.12 1.00
(0, 2.34) (0, 2.53) (0, 2.05) (0, 2.00) (0, 2.83) (0.18, 2.33) (0, 2.50) (0, 2.05) (0.02, 2.71)
(n) 147 137 130 127 118 112 105 86 84
E-HD 0.57 0.57* 0.5** 0.50 0.76** 0.81* 1.07 0.86 0.62*
(0, 2.14) (0, 1.53) (0, 1.21) (0, 1.34) (0, 1.50) (0.03, 1.62) (0.06, 1.90) (0, 1.87) (0, 1.62)
(n) 159 159 151 145 130 124 120 115 104
Fatigue Grade C-HD 2.9 ± 1.0 2.8 ± 1.1 2.6 ± 1.1 3.0 ± 1.2 2.8 ± 1.2 2.7 ± 1.2 2.8 ± 1.2 2.9 ± 1.1 2.9 ± 1.1
(n) 148 136 124 123 111 112 103 79 74
E-HD 2.9 ± 1.1 3.0 ± 1.0 2.9 ± 1.2 2.9 ± 1.3 2.9 ± 1.3 3.1 ± 1.1* 2.9 ± 1.4 3.0 ± 1.3 3.2 ± 1.1
(n) 161 152 139 136 124 120 118 106 96
Pruritus Intensity Grade C-HD 3.4 ± 0.9 3.2 ± 0.9 3.1 ± 1.0 3.2 ± 1.0 3.1 ± 1.1 3.1 ± 1.0 3.1 ± 1.0 3.2 ± 0.9 3.0 ± 1.0
(n) 148 136 124 123 110 112 103 79 74
E-HD 3.2 ± 0.9* 3.2 ± 1.1 3.4 ± 0.9 3.5 ± 0.9 3.2 ± 1.0 3.4 ± 0.9 3.3 ± 1.0* 3.4 ± 0.9 3.3 ± 0.9*
(n) 161 152 139 136 124 120 118 106 96
Puriritus Frequency Grade C-HD 3.2 ± 1.0 2.9 ± 1.1 2.9 ± 1.1 2.9 ± 1.2 2.9 ± 1.2 2.9 ± 1.2 2.9 ± 1.1 3.1 ± 1.1 2.8 ± 1.2
(n) 148 135 124 123 111 112 103 79 74
E-HD 3.0 ± 1.1 3.1 ± 1.2 3.2 ± 1.1 3.3 ± 1.0 3.1 ± 1.1 3.3 ± 1.0 3.2 ± 1.1 3.3 ± 1.1 3.2 ± 1.1*
(n) 161 152 139 136 124 120 118 106 96

vs. C-HD; *p < 0.05, **p < 0.01

MBP, mean blood pressure; CTR, cardiothoracic ratio; DDD, defined daily dose of anti-hypertensive agents.

C-HD, conventional haemodialysis; E-HD, electrolyzed water haemodialysis; WBC, white blood cell; BUN, blood urea nitrogen; Ca, serum Calcium; Pi, serum phosphate; CRP, C-reactive protein.

Changes in post-dialysis systolic blood pressure, and prescription of antihypertensive agents during the study. Patients with post-dialysis SBP ≥ 140 mmHg (n = 139) at baseline (0 month): changes in post-dialysis SBP (a), and changes in DDD (b); Patients with post-dialysis SBP < 140 mmHg (n = 168) at baseline: changes in post-dialysis SBP (c), and changes in DDD (d). Abbreviations: C-HD, conventional haemodialysis; E-HD, electrolyzed water haemodialysis; SBP, systolic blood pressure; DDD, daily defined dose of antihypertensive agents. (a,c) There were significant differences in post-dialysis SBP (6 months; p < 0.05), and DDD (6, 12, 18 months; p < 0.05, respectively) between the two groups. (b,d) No differences were observed in post-dialysis SBP or DDD between the two groups.

### Composite events summary and multivariate analysis of risk factors for events

During the mean observation period of 3.28 years, there were 91 events: 50 in the C-HD group and 41 in the E-HD group (Table 3). In Cox proportional hazards model analysis, possible risk factors for the primary endpoints, which were identified via p-values < 0.1, were depicted, e.g., E-HD dialysis modality, age, history of cardio-cerebrovascular disease (CVD), serum albumin, and CRP. Multivariate analysis after adjusting for these factors revealed E-HD as an independent significant factor for the primary event (hazard ratio [HR] 0.59 [95% confidence interval [CI]: 0.38–0.92]) (Fig. 3 and Table 4).

### Table 3

Summary of events in the two groups.

C-HD E-HD
Observation vintage (patient⋅year) 467 544
Number of Primary events 50 41
(all causes of deaths and non-lethal CVD events)
Cardiac events including death 29 20
Congestive heart failure 11 8
Ischemic heart disease 13 9
Aortic aneurysm rupture 1 1
Sudden cardiac arrest 4 2
Apoplexy including death (bleeding/infarction) 6 (1/5) 10 (2/8)
Primary events rate (1000 patients·year: 95%CI) 107.1 (81.2–141.1) 75.4 (55.6–102.2)
Number of deaths 17 20
Deaths rate (1000 patients·year: 95%CI) 36.4 (22.7–58.3) 36.8 (23.8–56.8)

C-HD, conventional haemodialysis; E-HD, electrolyzed water haemodialysis.

PAD, peripheral artery disease (with surgical procedure).

Cox proportional hazards model demonstrating events-free differences between patients on C-HD and those on E-HD. Treatment with E-HD was an independent predicting factor for events (hazard ratio:0.593; p < 0.05). Abbreviations: C-HD, conventional haemodialysis; E-HD, electrolyzed water haemodialysis.

### Table 4

Cox proportional hazards model analysis for the composite primary endpoints.

Univariate HR 95%CI P value Multivariate HR 95%CI P value
E-HD 0.687 0.454-1.039 0.076 0.593 0.384–0.916 0.019
HD vintage 1.000 0.997–1.002 0.824
Age 1.036 1.017–1.055 0.000 1.014 0.993–1.036 0.183
Gender (female) 0.698 0.454–1.074 0.102
History of CVD 3.085 2.040–4.665 0.000 3.037 1.977–4.665 0.000
non DM 0.865 0.569–1.314 0.497
BMI 0.987 0.933–1.044 0.644
Pre SBP 0.999 0.990–1.007 0.783
Albumin 0.195 0.101–0.377 0.000 0.328 0.160–0.674 0.002
CRP 1.266 1.017–1.576 0.035 1.323 1.005–1.740 0.046
Hg 0.893 0.741–1.075 0.230

E-HD, electrolyzed water haemodialysis; HD, haemodialysis; CVD, cardio-cerebral vascular disease; DM, diabetes mellituss; BMI, body mass index; Pre SBP, pre-dialysis systolic blood pressure; CRP, C-reactive protein; Hg, hemoglobin.

## Discussion

This prospective observational study primarily aimed to examine the clinical effects of the addition of H2to HD dialysate (an average of 30–80 ppb of H2), which was delivered continuously through the dialyzer membrane to the blood during treatment, as reported elsewhere. During the mean observation period of 3.28 years, the study results revealed E-HD as an independent significant factor for reducing the risk of the primary events of all-cause mortality and development of non-lethal cardio-cerebrovascular events. In the study, all HD systems employed an endotoxin-eliminating filter system. Thus, the different clinical profiles between the two groups, patients on E-HD and those on C-HD, reflects the influence of H2 during HD.

The mechanisms by which E-HD delivers clinical benefits remain to be elucidated, since there were no differences in dialysis-related clinically relevant parameters between the two groups during the study. However, we could speculate several possibilities. The observation that the amelioration of post-dialysis hypertension (SBP ≥ 140 mmHg) in E-HD patients may suggest an idea to elucidate the benefits of E-HD, because intra-dialysis systolic hypertension, as well as high SBP, are well-known risk factors for all-cause mortality in HD patients,. On the other hand, low SBP (<110 mmHg) has also been reported as a risk for excessive mortality. Interestingly enough, there were no differences during the course of the study in post-dialysis SBP levels among the patients with SBP < 140 mmHg at baseline (Fig. 2). Furthermore, there were no differences between the two groups in the proportion of patients with SBP < 110 mmHg (Supplementary Fig. S1). Thus, taken together the observations, the improved post-dialysis BP may have played a role, at least partially, for the better outcomes in patients with post-dialysis hypertension

Other possible mechanisms could be suggested in the previous studies, i.e., increased reduced albumin redox status by acute as well as long-term E-HD,, improved patients’ anti-oxidative capacity, amelioration of micro inflammation with reduction of pro-inflammatory cytokines,, and suppression of T-cell damage. These possible mechanisms need to be clarified in the context of patients’ clinical outcomes in the future.

The mitigating effect on elevated SBP, as observed in the present study and previous studies, is very unique. We speculate that the primary mechanism of BP reduction could not be attributed to changes in fluid volume, since there were no significant differences in body weight after HD. Rather, the primary mechanism of BP reduction might be related to vasodilation or to a reduction in vascular resistance. Recent studies in deoxycortisterone acetate (DOCA)-salt hypertension have revealed a crucial role of superoxide anion release from macrophages in mesenteric peri-arteries, due in part to impaired function of the Alpha 2-adrenergic autoreceptors, which provide negative feedback on the release of norepinephrine from the sympathetic nerves associated with the mesenteric arteries. The mesenteric arteries constitute a major resistance arterial bed for BP regulation. In addition, one fourth of the systemic blood volume is present in the splanchnic circulation. Therefore, an increase in arteriolar resistance will elevate the arterial BP, and an increase in the mesenteric venomotor tone will lead to an increase in the cardiac venous return and the cardiac load due to a decrease in the venous capacity,. The combination of these two pathological processes results in a severe cardiac load. Interestingly, a recent study showed that the chemokine (C-C motif) receptor type 2 blockade suppresses vascular macrophage infiltration and reduces blood pressure. Upon the observation that MCP-1 decreased in E-HD patients in the previous study, it is possible to speculate the possible action of E-HD on macrophage of patients. The question of whether the HD procedure activates the residential macrophages, or activates extrinsic macrophages to infiltrate the mesenteric vascular area, needs to be addressed.

There are several issues and limitations in this study. First, the observed results in the E-HD group were slightly complicated, i.e., the rate of the primary composite endpoint was lower in the E-HD group than in the C-HD group, although the rate of death was not different between the groups. In univariate analysis of the Cox proportional hazards model, E-HD was not a strong factor for the primary endpoint, although multivariate analysis showed E-HD to be a strong factor after adjusting for confounding factors. Regarding the reasons for this, we speculate that a potential bias existed in the patients who were allocated to the E-HD group in that these patients had a relatively higher incidence of CVD history. This may have influenced the results of the univariate-analysis, since the presence of a CVD history was the most influential risk factor for the occurrence of the primary endpoint. To clarify this point, we performed a sub-analysis on this profile according to the presence or absence of CVD history. And it was revealed that E-HD was a significant factor for reducing the risk of primary endpoint in patients without history of CVD (HR: 0.455; p = 0.010) by univariate as well as multivariate analysis (Supplementary Tables S1 and 2), which indicates the clinically significant impact of E-HD.

Second issue is the levels of H2 of HD solution. The H2 levels of the present dialysates were in the range of 30–80 ppb, and no adverse effects were observed with respect to an H2 load within this range. Upon the report that there are generation of H2 in average of 24 ml/min in healthy human (approximately over 15 mmol daily) in the colon, and that they are absorbed into body, the given H2 during the single session of HD, which we estimated approximately as much as 2.5 mmol, seemed to be within the physiological range. Therefore, it remains unknown whether the applied H2 levels were best in regards to provide clinical effects, and higher levels of H2 may offer additional clinical benefits without any adverse effects needs to be investigated.

Third, we could not conclude the influence of E-HD on clinical symptoms in this study. Of note is that during the clinical course, post-dialysis hypertension was ameliorated with significant reductions of anti-hypertensive agents in the patients on E-HD. However, patient selection in the present study was conducted according to the attending physician’s preference; therefore, the observed phenomena such as decrease in BP and improved subjective symptoms of fatigue and pruritus during the course, have remained speculative.

And lastly, there was a statistical difference in the age between the two groups in the present study, e.g. the E-HD group was 3.4 years younger than C-HD. Although we employed the age for multivariate analysis of Cox proportional hazards model analysis, this might have influenced the event rate in the real world. A randomized clinical study is critically needed to address these issues in the future.

H2 as biological gas has potentials in clinical medicine. However H2 volatile gas, is not easy to handle in the clinical setting. The technique of water electrolysis has made it possible to apply H2 very safe to generate H2 dissolved water for real HD therapy. We think that this innovative treatment could open a new therapeutic possibility beyond the conventional HD.

## Method

### Study design and participants

A non-randomized, non-blinded, prospective observational study was conducted to evaluate the clinical impact of the E-HD system (UMIN-ICDR Clinical Trial: Study Title: “Prospective observational study of the clinical effect of haemodialysis using electrolyzed water”; Unique ID issued by UMIN: UMIN000004857, Date of disclosure of the study information: 2011/01/11, Link to view the page (ICDR): https://upload.umin.ac.jp/cgi-bin/icdr_e/ctr_view.cgi?recptno = R000005491).

The primary composite endpoints included all-cause mortality, and concomitant disease such as cardiac disease (heart failure or myocardial infarction requiring hospitalization, coronary artery disease requiring invasive therapy), stroke (symptomatic cerebral hemorrhage or cerebral infarction confirmed by diagnostic imaging), and obstructive arteriosclerosis requiring leg amputation.

The study used a non-randomized design, and the candidate patients were selected by decision of the patient’s physician. In two centers (KH and NMH), candidates for the E-HD group were selected by chief physicians; subsequently, matched control patients in the C-HD group were selected from the rest of the patients in the respective centers in terms of demographic background such as age and sex. In two of the study centers (HMC and HHC), all patients were selected for the E-HD group since the centers were to employ a central E-HD system to completely replace the conventional HD system. In three study centers in which the E-HD system was not available (NH, TJC, GJC), more than one patient was selected as part of the matched control group to the E-HD group of the above four centers in terms of age and sex as much as possible. Patients who were receiving on-line hemodiafiltration or combination therapy with peritoneal dialysis, and potential subjects with serious disease at the time of enrollment, i.e., severe heart failure (New York Heart Association III/IV), severe liver disease, psychological problems, dementia, malignant disease within the previous 3 months, or an evidently poor systemic condition with an evidently poor short-term prognosis, were excluded from this study. History of CVD included cardiac disease, stroke (these definitions were comparable to those of the primary composite endpoints mentioned above), and symptomatic peripheral arterial disease requiring medical intervention.

The study was approved by the Ethics Committee of Fukushima Medical University (No. 1155: Supplementary file of study protocol), and the clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all patients registered.

### Data collection

All data generated or analysed during this study are included in this published article.

### Statistical Methods

The target sample size of the original study (n = 70 < each) was based on an estimated event-free rate of 10% differences at 3 years between groups with 1:1 ratio between them, and calculated from the rationale that a statistical power of 90% and the alpha level 0.05, using a two-sided log-rank test.

All values are expressed as the mean ± standard deviation (SD) or median (interquartile range) as appropriate. For comparisons between the two groups, Student’s t-test or the Mann-Whitney U test was used for continuous variables and chi-square test or Fisher’s exact test was used for nominal variable, as appropriate. Values of p < 0.05 were considered statistically significant. Data were statistically analyzed using IBM SPSS Statistics version 22.0 for Windows (Chicago, IL, USA).

### H2 delivery HD system

Figure 4 details of the system have been reported previously,. Briefly, test solutions were prepared as follows: pre-filtered water was processed using activated charcoal filtration and water softening to supply the HD-24K water electrolysis system (Nihon Trim, Osaka, Japan), where water was electrolyzed by direct current supply to the anode and cathode electrode plates. Water on the anode side was drained, and water from the cathode side (electrolyzed water) was collected to supply the reverse osmosis equipment (MH500CX; Japan Water System, Tokyo, Japan) at 500 mL/min. The intensity of electrolysis was adjusted to maintain a pH of 10.0. The reverse osmosis water produced by the water electrolysis system was supplied to prepare the HD solution. The composition of the inflow H2-HD solution was the same as the standard HD solution with the exception of the presence of dissolved H2 in the H2-HD, and there were no differences in terms of electrolytes levels and pH, as compared to the standard HD solution, as reported elsewhere,. Whereas regarding H2 levels of control group, dialysate and blood H2 levels were less than 1 ppb.

Manufacturing process of haemodialysis solution in the E-HD and H2 dynamics during treatment by E-HD. Abbreviations: E-HD, electrolyzed water haemodialysis; e-, electron; AVF, arterio-venous fistula.

The present E-HD system could deliver H2 (30–80 ppb)-enriched dialysis solution. H2 levels of inflow blood and HD solution reached an equivalent state in the dialyzer, and the H2 level of outflow blood from dialyzer showed approximately the same as that of inflow H2-HD solution under QB 200 ml/min and QD 500 ml/min. Therefore, H2 load to patient is determined by time of HD treatment and H2 levels of HD solution if QB and QD are fixed, i.e., it is estimated that about 1.2 mmol of H2 is loaded in case of 4 hour-treatment, and HD solution with 50 ppb H2. Regarding the H2 dynamics in the body, previous studies,revealed that no changes were found in H2 levels of inflow blood after 4-hour treatment, and there were increases of constant H2 levels in expired air of patients by treatment, and they soon returned to the basal levels by stop of treatment. Therefore, it is supposed that delivered H2 into blood during the HD treatment is mostly excreted from lung during the time on HD.

## Acknowledgements

The present study was conducted by fund from Nihon Trim Co., Ltd. (www.nihon-trim.co.jp; Osaka, Japan). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

. 2018; 8: 254.
Published online 2018 Jan 10.
PMCID: PMC5762770
PMID: 29321509

## Author Contributions

M.N. wrote the main manuscript text and prepared all figures. M.N., N.I., S.K., R.N., M.M. and S.I. organized the study group. N.I., H.S., H.H., R.Y., K.T., N.O. and H.N. collected data. M.N. and Y.M. analyzed the data. M.N. and S.I. supervise the progress of research of all aspects.

## Competing Interests

The authors declare that they have no competing interests.

## Footnotes

Electronic supplementary material

Supplementary information accompanies this paper at 10.1038/s41598-017-18537-x.

## Associated Data

Supplementary Materials

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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# Alkaline ionized water improves exercise-induced metabolic acidosis and enhances anaerobic exercise performance in combat sport athletes

note: this article may be useful to cancer patients as it is well known that most cancer cells use anaerobic glycolysis to produce ATP energy and excrete lactic acid
Abstract

Hydration is one of the most significant issues for combat sports as athletes often use water restriction for quick weight loss before competition. It appears that alkaline water can be an effective alternative to sodium bicarbonate in preventing the effects of exercise-induced metabolic acidosis. Therefore, the main aim of the present study was to investigate, in a double blind, placebo controlled randomized study, the impact of mineral-based highly alkaline water on acid-base balance, hydration status, and anaerobic capacity. Sixteen well trained combat sport athletes (n = 16), were randomly divided into two groups; the experimental group (EG; n = 8), which ingested highly alkaline ionized water for three weeks, and the control group (CG; n = 8), which received regular table water. Anaerobic performance was evaluated by two double 30 s Wingate tests for lower and upper limbs, respectively, with a passive rest interval of 3 minutes between the bouts of exercise. Fingertip capillary blood samples for the assessment of lactate concentration were drawn at rest and during the 3rd min of recovery. In addition, acid-base equilibrium and electrolyte status were evaluated. Urine samples were evaluated for specific gravity and pH. The results indicate that drinking alkalized ionized water enhances hydration, improves acid-base balance and anaerobic exercise performance.

## Introduction

Despite numerous scientific data, there is still no conclusive answer regarding what and how much we should drink to optimize sports performance. Until the middle of the 20th century, the recommendation was to avoid drinking to optimize performance. The first drinking guidelines were introduced by the ACSM to avoid heat stress in 1975, while hydration and performance were first addressed only in 1996 []. At that time, athletes were encouraged to drink the maximum amount of fluids during exercise that could be tolerated without gastrointestinal discomfort and up to the rate lost through sweating. Depending on the type of exercise and the environment, volumes from 0.6 to 1.2 L per hour were recommended. These drinking guidelines have been questioned recently, and other issues such as over hydration and hyponatremia have been addressed [].

The inconsistency of the results regarding hydration and sports performance arise from differences in experimental protocols. In studies in which dehydration develops during exercise, fluid loss of up to 4% body mass does not compromise performance, while in studies that induced dehydration prior to exercise, performance impairments have been observed after dehydration as low as 1–2% body mass []. Several comprehensive reviews on the influence of dehydration on muscle endurance, strength, anaerobic capacity, jumping performance and skill performance in team sport games have revealed negative effects of dehydration ≥ 2% body mass []. Hydration is one of the most significant issues for combat sports, as athletes often use water restriction for quick weight loss before competition. During tournaments lasting several hours, combat sport athletes sweat immensely and increase their core temperature affecting muscle strength, reducing motor cortex activation, peripheral stimulus as well as the speed of reaction and power output [].

Considering the vast amounts of fluids used during exercise, water seems to be the most often form of hydration. Water comes in different forms, with specific properties depending on its mineral content. The pH of water, as well as the proportions between SO42- and HCO3 determines hydration status and other therapeutic properties []. Drinking hydrogen rich water in human nutrition is a rather new concept, and it is recently suggested for medical purposes and hydration during exercise []. Alkaline ionized water is being marketed as a nutritional aid for the general public for acidity-lowering, antioxidant, and antiaging properties. Some of the animal and human research has confirmed its effectiveness as an alkalizing agent in the treatment of metabolic acidosis []. However, metabolic acidosis that occurs during high intensity exercise is a distinct form of metabolic alteration, when cells are forced to rely on anaerobic ATP turnover that leads to proton release and a decrease in blood pH that can impair performance [].

Anaerobic exercise metabolism leads to the production of lactic acid in the working muscles. Part of the produced lactic acid is released to the blood, reducing blood pH, and disturbing acid—base balance. Several studies have provided evidence that hydrogen ions are released from the muscles in excess of lactate after intense exercise []. Two mechanisms have been proposed to explain this phenomenon. It seems that hydrogen ions are released both by a sodium-hydrogen ion exchanger and by a lactic acid transporter []. Since red blood cells have a higher buffering capacity than blood plasma, the lactate generated during exercise largely remains in the plasma while hydrogen ions are transferred to the red blood cells and buffered by hemoglobin []. One of the objectives of training and supplementation in high intensity anaerobic sports disciplines is to increase the buffering capacity of the blood and tissues []. The use of sodium bicarbonate has proven effective in speed endurance and strength endurance sports, yet its use has been limited due to the possibility of gastrointestinal distress, metabolic alkalosis, and even edema due to sodium overload []. It appears that alkaline water can be an effective alternative to sodium bicarbonate in preventing exercise-induced metabolic acidosis []. Contrary to bicarbonate, alkaline water can be used on an everyday basis and has no known side effects. However, there are only few cross-sectional or longitudinal studies on the impact of alkaline water ingestion in combat sport athletes. Therefore, the main objective of the current study was to investigate in a double blind, placebo controlled randomized study, the impact of mineral-based highly alkaline water on acid-base balance, hydration status, and anaerobic capacity in experienced combat sport athletes subjected to a very intense exercise protocol.

## Materials and methods

### Subjects

Sixteen very well-trained males, who trained and competed in combat sports for at least 7.6 years, participated in the study. The athletes constituted a homogenous group in regard to age (average age of 22.3 ± 0.5 years), somatic characteristics, as well as aerobic and anaerobic performance (Table 1). The subjects (n = 16) were randomly divided into two groups, the experimental group (EG; n = 8), which received highly alkaline ionized water, and the control group (CG; n = 8), which was hydrated with table water. All subjects had valid medical examinations and showed no contraindications to participate in the study. The athletes were informed verbally and in writing of the experimental protocol, the possibility to withdraw at any stage of the experiment, and gave their written consent for participation. The study was approved by the Research Ethics Committee of the Academy of Physical Education in Katowice, Poland.

### Table 1

Characteristics of the study participants.
Variables Experimental Group
(n = 8)
Control Group
(n = 8)
Age (yrs.) 22.7±3.2 22.4 ± 2.8
Height (cm) 181.2±2.1 178.3±4.9
Body mass (kg) 81.8±3.2 79.2 ±2.6
FM (%) 10.2±2.1 10.8±2.4
Wt—upper limbs (J/kg) 138±14 136±19
Wt—lower limbs (J/kg) 276±04 283±26
Pmax–lower limbs (W/kg)
Pmax–upper limbs (W/kg)
19.8±0.9
8.9±1.1
20.2±1.6
8.7±0.4
VO2max (ml/kg/min) 64.7±2.8 62.6±3.2

### Diet and hydration protocol

Energy intake, as well as macro and micronutrient an intake of all subjects was determined by the 24 h nutrition recall 3 weeks before the study was initiated. The participants were placed on an isocaloric (3455 ± 436 kcal/d) mixed diet (55% carbohydrates, 20% protein, 25% fat) prior and during the investigation. The pre-trial meals were standardized for energy intake (600 kcal) and consisted of carbohydrate (70%), fat (20%) and protein (10%). During the experiment, and 3 weeks before the commencement of the study, the participants did not take any medications or supplements. Throughout the experiment water intake was individualized based on the recommendation of the National Athletic Trainers Association and averaged 2.6–3.2 L per day. In our study we used water which had a pH of 9.13 which is highly alkaline compared to other commercially available products. The water ingested during the experiment contained 840 mg/dm3 of permanent ingredients, and was classified as medium mineral content. The bicarbonate ion HCO3 (357.8 mg/dm3) and carbonate ion CO32- (163.5 mg/dm3) consisted the dominant anions. Sodium (Na+ 254.55 mg/dm3) dominated among cations. The water contained bicarbonate, carbonate-sodium (HCO3, CO3Na+). The chemical properties of both types of water used in the experiment (alkaline and table water) are presented in Table 2.

### Table 2

Chemical properties of water used in the study.
Variable Measurement Unit Alkaline Water Table Water
pH pH 9.13 ± 0.04 5.00 ± 0.08
CO32- mg/dm3 163.5 ± 6.3 14.98 ± 0.66
HCO3 mg/dm3 357.8 ± 6.14 3.62 ± 0.12
Cl mg/dm3 26.4 ± 2.3 0.41 ± 0.03
SO42- mg/dm3 7.81± 1.2 1.60 ± 0.09
Na+ mg/dm3 254.55 ± 7.1 1.21 ± 0.05
K+ mg/dm3 0.91 ± 0.04 0.30 ± 0.03
Ca2+ mg/dm3 10.00 ± 1.6 1.21 ± 0.05
Mg2+ mg/dm3 0.37 ± 0.03 0.40 ± 0.04

Note: Data shows mean values ± SD of three analysis of each type of water

### Study protocol

The experiment lasted 3 weeks, during which two series of laboratory analyses were performed. The tests were carried out at baseline and after three weeks of hydration with alkaline or table water. The study was conducted during the preparatory period of the annual training cycle, when a high volume of work dominated the daily training loads. The participants refrained from exercise for 2 days before testing to minimize the effect of fatigue.

The subjects underwent medical examinations and somatic measurements. Body composition was evaluated in the morning, between 8.00 and 8.30 am. The day before, the participants had the last meal at 20.00. They reported to the laboratory after an overnight fast, refraining from exercise for 48h. The measurements of body mass were performed on a medical scale with a precision of 0.1 Kg. Body composition was evaluated using the electrical impedance technique (Inbody 720, Biospace Co., Japan). Anaerobic performance was evaluated by a two double 30-second Wingate test protocol for lower and upper limbs respectively, with a passive rest interval of 3 minutes between the bouts of exercise. The test was preceded by a 5 min warm-up with a resistance of 100 W and cadence within 70–80 rpm for lower limbs and 40 W and 50–60 rpm for the upper limbs. Following the warm-up, the test trial started, in which the objective was to reach the highest cadence in the shortest possible time, and to maintain it throughout the test. The lower limb Wingate protocol was performed on an Excalibur Sport ergocycle with a resistance of 0.8 Nm·Kg-1 (Lode BV, Groningen, Netherland). The upper body Wingate test was carried out on a rotator with a flying start with a load of 0.45 Nm·Kg-1 (Brachumera Sport, Lode, Netherland). Each subject completed 4 test trials with incomplete rest intervals. The variables of peak power–Pmax (W/Kg) and total work performed–Wt (J/Kg), were registered and calculated by the Lode Ergometer Manager (LEM, software package, Netherland).

### Biochemical assays

To determine lactate concentration (LA), acid-base equilibrium and electrolyte status the following variables were evaluated: LA (mmol/L), blood pH, pCO2 (mmHg), pO2 (mmHg), HCO3- act (mmol/L), HCO3-std, (mmol/L), BE (mmol/L), O2SAT (mmol/L), ctCO2 (mmol/L), Na+ (mmol/L), and K+ (mmol/L). The measurements were performed on fingertip capillary blood samples at rest and after 3 minutes of recovery. Determination of LA was based on an enzymatic method (Biosen C-line Clinic, EKF-diagnostic GmbH, Barleben, Germany). The remaining variables were measured using a Blood Gas Analyzer GEM 3500 (GEM Premier 3500, Germany).

Urine samples were taken at rest, after an overnight fast, at baseline and at the conclusion of the investigation. They were placed in a plastic container and mixed with 5 ml/L of 5% solution of isopropyl alcohol and thymol for preservation. Urine samples were assayed for the presence of blood and proteins. Specific gravity was determined using the Atago Digital refractometer (Atago Digital, USA). Urine pH was determined based on the standardized Mettler Toledo potentiometer (Mettler Toledo, Germany).

### Statistical analysis

The Shapiro-Wilk, Levene and Mauchly´s tests were used to verify the normality, homogeneity and sphericity of the sample’s data variances, respectively. Verifications of the differences between analyzed variables before and after water supplementation and between the EG and CG were performed using ANOVA with repeated measures. Effect sizes (Cohen’s d) were reported where appropriate. Parametric effect sizes were defined as large for d > 0.8, as moderate between 0.8 and 0.5, and as small for < 0.5 (Cohen 1988; Maszczyk et al., 2014, 2016). Statistical significance was set at p<0.05. All statistical analyses were performed using Statistica 9.1 and Microsoft Office, and were presented as means with standard deviations.

## Results

All participants completed the described testing protocol. All procedures were carried out in identical environmental conditions with an air temperature of 19.2°C and humidity of 58% (Carl Roth hydrometer, Germany).

The repeated measures ANOVA between the experimental and control group and between the baseline and post-intervention period (3 weeks of alkaline and table water ingestion) revealed statistically significant differences for thirteen variables (Table 3).

### Table 3

Statistically significant differences between the experimental and control groups at baseline and after 3 weeks of intervention (alkaline vs table water).
Variables d p F
Wingate Lower Limbs Average Power Exp. 0.884 0.001 21.161
Wingate Upper Limbs Average Power Exp. 0.587 0.011 8.528
Wingate UL Peak Power Exp. 0.501 0.026 6.228
Wingate LL Total Work Exp. 0.567 0.045 4.822
Wingate UL Total Work Exp. 0.522 0.011 8.459
LA rest 0.534 0.008 9.429
LA post exr 0.618 0.003 13.382
pH rest 0.834 0.001 120.159
HCO3 rest 0.844 0.001 109.250
HCO3 post exr 0.632 0.002 14.724
K+ post exr 0.501 0.040 5.154
Urine pH 0.589 0.017 7.298
SG 0.884 0.001 19.707

Note: d—effect size; p—statistical significance

F–value of analysis of variance function

Post-hoc tests revealed a statistically significant increase in mean power when comparing the values (7.98 J/kg to 9.38 J/kg with p = 0.001) at baseline vs. at the conclusion of the study in the experimental group supplemented with alkaline water. In contrast, the control group which received table water did not reveal any statistically significant results.

Similar changes were observed for Upper Limb Average Power (from 4.32 J/kg to 5.11 J/kg with p = 0.011) and Upper Limb Peak Power (from 7.90 J/kg to 8.91 J/kg with p = 0.025) in the experimental group. The post-hoc tests also showed statistically significant increases in values for Lower Limb Total Work (from 276.04 J/kg to 292.96 J/kg with p = 0.012) and Upper Limb Total Work (from 138.15 J/kg to 156.37 J/kg with p = 0.012) when baseline and post intervention values were compared. The changes in the control group were not statistically significant. These results are presented in Fig 1.

Differences between the control and experimental groups in total work of the lower and upper limbs (30s Wingate test) at baseline and after 3 weeks of alkaline or table water ingestion.

Note: * statistically significant values.

Post-hoc tests also revealed statistically significant decreases in LA concentration at rest (from 1.99 mmol/L to 1.30 mmol/L with p = 0.008), and a significant increase in post exercise LA concentration (from 19.09 mmol/L to 21.20 mmol/L with p = 0.003) in the experimental group ingesting alkaline water.

Additionally, a significant increase in blood pH at rest (from 7.36 to 7.44 with p = 0.001), HCO3 at rest (from 23.87 to 26.76 with p = 0.001), and HCO3 post exercise (from 12.90 to 13.88 with p = 0.002) were observed in the experimental group. The other significant changes occurred in the post exercise concentration of K+ (from 4.15 to 4.41 with p = 0.039), in urine pH (from 5.75 to 6.62 with p = 0.017), and a decrease in the value of SG (from 1.02 to 1.00 with p = 0.001), all in the experimental group supplemented with alkaline water.

## Discussion

Acid-base equilibrium within the human body is tightly maintained through the blood and tissue buffering systems, the diffusion of carbon dioxide from the blood to the lungs via respiration, and the excretion of hydrogen ions from the blood to urine by the kidneys. These mechanisms also regulate acid-base balance following high intensity exercise. Metabolic acidosis is a consequence of exercise induced ionic changes in contracting muscles. Increased intramuscular acidity impairs muscle contractibility, significantly limiting high intensity exercise performance []. Importantly, acid-base equilibrium can be influenced by dietary supplementation.

In the present study, we investigated the effect of mineral-based alkaline water on acid-base balance, hydration status and anaerobic performance of competitive combat sport athletes. The study participants were experienced athletes (Table 1), capable of performing extreme anaerobic efforts. We have chosen such an approach for two reasons. First, it is well-documented that consumption of alkalizing water can have a significant effect on the hydration status, acid-base balance, urine and blood pH [], as well as Ca metabolism and bone resorption markers []. However, the majority of these research reports have been performed on sedentary individuals [] or on subjects with self-reported physical activity []. Second, alkalization by alkaline water has been mostly discussed in the context of dehydration and aerobic performance []. Therefore, our study is novel by including both well trained combat sport athletes and the use of an extremely intensive anaerobic exercise protocol.

### Acid-base balance and hydration status

The exchange of ions, CO2, and water between the intracellular and extracellular compartments helps to restore acid-base balance following intensive exercise. There is sufficient data indicating that, supplements that modify the blood buffering system affect high-intensity exercise performance []. In humans, especially well trained athletes muscle pH may decrease from 7.0 at rest to values as low as 6.4–6.5 during exercise []. Ergogenic aids that help buffer protons attenuate changes in pH and enhance the muscle’s buffering capacity. This in turn allows for a greater amount of lactate to accumulate in the muscle during exercise.

The results of our study are in line with the available literature regarding the impact of alkaline water on blood and urine pH at rest []. However, novel results of the present research are related to the changes in HCO3- after exercise in athletes ingesting alkaline water. Bicarbonate-CO2 accounts for more than 90% of the plasma buffering capacity. Supplementation can increase bicarbonate concentration in the blood and its pH. Since bicarbonate concentration is much lower in the muscles (10 mmol/L) than in the blood (25 mmol/L), the low permeability of charged bicarbonate ions precludes any immediate effects on muscle acid-base status []. These results confirm the view that an appropriate hydration status is necessary for active bicarbonate ion transport.

Several lines of evidence support the negative impact of dehydration (>2% body mass) on muscle endurance, strength, and anaerobic performance []. On the other hand, literature data indicates that consumption of alkaline water following a dehydrating bout of cycling exercise was shown to rehydrate cyclists faster and more completely compared to table water. Following consumption of alkaline water, the cyclists demonstrated lower total urine output, their urine was more concentrated (i.e., with higher specific gravity), and the total blood protein concentration was lower, indicating improved hydration status [].

Our previous study revealed that the use of water with alkalizing properties exhibits a significant potential for hydration during anaerobic exercise []. The results of the present study confirm a decrease in urine specific gravity (from 1.02 to 1.00, with p = 0.001) and an increase in urine pH as the result of consumption of alkaline water. These results illustrate that the habitual consumption of highly alkaline water can markedly improve hydration status.

### Anaerobic performance

The current investigation demonstrated a significant increase in anaerobic capacity (Wt−J/Kg) of athletes in the experimental group supplemented with alkaline water. The improvements in Wt following alkaline water consumption were influenced by positive changes in blood pH and bicarbonate. This phenomenon could be explained by the ergogenic effects of high alkalization and mineral ingredients.

High intensity exercise in which anaerobic glycolysis provides ATP for muscle contraction leads to an equal production of lactate and hydrogen ions. Most of the released hydrogen ions are buffered; however, a small portion (~0.001%) that remains in the cytosol causes a decrease in muscle pH and an impairment of exercise. Lactate efflux [] and its oxidation are accompanied by a similar removal of hydrogen ions. The results of the current study demonstrated a statistically significant decrease in lactate concentration at rest (from 1.99 mmol/L to 1.30 mmol/L, p = 0.008), and a significant increase post exercise (from 19.09 mmol/L to 21.20 mmol/L, p = 0.003) when compared to the baseline levels with the values recorded at the end of alkaline water supplementation. The extremely intense 4 x 30s upper/lower limb Wingate test protocol employed in our study, with only short rest intervals between each bout of exercise, was a likely reason that less of the total lactate produced in the muscles was transported to the blood [].

Muscle blood flow determines lactate efflux from the muscle [], and is dependent on the activity of lactate transport proteins [], the extracellular buffering capacity [], and the extracellular lactate concentration []. Thus, our results on lactate concentration are in agreement with the view that anaerobic performance (i.e., Wt−J/Kg, WAvr−J/Kg) depends on counter-regulatory variables. Indeed, we demonstrated that changes in resting blood pH and HCO3 significantly improved anaerobic performance.

Another variable that can affect anaerobic performance includes blood viscosity. Weidmann et al. (2016) showed that the intake of highly alkaline water decreased blood viscosity by 6.30%, compared to table water (3.36%) in 100 recreationally active female and male subjects. Therefore, it may be possible that the excess of metabolic end-products (namely, H+ and Pi), which disturb cellular homeostasis and muscle contraction, are more effectively transported. The available literature data does not specify clearly which components of buffering capacity are altered by the above changes. It must be indicated, that there are several methods available to determine muscle buffering capacity. Due to the methodological complexity, none of these methods are free from criticism. In most studies buffering capacity has been determined in vitro by titration, which does not include trans-membrane transport of acid-base substances or dynamic buffering by biochemical processes occurring in vivo [].

Most studies show a documented ergogenic effect of bicarbonate loading during exhaustive exercise lasting 1–7 min, when anaerobic glycolysis plays a major role in energy provision []. The rationale for the ergogenic effect of bicarbonate is that the increase in extracellular pH and bicarbonate will enhance the efflux of lactate and H+ from muscle. There is also evidence that the ergogenic effect of bicarbonate is more pronounced during repeated sprints than during sustained exercise [].

Different strategies used for improving buffering capacity of tissues and blood do not allow for a direct comparison. Despite this, there appears to exist an ergogenic effect in response to NaHCO3, what may explain the large effect size noted by Tobias et al. []. In our research we obtained large effect sizes with regards to 4 variables (Average power of the lower limbs, resting HCO3, resting blood pH and urine SG).

## Conclusions

The results of the present study indicate that drinking alkalized water improves hydration status, acid-base balance, and high intensity anaerobic exercise performance. It appears that both greater muscle buffering capacity and enhanced removal of protons, resulting in increased glycolytic ATP production, may be responsible for these effects. Considering the energy demands and the intense sweat rate of combat sport athletes, the authors recommend the daily intake of 3–4 L of highly alkaline mineralized water to improve hydration and anaerobic performance during training and competition.

## Supporting information

Data for Fig 1.

(XLSX)

#### S2 Table

Stress test data.

(XLSX)

Water data.

(XLSX)

## Acknowledgments

This work was supported by the Ministry of Science and Higher Education of Poland under Grant NRSA3 03953 and NRSA4 040 54.

## Funding Statement

This work was supported by the Ministry of Science and Higher Education of Poland under Grant NRSA3 03953 and NRSA4 040 54.

## Data Availability

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

. 2018; 13(11): e0205708.
Published online 2018 Nov 19.
PMCID: PMC6242303
PMID: 30452459
###### Alkaline ionized water improves exercise-induced metabolic acidosis and enhances anaerobic exercise performance in combat sport athletes
Jakub ChyckiConceptualizationInvestigationMethodologyWriting – original draft,1,* Anna KurylasData curationMethodologyProject administration,1 Adam MaszczykData curationValidationVisualization,2Artur GolasData curationFormal analysis,1 and Adam ZajacConceptualizationInvestigationMethodologyWriting – original draft1
Michal Toborek, Editor
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

## Associated Data

Supplementary Materials
Data Availability Statement
All relevant data are within the paper and its Supporting Information files.

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# alkaline ionized water and longevity

## Abstract

The biological effect of alkaline water consumption is object of controversy. The present paper presents a 3-year survival study on a population of 150 mice, and the data were analyzed with accelerated failure time (AFT) model. Starting from the second year of life, nonparametric survival plots suggest that mice watered with alkaline ionized water showed a better survival than control mice. Interestingly, statistical analysis revealed that alkaline ionized water provides higher longevity in terms of “deceleration aging factor” as it increases the survival functions when compared with control group; namely, animals belonging to the population treated with alkaline ionized water resulted in a longer lifespan. Histological examination of mice kidneys, intestine, heart, liver, and brain revealed that no significant differences emerged among the three groups indicating that no specific pathology resulted correlated with the consumption of alkaline ionized water. These results provide an informative and quantitative summary of survival data as a function of watering with alkaline ionized water of long-lived mouse models.

## 1. Introduction

Alkaline water, often referred to as alkaline ionized water (AKW), is commercially available and is mainly proposed for electrolyte supplementation during intensive perspiration. Early studies on animal models reported that alkaline ionized water supplementation may exert positive effects on body weight improvement and development in offspring []. Even biochemical markers were analyzed, suggesting that alkaline ionized water intake can cause elevation of metabolic activity. In particular, hyperkaliemia was observed in 15-week-old rats and pathological changes of necrosis in myocardial muscle were found [].

More recently, studies were carried out on alkaline ionized/electrolysis reduced water (ARW), referring to electrolyzed water produced from minerals, such as magnesium and calcium, which is characterized by supersaturated hydrogen, high pH, and a negative redox potential ORP. This hydrogen-rich functional water has been introduced as a therapeutic strategy for health promotion and disease prevention [].

Alkaline ionized/ electrolyzed reduced water have been shown to exert a suppressive effect on free radical levels in living organisms, thereby resulting in disease prevention []. Various biological effects, such as antidiabetic and antioxidant actions [], DNA protecting effects [], and growth-stimulation activities [], were documented.

Although a variety of bioactive functions have been reported, the effect of alkaline water on lifespan and longevity in vivo is still unknown. Animal alkalization has been shown to be well tolerated and to increase tumor response to metronomic chemotherapy as well the quality of life in pets with advanced cancer []. Therefore, we performed a study based on survival rate experiments, which play central role in aging research and are generally performed to evaluate whether specific interventions may alter the aging process and lifespan in animal models.

## 2. Materials and Methods

Biological effects of alkaline ionized water were evaluated on a selected population of 150 mice (CD1, by Charles River, Oxford, UK). Pathogen-free mice were purchased and placed in a specific breeding facility. No other animal was present in the room. Contact with animal caretakers was minimized to feeding and watering. The population was divided into 3 groups, each consisting of 50 individuals, as follows:

1. Group A: 50 mice conventionally fed and watered with alkaline ionizefd water produced by the Water Ionizer (mod. NT010) by Asiagem (Italy). The Water Ionizer is a home treatment device for producing alkaline drinking water.
2. Group B: 50 mice conventionally fed and watered with alkalized water obtained by dilution of a concentrated alkaline solution (AlkaWater by Asiagem, Italy). AlkaWater is a concentrated alkaline solution for preparing alkaline drinking water.
3. Group C: 50 mice conventionally fed and watered as conventional (control group) with tap water. The local water supply was evaluated weekly for assuring the absence of toxins and pathogens. The pH values were in the 6.0–6.5 range.

All procedures involving animals were conducted in accordance with the Italian law on experimental animals and were approved by the Ethical Committee for Animal Experiments of the University of Padua and the Italian health Ministry (Aut. no. 39ter/2011). Efforts were made to minimize animal suffering.

### 2.1. Histological Examination

Treated aged mice were sampled postmortem and subjected to histological examination. Animals belonging to the populations treated with alkaline water, A and B, were sacrificed after 24 months and compared to mice treated with tap water. Samples from kidneys, intestine, heart, liver, and brain were fixed in 10% neutral buffered formalin, and 4 μm sections were analyzed by optical microscopy.

### 2.2. Statistical Analysis

In order to investigate the biological influence of alkaline water on mouse longevity, we employed the accelerated failure time model (AFT) [], which allows formally exploring the possible effect on survival curves of the applied three-level treatment, that is, examining the role of group membership as a covariate of lifespan. As a more robust alternative to the commonly used proportional hazards models, such as the Cox model, the use of AFT models is advised in the field of survival analysis when the goal is to investigate if a covariate may affect the lifespan in a way that the life cycle may pass more or less rapidly. In fact, whereas a proportional hazard model assumes that the effect of a covariate is constant over time, an AFT model assumes that the effect of a covariate is to accelerate or decelerate the life course.

The relevance of AFT model for biomedical studies has been already recognized in the literature []. With more specific reference to the issue of aging, Swindell [] observed that some genetic manipulations were found to have a multiplicative effect on survivorship which were well characterized by the AFT model “deceleration factor.” Moreover, Swindell [] argued also that the AFT model should be utilized more widely in aging research since it provides useful tools to maximize the insight obtained from experimental studies of mouse survivorship.

To perform all calculations, we applied a parametric survival analysis approach using a class of 3-parameter AFT distribution models implemented within the statistical software Minitab, version 17.2.1 []. More specifically, we employed three types of random distributions, namely, log-logistic, log-normal, and generalized Weibull.

## 3. Results

The experiment consisted in an initial 15-day acclimatization period. After acclimatization, animals (50, group A) were watered with alkaline ionized  water (pH 8.5), obtained by the Water Ionizer ,  whereas group B animals (50) were watered with water alkalized at pH 8.5 by a concentrated alkaline solution  for 15 days. Group C animals (50), control group, were watered with the local water supply. This period has been identified to gradually accustom the animals treated with alkaline water. At the end of the second period of acclimatization, group A and B animals were watered with alkaline ionized water at pH 9.5, while animals of group C were watered with local tap water.

After the first year, the most aggressive individuals were moved to other cages within the same group and an environmental enrichment protocol was employed in order to decrease the hyperactivity. This phenomenon was observed especially in animals of groups A and B.

Table 1 reported basic statistics on mice survival of treated and control animals.

### Table 1

Basic statistics on mice survival by treatment level.

Treatment level Mortality rate
%
Lifespan mean (std. dev.)
Days
Group A 88 679 (209)
Group B 92 671 (180)
Group C 96 667 (185)

Regarding group A, animals (50) were watered with alkaline ionized water (pH 8.5), obtained by the Water Ionizer (Asiagem, Italy). As for group B, animals (50) were watered with water alkalized at pH 8.5 by a concentrated alkaline solution for 15 days. Regarding group C, animals (50), control group, were watered with the local water supply.

A first look on experimental data is provided in Figure 1, where nonparametric hazard and survival plots seem to suggest that even if no macroscopic difference emerges, starting from the second year of life mice watered with alkaline ionized  Water  and those treated with AlkaWater overwhelmed control mice.

Nonparametric hazard and survival plots by treatment level. Group A: animals (50) were watered with alkaline ionized water (pH 8.5), obtained by the Water Ionizer  Group B: animals (50) were watered with water alkalized at pH 8.5 by a concentrated alkaline solution  for 15 days. Group C: animals (50), control group, were watered with the local water supply.

In order to explore the possible effect of different treatments, that is, to examine the role of group membership on longevity, we applied a parametric survival analysis approach using a class of 3-parameter survival distributions that represent flexible accelerated failure time, AFT models. First of all, using the Anderson-Darling goodness-of-fit statistic, we compared three specific survival distributions, that is, log-logistic (AD = 6.397), log-normal (AD = 6.519), and generalized Weibull (AD = 6.447). Since the best fitting was shown by log-logistic model, we adopted this one as final survival distribution model. The straight lines in the log-logistic distribution QQ plots (Figures 2(a) and 2(b)) indicate that this distribution provides a suitable fit to our survival data.

QQ plots using the 3-parameter log-logistic distribution model. (a) Treatment A survival time quantiles (vertical axis) versus treatment C survival time quantiles (horizontal axis); (b) treatment B survival time quantiles (vertical axis) versus treatment C survival time quantiles (horizontal axis).

Finally, by including our treatment as covariate, we performed a parametric distribution analysis whose results are graphically represented in Figure 3.

Distribution plot results using the 3-parameter log-logistic model. Group A: animals (50) were watered with alkaline ionized water (pH 8.5), obtained by the Water Ionizer . Group B: animals (50) were watered with water alkalized at pH 8.5 by a concentrated alkaline solution for 15 days. Group C: animals (50), control group, were watered with the local water supply.

Starting with the second year of life, it is worth noting that both alkaline water treated groups denote a decreasing hazard curve over time, while the corresponding curve for control group is monotonically increasing. To more formally compare the treatment levels, the proposed analysis provided also suitable pvalues. Since the p values related on the null hypotheses of equality of location, scale and threshold parameters were, respectively, less than 0.001 (for both locations and scales) and 0.634 (for thresholds) at a 5% significance level; we can state that there is enough experimental evidence to conclude that the treatment significantly affects the mice longevity; in particular the alkaline ionized water provides a benefit to longevity in terms of “deceleration aging factor” as it decreases the hazard functions when compared with the control group. Note that the treatment effect cannot be directly related to no one of the three distribution parameters. Anyway, using the estimated parameters, it should be possible to provide an estimate for the effect of each treatment on survivorship: setting the reference survival time to 1000, 1200, and 1400 days, Table 2 summarizes the estimated point and 95% interval survival probabilities by each treatment level.

### Table 2

Table of survival probabilities by treatment level. The probabilities, along with their related 95% confidence interval limits, were calculated using the normal approximation.

Treatment level Time (days) Estimated probability Lower 95% CI limit Upper 95% CI limit
A 1000 0.116 0.056 0.226
1200 0.046 0.014 0.140
1400 0.020 0.004 0.098

B 1000 0.055 0.021 0.137
1200 0.013 0.003 0.066
1400 0.004 0.000 0.039

C 1000 0.049 0.022 0.106
1200 0.008 0.002 0.027
1400 0.001 0.000 0.007

As final remark, it should be noted that even if our parametric AFT survival analysis was performed using the log-logistic distribution, our conclusions are consistent with results obtained using the generalized Weibull distribution, while via log-normal distribution no significant effect was found.

### 3.1. Histological Examination

No significant differences emerged from the histological examination among the three groups. In all examined samples, renal tissue was characterized by a mild-to-moderate lymphoplasmacytic interstitial infiltrate and few occasional glomerular changes as glomerular size reduction and increasing of Bowman’s space (Figure 4).

Kidney, a specific chronic nephropathy. Focal interstitial mainly lymphocytic infiltrate (upright) and a sclerotic glomerulus (middle right). Hematoxylin and Eosin.

Final diagnosis was mild chronic progressive nephropathy for the three analyzed mouse groups.

The microscopic examination of the liver revealed a multifocal nodular pattern of the parenchyma and diffuse mild-to-moderate hepatocellular cytoplasmic hydropic degeneration with multifocal binucleation in all explored animals (Figure 5).

Liver, aging change. Hepatocellular abundant dishomogeneous cytoplasm, binucleation (center), variably sized nuclei, and a nuclear pseudoinclusion cyst (arrow). Hematoxylin and Eosin.

Mild-to-moderate anisokaryosis was the most relevant alteration, with few pleomorphic nuclei and frequent intranuclear pseudoinclusions and karyomegaly. A specific mild perivascular infiltrate was occasionally present. Final diagnosis was mild-to-moderate diffuse hepatopathy with multifocal hyperplastic hyperplasia.

The pulmonary parenchyma showed mild multifocal areas of interstitial thickening of the interalveolar septa due to moderate congestion and mild cellular mixed infiltrate (Figure 6). Mild areas of emphysema were detected at the periphery of the parenchyma. Final diagnosis was multifocal very mild atelectasis and mild vicarious emphysema.

Lung, mild atelectasis. Very mild multifocal interstitial thickening of the alveolar septa associated with congestion and mild cellular increase. Hematoxylin and Eosin.

At the same time, no relevant histopathologic histological changes have been noticed in intestine (Figure 7), brain, and heart.

Intestine. Longitudinal section of duodenum showing uniformly thin and elongated villi. Hematoxylin and Eosin.

## 4. Discussion

The present work presents a 3-year survival study on a population of 150 mice and the data were analyzed with accelerated failure time (AFT) model. Kaplan-Meier statistical analysis of the survival data indicates the possibility of a positive effect of alkaline ionized water on mouse lifespan and AFT model allowed evaluating differences starting from the second year of the survival curves. These results provide an informative and quantitative summary of survival data as a function of watering with alkaline ionized water on long-lived mouse models. It should be pointed out that, from the standpoint of aging research, this statistical approach presents appealing properties and provides valuable tools for the analysis of survival. The observation of tissues of deceased animals was performed for the assessment of the state of internal organs to be compared with similar analyses of untreated animals. The renal lesions observed at histology were specific and common for the three animal groups. Chronic progressive nephropathy has been well described as normal aging change in mice []. In our cases animals did not show any clinical sign of nephropathy or any other histological evidence of specific kidney disease and we ascribed the lesions to the aging process [].

The examined livers were also affected by typical lesions of mature subjects, such as hyperplastic nodules. Furthermore, well known aging changes were individuated in the hepatocytes, such as karyomegaly, nuclear pleomorphism, and pseudoinclusions cysts [].

## 5. Conclusions

A 3-year survival study on a population of 150 mice was carried out in order to investigate the biological effect of alkaline water consumption. Firstly, nonparametric hazard and survival plots suggest that mice watered with alkaline ionized water overwhelmed control mice. Secondly, data were analyzed with accelerated failure time (AFT) model inferring that a benefit on longevity, in terms of “deceleration aging factor,” was correlated with the consumption of alkaline ionized water. Finally, histological examination of mice kidneys, intestines, hearts, livers, and brains was performed in order to verify the risk of diseases correlated to alkaline watering. No significant damage, but aging changes, emerged; organs of alkaline watered animals resulted to be quite superimposable to controls, shedding a further light in the debate on alkaline water consumption in humans.

## Acknowledgments

This paper is dedicated to the memory of Tommaso Nicoletti. The authors are grateful to Rocco Palmisano for original ideas and support. The authors would like to thank Asiagem (Italy) for partial support and Ludovico Scenna, Carlo Zatti, and Silvano Voltan for their scientific and professional contribution.

## Competing Interests

The authors declare that there are no competing financial interests.

Published online 2016 May 31.
PMCID: PMC4906185
PMID: 27340414

## References

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3. Watanabe T., Kishikawa Y., Shirai W. Influence of alkaline ionized water on rat erythrocyte hexokinase activity and myocardium. Journal of Toxicological Sciences1997;22(2):141–152. doi: 10.2131/jts.22.2_141. [PubMed] [CrossRef]
4. Jin D., Ryu S. H., Kim H. W., et al. Anti-diabetic effect of alkaline-reduced water on OLETF rats. Bioscience, Biotechnology and Biochemistry2006;70(1):31–37. doi: 10.1271/bbb.70.31. [PubMed] [CrossRef]
5. Hanaoka K., Sun D., Lawrence R., Kamitani Y., Fernandes G. The mechanism of the enhanced antioxidant effects against superoxide anion radicals of reduced water produced by electrolysis. Biophysical Chemistry2004;107(1):71–82. doi: 10.1016/j.bpc.2003.08.007. [PubMed] [CrossRef]
6. Shirahata S., Kabayama S., Nakano M., et al. Electrolyzed-reduced water scavenges active oxygen species and protects DNA from oxidative damage. Biochemical and Biophysical Research Communications1997;234(1):269–274. doi: 10.1006/bbrc.1997.6622. [PubMed] [CrossRef]
7. Spugnini E. P., Buglioni S., Carocci F., et al. High dose lansoprazole combined with metronomic chemotherapy: a phase I/II study in companion animals with spontaneously occurring tumors. Journal of Translational Medicine2014;12:p. 225. doi: 10.1186/s12967-014-0225-y.  [PubMed] [CrossRef]
8. Collett D. Modelling Survival Data in Medical Research. 3rd. Chapman & Hall/CRC; 2014. (Texts in Statistical Science).
9. Swindell W. R. Accelerated failure time models provide a useful statistical framework for aging research. Experimental Gerontology2009;44(3):190–200. doi: 10.1016/j.exger.2008.10.005. [PubMed] [CrossRef]
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Articles from Evidence-based Complementary and Alternative Medicine : eCAM are provided here courtesy of Hindawi Limited

# Studies and observations on the health effects of drinking electrolyzed-reduced alkaline water ( ionized)

B. Rubik Institute for Frontier Science, Oakland, California, USA

Abstract

Studies and observations on the health effects of drinking electrolyzed-reduced alkaline(ionized ) water Municipal drinking water, prefiltered and treated by partial electrolysis, followed by collecting the cathodic water that is alkaline (pH 8.5 to 9.5), shows a high negative oxidative reductive potential (ORP) (-150 to -250 mV) compared to untreated tap water (+150 mV) as well as smaller molecular clusters.

A growing body of literature indicates beneficial effects from drinking this electrolyszed (ionized) alkaline water by patients with diabetes and kidney disease, with improved outcomes and fewer medical complications. Additional studies suggest that this water increases the activity of a key detoxifying enzyme in the body, superoxide dismutase, which is central to protecting against free radical damage both in aging and chronic degenerative disease. Recent published studies on the health benefits of this drinking (alkaline ionized/electrolyzed reduced ) water are summarized. Evidence from live blood analysis from a case study suggests that drinking reduced alkaline water reduces blood cell stickiness, aggregation, and early clotting. Results suggest that long term consumption of this water slows the effects of aging and may improve the peripheral circulation; serve as an adjunct therapy for diabetes and kidney disorders; and help prevent cardiovascular and other chronic diseases.

### 1 Introduction

Water, which constitutes over 70% of the human body, is involved in virtually every function of life. It is an essential but often underrated necessity that is involved in most biochemical reactions; a constituent of the bodily fluids – blood, lymph, cerebrospinal fluid, saliva and other digestive fluids; joint lubrication; detoxification; and maintaining the blood pressure. Yet it is far more than just a constituent. A new science of water is emerging in which the structure and dynamics of water is much more complex than was previously thought. Water is a complex dynamic liquid sensitive and responsive to its environment. We are now in the midst of a paradigm shift in which liquid water is seen as an active agent rather than a passive constituent or universal solvent of life. For example, water and living systems are equally sensitive to a single quantum of magnetic flux [1]. Water shows long-range ordering features around cell membranes, where is it more like an epitaxial liquid crystal with distinct properties in those regions that radically distinguish it from bulk water [2] while chemically still remaining H2O. It used to be thought that water was passive, “dancing to the tune of biomolecules”, but now, water is considered the “matrix of life.” The greatest physiological change with aging is not within our biomolecules, but in the loss of water from the body. The body of a young infant consists of over 80% water, but that of a person over 70 years old is typically comprised of less than 60% water. There are many concerns about drinking water quality worldwide today. Many people choose to drink commercially bottled water for various reasons, but its healthfulness is questionable. Some device manufacturers claim that water treated by a vortex, electromagnetic fields, or other physical means to “energize” it, is more healthful than untreated waters and may also slow the aging process. In this paper, we review published evidence for the impact on health of drinking a particular type of water called “electrolyzed-reduced”(ionized alkaline) water and show observations from the blood.

### 2 Background and literature review

Alkaline mineral water with a relatively high negative oxidative reductive potential (ORP = -150 to -300 millivolts) and a pH over 8.0 is characteristic of natural mountain streams and certain deep wells. Aside from these natural sources, where can one find water with these properties? If this natural water were to be bottled, it would lose its high negative ORP and may react with plastic bottles that contain phthalates, toxic carcinogens. Nonetheless, one can generate water with these properties at point-of-use by using a device called a water ionizer. This is a commercially available water treatment system for the home that first filters the water through a multi-stage water filter to remove chlorine, chloramine, and other contaminants, and then performs partial electrolysis of the filtered water over platinum-coated titanium electrodes with a DC electric field. This yields 2 fractions: oxidized acidic water from the anode, and reduced alkaline water from the cathode. The reduced alkaline water appears to match most closely natural mountain spring water at the source in its physical properties and taste. This water is the subject of this paper. It has been called by various names: reduced, electrolyzed-reduced, and alkaline ionized water, to name a few. In this paper, we refer to it as ERW, “electrolyzed-reduced water.” The ERW fraction retains the alkaline ionic minerals from the tap water, including calcium and magnesium, which are important minerals for health, has a high negative ORP, an alkaline pH, a low level of dissolved oxygen, and is microstructured, with 5 to 6 molecules of water per cluster. It also has a lower surface tension than the starting tap water, which makes it a better solvent and may improve hydration.

Most of the studies on ERW have been conducted in Japan, Korea, and China. Not all of the research reports have been translated into English. Peer-reviewed studies from various laboratories worldwide show that ERW “electrolyzed-reduced” water aka (ionized alkaline) water , with its high negative ORP, scavenges free radical chemical species, protecting from oxidative damage. This, along with its alkalinity and microstructure, yield numerous health benefits. In addition, clinical reports, with or without controlled studies supporting them, further suggest that ERW produces declines in blood sugar levels in diabetic patients; improvements in peripheral circulation in diabetic gangrene; improvements in intestinal flora; declines in uric acid levels in patients with gout; improvements in liver function tests in hepatic disorders; improvements in gastroduodenal ulcer with prevention of recurrences; improved hydration and fluid replacement; and improvements in blood pressure in the case of either hypertension or hypotension. Here we summarize some of the key findings from the peer-reviewed literature on humans and other biological systems.

#### 2.1 Active reducing agent and protection against oxidative stress

Shirahata et al. studied the properties of ERW “electrolyzed-reduced” water aka (ionized alkaline) water and reported that it showed a superoxide dismutase-like activity in protecting against oxidative damage, alleviating oxidative damage of DNA molecules and other species in vitro [3]. This antioxidant effect of ERW “electrolyzed-reduced” water aka (ionized alkaline) water has been verified [4]. The nature of the reducing species (antioxidant) in ERW “electrolyzed-reduced” water aka (ionized alkaline) water has been proposed to be active hydrogen and/or molecular hydrogen, but it is NOT the same as ordinary hydrogen gas and remains unresolved [5, 6]. One group reports that ERW contains both atomic and molecular hydrogen [7]. ERW “electrolyzed-reduced” water aka (ionized alkaline) water prevented oxidative cleavage of proteins and also stimulated the activity of free radical scavenger, ascorbic acid(vitamin C) [8]. Rats, upon drinking ERW “electrolyzed-reduced” water aka (ionized alkaline) water for just one week, showed significantly reduced amounts of peroxidized lipid in their urine, suggesting reduced oxidative stress in the rats [9]. These studies document that ERW has strong antioxidant activity. Antioxidant activity is important to protect cells and biomolecules from the toxic effects of oxidative damage associated with reactive oxygen species such as superoxide radicals that are associated with the biochemistry of inflammation and implicated as underlying factors in chronic disease.

#### 2.2 Prolonged lifespan in nematodes and mice

It is well accepted that oxidative damaged mice compared to control mice fed tap water [10]. Landis and Tower showed that enhanced activity of superoxide dismutase, as has been demonstrated by various investigators using ERW, can reduce oxidative damage and extend life span [11]. A study on ERW used in the aqueous medium of the nematode (worm), C. Elegans in laboratory cultures showed that it significantly extended its lifespan, which has been interpreted to be at least in part due to the reactive oxygen species (ROS)-scavenging action of ERW [12].

#### 2.3 Studies on kidney disease and use of hemodialysis

In end-stage kidney-diseased patients on dialysis, ERW “electrolyzed-reduced” water aka (ionized alkaline) water appears to have a beneficial effect on reduction of hemodialysis-induced oxidative stress. Huang et al. studied the reactive oxygen species in the plasma of these patients and found that ERW “electrolyzed-reduced” water aka (ionized alkaline) water diminished hemodialysis-enhanced peroxide levels, and minimized oxidized and inflammatory markers (C-reactive protein and interleukin-6) after 1 month of drinking ERW “electrolyzed-reduced” water aka (ionized alkaline) water . These findings suggest that cardiovascular complications (stroke and heart attack) in these kidney dialysis patients might be prevented by ERW [13].

Another study investigated use of ERW directly in the hemodialysis process of 8 kidney patients, and found that the viability of patients’ polymorphonuclear leukocytes was better preserved [14].

#### 2.4 Studies on diabetes and blood glucose levels

Reactive oxygen species (ROS), such as superoxide and other free radical oxygen species, are known to cause reduction of glucose update by inhibiting the insulin-signaling pathway in cultured cells. Therefore, the scavenging of ROS is important to the control of diabetes. ERW scavenged intracellular ROS and stimulated glucose uptake in the presence or absence of insulin in rat L6 skeletal muscle cells and mouse 3T3/L1 adipocytes. This insulin-like activity of ERW was inhibited by wortmannin, a specific inhibitor of PI-3 kinase, a key molecule in insulin signalling pathways. ERW protected insulin responsive cells from sugar toxicity and improved the damaged sugar tolerance of type II diabetes model mice. This suggests that ERW may improve the status of those with insulin-independent diabetes mellitus [15]. Oxidative stress is produced under diabetic conditions and involved in progression of pancreatic beta-cell dysfunction. ERW in diabetic mice improved islet beta-cell function, resulting in increased release of circulating insulin and improved insulin sensitivity in both type I and type II diabetes [16, 17]. In a study on Otsuka Long-Evans Tokushima Fatty (OLETF) rats, ERW given to one group showed significantly lower blood glucose levels than controls given tap water. Moreover, blood levels of triglycerides and total cholesterol also decreased in the rats fed ERW [18].

A study on 411 type II diabetes patients whose average age was 71.5 years, who drank natural reduced water from the Nordenau Spring in Germany, up to 2 liters per day over 6 days, showed that 186 (45%) responded positively, with reduced blood glucose, improved cholesterol, LDL, HDL, and serum creatinine levels. 70.6% of a random sample of 136 of the patients also showed a decrease in blood ROS [19].

Recent bioelectrical impedance analysis studies showed that diabetics have a lower ratio of intracellular water (ICW) to extracellular water (ECW).

336 type II diabetics were recruited in a randomized, double-blind trial. The subjects received 250 ml of ERW or distilled water twice daily for 4 weeks. Results show that ERW consumption improved cell water distribution (ICW/ECW), basal metabolism rate, and cell capacitance during the 4-week period. The authors speculate that the relatively small size of the water molecule clusters in ERW may underlie the beneficial findings of improved cell structure and function [20].

#### 2.5 Stimulation of anaerobic microflora in the human gut

The high negative ORP of ERW favors the growth of key anaerobic bacteria in the human gut that are important for normal intestinal microflora, health of the colon, and optimum nutrition [21]. 2.6 Lack of toxicity in microbes, cells, and animals ERW used up to a concentration of 100% in the Ames Test with Salmonella typhimurium did not show any bacterial mutations, either in the presence or absence of rat liver for exogenous metabolic activation. Similarly, ERW did not induce any chromosome aberrations in Chinese hamster lung fibroblast cells with or without rat liver, for up to 24 hours. Rats administered ERW at a dose of 20 mL/kg/day for 28 days via intragastric infusion did not show any clinical symptoms or toxic changes. These results demonstrate the expected safety for a 60 kg human to drink at least 1.2L/day of ERW [22]. Developing animals are the most sensitive to biological agents and are often used in studies to investigate toxicity. Thus, ERW was given to pregnant and also lactating rats to look for any effects. Development of rat fetuses and offspring were normal, and ERW increased the weight of the animals over controls. ERW was also found to have positive biological effects on postnatal growth. Moreover, postnatal morphological development was also accelerated [23]. No significant difference in milk yield or suckled milk volume was noted. It is suspected that the water-hydrated calcium cations transferred to the fetus through the placenta and to the offspring through the milk, might be the cause of the increased body weight, since calcium plays a key role in skeletal formation [24].

#### 2.7 Inhibition of cancer but not normal cells

It is known that tumor cells produce ROS more abundantly than normal cells. It is also well known that antioxidants can inhibit tumor cell proliferation, which indicates an important role of ROS in mediating the loss of growth control. Human tongue carcinoma cells were shown to be significantly inhibited for either colony formation or colony sizes by ERW in cell cultures without inhibition to normal human tongue epithelial cells. ERW also caused growth inhibition, cell degeneration, and inhibition of invasion to human fibrosarcoma cells HT-1080. These studies suggest that ERW may help prevent tumor progression and invasion [25]. In vitro examination of leukemia cells (HL-60) treated with ERW showed enhanced mitochondrial damage and cell apoptosis. However, normal peripheral blood mononuclear cells showed no cytotoxic effect from ERW [26]. ERW also suppressed the growth rate of cancer cells transplanted into mice, demonstrating anti-cancer effects in vivo.

#### 2.8 Protection of liver from toxic agents

Mice with carbon tetrachloride-induced liver damage given ERW showed significantly lowered serum levels of hepatic enzyme markers and increased activities of superoxide dismutase and other key detoxifying enzymes. The effects of ERW were similar to silymarin, an extract from milk thistle well known for its hepato-protective properties. Results suggest that ERW may be used to protect the liver against toxins that induce oxidative damage [27].

#### 2.9 Conclusions from the literature review

These studies show impressive health benefits in humans and other biological systems from consumption of ERW over a very short time, and without any toxic effects observed. Clearly, ERW is a useful adjunct for treating ROS-associated diseases, including diabetes, kidney disease, cancer, and cardiovascular disease. In addition, due to its anti-aging effects in scavenging oxygen free radicals, ERW appears to be an excellent choice for regular water consumption, although its antioxidant activity is unstable upon storage. Nonetheless, it is easily produced from tap water at point-of-use.

### 3 Observations from live blood analysis

The blood is the most easily monitored tissue that can show rapid changes that correlate with health and disease. We have observed that persons drinking ERW show exceptionally clean biological terrains as monitored by live blood analysis. Live blood analysis is the visual examination of a small droplet of fresh capillary blood typically taken from the fingertip, put onto a glass slide, and immediately observed under a high-powered light microscope equipped with a dark-field condenser. This method offers a visual perspective of the blood cells and plasma at high magnification enhanced by modern optical techniques. It provides an assessment of the ecology of the blood, the “biological terrain”. Live blood analysis is used clinically to look for the malaria and Lyme disease parasites. Here we discuss it as a tool to assess blood cell stickiness, clumping, and coagulation and clotting processes, which are related to the activation of the inflammatory cascade.

A microphotograph from live blood analysis is shown in Figure 1. This is a photograph of normal healthy blood of a fasted female, age 37. The red blood cells (RBCs) are seen as single, free, round cells. Only a few platelet aggregates are seen in the plasma as grey areas. No RBC stickiness and no other clotting factors are found throughout the blood sample.

Figure 1: Normal healthy blood from female, age 37.

By contrast, Figure 2 shows the blood of a male, 65 years old. This blood is also typical of that found in many elderly persons. The RBCs are sticky and tightly clumped together in rouleau (rolls of coins seen on edge). Fibrin (white threads) is present, indicating that blood coagulation and clotting have been activated. This is the picture of systemic inflammation. Peripheral circulation was also impaired for this subject, because only single RBC can move freely through the smallest capillaries. Poor circulation in the extremities is a common complaint of the elderly.

Figure 2: Unhealthy blood from male, 65, showing blood congestion and clotting.

Following this test, the subject, M, age 65, drank 1 to 1.5 liters/day of ERW from a water ionizer for 6 months but made no other changes in diet or lifestyle. Figure 3 shows the blood from the same person after 6 months. The RBC stickiness, aggregation, and clotting factors are no longer present. It is particularly striking to see this change in an older person’s blood. Although this is a single case presented here, numerous other cases have been observed as well.

### 4 Conclusions

Chronic inflammation is considered to be one of the main underlying factors of virtually all of the chronic degenerative diseases, including cancer, cardiovascular, and autoimmune diseases. From observing changes in the biological terrain apparently due to consumption of ERW, it appears that ERW may be a useful intervention to mitigate activation of the clotting and inflammatory pathways. Long-term consumption of ERW may improve the blood circulation and possibly help prevent the chronic diseases of our times. Acid-alkaline balance is another key to health and wellness [28]. Metabolism of food leads to acid wastes, yet the biological terrain needs to be alkaline, pH 7.2-7.4. Drinking alkaline water such as ERW can contribute to neutralizing acid waste and maintaining proper pH balance in the body. In conclusion, a growing body of scientific and clinical literature shows increasing support for ERW as a “functional” drinking water that scavenges free radicals, diminishes systemic inflammation, and is a useful adjunct for treating ROS-associated diseases, including diabetes, kidney disease, cancer, and cardiovascular disease. From observations of the blood, it appears to mitigate early blood clotting and systemic inflammation seen as sticky, aggregated RBCs and fibrin. Collectively, this evidence points to ERW alkaline ionized water as a healthy drinking water

References

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[22] Saitoh, Y., Harata, Y., Mizukashi, F., Nakajima, M., Miwa, N. Biological safety of neutral-pH hydrogen-enriched electrolyzed water upon mutagenicity, genotoxicity, and subchronic oral toxicity. Toxicology and Industrial Health 26(4), pp. 203-216, 2010.

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# Effects of hydrogen rich water on prolonged intermittent exercise

Peak power output (PPO), also known as “peak work rate” is a common measure of exercise intensity.

The Authors of this 2-weeks ‘ hydrogen /placebo water’ study (a cross over single-blind protocol -see footnotes for references) tested  8 trained male cyclists and measured multiple parameters(including mean and peak power output) on a regular basis(before and after 2 weeks cycle )

Notes:

1 The athletes were given were provided daily with 2 liters of placebo normal water (pH 7.6, ORP +230 mV, H2 0 ppb) or 2 liters hydrogen rich water(pH 9.8, ORP -180 mV, H2 450 ppb)

2  AlkaViva H2 ionizers such as Vesta/Delphy H2 can produce up to 1600 ppb at ph<10  ).

The authors found that while in the placebo group, Peak Power Output  in absolute values decreased significantly at the last couple of sprints and in relative values and ΔPPO decreased significantly  in more than a couple of sprints,  it remained unchanged in the group that drunk hydrogen rich water . Thus they conclude drinking 2  liters of hydrogen rich water per day over a 2 week period may help to maintain peak power output in intense exercise such as repetitive sprints to exhaustion over 30 minutes.

PMID:28474871
DOI:10.23736/S0022-4707.17.06883-9

2018 May;58(5):612-621. doi: 10.23736/S0022-4707.17.06883-9. Epub 2017 Apr 26.

# Protective effect of hydrogen-rich water on liver function of colorectal cancer patients during chemotherapy

The study published in 2017  was conducted to investigate the protective effect of hydrogen-rich water on the liver function of colorectal cancer (CRC) patients treated with mFOLFOX6 chemotherapy.

A controlled, randomized, single-blind clinical trial was designed.

A total of 152 patients with colorectal cancer were recruited by the Department of Oncology of Taishan Hospital (Taian, China) between June 2010 and February 2016, among whom 146 met the inclusion criteria. Subsequently, 144 patients were randomized into the treatment with hydrogen water(n=80) and placebo (n=64) groups. At the end of the study, 76 patients in the hydrogen water treatment group and 60 patients in the placebo group were included in the final analysis.

The 80 patients group started drinking hydrogen-rich water 1 day prior to chemotherapy until the end of the cycle, for a total of 4 days, with a total intake of 1,000 ml hydrogen-rich water per day in 4 doses (250 ml hydrogen-rich water each). Hydrogen-rich water was consumed 0.5 h after a meal and before bedtime.

The patients did not discontinue consuming hydrogen-rich water during the entire course of chemotherapy.

The other 64 placebo patients consumed distilled water, with a daily intake of 1,000 ml in 4 doses (250 ml each).

The changes in liver function after the chemotherapy, such as altered levels of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase, indirect bilirubin (IBIL) and direct bilirubin, were observed. The damaging effects of the mFOLFOX6 chemotherapy on liver function were mainly represented by increased ALT, AST and IBIL levels. The hydrogen-rich water group exhibited no significant differences in liver function before and after treatment, whereas the placebo group exhibited significantly elevated levels of ALT, AST and IBIL. Thus, hydrogen-rich water appeared to alleviate the mFOLFOX6-related liver injury

PMID:29142752
PMCID:PMC5666661
DOI:10.3892/mco.2017.1409
2017 Nov;7(5):891-896. doi: 10.3892/mco.2017.1409. Epub 2017 Sep 1.
###### Protective effect of hydrogen-rich water on liver function of colorectal cancer patients treated with mFOLFOX6 chemotherapy.
Yang Q1Ji G1Pan R1Zhao Y2Yan P3.

### Author information

1
Department of Oncology, Shandong Provincial Taishan Hospital, Taian, Shandong 271000, P.R. China.
2
Department of Pathology, Taishan Medical University, Taian, Shandong 271000, P.R. China.
3
Department of Oncology, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong 250013, P.R. China.

# haemodialysis system using dissolved dihydrogen (H2) in water produced by electrolysis: a clinical trial

### Abstract

#### BACKGROUND:

Chronic inflammation in haemodialysis (HD) patients indicates a poor prognosis. However, therapeutic approaches are limited. Molecular hydrogen gas (H(2)) ameliorates oxidative and inflammatory injuries to organs in animal models. We developed an HD system using a dialysis solution with high levels of dissolved molecular hydrogen H(2) and examined the clinical effects.

#### METHODS:

Dialysis solution with molecular hydrogen H(2) (average of 48 ppb) was produced by mixing dialysate concentrates and reverse osmosis water containing dissolved molecular hydrogen H(2) generated by a water electrolysis technique. Subjects comprised 21 stable patients on standard HD who were switched to the test HD for 6 months at three sessions a week.

#### RESULTS:

During the study period, no adverse clinical signs or symptoms were observed.

A significant decrease in systolic blood pressure (SBP) before and after dialysis was observed during the study, and a significant number of patients achieved SBP <140 mmHg after HD (baseline, 21%; 6 months, 62%; P < 0.05). Changes in dialysis parameters were minimal, while significant decreases in levels of plasma monocyte chemoattractant protein 1 (P < 0.01) and myeloperoxidase (P < 0.05) were identified.

#### CONCLUSIONS:

Adding molecular hydrogen H(2) to haemodialysis solutions ameliorated inflammatory reactions and improved BP control. This system could offer a novel therapeutic option for control of uraemia.

2010 Sep;25(9):3026-33. doi: 10.1093/ndt/gfq196. Epub 2010 Apr 12.

### Author information

PMID:
20388631
DOI:
10.1093/ndt/gfq196

https://www.ncbi.nlm.nih.gov/pubmed/20388631

# Molecular Hydrogen as an Emerging Therapeutic Medical Gas for Neurodegenerative and Other Diseases

## Abstract

Effects of molecular hydrogen (water ) on various diseases have been documented for 63 disease models and human diseases in the past four and a half years(by 2012(. Most studies have been performed on rodents including two models of Parkinson’s disease and three models of Alzheimer’s disease. Prominent effects are observed especially in oxidative stress-mediated diseases including neonatal cerebral hypoxia; Parkinson’s disease; ischemia/reperfusion of spinal cord, heart, lung, liver, kidney, and intestine; transplantation of lung, heart, kidney, and intestine. Six human diseases have been studied to date: diabetes mellitus type 2, metabolic syndrome, hemodialysis, inflammatory and mitochondrial myopathies, brain stem infarction, and radiation-induced adverse effects.

Two enigmas, however, remain to be solved. First, no dose-response effect is observed. Rodents and humans are able to take a small amount of hydrogen by drinking hydrogen-rich water, but marked effects are observed. Second, intestinal bacteria in humans and rodents produce a large amount of hydrogen, but an addition of a small amount of hydrogen exhibits marked effects. Further studies are required to elucidate molecular bases of prominent hydrogen (water ) effects and to determine the optimal frequency, amount, and method of hydrogen administration for each human disease

## 1. Introduction

Molecular hydrogen (H2) is the smallest gas molecule made of two protons and two electrons. Hydrogen is combustible when the concentration is 4–75%. Hydrogen, however, is a stable gas that can react only with oxide radical ion (•O) and hydroxyl radical (•OH) in water with low reaction rate constants []:

O+H2H+OHk=8.0×107M1s1OH+H2H+H2Ok=4.2×107M1s1H+OHH2Ok=7.0×109M1s1.
(1)

The reaction rate constants of  •O and •OH with other molecules are mostly in the orders of 109 to 1010 M−1·s−1, whereas those with H2 are in the order of 107 M−1·s−1. Hydrogen, however, is a small molecule that can easily dissipate throughout the body and cells, and the collision rates of hydrogen with other molecules are expected to be very high, which is likely to be able to overcome the low reaction rate constants []. Hydrogen is not easily dissolved in water, and 100%-saturated hydrogen water contains 1.6 ppm or 0.8 mM hydrogen at room temperature( our note: see AlkaViva Vesta H2 water ionizer performance below)

In 1995, hydrogen was first applied to human to overcome high-pressure nervous syndrome in deep sea diving []. Hydrogen was used to reduce nitrogen (N2) toxicity and to reduce breathing resistance in the deep sea. In 2001, being prompted by the radical-scavenging activity of hydrogen, Gharib and colleagues examined an effect of molecular hydrogen on a mouse model of schistosomiasis-associated chronic liver inflammation []. Mice were placed in a chamber with 70% hydrogen gas for two weeks. The mice exhibited decreased fibrosis, improvement of hemodynamics, increased nitric oxide synthase (NOS) II activity, increased antioxidant enzyme activity, decreased lipid peroxide levels, and decreased circulating tumor-necrosis-factor-(TNF-)  α  levels. Although helium gas also exerted some protective effects in their model, the effect of helium gas was not recapitulated in a mouse model of ischemia/reperfusion injury of the liver [].

## 2. Effects of Hydrogen (water ) Have Been Reported in 63 Disease Models and Human Diseases

A major breakthrough in hydrogen research occurred after Ohsawa and colleagues reported a prominent effect of molecular hydrogen on a rat model of cerebral infarction in June 2007 []. Rats were subjected to left middle cerebral artery occlusion. Rats placed in 2–4% hydrogen gas chamber showed significantly smaller infarction volumes compared to controls. They attributed the hydrogen effect to the specific scavenging activity of hydroxyl radical (•OH). They also demonstrated that hydrogen scavenges peroxynitrite (ONOO) but to a lesser extent.

As have been previously reviewed [], effects of molecular hydrogen on various diseases have been reported since then. The total number of disease models and human diseases for which molecular hydrogen has been proven to be effective has reached 63 (by 2012)(Table 1). The number of papers is increasing each year (Figure 1). Among the 87 papers cited in Table 1, 21 papers showed an effect with inhalation of hydrogen gas, 23 with drinking hydrogen-rich water, 27 with intraperitoneal administration or drip infusion of hydrogen-rich saline, 10 with hydrogen-rich medium for cell or tissue culture, and 6 with the other administration methods including instillation and dialysis solution. In addition, among the 87 papers, 67 papers showed an effect in rodents, 7 in humans, 1 in rabbits, 1 in pigs, and 11 in cultured cells or cultured tissues.

Number of papers that report effects of molecular hydrogen since 2007 shown in Table 1.

### Table 1

Sixty-three disease models and human diseases for which beneficial effects of hydrogen have been documented.

Brain
Cerebral infarction [] Rodent, human Gas, saline
Cerebral superoxide production [] Rodent Water
Restraint-induced dementia [] Rodent Water
Alzheimer’s disease [] Rodent Saline
Senile dementia in senescence-accelerated mice [] Rodent Water
Parkinson’s disease [] Rodent Water
Hemorrhagic infarction [] Rodent Gas
Brain trauma [] Rodent Gas
Carbon monoxide intoxication [] Rodent Saline
Transient global cerebral ischemia [] Rodent Gas
Deep hypothermic circulatory arrest-induced brain damage [] Rodent Saline
Surgically induced brain injury [] Rodent Gas
Spinal Cord
Spinal cord injury [] Rodent Saline
Spinal cord ischemia/reperfusion [] Rabbit Gas
Eye
Glaucoma [] Rodent Instillation
Corneal alkali-burn [] Rodent Instillation
Ear
Hearing loss [] Tissue, rodent Medium, water
Lung
Oxygen-induced lung injury [] Rodent Saline
Lung transplantation [] Rodent Gas
Paraquat-induced lung injury [] Rodent Saline
Radiation-induced lung injury [] Rodent Water
Burn-induced lung injury [] Rodent Saline
Intestinal ischemia/reperfusion-induced lung injury [] Rodent Saline
Heart
Acute myocardial infarction [] Rodent Gas, saline
Cardiac transplantation [] Rodent Gas
Sleep apnea-induced cardiac hypoxia [] Rodent Gas
Liver
Schistosomiasis-associated chronic liver inflammation [] Rodent Gas
Liver ischemia/reperfusion [] Rodent Gas
Hepatitis [] Rodent Intestinal gas
Obstructive jaundice [] Rodent Saline
Carbon tetrachloride-induced hepatopathy [] Rodent Saline
Kidney
Cisplatin-induced nephropathy [] Rodent Gas, water
Hemodialysis [] Human Dialysis solution
Kidney transplantation [] Rodent Water
Renal ischemia/reperfusion [] Rodent Saline
Melamine-induced urinary stone [] Rodent Water
Chronic kidney disease [] Rodent Water
Pancreas
Acute pancreatitis [] Rodent Saline
Intestine
Intestinal transplantation [] Rodent Gas, medium, saline
Ulcerative colitis [] Rodent Gas
Intestinal ischemia/reperfusion [] Rodent Saline
Blood vessel
Atherosclerosis [] Rodent Water
Muscle
Inflammatory and mitochondrial myopathies [] Human Water
Cartilage
NO-induced cartilage toxicity [] Cells Medium
Metabolism
Diabetes mellitus type I [] Rodent Water
Diabetes mellitus type II [] Human Water
Metabolic syndrome [] Human, rodent Water
Diabetes/obesity [] Rodent Water
Perinatal disorders
Neonatal cerebral hypoxia [] Rodent, pig Gas, saline
Preeclampsia [] Rodent Saline
Inflammation/allergy
Type I allergy [] Rodent Water
Sepsis [] Rodent Gas
Zymosan-induced inflammation [] Rodent Gas
LPS/IFNγ-induced NO production [] Cells Gas
Cancer
Growth of tongue carcinoma cells [] Cells Medium
Lung cancer cells [] Cells Medium
Radiation-induced thymic lymphoma [] Rodent Saline
Others
UVB-induced skin injury [] Rodent Bathing
Decompression sickness [] Rodent Saline
Viability of pluripotent stromal cells [] Cells Gas
Radiation-induced cell damage [] Cells Medium
Oxidized low density lipoprotein-induced cell toxicity [] Cells Medium
High glucose-induced oxidative stress [] Cells Medium

Two papers, however, showed that hydrogen was ineffective for two disease models (Table 2). One such disease was moderate to severe neonatal brain hypoxia [], although marked effects of hydrogen gas [] and intraperitoneal administration of hydrogen-rich saline [] on neonatal brain hypoxia have been reported in rats [] and pigs []. We frequently observe that therapeutic intervention that is effective for mild cases has little or no effect on severe cases, and hydrogen is unlikely to be an exception. Another disease is muscle disuse atrophy []. Although oxidative stress is involved in the development of muscle disuse atrophy, oxidative stress may not be a major driving factor causing atrophy and thus attenuation of oxidative stress by hydrogen may not be able to exhibit a beneficial effect.

### Table 2

Two disease models for which hydrogen has no effect.

Brain

Moderate to severe neonatal brain hypoxia [] Rodent Gas

Muscle

Muscle disuse atrophy [] Rodent Water

Effects of molecular hydrogen have been observed essentially in all the tissues and disease states including the brain, spinal cord, eye, ear, lung, heart, liver, kidney, pancreas, intestine, blood vessel, muscle, cartilage, metabolism, perinatal disorders, and inflammation/allergy. Among them, marked effects are observed in ischemia/reperfusion disorders as well as in inflammatory disorders. It is interesting to note, however, that only three papers addressed effects on cancers. First, molecular hydrogen caused growth inhibition of human tongue carcinoma cells HSC-4 and human fibrosarcoma cells HT-1080 but did not compromise growth of normal human tongue epithelial-like cells DOK []. Second, hydrogen suppressed the expression of vascular endothelial growth factor (VEGF), a key mediator of tumor angiogenesis, in human lung adenocarcinoma cells A549, which was mediated by downregulation of extracellular signal-regulated kinase (ERK) []. Third, hydrogen protected BALB/c mice from developing radiation-induced thymic lymphoma []. Elimination of radical oxygen species by hydrogen should reduce a probability of introducing somatic mutations. Unlike other disease models, cancer studies were performed only with cells in two of the three papers. Hydrogen is likely to have a beneficial effect on cancer development by suppressing somatic mutations, but an effect on cancer growth and invasion needs to be analyzed further in detail.

## 3. Effects of Molecular Hydrogen on Rodent Models of Neurodegenerative Diseases

Parkinson’s disease is caused by death of dopaminergic neurons at the substantia nigra pars compact of the midbrain and is the second most common neurodegenerative disease after Alzheimer’s disease. Parkinson’s disease is caused by two mechanisms: excessive oxidative stress and abnormal ubiquitin-proteasome system []. The neurotransmitter, dopamine, is a prooxidant by itself and dopaminergic cells are destined to be exposed to high concentrations of radical oxygen species. An abnormal ubiquitin-proteasome system also causes aggregation of insoluble  α-synuclein in the neuronal cell body that leads to neuronal cell death. We made a rat model of hemi-Parkinson’s disease by stereotactically injecting catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in the right striatum []. Ad libitum administration of hydrogen-rich water starting one week before surgery completely abolished the development of hemi-Parkinson’s symptoms. The number of dopaminergic neurons on the toxin-injected side was reduced to 40.2% of that on the control side, whereas hydrogen treatment improved the reduction to 83.0%. We also started giving hydrogen-rich water three days after surgery, and hemi-Parkinson’s symptoms were again suppressed, but not as much as those observed in pretreated rats. The number of dopaminergic neurons on the toxin-injected side was 76.3% of that on the control side. Pretreated rats were also sacrificed 48 hrs after toxin injection, and the tyrosine hydroxylase activity at the striatum, where dopaminergic neurons terminate, was decreased in both hydrogen and control groups. This indicated that hydrogen did not directly detoxicate 6-OHDA but exerted a delayed protective effect for dopaminergic cells. Fujita and colleagues also demonstrated a similar prominent effect of hydrogen-rich water on an MPTP-(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-) induced mouse model of Parkinson’s disease []. MPTP is a neurotoxin that blocks complex I of the mitochondrial electron transport system and causes Parkinson’s disease in mice and humans. It is interesting to note that the concentration of hydrogen that they used for the MPTP mice was only 0.08 ppm (5% saturation), which is the second lowest among all the trials published to date for rodents and humans. The lowest hydrogen concentration ever tested is 0.048 ppm in the dialysis solution for patients receiving hemodialysis [].

Alzheimer’s disease is the most common neurodegenerative disease and is characterized by abnormal aggregation of β-amyloid (Aβ) and tau, the large aggregates of which are recognizable as senile plaques and neurofibrillary tangles, respectively []. Effects of molecular hydrogen on Alzheimer’s disease have been studied in three rodent models. First, Nagata and colleagues made a mouse model of dementia by restricting movement of mice for 10 hrs a day []. They analyzed cognitive functions through passive avoidance learning, object recognition tasks, and the Morris water maze and demonstrated that ad libitum administration of hydrogen-rich water efficiently ameliorated cognitive impairment. They also showed that neural proliferation in the dentate gyrus was restored by hydrogen water . Second, Li and colleagues made a rat model of Alzheimer’s disease by intracerebroventricular injection of Aβ1-42 []. They analyzed cognitive functions by the Morris water maze open field tasks, and electrophysiological measurement of long-term potentiation (LTP) and found that intraperitoneal injection of hydrogen-rich saline for 14 days efficiently ameliorated cognitive decline and preserved LTP. The same team later reported that the protective effects were mediated by suppression of abnormal activation of IL1β, JNK, and NFκB []. Third, Gu and colleagues used a senescence-accelerated mouse strain (SAMP8) that exhibits early aging syndromes including impairment in learning ability and memory []. Ad libitum administration of hydrogen-rich water for 30 days prevented cognitive decline, which was examined by the Morris water maze. Additionally, ad libitum drinking of hydrogen water for 18 weeks showed efficient amelioration of hippocampal neurodegeneration.

Cerebrovascular diseases are the most frequently reported neurological diseases for which hydrogen (water )has prominent effects. As stated in Section 2, current hydrogen (water ) research has broken out after Ohsawa reported a prominent effect of 2–4% hydrogen for a rat model of left cerebral artery occlusion in 2007 [].

In addition to neurodegenerative disorders of Parkinson’s disease and Alzheimer’s disease, effects of molecular hydrogen (water ) have been reported in eight other brain diseases listed under the categories of “brain” and “perinatal disorders” in Table 1. The brain consumes a large amount of oxygen and is predisposed to be exposed to a large amount of radical oxygen species especially under pathological conditions. Molecular hydrogen is thus likely to exert a prominent beneficial effect on brain diseases.

## 4. Molecular Hydrogen Is Effective for Six Human Diseases(known by 2012)

As in other therapeutic modalities, effects of molecular hydrogen have been tested mostly on rodents but have also been studied in six human diseases( by 2012). The reported human diseases include diabetes mellitus type II [], metabolic syndrome [], hemodialysis [], inflammatory and mitochondrial myopathies [], brain stem infarction [], and radiation-induced adverse effects for liver tumor []. These studies are reviewed in detail here. In addition, a therapeutic trial for Parkinson’s disease is currently in progress and exhibits favorable responses as far as we know, but the details are not yet disclosed.

First, Kajiyama and colleagues performed a randomized, double-blind, placebo-controlled, crossover study in 30 patients with diabetes mellitus type II and 6 patients with impaired glucose tolerance []. The patients consumed either 900 mL of hydrogen-rich water or placebo water for 8 weeks, with a 12-week washout period. They measured 13 biomarkers to estimate lipid and glucose metabolisms at baseline and at 8 weeks after hydrogen water treatment. All the biomarkers were favorably changed with hydrogen, but statistical significance was observed only in improvement of electronegative charge-modified low-density lipoprotein-(LDL-) cholesterol, small dense LDL, and urinary 8-isoprostanes. In four of six patients with impaired glucose tolerance, hydrogen normalized the oral glucose tolerance test. Lack of statistical significance in their studies was likely due to the small number of patients and the short observation period. Lack of statistical significance, however, may also suggest a less prominent effect in human diabetes mellitus compared to rodent models [].

Second, Nakao and colleagues performed an open-label trial in 20 subjects with potential metabolic syndrome []. Hydrogen-rich water was produced by placing a metallic magnesium stick in water, which yielded 0.55–0.65 mM hydrogen water (70–80% saturation). The participants consumed 1.5–2.0 liters of hydrogen water per day for 8 weeks and showed a 39% increase in urinary superoxide dismutase (SOD), an enzyme that catalyzes superoxide anion (O2); a 43% decrease in urinary thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation; an 8% increase in high-density-lipoprotein-(HDL-) cholesterol; a 13% decrease in total cholesterol/HDL-cholesterol. The aspartate aminotransferase (AST) and alanine transaminase (ALT) levels remained unchanged, whereas the gamma glutamyl transferase (GGT) level was increased by 24% but was still within a normal range. Although the study was not double blinded and placebo controlled, improvements in biomarkers were much more than those in other hydrogen water studies in humans. As this study used a large amount of hydrogen water, the amount of hydrogen might have been a critical determinant. Alternatively, excessive hydration might have prevented the participants from excessive food intake.

Third, Nakayama and colleagues performed an open-label placebo-controlled crossover trial of 12 sessions of hemodialysis in eight patients [] and an open-label trial of 78 sessions of hemodialysis in 21 patients []. In both studies, continuous sessions of hemodialysis with hydrogen-rich dialysis solution decreased systolic blood pressure before and after dialysis. In the short-term study, plasma methylguanidine was significantly decreased. In the long-term study, plasma monocyte chemoattractant protein 1 and myeloperoxidase were significantly decreased.

Fourth, we performed an open-label trial of 1.0 liter of hydrogen water per day for 12 weeks in 14 patients with muscular diseases including muscular dystrophies, polymyositis/dermatomyositis, and mitochondrial myopathies, as well as a randomized, double-blind, placebo-controlled, crossover trial of 0.5 liter of hydrogen water or dehydrogenized water per day for 8 weeks in 22 patients with dermatomyositis and mitochondrial myopathies []. In the open-label trial, significant improvements were observed in lactate-to-pyruvate ratio, fasting blood glucose, serum matrix metalloproteinase-3 (MMP3), and triglycerides. Especially, the lactate-to-pyruvate ratio, which is a sensitive biomarker for the compromised mitochondrial electron transport system, was decreased by 28% in mitochondrial myopathies. In addition, MMP3, which represents the activity of inflammation, was decreased by 27% in dermatomyositis. In the double-blind trial, a statistically significant improvement was observed only in serum lactate in mitochondrial myopathies, but lactate-to-pyruvate ratio in mitochondrial myopathies and MMP3 in dermatomyositis were also decreased. Lack of statistical significance with the double-blind study was likely due to the shorter observation period and the lower amount of hydrogen compared to those of the open-label trial.

Fifth, Kang and colleagues performed a randomized placebo-controlled study of 1.5–2.0 liters of 0.55–0.65 mM hydrogen water per day for 6 weeks in 49 patients receiving radiation therapy for malignant liver tumors. Hydrogen water suppressed the elevation of total hydroperoxide levels, maintained serum antioxidant capacity, and improved the quality of life (QOL) scores. In particular, hydrogen water efficiently prevented loss of appetite. Although the patients were randomly assigned to the hydrogen and placebo groups, the study could not be completely blinded because hydrogen water was produced with a metallic magnesium stick, which generated hydrogen bubbles.

Sixth, Ono and colleagues intravenously administered hydrogen along with Edaravone, a clinically approved radical scavenger, in 8 patients with acute brain stem infarction and compared MRI indices of 26 patients who received Edaravone alone []. The relative diffusion-weighted images (rDWIs), regional apparent diffusion coefficients (rADCs), and pseudonormalization time of rDWI and rADC were all improved with the combined infusion of Edaravone and hydrogen.

No adverse effect of hydrogen has been documented in the six human diseases described above. Among the six diseases, the most prominent effect was observed in subjects with metabolic syndrome, who consumed 1.5–2.0 liters of hydrogen water per day [].

The amount of hydrogen water may be a critical parameter that determines clinical outcome.

It is also interesting to note that lipid and glucose metabolisms were analyzed in three studies and all showed favorable responses to hydrogen [].

Update : since 2012 more clinical trials have been performed.
Acarbose/MOLECULAR HYDROGEN TREATMENT AND THE RISK OF CARDIOVASCULAR DISEASE AND HYPERTENSION IN PATIENTS WITH IMPAIRED GLUCOSE TOLERANCE: THE STOP-NIDDM TRIAL

Molecular  Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL function in patients with potential metabolic syndrome

Improvement of psoriasis-associated arthritis and skin lesions by treatment with molecular hydrogen: A report of three cases.

Molecular hydrogen(H2) treatment for acute erythymatous skin diseases. A report of 4 patients with safety data and a non-controlled feasibility study with H2 concentration measurement on two volunteers

MOLECULAR HYDROGEN WATER FOR PATIENTS WITH PRESSURE ULCER – EFFECTS ON NORMAL HUMAN SKIN WOUNDS

MOLECULAR HYDROGEN WATER FOR PATIENTS WITH RHEUMATOID ARTHRITIS: AN OPEN-LABEL PILOT STUDY

EFFECTIVENESS OF ORAL AND TOPICAL MOLECULAR HYDROGEN FOR SPORTS-RELATED SOFT TISSUE INJURIES

MOLECULAR HYDROGEN WATER BENEFITS FOR ATHLETES, EXERCISE, MUSCLE FATIGUE

MOLECULAR HYDROGEN WATER FOR VASCULAR ENDOTELIAL FUNCTION

MOLECULAR HYDROGEN WATER-  PERIODONTITIS TREATMENT
Please see this section:Hydrogen water

## 5. Molecular Bases of Hydrogen Effects

Effects of hydrogen on various diseases have been attributed to four major molecular mechanisms: a specific scavenging activity of  hydroxyl radical, a scavenging activity of peroxynitrite, alterations of gene expressions, and signal-modulating activities. The four mechanisms are not mutually exclusive and some of them may be causally associated with other mechanisms.

The first molecular mechanism identified for hydrogen was its specific scavenging activity of hydroxyl radical []. Indeed, oxidative stress markers like 8-OHdG, 4-hydroxyl-2-nonenal (4-HNE), malondialdehyde (MDA), and thiobarbituric acid reactive substances (TBARSs) are decreased in all the examined patients and rodents. As hydrogen can easily dissipate in exhalation, hydrogen in drinking water is able to stay in human and rodent bodies in less than 10 min (unpublished data). Hydrogen, however, can bind to glycogen, and the dwell time of hydrogen is prolonged in rat liver after food intake []. A question still remains if mice and humans can take a sufficient amount of hydrogen that efficiently scavenges hydroxyl radicals that are continuously generated in normal and disease states.

Another molecular mechanism of hydrogen effect is its peroxynitrite-(ONOO-) scavenging activity []. Although hydrogen cannot eliminate peroxynitrite as efficiently as hydroxyl radical in vitro [], hydrogen can efficiently reduce nitric-oxide-(NO-) induced production of nitrotyrosine in rodents []. NO is a gaseous molecule that also exerts therapeutic effects including relaxation of blood vessels and inhibition of platelet aggregation []. NO, however, is also toxic at higher concentrations because NO leads to ONOO-mediated production of nitrotyrosine, which compromises protein functions. A part of hydrogen effects may thus be attributed to the reduced production of nitrotyrosine.

Expression profiling of rat liver demonstrated that hydrogen has a minimal effect on expression levels of individual genes in normal rats []. Gene ontology analysis, however, revealed that oxidoreduction-related genes were upregulated. In disease models of rodents, expression of individual genes and proteins is analyzed. In many disease models, hydrogen downregulated proinflammatory cytokines including tumor necrosis-factor-(TNF-)  α, interleukin-(IL-) 1β, IL-6, IL-12, interferon-(IFN-)  γ, and high mobility group box 1 (HMGB1) []. Hydrogen also downregulated nuclear factors including nuclear factor kappa B (NFκB), JNK, and proliferation cell nuclear antigen (PCNA) []. Caspases were also downregulated []. Other interesting molecules studied to date include vascular endothelial growth factor (VEGF) []; MMP2 and MMP9 []; brain natriuretic peptide []; intercellular-adhesion-molecule-1 (ICAM-1) and myeloperoxidase []; B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) []; MMP3 and MMP13 []; cyclooxygenase 2 (COX-2), neuronal nitric oxide synthase (nNOS), and connexins 30 and 43 []; ionized calcium binding adaptor molecule 1 (Iba1) []; fibroblast growth factor 21 (FGF21) []. Most molecules, however, are probably passengers that are secondarily changed by hydrogen administration, and some are potentially direct targets of hydrogen effects, which need to be identified in the future.

Using rat RBL-2H3 mast cells, we demonstrated that hydrogen attenuates phosphorylation of FcεRI-associated Lyn and its downstream signaling molecules []. As phosphorylation of Lyn is again regulated by the downstream signaling molecules and makes a loop of signal transduction pathways, we could not identify the exact target of hydrogen. Our study also demonstrated that hydrogen ameliorates an immediate-type allergic reaction not by radical-scavenging activity but by direct modulation of signaling pathway(s). In addition, using murine RAW264 macrophage cells, we demonstrated that hydrogen reduces LPS/IFNγ-induced NO production []. We found that hydrogen inhibits phosphorylation of ASK1 and its downstream signaling molecules, p38 MAP kinase, JNK, and IκBα  without affecting ROS production by NADPH oxidase. Both studies point to a notion that hydrogen is a gaseous signal modulator. More animal and cells models are expected to be explored to confirm that hydrogen exerts its beneficial effect as a signal modulator.

6. Enigmas of Hydrogen Effects

Two enigmas remain to be solved for hydrogen effects. First, no dose-response effect of hydrogen has been observed. Hydrogen has been administered to animals and humans in the forms of hydrogen gas, hydrogen-rich water, hydrogen-rich saline, instillation, and dialysis solution (Table 1). Supposing that a 60-kg person drinks 1000 mL of saturated hydrogen-rich water (1.6 ppm or 0.8 mM) per day, 0.8 mmoles of hydrogen is consumed by the body each day, which is predicted to give rise to a hydrogen concentration of 0.8 mmoles/(60 kg × 60%) = 0.022 mM (2.8% saturation = 0.022 mM/0.8 mM). As hydrogen mostly disappears in 10 min by dissipation in exhalation (unpublished data), an individual is exposed to 2.8% hydrogen only for 10 min. On the other hand, when a person is placed in a 2% hydrogen environment for 24 hrs, body water is predicted to become 2% saturation (0.016 mM). Even if we suppose that the hydrogen concentration after drinking hydrogen water remains the same for 10 min, areas under the curves of hydrogen water and 2% hydrogen gas are 0.022 mM × 1/6 hrs and 0.016 mM × 24 hrs, respectively. Thus, the amount of hydrogen given by 2% hydrogen gas should be 104 or more times higher than that given by drinking hydrogen water. In addition, animals and patients are usually not able to drink 100%-saturated hydrogen water. If the hydrogen concentration is 72% of the saturation level, the peak concentrations achieved by drinking hydrogen water and 2% hydrogen gas should be identical (0.022 mM × 72% = 0.016 mM). Nevertheless, hydrogen water is as effective as, or sometimes more effective than, hydrogen gas.

In addition, orally taken hydrogen can be readily distributed in the stomach, intestine, liver, heart, and lung but is mostly lost in exhalation. Thus, hydrogen concentrations in the arteries are predicted to be very low. Nevertheless, marked hydrogen effects are observed in the brain, spinal cord, kidney, pancreas muscle, and cartilage, where hydrogen is carried via arteries.

The second enigma is intestinal production of hydrogen gas in rodents and humans. Although no mammalian cells can produce hydrogen endogenously, hydrogen is produced by intestinal bacteria carrying hydrogenase in both rodents and humans. We humans are able to make a maximum of 12 liters of hydrogen in our intestines []. Specific-pathogen-free (SPF) animals are different from aseptic animals and carry intestinal bacteria that produce hydrogen. The amount of hydrogen taken by water or gas is much less than that produced by intestinal bacteria, but the exogenously administered hydrogen demonstrates a prominent effect.

In a mouse model of Concanavalin A-induced hepatitis, Kajiya and colleagues killed intestinal bacteria by prescribing a cocktail of antibiotics []. Elimination of intestinal hydrogen worsened hepatitis. Restitution of a hydrogenase-negative strain of E. coli had no effects, whereas that of a hydrogenase-positive strain of E. coli ameliorated hepatitis. This is the only report that addressed a beneficial effect of intestinal bacteria, and no human study has been reported to date. Kajiya and colleagues also demonstrated that drinking hydrogen-rich water was more effective than the restitution of hydrogenase-positive bacteria. If intestinal hydrogen is as effective as the other hydrogen administration methods, we can easily increase hydrogen concentrations in our bodies by an  α-glucosidase inhibitor, acarbose [], an ingredient of curry, turmeric [], or a nonabsorbable synthetic disaccharide, lactulose []. The enigma of intestinal bacteria thus needs to be solved in the future.

## 7. Summary and Conclusions

Effects of hydrogen have been reported in 63 disease models and human diseases (Table 1). Only two diseases of cerebral infarction and metabolic syndrome have been analyzed in both rodents and humans.

Lack of any adverse effects of hydrogen enabled clinical studies even in the absence of animal studies. Some other human studies including Parkinson’s disease are currently in progress, and promising effects of hydrogen are expected to emerge for many other human diseases. We also have to elucidate molecular bases of hydrogen effects in detail.

## 8. Added Note in Proof

We recently reported a line of evidence that molecular hydrogen has no dose-response effect in a rat model of Parkinson’s disease [].

. 2012; 2012: 353152.
Published online 2012 Jun 8. doi:  [10.1155/2012/353152]
PMCID: PMC3377272
PMID: 22720117

# Molecular Hydrogen as an Emerging Therapeutic Medical Gas for Neurodegenerative and Other Diseases

1Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan
2Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Aichi 487-8501, Japan
3Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
Received 2012 Jan 11; Revised 2012 Mar 24; Accepted 2012 Apr 13.

## Acknowledgments

Works performed in the authors’ laboratories were supported by Grants-in-Aid from the MEXT and MHLW of Japan and from the Priority Research Project of Aichi.

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# A Randomized Double-Blind, Placebo-Controlled Pilot Study

## Abstract

### Objectives

The purpose of this study was to investigate whether the ingestion of alkaline-reduced water (ARW) is helpful in improving the symptoms of diarrhea-predominant irritable bowel syndrome (IBS).

### Methods

Twenty-seven patients (male, 25.9%; mean, 41.7 years old) with diarrhea-predominant IBS were randomly allocated to two groups. For eight weeks, the ARW group (n = 13) ingested at least 2 liters/day of ARW, while the control group (n = 14) ingested placebo water. IBS symptom scores (quality-of-life, abdominal pain/discomfort), stool form, and frequency were assessed before and after treatment via questionnaires.

### Results

Eight patients (61.5%) in the ARW group and six patients (42.9%) in the control group indicated that their symptoms had improved in more than four out of the eight weeks of treatment (p = 0.449). The IBS quality-of-life score significantly improved from 57.2 to 30.8 in the ARW group; this improvement was significantly greater than the slight improvement from 48.7 to 42.2 observed in the control group (p = 0.029). The abdominal pain score improved from 1.8 to 0.9 in the ARW group and from 1.8 to 1.1 in the control group, with no significant group difference (p = 0.232).

### Conclusions

Drinking ARW for eight weeks improves the quality of life in patients with diarrhea-predominant IBS.

## 1. Introduction

Irritable bowel syndrome (IBS) is a functional intestinal disorder accompanied by abdominal pain and bowel habit changes, without evidence of an underlying injury. It is a very common disease, occurring in about 11% of people worldwide []. According to the Korean National Health Insurance System database, 5.1% of men and 6.9% of women were diagnosed with IBS []. IBS is one of the most common illnesses in primary care, with a repeated cycle of deterioration and relief over the years. Improving symptoms through appropriate treatment is important; IBS lowers the quality of life and increases medical costs []. Patients with IBS also suffer from anxiety, major depressive disorder, and chronic fatigue syndrome []. However, the cause and mechanisms underlying the various symptoms are not entirely understood. Many hypotheses have been proposed, including small bowel bacterial overgrowth syndrome, genetic factors, food hypersensitivities, gastrointestinal motility disorders, gut-brain axis alterations, hypersensitivity of the intestine, and psychosocial factors []. Recent studies indicate that the intestinal microbiota is one of the important factors affecting the onset of IBS [].

Various drugs have been used to improve symptoms, including antacids, antispasmodics, and drugs that stimulate gastrointestinal motility (prokinetic agents). However, with a lack of convincing evidence for a pathophysiological basis, conventional therapies have not achieved complete symptom improvement. Therefore, several alternative therapeutic methods, including dietary changes, probiotics, and other medications, have been proposed []. Furthermore, mineral water with various electrolyte compositions has been utilized in the treatment of functional gastrointestinal diseases; mineral water supplements have been reported to improve functional dyspepsia associated with IBS by controlling gastric acid output and intestinal transit time []. In addition, carbonated water not only attenuates the hunger but also improves dyspeptic symptoms and heartburn []. Drinking sulfur-rich mineral water for more than three weeks was found to be effective in treating constipation by increasing frequency of bowel movements []. Bicarbonate-containing alkaline-reduced water (ARW) has also been hypothesized to affect various digestive functions. Although animal studies have provided evidence that ARW is effective in treating functional bowel disease, human studies are lacking []. Therefore, the purpose of this randomized double-blind pilot study was to evaluate the effect of ARW ingestion on diarrhea-predominant IBS.

## 2. Methods

### 2.1. Ethical Approval

This study was conducted in accordance with the ethical principles for medical research involving human subjects in the Declaration of Helsinki. This study was approved by the Seoul National University Bundang Hospital Medical Ethics Committee (IRB number: E-1405/250-002) and aspires to protect the lives, health, privacy, and dignity of the research participants. Thus, the purpose and characteristics of the clinical trial were fully explained to the participants. Only patients who voluntarily signed an informed consent were included, and patients were allowed to stop participating at any time during the trial. All results obtained in this clinical study are confidential.

### 2.2. Study Population

Men and women aged 18–75 years who met Rome III criteria [] for diarrhea-predominant IBS, had no underlying disease of the colon on a sigmoidoscopy or colonoscopy performed within 5 years prior to screening, and could understand and respond to the symptom questionnaires were included. Rome III criteria for IBS involve recurrent abdominal pain or discomfort at least 3 days/month in the last 3 months with two or more of the following: improvement with defecation, onset associated with a change in stool frequency, or onset associated with a change in stool form []. Diarrhea-predominant IBS involves loose or watery stools in more than 25% of bowel movements and hard or lumpy stool in less than 25% of bowel movements.

The following were excluded: patients with a psychiatric history; patients with untreated malignant tumors; patients with severe liver or kidney disease (AST, ALT levels 3-fold greater than the normal upper limit, and serum creatinine levels 1.5-fold greater than the normal upper limit); patients with severe heart failure; patients with acute gastrointestinal tract infection within the last 3 months. In addition, patients who were taking medications during the study period that could affect the results were also excluded. This included drugs that might influence IBS symptoms, such as antispasmodics, laxatives, prokinetics, anticholinergics, antianxiety drugs, antidepressants, analgesics, thyroid hormone, antibiotics, and steroids.

It is difficult to predict the therapeutic response rate between the test group and the control group since similar studies related to ARW have not existed before. This is a small-scale preliminary pilot study to investigate feasibility, adverse events, and improvement before a full-scale research project. This study was planned with 30 participants per group, which is the minimum number of participants recommended in a pilot study []. Given an estimated dropout rate of 15%, at least 35 people per group were planned to be enrolled.

### 2.3. Randomization and Allocation

Patients who were diagnosed with diarrhea-predominant IBS by Rome III criteria were equally allocated to experimental and control groups. Randomization was performed using a 1 : 1 computerized block randomization with a predetermined random code. Because both the investigator and the patients were blinded, a research coordinator performed the random assignment. The research coordinator did not provide information on randomization to the patients and researchers until the end of the study. Neither the participants nor the researchers could distinguish group assignments.

### 2.4. Study Design

A flowchart of the study design is provided in Figure 1. Patients completed screening tests (blood, urinalysis, colonoscopy, and a past medical history questionnaire) 1–3 weeks before participating in the study. Laboratory evaluation included assessments of liver function (albumin, total bilirubin, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels), kidney function (creatinine and blood urea nitrogen levels), electrolytes (sodium, potassium, chloride, calcium, and inorganic phosphorus levels), and the complete blood count (CBC).

Schedule of patients participating in the study.

Baseline questionnaires on the IBS quality of life, abdominal pain/discomfort, stool form, and stool frequency were completed at the start of the study. The IBS quality-of-life questionnaire is an indicator of abdominal discomfort and consists of 34 items (each recorded as 1–5 points: 1: not at all, 2: slightly, 3: moderately, 4: quite a bit, and 5: extremely) []. Symptom scores for abdominal pain/discomfort were rated on a scale of 0–4 (0: asymptomatic, 1: mild, 2: moderate, 3: severe, and 4: very severe) and were based on the worst level of the day. Abdominal discomfort was defined as an uncomfortable sensation not described as pain. Stool form was assessed using the Bristol stool scale, which is a diagnostic tool designed to classify the form of human feces into seven categories. In general, types 1 and 2 (hard or lumpy stool) indicate constipation, and types 5–7 (soft or watery stool) indicate diarrhea []. In addition, the number of bowel movements was recorded daily.

The experimental group ingested ARW from an installed test device, while the control group ingested placebo water from a sham device. Both groups were instructed to ingest more than 2 liters per day for eight weeks. Participants visited the hospital every two weeks and completed self-administered questionnaires on compliance, adverse effects, the amount ingested, symptom scores (abdominal pain/discomfort), stool form, and the number of daily bowel movements. Questionnaires on the IBS quality of life were completed only at the end of the eighth week. If adverse events occurred during the trial period, participants were instructed to stop the medication immediately and visit an outpatient clinic.

The primary outcome was the proportion of participants with adequate symptomatic improvement in more than four weeks of the 8-week treatment period. The secondary outcomes were changes in IBS quality of life, symptom scores (abdominal pain/discomfort), and stool form/frequency.

### 2.5. Research Equipment

ARW with a pH of 8.5–10.0 was produced using an alkali water ionizer (Kim Young Kwi alkali water ionizer, KYK33000). Placebo water was prepared using a sham device (model name: sham KYK33000), which was not able to generate ARW, but had the same appearance as that of the test apparatus. The devices were installed at the patient’s home and patients were allowed to drink water as needed.

### 2.6. Statistical Analyses

Statistical analyses were performed using SPSS for Windows (ver. 22.0, IBM Corporation, Chicago, IL, USA) and STATA software (ver. 14.0, STATA Corporation, College Station, TX, USA). Group differences were evaluated using Student’s t-test for continuous variables and the Chi-square or Fisher’s exact test for categorical variables. Group differences in treatment-related changes in variables related to IBS (abdominal pain/frequency, stool form, and frequency of bowel movements) were evaluated using a linear mixed model with an interaction term between group and time (before and after treatment). Changes in the IBS quality-of-life score were evaluated using the paired t-test. Two-sided p values less than 0.05 were considered statistically significant.

## 3. Results

### 3.1. Baseline Characteristics

Only 29 were enrolled in the study and 2 dropped out during the study; because the patients were burdened with drinking more than 2 liters of water a day for a long time, we failed to enroll the intended 70 patients. Finally, 13 patients in the ARW group and 14 patients in the control group completed the study (Figure 2). There were no significant group differences in baseline characteristics (Table 1). Ten out of thirteen (76.9%) patients in the ARW group and ten out of fourteen (71.4%) patients in the control group were women. The mean age in the ARW group was slightly higher compared to that of the control group, but without statistical significance (43.3 versus 40.1, p = 0.584). At the beginning of the study, IBS symptom scores (quality-of-life, abdominal pain/discomfort), Bristol stool form, and stool frequency were not significantly different between the two groups. In addition, the consumption of water was similar in the two groups (ARW group: 2,124 ± 900 ml/day; control group: 2,052 ± 648 ml/day, p = 0.834).

CONSORT flow diagram of patient recruitment.

### Table 1

Characteristics of baseline demographics of patients.

Alkaline-reduced water group
(n = 13)
Control group
(n = 14)
p value
Female, n (%) 10 (76.9%) 10 (71.4%) 0.745
Mean age ± SD (years) 43.3 ± 14.4 40.1 ± 15.7 0.584
Initial symptom scores
Quality-of-life score 57.2 ± 28.0 48.7 ± 26.4 0.428
Abdominal pain 1.8 ± 0.9 1.8 ± 0.8 0.983
Abdominal discomfort 1.8 ± 0.8 2.1 ± 0.8 0.362
Stool form (BSFS) 5.3 ± 0.5 5.3 ± 1.4 0.939
Stool frequency/day 2.6 ± 1.2 1.9 ± 1.0 0.130
Amount of water (ml/day) 2,124 ± 900 2,052 ± 648 0.834

SD: standard deviation; BSFS: Bristol stool form scale.

### 3.2. Primary Outcome Measure

Table 2 shows the number of responders (a favorable symptom improvement in more than four weeks of the eight-week treatment period) and nonresponders in each group. Although the proportion of patients responding to the treatment was higher in the ARW group (8/13, 61.5%) than in the control group (6/14, 42.9%), the difference was not statistically significant (Fisher’s exact test, p = 0.449).

### Table 2

Proportion of responders who showed symptomatic improvement after treatment (primary outcome measure).

Alkaline-reduced water group (n = 13) Control group (n = 14) p value
Responder, n (%) 8 (61.5%) 6 (42.9%) 0.449
Nonresponder, n (%) 5 (38.5%) 8 (57.1%)

### 3.3. Secondary Outcome Measures

After eight weeks of treatment, the IBS quality-of-life score had improved from 57.2 to 30.8 points in the ARW group and from 48.7 to 42.2 in the control group (Table 3), with a significant group difference (Figure 3(a)p = 0.029). The abdominal pain score improved from 1.8 to 0.9 in the ARW group and from 1.8 to 1.1 in the control group, without a statistically significant group difference (Figure 3(b)p = 0.232). Abdominal discomfort, stool form, and stool frequency were somewhat improved in the ARW group; however, there were no significant group differences (Figures 3(c)3(e)).

Graph of change before and after treatment of IBS. (a) Quality-of-life score. (b) Abdominal pain score. (c) Abdominal discomfort score. (d) Bristol stool form scale. (e) Stool frequency per day.

### Table 3

Symptom scores of patients before and after treatment (secondary outcome measures).

Alkaline-reduced water group (n = 13) Control group
(n = 14)
p value
Quality-of-life score Week 0 57.2 ± 28.0 48.7 ± 26.4 0.428
Week 8 30.8 ± 24.9 42.2 ± 36.3 0.353

Abdominal pain Week 0 1.8 ± 0.9 1.8 ± 0.8 0.983
Week 2 1.6 ± 1.0 1.7 ± 0.8 0.796
Week 4 1.0 ± 0.9 1.4 ± 0.8 0.324
Week 6 0.8 ± 0.8 1.3 ± 0.7 0.123
Week 8 0.9 ± 0.8 1.1 ± 0.6 0.480

Abdominal discomfort Week 0 1.8 ± 0.8 2.1 ± 0.8 0.362
Week 2 1.9 ± 1.1 1.9 ± 0.7 0.964
Week 4 1.4 ± 1.2 1.6 ± 0.8 0.688
Week 6 1.0 ± 0.7 1.5 ± 0.8 0.113
Week 8 1.2 ± 0.9 1.3 ± 0.7 0.777

Stool form (BSFS) Week 0 5.3 ± 0.5 5.3 ± 1.4 0.939
Week 2 4.9 ± 0.8 5.1 ± 0.8 0.546
Week 4 4.5 ± 0.8 4.6 ± 1.0 0.791
Week 6 4.5 ± 0.8 4.4 ± 1.3 0.747
Week 8 4.7 ± 0.9 4.4 ± 1.0 0.313

Stool frequency/day Week 0 2.6 ± 1.2 1.9 ± 1.0 0.130
Week 2 2.5 ± 1.1 1.8 ± 0.7 0.073
Week 4 2.1 ± 0.9 1.7 ± 0.7 0.198
Week 6 2.0 ± 0.8 1.7 ± 0.9 0.349
Week 8 2.1 ± 0.9 1.7 ± 0.7 0.213

Week 0: the time of randomization; BSFS: Bristol stool form scale.

One of the patients in the control group visited the emergency room due to vomiting and abdominal pain during the second week of the study, but improved with conservative treatment. There were no specific adverse effects associated with ARW ingestion during the eight weeks of the trial.

## 4. Discussion

IBS is one of the most common gastrointestinal disorders in the general population []. In addition, because the effects of medications are often temporary, patients may increase the dose of the medication or take several medications, resulting in the occurrence of side effects. Thus, interest in alternative therapies that do not have side effects (even after long-term use) is growing []. Numerous animal studies have investigated the ability of controlling the electrolyte balance or acidity of the drinking water to treat functional gastrointestinal disorders. For example, animal studies have shown ARW to be effective in treating gastritis because it permanently denatures pepsin []. In addition, an animal study demonstrated that ingestion of more than 1.5 liters of bicarbonate-alkaline mineral water for 30 days improves dyspeptic symptoms []. It has also been suggested that a regular course of crenotherapy with bicarbonate-alkaline mineral water can be used to treat functional dyspepsia, improving gastrointestinal motility and secretory function by modulating the secretion of peptide hormones and regulating the movement of digestive organs []. These studies support the hypothesis that ARW can effectively treat IBS; however, prior to the present study, there were no supporting human clinical trials. Given this preclinical basis, we aimed to investigate whether ARW ingestion for eight weeks improved the symptoms of IBS.

This randomized controlled, double-blind, placebo-controlled study was designed to determine whether the ingestion of ARW could improve the quality of life, abdominal pain/discomfort, stool form, and stool frequency in diarrhea-predominant IBS. In terms of the primary endpoint, the proportion of responders (IBS patients who had improved symptoms in more than four weeks of the 8-week treatment period) was higher in the ARW group than in the control group, but the group difference was not statistically significant. This is likely due to the small number of patients who completed the trial; however, it is hard to predict an effect size, as no similar studies exist. We believe that a positive result could be obtained in a larger-scale study. In contrast to the primary outcome, a significant group difference was observed in the secondary outcomes. The IBS-related quality-of-life and abdominal pain scores were decreased to a greater extent with ARW ingestion compared to those with the ingestion of placebo water. This is a meaningful result because it demonstrates that it is possible to reduce IBS symptoms simply by ingesting water with a different pH, without taking any other medication. In addition, ARW has few adverse effects; thus, it shows potential in becoming an important complementary therapy for functional bowel disease. However, there were no significant group differences in the stool form and frequency improvements. At the beginning of the study, the frequency of bowel movements in both groups was 2-3 times a day, which is less than that for the definition of diarrhea (more than three times a day). Thus, the patients in both groups mainly had mild diarrhea, which may explain the lack of a significant change in symptom scores with treatment.

The mechanism by which ARW improves IBS symptoms remains unclear. ARW refers to water with a pH of at least 8.4; in contrast, most tap or bottled water has a pH between 6.7 and 7.4 []. ARW is thought to increase the pH level of the stomach given its large amount of bicarbonate ions. Interestingly, just infusing a small amount (0.1 mol/L) of acid into the stomach can aggravate indigestion in most people []. In addition, acidification of the duodenum exacerbates dyspeptic symptoms by inducing proximal gastric relaxation and inhibiting gastric accommodation to a meal []. In one animal study, duodenal acidification-induced gastric hypersensitivity could be the cause of dyspepsia in patients with IBS and serotonin 5-HT3 receptors play a key role []. Furthermore, in patients with pancreatic insufficiency, such as cystic fibrosis, the small intestine is exposed to an acidic environment, resulting in impaired absorption. Rapid neutralization of gastric acid in the proximal portion of the duodenum and tight regulation of the gastrointestinal pH play important roles in maintaining nutrient absorption and function in the intestines []. In addition, mineral water with a unique electrolyte composition may help improve the symptoms of indigestion []. Carbonated water could regulate gastrointestinal motility diseases by stimulating bile flow and pancreatic exocrine secretion. Furthermore, drinking carbonated water for more than 15 days has been shown to improve gallbladder muscle contractions []. The ingestion of water containing a lot of mineral salts has been shown to improve gastric emptying in patients with indigestion []. It is presumed that the various ions contained in mineral water directly or indirectly (via neuroendocrine secretion of vasointestinal peptides) stimulate the smooth muscle involved in gastrointestinal motility. These actions appear to improve the symptoms of IBS by improving intestinal transit time and excretory capacity. These actions are thought to not only reduce the bowel transit time, but also promote gastrointestinal hormone secretion, thereby improving abdominal bloating. We expect that large-scale studies on ARW with various electrolytic compositions will proceed in the future.

Gut microbiota appear to be one of the important factors contributing to the cause and pathophysiology of IBS []. Postinfectious IBS should be suspected when the patient complains of dyspepsia or abdominal discomfort after acute gastroenteritis []. Postinfectious IBS is thought to be due to persistent low-grade inflammation and alteration of gut flora intestinal microorganisms. The composition of gut microbiota is also associated with the pathophysiology of IBS and the host immune response []. Abundance of Cyanobacteria is associated with bloating, satiety, and increased abdominal discomfort. The amount of Proteobacteria is associated with pain threshold []. Therefore, it was suggested that probiotics, antibiotics, and fecal microbiota transplantation might be effective in the treatment of IBS []. Many gastrointestinal disorders, including IBS, are caused by an imbalance of residential microflora of the intestinal tract. Human intestinal microbiota consist of 96–99% anaerobes and 1–4% aerobes. Microorganisms have their own intrinsic reduction potential (Eh) for each species, and aerobic and anaerobic bacteria grow at different oxidation-reduction potentials. Aerobic bacteria require a positive potential of +400 mV and facultative anaerobic bacteria require negative electric potential between −300 and −400 mV. Electrochemically generated reduced water has a negative potential of 0 to −300 mV, while the tap water has a potential of +300 to +450 mV []. By drinking reduced water, it is possible to improve symptoms of functional bowel disease by accelerating the growth of anaerobic bacteria (Lactobacilli andBifidobacteria) and inhibiting the growth of aerobic pathogens.

The present study has some limitations. First, the statistical power was weak because of the small sample size. Second, patients with IBS tend to be somewhat less adherent due to the distrust of conventional therapies and hospitals. Third, although IBS is a highly prevalent disease, there were some difficulties in recruiting patients. Because the participants expressed difficulty in drinking more than 2 liters of water a day, we could not enroll as many patients as intended. In addition, subjects were already taking several medications before participating in the study, so it was not easy to stop them all and treat them with ARW only for 8 weeks. Patients’ compliance should be taken into account when designing large-scale studies on this topic in the future. Fourth, the lifestyle and diet were not controlled except for the medications. These confounding factors may be somewhat offset in the randomization process. Despite these limitations, the main strength of the present study is its randomized, double-blind, placebo-controlled design. Moreover, ARW is a simple and inexpensive treatment that physicians can easily consider in the treatment of IBS. To our knowledge, this is the first study to show whether ARW can improve IBS in humans, irrespective of the mechanism.

In conclusion, the present study suggests that ingestion of ARW can improve the quality of life and reduce abdominal pain in patients with diarrhea-predominant IBS. We hope that this pilot study provides a cornerstone for future large-scale trials on the effectiveness of ARW in the treatment of IBS.

Published online 2018 Apr 15. doi:  [10.1155/2018/9147914]
PMCID: PMC5925025
PMID: 29849734
###### Effects of Alkaline-Reduced Drinking Water on Irritable Bowel Syndrome with Diarrhea: A Randomized Double-Blind, Placebo-Controlled Pilot Study
1Department of Internal Medicine and Seoul National University Bundang Hospital, Seongnam, Gyeonggi-do, Republic of Korea
2Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea
Dong Ho Lee: rk.oc.oohay@nhojlhd
Received 2017 Nov 3; Revised 2018 Mar 4; Accepted 2018 Mar 6.

## Acknowledgments

Statistical analysis support was provided by the Medical Science Research Institute in Seoul National University Bundang Hospital. This study was funded by a grant from Seongnam Industry Promotion Agency’s 2014 Medibio products clinical trial support program.

## Conflicts of Interest

The authors declare that they have no conflicts of interest.

## Authors’ Contributions

Dong Woo Shin analyzed the data and drafted the manuscript. Hyuk Yoon and Dong Ho Lee designed the study and revised the manuscript. Hyun Soo Kim, Yoon Jin Choi, Cheol Min Shin, Young Soo Park, and Nayoung Kim critically reviewed the manuscript. Dong Woo Shin and Hyuk Yoon have contributed equally to this work.

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Articles from Evidence-based Complementary and Alternative Medicine : eCAM are provided here courtesy of Hindawi Limited

# Hydrogen-rich water for improvements of mood, anxiety, and autonomic nerve function in daily life

## Abstract

Health and a vibrant life are sought by everyone. To improve quality of life (QOL), maintain a healthy state, and prevent various diseases, evaluations of the effects of potentially QOL-increasing factors are important. Chronic oxidative stress and inflammation cause deteriorations in central nervous system function, leading to low QOL. In healthy individuals, aging, job stress, and cognitive load over several hours also induce increases in oxidative stress, suggesting that preventing the accumulation of oxidative stress caused by daily stress and daily work contributes to maintaining QOL and ameliorating the effects of aging. Hydrogen has anti-oxidant activity and can prevent inflammation, and may thus contribute to improve QOL. The present study aimed to investigate the effects of drinking hydrogen-rich water (HRW) on the QOL of adult volunteers using psychophysiological tests, including questionnaires and tests of autonomic nerve function and cognitive function. In this double-blinded, placebo-controlled study with a two-way crossover design, 26 volunteers (13 females, 13 males; mean age, 34.4 ± 9.9 years) were randomized to either a group administered oral HRW (600 mL/d) or placebo water (PLW, 600 mL/d) for 4 weeks. Change ratios (post-treatment/pre-treatment) for K6 score and sympathetic nerve activity during the resting state were significantly lower after HRW administration than after PLW administration. These results suggest that HRW may reinforce QOL through effects that increase central nervous system functions involving mood, anxiety, and autonomic nerve function.

## Introduction

Health and a vibrant life are much craved by everyone. To improve quality of life (QOL), maintain a healthy state, and prevent the onset of various diseases, evaluation of interventional effects for improving QOL is important. The high metabolic rate of the brain results in the generation of disproportionate amounts of reactive oxygen and nitrogen species, leading to increased oxidative stress. Increased oxidative stress and lipid peroxidation initiate a cascade of proinflammatory signals, leading to inflammation. Altered homeostasis of oxidation, inflammation, and protein aggregation has been suggested to contribute to the death of neurons, which is directly related to impairments in various cognitive domains. As such, chronic oxidative stress and inflammation may cause deteriorations in the function of the central nervous system, leading to reductions in QOL. Hydrogen has antioxidant activity and can prevent inflammation.,, The distribution of hydrogen throughout the brain and body indicates actions both in the central and peripheral nervous systems. Previous clinical studies have shown that hydrogen-rich water (HRW) reduces concentrations of markers of oxidative stress in patients with metabolic syndrome,,improves lipid and glucose metabolism in patients with type 2 diabetes, improves mitochondrial dysfunction in patients with mitochondrial myopathies, and reduces inflammatory processes in patients with polymyositis/dermatomyositis. In another study, exercise-induced declines in muscle function among elite athletes were also improved by administering HRW. Although such findings suggest that HRW may help alleviate symptoms of several diseases and increase the physical performance of athletes, the effects of prolonged HRW ingestion on the QOL of individuals in the general population remain unknown.

Some reports have demonstrated that oxidative stress is associated with QOL in patients with chronic obstructive pulmonary disease and cervical cancer., During oncological treatment among patients with cervical cancer, antioxidant supplementation was found to be effective in improving QOL. In addition, Kang et al. reported that treatment with HRW for patients receiving radiotherapy for liver tumors decreased oxidative stress and improved QOL. Although the association between oxidative stress and QOL in healthy individuals is still unclear, aging, job stress, and cognitive load over the course of several hours in healthy individuals have also been found to induce increases in oxidative stress,,,, suggesting that preventing the accumulation of oxidative stress caused by daily stress and daily work may contribute to the maintenance of QOL and amelioration of the effects of aging. Continuous HRW intake might therefore be expected to reduce accumulation of oxidative stress, thus helping to prevent decreases in QOL.

The aim of the present study was to investigate the effects of drinking 600 mL of HRW per day for 4 weeks on the QOL of adult volunteers using questionnaires for sleep, fatigue, mood, anxiety, and depression, an autonomic function test, and a higher cognitive function test.

## Subjects and Methods

### Subjects

Thirty-one adult volunteers between 20 and 49 years old participated in this double-blinded, randomized, placebo-controlled study with a two-way crossover design. Exclusion criteria comprised: history of chronic illness; chronic medication or use of supplemental vitamins; employment in shift work; pregnancy; body mass index ≤ 17 or ≥ 29 kg/m2; food allergy; history of smoking; or history of drinking excessive amounts of alcohol (≥ 60 g/day). Shift workers were excluded because the water was administered at breakfast and dinner, the timings of which are irregular among shift workers. In addition, the mental and physical conditions of shift workers can be greatly affected by the shift schedule for the preceding 2 days, which may impact the results obtained from the questionnaires used in this study. Before each experiment, participants were asked to refrain from drinking alcohol, since drinking excessive amounts of alcohol carries significant risks of fluctuations in physical condition. All experiments were conducted in compliance with national legislation and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association (the Declaration of Helsinki) and registered to the UMIN Clinical Trials Registry (No. UMIN000022382). The study protocol was approved by the Ethics Committee of Osaka City University Center for Health Science Innovation (OCU-CHSI-IRB No. 4), and all participants provided written informed consent for participation in the study.

### Study design

Time course of the experiments.

Note: Participants were randomly divided into two study groups. The experiment consisted of 4 weeks of hydrogen-rich water (HRW) administration or placebo water (PLW) administration, a 4-week washout period, and then another 4 weeks of PLW administration or HRW administration. Before (pre) and after (post) each period of HRW or PLW administration, subjective and objective measurements for quality of life were obtained, such as results for sleep, mood, anxiety, feelings of depression, autonomic nerve function, and cognitive function.

### Questionnaire

Severity of fatigue was measured using the Chalder Fatigue Scale (CFS) and a modified version of the Osaka City University Hospital Fatigue Scale. Mood and anxiety were evaluated using the K6 scale.Symptoms of depression were measured using the Center for Epidemiologic Studies Depression Scale.General sleepiness and daytime sleepiness scores were calculated using the Pittsburgh Sleep Quality Index (PSQI) and the Epworth Sleepiness Scale, respectively. The reliability and validity of the Japanese versions of these questionnaires have been confirmed.,,,,,

### Autonomic function test

Participants underwent simultaneous electrocardiography and photoplethysmography using a Vital Monitor 302 system (Fatigue Science Laboratory, Osaka, Japan) while sitting quietly with their eyes closed for 3 minutes. These data were analyzed using MemCalc software (GMS, Tokyo, Japan). Frequency analyses for R-R interval variation from electrocardiography and a-a interval variation as the second derivative of photoplethysmography (accelerated plethysmography) were performed using the maximum entropy method, which is capable of estimating the power spectrum density from short time series data, and is adequate for examining changes in heart rate variability under different conditions of short duration.,The power spectrum resolution was 600 Hz. For frequency analyses, the low-frequency component power (LF) was calculated as the power within a frequency range of 0.04–0.15 Hz, and the high-frequency component power (HF) was calculated as that within a frequency range of 0.15–0.4 Hz. HF is vagally mediated,,, whereas LF originates from a variety of sympathetic and vagal mechanisms., Some review articles,, mentioned that LF reflects sympathetic nerve activity and is used as a marker of sympathetic nerve activity in original articles. Before autonomic nerve function testing was conducted for 3 minutes, a practice test was conducted for a period of 1 minute, in accordance with previous studies.,, The reliability of these tests has been confirmed.,

### Cognitive function test

Since previous studies have revealed that a switching attention task is useful for evaluating reduced performance under fatigue conditions,,, we used task E of the modified advanced trail making test (mATMT) as a switching attention task for evaluating executive function., Circles with numbers (from 1 to 13) or kana (Japanese phonograms, 12 different letters) were shown in random locations on a screen, and participants were required to use a computer mouse to alternately touch the numbers and kana; this task thus required switching attention. When participants touched a target circle, it remained in the same position, but its color changed from black to yellow. Participants were instructed to perform the task as quickly and correctly as possible, and continuously performed this task for 5 minutes. We evaluated three indices of task performance: the total count of correct responses (number of correctly touched numbers and letters); the total count of errors (number of incorrectly touched numbers and letters); and the motivational response (reaction time from a finished trial to the next trial). Based on our previous study, before participants performed task E of the mATMT on each experimental day, they practiced for a period of 1 minute. The reliability of this test has been confirmed.,

### Blood sample analyses

Blood samples were collected from the brachial vein. The amount of blood sampled was 13 mL per experimental day. We thus collected blood samples on four occasions (once per experimental day) in the study. Blood samples for serum analyses were centrifuged at 1,470 × g for 5 minutes at 4°C. The concentration of high-sensitivity C-reactive protein (hs-CRP) in each serum sample was assessed by particle-enhanced immunonephelometry using a BNII analyzer (BN II ProSpec; Siemens, Munich, Germany). Oxidative activity in each serum sample was assessed with the reactive oxygen metabolites-derived compounds (d-ROMs) test (Diacron International, Grosseto, Italy), while anti-oxidative activity was measured with the biological anti-oxidant potential (BAP) test (Diacron International) using a JCABM1650 automated analyzer (JEOL, Tokyo, Japan). The concentrations of ROMs are expressed in Carratelli units (1 CARR U = 0.08 mg of hydrogen peroxide/dL). The oxidative stress index (OSI) was calculated using the following formula: OSI = C × (d-ROMs/BAP), where C denotes a coefficient for standardization to set the mean OSI in healthy individuals at 1.0 (C = 8.85). All supernatants were stored at -80°C until analyzed. Assays for hs-CRP were performed at LSI Medience Corporation (Tokyo, Japan) and those for serum d-ROMs and BAP were performed at Yamaguchi University Graduate School of Medicine.

### Daily daytime activity and daily journal

Daily daytime activity, representing the expenditure of calories and amount of physical activity (METs × time) was recorded using an Active style Pro HJA-350IT pedometer (OMRON, Kyoto, Japan). A daily journal was kept for 4 weeks, and included information on fatigue (based on a visual analogue scale from 0, representing “no fatigue”, to 100, representing “total exhaustion”) just after waking up and before bedtime, sleeping times, physical condition (1, good; 2, normal; or 3, bad), and special events (if the day was different from a usual day: 1, no; or 2, yes). We carefully checked the daily journal every four weeks, on the 2nd, 3rd, and 4th experimental days.

### Statistical analyses

First, we tested the normality (parametric or non-parametric distributions) of each measured parameter using the Kolmogorov-Smirnov test. Values are presented as the mean ± standard deviation or median and interquartile range based on the results of Kolmogorov-Smirnov test. The Wilcoxon signed-rank test for non-parametric parameters and paired t-test for differences between HRW and PLW administrations after two-way repeated-measurement analysis of variance for parametric parameters were conducted. If significant changes were observed by comparisons within each condition (pre- vs. post-HRW; pre- vs. post-PLW) or between post-treatment values (post-HRW vs. post-PLW), then we compared change ratios between post-HRW/pre-HRW and post-PLW/pre-PLW using the Wilcoxon signed-rank test or paired t-test. All P values were two-tailed, and those less than 0.05 were considered statistically significant. Statistical analyses were performed using IBM SPSS Statistical Package version 20.0 (IBM, Armonk, NY, USA).

## Results

### General results

During the study, we excluded five participants from data analyses due to symptoms of hay fever, prolonged medication use because of a cold, insufficient intake of HRW or PLW intake (≥ 85%), or a frequency of special events ≤ 15% as recorded in the daily diary. We thus analyzed data from a total of 26 participants (13 females, 13 males; mean age, 34.4 ± 9.9 years; mean body mass index, 21.5 ± 2.6 kg/m2). No side, order, and carry-over effects were observed from the oral administrations of HRW and PLW in any participant.

### Questionnaire results

Results from the questionnaires are summarized in Table 1. No questionnaire scores at baseline (pre) showed any significant differences between HRW and PLW administration groups. With HRW administration, scores for K6, CFS, and PSQI were significantly decreased after the 4-week administration period. In addition, the change ratio (post/pre) for K6 score was significantly lower in the HRW administration group than in the PLW administration group (Figure 2). No significant changes were seen in any other questionnaire scores (modified version of the Osaka City University Hospital Fatigue Scale, Center for Epidemiologic Studies Depression Scale or Epworth Sleepiness Scale) after HRW administration and no significant changes in any of the scores were seen after PLW administration. Likewise, these scores did not differ significantly between HRW and PLW after administration.

### Table 1

Changes in parameters related to quality of life due to hydrogen-rich water (HRW) or placebo water (PLW) administration

Comparison of change ratios (post-treatment/pre-treatment) for parameters related to quality of life with administration of hydrogen-rich water (HRW) or placebo water (PLW) for 4 weeks.

Note: Change ratios for K6 score for mood (A) and anxiety and the low-frequency component power (LF) for autonomic nerve function (B). *P < 0.05.

### Autonomic function results

Results for the autonomic nerve function are summarized in Table 1. LF, HF, and LF/HF ratio at baseline (pre) did not differ significantly between HRW and PLW administrations, indicating similar autonomic nerve function in the two groups before water intake. Although the HF and LF/HF ratio were not significantly affected by 4-week administrations of HRW or PLW, LF after HRW administration was significantly lower than that after PLW administration. The change ratio (post/pre) for LF was also significantly lower in the HRW administration group than in the PLW administration group (Figure 2).

### Cognitive function results

Results for the cognitive function test are shown in Table 1. Motivational response and total counts of correct responses and errors at baseline (pre) did not differ significantly between HRW and PLW administrations, indicating similar cognitive function between groups before water intake. Motivational response after HRW administration was significantly faster than that before HRW administration. The change ratio (post/pre) for motivational response was not significantly different in the HRW administration group than in the PLW administration group. No significant differences in motivational response, total counts of correct responses, or errors after water administration were seen between HRW- and PLW-administered conditions.

### Blood sample results

No significant differences were seen in any blood parameters (hs-CRP, d-ROMs, BAP, and OSI) before HRW or PLW administration (Table 1), indicating the comparability of the two groups before water intake. After HRW and PLW administrations, we again found no significant differences in these blood parameters.

### Daily daytime activity and daily journal results

The daily expenditure of calories and amount of physical activity during the 4-week administration periods did not differ significantly between HRW and PLW administration conditions (Table 2). Similarly, visual analogue scale scores for fatigue just after waking and before bedtime, sleeping times, physical condition, and counts of special events were comparable between HRW and PLW administration conditions (Table 2), indicating that living habits were successfully controlled during the experimental period in the two groups.

### Table 2

Daily daytime activity and data recorded in the daily journal during the hydrogen-rich water (HRW) or placebo water (PLW) administration period (4 weeks)

## Discussion

The present findings suggest that HRW administration for 4 weeks may have improved the QOL of adult volunteers in terms of improved mood and anxiety and reduced activity of the sympathetic nervous system at rest.

In terms of associations between hydrogen and the central nervous system, a report by Ohsawa et al. was the first to demonstrate that molecular hydrogen acts, at least in part, as an anti-oxidant as it binds to hydroxyl ions produced in central nervous system injuries. Previous studies have proposed that HRW administration has neuroprotective effects and anti-aging effects on periodontal oxidative damage in healthy aged rats. In a rat model of Alzheimer’s disease, hydrogen-rich saline prevented neuroinflammation and oxidative stress, and improved memory function. In terms of the association between HRW and QOL, only one study reported that HRW administration for 6 weeks improved QOL scores in patients treated with radiotherapy for liver tumors. Although reports on the effects of HRW administration in healthy populations have not been accumulated, job stress, and acute fatigue caused by mental and physical loading for several hours, have been shown to enhance oxidative stress. As for physical fatigue, in order to alleviate acute physical fatigue in healthy volunteers not including athletes, we have previously demonstrated that treatment with antioxidant supplements is effective.,, The present study provided new findings that HRW affects not only physical condition but also mental conditions such as mood, anxiety, and autonomic nerve function. One of the advantages of HRW is the ability to cross the blood-brain barrier, offering high potential to reduce oxidative stress in the brain. A previous study in rats found that levels of malondialdehyde, a marker of oxidative stress, were around 4.8-fold higher in the brain than in the blood (plasma). These results suggest that HRW may be effective for reducing accumulated oxidative stress in the brain in daily life, potentially contributing to the maintenance of central nervous system activity and preventing decreases in QOL.

In the present study, mood and anxiety levels improved after HRW administration. These negative emotions are also known to be involved in conditions related to oxidative stress; social phobia,,depression, anxiety,, and other neuropsychiatric disorders have been shown to be associated with increased oxidative stress. Neuroinflammation is also related to fatigue, mood, anxiety, and sleep.,,, In older mice, HRW administration succeeded in suppressing depression-like behaviors. These findings suggest that administration of HRW for 4 weeks may be effective for controlling such negative emotions by reducing oxidative stress and inflammation of the central nervous system. Increasing evidence suggests that oxidative stress and inflammation in neurons are involved in the pathological manifestations of many neurological and neuropsychiatric disorders, and HRW administration may thus help alleviate the symptoms of these disorders. Previous study revealed that oxidative stress of the brain causes cognitive and motivational deficits in a mouse model of neuropsychiatric disorder (schizophrenia). In the present study, motivational response of cognitive function test was improved by prolonged HRW intake, suggesting that a reduction of oxidative stress in the brain by the intake of HRW may increase motivational performance of cognitive task.

Stressors can enhance sympathetic hyperactivity, promote oxidative stress, and boost pro-inflammatory cytokine production.,, Autonomic nerve function is thus closely associated with oxidative stress and inflammation. Attenuation of sympathetic nervous system activity during the resting state in adult volunteers may therefore be the result of decreases in inflammation and oxidative stress as an effect of prolonged HRW administration. However, the lack of changes in oxidative stress markers noted in the present study after HRW intake for 4 weeks could be due to the low severity of oxidative stress in the participants. Actually, serum d-ROMs (307.1 ± 49.4 CARR U) and BAP (2,549 ± 194 µM) concentrations at the first measurement point in the present study were within normal ranges based on the results of serum d-ROMs (286.9 ± 100.2 CARR U) and BAP (2,541 ± 122 µM) concentrations measured in 312 healthy participants in our previous study. However, levels of oxidative stress fluctuate depending on daily work load and stress. In addition, the rat study by García-Niño et al. that found malondialdehyde levels around 4.8-fold higher in the brain than in plasma indicate that oxidative stress in the brain is more severe. Daily administration of HRW for 4 weeks may thus contribute to attenuation of and prevention from the cumulative oxidative stress in the brain. Mood, anxiety, and autonomic nerve function could thus potentially be improved. Although the range of sympathetic nerve activity in the present study considers to be normal based on our previous studies,, sympathetic nerve activity also fluctuates depending on daily work load and stress. Therefore, lower sympathetic nerve activity of resting state may contribute to suppress an excessive increase in sympathetic nerve activity after the daily work load and stress.

We conducted this study with a limited number of participants. Before our results can be generalized, studies involving larger numbers of participants are essential.

Although we mainly examined the effects of HRW on the central nervous system, we did not directly evaluate the dynamics of inflammation and oxidation in the brain. Neuroimaging studies using positron emission tomography and magnetic resonance imaging are thus underway in our laboratory to identify the mechanisms underlying the effects of HRW intake on the central nervous system that can improve QOL.

In conclusion, HRW administration for 4 weeks in adult volunteers improved mood, anxiety, and autonomic nerve function, suggesting that HRW administration may offer an effective method to reinforce QOL and maintain good health. In a further study, we will try to identify the effects of HRW administration in participants with ongoing stress or chronic fatigue.

. 2017 Oct-Dec; 7(4): 247–255.
Published online 2018 Jan 22. doi:  [10.4103/2045-9912.222448]
PMCID: PMC5806445
PMID: 29497485

## Acknowledgments

We would like to thank Ms. Mika Furusawa for her excellent technical assistances and Forte Science Communications for editorial help with this manuscript.

## Footnotes

Conflicts of interest

This work was presented at Japanese Society of Fatigue Science, Yamaguchi City, Japan on May 16, 2016. Yasuyoshi Watanabe received funding for the present study from Melodian Corporation. The other authors have no conflicts of interest to declare.

Research ethics

All experiments were conducted in compliance with national legislation and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association (the Declaration of Helsinki) and registered to the UMIN Clinical Trials Registry (UMIN000022382). The study protocol was approved by the Ethics Committee of Osaka City University Center for Health Science Innovation (OCU-CHSI-IRB No. 4).

Declaration of participant consent

The authors certify that they have obtained all appropriate participant consent forms. In the form the participants have given their consent for their images and other clinical information to be reported in the journal. The participants understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Data sharing statement

Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check

Checked twice by iThenticate.

Peer review

Externally peer reviewed.

Open peer reviewers

Lei Huang, Loma Linda University, USA; Qin Hu, Shanghai Jiao Tong University, China.

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# Modulation of the oxidative plasmatic state in gastroesophageal reflux disease with the addition of molecular hydrogen rich water : A new biological vision

## Abstract

Gastroesophageal reflux disease (GERD), a clinical condition characterized by reflux of gastroduodenal contents in the oesophagus, has proved to demonstrate a strong link between oxidative stress and the development of GERD. Proton pump inhibitors (PPIs) have been universally accepted as first‐line therapy for management of GERD. The potential benefits of electrolysed reduced water (ERW), rich in molecular hydrogen, in improving symptoms and systemic oxidative stress associated with GERD was assessed. The study was performed on 84 GERD patients undergoing control treatment (PPI + tap water) or experimental treatment (PPI + ERW) for 3 months. These patients were subjected to the GERD‐Health Related Quality of Life Questionnaire as well as derivatives reactive oxigen metabolites (d‐ROMs) test, biological antioxidant potential (BAP) test, supe