Molecular Hydrogen for Medicine : The Art of Ancient Life Revived [1st ed.] 9789811571565, 9789811571572

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Yuh Fukai

Molecular Hydrogen for Medicine The Art of Ancient Life Revived

Molecular Hydrogen for Medicine

Yuh Fukai

Molecular Hydrogen for Medicine The Art of Ancient Life Revived

Yuh Fukai Department of Physics Prof. Emer. of Chuo University Tokyo, Japan

ISBN 978-981-15-7156-5 ISBN 978-981-15-7157-2 https://doi.org/10.1007/978-981-15-7157-2

(eBook)

“Revised translation from the Japanese language edition: Suiso Bunshi wa Kanari Sugoi” by Yuh Fukai # 2017 published by Kobunsha Co., Ltd. All rights reserved. # The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

This is a book of introduction to Molecular Hydrogen Medicine, a young field of medicine initiated by a seminal paper of Ohsawa and others in 2007. They reported that inhalation of hydrogen gas led to the elimination of harmful reactive oxygen species in rats and proceeded further to demonstrate various physiological effects and possible medical applications of hydrogen gas. After vigorous research activities of many people that ensued, their discovery was firmly established, and numerous clinical projects were started, yielding eye-opening results in many cases. Unfortunately, however, the vast array of research results remains mostly unknown to the public, and there are even many skeptics among medical specialists. In fact, the discovery of Ohsawa et al. came as a surprise to most people in biochemistry and medicine because hydrogen gas was generally regarded to be an inert gas doing nothing to the living body. This “common sense” of biochemistry, namely the inactivity of molecular hydrogen, has been an obstacle for Molecular Hydrogen Medicine to be duly appreciated from the medical community at large. Clearly, something must be done to remove this obstacle. I am not an expert in Molecular Hydrogen Medicine and do not even belong to the medical community. I spent most of my life doing research on hydrogen in physics, chemistry, materials science, and Earth science, but I have always kept my eye on whatever is related to hydrogen, including of course Molecular Hydrogen Medicine. Now I realize that there is an important role for me to play in this occasion, a role of fair and critical guide to Molecular Hydrogen Medicine. There is an urgent need for an accurate explanation of the present state of science and medicine of molecular hydrogen to the public, but no one engaged in research dared to take on such a job; they are too busy in pursuing their own problems. So I decided to do it myself. This book is currently the only monograph written on this subject and hopefully a useful guide to Molecular Hydrogen Medicine. In fact, it is not this sense of mission alone that brought me to this job of writing. With some knowledge of Earth science, especially that the life on Earth was born in the world of hydrogen, I have come to an idea that a hydrogen machinery engraved in our body, disclosed by Molecular Hydrogen Medicine, must have been inherited, at least in part, from our distant ancestors in the hydrogen era. Only from this point of view, Molecular Hydrogen Medicine could be placed legitimately in the broad perspective of life on Earth. This is an attempt I wish to make in writing this book. v

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Preface

This book is based on “Marvelous Medical Actions of Molecular Hydrogen” (Kobunsha, Tokyo in Japanese) published in 2017, which has been revised to a large extent to include recent progress. I hope that in reading this book, the readers will gain an awareness of Molecular Hydrogen Medicine and recognize the depth and breadth of the world of life with hydrogen. I received support from Dr. Ikuroh Ohsawa, one of the founding fathers of Molecular Hydrogen Medicine and a specialist in the field, in every phase of writing. I wish to express my heartfelt thanks for his kind support. I also wish to thank Dr. Moto Fukai for careful reading of the manuscript. I acknowledge some assistance from Editage (www.editage.com) for English language editing. April 2020

Yuh Fukai

Introduction

Molecular Hydrogen Medicine is a field of medicine in which physiological actions of molecular hydrogen are utilized for medical purposes. There, molecular hydrogen H2 is supplied to patients either as hydrogen gas or hydrogen water (aqueous solution of H2), by breathing, drinking, or injection. Some might think that, since hydrogen is a major constituent of the human body, the intake of small amounts of hydrogen should not cause any significant effects. However, this is not the case. Whereas hydrogen in the body exists exclusively as compounds (i.e. water, proteins, fats, carbohydrates, etc.), hydrogen in molecular form ( molecular H2 for short) is almost non-existent; therefore, its introduction from outside, though small in amounts, may produce situations totally unknown to the body. And indeed, as research activities progressed, it has come to be recognized that molecular H2 exerts therapeutic and preventive effects on many diseases, including cardiovascular diseases, metabolic syndromes, rheumatoid arthritis, radiation damage, to name a few. The efficacy is far beyond any expectations. Hydrogen exhibits different properties in different bonding states, and only in the molecular form, it exerts these unique physiological effects. In Part I of the book “What Is Molecular Hydrogen Medicine?” a consistent description of Molecular Hydrogen Medicine is given, from its birth in 2007 to the present state and its future prospect, encompassing animal experiments and numerous clinical applications. In this young field of research, however, it is essential to make a critical evaluation and selection of existing data. Therefore, the primary aim of Part I is not to provide a comprehensive review of existing data, but a presentation of well-scrutinized set of data that delineate the possible extension and in-depth understanding of this area of medicine. Part II, “Tracing the History of Life and Hydrogen” tries to place Molecular Hydrogen Medicine in the context of the evolution of life from ancient hydrogen age. It is believed that first life on Earth originated in the sea, where hydrogen was utilized as an energy source. Subsequently, oxygen was produced by cyanobacteria and living organisms developed the ability to use this oxygen and then moved onto land. It is unlikely that the memory of the “hydrogen era,” from which life evolved, has been completely lost in these processes. Thus, it is believed that in order to

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Introduction

understand the functions of the human body, the process of how we have reached the present state after the long history of life must be understood. Such an approach should provide new and exciting research perspectives. Observation of our current form alone, however hard we may try, has its own limitations. In short, I wish to lay out the story of how humans rediscovered the value of hydrogen after our long evolutionary history.

Contents

Part I 1

2

3

What is Molecular Hydrogen Medicine?

The Power of Hydrogen Molecules Uncovered . . . . . . . . . . . . . . . 1.1 Early Sporadic Reports of Hydrogen Effects . . . . . . . . . . . . . . 1.1.1 How the Story Started in Japan . . . . . . . . . . . . . . . . . 1.2 Ohsawa, Ohta, and the Dawn of Molecular Hydrogen Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 Molecular Hydrogen Selectively Eliminates ROS . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . .

3 3 4

. . .

4 5 11

Development of Molecular Hydrogen Medicine . . . . . . . . . . . . . . . 2.1 Therapeutic Hydrogen Effects More Extended than Expected . . 2.1.1 Possible Therapeutic Effects of Molecular Hydrogen . 2.1.2 Preventive Effects of Molecular Hydrogen . . . . . . . . . 2.2 Origin of Fatigue and Aging . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 The Mechanism of Fatigue . . . . . . . . . . . . . . . . . . . . 2.2.2 The Mechanism of Aging . . . . . . . . . . . . . . . . . . . . . 2.3 The Current State of Molecular Hydrogen Medicine . . . . . . . . 2.3.1 Establishment of the Japanese Society for Medical and Biological Research on Molecular Hydrogen . . . . 2.3.2 Major Research on the Medical Effects of Hydrogen Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3 The Current State of Human Clinical Trials . . . . . . . . 2.4 Major Results of Human Clinical Trials . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . .

13 13 14 36 38 38 39 40

.

41

. . . .

42 42 46 57

From the Front-Line of Research: Interviews . . . . . . . . . . . . . . . . . 3.1 Interview with Dr. Ikuroh Ohsawa of the Tokyo Metropolitan Institute of Gerontology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Interview with Dr. Motoaki Sano of Department of Cardiology, Center for Molecular Hydrogen Medicine, Keio University . . . . 3.3 Interview with Dr. Hirohisa Ono of the Neurosurgery Department, Nishijima Hospital . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61 62 65 68 71 ix

x

Contents

4

Physiological Effects of the Hydrogen Molecules . . . . . . . . . . . . . . 4.1 Entry of Hydrogen Molecules into Human Body . . . . . . . . . . . 4.2 Hydrogen Molecules Are Harmless . . . . . . . . . . . . . . . . . . . . 4.3 The Mechanisms of Action on Diseases . . . . . . . . . . . . . . . . . 4.3.1 Direct Scavenging of Hydroxyl Radicals . . . . . . . . . . 4.3.2 Nrf2/HO-1 Pathway . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3 Actions Mediated by Intestinal Microbiota . . . . . . . . . 4.3.4 Additional Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . .

73 73 77 78 78 80 84 86 87

5

Some Fundamental Properties of Hydrogen and Water . . . . . . . . 5.1 The Solubility of Hydrogen in Water . . . . . . . . . . . . . . . . . . . 5.2 Hydrogen Bonding in Water . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Tunneling Mechanism in Atomic/Molecular Migration . . . . . . 5.3.1 What Is the Tunneling Mechanism? . . . . . . . . . . . . . . 5.3.2 Tunneling Motion of Hydrogen Atoms . . . . . . . . . . . 5.3.3 Tunneling Motion of Hydrogen Molecules . . . . . . . . . 5.4 The Grotthuss Mechanism for the Migration of Hydrogen in Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . .

91 91 92 94 95 96 98

Preparation, Handling, and Usage of Hydrogen . . . . . . . . . . . . . . 6.1 Hydrogen Gas and Hydrogen Water for Medical Use . . . . . . . 6.1.1 How to Administer Hydrogen . . . . . . . . . . . . . . . . . . 6.1.2 Hydrogen Gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.3 Hydrogen Water for Medical Use . . . . . . . . . . . . . . . 6.2 Hydrogen Water as Supplement . . . . . . . . . . . . . . . . . . . . . . . 6.2.1 Difference from Carbonated Water . . . . . . . . . . . . . . 6.2.2 Permeation of H2 in Metals: Aluminum (Al) Containers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.3 How to Produce Hydrogen Water as Supplements . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . .

Functions of Heavy Water in Living Organisms . . . . . . . . . . . . . . 7.1 Physiological Actions of Heavy Water . . . . . . . . . . . . . . . . . . 7.1.1 What Happens to Small Animals . . . . . . . . . . . . . . . . 7.1.2 What Happens at the Cellular Level? . . . . . . . . . . . . . 7.1.3 Why Does the Physiological Action of Heavy Water Occur? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Medical Applications of Heavy Water: A Focus on Organ Transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1 Applications in Organ Preservation . . . . . . . . . . . . . . 7.2.2 Development of Heavy Water Preservation Solutions . 7.2.3 High-Performance Deuterated Water Solution Dsol . . 7.3 From the research Field: An Interview . . . . . . . . . . . . . . . . . .

. . . .

6

7

. 99 . 104 105 105 105 106 108 110 112

. 112 . 114 . 115 117 117 118 119

. 120 . . . . .

121 121 122 125 130

Contents

xi

7.3.1

Interview with Dr. Moto Fukai, Department of Transplant Surgery, Graduate School of Medicine, Hokkaido University . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 7.4 The iPS Cells and Autophagy in the Context of Transplant Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 8

The Future of Molecular Hydrogen Medicine . . . . . . . . . . . . . . . . . 141

Part II

Tracing the History of Life and Hydrogen

9

The Genesis of Life in the World of Hydrogen, Eons Ago . . . . . . . 9.1 Origin of Life on Earth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Ancient Organisms Lived in a Hydrogen World . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . .

145 145 148 151

10

Drastic Changes of Living Organisms in the Oxygen Age . . . . . . . 10.1 How Was Oxygen Produced? . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Birth of Oxygen-Breathing Organisms . . . . . . . . . . . . . . . . . . 10.3 Multicellular Organisms Conquer Land . . . . . . . . . . . . . . . . . 10.4 Evolution of Biological Molecules (Biomaterials) . . . . . . . . . .

. . . . .

153 153 154 156 158

11

Since the Appearance of Humans . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Traces of the Ancient Ocean Retained in the Human Body . . . 11.2 Circulation of Water and Oxygen in the Human Body . . . . . . . 11.3 Living with Oxygen Is Difficult: How to Manage ROS . . . . . . 11.3.1 What Is Reactive Oxygen Species? . . . . . . . . . . . . . . 11.3.2 How to Live with ROS . . . . . . . . . . . . . . . . . . . . . . . 11.4 Memories of the Hydrogen Age Retained in the Present Living Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .

159 159 161 166 166 167

. 170 . 171

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Index of Proper Names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

Part I What is Molecular Hydrogen Medicine?

A consistent description is given of Molecular Hydrogen Medicine, from its birth in 2007 to the present state and its future prospect, encompassing animal experiments and numerous clinical applications. Out of vast number of research papers on various physiological and therapeutic effects of molecular hydrogen, a limited number of well-qualified papers including animal experiments and clinical trials have been selected and elucidated. A brief description is given of the mechanisms of action presently under intensive studies. Some fundamental properties of molecular hydrogen (hydrogen gas and hydrogen water) are explained, together with its preparation, handling and methods of administration to provide basic information necessary for Molecular Hydrogen Medicine. Application of heavy water for organ transplantation, though slightly out of context, is included as a recent topic in the Hydrogen Medicine. Key players of Molecular Hydrogen Medicine are hydrogen gas and hydrogen water. Hydrogen water is simply water in which hydrogen gas has been dissolved, in other words, a mixture of hydrogen molecules and water molecules. As hydrogen water has been found to exhibit nearly the same effects as hydrogen gas, the actual effective component is thought to be the hydrogen molecule. Usually, molecular hydrogen is so stable that it does not undergo any chemical reaction with any substance at room temperature and is therefore regarded to be an inert gas for living bodies. Molecular Hydrogen Medicine is peculiar in that, in contradistinction to this common sense of biochemistry, it aims to investigate physiological effects exerted by molecular hydrogen and its possible clinical applications. This is a new field of research having profound implications for life science.

1

The Power of Hydrogen Molecules Uncovered

Abstract

After some sporadic reports, a seminal paper of Ohsawa et al. (Nat. Med. 13:688–694, 2007) was published that clearly demonstrated physiological and therapeutic effects of molecular hydrogen (MH), and opened a new area of medicine to be called Molecular Hydrogen Medicine (MHM).

1.1

Early Sporadic Reports of Hydrogen Effects

In the experiment of Malcolm Dole et al. (1975), mice with skin cancer (squamous cell carcinomas) were reared in an environment with 0.8 MPa (8 atm) of hydrogen gas (and an appropriate amount of oxygen) for 2 weeks, which led to the shrinkage of the cancer cells. This was an important discovery demonstrating the physiological effects of molecular hydrogen, but attracted little attention at that time, and no follow-up experiments were carried out. The next time the physiological effects of hydrogen gas appeared in print was 20 years later in an article titled “Gas Therapy” in the “Daedalus” column of Nature Magazine (Jones 1996). This article suggested that inhalation of hydrogen gas could rapidly eliminate hydroxyl radicals within the body, and that hydrogen should be entirely harmless to the body and be completely excreted without being accumulated, and therefore should be an ideal gas therapy. The article stated that hydrogen could also be dissolved in water and taken orally. This might appear to be an excellent prediction, but in fact, Jones apparently wrote the article as a fantasy. Daedalus appears in Greek mythology as an inventor who made wings for his son, Icarus, and the column was named after him because it was science fiction. Subsequently, Gharib et al. (2001) attempted to test Jones’ fantasy in real life. They reared mice with chronic liver tumors in an environment with an additional 0.7 MPa of hydrogen gas (of a total of 0.8 MPa) for two weeks and discovered that # The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2020 Y. Fukai, Molecular Hydrogen for Medicine, https://doi.org/10.1007/978-981-15-7157-2_1

3

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The Power of Hydrogen Molecules Uncovered

the liver damage in these mice was markedly reduced. Jones and Gharib unknowingly rediscovered the pioneering research of Dole et al. (1975). However, this rediscovery, too, was forgotten, having failed to attract any attention. In fact, prior to these sporadic reports, a company called COMEX (Marseille, France) had started intensive studies to utilize hydrogen for deep sea diving technologies. After performing various experiments on cells and animals, they succeeded in developing a high-pressure mixture gas containing hydrogen which is effective for the prevention of diving disease. This may be regarded to be the very first achievement related to Molecular Hydrogen Medicine.

1.1.1

How the Story Started in Japan

Hydrogen research in Japan started in an entirely different way. In Japan, there were people, including physicians, who worked fervently to emphasize the health benefits of “reduced water” produced by the electrolysis of normal water. Reading early publications on the subject, one can see numerous examples of its benefits. The effects mentioned include improved fitness, smoother skin, improved diabetes symptoms, and anti-cancer effects. In all the cases, however, there was a lack of stringency required for scientific research, and therefore, these reports cannot be taken at face value. Nonetheless, some of the data generated cannot be completely dismissed. For example, MiZ Co., Ltd. reported that electrolyzed water suppressed oxidative liver damage (Yanagihara et al. 2005). Having observed the controversy in this research field continuing for years, the research group of Shigeo Ohta and Ikuroh Ohsawa of the Nippon Medical School decided to intervene. As experts in biochemical and cellular mitochondrial physiology they were determined to settle the issue of this “miracle water” through cautiously designed experiments.

1.2

Ohsawa, Ohta, and the Dawn of Molecular Hydrogen Medicine

Ohta had been studying the functions of mitochondria for more than thirty years. However, with growing awareness of the harmful effects of reactive oxygen species (ROS) generated by mitochondria, including aging and various diseases, Ohta came to focus his attention on these effects. In 2005 Ohta’s research group began investigating the effect of hydrogen on ROS. As a standard procedure in biochemical research, they started from the cellular level and moved to the organs, and then to the whole body. They started with animal experiments, aiming to progress to clinical applications. In this section, I introduce their research which paved the way for Molecular Hydrogen Medicine.

1.2 Ohsawa, Ohta, and the Dawn of Molecular Hydrogen Medicine

1.2.1

5

Molecular Hydrogen Selectively Eliminates ROS

Not long after starting their cellular experiments, the Ohta group were surprised by the remarkable effects of hydrogen. Later, Ohta wrote the following: Soon after we started, we were shocked by what we saw. The first results were such a surprise that I stopped in my tracks. It was the third day of the experiment and, without thinking, I yelled: Look! It’s amazing! In the culture media with hydrogen, ROS induced in cells didn’t do any harm. The cells were all alive. It was a revolutionary discovery. . .

This was the first experiment that demonstrated the effects of hydrogen on living cells. Stimulating the formation of ROS in cells using drugs usually causes the cells to shrink and become spherical in shape as their metabolism deteriorates, and they eventually die. However, when hydrogen was dissolved in the culture medium, the cells’ functions were unaffected and cell death was greatly reduced (Fig. 1.1). Microscopic observations showed that hydrogen could enter all organelles within the cell (nucleus, cytoplasm, mitochondria, etc.), in contrast to traditional antioxidants which are unable to enter the cell. Their subsequent experiments led to another important discovery that the effect of hydrogen was very selective (Ohsawa et al. 2007). As shown in Fig. 1.2, the scavenging effect of molecular hydrogen in solution was different for different ROS; particularly large for •OH, much smaller for ONOO–, and negligibly small for other ROS. This implies that molecular H2 eliminates only harmful ROS, leaving other ROS playing important roles in cell signaling intact. This is a completely different behavior from existing antioxidants (e.g., vitamin C), which eliminate both beneficial and harmful ROS indiscriminately. The antioxidant effects of molecular H2 were also confirmed by other in vitro experiments. Figure 1.3 shows that the increase of 8-OHdG (8-hydroxy-

Without H2

With H2

Red: dead cells

Green: live cells

Fig. 1.1 Molecular hydrogen H2 protects PC12 cells from reactive oxygen species (hydroxyl radicals •OH). Number of cells 1 h after induction of •OH; in culture medium without H2 (left), and with H2 (right) (I. Ohsawa 2007, private communication)

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The Power of Hydrogen Molecules Uncovered

Fig. 1.2 Molecular H2 dissolved in saline scavenges •OH selectively in cell free system. (a) Formation of •OH by Fenton reaction at 0.8 mM H2; Baseline 1, without H2O2, and Baseline 2, without ferrous perchlorate. (b) ~ (f) levels of ROS concentrations after incubation with 0.6 mM of H2: (b) •OH, (c) ONOO
, (d) O2
•, (e) H2O2, (f) NO
•. Mean value  SD, (n ¼ 6). P < 0.05 (Ohsawa et al. 2007)

Fig. 1.3 Molecular H2 protects cultured PC12 cells by scavenging •OH radicals. Suppression by molecular H2 of oxidation (induced by antimycin A) of nuclear DNA measured by 8-OHdG (P < 0.05) and peroxidation of lipids measured by 4-HNE conjugate (P < 0.01). Mean  SD (Ohsawa et al. 2007)

1.2 Ohsawa, Ohta, and the Dawn of Molecular Hydrogen Medicine

7

H2 conc. in inhaled gas 0%

2%

Fig. 1.4 Brain injury by ischemia/reperfusion in rats was reduced by inhalation of H2 gas. The infarct size 1 day after the I-R (white area) was reduced by inhalation of 2% H2 gas during reperfusion (Ohsawa et al. 2007)

20 -deoxyguanosine) and 4-HNE (4-hydroxyl-2-nonenal) produced by peroxidation of DNA and lipids, respectively, was suppressed effectively by molecular H2. These discoveries strongly suggested that hydrogen should have the potential to act as a new antioxidant. Then, with clinical applications in mind, they proceeded to investigate the effects of hydrogen in ischemia-reperfusion (I-R) injury, in which serious oxidative damage usually occurs. I-R injury occurs in circumstances such as organ transplant, where blood (with oxygen) is temporarily removed from an organ and subsequently poured into the organ again (reperfused) after the transplant. Supplying oxygen after a period of oxygen deprivation results in the production of large quantities of ROS. This is a serious problem because the ROS can then cause damage in internal organs. In their experiment, the cerebral arteries of rats were blocked to stop blood flow for 90 min, and the effect of hydrogen on brain damage upon reperfusion after 30 min was investigated. The results were eye opening. By having the rats breathe 2–4% H2 gas, the region of the brain damaged after1 day was reduced to nearly half (Fig. 1.4). Additionally, reductions in body temperature, body weight, and motor function as a result of I-R tended to recover after 1 week of hydrogen treatment. Thus, inhalation of H2 gas suppressed temporary brain damage due to I-R, as well as the associated secondary diseases. These experiments demonstrated, beyond any doubt, that H2 molecules administered as a gas or dissolved in water passed through tissues and biological membranes and eliminated hydroxyl radicals throughout the body, the most harmful ROS. Subsequently, their group demonstrated by experiments with mice that hepatic I-R injury could be suppressed by inhalation of H2 gas (Fukuda et al. 2007). The procedure was as follows: occlusion for 90 min, reperfusion for 180 min, and inhalation of H2 gas (1–4%) for the last 190 min. Figure 1.5(a) shows that the vacuolization induced by hepatic injury was suppressed effectively by H2 gas, whereas He gas exerted no effects. Figure 1.5(b) shows that the inhalation of H2 gas reduced the level of MDA (malondialdehyde), a marker of oxidative stress, to

8

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The Power of Hydrogen Molecules Uncovered

Fig. 1.5 Molecular H2 suppresses hepatic injury in mice. Effects of molecular H2 on (a) vacuolization in hepatic tissues (n ¼ 6, *P < 0.001), (b) oxidative stress marker MDA (n ¼ 6, **P < 0.0001), and (c) a marker of hepatic injury, serum ALT (n ¼ 6, *P < 0.05, **P < 0.005) (Fukuda et al. 2007)

almost normal level (without I-R treatment), and in Fig. 1.5(c) are shown the concomitant changes of serum ALT (alanine aminotransferase), a biomarker of hepatic injury. The effect of molecular H2 was very similar. These experiments came as a big surprise to most biochemists, because, due to its high chemical stability, H2 gas had been regarded as an inert gas to living bodies, including humans. However, as their experiments were so well designed and carefully performed that there was no room for any doubt in the result. And indeed, their results were confirmed by numerous follow-up experiments. Thus, the seminal paper of Ohsawa et al. (2007) entitled “Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals” attracted widespread attention, and research into possible clinical applications of hydrogen, now called “Molecular Hydrogen Medicine,” commenced. Column 1: When Hydrogen Has Come to be Known—the World as Seen by Lavoisier The word hydrogen means literally “the element of water,” which in fact consists of hydrogen and oxygen. This fact is now widely known, but was not known until the end of the eighteenth century when the French chemist Antoine Lavoisier conducted experiments to confirm for the first time that hydrogen is present in nature as an element and named it hydrogène (Fig. 1.6). Lavoisier’s discovery conflicted with the phlogiston theory that predominated the academic community of the time, and more than 20 years passed before it was accepted as such. In the phlogiston theory, all flammable substances contain weightless and invisible phlogiston, and upon combustion, phlogiston leaves the substance and incombustible ash remains. In 1766, Henry Cavendish (UK) discovered a flammable gas that was lighter than air and suggested that it was phlogiston. This flammable gas (later known as (continued)

1.2 Ohsawa, Ohta, and the Dawn of Molecular Hydrogen Medicine

9

Fig. 1.6 Lavoisier is the father of ‘hydrogène’. In his monumental work, “Traité Élémentaire de Chimie”, published in 1789, he gave this name to a gaseous substance that produced water when combined with oxygen, a composite of Greek hydro (water) and gene (make)

Column 1: When Hydrogen Has Come to be Known—the World as Seen by Lavoisier (continued) hydrogen), and “living gas” (oxygen) comprising 1/5th of air reacted to produce water; thus, water was considered a “living gas” (oxygen) attached with phlogiston. Cavendish did not realize that the “flammable gas” was an element, being obsessed with the abstract notion of phlogiston at that time. In contrast, Lavoisier adhered to concrete evidence. In the introduction of Lavoisier’s masterpiece “Traite Élémentaire de Chimie” (Elementary Treatise on Chemistry) published in 1789, he stated: “We only need to progress from learning what is known to what is unknown. The strict rule that I follow is never to form any conclusions which are not fully warranted by experiment, nor to provide any additional information in the absence of facts” (“Lavoisier 1743-1794” by Grimaux, Paris 1888). (continued)

10

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The Power of Hydrogen Molecules Uncovered

Column 1: When Hydrogen Has Come to be Known—the World as Seen by Lavoisier (continued) Thus, from the results of his extensive experiments, knowledge that had not been predicted until then was gained, specifically that mass remains unchanged by chemical reactions. In experiments with water, Lavoisier observed that steam flow on red-hot iron powder increased the weight of iron and led to the production of a lighter gas. He considered that this weight gain of iron was equivalent to the weight of oxygen in the air and concluded that water contains 85% oxygen and 15% hydrogen (light gas) by weight. Conversely, after mixing oxygen and hydrogen at a volume ratio of 1:2 (weight ratio of 84:16), he demonstrated that igniting the mixture produced water. According to our current knowledge of water, the volume ratio of oxygen to hydrogen is 1:2 and the weight ratio is 8:1. Given the difficulty of measuring the weight of light gases such as hydrogen, Lavoisier’s experiments were highly accurate. It was from these studies that hydrogen was firmly characterized as an element. Lavoisier’s contribution to the understanding of combustion proceeded still further. He showed that when charcoal was combusted, it reacted with oxygen and produced a gas, which is now identified as carbon dioxide (CO2). Furthermore, he noted that in the gas exhaled by humans, oxygen was lost, and CO2 was generated. These observations led him to conclude that respiration was a slowly occurring combustion process. This finding was further advanced by the later invention of the calorimeter, which was used to establish relationships among and between movement, heat, respiration, perspiration, and digestion. These discoveries launched a new research field of physiology. Years later, Lavoisier wrote a short comment on his unfinished studies on the chemistry of plants and animals. He stated that plants extract substances necessary for forming living organisms from the atmosphere and water, from the mineral kingdom generally speaking. Animals sustain their bodies by eating plants or animals that eat plants. Therefore, substances that are formed in animals are originally derived from the air and the mineral kingdom. Finally, fermentation, putrefaction, and combustion return the elements borrowed by animals and plants to the mineral kingdom. In what way does nature govern this wonderful cycle between these three kingdoms? How does nature construct combustible, fermentable, and perishable substances from materials that do not have such properties? As of now, this is an unfathomable mystery. However, combustion and putrefaction are certainly the methods to return materials forming plants and animals to the mineral kingdom. Thus, the constitution of substances as plants and animals must be the opposite phenomenon of combustion and putrefaction. Modern chemistry, which was initiated by Lavoisier, has greatly developed since then. Subsequently, biochemistry, which examines the structures and (continued)

References

11

Fig. 1.7 Antoine Laurent Lavoisier (1743–1794). A picture taken of the portrait displayed in Conciergerie, where he had been imprisoned before sent to the guillotine

Column 1: When Hydrogen Has Come to be Known—the World as Seen by Lavoisier (continued) reactions of diverse biological substances that support life activities, has become a colossal field and is continuing to advance. Figure 1.7 shows a portrait of Antoine Laurent Lavoisier exhibited at the Conciergerie in Paris, where he was imprisoned at the time of French Revolution. Lavoisier was then subjected to a revolutionary trial for his participation in tax collection, and sentenced to capital punishment and guillotined on May 8, 1794. He was 50 years old at the time of death. His friend, a mathematician Joseph-Louis Lagrange, stated: “It took them only an instant to cut off his head, but one hundred years might not suffice to reproduce its like.” Although Lavoisier’s death was premature, so were the deaths of many others at that time, and as many as 2 million unnamed people shed blood as compensation for social transformation.

References M. Dole, F.R. Wilson, W.P. Fife, Hyperbaric hydrogen therapy: A possible treatment for cancer. Science 190, 152–154 (1975) K. Fukuda, S. Asoh, M. Ishikawa, Y. Yamamoto, I. Ohsawa, S. Ohta, Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress. Biochem. Biophys. Res. Commun. 361, 670–674 (2007) B. Gharib, S. Hanna, O.M.S. Abdallahi, H. Lepidi, B. Gardette, M. de Reggi, Anti-inflammatory properties of molecular hydrogen: investigation on parasite-induced liver inflammation. Compte Rendu des Académie des Sciences, Life Sci 324, 719–724 (2001) É. Grimaux, Lavoisier 1743-1794 (Paris, 1888) D. Jones, Gas therapy. Nature 383, 676 (1996) A.L. Lavoisier, Traité Élémentaire de Chimie (Paris, 1789) I. Ohsawa, M. Ishikawa, K. Takahashi, M. Watanabe, K. Nishimaki, K. Yamagata, K. Katsura, Y. Katayama, S. Asoh, S. Ohta, Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat. Med. 13, 688–694 (2007) T. Yanagihara, K. Arai, K. Miyamae, B. Sato, T. Shudo, M. Yamada, M. Aoyama, Electrolyzed hydrogen-saturated water for drinking use elicits an antioxidative effects: a feeding test with rats. Biosci. Biotechnol. Biochem. 69, 1985–1987 (2005)

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Development of Molecular Hydrogen Medicine

Abstract

As research activities progressed, effects of molecular hydrogen (MH) proved to be more extended than previously expected. In parallel with exploratory experiments with animals, human clinical trials have also been performed and yielded positive results in most cases. Here I begin with animal experiments on ischemia-reperfusion injury, peritonitis to sepsis, wound healing, radiation injuries, organ transplantation, metabolic syndrome and lung diseases, and then proceed to describe clinical trials conducted in Japan including recovery from acute erythematous skin disease, cardiac arrest, stroke, brain disorder (Parkinson’s and Alzheimer’s diseases), diabetes, fatigue and aging, rheumatoid arthritis, blood dialysis, cataract surgery, periodontitis, and finally, recent results from other countries regarding radiation injuries and COVID-19 pneumonia. This is a main chapter of the book.

2.1

Therapeutic Hydrogen Effects More Extended than Expected

ROS are associated with illnesses in almost all organs, including the central nerve system, respiratory, circulatory, digestive, vascular, and endocrinological systems, as well as the eyes, nose, teeth, bone, and skin. Hydrogen has been shown to work effectively in many of these situations. The extent to which hydrogen influences physiological processes has exceeded expectations. Moreover, later research showed that hydrogen has not only antioxidant effects, but also anti-inflammatory, antiallergen, and metabolic enhancing effects. Figure 2.1 presents a list of studies that have shown the efficacy of hydrogen. Although medical advancements often face a major hurdle in ensuring safety when advancing from animal to human clinical trials, in the case of hydrogen, numerous # The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2020 Y. Fukai, Molecular Hydrogen for Medicine, https://doi.org/10.1007/978-981-15-7157-2_2

13

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Metabolic syndrome Diabetes Hyperlipidemia Arteriosclerosis Hypertension Obesity Aging

Fatigue

Radiation damage

Development of Molecular Hydrogen Medicine

Inflammation Rheumatism Sepsis Periodontitis

Administration of H2-water, H2-gas Injection of H2-saline

Vascular diseases Atherosclerosis Atheroma

Ischemia/reperfusion injury Cerebral/myocardial infarction Organ transplantation Post-cardiac arrest Neurological diseases Dementia Alzheimer’s disease Parkinson’s disease Depression Anesthetic

Hemodialysis Ventilation Diver’s Disease

Fig. 2.1 Potential medical applications of molecular H2; the areas where various therapeutic and preventive effects of H2 have been observed (Ohta 2014)

clinical trials have also been conducted owing to the fact that it is not harmful to the human body. Here, we introduce a summary of studies that have been carried out mostly in Japan.

2.1.1

Possible Therapeutic Effects of Molecular Hydrogen

Ischemia-Reperfusion Injury According to the experiments conducted at Keio University, when a rat heart was temporarily stopped and started working again, inhalation of H2 gas improved the survival rate after 7 days from 38% to 86%. Cranial nerve injuries caused by ischemia were clearly reduced (Hayashida et al. 2014). Keio University has since been organizing a large-scale clinical trial in which H2 gas is administered to patients with acute myocardial infarction (MCI) and patients that have been resuscitated after cardiac arrest. More recent results are described in Sect. 2.3. It has also been reported that the sort of I-R injury seen in rat hearts, lungs, and small intestines at the time of organ transplant can be ameliorated through inhalation of H2 gas. Kawamura et al. (2010) investigated transplant-induced I-R injury of rats’ lungs. Grafts were perfused with and stored in dextran solution at 4
C for 6 h, and after transplantation, the recipients were placed in four different atmospheres, 100% O2, 98% O2 + 2% (N2, He, H2) during surgery and 1 h after reperfusion. Figure 2.2 shows the partial pressure of O2 and CO2 in the graft pulmonary vein 2 h after reperfusion. Gas exchange function was deteriorated in O2 and 98% O2 + 2% (N2, He), which was significantly improved in 98% O2 + 2% H2. The cold I-R injury was accompanied by increased expression of several proinflammatory cytokines, as shown in Fig. 2.3 (a) and (b), and this increase was attenuated in 2% H2 atmosphere.

2.1 Therapeutic Hydrogen Effects More Extended than Expected

15

Fig. 2.2 Gas exchange function of the lung graft of rats. (a) partial pressure of O2, and (b) partial pressure of CO2 for the pulmonary vein of the transplanted left lung 2 h after reperfusion. Lung transplant (LTx) recipients treated with four different atmospheres, 100% O2, 98% O2 + 2% (N2, He, H2). n ¼ 6 ~ 8, {P < 0.05 vs. LTx O2, N2, He (Kawamura et al. 2010)

b. IL-1

％GAPDH

a. TNF-

†

†

c. Bcl -2

†

d. Bcl- xl

†

e. Bax

†

Fig. 2.3 Assessment of inflammation and apoptosis in the lung graft of rats. Indicators of inflammation (a. TNF-α and b. IL-1β) measured 2 h after 6 h I-R injury. Indicators of apoptosis (c. Bcl-2, d. Bcl-xl, e. Bax) measured 6 h after 6 h I-R injury. n ¼ 5, {P < 0.05 vs. LTx N2, He. GAPDH glyceraldehyde-3-phosphate dehydrogenase (Kawamura et al. 2010)

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Fig. 2.4 Hydrogen treatment increased HO-1 in the lung graft of rats before transplantation. N2 (98% O2 + 2%N2), H2 (98% O2 + 2%H2). n ¼ 5, {P < 0.01 (Kawamura et al. 2011)

(c) and (d) show that H2-treatment promotes the production of anti-apoptotic proteins B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl), while suppressing the proapoptotic Bcl-2-associated X-protein (Bax) (e). These results clearly indicate that molecular H2 has both antioxidative and anti-apoptotic effects. They proceeded further to demonstrate that preloaded H2 prior to procurement effectively protected grafts from I-R injury (Kawamura et al. 2011). Figure 2.4 shows the increase of HO-1 (heme oxygenase 1) expression in the graft of rat’s lungs caused by H2 loading before transplantation. This increase has strong inverse correlation with the deterioration of gas exchange function after transplantation. Because HO-1 is an inducible antioxidant enzyme, this appears to indicate that HO-1 induction is one of the mechanisms underlying the protective effects of H2. It was reported that induction of HO-1 in kidney, heart, and liver graft prior to transplantation provided graft protection after reperfusion. The role of HO-1 in the mechanism of action of molecular H2 on diseases is described in Sect. 4.3. The process of I-R injury during liver transplantation in rats was studied in greater detail at Hokkaido University (Shimada et al. 2016). After cold storage for 48 h in University of Wisconsin (UW) solution, the graft was circulated with oxygenated buffer with or without H2 dissolution at 37
C on an isolated perfused rat liver (IPRL) apparatus. The state of the graft after 90-min reperfusion was examined by several different measurements, as shown in Figs. 2.5 and 2.6. Results obtained for H2(+) and H2() groups are compared with those of control (CT) group without cold storage. As shown in Fig. 2.5, after cold storage, portal vein (inlet) resistance, portal vein pressure needed to sustain the perfusate’s flow, was markedly increased in the H2() group, which was ameliorated in the H2(+) group. The results indicate that the

Portal vein resistance cm H2O/(mL/min/g liver)

2.1 Therapeutic Hydrogen Effects More Extended than Expected

8

†

†

†

†

†

†

17

†

6

H2(-)

4

* * * * * * * H (+) 2

CT 2 30

40

50

60

70

80

90

Time (min)

Fig. 2.5 Portal vein resistance (PVR) of brain-dead rat’s liver. Pressure needed to sustain the perfusate’s flow into rat’s liver was measured between 30 and 90 min after starting reperfusion. In the control group (CT), livers were reperfused immediately, without preservation. Deterioration as compared to the control group was ameliorated significantly in the H2-added perfusate. P < 0.05 (Shimada et al. 2016)

(b) Bile production

*

10

† 5

120

10 100

[ µL/g ]

†

Bile production rate (relative)

Oxygen consumption rate (relative)

(a) OCR

†

808

*

606 404

†

202

0

CT (–) (+) H2

00

CT (–) (+) H2

Fig. 2.6 Function of brain-death rat’s liver after 90-min reperfusion; (a) viability, (b) bile production. Both viability (oxygen consumption rate) and bile production were deteriorated during cold storage and subsequent reperfusion, but ameliorated significantly by reperfusion with H2-added solution. P < 0.05 (Shimada et al. 2016)

hepatic microcirculatory disturbance had progressed during cold storage, but recovered to a large extent in the first 30 min of reperfusion in the H2(+) group. Note: Portal vein resistance is defined by portal vein pressure (in cm H2O) divided by perfusate’s flow rate (in mL/min/g liver). Usually, flow rates were measured at fixed time intervals under constant portal vein pressure.

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Development of Molecular Hydrogen Medicine

Fig. 2.7 Portal vein resistance of rat’s liver after cardiac death. Up to 50 min of reperfusion, deterioration and its amelioration in the H2-added perfusate were very similar to brain-death grafts (Fig. 2.5) but tended to deviate thereafter. P < 0.05 (Ishikawa et al. 2018)

Figure 2.6 shows the liver function after 90-min reperfusion; (a) Oxygen consumption rate, a measure of metabolic activity (viability), reflecting the respiratory function of mitochondria, and (b) Bile production, one of the major liver functions. Both functions were deteriorated during cold storage, but ameliorated to some extent during reperfusion in the H2(+) group. Subsequently, the effect of H2 was tested with rats’ liver retrieved after cardiac death (DCD graft) (Ishikawa et al. 2018). Use of DCD grafts had been precluded due to rapid progression of injuries triggered by warm ischemia, but post-reperfusion H2 treatment was found to ameliorate the deterioration to a large extent. As shown in Fig. 2.7, portal vein resistance was nearly the same as in the brain-death (DBD) grafts (Fig. 2.5), followed by slow increase thereafter. Amelioration of viability and bile production was also very similar to DBD grafts, though slightly smaller. This is a very important discovery for transplant medicine because it might open the possibility of allowing the use of DCD grafts. Exploiting heavy water in organ transplantation is described in Chap. 7. Note: Statistical analysis of experimental data—The P-value In the field of biology and medicine, quite unlike physics and chemistry, experiments/observations often give a rather large scatter of data. Even after removing all conceivable errors, there remain a scatter of unspecified origin, due to inevitable difference between the subjects, including both innate and environmental. Therefore, in biology and medicine it is vitally important to critically evaluate the quality of data, which is expressed by two parameters: standard deviation and P-value. The scatter of data is represented by the standard deviation, usually inscribed as error bars in graphical presentations. It is defined by σ ¼ {[(X1  Xav)2 + . . . . + (Xn  Xav)2]/(n  1)}½ where Xav is the average value Xav ¼ (X1 + X2 + . . . + Xn)/n, n being the total number of measurements.

2.1 Therapeutic Hydrogen Effects More Extended than Expected

19

Fig. 2.8 Explanation of Pvalue. (a) Distribution of actual data set {Xk} with the average value Xav, approximately represented by normal distribution function. (b) Distribution of hypothetical data with the average value of zero. The shaded area (the P-value) is the probability of obtaining the erroneous conclusion that the average value is larger than Xav instead of zero

Due to the presence of these errors, any conclusion to be drawn from such experiments/observations should be made with some reservations. To put it more precisely, any statement should be presented with the probability of the statement being in error due to the scatter of data of unspecified origin. This is the P-value (probability value). The P-value can be calculated from the theory of statistics, with the assumption that the scatter is random in origin and the sample size (number of subjects) is sufficiently large. In order to explain the P-value, let us examine the case whether administration of H2 water instead of normal water increases the H2 concentration in the blood. For this purpose, we prepare two groups, HW (H2 water) group (A) and control group (B), each consisting of n subjects, and obtain two sets of measured data {Ai} and {Bj}, with average values Aav and Bav, respectively. Let us construct a new set of data consisting of n differences Ai  Bj of arbitrary combination of i and j, which we write {Xk}. The distribution of data is shown in Fig. 2.8a. If the origin of scatter is random, and sample size is sufficiently large, the distribution of Xk approaches the welldefined bell-shaped function called normal distribution function, of which the center is located at Xav ¼ Aav  Bav, the width is determined by σ, and the area under the curve is normalize to unity. What we wish to know is, whether the difference between Aav and Bav is statistically significant. To answer this question in quantitative terms, we consider the hypothetical case where there is no difference between the two groups (a null hypothesis). Although the average value should be zero by definition, there is a finite probability that we obtain a non-zero value “erroneously” due to distribution of errors. In the case of null hypothesis, the probability of obtaining “erroneously” average values larger than Aav  Bav is given by the shaded area in Fig. 2.8b. This is the P-value. If this value is smaller than a pre-set value, say Pc ¼ 0.05, then we can say that the probability of Xav accidentally becoming zero is sufficiently small. In other words, difference

20

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Development of Molecular Hydrogen Medicine

between {Ai} and {Bj} is statistically significant. The level of significance Pc is set by the judgment of the researcher, depending on the problem under consideration; for biological and medical problems, Pc is usually set at 0.05 or 0.01. P-value is certainly helpful for judging the quality of the data, but it does not “prove” anything. It only provides a measure of likelihood of the correctness of the statement, calculated under a set of assumptions and approximations, often excluding other possible factors. As frequently claimed, caution must be made against misinterpretation and misuse of the P-value.

Injuries Due to Glaucoma and Cataract Surgery Hydrogen can suppress retinal injuries caused by high ocular pressure in glaucoma (Oharazawa et al. 2010). In rats’ experiment, I-R injuries induced by temporarily raising the ocular pressure was found to be suppressed by administration of a saline solution saturated with hydrogen (saturated H2 saline). Figure 2.9 illustrates this effect. Cell death normally starts 1 day later, advances in 1 week, and results in retinal atrophy, but the administration of hydrogen significantly reduced cell death. Elsewhere, experiments in rats and rabbits have shown that eye drops containing hydrogen water (H2 water) are also effective for cataracts and diabetic retinopathy. Subsequently, Igarashi et al. (2016) showed that corneal endothelial damage that sometimes happens in cataract surgery could be mitigated by using H2-dissolved irrigating solution. As a measure of corneal damage, the opacity was measured 5 h after surgery, as shown in Fig. 2.10. The suppression of damage in the H2 group is apparent. The mechanism of action of H2 in this case is ascribed to the elimination of hydroxyl radicals produced by ultrasound in phacoemulsification process (see Sect. 4.3). Acute Erythematous Skin Diseases Nishijima Hospital in Numazu, Japan reported a case of acute erythematous skin diseases (Ono et al. 2012). They are a terrifying illness where one suddenly develops red spots all over the body, leading to serious symptoms over time, and even death. The cause of the illness is said to be a rapid increase of ROS due to an unknown trigger. When a patient was received in the hospital, he was in a very serious condition. Many erythema and ulcers were apparent on his face, and he could not open the left eye and could hardly open his mouth. Treatment with H2 saline via drip infusion led to a remarkable recovery: He became able to open both his eyes and mouth 3 days later, and 6 days later the erythema disappeared and the patient was completely cured. Although this is admittedly a single case study, it is an example of dramatic effects hydrogen administration could provide in some cases. Peritonitis to Sepsis Zhang et al. (2014) demonstrated the effectiveness of H2 water on acute peritonitis in rats. Peritonitis is inflammation of the peritoneum, a membrane lining the wall of the abdomen, in most cases caused by bacterial infection from a rupture of appendix, stomach ulcer or perforated intestines. Without immediate treatment, acute peritonitis can lead to life-threatening infection throughout the body, to lethal sepsis, for example.

2.1 Therapeutic Hydrogen Effects More Extended than Expected

Normal

21

I/R injury Without H2

With H2

After 1 day

After 7 days

50 μm Fig. 2.9 H2-loaded eye-drops protected retina of rats injured by ischemia/reperfusion; crosssectional views. Upper panels; cell death (black dots) 1 day after I-R injury. Lower panels; shrinkage after 7 days (Oharazawa et al. 2010)

Their experimental procedure was as follows: administration of H2 water for 7 days, induction of peritonitis on day 8, administration of H2 water for 3 days, and sacrificing for analysis on day 11. H2 water (1.2–1.6 ppm) of 6 mL was administered daily by gavage. Visceral peritoneum injuries were alleviated by administration of H2 water, and survival rates 3 days after the induction by cecal ligation and puncture were increased by ~10% (Fig. 2.11). Essentially the same results were obtained by other two methods of induction (intraperitoneal injection of lipopolysaccharide and rats’ feces). They also observed that inflammation indices (interleukin-6, etc.) decreased significantly after 1–2 days. From these results they concluded that H2

22

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Development of Molecular Hydrogen Medicine

Fig. 2.10 Corneal edema caused by cataract surgery is suppressed by H2. Opaqueness caused by phacoemulsification in rabbits was measured with normal irrigation solution (control) and H2-dissolved solution (H2 group). P < 0.005 by the unpaired t-test (Igarashi et al. 2016)

Fig. 2.11 Effects of H2 water (HW) on the survival rate after induction of acute peritonitis in a rat model (Zhang et al. 2014)

water has a potential protective effect on acute peritonitis and suggested that a combination of H2 water and traditional antibiotics should be useful for clinical applications. Improvement of the survival rate by 30% was observed in a subsequent experiment of Ikeda et al. (2018) on mice, corresponding to larger H2 dosages (slightly more than ten times). More details of their paper, focused on bacterial translocation, are described in Sect. 4.3.

2.1 Therapeutic Hydrogen Effects More Extended than Expected

23

Fig. 2.12 Healing of palatal wounds in rats. Wound closure with time (in areal %) is shown for H2 water (HW)- and control groups. P < 0.01 (Tamaki et al. 2016)

Wound Healing Wound healing is a sequence of orderly events—hemostasis, inflammation, proliferation, and maturation of the tissue, a process to recover the structure and function of tissues lost by injury. Tamaki et al. (2016) examined the effect of H2 water on the wound healing in a rat model. A circular full-thickness excisional wound of 3.5 mm in diameter was created in the center of the oral palatal region, and pictures were taken on day 0, 1, 2, 3, and 7 after the operation to measure the wound area. During the period, distilled water for the control group (CL) and H2 water of initial concentration 5–7 ppm for the H2 water group, each consisting of 12 rats, was given. The results are shown in Fig. 2.12. The healing is clearly accelerated in the H2 water group. The mechanism of the acceleration examined on the molecular level is described in Sect. 4.3. Radiation Injuries Radiation exerts Janus-faced actions towards living bodies: It is utilized for radiotherapy where malignant tumors are eliminated by irradiation, whereas it exerts various detrimental effects on living tissues and organs. It even acts as a carcinogen. Radiation-induced injuries are divided into two categories; direct effects in which the energetic radiation hits biomolecules along the path and destroys them, and indirect effects mediated by ROS produced by radiation, more specifically by increased production of hydroxyl radicals. Given this knowledge, radioprotective effects of molecular hydrogen could be naturally expected. The survival rate of mice after the whole-body radiation of γ-rays was increased significantly by pre-administration of H2 water (Qian et al. 2010). Biochemical measurements revealed the process following the local irradiation of the heart

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Development of Molecular Hydrogen Medicine

Fig. 2.13 Pre-administration of H2 water (HW) suppresses γ-ray induced myocardial injuries. Injuries measured by myocardial MDA and 8-OHdG in DNA of γ-ray irradiated mice hearts; mean  SEM (n ¼ 8), P < 0.01 (Qian et al. 2010)

Fig. 2.14 Pre-administration of H2 water (HW) (0.6 mM) suppresses the decease of antioxidant SOD and GSH in γ-ray irradiated mice hearts. Mean  SEM (n ¼ 8), P < 0.01 (Qian et al. 2010)

(6 Gy). Figure 2.13 shows the increase of MDA and 8-OHdG produced by oxidative modification of membrane lipids and DNA, respectively. Note that the increase was suppressed by pre-administration of H2 water. Damage on endogenous antioxidant defense system was also ameliorated by H2 water, as shown in Fig. 2.14. Both SOD (superoxide dismutase, enzymatic antioxidant) and GSH (glutathione, non-enzymatic antioxidant) activities were deteriorated by radiation, but the deterioration was suppressed by pre-administration of H2 water. Subsequently, results of more detailed experiments were reported by Terasaki et al. (2011). They started with radioprotective effects of H2 on human cells (epithelial cell line A549), cultured in H2-rich (H2+) and N2-rich (H2–) medium and irradiated with X-rays (10 Gy). Figure 2.15 (a) shows a percentage of dead cells counted under microscope. The radiation-induced death was significantly reduced in the H2-treated case. The trend is reproduced by apoptotic markers active caspase 3 (b) and Bax (c).

2.1 Therapeutic Hydrogen Effects More Extended than Expected

Cell death (%)

30

a.

25

c.

b.

20

10

0

Radiation H2

Radiation (Gy) H2

Radiation (Gy) H2

Fig. 2.15 Effects of molecular H2 on X-ray induced apoptosis of cultured human cells. Irradiation of X-rays (10 Gy) is given to human lung epithelial cells in H2-rich (H2+) and N2-rich (H2–) medium. (a) percentage of dead cells 36 h after irradiation, (b) concentrations of active caspase 3, and (c) marker protein Bax expressed relative to GAPDH (control). Measured 24 h after irradiation. Data represent mean  SE. n ¼ 5, *P < 0.05 (Terasaki et al. 2011)

Fig. 2.16 Molecular H2 suppresses X-ray induced damage in mice lungs. 8-OHdG and TUNEL levels (indicator of oxidative stress and apoptosis, respectively) measured 1 d after irradiation (n ¼ 6) and the concentration of MDA (indicator of lipid peroxidation) measured 7 d after irradiation (n ¼ 5) are shown. *P < 0.05, **P < 0.01 (Terasaki et al. 2011)

Their roles in the apoptotic process are as follows: Bax translocation from the cytosol to mitochondria triggers death signals, which lead to the activation of caspase 3, followed by the cleavage of the DNA repair enzyme, and eventually DNA fragmentation as an apoptotic reaction. These changes show strong correlation with concentrations of •OH measured directly by fluorescence, and indirectly by concentrations of 8-OHdG in DNA and 4-NHE in cytoplasm. These results indicate that oxidative stress is a major cause of radiation-induced injuries. Then, they proceeded to irradiation of the lungs of mice, and found that administration of 3% H2 gas at the time of irradiation (15 Gy on whole thorax) plus H2 water (0.4–0.6 mM) in the following period suppressed acute symptoms as well as chronic fibrosis after 5 months. Some representative biochemical data in the acute phase (< 1 week) are shown in Fig. 2.16, and histological observations of fibrosis after

2

H2(-)

H2(+)

Control

Development of Molecular Hydrogen Medicine

Type III collagen in FOV (%)

26

10 7.5 5 2.5 0

Irradiation – – H2

+ –

+ +

Fig. 2.17 Molecular H2 suppresses X-ray induced fibrosis of mice lungs. Histological observation 5 months after irradiation. Control group; without irradiation, with administration of pure water. Test group; with irradiation, with H2 water (H2(+)) or pure water (H2()). Scale bars ¼ 100 μm. The graph shows the collagen % in FOV, n ¼ 6, *P < 0.05, **P < 0.01 (Terasaki et al. 2011)

5 months are shown in Fig. 2.17. These results support the observations of Qian et al. on the heart of mice (Figs. 2.13 and 2.14). Radiation produces peculiar injuries in the skin which is a defense barrier of a living body. As it is exposed to more intense radiation than any other organs inside, there is a pressing need for effective means of prevention and remedy of radiationinduced skin injuries. Mei et al. (2014) tested for the efficacy of H2 saline administration for dermatitis induced by irradiation of the head-and-neck area of rats. Rats were treated intraperitoneally with physiological saline with or without H2, 5 min before irradiation, and radiation-induced skin dermatitis was observed during 2–3 weeks following radiation. The effect of H2 saline administration was confirmed below the dose of 20 Gy, and biochemical effects similar to Figs. 2.15 and 2.16 were observed. Above 25 Gy, however, molecular hydrogen could not exert any beneficial effects. Observed changes of plasma MDA, SOD, and GSH were similar to those observed in the γ-ray irradiated heart of mice (Figs. 2.15 and 2.16). Numerous animal experiments performed since, regarding H2 effects on radiation-induced injuries of bone marrow, testis, lymphoma, etc. have been reviewed by Qian et al. (2013). Subsequently, efforts to elucidate the radiationinduced biochemical processes have been made (Kura et al. 2019), as described in Sect. 2.4. Human clinical experiments on radiation injuries are also described in Sect. 2.4.

Metabolic Syndrome Hydrogen is effective for the treatment of metabolic syndrome. Metabolic syndrome is a collective name given to lifestyle diseases such as diabetes, dyslipidemia, and

2.1 Therapeutic Hydrogen Effects More Extended than Expected

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Fig. 2.18 H2 water suppressed obesity of mice. The appearance of db/db (obesity-susceptible) mice reared for 3 months: Left, with administration of ordinary water; right, with hydrogen water (Kamimura et al. 2011)

hypertension caused by overweight and obesity, inactive lifestyle, and insulin resistance. When these diseases advance, there is a risk of arteriosclerotic diseases such as angina, mild cognitive impairment (MCI), and strokes; thus, precautionary measures are required. Kajiyama et al. (2008) continuously administered 900 mL/day of H2 water to diabetic patients for 8 weeks and studied the effect, and found that fat and sugar metabolism improved, leading to significant alleviation of the symptoms. This is the first publication in which the effect of hydrogen was investigated in the clinic. Various other details have come to light in ensuing animal experiments. Obese mice treated with saturated H2 water for 3 months showed clear reduction in weight (Fig. 2.18), a 40% reduction in liver fat, and furthermore, decreased blood glucose, insulin, and neutral fat levels (Fig. 2.19) (Kamimura et al. 2011). There are also reports suggesting that H2 water reduces erectile dysfunction and retinopathy in the eyes, which are complications of diabetes. It was also later confirmed that H2 water suppresses fatty acid uptake by the liver, inflammation, and the incidence of cancer. Hydrogen also acts in the blood vessels. The manifestation of arteriosclerosis was suppressed in model mice with arteriosclerosis that were treated by oral administration of H2 water. While arteriosclerosis begins due to injuries to the endothelium (membrane in contact with the blood) that occur due to the deposition of cholesterol, fat, calcium, and other substances in the blood vessels, H2 water helped reduce the amount of these deposits (Ohsawa et al. 2008). Many animal experiments were performed thereafter, and clinical studies of the effects of H2 water on hepatitis and diabetes have been conducted in the Tokyo Metropolitan Institute of Gerontology, Okayama University and in Tohoku University, Japan. H2 water is also expected to be effective for improving the general condition of metabolic syndrome.

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Fig. 2.19 Drinking H2 water reduced the oil accumulation in the liver. Administration of H2 water reduced the oil accumulation; for wild-type mice given fatty diet for 1 and 2 weeks (P < 0.01), and for db/db (obesity- susceptible) mice for 3 months (P < 0.05) (Kamimura et al. 2011)

Brain Impairments Hydrogen works well to reduce functional impairment in the nervous system, specifically in the brain. Not only does it help alleviate I-R injury and injuries from surgical intervention in the brain, hydrogen is also known to influence higher-order functions in the brain. Keeping mice locked in a narrow space for a long period causes memory impairment due to oxidative stress; but when H2 water, instead of ordinary water, is administered during this time, the impairment is alleviated and deterioration of the learning function is also reduced (Nagata et al. 2009). There are also results from animal experiments suggesting that H2 water is effective against Alzheimer’s disease (Li et al. 2010). These findings suggest that hydrogen works effectively on the hippocampus, which is responsible for the memory and cognitive functions of the brain. In practice, an experiment conducted later using aging-promoted mice showed that providing H2 water suppressed the advancement of memory impairment, as well as atrophy of the hippocampus due to nerve cell death (Nishimaki et al. 2018). In Parkinson’s disease, death of the neurons (nerve cells) controlling the movement leads to brain atrophy and unsteady movement, and in extreme cases, the disease also involves intellectual impairment. Parkinson’s disease manifests due to lack of dopamine secretion in the brain, the cause of which is unclear. Even levodopa, a symptomatic therapy provided to supplement dopamine levels in the body, results in side effects after several years of use. Remarkably, H2 water was shown to have a significant effect on Parkinson’s disease, at least in the rodent models of the disease. According to studies conducted at Nagoya University, rats with 6-hydroxydopamine (dopamine antagonist)-induced neurodegeneration in the lateral substantia nigra behave abnormally by spinning around in circles when

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stimulated with methamphetamine, but these abnormal movements did not occur at all when the animals were given H2 water in advance. The effect was maintained even when H2 water was given 3 days after drug administration (Fu et al. 2009). In a detailed mouse experiment conducted independently at Kyushu University, administration of H2 water helped suppress MPTP (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine)-induced changes in the cells and function of the brain that are normally associated with Parkinson’s disease (Fujita et al. 2009).

Lung Diseases Causes and symptoms of lung injuries/diseases are highly variable, and some of the lung diseases that exhibit high incidence and mortality constitute world’s major cause of death. Here I describe some results of animal experiments that demonstrated the efficacies of molecular H2 in these complex diseases. I deal mostly with acute lung injuries/diseases, adding a description on one chronic lung disease COPD. Acute lung injury (ALI) is a collective name given to a group of diseases of similar symptoms arising from many different causes. Hyperoxic injury caused by breathing a gas of high oxygen concentrations is one of the ALIs, which is a leading cause of morbidity and mortality in critically ill patients. Effects of H2 on hyperoxic injuries were studied by Sun et al. (2011) on rats’ model. They reared rats in hyperoxia (>98% O2) for 60 h to test for the effect of H2-dissolved saline injection. During this period, cell apoptosis increased from 5% in normoxia to ~30% in hyperoxia, which was reduced to ~20% by injection of H2-saline. Figures 2.20a–c show the variation of oxidative stress markers, MDA, SOD activity, and 8-OHdG. Clearly, oxidative stress is increased in hyperoxia, which is reduced by injection of H2-saline. (Note: for the Norm () group, actually no injection was made; in other 3 groups, 4 injections were made every 12 h.) (d) and (e) show that proinflammatory cytokines TNF-α (tumor necrosis factor α) and IL-1β (interleukin 1β) are increased in hyperoxia, which is suppressed by injection of H2-saline. A marker of neutrophil recruitment MPO (myeloperoxidase) varies in a similar way (f). Mechanism of hyperoxic injury is believed to be such that ROS produced on exposure to hyperoxia invoke pulmonary cells to increase the secretion of cytokines to recruit leukocytes to the lung, which in turn produce additional ROS to form a vicious cycle (Barazzone et al. 2000). Suppression of MPO shown in (f) indicates that the vicious cycle is effectively interrupted by H2. The preventive effect of H2 on lung injury may be the consequence of suppression of the excessive inflammatory response and its downstream cascade. Audi et al. demonstrated that SPECT (single-photon emission computed tomography) can be utilized for imaging the distribution of oxidative stress in the lung (Audi et al. 2015), and applied the technique for in vivo observation of endothelial cell death in hyperoxia-induced ALI in rats (Audi et al. 2017). Radioactive Tc compound 99mTc-HMPAO (hexamethylpropyleneamine oxide) and 99mTcduramycin administered via the femoral vein catheter accumulate on the surface of dead cells, and serve as a marker of cell death. Representative images taken with 99m Tc-duramycin after 60 h of treatment are shown in Fig. 2.21: in normoxia (left),

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Fig. 2.21 Representative images of 99mTc duramycin distribution in rats’ lungs in a normoxia (left), hyperoxia for 60 h (center), and in hyperoxia + H2 for 60 h (right). Pictures were taken 20 min after injection. Lung ROI is determined from the 99mTc-MAA image with the dashed horizontal boundary to avoid liver contribution. (Audi et al. 2017)

hyperoxia (middle), and hyperoxia + H2 (right). Contours of the lung were obtained by subsequent injection of radioactive solution 99mTc-MAA. The results indicate that molecular H2 exhibits anti-apoptotic effect in addition to antioxidative effect. The authors suggest the utility of SPECT biomarker for in vivo assessment of the process of ALI.

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Fig. 2.22 Concentrations of MDA, IL-1β and MPO activity in burned rats lungs. S + N sham + normal saline; B + N burn + normal saline; B + H burn + H2-saline; B + E burn + edaravone. n ¼ 8, P < 0.01 vs. B + N group (Fang et al. 2011)

Kawamura et al. (2013) proceeded to investigate the underlying mechanism (pathway) of H2 effects on hyperoxic injury. Details of their work are described in Sect. 4.3. Fang et al. (2011) studied ALI induced by extensive burns in rat model. Rats were given full-thickness burn (30% TBSA) test for the effect of H2-saline and edaravone. At 12 h postburn, the lung function as measured by oxygen concentration in arterial blood was decreased to 74  11%, which was ameliorated to 86  6% by H2-saline and 87  7% by edaravone. They assessed the effect of H2-saline injection by measuring oxidative stress (concentration of •OH, MDA, 8-OHdG, and protein carbonyl) and inflammatory mediators (IL-1β, IL-6, and TNF-α), and found that all these indicators increased by burns, but ameliorated by H2-saline and edaravone. Measured data for MDA and IL-1β are shown in Fig. 2.22a, b, respectively. Other indicators varied in nearly the same way. These results clearly indicate that the burn injury accompanied with severe inflammation was caused by oxidative stress, which was suppressed by H2-saline and edaravone. Figure 2.22c shows the increase of an enzyme MPO, existing predominantly in neutrophils, was suppressed by H2-saline and edaravone. In other words, H2-saline and edaravone act to break progression of inflammatory cascades by suppressing the accumulation of neutrophils. Kohama et al. (2015) studied H2 effects on ALI induced by hemorrhage shock and resuscitation (HS/R). Despite recent advances in intensive care, lung injury after HS/R is still among the most common causes of death after trauma. In their experiment on rats’ model, HS/R was produced by withdrawing blood through femoral artery to a blood pressure of 30  5 mmHg for 60 min, and then shed blood was reinfused. After this procedure, the rats were kept in a chamber filled with air or 1.3% H2 gas for 1–6 h before being sacrificed. Some results of their measurements are shown in Fig. 2.23. Clearly, damage caused by HS/R was ameliorated by H2 inhalation. (a) The respiratory function as measured by blood O2 concentration was severely damaged by HS/R, but recovered significantly by H2 inhalation. (b) Number of neutrophils, predominant infiltering cells in the lung, was nearly doubled by HS/R, but recovered to a normal level by H2 inhalation. This indicates that cellular infiltration was reduced effectively by H2

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Fig. 2.23 H2 inhalation protects rats’ lungs from hemorrhagic shock and rescucitation. (a) partial pressure of O2 in blood, (b) number of neutrophils and (c) concentration of MDA in lung tissue, measured after 3 h. N2: air; H2: air + H2. n ¼ 6, *P < 0.05 (Kohama et al. 2015)

inhalation. (c) Elevation of tissue MDA levels caused by HS/R was reduced by H2 inhalation. This indicates that the damage was caused primarily by oxidative stress, and at the same time suggests that formation of blood clots, one of the serious features of ALI, was effectively mitigated by H2 inhalation. All measured indicators of inflammation, IL-1β, IL-6, TNF-α, ICAM-1 (intercellular adhesion molecule 1), iNOS (inducible nitric oxide synthase), CCL2 (chemokine (C-C motif) ligand 2) were increased by HS/R, and the increase was suppressed by inhalation of H2. Activated p65 protein was increased in the nucleus by HS/R, which was reduced by H2 administration. This indicates that the NF-κB (nuclear factor κB) signaling pathway which induces upregulation of inflammatory mediators was blocked by molecular H2. The authors claim, in conclusion, that inhalation of H2 may exert potent therapeutic effects against ALI induced by HS/R by attenuating the activation of inflammatory cascades. Among the drugs that induce ALI, lipopolysaccharide (LPS) is the most widely used for animal model experiments. It has been very helpful in exploring the mechanisms of the diseases and providing information for the discovery of new biomarkers and drug targets. Here I summarize the effect of molecular H2 on some characteristic symptoms and underlying mechanisms of ALI, established heretofore with a help of LPS. (Qiu et al. 2011; Xie et al. 2012; Zhang et al. 2015). The major pathological changes of ALI include impaired gas exchange (respiratory function), neutrophil accumulation, increased vascular permeability (leakage) and parenchyma injury (tissue damage), and molecular H2 ameliorates most of these dysfunctions. Various biomarkers (indicators) have been utilized for measuring oxidative stress, antioxidative activity, inflammation, apoptosis and autophagy, and identifying possible pathways. Thus, by far the most important cause of damage is found to be the oxidative stress, for the reduction of which molecular H2 is very effective. Molecular H2 also exerts anti-inflammatory and anti-apoptotic effects. Qiu et al. (2011) observed that H2 gas inhalation prevented apoptosis through downregulation

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of genes Bax and caspase 3, and upregulation of Bcl-xl, and suggested that Bcl-2/ Bcl-xl/caspase 3 pathway may be the key mechanism of protective effects of H2. Zhang et al. (2015) noted further that a marker of autophagy LC3I/II (light chain 3 protein) is increased by LPS, which is suppressed by molecular H2. Concomitant changes are observed for p38 expression, which is linked to multiple cellular processes including apoptosis and autophagy. They suggested, based on these observations, that p38MAPK (mitogen-activated protein kinase) pathway is playing an important role in the protective effect of H2. Parenthetically, let me add a few words on the use of LPS for animal model experiments on lungs. Generally speaking, there is always a danger in translating the results of druginduced animal experiments to actual human diseases. As both pathogens and subjects are different, there is no guarantee that the nature of the diseases is the same. In fact, LPS is not an artificial drug developed for simulating human ALI but a natural pathogen, an endotoxin existing in gram-negative bacterial cell walls. The values and limitations of LPS in pulmonary research are discussed by Chen et al. (2010). In their recent paper, Terasaki et al. (2019) reported that respiratory impairment caused by anti-tumor drug gefitinib in mice model was effectively ameliorated by administration of H2 water without interfering with its anti-tumor activity. Despite excellent clinical response, gefitinib might promote severe and lethal interstitial lung disease, which restricts the therapeutic efficacy of this agent. Their experimental procedure was as follows: In order to raise the background sensitivity to ALI, naphthalene (dissolved in corn oil) was injected to all subjects intraperitoneally at day 0, then, the subjects were divided into two groups, with and without daily administration of gefitinib (dissolved in 1% Tween 80), and biochemical response of the two groups was measured with and without administration of H2 water (80% saturation). The study period was 14 days. Figure 2.24 (a) shows the change of body weight caused by coadministration of naphthalene and gefitinib, with and without administration of H2 water. The drug-induced weight loss was significantly suppressed by H2 administration. The variation of IL-6, shown in (b) and (c) for 7 days and 14 days after injection, indicates that concomitant inflammation was also suppressed by H2 administration. Possible interference of H2 with gefitinib was examined, both in vitro and in vivo, and results are shown in Fig. 2.25. In in vitro experiments, human cancer cells A539 and H1975 were cultured with three different concentrations of gefitinib, in two different ways of H2 administration: pre-administration and coadministration. In pre-administration, cells were incubated for 30 min in the flow of H2-containing gas before being transferred to gefitinib-containing medium. Results (a–d) show that, irrespective of H2 treatments, anti-cancer activity of gefitinib is the same. Results of in vivo experiments with mice are shown in (e–f). A549 cancer cells were transfected onto the dorsal flank of nude mice 7 days before gefitinib treatment, and subsequent

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Fig. 2.24 Suppression of naphthalene/gefitinib - induced damages in mice by H2-administration. (a) body-weight changes during the study period of 14 days for five different conditions (O corn oil, N naphthalene, G gefitinib, CW control water, HW H2-water). n ¼ 8 ~ 11, **P < 0.01. Concentrations of IL-6 7 days (b) and 14 days (c) after injection. n ¼ 7 ~ 8, *P < 0.05, **P < 0.01 (Terasaki et al. 2019)

changes of tumor volume and body weight were measured. Note, the anti-tumor activity of gefitinib is not affected by H2. Thus, drinking H2 water has the potential to improve the quality of life during gefitinib therapy by mitigating the pulmonary toxicity without impairing the antitumor activity. Let me now turn to chronic lung diseases. One of the most serious chronic lung diseases is COPD (chronic obstructive pulmonary disease), which ranked third in the

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world’s cause of death in 2020. COPD is characterized by chronic inflammation of peripheral airways and emphysema, accompanied with enlarged alveolar airspaces and destruction of the lung parenchyma. The pathogenesis of COPD is complex, due to the involvement of many factors: recurrent inflammation, oxidative stress (oxidant/antioxidant imbalance), protease/anti-protease imbalance, apoptosis, enhanced senescence of structural cells, and defense repair processes. In spite of these complexities, however, a major risk factor in the development of COPD is known to be cigarette smoking (CS). Up to now, a number of papers have reported beneficial effects of H2 on some aspects of COPD. Here, instead of enumerating those pieces of experiments, I prefer to focus on the result of well-organized experiments of Suzuki et al. (2017), addressed to clarifying underlying mechanisms of COPD on a specially prepared mouse model. The experiments were performed on SMP30-KO (senescence marker protein 30 knockout) mice, the mice lacking the ability to produce vitamin C (VC). Almost all species, not including humans and other primates, have an ability to synthesize VC from glucose. They reported previously that SMP30, a gluconolactonase involved in VC biosynthesis that decreases with aging in rats and mice protected mouse lungs from the oxidative stress associated with aging

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and smoking, and that SMP30-KO mice have a shorter lifespan, during which they develop emphysema within 8 weeks of exposure to CS. Recognizing that the control of VC is essential for investigating COPD, they organized the experiments as follows: Mice were reared in VC-controlled condition for 3 months, subsequently under VC-deficient condition for 1 month, before being assigned to four different experimental conditions: (1) air-exposed, (2) air-exposed with administration of H2 water, (3) CS-exposed, and (4) CS-exposed with H2 water. Each group (consisting of 12 subjects) were exposed to given atmosphere for 8 weeks before being sacrificed. Fig. 2.26 (a) shows the destructive index (DI), a measure of destruction of alveolar walls, calculated from histologic images of SMP30-KO mice. CS-induced damage is significantly attenuated by administration of H2 water. The variation of γH2AX (phosphorylated histon) and 8-OHdG, indicators of DNA damage in the lungs, are shown in (b) and (c), respectively. The variation is similar to (a). Figure 2.26 (d–f) show that levels of senescence markers, protein p16, p21, and β-galactosidase, are all strongly enhanced by CS, which are suppressed markedly by H2 water. The authors call attention to the fact that γH2AX is a reliable and sensitive indicator of DNA double strand breaks (DSBs), the most severe damage that induces apoptosis, cellular senescence, proinflammatory responses, and oncogenesis, and suggest that CS-induced DSBs and DNA oxidation may be a core mechanism for pulmonary emphysema development. Admittedly, the effect of CS demonstrated in their experiment is only a part of the story, but its elucidation to this level is certainly a big step towards preventive and therapeutic measures for the intractable disease COPD. In closing this section, let me add reference to two most recent papers describing H2 effects on chronic lung diseases: hypoxia/re-oxygenation injury (Chen et al. 2018) and pulmonary fibrosis (Gao et al. 2019).

2.1.2

Preventive Effects of Molecular Hydrogen

As described in the foregoing section, administration of molecular H2 prior to irradiation ameliorated radiation injuries, and this radioprotective effect was maintained for long periods of time, for 100 days for example. This is a clear indication that molecular H2 helped to establish a state to cope with radiation, most probably by triggering innate antioxidative system. The biochemistry of such antioxidative system is under intensive studies (See, Section 4.3). Preventive effects of molecular H2 were also noted in the case of Parkinson’s disease. When mice with drug-induced Parkinson’s disease were treated with H2 water 1 week before disease induction, the symptoms were less severe than when it was administered following disease onset, and there was also less neuronal cell death in the brain (Fu et al. 2009). This shows that the administration of H2 water may be helpful for preventing Parkinson’s disease. Furthermore, according to recent data by Iketani et al. (2017), while the three-day survival rate of mice with drug-induced

Fig. 2.26 Effects of H2-water on cigarette smoke–induced lung injury in SMP30-deficient mice. Senescence marker protein 30 knockout (SMP30-KO) mice were reared for 8 weeks in two different atmospheres (Air, Air + CS(cigarette smoke)), with or without administration of H2-water. (a) destructive index calculated from histologic image, (b) γH2AX, (c) 8-OHdG, (d) p16, (e) p21, (f) β-galactosidase. All these indicators of lung damage were enhanced by cigarette smoking, but suppressed significantly by H2-administration. n ¼ 12, *P < 0.05, **P < 0.01 (Suzuki et al. 2017)

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sepsis is 25%, the survival rate improved to approximately 60% when the mice were continuously given H2 water. Notably, even when the mice were treated with H2 water for 3 days prior to drug administration, the survival rate increased to 50%, thereby showing that H2 water can also prevent sepsis. While all these studies concern mice, if these findings can be translated to humans, this would be a major revelation. Drinking H2 water regularly may help prevent Parkinson’s disease, which has no effective treatment, while also suppressing the incidence of sepsis that accounts for 20–30% of causes of deaths among patients that enter the intensive care unit in hospitals. Hydrogen’s ability to prevent diseases will likely develop as an important field of Molecular Hydrogen Medicine in the future.

2.2

Origin of Fatigue and Aging

2.2.1

The Mechanism of Fatigue

Although fatigue occurs after working too much or exercising too hard, we eventually recover from such fatigue. This is the normal response of the body. What then, does it mean to be “tired”? In fact, the cause of fatigue has only recently been properly understood. The previous school of thought proposed that accumulation of lactic acid with exercise leads to fatigue. This is wrong. What really happens is that exercise leads to the formation and accumulation of superoxide anions, one of the ROS, resulting in fatigue. In the body, some superoxide anions are always produced during metabolism, but decomposed by the action of enzyme superoxide dismutase (SOD) (Sect. 11.3) and rendered harmless. However, if this balance is destroyed due to excessive exercise, the superoxide anion cannot be processed completely, resulting in “fatigue.” Numerous experiments have been performed on the effect of hydrogen for mitigating fatigue. Animal experiments with racehorses and sports medicine studies with athletes have demonstrated that ROS are generated during exercise, and the ensuing muscle fatigue is mitigated when H2 water is administered (orally or intravenously) prior to exercise. In Japan, full-scale research into fatigue started in the 1990s, and in 2003, an industry-government-academia collaboration centered in Osaka, called the “AntiFatigue Project” was organized; since then, Osaka City University has served as the base of growing research. Data from this collaboration has revealed that fatigue is recognized when waste products generated by ROS accumulate in the body. These wastes create a series of proteins called fatigue factors that then act on the brain. The signal creates a fatigue restoring factor, repairing cell damage caused by ROS. The details of the active substance are still unknown, but the cranial orbital frontal cortex was shown to be the site that senses and deals with fatigue. This research has continued since, and a detailed report on the anti-fatigue effect of H2 water was published (Mizuno et al. 2018; Watanabe et al. 2018). According to this report, drinking H2 water for 4 weeks alleviates fatigue in the form of

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drowsiness, nervousness, and decreased work efficiency, while improving motivation and relaxation. Improvement in the autonomic nerve function and cognitive function (response time) has also been observed. Thus, H2 water has been shown to alleviate both physical and mental fatigue, suggesting it is useful for everyday life. Popular anecdotal accounts such as “I became less easily tired after I started drinking H2 water” and “my body felt lighter” are not merely psychological phenomena, as the ability of H2 water to mitigate fatigue and aid in recovery has been scientifically verified. Incidentally, there are concerns about potential disruptions in the sports world if the effect of hydrogen on muscle fatigue is found to be significant. Athletic records would surely tumble one after the other if all athletes began drinking H2 water; however, since hydrogen has been approved as a food additive, H2 water should not be considered a prohibited substance. Furthermore, as described in Sect. 4.1, ingested hydrogen is quickly discharged leaving no trace. Therefore, there is no way to test for it. The “hydrogen effect” on sports and athletic records may become an important issue in the future.

2.2.2

The Mechanism of Aging

Although the life expectancy of humans is said to be 120 years, most people fail to achieve this age because the organs necessary for maintaining life tend to fail before we reach the age of 120. Simply stated, people generally do not live until the age of 120 due to illnesses. Statistically speaking, the average life expectancy of a Japanese person has increased rapidly, now exceeding 80 years, compared to only around 40 years in 1900. The rate of increase for life expectancy in Japan of 2 months per year is a common trend in developed countries. This change is due to the reduction in deaths due to infectious diseases as a result of medical advances, as well as reduced morbidity due to nutritional improvements. As a result, the top three most common causes of death in the world’s high-income regions in 2016 are ischemic heart disease (myocardial infarction, etc.), strokes, and dementia (Alzheimer’s disease, etc.), which are caused by aging of the blood vessels, heart, and brain. For example, as dementia normally progresses slowly over the course of several decades, the disease is now more prevalent due to the extended life expectancy. Around 10% of elderly people at the age of 80 are living with dementia, 40% at 90, and 90% at 100. This means that the lifespan of individuals is approaching the inherent lifespan of the organs. The mechanism by which aging occurs in organs has been studied intensively. While aging was previously thought to be unavoidable, as we have started to understand the causes of aging, now it seems that this paradigm is not necessarily true. We are beginning to think that aging can be prevented. The cause of aging is primarily a decline in bodily function due to accumulated injuries from ROS. Aging manifests through a variety of different symptoms, such as the formation of wrinkles in the skin, senile plaques, hardening of blood vessels, and

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the onset of psychological disabilities, such as senility, and physical disabilities. It also manifests as an accumulation of damage over many years, involving many environmental factors, which makes it difficult to explain. However, these are believed to stem from a single main cause; the accumulated damage from ROS. Thus, a reduction of ROS by H2 water may help prevent aging. In fact, there is a report claiming that the progression of aging was suppressed in a rapidly aging line of mouse when treated with H2 water. While H2 water is certainly not a drug for “eternal longevity,” we expect that it can play a role in slowing down the aging process and allow for the possibility of attaining lifespans close to the maximum life expectancy of 120 years old. Molecular H2 related therapies are rapidly being developed, with active studies now underway into not only the treatment of diseases, but also disease prevention, as well as fatigue prevention and anti-aging. The molecular H2 healthcare revolution has only just begun.

2.3

The Current State of Molecular Hydrogen Medicine

As of 2018, around 700 papers have been published on the physiological and medical effects of hydrogen. The number of publications up to June 2015 is shown in Fig. 2.27 (Ichihara et al. 2015). A general trend is that, for a few years following the paper of Ohsawa et al. in 2007, there was a period of rapid expansion, which stabilized around 2012. During this period of expansion, the number of papers increased drastically, especially in China, including papers of various scientific levels. In writing this book, I decided to focus on research conducted in Japan, of which I am better informed, and by which I believe I can provide a reasonably precise description of the current state of hydrogen medicine. Fig. 2.27 Temporal profile of research publications on therapeutic effects of molecular H2 (2007–2015); breakdown by countries (Ichihara et al. 2015)

2.3 The Current State of Molecular Hydrogen Medicine

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Establishment of the Japanese Society for Medical and Biological Research on Molecular Hydrogen

In 2011, a small group of researchers started a workshop, “Symposium on Molecular Hydrogen Medicine”, and met once a year for mutual communication of their research. In May 2016, the workshop was restructured into an academic society, The Japanese Society for Medical and Biological Research on Molecular Hydrogen. The aim of the society is “to contribute to the development and enhancement of medical research, research into clinical applications, and biological research related to molecular hydrogen, and the applications thereof, and, in so doing, to contribute to improvements in health and medical treatments as well as the welfare of mankind.” The following address of the director Shigeo Ohta is recorded on the homepage of the Society: “Since the publication of our paper suggesting the possible medical applications of molecular hydrogen in Nature Medicine in 2007, medical research into molecular hydrogen has been pursued extensively and intensively. In these research activities, Japanese researchers have made various important contributions, not only on the antioxidant action of molecular hydrogen, but also its anti-inflammatory effects, anti-apoptotic (cell death) effects, and energy-metabolism-promoting effects. As the synergistic effects of these actions have come to be uncovered, molecular hydrogen is recognized to be a promising agent for the treatment and prevention of various diseases. Research into the mechanisms that manifest these various effects of molecular hydrogen is also progressing. Although admittedly in the very early stage, clinical research is ongoing at more than 10 university hospitals, leading to a growing consensus that molecular hydrogen will contribute to actual medical treatments in the near future. In addition to its possible roles in disease treatment, molecular hydrogen is believed to help promote health in healthy individuals as well and is expected to have some cosmetic effects. Research has also been extended to study the effects of molecular hydrogen in plants, in addition to animals including humans. Five years after starting the “Symposium on Molecular Hydrogen Medicine”, recognizing various achievements made during this period, we decided at the fifth symposium to establish The Japanese Society for Medical and Biological Research on Molecular Hydrogen. The society aims to primarily work towards adoption of hydrogen medical treatments and to further our research activities beyond medicine and to the wider area of biology. In our broader perspective, our intention is to contribute to the prosperity and welfare of mankind through hydrogen research.”

Based on the growing research activities in Japan, China, and Korea, the International Society for Hydrogen Medicine and Biology was established in December 2017, and the international conference is held annually, in China in 2018, Korea in 2019, and Japan in 2020.

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Major Research on the Medical Effects of Hydrogen Molecules

As overview of the area of Molecular Hydrogen Medicine, Table 2.1 provides a list of experimental work classified according to the object under study (i.e. disease and organ). After such comprehensive survey of hydrogen effects on animals, cases of favorable results have been selected and assigned to clinical trials in humans, and now, a fairly complete picture of this field is coming into view. In the meantime, basic research of hydrogen effects in physiology and biology, from cellular- to individual-level, has also been performed. For detailed and comprehensive reviews of the field, readers are referred to the following papers: Dixon et al. (2013), Ohta (2014), Ichihara et al. (2015), Sun et al. (2015), Ohta (2015), and Iketani and Ohsawa (2017).

2.3.3

The Current State of Human Clinical Trials

Here I wish to make some general comments on human clinical trials as compared to animal experiments, before going to describe some representative results in the next chapter. The research performed to date has mostly been conducted in mice and rats; only a limited number of experiments have been conducted on large animals or humans. As shown in Fig. 2.28, of the papers published until June 2015, one eighth were on humans. One might expect that, when good results are obtained from small animals, similar results may also be obtained for humans. However, this is not always the case. Roughly speaking, small mammals and humans have similar constitution and physiology, but there are differences originating from the difference on the DNA level. Thus, there is no guarantee that humans react to hydrogen in the same way, and clinical experiments with humans are indispensable. Clinical studies are, however, very demanding because they require labor and costs far beyond those of animal experiments. In addition, they must be conducted with appropriate ethical considerations of the subjects. Being a subject in a clinical study does not necessarily mean that he or she will gain any benefits. No matter how much precaution is taken for safety, 100% safety cannot be guaranteed for an experiment performed for the first time. Moreover, in a double-blind study (see, the Note below), half of subjects are given a “placebo” as a control, which has no effect whatsoever. All these procedures must be explained to the subjects and written consent must be obtained before they are enrolled in the study. This is a procedure that must be strictly followed even when only a few milliliters of blood is taken, and is stringently supervised by an ethical review board including third-party members. As researchers are apt to be hasty in the pursuit of results, the role of clinical research coordinators is to guarantee that clinical trials to be performed safely, ethically, and scientifically. This is an important role of the clinical trial review board, in addition to its primary objective to examine and acknowledge that the project itself is appropriate and effective. Note: Randomized double-blind placebo-controlled test When investigating the efficacy of a drug in a clinical trial, subjects are divided into two groups: one receives

2.3 The Current State of Molecular Hydrogen Medicine

43

Table 2.1 The diseases examined for the effect of molecular hydrogen A. Animal experiments • Blood vessels/blood: Sepsis, arteriosclerosis, vascular disorder, vascular endothelial function, platelet aggregation, and blood fluidity • Bone: Osteopenia • Brain: Cerebral infarction, cerebral hemorrhage, cerebral ischemic disorder, brain damage after cardiopulmonary resuscitation, carbon monoxide poisoning, subarachnoid hemorrhage, and cognitive disorder due to cerebral infarction, ischemia, sepsis, trauma, restriction, aging, Alzheimer’s disease, Parkinson’s disease, ALS, and depression • Cancer: Tongue cancer cell proliferation, cancer cell killing effect, anti-tumor effect, and enhancement of anticancer agent effect • Ears: Hearing impairment and noise-induced hearing loss • Eyes: Cataracts, retinopathy, and ischemic reperfusion injury of the retina • Heart: Acute myocardial infarction, diabetic cardiomyopathy, heart transplant, sleep apneainduced cardiac hypoxia, disorders caused by cardiopulmonary resuscitation, and ischemic reperfusion injury • Inflammation/allergy: Type 1 allergy and sepsis • Intestines: Ulcerative colitis and ischemic reperfusion injury after intestinal transplantation. • Kidneys: Hemodialysis, peritoneal dialysis, chronic kidney disease, kidney transplant, and ischemic reperfusion injury • Liver: Hepatitis, hepatitis B, jaundice, liver cirrhosis, liver regeneration, and ischemic reperfusion injury • Lungs: Oxygen-induced, radiation-induced, burn-induced, sepsis-induced, surgery induced, and tobacco-induced lung injury, lung transplant, ischemic reperfusion injury, and pulmonary reperfusion after cardiac death • Metabolism: Diabetes, diabetic retinitis/erectile dysfunction, metabolic syndrome, and hypercholesterolemia • Muscle: Ischemic reperfusion injury to the skeletal muscle, muscular dystrophy, and oxidative stress due to exercise • Nerve/spinal cord: Spinal cord damage, neuropathic pain and ischemic reperfusion injury • Pancreas: Acute pancreatitis and ischemic reperfusion injury after transplant • Perinatal abnormalities: Brain damage due to maternal hypoxia, fetal hippocampal disorder, neonatal hypoxic encephalopathy, neonatal hyperoxia-induced retinopathy, and neonatal neurovascular disorder • Skin: UV/radiation-induced skin damage, atopic dermatitis, inflammation due to burns, and ischemic reperfusion injury. • Stomach: Stress-induced gastric ulcer and aspirin-induced gastric ulcer • Teeth: Periodontitis and aging of periodontal cells • Testis/ovary: Radiation damage to reproductive cells and ischemic reperfusion injury B. Clinical trials in humans • Blood vessel/blood: Vascular epithelial function and blood antioxidant potential • Brain: Cerebral infarction, subarachnoid hemorrhage, damage after cardiopulmonary resuscitation, Parkinson’s disease, Alzheimer’s disease, and mild cognitive impairment • Eyes: Cataracts surgery and retinopathy • Fatigue: Oxidative stress and acidosis from exercise, and anti-fatigue effect • Heart: Myocardial infarction and damage after cardiopulmonary resuscitation • Intestines: Intestinal obstruction and enteric bacteria • Joints: Rheumatoid arthritis and psoriatic arthritis (continued)

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Table 2.1 (continued) • Kidneys: Hemodialysis and peritoneal dialysis • Liver: Hepatitis B and radiation damage from liver tumor treatment • Lungs: Lung transplant • Metabolism: Diabetes, metabolic syndrome, and hypercholesterolemia • Muscle: Inflammatory and mitochondrial myositis, muscle fatigue, and soft tissue disorder • Radiation damage (liver, skin) • Skin: Skin damage due to UV irradiation, acute erythematous skin disorder, and bedsore • Teeth: Periodontitis Compiled from the reference list of review papers: Ohta (2011), Ichihara et al. (2015) and Iketani and Ohsawa (2017)

Fig. 2.28 Research publications on biological effects of molecular H2 (2007–2015); breakdown by biological species (Ichihara et al. 2015)

Rodent Human Cell lines Plants Pig Rabbit Others

the experimental drug while the other receives a placebo. Then, comparison between the two groups is made using the theory of statistics. Where this division of subjects into two groups is performed randomly by a third party such that neither the subjects nor the observers know which group is which, it is termed a randomized, doubleblind trial, or a double-blind test. The aim of this procedure is to avoid the placebo effect (i.e. where an effect is found not because the drug is effective, but because the subject expects it to be effective) and observer bias (i.e. the possibility that the observer unconsciously influences the results because of their expectations). Performing comparative trials with a placebo in this manner and obtaining consent from subjects is necessary to maintain the credibility of the research. Because there is usually considerable variation in data from clinical trials with people, it is necessary to enroll a larger number of specimens/subjects compared to animal experiments. While individual differences in animal experiments is minimized by using animals bred for a specific purpose, individual differences are unavoidable with human subjects. A crude measure is, to obtain similar statistical precision as in experiments with ten mice; a clinical experiment with humans requires 100 subjects or more.

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Table 2.2 Registered clinical trials for hydrogen effects (selected) Enrollment 2009/07/17

2013/02/13

2015/09/22

2015/11/06

2011/12/04 2013/11/22

2014/07/01

2016/01/01

2011/06/02 2014/07/29

2013/05/01 2014/08/01 2014/10/27 2016/11/24 2015/07/03

2015/10/30

Title An interventional trial for mild cognitive impairment using hydrogenrich water Randomized, double-blind, placebocontrolled trial on molecular hydrogen water in Parkinson’s disease The effect of smell dysfunction using hydrogen gas for Parkinson’s disease recruiting The effect of hydrogen-rich water drinking to gut peptide of patients of Parkinson’s disease and healthy control Inhalation of H2 gas in patients with acute myocardial infarction The effect and safety of H2 inhalation for patients with cardiac arrest syndrome The efficacy of hydrogen gas inhalation in patients with ST elevation acute myocardial infarction Efficacy of inhaled hydrogen on neurological outcome following brain ischemia during post-cardiac arrest care Hydrogen for cardiac ischemia Effects of intravenous infusion of hydrogen-rich fluid combined with intra-cisternal infusion of magnesium sulfate in severe aneurysmal subarachnoid hemorrhage: a randomized controlled trial Effect of hydrogen-rich water on chronic obstructive pulmonary disease The safety and effect of inhalation of hydrogen gas after lung transplantation The efficacy of hydrogen eye drop for retinal artery occlusion Effect of H2 gas for the phatoemulsification of cataract surgery Investigation for effects of hydrogen water on lipid and glucose metabolism in diabetes mellitus Electrolysis hydrogen water improves insulin resistance in type 2 diabetes

Institution Univ. of Tsukuba

Status Completed

Juntendo Univ.

Completed

Nagoya Univ.

No longer

Nagoya Univ.

Preinitiation

Keio Univ.

Completed

Keio Univ.

Completed

National Institute for Global Health and Medicine Keio Univ.

No longer recruiting

National Defense Medical College National Defense Medical College

Open public recruiting Open public recruiting

Juntendo Univ.

Completed

Osaka Univ.

Enrolling by invitation Open public recruiting Enrolling by invitation Open public recruiting

Nippon Medical School Nippon Medical School Tokyo Metropolitan Institute of Gerontology Tohoku Univ.

Open public recruiting

No longer recruiting (continued)

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Table 2.2 (continued) 2016/12/05 2016/03/15 2016/02/23 2017/01/06

2017/09/28 2016/05/20 2016/08/10

2017/09/11

Investigation of the clinical effect of hemodialysis using electrolyzed water Digestive disease and hydrogen concentration in breath Effect of hydrogen tablet on blood pressure and endothelial function Effect of hydrogen-rich water on the level of reactive oxygen species in healthy volunteer Evaluation of hydrogen-rich water to pre-metabolic syndrome participants The anti-fatigue effects of hydrogenrich water Hydrogen gas concentrations in the expired air after intake of hydrogendissolved water and water containing indigestible sugars Efficacy of drinking hydrogen-water on exercise tolerance and fatigue School

Osaka National Hospital Nagoya Univ.

No longer recruiting Preinitiation

Tokorozawa Heart Center Kochi Medical School

Open public recruiting Preinitiation

Kochi Medical School Osaka City Univ.

Open publicrecruiting Completed

Kitasato Univ.

Preinitiation

Nippon Medical

Preinitiation

Extracted from the data base of UMIN (University hospital Medical Information Network); https:// www.umin.ac.jp/english/

To establish a plan for any clinical experiment, the methodology must be well designed to investigate the efficacy and safety of the new treatment method, and cooperation from many patients must be acquired. Since all advanced medical treatments must pass through this process, it is natural that the process of developing a novel treatment, that is proven to be effective and eventually approved, is a lengthy one. Around 80 clinical research projects of Molecular Hydrogen Medicine are being planned or implemented by universities, research institutes, and public hospitals in Japan; these can be viewed through the University Hospital Medical Information Network (UMIN). For the interest of readers, some major projects are shown in Table 2.2. There are, in addition, physician-initiated clinical trials that can be learned from the Japan Medical Association’s Center for Clinical Trials (JMACCT).

2.4

Major Results of Human Clinical Trials

In the following, I will describe some representative results of clinical trials performed to date. Recovery from Cardiac Arrest Keio University is performing clinical trials to investigate the effect of H2 gas on patients who have suffered acute heart attacks and receiving resuscitation treatments after cardiac arrest.

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Based on successful experiments with rats, in 2011, Motoaki Sano and his collaborators started a clinical trial of the inhalation of H2 gas as part of a hypothermia therapy during coronary angioplasty in acute heart attack patients transported to the emergency outpatient department. An acute heart attack occurs when a blood clot is suddenly formed in the coronary arteries of the heart, stopping blood flow and causing cell death in the heart muscle. If not reperfused within 2 h, cell death will proceed rapidly. To enroll sufficient number of subjects (40), the schedule was extended until September 2016, and the results confirmed the expectation that inhalation of H2 gas could be effective for suppressing the I-R injury during surgery under this condition. In fact, major technological advancements were made during this period. A hydrogen-gas inhalation device must always be on alert during cardiac surgery so that a hydrogen-containing gas can be supplied when restarting the blood circulation. However, the handling of a mixed gas of hydrogen and oxygen in such an enclosed space as an operating theater increases the risk of explosion which could be triggered by a single tiny spark of electrical discharge. Such a risk is very difficult to manage in the presence of various medical equipment, but the crews of Keio University Hospital have established a method to safely handle hydrogen in such an environment. Keio University Hospital is also proceeding with another clinical trial where hydrogen will be administered to patients after the resumption of the pulse by a resuscitation device. This project, initiated in 2013, also showed hydrogen administration to have considerable efficacy. Specifically, the risk of many disorders that occur within 1 week of cardiac arrest was reduced. Of the five patients who were taken to the emergency department in 2015, four (one was excluded due to cardiac arrest caused by sepsis) showed almost no neurological after-effects and were able to walk out of the hospital unassisted at discharge. In 2015, Keio University established the “Center for Molecular Hydrogen Medicine,” with an aim of creating a nationwide organization engaged in the medical applications of hydrogen. The center’s first major project was the “Trial of Hydrogen Inhalation for Patients After Out-of-Hospital Cardiac Arrest” by the “HYBRID Research Group,” comprising a large number of organizations. At present, about 130,000 cases of out-of-hospital cardiac arrest occur in Japan each year. While the improvement in cardiopulmonary resuscitation has increased survival, rehabilitation of patients suffering secondary disorders caused by I-R injury of the brain and heart remains rather unsatisfactory. If the I-R injury could be prevented by administration of hydrogen, this would be a real godsend. In this project, the start of which was coordinated in 2016 by the emergency medicine specialist Masaru Suzuki, the number of subjects planned was as large as 230, with the expectation of a largescale, nationwide support. Indeed, as of February 2019, 19 hospitals in Japan are enrolled in the project, and the results obtained by a unified protocol are being accumulated. It will be at least several years before the final report is published, but there is scarcely any doubt that the project could be anything less than successful.

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Stroke Hirohisa Ono and others of the Neurosurgery Department of Nishijima Hospital, Numazu, Japan have implemented measures for administering H2 gas in acute stroke patients who arrive by ambulance. A stroke occurs when a blood clot in the vessels of the brain causes ischemic injury, and it requires emergency treatment similar to that for a heart attack. Nishijima Hospital achieved favorable results by administering 3% H2 gas for 1 h twice daily to acute cerebral infarction patients within 24 h of stroke (Ono et al. 2017). Fifty patients were randomly divided into two groups: one was given standard treatment and the other standard treatment plus H2 gas for 7 days. In the group treated with hydrogen, the spread of injured brain regions diagnosed by magnetic resonance imaging (MRI) was suppressed (Fig. 2.29), deterioration of brain function was reduced, and the ability to lead a normal lifestyle was clearly improved. This might appear to be simply a clinical application of the study by Ohsawa and others that showed hydrogen administration reduced brain damage in rats; however, there is a big difference. In the experiment with rats, administering hydrogen limited the onset of brain damage, whereas in this clinical study the progress of pre-existing injuries was reduced. Brain cells weaken and begin to die in even a few minutes

H2 group Control group

Damaged area (from MRI)

1000

Max 75% Median Min 75%

800 600 400 200 0

1

3

5

Days

7

10

14

Fig. 2.29 Inhalation of H2 gas improved acute cerebral infarction. Temporal variation of cerebral infarction areas measured by MRI. A group administered with H2 for the first 7 days (blue) showed consistently smaller infarction in comparison to control group (orange) (Ono et al. 2017)

2.4 Major Results of Human Clinical Trials

49

without oxygen. However, hydrogen administration prevented death of the dying cells surrounding the injured regions. Until relatively recently (around 1990), it was thought that adult brain cells do not proliferate, but this is not true. It is suggested that adult brain cells can be produced from “stem cells” as required. They exist throughout the body in a dormant state and, after being signaled, change (i.e. differentiate) into cells with specific functions. In fact, it was observed that, when a part of tissue is damaged, nearby stem cells are activated and regenerate damaged cells. It is suggested that the suppression of ischemic injury by hydrogen may be due to its ability to enhance the potential of stem cells. Brain Disorders: Parkinson’s and Alzheimer’s Diseases The brain is a control center that governs various functions from the maintenance of the body to high-level neurological activity. It processes information sent via the nervous system from various sensory organs, integrates this information, makes judgments, and then sends commands. In so doing, various information is processed by nerve cells (neurons) located throughout the brain. The neurons in the brain, which are estimated to number 100 billion, form a network in which information is exchanged in order to determine the response to a given input. Therefore, if a single part of the brain is injured, problems may occur in the entire system. In the following, I will introduce some of the research findings related to Parkinson’s disease and Alzheimer’s disease, which are typical neurodegenerative disorders. Researchers at Juntendo University examined the effects of H2 water in Parkinson’s disease (Yoritaka et al. 2013). Eighteen patients with similar histories of Parkinson’s disease were selected (excluding those of possible effects of other diseases) and tested by a randomized double-blind placebo-controlled method for a period of 48 weeks. They were randomly assigned to one of two groups; the first group administered with saturated H2 water and the second group a placebo water. The placebo water, plain water without hydrogen, was prepared by a placebo machine indistinguishable by appearance from normal machines for producing H2 water. Patients in each group consumed 1 L of their assigned water daily in addition to their usual doses of levodopa. Results revealed that, while symptoms gradually worsened in patients treated with placebo water, patients treated with H2 water showed clearly reduced disease progression. The difference was statistically significant. Although the results of this small-scale pilot study are preliminary in nature, it is expected that a full-scale study involving large numbers of patients may provide conclusive evidence of therapeutic effects of H2 water on Parkinson’s disease. With regards to Alzheimer’s disease, an eye-opening clinical trial was performed recently by Hirohisa Ono and others—albeit on a small scale (Ono et al. 2017, private communication). They investigated the effects of administering 3% H2 gas for 1 h twice daily for four to 7 months to patients of Alzheimer’s disease for whom standard treatment had not been effective in halting disease progression. During the

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H2

Period (day)

91

121

149

212

279

Fig. 2.30 Inhalation of H2 gas improved the patients with Alzheimer’s disease; recovery of tract fibers (neuro-bundles) passing through hippocampus observed by MRI. A gradual brightening of the (diffusion tensor imaging) signal to 149 days indicates the recovery of blood flow along the tract fibers caused by inhalation of H2 (Ono et al. 2017, private communication)

period of hydrogen treatment, standard treatment was continued without alteration. The results showed overall improvements in patients treated with hydrogen compared to those who were not, whose symptoms became worse. Moreover, in the diagnosis by MRI, evidence of regeneration of nerve fiber bundles around the hippocampus was observed (see Column 1, Chap. 1). A special method of MRI called diffusion tensor imaging with specific measurement conditions (FA parameter value) enables visualization of one-dimensional flow (that is, flow along vessels) of water molecules. Figure 2.30 shows the results of MRI imaging. The parts in white are the nerve fiber bundles (tract fibers) that send signals from the hippocampus to other parts of the brain. A gradual increase in brightness can be seen up to day 149, which has been interpreted as an increase in one-dimensional flow due to the regeneration of cell axons in tract fibers caused by hydrogen administration. The brain neural network, which had been in decline in these patients such that normal functioning had begun to cease, was restored by the administration of hydrogen, making signal transmission easier. However, the number of subjects in this clinical trial was small (16 peoples) and the hydrogen treatment period was not sufficiently long. Clinical trials for chronic diseases like Alzheimer’s require a very long treatment period, but it is difficult to obtain the cooperation of impaired patients for such long periods. Future large-scale, long-term clinical trials will be indispensable for establishing the medical effects of hydrogen. Since hydrogen was found to be effective against Parkinson’s disease and Alzheimer’s disease in animal experiments, there are great expectations for its efficacy in humans; unfortunately, the progress of human clinical trials to date has been rather limited.

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51

Rheumatoid Arthritis Toru Ishibashi and his collaborators of the Orthopedic Department at Haradoi Hospital in Fukuoka reported significant improvements in symptoms of rheumatoid arthritis patients after treatment with H2 water (Ishibashi et al. 2012, 2014). Rheumatoid arthritis is a progressive, intractable disease that is common in women of middle age. It makes it difficult to move joints throughout the body, causing intense pain due to inflammation and deformation of the bones and cartilage. Rheumatoid arthritis is caused by abnormal excitation of the defense response of the immune system; however, further details remain unknown. At Haradoi Hospital, 530 mL of high-concentration (4–5 ppm) H2 water was administered to 20 patients each day for 4 weeks, followed by another 4 weeks without treatment and subsequent resumption of treatment for 4 weeks. This treatment led to improved symptoms probably due to reduction of ROS in the body. The effect was particularly marked in five patients with early stage of rheumatoid arthritis; the symptoms were eliminated in four of them. Ishibashi and his collaborators later performed a rigorous clinical trial by administering physiological saline, into which hydrogen had been dissolved, and obtained similar results. Given that there are currently no effective treatments for rheumatoid arthritis, the efficacy of H2 water may give hope to many patients. Blood Dialysis There are more than 13 million patients with chronic kidney disease in Japan, which is being called the new national disease. This disease does progressive harm to the entire body as kidney functions, such as water content modulation and excretion of waste products from blood, deteriorate. The cause of this disease is poorly understood and there are currently no effective treatments. If chronic kidney disease progresses, it becomes necessary to perform artificial dialysis, in which blood is taken out of the body for processing. As of 2017, 300 thousand patients in Japan regularly receive hemodialysis treatments in hospitals. The treatment usually requires 2–3 days a week and ~ 4 hours a day—a big burden for the patients. The efficacy of hemodialysis is, however, rather limited, and the rate of mortality of patients receiving dialysis amounts to nearly 10%. Nakayama et al. discovered that kidney functions were improved when hydrogen was added to dialysis solution. In their system, hydrogen of 0.03–0.08 ppm was transferred through the membrane filter from dialysis solution to regenerated blood, which was sent out for circulation for the duration of several hours (Nakayama et al. 2010). With this success, they proceeded to organize a large-scale clinical trial including around 300 subjects (seven different institutions) extending to the period of 5 years, and demonstrated the improved performance of H2-dissolved dialysis solution (Nakayama et al. 2018). The subjects were divided into two groups: H2 group and control group. In the H2 group, by delivering H2-dissolved solution (H2 dialysate), various indicators of kidney function were ameliorated, as well as prognosis after the treatment. Overall, occurrence of primary events during the

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observation period, including all causes of death and non-lethal cardiac-cerebrovascular diseases, was reduced by 41% in the H2 group. The authors claim that this novel treatment may be easily introduced to hospitals/ clinics because it requires only small modifications of the existing dialyzer. The constitution and performance of the new dialyzer is described in Sect. 6.1. It must be admitted, however, that there remains a big question: How could it be possible that such low concentrations of hydrogen bring about such notable effects? The amount of H2 delivered in the single session of hemodialysis is only 1/10 of H2 produced daily by intestinal bacteria and dissolved in the blood. One possible answer may be that hydrogen acts to repair injuries in the blood incurred in the process of filtering in the machine, instead of exerting some therapeutic effects in the body. Whatever the mechanism, the benefit of this innovation should be unequivocal. Cataract Surgery Based on the success of their animal experiment, Igarashi et al. (2019) proceeded to a prospective clinical trial to test the hydrogen effects for preventing corneal endothelial damage in cataract surgery. Thirty-two patients of bilateral cataract were recruited, and phacoemulsification was performed in both eyes of each patient. A conventional irrigation solution was used in one eye, and H2-dissolved one for the other eye. Irrigation was performed continuously (10 mL/min) for 30 min, during which time the hydrogen concentration stayed nearly constant (0.99–0.86 ppm). Endothelial cell density (ECD) was measured at the center of the cornea before operation, 1 day, 1 week, and 3 weeks after operation. The reduction ratio measured from preoperative ECD, which is a measure of corneal damage, is shown in Fig. 2.31. The reduction ratio in the H2 group is significantly lower than the control group, and in addition, its progression stopped at 1 day, in contrast to the control group, in which the damage was still progressing after 3 weeks. Though small in sample size, this pilot study confirmed the effectiveness of the H2-dissolved irrigation solution in preventing corneal endothelial damage in cataract surgery. Periodontitis It is presumed that periodontal disease is by far the most common disease, from which more than half of grown-up population in the world are suffering. Periodontitis is a chronic disease of the supporting tissues of teeth, characterized by gingival bleeding, periodontal pocket formation, connecting tissue destruction, and eventually, alveolar bone loss. The disease is caused by oral bacteria; among ca. 800 bacteria species, Porphyromonas gingivalis and a few others are known to be responsible. Proliferation of P. gingivalis occurs explosively when heme-Fe is supplied by bleeding and sustained as long as bleeding continues in the pocket. The reason why periodontal disease is attracting increasing attention in these years is because large-scale epidemic studies from the late 1980s clearly revealed its close correlation with many systemic diseases: cardiovascular diseases (especially, coronary heart disease), pulmonary diseases, renal dysfunction, diabetes, osteoporosis, adverse pregnancy outcomes, etc. (Garcia et al. 2000), and more recently,

2.4 Major Results of Human Clinical Trials

53

Fig. 2.31 Reduction of corneal endothelial cell density (a measure of damage) after phacoemulsification: A clinical trial. The reduction rate was significantly smaller with H2-dissolved irrigation solution (white box) compared to the control group with normal solution (black box). * P < 0.05, ** P < 0.01 by paired t-test. NS means not significant (Igarashi et al. 2019)

Alzheimer’s disease (Dominy et al. 2019). Details of these oral-systemic relationships are yet to be clarified. To provide a measure of concomitant risk, we may quote the result of metaanalysis performed in the USA including ca. 10,000 people of 25–74 years old: People with periodontitis had nearly 50% higher risk of mortality in comparison to people with no disease, and the risk increased as periodontal status were more serious. For improving the state of periodontitis, administration of some antioxidants has been known to be effective, both for animals and humans. Thus, on the simplistic assumption that oxidative stress was a major cause of periodontitis, H2 water was administered to rats, and found to suppress the progression of the disease. This success led to the pilot study of Azuma et al. (2015). They recruited 13 patients with mild periodontitis and divided them into the control group (n ¼ 6) and the HW group (n ¼ 7), and tested the effect of H2 water (HW) for the period of 8 weeks. The H2 water (of concentration ~ 1 ppm) was given to the subjects 4–5 times (a total of 1000 mL) per day. Clinical data and serum samples were obtained at time zero, 2, 4, and 8 weeks. The effect of H2 water was very clear: Whereas the pocket depth of 4–6 mm remained essentially unchanged in the control group, it decreased by 1.3 mm in the HW group. Although the scatter of data was

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rather large due to small sample size, the difference between the groups was statistically significant. The increase of the antioxidant capacity of the serum was also observed in the HW group. This was admittedly a small-scale clinical experiment limited to mild periodontitis, but strongly suggests the efficacy of H2 water administration as a non-surgical treatment for improving periodontitis. Radiation Injuries in Humans Radiotherapy is a primary method to manage malignant tumors (including cancer), which, however, is inevitably accompanied with various side effects. Radiationinduced effects such as fatigue, decreased physical strength, appetite loss, and tasting disorder impair the quality of life (QOL) of patients in many cases, and even lead to chronic cardiovascular disorders in some cases. Thus, alleviation of such side effects has been a long-standing issue in radiotherapy. Successful alleviation of radiation injuries in animal experiments provided a clue for the solution. Kang et al. (2011) in Korea showed by randomized, placebo-controlled study on 49 patients that administration of H2 water (0.55–0.65 mM) for 6 weeks following the irradiation of liver cancer (total dose of 50–65 Gy) reduced the symptoms such as anorexia and dysgeusia, hence appreciably improved the QOL. Figure 2.32 shows the result of biochemical measurements. The level of dROM (derivatives of reactive oxidative metabolites) increased by radiation was suppressed by administration of H2 water, and the level of BAP (biological antioxidant power) decreased by radiation was increased by H2 water. They interpreted these results as being primarily due to the reduction of radiation-induced oxidative stress by H2 water.

Fig. 2.32 Administration of H2 water mitigates oxidative stress in patients receiving radiotherapy for liver tumors. Effects on dROM concentration, the total level of peroxide metabolites, and BAP, the serum antioxidant capacity (n ¼ 49), (Kang et al. 2011)

2.4 Major Results of Human Clinical Trials

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Advancements of technology towards the end of last century enabled the radiation to be focused in a small targeted volume, and in consequence avoided unintended irradiation of surrounding healthy tissues. Thus, the occurrence of acute radiation injuries caused by large total doses was practically eliminated. It has come to be recognized, however, that there remain some chronic diseases that become manifest years or decades after radiation. Among them, cardiovascular diseases are the major target of research in recent years. They are induced by modest doses of ~3 Gy, less than 10% of the tolerant doses of other radiation-induced heart diseases, and responsible for one-fourth to one-third of the mortality of cancer survivors receiving breast or mediastinal radiotherapy. There, fibrosis is the main damaging process. For the chronic phase of radiation-induced cardiovascular diseases (RICVD), a consistent description of the symptoms is still lacking, due to various complexities that arise over long periods of time. It is difficult to organize clinical studies extending over decades, and in addition, analysis of cohort studies is complicated by big changes in the instrumentation and regimen of radiotherapy that overlap the period of personal histories of medication. In spite of these difficulties, however, it appears almost certain that progression from acute to chronic phase is fueled by persistent states of inflammation and oxidative stress. Accepting this, administration of molecular H2 would naturally come into view: Daily intake of H2 water may be implemented easily (and cheaply.) A comprehensive review of the pathophysiology of RICVD was written by Cuomo et al. (2016), and possible application of molecular H2 was proposed by LeBaton et al. (2019). These protective effects of hydrogen will undoubtedly be good news for people who work under radiation. For example, cabin crews on airplanes are exposed to large amounts of cosmic radiation (particularly in the Arctic route). The dose of cosmic radiation is much higher in space, and therefore, it is essential for people spending any time in space to be well protected from radiation. Drinking H2 water can, almost certainly, help prevent and treat radiation injuries. COVID-19 Pneumonia Currently, we are all under serious threat of a pandemic COVID-19, caused by a novel coronavirus named SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). It emerged in Wuhan, China, sometime in the fall of 2019, and has rapidly spread over the world. As of September 25, the novel virus infected nearly 32 million people and killed more than 980,000 (WHO 2020). One of the most intriguing features of COVID-19 is that the symptoms are widely variable: Most patients exhibit only mild symptoms similar to common cold and recover in a few weeks, but a small fraction of patients suffer from respiratory syndromes (difficulty breathing; pneumonia), which sometimes even lead to lethal conditions. In pneumonia, tiny sacs (alveoli) normally filled with air become clogged, resulting in the deterioration of gas exchange function. For COVID-19 pneumonia, quite unlike ordinary pneumonia of bacterial origin curable by antibiotics, there is at

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present no effective way of medication. What can be done is supportive care to mitigate respiratory distress and/or hypoxemia, by delivery of oxygen through a mask or nasal tubes, or a breathing machine (ventilator) in case of severity. A last measure is to supply oxygen directly to the blood by a machine called ECMO (extracorporeal membrane oxygenation). The purpose of these supportive cares is to keep the patients alive until their lungs begin to heal. The difficulty is that, in severe patients, the clogging occurs in many regions of the lungs simultaneously, with a speed too rapid for any supportive cares to keep up. Typically, it takes 5 days from the first symptom to dyspnea, and another 8 days for the development of acute respiratory syndromes. Here I wish to give a brief description of what little has been done to explore possible roles of hydrogen in fighting against COVID-19. Recently, a paper from China reported first results on the efficacy of hydrogen gas in ameliorating conditions of patients in the initial stage of COVID-19 pneumonia (Guan et al. 2020). Some 90 patients with dyspnea were recruited and divided into two groups; an H2-group administered with a mixed gas (H2/O2 = 66%/33%) through nasal tubes and a control group given standard oxygen therapy, and progression of respiratory symptoms was assessed by six different diagnostic scales. Relative improvements of the scores in the H2-group were recorded from as early as day 2 of the treatment. It is expected that such beneficial effects of hydrogen, if confirmed by further experiments, would provide effective means of medication for COVID-19 pneumonia. Mechanism of action of hydrogen was not identified in this clinical trial as it was not of primary interest in combatting against the life-threatening disease. The mechanism, we presume, comes mainly from the anti-inflammatory effects of H2 as observed in many other cases including acute lung injuries. Like other viral diseases, SARS, MERS, bird flu, etc., the occurrence of acute syndromes in the later stage of COVID-19 is believed to be the consequence of a cytokine storm, a burst of pro-inflammatory cytokines (for a review, see Song et al. 2020). Cytokine is a general name for a group of numerous small proteins distributed over the body, mediating various interactions/communications between cells. Usually, in case of viral infection, appropriate number and species of cytokine molecules are summoned and accumulated on demand from the immune system to fight against the infection, accompanied with some local inflammation as a side effect. (In fact, the inflammation is an inevitable part of response of the immune system for its function; otherwise, pathogens could hardly be eliminated.) In some exceptional cases, however, when the balance between pro-inflammatory and anti-inflammatory cytokines is shifted towards the pro-inflammatory side, inflammation is amplified, and additional cytokines are induced, resulting in a vicious cycle (a positive feedback loop) of cytokine formation and inflammation. Then, the process becomes uncontrollable with explosive increase of pro-inflammatory cytokines (cytokine storm) until it damages the whole body by multiple organ failure. Such extreme imbalance of pro-inflammatory and anti-inflammatory cytokines was indeed observed in the case of SARS. The vicious cycle, once started, is extremely hard to stop. Therefore, the most effective way to alleviate it is to prevent its initiation by suppressing the

References

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inflammation by some means. The anti-inflammatory effect of hydrogen noted in the clinical trial described above arouses a renewed interest in this context. Suppression of inflammation in the initial stage of COVID-19 pneumonia may serve as a preventive measure to alleviate its progression to more serious stages. Admittedly, the argument presented here is tentative (and speculative), but in the absence of effective medications for COVID-19 a possible role of Molecular Hydrogen Medicine suggested here is believed to be worth pursuing further.

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W.J. Guan, C.H. Wei, A.L. Chen, X.C. Sun, G.Y. Guo, X. Zou, J.D. Shi, P.Z. Lai, Z.G. Zheng, N.S. Zhong, Hydrogen/oxygen mixed gas inhalation improves disease severity and dyspnea in patients with Coronavirus disease 2019 in a recent multicenter, open-label clinical trial. J. Thorac. Dis. 12, 3448–3452 (2020). https://doi.org/10.21037/jtd-2020-057 K. Hayashida, M. Sano, N. Kamimura, T. Yokota, M. Suzuki, S. Ohta, K. Fukuda, et al., Hori, hydrogen inhalation during normoxic resuscitation improve neurological outcome in a rat model of cardiac arrest independently of targeted temperature management. Circulation 130, 2173–2180 (2014) M. Ichihara, S. Sobue, M. Ito, M. Ito, M. Hirayama, K. Ohno, et al., Beneficial biological effects and the underlying mechanisms of molecular hydrogen – Comprehensive review of 321 original articles. Med Gas Res 5, 12 (2015) T. Igarashi, I. Ohsawa, M. Kobayashi, T. Igarashi, H. Suzuki, M. Iketani, H. Takahashi, et al., Hydrogen prevents corneal endothelial damage in phacoemulsification cataract surgery. Sci. Rep. 6, 31190 (2016) I. Igarashi, I. Ohsawa, M. Kobayashi, Y. Umemoto, T. Arima, H. Suzuki, T. Igarashi, T. Otsuka, H. Takahashi, et al., Effects of hydrogen in prevention of corneal endothelial damage during phacoemulsification: A prospective randomized clinical trial. Am J. Ophthalmol. 207, 10–17 (2019) M. Ikeda, K. Shimizu, H. Ogura, T. Kurakawa, E. Umemoto, D. Motooka, S. Nakamura, N. Ichimaru, K. Takeda, S. Takahara, S. Hirano, T. Shimazu, et al., Hydrogen-rich saline regulates intestinal barrier dysfunction, dysbiosis, and bacterial translocation in a murine model of sepsis. Shock 50, 640–647 (2018) M. Iketani, I. Ohsawa, Molecular hydrogen as a neuroprotective agent. Curr. Neuropharmacol. 15, 324–331 (2017) M. Iketani, J. Ohshiro, T. Urushibara, M. Takahashi, T. Arai, H. Kawaguchi, I. Ohsawa, et al., Preadministration of hydrogen-rich water protects against lipopolysaccharide-induced sepsis and attenuates liver injury. Shock 48, 85–93 (2017) T. Ishibashi, B. Sato, M. Rikitake, T. Seo, R. Kurokawa, Y. Hara, Y. Naritomi, H. Hara, T. Nagao, et al., Consumption of water containing a high concentration of molecular hydrogen reduces oxidative stress and disease activity in patients with rheumatoid arthritis: An open-label pilot study. Med. Gas Res. 2, 27 (2012) T. Ishibashi, B. Sato, S. Shibata, T. Sakai, Y. Hara, Y. Naritomi, S. Koyanagi, H. Hara, T. Nagao, et al., Therapeutic efficacy of infused molecular hydrogen in saline on rheumatoid arthritis: A randomized, double-blind, placebo-controlled pilot study. Int. Immunopharmacol. 21, 468–473 (2014) T. Ishikawa, S. Shimada, M. Fukai, T. Kimura, K. Umemoto, K. Shibata, M. Fujiyoshi, S. Fujiyoshi, T. Hayasaka, N. Kawamura, N. Kobayashi, T. Shimamura, A. Taketomi, et al., Post-reperfusion hydrogen gas treatment ameliorates ischemia reperfusion injury in rat livers from donors after cardiac death: A preliminary study. Surg. Today (2018). https://doi.org/10. 1007/s00595-018-1693-0 S. Kajiyama, G. Hasegawa, M. Asano, H. Hosoda, M. Fukui, N. Kitawaki, S. Imai, K. Nakano, M. Ohta, T. Adachi, H. Obayashi, T. Yoshikawa, et al., Supplementation of hydrogen-rich water improves lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance. Nutr. Res. 28, 137–143 (2008) N. Kamimura, K. Nishimaki, I. Ohsawa, S. Ohta, et al., Molecular hydrogen improves obesity and diabetes by inducing hepatic FGF21 and stimulating energy metabolism in db/db mice. Obesity 19, 1396–1403 (2011) K.M. Kang, Y.N. Kang, I.B. Choi, Y. Gu, T. Kawamura, Y. Toyoda, A. Nakao, et al., Effects of drinking hydrogen-rich water on the quality of life of patients treated with radiotherapy for liver tumors. Med. Gas Res. 1, 11 (2011) T. Kawamura, C.-S. Huang, N. Tochigi, S. Lee, N. Shigemura, T.R. Billiar, M. Okamura, A. Nakao, Y. Toyoda, et al., Inhaled hydrogen gas therapy for prevention of lung transplant-induced ischemia/reperfusion injury in rats. Transplantation 90, 1344–1351 (2010)

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T. Kawamura, C.-S. Huang, X. Peng, K. Masutani, N. Shigemura, T.R. Billiar, M. Okamura, Y. Toyoda, A. Nakao, et al., The effect of donor treatment with hydrogen on lung allograft function in rats. Surgery 150, 240–249 (2011) T. Kawamura, N. Wakabayashi, N. Shigemura, C.-S. Huang, K. Masutani, Y. Tanaka, K. Noda, X. Peng, T. Takahashi, T.R. Billiar, M. Okamura, Y. Toyoda, T.W. Kensler, A. Nakao, et al., Hydrogen reduces hyperoxic lung injury via the Nrf2 pathway in vivo. Am. J. Physiol. Lung cell Molecul. Physiol. 304, L646–L656 (2013) K. Kohama, H. Yamashita, M. Aoyama-Ishikawa, T, Takahashi, T. R. Billiar, T. Nishimura, J. Kotani, A Nakao, et al. , Hydrogen inhalation protects against acute lung injury induced by hemorrhagic shock and resuscitation, Surgery 158, 399–407 (2015) B. Kura et al., Molecular hydrogen: Potential in mitigating oxidative-stress-induced radiation injury. Can. J. Physiol. Pharmacol. 97, 287–292 (2019) T.W. LeBaron, B. Kura, B. Kalocayova, N. Tribulova, J. Slezak, et al., A new approach for the prevention and treatment of cardiovascular disorders. Molecular hydrogen significantly reduces the effects of oxidative stress. Molecules 24, 2076 (2019) J. Li, C. Wang, J.H. Zhang, J.-M. Cai, Y.-P. Cao, X.-J. Sun, et al., Hydrogen-rich saline improves memory function in a rat model of amyloid-beta-induced Alzheimer’s disease by reduction of oxidative stress. Brain Res. 1328, 152–161 (2010) K. Mei, S. Zhao, L. Qian, B. Li, J. Ni, J. Cai, et al., Hydrogen protects rats from dermatitis caused by local radiation. J. Dermatol. Treat. 25, 182–188 (2014) K. Mizuno, A.T. Sasaki, K. Ebisu, K. Tajima, O. Kajimoto, J. Nojima, H. Kuratsune, H. Hori, Y. Watanabe, et al., Hydrogen-rich water for improvements of mood, anxiety, and autonomic nerve function in daily life. Med. Gas Res. 7, 247–255 (2018) K. Nagata, N. Nakashima-Kamimura, T. Mikami, I. Ohsawa, S. Ohta, et al., Consumption of molecular hydrogen prevents the stress-induced impairments in hippocampus-dependent learning tasks during chronic physical restraint in mice. Neuropsychopharmacology 34, 501–508 (2009) M. Nakayama, H. Nakano, H. Hamada, N. Itami, R. Nakazawa, S. Ito, et al., A novel bioactive haemodialysis system using dissolved dihydrogen (H2) produced by water electrolysis: A clinical trial. Nephrol. Dial. Transplant. 25, 3026–3033 (2010) M. Nakayama, N. Itami, H. Suzuki, H. Hamada, R. Yamamoto, K. Tsunoda, N. Osaka, H. Nakao, Y. Maruyama, S. Kabayama, R. Nakazawa, M. Miyazaki, S. Ito, et al., Novel haemodialysis (HD) treatment employing molecular hydrogen(H2)-enriched dialysis solution improves prognosis of chronic dialysis patients: A prospective observational study. Sci. Rep. 8, 254 (2018) K. Nishimaki, T. Asada, I. Ohsawa, E. Nakajima, C. Ikejima, T. Yokota, N. Kamimura, S. Ohta, et al., Effects of molecular hydrogen assessed by an animal model and a randomized clinical study on mild cognitive impairment. Curr. Alzheimer Res. 15, 482–492 (2018) H. Oharazawa, T. Igarashi, T. Yokota, H. Fujii, H. Suzuki, M. Machide, H. Takahashi, S. Ohta, I. Ohsawa, et al., Protection of the retina by rapid diffusion of hydrogen: Administration of hydrogen-loaded eye drops in retina ischemia-reperfusion injury. Investig. Ophthalmol. Vis. Sci. 51, 487–592 (2010) I. Ohsawa, K. Nishimaki, K. Yamagata, M. Ishikawa, S. Ohta, et al., Consumption of hydrogen water prevents atherosclerosis in apolipoprotein E knockout mice. Biochem. Biophys. Res. Commun. 377, 1195–1198 (2008) S. Ohta, Recent progress toward hydrogen medicine: Potential of molecular hydrogen for preventive and therapeutic applications. Curr. Pharm. Des. 17, 2241–2252 (2011) S. Ohta, Molecular hydrogen as a preventive and therapeutic medical gas: Initiation, development and potential of hydrogen medicine. Pharmacol. Ther. 144, 1–11 (2014) S. Ohta, Molecular hydrogen as a novel antioxidant: Overview of the advantages of hydrogen for medical applications. Methods Enzymol 555, 289–317 (2015) H. Ono, Y. Nishijima, N. Adachi, M. Sakamoto, Y. Kudo, J. Nakazawa, K. Kaneko, A. Nakao, et al., Hydrogen (H2) treatment for acute erythematous skin diseases. A report of 4 patients with safety data and a non-controlled feasibility study with H2 concentration measurement on two volunteers. Med. Gas Res. 2, 14 (2012)

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H. Ono, Y. Nishijima, S. Ohta, M. Sakamoto, K. Kinose, T. Horikosi, M. Tamaki, H. Takeshita, T. Futatuki, W. Ohishi, T. Ishiguro, S. Okamoto, S. Ishii, H. Takanami, et al., Hydrogen gas inhalation treatment in acute cerebral infarction: A randomized controlled clinical study on safety and neuroprotection. J. Stroke Cerebrovasc. Dis. 26, 2587–2594 (2017) L. Qian, F. Cao, J. Cui, Y. Wang, Y. Huang, Y. Chuai, L. Zaho, H. Jiang, J. Cai, et al., The potential cardioprotective effects of hydrogen in irradiated mice. J. Radiat. Res. (Tokyo) 51, 741–747 (2010) L. Qian, J. Shen, Y. Chuai, J. Cai, et al., Hydrogen as a new class of radioprotective agent. Int. J. Biol. Sci. 9, 887–894 (2013) X. Qiu, H. Li, H. Tang, Y. Jin, W. Li, Y. Sun, P. Feng, X. Sun, P. Feng, X. Sun, Z. Xie, et al., Hydrogen inhalation ameliorates lipopolysaccharide-induced acute lung injury in mice. Int. Immunopharmacol. 11, 21302137 (2011) S. Shimada, K. Wakayama, M. Fukai, T. Shimamura, T. Ishikawa, D. Fukumori, M. Shibata, K. Yamashita, T. Kimura, S. Todo, I. Ohsawa, A. Taketomi, et al., Hydrogen gas ameliorates hepatic injury after prolonged cold preservation in isolated perfused rat liver. Artif. Organs 40, 1128–1136 (2016) P. Song, W. Li, J. Xie, Y. Hou, C. You, Cytokine storm induced by SAES-CoV-2. Clin. Chim. Acta 509, 280–287 (2020). https://doi.org/10.1016/j.cca.2020.06.017 Q. Sun, J. Cai, S. Liu, Y. Lin, W. Xu, H. Tao, X. Sun, et al., Hydrogen-rich saline provides protection against hyperoxic lung injury. J. Surg. Res. 165, e43–e46 (2011) X. Sun, S. Ohta, A. Nakao, et al., Hydrogen molecular biology and medicine (Springer, Dordrecht, 2015) Y. Suzuki, T. Sato, M. Sugimoto, H. Baskoro, K. Karasutani, A. Mitsui, F. Nurwidya, N. Amano, Y. Kodama, S. Hirano, A. Ishigami, K. Seyama, K. Terasaki, et al., Hydrogen-rich water prevents cigarette smoke-induced pulmonary emphysema in SMP30 knockout mice. Biochem. Biophys. Res. Commun. 492, 74–81 (2017) N. Tamaki, R.C. Orihuela-Campos, M. Fukui, H.-O. Ito, et al., Hydrogen-rich water intake accelerates oral palatal wound healing via activation of the Nrf2/antioxidant defense pathways in a rat model. Oxidat Med. Cell. Longevity 2016, 5679040 (2016). https://doi.org/10.1155/ 2016/5679040 T. Terasaki, I. Ohsawa, M. Terasaki, M. Takahashi, S. Kunugi, K. Dedong, H. Urushiyama, S. Amenomori, M. Kaneko-Togashi, N. Kuwahara, A. Ishikawa, N. Kamimura, S. Ohta, Y. Fukuda, et al., Hydrogen therapy attenuates irradiation-induced lung damage by reducing oxidative stress. Am. J. Physiol. Lung Cell Molecul. Physiol. 301, L415–L426 (2011) Y. Terasaki, T. Suzuki, K. Tonaki, M. Terasaki, N. Kumahara, J. Ohsiro, M. Iketani, M. Takahashi, M. Hamanoue, Y. Kajimoto, S. Hattori, H. Kawaguchi, A. Shimizu, I. Ohsawa, et al., Molecular hydrogen attenuates gefitinib-induced exacerbation of naphthalene-evoked acute lung injury through a reduction in oxidative stress and inflammation. Lab. Investig. 99, 793–806 (2019) K. Watanabe, S. Tsuji, N. Hiramatsu, T. Monden, O. Kajimoto, et al., Effects of hydrogen-rich water on attenuating fatigue induced by daily activities or mental tasks. Japan Pharmacol Ther 46, 581–597 (2018) (in Japanese) WHO COVID-19 Situation Report, 25 September 2020 K. Xie, Y. Yu, Y. Huang, L. Zheng, J. Li, H. Chen, H. Han, L. Hou, G. Gong, G. Wang, et al., Molecular hydrogen ameliorates lipopolysaccharide-induced acute lung injury in mice through reducing inflammation and apoptosis. Shock 37, 548–555 (2012) A. Yoritaka, M. Takanashi, M. Hirayama, T. Nakahara, S. Ohta, N. Hattori, et al., Pilot study of H2 therapy in Parkinson’s disease: A randomized double-blind placebo-controlled trial. Mov. Disord. 28, 836–839 (2013) J. Zhang, Q. Wu, S. Song, Y. Wan, R. Zhang, M. Tai, C. Liu, et al., Effect of hydrogen-rich water on acute peritonitis of rat models. Int. Immunopharmacol. 21, 94–101 (2014) Y. Zhang, Y. Liu, J. Zhang, et al., Saturated hydrogen saline attenuates endotoxin-induced lung dysfunction. J. Surg. Res. 198, 41–49 (2015)

3

From the Front-Line of Research: Interviews

Abstract

From what has been described thus far, the readers have probably learned how Molecular Hydrogen Medicine emerged and developed as a new field of medicine, and what its current situation is. Since many medical effects of molecular H2 were far beyond expectations, the reports were often viewed with suspicion. Even now, some people in medical community appear rather skeptical. In order to solve such misunderstandings, in this book, I have made the effort to provide an introduction as precisely and objectively as possible. However, there are things that could hardly be conveyed by a third-person like myself acting as an intermediary, the seriousness and enthusiasm of researchers on the front-line of research.

Recognizing this, I decided to post a series of interviews with four top researchers, who kindly agreed to: – Dr. Ikuroh Ohsawa, Tokyo Metropolitan Institute of Gerontology. – Dr. Motoaki Sano, Department of Cardiology, School of Medicine, Keio University. – Dr. Hirohisa Ono, Department of Neurosurgery, Nishijima Hospital, – with Column 2 Alzheimer’s disease, – Dr. Moto Fukai, Department of Transplant Surgery, Graduate School of Medicine, Hokkaido University. Among these, the interview with Dr. Fukai (Hokkaido University) is found in Chap. 7 as it concerns heavy water medicine.

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2020 Y. Fukai, Molecular Hydrogen for Medicine, https://doi.org/10.1007/978-981-15-7157-2_3

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From the Front-Line of Research: Interviews

Interview with Dr. Ikuroh Ohsawa of the Tokyo Metropolitan Institute of Gerontology

Q: It has been 10 years since your paper with Dr. Shigeo Ohta reported that hydrogen removed hydroxyl radicals in the living body. Please tell us about how this momentous research started and progressed. A: What triggered this research was a vendor who brought a hydrogen water manufacturing device to the Ohta Laboratory of Nippon Medical School, where I was affiliated at the time. I think it was before the summer of 2004. There was a red hydrogen canister connected to the device and hydrogen could be dissolved in water fairly easily. I knew that the reactivity of hydrogen is very low at room temperature and pressure, and based on what I learned from microbiology during my time as a student, I also knew that some bacteria have an enzyme called hydrogenase that converts hydrogen to energy, which is absent in higher organisms. So, I wondered what could be the use of hydrogen for humans. However, as an experimental researcher, I thought I had to see it for myself. I dissolved hydrogen into the culture solution and applied oxidative stress damage to cells. To my surprise, cellular damage decreased! Q: Research about the medicinal effect of hydrogen spread extensively after that. How did your own research progress from there? A: To build a theory based on observable facts, experiments need to be conducted under strict test conditions. We examined many characteristics of hydrogen, including how much hydrogen is dissolved in water, how long it takes to dissolve, whether or not there are impurities in hydrogen gas, whether or not the pH of water (i.e. concentration of hydrogen ions) changes as hydrogen gas is dissolved, and whether or not the concentration of the existing oxygen, which decreases with hydrogen, was corrected properly. At first, I wanted to know the concentration of trace hydrogen dissolved in a few milliliters of aqueous solution, but I could not. I then found a company in Denmark that made a very thin hydrogen electrode, which had been developed to measure the hydrogen concentration in hydrothermal vents found in the sea. After careful evaluations, I concluded that hydrogen actually suppressed the oxidative stress in cells. This is when I was convinced that “something was happening.” Why does hydrogen suppress the oxidative stress damage in cells? Together with Dr. Ohta, I came up with a hypothesis that hydrogen must be able to react with hydroxyl radicals, which is the most reactive of all ROS to cause oxidative stress. Fortunately, a convenient fluorescent reagent for its quantification had been developed by Dr. Nagano of the University of Tokyo. I remember that our first presentation of the physiological effect of hydrogen was made at the annual meeting of the Japanese Biochemical Society, in Kobe. Most attendees including expert researchers in mitochondria and oxidative stress did not understand what we were talking about. It seemed that as soon as they heard “hydrogen,” they thought of hydrogen ions (i.e. protons), not hydrogen molecules. Someone asked a question assuming that hydrogen molecules dissociated into ions when dissolved in water, which in reality does not occur. Nobody thought that hydrogen molecules could have any physiological effects. Since then, we have

3.1 Interview with Dr. Ikuroh Ohsawa of the Tokyo Metropolitan Institute of. . .

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decided to carefully explain the difference between hydrogen molecules, protons, and hydrogen atoms. The first animal experiment was a brain I-R injury experiment (a model of cerebral infarction) using rats. After reperfusion, large quantities of hydroxyl radicals were generated immediately, resulting in brain cell death. We then examined what happened when rats inhaled hydrogen gas. These experiments were highly technical, but Dr. Katsura and Mr. Takahashi of Nippon Medical School were very skillful and helped us in these experiments. We administered hydrogen gas instead of hydrogen water because we wanted to give large quantities of hydrogen and inhalation was the easiest way. Following inhalation, hydrogen is dissolved in the blood via the lungs and reaches the brain; the problem was the concentration. Unfortunately, the explosion limit of hydrogen in the air is around 4%, so we had no choice but to dilute the gas to below this limit. After the rats inhaled the gas, we observed that the brain cell death, caused by the I-R injury, was suppressed. We achieved a good result with a concentration of 2% hydrogen gas. The effect was, however, smaller at a concentration of 8%. If we had given a gas of high hydrogen concentrations from the beginning, it would have taken us longer to discover this effect. The results of this research were applied in the very early stage to patients with cardiopulmonary arrest thanks to the efforts of Dr. Sano of Keio University. My utmost gratefulness to Dr. Sano for his foresight and proactiveness. The research was also applied in the field of ophthalmology. Dr. Igarashi and Dr. Takahashi of Nippon Medical School developed a surgical approach that used eye perfusion solutions containing hydrogen. The study targeted patients undergoing cataract surgery, in which hydroxyl radicals generated during the surgery were reduced by an eye perfusion solution containing hydrogen, continuously supplied to the eye. In view of very small amounts of hydrogen dissolved in hydrogen water, I was skeptical about its effect, even after we had demonstrated the effect of hydrogen gas. The quantity of hydrogen that can be ingested from hydrogen water should be significantly lower than what can be inhaled as a gas. Additionally, most of hydrogen would be excreted within 30 minutes of drinking. But, as experimental researchers, we decided to examine the possible effect of hydrogen water without any preconceptions. We let mice of arteriosclerosis model to drink hydrogen water for 4 months, and the results were remarkable. Mr. Ishikawa, who was observing the accumulated matter (atheroma) in blood vessels, noted that the atheroma in the aorta had decreased significantly. Since then, many researchers in Japan and abroad have confirmed the effect of hydrogen water in both animal experiments and clinical studies, including research on the effect of hydrogen water in Parkinson’s disease, led by Dr. Kinji Ohno of Nagoya University and Dr. Mami Noda of Kyushu University. When I was first shown the raw data from Dr. Ohno’s study, I was amazed. The effect was so dramatic that I could hardly believe the results. During this time, Dr. Noda carried out her experiments independently and reached the same conclusion. Both papers were published around the same time (Fu et al. 2009; Fujita et al. 2009). Hydrogen

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water has a dramatically positive effect on Parkinson’s disease, at least in animal models. Having seen this, Dr. Hattori of Juntendo University started a clinical study. Q: How is “Molecular Hydrogen Medicine” positioned in the Tokyo Metropolitan Institute of Gerontology, which you are affiliated with? A: The mission of our institute is to study aging. We aim to study how we can live healthy, long-lived lives. To achieve this, one needs to reduce diseases associated with old age, which is why we are studying Molecular Hydrogen Medicine. We asked ourselves who would benefit from hydrogen and what was the best way to take hydrogen; inhaling, drinking, or ingesting. To look for answers, we needed to understand how molecular hydrogen acts in the body. Apparently, reducing hydroxyl radicals alone is not enough to explain the several disease-improving effects that hydrogen water has shown. And indeed, it had been reported that hydrogen could change the expression of genes that suppress inflammation in addition to oxidative stress (the antioxidant gene Nrf2, for example). So instead of seeking for recipes for individual diseases, we decided to look into what changes occur in the body as we consume hydrogen. In this connection, I wish to mention our recent study, namely, the prophylactic effect of hydrogen. In our experiment on mice, we induced sepsis, which was severe enough for most mice to die within days. However, when they were given hydrogen water for 3 days before sepsis was induced, more than half of them survived. This experiment clearly showed the prophylactic effect of hydrogen water. How does this happen? We found that antioxidant enzymes in the mice livers were significantly increased by drinking hydrogen water, which helped to reduce the oxidative stress due to sepsis (Iketani et al. 2017). We called this the “hormesis-like effect of hydrogen,” which refers to a phenomenon where a harmful stimulus can be made beneficial if the stimulus is kept mild. This suggests that, if we drink hydrogen water on daily basis, the condition could be kept mild even when we get sick. As staff in a medical institution, we are all aware that clinical research is one of our important missions. Usually, in developing new drugs, most clinical research is conducted by pharmaceutical companies, but they are reluctant to get involved in supplying hydrogen as a drug, because hydrogen cannot be patented as a drug and, therefore, does not bring any profit to them. On the other hand, food companies cannot make use of the beneficial effects of hydrogen because they do not have the expertise to handle it. Under these circumstances, basic as well as the clinical research into hydrogen is put in the hand of us researchers. We are now asking diabetic patients to drink hydrogen water to examine whether their condition improves. Q: Please tell us a little more about the “clinical research project of the effect of hydrogen water on diabetes” that you mentioned. A. If we take a look at the effect of hydrogen water reported thus far, we recognize that its significant effects have been demonstrated for diseases caused by drugs, allergies, and organ transplantation. For example, both the animal models of Parkinson’s disease and sepsis are produced by drug administration. Additionally, we were able to observe a clear effect of hydrogen in animal models of atopic dermatitis and on chronic nephropathy that occurs after kidney transplantation. In

3.2 Interview with Dr. Motoaki Sano of Department of Cardiology, Center for. . .

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both cases, hydrogen suppressed the inflammation. In recent years, the main cause of type 2 diabetes is thought to be chronic inflammation, so we wanted to investigate whether hydrogen water could improve this condition. Thus, we have asked patients from the Aizu Central Hospital in Fukushima to drink 300 mL of hydrogen water daily during a 3-month period. Data are currently being analyzed and we will publish the results as soon as possible. Q: Please tell us your future dreams about Molecular Hydrogen Medicine. I would also like to hear your opinion on how we should respond to the widespread “hydrogen water boom” in Japan. A: I hope that, in the future, people are able to drink hydrogen water when needed. I also hope that hydrogen water will be available in first-aid kits, operating rooms, hospital rooms, caregiving facilities, and in people’s homes, for us to achieve long and healthy lives. In many cases, molecular hydrogen treatments can prevent and improve diseases where conventional treatments have failed, including intervention after cardiopulmonary arrest and as a treatment of Parkinson’s disease. We believe that accumulating these results in a reliable manner is the only way to establish and incite wide application of Molecular Hydrogen Medicine. We are very pleased that we can make contributions to the mankind with our research. The merits and demerits of the current hydrogen water boom are clear. For one thing, many people have come to know what hydrogen water is and the health benefits it may bring about, whereas misleading information disseminated by certain distributors has given a strange image of hydrogen water. Numerous high-level publications have contributed to the progress of Molecular Hydrogen Medicine, but unfortunately, the mechanism of action still remains unclear in many cases. In fact, the mechanism of action of medical gases is a very difficult problem: not only hydrogen but also the action of common anesthetic gases is still under discussion. I have been drinking hydrogen water every morning, for the past 10 years. Various data from animal experiments and clinical research have convinced me that I should keep drinking it. I drink hydrogen water of at least 50% saturation. For those who want to drink hydrogen water, I recommend you purchase it from reliable manufacturers and consume it promptly. I should add that, as we still lack adequate information of its side effects, we cannot recommend its use indiscriminately at this time. To be on the safe side, we do not recommend feeding hydrogen water to babies/infants and pregnant women (Recorded February 9, 2017).

3.2

Interview with Dr. Motoaki Sano of Department of Cardiology, Center for Molecular Hydrogen Medicine, Keio University

Q: For many people, I think “cardiac arrest” means death, but I have heard that the emergency outpatient department of Keio University Hospital succeeded in resuscitating cardiac arrest patients, and by giving hydrogen gas, led them to complete recoveries. Is such a thing really possible?

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A: Hydrogen gas is not a magic bullet, so such a thing is, of course, not possible. Please do not misunderstand. The medical efficacy of hydrogen gas has been reported many times in animal experiments and clinical research. Included are acute diseases such as strokes and heart attacks, and chronic diseases such as metabolic syndrome, high blood pressure, and neurodegenerative diseases, and so on. However, because there has not been sufficient objective evaluation of those effects through clinical trials with people, the use of hydrogen gas is not approved for medical purposes yet. This is the reason why we decided to verify scientifically whether the treatment by breathing hydrogen gas could improve the state of consciousness and physical function of patients after they recovered from cardiac arrest by conventional methods. Q: I imagine you went through exceedingly careful preparations to introduce this unfamiliar technique of breathing hydrogen gas in the emergency outpatient department, where there is a constant race against time and the way how things will turn out is unpredictable. Could you tell me about the difficulties you have faced in that regard? A: Clinical research is indispensable for the progress of medical treatments. However, the top priority there must be the welfare of the patients, above any scientific or societal benefits. Furthermore, as hydrogen gas is not approved as a drug, we paid meticulous attention to designing the clinical trial by rigorously following the Ethical Guidelines for Clinical Trials, to obtain the understanding and cooperation of society, and contribute to the betterment of society in return. We proceeded carefully when preparing the research plan and frequently sought guidance from specialists from other divisions (ICU, monitoring, auditing, statistics, and the like) involved in advanced medical care reviews at Keio University Hospital Clinical and Translational Research Center and the Ministry of Health, Labor, and Welfare. This is how hydrogen therapy was first adopted in the emergency medicine. Q: I learned that The Center for Molecular Hydrogen Medicine was established in 2015. Could you tell me about its purpose and its activities? A: The results of preliminary clinical trials to date have given credence to the medical effects of hydrogen gas, so we decided to take the next step. By demonstrating the medical effects of hydrogen in various critical care settings, we have attained a pioneering, strategic position in the realization of Molecular Hydrogen Medicine. In the future, I hope to strengthen the collaboration between different expertise to implement a system for investigating the production, management, and effects of hydrogen gas. In addition, we wish to create an environment that facilitates cooperation between industry and academia. Our final goal is to obtain approval for hydrogen gas and medical devices that utilize hydrogen from the Japanese Pharmaceutical Affairs Act. Recognizing the situation that medical effects of hydrogen are not correctly known, and properly appreciated, I also try to make it visible to society that we are seriously engaged in this research. The Center is located at Keio University Hospital where the research is being actively pursued, with the participation of experts from various fields such as internal medicine, emergency medicine, anesthesia, medicinal chemistry, and electrical engineering.

3.2 Interview with Dr. Motoaki Sano of Department of Cardiology, Center for. . .

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Q: I have heard that you are currently trying to start a large-scale clinical trial called the “Trial of Hydrogen Inhalation for Patients After Out-of-Hospital Cardiac Arrest.” Could you please explain this trial? Also, I wish to know if, in the future, hydrogen gas inhalation will come to be used as a standard method in emergency outpatient departments, not only at Keio University Hospital, but also nationwide. A: Post-cardiac arrest syndrome is a condition that occurs after the heart has suddenly stopped beating and, although the heartbeat may be restarted using emergency resuscitation techniques, damage to the heart muscle has already occurred, leading to impaired function of various organs, including the brain. The post-cardiac arrest can result in lasting, higher-order disabilities, often leaving patients bedridden or in a so-called vegetative state. Only a small number of patients achieve a complete recovery, able to return to society without secondary conditions. According to a 2015 review by the Fire and Disaster Management Agency, the 1-month survival rate among the 25,000 people who experienced out-of-hospital cardiac arrest in Japan each year is only 12%. The proportion of people who recover from postcardiac arrest syndrome and return to society is around 8%. The mechanism of post-cardiac arrest syndrome is considered to be reperfusion injury. The only effective treatment widely adopted to minimize this damage is hypothermia therapy, namely, to keep the body temperature low, between 32  C and 36  C, while administering an anesthetic. However, as the effectiveness of this method is limited, there is a demand for additional, more effective treatments. Therefore, we decided to perform a clinical test of the hydrogen inhalation, and evaluate its efficacy by a large-scale, well-organized procedure. The effectiveness of breathing hydrogen gas for post-cardiac arrest had been known in animal experiments but was not proven with certainty in humans. So, we started with developing a method to administer hydrogen gas safely to patients with post-cardiac arrest syndrome and succeeded in establishing the technology. The method of hydrogen administration involves adding hydrogen to the mixture of gases (oxygen and nitrogen) used in artificial respiration while performing the conventional hypothermia therapy. In this clinical trial, we plan to evaluate the recovery of consciousness and activities of daily life after 90 days and compare the result with the extent of brain function recovery. This is the world’s first full-scale clinical trial to verify the efficacy of the hydrogen inhalation method. If the efficacy of hydrogen gas treatment is verified by this clinical trial, then hopefully this will increase the chances of hydrogen gas to be approved for use in other cases as well, including, for example, strokes and heart attacks. Since the hydrogen inhalation does not require any costly devices, the treatment could be introduced very easily. I would expect that the hydrogen treatment will spread rapidly in emergency medicine and then be disseminated to other areas as well. I believe that this clinical trial opens the possibility of saving the lives of many patients, and in this way, makes Japan a world leader in this revolutionary treatment of hydrogen medicine. (Recorded February 16, 2017).

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From the Front-Line of Research: Interviews

Interview with Dr. Hirohisa Ono of the Neurosurgery Department, Nishijima Hospital

Q: You have more than 40 years of experience in treatment and research in neurosurgery. I think there have been big changes in the science and medicine of the brain, so I would ask you to describe your own experience during this period. A: I started working in this field in the early 1960s, for 3 years as a member of staff in the neurosurgery service established as a part of the surgical department at Nagasaki University. During that time, common brain surgeries were craniotomies for traffic-accident-related brain hemorrhages and for brain tumors. We used cerebral angiograms, ultrasound, and similar tools for non-invasive diagnosis, but it was difficult to locate the damaged regions accurately, and we often needed to open the skull without sufficient knowledge in advance. This situation changed completely when I became a staff member at the University of Oregon Medical School, where X-ray computerized tomography (CT) was available. In addition, in 1983, the MRI machine was also introduced, and it became possible to see brain lesions of some millimeters in size, without placing any burden on the patient. MRI can provide various kinds of data, not only on the location of tissue lesions but also on the flow of blood, from the signals produced by hydrogen in various different states in the body. In this way, neurological medicine and treatments have changed significantly. It is no exaggeration to say this was revolutionary. CT and MRI have become indispensable methods, particularly for the science and treatment of the brain. It is no surprise that Nobel Prize in Physiology or Medicine was awarded to the development of the medical technologies of CT and MRI. (For CT, G. N. Housefield and A. M. Cormack in 1979; for MRI, P. Lauterbar and P. Mansfield in 2003). Subsequently, other special imaging techniques called positron emission tomography (PET) and single photon emission computed tomography (SPECT) were developed and advanced the treatment of patients with brain disorders still further. As for myself, after working in the hospital of the University of Oregon in the USA for around 30 years, from 1982, I returned to Japan to work in Nishijima Hospital in Numazu. During these times, I have constantly been working in the clinical setting as a neurosurgeon, but I have also been engaged in research as well, culturing of brain tumor tissues, and later stem cells. Stem cells are undifferentiated cells, baby cells not given specific roles yet but later grow into cells having specific roles. Until 1980, it was widely believed that for adult higher animals, the brain’s nerve cells (neurons) were no longer reproduced and that once injury occurs, recovery was not possible. Actually, it was reported in 1965 that neurons in the hippocampus of rat brains could be regenerated. Undoubtedly, dormant stem cells were playing a role there. Subsequently, it was found that stem cells exist throughout the human body, and making use of them in regenerative treatments has become a hot topic. I am continuing such a research into human brain stem cells. Looking back, I entered this field soon after the structure of the DNA molecule was elucidated, which led to the reformation of biology from its very foundation. Its

3.3 Interview with Dr. Hirohisa Ono of the Neurosurgery Department, Nishijima. . .

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effects are really profound in all the areas of biology and medicine, and neurology was no exception. I feel very pleased that I happened to be born into such a time. Q: Given that Nishijima Hospital in Numazu, your current working place, is a private hospital that treats outpatients, I imagine there were difficulties in adopting a totally new method like molecular hydrogen treatment there. Could you tell me about your motives for becoming actively engaged under these circumstances? A: Nishijima Hospital has seven departments, with a focus on the Neurosurgery, Orthopedics, Neurology, and Cardiovascular/Intravascular Treatment Departments. It is a general hospital with 150 beds, but more than this, it is a core hospital in the region, maintaining the motto “we never turn down an emergency patient”, admitting more than 2000 patients who arrive by ambulance each year. The director, Dr. Yoji Nishijima, is also a neurosurgeon and his stance is to proactively adopt new treatments and devices wherever possible, so we introduced molecular hydrogen treatments at quite an early stage. I got engaged in molecular hydrogen treatments with the strong encouragement of Dr. Nishijima. Fortunately, as the administration of hydrogen is known to cause no side effects, we administered it to patients with severe conditions, for whom recovery seemed unlikely, and this produced much better results than we had expected. Acute cutaneous erythema and secondary symptoms of strokes described in the text are just such examples. Before everything, rather than spending time on basic pathology, all of us working in the clinical settings want to see the patients get better, and try to do whatever we can to find some prescriptions/therapies, if not available at present. The hydrogen therapy was certainly one of them, fortunately a successful one. I feel really happy when a patient who had severe facial cutaneous erythema comes to the hospital after recovery, from time to time, and shows me his healthy face. Performing a large clinical trial is difficult at a single private hospital because it requires so much extra of the already-busy staff. The clinical trial with 50 patients to investigate secondary conditions associated with acute strokes is by no means sufficient, but it was the best we could do. For this, the cooperative stance that has always been present in emergency medicine was very helpful. Sometimes, there are discoveries that can only be made on the front of emergency medicine. The acute cutaneous erythema case was one such example. A patient, who just happened to be brought to us for acute facial cutaneous erythema, was treated with hydrogen and recovered remarkably. Certainly, this is only a single case report, but I think the result is very significant. It surely opens the way for treating this intractable disease. For the mechanism of this hydrogen treatment, I hope those involved in basic research will give us explanations some day in the future. (Recorded October 31, 2018). Many researchers have been involved in the development of this field. Without giving in to the incomprehension and animosity of those around them, they have opened the door to the unknown world one by one. Find out from these interviews their sense of mission as medical scientists and researchers. Molecular Hydrogen Medicine will eventually be established as a field of medicine. This record will certainly remain as a historical testimony.

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Fig. 3.1 Shape of neurons (neural cells) in the brain (schematic). Signals received by dendrites are processed in the cell and transmitted to other neurons via extended axons. In this way, a huge number of neurons are connected with each other to form a neural network

Column 2: Alzheimer’s Disease Dementia increases in the elderly and is becoming a big social problem in the developed countries where life expectancy is increasing. As of 2018, six million people in Japan and nearly the same number of people in the USA were affected by dementia. The number is estimated to be 47 million globally and predicted to be 130 million by 2050. Among the four types of dementia having different causes and symptoms, the most common is Alzheimer’s disease, which accounts for approximately 60% of all dementia cases. In Alzheimer’s disease, the hippocampal cells inside the cerebral surface (cerebral cortex) gradually die, resulting in memory and judgment disorders. Lesions form first among the brain cells as protein agglomerations known as amyloid beta. Subsequently, rolled thread-like tau proteins form in the cells, and the resulting cell death leads to cognitive impairments. The schematic of a neuron (Fig. 3.1) depicts the process by which the dendrites receive signals from the surrounding cells and process these to create and send new signals to other cells via elongated axons. The succession of signal transmission in the network of neurons this way eventually activates the brain system. In Alzheimer’s disease, these signals are interrupted by atrophy and death of the hippocampal axons, which causes damage on the brain function. Tremendous numbers of studies have been conducted since the first case was reported by Alois Alzheimer in 1906, and in the past 20 years, research has progressed to the genetic and molecular levels. In spite of these efforts, the precise causes of cell death remain unknown. (continued)

References

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Column 2: Alzheimer’s Disease (continued) Despite large financial investments into drug development for dementia, exceeding several trillion Japanese yen (more than 10 billion US dollars), very few pharmaceutical companies have succeeded in delaying the progression of dementia, not to speak of cures or fundamental treatments. In fact, some drugs succeeded in removing amyloid beta, but failed to cure the disease. Thus, it is thought that amyloid beta deposition may not be the only causative factor, although it is certainly one of the characteristic features of Alzheimer’s disease. Lesions seem to start well before amyloid beta begins to deposit (as early as 20 years), but the very initial causes are unidentified yet. Thus, with regard to Alzheimer’s disease, conventional strategies of drug developments based on the assumption of specific mechanisms of action are losing credence, which obliged many companies to withdraw from this field of research and development. Recently, an alternative view has been growing that Alzheimer’s disease is inherently a multifactorial disease. For example, in the USA, Dale Bredesen gained attention by identifying 36 causative factors for Alzheimer’s disease and developed a personalized method of treatment, tailored according to these causative factors in each patient. These treatments have been reported to improve the medical conditions of several hundred patients (Bredesen 2017). The successful treatment of dementia by Ono and his collaborators, previously described, may also be the consequence of multi-functional action of hydrogen. The recognition of multifactorial nature of Alzheimer’s disease is now shedding new light on the approaches of clinical doctors, such as Bredesen and Ono, who are well aware of the complexity of the disease and constructed new therapeutic systems based on their experience. Their interpretation of the disease may not be entirely correct, but in the absence of precise knowledge of the underlying causes of Alzheimer’s disease, it cannot be helped. Nevertheless, we may expect that breakthroughs might be achieved from such clinical approaches, which are very different from conventional treatments and drug developments. We may hope that the enthusiasm of clinicians to cure patients may lead to novel interventions.

References D.E. Bredesen, The End of Alzheimer’s (Penguin Random House LLC, New York, 2017) Y. Fu, M. Ito, Y. Fujita, M. Ito, M. Ichikawa, A. Masuda, Y. Suzuki, S. Maesawa, Y. Kajita, M. Hirayama, I. Ohsawa, S. Ohta, K. Ohno, Molecular hydrogen is protective against 6-OHDA induced nigrostriatal degeneration in a rat model of Parkinson’s disease. Neurosci. Lett. 453, 81–85 (2009)

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K. Fujita, T. Seike, N. Yutsudo, M. Ohno, H. Yamada, H. Yamaguchi, K. Sakumi, Y. Yamakawa, M.A. Kido, A. Takaki, T. Katafuchi, Y. Tanaka, Y. Nakabeppu, M. Noda, Hydrogen in drinking water reduces dopaminergic neuronal loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydrophyridine mouse model of Parkinson’s disease. PLoS One 4, e7247 (2009) M. Iketani, J. Ohshiro, T. Urushibara, M. Takahashi, T. Arai, H. Kawaguchi, I. Ohsawa, Preadministration of hydrogen-rich water protects against lipopolysaccharide-induced sepsis and attenuates liver injury. Shock 48, 85–93 (2017)

4

Physiological Effects of the Hydrogen Molecules

Abstract

Following the introduction to the medical effects of H2 molecules, this section summarizes some basic physiological effects of molecular H2 and its mechanism of action on diseases. It should be noted that the molecular hydrogen discussed here is a completely different entity from ions. Positive hydrogen ions (i.e. the proton H+ or the oxonium ion H3O+) and negative hydrogen ions (i.e. the hydroxide ion OH
) commonly exist in acidic and alkaline solutions, but molecular H2 does not ordinarily exist. In addition, the properties of these ions are completely different from molecular H2. It is in the form of molecular H2 that hydrogen exerts such physiological effects.

4.1

Entry of Hydrogen Molecules into Human Body

Hydrogen molecules taken into the body by breathing H2 gas or drinking H2 water are transported to every part of the body by blood flow and subsequent permeation through the tissues. Figure 4.1 shows first results on rats observed after inhalation of H2-containing gases (Ohsawa et al. 2007). The concentration of hydrogen in the blood increased proportionally to the gaseous hydrogen concentration. The concentration was lower in venous blood than in arterial blood. Generally, concentrations reached after breathing H2 gas are higher than after drinking H2 water. After drinking H2 water, concentrations temporarily increase in the stomach, small intestine and liver, but the average concentration in the body remains low. Figure 4.2 shows the temporal variation of the hydrogen concentration in the blood, liver and kidney tissues in rats after administration of H2 water (Sobue et al. 2015). The concentration reached maximum in ~2 min in the blood, and ~5 min in the liver and kidney. Decrease of hydrogen concentration from atrial to aortic blood is mainly due to gas # The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2020 Y. Fukai, Molecular Hydrogen for Medicine, https://doi.org/10.1007/978-981-15-7157-2_4

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Fig. 4.1 Inhalation of H2 gas by rats increased the H2 concentration in the blood. H2 concentration was measured after 1 h of H2-mixed gas inhalation. The concentration increased in proportion to the H2 concentration in the gas, lower in the vein than in the artery (Ohsawa et al. 2008)

Fig. 4.2 H2 concentration in the blood, liver and kidney after administration of 4 mL of H2 water (1.4 ~ 1.6 ppm) to rats. 1 μM ¼ 0.002 ppm (Sobue et al. 2015)

4.1 Entry of Hydrogen Molecules into Human Body

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Fig. 4.3 H2 concentration in different organs and bloods in rats after starting inhalation of H2-containing gas; organs with inhalation of 3% H2 gas (Yamamoto et al. 2019). Atrial blood (○) and aortic blood (□) with 2% H2 gas (Sobue et al. 2015). 1 μM ¼ 0.002 ppm

exchange in the lung. Note the big difference between the organs: the concentration in the kidney is much smaller than in the liver. Recently, Yamamoto et al. (2019) measured the temporal variation of hydrogen concentration in various tissues (organs) in rats during inhalation of H2 gas. The concentration was measured continuously by inserting a microsensor in the brain, liver, kidney, mesentery fat, and thigh muscle, after starting inhalation of 3% H2 gas at a rate of 0.2 L/min. As shown in Fig. 4.3, time to reach constant steady state was