Molecular hydrogen in health and disease

Hydrogen: From Stars to Fuel to Medicine

Tyler W. LeBaron, Randy Sharpe, Felix A. Pyatakovich,
and Mikhail Yu. Artamonov
Molecular Hydrogen Institute, Enoch, UT, USA

Abstract
Hydrogen gas has garnered significant attention in recent years due to its remarkable antioxidant and anti-inflammatory properties. However, the extensive research on hydrogen’s applications in the energy sector often overshadows its potential as a medically and biologically active gas. Surprisingly, investigations into the biomedical aspects of H2 trace back to as early as 1793. Hydrogen exhibits exceptional pharmacokinetics, swiftly traversing cellular biomembranes, including the blood–brain and testes barriers, to access subcellular organelles. Following ingestion, hydrogen follows the path of least resistance through the circulatory system and is primarily eliminated through exhalation. Despite the intricate molecular mechanisms and precise targets remaining elusive, the antioxidant effects of hydrogen involve the upregulation of endogenous antioxidants via the activation of the Nrf2/keap1 pathway. Recent research highlights the potential role of Fe-porphyrin as a redox-related biosensor, facilitating hydrogen’s reactions with hydroxyl radicals and triggering additional signal transduction processes. Moreover, this review delves into the physicochemical properties of hydrogen, particularly emphasizing its molar solubility, considerations regarding the term saturation, and other unique characteristics of H2 are discussed. The expanding knowledge and research surrounding the history of H2 underscore its transformative potential in biomedical applications and pave the way for future advancements in harnessing its therapeutic properties.

An Exploration of the Direct Biological Targets of Molecular Hydrogen

John T. Hancock, Jennifer E. May, Tyler W. LeBaron, Rajalaksmi Punampalam und Grace Russel
School of Applied Sciences, College of Health, Science and Society, UWE, Coldharbour Lane, Bristol, BS16 1QY, UK

Abstract
Molecular hydrogen (H2), supplied either as a gas or in a solution, has been gaining popularity as a treatment for a variety of conditions and diseases. For example, it has been suggested to be beneficial for neurodegenerative diseases, to ease the injuries caused by restoration of blood flow to previously ischaemic tissues, and even to alleviate the symptoms of COVID-19. It has also been suggested as an ergogenic sports supplement. However, the exact mode of action of H2 has yet to be definitively unravelled. It has been suggested that H2 acts as an antioxidant and, in particular, as a scavenger of hydroxyl radicals (·OH). This might be the case, but it is unlikely that this is the only mode of action of H2 in biological systems. Here we discuss some of the possible mechanisms by which H2 may have an effect, which may explain how it is acting in a medical context.

Prospects of hydrogen medicine due to its effects on mitochondrial function

Shin-ichi Hirano, Yusuke Ichikawa, Bunpei Sato, Yoshiyasu Takefuji, Xiao-Kang Li und Fumitake Satoh
Division of Transplantation Immunology, National Institute for Child Health and Development, Tokyo, Japan

Abstract
Mitochondria evolved from aerobic bacteria through endosymbiosis. Through this symbiosis, eukaryotes acquired an efficient energy production system, but at the cost of being exposed to oxidative stress by reactive oxygen species (ROS). Molecular hydrogen (H2) has recently been identified as an antioxidant that selectively reduces ROS such as hydroxyl radicals and peroxynitrite, which are strong oxidizing agents, and its clinical application is progressing. In this work, the efficacy of H2 was investigated in experimental models and in various chronic inflammatory diseases in humans and shown to exert its effect via protection of mitochondrial function. Protection by H2 may occur through the regulation of mitochondrial ROS. As mitochondrial dysfunction has been identified in many common diseases such as metabolic and neurodegenerative diseases, the development of technologies and compounds that protect or activate mitochondrial function will be necessary for the future of medicine. H2 may be a candidate for future medicine due to its effects on mitochondrial function.

Molecular Hydrogen: A New Treatment Strategy of Mitochondrial Disorders

Anna Gvozdjáková, Jarmila Kucharská, Zuzana Sumbalová, Zuzana Rausová, Branislav Kura, Barbora Bartolˇciˇcová und Ján Slezák
Faculty of Medicine, Pharmacobiochemical Laboratory of 3rd Department of Internal Medicine, Comenius University in Bratislava, Sasinkova 4, 811 08, Bratislava, Slovakia

Abstract
Disturbances of mitochondrial function and oxidative stress are considered to be the molecular basis of the origin and development of various diseases, including mitochondrial diseases. The beneficial effect of molecular hydrogen (H2) has been proven in the prevention and supportive therapy of patients with cardiovacular disease, Parkinson’s disease, in patients with metabolic syndrome, in respiratory system disease, in oncology patients treated with radiation, in cerebral infarction, in diabetes mellitus, in rheumatoid arthritis. Exact molecular mechanisms of H2 on mitochondrial level are not fully understood. We proposed new mechanism of the H2 effect in mitochondrial respiratory chain function. H2 may be a donor of both electron and proton to the Q-cycle of the mitochondrial respiratory chain and thus can preserve coenzyme Q level with the subsequent ATP production via oxidative phosphorylation. H2 was shown to alter the direction of the electron flow of mitochondrial respiratory chain system, which depends on NAD+/NADH ratio. We also found beneficial effect of H2 on platelet mitochondrial bioenergy function in patients with NAFLD. The application of H2 appears to be a new treatment strategy for targeted therapy of mitochondrial disorders.

Autonomic Cardiac Regulation in Response to Exercise and Molecular Hydrogen Administration in Well-Trained
Athletes

Michal Botek, Jakub Krejˇcí, Barbora Sládeˇcková und Andrew McKune
Department of Natural Sciences in Kinanthropology, Faculty of Physical Culture, Palacký University Olomouc, Olomouc, Czech Republic

Abstract
Exercise induces considerable changes in the autonomic nervous system (ANS). The main objective of this chapter was to determine whether H2 administration through the hydrogen rich water (HRW) can affect ANS activity during two experimental exercise protocols in well-trained athletes. Both experiments were designed as randomized, double-blind, placebo-controlled crossover trials. Study A (12 fin-swimmers) assessed ANS responses before and during a simulated competitive day, and Study B (12 soccer players) assessed heart rate (HR) responses following a repeated sprint ability protocol (15 × 30 m). The heart rate variability method was performed to determine ANS activity for 5 min in standing and supine position using the DiANS PF8 system, and HR recovery was evaluated using the HR monitor at 1 and 3 min post exercise. Study A showed that three days of HRW administration induced a significant decline in vagal activity and HR stimulation in elite fin-swimmers solely in the standing position during the pre-competition phase of the simulated competition day. Study B showed that acute HRW administration can improve HR recovery of team sport athletes performing maximal repeated sprints that may translate to improved performance during training and competition. Therefore, it appears that H2 may be considered a promising dietary supplement in the future.

The Clinical Use of Hydrogen as a Medical Treatment

Yunbo Xie and Guohua Song
School of Basic Medical Sciences, Shandong First Medical University and Shandong Academy of Medical Science, Jinan, 250117, China

Abstract
It has been demonstrated that hydrogen molecules possess biological effects, and furthermore, they are colorless, non-toxic, and have a small molecular weight, enabling them to traverse the blood–brain barrier. This review synthesizes more than 100 publications on the use of hydrogen therapy in clinical ailments and categorizes the applications of hydrogen medicine into nine major systemic diseases based on the International Classification of Diseases (ICD)-11. The efficacy of hydrogen therapy is influenced by the in vivo metabolic kinetics associated with different administration routes. In this review, we examine the utilization of hydrogen molecules via various delivery methods and their impact on the treatment of clinical diseases, along with the mechanisms underlying their biological effects.

Homeostatic and Endocrine Response Underlying Protective
Effects by Molecular Hydrogen

Mami Noda and Eugene Iv. Nazarov
Institute of Mitochondrial Biology and Medicine of Xi’an Jiaotong University School of Life Science and Technology, Xi’an, China

Abstract
Molecular hydrogen (H2) has multiple properties such as anti-apoptotic, anti-inflammatory and anti-oxidative properties, having wide variety of beneficial outcomes. Effects of H2 are often slow to show the effects. The amount of H2 in drinking water is far less than that of inhalation. H2, especially in drinking water, can be used for therapy but rather for maintenance and improvement of the health condition or prevention of disorders. From mechanistic point of view, the problem is that pharmacokinetic of H2 in drinking water is largely unknown, especially in the brain. This chapter focuses the long-term intake of H2 and the mechanism is discussed from homeostatic and endocrinal point of view, trying to understand why H2 has such a wide variety of effects. H2 stimulates the production of gastrointestinal hormone, ghrelin, which stimulates growth hormone releasing, affects hypothalamic–pituitary–adrenal (HPA) axis, and also seems to affect hypothalamic–pituitary–gonadal (HPG) axis. Due to these endocrinological effects, H2 has a corrective effect on the neuro-immuno-endocrine system and determines the functioning of the body’s homeostatic system.

Radiation-Induced Heart Disease: Potential Role
for Molecular Hydrogen

Branislav Kura, Patricia Pavelkova, Barbora Kalocayova und Jan Slezak
Centre of Experimental Medicine, Institute for Heart Research, Slovak Academy of Sciences, Dubravska cesta 9, 841 04, Bratislava, Slovakia

Abstract
Radiation-induced heart disease (RIHD) is a common complication of mediastinal radiotherapy. RIHD includes structural and functional abnormalities of the pericardium, coronary vessels, myocardium, valves, and conduction system. The underlying pathological mechanisms are complex, mainly related to endothelial cell injury, oxidative stress, and inflammation. Radiation can cause cardiomyocyte death, tissue fibrosis and ultimately may end up with heart failure. To overcome these complications, there is a need to look for specific therapeutic interventions, which are still missing. Molecular hydrogen (H2) has been recognized as a molecule with antioxidant, anti-inflammatory, and anti-apoptotic protective effects in different disease settings. In vitro as well as in vivo studies demonstrated that H2 exerted preventive or therapeutic effects on radiation-induced injury, including RIHD. H2 could be effective to mitigate RIHD through various mechanisms, e.g. selective neutralization of hydroxyl radicals, protection against inflammatory and apoptotic damage, anti-fibrotic and anti-hypertrophic effects, etc. More research is needed to elucidate further mechanisms of H2 action, and to verify the effectiveness of H2 therapy in clinical trials.

Short-Lasting Supplementation with Molecular Hydrogen and Vitamin E Upregulates Myocardial Connexin-43 in Irradiated and Non-irradiated Rat Heart

Barbara Szeiffova Bacova, Katarina Andelova, Matus Sykora, Branislav Kura, Barbora Kalocayova, Jan Slezak und Narcis Tribulova
Centre of Experimental Medicine SAS, Institute for Heart Research, Dúbravská cesta 9, 841 04, Bratislava, Slovak Republic

Abstract
In this study we aimed to explore whether supplementation with antioxidants, molecular H2 and vitamin E, may impact gap junction channel protein, connexin-43, in left ventricular tissue of irradiated rat hearts. Experiments were performed using 3-month-old male Wistar rats that were randomized into 6 groups. (1) Intact rats; (2) Intact rats treated with molecular H2 (4% H2 in inhalation chamber, 3 × 30 min per day) (3) intact rats supplemented with vitamin E (30 mg/kg body weight); (4) Irradiated rats after exposure to single dose of 10 Gy; (5) Irradiated rats treated with molecular H2; (6) Irradiated rats treated with vitamin E. Left heart myocardium was used for analysis, at 9 days after irradiation. Findings indicate that treatment with molecular H2 and vitamin E up-regulates connexin-43 and PKCε not only in irradiated but also in intact rat hearts. It is challenging for further research to elucidate mechanisms.

Molecular Hydrogen: A New Protective Tool Against
Radiation-Induced Toxicity

Jana Vlkovicova, Branislav Kura, Patricia Pavelkova und Barbora Kalocayova
Centre of Experimental Medicine, Institute for Heart Research, Slovak Academy of Sciences, Dubravska cesta 9, 841 04, Bratislava, Slovak Republic

Abstract
Disturbances of mitochondrial function and oxidative stress are considered to be the molecular basis of the origin and development of various diseases, including mitochondrial diseases. The beneficial effect of molecular hydrogen (H2) has been proven in the prevention and supportive therapy of patients with cardiovacular disease, Parkinson’s disease, in patients with metabolic syndrome, in respiratory system disease, in oncology patients treated with radiation, in cerebral infarction, in diabetes mellitus, in rheumatoid arthritis. Exact molecular mechanisms of H2 on mitochondrial level are not fully understood. We proposed new mechanism of the H2 effect in mitochondrial respiratory chain function. H2 may be a donor of both electron and proton to the Q-cycle of the mitochondrial respiratory chain and thus can preserve coenzyme Q level with the subsequent ATP production via oxidative phosphorylation. H2 was shown to alter the direction of the electron flow of mitochondrial respiratory chain system, which depends on NAD+/NADH ratio. We also found beneficial effect of H2 on platelet mitochondrial bioenergy function in patients with NAFLD. The application of H2 appears to be a new treatment strategy for targeted therapy of mitochondrial disorders.

Role of Matrix Metalloproteinases in Effects of MolecularHydrogen

Barbora Bot’anská, Viktória Pecníková, Branislav Kura, Ján Slezák und Miroslav Baranˇcík
Centre of Experimental Medicine, Institute for Heart Research, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, 841 04, Slovakia

Abstract
Molecular hydrogen plays a role in modulating several cellular functions and in several diseases were documented its pleiotropic therapeutic effects. Several lines of evidence indicates that an important role in mechanisms involved in molecular hydrogen effects play modulation of cellular antioxidant defenses, including intracellular and extracellular redox signaling. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are involved in regulation of several cellular functions, in particular the modulation of extracellular matrix turnover. Moreover, oxidative stress-induced dysregulation of MMPs activities plays a crucial role in the development of pathological changes. Since it was described regulatory role of molecular hydrogen in modulation of several MMPs these enzymes represent potential targets of molecular hydrogen action. Our data demonstrated potential negative role of MMP-2 and MMP-28 in development of pathological changes induced by mediastinal irradiation (MI) of rats. Molecular hydrogen possessed beneficial effects against MI-induced changes in both matrix metalloproteinases. To the potential role of MMPs in protective effects of molecular hydrogen point also findings that inhalation of hydrogen-rich air was associated with protection against post-transplant complications and led to a partial reversal of MMP-9 activation. In summary, the modulation of matrix metalloproteinases with molecular hydrogen could be a promising strategy for the treatment of various diseases in which oxidative stress and tissue remodeling play an important role.

Perioperative Mitigation of Oxidative Stress with Molecular Hydrogen During Simulated Heart Transplantation in Pigs

Branislav Kura, Barbara Szeiffova Bacova, Miroslav Barancik, Matus Sykora, Ludmila Okruhlicova, Narcisa Tribulova, Roberto Bolli, Barbora Kalocayova, Tyler W. LeBaron, Katarina Andelova und Jan Slezak
Centre of Experimental Medicine, Institute for Heart Research, Slovak Academy of Sciences, Dúbravská Cesta 9, Bratislava, 84104, Slovak Republic

Abstract
Heart transplantation is now a routine method for severe heart failure treatment. It is critical to focus on preventing ischemia–reperfusion damage and mitigating oxidative stress to achieve successful outcomes. However, prolonged anesthesia, hyperoxia, and defibrillations contribute to an increase of ROS/RNS and disrupt the redox homeostasis, which poses a serious risk factor. Numerous publications have confirmed the remarkable antioxidant, anti-apoptotic, and anti-inflammatory properties of molecular hydrogen. In our simulated heart transplantation experiment, we demonstrate that administering 2% hydrogen gas during anesthesia and extracorporeal circulation (ECC) significantly alleviates oxidative stress-induced damage. This is evidenced by a significant decrease in markers of ischemia, lipid peroxidation, and inflammation. The restoration of the pumping activity in the implanted pig hearts showed improvement, with a reduced need for repeated defibrillations. The administration of H2 during graft collection and transplantation significantly enhances the function of the transplanted heart and the overall condition of the recipient. Hydrogen administered by conventional ventilators and ECC oxygenators represents an innovative therapy that can significantly improve current transplantation techniques.

Application of Hydrogen in Hemodialysis: A Brief Review
with Emphasis on the Quantification of Dissolved H2

Foivos Leonidas Mouzakis, Lal Babu Khadka, Miguel Pereira da Silva und Khosrow Mottaghy
Institute of Physiology, RWTH Aachen University Hospital, Aachen, Germany

Abstract
Chronic kidney disease patients frequently manifest signs of oxidative and inflammatory stress associated with the lifesaving haemodialysis therapy itself. Lately, hydrogen has gained traction as an antioxidant with a deluge of reports bearing evidence of its potential in treating a plethora of medical conditions. Among the various animal models and clinical studies communicated, a novel hydrogen administration method revolving around H2-rich dialysate stands out. Over the past decade, hydrogen enriched haemodialysis (E-HD) has steadily been gaining ground thanks to its ameliorating effect on oxidative and inflammatory complications arising during haemodialysis. To complement this modality, a hydrogen water monitoring system (HWMS) has been developed by Pureron Japan Co., Ltd to assist in determining the levels of dissolved hydrogen in liquids. Preliminary investigations have validated the sensitivity and accuracy of the contactless hydrogen sensor and its applicability in various clinical settings. Nevertheless, a few setbacks such as the long response time, and range-specific accuracy prompted further examination and in-depth analysis of its capabilities. To achieve that, three such sensors have been integrated in an in vitro haemodialysis circuit to monitor H2 concentration at the dialyzer’s inlet–outlet. Moreover, experiments have been conducted using a regular dialyzer as well as a modified one with a purely diffusive coating, in order to discern the nature of hydrogen transfer through the capillary membrane and distinguish between ultrafiltration and diffusion.

Hydrogen as a Potential Therapeutic Approach
in the Treatment of Cancer: From Bench to Bedside

Arian Karimi Rouzbehani, Golnaz Mahmoudvand, Zahra Goudarzi, Arshia Fakouri, Simin Farokhi, Saeideh Khorshid Sokhangouy, Elnaz Ghorbani, Amir Avan, Elham Nazari und Majid Khazaei
Department of Health Information Technology and Management, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Abstract
Cancer is still remained among the leading cause of death worldwide, mainly due to the metastatic spread and chemotherapy resistance. In turn chemotherapy is associated with several side effects. Therefore, there is a need to develop new therapies for the management of this condition, reducing toxicity and drug resistance. Recently, hydrogen molecule has been reported as a novel therapeutic approach for many diseases including cancer. there is growing body of data showing the anti-tumor, antioxidant, and anti-inflammatory activity of hydrogen therapy in cancer. Also, various studies have reported the effectiveness of hydrogen therapy in cancer in preclinical setting, However, it is important to note that these studies are still preliminary, and more research is needed to determine the safety and efficacy of hydrogen therapy for cancer treatment. Additionally, while some studies suggest that hydrogen therapy may have potential benefits for certain types of cancer, it is not a cure or replacement for conventional cancer treatments such as chemotherapy or radiation therapy. while there is some evidence to suggest that hydrogen molecules may have therapeutic potential for cancer treatment, more research is needed before any definitive conclusions can be drawn. In this chapter, we provide an overview on the antiproliferative activity of hydrogen and its potential molecular mechanisms of actions followed by recent reports on its potential side effects.

The Role of the Smallest Molecule Hydrogen Overcoming
Ageing-Related Disease

Wenjing He, Md. Habibur Rahman, Chaodeng Mo, Arounnapha Vongdouangchanh, Cheol-Su Kim und Kyu-Jae Lee
Department of Convergence Medicine, Yonsei University Wonju College of Medicine, Gangwon-Do, Wonju, 26426, Republic of Korea

Abstract
Ageing is an inevitable process that increases the probability of chronic disease that involves an evolving decline in organism function over time. The length of human life is closely related to ageing, which can cause a deterioration of physiological functions and lead to the development of various chronic diseases. Many theories have been put forward to illustrate the ageing mechanisms, with the free oxidative stress theory being the most well-known. Aging is caused by the excessive accumulation of cells or tissues related to oxidative damage caused by ROS. As everyone knows that oxidative stress is closely related to aging, antioxidants may be potentially valuable in the treatment of senescence disease. Hydrogen (H2) is a colorless, tasteless small molecule that plays a major role in eliminating harmful free radicals and reducing oxidative damage in vivo, such as anti-oxidation, anti-inflammation, and anti-apoptosis. Additionally, H2 can be utilized to prevent and cure several aging-related diseases, including cancer, Alzheimer’s disease, and gastrointestinal diseases. Understanding the aging process is crucial for comprehending how to slow the aging process and the development of aging-related diseases. This chapter summarized the preventive and treatment applications of molecular H2 in anti-ageing and underlying mechanisms in aging-related diseases.

Dihydrogen and Hepatic Function: Systematic Review
and Meta-analysis

Nikola Todorovic und Sergej M. Ostojic
Applied Bioenergetics Lab, Faculty of Sport and Physical Education, University of Novi Sad, 21000, Novi Sad, Serbia

Abstract
Molecular hydrogen (H2, dihydrogen) is an innovative experimental agent that could foster beneficial effects in a plethora of human conditions, including liver disorders. Recent trials demonstrated the positive effects of dihydrogen in several acute and chronic liver diseases, including chronic hepatitis B, non-alcoholic fatty liver disease, and liver cancer. Still, no systematic review or meta-analysis investigated the effects of dihydrogen intake on the hepatic function panel. For this report, we searched three relevant databases (PubMed, Web of Science, Scopus) from inception until December 24, 2022, using PRISMA guidelines. A literature search yielded 365 publications in total. After removing duplicates and studies that did not meet the inclusion criteria, 12 studies (published from 2008 to 2022) were included in this analysis. We found that serum malondialdehyde levels were significantly decreased following dihydrogen intake (pooled standardized mean difference = − 0.97 [95% confidence interval [CI], from − 1.65 to − 0.19; P = 0.01), with results indicating a large effect of dihydrogen intervention. A strong trend for a reduction in the liver function tests (including aspartate aminotransferase and alanine transaminase) has also been observed after dihydrogen administration (P < 0.20). Our findings suggested that dihydrogen can favorably affect the hepatic function panel; additional well-sampled interventional trials are highly warranted to corroborate our results.[/fusion_li_item][fusion_li_item]

Hydrogen-Rich Water Using as a Modulator of Gut
Microbiota and Managing the Inflammatory Bowel Disease

Atieh Yaghoubi, Saman Soleimanpour und Majid Khazaei
School of Applied Sciences, Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Abstract
Molecular hydrogen (H2) is well known as a colorless gas, and water enriched with H2 (HRW) is an innovative, beneficial beverage for human health that improves gut microbiota management and the viability of the intestinal. Drinking water enriched with hydrogen has been found to have therapeutic effects on inflammatory bowel diseases (IBDs). The low molecular weight of H2 enables it to easily diffuse and permeate cell membranes to exert various biological effects. In addition, H2 may control the immune system, antioxidant and anti-inflammatory activities (metabolism of mitochondrial energy), and cell death processes (apoptosis, autophagy, and pyroptosis) by reducing excessive reactive oxygen species generation and altering nuclear transcription factors. The fundamental mechanism of H2 is still not fully understood. Given its safety and possible usefulness, H2 has a promising future as a treatment for a variety of illnesses, including IBD. This review aimed to comprehensively highlight the current knowledge in the fields of H2 function in antioxidative, anti-inflammatory, and anti-apoptotic effects as well as its underlying mechanism, with a focus on IBD, and also offer recommendations for using H2 medically to treat IBD.

Effects of Molecular Hydrogen in the Pathophysiology
and Management of Metabolic and Non-communicable Diseases

Ram B. Singh, Alex Tarnava, Jan Fedacko, Gizal Fatima, Sunil Rupee und Zuzana Sumbalova
Halberg Hospital and Research Institute, Moradabad, India

Abstract
The sustainable development goals (SDG) of the UNO would be difficult to achieve without prevention of non-communicable diseases (NCDs). Western diet and lifestyle, which are major risk factors of NCDs, are known to cause oxidative stress and decrease in production of molecular hydrogen in the intestines which leads to a decline in endogenous antioxidant status in the body, leading to increase in systemic inflammation. There are gaps in the knowledge about the role molecular hydrogen plays, in the treatment of these diseases This review aims to discuss the role of hydrogen in the pathogenesis and prevention of NCDs. Molecular hydrogen (H2) has been studied extensively as a therapeutic gas, with an estimated 2000 publications to date, exploring its potential therapeutic use in 170 disease models across every organ in the mammalian body. Hydrogen therapy can be administered through several methods, such as H2 inhalation, dissolving H2 gas in water to make hydrogen-rich water (HRW) for oral consumption or topical application, or hydrogen-rich saline. The exact mechanism of action of molecular hydrogen is not known but it is in itself a potential antioxidant that can also inhibit hydroxyl and nitrosyl radicals in the cells and tissues. Hydrogen is known to cause a marked decline in oxidative stress, and inflammation that are crucial in the pathogenesis of NCDs. Hydrogen therapy has been found protective against NCDs, including, metabolic diseases, cardiovascular diseases (CVDs), neurodegenerative diseases, chronic kidney disease, cancer, and chronic lung diseases.

Consumption of Hydrogen-Treated Foods Provides
Nutritional and Health Benefits

Duried Alwazeer
Department of Nutrition and Dietetics, Faculty of Health Sciences, Iğdır University, 76000, Iğdır, Turkey
Research Center for Redox Applications in Foods (RCRAF), Iğdır University, 76000, Iğdır, Turkey
Application, and Research Center, Innovative Food Technologies Development, Iğdır University, 76000, Iğdır, Turkey

Abstract
Molecular hydrogen (H2) was proven to be a therapeutic agent for many diseases. In parallel with its biomedical benefits, H2 has been shown in recent reports to have many applications in horticultural and food industry fields. Many hydrogen-infused products including hydrogen-rich water (HRW), hydrogen-infused beverages, and hydrogen-incorporated atmosphere products have been commercialized or at least studied at the laboratory scale. The hydrogen-treated crops possess preservative properties of phytochemicals e.g. anthocyanins and flavonoids and longer shelf life. The consumption of hydrogen-treated crops and hydrogen-infused beverages can provide many health and nutritional benefits to the consumer. The present report discusses the recent knowledge on the use of molecular hydrogen in crop production and food processing as well as their potential benefits on the health and nutrition of the consumer.

Differential Effects of Carbohydrates on the Generation of Hydrogen and Methane in Lowand High-Methane-Producing Rats

Oleg S. Medvedev, Anastasiia Yu. Ivanova, Margarita A. Belousova, Stepan V. Toshchakov, Anastasia S. Krylova, Ivan V. Shirokov, Olga N. Obolenskaya, Tatiana A. Kuropatkina, Grigorii N. Bondarenko und Ilya B. Gartseev
Faculty of Medicine, Lomonosov Moscow State University, Moscow, 119991, Russia
Institute of Experimental Cardiology, National Medical Research Centre of Cardiology, Moscow, 121552, Russia

Abstract
The goal of the study was to check the hypothesis that low- and high-methane producers will react differently to the administration of non-digestible carbohydrates. It was discovered in our previous studies that Wistar rats from the Puschino nursery (SPF status) were low methane producers, whereas conventional rats from Stolbovaya nursery were high methane producers. Hydrogen and methane breath tests and the taxonomic content of the gut microbiota were evaluated in 25 rats from each nursery. Samples of the exhaled air were taken from awake rats using nose-only apparatus and analyzed by gas chromatography. The taxonomic content of the gut microbiota was evaluated in each rat by the 16S rRNA method. Lactulose, Guar Gum, and inulin were administered by gavage to each rat in 1-week time intervals. Levels of hydrogen and methane in the samples of exhaled air were measured during 8 h after carbohydrate administration. Taxonomic microbiome compositions were quite different between groups. Low methane-producing rats had low alpha and beta diversity, higher abundance of Christensenellaceae and Akkermansia bacteria, lower abundance of Helicobacteraceae, and absence of Methanobacteriaceae) that show similarity to the microbiome of the newborns and children. High methane-producing rats (from Stobovaya nursery) had a much higher diversity of microbiota bacteria, a higher abundance of hydrogen-consuming microorganisms, like Helicobacteraceae and Methanobacteriaceae, and close to the microbiota composition in the elderly. The gavage of carbohydrates in low-methane-producing rats was followed just by the increase in hydrogen level in exhaled air, whereas the same carbohydrates evoked an increase in methane level only. We speculate that the administration of the exogenous hydrogen (hydrogen-rich water) will be more efficient in increasing the antioxidant defense in the elderly because taking the food fibers is not followed by the increase of hydrogen level in the blood.

Natural Biomolecules, Plant Extracts and Molecular
Hydrogen—New Antioxidant Alternatives in the Management
of Male Infertility

Eva Tvrdá, Michal Duraˇ ˇ cka, and Eva Ivanišová
Faculty of Biotechnology and Food Sciences, Institute of Biotechnology, Slovak
University of Agriculture, Tr. A. Hlinku 2, 949 76, Nitra, Slovakia

Abstract
Oxidative stress resulting from an imbalance between the levels of reactive oxygen species (ROS) and antioxidants, plays a pivotal role in the pathophysiology of male sub- or infertility. Substantial evidence highlights the importance of oral antioxidant therapy in the management of male reproductive dysfunction. Nevertheless, studies focused on traditional antioxidant supplement have often come to inconclusive or contradictory results. In the meantime, scientific focus has shifted to alternative remedies such as plant extracts, plant-based biomolecules as well as molecular hydrogen which present with numerous health benefits and powerful antioxidant properties. As such, the focus of this chapter is to present recent evidence assessing the in vivo effects of alternative antioxidant remedies on the structural, functional, and oxidative indicators of male reproductive function with a special emphasis on rats as attractive animal models.

Comparison of Free-Radical Scavenging Activity of Various
Sources of Molecular Hydrogen

Katarína Valachová, Branislav Kura, Ján Slezák, Mojmír Mach und Ladislav Šoltés
Centre of Experimental Medicine, Slovak Academy of Sciences, Dúbravská Cesta 9, 84104, Bratislava, Slovakia

Abstract
Molecular hydrogen is well-known for its antioxidative and antiinflammatory properties in numerous in vitro and in vivo experiments. High-molar-mass hyaluronan (HMM HA), a natural polysaccharide present in tissues of all vertebrates, was used as a marker of the polysaccharide degradation by reactive oxygen species. The radical scavenging capacity of various sources of molecular hydrogen dissolved in water was assessed by the DPPH assay. In experiments using rotational viscometry, HMM HA was oxidatively degraded by cupric ions (1 µM) and ascorbic acid (100 µM). Further, the effects of molecular hydrogen from various sources were assessed before beginning •OH radical-induced HA degradation or one hour later, when alkyloxy- and alkylperoxy-type radical-induced HA degradation prevailed. The results of the DPPH assay showed that of the examined samples, only H2 tablet dissolved in both distilled and drinkable water mildly scavenged DPP•. In contrast, experiments from rotational viscometry showed that the inhibition of reactive oxygen species induced-hyaluronan degradation was observed when examining molecular hydrogen saturated in both distilled and drinkable water.

Development of a Preclinical Tool for Measuring Percutaneous
Transfer of Dihydrogen, with a View to Optimizing Medical
Devices Adapted to Focal Therapies in Dermatology

C. Salomez-Ihl, S. Tanguy, F. Boucher, V. Pascal Mousselard,
P. Bedouch, A. Stephanou, J. P. Alcaraz und P. Cinquin
University of Grenoble Alpes, CNRS, UMR 5525, VetAgro Sup, Grenoble INP, TIMC, 38000, Grenoble, France
Centre for Clinical Investigation Technological Innovation, INSERM CIC803, Grenoble University Hospital, 38000, Grenoble, France

Abstract
Numerous studies have demonstrated the efficacy of dihydrogen (H2) in dermatological pathologies, with no attributed adverse effects. However, there is no formal proof of percutaneous transfer of H2 through the skin. The aim of the present study is to demonstrate this transfer and to characterize the H2 diffusion coefficient in the skin. Rat abdominal skin is affixed to the center of a diffusion cell. In a first compartment, a gas mixture containing different proportions of H2 combined with air is injected. A second compartment contains either air or physiological medium + air. A H2 sensor is connected to the second compartment and records H2 concentration. A bi-exponential model of H2 concentration is then fitted. The mean value of the flow is 0.40 nmol.s−1.cm−2, and the mean value of the H2 diffusion coefficient is 4.2 10–5 cm2.s−1, which is very close to H2 diffusion coefficient in water (D = 4.58 10–5 cm2.s−1). Our model confirms the major interest of designing new approaches to deliver H2 for dermatology applications, because the local concentration of H2 in the target zones will be by several orders of magnitude higher than what can be achieved with oral administration.

Intraosseous Administration of Molecular Hydrogen: A Novel
Technique—From Molecular Effects to Tissue Regeneration

Mikhail Yu. Artamonov, Tyler W. LeBaron, Evgeniy L. Sokov, Lyudmila E. Kornilova, Felix A. Pyatakovich und Inessa A. Minenko
MJA Research and Development Inc., East Stroudsburg, PA, 18301, USA

Abstract
In recent decades, molecular hydrogen has been shown to have diverse biological effects. By the end of 2022, more than 2000 articles have been published in the field of hydrogen medicine, many of which are original studies. There is some preliminary evidence on the regenerative effect of molecular hydrogen when administered by inhalation or via drinking hydrogen-rich water. Here we propose a novel method of administering hydrogen via intraosseous route, which potentially enhances the tissue regenerative effect. The purpose of this review was to systematize ideas about the nature, characteristics, and mechanisms of the influence of molecular hydrogen on various types of cells, including stem cells, as well as to introduce the hypothesis of the potential advantages of intraosseous administration route. The molecular, cellular, tissue and systemic effects of hydrogen are also reviewed. The existing literature indicates that the molecular and cellular effects of hydrogen qualify it to be a potentially effective agent in regenerative medicine. Administration of molecular hydrogen via intraosseous route may serve as an optimal way to stimulate a bone marrow stem cell pool.

Perspective of Nanomaterials and Nanomedicine Procedures in Molecular Hydrogen Therapy

Štefan Luby
Institute of Physics, Slovak Academy of Sciences, Dubravska cesta 9, 84511, Bratislava, Slovakia

Abstract
In parallel with the development of nanoscience, nanomedicine and its branches were established. Its remarkable development is evidenced by milestones, awarded several times with Nobel prizes. Milestones also include the renaissance of molecular hydrogen therapy from 2007, which uses H2 as an effective antioxidant in preventive and therapeutic applications. In this chapter, in connection with the developed targeted delivery of drugs, we are dedicated to its modification in the form of local generation and release of hydrogen from nanocarriers such as nanoparticles, nanorods etc. The results are already reported in Alzheimer’s disease and cancer treatment, wound healing, etc. Hydrogen therapy requires support and service which includes safe solid state storage of hydrogen to avoid potential explosions, where, in addition to hydrides such as MgH2, functionalized nanomaterials from the carbon family—as graphene or carbon nanotubes are applied. Attention must also be paid to the leakage of hydrogen and its presence in the environment, where nanosensors based on metal oxides and especially graphene-based sensors are the promising solution. Considering the numerous clinical trials and studies, H2 therapy can today be situated at the rise part of the Gartner’s cycle heading for the production plateau.

The Emergence, Development, and Future Mission of Hydrogen Medicine and Biology

Shigeo Ohta
Department of Neurology Medicine, Juntendo University Graduate School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan
Institute for Advanced Medical Sciences, Nippon Medical University, 1-25-16 Nezu, Bunkyo-ku, Tokyo, 113-8602, Japan

Abstract
Molecular hydrogen (H2) has emerged as a promising therapeutic and preventive medical gas. In 2007, we overturned the conventional concept that H2 had no effect on mammalian cells, leading to the emergence of the field of “hydrogen medicine and biology”. H2 has numerous advantages, including high efficacy, lack of adverse effects, and multiple functions, such as anti-oxidation, anti-inflammation, anti-cell death, and energy metabolism stimulation. Moreover, H2 is beneficial from healthy individuals to severe chronic and acute diseases. The target molecule of H2 was identified as the oxidized form of porphyrins, subsequent hydride form acts as a catalyst to stimulate the selective reaction of H2 with hydroxyl radical. Various species of porphyrins are widely and abundantly distributed throughout the body. Thus, repairing the oxidized porphyrins by H2 may result in multiple benefits across various cells. H2 suppresses the free radical chain reaction and modifies signaling mediators involved in lipid peroxides. H2 indirectly regulates hormones and cytokines through various signal transduction pathways. H2 has the potential to address a wide range of issues, including cardiac arrest, Alzheimer’s-type dementia, metabolic syndrome, advanced-stage of cancer, inflammatory cytokine storms, healthcare, beauty, and agriculture. The mission of H2 medicine is to overcome these unsolved ailments.