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Safety & Health Benefits of Hydrogen Water – Nobel Prize Nominee, Dr. GARTH NICOLSON


Safety & Health Benefits of Hydrogen Water – Nobel Prize Nominee, Dr. GARTH NICOLSON

Molecular Hydrogen CANCER treatment

Gas signaling molecules (GSMs), composed of oxygen, carbon monoxide, nitric oxide, hydrogen sulfide, etc., play critical roles in regulating signal transduction and cellular homeostasis. Interestingly, through various administrations, these molecules also exhibit potential in cancer treatment. Recently, hydrogen gas (formula: H2) emerges as another GSM which possesses multiple bioactivities, including anti-inflammation, anti-reactive oxygen species, and anti-cancer. Growing evidence has shown that hydrogen gas can either alleviate the side effects caused by conventional chemotherapeutics, or suppress the growth of cancer cells and xenograft tumor, suggesting its broad potent application in clinical therapy. In the current review, we summarize these studies and discuss the underlying mechanisms. The application of hydrogen gas in cancer treatment is still in its nascent stage, further mechanistic study and the development of portable instruments are warranted.

Introduction

Gaseous signaling molecules (GSMs) refer to a group of gaseous molecules, such as oxygen (1), nitric oxide (2), carbon monoxide (3), hydrogen sulfide (4), sulfur dioxide (56), ethylene (78), etc. These gaseous molecules possess multiple critical functions in regulating cell biology in vivo via signal transduction (9). More importantly, certain GSMs could serve as therapeutic agents in primary cancer, as well as in multidrug-resistant cancer treatment when used by directly or certain pharmaceutical formulations (913). In addition, some of these GSMs can be generated in body via different bacteria or enzymes, such as nitric oxide, hydrogen sulfide, indicating that they are more compatible molecules that may exhibit less adverse effects compared with conventional chemotherapeutics (91415). Recently, hydrogen gas has been recognized as another important GSM in biology, exhibiting appealing potential in health care for its role in preventing cell injury from various attacking (1619).

With the formula of H2, hydrogen gas is the lightest molecule in the nature which only accounts for about 0.5 parts per million (ppm) of all the gas. Naturally, hydrogen gas is a colorless, odorless, tasteless, non-toxic, highly combustible gas which may form explosive mixtures with air in concentrations from 4 to 74% that can be triggered by spark, heat, or sunlight. Hydrogen gas can be generated in small amount by hydrogenase of certain members of the human gastrointestinal tract microbiota from unabsorbed carbohydrates in the intestine through degradation and metabolism (2021), which then is partially diffused into blood flow and released and detected in exhaled breath (20), indicating its potential to serve as a biomarker.

As the lightest molecule in natural, hydrogen gas exhibits appealing penetration property, as it can rapidly diffuse through cell membranes (2223). Study in animal model showed that, after orally administration of hydrogen super-rich water (HSRW) and intra-peritoneal administration of hydrogen super-rich saline (HSRS), the hydrogen concentration reached the peak at 5 min; while it took 1 min by intravenous administration of HSRS (23). Another in vivo study tested the distribution of hydrogen in brain, liver, kidney, mesentery fat, and thigh muscle in rat upon inhalation of 3% hydrogen gas (24). The concentration order of hydrogen gas, when reached saturated status, was liver, brain, mesentery, muscle, kidney, indicating various distributions among organs in rats. Except the thigh muscle required a longer time to saturate, the other organs need 5–10 min to reach Cmax (maximum hydrogen concentration). Meanwhile, the liver had the highest Cmax (24). The information may direct the future clinical application of hydrogen gas.

Although hydrogen gas was studied as a therapy in a skin squamous carcinoma mouse model back in 1975 (25), its potential in medical application has not been vastly explored until 2007, when Oshawa et al. reported that hydrogen could ameliorate cerebral ischemia-reperfusion injury by selectively reducing cytotoxic reactive oxygen species (ROS), including hydroxyl radical (•OH) and peroxynitrite (ONOO-) (26), which then provoked a worldwide attention. Upon various administrative formulations, hydrogen gas has been served as a therapeutic agent for a variety of illnesses, such as Parkinson’s disease (2728), rheumatoid arthritis (29), brain injury (30), ischemic reperfusion injury (3132), and diabetes (3334), etc.

More importantly, hydrogen has been shown to improve clinical end-points and surrogate markers, from metabolic diseases to chronic systemic inflammatory disorders to cancer (17). A clinical study in 2016 showed that inhalation of hydrogen gas was safe in patients with post-cardiac arrest syndrome (35), its further therapeutic application in other diseases became even more appealing.

In the current review, we take a spot on its application in cancer treatment. Typically, hydrogen gas may exert its bio-functions via regulating ROS, inflammation and apoptosis events.

Hydrogen Gas Selectively Scavenges Hydroxyl Radical and Peroxynitrite, and Regulates Certain Antioxidant Enzymes

By far, many studies have indicated that hydrogen gas doesn’t target specific proteins, but regulate several key players in cancer, including ROS, and certain antioxidant enzymes (36).

ROS refers to a series of unstable molecules that contain oxygen, including singlet oxygen (O2•), hydrogen peroxide (H2O2), hydroxyl radical (•OH), superoxide (O2•O2-), nitric oxide (NO•), and peroxynitrite (ONOO), etc. (3738). Once generated in vivo, due to their high reactivity, ROS may attack proteins, DNA/RNA and lipids in cells, eliciting distinct damage that may lead to apoptosis. The presence of ROS can produce cellular stress and damage that may produce cell death, via a mechanism known as oxidative stress (3940). Normally, under physical condition, cells including cancer cells maintain a balance between generation and elimination of ROS, which is of paramount importance for their survival (4142). The over-produced ROS, resulted from imbalance regulatory system or outer chemical attack (including chemotherapy/radiotherapy), may initiate inner apoptosis cascade, causing severely toxic effects (4345).

Hydrogen gas may act as a ROS modulator. First, as shown in Ohsawa et al.’s study, hydrogen gas could selectively scavenge the most cytotoxic ROS, •OH, as tested in an acute rat model of cerebral ischemia and reperfusion (26). Another study also confirmed that hydrogen gas might reduce the oxygen toxicity resulted from hyperbaric oxygen via effectively reducing •OH (46).

Second, hydrogen may induce the expression of some antioxidant enzymes that can eliminate ROS, and it plays key roles in regulating redox homeostasis of cancer cells (4247). Studies have indicated that upon hydrogen gas treatment, the expression of superoxide dismutase (SOD) (48), heme oxyganase-1 (HO-1) (49), as well as nuclear factor erythroid 2-related factor 2 (Nrf2) (50), increased significantly, strengthening its potential in eliminating ROS.

By regulating ROS, hydrogen gas may act as an adjuvant regimen to reduce the adverse effects in cancer treatment while at the same time doesn’t abrogate the cytotoxicity of other therapy, such as radiotherapy and chemotherapy (4851). Interestingly, due the over-produced ROS in cancer cells (38), the administration of hydrogen gas may lower the ROS level at the beginning, but it provokes much more ROS production as a result of compensation effect, leading to the killing of cancer cells (52).

Hydrogen Gas Suppresses Inflammatory Cytokines

Inflammatory cytokines are a series of signal molecules that mediate the innate immune response, whose dys-regulation may contribute in many diseases, including cancer (5355). Typical inflammatory cytokines include interleukins (ILs) excreted by white blood cells, tumor necrosis factors (TNFs) excreted by macrophages, both of which have shown close linkage to cancer initiation and progression (5659), and both of ILs and TNFs can be suppressed by hydrogen gas (6061).

Inflammation induced by chemotherapy in cancer patients not only causes severely adverse effects (6263), but also leads to cancer metastasis, and treatment failure (6465). By regulating inflammation, hydrogen gas can prevent tumor formation, progression, as well as reduce the side effects caused by chemotherapy/radiotherapy (66).

Hydrogen Gas Inhibits/Induces Apoptosis

Apoptosis, also termed as programed cell death, can be triggered by extrinsic or intrinsic signals and executed by different molecular pathways, which serve as one efficient strategy for cancer treatment (6768). Generally, apoptosis can be induced by (1) provoking the death receptors of cell surface (such as Fas, TNF receptors, or TNF-related apoptosis-inducing ligand), (2) suppressing the survival signaling (such as epidermal growth factor receptor, mitogen-activated protein kinase, or phosphoinositide 3-kinases), and (3) activating the pro-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins, or down-regulating anti-apoptosis proteins (such as X-linked inhibitor of apoptosis protein, surviving, and the inhibitor of apoptosis) (6970).

Hydrogen gas can regulate intracellular apoptosis by impacting the expression of apoptosis-related enzymes. At certain concentration, it can either serve as apoptosis-inhibiting agent via inhibiting the pro-apoptotic B-cell lymphoma-2-associated X protein (Bax), caspase-3, 8, 12, and enhancing the anti-apoptotic B-cell lymphoma-2 (Bcl-2) (71), or as apoptosis-inducing agent via the contrast mechanisms (72), suggesting its potential in protecting normal cells from anti-cancer drugs or in suppressing cancer cells.

Hydrogen Gas Exhibits Potential in Cancer Treatment

Hydrogen Gas Relieves the Adverse Effects Related to Chemotherapy/Radiotherapy

Chemotherapy and radiotherapy remain the leading strategies to treat cancer (7374). However, cancer patients receiving these treatments often experience fatigue and impaired quality of life (7577). The skyrocketed generation of ROS during the treatment is believed to contribute in the adverse effects, resulting in remarkable oxidative stress, and inflammation (414278). Therefore, benefited from its anti-oxidant and anti-inflammatory and other cell protective properties, hydrogen gas can be adopted as an adjuvant therapeutic regimen to suppress these adverse effects.

Under treatment of epidermal growth factor receptor inhibitor gefitinib, patients with non-small cell lung cancer often suffer with severe acute interstitial pneumonia (79). In a mice model treated with oral administration of gefitinib and intraperitoneal injection of naphthalene which induced severely lung injury due to oxidative stress, hydrogen-rich water treatment significantly reduced the inflammatory cytokines, such as IL-6 and TNFα in the bronchoalveolar lavage fluid, leading to a relieve of lung inflammation. More importantly, hydrogen-rich water didn’t impair the overall anti-tumor effects of gefitinib both in vitro and in vivo, while in contrast, it antagonized the weight loss induced by gefitinib and naphthalene, and enhanced the overall survival rate, suggesting hydrogen gas to be a promising adjuvant agent that has potential to be applied in clinical practice to improve quality of life of cancer patients (80).

Doxorubicin, an anthracycline antibiotic, is an effective anticancer agent in the treatment of various cancers, but its application is limited for the fatal dilated cardiomyopathy and hepatotoxicity (8182). One in vivo study showed that intraperitoneal injection of hydrogen-rich saline ameliorated the mortality, and cardiac dysfunction caused by doxorubicin. This treatment also attenuated histopathological changes in serum of rats, such as the serum brain natriuretic peptide (BNP), aspartate transaminase (AST), alanine transaminase (ALT), albumin and malondialdehyde (MDA) levels. Mechanistically, hydrogen-rich saline significantly lowered the ROS level, as well as inflammatory cytokines TNF-α, IL-1β, and IL-6 in cardiac and hepatic tissue. Hydrogen-rich saline also induced less expression of apoptotic Bax, cleaved caspase-3, and higher anti-apoptotic Bcl-2, resulting in less apoptosis in both tissues (71). This study suggested that hydrogen-rich saline treatment exerted its protective effects via inhibiting the inflammatory TNF-α/IL-6 pathway, increasing the cleaved C8 expression and Bcl-2/Bax ratio, and attenuating cell apoptosis in both heart and liver tissue (71).

Hydrogen-rich water also showed renal protective effect against cisplatin-induced nephrotoxicity in rats. In the studies, blood oxygenation level-dependent (BOLD) contrast magnetic resonance images (MRI) acquired in different treated group showed that the creatinine and blood urea nitrogen (BUN) levels, two parameters that related to nephrotoxicity, were significantly higher in cisplatin treated group than those in the control group. Hydrogen-rich water treatment could significantly reverse the toxic effects, and it showed much higher transverse relaxation rate by eliminating oxygen radicals (8384).

Another study showed that both inhaling hydrogen gas (1% hydrogen in air) and drinking hydrogen-rich water (0.8 mM hydrogen in water) could reverse the mortality, and body-weight loss caused by cisplatin via its anti-oxidant property. Both treatments improved the metamorphosis, accompanied with decreased apoptosis in the kidney, and nephrotoxicity as assessed by serum creatinine and BUN levels. More importantly, hydrogen didn’t impair the anti-tumor activity of cisplatin against cancer cell lines in vitro and in tumor-bearing mice (85). Similar results were also observed in Meng et al.’s study, as they showed that hydrogen-rich saline could attenuate the follicle-stimulating hormone release, elevate the level of estrogen, improve the development of follicles, and reduce the damage to the ovarian cortex induced by cisplatin. In the study, cisplatin treatment induced higher level of oxidation products, suppressed the antioxidant enzyme activity. The hydrogen-rich saline administration could reverse these toxic effects by reducing MDA and restoring the activity of superoxide dismutase (SOD), catalase (CAT), two important anti-oxidant enzymes. Furthermore, hydrogen-rich saline stimulated the Nrf2 pathway in rats with ovarian damage (86).

The mFOLFOX6 regimen, composed with folinic acid, 5-fluorouracil, and oxaliplatin, is used as first-line treatment for metastatic colorectal cancer, but it also confers toxic effects to liver, leading to bad quality of life of patient (8788). A clinical study was conducted in China by investing the protective effect of hydrogen-rich water on hepatic function of colorectal cancer patients (144 patients were enrolled and 136 of them were include in the final analysis) treated with mFOLFOX6 chemotherapy. The results showed that the placebo group exhibited damaging effects caused by mFOLFOX6 chemotherapy as measured by the elevated levels of ALT, AST and indirect bilirubin (IBIL), while the hydrogen-rich water combinational treatment group exhibited no differences in liver function during the treatment, probably due to its antioxidant activity, indicating it a promising protective agent to alleviate the mFOLFOX6-related liver injury (51).

Most of the ionizing radiation-induced adverse effects to normal cells are induced by hydroxyl radicals. The combination of radiotherapy with certain forms of hydrogen gas may be beneficial to alleviate these side effects (89). Indeed, several studies found that hydrogen could protect cells and mice from radiation (4890).

As tested in a rat model of skin damage established by using a 44 Gy electronic beam, the treated group by hydrogen-rich water exhibited higher lever of SOD activity and lower MDA and IL-6 in the wounded tissues than the control group and the distilled water group. Furthermore, hydrogen-rich water shortened the healing time and increased the healing rate of skin injury (48).

Gastrointestinal toxicity is a common side effect induced by radiotherapy, which impairs the life quality of cancer patients (91). As shown in Xiao et al.’s study in mice model, hydrogen-water administration via oral gavage increased the survival rate and body weight of mice which were exposed to total abdominal irradiation, accompanied with an improvement in gastrointestinal tract function and the epithelial integrity of the small intestine. Further microarray analysis revealed that hydrogen-water treatment up-regulated miR-1968-5p, which then up-regulated its target myeloid differentiation primary response gene 88 (MyD88, a mediator in immunopathology, and gut microbiota dynamics of certain intestinal diseases involving toll-like receptors 9) expression in the small intestine after total abdominal irradiation (92).

Another study conducted in clinical patients with malignant liver tumors showed that the consumption of hydrogen-rich water for 6 weeks reduced the level of reactive oxygen metabolite, hydroperoxide, and maintained the biologic antioxidant activity in the blood. Importantly, scores of quality of life during radiotherapy were significantly improved in hydrogen-rich water group compared to the placebo water group. Both groups exhibited similar tumor response to radiotherapy, indicating that the consumption of hydrogen-rich water reduced the radiation-induced oxidative stress while at the same time didn’t compromise anti-tumor effect of radiotherapy (93).

Hydrogen Gas Acts Synergistically With Thermal Therapy

Recently, one study found that hydrogen might enhance the effect of photothermal therapy. Zhao et al. designed the hydrogenated Pd nanocrystals (named as PdH0.2) as multifunctional hydrogen carrier to allow the tumor-targeted delivery (due to 30 nm cubic Pd nanocrystal) and controlled release of bio-reductive hydrogen (due to the hydrogen incorporated into the lattice of Pd). As shown in this study, hydrogen release could be adjusted by the power and duration of near-infrared (NIR) irradiation. Treatment of PdH0.2 nanocrystals plus NIR irradiation lead to higher initial ROS loss in cancer cells, and the subsequent ROS rebound was also much higher than that in normal cells, resulting in more apoptosis, and severely mitochondrial metabolism inhibition in cancer cells but not in normal cells. The combination of PdH0.2 nanocrystals with NIR irradiation significantly enhanced the anticancer efficacies of thermal therapy, achieving a synergetic anticancer effect. In vivo safety evaluation showed that the injection dose of 10 mg kg−1 PdH0.2 nanocrystals caused no death, no changes of several blood indicators, and no affected functions of liver and kidney. In 4T1 murine breast cancer tumor model and B16-F10 melanoma tumor model, the combined PdH0.2 nanocrystals and NIR irradiation therapy exhibited a synergetic anticancer effect, leading to remarkable tumor inhibition when compared with thermal therapy. Meanwhile, the combination group showed no visible damage to heart, liver, spleen, lung, and kidney, indicating suitable tissue safety and compatibility (52).

Hydrogen Gas Suppresses Tumor Formation

Li et al. reported that the consumption of hydrogen-rich water alleviated renal injury caused by Ferric nitrilotriacetate (Fe-NTA) in rats, evidenced by decreased levels of serum creatinine and BUN. Hydrogen-rich water suppressed the Fe-NTA-induced oxidative stress by lowering lipid peroxidation, ONOO, and inhibiting the activities of NADPH oxidase and xanthine oxidase, as well as by up-regulating antioxidant catalase, and restoring mitochondrial function in kidneys. Consequently, Fe-NTA-induced inflammatory cytokines, such as NF-κB, IL-6, and monocyte chemoattractant protein-1 were significantly alleviated by hydrogen treatment. More importantly, hydrogen-rich water consumption inhibited several cancer-related proteins expression, including vascular endothelial growth factor (VEGF), signal transducer and activator of transcription 3 (STAT3) phosphorylation, and proliferating cell nuclear antigen (PCNA) in rats, resulting in lower incidence of renal cell carcinoma and the suppression of tumor growth. This work suggested that hydrogen-rich water was a promising regimen to attenuate Fe-NTA-induced renal injury and suppress early tumor events (66).

Non-alcoholic steatohepatitis (NASH) due to oxidative stress induced by various stimuli, is one of the reasons that cause hepatocarcinogenesis (9495). In a mouse model, hydrogen-rich water administration lowered the hepatic cholesterol, peroxisome proliferator-activated receptor-α (PPARα) expression, and increased the anti-oxidative effects in the liver when compared with control and pioglitazone treated group (96). Hydrogen-rich water exhibited strong inhibitory effects to inflammatory cytokines TNF-α and IL-6, oxidative stress and apoptosis biomarker. As shown in NASH-related hepatocarcinogenesis model, in the group of hydrogen-rich water treatment, tumor incidence was lower and the tumor volumes were smaller than control and pioglitazone treated group. The above findings indicated that hydrogen-rich water had potential in liver protection and liver cancer treatment (96).

Hydrogen Gas Suppresses Tumor Growth

Not only working as an adjuvant therapy, hydrogen gas can also suppress tumor and tumor cells growth.

As shown in Wang et al.’s study, on lung cancer cell lines A549 and H1975 cells, hydrogen gas inhibited the cell proliferation, migration, and invasion, and induced remarkable apoptosis as tested by CCK-8, wound healing, transwell assays and flow cytometry. Hydrogen gas arrested the cell cycle at G2/M stage on both cell lines via inhibiting the expression of several cell cycle regulating proteins, including Cyclin D1, CDK4, and CDK6. Chromosomes 3 (SMC3), a complex required for chromosome cohesion during the cell cycle (97), was suppressed by hydrogen gas via ubiquitinating effects. Importantly, in vivo study showed that under hydrogen gas treatment, tumor growth was significantly inhibited, as well as the expression of Ki-67, VEGF and SMC3. These data suggested that hydrogen gas could serve as a new method for the treatment of lung cancer (98).

Due to its physicochemical characteristics, the use of hydrogen gas has been strictly limited in hospital and medical facilities and laboratories. Li et al. designed a solidified hydrogen-occluding-silica (H2-silica) that could stably release molecular hydrogen into cell culture medium. H2-silica could concentration-dependently inhibit the cell viability of human esophageal squamous cell carcinoma (KYSE-70) cells, while it need higher dose to suppress normal human esophageal epithelial cells (HEEpiCs), indicating its selective profile. This effect was further confirmed by apoptosis and cell migration assay in these two cell lines. Mechanistic study revealed that H2-silica exerted its anticancer via inducing H2O2 accumulation, cell cycle arrest, and apoptosis induction mediated by mitochondrial apoptotic pathways (72).

Recently, hydrogen gas was found to inhibit cancer stem cells (CSCs). Hydrogen gas reduced the colony formation and sphere formation of human ovarian cancer cell lines Hs38.T and PA-1 cells via inhibiting the proliferation marker Ki67, stem cell markers CD34, and angiogenesis. Hydrogen gas treatment significantly inhibited the proliferation, invasion, migration of both Hs38.T and PA-1 cells. More important, inhalation of hydrogen gas inhibited the tumor volume significantly as shown in the Hs38.T xenografted BALB/c nude mice model (99).

Another recent study also confirmed the effects of hydrogen gas in suppressing glioblastoma (GBM), the most common malignant brain tumor. In vitro study indicated that hydrogen gas inhibited several markers involved in stemness, resulting in the suppression of sphere formation, cell migration, invasion, and colony formation of glioma cells. By inhaling hydrogen gas (67%) 1 h, 2 times per day, the GBM growth was significantly inhibited, and the survival rate was improved in a rat orthotopic glioma model, suggesting that hydrogen might be a promising agent in the treatment of GBM (100).

Discussion

Hydrogen gas has been recognized as one medical gas that has potential in the treatment of cardiovascular disease, inflammatory disease, neurodegenerative disorders, and cancer (1760). As a hydroxyl radical and peroxynitrite scavenger, and due to its anti-inflammatory effects, hydrogen gas may work to prevent/relieve the adverse effects caused by chemotherapy and radiotherapy without compromise their anti-cancer potential (as summarized in Table 1 and Figure 1). Hydrogen gas may also work alone or synergistically with other therapy to suppress tumor growth via inducing apoptosis, inhibiting CSCs-related and cell cycle-related factors, etc. (summarized in Table 1).

TABLE 1

www.frontiersin.orgTable 1. The Summary of various formulation, application, mechanisms of H2 in cancer treatment.

FIGURE 1

www.frontiersin.orgFigure 1. Hydrogen in cancer treatment.

More importantly, in most of the research, hydrogen gas has demonstrated safety profile and certain selectivity property to cancer cells over normal cells, which is quite pivotal to clinical trials. One clinical trials (NCT03818347) is now undergoing to study the hydrogen gas in cancer rehabilitation in China.

By far, several delivery methods have proved to be available and convenient, including inhalation, drinking hydrogen-dissolved water, injection with hydrogen-saturated saline and taking a hydrogen bath (101). Hydrogen-rich water is non-toxic, inexpensive, easily administered, and can readily diffuse into tissues and cells (102), cross the blood-brain barrier (103), suggesting its potential in the treatment of brain tumor. Further portable devices that are well-designed and safe enough will be needed.

However, regarding its medicinal properties, such as dosage and administration, or possible adverse reactions and use in specific populations, less information is available. Its mechanism, target, indications are also not clear, further study are warranted.

NOTE:

Molecular hydrogen-rich water generally shows a more prominent effect than molecular hydrogen gas, although the amount of hydrogen taken up by hydrogen water is ~100 times less than that given by hydrogen gas [11].

We have showed that drinking molecular hydrogen water, but not continuous molecular hydrogen gas exposure, prevented development of 6-hydorxydopamine-induced Parkinson’s disease in rats [11].

https://water-ionizers.info/en/2017/09/05/modalities-of-molecular-hydrogen-administrationin-water-gas-or-saline-to-animals-humans-and-plants/

 

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REVIEW ARTICLE

Front. Oncol., 06 August 2019 | https://doi.org/10.3389/fonc.2019.00696

Hydrogen Gas in Cancer Treatment
Sai Li1Rongrong Liao2Xiaoyan Sheng2Xiaojun Luo3Xin Zhang1Xiaomin Wen3Jin Zhou2* and Kang Peng1,3*
  • 1Department of Pharmacy, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China
  • 2Nursing Department, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China
  • 3The Centre of Preventive Treatment of Disease, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China

Author Contributions

SL, XW, JZ, and KP: conceptualization. SL, RL, XS, XL, XZ, JZ, and KP: writing. SL, RL, and XS: revising.

Funding

This work was supported in part by grants from the Natural Science Foundation of Guangdong Province (2018A030313987) and Traditional Chinese Medicine Bureau of Guangdong Province (20164015 and 20183009) and Science and Technology Planning Project of Guangdong Province (2016ZC0059).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank Miss Ryma Iftikhar, Dhiviya Samuel, Mahnoor Shamsi (St. John’s University), and Mr. Muaz Sadeia for editing and revising the manuscript.

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Citation: Li S, Liao R, Sheng X, Luo X, Zhang X, Wen X, Zhou J and Peng K (2019) Hydrogen Gas in Cancer Treatment. Front. Oncol. 9:696. doi: 10.3389/fonc.2019.00696

Received: 02 May 2019; Accepted: 15 July 2019;
Published: 06 August 2019.

Edited by:

Nelson Shu-Sang Yee, Penn State Milton S. Hershey Medical Center, United States

Reviewed by:

Leo E. Otterbein, Beth Israelv Deaconess Medical Center and Harvard Medical School, United States
Paolo Armando Gagliardi, University of Bern, Switzerland

Copyright © 2019 Li, Liao, Sheng, Luo, Zhang, Wen, Zhou and Peng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Jin Zhou, zhou-jin-2008@163.com; Kang Peng, kds978@163.com

These authors share co-first authorship

AlkaViva EmcoTech/Jupiter alkaline ionized water as cancer treatment -clinical case , integrative oncology

AlkaViva EmcoTech/Jupiter alkaline ionized water as cancer treatment -clinical case , integrative oncology

Abstract

The present article describes the ongoing (partial) remission of a female patient (41 years old) from estrogen receptor (ER)-positive/progesterone receptor (PR)-negative metastatic breast cancer in response to a combination treatment directed towards the revitalization of the mitochondrial respiratory chain (oxidative phosphorylation), the suppression of NF-kappaB as a factor triggering the inflammatory response, and chemotherapy with capecitabine. The reduction of tumor mass was evidenced by a continuing decline of CA15-3 and CEA tumor marker serum levels and 18FDG-PET-CT plus magnetic resonance (MR) imaging. It is concluded that such combination treatment might be a useful option for treating already formed metastases and for providing protection against the formation of metastases in ER positive breast cancer. The findings need to be corroborated by clinical trials. Whether similar results can be expected for other malignant tumor phenotypes relying on glycolysis as the main energy source remains to be elucidated.

1. Introduction

Since Richard Nixon declared war on cancer about 30 years ago, much efforts have been made in order to overcome this dreadful disease. Enormous financial resources have been invested in cancer research in the last three decades, yet most metastasized solid malignant tumors are still considered incurable. Chemotherapy has been shown to be a potent (long lasting) treatment option against only a few solid cancers including testis cancer. The overall contribution of curative and adjuvant cytotoxic chemotherapy was assessed to be 2.3% in Australia and 2.1% in the United States of America with a five-year survival in adults based on data for 1998 []. Under chemotherapy, cancer cells can gradually develop drug resistance that is acquired, for instance, by overexpression of transporter proteins (e.g., those belonging to the ATP-binding cassette type) [,] and fractionation of the cancerous stem cells [] (which are less sensitive to exposure to cytostatics than more differentiated cancer cells), plus AKT [,] and NF-kappaB [,] overexpression as a compensatory response to administered cytotoxic drugs. Likewise, induced hypoxia may act as protective shield against tumor eradication by chemotherapeutics and radiation due to alterations of gene expression profiles related to hypoxia, which result in the inhibition of apoptosis [].

On the other hand, a plethora of “alternative” cancer therapies have been developed and applied in the past. Here, we report on a combination treatment, including chemotherapy, bisphosphonates, and complementary measures, aiming at the normalization of the cellular metabolism, vascular angiogenesis, cell life cycle, and cell proliferation activity.

2. Experimental

2.1. Chemicals/Dietary Supplements

Super Ubiquinol CoQ10, Life Extension, article nr. 01426, USA: www.lefeurope.com

Vitamin B2, tablets, 10 mg, Jenapharm®, Mibe GmbH, Germany

Vitamin B3, capsules, 54 mg, Allpharm, Germany, PZN 6605862

5-Loxin®capsules, 75 mg, (std. for acetyl-11-keto-β-boswellic acid (AKBA), minimum 30% on dry basis), Life Extension, article nr. 00939, USA, www.lefeurope.com

Linseed oil, Linosan Leinöl, Heirler Cenovis GmbH, D-78303 Radolfzell, Germany

Bio-Kefir, Andechser Natur, 1,5% fat, containing L(+) dextrorotatory lactic acid, Andechser Molkerei Scheitz GmbH, D-82346 Andechs, Germany, www.andechser-molkerei.de

Bio-Yoghurt, Andechser Natur, 0,1% fat, containing L. acidophilus and B. bifidus, Andechser Molkerei Scheitz GmbH, D-82346 Andechs, Germany, www.andechser-molkerei.de

Flaxseed, freshly ground

EPA/DHA: Mega EPA/DHA, capsules, Life Extension, article nr. 00625

Sodium selenite, Selenase®200 XXL, 200 μg selenium, biosyn Arzneimittel GmbH, D-70734 Fellbach, Germany

L-Carnitine: Multinorm® L-Carnitin aktiv, 250 mg L-carnitin plus 3 μg Vitamin B12, Sankt Pirmin® Naturprodukte GmbH, D-55218 Ingelheim, Germany

L-Carnitine, 300 mg capsules: Altapharma, Germany

Zinc, Unizink® 50, 50 mg zinc-bis(hydrogen-DL-aspartat), Kohler Pharma GmbH, D-64665 Alsbach-Hähnlein, Germany, PZN-3441621

Ibandronat Bondronat®, 6 mg/6 mL concentrate, Roche Pharma AG, D-79639 Grenzach-Wyhlen, Germany

Capecitabine, Xeloda®, Roche Pharma AG, D-79639 Grenzach-Wyhlen, Germany

Drinking water ion exchanger and filter, pHresh, EMCO TECH Co. Ltd., Korea

Vitamin D and vitamin A were sporadically taken.

2.2. Procedure

The mentioned chemicals/dietary supplements have been taken as follows:

Alkalized drinking water was prepared ad lib by using water ion exchanger and filter. The filtered water was boiled prior to use.

Capecitabine was taken orally at 3.65 g Xeloda®/70 kg body weight per day. Two weeks of treatment were followed by one week of therapy pause per cycle.

“Budwig diet”: the following items were mixed for preparing a full batch using a blender: 1 kg Bio-Yoghurt, 0.1% fat, 0.25 kg Bio-Kefir, 1.5% fat, 6 table spoons of linseed oil, 4 table spoons of linseed, to be freshly milled: A part of this full batch may be prepared daily (the daily dose per person was about 250 grams).

Taken together around noon: 400 mg of Ubiquinol CoQ10 (4 capsules à 100 mg), 10 mg vitamin B2 (Riboflavin), 50 mg vitamin B3 (Niacin)

Taken three times daily: 2 softgels of MEGA EPA/DHA (eicosapentaenoic acid/docosahexaenoic acid), including 720 mg of EPA and 480 mg of DHA per 2 capsules.

One capsule of 5-Loxin®, one dose of Multinorm® L-Carnitin aktiv (taken only during chemotherapy pause; during the chemotherapy 300 mg pure L-carnitine not containing vitamin B12 was ingested), one tablet of Unizink® 50, and one tablet of Selenase®200 XXL were taken daily. EPA/DHA are COX-2 inhibitors. Therefore, the heart and vascular functions should be checked by a physician on a regular basis (it has been found that members of synthetic COX-2 inhibitors have been found to increase thrombosis, stroke, and heart attack risk under certain conditions). Moreover, Q10/B2/B3 were not taken in combination with radiation (the antioxidant Q10 potentially quenches the oxidative damage caused by radiation). EPA and DHA have potentially blood thinning effect.

3. Results

3.1. Applied Methodology and Methods

It has been hypothesized by the author that a multi-factorial approach towards breast cancer treatment would result in a synergetic response and reduced likelihood of development of resistance to treatment. Accordingly, it was sought to combine complementary, non-antagonistic treatments, which have the theoretical potential to suppress tumorigenesis and proliferation, with a “conventional” treatment. The envisaged therapy modules were Budwig diet and normalization of the fatty acid dietary balance, alkaline therapy, suppression of the inflammatory signaling chain, revitalization of the mitochondrial respiratory chain, bone protection against osteoclast-effected resorption by bisphosphonates and AKBA, and finally chemotherapy in the form of the prodrug capecitabine as 5-fluorouracil precursor []. The latter has been the recommended treatment by the medical tumor board in charge.

The described efforts have concretely been undertaken for suppressing refractory breast cancer stage IV in a female patient (body mass index 24–26, 41 years old), having developed a ductal carcinoma in situ in 2007. After biopsy revealed an estrogen receptor positive and progesterone receptor negative breast cancer, followed by surgical resection of the invaded sentinel lymph nodes, a neoadjuvant chemotherapy (four cycles Epirubicin/Cyclophosphamide, followed by four cycles of Taxotere®) was applied. However, the tumor showed little response (the tumor regression grade according to Sinn was only 1). Thus, the first and second axillary lymph node levels were resected in the following, and the affected breast was ablated. No suspicious tumor marker levels have been observed after ablation. The resection area was furthermore treated with radiation (gamma rays). The post-operational therapy included firstly tamoxifen, clodronate (a bisphosphonate), and a GNRH analogue (Enantone-Gyn®).

However, in September 2008, the patient – alerted by pain in the spinal cord – underwent MRI imaging, which revealed multiple bone metastases, including in the spinal cord.

As a consequence, the medication was altered as follows by the medical board in charge: Letrozol (aromatase inhibitor, 2.5 mg/d) and Ibandronat (6 mg intravenous infusion per month) as bisphosphonate. However, the disease progressed and a staging (18FDG-PET-CT and MRI) in March 2009 revealed the formation of various liver metastases. Therefore, the medication was changed to capecitabine chemotherapy instead of anti-hormonal therapy, accompanied by continuation of administration of Ibandronat.

Together with this therapy change, the author recommended the complimentary ingestion of the following substances: “Budwig diet” (linseed oil, flaxseed, and yoghurt), EPA/DHA concentrate in the form of distilled fish oil, ubiquinol (Q10 in reduced form), and vitamins B2 and B3, later on also 5-Loxin®(AKBA). See above for further dosage and substance specifications.

3.2. Results

After about three months (June 2009) of continued intake of the above mentioned substances (besides 5-Loxin®), PET-CT showed no metabolic activity of the liver metastases any longer and reduced activity of the bone metastases under 18F-deoxyglucose as tracer in the PET. Concurrently, a decline of the tumor markers’ (CA 15-3 and CEA) serum concentration was observed.

At this time, as a further element, 5-Loxin® (AKBA) was introduced into the supplementation scheme for the reasons mentioned.

Nine months later, the MRI showed that three out of six initial liver metastases could no longer be imaged, and that the largest lesion had decreased from about 15 mm to about 7 mm. A further small liver metastasis remained unchanged in size. This situation is depicted in Figure 1. Again, no metabolic activity in 18FDG-PET-CT was detected for any of the liver metastases.

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Diffusion-weighted MRI of the liver showing two metastases in the right lobe in (a) June 2009, and (b) February 2010. One metastasis (arrow) decreased from 15 mm in diameter to 7 mm, while the other remained unchanged (courtesy of Prof. Dr. E. Rummeny, Klinikum Rechts der Isar, Technische Universität München, Technical University of Munich, Germany).

Moreover, the PET-CT (18F-deoxyglucose as PET tracer) showed, in addition, a reduction of the size and metabolic activity of bone metastases, accompanied by re-calcification of the lesions. The response to treatment correlated with markedly decreased tumor marker serum levels, with CEA concentration being close to the significance threshold of 4 ng/mL. The development of the tumor marker levels over time is displayed in Table 1 below. The decline of tumor marker concentrations has been found to correlate with cancer remission in clinical studies on breast cancer patients [,]. In addition, the initial concentration of CEA has been associated with the clinical disease outcome in breast cancer patients.

Table 1.

Development of the CEA and CA 15-3 serum concentrations over time; cut-off values were 4 ng/mL for CEA and 27 U/mL for CA15-3.

Date/Months after Therapy Start CA 15-3 (U/mL) Excess over Cut-off Value [%] CEA (ng/mL) Excess over Cut-off Value [%]
29 June 2009/3 49.3 82.6 31.4 684
13 September 2009/7 46.2 71.1 8.4 110
11 January 2010/10 37 37.0 4.1 2.5
19 April 2010/13 38.3 41.9 3.6 -10.8
12 July 2010/16 35.7 32.3 4.1 1.5

The latest 18FDG-PET-CT of August 2010 showed ongoing sclerosis of at least some of the bone lesions and stable disease.

4. Discussion and Conclusions

A plethora of complementary cancer treatments have been reported. Firstly, the intake of polysaccharides and proteoglucans, such as mushroom and yeast glucans [  ,  ], mistletoe lectins [,] and nerium oleander extracts, the latter also in combination with sutherlandia frutescens extracts [,], have been described. The activation of the immune system against cancer cells has been ascribed to all of these compounds.

Another approach employed against the proliferation of cancer is alkaline therapy, which addresses the cellular acid-base balance. It has been found that extracellular/interstitial cancer tissue is more acidic than healthy tissue due to excessive production of lactic acid stemming from the glycolysis of glucose []. Otto Warburg already suggested in the last century that (as a consequence of hypoxia often encountered in tumor tissues) cancer cells undergo excessive glycolysis instead of relying on the energetically by far more effective oxidative phosphorylation [,], a fact which could recently also be verified by biopsy analysis in breast cancer patients, revealing a marked decrease in β-F1-ATPase/HSP60 expression ratio during disease progression []. Lately, it has been suggested that the initiation of glycolysis could be triggered by AKT activation during tumor development [] and that the resulting acidification of the extra-cellular cancer tissue brings about survival advantages for cancer cells [,]. It has been found recently that T-cell development is markedly suppressed in acidified cancer tissue []. Alternative alkaline therapies applied for cancer treatment included the intake of sodium bicarbonate [], cesium chloride [], or alkaline diet, which is based on fruit and vegetables having high potassium content. A further approach was the ingestion of alkaline drinking water obtained from ion exchangers.

Another avenue towards cancer suppression has been established by the supplementation of (essential) polyunsaturated fatty acids, aiming at re-establishing cellular membrane functionality [] and fluidity []. In addition, the polyunsaturated omega-3 fatty acids eicosapentaenoic (EPA) and docosapentaenoic acid (DHA) have been found to have a direct bearing on gene expression level by e.g., deactivation of NF-kappaB and AKT by EPA and DHA in a mouse model []. Polyunsaturated omega-3 fatty acids have also been shown to possess anti-inflammatory properties, for instance. by suppression of NF-KappaB and cyclooxygenases [], or caused by the reduction of prostaglandin E2 biosynthesis via arachidonic acid due to a shift in the omega-6 fatty acid/omega-3 FA level towards omega-3 species (omega-6 fatty acids form the pool for the endogenous biosynthesis of E2 prostaglandin) [,].

In addition, a direct positive correlation between cytotoxic drug efficacy and DHA level in breast adipose tissue of patients has been observed [  ]. Also, recent clinical studies suggested that EPA/DHA supplementation may suppress cancer-related cachexia []. Whereas severe side effects have been reported for the prolonged administration of some synthetic COX-II inhibitors, including increased thrombosis, stroke, and heart attack risk, to our best knowledge no comparably grave effects have been reported for the prolonged intake of EPA/DHA (e.g. in the form of fish oil) in clinical studies. The side effects of fish oil therapy, including blood thinning, have recently been discussed, e.g., by Farooqui et al.[].

Likewise, Johanna Budwig established a cancer diet (the so-called “Budwig diet”), which includes inter alia the daily intake of linseed oil as a potent source of alpha-linolenic acid as essential omega-3 fatty acid []. Anecdotal cases of complete cancer remissions after continued Budwig diet have been reported []. To our best knowledge, no randomized clinical trials exploring the efficacy of the Budwig diet have been launched to date. The consequence of a continued Budwig diet is said to be an optimization of the dietary balance of omega-6/omega-3 fatty acids and reconstitution of physiologically intact cellular membrane composition by enhanced administration of polyunsaturated fatty acids as a substitute for peroxidized and saturated fatty acids in cellular membranes, thus increasing membrane fluidity. Furthermore, it has been hypothesized that polyunsaturated fatty acids may act as oxygen carriers []. The present-day Western diet results in an adverse ratio of about 15:1 of omega-6/omega-3 fatty acids, whereas a ratio of about 1:1 has been reported as paleolithic reference value for humans []. As a consequence, the endogenous high level of omega-6 fatty acids in humans fosters the increased biosynthesis of pro-inflammatory arachidonic acid from e.g., linoleic acid. Moreover, it has been hypothesized that cottage cheese, quark or yoghurt as second constituent of the Budwig diet refills the pool of sulfhydryl amino acids (which are essential for glutathione biosynthesis).

Warburg considered the glycolytic switch as being a final event in cancer formation, accompanied by irreversible genetic changes and the inactivation of the mitochondrial respiratory chain in cells, giving rise to their dedifferentiation []. However, recent studies suggest that this may not be the case: Dichloroacetate has been shown to be a potent inhibitor of pyruvate dehydrogenase kinase, thereby suppressing, as other agents, the glycolytic switch and thus fostering oxidative phosphorylation [,,]. As a consequence of such an apparent normalization of the cellular energy production, cancer remissions in animal trials and anecdotal reports of healing of malignant tumors in human patients have been reported lately [].

Moreover, investigations involving the administration of coenzyme Q10 directed towards the revitalization of the mitochondrial respiratory chain suggest that, indeed, the inhibition of the respiratory chain (Q10 is present in various complexes thereof) can be reversed or at least be halted: Folkers et al. reported that breast cancer patients taking 90 mg per day Q10 stayed in a state of constant disease, and did not develop new metastases. No patient in the group died, although about 20% (6/32) deaths were statistically expected in the observation period. When the dose of Q10 was augmented to 390 mg daily, five patients who already showed remission under 90 mg of Q10 per day went into apparently complete remission, including the eradication of liver metastases [,]. Cases of complete remission in response to high doses of Q10 for other cancer types, such as small cell bronchogenic carcinoma, have also been published by Folkers et al.[].

Likewise, Sachdanandam et al. recently reported on tumor control and remission caused by a combination treatment by coenzyme Q10, vitamins B2 and B3 (all of which are essential for the cellular energy generation) and tamoxifen in animal trials []. As a result, markedly lower levels of lipid peroxidation and cachexia over the tumor-induced non-treated control group was observed. Orienting clinical trials of Premkumar et al., involving 84 breast cancer patients, affirmed the anti-tumor action of said agent combination []. Inter alia, a decrease of the plasma concentration of urokinase plasminogen activator (UPA) by about 50% was observed, and the level of adhesion factors such as E-selectin and pro-angiogenic proteinase MMP-9 were found to be drastically decreased after only 90 days of treatment. Moreover, significantly reduced tumor marker levels (CA-15-3 and CEA) have been measured after 90 days of coenzyme Q10, vitamins B2 and B3 plus tamoxifen combination treatment []. UPA expression level was determined as correlating with the clinical outcome of breast cancer, and UPA inhibition has therefore been made the target of extensive research [].

Another approach addressing the stabilization of the course of breast cancer is the administration of bisphosphonates [] such as ibandronate, which stabilize the bone matrix and thus impede osteoclast-mediated bone lysis. In addition, certain bisphosphonates, such as the latter compound, have been shown to possess direct anti-tumor action in vitro and in vivo [,].

Finally, a further route towards the suppression of cancers is the suppression of nuclear factor kappa B (a gene transcription promoter involved in the inflammatory chain and in a tumor’s capability to invade, metastasize and evade apoptosis) []. NF-kappaB stimulates the expression of various pro-inflammatory genes [,], also in breast cancer []. Consequently, a number of approaches have been divulged lately which are concerned with inhibition of this factor. The different compounds, which hinder the activation of NF-kappaB, are e.g., EPA (see above), and 11-keto-17-hydroxy boswellic acid (AKBA) [], a compound which has been shown to abrogate the osteoclastogenesis by inhibition of NF-kappaB activation in vitro. AKBA was also shown to hinder the enzyme 5-lipoxygenase [], which plays a pivotal role in the biosynthesis of pro-inflammatory leucotrienes. Remarkably, it has been shown that NF-kappaB inhibitors effectively inhibited MCF-7 breast cancer stem-like cells [].

In the present case, refractory breast cancer, which had not or has poorly responded to initial chemo- and anti-hormonal therapy, showed drastic and ongoing response to a combination treatment including capecitabine and complementary treatment components; the latter include NF-kappaB blockers, and other inhibitors of the inflammatory chain, respiratory chain stimulants, plus alkaline therapy. The rationale for employing these agents has been explained in the preceding paragraphs. No resistance to the therapy was observed after 17 months, and the decrease of tumor marker levels correlated with imaging results. The obtained results are significant in view of the initial heavy disease progress and lack of relevant response to all preceding therapies.

The incremental contributions of each individual treatment element remain unclear. However, it is hypothesized that a synergetic action of the measures takes place. These have been selected by theoretical considerations in order to avoid potential antagonistic interferences, which could annihilate action. It should also be noted that concerns about the simultaneous intake of chemotherapeutics and antioxidants have been raised in the literature, especially in the context of cytostatics that have free radical formation as their believed primary mechanism of action. To our best knowledge, the primary mechanism of action of capecitabine, however, is not via free radicals but DNA synthesis and thymidylate synthase inhibition []. No antagonistic interaction with the remaining measures “base therapy” (addressing the immune suppression observed in the acidic tumor environment due to the purported suppression of T-cell development in the acidic tissue adjacent to tumors) and bone stabilization by bisphosphonates has been expected. On the contrary, the reported suppression of NF-kappaB expression by e.g. AKBA should reduce the RANKL-induced osteoclastogenesis, which is triggered by the transcription factor NF-kappaB [].

Whether all measures contribute to the observed results remains speculative. The progression-free interval of 17 months observed so far is encouraging in view of a median time to progression from 3–9 months reported for the first line treatment of metastatic breast cancer by capecitabine []. Randomized clinical trials appear to be indicated in view of the promising orienting results.

Note that ER positive/PR negative breast cancer constitutes a rather limited high risk subset within the broader patient collective suffering from luminal breast cancer. Lately, it has been hypothesized in the literature that the expression of progesterone receptor (genes) in breast cancer has a positive bearing on the disease malignity and outcome, correlating with a less aggressive phenotype, and that the expression of progesterone receptor genes may be hindered by AP-1 [,,]. AP-1 and NF-kappaB have been shown to bind to UPA promoter sequence and to cooperatively foster UPA expression. Consequently, it has been directly or indirectly suggested to therapeutically inhibit NF-kappaB in order to improve efficacy of antiestrogen treatment of patients associated to high risk hormone-dependent breast cancer [,].

Moreover, the reduced UPA expression mediated by Q10 described in the literature could also be a sign of a reduced activity of the transcription factor AP-1. At the same time, reduction of AP-1 activity could lead to a reversal of the blockage of the progesterone receptor expression caused by the inhibitory action of AP-1 and a consequent sensitization of ER-positive/PR-negative breast cancers to anti-estrogenic treatment by tamoxifen (compare to references [,]).

It is further hypothesized that the obtained orienting results hint (as already observed for DCA) at a revitalization of the mitochondrial respiratory chain at the expense of a pathologic increase of glycolysis. The reduction of glucose metabolism of the metastases was corroborated by reduced signal intensity in 18FDG-PET-CT scans during the treatment. Hence, the results are interpreted as a pointer towards the (at least partial) reversibility of the glycolytic switch and the associated changes in gene profile expression.

 

SEE ALL WATER IONIZERS – MOLECULAR HYDROGEN GENERATORS

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Link to Publisher's site
. 2011 Mar; 3(1): 1454–1466.
Published online 2011 Mar 17. doi: 10.3390/cancers3011454
PMCID: PMC3756422
PMID: 24212668

Clinical Response of Metastatic Breast Cancer to Multi-targeted Therapeutic Approach: A Single Case Report

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Acknowledgements

Thanks are due to E. Rummeny and J. Gaa, both Department of Radiology, Klinikum Rechts der Isar, Technische Universitat Munchen, for kindly providing the MRI images and the image analysis.

Disclaimer

The author does not suggest that breast cancer can be healed by applying the described measures. Moreover, the author disclaims all responsibilities and liabilities as consequence of a potential application of the described treatment steps, either taken separately or in any combination by patients, third parties, institutions, or other persons, and for the correctness of the provided information. Questions concerning the disclosed treatment will be answered to physicians and clinical academia in general, only.

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Articles from Cancers are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

 

Molecular Hydrogen water effects on Mild Cognitive Impairment

Abstract

Background:

Oxidative stress is one of the causative factors in the pathogenesis of neuro-degenerative diseases including mild cognitive impairment (MCI) and dementia. We previously reported that molecular hydrogen (H2) acts as a therapeutic and preventive antioxidant.

Objective:

We assess the effects of drinking H2 hydrogen-water (water infused with hydrogen gas H2) on oxidative stress model mice and human subjects with MCI.

Methods:

Transgenic mice expressing a dominant-negative form of aldehyde dehydrogenase 2 were used as a dementia model. The mice with enhanced oxidative stress were allowed to drink hydrogen H2-water.

For a ran-domized double-blind placebo-controlled clinical study, 73 subjects with mild cognitive impairment MCI drank ~300 mL of hydrogen H2-water (H2-group) or placebo water (control group) per day, and the Alzheimer’s Disease Assessment Scale-cognitive subscale (ADAS-cog) scores were determined after 1 year.

Results:

In mice, drinking hydrogen H2-water decreased oxidative stress markers and suppressed the decline of memory impairment and neurodegeneration. Moreover, the mean lifespan in the hydrogen H2-water group was longer than that of the control group.’

In MCI subjects, although there was no significant difference between the hydrogen water H2- and control groups in ADAS-cog score after 1 year, carriers of the apolipoprotein E4 (APOE4) geno-type in the H2-group were improved significantly on total ADAS-cog score and word recall task score (one of the sub-scores in the ADAS-cog score).

Conclusion:

H2-water may have a potential for suppressing dementia in an oxidative stress model and in the APOE4 carriers with MCI.

1. INTRODUCTION

Oxidative stress is one of the causative factors in the pathogenesis of major neurodegenerative diseases including Alzheimer’s disease (AD), mild cognitive impairment (MCI), and Parkinson disease (PD) []. Moreover, the genotype of apolipoprotein E4 (APOE4) is a genetic risk for AD, and the increased oxidative stress in the APOE4 carriers is considered as one of the modifiers for the risk [].

To explore effective dietary antioxidants to mitigate age-dependent neurodegeneration, it may be useful to construct model mice in which AD phenotypes would progress in an age-dependent manner in response to oxidative stress. We constructed transgenic DAL101 mice expressing a polymorphism of the mitochondrial aldehyde dehydrogenase 2 gene (ALDH2*2) []. ALDH2*2 is responsible for a deficiency in ALDH2 activity and is specific to North-East Asians []. We reported previously that ALDH2 deficiency is a risk factor for late-onset AD in the Japanese population, [] which was reproduced by Chinese and Korean studies in their respective populations []. DAL101 mice exhibited a decreased ability to detoxify 4-hydroxy-2-nonenal (4-HNE) in cortical neurons, and consequently an age-dependent neurodegeneration, cognitive decline, and a shortened lifespan [].

We proposed that molecular hydrogen (H2) has potential as a novel antioxidant, [] and numerous studies have strongly suggested its potential for preventive and therapeutic applications []. In addition to extensive animal experiments, more than 25 clinical studies examining the efficacy of molecular hydrogen H2 have been reported, [] including double-blind clinical studies. Based on these studies, the field of hydrogen medicine is growing rapidly.

There are several methods to administer hydrogen H2, including inhaling hydrogen gas (H2-gas), drinking hydrogen H2-dissolved water (H2-water), and injecting hydrogen H2-dissolved saline (hydrogen-rich saline) []. Drinking hydrogen H2-water prevented the chronic stress-induced impairments in learning and memory by reducing oxidative stress in mice [] and protects neural cells by stimulating the hormonal expression of ghrelin []. Additionally, injection of hydrogen-rich saline improved memory function in a rat model of amyloid-β-induced dementia by reducing oxidative stress []. Moreover, hydrogen inhalation during normoxic resuscitation improved neurological outcome in a rat model of cardiac arrest independently of targeted temperature management [].

In this study, we examined whether drinking hydrogen H2-water could suppress aging-dependent memory impairment induced by oxidative stress in DAL101 mice. Next, in a randomized double-blind placebo-controlled study, we investigated whether H2-water could delay the progression of MCI as assessed by the scores on the Alzheimer’s Disease Assessment Scale-cognition sub-scale (ADAS-cog) [] from baseline at 1-year. We found a significant improvement in cognition at 1 year in carriers with the APOE4 genotype in the H2-group using sub- and total ADAS-cog scores.

2. MATERIALS AND METHODS

2.1. Ethical Approval and Consent to Participate

This animal study was approved by the Animal Care and Use Committee of Nippon Medical School. The methods were carried out in “accordance” with the relevant guidelines and regulations.

The clinical study protocol was approved by the ethics committees of University of Tsukuba, and registered in the university hospital medical information network (UMIN) as UMIN000002218 on July 17, 2009 at https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=history&action =list&type= summary&recptno= R000002-725&language=J.

Participants were enrolled from July 2009. All patients provided written informed consent prior to research investigations, which were conducted according to the Declaration of Helsinki and subsequent revisions.

2.2. Transgenic DAL101 Mice

Transgenic mice (DAL101) that express a transgene containing a mouse version of ALDH2*2 were constructed as described previously []. Since the number of mice used for each experiment was not consistent because of a breeding difficulty, the number of the mice used was specified. All mice were kept in a 12-hr light/dark cycle with ad libitum access to food and water. Examiners performed experiments in a blinded fashion. Since no significant decline was observed in cognitive impairment at the age of 18 months in wild-type mice with the same genetic background (C57BL/6), [] the effects of hydrogen H2-water were not assessed in this study.

2.3. Hydrogen Water

For animal experiments, saturated hydrogen H2-water was prepared as described previously []. In brief, hydrogen  H2 was dissolved in water under high pressure (0.4 MPa) to a supersaturated level, and the saturated H2-water was stored under atmospheric pressure in an aluminum bag with no headspace. As a control, H2-water was completely degassed by gentle stirring for one day. Mice were given water freely using closed glass vessels equipped with an outlet line containing two ball bearings, which kept the water from being degassed. The vessel was freshly refilled with H2-water 6 days per week at 2:00 pm. The hydrogen H2-concentration was still more than 0.3 mM on the next day.

For this clinical study, commercially available hydrogen H2-water was a gift from Blue Mercury, Inc. (Tokyo, Japan). The hydrogen H2-water (500 mL) was packed in an aluminum pouch with no headspace to maintain H2 concentration, and sterilized at 80°C for 30 min. The concentration of hydrogen H2 was measured using a hydrogen sensor (Unisense, Aarhus N, Denmark), and used if the value was more than 0.6 mM. Placebo water packed in an identical package (500 mL) was also provided by Blue Mercury Inc. This company played no role in collection of data, management, analysis, or interpretation of the data. One package with 500 mL of placebo or hydrogen H2-water per day was provided after showing previous empty packages, by which self-reported compliance rates in the intervention group were calculated as the volume of hydrogen  H2-water at 1-year.

2.4. Measurement of Oxidative Stress

As an oxidative stress marker, 8-OHdG [] was measured using urine samples, which were collected between 9:00 and 10:00 am as described previously [], by using a competitive enzyme-linked immunoassay (New 8-OHdG check; Japan Institute for the Control of Aging, Shizuoka, Japan). The values were normalized by urinary creatinine concentration, which was assayed using a standard kit (Wako, Kyoto, Japan). As an additional oxidative stress marker in the brain, accumulated MDA was determined using a Bioxytech MDA-586 Assay Kit (Percipio Biosciences, CA, USA). Malondialdehyde(MDA)levels were normalized against protein concentrations.

2.5. Measurement of Memory Impairment: Object Recognition Task

Learning and memory abilities were examined using objection recognition task (ORT) []. A mouse was habituated in a cage for 4 h, and then two different-shaped objects were presented to the mouse for 10 min as training. The number of times of exploring and/or sniffing each object was counted for the first 5 min (Training test). The frequencies (%) in training test were considered as the backgrounds. To test memory retention after 1 day, one of the original objects was replaced with a novel one of a different shape and then times of exploration and/or sniffing was counted for the first 5 min (Retention test). When mice would lose learning and memory abilities, the frequencies of exploration and/or sniffing of each object should be equal (about 50%) in the training session, indicating that mice showed a similar interest in each object because of lack of memory for the objects. Learning and memory abilities were evaluated as the subtraction of the frequencies (%) in the retention test from each background (Training test).

2.6. Measurement of Memory Impairment: Passive Avoidance Task (PA)

The apparatus consisted of two compartments, one light and the other dark, separated by a vertical sliding door []. On day 1, we initially placed a mouse in the light compartment for 20 s. After the door was opened, the mouse could enter the dark compartment (mice instinctively prefer being in the dark). On day 2, the mouse was again placed in the light section to allow the mouse to move into the dark section. After the mouse entered the dark compartment, the door was closed. After 20 s, the mouse was given a 0.3 mA electric shock for 2 s. The mouse was allowed to recover for 10 s, and was then returned to the home cage. On day 3, 24h after the shock, the mouse was again placed in the light section with the door opened to allow the mouse to move into the dark section. We examined the latency time for stepping through the door. Learning and memory abilities were assessed as the subtraction of the latency times after the electric shock from each background (before).

2.7. Immunostaining of the Hippocampal CA1 Region

To examine neuronal loss and glial activation, the hippocampus region was stained with a pyramidal neuron-specific anti-NeuN antibody (clone A60; Merck Millipore, Darmstadt, Germany), an astrocyte-specific anti-glial fibrillary acidic protein (anti-GFAP) antibody (Thermo Scientific, MA, USA) or a microglia-specific anti-IbaI antibody (Wako). Mice were transcardially perfused to be fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) under anesthesia, and their brains were cryoprotected with 30% sucrose, and then frozen brain was sectioned at 8 μm thickness. After incubation with each primary antibody, sections were treated with secondary antibodies (Vector Laboratories, CA, USA) and their immunereactivity was visualized by the avidin-biotin complex method (Vector Laboratories).

2.8. Subjects of the Clinical Study

This study was a randomized, double-blind, placebo-controlled trial undertaken as a part of Tone project, an ongoing epidemiological study conducted in Tone Town, Ibaraki, Japan as described in detail previously []. This town is located approximately 40 km northeast of central Tokyo and consists of 22 districts. The baseline survey of the Tone project included 1,032 participants in July 2009, and subjects of the present study were recruited from these participants.

Eligibility criteria are age 67 years or older, being able to give written informed consent for participation in the present study, with a diagnosis of MCI, being able to observe the following requirement: good compliance with water consumption; participation in the scheduled examinations for assessment; keeping a log-diary recording consumption of the water, with a modified Hachinski Ischemic score of 4 or less and a 15-item Geriatric Depression Scale score of 6 or less. In brief, 3 months before this clinical study, all participants underwent a group assessment which used a set of 5 tests that measured the following cognitive domains: attention; memory; visuospatial function; language; and reasoning as described previously []. Objective impairment in at least 1 cognitive domain based on the average of the scores on the neuropsychological measures within that domain and 1 SD cut-off using normative corrections for age, years of education, and sex.

Exclusion criteria were having “The Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV TR” criteria for dementing illnesses, a serious or unstable illnesses, a history within the past 5 years of serious infectious disease affecting the brain and/or malignant diseases, a history of alcohol or drug abuse or dependence (on DSM-IV TR) within the past 5 years, and receiving any types of anti-Alzheimer drugs and recent (within 4 weeks) initiation of medications that affect the central nervous system. When the score of Mini Mental State Examination (MMSE) [] was less than 24, the subjects were excluded.

In this study, subjects were randomly assigned to either to an intervention group, who received H2-water every-day for 1 year, or a control group, who received placebo water. The allocation sequence was determined by computer-generated random numbers that were concealed from the investigators and subjects. Drs. Nakajima and Ikejima generated the random allocation sequence, enrolled participants, and assigned participants to interventions. Any participants and care providers were blindly masked.

In the original protocol, we planed to administer H2-water for 2 years and assess the secondary outcomes; however, we had to stop the project in 2011 by the Tsunami-disaster and could not obtained the 2-year data and secondary outcomes.

The APOE4 genotype was determined as described [].

2.9. Statistical Considerations

All statistical analyses were performed by an academic biostatistician using SAS software version 9.2 (SAS Institute Inc, Cary, NC, USA). Results were considered significant at p < 0.05.

For the comparison of two groups in learning and memory abilities, and lifespans, unpaired two-tailed Student’s t-test was used for the comparison of H2-group with control group. For the other animal experiments, one-way analysis of variance (ANOVA) with Tukey-Kramer or Dunnett post hoc analysis was applied unless otherwise mentioned.

For the clinical trial, we planned to recruit a total of 120 patients, which would provide 90% power to detect an effect size of 0.6 using a two-sided test with a 5% significance level, but the actual sample size for the primary analysis was 73, leading to 70% power in the same setting. End-points were scores in the Japanese version of ADAS-cog at 1-year, and the changes were evaluated by Mann-Whitney’s U test (non-parametric analysis) as well as Student’s t-test (parametric analysis).

3. RESULTS

3.1. Hydrogen-water Reduced Oxidative Stress in DAL Mice

Male DAL101 mice were given H2– or control water to drink ad libitum from the age of 1 month, and continued until the age of 18 months. The H2-water DAL101 group showed a significant decrease in the level of an oxidative stress marker, urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG)[] at the age of 14months (Suppl. Fig. S1A). Moreover, DAL101 mice increased oxidative stress in the brain as measured by the level of MDA as an alternative oxidative stress marker, and H2-water showed a significant recovery of this increased level of MDA in DAL101 mice (Suppl. Fig. S1B).

3.2. Hydrogen Water Suppressed a Decline in Learning and Memory Impairment

We examined learning and memory abilities using ORT []. As described in MATERIALS AND METHODS, learning and memory abilities were evaluated as the subtraction of the frequency (%) in Retention test from each background (Training test). Mice were provided with control or H2-water from the age of 1 month. At the age of 14 months, the H2-group significantly memorized the original objects and showed the preference for the novel object more than the control group (Fig. 1A1A 14-month-old).

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Hydrogen water prevented cognitive decline. H2-water was provided from the age of 1 month (A, C), and from the age of 8 months (B). The mice were subjected to the first objection recognition task (ORT) at the age of 14 months (A, B, 14-month-old) and the second ORT at the age of 18 months (A, B, 18-month-old).

The recognition indexes were obtained as the frequency (%) of exploring and/or sniffing the object that would be replaced or the novel one that had been replaced. ΔRecognition index (%) indicates the frequencies in Retention test of ORT after the subtraction of those in Training test (background). WT, wild-type; (DAL, H2-),

DAL101 mice drinking degassed control water; (DAL, H2+), DAL101 mice drinking hydrogen water. Data are shown as the mean ± SEM. n = 9, *p < 0.05, **p < 0.01 by Student’s t-test. (C) The mice were subjected to a passive avoidance task. Step-through latencies before and after the electric shock are obtained and ΔStep-through latency (s) indicates the subtraction of Step-through latencies after from before the electric shock. WT, wild-type (n = 10); DAL, H2-, DAL101 mice receiving degassed control water (n = 8); and DAL, H2+, DAL101 mice receiving H2-water (n = 8). Data are shown as the mean ± SEM. *p < 0.05.

At the age of 18 months, the mice were subjected to the second ORT, which can be done by using different objects at the age of 18 months []. The aged DAL101 mice drinking H2-water still significantly memorized the original objects and preferred the novel one more than the control group (Fig. 1A1A 18-month-old).

Next, to test the drinking effects of H2-water from the later stage, we started giving H2-water to male DAL101 mice at the age of 8 months instead of 1 month, and subjected to ORT at the age of 14 months (Fig. 1B1B 14-month-old) and the second ORT at the age of 18 months (Fig. 1B1B 18-month-old). Even when the mice began to drink at the age of 8 months, H2-water significantly suppressed the decline in the learning and memory abilities at the age of 18 months as well as at the age of 14 months (Fig. 1B1B).

Moreover, we subjected the mice to PA [] at the age 18 months as an alternative method. One day after a 0.3 mA electric shock for 2 s was given, wild-type C57BL/6 mice memorized the shock as evaluated by the subtraction of the latency time (s) to re-enter the dark compartment from each background (Fig. 1C1C). The H2-water group significantly suppressed the decline in learning and memory more than the control group (Fig. 1C1C).

Thus, drinking hydrogen H2-water suppressed the learning and memory impairment in the oxidative stress mice.

3.3. Hydrogen-water Suppressed Neurodegeneration

To examine whether hydrogen H2-water could prevent neurodegeneration in aged DAL101 mice, we stained the hippocampus with a neuron-specific anti-NeuN antibody (Fig. 2A2A). Neurodegeneration was evaluated by glial activations using an anti-GFAP antibody and a microglia-specific anti-Iba-I antibody. Immune-positive cells per field of view (FOV) were counted in the CA1 region (Fig. 2B2B).

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Hydrogen water suppressed neurodegeneration. (A) The hippocampal CA1 region was stained with antibodies against NeuN (a neuronal marker), GFAP (an astrocytic marker) or Iba-1 (a microglial marker) (Scale bars: 50 µm). Right panels show magnified images of the squares in the left panels (Scale bars: 10 µm). (B) Cells positive for anti-NeuN, anti-GFAP and anti-Iba-I antibodies per field of view (FOV) were counted in the CA1 region (n = 5). Data are shown as the mean ± SD. *p < 0.05, **p < 0.01 (wild-type vs DAL), #p < 0.05 (H2-water vs. control water in DAL).

The number of neurons was decreased in the control DAL101 group as the comparison with wild type group, and the H2-DAL101 group showed a trend in recovery of the decrease (Fig. 2A2A). As has been described previously, [] the control DAL101 mice exhibited an increase in glial activation, and the H2-water group suppressed the enhanced glial activation in the CA1 region (Fig. 22, GFAP and Iba-I).

3.4. Hydrogen-water Extended the Average Lifespan of Mice

DAL101 mice showed a shorter lifespan, which has also been described previously []. To examine whether consumption of hydrogen H2-water attenuated the shortened lifespan, female DAL101 mice started drinking control or H2-water at the age of 1 month. Although hydrogen H2-water did not extend the maximum lifespan (Fig. 3A3A), hydrogen H2-water significantly extended the mean of lifespan of DAL101 mice (Fig. 3B3B).

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Extension of the average lifespan by continuous drinking H2-water. (A) Kaplan-Meier curve representing the survival of female C57BL/6 mice (wild-type), female DAL101 mice drinking control water (control water) and H2-water (H2-water). (B) Each dot indicates the lifespan of each mouse. The bars indicate the average lifespan of each group. *p < 0.05 (p = 0.036) by Student’s t-test.

3.5. A Randomized, Placebo Controlled Clinical Study

Fig. (44) shows the profile on the recruitment, randomization, and follow-up of this study. A total of 81 subjects of the 1,032 participants were randomized; however, 3 in the control group and 5 in the intervention group were diagnosed as ineligible after randomization and not included in this analysis. Baseline characteristics and lifestyle factors were balanced between the study groups (Table 11). Random assignment was stratified by age of ~74 years and MMSE score of ~28 points. The average compliance rate of drinking water was estimated as 64% in both groups at 1-year, meaning the subjects drank 320 mL/day on the average. The mean total ADAS-cog scores in the H2– and control groups were 8.04 and 7.89, respectively, with no significance.

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Profile of the recruitment, randomization, and follow-up of this study. This study was a randomized, double-blind, placebo-controlled trial undertaken as a part of Tone project, an ongoing epidemiological study conducted in Tone Town, Ibaraki, Japan [].

Table 1

Background characteristics of 73 subjects with mild cognitive impairment.

Control (n=38) Intervention (n=35)
Mean SD or % Mean SD or %
Woman * 20 (52.6%) 19 (54.3%)
Age (years) 74.45 5.44 73.97 5.11
Body mass index (kg/m2) 23.55 2.59 23.19 4.08
Systolic blood pressure (mmHg) 131.26 12.35 135.14 13.31
Diastolic blood pressure (mmHg) 77.92 7.13 78.89 9.53
Education (years) 11.26 2.71 11.57 2.83
Current alcohol drinker * 19 (50.0%) 14 (40.0%)
Current smoker * 4 (10.5%) 5 (14.3%)
Current exercise habit * 27 (71.1%) 22 (62.9%)
APOE4 carrier * 6 (15.7%) 7 (20.0%)
Family history * 2 (5.3%) 2 (5.7%)
Comorbidity *
Hypertension 15 (39.5%) 14 (40.0%)
Diabetes mellitus 4 (10.5%) 5 (14.3%)
Dyslipidemia 4 (10.5%) 4 (11.4%)
Stroke 2 (5.3%) 1 (2.9%)
Depression 1 (2.6%) 2 (5.7%)
MMSE 28.08 1.66 27.83 1.74
ADAS-cog 7.89 3.19 8.04 3.47

* indicates frequency (%).

After 1 year, no observable harms or unintended effects in each group were found, and there was a trend to improve total ADA-cog score both in the H2– and control-groups (Suppl. Table S1), probably because of interventions such as moderate exercise by the Tone project. Moreover, the subjects in the H2-group had more trends for the improvement than those in the control-groups although there was no significance (Suppl. Table S1). However, when we pay attention to score-changes in carriers of the APOE4 genotype, the total ADAS-cogs and word recall task scores (one of the sub-scores) significantly improved as assessed by the distribution of the score change in each subject (Fig. 55). In the APOE4 carriers, the hydrogen  water H2-group significantly improved, whereas the control group slightly worsened. Moreover, Fig. (66) shows the score change of each subject as an alternative presentation. Although the subjects in the control group did not improved, six and five out of 7 subjects improved on the total ADAS score and word recall task scores, respectively, in the hydrogen water H2-group of the APOE4 carriers.

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Distribution of changes of sub- and total-ADAS-cog score. Distribution of change of word recall task score (A), a sub-score of ADAS-cog, and (B) total ADAS-cogs score in APOE4 non-carriers (left) and APOE4 carriers (right). Each dot indicates the change of individual subjects. The difference between the H2- and control groups was significant in APOE4 carriers by a non-parametric analysis as well as a parametric analysis. (Ap = 0.036 (by Student’s t-test) and p =0.047 (by Mann-Whitney’s U test) and (Bp = 0.037 (by Student’s t-test) and p = 0.044 (by Mann-Whitney’s U test) for (A) and (B), respectively. Middle bars in lozenges indicate median values.

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Changes in a sub-sore and total ADAS-cog score of each subject in the APOE4 carriers. Each line indicates the 1-year change in the word recall task score (A) and total ADAS-cog score (B) of a subject in the APOE4 carriers. * indicates p < 0.05 as shown in the legend of Fig. 5.

DISCUSSION

Age-dependent neurodegenerative disorders are involved in oxidative stress. In this study, we showed that drinking hydrogen H2-water suppressed the biochemical, behavioral, and pathological decline in oxidative stress mice. The score of ADAS-cog [] is the most widely used general cognitive measure in clinical trials of AD []. The ADAS-cog score assesses multiple cognitive domains including memory, language, praxis, and orientation. Overall, the ADAS-cog has proven successful for its intended purpose. The present clinical study shows that drinking hydrogen H2-water significantly improved the ADAS-cog score of APOE4 genotype-carriers.

We have previously showed that DAL101 mice show age-dependent neurodegeneration and cognitive decline and the shorten lifespan []. DAL101 mice exhibit dementia phenotypes in an age-dependent manner in response to an increasing amount of oxidative stress []. Oxidative stress enhances lipid peroxidation, leading to the formation of highly reactive α, β-unsaturated aldehydes, such as MDA and 4-HNE []. The accumulation of 4-HNE-adducted proteins in pyramidal neurons has been observed in the brains of patients with AD and PD []. The decline of ALDH2*2 ability failed to detoxify cytotoxic aldehydes, and consequently increases in oxidative stress [].

Moreover, double-transgenic mice were constructed by crossing DAL101 mice with Tg2576 mice, which express a mutant form of human amyloid precursor protein (APP). They showed accelerated amyloid deposition, tau phosphorylation, and gliosis, as well as impaired learning and memory abilities. The lifespan of APP/DAL mice was significantly shorter than that of APP and DAL101 mice []. Thus, these model animals may be helpful to explore antioxidants that could be able to prevent age-dependent dementia. Indeed, a diet containing Chlorella showed mitigated effects on cognitive decline in DAL101 [].

One of the most potent risk factors for AD is carrier status of the APOE4 genotype, and the roles of APOE4 on the progression of AD have been extensively examined from various aspects []. APOE4 also increase the number of atherogenic lipoproteins, and accelerate atherogenesis []. The increased oxidative stress in APOE4 carriers is considered as one of the modifiers for the risk []. A combination of antioxidants improved cognitive function of aged subjects after 3 years, especially in APOE4 carriers []. This previous clinical result agrees with the present study. hydrogen H2 acts as an efficient antioxidant inside cells owing to its ability to rapidly diffuse across membranes []. Moreover, as a secondary anti-oxidative function, H2 seems to activate NF-E2-related factor 2 (Nrf2), [] which reduces oxidative stress by expression a variety of antioxidant enzymes []. We reported that drinking hydrogen H2-water prevented arteriosclerosis using APOE knockout mice, a model of the spontaneous development of atherosclerosis accompanying a decrease in oxidative stress []. Thus, it is possible that drinking H2-water improves vascular damage by decreasing oxidative stress as a direct or indirect antioxidant, leading to the improvement of a demintia model and MCI subjects. In this study, we focused on the genotype of APOE-isoforms; however, the polymorphism of the APOE gene in the promoter region influences the expression of the APOE gene []. Thus, it will be important to examine the effect of hydrogen H2-water under this polymorphism.

For mitigating AD, significant attention has been given to regular, moderate exercise to help reduce the risk of dementia and prevent MCI from developing in aging patients [ – ]. Moderate exercise enhances energy metabolism and suppresses the expression of pro-inflammatory cytokines, [] and protects vascular systems [].molecular hydrogen H2 exhibits multiple functions by a decrease in the levels of pro-inflammatory cytokines and an increase in energy metabolism in addition to anti-oxidative roles. To exert multiple functions, molecular hydrogen H2 regulates various signal transduction pathways and the expression of many genes []. For examples,molecular hydrogen H2 protects neural cells and stimulates energy metabolism by stimulating the hormonal expression of ghrelin [] and fibroblast growth factor 21, [] respectively. In contrast, molecular hydrogen H2 relieves inflammation by decreasing pro-inflammatory cytokines []. Thus, the combination of these functions of molecular hydrogen H2 on anti-inflammation and energy metabolism-stimulation might prevent the decline in brain function, [] both of which are improved by regular and moderate exercise. Thus, it is possible that the multiple functions of molecular hydrogen H2, including energy metabolism-stimulation and anti-inflammation, may contribute to the improvement of the dementia model and the MCI subjects.

As an alternative aspect, molecular hydrogen H2 suppresses the nuclear factor of activated T cell (NFAT) transcription pathway to regulate various gene expression patterns []. NFAT signaling is altered in AD and plays an important role in driving amyloid β-mediated neurodegeneration []. Moreover, the NFAT transcriptional cascade contributes to amyloid β synaptotoxicity []. Additionally, an active involvement of the NFAT-mediated signaling pathway in α-syn-mediated degeneration of neurons in PD []. Indeed, patients with PD improved by drinking molecular hydrogen H2-water as revealed by a double-blind, placebo-controlled clinical study, [] and a larger scale of a clinical trial is under investigation []. Thus, the beneficial effects of molecular hydrogen H2 on the neurodegenerative diseases may be explained by the suppression of NFAT transcriptional regulation.

CONCLUSION

The present study suggests a possibility for slowing the progress of dementia by drinking molecular hydrogen H2-water by means of animal experiments and a clinical intervention study for APOE4 carriers; however, a longer and larger scale of trials will be necessary to clarify the effect of H2-water on MCI.

PMCID: PMC5872374
PMID: 29110615
Effects of Molecular Hydrogen Assessed by an Animal Model and a Randomized Clinical Study on Mild Cognitive Impairment
This is an open access article licensed under the terms of the Creative Commons Attribution-Non-Commercial 4.0 International Public License (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/legalcode), which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

Associated Data

Supplementary Materials

ACKNOWLEDGEMENTS

We thank Blue Mercury, Inc. (Tokyo, Japan) for providing H2-water and placebo water, Ms. Hiroe Murakoshi for technical assistance and Ms. Suga Kato for secretarial work. Financial support for this study was provided by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23300257, 24651055, and 26282198 to S.O.; 23500971 and 25350907 to K.N.). Financial support for this study was provided by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23300257, 24651055, and 26282198 to S.O.; 23500971 and 25350907 to K.N.).

LIST OF ABBREVIATIONS

APOE4 Apolipoprotein E4
MCI Mild cognitive Impairment
ALDH2 Aldehyde Dehydrogenase 2
ADAS-cog Alzheimer’s Disease Assessment Scale-cognitive subscale
AD Alzheimer’s Disease
PD Parkinson’s Disease
DAL101 Dominant Negative Type 101 of the ALDH2 Mutant Polymorphism (ALDH2*2)
4-HNE 4-Hydroxy-2-nonenal
8-OHdG 8-Hydroxy-2’-deoxyguanosine
MDA Malondialdehyde
ORT Object Recognition Task
PA Passive Avoidance Task
GFAP Glial Fibrillary Acidic Protein
PBS Phosphate-buffered Saline
ANOVA One-way Analysis of Variance
CI Confidence Interval
MMSE Mini Mental State Examination
FOV Field of View
APP Amyloid Precursor Protein
Nrf2 NF-E2-related Factor 2
NFAT Nuclear Factor of Activated T Cell

 

SUPPLEMENTARY MATERIAL

Supplementary material is available on the publisher’s web site along with the published article.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The animal study was approved by the Animal Care and Use Committee of Nippon Medical School.

The human clinical study protocol was approved by the ethics committees of University of Tsukuba.

HUMAN AND ANIMAL RIGHTS

All animal research procedures followed were in accordance with the standards set forth in the eighth edition of Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences, The National Academies Press, Washington, D.C.).

All human material was obtained in accordance with the standards set forth in the Declaration of Helsinkiprinciples of 1975, as revised in 2008 (http://www.wma.net/en/10ethics/10helsinki/<http://www.wma.net/en/10ethics/10helsinki/>).

Consent for Publication

All the patients provided written informed consent priority to research investigations.

CONFLICT OF INTEREST

We declare that there is no actual and potential conflict of interest on this study. Although SO was a scientific advisor of Blue Mercury, Inc. (Tokyo, Japan) from 2,005 to 2,008, there was no involvement during this study.

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molecular hydrogen water benefits for recovering from ACUTE BRAIN STEM INFARCT – clinical trial

Background

In acute stage of cerebral infarction, MRI indices (rDWI & rADC) deteriorate during the first 3-7 days after the ictus and then gradually normalize in approximately 10 days (pseudonormalization time), although the tissue is already infarcted. Since effective treatments improve these indices significantly and in less than the natural pseudonormalization time, a combined analysis of these changes provides an opportunity for objective evaluation on the effectiveness of various treatments for cerebral infarction. Hydroxyl radicals are highly destructive to the tissue and aggravate cerebral infarction. We treated brainstem infarction patients in acute stage with hydroxyl radical scavengers (Edaravone and hydrogen) by intravenous administration and evaluated the effects of the treatment by a serial observation and analysis of these MRI indices. The effects of the treatment were evaluated and compared in two groups, an Edaravone alone group and a combined group with Edaravone and hydrogen, in order to assess beneficial effects of addition of hydrogen.

Methods

The patients were divided in Edaravone only group (E group. 26 patients) and combined treatment group with Edaravone and hydrogen enriched saline (EH group. 8 patients). The extent of the initial hump of rDWI, the initial dip of rADC and pseudo-normalization time were determined in each patient serially and averages of these data were compared in these two groups and also with the natural course in the literatures.

Results

The initial hump of rDWI reached 2.0 in the E group which was better than 2.5 of the natural course but was not as good as 1.5 of the EH group. The initial dip of rADC was 0.6 in the E group which was close to the natural course but worse than 0.8 of the EH group. Pseudonormalization time of rDWI and rADC was 9 days only in EH group but longer in other groups. Addition of hydrogen caused no side effects.

Conclusions

Administration of hydroxyl radical scavengers in acute stage of brainstem infarction improved MRI indices against the natural course. The effects were more obvious and significant in the EH group. These findings may imply the need for more frequent daily administration of hydroxyl scavenger, or possible additional hydrogen effects on scavenger mechanisms.

Background

Clinical care of cerebral infarction patients begins with visual evaluation of MRI (magnetic resonance image). It is well known now that the diffusion based MRI sequences can detect the abnormality within minutes after the onset of severe ischemia in the brain tissue. However, the differences in the MRI scan machinery, display software and filing methods may make the visual interpretation of the MRI images sometimes inconsistent. The diffusion data are more useful when presented as a comparison to those in the identical area of the other side of the brain, because in this way, all the hardware related inconsistency can be removed. The comparison utilizes a ratio of the MRI data, particularly the data capable of determining the degree of water molecule diffusion in the tissue such as DWI (Diffusion Weighted Image) and ADC (Apparent Diffusion Coefficient). The ratio is calculated by dividing the data in the pathological side by those in the normal side and designated as rDWI (relative DWI) and rADC (relative ADC).

The cells in severely ischemic brain tissue swell due to accumulation of water and electrolytes in the cells, immediately after the Na pump fails. The swelling reduces the extracellular space where the free motion of water molecules was a major source of the tissue diffusion. Thus, MRI indices (rADC and rDWI) deteriorate within minutes after the Na pump failure and continue to get worse for the first 3 to 5 days in the infarcted brain tissue [], unless recanalization or restoration of blood flow occurs []. The deterioration of the indices is characterized by the initial rDWI increase (initial hump) up to 2.5 or higher and the initial rADC decrease (initial dip) down to 0.6 or below [], reaching to a lowest value on Day3 []. Then, both indices gradually return to close to a normal level or 1.0, despite of the fact that the tissue is already infarcted (pseudonormalization) in 10 to 11 days (pseudo normalization time) after the ischemic ictus in the white matter []. After the pseudonormalization, rADC continues to increase (late hike) for many months [,]. However, recanalization treatment alters this natural course dramatically and the hump and the dip of diffusion related MRI indices may not appear at all and the pseudonormalization time shortens significantly down to 24 hrs or less after the treatment [,], only when the recanalization successfully restores the blood flow in the area. Although recanalization treatment such as with tPA (tissue plasminogen activator) is the most potent treatment of all for acute cerebral infarction, the treatment needs to be started within 3 hrs after the onset of the symptoms and has to satisfy rigid criteria. Therefore, except for few lucky tPA treated patients, the majority of the acute cerebral infarct patients are currently treated with diverse medications, including scavengers of reactive oxygen species (ROS). The ROS aggravate the ischemic tissue by a self-propagating chain reaction of depriving another electron from near-by molecules. In Japan, Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) [] is the only medication approved since 2001 for the use in acute stage of cerebral infarction patients as a scavenger of hydroxyl radicals and a neuroprotectant [].

However, in our preliminary study, the treatment of acute cerebral infarction with Edaravone improved the initial hump and the initial dip of the MRI indices only slightly and it shortened the pseudo normalization time but rather mildly. Edaravone is known to have a rather short t1/2 beta, or elimination half life of the drug level, particularly in elderly patients who occupy a majority of cerebral infarction population. In addition, Cmax, or maximum drug concentration in the blood, of the Edaravone, with currently approved intravenous administration of 30 mg remains at about 1/10 level of a standard 1-10 micromole concentration used in many in vitro experiments. In addition, because of possible side effects, Edaravone may not be given to the patients who have compromised liver or kidney function and also not more than twice a day according to the governmental approval. On the other hand, molecular hydrogen, which is well known to have potent scavenger actions against hydroxyl radicals and related harmful oxidation [] had no risk of complications in our preliminary study even on the patients who had already established kidney or liver disease. Our current study was designed to supplement possible low and short blood level of Edaravone with hydrogen for the treatment of acute cerebral infarction. The effects of the supplementing with hydrogen were evaluated by comparing the results of the treatment in a group treated with Edaravone only (E group) and in a combined Edaravone and hydrogen group (EH group) and also against the natural course published in the literatures []. Since subtle neurological changes after cerebral infarction during the acute stage are sometimes difficult to substantiate, a totally objective method using MRI indices, rADC and rDWI, was adopted for the evaluation. These indices were calculated at the infarction sites of the patients serially and averaged and compared daily in the two groups. In addition, regular neurological evaluation of the patients was done mainly with NIHSS (NIH stroke score).

Methods

Patients

Consecutive 34 patients who were diagnosed as having cerebral infarction of BAD type (branch atheromatous disease) in the brainstem were enrolled in the study. All of these patients lived in the local area of our hospital and were brought in within 4 to 24 hours after the onset of the symptoms. The first 26 patients were treated with Edaravone alone (E group) and the following 8 patients received hydrogen-rich intravenous fluid in addition to Edaravone (EH group). For the EH group of 8 patients, intravenous Edaravone (30 mg Edaravone Kit) was given at 6 AM and 6 PM as a regular schedule and hydrogen-rich intravenous solutions were added at 10 AM and 4 PM. These treatments lasted for 7 days. Neurological status was recorded essentially with NIHSS and compared at the time of admission and discharge from the hospital. The neurological evaluation was based upon the NIHSS method and was equally performed in the two groups. Since the dramatic and substantial improvements in clinical conditions and MRI indices after recanalization may overwhelm any effects of other medications, only those patients who were diagnosed as stroke due to branch atheromatous disease (BAD), which is a non-recanalization type cerebral infarction, in the brainstem were recruited. BAD involves perforating arteries particularly at lateral striate artery (LSA) region or at parapontine artery (PPA) region and is known as a type of progressive stroke [] also.

The informed consent in a form approved by the Nishijima Hospital Ethics Committee was obtained from all the patients before the treatment or from their legal guardians when the patients could not sign the consent, by the time of initiation of the treatment.

Production of hydrogen-rich intravenous fluid

Regular intravenous fluid bags were immersed, without opening the bag and without adding any alteration on the bag, in a hydrogen water tank which is capable of producing hydrogen-rich water up to 1.6 ppm concentration (Miz.Co, Fujisawa, Japan, Patent No.4486157, Patent Gazette of Japan 2010). The hydrogen concentration increased in the bag by diffusion through the totally intact wall of the plastic bag to more than 250 micromole/L and to saturation, depending upon the duration of the immersion and temperature. A saline bag of 250 ml size (Terumo Co. Tokyo, Japan) and a maltose solution bag of 200 ml size (Airomu Co. Atsugi, Japan) were chosen according to the highest diffusibility of the bag wall we could find.

MRI analysis

MRI signal intensities in DWI and ADC of each infarction site were observed first and then, serial changes of these images were compared in the E group and the EH group. The DWI and ADC signal intensities were also compared with those in the exactly same area of the other side of the brain and the ratio was calculated as rDWI (relative DWI) and rADC (relative ADC). Averages of these indices were compared in the two groups and also with the previous publications by using the data in the literature [] for a statistical significance. A special attention was paid for the determination of abnormal area. Firstly, all of the MRI images of the patient were reviewed and the largest area of the abnormality was chosen to be the site and size of the lesion for the calculation and the pixel size of the area were recorded. Then, the area was copied on a transparent film together with surrounding recognizable structures as a template, which was used for calculation of the remainder of MRIs. This is to prepare, in case of size changes of the abnormality or even disappearance of the abnormality, to calculate the indices exactly in the same area and in a same manner. If an ADC map was not distinct enough by the naked eye, then the DWI template was used to define the area of abnormality. The MRI scan was taken on the day of admission (Day1) and follow-up MRIs were scheduled to be taken every other day but this could not be accomplished in every patient when other tests such as patient’s vascular evaluation or cardiopulmonary function test were thought to be more urgent.

The study was approved by Nishijima Hospital Ethics Committee and the production of hydrogen rich IV fluid as “Hospital Preparation” and its clinical use in Nishijima Hospital, were conducted upon the advice from Nishijima Hospital Pharmacists Council and Japanese Welfare-Labour Administration (Tokai-Hokuriku District Bureau) and Sizuoka Prefectural Administration (Pharmaceutical Affair, Regulatory Audit Section).

Results

MRI images (DWI and ADC) of infarction areas and comparison of the images in the E group (treated with Edaravone only, Figure Figure11 upper) and the EH group (treated with a combination of Edaravone and hydrogen, Figure Figure11 lower)

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Serial MRI changes in the upper brain stem lesion slices (1st & 3rd row) and lower brain stem lesion slices (2nd and 4th row) of DWI (1st & 2nd row) and ADC (3rd & 4th row) imagesupper. Serial MRI of a representative patient in E group on Day 1, 3, 6 (left to right). The lesion involved two adjacent slices at the upper (1st row) and lower (2nd row) brain stem. The DWI signal intensity (whiteness) of the upper slice increased on Day3 (presence of the initial hump), but remained almost unchanged on Day 1,3& 6 in the lower slice (2nd row) by the naked eye. The reduced ADC signal intensity (blackness) of the same lesion was seen even on Day6, particularly in the lower lesion slice (4th row). lower. Serial MRI of a representative patient in EH group on Day1, 2, 7, 9 (left to right). The lesions also involved two adjacent slices. The DWI signal intensity of the upper slice (1st row) was seen on Day1 but was invisible on the Day2 &7 (absence of the initial hump). The initial hump was seen only in the anterior part of the lower lesion slice (2nd row) but not in the posterior-lateral extension of the lesion towards the cerebellum which had disappeared on Day2 & 7(absence of the initial hump). The ADC signal was clearly darker in the lower brainstem lesion (4th row) on Day 2 but disappearing on Day7 and became grey colour on Day9 (shortened pseudonormalization time and late hike, 4th row, right end).

The results were firstly evaluated by MRI images (DWI and ADC) without indices (Figure (Figure1).1). The DWI images generally showed increased signal intensity (appeared with more whiteness) at the infarction sites in both groups. The ADC images, on the other hand, showed decreased signal intensity (appeared with more blackness) at the lesion sites, which were rather difficult to see as compared to the lesions in DWI images. These signal intensities of the lesions in the E group and the EH group differed obviously in many cases but in some cases, the differences were rather subtle when compared by single images and by the naked eye. However, when these single images were arranged serially, the differences between the two groups became more apparent and the initial hump, the initial dip and the pseudonormalization time could be assessed even without the indices, after getting used to the visual evaluation. In the E group, the DWI signal intensities increased from Day3 to Day7 in most cases (Figure (Figure11 upper, 1st row) and the change was confirmed to be the initial hump by the rDWI. However, in the EH group, the increase was significantly less and in some cases, no increase was seen at all (absence of the initial hump, Figure Figure11 lower, 1st row). In addition, in the E group, the increase lasted longer than 9 days, which was regarded as the lack of shortening of the pseudonormalization time (Figure (Figure11 upper, 2nd row) and this was also confirmed by indices. In the EH group, however, the increase returned to a normal level by Day 9 in many cases (the shortened pseudonormalization time, Figure Figure11 lower, 1st and 2nd row).

The ADC images when observed in a serial manner also showed substantial differences between the E group and the EH group. The degree of reduction of the ADC signal intensities at the lesion sites was less in EH group (Figure (Figure11 lower, 3rd and 4th row) and then, increased to the normal level within Day9, which qualified for the shortening of the pseudonormalization time. On the contrary, in the E group, the ADC image at the lesion site was darker and lasted longer without returning to a normal level within 9 days (lack of shortening of the pseudonormalization time, Figure Figure11 upper, 3rd and 4th row). The dark ADC intensity at the lesion site became greyish in colour after 9 days in the EH group and the whiteness gradually increased further (late hike) afterwards. In many lesions where the differences were not obvious by the naked eye, the evaluation by the indices still demonstrated significant differences. For an example, in the upper brain stem lesion of the E group (Figure (Figure11 upper, 1st row), the initial hump was not too obvious by the naked eye but the indices (rDWI) were above the normal level of 1.20 on Day3 and Day5 (1.54 and 1.30, respectively), indicating the presence of the initial hump. Since ADC images are more difficult to evaluate by the naked eye, the lack of the pseudonormalization of the lesions such as in the Figure Figure11 upper, 3rd and 4th row could only be evaluated by the indices (rADC), which, at these lesions, had changed from 0.48 to 0.31 to 065 (3rd row) and 0.79 to 0.39 to 0.82 (4th row) on Day1, Day3 and Day6, respectively. All of these indices were below the normal level of 0.9 and remained depressed longer than Day10 and therefore the changes were regarded as showing the lack of the pseudonormalization (or failure of shortening of the pseudonormalization time). On the other hand, the presence of shortened pseudonormalization time in the EH group was shown by the both indices as in Figure Figure11 lower lesions. The lesions showed the initial hump of rDWI (2nd row, 2.03) and the initial dip of rADC (4th row, 0.54) on Day2 but these data improved to 1.14 (rDWI, as compared to the normal value of less than 1.2) and to 2.50 (rADC, as compared to the normal value of more than 0.9), by the Day9 (therefore, the shortened pseudonormalization time and late hike).

Serial rDWI averages in the E group (treated with Edaravone only) and in the EH group (treated with a combination of Edaravone and hydrogen (Figure (Figure22 upper)

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Serial changes in rDWI (upper) and rADC (lower)upper: Daily averages of rDWI in the E group patients showed a mild initial hump (Day4 to Day8, up to 2.2) but remained less than a natural course (rDWI of 2.5, Huang et al []). In the EH group, the initial hump was not seen (p < 0.05 on the Day 5, 8 and 9). No shortening of the pseudonormalization time was seen in E group (the rDWI average remained above 1.2 by Day9). In the EH group, the rDWI averages on Day 8 reached the normal level of 1.2 (shortened pseudonormalization time). Lower: Daily averages of rADCs in the E group patients showed a mild initial dip (Day4 to 7). In the EH group, the initial dip was rather short lived on Day 5 but no data available on Day6 & 7. No pseudonormalization of the rADC was noted within 9 days in the E group. In the EH group, however, the shortening was seen on Day 9. Then, the rADC of EH group increased (late hike). The differences of the rADC in the two groups reached a statistical significance on the Day5, 8 and 9.

Daily averages of rDWI in the E group patients showed a definite initial hump (above 1.2) between Day4 and Day8. However, the highest rDWI averages of the E group remained at 2.1 levels and did not deteriorate as high as 2.5, as in the natural course [] and the difference was statistically significant on Day4 (Figure (Figure22 upper). On the other hand, the initial hump was not seen in the EH group and the difference was significant (p < 0.05) on the Day5, 8 and 9 (absence of the initial hump). The rDWI averages of the E group did not fall below a normal level of 1.2 by Day10 and thus failed to shorten the pseudonormalization time. However, in the EH group, the rDWI averages on Day8 and Day9 reached 1.2 or less and thus qualified for the shortening of the pseudonormalization time. These findings indicate that the treatment in the E group did not abolish the initial hump and did not shorten the pseudonormalization. However, both conditions were accomplished in the EH group and in this sense, although the differences may appear rather minuscule, the results of the treatment in EH group was superior to those of the E group, when evaluated by the rDWI. The degree of the initial hump of the E group was significantly less and better than that of the natural course, however.

Serial rADC averages in the E group (treated with Edaravone only) and in the EH group (treated with a combination of Edaravone and hydrogen) (Figure (Figure22 lower)

Daily averages of rADCs in the E group patients showed the initial dip on the Day4 and Day5. In the EH group, however, the initial dip appeared to be delayed and rather short lived on the Day5 and possibly on the Day6 or Day7 but no data available during this period. These patients were usually scheduled for MRA (MRI angiogram) of the cervical carotid artery on the Day3 and other cardiopulmonary studies on Day6 or Day7 and the lack of the MRI data on these hospital days made it difficult to assert the duration of the short lived initial dip. Definite pseudonormalization of the rADC was not noted within 10 days in the E group while in the EH group, the shortening of the pseudonormalization time was seen on Day9. The rADC of the EH group increased gradually afterwards (late hike). The difference of the daily averages between the E group and the EH group reached a statistical significance on the Day5, 8 and 9. The results of the treatment in EH group were, therefore, superior to those of E group when evaluated by the rADC also.

Neurological outcomes in the E group (treated with Edaravone only) and in the EH group (treated with a combination of Edaravone and hydrogen)

The neurological conditions of the patients recorded on the Day1 and at the time of discharge from the hospital were compared. There were 4, 2 and 20 patients, who were regarded as improved, worse and unchanged, respectively, in the E group. However, all of the patients in the EH group were regarded as unchanged, except one patient who had a very high blood sugar from uncontrolled diabetes and got worse. The neurological evaluation was based upon NIHSS and if the score did not show any change, then, the result of the MMT was added. The difference of the neurological changes in the two groups was statistically not significant.

Discussion

MRI analysis

Since MRI scan is an essential part of the diagnosis of the cerebral infarction patients, the effects of the infarction treatment have frequently been evaluated by the MRI scan also. Previous publications utilized the area of DWI abnormality as an equivalent to the size of infarction. However, it is now well known that areas of the DWI abnormality are consisted of heterogeneous tissues and all of the area of DWI abnormalities may not progress to infarction. The increase in the size and density of the DWI abnormality may not reflect worsening and/or expansion of the infarction because the DWI data include T2 sequence of the MRI. Therefore, the increase may simply reflect the increase in water content of the area from vasogenic edema or from proliferated primitive and leaky neovasculature and the phenomena are inclusively called “T2 shine through” []. Therefore, if the effects of the treatment were analyzed only by the increase or decrease of the size and density of the DWI abnormality, the analysis may falsely conclude the treatment to be ineffective or effective, respectively. The ADC is not influenced by the T2 change and more valuable than DWI. However, since the ischemic tissue abnormality reduces the ADC data and this makes the area of the ADC abnormality very difficult to discern from the surrounding tissue. Therefore, the analysis of the effects of the treatment based upon the size of the DWI/ADC abnormality was thought to be inappropriate and we adopted the current technique. The technique is to calculate the average number of DWI/ADC raw data within the identical area of the brain within the recorded pixel size in all the MRI images obtained during the hospitalization by using a specific template made for each patient. This appeared to have accomplished the calculation in exactly same area of the same size in a consistent manner. This technique has been utilized in pharmacological evaluation of medications in the ischemic brain in the past but mainly in the animal experiments, probably due to difficulty in obtaining frequent MRI scans in clinical settings.

Our study included only brainstem infarction cases because of ease of defining the perimeter of the lesion for the calculation. The brainstem infarctions are usually round or oval in shape and small and very discrete from the surrounding tissue. In addition, the tissue is mainly consisted of white matter and devoid of CSF space. The MRI indices are influenced by the heterogeneity of the tissue [] and particularly by the presence of CSF space in the tissue as in the cerebral cortical lesions.

Neurological evaluation of brainstem infarction patients with NIHSS

All of the patients in the EH group were regarded as neurologically unchanged except one patient after the combined treatment with Edaravone and hydrogen, based upon the NIHSS. However, all of these patients in the EH group except one were satisfied with significant improvement of their preadmission symptoms by the time of discharge from the hospital. NIHSS is the most reliable and most accepted neurological scoring system for stroke patients which is calculated and recorded after performing well described and rather simple neurological examinations. However, these examinations are heavily weighted for the evaluation of anterior circulation stroke. Major symptoms of our brainstem stroke patients were due to posterior circulation abnormality and included dizzy sensation, vertigo without nystagmus, vague and subjective paresthesia of one side of the body with normal touch sensation, difficulty in walking from some swaying and staggering sensation but with normal knee to heel tests, normal diadochokinesis and normal muscle strength, in addition to some sensation of swallowing difficulty with normal gag reflex etc. None of these symptoms are calculable by NIHSS and therefore, the patient’s satisfaction in the EH group was not reflected as improvement in the NIHSS.

Effects of hydroxyl radical scavengers, Edaravone and hydrogen on cerebral infarction

The beneficial effects of Edaravone in the treatment of cerebral infarction have been well established []. Edaravone is known for its unique property with both water and lipid solubility and has potent scavenger action against hydroxyl and peroxynitrite radicals and ROS []. It acts also in reducing the brain edema of the ischemic brain tissue by protecting endothelial cells from ROS and by keeping integrity of the blood brain barrier and also by reducing the inflammatory responses in the ischemic area of the brain []. Initially, Edaravone was thought to be a simple quencher of the radicals but later many neuroprotective properties were found [,], and effectiveness in many organs and many disease conditions are added [,]. Currently, it is recognized as a most effective scavenger of radicals and also neuroprotective agents in Japanese neurosurgical community but additional clinical studies were discussed in the U.S.A [].

Hydrogen is also known as a potent scavenger of the hydroxyl and peroxynitrite radicals and does not affect NO production which is advantageous to the ischemic brain tissue. The investigational and clinical interests have been promulgated recently by epochal articles [] and a review []. Direct actions of hydrogen on extracellular and intracellular hydroxyl radical provide protection of mitochondria and nuclear DNA but hydrogen does not harm other cellular elements which relate to signal transduction. When hydrogen was given during reperfusion in an animal ischemic brain model, it protected ischemia-reperfusion injury of the brain, although only when hydrogen was given during the reperfusion but not during the ischemic period. However, these effects were actually better than those of Edaravone and FK506 combination []. Since FK506 alone is known to decrease the ischemic brain size, it is remarkable that hydrogen superseded the effects of the combination. In addition, hydrogen demonstrated extended effectiveness in many other organs and in various situations such as in diabetes[], intestinal grafts[], tumor growth inhibition [], allograft nephropathy[], cardiac ischemia/reperfusion[], sepsis [], liver injury [], haemodialysis[], spinal cord injury[], an animal model of Parkinson’s disease[] and Alzheimer’s disease[], in addition to health promotion []. Therefore, there is nothing to indicate that hydrogen is inferior to Edaravone for the treatment of cerebral infarction and it is quite possible that a single use of hydrogen is as effective as Edaravone treatment and probably much safer. However, it would be an unethical conduct until larger controlled clinical studies accumulate more evidences, because of limitations of our study. However, if the advantages in the EH group of current study were substantiated in the future studies, the advantages may be due to the increased frequency of administration of the radical scavengers as was in EH group (4 times per day vs. 2 times per day), and/or direct hydrogen effects on the inflammatory cells, chemokines and growth and antiapoptoic factors and/or a direct neutralizing action on the residual radical substances of intermediate Edaravone metabolites in ischemic and hypoxic brain tissue. Edaravone putatively provides electrons and becomes a radical by itself until it reacts with oxygen and then changes, through Edaravone peroxyl radical, to a non-radical material, 2-oxo-3-(phenylhydrazono)-butanoic acid (OPB) [] which may accumulate in the brain eventually. Hydrogen may have interacted with those intermediate radical products favourably and provided better MRI changes in our study. At the beginning of this study, our concerns included the government approved and recommended Edaravone dose (60 mg/day for 2 weeks = 840 mg) and subsequent blood level dynamics. It is interesting that a currently on going Phase 2 study in Europe increased the Edaravone doses from 840 mg to 1000 mg and 2000 mg []. The results of the study may solve some of our concerns.

The limitations of our study include a non-controlled way of patient selection, inclusion of rather small number of the patients particularly in the combined group, use of current NIHSS for neurological evaluation for the brainstem infarction, lack of long term follow-up etc. We are organizing a new study to improve these limitations currently.

Conclusions

Administration of hydroxyl radical scavengers in acute stage of brainstem infarction improved MRI indices (rDWI, rADC) against the natural course. The favourable effects were more obvious and significant in the EH group (a combined group of Edaravone and hydrogen) as compared to the E group (Edaravone alone group). These findings may imply the need for more frequent daily administration of hydroxyl radical scavenger, or possible presence of additional hydrogen effects on scavenger mechanisms

 

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Link to Publisher's site
. 2011; 1: 12.
Published online 2011 Jun 7. doi: 10.1186/2045-9912-1-12
PMCID: PMC3231971
PMID: 22146068
Improved brain MRI indices in the acute brain stem infarct sites treated with hydroxyl radical scavengers, Edaravone and hydrogen, as compared to Edaravone alone. A non-controlled study
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Competing interests

The authors declare that they have no competing interests and were not compensated at all by any pharmaceutical and biotechnology company or any other companies to contribute this article to the peer-reviewed scientific literature.

Authors’ contributions

The authors equally contributed to the production of this article and have read and approved the final manuscript.

Acknowledgements

The authors would like to thank Miz Company for technical assistance for setting up the hydrogen water tank and initial measurement of hydrogen concentration in the intravenous fluid bag.

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role of molecular hydrogen H2 water in the regression of HYPERCHOLESTEROLEMIA and ATHEROSCLEROSIS

Abstract

CONTEXT:

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

OBJECTIVE:

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

DESIGN:

This was a case-control study.

SETTING:

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

PATIENTS:

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

RESULTS:

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

CONCLUSIONS:

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

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

Author information

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

 

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

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

We have found that molecular hydrogen (dihydrogen; H2) water has beneficial lipid-lowering effects in high-fat diet-fed Syrian golden hamsters.

The objective of this study was to characterize the effects of molecular hydrogen H2-rich water (0.9-1.0 l/day) on the content, composition, and biological activities of serum lipoproteins on 20 patients with potential metabolic syndrome.

Serum analysis showed that consumption of molecular hydrogen H2-rich water for 10 weeks resulted in decreased serum total-cholesterol (TC) and LDL-cholesterol (LDL-C) levels.

Western blot analysis revealed a marked decrease of apolipoprotein (apo)B100 and apoE in serum.

In addition, we found molecular hydrogen water H2 significantly improved HDL functionality assessed in four independent ways, namely:

i) protection against LDL oxidation,

ii) inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to endothelial cells,

iii) stimulation of cholesterol efflux from macrophage foam cells, and

iv) protection of endothelial cells from TNF-α-induced apoptosis.

Further, we found consumption of molecular hydrogen H2-rich water resulted in an increase in antioxidant enzyme superoxide dismutase and a decrease in thiobarbituric acid-reactive substances in whole serum and LDL.

In conclusion, supplementation with molecular hydrogeb H2-rich water seems to decrease serum LDL-C and apoB levels, improve dyslipidemia-injured HDL functions, and reduce oxidative stress, and it may have a beneficial role in prevention of potential metabolic syndrome

Song G1, Li M, Sang H, Zhang L, Li X, Yao S, Yu Y, Zong C, Xue Y, Qin S. Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL function in patients with potential metabolic syndrome.
1, Key Laboratory of Atherosclerosis in Universities of Shandong, Shandong, China.
PMID: 23610159
PMCID: PMC3679390
DOI: 10.1194/jlr.M036640
[Indexed for MEDLINE]

Free PMC Article

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5525017/

 

Molecular hydrogen (water) benefits/effects for METABOLIC SYNDROME

 

The objective of this study was to examine the effectiveness of molecular hydrogen rich water (1.5-2 L/day; molecular hydrogen concentration; 0.55-0.65 mM)) in an open label, 8-week study on 20 human subjects with potential metabolic syndrome.

Metabolic syndrome is characterized by cardiometabolic risk factors that include obesity, insulin resistance, hypertension and dyslipidemia. Oxidative stress is known to play a major role in the pathogenesis of metabolic syndrome.

The consumption of hydrogen rich water for 8 weeks resulted in a 39% increase (p<0.05) in antioxidant enzyme superoxide dismutase (SOD) and a 43% decrease (p<0.05) in thiobarbituric acid reactive substances (TBARS) in urine.

Further, subjects demonstrated an 8% increase in high density lipoprotein (HDL)-cholesterol and a 13% decrease in total cholesterol/HDL-cholesterol from baseline to week 4.

There was no change in fasting glucose levels during the 8 week study.

In conclusion, drinking molecular hydrogen rich water represents a potentially novel therapeutic and preventive strategy for metabolic syndrome.

 

 

 2010 Mar;46(2):140-9. doi: 10.3164/jcbn.09-100. Epub 2010 Feb 24.
Effectiveness of hydrogen rich water on antioxidant status of subjects with potential metabolic syndrome-an open label pilot study.

Heart, Lung and Esophageal Surgery Institute, Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, 15213, USA.

molecular hydrogen water improves lipid and glucose metabolism in patients with TYPE 2 DIABETES or impaired glucose tolerance

Supplementation of molecular hydrogen-rich water improves lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance.

It is well established that molecular hydrogen (water) has a selective oxidation/free radical reducing action.

Oxidative stress is recognized widely as being associated with various disorders including diabetes, hypertension, and atherosclerosis.

We therefore investigated the effects of molecular hydrogen-rich water intake on lipid and glucose metabolism in patients with either type 2 diabetes mellitus (T2DM) or impaired glucose tolerance (IGT).

We performed a randomized, double-blind, placebo-controlled, crossover study in 30 diabetes patients with T2DM controlled by diet and exercise therapy and 6 patients with IGT.

The diabetes patients consumed either 900 mL/d of  hydrogen-rich pure water or 900 mL of placebo pure water for 8 weeks, with a 12-week washout period. Several biomarkers of oxidative stress, insulin resistance, and glucose metabolism, assessed by an oral glucose tolerance test, were evaluated at baseline and at 8 weeks.

Intake of  hydrogen-rich water was associated with significant decreases in the levels of modified low-density lipoprotein (LDL) cholesterol (ie, modifications that increase the net negative charge of LDL), small dense LDL, and urinary 8-isoprostanes by 15.5% (P < .01), 5.7% (P < .05), and 6.6% (P < .05), respectively.

Hydrogen-rich water intake was also associated with a trend of decreased serum concentrations of oxidized LDL and free fatty acids, and increased plasma levels of adiponectin and extracellular-superoxide dismutase. In 4 of 6 patients with IGT, intake of molecular hydrogen-rich water normalized the oral glucose tolerance test.

In conclusion, these results suggest that supplementation with molecular hydrogen-rich water may have a beneficial role in prevention of T2DM and insulin resistance.

hydrogen-enriched water for mitochondrial and inflammatory MYOPATHIES

Molecular hydrogen has prominent effects on more than 30 animal models especially of oxidative stress-mediated diseases and inflammatory diseases. In addition, hydrogen effects on humans have been reported in diabetes mellitus type 2, hemodialysis, metabolic syndrome, radiotherapy for liver cancer, and brain stem infarction. Molecular hydrogen effects are ascribed to specific radical-scavenging activities that eliminate hydroxyl radical and peroxynitrite, and also to signal-modulating activities, but the detailed molecular mechanisms still remain elusive. Molecular hydrogen is a safe molecule that is largely produced by intestinal bacteria in rodents and humans, and no adverse effects have been documented.

Methods

We performed open-label trial of drinking 1.0 liter per day of molecular hydrogen-enriched water for 12 weeks in 5 patients with progressive muscular dystrophy (PMD), 4 patients with polymyositis/dermatomyositis (PM/DM), and 4 patients with mitochondrial myopathies (MM), and measured 18 serum parameters as well as urinary 8-isoprostane every 4 weeks.

We next conducted randomized, double-blind, placebo-controlled, crossover trial of 0.5 liter per day of hydrogen-enriched water or placebo water for 8 weeks in 10 patients with dermatomyositis  DM and 12 patients with mitochondrial myopathies MM, and measured 18 serum parameters every 4 weeks.

Results

In the open-label trial, no objective improvement or worsening of clinical symptoms was observed. We, however, observed significant effects in lactate-to-pyruvate ratios in progressive muscular dystrophy PMD and mitochondrial myopathies MM, fasting blood glucose in progressive muscular dystrophy PMD, serum matrix metalloproteinase-3 (MMP3) in polymyositis/dermatomyositis PM/DM, and serum triglycerides in polymyositis/dermatomyositis PM/DM.

In the double-blind trial, no objective clinical effects were observed, but a significant improvement was detected in lactate in mitochondrial myopathies  MM. Lactate-to-pyruvate ratios in  mitochondrial myopathies MM and MMP3 in dermatomyositis DM also exhibited favorable responses but without statistical significance.

No adverse effect was observed in either trial except for hypoglycemic episodes in an insulin-treated MELAS patient, which subsided by reducing the insulin dose.

Conclusions

Molecular hydrogen-enriched water improves mitochondrial dysfunction in mitochondrial myopathies  MM and inflammatory processes in polymyositis/dermatomyositis  PM/DM.

Less prominent effects with the double-blind trial compared to the open-label trial were likely due to a lower amount of administered molecular hydrogen and a shorter observation period, which implies a threshold effect or a dose-response effect of molecular hydrogen.

Background

Ohsawa and colleagues first reported an effect of molecular hydrogen gas on cerebral infarction in June 2007 []. Effects of molecular hydrogen administered in the forms of inhaled gas, drinking water, instillation, and intraperitoneal injection have been reported for 31, 4, and 5 diseases in animal models, cells, and humans, respectively [].

Molecular hydrogen exhibits prominent effects especially on oxidative stress-mediated diseases and inflammatory diseases in rodents. Molecular Hydrogen scavenges hydroxyl radicals and less efficiently peroxynitrite []. The radical-scavenging activities, however, are unlikely to be an exclusive mechanism, because the amount of radical oxygen species generated in rodents and humans is much more than the amount of molecular hydrogen molecules taken up by the body. Indeed, the amount of molecular hydrogen taken up by drinking molecular hydrogen-enriched water (HEW) is 100 or more times less than that by inhaling 2% molecular hydrogen gas, but drinking molecular hydrogen-enriched water HEW exhibits beneficial effects as good as or even better than inhaling 2% molecular hydrogen gas in rodents [], which suggests the lack of a simple dose-response effect.

Our previous study on type 1 allergy also indicates that molecular hydrogen suppresses type 1 allergy by acting as a gaseous signal modulator not as a free radical scavenger [].

Effects of molecular hydrogen in humans have been examined in five studies.

First, a randomized, double-blind, placebo-controlled crossover study of 900 ml/day of molecular hydrogen-enriched water HEW for 8 weeks in 30 patients with diabetes mellitus type 2 demonstrated significant decreases of electronegative charge-modified LDL cholesterol, small dense LDL, and urinary 8-isoprostanes [].

Second, an open-label trial of electrolyzed molecular hydrogen-enriched hemodialysis solution in 9 patients for 4 months [] and 21 patients for 6 months [] showed significant decreases of systolic blood pressure before and after dialysis, as well as of plasma monocyte chemoattractant protein 1 and myeloperoxidase.

see also alkaline ionized water / molecular hydrogen water and hemodialisys

Third, an open-label trial of 1.5-2.0 liters per day of moelcualr hydrogen enriched water HEW for 8 weeks in 20 subjects with metabolic syndrome exhibited a 39% increase of urinary superoxide dismutase (SOD), a 43% decrease of urinary thiobarbituric acid reactive substances (TBARS), an 8% increase of high density lipoprotein (HDL)-cholesterol, and a 13% decrease of total cholesterol/HDL-cholesterol ratio [].

Fourth, a randomized placebo-controlled study of 1.5-2.2 liters/day of molecular hydrogen enriched water HEW for 6 weeks in 49 patients receiving radiotherapy for malignant liver tumors showed marked improvements of QOL scores [].As the study was not blinded, subjective QOL scores tended to be overestimated by a placebo effect, but objective markers for oxidative stress were also significantly decreased.

Fifth, drip infusion of hydrogen-enriched saline in combination with Edaravone, a clinically approved radical scavenger for cerebral infarction, for 7 days in 8 patients with brain stem infarction was compared to 24 such patients receiving Edaravone alone []. Although the study was not randomized and not blinded, MRI markers of patients infused with molecular hydrogen showed significant improvements and accelerated normalization.

Being prompted by the prominent effects of molecular hydrogen on inflammatory diseases and oxidative stress-mediated diseases especially in rodents, we performed an open-label trial of drinking 1.0 liter per day of molecular hydrogen enriched water  HEW for 12 weeks in 14 patients with muscle diseases, and identified improvement in four parameters: (i) a decrease of the lactate-to-pyruvate ratio in mitochondrial myopathies (MM) and progressive muscular dystrophy (PMD); (ii) a decrease of serum matrix metalloproteinase-3 (MMP3) in polymyositis/dermatomyositis (PM/DM), (iii) a decrease of fasting glucose in PMD, and (iv) a decrease of serum triglycerides in polymyositis/dermatomyositis  PM/DM.

We then conducted a randomized, double-blind, placebo-controlled, crossover trial of 0.5 liter per day of molecular hydrogen enriched water HEW for 8 weeks in 12 mitochondrial myopathies MM and 10 dermatomyositis DM cases. We observed that molecular hydrogen enriched water HEW significantly improved serum lactate in mitochondrial myopathies MM. In both studies, some patients reported subjective improvement of fatigability, diarrhea, and myalgia, but others reported floating sensation and worsening of diarrhea.

We observed no objective improvement or worsening of clinical symptoms during each study.

Our studies imply that molecular hydrogen enriched water HEW improves clinical parameters in mitochondrial myopathies  MM and polymyositis/dermatomyositis  PM/DM, but 0.5 liter/day for 8 weeks is likely to be insufficient to demonstrate statistically significant effects.

Patients and methods

Patients

For the open-label trial, we recruited 5 patients with progressive muscular dystrophy PMD, 4 patients with polymyositis/dermatomyositis PM/DM, and 5 patients with mitochondrial myopathies MM.

The progressive muscular dystrophy PMD patients comprised 1 male with Miyoshi myopathy and 4 females with limb girdle muscular dystrophy type 2B with an average age and SD of 50.4 ± 15.9 years (range 25 – 66).

The polymyositis/dermatomyositis PM/DM patients comprised 2 males and 2polymyositis/dermatomyositis females with an average age of 53.8 ± 24.8 years (range 29 – 83). All the polymyositis/dermatomyositis PM/DM cases were taking 5 – 10 mg of prednisolone per day and were well controlled.

The mitochondrial myopathies MM patients comprised 4 cases with MELAS (2 males and 2 females with an average age of 45.8 ± 12.3 years, range 37 – 64) and a 54-year-old female with chronic progressive external ophthalmoplegia (CPEO).

For the randomized, double-blind, placebo-controlled, crossover trial, we recruited 12 patients with mitochondrial myopathies MM and 10 patients with dermatomyositis DM.

The mitochondrial myopathies  MM patients comprised 5 cases with MELAS (2 males and 3 females with an average age of 44.6 ± 17.6 years, range 20 – 65), as well as 7 cases with CPEO (3 males and 4 females with an average age of 49.1 ± 11.1 years, range 29 – 61).

The dermatomyositis DM patients comprised 3 males and 7 females with an average age of 49.6 ± 13.7 years (range 32 – 66). All thdermatomyositis e DM patients were well controlled with 5 – 10 mg prednisolone per day.

3 mitochondrial myopathies MM and 3 DM dermatomyositis patients participated in both trials. Both trials were approved by the Ethical Review Board of the Aichi Medical University. Informed consent was obtained from each patient.

Protocols

We purchased 500 ml molecular hydrogen enriched water HEW or placebo water in aluminum pouch from Blue Mercury Inc. (Tokyo, Japan). We measured molecular hydrogen concentrations using an H2-N molecular hydrogen needle sensor attached to a PA2000 2-Channel Picoammeter (Unisense Science, Aarhus, Denmark). The molecular hydrogen concentrations were ~0.5 ppm (~31% saturation). We also confirmed that molecular hydrogen in placebo water was undetectable with our system. For each trial, we instructed patients to evacuate the air from the pouch and to close a plastic cap tightly every time after they drink water to keep the molecular hydrogen concentration as high as possible.

For the open-label trial, patients took 1.0 liter per day of molecular hydrogen enriched water HEW in five to ten divided doses for 12 weeks. We measured 18 serum and one urinary parameters and recorded clinical symptoms at 0, 4, 8, 12, 16 weeks.

For the double-blind trial, patients took 0.5 liter per day of molecular hydrogen enriched water HEW or placebo water in two to five divided doses for 8 weeks. Between the 8-week trials with molecular hydrogen enriched water HEW and placebo, we placed a 4-week washout period. We measured 18 serum parameters and recorded clinical symptoms at 0, 4, 8, 12, 16, 20, 24 weeks. In the double-blind trial, we did not measure urinary 8-isoprostane levels.

The data were statistically analyzed using one-way repeated measures ANOVA for the open-label trial and two-way repeated measures ANOVA for the double-blind trial, both followed by the Bonferroni’s multiple comparison test using Prism version 4.0c (Graphpad Software, San Diego, CA).

Results

Open-label trial

Fourteen patients with PMD, PM/DM, and MM participated in the study and no patient dropped out of the study. Patients took 1.0 liter of molecular hydrogen enriched water HEW for 12 weeks and we measured 18 serum and one urinary parameters every 4 weeks (Table (Table1).1).

We observed no objective improvement or worsening of clinical symptoms during the study. All the patients reported increased micturition frequency. Two MELAS patients reported improvement of fatigability, and another MELAS patient complained mild occasional floating sensation. We estimated statistical significance using one-way repeated measures ANOVA analysis and detected five parameters (Figure (Figure1).1). Serum lactate-to-pyruvate (L/P) ratios of MM patients were high before the study, and were decreased during the study (Figure (Figure1A).1A). Serum L/P ratios and fasting glucose levels of PMD patients were elevated after the study, but the values were still within normal ranges (Figures (Figures1B1B and and1C).1C). Serum MMP3 levels of DM patients were decreased down to 72.9% of those before HEW, which were again increased after the study (Figure (Figure1D).1D). Serum triglyceride levels of DM patients were elevated after the study (Figure (Figure1E1E).

Table 1

Open-label trial of HEW in 14 myopathic patients

Figure 1

Temporal profiles of four parameters that demonstrate statistical significance by one-way repeated measures ANOVA in the open-label trial. Ratios of serum lactate/pyruvate (L/P) in 5 mitochondrial myopathies (MM) patients (A) and 4 progressive muscular 

Randomized, double-blind, placebo-controlled, crossover trial

Twelve MM and ten DM patients participated in the study and no patient dropped out of the study. Patients took 0.5 liter of molecular hydrogen enriched water HEW or placebo water for 8 weeks and we measured 18 serum parameters every 4 weeks (Table (Table2).2). An MM patient reported increased micturition frequency on HEW. A DM patient reported subjective improvement of fatigability and diarrhea on molecular hydrogen enriched water HEW, but an MM patient rather complained increased diarrhea at first on molecular hydrogen enriched water HEW. Another DM patient reported an improvement of myalgia on molecular hydrogen enriched water HEW. A MELAS patient had hypoglycemic episodes only on molecular hydrogen enriched water HEW, but the episodes subsided after the insulin dose was decreased. We observed no objective improvement or worsening of clinical symptoms during the study. Two-way repeated measures ANOVA analysis revealed that only serum lactate levels were significantly decreased in MM by molecular hydrogen enriched water HEW (Figure (Figure2A).2A). Temporal profiles of serum L/P ratios in MM (Figure (Figure2B)2B) and of serum MMP3 levels in DM (Figure (Figure2C)2C) also demonstrated favorable responses to molecular hydrogen enriched water HEW but without statistical significance.

Table 2

Randomized, double-blind, placebo-controlled, crossover trial of molecular hydrogen enriched water HEW in 10 DM and 12 MM patients

Figure 2

Temporal profiles of three parameters in the double-blind trial. Serum lactate (A) and L/P ratios (B) in 12 mitochondrial myopathies (MM) patients. (C) Serum MMP3 in 10 dermatomyositis (DM) patients. Patients took molecular hydrogen enriched water HEW or placebo for 8 weeks. Means and 

Discussion

We performed open-label and double-blind studies of molecular hydrogen enriched water HEW on myopathic patients. In the open-label study, we observed statistical significance of molecular hydrogen water effects in four parameters: L/P ratios in MM and PMD; fasting glucose in PMD; MMP3 in PM/DM; and triglycerides in PM/DM (Figure (Figure1).1).

In the double-blind study, serum lactate levels were significantly improved in MM. L/P ratios in MM and MMP3 in DM were also improved but without statistical significance (Figure (Figure2).2).

Small numbers of participants in both the open-label and double-blind studies might have failed to disclose statistically significant effects of molecular hydrogen enriched water HEW.

In MM, the mitochondrial electron transfer system (mETS) is compromised by mutations in mitochondrial DNA []. This results in a decreased influx of NADH into mETS and elevates NADH levels in the cytoplasm, which facilitates conversion of pyruvate to lactate by lactate dehydrogenase. Thus, lactate and L/P ratio are useful surrogate markers to estimate functions of mETS, and are usually abnormally elevated in MM []. Defective mETS also causes leakage of electrons from mitochondrial inner membranes and increases production of reactive oxygen species (ROS), which further damages mETS [,]. Reduction of the L/P ratios in the open-label and double-blind studies suggests that hydrogen alleviates mETS dysfunction in MM either by scavenging ROS or by yet unidentified signaling mechanisms.

MMP3 belongs to a family of calcium-dependent zinc proteinases induced by cytokines and secreted by inflammatory cells. MMPs enhance T-cell migration and adhesion, and also degrade the extracellular matrix proteins []. MMP3 is increased in a fraction of DM patients []. MMP3 may facilitate lymphocyte adhesion and enhance T-cell-mediated cytotoxicity by degrading extracellular matrix proteins in DM.

molecular hydrogen water improved serum MMP3 levels in the open-label and double-blind studies, which is expected to ameliorate pathogenic inflammatory processes that culminates in muscle fiber destruction.

We observed less prominent effects with the double-blind study compared to the open-label study. The lack of statistically significance in the double-blind study is possibly due to a lower amount of molecular hydrogen enriched water HEW (1.0 vs. 0.5 liter per day) and to a shorter observation period (12 vs. 8 weeks). In the open-label study, drinking 1.0 liter of molecular hydrogen enriched water HEW was not readily accommodated by most myopathic patients. molecular hydrogen does not show simple dose-response relationship in rodents [], and ad libitum administration of even 5%-saturated molecular hydrogen enriched water HEW markedly attenuates development of Parkinson’s disease in mice []. We thus reduced the amount of hydrogen to 0.5 liter in the double-blind trial, and also shortened the observation period to minimize the burden on the participants. This, however, might have masked effects of molecular hydrogen enriched water HEW. Indeed, when we compare studies of diabetes mellitus type 2 [], the current open-label trial, and metabolic syndrome [], the participants took 0.9, 1.0, and 1.5-2.0 liters of molecular hydrogen enriched water HEW, respectively. Ratios of total cholesterol/HDL-cholesterol are available at 8 weeks in all the studies, and are changed to 103.8%, 98.6%, and 95.8%, respectively, of those before molecular hydrogen administration, which is in accordance with a dose-response effect of molecular hydrogen enriched water HEW. Additionally, among the two previous studies [,] and the current open-label and double-blind studies, the most prominent effects are observed with 1.5-2.0 liters of molecular hydrogen enriched water HEW. As drinking a large amount of molecular hydrogen enriched water HEW is not easily accommodated by most patients especially in winter, a threshold effect and/or a dose-response effect should be further elaborated for each pathological state.

Conclusions

molecular hydrogen enriched water HEW is effective for mitochondrial dysfunction in MM and inflammatory processes in DM.

molecular hydrogen may have a threshold effect or a dose-response effect and 1.0 liter or more per day of molecular hydrogen enriched water HEW is likely to be required to exert beneficial effects.

 

Abbreviations

HEW: hydrogen-enriched water; PMD: progressive muscular dystrophy; PM: polymyositis; DM: dermatomyositis; MM: mitochondrial myopathies; CPEO: chronic progressive external ophthalmoplegia; MELAS: mitochondrial myopathy with lactic acidosis and stroke-like episodes; MMP3: matrix metalloproteinase-3.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

TI and KS examined patients and acquired data. MIand TI organized data and performed statistical analysis. MIand KO wrote the paper. MI4, MI5, and KO conceived the study. All authors read and approved the final manuscript.

Acknowledgements

We would like to thank the patients for their participation in these studies. We thank Fumiko Ozawa for her technical assistance. This work was supported by Grants-in-Aid from the Ministry of Health, Labor, and Welfare of Japan and the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Articles from Medical Gas Research are provided here courtesy of Wolters Kluwer — Medknow Publications
Logo of mgr
. 2011; 1: 24.
Published online 2011 Oct 3. doi:  10.1186/2045-9912-1-24
PMCID: PMC3231939
Open-label trial and randomized, double-blind, placebo-controlled, crossover trial of hydrogen-enriched water for mitochondrial and inflammatory myopathies

 

Drinking hydrogen water enhances ENDURANCE and relieves psychometric FATIGUE: a randomized, double-blind, placebo-controlled study 

Drinking hydrogen water enhances endurance and relieves psychometric fatigue: a randomized, double-blind, placebo-controlled study

Abstract

Acute physical exercise increases reactive oxygen species in skeletal muscle, leading to tissue damage and fatigue. Molecular hydrogen (H2) acts as a therapeutic antioxidant directly or indirectly by inducing antioxidative enzymes.

Here, we examined the effects of drinking hydrogen H2 water (H2-infused water) on psychometric fatigue and endurance capacity in a randomized, double-blind, placebo-controlled fashion.

In Experiment 1, all participants(humans) drank only placebo water in the first cycle ergometer exercise session, and for comparison they drank either hydrogen H2 water or placebo water 30 min before exercise in the second examination.In these healthy non-trained participants (n = 99), psychometric fatigue judged by visual analogue scales was significantly decreased in the hydrogen H2 water group after mild exercise. When each group was divided into 2 subgroups, the subgroup with higher visual analogue scale values was more sensitive to the effect of hydrogen water H2.

In Experiment 2, trained participants (n = 60) were subjected to moderate exercise by cycle ergometer in a similar way as in Experiment 1, but exercise was performed 10 min after drinking hydrogen H2 water. Endurance/fatigue were significantly improved/relieved in the hydrogen water H2 group as judged by maximal oxygen consumption and Borg’s scale, respectively.

Taken together, drinking hydrogen H2 water just before exercise exhibited anti-fatigue and improved endurance effects.

PMID:31251888
DOI:10.1139/cjpp-2019-0059
 2019 Jun 28:1-6. doi: 10.1139/cjpp-2019-0059. [Epub ahead of print]
Drinking hydrogen water enhances endurance and relieves psychometric fatigue: a randomized, double-blind, placebo-controlled study 1.

Author information

1 Department of Health and Sports Science, Nippon Medical School, Musashino, Tokyo 180-0023, Japan.
2 Fitness Club, Asahi Big S Mukogaoka, Kawasaki-city, Kanagawa pref. 214-0014, Japan.
3 Hydrogen Health Medical Laboratory, Co., Ltd., Arakawa-ku, Tokyo 116-0001, Japan.
4 Slovak Academy of Sciences, Centre of Experimental Medicine, Institute for Heart Research, Bratislava 84005, Slovak Republic.
5 Molecular Hydrogen Institute, Enoch, UT 84721, USA.
6Department of Neurology Medicine, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan.

Hydrogen-rich water affected blood alkalinity in physically active men

Abstract

Possible appliance of effective and safe alkalizing agent in the treatment of metabolic acidosis could be of particular interest to humans experiencing an increase in plasma acidity, such as exercise-induced acidosis.

In the present study we tested the hypothesis that the daily oral intake of 2L of hydrogen-rich water (HRW) for 14 days would increase arterial blood alkalinity at baseline and post-exercise as compared with the placebo.

This study was a randomized, double blind, placebo-controlled trial involving 52 presumably healthy physically active male volunteers. Twenty-six participants received hydrogen-rich water HRW and 26 a placebo (tap water) for 14 days.

Arterial blood pH, partial pressure for carbon dioxide (pCO2), and bicarbonates were measured at baseline and postexercise at the start (day 0) and at the end of the intervention period (day 14).

Intake of hydrogen-rich water HRW significantly increased fasting arterial blood pH by 0.04 (95% confidence interval; 0.01 – 0.08; p < 0.001), and postexercise pH by 0.07 (95% confidence interval; 0.01 – 0.10; p = 0.03) after 14 days of intervention.

Fasting bicarbonates were significantly higher in the hydrogen-rich water HRW trial after the administration regimen as compared with the preadministration (30.5 ± 1.9 mEq/L vs. 28.3 ± 2.3 mEq/L; p < 0.0001).

No volunteers withdrew before the end of the study, and no participant reported any vexatious side effects of supplementation.

These results support the hypothesis that hydrogen-rich water HRW administration is safe and may have an alkalizing effect in young physically active men.

 2014;22(1):49-60. doi: 10.1080/15438627.2013.852092.
PMID: 24392771
Hydrogen-rich water affected blood alkalinity in physically active men.
1 a Center for Health, Exercise and Sport Sciences , Stari DIF , Belgrade , Serbia.

hydrogen water , SPORTS , ANTIOXIDANTS and GUT FLORA

Abstract

Expending a considerable amount of physical energy inevitably leads to fatigue during both training and competition in football. An increasing number of experimental findings have confirmed the relationship between the generation and clearance of free radicals, fatigue, and exercise injury. Recently, hydrogen was identified as a new selective antioxidant with potential beneficial applications in sports. The present study evaluated the effect of 2-month consumption of hydrogen-rich water on the gut flora in juvenile female soccer players from Suzhou. As demonstrated by enzyme linked immunosorbent assay and 16S rDNA sequence analysis of stool samples, the consumption of hydrogen-rich water for two months significantly reduced serum malondialdehyde, interleukin-1, interleukin-6, tumour necrosis factor-α levels; then significantly increased serum superoxide dismutase, total antioxidant capacity levels and haemoglobin levels of whole blood. Furthermore, the consumption of hydrogen-rich water improved the diversity and abundance of the gut flora in athletes. All examined indices, including the shannon, sobs, ace, and chao indices, were higher in the control group than those proposed to result from hydrogen-rich water consumption prior to the trial, but these indices were all reversed and were higher than those in the controls after the 2-month intervention. Nevertheless, there were some differences in the gut flora components of these two groups before the trial, whereas there were no significant changes in the gut flora composition during the trial period. Thus, the consumption of hydrogen-rich water for two months might play a role modulating in the gut flora of athletes based on its selective antioxidant and anti-inflammatory activities. The study protocol was approved by the ethics committee of the Suzhou Sports School (approved number: SSS-EC150903).

Introduction

A number of studies have confirmed that the occurrence of exercise-induced fatigue is closely related to the level of oxidative stress in the body., The lipid peroxidative damage caused by the accumulation of free radicals in the body and the corresponding chain reaction are considered important factors responsible for decreased function of the body.,,

The antioxidant capacity of professional athletes is much higher than that of ordinary people, and athletes develop a greater ability to withstand the accumulation of free radicals and oxidative damage generated in sports. However, there are still many problems regarding protection against and alleviation and removal of the oxidative stress reaction induced by free radical accumulation in the aftermath of exercise and sports. Currently, the effects of antioxidants used in exercise practice vary, and studies have indicated that some of these substances may induce more significant skeletal muscle injury in athletes.,, Therefore, the search for safe and effective selective antioxidants has become an important research endeavour.

The selective antioxidant activity of hydrogen was first reported in 2007 by Ohsawa et al. Thereafter, a significant number of studies confirmed that hydrogen-rich water, prepared by dissolving hydrogen in water, shows selective antioxidant activity. Currently, sports science researchers are paying increasing attention to the selective antioxidant, anti-inflammatory, and anti-apoptotic effects of hydrogen and its regulation of the alkalinizing environment of the body., The beneficial protective effect of hydrogen-rich water has gradually been confirmed in both animal and human experiments.

The human symbiotic gut flora, considered the body’s “second genome”, has significant effects on human health., In recent years, studies have confirmed that imbalance of the intestinal flora is directly related to oxidative stress., The results of human experiments on athletes have shown that a greater exercise intensity results in increased oxidative stress in the body and, thus, a higher incidence of gastrointestinal stress symptoms. Therefore, in the training process, athletes should drink a sufficient amount of selective antioxidant hydrogen-rich water to regulate their gut flora, which might have a protective effect on the gastrointestinal tract and reduce stress reactions.

Participants and Methods

Participants and grouping

Thirty-eight juvenile female football players from the Suzhou Sports School showing a healthy status and absence of sports injury, without any obvious food preference, and with no significant reported intake of nutritional supplements and antibiotics for 3 months were randomly divided into two groups: the control group (n = 10) and the hydrogen-rich water treatment group (n = 28) (Figure 1). Written informed consent was obtained from each participant prior to admission to the protocol, and the study protocol was approved by the ethics committee of the Suzhou Sports School (approved number: SSS-EC150903). This study follows the Consolidated Standards of Reporting Trials (CONSORT) guidelines. During the experiment, the athletes in the hydrogen-rich water treatment group drank hydrogen-rich water in an amount equivalent to the amount of normal water they had previously consumed daily, while athletes in the control group continued to drink standard water in amounts consistent with their previous habits. The experiment lasted for 2 months. The basic information of the subjects is shown in Table 1.

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Trial flow chart.

Table 1

Characteristics of all subjects

Characteristics Control group (n = 10) Hydrogen-rich water treatment group (n = 28)
Age (year) 13.7±1.06 12.18±0.86
Height (cm) 159.1±5.51 149.32±8.69
Body weight (kg) 48.97±4.56 40.15±7.56
Training period (year) 3.4±1.51 1.21±0.6

Note: Data as expressed as the mean ± SD.

Sample collection

During the experiment, the athletes followed their previous dietary and resting regimes and other aspects of their normal daily routine. The training content, exercise intensity, frequency of exercise, and other parameters were consistent with the routine training regimen of the athletes.

Blood sample test

We collected 5 mL samples of venous blood (fasting) from all 38 athletes at a predetermined time in the morning, and 100 μL of whole blood was sampled for the measurement of haematological parameters in a blood cell analyser. The remaining blood samples were centrifuged at 3000 × g for 5 minutes. The serum samples were then collected and analysed with an automatic biochemical analysis apparatus to determine hemoglobin (HGB), blood urea nitrogen (BUN) and creatine kinase (CK). Then, the serum samples were analysed for oxidative response indices (malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC)) and inflammatory indices (interleukin-1 (IL-1), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-α)) using enzyme linked immunosorbent assay.

16S rDNA sequencing analysis of gut flora samples

Faecal flora samples were collected from all 38 athletes according to the specifications for stool sampling and stored at –80°C. The subsequent DNA sample extraction and 16S rDNA sequencing analysis were performed with the assistance of the Novagene Genomics Institute.

Statistical analysis

SPSS 19.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. The results were expressed as the mean ± SD. Significant differences between the two groups were analysed with repeated measured one-way analysis of variance, and the significance level was set at P < 0.05.

Results

Effects of long-term consumption of hydrogen-rich water on routine indices of juvenile female football players

Hemoglobin

After 4 weeks, HGB decreased from 134.3 ± 12.95 g/L to 124.00 ± 17.75 g/L in the control group, while that in the hydrogen-rich water treatment group decreased from 138.74 ± 9.38 g/L to 129.59 ± 8.57 g/L. After 8 weeks, HGB increased from 124.00 ± 17.75 g/L to 131.6 ± 25.31 g/L in the control group, while that in the hydrogen-rich water treatment group increased from 129.59 ± 8.57 g/L to 139.89 ± 7.02 g/L (Figure 2A). The increasing trend and amplitude of HGB were more significant in the hydrogen-rich water treatment group (P = 0.032).

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Changes in HGB, BUN and CK before and after hydrogen-rich water consumption.

Note: (A) The shift of HGB before and after hydrogen-rich water consumption; (B) The shift of BUN before and after hydrogen-rich water consumption; (C) The shift of CK before and after hydrogen-rich water consumption. HGB: Hemoglobin; BUN: blood urea nitrogen; CK: creatine kinase.

Blood urea nitrogen

After 4 weeks, the level of BUN increased from 4.73 ± 0.88 to 4.83 ± 0.81 mM in the control group, while that in the hydrogen-rich water treatment group changed from 5.19 ± 0.85 to 5.17 ± 1.03 mM. After 8 weeks, the level of BUN in the control group continued to increase, from 4.83 ± 0.81 to 5.29 ± 0.97 mM, while that in the hydrogen-rich water treatment group decreased from 5.17 ± 1.03 to 4.42 ± 0.95 mM (Figure 2B). There was a more distinct difference between the two groups (P = 0.887).

Creatine kinase

After 4 weeks, CK in the control group increased from 157.3 ± 17.37 to 171.3 ± 31.96 IU, while that in the hydrogen-rich water treatment group decreased from 149.3 ± 30.43 to 135.85 ± 24.44 IU (Figure 2C). After 8 weeks, CK decreased from 171.3 ± 31.96 to 129.7 ± 30.05 IU in the control group and from 135.85 ± 24.44 to 119.85 ± 29.93 IU in the hydrogen-rich water treatment group (P = 0.061).

Compared with HGB and BUN, CK was more sensitive to changes in the exercise load. These results suggest that the hydrogen-rich water treatment exerted a somewhat effect to enhance the whole blood HGB level of the athletes.

Effects of long-term consumption of hydrogen-rich water on oxidative response indices of juvenile female football players

Malondialdehyde

After 4 weeks, serum MDA decreased from 24.77 ± 7.32 to 16.67 ± 4.19 μM in the control group, while that decreased from 22.39 ± 6.20 to 13.80 ± 3.33 μM in the hydrogen-rich water treatment group. After 8 weeks, serum MDA changed from 16.67 ± 4.19 to 15.79 ± 3.07 μM in the control group and from13.80 ± 3.33 to 12.69 ± 1.94 μM in the hydrogen-rich water treatment group, with significant differences being observed between the two groups (P = 0.000; Figure 3A).

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Changes in MDA, SOD and T-AOC before and after hydrogen-rich water consumption.

Note: (A) The shift of MDA before and after hydrogen-rich water consumption; (B) The shift of SOD before and after hydrogen-rich water consumption; (C) The shift of T-AOC before and after hydrogen-rich water consumption. MDA: Malondialdehyde; SOD: superoxide dismutase; T-AOC: total antioxidant capacity.

Superoxide dismutase

After 4 weeks, the serum SOD level increased from 10.14 ± 2.60 to 13.14 ± 2.18 U/mL in the control group and from 11.09 ± 3.17 to 14.07 ± 1.91 U/mL in the hydrogen-rich water treatment group. After 8 weeks, the serum SOD level in the control group decreased from 13.14 ± 2.18 to 13.01 ± 1.08 U/mL, while that in the hydrogen-rich water treatment group decreased from 14.07 ± 1.91 to 13.69 ± 2.10 U/mL, with significant differences between the two groups (P = 0.027; Figure 3B).

Total antioxidant capacity

After 4 weeks, serum T-AOC increased from 0.8 ± 0.08 to 1.11 ± 0.17 μM in the control group, while serum T-AOC in the hydrogen-rich water treatment group changed from 0.87 ± 0.11 to 1.17 ± 0.13 μM. After 8 weeks, T-AOC changed from 1.17 ± 0.13 to 0.84 ± 0.09 μM in the control group and from 1.17 ± 0.13 to 0.9 ± 0.13 μM in the hydrogen-rich water treatment group, with significant differences between the two groups (P = 0.004, Figure 3C).

These results suggest that the hydrogen-rich water treatment exerted an anti-oxidative effect.

Effects of long-term consumption of hydrogen-rich water on inflammatory indices of juvenile female football players

Interleukin-1

After 4 weeks, the level of serum IL-1 in the control group increased from 24.77 ± 7.32 to 32.56 ± 7.61 μM, and that in the hydrogen-rich water treatment group increased from 24.79 ± 8.94 to 29.32 ± 7.09 μM. After 8 weeks, the IL-1 level increased from 32.56 ± 7.61 to 42.94 ± 6.24 μM in the control group and from 29.32 ± 7.09 μM to 34.47 ± 6.22 μM in the hydrogen-rich water treatment group, with significant differences between the two groups (P = 0.002, Figure 4A).

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Changes in IL-1, IL-6 and TNF-α before and after hydrogen-rich water consumption.

Note: (A) The shift of IL-1 before and after hydrogen-rich water consumption; (B) The shift of IL-6 before and after hydrogen-rich water consumption; (C) The shift of TNF-α before and after hydrogen-rich water consumption. IL: Interleukin; TNF-α: tumour necrosis factor alpha.

Interleukin-6

After 4 weeks, the level of serum IL-6 decreased from 19.48 ± 2.16 to 10.53 ± 1.62 ng/L in the control group and from 17.72 ± 2.1 to 8.74 ± 2.57 ng/L in the hydrogen-rich water treatment group. After 8 weeks, the level of serum IL-6 in the control group increased from 10.53 ± 1.62 ng/L to 24.88 ± 6.11 ng/L, while that in the hydrogen-rich water treatment group increased from 8.74 ± 2.57 to 12.37 ± 3.2 ng/L, with significant differences between the two groups (P = 0.000, Figure 4B).

Tumour necrosis factor-α

After 4 weeks, serum TNF-α increased from 20.04 ± 7.99 to 60.57 ± 10.09 μM in the control group and increased from 20.44 ± 7.75 to 49.46 ± 11.59 μM in the hydrogen-rich water treatment group. After 8 weeks, serum TNF-α increased from 60.57 ± 10.09 to 132.24 ± 10.46 μM in the control group and from 49.46 ± 11.59 to 107.00 ± 13.89 μM in the hydrogen-rich water treatment group, with significant differences between the two groups (P = 0.000, Figure 4C).

These results suggest that the hydrogen-rich water treatment exerted an anti-inflammatory effect.

Effects of long-term consumption of hydrogen-rich water on gut flora components of juvenile female football players

Classification by phylum

In the samples collected from the athletes after pre-treatment with hydrogen-rich water, the number of Actinobacteria in the control group was higher than that in the treatment group, and the number of Bacteroides in the control group was slightly lower than that in the hydrogen-rich water treatment group. Moreover, the number of Clostridia in the control group was slightly higher than that in the hydrogen-rich water treatment group. However, there were no significant differences in the numbers of these bacterial groups after 2 months of hydrogen-rich water treatment.

Classification by class

In samples collected from the athletes after pre-treatment with hydrogen-rich water, the number of Actinobacteria in the control group was higher than that in the hydrogen-rich water treatment group, while the number of Bacteroides in the control group was slightly lower than that in the hydrogen-rich water treatment group, and the numbers of ClostridiaCoriobacteria, and Erysipelotrichia in the control group were higher than those in the hydrogen-rich water treatment group. However, there was no significant difference in the numbers of these bacterial groups after 2 months of hydrogen-rich water treatment.

Classification by order

In samples collected from the athletes after pre-treatment with hydrogen-rich water, the number of Actinobacteria in the control group was higher than that in the hydrogen-rich water treatment group, while the number of Bacteroides in the control group was slightly lower than that in the hydrogen-rich water treatment group, and the numbers of Clostridia and Coriobacteria in the control group were higher than those in the hydrogen-rich water treatment group. The number of Erysipelotrichia in the control group was higher than that in the hydrogen-rich water treatment group, although this difference was not significant. Nevertheless, there were no significant differences in the numbers of related bacteria after 2 months of hydrogen-rich water treatment.

Classification by family

In samples collected from the athletes after pre-treatment with hydrogen-rich water, the numbers of Acidaminococcaceae, Bacteriodaceae, Bifidobacteriaceae, Coriobacteriaceae, Desulforibrionaceae, Erysipelotrichaceae and Ruminococcaceae were higher than those in the hydrogen-rich water treatment group, with differences being observed in the number of Bifidobacteriaceae, Ruminococcaceae, Coriobacteriaceae and Erysipelotrichaceae. There was no difference in the number of Lachnospiraceae between the two groups. The number of Prevotellaceae in the hydrogen-rich water treatment group was higher than that in the control group. However, there were no significant differences in the number of these bacterial groups after 2 months of hydrogen-rich water treatment.

Classification by genus

In samples collected from the athletes after pre-treatment with hydrogen-rich water, the numbers of Bifidobacterium and Oscillibacter in the control group were higher than those in the hydrogen-rich water treatment group, with a difference being observed in the number of Bifidobacteriaceae. The number of Prevotella in the hydrogen-rich water treatment group was higher than that in the control group, although this difference was not significant. There were no significant differences in the number of these bacterial groups after 2 months of hydrogen-rich water treatment.

Effects of long-term consumption of hydrogen-rich water on gut flora diversity and abundance in juvenile female football players

The actual number of operational taxonomic units (sobs) and the ace, chao and shannon indices were determined, and then a dilution curve was drawn. The recorded changes indicated that the sobs, ace, chao and shannon indices of the control group were all higher than those of the hydrogen-rich water treatment group, suggesting that the abundance and diversity of the gut flora in the control group were higher than those in the hydrogen-rich water treatment group.

After 1 month of hydrogen-rich water treatment, the sobs, ace, and chao indices were higher in the hydrogen-rich water treatment group than those in the control group. The trend was slightly reversed, indicating that the abundance of gut flora was higher in the hydrogen-rich water treatment group than in the control group. The shannon index of the treatment group at that time was essentially the same as that in the control group, indicating that treatment with hydrogen-rich water could also enhance the diversity of the gut flora. After 2 months of hydrogen-rich water treatment, the sobs, ace, chao and shannon indices were much higher than those in the control group (P = 0.479, P = 0.710, P = 0.369, P = 0.369). Indicating that treatment with hydrogen-rich water can enhance the gut flora abundance and diversity of the gut flora (Figure 5).

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Changes in gut flora diversity and abundance before and after hydrogen-rich water consumption.

Note: (A) The shift of sobs before and after hydrogenrich water consumption; (B) The shift of ace index before and after hydrogen-rich water consumption; (C) The shift of chao index before and after hydrogen-rich water consumption; (D) The shift of shannon index before and after hydrogen-rich water consumption.

Discussion

The existing experimental and clinical studies have shown that animals or humans need only to breathe hydrogen or drink or inject hydrogen-rich water to protect the heart, brain, liver, kidney, lung, and small intestine from ischaemia/reperfusion oxidative injury or inflammatory injury after cardiac organ transplantation.,

The potential biological effects of hydrogen in sports have drawn much attention from researchers in sports science. The beneficial protective effects of hydrogen-rich water on the body have been gradually confirmed in both animal and human experiments. Ostojic summarized the current applications of hydrogen in the field of sports, emphasizing that hydrogen

1) can effectively remove a large number of harmful free radicals generated through movement, thus enhancing the antioxidant capacity;

2) is an effective alkalizing agent in the internal environment that can effectively inhibit blood acidification induced by lactic acid accumulation in sports; and

3) is an important gas signalling molecule that can participate in physiological regulatory processes such as anti-inflammatory, anti-apoptotic, and anti-autophagy processes., This regulation does not involve the same signalling pathway as antioxidative stress.

Analysis of the effect of long-term consumption of hydrogen-rich water on routine indices of juvenile female football players

HGB is one of the classic indicators reflecting the level of endurance exercise. The shift of HGB after 4 weeks was caused by increases in the amount or intensity of exercise and seasonal factors during winter training. The HGB level began to gradually increase, suggesting that the athletes had adapted well to the winter training load. The increase in the HGB level was higher overall in the hydrogen-rich water treatment group suggested that long-term hydrogen-rich water treatment could help increase the HGB level.

Urea nitrogen is the final product of protein metabolism. The participation of protein catabolism in the energy supply is enhanced during long-term and high-intensity exercise, thus increasing the amount of urea nitrogen in the blood and urine with increased decomposition of proteins and amino acids. The shift of the BUN level of all 38 athletes increased slightly due to winter training and seasonal factors. After 8 weeks, the decrease in the serum urea nitrogen level and the increase in the HGB level indicated that long-term hydrogen-rich water treatment has beneficial effects on the physiological functions of athletes.

CK is the key enzyme in energy metabolism in skeletal muscle cells, whose activity directly affects the short-term maximum intensity of the exercise capacity. After a high-intensity muscle load, muscle soreness and serum CK levels are highly and positively correlated. Clarke et al. found that the level of CK in the serum of professional rugby athletes is markedly high. CK is an important index reflecting the exercise load, particularly that suffered by the skeletal muscle. Thus, CK could indirectly reflect the levels of injury and active repair of the skeletal muscle ultrastructure.

After 8 weeks, the level of serum CK in both the control and hydrogen-rich water treatment groups continued to decrease.

Analysis of the effect of long-term consumption of hydrogen-rich water on the serum oxidative response of juvenile female football players

Tsubone et al. compared the effects of drinking hydrogen-rich water on the levels of oxidative stress and antioxidant metabolites in the serum of British thoroughbred horses and found that hydrogen-rich water treatment had a beneficial antioxidant effect. Aoki et al. conducted studies on football players and showed that drinking hydrogen-rich water for 1 week could reduce exercise fatigue and lactic acid accumulation after exercise but had no significant effect on the oxidative response index.

Li et al. showed that hydrogen-rich water could significantly prolong the duration of exercise before exhaustion in rats and improve their exercise capacity, indicating a significant anti-fatigue effect.

Zhao and Zhang showed that supplementation of hydrogen-rich water at different times before, during, and after exercise exerted significant protective effects against oxidative stress injury in swimming athletes during high-intensity exercise. This supplementation of hydrogen-rich water can reduce the production of excessive free radicals and enhance the activity of antioxidant enzymes and the antioxidant capacity of the body, thereby promoting physical recovery after high-intensity exercise. Hu and Zhang showed that high-intensity intermittent training increases the concentration of O2 , •OH and H2O2. Hydrogen-rich water can significantly enhance the body’s inhibition of O2  and •OH, showing a higher rate of •OH inhibition, fully reflecting its selective antioxidant effect.

Li et al. found that hydrogen-rich water treatment could effectively reduce oxidative stress injury induced in skeletal muscle by severe exercise while improving the muscle ultrastructure.

Wang et al. reported that hydrogen-rich water treatment could up-regulate the expression of SIRT3, enhance the activity of antioxidant enzymes, and reduce the inflammatory response after centrifugal exercise.

MDA is one of the classic indicators reflecting the level of lipid peroxidation. After 8 weeks, the difference of serum MDA between the two groups was significant, it suggested that long-term hydrogen-rich water treatment exerts an antioxidant effect.

SOD is one of the classic indicators reflecting the free radical-scavenging antioxidant capacity. The SOD levels of both the control and hydrogen-rich water treatment groups slightly increased after 4 weeks. And the mean serum SOD level of the hydrogen-rich water treatment group was consistent higher than the control group after 8 weeks.

Serum antioxidant substances can be divided into the enzymatic antioxidant system and the non-enzymatic antioxidant system. The enzymatic antioxidant system mainly involves substances such as SOD, glutathione peroxidase, glutathione reductase, and catalase. The non-enzymatic antioxidant system mainly involves water-soluble substances, such as vitamin C, bilirubin, fat-soluble vitamin E, coenzyme Q, carotenoids, and flavonoid antioxidants. In terms of their function, serum antioxidant substances can be divided into three types: preventive antioxidants; capture-type antioxidants; and repair and regeneration antioxidants. The total antioxidant capacity represents the sum of the above substances and functions.

The observed changes in serum T-AOC suggested that 4 weeks of hydrogen-rich water treatment group indeed improved the free radical-scavenging ability of antioxidants. These results suggest that long-term hydrogen-rich water treatment exerts an antioxidant effect.

Analysis of the effect of long-term consumption of hydrogen-rich water on serum inflammatory indices of juvenile female football players

Inflammatory factors will increase, and inflammation will intensify during exercise due to increases in energy consumption, free radicals, and intensification of oxidative stress. However, there are three anti-inflammatory mechanisms that may be deployed in the course of exercise.

1) Exercise can increase energy consumption, thereby reducing visceral fat volume and alleviating the infiltration of fat into inflammatory lymphocytes.

2) Exercise can effectively increase the production and release of muscle-derived anti-inflammatory cytokines during skeletal muscle contraction; skeletal muscle accounts for 35–45% of the total body weight, and the regulatory effects of this major endocrine organ on human homeostasis cannot be ignored.

3) Exercise can effectively reduce toll-like receptor expression on the membrane surface of monocytes and macrophages, which may lead to a decreased downstream response, including reduced secretion of inflammatory agents, decreased expression of compatibility complexes in major organs, and decreases in co-stimulatory Mecules.,

These three effects can ensure that the levels of inflammatory agent factors in athletes participating in strenuous exercise will not increase and may even decrease. However, the effect of oxidative stress on the body will not be weakened. After 8 weeks of hydrogen-rich water treatment, the levels of IL-1, IL-6 and TNF-α in the hydrogen-rich water treatment group were lower than those in the control group and with significant differences between the two groups. Compared with the abovementioned changes in the oxidative stress indices, long-term hydrogen-rich water treatment showed a stronger anti-inflammatory effect in addition to an antioxidant effect.

Analysis of the effect of long-term consumption of hydrogen-rich water on gut flora components of juvenile female football players

Analysis of the structural components of the gut flora at different levels of classification in the two groups showed some differences between the two groups at different stages of the experiment. However, there were no significant changes in the structural components of flora between the two groups in terms of the oxidative response and the anti-inflammatory effect. These results suggest that two months of hydrogen-rich water treatment did not significantly change the structural components of the gut flora of the juvenile female football players. Differences in the composition of the flora between the two groups are an expected result of differences in age, particularly regarding the number of training years.

In 2007, Ohsawa et al. suggested that the selective antioxidant activity of hydrogen-rich water, and particularly its selective elimination of •OH, is superior to that of traditional antioxidants, while its overall antioxidant capacity is much lower than that of traditional antioxidants. Therefore, the effect of 2 months of hydrogen-rich water treatment on the regulation of gut flora was also much lower than that of established supplements such as resveratrol, grape antioxidant dietary fibre, selenium supplements, anthocyanin, and pomegranate peel polyphenols.,,,,,

Analysis of the effect of long-term consumption of hydrogen-rich water on gut flora diversity and abundance in juvenile female football players

As a complex and variable micro-ecological system, the gut flora is constantly undergoing changes in its dynamic equilibrium. The richness and diversity of its components are important indicators of the health of this ecological system. The richness of the gut flora in patients with inflammatory bowel disorder is decreased in elderly and obese individuals. Le Chatelier et al. compared the composition of the gut flora of 123 non-obese and 169 obese Danes and found that the gut flora richness of these two groups differed, as did the number of genes in their gut flora. Individuals with lower gut flora richness were found to exhibit more significant obesity characteristics, insulin resistance, and lipid metabolic disorders as well as more severe inflammatory phenotypes.,

As a strong stressor, long-term and high-intensity professional sports training eventually has a corresponding impact on the gut flora. Clarke et al. found that professional rugby athletes exhibited a more abundant gut flora in their intestines compared with control groups of individuals with a body mass index (BMI) < 25 or BMI > 28. In samples from the professional rugby athletes, the total microorganisms identified came from 22 phyla, 68 families, and 113 genera. In the control group with a BMI < 25, a total of 11 phyla, 33 families, and 65 genera of microorganisms were detected, whereas the microorganisms in the control group with a BMI > 28 came from 9 phyla, 33 families, and 61 genera. The richness and diversity of the gut flora were lowest in obese individuals, while the professional athletes exhibited the highest richness and diversity levels.

Before treatment with hydrogen-rich water, the richness and diversity of the gut flora were higher in the control group (3.4 ± 1.51 years of training) than in the treatment group (1.21 ± 0.6 years of training), and the training period was the main factor leading to this difference. Individuals who had a longer training period exhibited a higher richness and diversity in their gut flora; this trend is consistent with the results of Clarke et al.

After 4 weeks of treatment with hydrogen-rich water, the trend was slightly reversed. The richness and diversity of the gut flora were higher in athletes who had a shorter training period than those who had a longer training period. This finding indicated that drinking hydrogen-rich water for a long period of time may plays an important role in enhancing the richness and diversity of the gut flora. At the same time, the levels of serum MDA, IL-1, IL-6 and TNF-α decreased in the treatment group, and the SOD, T-AOC level increased. Such changes are closely related to changes in the richness and diversity of the gut flora.

After 8 weeks of treatment with hydrogen-rich water, the richness and diversity of the gut flora were still higher in athletes who had a shorter training period than in control individuals who had a longer training. Additionally, the serum levels of MDA, IL-1, IL-6 and TNF-α decreased, and the levels of HGB SOD, T-AOC level increased to various degrees in the hydrogen-rich water treatment group. The trend of favourable changes of motor function indices, the oxidative response index, and inflammatory factor indices were almost consistent with the changes in the richness and diversity of the gut flora.

The above results showed that long-term consumption of hydrogen-rich water not only exerts certain antioxidant and anti-inflammatory effects but also enhances the diversity and abundance of the gut flora of the subjects.

Footnotes

Funding: The study was supported by the National Basic Research Project of China (973 Program), No. 2012CB518200 (to ZCG), the General Program of the Natural Science Foundation of China, No. 81371232, 81573251 (to ZCG), and the Special Key Programs for Science and Technology of China, No. 2012ZX09102301-016 and 2014ZX09J14107-05B (to ZCG).

Conflicts of interest

There is no conflict of interest.

Financial support

The study was supported by The National Basic Research Project of China (973 Program), No. 2012CB518200, the General Program of the Natural Science Foundation of China, No. 81371232, 81573251, and the Special Key Programs for Science and Technology of China, No. 2012ZX09102301-016, 2014ZX09J14107-05B.

Institutional review board statement

The institutional review board approval of Suzhou Sports School was obtained for this study.

Declaration of participant consent

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

Reporting statement

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

Biostatistics statement

The statistical methods of this study were reviewed by the biostatistician of the State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center, Beijing, China.

Copyright license agreement

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

Data sharing statement

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

Plagiarism check

Checked twice by iThenticate.

Peer review

Externally peer reviewed.

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Link to Publisher's site
. 2018 Oct-Dec; 8(4): 135–143.
Published online 2019 Jan 9. doi: 10.4103/2045-9912.248263
PMCID: PMC6352569
PMID: 30713665
Effects of the long-term consumption of hydrogen-rich water on the antioxidant activity and the gut flora in female juvenile soccer players from Suzhou, China
This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

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Articles from Medical Gas Research are provided here courtesy of Wolters Kluwer — Medknow Publications

molecular hydrogen water for patients with pressure ulcer – effects on normal human SKIN WOUNDS

 
Pressure ulcer (PU) is common in immobile elderly patients, and there are some research works to investigate a preventive and curative method, but not to find sufficient effectiveness. The aim of this study is to clarify the clinical effectiveness on wound healing in patients with pressure ulcer PU by molecular hydrogen-dissolved in water (HW) intake via tube-feeding (TF). Furthermore, normal human dermal fibroblasts OUMS-36 and normal human epidermis-derived cell line HaCaT keratinocytes were examined in vitro to explore the mechanisms relating to whether molecular hydrogen plays a role in wound-healing at the cellular level.

22 severely hospitalized elderly Japanese patients with pressure ulcer PU were recruited in the present study, and their ages ranged from 71.0 to 101.0 (86.7 ± 8.2) years old, 12 male and 10 female patients, all suffering from eating disorder and bedridden syndrome as the secondary results of various underlying diseases. All patients received routine care treatments for pressure ulcer PU in combination with molecular hydrogen water HW intake via TF for 600 mL per day, in place of partial moisture replenishment. On the other hand, HW was prepared with a hydrogen-bubbling apparatus which produces molecular hydrogen water HW with 0.8-1.3 ppm of dissolved hydrogen concentration (DH) and −602 mV to −583 mV of oxidation-reduction potential (ORP), in contrast to reversed osmotic ultra-pure water (RW), as the reference, with DH of < 0.018 ppm and ORP of +184 mV for use in the in vitro experimental research. In in vitro experiments, OUMS-36 fibroblasts and HaCaT keratinocytes were respectively cultured in medium prepared with molecular hydrogen water  HW and/or reversed osmotic ultra-pure water RW. Immunostain was used for detecting type-I collagen reconstruction in OUMS-36 cells. And intracellular reactive oxygen species (ROS) were quantified by NBT assay, and cell viability of HaCaT cells was examined by WST-1 assay, respectively.

Results

22 patients were retrospectively divided into an effective group (EG, n = 12) and a less effective group (LG, n = 10) according to the outcomes of endpoint evaluation and the healing criteria. Pressure Ulcers  hospitalized days in EffectiveGroup were significantly shorter than in LessseffectiveGroup (113.3 days vs. 155.4 days, p < 0.05), and the shortening rate was approximately 28.1%. Either in EG or in LG, the reducing changes (EG: 91.4%; LG: 48.6%) of wound size represented statistically significant difference versus before molecular hydrogen water HW intake (p < 0.05, p < 0.001). The in vitro data demonstrate that intracellular ROS as quantified by NBT assay was diminished by molecular hydrogen water HW, but not by reverse osmosys water ultraviolet-A (UVA)-irradiated HaCaT cells. Nuclear condensation and fragmentation had occurred for UVA-irradiated HaCaT cells in reverse osmosis water RW, but scarcely occurred in molecular hydrogen water HW as demonstrated by Hoechst 33342 staining. Besides, under UVA-irradiation, either the mitochondrial reducing ability of HaCaT cells or the type-I collagen construction in OUMS-36 cells deteriorated in reverse osmosis water  RW-prepared culture medium, but was retained in molecular hydrogen water HW-prepared culture medium as shown by WST-1 assay or immunostain, respectively.

Conclusions

molecular hydrogen water HW intake via TF was demonstrated, for severely hospitalized elderly patients with PressureUlcers, to execute wound size reduction and early recovery, which potently ensue from either type-I collagen construction in dermal fibroblasts or the promoted mitochondrial reducing ability and ROS repression in epidermal keratinocytes as shown by immunostain or NBT and WST-1 assays, respectively.

Introduction

PressureUlcer is common in the immobile elderly or other immobile patients suffering from diseases such as spinal cord injury, amyotrophic lateral sclerosis, multiple sclerosis, and muscular dystrophy, etc. Furthermore, aged and weak bedridden patients belong to a high risk population for Pressure Ulcers [1]. It is estimated that there are over one million elderly people who are suffering from the skin peculiarity and are facing the risk factors of Pressure Ulcers in USA [2]. Fundamentally, it is usually pointed out that social, psychological and financial expenses for Pressure Ulcers are immeasurable, patients and their families as well as health care providers are always receiving the mental strain [3].

For Pressure Ulcers , it is a primary research task to explore a cheap but effective preventive and curative method. Although various methods for prevention and treatment have been developed, they are far from sufficiently succeeding. While slightly delayed, basic studies are seen to steadily proceed in the same way as the clinical study. As the basic studies for wound healing, a lot of researchers are focusing on skin-constructing proteins such as collagen, elastin, laminin and fibronectin, and on metabolic activity and proliferating ability of dermal fibroblasts [45].

In relation to this matter, we had confirmed the fact that molecular hydrogen-dissolved in water HW, as an external use for skin, can promote the construction of the type-I collagen in fibroblastic cells of dermis [678]. We prepared molecular hydrogen-dissolved in water HW with a hydrogen-bubbling apparatus, exhibited a DH of 1.13 ppm and an ORP of −741 mV, in contrast a DH of < 0.01 ppm and an ORP of +150 mV for normal water [6]. Simultaneously, normal human dermal fibroblasts OUMS-36 and normal human epidermis-derived keratinocytes HaCaT were cultured using an immunostain, in addition, WST-8 and DAPI stains were conducted to examine the cytoprotective effects of molecular hydrogen-dissolved in water HW against UVA-ray irradiation. Six Japanese subjects were enrolled in a trial of molecular hydrogen water HW-bathing (DH, 0.2-0.4 ppm) every day for 3 months. The results obtained showed that molecular hydrogen water  HW-bathing significantly improved wrinkles on the back of the neck in four subjects on 90th day as compared to day 0. Thus the conclusion was achieved, in which molecular hydrogen water  HW can serve as a daily skin care routine to repress UVA-induced skin damages by ROS-scavenging and promotion of type-I collagen synthesis in dermis. On the other hand, many basic research studies demonstrated that molecular hydrogen water HW is widely applied to various diseases, as an oral intake for absorbing via the gastrointestinal tract [91011121314]. The researches obviously suggest that whether using a bathing type or oral intake type of treatment,molecular hydrogen water HW is still an effective method to repair the skin and scavenge the ROS [151617].

We theorized that a routine care treatment in combination with molecular hydrogen water  HW intake via TF for patients with PressureUlcers may improve wound healing and maintain a better health condition than before. The purpose of this study is to clarify the clinical effectiveness of wound healing for patients with PressureUlcers by means of an oral intake of molecular hydrogen water  HW via TF. Furthermore, OUMS-36 cells and HaCaT cells were examined to analyze the mechanisms relating to whether molecular hydrogen water  plays a role in wound healing at the cellular level, in vitro.

Methods

Clinical materials

Patients

Medical record data that were analyzed for this study were obtained from twenty-two elderly Japanese patients with Pressure Ulcers who were hospitalized and institutionalized in Kobayashi Hospital, Fukuyama City, Hiroshima Prefecture, Japan, which is a general hospital attached to a mixed long-term care facility. This study was approved by the Ethics Committee of Kobayashi Hospital.

The ages of PressureUlcers patients who we treated in this study ranged from 71.0 to 101.0 (86.7 ± 8.2) years old, and ten patients were women. On the time of admission, they had suffered from one or multiple diseases and complications, and almost all of them were bedridden elderly people at a high risk of PressureUlcers development, and all of them could not eat without other people’s aid. On the time of admission or during the hospitalization, all patients had been or were gradually appearing symptoms of PressureUlcers.

The types of diseases and complications in these patients, not only included eating disorder but 90% also showed the prevalence of being in the aged period, and 100% had impaired mobility. However, it must be emphasized that PressureUlcers incidence of new onset in Kobayashi Hospital remained approximately 2.10% in 2010–2011, persisted in low level. Because it was reported that average PressureUlcer incidence was 2.43% in a nationwide survey executed by Japanese Society of Pressure Ulcers [18].

Twenty-two patients were retrospectively grouped into EG (effective group, n = 12) and LG (less effective group, n = 10) according to the outcomes of endpoint evaluation and the healing criteria. Details with regard to the discharge from hospital for whether cure or not were analyzed, and baseline data were summarized (Table 1). In data processing, results of all patients were classified as stage I-IV according to the Guideline in 2009 of EPUAP (European Pressure Ulcer Advisory Panel) & NPUAP (National Pressure Ulcer Advisory Panel) that is used as assessment for the severity of PressureUlcers. Coincidentally, all patients in this study belonged to stage II or III.

Table 1

Characteristics of baseline data of PressureUlcers patients in two groups

Item

Effective group (EG)

Less effective group (LG)

Number of patients

12

10

Age (mean ± SD) at onset

87.9 ± 9.0

85.5 ± 7.3

  range

71.0-101.0

73.0-98.0

Gender (male/female)

4/8

8/2

Admission diagnosis

  PU

8

7

  Tumor

0

1

  Pneumonia

4

0

  COPD

0

1

  CIS

0

1

Hospitalized days (mean ± SD)

113.3 ± 89.6

155.4 ± 92.6

  range

32-379

63-335

DESIGN-Rating (mean ± SD) at onset

14.0 ± 5.4

12.7 ± 3.3

Wound size (mean ± SD) at onset

6.9 ± 0.9 cm2

6.3 ± 0.9 cm2

Locations*

  Total

16

12

  Back

3

0

  Sacrum

3

5

  Buttocks

3

2

  Ilium

1

3

  Greater trochanter

2

1

  Thigh

1

0

  Knee

1

0

  Heel

1

1

  Toes

1

0

Stages at onset (number of locations*)

  Stage II

6

4

  Stage III

10

8

Abbreviations: PU pressure ulcer, COPD chronic obstructive pulmonary disease, CIS cerebral infarction sequela, DESIGN-Ratingdepth, exudate, size, inflammation/infection, granulation, necrotic tissue.

*Some patients had multiple locations for pressure ulcers.

Clinical care treatments

Hospitalized routine care treatment

The treatment focused on preventing PressureUlcers from getting worse and on restoring healthy skin. According to the routine care treatments for all patients, nonsurgical therapies were selected, such as ointment, gauze dressing, wrapping, and bed-pad were used after washing by the acidic water disinfection. The skin care, pressure relief and nutritional support were aggressively used as a part of this care treatment [13]. The main care steps to treat PressureUlcers included:

  1. a.

    Managing the tissue load.

  2. b.

    Keeping the ulcer area clean and covered, and not letting it dry out.

  3. c.

    Body-position changes at least every 2 hours if the patient is confined to a bed, or as often as every 15 min if sitting in a wheelchair.

  4. d.

    To achieve positive nutritional nitrogen balance, patient consumed by TF approximately 30 to 35 calories per kg per day and 1.25 to 1.50 g of protein per kg per day.

Preparation for molecular hydrogen water HW

HW molecular hydrogen water was prepared with a molecular hydrogen-bubbling apparatus which consists mainly of a water supply section for manufacturing reverse osmosis water RW with less 0.018 ppm of DH and +184 mV of ORP, and molecular hydrogen water  HW with 0.8-1.3 ppm of DH and −602 mV to −583 mV of ORP. For comparing molecular hydrogen water  HW with reverse osmosys water RW, the water characteristic parameters were measured with the different dilution rates (Table 2, Figures 1 and 2). It must be emphasized that some stable characteristic indicators and the proprieties for innocuity and harmlessness of molecular hydrogen water were obtained from several separated in vivo and human experiments which we had reported [1920212223]. Meanwhile, via tube-feeding, Pressure Ulcers  patients were enforced to intake molecular hydrogen  water HW of 600 mL per day, in the morning and afternoon for approximately one hour, respectively, immediately after molecular hydrogen  water HW was manufactured everytime.

Table 2

Characteristic parameters obtained from hydrogen-dissolved water vs. reversed osmotic ultra-pure water

DH(ppm)

DO(ppm)

ORP (mV)

pH

Water temperature (°C)

Hydrogen-dissolved water (HW)

0.80-1.30

6.91

−602

7.40

24.1

Reversed osmotic ultra-pure water (RW)

< 0.018

8.26

+184

7.37

24.2

Abbreviations: DHdissolved hydrogen concentration, DOdissolved oxygen concentration, ORPoxidation-reduction potential.

Figure 1

Measurement results of diluting molecular hydrogen  water HW with reverse osmosys water RW. The dilution rates are showed as Figure 1. Figures 1a and –b show the ever-increasing tendencies on DO (dissolved oxygen concentration) and ORP (oxidation-reduction potential). Meanwhile, as shown by Figures 1c and –d, DH (dissolved hydrogen concentration) shows the ever-decreasing tendency which indicates the dissolved hydrogen in the hydrogen water was evaporated slowly by mixing with normal regular water. On the other hand, both molecular hydrogen  water HW with reverse osmosys water RW have been holding the temperature range of 23.2-24.1°C and pH 7.37-7.48 no matter from 1 to 11-fold dilution rate.

Clinical evaluations

The evaluative indices for clinical therapeutic effects on PRESSURE ULCERS consisted of the hospitalized days, wound size, classifications of PressureUlcers-stage and DESIGN- rating.

Hospitalized days

Because the increased length of hospitalized stay is an important index for a PressureUlcers patient of QOL (Quality of Life), the days from admission to discharge for twenty-two patients were counted.

Wound size

For obtaining precise objective information and monitoring the healing degree about wound, the medical-care staff measured the size, depth and area [24], utilized photography and diagrams for recording the shape and outline of the wound.

Classifications of PressureUlcers-stages

According to a well-known Panel Guideline established by EPUAP and NPUAP in 2009 [3], stage II includes the partial thickness for loss of skin involving epidermis, dermis or both. The ulcer is superficial and presents clinically as an abrasion or blister, but is not deeper than the dermis. On the other hand, stage III involves the full depth of the skin, and may extend into the subcutaneous tissue layer which has a relatively poor blood supply and can be difficult to heal [2526].

DESIGN-rating

DESIGN was an absolute evaluation tool and consumed as a clinical indicator to assess the quality of medical care. But, its score could not be compared the severity of PressureUlcers among different patients and their various ulcers. Because of this, the DESIGN-rating was invented to use as a simple and easy assessment of PressureUlcers [2728]. In our study, the DESIGN-rating score of every patient was recorded by the medical-care staff, at least once monthly.

In vitro experiments

Materials and methods

Normal human dermal fibroblastic cells OUMS-36

OUMS-36 cells were cultivated for 18 hours in molecular hydrogen  water HW or reverse osmosys water RW-prepared Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical Co. Ltd., Tokyo) supplemented with 10% FCS (fetal calf serum) (GIBCO) in a CO2incubator to be kept at 37°C and pH 7.1-7.4 in a moistened atmosphere of 5% CO2. The spent medium was replaced by the fresh molecular hydrogen  water HW or reverse osmosys water RW-prepared culture medium, and was at once irradiated with UVA ray at doses of 12 or 18 J/cm2, corresponding to the normal dose range for the human daily life. The resultant cells were stained for the nuclei with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Ultracruz Mounting Medium, sc-24941, Santa Cruz Biotechnology Inc., Santa Cruz, CA), and observed for type-I collagen reconstruction by immunostain using the first antibody directed against type-I collagen and the secondary antibody conjugated with FITC (fluorescein isothiocyanate), as observed with a fluorescence microscope (ECLIPSE E600, Nikon Corp., Tokyo) as previously described [6].

Normal human epidermis-derived keratinocytes HaCaT

HaCaT cells were similarly cultivated in molecular hydrogen  water HW or reverse osmosys water RW-prepared DMEM supplemented with 10% FCS (GIBCO), and similarly UVA-irradiated. The resultant cells were examined for cell viability by WST-1 methods using (phenyl)-5-(2-disulfophenyl)-2H-tetrazolium, monosodium salt as a redox indicator, and for ROS such as superoxide anion radicals by NBT (nitro blue tetrazorium) assay as previously described [6].

Statistical analysis

Either clinical study or in vitro research, the Student’s t-test was used to compare the difference in means ± SD between the control and treated groups using a Microsoft Office Excel 2010 software (Microsoft, Albuquerque, NM, USA) or a software package SPSS 11.0 (SPSS inc., Chicago, IL, USA) for Windows. A p-value that is below 0.05 was regarded to be statistically significant.

Results

The clinical results of routine care treatments in combination with molecular hydrogen  water HW via Tube for Feeding

The hospitalized days and the DESIGN-rating of PressureUlcers

For the PressureUlcer patients, the hospitalized days in EffectiveGroup were significantly shorter than in LesseffectiveGroup (113.3 days vs. 155.4 days, p < 0.05), and the PressureUlcers reduction rate was approximately 28.1% (Figure 3-a). Likewise, DESIGN-rating in EG was also decreased for comparing the onset with the endpoint (11.5 rates vs. 14.3 rates, p < 0.05) between pre-post evaluations including both in onset (evaluation in the initial time, at the day for the admission to hospital) and in endpoint (evaluation in the last time, at the day for the discharge from hospital or death day). In LG, no statistically significance was seen, in DESIGN-rating indicative of degree of severity for PU, between both of them (Figure 3-b).

Figure 3

Comparison of PU clinical effects for the hospitalized days and the DESIGN-rating in the effective group and in the less effective group. Figure 3a shows the period for the PU hospitalized days in EG was significantly shorter than in LG. Figure