Currently, inhalation of molecular hydrogen gas is being used in at least four clinical studies .
Recent public reports by China’s National Health Commission and the Chinese CDC and Prevention have recommended effective oxygen therapy measures as an element of general treatment in patients with COVID19 penumonia ( novel coronavirus pneumonia).2,3 A ratio of (2 H2 to 1 O2 ; molecular hydrogen2 :1 oxygen) was stated as the composition of the gas mixture for inhalation. While high oxygen levels are administered due to apparent lung dysfunction in coronavirus 2019 – COVID19, adding H2 to oxygen for a breathing mixture raises interesting questions because molecular hydrogen may have beneficial effects by itself.
A recent study suggested that hydrogen gas inhibits airway inflammation in patients with asthma,4 an effect thatmight improve the condition of inflammatory cytokines storm seen in COVID19.5
Dr. Zhong Nanshan, the epidemiologist who discovered the SARS coronavirus in 2003, recently recommended the inhalation of molecular hydrogen for COVID19 patients based on the preliminary clinical data .
Molecular hydrogen treatment resulted in significant amelioration of dyspnea in most patients with COVID19 in the pilot investigation.
Correspondingly, molecular hydrogen inhalation is included as a
recommendation by the National Health Commission of The People’s Republic of China’s protocol for COVID-19 .
Although the research is on inhalation of molecular hydrogen gas, ingestion of molecular hydrogen rich water HRW is also effective, and, at least in some cases, even more effective (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 .).
Though we do NOT state hydrogen water is a covid19 treatment, due to the urgency of the COVID19 pandemic, and the
safety and clinical viability of hydrogen water, it might be useful to clinically test hydrogen water thouroughlly for its potential use in treating CoronaVirus 2019 COVID19 patients
MUST READ ABOUT COVID19 TREATMENTS:
LeBaron TW (2020) A novel functional beverage for COVID-19 and other conditions: Hypothesis and preliminary data, increased blood flow, and wound healing J Transl Sci, 2020 doi: 10.15761/JTS.1000380 Volume 6: 6-6
We present the case of a lung cancer patient with multiple metastases (diagnosis established in 2015) who, after the failure of the targeted oral drugs (chemotherapy), multiple cerebral , bone , adrenal gland and liver metastases have reappeared .
4 months after molecular gas-hydrogen therapy as a UNIQUE TREATMENT, the size of brain metastases / tumors was significantly reduced, and the amount of intracranial fluid was significantly reduced.
12 months of SOLE treatment with molecular hydrogen, ALL brain tumors entered FULL REMISSION and the liver and lung metastases remained stable and the survival time was prolonged.
For copyright reasons I will not reproduce the entire article but I invite you to your read it here
Regarding administration of molecular hydrogen – can get a similar effect with hydrogenated water?!
The results Showed That although molecular hydrogen H 2 Concentrations in the brain either tilt to be high after inhalation or intravenous administration, have not been Observed Significant differences in comparison with the Concentrations after the consumption of molecular hydrogen H 2 -rich water and ip administration of molecular hydrogen H 2 -rich brine. Thus, although variations There have been based on the administration method, all Methods have been found to result in the presence of molecular hydrogen H 2 in the serum and brain tissue .
Liu et al. ( 39 ) Measured molecular hydrogen H 2 levels in the arteries, veins, and brain tissues after the inhalation of 2% molecular hydrogen H 2 gas. They found That arterial molecular hydrogen H 2 peaked at 30 min after administration, whereas brain tissue and venous molecular hydrogen H 2 peaked at 45 min after administration. They Reported That molecular hydrogen H 2 levels in arteries and similar Were brain tissues.
This demonstrated That molecular hydrogen H 2 migrates to the brain tissue Regardless of the method of administration ( Thus, the studies have been Might well as molecular hydrogen Performed Using INSTEAD of molecular hydrogen gas water or saline molecular hydrogen ).
Furthermore, keep in mind That crossing the blood-brain barrier ( BBB ) is a very difficult task to Achieve for many Substances, thus the FACT That molecular hydrogen H 2 Crosses the BBB and migrates to the brain tissue Regardless of the method of administration ( Including molecular hydrogen by drinking water – Which is the easiest method of administration molecular hydrogen ) That is a strong indicator of can benefit from one molecular hydrogen drinking water just as much as from any other method of administration molecular hydrogen
A dose-response effect of molecular hydrogen is molecular hydrogen Observed in drinking water-rich [ 94 , 97 ] .
A similar dose-response effect is inhaled Also Observed molecular hydrogen in the gas [ 1 , 17 , 98 ].
However, when molecular hydrogen Concentrations in drinking water in the inhaled gas and is compared, dose-response There is no effect.
Molecular hydrogen-rich water shows generally the 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 ] .
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.
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 (5, 6), ethylene (7, 8), 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 (9–13). 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 (9, 14, 15). 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 (16–19).
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 (20, 21), 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 (22, 23). 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 (27, 28), rheumatoid arthritis (29), brain injury (30), ischemic reperfusion injury (31, 32), and diabetes (33, 34), 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 (∙O−2•O2-), nitric oxide (NO•), and peroxynitrite (ONOO−), etc. (37, 38). 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 (39, 40). 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 (41, 42). 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 (43–45).
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 (42, 47). 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 (48, 51). 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 (53–55). 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 (56–59), and both of ILs and TNFs can be suppressed by hydrogen gas (60, 61).
Inflammation induced by chemotherapy in cancer patients not only causes severely adverse effects (62, 63), but also leads to cancer metastasis, and treatment failure (64, 65). 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 (67, 68). 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) (69, 70).
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 (73, 74). However, cancer patients receiving these treatments often experience fatigue and impaired quality of life (75–77). The skyrocketed generation of ROS during the treatment is believed to contribute in the adverse effects, resulting in remarkable oxidative stress, and inflammation (41, 42, 78). 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 (81, 82). 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 (83, 84).
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 (87, 88). 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 (48, 90).
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 (94, 95). 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).
Hydrogen gas has been recognized as one medical gas that has potential in the treatment of cardiovascular disease, inflammatory disease, neurodegenerative disorders, and cancer (17, 60). 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. The Summary of various formulation, application, mechanisms of H2 in cancer treatment.
Figure 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.
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 .
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 .
Sai Li1†, Rongrong Liao2†, Xiaoyan Sheng2†, Xiaojun Luo3, Xin Zhang1, Xiaomin Wen3, Jin 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
SL, XW, JZ, and KP: conceptualization. SL, RL, XS, XL, XZ, JZ, and KP: writing. SL, RL, and XS: revising.
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.
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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This is the first report demonstrating the benefits of drinking molecular hydrogen water in liver cancer patients receiving radiation therapy for malignant tumors. – Molecular hydrogen dissolved in water improved the QOL of (liver) cancer patients reciving radiotherapy – Molecular hydrogen water mitigated oxidative stress marker during radiotherapy – Molecular hydrogen water did NOT compromise the radiation cancer treatment efficacies -Molecular hydrogen water treatment did NOT alter liver function or blood composition during radiotherapy
This study examined whether molecular hydrogen (dissolved in water ) treatment, improved QOL in patients receiving radiotherapy.
Cancer patients receiving radiotherapy often experience fatigue and impaired quality of life (QOL).
Most radiation-induced symptoms are believed to be associated with increased oxidative stress and inflammation, due to the generation of reactive oxygen species (ROS) during radiotherapy, and may significantly affect the patient’s quality of life (QOL) .
Molecular hydrogen (dissolve in water) can be administered as a therapeutic medical gas, has selective ANTIoxidant( molecular hydrogen ( water ) neutralizes only bad free radicals while supporting the beneficial ones) & ANTIinflammatory (molecular hydrogen( water )reduces inflammation in tisues) properties.
Drinking liquids(i.e. : water) with dissolved molecular hydrogen represents a novel method of molecular hydrogen gas delivery that is easily translatable into clinical practice, with beneficial effects for several medical conditions, including atherosclerosis, type 2 diabetes, metabolic syndrome, and cognitive impairment during aging and in Parkinson’s disease [7–11].
A randomized, placebo-controlled study was performed to evaluate the effects of drinking molecular hydrogen-rich water on 49 patients receiving radiotherapy for malignant liver tumors.
The subjects were randomly assigned to groups to either drink molecular hydrogen-rich water for 6 weeks (n = 25) or drink water containing a placebo (n = 24).
Subjects were provided with four 500 mL bottles of drinking molecular hydrogen water per day .
Molecular hydrogen rich water had final molecular hydrogen concentration; 0.55~0.65 mM.
The subjects were expected to consume 100-300 mL of molecular hydrogen-rich water more than 10 times per day for a total minimum consumption of 1500 mL (1.5 L) and a maximum consumption of 2000 mL (2.0 L).
Oral intake of molecular hydrogen water or placebo water started on the first day of radiotherapy and continued for 6 weeks.
All participants received 5040-6500 cGy of radiotherapy for 7-8 weeks using a 6 MV system (Cyber Knife, Fanuc, Yamanashi, Japan).
All the liver cancer patients survived through the 6 week follow-up period when the QOL questionnaire was administered.
The Korean version of the European Organization for Research and Treatment of Cancer’s QLQ-C30 instrument was used to evaluate global health status and QOL. The concentration of derivatives of reactive oxidative metabolites and biological antioxidant power in the peripheral blood were assessed.
Results & Conclusions
The consumption of molecular hydrogen-rich water for 6 weeks reduced reactive oxygen metabolites in the blood and maintained blood oxidation potential. QOL scores during radiotherapy were SIGNIFICANTLY IMPROVED in patients treated with molecular hydrogen-rich water compared to patients receiving placebo water.
There was no difference in tumor response to radiotherapy between the two groups( meaning drinking molecular hydrogen water did not interfere with the desired antitumor effects of radiation therapy ).
Daily consumption of molecular hydrogen-rich water is a potentially novel, therapeutic strategy for improving QOL after radiation exposure.
Consumption of hydrogen-rich water reduces the biological reaction to radiation-induced oxidative stress without compromising anti-tumor effects.
Molecular hydrogen dissolved in water improved the QOL of (liver) cancer patients receiving radiotherapy
The QOL of the liver cancer patients who were given placebo water deteriorated significantly within the first month of radiotherapy (Figure1A)
Gastrointestinal (GI) symptoms are one of the most common complaints of patients undergoing radiotherapy and are considered to have a high impact on the patient’s QOL after 6 weeks of radiotherapy.
The patients consuming molecular hydrogen water experienced significantly less appetite loss and fewer tasting disorders compared to the patients consuming placebo water.
Liver cancer patients experience GI symptoms and decreased QOL during radiotherapy. These symptoms usually occur as a result of the body repairing damage to healthy cells, are particularly common towards the end of a course of radiation treatment, and can last for some time. The symptoms and their impact on QOL can be worsened by having to travel to the hospital each day.
Drinking molecular hydrogen-rich water improved the QOL of the liver cancer patients receiving radiotherapy and did not require additional hospital visits.
There were no differences between the groups in the QOL subscales for fatigue, depression, or sleep. No significant difference was seen in the mean scores for vomiting or diarrhea (Figure1B).
Placebo water and molecular hydrogen water improved the QOL of patients receiving radiotherapy. A. Weekly assessment of the patients’ QOL. B. Scoring system of GI symptoms after 6 weeks of radiotherapy with or without molecular hydrogen water.
Molecular hydrogen water mitigated oxidative stress marker during radiotherapy
Before treatment, there were no differences in total hydroperoxide levels, representative of total dROM levels, between the treatment groups.
Radiotherapy markedly increased total hydroperoxide levels in the patients consuming placebo water.
However, drinking molecular hydrogen water prevented this increase in total serum hydroperoxide, as determined by the dROM test(Figure2A), indicating DECREASED OXIDATIVE STRESS during radiotherapy in the liver cancer patients who consumed molecular hydrogen water.
Similarly, endogenous serum antioxidant activity significantly deteriorated during radiotherapy in the patients consuming placebo water, and biologic antioxidant activity was MAINTAINED in liver cancer patients who consumed molecular hydrogen-rich water, even after 6 weeks of radiotherapy(Figure2B).
Molecular hydrogen water mitigated oxidative stress marker during radiotherapy. Antioxidative effects in patients with placebo water (n = 24) and molecular hydrogen rich water (n = 25). The dROM level (A) represents the total level of peroxide metabolities, and BAP (B) reflects ...
Previous experimental studies have linked daily consumption of molecular hydrogen-rich water with improvement of a number of conditions in rodent models, including reducing atherosclerosis in apolipoprotein E knockout mice , alleviating cisplatin(chemotherapy)-induced nephrotoxicity , reducing vitamin C deficiency-induced brain injury , preventing chronic allograft nephropathy after renal transplantation , and ameliorating cognitive defects in senescence-accelerated mice  and a Parkinson’s disease model . In human studies, consumption of molecular hydrogen-rich water prevented adult-onset diabetes and insulin resistance , as well as oxidative stress in potential metabolic syndrome .
Radiotherapy is associated with an increase in ROS, followed by damage to DNA, lipids, and proteins, and activation of transcription factors and signal transduction pathways. It has been estimated that 60-70% of the ionizing radiation-induced cellular damage is caused by hydroxyl radicals .
Therefore, a number of trials with the goal of reducing adverse effects due to excess ROS production have been performed with antioxidants delivered during the course of radiotherapy. Supplementation with α-tocopherol improves the salivary flow rate and maintains salivary parameters . Treatment with the antioxidant enzyme superoxide dismutase prevented radiotherapy-induced cystitis and rectitis in bladder cancer patients receiving radiotherapy . In addition, the combined use of pentoxifylline and vitamin E reduced radiation-induced lung fibrosis in patients with lung cancer receiving radiotherapy .
Thus, in general, supplementation with antioxidants is likely to offer overall benefits in the treatment of adverse effects of radiotherapy.
However, not all antioxidants can afford radioprotection [22–24].
Furthermore, of significant concern is the finding that high doses of antioxidants administered as adjuvant therapy might compromise the efficacy of radiation treatment and increase of the risk of local recurrence of cancer [25,26].
Hence, the relatively lower toxicity associated with the use of these antioxidant agents is appealing, but not at the cost of poor tumor control.
In contrast, in this study, drinking molecular hydrogen-rich water did NOT affect radiotherapy’s anti-tumor effects.
Molecular hydrogen water did NOT compromise the radiation cancer treatment efficacies
Tumor response to radiotherapy was similar between the cancer treatment groups, and 12 of 24 (50.0%) liver cancer patients in the placebo group and 12 of 25 (48%) patients in molecular hydrogen water group exhibited either a completed response (CR) or a partial response (PR). There were no patients in either group with progressive disease (PD) during the follow-up period (3 months). Thus, drinking molecular hydrogen water did NOT compromise the anti-tumor effects of radiotherapy.
Our results may suggest that hydrogen water functions not only as an antioxidant, but also plays a protective role by inducing radioprotective hormones or enzymes.
Molecular hydrogen water treatment did NOT alter liver function or blood composition during radiotherapy
There were no significant differences in aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase (γ-GTP) and total cholesterol levels at week 0 and week 6, regardless of the type of water consumed(Table2), indicating that molecular hydrogen water consumption did NOT alter liver function.
Similarly, there were no significant differences in red blood cell count, white blood cell count, or platelet count between patients consuming molecular hydrogen water and patients consuming placebo water (Table3).
This finding may provide the foundation for a clinically applicable, effective, and safe strategy for the delivery of molecular hydrogen gas (dissolved in water) to mitigate radiation-induced cellular injury.
Oral intake of daily molecular hydrogen-supplemented water might be a prophylactic strategy to improve QOL of the (liver cancer ) patients receiving radiotherapy.
Although the mechanisms underlying the beneficial effects of molecular hydrogen-rich water during radiotherapy have not been clearly elucidated, drinking molecular hydrogen dissolved in water reduced dROM levels and maintained BAP levels in the serum, suggesting molecular hydrogen-rich water exhibits potent systemic antioxidant activity.
The safety of molecular hydrogen-rich water has also been determined as well as the optimal concentration of molecular hydrogen dissolved in water;
Daily intake of molecular hydrogen-rich water may be a promising approach for counteracting radiation-induced impairments to QOL.
This therapeutic use of molecular hydrogen is also supported by the work of Qian et al., who demonstrated that treating human lymphocyte AHH-1 cells with molecular hydrogen (saline) before irradiation significantly inhibited ionizing irradiation-induced apoptosis and increased cell viability in vitro.
They also showed that injection of molecular hydrogen-rich saline could protect the gastrointestinal endothelia from radiation-induced injury, decrease plasma malondialdehyde and intestinal 8-hydroxydeoxyguanosine levels, and increase plasma endogenous antioxidants in vivo .
In conclusion, our study demonstrated that drinking molecular hydrogen-rich water improved QOL and reduced oxidative markers in patients receiving radiotherapy for liver tumors.
This novel approach of oral intake of molecular hydrogen-rich water may be applicable to a wide range of radiation-related adverse symptoms.
Drinking solubilized molecular hydrogen (dissolved in water) on a daily basis is beneficial and would be quite easy to administer without complicating or changing a patient’s lifestyle
Radiotherapy is one of the major treatment options for malignant neoplasms. Nearly half of all newly diagnosed cancer patients will receive radiotherapy at some point during treatment and up to 25% may receive radiotherapy a second time . Radiotherapy adversely affects the surrounding normal cells . Acute radiation-associated side effects include fatigue, nausea, diarrhea, dry mouth, loss of appetite, hair loss, sore skin, and depression. Radiation increases the long-term risk of cancer, central nervous system disorders, cardiovascular disease, and cataracts. The likelihood of radiation-induced complications is related to the volume of the irradiated organ, the radiation dose delivered, the fractionation of the delivered dose, the delivery of radiation modifiers, and individual radiosensitivity . Most radiation-induced symptoms are believed to be associated with increased oxidative stress and inflammation, due to the generation of reactive oxygen species (ROS) during radiotherapy, and may significantly affect the patient’s quality of life (QOL) .
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The study published in 2017 was conducted to investigate the protective effect of hydrogen-rich water on the liver function of colorectal cancer (CRC) patients treated with mFOLFOX6 chemotherapy.
A controlled, randomized, single-blind clinicaltrial was designed.
A total of 152 patients with colorectal cancer were recruited by the Department of Oncology of Taishan Hospital (Taian, China) between June 2010 and February 2016, among whom 146 met the inclusion criteria. Subsequently, 144 patients were randomized into the treatment with hydrogen water(n=80) and placebo (n=64) groups. At the end of the study, 76 patients in the hydrogen water treatment group and 60 patients in the placebo group were included in the final analysis.
The 80 patients group started drinking hydrogen-rich water 1 day prior to chemotherapy until the end of the cycle, for a total of 4 days, with a total intake of 1,000 ml hydrogen-rich water per day in 4 doses (250 ml hydrogen-rich water each). Hydrogen-rich water was consumed 0.5 h after a meal and before bedtime.
The patients did not discontinue consuming hydrogen-rich water during the entire course of chemotherapy.
The other 64 placebo patients consumed distilled water, with a daily intake of 1,000 ml in 4 doses (250 ml each).
The changes in liver function after the chemotherapy, such as altered levels of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase, indirect bilirubin (IBIL) and direct bilirubin, were observed. The damaging effects of the mFOLFOX6 chemotherapy on liver function were mainly represented by increased ALT, AST and IBIL levels.The hydrogen-rich water group exhibited no significant differences in liver function before and after treatment, whereas the placebo group exhibited significantly elevated levels of ALT, AST and IBIL. Thus, hydrogen-rich water appeared to alleviate the mFOLFOX6-related liver injury
Molecular hydrogen (water) in the treatment of acute and chronic neurological conditions(i.e Alzheimer’s, Parkinson’s, etc. ): mechanisms of protection and routes of administration
We review the effects of molecular hydrogen water therapy in acute neuronal conditions and neurodegenerative diseases.
Molecular hydrogen water therapy /drinking water with dissolved molecular hydrogen may be useful for the prevention of neurodegenerative diseases and for reducing the symptoms of acute neuronal conditions.
Recently, the neuroprotective effects of treatment with molecular hydrogen (water) have been reported in both basic and clinical settings-as you will see below, we have examined the effects of molecular hydrogen H2 (water) treatment on acute central nervous system diseases and on chronic neurodegenerative diseases. We have also examined the various mechanism by which molecular hydrogen H2 exerts its neuroprotective effects .
Molecular hydrogen H2 acts as a scavenger for OH− and ONOO−, affects neuroinflammation, preserves mitochondrial energy production, and possesses neuroprotective properties.
Unlike more conventional drugs, molecular hydrogen H2 treatment, particularly the consumption of molecular hydrogen H2-rich water, has no known serious side effects and is effective for preventing the onset of neurodegenerative disease and aggravation of acute neuronal conditions – i.e.:
Molecular hydrogen water & Parkinson’s disease (PD)
Parkinson’s disease PD is a disorder that presents with extrapyramidal symptoms caused by the degeneration and loss of dopamine-producing cells in substantia nigra. Oxidative stress is known to be involved in the clinical condition of PD.(7) Moreover, the involvement of mitochondrial dysfunction in PD has been reported.(45)
The effects of molecular hydrogen H2 on Parkinson’s disease PD have been reported in animal models of PD as well as in clinical studies.(46–48)
In 2009, Fujita et al.(47) and Fu et al.(48) reported that consuming molecular hydrogen H2-rich water inhibits oxidative stress on the nigrostriatal pathway and prevents the loss of dopamine cells in a PD animal model. With the consumption of molecular hydrogen H2-rich-water-drinking, oxidative stress in the nigrostriatal pathway was inhibited and loss of dopamine cells was decreased. These results suggest that consuming molecular hydrogen H2-rich water could affect the onset of Parkinson’s Disease PD.
In recent years, the results of a clinical trial on the effects of consuming molecular hydrogen H2-rich water for Parkinson’s Disease PD have been reported.(49) A randomized double-blind study showed that consuming molecular hydrogen H2-rich water (1,000 ml/day) for 48 weeks significantly improved the total Unified Parkinson’s Disease Rating Scale (UPDRS) score of Parkinson’s disease PD patients treated with levodopa. A double-blind multi-center trial of molecular hydrogen H2 water is currently underway (Table 1).(50)
Molecular hydrogen water & Alzheimer’s disease (AD)
Alzheimer Disease AD, an age-related neurodegenerative disease, is the most common cause of dementia.(1,51) Pathologically, it is characterized by the deposition of Aβ protein outside nerve cells and the accumulation of phosphorylated tau protein inside nerve cells. There is also a marked loss of nervous cells in the cerebral cortex.(52) In recent years, oxidative stress and neuroinflammation have been reported to be involved in Alzheimer’s disease AD.(1,5) To date, reports have centered on the involvement of oxidative stress in brain parenchyma.(1,51,53)The accumulation of Aβ protein is strongly associated with the failure of Aβ clearance that is closely related to the pathogenesis of Alzheimer’s Disease AD.(5) It is known that low-density lipoprotein receptor-related protein 1 (LRP1) is involved in Aβ protein elimination. LRP dysfunction caused by oxidative stress and neuroinflammation is involved in the onset of Alzheimer’s Disease AD.(5) The regulation of oxidative stress and neuroinflammation may prevent the onset or progression of Alzheimer’s Disease AD. A number of reports have investigated the effects of molecular hydrogen H2 for the prevention of Alzheimer’s Disease AD onset.(51,53)
In a rat Alzheimer’s Disease AD model, it has been reported that the administration of molecular H2-rich saline (5 ml/kg, i.p., daily) inhibited oxidative stress, cytokine production, and nuclear factor-κB (NF-κB) production in the hippocampus and cerebral cortex, and improved impaired memory.(51,53)
It has been reported that consuming molecular hydrogen H2-rich water inhibits age-related brain alterations and spatial memory decline.(54)
The therapeutic effect of molecular hydrogen H2-rich water following Traumatic brain injury (TBI) and in posttraumatic onset of Alzheimer’s disease (AD) was investigated by Dohi et al. in 2014,(18) who investigated whether the consumption of molecular hydrogen H2-rich water 24 h prior to trauma can inhibit neuronal damage in a controlled cortical injury model using mice. The authors found that the expression of the phosphorylated tau proteins AT8 and Alz50 in the hippocampus and cortex was blocked in mice that consumed molecular hydrogen H2-rich water. Moreover, the activity of astrocytes and microglia were inhibited in mice Traumatic Brain Injury model consuming molecular hydrogen H2-rich water. The expression of genes induced by Traumatic Brain Injury, particularly those that are involved in oxidation/carbohydrate metabolism, cytokine release, leukocyte or cell migration, cytokine transport, and adenosine triphosphate (ATP) and nucleotide binding, was inhibited by consuming molecular hydrogen H2-rich water.
Dohi et al.(18) specifically reviewed the role of molecular hydrogen H2-rich water in neuroinflammation following brain trauma. The consumption of molecular hydrogen H2-rich water influenced the production of cytokines and chemokines in the damaged brain and inhibited the production of hypoxia inducible factor-1 (HIF-1), MMP-9, and cyclophilin A. However,molecular hydrogen H2-rich water did not affect the production of amyloid precursor protein (APP), Aβ-40, or Aβ-42. They also investigated the relationship between molecular hydrogen H2 and ATP production and reported that molecular hydrogen H2 increased basal respiration, reserve capacity, and nonmitochondrial respiration but did not increase aerobic ATP production. It has thus been demonstrated that the inhibitory effects of molecular hydrogen H2 on nerve damage are not solely due to its simple function as a free radical scavenger (Fig. 1 and and22).
Molecular hydrogen is well characterized as a selective scavenger of hydroxyl radicals and peroxynitrite.
Oxidative stress caused by reactive oxygen species is considered a major mediator of tissue and cell injuries in various neuronal conditions, including neurological emergencies and neurodegenerative diseases.
Oxidative stress caused by reactive oxygen species (ROS) is a major mediator of tissue and cellular injuries in various neuronal conditions, including neurological emergencies and neurodegenerative diseases.(1–7)
Control of oxidative stress is a major therapeutic strategy for various neuronal conditions.(6,8,9) There are many methods for controlling oxidative stress with the use of free radical scavengers being the most common approach.(6,8) Evidence from animal experiments support the notion that free radical scavengers and antioxidants dramatically reduce cerebral damage.(9) Edaravone (MCI-186), a novel free radical scavenger, was developed to prevent lipid peroxidation in pathological neurological conditions.(8,9)Edaravone is currently the only antioxidant drug approved for treating cerebral infarction that improves the functional outcome of ischemic stroke.(8) Brain hypothermia therapy (targeted temperature management) can also effectively control oxidative stress. Brain hypothermia therapy is effective in patients with various acute neuronal diseases.(6,10,11)
In 2007, Ohsawa et al.(12) reported that molecular hydrogen (H2) can act as an antioxidant to prevent and treat middle cerebral artery occlusion–reperfusion injury in rats. This effect has been supported by additional reports. Recently, the beneficial effect of molecular H2 has been reported in many other organs, including the brain.(13–17) The first major therapeutic effect of molecular hydrogen H2 was that of an antioxidant, combining with hydroxyl ions to produce water.(12) Recently, other biological mechanisms of molecular hydrogen H2 (anti-inflammatory, anti-apoptosis, anti-cytokine, DNA expression, and energy metabolism) have been proposed (Fig. 1 and and22).(18)Therefore, the biology of molecular hydrogen H2 is not simple. In this review, we discuss the role of molecular H2 in various neuronal conditions.
Effect of consumption of molecular hydrogen-rich water as functional water in pathophysiology of neurodegenerative diseases. ATP, adenosine triphosphate; miR-200, microRNA-200; ROS, reactive oxygen species.
Method and Route of Administration in Molecular hydrogen H2 Therapy
As a small (2 Da), uncharged molecule of hydrogen H2, would be expected to readily distribute throughout the body, including being able to easily penetrate cell membranes, However we are unable to determine the distribution of moleclar hydrogen H2 among organs and its concentrations in each organ and serum based on the administration methods and dosage. This problem was investigated in 2014.(55) A comparative review was conducted on the consumption of molecular hydrogen H2-rich water, i.p. or intravenous administration of molecular hydrogen H2-rich saline, and inhalation of molecular hydrogen H2 gas. The results showed that the highest concentrations are reached 1 min after intravenous administration and 5 min after oral administration. The highest concentration was reached 30 min after the inhalation of molecular hydrogen H2 gas and was maintained for some time. Although molecular hydrogen H2 concentrations in the brain tend to be high after either intravenous administration or inhalation, no significant differences have been observed in comparison with the concentrations after the consumption of molecular hydrogen H2-rich water and i.p. administration of molecular hydrogen H2-rich saline. Thus, although there have been variations based on the administration method, all methods have been found to result in the presence of molecular hydrogen H2 in the serum and brain tissue. Liu et al.(39) measured molecular hydrogen H2 levels in the arteries, veins, and brain tissues after the inhalation of 2% molecular hydrogen H2 gas. They found that arterial molecular hydrogen H2 peaked at 30 min after administration, whereas venous and brain tissue molecular hydrogen H2 peaked at 45 min after administration. They reported that molecular hydrogen H2 levels were similar in arteries and brain tissues.
This demonstrated that molecular hydrogen H2 migrates to the brain tissue regardless of the method of administration(Thus, the studies below might as well have been performed using molecular hydrogen water instead of molecular hydrogen gas or molecular hydrogen saline).
These results suggest that the consumption of molecular hydrogen H2-rich water prevents neurodegenerative disease and that molecular hydrogen H2-rich drinking water could be used to treat acute brain disorders (Fig. 1 and and22).
Molecular Hydrogen & Neurological Diseases
Molecular hydrogen & Ischemic brain injury
It has been reported that molecular hydrogen H2 prevents ischemic brain damage in animal experiments.(12,19–21) Ohsawa et al.(12) reported that inhalation of 2% molecular hydrogen H2 gas strongly suppressed infarct volume after middle cerebral artery ischemia–reperfusion in rats. In an electron spin resonance (ESR) study, they showed that molecular hydrogen H2 had hydroxyl radical scavenging activity. Hydroxynonenal (HNE) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) immunoreactivity was suppressed in the damaged brain after treatment with 2% molecular hydrogen H2. molecular hydrogen H2 inhalation reduced ischemic damage and hemorrhagic volume after transient middle crebral artery occlusion (MCAO) ischemia.(19) Free radical generation after ischemia induces matrix metalloproteinase (MMP) expression.(19,20) MMP-9 promotes hemorrhagic infarction by disrupting cerebral vessels.(20) molecular hydrogen H2 inhalation has been found to reduce MMP-9 expression in an MCAO rat model. molecular hydrogen H2 also has a neuroprotective effect against global ischemia. Ji et al.(21) reported that molecular hydrogen H2-rich saline injection [5 ml/kg intra-peritoneal (i.p.) administration] after global ischemia reduced neuronal cell death in hippocampal Cornet d’Ammon 1 (CA1) lesions in rats. Cerebral hypoxia–ischemia and neonatal asphyxia are major causes of brain damage in neonates. molecular hydrogen H2 gas inhalation and molecular hydrogen H2-rich saline injection provide early neuroprotection from neonatal neurological damage.(22) Nagatani et al.(23) reported that that an molecular hydrogen H2-enriched intravenous solution is safe for patients with acute cerebral infarction, including patients treated with tissue plasminogen activator (t-PA) therapy.
Metabolic syndrome is a strong risk factor of stroke. It has been reported that molecular hydrogen H2 therapy can improve metabolic syndrome in basic and clinical settings.(24–27) molecular hydrogen H2 therapy may reduce stroke in patients with metabolic syndrome involving diabetes mellitus.
Molecular hydrogen & Hemorrhagic stroke
Hemorrhagic stroke involving intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH) is a critical neuronal condition, and the mortality rate of hemorrhagic stroke is still high.(28–30) Manaenko et al.(28) reported a neuroprotective effect of molecular hydrogen H2 gas inhalation using an experimental ICH animal model.molecular hydrogen H2 gas inhalation suppresses redox stress and blood brain barrier (BBB) disruption by reducing mast cell activation and degranulation. Brain edema and neurological deficits were also suppressed. In SAH, there are several studies demonstrating the neuroprotective effect of molecular hydrogen H2 treatment.(29–31) A clinical trial has started in patients with SAH (Table 1).(32)
Clinical trials of molecular hydrogen in central nervous system (CNS) diseases
Molecular hydrogen & Traumatic brain injury (TBI)
The efficacy of molecular hydrogen H2 for treating TBI has been investigated in several studies.(18,33,34) Ji et al.(33) reported that in a rat TBI model,molecular hydrogen H2 gas inhalation has been found to protect BBB permeability and regulate posttraumatic brain edema, thereby inhibiting brain damage. molecular hydrogen H2 gas inhalation also inhibits the decrease in superoxide dismutase (SOD) activity and catalase (CAT) activity. These are antioxidant enzymes in posttraumatic brains that inhibit the production of malondialdehyde (MDA) and 8-iso-prostaglandin F2α (8-iso-PGF2α). Eckermann et al.(34) reported that in a surgical trauma mouse model involving right frontal lobectomy, molecular hydrogen H2 gas inhalation has been found to inhibit postoperative brain edema and improve the postoperative neurobehavioral score. The same report also showed that lipid peroxidation and the production of oxidative stress substances were not inhibited by molecular hydrogen H2 gas inhalation.(34)
Molecular Hydrogen & Spinal cord injury
Chen et al.(35) reviewed the effects of molecular hydrogen H2-rich saline administration (i.p.) in a rat traumatic spinal cord injury model. They found that posttraumatic neurological symptoms were improved by molecular hydrogen H2-rich saline treatment. Furthermore, molecular hydrogen H2-rich saline treatment has been found to reduce inflammatory cell infiltration, TdT-mediated dUTP nick and labeling (TUNEL)-positive cells, and hemorrhage. In addition, oxidative stress was inhibited and the expression of brain derived neurotrophic factor (BDNF) was increased.
The effects of molecular hydrogen H2 administration on spinal cord ischemia have also been reported.(36,37) Huang et al.(36)investigated the effects of molecular hydrogen H2 gas inhalation in a rabbit spinal cord ischemia–reperfusion model. They reviewed the effects of molecular hydrogen H2 inhalation with different concentrations (1, 2, and 4%) and reported that molecular hydrogen H2 gas inhalation at concentrations of 2% and 4% inhibited neuronal death. However, they did not observe significant differences between the two groups in terms of effects with 2% and 4% being equally effective.(36) It has been reported that the inhalation of 2% molecular hydrogen H2 gas inhibits apoptosis following spinal cord injury caused by ischemia–reperfusion. In addition, molecular hydrogen H2 gas inhalation regulates caspase-3 activity, the production of inflammatory cytokines, oxidative stress, and the decrease in endogenous antioxidant substances. Zhou et al.(37) also reported that molecular hydrogen H2-rich saline administration (i.p.) has beneficial effects on spinal cord ischemia–reperfusion injury in rabbits.
Other acute neurological conditions
In recent years, research has shown that there is a high incidence of comorbid central nervous system symptoms in sepsis cases.(38) Using a mice cecal ligation and puncture (CLP) model, Liu et al.(39) reported that molecular hydrogen H2 gas inhalation improves septic encephalopathy. They reported that 2%molecular hydrogen H2 gas inhalation inhibited post-CLP apoptosis, brain edema, BBB permeability, cytokine production, and oxidative stress in the CA1 hippocampus region as well as improves cognitive function. Nakano et al.(40) reported that maternal administration of molecular hydrogen H2 has a suppressive effect on fetal brain injury caused by intrauterine inflammation with maternal intraperitoneal injection of lipopolysaccharide (LPS).
The treatment of carbon monoxide (CO) poisoning encephalopathy, which is a common gas poisoning, is yet to be established.(41,42) Sun et al.(42) and Shen et al.(41) investigated the effects of molecular hydrogen H2-rich saline. They reported that in a CO poisoning model, the administration of molecular hydrogen H2-rich saline decreased glial activation, cytokine production, oxidative stress, and caspase 3 and 9 production as well as inhibited nerve cell death.
It is known that oxidative stress causes nerve cell impairments.(43) The consumption of molecular hydrogen H2-rich water inhibits oxidative stress and thereby inhibits the onset of stress-induced brain damage.(43)
Hypoxic brain injury caused by asphyxiation, hypoxic ischemic encephalopathy, neonatal asphyxia, and other similar hypoxia-mediated event is a common clinical condition in medical emergencies. Molecular hydrogen H2 treatment has been found to inhibit cell death in an in vitro hypoxia/reoxygenation model using immortalized mouse hippocampal (HT-22) cells. Molecular hydrogen H2 treatment increased phosphorylated Akt (p-Akt) and B-cell leukemia/lymphoma-2 (BCL-2), while it decreased Bax and cleaved caspase-3.(44) In recent years, it has been found that the microRNA-200 (miR-200) family regulates oxidative stress.(44) The inhibition of miR-200 suppresses H/R-induced cell death, reducing ROS production and MMP. Molecular hydrogen H2 treatment suppressed H/R-induced expression of miR-200. In Japan, a double blind randomized controlled trial for post cardiac arrest syndrome has started from 2017 (Table 1).
diatomic molecular hydrogen H2 dissolved in Alkaline Ionized Water Products (you could drink around 4 liters per day as an adult )
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Effects of molecular hydrogen (water ) on various diseases have been documented for 63 disease models and human diseases in the past four and a half years(by 2012(. Most studies have been performed on rodents including two models of Parkinson’s disease and three models of Alzheimer’s disease. Prominent effects are observed especially in oxidative stress-mediated diseases including neonatal cerebral hypoxia; Parkinson’s disease; ischemia/reperfusion of spinal cord, heart, lung, liver, kidney, and intestine; transplantation of lung, heart, kidney, and intestine. Six human diseases have been studied to date: diabetes mellitus type 2, metabolic syndrome, hemodialysis, inflammatory and mitochondrial myopathies, brain stem infarction, and radiation-induced adverse effects.
Two enigmas, however, remain to be solved. First, no dose-response effect is observed. Rodents and humans are able to take a small amount of hydrogen by drinking hydrogen-rich water, but marked effects are observed. Second, intestinal bacteria in humans and rodents produce a large amount of hydrogen, but an addition of a small amount of hydrogen exhibits marked effects. Further studies are required to elucidate molecular bases of prominent hydrogen (water ) effects and to determine the optimal frequency, amount, and method of hydrogen administration for each human disease
Molecular hydrogen (H2) is the smallest gas molecule made of two protons and two electrons. Hydrogen is combustible when the concentration is 4–75%. Hydrogen, however, is a stable gas that can react only with oxide radical ion (•O−) and hydroxyl radical (•OH) in water with low reaction rate constants :
The reaction rate constants of •O− and •OH with other molecules are mostly in the orders of 109 to 1010 M−1·s−1, whereas those with H2 are in the order of 107 M−1·s−1. Hydrogen, however, is a small molecule that can easily dissipate throughout the body and cells, and the collision rates of hydrogen with other molecules are expected to be very high, which is likely to be able to overcome the low reaction rate constants . Hydrogen is not easily dissolved in water, and 100%-saturated hydrogen water contains 1.6 ppm or 0.8 mM hydrogen at room temperature( our note: see AlkaViva Vesta H2 water ionizer performance below)
In 1995, hydrogen was first applied to human to overcome high-pressure nervous syndrome in deep sea diving . Hydrogen was used to reduce nitrogen (N2) toxicity and to reduce breathing resistance in the deep sea. In 2001, being prompted by the radical-scavenging activity of hydrogen, Gharib and colleagues examined an effect of molecular hydrogen on a mouse model of schistosomiasis-associated chronic liver inflammation . Mice were placed in a chamber with 70% hydrogen gas for two weeks. The mice exhibited decreased fibrosis, improvement of hemodynamics, increased nitric oxide synthase (NOS) II activity, increased antioxidant enzyme activity, decreased lipid peroxide levels, and decreased circulating tumor-necrosis-factor-(TNF-) α levels. Although helium gas also exerted some protective effects in their model, the effect of helium gas was not recapitulated in a mouse model of ischemia/reperfusion injury of the liver .
2. Effects of Hydrogen (water ) Have Been Reported in 63 Disease Models and Human Diseases
A major breakthrough in hydrogen research occurred after Ohsawa and colleagues reported a prominent effect of molecular hydrogen on a rat model of cerebral infarction in June 2007 . Rats were subjected to left middle cerebral artery occlusion. Rats placed in 2–4% hydrogen gas chamber showed significantly smaller infarction volumes compared to controls. They attributed the hydrogen effect to the specific scavenging activity of hydroxyl radical (•OH). They also demonstrated that hydrogen scavenges peroxynitrite (ONOO−) but to a lesser extent.
As have been previously reviewed [7, 8], effects of molecular hydrogen on various diseases have been reported since then. The total number of disease models and human diseases for which molecular hydrogen has been proven to be effective has reached 63 (by 2012)(Table 1). The number of papers is increasing each year (Figure 1). Among the 87 papers cited in Table 1, 21 papers showed an effect with inhalation of hydrogen gas, 23 with drinking hydrogen-rich water, 27 with intraperitoneal administration or drip infusion of hydrogen-rich saline, 10 with hydrogen-rich medium for cell or tissue culture, and 6 with the other administration methods including instillation and dialysis solution. In addition, among the 87 papers, 67 papers showed an effect in rodents, 7 in humans, 1 in rabbits, 1 in pigs, and 11 in cultured cells or cultured tissues.
Two papers, however, showed that hydrogen was ineffective for two disease models (Table 2). One such disease was moderate to severe neonatal brain hypoxia , although marked effects of hydrogen gas [10, 11] and intraperitoneal administration of hydrogen-rich saline  on neonatal brain hypoxia have been reported in rats [10, 12] and pigs . We frequently observe that therapeutic intervention that is effective for mild cases has little or no effect on severe cases, and hydrogen is unlikely to be an exception. Another disease is muscle disuse atrophy . Although oxidative stress is involved in the development of muscle disuse atrophy, oxidative stress may not be a major driving factor causing atrophy and thus attenuation of oxidative stress by hydrogen may not be able to exhibit a beneficial effect.
Two disease models for which hydrogen has no effect.
Effects of molecular hydrogen have been observed essentially in all the tissues and disease states including the brain, spinal cord, eye, ear, lung, heart, liver, kidney, pancreas, intestine, blood vessel, muscle, cartilage, metabolism, perinatal disorders, and inflammation/allergy. Among them, marked effects are observed in ischemia/reperfusion disorders as well as in inflammatory disorders. It is interesting to note, however, that only three papers addressed effects on cancers. First, molecular hydrogen caused growth inhibition of human tongue carcinoma cells HSC-4 and human fibrosarcoma cells HT-1080 but did not compromise growth of normal human tongue epithelial-like cells DOK . Second, hydrogen suppressed the expression of vascular endothelial growth factor (VEGF), a key mediator of tumor angiogenesis, in human lung adenocarcinoma cells A549, which was mediated by downregulation of extracellular signal-regulated kinase (ERK) . Third, hydrogen protected BALB/c mice from developing radiation-induced thymic lymphoma . Elimination of radical oxygen species by hydrogen should reduce a probability of introducing somatic mutations. Unlike other disease models, cancer studies were performed only with cells in two of the three papers. Hydrogen is likely to have a beneficial effect on cancer development by suppressing somatic mutations, but an effect on cancer growth and invasion needs to be analyzed further in detail.
3. Effects of Molecular Hydrogen on Rodent Models of Neurodegenerative Diseases
Parkinson’s disease is caused by death of dopaminergic neurons at the substantia nigra pars compact of the midbrain and is the second most common neurodegenerative disease after Alzheimer’s disease. Parkinson’s disease is caused by two mechanisms: excessive oxidative stress and abnormal ubiquitin-proteasome system . The neurotransmitter, dopamine, is a prooxidant by itself and dopaminergic cells are destined to be exposed to high concentrations of radical oxygen species. An abnormal ubiquitin-proteasome system also causes aggregation of insoluble α-synuclein in the neuronal cell body that leads to neuronal cell death. We made a rat model of hemi-Parkinson’s disease by stereotactically injecting catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in the right striatum . Ad libitum administration of hydrogen-rich water starting one week before surgery completely abolished the development of hemi-Parkinson’s symptoms. The number of dopaminergic neurons on the toxin-injected side was reduced to 40.2% of that on the control side, whereas hydrogen treatment improved the reduction to 83.0%. We also started giving hydrogen-rich water three days after surgery, and hemi-Parkinson’s symptoms were again suppressed, but not as much as those observed in pretreated rats. The number of dopaminergic neurons on the toxin-injected side was 76.3% of that on the control side.Pretreated rats were also sacrificed 48 hrs after toxin injection, and the tyrosine hydroxylase activity at the striatum, where dopaminergic neurons terminate, was decreased in both hydrogen and control groups. This indicated that hydrogen did not directly detoxicate 6-OHDA but exerted a delayed protective effect for dopaminergic cells.Fujita and colleagues also demonstrated a similar prominent effect of hydrogen-rich water on an MPTP-(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-) induced mouse model of Parkinson’s disease . MPTP is a neurotoxin that blocks complex I of the mitochondrial electron transport system and causes Parkinson’s disease in mice and humans. It is interesting to note that the concentration of hydrogen that they used for the MPTP mice was only 0.08 ppm (5% saturation), which is the second lowest among all the trials published to date for rodents and humans. The lowest hydrogen concentration ever tested is 0.048 ppm in the dialysis solution for patients receiving hemodialysis .
Alzheimer’s disease is the most common neurodegenerative disease and is characterized by abnormal aggregation of β-amyloid (Aβ) and tau, the large aggregates of which are recognizable as senile plaques and neurofibrillary tangles, respectively . Effects of molecular hydrogen on Alzheimer’s disease have been studied in three rodent models. First, Nagata and colleagues made a mouse model of dementia by restricting movement of mice for 10 hrs a day . They analyzed cognitive functions through passive avoidance learning, object recognition tasks, and the Morris water maze and demonstrated that ad libitum administration of hydrogen-rich water efficiently ameliorated cognitive impairment. They also showed that neural proliferation in the dentate gyrus was restored by hydrogen water . Second, Li and colleagues made a rat model of Alzheimer’s disease by intracerebroventricular injection of Aβ1-42 . They analyzed cognitive functions by the Morris water maze open field tasks, and electrophysiological measurement of long-term potentiation (LTP) and found that intraperitoneal injection of hydrogen-rich saline for 14 days efficiently ameliorated cognitive decline and preserved LTP. The same team later reported that the protective effects were mediated by suppression of abnormal activation of IL1β, JNK, and NFκB . Third, Gu and colleagues used a senescence-accelerated mouse strain (SAMP8) that exhibits early aging syndromes including impairment in learning ability and memory . Ad libitum administration of hydrogen-rich water for 30 days prevented cognitive decline, which was examined by the Morris water maze. Additionally, ad libitum drinking of hydrogen water for 18 weeks showed efficient amelioration of hippocampal neurodegeneration.
Cerebrovascular diseases are the most frequently reported neurological diseases for which hydrogen (water )has prominent effects. As stated in Section 2, current hydrogen (water ) research has broken out after Ohsawa reported a prominent effect of 2–4% hydrogen for a rat model of left cerebral artery occlusion in 2007 .
In addition to neurodegenerative disorders of Parkinson’s disease and Alzheimer’s disease, effects of molecular hydrogen (water ) have been reported in eight other brain diseases listed under the categories of “brain” and “perinatal disorders” in Table 1. The brain consumes a large amount of oxygen and is predisposed to be exposed to a large amount of radical oxygen species especially under pathological conditions. Molecular hydrogen is thus likely to exert a prominent beneficial effect on brain diseases.
4. Molecular Hydrogen Is Effective for Six Human Diseases(known by 2012)
As in other therapeutic modalities, effects of molecular hydrogen have been tested mostly on rodents but have also been studied in six human diseases( by 2012). The reported human diseases include diabetes mellitus type II , metabolic syndrome , hemodialysis [20, 28], inflammatory and mitochondrial myopathies , brain stem infarction , and radiation-induced adverse effects for liver tumor . These studies are reviewed in detail here. In addition, a therapeutic trial for Parkinson’s disease is currently in progress and exhibits favorable responses as far as we know, but the details are not yet disclosed.
First, Kajiyama and colleagues performed a randomized, double-blind, placebo-controlled, crossover study in 30 patients with diabetes mellitus type II and 6 patients with impaired glucose tolerance . The patients consumed either 900 mL of hydrogen-rich water or placebo water for 8 weeks, with a 12-week washout period. They measured 13 biomarkers to estimate lipid and glucose metabolisms at baseline and at 8 weeks after hydrogen water treatment. All the biomarkers were favorably changed with hydrogen, but statistical significance was observed only in improvement of electronegative charge-modified low-density lipoprotein-(LDL-) cholesterol, small dense LDL, and urinary 8-isoprostanes. In four of six patients with impaired glucose tolerance, hydrogen normalized the oral glucose tolerance test. Lack of statistical significance in their studies was likely due to the small number of patients and the short observation period. Lack of statistical significance, however, may also suggest a less prominent effect in human diabetes mellitus compared to rodent models [32, 33].
Second, Nakao and colleagues performed an open-label trial in 20 subjects with potential metabolic syndrome . Hydrogen-rich water was produced by placing a metallic magnesium stick in water, which yielded 0.55–0.65 mM hydrogen water (70–80% saturation). The participants consumed 1.5–2.0 liters of hydrogen water per day for 8 weeks and showed a 39% increase in urinary superoxide dismutase (SOD), an enzyme that catalyzes superoxide anion (O2−); a 43% decrease in urinary thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation; an 8% increase in high-density-lipoprotein-(HDL-) cholesterol; a 13% decrease in total cholesterol/HDL-cholesterol. The aspartate aminotransferase (AST) and alanine transaminase (ALT) levels remained unchanged, whereas the gamma glutamyl transferase (GGT) level was increased by 24% but was still within a normal range. Although the study was not double blinded and placebo controlled, improvements in biomarkers were much more than those in other hydrogen water studies in humans. As this study used a large amount of hydrogen water, the amount of hydrogen might have been a critical determinant. Alternatively, excessive hydration might have prevented the participants from excessive food intake.
Third, Nakayama and colleagues performed an open-label placebo-controlled crossover trial of 12 sessions of hemodialysis in eight patients  and an open-label trial of 78 sessions of hemodialysis in 21 patients . In both studies, continuous sessions of hemodialysis with hydrogen-rich dialysis solution decreased systolic blood pressure before and after dialysis. In the short-term study, plasma methylguanidine was significantly decreased. In the long-term study, plasma monocyte chemoattractant protein 1 and myeloperoxidase were significantly decreased.
Fourth, we performed an open-label trial of 1.0 liter of hydrogen water per day for 12 weeks in 14 patients with muscular diseases including muscular dystrophies, polymyositis/dermatomyositis, and mitochondrial myopathies, as well as a randomized, double-blind, placebo-controlled, crossover trial of 0.5 liter of hydrogen water or dehydrogenized water per day for 8 weeks in 22 patients with dermatomyositis and mitochondrial myopathies . In the open-label trial, significant improvements were observed in lactate-to-pyruvate ratio, fasting blood glucose, serum matrix metalloproteinase-3 (MMP3), and triglycerides. Especially, the lactate-to-pyruvate ratio, which is a sensitive biomarker for the compromised mitochondrial electron transport system, was decreased by 28% in mitochondrial myopathies. In addition, MMP3, which represents the activity of inflammation, was decreased by 27% in dermatomyositis. In the double-blind trial, a statistically significant improvement was observed only in serum lactate in mitochondrial myopathies, but lactate-to-pyruvate ratio in mitochondrial myopathies and MMP3 in dermatomyositis were also decreased. Lack of statistical significance with the double-blind study was likely due to the shorter observation period and the lower amount of hydrogen compared to those of the open-label trial.
Fifth, Kang and colleagues performed a randomized placebo-controlled study of 1.5–2.0 liters of 0.55–0.65 mM hydrogen water per day for 6 weeks in 49 patients receiving radiation therapy for malignant liver tumors.Hydrogen water suppressed the elevation of total hydroperoxide levels, maintained serum antioxidant capacity, and improved the quality of life (QOL) scores. In particular, hydrogen water efficiently prevented loss of appetite. Although the patients were randomly assigned to the hydrogen and placebo groups, the study could not be completely blinded because hydrogen water was produced with a metallic magnesium stick, which generated hydrogen bubbles.
Sixth, Ono and colleagues intravenously administered hydrogen along with Edaravone, a clinically approved radical scavenger, in 8 patients with acute brain stem infarction and compared MRI indices of 26 patients who received Edaravone alone .The relative diffusion-weighted images (rDWIs), regional apparent diffusion coefficients (rADCs), and pseudonormalization time of rDWI and rADC were all improved with the combined infusion of Edaravone and hydrogen.
No adverse effect of hydrogen has been documented in the six human diseases described above. Among the six diseases, the most prominent effect was observed in subjects with metabolic syndrome, who consumed 1.5–2.0 liters of hydrogen water per day .
The amount of hydrogen water may be a critical parameter that determines clinical outcome.
It is also interesting to note that lipid and glucose metabolisms were analyzed in three studies and all showed favorable responses to hydrogen [26, 27, 29].
Update : since 2012 more clinical trials have been performed.
Effects of hydrogen on various diseases have been attributed to four major molecular mechanisms: a specific scavenging activity of hydroxyl radical, a scavenging activity of peroxynitrite, alterations of gene expressions, and signal-modulating activities. The four mechanisms are not mutually exclusive and some of them may be causally associated with other mechanisms.
The first molecular mechanism identified for hydrogen was its specific scavenging activity of hydroxyl radical . Indeed, oxidative stress markers like 8-OHdG, 4-hydroxyl-2-nonenal (4-HNE), malondialdehyde (MDA), and thiobarbituric acid reactive substances (TBARSs) are decreased in all the examined patients and rodents. As hydrogen can easily dissipate in exhalation, hydrogen in drinking water is able to stay in human and rodent bodies in less than 10 min (unpublished data). Hydrogen, however, can bind to glycogen, and the dwell time of hydrogen is prolonged in rat liver after food intake . A question still remains if mice and humans can take a sufficient amount of hydrogen that efficiently scavenges hydroxyl radicals that are continuously generated in normal and disease states.
Another molecular mechanism of hydrogen effect is its peroxynitrite-(ONOO−-) scavenging activity . Although hydrogen cannot eliminate peroxynitrite as efficiently as hydroxyl radical in vitro , hydrogen can efficiently reduce nitric-oxide-(NO-) induced production of nitrotyrosine in rodents [34–38]. NO is a gaseous molecule that also exerts therapeutic effects including relaxation of blood vessels and inhibition of platelet aggregation . NO, however, is also toxic at higher concentrations because NO leads to ONOO−-mediated production of nitrotyrosine, which compromises protein functions. A part of hydrogen effects may thus be attributed to the reduced production of nitrotyrosine.
Expression profiling of rat liver demonstrated that hydrogen has a minimal effect on expression levels of individual genes in normal rats . Gene ontology analysis, however, revealed that oxidoreduction-related genes were upregulated. In disease models of rodents, expression of individual genes and proteins is analyzed. In many disease models, hydrogen downregulated proinflammatory cytokines including tumor necrosis-factor-(TNF-) α, interleukin-(IL-) 1β, IL-6, IL-12, interferon-(IFN-) γ, and high mobility group box 1 (HMGB1) [4, 23, 24, 36, 41–59]. Hydrogen also downregulated nuclear factors including nuclear factor kappa B (NFκB), JNK, and proliferation cell nuclear antigen (PCNA) [24, 44, 50, 55, 60–63]. Caspases were also downregulated [10, 55–57, 62, 64, 65]. Other interesting molecules studied to date include vascular endothelial growth factor (VEGF) ; MMP2 and MMP9 ; brain natriuretic peptide ; intercellular-adhesion-molecule-1 (ICAM-1) and myeloperoxidase ; B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) ; MMP3 and MMP13 ; cyclooxygenase 2 (COX-2), neuronal nitric oxide synthase (nNOS), and connexins 30 and 43 ; ionized calcium binding adaptor molecule 1 (Iba1) ; fibroblast growth factor 21 (FGF21) . Most molecules, however, are probably passengers that are secondarily changed by hydrogen administration, and some are potentially direct targets of hydrogen effects, which need to be identified in the future.
Using rat RBL-2H3 mast cells, we demonstrated that hydrogen attenuates phosphorylation of FcεRI-associated Lyn and its downstream signaling molecules . As phosphorylation of Lyn is again regulated by the downstream signaling molecules and makes a loop of signal transduction pathways, we could not identify the exact target of hydrogen. Our study also demonstrated that hydrogen ameliorates an immediate-type allergic reaction not by radical-scavenging activity but by direct modulation of signaling pathway(s). In addition, using murine RAW264 macrophage cells, we demonstrated that hydrogen reduces LPS/IFNγ-induced NO production . We found that hydrogen inhibits phosphorylation of ASK1 and its downstream signaling molecules, p38 MAP kinase, JNK, and IκBα without affecting ROS production by NADPH oxidase. Both studies point to a notion that hydrogen is a gaseous signal modulator. More animal and cells models are expected to be explored to confirm that hydrogen exerts its beneficial effect as a signal modulator.
6. Enigmas of Hydrogen Effects
Two enigmas remain to be solved for hydrogen effects. First, no dose-response effect of hydrogen has been observed. Hydrogen has been administered to animals and humans in the forms of hydrogen gas, hydrogen-rich water, hydrogen-rich saline, instillation, and dialysis solution (Table 1). Supposing that a 60-kg person drinks 1000 mL of saturated hydrogen-rich water (1.6 ppm or 0.8 mM) per day, 0.8 mmoles of hydrogen is consumed by the body each day, which is predicted to give rise to a hydrogen concentration of 0.8 mmoles/(60 kg × 60%) = 0.022 mM (2.8% saturation = 0.022 mM/0.8 mM). As hydrogen mostly disappears in 10 min by dissipation in exhalation (unpublished data), an individual is exposed to 2.8% hydrogen only for 10 min. On the other hand, when a person is placed in a 2% hydrogen environment for 24 hrs, body water is predicted to become 2% saturation (0.016 mM). Even if we suppose that the hydrogen concentration after drinking hydrogen water remains the same for 10 min, areas under the curves of hydrogen water and 2% hydrogen gas are 0.022 mM × 1/6 hrs and 0.016 mM × 24 hrs, respectively. Thus, the amount of hydrogen given by 2% hydrogen gas should be 104 or more times higher than that given by drinking hydrogen water. In addition, animals and patients are usually not able to drink 100%-saturated hydrogen water. If the hydrogen concentration is 72% of the saturation level, the peak concentrations achieved by drinking hydrogen water and 2% hydrogen gas should be identical (0.022 mM × 72% = 0.016 mM). Nevertheless, hydrogen water is as effective as, or sometimes more effective than, hydrogen gas.
In addition, orally taken hydrogen can be readily distributed in the stomach, intestine, liver, heart, and lung but is mostly lost in exhalation. Thus, hydrogen concentrations in the arteries are predicted to be very low. Nevertheless, marked hydrogen effects are observed in the brain, spinal cord, kidney, pancreas muscle, and cartilage, where hydrogen is carried via arteries.
The second enigma is intestinal production of hydrogen gas in rodents and humans. Although no mammalian cells can produce hydrogen endogenously, hydrogen is produced by intestinal bacteria carrying hydrogenase in both rodents and humans. We humans are able to make a maximum of 12 liters of hydrogen in our intestines [68, 69]. Specific-pathogen-free (SPF) animals are different from aseptic animals and carry intestinal bacteria that produce hydrogen. The amount of hydrogen taken by water or gas is much less than that produced by intestinal bacteria, but the exogenously administered hydrogen demonstrates a prominent effect.
In a mouse model of Concanavalin A-induced hepatitis, Kajiya and colleagues killed intestinal bacteria by prescribing a cocktail of antibiotics . Elimination of intestinal hydrogen worsened hepatitis. Restitution of a hydrogenase-negative strain of E. coli had no effects, whereas that of a hydrogenase-positive strain of E. coli ameliorated hepatitis. This is the only report that addressed a beneficial effect of intestinal bacteria, and no human study has been reported to date. Kajiya and colleagues also demonstrated that drinking hydrogen-rich water was more effective than the restitution of hydrogenase-positive bacteria. If intestinal hydrogen is as effective as the other hydrogen administration methods, we can easily increase hydrogen concentrations in our bodies by an α-glucosidase inhibitor, acarbose , an ingredient of curry, turmeric , or a nonabsorbable synthetic disaccharide, lactulose [68, 72, 73]. The enigma of intestinal bacteria thus needs to be solved in the future.
7. Summary and Conclusions
Effects of hydrogen have been reported in 63 disease models and human diseases (Table 1). Only two diseases of cerebral infarction and metabolic syndrome have been analyzed in both rodents and humans.
Lack of any adverse effects of hydrogen enabled clinical studies even in the absence of animal studies. Some other human studies including Parkinson’s disease are currently in progress, and promising effects of hydrogen are expected to emerge for many other human diseases. We also have to elucidate molecular bases of hydrogen effects in detail.
8. Added Note in Proof
We recently reported a line of evidence that molecular hydrogen has no dose-response effect in a rat model of Parkinson’s disease .
diatomic molecular hydrogen H2 dizzolved in Alkaline Ionized Water Products
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This article has been cited by other articles in PMC.
Works performed in the authors’ laboratories were supported by Grants-in-Aid from the MEXT and MHLW of Japan and from the Priority Research Project of Aichi.
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Articles from Oxidative Medicine and Cellular Longevity are provided here courtesy of Hindawi Limited
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.
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.
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.
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).
H2-water may have a potential for suppressing dementia in an oxidative stress model and in the APOE4 carriers with MCI.
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) [1, 2]. 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 [7, 8]. 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 [10–12]. In addition to extensive animal experiments, more than 25 clinical studies examining the efficacy of molecular hydrogen H2 have been reported, [11, 12] 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) [18, 19] 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.
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 [23, 24]. 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).
3.1. 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. 1A1A14-month-old).
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. 1A1A18-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. 1B1B14-month-old) and the second ORT at the age of 18 months (Fig. 1B1B18-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).
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).
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.
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 [23, 24].
Background characteristics of 73 subjects with mild cognitive impairment.
SD or %
SD or %
Body mass index (kg/m2)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Current alcohol drinker *
Current smoker *
Current exercise habit *
APOE4 carrier *
Family history *
* 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.
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. (A) p = 0.036 (by Student’s t-test) and p =0.047 (by Mann-Whitney’s U test) and (B) p = 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.
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.
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 [27, 28]. 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 [34, 35]. 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 [40 – 42]. Moderate exercise enhances energy metabolism and suppresses the expression of pro-inflammatory cytokines,  and protects vascular systems [40, 44, 45].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.
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.
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.
This article has been cited by other articles in PMC.
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.).
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.).
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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Health and a vibrant life are sought by everyone. To improve quality of life (QOL), maintain a healthy state, and prevent various diseases, evaluations of the effects of potentially QOL-increasing factors are important. Chronic oxidative stress and inflammation cause deteriorations in central nervous system function, leading to low QOL. In healthy individuals, aging, job stress, and cognitive load over several hours also induce increases in oxidative stress, suggesting that preventing the accumulation of oxidative stress caused by daily stress and daily work contributes to maintaining QOL and ameliorating the effects of aging. Hydrogen has anti-oxidant activity and can prevent inflammation, and may thus contribute to improve QOL. The present study aimed to investigate the effects of drinking hydrogen-rich water (HRW) on the QOL of adult volunteers using psychophysiological tests, including questionnaires and tests of autonomic nerve function and cognitive function. In this double-blinded, placebo-controlled study with a two-way crossover design, 26 volunteers (13 females, 13 males; mean age, 34.4 ± 9.9 years) were randomized to either a group administered oral hydrogen-rich water HRW (600 mL/d) or placebo water (PLW, 600 mL/d) for 4 weeks. Change ratios (post-treatment/pre-treatment) for K6 score and sympathetic nerve activity during the resting state were significantly lower after hydrogen-rich water HRW administration than after PLW administration. These results suggest that hydrogen-rich water HRW may reinforce QOL through effects that increase central nervous system functions involving mood, anxiety, and autonomic nerve function.
Health and a vibrant life are much craved by everyone. To improve quality of life (QOL), maintain a healthy state, and prevent the onset of various diseases, evaluation of interventional effects for improving QOL is important. The high metabolic rate of the brain results in the generation of disproportionate amounts of reactive oxygen and nitrogen species, leading to increased oxidative stress.1 Increased oxidative stress and lipid peroxidation initiate a cascade of proinflammatory signals, leading to inflammation. Altered homeostasis of oxidation, inflammation, and protein aggregation has been suggested to contribute to the death of neurons, which is directly related to impairments in various cognitive domains. As such, chronic oxidative stress and inflammation may cause deteriorations in the function of the central nervous system, leading to reductions in QOL. Hydrogen has antioxidant activity and can prevent inflammation.2,3,4 The distribution of hydrogen throughout the brain and body indicates actions both in the central and peripheral nervous systems. Previous clinical studies have shown that hydrogen-rich water (HRW) reduces concentrations of markers of oxidative stress in patients with metabolic syndrome,5,6improves lipid and glucose metabolism in patients with type 2 diabetes,7 improves mitochondrial dysfunction in patients with mitochondrial myopathies, and reduces inflammatory processes in patients with polymyositis/dermatomyositis.8 In another study, exercise-induced declines in muscle function among elite athletes were also improved by administering hydrogen-rich water HRW.9 Although such findings suggest that hydrogen-rich water HRW may help alleviate symptoms of several diseases and increase the physical performance of athletes, the effects of prolonged hydrogen-rich water HRW ingestion on the QOL of individuals in the general population remain unknown.
Some reports have demonstrated that oxidative stress is associated with QOL in patients with chronic obstructive pulmonary disease and cervical cancer.10,11 During oncological treatment among patients with cervical cancer, antioxidant supplementation was found to be effective in improving QOL.11 In addition, Kang et al.12 reported that treatment with hydrogen-rich water HRW for patients receiving radiotherapy for liver tumors decreased oxidative stress and improved QOL. Although the association between oxidative stress and QOL in healthy individuals is still unclear, aging, job stress, and cognitive load over the course of several hours in healthy individuals have also been found to induce increases in oxidative stress,13,14,15,16 suggesting that preventing the accumulation of oxidative stress caused by daily stress and daily work may contribute to the maintenance of QOL and amelioration of the effects of aging. Continuous hydrogen-rich water HRW intake might therefore be expected to reduce accumulation of oxidative stress, thus helping to prevent decreases in QOL.
The aim of the present study was to investigate the effects of drinking 600 mL of hydrogen-rich water HRW per day for 4 weeks on the QOL of adult volunteers using questionnaires for sleep, fatigue, mood, anxiety, and depression, an autonomic function test, and a higher cognitive function test.
Subjects and Methods
Thirty-one adult volunteers between 20 and 49 years old participated in this double-blinded, randomized, placebo-controlled study with a two-way crossover design. Exclusion criteria comprised: history of chronic illness; chronic medication or use of supplemental vitamins; employment in shift work; pregnancy; body mass index ≤ 17 or ≥ 29 kg/m2; food allergy; history of smoking; or history of drinking excessive amounts of alcohol (≥ 60 g/day). Shift workers were excluded because the water was administered at breakfast and dinner, the timings of which are irregular among shift workers. In addition, the mental and physical conditions of shift workers can be greatly affected by the shift schedule for the preceding 2 days, which may impact the results obtained from the questionnaires used in this study. Before each experiment, participants were asked to refrain from drinking alcohol, since drinking excessive amounts of alcohol carries significant risks of fluctuations in physical condition. All experiments were conducted in compliance with national legislation and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association (the Declaration of Helsinki) and registered to the UMIN Clinical Trials Registry (No. UMIN000022382). The study protocol was approved by the Ethics Committee of Osaka City University Center for Health Science Innovation (OCU-CHSI-IRB No. 4), and all participants provided written informed consent for participation in the study.
We used a double-blinded, placebo-controlled study with a two-way crossover design, as summarized in Figure 1. After admission to the study, participants were randomized in a double-blinded manner to receive hydrogen-rich water HRW in an aluminum pouch (0.8–1.2 ppm of hydrogen, 300 mL/pouch; Melodian Corporation, Yao, Japan) or placebo water (PLW), representing mineral water from the same source (i.e., same components without hydrogen) in an aluminum pouch (0 ppm of hydrogen, 300 mL/pouch; Melodian Corporation) twice a day for 4 weeks. Fifteen participants were administered PLWfirst, and then hydrogen-rich water HRW. The remaining 16 participants were administered HRW hydrogen-rich water first, and then PLW. Participants consumed water within 5 minutes twice a day, at breakfast and dinner in their home, and confirmed the water intake at breakfast and dinner in a daily journal for 4 weeks. We assessed the intake rate of water by checking the daily journal every 4 weeks, on the 2nd and 4th experimental days. No participants reported any difference in taste between hydrogen-rich water HRW and PLW. Previous studies have reported interventional effects of administering hydrogen-rich water HRW to humans at hydrogen concentrations under 1.3 ppm.5,12 We therefore used a similar concentration of 0.8–1.2 ppm in the present study. Absolute volumes (600 mL) of hydrogen-rich water HRW and PLW were provided to participants rather than a volume proportional to body mass, based on previously reported results.5,6,7,12 The duration of supplementation was set based on previous findings with hydrogen-rich water HRW administration for 2–8 weeks.5,12,17 A 4-week washout period was provided between hydrogen-rich water HRW and PLW administrations based on a previous study.8The day before starting each experiment, participants were told to finish dinner by 21:00, and were required to fast overnight to avoid any influence of diet on concentrations of measured parameters (markers of inflammation and oxidative stress) in blood samples. At 09:00 the next day, participants completed the questionnaires after confirming that they had refrained from drinking alcohol, had finished dinner by 21:00, and had fasted overnight. Autonomic nerve function was measured at 09:30. Cognitive function testing was conducted at 09:45. Blood samples were collected at 10:00. These measurements were performed a total of four times for each participant, before (pre) and after (post) each of the two 4-week administration periods. From 24 hours (the day before the visit day) before each visit for measurements, participants were told to refrain from drinking alcohol or performing strenuous physical activity and to follow their normal diets, drinking habits, and sleeping hours. During the 4-week PLW or hydrogen-rich water HRW administration periods, daily daytime activity (amount of physical exertion) of participants was measured using a pedometer and participants kept a daily journal to record drinking volume and times of PLW or HRW intake, physical condition (e.g., pain, lassitude, and indefinite complaints), sleeping times, etc.
Note: Participants were randomly divided into two study groups. The experiment consisted of 4 weeks of hydrogen-rich water (HRW) administration or placebo water (PLW) administration, a 4-week washout period, and then another 4 weeks of PLW administration or hydrogen-rich water HRW administration. Before (pre) and after (post) each period of hydrogen-rich water HRW or PLW administration, subjective and objective measurements for quality of life were obtained, such as results for sleep, mood, anxiety, feelings of depression, autonomic nerve function, and cognitive function.
Severity of fatigue was measured using the Chalder Fatigue Scale (CFS)18 and a modified version of the Osaka City University Hospital Fatigue Scale.19 Mood and anxiety were evaluated using the K6 scale.20Symptoms of depression were measured using the Center for Epidemiologic Studies Depression Scale.21General sleepiness and daytime sleepiness scores were calculated using the Pittsburgh Sleep Quality Index (PSQI)22 and the Epworth Sleepiness Scale,23 respectively. The reliability and validity of the Japanese versions of these questionnaires have been confirmed.19,24,25,26,27,28
Autonomic function test
Participants underwent simultaneous electrocardiography and photoplethysmography using a Vital Monitor 302 system (Fatigue Science Laboratory, Osaka, Japan) while sitting quietly with their eyes closed for 3 minutes. These data were analyzed using MemCalc software (GMS, Tokyo, Japan). Frequency analyses for R-R interval variation from electrocardiography and a-a interval variation as the second derivative of photoplethysmography (accelerated plethysmography) were performed using the maximum entropy method, which is capable of estimating the power spectrum density from short time series data, and is adequate for examining changes in heart rate variability under different conditions of short duration.29,30The power spectrum resolution was 600 Hz. For frequency analyses, the low-frequency component power (LF) was calculated as the power within a frequency range of 0.04–0.15 Hz, and the high-frequency component power (HF) was calculated as that within a frequency range of 0.15–0.4 Hz. HF is vagally mediated,31,32,33 whereas LF originates from a variety of sympathetic and vagal mechanisms.30,34 Some review articles35,36,37 mentioned that LF reflects sympathetic nerve activity and is used as a marker of sympathetic nerve activity in original articles. Before autonomic nerve function testing was conducted for 3 minutes, a practice test was conducted for a period of 1 minute, in accordance with previous studies.38,39,40 The reliability of these tests has been confirmed.41,42
Cognitive function test
Since previous studies have revealed that a switching attention task is useful for evaluating reduced performance under fatigue conditions,43,44,45 we used task E of the modified advanced trail making test (mATMT) as a switching attention task for evaluating executive function.46,47 Circles with numbers (from 1 to 13) or kana (Japanese phonograms, 12 different letters) were shown in random locations on a screen, and participants were required to use a computer mouse to alternately touch the numbers and kana; this task thus required switching attention. When participants touched a target circle, it remained in the same position, but its color changed from black to yellow. Participants were instructed to perform the task as quickly and correctly as possible, and continuously performed this task for 5 minutes. We evaluated three indices of task performance: the total count of correct responses (number of correctly touched numbers and letters); the total count of errors (number of incorrectly touched numbers and letters); and the motivational response (reaction time from a finished trial to the next trial). Based on our previous study,47 before participants performed task E of the mATMT on each experimental day, they practiced for a period of 1 minute. The reliability of this test has been confirmed.43,44
Blood sample analyses
Blood samples were collected from the brachial vein. The amount of blood sampled was 13 mL per experimental day. We thus collected blood samples on four occasions (once per experimental day) in the study. Blood samples for serum analyses were centrifuged at 1,470 × g for 5 minutes at 4°C. The concentration of high-sensitivity C-reactive protein (hs-CRP) in each serum sample was assessed by particle-enhanced immunonephelometry using a BNII analyzer (BN II ProSpec; Siemens, Munich, Germany). Oxidative activity in each serum sample was assessed with the reactive oxygen metabolites-derived compounds (d-ROMs) test (Diacron International, Grosseto, Italy), while anti-oxidative activity was measured with the biological anti-oxidant potential (BAP) test (Diacron International) using a JCABM1650 automated analyzer (JEOL, Tokyo, Japan).48 The concentrations of ROMs are expressed in Carratelli units (1 CARR U = 0.08 mg of hydrogen peroxide/dL).49 The oxidative stress index (OSI) was calculated using the following formula: OSI = C × (d-ROMs/BAP), where C denotes a coefficient for standardization to set the mean OSI in healthy individuals at 1.0 (C = 8.85).45 All supernatants were stored at -80°C until analyzed. Assays for hs-CRP were performed at LSI Medience Corporation (Tokyo, Japan) and those for serum d-ROMs and BAP were performed at Yamaguchi University Graduate School of Medicine.
Daily daytime activity and daily journal
Daily daytime activity, representing the expenditure of calories and amount of physical activity (METs × time) was recorded using an Active style Pro HJA-350IT pedometer (OMRON, Kyoto, Japan). A daily journal was kept for 4 weeks, and included information on fatigue (based on a visual analogue scale from 0, representing “no fatigue”, to 100, representing “total exhaustion”) just after waking up and before bedtime, sleeping times, physical condition (1, good; 2, normal; or 3, bad), and special events (if the day was different from a usual day: 1, no; or 2, yes). We carefully checked the daily journal every four weeks, on the 2nd, 3rd, and 4th experimental days.
First, we tested the normality (parametric or non-parametric distributions) of each measured parameter using the Kolmogorov-Smirnov test. Values are presented as the mean ± standard deviation or median and interquartile range based on the results of Kolmogorov-Smirnov test. The Wilcoxon signed-rank test for non-parametric parameters and paired t-test for differences between hydrogen-rich water HRW and PLW administrations after two-way repeated-measurement analysis of variance for parametric parameters were conducted. If significant changes were observed by comparisons within each condition (pre- vs. post-HRW; pre- vs. post-PLW) or between post-treatment values (post-HRW vs. post-PLW), then we compared change ratios between post-HRW/pre-HRW and post-PLW/pre-PLW using the Wilcoxon signed-rank test or paired t-test. All P values were two-tailed, and those less than 0.05 were considered statistically significant. Statistical analyses were performed using IBM SPSS Statistical Package version 20.0 (IBM, Armonk, NY, USA).
During the study, we excluded five participants from data analyses due to symptoms of hay fever, prolonged medication use because of a cold, insufficient intake of hydrogen-rich water HRW or PLW intake (≥ 85%), or a frequency of special events ≤ 15% as recorded in the daily diary. We thus analyzed data from a total of 26 participants (13 females, 13 males; mean age, 34.4 ± 9.9 years; mean body mass index, 21.5 ± 2.6 kg/m2). No side, order, and carry-over effects were observed from the oral administrations of hydrogen-rich water HRW and PLW in any participant.
Results from the questionnaires are summarized in Table 1. No questionnaire scores at baseline (pre) showed any significant differences between hydrogen-rich water HRW and PLW administration groups. With HRW administration, scores for K6, CFS, and PSQI were significantly decreased after the 4-week administration period. In addition, the change ratio (post/pre) for K6 score was significantly lower in the hydrogen-rich water HRW administration group than in the PLW administration group (Figure 2). No significant changes were seen in any other questionnaire scores (modified version of the Osaka City University Hospital Fatigue Scale, Center for Epidemiologic Studies Depression Scale or Epworth Sleepiness Scale) after hydrogen-rich water HRW administration and no significant changes in any of the scores were seen after PLW administration. Likewise, these scores did not differ significantly between HRW and PLW after administration.
Changes in parameters related to quality of life due to hydrogen-rich water (HRW) or placebo water (PLW) administration
Comparison of change ratios (post-treatment/pre-treatment) for parameters related to quality of life with administration of hydrogen-rich water (HRW) or placebo water (PLW) for 4 weeks.
Note: Change ratios for K6 score for mood (A) and anxiety and the low-frequency component power (LF) for autonomic nerve function (B). *P < 0.05.
Autonomic function results
Results for the autonomic nerve function are summarized in Table 1. LF, HF, and LF/HF ratio at baseline (pre) did not differ significantly between hydrogen-rich water HRW and PLW administrations, indicating similar autonomic nerve function in the two groups before water intake. Although the HF and LF/HF ratio were not significantly affected by 4-week administrations of hydrogen-rich water HRW or PLW, LF after hydrogen-rich water HRW administration was significantly lower than that after PLW administration. The change ratio (post/pre) for LF was also significantly lower in the hydrogen-rich water HRW administration group than in the PLW administration group (Figure 2).
Cognitive function results
Results for the cognitive function test are shown in Table 1. Motivational response and total counts of correct responses and errors at baseline (pre) did not differ significantly between hydrogen-rich water HRW and PLW administrations, indicating similar cognitive function between groups before water intake. Motivational response after hydrogen-rich water HRW administration was significantly faster than that before hydrogen-rich water HRW administration. The change ratio (post/pre) for motivational response was not significantly different in the hydrogen-rich water HRW administration group than in the PLW administration group. No significant differences in motivational response, total counts of correct responses, or errors after water administration were seen between hydrogen-rich water HRW- and PLW-administered conditions.
Blood sample results
No significant differences were seen in any blood parameters (hs-CRP, d-ROMs, BAP, and OSI) before hydrogen-rich water HRW or PLW administration (Table 1), indicating the comparability of the two groups before water intake. After hydrogen-rich water HRW and PLW administrations, we again found no significant differences in these blood parameters.
Daily daytime activity and daily journal results
The daily expenditure of calories and amount of physical activity during the 4-week administration periods did not differ significantly between hydrogen-rich water HRW and PLW administration conditions (Table 2). Similarly, visual analogue scale scores for fatigue just after waking and before bedtime, sleeping times, physical condition, and counts of special events were comparable between hydrogen-rich water HRW and PLW administration conditions (Table 2), indicating that living habits were successfully controlled during the experimental period in the two groups.
Daily daytime activity and data recorded in the daily journal during the hydrogen-rich water (HRW) or placebo water (PLW) administration period (4 weeks)
The present findings suggest that hydrogen-rich water HRW administration for 4 weeks may have improved the QOL of adult volunteers in terms of improved mood and anxiety and reduced activity of the sympathetic nervous system at rest.
In terms of associations between hydrogen and the central nervous system, a report by Ohsawa et al.4 was the first to demonstrate that molecular hydrogen acts, at least in part, as an anti-oxidant as it binds to hydroxyl ions produced in central nervous system injuries. Previous studies have proposed that hydrogen-rich water HRW administration has neuroprotective effects50 and anti-aging effects on periodontal oxidative damage in healthy aged rats.51 In a rat model of Alzheimer’s disease, hydrogen-rich saline prevented neuroinflammation and oxidative stress, and improved memory function.52 In terms of the association between hydrogen-rich water HRW and QOL, only one study reported that HRW hydrogen-rich water administration for 6 weeks improved QOL scores in patients treated with radiotherapy for liver tumors.12 Although reports on the effects of HRW hydrogen-rich water administration in healthy populations have not been accumulated, job stress14,15 and acute fatigue caused by mental and physical loading for several hours16,53 have been shown to enhance oxidative stress. As for physical fatigue, in order to alleviate acute physical fatigue in healthy volunteers not including athletes, we have previously demonstrated that treatment with antioxidant supplements is effective.54,55,56 The present study provided new findings that HRW hydrogen-rich water affects not only physical condition but also mental conditions such as mood, anxiety, and autonomic nerve function. One of the advantages of HRW hydrogen-rich water is the ability to cross the blood-brain barrier, offering high potential to reduce oxidative stress in the brain. A previous study in rats found that levels of malondialdehyde, a marker of oxidative stress, were around 4.8-fold higher in the brain than in the blood (plasma).57 These results suggest that HRW hydrogen-rich water may be effective for reducing accumulated oxidative stress in the brain in daily life, potentially contributing to the maintenance of central nervous system activity and preventing decreases in QOL.
In the present study, mood and anxiety levels improved after HRW hydrogen-rich water administration. These negative emotions are also known to be involved in conditions related to oxidative stress; social phobia,58,59depression,60 anxiety,61,62 and other neuropsychiatric disorders63 have been shown to be associated with increased oxidative stress. Neuroinflammation is also related to fatigue, mood, anxiety, and sleep.64,65,66,67 In older mice, HRW hydrogen-rich water administration succeeded in suppressing depression-like behaviors.68 These findings suggest that administration of HRW hydrogen-rich water for 4 weeks may be effective for controlling such negative emotions by reducing oxidative stress and inflammation of the central nervous system. Increasing evidence suggests that oxidative stress and inflammation in neurons are involved in the pathological manifestations of many neurological and neuropsychiatric disorders, and HRW hydrogen-rich water administration may thus help alleviate the symptoms of these disorders. Previous study revealed that oxidative stress of the brain causes cognitive and motivational deficits in a mouse model of neuropsychiatric disorder (schizophrenia).69 In the present study, motivational response of cognitive function test was improved by prolonged HRW hydrogen-rich water intake, suggesting that a reduction of oxidative stress in the brain by the intake of HRW hydrogen-rich water may increase motivational performance of cognitive task.
Stressors can enhance sympathetic hyperactivity, promote oxidative stress, and boost pro-inflammatory cytokine production.70,71,72 Autonomic nerve function is thus closely associated with oxidative stress and inflammation. Attenuation of sympathetic nervous system activity during the resting state in adult volunteers may therefore be the result of decreases in inflammation and oxidative stress as an effect of prolonged HRW hydrogen-rich water administration. However, the lack of changes in oxidative stress markers noted in the present study after HRW intake for 4 weeks could be due to the low severity of oxidative stress in the participants. Actually, serum d-ROMs (307.1 ± 49.4 CARR U) and BAP (2,549 ± 194 µM) concentrations at the first measurement point in the present study were within normal ranges based on the results of serum d-ROMs (286.9 ± 100.2 CARR U) and BAP (2,541 ± 122 µM) concentrations measured in 312 healthy participants in our previous study.48 However, levels of oxidative stress fluctuate depending on daily work load and stress. In addition, the rat study by García-Niño et al.57 that found malondialdehyde levels around 4.8-fold higher in the brain than in plasma indicate that oxidative stress in the brain is more severe. Daily administration of HRW hydrogen-rich water for 4 weeks may thus contribute to attenuation of and prevention from the cumulative oxidative stress in the brain. Mood, anxiety, and autonomic nerve function could thus potentially be improved. Although the range of sympathetic nerve activity in the present study considers to be normal based on our previous studies,73,74 sympathetic nerve activity also fluctuates depending on daily work load and stress.35 Therefore, lower sympathetic nerve activity of resting state may contribute to suppress an excessive increase in sympathetic nerve activity after the daily work load and stress.
We conducted this study with a limited number of participants. Before our results can be generalized, studies involving larger numbers of participants are essential.
Although we mainly examined the effects of HRW hydrogen-rich water on the central nervous system, we did not directly evaluate the dynamics of inflammation and oxidation in the brain. Neuroimaging studies using positron emission tomography and magnetic resonance imaging are thus underway in our laboratory to identify the mechanisms underlying the effects of HRW intake on the central nervous system that can improve QOL.
In conclusion, HRW hydrogen-rich water administration for 4 weeks in adult volunteers improved mood, anxiety, and autonomic nerve function, suggesting that HRW hydrogen-rich water administration may offer an effective method to reinforce QOL and maintain good health. In a further study, we will try to identify the effects of HRW hydrogen-rich water administration in participants with ongoing stress or chronic fatigue.
We would like to thank Ms. Mika Furusawa for her excellent technical assistances and Forte Science Communications for editorial help with this manuscript.
Conflicts of interest
This work was presented at Japanese Society of Fatigue Science, Yamaguchi City, Japan on May 16, 2016. Yasuyoshi Watanabe received funding for the present study from Melodian Corporation. The other authors have no conflicts of interest to declare.
All experiments were conducted in compliance with national legislation and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association (the Declaration of Helsinki) and registered to the UMIN Clinical Trials Registry (UMIN000022382). The study protocol was approved by the Ethics Committee of Osaka City University Center for Health Science Innovation (OCU-CHSI-IRB No. 4).
Declaration of participant consent
The authors certify that they have obtained all appropriate participant consent forms. In the form the participants have given their consent for their images and other clinical information to be reported in the journal. The participants understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Data sharing statement
Datasets analyzed during the current study are available from the corresponding author on reasonable request.
Checked twice by iThenticate.
Externally peer reviewed.
Open peer reviewers
Lei Huang, Loma Linda University, USA; Qin Hu, Shanghai Jiao Tong University, China.
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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.
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.
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.
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.
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 [5,6]. 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 [7,8], 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 [1–6]. 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).
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 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).
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)
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) images. upper. 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)
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.
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 [17,18], and effectiveness in many organs and many disease conditions are added [19,20]. 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.
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
up to 1.6 ppm H2 diatomic molecular hydrogen H2 dizzolved in Alkaline Ionized Water Products
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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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