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 ).(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. and ).
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. and ).(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.
Beneficial effects of molecular hydrogen in pathophysiology of various acute neuronal conditions. ATP, adenosine triphosphate; miR-200, microRNA-200; ROS, reactive oxygen species.
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. and ).
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 ).(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 ).
|AD ||Alzheimer’s disease |
|APP ||amyloid precursor protein |
|ATP ||adenosine triphosphate |
|BBB ||blood brain barrier |
|CA1 ||Cornet d’Armon 1 |
|CLP ||cecal ligation and puncture |
|CO ||carbon monoxide |
|ICH ||intracerebral hemorrhage |
|LRP ||lipoprotein receptor-related protein |
|MCAO ||middle cerebral artery occlusion |
|miR-200 ||microRNA-200 |
|MMP ||matrix metalloproteinase |
|PD ||Parkinson’s disease |
|ROS ||reactive oxygen species |
|SAH ||subarachnoid hemorrhage |
|TBI ||traumatic brain injury |
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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 :
∙O−+ H2→H∙+OH− k=8.0×107 M−1⋅s−1∙OH+H2→H∙+H2O k=4.2×107 M−1⋅s−1H∙+∙OH→H2O k=7.0×109 M−1⋅s−1.
The reaction rate constants of •O− and •OH with other molecules are mostly in the orders of 109 to 1010 M−1·s−1, whereas those with H2 are in the order of 107 M−1·s−1. Hydrogen, however, is a small molecule that can easily dissipate throughout the body and cells, and the collision rates of hydrogen with other molecules are expected to be very high, which is likely to be able to overcome the low reaction rate constants . Hydrogen is not easily dissolved in water, and 100%-saturated hydrogen water contains 1.6 ppm or 0.8 mM hydrogen at room temperature( our note: see AlkaViva Vesta H2 water ionizer performance below)
In 1995, hydrogen was first applied to human to overcome high-pressure nervous syndrome in deep sea diving . Hydrogen was used to reduce nitrogen (N2) toxicity and to reduce breathing resistance in the deep sea. In 2001, being prompted by the radical-scavenging activity of hydrogen, Gharib and colleagues examined an effect of molecular hydrogen on a mouse model of schistosomiasis-associated chronic liver inflammation . Mice were placed in a chamber with 70% hydrogen gas for two weeks. The mice exhibited decreased fibrosis, improvement of hemodynamics, increased nitric oxide synthase (NOS) II activity, increased antioxidant enzyme activity, decreased lipid peroxide levels, and decreased circulating tumor-necrosis-factor-(TNF-) α levels. Although helium gas also exerted some protective effects in their model, the effect of helium gas was not recapitulated in a mouse model of ischemia/reperfusion injury of the liver .
2. Effects of Hydrogen (water ) Have Been Reported in 63 Disease Models and Human Diseases
A major breakthrough in hydrogen research occurred after Ohsawa and colleagues reported a prominent effect of molecular hydrogen on a rat model of cerebral infarction in June 2007 . Rats were subjected to left middle cerebral artery occlusion. Rats placed in 2–4% hydrogen gas chamber showed significantly smaller infarction volumes compared to controls. They attributed the hydrogen effect to the specific scavenging activity of hydroxyl radical (•OH). They also demonstrated that hydrogen scavenges peroxynitrite (ONOO−) but to a lesser extent.
As have been previously reviewed [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)(). The number of papers is increasing each year (). Among the 87 papers cited in , 21 papers showed an effect with inhalation of hydrogen gas, 23 with drinking hydrogen-rich water, 27 with intraperitoneal administration or drip infusion of hydrogen-rich saline, 10 with hydrogen-rich medium for cell or tissue culture, and 6 with the other administration methods including instillation and dialysis solution. In addition, among the 87 papers, 67 papers showed an effect in rodents, 7 in humans, 1 in rabbits, 1 in pigs, and 11 in cultured cells or cultured tissues.
Number of papers that report effects of molecular hydrogen since 2007 shown in .
Sixty-three disease models and human diseases for which beneficial effects of hydrogen have been documented.
|Diseases ||Species ||Administration |
|Brain || || |
| Cerebral infarction [6, 30, 55, 56] ||Rodent, human ||Gas, saline |
| Cerebral superoxide production  ||Rodent ||Water |
| Restraint-induced dementia  ||Rodent ||Water |
| Alzheimer’s disease [23, 24] ||Rodent ||Saline |
| Senile dementia in senescence-accelerated mice  ||Rodent ||Water |
| Parkinson’s disease [18, 19] ||Rodent ||Water |
| Hemorrhagic infarction  ||Rodent ||Gas |
| Brain trauma  ||Rodent ||Gas |
| Carbon monoxide intoxication  ||Rodent ||Saline |
| Transient global cerebral ischemia  ||Rodent ||Gas |
| Deep hypothermic circulatory arrest-induced brain damage  ||Rodent ||Saline |
| Surgically induced brain injury  ||Rodent ||Gas |
|Spinal Cord || || |
| Spinal cord injury  ||Rodent ||Saline |
| Spinal cord ischemia/reperfusion  ||Rabbit ||Gas |
|Eye || || |
| Glaucoma  ||Rodent ||Instillation |
| Corneal alkali-burn  ||Rodent ||Instillation |
|Ear || || |
| Hearing loss [80–82] ||Tissue, rodent ||Medium, water |
|Lung || || |
| Oxygen-induced lung injury [53, 60, 83, 84] ||Rodent ||Saline |
| Lung transplantation  ||Rodent ||Gas |
| Paraquat-induced lung injury  ||Rodent ||Saline |
| Radiation-induced lung injury [87–89] ||Rodent ||Water |
| Burn-induced lung injury  ||Rodent ||Saline |
| Intestinal ischemia/reperfusion-induced lung injury  ||Rodent ||Saline |
|Heart || || |
| Acute myocardial infarction [36, 65, 91] ||Rodent ||Gas, saline |
| Cardiac transplantation  ||Rodent ||Gas |
| Sleep apnea-induced cardiac hypoxia  ||Rodent ||Gas |
|Liver || || |
| Schistosomiasis-associated chronic liver inflammation  ||Rodent ||Gas |
| Liver ischemia/reperfusion  ||Rodent ||Gas |
| Hepatitis  ||Rodent ||Intestinal gas |
| Obstructive jaundice  ||Rodent ||Saline |
| Carbon tetrachloride-induced hepatopathy  ||Rodent ||Saline |
| Radiation-induced adverse effects for liver tumors  ||Human ||Water |
|Kidney || || |
| Cisplatin-induced nephropathy [92–94] ||Rodent ||Gas, water |
| Hemodialysis [20, 28] ||Human ||Dialysis solution |
| Kidney transplantation  ||Rodent ||Water |
| Renal ischemia/reperfusion  ||Rodent ||Saline |
| Melamine-induced urinary stone  ||Rodent ||Water |
| Chronic kidney disease  ||Rodent ||Water |
|Pancreas || || |
| Acute pancreatitis  ||Rodent ||Saline |
|Intestine || || |
| Intestinal transplantation [41, 45, 59] ||Rodent ||Gas, medium, saline |
| Ulcerative colitis  ||Rodent ||Gas |
| Intestinal ischemia/reperfusion  ||Rodent ||Saline |
|Blood vessel || || |
| Atherosclerosis  ||Rodent ||Water |
|Muscle || || |
| Inflammatory and mitochondrial myopathies  ||Human ||Water |
|Cartilage || || |
| NO-induced cartilage toxicity  ||Cells ||Medium |
|Metabolism || || |
| Diabetes mellitus type I  ||Rodent ||Water |
| Diabetes mellitus type II  ||Human ||Water |
| Metabolic syndrome [27, 99] ||Human, rodent ||Water |
| Diabetes/obesity  ||Rodent ||Water |
|Perinatal disorders || || |
| Neonatal cerebral hypoxia [10–12] ||Rodent, pig ||Gas, saline |
| Preeclampsia  ||Rodent ||Saline |
|Inflammation/allergy || || |
| Type I allergy  ||Rodent ||Water |
| Sepsis  ||Rodent ||Gas |
| Zymosan-induced inflammation  ||Rodent ||Gas |
| LPS/IFNγ-induced NO production  ||Cells ||Gas |
|Cancer || || |
| Growth of tongue carcinoma cells  ||Cells ||Medium |
| Lung cancer cells  ||Cells ||Medium |
| Radiation-induced thymic lymphoma  ||Rodent ||Saline |
|Others || || |
| UVB-induced skin injury  ||Rodent ||Bathing |
| Decompression sickness  ||Rodent ||Saline |
| Viability of pluripotent stromal cells  ||Cells ||Gas |
| Radiation-induced cell damage [104, 105] ||Cells ||Medium |
| Oxidized low density lipoprotein-induced cell toxicity  ||Cells ||Medium |
| High glucose-induced oxidative stress  ||Cells ||Medium |
Two papers, however, showed that hydrogen was ineffective for two disease models (). 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.
|Diseases ||Species ||Administration |
|Brain || || |
|Moderate to severe neonatal brain hypoxia  ||Rodent ||Gas |
|Muscle || || |
|Muscle disuse atrophy  ||Rodent ||Water |
Effects of molecular hydrogen have been observed essentially in all the tissues and disease states including the brain, spinal cord, eye, ear, lung, heart, liver, kidney, pancreas, intestine, blood vessel, muscle, cartilage, metabolism, perinatal disorders, and inflammation/allergy. Among them, marked effects are observed in ischemia/reperfusion disorders as well as in inflammatory disorders. It is interesting to note, however, that only three papers addressed effects on cancers. First, molecular hydrogen caused growth inhibition of human tongue carcinoma cells HSC-4 and human fibrosarcoma cells HT-1080 but did not compromise growth of normal human tongue epithelial-like cells DOK . Second, hydrogen suppressed the expression of vascular endothelial growth factor (VEGF), a key mediator of tumor angiogenesis, in human lung adenocarcinoma cells A549, which was mediated by downregulation of extracellular signal-regulated kinase (ERK) . Third, hydrogen protected BALB/c mice from developing radiation-induced thymic lymphoma . Elimination of radical oxygen species by hydrogen should reduce a probability of introducing somatic mutations. Unlike other disease models, cancer studies were performed only with cells in two of the three papers. Hydrogen is likely to have a beneficial effect on cancer development by suppressing somatic mutations, but an effect on cancer growth and invasion needs to be analyzed further in detail.
3. Effects of Molecular Hydrogen on Rodent Models of Neurodegenerative Diseases
Parkinson’s disease is caused by death of dopaminergic neurons at the substantia nigra pars compact of the midbrain and is the second most common neurodegenerative disease after Alzheimer’s disease. Parkinson’s disease is caused by two mechanisms: excessive oxidative stress and abnormal ubiquitin-proteasome system . The neurotransmitter, dopamine, is a prooxidant by itself and dopaminergic cells are destined to be exposed to high concentrations of radical oxygen species. An abnormal ubiquitin-proteasome system also causes aggregation of insoluble α-synuclein in the neuronal cell body that leads to neuronal cell death. We made a rat model of hemi-Parkinson’s disease by stereotactically injecting catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in the right striatum . Ad libitum administration of hydrogen-rich water starting one week before surgery completely abolished the development of hemi-Parkinson’s symptoms. The number of dopaminergic neurons on the toxin-injected side was reduced to 40.2% of that on the control side, whereas hydrogen treatment improved the reduction to 83.0%. We also started giving hydrogen-rich water three days after surgery, and hemi-Parkinson’s symptoms were again suppressed, but not as much as those observed in pretreated rats. The number of dopaminergic neurons on the toxin-injected side was 76.3% of that on the control side. Pretreated rats were also sacrificed 48 hrs after toxin injection, and the tyrosine hydroxylase activity at the striatum, where dopaminergic neurons terminate, was decreased in both hydrogen and control groups. This indicated that hydrogen did not directly detoxicate 6-OHDA but exerted a delayed protective effect for dopaminergic cells. Fujita and colleagues also demonstrated a similar prominent effect of hydrogen-rich water on an MPTP-(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-) induced mouse model of Parkinson’s disease . MPTP is a neurotoxin that blocks complex I of the mitochondrial electron transport system and causes Parkinson’s disease in mice and humans. It is interesting to note that the concentration of hydrogen that they used for the MPTP mice was only 0.08 ppm (5% saturation), which is the second lowest among all the trials published to date for rodents and humans. The lowest hydrogen concentration ever tested is 0.048 ppm in the dialysis solution for patients receiving hemodialysis .
Alzheimer’s disease is the most common neurodegenerative disease and is characterized by abnormal aggregation of β-amyloid (Aβ) and tau, the large aggregates of which are recognizable as senile plaques and neurofibrillary tangles, respectively . Effects of molecular hydrogen on Alzheimer’s disease have been studied in three rodent models. First, Nagata and colleagues made a mouse model of dementia by restricting movement of mice for 10 hrs a day . They analyzed cognitive functions through passive avoidance learning, object recognition tasks, and the Morris water maze and demonstrated that ad libitum administration of hydrogen-rich water efficiently ameliorated cognitive impairment. They also showed that neural proliferation in the dentate gyrus was restored by hydrogen water . Second, Li and colleagues made a rat model of Alzheimer’s disease by intracerebroventricular injection of Aβ1-42 . They analyzed cognitive functions by the Morris water maze open field tasks, and electrophysiological measurement of long-term potentiation (LTP) and found that intraperitoneal injection of hydrogen-rich saline for 14 days efficiently ameliorated cognitive decline and preserved LTP. The same team later reported that the protective effects were mediated by suppression of abnormal activation of IL1β, JNK, and NFκB . Third, Gu and colleagues used a senescence-accelerated mouse strain (SAMP8) that exhibits early aging syndromes including impairment in learning ability and memory . Ad libitum administration of hydrogen-rich water for 30 days prevented cognitive decline, which was examined by the Morris water maze. Additionally, ad libitum drinking of hydrogen water for 18 weeks showed efficient amelioration of hippocampal neurodegeneration.
Cerebrovascular diseases are the most frequently reported neurological diseases for which hydrogen (water )has prominent effects. As stated in Section 2, current hydrogen (water ) research has broken out after Ohsawa reported a prominent effect of 2–4% hydrogen for a rat model of left cerebral artery occlusion in 2007 .
In addition to neurodegenerative disorders of Parkinson’s disease and Alzheimer’s disease, effects of molecular hydrogen (water ) have been reported in eight other brain diseases listed under the categories of “brain” and “perinatal disorders” in . 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.
Acarbose/MOLECULAR HYDROGEN TREATMENT AND THE RISK OF CARDIOVASCULAR DISEASE AND HYPERTENSION IN PATIENTS WITH IMPAIRED GLUCOSE TOLERANCE: THE STOP-NIDDM TRIAL
Molecular Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL function in patients with potential metabolic syndrome
Improvement of psoriasis-associated arthritis and skin lesions by treatment with molecular hydrogen: A report of three cases.
Molecular hydrogen(H2) treatment for acute erythymatous skin diseases. A report of 4 patients with safety data and a non-controlled feasibility study with H2 concentration measurement on two volunteers
MOLECULAR HYDROGEN WATER FOR PATIENTS WITH PRESSURE ULCER – EFFECTS ON NORMAL HUMAN SKIN WOUNDS
MOLECULAR HYDROGEN WATER FOR PATIENTS WITH RHEUMATOID ARTHRITIS: AN OPEN-LABEL PILOT STUDY
EFFECTIVENESS OF ORAL AND TOPICAL MOLECULAR HYDROGEN FOR SPORTS-RELATED SOFT TISSUE INJURIES
MOLECULAR HYDROGEN WATER BENEFITS FOR ATHLETES, EXERCISE, MUSCLE FATIGUE
MOLECULAR HYDROGEN WATER FOR VASCULAR ENDOTELIAL FUNCTION
MOLECULAR HYDROGEN WATER- PERIODONTITIS TREATMENT
5. Molecular Bases of Hydrogen Effects
Effects of hydrogen on various diseases have been attributed to four major molecular mechanisms: a specific scavenging activity of hydroxyl radical, a scavenging activity of peroxynitrite, alterations of gene expressions, and signal-modulating activities. The four mechanisms are not mutually exclusive and some of them may be causally associated with other mechanisms.
The first molecular mechanism identified for hydrogen was its specific scavenging activity of hydroxyl radical . Indeed, oxidative stress markers like 8-OHdG, 4-hydroxyl-2-nonenal (4-HNE), malondialdehyde (MDA), and thiobarbituric acid reactive substances (TBARSs) are decreased in all the examined patients and rodents. As hydrogen can easily dissipate in exhalation, hydrogen in drinking water is able to stay in human and rodent bodies in less than 10 min (unpublished data). Hydrogen, however, can bind to glycogen, and the dwell time of hydrogen is prolonged in rat liver after food intake . A question still remains if mice and humans can take a sufficient amount of hydrogen that efficiently scavenges hydroxyl radicals that are continuously generated in normal and disease states.
Another molecular mechanism of hydrogen effect is its peroxynitrite-(ONOO−-) scavenging activity . Although hydrogen cannot eliminate peroxynitrite as efficiently as hydroxyl radical in vitro , hydrogen can efficiently reduce nitric-oxide-(NO-) induced production of nitrotyrosine in rodents [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 (). 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 (). 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 .
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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