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Molecular hydrogen (water) in the treatment of acute and chronic neurological conditions(Alzheimer’s, Parkinson’s,etc): mechanisms of protection and routes of administration

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.() Moreover, the involvement of mitochondrial dysfunction in PD has been reported.()

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.()

In 2009, Fujita et al.() and Fu et al.() 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.() 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).()

 

Molecular hydrogen water &  Alzheimer’s disease (AD)

Alzheimer Disease AD, an age-related neurodegenerative disease, is the most common cause of dementia.(,) 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.() In recent years, oxidative stress and neuroinflammation have been reported to be involved in Alzheimer’s disease AD.(,) To date, reports have centered on the involvement of oxidative stress in brain parenchyma.(,,)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.() 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.() 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.(,)

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.(,)

It has  been reported that consuming molecular hydrogen H2-rich water inhibits age-related brain alterations and spatial memory decline.()

 

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,() 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.() 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.()

Control of oxidative stress is a major therapeutic strategy for various neuronal conditions.(,,) There are many methods for controlling oxidative stress with the use of free radical scavengers being the most common approach.(,) Evidence from animal experiments support the notion that free radical scavengers and antioxidants dramatically reduce cerebral damage.() Edaravone (MCI-186), a novel free radical scavenger, was developed to prevent lipid peroxidation in pathological neurological conditions.(,)Edaravone is currently the only antioxidant drug approved for treating cerebral infarction that improves the functional outcome of ischemic stroke.() Brain hypothermia therapy (targeted temperature management) can also effectively control oxidative stress. Brain hypothermia therapy is effective in patients with various acute neuronal diseases.(,,)

In 2007, Ohsawa et al.() 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.() The first major therapeutic effect of molecular hydrogen H2 was that of an antioxidant, combining with hydroxyl ions to produce water.() 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).()Therefore, the biology of molecular hydrogen H2 is not simple. In this review, we discuss the role of molecular H2 in various neuronal conditions.

Fig. 1

Beneficial effects of molecular hydrogen in pathophysiology of various acute neuronal conditions. ATP, adenosine triphosphate; miR-200, microRNA-200; ROS, reactive oxygen species.

Fig. 2

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.() 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.() 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.(,) Ohsawa et al.() 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.() Free radical generation after ischemia induces matrix metalloproteinase (MMP) expression.(,) MMP-9 promotes hemorrhagic infarction by disrupting cerebral vessels.() 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.() 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.() Nagatani et al.() 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.() 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.() Manaenko et al.() 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.() A clinical trial has started in patients with SAH (Table 1).()

Table 1

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.(,,) Ji et al.() 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.() 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.() 

Molecular Hydrogen & Spinal cord injury

Chen et al.() 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.(,) Huang et al.()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.() 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.() 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.() Using a mice cecal ligation and puncture (CLP) model, Liu et al.() 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.() 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.(,) Sun et al.() and Shen et al.() 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.() The consumption of molecular hydrogen H2-rich water inhibits oxidative stress and thereby inhibits the onset of stress-induced brain damage.()

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.() In recent years, it has been found that the microRNA-200 (miR-200) family regulates oxidative stress.() 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).

 

abbreviations

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

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Molecular Hydrogen water effects on Mild Cognitive Impairment

Abstract

Background:

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

Objective:

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

Methods:

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

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

Results:

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

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

Conclusion:

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

1. INTRODUCTION

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

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

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

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

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

2. MATERIALS AND METHODS

2.1. Ethical Approval and Consent to Participate

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

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

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

2.2. Transgenic DAL101 Mice

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

2.3. Hydrogen Water

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

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

2.4. Measurement of Oxidative Stress

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

2.5. Measurement of Memory Impairment: Object Recognition Task

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

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

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

2.7. Immunostaining of the Hippocampal CA1 Region

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

2.8. Subjects of the Clinical Study

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

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

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

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

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

The APOE4 genotype was determined as described [].

2.9. Statistical Considerations

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

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

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

3. RESULTS

3.1. Hydrogen-water Reduced Oxidative Stress in DAL Mice

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

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

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

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

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

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

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

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

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

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

3.3. Hydrogen-water Suppressed Neurodegeneration

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

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

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

3.4. Hydrogen-water Extended the Average Lifespan of Mice

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

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

3.5. A Randomized, Placebo Controlled Clinical Study

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

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

Table 1

Background characteristics of 73 subjects with mild cognitive impairment.

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

* indicates frequency (%).

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

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

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

DISCUSSION

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

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

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

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

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

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

CONCLUSION

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

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

Associated Data

Supplementary Materials

ACKNOWLEDGEMENTS

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

LIST OF ABBREVIATIONS

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

 

SUPPLEMENTARY MATERIAL

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

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

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

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

HUMAN AND ANIMAL RIGHTS

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

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

Consent for Publication

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

CONFLICT OF INTEREST

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

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

Background

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

Methods

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

Results

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

Conclusions

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

Background

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

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

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

Methods

Patients

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

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

Production of hydrogen-rich intravenous fluid

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

MRI analysis

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

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

Results

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

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

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

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

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

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

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

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

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

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

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

Discussion

MRI analysis

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

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

Neurological evaluation of brainstem infarction patients with NIHSS

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

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

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

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

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

Conclusions

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

 

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

Competing interests

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

Authors’ contributions

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

Acknowledgements

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

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

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

Abstract

CONTEXT:

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

OBJECTIVE:

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

DESIGN:

This was a case-control study.

SETTING:

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

PATIENTS:

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

RESULTS:

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

CONCLUSIONS:

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

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

Author information

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

 

Effectiveness of oral and topical molecular hydrogen for SPORTS-related soft tissue INJURIES

Because molecular hydrogen (water) therapy has been found beneficial for the treatment of inflammation, ischemia-reperfusion injury, and oxidative stress in humans, it seems useful to evaluate the effects of exogenously administered molecular hydrogen as an element in the immediate management of sports-related soft tissue injuries. The main aim of this pilot study was to examine the effects of 2-week administration of molecular hydrogen on the biochemical markers of inflammation and functional recovery in male professional athletes after acute soft tissue injury.

METHOD:

During the 2013 season (from March to May), 36 professional athletes were recruited as participants and examined by a certified sports medicine specialist in the first 24 hours after an injury was sustained. Subjects were allocated to 3 randomly assigned trials in a single-blind design. Those in the control group received a traditional treatment protocol for soft tissue injury. Subjects in the first experimental group followed the same procedures as the control group but with additional administration throughout the study of oral molecular hydrogen-rich tablets (2 g per day). Subjects in the second experimental group also followed the procedures of the control group, with additional administration throughout the study of both oral molecular hydrogen-rich tablets (2 g per day) and topical molecular hydrogen-rich packs (6 times per day for 20 minutes). Participants were evaluated at the time of the injury report and at 7 and 14 days after baseline testing.

RESULTS:

Oral and topical molecular hydrogen intervention was found to augment plasma viscosity decrease as compared with the control group (P = 0.04). Differences were found for range-of-motion recovery between the 3 groups; oral and topical molecular hydrogen intervention resulted in a faster return to normal joint range of motion for both flexion and extension of the injured limb as compared with the control intervention (P < 0.05).

CONCLUSION:

These preliminary results support the hypothesis that the addition of molecular hydrogen to traditional treatment protocols is potentially effective in the treatment of soft tissue injuries in male professional athletes.

Trial identification: Clinicaltrials.gov number NCT01759498.

PMID:25295663
DOI: 10.3810/pgm.2014.09.2813
 2014 Sep;126(5):187-95. doi: 10.3810/pgm.2014.09.2813.
Effectiveness of oral and topical hydrogen for sports-related soft tissue injuries.

1Center for Health, Exercise, and Sport Sciences, Stari DIF, Belgrade, Serbia. sergej.ostojic@chess.edu.rs.

Molecular hydrogen(H2) treatment for ACUTE ERYTHYMATOUS SKIN DISEASES

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

Background

We have treated 4 patients of acute erythematous skin diseases with fever and/or pain by molecular hydrogen H2 enriched intravenous fluid. We also added data from two volunteers for assessing the mode of molecular hydrogen  H2 delivery to the skin for evaluation of feasibility of molecular hydrogen  H2 treatment for this type of skin diseases.

Methods

All of the four patients received intravenous administration of 500 ml of molecular hydrogen  H2 enriched fluid in 30 min for more than 3 days except in one patient for only once. From two volunteers (one for intravenous molecular hydrogen  H2 administration and the other for molecular hydrogen  H2 inhalation), blood samples were withdrawn serially and air samples were collected from a heavy duty plastic bag covering a leg, before, during and after molecular hydrogen  H2 administration. These samples were checked for molecular hydrogen  H2 concentration immediately by gas chromatography. Multiple physiological parameters and blood chemistry data were collected also.

Results

Erythema of these 4 patients and associated symptoms improved significantly after the molecular hydrogen  H2 treatment and did not recur.

Administration of molecular hydrogen  H2 did not change physiological parameters and did not cause deterioration of the blood chemistry. The molecular hydrogen H2 concentration in the blood from the volunteers rapidly increased with molecular hydrogen  H2 inhalation and slowly decreased with cessation of molecular hydrogen H2 particularly in the venous blood, while molecular hydrogen  H2 concentration of the air from the surface of the leg showed much slower changes even after molecular hydrogen  H2 inhalation was discontinued, at least during the time of sample collection.

Conclusion

An improvement in acute erythemtous skin diseases followed the administration of molecular hydrogen H2 enriched fluid without compromising the safety. The molecular hydrogen H2 delivery study of two volunteers suggested initial direct delivery and additional prolonged delivery possibly from a slowly desaturating reservoir in the skin to the surface.

Introduction

Severe and acute erythematous skin diseases usually require immediate medical attention, particularly when the symptoms involve severe pain and/or fever. Treatment may have to be initiated before spending enough time and effort for investigating real causes of the rush or functional state of the other organs and the steroid agents tend to be the first choice of the treatment. However, the complications from the general use of steroid have been well known and therefore, non-dermatological clinics like ours frequently encounter difficulty in finding quick remedies with minimal side effects. Erythema is reddening of the skin due to inflammatory mechanisms either as primary culprits or secondary features and locally released inflammatory cytokines such as TNF-α, IL-1,8, GM-CSF etc., stimulate phagocytes and inflammatory cells and results in production of ROS (reactive oxygen species)[12]. The interaction between the ROS and nitric oxide leads to the formation of peroxynitrite radicals and also by the iron-mediated Fenton reaction, hydroxyl radicals, both of which are highly reactive and destructive to the cell membrane and mitochondria and polyunsaturated fatty acids(PUFAs) [3]. However, ROS dismutases, which are abundant in the skin and also currently available medications are ineffective to neutralize these most destructive radicals except Edaravone [4], of which use is strictly limited for the treatment of acute cerebral infarction patients with normal kidney and liver function.

molecular hydrogen  H2 may be useful in these situations because it immediately and simultaneously neutralizes both peroxynitrites and hydroxyl radicals [5] and also molecular hydrogen  H2 is known to cause no significant side effects since it is produced in the human intestine as a fermentation process, although not continuously[6].

We report four cases of acute erythematous skin disease patients who were suffering from skin rash and also from associated symptoms such as severe pain and/or fever. They were treated with regular medications first and when the conventional treatments failed, then, intravenous fluids which contained molecular hydrogen  H2 were added after a proper consent form was signed. However, molecular hydrogen  H2 administration may not be therapeutic unless enough concentration stays at the surface layer of the skin for a sufficient period and the concentration should be higher than that of internally produced molecular hydrogen  H2. Two volunteers participated in a molecular hydrogen  H2 delivery study where molecular hydrogen  H2 concentration in the blood and in the air at the surface of the skin was measured before, during and after molecular hydrogen  H2 administration by inhalation or by intravenous fluid infusion.

Methods

Patients and volunteers

Before recruiting the patients and volunteers to the current study, a complete PARQ conference was given to all of the patients and their family and to volunteers. Our specific consent form, which had been approved by the Nishijima Hospital Ethics Committee and the Nishijima Hospital Pharmacists Council, was signed before the study with clear understanding of the nature of the study.

Case history of 4 patients

Case 1

48 y.o. male who was in good health until 5 days prior to the admission to Nishijima Hospital when severe pain and skin rash involving his left side of the face made him to visit an emergency service where he was diagnosed as having herpes simplex infection and was treated with antivirus agents and pain medications. However, the pain increased and the left side of the face became numb. In addition, blisters in the erythematous area coalesced and formed ulcer-like appearance. The patient also noticed left ptosis and double vision and became unable to open the mouth, which made oral intake impossible. The patient was admitted to the hospital for deteriorated general condition with dehydration, severe pain and fever. On admission, the patient was found to have partial paralysis of the left 3 rd, 5th and 6th cranial nerves in addition to severe erythema with edema and small ulcers, covering the left side of the face and frontal region. The hydration treatment was initiated with 3 bags of 500 ml glucose and electrolyte solution and continued for 6 days with a decreasing dose during the hospitalization. Initially, two bags of these solutions (500 ml) had been enriched with molecular hydrogen H2. No antibiotic was given. Before the infusion therapy, the patient was unable to open his left eye and the mouth (Figure 1, upper left). The picture of Figure 1 upper right was taken after the patient was asked to open his left eye and the mouth. The patient was unable to do so, except for minimal opening of the mouth. However, 3 days after the admission and molecular hydrogen  H2 infusion, the patient’s condition remarkably improved, including erythema, ulcers, pain level, opening the eye and mouth (Figure 1, lower left) and the patient became afebrile. Since cranial nerve functions recovered also and he became able to take oral soft nutrients, intravenous hydration was decreased to 2 bags of molecular hydrogen  H2- enriched glucose-electrolyte solution (esuron B,200 ml/bag), daily. By the 6th hospital day, the patient was eating a regular food and his dehydration was corrected. He had no pain and the severe inflammation of the skin disappeared. The patient discharged home and no return of the skin erythema noted during a follow-up period (Figure 1, lower right).

Figure 1

Erythematous skin disease, Case 1. Before the molecular hydrogen  H2 treatment with severe erythema and edema (upper left), the patient was unable to open his left eye and the mouth except for a minimal degree with a maximal effort (upper right). Improved conditions, 3 days after the molecular hydrogen  H2 treatment (lower left) with opening eye and mouth. The severe inflammation of the skin almost disappeared in 6 days after molecular hydrogen  H2 treatment (lower right) and was discharged home and no return of the skin erythema noted during a follow-up period.

Case 2

67 y.o male lapsed into coma after a large basilar artery aneurysm rupture and subarachnoid hemorrhage. After the aneurysm was surgically clipped, the patient remained comatose and developed pneumonia and cystitis, with deterioration of the liver and kidney function. After multiple medications including antibiotic and anticonvulsant, his general condition had been stabilized until 2 months after the surgery when he became febrile and developed severe skin abnormality. The abnormality consisted of erythematous papules, severe skin edema, blisters and vesicles and shedding of the skin. The Stevens-Johnson syndrome was suspected and he was transported to a general hospital with dermatology department. However, the patient was sent back with several diagnosis such as drug erythema, thrombocytopenia, possible trichophyton infection etc. and use of steroid and antifungal cream were recommended but not systemic steroid. However, application of these creams further deteriorated the skin condition despite of discontinuation of suspected drugs and finally, it was decided to use molecular hydrogen  H2-enriched intravenous fluid. After a complete PARQ with the patient’s family who signed a consent,molecular hydrogen H2-enriched saline solution (500 ml) was given twice a day. Redness of the skin started fading and swelling and hardness of the skin from severe edema significantly improved in 3 days. His high fever subsided. After one week of the hydrogen treatment, the skin lesions almost disappeared (Figure 2, lower left) and general condition improved also. Although the patient remained comatose after the treatment and expired approximately 4 months after the surgery, the skin lesions did not recur.

Figure 2

Erythematous skin disease, Case 2, 3 and 4. Erythematous skin lesion of the case 3 in the entire face (upper left) started improving approximately 30 min after the molecular hydrogen  H2 infusion in the left side of the face first (upper-middle) and then in about one hour, the whole face improved (upper right). Severe swelling and erythema of case 2 subsided in 7 days after molecular hydrogen  H2 treatment (lower left). Finer papules of case 4 started coalescing (lower middle). In 3 days after molecular hydrogen H2 treatment (lower right), significant improvement was noted and the skin lesion did not recur.

Case 3

48 y.o female started feeling hot sensation in her face and developed erythema in the entire face (Figure 2, upper left) after a CT scan study with contrast enhancement for cerebral aneurysm. Drug eruption was suspected and a minophagenC solution (Minophagen Pharmaceutical Co.) which had been effective in these situations, was given intravenously. However, the erythema did not subside and the patient developed fever (38.5C), headaches and nausea. As an emergency measure, two bags of a 250 ml of saline solution (Terumo Co.), which had been enriched with molecular hydrogen  H2 was given. Approximately 30 min. after the infusion, the erythema started fading in the left side of the face first (Figure 2, upper middle) and then in about one hour, the whole face improved (Figure 2, upper right) and her body temperature started coming down in about one hour. At that point, the infusion stopped and the patient returned home. No recurrence of the skin rush nor fever was noted during a follow-up period.

Case 4

62 y.o. male had been intubated and mechanically ventilated with stable vital signs after severe subarachnoid hemorrhage from a ruptured cerebral aneurysm until 7 days after the ictus when the patient developed high fever and erythema which consisted of finer papules without fusing together. Initially, the patient was treated with local ointments with steroid but the erythema spread in the whole body and started coalescing (Figure 2, lower middle). In 3 days after molecular hydrogen  H2-enriched saline solution was given twice a day intravenously, the skin lesion started fading (Figure 2, lower right) and the elevated body temperature normalized.

Volunteers

Two volunteers who were already in Nishijima hospital with different medical conditions agreed to let the study to use molecular hydrogen H2 and their arterial access port and venous port which had been established for their medical treatment. The blood samples (1 ml at each time) were withdrawn from these ports, before, during and after molecular hydrogen  H2 administration by intravenous infusion of 500 cm3 of saline or by inhalation of 2% molecular hydrogen  H2 gas for 20 min followed by inhalation of 4% H2 gas. Both patients and their family understood perfectly that the study will not provide any benefit to them directly but possibly for the future of molecular hydrogen  H2 treatment research. All the proper PARQ and signing of the consent form had been done before the initiation of the study.

Results

Erythema of these 4 patients and associated symptoms, such as intensive pain in the face with neurological deficits and skin ulcers (case 1), fever and edematous hardening of the entire body, particularly in the extremities with skin ulcers (Case 2), rather mild but with acute fever and nausea and headache (case 3), mild but worsening and spreading skin lesions with fever (case 4), all improved significantly after the molecular hydrogen H2 treatment and did not recur.

The molecular hydrogen  H2 delivery study of two volunteers showed that the concentration of molecular hydrogen  H2 in the blood rapidly increased with molecular hydrogen H2 inhalation and slowly decreased with cessation of molecular hydrogen H2, particularly in the venous blood. However, molecular hydrogen  H2 concentration of the air samples in the plastic bag covering a leg showed much slower changes and continued to increase even after molecular hydrogen  H2 inhalation was discontinued, at least during the time of sample collection (Figure 5). The blood level of molecular hydrogen H2 was significantly higher when molecular hydrogen  H2 was given by inhalation as compared to via intravenous route.

Administration of molecular hydrogen  H2 did not change physiological parameters and did not cause significant deterioration of the blood chemistry, although some of these patients already had severe abnormalities before the molecular hydrogen  H2 treatment such as thrombocytopenia of case 2

The safety monitoring with physiological parameters and laboratory studies showed no ill effects on those multiple indices and organ function such as kidney and liver function, by this method of molecular hydrogen  H2 administration (Table 1). Even in the case 2 with thrombocytopenia, no other hematological worsening was noted. Clinical symptoms of the skin diseases of all four patients improved rather rapidly and significantly. Therefore, it may be reasonable to assume that molecular hydrogen H2 infusion in these situations was quite safe and effective.

In summary, erythema of these 4 patients and associated symptoms, such as intensive pain in the face with neurological deficits and skin ulcers, fever and edematous hardening of the entire body, rather mild but with acute fever and nausea and headache, mild but worsening and spreading skin lesions with red rush all improved significantly after the molecular hydrogen H2 treatment and did not recur.

The molecular hydrogen H2 delivery study of two volunteers showed that the concentration of molecular hydrogen H2 in the blood rapidly increased with molecular hydrogen H2 inhalation and slowly decreased with cessation of molecular hydrogen H2, particularly in the venous blood. However, molecular hydrogen H2 concentration of the air samples in the plastic bag covering a leg showed much slower changes and continued to increase even after molecular hydrogen  H2 inhalation was discontinued, at least during the time of sample collection.

The blood level of molecular hydrogen H2 was significantly higher when molecular hydrogen  H2 was given by inhalation as compared to via intravenous route.

complete article  https://medicalgasresearch.biomedcentral.com/articles/10.1186/2045-9912-2-14

REMEMBER:

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

 

 

 

https://medicalgasresearch.biomedcentral.com/articles/10.1186/2045-9912-2-14
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
  • Hirohisa OnoEmail author,
  • Yoji Nishijima,
  • Naoto Adachi,
  • Masaki Sakamoto,
  • Yohei Kudo,
  • Jun Nakazawa,
  • Kumi Kaneko and
  • Atsunori Nakao
Contributed equally
Medical Gas Research20122:14

https://doi.org/10.1186/2045-9912-2-14

Received: 25 December 2011

Accepted: 20 May 2012

Published: 20 May 2012

Notes

Declarations

Acknowledgements

The authors would like to thank Miz Company for technical assistance for setting up the hydrogen water tank and initial measurement of H2 concentration in the intravenous fluid bag.

Authors’ Affiliations

(1)

Department of Neurosurgery, Nishijima Hospital

(2)

Department of Surgery, University of Pittsburgh

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Copyright

© Ono et al.; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

molecular hydrogen water PERIODONTITIS

Oxidative stress is involved in the pathogenesis of periodontitis. A reduction of oxidative stress by drinking molecular hydrogen-rich water (HW) might be beneficial to periodontal health.

In this pilot study, we compared the effects of non-surgical periodontal treatment with or without drinking molecular hydrogen-rich water HW on periodontitis.

13 patients (3 women, 10 men) with periodontitis were divided into two groups: The control group (n = 6) or the molecular hydrogen-rich water HW group (n = 7). In the molecular hydrogen-rich water HW group, participants consumed molecular hydrogen-rich water HW 4-5 times/day for eight weeks. At two to four weeks, all participants received non-surgical periodontal treatment. Oral examinations were performed at baseline, two, four and eight weeks, and serum was obtained at these time points to evaluate oxidative stress. At baseline, there were no significant differences in periodontal status between the control and molecular hydrogen-rich water HW groups. The molecular hydrogen-rich water HW group showed greater improvements in probing pocket depth and clinical attachment level than the control group at two, four and eight weeks (p < 0.05). The molecular hydrogen-rich water HW group also exhibited an increased serum level of total antioxidant capacity at four weeks, compared to baseline (p < 0.05). Drinking molecular hydrogen-rich water HW enhanced the effects of non-surgical periodontal treatment, thus improving periodontitis.

PMID:26783840
PMCID:PMC4665424
DOI:10.3390/antiox4030513

 

 2015 Jul 9;4(3):513-22. doi: 10.3390/antiox4030513.
Drinking Hydrogen-Rich Water Has Additive Effects on Non-Surgical Periodontal Treatment of Improving Periodontitis: A Pilot Study.

Author information

1
Departments of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. tetsuji@md.okayama-u.ac.jp.
2
Departments of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. de18053@s.okayama-u.ac.jp.
3
Departments of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. dekuni7@md.okayama-u.ac.jp.
4
Departments of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. de18019@s.okayama-u.ac.jp.
5
Departments of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. de18017@s.okayama-u.ac.jp.
6
Center for Innovative Clinical Medicine, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. t-maru@md.okayama-u.ac.jp.
7
Departments of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. tomofu@md.okayama-u.ac.jp.
8
Departments of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. mmorita@md.okayama-u.ac.jp.