Oxidative stress is one of the causative factors in the pathogenesis of neuro-degenerative diseases including mild cognitive impairment (MCI) and dementia. We previously reported that molecular hydrogen (H2) acts as a therapeutic and preventive antioxidant.
We assess the effects of drinking H2 hydrogen-water (water infused with hydrogen gas H2) on oxidative stress model mice and human subjects with MCI.
Transgenic mice expressing a dominant-negative form of aldehyde dehydrogenase 2 were used as a dementia model. The mice with enhanced oxidative stress were allowed to drink hydrogen H2-water.
For a ran-domized double-blind placebo-controlled clinical study, 73 subjects with mild cognitive impairment MCI drank ~300 mL of hydrogen H2-water (H2-group) or placebo water (control group) per day, and the Alzheimer’s Disease Assessment Scale-cognitive subscale (ADAS-cog) scores were determined after 1 year.
In mice, drinking hydrogen H2-water decreased oxidative stress markers and suppressed the decline of memory impairment and neurodegeneration. Moreover, the mean lifespan in the hydrogen H2-water group was longer than that of the control group.’
In MCI subjects, although there was no significant difference between the hydrogen water H2- and control groups in ADAS-cog score after 1 year, carriers of the apolipoprotein E4 (APOE4) geno-type in the H2-group were improved significantly on total ADAS-cog score and word recall task score (one of the sub-scores in the ADAS-cog score).
H2-water may have a potential for suppressing dementia in an oxidative stress model and in the APOE4 carriers with MCI.
Oxidative stress is one of the causative factors in the pathogenesis of major neurodegenerative diseases including Alzheimer’s disease (AD), mild cognitive impairment (MCI), and Parkinson disease (PD) [1, 2]. Moreover, the genotype of apolipoprotein E4 (APOE4) is a genetic risk for AD, and the increased oxidative stress in the APOE4 carriers is considered as one of the modifiers for the risk .
To explore effective dietary antioxidants to mitigate age-dependent neurodegeneration, it may be useful to construct model mice in which AD phenotypes would progress in an age-dependent manner in response to oxidative stress. We constructed transgenic DAL101 mice expressing a polymorphism of the mitochondrial aldehyde dehydrogenase 2 gene (ALDH2*2) . ALDH2*2 is responsible for a deficiency in ALDH2 activity and is specific to North-East Asians . We reported previously that ALDH2 deficiency is a risk factor for late-onset AD in the Japanese population,  which was reproduced by Chinese and Korean studies in their respective populations [7, 8]. DAL101 mice exhibited a decreased ability to detoxify 4-hydroxy-2-nonenal (4-HNE) in cortical neurons, and consequently an age-dependent neurodegeneration, cognitive decline, and a shortened lifespan .
We proposed that molecular hydrogen (H2) has potential as a novel antioxidant,  and numerous studies have strongly suggested its potential for preventive and therapeutic applications [10–12]. In addition to extensive animal experiments, more than 25 clinical studies examining the efficacy of molecular hydrogen H2 have been reported, [11, 12] including double-blind clinical studies. Based on these studies, the field of hydrogen medicine is growing rapidly.
There are several methods to administer hydrogen H2, including inhaling hydrogen gas (H2-gas), drinking hydrogen H2-dissolved water (H2-water), and injecting hydrogen H2-dissolved saline (hydrogen-rich saline) . Drinking hydrogen H2-water prevented the chronic stress-induced impairments in learning and memory by reducing oxidative stress in mice  and protects neural cells by stimulating the hormonal expression of ghrelin . Additionally, injection of hydrogen-rich saline improved memory function in a rat model of amyloid-β-induced dementia by reducing oxidative stress . Moreover, hydrogen inhalation during normoxic resuscitation improved neurological outcome in a rat model of cardiac arrest independently of targeted temperature management .
In this study, we examined whether drinking hydrogen H2-water could suppress aging-dependent memory impairment induced by oxidative stress in DAL101 mice. Next, in a randomized double-blind placebo-controlled study, we investigated whether H2-water could delay the progression of MCI as assessed by the scores on the Alzheimer’s Disease Assessment Scale-cognition sub-scale (ADAS-cog) [18, 19] from baseline at 1-year. We found a significant improvement in cognition at 1 year in carriers with the APOE4 genotype in the H2-group using sub- and total ADAS-cog scores.
2. MATERIALS AND METHODS
2.1. Ethical Approval and Consent to Participate
This animal study was approved by the Animal Care and Use Committee of Nippon Medical School. The methods were carried out in “accordance” with the relevant guidelines and regulations.
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 [23, 24]. This town is located approximately 40 km northeast of central Tokyo and consists of 22 districts. The baseline survey of the Tone project included 1,032 participants in July 2009, and subjects of the present study were recruited from these participants.
Eligibility criteria are age 67 years or older, being able to give written informed consent for participation in the present study, with a diagnosis of MCI, being able to observe the following requirement: good compliance with water consumption; participation in the scheduled examinations for assessment; keeping a log-diary recording consumption of the water, with a modified Hachinski Ischemic score of 4 or less and a 15-item Geriatric Depression Scale score of 6 or less. In brief, 3 months before this clinical study, all participants underwent a group assessment which used a set of 5 tests that measured the following cognitive domains: attention; memory; visuospatial function; language; and reasoning as described previously . Objective impairment in at least 1 cognitive domain based on the average of the scores on the neuropsychological measures within that domain and 1 SD cut-off using normative corrections for age, years of education, and sex.
Exclusion criteria were having “The Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV TR” criteria for dementing illnesses, a serious or unstable illnesses, a history within the past 5 years of serious infectious disease affecting the brain and/or malignant diseases, a history of alcohol or drug abuse or dependence (on DSM-IV TR) within the past 5 years, and receiving any types of anti-Alzheimer drugs and recent (within 4 weeks) initiation of medications that affect the central nervous system. When the score of Mini Mental State Examination (MMSE)  was less than 24, the subjects were excluded.
In this study, subjects were randomly assigned to either to an intervention group, who received H2-water every-day for 1 year, or a control group, who received placebo water. The allocation sequence was determined by computer-generated random numbers that were concealed from the investigators and subjects. Drs. Nakajima and Ikejima generated the random allocation sequence, enrolled participants, and assigned participants to interventions. Any participants and care providers were blindly masked.
In the original protocol, we planed to administer H2-water for 2 years and assess the secondary outcomes; however, we had to stop the project in 2011 by the Tsunami-disaster and could not obtained the 2-year data and secondary outcomes.
The APOE4 genotype was determined as described .
2.9. Statistical Considerations
All statistical analyses were performed by an academic biostatistician using SAS software version 9.2 (SAS Institute Inc, Cary, NC, USA). Results were considered significant at p < 0.05.
For the comparison of two groups in learning and memory abilities, and lifespans, unpaired two-tailed Student’s t-test was used for the comparison of H2-group with control group. For the other animal experiments, one-way analysis of variance (ANOVA) with Tukey-Kramer or Dunnett post hoc analysis was applied unless otherwise mentioned.
For the clinical trial, we planned to recruit a total of 120 patients, which would provide 90% power to detect an effect size of 0.6 using a two-sided test with a 5% significance level, but the actual sample size for the primary analysis was 73, leading to 70% power in the same setting. End-points were scores in the Japanese version of ADAS-cog at 1-year, and the changes were evaluated by Mann-Whitney’s U test (non-parametric analysis) as well as Student’s t-test (parametric analysis).
3.1. Hydrogen-water Reduced Oxidative Stress in DAL Mice
Male DAL101 mice were given H2– or control water to drink ad libitum from the age of 1 month, and continued until the age of 18 months. The H2-water DAL101 group showed a significant decrease in the level of an oxidative stress marker, urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG) at the age of 14months (Suppl. Fig. S1A). Moreover, DAL101 mice increased oxidative stress in the brain as measured by the level of MDA as an alternative oxidative stress marker, and H2-water showed a significant recovery of this increased level of MDA in DAL101 mice (Suppl. Fig. S1B).
3.2. Hydrogen Water Suppressed a Decline in Learning and Memory Impairment
We examined learning and memory abilities using ORT . As described in MATERIALS AND METHODS, learning and memory abilities were evaluated as the subtraction of the frequency (%) in Retention test from each background (Training test). Mice were provided with control or H2-water from the age of 1 month. At the age of 14 months, the H2-group significantly memorized the original objects and showed the preference for the novel object more than the control group (Fig. 1A1A 14-month-old).
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).
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).
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.
|Control (n=38)||Intervention (n=35)|
|Mean||SD or %||Mean||SD or %|
|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|
|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%)|
* 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.
Age-dependent neurodegenerative disorders are involved in oxidative stress. In this study, we showed that drinking hydrogen H2-water suppressed the biochemical, behavioral, and pathological decline in oxidative stress mice. The score of ADAS-cog  is the most widely used general cognitive measure in clinical trials of AD [27, 28]. The ADAS-cog score assesses multiple cognitive domains including memory, language, praxis, and orientation. Overall, the ADAS-cog has proven successful for its intended purpose. The present clinical study shows that drinking hydrogen H2-water significantly improved the ADAS-cog score of APOE4 genotype-carriers.
We have previously showed that DAL101 mice show age-dependent neurodegeneration and cognitive decline and the shorten lifespan . DAL101 mice exhibit dementia phenotypes in an age-dependent manner in response to an increasing amount of oxidative stress . Oxidative stress enhances lipid peroxidation, leading to the formation of highly reactive α, β-unsaturated aldehydes, such as MDA and 4-HNE . The accumulation of 4-HNE-adducted proteins in pyramidal neurons has been observed in the brains of patients with AD and PD . The decline of ALDH2*2 ability failed to detoxify cytotoxic aldehydes, and consequently increases in oxidative stress .
Moreover, double-transgenic mice were constructed by crossing DAL101 mice with Tg2576 mice, which express a mutant form of human amyloid precursor protein (APP). They showed accelerated amyloid deposition, tau phosphorylation, and gliosis, as well as impaired learning and memory abilities. The lifespan of APP/DAL mice was significantly shorter than that of APP and DAL101 mice . Thus, these model animals may be helpful to explore antioxidants that could be able to prevent age-dependent dementia. Indeed, a diet containing Chlorella showed mitigated effects on cognitive decline in DAL101 .
One of the most potent risk factors for AD is carrier status of the APOE4 genotype, and the roles of APOE4 on the progression of AD have been extensively examined from various aspects [34, 35]. APOE4 also increase the number of atherogenic lipoproteins, and accelerate atherogenesis . The increased oxidative stress in APOE4 carriers is considered as one of the modifiers for the risk . A combination of antioxidants improved cognitive function of aged subjects after 3 years, especially in APOE4 carriers . This previous clinical result agrees with the present study. hydrogen H2 acts as an efficient antioxidant inside cells owing to its ability to rapidly diffuse across membranes . Moreover, as a secondary anti-oxidative function, H2 seems to activate NF-E2-related factor 2 (Nrf2),  which reduces oxidative stress by expression a variety of antioxidant enzymes . We reported that drinking hydrogen H2-water prevented arteriosclerosis using APOE knockout mice, a model of the spontaneous development of atherosclerosis accompanying a decrease in oxidative stress . Thus, it is possible that drinking H2-water improves vascular damage by decreasing oxidative stress as a direct or indirect antioxidant, leading to the improvement of a demintia model and MCI subjects. In this study, we focused on the genotype of APOE-isoforms; however, the polymorphism of the APOE gene in the promoter region influences the expression of the APOE gene . Thus, it will be important to examine the effect of hydrogen H2-water under this polymorphism.
For mitigating AD, significant attention has been given to regular, moderate exercise to help reduce the risk of dementia and prevent MCI from developing in aging patients [40 – 42]. Moderate exercise enhances energy metabolism and suppresses the expression of pro-inflammatory cytokines,  and protects vascular systems [40, 44, 45].molecular hydrogen H2 exhibits multiple functions by a decrease in the levels of pro-inflammatory cytokines and an increase in energy metabolism in addition to anti-oxidative roles. To exert multiple functions, molecular hydrogen H2 regulates various signal transduction pathways and the expression of many genes . For examples,molecular hydrogen H2 protects neural cells and stimulates energy metabolism by stimulating the hormonal expression of ghrelin  and fibroblast growth factor 21,  respectively. In contrast, molecular hydrogen H2 relieves inflammation by decreasing pro-inflammatory cytokines . Thus, the combination of these functions of molecular hydrogen H2 on anti-inflammation and energy metabolism-stimulation might prevent the decline in brain function,  both of which are improved by regular and moderate exercise. Thus, it is possible that the multiple functions of molecular hydrogen H2, including energy metabolism-stimulation and anti-inflammation, may contribute to the improvement of the dementia model and the MCI subjects.
As an alternative aspect, molecular hydrogen H2 suppresses the nuclear factor of activated T cell (NFAT) transcription pathway to regulate various gene expression patterns . NFAT signaling is altered in AD and plays an important role in driving amyloid β-mediated neurodegeneration . Moreover, the NFAT transcriptional cascade contributes to amyloid β synaptotoxicity . Additionally, an active involvement of the NFAT-mediated signaling pathway in α-syn-mediated degeneration of neurons in PD . Indeed, patients with PD improved by drinking molecular hydrogen H2-water as revealed by a double-blind, placebo-controlled clinical study,  and a larger scale of a clinical trial is under investigation . Thus, the beneficial effects of molecular hydrogen H2 on the neurodegenerative diseases may be explained by the suppression of NFAT transcriptional regulation.
The present study suggests a possibility for slowing the progress of dementia by drinking molecular hydrogen H2-water by means of animal experiments and a clinical intervention study for APOE4 carriers; however, a longer and larger scale of trials will be necessary to clarify the effect of H2-water on MCI.
Effects of Molecular Hydrogen Assessed by an Animal Model and a Randomized Clinical Study on Mild Cognitive Impairment
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
|MCI||Mild cognitive Impairment|
|ALDH2||Aldehyde Dehydrogenase 2|
|ADAS-cog||Alzheimer’s Disease Assessment Scale-cognitive subscale|
|DAL101||Dominant Negative Type 101 of the ALDH2 Mutant Polymorphism (ALDH2*2)|
|ORT||Object Recognition Task|
|PA||Passive Avoidance Task|
|GFAP||Glial Fibrillary Acidic Protein|
|ANOVA||One-way Analysis of Variance|
|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 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.
Oxidative stress (OS) related to glucose degradation products such as methylglyoxal is reportedly associated with peritoneal deterioration in patients treated with peritoneal dialysis (PD). However, the use of general antioxidant agents is limited due to their harmful effects. This study aimed to clarify the influence of the novel antioxidant molecular hydrogen (H2) on peritoneal OS using albumin redox state as a marker.
Effluent and blood samples of 6 regular PD patients were obtained during the peritoneal equilibrium test using standard dialysate and hydrogen-enriched dialysate. The redox state of albumin in effluent and blood was determined using high-performance liquid chromatography.
Mean proportion of reduced albumin (ƒ(HMA)) in effluent was significantly higher in H2-enriched dialysate (62.31 ± 11.10%) than in standard dialysate (54.70 ± 13.08%). Likewise, serum ƒ(HMA) after administration of hydrogen-enriched dialysate (65.75 ± 7.52%) was significantly higher than that after standard dialysate (62.44 ± 7.66%).
Trans-peritoneal administration of H2 reduces peritoneal and systemic OS.
Peritoneal deterioration is one of the most serious complications of peritoneal dialysis (PD) therapy, leading to ultrafiltration failure and the more severe complication of encapsulating peritoneal sclerosis (EPS). As the duration of PD increases, so does the risk of peritoneal deterioration . More than 40% of patients in Japan who were on PD treatment for longer than 8 years stopped it due to the progression of peritoneal damage . The pathological mechanisms of peritoneal damage are multi-factorial, but accumulated data have revealed the critical role of glucose degradation end-products (GDPs), i.e., chemically reactive carbonyl compounds. Methylglyoxal (MG) is one of the representative toxic GDPs, causing detrimental effects due to its rapid and indiscriminate oxidative nature , and its production of toxic reactive oxygen species (ROS) such as hydroxyl radical, methyl radical, and undetermined carbon-centered radicals . These used to be present in conventional dialysate, and also enter into the dialysate from uremic plasma . Bio-compatible low-GDP dialysate is currently available, but a Japanese multicenter nationwide study, the NEXT-PD study , revealed the occurrence of EPS even with the use of low-GDP solutions [under submission]. This indicates the need for novel therapeutic approaches to suppress possible insults from enhanced oxidative stress (OS) due to uremic oxidants in the peritoneal cavity.
Recently, the novel role of molecular hydrogen (H2) as an antioxidant has been revealed. H2 eliminates the hydroxyl radical in cultured cells and living organisms . Interestingly, H2 does not influence other ROS, including superoxide, peroxide, and nitric oxide; these ROS play important physiological roles in body . In humans, the safety of H2 has been tested, particularly in the field of deep diving. In contrast to general drugs, which usually have some harmful effects, no toxicity was found even at high concentrations of H2. H2 thus has therapeutic potential for pathological states related to ROS .
The present study tested the effects of peritoneal dialysate containing a high concentration of molecular hydrogen (H2-enriched dialysate) as a novel anti-oxidant among patients treated with PD. As a result, we demonstrated that the use of hydrogen-enriched dialysate could reduce not only peritoneal, but also systemic OS in clinical settings.
Preparation of hydrogen-enriched dialysate
Hydrogen-enriched dialysate was prepared using MiZ nondestructive hydrogen dissolver (MiZ, Kanagawa, Japan), as reported elsewhere . When commercial peritoneal dialysate is immersed in H2-enriched water, hydrogen permeates through the container, resulting in the H2 concentration of dialysate gradually increasing in a time-dependent manner (Figure 1). We prepared H2-enriched dialysate using this apparatus by immersing commercial peritoneal dialysate bags for more than 2 hr. Hydrogen-enriched dialysate was then applied as a test solution for peritoneal equilibrium testing.
Six male PD patients were studied (mean age, 55 years; range, 44–71 years; length of PD, 39 ± 17 months; weight, 68.1 ± 16.1 kg; height, 166.2 ± 5.6 cm). The pathology underlying end-stage renal disease was as follows: chronic glomerulonephritis, n = 3; diabetic nephropathy, n = 2; and hypertensive nephropathy, n = 1. Patients with active infection, bleeding, liver dysfunction, collagen disease, systemic vasculitis, cardiovascular accident within 6 months, or malignancy were excluded from this study. Performance status of all patients was class 1 according to American Heart Association criteria . All patients had been receiving daily continuous ambulatory PD (3–4 bags/day) using neutral low-GDP dextrose solution. The ethics committee of Fukushima Medical University approved this study protocol (Acceptance No. 1362) and written informed consent was obtained from all patients prior to enrollment.
Patients underwent a simplified peritoneal equilibration test (fast PET) using standard dialysate, then underwent fast PET using hydrogen-enriched dialysate 2 weeks later. Fast PET was conducted in accordance with the method of Twardowski . In brief, peritoneal dialysate (2 L of 2.5% dextrose-dialysate) was intraperitoneally infused with a Tenckhoff catheter, and the entire volume of dialysate was drained from the body after 240 min. The drained effluent was mixed well and 2 mL was collected as an effluent sample. Blood samples were obtained before and after fast PET, then 2 mL of serum was drawn after centrifugation and stored at −80°C for 1–4 weeks until analysis. Samples of serum and effluent collected to measure albumin redox were stored at −80°C for 1–4 weeks until analysis. During fast PET, blood pressure, cardiac pulse, and hydrogen concentration in the breath were measured repeatedly every 60 min. Breath hydrogen concentration was also measured in three cases just after, 15 min after, and 30 min after infusion of H2-enriched dialysate. Breath hydrogen concentration was measured using a biological gas (gas in the oral cavity) H2 measurement apparatus BGA-1000D (Aptec, Kyoto, Japan).
Measurement of albumin redox state
Human serum albumin (HSA) is a protein composed of 585 amino acids. The amino residue at position 34 from the N-terminus is a cysteine, containing a mercapto group (SH group). This mercapto group deoxidizes other substances according to the degree of surrounding OS and is itself oxidized. From the perspective of cysteine residues, HSA is a mixture of human mercaptoalbumin (HMA) in which the mercapto group is not oxidized, human non-mercaptoalbumin-1 in which disulfide bond formation is reversibly oxidized mainly by cysteine (HNA-1), and human non-mercaptoalbumin-2 which is strongly oxidized and forms a sulfinic (−SO2H) or sulfonic (−SO3H) group.
The redox state of HSA was determined using high-performance liquid chromatography (HPLC), as previously reported . The HPLC system consisted of an autosampler (AS-8010; Tosoh, Tokyo, Japan; injection volume, 2 μL) and double-plunger pump (CCPM; Tosoh) in conjunction with a system controller (CO-8011; Tosoh). Chromatographs were obtained using a UV6000LP photodiode alley detector (detection area, 200–600 nm with 1-nm step; Thermo Electron, Waltham, MA, USA). A Shodex-Asahipak ES-502N 7C column (10 × 0.76 cm I.D., DEAE-form for ion-exchange HPLC; Showa Denko, Tokyo, Japan; column temperature, 35 ± 0.5°C) was used in this study. Elusion was performed as linear gradient elusion with graded ethanol concentrations (0 to 1 min, 0%; 1 to 50 min, 0 → 10%; 50 to 55 min, 10 → 0%; 55 to 60 min, 0%) for serum in 0.05 M sodium acetate and 0.40 M sodium sulfate mixture (pH 4.85) at a flow rate of 1.0 mL/min. De-aeration of the buffer solution was performed by bubbling helium.
HPLC profiles obtained from these procedures were subjected to numerical curve fitting with PeakFit version 4.05 simulation software (SPSS Science, Chicago, IL, USA), and each peak shape was approximated by a Gaussian function. Values for fractions of HMA, HNA-1, and HNA-2 to total HSA were then calculated (ƒ(HMA), ƒ(HNA-1), and ƒ(HNA-2), respectively).
Values are expressed as mean ± standard deviation unless otherwise stated. StatView version 5.0 statistical software (SAS Institute, Cary, NC, USA) was used for statistical analysis. The significance of collected data was evaluated using a paired t-test or 1-factor repeated-measures analysis of variance (ANOVA) followed by Scheffe’s test as a post-hoc test, as appropriate. For magnitude of correlation, Pearson’s correlation coefficient (R) was used. Differences or correlations were considered significant for values of P < 0.05.
Table 1 shows changes in blood pressure, heart rate, and breath hydrogen concentration during fast PET. Regarding blood pressure and heart rate, no significant difference was seen between standard and H2-enriched dialysate (paired t-test). No significant changes were observed during fast PET in either standard or H2-enriched dialysate (1-factor repeated-measures ANOVA).
|Standard dialysate||H2-enriched dialysate|
|Blood pressure mmHg || || |
| 0 min ||130 ± 12 / 79 ± 10 ||135 ± 13 / 81 ± 10 |
| 60 min ||130 ± 11 / 79 ± 5 ||131 ± 14 / 82 ± 12 |
| 120 min ||125 ± 9 / 79 ± 7 ||134 ± 8 / 80 ± 14 |
| 180 min ||123 ± 12 / 75 ± 12 ||136 ± 5 / 78 ± 12 |
| 240 min ||128 ± 9 / 78 ± 7 ||132 ± 9 / 81 ± 13 |
|Pulse /min || || |
| 0 min ||81 ± 7 ||82 ± 12 |
| 60 min ||76 ± 6 ||79 ± 12 |
| 120 min ||74 ± 6 ||78 ± 14 |
| 180 min ||77 ± 4 ||78 ± 17 |
| 240 min ||78 ± 7 ||81 ± 15 |
|Breath H2 ppm || || |
| 0 min ||4.7 ± 6.6 ||3.2 ± 2.0 |
| 60 min ||1.8 ± 1.3 ||8.3 ± 7.5* |
| 120 min ||3.0 ± 1.7 ||8.5 ± 11.0 |
| 180 min ||4.2 ± 2.8 ||5.8 ± 4.8 |
|240 min||5.5 ± 6.7||7.2 ± 4.6|
*; p < 0.05 vs. standard dialysate.
Changes in breath hydrogen concentration in all cases are shown in Table 1 and Figure 2 (A, B). Although no significant changes were observed during fast PET in both standard and H2-enriched dialysate, the hydrogen concentration at 60 min was significantly higher in H2-enriched dialysate than in standard dialysate.
Breath hydrogen concentrations before, just after, 15 min after, and 30 min after administration of H2-enriched dialysate in three cases are shown in Figure 2C. Hydrogen concentrations were significantly higher just after and 15 min after administration (22.7 ± 5.7 and 15.3 ± 3.5 ppm, respectively) than before administration (4.0 ± 1.7 ppm).
Figure 3 shows the redox state of albumin in effluent fluid. The mean proportion of HMA (ƒ(HMA)) was significantly higher in H2-enriched dialysate (62.31 ± 11.10%) than in standard dialysate (54.70 ± 13.08%). In contrast, ƒ(HNA-1) was significantly lower in H2-enriched dialysate (34.26 ± 10.24%) than in standard dialysate (41.36 ± 12.04%). Like ƒ(HNA-1), ƒ(HNA-2) was significantly lower in H2-enriched dialysate (3.43 ± 0.92%) than in standard dialysate (3.94 ± 1.13%). These results suggest that the use of H2-enriched dialysate reduced peritoneal OS. Regarding the result of fast PET (D/P-Cre, drained volume) and effluent creatinine, albumin, interleukin 6 and carbohydrate antigen 125 levels, no differences were evident between standard and H2-enriched dialysate (Table 2).
|Standard dialysate||H2-enriched dialysate|
|Creatinine mg/dL ||10.53 ± 2.27 ||10.03 ± 2.19 |
|Parameter of fast PET || || |
| D/P-Cre ||0.71 ± 0.12 ||0.66 ± 0.11 |
| Drained volume mL/4 hr ||470 ± 184 ||442 ± 130 |
|Effluent test || || |
| Albumin mg/L ||408 ± 175 ||402 ± 145 |
| Interleukin-6 pg/mL ||6.0 ± 3.3 ||5.5 ± 2.3 |
|CA125 U/mL||18.8 ± 8.5||19.5 ± 5.0|
Figure 4 shows the redox state of albumin in serum before and after fast PET. The serum ƒ(HMA) level after administration of H2-enriched dialysate (65.75 ± 7.52%) was significantly higher than that after standard dialysate (62.44 ± 7.66%). In contrast, ƒ(HNA-1) after administration of H2-enriched dialysate (31.12 ± 6.73%) was significantly lower than that of standard dialysate (34.73 ± 7.02%). These results suggest that use of H2-enriched dialysate reduced not only peritoneal, but also systemic OS. No significant difference was seen between effluent and serum ƒ(HMA) levels after administration of H2-enriched dialysate (65.31 ± 11.10% and 62.71 ± 7.52%, respectively), while effluent ƒ(HMA) after administration of standard dialysate was significantly lower than serum ƒ(HMA) before administration of standard dialysate (54.70 ± 13.08% and 62.96 ± 8.34%, respectively; P = 0.0339), suggesting that intraperitoneal oxidation of albumin was suppressed by H2-enriched dialysate.
Several reports have suggested that OS participates in peritoneal deterioration, with findings such as strong cytoplasmic staining of 8-hydroxy-2′-deoxyguanosine in peritoneal biopsy specimens of long-term PD patients , amplified protein kinase C signaling and fibronectin expression due to enhanced ROS in cultured human mesothelial cells . In terms of the central role of enhanced OS in PD peritoneal damage, Gunal et al.  showed that oral supplementation with the anti-oxidative agent trimetazidine inhibited morphological and functional deterioration of the peritoneum in a PD rat model. However, regarding suppressing OS, no clinical approaches have been available for PD treatment so far.
The present study aimed to test the therapeutic possibility of using dissolved hydrogen in the dialysate to suppress intra-cavity OS in the clinical setting. This study examined the redox state of albumin as a marker of OS. Since the change in redox state of albumin is a physiological and direct reaction, it is appropriate when evaluating real-time OS and/or detecting rapid changes in OS, as compared to other OS markers such as 8-hydroxy-2′–deoxyguanosine, oxidized low-density lipoprotein and F2 isoprotanes, all of which are in vivo by-products during the process of oxidation.
This pilot study of 6 patients clearly demonstrated that single administration of H2-enriched dialysate increased levels of both peritoneal and plasma ƒ(HMA) without any detrimental effects.
Intraperitoneal administration of H2 altered the local redox state, which may indicate the therapeutic potential of delivering H2 directly to the abdominal cavity in respect to the amelioration of peritoneal damage by PD treatment. On the other hand, interestingly, significant increases in serum ƒ(HMA) levels were seen on intraperitoneal administration of H2. Rapid changes in hydrogen concentration of expired gas after the administration of H2-enriched dialysate may mean that molecular hydrogen in dialysate is rapidly distributed to the body to suppress systemic OS. Another possibility is that increased ƒ(HMA) in the cavity may be recruited into systemic circulation through the abdominal lymphatic drainage. The exact mechanisms underlying increased serum ƒ(HMA) need to be addressed in the future.
In addition, the mechanisms of increased ƒ(HMA) and decreased ƒ(HMA1) by H2 have remained unclear in this study. However, molecular hydrogen is known to directly reduce levels of the cytotoxic hydroxyl radical , through several possible mechanisms, such as regulation of particular metalloproteins by bonding, or metalloprotein-hydrogen interactions . Whether H2 directly reacts with the mercapto-residue of albumin, or H2 indirectly modifies it, should be clarified in the future.
Satisfactory anti-oxidative capability of drinking H2-enriched water without any detrimental effects has been reported, in both experimental [19–23] and clinical settings, e.g., type II diabetes mellitus , metabolic syndrome , myopathies (progressive muscular dystrophy and polymyositis/dermatomyositis) , and rheumatoid arthritis . In addition, we also reported the clinical feasibility of applying H2-enriched water as dialysate for hemodialysis treatment [28,29]. Given these reports and our present findings, H2-enriched peritoneal dialysate could be of interest in clinical trials with respect to peritoneal preservation. Furthermore, therapeutic effects seem plausible in terms of the prevention of cardiovascular events in patients, since low f(HMA) has been a significant risk factor for cardiovascular mortality among patients treated with PD  and HD .
In summary, single administration of H2-enriched dialysate reduced peritoneal and systemic OS without any detrimental effects. A longitudinal study is warranted to ensure clinically beneficial effects, such as suppression of peritoneal deterioration and cardiovascular damage.
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Transperitoneal administration of dissolved hydrogen for peritoneal dialysis patients: a novel approach to suppress oxidative stress in the peritoneal cavity
The authors declare that they have no competing interests.
HT, YH, and WJZ carried out the selections of patients, and the sample collections. HT drafted the manuscript. YM, TT, and SE carried out the measurements of samples. SK, and TW contributed to the study as senior advisers. BS carried out the set-up of equipment system for study. MN organized the study project, and drafted the final manuscript. All authors read and approved the final manuscript.
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Articles from Medical Gas Research are provided here courtesy of Wolters Kluwer — Medknow Publications
Molecular Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL function in patients with potential metabolic syndrome
We have found that molecular hydrogen (dihydrogen; H2) water has beneficial lipid-lowering effects in high-fat diet-fed Syrian golden hamsters.
The objective of this study was to characterize the effects of molecular hydrogen H2-rich water (0.9-1.0 l/day) on the content, composition, and biological activities of serum lipoproteins on 20 patients with potential metabolic syndrome.
Serum analysis showed that consumption of molecular hydrogen H2-rich water for 10 weeks resulted in decreased serum total-cholesterol (TC) and LDL-cholesterol (LDL-C) levels.
Western blot analysis revealed a marked decrease of apolipoprotein (apo)B100 and apoE in serum.
In addition, we found molecular hydrogen water H2 significantly improved HDL functionality assessed in four independent ways, namely:
i) protection against LDL oxidation,
ii) inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to endothelial cells,
iii) stimulation of cholesterol efflux from macrophage foam cells, and
iv) protection of endothelial cells from TNF-α-induced apoptosis.
Further, we found consumption of molecular hydrogen H2-rich water resulted in an increase in antioxidant enzyme superoxide dismutase and a decrease in thiobarbituric acid-reactive substances in whole serum and LDL.
In conclusion, supplementation with molecular hydrogeb H2-rich water seems to decrease serum LDL-C and apoB levels, improve dyslipidemia-injured HDL functions, and reduce oxidative stress, and it may have a beneficial role in prevention of potential metabolic syndrome
Song G1, Li M, Sang H, Zhang L, Li X, Yao S, Yu Y, Zong C, Xue Y, Qin S. Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL function in patients with potential metabolic syndrome.
- 1, Key Laboratory of Atherosclerosis in Universities of Shandong, Shandong, China.
- PMID: 23610159
- PMCID: PMC3679390
- DOI: 10.1194/jlr.M036640
- [Indexed for MEDLINE]
Modulation of the oxidative plasmatic state in gastroesophageal reflux disease with the addition of molecular hydrogen rich water : A new biological vision
Gastroesophageal reflux disease (GERD), a clinical condition characterized by reflux of gastroduodenal contents in the oesophagus, has proved to demonstrate a strong link between oxidative stress and the development of GERD. Proton pump inhibitors (PPIs) have been universally accepted as first‐line therapy for management of GERD. The potential benefits of electrolysed reduced water (ERW), rich in molecular hydrogen, in improving symptoms and systemic oxidative stress associated with GERD was assessed. The study was performed on 84 GERD patients undergoing control treatment (PPI + tap water) or experimental treatment (PPI + ERW) for 3 months. These patients were subjected to the GERD‐Health Related Quality of Life Questionnaire as well as derivatives reactive oxigen metabolites (d‐ROMs) test, biological antioxidant potential (BAP) test, superoxide anion, nitric oxide and malondialdehyde assays, which were all performed as a proxy for the oxidative/nitrosative stress and the antioxidant potential status. Spearman’s correlation coefficient was used to evaluate the correlation between scores and laboratory parameters. Overall results demonstrated that an optimal oxidative balance can be restored and GERD symptoms can be reduced rapidly via the integration of ERW in GERD patients. The relative variation of heartburn and regurgitation score was significantly correlated with laboratory parameters. Thus, in the selected patients, combination treatment with PPI and ERW improves the cellular redox state leading to the improvement of the quality of life as demonstrated by the correlation analysis between laboratory parameters and GERDsymptoms.
Generally, oxidative stress can be easily defined as the condition arising from the imbalance between toxic reactive oxygen species (ROS) and the antioxidant systems.1 As the first step in generating persistent ROS, the majority of superoxide anion radicals (·O2−) are generated in mitochondria by electron leakage from the electron transport chain. Superoxide dismutase converts the superoxide anion to hydrogen peroxide (H2O2), which is metabolized by glutathione peroxidase and catalase to generate water. Highly reactive hydroxyl radicals (·OH) are generated from H2O2 via the Fenton or Weiss reaction in the presence of catalytically active metals, such as Fe2+ and Cu2+.2 In the last few years, molecular hydrogen (H2) has been pointed out to be a preventive and therapeutic antioxidant. Several research articles have confirmed the efficacy of H2 both in vitro than in various animal models.3 H2, because of its physicochemical properties of solubility, neutrality and small size, has some high distribution properties allowing it to quickly penetrate bio‐membranes and get to intracellular compartments, where it can carry out its biological effects. Ohsawa et al4 first reported that pre‐treatment with H2 inhalation ameliorates brain lesions after cerebral infarction in rats. Emerging data have shown that H2‐rich water has beneficial effects on oxidative stress‐related diseases such as cancer, arteriosclerosis, diabetes, neurodegenerative diseases and the side effects of haemodialysis.5 Moreover, it was reported that H2 treatment resulted in significantly improved gastrointestinal (GI) transit, protected organs from tissue damage induced by ischaemia reperfusion and effectively ameliorated stress‐associated gastric mucosal damage via its anti‐inflammatory, antioxidant and anti‐apoptotic effects.6, 7, 8 A new technology based on electrolysis of water has been suggested for clinical amelioration of several pathologies. Electrolysed reduced water (ERW), rich in H2, generated at the cathode during water electrolysis, has a high pH, low dissolved oxygen and an extremely negative redox potential (ORP).9 Moreover, in our recent study, we have demonstrated that human histiocytic lymphoma cells line U937 cultured in an ERW‐medium could alleviate H2O2‐induced cytotoxicity of cells through the modulation of cellular redox state.10 Gastroesophageal reflux disease (GERD) is a clinical condition in which the reflux of gastric contents into the oesophagus induces complications and complex symptoms, impairing quality of life.11, 12 Even if the gastric mucosal acts as a protective barrier, pathogens and ingested materials can induce an unbalance of the redox cell state and GI inflammatory responses.13 In fact, several studies have highlighted that oxidative stress is involved in the development and progression of several GI disorders such as GERD, enteritis, gastritis, peptic ulcer, GI cancers and colitis.14, 15 ROS are produced within the GI tract, but their involvement in pathophysiology of GERD have not been well investigated.13, 16 The production of ROS in cell systems is attributable to the activity of many enzymes such as peroxidases, xanthine oxidase, NADPH oxidase, NADPH oxidase isoforms, glucose oxidase, lipoxygenases, myeloperoxidase and cyclooxygenases.13, 17 Proton pump inhibitors (PPIs) have been universally accepted as a first‐line therapy for management of GERD and are among the most commonly prescribed medicines for gastroesophageal reflux and peptic ulcer disease.18PPIs block acid production irreversibly inhibiting H+/K+ adenosine triphosphatase in gastric parietal cell.19 Omeprazole, the first drug in this class, was introduced in 1989 and was followed by lansoprazole (1995), pantoprazole (2000), esomeprazole (2001) and dexlansoprazole (2009). Current guidelines recommend empiric therapy with PPIs for patients suspected of having GERD.11 Despite their efficacy, several studies have shown that a significant proportion of GERD patients are either partial or non‐responders to PPI therapy. In a recent article in JAMA Internal Medicine, some researchers report data on the negative effects of the often overuse of PPIs, widely used in the United States (as well as in Italy, as pointed out by OsMed data).20 A series of systematic reviews have brought further evidence to support the thesis that PPIs are overprescribed and are associated with a number of adverse effects. Numerous observational studies have documented probable causal links with the use of PPIs and adverse reactions, including acute and chronic kidney disease, fractures, hypomagnesaemia, bacterial infections and cardiovascular risk.21, 22, 23, 24, 25 Thus, GERD reduces the quality of life and significantly affects the health care system.26 For these reasons, the aim of this study was to assess the efficacy of H2‐rich water, called ERW, in modulating the symptoms and systemic oxidative stress associated with GERD. We hypothesize that the ERW could be considered as supplementary treatment for GERD, because it could reduce the heartburn and regurgitation in turn enhancing the well‐being of the patients. Thus, the goal of our study was to investigate whether ERW reduces the plasmatic level of oxidative stress in ex vivo peripheral blood mononuclear cells (PBMCs) of GERD patients, relating to scores GERD, as total score (TS), heartburn score (HS) and regurgitation score (RS). Altogether 84 patients reporting moderate to severe heartburn and regurgitation symptoms underwent control treatment (PPI + tap water) or experimental treatment (PPI + ERW) for 3 months. Our findings demonstrated that experimental treatment improves the oxidative balance through a reduction in typical GERD symptoms such as heartburn and regurgitation compared to control treatment.
2. MATERIALS AND METHODS
2.1. Electrolysed reduced water
Electrolysed reduced water was prepared as described previously using the medical device Alka vitha.10The apparatus for the electrolysis of water consists of an active carbon filter (0.2 μm) for water purification and a Pt‐coated Ti electrode for water electrolysis. Furthermore, the apparatus has a pH control system (pH 8.10‐11.60) and Eh values from −200 to −800 mV. The Eh represents the redox potential of an aqueous solution, and it is a measure of the reductive power ability of dissolved molecular hydrogen (H2).
We enrolled a group of drug‐naïve patients with a diagnosis of GERD. The diagnosis was carried out in accordance with the guidelines for GERD.27 The study was conducted in compliance with the “ethical principles for medical research involving human subjects” of the Helsinki Declaration. The local ethics committee has revised and finally approved this study (trial registration: number CE; 992 of 2015/07/07). The individuals were patients of the “Sant.ma Annunziata” Hospital of Chieti between September 2015 and March 2016. The study follow‐up ended on June 2016. The study included adults (age ≥ 18 years) who had a diagnosis of GERD, with a history of frequent episodes of GERD‐related symptoms (regurgitation, heartburn, retrosternal pain) for more than a month prior to the study screening. Patients were excluded from the study if they had experienced one of the following conditions within the previous 3 months: acute infections, vascular access thrombosis, acute myocardial infarction, stroke, diabetes, clinically relevant bleedings, major surgical procedures, blood transfusions, systemic inflammatory of metabolic diseases, active malignancies, smoking habit and participation in other experimental clinical studies. Moreover, patients were also excluded if they suffered from any type of GI disorders, gastroduodenal ulcers, Barrett’s oesophagus, use of concomitant therapy, as well as alcohol or drug abuse. Patients with a BMI of <20 and >33 kg/m2, as well as unusual dietary habits (eg vegetarians), were also excluded. The participants of study were subjected to a blood sample and submitted to GERD‐Health Related Quality of Life Questionnaire (GERD‐HRQL), to define successful response both clinically and systemically to the 3‐month dose of PPI or ERW + PPI. C‐reactive protein (CRP) was measured as a non‐specific marker for inflammation. All the patients underwent 2 monitoring visits, at baseline (t 0) and after 3 months (t 1).
2.3. GERD‐Health Related Quality of Life Questionnaire (GERD‐HRQL)
The Gastroesophageal Reflux Disease‐Health Related Quality of Life (GERD‐HRQL) instrument is a self‐administered questionnaire introduced to provide a quantitative method of measuring frequency and severity of GI symptoms in gastroesophageal reflux disease (GERD). The purpose of GERD‐HRQL was to measure symptomatic change as a result of medical or surgical treatment of GERD. The GERD‐HRQL instrument is practical and generally administered by simply handing it to the patient during a screening visit.28 The questionnaire measuring 16 items (6 related to heartburn, 2 to dysphagia, 6 to regurgitation, 1 to the impact of medication on daily life and 1 on the satisfaction level) on the VAS scale from 0 (no symptoms) to 5 (worst symptoms). The results are expressed as TS, heartburn score (HS) and RS. TS was calculated by summing the individual scores to questions 1‐15 with scores ranging from 0 (no symptoms) to 75 (worst symptoms). HS was calculated by summing the individual scores to questions 1‐6 with scores ranging from 0 (no heartburn symptoms) to 30 (worst heartburn symptoms). RS was calculated by summing the individual scores to questions 10‐15 with scores ranging from 0 (no regurgitation symptoms) to 30 (worst regurgitation symptoms). Satisfaction level‐related quality of life was measured considering the responses at treatment experience assessing in satisfied, neutral and not satisfied.
2.4. Isolation of human peripheral blood mononuclear cells
Blood samples for laboratory screening were collected at t0 (before administration of ERW or tap water + PPI) and t1 (at study end‐point) in 4‐mL endotoxin‐free Heparin tubes (Vacutainer; Becton Dickinson, NJ, USA). Venipuncture was performed in the morning (08.00‐10.00 am.) after an overnight fast and before breakfast. Tubes were kept at room temperature and transported to the laboratory for processing within 1 hour of collection. PBMCs were isolated by density‐gradient centrifugation through Ficoll‐Hypaque (Pharmacia) as described previously.29 Cell viability in each culture was assessed by Trypan blue die exclusion. All solutions were prepared using pyrogen‐free water and sterile polypropylene plastic‐ware and were free of detectable LPS (<0.1 EU/mL), as determined by the Limulus amoebocyte lysate assay (sensitivity limit 12 pg/mL; Associates of Cape Cod, MA, USA). All reagents used were tested before use for mycoplasma contamination (minimum detection level 0.1 μg/mL) (Whittaker Bioproducts, Walkersville, MD, USA) and found negative. The same batches of serum and medium were used in all experiments. After 24 hours incubation, samples were centrifuged at 400 g for 10 minutes at room temperature and supernatants were collected and stored at −80°C until assay. The PBMCs yield per ml of blood was approximately 1 × 106 cells. The plasma was obtained by blood centrifugation as described previously and was kept frozen at −20°C.30
2.5. Assessment of oxidative stress
Plasma was tested for total oxidant capacity and antioxidant potential using a derivatives reactive oxygen metabolites (d‐ROMs) and a biological antioxidant potential (BAP) test kit (Diacron International s.r.l., Grosseto, Italy), respectively.
2.5.1. d‐ROMs test
The test is based on the concept that the amount of organic hydroperoxides present in serum is related to the free radicals from which they are formed. Serum sample is dissolved in an acidic buffer (pH 4.8). The d‐ROMs test is based on the ability of a plasma sample to oxidize the chromogen substrate (N‐N‐diethylparaphenilendiamine) to its radical cation; the reaction is monitored photometrically at 37°C at 505 nm, and the results are expressed as Carratelli Units (CARR U, ΔAbs5050 nm/min), where 1 U‐CARR. corresponds to 0.8 mg/L H2O2. The normal values of the test are between 250 and 300 U‐CARR. (Carratelli Units Values) outside this range are considered indicative of an alteration in the equilibrium between pro‐oxidant and antioxidant capability of patients. Values >300 U‐CARR. indicate a condition of oxidative stress.
2.5.2. BAP assay
Through this test, the components of the antioxidant plasma barrier were measured directly by the active scavengers. The BAP test was performed according to the manufacturer’s instructions (Diacron). A chromogen reagent containing trivalent iron was added to a plasma sample. BAP assay is based on the ability of a plasma sample to reduce Fe3+ to its colourless ferrous derivative (Fe 2). The reaction is monitored by photometric reading at 37°C at 505 nm, and the results are expressed in μEq/L of reduced iron using vitamin C as a standard. The optimal value of a BAP test is >2200 μEq/L. Values lower than 2.200 μEq/L indicate a reduced “biological potential” and hence a decreased effectiveness of the antioxidant plasma barrier, according to an arbitrary scale of severity.
2.5.3. Nitro blue tetrazolium (NBT) assay
The production of intracellular superoxide anion was performed using nitro blue tetrazolium (NBT) (Sigma‐Aldrich SRL, Milano, Italy, Catalog No: N6639) as described previously.31 After PBMC extraction, cells were incubated with NBT (0.1 mg/mL) in culture medium for 3 hours at 37°C; and were further washed 3 times with methanol. The amount of NBT‐formazan produced is an index of O2 − intracellular level. After the solubilization of crystals in 200 mL of KOH 2M/DMSO solution, the quantization was determined spectrophotometrically (Spec‐traMaxH 190; Molecular Devices) at 630 nm. The results were expressed as nmol/mL of O2 − released.
2.5.4. Griess assay
The assay was carried out as described previously.32 Two ×106 cells were seeded in 6 wells/plates, and nitrite was measured in culture supernatants as an indicator of the nitric oxide production. Aliquots of the culture supernatant were mixed with an equal volume of the Griess reagent (Sigma‐Aldrich, USA; Catalog No: G4410) and absorbance was determined at 540 nm using a microplate reader. Sodium nitrite, at concentrations of 0 to 100 μM, was used as a standard to assess nitrite concentrations.
2.6. Measurement of CRP
The amount of circulating CRP levels was assayed using specific ELISA development systems (Diagnostics Biochem Canada Inc, Neptune Crescent, London, ON, Canada, Catalog No: CAN‐CRP‐4360). The experiments were performed in triplicate according to the manufacturer’s instructions. CRP values are expressed as mg/L. The CRP assay sensitivity was <10 ng/mL. The intra‐ and inter‐assay reproducibility was >90%. Triplicate values that differed from the mean by more than 10% were considered suspect and were repeated.
2.7. Measurement of malondialdehyde (MDA)
MDA levels were assayed using specific ELISA development systems (Elabscience; Catalog No: E‐EL‐0060). Plates were scanned using a specialized charge coupled device cooled tool. The integrated density values of the spots of known standards were used to generate a standard curve. Density values for unknown samples were determined using the standard curve for each patient to calculate the real values in pg/mL. All steps were performed in triplicate and at room temperature. The MDA assay sensitivity was <18.75 ng/mL. The intra‐ and inter‐assay reproducibility was >90%. Triplicate values that differed from the mean by more than 10% were considered suspect and were repeated.
2.8. Statistical analysis
The quantitative variables were summarized as mean and standard deviation (SD) or median and interquartile range (IQR), according to their distribution. Qualitative variables were summarized as frequency and percentage. A Shapiro‐Wilk’s test was performed to evaluate the departures from normality distribution for each variable. An analysis of variance (ANOVA) for repeated measures was performed to evaluate the effect of time (baseline vs post‐therapy), group (PPI vs PPI + ERW) and their interaction on laboratory parameters. Chi‐square test was performed to evaluate differences in distribution of d‐ROMs test and BAP test between groups when analysed as categorical data. A Friedman’s test was performed to evaluate the differences in GERD total scores, heartburn score and regurgitation score from baseline to post‐therapy. Mann‐Whitney U‐test was performed to evaluate differences in score relative variation between groups. Spearman’s correlation coefficient (Ρ) was performed to evaluate the correlation among laboratory parameters and scores. The false discovery rate correction (FDR) was used to control the family‐wise type I error rate and an FDR‐adjusted P‐value < .05 was determined to be statistically significant. Statistical analysis was performed using IBM® SPSS Statistics v 20.0 software (SPSS Inc, Chicago, IL, USA).
As reported in Figure Figure1,1, 139 patients took part in the study, 7 of these withdrew while 38 were excluded after the screening interview. In the end, 84 consecutive individuals were included in the study. After giving their written informed consent, the patients were assigned to the control treatment (PPI + tap water) or to the experimental treatment (PPI + ERW) for 3 months. According to the protocol, on a daily basis, the participants drank 1.500 mL of ERW containing dissolved H2 or tap water. All patients included into the experimental treatment received the medical device for the time set for the study. Firstly, all patients received a shock treatment of pantoprazole, 40 mg⁄d, orally for 4 weeks and then 20 mg⁄d for 8 weeks. Pantoprazole was taken 30 minutes before breakfast for a period of 3 months. Of the 84 patients with GERD who were enrolled in this survey, 44 patients were female and 40 patients were male. The mean age of the patients was 51.95 ± 10.90 years, ranging from 23 to 71 years of age. The patients were randomized into PPI (control group‐CG‐) and PPI + ERW (experimental group‐EG‐) groups. Of the 40 patients included in the control group (CG), the mean age as 52.3 ± 10.7 years, 18 patients were male (45%) and 22 patients were female (55%). Of the 44 individuals included in the EG, with mean age of 51.6 ± 11.1 years, 22 patients were male (50%) and 22 patients were female (50%). Statistical analysis showed no statistical differences between the 2 groups regarding age, gender and BMI.
3.2. Quality of life outcome
The typical symptoms of GERD include heartburn and regurgitation, occurring both during the night, frequently waking the patient up from sleep, and during the day, frequently associated with meals which have a great impact on a patients’ quality of life.33 Table 1 shows the difference of the frequency of GERD presentations, before and after treatment among all the patients. As the table shows, the frequency of presentations decreased in both CG and EG groups after treatment. Baseline GERD total scores were 63.0 (53.8‐71.0) and 56.5 (47.3‐64.8) in the CG and EG groups, respectively (P < .05). Post‐treatment results were 38.0 (30.0‐46.0) and 27.5 (19.5‐37.8) in the CG and EG groups, respectively (P < .001), with a relative variation of 0.4 and 0.5, respectively (P = .013). Baseline HS and RS were, respectively, 25.0 (20.3‐27.0) and 25.0 (21.3‐27.0) for the CG and 23.5(20.0‐26.0) and 25.0 (21.3‐28.0) for the EG groups. Post‐treatment results were, respectively, 15.0 (12.0‐19.0) and 15.5 (12.0‐18.0) for the CG and 7.0 (4.0‐12.0) and 7.5 (4.0‐11.0) for the EG. The effect of time was significant for all considered scales (P < .001). Relative variation of HS and RS were, respectively, −0.4 for the CG, −0.7 for the EG group (both P < .001). At 3 months’ follow‐up, the median GERD‐HRQL scores improved significantly after treatment both in CG and in EG groups (38.0 CG vs 27.5 EG), but the statistical analysis revealed that in the patients that associate with the intake of PPI also ERW there is a better significance in relation to HS and RS parameters (P < .001). In summary, treatment with ERW + PPI, for 3 months, gave significantly better symptom control than PPI treatment. Finally, in our study, 75% of the patients studied report a good satisfaction level after ERW treatment. Taken together the results showed that there was significant increase in quality of life at 3 months after supplementation with ERW when compared to baseline (P < .005).
|TS Item GERD|
|CG||63.0 (53.8; 71.0)||38.0 (30.0; 46.0)||−0.4 (−0.5; −0.2)||<.001||.013|
|EG||56.5 (47.3; 64.8)||27.5 (19.5; 37.8)||−0.5 (−0.7; −0.4)|
|CG||25.0 (20.3; 27.0)||15.0 (12.0; 19.0)||−0.4 (−0.5; −0.2)||<.001||<.001|
|EG||23.5 (20.0; 26.0)||7.0 (4.0; 12.0)||−0.7 (−0.9; −0.5)|
|CG||25.0 (21.3; 27.0)||15.5 (12.0; 18.0)||−0.4 (−0.5; −0.3)||<.001||<.001|
|EG||25.0 (21.3; 28.0)||7.5 (4.0; 11.0)||−0.7 (−0.8; −0.5)|
CG, control group (PPI + TAP water); EG, experimental group (PPI + ERW); TS, total score; HS, heartburn score; RS, regurgitation score; ERW, electrolysed reduced water; PPI, proton pump inhibitors.
Bolded P‐values are significant after FDR correction.
3.3. Effect of ERW on oxidative stress in GERD patients
Laboratory parameters trends in the CG and EG groups during follow‐up are reported in Table 2. Several studies have been highlighted that inflammatory cytokines and oxidative stress are involved in the development and progression of GERD.34 Our results confirmed that patients affected by GERD presented higher levels of systemic nitrosative and oxidative stress at baseline. On recruitment, the mean values of nitric oxide, MDA and O2 − were 61.75 ± 24.90 nmol/mL/106 cells, 193.45 ± 121.20 pg/mL and 89.66 ± 24.60 nmol/mL, respectively. Moreover, the analysis of the balance between ROS and antioxidant barrier demonstrated that the values of d‐ROMs and BAP test in GERD patients at baseline were 394.05 ± 110.65 U‐CARR and 847.15 ± 443.05 μEq/L, respectively. Our data are consistent with Wetscher et al35, who observed that free radicals/active oxygen species are involved in the pathogenesis of reflux oesophagitis. After treatment, the balance between ROS and antioxidant barrier were generally found to have progressively returned to normal range. Indeed, the follow‐up visit at 3 months after treatment (t1) revealed an average reduction in the value of the d‐ROMs test and an average increase in the value of the BAP test. ANOVA test for repeated measures indicated a significant difference for nitric oxide level (P = .025) and BAP test (P < .001) between 2 groups. Nitric oxide levels were significantly decreased in EG vs. CG (57.2 ± 12.29 vs 41.1 ± 14.9; P‐value < .001). These data are supported by the remarkable increase in the antioxidant barrier in EG patients compared to controls (798.1 ± 339.3 vs 1796.7 ± 467.2; P‐value < .001). Significant effect of period (P < .001) was found for all laboratory parameters. Interaction group × period was significant for all parameters (P < .001) except for CRP. These values indicated a positive modulation of the pro‐oxidant/antioxidant balance with a reduction in oxidative damage in GERD patients. In addition, we analysed the severity of oxidative stress and of antioxidant barrier impairment (Table 3). On recruitment, about the same percentage of the patients belonging to CG and EG exhibited highly oxidative stress (>500 U‐CARR). Moreover, at the t0, 92.5% of patients belonging to CG and 88.6% of patients in EG had a very strong reduction in the antioxidant barrier (BAP test value < 1400). After 3 months of treatment (t1), no changes in antioxidant barrier were observed in the CG. Notably, in the EG, 23.3% of patients fall within the optimum range of antioxidant barrier and the 53.5% have an optimal value of plasmatic oxidative stress.
|CG||2.3 ± 2.2||1.6 ± 1.6||<.001||.839||.455|
|EG||2.2 ± 1.6||1.4 ± 1.1|
|NO (nmol/mL/106 cells)|
|CG||59.3 ± 13.6||57.2 ± 12.9||<.001||.025||<.001|
|EG||64.2 ± 11.3||41.1 ± 14.9|
|CG||190.3 ± 106.8||203.0 ± 112.0||.001||.084||<.001|
|EG||196.6 ± 135.4||117.9 ± 91.6|
|d‐ROMs test (U‐CARR)|
|CG||385.1 ± 86.4||380.9 ± 71.6||<.001||.062||<.001|
|EG||403.0 ± 134.9||292.2 ± 89.2|
|Biological antioxidant potential test (μEq/L)|
|CG||839.2 ± 441.2||798.1 ± 339.3||<.001||<.001||<.001|
|EG||855.1 ± 444.9||1796.7 ± 467.2|
|O2 − (nmol/mL)|
|CG||83.53 ± 21.00||78.1 ± 14.3||<.001||.218||<.001|
|EG||95.8 ± 28.2||57.1 ± 21.2|
CG, control group; EG, experimental group; CRP, C‐reactive protein; NO, nitric oxide; MDA, malondialdehyde; O2−, superoxide anion; d‐ROMs, derivatives reactive oxygen metabolites; ERW, electrolysed reduced water; PPI, proton pump inhibitors.
Bolded P‐values are significant after FDR correction.
Probability that effect on the addressed variable is influenced by: *period. For each variable, the differences have been tested between the means of each period of the 2 groups (CG and EG); **groups. For each variable, the differences have been tested between the means of PPI group in 2 time (Baseline and post‐treatment) and the means of the EG group in 2 time; ***probability that the effects of period is greater in one distinct group (interaction period × group).
|CG n (%)||EG n (%)||χ2 P‐value||CG n (%)||EG n (%)||χ2 P‐value|
|d‐ROMs test (U‐CARR)|
|<300||8 (20.0)||9 (20.5)||.290||4 (10.0)||23 (53.5)||<.001|
|300‐320||3 (7.5)||3 (6.8)||3 (7.5)||3 (7.0)|
|321‐340||2 (5.0)||5 (11.4)||5 (12.59||5 (11.6)|
|341‐400||12 (30.0)||6 (13.6)||13 (32.5)||9 (20.9)|
|401‐500||12 (3.0)||12 (27.3)||14 (35.5)||1 (2.3)|
|>500||3 (7.5)||9 (20.5)||1 (2.5)||2 (4.7)|
|BAP test (μEq/L)|
|2000‐1801||3 (7.5)||3 (6.8)||0||9 (20.9)|
|1800‐1601||0||0||1 (2.5)||8 (18.6)|
|1600‐1401||0||2 (4.5)||2 (5.0)||3 (7.0)|
|≤1400||37 (92.5)||39 (88.6)||37 (92.5)||9 (20.9)|
CG, control group; EG, experimental group; d‐ROMs, derivatives reactive oxygen metabolites; ERW, electrolysed reduced water; PPI, proton pump inhibitors.
χ2 P‐value = Chi‐squared test. p value < 0,05 are considerated statistically significant.
3.4. Correlation between laboratory parameters and GERD
Spearman’s correlation coefficient was used to evaluate the link among scores and laboratory parameters. TS relative variations correlated with laboratory parameters relative variations, except for BAP test, as shown in Table 4. HS and RS relative variations were significantly correlated with laboratory parameters variation, except for PCR. BAP was significantly associated with HS and RS reduction (ρ = −.439 and −.505, respectively).
|NO (μmol/L/106 cells)|
|d‐ROMs test (U‐CARR)|
|BAP test (μEq/L)|
|O2 − (nmol/mL)|
CRP, C‐reactive protein; NO, nitric oxide; MDA, malondialdehyde; O2 −, superoxide anion; TS, total score; HS, heartburn score; RS, regurgitation score; BAP, biological antioxidant potential.
Bolded P‐values are significant after FDR correction.
GERD is characterized by a number of symptoms, the 2 most common being frequent heartburn and regurgitation.11 For these patients, proton pump inhibitors (PPIs) have been widely adopted as first‐line therapy management of GERD and represent the gold standard therapy. PPIs act by blocking the proton pump of the gastric parietal cells, thus inhibiting a large percentage of acid secretion over 24 hours. Nowadays, there is no evidence that PPIs therapy can prevent the onset of erosion and its progression to pathological lesion.24, 36 The oesophageal mucosa has the intrinsic capacity to resist pathogenic damage, which makes it suitable to self‐protection and regeneration. This intrinsic capacity of regeneration could be the basis of the metaplasia. From the point of view of cell growth, unfortunately oesophageal epithelium is less studied. There are at least 3 different levels of intrinsic defence in the oesophageal mucosa. The first level is pre‐epithelial and is represented by the surfactant, a liquid film deposited on the mucous membrane, which because of its visco‐elastic properties, mechanically protects the epithelium and avoids that the lytic substances come into contact with it. The second level is intra‐epithelial and it is represented by the layer of epithelial cells, which through their relative tight junctions prevent the penetration of H+ions. The third level is post‐epithelial and is represented by the regulatory mechanism of cell tropism.37, 38 When there is a correct tissue blood flow, the tissue oxygenation and the process of neutralization of free radicals play a role in the maintenance of an effective tissue homeostasis.39 In patients with GERD, an adequate blood supply ensures hyperaemia, which leads to infiltration of neutrophils and eosinophils cells in the oesophageal mucosa, causing cell necrosis. In recent years, oxidative stress has been postulated to be an important factor in the pathogenesis and development of lifestyle‐related disease, such as gastroesophageal reflux.21 It is strongly agreed that ROS and reactive nitrogen species (RNS) are generated during inflammation and are considered to contribute to flogosis leading to carcinogenesis.40 In fact, chronic inflammation during GERD is an important risk factor of Barrett’s oesophagus (BE) and oesophageal carcinogenesis.3 The goal of reflux treatment is not necessarily, the complete absence of symptoms, the healing of major oesophageal lesion and the prevention of complications.41 ROS and RNS can induce the formation of a variety of molecule markers of oxidative and nitrosative damage, such as the production of superoxide anion (O2 −) and nitric oxide (NO). In the condition of oxidative stress, nitric oxide was produced through the activation of inducible isoform iNOS with formation to elevate concentration of nitric oxide and thus of peroxynitrite (ONOO−). As nitric oxide is a main signalling molecule in cells, its overproduction may lead to pathological effects in several organ systems.29, 42, 43Wide quantities of nitric oxide were found in human gastro‐junction, and it can diffuse epithelial mucosa and contribute to the increase in the GERD pathological condition. ROS levels have been reported to be increased in oesophagitis compared to healthy controls in both patients and murine models and are hypothesized to mediate mucosal damage and drive disease progression.44, 45 Administration of many antioxidants have been shown to prevent mucosal damage in models of oesophagitis suggesting that antioxidant treatment should be considered as a therapy in the treatment of oesophagitis.33, 45 Alternative treatments are commonly used for various disorders and are often taken on‐demand. There is an increasing use of complementary and alternative medicine that, in contrast to drugs, is believed to be harmless.41, 46Medical research has shown in some studies that the H2 molecule can have an antioxidant and cytoprotective role in several diseases. With the recent progress of H2 science, considering the report that H2 gas could reduce cytotoxic oxygen radicals, therapeutic application of H2 has become a clinical challenge. Recently, several studies have revealed that ERW, enriched of H2, has a unique biological capacity to act as an antioxidant and anti‐inflammatory substance.47, 48 The consumption of ERW has also been shown to exhibit scavenging activity.49 Kashiwagi et al50 showed in a recent study how ERW supplies a DNA protection from free radicals damage. Many have reported in the last few years that GERD is a complex inflammatory disease characterized by the recruitment of factors related to inflammation such as chemokines, cytokines, oxidative stress, growth factors and inflammatory cells.34, 51 Our hypothesis is that H2, being an extremely volatile and permeable gas, crosses the plasma membrane with the ability to react with toxic radicals neutralizing them. In this new original study, we recruited 84 patients with GERD, divided into 2 groups, control group (CG) and an EG. The statistical analysis shows that in the 2 groups studied, PPI therapy improves GERD‐related symptomatology (Table 1). Supplementation of standard therapy with ERW gave significantly better symptom control than PPI treatment. In GERD patients, it was noted how problems linked to drinking, eating, pain, sleeping, compromises life’s quality. In actual fact, it is known that people with this disorder have a lower quality of life than those without GERD. Our results demonstrated that the reduction in clinical symptoms such as heartburn and regurgitation leads to a statistical improvement of the quality of life, as demonstrated by the analysis of satisfaction levels, at 3 months after supplementation with ERW when compared to baseline. In addition, we observed a higher significant difference between the 2 groups at t1, not only in reducing of clinical symptoms, but also an elevated reduction in MDA level, a clear index of a considerable decrease in lipid peroxidation (Table 2). These results also supported a marked reduction in nitric oxide production which was statistically significative in EG respect to the CG. The assessment of oxidative stress is an important but technically challenging procedure in medical and biological research. Jiménez et al52 reported that a decrease in antioxidant activity leading to increased mucosal levels of superoxide anion and peroxynitrite radicals may contribute to the development of oesophageal damage and Barrett’s oesophagus in patients with GERD. Accordingly, our results demonstrated that GERD is associated with a clear alteration of cellular redox state, which is characterized by a profound increase in O2 − production, an increase in nitric oxide and MDA levels (Table 2). To confirm these data, we evaluated derivate reactive oxygen metabolites (d‐ROMs) and BAP in GERD patients. We noted that after treatment, reduction in oxidative stress in plasma is present in both groups, but notably, in the EG, 23.3% of patients return to the optimum range of antioxidant barrier (<2200 μEq/L), while the 92.5% of CG patients have a strongly compromised antioxidant barrier (Table 3). Furthermore, increased BAP test was significantly associated with HS and RS reduction (ρ = −.439 and −.505, Table 4). Thus, the combination of ERW and PPI was shown to be effective in decreasing the scores of GERD and in decreasing oxidative injury‐mediated by nitric oxide and O2 − in GERD patients. These findings signify that ERW supplementation and subsequent ROS reduction together could be used to improve oesophageal damage. These new results, along with our previous results, are in accordance with in vitro research experiments by Hamasaki and his group, which made evident that ERW neutralizes ROS, in a very similar process to the action of SOD and CAT enzymes.53 As GERD is characterized by excessive production of free radicals in the GI system exceeding the endogenous system’s capability to neutralize and eliminate them, we conclude that oxidative stress should be modulated to maintain cellular homeostasis. Therefore, balanced redox status through the optimal modulation of oxidative stress or homeostasis could be essential in considering antioxidant therapy for the prevention of inflammation‐based GI disorder. Our results demonstrate that in GERD patients, combination treatment with PPI and ERW improves the cellular redox state leading to the improvement of the quality of life as demonstrated by the correlation analysis between laboratory parameters and GERD. H2 easily penetrates cells by diffusion and, without disturbing metabolic redox reactions, reduces oxidative stress because of its ability to react with strong oxidants. Our hypothesis is that H2, acting as a scavenger against the ·O2− and the ·OH, neutralizes the toxicity induced by these radical species with consequent reduction in the formation of ONOO−. This leads to a significant lowering in the oxidative systemic damage, which results in a minor infiltration of the inflammatory cells thus in lowering the local hyperaemia and returning the redox cell balance. The increase in the plasma antioxidant barrier and the reduction in free radicals lead to a reduction in the flogosis, decreasing patient symptomatology and improving quality of life. Moreover, GERD is linked to exclusive use of therapy with PPIs as well as a correct lifestyle, and this entails considerable expenditure on health care system. This treatment, for a large number of patients, is not efficient (PPIs non‐responders) and one must not exclude the adverse effects of its prolonged use. Clinicians must be aware of the potential risks and ensure the supervision of the prescriptions of PPIs use must be tailored, using a personalized therapy. Our study is innovative and of great social impact because it highlights that in GERD patients, using a combination regimen with PPI and ERW, rich in molecular hydrogen (H2), as a therapy, can provide systemic changes such as a reduction in heartburn and regurgitation symptoms as well as a major improvement of the quality of life. The future perspectives may be based on the hypothesis of using ERW as neoadjuvant/coadjuvant therapy with PPI at decreasing doses for the treatment of GERD.
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CONFLICTS OF INTEREST
We state that there is no conflict of interest and declare that we have no financial and personal relationship with other people or organizations that could influence this work.
This work is supported by the Italian Ministry for the University and Research. We thank Marco Reato for providing the medical device Alka vitha.
Franceschelli S, Gatta DMP, Pesce M, et al. Modulation of the oxidative plasmatic state in gastroesophageal reflux disease with the addition of rich water molecular hydrogen: A new biological vision. J Cell Mol Med. 2018;22:2750–2759. https://doi.org/10.1111/jcmm.13569
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