Modulation of the oxidative plasmatic state in gastroesophageal reflux disease with the addition of rich water molecular hydrogen: A new biological vision

Abstract 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 GERD symptoms.


| INTRODUCTION
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 ( AE O 2 À ) are generated in mitochondria by electron leakage from the electron transport chain. Superoxide dismutase converts the superoxide anion to hydrogen peroxide (H 2 O 2 ), which is metabolized by glutathione peroxidase and catalase to generate water. Highly reactive hydroxyl radicals ( AE OH) are generated from H 2 O 2 via the Fenton or Weiss reaction in the presence of catalytically active metals, such as Fe 2+ and Cu 2+ . 2 In the last few years, molecular hydrogen (H 2 ) has been pointed out to be a preventive and therapeutic antioxidant. Several research articles have confirmed the efficacy of H 2 both in vitro than in various animal models. 3 H 2 , 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 al 4 first reported that pre-treatment with H 2 inhalation ameliorates brain lesions after cerebral infarction in rats. Emerging data have shown that H 2 -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 H 2 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 H 2 , 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 H 2 O 2 -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. 18 PPIs 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

| Patients
We enrolled a group of drug-na€ ıve 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 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 ).

| 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

| Isolation of human peripheral blood mononuclear cells
Blood samples for laboratory screening were collected at t 0 (before administration of ERW or tap water + PPI) and t 1 (at study endpoint) 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 plasticware 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 lg/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 À80°C until assay. The PBMCs yield per ml of blood was approximately 1 9 10 6 cells. The plasma was obtained by blood centrifugation as described previously and was kept frozen at À20°C. 30

| 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.

| 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 indicate a condition of oxidative stress.

| Griess assay
The assay was carried out as described previously. 32 Two 910 6 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 lM, was used as a standard to assess nitrite concentrations.

| Patients
As reported in Figure 1, 139

| 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, for the CG, À0.7 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).

| 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

| 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 (q = À.439 and À.505, respectively). Differences between PPI and PPI + ERW therapy group assessed by Mann-Whitney U-test.

| DISCUSSION
Bolded P-values are significant after FDR correction.
T A B L E 2 ANOVA for repeated measures performed to evaluate pre-and post-therapy parameters between PPI + TAP water (CG) and PPI + ERW (EG) therapy group , 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 9 group). 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 (O 2 À ) 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 À ). As nitric oxide is a main signalling molecule in cells, its overproduction may lead to pathological effects in several organ systems. 29,42,43 Wide 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 , superoxide anion; TS, total score; HS, heartburn score; RS, regurgitation score; BAP, biological antioxidant potential. Bolded P-values are significant after FDR correction. a clear alteration of cellular redox state, which is characterized by a profound increase in O 2 À 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 lEq/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 (q = À.439 and À.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 O 2 À 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 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 (H 2 ), 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.

ACKNOWLEDG EMENTS
This work is supported by the Italian Ministry for the University and Research. We thank Marco Reato for providing the medical device Alka vitha.

CONFLI CTS 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.