Hydration is one of the most significant issues for combat sports as athletes often use water restriction for quick weight loss before competition. It appears that alkaline water can be an effective alternative to sodium bicarbonate in preventing the effects of exercise-induced metabolic acidosis. Therefore, the main aim of the present study was to investigate, in a double blind, placebo controlled randomized study, the impact of mineral-based highly alkaline water on acid-base balance, hydration status, and anaerobic capacity. Sixteen well trained combat sport athletes (n = 16), were randomly divided into two groups; the experimental group (EG; n = 8), which ingested highly alkaline ionized water for three weeks, and the control group (CG; n = 8), which received regular table water. Anaerobic performance was evaluated by two double 30 s Wingate tests for lower and upper limbs, respectively, with a passive rest interval of 3 minutes between the bouts of exercise. Fingertip capillary blood samples for the assessment of lactate concentration were drawn at rest and during the 3rd min of recovery. In addition, acid-base equilibrium and electrolyte status were evaluated. Urine samples were evaluated for specific gravity and pH. The results indicate that drinking alkalized ionized water enhances hydration, improves acid-base balance and anaerobic exercise performance.
Despite numerous scientific data, there is still no conclusive answer regarding what and how much we should drink to optimize sports performance. Until the middle of the 20th century, the recommendation was to avoid drinking to optimize performance. The first drinking guidelines were introduced by the ACSM to avoid heat stress in 1975, while hydration and performance were first addressed only in 1996 . At that time, athletes were encouraged to drink the maximum amount of fluids during exercise that could be tolerated without gastrointestinal discomfort and up to the rate lost through sweating. Depending on the type of exercise and the environment, volumes from 0.6 to 1.2 L per hour were recommended. These drinking guidelines have been questioned recently, and other issues such as over hydration and hyponatremia have been addressed .
The inconsistency of the results regarding hydration and sports performance arise from differences in experimental protocols. In studies in which dehydration develops during exercise, fluid loss of up to 4% body mass does not compromise performance, while in studies that induced dehydration prior to exercise, performance impairments have been observed after dehydration as low as 1–2% body mass . Several comprehensive reviews on the influence of dehydration on muscle endurance, strength, anaerobic capacity, jumping performance and skill performance in team sport games have revealed negative effects of dehydration ≥ 2% body mass [4, 5, 6]. Hydration is one of the most significant issues for combat sports, as athletes often use water restriction for quick weight loss before competition. During tournaments lasting several hours, combat sport athletes sweat immensely and increase their core temperature affecting muscle strength, reducing motor cortex activation, peripheral stimulus as well as the speed of reaction and power output .
Considering the vast amounts of fluids used during exercise, water seems to be the most often form of hydration. Water comes in different forms, with specific properties depending on its mineral content. The pH of water, as well as the proportions between SO42- and HCO3– determines hydration status and other therapeutic properties . Drinking hydrogen rich water in human nutrition is a rather new concept, and it is recently suggested for medical purposes and hydration during exercise [8–10]. Alkaline ionized water is being marketed as a nutritional aid for the general public for acidity-lowering, antioxidant, and antiaging properties. Some of the animal and human research has confirmed its effectiveness as an alkalizing agent in the treatment of metabolic acidosis [11, 12]. However, metabolic acidosis that occurs during high intensity exercise is a distinct form of metabolic alteration, when cells are forced to rely on anaerobic ATP turnover that leads to proton release and a decrease in blood pH that can impair performance [8, 13].
Anaerobic exercise metabolism leads to the production of lactic acid in the working muscles. Part of the produced lactic acid is released to the blood, reducing blood pH, and disturbing acid—base balance. Several studies have provided evidence that hydrogen ions are released from the muscles in excess of lactate after intense exercise . Two mechanisms have been proposed to explain this phenomenon. It seems that hydrogen ions are released both by a sodium-hydrogen ion exchanger and by a lactic acid transporter . Since red blood cells have a higher buffering capacity than blood plasma, the lactate generated during exercise largely remains in the plasma while hydrogen ions are transferred to the red blood cells and buffered by hemoglobin . One of the objectives of training and supplementation in high intensity anaerobic sports disciplines is to increase the buffering capacity of the blood and tissues . The use of sodium bicarbonate has proven effective in speed endurance and strength endurance sports, yet its use has been limited due to the possibility of gastrointestinal distress, metabolic alkalosis, and even edema due to sodium overload [8, 18]. It appears that alkaline water can be an effective alternative to sodium bicarbonate in preventing exercise-induced metabolic acidosis [8, 19]. Contrary to bicarbonate, alkaline water can be used on an everyday basis and has no known side effects. However, there are only few cross-sectional or longitudinal studies on the impact of alkaline water ingestion in combat sport athletes. Therefore, the main objective of the current study was to investigate in a double blind, placebo controlled randomized study, the impact of mineral-based highly alkaline water on acid-base balance, hydration status, and anaerobic capacity in experienced combat sport athletes subjected to a very intense exercise protocol.
Materials and methods
Sixteen very well-trained males, who trained and competed in combat sports for at least 7.6 years, participated in the study. The athletes constituted a homogenous group in regard to age (average age of 22.3 ± 0.5 years), somatic characteristics, as well as aerobic and anaerobic performance (Table 1). The subjects (n = 16) were randomly divided into two groups, the experimental group (EG; n = 8), which received highly alkaline ionized water, and the control group (CG; n = 8), which was hydrated with table water. All subjects had valid medical examinations and showed no contraindications to participate in the study. The athletes were informed verbally and in writing of the experimental protocol, the possibility to withdraw at any stage of the experiment, and gave their written consent for participation. The study was approved by the Research Ethics Committee of the Academy of Physical Education in Katowice, Poland.
|Variables||Experimental Group |
(n = 8)
|Control Group |
(n = 8)
|Age (yrs.)||22.7±3.2||22.4 ± 2.8|
|Body mass (kg)||81.8±3.2||79.2 ±2.6|
|Wt—upper limbs (J/kg)||138±14||136±19|
|Wt—lower limbs (J/kg)||276±04||283±26|
|Pmax–lower limbs (W/kg) |
Pmax–upper limbs (W/kg)
Diet and hydration protocol
Energy intake, as well as macro and micronutrient an intake of all subjects was determined by the 24 h nutrition recall 3 weeks before the study was initiated. The participants were placed on an isocaloric (3455 ± 436 kcal/d) mixed diet (55% carbohydrates, 20% protein, 25% fat) prior and during the investigation. The pre-trial meals were standardized for energy intake (600 kcal) and consisted of carbohydrate (70%), fat (20%) and protein (10%). During the experiment, and 3 weeks before the commencement of the study, the participants did not take any medications or supplements. Throughout the experiment water intake was individualized based on the recommendation of the National Athletic Trainers Association and averaged 2.6–3.2 L per day. In our study we used water which had a pH of 9.13 which is highly alkaline compared to other commercially available products. The water ingested during the experiment contained 840 mg/dm3 of permanent ingredients, and was classified as medium mineral content. The bicarbonate ion HCO3– (357.8 mg/dm3) and carbonate ion CO32- (163.5 mg/dm3) consisted the dominant anions. Sodium (Na+ 254.55 mg/dm3) dominated among cations. The water contained bicarbonate, carbonate-sodium (HCO3–, CO3–Na+). The chemical properties of both types of water used in the experiment (alkaline and table water) are presented in Table 2.
|Variable||Measurement Unit||Alkaline Water||Table Water|
|pH||pH||9.13 ± 0.04||5.00 ± 0.08|
|CO32-||mg/dm3||163.5 ± 6.3||14.98 ± 0.66|
|HCO3–||mg/dm3||357.8 ± 6.14||3.62 ± 0.12|
|Cl–||mg/dm3||26.4 ± 2.3||0.41 ± 0.03|
|SO42-||mg/dm3||7.81± 1.2||1.60 ± 0.09|
|Na+||mg/dm3||254.55 ± 7.1||1.21 ± 0.05|
|K+||mg/dm3||0.91 ± 0.04||0.30 ± 0.03|
|Ca2+||mg/dm3||10.00 ± 1.6||1.21 ± 0.05|
|Mg2+||mg/dm3||0.37 ± 0.03||0.40 ± 0.04|
Note: Data shows mean values ± SD of three analysis of each type of water
The experiment lasted 3 weeks, during which two series of laboratory analyses were performed. The tests were carried out at baseline and after three weeks of hydration with alkaline or table water. The study was conducted during the preparatory period of the annual training cycle, when a high volume of work dominated the daily training loads. The participants refrained from exercise for 2 days before testing to minimize the effect of fatigue.
The subjects underwent medical examinations and somatic measurements. Body composition was evaluated in the morning, between 8.00 and 8.30 am. The day before, the participants had the last meal at 20.00. They reported to the laboratory after an overnight fast, refraining from exercise for 48h. The measurements of body mass were performed on a medical scale with a precision of 0.1 Kg. Body composition was evaluated using the electrical impedance technique (Inbody 720, Biospace Co., Japan). Anaerobic performance was evaluated by a two double 30-second Wingate test protocol for lower and upper limbs respectively, with a passive rest interval of 3 minutes between the bouts of exercise. The test was preceded by a 5 min warm-up with a resistance of 100 W and cadence within 70–80 rpm for lower limbs and 40 W and 50–60 rpm for the upper limbs. Following the warm-up, the test trial started, in which the objective was to reach the highest cadence in the shortest possible time, and to maintain it throughout the test. The lower limb Wingate protocol was performed on an Excalibur Sport ergocycle with a resistance of 0.8 Nm·Kg-1 (Lode BV, Groningen, Netherland). The upper body Wingate test was carried out on a rotator with a flying start with a load of 0.45 Nm·Kg-1 (Brachumera Sport, Lode, Netherland). Each subject completed 4 test trials with incomplete rest intervals. The variables of peak power–Pmax (W/Kg) and total work performed–Wt (J/Kg), were registered and calculated by the Lode Ergometer Manager (LEM, software package, Netherland).
To determine lactate concentration (LA), acid-base equilibrium and electrolyte status the following variables were evaluated: LA (mmol/L), blood pH, pCO2 (mmHg), pO2 (mmHg), HCO3- act (mmol/L), HCO3-std, (mmol/L), BE (mmol/L), O2SAT (mmol/L), ctCO2 (mmol/L), Na+ (mmol/L), and K+ (mmol/L). The measurements were performed on fingertip capillary blood samples at rest and after 3 minutes of recovery. Determination of LA was based on an enzymatic method (Biosen C-line Clinic, EKF-diagnostic GmbH, Barleben, Germany). The remaining variables were measured using a Blood Gas Analyzer GEM 3500 (GEM Premier 3500, Germany).
Urine samples were taken at rest, after an overnight fast, at baseline and at the conclusion of the investigation. They were placed in a plastic container and mixed with 5 ml/L of 5% solution of isopropyl alcohol and thymol for preservation. Urine samples were assayed for the presence of blood and proteins. Specific gravity was determined using the Atago Digital refractometer (Atago Digital, USA). Urine pH was determined based on the standardized Mettler Toledo potentiometer (Mettler Toledo, Germany).
The Shapiro-Wilk, Levene and Mauchly´s tests were used to verify the normality, homogeneity and sphericity of the sample’s data variances, respectively. Verifications of the differences between analyzed variables before and after water supplementation and between the EG and CG were performed using ANOVA with repeated measures. Effect sizes (Cohen’s d) were reported where appropriate. Parametric effect sizes were defined as large for d > 0.8, as moderate between 0.8 and 0.5, and as small for < 0.5 (Cohen 1988; Maszczyk et al., 2014, 2016). Statistical significance was set at p<0.05. All statistical analyses were performed using Statistica 9.1 and Microsoft Office, and were presented as means with standard deviations.
All participants completed the described testing protocol. All procedures were carried out in identical environmental conditions with an air temperature of 19.2°C and humidity of 58% (Carl Roth hydrometer, Germany).
The repeated measures ANOVA between the experimental and control group and between the baseline and post-intervention period (3 weeks of alkaline and table water ingestion) revealed statistically significant differences for thirteen variables (Table 3).
|Wingate Lower Limbs Average Power Exp.||0.884||0.001||21.161|
|Wingate Upper Limbs Average Power Exp.||0.587||0.011||8.528|
|Wingate UL Peak Power Exp.||0.501||0.026||6.228|
|Wingate LL Total Work Exp.||0.567||0.045||4.822|
|Wingate UL Total Work Exp.||0.522||0.011||8.459|
|LA post exr||0.618||0.003||13.382|
|HCO3– post exr||0.632||0.002||14.724|
|K+ post exr||0.501||0.040||5.154|
Note: d—effect size; p—statistical significance
F–value of analysis of variance function
Post-hoc tests revealed a statistically significant increase in mean power when comparing the values (7.98 J/kg to 9.38 J/kg with p = 0.001) at baseline vs. at the conclusion of the study in the experimental group supplemented with alkaline water. In contrast, the control group which received table water did not reveal any statistically significant results.
Similar changes were observed for Upper Limb Average Power (from 4.32 J/kg to 5.11 J/kg with p = 0.011) and Upper Limb Peak Power (from 7.90 J/kg to 8.91 J/kg with p = 0.025) in the experimental group. The post-hoc tests also showed statistically significant increases in values for Lower Limb Total Work (from 276.04 J/kg to 292.96 J/kg with p = 0.012) and Upper Limb Total Work (from 138.15 J/kg to 156.37 J/kg with p = 0.012) when baseline and post intervention values were compared. The changes in the control group were not statistically significant. These results are presented in Fig 1.
Post-hoc tests also revealed statistically significant decreases in LA concentration at rest (from 1.99 mmol/L to 1.30 mmol/L with p = 0.008), and a significant increase in post exercise LA concentration (from 19.09 mmol/L to 21.20 mmol/L with p = 0.003) in the experimental group ingesting alkaline water.
Additionally, a significant increase in blood pH at rest (from 7.36 to 7.44 with p = 0.001), HCO3– at rest (from 23.87 to 26.76 with p = 0.001), and HCO3– post exercise (from 12.90 to 13.88 with p = 0.002) were observed in the experimental group. The other significant changes occurred in the post exercise concentration of K+ (from 4.15 to 4.41 with p = 0.039), in urine pH (from 5.75 to 6.62 with p = 0.017), and a decrease in the value of SG (from 1.02 to 1.00 with p = 0.001), all in the experimental group supplemented with alkaline water.
Acid-base equilibrium within the human body is tightly maintained through the blood and tissue buffering systems, the diffusion of carbon dioxide from the blood to the lungs via respiration, and the excretion of hydrogen ions from the blood to urine by the kidneys. These mechanisms also regulate acid-base balance following high intensity exercise. Metabolic acidosis is a consequence of exercise induced ionic changes in contracting muscles. Increased intramuscular acidity impairs muscle contractibility, significantly limiting high intensity exercise performance . Importantly, acid-base equilibrium can be influenced by dietary supplementation.
In the present study, we investigated the effect of mineral-based alkaline water on acid-base balance, hydration status and anaerobic performance of competitive combat sport athletes. The study participants were experienced athletes (Table 1), capable of performing extreme anaerobic efforts. We have chosen such an approach for two reasons. First, it is well-documented that consumption of alkalizing water can have a significant effect on the hydration status, acid-base balance, urine and blood pH [8, 10], as well as Ca metabolism and bone resorption markers . However, the majority of these research reports have been performed on sedentary individuals  or on subjects with self-reported physical activity . Second, alkalization by alkaline water has been mostly discussed in the context of dehydration and aerobic performance . Therefore, our study is novel by including both well trained combat sport athletes and the use of an extremely intensive anaerobic exercise protocol.
Acid-base balance and hydration status
The exchange of ions, CO2, and water between the intracellular and extracellular compartments helps to restore acid-base balance following intensive exercise. There is sufficient data indicating that, supplements that modify the blood buffering system affect high-intensity exercise performance . In humans, especially well trained athletes muscle pH may decrease from 7.0 at rest to values as low as 6.4–6.5 during exercise . Ergogenic aids that help buffer protons attenuate changes in pH and enhance the muscle’s buffering capacity. This in turn allows for a greater amount of lactate to accumulate in the muscle during exercise.
The results of our study are in line with the available literature regarding the impact of alkaline water on blood and urine pH at rest [9, 19, 25]. However, novel results of the present research are related to the changes in HCO3- after exercise in athletes ingesting alkaline water. Bicarbonate-CO2 accounts for more than 90% of the plasma buffering capacity. Supplementation can increase bicarbonate concentration in the blood and its pH. Since bicarbonate concentration is much lower in the muscles (10 mmol/L) than in the blood (25 mmol/L), the low permeability of charged bicarbonate ions precludes any immediate effects on muscle acid-base status . These results confirm the view that an appropriate hydration status is necessary for active bicarbonate ion transport.
Several lines of evidence support the negative impact of dehydration (>2% body mass) on muscle endurance, strength, and anaerobic performance . On the other hand, literature data indicates that consumption of alkaline water following a dehydrating bout of cycling exercise was shown to rehydrate cyclists faster and more completely compared to table water. Following consumption of alkaline water, the cyclists demonstrated lower total urine output, their urine was more concentrated (i.e., with higher specific gravity), and the total blood protein concentration was lower, indicating improved hydration status .
Our previous study revealed that the use of water with alkalizing properties exhibits a significant potential for hydration during anaerobic exercise . The results of the present study confirm a decrease in urine specific gravity (from 1.02 to 1.00, with p = 0.001) and an increase in urine pH as the result of consumption of alkaline water. These results illustrate that the habitual consumption of highly alkaline water can markedly improve hydration status.
The current investigation demonstrated a significant increase in anaerobic capacity (Wt−J/Kg) of athletes in the experimental group supplemented with alkaline water. The improvements in Wt following alkaline water consumption were influenced by positive changes in blood pH and bicarbonate. This phenomenon could be explained by the ergogenic effects of high alkalization and mineral ingredients.
High intensity exercise in which anaerobic glycolysis provides ATP for muscle contraction leads to an equal production of lactate and hydrogen ions. Most of the released hydrogen ions are buffered; however, a small portion (~0.001%) that remains in the cytosol causes a decrease in muscle pH and an impairment of exercise. Lactate efflux  and its oxidation are accompanied by a similar removal of hydrogen ions. The results of the current study demonstrated a statistically significant decrease in lactate concentration at rest (from 1.99 mmol/L to 1.30 mmol/L, p = 0.008), and a significant increase post exercise (from 19.09 mmol/L to 21.20 mmol/L, p = 0.003) when compared to the baseline levels with the values recorded at the end of alkaline water supplementation. The extremely intense 4 x 30s upper/lower limb Wingate test protocol employed in our study, with only short rest intervals between each bout of exercise, was a likely reason that less of the total lactate produced in the muscles was transported to the blood .
Muscle blood flow determines lactate efflux from the muscle , and is dependent on the activity of lactate transport proteins , the extracellular buffering capacity , and the extracellular lactate concentration . Thus, our results on lactate concentration are in agreement with the view that anaerobic performance (i.e., Wt−J/Kg, WAvr−J/Kg) depends on counter-regulatory variables. Indeed, we demonstrated that changes in resting blood pH and HCO3– significantly improved anaerobic performance.
Another variable that can affect anaerobic performance includes blood viscosity. Weidmann et al. (2016) showed that the intake of highly alkaline water decreased blood viscosity by 6.30%, compared to table water (3.36%) in 100 recreationally active female and male subjects. Therefore, it may be possible that the excess of metabolic end-products (namely, H+ and Pi), which disturb cellular homeostasis and muscle contraction, are more effectively transported. The available literature data does not specify clearly which components of buffering capacity are altered by the above changes. It must be indicated, that there are several methods available to determine muscle buffering capacity. Due to the methodological complexity, none of these methods are free from criticism. In most studies buffering capacity has been determined in vitro by titration, which does not include trans-membrane transport of acid-base substances or dynamic buffering by biochemical processes occurring in vivo .
Most studies show a documented ergogenic effect of bicarbonate loading during exhaustive exercise lasting 1–7 min, when anaerobic glycolysis plays a major role in energy provision . The rationale for the ergogenic effect of bicarbonate is that the increase in extracellular pH and bicarbonate will enhance the efflux of lactate and H+ from muscle. There is also evidence that the ergogenic effect of bicarbonate is more pronounced during repeated sprints than during sustained exercise .
Different strategies used for improving buffering capacity of tissues and blood do not allow for a direct comparison. Despite this, there appears to exist an ergogenic effect in response to NaHCO3–, what may explain the large effect size noted by Tobias et al. . In our research we obtained large effect sizes with regards to 4 variables (Average power of the lower limbs, resting HCO3–, resting blood pH and urine SG).
The results of the present study indicate that drinking alkalized water improves hydration status, acid-base balance, and high intensity anaerobic exercise performance. It appears that both greater muscle buffering capacity and enhanced removal of protons, resulting in increased glycolytic ATP production, may be responsible for these effects. Considering the energy demands and the intense sweat rate of combat sport athletes, the authors recommend the daily intake of 3–4 L of highly alkaline mineralized water to improve hydration and anaerobic performance during training and competition.
Stress test data.
This work was supported by the Ministry of Science and Higher Education of Poland under Grant NRSA3 03953 and NRSA4 040 54.
This work was supported by the Ministry of Science and Higher Education of Poland under Grant NRSA3 03953 and NRSA4 040 54.
All relevant data are within the paper and its Supporting Information files.
Alkaline ionized water improves exercise-induced metabolic acidosis and enhances anaerobic exercise performance in combat sport athletes
Articles from PLoS ONE are provided here courtesy of Public Library of Science
The biological effect of alkaline water consumption is object of controversy. The present paper presents a 3-year survival study on a population of 150 mice, and the data were analyzed with accelerated failure time (AFT) model. Starting from the second year of life, nonparametric survival plots suggest that mice watered with alkaline ionized water showed a better survival than control mice. Interestingly, statistical analysis revealed that alkaline ionized water provides higher longevity in terms of “deceleration aging factor” as it increases the survival functions when compared with control group; namely, animals belonging to the population treated with alkaline ionized water resulted in a longer lifespan. Histological examination of mice kidneys, intestine, heart, liver, and brain revealed that no significant differences emerged among the three groups indicating that no specific pathology resulted correlated with the consumption of alkaline ionized water. These results provide an informative and quantitative summary of survival data as a function of watering with alkaline ionized water of long-lived mouse models.
Alkaline water, often referred to as alkaline ionized water (AKW), is commercially available and is mainly proposed for electrolyte supplementation during intensive perspiration. Early studies on animal models reported that alkaline ionized water supplementation may exert positive effects on body weight improvement and development in offspring [1, 2]. Even biochemical markers were analyzed, suggesting that alkaline ionized water intake can cause elevation of metabolic activity. In particular, hyperkaliemia was observed in 15-week-old rats and pathological changes of necrosis in myocardial muscle were found .
More recently, studies were carried out on alkaline ionized/electrolysis reduced water (ARW), referring to electrolyzed water produced from minerals, such as magnesium and calcium, which is characterized by supersaturated hydrogen, high pH, and a negative redox potential ORP. This hydrogen-rich functional water has been introduced as a therapeutic strategy for health promotion and disease prevention .
Alkaline ionized/ electrolyzed reduced water have been shown to exert a suppressive effect on free radical levels in living organisms, thereby resulting in disease prevention . Various biological effects, such as antidiabetic and antioxidant actions , DNA protecting effects , and growth-stimulation activities , were documented.
Although a variety of bioactive functions have been reported, the effect of alkaline water on lifespan and longevity in vivo is still unknown. Animal alkalization has been shown to be well tolerated and to increase tumor response to metronomic chemotherapy as well the quality of life in pets with advanced cancer . Therefore, we performed a study based on survival rate experiments, which play central role in aging research and are generally performed to evaluate whether specific interventions may alter the aging process and lifespan in animal models.
2. Materials and Methods
Biological effects of alkaline ionized water were evaluated on a selected population of 150 mice (CD1, by Charles River, Oxford, UK). Pathogen-free mice were purchased and placed in a specific breeding facility. No other animal was present in the room. Contact with animal caretakers was minimized to feeding and watering. The population was divided into 3 groups, each consisting of 50 individuals, as follows:
- Group A: 50 mice conventionally fed and watered with alkaline ionizefd water produced by the Water Ionizer (mod. NT010) by Asiagem (Italy). The Water Ionizer is a home treatment device for producing alkaline drinking water.
- Group B: 50 mice conventionally fed and watered with alkalized water obtained by dilution of a concentrated alkaline solution (AlkaWater by Asiagem, Italy). AlkaWater is a concentrated alkaline solution for preparing alkaline drinking water.
- Group C: 50 mice conventionally fed and watered as conventional (control group) with tap water. The local water supply was evaluated weekly for assuring the absence of toxins and pathogens. The pH values were in the 6.0–6.5 range.
All procedures involving animals were conducted in accordance with the Italian law on experimental animals and were approved by the Ethical Committee for Animal Experiments of the University of Padua and the Italian health Ministry (Aut. no. 39ter/2011). Efforts were made to minimize animal suffering.
2.1. Histological Examination
Treated aged mice were sampled postmortem and subjected to histological examination. Animals belonging to the populations treated with alkaline water, A and B, were sacrificed after 24 months and compared to mice treated with tap water. Samples from kidneys, intestine, heart, liver, and brain were fixed in 10% neutral buffered formalin, and 4 μm sections were analyzed by optical microscopy.
2.2. Statistical Analysis
In order to investigate the biological influence of alkaline water on mouse longevity, we employed the accelerated failure time model (AFT) , which allows formally exploring the possible effect on survival curves of the applied three-level treatment, that is, examining the role of group membership as a covariate of lifespan. As a more robust alternative to the commonly used proportional hazards models, such as the Cox model, the use of AFT models is advised in the field of survival analysis when the goal is to investigate if a covariate may affect the lifespan in a way that the life cycle may pass more or less rapidly. In fact, whereas a proportional hazard model assumes that the effect of a covariate is constant over time, an AFT model assumes that the effect of a covariate is to accelerate or decelerate the life course.
The relevance of AFT model for biomedical studies has been already recognized in the literature . With more specific reference to the issue of aging, Swindell  observed that some genetic manipulations were found to have a multiplicative effect on survivorship which were well characterized by the AFT model “deceleration factor.” Moreover, Swindell  argued also that the AFT model should be utilized more widely in aging research since it provides useful tools to maximize the insight obtained from experimental studies of mouse survivorship.
To perform all calculations, we applied a parametric survival analysis approach using a class of 3-parameter AFT distribution models implemented within the statistical software Minitab, version 17.2.1 . More specifically, we employed three types of random distributions, namely, log-logistic, log-normal, and generalized Weibull.
The experiment consisted in an initial 15-day acclimatization period. After acclimatization, animals (50, group A) were watered with alkaline ionized water (pH 8.5), obtained by the Water Ionizer , whereas group B animals (50) were watered with water alkalized at pH 8.5 by a concentrated alkaline solution for 15 days. Group C animals (50), control group, were watered with the local water supply. This period has been identified to gradually accustom the animals treated with alkaline water. At the end of the second period of acclimatization, group A and B animals were watered with alkaline ionized water at pH 9.5, while animals of group C were watered with local tap water.
After the first year, the most aggressive individuals were moved to other cages within the same group and an environmental enrichment protocol was employed in order to decrease the hyperactivity. This phenomenon was observed especially in animals of groups A and B.
Table 1 reported basic statistics on mice survival of treated and control animals.
|Treatment level||Mortality rate |
|Lifespan mean (std. dev.) |
|Group A||88||679 (209)|
|Group B||92||671 (180)|
|Group C||96||667 (185)|
Regarding group A, animals (50) were watered with alkaline ionized water (pH 8.5), obtained by the Water Ionizer (Asiagem, Italy). As for group B, animals (50) were watered with water alkalized at pH 8.5 by a concentrated alkaline solution for 15 days. Regarding group C, animals (50), control group, were watered with the local water supply.
A first look on experimental data is provided in Figure 1, where nonparametric hazard and survival plots seem to suggest that even if no macroscopic difference emerges, starting from the second year of life mice watered with alkaline ionized Water and those treated with AlkaWater overwhelmed control mice.
In order to explore the possible effect of different treatments, that is, to examine the role of group membership on longevity, we applied a parametric survival analysis approach using a class of 3-parameter survival distributions that represent flexible accelerated failure time, AFT models. First of all, using the Anderson-Darling goodness-of-fit statistic, we compared three specific survival distributions, that is, log-logistic (AD = 6.397), log-normal (AD = 6.519), and generalized Weibull (AD = 6.447). Since the best fitting was shown by log-logistic model, we adopted this one as final survival distribution model. The straight lines in the log-logistic distribution QQ plots (Figures 2(a) and 2(b)) indicate that this distribution provides a suitable fit to our survival data.
Finally, by including our treatment as covariate, we performed a parametric distribution analysis whose results are graphically represented in Figure 3.
Starting with the second year of life, it is worth noting that both alkaline water treated groups denote a decreasing hazard curve over time, while the corresponding curve for control group is monotonically increasing. To more formally compare the treatment levels, the proposed analysis provided also suitable pvalues. Since the p values related on the null hypotheses of equality of location, scale and threshold parameters were, respectively, less than 0.001 (for both locations and scales) and 0.634 (for thresholds) at a 5% significance level; we can state that there is enough experimental evidence to conclude that the treatment significantly affects the mice longevity; in particular the alkaline ionized water provides a benefit to longevity in terms of “deceleration aging factor” as it decreases the hazard functions when compared with the control group. Note that the treatment effect cannot be directly related to no one of the three distribution parameters. Anyway, using the estimated parameters, it should be possible to provide an estimate for the effect of each treatment on survivorship: setting the reference survival time to 1000, 1200, and 1400 days, Table 2 summarizes the estimated point and 95% interval survival probabilities by each treatment level.
|Treatment level||Time (days)||Estimated probability||Lower 95% CI limit||Upper 95% CI limit|
As final remark, it should be noted that even if our parametric AFT survival analysis was performed using the log-logistic distribution, our conclusions are consistent with results obtained using the generalized Weibull distribution, while via log-normal distribution no significant effect was found.
3.1. Histological Examination
No significant differences emerged from the histological examination among the three groups. In all examined samples, renal tissue was characterized by a mild-to-moderate lymphoplasmacytic interstitial infiltrate and few occasional glomerular changes as glomerular size reduction and increasing of Bowman’s space (Figure 4).
Final diagnosis was mild chronic progressive nephropathy for the three analyzed mouse groups.
The microscopic examination of the liver revealed a multifocal nodular pattern of the parenchyma and diffuse mild-to-moderate hepatocellular cytoplasmic hydropic degeneration with multifocal binucleation in all explored animals (Figure 5).
Mild-to-moderate anisokaryosis was the most relevant alteration, with few pleomorphic nuclei and frequent intranuclear pseudoinclusions and karyomegaly. A specific mild perivascular infiltrate was occasionally present. Final diagnosis was mild-to-moderate diffuse hepatopathy with multifocal hyperplastic hyperplasia.
The pulmonary parenchyma showed mild multifocal areas of interstitial thickening of the interalveolar septa due to moderate congestion and mild cellular mixed infiltrate (Figure 6). Mild areas of emphysema were detected at the periphery of the parenchyma. Final diagnosis was multifocal very mild atelectasis and mild vicarious emphysema.
At the same time, no relevant histopathologic histological changes have been noticed in intestine (Figure 7), brain, and heart.
The present work presents a 3-year survival study on a population of 150 mice and the data were analyzed with accelerated failure time (AFT) model. Kaplan-Meier statistical analysis of the survival data indicates the possibility of a positive effect of alkaline ionized water on mouse lifespan and AFT model allowed evaluating differences starting from the second year of the survival curves. These results provide an informative and quantitative summary of survival data as a function of watering with alkaline ionized water on long-lived mouse models. It should be pointed out that, from the standpoint of aging research, this statistical approach presents appealing properties and provides valuable tools for the analysis of survival. The observation of tissues of deceased animals was performed for the assessment of the state of internal organs to be compared with similar analyses of untreated animals. The renal lesions observed at histology were specific and common for the three animal groups. Chronic progressive nephropathy has been well described as normal aging change in mice [11, 12]. In our cases animals did not show any clinical sign of nephropathy or any other histological evidence of specific kidney disease and we ascribed the lesions to the aging process [11, 12].
The examined livers were also affected by typical lesions of mature subjects, such as hyperplastic nodules. Furthermore, well known aging changes were individuated in the hepatocytes, such as karyomegaly, nuclear pleomorphism, and pseudoinclusions cysts [11, 12].
A 3-year survival study on a population of 150 mice was carried out in order to investigate the biological effect of alkaline water consumption. Firstly, nonparametric hazard and survival plots suggest that mice watered with alkaline ionized water overwhelmed control mice. Secondly, data were analyzed with accelerated failure time (AFT) model inferring that a benefit on longevity, in terms of “deceleration aging factor,” was correlated with the consumption of alkaline ionized water. Finally, histological examination of mice kidneys, intestines, hearts, livers, and brains was performed in order to verify the risk of diseases correlated to alkaline watering. No significant damage, but aging changes, emerged; organs of alkaline watered animals resulted to be quite superimposable to controls, shedding a further light in the debate on alkaline water consumption in humans.
This paper is dedicated to the memory of Tommaso Nicoletti. The authors are grateful to Rocco Palmisano for original ideas and support. The authors would like to thank Asiagem (Italy) for partial support and Ludovico Scenna, Carlo Zatti, and Silvano Voltan for their scientific and professional contribution.
The authors declare that there are no competing financial interests.
Alkaline Water and Longevity: A Murine Study
Articles from Evidence-based Complementary and Alternative Medicine : eCAM are provided here courtesy of Hindawi Limited
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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