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Alkaline ionized water improves exercise-induced metabolic acidosis and enhances anaerobic exercise performance in combat sport athletes

note: this article may be useful to cancer patients as it is well known that most cancer cells use anaerobic glycolysis to produce ATP energy and excrete lactic acid  
Abstract

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.

Introduction

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 []. 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 []. 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 []. 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 [].

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 []. It appears that alkaline water can be an effective alternative to sodium bicarbonate in preventing exercise-induced metabolic acidosis []. 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

Subjects

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.

Table 1

Characteristics of the study participants.
Variables Experimental Group
(n = 8)
Control Group
(n = 8)
Age (yrs.) 22.7±3.2 22.4 ± 2.8
Height (cm) 181.2±2.1 178.3±4.9
Body mass (kg) 81.8±3.2 79.2 ±2.6
FM (%) 10.2±2.1 10.8±2.4
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)
19.8±0.9
8.9±1.1
20.2±1.6
8.7±0.4
VO2max (ml/kg/min) 64.7±2.8 62.6±3.2

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, CO3Na+). The chemical properties of both types of water used in the experiment (alkaline and table water) are presented in Table 2.

Table 2

Chemical properties of water used in the study.
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

Study protocol

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).

Biochemical assays

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).

Statistical analysis

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.

Results

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).

Table 3

Statistically significant differences between the experimental and control groups at baseline and after 3 weeks of intervention (alkaline vs table water).
Variables d p F
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 rest 0.534 0.008 9.429
LA post exr 0.618 0.003 13.382
pH rest 0.834 0.001 120.159
HCO3 rest 0.844 0.001 109.250
HCO3 post exr 0.632 0.002 14.724
K+ post exr 0.501 0.040 5.154
Urine pH 0.589 0.017 7.298
SG 0.884 0.001 19.707

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.

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Differences between the control and experimental groups in total work of the lower and upper limbs (30s Wingate test) at baseline and after 3 weeks of alkaline or table water ingestion.

Note: * statistically significant values.

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.

Discussion

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 [], 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 []. 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.

Anaerobic performance

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).

Conclusions

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.

Supporting information

S1 Table

Data for Fig 1.

(XLSX)

S2 Table

Stress test data.

(XLSX)

S3 Table

Water data.

(XLSX)

Acknowledgments

This work was supported by the Ministry of Science and Higher Education of Poland under Grant NRSA3 03953 and NRSA4 040 54.

Funding Statement

This work was supported by the Ministry of Science and Higher Education of Poland under Grant NRSA3 03953 and NRSA4 040 54.

Data Availability

All relevant data are within the paper and its Supporting Information files.

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PLoS One View this Article Submit to PLoS Get E-mail Alerts Contact Us Public Library of Science (PLoS)
. 2018; 13(11): e0205708.
Published online 2018 Nov 19. doi: 10.1371/journal.pone.0205708
PMCID: PMC6242303
PMID: 30452459
Alkaline ionized water improves exercise-induced metabolic acidosis and enhances anaerobic exercise performance in combat sport athletes
Jakub ChyckiConceptualizationInvestigationMethodologyWriting – original draft,1,* Anna KurylasData curationMethodologyProject administration,1 Adam MaszczykData curationValidationVisualization,2Artur GolasData curationFormal analysis,1 and Adam ZajacConceptualizationInvestigationMethodologyWriting – original draft1
Michal Toborek, Editor
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Associated Data

Supplementary Materials
Data Availability Statement
All relevant data are within the paper and its Supporting Information files.

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alkaline ionized water and longevity

Abstract

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.

1. Introduction

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 []. 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:

  1. 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.
  2. 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.
  3. 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.

3. Results

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.

Table 1

Basic statistics on mice survival by treatment level.

Treatment level Mortality rate
%
Lifespan mean (std. dev.)
Days
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.

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Nonparametric hazard and survival plots by treatment level. Group A: animals (50) were watered with alkaline ionized water (pH 8.5), obtained by the Water Ionizer  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.

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.

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QQ plots using the 3-parameter log-logistic distribution model. (a) Treatment A survival time quantiles (vertical axis) versus treatment C survival time quantiles (horizontal axis); (b) treatment B survival time quantiles (vertical axis) versus treatment C survival time quantiles (horizontal axis).

Finally, by including our treatment as covariate, we performed a parametric distribution analysis whose results are graphically represented in Figure 3.

An external file that holds a picture, illustration, etc. Object name is ECAM2016-3084126.003.jpg

Distribution plot results using the 3-parameter log-logistic model. Group A: animals (50) were watered with alkaline ionized water (pH 8.5), obtained by the Water Ionizer . 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.

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.

Table 2

Table of survival probabilities by treatment level. The probabilities, along with their related 95% confidence interval limits, were calculated using the normal approximation.

Treatment level Time (days) Estimated probability Lower 95% CI limit Upper 95% CI limit
A 1000 0.116 0.056 0.226
1200 0.046 0.014 0.140
1400 0.020 0.004 0.098

B 1000 0.055 0.021 0.137
1200 0.013 0.003 0.066
1400 0.004 0.000 0.039

C 1000 0.049 0.022 0.106
1200 0.008 0.002 0.027
1400 0.001 0.000 0.007

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).

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Kidney, a specific chronic nephropathy. Focal interstitial mainly lymphocytic infiltrate (upright) and a sclerotic glomerulus (middle right). Hematoxylin and Eosin.

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).

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Liver, aging change. Hepatocellular abundant dishomogeneous cytoplasm, binucleation (center), variably sized nuclei, and a nuclear pseudoinclusion cyst (arrow). Hematoxylin and Eosin.

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.

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Lung, mild atelectasis. Very mild multifocal interstitial thickening of the alveolar septa associated with congestion and mild cellular increase. Hematoxylin and Eosin.

At the same time, no relevant histopathologic histological changes have been noticed in intestine (Figure 7), brain, and heart.

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Intestine. Longitudinal section of duodenum showing uniformly thin and elongated villi. Hematoxylin and Eosin.

4. Discussion

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 []. 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 [].

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 [].

5. Conclusions

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.

Acknowledgments

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.

Competing Interests

The authors declare that there are no competing financial interests.

Published online 2016 May 31. doi: 10.1155/2016/3084126
PMCID: PMC4906185
PMID: 27340414
Alkaline Water and Longevity: A Murine Study

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