Effects of hydrogen-rich water prepared by alternating-current-electrolysis on antioxidant activity, DNA oxidative injuries, and diabetes-related markers

Written by: Ryoko Asada, PhD,1,#* Kenji Tazawa,2,# Shinkichi Sato,3 and Nobuhiko Miwa4

September 30, 2020

Abstract

Hydrogen-rich water is conventionally prepared by direct current-electrolysis but has been not or scarcely prepared by alternating current (AC)--electrolysis. The AC preparations from tap water for 20–30 minutes exhibited a dissolved hydrogen concentration of 1.55 mg/L, which was close to the theoretical maximum value of 1.6 mg/L. These preparations also displayed an oxidation-reduction potential of –270 mV (tap water: +576 mV) and pH of 7.7–7.8, being closer to physiological values of body fluids than general types of direct current-electrolytic hydrogen-rich water. We examined whether AC-electrolytic hydrogen-water is retained for hydrogen-abundance after boiling or for antioxidant abilities, and whether the oral administration of this water is clinically effective for diabetes and prevention against systemic DNA-oxidative injuries. 5,5-Dimethyl-1-pyrroline-N-oxide spin trapping and electron spin resonance revealed that the hydrogen-rich water generated by AC-electrolysis exhibited hydroxyl-radical-scavenging activities. Laser nanoparticle tracking method revealed that nanoparticle suspensions as abundant as 5.4 × 10 ⁷ /mL were efficiently retained (up to 3.5 × 10 ⁷ /mL) even after boiling for 10 minutes, being thermodynamically contrary to Henry's law. Oral intake of hydrogen-rich water, 1500 mL per day, lasted for 8 weeks in nine people with the diabetes-related serum markers beyond the normal ranges. The subjects exhibited significant tendencies for the decreased fasting blood glucose and fructosamine, and for the increased 1,5-anhydro-D-glucitol, concomitantly with significant decreases in urinary 8-hydroxy-2-deoxyguanosine contents and its rate of generation. Hydrogen-rich water prepared by AC-electrolysis may be effective in improving diverse diabetes-related markers and systemic DNA oxidative injuries through the formation of abundant heat-resistant nanobubbles and the increased hydrogen concentrations. The study protocol was officially approved by the Medical Ethics Committee of the Japanese Center for Anti-Aging Medical Sciences (approval No. 01S02) on September 15, 2009.

Keywords: alternating current-electrolysis, antioxidant activity, diabetes, DNA-oxidative injuries, hydrogen-rich water, reactive oxygen species.

Introduction

Recently reported health benefits of hydrogen-rich water include skin beauty and prevention of metabolic syndrome. 1 , 2 , 3 , 4 Many lifestyle-related chronic diseases are primarily caused or exacerbated by reactive oxygen species (ROS), which can be efficiently scavenged by hydrogen-rich water with the ability to permeate deeply into human tissues. 5 , 6 , 7 Hydrogen-rich water is expected to have preventive or therapeutic effects on oxidative stress-related diseases, such as diabetes, cancer, arteriosclerosis, hypertension, apoplexy, and aging. 8 , 9 , 10 , 11 , 12 , 13 Over the past few years, several studies in Japan have reported on hydrogen-water-manufacturing equipment. 14 , 15 , 16

Hydrogen-rich water can be prepared using diverse methods such as electrolysis by alternating current (AC) or direct current (DC), treatment with a magnetic field, bubbling of hydrogen gas in water, and treatment with certain minerals or rocks. 17 , 18 The present study examined the use of AC electrolysis delivered by the “Hayakawa Method,” a patented method approved by the Japan Patent Office (patient No. 2,615,308 and 2,623,204) and United States Patent Office (patient No. 5,435,894) ( Figure 1A ). The apparatus is composed of three electrodes: a grounding electrode and a pair of conventional electrodes, which generates the reduced hydrogen bubble rich water in the vicinity ( Figure 1B ). The first and second high-frequency switches are connected to DC voltage sources through a variable resistor. The electrolysis apparatus delivers high-frequency AC-electrolysis at an interchange cycle of 30 kHz. The level of the signal output from the high-frequency oscillator to the high-frequency switching commander circuit should preferably be within a range of 20 to 50 kHz and 10 to 50 V, from the viewpoint of safety. A specified frequency and voltage were selected within these ranges, in response to the quality of water to be treated. The process and apparatus are suitable for improving the taste of drinking water such as tap water, well water, and commercially available water. The success of the method runs counter to the notion that water cannot be electrolyzed by AC. The purpose of this study is to explore whether AC-electrolytic hydrogen-rich water is retained for hydrogen-abundance after boiling or for antioxidant abilities, and whether the oral administration of this water is clinically effective for diabetes and prevention against systemic DNA-oxidative injuries.

Preparation and storage of hydrogen-rich water

Hydrogen-rich water was prepared using the AQUOLIA SWM300 AC-electrolysis apparatus (Silver Seiko Co. Ltd., Tokyo, Japan). Diverse water parameters were measured as follows: dissolved hydrogen (DH) concentrations were determined using an ENH-1000 DH meter (TRUSTLEX Co. Ltd., Tokyo, Japan). Oxidation-reduction potentials (ORP) were measured using a model 9300-10D ORP meter (HORIBA Ltd., Kyoto, Japan). Dissolved oxygen (DO) concentrations were determined using a model 9520-10D DO meter (HORIBA Ltd.), and pH was measured using a model 9620-10D pH meter (HORIBA Ltd., Kyoto, Japan).

The AC-electrolytically prepared hydrogen-rich water was sealed in a screw-cap-attached aluminum-made bottle with removal of gapped internal air, and left overnight below 25°C without vibration, resulting in the retention over 99% of DH. The DH values were kept over 98% at 4°C for more than 30 days, demonstrating the long-term stability of this water.

Characterization of hydrogen-rich water

Analysis for trace-metal elements in hydrogen-rich water was performed by inductively coupled plasma-mass spectrometry. Hydrogen-rich water was prepared by a 30-minute electrolysis of tap water that had been left overnight. Trace-metal elemental analysis was done using a model ICPMS-2030 (Shimadzu Corporation, Kyoto, Japan).

Nano-particle distributions were estimated three times by nanoparticle tracking analysis with a NanoSight apparatus type-LM10V-HS (Malvern Co. Ltd., Salisburg, UK) equipped with a CMOS camera under irradiation with a violet-colored laser beam (405 nm, < 60 mW), where polystyrene latex particles at 100 nm-diameter were used as the standard particles.

The sample solution designed for examination of its ROS-scavenging ability was mixed with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) reagent (Labotec Co., Tokyo, Japan), and was immediately poured in a flat quartz cell, which was, after 2 minutes, set in the electron spin resonance (ESR) apparatus (FR-30, JEOL Ltd., Tokyo, Japan), and then ESR measurement was started and evaluated as the relative signal intensity of DMPO adducts as compared to the internal standard Manganese(II) oxide (MnO).

Effect of oral administration of hydrogen-rich water on diabetes-related markers

Ten Japanese adults who provided the informed consent were selected based on their fasting blood glucose (FBG) levels that exceeded 6.1 mM and was below approximately 7.8 mM. FBG levels greater than 7.0 mM were considered to be beyond the regular values. The subjects were not taking any medicines that could affect FBS levels, such as hypoglycemic drugs. One person dropped out for clinical reasons, resulting in a total of nine persons finally enrolled in the study (five males and four females aged between 39 and 63 years, with an average age of 52.1 years). The subjects were orally administered 1500 mL of hydrogen-rich water per day for 8 weeks. The water was freshly prepared by AC-electrolysis of tap water left overnight. The experiment was performed as an open clinic test at the Surugadai Clinic in Tokyo (n = 4) and the Yokohama Minoru Clinic (n = 6) in Yokohama. The research was officially approved by the Medical Ethics Committee of the Japanese Center for Anti-Aging Medical Sciences (approval No. 01S02) on September 15, 2009 ( Additional file 1 ), a non-profit organization authenticated by the Hiroshima Prefecture Government of Japan. The approval specified that the research would be noninvasive (such as lack of hemorrhage), painless, with no physical trace/sign, and would be executed using a commercially available apparatus.

Estimation of diabetes-related clinical examination parameters

A glucose oxidase/horseradish peroxidase-colorimetric method 19 for fetal bovine serum was done using a serum glucose test kit (Cosmo Bio Co. Ltd., Tokyo, Japan). 1,5-Anhydro-D-glucitol (1,5-AG) was measured using a 1,5-anhydroglucitol test kit based on adenosine diphosphate-dependent hexokinase (Determiner L; Kyowa Medix Co. Ltd., Tokyo, Japan). Fructosamine was measured using a modified-nitro blue tetrazolium colorimetric method incorporating K-assay reagent (Funakoshi Co. Ltd., Tokyo, Japan). Hemoglobin A1c (HbA1c) was measured using an International Federation of Clinical Chemistry method 20 that utilized a glycohemoglobin A1c kit with an anti-human-HbA1c polyclonal antibody (Wako Pure Chemical Co. Ltd., Osaka, Japan). An enzyme-linked immunosorbent assay for urinary 8-hydroxydeoxyguanosine (8-OHdG) utilized a high-sensitivity check kit (Nikken Seil Co. Ltd., Shizuoka, Japan). A picric acid-colorimetric method for creatinine utilized a LabAssay kit (Wako Pure Chemicals, Osaka, Japan). Uric acid was measured using a 2, 4, 6-tripyridyl-s-triazine based colorimetric method 21 (BioAssay System kit; Funakoshi Co. Ltd.).

Clinical examination for blood and urine

The subjects periodically underwent diverse clinical examinations before examination, and 4 and 8 weeks after intervention. Blood biochemical parameters that were measured including triglyceride, total cholesterol, high-density lipoprotein cholesterol, total phosphorus, albumin, total bilirubin, direct bilirubin, glutamic-oxaloacetic transaminase, glutamic pyruvic transaminase, gamma-glutamyltransferase, alkaline phosphatase, lactate dehydrogenase, lactic acid, pyruvic acid, Na, K, Cl, Cr, blood urea nitrogen, uric acid, FBG, fructosamine, HbA1c, 1,5-AG, total ketone body, 3-hydroxybutyric acid, and acetoacetic acid. Urine biochemical parameters that were measured included proteins, glucose, ketone body, urobilinogen, and 8-OHdG. Physical parameters that were included systolic blood pressure, diastolic blood pressure, pulse, and body weight.

Safety for clinical use of hydrogen-rich water

The safety of orally administered hydrogen-rich water was assessed by clinical examination tests, such as physical, blood and urine biochemical tests. Safety was indicated by the absence of abnormalities or harmful events, with the exception of the pre-existing diabetes-related symptoms. This procedure was conducted for all nine out of ten subjects who participated. No problems were evident, except for one person who showed marked alterations in the degree of exercise and amount of food consumed. No abnormality was observed for the averages and standard deviations of the urinary 8-OHdG values, together with baseline values, for urine qualitative tests.

Statistical analyses

All the data were processed by the statistical significance analysis using the SPSS program, Ver. 11.0 (SPSS, Chicago, IL, USA) or the Wilcoxon signed-rank test, and were typically expressed as the converged average values or with probability-values, which were obtained from the experiments repeated for three to four-times. In tests for examinations of the two-sided hypothesis, the statistical significance and the statistically significant tendency were indicated at P ≤ 0.01 and 0.01 < P ≤ 0.05, respectively. Quantitative variables were expressed by estimating statistical values that included the number of subjects or experimental frequencies (number), mean, for all the clinical examinations such as urinary 8-OHdG, FBG, 1,5-AG, HbA1c and fructosamine, and standard deviation (SD), for urinary 8-OHdG, 8-OHdG/creatinine and 8-OHdG forming speed indicating the P values. The qualitative variables were described using frequency tables. The baselines for the test variables were defined as initial values observed before intake of hydrogen-rich water. As indicators for effectiveness of the estimation, descriptive statistic values including the subject number, average and SD were expressed. The changes in clinical examination parameters before and after intake of hydrogen-rich water were evaluated using the Wilcoxon signed-rank test.

Parameters of AC-electrolyzed hydrogen-rich water

The water-characteristic parameters of hydrogen-rich water produced by AC-electrolysis of tap water according to the Hayakawa Method ( Figure 1 ) were analyzed. DH rose to 1.55 mg/L after 30 minutes of electrolysis, representing an approximately 20-fold increase from the DH value of 0.08 mg/L prior to electrolysis ( Figure 2A ). The value following electrolysis was very close to the maximum DH value of 1.6 mg/mL that is attained under normal temperature and pressure. The ORP decreased from +560 mV at the start of electrolysis to –270 mV after 30 minutes of electrolysis, which indicated the intensified reducing power ( Figure 2B ). The DO value rose to 14.6 mg/L for 30 minutes of electrolysis, and was approximately 1.5-fold higher than the initial DO value of 9.36 mg/L ( Figure 2C ), indicating that aerobic water quality can promote intracellular aerobic metabolism. The pH changed within a narrow range of approximately 7.7 to 7.8 ( Figure 2D ), which was physiologically compatible with the human body, in contrast to pH values higher than 8 that have been observed for most of other types of DC-electrolytic hydrogen-rich water.

Trace-metal content in the AC-electrolyzed hydrogen-rich water

The trace-metal elements in the AC-electrolyzed hydrogen-rich water from tap water that had been left overnight were analyzed by inductively coupled plasma-mass spectrometry ( Figure 3 ). The zinc (Zn) content increased to 1.37 μg/L. The level of Zn is non-toxic and is typical of beneficial trace-elements. Surplus Zn is typically secreted into the pancreatic liquid. 22 Titanium (Ti) was increased to 2.20 μg/L. Ti is a biocompatible element that is sufficiently corrosion-resistant to be utilized for diverse artificial organs that are biologically safe. 23 Platinum (Pt) was absent before electrolysis, but became detectable at a concentration of 0.51 μg/L, suggesting the elution of Pt ions from the electrode surface into the electrolysis fluid. Pt colloid, which is commercially available as an antioxidant oral supplement, prolonged the generation of hydrogen bubbles via the formation of a Pt-hydrogen complex. The iron (Fe) content increased to 3.66 μg/L, which was much lower than the permissible amount of 45 mg/d according to the Dietary Reference Intake indices of the American Science Academy. 24

Nanobubbles and nanoparticles in the AC-electrolyzed hydrogen-rich water

Nanobubbles generated after AC-interchange electrolysis by the Hayakawa Method were estimated by nanoparticle tracking analysis, where Brownian movement of the nanoparticles was visualized using a laser beam, and the number and diameter of nanoparticles as well as the particle size distribution were evaluated ( Figure 4A ). The total number of nanoparticles was determined to be 5.4 × 10 ⁷ /mL ( Figure 4B ). Even after boiling, 3.5 × 10 ⁷ /mL nanoparticles were retained ( Figure 4C ), representing a retention rate of 64.8%. This rate is unexpectedly high as normal-size bubbles, but is not applicable to nanobubble gas, since Henry's rule dictates that gas-solubility is lower at higher temperatures. 25

In the Hayakawa Method, electrolysis with an AC of 5 kHz is required with an oscillation (plus-minus) of 5000 times per second with hydrogen molecules gas (bubble: H ₂ ) of the nano-unit. The method with 30,000 interchanges times with the three electrodes (including the ground electrode) yielded a large quantity of hydrogen gas nanobubbles for AC-electrolysis at 30 kHz. The nanobubbles in water remained in the bulk space. They did not float up or sediment, and were retained for a long time without the fusion of mutual bubbles due to electrostatic repulsion among the negative-charges on the bubble-surface. These nanobubbles persisted for a few days.

Hydrogen molecules of nanobubbles can easily penetrate the cell membrane or skin corneum, and can reach the intracellular space and diverse organs. At these destinations, it can be converted to a harmless water molecule after removal of ROS.

Physiological benefits of AC-electrolyzed hydrogen-rich water

Hydrogen-rich water prepared by 30 minutes of AC-electrolysis was shown to appreciably scavenge superoxide anion radicals (•O ₂ ^– ) generated by hypoxanthine-xanthine oxidase enzymatic reaction, according to DMPO spin trapping and ESR). Hydroxyl radicals (•OH) generated by the Fenton reaction were markedly scavenged by the hydrogen-rich water, as also determined using DMPO-spin trapping/ESR ( Figure 5 ).

Effect of AC-electrolyzed hydrogen-rich water on diabetes-related serum markers

8-OHdG is an established urine marker for DNA oxidative injury. 26 8-OHdG was decreased significantly after the 8-week daily consumption of hydrogen-rich water (P = 0.028), suggesting the systemic decrease of •OH by hydrogen absorbed into human body fluid ( Figure 6A ). The 8-OHdG/creatinine values were unchanged after 4 weeks and were decreased after 8 weeks ( Figure 6B ). The rate of 8-OHdG generation decreased from 4.2 ng/kg per hour before intake to 2.5 ng/kg per hour after 8 weeks ( Figure 6C ). The difference was not statistically significant.

FBG levels were evaluated for the nine diabetic patients who consumed the hydrogen-rich water (1500 mL per day for 8 weeks) prepared by AC-electrolysis of tap water. FBG levels decreased significantly (P = 0.051) after the 8-week regimen ( Figure 6D ).

1,5-AG is a marker reflecting the average concentrations of blood glucose. 27 The 1,5-AG value of 9.0 μg/mL prior to intake of hydrogen-rich water was increased slightly to 9.4 μg/mL at 4 weeks and 9.5 μg/mL at 8 weeks ( Figure 6E ). Although the slight increases in 1,5-AG were not statistically significant, the desired blood level of 1,5-AG as an indicator for glucose reabsorption in the kidney should normally exceed 12 µg/mL, whereas 1,5-AG in blood is known to be 10 µg/mL in case of the past blood glucose of 10.3 mM, being doubted for diabetes. More than 99% of blood 1,5-AG is reabsorbed in the kidney. However, the reabsorption of 1,5-AG, a structural analogue to glucose, is competitively inhibited by secreted urinary glucose, because of the abundant blood glucose. This phenomenon is an extremely fast response to urinary glucose secretion, enabling it to estimate blood glucose concentration within several days. 28

HbA1c levels tended to decline after 4 weeks of intake, but were unchanged from the baseline after 8 weeks. The findings indicate the necessity for long-term intake (more than 8 weeks) because HbA1c values reflect the average concentrations of blood glucose for the past 1 to 2 months and taking into consideration that the initial HbA1c values were up to 6.4% ( Figure 6F ).

ROS generation has been implicated in the development and progression of diabetes, since saccharified proteins, such as HbA1c, increase proportionately with ROS generation. 12 Diabetic patients with higher FBG levels were orally administered hydrogen-rich water for 8 weeks, and examined for temporal changes in serum levels of FBG, HbA1c, fructosamine, 1,5-AG, and uric acid, and urinary levels of 8-OHdG, 8-OHdG/creatinine and 8-OHdG formation rate. The levels of fructosamine, a marker for serum glucose over a period of 1 to 2 weeks, was significantly decreased (P = 0.008) after 4 weeks of intake ( Figure 6G ).

Statistically significant changes were evident for platelet, total phosphorus, albumin, total bilirubin, aspartate transaminase and gamma-glutamyltransferase. Statistically significant tendency was observed for direct bilirubin, lactic acid, pyruvic acid and Na. These values were within the margin of deviation from baseline values, before and after the intake test, and did not exceed the levels indicative of abnormal changes, suggesting the safety of the 8-week regimen of 1500 mL/d of the AC-electrolytic hydrogen-rich water.

Discussion

The hydrogen-rich water prepared by the Hayakawa Method displayed near-neutral pH that is compatible for human use; low ORP (–270 mV) indicative of intensified reducing power; DH of approximately 1.55 mg/L (which is very similar to the theoretical maximum DH of 1.6 mg/L under normal temperature and pressure conditions); low surface-tension and high osmotic capability indicative of easy absorption into cells; binding of hydrogen molecules to Pt nano-colloid that is of sizes for nanometers in a diameter, facilitating the dissociation to the atomic hydrogen; scavenging of ROS, particularly •OH; and an appropriate balance of positive and negative mineral ions.

Harmful trace-metal elements were not generated at detectable levels by AC-electrolysis. The material of the applying electrodes was selected by taking into consideration the quality of treated water. Zn, lithium oxide, magnesium (Mg) alloys (with Zn), copper, Fe, stainless steel and Ti are candidate materials for the electrodes. Of these, Zn and Mg alloys are preferable for obtaining the drinking water. Electrodes composed of Zn and Mg gradually dissolve from the surface to enrich the water. Of the diverse types of ROS, superoxide anion radicals (•O ₂ ^– ) and hydrogen peroxide can be removed by intracellular enzymes, such as superoxide dismutase and catalase/glutathione-peroxidase, respectively. In contrast, •OH cannot be scavenged by any human-endogenous enzymes, resulting in injury to cellular DNA and the plasma membrane. 29 , 30 Hydrogen-rich water prepared by AC-electrolysis, apart from the conventional DC-electrolysis, scavenges •OH and possibly protects cells against oxidative injury. In contrast, DNA cannot be directly protected from attack by •OH, resulting in elimination of some 8-OHdG fragments from the body in the urine. The relative amounts of 8-OHdG are an appropriate marker for the extent of cellular injury. Repression of urinary 8-OHdG by hydrogen-rich water that was prepared by AC-electrolysis, suggests that cells can be protected against attack from •OH through cellular conversion of the oxidative state to the reductive state, by means of the antioxidant ability of hydrogen bubbles.

The development and progression of diabetes may be correlated to ROS and peroxylipid, especially in terms of metabolic activities of glucose-taking tissues and insulin-secreting ability of the pancreatic langerhans islet β-cells. Peroxylipid values in serum are linked with the progression of diabetes, whereas blood glucose values are improved by the intake of antioxidant foods having an superoxide dismutase-like action. Furthermore, participation of ROS in diabetes may also be possible because ROS is generated from some glycoproteins, for example, HbA1c which increase as diabetes develops and progresses. 31

During hyperglycemia, the glucose oxidative metabolism and the promoted metabolism of arachidonic acid may generate ROS, such as superoxide anion radicals (•O ₂ ^– ) and •OH, which injure endotheliocytes and lead to the accumulation of oxidative low-density lipoprotein, which in turn promotes the progression of diabetes and the diverse complications associated with the disease. 32

As intrinsic improvement for diabetes, glucose transporter-4, which is located in the endoplasmic reticulum membrane, may be translocated into the cell membrane, and subsequently functions in the intracellular intake of extracellular glucose. Glucose transporter-4 location requires a higher pH of as little as slight as 0.2 pH unit in the extracellular liquid, which occurs by the activation of the Na ⁺ /H ⁺ channel, due to the increased abundance of hydrogen bubbles in the intracellular liquid, in addition to insulin. The translocation of glucose transporter-4 from the endoplasmic reticulum to the cell membrane is disturbed even if insulin exists when the Na ⁺ /H ⁺ channel is perturbed. 33

The present study explored the effects of hydrogen-rich water intake on the FBG levels of the nine participants with relatively high blood glucose levels. The subjects drank 1500 mL of hydrogen-rich water each day for 8 weeks. HbA1c levels, fructosamine levels, 1,5-AG values and FBG levels were measured to gauge their glycemic control. The findings are presented in Figure 7 . Chronologically, the glucose status was first evaluated with HbA1c reflecting blood glucose values for one to two months before the examination, then with fructosamine levels 2 weeks before, with 1,5-AG values a few days before, and finally with the FBG value at the time of examination.

In diabetes, abundant glucose in the blood has been correlated to higher glucose concentrations in erythrocytes, 12 suggesting the possibility for the decreased metabolism of glucose. In contrast, active consumption of glucose might be restored, as shown presently by the decreases in FBG and fructosamine during the 8-week daily consumption of hydrogen-rich water prepared by AC-electrolysis.

As concerned with a limitation range for reliability to the present study, diverse material- or antioxidant-properties of AC-electrolytically prepared hydrogen-water were thoroughly elucidated in terms of water-property parameters such as DH, DO, ORP and pH, ROS-scavenging abilities and particle-size distributions. In contrast, from viewpoints of hydrogen-intervening clinic examination for diabetes, the diverse time-series diabetes-related indicators, such as FBS for the current time, 1,5-AG for several days before, fructosamine for 2 weeks before and HbA1c for 2 months before, were examined in the present study, indicating the data reliability that was comprehensively attributed to the systemically chronological sequence. Based on these results, hydrogen-rich water prepared by AC-electrolysis may be effective in improving diverse diabetes-related markers and systemic DNA oxidative injuries through the formation of abundant heat-resistant nanobubbles and the increased hydrogen concentrations.

Financial support

The study was supported in part by a Grant-in-Aid #0204 for AntiAging Medical Sciences from Japanese Center for AntiAging MedSciences, Hiroshima, Japan (to RA).

Institutional review board statement

This study was approved by the Medical Ethics Committee of the Japanese Center for Anti-Aging Medical Sciences (approval No. 01S02) on September 15, 2009.

Declaration of patient consent

The authors certify that they have obtained all patient consent forms. In the form, patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published.

Biostatistics statement

The statistical methods of this study were reviewed by the epidemiologist of Osaka Prefecture University, Japan.

Copyright license agreement

The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement

Personal names of subjects or personal specific information will not be announced even upon presentation of the research results. Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check:

Checked twice by iThenticate.

Peer review:

Externally peer reviewed.

Funding:

The study was supported in part by a Grant-in-Aid #0204 for AntiAging Medical Sciences from Japanese Center for AntiAging MedSciences, Hiroshima, Japan (to RA).

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