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

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 exhibit 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 × 10⁷/mL were efficiently retained (up to 3.5 × 10⁷/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: DNA-oxidative injuries; alternating current-electrolysis; antioxidant activity; diabetes; hydrogen-rich water; reactive oxygen species.

Figure 1

The principle of alternating current (AC) electrolysis for preparation of hydrogen-rich water. Note: (A) Generation of nanobubbles by AC electrolysis. The method used the Hayakawa Method (Patent No. for the Japan Patent Office: 5,435,894 2,615,308, 2,623,204). The high frequency switching circuit was selected within a range of 20–50 kHz and 10–50 V. (B) Polarization of water molecules and migration of protons towards the cathodal electrode interchanged at a cycle of 30 kHz in an AC-electrolysis tub.

Figure 2

Time courses in changes of various water-property parameters. Note: (A–D) The time course of DH (A), ORP (B), DO (C), and pH (D) values in hydrogen-rich water generated by alternating current-electrolysis of tap water. The pH values were stable between 7.7 and 7.9. Data were expressed as the means for three measurements. DH: Dissolved hydrogen; DO: dissolved oxygen; ORP: Oxidation-reduction potential.

Figure 3

Trace-metal elements content in hydrogen-rich water generated by alternating current-electrolysis of tap water for 30 minutes, as analyzed by inductively coupled plasma-mass spectrometry method. Note: Data are expressed as the means for three measurements.

Figure 4

Particle-size distributions in alternating current-electrolytically prepared hydrogen-rich water. Note: (A) Visualization of Brownian movement of nanoparticles by nanoparticle tracking analysis (NTA) using a laser beam tracing NanoSight LM20/NTA2.3 apparatus. (B) Size distribution (diameters versus numbers) of nanoparticles in hydrogen-rich water generated by alternating current-electrolysis of tap water for 30 minutes as measured by NTA. (C) Size distribution in hydrogen-rich water generated as in B and subjected to boiling and cooling. Nanoparticles after boiling were decreased to 1.9 × 10⁷/mL, at a rate of 35.2%.

Figure 5

ESR spectra of superoxide anion radicals (•O₂ ^–) and hydroxyl radicals (•OH) upon addition of alternating current-electrolytically prepared hydrogen-rich water. Note: (A, B) An ability of hydrogen-rich water to scavenge superoxide anion radicals, as evaluated by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-spin trapping/electron spin resonance (ESR) method. (A) superoxide anion radicals generated by hypoxanthine-xanthine oxidase enzymatic reaction. (B) superoxide anion radicals immediately after addition of hydrogen-rich water that was freshly prepared by 20-minute AC-electrolysis. (C, D) An ability of hydrogen-rich water to scavenge hydroxyl radicals, as evaluated by DMPO-spin trapping/ESR method. (C) hydroxyl radicals generated by the Fenton reaction. (D) ESR spectrum of hydroxyl radicals immediately after addition of hydrogen-rich water that was freshly prepared by 20-minute AC-electrolysis.

Figure 6

Effect of alternating current-electrolyzed hydrogen-rich water on diabetes-related serum markers in nine subjects with higher blood sugar levels. Note: (A) 8-Hydroxydeoxyguanosine (8-OHdG) level in urine; (B) 8-OHdG/creatinine (Cre) in urine; (C) 8-OHdG formation per hour in urine; (D) fasting blood glucose; (E) 1,5-anhydro-D-glucitol (1,5-AG) in blood; (F) hemoglobin A1c (HbA1c); (G) fructosamine in blood. Data are expressed as mean, and analyzed by Wilcoxon signed-rank test. *P < 0.05.

Figure 7

Time schedule that can predict blood glucose level before measurement by measuring markers related to diabetes. Note: Hemoglobin A1c (HbA1c) levels, fructosamine levels, 1,5-anhydro-D-glucitol (1,5-AG) values, and fasting blood glucose levels were measured in the nine subjects who drank 1500 mL of hydrogen-rich water every day for 8 weeks, to trace their glycemic control.