Presence of Hydrogen Peroxide, a Source of Hydroxyl Radicals, in Acid Electrolyzed Water

Takayuki Mokudai,Taro Kanno,Yoshimi Niwano,Keisuke Nakamura,Ching-Hong Yang

doi:10.1371/journal.pone.0046392

Abstract

BackgroundAcid electrolyzed water (AEW), which is produced through the electrolysis of dilute sodium chloride (NaCl) or potassium chloride solution, is used as a disinfectant in various fields because of its potent antimicrobial activity. The hydroxyl radical, an oxygen radical species, is often suggested as a putative active ingredient for AEW antimicrobial activity.Methodology/Principal FindingsThe aim of the present study is to detect hydroxyl radicals in AEW. The hydroxyl radicals in AEW prepared under different conditions were determined using an electron spin resonance (ESR) technique. A signal from 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH, an adduct of DMPO and the hydroxyl radical, was detected in AEW prepared by double or triple electrolyses of 1% NaCl but not of 0.1% NaCl solution. Then the presence of hydrogen peroxide as a proposed source of hydroxyl radicals was examined using a combination of ESR and a Fenton reaction. The DMPO-OH signal was clearly detected, even in AEW prepared by single electrolysis of 0.1% NaCl solution, when ferrous sulfate was added to induce a Fenton reaction, indicating the presence of hydrogen peroxide in the AEW. Since sodium formate, a hydroxyl radical scavenger, did not affect the bactericidal activity of AEW, it is concluded that the radical is unlikely to contribute to the antimicrobial activity of AEW, although a small amount of the radical is produced from hydrogen peroxide. Dimethyl sulfoxide, the other hydroxyl radical scavenger used in the present study, canceled the bactericidal activity of AEW, accompanied by complete depletion of free available chlorine, suggesting that hypochlorous acid is probably a major contributor to the antimicrobial activity.ConclusionsIt is strongly suggested that although hydrogen peroxide is present in AEW as a source of hydroxyl radicals, the antimicrobial activity of AEW does not depend on these radicals.

Highlights

It is strongly suggested that hydrogen peroxide is present in Acid electrolyzed water (AEW) as a source of hydroxyl radicals, the antimicrobial activity of AEW does not depend on these radicals
In a study where an electron spin resonance (ESR) spin-trapping technique was applied to AEW using two spin traps, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and N-[(1-oxido-4-pyridinio)methylene]-t-butylamine N-oxide, the spin adducts of hydroxyl radical were observed when electrolyte was dissolved in tap water but not in pure water [24]
When 1% (w/v) NaCl solution was used as the electrolyte solution, a clear signal from DMPO-OH was detected in the AEW, especially that obtained by triple electrolyses, and the concentration of DMPO-OH was calculated to be 6.2 mM, using 20 mM 4-hydroxy-2,2,6,6-tetramethylpiperidine (TEMPOL) as a standard

Summary

Introduction

Acid electrolyzed water (AEW), which is produced by electrolysis of dilute sodium chloride (NaCl) or potassium chloride solution in the anode side of an instrument where the anode and cathode are separated by an ion-permeable diaphragm, has been used mainly in the agricultural and medical fields as a disinfectant in farm and food hygiene [1,2,3,4,5,6,7,8], and disinfection of medical instruments such as dialyzers [9], patient-used endoscopes [10], and dentures [11] because of the potent antimicrobial potential of AEW [12,13,14,15,16]. The hydroxyl radical, an oxygen radical species, is often suggested as a putative active ingredient for AEW antimicrobial activity [19]. The addition of DMPO to AEW generated an ESR signal from 5,5-dimethyl-2pyrrolidone-N-oxyl (DMPOX) but not ESR signals from DMPOOH (a spin adduct of DMPO and a hydroxyl radical) and DMPOOOH (a spin adduct of DMPO and a superoxide anion), suggesting that hypochlorous acid oxidizes the spin-trap DMPO, with the formation of DMPOX. Acid electrolyzed water (AEW), which is produced through the electrolysis of dilute sodium chloride (NaCl) or potassium chloride solution, is used as a disinfectant in various fields because of its potent antimicrobial activity. The hydroxyl radical, an oxygen radical species, is often suggested as a putative active ingredient for AEW antimicrobial activity

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