title: Determination of H2O2Generation by pHPA Assay.
abstract: The production of reactive oxygen species, including H2O2, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H2O2release from cells (1-5). This assay takes advantage of H2O2-mediated oxidation of horseradish peroxidase (HRP) to Complex I, which, in turn, oxidizes p-hydroxyphenylacetic acid (pHPA) to a stable, fluorescent pHPA dimer (2,2'-dihydroxy-biphenyl-5,5' diacetate [(pHPA)2]). The H2O2-dependent HRP-mediated oxidation of pHPA is a sensitive and specific assay for quantifying H2O2release from cells. This assay can measure H2O2release from whole cells, mitochondria, or the NADPH oxidase.
authors.affiliation: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA 35294.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Larson-Casey
authors.affiliation: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA 35294; Birmingham Veterans Administration Medical Center, Birmingham, AL, USA 35294.
authors.forename: A Brent
authors.initials: AB
authors.lastname: Carter