title: Measurement of Superoxide Production and NADPH Oxidase Activity by HPLC Analysis of Dihydroethidium Oxidation.
abstract: The fluorogenic probe dihydroethidium (DHE) is widely used for detecting intracellular superoxide. DHE oxidation by superoxide generates specifically the compound 2-hydroxyethidium (2-E+OH), so that 2-E+OH detection confers specificity to superoxide assessment among many other reactive oxygen species. However, DHE oxidation in biological systems leads to formation of other fluorescent products, particularly ethidium, usually formed at higher quantities than 2-E+OH. Since both 2-E+OH and ethidium are fluorescent, their identification and quantification is possible only after their physical separation by HPLC. Here we describe the detailed procedures for superoxide measurement in cells (adhered or not) and fresh tissues fragments, followed by acetonitrile extraction and simultaneous fluorescent detection of 2-E+OH and ethidium and absorbance detection of remaining unreacted DHE. In addition we report the use of DHE/HPLC for measuring NADPH oxidase activity in enriched-membrane fraction isolated from cells or tissues. These methods can improve accuracy and precision of quantitative superoxide measurements in biological samples.
authors.affiliation: Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine, Av. Eneas Carvalho Aguiar, 44, Annex II, 9th Floor, CEP 05403-000, São Paulo, Brazil. denisef@usp.br.
authors.forename: Denise C
authors.initials: DC
authors.lastname: Fernandes
authors.affiliation: Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine, Av. Eneas Carvalho Aguiar, 44, Annex II, 9th Floor, CEP 05403-000, São Paulo, Brazil.
authors.forename: Renata C
authors.initials: RC
authors.lastname: Gonçalves
authors.affiliation: Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine, Av. Eneas Carvalho Aguiar, 44, Annex II, 9th Floor, CEP 05403-000, São Paulo, Brazil.
authors.forename: Francisco R M
authors.initials: FR
authors.lastname: Laurindo
mesh_terms: D000097:Acetonitriles / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004996:Ethidium / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D019255:NADPH Oxidases / Q000378:metabolism*; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
chemical_list: C506392:2-hydroxyethidium; D000097:Acetonitriles; D017382:Reactive Oxygen Species; C067883:dihydroethidium; D013481:Superoxides; D019255:NADPH Oxidases; D004996:Ethidium; C032159:acetonitrile
keywords: 2-hydroxyethidium; Dihydroethidium; Ethidium; Fluorescence; HPLC; Hydroethidine; NADPH oxidase; Oxidants; Reactive species; Superoxide