title: Carboxylesterase converts Amplex red to resorufin: Implications for mitochondrial H2O2 release assays.
abstract: Amplex Red is a fluorescent probe that is widely used to detect hydrogen peroxide (H2O2) in a reaction where it is oxidised to resorufin by horseradish peroxidase (HRP) as a catalyst. This assay is highly rated amongst other similar probes thanks to its superior sensitivity and stability. However, we report here that Amplex Red is readily converted to resorufin by a carboxylesterase without requiring H2O2, horseradish peroxidase or oxygen: this reaction is seen in various tissue samples such as liver and kidney as well as in cultured cells, causing a serious distortion of H2O2 measurements. The reaction can be inhibited by Phenylmethyl sulfonyl fluoride (PMSF) at concentrations which do not disturb mitochondrial function nor the ability of the Amplex Red-HRP system to detect H2O2.In vitro experiments and in silico docking simulations indicate that carboxylesterases 1 and 2 recognise Amplex Red with the same kinetics as carboxylesterase-containing mitochondria. We propose two different approaches to correct for this problem and re-evaluate the commonly performed experimental procedure for the detection of H2O2 release from isolated liver mitochondria. Our results call for a serious re-examination of previous data.
authors.affiliation: Institute for Cell & Molecular Biosciences and Newcastle University Institute for Ageing, Ageing Research Laboratories, Campus for Ageing and Vitality, Newcastle University, Newcastle upon Tyne NE4 5PL, UK.
authors.forename: Satomi
authors.initials: S
authors.lastname: Miwa
authors.affiliation: Newcastle University Protein and Proteome Analysis, Devonshire Building, Devonshire Terrace, Newcastle upon Tyne NE1 7RU, UK.
authors.forename: Achim
authors.initials: A
authors.lastname: Treumann
authors.affiliation: Institute for Cell & Molecular Biosciences and Newcastle University Institute for Ageing, Ageing Research Laboratories, Campus for Ageing and Vitality, Newcastle University, Newcastle upon Tyne NE4 5PL, UK.
authors.forename: Amy
authors.initials: A
authors.lastname: Bell
authors.affiliation: Department of Pharmaceutical Sciences, University of Milan, via Mangiagalli, 25, I-20133 Milan, Italy.
authors.forename: Giulio
authors.initials: G
authors.lastname: Vistoli
authors.affiliation: Institute for Cell & Molecular Biosciences and Newcastle University Institute for Ageing, Ageing Research Laboratories, Campus for Ageing and Vitality, Newcastle University, Newcastle upon Tyne NE4 5PL, UK; Bioimaging Unit, Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
authors.forename: Glyn
authors.initials: G
authors.lastname: Nelson
authors.affiliation: Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: Sam
authors.initials: S
authors.lastname: Hay
authors.affiliation: Institute for Cell & Molecular Biosciences and Newcastle University Institute for Ageing, Ageing Research Laboratories, Campus for Ageing and Vitality, Newcastle University, Newcastle upon Tyne NE4 5PL, UK. Electronic address: t.vonzglinicki@ncl.ac.uk.
authors.forename: Thomas
authors.initials: T
authors.lastname: von Zglinicki
mesh_terms: D000818:Animals; D043182:Carboxylesterase / Q000378:metabolism*; D002384:Catalysis; D006735:Horseradish Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008930:Mitochondria, Liver / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology
chemical_list: D010078:Oxazines; C470430:Amplex Red; D010664:Phenylmethylsulfonyl Fluoride; C014180:resorufin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D043182:Carboxylesterase
keywords: Amplex Red; Carboxylesterase; Liver; Measurement; Mitochondria; Reactive oxygen species