title: Detailed protocol to assess in vivo and ex vivo myeloperoxidase activity in mouse models of vascular inflammation and disease using hydroethidine.
abstract: Myeloperoxidase (MPO) activity contributes to arterial inflammation, vascular dysfunction and disease, including atherosclerosis. Current assessment of MPO activity in biological systems in vivo utilizes 3-chlorotyrosine (3-Cl-Tyr) as a biomarker of hypochlorous acid (HOCl) and other chlorinating species. However, 3-Cl-Tyr is formed in low yield and is subject to further metabolism. Recently, we reported a method to selectively assess MPO-activity in vivo by measuring the conversion of hydroethidine to 2-chloroethidium (2-Cl-E(+)) by liquid chromatography with tandem mass spectrometry (LC-MS/MS) (J. Biol. Chem., 289, 2014, pp. 5580-5595). The hydroethidine-based method has greater sensitivity for MPO activity than measurement of 3-Cl-Tyr. The current methods paper provides a detailed protocol to determine in vivo and ex vivo MPO activity in arteries from mouse models of vascular inflammation and disease by utilizing the conversion of hydroethidine to 2-Cl-E(+). Procedures for the synthesis of standards, preparation of tissue homogenates and the generation of 2-Cl-E(+) are also provided in detail, as are the conditions for LC-MS/MS detection of 2-Cl-E(+).
authors.affiliation: Vascular Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia.
authors.forename: Jihan
authors.initials: J
authors.lastname: Talib
authors.affiliation: Vascular Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia.
authors.forename: Ghassan J
authors.initials: GJ
authors.lastname: Maghzal
authors.affiliation: Vascular Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Cheng
authors.affiliation: Vascular Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia; School of Medical Sciences, University of New South Wales, Sydney, Australia. Electronic address: r.stocker@victorchang.edu.au.
authors.forename: Roland
authors.initials: R
authors.lastname: Stocker
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D002853:Chromatography, Liquid; D004195:Disease Models, Animal; D054879:Halogenation; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006997:Hypochlorous Acid / Q000378:metabolism; D007249:Inflammation / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000235:genetics*; D010617:Phenanthridines / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000031:analogs & derivatives / Q000302:isolation & purification / Q000378:metabolism; D014652:Vascular Diseases / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
chemical_list: D015415:Biomarkers; D010617:Phenanthridines; C058428:hydroethidine; D014443:Tyrosine; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide; D009195:Peroxidase; C087259:3-chlorotyrosine
keywords: 3-Chlorotyrosine; Arteries; Atherosclerosis; Biomarker; Hydroethidine; Hypochlorous acid; Inflammation; Mass spectrometry; Myeloperoxidase