title: Assessment of myeloperoxidase activity by the conversion of hydroethidine to 2-chloroethidium.
abstract: Oxidants derived from myeloperoxidase (MPO) contribute to inflammatory diseases. In vivo MPO activity is commonly assessed by the accumulation of 3-chlorotyrosine (3-Cl-Tyr), although 3-Cl-Tyr is formed at low yield and is subject to metabolism. Here we show that MPO activity can be assessed using hydroethidine (HE), a probe commonly employed for the detection of superoxide. Using LC/MS/MS, (1)H NMR, and two-dimensional NOESY, we identified 2-chloroethidium (2-Cl-E(+)) as a specific product when HE was exposed to hypochlorous acid (HOCl), chloramines, MPO/H2O2/chloride, and activated human neutrophils. The rate constant for HOCl-mediated conversion of HE to 2-Cl-E(+) was estimated to be 1.5 × 10(5) M(-1)s(-1). To investigate the utility of 2-Cl-E(+) to assess MPO activity in vivo, HE was injected into wild-type and MPO-deficient (Mpo(-/-)) mice with established peritonitis or localized arterial inflammation, and tissue levels of 2-Cl-E(+) and 3-Cl-Tyr were then determined by LC/MS/MS. In wild-type mice, 2-Cl-E(+) and 3-Cl-Tyr were detected readily in the peritonitis model, whereas in the arterial inflammation model 2-Cl-E(+) was present at comparatively lower concentrations (17 versus 0.3 pmol/mg of protein), and 3-Cl-Tyr could not be detected. Similar to the situation with 3-Cl-Tyr, tissue levels of 2-Cl-E(+) were decreased substantially in Mpo(-/-) mice, indicative of the specificity of the assay. In the arterial inflammation model, 2-Cl-E(+) was absent from non-inflamed arteries and blood, suggesting that HE oxidation occurred locally in the inflamed artery. Our data suggest that the conversion of exogenous HE to 2-Cl-E(+) may be a useful selective and sensitive marker for MPO activity in addition to 3-Cl-Tyr.
authors.affiliation: From the 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.forename: Katie M
authors.initials: KM
authors.lastname: Cergol
authors.forename: Sudhir R
authors.initials: SR
authors.lastname: Shengule
authors.forename: Cacang
authors.initials: C
authors.lastname: Suarna
authors.forename: Darren
authors.initials: D
authors.lastname: Newington
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Kettle
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Payne
authors.forename: Roland
authors.initials: R
authors.lastname: Stocker
mesh_terms: D000818:Animals; D001167:Arteritis / Q000201:enzymology / Q000235:genetics; D004195:Disease Models, Animal; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D051379:Mice; D018345:Mice, Knockout; D016877:Oxidants / Q000737:chemistry*; D010538:Peritonitis / Q000201:enzymology / Q000235:genetics; D009195:Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010617:Phenanthridines / Q000737:chemistry*
chemical_list: D016877:Oxidants; D010617:Phenanthridines; C058428:hydroethidine; D006861:Hydrogen Peroxide; D009195:Peroxidase
keywords: 3-Chlorotyrosine; Chloramines; Dihydroethidium; HPLC; Hypochlorous Acid; Inflammation; Mass Spectrometry (MS); Neutrophil; Oxidative Stress