Assessment of Myeloperoxidase Activity by the Conversion of Hydroethidine to 2-Chloroethidium [Molecular Bases of Disease]

January 16th, 2014 by Maghzal, G. J., Cergol, K. M., Shengule, S. R., Suarna, C., Newington, D., Kettle, A. J., Payne, R. J., Stocker, R.

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, 1H NMR and 2D 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 x 105 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 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 concentration (17 versus 0.3 pmol/mg protein) and 3-Cl-Tyr could barely be detected. Similar to the situation with 3-Cl-Tyr, tissue levels of 2-Cl-E+ were decreased substantially in Mpo−/−, 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.