Detailed protocol to assess in vivo and ex vivo myeloperoxidase activity in mouse models of vascular inflammation and disease using hydroethidine

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+.