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

作者：Talib Jihan; Maghzal Ghassan J; Cheng David; Stocker Roland^*

来源：Free Radical Biology and Medicine, 2016, 97: 124-135.

DOI：10.1016/j.freeradbiomed.2016.05.004

• 3-Chlorotyrosine
• Myeloperoxidase
• Mass spectrometry
• Hypochlorous acid
• Hydroethidine
• Arteries
• Inflammation
• Atherosclerosis
• Biomarker

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-CI-Tyr) as a biomarker of hypochlorous acid (HOCI) 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-CI-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-CI-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-CI-E+ Procedures for the synthesis of standards, preparation of tissue homogenates and the generation of are also provided in detail, as are the conditions for LC-MS/MS detection of 2-CI-E+.

• 出版日期2016-8