An improved method for the detection of myeloperoxidase chlorinating activity in biological systems using the redox probe hydroethidine

Conversion of the redox probe hydroethidine (HE) to 2-chloroethidium (2-Cl-E+) by myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) provides comparable specificity and superior sensitivity to measurement of 3-chlorotyrosine (3-Cl-Tyr), the gold standard biomarker for MPO chlorinating activity in biological systems. However, a limitation of the former method is the complex mixture of products formed by the reaction of HE with reagent HOCl, coupled with the difficult purification of 2-Cl-E+ from this mixture for analytical purposes. This limitation prompted us to test whether 2-Cl-E+ could be formed by reaction of HE with the strong and widely used chlorinating agent, N-chlorosuccinimide (NCS). Unexpectedly, such reaction yielded 2-chlorohy-droethidine (2-Cl-HE) as the major product in addition to 2-Cl-E+, as assessed by high performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR). 2-Cl-HE was also observed to be the major chlorination product formed from HE with both reagent and enzymatically generated HOCl, just as it was formed ex vivo in different healthy and diseased mouse and human tissues upon incubation with glucose/glucose oxidase to generate a flux of hydrogen peroxide (H2O2). Quantification of 2-Cl-HE plus 2-Cl-E+ improved the sensitivity of the HE-based method compared with measurement of only 2-Cl-E+. Moreover, 2-chlorodimidium (2-Cl-D+) was developed as a practical internal standard instead of the previously used in-ternal standard, deuterated 2-Cl-E+ (d5-2-Cl-E+). Overall, the present study describes an improved method for the detection of MPO/chlorinating activity in biological systems of health and disease.