A proposed enzyme-linked hydrogen peroxide radical assay system (HORAS)

Reactive Oxygen Species (ROS) are chemically reactive molecules that contain oxygen. ROS are naturally generated as a byproduct during mitochondrial oxidative metabolism as well as by cellular responses to a variety of inflammatory stimuli. Intracellularly formed ROS plays an important role in maintaining homeostasis and in cell signaling but, ROS are challenging to quantify. Phagocytic cells such as macrophages may produce H2O2 during the action of bacterial engulfment. Here UV-Vis versus LC-ESI-MS detection methods for an enzyme-linked, cellular assay of H2O2 production in cultured macrophages are compared. In the presence of Horseradish Peroxidase (HRP), Amplex Red (AR) reacts with H2O2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product resorufin that can be measured by UV/Vis at an absorbance of 570 nm or by LC-ESI-MS at 214 m/z [M+H]+. RAW 264.7 macrophages were stimulated by microscopic foreign particles, with the addition of 0.1mM of Amplex Red substrate and 10 ng/mL of HRP to the cellular media to enzymatically detect H2O2 production. The oxidation product resorufin can be detected by the colorimetric method as low as 50 pmol while liquid chromatography with electrospray ionization and mass spectrometry (LC-ESI-MS) was able to detect as little as 0.2 pmol in vitro. Thus, it was possible to measure low levels of H2O2 released by cells using an enzyme coupled cellular assay with LC-ESI-MS.