Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine

Various detection methods of the specific product of reaction of superoxide (O(2)(*-)) with hydroethidine (HE), namely 2-hydroxyethidium (2-OH-E(+)), and with its mitochondria-targeted analog are described. The detailed protocol for quantification of 2-OH-E(+), the unique product of HE/O(2)(*-) in cellular systems, is presented. The procedure includes cell lysis, protein precipitation using acidified methanol and HPLC analysis of the lysate. Using this protocol, we determined the intracellular levels of 2-OH-E(+) and E(+) in the range of 10 and 100 pmol per mg protein in unstimulated macrophage-like RAW 264.7 cells. In addition to HE, 2-OH-E(+) and E(+), we detected several dimeric products of HE oxidation in cell lysates. As several oxidation products of HE are formed, the superoxide-specific product, 2-OH-E(+) needs to be separated from other HE-derived products for unequivocal quantification.