Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*} ). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E ⁺ ). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*} formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5- tert -butoxycarbonyl-5-methyl-1-pyrroline N -oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*} that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E ⁺ . However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*} with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*} . Analysis of the fluorescence characteristics of ethidium (E ⁺ ) and 2-OH-E ⁺ strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*} . We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable reactive oxygen species for detecting intracellular \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*} .

This article provides a succinct but limited overview of the protective and deleterious effects of reactive oxygen and nitrogen species in a clinical context. Reactive oxygen species include superoxide, hydrogen peroxide, single oxygen and lipid peroxides. Reactive nitrogen species include species derived from nitric oxide. This review gives a brief overview of the reaction chemistry of these species, the role of various enzymes involved in the generation and detoxification of these species in disease mechanisms and drug toxicity and the protective role of dietary antioxidants. I hope that the graphical review will be helpful for teaching both the first year medical and graduate students in the U.S. and abroad the fundamentals of reactive oxygen and nitrogen species in redox biology and clinical medicine.