Superoxide (O₂^•−) has been implicated in the pathogenesis of many human diseases, but detection of the O₂^•− radicals in biological systems is limited due to inefficiency of O₂^•− spin trapping and lack of site-specific information. This work studied production of extracellular, intracellular and mitochondrial O₂^•− in neutrophils, cultured endothelial cells and isolated mitochondria using a new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O₂^•−, producing stable nitroxides and allowing site-specific O₂^•− detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O₂^•−. Inhibition of EPR signal by SOD2 over-expression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O₂^•− both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected an increase in cytoplasm O₂^•− stimulated by PMA, but only CM-H and mitoTEMPO-H showed an increase in rotenone-induced mitochondrial O₂^•−. These data show that a new set of hydroxylamine spin probes provide unique information about site-specific production of the O₂^•− radical in extracellular or intracellular compartments, cytoplasm or mitochondria.

Cyclic voltammetry is a widely used and powerful tool for sensitively and selectively measuring hydrogen peroxide (H₂O₂). Herein, voltammetry was combined with electron paramagnetic resonance spectroscopy to identify and define the role of an oxygen-centered radical liberated during the oxidation of H₂O₂. The spin-trap reagents, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO), were employed. Spectra exhibit distinct hyperfine patterns that clearly identify the DMPO^•–OH and EMPO^•–OH adducts. Multiple linear regression analysis of voltammograms demonstrated that the hydroxyl radical is a principal contributor to the voltammetry of H₂O₂, as signal is attenuated when this species is trapped. These data incorporate a missing, fundamental element to our knowledge of the mechanisms that underlie H₂O₂ electrochemistry.