Nitric oxide decreases the stability of DMPO spin adducts

The effect nitric oxide (NO^{{radical dot}}) on the stability of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) adducts has been investigated using EPR spectroscopy. We report that the DMPO/HO^{{radical dot}} adduct, generated by porcine pulmonary artery endothelial cells in the presence of H₂O₂ and DMPO, or by a Fenton system (Fe(II) + H₂O₂) is degraded in the presence of the NO^{{radical dot}}-donor, 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) or by bolus addition of an aqueous solution of NO^{{radical dot}}. A similar effect of DEANO was observed on other DMPO adducts, such as DMPO/^{{radical dot}}CH₃ and DMPO/^{{radical dot}}CH(CH₃)OH, generated in cell-free systems. Measurements of the loss of DMPO/HO^{{radical dot}} in the presence of DEANO in aerated and oxygen-free buffers showed that in both of these settings the process obeys first-order kinetics and proceeds with similar efficacy. This indicates that direct interaction of the nitroxide with NO^{{radical dot}}, rather than with NO₂^{{radical dot}} (formed from NO^{{radical dot}} and O₂ in aerated media), is responsible for destruction of the spin adduct. These results suggest that the presence of NO^{{radical dot}} may substantially affect the quantitative determination of DMPO adducts. We also show that NO₂^{{radical dot}} radicals, generated by a myeloperoxidase/H₂O₂/nitrite system, also degrade DMPO/HO^{{radical dot}}. Because DMPO is frequently used to study generation of superoxide and hydroxyl radicals in biological systems, these observations indicate that extra caution is required when studying generation of these species in the presence of NO^{{radical dot}} or NO₂^{{radical dot}} radicals.