Reactions of nitric oxide and peroxynitrite with organic molecules and ferrihorseradish peroxidase: Interference with the determination of hydrogen peroxide

Abstract

Endothelial, inflammatory, and other cell types, in addition to forming reactive oxygen species, under proper stimulation release free radicals such as nitric oxide ('NO) and strong oxidants such as peroxynitrite (ONOO^-), which is the product of the reaction of nitric oxide with superoxide. Several methods for the detection of H₂O₂ are based on the ferrihorseradish peroxidase catalyzed oxidation of organic molecules. We investigated the reactions of nitric oxide and peroxynitrite with organic molecules as well as with ferrihorseradish peroxidase and examined the potential interference with the detection of H₂O₂. Peroxynitrite at low concentrations (010 μM) induced a concentration-dependent oxidation of 1,2 phenylenediamine, 3-3′ dimethoxybenzidine (odianisidine) and para-hydroxyphenylacetic acid (pHPA). With the exception of pHPA, the oxidation of the above compounds by peroxynitrite was not affected by the presence of ferrihorseradish peroxidase. The yield of HPA-dimmer, the oxidation product of pHPA by peroxynitrite, was decreased because ferrihorseradish peroxidase catalyzed the formation of a different product, 3-nitro-HPA. Nitrogen oxides, formed by the reaction of nitric oxide with oxygen, oxidized the aromatic amines o-phenylenediamine and o-dianisidine. A 10-fold excess of nitric oxide over H₂O₂ decreased the yield of pHPA and dihydrorhodamine 123 (DHR123) by 58 and 72%, respectively, as compared to H₂O₂ plus ferrihorseradish peroxidase. The inhibition of pHPA oxidation by nitric oxide was due in part to the reaction of nitric oxide with compound I and compound II and in part due to the reaction with the phenoxyl radical. These data suggest that the simultaneous generation of nitric oxide and peroxynitrite can interfere with the detection of H₂O₂. The overestimation or underestimation of the H₂O₂ detected is dependent upon the organic molecule utilized for detection and by the relative rate of nitric oxide, superoxide, and peroxynitrite generation.