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

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 (0-10 microM) induced a concentration-dependent oxidation of 1,2 phenylenediamine, 3-3' dimethoxybenzidine (o-dianisidine) 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 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.