REACTIONS OF NITRIC-OXIDE AND PEROXYNITRITE WITH ORGANIC-MOLECULES AND FERRIHORSERADISH PEROXIDASE - INTERFERENCE WITH THE DETERMINATION OFHYDROGEN-PEROXIDE

Endothelial, inflammatory, and other cell types, in addition to formin g reactive oxygen species, under proper stimulation release free radic als such as nitric oxide ((NO)-N-.) and strong oxidants such as peroxy nitrite (ONOO-), which is the product of the reaction of nitric oxide with superoxide. Several methods for the detection of H2O2 are based o n the ferrihorseradish peroxidase catalyzed oxidation of organic molec ules. 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. Perox ynitrite at low concentrations (0-10 mu M) induced a concentration-dep endent oxidation of 1,2 phenylenediamine, 3-3' dimethoxybenzidine (o-d ianisidine) and para-hydroxyphenylacetic acid (pHPA). With the excepti on 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 de creased because ferrihorseradish peroxidase catalyzed the formation of a different product, 3-nitro-HPA. Nitrogen oxides, formed by the reac tion of nitric oxide with oxygen, oxidized the aromatic amines o-pheny lenediamine and o-dianisidine. A 10-fold excess of nitric oxide over H 2O2 decreased the yield of pHPA and dihydrorhodamine 123 (DHR123) by 5 8 and 72%, respectively, as compared to H2O2 plus ferrihorseradish per oxidase. The inhibition of pHPA oxidation by nitric oxide was due in p art to the reaction of nitric oxide with compound I and compound II an d in part due to the reaction with the phenoxyl radical. These data su ggest that the simultaneous generation of nitric oxide and peroxynitri te can interfere with the detection of H2O2. The overestimation or und erestimation of the H2O2 detected is dependent upon the organic molecu le utilized for detection and by the relative rate of nitric oxide, su peroxide, and peroxynitrite generation.