The reaction of oxyhemoglobin with phenylhydrazine has received considerable attention for many decades. The basis for this interest stems from the ability of phenylhydrazine and hydrazine-based drugs to induce hemolytic anemia. Considerable evidence obtained from in vitro electron spin resonance (ESR) experiments implicates free radicals in the events leading to red blood cell hemolysis. However, until this report, no corroborating ESR evidence for in vivo free radical formation has been presented. We have successfully employed ESR to detect the formation of a radical adduct in the blood of rats which received an intragastric dose of phenylhydrazine followed by an intraperitoneal injection of the spin trap 5,5-dimethyl-l-pyrroline N-oxide (DMPO). The results of a series of experiments with sulfhydryl reagents and C-13-labelled phenylhydrazine led us to assign this DMPO radical adduct to the trapping of a hemoglobin-derived thiyl free radical. In addition to phenylhydrazine the hydrazine-based drugs isoniazid, iproniazid, phenelzine, and hydralazine were examined. Of the four drugs, only phenelzine and iproniazid were able to induce the formation of the DMPO/hemoglobin thiyl free radical adduct in vivo, whereas only phenelzine and hydralazine were able to form this adduct in vitro. We were able to decrease the in vivo iproniazid-induced adduct formation by pretreating the rats with bis-para-nitrophenylphosphate, an arylamidase inhibitor. Our results support the idea that iproniazid is hydrolyzed in the liver to a more reactive metabolite, most likely isopropylhydrazine, which is subsequently released into the blood stream. DMPO/hemoglobin thiyl free radical formation is not limited to hydrazines, but forms when either hydroperoxides or aromatic hydroxylamines react with oxyhemoglobin. This species may be in vivo indicator of free radical damage to red blood cells.
