Detection of free radical metabolite formation using in vivo EPR spectroscopy: Evidence of rat hemoglobin thiyl radical formation following administration of phenylhydrazine

The spin-trapping technique in conjunction with a low-frequency electron paramagnetic (or spin) resonance (EPR or ESR) spectrometer was used to detect the hemoglobin thiyl free radical in living rats using a whole body resonator. The hemoglobin thiyl free radical was formed following the intragastric administration of phenylhydrazine at the LD₅₀ dose of 188 mg/kg. The hemoglobin thiyl free radical was then trapped by preinjected 5,5- dimethyl-1-pyrroline N-oxide (DMPO), which formed the DMPO/hemoglobin thiyl- free radical adduct in the blood. The time course of the in vivo formation and disappearance of the spin adduct was followed. The DMPO/hemoglobin thiyl free radical was detected in blood samples using 9.5 GHz (X-band) and 1.1 GHz (L-band) EPR at room temperature and 77 K. Pretreatment of rats with ascorbate and diethylmaleate (DEM) decreased the signal intensity of the DMPO/hemoglobin thiyl free radical spin adduct. The incubation of ascorbate or DEM at 37°C with rat blood containing preformed DMPO/hemoglobin thiyl radical adduct showed that there was no effect of DEM on the free radical concentration, while ascorbate reduced the radical adduct. This study provided direct evidence of the formation of the DMPO/hemoglobin thiyl free radical in vivo and enabled us to study this formation in living animals free of any artifacts that can occur when using ex vivo methods.