Detection of Short-Lived Free Radicals by Low-Frequency Electron Paramagnetic Resonance Spin Trapping in Whole Living Animals

We report the direct observation of sulfur trioxide anion free radical (SO.3−) from the body of live mice by using a low-frequency (1.2 GHz, L-band) EPR spin trapping method. Intraperitoneal injection of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (15 mmol/kg) used as trapping agent was followed by sequential iv injections of sodium sulfite (2-5 mmol/kg) and sodium dichromate (0.3 mmol/kg). The mouse was placed in an L-band loop gap resonator. The observed spectra consisted of two superposed components: DMPO/SO.3− (aN = 14.0, aH = 16.0) and Cr(V) (g = 1.9798). The time course for the generation and reduction of DMPO/SO.3− was followed. The signal intensity reached a maximum at about 7 min. In vitro experiments carried out at 9.15 GHz confirmed the production of SO.3− under the conditions used for in vivo studies; the hyperfine splittings of DMPO/SO.3− produced in the in vitro experiments were the same as those produced in the in vivo studies. The production of SO.3− is supported by oxygen uptake studies, which show that the prior addition of 300 mM DMPO inhibits oxygen consumption by SO.3−.