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 (SO₃ ^º-) 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/SO₃ ^º- (α(N) = 14.0, α(H) = 16.0) and Cr(V) (g = 1.9798). The time course for the generation and reduction of DMPO/SO₃ ^º- 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 SO₃ ^º- under the conditions used for in vivo studies; the hyperfine splittings of DMPO/SO₃ ^º- produced in the in vitro experiments were the same as those produced in the in vivo studies. The production of SO₃ ^º- is supported by oxygen uptake studies, which show that the prior addition of 300 mM DMPO inhibits oxygen consumption by SO₃ ^º-.