Evidence against the 1:2:2:1 quartet DMPO spectrum as the radical adduct of the lipid alkoxyl radical.

It was reported that the electron paramagnetic resonance (EPR) spectrum of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/lipid alkoxyl radical exhibited a quartet with 1:2:2:1 relative intensity that is identical to that of DMPO/hydroxyl radical (K. M. Schaich and D. C. Borg, 1990, Free Radicals Res. Commun. 9, 267-278). We repeated these EPR experiments using HPLC separation of radical adducts and isotope substitution. We found that the HPLC/EPR chromatogram of the radical adduct with a 1:2:2:1 quartet obtained by the reduction of methyl linoleate hydroperoxide (MLOOH) with Fe2+ exhibited identical retention time to that of the DMPO/OH radical adduct obtained from the Fenton reaction in two different solvent systems. Upon performing the same reaction in 17O-enriched water, the 17O-hyperfine coupling constants due to DMPO/17OH were identified. Ultimately, approximately 80-90% of the total DMPO/OH is derived from water by an iron-dependent nucleophilic addition reaction. Initially, a water-independent mechanism also significantly contributes to DMPO/OH formation. Regardless of its mechanism of formation, the 1:2:2:1 quartet radical adduct of DMPO formed during the reduction of MLOOH by Fe2+ is in fact DMPO/OH.