EPR detection of lipid-derived free radicals from PUFA, LDL, and cell oxidations

We have used the spin trap 5,5-dimethyl-pyrroline-1-oxide (DMPO) and EPR to detect lipid-derived radicals (L-d(.)) during peroxidation of polyunsatura ted fatty acids (PUFA), low-density lipoprotein (LDL) and cells (K-562 and MCF-7). All oxygen-centered radical adducts of DMPO from our oxidizable tar gets have short lifetimes (<20 min). We hypothesized that the short lifetim es of these spin adducts are due in part to their reaction with radicals fo rmed during lipid peroxidation. We proposed that stopping the lipid peroxid ation processes by separating oxidation-mediator from oxidation-substrate w ith an appropriate extraction would stabilize the spin adducts. To rest thi s hypothesis we used ethyl acetate to extract the lipid-derived radical add ucts of DMPO (DMPO/L-d(.)) from an oxidizing docosahexaenioc acid (DHA) sol ution: Folch extraction was used for LDL and cell experiments. The lifetime s of DMPO spin adducts post-extraction are much longer (>10 h) than the spi n adducts detected without extraction. in iron-mediated DHA oxidation we ob served three DMPO adducts in the aqueous phase and two in the organic phase . The aqueous phase contains DMPO/HO. a(N) approximate to a(H) approximate to 14.8 G) and two carbon-centered radical adducts (a(1)(N) approximate to 15.8 G, a(1)(H) approximate to 22.6 G; a(2)(N) approximate to 15.2 G, a(2)( H) approximate to 18.9 G). The organic phase contains two long-chain lipid radical adducts (a(N) approximate to 13.5 G, a(H) approximate to 10.2 G; an d a(N) approximate to 12.8 G; a(H) approximate to 6.85 G, 1.9 G). We conclu de that extraction significantly increases the lifetimes of the spin adduct s, allowing detection of a variety of lipid-derived radicals by EPR. (C) 20 00 Elsevier Science Inc.