Spin-trapping study of free radical penetration into liposomal membranes

Abstract

Despite the recognized role of the hydroxyl radical in human pathology, experimental data regarding the membrane penetration of this highly reactive species is lacking. We have attempted to study this question utilizing the EPR spin-trapping technique with two new hydrophobic analogues of 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), 2,2-dimethyl-4-phenyl-2H-imidazole 1-oxide (DMPIO, 1) and its 2-methyl-2-nonyl analogue (MNPIO, 2). EPR spectra were obtained for these spin-traps with HO˙, CH₃˙, HOCH₂˙, CH₃(OH)CH˙ and CH₃CH₂CH₂(OH)CH˙ radicals in phosphate buffer, in sodium dodecyl sulfate (SDS) micelles and, for DMPIO only, in dimyristoylphosphatidylcholine liposomes. The data suggest that these radicals do not penetrate into the lipid phase. In the case of the mildly lipophilic DMPIO, the spin-adduct signals observed result either from a rapid exchange of spin-adduct between the lipid and water phases or, more likely, from the fact that the aminoxyl, group of the spin-adduct is located at the interface and, hence, available for interaction with radicals and paramagnetic ions. The highly lipophilic MNPIO, on the other hand, resides deep in the lipid bilayer of liposomes and hence no spin-trapping is observed.