Vitamin E Forms Domains with Pufa in Membranes: An EPR Spin Trapping Study of Lipid Peroxidation in Model Membranes

Abstract

Spin trapping in conjunction with EPR spectroscopy is a widely used technique for measurement of short-lived free radical species of biological interest due to its high sensitivity and specificity. In spin trapping a molecule (the spin trap) reacts with the free radical producing a spin adduct that is sufficiently stable to be detected by EPR. In this study, we employ lipid-soluble N-tert-butyl-α-phenylnitrone (PBN) as our spin trap. PBN spin adducts detected by EPR are believed to be lipid hydroperoxides that are secondary species resulting from free radical attack on cell membranes. Thus the presence of the spin-adducts is reflective of ROS levels. Lipid auto oxidation will be monitored in model membranes containing 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC) with various concentrations of α-tocopherol, the major lipid soluble antioxidant in membranes and a primary component of vitamin E. Preliminary data of PDPC without α-tocopherol show auto oxidation that leads to a free radical cascade. By measuring the reduction in lipid peroxidation due to the presence of α-tocopherol, we test the hypothesis that the vitamin co-localizes with polyunsaturated lipids in membrane domains to ensure its close proximity to the most vulnerable lipid species.