EPR Kinetic Studies of the LDL Oxidation Process Driven by Free Radicals

Abstract

Oxidation is widely assumed to play a causal role in the pathogenesis of atherosclerosis. Oxidative modification of low density lipoprotein (LDL) can be followed by analyzing the lag phase of the conjugated diene formation at 234 nm in LDL exposed to Cu²⁺. This procedure is restricted to isolated LDL fractions. To make this assay applicable to different biological systems, the present paper introduces a method to determine the time course of lipid peroxidation by measuring the EPR signal intensity and thereby the concentration of the radicals formed. Stable radical spin adducts were generated using the spin trap PBN (N-tert.-butyl-α-phenylnitrone) and were detected by EPR spectroscopy. Comparing the specific formation of radicals and the generation of conjugated dienes as measured by UV absorbance revealed analogous lag, propagation and decomposition phases.

Key Words:

• Antioxidants
• copper
• EPR
• LDL
• PBN
• spin trapping