Preparation of lipid hydroperoxide-free low-density lipoproteins

Lipid hydroperoxides formed during low density lipoproteins (LDL) isolation are important determinants of subsequent LDL resistance to metal ion-dependent oxidation and can mask or alter effects of LDL antioxidant manipulation. Thus, of paramount importance in studying the oxidation of LDL ex vivo is the prevention of adventitious oxidation during its isolation and handling in preparation for such assays. This chapter describes a method for isolating LDL in a manner that prevents artifactual oxidation, yielding an LDL preparation containing the same amounts of lipid hydroperoxides, if any, as LDL in plasma. Modifications of the method described by Redgrave have been used combining standard fixed angle or swinging bucket rotors with one or two centrifugations of 20 h or more. More recently, with the advent of near-vertical and vertical rotors, simple two-step gradients have been employed with durations of ultracentrifugation as brief as 25 min for isolation of LDL. These methods described by Chung and colleagues have several advantages, including significantly shorter centrifugation times and ease of preparation with underlaying of density adjusted plasma in a simple two-step gradient; the methods also minimize apoprotein redistribution and oxidation of lipoproteins that may occur during longer periods of ultracentrifugation.