The plasma HDLs represent a major class of cholesterol-transporting lipoprotein that can be divided into two distinct subfractions, HDL 2 and HDL 3 , by ultracentrifugation. Existing methods for the subfractionation of HDL requires lengthy ultracentrifugations, making them unappealing for large-scale studies. We describe a method that subfractionates HDL from plasma in only 6 h, representing a substantial decrease in total isolation time. The subfractions so isolated were assessed for a variety of lipid and protein components, in addition to their susceptibility to oxidation, both alone and in combination with VLDL and LDL. We report for the first time a prooxidant role for HDL during VLDL oxidation, in which HDL donates preformed hydroperoxides to VLDL in a cholesteryl ester transfer protein (CETP)-dependent process. Examination of the participation of HDL in LDL oxidation has reinforced its classic role as a potent antioxidant. Furthermore, we have also implicated the second major HDL-associated enzyme, LCAT, in these processes, whereby it acts as a potent prooxidant during VLDL oxidation but as an antioxidant during LDL oxidation. Thus, we have identified a potentially duplicitous role for HDL in the pathogenesis of atherosclerosis, attributable to both CETP and LCAT. High density lipoproteins are a heterogeneous group of particles whose principal physiological role is that of reverse cholesterol transport, but they also exert important antiinflammatory and antithrombotic effects (1). They can be classified according to their chemical and physical characteristics, including hydrated density (1.063-1.210 kg/l), flotation rate (0-9 Svedbergs), diameter (7-12 nm), and electrophoretic mobility [mainly a-migrating, although some nascent HDLs also display preb mobility, with the latter particles containing only apolipoprotein A-I (apoA-I)]. Analysis of material found in the 1.063-1.210 kg/l density interval by moving-boundary ultracentrifugation reveals a bimodal distribution of particles exhibiting a Schlieren pattern: an effect attributable to a change in the index of refraction of a light beam, caused by differences in particle concentration. This provides evidence for the subdivision of HDL into at least two distinct subfractions: HDL 2 and HDL 3 . Further studies of these subfractions reveal that the larger and less dense particle (HDL 2 ) is formed by the action of the enzyme LCAT upon the smaller, denser particle (HDL 3 ). LCAT is also involved in the formation of the HDL 3 subfraction, by rendering it largely cholesteryl ester-replete. These and additional observations relating to HDL's physiological roles have encouraged investigation into HDL and its subfractions, especially in relation to their antiatherogenic properties. A variety of established techniques have been adapted to subfractionate HDL, including precipitation (2, 3), electrophoresis (4, 5), affinity chromatography (6), and ultracentrifugation (7-14). The last of these methods, ultracentrifugation, is by far the most routinely used me...
