HDL antioxidant effects as assessed using a nonexchangeable probe to monitor particle-specific peroxidative stress in LDL-HDL mixtures.

High density lipoproteins (HDL) have been reported to inhibit oxidation of low density lipoproteins (LDL) based in part on observations that oxidative changes occur more slowly in LDL-HDL mixtures than in LDL alone. In the current studies, we developed an approach to discern particle-specific oxidation kinetics within mixed particle systems using the oxidation-labile fluorescent probe parinaric acid cholesteryl ester (PnCE) and applied this to the study of HDL inhibition effects. PnCE was introduced into acceptor lipoproteins by cholesteryl ester transfer protein (CETP)-mediated transfer from donor microemulsions. Incubation of PnCE-containing LDL and HDL with non-probe-containing HDL and LDL, respectively, followed by measurement of reisolated fractions, indicated that PnCE does not transfer appreciably between lipoprotein fractions. Oxidative loss of lipoprotein-associated PnCE occurred essentially in tandem with changes in conjugated dienes, suggesting that PnCE loss reflects the course of peroxidation of endogenous lipoprotein lipids. Using PnCE to separately monitor LDL- and HDL-specific oxidation within LDL-HDL mixtures, we obtained direct evidence that HDL inhibits both Cu(2+)- and Fe(3+)-induced peroxidation of LDL-associated lipids. Notably, in the presence of Cu2+, loss of HDL-associated PnCE fluorescence also was inhibited in LDL-HDL co-incubations, suggesting that LDL exert an antioxidant effect under these conditions as well. Thus, results obtained using this new methodology are consistent with previously reported antioxidant effects of HDL, but indicate that the behavior of individual lipoprotein particles may be more complicated than can be predicted from the collective behavior of the lipoprotein mixture.