Greater selective uptake by Hep G2 cells of high-density lipoprotein cholesteryl ester hydroperoxides than of unoxidized cholesteryl esters

Abstract

We have observed recently that high-density lipoproteins (HDL) are the predominant carriers of cholesteryl ester hydroperoxides (CEOOH), the major class of lipid hydroperoxides detectable at nanomolar concentrations in the plasma of healthy fasting humans. The present study investigates the effect of such very low levels of CEOOH in apolipoprotein E-free HDL3 on lipoprotein particle metabolism and ‘selective uptake’ of its CE by human Hep G2 cells. Minimal oxidation with aqueous peroxyl radicals had a negligible effect on the binding, internalization and degradation of 125I-labelled HDL3. In contrast, with an increasing degree of radical-mediated oxidation of labelled HDL3, [3H]cholesteryl linoleate ([3H]Ch18:2) was taken up at an increasingly greater rate than were 125I-apoproteins. When [3H]cholesteryl linoleate hydroperoxide ([3H]Ch18:2-OOH was incorporated into unoxidized HDL3 by exchange from donor liposomes, it was taken up at a more than 8-fold higher rate than was incorporated [3H]Ch18:2. The same degree of preferential uptake of oxidized CE was observed when HDL3 was used that was doubly labelled with [3H]Ch18:2-OOH and cholesteryl [14C]oleate ([14C]Ch18:1). In both situations, uptake of [3H]Ch18:2-OOH exceeded that of 125I-apolipoprotein A-I some 40-fold. This increased selective uptake of [3H]Ch18:2-OOH from very mildly oxidized HDL3 was accompanied by a parallel increase in the intracellular levels of labelled free cholesterol. In contrast, lipid hydroperoxides were not detectable within Hep G2 cells, suggesting efficient detoxification of CEOOH by these cells. Neither the increased selective uptake of Ch18:2-OOHs nor the levels of intracellular free cholesterol were influenced by the presence of 50 microM chloroquine, suggesting extralysosomal hydrolysis of oxidized CEs. These results show that the selective uptake of HDL CEOOH by Hep G2 cells is more efficient than that of unoxidized CE, and support a protective role for rapid selective uptake in the removal of circulating HDL CEOOH.