Accumulation and metabolism of low density lipoprotein-derived cholesteryllinoleate hydroperoxide and hydroxide by macrophages

Cholesteryl linoleate hydroperoxide (CLOOH) and hydroxide (CLOH) are presen t in human atheroma. The intracellular metabolism of low density lipoprotei n (LDL)-derived CLOOH and CLOH remain undefined because extensive free radi cal-mediated LDL oxidation, which modifies LDL apolipoprotein B sufficientl y to allow endocytosis by the scavenger receptor (ScR), also degrades CLOOH and CLOH. This problem was approached by first acetylating LDL lysine resi dues (AcLDL) to achieve protein modification, then exposing AcLDL to the aq ueous radical donor 2,2'-azobis(2-amidinopropane) HCl (AAPH), to generate m ildly oxidized AcLDL (OxAcLDL), Murine peritoneal macrophages incubated wit h OxAcLDL accumulated large quantities of CE and small, non-toxic quantitie s of CLOOH and CLOH in a time- and concentration-dependent manner, and accu mulation was inhibited by fucoidin, Inhibition of acyl CoA: cholesterol acy ltransferase during loading did not inhibit the accumulation of either CLOO H or CLOH, whereas NH4Cl decreased intracellular clearance of accumulated C LOOH from 68.3 +/- 1.7% to 35.3 +/- 1.0% over 12 h, suggesting lysosomal or pre-lysosomal accumulation. Intracellular clearance of unoxidized lipoprot ein-derived CE decreased from 84.0 +/- 5,9% to 43.1 +/- 2.3% over 12 h when cells were loaded with AcLDL or OxAcLDL, respectively. Aggregation of mild ly oxidized LDL, even without acetylation, also promoted cellular accumulat ion of CLOOH and CLOH. We conclude that intracellular accumulation of chole steryl linoleate hydroperoxide and cholesteryl linoleate hydroxide can foll ow charge modification or aggregation of mildly oxidized LDL, and that LDL- derived oxidation products may inhibit hydrolysis of LDL-derived CE in foam cell macrophages.