Cholesteryl linoleate hydroperoxide (CLOOH) and
hydroxide (
CLOH) are present in human
atheroma. The intracellular metabolism of
low density lipoprotein (
LDL)-derived CLOOH and
CLOH remain undefined because extensive
free radical-mediated
LDL oxidation, which modifies
LDL apolipoprotein B sufficiently to allow endocytosis by the
scavenger receptor (ScR), also degrades CLOOH and
CLOH. This problem was approached by first acetylating
LDL lysine residues (
AcLDL) to achieve
protein modification, then exposing
AcLDL to the aqueous radical donor
2,2'-azobis(2-amidinopropane) HCl (
AAPH), to generate mildly oxidized
AcLDL (OxAcLDL). Murine peritoneal macrophages incubated with OxAcLDL accumulated large quantities of CE and small, non-toxic quantities of CLOOH and
CLOH in a time- and concentration-dependent manner, and accumulation was inhibited by
fucoidin. Inhibition of
acyl CoA: cholesterol acyltransferase during loading did not inhibit the accumulation of either CLOOH or
CLOH, whereas NH4Cl decreased intracellular clearance of accumulated CLOOH from 68.3 +/- 1.7% to 35.3 +/- 1.0% over 12 h, suggesting lysosomal or pre-lysosomal accumulation. Intracellular clearance of unoxidized
lipoprotein-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 mildly
oxidized LDL, even without acetylation, also promoted cellular accumulation of CLOOH and
CLOH. We conclude that intracellular accumulation of
cholesteryl linoleate hydroperoxide and
cholesteryl linoleate hydroxide can follow 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.