Abstract
Accumulation of the insoluble lipid-protein complex, ceroid, is a characteristic of atherosclerotic plaques. To determine whether deficient processing of cholesteryl esters in oxidized (ox) low density lipoprotein (LDL) contributes to ceroid formation, we studied the hydrolysis of internalized [3H] cholesteryl linoleate (CL) in oxLDL by mouse peritoneal macrophages (MPM). The hydrolysis by MPM of [3H]CL incorporated into oxLDL or LDL did not differ, suggesting that products of lipid and/or apoB oxidation had no impact on the lysosomal hydrolysis of [3H]CL. To evaluate the hydrolysis of oxCL by MPM, we subjected extensively ox[3H]CL to fractionation by TLC. The predominant fraction (D) consisted of sterols and oxysterols esterified to scission products of oxidized fatty acids containing terminal carbonyl groups, i.e., lipid core aldehydes. The extent of hydrolysis of [3H]-fraction D by MPM cultures, as well as by MPM extracts at pH 4.0, was significantly reduced when compared to the hydrolysis of intact [3H]CL. Fraction D also formed complexes with serum proteins, and the purified core aldehyde, cholesteryl 9-oxononanoate reacted with epsilon-amino group of lysines. Finally, several cholesteryl ester aldehydes were detected in lipid extracts of human atheroma. These results suggest that decomposition products of extensively oxidized cholesteryl linoleate that are also present in atherosclerotic lesions, are not adequately degraded by mouse peritoneal macrophage lysosomes and could interact with proteins to form ceroid.
Highlights
Accumulation of the insoluble lipid-protein complex, ceroid, is a characteristic of atherosclerotic plaques
To explore whether components within an oxidized LDL (oxLDL) particle affect the intracellular hydrolysis of cholesteryl linoleate (CL), we compared the hydrolysis by mouse peritoneal macrophages (MPM) of ['HICL incorpe rated into oxLDLwith that of ["HICLincorporated into either native low density lipoprotein (LDL) or acetylated LDL (acLDL)
The rate of hydrolysiswas not reduced for ['HICL incorporated into oxLDL when compared to that of vortexed LDL (vxLDL) or acLDL
Summary
Materials [1,2,6,7-SH(N)]cholesteryl linoleate was obtained from NEN Research Products (Boston, MA). Aliquots of MPM extracts (10-50 pg) were placed into a borosilicate test tube containing 100 pl of 100 mM acetate buffer (pH 4.0), and the mixture was pre-incubated for 10-15 min at room temperature. Lipid extracts were dried in borosilicate test tubes and 250-pl aliquots of 0.5 mM NaH2P0, buffer (pH ’7.5)containing 310 mM sucrose and 5 pl of a stock solution of cholesterol oxidase (CO) (2 units/ml final concentration) were added to each sample. The chloroform layer, which contained the DNPH derivatives of the core aldehydes along with other oxidation products of the cholesteryl linoleate, was subjected to high-performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC;/ESI/MS) directly or after initial separation by TLC [23]. ‘The chlor-oforrri extracts were blown down under nitrogen and rcdissolved in chloroform-methanol containing dinitrophenylhydrazine as previously described [23].The DNPH derivatives along with the unreacted lipids were subjected to reversed phase HPLC/ESI/MS. Samples were diluted in 1% SDS to minimize turbidi ty
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