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CA Curcio, K Bradley, C Guidry, M Kirk, L Wilson, S Barnes, HS Kruth, CC Y Chang, TY Chang; A Local Source for Esterified Cholesterol (EC) in Human Bruch’s Membrane (BrM) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):862.
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Purpose: To determine if EC in normal, aged BrM derives from directly infused plasma low-density lipoprotein (LDL) or from local cells with acyl co-A cholesterol acyl transferase (ACAT)-1 or -2. EC in LDL and EC in cells with ACAT is enriched in cholesteryl linoleate (Ch-18:2) and cholesteryl oleate (Ch-18:1), respectively. Methods: Ch-18:1 and Ch-18:2 were assayed by mass spectrometry in macular BrM, cornea, and sclera (10 donor eyes, greater than 60 yr), cholesterol-enriched human monocyte-macrophages, and LDL. Ch-16:0 (palmitate), Ch-18:0 (stearate), Ch-18:1, Ch-18:2, Ch-20:4 (arachidonate) and the corresponding free fatty acids (FFA) were assayed in macular BrM and RPE (n=6). Lipids were extracted with chloroform and methanol and separated by reversed phase HPLC using an isopropanol gradient in 10 mM ammonium acetate. Ion mass spectra (positive for ammonium ion adducts of cholesteryl esters and negative for FFA) were compared to standards. Protein extracts from human retinal pigment epithelium (RPE) and from HepG2 (hepatocyte) and Caco2 (enterocyte) cell lines underwent western blot analysis using antibodies to ACAT-1 and ACAT-2. Results: Ch-18:2/ Ch-18:1 ratios for LDL, cornea, sclera, BrM, and macrophages were 3.0, 2.0, 3.0, 0.35, and 0.59, respectively. RPE contained 30-40X less EC than BrM, normalized for area. Proportions of specific cholesteryl esters, relative to Ch-18:1 (Ch-16:0/ Ch-18:0/ Ch-18:2/ Ch-20:4), were similar in BrM (0.80/ 0.20/ 0.30/ 0.01) and RPE (0.84/ 0.28/ 0.30/ 0.01). FFA in BrM and RPE were much lower than the corresponding cholesteryl esters. Stearate was 3-5X more abundant than other FFA. Western blot analysis revealed bands at 50KD and 46KD, consistent with ACAT-1 and -2, respectively, in RPE. Conclusion: EC in BrM does not resemble plasma LDL but rather the output of cells with ACAT activity. EC in RPE is less abundant than that in BrM but similar in composition. RPE contains ACAT-1 and -2 proteins. The small quantities of free oleate and palmitate, the preferred substrates of ACAT-1 and -2, respectively, suggest that available substrate is efficiently utilized. This is the first evidence that human RPE has constitutive pathways for cholesterol processing and release. Formation of cholesterol-enriched drusen and basal deposits associated with age-related maculopathy may involve abnormalities in these pathways.
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