Investigation of lipid-related components in sub-retinal pigment epithelial deposits of normal and age-related maculopathy eyes

Apolipoproteins E, B and C-III (apo E, B, C-III), and cholesterol were localized in macular and peripheral Bruch's membrane (BrM), and deposits (basal deposits and drusen), of normal and age related maculopathy (ARM) eyes; the presence of apolipoproteins in human retinal pigment epithelium (RPE) extracts was examined. Normal and ARM donor eyes, preserved <4 hr after death, were cryosectioned at 10 mum thickness. ARM eyes contained RPE changes, basal deposits and/or drusen >63 mum. Apolipoproteins were identified through indirect immunofluorescence using polyclonal antibodies. Antibody specificity was confirmed through Western blot analysis of human plasma. The histochemical stain filipin was used to demonstrate unesterified cholesterol (UC) or esterified cholesterol (EC) following extraction and hydrolysis. Western blot analysis was also used to screen human RPE extracts for apolipoproteins. Histopathologic examination of eyes revealed many peripheral and few macular deposits in normal eyes; peripheral and macular deposits in ARM eyes. Apo E and B was present in peripheral deposits of normal and ARM eyes, but varied in pattern and extent in macular deposits. Apo C was absent from deposits. EC/UC and apo B co-localized in peripheral deposits of normal and ARM eyes. While EC/UC was present in all macular apo B-positive deposits of ARM eyes, the converse was not the case: apo B was not present in all EC/UC-rich deposits. Apo E was present in normal and ARM BrM. Apo B was absent from BrM except in association with deposits. Apo C-III was not present in BrM. Human RPE extract contained apo E but not apo B or C-III. Since apo B is the principal protein in low-density lipoproteins (LDL) and LDL transports cholesterol in plasma, it is plausible that apo B deposition in deposits indicates LDL retention. The partial colocalization of apo B and EC/UC suggests that LDL may be one means by which cholesterol accumulates in deposits. While peripheral deposits in our eyes consistently contained apo E and B, the apolipoprotein content of macular deposits was more variable. To our knowledge this is the first demonstration of differences in biochemical constituents of basal deposits and drusen between the macula and periphery. Results of the Western blot analysis suggest that RPE is a potential local source for apo E but not apo B or C-III, consistent with previous reports of apo E mRNA in RPE.