Identification and Quantitation of Lipid Compounds in Human RPE Lipofuscin by Nano-Electrospray Ionization Tandem Mass Spectrometry (nano-ESI-MS/MS)

Abstract

Abstract: : Purpose: Lipofuscin (LF) accumulates in the lysosomal compartment of retinal pigment epithelial (RPE) cells with age and is a common downstream pathogenetic pathway in association with various monogenetic and complex retinal diseases including Stargardt disease and age-related macular degeneration (ARMD). We have shown that individual components of LF such as A2-E may interfere with RPE cell function and have analyzed the LF-proteome. To identify further compounds in order to better understand lipofuscinogenesis and its possible toxic effects, we determined LF-lipid profiles in young and old human donor eyes. Methods: After homogenization and fractionation by gradient ultracentrifugation of RPE cells from 10 pairs of human aged donors (70-95 y/o) and 2 pairs of young donors (20+22 y/o) LF was isolated and subjected to lipid extraction. Lipid extracts were analysed by nano-ESI-MS/MS. Quantitation of lipids was performed by addition of lipid standards prior to extraction. Cholesterol was quantified after derivatization of the uncharged molecule to its sulfate ester. Results: LF particles of aged donors show significantly reduced levels of phosphatidylcholine (50%), sphingomyelin (60%), cholesterol (55%) and ceramides (80%) per µg protein as compared to young donors. Among the lipids quantified so far, cholesterol is the major lipid component, contributing 60 mol%. As the total phospholipid/protein ratio is only reduced by 18 %, these results indicate profound changes in the phospholipid composition. Whereas in most lipids the molecular species distribution remains constant, a significant change was detected in phosphatidylethanolamine and glucosylceramide species with a shift towards short chain fatty acids. Conclusions: Analysis of LF granules by nano-ESI-MS/MS revealed striking differences in the lipid composition between young and aged eyes. The altered lipid composition may be due to alterations in lipid degradation and are likely to evoke profound changes in overall lipid homoeostasis of RPE cells. While previous morphological and fundus autoflourescence studies show an overall, although topographically variable, increase of PRE-LF with age and disease, the results indicate that composition of LF-granules can vary markedly. This technique can now be used to characterize LF compositional differences also in association with various heterogenous retinal diseases.

Keywords: age-related macular degeneration • lipids • retinal pigment epithelium