Abstract
A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1–F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.
Highlights
Lipofuscin, a hallmark of aging [1], is formed in the process of a conserved catabolic mechanism known as autophagy [2,3] in which cellular components are digested in lysosomes and the undegradable material accumulates to form a polymorphous pigment waste, collectively called lipofuscin [1,4].The molecular composition of lipofuscin is highly heterogeneous, depending on cell types and metabolic origins [5,6]
retinal pigment epithelium (RPE) cells from human donor eyes were lysed and granules were fractionated by discontinuous sucrose density gradient centrifugation with four sucrose concentrations (2, 1.4, 1.2, and 1.0 M)
Four fractions were visible at the interfaces of 0–1.0, 1.0–1.2, 1.2–1.4, and 1.4–2.0 M sucrose, designated as Fraction 1 (F1), F2, F3, and F4, respectively
Summary
Lipofuscin, a hallmark of aging [1], is formed in the process of a conserved catabolic mechanism known as autophagy [2,3] in which cellular components are digested in lysosomes and the undegradable material accumulates to form a polymorphous pigment waste, collectively called lipofuscin [1,4]. The molecular composition of lipofuscin is highly heterogeneous, depending on cell types and metabolic origins [5,6]. In phagocytes, such as macrophages and retinal pigment epithelium (RPE), lipofuscin is formed by undegradable waste from phagocytosed materials [1]. It has long been suggested that lipofuscin in RPE plays a role in retinal diseases, including age-related macular degeneration (AMD) and Stargardt disease [6,11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
