A2E Distribution in RPE Granules in Human Eyes

Molecules. 2020 Mar 20;25(6):1413. doi: 10.3390/molecules25061413.

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.

Keywords: A2E (N-retinylidene-N-retinylethanolamine); RPE lipofuscin; lipofuscin fluorescence; liquid chromatography/mass spectrometry (LC/MS), melanin; retina.

MeSH terms

  • Aged
  • Aged, 80 and over
  • Cell Fractionation
  • Chromatography, Liquid
  • Cytoplasmic Granules / metabolism*
  • Female
  • Fluorescent Dyes / metabolism
  • Humans
  • Lipofuscin / metabolism
  • Male
  • Retinal Pigment Epithelium / metabolism*
  • Retinal Pigments / chemistry
  • Retinal Pigments / metabolism*
  • Retinoids / chemistry
  • Retinoids / metabolism*
  • Spectrum Analysis
  • Tandem Mass Spectrometry

Substances

  • A2-E (N-retinylidene-N-retinylethanolamine)
  • Fluorescent Dyes
  • Lipofuscin
  • Retinal Pigments
  • Retinoids