Abstract
Purpose :
A prominent aging change in human retinal pigment epithelium (RPE) under investigation for its role in age-related macular degeneration (AMD) is the accumulation of lipofuscin. Prior compositional analyses of lipofuscin revealed the fluorescent bisretinoid A2E, yet imaging mass spectrometry results suggest the presence of additional fluorophores of unknown origin. The purpose of this study is to identify and characterize new retinal fluorophores.
Methods :
Human eyes (n=3) (white donors > 80 years of age) were obtained from the Advancing Sight Network. The retina was carefully removed, extracted using Folch extraction solvent, and fluorophores were separated using high performance thin layer chromatography (HPTLC) RP-18 plates using chloroform: methanol 1:1(v/v) with TFA as mobile phase. After visualization with UV absorbance and autofluorescence microscopy, fluorescent bands were separated manually and subjected to MALDI analysis using a solariX 9.4T FT ICR mass spectrometer (Bruker Daltonics) and LC-MS/MS using a Q-Exactive HF instrument (Thermo Scientific).
Results :
Seven well-separated bands having UV absorbance and fluorescence properties were detected by TLC. A2E was identified in band 5 based on accurate mass measurement but was not located in the most fluorescent band. Mass spectra obtained from MALDI revealed the presence of high molecular weight species ranging from m/z 700-1100 in the intensely fluorescent bands (bands 3, 6 and 7). The molecular identity of m/z 782.557, 828.531, 711.980, 758.490 and 797.888 obtained from the most intensely fluorescent bands is currently under investigation using tandem mass spectrometry.
Conclusions :
A TLC method was developed to separate retina extracts with separated bands having differing absorbance and fluorescence properties compared to A2E. The strategy will help us in the identification of fluorophores of unknown origin which may have implications in the onset of AMD.
This is a 2021 ARVO Annual Meeting abstract.