Abstract
Purpose :
DHA, 22:6n3, is essential for photoreceptors (PRs), retinal pigment epithelium (RPE), and synapses. PRs take up 22:6 and incorporate it in phosphatidylcholine (PC), but it is also elongated to 32-38 carbons(C), to form Very Long Chain-PolyUnsaturated Fatty Acids (VLC-PUFAs), which incorporate in the same PCs. With stress 22:6 and VLC-PUFAs are released, changed to neuroprotective Neuroprotectin D1 (NPD1) and an Elovanoid (ELV), respectively, inhibiting apoptosis. We are interested in mitigating PR loss; we characterize docosanoids (22:6-derived molecules) control by mass spec.
LC-MS/MS analysis reveals molecular content and sample quantification, 22:6/VLC/PUFA-PCs, but cannot localize molecules; but, MALDI-IMS (Matrix-Assisted Laser Desorption/Ionization-imaging mass spectrometry) visualizes molecules in sections but cannot quantify. We utilize both methods.
Methods :
Retinas are removed for extraction and LC-MS/MS quantification. Also, eyecups are sectioned and a 2,5 dihydroxybenzoic acid matrix added to sections by sublimation. Sections are scanned (Synapt G2-Si mass spectrometer and MALDI source (Waters)) for data collection. MALDI spectra are collected from regions of interest, prominent peaks detected, and m/zs identified. Spectra from 2 different regions are compared; 1 spectrum subtracted from the other to produce a difference spectrum; upward and downward peaks reveal most abundant molecules (m/z) from each area. Full fragmentation confirms structure. Section images are constructed with BioMap.
Results :
We identified rod cell-specific PCs with 22:6n3 and VLC-PUFAs in human normal and macular degeneration (MD) peripheral retinas by MALDI. MD retinas showed selective peripheral VLC-PUFA-containing PC loss. LC-MS/MS showed arachidonic acid (20:4n6, PC(20:4/20:4) PLs remained constant in normal and MD peripheries; PC44:12, 22:6/22:6n3, was depressed in peripheral PCs. MD 54 & 56C PCs and 32 & 34C PC-VLC-PUFAs were reduced.
Conclusions :
Reduced retinal 22:6 impairs synthesis of VLC-PUFAs, which are abundant in rods/peripheral retina. VLC-PUFA reduction affects rod survival which may contribute to MD. Insufficient VLC-PUFA-containing PCs impede synthesis and availability of the pro-homeostatic and neuroprotective mediators NPD1 and ELVs, affecting rod PRs, and initiating onset of PR/RPE impairments in MD.
This is a 2021 Imaging in the Eye Conference abstract.