Abstract
Purpose :
Docosahexaenoic acid (DHA) elongation in the inner segment of photoreceptors leads to synthesis of very-long-chain polyunsaturated fatty acid (VLC-PUFA) omega-3s (n-3) by ELOVL4. Mutations of this enzyme cause certain forms of retinal degenerations. We found that genetic ablation of adiponectin receptor 1 (AdipoR1) results in retinal degeneration (Rice et al., Nature Comm, 2015) preceded by a remarkable downregulation of the VLC-PUFA omega-3 molecular species of phosphatidylcholine. These observations support our previous hypothesis that VLC-PUFA omega-3s are precursors of novel bioactive mediators. Here we report the identification of 32:6,n-3 and of 34:6,n-3 hydroxylated derivatives, their structural confirmation, and their biological activity in retinal pigment epithelial (RPE) cells.
Methods :
Human RPE cells were cultured with 32:6 and 34:6. After cells and media were collected, lipids were extracted and loaded onto a liquid chromatography-mass spectrometer (LC-MS/MS) for analysis. We analyzed fatty acids, precursors of elovanoids (27-hydroxy-FA32:6 and 29–hydroxyl-FA34:6), and elovanoids (20,27-dihydroxy-FA32:6 and 22,29-dihydroxy-FA34:6). Elovanoids were synthesized, and matching with deuterium-labeled derivatives was performed.
Results :
Both elovanoids and pre-cursors of elovanoids were detected on the cells under oxidative stress. We used m/z 499 -> 93 and 499 -> 401 MRM transitions for 20,27-dihydroxy-FA32:6, and m/z 527 -> 93 and 527 -> 429 transitions for 22,29-dihydroxy-FA34:6 for detection. For corresponding precursors, we used m/z 483 -> 385 for 27-hydroxy-FA32:6, and m/z 511 -> 413 for 29–hydroxyl-FA34:6. For further identification, we performed full fragmentation on elovanoids and found good matches to the standards.
Conclusions :
In RPE cells, VLC-PUFAs (32:6,n-3 and 34:6,n-3) produce derivatives through a lipoxygenation step, and we proposed the name elovanoids. We also identified stable hydroxyl-intermediates of their synthesis. Elovanoids also are released to the extracellular space, therefore they might exert autocrine and paracrine bioactivity. Clearly they are remarkable inducers of cell survival under uncompensated oxidative stress conditions. Thus RPE and photoreceptors undergoing uncompensated oxidative stress, might need elovanoids to contribute to sustaining functional integrity.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.