(P23-040-23) Detection of Epoxides and Vicinal Diols of Bis-Allylic Deuterated Docosahexaenoic Acid in a Retinal Degeneration Model by LC/MS/MS

Objectives: Retinal diseases such as age-related macular degeneration are increasingly prevalent, and new therapeutic options are needed. Retina is rich in docosahexaenoic acid (DHA), an oxidatively labile fatty acid required for retinal response to light. In a mouse model of retinal degeneration, we showed that feeding mice bis-allylic deuterated DHA (D-DHA) prior to intraocular iron injections completely protected the retina from damage. Epoxy and vicinal diol products of DHA derived from the cytochrome P450 (CYP450) pathway have been linked to retinal disease pathogenesis. We searched in retinal tissue to discover whether these products from D-DHA and natural DHA (H-DHA) could be detected.

Methods: Rats were fed a D-DHA or H-DHA diet for two months and exposed to fluorescent bright light stress (2700-lux for 6 h) one week before sacrifice. Fatty acid methyl esters were generated from single eyecups containing retinas and run on a GC-MS for fatty acid profiling. Additional eyecup samples were homogenized in ice-cold methanol, diluted to 10% methanol, and extracted with SPE columns in preparation for DHA-derived epoxy and vicinal diol profiling by LC-MS/MS. A Sciex® ExionLC® coupled to a model 7500 triple quadrupole ion trap was used for separations and run in MRM and IDA (data-dependent) modes.

Results: GC-MS analysis showed that an array of D-DHA isotopologues, bis-allylic D8-D13 DHA, were present in the eyecup tissue. D-DHA comprised over 50% of all DHA in the tissue. LC-MS/MS analysis revealed the presence of epoxides (EpDPA) and vicinal diols (DiHDPA) at the 7-8, 10-11, and 19-20 positions for the H-DHA and D-DHA parent molecules. The relative abundance of the H versus D peaks showed differences, with some dominated by the H isotopologue and others by the D isotopologues within one compound class. For metabolites related by a hydrolysis event, e.g., 19,20-EpDPA → 19,20-DiHDPA, relative abundances differ implying different rates of enzyme-driven hydrolysis.

Conclusions: D-DHA shows great promise as a therapeutic for retinal diseases driven by oxidative stress. This is the first time D-DHA derived bis-allylic deuterated oxylipins generated from the CYP450 pathway have been reported. D-DHA is metabolized to these oxylipins, illuminating a potential mechanism of action for D-DHA as a retinal disease therapeutic.

Funding Sources: University of Texas at Austin