Detection and relative quantification of ambient hydroperoxides of DHA phospholipids in retina and brain

Abstract: Neurodegeneration is thought to be initiated at least in part by reactive oxygen species (ROS) attacking highly unsaturated fatty acids (HUFA), which then enter a runaway lipid peroxidation cycle. The main HUFAs in neural tissue are omega-3 DHA and omega-6 arachidonic acid. We sought to measure ambient levels of lipid hydroperoxides in healthy cow retinas and rodent brains as a first step to understanding how to mitigate oxidation.
We first constructed a simple apparatus to oxidize bovine retinal lipids on the benchtop to obtain standards for analytical parameter development. Bovine retina fatty acid profiles were first established with routine gas chromatography (GC) methods and used to customize mass spectrometry scanning for major HUFA-carrying PLs. Targeted multiple reaction monitoring (MRM) scanning via triple quadrupole tandem mass spectrometry detected native (unoxidized) and oxidation-damaged PL regardless of the position of the O or O₂ addition along the acyl chains and enabled quantification of relative signals from intact native and oxidized PL (5%−10% CV). MRM-triggered information-dependent acquisition (IDA) spectra confirmed the structure of peroxidized PLs, revealing that peroxidized species (+O−OH) dominated over single O-added species in vitro and in vivo. Positive identification and relative quantification are reported for 12 selected in vivo native and peroxidized phosphatidylcholines and phosphatidylethanolamines. Similar studies were undertaken with oxidation-resistant bis-allylic deuterated DHA (D-DHA); our results indicated that these molecules oxidize by single O addition, which may explain their bioactivity. These results enable future studies of the initial peroxidation due to toxins, genetics, or other initiating events influencing in vivo oxidation levels and potentially the effectiveness of strategies to mitigate this mechanism of action.