In vivo peroxidized phospholipids detected in bovine retina via targeted LC-MS/MS

Neural tissue is rich in highly unsaturated fatty acids (HUFA) that are particularly susceptible to oxygen attack. While some oxidation is a regulated byproduct of metabolism, the lipid peroxidation reaction (LPO) is a non-enzymatic and unregulated chain reaction that destroys HUFAs and overwhelms mechanisms that combat oxidation. Increased oxidative stress is a shared characteristic of neurodegenerative diseases and is present at the earliest stages. Thus, peroxidized neural phospholipids are hypothetical targets for understanding the pathogenesis of neurodegeneration and identifying therapeutic strategies. In this study we demonstrate detection and relative quantification of intact in vivo peroxidized phospholipids (oxPL) in real complex tissue: bovine retina. Routine fatty acid quantification via GC-FID and GC-MS/MS establishes the fatty acid pool that makes up the glycerolipid profile of bovine retina. The quantified fatty acid pool is used to customize a phospholipid library reflecting the profile present in retina and are confirmed with LC-MS/MS. Analysis of intact oxPLs is done with a Sciex 7500 Triple Quadrupole/Qtrap coupled with hydrophilic interaction liquid chromatography (HILIC). We use targeted independent data acquisition (IDA) multiple reaction monitoring (MRM) to identify and quantify intact in vivo oxPLs on a relative basis. Signals are calibrated to generalized autoxidized oxPL standards prepared in-house as analytical references according to methods introduced recently (AOCS 2023).
We detected and quantified a profile of 26 bioactive HUFA-esterified in vivo oxPLs in retina. MS/MS spectra confirm oxidation of acyl chains in detected oxPLs. Quantification reveals peroxidation levels with a reproducibility of 5-10% RSD within a biological extract of native and oxPL. This methodology can be used to monitor relative amounts of oxidation in future studies where the effects of therapeutic treatments and mechanistic studies in neurodegenerative models are tested.