Intracellular distribution of bis-allylic deuterated linoleic acid into the lipidome of human keratinocytes

Polyunsaturated fatty acids (PUFA) are particularly susceptible to free radical-induced lipid peroxidation (LPO). Specific deuteration at bis-allylic positions of PUFA (D-PUFA) has been recently proposed as a way to inhibit the LPO. Here, a high mass resolution untargeted lipidomic analysis protocol was applied to examine the changes in the lipidome of keratinocytes supplemented with bis-allylic deuterated linoleic acid (D₂-LA). Incorporation of D₂-LA occurs preferentially in membrane phospholipids such as phosphatidylcholine and phosphatidylethanolamine, followed by triglycerides. However, the relative contribution of D₂-LA among membrane lipids is highest in cardiolipin (60%) followed by its precursor phosphatidylglycerol (50%). Cardiolipins are enriched in PUFA and exclusively located in mitochondrial membranes, thus representing major targets for lipid peroxidation. These findings indicate that D₂-LA supplementation is linked to the preservation of mitochondrial function under oxidative stress. Finally, our study highlights the suitability of high mass resolution lipidomic analysis to investigate lipid metabolism at the level of individual molecular species in stable isotope tracing experiments.