Mapping lipid C=C isomer profiles of human gut bacteria by a novel structural lipidomics workflow assisted by chemical epoxidation

The unsaturated lipids produced by human gut bacteria exhibit extraordinary structural diversity, largely attributed to the isomerism of the carbon-carbon double bond (C=C) in terms of position and stereochemistry. Characterizing these distinct C=C configurations poses a significant challenge in the research field, primarily due to limitations in current bioanalytical methodologies. In this study, we developed a novel structural lipidomic workflow by integrating an epoxidation protocol using meta-chloroperoxybenzoic acid for C=C derivatization and liquid chromatography-tandem mass spectrometry for C=C characterization. We utilized this workflow to quantitatively assess over 50 C=C positional and cis/trans isomers of fatty acids and phospholipids from selected human gut bacteria. The strain-specific isomer profiles revealed unexpected and remarkable productivity of trans-10-octadecenoic acid by Enterococcus faecalis, Bifidobacterium longum, and Lacto-bacillus acidophilus, among numerous other trans fatty acid isomers produced by gut bacteria. Isotope-tracking experiments suggest that gut bacteria produce trans-10-octadecenoic acid through isomeric biotransformation of oleic acid in vitro and that such isomeric biotransformation of dietary oleic acid is dependent on the presence of gut bacteria in vivo.