Mapping Lipid C═C Isomer Profiles of Human Gut Bacteria through a Novel Structural Lipidomics Workflow Assisted by Chemical Epoxidation

The unsaturated lipids produced by human gut bacteria have an extraordinary range of structural diversity, largely because of the isomerism of the carbon-carbon double bond (C═C) in terms of its position and stereochemistry. Characterizing distinct C═C configurations poses a considerable challenge in research, primarily owing to limitations in current bioanalytical methodologies. This study developed a novel structural lipidomics workflow by combining MELDI (<i>meta-</i>chloroperoxybenzoic acid epoxidation for lipid double-bond identification) with liquid chromatography-tandem mass spectrometry for C═C characterization. We utilized this workflow to quantitatively assess more than 50 C═C positional and <i>cis/trans</i> isomers of fatty acids and phospholipids from selected human gut bacteria. Strain-specific isomer profiles revealed unexpectedly high productivity of <i>trans</i>-10-octadecenoic acid by <i>Enterococcus faecalis</i>, <i>Bifidobacterium longum</i>, and <i>Lactobacillus acidophilus</i> among numerous <i>trans</i>-fatty acid isomers produced by gut bacteria. Isotope-tracking experiments suggested that gut bacteria produce <i>trans</i>-10-octadecenoic acid through the isomeric biotransformation of oleic acid <i>in vitro</i> and that such isomeric biotransformation of dietary oleic acid is dependent on the presence of gut bacteria <i>in vivo</i>.