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 (meta-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 cis/trans isomers of fatty acids and phospholipids from selected human gut bacteria. Strain-specific isomer profiles revealed unexpectedly high productivity of trans-10-octadecenoic acid by Enterococcus faecalis, Bifidobacterium longum, and Lactobacillus acidophilus among numerous trans-fatty acid isomers produced by gut bacteria. Isotope-tracking experiments suggested that gut bacteria produce trans-10-octadecenoic acid through the 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.人类肠道细菌产生的不饱和脂质具有极为丰富的结构多样性，这主要源于碳-碳双键（CC）的位置异构与立体化学异构。精准表征不同的CC构型在研究中颇具挑战，这主要受制于当前生物分析方法的局限性。本研究开发了一种全新的结构脂质组学工作流程，将MELDI（间氯过氧苯甲酸环氧化脂质双键鉴定法，meta-chloroperoxybenzoic acid epoxidation for lipid double-bond identification）与液相色谱-串联质谱相结合，用于CC构型的表征。我们利用该工作流程，对筛选得到的人类肠道细菌所产生的脂肪酸与磷脂中超过50种CC位置异构体和顺/反式异构体进行了定量分析。菌株特异性的异构体谱图分析表明，在肠道细菌产生的众多反式脂肪酸异构体中，粪肠球菌、长双歧杆菌与嗜酸乳杆菌可意外高效地生成反-10-十八碳烯酸。同位素示踪实验证实，肠道细菌可在体外通过油酸的异构生物转化生成反-10-十八碳烯酸；而膳食来源油酸的此类异构生物转化过程，在体内依赖于肠道细菌的存在。