Discovering Novel Short‐ and Medium‐Chain Esters of Hydroxy Fatty Acids in Human Fecal Samples Using Untargeted Liquid Chromatography/Mass Spectrometry

Exploring novel metabolites produced by host gut microbiome communication is crucial for understanding their roles in various disease pathologies. We previously uncovered a novel class of lipids, short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs), in mouse fecal samples and demonstrated their promising physiological functions in mammals. However, the discovery of SFAHFAs in human samples remains unexplored. This study aimed to analyze the SFAHFAs and their structural analogs in human fecal samples using liquid chromatography/mass spectrometry. We identified 26 isomeric lipid species, including SFAHFAs and novel medium-chain fatty acid esters of hydroxy fatty acids (MFAHFAs). The detected SFAHFAs and MFAHFAs were characterized by accurate mass measurements using MSn analysis. The results were validated by matching the mass spectral fragmentation and retention time with authentic standards. Two new MFAHFAs, enanthic acid and caprylic acid esters of long-chain hydroxy fatty acids (C24 and C26), were detected and characterized for the first time in human fecal samples. Among the 26 isomeric lipid species, SFAHFA 2:0/24:0 or 4:0/22:0 and SFAHFA 2:0/24:1 were most abundant among the saturated and unsaturated SFAHFAs, respectively. This study offers the first insights into detecting and characterizing novel gut microbial lipids in human fecal samples. Further investigations are essential to recognize the metabolism and function of these lipids in the human gut.