D₂O-Enabled Chemical Beaconing: Tracking Steroid Metabolism by Pooled Gut Microbiota

The gut microbiota actively metabolizes steroid hormones, but the mechanisms of these transformations-particularly the sequence of enzymatic reactions and the source of hydrogen atoms-remain poorly understood. Conventional analytical approaches are hampered by the complex fecal matrix, isomeric metabolites, and the lack of authentic reference standards. Here we present a strategy based on parallel incubation of steroids with pooled human gut microbiota in H₂O and D₂O, followed by HPLC-HRMS, with the aim of determining the sequence of reductive steps and tracking the incorporation of atoms into steroid metabolites. Using a pooled fecal inoculum from multiple donors (n = 18) and three steroid substrates, we demonstrate that the characteristic mass shift (1.0063 Da per deuterium atom) enables detection of metabolites and distinguishes multi-step enzymatic reactions in the microbial community without recombinant enzymes and authentic standards. Progesterone and 19-hydroxy-4-androstene-3,17-dione underwent sequential two-step reduction incorporating up to three deuterium atoms, while 17α-hydroxypregnenolone followed a three-step pathway incorporating up to four deuterium atoms, consistent with localization to the A-ring. This workflow provides a practical tool for investigating gut microbial steroid metabolism in the context of human physiology and disease, with potential applications in monitoring microbial activity in endocrine disorders, inflammatory bowel disease, and neuropsychiatric conditions.