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

Area of Science:

• Microbiology
• Metabolomics
• Endocrinology

Background:

• Gut microbiota significantly influences steroid hormone metabolism.
• Mechanisms, including enzymatic sequences and hydrogen sources, are poorly understood.
• Conventional methods face challenges due to complex matrices and lack of standards.

Purpose of the Study:

• To develop and validate a novel strategy for elucidating gut microbial steroid metabolism.
• To determine the sequence of reductive enzymatic reactions and track atom incorporation.
• To overcome limitations of existing analytical approaches.

Main Methods:

• Parallel incubation of steroid substrates with pooled human gut microbiota in H2O and D2O.
• High-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) analysis.
• Utilizing deuterium labeling to track atom incorporation and infer reaction sequences.

Main Results:

• A characteristic mass shift (1.0063 Da/deuterium atom) enabled metabolite detection and differentiation of multi-step reactions.
• Progesterone and 19-hydroxy-4-androstene-3,17-dione showed two-step reductions with up to three deuterium atoms incorporated.
• 17α-hydroxypregnenolone underwent a three-step pathway incorporating up to four deuterium atoms, suggesting A-ring localization.

Conclusions:

• The developed workflow effectively elucidates gut microbial steroid metabolism without recombinant enzymes or standards.
• This method provides a practical tool for studying steroid transformations in the gut microbiome.
• Potential applications include monitoring microbial activity in various human diseases.