Fecal Metabolite Quantitation for Rapid Assessment of the Gut Microbiome

Mullowney, M. W.; Moran, A.; Hernandez, A.; McMillin, M.; Rose, A.; Moran, D.; Little, J.; Nguyen, A. B.; Patel, B. K.; Lehmann, C. J.; Odenwald, M. A.; Pamer, E. G.; Yeo, K.-T. J.; Sidebottom, A. M.

doi:10.1101/2024.12.13.628394

bioRxiv · 10.1101/2024.12.13.628394

Fecal Metabolite Quantitation for Rapid Assessment of the Gut Microbiome

Mullowney, M. W.·Moran, A.·Hernandez, A.·McMillin, M.·Rose, A.·Moran, D.·Little, J.·Nguyen, A. B.·Patel, B. K.·Lehmann, C. J.·Odenwald, M. A.·Pamer, E. G.·Yeo, K.-T. J.·Sidebottom, A. M.

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Abstract

The intestinal microbiome is composed of myriad microbial species that produce metabolites that impact host health. While loss of bacterial species and beneficial metabolites from the fecal microbiome is associated with development of a range of diseases and medical complications, there are currently no diagnostic tests that rapidly identify individuals with microbiome deficiencies. Reduced concentrations of fecal butyrate and deoxycholic acid are associated with adverse clinical outcomes and result from the loss of a subset of health-associated bacterial species. We present a rapid diagnostic test based on 3-nitrophenylhydrazine derivatization and ultrahigh-performance liquid chromatography-mass spectrometry that measures fecal butyrate and deoxycholic acid concentrations as markers of microbiome function. A matrix-matched calibration curve was developed using a simulated fecal mixture to optimize accuracy and facilitate adherence to clinical laboratory regulations. The assay showed an analytical measurement range from 4.3-3030.1 {micro}M (LLOQ = 9.75 {micro}M) for butyrate and from 0.9- 64.9 {micro}M (LLOQ = 0.9 {micro}M) for deoxycholic acid. Precision demonstrated a coefficient of variation <15% at all QC levels tested. The assay can be performed in under an hour from extraction to provision of quantitative results, enabling the rapid identification of patients with defective microbiome function.

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Mullowney, M. W., Moran, A., Hernandez, A., McMillin, M., Rose, A., Moran, D., Little, J., Nguyen, A. B., Patel, B. K., Lehmann, C. J., Odenwald, M. A., Pamer, E. G., Yeo, K.-T. J., Sidebottom, A. M.. 2024-12-17. Fecal Metabolite Quantitation for Rapid Assessment of the Gut Microbiome. https://doi.org/10.1101/2024.12.13.628394

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