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. 2023 Jan 31;14:512. doi: 10.1038/s41467-023-36138-3

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Urine metabolites dependent on C. sporogenes colonization that are significantly different between MCAD^−/− (positive fold change, red) and MCAD^+/+ mice (negative, gray). Metabolites are colored if significant (q value < 0.05) and exhibit a greater than two-fold change (paired t-tests with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli). Named metabolites are colored in blue. b Total ion chromatograms and MS/MS fragmentation spectra for validation of four predicted metabolites in a. Standards are shown above; biological samples, below in blue. c Conversion of tyrosine to 3-(4-hydroxyphenyl)propionic acid by C. sporogenes and predicted conversion to 4-hydroxyhippuric acid via host MCAD and glycine conjugation yielding 3-(4-hydroxyphenyl)propionylglycine in MCAD^−/−. d 4-OH-PPA and 4-OH-PPG accumulate in the absence of MCAD, whereas 4-OH-hippuric acid is higher in MCAD^+/+ mice (4-OH-PPA and 4-OH-hippuric acid: unpaired two-tailed Student’s t-test, 4-OH-PPG: two-sided Mann-Whitney). e C. sporogenes metabolism of leucine produces isocaproic acid; predicted products based on MCAD activity in the host. f Isocaproylglycine accumulates in MCAD-deficient mice, indicating isocaproic acid undergoes host β-oxidation under homeostatic conditions (unpaired two-tailed Student’s t-tests). For d and f: n = 14 mice/GF group, n = 11 MCAD^+/+ Cs-colonized, n = 12 MCAD^−/− Cs-colonized mice; data were combined across two independent experiments. Boxes denote the median with inter-quartile distance, whiskers indicate maxima and minima. g Four previously demonstrated compounds in host circulation are the product of canonical host phase I (flavin monooxygenases) and phase II (sulfation, glutamine or glycine conjugation) metabolism of microbially-produced metabolites. Host β-oxidation (red) by MCAD generates hippuric acid and 4-hydroxyhippuric acid, two high-abundance compounds in the host.