Choline Metabolism to the Proatherogenic Metabolite Trimethylamine Occurs Primarily in the Distal Colon Microbiome In Vitro
Abstract
1. Introduction
2. Materials and Methods
2.1. Preparation of Fecal Slurry
2.2. MiGut Model Setup
2.3. Choline-d9 Fermentations
2.3.1. Experiment 1 Timeline
2.3.2. Experiment 2 Timeline
2.4. Measurement of Choline-d9 and TMA-d9
2.5. Microbiome Analysis
2.5.1. DNA Extraction and Sequencing
2.5.2. Metagenomic Sequence Analysis
2.6. cutC Quantitative PCR
2.7. Data Analysis and Statistics
3. Results
3.1. Choline-d9 Conversion to TMA-d9 Differs by Simulated Colon Region
3.2. Microbiome Characterization
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Amini, M.; Zayeri, F.; Salehi, M. Trend Analysis of Cardiovascular Disease Mortality, Incidence, and Mortality-to-Incidence Ratio: Results from Global Burden of Disease Study 2017. BMC Public Health 2021, 21, 401. [Google Scholar] [CrossRef] [PubMed]
- Roth, G.A.; Mensah, G.A.; Johnson, C.O.; Addolorato, G.; Ammirati, E.; Baddour, L.M.; Barengo, N.C.; Beaton, A.Z.; Benjamin, E.J.; Benziger, C.P.; et al. Global Burden of Cardiovascular Diseases and Risk Factors, 1990–2019: Update from the GBD 2019 Study. J. Am. Coll. Cardiol. 2020, 76, 2982–3021. [Google Scholar] [CrossRef] [PubMed]
- Calazans, J.A.; Permanyer, I. Levels, Trends, and Determinants of Cause-of-Death Diversity in a Global Perspective: 1990–2019. BMC Public Health 2023, 23, 650. [Google Scholar] [CrossRef]
- Witkowski, M.; Weeks, T.L.; Hazen, S.L. Gut Microbiota and Cardiovascular Disease. Circ. Res. 2020, 127, 553–570. [Google Scholar] [CrossRef]
- Talmor-Barkan, Y.; Bar, N.; Shaul, A.A.; Shahaf, N.; Godneva, A.; Bussi, Y.; Lotan-Pompan, M.; Weinberger, A.; Shechter, A.; Chezar-Azerrad, C.; et al. Metabolomic and Microbiome Profiling Reveals Personalized Risk Factors for Coronary Artery Disease. Nat. Med. 2022, 28, 295–302. [Google Scholar] [CrossRef]
- Kazemian, N.; Mahmoudi, M.; Halperin, F.; Wu, J.C.; Pakpour, S. Gut Microbiota and Cardiovascular Disease: Opportunities and Challenges. Microbiome 2020, 8, 36. [Google Scholar] [CrossRef] [PubMed]
- Wang, Z.; Klipfell, E.; Bennett, B.J.; Koeth, R.; Levison, B.S.; DuGar, B.; Feldstein, A.E.; Britt, E.B.; Fu, X.; Chung, Y.-M. Gut Flora Metabolism of Phosphatidylcholine Promotes Cardiovascular Disease. Nature 2011, 472, 57–63. [Google Scholar] [CrossRef]
- Tang, W.W.; Wang, Z.; Levison, B.S.; Koeth, R.A.; Britt, E.B.; Fu, X.; Wu, Y.; Hazen, S.L. Intestinal Microbial Metabolism of Phosphatidylcholine and Cardiovascular Risk. N. Engl. J. Med. 2013, 368, 1575–1584. [Google Scholar] [CrossRef]
- Jameson, E.; Quareshy, M.; Chen, Y. Methodological Considerations for the Identification of Choline and Carnitine-Degrading Bacteria in the Gut. Methods 2018, 149, 42–48. [Google Scholar] [CrossRef]
- Koeth, R.A.; Wang, Z.; Levison, B.S.; Buffa, J.A.; Org, E.; Sheehy, B.T.; Britt, E.B.; Fu, X.; Wu, Y.; Li, L. Intestinal Microbiota Metabolism of L-Carnitine, a Nutrient in Red Meat, Promotes Atherosclerosis. Nat. Med. 2013, 19, 576–585. [Google Scholar] [CrossRef]
- Koeth, R.A.; Levison, B.S.; Culley, M.K.; Buffa, J.A.; Wang, Z.; Gregory, J.C.; Org, E.; Wu, Y.; Li, L.; Smith, J.D.; et al. γ-Butyrobetaine Is a Proatherogenic Intermediate in Gut Microbial Metabolism of L-Carnitine to TMAO. Cell Metab. 2014, 20, 799–812. [Google Scholar] [CrossRef] [PubMed]
- Seim, H.; Löster, H.; Claus, R.; Kleber, H.-P.; Strack, E. Formation of γ-Butyrobetaine and Trimethylamine from Quaternary Ammonium Compounds Structure-Related to l-Carnitine and Choline by Proteus Vulgaris. FEMS Microbiol. Lett. 1982, 13, 201–205. [Google Scholar] [CrossRef]
- Meyer, K.A.; Shea, J.W. Dietary Choline and Betaine and Risk of CVD: A Systematic Review and Meta-Analysis of Prospective Studies. Nutrients 2017, 9, 711. [Google Scholar] [CrossRef]
- Cashman, J.R.; Camp, K.; Fakharzadeh, S.S.; Fennessey, P.V.; Hines, R.N.; Mamer, O.A.; Mitchell, S.C.; Preti, G.; Schlenk, D.; Smith, R.L. Biochemical and Clinical Aspects of the Human Flavin-Containing Monooxygenase Form 3 (FMO3) Related to Trimethylaminuria. Curr. Drug Metab. 2003, 4, 151–170. [Google Scholar] [CrossRef]
- Shimizu, M.; Koibuchi, N.; Mizugaki, A.; Hishinuma, E.; Saito, S.; Hiratsuka, M.; Yamazaki, H. Genetic Variants of Flavin-Containing Monooxygenase 3 (FMO3) in Japanese Subjects Identified by Phenotyping for Trimethylaminuria and Found in a Database of Genome Resources. Drug Metab. Pharmacokinet. 2021, 38, 100387. [Google Scholar] [CrossRef]
- Roberts, A.B.; Gu, X.; Buffa, J.A.; Hurd, A.G.; Wang, Z.; Zhu, W.; Gupta, N.; Skye, S.M.; Cody, D.B.; Levison, B.S.; et al. Development of a Gut Microbe–Targeted Nonlethal Therapeutic to Inhibit Thrombosis Potential. Nat. Med. 2018, 24, 1407–1417. [Google Scholar] [CrossRef] [PubMed]
- Wang, Z.; Roberts, A.B.; Buffa, J.A.; Levison, B.S.; Zhu, W.; Org, E.; Gu, X.; Huang, Y.; Zamanian-Daryoush, M.; Culley, M.K. Non-Lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis. Cell 2015, 163, 1585–1595. [Google Scholar] [CrossRef] [PubMed]
- Iglesias-Carres, L.; Krueger, E.S.; Herring, J.A.; Tessem, J.S.; Neilson, A.P. Potential of Phenolic Compounds and Their Gut Microbiota-Derived Metabolites to Reduce TMA Formation: Application of an In Vitro Fermentation High-Throughput Screening Model. J. Agric. Food Chem. 2022, 70, 3207–3218. [Google Scholar] [CrossRef]
- Iglesias-Carres, L.; Essenmacher, L.A.; Racine, K.C.; Neilson, A.P. Development of a High-Throughput Method to Study the Inhibitory Effect of Phytochemicals on Trimethylamine Formation. Nutrients 2021, 13, 1466. [Google Scholar] [CrossRef]
- Iglesias-Carres, L.; Bruno, A.; D’Antuono, I.; Linsalata, V.; Cardinali, A.; Neilson, A.P. In Vitro Evidences of the Globe Artichoke Antioxidant, Cardioprotective and Neuroprotective Effects. J. Funct. Foods 2023, 107, 105674. [Google Scholar] [CrossRef]
- Iglesias-Carres, L.; Racine, K.C.; Neilson, A.P. Phenolic-Rich Beverages Reduce Bacterial TMA Formation in an Ex Vivo–In Vitro Colonic Fermentation Model. Food Funct. 2022, 13, 8022–8037. [Google Scholar] [CrossRef]
- Bresciani, L.; Dall’Asta, M.; Favari, C.; Calani, L.; Rio, D.D.; Brighenti, F. An in Vitro Exploratory Study of Dietary Strategies Based on Polyphenol-Rich Beverages, Fruit Juices and Oils to Control Trimethylamine Production in the Colon. Food Funct. 2018, 9, 6470–6483. [Google Scholar] [CrossRef]
- Day-Walsh, P.; Shehata, E.; Saha, S.; Savva, G.M.; Nemeckova, B.; Speranza, J.; Kellingray, L.; Narbad, A.; Kroon, P.A. The Use of an In-Vitro Batch Fermentation (Human Colon) Model for Investigating Mechanisms of TMA Production from Choline, l-Carnitine and Related Precursors by the Human Gut Microbiota. Eur. J. Nutr. 2021, 60, 3987–3999. [Google Scholar] [CrossRef] [PubMed]
- Orman, M.; Bodea, S.; Funk, M.A.; Campo, A.M.-D.; Bollenbach, M.; Drennan, C.L.; Balskus, E.P. Structure-Guided Identification of a Small Molecule That Inhibits Anaerobic Choline Metabolism by Human Gut Bacteria. J. Am. Chem. Soc. 2019, 141, 33–37. [Google Scholar] [CrossRef] [PubMed]
- Venema, K. The TNO In Vitro Model of the Colon (TIM-2). In The Impact of Food Bioactives on Health: In Vitro and Ex Vivo Models; Verhoeckx, K., Cotter, P., López-Expósito, I., Kleiveland, C., Lea, T., Mackie, A., Requena, T., Swiatecka, D., Wichers, H., Eds.; Springer: Cham, Switzerland, 2015; ISBN 978-3-319-15791-7. [Google Scholar]
- Van de Wiele, T.; Van den Abbeele, P.; Ossieur, W.; Possemiers, S.; Marzorati, M. The Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). In The Impact of Food Bioactives on Health: In Vitro and Ex Vivo Models; Verhoeckx, K., Cotter, P., López-Expósito, I., Kleiveland, C., Lea, T., Mackie, A., Requena, T., Swiatecka, D., Wichers, H., Eds.; Springer: Cham, Switzerland, 2015; ISBN 978-3-319-15791-7. [Google Scholar]
- Davis Birch, W.A.; Moura, I.B.; Ewin, D.J.; Wilcox, M.H.; Buckley, A.M.; Culmer, P.R.; Kapur, N. MiGut: A Scalable in Vitro Platform for Simulating the Human Gut Microbiome—Development, Validation and Simulation of Antibiotic-Induced Dysbiosis. Microb. Biotechnol. 2023, 16, 1312–1324. [Google Scholar] [CrossRef]
- Ferrell, M.; Bazeley, P.; Wang, Z.; Levison, B.S.; Li, X.S.; Jia, X.; Krauss, R.M.; Knight, R.; Lusis, A.J.; Garcia-Garcia, J.C. Fecal Microbiome Composition Does Not Predict Diet-Induced TMAO Production in Healthy Adults. J. Am. Heart Assoc. 2021, 10, e021934. [Google Scholar] [CrossRef]
- Wang, Z.; Hazen, J.; Jia, X.; Org, E.; Zhao, Y.; Osborn, L.J.; Nimer, N.; Buffa, J.; Culley, M.K.; Krajcik, D.; et al. The Nutritional Supplement L-Alpha Glycerylphosphorylcholine Promotes Atherosclerosis. Int. J. Mol. Sci. 2021, 22, 13477. [Google Scholar] [CrossRef] [PubMed]
- Rath, S.; Heidrich, B.; Pieper, D.H.; Vital, M. Uncovering the Trimethylamine-Producing Bacteria of the Human Gut Microbiota. Microbiome 2017, 5, 1–14. [Google Scholar] [CrossRef]
- Jameson, E.; Doxey, A.C.; Airs, R.; Purdy, K.J.; Murrell, J.C.; Chen, Y. Metagenomic Data-Mining Reveals Contrasting Microbial Populations Responsible for Trimethylamine Formation in Human Gut and Marine Ecosystems. Microb. Genom. 2016, 2, e000080. [Google Scholar] [CrossRef]
- Ramireddy, L.; Tsen, H.-Y.; Chiang, Y.-C.; Hung, C.Y.; Chen, F.-C.; Yen, H.-T. The Gene Expression and Bioinformatic Analysis of Choline Trimethylamine-Lyase (CutC) and Its Activating Enzyme (CutD) for Gut Microbes and Comparison with Their TMA Production Levels. Curr. Res. Microb. Sci. 2021, 2, 100043. [Google Scholar] [CrossRef]
- Rath, S.; Rud, T.; Pieper, D.H.; Vital, M. Potential TMA-Producing Bacteria Are Ubiquitously Found in Mammalia. Front. Microbiol. 2020, 10, 02966. [Google Scholar] [CrossRef] [PubMed]
- Kashyap, J.; Ringiesn, J.R.; Schwab, N.; Ferguson, D.J. Isolation and Characterization of a Novel Choline Degrading Citrobacter Amalonaticus Strain from the Human Gut. Curr. Res. Microb. Sci. 2022, 3, 100157. [Google Scholar] [CrossRef]
- Ramezani, A.; Nolin, T.D.; Barrows, I.R.; Serrano, M.G.; Buck, G.A.; Regunathan-Shenk, R.; West, R.E.; Latham, P.S.; Amdur, R.; Raj, D.S. Gut Colonization with Methanogenic Archaea Lowers Plasma Trimethylamine N-Oxide Concentrations in Apolipoprotein e−/− Mice. Sci. Rep. 2018, 8, 14752. [Google Scholar] [CrossRef] [PubMed]
- de la Cuesta-Zuluaga, J.; Spector, T.D.; Youngblut, N.D.; Ley, R.E. Genomic Insights into Adaptations of Trimethylamine-Utilizing Methanogens to Diverse Habitats, Including the Human Gut. mSystems 2021, 6, e00939-20. [Google Scholar] [CrossRef] [PubMed]
- Goodrich, K.M.; Smithson, A.T.; Ickes, A.K.; Neilson, A.P. Pan-Colonic Pharmacokinetics of Catechins and Procyanidins in Male Sprague–Dawley Rats. J. Nutr. Biochem. 2015, 26, 1007–1014. [Google Scholar] [CrossRef]
- van Wijck, K.; van Eijk, H.M.H.; Buurman, W.A.; Dejong, C.H.C.; Lenaerts, K. Novel Analytical Approach to a Multi-Sugar Whole Gut Permeability Assay. J. Chromatogr. B 2011, 879, 2794–2801. [Google Scholar] [CrossRef]
- van Wijck, K.; Verlinden, T.J.M.; van Eijk, H.M.H.; Dekker, J.; Buurman, W.A.; Dejong, C.H.C.; Lenaerts, K. Novel Multi-Sugar Assay for Site-Specific Gastrointestinal Permeability Analysis: A Randomized Controlled Crossover Trial. Clin. Nutr. 2013, 32, 245–251. [Google Scholar] [CrossRef]
- Casso, A.G.; VanDongen, N.S.; Gioscia-Ryan, R.A.; Clayton, Z.S.; Greenberg, N.T.; Ziemba, B.P.; Hutton, D.A.; Neilson, A.P.; Davy, K.P.; Seals, D.R.; et al. Initiation of 3,3-Dimethyl-1-Butanol at Midlife Prevents Endothelial Dysfunction and Attenuates in Vivo Aortic Stiffening with Ageing in Mice. J. Physiol. 2022, 600, 4633–4651. [Google Scholar] [CrossRef]
- Winslow, C.J.; Nichols, B.L.B.; Novo, D.C.; Mosquera-Giraldo, L.I.; Taylor, L.S.; Edgar, K.J.; Neilson, A.P. Cellulose-Based Amorphous Solid Dispersions Enhance Rifapentine Delivery Characteristics in Vitro. Carbohydr. Polym. 2018, 182, 149–158. [Google Scholar] [CrossRef]
- Gilley, A.D.; Arca, H.C.; Nichols, B.L.B.; Mosquera-Giraldo, L.I.; Taylor, L.S.; Edgar, K.J.; Neilson, A.P. Novel Cellulose-Based Amorphous Solid Dispersions Enhance Quercetin Solution Concentrations in Vitro. Carbohydr. Polym. 2017, 157, 86–93. [Google Scholar] [CrossRef]
- Goodrich, K.M.; Neilson, A.P. Simultaneous UPLC-MS/MS Analysis of Native Catechins and Procyanidins and Their Microbial Metabolites in Intestinal Contents and Tissues. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2014, 958, 63–74. [Google Scholar] [CrossRef] [PubMed]
- Martínez-del Campo, A.; Bodea, S.; Hamer, H.A.; Marks, J.A.; Haiser, H.J.; Turnbaugh, P.J.; Balskus, E.P. Characterization and Detection of a Widely Distributed Gene Cluster That Predicts Anaerobic Choline Utilization by Human Gut Bacteria. mBio 2015, 6, e00042-15. [Google Scholar] [CrossRef] [PubMed]
Compound | MW | MS/MS Transition | CV (V) | CE (eV) |
---|---|---|---|---|
Choline-d9 | 113.2 | 113.3 > 69.1 | 40 | 16 |
Choline-1-13C-1,1,2,2-d4 | 109.2 | 109.3 > 60.3 | 36 | 18 |
Ethyl betaine-d9 a | 155.2 | 155.3 > 127.2 | 34 | 20 |
Ethyl betaine-13C3-15N a | 150.2 | 150.3 > 122.2 | 34 | 18 |
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Buckley, A.M.; Zaidan, S.; Sweet, M.G.; Ewin, D.J.; Ratliff, J.G.; Alkazemi, A.; Davis Birch, W.; McAmis, A.M.; Neilson, A.P. Choline Metabolism to the Proatherogenic Metabolite Trimethylamine Occurs Primarily in the Distal Colon Microbiome In Vitro. Metabolites 2025, 15, 552. https://doi.org/10.3390/metabo15080552
Buckley AM, Zaidan S, Sweet MG, Ewin DJ, Ratliff JG, Alkazemi A, Davis Birch W, McAmis AM, Neilson AP. Choline Metabolism to the Proatherogenic Metabolite Trimethylamine Occurs Primarily in the Distal Colon Microbiome In Vitro. Metabolites. 2025; 15(8):552. https://doi.org/10.3390/metabo15080552
Chicago/Turabian StyleBuckley, Anthony M., Sarah Zaidan, Michael G. Sweet, Duncan J. Ewin, Juanita G. Ratliff, Aliyah Alkazemi, William Davis Birch, Ashley M. McAmis, and Andrew P. Neilson. 2025. "Choline Metabolism to the Proatherogenic Metabolite Trimethylamine Occurs Primarily in the Distal Colon Microbiome In Vitro" Metabolites 15, no. 8: 552. https://doi.org/10.3390/metabo15080552
APA StyleBuckley, A. M., Zaidan, S., Sweet, M. G., Ewin, D. J., Ratliff, J. G., Alkazemi, A., Davis Birch, W., McAmis, A. M., & Neilson, A. P. (2025). Choline Metabolism to the Proatherogenic Metabolite Trimethylamine Occurs Primarily in the Distal Colon Microbiome In Vitro. Metabolites, 15(8), 552. https://doi.org/10.3390/metabo15080552