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Original article
Identification of TMAO-producer phenotype and host–diet–gut dysbiosis by carnitine challenge test in human and germ-free mice
  1. Wei-Kai Wu1,2,
  2. Chieh-Chang Chen3,
  3. Po-Yu Liu4,5,
  4. Suraphan Panyod2,6,
  5. Ben-Yang Liao7,
  6. Pei-Chen Chen2,
  7. Hsien-Li Kao3,
  8. Han-Chun Kuo8,9,
  9. Ching-Hua Kuo8,9,
  10. Tina H T Chiu10,
  11. Rou-An Chen2,
  12. Hsiao-Li Chuang11,
  13. Yen-Te Huang11,
  14. Hsin-Bai Zou12,
  15. Cheng-Chih Hsu12,
  16. Ting-Yan Chang7,
  17. Chin-Lon Lin13,
  18. Chi-Tang Ho14,
  19. Hon-Tsen Yu4,5,
  20. Lee-Yan Sheen2,
  21. Ming-Shiang Wu3,6
  1. 1 Department of Internal Medicine, National Taiwan University Hospital Bei-Hu Branch, Taipei, Taiwan
  2. 2 Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan
  3. 3 Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
  4. 4 Department of Life Science, National Taiwan University, Taipei, Taiwan
  5. 5 Genome and Systems Biology Degree Program, Academia Sinica, Taipei, Taiwan
  6. 6 Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
  7. 7 Institute of Population Health Sciences, National Health Research Institutes, Miaoli, Taiwan
  8. 8 The Metabolomics Core Laboratory, Center of Genomic Medicine, National Taiwan University, Taipei, Taiwan
  9. 9 School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
  10. 10 Department of Nutrition Therapy, Dalin Tzu Chi Hospital, Chiayi, Taiwan
  11. 11 National Applied Research Laboratories, National Laboratory Animal Center, Taipei, Taiwan
  12. 12 Department of Chemistry, National Taiwan University, Taipei, Taiwan
  13. 13 Department of Internal Medicine, Dalin Tzu Chi Hospital, Taipei, Taiwan
  14. 14 Department of Food Science, Rutgers University, New Burnswick, New Jersey, USA
  1. Correspondence to Professor Lee-Yan Sheen, Institute of Food Science and Technology, National Taiwan University, Taipei 106, Taiwan; lysheen{at} and Professor Ming-Shiang Wu, Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan; mingshiang{at}


Objective The gut microbiota-derived metabolite, trimethylamine N-oxide (TMAO) plays an important role in cardiovascular disease (CVD). The fasting plasma TMAO was shown as a prognostic indicator of CVD incident in patients and raised the interest of intervention targeting gut microbiota. Here we develop a clinically applicable method called oral carnitine challenge test (OCCT) for TMAO-related therapeutic drug efforts assessment and personalising dietary guidance.

Design A pharmacokinetic study was performed to verify the design of OCCT protocol. The OCCT was conducted in 23 vegetarians and 34 omnivores to validate gut microbiota TMAO production capacity. The OCCT survey was integrated with gut microbiome, host genotypes, dietary records and serum biochemistry. A humanised gnotobiotic mice study was performed for translational validation.

Results The OCCT showed better efficacy than fasting plasma TMAO to identify TMAO producer phenotype. The omnivores exhibited a 10-fold higher OR to be high TMAO producer than vegetarians. The TMAO-associated taxa found by OCCT in this study were consistent with previous animal studies. The TMAO producer phenotypes were also reproduced in humanised gnotobiotic mice model. Besides, we found the faecal CntA gene was not associated with TMAO production; therefore, other key relevant microbial genes might be involved. Finally, we demonstrated the urine TMAO exhibited a strong positive correlation with plasma TMAO (r=0.92, p<0.0001) and improved the feasibility of OCCT.

Conclusion The OCCT can be used to identify TMAO-producer phenotype of gut microbiota and may serve as a personal guidance in CVD prevention and treatment.

Trial registration number NCT02838732; Results.

  • gut microbiota
  • trimethylamine n-oxide
  • oral carnitine challenge test
  • cardiovascular disease

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  • Contributors W-KW designed the animal and human experiments, assisted in statistical analyses and drafted the manuscript. C-CC and H-LK assisted human specimen collection. P-YL and SP assisted in experiment, performed bioinformatics and statistical analysis. B-YL, T-YC and H-TY revised bioinformatics and statistical analysis. THTC and C-LL assisted FFQ design and analysed dietary assessments. H-LK, C-HK, C-TH and H-BZ performed metabolomics study and assisted with mass spectrometry analysis. H-LC and Y-TH provided the humanized mouse platform. P-CC and R-AC assisted the experiment and animal study. C-TH critically reviewed the manuscript. L-YS and M-SW designed the experiments, provided the funding for the study and critically revised the manuscript.

  • Funding This study was funded by grants from the Ministry of Science and Technology (Taiwan) (106-2314-B-002-039-MY3 and 107-2321-B-002-017) and partially supported by a grant from the Ministry of Science and Technology (Taiwan) (106-3114-B-002-003), as well as a portion of a grant from the Program for Translational Innovation of Biopharmaceutical Development-Technology Supporting Platform Axis (106-0210-01-10-01 and 107-0210-01-19-04). This work was also partially financially supported by the ‘Center of Precision Medicine’ from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education in Taiwan (Grant Number: NTU- 107L9014-1) and the Ministry of Science and Technology, Executive Yuan, ROC, Taiwan (Grant Number: TCTC-TR2 106-2321-B-002-025). This work was also supported by the research grant from National Taiwan University Hospital (107-T11).

  • Competing interests None declared.

  • Patient consent Obtained.

  • Ethics approval Institutional Review Board of National Taiwan University Hospital.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data sharing statement NCBI Sequence Read Archive: 16s rDNA amplicon of human omnivore (SRX4119476 – SRX4119509) and vegetarian (SRX4119396 – SRX4119418).