Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease

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Metabolomics studies hold promise for the discovery of pathways linked to disease processes. Cardiovascular disease (CVD) represents the leading cause of death and morbidity worldwide. Here we used a metabolomics approach to generate unbiased small-molecule metabolic profiles in plasma that predict risk for CVD. Three metabolites of the dietary lipid phosphatidylcholine—choline, trimethylamine N-oxide (TMAO) and betaine—were identified and then shown to predict risk for CVD in an independent large clinical cohort. Dietary supplementation of mice with choline, TMAO or betaine promoted upregulation of multiple macrophage scavenger receptors linked to atherosclerosis, and supplementation with choline or TMAO promoted atherosclerosis. Studies using germ-free mice confirmed a critical role for dietary choline and gut flora in TMAO production, augmented macrophage cholesterol accumulation and foam cell formation. Suppression of intestinal microflora in atherosclerosis-prone mice inhibited dietary-choline-enhanced atherosclerosis. Genetic variations controlling expression of flavin monooxygenases, an enzymatic source of TMAO, segregated with atherosclerosis in hyperlipidaemic mice. Discovery of a relationship between gut-flora-dependent metabolism of dietary phosphatidylcholine and CVD pathogenesis provides opportunities for the development of new diagnostic tests and therapeutic approaches for atherosclerotic heart disease.

At a glance


  1. Strategy for metabolomics studies to identify plasma analytes associated with cardiovascular risk.
    Figure 1: Strategy for metabolomics studies to identify plasma analytes associated with cardiovascular risk.

    a, Overall schematic to identify plasma analytes associated with cardiac risk over the ensuing 3-year period. CVA, cerebrovascular accident; HPLC, high-performance liquid chromatography; MI, myocardial infarction. b, Odds ratio (OR) and 95% confidence intervals (CI) of incident (3-year) risk for MI, CVA or death of the 18 plasma analytes that met all selection criteria in both Learning and Validation Cohorts; odds ratio and 95% confidence intervals shown are for the highest versus lowest quartile for each analyte. Filled circles represent the analytes (m/z = 76, 104, 118) focused on in this study. m/z, mass to charge ratio.

  2. Identification of metabolites of dietary PC and an obligatory role for gut flora in generation of plasma analytes associated with CVD risks.
    Figure 2: Identification of metabolites of dietary PC and an obligatory role for gut flora in generation of plasma analytes associated with CVD risks.

    a, Summary schematic indicating structure of metabolites and routes (oral or i.p.) of formation observed in choline challenge studies in mice using the indicated isotope-labelled choline. The m/z in plasma observed for the isotopomers of the choline metabolites are shown. b, Plasma levels of d9 metabolites after i.p. challenge with d9(trimethyl)-dipalmitoylphosphatidylcholine (d9-DPPC). c, d9-TMAO production after oral d9-DPPC administration in mice, following suppression of gut flora with antibiotics (3 weeks), and then following placement (4 weeks) into conventional cages with non-sterile mice (‘conventionalized’). Data are presented as mean±standard error (s.e.) from four independent replicates.

  3. Plasma levels of choline, TMAO and betaine are associated with atherosclerosis risks in humans and promote atherosclerosis in mice.
    Figure 3: Plasma levels of choline, TMAO and betaine are associated with atherosclerosis risks in humans and promote atherosclerosis in mice.

    ac, Spline models of the logistic regression analyses reflecting risk of CVD (with 95% CI) according to plasma levels of choline, TMAO and betaine in the entire cohort (n = 1,876 subjects). d, Comparison in aortic lesion area among 20-week-old female C57BL/6J Apoe−/− mice fed with chow diet supplemented with the indicated amounts (wt/wt) of choline or TMAO from time of weaning (4 weeks). e, Relationship between plasma TMAO levels and aortic lesion area. f, Relationship between fasting plasma levels of TMAO versus CAD burden among subjects (N = 1,020). Boxes represent 25th, 50th and 75th percentile, and whiskers 5th and 95th percentile plasma levels. Single, double and triple coronary vessel disease refers to number of major coronary vessels demonstrating≥50% stenosis on diagnostic coronary angiography.

  4. Hepatic Fmo genes are linked to atherosclerosis and dietary PC metabolites enhance macrophage scavenger receptor expression.
    Figure 4: Hepatic Fmo genes are linked to atherosclerosis and dietary PC metabolites enhance macrophage scavenger receptor expression.

    ac, Correlation between hepatic Fmo3 expression and aortic lesion, plasma HDL cholesterol and TMAO in female mice from the F2 intercross between atherosclerosis-prone C57BL/6J Apoe−/− and atherosclerosis-resistant C3H/HeJ Apoe−/− mice. d, Correlation between human hepatic FMO3 expression and plasma TMAO. e, Effect of Fmo3 genotype (SNP rs3689151) on aortic sinus atherosclerosis in male mice from the C57BL6/J Apoe−/− and C3H/HeJ Apoe−/− F2 intercross. f, g, Quantification of scavenger receptor CD36 and SR-A1 surface protein levels in macrophages harvested from C57BL/6J mice (13 week) after three weeks of standard chow versus chow supplemented with the indicated amounts (wt/wt) of choline, TMAO or betaine. Data are presented as mean±s.e. from the indicated numbers of mice in each group.

  5. Obligatory role of gut flora in dietary choline enhanced atherosclerosis.
    Figure 5: Obligatory role of gut flora in dietary choline enhanced atherosclerosis.

    a, Choline supplementation promotes macrophage foam cell formation in a gut-flora-dependent fashion. C57BL/6J Apoe−/− mice at time of weaning (4 weeks) were provided drinking water with or without broad-spectrum antibiotics (Abx), and placed on chemically defined diets similar in composition to normal chow (control diet, 0.08±0.01% total choline, wt/wt) or normal chow with high choline (choline diet, 1.00%±0.01% total choline, wt/wt). Resident peritoneal macrophages were recovered at 20 weeks of age. Typical images of Oil-red-O/haematoxylin-stained macrophages in each diet group are shown. b, Foam cell quantification from peritoneal macrophages recovered from mice in studies described in panel a. c, Macrophage cellular cholesterol content. d, Representative Oil-red-O/haematoxylin-stained aortic root sections from female C57BL/6J Apoe−/− mice fed control and high-choline diets in the presence or absence of antibiotics. e, f, Aortic lesion area in 20 week old C57BL/6J Apoe−/− mice off or on antibiotics and fed with control or choline diet. g, Aortic macrophage quantification with anti-F4/80 antibody staining. h, Quantification of the scavenger receptor CD36 in aorta within the indicated groups. Error bars represent s.e.m. from the indicated numbers of mice.

  6. Gut-flora-dependent metabolism of dietary PC and atherosclerosis.
    Figure 6: Gut-flora-dependent metabolism of dietary PC and atherosclerosis.

    Schematic summary illustrating newly discovered pathway for gut-flora-mediated generation of pro-atherosclerotic metabolite from dietary PC.


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Author information


  1. Department of Cell Biology, Cleveland Clinic, Cleveland, Ohio 44195, USA

    • Zeneng Wang,
    • Elizabeth Klipfell,
    • Robert Koeth,
    • Bruce S. Levison,
    • Brandon DuGar,
    • Ariel E. Feldstein,
    • Earl B. Britt,
    • Xiaoming Fu,
    • Yoon-Mi Chung,
    • Jonathan D. Smith,
    • W. H. Wilson Tang,
    • Joseph A. DiDonato &
    • Stanley L. Hazen
  2. Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic, Cleveland, Ohio 44195, USA

    • Zeneng Wang,
    • Elizabeth Klipfell,
    • Bruce S. Levison,
    • Ariel E. Feldstein,
    • Earl B. Britt,
    • Xiaoming Fu,
    • Yoon-Mi Chung,
    • W. H. Wilson Tang,
    • Joseph A. DiDonato &
    • Stanley L. Hazen
  3. Department of Medicine/Division of Cardiology, BH-307 Center for the Health Sciences, University of California, Los Angeles, California 90095, USA

    • Brian J. Bennett &
    • Aldons J. Lusis
  4. Department of Mathematics, Cleveland State University, Cleveland, Ohio 44115, USA

    • Yuping Wu
  5. Bariatric and Metabolic Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA

    • Phil Schauer
  6. Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio 44195, USA

    • Jonathan D. Smith,
    • W. H. Wilson Tang &
    • Stanley L. Hazen
  7. Department of Preventive Medicine and Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California 90089, USA

    • Hooman Allayee


Z.W. performed metabolomics analyses, and biochemical, cellular, animal model and mass spectrometry studies. He assisted with statistical analyses, and assisted in both drafting and critical review of the manuscript. E.K., B.D. and J.D.S. assisted with performance of animal models and their analyses. B.S.L. synthesized d9-DPPC and assisted in metabolomics/mass spectrometry analyses. B.J.B., H.A. and A.J.L. performed the mouse eQTL experiments and analyses, and assisted in both drafting and critical review of the manuscript. A.J.L. provided some funding for the study. R.K., E.B.B., X.F. and Y.-M.C. performed mass spectrometry analyses of clinical samples. Y.W. performed statistical analysis. A.E.F. and P.S. helped with collection of human liver biopsy material and interpretation of biochemical and pathological examination of animal liver for steatosis. W.H.W.T. assisted in GeneBank study design and enrolment, as well as analyses of clinical studies and critical review of the manuscript. J.A.D. assisted in clinical laboratory testing for human clinical studies, animal model experimental design, and critical review of the manuscript. S.L.H. conceived of the idea, designed experiments, assisted in data analyses, the drafting and critical review of the manuscript, and provided funding for the study.

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