Intestinal microbiota metabolism of l-carnitine, a nutrient in red meat, promotes atherosclerosis

Journal name:
Nature Medicine
Volume:
19,
Pages:
576–585
Year published:
DOI:
doi:10.1038/nm.3145
Received
Accepted
Published online

Abstract

Intestinal microbiota metabolism of choline and phosphatidylcholine produces trimethylamine (TMA), which is further metabolized to a proatherogenic species, trimethylamine-N-oxide (TMAO). We demonstrate here that metabolism by intestinal microbiota of dietary l-carnitine, a trimethylamine abundant in red meat, also produces TMAO and accelerates atherosclerosis in mice. Omnivorous human subjects produced more TMAO than did vegans or vegetarians following ingestion of l-carnitine through a microbiota-dependent mechanism. The presence of specific bacterial taxa in human feces was associated with both plasma TMAO concentration and dietary status. Plasma l-carnitine levels in subjects undergoing cardiac evaluation (n = 2,595) predicted increased risks for both prevalent cardiovascular disease (CVD) and incident major adverse cardiac events (myocardial infarction, stroke or death), but only among subjects with concurrently high TMAO levels. Chronic dietary l-carnitine supplementation in mice altered cecal microbial composition, markedly enhanced synthesis of TMA and TMAO, and increased atherosclerosis, but this did not occur if intestinal microbiota was concurrently suppressed. In mice with an intact intestinal microbiota, dietary supplementation with TMAO or either carnitine or choline reduced in vivo reverse cholesterol transport. Intestinal microbiota may thus contribute to the well-established link between high levels of red meat consumption and CVD risk.

At a glance

Figures

  1. TMAO production from l-carnitine is a microbiota-dependent process in humans.
    Figure 1: TMAO production from l-carnitine is a microbiota-dependent process in humans.

    (a) Structure of carnitine and scheme of carnitine and choline metabolism to TMAO. l-Carnitine and choline are both dietary trimethylamines that can be metabolized by microbiota to TMA. TMA is then further oxidized to TMAO by flavin monooxygenases (FMOs). (b) Scheme of the human l-carnitine challenge test. After a 12-h overnight fast, subjects received a capsule of d3-(methyl)-carnitine (250 mg) alone, or in some cases (as in data for the subject shown) also an 8-ounce steak (estimated 180 mg l-carnitine), whereupon serial plasma and 24-h urine samples were obtained for TMA and TMAO analyses (visit 1). After a weeklong regimen of oral broad-spectrum antibiotics to suppress the intestinal microbiota, the challenge was repeated (visit 2), and then again a final third time after a ≥3-week period to permit repopulation of intestinal microbiota (visit 3). (c,d) LC-MS/MS chromatograms of plasma TMAO (c) and d3-TMAO (d) in an omnivorous subject using specific precursor right arrow product ion transitions indicated at t = 8 h for each visit. (e) Stable-isotope-dilution LC-MS/MS time course measurements of d3-labeled TMAO and carnitine in plasma collected from sequential venous blood draws at the indicated time points. Data shown in ce are from a representative female omnivorous subject who underwent carnitine challenge. Data are organized vertically to correspond with the visit schedule indicated in b.

  2. The formation of TMAO from ingested l-carnitine is negligible in vegans, and fecal microbiota composition associates with plasma TMAO concentrations.
    Figure 2: The formation of TMAO from ingested l-carnitine is negligible in vegans, and fecal microbiota composition associates with plasma TMAO concentrations.

    (a,b) Data from a male vegan subject in the carnitine challenge consisting of co-administration of 250 mg d3-(methyl)-carnitine and an 8-ounce sirloin steak and, for comparison, a representative female omnivore who frequently consumes red meat. Plasma TMAO and d3-TMAO were quantified after l-carnitine challenge (a) and in a 24-h urine collection (b). Urine TMAO and d3-TMAO reported as ratio with urinary creatinine (Cr) to adjust for urinary dilution. Data are expressed as means ± s.e.m. (c) Baseline fasting plasma concentrations of TMAO and d3-TMAO from male and female vegans and vegetarians (n = 26) and omnivores (n = 51). Boxes represent the 25th, 50th, and 75th percentiles and whiskers represent the 10th and 90th percentiles. (d) Plasma d3-TMAO concentrations in male and female vegans and vegetarians (n = 5) and omnivores (n = 5) participating in a d3-(methyl)-carnitine (250 mg) challenge without concomitant steak consumption. The P value shown is for the comparison of the area under the curve (AUC) of groups using the Wilcoxon nonparametric test. Data points represent mean ± s.e.m. of n = 5 per group. (e) Baseline TMAO plasma concentrations associate with enterotype 2 (Prevotella) in male and female subjects with a characterized gut microbiome enterotype. Boxes represent the 25th, 50th (middle lines) and 75th percentiles, and whiskers represent the 10th and 90th percentiles. (f) Plasma TMAO concentrations (plotted on x axes) and the proportion of taxonomic operational units (OTUs, plotted on y axes), determined as described in Supplementary Methods. Subjects were grouped by dietary status as either vegan or vegetarian (n = 23) or omnivore (n = 30). P value shown is for comparisons between dietary groups using a robust Hotelling T2 test. Data are expressed as means ± s.e.m. for both TMAO concentration (x axis) and the proportion of OTUs (y axis).

  3. The metabolism of carnitine to TMAO is an inducible trait and associates with microbiota composition.
    Figure 3: The metabolism of carnitine to TMAO is an inducible trait and associates with microbiota composition.

    (a) d3-carnitine challenge of mice on either an l-carnitine–supplemented diet (1.3%) for 10 weeks and compared to age-matched normal chow–fed controls. Plasma d3-TMA and d3-TMAO were measured at the indicated times after d3-(methyl)-carnitine administration by oral gavage using stable-isotope-dilution LC-MS/MS. Data points represent mean ± s.e.m. of n = 4 per group. (b) Correlation heat map demonstrating the association between the indicated microbiota taxonomic genera and TMA and TMAO concentrations (all reported as mean ± s.e.m. in μM) of mice grouped by dietary status (chow, n = 10 (TMA, 1.3 ± 0.4; TMAO, 17 ± 1.9); and l-carnitine, n = 11 (TMA, 50 ± 16; TMAO, 114 ± 16). Red denotes a positive association, blue a negative association, and white no association. A single asterisk indicates a significant FDR-adjusted association of P ≤ 0.1, and a double asterisk indicates a significant FDR-adjusted association of P ≤ 0.01. (c) Plasma TMAO and TMA concentrations determined by stable-isotope-dilution LC-MS/MS (plotted on x axes) and the proportion OTUs (plotted on y axes). Statistical and laboratory analyses were performed as described in Supplementary Methods. Data are expressed as means ± s.e.m. for both TMAO or TMA concentrations (x axis) and the proportion of OTUs (y axis).

  4. Relationship between plasma carnitine concentration and CVD risks.
    Figure 4: Relationship between plasma carnitine concentration and CVD risks.

    (ac) Forrest plots of the odds ratio of CAD (a), PAD (b) and CVD (c) and quartiles of carnitine before (closed circles) and after (open circles) logistic regression adjustments with traditional cardiovascular risk factors, including age, sex, history of diabetes mellitus, smoking, systolic blood pressure, LDL cholesterol and HDL cholesterol. Bars represent 95% confidence intervals. (d) Relationship of fasting plasma carnitine concentrations and angiographic evidence of CAD. Boxes represent the 25th, 50th and 75th percentiles of plasma carnitine concentration, and whiskers represent the 10th and 90th percentiles. The Kruskal-Wallis test was used to assess the degree of CAD (none, single-, double- or triple-vessel disease) association with plasma carnitine concentrations. (e) Forrest plot of the hazard ratio of MACE and quartiles of carnitine unadjusted (closed circles) and after adjusting for traditional cardiovascular risk factors (open circles), or traditional cardiac risk factors plus creatinine clearance, history of myocardial infarction, history of CAD, burden of CAD (one-, two- or three-vessel disease), left ventricular ejection fraction, baseline medications (angiotensin-converting enzyme (ACE) inhibitors, statins, beta blockers and aspirin) and TMAO levels (open squares). Bars represent 95% confidence intervals. (f) Kaplan-Meier plot and hazard ratios with 95% confidence intervals for unadjusted model, or following adjustments for traditional risk factors as in e. Median plasma concentration of carnitine (46.8 μM) and TMAO (4.6 μM) within the cohort were used to stratify subjects as having 'high' (≥median) or 'low' (<median) values.

  5. Dietary l-carnitine accelerates atherosclerosis and inhibits reverse cholesterol transport in a microbiota dependent fashion.
    Figure 5: Dietary l-carnitine accelerates atherosclerosis and inhibits reverse cholesterol transport in a microbiota dependent fashion.

    (a) Representative oil red O–stained aortic roots (counterstained with hematoxylin) of 19-week-old Apoe−/− female mice on the indicated diets in the presence versus absence of antibiotics (ABS) as described in the Online Methods. (b) Quantification of mouse aortic root plaque lesion area. Apoe−/− female mice at 19 weeks of age were started on the indicated diets at the time of weaning (4 weeks of age) before killing, and lesion area was quantified as described in the Online Methods. (c) Carnitine, TMA and TMAO concentrations as determined using stable-isotope-dilution LC-MS/MS analysis of plasma recovered from mice at the time of killing. (d) RCT (72-h stool collection) in adult female (>8 weeks of age) Apoe−/− mice on normal chow versus diet supplemented with either l-carnitine or choline, as well as after suppression of microbiota using cocktail of antibiotics (+ ABS). Also shown are RCT (72-h stool collection) results in adult female (>8 weeks of age) Apoe−/− mice on normal chow versus diet supplemented with TMAO. (e,f) Relative mRNA levels (to Actb) of mouse liver candidate genes involved in bile acid synthesis or transport. Data are expressed as means ± s.e.m.

  6. Effect of TMAO on cholesterol and sterol metabolism.
    Figure 6: Effect of TMAO on cholesterol and sterol metabolism.

    (a,b) Measurement of total bile acid pool size and composition (a) and cholesterol absorption (b) in adult female (>8 weeks of age) Apoe−/− mice on normal chow diet versus diet supplemented with TMAO for 4 weeks. Data are expressed as means ± s.e.m. (c) Summary scheme outlining the proposed pathway by which microbiota participate in atherosclerosis. The microbiota metabolizes dietary l-carnitine and choline to form TMA and TMAO. TMAO affects cholesterol and sterol metabolism in macrophages, liver and intestine.

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Author information

Affiliations

  1. Department of Cellular & Molecular Medicine, Cleveland Clinic, Cleveland, Ohio, USA.

    • Robert A Koeth,
    • Zeneng Wang,
    • Bruce S Levison,
    • Jennifer A Buffa,
    • Brendan T Sheehy,
    • Earl B Britt,
    • Xiaoming Fu,
    • Lin Li,
    • Jonathan D Smith,
    • Joseph A DiDonato,
    • W H Wilson Tang &
    • Stanley L Hazen
  2. Center for Cardiovascular Diagnostics & Prevention, Cleveland Clinic, Cleveland, Ohio, USA.

    • Robert A Koeth,
    • Zeneng Wang,
    • Bruce S Levison,
    • Jennifer A Buffa,
    • Earl B Britt,
    • Xiaoming Fu,
    • Lin Li,
    • Jonathan D Smith,
    • Joseph A DiDonato,
    • W H Wilson Tang &
    • Stanley L Hazen
  3. Department of Medicine, Division of Cardiology, David Geffen School of Medicine, University of California–Los Angeles, Los Angeles, California, USA.

    • Elin Org &
    • Aldons J Lusis
  4. Department of Mathematics, Cleveland State University, Cleveland, Ohio, USA.

    • Yuping Wu
  5. Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio, USA.

    • Jonathan D Smith,
    • W H Wilson Tang,
    • Frederic D Bushman &
    • Stanley L Hazen
  6. Department of Microbiology, Center for Clinical Epidemiology and Biostatistics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Jun Chen,
    • Hongzhe Li &
    • James D Lewis
  7. Division of Gastroenterology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Gary D Wu
  8. Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • James D Lewis
  9. Department of Pathology, Section on Lipid Sciences, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.

    • Manya Warrier &
    • J Mark Brown
  10. Children's Hospital Oakland Research Institute, Oakland, California, USA.

    • Ronald M Krauss

Contributions

R.A.K. participated in laboratory, mouse and human studies, assisted in statistical analyses, helped design the experiments and drafted the manuscript. Z.W. performed the initial metabolomics study and assisted with mouse and mass spectrometry analyses. B.S.L. synthesized d3- and d9-carnitine for studies, assisted with mass spectrometry analyses and helped draft the manuscript. E.B.B. and X.F. assisted in performance of mass spectrometry analyses of the large human clinical cohort study. Y.W. and L.L. performed the statistical analyses and critically reviewed the manuscript. J.D.S. helped with aortic root atherosclerosis analyses and critical review of the manuscript. J.A.D. assisted in experimental design. J.A.B. and B.T.S. assisted in laboratory and mouse experiments. E.O. and A.J.L. performed and helped interpret mouse cecal microbiota analyses. J.C., F.D.B., H.L., G.D.W., J.D.L. and R.M.K. assisted in human subject microbiota analyses and helped interpret human microbiota data. M.W. and J.M.B. assisted with measurement of bile acid pool size and helped with critical review of the manuscript. W.H.W.T. helped with human studies and critical review of the manuscript. S.L.H. conceived of the idea, helped design the experiments, provided the funding for the study and helped draft and critically revise the manuscript.

Competing financial interests

Z.W. and B.S.L. are named as co-inventors on pending patents held by the Cleveland Clinic relating to cardiovascular diagnostics and have the right to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics from Liposciences. W.H.W.T. received research grant support from Abbott Laboratories and served as a consultant for Medtronic and St. Jude Medical. S.L.H. and J.D.S. are named as co-inventors on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics patents. S.L.H. has been paid as a consultant or speaker by the following companies: Cleveland Heart Lab., Esperion, Liposciences, Merck & Co. and Pfizer. He has received research funds from Abbott, Cleveland Heart Lab., Esperion and Liposciences and has the right to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics from Abbott Laboratories, Cleveland Heart Lab., Frantz Biomarkers, Liposciences and Siemens.

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