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Clinical Trial
. 2019 Jan 2;129(1):373-387.
doi: 10.1172/JCI94601. Epub 2018 Dec 10.

l-Carnitine in omnivorous diets induces an atherogenic gut microbial pathway in humans

Robert A Koeth  1   2   3 , Betzabe Rachel Lam-Galvez  4 , Jennifer Kirsop  1   2 , Zeneng Wang  1   2 , Bruce S Levison  1 , Xiaodong Gu  1   2 , Matthew F Copeland  4 , David Bartlett  1 , David B Cody  4 , Hong J Dai  5 , Miranda K Culley  1 , Xinmin S Li  1   2 , Xiaoming Fu  1   2 , Yuping Wu  6 , Lin Li  1   2 , Joseph A DiDonato  1   2 , W H Wilson Tang  1   2   3 , Jose Carlos Garcia-Garcia  4 , Stanley L Hazen  1   2   3
Affiliations
Clinical Trial

l-Carnitine in omnivorous diets induces an atherogenic gut microbial pathway in humans

Robert A Koeth et al. J Clin Invest. .

Abstract

Background: l-Carnitine, an abundant nutrient in red meat, accelerates atherosclerosis in mice via gut microbiota-dependent formation of trimethylamine (TMA) and trimethylamine N-oxide (TMAO) via a multistep pathway involving an atherogenic intermediate, γ-butyrobetaine (γBB). The contribution of γBB in gut microbiota-dependent l-carnitine metabolism in humans is unknown.

Methods: Omnivores and vegans/vegetarians ingested deuterium-labeled l-carnitine (d3-l-carnitine) or γBB (d9-γBB), and both plasma metabolites and fecal polymicrobial transformations were examined at baseline, following oral antibiotics, or following chronic (≥2 months) l-carnitine supplementation. Human fecal commensals capable of performing each step of the l-carnitine→γBB→TMA transformation were identified.

Results: Studies with oral d3-l-carnitine or d9-γBB before versus after antibiotic exposure revealed gut microbiota contribution to the initial 2 steps in a metaorganismal l-carnitine→γBB→TMA→TMAO pathway in subjects. Moreover, a striking increase in d3-TMAO generation was observed in omnivores over vegans/vegetarians (>20-fold; P = 0.001) following oral d3-l-carnitine ingestion, whereas fasting endogenous plasma l-carnitine and γBB levels were similar in vegans/vegetarians (n = 32) versus omnivores (n = 40). Fecal metabolic transformation studies, and oral isotope tracer studies before versus after chronic l-carnitine supplementation, revealed that omnivores and vegans/vegetarians alike rapidly converted carnitine to γBB, whereas the second gut microbial transformation, γBB→TMA, was diet inducible (l-carnitine, omnivorous). Extensive anaerobic subculturing of human feces identified no single commensal capable of l-carnitine→TMA transformation, multiple community members that converted l-carnitine to γBB, and only 1 Clostridiales bacterium, Emergencia timonensis, that converted γBB to TMA. In coculture, E. timonensis promoted the complete l-carnitine→TMA transformation.

Conclusion: In humans, dietary l-carnitine is converted into the atherosclerosis- and thrombosis-promoting metabolite TMAO via 2 sequential gut microbiota-dependent transformations: (a) initial rapid generation of the atherogenic intermediate γBB, followed by (b) transformation into TMA via low-abundance microbiota in omnivores, and to a markedly lower extent, in vegans/vegetarians. Gut microbiota γBB→TMA/TMAO transformation is induced by omnivorous dietary patterns and chronic l-carnitine exposure.

Trial registration: ClinicalTrials.gov NCT01731236.

Funding: NIH and Office of Dietary Supplements grants HL103866, HL126827, and DK106000, and the Leducq Foundation.

Keywords: Atherosclerosis; Cardiology; Cardiovascular disease; Vascular Biology.

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Conflict of interest statement

Conflict of interest: ZW, BSL, and SLH are named as co-inventors on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics, and have the right to receive royalty payment for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland HeartLab, Quest Diagnostics, and Procter & Gamble. SLH also reports having been paid as a consultant by Procter & Gamble, and having received research funds from Procter & Gamble and Roche. BRLG, MFC, DBC, HJD, and JCGG are employees of Procter & Gamble and are named as co-inventors on patents relevant to this work.

Figures

Figure 1
Figure 1. γBB and TMAO production from l-carnitine is a gut microbiota–dependent process in humans.
Subjects (n = 5) ingested a capsule containing d3-l-carnitine (250 mg; t0), after which serial plasma aliquots were obtained at the times shown (Baseline, filled circles). After a week-long regimen of oral broad-spectrum antibiotics to suppress the intestinal microbiota, the oral l-carnitine challenge was repeated (+ Abx, open circles). Stable isotope dilution LC-MS/MS was used to quantify d3-TMAO (A), d3-γBB (B), and d3-l-carnitine (C) in plasma collected from sequential venous blood draws at the noted times. Time points are represented as mean ± SEM plasma concentrations, and a zero-inflated linear mixed-effects model was used to compare subjects before and after antibiotic exposure.
Figure 2
Figure 2. TMAO is a gut microbiota–dependent product of γBB in humans.
Subjects (n = 6) received oral d9-γBB (250 mg; t0), and then serial plasma aliquots were obtained at the noted time points (Baseline, filled circles). After a week-long regimen of oral broad-spectrum antibiotics to suppress the intestinal microbiota, the oral d9-γBB challenge was repeated (+ Abx, open circles). Stable isotope dilution LC-MS/MS was used to quantify d9-TMAO (A), d9-γBB (B), and d9-l-carnitine (C) in plasma collected from sequential venous blood draws at the noted times. Time points are mean ± SEM plasma concentrations, and a zero-inflated linear mixed-effects model was used to compare subjects before and after antibiotic exposure.
Figure 3
Figure 3. γBB is a major gut microbiota metabolite of l-carnitine, and TMA formation from γBB is influenced by dietary habits.
(A) Plasma d3-TMAO and d3-γBB concentrations in vegans/vegetarians (d3-TMAO, n = 9; d3-γBB, n = 9) versus omnivores (d3-TMAO, n = 15; d3-γBB, n = 12) participating in an oral d3-l-carnitine (250 mg) challenge. The left panel illustrates the marked differences in d3-TMAO generation previously reported in omnivores versus vegans/vegetarians. The right panel shows a small difference in plasma d3-γBB concentration between omnivores and vegans/vegetarians. Data represent mean ± SEM. A Mann-Whitney test was used to compare the AUCs between dietary groups. (B) Box-and-whisker plots of fasting plasma concentrations of γBB from subjects (n = 9) before versus after 1 week of oral broad-spectrum antibiotics to suppress gut microbiota. Boxes represent the 25th, 50th, and 75th percentiles, and whiskers represent the 10th and 90th percentiles. Differences were assessed using a Wilcoxon matched-pairs test. (C) Fasting plasma concentrations of γBB in vegans/vegetarians (n = 32) versus omnivores (n = 40). Boxes represent the 25th, 50th, and 75th percentiles, and whiskers represent the 10th and 90th percentiles. A Mann-Whitney test was used to assess differences between groups. (D) Baseline human fecal metabolite studies in vegans/vegetarians and omnivores (n = 10 each group). Fecal samples were incubated anaerobically with d3-l-carnitine, and d3-TMA and d3-γBB were quantified by LC-MS/MS. Data are expressed as mean ± SEM. A Mann-Whitney test was used to assess differences between groups. (E) Baseline human fecal metabolite studies in vegans/vegetarians (n = 10) versus omnivores (n = 10). Fecal samples were incubated with d3-l-carnitine or d9-γBB as indicated. Metabolites were quantified by LC-MS/MS. Data are expressed as mean ± SEM. A Mann-Whitney test was used to assess differences between groups.
Figure 4
Figure 4. l-Carnitine supplementation enhances the synthetic capacity of gut microbiota to form TMAO.
(A) Plasma d3-TMAO concentrations in sequential venous blood draws after oral d3-l-carnitine challenge in vegans (n = 7) and omnivores (n = 8) at baseline, visit 1 (V1, 1 month), and visit 2 (V2, 2–3 months). Data represent mean ± SEM. A zero-inflated linear mixed-effects model reveals that plasma d3-TMAO is significantly higher after l-carnitine supplementation. (B) Plasma TMAO concentrations in vegans (n = 7) and omnivores (n =10) at baseline and following daily l-carnitine supplementation at visit 2 (V1, 1 month), and visit 3 (V2, 2 months). Boxes represent the 25th, 50th, and 75th percentiles, and whiskers represent the 10th and 90th percentiles. A repeated-measures 1-way ANOVA test was used to assess differences between baseline visits. (C) Plasma d3-γBB concentrations in sequential venous blood draws after oral d3-l-carnitine challenge in vegans/vegetarians (n = 7) and omnivores (n = 5) at baseline, visit 1 (V1, 1 month), and visit 2 (V2, 2 months). Data represent mean ± SEM. A zero-inflated linear mixed-effects model reveals that plasma d3-γBB production is not significantly higher after l-carnitine supplementation in vegans/vegetarians or omnivores.
Figure 5
Figure 5. l-Carnitine supplementation does not enhance the synthetic capacity of gut microbiota to produce γBB from l-carnitine, but does enhance gut microbiota–dependent transformation of γBB into TMA.
At baseline and after l-carnitine supplementation (l-Carn; 2–3 months), human fecal metabolite studies (n = 6 omnivores and n = 7 vegans/vegetarians) in the conversion of d9-l-carn to d9-TMA (A), d9-l-carn to γBB (B), or d9-γBB to d9-TMA (C). Fecal samples were incubated with either d9-l-carnitine or d9-γBB as described in Methods, and d9-TMA and d9-γBB were quantified by LC-MS/MS. A Wilcoxon matched-pairs test was used to assess for differences between groups.
Figure 6
Figure 6. l-Carnitine catabolism to TMA involves multiple microorganisms.
(A) l-Carnitine–enriched fecal communities were plated on solid media. Single microbial colonies and subcommunities of up to 3 and 4+ colonies were picked and tested for their l-carnitine→TMA activity. The top producing community was refractionated to select the highest TMA-producing subcommunity. (B) Species pools (SP1 through SP5) were evaluated alone or in combination for l-carnitine→TMA activity in n = 2 replicate values. Data are expressed as the mean. (C) Combinations of individual members of SP2 and SP5 were evaluated for TMA production. Concentrations of d9-TMA were determined by stable isotope dilution LC-MS/MS.
Figure 7
Figure 7. Anaerobic microbial l-carnitine consumption is associated with the presence of the microbial caiTABCDE genes.
(A) Individual SP2-71 microbes were evaluated alone or in combination with E. lenta SP5-62 for l-carnitine→TMA activity. Data are expressed as the mean of the d9-l-carnitine peak ratio and d9-TMA concentration in n = 2 replicate values. (B) E. lenta genome mining revealed the presence of the caiTABCDE gene operon. Data are expressed as the mean. (C) l-Carnitine→γBB pathway involving enzymes encoded by the caiTABCDE gene operon. Crotono, crotonobetaine.
Figure 8
Figure 8. CaiTABCDE gene–expressing microbes consume l-carnitine, but only make TMA in the presence of E. timonensis.
Concentrations of l-carnitine and TMA were determined by stable isotope dilution LC-MS/MS in n = 3 replicate values. Data are expressed as mean ± SEM.
Figure 9
Figure 9. Anaerobic microbial l-carnitine catabolism generates γBB as an intermediate.
(A) Kinetic changes of l-carnitine, γBB, and TMA were determined in cultures of P. penneri alone or in combination with E. timonensis, SP2-71.3, supplemented with l-carnitine (top panel) or γBB (bottom panel). Concentrations were determined in n = 2 replicate values. Data are expressed as the mean. (B) Selected human fecal communities from 4 different donors were studied for their l-carnitine→γBB→TMA activity sampling (n = 1) every 4 hours for 32 hours. Concentrations of l-carnitine, γBB, and TMA were determined by stable isotope dilution LC-MS/MS. All human subjects are displayed in Supplemental Figure 6.
Figure 10
Figure 10. Scheme of l-carnitine and γBB metabolism in humans, links to gut microbial TMAO generation, and adverse cardiometabolic phenotypes.
FMO, flavin-containing monooxygenase.
See this image and copyright information in PMC

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References

    1. Brown JM, Hazen SL. The gut microbial endocrine organ: bacterially derived signals driving cardiometabolic diseases. Annu Rev Med. 2015;66:343–359. doi: 10.1146/annurev-med-060513-093205. - DOI - PMC - PubMed
    1. Jonsson AL, Bäckhed F. Role of gut microbiota in atherosclerosis. Nat Rev Cardiol. 2017;14(2):79–87. doi: 10.1038/nrcardio.2016.183. - DOI - PubMed
    1. Blaser MJ. The microbiome revolution. J Clin Invest. 2014;124(10):4162–4165. doi: 10.1172/JCI78366. - DOI - PMC - PubMed
    1. Zhu W, et al. Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk. Cell. 2016;165(1):111–124. doi: 10.1016/j.cell.2016.02.011. - DOI - PMC - PubMed
    1. Wang Z, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 2011;472(7341):57–63. doi: 10.1038/nature09922. - DOI - PMC - PubMed

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