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Cited by 546Gut microbiota and healthPolyamine Metabolism and ApplicationsNutritional Studies and Diet

γ-Butyrobetaine Is a Proatherogenic Intermediate in Gut Microbial Metabolism of L-Carnitine to TMAO

Open access
Koeth, Robert A., Levison, Bruce S., Culley, Miranda K., Buffa, Jennifer A., Wang, Zeneng, Gregory, Jill C., Org, Elin, Wu, Yuping +6 more·2014·Cell Metabolism
DOI 10.1016/j.cmet.2014.10.006PMC PMC4255476
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Abstract

L-carnitine, a nutrient in red meat, was recently reported to accelerate atherosclerosis via a metaorganismal pathway involving gut microbial trimethylamine (TMA) formation and host hepatic conversion into trimethylamine-N-oxide (TMAO). Herein, we show that following L-carnitine ingestion, γ-butyrobetaine (γBB) is produced as an intermediary metabolite by gut microbes at a site anatomically proximal to and at a rate ∼1,000-fold higher than the formation of TMA. Moreover, we show that γBB is the major gut microbial metabolite formed from dietary L-carnitine in mice, is converted into TMA and TMAO in a gut microbiota-dependent manner (like dietary L-carnitine), and accelerates atherosclerosis. Gut microbial composition and functional metabolic studies reveal that distinct taxa are associated with the production of γBB or TMA/TMAO from dietary L-carnitine. Moreover, despite their close structural similarity, chronic dietary exposure to L-carnitine or γBB promotes development of functionally distinct microbial communities optimized for the metabolism of L-carnitine or γBB, respectively.

AI snapshot

Written from the paper’s full text.

TL;DR

This paper identifies γ-butyrobetaine (γBB) as a gut microbial intermediate in the conversion of dietary L-carnitine to trimethylamine (TMA) and trimethylamine-N-oxide (TMAO), and shows that γBB supplementation accelerates atherosclerosis in mice in a microbiota-dependent manner. It also identifies a microbial enzyme complex, yeaW/X, that can produce TMA from γBB and shows that chronic L-carnitine versus γBB feeding selects functionally distinct gut microbial communities.

Key findings

  • In L-carnitine-supplemented mice, plasma γBB increased nearly 100-fold over chow-fed controls and was almost eliminated by oral antibiotics; γBB concentrations were about twofold higher than carnitine, TMA, or TMAO. Germ-free mice produced no detectable d3-γBB after labeled L-carnitine until conventionalized.
  • Ex vivo gut segment incubations showed γBB formation from L-carnitine mainly in jejunum and ileum, while TMA formation occurred mainly in cecum; d3-γBB production from d3-carnitine was about 1,000-fold higher than d3-TMA, and TMA formation from γBB was about 1.5-fold higher than from equimolar L-carnitine.
  • Oral d9-γBB generated d9-TMA and d9-TMAO in conventional mice but not in antibiotic-treated or germ-free mice; capacity returned after recolonization. A γBB-supplemented diet (1.3%) increased aortic root plaque area by 50% versus chow, and this increase was absent when gut microbes were suppressed despite high plasma γBB.
  • Recombinant E. coli yeaW/X complex showed TMA lyase activity and acted on multiple substrates including γBB, L-carnitine, choline, and betaine. Chronic L-carnitine versus γBB diets produced distinct microbial taxa associations; for example, Akkermansia associated with TMAO in γBB-fed mice, while Parasutterella, Prevotella, and Bacteroides associated with γBB levels in L-carnitine-fed mice.

Methods

  • Mouse experiments used C57BL/6J and Apoe−/− mice, plus germ-free Swiss Webster mice, with diets supplemented with L-carnitine or γBB (1.3% gm/gm or in drinking water), antibiotic cocktails to suppress gut microbes, oral gavage of deuterium-labeled tracers, plasma LC/MS/MS quantification, and aortic root plaque measurement at 19 weeks.
  • Ex vivo gut segment incubations used d9-γBB and d3-L-carnitine under anaerobic conditions; plasma clearance studies injected labeled trimethylamines intravenously; microbial composition was assessed by 16S rRNA gene sequencing from cecum, jejunum, and ileum.
  • The yeaW/X enzyme complex was identified by bioinformatic search of E. coli genes, cloned, expressed, purified, and tested for TMA lyase activity.

Limitations

  • The authors note that ex vivo gut segment incubations lack neuro-endocrine inputs and the dynamic metabolism of the intact gastrointestinal tract, so conclusions from those experiments should be considered in that context.
  • The authors state that a causal role for TMAO in human atherosclerosis remains unproven, that no current studies directly link γBB to human atherosclerosis, and that long-term safety studies with supplemental γBB, choline, and carnitine are warranted.
  • Evident from the text: the atherosclerosis and microbial findings are from mouse models using dietary supplementation and antibiotic suppression, so human relevance is not established; also, mildronate was found to be nonspecific and inhibited several microbial reactions, limiting its use as a tool.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.cmet.2014.10.006

Cites

36 of 36 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 31 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    After antibiotic withdrawal and recolonization, mice regained the capacity to produce d9-TMA and d9-TMAO from oral d9-γBB.

    “After withdrawal of the oral antibiotics and a period of housing the mice in conventional cages to permit recolonization, there was reacquisition of the capacity to produce d9-TMA and d9-TMAO from oral d9-γBB”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The oral antibiotic cocktail suppresses gut microbial metabolism without directly affecting host atherosclerosis or trimethylamine/TMAO metabolism.
  • Findingown data

    No detectable d3-isotopologues of betaine, glycine, or 3-dehydrocarnitine were produced from d3-carnitine in plasma or ex vivo gut segment incubations under aerobic or anaerobic conditions.

    “We observed no detectable production of d3-isotopologues of betaine, glycine, or 3-dehydrocarnitine in either plasma (multiple time points between 0–24 hours) of C57BL/6J female mice challenged (gastric gavage) with d3-carnitine or in the ex vivo gut segment incubation experiments under either anaerobic or aerobic conditions (Figure S3, and data not shown).”
    Quote in the paperSubstantial claim
  • Findingown data

    Germ-free mice given oral L-d3(methyl)-carnitine had no detectable plasma d3-γBB.

    “Serial plasma measurements following oral L-d3(methyl)-carnitine showed no detectable d3-γBB (Figure 1D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Germ-free mice lack gut microbes, and conventionalization restores a microbiota capable of normal gut microbial metabolism.
  • Findingown data

    Production of trans crotonobetaine from carnitine was almost two orders of magnitude lower than production of γBB or TMA/TMAO.

    “However, the production of trans crotonobetaine was significantly (almost 2 orders of magnitude) less than the production of γBB or TMA/TMAO from carnitine.”
    Quote in the paperSubstantial claim
  • Findingown data

    In Apoe−/− mice, a γBB-supplemented diet (1.3% gm/gm) increased aortic root atherosclerotic plaque total area by 50% compared with chow-fed littermate controls.

    “A significant 50% increase in total area of plaque was noted within the γBB diet supplemented mice compared to their littermate controls on chow diet (Figure 4A,B).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 20
  • May combine two claims. 29

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    A similar modest significant increase in plasma triglycerides occurred in antibiotic-treated γBB-supplemented mice versus chow plus antibiotic controls.

    “A similar modest significant increase in plasma triglyceride concentrations, however, was also noted within antibiotic treated mice in the γBB-supplemented dietary arm versus controls (chow +ABS; Table S1).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    No other pro-atherosclerotic alterations were found; HDL cholesterol and glucose levels were comparable among groups.

    “Further analyses of plasma showed no other pro-atherosclerotic alterations, with comparable HDL cholesterol and glucose levels observed among the different groups of mice.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The small bowel, especially ileum and jejunum, was a major initial site of γBB production from L-carnitine, anatomically proximal to TMA production sites.

    “Second, the small bowel (jejunum and especially ileum) served as a major initial site of production of γBB from L-carnitine, anatomically proximal to those for TMA production (Figure 3A, right panel).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After oral or parenteral d9-carnitine, plasma d9-carnitine levels were similar within about a factor of two, but plasma d9-TMAO was more than 100-fold lower in the parenteral group.

    “While the plasma levels of d9-carnitine achieved in the two groups of mice were on the whole similar (i.e. within approximately a factor of two), the concentrations of plasma d9-TMAO observed were dramatically reduced (>100-fold less) in the mice that received parenteral d9-carnitine.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Akkermansia was significantly associated with TMAO production in γBB-supplemented mice in both cecum and ileum, but not significantly associated with TMAO levels in carnitine-supplemented mice.

    “Also of note, the genre Akkermansia was significantly associated with TMAO production in γBB supplemented mice in both the cecum and ilium, but was not significantly associated (all gut segment examined) with TMAO levels in carnitine supplemented mice.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Plasma clearance rates of γBB, TMA, and TMAO were within a factor of 2 of one another, and γBB was eliminated faster than TMA or TMAO.

    “Plasma clearance rates for trimethylamines were also determined, and observed to be within a factor of 2 of one another, with elimination of γBB being faster than that of either TMA or TMAO (Figure S2).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    Modest trans crotonobetaine production occurred from carnitine in ex vivo gut segments and after oral ingestion.

    “We did detect modest production of trans crotonobetaine in the ex vivo gut segment experiments, as well as following oral (gastric gavage) ingestion (Figure S3 and S4).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    TMA and TMAO production in γBB-supplemented mice showed no association with Bacteroides or Proteobacteria phyla, which co-associated with plasma γBB in L-carnitine-fed mice.

    “Interestingly, TMA and TMAO production in the γBB-supplemented mice showed no association with bacteria from the Bacteroides or Proteobacteria phyla, taxa that co-associated with plasma γBB levels in L-carnitine fed mice (Table S2).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    γBB-supplemented mice receiving antibiotics had the highest plasma γBB concentrations observed yet showed no increase in atherosclerotic plaque.

    “In fact, γBB-supplemented mice receiving antibiotics had the highest plasma γBB concentrations observed, yet showed no increase in atherosclerosis plaque (Figure 4A,B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The oral antibiotic cocktail suppresses gut microbial metabolism without directly affecting host atherosclerosis or trimethylamine/TMAO metabolism.

  • May combine two claims.

    Findingcitation

    In L-carnitine-supplemented mice, production of d3-TMA and d3-TMAO after oral d3-carnitine was significantly induced, and plasma d3-carnitine was significantly lower.

    “As previously described (Koeth et al., 2013), production of both d3-TMA and d3-TMAO were also significantly induced in the L-carnitine supplemented mice, and plasma concentrations of d3-carnitine were significantly lower (consistent with a large portion of dietary L-carnitine being catabolized at greater efficiency by gut microbes following the chronic dietary exposure) (Figure 5A).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Koeth et al., 2013 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Germ-free mice given oral d9-γBB produced no detectable d9-TMA or d9-TMAO but did produce d9-carnitine.

    “As observed in the antibiotic suppressed C57BL/6J mice, no detectable d9-TMA or d9-TMAO were formed following gastric gavage of mice with d9-γBB, whereas d9-carnitine was produced, consistent with the endogenous (host) pathway.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Germ-free mice lack gut microbes, and conventionalization restores a microbiota capable of normal gut microbial metabolism.

  • May combine two claims.

    Findingown data

    In the small bowel jejunum, Staphylococcus was the only taxon significantly associated with γBB levels, and it was not significantly associated with γBB in the cecum.

    “Further, the only microbial taxa in the small bowel (jejunum) that was significantly (FDR adjusted) associated with γBB levels was staphylococcus, which in cecum was not significantly associated with γBB levels (Table S2).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    Distinct cecal microbial taxa associated with TMA or TMAO production from dietary L-carnitine in past and present studies, or from dietary γBB in present studies, are known to be predominantly anaerobic species.

    “are known to be predominantly populated with anaerobic species”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Koeth et al., 2013 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Only a minimal level (2 orders of magnitude less) of d3-trans crotonobetaine was detected after d3-carnitine ingestion, and gut segment studies show similarly modest trans crotonobetaine production.

    “Only a minimal (2 orders of magnitude less) level of d3-trans crotonobetaine was detected following ingestion of d3-carnitine, and our studies with gut segments show similarly modest trans crotonobetaine production.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Ex vivo gut segment incubations reflect in vivo gut microbial metabolism sufficiently to support conclusions about anatomical distribution and pathway contributions.

  • May combine two claims.

    Findingown data

    Mice with the highest plasma TMAO concentrations had the most plaque at the aortic root, whereas those with suppressed gut microbes and lacking TMA/TMAO showed no increase in atherosclerosis.

    “mice with the highest plasma concentrations of TMAO had the most plaque at the aortic root, whereas those with suppressed gut microbes and lacking TMA/TMAO showed no increase in atherosclerosis”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Oral antibiotic treatment suppresses gut microbes and TMA/TMAO production without directly affecting atherosclerotic plaque formation.

  • May combine two claims.

    Findingown data

    Chronic L-carnitine supplemented mice demonstrated marked induced synthetic capacity to produce TMAO from L-carnitine but not γBB.

    “chronic L-carnitine supplemented mice demonstrated marked induced synthetic capacity to produce TMAO from L-carnitine but not γBB”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mice chronically supplemented with γBB demonstrated enhanced TMAO formation from oral γBB but not L-carnitine.

    “mice chronically supplemented with γBB demonstrated enhanced TMAO formation from oral γBB but not L-carnitine”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Neither carnitine nor γBB is likely the direct pro-atherogenic species, because both remained high in γBB-fed mice on antibiotics.

    “Analyses of plasma levels of carnitine, γBB, TMA and TMAO showed neither carnitine nor γBB were likely the direct pro-atherogenic species since they remained high in the γBB-fed mice on antibiotics (Figure 4C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The oral antibiotic cocktail suppresses gut microbial metabolism without directly affecting host atherosclerosis or trimethylamine/TMAO metabolism.

  • May combine two claims.

    Interpretationown data

    Carnitine synthesized endogenously from γBB enters the circulation, largely bypasses the gut lumen and microbes, and does not significantly contribute to TMA/TMAO generation.

    “Thus, carnitine synthesized via the endogenous pathway from γBB enters the circulation and essentially bypasses the gut lumen (and microbes), and does not significantly contribute to observed TMA/TMAO generation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    Oral d9-γBB is absorbed and converted to d9-carnitine in normal chow-fed mice, and endogenous L-carnitine biosynthesis is suppressed in L-carnitine-supplemented mice.

    “This latter data is consistent with oral d9-γBB being absorbed and converted into d9-carnitine in normal chow fed mice, and suppression of the endogenous L-carnitine biosynthesis pathway in mice supplemented with L-carnitine (Rebouche, 1983) (Figure 5B).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Rebouche, 1983 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Chronic L-carnitine exposure induces functional specificity for gut microbial metabolism of L-carnitine but not of the downstream metabolite γBB.

    “These results indicate that there is a remarkable functional specificity induced following chronic L-carnitine exposure for gut microbe-dependent metabolism of the proximal dietary nutrient provided in abundance, L-carnitine, but not the more downstream metabolite, γBB (Figure 5B).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Gut microbial metabolism of phosphatidylcholine produces TMAO via initial TMA formation and host hepatic flavin monooxygenase-mediated conversion.

    “Specifically, gut microbial metabolism of phosphatidylcholine, the major dietary source of choline, was first shown to produce a proatherogenic metabolite, trimethylamine-N-oxide (TMAO), via initial formation of trimethylamine (TMA), followed by host hepatic flavin monooxygenase mediated conversion to TMAO (Wang et al., 2011) (Figure S1; Gut Pathway1).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Wang et al., 2011 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A prior study linked γBB to TMA production in culture with the bacterial isolate Acinetobacter calcoaceticus.

    “Additionally, there is a study linking γBB to TMA production in culture with the bacterial isolate Acinetobacter calcoaceticu (Kleber et al., 1977).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Kleber et al., 1977

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Most studies consider the γBB-to-L-carnitine pathway unidirectional, catalyzed by γBB hydroxylase, with γBB produced during carnitine biosynthesis rather than catabolism.

    “Most studies consider the biochemical pathway of γBB → L-carnitine as unidirectional, catalyzed by the endogenous enzyme γBB hydroxylase (Rebouche and Engel, 1980), with γBB being a metabolite produced during L-carnitine biosynthesis, not catabolism (Figure S1; Endogenous Pathway).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Rebouche and Engel, 1980

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Prior studies demonstrated that gut microbes can convert carnitine into γBB.

    “There have been previous studies demonstrating that gut microbes can convert carnitine into γBB (Rebouche et al., 1984; Seim et al., 1985).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Rebouche et al., 1984 source

      Not checked in the free run.

    • cites Seim et al., 1985 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    An early rat study reported that orally ingested radioactively labeled L-carnitine can produce γBB.

    “Interestingly, an early study in rats examining orally ingested radioactively labeled L-carnitine reported that γ-butyrobetaine (γBB), another trimethylamine, can be produced (Rebouche et al., 1984) (Figure S1; Gut Pathway 3, first reaction).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Rebouche et al., 1984 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    TMA and TMAO do not significantly participate as intermediates in γBB formation.

    “arguing these more terminal trimethylamine catabolites do not significantly participate as an intermediate in the formation of γBB”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Ex vivo gut segment incubations reflect in vivo gut microbial metabolism sufficiently to support conclusions about anatomical distribution and pathway contributions.

  • May combine two claims.

    Premisereasoning

    No prior studies linked γBB to TMA generation in vivo or the sequential catabolism of L-carnitine into TMA and TMAO via γBB.

    “However, no studies have either linked γBB to TMA generation in vivo, or the sequential catabolism of L-carnitine into TMA and TMAO via the intermediate γBB.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Ex vivo gut segment incubations have the inherent limitation of not being subject to neuro-endocrine inputs or the dynamic metabolism of the intact GI tract within a host.

    “the ex vivo gut segment incubations have the inherent limitation of not being subject to neuro-endocrine inputs, nor the dynamic metabolism of the intact GI tract within a host”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    γBB serves as the proximal biosynthetic intermediate in endogenous L-carnitine production, which begins with dietary lysine and also utilizes methionine.

    “γBB serves as the proximal biosynthetic intermediate in endogenous L-carnitine production that begins with dietary lysine and also utilizes methionine (Figure 7) (Bremer, 1983; Rebouche, 1983; Rebouche and Chenard, 1991).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Bremer, 1983 source

      Not checked in the free run.

    • cites Rebouche, 1983 source

      Not checked in the free run.

    • cites Rebouche and Chenard, 1991

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    L-carnitine is mostly absorbed through active transport in the upper gastrointestinal tract, and only when that transport is saturated do appreciable levels reach more distal segments for gut microbial catabolism.

    “L-carnitine is mostly absorbed through active transport in the upper gastrointestinal tract and only when the active transport system is saturated will appreciable levels of L-carnitine reach the more distal segments of the gastrointestinal tract to be catabolized by the gut micobiota (Rebouche and Seim, 1998).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Rebouche and Seim, 1998 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Production of γBB from L-carnitine by a gut microbial process has been suggested.

    “However, the production of γBB from L-carnitine by a gut microbial process has been suggested (Rebouche and Chenard, 1991; Rebouche et al., 1984; Seim et al., 1985).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Rebouche and Chenard, 1991

      Not checked in the free run.

    • cites Rebouche et al., 1984 source

      Not checked in the free run.

    • cites Seim et al., 1985 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Host genetic factors influence gut microbial communities, while diet shapes gut inhabitant composition.

    “host genetic factors influence gut microbial communities, while diet shapes gut inhabitant composition (Wu et al., 2011)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Wu et al., 2011

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Host genetic factors, dietary and other environmental exposures, and microbial factors collectively contribute to the overall metabolic network or 'meta-metabolome' that impacts atherosclerosis expression.

    “the interaction between host genetic factors, dietary and other environmental exposures, and microbial factors collectively contribute to the overall metabolic network or ‘meta-metabolome’ that impacts atherosclerosis expression in the present example”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    L-carnitine is another dietary source for gut microbe-dependent TMAO formation and enhanced atherosclerosis.

    “Subsequent studies identified an alternative trimethylamine nutrient found predominantly in red meat, L-carnitine, as another dietary source for gut microbe-dependent formation of TMAO and enhanced atherosclerosis (Koeth et al., 2013); (Figure S1; Gut Pathway2).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Koeth et al., 2013 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Mammals completely lack the capacity to catabolize L-carnitine.

    “Early studies demonstrated that mammals completely lack the capacity to catabolize L-carnitine (Bremer, 1983; Kleber, 1997; Rebouche and Chenard, 1991).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Bremer, 1983 source

      Not checked in the free run.

    • cites Kleber, 1997 source

      Not checked in the free run.

    • cites Rebouche and Chenard, 1991

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The small increases in d3-TMA production paralleled the kinetics of d3-γBB production and may result from microbial metabolism of d3-γBB formed within the gut.

    “The small increases in d3-TMA production paralleled the kinetics of production of d3- γBB, and may thus be the result of microbial metabolism of d3-γBB formed within the gut (luminal)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    A causal role between TMAO and atherosclerosis and its adverse events in humans remains to be proven.

    “a causal role between TMAO and atherosclerosis and its adverse events in humans remains to be proven”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Gut microbial metabolism of γBB to TMA/TMAO, and not γBB itself directly, is associated with enhanced atherosclerosis.

    “the present data clarify that gut microbial metabolism of γBB to TMA/TMAO, and not γBB itself directly, is associated with enhanced atherosclerosis”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Oral antibiotic treatment suppresses gut microbes and TMA/TMAO production without directly affecting atherosclerotic plaque formation.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All animal studies were performed under approval of the Animal Research Committee of the Cleveland Clinic.

    “All animal studies were performed under approval of the Animal Research Committee of the Cleveland Clinic.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Preceding dietary habits can greatly influence gut microbial composition.

    “Previous studies from our laboratory (Koeth et al., 2013) and others (Muegge et al., 2011; Wu et al., 2011; Zimmer et al., 2012) have demonstrated that preceding dietary habits can greatly influence the gut microbial composition.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Koeth et al., 2013 source

      Not checked in the free run.

    • cites Muegge et al., 2011 source

      Not checked in the free run.

    • cites Wu et al., 2011

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    No current studies directly link γBB to human atherosclerosis.

    “Although there are no current studies directly linking γBB to human atherosclerosis”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Bacterial isolates grown on L-carnitine can induce enzymes involved in carnitine catabolism.

    “there are data to show that bacterial isolates grown on L-carnitine can induce enzymes involved in carnitine catabolism (Aurich, 1966; Seim et al., 1982b)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Aurich, 1966

      Not checked in the free run.

    • cites Seim et al., 1982b

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    The purity (>98%) and identity of all synthesized compounds were confirmed by NMR, mass spectrometry, and thin layer chromatography.

    “The purity (>98%) and identity of all compounds were confirmed by NMR, mass spectrometry, and thin layer chromatography.”
    Quote in the paperSubstantial claim
44 of 44

  • The paper relies on this without support.

    The oral antibiotic cocktail suppresses gut microbial metabolism without directly affecting host atherosclerosis or trimethylamine/TMAO metabolism.

    “in half the mice, gut microbes were suppressed by incorporating within the drinking water a cocktail of poorly absorbed antibiotics (Methods).”
    “Importantly, no increase in total plaque area was observed in mice on the γBB supplemented diet in which gut microbes was suppressed by the antibiotics cocktail (+ABS).”

  • The paper relies on this without support.

    Germ-free mice lack gut microbes, and conventionalization restores a microbiota capable of normal gut microbial metabolism.

    “To directly establish a role for gut microbes in γBB formation from dietary L-carnitine, synthetic L-d3(methyl)-carnitine was again provided by gastric gavage, but this time into germ-free mice.”
    “following housing in conventional cages to permit gut colonization with microbes”

  • The paper relies on this without support.

    Oral antibiotic treatment suppresses gut microbes and TMA/TMAO production without directly affecting atherosclerotic plaque formation.

    “mice supplemented with γBB and concomitantly treated with oral antibiotics to suppress gut microbes (and TMA/TMAO production) did not have an increase in aortic root atherosclerotic plaque area”

  • The paper relies on this without support.

    The only structural difference between γBB and L-carnitine is a hydroxyl group at carbon 3 of carnitine.

    “The only structural difference between γBB and L-carnitine is the presence of a hydroxyl group at carbon 3 in carnitine (Fig. 7).”

  • The paper relies on this without support.

    Ex vivo gut segment incubations reflect in vivo gut microbial metabolism sufficiently to support conclusions about anatomical distribution and pathway contributions.

    “the ex vivo gut segment incubations have the inherent limitation of not being subject to neuro-endocrine inputs, nor the dynamic metabolism of the intact GI tract within a host”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • In mice, supplementation with dietary choline, carnitine, precarnitine, or TMAO alone enhances macrophage cholesterol accumulation and atherogenic plaque formation.

    “In studies in mice, supplementation with dietary choline, carnitine, precarnitine (γ‐butyrobetaine), or even TMAO alone was sufficient to enhance macrophage cholesterol accumulation and atherogenic plaque formation, supporting a causal relationship.1, 2, 11”

    From Trimethylamine N‐Oxide Promotes Vascular Inflammation Through Signaling of Mitogen‐Activated Protein Kinase and Nuclear Factor‐κB (2016) · cites this paper as [11]

  • TMAO is derived primarily from dietary choline and carnitine through gut microbiota metabolism to TMA, which is absorbed and oxidized in the liver primarily by FMO3 to TMAO.

    “TMAO is derived primarily from dietary choline and carnitine through the action of gut microbiota, which metabolize these constituents to trimethylamine (TMA).1, 11, 12 TMA is absorbed in turn and travels via the portal circulation to the liver, where it is oxidized by flavin‐containing monoxygenases, primarily FMO3, to TMAO.1, 11, 12”

    From Trimethylamine N‐Oxide Promotes Vascular Inflammation Through Signaling of Mitogen‐Activated Protein Kinase and Nuclear Factor‐κB (2016) · cites this paper as [11]

  • Animal studies show that dietary TMAO is pro-atherogenic, and dietary precursors such as choline, carnitine, and gamma butyrobetaine accelerate atherosclerosis only when gut microbiota and TMA/TMAO generation are intact.

    “Animal model studies show direct provision of TMAO within the diet is pro-atherogenic, and similarly, provision of its dietary precursors (e.g. choline, carnitine, gamma butyrobetaine) also accelerates atherosclerosis development, but only in the setting of intact gut microbiota and TMA/TMAO generation (Wang et al., 2011; Koeth et al., 2013; Koeth et al, 2014).”

    From Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk (2016) · cites this paper as Koeth et al, 2014

  • The TMAO meta-organismal pathway has clinical and mechanistic links with atherosclerotic plaque development and whole-body cholesterol and sterol metabolism.

    “The TMAO meta-organismal pathway has been shown to possess numerous clinical and mechanistic links (Figure 7A) with both atherosclerotic plaque development and whole body cholesterol and sterol metabolism”

    From Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk (2016) · cites this paper as Koeth et al., 2014

  • Some TMA-containing compounds can participate in TMA/TMAO formation in a gut microbiota-dependent manner and can be generated by gut microbiota metabolism of L-carnitine.

    “Some of these TMA containing compounds can participate in TMA/TMAO formation in a gut microbiota-dependent manner, and also be generated by gut microbiota metabolism of L-carnitine.1,3,16,19”

    From Impact of chronic dietary red meat, white meat, or non-meat protein on trimethylamine N-oxide metabolism and renal excretion in healthy men and women (2019) · cites this paper as [16]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • γBB supplementation is described as both food-based and water-based

    Major

    The atherosclerosis results describe γBB as a 1.3% mass-by-mass addition to the diet, whereas the methods say it was added at 1.3% to drinking water. These are different delivery routes and do not specify the same dose.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 4

  1. Englard, 1983, Production of trimethylamine from structurally related trimethylammonium compounds by resting cell suspensions of gamma-butyrobetaine and D,L-carnitine-grown Acinetobacter calcoaceticus and Pseudomonas putida, Arch. Microbiol., 135, 305, 10.1007/BF00413486 · DOI
  2. Heinz, 1996, Synthesis of [methyl-14C]crotonobetaine from DL-[methyl-14C]carnitine, J. Labelled Comp. Radiopharm., 38, 179, 10.1002/(SICI)1099-1344(199602)38:2<179::AID-JLCR825>3.0.CO;2-X · DOI
  3. Micha, 2010, Red and processed meat consumption and risk of incident coronary heart disease, stroke, and diabetes mellitus: a systematic review and meta-analysis, Circulation, 121, 2271, 10.1161/CIRCULATIONAHA.109.924977 · DOI · Paper page
  4. Seim, 1977, [Utilization of trimethylammonium-compounds by Acinetobacter calcoaceticus (author’s transl)], Arch. Microbiol., 112, 201

Cited by

Sorted by citations

Authors
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., Tang, W. H. W., DiDonato, J. A., Lusis, A. J., & Hazen, S. L.
Affiliations
  • Cleveland Clinic (US)
  • University of California, Los Angeles (US)
  • Cleveland State University (US)
Corresponding author
Hazen, Stanley L.
ORCID
Venue
Cell Metabolism
Publisher
Elsevier BV
Published
2014-11
Volume
20
Issue
5
Pages
799-812
ISSN
1550-4131 (ISSN-L)
DOI
10.1016/j.cmet.2014.10.006
PMC
PMC4255476
OpenAlex
W2044638285
Cited by
546
References
36
Citations by year
2026: 22 · 2025: 59 · 2024: 38 · 2023: 52 · 2022: 77 · 2021: 66 · 2020: 58 · 2019: 39 · 2018: 46 · 2017: 31 · 2016: 33 · 2015: 22 · 2014: 2
FWCI
12.04
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Metabolism (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • EngagedScholarship @ Cleveland State University (Cleveland State University) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • National Institutes of Health (T32 GM007250)
  • Office of Dietary Supplements (P20 HL113452, PO1 H28481, PO1 HL30568, R01 HL-094322, R01 HL098193, R01 HL103866)
  • Abbott Laboratories
  • St. Jude Medical
  • Fondation Leducq
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Polyamine Metabolism and Applications
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Nutritional Studies and Diet
Keywords
gamma-butyrobetaine, L-carnitine, trimethylamine, trimethylamine N-oxide, gut microbiota, atherosclerosis, microbial metabolism, red meat, microbial community composition
MeSH terms
Animals, Betaine, Carnitine, Female, Methylamines, Mice, Inbred C57BL, Gastrointestinal Tract, Atherosclerosis, Mice, Microbiota

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-05.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-05.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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