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

High plasma choline level is associated with a high risk of cardiovascular disease via trimethylamine-N-oxide (TMAO) produced by gut microbiota from choline.

“a growing body of evidence suggests that a high plasma choline level is associated with a high risk of cardiovascular disease via trimethylamine-N-oxide (TMAO) produced by gut microbiota from choline.”

L-carnitine, which is relatively high in red meat, might be metabolized by intestinal microbiota into proatherogenic compounds, trimethylamine-N-oxide, promoting atherosclerosis.

“L-carnitine, which is relatively high in red meat, might be metabolized by intestinal microbiota into proatherogenic compounds, trimethylamine-N-oxide, promoting atherosclerosis.38”

TMAO levels are associated with cardiovascular disease risks in a variety of cohorts.

“Numerous recent case–control and longitudinal studies have shown a striking association between trimethylamine N‐oxide (TMAO) levels and cardiovascular disease risks in a variety of cohorts.1, 2, 3, 4, 5, 6, 7, 8, 9, 10”

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”

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”

Isotope tracer studies before and after oral antibiotics affirmed that gut microbes are obligatory for TMAO generation in humans.

“Moreover, an obligatory role for gut microbes in TMAO generation in humans was affirmed by two distinct studies involving ingestion of either isotope labeled phosphatidylcholine or isotope labeled carnitine as tracer before versus following exposure to an oral cocktail of poorly absorbed antibiotics to suppress intestinal microbes (Tang et al., 2013; Koeth et al., 2013).”

Gut microbes take part in a metaorganismal pathway that contributes to atherosclerosis.

“recent studies reveal participation of gut microbes in a metaorganismal pathway that contributes to the development of atherosclerosis (Wang et al., 2011; Koeth et al., 2013; Tang et al., 2013; Gregory et al., 2015).”

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).”

Mechanistic work linking the TMAO production pathway to cardiovascular risk has focused on TMAO and FMO3 in atherosclerotic plaque development, sterol and glucose metabolism, and macrophage phenotype.

“Insights into the mechanisms through which the meta-organismal pathway responsible for TMAO production are associated with enhanced CVD risks have thus far focused on the involvement of TMAO and FMO3 in atherosclerotic plaque development, altered sterol and glucose metabolism, and changes in macrophage phenotype (Wang et al., 2011 ; Koeth et al., 2013; Miao et al., 2015 ; Shih et al., 2015; War”

Multiple clinical studies have linked TMAO levels to cardiovascular risk.

“in multiple clinical studies, TMAO levels have been shown to be associated with cardiovascular risks8–10, 13.”

Trimethylamine-N-oxide (TMAO) is produced by gut microbes from dietary choline, phosphatidylcholine and L-carnitine.

“Specifically, gut microbiota-mediated metabolism of phosphatidylcholine, choline or L-carnitine each have been shown to ultimately produced trimethylamine-N-oxide (TMAO), and in multiple clinical studies, TMAO levels have been shown to be associated with cardiovascular risks8–10, 13.”

Gut microbes are required for TMAO formation from trimethylamine-containing nutrients, including choline, phosphatidylcholine, L-carnitine, γ-butyrobetaine, and to a lesser extent betaine.

“Animal model and human clinical studies demonstrate an obligatory role for gut microbiota in TMAO formation from TMA-containing nutrients including choline and phosphatidylcholine9, 10, L-carnitine8, and more recently, γ-butyrobetaine15, and to a lesser degree, the choline oxidation metabolite betaine11.”

TMAO itself inhibits reverse cholesterol transport and alters cholesterol and sterol metabolism in multiple compartments, including suppression of bile acid pool size and altered composition.

“Prior mechanistic studies in mice reveal that TMAO itself inhibits reverse cholesterol transport, and also alters cholesterol and sterol metabolism in multiple different compartments, including suppression of bile acid pool size and altered composition.16”

Elevated TMAO levels predict increased prevalent cardiovascular disease risk and increased incident risk of major adverse cardiovascular events in large-scale human clinical studies.

“Moreover, elevated levels of TMAO have been shown to predict increased prevalent cardiovascular disease (CVD) risk, and increased incident risk of major adverse cardiovascular events (MACE ¼ myocardial infarction, stroke, and death) in large-scale human clinical studies.14,16,17”

Gut microbiota play an obligate role in TMAO formation from ingested choline, phosphatidylcholine, and carnitine in both animal models and humans.

“Gut microbiota have been shown to play an obligate role in TMAO formation from ingested choline, PC, and carnitine in both animal models and humans.14,16”

The authors' group has established the obligatory role of intestinal microbiota in TMAO production from dietary choline and other TMA nutrients in humans, and the association between elevated fasting TMAO and higher incident risks of long-term MACEs.

“We have also established the obligatory role of intestinal microbiota in the production of TMAO from dietary choline and other TMA nutrients in humans, 16,17 and the association between elevated fasting levels of TMAO and higher incident risks of long-term MACEs.17”

Host hepatic FMO3 is the main enzyme producing TMAO.

“host hepatic FMO3, the primary FMO responsible for TMAO production”

TMAO is associated with cardiovascular risk in clinical studies.

“is associated with cardiovascular risks in clinical studies”

TMAO promotes atherosclerosis in animal models.

“both enhances atherosclerosis in animal models”

Choline, phosphatidylcholine, and carnitine are dietary precursors for TMAO production in mice and humans.

“choline, phosphatidylcholine and carnitine, trimethylamine (TMA)-containing nutrients abundant in foods like meat, egg yolk and high fat dairy products, serve as dietary precursors for trimethylamine N-oxide (TMAO) generation in mice and humans”

FMO3 regulates host lipid and sterol metabolism and atherosclerosis development.

“important regulators in host lipid and sterol metabolism, as well as atherosclerosis development”

Mammalian TMAO formation proceeds in two metaorganismal steps: gut microbes make TMA, then host hepatic FMOs convert TMA to TMAO.

“TMAO formation in mammals occurs via a two-step metaorganismal pathway”

Multiple lines of evidence show that the meta-organismal pathway producing TMAO is mechanistically linked to susceptibility to atherosclerosis.

“Multiple lines of evidence show the meta-organismal pathway involved in TMAO production is mechanistically linked to atherosclerosis susceptibility (Bennett et al., 2013; Gregory et al., 2015; Koeth et al., 2013; Wang et al., 2011).”

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).”

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”

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).”

One route is direct cleavage of the C-N bond in L-carnitine to liberate TMA in the cecum and proximal large bowel, followed by host FMO3 conversion of TMA to TMAO.

“The first pathway represents the direct scission of the C-N bond in L-carnitine liberating TMA within the cecum and proximal large bowel, which is then converted into TMAO by host FMO3, as previously described (Koeth et al., 2013).”

Gut microbes are obligatory for metabolism of dietary L-carnitine into TMA and TMAO in animal models and human clinical studies.

“An obligatory role for gut microbes in the metabolism of dietary L-carnitine into TMA and TMAO has been established in both animal models and human clinical studies (Koeth et al., 2013); however, the pathways and enzyme(s) bacteria use to metabolize L-carnitine ultimately into TMA are poorly defined.”

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.”

Multiple human studies with distinct clinical populations have confirmed a strong association between plasma TMAO levels and cardiovascular disease risks.

“multiple human studies with distinct clinical populations have now confirmed a strong association between plasma TMAO levels and cardiovascular disease risks (Koeth et al., 2013; Tang et al., 2013; Tang WHW, (2014); Wang et al., 2011; Wang et al., 2014)”

Dietary nutrients containing a trimethylamine moiety, specifically choline/phosphatidylcholine and L-carnitine, participate in the development of atherosclerotic heart disease.

“Our group recently discovered that certain dietary nutrients possessing a trimethylamine (TMA) moiety, namely choline/ phosphatidylcholine and L-carnitine, participate in the development of atherosclerotic heart disease.”

Elevated circulating carnitine, choline, or betaine levels are each associated with future MI, stroke, or death independent of traditional risk factors, but their prognostic value is primarily restricted to those with elevated TMAO.

“Thus, while elevated circulating carnitine, choline, or betaine levels (all substrates of gut microbiota and dietary precursors of TMA/TMAO production) were each associated with future risk of MI, stroke, or death independent of traditional risk factors, their prognostic values were primarily restricted to those with concomitantly elevated TMAO levels (39,55).”

TMAO induces alterations in bile acid pool size and composition.

“Alterations were also observed in bile acid pool size and composition induced by TMAO (39).”

TMAO levels are strongly linked to cardiovascular disease risks.

“a metabolite shown to be both mechanistically linked to atherosclerosis and whose levels are strongly linked to cardiovascular disease (CVD) risks.”

Dietary L-carnitine accelerates atherosclerosis in mouse models only when intact gut microbiota and TMA/TMAO generation are present.

“In additional studies, dietary L-carnitine was similarly shown to foster accelerated atherosclerosis in mouse models, but only in the presence of intact gut microbiota and TMA/TMAO generation (39).”

TMAO affects cholesterol and sterol metabolism in animal models, enhancing macrophage cholesterol accumulation and atherosclerosis development.

“TMAO has been shown to affect cholesterol and sterol metabolism in animal models, enhancing macrophage cholesterol accumulation and atherosclerosis development.”

In multiple human studies, elevated TMAO is independently associated with prevalent CVD and incident risks for MI, stroke, death, and revascularization.

“In multiple human studies, elevated TMAO has been independently associated with prevalent CVD and incident risks for MI, stroke, death, and revascularization.”

In larger cohorts, the prognostic value for CVD was largely confined to TMAO formation, especially from choline and L-carnitine.

“subsequent analyses in larger cohorts revealed that the prognostic value was largely confined to the formation of TMAO, especially from choline and L-carnitine (39,55).”

TMAO is mechanistically linked to atherosclerosis.

“a metabolite shown to be both mechanistically linked to atherosclerosis and whose levels are strongly linked to cardiovascular disease (CVD) risks.”

L-carnitine serves as a dietary precursor to gut microbial production of TMA and TMAO in mice and humans.

“Additional studies have shown that L-carnitine, an alternative TMA-containing nutrient found almost exclusively in red meat, similarly serves as a dietary precursor to gut microbial production of TMA and TMAO in both mice and humans (ref.39 and Figure 1).”

Chronic dietary exposure shifts gut microbial composition, giving a selective advantage to bacterial species that prefer L-carnitine as a carbon fuel source, increasing their proportion and TMA production potential.

“Thus, chronic dietary exposure (e.g., omnivore vs. vegan/vegetarian among humans, or normal chow vs. chow plus L-carnitine in mouse studies) shifts gut microbial composition, with a selective advantage for bacterial species that prefer L-carnitine as a carbon fuel source to increase in proportion within the community and amplify the potential to produce TMA.”

Intestinal microbiota are obligatory for generating TMAO from multiple dietary nutrients.

“These observations highlight the obligatory role of intestinal microbiota in the generation of TMAO from multiple dietary nutrients, and TMAO as the proatherogenic species likely promoting the striking associations noted between plasma levels and both prevalent and incident CVD risks.”

Gut microbial communities differ between vegetarians or vegans and omnivores.

“Similarly, studies from our group (39) and others (71) show that gut microbial communities differ in vegetarians and vegans compared with omnivores.”

Chronic dietary exposure can profoundly affect TMA synthetic capacity, whereas single acute dietary exposures affect TMA and TMAO production mainly by modulating precursor substrate availability.

“Chronic dietary exposure can produce a profound effect on TMA synthetic capacity, whereas single (acute) dietary exposures affect TMA and TMAO production predominantly via modulation of precursor substrate availability.”

Labeled betaine or phosphatidylcholine given in vivo produced d9-TMA and d9-TMAO directly in plasma and urine.

“In vivo studies in which trimethyl-d9-labeled betaine or PC were ingested showed a direct generation of d9-TMA and d9-TMAO within plasma and urine (37,39,55).”

Vegetarians and vegans have a much lower capacity to form TMA and TMAO from dietary L-carnitine and have lower plasma TMAO than omnivores.

“We previously showed that vegetarians and vegans have markedly reduced synthetic capacity to make TMA and TMAO from dietary L-carnitine and have lower plasma TMAO levels than omnivores (39).”

The microbiota converts dietary L-carnitine and choline into TMA and TMAO, which affects cholesterol and sterol metabolism in macrophages, liver, and intestine.

“The microbiota metabolizes dietary L-carnitine and choline to form TMA and TMAO, influencing cholesterol and sterol metabolism in macrophages, liver, and intestine (39).”

Intestinal microbiota, dietary trimethylamine-containing nutrients, and cardiovascular disease are linked.

“Our laboratory has elucidated a link between intestinal microbiota, dietary trimethylamine-containing nutrients ( e.g. choline, phosphatidylcholine, and carnitine), andcardiovasculardisease(5-7).”

Phosphatidylcholine and TMAO are both mechanistically linked to atherosclerosis development.

“phosphatidylcholine, the major dietary source of choline, and the gut microbe-dependent metabolite, trimethylamine N -oxide (TMAO), 4 are both mechanistically linked to atherosclerosis development (5, 6, 8)”

An obligatory role for gut microbes in TMAO formation in both mice and humans was shown.

“An obligatory role for gut microbes in TMAO formation in both mice (5, 6) and humans (7) was shown”

No studies to date have directly demonstrated transmission of atherosclerosis susceptibility by gut microbial transplantation.

“no studies to date have directly demonstrated transmission of atherosclerosis susceptibility by gut microbial transplantation.”

Dietary choline-induced and carnitine-induced enhancement in atherosclerosis in mice is prevented by suppression of gut microbes (and TMAO formation) with poorly absorbed broad spectrum oral antibiotics.

“both dietary choline-induced and carnitine-induced enhancement in atherosclerosis in mice is prevented by suppression of gut microbes (and TMAO formation) with poorly absorbed broad spectrum oral antibiotics (5, 6).”

Prevotella was recently reported to be associated with TMAO levels in cecal microbes from carnitine-supplemented mice.

“Proportions of Prevotella were strongly associated with TMAO levels ( r = 0.44, p = 0.0003; Fig. 10 A ), a genuswerecentlyreported was associated with TMAO levels in cecal microbes from carnitine-supplemented mice (6).”

Intestinal microbiota alterations are associated with major conditions like obesity, type-2 diabetes, cardiovascular disease, non-alcoholic fatty acid liver disease, cancer, and the response to antineoplastic therapy.

“Intestinal microbiota alterations are associated with major conditions like obesity, type-2 diabetes, cardiovascular disease, non-alcoholic fatty acid liver disease, cancer, and the response to antineoplastic therapy [3][4][5][6] .”

TMAO has been implicated in metabolic, cardiovascular, and cerebrovascular disease.

“The microbial-derived metabolite trimethylamine N-oxide (TMAO) has been implicated in metabolic [5], cardiovascular [6, 7], and cerebrovascular [8] disease.”

Elevated plasma TMAO levels are observed in subjects at risk for incident CVD and adverse CVD events including heart attack, stroke, and death.

“Elevated plasma TMAO levels are observed in subjects at risk for incident cardiovascular disease (CVD) development and adverse CVD events including heart attack, stroke, and death.[1][2][3][4]”

TMAO is a gut microbiota-generated metabolite with mechanistic links to atherosclerotic heart disease.

“Trimethylamine N-oxide (TMAO) is a gut microbiota-generated metabolite with mechanistic links to the pathogenesis of atherosclerotic heart disease.[1][2][3][4][5]”

TMAO is generated via a metaorganismal pathway beginning with gut microbiota-dependent formation of TMA from TMA-containing nutrient precursors.

“Trimethylamine N-oxide is generated via a metaorganismal pathway that begins with gut microbiota-dependent formation of trimethylamine (TMA) from TMA containing nutrient precursors (Figure 1A).[1][2][3]16”

Carnitine is enriched in red meat, and higher total choline content is found in beef and other meats, liver, and egg yolks.

“Carnitine is enriched in red meat, 3 and higher total choline content is found in beef and other meats, liver, and egg yolks.17”

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”

In small cross-sectional observational studies, plasma TMAO levels were modestly increased in omnivores relative to vegans/vegetarians.

“Interestingly, in small cross-sectional observational studies, plasma levels of TMAO were modestly increased in omnivores relative to vegans/vegetarians, and in recent clinical challenge studies, TMA and TMAO generation from oral carnitine was observed to be substantially reduced in vegans/vegetarians.3,19”

A diet enriched in meat, particularly red meat, has higher content of both choline and carnitine nutrient precursors for TMA and TMAO generation.

“Consequently, a diet enriched in meat, particularly red meat, has higher content of both choline and carnitine nutrient precursors for TMA and TMAO generation.1,3”

In recent clinical challenge studies, TMA and TMAO generation from oral carnitine was substantially reduced in vegans/vegetarians.

“Interestingly, in small cross-sectional observational studies, plasma levels of TMAO were modestly increased in omnivores relative to vegans/vegetarians, and in recent clinical challenge studies, TMA and TMAO generation from oral carnitine was observed to be substantially reduced in vegans/vegetarians.3,19”

High plasma TMAO levels are associated with risk for developing atherosclerosis.

“studies suggesting an association between high plasma TMAO levels and risk for developing atherosclerosis have increased markedly [7,13,14,15,16,17]”

In humans, elevated plasma TMAO levels are positively correlated with increased risk for major adverse cardiovascular events.

“In humans, recent clinical studies evidence a positive correlation between elevated plasma levels of TMAO and an increased risk for major adverse cardiovascular events.”

Elevated plasma TMAO levels have been found in people at risk of CVD, and these patients usually show greater concentrations of TMAO precursors.

“Indeed, elevated plasma TMAO levels have been found in people at risk of CVD. These patients usually show greater concentrations of the TMAO precursors too.”

TMAO lowers expression of Cyp7a1, the rate-limiting enzyme in cholesterol catabolism.

“In the study performed by Koeth et al., (2013) [14], TMAO lowered the expression of the main bile acid synthetic enzyme (Cyp7a1), which is the rate-limiting step in the catabolism of cholesterol.”

TMAO alters cholesterol and sterol metabolism in various compartments, including the bile acid compartment.

“Some investigations revealed that TMAO alters cholesterol and sterol metabolism in various compartments, including the bile acid compartment [7,14,15].”

TMAO could serve as a biomarker in humans to predict prevalence of CVD and increased incidence of major adverse cardiovascular events.

“In this context, it is thought that TMAO could serve as a biomarker in humans to predict prevalence of CVD and increased incidence of major adverse cardiovascular events (MACE), such as myocardial infarction, stroke and even death [7,14,52,53,54,55].”

Elevated TMAO has been observed in patients with cardiovascular disease and atherosclerosis, highlighting a potential downside to increased protein intake.

“Elevated TMAO has been observed in patients with cardiovascular disease and atherosclerosis, highlighting a potential downside to increased protein intake.[15-17,22, 37]”

Of the 22 studies, 2 only investigated combinations of TMAO and choline, betaine, or l-carnitine, so they were not included in the analysis for TMAO.

“Of the 22 studies, 2 only investigated combinations of TMAO and choline, betaine,9 or l‐carnitine4; thus we did not include the 2 studies4, 9 in the analysis for TMAO, although these studies were eligible for the analysis of TMAO precursors and risk of MACE.”

TMAO is mainly derived from choline, which is metabolized to TMA by microbiota and then to TMAO by hepatic flavin monooxygenase 3.

“TMAO is a small organic compound, mainly derived from choline (found in foods such as red meat, fish, poultry, and eggs), which is metabolized to produce trimethylamine (TMA) by microbiota1, 4 and then to TMAO by the hepatic enzyme, flavin monooxygenase 3.1, 5”

Gut microbiota are affected by environmental factors such as dietary intakes, which may in turn influence blood levels of TMAO and its precursors.

“Fourth, the gut microbiota are affected by environmental factors37, 38 such as dietary intakes,39, 40, 41 which may, in turn, influence blood levels of TMAO and its precursors.4”

Elevated concentrations of l-carnitine were only predictive of major adverse cardiovascular event risk among individuals with higher TMAO levels.

“In addition, it was noted that elevated concentrations of l‐carnitine were only predictive of MACE risk among individuals with higher TMAO levels.4”

In a study, plasma l-carnitine concentrations predicted major adverse cardiovascular events independent of traditional cardiovascular risk factors, but the significant association disappeared after adjustment for plasma TMAO concentration.

“A study4 showed that plasma l‐carnitine concentrations predicted MACE independent of traditional cardiovascular risk factors, but the significant association disappeared after adjustment for plasma TMAO concentration.”

Omnivorous subjects produced more trimethylamine oxide than vegans or vegetarians after ingesting L-carnitine, through an intestinal microbiota-dependent mechanism.

“omnivorous subjects produced more TMAO than vegans or vegetarians following ingestion of L-carnitine, via an intestinal microbiota-dependent mechanism59.”

A similar pathway converts dietary carnitine, which is high in red meat, into trimethylamine oxide.

“A similar pathway has been identified for conversion of dietary carnitine, which is high in red meat, into TMAO59.”

Carnitine can be synthesized endogenously from lysine and methionine in kidney, liver, and brain.

“Carnitine also can be synthesized endogenously from two essential amino acids, lysine and methionine (63).This occurs in kidney, liver, and brain (42).”

TMAO production after an L-carnitine challenge was significantly greater in omnivores than vegans, and specific bacterial taxa were associated with dietary status and plasma TMAO.

“TMAO production, following a challenge with L-carnitine, was significantly greater in omnivores versus vegans, and there were specific bacterial taxa associated with both dietary status and plasma TMAO concentration (63).”

After an L-carnitine challenge, people produced TMAO from L-carnitine in a gut microbe-dependent manner.

“After administration of an L-carnitine challenge test (consumption of a diet containing an 8-ounce sirloin steak and a capsule containing 250 mg of a heavy isotope-labeled L-carnitine), people produced TMAO from L-carnitine in a gut microbe-dependent manner (63).”

Antibiotic treatment prevented the proatherogenic effects of choline, γ-butyrobetaine, and carnitine in mice.

“The proatherogenic effects of choline, γ-butyrobetaine, and carnitine were prevented by treatment with antibiotics, suggesting that microbial metabolism of these precursors was required for the effect (63, 118).”

Plasma L-carnitine concentrations in subjects undergoing cardiac evaluation (n = 2,595) predicted increased risks for cardiovascular disease and major adverse cardiac events including myocardial infarction, stroke, and death.

“In addition, plasma concentrations of the TMAO precursor L-carnitine in subjects undergoing cardiac evaluation (n = 2,595) predicted increased risks for CVD and for major adverse cardiac events (myocardial infarction, stroke, and death) (63).”

In atherosclerosis-prone C57BL/6J Apoe-/- mice, a normal diet supplemented with choline, L-carnitine, or TMAO increased total aortic root atherosclerotic plaque area compared with control diet.

“Atherosclerosis-prone mice (C57BL/6J Apo e -/-) fed a normal diet supplemented with additional choline (118) and L-carnitine (63) or TMAO (118) had increased total aortic root atherosclerotic plaque area compared with mice fed a control diet (118).”

This cohort had a mean eGFR of 83 mL/min.

“This cohort had a mean eGFR of 83 mL/min (63).”

The prevention by antibiotics suggests microbial metabolism of these precursors was required for the proatherogenic effect.

“The proatherogenic effects of choline, γ-butyrobetaine, and carnitine were prevented by treatment with antibiotics, suggesting that microbial metabolism of these precursors was required for the effect (63, 118).”

Multiple human and animal studies support mechanistic and clinical prognostic associations between TMAO formation and cardiometabolic disease risks.

“multiple human and animal studies supporting both mechanistic and clinical prognostic associations between TMAO formation and cardiometabolic disease risks have been reported8–16.”

TMAO generation is dependent on the gut microbiota, which first metabolizes dietary choline to trimethylamine (TMA), and TMA is then metabolized to TMAO by flavin monooxygenase (FMO) enzymes in the liver.

“TMAO generation is dependent on the gut microbiota, which first metabolizes dietary choline to trimethylamine (TMA). Thereafter, the TMA is metabolized to TMAO by enzymes of the flavin monooxygenase (FMO) family in the liver (4).”

TMAO induces atherosclerosis by affecting cholesterol metabolism through inhibiting hepatic bile acid synthesis.

“TMAO has been shown to induce AS by affecting cholesterol metabolism through inhibiting hepatic bile acid (BA) synthesis (2–4).”

Antibiotics attenuate TMAO-caused atherosclerosis by reducing TMAO synthesis via blocking the gut microbiota-governed pathway from l-carnitine to TMA.

“It was found that antibiotics (Abs) could attenuate TMAO-caused AS by reducing TMAO synthesis via blocking the pathway of l-carnitine to TMA governed by the gut microbiota (4).”

TMAO contributes to the development of atherosclerosis, in part by affecting cholesterol metabolism through regulation of the bile acid synthesis pathway.

“TMAO appears to contribute to the development of AS, in part by affecting cholesterol metabolism through the regulation of the BA synthesis pathway (4).”

Treatment with antibiotics or germ-free conditions abolished dietary choline-driven TMAO generation and the development of atherosclerosis.

“Treatment with Abs, or germ-free conditions, abolished dietary choline-driven TMAO generation and the development of AS (2, 4).”

Enhanced atherosclerosis observed with dietary choline supplementation is entirely dependent on the gut microbiota.

“Enhanced AS, as observed by dietary choline supplementation, is entirely dependent on the gut microbiota.”

Antibiotics could attenuate TMAO-caused atherosclerosis by reducing TMAO synthesis via blocking the pathway of choline to TMA governed by the gut microbiota.

“Furthermore, it has been found that Abs could attenuate TMAO-caused AS by reducing TMAO synthesis via blocking the pathway of choline to TMA governed by the gut microbiota (4).”

Plasma TMAO is a metabolite strongly associated with atherosclerosis.

“Recently, plasma TMAO was identified as a metabolite strongly associated with AS (2–4).”

Feeding atherosclerosis-prone mice diets enriched in either choline or TMAO enhanced the development of atherosclerosis.

“Researchers confirmed that feeding AS-prone mice diets enriched in either choline or TMAO enhanced the development of AS.”

TMAO partially contributes to the development of atherosclerosis by regulating the major pathway of cholesterol metabolism and the bile acid synthetic pathway.

“Previous studies suggest that TMAO partially contributes to the development of AS by regulating the major pathway of cholesterol metabolism and the bile acid synthetic pathway.4, 5, 6”

TMAO contributes to the development of AS, in part, by affecting cholesterol metabolism through regulating the major pathway for cholesterol elimination from the body and the bile acid synthetic pathway at multiple levels.

“Researchers have confirmed that TMAO contributes to the development of AS, in part, by affecting cholesterol metabolism through regulating the major pathway for cholesterol elimination from the body and the bile acid synthetic pathway at multiple levels.4, 6”

In a cohort of more than 2000 cardiovascular disease patients, those in the highest quartile of plasma TMAO had a 2-fold risk of heart attack or stroke compared with those in the lowest quartile.

“Subjects from a cohort of .2000patients with cardiovascular disease, who were in the highest quartile for plasma TMAO concentrations, had a .2-foldrisk of a heart attack or stroke compared with subjects from the lowest quartile (108, 172).”

Follow-up targeted studies with choline challenge and microbiota characterization/manipulation showed that consuming foods rich in choline or carnitine (eggs, milk, liver, meat, fish) produced high TMAO concentrations in humans and animals that had the requisite microbiota for converting choline or carnitine to trimethylamine, the immediate TMAO precursor.

“Hazen and coworkers showed that consumption of foods rich in choline or carnitine, such as eggs, milk, liver, meat, or fish, produced high concentrations of TMAO in both humans and animals who possessed the requisite microbiota for metabolizing choline or carnitine to trimethylamine, the immediate precursor of TMAO (108, 171, 172).”

TMAO exacerbates atherosclerosis in a genetic knockout mouse model.

“TMAO exacerbates atherosclerosis in a genetic knockout mouse model, in part by promoting forward cholesterol transport and by inhibiting reverse cholesterol transport (8, 10, 19, 20).”

Serum TMAO levels are positively correlated with cardiovascular disease in humans.

“Recent human studies have established that the levels of TMAO in serum are positively correlated with impaired renal function, colorectal cancer, and cardiovascular disease (CVD) (8, 10, 17, 18).”

None of the identified TMA-producing strains generated TMA from l-carnitine under the same test conditions.

“None of the identified TMA-producing strains generated TMA from l-carnitine, another quaternary amine linked to TMAO accumulation, under the same test conditions (Table 1) (10).”

TMAO promotes forward cholesterol transport.

“TMAO exacerbates atherosclerosis in a genetic knockout mouse model, in part by promoting forward cholesterol transport and by inhibiting reverse cholesterol transport (8, 10, 19, 20).”

Serum TMAO levels are positively correlated with impaired renal function in humans.

“Recent human studies have established that the levels of TMAO in serum are positively correlated with impaired renal function, colorectal cancer, and cardiovascular disease (CVD) (8, 10, 17, 18).”

Serum TMAO levels are positively correlated with colorectal cancer in humans.

“Recent human studies have established that the levels of TMAO in serum are positively correlated with impaired renal function, colorectal cancer, and cardiovascular disease (CVD) (8, 10, 17, 18).”

TMAO inhibits reverse cholesterol transport.

“TMAO exacerbates atherosclerosis in a genetic knockout mouse model, in part by promoting forward cholesterol transport and by inhibiting reverse cholesterol transport (8, 10, 19, 20).”

Gut microbes can convert otherwise beneficial dietary compounds, such as choline, into metabolites that are detrimental to human health.

“Likewise, gut microbes can also convert otherwise beneficial dietary compounds, such as choline, into metabolites that are detrimental to human health (7–10).”

Long-term dietary habits are a dominant determinant of an individual's gut microbiota composition.

“long-term dietary habits are a dominant force in determining the composition of an individual’s gut microbiota”

Mice with low TMA-producing microbiota can become high TMA producers when fed L-carnitine for 10 weeks.

“mice that harbour microbiotas with low production of trimethylamine (TMA) could be converted into high producers when their diet was supplemented with the TMA-containing compound L-carnitine for 10 weeks27.”

Several diet-induced host phenotypes are sufficiently mediated by changes in the gut microbiota.

“For example, several diet-induced host phenotypes are sufficiently mediated by changes in the gut microbiota (85–88).”

L-carnitine present in red meat can be metabolized to trimethylamine oxide, which is associated with an increased incidence of atherosclerosis and obesity.

“Accordingly, L-carnitine present in red meat can be metabolized to trimethylamine oxide (TMAO), which is associated with an increased incidence of atherosclerosis [194] and obesity [195].”

L-carnitine- or phosphatidylcholine-supplemented diets induce higher atheroma formation and wider aortic lesions in ApoE- mice, and disease is ameliorated by antibiotic treatment.

“LC- or PC- supplemented diets were found to induce higher atheroma formation and wider aortic lesions in ApoE- mice, and disease was ameliorated by antibiotic treatment [68, 69].”

Recent studies reveal a link between microbiota and atherogenesis.

“a series of recent studies [68-70] revealed a link between microbiota and atherogenesis.”

Vegan or vegetarian subjects have a reduced or non-existent capacity to produce TMA upon L-carnitine supplementation, proposed to be because their bacteria cannot perform this conversion.

“Vegan or vegetarian subjects exhibited a reduced or non-existent capacity to produce TMA upon LC supplementation, and it was proposed that bacteria, presumably selected through the host’s diet, were incapable of performing this biochemical conversion [68].”

In humans, phosphatidylcholine or L-carnitine administration augments TMA plasma levels and increases cardiovascular disease risk; antibiotic treatment dramatically reduces TMA levels.

“Also in humans, PC or LC administration augmented TMA plasma levels, and increased the risk for cardiovascular disease; notably, antibiotic treatment dramatically reduced TMA levels in these subjects [68, 70].”

Dietary phosphatidylcholine and L-carnitine are transformed by the microbiota into pro-atherogenic trimethylamine.

“Dietary phosphatydilcholine (PC), a lipid broadly present in foods, and L-carnitine (LC), a molecule abundant in red meat, are transformed by the microbiota into pro-atherogenic trimethylamine (TMA).”

Bifidobacterium strains have generated the best results in gut-brain axis research.

“Bifidobacterium strains, which is common in the gut flora of many mammals, including humans, have generated the best results [134] though more research is needed to better understand the gut-brain axis.”

Gut microbiota from mice supplemented with L-carnitine had an altered cecum microbiota composition that metabolized trimethylamine into trimethylamine-N-oxide (TMAO), which is associated with atherosclerosis.

“Koeth et al [134] reported the gut microbiota from mice that were supplemented with L-carnitine had an altered cecum microbiota composition that metabolized trimethylamine into trimethylamine-N-oxide (TMAO), which is associated with atherosclerosis.”

Diets high in red meat lead to a higher risk in cardiovascular disease due to microbiota-dependent production of TMAO.

“The authors concluded that diets high in red meat lead to a higher risk in cardiovascular disease due to microbiota-dependent production of TMAO.”

The microbiota composition of vegans, vegetarians, omnivores and diets high in red meat consumption differ greatly.

“Consequently, the microbiota composition of vegans, vegetarians, omnivores and diets high in red meat consumption differ greatly [134].”

Bacteria metabolize animal and vegetable proteins differently.

“It is important to note that while bacteria do ferment amino acids, they metabolize animal and vegetable proteins differently [134].”

Excess conversion of choline to TMA can lead to atherosclerosis as a consequence of further metabolism by host hepatic processing to TMAO.

“excess conversion to TMA can lead to artherosclerosis as a consequence of further metabolism by host hepatic processing to TMAO (Zeisel and da Costa, 2009;Wang et al., 2011;Koeth et al., 2013;Craciun et al., 2014).”

TMA is produced from dietary carnitine and choline.

“the production of trimethylamine (TMA) from dietary carnitine and choline (Wang et al., 2011;Koeth et al., 2013).”

TMAO promotes atherogenesis through formation of foam cells and reduction of reverse cholesterol transport from atherosclerotic plaque.

“A mechanistic model has been formulated proposing that TMAO promotes atherogenesis through the formation of foam cells (lipid-laden macrophages) and a reduction of the reverse cholesterol transport from the atherosclerotic plaque [5].”

Individuals on an omnivorous diet produced elevated TMAO levels in comparison to vegans or vegetarians following ingestion of l-carnitine.

“individuals on an omnivorous diet produced elevated TMAO levels in comparison to vegans or vegetarians following ingestion of l-carnitine [5]”

A correlation between dietary habits and production of TMAO was previously established.

“a correlation between dietary habits and production of TMAO was previously established”

Trimethylamine N-oxide is a metaorganismal metabolite formed following ingestion of dietary nutrients abundant in a Western diet, such as lecithin, choline, and carnitine.

“The first studies revealing a potential causal link between the gut microbiome and CVD focused on trimethylamine N-oxide (TMAO), a metaorganismal metabolite formed following ingestion of dietary nutrients abundant in a Western diet (e.g. lecithin, choline, carnitine)7–9.”

Trimethylamine N-oxide has been shown to enhance atherosclerosis in most, but not all, mechanistic and animal model studies.

“TMAO has been shown to enhance atherosclerosis in most, but not all, mechanistic and animal model studies7, 9, 78–82.”

Generation of trimethylamine N-oxide in humans and mice occurs via a multistep, metaorganismal pathway starting with dietary precursors choline, phosphatidylcholine, and carnitine.

“Through these and other studies, generation of TMAO in humans and mice has been shown to occur via a multistep, metaorganismal pathway starting with the dietary precursors choline9, phosphatidylcholine8, 9, and carnitine7.”

Gut microbial community composition is critical because there is an initial obligatory role of gut microbes in trimethylamine/trimethylamine N-oxide generation.

“First, the gut microbial community composition is critical since there is an initial obligatory role of gut microbes in TMA(O) generation7, 9.”

Trimethylamine N-oxide suppressed bile acid pool size and therefore cholesterol clearance in the host.

“In addition, TMAO suppressed bile acid pool size and therefore cholesterol clearance in the host7.”

In atherosclerosis-prone ApoE−/− mice, dietary supplementation with choline led to augmented atherosclerotic lesion burden, higher aortic expression of scavenger receptors CD36 and scavenger receptor A, increased cholesterol-laden macrophage foam cell formation, and impaired in vivo reverse cholesterol transport.

“In atherosclerosis-prone ApoE−/− mice, dietary supplementation with choline led to augmented atherosclerotic lesion burden, higher aortic expression of scavenger receptors (CD36 and scavenger receptor A), increased cholesterol laden macrophage foam cell formation9, and impaired in vivo reverse cholesterol transport7.”

TMAO is either transported to tissues for accumulation as an osmolyte or cleared by the kidney.

“TMAO is then either transported to the tissues for accumulation as an osmolyte or, more commonly, cleared by the kidney [3,9,13,14].”

Human and animal studies suggest that several families of bacteria are involved in TMA/TMAO production, namely Deferribacteraceae, Anaeroplasmataceae, Prevotellaceae and Enterobacteriaceae.

“Human and animal studies suggest that several families of bacteria are involved in TMA/TMAO production, namely, Deferribacteraceae, Anaeroplasmataceae, Prevotellaceae [3] and Enterobacteriaceae [18,19].”

Levels of cholesterol transporters in macrophages did not change significantly following TMAO treatment.

“However, the levels of cholesterol transporters in macrophages did not change significantly following TMAO treatment [3].”

In mice with an intact intestinal microbiota, dietary supplementation with TMAO, carnitine or choline reduced in vivo reverse cholesterol transport.

“In mice with an intact intestinal microbiota, dietary supplementation with TMAO, carnitine or choline reduced in vivo reverse cholesterol transport [3].”

Mice supplemented with dietary TMAO showed a significant reduction in cholesterol absorption, as well as liver expression of bile acid synthetic enzymes Cyp7a1 and Cyp27a1.

“Also, mice supplemented with dietary TMAO showed a significant reduction in cholesterol absorption, as well as liver expression of bile acid synthetic enzymes cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1) and cytochrome P450, family 27, subfamily a, polypeptide 1 (Cyp27a1) [3].”

The liver's levels of bile acid synthetic enzymes (Cyp7a1 and Cyp27a1) and bile acid transporters (Oatp1, Oatp4, Mrp2, and Ntcp) were reduced in mice treated with TMAO.

“The liver’s levels of bile acid synthetic enzymes (Cyp7a1 and Cyp27a1) and bile acid transporters (Oatp1, Oatp4, Mrp2, and Ntcp) were reduced in mice that were treated with TMAO [3].”

Dietary supplementation with TMAO markedly reduces expression of intestinal cholesterol transporters Npc1L1 and Abcg5/8.

“Interestingly, dietary supplementation with TMAO markedly reduces the expression of intestinal cholesterol transporters Niemann-Pick C1-like1 (Npc1L1), and Abcg5/8, which transport cholesterol into and out of enterocytes, respectively [3].”

Consumption of diet high in carnitine or rich in choline could potentially lead to increased cardiovascular disease through generation of TMAO.

“Emerging evidence indicates that consumption of diet high in carnitine or rich in choline could potentially lead to increased cardiovascular disease through generation of TMAO [3].”

Dietary l-carnitine induces gut microbiota-dependent production of TMA and TMAO in an animal study.

“Dietary l-carnitine has been shown to induce gut microbiota-dependent production of TMA and TMAO in an animal study [3].”

Decreased consumption of l-carnitine and choline could decrease TMAO levels.

“Thus, decreased consumption of l-carnitine and choline could decrease TMAO levels [3].”

Blood TMAO level is positively correlated with long-term mortality risk in patients with atherosclerosis, heart failure, and chronic kidney disease.

“The blood TMAO level has been reported to be positively correlated with long‐term mortality risk in patients with atherosclerosis, heart failure, and chronic kidney disease.5, 6, 7”

Gut microbes transform dietary phosphatidylcholine and l-carnitine to trimethylamine, which is absorbed and further oxidized by hepatic flavin monooxygenase to trimethylamine-N-oxide (TMAO).

“Gut microbes transform phosphatidylcholine and l‐carnitine from ingested nutrients to trimethylamine, which is absorbed and further oxidized by hepatic flavin monooxygenase to trimethylamine‐N‐oxide (TMAO)”

TMAO promotes atherogenesis partly by promoting the formation of foam cells from macrophages.

“a metabolite that could promote atherogenesis partly by promoting the formation of foam cells from macrophages.4, 5”

The average and median TMAO levels of stroke/TIA patients were comparable to quartile 1 in a US cohort.

“The average and median TMAO levels of stroke/TIA patients were only 2.68 μmol/L and 1.91 μmol/L, respectively, which were comparable to quartile 1 in a US cohort.5”

Many cohort studies have suggested that an increased number of adverse outcomes are associated with high TMAO levels.

“many cohort studies have suggested that an increased number of adverse outcomes are associated with high TMAO levels,4, 5, 6, 7”

TMAO level has been suggested to be involved in atherosclerosis.

“Although the TMAO level has been suggested to be involved in atherosclerosis,4, 5”

Consumption of choline and L-carnitine rich diets offer the necessary precursors for the production of TMAO.

“Indeed, consumption of choline and L-carnitine rich diets offer the necessary precursors for the production of TMAO.2,3”

Trimethylamine N-oxide exerts pro-atherosclerotic effects via changes in macrophage and tissue sterol metabolism and endothelial cell activation.

“Trimethylamine N-oxide exerts pro-atherosclerotic effects via a variety of effects, including changes in macrophage and tissue sterol metabolism, and endothelial cell activation 1,3,23 .”

Adherence to a vegetarian or vegan diet compared with an omnivorous diet is associated with reduced plasma and urinary levels of TMAO.

“Moreover, adherence to a vegetarian or vegan diet compared with an omnivorous diet is reported to be associated with reduced plasma and urinary levels of TMAO.3,44”

TMAO is modifiable both with diet and with potential therapeutics under development.

“The importance of our findings is underlined by the modifiable nature of TMAO, both with diet, and with potential therapeutics under development, offering new prospects for treatment strategies at both levels of primary and secondary prevention.”

Metabolomics analysis shows that metabolism of dietary phosphatidylcholine by gut microbiota produces proatherogenic trimethylamine-N-oxide, which can accelerate atherosclerosis in mice.

“a metabolomics analysis shows that metabolism of dietary phosphatidylcholine by gut microbiota produces proatherogenic trimethylamine-N-oxide, which can accelerate atherosclerosis in mice.17,18”

Several recent studies have identified trimethylamine N-oxide, a metabolite of the gut microbiota, as an independent risk factor for cardiovascular disease.

“Several recent studies have identified trimethylamine N-oxide, a metabolite of the gut microbiota, as an independent risk factor for cardiovascular disease.17,18,34”

OCA reverses fibrosis in patients with nonalcoholic steatohepatitis (NASH).

“its ability to reverse fibrosis in patients with nonalcoholic steatohepatitis (NASH) was demonstrated in a proof-of-concept study followed by a randomized control trial (RCT)150,151.”

Few connections between the microbiome and vascular and cardiovascular diseases have been identified with mechanistic certainty.

“Despite an explosion of associative studies involving the microbiome and vascular and cardiovascular diseases, few connections have been identified with mechanistic certainty[2,3].”

The gut microbiome of human vegetarians produces significantly less TMA than the gut microbiome of omnivores when fed L-carnitine.

“the gut microbiome of human vegetarians produces significantly less TMA than the gut microbiome of omnivores when fed L-carnitine, another substrate for TMA synthesis.158”

The gut microbiota metabolizes choline, phosphatidylcholine, and l-carnitine to produce trimethylamine (TMA), which is oxidized in the liver into the proatherogenic metabolite trimethylamine-N-oxide (TMAO).

“Previous studies have shown that the gut microbiota metabolizes choline, phosphatidylcholine, and l-carnitine to produce trimethylamine (TMA), which is oxidized in the liver into the proatherogenic metabolite, trimethylamine-N-oxide (TMAO)11–13.”