Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease

The TMAO pathway promotes the development of atherosclerosis and thrombosis.

“The mechanism by which TMAO induces stroke is not well understood; however, it is widely established that the TMAO pathway promotes the development of atherosclerosis and thrombosis.11,26,27”

Dietary supplementation of mice with choline, betaine, and TMAO activates upregulation of multiple macrophage scavenger receptors and subsequent accumulation of cholesterol and foam cells.

“First, a metabolomics study reported that dietary supplementation of mice with choline, betaine, and TMAO activates upregulation of multiple macrophage scavenger receptors and subsequent accumulation of cholesterol and foam cells.11”

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.”

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”

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”

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”

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”

Circulating TMAO levels are associated with cardiovascular disease risks in multiple distinct clinical studies and cohorts.

“circulating levels of TMAO have been shown in multiple distinct clinical studies and cohorts to be associated with CVD risks”

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”

Hepatic FMO enzyme activity did not differ significantly between donor strains or among recipient groups after cecal microbial transplantation.

“In contrast, examination of hepatic FMO enzyme activity, which is responsible for converting TMA into TMAO (Wang et al., 2011; Bennett et al., 2013), revealed no significant differences between both donor mouse strains, and among the various recipient groups post cecal microbial transplantation (Figure S6, panel B).”

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).”

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).”

Dietary nutrients containing TMA, such as phosphatidylcholine, choline, and carnitine, can be used by gut microbes as a carbon source; the TMA produced travels to the liver and is converted by host hepatic FMOs to TMAO.

“Briefly, specific trimethylamine (TMA) containing dietary nutrients, such as phosphatidylcholine, choline, and carnitine, can be used by gut microbes as a carbon fuel source. A waste product produced is TMA, which is carried via the portal circulation to the liver where it is rapidly converted by a family of enzymes, host hepatic flavin monooxygenases (FMOs), into TMA N-oxide (TMAO) (Wang et al.,”

Multiple clinical studies have found associations between plasma TMAO and coronary atherosclerotic plaque burden and cardiovascular disease risk.

“Finally, an association between plasma TMAO levels and both the extent of coronary atherosclerotic plaque burden and CVD risks has been observed in multiple distinct clinical studies (Wang et al., 2011; Tang et al., 2013; Tang et al., 2014; Lever et al., 2014; Tang et al., 2015).”

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.”

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.”

TMAO was measured by stable isotope dilution HPLC with online electrospray ionization tandem mass spectrometry using d9-labeled internal standards.

“Trimethylamine-N-oxide (TMAO) levels were determined by stable isotope dilution high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/MS/MS) on an AB SCIEX 5500 triple quadrupole mass spectrometer (AB SCIEX, Framingham MA) using d9-(trimethyl)-labeled internal standards as previously described10, 21.”

Betaine was a far poorer precursor for TMAO than choline, producing 100-fold less d9-TMAO.

“Of note, however, compared with comparable oral challenge of d9-choline, 14 betaine was a far poorer precursor for TMAO, as it produced 100-fold less d9-TMAO (Figure 3A and B).”

Systemic levels of choline, betaine, and TMAO are each independently associated with the prevalence of cardiovascular disease.

“Moreover, systemic levels of choline, betaine, and TMAO each are independently associated with the prevalence of CVD. 14”

Choline, betaine, and TMAO are all metabolites formed following ingestion of dietary phosphatidylcholine.

“Choline, betaine, and TMAO are all metabolites formed following ingestion of dietary PC. 14,17”

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”

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”

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”

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”

Anaerobic choline degradation is thought to be the main intestinal source of TMA.

“Anaerobic choline degradation is believed to serve as the major source of TMA formation within the intestines”

A choline-enriched diet increases endogenous macrophage foam cell generation about 2-fold in Apoe−/− mice (p=0.02).

“As was previously observed (Wang et al., 2011), a choline-enriched diet promoted marked increase in endogenous macrophage foam cell generation (approximately 2-fold on choline-supplemented diet; p=0.02)”

Changes in bacterial composition serve as a primary driver of TMAO levels in other studies.

“Changes in bacterial composition have been shown to serve as a primary driver of TMAO levels in other studies (10,15).”

TMAO has pro-atherogenic properties.

“While TMAO has been shown to possess pro-atherogenic properties (10,11,17)”

Intestinal microbe-dependent generation of TMAO is mechanistically linked to increased risk for future cardiovascular events via dietary nutrients such as phosphatidylcholine, choline, and carnitine.

“Our group has recently described a mechanistic link between intestinal microbe-dependent generation of trimethylamine-N-oxide (TMAO) and increased risk for future cardiovascular events (death, myocardial infarction, and stroke) by a pathway involving dietary nutrients such as phosphatidylcholine, choline, and carnitine (10,11).”

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).”

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.”

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.”

A meta-organismal pathway was elucidated in which gut microbiota form TMA and host hepatic FMO3 forms TMAO.

“A meta-organismal pathway was elucidated involving gut microbiota-dependent formation of TMA and host hepatic flavin monooxygenase 3dependent (FMO3-dependent) formation of TMA-N-oxide (TMAO), a metabolite shown to be both mechanistically linked to atherosclerosis and whose levels are strongly linked to cardiovascular disease (CVD) risks.”

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.”

Plasma choline, betaine, and TMAO levels are associated with increased risk of CVD phenotypes in subjects presenting for cardiac risk evaluation (n=1,876).

“While plasma levels of all 3 metabolites (choline, betaine, and TMAO) identified in the original metabolomics study are associated with increased risk of CVD phenotypes in subjects presenting for cardiac risk evaluation (n = 1,876) (25), subsequent analyses in larger cohorts revealed that the prognostic value was largely confined to the formation of TMAO, especially from choline and L-carnitine (”

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.”

TMAO supplementation increased surface levels of scavenger receptors CD36 and SR-A1 on macrophages involved in cholesterol accumulation and foam cell formation.

“Moreover, enhanced protein surface levels were observed of previously implicated scavenger receptors (CD36 and SR-A1) on macrophages involved in cholesterol accumulation and foam cell formation (25).”

TMAO supplementation in apolipoprotein E-null mice enhanced macrophage foam cell formation in the artery wall and peritoneal cavity and promoted aortic root atherosclerotic plaque development.

“TMAO supplementation of apolipoprotein E-null mice was observed to foster enhanced macrophage foam cell formation in both the artery wall and the peritoneal cavity, as well as to promote aortic root atherosclerotic plaque development (25).”

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.”

An obligatory role for gut microbiota in TMA and TMAO formation from ingested phosphatidylcholine was confirmed in germ-free mice and human clinical investigations using egg yolk, isotope-labeled phosphatidylcholine, and oral antibiotics.

“An obligatory role for gut microbiota in both TMA and TMAO formation from ingested PC was confirmed in animal model studies, which included germ-free mice (25), as well as human clinical investigations involving ingestion of egg yolk, isotope-labeled PC, and a cocktail of oral antibiotics (37).”

In humans and mice, hepatic FMO3 expression is lower in males than females and can be induced by dietary bile acids through an FXR-dependent pathway.

“In both humans and mice, hepatic FMO3 expression was observed to be reduced in males compared with females (25,54) and could be induced by dietary bile acids through a mechanism that involves FXR (54).”

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)”

Plasma TMAO levels in subjects are associated with prevalent and incident cardiovascular disease risks.

“plasma levels in subjects are associated with prevalent and incident cardiovascular disease risks (5, 7, 9, 10).”

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”

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).”

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.”

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.”

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]”

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 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”

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”

Potential nutrient precursors for TMA production include c-butyrobetaine, crotonobetaine, and betaine.

“Beyond choline and carnitine, potential nutrient precursors for TMA production have been reported, including the carnitine related metabolites c-butyrobetaine and crotonobetaine, and the choline oxidation product betaine.1,16,19”

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”

The metaorganismal TMAO pathway is a potential candidate to explain the association between red meat ingestion and heightened CVD risks.

“One potential candidate for this is the metaorganismal TMAO pathway.1,14,15”

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.”

Production of TMA and TMAO is almost totally suppressed with broad-spectrum antibiotics such as Ciprofloxacin, Vancomycin or Metronidazole in rats and humans, but levels return to normal after one month of antibiotic withdrawal.

“some studies performed with antibiotics in rats and humans reveal that the production of TMA and TMAO is almost totally suppressed with the use of broad-spectrum antibiotics, such as Ciprofloxacin, Vancomycin or Metronidazole. However, the levels of TMAO return to normal after one month of the withdrawal of the antibiotics [7,52].”

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 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 increases expression in macrophages of scavenger receptors CD36 and SR-A1, which promote lipid accumulation and foam cell formation.

“Additionally, TMAO increases the expression in macrophages of scavenger receptors CD36 and SR-A1, which promote lipid accumulation and foam cell formation [7,17].”

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].”

In that study, macrophage foam cell formation was inhibited.

“Plasma TMAO levels were supressed and macrophage foam cell formation was inhibited [7].”

In mice, a mix of vancomycin, neomycin-sulphate, metronidazole and ampicillin inhibited dietary choline-enhanced atherosclerosis.

“Other studies performed in mice using a mix of vancomycin, neomycin-sulphate, metronidazole and ampicillin, showed an inhibition of dietary choline-enhanced atherosclerosis.”

In that study, plasma TMAO levels were suppressed.

“Plasma TMAO levels were supressed and macrophage foam cell formation was inhibited [7].”

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]”

Circulating concentrations of TMAO were predictive of prevalent cardiovascular diseases and future cardiovascular events in clinical cohorts.

“Recent studies by Wang and Tang et al reported that circulating concentrations of trimethylamine N‐oxide (TMAO), a metabolite derived from the gut microbiota, were predictive of prevalent cardiovascular diseases (CVDs)1 and future cardiovascular events3 in clinical cohorts.”

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”

Trimethylamine oxide promotes atherosclerosis in animal models, providing the first link between dietary lipid intake, the intestinal microbiota, and risk for atherosclerosis.

“TMAO was also found to promote the development of atherosclerosis in animal models, providing the first link between dietary lipid intake, the intestinal microbiota, and the risk for the development of atherosclerosis57.”

The intestinal microbiota may contribute to atherosclerosis by producing metabolites of the dietary lipid phosphatidylcholine that are associated with risk for coronary vascular disease.

“The intestinal microbiota may also contribute to development of atherosclerosis by producing metabolites of the dietary lipid phosphatidylcholine that are associated with the risk for coronary vascular disease.”

Wang et al. identified a novel pathway linking dietary lipid intake, the intestinal microbiota, and atherosclerosis.

“Wang et al. identified a novel pathway linking dietary lipid intake, the intestinal microbiota, and atherosclerosis (Figure 3)57, 58.”

Catabolism of choline by the intestinal microbiota forms trimethylamine, which the liver metabolizes into trimethylamine oxide; trimethylamine oxide is strongly associated with increased risk for coronary vascular disease in humans.

“Catabolism of choline by the intestinal microbiota results in the formation of the gas, trimethylamine (TMA), which is metabolized by the liver into trimethylamine oxide (TMAO)—a small molecule that is strongly associated with the increased risk for coronary vascular disease in humans.”

High circulating TMAO increases cholesterol influx into peritoneal macrophages, while cholesterol efflux or biosynthesis appears unchanged.

“Even though high concentrations of circulating plasma TMAO increase cholesterol influx to peritoneal macrophages (118), cholesterol efflux or cholesterol biosynthesis appears to be unchanged.”

Transcript levels of hepatic bile acid transporters Oatp1, Oatp4, Mrp2, and Ntcp were significantly reduced by TMAO feeding.

“Transcript levels of hepatic bile acid transporters, such as Oatp1, Oatp4, Mrp2, and Ntcp, also showed significant reductions in response to TMAO feeding (118).”

Dietary TMAO supplementation decreased intestinal cholesterol absorption, consistent with reduced Niemann-Pick C1-like 1 expression.

“Dietary supplementation of TMAO also decreased intestinal cholesterol absorption.The effect is consistent with reduced expression of the cholesterol transporter Niemann-Pick C1-like 1 but again does not explain reductions in RCT (118).”

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).”

TMAO feeding did not change hepatic cholesterol exporter expression, but it caused striking reductions in bile acid pool size and species and reduced expression of Cyp7a1 and Cyp27a1.

“TMAO feeding did not change the expression of hepatic cholesterol exporters, but striking reductions in bile acid pool size and species, along with reduced expression of the cytochrome P450 family of enzymes, Cyp7a1 and Cyp27a1, were noted (118).”

Modest but significant increases in cholesterol transporters such as Abca1 and Abcg1 were observed.

“In fact, modest but significant increases in many cholesterol transporters, such as Abca1 and Abcg1, were noted (118).”

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).”

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).”

Those macrophages had excess cholesterol accumulation and foam cell formation.

“These macrophages had excess cholesterol accumulation and foam cell formation (118).”

Peritoneal macrophages from C57BL/6J mice fed a TMAO-supplemented diet for more than three weeks showed increased mRNA levels of CD36 and SR-A1.

“Peritoneal macrophages obtained from C57BL/6J mice, fed a TMAO-supplemented diet for more than three weeks, showed increased mRNA levels of two macrophage scavenger receptors (CD36 and SR-A1), which are implicated in atherosclerosis (118).”

A link between dietary phosphatidylcholine, gut microbiota-dependent TMAO generation, and cardiovascular disease pathogenesis was first described less than a decade ago.

“a link between dietary phosphatidylcholine, a nutrient common in a Western diet, gut microbiota-dependent generation of the metabolite trimethylamine N-oxide (TMAO), and cardiovascular disease (CVD) pathogenesis, was first described7.”

TMAO is a microbiota-dependent metabolite produced from TMA-containing nutrients common in Western diets.

“Trimethylamine-N-oxide (TMAO), a microbiota-dependent metabolite derived from trimethylamine (TMA)-containing nutrients that are abundant in a Western diet”

Plasma trimethylamine-N-oxide (TMAO), a choline metabolite, is a novel and independent risk factor for promoting atherosclerosis.

“A recent metabolomics approach identified that plasma trimethylamine-N-oxide (TMAO), a choline metabolite, is a novel and independent risk factor for promoting AS (2, 3).”

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).”

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).”

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.”

Plasma TMAO is a metabolite strongly associated with atherosclerosis.

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

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).”

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”

Trimethylamine-N-oxide (TMAO) is a novel and independent risk factor for promoting atherosclerosis through inducing vascular inflammation.

“Trimethylamine‐N‐oxide (TMAO) has recently been identified as a novel and independent risk factor for promoting atherosclerosis through inducing vascular inflammation.”

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”

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).”

In an initial untargeted LC-MS/MS investigation, choline and its major metabolites betaine and TMAO were associated with cardiovascular disease risk, particularly TMAO.

“In their initial untargeted LC-MS/MS investigation (171), the authors showed that the nutrient choline, along with its major metabolites, betaine and TMAO, were associated with risks of cardiovascular disease, particularly TMAO.”

High TMAO levels linked to TMA oxidation by the human hepatic flavin monooxygenase FMO3 have been associated with cardiovascular diseases.

“These observations are of interest because high TMAO levels linked to TMA oxidation by the human hepatic flavin monooxygenase FMO3 have been associated with cardiovascular diseases (CVDs) (Tang et al., 2013; Wang et al., 2011), while it is known that RYGB is associated with a lower CVD risk (Sjöström et al., 2012).”

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).”

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 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).”

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).”

Once absorbed by the host, TMA is further metabolized by flavin monooxygenases 1 and 3 (FMO1 and FMO3) in the host liver to generate trimethylamine-N-oxide (TMAO).

“Once TMA is absorbed by the host, it is further metabolized by flavin monooxygenases 1 and 3 (FMO1 and FMO3) in the host liver to generate trimethylamine-N-oxide (TMAO) (8, 15, 16).”

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).”

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).”

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).”

Previous studies in conventionally raised mice demonstrated that FMO3 is expressed at higher levels in females than in males.

“consistent with previous studies in conventionally raised mice which demonstrated that FMO3 is expressed at higher levels in females than in males (8, 27).”

TMAO is linked to cholesterol accumulation in macrophages and foam cell deposition and to higher risk of a major adverse cardiac event.

“a metabolite linked to accumulation of cholesterol in macrophages and foam cell deposition127, and higher risk of a major adverse cardiac event128.”

Choline-containing compounds are metabolized by microbial glycyl radical enzymes to trimethylamine, which is absorbed and oxidized by hepatic flavin monooxygenases to TMAO.

“In the colon, choline-containing compounds undergo metabolism by microbial glycyl radical enzymes126 to form the intermediate gas trimethylamine (TMA; Fig. 4). In turn, TMA is absorbed and oxidized by hepatic flavin monooxygenases, forming TMA N-oxide (TMAO)”

TMAO reinforces cholesterol accumulation in macrophages and foam cell formation.

“and by reinforcing cholesterol accumulation in macrophages and foam cell formation.186”

TMAO augments atherosclerosis by upregulating macrophage scavenger receptors CD36 and SR-A1.

“TMAO augments arthrosclerosis by upregulating the macrophage scavenger receptors CD36 and SR-A1”

Recent studies reveal a link between microbiota and atherogenesis.

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

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].”

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).”

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).”

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 produced by gut microbiota from dietary quaternary amines, mainly choline and carnitine, is associated with atherosclerosis and severe cardiovascular disease.

“This is exemplified by trimethylamine (TMA), a microbial metabolite that is produced by various taxa of the gut microbiota from dietary quaternary amines, mainly choline and carnitine. TMA is absorbed via the intestinal epithelium and further oxidized to trimethylamine N-oxide (TMAO), which was associated with atherosclerosis and severe cardiovascular disease in several independent studies [1–4].”

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.”

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.”

Wang et al. first causally linked gut microbiota with oral intake of nutrient precursors, trimethylamine N-oxide production, and cardiovascular disease risk.

“Almost a decade ago, Wang et al. first causally linked gut microbiota with oral intake of nutrient precursors, TMAO production, and CVD risk (Figures 1 and 2)9.”

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 was shown to predict cardiovascular disease risks in multiple clinical cohorts and to facilitate accelerated atherosclerosis in animal models.

“Several analytes linked to phosphatidylcholine metabolism, including TMAO, were identified, and TMAO was shown to both predict CVD risks in multiple clinical cohorts, and facilitate accelerated atherosclerosis (as did nutrient precursors with an intact host gut microbiome) in animal models9.”

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.”

Trimethylamine produced by gut microbes is transported to the liver via the portal vein and readily metabolized by host hepatic flavin monooxygenases, mainly FMO3, into trimethylamine N-oxide.

“The TMA produced is then transported to liver via the portal vein and readily metabolized by host hepatic flavin monooxygenases (FMOs) (mainly FMO376) into TMAO9.”

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.”

Rodents show significant sexual dimorphism in FMO3 expression and TMAO levels, with females showing higher levels and greater atherosclerosis capacity.

“Rodents show a significant sexual dimorphism with respect to FMO3 expression, and TMAO levels, with females showing higher levels (and greater atherosclerosis capacity9, 76).”

A cluster of three phospholipid-associated molecules choline, betaine, and TMAO is associated with atherosclerosis.

“Using a metabolomics approach, Wang et al. discovered a cluster of three phospholipid-associated molecules choline, betaine, and TMAO, associated with atherosclerosis [32].”

In a murine model of atherosclerosis, plasma TMAO levels in apoE−/− mice positively correlated with atheroma burden.

“Further studies in a murine model of atherosclerosis demonstrated that plasma TMAO levels in apoE−/− mice positively correlated with atheroma burden [32].”

Diet containing choline, TMAO or betaine can enhance up-regulation of multiple macrophage scavenger receptors linked to atherosclerosis.

“Wang et al. showed that diet containing choline, TMAO or betaine could enhance up-regulation of multiple macrophage scavenger receptors that are linked to atherosclerosis [32,38].”

Trimethylamine N-oxide (TMAO) is a pro-atherogenic and pro-thrombotic metabolite produced from gut microbiota metabolism of dietary trimethylamine (TMA)-containing nutrients such as choline or carnitine.

“Systemic levels of trimethylamine N-oxide (TMAO), a pro-atherogenic and pro-thrombotic metabolite produced from gut microbiota metabolism of dietary trimethylamine (TMA)-containing nutrients such as choline or carnitine, predict incident cardiovascular event risks in stable primary and secondary prevention subjects.”

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 .”

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”

Trimethylamine and trimethylamine N-oxide, gut microbiota metabolites of dietary phosphatidylcholine, are associated with cardiovascular disease and with atherosclerosis.

“Trimethylamine (TMA) and TMA N-oxide, which are gut microbiota metabolites of dietary phosphatidylcholine, are known to be associated with cardiovascular disease and with the atherosclerosis process in particular.3,4”

In the OCA trial for NASH, increased blood lipid levels were observed.

“in the OCA trial for NASH, additional metabolic derangements such as increased blood lipid levels were also observed, in contrast to the findings of other proof of concept studies152,153.”

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.”