Gut microbiota-derived metabolite trimethylamine N-oxide in major adverse cardiovascular events: Mechanisms, risk assessment, and therapeutic strategies

Abstract

The relationship between trimethylamine N-oxide (TMAO), gut microbiome (GM), and major adverse cardiovascular events (MACE) has gained increasing interest in clinical research. TMAO, a metabolite formed by GM metabolism of dietary nutrients like choline and L-carnitine, has been implicated in the pathogenesis of cardiovascular diseases, including ischemic heart disease, acute myocardial infarction, stroke, and heart failure. Its role in promoting atherosclerosis, endothelial dysfunction, vascular inflammation, and thrombosis highlight its clinical significance. The evidence suggests that unhealthy diet habits and sedentary lifestyles promote GM dysbiosis, which increases TMAO production and MACE risk. Various interventions, like dietary changes, probiotics, and prebiotics, can improve GM to reduce circulating TMAO levels. These approaches show promise in mitigating inflammation and vascular damage associated with cardiovascular diseases. Future research is needed to assess long-term clinical outcomes and establish effective, personalized GM modulation protocols. This review draws upon recent reviewed studies from biomedical databases focusing on TMAO related mechanisms, GM dysbiosis, and MACE risk.

Key Words: Trimethylamine N-oxide; Major adverse cardiovascular event; Atherosclerosis; Prognosis biomarker; 3,3-dimethyl-1-butanol; Fluoromethylcholine

Core Tip: Trimethylamine N-oxide (TMAO) is increasingly proposed as a prognostic biomarker and therapeutic target in cardiovascular disease, yet its clinical signal is strongly shaped by renal function and cardiometabolic comorbidity. We synthesize evidence linking TMAO to risk stratification, including incremental value beyond GRACE in acute coronary syndrome, highlight mechanistic advances from microbiota-targeted trimethylamine-lyase inhibitors, 3,3-dimethyl-1-butanol/fluoromethylcholine, that improve vascular and cardiac phenotypes in preclinical models, and outline a precision-nutrition framework that tailors TMAO-lowering strategies to individual producer metabotypes.

INTRODUCTION

Major adverse cardiovascular events

Patients with a history of cardiovascular diseases (CVD) are at an increased risk of major adverse cardiovascular events (MACE), such as acute myocardial infarction (AMI), stroke, and heart failure (HF). These events are associated with increased hospitalization, high care costs, and finally impaired quality of life[1]. Worldwide, CVD accounts for more than 17.9 million deaths each year, 32% of all deaths with AMI and CVD responsible for approximately 85% of these fatalities[2].

Most CVD can be prevented by addressing environmental and behavioral risk factors, such as smoking, unhealthy eating habits, obesity, physical inactivity, alcoholism, and environmental pollution[2]. These factors directly affect both cardiovascular health and the composition and balance of gut microbiome (GM), suggesting an indirect but significant link between the environment, lifestyle, and cardiovascular risk. In this context, GM has emerged as a key factor in cardiovascular health. Through the production of metabolites such as trimethylamine N-oxide (TMAO), derived from nutrients like choline, L-carnitine, and betaine, elevated TMAO levels have been associated with an increased risk of MACE, positioning it as a potential prognostic biomarker in CVD patients.

GM functions

GM is an ecosystem of bacteria, fungi, viruses, and parasites residing in the gastrointestinal (GI) tract[3]. Established at birth and evolving throughout life, it maintains a commensal relationship with the host and is a fundamental part of the human body[4]. GM is determined by the host’s diet, environment, and activity patterns[5]. It is also essential for maintaining body homeostasis, modulating the immune system, and influencing its development and physiology. It also plays a relevant role in host metabolism, participating in nutrient metabolism, producing essential vitamins, and removing xenobiotics, among other functions[6]. When all these mechanisms fail, GM dysbiosis occurs, usually caused by an inadequate diet, constant stress, increased inflammatory markers, and the inappropriate use of drugs, mainly antibiotics. These disrupt the body’s homeostatic functions and can play a significant role in the pathophysiology of a plethora of metabolic and CVD[3].

The GM is primarily composed of the commensal bacterial phyla Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria, with Firmicutes and Bacteroidetes being the most abundant. These bacteria generate a wide variety of metabolic products, including trimethylamine (TMA), which is converted into TMAO, short-chain fatty acids, and bile acids. However, under conditions of GM dysbiosis, these metabolic processes become dysregulated. Dysbiosis is a shared feature of many risk factors[7].

TMAO biosynthesis and metabolism

As previously reported, TMAO is a small molecule formed by the TMA oxidation by flavin-containing hepatic monooxygenases[8]. TMA is produced by the GM from dietary precursors, primarily betaine, L-carnitine, and choline, and subsequently crosses the gut lumen and is transported through the portal circulation to the liver. TMA is then converted to TMAO by flavin monooxygenase (FMO). Of the five members of the FMO family, only FMO1 and FMO3 have the ability to oxidize TMA to TMAO, with FMO3 being the major isoform expressed in the human liver. Once produced, TMAO is removed from the systemic circulation primarily through urinary excretion, in its unmodified form, by means of filtration and active secretion mechanisms[9] (Figure 1).

Figure 1 Trimethylamine N-oxide metabolic pathway. Choline, betaine, and L-carnitine, present in animal-derived foods, are metabolized by the gut microbiome into trimethylamine (TMA) by gut microbiome enzyme complexes. TMA is absorbed and transported to the liver via the portal vein system, where it is oxidized by the coenzyme flavin monooxygenase 3 to TMA oxide. This metabolite is primarily excreted through the kidney. TMAO: Trimethylamine N-oxide; cutC/D: Choline utilization gene C/D; GbcA/B: Glycine betaine reductase components A/B; GrdH: Glycine reductase component H; cntA/B: Carnitine monooxygenase system; TMA: Trimethylamine; FMO3: Flavin monooxygenase 3.

It has been suggested that hepatic TMAO production could increase cardiovascular and mortality risk by promoting the development of atherosclerotic lesions[10,11]. Several families of bacteria from the phyla Firmicutes, Proteobacteria, and Actinobacteria have been identified as producing high TMAO concentrations when compared to Bacteroidetes. The most abundant genera have been identified as belonging to the families Clostridiaceae, Lachnospiraceae, and Veillonellaceae, as well as Clostridiales from the phylum Firmicutes.

The CVD patients have been found to have a different GM composition compared to healthy individuals[5], for example, plasma TMAO levels were shown to be associated with Prevotella and Peptococcaceae[12], and a positive correlation has been observed between Clostridium and TMAO formation, which could be considered as a marker of inflammation and CVD[13,14]. Furthermore, bacteria of the class Erysipelotrichia have been reported to promote atherosclerosis through the production of TMA and TMAO. Conversely, individuals who produce low levels of TMAO have higher numbers of Bacteroidales[13,15]. Finally, evidence suggests that subjects with a Bacteroidetes deficiency are at higher risk of coronary artery disease (CAD) compared to individuals with an adequate GM quality[5]. This relationship is reinforced by the observation that GM plays a crucial role in TMAO production and its link to inflammation and MACE risk[16].

Atherosclerosis and TMAO

Atherosclerosis is an inflammatory disease triggered by lipid accumulation and the deposition of fatty, fibrous, and calcified material in the innermost layer of large and medium-sized arteries[17]. Atherosclerosis develops through three fundamental processes: (1) The deposition of atherogenic lipids; (2) Proinflammatory conditions; and (3) Endothelial dysfunction. In this context, elevated low-density lipoprotein cholesterol (LDL-C) and reduced high-density lipoprotein cholesterol (HDL-C) levels favor the formation of foam cells from macrophages, a key process in atherogenesis[18]. This process is further amplified by the presence of lipopolysaccharide (LPS), an endotoxin derived from gram-negative bacteria that, upon crossing the altered GI barrier, enters the systemic circulation and activates monocytes and macrophages[19].

Once formed, foam cells secrete proinflammatory cytokines, mainly interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein 1, promoting further monocyte recruitment and perpetuating the inflammatory response[17]. Consequently, vascular smooth muscle cells proliferate, causing intimal thickening. As the process repeats, plaque thickness increases, forming a fibrous cap that, together with programmed leukocyte cell death and microcalcifications, favors the formation of a necrotic core (Figure 2). These plaques, more vulnerable to rupture, can negatively modify blood flow, induce thrombosis and impair or even block blood flow, leading to acute coronary syndromes (ACS)[17,18,20].

Figure 2 Role of trimethylamine N-oxide in atherosclerotic plaque formation and thrombosis. Increased circulating trimethylamine N-oxide promotes endothelial dysfunction, foamy macrophage accumulation, and an inflammatory environment favoring the expression of adhesion molecules (vascular cell adhesion molecule-1, intercellular adhesion molecule 1). In addition, it contributes to lipid imbalance, which increases the uptake of oxidized low-density lipoprotein cholesterol by macrophages, increases platelet reactivity, and alters vascular remodeling through collagen synthesis and calcification. They culminate in the formation and maturation of atherosclerotic plaque. TMAO: Trimethylamine N-oxide; c-LDL: Low-density lipoprotein cholesterol; oxc-LDL: Oxidized low-density lipoprotein cholesterol; c-HDL: High-density lipoprotein cholesterol; VCAM-1: Vascular cell adhesion molecule-1; ICAM-1: Intercellular adhesion molecule-1; P2Y12: Purinergic receptor P2Y12; ROS: Reactive oxygen species.

It has been proposed that TMAO is related to the atherosclerosis pathogenesis, activating proinflammatory pathways and making changes that promote endothelial dysfunction by activating signaling cascades. Additionally, evidence shows that TMAO induces mitogen-activated protein kinases, extracellular signal-regulated kinase and nuclear factor kappa B, whose signaling is activated in association with the G-protein beta-gamma subunit dimer, in endothelial cells[11,21]. Moreover, TMAO significantly increases inflammatory markers, including cyclooxygenase 2, IL-6, E-selectin, and intercellular adhesion molecule 1, which recruit leukocytes and induce vascular inflammation[21]. There is an increase in thrombosis through platelet hyperreactivity, which occurs since direct exposure of platelets to TMAO enhances stimulus-dependent platelet activation from multiple agonists through augmented Ca²⁺ release from intracellular stores[11,22] and atherosclerosis development promoted by the accumulation of cholesterol in macrophages, induced by the expression of scavenger-like receptors, CD36 and scavenger receptor A1[23], and decreased reverse cholesterol transport, as well as defects in cholesterol metabolic pathways (Figure 2). Atherosclerosis is one of the main causes of CVD, and given TMAO’s association with the atherosclerosis development, it may also be linked to CVD[11]. Furthermore, elevated TMAO levels have been correlated with an increased MACE risk[16].

TMAO AND PATHOPHYSIOLOGICAL PATHWAYS UNDERLYING MACE

Implications of TMAO in CAD

Growing evidence suggests that GM and oral microbiota (OM) play an essential role in cardiovascular health[24]. TMAO plays a pivotal role in the development of atherosclerosis. As mentioned above, this metabolite not only induces local inflammation that contributes to the progression of CVD, but also alters blood lipid levels, further accelerating this process[25]. A systematic review and meta-analysis by Hassan et al[5] reported that the increased abundance of the genera bacteria Acidaminococcus and Aggregatibacter may unfavorably modulate HDL-C levels, promoting lipid dysregulation and atherogenic risk. This lipid perturbation may contribute to the establishment of advanced arterial obstructions and myocardial ischemia. In addition, elevated TMAO concentrations have been shown to be associated with an increased risk of peripheral artery disease, CAD, and AMI[16,23]. Furthermore, it has been proposed that TMAO may also be involved in the instability and rupture of atherosclerotic plaques by inducing, in addition to inflammatory processes, endothelial dysfunction, further increasing cardiovascular risk[26].

In this context, CAD represents one of the most common clinical manifestations of the afore-mentioned disorders, characterized by the accumulation of plaque in the heart arteries. If not treated promptly, it can progress to ACS, such as AMI, or even stroke[27]. CAD is highly associated with complex metabolic disorders, including obesity, insulin resistance, and type 2 diabetes mellitus (T2DM)[28], so these metabolic changes represent an indication of the disease onset and progression[29] (Figure 3).

Figure 3 Components of major cardiovascular events. Elevated trimethylamine N-oxide levels contribute to the development of various cardiovascular diseases that are classified as major cardiovascular events. These factors derived from mechanisms such as inflammation, myocardial fibrosis, atherosclerosis, neuroinflammation, platelet activation, atherosclerotic plaque rupture and instability, and thrombus formation. TMAO: Trimethylamine N-oxide.

TMAO has gained relevance as a potential marker associated with CAD progression. In a prospective study conducted in China, patients diagnosed with CAD were found to have significantly higher plasma TMAO levels compared with subjects without the disease[29]. Similarly, another study in CAD patients documented that those with plaque rupture had significantly higher TMAO concentrations compared with those without plaque rupture (8.6 ± 4.8 μmol/L vs 4.2 ± 2.4 μmol/L)[30]. These findings support the hypothesis that circulating TMAO levels may mirror the progression and instability of coronary plaques.

TMAO as a prognostic biomarker in ACS

ACS are critical conditions characterized by coronary artery occlusion due to the rupture of atherosclerotic plaques. A portion of the thrombus can break off and block one of the coronary arteries, leading to diminished blood flow and causing AMI[31-33]. TMAO is closely linked to the ACS progression. Furthermore, TMAO concentrations in patients with moderate atherosclerosis were higher than those with mild atherosclerosis, suggesting a strong association between plasma TMAO concentrations and the severity of coronary atherosclerosis[33]. These findings reinforce the concept that TMAO could be a relevant risk factor for severe atherosclerosis and a predictive biomarker for ACS[29,33].

AMI is the leading cause of morbidity and mortality worldwide[34]. It is characterized by clinical evidence of ischemia, along with alterations in cardiac troponin levels. It is classified into ST-segment elevation myocardial infarction (STEMI) and non-STEMI[35]. The AMI pathophysiology involves a network of complex interactions, with atherosclerosis as a principal risk factor and multiple downstream processes, including endothelial dysfunction, vascular inflammation and thrombosis, culminating in intimal thickening and, ultimately, obstruction of coronary blood flow[14,36] (Figure 3). Within this multifactorial framework, the OM has been proposed as an additional contributor to acute coronary instability. The OM bacteria, such as Streptococcus intermedius, Streptococcus sanguis, Streptococcus anginosus, Tannerella forsythensis, Tannerella denticola, and Porphyromonas gingivalis, have been linked to ACS, with higher bacterial loads observed in patients, suggesting that these species may be associated with ACS development and could contribute to pro-atherothrombotic pathways[14]. On the other hand, OM has gained relevance in AMI due to its probable involvement in the TMAO production, showing an association between OM dysbiosis and elevated plasma levels of this metabolite in AMI patients. A study in patients with STEMI showed a positive correlation between TMAO levels and the abundance of the genera Porphyromonas, as well as Aggregatibacter. Furthermore, TMAO showed a positive association with triglycerides and a negative association with HDL-C[37], suggesting a potential link between OM dysbiosis, TMAO metabolism and atherogenic lipid profile in these patients.

In AMI patients, GM is altered and characterized by a lower abundance of the phylum Firmicutes and a slightly higher abundance of the phylum Bacteroidetes[34]. Beyond these broad taxonomic shifts, case-control data further suggest that ACS is accompanied by more specific, potentially pathogenic microbial signatures and host-barrier perturbations. One case-control study showed that patients with ACS had a nine-fold increase in gut Gammaproteobacteria, a class that includes gram-negative pathogens, opportunistic bacteria, and Enterobacteriaceae, along with increased gut permeability compared with controls. In the same cohort, compositional differences were also observed at genus/order level, with ACS patients showing higher levels of Prevotella and controls having higher levels of Bacteroidales overall. Notably an elevated Prevotella/Bacteroides ratio has been associated with a high-carbohydrate dietary pattern, which may further contribute to cardiometabolic risk[38]. Similarly, a prospective study, with 211 STEMI patients, documented that patients with plaque rupture had three times as many levels of TMAO compared to those without plaque rupture (3.33 μmol/L vs 1.21 μmol/L)[26]. For another hand, in a study with STEMI patients who presented STEMI, they measured plasma TMAO levels at baseline and again in the chronic phase (10 months later). TMAO levels increased significantly from the acute to the chronic phase of STEMI (median: 5.63-6.76 μmol/L). During a 5.4 year follow up, 17 patients experienced cardiovascular events, and chronic-phase TMAO levels independently predicted subsequent events. These findings underscore the potential prognostic value of chronic phase TMAO for risk stratification after STEMI[39].

Stroke, dysbiosis and TMAO

Strokes occur when blood flow to certain brain regions is interrupted, causing oxygen and nutrient deprivation and resulting in neurodegeneration[40]. This interruption can be caused by an arterial occlusion, leading to ischemic stroke, the most common form, or by intracranial bleeding, resulting in hemorrhagic stroke, which is generally associated with higher fatality[41]. Beyond vascular and hemostatic mechanisms, growing evidence indicates that stroke pathophysiology is influenced by systemic host-GM interactions. It has been shown that the central nervous system maintains constant interaction with the GM, giving rise to the gut-brain axis, where GM-derived metabolites can modulate brain development and physiological functions[42]. Accordingly, during cerebral ischemia, reduced oxygen and glucose delivery induce neuronal injury and triggers the release of proinflammatory factors such as TNF-α, IL-1 and IL-6, promoting neuroinflammation.

This compromises the integrity of the blood-brain barrier by altering the expression of tight junction proteins, increasing its permeability, and allowing the passage of immune cells and GM-derived metabolites, such as TMAO and LPS, into the brain. Consequently, GM, gut metabolites, and immune cells translocate from the leaky gut to the infarcted region of the brain through systemic circulation, exacerbating the stroke severity[40] (Figure 3).

TMAO levels have been associated with the incidence and severity of CVD, including infarct size and functional deficit, suggesting that TMAO may serve as a prognostic biomarker and risk stratifier for adverse outcomes including death[40]. Consistent with this broader vascular risk signal, evidence from stroke-focused cohorts has linked baseline TMAO levels to both incident and recurrent cerebrovascular events. Using data from the China Stroke Primary Prevention Trial, one analysis evaluated baseline serum TMAO concentrations and the risk of a first stroke and found that individuals with higher TMAO levels (≥ 1.79 μmol/L) had a 34% higher risk of a first stroke compared with those with lower levels (< 1.79 μmol/L), supporting an association between TMAO and the stroke risk in hypertensive patients[43].

Xue et al[44] conducted a prospective, multicenter study of patients with acute ischemic stroke, participants with high TMAO and choline levels had significantly higher cumulative recurrence rates than those with low TMAO and choline levels. Similarly, two prospective cohort studies involving patients with previous ischemic stroke further reinforced the association between TMAO levels and the risk of subsequent cardiovascular events, mainly AMI, recurrent stroke, and cardiovascular death, suggesting that it might be useful in predicting these outcomes[45]. In conclusion, TMAO has been identified as a significant risk factor in the development and severity of various diseases, including stroke.

HF and TMAO

HF is a clinical syndrome characterized by a decreased ability of the heart to pump or fill with blood, resulting in inadequate cardiac output[46]. This syndrome is a common complication of atherosclerotic CVD that can manifest itself as myocardial ischemia, vascular dysfunction, and fibrosis, leading to progressive diastolic dysfunction or progressive myocyte damage and cardiac remodeling that culminate in systolic dysfunction[47]. Key risk factors include CVD and components of metabolic syndrome (MetS)[48] (Figure 3).

HF is a multisystem disorder that affects the heart and circulation but also other systems, including the metabolic system[19]. In this context, the GI tract has acquired relevance as a key organ in the pathogenesis of chronic HF. Mesenteric venous congestion, characteristic of this condition, can increase gut permeability, promoting bacterial translocation and endotoxin release. This process contributes to sustained immune activation, evidenced by increased levels of CD14, TNF-α, proinflammatory cytokines such as IL-1 and IL-6, and adhesion molecules such as intercellular adhesion molecule 1[49-51]. Mechanistically, this GM-hypothesis of HF links hemodynamic derangements to barrier failure and systemic inflammation.

It has been proposed that activation of the sympathetic nervous system, although with a compensatory purpose in HF due to its inotropic and vasoconstrictor effect, may contribute to gut ischemia by reducing splanchnic blood flow[52]. Under these conditions, LPS can cross the compromised gut barrier and enter the systemic circulation, promoting the release of proinflammatory mediators. Sandek et al[19], reported that the gut permeability index, measured by the urinary lactulose/mannitol ratio, has been shown to be significantly higher in patients with chronic HF, mirroring a 35% increase in gut permeability. This barrier dysfunction provides a plausible substrate for GM-host interaction that may amplify systemic inflammation and metabolic perturbations in HF.

GM dysbiosis is increasingly recognized as a contributor to elevated TMAO levels and has been linked to the development and progression of HF[23]. Tang et al[47] proposed that gut congestion and interstitial edema could alter GM composition, thereby enhancing microbial TMA production and downstream TMAO production. Clinically, higher circulating TMAO levels have been associated with adverse cardiac structure and function, including impaired diastolic indices, such as increased left atrial volume, suggesting that its accumulation could affect tissue mechanics, through increased myocardial fibrosis, resulting in cardiac hypertrophy, and increased accompanying diastolic dysfunction[25,53]. These observations support the view that TMAO may act as a risk marker, and potentially a mechanistic mediator of HF development and progression[25].

As previously reported, subjects with a higher abundance of bacteria from the genera Prevotella, Clostridium, Ruminococcus and the family Lachnospiraceae, and a lower presence of bacteria from the phylum Bacteroidota, have been shown to have elevated plasma concentrations of TMAO[54]. The taxonomic patterns support the concept that compositional shifts in GM can translate into measurable differences in circulating microbial metabolites relevant to HF risk. Furthermore, indole-3-propionic acid, mainly produced by Clostridium sporogenes in GI tract, has been identified to regulate mitochondrial function at supra-physiological levels, by decreasing maximal respiration and respiratory reserve capacity. Such mitochondrial perturbations may contribute to cardiac remodeling and, over time, to HF progression[55].

In HF patients, especially those with HF with reduced ejection fraction, a decrease in GM diversity has been observed. Luedde et al[56] observed lower levels of Blautia, Collinsella and the Erysipelotrichaceae family, alongside increases in the abundance of Streptococcus and Veillonella has been reported[57]. Complementing these findings, Katsimichas et al[57] reported a higher proportion of Fusobacterium prausnitzii in chronic HF. More recently, Pasini et al[58] identified correlations between the presence of Candida, Campylobacter, Shigella, and Yersinia and HF severity.

In a prospective observational study, TMAO levels were significantly higher in patients with ischemic HF than in those with dilated cardiomyopathy or stable CAD, suggesting that both HF and ischemic etiology may contribute to elevated TMAO levels. Consistent with this clinical gradient, TMAO levels were associated with New York Heart Association functional class, a standard measure of HF severity, as well as congestion related hemodynamics indices, including pulmonary capillary wedge pressure and pulmonary artery pressure[59,60]. In line with these observations, another study showed higher TMAO levels than individuals without HF; however, the median TMAO level varied across comparison groups; for HF group TMAO levels were 5.0 μmol/L (interquartile range: 3.0-8.5 μmol/L), which was higher than that of the control cohort (3.5 μmol/L; interquartile range: 2.3-5.7 μmol/L). Underscoring inter-cohort heterogeneity in baseline exposure and patient characteristics.

Beyond cross-sectional severity markers, TMAO has also been linked to prognostic indicators. A significant correlation was observed between TMAO levels and brain-type natriuretic peptide levels, and elevated TMAO was associated with an increased risk of 5-year mortality risk[61]. Moreover, Tang et al[62], reported two prospective cohorts and found that circulating TMAO levels, along with related metabolites such as croto-betaine, betaine, carnitine, and choline, were associated with an increased risk of HF. Supporting a mechanistic link to cardiac structure and function, Tang et al[47] reported elevated TMAO levels, choline and betaine have been associated with higher N-terminal-pro brain-type natriuretic peptide levels and more advanced left ventricular diastolic dysfunction. Summarizing the literature data indicates that elevated TMAO levels are closely related to HF and its etiology, especially in patients with ischemic HF. These higher levels are associated with greater disease severity, as reflected in the New York Heart Association functional classification. Furthermore, elevated TMAO levels correlate with a higher risk of long-term mortality.

RISK FACTORS IN THE DEVELOPMENT OF CVD

MACE represents a group of serious complications, and its incidence rates are closely linked to various risk factors. These factors, categorized as modifiable and non-modifiable, include metabolic, cardiovascular, chronic, and autoimmune conditions[63]. Alterations in GM composition may contribute to the development and complications of CVD through mechanisms such as systemic inflammation, dysbiosis, and metabolite production.

Mediterranean diet vs western diets

Dietary patterns critically shape GM composition and function, with Western and Mediterranean diets exerting distinct effects on microbial diversity, host-microbe interactions, and finally TMAO metabolism. The Western diet, characterized by a high intake of red meat, saturated fats, refined sugars, and predominantly ultra-processed foods, markedly decreases overall GM diversity, promotes significant weight gain and a pro-inflammatory tone, and is associated with a higher relative abundance of Firmicutes alongside a decrease in Bacteroidetes[64,65]. Consistently, Western diet promotes choline and carnitine catabolism, leading to significantly elevated plasma and urinary TMAO concentrations[66,67].

In contrast, adherence to a Mediterranean-style diet, characterized by lower fat intake and high dietary fiber consumption, is associated with improved quality of life, diminished inflammatory markers, and an increased abundance of Bacteroidetes and anti-inflammatory metabolites[68,69]. The high fiber use supports beneficial, short-chain fatty acid-producing GM, whereas fiber deficiency promotes intestinal mucus degradation, barrier erosion, and intestinal inflammation[70]. Notably, dietary fiber supplementation may attenuate TMAO formation by significantly reducing the abundance of the GM choline utilization gene C (cutC) gene, suggesting a microbiota-mediated mechanism for lowering plasma TMAO levels[71,72]. Although the Mediterranean diet permits moderate red meat intake, consumption levels are critical, as lower red meat intake is associated with significantly reduced serum TMAO levels, highlighting the importance of moderation[73]. In sum, western diet promotes dysbiosis and elevated TMAO, whereas a fiber-rich Mediterranean diet supports a healthier GM, gut barrier integrity, and reduced metabolic risk.

High-fat diets as triggers of gut barrier failure

It has been reported that short-term high-fat feeding rapidly compromises gut barrier integrity and mucosal immunity, while promoting obesity, insulin resistance, and non-alcoholic steatohepatitis, in association with GM dysbiosis characterized by a shift from Bacteroidetes toward Firmicutes[74,75]. Likewise, chronic exposure to a high-fat diet enhances choline catabolism, leading to increased TMA/TMAO production and renal excretion, alongside reduced GM diversity[67,76]. Taken together, these findings suggest that high-fat consumption creates a permissive microbial and immunological environment that facilitates barrier dysfunction and enhanced TMAO production, contributing to adverse metabolic outcomes.

Age-related dysbiosis and TMAO

Aging is a gradual decline in homeostasis, functional capacity, leading to increased susceptibility to death[77]. Gut permeability increases with age as a consequence of GM alterations, promoting the translocation of microbial products into the circulation and triggering systemic inflammation, which has been linked to increased mortality[78-81]. Older individuals exhibit distinct GM profiles compared with younger populations; however, evidence is heterogeneous, with some studies reporting a higher abundance of Bacteroidetes and reduced Firmicutes, while others describe decreased Bacteroidetes abundance accompanied by lower overall GM diversity[82-84]. Moreover, advanced age is associated with an enrichment of TMA-producing bacteria carrying the cutC and cntA genes, greater availability of dietary precursors, and elevated circulating TMAO levels, contributing to a persistent inflammatory state and vascular aging[85-87]. In this context, TMAO emerges as a key link between the GM and pathological aging by promoting inflammation, gut barrier dysfunction and endothelial impairment.

Clinical implications of drug-associated dysbiosis

Antibiotics and their microbial regulation: Antibiotics can induce GM dysbiosis. In detail, broad-spectrum agents promote excessive inflammation and pathobiont expansion, leading to immune alterations and dysbiotic GM characterized by selective enrichment and widespread taxonomic loss. In contrast, antibiotics, such as metronidazole, appear to preserve anti-inflammatory capacity by enriching Lactobacillus and supporting innate immune responses[88-92]. Alternatively, antibiotic administration markedly decreases plasma TMAO levels, suggesting therapeutic potential; but, TMAO may also promote antibiotic resistance in certain bacteria, thereby limiting therapeutic efficacy[16,93,94]. Therefore, antibiotics exert complex and context-dependent effects on the GM, with broad-spectrum agents driving dysbiosis and immune disruption, while modulating microbial metabolites such as TMAO, underscoring both their therapeutic potential and limitations.

Metformin’s microbial footprint, inconsistent effects on dysbiosis and TMAO: Metformin, a first-line therapy for T2DM, has emerged as a potential modulator of TMAO concentrations but the evidence remains contrasting. While some studies report increased circulating TMAO concentrations alongside improved glucose control, others document that metformin reduces plasma TMAO levels and bacterial TMA production from choline, with or without accompanying changes in GM diversity[95-98]. However, some evidence indicates that metformin does not significantly affect serum TMAO levels or related metabolic and cardiac outcomes[99]. In sum, the metformin impact on TMAO levels and GM composition remains inconsistent. These discrepancies emphasize the context-dependent nature of metformin-GM interactions and the need for further investigation.

Systemic arterial hypertension

Systemic arterial hypertension (SAH) is the most prevalent risk factor for MACE[100,101] and, due to its ability to cause endothelial damage and vascular remodeling, remains one of the leading causes of premature mortality[102,103]. Recent advances suggest that GM composition is closely related to SAH[104-108], and animal models confirm this association[109-111]. High plasma TMAO concentrations increase the risk of SAH developing by 12%[112]. Since diet modulates GM and its metabolites[112,113], dietary and GM variations are the main determinants of circulating TMAO levels[114-116]. TMAO promotes hypertension by enhancing Ang II-induced vasoconstriction through activation of the protein kinase r-like endoplasmic reticulum kinase/reactive oxygen species/calcium/calmodulin-dependent protein kinase II/phospholipase C β3 signaling pathway, leading to increased intracellular Ca²⁺ and vascular contraction[117]. Maintaining blood pressure within optimal ranges reduces the risk of MACE[118-121]. Therefore, individualized SAH management and GM modulation are emerging as key strategies to prevent cardiovascular events.

T2DM

T2DM is a relevant cause of AMI and stroke[122]. TMAO is linked to T2DM, promoting insulin resistance in animal models, with elevated levels consistently associated with higher disease incidence, and FMO3 playing a key mechanistic role[123-128]. Moreover, high intake of red meat, eggs, and dairy products is associated with higher plasma TMAO concentrations[129,130] and, independently, a higher T2DM risk[131-133]. This is associated with GM dysbiosis, which not only contributes to T2DM development but also worsens its cardiometabolic complications. In T2DM patients undergoing percutaneous coronary intervention, elevated TMAO levels were associated with a higher risk of MACE and mortality[134], highlighting its prognostic value; however, in the ACCORD trial involving poorly controlled, high-cardiovascular-risk individuals, TMAO did not discriminate between those with and without MACE[135]. Thus, TMAO emerges as a biomarker and potential therapeutic target, although its clinical utility may depend on the patient’s glycemic profile and metabolic control.

Chronic kidney disease

TMAO has emerged as a microbiota-derived metabolite closely linked to chronic kidney disease (CKD), defined as abnormalities of kidney structure or function, present for a minimum of 3 months, with implications for health[136]. In this context, patients with CKD exhibit elevated circulating TMAO levels, which are associated with greater renal fibrosis, amplified inflammatory responses, and an increased risk of acute kidney injury-to-CKD progression[137,138]. Moreover, circulating TMAO levels and the TMAO/creatinine ratio increase in a CKD stage-dependent manner, while urinary TMAO levels decline, with the abundance of microbial TMA-producing genes, cutC and cntA, correlating with plasma TMAO and estimated glomerular filtration rate; in parallel, higher TMAO levels are associated with an increased risk of incident CKD and a more rapid annual decline in estimated glomerular filtration rate[139,140].

Dietary habits, aging and certain pharmacological interventions significantly influence GM dysbiosis and circulating TMAO levels. While western diets and high-fat intake promote dysbiosis and TMAO elevation, Mediterranean-style diets and high fiber consumption support a healthier GM and limit TMAO formation. Conditions such as CKD, SAH and T2DM are associated with elevated TMAO, highlighting the critical role of the GM and its metabolites in cardiometabolic risk. These findings emphasize the importance of targeting modifiable factors to prevent metabolic and cardiovascular complications.

PROGNOSTIC ASSESSMENT

CVD prognostic assessment underpins risk stratification and therapeutic decisions but incompletely captures biological heterogeneity and dynamic risk. In this scenario, TMAO has been proposed as a prognostic biomarker and potential causal risk factor through its links to key cardiovascular pathophysiological pathways.

Integrating TMAO into clinical models risk and prognosis stratification

Clinical risk models are tools that integrate clinical and laboratory variables to estimate the outcome probability. In ACS, the GRACE score[141] combines readily available variables to predict the risk of death and AMI. In this scenario, TMAO has been proposed as a complementary biomarker that could add predictive value. Suzuki et al[142] documented that TMAO independently predicted death and recurrent AMI at 2 years and improved 6-month GRACE stratification by down-classifying risk. Similarly, Tan et al[143] reported a prospective cohort of 444 STEMI patients undergoing primary percutaneous coronary intervention; plasma TMAO levels measured before the procedure and predicted 6-month MACE. Of note, adding myeloperoxidase plus TMAO to the GRACE score increased discrimination and improved reclassification, supporting the incremental prognostic value of TMAO for refining GRACE-based risk stratification. In 2017, Li et al[144], reported that higher TMAO levels predicted a markedly increased risk of 30-day and 6-month MACE, and predicted 7-year mortality, consistent with results from a study conducted a year earlier by the same group[145]. Notably, TMAO retained short-term prognostic value even among patients who were troponin T-negative at presentation, suggesting potential utility for early risk stratification before conventional biomarkers become positive.

In complex CAD, the SYNTAX score[146] is used to stratify atherosclerotic disease severity. Senthong et al[147] documented that plasma TMAO levels showed moderate-to-strong correlations with both the SINTAX and SINTAX-II scores, and after multivariable adjustment, higher TMAO levels remained independently associated with higher SINTAX and SINTAX-II scores. Multiple prospective cohorts indicate that plasma TMAO levels improve GRACE-based discrimination and reclassification.

In primary prevention, the Framingham Risk Score[148,149], and QRISK[150] are used to estimate cardiovascular risk in the general population. Sikora et al[151], in a pilot study in psoriasis, analysis indicated that TMAO was an independent predictor of higher Framingham-estimated risk. Similarly, Zou et al[152] reported that uremic TMAO was quantified independently associated with Framingham predicted 10-year cardiovascular event risk. Future prospective primary-prevention studies should test whether adding TMAO improves prediction beyond established scores such as Framingham and QRISK.

Limitations in TMAO as predictor of cardiovascular risk

Translating TMAO from an associative finding into a clinically actionable predictor faces two key challenges: Assay standardization and clarifying the biological meaning of an elevated concentration[153]. First, reported TMAO levels can vary across studies due to differences in sample type, pre-analytical conditions and analytical platforms, thereby limiting cross-study comparability. Second, a critical distinction remains between TMAO as a causal risk factor vs risk biomarker, since observed associations may be influenced by confounding or mediation.

In a 2018 meta-analysis of 11 prospective cohort studies[154], higher TMAO levels were linked to a 23% higher risk of cardiovascular events and a 55% higher risk of all-cause mortality. In addition, a 2017 meta-analysis[155] reported a dose-response relationship, estimating that in CVD populations all-cause mortality risk increases by 7.6% per 10 μmol/L increment in plasma TMAO, consistent with a graded association between TMAO levels and mortality. In 2024, it reported a significant positive association between TMAO and MACE, all-cause mortality, and recurrent AMI in patients with AMI; this finding contrasts with results reported in the PEGASUS-TIMI 54 trial[156]. Other systematic reviews and meta-analyses report broadly concordant association[157,158]. Overall, the meta-analytic literature supports an association between circulating TMAO and MACE although effect sizes and consistency may vary by population and study design.

However, more recently, Obeid et al[159], published an umbrella review summarizing evidence linking circulating TMAO levels with cardiovascular, cerebrovascular, and renal outcomes, including mortality. The main finding was that most reviews reported higher TMAO levels among individuals who experienced adverse outcomes. Consistent with this, like Hu et al[160] that patients with ischemic stroke tend to have higher TMAO levels, yet analyses did not support a causal TMAO effect on stroke occurrence. Notably, Obeid et al[159] emphasized that the underlying primary studies were predominantly conducted in clinically ill, high-risk populations with multiple comorbidities, which may confound causal inference and limit “pure” prognostic interpretation[159]. In addition, definitions of “elevated” TMAO varied substantially across studies, as previously reported[161,162], hindering the establishment of clinically meaningful thresholds for high-risk classification. TMAO interpretation in clinical cohorts is complicated by strong dependence on renal clearance[163-165] and cardiometabolic comorbidity like T2DM and MetS[166] which can confound or mediate observed associations. TMAO often reflects underlying cardiorenal risk rather than acting as an independent causal mediator. In summary, the current evidence remains insufficient to rightly determine whether TMAO is a causal mediator, a risk marker, or a reliable standalone prognostic factor, and higher-quality prospective studies and randomized interventions are needed to clarify its causal and predictive roles.

EMERGING STRATEGIES TO REDUCE TMAO

3,3-dimethyl-1-butanol

3,3-dimethyl-1-butanol (3,3-DMB), a choline analogue, inhibitor of microbial TMA-lyases that suppresses upstream TMA formation, and improves endothelial function reduced foam cell formation and atherosclerotic lesion development[167]. Brunt et al[168] further showed that 3,3-DMB mitigates diet-induced endothelial dysfunction and is associated with improved exercise tolerance. Chen et al[169] extended these findings to cardiac phenotypes by demonstrating that, lowers TMAO and prevents cardiac dysfunction, inflammation and fibrosis. Wang et al[170] similarly reported attenuation of adverse cardiac remodeling in a HF setting, collectively supporting that microbial TMA inhibition via 3,3-DMB can beneficially modulate atherosclerotic, vascular, and cardiac endpoints across multiple disease models, including CKD-associated endothelial dysfunction[171]. However, there is evidence suggesting limited or missing TMA/TMAO suppression by 3,3-DMB in certain non-cardiovascular contexts[170,172]. 3,3-DMB effects can be context-dependent and may not always be mediated by measurable suppression of the TMA/TMAO axis.

Inhibitors of the microbial cutC/D

Halomethylcholines, such as fluoromethylcholine and iodomethylcholine, are mechanism-based, irreversible inhibitors of the microbial cutC/D enzyme, representing promising microbiome-targeted compounds to lower TMAO[173]. These compounds are reported to be substantially more potent than 3,3-DMB in suppressing microbial TMA/TMAO at lower effective concentrations[174]. Future studies should prioritize well-controlled intervention studies that define dose-response relationships and the durability of TMAO suppression, establish safety and off-target consequences, and test whether microbiome-targeted cutC/D inhibition yields consistent benefit across diverse cardiometabolic and vascular phenotypes and in translational settings.

Direct inhibitors of FMO3

Direct hepatic FMO3 inhibition has mainly been explored as an experimental strategy to modulate the TMA/TMAO axis. Methimazole is a high-affinity FMO3 substrate and widely used tool inhibitor that can competitively suppress[175,176]. Dietary indole-derived compounds can also inhibit FMO activity[177]. Finally, phenylthiourea has been used to suppress clearance of FMO3-dependent substrates/metabolites[178].

It has been shown that regulation of hepatic FMO3 decreases TMA levels and increases circulating TMAO levels, whereas silencing it has the opposite effect, i.e., elevates TMA levels and reduces TMAO levels[179]. This phenomenon has been further evidenced in studies where hepatic FMO3 expression was decreased in LDL-C receptor knockout mice using an antisense oligonucleotide. In these experiments, FMO3 reduction resulted in a decrease in both plasma TMAO levels and atherosclerosis[180]. These findings highlight the FMO3 relevance in regulating TMAO and its role in preventing atherosclerosis, thus highlighting alternative therapeutic pathways for CVD.

Intermittent hypoxia-hyperoxia exposure

Another emerging approach, intermittent hypoxia-hyperoxia exposure (IHHE), consists of alternating short periods of reduced and enriched oxygen in order to induce beneficial physiological adaptations[181,182]. This treatment was initially studied in cardiac rehabilitation and fitness improvement[183], its impact on GM[184] and metabolism has now been explored[185,186]. Evidence on whether IHHE can alter GM or reduce TMAO is still under investigation. A prospective study evaluating IHHE in MetS patients with elevated baseline TMAO levels (≥ 5 μmol/L) showed a significant decrease after the intervention. Furthermore, the reduction in total cholesterol and LDL-C was more notable in subjects with normal TMAO levels at baseline[187]; this suggests that controlled hypoxia could have beneficial cardiometabolic effects, especially in individuals with elevated TMAO. Further studies are required to confirm its effectiveness.

NUTRITIONAL INTERVENTIONS TARGETING TMAO TO PREVENT CARDIOVASCULAR EVENTS

Alterations in glucose metabolism

TMAO has been reported to be associated with glucose metabolism and independently predicts the risk of CVD and mortality[154]. In a clinical study in obese subjects at risk of insulin resistance, supplementation with flavonoids from tea and cocoa failed to significantly reduce TMAO levels[188] (Table 1). However, a pilot randomized controlled trial in adults at elevated risk for T2DM. Six weeks of inulin supplementation did not decrease fasting or postprandial plasma TMAO, indicating that simply increasing fermentable fiber is not consistently sufficient to suppress the TMO/TMAO axis[189] (Table 1). In contrast, a clinical trial in prediabetic subjects[190] showed that regular pistachio consumption reduced urinary metabolites related to GM, including TMAO, suggesting a potential beneficial effect on the T2DM risk (Table 1). Similarly, a prospective study showed that a vegan diet[191] significantly reduced TMAO levels, although these levels increased after participants resumed a normal diet (Table 1). These findings support a vegan diet as an effective intervention for reducing this metabolite.

Table 1 Evidence on interventions for trimethylamine N-oxide modification.

Ref.	Intervention	Mechanism	Sample type	Duration	Population	Results
Specific diets and foods
Argyridou et al[191], 2021	Vegan	Reduces dietary precursors	Plasma	8 weeks	Overweight patients over 18 years of age with untreated HbA1c ≥ 5.7% and ≤ 8%	TMAO decrease
Diao et al[211], 2024	DASH	GM remodeling and concurrent lowering LPS	Plasma	12 weeks	Overweight and obese, ages 20-55 years	TMAO decrease; Firmicutes/Bacteroidetes ratio decrease
Hernández-Alonso et al[190], 2017	Pistachios (57 g/day)	Modulation of GM related metabolism chronic pistachio intake	Urinary	15 days	BMI ≤ 35 kg/m2 and glucose levels between 100 and 125 mg/dL, ages 25-65 years	TMAO decrease
Haas et al[214], 2022	Red wine 250 mL/day	Redox homeostasis	Plasma	3 weeks	CAD, average age 60 years	TMAO (ns)
Thomas et al[192], 2022	Consume three eggs per day (400 mg/day)	Dietary choline (phosphatidylcholine), influences GM-dependent TMA generation	Plasma	4 weeks	MetS, ages 35-70 years	TMAO (-); antioxidants increase
Angiletta et al[188], 2018	Cocoa drink (28 g) and green tea (1.2 g)	Nutritionally relevant flavonol doses could reduce TMAO production	Plasma	5 days	Obesity and risk of insulin resistance, ages 25-55 years	TMAO (-)
Bergeron et al[222], 2016	CHO and resistant starches	GM remodeling and CHO diet-GM interaction influence TMAO production, not simply precursor intake	Plasma	56 days	Men > 20 years and postmenopausal women, BMI ≥ 20 and ≤ 35 kg/m2	TMAO increase
Prebiotics
Baugh et al[189], 2018	Inulin 10 g/day added to a standardized diet	Modulate GM and thereby reduce microbial TMA production	Plasma	6 weeks	Sedentary overweight/obese adults elevated risk for T2DM	TMAO (ns); TMA moieties (ns)
Probiotics
Malik et al[216], 2018	Lactobacillus plantarum 299v (20 billion CFU once daily)	Changes in GM derived metabolites (not TMAO)	Plasma	6 weeks	History of stable CAD, age 40-75 years	TMAO (-); propionate increase; acetate decrease; Lactobacillus in stool increase; endothelial function increase
Spasova et al[215], 2024	Lactobacillus plantarum GLP3 (1 × 109 CFU)	GM modulation (increase in Lactobacillus spp.) with plausible contribution from prebiotic and polyphenol	Plasma	12 weeks	History of CAD, age 58.5 ± 8.16 years	TMAO decrease
Tripolt et al[217], 2015	Lactobacillus casei Shirota (6.5 × 109 CFU 3 ×/day)	GM modulation could alter microbial TMA-formation	Plasma	12 weeks	MetS, age 52 ± 11 years	TMAO (ns)
Awoyemi et al[224], 2021	Saccharomyces boulardii 250 mg capsules twice daily and rifaximin 550 mg orally twice daily	GM modulation affects composition and function reducing microbial-linked metabolites (TMAO)	Plasma	3 months	HFrEF and NYHA functional class II or III, average age 60 years	TMAO (-)
Other interventions
Smits et al[200], 2018	Vegan donor FMT (autologous)	GM replacement strategy to reduce GM conversion of choline/carnitine to TMA	Plasma	2 weeks	MetS, no history of CVD, aged 21-69 years	TMAO decrease
Bestavashvili et al[187], 2023	IHHE (total 15 sessions)	Hypoxic conditioning antioxidant/anti-inflammatory signaling and improving gut-liver axis	Stool and plasma	3 weeks	MetS, age 29-74 years	TMAO (ns)

DASH: Dietary Approaches to Stop Hypertension; CHO: Carbohydrate; CFU: Colony forming unit; FMT: Fecal microbiota transplantation; IHHE: Intermittent hypoxia-hyperoxia exposure; GM: Gut microbiome; LPS: Lipopolysaccharide; TMA: Trimethylamine; TMAO: Trimethylamine N-oxide; HbA1c: Glycated hemoglobin; BMI: Body mass index; CAD: Coronary artery disease; MetS: Metabolic syndrome; T2DM: Type 2 diabetes mellitus; HFrEF: Heart failure with reduced ejection fraction; NYHA: New York Heart Association; CVD: Cardiovascular diseases; ns: Not significant.

MetS

In a clinical trial, daily egg intake did not increase plasma TMAO levels nor significantly alter GM compared to a choline supplement, although it increased antioxidant levels of lutein and zeaxanthin[192]. Probiotics have emerged as a complementary strategy to modulate GM[193-195] in metabolic and cardiovascular pathologies[196]. Likewise, fecal microbiota transplantation (FMT), studied in different diseases[197-199], showed, in a pilot study with vegan donors for subjects with MetS, partial changes in GM without effect on TMAO levels and vascular inflammation[200] (Table 1). Together, these findings show the complexity of modulating the GM-TMAO axis in MetS. In detail, probiotics require more specific combinations and FMT requires more intensive or repeated strategies to influence cardiometabolic parameters.

Interventions for obesity and overweight

Obesity is a global health concern associated with an increased risk of CVD[201,202]. Dietary interventions[203,204] and the adoption of healthy lifestyles[205,206] can modulate GM, offering an effective complementary therapeutic option to mitigate metabolic effects. It has been reported that reducing dietary choline and L-carnitine was associated with improvements in insulin resistance with a low-calorie weight-loss diet. Furthermore, increased TMAO levels were associated with less improvement in those following a high-fat diet[207]. A combined intervention of a low-calorie diet and exercise was assessed to determine whether it could reduce TMAO levels more effectively compared to diet alone in obese women[208]. Similarly, other studies involving comparable populations and interventions found that although TMAO levels do not significantly change, it is suggested that a combined intervention may be useful in decreasing TMAO production[209,210] (Table 1). Approaches that combine exercise and diet show clear metabolic benefits and could be useful in reducing TMAO. There are specialized diets, such as the Dietary Approaches to Stop Hypertension[211] that have shown a significant decrease in TMAO levels and the Firmicutes/Bacteroidetes index, which influences TMAO levels (Table 1). The cardiovascular benefits of the Dietary Approaches to Stop Hypertension diet may have a more favorable impact on microbial metabolites involved in cardiovascular risk.

TMAO reduction as prevention of CAD

CAD progresses with high TMAO levels, so reducing this metabolite is a key therapeutic goal. A study evaluated how the consumption of products rich in L-carnitine and phosphatidylcholine influences TMAO levels in CAD patients. Compared with the control group, CAD patients more frequently consumed red meat and dairy products and had higher blood concentrations of this metabolite and a greater abundance of TMA-producing bacteria[212]. Wine consumption has been associated with cardiovascular benefits[213] (Table 1), although no changes in TMAO levels have been observed, changes in GM and an improvement in redox balance were observed[214]. A study in men with a history of atherosclerotic disease evaluated the effect of Lactobacillus plantarum GLP3 supplementation, and patients showed a significant reduction in TMAO levels[215] (Table 1). In contrast, Malik et al[216] reported that supplementation with the probiotic Lactobacillus plantarum 299v improved vascular endothelial function and reduced inflammatory biomarkers, despite no measurable reduction in circulating TMAO. (Table 1). In another hand, supplementation with Lactobacillus casei Shirota produced a non-significant reduction in TMAO (Table 1). The variability in results with probiotics, prebiotics, and other modulators indicates that many formulations have yet to consistently alter this metabolite[217] (Table 1). Reducing TMAO levels in CAD patients is not only possible but also relevant to prevent cardiovascular risk.

Dietary interventions that most consistently lower circulating TMAO

The most consistent TMAO lowering occurs when red meat is restricted and protein is shifted toward non-meat sources[218]. Complementary evidence shows that plant-forward substitutions can reduce TMAO over 8 weeks[219]. Argyridou et al[191] suggests rapid responsiveness to vegan diet, with fasting TMAO reductions detectable by week 1 and maintained at week 8. By contrast, fiber supplementation shows context-dependent effects; it showed a potential effect in individuals with lower habitual meat intake[220]. Finally, broader pattern changes such as a 6-month Mediterranean diet not uniformly lower fasting TMAO[221]. Same results reported Bergeron et al[222], evaluated diets varying in resistant starch and carbohydrate contests and concluded that high resistant-starch intake increased plasma TMAO levels.

TMAO decrease clinically meaningful?

Interventional evidence is still lacking to demonstrate that lowering TMAO reduces MACE, most clinical studies are not good quality[191]. In PEGASUS-TIMI 54, higher baseline TMAO related to certain outcomes, yet ticagrelor’s efficacy was consistent irrespective of TMAO levels, illustrating that a TMAO risk signal does not automatically translate into a TMAO-dependent treatment effect[156]. Notably, TMAO levels can also increase in contexts not clearly harmful, such as after fish intake and “healthy” diets enriched in marine n-3 fatty acids[13,223], complicating a superficial “lower is better” interpretation. Meanwhile, dietary manipulation can meaningfully shift TMAO levels within weeks[218,219], but these remain surrogate-endpoint demonstrations rather than proof of MACE reduction. Ongoing registered studies targeting the pathway are primarily designed around TMAO modulation, not MACE endpoints, including trials in CKD (ClinicalTrials ID: NCTNCT03152097 and NCT03348592), and mechanistic nutrition designs (ClinicalTrials ID: NCT06518343).

FUTURE PERSPECTIVES

Several strategies to decrease TMAO levels have shown promise. Ranging from dietary modifications and targeted nutrient substitution to GM directed approaches, such as the use of probiotics, prebiotics and more experimental tools such as 3,3-DMB, fluoromethylcholine and FMT. Collectively, these interventions underscore the potential to modulate GM and its metabolite output as a complementary avenue for CVD prevention and management.

Yet, the process of translating findings faces some limitations due to significant variations in dietary intake, kidney function, comorbidities, prescribed medications, and baseline GM. All these factors can greatly affect levels of circulating TMAO, making it difficult to draw causal relationships and compare results across different studies. Thus, upcoming investigations should focus on meticulously controlled and adequately sized human trials that unify sampling methods, pre-examination procedures, and testing systems; incorporate microbial “producer” profiling and functional outputs, such as cutC/D capability, in association with host factors and evaluate if strategies targeting TMAO provide additional predictive value beyond current risk assessment scores and, importantly, lead to enhancements in significant clinical outcomes. Setting a consistent clinical threshold and showing improved outcomes in forward-looking trials will be crucial for transitioning TMAO from merely a strong association to a clinically relevant biomarker and treatment focus.

CONCLUSION

Risk factors and unhealthy lifestyle choices can disrupt GM balance, negatively affecting cardiovascular health. When dysbiosis occurs, metabolites such as TMAO are generated, which plays a role in the activation of pathophysiological mechanisms. Several studies have shown that elevated TMAO levels are associated with the progression and poor prognosis of ACS, CVA, and HF, due to its ability to cross biological barriers such as the gut and the blood-brain barrier and directly influence cardiac or brain function. The apparent clinical benefit often mirrors implicit assumptions, that lowering TMAO certainly modifies risk, that studies are directly comparable, and that TMAO is an independent causal marker, in reality, context like diet, renal function, comorbidities, methodological heterogeneity, and reliance on surrogate endpoints may account for discordant findings without, by themselves, excluding a biologically relevant role for the TMA/TMAO axis. Future progress will depend on standardized pre-analytical and analytical workflows, functional stratification of GM producer phenotypes, and adequately powered prospective trials designed to test whether TMAO-targeted interventions provide incremental prognostic value and translate into decreases of adjudicated MACE.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Brenda Rosario Sandoval Meza, part of the Medical Translation Area at the Research Division, Faculty of Medicine, Universidad Nacional Autónoma de México for translating and proofreading this paper.