Trimethylamine N-oxide (TMAO)-Sources, Metabolism, Implications, and Detection Methods

What is Trimethylamine N-oxide (TMAO)?

Trimethylamine N-oxide (TMAO) is a small organic molecule, classified as an amine oxide. Structurally, it consists of a nitrogen atom bonded to three methyl groups and one oxygen atom, rendering the chemical formula (CH₃)₃NO. TMAO is highly soluble in water and has been studied extensively for its role in various biological processes and its potential impact on human health.

Sources and Formation of TMAO

Dietary Sources

Choline and Phosphatidylcholine

Choline is an essential nutrient found in various foods, including eggs, liver, and soybeans. Phosphatidylcholine, a major component of cell membranes, is abundant in animal products like red meat and dairy. Both choline and phosphatidylcholine are precursors for trimethylamine (TMA), which is a direct precursor of TMAO.

• Eggs: Rich in choline, eggs are a significant dietary source that contributes to TMA formation.
• Red Meat: Contains high levels of phosphatidylcholine, which is metabolized to TMA.
• Fish: While fish contain TMAO directly, they also provide substrates for TMA production.

Carnitine

Carnitine, another significant precursor to TMA, is prevalent in red meats, particularly beef and lamb. When ingested, carnitine undergoes microbial metabolism in the gut, similar to choline, resulting in the production of TMA.

Betaine

Betaine, found in foods like spinach, beets, and whole grains, can also be a precursor to TMA. It is metabolized by gut bacteria, although it plays a less prominent role compared to choline and carnitine.

Microbial Contribution

Gut Microbiota

The human gut hosts a complex community of microorganisms, collectively known as the gut microbiota. These microorganisms possess specialized enzymes capable of metabolizing dietary choline, phosphatidylcholine, carnitine, and betaine into TMA. Specific bacterial genera, such as Escherichia, Enterococcus, and Clostridium, are known to contribute significantly to TMA production. The presence and activity of these bacteria can vary widely among individuals, influenced by factors such as diet, genetics, and overall gut health.

Enzymatic Conversion

The key enzymatic step in the microbial conversion involves choline TMA-lyase, which cleaves choline into TMA and acetaldehyde. For carnitine, the enzyme involved is carnitine monooxygenase, which similarly converts carnitine into TMA.

Hepatic Conversion

Once TMA is produced in the gut, it is absorbed into the bloodstream and transported to the liver. Here, it undergoes a critical transformation to TMAO, facilitated by the flavin-containing monooxygenase (FMO) enzyme family, particularly FMO3.

FMO3 Enzyme

FMO3 is the predominant enzyme responsible for oxidizing TMA to TMAO in the liver. This enzyme utilizes oxygen and NADPH (nicotinamide adenine dinucleotide phosphate) to catalyze the conversion, producing TMAO and water as by-products. Genetic variations in the FMO3 gene can significantly impact TMAO levels, influencing individual susceptibility to diseases associated with high TMAO levels.

Factors Influencing TMAO Formation

Diet

Dietary intake of choline, phosphatidylcholine, and carnitine directly affects the levels of TMA and subsequently TMAO. Diets high in red meat, eggs, and certain fish can lead to increased TMAO production due to their rich content of these precursors.

Gut Microbiota Composition

The composition and activity of gut microbiota are critical determinants of TMA production. Probiotic and prebiotic interventions can modulate the gut microbiota, potentially altering TMA and TMAO levels. For example, increasing the abundance of TMA-producing bacteria through diet can elevate TMAO levels, whereas reducing these bacteria can have the opposite effect.

Genetic Factors

Genetic variations, particularly in the FMO3 gene, can influence an individual's capacity to convert TMA to TMAO. Certain polymorphisms in the FMO3 gene are associated with reduced enzyme activity, leading to lower TMAO production and potentially altered risk profiles for diseases linked to TMAO.

Health Status

Conditions such as liver disease, which can impair the function of the FMO3 enzyme, and renal disease, which affects the excretion of TMAO, can significantly impact TMAO levels in the body. Additionally, disruptions in gut health, such as dysbiosis, can alter the production of TMA from dietary precursors.

Metabolism of TMAO

Absorption and Distribution

Intestinal Absorption

After TMA is produced in the gut by microbial action on dietary precursors like choline, phosphatidylcholine, and carnitine, it is absorbed into the bloodstream. The absorption of TMA occurs primarily in the small intestine through passive diffusion, owing to its small molecular size and lipophilic nature. Some active transport mechanisms may also play a role, but passive diffusion is the predominant mode.

Hepatic Conversion

Once absorbed, TMA is transported to the liver via the portal circulation. In the liver, flavin-containing monooxygenase 3 (FMO3) catalyzes the oxidation of TMA to TMAO. This conversion is crucial for detoxifying TMA, which can be toxic at high concentrations. The enzymatic reaction involves:

• Substrate: Trimethylamine (TMA)
• Enzyme: Flavin-containing monooxygenase 3 (FMO3)
• Co-substrates: NADPH and oxygen
• Products: TMAO and NADP+

Systemic Circulation

Following its formation in the liver, TMAO enters the systemic circulation. It is a highly water-soluble molecule, which facilitates its transport through the bloodstream. TMAO can cross various biological membranes and is distributed widely throughout the body, reaching different tissues and organs.

Tissue Distribution

TMAO is found in various tissues, reflecting its systemic distribution. Studies have shown that TMAO can accumulate in:

• Kidneys: Due to its role in excretion, TMAO is present in high concentrations in the kidneys.
• Heart: Given its links to cardiovascular health, TMAO distribution in the heart is of particular interest.
• Brain: TMAO can cross the blood-brain barrier, indicating potential neurological implications.
• Liver: As the site of its synthesis, the liver naturally contains significant amounts of TMAO.

Excretion

Renal Clearance

The primary route of TMAO excretion is through the kidneys. Renal clearance involves filtration at the glomerulus, followed by reabsorption and secretion processes in the renal tubules. The efficiency of TMAO excretion depends on several factors, including kidney function and hydration status.

• Glomerular Filtration: TMAO is freely filtered from the blood into the glomerular filtrate due to its small size and high water solubility.
• Tubular Reabsorption and Secretion: Some reabsorption of TMAO occurs in the renal tubules, but active secretion mechanisms also exist to enhance its excretion.

Urinary Excretion

TMAO is primarily excreted in urine. The urinary excretion rate of TMAO can serve as a biomarker for its systemic levels and, by extension, for dietary intake and gut microbial activity. Factors affecting urinary TMAO levels include:

• Dietary Intake: High intake of choline, phosphatidylcholine, and carnitine can increase urinary TMAO excretion.
• Renal Function: Impaired kidney function can lead to decreased clearance and elevated TMAO levels in the blood.

Regulatory Mechanisms

Genetic Factors

Genetic variations, particularly polymorphisms in the FMO3 gene, can significantly influence TMAO metabolism. These polymorphisms can result in differences in enzyme activity, affecting the rate of TMA oxidation and subsequently TMAO levels. Individuals with certain genetic variants may have higher or lower baseline TMAO levels.

Dietary Influences

Diet plays a pivotal role in regulating TMAO metabolism. Diets rich in red meat, eggs, and fish provide ample substrates for TMA production, subsequently leading to higher TMAO levels. Conversely, plant-based diets, which are lower in choline and carnitine, tend to result in lower TMAO levels.

Microbiome Composition

The composition of the gut microbiota is another critical regulator of TMAO metabolism. The presence of specific bacterial strains that efficiently convert dietary precursors to TMA can influence TMAO production. Probiotics, prebiotics, and antibiotics can alter gut microbiota composition, thereby impacting TMAO levels.

Environmental Factors

Environmental factors such as age, sex, and overall health status also affect TMAO metabolism. For instance, age-related changes in kidney function can alter TMAO clearance, while sex differences in liver enzyme activity can affect TMAO synthesis.

Physiological Roles of Trimethylamine N-oxide (TMAO)

TMAO and Cardiovascular Diseases

Cardiovascular disease (CVD) stands as a leading cause of mortality and morbidity worldwide. The correlation between TMAO levels and CVD has been a central focus of research, with multiple studies indicating a significant positive association between elevated TMAO levels and cardiovascular ailments, including hypertension, heart failure, and atherosclerosis. Furthermore, TMAO levels can serve as predictive markers for cardiovascular disease risk.

The relationship between TMAO and cardiovascular diseases has been investigated across various cohorts. For instance, studies have examined the correlation between the consumption of red and processed meats and the incidence of atherosclerotic cardiovascular disease (ASCVD). It was suggested that these associations are partially mediated by the levels of TMAO-related metabolites in plasma. In a cohort study comprising over 3931 individuals aged 65 and above from a community in the United States, continuous measurements of animal-derived food intake and TMAO-related metabolites were conducted over a 12.5-year follow-up period. The findings revealed a correlation between TMAO levels and cardiovascular event risk. Upon the consumption of high-fat foods, food residues enter the intestines where gut bacteria metabolize them into TMA, which is subsequently oxidized by flavin-containing monooxygenase (FMO) in the liver to form TMAO. TMAO functions by inhibiting cholesterol degradation in the blood, leading to cholesterol deposition in arterial walls and consequent thickening and hardening.

The mechanism by which TMAO contributes to cardiovascular disease may involve increased cholesterol accumulation in macrophages. Other potential mechanisms include prolonging the hypertensive effects of angiotensin II and enhancing platelet activation, which may contribute to increased platelet reactivity and thrombosis.

TMAO and Liver Diseases

Non-alcoholic fatty liver disease (NAFLD) represents a significant health burden globally. Several clinical studies have demonstrated a notable association between blood TMAO levels and the occurrence of NAFLD and non-alcoholic steatohepatitis (NASH). Elevated TMAO levels may adversely affect the progression of NAFLD, suggesting that strategies aimed at reducing TMA and/or TMAO could be utilized for the treatment or prevention of NAFLD.

TMAO can promote the development of NAFLD through various mechanisms. For instance, in high-fat diet (HFD) mouse models, TMAO upregulates glucose metabolism and increases insulin resistance. Additionally, TMAO may promote insulin resistance by increasing the levels of serum inflammatory cytokine CCL2. Moreover, TMAO can affect lipid metabolism and cholesterol homeostasis by reducing cholesterol conversion to bile acids.

TMAO and Kidney Diseases

In addition to its association with cardiovascular diseases, TMAO has been found to be associated with the development of chronic kidney disease (CKD). Integration of data from numerous clinical studies involving a large cohort of participants revealed a significant negative correlation between TMAO levels and glomerular filtration rate. Notably, patients with advanced CKD exhibited significantly higher TMAO levels, indicating a close relationship between TMAO and renal impairment. Animal studies have also suggested a close association between kidney function and TMAO levels. However, the mechanisms by which TMAO may exacerbate kidney damage require further investigation.

TMAO and Type 2 Diabetes

TMAO has also been implicated in type 2 diabetes mellitus (T2DM). Research has shown that circulating TMAO levels in diabetic animal models are tenfold higher compared to normal mice. Similarly, elevated TMAO levels have been observed in T2DM patients, suggesting that TMAO may be a significant risk factor for diabetes. The exact mechanisms underlying the effects of TMAO on T2DM pathogenesis remain unclear. Studies suggest that the addition of TMAO to a high-fat diet, compared to a high-fat diet alone, disrupts insulin signaling pathways in mouse liver and increases inflammation in adipose tissue. Moreover, higher cardiometabolic risks in T2DM patients may be associated with alterations in TMAO metabolism.

TMAO and Cancer

Elevated circulating TMAO levels have been associated with an increased risk of certain cancers. Studies have demonstrated a positive correlation between TMAO levels and colorectal cancer risk, particularly in postmenopausal women with low vitamin B12 levels. Genome-wide association studies have identified a close relationship between TMAO and colorectal cancer, indicating shared genetic pathways. Additionally, research has shown that TMAO can activate anti-tumor immunity and enhance the efficacy of immunotherapy in triple-negative breast cancer, suggesting TMAO as a potential target for new immunotherapeutic strategies.

TMAO Promotes Antitumor Immunity in Triple-Negative Breast Cancer (Wang et al., 2022)

TMAO in Aquaculture and Nutrition

Furthermore, TMAO serves as a feed additive in aquaculture, promoting muscle growth in fish and poultry. It is also a naturally occurring endogenous substance in seafood and serves as a biochemical indicator of fish freshness. While TMAO itself is odorless, its breakdown into TMA releases a strong fishy odor, indicating spoilage. Higher TMA levels indicate decreased freshness.

These findings underscore the diverse physiological roles of TMAO and its implications in various health conditions, highlighting the importance of further research to elucidate its mechanisms and develop targeted interventions.

TMAO Detection Methods and Sample Types

The complete quantitative analysis of TMAO and related metabolites provides reliable evidence for the diagnosis, prediction, pathogenesis research, and efficacy evaluation of related diseases. Currently, high-throughput detection platforms for TMAO and its metabolites primarily rely on ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. These methods enable qualitative and quantitative detection of TMAO and its related metabolites, ensuring good separation of various isomers. The corresponding sample types mainly include serum, plasma, feces, urine, tissues, and intestinal contents.

Reference

1. Wang, Hai, et al. "The microbial metabolite trimethylamine N-oxide promotes antitumor immunity in triple-negative breast cancer." Cell metabolism 34.4 (2022): 581-594.