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Trimethylamine N-oxide (TMAO): a new attractive target to decrease cardiovascular risk
  1. Ione Swanepoel1,
  2. April Roberts1,
  3. Chelsea Brauns1,
  4. Devahuti R Chaliha2,
  5. Veronica Papa3,4,
  6. Raymond D Palmer5,
  7. Mauro Vaccarezza6,7
  1. 1School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, Western Australia, Australia
  2. 2Curtin Health Innovation Research Institute (CHIRI), Faculty of Health Sciences, Curtin University, Bentley, Western Australia, Australia
  3. 3Sport Sciences and Wellness, University of Naples Parthenope, Naples, Campania, Italy
  4. 4FAPAB Research Center, Avola, Siracusa, Italy
  5. 5Longevity Experts, Helium-3 Biotech, South Perth, Western Australia, Australia
  6. 6School of Pharmacy and Biomedical Sciences, Curtin University, Perth, Western Australia, Australia
  7. 7Curtin Medical School, Curtin Health Innovation Research Institute, Bentley, Perth, Western Australia, Australia
  1. Correspondence to Dr. Mauro Vaccarezza, Curtin Medical School, Curtin University, Perth, WA 6102, Australia; mauro.vaccarezza{at}curtin.edu.au

Abstract

Cardiovascular disease (CVD) is one of the greatest disease burdens and takes the lives of many each year. There are many risk factors both modifiable and non-modifiable which contribute to the onset and progression of the disease. Trimethylamine N-oxide (TMAO) in recent years has been found to have a correlation with CVD onset. Those with increased levels of the metabolite have a markedly increased risk of future development of cardiometabolic disorders.

This literature review aimed to critique past studies undertaken to find a consensus of the significance of the interrelationship between TMAO and cardiovascular risk. A definite link between TMAO levels and a CVD outcome was found. The majority of the literature stated the relationship with evidence; however, there is still some uncertainty as to why and how the correlation occurs. Further study needs to be done to further dissect and understand the relationship between TMAO and CVD risk.

  • microbiology
  • pathology
  • biophysics
  • adult cardiology
  • coronary heart disease
  • vascular medicine

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Introduction

With a total of 17 million lives lost each year, cardiovascular disease (CVD) continues to be a leading cause of mortality worldwide.1 Aside from mortality risk, CVD is a major contributor to hospital overcrowding.1 The composition of the gut microbiome influences an individual’s long-term susceptibility to CVD progression.2–5 Trimethylamine N-oxide (TMAO), being a biologically active gut microbe-derived metabolite, is a recognised contributor to the increase in cardiometabolic risk when found in high concentrations in the body.2 Intestinal microbiota have the ability to initiate the production of TMAO from several trimethylamine (TMA)-containing molecules (primarily choline and carnitine) in food.3–5 The gut microbiota can metabolise such precursors to form TMA, which can then be catalysed via flavin-containing monooxygenase 3 (FMO3) to produce TMAO in the liver.3

The aim of this study was to find the underlying reasons for why TMAO causes an increase in cardiovascular risk, how TMAO levels are dependent on individual diet and lifestyle factors and what can be done with this information going forward. This information can then be used to determine if TMAO is a possible target for future therapeutic prevention and treatment for CVDs such as hypertension, arteriosclerosis (including complications such as myocardial infarction and stroke) and heart failure.

Methods

A literature review was undertaken using PubMed, MEDLINE, ProQuest and Google Scholar as databases to perform a comprehensive search on the correlation between TMAO and cardiovascular risk. The only alternative search for trimethylamine n-oxide was TMAO. Keywords used for cardiovascular risk included cardiometabolic risk, cardiac risk, heart disease, atherosclerosis and CVD. The limitations placed on the search results were from 2010 to 2020 as well as refining the language to English only. Both animal and human studies were referenced. From all the results garnished, the titles and abstracts were analysed for their relevance to the topic and only those that specifically addressed the topic were included in the final reference list.

Results and discussion

Cardiovascular risk

In recent studies, it has been confirmed that the gut-derived metabolite TMAO has been found to increase CVD risk,4 although the exact mechanism of how this occurs remains unknown.2 Many studies have looked to provide explanations for this, including TMAOs’ inflammatory and prothrombotic effects.4–8

Hypertension

The study by Ge et al9 was one of the only studies that examined the association between TMAO concentration and the prevalence of hypertension. By comparing patients with low TMAO concentrations, they found that those with elevated TMAO had a 12% higher risk of having hypertension.9 The authors highlight that the positive correlation could be due to an increased production of TMA, a precursor of TMAO and a higher degree of colon TMAO permeability.9 In addition, the study reported that a TMAO infusion could lengthen the hypertensive effect and play a role in promoting inflammatory responses associated with CVDs.9

Atherosclerosis and its complications

Heianza et al10 examined the association between TMAO and its precursors with major cardiovascular events and mortality in over 19 000 participants. It was found that those with higher TMAO concentrations had a 62% higher risk of experiencing a cardiovascular event.10 Heianza et al suggested that this could be due to TMAO increasing atherosclerotic development by having cholesterol modulator effects and also the ability to influence platelet activity.10 Alongside this, it stated that TMAO can cause aortic cell inflammation, contributing to the production of an atherosclerotic plaque.10

Cheng et al11 analysed isolated coronary artery endothelial cells to determine if TMAO was associated with increased levels of tissue factor (TF). TF has been shown to initiate a coagulation pathway which can result in atherosclerotic plaque formation and eventual rupture.11 The study found that high levels of TMAO promoted the expression of TF and therefore were strongly associated with thrombus production.11 The results were then confirmed in patients with ST-elevation myocardial infarction (STEMI).11

The study by Matsuzawa et al12 also looked at the effects of TMAO levels and its correlation with post-STEMI outcomes. It was found that TMAO levels significantly increased the acute and chronic phases of STEMI.12 During the chronic phase, TMAO levels were found to be higher than the acute phase and were able to predict future cardiovascular outcomes.12 While the acute phase showed no insight into cardiovascular outcome predictions, the chronic phase directly correlated high levels of TMAO to be indicative of increased risk of cardiovascular events post-STEMI. The increased levels in the chronic phase are also said to be associated with coronary plaque progression.12 It is also worth noting that chronic phase TMAO levels were found to be higher in older patients.12

Haghikia et al13 undertook two independent cohort studies with the aim to examine the relationship between TMAO levels and the incidence of cardiovascular events in patients who reccently had first-time ischaemic stroke. The results showed that there was a link between higher TMAO plasma concentrations and an increased incidence of cardiovascular events.13 Alternatively, Haghikia et al reported that there was a positive correlation found between TMAO and proinflammatory monocytes (CD14++CD16+).13 These have been shown to increase inflammatory processes by the release of cytokines while also contributing to the upregulation of adhesive molecules that could lead to thrombus formation.13 Similarly, Zhai et al14 aimed to examine whether high TMAO concentrations were associated with a higher chance of poor clinical outcomes occurring in patients who had ischaemic stroke. The prospective study followed up 225 admitted patients, reporting that 167 patients had experienced unfavourable outcomes after 3 months.14 These results correlated with blood levels that exhibited a high TMAO concentration compared with that of those who did not experience a poor clinical outcome in that time.14 The study proposed this was due to TMAO increasing cytokine activation, producing long-term inflammation and increased platelet reactivity which led to adverse outcomes in patients with ischaemic stroke.14

The study by Nie et al15 aimed to examine the association between TMAO and risk of first stroke in hypertensive patients. It was found that there was a positive correlation between TMAO plasma levels and stroke risk.15 Interestingly, the study also concluded that there is a stronger association of TMAO with haemorrhagic stroke, compared with ischaemic stroke, stating that increased TMAO levels induce vascular inflammation and cell dysfunction through the production of oxygen radicals, which ultimately contribute to haemorrhagic stroke.15

Alternatively, the prospective study by Senthong et al16 analysed the relationship between TMAO plasma levels and mortality in patients with coronary artery disease. Senthong et al observed that increased TMAO levels was a strong prognostic value and was associated with a higher mortality risk.16 They concluded that TMAO is a proatherogenic metabolite which may increase plaque formation and potentially decrease the reverse transport of cholesterol, demonstrating the link to cardiovascular events in patients with coronary heart disease.16 A more recent study by Guo et al17 found that the association between TMAO and coronary artery disease (CAD) and severe artery stenosis was sex-related, with a predominance of TMAO alone as a valuable predictor of CAD and artery stenosis in men, whereas a more comprehensive panel of TMAO, choline, L-carnitine and betaine could be a useful biomarker of CAD and artery stenosis in both sexes.17 More recent data seem to confirm the role of TMAO in coronary artery disease18: long-term increases in TMAO were associated with higher CVD risk, and repeated assessment of TMAO over 10 years improved the identification of people with a higher risk of CVD.18 Of note, there have been conflicting reports of its association with atherosclerosis and thrombosis, with some even showing an inverse association (reviewed in Hardin et al4). A very recent publication seems to reconcile contradictory data on TMAO. Koay et al19 found no direct association of plasma TMAO and the extent of atherosclerosis, both in mice and humans. However, using a mouse model of plaque instability, they showed an association of TMAO plasma levels with atherosclerotic plaque instability.19 The latter finding supports TMAO as a valuable marker of CVD risk.

Heart failure

The study by Li et al20 looked at many different mechanisms to which TMAO induces cardiac events. The study focused on exploring the effects on cardiac hypertrophy and fibrosis which are common pathological features of heart failure and other CVDs.20 Through a multitude of different experiments both in vivo (involving rats) and in vitro it was found that TMAO causes poor cardiac remodelling as well as an increase in cardiomyocyte size. In addition, it is suggested that TMAO has a regulatory effect over cardiac hypertrophy, an intermediate and determinant factor in heart failure.20

Organ et al21 looked into the effects of diets high in choline or TMAO on cardiac hypertrophy as well as heart failure post-transverse aortic constriction (TAC). Heart failure severity was found to be increased in mice fed diets consisting of choline or TMAO. Post-TAC, poor heart remodelling as well as an acceleration in heart failure progression was discovered, further suggesting the significance of dietary choline on heart failure severity.21 Clinical consequences of heart failure, including cardiac enlargement and pulmonary oedema, were significantly increased in mice, suggesting its pathological contribution to the progression of the disease.21 Correspondingly, Seldin et al22 fed mice choline diets showing similar results as well as an elevated inflammatory gene expression. Seldin et al also tested the response to an acute TMAO injection in mice and noted an activation of signalling cascades as well as an increase in gene expression of inflammatory markers.22

In a prospective cohort case–control study, Zheng et al23 looked into the association between levels of TMAO and the risk of CVD in a community-based setting. Assessing rural Chinese adults as a community-based general population, Zheng et al found an association with increased TMAO levels and risk of CVD.23 Zheng et al built a firm case for the potential clinical use of TMAO as a possible modifiable marker of CVD risk due to the association of increased risk which links CVD with higher levels of TMAO.23

A prospective observational study by Trøseid et al24 found the highest levels of TMAO correlated with patients diagnosed with ischaemic heart disease. Approximately 50% of the patients assessed with elevated levels (toward the upper limits) of TMAO died or required a transplant within the 5.2-year follow-up. This same study looked at the metabolic and inflammatory markers in heart failure, specifically an elevation in TMAO, choline and betaine, and how they relate to the disease severity.24 It was found that only TMAO directly influenced disease severity, causing adverse reactions during patient follow-ups.24 However, according to the study by Tang et al,25 elevated levels of TMAO, choline and betaine can predict higher risks of adverse cardiovascular events in the next 5 years. A higher risk of advanced left ventricular dysfunction and suboptimal long-term outcomes in heart failure are associated with increased levels of these microbiota metabolites.25 Tang et al also points out a correlation of an even higher TMAO level which is seen in patients with heart failure who also had diabetes mellitus.25 Recent careful meta-analysis of prospective clinical studies confirmed the link between elevated TMAO levels and poorer prognosis in patients with heart failure.26

Potential prevention and interventional approaches to decrease CVD: diet

TMAO is derived in the gut from microbial metabolism of L-carnitine and choline.27 28 Metabolism produces TMA, which then undergoes conversion to TMAO once acted on by monooxygenase 3 in the liver and, once produced, can promote foam cell formation, deregulated cholesterol metabolism and impaired cholesterol transport.1 29 30 Existing literature shows that raised TMAO plasma levels strongly correspond to diets that are high in red meat, fish and dairy consumption.28 31–35 Urine concentration of TMAO was markedly higher in rats that were fed beef as their main source of food when compared with chicken.33 36 Studies also found that high beef consumption stimulated oxidative stress in the gastrointestinal tract, leading to greater levels of inflammation attributed to the increased levels of TMAO.33 36

The three proposed mechanisms for how TMAO may be increased in individuals who consume high volumes of red meat are as follows: red meat contains the precursors including TMA required for conversion to TMAO in the gut, the increased production of both TMA and TMAO from L-carnitine, and the reduction of TMAO excretion by the kidneys, all of which raise TMAO levels.1 28 34 Some studies have attributed the cause of an increased incidence of atherosclerosis among individuals with raised TMAO levels to TMAO’s effect on reverse cholesterol transport, whereby cholesterol homeostasis is impaired, and that its elimination pathway is heavily impacted on.1 30 While fish has been known for its cardioprotective effect owing to its high omega 3 content, fish was also found to have the greatest source of TMAO, especially deep-sea species, and therefore was found to have the greatest increase in urine-excreted TMAO.1–3 More TMAO was found in the urine of humans who consumed fish as to those who ate red meat.32 33 A potential explanation for this could be because red meat has many TMA precursors for TMAO’s production in the gut, allowing a greater amount of TMAO to be manufactured inside the gut and then compounded by less TMAO being excreted, whereas in fish, TMAO is already produced on consumption and therefore less TMAO is produced inside the body due to the lack of precursors, resulting in more being excreted through the urine.1 32 This may potentially also explain why red meat consumption has a greater adverse impact on cardiovascular health than fish.1

The pathogenicity and progression of many cardiovascular risk factors are directly related to oxidative stress. Malondialdehyde is overproduced when too many free radicals are formed due to oxidative stress.37 During the digestion of red meat, toxic oxidation products are formed, including both malondialdehyde and 4-hydroxy-nonenal.34 36 Epidemiological studies have also found that raised C reactive protein, a common marker for inflammation in the body, has been attributed to diets high in red meat.1 Multiple studies have shown the negative impacts of TMAO on cardiometabolic function; however, TMAO involvement has also been questioned in some studies due to evidence of TMAO having protective effects on the body and the fact that fish, which delivers the greatest levels of TMAO into the body, is also associated with lower CVD risk.1 28 However, multiple studies have demonstrated the association between CVD risk and TMAO, through evidence of oxidative stress markers, correlation between TMAO and CVD disease outcome studies and extensive research on mice.31 32 The differing results could be attributed to TMAO being at high concentrations in the studies that show a negative impact of TMAO, while studies that show a more protective effect of TMAO may have been due to the presence of the metabolite being at lower concentrations.32

Potential drug targets

A study by Roberts et al38 showed a potential for a new antithrombotic therapy regimen by reducing TMAO by inducing a lack of bleeding risk associated with current antiplatelet drugs. Roberts et al looked at the effects of 3,3-dimethyl-1-butanol on inhibiting TMA lyase activity from microbial choline in vivo.38 Extensive research optimisation led to the further identification of iodomethylcholine (IMC) and fluoromethylcholine (FMC) as specific inhibitors of TMA lyase, the latter being the most potent compound.38 IMC and FMC retained favourable pharmacological properties in vivo and suggest a new valuable target for the treatment of subjects at risk of thrombotic complications and heightened CVD risk.38 For their animal study, Roberts et al used arteriosclerotic-prone mice on controlled diets, with one group having the addition of choline to increase TMAO levels, each of which was administered a single oral dose of the experimental drug. Results of the study showed promise with a significant decline in TMAO levels for up to 3 days as well as a reduction in platelet responsiveness and the formation of thrombus.38 In addition to these results, they observed no signs of potential toxicity or increased bleeding risk.38 TMAO increases platelet response and thrombosis; thus, targeting the TMA-generating enzymes leads us to the development of potential time-dependent irreversible inhibitors of these mechanisms.38 Wang et al39 also looked at the potential use of 3,3-diemthyl-1-butanol (DMB), a choline structural analogue in reducing TMAO plasma levels. DMB was found to prevent endogenous macrophage foam cell formation and atherosclerotic lesion formation in mice.39 DMB may be considered a potential treatment in CVD as an alternative to the studies looking into FMO3 enzyme inhibition, which has been associated with side effects, including hepatic inflammation and fish odour syndrome due to TMA accumulation.39

Alternatively, Konop et al40 demonstrated the use of enalapril (ACE inhibitory drug) in rats to significantly lower TMA and TMAO levels in plasma. Further studies would be beneficial to determine if this trend is seen with other ACE inhibitors and what biochemical pathways are involved with the increased excretion of the microbial metabolite in these drugs.40 Of note, other compounds already in use in the clinic (such as the widely used antidiabetic drug metformin and meldonium, an antiischaemic drug used in Eastern European countries) have been reported to decrease microbial production of TMA and plasma TMAO in some animal models.41 42 Further studies are warranted to better understand the role and the mechanisms of old and new molecules in modulating TMA production and TMAO levels with the aim to reduce CVD risk.

Conclusions

The significance of the correlation between TMAO and the increased risk of CVD development means that detection of TMAO will eventually act as a biomarker for earlier diagnosis of cardiometabolic diseases.4 5 19 28 Therefore, future research should be conducted to develop medicines that aim to decrease TMAO levels and thereby reduce the risk of cardiovascular complications that are posed by this menacing metabolite.4 5 28 Many studies have demonstrated the strong association between increased CVD risks and elevated TMAO levels; however, further research needs to be done to determine why the relationship between TMAO and increased cardiovascular risk exists and if there is a possibility to potentially use this inter-relationship in the prevention and treatment of CVD.28 35 For now, the primary influencing factor which delivers a great impact on decreasing TMAO levels and by extension decreasing cardiovascular risk is by modifying an individual’s diet through recommendations of limiting phosphatidylcholine-rich food sources.3 4 31–35 Moreover, further study of microbial metabolites (related or not to TMAO) should extend our understanding of their role (and potential druggability) in CVD risk.5 43

Main message

  • Diet is a recognized important risk factor for cardiovascular disease. In addition to classical risk related to lipids and sugar intake, recent data suggests that other diet components increase CVD risk. TMAO is one of these new molecules derived from food intake. Increasing knowledge of TMAO formation and metabolism could pave the way to new treatments aimed to decrease CVD risk.

Current research question

  • We need to better delineate TMAO metabolism and how TMAO is detrimental for CVD and in particular the mechanisms behind TMAO- driven platetele reactivity.

Key references

  1. Zhu W, Gregory JC, Org E, et al. Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk. Cell 2016;165:111–24.

  2. Li XS, Obeid S, Klingenberg R, et al. Gut microbiota-dependent trimethylamine N oxide in acute coronary syndromes: a prognostic marker for incident cardiovascular events beyond traditional risk factors. Eur Heart J 2017;38:814–24.

  3. Koay YC, Chen YC, Wali JA. Plasma Levels of TMAO can be Increased with ’Healthy’ and ’Unhealthy’ Diets and Do Not Correlate with the Extent of Atherosclerosis but with Plaque Instability. Cardiovasc Res 2020:cvaa094.

  4. Koeth RA, Wang Z, Levison BS, et al. Intestinal microbiota metabolism of L-carnitine,a nutrient in red meat, promotes atherosclerosis. Nat Med 2013;19:576–85.

  5. Roberts AB, Gu X, Buffa JA, et al. Development of a gut microbetargeted nonlethal therapeutic to inhibit thrombosis potential. Nat Med 2018;24:1407–17.

Self-assessment questions

  1. Trimethylamine-N-oxide (TMAO) is a consistent biomarker of cardiovascular risk.

    • True.

    • False.

  2. Levels of TMAO are not determined by the diet of a given individual.

    • False.

    • True.

  3. In considering cardiovascular risk, TMAO levels are predictive only of atherosclerosis and coronary disease risk.

    • True.

    • False.

  4. Gut microbiota is the main source of TMAO, derived from diet-introduced precursors.

    • False.

    • True.

  5. Inhibition of Gut microbiota TMAO-producing enzymes is a viable and proven option to decrease TMAO levels in humans.

    • True.

    • False.

Answers

  1. True.

  2. False.

  3. False.

  4. True.

  5. True.

Acknowledgments

The authors thank Dr Leanne Chalmers (School of Pharmacy and Biomedical Sciences, Curtin University) for her help, feedback and support.

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Footnotes

  • Twitter @VeronicaPapa6

  • Contributors The topic was devised by IS, AR and CB under the conceptualisation and supervision of MV. IS, AR, CB and MV undertook the literature searching, created the outline of the article and wrote the draft. MV, DRC, VP and RDP provided feedback on the draft. IS, AR, CB and MV then revised the article. CB undertook final proofreading with further contribution of DRC and RDP. Final review of the manuscript and final version was implemented by MV with contribution of VP. All the authors read and approved the final version of the article for submission.

  • Funding None

  • Competing interests RDP is the chief scientific officer of Longevity Experts, Helium-3 Biotech.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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