# TMAO: Why It Increases & Factors That May Reduce TMAO levels

MIAMI – Researchers are one step closer to developing “drugs for bugs” – agents that target the gut microbiome to prevent and treat cardiometabolic diseases, Stanley L. Hazen, MD, PhD, said at the 2019 Gut Microbiota for Health World Summit.

“Each person experiences a meal differently through the filter of their gut microbiome, which helps explain individual differences in susceptibility to disease,” said Dr. Hazen of Cleveland Clinic. “In the future, our medicine cabinets will have drugs in them that not only affect us, but also target the microbial enzymes that affect levels of metabolites TMAO.”

Trimethylamine N-oxide (TMAO) is produced by gut bacteria. High levels (in one study, approximately 6.

2 micromolar) significantly increase the risk of major adverse cardiovascular events even after controlling for traditional demographic and clinical risk factors.

Studies indicate that TMAO alters cholesterol and bile acid metabolism, upregulates inflammatory pathways, and promotes foam cell formation, all of which worsen atherosclerosis. In addition, TMAO increases clotting risk by enhancing platelet reactivity.

“Reducing the amount of animal products in one’s diet helps reduce TMAO levels,” said Dr. Hazen. Certain fish – mainly those found in deep, cold water, such as cod – are high in TMAO. However, a bigger culprit in the United States is red meat, which contains two major TMAO precursors – choline and carnitine. In a recent study, Dr.

Hazen and his associates gave 113 healthy volunteers three isocaloric diets in random order red meat, white meat, or plant-based protein. After 4 weeks, eating the daily equivalent of 8 ounces of steak or two quarter-pound beef patties nearly tripled plasma TMAO levels (P less than .05) from baseline.

The white meat and vegetarian diets showed no such effect.

Crucially, the effect of red meat was reversible – TMAO levels fell significantly within 4 weeks after participants stopped consuming red meat. Eating red meat low in saturated fat did not prevent TMAO levels from rising, Dr. Hazen noted at the meeting at the meeting sponsored by the American Gastroenterological Association and the European Society for Neurogastroenterology and Motility.

In a second study, Dr. Hazen and his associates identified a two-step process by which gut bacteria metabolize carnitine to TMAO. The second step was greatly enhanced in individuals who eat red meat, suggesting a possible therapeutic target.

In a third study, they found that high TMAO levels in mice fell significantly with a single oral dose of a second-generation inhibitor of trimethylamine lyase, the enzyme used by gut bacteria to convert choline to TMAO.

The inhibitory effect was irreversible, did not reduce the viability of commensal microorganisms, and significantly lowered platelet hyperreactivity and clot formation.

Such results are exciting, but “drugs for bugs” will exhibit varying effects depending on which gut species are present at baseline, Dr. Hazen explained. Investigators will need to understand and account for these differences before therapies for the microbiome can enter the clinic. For now, a blood test for TMAO is available and can help clinicians tailor their suggestions on what to eat.

Dr. Hazen disclosed a consulting relationship with Proctor & Gamble, royalties for patents from Proctor & Gamble, Cleveland Heart Lab, and Quest Diagnostics, and research support from AstraZeneca, Pfizer, Roche Diagnostics, and Proctor & Gamble.

Source: https://www.mdedge.com/internalmedicine/article/197940/gastroenterology/studies-link-tmao-microbiome-reveal-new-heart

## How TMAO Fooled Us

In 2011, a group of researchers published a paper in Nature,1 linking the compound trimethylamine N-oxide (TMAO) to cardiovascular disease (CVD). Since then, TMAO’s role in the pathology of CVD has accumulated more and more evidence, convincing several skeptics along the way of its importance.

For those unfamiliar, TMAO is a metabolite produced by gut microbes from dietary substrates such as choline, betaine, and carnitine, which are all found in animal products. These dietary substrates are converted by certain microbes into the gas trimethylamine (TMA), which is then oxygenated by certain liver enzymes to form TMAO (chemical structure shown below).

Trimethylamine N-oxide (TMAO) structure

We know that microbes are necessary for this process because experiments have shown that oral antibiotics suppress TMAO production, even in the presence of these dietary substrates.2 Once formed, TMAO is then transported to tissues in the body, where it accumulates, while some of it is cleared by the kidneys.

There are several mechanisms by which TMAO is believed to cause cardiovascular disease, but the main one involves higher levels of it increasing scavenger receptors in macrophages.3 More scavenger receptors increase the lihood of these macrophages binding low-density lipoprotein (LDL) and forming foam cells, named for their… foamy appearance.

The creation of foam cells and their localization to fatty deposits in blood vessels can disrupt cholesterol influx, esterification, efflux, and promote inflammation, all increasing the risk of CVD.4 This process can be seen in the image below, taken from Tang et al.2

Tang WW, Wang Z, Levison BS, et al. Intestinal Microbial Metabolism of Phosphatidylcholine and Cardiovascular Risk. New England Journal of Medicine. 2013;368(17):1575-1584. doi:10.1056/NEJMoa1109400

Several observational studies have found associations between TMAO levels and CVD risk, but more importantly, three recent systematic reviews and meta-analyses of cohort studies have also found increases in all-cause mortality and CVD events.5–8

If we assume, just for the sake of this discussion, that the biological mechanisms are valid and that the results of the cohort studies are not seriously plagued by stochastic error and bias (which is ironic, given how much I’ve discussed measurement error and multiplicity in nutritional epidemiology), then we may visualize our knowledge with the following causal diagram.

To recap, certain dietary substrates, found mostly in animal products, get converted into TMA by gut microbes and then into TMAO by liver enzymes, which then contribute to the production of foam cells, which when excessively localized, wreak all sorts of havoc.

This evidence has convinced many clinicians and researchers to discourage the consumption of animal products, especially red meat, a source of carnitine. Some cardiologists have even recommended that patients get their blood TMAO levels measured to predict their CVD risk, meaning these health professionals see it as a biomarker with clinical validity and utility.

In the words of Kraus (2018),9 clinical validity for a biomarker is:

How well the test measures a clinical feature of a disease, disease outcome or treatment outcome — is required to demonstrate the relevance of the test to the clinical condition as a guide to clinical decision-making.

Kraus VB. Biomarkers as drug development tools: Discovery, validation, qualification and use. Nature Reviews Rheumatology. 2018;14(6):354. doi:10.1038/s41584-018-0005-9

Kraus (2018)9 describes clinical utility as,

How well the test improves patient outcomes, confirms or changes a diagnosis, determines appropriate therapy or identifies individuals at risk of a disease — is required to determine how well a test balances benefits and harms when used in patient management. A process separate from biomarker qualification governs the approval of a biomarker as a medical test.

All of these characteristics are more ly to be met if the biomarker is in the causal pathway of the disease.

Kraus VB. Biomarkers as drug development tools: Discovery, validation, qualification and use. Nature Reviews Rheumatology. 2018;14(6):354. doi:10.1038/s41584-018-0005-9

So, if we assume that TMAO is in the causal pathway of CVD, that it can accurately predict CVD risk, and that reducing TMAO levels via certain interventions also lowers the risk of cardiovascular disease, then we’ve struck gold. Unfortunately, the literature on TMAO as a biomarker is preliminary and mixed.10–13

However, that hasn’t stopped researchers from devising strategies to reduce TMAO formation. For example, take a look at some proposed interventions in the table below by Velasquez et al.3 While many of these may seem low-cost interventions with minimal side effects, some are clearly not ( taking antibiotics) and this is all assuming TMAO is a valid marker in the causal pathway. So is it?

 Antibiotics Decreased TMAO plasma levels Microbiome Therapy Decreased TMA formation in the gut Reduced L-carnitine and choline consumption Decrease TMAO level Resveratrol Decreased TMA and TMAO plasma levels Meldonium Reduced TMA production by the intestinal microbiota bacteria 3,3-dimethyl-1-butanol Inhibition of TMA formation through inhibition of microbial TMA lyases FMO3 enzyme inhibition Prevented the oxidization of TMA to TMAO

Several characteristics of TMAO conflict with known findings. For example, fish and other seafood contain some of the highest amounts of free TMAO,14–16 yet several prospective studies have found these foods to be associated with positive CVD outcomes.

One possible explanation may be that these observational studies are simply unreliable and suffer from far too much measurement error ($$W_{ij}=X_{i}+ \epsilon_{ij}$$)17 and analytical flexibility.18 However, even randomized controlled crossover trials have shown that lean white fish reduces lipids that are risk factors for cardiovascular disease.19

Some have argued that the omega-3 fatty acids and other compounds in fish offset the harmful effects of TMAO, however, much of this is speculative.

Those who remain skeptical of TMAO believe that it is a confounder and that it is the presence of certain microbes that contributes to the pathology of CVD, and these microbes also happen to produce TMAO.

14,20 We can see this perspective visualized with the causal diagram below.

It’s difficult to truly know the role of TMAO in the pathology of CVD without a randomized trial explicitly manipulating TMAO levels and looking at clinical endpoints. Luckily, a recently published study21 with a powerful design offers us the next best thing.

In observational studies where the exposure is usually nonrandom, the ability to infer cause and effect tends to be limited by hidden confounders, reverse causality, measurement error, and selection bias, even with the use of complex methods that attempt to make groups as comparable as possible. However, a relatively new study design, called Mendelian randomization, offers a way to address many of these issues.22

It uses a technique developed in econometrics called instrumental variable analysis, where certain instruments, such as genes associated with the exposure, are used as proxies for exposures such as TMAO, choline, betaine, and carnitine.

In observational studies, we cannot randomly sample or assign TMAO and its precursors (practically) and look at clinical endpoints, but we can rely on the first and second laws of Mendel’s inheritance, because during cellular meiosis, alleles are randomly segregated and assorted, making it unly that many inherited genotypes are associated with population-level confounders.

Figure from http://exammasters.ca/everything-need-know-meiosis/.

In a Mendelian randomization study, once the instruments (certain genes) are known to be valid, they are tested for associations with the outcomes of interest, such as cardiovascular disease events. This method is powerful since random assortment deals with many of the issues that statistical methods cannot.

This is the approach that a group of researchers recently used to test the associations between TMAO, choline, betaine, and carnitine, and outcomes such as CVD events, type 2 diabetes risk, and chronic kidney disease.

The researchers searched published databases of genome-wide association studies to look for genetic variants that were linked to the exposures (TMAO, choline, betaine, carnitine) in order to produce their instrument.

They selected single nucleotide polymorphisms (SNPs) that met the genome-wide association significance level (p < 0.000005), since these are more ly to be replicable.

These instruments were then tested for associations with the clinical outcomes. Sounds easy enough… or not. There are no free lunches, even in Mendelian randomization studies. In order for the instrument to be valid, it must meet certain assumptions:22

1. The association between the instrument (the genes) and the outcomes must only be through the exposure to which the genes are linked. That means if the genes/SNPs ( those that produce TMAO) affect the outcome (CVD) in other ways, the instrument is invalid.

2. The genes must truly be associated with the exposure of interest. That means the genes used in the instrument must truly explain a portion of the variance in the exposures (such as choline, betaine, carnitine, and TMAO).

3. The genes cannot be associated with unmeasured confounders that are associated with the exposure and the outcome.

These assumptions can be seen below, an image taken from the original paper.21

Jia J, Dou P, Gao M, et al. Assessment of Causal Direction Between Gut Microbiota-Dependent Metabolites and Cardiometabolic Health: Abi-Directional Mendelian Randomisation Analysis. Diabetes. June 2019:db190153. doi:10.2337/db19-0153

The researchers made sure their genetic variants met these assumptions by looking at the strength of the associations between the genetic variants and the metabolites and selected independent SNPs with the strongest associations (lowest P-value) for each variant.

The drawback of this approach is that it can also weaken the instruments since there are bound to be SNPs with lower test statistics that can still explain a notable amount of variance.

The authors explain how much metabolite variance is explained by their selected SNPs,

Our genetic analysis showed that 15.4% of betaine, 17.1% of carnitine, 8.0% of choline, and 9.6% of TMAO were explained by its SNPs.

The authors set their alpha level at P ≤ 0.0005 (0.05/100) after Bonferroni correction for multiple comparisons and took results ≤ 0.05 as suggestive of an association. They conducted an apriori power analysis with an alpha level of 0.05, to detect at least a 30% increase in the odds of cardiometabolic events for the dietary substrates and metabolites.

MR power calculation (http://cnsgenomics.com/shiny/mRnd) showed that we have 87%, 84%, 78%, 81% power to test significant (P

Source: https://lesslikely.com/nutrition/tmao-mendelian-randomization/

## Eat your vegetables (and fish): Another reason why they may promote heart health

Elevated levels of trimethylamine N-oxide (TMAO) — a compound linked with the consumption of fish, seafood and a primarily vegetarian diet — may reduce hypertension-related heart disease symptoms.

New research in rats finds that low-dose treatment with TMAO reduced heart thickening (cardiac fibrosis) and markers of heart failure in an animal model of hypertension.

The study is published ahead of print in the American Journal of Physiology — Heart and Circulatory Physiology and was chosen as an APSselect article for November.

TMAO levels in the blood significantly increase after eating TMAO-rich food such as fish and vegetables. In addition, the liver produces TMAO from trimethylamine (TMA), a substance made by gut bacteria.

The cause of high TMAO levels in the blood and the compound's effects on the heart and circulatory system are unclear, and earlier research has been contradictory.

It was previously thought that TMAO blood plasma levels — and heart disease risk — rise after the consumption of red meat and eggs.

However, “it seems that a fish-rich and vegetarian diet, which is beneficial or at least neutral for cardiovascular risk, is associated with a significantly higher plasma TMAO than red meat- and egg-rich diets, which are considered to increase the cardiovascular risk,” researchers from the Medical University of Warsaw in Poland and the Polish Academy of Sciences wrote.

The researchers studied the effect of TMAO on rats that have a genetic tendency to develop high blood pressure (spontaneously hypertensive rats). One group of hypertensive rats was given low-dose TMAO supplements in their drinking water, and another group received plain water.

They were compared to a control group of rats that does not have the same genetic predisposition and received plain water. The dosage of TMAO was designed to increase blood TMAO levels approximately four times higher than what the body normally produces.

The rats were given TMAO therapy for either 12 weeks or 56 weeks and were assessed for heart and kidney damage and high blood pressure.

TMAO treatment did not affect the development of high blood pressure in any of the spontaneously hypertensive rats. However, condition of the animals given the compound was better than expected, even after more than a year of low-dose TMAO treatment.

“A new finding of our study is that [a] four- to five-fold increase in plasma TMAO does not exert negative effects on the circulatory system.

In contrast, a low-dose TMAO treatment is associated with reduced cardiac fibrosis and [markers of] failing heart in spontaneously hypertensive rats,” the researchers wrote.

“Our study provides new evidence for a potential beneficial effect of a moderate increase in plasma TMAO on pressure-overloaded heart,” the research team wrote.

The researchers acknowledge that further study is needed to assess the effect of TMAO and TMA on the circulatory system.

However, an indirect conclusion from the study could underscore the heart-healthy benefits of following a Mediterranean-style diet rich in fish and vegetables.

Story Source:

Materials provided by American Physiological Society. Note: Content may be edited for style and length.

Journal Reference:

1. Tomasz Huc, Adrian Drapala, Marta Gawrys, Marek Konop, Klaudia Bielinska, Ewelina Zaorska, Emilia Samborowska, Aleksandra Wyczalkowska-Tomasik, Leszek Pączek, Michal Dadlez, Marcin Ufnal. CHRONIC, LOW-DOSE TMAO TREATMENT REDUCES DIASTOLIC DYSFUNCTION AND HEART FIBROSIS IN HYPERTENSIVE RATS.. American Journal of Physiology-Heart and Circulatory Physiology, 2018; DOI: 10.1152/ajpheart.00536.2018

Source: https://www.sciencedaily.com/releases/2018/11/181106073239.htm

## The Gut, the Heart, and TMAO

Science has long recognized that what we eat plays a critical role in our heart health. Now the details of this complex connection are coming into focus.

One of the more intriguing recent discoveries has to do with the role of the gut microbiome—the trillions of microbes that reside in the GI tract and influence health by helping digest food, making vitamins, and providing protection against disease-causing microorganisms. Recently Cleveland Clinic researchers reported findings from several studies involving people and animals, that the gut microbiome directly changes the function of blood platelets, influencing the risk for heart attack and stroke.

Here’s how it works: When people ingest certain nutrients, such as choline (abundant in red meat, egg yolks, and dairy products) and L-carnitine (found in red meat as well as some energy drinks and supplements), the gut bacteria that break it down produce a compound called trimethylamine (TMA). The liver then converts TMA into the compound, trimethylene N-oxide (TMAO).

The trouble with TMAO is that data show high levels contribute to a heightened risk for clot-related events such as heart attack and stroke—even after researchers take into account the presence of conventional risk factors and markers of inflammation that might skew the results.

In their most recent analysis, scientists showed that high blood levels of TMAO were associated with higher rates of premature death in a group of 2235 patients with stable coronary artery disease.

Those found to have higher blood levels of TMAO had a four-fold greater risk of dying from any cause over the subsequent five years.

The implications are intriguing. Taken together, the new studies suggest that positively altering the gut microbiota may help to reduce damage to blood vessels, resulting in a stronger cardiovascular system, and they point to targets for potential new heart disease therapies.

The insights also underscore the potential power of TMAO testing, which can help patients determine if their gut microbiome is contributing to their risk for heart disease and whether they might benefit from limiting foods that contain the building blocks of TMAO. TMAO tests are only available through the Cleveland Heartlab.

To lower your TMAO levels, consider minimizing the consumption of full-fat dairy products, including whole milk, egg yolk, cream cheese, and butter; both processed and unprocessed red meat (beef, pork, lamb, and veal), as well as nutritional supplements and energy drinks containing choline, phosphatidylcholine (lecithin), and/or L-carnitine. Vegetarians and vegans, who avoid meat products, for instance, produce little TMAO.

In general, consuming a diverse diet rich in plant foods and fiber may be helpful.

When Cleveland Clinic researchers fed mice a diet rich in TMAO producing nutrients, they identified a compound called DMB capable of minimizing TMAO produced from their gut microbiota.

In fact, when DMB was added to their drinking water, they found TMAO levels and the formation of arterial plaques both declined. DMB may be found naturally in many Mediterranean diet foods, including red wine and extra virgin olive oil.

Such an eating pattern may turn out to be key to cultivating a healthy human gut microbiome—one that will fend off myriad illnesses, including heart disease, America’s chief health threat.

Source: http://www.clevelandheartlab.com/blog/the-gut-the-heart-and-tmao/

## Trimethylamine-N-Oxide (TMAO) Predicts Cardiovascular Mortality in Peripheral Artery Disease

• Outcomes research
• Prognostic markers
• Risk factors

Peripheral artery disease (PAD) is a major cause of acute and chronic illness, with extremely poor prognosis that remains underdiagnosed and undertreated.

Trimethylamine-N-Oxide (TMAO), a gut derived metabolite, has been associated with atherosclerotic burden. We determined plasma levels of TMAO by mass spectrometry and evaluated their association with PAD severity and prognosis.

262 symptomatic PAD patients (mean age 70 years, 87% men) categorized in intermittent claudication (IC, n = 147) and critical limb ischemia (CLI, n = 115) were followed-up for a mean average of 4 years (min 1-max 102 months). TMAO levels were increased in CLI compared to IC (P  2.

26 µmol/L exhibited higher risk of cardiovascular death (sub-hazard ratios ≥2, P 2.26 µmol/L) to the considered models including the previously mentioned relevant covariates, improved risk prediction for CV mortality in symptomatic PAD patients as assessed by NRI in models 2, 3 and 4 (Table 4).

Table 4 Added predictive value of TMAO > 2.26 µmol/L for CV death in PAD patients.

We determined plasma levels of TMAO to assess their association with PAD severity and their possible use as prognostic markers in symptomatic PAD. TMAO was independently associated to PAD severity and CV-mortality, but not to all-cause death and MACE.

The analysis of circulating gut-microbiome derived products has identified TMAO, a secondary product after TMA is metabolized by the liver enzyme FMO3, as a possible prognostic marker in cardiometabolic diseases5,6,7.

In most cases increased levels of TMAO or its initial precursors, betaine, choline or L-carnitine, have been associated to atherosclerotic burden5,9,10,17 and worse outcome in arterial pathologies in large cohorts6,7,11,12,13.

In addition, the causal role of TMAO in atherosclerosis development has been further studied in murine models, showing accelerated plaque development with dietary supplementation of TMAO or its precursors (e.g.: choline, L-carnitine)5,11,15,18,19.

Nonetheless, some authors found no predictive value of high TMAO levels for CV events or mortality in smaller cohorts of patients with suspected coronary artery disease20 or receiving dialysis21, and the contribution of TMAO to early atherosclerosis development in healthy-middle-aged adults is unclear22.

Symptomatic PAD patients present a complex pathophysiology, frequently associated with comorbidities such as diabetes, hypertension, or CKD, that are suspected to greatly interfere with the metabolism of microbial derived products8. Indeed, TMAO homeostasis considerably depends on liver FMO3 activity16 and renal clearance8,23.

We found increased levels of TMAO in CLI patients compared to IC, and also determined hepatic enzymes for liver function assessment, finding no differences in transaminases between IC and CLI. Despite these results, we cannot exclude increased FMO3 activity in our cohort as no direct measurement of the enzymatic activity was performed. Moreover, other factors such as sex hormones, bile acids or insulin have been also described to regulate FMO3 activity and increase TMA oxidation24,25.

TMAO accumulation might be also related to worse kidney clearance23, since it has been described that patients with decreased kidney function present elevated levels of TMAO in circulation compared to those without CKD21,26,27,28.

A trend for reduced eGFR according to TMAO quartiles was reported in the KarMeN study29, while no association of TMAO with creatinine was observed in the EPIC-Heidelberg study30, suggesting an irrelevant role of the kidney function for TMAO homeostasis in healthy subjects.

In contrast, we found a negative correlation between TMAO and eGFR in a small cohort of people with no manifest cardiovascular disease, but with more than two cardiovascular risk factors, and older than those in the studies by Krüger et al. and Kühn et al.29,30.

These data suggest that individuals with mildly impaired kidney function might be more susceptible to the detrimental effects of TMAO accumulation.

In line with these observations and taking in consideration the high percentage of CKD patients in our PAD population (39%), we found a lineal-inverse association between TMAO and eGFR, and tested the possible interaction between the two according to disease severity.

No interaction between TMAO and eGFR was found, however, considering the important role of the kidney on TMAO homeostasis8, eGFR was included as covariate for further regression analysis. To assess the accuracy of TMAO levels for PAD patient stratification we established the association between TMAO and PAD severity after adjustment for traditional risk factors and described a cutoff value with diagnostic purposes.

Increased levels of plasma TMAO have been shown to predict future major adverse cardiac events including myocardial infarction, stroke, and death in different CV pathologies6,7,11,12,31 and a role for gut derived metabolites in thrombosis has been described in vivo, showing that intestinal microbes can directly modulate platelet hyperresponsiveness and clot formation rate via TMAO generation12,16. Even if most studies point towards TMAO as marker of worse outcome in CV pathologies, controversy remains when specific patient cohorts are evaluated. For example, no correlation between TMAO and increased risk for CV disease or MACE was observed in a group of end-stage renal disease patients21 and in subject with suspected CAD undergoing coronary angiography20. Mueller et al. speculated that those results could be confounded at least in part by impaired kidney function or poor metabolic control20 and encouraged the consideration of these parameters when interpreting the results. In addition, TMAO has been studied mainly in coronary pathologies, while it is prognostic value in other arterial localization, such as PAD, has been little investigated. We report an association between TMAO and CV-mortality, but not with all-cause death or MACE. Our results differ in part from previous data, describing an association of TMAO levels and global death in PAD14. However, differences between the studied populations regarding PAD definition and severity status should be considered when interpreting the data. Senthong et al. included by the term PAD the majority of non-coronary arterial territories14, while our cohort is restricted to symptomatic lower limb artery disease (mean ABI 0.55 ± 0.19). TMAO appears to be better for CV-mortality prognosis than for all-cause death, which might be important for patient evaluation.

### Limitations

The current study including 262 patients could be considered as small, however the high percentage of deaths (39% all-cause and 15% CV origin) provides the statistical power required to support our conclusions.

Events were recorded during a mean follow up of 4 years, reasonable to estimate early to medium-term mortality. Longer term studies should be designed to confirm the involvement of TMAO in PAD mortality. No causal relationship between high TMAO levels and CV-mortality can be inferred from our prospective study.

Death cause in some patients was unknown and we might have lost some cases related to CV events. Finally, the influence of two important variables for TMAO production, the use of antibiotics and modifications in dietary habits were not recorded and could not be included as confounding variables in our population.

FMO3 activity, which has been implicated in thrombosis risk16 and converts TMA in TMAO24, was not measured.

### Conclusions

We show increased TMAO levels according to PAD severity and an independent association between TMAO and elevated risk for CV-mortality.

The design of novel therapeutic strategies towards gut-derived metabolite control in vascular patients will need to consider not only intestinal bacterial function, but also the activity of key hepatic enzymes for TMA oxidation (FMO3), and renal function.

The clinical and demographic characteristics of the PAD cohort were previously described by Martinez-Aguilar et al.32,33. As the cohort has included new cases we include the complete description in supplemental material and methods. Control subjects were previously described by Marcos-Jubilar M et al.34.

### Baseline characteristics of PAD and control patients

PAD Patients [n = 262, mean age 70 years (SD: 11), 87% men] were prospectively enrolled at the outpatient service of the Department of Vascular Surgery of Complejo Hospitalario de Navarra between 2010 and 2017 (supplemental information). Blood samples were collected at the time of clinical evaluation and tested for biochemical parameters. Ankle brachial index (ABI) was measured at rest, in both lower limbs.

Fontaine classification was used for severity assessment as follows: intermittent claudication (IC, Fontaine class II, n = 147) diagnosed by hemodynamic study (Doppler ultrasound), and critical limb ischemia (CLI, n = 115) with lower limb rest pain and/or trophic lesions (Fontaine class III-IV) confirmed by imaging studies (arteriography, magnetic resonance angiography, or ultrasonography). Exclusion criteria were established as follows: patients with Fontaine class IV and infected-lesions, individuals with evidence of neoplastic disease, generalized or localized inflammatory disease (moderate or severe), severe chronic kidney disease, on haemodialysis, or receiving antinflammatory drugs.

Control subjects (n = 45) were enrolled at the outpatient service of the Department of Internal Medicine, Clínica Universidad de Navarra (April 2016-December 2017). Blood samples were collected at the time of clinical evaluation. Patients were included if older than 45 years, with ≥2 cardiovascular risk factors and no manifested cardiovascular disease at recruitment.

Exclusion criteria included active neoplastic disease, acute or chronic inflammatory disease of any aetiology, and intake of nonsteroidal anti-inflammatory or steroid drugs 2 weeks before blood withdrawal.

Samples and data from control patients were provided by the Biobank of the University of Navarra and were processed following standard operating procedures approved by the Ethical and Scientific Committees.

The study was approved by the Institutional Review Boards of Complejo Hospitalario de Navarra and Clínica Universidad de Navarra, according to the standards of the Declaration of Helsinki on medical research, and written informed consent was obtained from all patients who were enrolled in this study.

PAD patients were followed up for a mean period of 4 years (min 1 max 102 months) at the outpatient service of the Department of Vascular Surgery every 3, 6 or 12 months, depending on the severity of PAD. Death, either all-cause or cardiovascular, and MACE including amputation, stroke, myocardial infarction and all-cause death were recorded.

### TMAO determination

a previously reported approach by Awwad H.M., et al.35, a precise and reliable UHPLC-MS/MS method has been implemented in our laboratory for the quantification of TMAO in human plasma. Frozen citrate plasma samples, in which the corresponding stabilized TMA salt was formed, were utilized to perform these analyses.

Concentrations of the analyte in plasma samples was determined from calibration curves, which indicated a good linearity (r2 ≥ 0.999) within the studied concentration range (100 nM–10 μM), using peak area ratio of the analyte to its isotope.

The implemented UHPLC-MS/MS method has good analytical accuracy, precision and recovery; in fact, the recoveries ranged between 97% and 104% and, accuracy and precision of the assays were between 3.7% and 7.3% (reported in the Supplementary Table S1).

Details about standards and chemicals, calibration curve and sample preparation as well as UHPLC and MS system conditions are explicitly described in the Supplementary Information together with their corresponding Figures (Supplementary Figs S1 and S2).

### Statistical analysis

Normality was demonstrated by the Kolmogorov-Smirnov test. Non-normally distributed variables were log-transformed. Differences between two groups of subjects were tested by Student’s t test (normal unpaired data) or Mann-Whitney U test (nonparametric test). χ2 or Fisher’s exact test was used for categorical variables.

Association studies were performed by Pearson correlation test for continuous variables. Receiver Operating Characteristic (ROC) curves were plotted to assess disease severity (IC vs. CLI), and the cut-off value for TMAO established with the Youden Index.

Multivariable logistic regression models were adjusted for relevant covariates: age, sex, cigarette smoking, diabetes mellitus, hypertension, dyslipidemia, HDL-C, eGFR (

Source: https://www.nature.com/articles/s41598-019-52082-z