Trimethylamine N-oxide Precursor Research Articles

Interactions between the gut microbiome, associated metabolites and the manifestation and progression of heart failure with preserved ejection fraction in ZSF1 rats

BackgroundHeart failure with preserved ejection fraction (HFpEF) is associated with systemic inflammation, obesity, metabolic syndrome, and gut microbiome changes. Increased trimethylamine-N-oxide (TMAO) levels are predictive for mortality in HFpEF. The TMAO precursor trimethylamine (TMA) is synthesized by the intestinal microbiome, crosses the intestinal barrier and is metabolized to TMAO by hepatic flavin-containing monooxygenases (FMO). The intricate interactions of microbiome alterations and TMAO in relation to HFpEF manifestation and progression are analyzed here.MethodsHealthy lean (L-ZSF1, n = 12) and obese ZSF1 rats with HFpEF (O-ZSF1, n = 12) were studied. HFpEF was confirmed by transthoracic echocardiography, invasive hemodynamic measurements, and detection of N-terminal pro-brain natriuretic peptide (NT-proBNP). TMAO, carnitine, symmetric dimethylarginine (SDMA), and amino acids were measured using mass-spectrometry. The intestinal epithelial barrier was analyzed by immunohistochemistry, in-vitro impedance measurements and determination of plasma lipopolysaccharide via ELISA. Hepatic FMO3 quantity was determined by Western blot. The fecal microbiome at the age of 8, 13 and 20 weeks was assessed using 16s rRNA amplicon sequencing.ResultsIncreased levels of TMAO (+ 54%), carnitine (+ 46%) and the cardiac stress marker NT-proBNP (+ 25%) as well as a pronounced amino acid imbalance were observed in obese rats with HFpEF. SDMA levels in O-ZSF1 were comparable to L-ZSF1, indicating stable kidney function. Anatomy and zonula occludens protein density in the intestinal epithelium remained unchanged, but both impedance measurements and increased levels of LPS indicated an impaired epithelial barrier function. FMO3 was decreased (− 20%) in the enlarged, but histologically normal livers of O-ZSF1. Alpha diversity, as indicated by the Shannon diversity index, was comparable at 8 weeks of age, but decreased by 13 weeks of age, when HFpEF manifests in O-ZSF1. Bray–Curtis dissimilarity (Beta-Diversity) was shown to be effective in differentiating L-ZSF1 from O-ZSF1 at 20 weeks of age. Members of the microbial families Lactobacillaceae, Ruminococcaceae, Erysipelotrichaceae and Lachnospiraceae were significantly differentially abundant in O-ZSF1 and L-ZSF1 rats.ConclusionsIn the ZSF1 HFpEF rat model, increased dietary intake is associated with alterations in gut microbiome composition and bacterial metabolites, an impaired intestinal barrier, and changes in pro-inflammatory and health-predictive metabolic profiles. HFpEF as well as its most common comorbidities obesity and metabolic syndrome and the alterations described here evolve in parallel and are likely to be interrelated and mutually reinforcing. Dietary adaption may have a positive impact on all entities.Graphical abstract

The microbiome-derived metabolite TMAO drives immune activation and boosts responses to immune checkpoint blockade in pancreatic cancer.

The composition of the gut microbiome can control innate and adaptive immunity and has emerged as a key regulator of tumor growth, especially in the context of immune checkpoint blockade (ICB) therapy. However, the underlying mechanisms for how the microbiome affects tumor growth remain unclear. Pancreatic ductal adenocarcinoma (PDAC) tends to be refractory to therapy, including ICB. Using a nontargeted, liquid chromatography-tandem mass spectrometry-based metabolomic screen, we identified the gut microbe-derived metabolite trimethylamine N-oxide (TMAO), which enhanced antitumor immunity to PDAC. Delivery of TMAO intraperitoneally or via a dietary choline supplement to orthotopic PDAC-bearing mice reduced tumor growth, associated with an immunostimulatory tumor-associated macrophage (TAM) phenotype, and activated effector T cell response in the tumor microenvironment. Mechanistically, TMAO potentiated the type I interferon (IFN) pathway and conferred antitumor effects in a type I IFN-dependent manner. Delivering TMAO-primed macrophages intravenously produced similar antitumor effects. Combining TMAO with ICB (anti-PD1 and/or anti-Tim3) in a mouse model of PDAC significantly reduced tumor burden and improved survival beyond TMAO or ICB alone. Last, the levels of bacteria containing CutC (an enzyme that generates trimethylamine, the TMAO precursor) correlated with long-term survival in patients with PDAC and improved response to anti-PD1 in patients with melanoma. Together, our study identifies the gut microbial metabolite TMAO as a driver of antitumor immunity and lays the groundwork for potential therapeutic strategies targeting TMAO.

Trimethyllysine, a trimethylamine N-oxide precursor, predicts the presence, severity, and prognosis of heart failure

Background and aimsIntestinal flora metabolites are associated with cardiovascular (CV) diseases including heart failure (HF). The carnitine precursor trimethyllysine (TML), which participates in the generation of the atherogenic-related metabolite trimethylamine N-oxide (TMAO), was found to be related to poor prognosis in patients with CV diseases. The aim of the present study was to examine the relationship between TML and stable chronic HF.Methods and resultsIn total, 956 subjects including 471 stable chronic HF and 485 non-HF patients were enrolled in the present cohort study and subjects with stable HF were followed up for 2.0 ± 1.1 years. Serum levels of TML and TMAO were measured by liquid chromatography mass spectrometry in tandem. TML levels were significantly elevated in patients with HF compared with non-HF patients and were positively correlated with N-terminal pro-brain natriuretic peptide (NTproBNP) levels (r = 0.448, P < 0.001). TML was associated with the presence of HF after adjusting for age, sex, complications, traditional clinical factors, and TMAO (tertile 3 (T3), adjusted odds ratio (OR) 1.93, 95% confidence interval (CI) 1.19–3.13, and P = 0.007). In patients with HF, increased TML levels were associated with a composite endpoint of CV death and HF hospitalization during follow-up (T3, adjusted hazard ratio (HR) 1.93, 95% CI 1.27–2.93, and P = 0.002). Increased TML levels indicated a higher risk of CV death, re-hospitalization, and all-cause mortality.ConclusionSerum TML levels were associated with the presence and severity of HF in all subjects. High levels of TML can indicate complications and poor prognosis in HF patients.

Finasteride Alleviates High Fat Associated Protein-Overload Nephropathy by Inhibiting Trimethylamine N-Oxide Synthesis and Regulating Gut Microbiota.

Unhealthy diet especially high-fat diet (HFD) is the major cause of hyperlipidemia leading to deterioration of chronic kidney diseases (CKD) in patients. Trimethylamine N-oxide (TMAO) is a gut-derived uremic toxin. Our previous clinical study demonstrated that the elevation of TMAO was positively correlated with CKD progression. Finasteride, a competitive and specific inhibitor of type II 5a-reductase, has been reported recently to be able to downregulate plasma TMAO level thus preventing the onset of atherosclerosis by our research group. In this study, we established a protein-overload nephropathy CKD mouse model by bovine serum albumin (BSA) injection to investigate whether hyperlipidemia could accelerate CKD progression and the underlying mechanisms. Finasteride was administrated to explore its potential therapeutic effects. The results of biochemical analyses and pathological examination showed that HFD-induced hyperlipidemia led to aggravated protein-overload nephropathy in mice along with an elevated level of circulating TMAO, which can be alleviated by finasteride treatment possibly through inhibition of Fmo3 in liver. The 16 S rRNA sequencing results indicated that HFD feeding altered the composition and distribution of gut microbiota in CKD mice contributing to the enhanced level of TMAO precursor TMA, while finasteride could exert beneficial effects via promoting the abundance of Alistipes_senegalensis and Akkermansia_muciniphila. Immunofluorescence staining (IF) and qRT-PCR results demonstrated the disruption of intestinal barrier by decreased expression of tight junction proteins including Claudin-1 and Zo-1 in HFD-fed CKD mice, which can be rescued by finasteride treatment. Cytokine arrays and redox status analyses revealed an upregulated inflammatory level and oxidative stress after HFD feeding in CKO mice, and finasteride-treatment could alleviate these lesions. To summarize, our study suggested that finasteride could alleviate HFD-associated deterioration of protein-overload nephropathy in mice by inhibition of TMAO synthesis and regulation of gut microbiota.

Product of metabolic activity of intestinal microbium trimethylamine-N-oxide (TMAO)— biomarker of progression of atherosclerosis-copy in the heart of the heart

The literature data on the importance of intestinal microbiota as an endocrine organ— producer of biologically active metabolites, which perform key functions to maintain metabolic homeostasis of the whole organism, in particular the condition of the cardiovascular system, are analyzed. Clinical and experimental studies using a metabolomical approach have shown that the development of atherosclerotic CVD is often associated with elevated levels of one of the microbial metabolites, trimethylamine N-oxide (TMAO). TMAO may be a sensitive prognostic biomarker of complications of type 2 diabetes, including atherosclerosis and cardiovascular disease. The precursor of TMAO is trimethylamine (TMA), formed by intestinal bacteria from food phosphatidylcholine and L-carnitine. In the liver, TMA is converted to TMAO under the influence of hepatic flavin monooxygenase 3.The mechanisms of the proatherogenic effect of elevated levels of TMAO include effects on bile acid and cholesterol metabolism, platelet hyperactivation, stimulation of inflammatory processes and oxidative stress, induction of endothelial disfunction and endoplasmic reticulum stress. It has been established that TMAO, in conditions of chronic elevation, can contribute to cardiometabolic diseases. Elevated levels of TMAO in dysmetabolic conditions (obesity, type2 diabetes, atherosclerosis, or coronary heart disease) have been suggested to be largely associated with the gut microbiota profile. Therefore, regulating the ratio of intestinal microorganisms or their ability to form a precursor of TMAO— TMA, may be a way to develop new tools for the prevention and treatment of atherosclerosis and prevent the progression of cardiovascular complications, including in patients with type 2 diabetes. Studies have shown that inhibiting various stages of TMAO production can reduce TMAO levels and help treat atherosclerosis and diabetes.

Impact of Protein Sources in a Guideline-Based Omnivorous Diet on Circulating Biogenic Amine Metabolites: A Randomized Controlled-Feeding Trial

ObjectivesAge is a risk factor for chronic diseases—the leading causes of death and disability. An omnivorous diet compared to a plant-based diet was shown to increase blood Trimethylamine-N-oxide (TMAO)—a biogenic amine biomarker for atherosclerosis that may also increase the risk for insulin resistance and certain cancers. Worldwide, pork and chicken are the most popular animal proteins, however, poultry is perceived as healthier. It is unknown if pork intake differentially modifies the TMAO-response than poultry which was investigated here using a non-inferiority study design to avoid any potential negative results. MethodsIn a randomized, active-controlled, 2-arm-crossover trial (NCT03539666), healthy adults (n = 38/arm, age ≥ 50 y) consumed 156 g/day of lean pork or white-meat chicken as the main protein source as part of an omnivorous diet based on the 2015 USDA dietary guidelines. The feeding arms were matched for food ingredients, nutrients, and energy. Overnight-fasted samples were collected before and after each diet phase. LC/MS with isotope-labeled internal standards and 16S-rDNA sequencing were used for metabolite and microbiome measurements, respectively. Univariate and multivariate data were analyzed using supervised and unsupervised machine learning algorithms in R. ResultsA non-inferiority of pork to poultry for a circulating TMAO-response (97.5% CI, linear mixed model) was observed for the first time. In addition, global biogenic amine signature (346 metabolites, 95% CI), microbiota-dependent essential TMAO precursor—trimethylamine (97.5% CI), three dietary TMAO substrates (choline, betaine, and L-carnitine) as well as the microbiota composition (95% CI) underlying the observed TMAO-response were not different between the two proteins. TMAO phenotype varied at baseline; a higher baseline circulating TMAO concentration associated with distinct microbiota profiles (all, FDR corrected p < 0.05) and greater TMAO-response (p = 0.0001) independent of the dietary proteins. ConclusionsLean pork does not exacerbate TMAO-risk in omnivores 50 years or older. With 95% of Americans being omnivorous and a third of them fast aging, lean pork increases dietary protein options that may help improve adherence to dietary recommendations for greater healthspan. Funding SourcesNational Pork Board, National Institute of Food & Agriculture

Integrated metagenomics identifies a crucial role for trimethylamine-producing Lachnoclostridium in promoting atherosclerosis

Microbial trimethylamine (TMA)-lyase activity promotes the development of atherosclerosis by generating of TMA, the precursor of TMA N-oxide (TMAO). TMAO is well documented, but same can not be said of TMA-producing bacteria. This work aimed to identify TMA-producing genera in human intestinal microbiota. We retrieved the genomes of human-associated microorganisms from the Human Microbiome Project database comprising 1751 genomes, Unified Human Gastrointestinal Genome collection consisting 4644 gut prokaryotes, recapitulated 4930 species-level genome bins and public gut metagenomic data of 2134 individuals from 11 populations. By sequence searching, 216 TMA-lyase-containing species from 102 genera were found to contain the homologous sequences of cntA/B, yeaW/X, and/or cutC/D. We identified 13 strains from 5 genera with cntA sequences, and 30 strains from 14 genera with cutC showing detectable relative abundance in healthy individuals. Lachnoclostridium (p = 2.9e−05) and Clostridium (p = 5.8e−04), the two most abundant cutC-containing genera, were found to be much higher in atherosclerotic patients compared with healthy persons. Upon incubation with choline (substrate), L. saccharolyticum effectively transformed it to TMA at a rate higher than 98.7% while that for C. sporogenes was 63.8–67.5% as detected by liquid chromatography-triple quadrupole mass spectrometry. In vivo studies further showed that treatment of L. saccharolyticum and choline promoted a significant increase in TMAO level in the serum of ApoE−/− mice with obvious accumulation of aortic plaque in same. This study discloses the significance and efficiency of the gut bacterium L. saccharolyticum in transforming choline to TMA and consequently promoting the development of atherosclerosis.

The microbial gbu gene cluster links cardiovascular disease risk associated with red meat consumption to microbiota L-carnitine catabolism.

The heightened cardiovascular disease (CVD) risk observed with omnivores is thought to be linked, in part, to gut microbiota-dependent generation trimethylamine-N-oxide (TMAO) from L-carnitine, a nutrient abundant in red meat. Gut microbial transformation of L-carnitine into trimethylamine (TMA), the precursor of TMAO, occurs via the intermediate γ-butyrobetaine (γBB). However, the relationship between γBB, red meat ingestion and CVD risks, as well as the gut microbial genes responsible for the transformation of γBB to TMA, are unclear. Here we show plasma γBB levels in individuals from a clinical cohort (n=2,918) are strongly associated with incident CVD event risks. Culture of human fecal samples and microbial transplantation studies in gnotobiotic mice with defined synthetic communities showed that the introduction of Emergencia timonensis, a human gut microbe that can metabolize γBB into TMA, is sufficient to complete the carnitine→γBB→TMA transformation, elevate TMAO levels, and enhance thrombosis potential in recipients following arterial injury. RNAseq analyses of E. timonensis identified a 6 gene cluster, herein named gamma-butyrobetaine utilization gene cluster (gbu), which is upregulated in response to γBB. Combinatorial cloning and functional studies identified 4 genes (gbuA, gbuB, gbuC, and gbuE) that are necessary and sufficient to recapitulate the conversion of γBB to TMA when co-expressed in E. coli. Finally, reanalysis of samples (n=113) from a clinical randomized diet intervention study showed that the abundance of fecal gbuA correlates with plasma TMAO and a red meat-rich diet. Our findings reveal a microbial gene cluster that is critical to dietary carnitine→γBB→TMA→TMAO transformation in hosts and contributes to CVD risk.

Diet and APOE as moderators of the relationship between trimethylamine N‐oxide and biomarkers of Alzheimer's disease and glial activation

Dietary patterns appear to impact cognitive trajectories in aging, and gut microbiota have been implicated in Alzheimer's disease (AD) pathogenesis, potentially as modulators of neuroinflammation early in the disease. Diets featuring low meat and dairy consumption have been linked to reduced AD risk, and recently, the gut microbial metabolite trimethylamine N-oxide (TMAO) was found in cerebrospinal fluid (CSF) and linked to CSF biomarkers of AD. Because TMAO is largely derived from dietary sources of choline, carnitine, and betaine, we examined whether these precursors drive the association between TMAO and sTREM2, a marker for glial activation. Additionally, TMAO has been found to inhibit cholesterol metabolism, a strong risk factor for AD, which is further dysregulated by the APOE4 allele. Therefore, we aimed to determine whether TMAO-glial activation relationships are moderated by APOE4 carrier status.Participants from the Wisconsin Registry for Alzheimer's Prevention and the Wisconsin Alzheimer's Disease Research Center provided CSF samples (n=570, Table 1). sTREM2 and YKL-40 biomarkers were measured with the exploratory Roche NeuroToolKit assays, a panel of robust prototype immunoassays (Roche Diagnostics International Ltd). TMAO, carnitine, choline, and betaine relative abundance were obtained using Metabolon's UHPLC-MS/MS metabolomics platform. A subset of participants (n=159) completed the MIND diet questionnaire. Metabolite and biomarker levels were log-transformed for analysis; models were adjusted for age, sex, and APOE4 carrier status. Linear regression tested associations between intake of TMAO precursor-containing foods (red meat, butter, cheese, fish) and CSF levels of each TMAO precursor. Path analysis with Satorra-Bentler adjustments tested whether TMAO mediated precursor-biomarker relationships. Linear regression tested whether APOE4 carrier status moderated TMAO-biomarker relationships.Red meat and cheese consumption predicted levels of CSF carnitine (βs=0.009, -0.017; ps=0.0609, 0.0007; Figure 1 A, B; respectively). TMAO mediated the relationship between carnitine and sTREM2, although effects were marginal; several individual relationships throughout both path models showed strong associations (Tables 2, 3; Figures 2, 3). APOE4 carrier status did not significantly moderate TMAO-glial activation relationships.This study suggests that CSF carnitine reflects dietary intake, and may drive the TMAO-sTREM2 association previously identified. Future studies in animal models are required to confirm these results mechanistically.

Regulation of blood\u2013brain barrier integrity by microbiome-associated methylamines and cognition by trimethylamine N-oxide

BackgroundCommunication between the gut microbiota and the brain is primarily mediated via soluble microbe-derived metabolites, but the details of this pathway remain poorly defined. Methylamines produced by microbial metabolism of dietary choline and l-carnitine have received attention due to their proposed association with vascular disease, but their effects upon the cerebrovascular circulation have hitherto not been studied.ResultsHere, we use an integrated in vitro/in vivo approach to show that physiologically relevant concentrations of the dietary methylamine trimethylamine N-oxide (TMAO) enhanced blood-brain barrier (BBB) integrity and protected it from inflammatory insult, acting through the tight junction regulator annexin A1. In contrast, the TMAO precursor trimethylamine (TMA) impaired BBB function and disrupted tight junction integrity. Moreover, we show that long-term exposure to TMAO protects murine cognitive function from inflammatory challenge, acting to limit astrocyte and microglial reactivity in a brain region-specific manner.ConclusionOur findings demonstrate the mechanisms through which microbiome-associated methylamines directly interact with the mammalian BBB, with consequences for cerebrovascular and cognitive function.24wuPkhfjSm7S4zaEcHM79Video abstract

Proatherogenic gut-microbiome metabolite trimethylamine elicits a gut-liver circuit to regulate TMAO metabolism and atherosclerosis via mediation of CREBH

Abstract Introduction In humans, circulating metabolite Trimethylamine N-oxide (TMAO) is closely associated with higher risk of cardiovascular disease. Trimethylamine (TMA), a precursor of TMAO, is produced by gut microbiome using dietary components, i.e., choline and carnitine, as substrates. The gut-derived TMA is then transferred to the liver where it is further oxidized to TMAO by the flavin-containing monooxygenases (FMOs). The ER-resident transcription factor c-AMP responsive element binding protein H (CREBH/CREB3L3) is exclusively expressed in the liver and intestine. Perturbation of CREBH activity contributes to the development of hyperlipidemia and cardiovascular disease. Therefore, the Purpose of this study is to investigate the regulatory effect of a gut bacterium, Akkermansia muciniphila (A. muciniphila), on TMA and TMAO metabolism and the role of CREBH in this process. Methods Two groups of wild type (WT) and CREBH knockout (CREBH-KO) mice were inoculated with 200 μL of A. muciniphila (2×108 cfu/0.2 mL) in PBS or the vehicle (PBS) alone as control every other day through oral gavage for 2 weeks. Plasmas, liver and intestinal tissues were collected for metabolomics analysis, immunoblotting analysis and q-RT-PCR. Results Metabolomics analysis of the plasmas from the experimental mice revealed that increased colonization of A. muciniphila in the gut significantly reduced circulating TMA in the WT mice but not in CREBH-KO mice (P&amp;lt;0.05), suggesting that depletion of CREBH altered the microenvironment of gut microbiome which affected the metabolism of TMA by gut bacteria. In the livers, A. muciniphila treatment markedly reduced mRNA expression of FMO1 and FMO3 (P&amp;lt;0.05), which subsequently inhibited the enzymatic conversion of TMA to TMAO in hepatocytes. Immunoblotting analysis further revealed that LDL receptor was upregulated whereas ER stress markers, GRP94 and JNK1/2, were downregulated in the A. muciniphila treated KO mice, indicating an acceleration in lipoprotein (VLDL remnant) clearance from the circulation and the improvement of metabolic inflammation. In vitro, incubation of mouse hepatocytes AML12 with TMA (600 mM) for 12 hours stimulated expression of FMOs to facilitate the conversion of TMA to TMAO and induced lipotoxicity. Conclusion CREBH mediates the crosstalk between gut microbiome and liver metabolic system that regulates TMA and TMAO metabolism, which contributes to the induction of metabolic inflammation and atherogenesis. This novel finding may lend support to the therapeutic strategy of atherosclerosis. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

TMAO (trimethylamine n-oxide) as a potential biomarker of individual severe stress perception in posttraumatic stress disorder (PTSD)-vulnerable patients after acute myocardial infarction

Abstract Background Acute myocardial infarction is not only a somatic disease but potentially triggers psychological effects, too. Post-traumatic stress disorder (PTSD) is a common stress-related disorder. It is characterized by numerous symptoms, such as flashbacks, intrusions, nightmares and severe anxiety, as well as uncontrollable, intense and disturbing thoughts and feelings related to the traumatic experience. However, with regard to the development of PTSD, individual stress perception might be crucial since not every serious traumatic experience leads to PTSD. To date, almost no biological correlates of an individual's perception of stress have been identified as being associated with the long-term development of PTSD. Objective The aim of the study was to determine whether blood levels of TMAO vary immediately after AMI (1) in patients with or without depression, and (2) in patients with AMI induced PTSD symptomatology (subsyndromal PTSD and full PTSD). Furthermore, we investigated whether TMAO is a potential biomarker that might be useful in the prediction of PTSD symptomatology in the long term. Method A total of 114 AMI patients were assessed with standardized clinical psychiatric interviews based on the Hamilton Depression Scale (HAMD-17) after admission to the hospital and 6 months later. In addition, the CAPS-5 was used to explore PTSD symptoms (subsyndromal PTSD and full PTSD) 6 months after AMI. To assess patients' TMAO status, serum samples were collected at hospitalization and 6 months after AMI. Results Study participants with post-myocardial infarction PTSD symptomatology (subsyndromal PTSD and full PTSD) had significantly higher TMAO levels immediately after AMI than patients without PTSD symptoms (ANCOVA: TMAO (PTSD x time), F = 4.544, df = 1, p=0.035). In contrast, depressive symptomatology 6 months after AMI had no influence on TMAO levels (TMAO (depression x time), F = 0.083, df = 1, p=0.774). With the inclusion of additional clinical predictors in a hierarchical logistic regression model, TMAO becomes a significant predictor of PTSD symptomatology. Conclusions An elevated TMAO level immediately after AMI might reflect severe stress in PTSD-vulnerable patients, which might also lead to a short-term increased gut permeability to trimethylamine (TMA), the precursor of TMAO. Thus, elevated TMAO might be a biological correlate for stress that is associated with vulnerability to PTSD and might help to identify patients at increased risk. Funding Acknowledgement Type of funding sources: None.

Abstract P174: Sex Differences In The Pro-hypertensive Effect Of Carnitine, The Precursor Of Gut-derived Metabolite TMAO

Recent evidence reports sexually divergent mechanisms that differentially drive the severity of hypertension. Our data show that female Dahl Salt-Sensitive (SS) rats are significantly protected from salt-induced hypertension and renal injury and have stark differences in gut microbiota composition compared to males. Gut-derived metabolites are increasingly being recognized as mechanistic links between the gut microbiota and hypertension. One such metabolite is trimethylamine N-oxide (TMAO), which is derived from the bacterial metabolism of carnitine and is gaining notoriety for its role in cardiovascular disease. Metabolomics analysis in high salt-fed SS rats revealed a trend for increased TMAO (1.3-fold, p=0.11) in the serum of males compared to females (n=6). TMAO appears to be specifically derived from gut bacteria since oral antibiotic treatment nearly eliminated circulating TMAO levels in both males and females (99.3% and 88.9% reduction, respectively; p&lt;0.001). Interestingly, antibiotic treatment reduced salt-sensitive hypertension in males but not females. There was also a corresponding increase in the TMAO precursor carnitine (1.9-fold, p&lt;0.01) in the serum of males versus females. Thus, we hypothesized that administration of carnitine (400 mg/kg/day) in the drinking water would exacerbate salt-sensitive hypertension, renal damage, and gut inflammation in male and female SS rats challenged with high salt (4% NaCl). There was a trend for carnitine treatment to exacerbate mean arterial pressure in both males (160±9 vs 146±2 mmHg, n=4-6, p=0.22) and females (155±6 vs 139±2 mmHg, n=2, p=0.14) compared to vehicle. Despite elevated pressure in both sexes, carnitine-treated males exhibited greater increases in albuminuria (340±136 vs 194±29 mg/day, carnitine vs vehicle, p=0.28) than females (55±33 vs 26±5 mg/day). Carnitine treatment also significantly increased the number of CD3+ T cells in the colonic lamina propria (24.3±6.0 vs 2.4±0.5 x 10 6 cells/g tissue, n=5, p&lt;0.05) of male rats compared to vehicle. Together, these data identify gut microbiota-mediated carnitine/TMAO metabolism as a potentially detrimental pathway that promotes greater salt-sensitivity, renal damage, and gut inflammation in males versus females.

Trimethyllysine predicts all-cause and cardiovascular mortality in community-dwelling adults and patients with coronary heart disease

AimsTrimethyllysine (TML) is involved in carnitine synthesis, serves as a precursor of trimethylamine N-oxide (TMAO) and is associated with cardiovascular events in patients with established coronary heart disease (CHD). We prospectively examined circulating TML as a predictor of all-cause and cardiovascular mortality in community-dwelling adults and patients with CHD.Methods and resultsBy Cox regression modelling, risk associations were examined in 6393 subjects in the community-based Hordaland Health Study (HUSK). A replication study was conducted among 4117 patients with suspected stable angina pectoris in the Western Norway Coronary Angiography Cohort (WECAC). During a mean follow-up of 10.5 years in the HUSK-cohort, 884 (13.8%) subjects died, of whom 287 from cardiovascular causes. After multivariable adjustments for traditional cardiovascular risk factors, the hazard ratio (HR) [95% confidence interval (95% CI)] for all-cause mortality comparing the 4th vs. 1st TML-quartile was 1.66 (1.31–2.10, P < 0.001). Particularly strong associations were observed for cardiovascular mortality [HR (95% CI) 2.04 (1.32–3.15, P = 0.001)]. Corresponding risk-estimates in the WECAC (mean follow-up of 9.8 years) were 1.35 [1.10–1.66, P = 0.004] for all-cause and 1.45 [1.06–1.98, P = 0.02] for cardiovascular mortality. Significant correlations between plasma TML and TMAO were observed in both cohorts (rs ≥ 0.42, P < 0.001); however, additional adjustments for TMAO did not materially influence the risk associations, and no effect modification by TMAO was found.ConclusionsElevated TML-levels were associated with increased risk of all-cause and cardiovascular mortality both in subjects with and without established CHD.

Ttrimetyllysine and risk of new-onset atrial fibrillation in two large norwegian cohorts

Abstract Funding Acknowledgements Type of funding sources: None. Background/Aim Increased plasma trimetyllysine (TML), a methylated amino acid, has recently been linked to higher risk of acute myocardial infarction (AMI). TML is also a precursor of trimethylamine-N oxide (TMAO), which has been linked to increased cardiovascular risk, including that of atrial fibrillation (AF). We investigated the association between TML and new-onset AF in two large Norwegian cohorts. Methods The primary cohort consisted of 6396 participants in the community-based Hordaland Health Study (HUSK). The validation cohort consited of 2027 patients who underwent coronary angiography due to suspected stable angina pectoris in the Western Norway Coronary Angiography Cohort (WECAC). Information on new-onset AF was obtained by linking patient data to Norwegian public health registries. Risk associations were explored by Cox regression. Results During median (25th-75th percentile) follow-up of 10.9 (10.6-11.3) and 7.0 (6.3-8.6) years, 560 (8.8%) patients in the HUSK and 210 (10.4%) in the WECAC was diagnosed with AF. In the HUSK, the age and gender adjusted HR (95 % CI) for the 4th vs. 1st plasma TML quartiles 1.84 (1.37-2.48) p &amp;lt; 0.001. In multivariable models the association was only slightly attenuated. Correspondingsly, the age and gender adjusted HR (95% CI) for the 4th vs. 1st TML quartiles in the WECAC was 1.48 (0.96-2.27) p = 0.07. Testing for collinearity between TMAO and TML revealed variance inflation factors between 1.0-1.1 in HUSK and WECAC, thus ruling out collinearity. Conclusion Plasma TML was associated with new-onset AF among subjects from the general population, and the relationship was independent from established AF risk factors. A similar trend was also seen in patients with suspected stable angina pectoris, strengthening our findings, which motivate further studies to explore potential pathophysiological relationships between one-carbon metabolism and cardiac arrhythmias

Assessment of trimethylamine N-oxide (TMAO) as a potential biomarker of severe stress in patients vulnerable to posttraumatic stress disorder (PTSD) after acute myocardial infarction

ABSTRACT Background: Posttraumatic stress disorder (PTSD) is a frequently observed stress-related disorder after acute myocardial infarction (AMI) and it is characterized by numerous symptoms, such as flashbacks, intrusions and anxiety, as well as uncontrollable thoughts and feelings related to the trauma. Biological correlates of severe stress might contribute to identifying PTSD-vulnerable patients at an early stage. Objective: Aims of the study were (1) to determine whether blood levels of trimethylamine N-oxide (TMAO) vary immediately after AMI in patients with/without AMI-induced PTSD symptomatology, (2) to investigate whether TMAO is a potential biomarker that might be useful in the prediction of PTSD and the PTSD symptom subclusters re-experiencing, avoidance and hyperarousal, and (3) to investigate whether TMAO varies immediately after AMI in patients with/without depression 6 months after AMI. Method: A total of 114 AMI patients were assessed with the Hamilton-Depression Scale after admission to the hospital and 6 months later. The Clinician Administered PTSD Scale for DSM-5 was used to explore PTSD-symptoms at the time of AMI and 6 months after AMI. To assess patients’ TMAO status, serum samples were collected at hospitalization and 6 months after AMI. Results: Participants with PTSD-symptomatology had significantly higher TMAO levels immediately after AMI than patients without PTSD-symptoms (ANCOVA: TMAO(PTSD x time), F = 4.544, df = 1, p = 0.035). With the inclusion of additional clinical predictors in a hierarchical logistic regression model, TMAO became a significant predictor of PTSD-symptomatology. No significant differences in TMAO levels immediately after AMI were detected between individuals with/without depression 6 months after AMI. Conclusions: An elevated TMAO level immediately after AMI might reflect severe stress in PTSD-vulnerable patients, which might also lead to a short-term increase in gut permeability to trimethylamine, the precursor of TMAO. Thus, an elevated TMAO level might be a biological correlate for severe stress that is associated with vulnerability to PTSD.

Gut Microbiota-Related Effects of Tanhuo Decoction in Acute Ischemic Stroke.

Acute ischemic stroke (AIS) is a major cause of acquired adult disability and death. Our previous studies proved the efficacy and effectiveness of Tanhuo decoction (THD) on AIS. However, the therapeutic mechanism remains unclear. We recruited 49 AIS patients and 30 healthy people to explore the effects of THD+basic treatment on the poststroke gut microbiota of AIS patients using 16S rRNA sequencing, in which 23 patients received basic treatment (control group) and 26 patients received THD+basic treatment (THD group). By comparing the data before and after treatments, we found the THD group acquired better outcome than the control group on both clinical outcome indices and the characteristics of gut microbiota. In addition to the mediation on short-chain fatty acid- (SCFA-) producing bacteria in two groups, treatment in the THD group significantly decreased the lipopolysaccharide- (LPS-) producing bacteria to reduce LPS biosynthesis. Besides, the complexity of the cooccurrence of gut microbiota and the competition among LPS-producing bacteria and opportunistic pathogenetic bacteria were enhanced in the THD group. Treatment in the THD group also exhibited the potential in decreasing genes on the biosynthesis of trimethylamine (TMA), the precursor of Trimethylamine N-oxide (TMAO), and increasing genes on the degradation of TMA, especially increasing trimethylamine-corrinoid protein Co-methyltransferase (mttB) which catabolizes TMA to methane. These results hinted that THD+basic treatment might exert its efficacy by mediating the gut microbiota and microbial metabolites, including LPS and TMAO that aggravate the sterile inflammation and platelet aggregation. Moreover, the well-fitting regression model results in predicting the clinical outcome with the alteration of gut microbiota proved gut microbiota as a potential indicator of AIS and provided evidence of the communication between the gut and brain of AIS patients.

Trimethylamine N-oxide (TMAO) Is not Associated with Cardiometabolic Phenotypes and Inflammatory Markers in Children and Adults.

ABSTRACTBackgroundTrimethylamine N-oxide (TMAO) is a diet- and microbiome-derived metabolite and a proposed biomarker of adverse cardiometabolic outcomes. TMAO studies have mainly been conducted in individuals with cardiometabolic disease, and studies in population-derived samples are limited.ObjectiveWe aimed to investigate the associations between plasma TMAO concentrations and its precursors [carnitine, choline, betaine, and dimethylglycine (DMG)] with metabolic syndrome (MetS) scores, preclinical cardiovascular phenotypes, and inflammatory biomarkers (i.e. high-sensitivity C-reactive protein and serum glycoprotein acetyls) in a population-derived cohort of children and their parents.MethodsThe concentrations of TMAO and its precursors were quantified using UHPLC coupled with tandem MS (UHPLC/MS-MS) in 1166 children (mean age 11 y ± 0.5 y, 51% female) and 1324 adults (44 y ± 5.1 y, 87% female) participating in The Growing Up in Australia's Child Health CheckPoint Study. We developed multivariable fractional polynomial models to analyze associations between TMAO, its precursors, MetS (adjusted for sex and age), and cardiovascular phenotypes (adjusted for sex, age, BMI, household income, and the urinary albumin to creatinine ratio). Pearson's correlations were computed to identify associations between TMAO, its precursors, and inflammatory biomarkers.ResultsThe concentrations of TMAO precursors, but not TMAO itself, were associated with MetS, cardiovascular phenotypes, and inflammatory biomarkers in children and adults.ConclusionsTMAO precursors, but not TMAO itself, were associated with adverse cardiometabolic and inflammatory phenotypes in children and adults. TMAO precursor concentrations may better reflect cardiovascular health and inflammatory status within the wider population. Replication in other population settings and mechanistic studies are warranted.

Metformin decreases bacterial trimethylamine production and trimethylamine N-oxide levels in db/db mice

The current study aimed to explore whether metformin, the most widely prescribed oral medication for the treatment of type 2 diabetes, alters plasma levels of cardiometabolic disease-related metabolite trimethylamine N-oxide (TMAO) in db/db mice with type 2 diabetes. TMAO plasma concentration was up to 13.2-fold higher in db/db mice when compared to control mice, while in db/db mice fed choline-enriched diet, that mimics meat and dairy product intake, TMAO plasma level was increased 16.8-times. Metformin (250 mg/kg/day) significantly decreased TMAO concentration by up to twofold in both standard and choline-supplemented diet-fed db/db mice plasma. In vitro, metformin significantly decreased the bacterial production rate of trimethylamine (TMA), the precursor of TMAO, from choline up to 3.25-fold in K. pneumoniae and up to 26-fold in P. Mirabilis, while significantly slowing the growth of P. Mirabilis only. Metformin did not affect the expression of genes encoding subunits of bacterial choline-TMA-lyase microcompartment, the activity of the enzyme itself and choline uptake, suggesting that more complex regulation beyond the choline-TMA-lyase is present. To conclude, the TMAO decreasing effect of metformin could be an additional mechanism behind the clinically observed cardiovascular benefits of the drug.

Diabetes is Associated with Increased Passage of Bacterial Metabolite TMA, a TMAO Precursor, Through the Intestinal Barrier

Trimethylamine N‐oxide (TMAO) is suggested to be a marker of cardiovascular outcome in diabetic patients. However, mechanism of the TMAO increase remains unclear. TMAO is a product of liver oxidation of trimethylamine (TMA), a metabolite of gut bacteria. Diabetes is associated with gut barrier disruption, which may result in increased passage of bacterial metabolites to the bloodstream. We hypothesized that higher gut barrier permeability to TMA is responsible for TMAO elevation in diabetic patients with increased cardiovascular risk. We assessed gut permeability to TMA and TMA liver clearance in animal model of diabetes. Additionally we studied therapeutic potential of ACE‐I (angiotensin‐converting enzyme inhibitor) in restoring proper gut barrier function.12‐week‐old Sprague Dawley rats received either streptozotocin (65 mg/kg bw i.p.) (diabetic group, n=15, and ACE‐I group, n=12) or saline i.p. (control group, n=12). Fasting glucose level above 250mg/dl was considered a successful diabetes induction. Rats with ineffective diabetes induction were not included in the analysis. ACE‐I group was treated with enalapril (Polpharma, Poland), dissolved in drinking water 50mg/L (a dose of 6.5±0.3 mg/kg bw) for 4 weeks. Diabetic and control group were maintained on tap water. 4‐weeks after induction of diabetes, rats were anesthetized with urethane (1.5 g/kg bw i.p.) and blood samples from portal vein and inferior vena cava were collected. Rats were euthanized and tissues and colon content (stools) were harvested for histological and biochemical analysis, respectively. The concentration of TMA and TMAO in blood and stool samples were evaluated using liquid chromatography coupled with mass spectrometry. Gut permeability was estimated by the ratio of TMA stool concentration to TMA portal blood concentration. TMA liver clearance was calculated using the equation: 1−(systemic blood concentration/portal blood concentration).Histological examination revealed pronounced changes in intestinal morphology in rats with induced diabetes. Diabetic group showed significantly increased concentration of TMA in portal blood and increased gut permeability to TMA in comparison to control group and ACE‐I group (diabetic rats treated with enalapril). Additionally, diabetic group demonstrated higher liver clearance of TMA in comparison to healthy rats, which was also reflected by a trend towards higher concentration of TMAO in diabetic rats.Diabetes is associated with disruption of gut barrier, which results in increased permeability to TMA, and these changes are ameliorated by enalapril. Increased availability of TMA, a TMAO precursor, together with higher TMA liver clearance appear to be probable mechanisms of TMAO increase in diabetes. Given the potential harmful effects of TMA or TMAO our study indicates gut barrier as a therapeutic target with the promising use of ACE‐I.Support or Funding InformationThis work was supported by the Ministry of Science and Higher Education Republic of Poland, Diamond grant no: DI2017 009247.Selected parameters in experimental groups: diabetic rats (diabetes), diabetic rats on enalapril (ACE‐I) and rats without diabetes on tap water (control). Gut permeability was assessed by the ratio: TMA stool concentration/TMA portal blood concentration; liver clearance was calculated: 1‐(systemic blood concentration/portal blood concentration). TMA ‐ trimethylamine, TMAO ‐ trimethylamine N‐oxide. Values are means ± SE, * ‐ p&lt;0.05, by one‐way ANOVA followed by Duncan’s test.Figure 1