Increases In Trimethylamine N-oxide Research Articles

Distinct Plasma Metabolomic and Gut Microbiome Profiles after Gestational Diabetes Mellitus Diet Treatment: Implications for Personalized Dietary Interventions.

Gestational diabetes mellitus (GDM) triggers alterations in the maternal microbiome. Alongside metabolic shifts, microbial products may impact clinical factors and influence pregnancy outcomes. We investigated maternal microbiome-metabolomic changes, including over 600 metabolites from a subset of the "Choosing Healthy Options in Carbohydrate Energy" (CHOICE) study. Women diagnosed with GDM were randomized to a diet higher in complex carbohydrates (CHOICE, n = 18, 60% complex carbohydrate/25% fat/15% protein) or a conventional GDM diet (CONV, n = 16, 40% carbohydrate/45% fat/15% protein). All meals were provided. Diets were eucaloric, and fiber content was similar. CHOICE was associated with increases in trimethylamine N-oxide, indoxyl sulfate, and several triglycerides, while CONV was associated with hippuric acid, betaine, and indole propionic acid, suggestive of a healthier metabolome. Conversely, the microbiome of CHOICE participants was enriched with carbohydrate metabolizing genes and beneficial taxa such as Bifidobacterium adolescentis, while CONV was associated with inflammatory pathways including antimicrobial resistance and lipopolysaccharide biosynthesis. We also identified latent metabolic groups not associated with diet: a metabolome associated with less of a decrease in fasting glucose, and another associated with relatively higher fasting triglycerides. Our results suggest that GDM diets produce specific microbial and metabolic responses during pregnancy, while host factors also play a role in triglycerides and glucose metabolism.

Antibiotic-induced intestinal microbiota depletion can attenuate the acute kidney injury to chronic kidney disease transition via NADPH oxidase 2 and trimethylamine-N-oxide inhibition

Intestinal microbiota and their metabolites affect systemic inflammation and kidney disease outcomes. Here, we investigated the key metabolites associated with the acute kidney injury (AKI)-to chronic kidney disease (CKD) transition and the effect of antibiotic-induced microbiota depletion (AIMD) on this transition. In 61 patients with AKI, 59 plasma metabolites were assessed to determine the risk of AKI-to-CKD transition. An AKI-to-CKD transition murine model was established four weeks after unilateral ischemia-reperfusion injury (IRI) to determine the effects of AIMD on the gut microbiome, metabolites, and pathological responses related to CKD transition. Human proximal tubular epithelial cells were challenged with CKD transition-related metabolites and inhibitory effects of NADPH oxidase 2 (NOX2) signals were tested. Based on clinical metabolomics, plasma trimethylamine N-oxide (TMAO) was associated with a significant increased risk for AKI-to-CKD transition [adjusted odds ratio 4.389 (95% confidence interval 1.106–17.416)]. In vivo, AIMD inhibited a unilateral IRI-induced increase in TMAO, along with a decreased in apoptosis, inflammation, and fibrosis. The expression of NOX2 and oxidative stress decreased after AIMD. In vitro, TMAO induced fibrosis with NOX2 activation and oxidative stress. NOX2 inhibition successfully attenuated apoptosis, inflammation, and fibrosis with suppression of G2/M arrest. NOX2 inhibition (in vivo) showed improvement in pathological changes with a decrease in oxidative stress without changes in TMAO levels. Thus, TMAO is a key metabolite associated with the AKI-to-CKD transition and NOX2 activation was identified as a key regulator of TMAO-related AKI-to-CKD transition both in vivo and in vitro.

Connections between serum Trimethylamine N-Oxide (TMAO), a gut-derived metabolite, and vascular biomarkers evaluating arterial stiffness and subclinical atherosclerosis in children with obesity.

Childhood obesity leads to early subclinical atherosclerosis and arterial stiffness. Studying biomarkers like trimethylamine N-oxide (TMAO), linked to cardio-metabolic disorders in adults, is crucial to prevent long-term cardiovascular issues. The study involved 70 children aged 4 to 18 (50 obese, 20 normal-weight). Clinical examination included BMI, waist measurements, puberty stage, the presence of acanthosis nigricans, and irregular menstrual cycles. Subclinical atherosclerosis was assessed by measuring the carotid intima-media thickness (CIMT), and the arterial stiffness was evaluated through surrogate markers like the pulse wave velocity (PWV), augmentation index (AIx), and peripheral and central blood pressures. The blood biomarkers included determining the values of TMAO, HOMA-IR, and other usual biomarkers investigating metabolism. The study detected significantly elevated levels of TMAO in obese children compared to controls. TMAO presented positive correlations to BMI, waist circumference and waist-to-height ratio and was also observed as an independent predictor of all three parameters. Significant correlations were observed between TMAO and vascular markers such as CIMT, PWV, and peripheral BP levels. TMAO independently predicts CIMT, PWV, peripheral BP, and central SBP levels, even after adding BMI, waist circumference, waist-to-height ratio, puberty development and age in the regression model. Obese children with high HOMA-IR presented a greater weight excess and significantly higher vascular markers, but TMAO levels did not differ significantly from the obese with HOMA-IR<cut-offs. TMAO did not correlate to HOMA-IR and insulin levels but presented a negative correlation to fasting glucose levels. An increase in TMAO was shown to be associated with an increase in the probability of the presence of acanthosis nigricans. TMAO levels are not influenced by other blood biomarkers. Our study provides compelling evidence supporting the link between serum TMAO, obesity, and vascular damage in children. These findings highlight the importance of further research to unravel the underlying mechanisms of this connection.

The gut microbial metabolite trimethylamine N-oxide and cardiovascular diseases.

Morbidity and mortality of cardiovascular diseases (CVDs) are exceedingly high worldwide. Researchers have found that the occurrence and development of CVDs are closely related to intestinal microecology. Imbalances in intestinal microecology caused by changes in the composition of the intestinal microbiota will eventually alter intestinal metabolites, thus transforming the host physiological state from healthy mode to pathological mode. Trimethylamine N-oxide (TMAO) is produced from the metabolism of dietary choline and L-carnitine by intestinal microbiota, and many studies have shown that this important product inhibits cholesterol metabolism, induces platelet aggregation and thrombosis, and promotes atherosclerosis. TMAO is directly or indirectly involved in the pathogenesis of CVDs and is an important risk factor affecting the occurrence and even prognosis of CVDs. This review presents the biological and chemical characteristics of TMAO, and the process of TMAO produced by gut microbiota. In particular, the review focuses on summarizing how the increase of gut microbial metabolite TMAO affects CVDs including atherosclerosis, heart failure, hypertension, arrhythmia, coronary artery disease, and other CVD-related diseases. Understanding the mechanism of how increases in TMAO promotes CVDs will potentially facilitate the identification and development of targeted therapy for CVDs.

Trimethylamine N-oxide and its precursors in relation to blood pressure: A mendelian randomization study.

ObjectivePrevious studies have demonstrated that trimethylamine N-oxide (TMAO) and its precursors, including choline, betaine, and carnitine, are closely associated with blood pressure (BP) changes. Nevertheless, with the limitation of reverse causality and confounder in observational studies, such a relationship remains unclear. We aimed to assess the causal relationship of TMAO and its precursors with BP by the Mendelian Randomization (MR) approach.MethodIn this study, two-sample MR was used to reveal the causal effect of TMAO and its precursors on BP. Pooled data of TMAO and its precursors was from genome-wide association studies (GWAS) which includes summary data of human metabolome in 2,076 European participants from Framingham Heart Study. Summary-level data for BP was extracted from the International Consortium of Blood Pressure-Genome Wide Association Studies. Inverse variance weighted (IVW), MR Egger regression, Maximum likelihood, Weighted median, and MR pleiotropy residual sum and outlier test (MR-PRESSO) were used in this MR analysis.ResultsA total of 160 independent SNP loci were associated with TMAO and three precursors, including 58 associated with TMAO, 29 associated with choline, 44 associated with betaine, and 29 associated with carnitine, were selected. MR results suggested that a 1 unit increase in TMAO should be associated with a 1SD increase in systolic BP mmHg (beta: 0.039, SE, 0.072, p = 0.020). Additionally, our findings also indicated that a 1 unit increase in carnitine should be associated with a 1SD increase in systolic BP mmHg (beta: 0.055, SE: 0.075, p = 0.039). This result was also confirmed by sensitivity analysis methods such as Maximum likelihood, MR-PRESSO, and Weighted median. No effects of betaine or choline on systolic or diastolic BP were observed in the present study.ConclusionOur study provides evidence of a causal relationship of TMAO and its precursors with BP, suggesting that mediating the generation of TMAO would be beneficial for lowering BP.

Experimental Periodontitis Deteriorated Atherosclerosis Associated With Trimethylamine N-Oxide Metabolism in Mice.

BackgroundPeriodontitis is considered a risk factor for atherosclerosis, but the mechanism is not clear. It was reported that oral administration of Porphyromonas gingivalis altered the gut microbiota in mice. Gut dysbiosis and the intestinal metabolite trimethylamine N-oxide (TMAO) were verified to be associated with atherosclerosis. Therefore, the possible TMAO-related mechanism between periodontitis and atherosclerosis needs to be explored.MethodsExperimental periodontitis was established by oral administration of P. gingivalis for 2 months in ApoE−/− mice. Mouse hemi-mandibles were scanned using Micro-CT. Quantification of TMAO was performed using liquid chromatography–tandem mass spectrometry. Mouse feces were collected and the bacterial DNA was extracted, then the gut microbiota was analyzed using 16S rRNA genes. Atherosclerotic lesion areas were quantified. Livers, small intestines, and large intestines were analyzed for gene expression.ResultsAggravated atherosclerosis plaques were found in experimental periodontitis mice. Plasma TMAO, a pathogenic factor of atherosclerosis, was initially found to be increased in periodontitis mice. Changes in the composition and abundance of the intestinal microflora of periodontitis mice were found. Flavin monooxygenase 3 (FMO3), the catalyzing enzyme of TMAO in the liver, was significantly increased, accompanied by an increase of IL-6 in liver, the abnormal intestinal integrity and enhanced plasma LPS. The IL-6 and LPS were verified to be able to increase FMO3 in HepG2 cells.ConclusionOur research discovered that experimental periodontitis in ApoE−/− mice induced gut dysbiosis and an increase in TMAO. These results suggest a possible mechanism by which periodontitis may accelerate atherosclerosis by influencing the intestinal microbes and the metabolism, which were triggered by inflammation of the liver and intestine.

Changes in gut-microbiota-related metabolites and long-term improvements in lipoprotein subspecies in overweight and obese adults: the POUNDS lost trial.

Background/Objectives:Alterations in gut microbiota have been linked to obesity and impaired lipid metabolism. Lipoproteins are heterogeneous, and lipoprotein subspecies containing apolipoprotein C-III (apoCIII) have adverse associations with obesity and related cardiometabolic abnormalities. We investigated associations of weight-loss diet-induced decreases in atherogenic gut-microbial metabolites, trimethylamine N-oxide (TMAO) and L-carnitine, with improvements in atherogenic lipoproteins containing apoCIII among patients with obesity.Subjects/Methods:This study included overweight and obese adults who participated in a 2-year weight-loss dietary intervention, the POUNDS Lost trial. Blood levels of TMAO and L-carnitine were measured at baseline and 6 months after the intervention; 6-month changes in the metabolites were calculated. We evaluated 2-year changes in lipid profiles (n=395) and cholesterol [Chol] in lipoprotein (very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL)) subfractions defined by the presence or absence of apoCIII (n=277).Results:The initial (6-month) decrease in L-carnitine was significantly associated with long-term (2-year) reductions in non-HDL-Chol and LDL-Chol (p < 0.05). Also, the decrease in L-carnitine was significantly related to decreases in Chol in LDL with apoCIII (p=0.034) and Chol in [LDL + VLDL] with apoCIII (p=0.018). We found significant interactions between dietary fat and TMAO on changes in LDL-Chol (Pinteraction=0.013) and Chol in [LDL + VLDL] with apoCIII (Pinteraction=0.0048); a greater increase in TMAO was related to lesser improvements in the lipoprotein outcomes if participants consumed a high-fat compared to a low-fat diet.Conclusions:Changes in TMAO and L-carnitine induced by weight-loss diets were associated with long-term improvements in atherogenic lipoproteins containing apoCIII, implicating that these metabolic changes might be predictive of an individual’s response to the dietary treatment to modify the unfavorable lipid profiles in obese patients. Dietary fat intake might modify associations of TMAO changes with long-term improvements of atherogenic cholesterol metabolism in overweight and obese adults.

Fasting and postprandial trimethylamine N-oxide in sedentary and endurance-trained males following a short-term high-fat diet.

Gut bacteria release trimethylamine (TMA) from dietary substrates. TMA is absorbed and is subsequently oxidized in the liver to produce trimethylamine N‐oxide (TMAO). Plasma TMAO levels are positively correlated with risk for type 2 diabetes (T2D) and cardiovascular disease (CVD). High‐fat diet (HFD) consumption has been reported to increase fasting and postprandial TMAO in sedentary individuals. However, whether the increase in TMAO with consumption of an HFD is observed in endurance‐trained males is unknown. Healthy, sedentary (n = 17), and endurance‐trained (n = 7) males consumed a 10‐day eucaloric diet comprised of 55% carbohydrate, 30% total fat, and <10% saturated fat prior to baseline testing. Blood samples were obtained in a fasted state and for a 4‐hour high‐fat challenge (HFC) meal at baseline and then again following 5‐day HFD (30% carbohydrate, 55% total fat, and 25% saturated fat). Plasma TMAO and TMA‐moiety (choline, betaine, L‐carnitine) concentrations were measured using isocratic ultraperformance liquid chromatography‐tandem mass spectrometry. Age (23 ±3 vs. 22 ± 2 years) and body mass index (23.0 ± 3.0 vs. 23.5 ± 2.1 kg/m2) were similar (both p > 0.05) in the sedentary and endurance‐trained group, respectively. VO2max was significantly higher in the endurance‐trained compared with sedentary males (56.7 ± 8.2 vs. 39.9 ± 6.0 ml/kg/min). Neither the HFC nor the HFD evoked a detectable change in plasma TMAO (p > 0.05) in either group. Future studies are needed to identify the effects of endurance training on TMAO production.

Metagenomic analysis of gut microbiota reveals its role in trimethylamine metabolism in heart failure

BackgroundWe had previously reported an increase in trimethylamine N-oxide (TMAO) levels in patients with both compensated and decompensated heart failure (HF) and alteration in gut microbiota composition using 16S rRNA gene amplicon analysis. Although a metagenome-wide analysis showed that choline-TMA lyase levels increased in HF patients, which TMA generation pathway from choline, carnitine, or betaine contributes to the increase in TMAO levels in HF needs to be elucidated. MethodsWe conducted a metagenome-wide shotgun sequencing analysis of gut microbiota and measured the TMAO levels in plasma of 22 HF patients during the compensated phase and 11 age-, sex-, and comorbidity-matched control subjects, whose gut microbiota compositions were reported in a previous 16S rRNA-based analysis. ResultsThe abundance of cntA/B was positively correlated with TMAO, especially in HF patients, whereas that of cutC/D or betaine reductase was not correlated either in controls or HF patients. The abundance of cntA/B was mainly derived from the genera Escherichia and Klebsiella either in controls or HF patients. ConclusionTMAO levels in plasma depend on the abundance of cntA/B in HF. Although it is difficult to exclude the involvement of confounding factors, microbial dysbiosis connecting the abundance of cntA/B in the gut and the increase of TMAO in plasma can be a therapeutic target for HF.

Gut-microbe derived TMAO and its association with more progressed forms of AF: Results from the AF-RISK study.

IntroductionThe importance of gut microbiome in cardiovascular disease has been increasingly recognized. Trimethylamine N-oxide (TMAO) is a gut microbe-derived metabolite that is associated with cardiovascular disease, including atrial fibrillation (AF). The role of TMAO in clinical AF progression however remains unknown.Methods and resultsIn this study we measured TMAO and its precursor (betaine, choline, and L- carnitine) levels in 78 patients using plasma samples from patients that participated in the AF-RISK study. 56 patients suffered from paroxysmal AF and 22 had a short history of persistent AF. TMAO levels were significantly higher in patients with persistent AF, as compared to those with paroxysmal AF (median [IQR] 5.65 [4.7–9.6] m/z versus 4.31 [3.2–6.2] m/z, p < 0.05), while precursor levels did not differ. In univariate analysis, we observed that for every unit increase in TMAO, the odds for having persistent AF increased with 0.44 [0.14–0.73], p < 0.01. Conclusion: These results suggest that higher levels of TMAO are associated with more progressed forms of AF. We therefore hypothesize that increased TMAO levels may reflect disease progression in humans. Larger studies are required to validate these preliminary findings.Trial Registration number: Clinicaltrials.gov NCT01510210.

Dietary Choline Supplements, but Not Eggs, Raise Fasting TMAO Levels in Participants with Normal Renal Function: A Randomized Clinical Trial

BackgroundCholine is a dietary precursor to the gut microbial generation of the prothrombotic and proatherogenic metabolite trimethylamine-N-oxide (TMAO). Eggs are rich in choline, yet the impact of habitual egg consumption on TMAO levels and platelet function in human subjects remains unclear. MethodsHealthy volunteers (41% male, 81% Caucasian, median age 28 years) with normal renal function (estimated glomerular filtration rate >60) were recruited and assigned to 1 of 5 daily interventions for 4 weeks: 1) hardboiled eggs (n = 18); 2) choline bitartrate supplements (n = 20); 3) hardboiled eggs + choline bitartrate supplements (n = 16); 4) egg whites + choline bitartrate supplements (n = 18); 5) phosphatidylcholine supplements (n = 10). Fasting blood and urine samples were collected for quantification of TMAO, its precursors, and platelet aggregometry. ResultsParticipants’ plasma TMAO levels increased significantly in all 3 intervention arms containing choline bitartrate (all P < .0001), but daily ingestion of 4 large eggs (P = .28) or phosphatidylcholine supplements (P = .27) failed to increase plasma TMAO levels. Platelet reactivity also significantly increased in the 3 intervention arms containing choline bitartrate (all P < .01), but not with eggs (P = .10) or phosphatidylcholine supplements (P = .79). ConclusionsDespite high choline content in egg yolks, healthy participants consuming 4 eggs daily showed no significant increase in TMAO or platelet reactivity. However, choline bitartrate supplements providing comparable total choline raised both TMAO and platelet reactivity, demonstrating that the form and source of dietary choline differentially contributes to systemic TMAO levels and platelet responsiveness.

FECAL METABOLOMIC ANALYSIS OF SERT DEFICIENT MICE UNDER BASAL CONDITIONS AND CHRONIC COLITIS

Abstract Inflammatory bowel diseases (IBDs) are chronic inflammatory multifactorial diseases caused by genetic, immune, and environmental factors. A decrease in intestinal serotonin transporter (SERT), which controls the extracellular availability of serotonin (5-HT) has been implicated in IBD. We previously showed that SERT deletion in mice altered gut bacterial community structure. The gut microbiota-derived metabolites are functional intermediaries between the microbiota and host. Here, we investigated the impact of SERT deficiency on gut metabolites under basal conditions and chronic colitis mimicking human IBD. Methods: Global metabolic profiles were analyzed by “Metabolon” (Durham, NC) on fecal samples from wild type littermates (WT) and SERT KO mice given water or chronic DSS (2.5% DSS for 5 weeks, n=7–9/group). Data were analyzed by ANOVA contrasts (difference between groups) and by two-way ANOVA (P&amp;lt;0.05, q&amp;lt;0.01). Results: SERT KO exhibited more severe colitis vs WT as assessed by histological score, myeloperoxidase activity and colon length. There was more pronounced decrease in the mRNA of tight junction proteins (TJs) occludin-1 and ZO-1 in SERT KO DSS vs WT DSS intestine. Metabolic profiling revealed that SERT deficiency alone resulted in extremely low levels of fecal ectoine, a bacterial derived solute that maintains TJ proteins expression. DSS treatment of WT (but not SERT KO) resulted in a significant increase in microbial derived metabolites including phenylalanine, N-acetylphenylalanine, tyrosine derivatives, glutamate, glutamine and benzoate derivatives. An increase in Trimethylamine N-oxide (TMAO), the short chain fatty acids butyrate/isobutyrate, TCA cycle metabolites; and a decrease in several metabolites including spermidine and various primary and secondary bile acids occurred in DSS treated WT and SERT KO to varying degrees; suggesting that these pathways may contribute to the colitis severity in SERT KO mice. Several secondary bile acids, ketone bodies, the metabolites of pterin, riboflavin pathway (FAD and FMN) and fatty acid metabolism pathways were increased in SERT KO (basal and DSS) suggesting genotype related differences in microbial community. We recently showed an impairment of Aryl hydrocarbon receptor (AhR), an IBD susceptible gene, in SERT KO mice, which could be partly due to altered availability of ligands. Indeed, the bacterial derived AhR ligand tryptamine was extremely low in SERT KO (basal and DSS). DSS increased the host derived AhR ligand, kynurenine in WT, but not in SERT KO.Conclusion:These data highlight the impact of serotonergic machinery and SERT inhibition on host physiology and pathophysiology of IBD. The results provide unique insights into gut bacteria derived metabolites and may aid in the development of novel treatment for disorders with altered SERT and 5-HT availability (Supported by CCFA and NIH).

Gut microbiota-associated trimethylamine N-oxide and increased cardiometabolic risk in adults: a systematic review and dose-response meta-analysis.

Several studies suggest a relationship between trimethylamine N-oxide (TMAO) concentrations and increased cardiometabolic risk, but findings are controversial. The aim of this systematic review and meta-analysis was to summarize evidence of the relationship between circulating TMAO levels and risk of hypertension and increased serum lipids in a dose-response and 2-class meta-analysis of discrete and continuous variables. PubMed, Scopus, Cochrane, and ProQuest databases were searched. Observational studies that reported disease status of participants (≥ 18 years), type of sample in which TMAO was measured (serum or plasma), and results based on at least 2 categories of TMAO concentrations, including relative risks, hazard ratios, or odds ratios with 95%CIs for cardiometabolic risk factors in association with circulating TMAO levels were selected. Papers were reviewed independently by both authors. The Newcastle-Ottawa Scale was used to assess the quality of included studies. The following data were extracted: first author's name, publication year, study design, study location, demographic information of participants, and concentrations of circulating TMAO. Eighteen studies were included in the meta-analysis. There was a dose-response relationship between circulating TMAO and increased odds of hypertension in cohort studies (P for nonlinearity = 0.049), in plasma-derived TMAO samples (P for nonlinearity = 0.043), in patients with cardiovascular disease (P for nonlinearity = 0.048), and in apparently healthy individuals from community-based studies (P for nonlinearity = 0.005). Moreover, the highest category of TMAO concentrations was associated with a 2.36 mmHg increase in systolic blood pressure when compared with the lowest category. The dose-response meta-analysis of continuous variables revealed that an increase in TMAO is associated with reduced high-density lipoprotein cholesterol in apparently healthy individuals and reduced high-density lipoprotein cholesterol and increased total cholesterol in patients with cardiovascular disease. Circulating TMAO is positively associated with an increased risk of hypertension and other cardiometabolic disorders in adults. PROSPERO identification number CRD42019138296.

Abstract 16549: Trimethylamine N-Oxide-Derived Endothelial Microvesicles Induce a Dysfunctional Endothelial Cell Phenotype

Introduction: Gut microbiota-generated increases in trimethylamine N-oxide (TMAO) has been linked to atherosclerosis and major adverse cardiovascular events. Experimental and clinical studies suggest that TMAO impairs endothelial cell function resulting in a proatherogenic endothelial phenotype. Endothelial microvesicles (EMVs) are anucleate vesicles shed constitutively by the endothelium aiding in cell-to-cell communication, activate repair or defense mechanisms, and/or stimulate immune responses. Under pathologic conditions, however, EMVs are released in greater number and their functional phenotype is more likely to evoke dysfunctional cellular effects. The experimental aims of this study were to determine: 1) if TMAO stimulates EMV release from endothelial cells in vitro ; and 2) the effects of TMAO-generated EMVs on endothelial cell inflammation, apoptosis, autophagy and nitric oxide (NO) production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with TMAO (100 μmol) for 24 h. EMVs released into the supernatant from cells treated with TMAO or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were treated with either TMAO-derived or control EMVs for 24 h. Results: EMV release was significantly higher in cells treated with TMAO compared with control (55±3 vs 28±3 EMV/μL). TMAO-generated EMVs induced significantly higher release of interleukin (IL)-6 (33.9±2.6 vs 20.2±1.3 pg/mL) and IL-8 (45.4±2.6 vs 33.1±1.6 pg/mL) and active NF-κB p65 (Ser536) (17.4±1.3 vs 7.7±1.0 AU) expression than control EMVs. TMAO EMVs significantly increased cell expression of apoptotic proteins caspase-9 (198.5±19.4 vs 119.7±11.2 AU) and active caspase-3 (17.7±2.8 vs 11.3±0.7 AU) and markedly depressed eNOS activity (10.6±0.9 vs 6.9±0.6 AU) and NO production (7.7±0.4 vs 5.5±0.6 μmol/L). In addition, cell autophagy was dysregulated by TMAO EMVs; cell expression of Beclin-1 (53.8±6.4 vs 36.6±3.6 AU), p62 (34.0±0.8 vs 16.9±1.2 AU) and LC3BII/LC3BI (15.0±0.1 vs 7.5±0.8 AU) were significantly elevated in cells treated with TMAO vs control EMVs. Conclusions: TMAO-generated EMVs adversely affect major functional characteristics of endothelial cells potentially contributing to the proatherogenic profile of TMAO.

Abstract P205: Long-term Changes in Microbiota-dependent Metabolite Trimethylamine N-oxide and Risks of Hypertension and Coronary Heart Disease, and Their Modulation by Diet: The Nurses' Health Study and the POUNDS Lost Trial

Introduction: A gut microbiota-related metabolite, trimethylamine N-oxide (TMAO) has been related to risks of cardiovascular events, however, whether changes in TMAO may contribute to the incidence of hypertension remains unknown. Gut microbiome may be affected by sodium intake and dietary patterns linked to blood pressure control, while little is known about whether these dietary factors may modify associations between TMAO changes and risks of hypertension and cardiovascular events. Hypothesis: To test whether long-term changes in TMAO are associated with increased risks of hypertension and coronary heart disease (CHD), and whether sodium intake and adherence to the DASH (Dietary Approaches to Stop Hypertension) dietary pattern may modulate these associations. Methods: A prospective nested case-control study was performed in 730 women (including 362 incident cases of fatal CHD and nonfatal myocardial infarction). We measured blood levels of TMAO at 2 time points (in 1989-90 and 2000-01), and prospectively examined associations of 10-year changes in TMAO with CHD events after the date of the second blood collection (through 2016). For the hypertension outcome, we assessed prevalent hypertension at 2nd time point among total participants, as well as the incidence of hypertension over 10 years (between 1989-90 and 2000-01) among people who were initially free of hypertension (n=410). In overweight and obese participants (n=429) from a randomized dietary intervention, we also investigated whether diet-induced changes in TMAO were related to blood pressure changes considering 24-hr urinary sodium excretion as a marker of sodium intake. Results: Elevated TMAO levels were significantly associated with hypertension at the 2nd time point (relative risk (RR): 1.30 (95% CI: 1.09, 1.56) per 1 SD increment). Also, greater 10-year increases in TMAO were significantly associated with increased risks of incident hypertension and CHD, with evidence of significant modification by adherence to the DASH diet (P-interaction =0.02 for hypertension; P-interaction =0.01 for CHD). Among people with lower adherence to the DASH diet (i.e., having unhealthy dietary patterns and higher sodium intake), every 1 SD increase in TMAO was associated with a RR of 2.13 (1.30, 3.49) for hypertension, and a RR of 2.03 (1.42, 2.89) for CHD. Such significantly increased risks of hypertension and CHD by TMAO increases were not found for women with higher adherence to the DASH diet. In a dietary intervention, we also found similar associations; diet-induced changes in TMAO levels with blood pressure changes were modified by urinary sodium excretion. Conclusion: Increases in TMAO, an atherogenic microbial metabolite, were associated with higher risks of hypertension and CHD among women, and heart-healthy dietary patterns and sodium intake modified the unfavorable associations of TMAO with the risks of these diseases.

Long-Term Changes in Gut Microbial Metabolite Trimethylamine N-Oxide and Coronary Heart Disease Risk

BackgroundA gut-microbial metabolite, trimethylamine N-oxide (TMAO), has been associated with coronary atherosclerotic burden. No previous prospective study has addressed associations of long-term changes in TMAO with coronary heart disease (CHD) incidence. ObjectivesThe purpose of this study was to investigate whether 10-year changes in plasma TMAO levels were significantly associated with CHD incidence. MethodsThis prospective nested case-control study included 760 healthy women at baseline. Plasma TMAO levels were measured both at the first (1989 to 1990) and the second (2000 to 2002) blood collections; 10-year changes (Δ) in TMAO were calculated. Incident cases of CHD (n = 380) were identified after the second blood collection through 2016 and were matched to controls (n = 380). ResultsRegardless of the initial TMAO levels, 10-year increases in TMAO from the first to second blood collection were significantly associated with an increased risk of CHD (relative risk [RR] in the top tertile: 1.58 [95% confidence interval (CI): 1.05 to 2.38]; RR per 1-SD increment: 1.33 [95% CI: 1.06 to 1.67]). Participants with elevated TMAO levels (the top tertile) at both time points showed the highest RR of 1.79 (95% CI: 1.08 to 2.96) for CHD as compared with those with consistently low TMAO levels. Further, we found that the ΔTMAO-CHD relationship was strengthened by unhealthy dietary patterns (assessed by the Alternate Healthy Eating Index) and was attenuated by healthy dietary patterns (p interaction = 0.008). ConclusionsLong-term increases in TMAO were associated with higher CHD risk, and repeated assessment of TMAO over 10 years improved the identification of people with a higher risk of CHD. Diet may modify the associations of ΔTMAO with CHD risk.

Relationship between elevated plasma trimethylamine N-oxide levels and increased stroke injury.

To investigate whether elevated plasma trimethylamine N-oxide (TMAO) levels are associated with initial stroke severity and infarct volume. This cross-sectional study included 377 patients with acute ischemic stroke and 50 healthy controls. Plasma TMAO levels were assessed at admission. Stroke infarct size and clinical stroke severity were measured with diffusion-weighted imaging and the NIH Stroke Scale (NIHSS). Mild stroke was defined as an NIHSS score <6. Plasma TMAO levels were higher in patients with ischemic stroke than in healthy controls (median 5.1 vs 3.0 μmol/L; p < 0.001). Every 1-µmol/L increase in TMAO was associated with a 1.13-point increase in NIHSS score (95% confidence interval [CI] 1.04-1.29; p < 0.001) and 1.69-mL increase in infarct volume (95% CI 1.41-2.03; p < 0.001) after adjustment for vascular risk factors. At admission, 159 patients (42.2%) had experienced a mild stroke, and their plasma TMAO levels were lower compared to those with moderate to severe stroke (median 3.6 vs 6.5 µmol/L; p < 0.001). The area under the receiver operating characteristics curve of plasma TMAO level in predicting moderate to severe stroke was 0.794 (95% CI 0.748-0.839; p < 0.001), and the optimal cutoff value was 4.95 μmol/L. The sensitivity and specificity of TMAO levels ≥4.95 μmol/L for moderate to severe stroke were 70.2% and 79.9%, respectively. Patients with ischemic stroke had higher plasma TMAO levels compared to healthy controls. Higher plasma TMAO level at admission is an independent predictor of stroke severity and infarct volume in patients with acute ischemia.

Increased Trimethylamine N-Oxide Is Not Associated with Oxidative Stress Markers in Healthy Aged Women

Increased plasma trimethylamine N-oxide (TMAO) levels have been associated with cardiovascular diseases (CVD). L-carnitine induces TMAO elevation in human blood, and thus, it has been suggested as developing atherosclerosis. The aim of this study was to determine the relation between selected markers of oxidative stress and plasma TMAO concentration induced by L-carnitine supplementation for 24 weeks in healthy aged women. Twenty aged women were supplemented during 24 weeks with either 1500 mg L-carnitine-L-tartrate (n = 11) or isonitrogenous placebo (n = 9) per day. Fasting blood samples were taken from antecubital vein. L-carnitine supplementation induced an increase in TMAO, but not in γ-butyrobetaine (GBB). Moreover, there were no significant changes in serum ox-LDL, myeloperoxidase, protein carbonyls, homocysteine, and uric acid concentrations due to supplementation. Significant reduction in white blood cell counts has been observed following 24-week supplementation, but not attributable to L-carnitine. Our results in healthy aged women indicated no relation between TMAO and any determined marker of oxidative stress over the period of 24 weeks. At the same time, plasma GBB levels were not affected by L-carnitine supplementation. Further clinical studies of plasma GBB level as a prognostic marker are needed.

Fasting and Postprandial Trimethylamine N‐oxide in Sedentary and Endurance Trained Males

BackgroundGut bacteria release trimethylamine (TMA) from dietary substrates, e.g., choline, phosphatidylcholine, and L‐carnitine. TMA is absorbed and subsequently oxidized in the liver to produce trimethylamine N‐oxide (TMAO). TMAO is associated with type 2 diabetes (T2D) and increased risk of cardiovascular disease. Consumption of a high fat diet (HFD) is associated with increase in TMAO in sedentary individuals. However, whether the increase in TMAO with consumption in a HFD is observed in endurance trained individuals is unknown.MethodsHealthy, sedentary (n=17) and endurance trained (n=7) males consumed a 10‐day isocaloric lead‐in diet comprised of 55% carbohydrate, 30% total fat, &lt;10% saturated fat prior to baseline testing. Blood samples were obtained in the fasting state and every hour during a 4‐hour high fat challenge (HFC) meal (820 kcal; 25% carbohydrate, 63% fat [21% saturated fat]) at baseline and following 5‐day HFD (30% carbohydrate, 55% total fat, 25% saturated fat). Plasma TMAO and TMA‐moieties (choline, betaine, L‐carnitine) were measured using isocratic ultraperformance liquid chromatography‐tandem mass spectrometry.ResultsAge (23±3 vs. 22±2.1 yrs) and body mass index (23.4± 3.0 vs. 23.5±2.1 kg/m2) were similar (both P&gt;0.05) in the sedentary and endurance trained group, respectively. As expected, VO2max was significantly higher in the endurance trained compared with sedentary individuals (56.7±8.2 vs. 39.3±5.8 ml/kg/min). There were no significant differences in fasting (1.92 ± 0.8 μM vs. 2.09 ± 1.1 μM, P&gt;0.05) or postprandial TMAO between sedentary or trained individuals, respectively, at baseline. Neither fasting (1.49± 1.2 μM vs. 2.25 ± 1.4 μM, P&gt;0.05) or postprandial TMAO changed significantly with the HFD in the sedentary and endurance training individuals.ConclusionsNeither fasting nor postprandial TMAO changed significantly following the HFD in the endurance trained compared to sedentary individuals in the present study. Future studies are needed to identify effective interventions that target TMA‐releasing bacteria and reduce TMAO.Support or Funding InformationAmerican Diabetes Association and Translation Obesity Research Interdisciplinary Graduate Education ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 019: Changes in Gut Microbial Metabolites and Risk of Coronary Heart Disease in US Women: the Nurses’ Health Study

Introduction: Circulating levels of gut microbial metabolites, trimethylamine N-oxide (TMAO) and its nutrient precursors of choline and L-carnitine, have been suggested as risk factors for coronary heart disease (CHD). Whether changes in gut microbial metabolites are associated with the CHD incidence remains unknown. Hypothesis: We assessed the hypothesis that long-term (over 10 years) changes in plasma levels of TMAO, choline, and L-carnitine would be associated with the risk of CHD. Methods: This prospective nested case-control study included a total of 768 women (385 incident cases of fatal CHD and nonfatal myocardial infarction and 383 controls) who had data on blood concentrations of TMAO, choline, and L-carnitine at 2 time points, 10 years apart (1989-90, and 2000-01). We identified incident cases of CHD from the date of the second blood collection through 2014. Results: Higher TMAO levels at the second collection were associated with higher risk of CHD (relative risk (RR) 1.22 [95% CI: 1.05, 1.42] per 1 SD increment in log-transformed TMAO) in multivariate analyses controlling for traditional risk factors. Compared with women with low TMAO levels (lower two tertiles) at both time points, the RR was 1.74 (95% CI: 1.05, 2.89) for those with elevated levels of TMAO (the highest tertile) at both time points. Regardless of metabolite levels at the first collection, increases in TMAO or L-carnitine from the first to second collections were significantly associated with an increased risk of CHD (Fig. panels A-B). Women who had increases in both TMAO and L-carnitine had a particularly elevated risk of CHD (RR 2.12, 95% CI: 1.25, 3.59), as compared to those with decreases in both metabolites. Adherence to healthy dietary habits attenuated the associations of TMAO with CHD risk (Fig. panel C). Conclusions: Long-term increases in TMAO were significantly predictive of subsequent risk for CHD among women. The associations may be modified by the nutrient metabolite precursor, L-carnitine, and adherence to healthy dietary habits.