Plasma Trimethylamine N-oxide Concentrations Research Articles

Plasma concentration of trimethylamine-N-oxide and risk of gestational diabetes mellitus

The microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) has been reported as a novel and independent risk factor for the development of cardiovascular and metabolic diseases, but the association with gestational diabetes mellitus (GDM) remains unclear. The aim of this study was to investigate the association between plasma TMAO concentration and GDM in a 2-phase study. A 2-phase design was used in the current study. An initial phase included 866 participants (433 GDM cases and 433 matched controls) with fasting blood samples collected at the time of GDM screening (24-32 wk of gestation). An independent-phase study, with 276 GDM cases and 552 matched controls who provided fasting blood samples before 20 wk of gestation and who had GDM screened during 24-32 wk of gestation, was nested within a prospective cohort study. These 2 studies were both conducted in Wuhan, China, and the incidence of GDM in the cohort study was 10.8%. Plasma TMAO concentrations were determined by stable isotope dilution liquid chromatography-tandem mass spectrometry. GDM was diagnosed according to the American Diabetes Association criteria by using an oral-glucose-tolerance test. In the initial case-control study, the adjusted OR of GDM comparing the highest TMAO quartile with the lowest quartile was 1.94 (95% CI: 1.28, 2.93). Each SD increment of ln-transformed plasma TMAO was associated with 22% (95% CI: 5%, 41%) higher odds of GDM. In the nested case-control study, women in the highest quartile also had increased odds of GDM (adjusted OR: 2.06; 95% CI: 1.28, 3.31) compared with women in the lowest quartile, and the adjusted OR for GDM per SD increment of ln-transformed plasma TMAO was 1.26 (95% CI: 1.08, 1.47). Consistent findings from this 2-phase study indicate a positive association between plasma TMAO concentrations and GDM. Future studies are warranted to elucidate the underlying mechanisms. This trial was registered at www.clinicaltrials.gov as NCT03415295.

Plasma trimethylamine-N-oxide and related metabolites are associated with type 2 diabetes risk in the Prevención con Dieta Mediterránea (PREDIMED) trial

The role of trimethylamine-N-oxide (TMAO) in type 2 diabetes (T2D) is currently partially understood and controversial. The aim of this study was to investigate associations between TMAO and related metabolites with T2D risk in subjects at high risk of cardiovascular disease. This is a case-cohort design study within the Prevención con Dieta Mediterránea (PREDIMED) study, with 251 incident T2D cases and a random sample of 694 participants (641 noncases and 53 overlapping cases) without T2D at baseline (median follow-up: 3.8 y). We used liquid chromatography-tandem mass spectrometry to measure plasma TMAO, l-carnitine, betaine, lyso-phosphatidylcholine (LPC) and lyso-phosphatidylethanolamine (LPE) species, phosphocholine, α-glycerophosphocholine, and choline at baseline and after 1 y. We examined associations with the use of weighted Cox proportional hazard models, accounting for the weighted case-cohort design by the Barlow method. After adjustment for recognized T2D risk factors and multiple testing, individuals in the highest quartile of baseline TMAO and α-glycerophosphocholine had a lower risk of T2D [HR (95% CI): 0.52 (0.29, 0.89) and 0.46 (0.24, 0.89), respectively]. The HR (95% CI) comparing the extreme quartiles of betaine was 0.41 (0.23, 0.74). Similar trends were observed for C16:0 LPC, C18:1 LPC, C18:0 LPC, C20:4 LPC, C22:6 LPC, C18:1 LPC plasmalogen, and C16:0 LPE. After correcting for multiple comparisons, participants in the highest quartile of 1-y changes in oleic acid LPC plasmalogen concentrations had a lower T2D risk than the reference quartile. Whether the associations between plasma TMAO and certain metabolite concentrations with T2D risk reflect its pathophysiology or represent an epiphenomenon needs to be elucidated. This trial is registered at http://www.controlled-trials.com as ISRCTN35739639.

Inulin Supplementation Does Not Reduce Plasma Trimethylamine N-Oxide Concentrations in Individuals at Risk for Type 2 Diabetes.

Trimethylamine N-oxide (TMAO) is associated with type 2 diabetes (T2DM) and increased risk of adverse cardiovascular events. Prebiotic supplementation has been purported to reduce TMAO production, but whether prebiotics reduce fasting or postprandial TMAO levels is unclear. Sedentary, overweight/obese adults at risk for T2DM (n = 18) were randomized to consume a standardized diet (55% carbohydrate, 30% fat) with 10 g/day of either an inulin supplement or maltodextrin placebo for 6 weeks. Blood samples were obtained in the fasting state and hourly during a 4-h high-fat challenge meal (820 kcal; 25% carbohydrate, 63% fat; 317.4 mg choline, 62.5 mg betaine, 8.1 mg l-carnitine) before and after the diet. Plasma TMAO and trimethylamine (TMA) moieties (choline, l-carnitine, betaine, and γ-butyrobetaine) were measured using isocratic ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). There were no differences in fasting or postprandial TMAO or TMA moieties between the inulin and placebo groups at baseline (all p > 0.05). There were no significant changes in fasting or postprandial plasma TMAO or TMA moiety concentrations following inulin or placebo. These findings suggest that inulin supplementation for 6 weeks did not reduce fasting or postprandial TMAO in individuals at risk for T2DM. Future studies are needed to identify efficacious interventions that reduce plasma TMAO concentrations.

Effects of Egg Consumption and Choline Supplementation on Plasma Choline and Trimethylamine-N-Oxide in a Young Population

ABSTRACTBackground: Plasma trimethylamine-N-oxide (TMAO) concentrations have been associated with cardiovascular disease risk. Eggs are a rich source of choline, which is a precursor of TMAO.Objective: The effects of egg intake versus daily choline supplementation were evaluated on plasma choline and TMAO in a young, healthy population.Methods: Thirty participants (14 males, 16 females; 25.6 ± 2.3 years; body mass index = 24.3 ± 2.9 kg/m2) were enrolled in this 13-week crossover intervention. After a 2-week washout, participants were randomized to consume either 3 eggs/d or a choline bitartrate supplement (∼ 400 mg choline total in eggs or supplement) for 4 weeks. Following a 3-week washout, participants were switched to the alternate treatment. Dietary records were measured at the end of each period. Plasma TMAO and choline were measured at baseline and at the end of each dietary intervention. Gene expression of scavenger receptors associated with plasma TMAO were quantified at the end of each intervention.Results: Compared to the choline supplement, intake of total fat, cholesterol, selenium, and vitamin E were higher (p < 0.05), whereas carbohydrate intake was lower (p < 0.001) with consumption of 3 eggs/d. Fasting plasma choline increased 20% (p = 0.023) with egg intake, while no changes were observed with choline supplementation. Plasma TMAO levels were not different between dietary treatments or compared to baseline.Conclusions: Dietary choline appears to be more bioavailable via egg consumption when compared to a choline supplement. Plasma TMAO concentrations were not affected in healthy participants after 4 weeks of taking ∼400 mg/d choline either via eggs or choline supplementation.

Marked elevation in plasma trimethylamine-N-oxide (TMAO) in patients with mitochondrial disorders treated with oral l-carnitine

Oral supplementation with l-carnitine is a common therapeutic modality for mitochondrial disorders despite limited evidence of efficacy. Recently, a number of studies have demonstrated that a gut microbiota-dependent metabolite of l-carnitine, trimethylamine oxide (TMAO), is an independent and dose-dependent risk factor for cardiovascular disease (CVD). Given the limited data demonstrating efficacy with oral l-carnitine therapy and the newly raised questions of potential harm, we assessed plasma TMAO levels in patients with mitochondrial disease with and without oral l-carnitine supplementation. Nine subjects were recruited and completed the study. Eight out of 9 subjects at baseline had plasma TMAO concentrations <97.5th percentile (<15.5 μM). One subject with stage 3 renal disease, had marked elevation in plasma TMAO (pre 33.98 μm versus post 101.6 μm). Following at least 3 months of l-carnitine supplementation (1000 mg per day), plasma TMAO levels were markedly increased in 7out of 9 subjects; overall, plasma TMAO significantly increased 11.8-fold (p < 0.001) from a baseline median level of 3.54 μm (interquartile range (IQR) 2.55–8.72) to 43.26 (IQR 23.99–56.04) post supplementation. The results of this study demonstrate that chronic oral l-carnitine supplementation markedly increases plasma TMAO levels in subjects with mitochondrial disorders. Further studies to evaluate both the efficacy and long term safety of oral l-carnitine supplementation for the treatment of mitochondrial disorders are warranted.

Human plasma concentrations of trimethylamine N-oxide extrapolated using pharmacokinetic modeling based on metabolic profiles of deuterium-labeled trimethylamine in humanized-liver mice.

Medicinal carnitine-derived and dietary-derived malodorous trimethylamine and its non-malodorous metabolite trimethylamine N-oxide were historically regarded as nontoxic. Clinical and toxicological interest has recently arisen because of their potential association with atherosclerosis. We previously reported a human physiologically based pharmacokinetic (PBPK) model for trimethylamine and its primary metabolite, trimethylamine N-oxide, based on reported rat trimethylamine pharmacokinetics. However, rats are poor metabolizers with respect to trimethylamine N-oxygenation, and this species difference was investigated in vitro using substrate depletion rates in rat and human liver microsomes. The current study investigated the pharmacokinetics of deuterium-labeled trimethylamine orally administered to immunodeficient humanized-liver mice transplanted with commercially available human hepatocytes. Trimethylamine N-oxide was extensively formed in vivo in humanized-liver mice, but not in control mice. The experimental pharmacokinetic data of deuterium-labeled trimethylamine and its N-oxide in humanized-liver mice were scaled up for application to a human PBPK model. The human plasma concentration curves generated by the resulting simple PBPK model were consistent with concentrations in humans reported in the literature. The model can also simulate human plasma levels of trimethylamine and trimethylamine N-oxide during treatment with the prescription medicine L-carnitine and in trimethylamine loading tests. The predicted plasma levels were in the ranges that occur under the consumption of daily dietary foodstuff; such levels are associated with few toxicological impacts. The present PBPK model for trimethylamine and trimethylamine N-oxide could estimate daily doses by both forward and reverse dosimetry and could facilitate risk assessment in humans.

Pressing the trimethylamine N-oxide narrative

We read with interest the correspondence titled “Gut Microbe-Generated Trimethylamine N-Oxide from Dietary Choline is Prothrombotic in Subjects”, published by Zhu et al . in the journal Circulation (1). This work builds on an emerging body of evidence which has suggested a role for the gut-derived metabolite, trimethylamine N -oxide (TMAO), in the development of cardiovascular disease (CVD). This current study is an important clinical test of hypotheses generated from innovative preclinical and observational evidence, and seeks to build upon the nature of TMAO’s relationship with CVD risk in humans. To assess this relationship, Zhu et al . enrolled 18 subjects (10 omnivores and 8 vegetarians) and provided choline bitartrate as a supplement (450 mg choline/day) for 2 months. Following choline supplementation, fasting plasma TMAO concentrations increased and were associated with increased ADP-induced platelet aggregation, an effect that was partially blunted by aspirin.

Trimethylamine N-oxide and platelets aggregation: insufficient evidence for causal inference in thrombosis

Trimethylamine N-oxide (TMAO) is an amine oxide generated in the liver from nutrients such as choline, betaine, or carnitine via an intermediate gut-bacteria driven metabolite, trimethylamine (TMA). Recently, Zhu et al . conducted a 2-month open-, non-placebo controlled intervention in vegetarians and omnivores using 450 mg total choline/day (1). Zhu et al . reported a significant increase in plasma TMAO concentrations (from 2.5 to 36.4 M in omnivores and from 2.6 to 27.2 M in vegetarians).

Trimethylamine N-Oxide, the Microbiome, and Heart and Kidney Disease.

Trimethylamine N-oxide (TMAO) is a biologically active molecule and is a putative promoter of chronic diseases including atherosclerosis in humans. Host intestinal bacteria produce its precursor trimethylamine (TMA) from carnitine, choline, or choline-containing compounds. Most of the TMA produced is passively absorbed into portal circulation, and hepatic flavin-dependent monooxygenases (FMOs) efficiently oxidize TMA to TMAO. Both observational and experimental studies suggest a strong positive correlation between increased plasma TMAO concentrations and adverse cardiovascular events, such as myocardial infarction, stroke, and death. However, a clear mechanistic link between TMAO and such diseases is not yet validated. Therefore, it is debated whether increased TMAO concentrations are the cause or result of these diseases. Here, we have tried to review the current understanding of the properties and physiological functions of TMAO, its dietary sources, and its effects on human metabolism. Studies that describe the potential role of TMAO in the etiology of cardiovascular and other diseases are also discussed.

Abstract 23: Gut Microbiota Related Plasma Metabolites and Risk of Cardiovascular Disease in the PREDIMED Study

Background: Metabolites associated with betaine and choline metabolism and the gut-microbiota-dependent metabolite trimethylamine N-oxide (TMAO) have been linked to the risk of cardiovascular disease (CVD). However, the relationship between plasma concentrations of other gut microbiota-related metabolites and major CVD endpoints remains unclear. Objectives: To evaluate the association between gut microbiota-related metabolites and risk of incident CVD and the potential modifying effect of Mediterranean diet (MedDiet) interventions. Methods: We designed a case-cohort study nested within the PREDIMED trial. We used liquid chromatography–tandem mass spectrometry to measure plasma gut microbiota-related metabolites. A score including the sum of quartile values of 8 metabolites was constructed (TMAO, betaine, choline, phosphocholine, alphaglycerophosphocholine, proline, hydroxyproline, allantoin). The primary outcome was a composite of myocardial infarction, stroke, and cardiovascular death. Blood samples from a randomly selected PREDIMED sub-cohort (n=751) and all available incident CVD cases (n=229) after 4.8-y of follow-up were included in the analysis. We used weighted Cox regression models to estimate multivariable-adjusted hazard ratios (HR) and their 95% confidence intervals (CI). Models were adjusted for age, sex, BMI, family history of premature heart disease, and smoking, physical activity (metabolic equivalent tasks in min/d), hypertension, dyslipidemia, diabetes and was stratified by intervention group. Results: Baseline plasma concentrations of choline and hydroxyproline were associated with higher CVD risk independent of traditional risk factors, while no significant association between plasma concentrations of TMAO and CVD was found. The HRs comparing extreme quartiles (lowest quartile as the reference) were 1.72 (95% CI: 1.05, 2.81; P for trend=0.01) and 1.65 (95% CI: 1.03, 2.65; P for trend =0.04), respectively. The microbiota metabolite score was associated with a 2.13-fold higher risk of CVD across extreme quartiles (95% CI: 1.32, 3.43; P for trend &lt;0.001) and a 1.99-fold higher risk of stroke (95% CI: 1.08, 3.65; P for trend=0.02). Baseline betaine/choline ratio was inversely associated with CVD. Compared to participants with a score below the median and randomized to the Mediterranean diet, the HR of developing CVD was 2.56 (95% CI: 1.59, 4.11) for participants with a gut microbiota score above the median and randomized to the control group. Conclusions: Plasma gut microbiota-related metabolites were associated with an increased risk of CVD in a Mediterranean population at high cardiovascular risk, independent of traditional CVD risk factors.

Intake of up to 3 Eggs/Day Increases HDL Cholesterol and Plasma Choline While Plasma Trimethylamine-N-oxide is Unchanged in a Healthy Population.

Eggs are a source of cholesterol and choline and may impact plasma lipids and trimethylamine-N-oxide (TMAO) concentrations, which are biomarkers for cardiovascular disease (CVD) risk. Therefore, the effects of increasing egg intake (0, 1, 2, and 3 eggs/day) on these and other CVD risk biomarkers were evaluated in a young, healthy population. Thirty-eight subjects [19 men/19 women, 24.1±2.2 years, body mass index (BMI) 24.3±2.5kg/m2] participated in this 14-week crossover intervention. Participants underwent a 2-week washout with no egg consumption, followed by intake of 1, 2, and 3 eggs/day for 4weeks each. Anthropometric data, blood pressure (BP), dietary records, and plasma biomarkers (lipids, glucose, choline, and TMAO) were measured during each intervention phase. BMI, waist circumference, systolic BP, plasma glucose, and plasma triacylglycerol did not change throughout the intervention. Diastolic BP decreased with egg intake (P<0.05). Compared to 0 eggs/day, intake of 1 egg/day increased HDL cholesterol (HDL-c) (P<0.05), and decreased LDL cholesterol (LDL-c) (P<0.05) and the LDL-c/HDL-c ratio (P<0.01). With intake of 2-3 eggs/day, these changes were maintained. Plasma choline increased dose-dependently with egg intake (P<0.0001) while fasting plasma TMAO was unchanged. These results indicate that in a healthy population, consuming up to 3 eggs/day results in an overall beneficial effect on biomarkers associated with CVD risk, as documented by increased HDL-c, a reduced LDL-c/HDL-c ratio, and increased plasma choline in combination with no change in plasma LDL-c or TMAO concentrations.

Intra-individual variation of plasma trimethylamine-N-oxide (TMAO), betaine and choline over 1 year.

Circulating trimethylamine-N-oxide (TMAO) has been implicated in the development of cardiovascular and chronic kidney diseases (CKD). However, while higher TMAO levels have been associated with increased risks of cardiovascular or renal events in first prospective studies, it remained unclear how much plasma TMAO concentrations vary over time. We measured fasting plasma levels of TMAO and two of its precursors, betaine and choline by LC-MS, in two samples of 100 participants of the European Investigation into Cancer and Nutrition (EPIC)-Heidelberg study (age range: 47-80 years, 50% female) that were collected 1 year apart, and assessed their intra-individual variation by Spearman's correlation coefficients (ρ). Correlations of metabolite concentrations over 1 year were at ρ=0.29 (p=0.003) for TMAO, ρ=0.81 (p<0.001) for betaine, and ρ=0.61 (p<0.001) for choline. Plasma levels of TMAO were not significantly associated with food intake, lifestyle factors, or routine biochemistry parameters such as C-reactive protein (CRP), low-density lipoprotein (LDL)-cholesterol, or creatinine. In contrast to fasting plasma concentrations of betaine and choline, concentrations of TMAO were more strongly affected by intra-individual variation over 1 year in adults from the general population. The modest correlation of TMAO levels over time should be considered when interpreting associations between TMAO levels and disease endpoints.

Trimethylamine-N-oxide

This article evaluates the link between trimethylamine-N-oxide (TMAO) and bile acids and the consequent impact on the development of atherosclerosis. Elevation in plasma TMAO concentrations is associated with an increased risk of cardiovascular disease in many different patient cohorts. In addition to the recently identified direct effects of TMAO on the development of atherosclerosis, other components involved in TMAO metabolism may also have an impact. Furthermore, the relationship between TMAO and bile acid regulation is emerging as a possible mediator of atherosclerosis. Studies that are emerging highlight the mechanistic relationship of TMAO to the development atherosclerosis in addition to its role as disease biomarker. The interplay between TMAO and bile acid metabolism mediated through multiple factors, such as the gut microbiome, farnesoid X receptor signaling, and flavin monooxygenase 3 activity may help identify another pathway by which atherosclerosis occurs. In this review, we discuss the most recent data regarding atherosclerosis, TMAO, and bile acid metabolism.

Plasma trimethylamine N-oxide concentration is associated with choline, phospholipids, and methyl metabolism1–3

AbstractBackground: Elevated plasma concentrations of the gut bacteria choline metabolite trimethylamine N-oxide (TMAO) are associated with atherosclerosis. However, the determinants of TMAO in humans require additional assessment.Objective: We examined cardiometabolic risk factors and pathways associated with TMAO concentrations in humans.Design: A total of 283 individuals (mean ± SD age: 66.7 ± 9.0 y) were included in this observational study. Plasma concentrations of trimethylamine, TMAO, choline, lipids, phospholipids, and methyl metabolites were measured.Results: Study participants were divided into 4 groups by median concentrations of TMAO and choline (4.36 and 9.7 μmol/L, respectively). Compared with the group with TMAO and choline concentrations that were less than the median (n = 82), the group with TMAO and choline concentrations that were at least the median (n = 83) was older and had lower high-density lipoprotein (HDL) cholesterol, phospholipids, and methylation potential, higher creatinine, betaine, S-adenosylhomocysteine (SAH), and S-adenosylmethionine (SAM), and higher percentages of men and subjects with diabetes. The difference in plasma TMAO concentrations between men and women (7.3 ± 10.0 compared with 5.4 ± 5.6 μmol/L, respectively) was NS after adjustment for age and creatinine (P = 0.455). The TMAO:trimethylamine ratio was higher in men (P < 0.001). Diabetes was associated with significantly higher plasma TMAO concentration (8.6 ± 12.2 compared with 5.4 ± 5.2 μmol/L) even after adjustments. Sex and diabetes showed an interactive effect on trimethylamine concentrations (P = 0.010) but not on TMAO concentrations (P = 0.950). Positive determinants of TMAO in a stepwise regression model that applied to the whole group were SAH, trimethylamine, choline, and female sex, whereas plasma phosphatidylcholine was a negative determinant.Conclusions: High TMAO and choline concentrations are associated with an advanced cardiometabolic risk profile. Diabetes is related to higher plasma TMAO concentrations but also to alterations in interrelated pathways such as lipids, phospholipids, and methylation. Elevated plasma TMAO concentrations likely reflect a specific metabolic pattern characterized by low HDL and phospholipids in addition to hypomethylation. This trial was registered at clinicaltrials.gov as NCT02586181 and NCT02588898.

Plasma Concentrations of Trimethylamine-N-oxide Are Directly Associated with Dairy Food Consumption and Low-Grade Inflammation in a German Adult Population

Background: Trimethylamine-N-oxide (TMAO) is a metabolite of carnitine, choline, and phosphatidylcholine, which is inversely associated with survival of cardiovascular disease (CVD) patients.Objective: We examined the associations of diet with plasma concentrations of TMAO, choline, and betaine and the associations of TMAO with plasma concentrations of various cytokines.Methods: Plasma TMAO, choline, and betaine concentrations were measured using LC-high resolution mass spectrometry in 271 participants, ≥18 y old, of the Second Bavarian Food Consumption Survey, conducted in 2002 and 2003. Food consumption was assessed using at least two 24-h dietary recalls. Cytokines were measured in plasma with enzyme-linked immunosorbent assays. Geometric mean concentrations of TMAO, choline, and betaine by categories of meat, dairy food, egg, and fish consumption were computed, adjusted for sex, age, and BMI. Multivariable-adjusted geometric mean concentrations of cytokines [tumor necrosis factor-α (TNF-α), soluble TNF receptors (sTNF-R) p55, sTNF-R p75, interleukin-6 (IL-6), and C-reactive protein (CRP)] were computed by quartiles of TMAO concentration using general linear models.Results: Meat, egg, or fish consumption was not associated with TMAO, choline, or betaine concentrations (all P-trend ≥ 0.05). With increases in milk and other dairy food consumption, the plasma TMAO concentration increased [geometric mean bottom quartile of milk consumption: 2.08 μM (95% CI: 1.69, 2.57 μM); compared with top quartile: 3.13 μM (95% CI: 2.56, 3.84 μM); P-trend = 0.008]. Participants in the top TMAO quartile had higher plasma concentrations of TNF-α, sTNF-R p55, and sTNF-R p75 than participants in the bottom quartile (percentage difference ranging between 14.4% and 17.3%; all P-trend < 0.05), but there were no differences in plasma concentrations of CRP and IL-6 (all P-trend ≥ 0.05).Conclusions: Results of this study conducted among healthy adults from the general population do not indicate a strong effect of diet on plasma concentrations of TMAO, choline, or betaine, with the exception of a positive association between dairy food consumption and plasma TMAO concentrations. Also, plasma TMAO concentrations were positively associated with inflammation. Whether habitual diet is strongly linked to the plasma TMAO concentration, a potential marker of CVD risk, needs to be determined in further studies.

Plasma levels of trimethylamine-N-oxide are confounded by impaired kidney function and poor metabolic control

BackgroundAfter ingestion of phosphatidylcholine, l-carnitine or betaine, trimethylamine-N-oxide (TMAO) is formed by gut microbiota and liver enzymes. Elevated TMAO plasma levels were associated with increased cardiovascular risk and other diseases. Also betaine and choline itself were recently associated with increased cardiovascular risk. MethodsA newly developed LC-HRMS method was applied to measure the plasma concentrations of TMAO, betaine and choline in a cohort of 339 patients undergoing coronary angiography for the evaluation of suspected coronary artery disease. ResultsBetaine concentrations in males were significantly higher than in females (42.0 vs. 35.9 μmol/L; p < 0.001). Plasma concentrations of TMAO but not of betaine or choline were higher in patients with diabetes compared to euglycemic patients (2.39 vs. 0.980 μmol/L; p = 0.001) as well as in patients with metabolic syndrome as compared to patients without metabolic syndrome (2.37 vs. 1.43 μmol/L; p = 0.002). Plasma concentrations of TMAO or choline increased significantly with decreasing renal function (Spearman's rho: −0.281; p < 0.001). However, plasma levels of TMAO or betaine were associated with neither a history of myocardial infarction nor the angiographically assessed presence of coronary heart disease, nor incident cardiovascular events during 8 years of follow-up. Plasma levels of choline were significantly lower in patients with a history of acute myocardial infarction as compared to those without such history (10.0 vs. 10.8 μmol/L; p = 0.045). ConclusionsPlasma levels of TMAO are confounded by impaired kidney function and poor metabolic control but are not associated with the history, presence or incidence of symptoms or events of coronary heart disease.

Probiotic supplementation and trimethylamine-N-oxide production following a high-fat diet.

The objective of this study was to test the hypothesis that the multi-strain probiotic VSL#3 would attenuate the increase in fasting plasma concentrations of trimethylamine-N-oxide (TMAO) following a high-fat diet. Nineteen healthy, non-obese males (18-30 years) participated in the present study. Following a 2-week eucaloric control diet, subjects were randomized to either VSL#3 (900 billion live bacteria) or placebo (cornstarch) during the consumption of a hypercaloric (+1,000 kcalday(-1) ), high-fat diet (55% fat) for 4 weeks. Plasma TMAO, L-carnitine, choline, and betaine (UPLC-MS/MS) were measured at baseline and following a high-fat diet. Plasma TMAO significantly increased 89% ± 66% vs. 115% ± 61% in both the VSL#3 and placebo groups, respectively; however, the magnitude of change in plasma TMAO was not different (P > 0.05) between them. Plasma L-carnitine, choline, and betaine concentrations did not increase following the high-fat diet in either group. A high-fat diet increases plasma TMAO in healthy, normal-weight, young males. However, VSL#3 treatment does not appear to influence plasma TMAO concentrations following a high-fat diet. Future studies are needed to determine whether other therapeutic strategies can attenuate the production of TMAO.

Short-term high-fat diet increases postprandial trimethylamine-N-oxide in humans

The gut microbiota plays an obligatory role in the metabolism of nutrients containing trimethylamine moieties, such as l-carnitine and choline, leading to the production of the proatherogenic trimethylamine-N-oxide (TMAO). We hypothesized that a short-term, high-fat diet would increase fasting and postprandial plasma concentrations of TMAO in response to a high-fat meal challenge. Following a 2-week eucaloric control diet, 10 nonobese men (18-30 years) consumed a eucaloric, high-fat diet (55% fat) for 5 days. Plasma TMAO was measured after a 12-hour fast and each hour after for 4 hours following a high-fat meal (63% fat) at baseline and after the high-fat diet using ultraperformance liquid chromatography/ tandem mass spectrometry. Fasting plasma TMAO did not increase significantly following the high-fat diet (1.83 ± 0.21 vs 1.6 ± 0.24 μmol/L). However, plasma TMAO was higher at hour 1 (2.15 ± 0.28 vs 1.7 ± 0.30 μmol/L), hour 2 (2.3 ± 0.29 vs 1.8 ± 0.32 μmol/L), hour 3 (2.4 ± 0.34 vs 1.58 ± 0.19 μmol/L), and hour 4 (2.51 ± 0.33 vs 1.5 ± 0.12 μmol/L) (all P < .05) following the high-fat diet as compared with the baseline postprandial response. In conclusion, a short-term, high-fat diet does not increase fasting plasma TMAO concentrations but appears to increase postprandial TMAO concentrations in healthy, nonobese, young men. Future studies are needed to determine the mechanisms responsible for these observations.

Abstract 311: Network Analysis of Pathways Associated with Genetic Regulation of Trimethylamine-N-Oxide

Common complex diseases, such as atherosclerosis, diabetes and obesity, are the result of genetic, environmental and gene/environmental interactions. Genetic reference panels are powerful tools to interrogate how the environment interacts with the underlying genetic architecture to influence disease susceptibility. Recently 2 genetic reference panels have been created: the Diversity Outbred (DO) mouse population and Collaborative Cross (CC). The mice comprising the CC/DO are mosaics of C57BL6/J, A/J, NOD/ShiLtJ, NZO/HiLtJ, WSB/EiJ, CAST/EiJ, PWK/PhJ, and 129S1/SvImJ. Both the DO and CC populations have tremendous genetic diversity, containing approximately 45 million segregating single-nucleotide polymorphisms (SNPs). Our primary goal was to characterize the genetic regulation of risk factors for atherosclerosis susceptibility in the 8 founder strains of the DO/CC reference panels. Using a systems genetic approach, we perform integrative analysis of clinical traits, targeted metabolomics and RNA expression. We quantitated genome wide mRNA levels using RNA sequencing followed by network analysis. The network was then related to clinical traits and a targeted panel of 5 metabolites (trimethylamine-N-oxide (TMAO), Betaine, Choline and Creatinine) to identify key groups of genes (called modules). We performed weighted gene network co-expression analysis (WGCNA) using 17,122 genes across 46 mice (N = 5-8 mice per strain). The network contains 47 independent co-expression modules and associated these modules with plasma lipoproteins, liver triglyceride levels and the novel plasma risk factors TMAO and Betaine. For example, the turquoise module was most strongly associated with TMAO levels (p &lt; 2 x 10-8) and the black module was associated with plasma Betaine concentrations. Both modules contain several candidate genes and enrichment analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID ) v6.7 Studies are ongoing to validate the key molecular drivers of the network with a particular interest in the transcriptional network regulating plasma TMAO and Betaine concentrations.