Trimethylamine N-oxide Precursor Research Articles

Ranitidine and finasteride inhibit the synthesis and release of trimethylamine N-oxide and mitigates its cardiovascular and renal damage through modulating gut microbiota.

Trimethylamine N-oxide (TMAO) leads to the development of cardiovascular and chronic kidney diseases, but there are currently no potent drugs that inhibit the production or toxicity of TMAO. In this study, high-fat diet-fed ApoE-/- mice were treated with finasteride, ranitidine, and andrioe. Subsequently, the distribution and quantity of gut microbiota in the faeces of the mice in each group were analysed using 16S rRNA sequencing of the V3+V4 regions. Pathological examination confirmed that both ranitidine and finasteride reduced atherosclerosis and renal damage in mice. HPLC analysis also indicated that ranitidine and finasteride significantly reduced the synthesis of TMAO and the TMAO precursor delta-Valerobetaine in their livers. The 16S rRNA sequencing showed that all 3 drugs significantly increased the richness and diversity of gut microbiota in the model mice. Bioinformatic analysis revealed that the faeces of mice treated with ranitidine and finasteride, had significant increases in the number of microbes in the families g_Helicobacter, f_Desulfovibrionaceae, Mucispirillum_schaedleri_ASF457, and g_Blautia, whereas the relative abundances of microbes in the families Enterobacter_sp._IPC1-8 and g_Bacteroides were significantly reduced. The microbiota metabolic pathways, such as nucleotide and cofactor and vitamin metabolism were also significantly increased, whereas the activities of metabolic signalling pathways related to glycan biosynthesis and metabolism and cardiovascular diseases were significantly reduced. Therefore, our study indicates that in addition to their known pharmacological effects, ranitidine and finasteride also exhibit potential cardiovascular and renal protective effects. They inhibit the synthesis and metabolism of TMAO and delay the deposition of lipids and endotoxins through improving the composition of the gut microbiota.

Abstract P3021: Trimethylamine but Not Trimethylamine N-Oxide Increases Blood Pressure in Rats, Affects Viability of Vascular Smooth Muscle Cells and Degrades Protein Structure

Increased plasma level of trimethylamine N-oxide (TMAO), a liver metabolite of gut bacteria-produced trimethylamine (TMA), has been suggested to increase cardiovascular risk. Mechanisms of TMAO increase in plasma and biological effects of TMAO are obscure. We evaluated the impact of heart failure (HF) on plasma levels of TMA and TMAO, and biological effects of the molecules. Gut permeability was evaluated in male, 58-week-old Spontaneously Hypertensive Heart Failure (SHHF) and Wistar-Kyoto (WKY) rats. TMA and TMAO levels were assessed using LC-MS. Mean arterial blood pressure (MABP) was measured in anaesthetized, male, 16-week-old WKY treated intravenously with either 0.9% NaCl (vehicle), TMA or TMAO. The cytotoxicity of TMA and TMAO in human vascular smooth muscle cells (hVSMCs) was evaluated using MTT assay. The effect of TMA and TMAO on protein structure of bovine albumin was assessed by fluorescence correlation spectroscopy. WKY showed no pathological changes in the circulatory system. SHHF showed HF with reduced ejection fraction. In comparison to WKY, SHHF had a significantly higher plasma level (μM) of TMA (99.05±6.40 vs 149.30±21.87) and TMAO (5.22±0.61 vs 6.69±0.67). SHHF had a significantly higher plasma-to-stool ratio of TMA, decreased intestinal blood flow and morphological alterations in the colon indicating the increased gut-to-blood penetration of TMA due to HF-induced leaky gut. In WKY treatment with the vehicle and TMAO did not affect MABP. In contrast, TMA (at equimolar doses to TMAO) significantly increased MABP by 5-40 mmHg in a dose dependent manner. In vitro, TMA at a concentration of 500μM reduced hVSMCs viability. TMAO at a concentration of 100mM was not cytotoxic whereas TMA at the same concentration killed cells within 24h. Finally, the incubation of albumin with TMA but not with TMAO resulted in the degradation of the protein structure. In conclusion, HF rats show increased plasma TMA and TMAO, which results from increased gut-to-blood penetration of TMA, a TMAO precursor. TMA but not TMAO affects hemodynamics in rats, reduces viability of cells and degrades protein structure. Therefore, TMA but not TMAO may exert deleterious effects on the circulatory system. Further clinical studies should evaluate not only TMAO but also TMA.

TMA, A Forgotten Uremic Toxin, but Not TMAO, Is Involved in Cardiovascular Pathology.

Trimethylamine-N-oxide (TMAO) has been suggested as a marker and mediator of cardiovascular diseases. However, data are contradictory, and the mechanisms are obscure. Strikingly, the role of the TMAO precursor trimethylamine (TMA) has not drawn attention in cardiovascular studies even though toxic effects of TMA were proposed several decades ago. We assessed plasma TMA and TMAO levels in healthy humans (HH) and cardiovascular patients qualified for aortic valve replacement (CP). The cytotoxicity of TMA and TMAO in rat cardiomyocytes was evaluated using an MTT test. The effects of TMA and TMAO on albumin and lactate dehydrogenase (LDH) were assessed using fluorescence correlation spectroscopy. In comparison to HH, CP had a two-fold higher plasma TMA (p < 0.001) and a trend towards higher plasma TMAO (p = 0.07). In CP plasma, TMA was inversely correlated with an estimated glomerular filtration rate (eGFR, p = 0.002). TMA but not TMAO reduced cardiomyocytes viability. Incubation with TMA but not TMAO resulted in the degradation of the protein structure of LDH and albumin. In conclusion, CP show increased plasma TMA, which is inversely correlated with eGFR. TMA but not TMAO exerts negative effects on cardiomyocytes, likely due to its disturbing effect on proteins. Therefore, TMA but not TMAO may be a toxin and a marker of cardiovascular risk.

Circulating Gut Microbiota Metabolite Trimethylamine N-Oxide (TMAO) and Changes in Bone Density in Response to Weight Loss Diets: The POUNDS Lost Trial.

Type 2 diabetes is related to obesity and altered bone health, and both are affected by gut microbiota. We examined associations of weight loss diet-induced changes in a gut microbiota-related metabolite trimethylamine N-oxide (TMAO), and its precursors (choline and l-carnitine), with changes in bone mineral density (BMD) considering diabetes-related factors. In the 2-year Preventing Overweight Using Novel Dietary Strategies trial (POUNDS Lost), 264 overweight and obese participants with measurement of BMD by DXA scan were included in the present analysis. The participants were randomly assigned to one of four diets varying in macronutrient intake. Association analysis was performed in pooled participants and different diet groups. Changes in blood levels of TMAO, choline, and l-carnitine from baseline to 6 months after the dietary intervention were calculated. We found that a greater reduction in plasma levels of TMAO from baseline to 6 months was associated with a greater loss in whole-body BMD at 6 months and 2 years (P = 0.03 and P = 0.02). The greater reduction in TMAO was also associated with a greater loss in spine BMD (P = 0.005) at 2 years, independent of body weight changes. The associations were not modified by baseline diabetes status and glycemic levels. Changes in l-carnitine, a precursor of TMAO, showed interactions with dietary fat intake in regard to changes of spine BMD and hip BMD at 6 months (all P < 0.05). Participants with the smallest decrease in l-carnitine showed less bone loss in the low-fat diet group than the high-fat diet group (P spine = 0.03 and P hip = 0.02). TMAO might protect against BMD reduction during weight loss, independent of diet interventions varying in macronutrient content and baseline diabetes risk factors. Dietary fat may modify the relation between change in plasma l-carnitine level and changes in BMD. Our findings highlight the importance of investigating the relation between TMAO and bone health in patients with diabetes.

Trimethyllysine, a trimethylamine N-oxide precursor, provides near- and long-term prognostic value in patients presenting with acute coronary syndromes.

Trimethyllysine (TML) serves as a nutrient precursor of the gut microbiota-derived metabolite trimethylamine N-oxide (TMAO) and is associated with incident cardiovascular (CV) events in stable subjects. We examined the relationship between plasma TML levels and incident CV events in patients presenting with acute coronary syndromes (ACS). Plasma levels of TML were quantified in two independent cohorts using mass spectrometry, and its relationship with CV events was investigated. In a Cleveland Cohort (N = 530), comprised of patients presenting to the emergency department with chest pain and suspected ACS, TML was associated with major adverse cardiac events (MACE, myocardial infarction, stroke, need for revascularization, or all-cause mortality) over both 30 days [3rd tertile (T3), adjusted odds ratio (OR) 1.77, 95% confidence interval (CI) 1.04-3.01; P < 0.05] and 6 months (T3, adjusted OR 1.95, 95% CI 1.15-3.32; P < 0.05) of follow-up independent of traditional CV risk factors and indices of renal function. Elevated TML levels were also associated with incident long-term (7-year) all-cause mortality [T3, adjusted hazard ratio (HR) 2.52, 95% CI 1.50-4.24; P < 0.001], and MACE even amongst patients persistently negative for cardiac Troponin T at presentation (e.g. 30-day MACE, T3, adjusted OR 4.49, 95% CI 2.06-9.79; P < 0.001). Trimethyllysine in combination with TMAO showed additive significance for near- and long-term CV events, including patients with 'negative' high-sensitivity Troponin T levels. In a multicentre Swiss Cohort (N = 1683) comprised of ACS patients, similar associations between TML and incident 1-year adverse cardiac risks were observed (e.g. mortality, adjusted T3 HR 2.74, 95% CI 1.28-5.85; P < 0.05; and MACE, adjusted T3 HR 1.55, 95% CI 1.04-2.31; P < 0.05). Plasma TML levels, alone and together with TMAO, are associated with both near- and long-term CV events in patients with chest pain and ACS.

Abstract P028: Gut Microbiota-Related Metabolites and Long-Term Bone Health in Response to Weight-Loss Diets: The POUNDS Lost Trial

Introduction: Weight-loss diet interventions affect gut microbiota-related metabolites, such as trimethylamine N-oxide (TMAO) and its precursors (choline and L-carnitine), and improve cardiometabolic status. However, little is known about how these metabolites may affect bone mineral density (BMD) during weight loss. Hypothesis: We assessed the hypothesis that changes in gut microbiota-related metabolites are related to changes in BMD during weight loss. Methods: A total of 264 overweight and obese participants with measurement of BMD by dual-energy X-ray (DEXA) scan were included in the present analysis. The participants were randomly assigned to one of four diets varying in macronutrient intakes. We investigated the associations of changes in plasma levels of TMAO, choline, and L-carnitine from baseline to 6 months with changes in BMD. Results and Conclusions: We found that changes in plasma levels of TMAO from baseline to 6 months were positively related to changes of whole body ( P =0.011) and spine ( P =0.002) BMD over the 2-year intervention, independent of weight change. The association with whole body BMD was also significant at 6 months. The correlations between changes in TMAO and BMD were not modified by dietary macronutrients; while changes in TMAO precursor L-carnitine showed interactions with dietary fat intake on changes of spine and trochanter BMD (all P &lt;0.05). Participants with the least L-carnitine decrease showed less BMD reduction by eating a low-fat diet ( P spine =0.031 and P trochanter =0.017). In conclusion, our results suggest that decrease in TMAO during weight loss may be related to reduction in BMD.

Relation of choline intake with blood pressure in the National Health and Nutrition Examination Survey 2007–2010

Dietary choline is a precursor of trimethylamine N-oxide (TMAO), a metabolite that has been associated with an increased risk of cardiovascular disease. The mechanism underlying this association is unknown, but may include TMAO effects on blood pressure (BP). This study assessed the association of choline intake with hypertension and BP in US adults through the use of NHANES 2007-2010 data. This cross-sectional study was conducted in nonpregnant individuals aged ≥20 y. Choline intake was assessed with the use of two 24-h recalls. Outcomes were BP and hypertension status, which was assessed through the use of questionnaires and BP measurements. Modifying factors (e.g., sex, race/ethnicity) and dietary compared with supplemental sources of choline intake were also investigated. The associations of total (dietary+supplemental) and dietary choline intake with the prevalence odds of hypertension differed by sex (n=9227; P-interaction=0.04 and 0.03, respectively). In women, both total and dietary choline intake tended to be inversely associated with hypertension (n=4748; prevalence OR per 100 mg of choline intake: 0.89; 95% CI: 0.77, 1.02; P<0.10 for both total and dietary choline). No association was observed in men (n=4479; P=0.54 and 0.49 for total choline and dietary choline, respectively). Use of choline supplements was inversely associated with hypertension in both sexes (user compared with nonuser; OR: 0.68; 95% CI: 0.49, 0.92; P=0.01). There was little to no association of total, dietary, or supplemental choline intake with systolic or diastolic BP (n=6,554; the mean±SEM change in BP associated with a 100-mg difference in total choline was -0.26±0.22 mm Hg for systolic BP and -0.29±0.19 mm Hg for diastolic BP). Cross-sectional NHANES data do not support thehypothesis of a positive association between choline intake and BP.

Egg Intake in Chronic Kidney Disease.

Patients with chronic kidney disease (CKD) are often instructed to adhere to a renal-specific diet depending on the severity and stage of their kidney disease. The prescribed diet may limit certain nutrients, such as phosphorus and potassium, or encourage the consumption of others, such as high biological value (HBV) proteins. Eggs are an inexpensive, easily available and high-quality source of protein, as well as a rich source of leucine, an essential amino acid that plays a role in muscle protein synthesis. However, egg yolk is a concentrated source of both phosphorus and the trimethylamine N-oxide precursor, choline, both of which may have potentially harmful effects in CKD. The yolk is also an abundant source of cholesterol which has been extensively studied for its effects on lipoprotein cholesterol and the risk of cardiovascular disease. Efforts to reduce dietary cholesterol to manage dyslipidemia in dialysis patients (already following a renal diet) have not been shown to offer additional benefit. There is a paucity of data regarding the impact of egg consumption on lipid profiles of CKD patients. Additionally, egg consumption has not been associated with the risk of developing CKD based on epidemiological studies. The egg yolk also contains bioactive compounds, including lutein, zeaxanthin, and vitamin D, which may confer health benefits in CKD patients. Here we review research on egg intake and CKD, discuss both potential contraindications and favorable effects of egg consumption, and describe the need for further research examining egg intake and outcomes in the CKD and end-stage renal disease population.

Increased plasma trimethylamine-N-oxide is associated with incident atrial fibrillation

BackgroundPlasma trimethylamine-N-oxide (TMAO) is associated with cardiovascular disease; however specific relationships with cardiac arrhythmias are unknown. We evaluated the association between plasma TMAO and incident atrial fibrillation (AF). MethodsRisk associations were explored among 3797 patients with suspected stable angina in the Western Norway Coronary Angiography Cohort (WECAC) and verified in 3143 elderly participants in the community-based Hordaland Health Study (HUSK). Information on endpoints was obtained from nationwide registries. ResultsMedian follow-up was 7.3 and 10.8 years in the WECAC and HUSK cohorts, respectively, and 412 (10.9%) and 484 (15.4%) subjects were registered with incident AF. The age and gender adjusted HRs were 1.16, 95% CI 1.05–1.28 and 1.10, 95% CI 1.004–1.19 per 1 SD increase in log-transformed plasma TMAO. Adjusting for hypertension, BMI, smoking, diabetes, or intake of total choline, a TMAO precursor, did not materially influence the risk associations. Among patients in WECAC, further extensive adjustment for other AF risk factors yielded similar results. Adding TMAO to traditional AF risk factors (age, gender, hypertension, BMI, smoking and diabetes) yielded a continuous net reclassification improvement of 0.108, 95% CI 0.015–0.202 and 0.139, 95% CI 0.042–0.235. ConclusionsPlasma TMAO was associated with and improved reclassification of incident AF in two independent Norwegian cohorts with long-term follow-up. The relationship was independent of traditional AF risk factors, as well as of dietary choline intake. Our findings motivate further studies to explore endogenous metabolic factors influencing the relationship between TMAO and cardiovascular disease.

Ruminant meat and milk contain δ-valerobetaine, another precursor of trimethylamine N-oxide (TMAO) like γ-butyrobetaine

Quaternary ammonium compounds containing N-trimethylamino moiety, such as choline derivatives and carnitine, abundant in meat and dairy products, are metabolic precursors of trimethylamine (TMA). A similar fate is reported for Nε-trimethyllysine and γ-butyrobetaine. With the aim at investigating the metabolic profile of such metabolites in most employed animal dietary sources, HPLC-ESI-MS/MS analyses on ruminant and non-ruminant milk and meat were performed. Results demonstrate, for the first time, the presence of δ-valerobetaine, occurring at levels higher than γ-butyrobetaine in all ruminant samples compared to non-ruminants. Demonstration of δ-valerobetaine metabolic origin, surprisingly, showed that it originates from rumen through the transformation of dietary Nε-trimethyllysine. These results highlight our previous findings showing the ubiquity of free Nε-trimethyllysine in vegetable kingdom. Furthermore, δ-valerobetaine, similarly to γ-butyrobetaine, can be degraded by host gut microbiota producing TMA, precursor of the proatherogenic trimethylamine N-oxide (TMAO), unveiling its possible role in the biosynthetic route of TMAO.

High salt intake increases plasma trimethylamine N-oxide (TMAO) concentration and produces gut dysbiosis in rats.

A high-salt diet is considered a cardiovascular risk factor; however, the mechanisms are not clear. Research suggests that gut bacteria-derived metabolites such as trimethylamine N-oxide (TMAO) are markers of cardiovascular diseases. We evaluated the effect of high salt intake on gut bacteria and their metabolites plasma level. Sprague Dawley rats ages 12-14 wk were maintained on either water (controls) or 0.9% or 2% sodium chloride (NaCl) water solution (isotonic and hypertonic groups, respectively) for 2 wk. Blood plasma, urine, and stool samples were analyzed for concentrations of trimethylamine (TMA; a TMAO precursor), TMAO, and indoxyl sulfate (indole metabolite). The gut-blood barrier permeability to TMA and TMA liver clearance were assessed at baseline and after TMA intracolonic challenge test. Gut bacterial flora was analyzed with a 16S ribosomal ribonucleic acid (rRNA) gene sequence analysis. The isotonic and hypertonic groups showed a significantly higher plasma TMAO and significantly lower 24-hr TMAO urine excretion than the controls. However, the TMA stool level was similar between the groups. There was no significant difference between the groups in gut-blood barrier permeability and TMA liver clearance. Plasma indoxyl concentration and 24-hr urine indoxyl excretion were similar between the groups. There was a significant difference between the groups in gut bacteria composition. High salt intake increases plasma TMAO concentration, which is associated with decreased TMAO urine excretion. Furthermore, high salt intake alters gut bacteria composition. These findings suggest that salt intake affects an interplay between gut bacteria and their host homeostasis.

Hypertension in rats is associated with an increased permeability of the colon to TMA, a gut bacteria metabolite.

An increased blood trimethylamine N-oxide (TMAO) has emerged as a marker of cardiovascular mortality, however, the mechanisms of the increase are not clear. We evaluated if hypertension was associated with changes in the colon permeability to trimethylamine (TMA), a TMAO precursor. We did experiments on male, 24-26-week-old normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and SHR treated with enalapril, an antihypertensive drug (SHR-E). To check the colon permeability and liver TMA clearance, blood was collected from the portal vein and hepatic veins confluence, at baseline and after the intracolonic administration of TMA. Arterial blood pressure (BP) and intestinal blood flow (IBF) recordings and histological assessment of the colon were performed. SHR showed an increased gut-blood barrier permeability to TMA. Namely, at baseline SHR had a higher BP and portal blood TMA, but a lower IBF than WKY. After the intracolonic administration of TMA, SHR had a significantly higher portal blood TMA and higher TMA liver clearance than WKY. In SHR the arteriolar walls of the colon mucosa were significantly thicker than in WKY. Furthermore, SHR showed a significant decrease in the height of the mucosa. In contrast, SHR-E had lower portal blood TMA, lower BP and smaller thickness of arteriolar walls, but higher IBF than SHR, which indicates improved function of the gut-blood barrier in SHR-E. All groups had similar immunostaining of occludin and zonula occludens-1, markers of tight junctions. In conclusion, hypertensive rats show an increased permeability of the colon to TMA, which is accompanied by morphological and hemodynamic alterations in the colon. Therefore, cardiovascular diseases may be characterized by an increased permeability of the gut-blood barrier to bacterial metabolites such as TMA.

Gut Microbiota-Dependent Trimethylamine-N-oxide and Serum Biomarkers in Patients with T2DM and Advanced CKD.

Trimethylamine-N-oxide (TMAO) is a product of dietary, gut microbiome, and tissues metabolism. Elevated blood TMAO levels are associated with heart attack, stroke and chronic kidney disease (CKD). The purpose of our study was to investigate the gut microbiota associated with trimethylamine (TMA) production, the precursor of TMAO, and the serum levels of TMAO and inflammatory biomarkers associated with type 2 diabetes mellitus (T2DM) and CKD. Twenty adults with T2DM and advanced CKD and 20 healthy adults participated in the study. Analyses included anthropometric and metabolic parameters, characterization of TMA producing gut microbiota, and concentrations of TMAO, lipopolysaccharides (LPS) endotoxin, zonulin (Zo) gut permeability marker, and serum inflammatory and endothelial dysfunction biomarkers. Diversity of the gut microbiota was identified by amplification of V3–V4 regions of the 16S ribosomal RNA genes and DNA sequencing. TMAO was quantified by Mass Spectrometry and serum biomarkers by ELISA. The significance of measurements justified by statistical analysis. The gut microbiome in T2DM-CKD patients exhibited a higher incidence of TMA-producing bacteria than control, p < 0.05. The serum levels of TMAO in T2DM-CKD patients were significantly higher than controls, p < 0.05. TMAO showed a positive correlation with Zo and LPS, inflammatory and endothelial dysfunction biomarkers. A positive correlation was observed between Zo and LPS in T2DM-CKD subjects. An increased abundance of TMA-producing bacteria in the gut microbiota of T2DM-CKD patients together with excessive TMAO and increased gut permeability might impact their risk for cardiovascular disease through elevation of chronic inflammation and endothelial dysfunction.

Trimethylamine-N-oxide (TMAO) response to animal source foods varies among healthy young men and is influenced by their gut microbiota composition: A randomized controlled trial.

Trimethylamine-N-oxide (TMAO), a metabolite linked to the gut microbiota, is associated with excess risk of heart disease. We hypothesized that (i) TMAO response to animal source foods would vary among healthy men and (ii) this response would be modified by their gut microbiome. A crossover feeding trial in healthy young men (n = 40) was conducted with meals containing TMAO (fish), its dietary precursors, choline (eggs) and carnitine (beef), and a fruit control. Fish yielded higher circulating and urinary concentrations of TMAO (46-62 times; p < 0.0001), trimethylamine (8-14 times; p < 0.0001), and dimethylamine (4-6-times; P<0.0001) than eggs, beef, or the fruit control. Circulating TMAO concentrations were increased within 15 min of fish consumption, suggesting that dietary TMAO can be absorbed without processing by gut microbes. Analysis of 16S rRNA genes indicated that high-TMAO producers (≥20% increase in urinary TMAO in response to eggs and beef) had more Firmicutes than Bacteroidetes (p = 0.04) and less gut microbiota diversity (p = 0.03). Consumption of fish yielded substantially greater increases in circulating TMAO than eggs or beef. The higher Firmicutes to Bacteroidetes enrichment among men exhibiting a greater response to dietary TMAO precursor intake indicates that TMAO production is a function of individual differences in the gut microbiome.