Plasma Trimethylamine N-oxide Levels Research Articles

Trimethylamine N-oxide as a Novel Biomarker for Left Ventricular Diastolic Dysfunction and Functional Remodeling in ST-Elevation Myocardial Infarction Patients

Abstract Background Over the last three decades, the mortality from ST-elevation myocardial infarction (STEMI) has halved. However, the incidence of heart failure or persistent angina remains high after STEMI, and the post-STEMI left ventricular (LV) systolic and diastolic function remains unpredictable. Thus, there is an unmet need to identify patients at risk of LV diastolic dysfunction or adverse remodeling. Purpose This study aims to investigate the potential of Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite associated with heart failure, and atherosclerosis, as a biomarker for adverse LV remodeling and diastolic dysfunction following STEMI. Methods In this prospective, observational cohort study, 210 STEMI patients with multivessel coronary artery disease (CAD) were enrolled after primary PCI. At 3 months follow-up, all patients underwent complete revascularization with FFR-guided PCI. Plasma TMAO, C-reactive protein (CRP), and brain natriuretic peptide (BNP) levels at 3 months follow-up and peak troponin level at primary PCI were collected. Echocardiographies were performed within 24 hours of STEMI and at 12-month follow-ups. Diastolic dysfunction was defined according to the ASE 2016 guidelines.(1) Functional LV remodeling (FLVR) was categorized into 4 categories according to Chimed et al.(2) Receiver operating characteristic (ROC) curve analysis, including the Delong test and Youden index, was performed to compare the biomarkers' diagnostic value and determine the optimal cutoffs for predicting diastolic dysfunction and FLVR, respectively. Results Of the 210 patients enrolled, 85 (40.48%) were female, with a median age of 65 years [interquartile range (IQR): 58.00, 76.00] and a median body mass index (BMI) of 27.39 kg/m² (IQR: [24.56, 30.69]). The area under the ROC curve for predicting post-PCI LV diastolic dysfunction ≥ grade II was 0.72 (95% CI 0.63–0.81; p<0.001) for TMAO (Figure 1). The optimal TMAO cutoff value of 3.80 yielded a sensitivity of 0.75 (95% CI 0.63–0.86) and a specificity of 0.82 (95% CI 0.76–0.88). For FLVR grade 3 or 4, the area under the ROC curve was 0.62 (95% CI 0.50–0.74) and 0.72 (95% CI 0.62 -0.83) for TMAO and BNP, respectively (Figure 2), but the AUC was not significantly different (p= 0.191). In multivariable logistic regression analysis, TMAO was independently associated with FLVR grade 3 or 4 (OR 1.18, 95% CI 1.00-1.38, p = 0.046) and diastolic dysfunction ≥grade 2 (OR 1.30, 95% CI 1.12-1.50, p < 0.001). Conclusion The novel parameter of TMAO is a useful predictor for LV diastolic dysfunction after STEMI. TMAO is independently associated with an increased risk of LV diastolic dysfunction and worse FLVR. These results suggest that monitoring plasma TMAO levels could be an effective and cost-efficient strategy for monitoring patients with an elevated risk of LV diastolic dysfunction or adverse LV remodeling.Figure 1Figure 2

Protective Role of (-)-Epicatechin on Trimethylamine-N-Oxide (TMAO)-Induced Cardiac Hypertrophy via SP1/SIRT1/SUMO1 Signaling Pathway.

(-)-Epicatechin (EPI) is beneficial for cardiovascular health. Trimethylamine N-oxide (TMAO), a gut microbe-derived food metabolite, is strongly associated with the risk of cardiovascular diseases. However, the effects and underlying mechanisms of EPI on TMAO-induced cardiac hypertrophy remain unclear. This study aimed to determine whether EPI inhibits TMAO-induced cardiac hypertrophy. Plasma levels of TMAO in control participantsand patients with cardiac hypertrophy were measured and analyzed. Male C57BL/6 mice were randomly divided into control group, TMAO group, EPI group and TMAO + EPI group. According to the groups assignments, mice received intraperitoneal (i.p.) injection of normal saline or i.p. injection of TMAO (150mg/kg/day) for 14days. The EPI group was given intragastric (i.g.) administration of EPI alone (1mg/kg/day) for 21days, and TMAO + EPI group received i.g. administration of EPI for 7days before starting i.p. injection of TMAO, continuing until the end of the TMAO treatment. Histological analyses of the mice's hearts was accessed by H&E and Masson staining. In vitro, H9c2 cells were induced to hypertrophy by TMAO (10µM) for 24h and were pre-treated with or without EPI (10µM) for 1h. Protein level of cardiac hypertrophy markers and Sp1/SIRT1/SUMO1 pathway were determined by western blot. The plasma level of TMAO was 2.66 ± 1.59μmol/L in patients with cardiac hypertrophy and 0.62 ± 0.30μmol/L in control participants. EPI attenuated TMAO-induced hypertrophy in H9c2 cells. In vivo, TMAO induced cardiac hypertrophy and impaired the cardiac function of mice. Pathological staining showed that TMAO induced cardiac hypertrophy and collagen deposition in mice. EPI treatment improved the cardiac function, inhibited the myocardial hypertrophy induced by TMAO. EPI significantly attenuated the TMAO-induced upregulation of ANP and BNP and the downregulation of SP1, SIRT1 and SUMO1 in vivo and in vitro. EPI may suppress TMAO-induced cardiac hypertrophy by activating the Sp1/SIRT1/SUMO1 signaling pathway.

Association between plasma trimethylamine N-oxide and coronary heart disease: new insights on sex and age differences.

Elevated plasma trimethylamine N-oxide (TMAO) is related to atherosclerosis. Whether the relationship of TMAO and coronary heart disease (CHD) is influenced by sex or age is uncertain. We aim to explore the sex and age differences in the relationship between plasma TMAO and CHD risk and severity. A case-control study was conducted in patients undergoing elective coronary angiography. Matched by sex, age (±2 years), and operation date (±180 days), a total of 429 CHD case-control pairs were included. Plasma TMAO was quantified using liquid chromatography-tandem mass spectrometry. Logistic regression analyses were performed to evaluate the association between plasma TMAO and CHD risk and severity. The overall median (interquartile range) plasma TMAO level was 0.11 (0.06-0.18) μg/ml. After stratification by sex and age, and adjustment for common CHD risk factors, the association between TMAO and CHD risk was significant in the older (≥65 years) male subgroup [odds ratios (OR) = 1.57, 95% confidence interval (CI): 1.09-2.28, P = 0.016], but not in other sex-age subgroups (all P > 0.05). The relationship of plasma TMAO and CHD risk was modified by age (adjusted P interaction = 0.001) in male individuals. Plasma TMAO was also associated with a higher risk of multi-vessel disease in male patients with CHD (OR = 1.65, 95% CI: 1.18-2.32, P = 0.004), but not in females. Plasma TMAO is significantly positively associated with the risk and severity of CHD in Chinese men. Age has an interactive effect on the relationship between plasma TMAO and CHD risk in men. Our findings warrant further investigation.

A-001 The Postprandial Levels of the Metabolites in the Metaorganismal Trimethylamine N-Oxide (TMAO) Pathway are Altered in A Diet-, Microbe-, and Sex-Specific Manner

Abstract Background Cardiovascular disease (CVD) remains the leading cause of death in developed countries. Elevated levels of the gut-microbe-associated metabolite trimethylamine-N-oxide (TMAO) have been associated with increased risk for CVD mortality in many large independent studies. In fact, large laboratory corporations such as Labcorp and Quest Diagnostic now offer TMAO diagnostic tests for the assessment of CVD risk and as a marker for disease-associated dysbiosis, using samples obtained from fasting patients. Although the strong association between TMAO levels and CVD risk has been established in fasting blood samples, here we are investigating the potential for a single meal to dynamically alter TMAO-related metabolites based on sex, diverse dietary substrates, and microbial influences. Methods 35 healthy participants were randomized to one of four study groups with approximately 8-9 subjects in each arm. Half of the participants were randomly assigned to receive a three-day broad spectrum antibiotic regimen (ciprofloxacin, metronidazole, and vancomycin) while the others received no antibiotics. These two groups were further subdivided into a group consuming a highly processed meal (originating from local fast food restaurants) or alternatively a whole food meal (containing a variety of fruits, vegetables, and healthy fiber). Blood samples were taken at baseline (after an overnight fast), and then postprandially at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours after meal ingestion. Targeted plasma metabolites were quantified using a stable isotope dilution, liquid chromatography - tandem mass spectrometry (LC-MS/MS) method Results While plasma TMAO levels between food groups did not change significantly within the total cohort, there were clear individualized responses to either highly processed or whole food meals. Separation based on sex showed that TMAO levels were significantly reduced in females at 6 hours but remained steady in males for both food groups. Particularly, in the processed food group, TMAO levels were significantly lower in females than males at the 6-hour time point. Neither of these observations held true for TMAO precursors choline, carnitine, or betaine. However, plasma levels of a dietary precursor for TMAO, γ-butyrobetaine, showed clear diet-microbe-host interactions. For males in the processed food group, plasma γ-butyrobetaine levels were significantly increased in subjects on broad spectrum antibiotics. Conclusions Our results show the postprandial levels of TMAO, and its nutrient precursors dynamically change in a diet-, microbe-, and sex-dependent manner. These findings provide new insights into the postprandial levels of TMAO-related metabolites and may inform precision nutritional approaches in those who could benefit from TMAO-lowering strategies.

Trimethylamine-N-oxide and 5-year mortality: the role of gut microbiota-generated metabolite from the CORE-Thailand cohort

The gut microbiota metabolite trimethylamine-N-oxide (TMAO)—derived from dietary phosphatidylcholine—is mechanistically linked to cardiovascular disease (CVD) and increased cardiovascular risk. This study examined the relationship between fasting plasma TMAO levels and 5-year all-cause mortality in a cohort of patients at high risk of cardiovascular events (CORE-Thailand Registry). Of the 134 patients, 123 (92%) had established cardiovascular disease, and 11 (8%) had multiple risk factors. Fasting plasma TMAO levels were measured using nuclear magnetic resonance spectroscopy. Within this prospective cohort study, the median TMAO was 3.81 μM [interquartile range (IQR) 2.89–5.50 μM], with a mean age of 65 ± 11 years; 61% were men, and 39.6% had type II diabetes. Among 134 patients, 65 (49%) were identified as the high-TMAO group (≥ 3.8 μM), and 69 (51%) were identified as the low-TMAO group (< 3.8 μM). After a median follow-up of 58.8 months, the high-TMAO group was associated with a 2.88-fold increased mortality risk. Following adjustment for traditional risk factors, high-sensitivity cardiac troponin-T, estimated glomerular filtration rate, angiotensin-converting enzyme (ACEI), or angiotensin-receptor blocker (ARB) use, the high-TMAO group remained predictive of 5-year all-cause mortality risk (the high-TMAO vs. the low-TMAO group, adjusted hazard ratio 2.73, 95% CI 1.13–6.54; P = 0.025). Among Thai patients at high risk of cardiovascular events, increased plasma TMAO levels portended greater long-term mortality risk.

Effect analysis of trimethylamine N-oxide and its precursors on susceptibility to pancreatic diseases

To investigate the causal relationship between trimethylamine N-oxide (TMAO) and its precursors (betaine, carnitine, and choline) and pancreatic diseases based on the Mendelian randomization (MR) method. Genome-wide association study data of TMAO, betaine, carnitine, choline, acute pancreatitis (AP), chronic pancreatitis (CP), pancreatic cancer (PC), and circulating immune cell characteristics (white blood cell, lymphocyte, monocyte, neutrophil, eosinophil and basophil) were collected. According to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)-MR reporting guidelines, the available genetic variants [single nucleotide polymorphism (SNP)] were strictly screened. The causal relationship between exposure (TMAO and its precursors) and outcomes (pancreatic diseases and circulating immune cell characteristics) was evaluated using inverse variance weighting (IVW), MR-Egger regression and weighted median. The reliability of the results was evaluated by sensitivity analysis based on MR-Egger regression, MR-PRESSO, Cochrane's Q test and leave-one-out method. A total of 36 SNP associated with TMAO and its precursors were included. Five of these were associated with TMAO, 13 with betaine, 12 with carnitine, and 6 with choline. (1) MR analysis showed that TMAO may increase the risk of AP [odds ratio (OR) = 1.100, 95% confidence interval (95%CI) was 1.008-1.200, P = 0.032], and choline may reduce the risk of alcoholic acute pancreatitis (AAP; OR = 0.743, 95%CI was 0.585-0.944, P = 0.015). The analysis results of MR-Egger regression and weighted median were consistent with the IVW results. There is no evidence to support a causal relationship between TMAO and its precursors and the risk of CP and PC. Sensitivity analysis indicated that SNP analyzed by MR showed no heterogeneity and low pleiotropy. The leave-one-out method analysis determined that after excluding any SNP, the effect intervals of the remaining SNP on the results were similar to the overall effect intervals, which suggested the robustness of MR results. (2) There was a positive causal relationship between plasma TMAO level and circulating monocyte count (OR = 1.017, 95%CI was 1.000*-1.034, P = 0.048, * represented that the data was obtained by correcting to 3 decimal places from 1.000 1). The causal effect obtained by MR-Egger regression and weighted median analysis was consistent with the results of IVW. Sensitivity analysis illustrated SNP analyzed by MR showed no heterogeneity and pleiotropy. The leave-one-out method analysis determined that after excluding any SNP, the effect intervals of the remaining SNP on the results were similar to the overall effect intervals, which suggested the robustness of MR results. TMAO and choline may change the risk of AP, and TMAO may contribute to the increase of circulating monocyte count in AP.

Potential Trimethylamine (TMA)-Producing Bacteria in patients with chronic kidney disease undergoing hemodialysis.

Trimethylamine (TMA), produced by gut microbiota, is the precursor of trimethylamine-N-oxide (TMAO), a uremic toxin that accumulates in patients with chronic kidney disease (CKD). Elevated TMAO plasma levels are associated with cardiovascular complications and CKD progression. To evaluate the association between gut microbiota composition and TMAO plasma levels in CKD patients undergoing hemodialysis (HD). This is a cross-sectional study with 25 patients evaluated (60% female, 53 (18) years, body mass index (BMI) 25.8 (6.75)Kg/m2). They were divided into two groups according to their TMAO plasma levels: normal (≤ 7.4μM) and high (> 7.4μM). Uremic toxins such as indoxyl sulfate (IS), p-cresyl sulfate (pCS), and indol acetic acid (IAA) were measured with RP-HPLC, and TMAO plasma levels were quantified using LC-MS/MS. Fecal DNA was extracted with a commercial kit, PCR amplified the V4 region of the 16S rRNA gene, and short-read sequencing was performed on the Illumina platform. Dietary intake, anthropometric measurements, and inflammation markers were also evaluated. Nrf2, NF-κB, IL-1β, and NLRP3 mRNA expressions were measured from peripheral blood mononuclear cells (PBMC) using quantitative real-time polymerase chain reaction (qPCR). There were significant positive correlations between TMAO and plasma levels of pCS, NLPR3 inflammasome mRNA expression, serum phosphorus levels, and negative correlations with dietary lipid intake. The group with TMAO > 7.4μM showed an increase in the microbiome abundance of Saccharibacteria (genus incertae sedis), Colidextribacter, Dorea, and Staphylococci genera, and a decrease in abundance in the genera Lachnospira, Lactobacilli, and Victivallis. TMAO plasma level was positively correlated with the abundance of bacteria of the genera Colidextribacter and Helicobacter and was negatively correlated with Sphingomanos, Lachnospira, Streptomyces, and Bacillus genera. Saccharibacteria (genus incertae sedis), Colidextribacter, Dorea, and Staphylococci genera showed higher abundance in patients with high TMAO levels. In addition, we observed that elevated plasma TMAO levels are associated with inflammation markers, dietary lipid intake, and serum phosphorus levels in patients undergoing HD.

Simultaneous determination of choline, L-carnitine, betaine, trimethylamine, trimethylamine N-oxide, and creatinine in plasma, liver, and feces of hyperlipidemic rats by UHPLC-MS/MS

BackgroundDue to the close correlation between choline, L-carnitine, betaine and their intestinal microbial metabolites, including trimethylamine (TMA) and trimethylamine N-oxide (TMAO), and creatinine, there has been an increasing interest in the study of these compounds in vivo. MethodsIn this study, a rapid stable isotope dilution (SID)-UHPLC-MS/MS method was developed for the simultaneous determination of choline, L-carnitine, betaine, TMA, TMAO and creatinine in plasma, liver and feces of rats. The method was validated using quality control (QC) samples spiked at low, medium and high levels. Second, we applied the method to quantify the effects of Rosa Roxburghii Tratt juice (RRTJ) on plasma, liver, and fecal levels of choline, L-carnitine, betaine, TMA, TMAO, and creatinine in high-fat diet-induced hyperlipidemic rats, demonstrating the utility of the method. ResultsThe limits of detection (LOD) were 0.04–0.027 µM and the limits of quantification (LOQ) were 0.009–0.094 µM. The linear ranges for each metabolite in plasma were choline1.50–96 µM; L-carnitine: 2–128 µM; betaine: 3–192 µM; TMA: 0.01–40.96 µM; TMAO: 0.06–61.44 µM and creatinine: 1–64 µM (R2 ≥ 0.9954). The linear ranges for each metabolite in liver were Choline: 12–768 µM; L-carnitine: 1.5–96 µM; betaine: 10–640 µM; TMA: 0.5–32 µM; TMAO: 0.02–81.92 µM and creatinine: 0.2–204.8 µM (R2 ≥ 0.9938). The linear ranges for each metabolite in feces were choline: 1.5–96 µM; L-carnitine: 0.01–40.96 µM; Betaine: 1.5–96 µM; TMA: 1–64 µM; TMAO: 0.02–81.92 µM and Creatinine: 0.02–81.92 µM (R2 ≥ 0.998). The intra-day and inter-day coefficients of variation were < 8 % for all analytes. The samples were stabilized after multiple freeze–thaw cycles (3 freeze–thaw cycles), 24 h at room temperature, 24 h at 4 °C and 20 days at −80 °C. The samples were stable. The average recovery was 89 %-99 %. This method was used to quantify TMAO and its related metabolites and creatinine levels in hyperlipidemic rats. The results showed that high-fat diet led to the disorder of TMAO and its related metabolites and creatinine in rats, which was effectively improved after the intervention of Rosa Roxburghii Tratt juice（RRTJ）. ConclusionsA method for the determination of choline, L-carnitine, betaine, TMA, TMAO and creatinine in plasma, liver and feces samples was established, which is simple, time-saving, high precision, accuracy and recovery.

#1801 Gut dysbiosis contributes to TMAO accumulation in CKD

Abstract Background and Aims Chronic kidney disease (CKD) patients face a substantial morbidity risk, especially related to cardiovascular complications. The imbalance of circulating metabolites influences inter-organ crosstalk and disease pathophysiology. In this regard, metabolites of microbial origin have gained increasing attention. Trimethylamine N-oxide (TMAO) originates from microbial Trimethylamine (TMA) metabolism and is found at high concentrations in the blood of CKD patients. A high TMAO concentration is known to be an independent risk factor for cardiovascular disease. This study aims to investigate the extent to which high concentrations of TMAO in the blood are due to increased bacterial production or are mainly a consequence of impaired renal clearance. Methods We used existing biomaterials and clinical data of a pediatric CKD cohort, consisting of patients enrolled at CKD stage G3-5, including G5 patients treated with hemodialysis (HD), as well as patients after kidney transplantation (KT) and individuals with normal kidney function. We analyzed TMAO in plasma and urine samples using LC-MS/MS and developed a novel gene targeting assay (GTA) that quantifies the abundance of the bacterial target genes (CutC, CntA) encoding for the key enzymes of microbial TMA synthesis in each fecal sample. Results Plasma TMAO levels were stage-dependently elevated in CKD, with HD patients showing the highest levels (75 ± 46 µmol/l). While urinary TMAO levels were lower in the CKD groups, TMAO/creatinine ratios and fractional excretion were not significantly different in CKD compared to controls. Fecal microbial gene abundance of both CutC and CntA genes was elevated across CKD stages and correlated with both plasma TMAO levels and eGFR. Although KT patients exhibited an eGFR range comparable to controls, they still showed higher plasma TMAO levels and elevated TMAO-associated gene abundances. In a regression analysis, this gene abundance was associated with plasma TMAO independent of eGFR in a model adjusted for age, sex, and BMI. Using a likelihood ratio test, we showed that adding fecal microbial gene abundance to the model provided a significantly better fit compared to only eGFR, age, sex, and BMI. Conclusion Our data indicate that the production of TMA by intestinal bacteria contributes significantly to the accumulation of TMAO in patients with CKD and after kidney transplantation. This finding opens the possibility for new potential interventions in the management of patients with CKD.

Effect of three oral pathogens on the TMA-TMAO metabolic pathway.

Trimethylamine-N-oxide (TMAO) is produced by hepatic flavin-containing monooxygenase 3 (FMO3) from trimethylamine (TMA). High TMAO level is a biomarker of cardiovascular diseases and metabolic disorders, and it also affects periodontitis through interactions with the gastrointestinal microbiome. While recent findings indicate that periodontitis may alter systemic TMAO levels, the specific mechanisms linking these changes and particular oral pathogens require further clarification. In this study, we established a C57BL/6J male mouse model by orally administering Porphyromonas gingivalis (P. gingivalis, Pg), Fusobacterium nucleatum (F. nucleatum, Fn), Streptococcus mutans (S. mutans, Sm) and PBS was used as a control. We conducted LC-MS/MS analysis to quantify the concentrations of TMAO and its precursors in the plasma and cecal contents of mice. The diversity and composition of the gut microbiome were analyzed using 16S rRNA sequencing. TMAO-related lipid metabolism and enzymes in the intestines and liver were assessed by qPCR and ELISA methods. We further explored the effect of Pg on FMO3 expression and lipid molecules in HepG2 cells by stimulating the cells with Pg-LPS in vitro. The three oral pathogenic bacteria were orally administered to the mice for 5 weeks. The Pg group showed a marked increase in plasma TMAO, betaine, and creatinine levels, whereas no significant differences were observed in the gut TMAO level among the four groups. Further analysis showed similar diversity and composition in the gut microbiomes of both the Pg and Fn groups, which were different from the Sm and control groups. The profiles of TMA-TMAO pathway-related genera and gut enzymes were not significantly different among all groups. The Pg group showed significantly higher liver FMO3 levels and elevated lipid factors (IL-6, TG, TC, and NEFA) in contrast to the other groups. In vitro experiments confirmed that stimulation of HepG2 cells with Pg-LPS upregulated the expression of FMO3 and increased the lipid factors TC, TG, and IL-6. This study conclusively demonstrates that Pg, compared to Fn and Sm, plays a critical role in elevating plasma TMAO levels and significantly influences the TMA-TMAO pathway, primarily by modulating the expression of hepatic FMO3 and directly impacting hepatic lipid metabolism.

Deciphering the causal relationship between plasma and cerebrospinal fluid metabolites and glioblastoma multiforme: a Mendelian Randomization study.

Glioblastoma Multiforme (GBM) is one of the most aggressive and fatal brain cancers. The study of metabolites could be crucial for understanding GBM's biology and reveal new treatment strategies. The GWAS data for GBM were sourced from the FinnGen database. A total of 1400 plasma metabolites were collected from the GWAS Catalog dataset. The cerebrospinal fluid (CSF) metabolites data were collected from subsets of participants in the WADRC and WRAP studies. We utilized the inverse variance weighting (IVW) method as the primary tool to explore the causal relationship between metabolites in plasma and CSF and glioblastoma, ensuring the exclusion of instances with horizontal pleiotropy. Additionally, four supplementary analytical methods were applied to reinforce our findings. Aberrant results were identified and omitted based on the outcomes of the leave-one-out sensitivity analysis. Conclusively, a reverse Mendelian Randomization analysis was also conducted to further substantiate our results. The study identified 69 plasma metabolites associated with GBM. Of these, 40 metabolites demonstrated a significant positive causal relationship with GBM, while 29 exhibited a significant negative causal association. Notably, Trimethylamine N-oxide (TMAO) levels in plasma, not CSF, were found to be a significant exposure factor for GBM (OR = 3.1627, 95% CI = (1.6347, 6.1189), P = 0.0006). The study did not find a reverse causal relationship between GBM and plasma TMAO levels. This research has identified 69 plasma metabolites potentially associated with the incidence of GBM, among which TMAO stands out as a promising candidate for an early detectable biomarker for GBM.

The Gut Microbial Metabolite Trimethylamine N -oxide, Incident CKD, and Kidney Function Decline.

Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite of dietary phosphatidylcholine and carnitine. Experimentally, TMAO causes kidney injury and tubulointerstitial fibrosis. Little is known about prospective associations between TMAO and kidney outcomes, especially incident CKD. We hypothesized that higher plasma TMAO levels would be associated with higher risk of incident CKD and greater rate of kidney function decline. We included 10,564 participants from two community-based, prospective cohorts with estimated glomerular filtration rate (eGFR) ≥ 60 mL/min/1.73m2 to assess incident CKD. TMAO was measured using targeted mass spectrometry at baseline and one follow-up visit. Creatinine and Cystatin C were measured up to 4 times during follow-up and used to compute eGFR. Incident CKD was defined as an eGFR decline ≥ 30% from baseline and a resulting eGFR<60 ml/min/1.73 m2. Time-varying Cox models assessed the association of serial TMAO measures with incident CKD, adjusting for sociodemographic, lifestyle, diet, and cardiovascular disease risk factors. Linear mixed models assessed the association with annualized eGFR change in 10,009 participants with at least one follow-up eGFR measure without exclusions for baseline eGFR levels. During a median follow-up of 9.4 years (interquartile range: 9.1-11.6 years), 979 incident CKD events occurred. Higher TMAO levels associated with higher risk of incident CKD (2nd to 5th vs. 1st quintile HR[95%CI]= 1.65 [1.22-2.23], 1.68 [1.26-2.25], 2.28 [1.72-3.02], and 2.24[1.68-2.98], respectively) and greater annualized eGFR decline ( 2nd to 5th vs. 1st quintile annualized eGFR change= -0.21 [-0.32, -0.09], -0.17 [-0.29, -0.05], -0.35 [-0.47, -0.22], and -0.43[-0.56, -0.30], respectively) with monotonic dose-response relationships. These associations were consistent across different racial/ethnic groups examined. The association with eGFR decline was similar to or larger than that seen for established CKD risk factors including diabetes, per 10 mmHg of higher systolic blood pressure, per 10 years of older age, and Black race. In community-based US adults, higher serial measures of plasma TMAO were associated with higher risk of incident CKD and greater annualized kidney function decline.

Trimethylamine-N-oxide aggravated sympathetic activation in D-galactose induced aging rats by down-regulating P2Y12 receptor in microglia

This study aimed to determine whether trimethylamine N-oxide (TMAO) was involved in sympathetic activation in aging and the underline mechanisms. Our hypothesis is that TMAO reduces P2Y12 receptor and induces microglia-mediated inflammation in the paraventricular nucleus, then leading to sympathetic activation in aging. Eighteen young adults (20-40 years old) and sixteen old adults (65-90 years old) were involved in the study. Aging rats were established by injecting D-galactose (200 mg/kg/d) subcutaneously for 12 weeks. TMAO (120 mg/kg/d) or 1% 3, 3-dimethyl-l-butanol (DMB, a structural analog of choline, inhibiting TMA formation) was administrated in drinking water for 12 weeks to investigate their effects on neuroinflammation and sympathetic activation in aging rats. Heart rate variability was analyzed by 24 h ambulatory ECG multichannel recording system (CT-083S Holter ECG recorder, BENEWARE, Hangzhou) in young and old adults. The PowerLab 15T data acquisition system (AD Instruments, Australia) was used to record blood pressure and renal sympathetic nerve activity in the rats. Plasma levels of TMAO, noradrenaline, and IL-1β in old adults were higher than those in young group. In addition, standard deviation of all normal to normal intervals and standard deviation of the average of normal to normal intervals were lower in old adults and negative correlated with TMAO, which indicating sympathetic activation in old adults and related to the increased TMAO. Rats treatment with D-gal showed increased senescence-associated protein, microglia-mediated inflammation levels and decreased P2Y12 receptor in the paraventricular nucleus. Plasma levels of TMAO, noradrenaline, and IL-1β were increased, accompanied by enhanced renal sympathetic nerve activity. While TMAO supplementation aggravated the above phenomenons, and DMB abated them. These findings suggested that TMAO might contribute to the sympathetic hyperactivity of aging via downregulating P2Y12 receptor in microglia and increasing inflammation in the paraventricular nucleus. These results may provide a new target for the prevention and treatment of aging and aging-related diseases. This work was supported by the Program for the National Natural Science Foundationof China (32271155 and 31671185), the Science and Technology Project of the Hebei Education Department (ZD2021076), the Youth Top Talent Foundation of Hebei Province of China and the Program of Clinical Medicine Innovation Research Team of Hebei Medical University (2022LCTD-A1). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Empagliflozin Mitigates TMAO-Induced NLRP3 Inflammation and Alters Transporter Expression in Renal Proximal Tubular Cells

The gut-microbe-derived metabolite, trimethylamine N-oxide (TMAO), not only exhibits elevated levels but also serves as a predictor of higher mortality in patients with chronic kidney disease (CKD). In additional to CKD, elevated TMAO levels were also observed in organic anion transporter 3 (OAT 3) knockout mice and in the presence of trimethoprim, an antibiotic known for inhibiting multiple transporters on renal proximal tubule (PT) cells. These findings suggest that plasma TMAO level is a marker for diminished transporter function in PT cells. Moreover, the pro-inflammatory effects of TMAO on PT cells is relatively underexplored. Besides reno-protective effect, a recent study has shown that the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin attenuates hyperuricemia by enhancing efflux transporter function. Therefore, we propose that empagliflozin can ameliorate TMAO-induced inflammation in PT cells by regulating the expression of membrane transporters. Western blotting results revealed that empagliflozin mitigated TMAO-induced NLRP3 inflammatory cytokines IL-1β, and IL-18. Additionally, empagliflozin reduced the elevated serum levels of TMAO in a CKD mouse model resulting from 5/6 nephrectomy (5/6Nx). Immuno-fluorescent staining demonstrated that TMAO decreased the expression of the basolateral transporter organic anion transporter 1 (OAT1) but increased the expression of apical membrane transporters ATP-binding cassette super-family G member 2 (ABCG2) on the PT cells of 5/6Nx mice. Our in vitro study indicated that TMAO reduced the expression of basolateral membrane transporters OAT1 and OAT3. But empagliflozin significantly increased the expression of OAT3 and ABCG2 in TMAO-treated HK-2 cells. In summary, TMAO induced NLRP3 inflammation in PT cells, while empagliflozin ameliorated the inflammation. Empagliflozin also lowered TMAO levels and influenced transporter expressions in a CKD mouse model. In conclusion, we have shown that empagliflozin mitigate TMAO-induced NLRP3 inflammation. Beyond inhibiting SGLT2, empagliflozin possesses additional effect on the expression of membrane transporters in PT, particularly in CKD mice. This research was funded by Chang Gung Medical Foundation CMRPG8K0011 and CMRPG8M0311. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Trimethylamine-N-Oxide and Related Metabolites: Assessing Cardiovascular Risk in the Dallas Heart Study

ObjectiveTo evaluate the association between trimethylamine N-oxide (TMAO) and related metabolites with adverse cardiovascular events in a multiethnic urban primary prevention population. MethodsWe performed a case-control study of 361 participants of the Dallas Heart Study, including 88 participants with an incident atherosclerotic cardiovascular disease (ASCVD) event and 273 controls matched for age, sex, and body mass index without an ASCVD event during 12 years of follow-up (January 1, 2000, through December 31, 2015). Plasma levels of TMAO, choline, carnitine, betaine, and butyrobetaine were measured by mass spectrometry. The differential odds for incident ASCVD by metabolite levels between cases and controls were compared by a conditional logistic regression model adjusted for cardiovascular risk factors. ResultsParticipants with incident ASCVD had higher levels of TMAO and related metabolites compared with those without ASCVD (P<.05 for all). Those with plasma TMAO concentrations in quartile 4 had a more than 2-fold higher odds of ASCVD compared with those in quartile 1 (odds ratio, 2.77 [95% CI, 1.05 to 7.7; P=.04] for hard ASCVD and 2.41 [95% CI, 1.049 to 5.709; P=.04]). Similar trends were seen with the related metabolites choline, betaine, carnitine, and butyrobetaine. ConclusionOur results suggest that TMAO and related metabolites are independently associated with ASCVD events. Although further studies are needed, measurement of TMAO and related metabolites may have a role in ASCVD risk stratification for primary prevention.

Unraveling interindividual variation of trimethylamine N-oxide and its precursors at the population level.

Trimethylamine N-oxide (TMAO) is a circulating microbiome-derived metabolite implicated in the development of atherosclerosis and cardiovascular disease (CVD). We investigated whether plasma levels of TMAO, its precursors (betaine, carnitine, deoxycarnitine, choline), and TMAO-to-precursor ratios are associated with clinical outcomes, including CVD and mortality. This was followed by an in-depth analysis of their genetic, gut microbial, and dietary determinants. The analyses were conducted in five Dutch prospective cohort studies including 7834 individuals. To further investigate association results, Mendelian Randomization (MR) was also explored. We found only plasma choline levels (hazard ratio [HR] 1.17, [95% CI 1.07; 1.28]) and not TMAO to be associated with CVD risk. Our association analyses uncovered 10 genome-wide significant loci, including novel genomic regions for betaine (6p21.1, 6q25.3), choline (2q34, 5q31.1), and deoxycarnitine (10q21.2, 11p14.2) comprising several metabolic gene associations, for example, CPS1 or PEMT. Furthermore, our analyses uncovered 68 gut microbiota associations, mainly related to TMAO-to-precursors ratios and the Ruminococcaceae family, and 16 associations of food groups and metabolites including fish-TMAO, meat-carnitine, and plant-based food-betaine associations. No significant association was identified by the MR approach. Our analyses provide novel insights into the TMAO pathway, its determinants, and pathophysiological impact on the general population.

High-throughput metabolomics identifies new biomarkers for cervical cancer

BackgroundCervical cancer (CC) is a danger to women’s health, especially in many developing countries. Metabolomics can make the connection between genotypes and phenotypes. It provides a wide spectrum profile of biological processes under pathological or physiological conditions.MethodIn this study, we conducted plasma metabolomics of healthy volunteers and CC patients and integratively analyzed them with public CC tissue transcriptomics from Gene Expression Omnibus (GEO).ResultHere, we screened out a panel of 5 metabolites to precisely distinguish CC patients from healthy volunteers. Furthermore, we utilized multi-omics approaches to explore patients with stage I-IIA1 and IIA2-IV4 CC and comprehensively analyzed the dysregulation of genes and metabolites in CC progression. We identified that plasma levels of trimethylamine N-oxide (TMAO) were associated with tumor size and regarded as a risk factor for CC. Moreover, we demonstrated that TMAO could promote HeLa cell proliferation in vitro. In this study, we delineated metabolic profiling in healthy volunteers and CC patients and revealed that TMAO was a potential biomarker to discriminate between I-IIA1 and IIA2-IV patients to indicate CC deterioration.ConclusionOur study identified a diagnostic model consisting of five metabolites in plasma that can effectively distinguish CC from healthy volunteers. Furthermore, we proposed that TMAO was associated with CC progression and might serve as a potential non-invasive biomarker to predict CC substage.ImpactThese findings provided evidence of the important role of metabolic molecules in the progression of cervical cancer disease, as well as their ability as potential biomarkers.

Research progress in the relationship between gut microbiota metabolite trimethylamine N-oxide and ischemic stroke.

Ischemic stroke (IS) is a severe cerebrovascular disease that seriously endangers human health. Gut microbiota plays a key role as an intermediate mediator in bidirectional regulation between the brain and the intestine. In recent years, trimethylamine N-oxide (TMAO) as a gut microbiota metabolite has received widespread attention in cardiovascular diseases. Elevated levels of TMAO may increase the risk of IS by affecting IS risk factors such as atherosclerosis, atrial fibrillation, hypertension, and type 2 diabetes. TMAO exacerbates neurological damage in IS patients, increases the risk of IS recurrence, and is an independent predictor of post-stroke cognitive impairment (PSCI) in patients. Current research suggests that the mechanisms of TMAO action include endothelial dysfunction, promoting of foam cell formation, influence on cholesterol metabolism, and enhancement of platelet reactivity. Lowering plasma TMAO levels through the rational use of traditional Chinese medicine, dietary management, vitamins, and probiotics can prevent and treat IS.

Trimethylamine N-Oxide as a Potential Biomarker for Cardiovascular Disease: Its Association with Dietary Sources of Trimethylamine N-Oxide and Microbiota.

Trimethylamine N-oxide (TMAO), the oxidized form of trimethylamine (TMA), was previously thought to be a waste product but is now considered an important risk factor for cardiovascular disease (CVD) and its comorbidities. Foods or supplements containing choline and carnitine are major precursors of TMA in the diet and are metabolized by gut microbiota. Trimethylamine N-oxide is produced through the oxidation of this compound by flavin-containing monooxygenase (FMO) in the liver. The organ responsible for the removal of TMAO from body fluids is the kidneys. Therefore, plasma TMAO levels are influenced by multiple complex factors, especially the amount of TMA precursors and dietary TMAO sources in the diet, the dominant genera in the gut microbiota, FMO3 enzyme activity, and kidney functions. Among these, the quantity of TMAO and its precursors in the diet and microbiota can be considered modifiable risk factors. However, discussions continue regarding how plasma TMAO levels reach pathological levels and their role (consequence or cause) in CVD. This review presents the current scientific evidence on the relationship and underlying mechanisms between CVD and TMAO and provides an overview of the association of plasma TMAO levels with modifiable risk factors, such as dietary TMAO precursors, dietary TMAO sources, and microbiota.

Gut microbial metabolite trimethylamine N-oxide induces aortic dissection

Aortic dissection (AD) is the most catastrophic vascular disease with a high mortality rate. Trimethylamine N-oxide (TMAO), a gut microbial metabolite, has been implicated in the pathogenesis of cardiovascular diseases. However, the role of TMAO in AD and the underlying mechanisms remain unclear. This study aimed to explore the effects of TMAO on AD. Plasma and fecal samples from patients with AD and healthy individuals were collected to analyze TMAO levels and gut microbial species, respectively. The plasma levels of TMAO were significantly higher in 253 AD patients compared with those in 98 healthy subjects (3.47, interquartile range (IQR): 2.33 to 5.18 μM vs. 1.85, IQR: 1.40 to 3.35 μM; p < 0.001). High plasma TMAO levels were positively associated with AD severity. An increase in the relative abundance of TMA-producing genera in patients with AD was revealed using 16S rRNA sequencing. In the angiotensin II or β-aminopropionitrile-induced rodent model of AD, mice fed a TMAO-supplemented diet were more likely to develop AD compared to mice fed a normal diet. Conversely, TMAO depletion mitigated AD formation in the BAPN model. RNA sequencing of aortic endothelial cells isolated from mice administered TMAO revealed significant upregulation of genes involved in inflammatory pathways. The in vitro experiments verified that TMAO promotes endothelial dysfunction and activates nuclear factor (NF)-κB signaling. The in vivo BAPN-induced AD model confirmed that TMAO increased aortic inflammation. Our study demonstrates that the gut microbial metabolite TMAO aggravates the development of AD at least in part by inducing endothelial dysfunction and inflammation. This study provides new insights into the etiology of AD and ideas for its management.