Trimethylamine N-oxide Levels Research Articles

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.

Targeting the Gut-Kidney Axis in Diarrhea with Kidney-Yang Deficiency Syndrome: The Role of Sishen Pills in Regulating TMAO-Mediated Inflammatory Response.

BACKGROUND Sishen Pills (SSPs) are commonly used to treat diarrhea with kidney-yang deficiency syndrome. Trimethylamine-N-oxide (TMAO) is produced through the metabolism of gut microbiota and can participate in diarrhea in kidney-yang deficiency syndrome by mediating the "gut-kidney axis" to transmit inflammatory factors. This study combined network pharmacology with animal experiments to explore whether SSPs can treat diarrhea with kidney-yang deficiency syndrome by affecting the interaction between TMAO and gut microbiota. MATERIAL AND METHODS A mouse model of diarrhea with kidney-yang deficiency syndrome was constructed by using adenine and Folium sennae decoction, and SSP decoction was used for treatment. This study utilized network pharmacology to predict the potential mechanisms of SSPs in treating diarrhea with kidney-yang deficiency syndrome. 16S rRNA high-throughput sequencing was used to analyze gut mucosal microbial characteristics. ELISA was used to measure TMAO, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), interleukin-1ß (IL-1ß), and transforming growth factor-ß1 (TGF-ß1) levels. We performed Masson and immunohistochemical (Occludin, ZO-1) staining of kidney and small intestinal tissues. The fluorescein diacetate (FDA) hydrolysis spectrophotometric method was used to assess the microbial activity in contents of the small intestine. RESULTS Network pharmacology analysis revealed that SSPs can modulate 108 target points involved in the development of diarrhea, including IL-1ß and TNF. The experimental results demonstrated that SSP decoction significantly improved the general behavioral profiles of the mice, and also reduced TMAO, NLRP3, IL-1ß, and TGF-ß1 levels (P<0.05). Correlation analysis revealed significant positive correlations between TMAO concentrations and NLRP3, IL-1ß and TGF-ß1 levels (P<0.05). Pathological analysis revealed improvements in renal fibrosis and increased expression of the Occludin and ZO-1 proteins in intestinal tissue. In the SSP group, there was a significant increase in microbial activity (P<0.001). According to the sequencing results, the characteristic bacteria of the SSP and NR groups included Succinatimonas hippei, uncultured Solirubrobacter sp., and Clostridium tyrobutyricum. Furthermore, TMAO, NLRP3, IL-1ß, and TGF-ß1 were significantly positively correlated (P<0.05) with Succinatimonas hippei and Clostridium tyrobutyricum. By modulating Firmicutes, Succinatimonas hippei, and Clostridium tyrobutyricum, SSP decoction lowers TMAO levels to alleviate diarrhea with kidney-yang deficiency syndrome. CONCLUSIONS TMAO likely plays a significant role in the "gut-kidney axis" of diarrhea with kidney-yang deficiency syndrome. By adjusting gut microbiota to reduce the inflammatory response that is transmitted through the "gut-kidney axis" as a result of elevated TMAO levels, SSP decoction can alleviate diarrhea with kidney-yang deficiency syndrome.

Circulating TMAO, the gut microbiome and cardiometabolic disease risk: an exploration in key precursor disorders

BackgroundElevations in the gut metabolite trimethylamine-N-oxide (TMAO) have been linked to cardiovascular and metabolic diseases. Whether elevated TMAO levels reflect early mechanistic involvement or a sequela of evolving disease awaits elucidation. The purpose of this study was to further explore these potential associations.MethodsWe investigated relationships between circulating levels of TMAO and its pre-cursor substrates, dietary factors, gut microbiome profiles and disease risk in individuals with a Healthy BMI (18.5 < BMI < 25, n = 41) or key precursor states for cardiometabolic disease: Overweight (25 < BMI < 30 kg/m2, n = 33), Obese (BMI > 30, n = 27) and Metabolic Syndrome (MetS; ≥ 3 ATPIII report criteria, n = 39).ResultsUnexpectedly, plasma [TMAO] did not vary substantially between groups (means of 3–4 µM; p > 0.05), although carnitine was elevated in participants with MetS. Gut microbial diversity and Firmicutes were also significantly reduced in the MetS group (p < 0.05). Exploratory analysis across diverse parameters reveals significant correlations between circulating [TMAO] and seafood intake (p = 0.007), gut microbial diversity (p = 0.017–0.048), and plasma [trimethylamine] (TMA; p = 0.001). No associations were evident with anthropometric parameters or cardiometabolic disease risk. Most variance in [TMAO] within and between groups remained unexplained.ConclusionsData indicate that circulating [TMAO] may be significantly linked to seafood intake, levels of TMA substrate and gut microbial diversity across healthy and early disease phenotypes. However, mean concentrations remain < 5 µM, with little evidence of links between TMAO and cardiometabolic disease risk. These observations suggest circulating TMAO may not participate mechanistically in cardiometabolic disease development, with later elevations likely a detrimental sequela of extant disease.

Trimethylamine N-Oxide in Aquatic Foods.

Trimethylamine N-oxide (TMAO), a characteristic nonprotein nitrogen compound, is widely present in seafood, which exhibits osmoregulatory effects for marine organisms in vivo and plays an important role in aquaculture and aquatic product preservation. However, much attention has been focused on the negative effect of TMAO since it has recently emerged as a putative promoter of chronic diseases. To get full knowledge and maximize our ability to balance the positive and negative aspects of TMAO, in this review, we comprehensively discuss the TMAO in aquatic products from the aspects of physiological functions for marine organisms, flavor, quality, the conversion of precursors, the influences on human health, and the seafood ingredients interaction consideration. Though the circulating TMAO level is inevitably enhanced after seafood consumption, dietary seafood still exhibits beneficial health effects and may provide nutraceuticals to balance the possible adverse effects of TMAO.

Effect of dapagliflozin on ferroptosis through the gut microbiota metabolite TMAO during myocardial ischemia–reperfusion injury in diabetes mellitus rats

Dapagliflozin (DAPA) demonstrates promise in the management of diabetic mellitus (DM) and cardiomyopathy. Trimethylamine N-oxide (TMAO) is synthesized by the gut microbiota through the metabolic conversion of choline and phosphatidylcholine. Ferroptosis may offer novel therapeutic avenues for the management of diabetes and myocardial ischemia–reperfusion injury (IRI). However, the precise mechanism underlying ferroptosis in cardiomyocytes and the specific role of TMAO generated by gut microbiota in the therapeutic approach for DM and myocardial IRI utilizing DAPA need to be further explored. Nine male SD rats with specific pathogen-free (SPF) status were randomly divided equally into the normal group, the DM + IRI (DIR) group, and the DAPA group. The diversity of the gut microbiota was analyzed using 16S rRNA gene sequencing. Additionally, the Wekell technique was employed to measure the levels of TMAO in the three groups. Application of network pharmacology to search for intersection targets of DAPA, DIR, and ferroptosis, and RT-PCR experimental verification. Ultimately, the overlapping targets that were acquired were subjected to molecular docking analysis with TMAO. The changes of Bacteroidetes and Firmicutes in the gut microbiota of DIR rats were most significantly affected by DAPA. Escherichia-Shigella and Prevotella_9 within the phylum Bacteroidetes could be identified as the primary effects of DAPA on DIR. Compared with the normal group, the TMAO content in the DIR group was significantly increased, while the TMAO content in the DAPA group was decreased compared to the DIR group. For the network pharmacology analysis, DAPA and DIR generated 43 intersecting target genes, and then further intersected with ferroptosis-related genes, resulting in 11 overlapping target genes. The mRNA expression of ALB, HMOX1, PPARG, CBS, LCN2, and PPARA decreased in the DIR group through reverse transcription polymerase chain reaction (RT-PCR) validation, while the opposite trend was observed in the DAPA group. The docking score between TMAO and DPP4 was − 5.44, and the MM-GBSA result of − 22.02 kcal/mol. It epitomizes the finest docking performance among all the target genes with the lowest score. DAPA could reduce the levels of metabolite TMAO produced by gut microbiota, thereby regulating related target genes to decrease ferroptosis in DIR cardiomyocytes.

α-Synuclein Overexpression and the Microbiome Shape the Gut and Brain Metabolome in Mice.

Pathological forms of the protein α-synuclein contribute to a family of disorders termed synucleinopathies, which includes Parkinson's disease (PD). Most cases of PD are believed to arise from gene-environment interactions. Microbiome composition is altered in PD, and gut bacteria are causal to symptoms and pathology in animal models. To explore how the microbiome may impact PD-associated genetic risks, we quantitatively profiled nearly 630 metabolites from 26 biochemical classes in the gut, plasma, and brain of α-synuclein-overexpressing (ASO) mice with or without microbiota. We observe tissue-specific changes driven by genotype, microbiome, and their interaction. Many differentially expressed metabolites in ASO mice are also dysregulated in human PD patients, including amine oxides, bile acids and indoles. Notably, levels of the microbial metabolite trimethylamine N-oxide (TMAO) strongly correlate from the gut to the plasma to the brain, identifying a product of gene-environment interactions that may influence PD-like outcomes in mice. TMAO is elevated in the blood and cerebral spinal fluid of PD patients. These findings uncover broad metabolomic changes that are influenced by the intersection of host genetics and the microbiome in a mouse model of PD.

Gut microbiota metabolites and risk of major adverse cardiovascular events and death: A systematic review and meta-analysis.

Gut microbial metabolites such as trimethylamine N-oxide (TMAO) and its precursors, namely betaine, L-carnitine, and choline, have been implicated as risk factors for cardiovascular events and mortality development. Therefore, we aim to perform a systematic review and meta-analysis to assess the validity of these associations. MEDLINE and Scopus were queried from their inception to August 2023 to identify studies that quantified estimates of the associations of TMAO with the development of major adverse cardiovascular events (MACE) or death. A random-effects meta-analysis was conducted to pool unadjusted or multivariable-adjusted hazard ratios (HR) and their 95% confidence intervals. The primary endpoint was the risk of MACE and all-cause death. 30 prospective observational studies (n = 48 968) were included in the analysis. Elevated TMAO levels were associated with a significantly greater risk of MACE and all-cause death compared to low TMAO levels (HR: 1.41, 95% CI 1.2-1.54, P < .00001, I2 = 43%) and (HR: 1.55, 95% CI 1.37-1.75, P < .00001, I2 = 46%), respectively. Furthermore, high levels of either L-carnitine or choline were found to significantly increase the risk of MACE. However, no significant difference was seen in MACE in either high or low levels of betaine. Elevated concentrations of TMAO were associated with increased risks of MACE and all-cause mortality. High levels of L-carnitine/choline were also significantly associated with an increased risk of MACE. However, no significant difference was found between high or low levels of betaine for the outcome of MACE.

Dynamic Changes in Gut Microbiota-Derived Metabolite Trimethylamine-N-Oxide and Risk of Type 2 Diabetes Mellitus: Potential for Dietary Changes in Diabetes Prevention.

A gut-microbial metabolite, trimethylamine N-oxide (TMAO), has been associated with type 2 diabetes mellitus (T2DM). Few previous prospective studies have addressed associations between the changes in TMAO and T2DM incidence. Data were derived from a longitudinal cohort conducted from 2019 to 2021 in rural areas of Fuxin County, Liaoning Province, China, and 1515 diabetes-free participants aged above 35 years were included. The concentrations of serum TMAO and its precursors were measured at two time points, namely in 2019 and 2021. TMAO and TMAO changes (ΔTMAO) were separately tested in a logistic regression model. For further examination, the odds ratios (ORs) for T2DM were calculated according to a combination of TMAO levels and ΔTMAO levels. During a median follow-up of 1.85 years, 81 incident cases of T2DM (5.35%) were identified. Baseline TMAO levels exhibited a nonlinear relationship, first decreasing and then increasing, and only at the highest quartile was it associated with the risk of T2DM. The OR for T2DM in the highest quartile of serum TMAO was 3.35 (95%CI: 1.55-7.26, p = 0.002), compared with the lowest quartile. As for its precursors, only choline level was associated with T2DM risk and the OR for T2DM in the Q3 and Q4 of serum choline was 3.37 (95%CI: 1.41-8.05, p = 0.006) and 4.72 (95%CI: 1.47-15.13, p = 0.009), respectively. When considering both baseline TMAO levels and ΔTMAO over time, participants with sustained high TMAO levels demonstrated a significantly increased risk of T2DM, with a multivariable-adjusted OR of 8.68 (95%CI: 1.97, 38.34). Both initial serum TMAO levels and long-term serum TMAO changes were collectively and significantly associated with the occurrence of subsequent T2DM events. Interventions aimed at normalizing TMAO levels, such as adopting a healthy dietary pattern, may be particularly beneficial in T2DM prevention.

#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.

Donepezil ameliorates gut barrier disruption in doxorubicin-treated rats

An impact of donepezil against doxorubicin-induced gut barrier disruption and gut dysbiosis has never been investigated. Twenty-four male Wistar rats were divided into three groups. Each group was treated with either vehicle as a control, doxorubicin, or doxorubicin-cotreated with donepezil. Heart rate variability was assessed to reflect the impact of doxorubicin and donepezil. Then, animals were euthanized, and the ileum and its contents were collected in each case to investigate the gut barrier and gut microbiota, respectively. The microbiota-derived endotoxin, trimethylamine N-oxide (TMAO), and short-chain fatty acids (SCFAs) in the serum were determined. An increase in the sympathetic tone, endotoxins, and TMAO levels with disruption of the gut barrier and a decrease in SCFAs levels were observed in doxorubicin-treated rats. Gut microbiota of doxorubicin-treated rats was significantly different from that of the control group. Donepezil treatment significantly decreased the sympathetic tone, restored the gut barrier, and reduced endotoxin and TMAO levels in doxorubicin-treated rats. Nonetheless, donepezil administration did not alter the gut microbiota profile and levels of SCFAs in doxorubicin-treated rats. Doxorubicin impaired the autonomic balance and the gut barrier, and induced gut dysbiosis, resulting in gut toxicity. Donepezil partially improved the doxorubicin-induced gut toxicity through balancing the autonomic disturbance.

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.

In-hospital trimethylamine N-oxide (TMAO) variation predicts major adverse cardiovascular events in myocardial infarction survivors

Abstract Funding Acknowledgements None. Background Trimethylamine N-oxide (TMAO), a gut microbiota result of the fermentation of animal foods rich in choline, L-carnitine and phosphatidylcholine (red meat, egg yolk, dairy food), is associated with alterations in lipid metabolism, increased foam cell formation, promotion of inflammation, oxidative stress, platelet aggregation and thrombosis. To date the data evaluating TMAO as a predictor of mortality and ischemic events in patients with acute myocardial infarction (AMI) based on one-time measurement of TMAO levels lead to conflicting results. TMAO levels are fluctuating and in addition to diet, genetic factors, general condition and some co-morbidities affect its circulating levels. Purpose Since, there is a need for better risk stratification of AMI patients before the hospital discharge for more personalised treatment, we evaluated in-hospital variation in TMAO levels and its impact on outcome. Methods Blood samples from 149 consecutive AMI patients were collected on admission and on discharge. The endpoint was a three-point major adverse cardiovascular events (MACE) composed as all-cause mortality, reinfarction and development of heart failure. Results The majority of AMI patients were male (77%), hospitalized for first coronary event, with mean age 64(11) years. Median circulating TMAO level was significantly higher on admission than on discharge, (7.81 [3.47 –19.98] vs 3.45 [2.3 – 4.78] μM, respectively, p&amp;lt;0.001). In order to evaluate the impact of TMAO variation during the hospital stay on outcome, we estimated TMAO cut-off value by continuous hazard ratio analysis; values above the 3.45 μM were associated with incident of MACE in a linear fashion (Fig. 1a). Subsequently, we stratified AMI patients in 2 groups: 1) LL/HL (low-low/high-low) that included 75 (50.3%) patients whose TMAO levels remained low or were high and decreased during hospitalization and 2) HH/LH (high-high/low-high) including 74 patients (49.7%) patients whose TMAO levels remained elevated or were initially low, but rose above the estimated threshold during hospitalisation (Fig. 1b). Thirty-two (21.5%) patients reached the combined endpoint during the median follow-up of 30 months. The Kaplan-Meier analysis demonstrated that patients from the HH/LH group had a higher likelihood of experiencing MACE (p=0.05) (Fig. 2a). The multivariate Cox analysis revealed that patients in the HH/LH group had over twice the risk of MACE compared to those in the LL/HL group. Furthermore, older age and worse left ventricular systolic function were identified as independent predictors of MACE during long-term follow-up (Fig. 2b). Conclusions AMI patients with permanently elevated or increasing levels of TMAO have a worse outcome. The novelty and the strength of our study is the fact that the direction of variation of TMAO during the index hospitalisation identifies patients at higher risk of MACE during 30 months follow-up.

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.

Exploring the relationship between trimethylamine N-Oxide levels, kidney function, and diabetes

Exploring the relationship between trimethylamine N-Oxide levels, kidney function, and diabetes

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.

Intra-hospital variation of gut microbiota product, trimethylamine N-oxide (TMAO), predicts future major adverse cardiovascular events after myocardial infarction

ObjectiveTrimethylamine N-oxide (TMAO) has been associated with atherosclerosis and poor outcome. We evaluated the prognostic impact of intra-hospital TMAO variation on patient outcome. Methods and ResultsBlood samples from 149 patients with acute myocardial infarction (AMI) were taken on admission and discharge. Plasma TMAO was determined by HPLC-MS. The endpoint was a composite three-point MACE (major adverse cardiovascular events), including all-cause mortality, re-infarction, or heart failure (HF) development. Median TMAO concentration on admission was significantly higher than on discharge (respectively, 7.81 [3.47–19.98] vs 3.45 [2.3–4.78] μM, p < 0.001). After estimating the 3.45 μM TMAO cut-off with the analysis of the continuous hazard ratio, we divided our cohort into two groups. The first group included 75 (50.3%) patients whose TMAO levels remained below or decreased under cut-off (low-low/high-low; LL/HL), while the second group included 74 (49.7%) patients whose TMAO levels remained high or increased above the cut-off during hospitalisation (high-high/low-high; HH/LH). During the median 30-month follow-up, 21.5% of patients experienced the composite endpoint. At Kaplan-Meier analysis, a trend of increasing MACE risk was observed in patients in the HH/LH group (p = 0.05). At multivariable Cox analysis, patients from the HH/LH group had more than two times higher risk of MACE during the follow-up than the LL/HL group (HR = 2.15 [95% CI, 1.03–4.5], p = 0.04). Other independent predictors of MACE were older age and worse left ventricular systolic function. ConclusionIn patients with AMI, permanently high or increasing TMAO levels during hospitalisation are associated with a higher risk of MACE during long-term follow-up.

Abstract 128: Blood TMAO Predicts Abdominal Aortic Aneurysm Growth Rate And Need For Surgical Intervention

Background: Patients known to have abdominal aortic aneurysms (AAAs) undergo surveillance imaging to predict both the risk for acute aortic syndromes including aortic dissection and aortic rupture, and the need for surgical intervention. Aortic rupture is a feared consequence of rapidly expanding AAAs. Aims: A blood biomarker that predicts risk for AAA and distinguishes slow from fast AAA growth does not exist but could be a valuable tool for risk stratification. We recently reported systemic levels of the gut microbe-generated metabolite trimethylamine N-oxide (TMAO) are associated with the presence of AAA in humans, and directly contribute to AAA progression in animal models. Methods: We leveraged access 805 patients in two cohorts from Europe (n=237) and the United States (n=658) undergoing serial aortic imaging surveillance. Blood TMAO concentration was measured by stable isotope dilution mass spectrometry. Results: TMAO was positively associated with larger baseline and follow-up abdominal aortic diameter beyond traditional factors (Adjusted OR 3.87, 95% CI, 2.70-5.58, P&lt;0.001). In addition, TMAO predicted rapid AAA growth (HR 1.81, 95%CI, 1.23-2.67, P=0.0029). When TMAO was added to a predictive model of traditional risk factors for AAA, we saw a significant improvement in the net reclassification index (NRI, 0.43, P &lt; 0.001; C statistic, 0.77 (0.72–0.81) with TMAO vs. NRI 0.73 (0.69–0.78) without TMAO, P =0.005). Finally, including TMAO in a predictive model for assessing need for surgical intervention among AAA patients significantly improved the prognostic performance of the model (NRI, 0.44, P &lt; 0.001; C statistic, 0.78 (0.73–0.82) with TMAO vs. 0.75 (0.70–0.79) without TMAO, P =0.004) (Fig. 1). Conclusions: Elevated TMAO levels predict increased risk of AAA, and identify patients with fast-growing AAA and those who may benefit from early surgical intervention to prevent acute aortic syndromes.

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.

Abstract 3090: Oral Administration Of Akkermansia Muciniphila Ameliorates Venous Wall Fibrosis: Therapeutic Implication Of A Novel Probiotic Therapy Against Post-thrombotic Syndrome

Introduction: Post-thrombotic syndrome (PTS) is the most common sequelae of deep vein thrombosis (DVT), manifested as venous insufficiency in up to 50% of patients. Unfortunately, none of the existing therapies, including anticoagulants and thrombolytics, have shown definitive effects for PTS, presenting a vital unmet clinical need. Akkermansia Muciniphila (AKK) , a gut commensal anaerobic bacterium, has been recognized with pleiotropic benefits in a wide range of diseases including cardiometabolic disorders. However, its therapeutic and mechanistic implications in thromboembolic diseases and the subsequent PTS remain unknown. We hypothesize that probiotic therapy with live AKK after DVT formation could ameliorate venous wall fibrosis, indicative of PTS. Methods: In male specific-pathogen free c57b6/j mice (10-12 weeks), DVT was experimentally created via partial ligation of inferior vena cava (IVC) as previously established, leading to progressive venous fibrosis. For the probiotic treatment, AKK (strain BAA-835) was cultured using BHI broth in an anaerobic Coy chamber, followed by daily oral gavage to mice beginning from day 1 post-DVT induction. At day 14 post-procedure, IVC was collected for Masson Trichrome and immunofluorescent staining, followed by quantitation of venous collagen deposition/fibrosis and phosphorylation of endoplasmic reticulum (ER) stress PERK kinase. Endpoint plasma specimens were collected to quantify the circulating levels of trimethylamine N-oxide (TMAO). Results: Post-DVT probiotic therapy with live AKK effectively mitigated venous wall fibrosis in comparison to BHI culture media gavage alone (fibrotic area: 61.12±6.64 vs 53.17±5.99; p&lt;0.05). TMAO, the only microbial metabolite associated with thromboembolic risk, was significantly reduced in plasma following AKK probiotic therapy. Furthermore, phosphorylation of ER stress PERK, which is directly activated by TMAO and critically contributes to myofibroblast activation and organ fibrosis, was inhibited in the AKK -treated IVC (435.09±26.49 vs 326.86±17.61; p&lt;0.01). Conclusion: Our current study supports the potential use of Post-DVT AKK probiotic therapy for the treatment of PTS, possibly via modulating the TMAO-PERK axis.