Serum Trimethylamine N-oxide Levels Research Articles

Development of a novel method for the simultaneous detection of trimethylamine N-oxide and creatinine in the saliva of patients with chronic kidney disease – Its utility in saliva as an alternative to blood

Chronic kidney disease (CKD) is associated with increased levels of creatinine and other uremic toxins (UTs), which impaired kidneys cannot filtrate. Typically, CKD is diagnosed by calculating the estimated glomerular filtration rate using serum creatinine or cystatin C levels. In pursuit of more sensitive and reliable biomarkers of kidney dysfunction, scientific attention has turned towards other UTs, such as trimethylamine N-oxide (TMAO), successfully quantified in standard matrices, blood and urine. However, less invasive monitoring of kidney function can be performed using an alternative diagnostic biofluid, saliva, which has been shown to contain clinically relevant concentrations of renal function markers. Accurate quantitative estimation of serum biomarkers using saliva measurements can only be achieved provided that there is a tight saliva-serum correlation for the analyte of interest. Therefore, we aimed to verify the correlation between saliva and serum levels of TMAO in CKD patients using newly developed and validated quantitative liquid chromatography coupled to mass spectrometry (LC-MS) method for simultaneous detection of TMAO, and creatinine – the conventional marker of renal impairment. Secondly, we applied this method to quantify TMAO and creatinine levels in the resting saliva of CKD patients collected with a standardised method involving swab-based collectors. A good linear correlation was obtained between the concentration of creatinine in serum and resting saliva of CKD patients (r = 0.72, p = 0.029) and even better in the case of TMAO (r = 0.81, p = 0.008). The analysed validation criteria were fulfilled. No significant influence of the type of swab in the Salivette® device on creatinine and TMAO concentrations in saliva was detected. Our study indicates that saliva can be successfully used in the non-invasive monitoring of renal failure in CKD by measuring salivary TMAO concentrations.

HIGH SERUM TRIMETHYLAMINE N-OXIDE LEVEL IS ASSOCIATED WITH ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH HYPERTENSION

Objective: Trimethylamine N-oxide (TMAO) is a metabolite derived from gut microbial metabolism and dietary intake and plays important roles in cardiovascular disease pathogenesis. Endothelial dysfunction is associated with risk factors of cardiovascular disease in patients with hypertension. The present study aimed to determine the correlation between serum TMAO and endothelial function, measured by vascular reactivity index (VRI) in patients with hypertension. Design and method: The present study collected fasting blood samples from 110 patients with hypertension. Serum TMAO levels were determination of by high-performance liquid chromatography–mass spectrometry. The endothelial function and VRI values were measured using digital thermal monitoring test, and VRI values of >2.0, 1.0–1.9 and <1.0 indicated good, intermediate and poor vascular reactivity, respectively. Results: Ten hypertensive patients (9.1%) were categorized as poor vascular reactivity (VRI < 1.0), 57 hypertensive patients (51.8%) were categorized as intermediate vascular reactivity (1.0 < VRI < 2.0), and 43 hypertensive patients had good vascular reactivity. Older age (P = 0.010), higher levels of serum total cholesterol (TCH, P = 0.007), low-density lipoprotein cholesterol (LDL-C, P = 0.009), and TMAO levels (P < 0.001) were associated with poor vascular reactivity. Furthermore, after multivariable forward stepwise linear regression analysis noted that logarithmically transformed serum level of TMAO (log-TMAO, adjusted R2 change = 0.274, P < 0.001) was significantly and independently associated with VRI values in hypertensive patients. Compared with good vascular reactivity index, patients with vascular reactivity dysfunction (intermediate vascular reactivity and poor vascular reactivity; odds ratio (OR) = 1.100; 95% confidence interval (CI) = 1.047–1.155; P < 0.001) or poor vascular reactivity (OR = 1.580, 95% CI = 1.002–2.492, P = 0.049) also noted TMAO was positively associated with poor vascular reactivity after multivariate logistic regression analysis. Conclusions: Serum TMAO levels were negatively associated with VRI and associated with endothelial dysfunction in patients with hypertension.

The Relationship of Serum Trimethylamine N-Oxide Levels with Carotid Intima-Media Thickness and Disease Activity in Psoriasis Patients.

Psoriasis is an inflammatory disease that can cause cardiovascular comorbidities. Some recent studies have indicated that impaired gut microbiota and metabolites may be associated with inflammatory diseases. In this study, the relationship between serum trimethylamine n-oxide (TMAO, a gut bacterial metabolite) level and carotid intima-media thickness (CIMT) and disease severity in psoriasis patients was investigated. Age- and gender-matched 73 patients and 72 healthy controls were included in the study. In both groups serum trimethylamine n-oxide(TMAO), oxidized low-density lipoprotein (ox-LDL), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride, total cholesterol, high-sensitivity C-reactive protein (hs-CRP), creatinine, aspartate aminotransferase (AST) and alanine aminotransferase(ALT) levels were recorded and the carotid intima-media thickness (CIMT) was measured by B-mode ultrasonography by a cardiologist. TMAO, hs-CRP, oxidized-LDL, triglyceride and CIMT levels were statistically higher in the patient group. HDL levels were statistically higher in the control group. There was no significant difference between the two groups in terms of total cholesterol and LDL-C levels. In partial correlation analyzes in the patient group, positive correlations were observed between TMAO and CIMT, LDL-C and total cholesterol levels. Linear regression analysis showed that TMAO levels positively predicted CIMT levels. This study confirmed that psoriasis is a risk factor for the development of cardiovascular disease and that elevated serum TMAO levels in these patients indicate the presence of intestinal dysbiosis. Furthermore, TMAO levels were found to be a predictor of the risk of developing cardiovascular disease in psoriasis patients.

The Association of Circulating L-Carnitine, γ-Butyrobetaine and Trimethylamine N-Oxide Levels with Gastric Cancer.

Our study aimed to evaluate the association between gastric cancer (GC) and higher concentrations of the metabolites L-carnitine, γ-butyrobetaine (GBB) and gut microbiota-mediated trimethylamine N-oxide (TMAO) in the circulation. There is evidence suggesting that higher levels of TMAO and its precursors in blood can be indicative of either a higher risk of malignancy or indeed its presence; however, GC has not been studied in this regard until now. Our study included 83 controls without high-risk stomach lesions and 105 GC cases. Blood serum L-carnitine, GBB and TMAO levels were measured by ultra-high-performance liquid chromatography-mass spectrometry (UPLC/MS/MS). Although there were no significant differences between female control and GC groups, we found a significant difference in circulating levels of metabolites between the male control group and the male GC group, with median levels of L-carnitine reaching 30.22 (25.78-37.57) nmol/mL vs. 37.38 (32.73-42.61) nmol/mL (p < 0.001), GBB-0.79 (0.73-0.97) nmol/mL vs. 0.97 (0.78-1.16) nmol/mL (p < 0.05) and TMAO-2.49 (2.00-2.97) nmol/mL vs. 3.12 (2.08-5.83) nmol/mL (p < 0.05). Thus, our study demonstrated the association between higher blood levels of L-carnitine, GBB, TMAO and GC in males, but not in females. Furthermore, correlations of any two investigated metabolites were stronger in the GC groups of both genders in comparison to the control groups. Our findings reveal the potential role of L-carnitine, GBB and TMAO in GC and suggest metabolic differences between genders. In addition, the logistic regression analysis revealed that the only significant factor in terms of predicting whether the patient belonged to the control or to the GC group was the blood level of L-carnitine in males only. Hence, carnitine might be important as a biomarker or a risk factor for GC, especially in males.

Dietary inclusion of nitrite-containing frankfurter exacerbates colorectal cancer pathology and alters metabolism in APCmin mice

Colorectal cancer (CRC) is the second most prevelant malignancy in Europe and diet is an important modifiable risk factor. Processed meat consumption, including meats with preservative salts such as sodium nitrite, have been implicated in CRC pathogenesis. This study investigated how the CRC pathology and metabolic status of adenomatous polyposis coli (APC) multiple intestinal neoplasia (min) mice was perturbed following 8 weeks of pork meat consumption. Dietary inclusions (15%) of either nitrite-free pork, nitrite-free sausage, or nitrite-containing sausage (frankfurter) were compared against a parallel control group (100% chow). Comprehensive studies investigated: gastrointestinal tract histology (tumours), aberrant crypt foci (ACF), mucin deplin foci (MDF), lipid peroxidation (urine and serum), faecal microbiota, and serum metabolomics (599 metabolites). After 8 weeks mice consuming the frankfurter diet had 53% more (P = 0.014) gastrointestinal tumours than control, although ACF and MDF did not differ. Urine and serum lipid peroxidation markers were 59% (P = 0.001) and 108% (P = 0.001) higher, respectively in the frankfurter group. Gut dysbiosis was evident in these mice with comparably fewer Bacteriodes and more Firmicutes. Fasting serum levels of trimethylamine N-oxide (TMAO) and numerous triglycerides were elevated. Various serum phosphotidylcholine species were decreased. These results demonstrate that nitrite-containing sausages may exaccerbate the development of CRC pathology in APCMin mice to a greater extent than nitrite-free sausages, and this is associated with greater lipid peroxidation, wide-ranging metabolic alternation and gut dysbiosis.

Higher serum trimethylamine-N-oxide levels are associated with increased abdominal aortic calcification in hemodialysis patients

Introduction Vascular calcification (VC) is high prevalent and predicts cardiovascular mortality in dialysis patients. The mechanisms are not known clearly. Trimethylamine-N-oxide (TMAO), a gut-microbiota derivate metabolite, is also associated with cardiovascular outcomes in hemodialysis (HD) patients. This study aims to evaluate serum TMAO levels and establish their relation to VC in HD patients. Methods Serum TMAO concentrations were measured by high-performance liquid chromatography–mass spectrometry. Vascular calcification was evaluated by abdominal aortic calcification (AAC) scores. Taking the AAC score value 5.5 as the cutoff value, the participants were divided into the high AAC score group and the low AAC score group. Results A total of 184 HD patients and 39 healthy controls were enrolled in this cross-sectional study. Serum Ln(TMAO) (the natural logarithm of TMAO) concentrations were significantly higher in HD patients than that of control subjects (1.82 ± 0.62 vs. −1.60 ± 0.77, p < 0.001). Compared with the group with low AAC scores, the HD patients with high AAC scores showed significantly higher serum Ln(TMAO) levels (2.09 ± 0.55 vs. 1.67 ± 0.54, p < 0.001). In the multivariate regression analysis, serum Ln(TMAO), HD vintage, with diabetic mellitus, age and plasma intact parathyroid hormone (iPTH) were independent determinant factors for VC in HD patients. Conclusions Higher serum TMAO levels, older age, longer HD vintage, higher plasma iPTH and with diabetes mellitus were independent risk factors for VC in HD patients. The underlying mechanism deserves further investigations and the finding hints at a new target for the treatment of VC.

Dietary choline, via gut microbe- generated trimethylamine-N- oxide, aggravates chronic kidney disease-induced cardiac dysfunction by inhibiting hypoxia-induced factor 1α.

Chronic kidney disease (CKD) is a global public health problem that shortens lifespan primarily by increasing the risk of cardiovascular diseases. Trimethylamine-N-oxide (TMAO), a gut microbiota-derived toxin produced by metabolizing high-choline or carnitine foods, is associated with cardiovascular events in patients with CKD. Although the deleterious effect of TMAO on CKD-induced cardiac injury has been confirmed by various researches, the mechanisms remain unclear. Here, we tested the hypothesis that TMAO aggravates CKD-induced cardiac injury and explores the potential mechanism. CD1 mice underwent 5/6 nephrectomy to induce CKD, and then fed with a diet supplemented with choline (1.2% total) for 8weeks. Serum TMAO levels were elevated in CKD mice compared with SHAM group, and higher TMAO levels were found in choline-supplemented CKD mice compared with CKD group. Dietary choline aggravated CKD-induced cardiac dysfunction, and reducing TMAO levels via medicinal charcoal tablets improved cardiac dysfunction. RNA-seq analysis revealed that dietary choline affected cardiac angiogenesis in CKD mice. Reduced cardiac capillary density and expressions of angiogenesis-related genes were observed in choline-treated CKD mice. Furthermore, dietary choline inhibited cardiac Hif-1α protein level in CKD mice, and Hif-1α stabilizer FG-4592 could improve cardiac angiogenesis and dysfunction in CKD mice on a high-choline diet. In conclusion, these data indicate that dietary choline, via gut microbe-generated TMAO, inhibits cardiac angiogenesis by reducing Hif-1α protein level, ultimately aggravates cardiac dysfunction in CKD mice.

Gut microbiota may mediate the impact of chronic apical periodontitis on atherosclerosis in apolipoprotein E-deficient mice.

There are growing evidences linking chronic apical periodontitis (CAP) to atherosclerosis. Gut microbiota is found to be involved in the development of atherosclerosis. Recent studies have shown that CAP could change the diversity and composition of the gut microbiota. It was therefore, we hypothesized that gut microbiota and its metabolites could mediate the impact of CAP on atherosclerosis. Twenty-four 5-week-old lipoprotein E knockout (apoE-/- ) mice were randomly divided into four groups: the CAP group, Con group, Co-CAP (cohoused with CAP) and Co-Con (cohoused with Con) group. In the CAP group, sterile cotton wool containing P.gingivalis was placed into the exposed pulp chamber, followed by coronal resin-based composite restoration of the bilateral maxillary first and second molars. In the Con group, a sham operation was performed. Biweekly, mice in the CAP group were anaesthetised to check the sealing of coronal access. Meanwhile, the animals in the Con group were anaesthetised. The cohousing approach was used to introduce gut microbiota from the CAP and Con groups into the Co-CAP and Co-Con groups, respectively. Alterations in the abundance and diversity of the gut microbiota were detected using 16S rRNA sequencing, Oil-red O staining was used to demonstrate the extent of lesions, and serum levels of trimethylamine N-oxide (TMAO), and immunohistochemistry of flavin-containing monooxygenase 3 (FMO3) in liver were used to assess TMAO-related metabolic alterations. Alterations of alpha and beta diversity were shown both in the CAP and the Co-CAP groups. Moreover, the percentage of atherosclerotic lesion area increased in the CAP and Co-CAP groups (p < .05). Linear discriminant analysis effect size (LEfSe) at the family level found the increases of Lachnospiraceae and Ruminococcaceae (p < .05), which were positively correlated with serum TMAO levels (p < .05). In the redundancy analysis technique (RDA), serum levels of TMAO were positively associated with the atherosclerotic lesions. Co-occurrence analysis revealed that the relative abundances of Lachnospiraceae and Porphyromonadacae were positively correlated with both the percentage of lesion area and TMAO level (p < .05). Thus, within the limitations of this study, the data suggest that the gut microbiota can mediate the effects of CAP on atherosclerosis.

Correlation between serum trimethylamine-N-oxide concentration and protein energy wasting in patients on maintenance hemodialysis

Objectives Chronic kidney disease (CKD) is a serious health problem that is associated with several systemic changes, including protein energy wasting (PEW). However, the exact mechanism of PEW in CKD remains unclear. As one of the important intestinal flora metabolites and uremic toxins, trimethylamine-N-oxide (TMAO) is involved in CKD-associated mortality, which might play a role in the development of PEW in CKD patients especially in patients on maintenance hemodialysis (MHD). However, this possibility has not been investigated. Methods PEW was diagnosed in a group of CKD patients on MHD according to the criteria of the International Society of Renal Nutrition and Metabolism. Serum TMAO concentration was assessed by high-performance liquid chromatography and mass spectrometry. The association between TMAO concentration and PEW was assessed using linear regression and logistic analysis after adjustment for confounding factors, including basic characteristics, comorbidities, and laboratory findings. Results The circulating TMAO level was higher in the MHD patients than in control (healthy) individuals (5653.76 ± 2853.51 vs. 254.92 ± 197.88 ng/mL, p < 0.001). Further, after the MHD patients were screened for PEW, those with PEW were found to have significantly higher serum TMAO levels than those without PEW (6760.9 vs. 4016.1 ng/mL, p < 0.001). Further, the serum TMAO concentration exhibited a significant negative correlation with body mass index (BMI) and dietary protein intake. In the logistic regression analysis, after adjustment for confounding factors, the serum TMAO concentration was still significantly correlated with PEW occurrence. Conclusions The circulating TMAO level is significantly correlated with the prevalence of PEW in MHD patients. TMAO might be a potential target in the prevention and treatment of PEW in CKD especially ESRD.

Monosodium glutamate consumption reduces the renal excretion of trimethylamine N-oxide and the abundance of Akkermansia muciniphila in the gut

We previously demonstrated that monosodium glutamate (MSG) consumption increases trimethylamine (TMA) level in the renal tissue as well as dimethylamine and methylamine levels in urine of rats, suggesting the effects of MSG on humans. To better define the findings, we investigated whether MSG consumption alters serum trimethylamine N-oxide (TMAO) level, and as a consequence, induces kidney injury in the rat model. Adult male Wistar rats (n = 40) were randomized to be fed with a standard diet (control group) or a standard diet with 0.5, 1.5 or 3.0 g% MSG corresponding to 7, 21, or 42 g/day in 60 kg man, respectively in drinking water (MSG-treated groups), or a standard diet with 3.0 g% MSG in drinking water which was withdrawn after 4 weeks (MSG-withdrawal group). Blood and urine samples were collected to analyze the TMAO levels using 1H NMR and markers of kidney injury. Fecal samples were also collected for gut microbiota analysis. We found serum TMAO levels increased and urinary TMAO excretion decreased during MSG consumption, in parallel with the increase of the neutrophil gelatinase-associated lipocalin (NGAL) excretion which subsided with the withdrawal of MSG. The fecal 16 S rRNA analysis during MSG consumption showed gut microbiota changes with a consistent suppression of Akkermansia muciniphila, a mucin producing bacteria, but not of TMA-producing bacteria. In conclusions, our findings suggested that prolonged high dose MSG consumption may cause TMAO accumulation in the blood via reduction of renal excretion associated with acute kidney injury. The mechanisms by which MSG reduced TMAO excretion require further investigation.

All-Cause Mortality and Trimethylamine N-Oxide Levels in Patients with Cardiovascular Disease

Introduction: Trimethylamine N-oxide (TMAO) is an organic compound with a well-established involvement in the pathogenesis of cardiovascular disease (CVD). However, data on the links between TMAO levels and cardiovascular mortality in Polish patients are lacking. Objectives: We aimed to assess the relationship between serum TMAO levels and 5-year mortality in Polish patients with CVD. Patients and Methods: We retrospectively assessed serum TMAO levels in 1,036 consecutive patients (median age, 62 years; men, 61%) hospitalized between 2013 and 2015. Correlations between TMAO levels and 5-year mortality as well as anthropometric and biochemical parameters were assessed for the whole population and the subgroups of patients with acute coronary syndrome, stable coronary syndrome (SCS), chronic heart failure (HF), and atrial fibrillation (AF). Results: In the univariate analysis, increased TMAO levels predicted 5-year mortality without clinically significant power (hazard ratio [HR], 1.01; 95% CI: 1.006–1.018; p < 0.0001). However, even this weak effect was lost in the multivariate analysis after adjustment for age, sex, comorbidities, and laboratory parameters. In the whole study group, TMAO levels in the fourth quartile of concentration (>6.01 µM) predicted 5-year mortality only in the univariate analysis (HR: 1.55; 95% CI: 1.34–1.79; p < 0.0001). In subgroup univariate analysis, TMAO levels predicted 5-year mortality in patients with SCS, chronic HF, and AF. Conclusions: Despite the promising results of previous studies, our study shows that the level of TMAO has at most moderate value in predicting all-cause mortality. TMAO levels depend on other clinical variables, which limits the use of TMAO as an independent predictor of mortality in these patients.

Serum Trimethylamine N-Oxide Level Is Associated with Peripheral Arterial Stiffness in Advanced Non-Dialysis Chronic Kidney Disease Patients.

Trimethylamine N-oxide (TMAO) is a gut-derived uremic toxin involved in cardiovascular diseases (CVD). Peripheral arterial stiffness (PAS), measured by the brachial-ankle pulse wave velocity (baPWV) is a valuable indicator of the existence of CVD alongside other diseases. The study recruited 157 patients with chronic kidney disease (CKD) stages 3 to 5, and aimed to determine the correlation between serum TMAO and PAS, defined as a baPWV of >18.0 m/s. Patients with CKD who were diagnosed with PAS (68 patients, 43.3%) were older, had a higher percentage of hypertension or diabetes mellitus, higher systolic blood pressure, and higher fasting glucose, C-reactive protein, and TMAO levels. Furthermore, besides old age and SBP, patients with CKD who had higher serum TMAO were more likely to have PAS, with an odds ratio of 1.016 (95% confidence interval = 1.002–1.029, p = 0.021) by multivariate logistic regression analysis. Correlation analysis demonstrated that serum TMAO was positively correlated with C-reactive protein level and either left or right baPWV. Thus, we supposed that serum TMAO levels were associated with PAS in patients with advanced non-dialysis CKD.

Increased Serum Trimethylamine N-Oxide Level in Type 2 Diabetic Patients with Mild Cognitive Impairment.

PurposeTrimethylamine N-oxide (TMAO) is a metabolite of phosphatidylcholine in red meat and other diets, which is associated with cardiovascular and other diseases. The aim of this study is to evaluate the associations of serum TMAO with mild cognitive impairment (MCI) in the Chinese type 2 diabetes mellitus (T2DM) population.Materials and MethodsA total of 253 hospitalized T2DM patients and 150 healthy controls were included in this cross-sectional study. Montreal Cognitive Assessment (MoCA) assessed the cognition function, and the 253 T2DM patients were divided into 74 subjects with MCI and 179 with non-MCI. Demographic data and biochemical test results were evaluated. Serum TMAO level was measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).ResultsA higher serum TMAO level was observed in T2DM patients compared with the healthy controls (P < 0.001). Among all T2DM patients, the MCI group (n = 74) showed higher serum TMAO levels than the non-MCI group. Spearman correlation test showed that TMAO levels were significantly positively correlated with age (r = 0.147, P = 0.019), body mass index (BMI) (r = 0.153, P = 0.015), diabetes duration (r = 0.160, P = 0.011), HbA1c (r = 0.138, P = 0.029), triglyceride (TG) (r = 0.138, P = 0.029), creatinine (r = 0.184, p = 0.003), hs-CRP (r = 0.243, P < 0.001), and were negatively correlated with HDL-C (r = −0.144, P = 0.022), BDNF (r = −0.165, p = 0.009), and MoCA (r = −0.386, P < 0.001) score (all P < 0.05). Multivariable Logistic regression identified high serum TMAO level as a significant independent factor of MCI in the T2DM patients (OR = 1.404, 95% CI = 1.255–1.571; P < 0.001).ConclusionOur study showed that T2DM patients with MCI have elevated serum TMAO levels.

Citrulline and long-term mortality in patients with cardiovascular disease.

Cardiovascular disease (CVD) is associated with intestinal barrier dysfunction and increased intestinal permeability. Increased intestinal permeability to gut microbial metabolites may accelerate the progression of CVD. Plasma citrulline levels are a marker of functional enterocyte mass, and reduced citrulline levels indicate intestinal epithelial damage. Citrulline was reported as a useful prognostic marker in critically ill patients. However, data are lacking on the association of citrulline with long-term mortality in patients with CVD and with the levels of trimethylamine N-oxide (TMAO), a microbiota-derived metabolite which has been implicated in the pathogenesis of CVD. To assess the effect of citrulline levels, a marker of intestinal barrier disruption, on long-term mortality in patients with CVD. Moreover, the relationship between the concentrations of 2 biomarkers - citrulline and TMAO - was assessed. Serum citrulline levels were retrospectively assessed in 1036 consecutive patients with CVD (median age: 62 years; 61% men) hospitalized between 2013 and 2015. Associations of citrulline levels with 5-year mortality rates as well as anthropometric and biochemical parameters were evaluated for the entire study group and in subgroups of patients with acute coronary syndrome (ACS), chronic coronary syndrome, chronic heart failure (chronic HF), and atrial fibrillation (AF). Correlations between serum citrulline and TMAO levels were assessed. The median citrulline level in the study population was 22.5 μM (interquartile range (IQR): 17.8-27.9). Citrulline levels were not associated with 5-year mortality in patients with CVD (hazard ratio (HR) = 0.99; 95% confidence interval (95% CI): 0.97-1.00; p = 0.49). Median citrulline levels differed significantly between deceased patients and survivors at 5 years in patients with ACS (p = 0.025). There were no significant correlations between citrulline and TMAO levels (Kendall's tau = 0.027). Decreasing citrulline levels do not predict long-term mortality of hospitalized patients with CVD. Moreover, they are not associated with the serum levels of TMAO in these patients.

No association in maternal serum levels of TMAO and its precursors in pre-eclampsia and in non-complicated pregnancies.

Only a few studies have explored the role of microbiota-dependent metabolite trimethylamine N-oxide (TMAO) in non-complicated pregnancy and in pre-eclampsia (PE). We enrolled 139 PE and 29 healthy pregnant women in a nested case control study. We hypothesized that elevated levels of circulating TMAO and its precursors choline and glycine betaine in the late second or in third trimester might contribute to the PE and are associated with the onset of the disease and clinical features such as elevated blood pressure. The association with a few available lifestyle factors (use of fish and physical activity) was also evaluated. In contrast with the previous findings, there was no difference in TMAO concentration between PE and healthy women. In addition, TMAO concentration was not associated with any of the PE related clinical features, angiogenic or inflammatory markers. In future, it is crucial to obtain longitudinal data on TMAO in both non-complicated and in PE pregnancies before we could have more detailed understanding of TMAO.

Bioinformatic exploration of trimethylamine N-oxide metabolism in human gut bacteria

Trimethylamine N-oxide (TMAO) is a microbial metabolite that has been shown to have protective effects on the blood–brain barrier, while elevated serum levels of TMAO and its precursors have been linked to cardiometabolic diseases in Western populations. Previous work examined the prevalence of TorA to determine which groups of bacteria were responsible for the metabolism of TMAO in the human gut. This study examined 6 TMAO metabolism pathways to provide a more in-depth analysis of bacterial TMAO metabolism. These results were then filtered for hits with &gt;90% coverage and &gt;70% identity. Results showed that Tor proteins were largely limited to members of the Enterobacteriaceae, mostly appearing in Escherichia coli and Citrobacter spp. &gt;1% of 9898 Klebsiella spp. genomes examined encode any Tor proteins, despite previous work highlighting Klebsiella spp. as one of the prevalent genera encoding TorA. Dms proteins were much more prevalent than TorA in other genera of bacteria, along with MsrP and BisC. 118 of the HGRGs were found to encode for at least 1 TMAO metabolism protein. Overall, this work highlights the need for more comprehensive methods to be used to examine large genomic and metagenomic datasets and the need for in vitro work to be done alongside in silico analyses to improve functional annotations and our understanding of the roles of gut bacteria.

Diet-Induced High Serum Levels of Trimethylamine-N-oxide Enhance the Cellular Inflammatory Response without Exacerbating Acute Intracerebral Hemorrhage Injury in Mice.

Trimethylamine-N-oxide (TMAO), an intestinal flora metabolite of choline, may aggravate atherosclerosis by inducing a chronic inflammatory response and thereby promoting the occurrence of cerebrovascular diseases. Knowledge about the influence of TMAO-related inflammatory response on the pathological process of acute stroke is limited. This study was designed to explore the effects of TMAO on neuroinflammation, brain injury severity, and long-term neurologic function in mice with acute intracerebral hemorrhage (ICH). We fed mice with either a regular chow diet or a chow diet supplemented with 1.2% choline pre- and post-ICH. In this study, we measured serum levels of TMAO with ultrahigh-performance liquid chromatography-tandem mass spectrometry at 24 h and 72 h post-ICH. The expression level of P38-mitogen-protein kinase (P38-MAPK), myeloid differentiation factor 88 (MyD88), high-mobility group box1 protein (HMGB1), and interleukin-1β (IL-1β) around hematoma was examined by western blotting at 24 h. Microglial and astrocyte activation and neutrophil infiltration were examined at 72 h. The lesion was examined on days 3 and 28. Neurologic deficits were examined for 28 days. A long-term choline diet significantly increased serum levels of TMAO compared with a regular diet at 24 h and 72 h after sham operation or ICH. Choline diet-induced high serum levels of TMAO did not enhance the expression of P38-MAPK, MyD88, HMGB1, or IL-1β at 24 h. However, it did increase the number of activated microglia and astrocytes around the hematoma at 72 h. Contrary to our expectations, it did not aggravate acute or long-term histologic damage or neurologic deficits after ICH. In summary, choline diet-induced high serum levels of TMAO increased the cellular inflammatory response probably by activating microglia and astrocytes. However, it did not aggravate brain injury or worsen long-term neurologic deficits. Although TMAO might be a potential risk factor for cerebrovascular diseases, this exploratory study did not support that TMAO is a promising target for ICH therapy.

Rhubarb Enema Decreases Circulating Trimethylamine N-Oxide Level and Improves Renal Fibrosis Accompanied With Gut Microbiota Change in Chronic Kidney Disease Rats.

Objectives: Trimethylamine N-oxide (TMAO), a metabolic product of gut flora, is increased in chronic kidney disease (CKD) subjects and is recognized as one type of uremic toxins which is associated with poor cardiovascular outcomes and kidney function loss. Previous studies have suggested that rhubarb enema could reduce circulating uremic toxins such as urea, creatinine, and indoxyl sulfate and also regulate the intestinal microbiota. However, whether rhubarb enema retards kidney dysfunction by reducing circulating TMAO and its underlying mechanism, are still unclear. The present study aims to investigate the impact of rhubarb enema on TMAO and its precursors, as well as on the intestinal microbiota in 5/6 nephrectomized (5/6Nx) CKD rats. Design: Rats in the treatment groups were given rhubarb enema after modeling. At the end of the study, blood, feces, and kidney tissues were collected and processed for biochemical analyses, histological and western blot analyses, 16S rRNA sequence and untargeted metabolomic analyses. Results: Rhubarb enema reduced serum TMAO and trimethylamine (TMA) levels, inhibited the expression of inflammatory markers (interleukin-6, tumor necrosis factor α and Interferon-γ) and alleviated tubular atrophy, monocyte infiltration and interstitial fibrosis in 5/6Nx CKD rats. Moreover, rhubarb enema significantly increased the abundance of some symbiotic bacteria and probiotics, while reduced the abundance of some potential pathogens at the genus level. In addition, Spearman’s correlation analysis revealed that lachnospiraceae and romboutsia were positively correlated with TMAO. Conclusion: Rhubarb enema decreases circulating TMAO level and improves renal fibrosis in 5/6Nx CKD rats, which may be related to the regulation of intestinal microbial community.

Trimethylamine N-oxide promotes hyperoxaluria-induced calcium oxalate deposition and kidney injury by activating autophagy

Calcium oxalate (CaOx) is the most common component of kidney stones. Oxidative stress, inflammation and autophagy-induced cell death are the major causes of CaOx crystal deposition and CaOx crystal deposition can further lead to kidney injury. Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, plays an important role in the pathogenesis of many diseases, such as atherosclerosis, diabetes and chronic kidney disease, but the effect of TMAO on hyperoxaluria-induced CaOx crystal deposition and kidney injury remains unknown. We hypothesize that TMAO aggravates CaOx crystal deposition via promoting CaOx-mediated cell death. C57Bl/6 mice were given high-oxalate diet as a model of hyperoxaluria. TMAO was provided via drinking water. Serum TMAO levels increased 15 days after CaOx treatment (6.30 ± 0.17 μmol/L vs. 34.65 ± 8.95 μmol/L). High-oxalate diet induced inflammation, CaOx deposition and kidney injury, which TMAO aggravated. In accordance, TMAO intensified high-oxalate diet induced oxidative stress, autophagy and apoptosis. Moreover, TMAO enhanced CaOx crystal adhesion to HK-2 cells and reduced cell viability (from 88.9 ± 1.6% to 75.0 ± 2.7%). Protein kinase R-like endoplasmic reticulum kinase (PERK) may mediate these TMAO effects, as TMAO promoted PERK phosphorylation. Consistently, PERK knockdown alleviated TMAO-evoked CaOx-autophagy, apoptosis and oxidative stress in HK-2 cells. In conclusion, TMAO can aggravate hyperoxaluria-induced kidney injury by triggering the PERK/ROS pathway, which enhances autophagy, apoptosis and inflammation, and facilitates CaOx crystal deposition in renal tubular cells.

Changes in Trimethylamine-N-oxide Levels in Obese Patients following Laparoscopic Roux-en-Y Gastric Bypass or Sleeve Gastrectomy in a Korean Obesity Surgical Treatment Study (KOBESS).

Trimethylamine N-oxide (TMAO), a gut microbe-dependent metabolite, has been implicated as a novel risk factor for cardiovascular events related to obesity and type 2 diabetes mellitus (T2DM). The aim of the study was to test the hypothesis if TMAO is associated with the reduction of cardiovascular disease in the Korean obese patients who underwent bariatric surgery. From a subgroup of a multicenter, nonrandomized, controlled trial, titled KOBESS, 38 obese patients, 18 with and 20 without T2DM, who underwent Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG) were investigated. Bariatric surgery is indicated for Korean patients with a body mass index (BMI) ≥ 35 kg/m2 or for Korean patients with a BMI ≥ 30 kg/m2 who have comorbidities. Serum levels of TMAO and its precursors, betaine, carnitine, and choline were measured before and six months after bariatric surgery. The levels of TMAO and its precursors did not differ between obese patients with T2DM and non-T2DM at baseline. However, TMAO increased more than twofold in patients with T2DM after RYGB surgery, but not in patients without T2DM. Choline levels were decreased by half in all patients after RYGB. In patients with T2DM who underwent SG, TMAO, betaine, and carnitine levels did not change after the surgery. Furthermore, in obese patients who underwent bariatric surgery, increased TMAO levels were associated with both T2DM and RYGB, while reduced choline levels were associated with RYGB. These associations need to be further elucidated in follow-up studies to gain further insights into the relationship between TMAO levels and bariatric surgery outcomes.