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Simultaneous quantification of trimethylamine N-oxide, trimethylamine, choline, betaine, creatinine, and propionyl-, acetyl-, and L-carnitine in clinical and food samples using HILIC-LC-MS.

ABSTRACT: Trimethylamine-N-oxide (TMAO), a microbiome-derived metabolite from the metabolism of choline, betaine, and carnitines, is associated to adverse cardiovascular outcomes. A method suitable for routine quantification of TMAO and its precursors (trimethylamine (TMA), choline, betaine, creatinine, and propionyl-, acetyl-, and L-carnitine) in clinical and food samples has been developed based on LC-MS. TMA was successfully derivatized using iodoacetonitrile, and no cross-reactions with TMAO or the other methylamines were detected. Extraction from clinical samples (plasma and urine) was performed after protein precipitation using acetonitrile:methanol. For food samples (meatballs and eggs), water extraction was shown to be sufficient, but acid hydrolysis was required to release bound choline before extraction. Baseline separation of the methylamines was achieved using a neutral HILIC column and a mobile phase consisting of 25 mmol/L ammonium formate in water:ACN (30:70). Quantification was performed by MS using external calibration and isotopic labelled internal standards. The assay proved suitable for both clinical and food samples and was linear from ≈ 0.1 up to 200 μmol/L for all methylamines except for TMA and TMAO, which were linear up to 100 μmol/L. Recoveries were 91-107% in clinical samples and 76-98% in food samples. The interday (n=8, four duplicate analysis) CVs were below 9% for all metabolites in clinical and food samples. The method was applied successfully to determine the methylamine concentrations in plasma and urine from the subjects participating in an intervention trial (n=10) to determine the effect of animal food ingestion on methylamine concentrations.

SUBMITTER: Hefni ME

PROVIDER: S-EPMC8405501 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundTrimethylamine N-oxide (TMAO), a compound derived from diet and metabolism by the gut microbiome, has been associated with several chronic diseases, although the mechanisms of action are not well understood and few human studies have investigated microbes involved in its production.ObjectivesOur study aims were 1) to investigate associations of TMAO and its precursors (choline, carnitine, and betaine) with inflammatory and cardiometabolic risk biomarkers; and 2) to identify fecal microbiome profiles associated with TMAO.MethodsWe conducted a cross-sectional analysis using data collected from 1653 participants (826 men and 827 women, aged 60-77 y) in the Multiethnic Cohort Study. Plasma concentrations of TMAO and its precursors were measured by LC-tandem MS. We also analyzed fasting blood for markers of inflammation, glucose and insulin, cholesterol, and triglycerides (TGs), and further measured blood pressure. Fecal microbiome composition was evaluated by sequencing the 16S ribosomal RNA gene V1-V3 region. Associations of TMAO and its precursors with disease risk biomarkers were assessed by multivariable linear regression, whereas associations between TMAO and the fecal microbiome were assessed by permutational multivariate ANOVA and hurdle regression models using the negative binomial distribution.ResultsMedian (IQR) concentration of plasma TMAO was 3.05 μmol/L (2.10-4.60 μmol/L). Higher concentrations of TMAO and carnitine, and lower concentrations of betaine, were associated with greater insulin resistance (all P < 0.02). Choline was associated with higher systolic blood pressure, TGs, lipopolysaccharide-binding protein, and lower HDL cholesterol (P ranging from <0.001 to 0.03), reflecting an adverse cardiometabolic risk profile. TMAO was associated with abundance of 13 genera (false discovery rate < 0.05), including Prevotella, Mitsuokella, Fusobacterium, Desulfovibrio, and bacteria belonging to the families Ruminococcaceae and Lachnospiraceae, as well as the methanogen Methanobrevibacter smithii.ConclusionsPlasma TMAO concentrations were associated with a number of trimethylamine-producing bacterial taxa, and, along with its precursors, may contribute to inflammatory and cardiometabolic risk pathways.

Project description:Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport.IMPORTANCEVibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.

Project description:BackgroundImpaired wound healing represents a high cost for health care systems. Endothelial dysfunction characterizes dermal microangiopathy and contributes to delayed wound healing and chronic ulcers. Endothelial dysfunction impairs cutaneous microvascular blood flow by inducing an imbalance between vasorelaxation and vasoconstriction as a consequence of reduced nitric oxide (NO) production and the increase of oxidative stress and inflammation. Propionyl-L-carnitine (PLC) is a natural derivative of carnitine that has been reported to ameliorate post-ischemic blood flow recovery.Methods and resultsWe investigated the effects of PLC in rat skin flap and cutaneous wound healing. A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS) reduction, inducible nitric oxide synthase (iNOS) and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and reduction of NADPH-oxidase 4 (Nox4) expression. In serum-deprived human dermal microvascular endothelial cell cultures, PLC ameliorated endothelial dysfunction by increasing iNOS, PlGF, VEGF receptors 1 and 2 expression and NO level. In addition, PLC counteracted serum deprivation-induced impairment of mitochondrial β-oxidation, Nox4 and cellular adhesion molecule (CAM) expression, ROS generation and leukocyte adhesion. Moreover, dermal microvascular endothelial cell dysfunction was prevented by Nox4 inhibition. Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction.ConclusionPLC treatment improved rat skin flap viability, accelerated wound healing and dermal angiogenesis. The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction. Antioxidant therapy and pharmacological targeting of endothelial dysfunction may represent a promising tool for the treatment of delayed wound healing or chronic ulcers.

Project description:BACKGROUND:Intestinal microbiota have been found to be linked to cardiovascular disease via conversion of the dietary compounds choline and carnitine to the atherogenic metabolite TMAO (trimethylamine-N-oxide). Specifically, a vegan diet was associated with decreased plasma TMAO levels and nearly absent TMAO production on carnitine challenge. METHODS AND RESULTS:We performed a double-blind randomized controlled pilot study in which 20 male metabolic syndrome patients were randomized to single lean vegan-donor or autologous fecal microbiota transplantation. At baseline and 2 weeks thereafter, we determined the ability to produce TMAO from d6-choline and d3-carnitine (eg, labeled and unlabeled TMAO in plasma and 24-hour urine after oral ingestion of 250 mg of both isotope-labeled precursor nutrients), and fecal samples were collected for analysis of microbiota composition. 18F-fluorodeoxyglucose positron emission tomography/computed tomography scans of the abdominal aorta, as well as ex vivo peripheral blood mononuclear cell cytokine production assays, were performed. At baseline, fecal microbiota composition differed significantly between vegans and metabolic syndrome patients. With vegan-donor fecal microbiota transplantation, intestinal microbiota composition in metabolic syndrome patients, as monitored by global fecal microbial community structure, changed toward a vegan profile in some of the patients; however, no functional effects from vegan-donor fecal microbiota transplantation were seen on TMAO production, abdominal aortic 18F-fluorodeoxyglucose uptake, or ex vivo cytokine production from peripheral blood mononuclear cells. CONCLUSIONS:Single lean vegan-donor fecal microbiota transplantation in metabolic syndrome patients resulted in detectable changes in intestinal microbiota composition but failed to elicit changes in TMAO production capacity or parameters related to vascular inflammation. CLINICAL TRIAL REGISTRATION:URL: http://www.trialregister.nl. Unique identifier: NTR 4338.

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Simultaneous quantification of trimethylamine N-oxide, trimethylamine, choline, betaine, creatinine, and propionyl-, acetyl-, and L-carnitine in clinical and food samples using HILIC-LC-MS.

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