Conversion Of Trimethylamine Research Articles

Gut Microbiome and Kidney Disease: Reconciling Optimism and Skepticism.

Gut Microbiome and Kidney Disease: Reconciling Optimism and Skepticism.

Proatherogenic gut-microbiome metabolite trimethylamine elicits a gut-liver circuit to regulate TMAO metabolism and atherosclerosis via mediation of CREBH

Abstract Introduction In humans, circulating metabolite Trimethylamine N-oxide (TMAO) is closely associated with higher risk of cardiovascular disease. Trimethylamine (TMA), a precursor of TMAO, is produced by gut microbiome using dietary components, i.e., choline and carnitine, as substrates. The gut-derived TMA is then transferred to the liver where it is further oxidized to TMAO by the flavin-containing monooxygenases (FMOs). The ER-resident transcription factor c-AMP responsive element binding protein H (CREBH/CREB3L3) is exclusively expressed in the liver and intestine. Perturbation of CREBH activity contributes to the development of hyperlipidemia and cardiovascular disease. Therefore, the Purpose of this study is to investigate the regulatory effect of a gut bacterium, Akkermansia muciniphila (A. muciniphila), on TMA and TMAO metabolism and the role of CREBH in this process. Methods Two groups of wild type (WT) and CREBH knockout (CREBH-KO) mice were inoculated with 200 μL of A. muciniphila (2×108 cfu/0.2 mL) in PBS or the vehicle (PBS) alone as control every other day through oral gavage for 2 weeks. Plasmas, liver and intestinal tissues were collected for metabolomics analysis, immunoblotting analysis and q-RT-PCR. Results Metabolomics analysis of the plasmas from the experimental mice revealed that increased colonization of A. muciniphila in the gut significantly reduced circulating TMA in the WT mice but not in CREBH-KO mice (P<0.05), suggesting that depletion of CREBH altered the microenvironment of gut microbiome which affected the metabolism of TMA by gut bacteria. In the livers, A. muciniphila treatment markedly reduced mRNA expression of FMO1 and FMO3 (P<0.05), which subsequently inhibited the enzymatic conversion of TMA to TMAO in hepatocytes. Immunoblotting analysis further revealed that LDL receptor was upregulated whereas ER stress markers, GRP94 and JNK1/2, were downregulated in the A. muciniphila treated KO mice, indicating an acceleration in lipoprotein (VLDL remnant) clearance from the circulation and the improvement of metabolic inflammation. In vitro, incubation of mouse hepatocytes AML12 with TMA (600 mM) for 12 hours stimulated expression of FMOs to facilitate the conversion of TMA to TMAO and induced lipotoxicity. Conclusion CREBH mediates the crosstalk between gut microbiome and liver metabolic system that regulates TMA and TMAO metabolism, which contributes to the induction of metabolic inflammation and atherogenesis. This novel finding may lend support to the therapeutic strategy of atherosclerosis. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Use of Flavin-Containing Monooxygenases for Conversion of Trimethylamine in Salmon Protein Hydrolysates.

Enzymatic processing of fish by-products for recovery of peptides (hydrolysates) is a promising technology to reach food grade ingredients of high nutritional quality. Despite this, their bitter taste and "fish" odor block implementation in food products and limit their economic potential. Trimethylamine (TMA) is a known contributor to malodor in fish. Current strategies to mask or remove the odor either are not effective or give rise to undesirable side effects. As an alternative approach to remediate TMA, we propose a novel enzymatic strategy to convert TMA into the odorless trimethylamine N-oxide (TMAO) using TMA monooxygenases (Tmms). We identified a diverse set of bacterial Tmms using a sequence similarity network. Purified, recombinant enzymes were assessed for their biocatalytic capacity by monitoring NADPH consumption and TMAO generation. Selected Tmms were subjected to biochemical characterization and investigated for their ability to oxidize TMA in an industry-relevant substrate. From the 45 bacterial Tmm candidates investigated, eight enzymes from four different taxa were selected for their high activity toward TMA. The three most active enzymes were shown to vary in temperature optimum, with the highest being 45°C. Enzymatic activity dropped at high temperatures, likely due to structural unfolding. The enzymes were all active from pH 6.0 to 8.5, with functional stability being lowest around the optimal pH. All three Tmms, given sufficient NADPH cofactor, were found to generate TMAO in the TMA-rich salmon protein hydrolysate. The Tmms serve as unique starting points for engineering and should be useful for guiding process development for marine biorefineries.IMPORTANCE Enzyme-based conversion of marine biomass to high-quality peptide ingredients leaves a distinct smell of "fish" caused by the presence of trimethylamine, which limits their economic potential. We suggest an enzymatic solution for converting trimethylamine to the odorless trimethylamine N-oxide as a novel strategy to improve the smell quality of marine protein hydrolysates. Following a systematic investigation of 45 putative bacterial trimethylamine monooxygenases from several phyla, we expand the repertoire of known active trimethylamine monooxygenases. As a proof-of-concept, we demonstrate that three of these enzymes oxidized trimethylamine in an industry-relevant salmon protein hydrolysate. Our results add new oxidoreductases to the industrial biocatalytic toolbox and provide a new point of departure for enzyme process developments in marine biorefineries.

No effect of plasma trimethylamine N-Oxide (TMAO) and plasma trimethyllysine (TML) on the association between choline intake and acute myocardial infarction risk in patients with stable angina pectoris

Plasma concentrations of trimethylamine N-oxide (TMAO) have been linked to cardiovascular disease (CVD) risk and mortality. TMAO is formed through the bacterial conversion of trimethylamine which is obtained either directly from food, generated from dietary precursors (e.g. choline) or derived from endogenous trimethyllysine (TML) production. In a previous article, we reported an increased risk of acute myocardial infarction with increased total choline intake in patients with stable angina pectoris. Due to the close link between TMAO, TML, choline metabolism and possibly CVD, we investigated whether plasma TMAO and TML modified the effect of total choline intake on acute myocardial infarction (AMI) risk in a post-hoc analysis. We found plasma TMAO and TML do not modify the association between higher dietary choline intake and increased AMI risk. Additionally, this association is not mediated via TMAO. • No effect modification by trimethylamine-N-oxide on the association between choline intake and acute myocardial infarction. • No effect modification by trimethyllysine on the association between choline intake and acute myocardial infarction. • No indirect effect of trimethylamine-N-oxide on the association between choline intake and acute myocardial infarction.

Trimethylamine N-Oxide Metabolites in Early Pregnancy and Risk of Gestational Diabetes: A Nested Case-Control Study.

This study aimed to investigate the associations between trimethylamine N-oxide (TMAO) and related metabolites in early pregnancy and the risk of gestational diabetes mellitus (GDM). A prospective cohort of 22,302 pregnant women from 2010 to 2012 in Tianjin, China, was used to perform a nested case-control study. A total of 243 women with GDM and 243 women without GDM matched by maternal age (±1 year) were used as cases and controls, respectively. Conditional logistic regression and restricted cubic spline were used to examine the full-range risk associations between individual TMAOs metabolites at the first antenatal care visit with GDM. Trimethylamine conversion ratio (TMAR) was defined as trimethylamine (TMA)/its precursors, and trimethylamine N-oxide conversion ratio (TMAOR) was defined as TMAO/TMA. An additive interaction between high TMAR and low TMAOR indicates a state of TMA accumulation, and a mathematical interaction between high TMAR and high TMAOR indicates accumulation of TMAO. TMA was linearly associated with GDM, whereas TMA precursors and TMAO were inversely associated with GDM with clear threshold effects, i.e., 16 nmol/mL for TMAO, 200 nmol/mL for betaine, 112 nmol/mL for l-carnitine, and 110 and 270 nmol/mL for cholinechloride (a U-shaped relationship). Copresence of TMAR >0.35 and TMAOR ≤0.15 was associated with a markedly higher OR (11.16; 95% CI, 5.45 to 22.8), compared with TMAR >0.35 only (OR = 1.71; 95% CI, 0.42 to 6.95) or TMAOR ≤0.15 only (OR = 2.06; 95% CI, 1.09 to 3.90), with a significant additive interaction. However, the mathematical interaction was nonsignificant. TMAO metabolites in the early pregnancy were associated with the risk of GDM, whereas TMA was more likely to play a causal role in GDM.

The genetic and biochemical basis of trimethylaminuria in an Irish cohort.

BackgroundInherited trimethylaminuria (TMAU), a rare genetic disorder of hepatic metabolism of trimethylamine (TMA) causing excessive accumulation of malodorous trimethylamine (TMA), is a socially distressing disorder. Diagnosis is made by biochemical analysis of urine, with the calculation of flavin monooxygenase trimethylamine conversion capacity. Genetic testing, sequencing the entire coding region of the FMO3 gene has been recommended for affected individuals who convert less than 90% of the total TMA load to TMAO.MethodsGenetic analysis was undertaken for 13 Irish patients with TMAU of varying phenotypic severity (three severe, six moderate, and four mild).ResultsA genetic diagnosis was made for seven patients, including for five of the nine moderate to severely affected cases. We noted the c.913G>T;p.(Glu305*) and c.458C>T;p.(Pro153Leu) mutations in this Irish population with severe TMAU which is consistent with our earlier findings in Australian and North American families of Irish and British descent.Three individuals were noted to be homozygous for the common variant haplotype c.472G>A;923A>G;p.(Glu158Lys);(Glu308Gly). We also identified three novel variants in this population, which are likely to be pathogenic: c.682G>A;p(Gly228Ser), c.694G>T:p(Asp232Tyr), and c.989G>A;p.(Gly330Glu).ConclusionUrinary biochemical analysis probably remains the first line diagnostic approach to classify the various types of TMAU. FMO3 gene analysis is likely only to be informative for certain presentations of TMAU.

Goose FMO3 gene cloning, tissue expression profiling, polymorphism detection and association analysis with trimethylamine level in the egg yolk

Flavin-containing monooxygenase 3 (FMO3) plays a critical role in catalyzing the conversion of trimethylamine (TMA) to trimethylamine-N-oxide (TMAO) in vivo. Despite the well-documented association between FMO3 mutations and a ‘fishy’ off-flavor eggs in chicken and quail, little information is available regarding the molecular characteristic of goose (Anser cygnoides) FMO3 and its relationship with the yolk TMA content. To fill these gaps, we cloned the full-length cDNA sequence of goose FMO3, which comprised 1851bp encoding 531 amino acids. FMO3 mRNA was dramatically expressed in liver than in other tissues in the geese. Eight single nucleotide polymorphisms (SNPs) were detected in the entire coding region. The CC genotype at the T669C site, GG at the A723G site, and AA at the G734A site of FMO3 were highly significantly associated with elevated TMA content in goose egg yolk (P<0.001). Carriers of the A allele of G734A or C allele of T885C had yolk TMA content that had a high probability of being elevated after feeding with additional choline chloride (P=0.0429, OR=4.1300, 95%CI=1.0390–16.4270, and P=0.0251, OR=4.6060, 95%CI=1.1620–18.2620, respectively). This work lays a foundation for studying the function of FMO3 and yolk TMA content in goose. However, studies using larger sample sizes and more goose breeds are required to determine whether the fishy off-flavor trait exists in goose.

TO ASSESS THE POTENTIAL OF CURCUMIN AGAINST GUT MICROBIOTA-INDUCED ALTERATION IN CHOLINE METABOLISM IN C57BL/6J MICE

Objective: The present study was focused on assessing the antimicrobial activity of curcumin against Providencia rettgeri, a gut microbe, which was further corelated to the effect of curcumin on choline metabolism and its role in preventing atherosclerosis and on the evaluation of potential of curcumin on the inhibition of conversion of choline to trimethylamine (TMA), and further its conversion to trimethylamine-N-oxide (TMAO) in liver.Methods: The minimum inhibitory concentration of curcumin was determined using Cup-plate method. The estimation of trimethylamine was performed using high performance thin layer chromatography (HP-TLC) technique. Estimation of blood serum parameters were performed using kits supplied by Span-diagnostics for estimation of triglycerides, total cholesterol and high density lipoprotein (HDL) cholesterol. All surgical procedures on animals including blood withdrawal and isolation of livers were carried out in accordance with CPCSEA guidelines.Results: The minimum inhibitory concentration of curcumin was found to be 10 µg/ml. There was a significant increase (p&lt;0.001) in the mean triglyceride, total cholesterol, low-density and very low-density lipoprotein, along with a significant decrease (p&lt;0.001) in high-density lipoprotein in choline-fed mice. Also, there was significant decrease (p&lt;0.001) in the mean triglyceride, total cholesterol, low-density and very low-density lipoprotein, along with a significant increase (p&lt;0.001) in high-density lipoprotein cholesterol in choline-fed mice treated with curcumin. Further, there was a decrease in the Flavin mono-oxygenase (FMO3) activity in the mice livers treated with curcumin by 21.57%.Conclusion: It was concluded that curcumin has the potential to inhibit gut microbiota thus prevention the conversion of choline to trimethylamine, has serum lipid lowering effect in female C57BL/6J mice and has an inhibitory effect on hepatic FMO3 thus preventing the conversion of trimethylamine to trimethylamine-N-oxide.

Involvement of methyltransferases enzymes during the energy metabolism of the anaerobic archaeon methanosarcina semesiae sp. Nov.

The methyl group transfer from dimethylsulfide (DMS), trimethylamine and methanol to 2-mercaptoethanesulfonic acid (coenzyme M) were investigated from cell extracts of Methanosarcina semesiae sp. nov. to evaluate whether the enzyme systems involved were constitutive or inductive. The extracts from cells grown on DMS exhibited methanogenic activity exclusively with DMS and methanethiol. Likewise when cells were pre-grown on trimethylamine or methanol the extracts only produced methane from the respective metaboilic substrate. Dimethylsulfide:methyl-coenzyme M transferase activity was dependent on ATP, but hydrogen did not stimulate activity. The fact that ATP could be replaced by the reductant Ti(III)-citrate indicates that reductive activation of methyl transfer reaction in DMS conversion proceeds in a manner similar to methyltransferasesinvolved in methanol and trimethylamine conversion, but with a different reduction source. This source appears to be limited since sometimes the cell extracts were totally inactive in the presence of ATP, while still being activated with Ti(III)-citrate. It was concluded that enzymes involved inmethyl transfer reactions are specific for each substrate; DMS, trimethylamine and methanol and have to be induced. Further investigations are recommended to corroborate the current study.

Functional characterization of genetic variants of human FMO3 associated with trimethylaminuria

Impaired conversion of trimethylamine to trimethylamine N-oxide by human flavin containing monooxygenase 3 (FMO3) is strongly associated with primary trimethylaminuria, also known as ‘fish-odor’ syndrome. Numerous non-synonymous mutations in FMO3 have been identified in patients suffering from this metabolic disorder (e.g., N61S, M66I, P153L, and R492W), but the molecular mechanism(s) underlying the functional deficit attributed to these alleles has not been elucidated. The purpose of the present study was to determine the impact of these disease-associated genetic variants on FMO3 holoenzyme formation and on steady-state kinetic parameters for metabolism of several substrates, including trimethylamine. For comparative purposes, several common allelic variants not associated with primary trimethylaminuria (i.e., E158K, V257M, E308G, and the E158K/E308G haplotype) were also analyzed. When recombinantly expressed in insect cells, only the M66I and R492W mutants failed to incorporate/retain the FAD cofactor. Of the remaining mutant proteins P153L and N61S displayed substantially reduced (<10%) catalytic efficiencies for trimethylamine N-oxygenation relative to the wild-type enzyme. For N61S, reduced catalytic efficiency was solely a consequence of an increased Km, whereas for P153L, both Km and kcat were altered. Similar results were obtained when benzydamine N-oxygenation was monitored. A homology model for FMO3 was constructed based on the crystal structure for yeast FMO which places the N61 residue alone, of the mutants analyzed here, in close proximity to the FAD catalytic center. These data demonstrate that primary trimethylaminuria is multifactorial in origin in that enzyme dysfunction can result from kinetic incompetencies as well as impaired assembly of holoprotein.

Transient trimethylaminuria related to menstruation

BackgroundTrimethylaminuria, or fish odor syndrome, includes a transient or mild malodor caused by an excessive amount of malodorous trimethylamine as a result of body secretions. Herein, we describe data to support the proposal that menses can be an additional factor causing transient trimethylaminuria in self-reported subjects suffering from malodor and even in healthy women harboring functionally active flavin-containing monooxygenase 3 (FMO3).MethodsFMO3 metabolic capacity (conversion of trimethylamine to trimethylamine N-oxide) was defined as the urinary ratio of trimethylamine N-oxide to total trimethylamine.ResultsSelf-reported Case (A) that was homozygous for inactive Arg500stop FMO3, showed decreased metabolic capacity of FMO3 (i.e., ~10% the unaffected metabolic capacity) during 120 days of observation. For Case (B) that was homozygous for common [Glu158Lys; Glu308Gly] FMO3 polymorphisms, metabolic capacity of FMO3 was almost ~90%, except for a few days surrounding menstruation showing < 40% metabolic capacity. In comparison, three healthy control subjects that harbored heterozygous polymorphisms for [Glu158Lys; Glu308Gly] FMO3 or homozygous for wild FMO3 showed normal (> 90%) metabolic capacity, however, on days around menstruation the FMO3 metabolic capacity was decreased to ~60–70%.ConclusionTogether, these results indicate that abnormal FMO3 capacity is caused by menstruation particularly in the presence, in homozygous form, of mild genetic variants such as [Glu158Lys; Glu308Gly] that cause a reduced FMO3 function.

Physiology and kinetics of trimethylamine conversion by two methylotrophic strains in continuous cultivation systems

The change of dilution rate (D) on both Methylophilus methylotrophus NCIMB11348 and Methylobacterium sp. RXM CCMI908 growing in trimethylamine (TMA) chemostat cultures was studied in order to assess their ability to remove odours in fish processing plants. M. methylotrophus NCIMB11348 was grown at dilution rates of 0.012–0.084 h−1 and the biomass level slightly increased up to values of D around 0.07 h−1. The maximum cell production rate was obtained at 0.07 h−1 corresponding to a maximum conversion of carbon into cell mass (35%). The highest rate of TMA consumption was 3.04 mM h−1 occurring at D=0.076 h−1. Methylobacterium sp. RXM CCMI908 was grown under similar conditions. The biomass increased in a more steep manner up to values of D around 0.06 h−1. The maximum cell production rate (0.058 g l−1h−1) was obtained in the region close to 0.06 h−1 where a maximum conversion of the carbon into cell mass (40%) was observed. The maximum TMA consumption was 2.33 mM h−1 at D=0.075 h−1. The flux of carbon from TMA towards cell synthesis and carbon dioxide in both strains indicates that the cell is not excreting products but directing most of the carbon source to growth. Carbon recovery levels of approximately 100% show that the cultures are carbon-limited. Values for theoretical maximum yields and maintenance coefficients are presented along with a kinetic assessment based on the determination of the substrate saturation constant and maximum growth rate for each organism.

A bubble column continuous fermentation system for trimethylamine conversion by Aminobacter aminovorans

The effect of changing dilution rate on Aminobacter aminovorans grown in chemostat cultures was studied. The microorganism was cultivated at dilution rates between 0.015–0.12 h −1. The biomass level remained fairly constant up to values of D around 0.07 h −1. At higher dilution rates, cell density began to decrease which indicated that the growth rate was leading to a wash-out of the culture. The maximum cell production rate (0.08 g l −1 h −1) was obtained in the region between 0.09 and 0.11 h −1 corresponding to a maximum conversion of the carbon into cell mass (65%). The highest rate of trimethylamine consumption was 1.79 m m h −1 occurring at the D = 0.062 h −1. The biomass productivity profile follows a classical pattern suggesting a culture wash-out near 0.15 h −1. The flux of carbon from trimethylamine toward cell synthesis and carbon dioxide production was expressed in terms of carbon recovery. Data show that the flux of carbon to cells and CO 2 drops from 100% to values between 66.4–74.1% in the region D = 0.027 h −1 to D = 0.07 h −1. The decrease in carbon recovery suggested a shift toward a different growth limitation; however, in the maximum dilution rate region (0.1 h −1) and above, the levels of carbon recovery increased to values closer to carbon limitation. The data is consistent with an industrial trimethylamine transformation using A. aminovorans particularly when the lower growth rates (0.035 h −1 or below) are used.

Oxidation of trimethylamine to the level of formaldehyde byMethanosarcina barkeriis dependent on the protonmotive force

The conversion of trimethylamine to methane, carbon dioxide and ammonia as catalyzed by cell suspensions of Methanosarcina barkeri was coupled to the generation of a protonmotive force and to the synthesis of ATP. Methanogenesis as well as ATP formation and protonmotive force generation was abolished by the uncoupler tetrachloro-salicylanilide (TCS). Inhibition of methane formation was reversed by addition of formaldehyde, which was predominantly oxidized to carbon dioxide, whereas trimethylamine was predominantly reduced to methane and ammonia under these conditions. Cell extracts of M. barkeri were unable to convert trimethylamine to methane, carbon dioxide and ammonia independent from the presence or absence of ATP.

Methanogenesis from trimethylamine + H 2 by Methanosarcina barkeri is coupled to ATP formation by a chemiosmotic mechanism

Cell suspensions of Methanosarcina barkeri catalyzed the conversion of trimethylamine and molecular hydrogen to methane according to the equation (CH 3) 3NH + + 3 H 2 → 3 CH 4 + NH + 4. The onset of methane formation resulted in an increase of the intracellular ATP content from 2 to 4.6 nmol/mg protein and in the generation of a protonmotive force ( Δp) of −130 mV, of which the Δψ contributed 90%. The addition of the uncoupler led to a drastic decrease of the intracellular ATP content and the Δψ, but stimulated methanogenesis. The ATPase inhibitor DCCD caused a rapid exhaustion of the ATP pool and inhibited methane formation, whereas Δψ was not affected. The inhibition of methane formation by DCCD could be relieved by addition of TCS, indicating a chemiosmotic coupling between methane formation according to the above equation and ATP synthesis.

Thermal conversion of trimethylamine- N-oxide to trimethylamine and dimethylamine in squids

The levels of dimethylamine-nitrogen (DMA-N), trimethylamine-nitrogen (TMA-N) and trimethylamine- N-oxide-nitrogen (TMAO-N) were determined in five species of dried squid. Each sample contained extremely high levels of TMAO-N (2558–8064 ppm) and moderate amounts of TMA-N (121–503 ppm) and DMA-N (124–373 ppm). Over 90% of TMAO-N in squid was converted to TMA-N and DMA-N after heating at 200°C for 1 hr; approximately 50% of the volatile TMA-N and DMA-N was lost during the course of the heating. The thermal conversions were accelerated by heat, and possibly involved catalysis by certain tissue constituents. Squids are a popular seafood in most oriental countries, but before appearing on the market they are subjected to a long food-processing procedure. Therefore, a high concentration of TMAO in squids is an important problem, for food technology as well as toxicology.