Suppression of intestinal microbiota-dependent production of pro-atherogenic trimethylamine N-oxide by shifting L-carnitine microbial degradation

Aims

Trimethylamine-N-oxide (TMAO) is produced in host liver from trimethylamine (TMA). TMAO and TMA share common dietary quaternary amine precursors, carnitine and choline, which are metabolized by the intestinal microbiota. TMAO recently has been linked to the pathogenesis of atherosclerosis and severity of cardiovascular diseases. We examined the effects of anti-atherosclerotic compound meldonium, an aza-analogue of carnitine bioprecursor gamma-butyrobetaine (GBB), on the availability of TMA and TMAO.

Main methods

Wistar rats received L-carnitine, GBB or choline alone or in combination with meldonium. Plasma, urine and rat small intestine perfusate samples were assayed for L-carnitine, GBB, choline and TMAO using UPLC-MS/MS. Meldonium effects on TMA production by intestinal bacteria from L-carnitine and choline were tested.

Key findings

Treatment with meldonium significantly decreased intestinal microbiota-dependent production of TMA/TMAO from L-carnitine, but not from choline. 24 hours after the administration of meldonium, the urinary excretion of TMAO was 3.6 times lower in the combination group than in the L-carnitine-alone group. In addition, the administration of meldonium together with L-carnitine significantly increased GBB concentration in blood plasma and in isolated rat small intestine perfusate. Meldonium did not influence bacterial growth and bacterial uptake of L-carnitine, but TMA production by the intestinal microbiota bacteria K. pneumoniae was significantly decreased.

Significance

We have shown for the first time that TMA/TMAO production from quaternary amines could be decreased by targeting bacterial TMA-production. In addition, the production of pro-atherogenic TMAO can be suppressed by shifting the microbial degradation pattern of supplemental/dietary quaternary amines.     

Trimethylaminuria (fish odor syndrome): Genotype characterization among Portuguese patients

Trimethylaminuria (TMAu) or “fish odor syndrome” is a metabolic disorder characterized by the inability to convert malodorous dietarily-derived trimethylamine (TMA) to odorless TMA N-oxide by the flavin-containing monooxygenase 3 (FMO3). Affected individuals unable to complete this reaction exude a “fishy” body odor due to the secretion of TMA in their corporal fluids leading to a variety of psychosocial problems. Interindividual variability in the expression of FMO3 gene may affect drug and foreign chemical metabolism in the liver and other tissues. Therefore, it is important to screen for common TMAu mutations but also extend the search to other genetic variants in order to correlate genotype and disease-associated phenotypes.     

Anaerobic growth of halophilic archaeobacteria by reduction of dimethysulfoxide and trimethylamine N-oxide

Abstract Most representatives of the halophilic arachaeobacterial genera Halobacterium, Haloarcula and Haloferax tested were able to reduce dimethylsulfoxide (DMSO) to dimethylsulfide (DMS) and trimethylamine N -oxide (TMAO) to trimethylamine (TMA) under (semi)anaerobic conditions. In most cases the reduction of DMSO and TMAO was accompanied by an increase in cell yield. The ability to reduce DMSO or TMAO was not correlated to reduced DMSO or TMAO was not correlated with the ability to reduce nitrate to nitrite. Anaerobic respiration with DMSO and TMAO as electron acceptor supplies the halophilic archeobacteria with an additional mode of energy generation in the absence of molecular oxygen.     

Trimethylaminuria: causes and diagnosis of a socially distressing condition

Trimethylaminuria is a disorder in which the volatile, fish-smelling compound, trimethylamine (TMA) accumulates and is excreted in the urine, but is also found in the sweat and breath of these patients. Because many patients have associated body odours or halitosis, trimethylaminuria sufferers can meet serious difficulties in a social context, leading to other problems such as isolation and depression. TMA is formed by bacteria in the mammalian gut from reduction of compounds such as trimethylamine-N-oxide (TMAO) and choline. Primary trimethylaminuria sufferers have an inherited enzyme deficiency where TMA is not efficiently converted to the non-odorous TMAO in the liver. Secondary causes of trimethylaminuria have been described, sometimes accompanied by genetic variations. Diagnosis of trimethylaminuria requires the measurement of TMA and TMAO in urine, which should be collected after a high substrate meal in milder or intermittent cases, most simply, a marine-fish meal. The symptoms of trimethylaminuria can be improved by changes in the diet to avoid precursors, in particular TMAO which is found in high concentrations in marine fish. Treatment with antibiotics to control bacteria in the gut, or activated charcoal to sequester TMA, may also be beneficial.     

Serum pharmacokinetics of choline,trimethylamine, and trimethylamine-N-oxide after oral gavage of phosphatidylcholines with different fatty acid compositions in mice

Little is known about the pharmacokinetics of phosphatidylcholine (PC)-derived choline, trimethylamine (TMA), and trimethylamine-N-oxide (TMAO). We therefore aim to investigate serum choline, TMA, and TMAO pharmacokinetics following different PCs gavage and compare the difference between PC emulsions and liposomes (SOL). Serum choline, TMA, and TMAO levels were measured after orally gavaged egg yolk PC emulsion (EGE), squid PC emulsion (SQE), soybean PC emulsion (SOE), and SOL in fasted mice. Time to reach peak concentration (Tmax) and productions for TMA and TMAO were more slow and less in SQE group compared with EGE and SOE groups. Tmax for choline, TMA, and TMAO prolonged, and the productions of them were significantly declined in SOL group compared to SOE group. These findings indicated that marine source squid PC could counter-regulate the potential risks of TMAO generation, and the use of liposome as the form of PC supplementary may eliminate TMAO production.     

Mutation, polymorphism and perspectives for the future of human flavin-containing monooxygenase 3

Flavin-containing monooxygenases (FMOs) catalyze NADPH-dependent monooxygenation of soft-nucleophilic nitrogen, sulfur, and phosphorous atoms contained within various drugs, pesticides, and xenobiotics. Flavin-containing monooxygenase 3 (FMO3) is responsible for the majority of FMO-mediated xenobiotic metabolism in the adult human liver. Mutations in the FMO3 gene can result in defective trimethylamine (TMA) N-oxygenation, which gives rise to the disorder known as trimethylaminuria (TMAU) or "fish-odour syndrome". To date 18 mutations of FMO3 gene have been reported that cause TMAU, and polymorphic variants of the gene have also been identified. Interindividual variability in the expression of FMO3 may affect drug and foreign chemical metabolism in the liver and other tissues. It is important therefore to study how base sequence variation of the FMO3 gene might affect the ability of individuals and different ethnic population groups to deal with the variety of environmental chemicals and pharmaceutical products that are substrates for FMO3.     

FMO3 allelic variants in Sicilian and Sardinian populations: Trimethylaminuria and absence of fish-like body odor

The N-oxygenation of amines by the human flavin-containing monooxygenase (form 3) (FMO3) represents an important means for the conversion of lipophilic nucleophilic heteroatom-containing compounds into more polar and readily excreted products. In healthy individuals, virtually all Trimethylamine (TMA) are metabolized to Trimethylamine N-oxide (TMAO). Several single nucleotide polymorphisms (SNPs) of the FMO3 gene have been described and result in an enzyme with decreased or abolished functional activity for TMA N-oxygenation thus leading to TMAU, or fish-like odor syndrome. Three coding region variants, c. G472A (p.E158K) in exon 4, c. G769A (p.V257M) in exon 6, and c.A923G (p.E308G) in exon 7, are common polymorphisms identified in all population examined so far and are associated with normal or slightly reduced TMA N-oxygenation activity. However, simultaneous occurrence of 158K and 308G variants results in a more pronounced decrease in FMO3 activity. A fourth polymorphism, c. G1424A (p.G475D) in exon 9, less common in the general population, was observed in individuals suffering severe or moderate trimethylaminuria.     

A microbial biosensor for trimethylamine using Pseudomonas aminovorans cells

A biosensor system based on the difference in the oxygen uptake response of two microbial electrodes was developed to monitor trimethylamine (TMA). The first electrode, constructed using Pseudomonas aminovorans grown on TMA, was sensitive to TMA, trimethylamine N-oxide (TMAO), dimethylamine (DMA) and monomethylamine (MMA). The second electrode responding to TMAO, DMA and MMA was prepared using Ps. aminovorans grown on TMAO. The difference in oxygen uptake was linearly related to the TMA concentration in the range of 5-26 microM. The minimum detectable level was 2.6 microM and the relative standard deviation was determined to be 14% for 16 repeated analyses. When operated and stored at 30 degrees C, the response of the system was stable for only 2 days. However, when the biosensor system was operated at 30 degrees C but stored overnight at 4 degrees C, the system was stable up to 20 days. The biosensor system was applicable for the determination of TMA in fish tissue extracts and the results compared well with those determined by HPLC.     

Mechanism of biosynthesis of trimethylamine oxide from choline in the teleost tilapia, Oreochromis niloticus, under freshwater conditions

The mechanism of biosynthesis of trimethylamine oxide (TMAO) from dietary precursors in the teleost tilapia (Oreochromis niloticus) was investigated. Diets supplemented with quaternary ammonium choline, glycine betaine, carnitine or phosphatidylcholine were administered and significant increases in TMAO levels in the muscle were only observed with choline. [Methyl-14C] and [1,2-14C] cholines were given through dietary and intraperitoneal injection routes, but 14C-TMAO was detected only in fish with dietary administration of [methyl-14C] choline. Dietary treatment with [15N] choline resulted in the formation of [15N] TMAO in the muscle. The incorporation of radioactivity into TMAO was also observed both following dietary administration and intraperitoneal injection of [14C] trimethylamine (TMA). When choline was introduced into the isolated intestine, marked increases in TMA levels occurred. These increases were significantly suppressed in the presence of penicillin. [14C]-TMA derived from [methyl-14C] choline was detected in the cavity of the isolated intestine. The introduction of [15N] choline into the intestinal cavity resulted in the formation of [15N] TMA. TMA mono-oxygenase activities were detected in the liver and kidney. We conclude that tilapia possess the ability to produce TMAO from choline, which is related to intestinal microorganisms and tissue mono-oxygenase under freshwater conditions.     

The measurement of dimethylamine, trimethylamine, and trimethylamine N-oxide using capillary gas chromatography-mass spectrometry

We have developed a method for measuring dimethylamine (DMA), trimethylamine (TMA), and trimethylamine N-oxide (TMAO) in biological samples using gas chromatography with mass spectrometric detection. DMA, TMA, and TMAO were extracted from biological samples into acid after internal standards (labeled with stable isotopes) were added. p-Toluenesulfonyl chloride was used to form the tosylamide derivative of DMA. 2,2,2-Trichloroethyl chloroformate was used to form the carbamate derivative of TMA. TMAO was reduced with titanium(III) chloride to form TMA, which was then analyzed. The derivatives were chromatographed using capillary gas chromatography and were detected and quantitated using electron ionization mass spectrometry (GC/MS). Derivative yield, reproducibility, linearity, and sensitivity of the assay are described. The amounts of DMA, TMA, and TMAO in blood, urine, liver, and kidney from rats and humans, as well as in muscle from fishes, were determined. We also report the use of this method in a pilot study characterizing dimethylamine appearance and disappearance from blood in five human subjects after ingesting [13C]dimethylamine (0.5 mumol/kg body wt). The method we describe was much more reproducible than existing gas chromatographic methods and it had equivalent sensitivity (detected 1 pmol). The derivatized amines were much more stable and less likely to be lost as gases when samples were stored. Because we used GC/MS, it was possible to use stable isotopic labels in studies of methylamine metabolism in humans.     

Metabolism of trimethylamines in kelp bass (Paralabrax clathratus) and marine and freshwater pink salmon (Oncorhynchus gorbuscha)

Summary ³H or¹⁴C labeled tracers were used to investigate the metabolism of trimethylamine (TMA), trimethylamine oxide (TMAO), choline, and betaine in free swimming kelp bass (Paralabrax clathratus). An indwelling cannula in the ventral aorta was used to administer tracer and withdraw blood samples. The concentrations of TMA and TMAO were determined in liver, muscle, and plasma. The TMA liver content is higher than that of muscle (0.85 vs 0.01 moles/g wet tissue) while the amount of TMAO in muscle greatly exceeds its liver concentration (60 vs 0.04 moles/g wet tissue). Prolonged fasting (21 and 75 days) or feeding the fish a squid diet containing high levels of TMAO did not alter the tissue concentrations of TMA or TMAO, suggesting that these compounds are endogenous in origin and that their tissue concentrations are subject to regulation. Comparison of the radiospecific activities of TMA and TMAO, and the administered TMA tracer suggest that TMA is channled directly to TMAO in the liver without equilibration in the hepatic TMA pool. The conversion kinetics of TMA to TMAO and the distribution of these amines in liver and muscle with time suggest that labeled TMA is rapidly taken up into a sequestered pool from which it is slowly released, oxidized to TMAO in the liver, and then transported via the circulation to the muscle mass. The location of this proposed sequestered TMA pool was not determined. Experiments with labeled choline and betaine suggest that these compounds are interconverted in the liver and that enzymes are present for conversion of choline betaine TMA TMAO. Labeled dimethylamine (DMA) was not metabolized and is, therefore, probably not a precursor of TMA and TMAO. [¹⁴C]Trimethylamine (TMA) was also used to investigate the possible role of trimethylamine oxide (TMAO) as an osmoregulatory compound in migrating prespawning cannulated Pacific pink salmon (Oncorhynchus gorbuscha) taken from marine or fresh water environments. Marine and fresh water salmon oxidized administered [¹⁴C]TMA to TMAO; labeled metabolites other than TMA and TMAO were not detected. Four hours after [¹⁴C]TMA injection about 10% of the administered dose was present in muscle as labeled TMAO and about 33% as TMA. Unlike our finding in kelp bass, [¹⁴C]TMAO was not recovered in liver, although low amounts of labeled TMA were found (0.4% of administered dose). Labeled TMA and TMAO, however, were detected in liver after [¹⁴C]betaine adminstration to a marine salmon, indicating that TMA-mono-oxygenase is present in salmon liver. The presence of labeled choline indicates that choline and betaine are interconverted as in kelp bass. The amount of [¹⁴C]TMA oxidized to [¹⁴C]TMAO and then accumulated in the muscle mass is the same in marine and fresh water salmon, as is the amount of chemical TMAO present (4.6 moles/g muscle).     

Elevated levels of trimethylamine oxide in deep-sea fish: evidence for synthesis and intertissue physiological importance

Tissue levels of trimethylamine oxide (TMAO) were compared for seven teleost and two elasmobranch species captured from three depth ranges: shallow (<150 m), moderate (500-700 m), and deep (1,000-1,500 m). Within the teleosts, the deep-caught species had significantly greater TMAO content than shallow- or moderate-caught species. In all teleosts, muscle had substantially more TMAO than all other tissues. Kidney or, in some cases, liver had elevated trimethylamine (TMA) content, 2.20-9.65 mmol/kg, along with appreciable trimethylamine oxidase (TMAoxi) activity, suggesting active TMAO synthesis. No correlation was found between TMAoxi activity and TMAO content. The elasmobranchs in this study, Squalus acanthias and Centroscyllium fabricii from shallow and deep water, respectively, were both squaliform sharks. The deep-caught species had significantly more TMAO in all tissues than the shallow species. Furthermore, urea was significantly less in the deep species in all tissues except liver, while the urea:TMAO ratio was significantly less in all tissues. As with teleosts, the TMAO content of muscle was substantially higher for both elasmobranchs than in all other tissues. TMAoxi was below levels of detection in both elasmobranch species, suggesting that TMAO is obtained solely from the diet. This study expands the trend of increased muscle TMAO in deep-sea fish to a variety of other tissues. The accumulation of TMAO in various tissues in deep-sea teleosts and the accumulation of TMAO and concurrent urea decrease in a deep-sea elasmobranch in comparison to a shallow water species strongly support the contention that TMAO is of physiological importance in deep-sea fish.     

Maintenance and accumulation of trimethylamine oxide by winter skate (Leucoraja ocellata): reliance on low whole animal losses rather than synthesis

Trimethylamine oxide (TMAO) is typically accumulated as an organic osmolyte in marine elasmobranchs to levels second only to urea (which can reach >400 mM); however, little is known about the whole animal regulation of TMAO in elasmobranchs. In the present study on the winter skate (Leucoraja ocellata), we determine whether this species can maintain levels of TMAO in the absence of feeding, and if so, is this due to endogenous synthesis or low whole animal losses. Winter skates maintain plasma TMAO levels for up to 45 days without feeding. The liver displays methimazole oxidation, which is consistent with the presence of flavin-containing monooxygenase (E.C. 1.14.13.8) activity, the class of enzymes responsible for the physiological oxygenation of trimethylamine (TMA) to TMAO in mammals. However, no evidence for TMA oxygenation by winter skates was found using in vivo or in vitro techniques, indicating no significant capacity for endogenous TMAO synthesis. Fed skates displayed low, but measurable ( approximately 4-13 micromol.kg(-1).h(-1)), efflux of TMAO (plus TMA), whereas fasted skates did not. Using the loss of injected [14C]TMAO, it was determined that whole animal TMAO losses are likely <1% of whole body TMAO per day. These results demonstrate that winter skates utilize low whole animal TMAO losses, rather than endogenous synthesis, to maintain TMAO levels when not feeding.     

Enzymatic and physiological properties of the tungsten-substituted molybdenum TMAO reductase from Escherichia coli

The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a molybdoenzyme that catalyses the reduction of the TMAO to trimethylamine (TMA) with a redox potential of +130 mV. We have successfully substituted the molybdenum with tungsten and obtained an active tungsto-TMAO reductase. Kinetic studies revealed that the catalytic efficiency of the tungsto-substituted TMAO reductase (W-TorA) was increased significantly (twofold), although a decrease of about 50% in its kcat was found compared with the molybdo-TMAO reductase (Mo-TorA). W-TorA is more sensitive to high pH, is less sensitive to high NaCl concentration and is more heat resistant than Mo-TorA. Most importantly, the W-TorA becomes capable of reducing sulphoxides and supports the anaerobic growth of a bacterial host on these substrates. The evolutionary implication and mechanistic significance of the tungsten substitution are discussed.     

Microcantilevers modified by specific peptide for selective detection of trimethylamine

We report a biosensor based on a microcantilever that is modified by a specific peptide for highly selective detection of trimethylamine (TMA). The assay is based on binding-induced bending of the peptide functionalized microcantilevers. The sensor is selectively responsive to TMA. The amplitude of microcantilever bending at equilibrium is a function of the concentration of TMA with a dynamic range from 8 ppm to 800 ppm. The detection limit is approximately 8 ppm. There is a good intra-sensor and an acceptable inter-sensor reproducibility as evidenced by the standard deviation of 5% and 15%, respectively.     

Transmission of Atherosclerosis Susceptibility with Gut Microbial Transplantation

Recent studies indicate both clinical and mechanistic links between atherosclerotic heart disease and intestinal microbial metabolism of certain dietary nutrients producing trimethylamine N-oxide (TMAO). Here we test the hypothesis that gut microbial transplantation can transmit choline diet-induced TMAO production and atherosclerosis susceptibility. First, a strong association was noted between atherosclerotic plaque and plasma TMAO levels in a mouse diversity panel (n = 22 strains, r = 0.38; p = 0.0001). An atherosclerosis-prone and high TMAO-producing strain, C57BL/6J, and an atherosclerosis-resistant and low TMAO-producing strain, NZW/LacJ, were selected as donors for cecal microbial transplantation into apolipoprotein e null mice in which resident intestinal microbes were first suppressed with antibiotics. Trimethylamine (TMA) and TMAO levels were initially higher in recipients on choline diet that received cecal microbes from C57BL/6J inbred mice; however, durability of choline diet-dependent differences in TMA/TMAO levels was not maintained to the end of the study. Mice receiving C57BL/6J cecal microbes demonstrated choline diet-dependent enhancement in atherosclerotic plaque burden as compared with recipients of NZW/LacJ microbes. Microbial DNA analyses in feces and cecum revealed transplantation of donor microbial community features into recipients with differences in taxa proportions between donor strains that were transmissible to recipients and that tended to show coincident proportions with TMAO levels. Proportions of specific taxa were also identified that correlated with plasma TMAO levels in donors and recipients and with atherosclerotic lesion area in recipients. Atherosclerosis susceptibility may be transmitted via transplantation of gut microbiota. Gut microbes may thus represent a novel therapeutic target for modulating atherosclerosis susceptibility.     

The effect of oxygen on denitrification during steady-state growth of Paracoccus halodenitrificans

Dimethylsulphoxide (DMSO) and trimethylamine oxide (TMAO) sustained anaerobic growth of Proteus vulgaris with the non-fermentable substrate lactate. Cytoplasmic membrane vesicles energized by electron transfer from formate to DMSO displayed anaerobic uptake of serine, which was hindered by metabolic inhibitors known to destroy the proton motive force. This showed that DMSO reduction was coupled with a chemiosmotic mechanism of energy conversion; similar data for TMAO respiration have been presented previously. All biochemical tests applied indicated that the oxides were reduced by the same reductase system. The DMSO and TMAO reductase activities showed the same mobility on ion-exchange chromatography, and polyacrylamide disc gel electrophoresis (pH 8.9), gradient gel electrophoresis, and gel isoelectric focusing; mol. wt. and pI determined were 95,000 and 4.6, respectively. DMSO inhibited reduction of [¹⁴C]TMAO in vesicles. The reductase was inducible to a certain extent; both oxides being equally efficient as inducers. TMAO was reduced at a higher rate than DMSO, explaining faster growth of cells and increased uptake of serine in vesicles with TMAO as electron acceptor. Comparative studies with Escherichia coli also gave evidence for common TMAO and DMSO reductase systems.Abbreviations TMAO trimethylamine oxide - DMSO dimethylsulphoxide     

Anaerobic Fish Spoilage by Bacteria II. Kinetics of Bacterial Growth and Substrate Conversions in Herring Extracts

The time course of the conversions of chemical components in herring extracts during anaerobic growth of Proteus sp., str. NTHC 153, Aeromonas sp., str. NTHC 154, and Enterobacter sp., str. NTHC 151 (Strøm & Larsen 1979) has been studied. When the Proteus sp. or the Aeromonas sp. were inoculated into the herring extracts and incubated at 15°C under anaerobic conditions, the sugar components (i.e. mainly ribose, free and bound) were the first substrates utilized. These compounds were converted to acetate and CO₂ by the use of trimethylamine oxide (TMAO) as an external hydrogen acceptor. Growth of bacteria ceased when all TMAO was reduced to trimethylamine (TMA). By adding an extra amount of TMAO to the herring extracts an increased growth of the Proteus sp. and the Aeromonas sp. ensued. The increased growth occurred concomitantly with a further conversion of TMAO to TMA and of lactate to acetate and CO₂. The Enterobacter sp., which did not utilize lactate, did not give an increased growth in herring extracts enriched with TMAO.     

Measurement of trimethylamine and trimethylamine N-oxide independently in urine by fast atom bombardment mass spectrometry

We report a method based upon fast atom bombardment mass spectrometry (FAB-MS) and stable isotope dilution techniques for the measurement of urinary trimethylamine (TMA) and trimethylamine N-oxide (TMAOx). TMA is extracted from urine that was spiked with (15)N-labeled TMA. The extracted TMA isotopomers are quaternized with trideuteromethyl iodide and analyzed in FAB-MS with hexaethylene glycol as matrix. TMAOx is measured by evaporation of another sample of the urine spiked with (15)N-labeled TMAOx on the FAB probe and analyzed as for the TMA. The method allows the ready and simple distinguishing of controls and patients with TMAuria, and is useful in monitoring patients with the disorder. We give examples of its use in determining normal control ranges for these metabolites and in evaluating patients.