Measurement of urinary trimethylamine and trimethylamime oxide by direct infusion electrospray quadrupole time-of-flight mass spectrometry

Urinary trimethylamine (TMA) and its oxide (TMAOx) are measured separately and as a mixture using ¹⁵N-labeled internal standards and direct infusion electrospray with a quadrupole time-of-flight (Q-ToF) instrument. TMA is quaternized with trideuteromethyl iodide to avoid inclusion of endogenous tetramethylammonium ion in the TMA measurement, whereas TMAOx is measured as the protonated molecule. Measurements reported as percentage TMA made with separate and combined samples agree within 6% of the measured values and demonstrate that both TMA and TMAOx can be measured simultaneously in a single analysis. Moreover, the analysis is simpler and less tedious and time-consuming than some earlier methods.     

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.     

The identification of trimethylamine excess in man: quantitative analysis and biochemical origins

The underlying biochemical causes of chronic odor problems in humans have attracted only a few investigators. This may be due, in part, to intermittent odor complaints, to the psychological problems often associated with these patients, or to the low incidence of a true metabolic disorder. One cause of intense odor is an excessive excretion of trimethylamine (TMA) in sweat, breath, and urine and was reported to be due to a defect in the liver enzyme that converts this volatile amine to its N-oxide. Other investigators have reported TMA excess as a result of liver, kidney, and/or gastrointestinal dysfunction. We report on the development of an analytical technique for urine that can be used to identify those individuals whose chronic odor is caused by a defect in their TMA pathway. A simple extension of the basic TMA analysis can be used to measure the concentration of TMA N-oxide in the affected patients. These data can, in turn, be used to demonstrate the level of activity of the N-oxide-forming enzyme in these subjects. The results obtained from using this test on more than 50 subjects indicate, in addition to a normal population, at least two types of patients with TMA excess. One group has excess TMA excretion with low activity of the N-oxide and another group shows excess TMA excretion with normal N-oxide activity.     

Effects of the dietary supplements, activated charcoal and copper chlorophyllin, on urinary excretion of trimethylamine in Japanese trimethylaminuria patients

Trimethylaminuria (TMAU) is a metabolic disorder characterized by the inability to oxidize and convert dietary-derived trimethylamine (TMA) to trimethylamine N-oxide (TMAO). This disorder has been relatively well-documented in European and North American populations, but no reports have appeared regarding patients in Japan. We identified seven Japanese individuals that showed a low metabolic capacity to convert TMA to its odorless metabolite, TMAO. The metabolic capacity, as defined by the concentration of TMAO excreted in the urine divided by TMA concentration plus TMAO concentration, in these seven individuals ranged from 70 to 90%. In contrast, there were no healthy controls examined with less than 95% of the metabolic capacity to convert TMA to TMAO. The intake of dietary charcoal (total 1.5 g charcoal per day for 10 days) reduced the urinary free TMA concentration and increased the concentration of TMAO to normal values during charcoal administration. Copper chlorophyllin (total 180 mg per day for 3 weeks) was also effective at reducing free urinary TMA concentration and increasing TMAO to those of concentrations present in normal individuals. In the TMAU subjects examined, the effects of copper chlorophyllin appeared to last longer (i.e., several weeks) than those observed for activated charcoal. The results suggest that the daily intake of charcoal and/or copper chlorophyllin may be of significant use in improving the quality of life of individuals suffering from TMAU.     

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.     

Voltammetric detection of trimethylamine using immobilized trimethylamine dehydrogenase on an electrodeposited goldnanoparticle electrode

A voltammetric enzyme electrode was developed based on nicotinamide-independent trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), which catalyses the oxidation of trimethylamine (TMA) to dimethylamine and formaldehyde. A quaternized osmium hydrogel polymer, poly(vinylimidazole-[Os(4,4′-dimethyl-2,2′-bipyridine)₂Cl]^+/2+) with ethylamine (PVI-Os-EA), was prepared as a potential redox mediator in an electrochemical biosensor. TMA was detected using TMADH that was co-immobilized with an osmium hydrogel polymer on electrodeposited gold nanoparticles (Au-NPs) on screen-printed carbon electrodes (SPCEs). The Au-NPs deposited onto SPCEs provided about a three times higher electrochemical response compared to that of a planar gold electrode. As TMA was catalyzed by wired TMADH, the electrical signal was monitored at 0.3 V versus Ag/AgCl by cyclic voltammetry and chronoamperometry. The anode currents increased linearly in proportion to the TMA concentration over the 0 ∼ 2.5 mM range with a detection limit of 1 μM (R = 0.9972).     

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.     

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.     

Structural mechanism for bacterial oxidation of oceanic trimethylamine into trimethylamine N‐oxide

Trimethylamine (TMA) and trimethylamine N‐oxide (TMAO) are widespread in the ocean and are important nitrogen source for bacteria. TMA monooxygenase (Tmm), a bacterial flavin‐containing monooxygenase (FMO), is found widespread in marine bacteria and is responsible for converting TMA to TMAO. However, the molecular mechanism of TMA oxygenation by Tmm has not been explained. Here, we determined the crystal structures of two reaction intermediates of a marine bacterial Tmm (RnTmm) and elucidated the catalytic mechanism of TMA oxidation by RnTmm. The catalytic process of Tmm consists of a reductive half‐reaction and an oxidative half‐reaction. In the reductive half‐reaction, FAD is reduced and a C4a‐hydroperoxyflavin intermediate forms. In the oxidative half‐reaction, this intermediate attracts TMA through electronic interactions. After TMA binding, NADP⁺ bends and interacts with D317, shutting off the entrance to create a protected micro‐environment for catalysis and exposing C4a‐hydroperoxyflavin to TMA for oxidation. Sequence analysis suggests that the proposed catalytic mechanism is common for bacterial Tmms. These findings reveal the catalytic process of TMA oxidation by marine bacterial Tmm and first show that NADP⁺ undergoes a conformational change in the oxidative half‐reaction of FMOs.     

Alphaproteobacteria facilitate Trichodesmium community trimethylamine utilization

In the surface waters of the warm oligotrophic ocean, filaments and aggregated colonies of the nitrogen (N)-fixing cyanobacterium Trichodesmium create microscale nutrient-rich oases. These hotspots fuel primary productivity and harbour a diverse consortium of heterotrophs. Interactions with associated microbiota can affect the physiology of Trichodesmium, often in ways that have been predicted to support its growth. Recently, it was found that trimethylamine (TMA), a globally abundant organic N compound, inhibits N₂ fixation in cultures of Trichodesmium without impairing growth rate, suggesting that Trichodesmium can use TMA as an alternate N source. In this study, ¹⁵N-TMA DNA stable isotope probing (SIP) of a Trichodesmium enrichment was employed to further investigate TMA metabolism and determine whether TMA-N is incorporated directly or secondarily via cross-feeding facilitated by microbial associates. Herein, we identify two members of the marine Roseobacter clade (MRC) of Alphaproteobacteria as the likely metabolizers of TMA and provide genomic evidence that they converted TMA into a more readily available form of N, e.g., ammonium (NH₄⁺), which was subsequently used by Trichodesmium and the rest of the community. The results implicate microbiome-mediated carbon (C) and N transformations in modulating N₂ fixation and thus highlight the involvement of host-associated heterotrophs in global biogeochemical cycling.     

Electrochemical sensing of trimethylamine based on polypyrrole-flavin-containing monooxygenase (FMO3) and ferrocene as redox probe for evaluation of fish freshness

Amperometric and impedimetric biosensor for detecting trimethylamine (TMA) which represents good parameters for estimating fish freshness has been developed. The biosensor is based on a conducting polypyrrole substituted with ferrocenyl, where flavin-containing monooxygenase 3 (FMO3) enzyme was immobilised by covalent bonding. FMO3 catalyzes the monooxygenation TMA to trimethylamine N-oxide (TMO). For catalysis FMO require flavin adenine (FAD) as a prosthetic group, NADPH as a cofactor and molecular oxygen as cosubstrate. Ferrocenyl group substituted on the polypyrrole matrix will serve as redox probe for monitoring the response of the biosensor to TMA. The construction of the biosensor was characterized by FT-IR, cyclic voltammetry and impedance measurements. Detection is done through the analysis of the current of oxidation signal of the ferrocenyl groups and compared to the measurement of impedance related to the electrical properties of the layers. Amperometric and impedimetric response were measured as a function of TMA concentration in range of 0.4 μgm L(-1)-80 μgm L(-1) (6.5 μmol L(-1)-1.5 mmol L(-1)). Amperometric measurements show a decrease in current response which is in correlation with the increase of the charge transfer resistance demonstrated by impedance. Calibration curve obtained by impedance spectroscopy shows a high sensitivity with a dynamic range from (0.4 μgm L(-1) to 80 μgm L(-1)). We demonstrated, using ferrocene as redox probe for catalytic reaction of FMO3, that high sensitivity and dynamic range was obtained. The biosensor was stable during 16 days. The biosensor shows high selectivity and its sensitivity to TMA in real samples was evaluated using fish extract after deterioration during storage.     

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.     

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.     

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.     

Redox cycles in trimethylamine dehydrogenase and mechanism of substrate inhibition.

The steady-state reaction of trimethylamine dehydrogenase (TMADH) with the artificial electron acceptor ferricenium hexafluorophosphate (Fc(+)) has been studied by stopped-flow spectroscopy, with particular reference to the mechanism of inhibition by trimethylamine (TMA). Previous studies have suggested that the presence of alternate redox cycles is responsible for the inhibition of activity seen in the high-substrate regime. Here, we demonstrate that partitioning between these redox cycles (termed the 0/2 and 1/3 cycles on the basis of the number of reducing equivalents present in the oxidized/reduced enzyme encountered in each cycle) is dependent on both TMA and electron acceptor concentration. The use of Fc(+) as electron acceptor has enabled a study of the major redox forms of TMADH present during steady-state turnover at different concentrations of substrate. Reduction of Fc(+) is found to occur via the 4Fe-4S center of TMADH and not the 6-S-cysteinyl flavin mononucleotide: the direction of electron flow is thus analogous to the route of electron transfer to the physiological electron acceptor, an electron-transferring flavoprotein (ETF). In steady-state reactions with Fc(+) as electron acceptor, partitioning between the 0/2 and 1/3 redox cycles is dependent on the concentration of the electron acceptor. In the high-concentration regime, inhibition is less pronounced, consistent with the predicted effects on the proposed branching kinetic scheme. Photodiode array analysis of the absorption spectrum of TMADH during steady-state turnover at high TMA concentrations reveals that one-electron reduced TMADH-possessing the anionic flavin semiquinone-is the predominant species. Conversely, at low concentrations of TMA, the enzyme is predominantly in the oxidized form during steady-state turnover. The data, together with evidence derived from enzyme-monitored turnover experiments performed at different concentrations of TMA, establish the operation of the branched kinetic scheme in steady-state reactions. With dimethylbutylamine (DMButA) as substrate, the partitioning between the 0/2 and 1/3 redox cycles is poised more toward the 0/2 cycle at all DMButA concentrations studied-an observation that is consistent with the inability of DMButA to act as an effective inhibitor of TMADH.     

The involvement of trimethylamine oxide in fish meal in the production of egg taint

Endogenous trimethylamine (TMA) oxidation was inhibited by giving (±)-5-vinyl-2-oxazolidenethione to laying hens that had been bred for low TMA oxidase activity. The addition of TMA oxide to the diet (5 g kg^?1) immediately produced an enormous increase in the TMA content of their eggs and a strong crab-like taint. Hens from another flock whose eggs were tainted when they were previously fed on capelin meal as a protein supplement (100 g kg^?1) again showed this abnormality when TMA oxide was added to the diet (0.5 g kg^?1) to simulate the amounts supplied by the meal. Tests with intravenous ¹⁴C-TMA demonstrated that their ability to oxidise TMA was lower than that of unaffected hens. Dietary TMA oxide and intravenous TMA reduced the oxidation of the test dose of ¹⁴C-TMA. The oxide had no effect when given intravenously and did not inhibit TMA oxidase in vitro. It was concluded that TMA oxide is an important source of TMA in fish meal and that tainting occurs when hens with inherently low TMA oxidase activity are overloaded with TMA derived from dietary TMA oxide and choline by the action of enteric bacteria. The sporadic occurrence of the taint in the field may be due partly to wide variations in the oxide content of fish meals.     

An improved gas--liquid chromatographic method for analysis of trimethylamine in urine.

An established gas-liquid chromatographic method for analysis of trimethylamine in urine was substantially improved by the use of an exchangeable lithium hydroxide, kieselguhr precolumn. Biological samples were acidified immediately upon collection and were analyzed by direct injection onto the precolumn. Using this technique, a normal urinary level of trimethylamine was found to be 5.0 ± 3.1 μmol/mmol creatinine in a group of 17 adults.     

Determination of trimethylamine in fish by capillary electrophoresis with electrogenerated tris(2,2'-bipyridyl)ruthenium(II) chemiluminescence detection.

A capillary electrophoresis with electrogenerated chemiluminescence (CE-ECL) method for the determination of trimethylamine (TMA) in fish was studied. In the presence of TMA, ECL from the reaction of analyte and in situ generated tris(2,2'-bipyridyl)ruthenium(III) [Ru(bpy)(3) (3+)] at electrode surface could be produced. The ECL detection was performed using a Pt working electrode biased at 1.23 V (vs. Ag/AgCl) potential in a 10 mmol/L sodium borate buffer solution, pH 9.2, containing 3 mmol/L Ru(bpy)(3) (2+). A linear calibration curve (correlation coefficient = 0.9996) was obtained in the range 8 x 10(-5)-4 x 10(-8) mol/L for TMA concentration. Recoveries obtained were in the range 98.78-101.46%. The method was successfully applied for the assay of TMA in fish, in combination with solid phase extraction (SPE) disks for sample clean-up and enrichment.     

Sensitive assay of trimethylamine N-oxide in liver microsomes by headspace gas chromatography with flame thermionic detection

To compare the trimethylamine N-oxygenase activity of liver microsomes from house musk shrew (Suncus murinus) and rat, a sensitive method for the quantitation of trimethylamine (TMA) N-oxide was developed using gas chromatography with flame thermionic detection. The limit of quantification was 0.5 μM and the calibration curve was linear at least up to 5 μM in incubations containing liver microsomal preparations from Suncus. The intra-day RSD values ranged from 10.4 to 12.8 at 0.5 μM and from 3.5 to 6.7 at 5 μM. The inter-day RSD values were 11.6 and 6.5 at 0.5 and 5 μM, respectively. This method provides a sensitive assay for TMA N-oxygenase activity in liver microsomes. Using this method we found that Suncus was capable of N-oxidizing trimethylamine at a very slow rate.     

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.