To Trimethylamine N-oxide Research Articles

Nutrition in Stroke Prevention

Nutrition is far more important than most physicians and patients recognize. Maintaining a healthy weight, reducing dietary cholesterol, avoiding egg yolks, and consuming a Cretan Mediterranean diet substantially reduce the risk for stroke. Besides the very high cholesterol content of egg yolks (more than the recommended daily intake of 200 mg of cholesterol in a single large egg yolk), it is now apparent that phosphatidylcholine (lecithin, 250 mg per egg yolk) is converted by intestinal bacteria to trimethylamine, which in turn is oxidized in the liver to trimethylamine n-oxide (TMAO), which is pro-atherosclerotic in animal models and increases the risk for cardiovascular events in patients at risk for coronary disease. Unrecognized metabolic deficiency of vitamin B12 is very common, and frequently missed, because a ‘normal’ serum B12 is not a reliable test for adequacy of B12. Besides neuropathy, myelopathy, and dementia, B12 deficiency increases the risk for stroke by raising levels of total homocysteine (tHcy). Despite widespread misunderstanding of the complex issues, it is increasingly clear that B vitamin therapy to reduce homocysteine does reduce the risk for stroke. However adequacy of B12 dosing and renal impairment determine the response to B vitamin therapy: cyanocobalamin is beneficial in patients with normal kidney function, but harmful in those with renal impairment (GFR<50). Methylcobalamin should be used in patients with renal impairment.

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.The aim of this study was to determine the allelic and genotypic distributions of these four FMO3 variants in 528 healthy individuals collected from the Sicilian and Sardinian populations together with haplotype and linkage analyses. Finally, we present data on the genotype–phenotype correlation by ESI-MS/MS TMA/TMAO urinary determination in 158KK/308EG individuals.Variant 158K shows the same frequency in Sicilian and Sardinian populations while variant 257M was not observed in the Sardinian sampling. No significant differences were found for 308G and 475D variants among two populations. Cis-linkage between 158K and 308G was confirmed with the compound variant (158K–308G) being found in a proportion of 0.9% and 0.3% of Sicilian subjects, and 0.01% and 0.5% in Sardinian population.Urinary determination of TMA/TMAO ratio in 158KK/308EG individuals showed a considerable reduction in FMO3 activity although they do not show the classical features of trimethylaminuria as a strong body odor and breath.Our data support the conclusion that trimethylaminuria is not always accompanied by a fish-like odor, despite the coexistence in the same individual of the two variants 158K and 308G, and other factors account for the expression of that phenotype.

Menopause status explains large individual variation in cardiovascular disease risk marker response to different dietary choline intake levels

Excess dietary choline has been implicated in increased cardiovascular disease (CVD) risk, a process mediated by bacterial catabolism of choline to trimethylamine N‐oxide (TMAO). To determine whether variation in dietary choline leads to differences in plasma TMAO and CVD risk in human subjects, we enrolled twenty female participants in a two‐month, controlled dietary intervention study where they were exposed to three different intake levels of choline‐equivalents (choline, phosphatidylcholine and other choline‐containing compounds). Plasma samples collected at the end of each exposure were analyzed using liquid chromatography time‐of‐flight mass spectrometry to determine TMAO levels. Plasma oxidized low density lipoprotein (oxLDL), a marker for CVD risk, was measured using sandwich ELISA to establish whether changes in dietary choline levels were associated changes in CVD risk. Our analysis identified significant dose‐response relationships between choline equivalent intake and both plasma TMAO response (P=2.15e‐05 – MANOVA) and plasma oxLDL levels (P=0.0285). TMAO and oxLDL response variation were largely explained by menopause status (P=0.0003 and P=0.0014 respectively). These results suggest the possibility that bacteria‐mediated TMAO production may be important in the increased CVD risk observed in post‐menopausal women. NIH DK055895, DK056350 and RR00046 supported this work.

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 0.4μgmL−1–80μgmL−1 (6.5μmolL−1–1.5mmolL−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 (0.4μgmL−1 to 80μgmL−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.

G protein-coupled receptor mediated sensing of TMA

A new approach for the detection of trimethylamine (TMA) using recombinant Xenopus laevis melanophores was developed. The cells were genetically modified to express the mouse trace amine-associated receptor 5 (mTAAR5), a G protein-coupled receptor from the olfactory epithelium, which conferred high sensitivity to TMA. A focused chemical screen allowed the discovery of additional, previously unknown stimuli of mTAAR5. The cell-based sensor demonstrated no sensitivity to trimethylamine N-oxide (TMAO), making it suitable for a convenient evaluation of TMA levels in fish tissue extracts. The developed gas measurement platform was able to detect TMA from 1 to 100 ppm within thirty-five minutes.

G protein-coupled receptor mediated trimethylamine sensing

A new approach for the detection of trimethylamine (TMA) using a recombinant cell line of Xenopus laevis melanophores was developed. The cells were genetically modified to express the mouse trace amine-associated receptor 5 (mTAAR5), a G protein-coupled receptor from the mouse olfactory epithelium, which conferred high sensitivity to TMA. Cellular responses to TMA were analyzed by two different techniques, either by absorbance measurements using a microplate reader or by cellular imaging via an inverted microscope. A focused chemical screen allowed the discovery of additional, previously unknown stimuli of mTAAR5. The developed cell-based sensor demonstrated no sensitivity to trimethylamine N-oxide (TMAO), making it suitable for a straightforward evaluation of TMA levels in fish tissue extracts. For the detection of TMA vapor, the cells were covered with agarose, which allowed for intact cell viability for at least 6h in air. The developed gas measurement platform was able to detect TMA from 1 to 100ppm within 35min.

Trimetilaminuria: el síndrome de olor a pescado

Primary trimethylaminuria, or fish odor syndrome, is a congenital metabolic disorder characterized by a failure in the hepatic trimethylamine (TMA) oxidation route to trimethylamine N-oxide (TMANO). TMA is mostly derived from dietary precursors such as choline, carnitine and TMANO. The presence of abnormal amounts of TMA in the urine, sweat, exhaled air and other body secretions confers a very unpleasant body odor resembling that of decaying fish. As a consequence, patients can suffer from serious psychosocial sequelae. We present a case of primary trimethylaminuria with the aim of raising awareness about this condition.

Terminal Electron Acceptors Influence the Quantity and Chemical Composition of Capsular Exopolymers Produced by Anaerobically Growing Shewanella spp.

Bacterial exopolymers perform various roles, including acting as a carbon sink, a protective layer against desiccation or antimicrobial agents, or a structural matrix in biofilms. Despite such varied roles, little is known about the heterogeneity of bacterial exopolymer production under varying growth conditions. Here we describe experiments designed to characterize the quantity and quality of exopolymers produced by two commonly studied members of the widely distributed genus Shewanella. Electrokinetic, spectroscopic, and electron microscopic techniques were employed to demonstrate that cell surfaces of Shewanella oneidensis MR-1 (electrophoretic softness, lambda(-1), range from 0.4 to 2.6 nm) are associated with less extracellular polymeric material than surfaces of Shewanella putrefaciens 200R (lambda(-1) range from 1.6 to 3.0 nm). Both species exhibit similar responses to changes in electron acceptor with nitrate- and fumarate-grown cells producing relatively little exopolymer compared to trimethylamine N-oxide (TMAO)-grown cells. In S. oneidensis, the increase in exopolymers has no apparent effect upon cell-surface fixed charge density (-7.7 to -8.7 mM), but for S. putrefaciens a significant drop in fixed charge density is observed between fumarate/nitrate-grown cells (-43 mM) and TMAO-grown cells (-20.8 mM). For both species, exopolymers produced during growth on TMAO have significant amide functionality, increasing from approximately 20-25% of C-containing moieties in nitrate-grown cells to over 30% for TMAO-grown cells (determined from X-ray photoelectron spectroscopy). The increased exopolymer layer associated with TMAO-grown cells appears as a continuous, convoluted layer covering the entire cell surface when viewed by low-temperature, high-resolution scanning electron microscopy. Such significant changes in cell-surface architecture, dependent upon the electron acceptor used for growth, are likely to influence a variety of cell interactions, including aggregation and attachment to surfaces, and the binding of aqueous metal species.

Enhancement of Polyunsaturated Fatty Acid Production by Tn5 Transposon in Shewanella baltica

Transposon Tn5 mutagenesis was used to generate random mutations in Shewanella baltica MAC1, a polyunsaturated fatty acid (PUFA)-producing bacterium. Three mutants produced 3-5 times more eicosapentaenoic acid (EPA 20:5 n-3) compared to the wild type at 10 degrees C. One of the mutants produced 0.3 mg EPA g(-1) when grown at high temperature (30 degrees C). Moreover, 2 mg docosahexaenoic acid (DHA 22:6 n-3) g(-1) was produced by S. baltica mutants at 4 degrees C. Sequencing of insertion mutation(s) showed 96% homology to trimethylamine N-oxide (TMAO) reductase gene and 85% homology to rRNA operons of E. coli. Tn5 transposon mutagenesis therefore is a suitable technique to increase PUFA formation in bacteria.

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.

Trimetilaminuria o síndrome del olor a pescado. Nueva mutación génica y primer caso documentado en España

Trimethylaminuria or fish odor syndrome is a metabolic disorder characterized by a failure in the oxidation route from trimethylamine (TMA) to trimethylamineN-oxide (TMA-O). Primary trimethylaminuria is an inherited autosomic recessive disease due to mutations in the human FMO3 gene. High levels of free TMA in urine and other body fluids confer an unpleasant body odor resembling that of fish. Here we report a case of primary trimethylaminuria in a 4-year-old girl. A 4-year-old girl who presented with a strong corporal scent resembling that of fish from the age of 9 months agreeing with the introduction of fish in the diet. The patient did not have other relevant personal history and had a correct psychomotor and growing development. Liver function, urea and creatinine levels were normal. The biochemical diagnosis was done by spectrometry, measuring the amount of TMA and TMA-O prior to and after fish intake. Genetic analysis evinced that the patient was homozygous for a novel mutation in exon 3, R51G (c. 151A > G). Both parents were heterozygous. R51G (c. 151 A > G) mutation had not been found in other patients with trimethylaminuria.

An unorthodox sensor protein (TorS) mediates the induction of the tor structural genes in response to trimethylamine N-oxide in Escherichia coli.

We isolated and characterized three spontaneous mutations leading to trimethylamine N-oxide (TMAO)-independent expression of the tor operon encoding the TMAO-reductase anaerobic respiratory system in Escherichia coli. The mutations lie in a new for regulatory gene, the torS gene, which probably encodes a sensor protein of a two-component regulatory system. One mutation, which leads to full TMAO-constitutive expression, is a 3-amino-acid deletion within the potential N-terminal periplasmic region, suggesting that this region contains the TMAO-detector site. For the other two mutations, a further induction of the tor operon is observed when TMAO is added. Both are single substitutions and affect the linker region located between the detector and the conserved transmitter domains. Thus, as proposed for other sensors, the TorS linker region might play an essential role in propagating conformational changes between the detector and the cytoplasmic signalling regions. The TorS histidine kinase is an unorthodox sensor that contains a receiver and a C-terminal alternative transmitter domain in addition to the domains found in most sensors. Previously, we showed that TMAO induction of the for operon requires the TorR response regulator and the TorT periplasmic protein. Additional genetic data confirm that torS encodes the sensor partner of TorR and TorT. First, insertion within torS abolishes tor operon expression whatever the growth conditions. Second, overexpressed TorR bypasses the requirement for torS, whereas the torT gene product is dispensable for tor operon expression in a torS constitutive mutant. This supports a signal-transduction cascade from TorT to TorR via TorS.

The N-oxidation of trimethylamine in a Jordanian population.

The ability to oxidise trimethylamine (TMA) to trimethylamine N-oxide (TMAO) is distributed polymorphically within a British white population with the majority of individuals excreting greater than 90% of total urinary TMA as TMAO. The opposite extreme is characterised by a rare inborn error of TMA N-oxidation known as the fish-odour syndrome. However there is a lack of information regarding inter-individual variability in the N-oxidation of TMA in other ethnic groups. In this study the urinary excretion of TMA and TMAO was determined over a period of 24 h in 82 Jordanian subjects. A frequency distribution histogram of % of total urinary TMA excreted as TMAO revealed that the majority of subjects excreted greater than 80% of the total urinary TMA as TMAO, however eight subjects (9.7%) excreted less than 80% of the total TMA as TMAO. In a previous study of 169 white British subjects only one (0.6%) excreted less than 80% of the total TMA as TMAO. The results suggest that the prevalence of compromised ability to N-oxidise TMA may be higher in a Jordanian population than in a British population.

スルメイカ塩辛の菌相特性に及ぼすトリメチルアミンオキシドの影響

Since no means of sterilization is used throughout the manufacturing process of the salted and ripened squid meat product “Ika-Shiokara, ” the quality of the product depends on its associated microbial flora. In the early stage of “Ika-Shiokara” ripening using 10% salts, viable bacteria occured at a level of about 105%/ml of fluid, and then increased gradually with time. The dominant species of bacteria in “Ika-Shiokara” was Staphylococcus epidermidis, but Staphylococcus aureus which closely resembles S. epidermidis in many respects was not detected in all the stages of ripening process. The result suggests that growth of S. aureus was inhibited by some component of “Ika-Shiokara”. We paid attention to trimethylamine-N-oxide (TMAO) and trimethylamine (TMA) which were the characteristic components derived from squid meat. A high amount of TMAO (500-600mg/100ml) was detected in raw squid and in the early ripening process of “Ika-Shiokara”, and it inhibited the growth of S. aureus at this level of concentration but not of S. epidermidis in the nutrient broth.

Identification of cytochromes involved in electron transport to trimethylamine N-oxide/dimethylsulphoxide reductase in Rhodobacter capsulatus

The role of cytochromes in the electron-transport pathway to trimethylamine N -oxide (TMAO)/dimethylsulphoxide (DMSO) reductase in the photosynthetic bacterium Rhodobacter capsulatus was investigated. Reduced-minus-oxidized difference spectra in intact cells with TMAO or DMSO as oxidant revealed cytochrome absorbance changes with a maximum at 559 nm and a shoulder between 548 nm and 556 nm. The former change indicates a role for a 6-type cytochrome and the latter for a c-type cytochrome, both of which are distinct from the cytochrome bc 1 complex. Cytochrome c -556 was identified in a bacterial periplasmic fraction as a redox component which couldbe oxidised by TMAO or DMSO. Cytochrome c -556 was the only cytochrome species which co-fractionated with TMAO/DMSO reductase following gel filtration of a post-chromatophore supernatant produced after French presstreatment of intact cells. The mid-point redox potential (pH 7.6) of cytochrome c -556 was + 105 mV ( n = 1). It is suggested that TMAO/DMSO reductase and cytochrome c -556 form a structural and functional association in the periplasm of Rhodobacter capsulatus .

Calcium carbonate precipitation by marine bacteria

Calcium carbonate precipitation by bacterial activity was noted in seawater collected from offshore Bermuda as well as from Halifax Harbor, provided that a minimum of 70 mg/liter of organic matter was added in addition to trimethylamine‐N‐oxide (TMAO) or sodium nitrate (final minimum concentrations, 1.33 and 0.17 g/liter, respectively). Observations made during these investigations indicated that the pH was the most important factor in the initiation of carbonate precipitation; the addition of calcium acetate, sodium carbonate, calcium sulfate, or ammonium carbonate did not result in carbonate precipitation. Precipitation was noted in cultures only when the pH exceeded 8.3. Precipitation occurred in the absence of bacteria when the culture was filtered after the pH increased to above 8.3 (2 days after nutrient amendment). Filter‐sterilized seawater, adjusted to pH 8.4 or above with NH4OH, also precipitated carbonates. A study of 53 random isolates from Bermuda waters and sediments revealed that only 20% o...