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.     

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).     

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.     

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.     

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.     

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.     

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.     

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.     

Investigations on the effects of rape oil quality, choline and methionine concentration in diets for laying hens on the trimethylamine content of the eggs, on trimethylamine metabolism and on laying performance

An experiment was conducted to study the effects of graded levels of choline addition (0, 500, 1000 and 4000 mg/kg diet) in laying hen diets prepared either with degummed or refined rape oil on the performance, sensory properties and trimethylamine (TMA) contents of the eggs. Furthermore, the diets containing no supplemented choline or 4000mg choline/kg diet were tested with adequate or inadequate methionine supply (4.2 vs. 2.8 g methionine/kg diet). TMA metabolism and N-balance were measured for the latter diet types, but only with the diets containing refined rape oil. Therefore, a total of 12 and 4 diets were tested in the feeding (n = 60) and balance study (n = 9). Laying performance (23 -75 weeks of age) was not significantly influenced by increasing choline additions with the exception of feed-to-egg mass ratio which decreased significantly linearly (P(linear) = 0.003). However, a significant interaction between choline addition and laying month was detected which was caused by a depression of performance of the unsupplemented control group occurring from the sixth laying month. The most obvious effect of an inadequate methionine supply was a temporary drop in performance between the third and sixth laying months. The mean TMA-concentration in pooled egg yolks [microg/g] increased with dietary choline concentration [mg/kg] in an exponentially related fashion (y = 1.14 + 4E(-10) x x(2.71), r2 = 0.962) and suggested only a minor influence of total dietary choline on TMA content up to approximately 2000mg choline/kg. Individual TMA-concentrations varied greatly from 0.4 - 1.5 microg/g, from 2.2 - 34 microg/g and from 18.4 - 75 microg/g for eggs with a normal, aberrant and heavily aberrant odour, respectively. It is concluded that a total choline concentration of at least approximately 1500 mg/kg is necessary to maintain a maximal laying performance. An inadequate methionine supply cannot be compensated by an increased addition of choline. Neither degummed nor refined rape oil influenced the TMA content of eggs.     

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.     

Effect of salt stress on the metabolism of ethanolamine and choline in leaves of the betaine-producing mangrove species Avicennia marina

Glycinebetaine synthesis from [methyl-14C]choline and [1,2-14C]ethanolamine in leaf disks of Avicennia marina, was increased by salt stress (250 and 500 mM NaCl). After 18 h incubation with [methyl-14C]choline, phosphocholine and CO(2) were found to be heavily labelled. Phosphocholine contained 39% of the total radioactivity taken up by non-salinised (control) leaf disks and 15% of the total for salinised leaf disks stressed with 500 mM NaCl. Eighteen and 49% of the radioactivity absorbed by control and salinised disks, respectively, were released as CO(2). Metabolic studies of [1,2-14C]ethanolamine revealed that the radioactivity taken up by the leaf disks was recovered as the following compounds after 18 h: phosphorylated compounds (mainly phosphoethanolamine, phosphodimethylethanolamine and phosphocholine) (40-50%); choline (1-2%); glycinebetaine (3-5%); lipids (20-28%); CO(2) (6-10%). Unlike glycinebetaine, incorporation into phosphorylated compounds and lipids were reduced by salt stress. Incorporation of [methyl-14C]S-adenosyl-L-methionine (SAM) into choline, phosphocholine and glycinebetaine in leaf disks was stimulated by salt stress. In vitro activities of adenosine kinase and adenosine nucleosidase, which are implicated in stimulating the SAM regeneration cycle, increased after the leaf disks were incubated with 250 and 500 mM NaCl for 18 h. Changes in metabolism involving choline and glycinebetaine due to salt stress are discussed.     

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.     

Measurement of the formation of betaine aldehyde and betaine in rat liver mitochondria by a high pressure liquid chromatography-radioenzymatic assay.

A new assay procedure for measurement of rat liver mitochondrial choline dehydrogenase was developed. Oxidation of [methyl-14C]choline to [methyl-14C]betaine aldehyde and [methyl-14C]betaine was measured after isolating these compounds using HPLC. We observed that NAD+ was required for conversion of betaine aldehyde to betaine in rat liver mitochondria. In the absence of this cofactor, oxidation of choline led to the accumulation of betaine aldehyde. The apparent Km of the mitochondrial choline dehydrogenase for choline was 0.14-0.27 mM, which is significantly lower than previously reported. A partially purified preparation of choline dehydrogenase catalyzed betaine aldehyde formation only in the presence of exogenous electron acceptors (e.g., phenazine methosulfate). This preparation failed to catalyze the formation of betaine even in the presence of NAD+, indicating that betaine aldehyde dehydrogenase may be a separate enzyme from choline dehydrogenase.     

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.     

Synthesis of phosphatidylcholines in rat hepatocytes. Possible regulation by norepinephrine via an alpha-adrenergic mechanism

The effect of norepinephrine on phosphatidylcholine and phosphatidylethanolamine formation was investigated in short-term incubations with freshly isolated rat hepatocytes. In the presence of dl-propranolol, norepinephrine decreases the incorporation of [methyl-14C]choline into phosphatidylcholines in a dose-dependent manner. At a concentration of 50 microM, norepinephrine (plus 20 microM propranolol) inhibits the incorporation of [methyl-14C]choline over a wide range of choline concentrations (59% inhibition at 5 microM choline; 34% inhibition at 1 mM choline). Norepinephrine also decreases the incorporation rates of [1-14C]palmitic acid and [1-14C]oleic acid into phosphatidylcholines. The effect of norepinephrine is mediated through an alpha-adrenergic receptor. Norepinephrine (plus propranolol) does not decrease the uptake or phosphorylation rate of [methyl-14C]choline. Pulse-label and pulse-chase studies indicate that the conversion rate of phosphocholine to CDP-choline, catalyzed by CTP:phosphocholine cytidylyltransferase, is diminished by norepinephrine. In contrast with the inhibitory effect of norepinephrine on phosphatidylcholine synthesis, this hormone stimulates the formation of phosphatidylethanolamines from [1,2-14C]ethanolamine. This increased incorporation rate is apparent at ethanolamine concentrations above 25 microM. A combination of norepinephrine and propranolol decreases, however, the synthesis of phosphatidylcholines from [1,2-14C]ethanolamine. The results indicate that alpha-adrenergic regulation dissociates the synthesis of phosphatidylcholines from that of phosphatidylethanolamines.     

Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.     

Enalapril decreases rat plasma concentration of TMAO,a gut bacteria-derived cardiovascular marker

Introduction: Increased plasma level of trimethylamine N-oxide (TMAO), a bacterial metabolite of choline, is associated with an increased cardiovascular risk. Indoxyl sulfate, a bacterial metabolite of tryptophan, is thought to be associated with higher mortality in cardiorenal syndrome. We hypothesized that enalapril, a well-established drug reducing cardiovascular mortality, may affect the plasma level of gut bacteria-derived metabolites and gut bacteria composition.

Materials and methods: 14–16-week-old Wistar rats were maintained either on water (controls) or water solution of enalapril for two weeks (5.3 or 12.6?mg/kg b.w.). Blood plasma and urine were analyzed for the concentration of TMAO and indoxyl sulfate using liquid chromatography coupled with triple-quadrupole mass spectrometry. Gut bacteria composition was analyzed with 16S rRNA gene sequence analysis.

Results: Rats treated with enalapril showed a significantly lower plasma TMAO level and a trend towards higher 24?h urine excretion of TMA and TMAO. Plasma indoxyl level was similar between the groups. There was no significant difference between the groups in gut bacteria composition.

Conclusions: Enalapril decreases rat plasma TMAO, but does not affect the plasma level of indoxyl sulfate and gut bacteria composition. The enalapril-induced decrease in plasma TMAO level may be of therapeutic and diagnostic importance.     

Early increase in phosphatidyl choline synthesis by choline and transmethylation pathways in spreading fibroblasts

Phosphatidyl choline (PC) synthesis in trypsinized and reattaching fibroblasts during the spreading state was studied by incorporation of [14C]choline and [methyl-14C]methionine. The choline and phosphatidyl-ethanolamine (PE) transmethylation pathways were both transiently increased about 2-fold during the first 2 h after replating. Maximum increase appeared to be simultaneous with maximum spreading. Incorporation of [32P]orthophosphate showed that the increase in PC synthesis was specific and most probably related to establishment of cell-substrate adhesion sites.     

Investigations on the effects of rape oil quality,choline and methionine concentration in diets for laying hens on the trimethylamine content of the eggs,on trimethylamine metabolism and on laying performance

Abstract

An experiment was conducted to study the effects of graded levels of choline addition (0, 500, 1000 and 4000 mg/kg diet) in laying hen diets prepared either with degummed or refined rape oil on the performance, sensory properties and trimethylamine (TMA) contents of the eggs. Furthermore, the diets containing no supplemented choline or 4000 mg choline/kg diet were tested with adequate or inadequate methionine supply (4.2 vs. 2.8 g methionine/kg diet). TMA metabolism and N-balance were measured for the latter diet types, but only with the diets containing refined rape oil. Therefore, a total of 12 and 4 diets were tested in the feeding (n = 60) and balance study (n = 9). Laying performance (23 – 75 weeks of age) was not significantly influenced by increasing choline additions with the exception of feed-to-egg mass ratio which decreased significantly linearly (p _linear = 0.003). However, a significant interaction between choline addition and laying month was detected which was caused by a depression of performance of the unsupplemented control group occurring from the sixth laying month. The most obvious effect of an inadequate methionine supply was a temporary drop in performance between the third and sixth laying months. The mean TMA-concentration in pooled egg yolks [μg/g] increased with dietary choline concentration [mg/kg] in an exponentially related fashion (y = 1.14 + 4E^?10 ? x^2.71, r² = 0.962) and suggested only a minor influence of total dietary choline on TMA content up to approximately 2000 mg choline/kg. Individual TMA-concentrations varied greatly from 0.4 – 1.5 μg/g, from 2.2 – 34 μg/g and from 18.4 – 75 μg/g for eggs with a normal, aberrant and heavily aberrant odour, respectively. It is concluded that a total choline concentration of at least approximately 1500 mg/kg is necessary to maintain a maximal laying performance. An inadequate methionine supply cannot be compensated by an increased addition of choline. Neither degummed nor refined rape oil influenced the TMA content of eggs.     

Formation of trimethylamine from dietary choline by Streptococcus sanguis I, which colonizes the mouth

Choline is a component of the normal diet, and when humans ingest large amounts they excrete trimethylamine (which can impart a fishy body odor). In the presence of nitrite, trimethylamine can be converted to dimethylnitrosamine, a potent carcinogen. Bacteria in the large intestine metabolize choline to form trimethylamine. We determined that a bacterium normally present in the oral cavity also has this capacity. Mixed bacterial flora cultured from dental plaque and saliva converted choline to trimethylamine. The only organism with trimethylamine-forming capability isolated from these mixed cultures was identified as Streptococcus sanguis I (a facultative anaerobe). The other products formed when choline was cleaved were ethanol and acetate. The formation of trimethylamine by S. sanguis I was enzyme-mediated. Activity was destroyed by heating at 100 degrees C, and obeyed Michaelis-Menten kinetics (K(apparent) for choline = 184 +/- 58 microM; V(max apparent) = 1.7 +/- 0.1 micromol/mg protein/h). Activity was maximal at pH 7.5 to 8.5, was membrane-bound, and required a divalent metal cation (cobalt or iron). More trimethylamine was produced by bacteria incubated under a nitrogen than under an aerobic atmosphere. Activity was inhibited by deanol, betaine aldehyde, hemicholinium-3, iodoacetate, semicarbazide, and 2,4-dinitrophenol, and was enhanced by sulfhydryl-reducing agents (glutathione, 2-mercaptoethanol, DL-dithiothreitol) and sodium bisulfite. The enzyme activity that we describe in S. sanguis I is similar to that previously described in the anaerobic bacteria isolated from intestinal flora.