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.     

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.     

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.     

Dietary phosphatidylcholine supplementation reduces atherosclerosis in Ldlr−/− male mice2

Choline is an essential nutrient required for various biological processes. Eggs, dairy, and meat are rich in phosphatidylcholine (PC), whereas cereal and legumes are rich in free choline. Excess dietary choline leads to increase plasma trimethylamine N-oxide (TMAO). Epidemiological studies suggest that plasma TMAO is a biomarker for atherosclerosis and it has been suggested that a lower intake of eggs and meat would reduce choline consumption and thus reduce atherosclerosis development. To investigate whether the form of dietary choline influences atherosclerosis development in Ldlr^−/−, we randomly fed Ldlr^−/−male mice (aged 8 – 10 wk) one of the three 40% (calories) high fat diets (with 0.5% w/w of cholesterol): Control (0.1% w/w free-choline, CON), choline-supplemented (0.4% free-choline, CS), or PC-supplemented (0.1% free-choline and 0.3% choline from PC, PCS). After 12-wk of dietary intervention, the animals were euthanized and tissues and blood collected. Aortic atherosclerotic plaque area, plasma choline, lipid metabolites, and spleen and peripheral blood cell phenotypes were quantified. Surprisingly, the PCS group had significantly lower atherosclerotic lesions while having 2-fold higher plasma TMAO levels compared with both CON and CS groups (P<0.05). In the fasting state, we found that PCS decreased plasma very low-density lipoprotein-cholesterol (VLDL-C) and apolipoprotein B48 (APOB48), and increased plasma high-density lipoprotein-cholesterol (HDL-C). However, very low-density lipoprotein (VLDL) secretion was not affected by dietary treatment. We observed lower levels of circulating pro-atherogenic chemokines in the PCS group. Our study suggests that increased dietary PC intake does not induce a pro-atherogenic phenotype.     

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.     

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.     

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.     

FMO3 and its metabolite TMAO contribute to the formation of gallstones

Trimethylamine-N-oxide (TMAO) is a metabolite derived from trimethylamine (TMA), which is first produced by gut microbiota and then oxidized by flavin-containing monooxygenase 3 (FMO3) in the liver. TMAO may contribute to the development of diseases such as atherosclerosis because of its role in regulating lipid metabolism. In this study, we found that high plasma TMAO levels were positively associated with the presence of gallstone disease in humans. We further found increased hepatic FMO3 expression and elevated plasma TMAO level in a gallstone-susceptible strain of mice C57BL/6J fed a lithogenic diet (LD), but not in a gallstone-resistant strain of mice AKR/J. Dietary supplementation of TMAO or its precursor choline increased hepatic FMO3 expression and plasma TMAO levels and induced hepatic canalicular cholesterol transporters ATP binding cassette (Abc) g5 and g8 expression in mice. Up-regulation of ABCG5 and ABCG8 expression was observed in hepatocytes incubated with TMAO in vitro. Additionally, in AKR/J mice fed a LD supplemented with 0.3% TMAO, the incidence of gallstones rose up to 70% compared with 0% in AKR/J mice fed only a LD. This was associated with increased hepatic Abcg5 and g8 expression induced by TMAO. Our study demonstrated TMAO could be associated with increased hepatic Abcg5/g8 expression, biliary cholesterol hypersecretion and gallstone formation.     

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.     

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.     

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.     

The role of Methanomassiliicoccales in trimethylamine metabolism in the rumen of dairy cows

A considerable amount of trimethylamine (TMA) is likely generated in the rumen; however, its metabolism is still unclear. This study aimed to investigate the role of Methanomassiliicoccales (Mmc) in TMA metabolism in the rumen of dairy cows. Three experiments, two rumen in vitro fermentation trials and one dairy cow in vivo trial, were conducted. Four groups were set in Experiment 1: control, nitroglycerin (NG, a methanogen inhibitor), TMA (7.2 mmol/L), and TMA + NG. The methanogenic activity was completely inhibited in the NG group, and no methane production was observed in the NG and TMA + NG groups. The TMA content hardly reduced in the TMA + NG group (6.9 mmol/L) following a 2 d-incubation; in contrast, it demonstrated a significant reduction by 47.2% in the TMA group. Methanogen 16S rRNA gene sequencing and real-time PCR showed that the relative abundance of Mmc increased in the TMA group (P = 0.005). The increase was mainly attributed to two species-level taxa, Group 9 sp. ISO4-G1 and Group 10 sp. Four groups were set in Experiment 2: control, NG, choline (choline chloride, 7.2 mmol/L), and choline + NG. Choline was completely degraded in 24 h, and the TMA content reached the peak point (7.3 mmol/L) in the fermentation culture. The TMA content remained relatively stable in the choline + NG group following the peak point. However, it started to decrease after 24 h in the choline group, corresponding to the rapid increase in methane production and the abundance of Mmc. Eight mid-lactating, rumen-fistulated Holstein cows were randomly assigned to the control (n = 4) or choline (n = 4) group in Experiment 3: In the choline group, cows were gradually supplemented with 100–250 g/(cow·d) of choline chloride over 4 weeks. Compared to the control group, TMA accumulated in the rumen fluid, and the abundance of Mmc 16S rRNA gene and choline-degrading bacterial cutC gene increased in the rumen content in the choline group (P < 0.050). The trimethylamine N-oxide content in the plasma and milk of the dairy cows was approximately 10 times higher in the choline group than that in the control at the end of the experiment. These findings revealed that Mmc played an important role in the elimination of TMA in the rumen. The accumulation of TMA in the rumen would lead to a large amount of TMA absorbed into the blood stream of the dairy cows.     

Interactions of monovalent cations with sodium channels in squid axon. II. Modification of pharmacological inactivation gating

The time-, frequency-, and voltage-dependent blocking actions of several cationic drug molecules on open Na channels were investigated in voltage-clamped, internally perfused squid giant axons. The relative potencies and time courses of block by the agents (pancuronium [PC], octylguanidinium [C8G], QX-314, and 9-aminoacridine [9-AA]) were compared in different intracellular ionic solutions; specifically, the influences of internal Cs, tetramethylammonium (TMA), and Na ions on block were examined. TMA+ was found to inhibit the steady state block of open Na channels by all of the compounds. The time-dependent, inactivation-like decay of Na currents in pronase-treated axons perfused with either PC, 9-AA, or C8G was retarded by internal TMA+. The apparent dissociation constants (at zero voltage) for interaction between PC and 9-AA with their binding sites were increased when TMA+ was substituted for Cs+ in the internal solution. The steepness of the voltage dependence of 9-AA or PC block found with internal Cs+ solutions was greatly reduced by TMA+, resulting in estimates for the fractional electrical distance of the 9-AA binding site of 0.56 and 0.22 in Cs+ and TMA+, respectively. This change may reflect a shift from predominantly 9-AA block in the presence of Cs+ to predominantly TMA+ block. The depth, but not the rate, of frequency-dependent block by QX-314 and 9-AA is reduced by internal TMA+. In addition, recovery from frequency-dependent block is not altered. Elevation of internal Na produces effects on 9-AA block qualitatively similar to those seen with TMA+. The results are consistent with a scheme in which the open channel blocking drugs, TMA (and Na) ions, and the inactivation gate all compete for a site or for access to a site in the channel from the intracellular surface. In addition, TMA ions decrease the apparent blocking rates of other drugs in a manner analogous to their inhibition of the inactivation process. Multiple occupancy of Na channels and mutual exclusion of drug molecules may play a role in the complex gating behaviors seen under these conditions.     

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.     

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.     

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.     

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.     

Effects of Sterylglycosides from Soybean on Lipid Indices in the Plasma,Liver, and Feces of Rats

The effects of soybean oil (SOO, control), soybean lecithin (SOL), and of sterylglycocides (STG) and phospholipids (PL) fractionated from SOL on lipid indices in the plasma, liver, and feces were examined for male Wistar rats fed with diets containing these lipids for 3 weeks. The body weight gain and liver weight decreased or tended to be reduced in the animals given the diet containing a 5% STG mixture (STGM) compared with the values in the other dietary groups. The plasma lipid concentration in general declined in the rats fed with the diets supplemented with 5% SOL, STGM, or the PL mixture (PLM), and with 1% of STGM, acylated STG (ASTG), or non-acylated STG (NSTG). The triacylglycerol level was significantly depressed in the rats fed with the diets including 1 or 5% of STGM, ASTG, or NSTG when compared to the level of the SOO—fed animals. The total cholesterol and triacylglycerol contents in the liver were lower in the rats provided with the diets containing 5% of SOL, PC, or PLM than in the SOO- or STGM-diet-fed animals. The rats given the diets supplemented with 1 or 5% of STGM, ASTG, or NSTG had a decreased content of liver triacylglycerol compared with the content of the SOO—fed animals. The amounts of total lipids and total cholesterol excreted into the feces were higher in the rats fed with the diets supplemented with 5% SOL, or with 1% of STGM, ASTG, or NSTG, or especially with 5% STGM than in the SOO—fed animals. The present results suggest that STG suppressed the absorption of cholesterol and fatty acids in the intestines.     

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.