Abstract
PurposePlasma trimethylamine-N-oxide (TMAO) levels have been shown to correlate with increased risk of metabolic diseases including cardiovascular diseases. TMAO exposure predominantly occurs as a consequence of gut microbiota-dependent trimethylamine (TMA) production from dietary substrates including choline, carnitine and betaine, which is then converted to TMAO in the liver. Reducing microbial TMA production is likely to be the most effective and sustainable approach to overcoming TMAO burden in humans. Current models for studying microbial TMA production have numerous weaknesses including the cost and length of human studies, differences in TMA(O) metabolism in animal models and the risk of failing to replicate multi-enzyme/multi-strain pathways when using isolated bacterial strains. The purpose of this research was to investigate TMA production from dietary precursors in an in-vitro model of the human colon.MethodsTMA production from choline, l-carnitine, betaine and γ-butyrobetaine was studied over 24–48 h using an in-vitro human colon model with metabolite quantification performed using LC–MS.ResultsCholine was metabolised via the direct choline TMA-lyase route but not the indirect choline–betaine-TMA route, conversion of l-carnitine to TMA was slower than that of choline and involves the formation of the intermediate γ-BB, whereas the Rieske-type monooxygenase/reductase pathway for l-carnitine metabolism to TMA was negligible. The rate of TMA production from precursors was choline > carnitine > betaine > γ-BB. 3,3-Dimethyl-1-butanol (DMB) had no effect on the conversion of choline to TMA.ConclusionThe metabolic routes for microbial TMA production in the colon model are consistent with observations from human studies. Thus, this model is suitable for studying gut microbiota metabolism of TMA and for screening potential therapeutic targets that aim to attenuate TMA production by the gut microbiota.Trial registration numberNCT02653001 (http://www.clinicaltrials.gov), registered 12 Jan 2016.
Highlights
It is well established that the human gut microbiota produce substrates that are both beneficial and deleterious to health [1,2,3]
Betaine, l-carnitine and γ-BB are at all metabolised to TMA in the multi-species batch fermentation human in-vitro colon model, we compared the increase in the concentrations of TMA over 24 h to the increase in the untreated ‘Blank’ fermentations
The main findings were that (i) TMA was produced from all three dietary precursors, as well as from the metabolic intermediate γ-BB, (ii) that the relative rates of production of TMA from the substrates was choline > carnitine > γ-BB > betaine, (iii) that all the previously described metabolic routes to TMA production reported in humans were replicated in this in-vitro pH-controlled batch colon model and (iv) that no metabolism to TMA occurred from any substrate when fermentations were performed in the absence of pH control
Summary
It is well established that the human gut microbiota produce substrates that are both beneficial and deleterious to health [1,2,3]. Another study incorporating experiments with single bacterial strains, live human faecal microbes and mouse caecal cell lysates provided evidence that the metabolism of choline to TMA could be inhibited by a choline analogue 3,3-dimethyl-1-butanol (DMB) [21]. It was shown that the concentration of TMAO in plasma peaked 24 h (h) after oral l-carnitine administration [22] Using both human and animal studies, Koeth et al.provided evidence that γ-BB is an obligate intermediate formed during the metabolism of l-carnitine to TMA with the major site for γ-BB production being the small bowel, proximal to that of TMA production [22]
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