The use of an in-vitro batch fermentation (human colon) model for investigating mechanisms of TMA production from choline, L-carnitine and related precursors by the human gut microbiota

Abstract

Purpose: Plasma 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.

Methods: TMA 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.

Results: Choline 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.

Conclusion: The 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 number: NCT02653001 ( http://www.clinicaltrials.gov ), registered 12 Jan 2016.

Keywords: Betaine; Cardiovascular disease; Carnitine; Fish odour syndrome; Human gut microbiota; Lecithin; Metabolic disease; Phosphatidylcholine; TMAO; γ-Butyrobetaine.

Conflict of interest statement

None of the authors declared a conflict of interest.

© 2021. The Author(s).

Figures

Fig. 1

Change in TMA between 0 and 24 h in each batch fermentation seeded with faecal samples of five different donors. Fermentation stratified by substrate added. Colours correspond to donors and marker shapes to individual experiments. There is a high intra-class correlation between replicate fermentations within experiments, but little intra-class correlation within donors beyond this. Variation in the levels of TMA produced across experiments is high for each substrate, despite similar levels of substrate being utilised

Fig. 2

Estimated mean TMA produced from each substrate over 24 h. Estimates were obtained by mixed effects regression models of the difference between TMA concentration at 0 and 24 h (as described in the statistical methods). The effect of each substrate is calculated with correction for the production of TMA observed in blank vessel without added substrates. Error bars represent 95% confidence intervals

Fig. 3

The average trajectory of all metabolites. With no added substrate (a) and following supplementation with each substrate (b–e). Error bars represent standard errors

Fig. 4

The effects of DMB on TMA production from choline in three independent experiments. The graph is stratified by experiment to enable a direct comparison between paired fermentations with and without DMB added. There was no effect of adding DMB on TMA production or choline concentration at any stage over the time period tested

Fig. 5

The fermentation of TMA substrates under anaerobic conditions without pH control. There was no fermentation of any of the substrate and no TMA production in anaerobic conditions without pH control, although only a few experiments were carried out (n = 3 for choline and 2 for betaine, l-carnitine and γ-BB)

Fig. 6

Pathways for the metabolism of choline, betaine, l-carnitine and γ-BB by human gut microbiota. This is based on data reported here and previously by others [, , , –22, 32]. In humans, choline is metabolised to TMA via the choline TMA-lyase pathway, betaine is not formed as an intermediate of choline to produce TMA via the choline dehydrogenase (CHDH)/betaine aldehyde dehydrogenase (BADH) > betaine reductase pathway, nor is it formed as an intermediate of l-carnitine via the l-carnitine dehydrogenase pathway. There is no direct conversion of l-carnitine to TMA via the Rieske-type C l-carnitine oxygenase/reductase pathway, instead l-carnitine is first converted to γ-BB by γ-butyrobetainyl-CoA:carnitine CoA transferases which is then converted to TMA by the l-carnitine TMA lyases. It is possible that betaine may be converted to dimethylglycine by glycine betaine transmethylase and then to TMA by decarboxylation although the evidence for this is weak. Dashed black lines are pathways shown not to be functional in this model; solid green lines indicate pathways we have demonstrated to be important for TMA production in the in-vitro human colonic fermentation model