Development of a High Throughput Method to Study the Inhibitory Effect of Phytochemicals on Trimethylamine Formation

Abstract

Choline is metabolized by the gut microbiota into trimethylamine (TMA), the precursor of pro-atherosclerotic molecule trimethylamine N-oxide (TMAO). Reduction of TMA formation has been shown to provide to cardioprotective effects, and some phytochemicals may produce such reduction. This study aimed to develop an optimized, high-throughput anaerobic fermentation methodology to study inhibition of choline microbial metabolism into TMA by phenolic compounds with healthy human fecal starter. Optimal fermentation conditions were: 20 % fecal slurry (1:10 in PBS), 100 M choline, and 12 h fermentation. Also, 10 mM of 3,3-dimethyl-1-butanol (DMB) was defined as a positive TMA production inhibitor, achieving a ~50 % reduction in TMA production. Gallic acid and chlorogenic acid reported higher TMA inhibitory potential (maximum of 80 -90 % in. TMA production inhibition), with IC50 around 5 mM. Nor DMB neither gallic acid and chlorogenic acid reduced TMA production through cytotoxic effects, indicating mechanisms such as altered TMA lyase activity or expression.

Phenolic-rich beverages reduce bacterial TMA formation in an ex vivo-in vitro colonic fermentation model

“…Growth media was prepared according to our optimized methodology 9 , which was originally adapted from Alqurashi et al 22 . Further details can be found in Supplementary materials and methods.…”

“…Humidity ranged between 41 and 47 % during fermentation. Due to the presence of endogenous choline and other TMA-lyase substrates which lead to the production of TMA in the absence of exogenous choline 9 , choline-d9 at a final concentration of 100 M was added as the substrate. The resulting TMA-d9 was quantified to evaluate the effects of the treatments, which reduces the impact of background TMA production on assay results (Figure S1).…”

“…TMAO then enters systemic circulation, where it exerts its proatherogenic and pro-thrombotic action 4,6 , and finally reaches the kidney, where it is eliminated in urine. The primary substrate for TMA production is choline 4,9,10 , a semi-essential nutrient involved in neurotransmitter synthesis and a component of lipoproteins and membrane lipids 11 . Thus, a logical approach to reduce circulating TMAO levels is to inhibit microbial conversion of choline into TMA 4 .…”

“…We recently developed an ex vivo-in vitro colonic fermentation model that allows identification of lead compounds with promising TMA-formation inhibition activity 9 . So far, we have identified catechin, epicatechin and chlorogenic acid as phenolic compounds with inhibitory effects at a dose of 2 mM 9,12 .…”

“…We recently developed an ex vivo-in vitro colonic fermentation model that allows identification of lead compounds with promising TMA-formation inhibition activity 9 . So far, we have identified catechin, epicatechin and chlorogenic acid as phenolic compounds with inhibitory effects at a dose of 2 mM 9,12 . Catechin and epicatechin can be found in foodstuffs such as tea and cocoa 13,14 , and one of the main dietary sources of chlorogenic acid is coffee 15 .…”