Background/Objectives: Gut microbial metabolism of choline and related quaternary amines to trimethylamine (TMA) is the first step in the production of trimethylamine N-oxide (TMAO), a circulating metabolite that contributes to the development of atherosclerosis and other forms of cardiovascular disease (CVD). No data exist on regional differences in TMA production within the colon due to difficulties studying gut regions in vivo. A better understanding of TMA production by gut microbiota is needed to develop strategies to limit TMA production in the gut and TMAO levels in circulation with the goal of reducing CVD risk. Methods: We employed our novel three-compartment MiGut in vitro model, which establishes three distinct microbial ecologies mimicking the proximal, mid, and distal colon, to study conversion of choline to TMA by human gut microbiota using isotopically labelled substrate. Results: Choline-d₉ was almost completely converted to TMA-d₉ in vessels 2–3 (mimicking the mid and distal colon) within 6–8 h, but little conversion occurred in vessel 1 (mimicking the proximal colon). Abundance of cutC, part of the cutC/D gene cluster responsible for choline conversion to TMA, was highest in vessel 1 vs. 2–3, suggesting that its expression or activity may be suppressed in the proximal colon. Another possibility is that the viability/activity of bacteria expressing cutC could be suppressed in the same region. Conclusions: This novel finding suggests that while bacteria capable of converting choline to TMA exist throughout the colon, their activity may be different in distinct colon regions. The regional specificity of TMA production, if confirmed in vivo, has implications for both basic microbial ecology related to CVD and the development of strategies to control TMA and TMAO production, with the goal of lowering CVD risk. These findings warrant further study in vitro and in vivo.

Circulating concentrations of the gut microbial-mammalian metabolite trimethylamine N-oxide (TMAO) is linked to atherosclerosis risk. TMAO biosynthesis begins when dietary choline is converted to trimethylamine (TMA) by gut microbial TMA lyase. TMA is transported to the liver, where flavin-containing monooxygenases convert it to TMAO. While dietary modifications regulate TMAO production, the impact of different intake methods, including oral gavage, dietary supplementation, and conditions such as fasting versus non-fasting, has not been fully explored. Twelve female Sprague-Dawley rats were divided into three diet groups ( n = 4 per group): no-choline (0% choline), low-choline (0.08% choline), and high-choline (1% choline). Choline and TMAO fasting and non-fasting blood concentrations, and their kinetics following an acute choline challenge, were assessed before and after a 2-week dietary intervention with the distinct choline dietary levels. Fasting choline was under tight control, with little effect of dietary choline. Non-fasting choline was more variable, with high dietary choline reflected in higher blood choline. Greater levels of dietary choline were reflected in significantly greater levels of TMAO, particularly for non-fasting levels. Kinetic profiling demonstrated additional information regarding the appearance and clearance of these compounds from blood. These results suggest that acute oral choline gavage is likely most suitable for studies targeting acute (direct) inhibitors, whereas a choline-rich diet with assessment of fasting and non-fasting blood levels is more suitable for studying alterations to TMAO production capacity. Future research should examine the impact on atherosclerosis biomarkers and microbiome diversity to deepen the understanding of TMAO regulation and its cardiovascular implications.

Background/Objectives: Gut microbial metabolism of choline and related quaternary amines to trimethylamine (TMA) is the first step in production of trimethylamine N-oxide (TMAO), a circulating metabolite that contributes to the development of atherosclerosis and other forms of cardiovascular disease (CVD). No data exist on regional differences in TMA production within the colon due to difficulties studying gut regions in vivo. A better understanding of TMA production by gut microbiota is needed to develop strategies to limit TMA production in the gut and TMAO levels in circulation with the goal of reducing CVD risk. Methods: We employed our novel 3-compartment MiGut in vitro model, which establishes three distinct microbial ecologies mimicking the proximal, mid and distal colon to study conversion of choline to TMA by human gut microbiota using isotopically labelled substrate. Results: Choline-d9 was almost completely converted to TMA-d9 in vessels 2-3 (mimicking the mid and distal colon) within 6-8 h, but little conversion occurred in vessel 1 (mimicking the proximal colon). Abundance of cutC, part of the cutC/D gene cluster responsible for choline conversion to TMA, was highest in vessel 1 vs 2/3, suggesting that its expression or activity (or the viability/activity of bacteria expressing cutC) may be suppressed in the proximal colon. Conclusions: This novel finding suggests that, while bacteria capable of converting choline to TMA exist throughout the colon, their activity may be different in distinct colon regions. The regional specificity of TMA production, if confirmed in vivo, has implications for both basic microbial ecology related to CVD and development of strategies to control TMA and TMAO production with the goal of lowering CVD risk. These findings warrant further study in vitro and in vivo.