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) are 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 nonfasting, 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 nonfasting 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. Nonfasting 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 nonfasting 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 nonfasting 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.

In a COSMOS pilot study, daily cocoa extract supplementation (500 mg flavanols, including 80 mg (−)-epicatechin) did not significantly alter serum TMAO or choline, but provides insights for biomarker evaluation in dietary interventions.

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.

Elevated blood levels of trimethylamine N-oxide (TMAO) are linked to increased risk of atherosclerosis. TMAO is produced when gut bacteria metabolize quaternary amines such as choline to trimethylamine (TMA), which is converted to TMAO in the liver. Chlorogenic acid (CGA), a phenolic abundant in blueberries, inhibits TMA production. Blueberries may be a TMA- (and TMAO)-lowering food. CGA content in blueberries varies significantly. It remains unclear whether variations in CGA levels influence the TMA-lowering activity of different cultivars. We investigated the impact of blueberry CGA content on inhibition of choline-d9 conversion to TMA-d9 in our upper gastrointestinal and in vitro human fecal model. Preliminary experiments indicated near-total inhibition of TMA-d9 production when whole blueberries were tested. Blueberry pulp and sugars recapitulated this complete inhibition, whereas blueberry skins and a fiber had more moderate inhibition. We proceeded with skins (to avoid interferences from sugar-rich pulp, which would not be present in the colon in vivo) from 20 highbush blueberry genotypes, chosen for extremes in CGA content. CGA in whole berries was 2.6-146 mg/100 g fresh weight, while CGA in skins was 13.6-975 mg/100 g fresh weight. No differences were observed in TMA-d9 production among the 4 highest and 4 lowest CGA genotypes in kinetic curves or area under the curve (AUC) values when skin digesta were fermented with choline-d9. However, significant differences were observed between all genotypes compared to blank digesta, with ~19.4.% reduction in TMA-d9 AUCs, indicating that skins provides similar TMA-lowering benefits across genotypes. Levels of free CGA in fermenta of skin digesta were 0.05-0.3 μM, >1000-fold lower than the minimum effective dose we observed for pure CGA in vitro, suggesting that blueberry CGA content is not a crucial factor for lowering TMA. Fiber also does not account for most of the inhibitory activity of blueberry skins. Studies are needed to confirm this in vitro study and understand how blueberries inhibit TMA and potentially TMAO production in vivo.