Inhibition of pro-atherogenic trimethylamine production by human gut bacteria is not determined by varying chlorogenic acid content in highbush blueberries

McAmis, A. M.; Sweet, M. G.; Chadwick, S.; Ratliff, J. G.; Mengist, M. F.; Bassil, N. V.; Anandh Babu, P. V.; Iorizzo, M.; Neilson, A. P.

doi:10.1101/2025.06.20.660815

bioRxiv · 10.1101/2025.06.20.660815

Inhibition of pro-atherogenic trimethylamine production by human gut bacteria is not determined by varying chlorogenic acid content in highbush blueberries

McAmis, A. M.·Sweet, M. G.·Chadwick, S.·Ratliff, J. G.·Mengist, M. F.·Bassil, N. V.·Anandh Babu, P. V.·Iorizzo, M.·Neilson, A. P.

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Abstract

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 M, >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.

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McAmis, A. M., Sweet, M. G., Chadwick, S., Ratliff, J. G., Mengist, M. F., Bassil, N. V., Anandh Babu, P. V., Iorizzo, M., Neilson, A. P.. 2025-06-21. Inhibition of pro-atherogenic trimethylamine production by human gut bacteria is not determined by varying chlorogenic acid content in highbush blueberries. https://doi.org/10.1101/2025.06.20.660815

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