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

Authors: MCAMIS, ASHLEY - North Carolina State University; SWEET, MICHAEL - North Carolina State University; CHADWICK-CORBIN, SYDNEY - North Carolina State University; RATLIFF, JUANITA - North Carolina State University; MENGIST, MOLLA - Virginia State University; Bassil, Nahla; ANANDH BABU, PON VELAYUTHAM - University Of Utah; IORIZZO, MASSIMO - North Carolina State University; NIELSON, ANDREW - North Carolina State University

Submitted to: Food and Function
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/3/2025
Publication Date: 9/23/2025
Citation: Mcamis, A.M., Sweet, M.G., Chadwick-Corbin, S., Ratliff, J.G., Mengist, M.F., Bassil, N.V., Anandh Babu, P., Iorizzo, M., Nielson, A.P. 2025. Inhibition of pro-atherogenic trimethylamine production from choline by human gut bacteria is not determined by varying chlorogenic acid content in highbush blueberries. Food and Function. https://doi.org/10.1039/D5FO02676H.
DOI: https://doi.org/10.1039/D5FO02676H

Interpretive Summary: Blueberry is known as a super food with several health properties derived from phenolic compounds including chlorogenic acid (CGA). Cardiovascular diseases (CVDs) are the leading cause of death in the United States. Elevated blood levels of a gut microbiota-derived metabolite named trimethylamine N-oxide (TMAO), are linked to increased risk of CVDs. With no FDA-approved drugs available to reduce this harmful metabolite or its trimethylamine (TMA) precursor, dietary interventions present the most promising strategy. CGA inhibits the production of TMA and its content in blueberries varies depending on cultivar, growth conditions, and storage conditions. In this study, we investigated the impact of blueberry CGA content on the inhibition of TMA generation in our ex vivo-in vitro human fecal fermentation model. We tested blueberry skins (to avoid interferences from sugar-rich pulp) from 20 genetically distinct highbush blueberry genotypes, chosen based on their high and low CGA content. No significant differences were observed in TMA production among the 4 highest and 4 lowest CGA genotypes. However, significant differences were observed between all genotypes compared to controls, with ~19.4.% reduction in TMA production compared to blank control, indicating that phenolic-rich skin provides similar metabolite-lowering benefits across blueberry varieties. This suggests that the CGA content of genotypes is not a crucial factor for lowering metabolite conversion. Future studies, including rodent and human studies, are needed to confirm this in vitro study and understand why blueberries may inhibit TMAO, as in vitro studies have limitations.

Technical Abstract: Cardiovascular diseases (CVDs) account for ~32% of all deaths worldwide, and atherosclerosis-related conditions are the leading cause of death in the United States. Elevated blood levels of trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, are linked to increased risk of atherosclerosis. TMAO is produced when gut bacteria metabolize dietary quaternary amines to trimethylamine (TMA), which is converted to TMAO in the liver. With no FDA-approved drugs available to reduce TMA or TMAO, dietary interventions present the most promising strategy. Chlorogenic acid (CGA), a phenolic abundant in blueberries, inhibits TMA production. Blueberries may thus be a TMA- (and TMAO)-lowering food. CGA content in blueberries varies depending on cultivar, growth conditions, and storage conditions; it remains unclear whether these variations in CGA levels influence the TMA-lowering activity of different blueberry varieties. We investigated the impact of blueberry CGA content on the inhibition of choline-d9 conversion to TMA-d9 in our ex vivo-in vitro human fecal fermentation model. We tested blueberry skins (to avoid interferences from sugar-rich pulp) from 20 genetically distinct highbush blueberry genotypes, chosen based on their high and low CGA content. CGA levels in whole berries ranged from 2.6-146 mg/100 g fresh weight (FW), while CGA concentrations in blueberry skins ranged from 0.14-9.7 mg/g. No significant 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. However, significant differences were observed between all genotypes compared to controls, with ~19.4.% reduction in TMA-d9 AUCs compared to blank digesta, indicating that phenolic-rich skin provides similar TMA-lowering benefits across blueberry varieties. This suggests that the CGA content of genotypes is not a crucial factor for lowering TMA. Preliminary evidence suggests that fiber is also not the primary driver of the inhibitory activities of blueberry skins. Future studies, including rodent and human studies, are needed to confirm this in vitro study and understand why blueberries may inhibit TMA and potentially TMAO, as in vitro studies have limitations, and the mechanism of TMA production remains unclear.