Plants for Human Health Institute, North Carolina State University, Kannapolis, North Carolina, USA.
Plants for Human Health Institute, North Carolina State University, Kannapolis, North Carolina, USA; Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina, USA.
J Nutr Biochem. 2023 Oct;120:109428. doi: 10.1016/j.jnutbio.2023.109428. Epub 2023 Aug 5.
High circulating levels of trimethylamine N-oxide (TMAO) have been associated with cardiovascular disease risk. TMAO is formed through a microbiome-host pathway utilizing primarily dietary choline as a substrate. Specific gut microbiota transform choline into trimethylamine (TMA), and, when absorbed, host hepatic flavin-containing monooxygenase 3 (FMO3) oxidizes TMA into TMAO. Chlorogenic acid and its metabolites reduce microbial TMA production in vitro. However, little is known regarding the potential for chlorogenic acid and its bioavailable metabolites to inhibit the last step: hepatic conversion of TMA to TMAO. We developed a screening methodology to study FMO3-catalyzed production of TMAO from TMA. HepG2 cells were unable to oxidize TMA into TMAO due to their lack of FMO3 expression. Although Hepa-1 cells did express FMO3 when pretreated with TMA and NADPH, they lacked enzymatic activity to produce TMAO. Rat hepatic microsomes contained active FMO3. Optimal reaction conditions were: 50 µM TMA, 0.2 mM NADPH, and 33 µL microsomes/mL reaction. Methimazole (a known FMO3 competitive substrate) at 200 µM effectively reduced FMO3-catalyzed conversion of TMA to TMAO. However, bioavailable chlorogenic acid metabolites did not generally inhibit FMO3 at physiological (1 µM) nor supra-physiological (50 µM) doses. Thus, the effects of chlorogenic acid in regulating TMAO levels in vivo are unlikely to occur through direct FMO3 enzyme inhibition. Potential effects on FMO3 expression remain unknown. Intestinal inhibition of TMA production and/or absorption are thus likely their primary mechanisms of action.
高水平的三甲基胺 N-氧化物（TMAO）与心血管疾病风险相关。TMAO 通过微生物组-宿主途径形成，主要利用饮食中的胆碱作为底物。特定的肠道微生物将胆碱转化为三甲胺（TMA），当被吸收时，宿主肝脏黄素单加氧酶 3（FMO3）将 TMA 氧化为 TMAO。绿原酸及其代谢物在体外减少微生物 TMA 的产生。然而，关于绿原酸及其可利用的代谢物是否有可能抑制最后一步：TMA 在肝脏中转化为 TMAO，知之甚少。我们开发了一种筛选方法来研究 FMO3 催化 TMA 生成 TMAO。由于缺乏 FMO3 的表达，HepG2 细胞无法将 TMA 氧化为 TMAO。尽管 Hepa-1 细胞在用 TMA 和 NADPH 预处理时确实表达了 FMO3，但它们缺乏产生 TMAO 的酶活性。大鼠肝微粒体含有活性 FMO3。最佳反应条件为：TMA 50µM，NADPH 0.2mM，反应中肝微粒体 33µL/mL。200µM 的甲巯咪唑（一种已知的 FMO3 竞争性底物）可有效降低 FMO3 催化 TMA 转化为 TMAO 的作用。然而，在生理（1µM）和超生理（50µM）剂量下，绿原酸的可利用代谢物通常不会抑制 FMO3。因此，绿原酸在调节体内 TMAO 水平方面的作用不太可能通过直接抑制 FMO3 酶发生。其对 FMO3 表达的潜在影响仍不清楚。因此，其主要作用机制可能是抑制肠道 TMA 的产生和/或吸收。
