Cardiovascular disease (CVD) is the leading cause of death worldwide and in the US [1,2]. According to a recent estimate, global deaths from CVD are 19.8 million annually [3]. Multiple risk factors are associated with CVD, such as poor diet, smoking, obesity, lack of physical activity, etc. The composition and function of the gut microbiome are also associated with various cardiovascular events [4] through the production of diet derived microbial metabolites [5]. Gut microbes can produce both beneficial and harmful metabolites, such as short-chain fatty acids and trimethylamine N-oxide (TMAO) [5,6], respectively.

Trimethylamine N-oxide (TMAO) was identified as a novel risk factor for CVD, specifically atherosclerosis, in 2011 by the Hazen group at the Cleveland Clinic [7,8]. Since then, elevated blood TMAO has been reported as a CVD and mortality risk factor [9,10], although controversy remains regarding its utility as a predictor of CVD risk [11,12]. TMAO is produced by sequential action of gut microbiota and the liver [8]. First, gut bacteria possessing choline trimethylamine (TMA) lyase enzymes (cutC/D gene cluster) metabolize choline into TMA [8]. TMA is then absorbed into the circulation and oxidized to TMAO by hepatic flavin-containing monooxygenase 3 (FMO3) [13]. Other quaternary amines, such as carnitine, can also be metabolized to TMA by similar microbial enzymes.

Choline is a semi-essential nutrient, and thus eliminating or drastically reducing choline intake is an unwise, impractical and ineffective strategy to reduce TMA and TMAO production [14,15]. Currently, there is no FDA-approved drug to manage elevated TMAO [16,17]. One strategy that has shown promise is direct inhibition of TMA lyases. For example, 3,3-dimethyl-1-butanol (DMB), a choline analog, inhibits TMA lyase and has shown a nontoxic potential to inhibit TMAO formation and prevent atherosclerosis in vivo [18,19]. However, due to the lack of approved pharmaceuticals to date, complementary nondrug strategies are needed. Dietary interventions are a logical strategy to reduce TMAO formation with the goal of preventing or slowing atherosclerosis/CVD [20,21]. Previous studies have demonstrated the cardioprotective roles of plant-based dietary interventions, including berries [20,22]. For example, blueberry supplementation mediates vascular inflammation, improves vascular dysfunction, and reduces circulating TMAO concentrations [23,24]. Polyphenols such as chlorogenic acid (CGA) are abundant in blueberries and have the potential to reduce choline microbial metabolism into TMA [25,26]. Chronic CGA supplementation improves the gut microbiome and protects against CVD in animal models [25,27]. It is currently unknown how CGA-rich foods may reduce TMA and TMAO, but it may act by acute mechanisms (such as direct TMA lyase inhibition) requiring the inhibitor and substrate to be present simultaneously, or mechanisms requiring chronic exposure, including shifts in microbiome taxonomic distribution, altered levels of cutC/D, and/or changes to FMO3 expression. The key step in lowering TMAO is reducing the conversion of choline to TMA by gut bacteria [18]. We recently developed an ex vivo-in vitro methodology to screen compounds for the ability to reduce microbial biotransformation of choline into TMA [28,29]. Using our fecal fermentation method, we tested the ability of CGA to inhibit TMA production [30]. We found CGA reduces both choline use and TMA production [30].

In vivo studies are needed to validate the efficacy of compounds identified in vitro as having potential TMA- and TMAO-lowering activities. There are various methods in place to use choline administration models either in a chronic diet or acute oral challenge (by gavage) [31,32]. However, there is a lack of consensus on the utility of various potential TMAO measures such as blood vs. urinary levels, or single time point vs. kinetics over time. There is no clearly accepted method to evaluate the efficacy of interventions that may reduce choline conversion to TMA and TMAO. Given that such interventions might work via acute or chronic intervention, experimental conditions such as blood collection in a fasting or nonfasting state, or use of chronic dietary choline vs. acute exposure, could yield vastly different results [33]. In the present study, we assessed the plasma TMAO responses with different experimental conditions such as acute oral choline gavage, dietary choline intervention, fasting, and nonfasting conditions. We found a clear difference in circulating TMAO between treatments of fasting, oral gavage, and dietary choline intervention in the preclinical model. This outcome could be considered for future in vivo studies to evaluate the efficacy of therapeutic compounds on circulating TMAO.