Studies in germ-free mice and cross-sectional clinical studies in humans have suggested a role for the intestinal microbiota in the pathogenesis of atherosclerosis in patients with a diet rich in phosphatidylcholine (with major sources including eggs, liver, beef, and pork) through the formation of the metabolite trimethylamine and conversion to TMAO.7,15
In our study, we describe the generation of the proatherogenic metabolite TMAO from dietary phosphatidylcholine through the use of stable-isotope-tracer feeding studies. We further found a role for the intestinal microbiota in the production of TMAO through its suppression by means of oral broad-spectrum antibiotics and then reacquisition of trimethylamine and the production of TMAO from dietary phosphatidylcholine after the withdrawal of antibiotics and subsequent intestinal recolonization. Finally, we describe the potential clinical significance of this intestinal microbiota-dependent metabolite by showing that fasting plasma TMAO levels predict the risk of incident major adverse cardiovascular events independently of traditional cardiovascular risk factors and the presence or extent of coronary artery disease and within multiple low-risk subgroups, including both participants without angiographic evidence of substantial coronary artery disease (i.e., stenosis of <50% in major coronary vessels) and those with low-risk lipid and apolipoprotein levels. Our findings suggest that pathways that are dependent on the intestinal microbiota may contribute to the pathophysiology of atherosclerotic coronary artery disease and suggest potential therapeutic targets.
The intestinal microbiota have previously been implicated in complex metabolic diseases such as obesity.4–6,16–18
However, the involvement of microbiota in the inception of atherosclerosis in humans has only recently been suggested.7
The ability of oral broad-spectrum antibiotics to temporarily suppress the production of TMAO is a direct demonstration that intestinal microorganisms play an obligatory role in the production of TMAO from phosphatidylcholine in humans. Intestinal microbiota convert the choline moiety of dietary phosphatidylcholine into trimethylamine, which is subsequently converted into TMAO by hepatic flavin-containing monooxygenases ().20–23
The observed delay in the detection of plasma d9-TMAO levels after the ingestion of d9-phosphatidylcholine may reflect the time required for the conversion of trimethylamine into TMAO,24
since separate analyses monitoring trimethylamine and d9-trimethylamine production showed a time course consistent with a precursor-to-product relationship (data not shown). TMAO has been identified in fish as an important osmolite,25
and the ingestion of fish raises urinary TMAO levels. Nevertheless, the high correlation between urine and plasma levels of TMAO argues for effective urinary clearance of TMAO. Hence, an efficient excretion mechanism may provide protection by preventing the accumulation of TMAO and does not undermine the mechanistic link between TMAO and cardiovascular risk.
Pathways Linking Dietary Phosphatidylcholine, Intestinal Microbiota, and Incident Adverse Cardiovascular Events
Although an association between infectious organisms and atherosclerosis has previously been postulated, studies looking at the role of antimicrobial therapy in preventing disease progression have been disappointing.26,27
It is important to recognize that the choice of antimicrobial therapy in previous intervention trials was based largely on targeting postulated organisms rather than modulating the composition of intestinal microbiota or their metabolites. Furthermore, even if an antibiotic initially suppressed TMAO levels, the durability of that effect with long-term use remains unknown. In unpublished studies, we observed that long-term use of a single antibiotic (6 months of ciprofloxacin), which initially suppressed plasma TMAO levels in a rodent model, completely lost its suppressive effect, an observation that is consistent with the expansion of antibiotic-resistant intestinal microbiota. Thus, instead of suggesting that intestinal microbes should be eradicated with long-term use of antibiotics, our findings point to the possibility that plasma TMAO levels may identify a pathway within intestinal microbiota amenable to therapeutic modulation. For example, our data suggest that excessive consumption of dietary phosphatidylcholine and choline should be avoided; a vegetarian or high-fiber diet can reduce total choline intake.16
It should also be noted that choline is a semiessential nutrient and should not be completely eliminated from the diet, since this can result in a deficiency state. However, standard dietary recommendations, if adopted, will limit the intake of phosphatidylcholine- and cholinerich foods, since these foods are also typically high in fat and cholesterol content.1
An alternative potential therapeutic intervention is targeting the composition of the microbiota or biochemical pathways, with either a functional food such as a probiotic17
or a pharmacologic intervention. The latter hypothetically could take the form of treatment with an inhibitor to block specific microbial metabolic pathways or a short course of nonsystemic antibiotics to reduce the burden of TMAO-producing microbes, two therapies that have been used in the treatment of irritable bowel syndrome.28
Further studies are warranted to establish whether antimicrobial therapies can significantly reduce cardiovascular risk.
In conclusion, we have shown that intestinal microbes participate in phosphatidylcholine metabolism to form circulating and urinary TMAO. We also established a correlation between high plasma levels of TMAO and an increased risk of incident major adverse cardiovascular events that is independent of traditional risk factors, even in low-risk cohorts.