Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk

Abstract

Background: Recent studies in animals have shown a mechanistic link between intestinal microbial metabolism of the choline moiety in dietary phosphatidylcholine (lecithin) and coronary artery disease through the production of a proatherosclerotic metabolite, trimethylamine-N-oxide (TMAO). We investigated the relationship among intestinal microbiota-dependent metabolism of dietary phosphatidylcholine, TMAO levels, and adverse cardiovascular events in humans.

Methods: We quantified plasma and urinary levels of TMAO and plasma choline and betaine levels by means of liquid chromatography and online tandem mass spectrometry after a phosphatidylcholine challenge (ingestion of two hard-boiled eggs and deuterium [d9]-labeled phosphatidylcholine) in healthy participants before and after the suppression of intestinal microbiota with oral broad-spectrum antibiotics. We further examined the relationship between fasting plasma levels of TMAO and incident major adverse cardiovascular events (death, myocardial infarction, or stroke) during 3 years of follow-up in 4007 patients undergoing elective coronary angiography.

Results: Time-dependent increases in levels of both TMAO and its d9 isotopologue, as well as other choline metabolites, were detected after the phosphatidylcholine challenge. Plasma levels of TMAO were markedly suppressed after the administration of antibiotics and then reappeared after withdrawal of antibiotics. Increased plasma levels of TMAO were associated with an increased risk of a major adverse cardiovascular event (hazard ratio for highest vs. lowest TMAO quartile, 2.54; 95% confidence interval, 1.96 to 3.28; P<0.001). An elevated TMAO level predicted an increased risk of major adverse cardiovascular events after adjustment for traditional risk factors (P<0.001), as well as in lower-risk subgroups.

Conclusions: The production of TMAO from dietary phosphatidylcholine is dependent on metabolism by the intestinal microbiota. Increased TMAO levels are associated with an increased risk of incident major adverse cardiovascular events. (Funded by the National Institutes of Health and others.).

Figures

Figure 1. Effects of a Phosphatidylcholine Challenge and Administration of Antibiotics on Mean Levels of Trimethylamine-N-Oxide (TMAO) and Its d9 Isotopologue (d9-TMAO)

All 40 study participants underwent the first dietary phosphatidylcholine challenge (visit 1), which consisted of the ingestion of deuterium-labeled phosphatidylcholine (d9-phosphatidylcholine) and two hard-boiled eggs. Six participants then received broad-spectrum antibiotics for 1 week, followed by a second phosphatidylcholine challenge (visit 2). These same participants returned again at least 1 month after discontinuing the antibiotics for a third challenge (visit 3). Shown are the results of assays for TMAO (Panel A) and d9-TMAO (Panel B) after the phosphatidylcholine challenge, before and after the administration of antibiotics, with the intensity of stable-isotope-dilution assays measured by means of high-performance liquid chromatography with online electro-spray ionization tandem mass spectrometry. The arrows indicate retention time where authentic isotope-labeled TMAO standards elute. Also shown are the plasma levels of TMAO (Panel C) and d9-TMAO (Panel D) at each visit. The plasma levels of TMAO were markedly suppressed after the administration of antibiotics and subsequently reappeared after the cessation of antibiotics, indicating that the production of TMAO from dietary phosphatidylcholine is dependent on metabolism by the intestinal microbiota.

Figure 2. Kaplan–Meier Estimates of Major Adverse Cardiovascular Events, According to the Quartile of TMAO Level

Data are shown for 4007 participants in the clinical-outcomes study. The P value is for all comparisons.

Figure 3. Pathways Linking Dietary Phosphatidylcholine, Intestinal Microbiota, and Incident Adverse Cardiovascular Events

Ingested phosphatidylcholine (lecithin), the major dietary source of total choline, is acted on by intestinal lipases to form a variety of metabolic products, including the choline-containing nutrients glycerophosphocholine, phosphocholine, and choline. Choline-containing nutrients that reach the cecum and large bowel may serve as fuel for intestinal microbiota (gut flora), producing trimethylamine (TMA). TMA is rapidly further oxidized to trimethylamine-N-oxide (TMAO) by hepatic flavin-containing monooxygenases (FMOs). TMAO enhances the accumulation of cholesterol in macro-phages, the accumulation of foam cells in artery walls, and atherosclerosis, all factors that are associated with an increased risk of heart attack, stroke, and death. Choline can also be oxidized to betaine in both the liver and kid-neys. Dietary betaine can serve as a substrate for bacteria to form TMA and presumably TMAO.