Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry.

Trimethylamine-N-oxide (TMAO) levels in blood predict future risk for major adverse cardiac events including myocardial infarction, stroke, and death. Thus, the rapid determination of circulating TMAO concentration is of clinical interest. Here we report a method to measure TMAO in biological matrices by stable isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with lower and upper limits of quantification of 0.05 and >200µM, respectively. Spike and recovery studies demonstrate an accuracy at low (0.5µM), mid (5µM), and high (100µM) levels of 98.2, 97.3, and 101.6%, respectively. Additional assay performance metrics include intraday and interday coefficients of variance of <6.4 and <9.9%, respectively, across the range of TMAO levels. Stability studies reveal that TMAO in plasma is stable both during storage at -80°C for 5years and to multiple freeze thaw cycles. Fasting plasma normal range studies among apparently healthy subjects (n=349) show a range of 0.73-126µM, median (interquartile range) levels of 3.45 (2.25-5.79)µM, and increasing values with age. The LC/MS/MS-based assay reported should be of value for further studies evaluating TMAO as a risk marker and for examining the effect of dietary, pharmacologic, and environmental factors on TMAO levels.

Prognostic value of elevated levels of intestinal microbe-generated metabolite trimethylamine-N-oxide in patients with heart failure: refining the gut hypothesis.

BACKGROUND: Altered intestinal function is prevalent in patients with heart failure (HF), but its role in adverse outcomes is unclear. OBJECTIVES: This study investigated the potential pathophysiological contributions of intestinal microbiota in HF. METHODS: We examined the relationship between fasting plasma trimethylamine-N-oxide (TMAO) and all-cause mortality over a 5-year follow-up in 720 patients with stable HF. RESULTS: The median TMAO level was 5.0 µM, which was higher than in subjects without HF (3.5 µM; p < 0.001). There was modest but significant correlation between TMAO concentrations and B-type natriuretic peptide (BNP) levels (r = 0.23; p < 0.001). Higher plasma TMAO levels were associated with a 3.4-fold increased mortality risk. Following adjustments for traditional risk factors and BNP levels, elevated TMAO levels remained predictive of 5-year mortality risk (hazard ratio [HR]: 2.2; 95% CI: 1.42 to 3.43; p < 0.001), as well as following the addition of estimated glomerular filtration rate to the model (HR: 1.75; 95% CI: 1.07 to 2.86; p < 0.001). CONCLUSIONS: High TMAO levels were observed in patients with HF, and elevated TMAO levels portended higher long-term mortality risk independent of traditional risk factors and cardiorenal indexes.