Intestinal microbiota metabolism of choline/phosphatidylcholine produces trimethylamine (TMA) which is usually

Intestinal microbiota metabolism of choline/phosphatidylcholine produces trimethylamine (TMA) which is usually further metabolized to a proatherogenic species trimethylamine-N-oxide (TMAO). adverse cardiac events (MI stroke or death) but only among subjects with concurrently high TMAO levels. Chronic dietary L-carnitine supplementation in mice significantly altered cecal microbial composition markedly enhanced synthesis of TMA/TMAO and increased atherosclerosis but not following suppression of intestinal microbiota. Dietary supplementation of TMAO or either carnitine or choline in mice with intact intestinal microbiota significantly reduced reverse cholesterol transport value cutoff for association with CVD. However a hypothesis-driven examination of the data using less stringent criteria (no adjustment for multiple screening) revealed an analyte with appropriate molecular excess weight and retention time that was associated with cardiovascular event risk (= 0.04)(Supplementary Table 1). In further studies we were able to confirm the identity of the plasma analyte as L-carnitine and develop a quantitative Fruquintinib stable isotope dilution liquid Fruquintinib chromatography tandem mass spectrometry (LC/MS/MS) method for measuring endogenous L-carnitine in all subsequent investigations (Supplementary Figs. 1 2 3 Human gut microbiota is required to form TMAO from L-carnitine The participation of gut microbiota in TMAO production from dietary L-carnitine in humans has not yet been shown. In initial subjects (omnivores) an “L-carnitine challenge test” was developed that incorporated a major source of dietary L-carnitine (8 ounce sirloin steak corresponding to an estimated 180 mg L-carnitine)12 13 14 and a capsule made up Fruquintinib of 250 mg of a heavy isotope labeled L-carnitine (synthetic d3-(methyl)-L-carnitine). At baseline (Visit 1) post-prandial increases in d3-TMAO and d3-L-carnitine in plasma were readily detected and 24 hour urine selections also revealed d3-TMAO (Fig. 1b-e; Supplementary Fig. 4 5 Data shown in Fig. 1 and Supplementary Fig. 4 are tracings from a representative omnivorous subject of =0.74) levels. Several bacterial taxa remained significantly associated with plasma TMAO levels after false discovery rate (FDR) adjustment for multiple comparisons (Supplementary Fig. 8). When subjects were classified into previously reported enterotypes18 based upon fecal microbial composition individuals with an enterotype characterized by enriched proportions of the genus ((< 0.05) CIC (Fig. 3c; Supplementary Fig. 11). Interestingly a Fruquintinib direct comparison of genera recognized in humans versus mice that associated with plasma TMAO levels failed to identify common genera. These results are consistent with prior reports that microbes recognized from Fruquintinib your distal gut of the mouse represent genera that are typically not detected in humans15 20 Plasma levels of L-carnitine are associated with CVD We next investigated the relationship of fasting plasma levels of L-carnitine with CVD risks in Fruquintinib an independent large cohort of stable subjects (= 2 595 undergoing elective cardiac evaluation. Patient demographics laboratory values and clinical characteristics are provided in Supplementary Table 2. A significant dose – dependent association between L-carnitine levels and risk of prevalent coronary artery disease (CAD) peripheral artery disease (PAD) and overall CVD was noted (< 0.05) (Fig. 4a-c). Moreover these associations remained significant following adjustments for traditional CVD risk factors (< 0.05) (Fig. 4a-c). In further analyses plasma levels of L-carnitine were observed to be increased in subjects with significant (≥ 50% stenosis) angiographic evidence of CAD regardless of the extent (e.g. single versus multi-vessel) of CAD as revealed by diagnostic cardiac catheterization (< 0.001) (Fig. 4d). Figure 4 Relation between plasma carnitine and CVD risks The relationship between fasting plasma levels of carnitine and incident (3 year) risk for major adverse cardiac events (MACE = composite of death MI stroke and revascularization) was also examined. Elevated carnitine (4th quartile) levels were an independent predictor of MACE including following adjustments for traditional CVD risk factors (Fig. 4e). After further adjustment for both TMAO and a larger number of comorbidities that might be known at time of presentation (e.g. extent of CAD ejection fraction medications and estimated renal function) the significant.