We next sought to independently confirm the prognostic value of monitoring fasting plasma levels of TMAO, choline and betaine in a large independent clinical cohort distinct from subjects examined in both the Learning and Validation Cohorts. Stable subjects (N=1876) undergoing elective cardiac evaluations were enrolled. Clinical, demographic and laboratory characteristics of the cohort are provided in Supplementary Table 4a. Fasting plasma levels of TMAO, choline and betaine were quantified by LC/MS/MS using methods specific for each analyte (Supplementary Fig. 8). Elevated levels of choline, TMAO and betaine were all observed to show dose-dependent associations with the presence of CVD (Fig. 3a–c) and multiple individual CVD phenotypes including peripheral artery disease (PAD), coronary artery disease (CAD), and history of myocardial infarction (MI) (Supplementary Tables 5a–d for multilogistic regression models, and Supplementary Table 5e for Somers' Dxy correlation). The associations between increased risk of all CVD phenotypes monitored and elevated systemic levels of the three PC metabolites held true following adjustments for traditional cardiac risk factors and medications usage (Figs. 3a–c, Supplementary Tables 5a–e).