Abstract Trimethylamine N‐oxide (TMAO) is a circulating microbiome‐derived metabolite implicated in the development of atherosclerosis and cardiovascular disease (CVD). We investigated whether plasma levels of TMAO, its precursors (betaine, carnitine, deoxycarnitine, choline), and TMAO‐to‐precursor ratios are associated with clinical outcomes, including CVD and mortality. This was followed by an in‐depth analysis of their genetic, gut microbial, and dietary determinants. The analyses were conducted in five Dutch prospective cohort studies including 7834 individuals. To further investigate association results, Mendelian Randomization (MR) was also explored. We found only plasma choline levels (hazard ratio [HR] 1.17, [95% CI 1.07; 1.28]) and not TMAO to be associated with CVD risk. Our association analyses uncovered 10 genome‐wide significant loci, including novel genomic regions for betaine (6p21.1, 6q25.3), choline (2q34, 5q31.1), and deoxycarnitine (10q21.2, 11p14.2) comprising several metabolic gene associations, for example, CPS1 or PEMT. Furthermore, our analyses uncovered 68 gut microbiota associations, mainly related to TMAO‐to‐precursors ratios and the Ruminococcaceae family, and 16 associations of food groups and metabolites including fish‐TMAO, meat‐carnitine, and plant‐based food‐betaine associations. No significant association was identified by the MR approach. Our analyses provide novel insights into the TMAO pathway, its determinants, and pathophysiological impact on the general population.
