Microbiome-derived trimethylamine N-oxide (TMAO) has been associated with platelet hyperreactivity and subsequent atherogenesis. Whether physiological TMAO-levels influence platelet-derived lipid mediators remains unknown. Little is known about pre-analytic factors potentially influencing TMAO concentrations. We aimed at developing a quantitative LC-MS/MS method to investigate in-vivo and in-vitro pre-analytical factors in TMAO analysis to properly assess the proposed activating effect of TMAO on platelets. TMAO, betaine, carnitine, and choline were analyzed by HILIC-ESI-MS/MS within 6 min total run time. Method validation included investigation of reproducibility, recovery, sensitivity, and in-vitro pre-analytical factors. A 24-h monitoring experiment was performed, evaluating in-vivo pre-analytical factors like daytime or diet. Finally, the effects of different TMAO concentrations on platelet activation and corresponding alterations of platelet-derived eicosanoid release were analyzed. The method showed high reproducibility (CVs ≤ 5.3%), good recovery rates (96-98%), and negligible in-vitro pre-analytical effects. The influence of in-vivo pre-analytical factors on TMAO levels was not observable within the applied experimental conditions. We did not find any correlation between TMAO levels and platelet activation at physiological TMAO concentrations, whereas platelet-derived eicosanoids presented activation of the cyclooxygenase and lipoxygenase pathways. In contrast to previously published results, we did not find any indications regarding diet dependency or circadian rhythmicity of TMAO levels. Our results do not support the hypothesis that TMAO increases platelet responsiveness via the release of lipid-mediators.
Keywords: HILIC-MS/MS; platelet activation; platelet lipidomics; thromboxane; trimethylamine N-oxide.