NMR quantification of trimethylamine-N-oxide in human serum and plasma in the clinical laboratory setting

Background and objectives: Trimethylamine-N-oxide (TMAO) produced by gut microbiota metabolism of dietary choline and carnitine has been shown to be associated with increased risk of cardiovascular disease (CVD) and to provide incremental clinical prognostic utility beyond traditional risk factors for assessing a patient's CVD risk. The aim of this study was to develop an automated nuclear magnetic resonance (NMR) spectroscopy assay for quantification of TMAO concentration in serum and plasma using a high-throughput NMR clinical analyzer. Methods: Key steps in assay development included: (i) shifting the TMAO analyte peak to a less crowded region of the spectrum with a pH buffer/reagent, (ii) attenuating the broad protein background signal in the spectrum and (iii) using a non-negative least squares algorithm for peak deconvolution. Assay performance was evaluated according to Clinical and Laboratory Standards Institute guidelines. A method comparison study was performed to compare TMAO concentrations quantified by NMR and mass spectrometry (MS). Results: The within-run and within-lab imprecision ranged from 4.3 to 14.5%. Under the acquisition method employed, the NMR assay had a limit of blank, detection and quantitation of 1.6, 3.0 and 3.3 mu M, respectively. Linearity was demonstrated within the reportable range of 3.3 to 3000 mu M. TMAO measurements using the NMR assay, which involves minimal sample preparation, compared well with values obtained with the MS-based assay (R-2 = 0.98). Conclusions: The NMR based assay provides a simple and accurate measurement of circulating TMAO levels amenable to the high-throughput demands of the clinical chemistry laboratory. Moreover, assay performance enables the levels of TMAO to be quantified in serum or plasma at clinically actionable concentrations for the assessment of cardiovascular disease risks and individualized dietary monitoring.