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

Abstract

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 μM, respectively. Linearity was demonstrated within the reportable range of 3.3 to 3,000 μM. TMAO measurements using the NMR assay, which involves minimal sample preparation, compared well with values obtained with the MS-based assay (R2 = 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.

Fast LC-ESI-MS/MS analysis and influence of sampling conditions for gut metabolites in plasma and serum

“…Techniques that have been used to analyze gut metabolites are proton nuclear magnetic resonance ( 1 H-NMR) and mass spectrometry. There are only a few studies that analyzed TMAO with 1 H-NMR 12,13 . This is probably due to the difficulties that are faced during 1 H-NMR analysis of the TMAO metabolism, such as the inability to distinguish betaine signals from TMAO signals at pH values above 5.8 14 and the overlap of glucose signals with TMAO, betaine and choline 15 .…”

Impact on cardiometabolic risk of a weight loss intervention with higher protein from lean red meat: Combined results of 2 randomized controlled trials in obese middle-aged and older adults

“…Linear regression of the NMR subclass signal areas against serum lipid and apolipoprotein levels measured chemically in a large reference range study population (n 5 698) provided the conversion factors to generate NMR-derived concentrations of total cholesterol and triglycerides, HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), apolipoprotein A-I, and apolipoprotein B. NMR-derived concentrations of these parameters are highly correlated (r $ 0.95) with those measured by standard methods. Quantification of GlycA, 45 BCAA, 37 TMAO, 30 betaine, 46 and calculation of the LP-IR scores (values 1-100) 32 have been described previously.…”

“…The CVD risk markers included conventional lipids, a comprehensive lipoprotein profile (lipoprotein particle class and subclass numbers and average sizes), [23][24][25][26] GlycA (a measure of global inflammatory burden), 27,28 and the gut microbiome-related metabolite trimethylamine-N-oxide (TMAO). [29][30][31] Markers of type II diabetes risk included lipoprotein insulin resistance index (LP-IR; a high-throughput measure of insulin resistance based on 6 lipoprotein parameters that are altered in metabolic disease), [32][33][34][35][36] branched-chain amino acids (BCAA), 37,38 and fasting blood glucose, all of which have strong positive associations with incident type II diabetes, and betaine (an active metabolite of choline and a component of beets) that is inversely related to type II diabetes risk, CVD, and nonalcoholic fatty liver disease. [39][40][41] All biomarkers were assessed at baseline and after 6 months of dietary intervention, and an exploratory approach was used with respect to all outcome variables with the effect of higher protein intake with change in the set of cardiometabolic items of primary interest.…”

Facile Fluorescence Monitoring of Gut Microbial Metabolite Trimethylamine N-oxide via Molecular Recognition of Guanidinium-Modified Calixarene

“…To our knowledge, many researchers preferred utilization of liquid chromatography tandem mass spectrometry (LC-MS/MS) for determining TMAO, employing a stable isotopically labelled standard 10-16, which required synthetic stable isotope markers, specially trained personnel, and specialized and costly analytical instruments that were not widely available in clinical diagnostic laboratories. Other studies have employed proton nuclear magnetic resonance spectroscopy ( 1 H NMR) to determine TMAO, which underwent poor sensitivity, instability of determining results subjected to pH variation 17-19. To address these issues, the development of a novel, simple, inexpensive and rapid method for TMAO detection is urgently needed.…”