Quantitation of Trimethylamine N-Oxide and its Precursors by HILIC-MS/MS and Their Potential Role in Cognitive Behavior Alexander Gaudl (Presenter) Leipzig University Presenter Bio: Alex, the chemist, began his master thesis in 2012 at the institute of laboratory medicine, clinical chemistry and molecular diagnostics of Leipzig University concerning the quantitation of endocannabinoids in human plasma via LC-MS/MS. Although being drawn to analytical chemistry since his bachelor thesis, it was not until finishing his master’s degree that he had finally become the analytical chemist. His subsequently beginning doctorate let him delve deeper into LC-MS/MS analysis with the quantitation of steroid hormones in human body fluids and hair being his main focus. Now, he plans to take on the neuroendocrine axis by means of LC-MS/MS for eventually helping in comprehending the development of depressive disorders.

Authors: Alexander Gaudl (1), Benedikt Zöhrer (1), Madlen Sander (1), Uta Ceglarek (1)
(1) Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig University

Trimethylamine N-oxide (TMAO) enhances thrombocyte activity, affects cholesterol and bile acid metabolism and has already been associated with cardiovascular disease. Its role regarding cognitive behavior, however, remains open. To that matter and to establish normal concentration ranges we developed a new HILIC-MS/MS method for the quantitation of TMAO and its precursors Betaine, Carnitine, and Choline and applied it to a subset of the LIFE Adult study. A potential association with cognitive behavior was assessed using SISCO score.

Introduction

Trimethylamine N-oxide (TMAO), though already known to marine biologists for many years, recently found itself in the scientific spotlight through its association with cardiovascular disease (CVD). Because it affects cholesterol and bile acid metabolism, further association with cognitive disorders is assumable. Results from previous studies focusing on the cardiovascular angle were inconclusive regarding TMAOs role as a biomarker for CVD and studies concerning cognitive functioning are virtually absent. Furthermore, baseline TMAO levels appear to differ significantly between populations from different continents. We established a new HILIC-MS/MS method and determined normal ranges of TMAO and its precursors Betaine, Carnitine, and Choline by means of the LIFE Adult study (n=1000) (1). Furthermore, a potential association with cognitive behavior was assessed using SISCO score.

Methods

Plasma or serum (10 µl) was prepared by protein precipitation using 90 µl methanol including internal standards. Prior to tandem mass spectrometric analysis 10 µl of the supernatant was diluted with 990 µl Acetonitrile/0.2 M ammonium formate buffer 90/10 v/v representing the initial conditions of the chromatographic separation. Rapid hydrophilic interaction liquid chromatography (Kinetex© HILIC 50x2.1 mm, Phenomenex) was coupled to tandem mass spectrometric detection on a SCIEX QTRAP® 6500, applying electrospray ionization in positive ion mode. Injection volume was 5 µl and the total run time was 5 minutes. Deuterium-labeled analytes were used as internal standards.

Results

Between-run imprecision ranged from 4.7% to 6.8% and LLOQ were below 0.3 µM at thousandfold dilution. Recovery rates ranged from 97% to 102%. Preanalytical investigations regarding hands on time of whole blood, blood plasma and processed samples at different temperatures, as well as stability towards freezing and thawing of biological samples or influence of sample material showed high stability and low variance in general. Solely choline concentration showed a slight increase during storage of whole blood at room temperature. Preliminary base data for LIFE Adult gave median TMAO concentrations of 3.8 µmol/l (IQR: 2.8-5.5 µmol/l).

Conclusions & Discussion

We present a robust and highly reproducible HILIC-MS/MS method for the quantitation of TMAO and its precursors Betaine, Carnitine, and Choline. Furthermore, it proved its reliability being applied to about 1000 samples of the LIFE Adult study.

(1) Loeffler M, Engel C, Ahnert P, Alfermann D, Arelin K, Baber R, et al. The LIFE-Adult-Study: objectives and design of a population-based cohort study with 10,000 deeply phenotyped adults in Germany. BMC Public Health. 2015;15:691.