Combined Targeted and Untargeted Strategy for Quantification of Amino Acids and Acylcarnitines in Clinically Relevant Biofluids and Tissue Lysates Vera van der Velpen (Presenter) Metabolomics Unit, University of Lausanne Presenter Bio: Vera van der Velpen finished her BSc in Nutrition and Health and her MSc in Food Safety at Wageningen University before starting her PhD at the same university in 2009. In her PhD, she applied transcriptomics in two clinical trials to investigate the potential health effects of isoflavone supplementation in postmenopausal women. She moved on to the University of East Anglia in 2014 for her first PostDoc position, where she first applied mass spectrometry-driven metabolomics to measure exposure markers of blueberry consumption in a large clinical trial. Since September 2017, she is working as a PostDoc at the University of Lausanne in the group of Julijana Ivanisevic, where she is using metabolomics to study the changes in brain metabolism in Alzheimer’s disease.

Authors: Vera van der Velpen (1), Tony Teav (1), Héctor Gallart-Ayala (1), Florence Mehl (1,2), Clotilde Roux (3), Shing-Chi Liu (1), Beat Riederer (4,5), Hugues Henry (6) and Julijana Ivanisevic (1)
(1) Metabolomics Platform, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland (2) Vital-IT – Swiss Institute of Bioinformatics, Lausanne, Switzerland. (3) Clinical Chemistry Service, Lausanne University Hospital, Lausanne, Switzerland (4) Proteomic Unit, Centre for Psychiatric Neuroscience, Department of Psychiatry, CHUV, Lausanne, Switzerland (5) Department of Fundamental Neurosciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland (6) Innovation and Development Laboratory, Clinical Chemistry Service, Lausanne University Hospital, Lausanne, Switzerland

Amino acids and acylcarnitines are key players in energy metabolism, however, analytical methods for comprehensive and robust measurement of these compounds without derivatization or use of ion-pairing agents are scarce. We present a HILIC-based HRMS method for the absolute quantification of amino acids and acylcarnitines in a single run, while simultaneously taking advantage of HRMS in full scan mode to screen for additional derivatives (e.g. amines, hydroxylated acylcarnitines) and other polar metabolites (e.g. purines, pyrimidines, hexoses). Using a simple extraction method with internal standards, we have successfully evaluated our method with NIST plasma and examined a profile of these compounds across different biofluids (plasma, urine, CSF) and human brain tissue.

Introduction

Amino acids and acylcarnitines are key players in energy metabolism and their circulating levels are indicative of inborn errors of metabolism as well as acquired metabolic disorders later in life, such as diabetes and cancer (1). While amino acids are known as protein building blocks, they can also act as potent nutrient signals whose sensing regulates mTOR signaling and cellular growth (2). Acylcarnitines are essential for oxidative catabolism of fatty acids (i.e. β-oxidation); a process that generates two times more energy than can be produced from glucose (3). Increased levels of branched-chain amino acids and long chain-acylcarnitines have been reported as predictive and associated with insulin resistance and type 2 diabetes in several population studies (4, 5). Despite their physiological importance, analytical methods for combined and comprehensive measurement of amino acids and acylcarnitines are scarce and typically involve derivatization or the use of ion pairing agents (6). Moreover, these methods focus mainly on plasma and dry blood spot analysis for inborn error screening (7). Yet, the analysis of other biofluids (e.g. cerebrospinal fluid) and tissue extracts could bring the important insights at the organ level, in the context of acquired metabolic disorders, for understanding of peripheral changes in plasma. Here, we present a hydrophilic interaction liquid chromatography (HILIC)-based high-resolution mass spectrometry (HRMS) method for the absolute quantification of amino acids and acyl carnitines in full scan mode, allowing for parallel-untargeted analysis of many additional precursors and derivatives, together with other relevant polar metabolites.

Methods

A rapid and simple sample preparation method was optimized to extract the amino acids and acylcarnitines, whilst precipitating proteins, from a minimal amount of biofluid using a 1-step dilution with an internal standard mixture in acetonitrile (stable-isotope labeled amino acids, n=22 and acylcarnitines, n=12). For tissue, the samples were first homogenized (using a beat beater) and the resulting organic homogenate underwent sample preparation as described above. The supernatant extracts were directly injected into the LC-HRMS system.

An Acquity UPLC BEH Amide column was used for the simultaneous separation of amino acids and acylcarnitines in acidic conditions. The chromatographic system was coupled to a HRMS instrument (QExactive Focus) using ESI in positive mode and data was acquired in full scan mode (m/z 60 – 900) at mass resolving power of 70,000 FWHM. Specificity of the analysis was validated by simultaneous data acquisition in parallel reaction monitoring (PRM) mode.

Results

The optimized method allows for the absolute quantification of 40 amino acids, 12 saturated short, medium and long-chain acylcarnitines, in addition to 35 unsaturated, hydroxylated and dicarboxylated acylcarnitines. The accuracy of the developed method was evaluated using plasma reference material (Certificate of Analysis, NIST 1950) and accuracies between 80% and 100% were obtained for the entire panel of measured amino acids. Method was also validated in terms of quantitation precision with intraday and interday relative standard deviations of ≤ 12% and ≤ 20%, respectively. Subsequently, to demonstrate the versatility of our method and compare the profiles of measurable compounds across different sample types, the amino acid and acylcarnitine profiles were acquired for different biofluids, including pooled plasma, urine and CSF as well as human brain tissue from two different regions, hippocampus and frontal cortex.

For example, in human brain tissue, 37 amino acids and 12 acyl carnitines were quantified in both regions. Preliminary results revealed similar amino acid and acylcarnitine profiles in paired frontal cortex and hippocampus samples, with statistically significantly different results for α-aminobutyric acid and γ-aminobutyric acid.

In addition to quantitative amino acid and acylcarnitine analysis, data acquisition on a high-resolution instrument operating in full scan mode allowed for the identification and relative quantification of other polar metabolites. For instance, with this approach we could quantify other types of acylcarnitines, other amino acids and derivatives, as well as purines, pyrimidines, alkaloids and carbohydrates (e.g. hexoses, disaccharides). These results were demonstrated across different sample types.

Conclusions & Discussion

The presented method allows for comprehensive quantification of amino acids and acyl carnitines in several biological matrices, while the use of HRMS in full scan mode enables the collection of untargeted data for identification and quantification of other amino acids, unsaturated and hydroxylated acylcarnitines as well as other polar metabolites in the same run. Accuracy of our method was successfully evaluated with the reference NIST plasma and a valuable overview will be given regarding the composition/signature of these compounds in different biofluids and tissue extracts.

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