Elsevier

Journal of Chromatography B

Volumes 1009–1010, 15 January 2016, Pages 144-151
Journal of Chromatography B

Simultaneous quantification of cardiovascular disease related metabolic risk factors using liquid chromatography tandem mass spectrometry in human serum

https://doi.org/10.1016/j.jchromb.2015.12.019Get rights and content

Highlights

  • A rapid and precise LC–MS/MS method for simultaneous quantification of CVD related metabolic risk factors was developed.

  • The 16 metabolites were effectively separated within 5.5 min.

  • 123 healthy subjects were analyzed.

  • Significant associations between the metabolites and classic CVD risk factors were observed.

  • This method is simple and precise and is suitable for CVD risk assessment.

Abstract

Recent observations from metabonomic studies have consistently found that branched-chain amino acids (BCAAs), aromatic amino acids (AAAs), glutamine (Gln), glutamic acid (Glu), Gln/Glu ratio, carnitine, and several species of acylcarnitines and lysophosphatidylcholines (LPCs) are possible risk factors for metabolic diseases such as diabetes mellitus (DM) and cardiovascular diseases (CVD). We described here a simple and reliable method for simultaneous quantification of these metabolic risk factors by liquid chromatography tandem mass spectrometry (LC–MS/MS). Serum samples were extracted with isopropanol, and the extracted metabolites were separated by hydrophilic interaction liquid chromatography (HILIC) and detected with electrospary ionization (ESI) inpositive ion mode with multiple reaction monitor (MRM) mode. All the metabolites were effectively separated within 5.5 min. Analytical recoveries were in the range of 92.8–106.9%, with an average of 100.6%. The intra- run and total imprecisions for the measurement of these metabolites were 1.2–3.8% and 1.5–7.4%, respectively. Serum concentrations of the metabolites were analyzed in 123 apparently healthy volunteers. Significant associations between the metabolites and traditional CVD risk factors were observed. The newly developed LC–MS/MS method was simple, precise, and accurate and can be used as an efficient tool in CVD research and studies.

Introduction

Diabetes mellitus (DM) is considered one of the most serious and prevalent metabolic disorders whose prevalence in adults has doubled over nearly 3 decades [1]. A portfolio of abnormalities of metabolic and macrovascular homeostasis accompanied DM is believed to conspire to lead to accelerated cardiovascular disease (CVD) and premature deaths [2], [3]. Therefore, DM has been proposed as an independent risk factor for the development of CVD. Earlier identification of individuals at risk and effective interventions are particularly important for delaying or preventing the onset of DM and CVD [4].

Recent high-throughput metabonomic/metabolomic studies have allowed the characterization of hundreds of metabolites (low-molecular weight compounds) from human biospecimens [5]. These metabolites represent intermediates and end products of metabolic pathways that reflect physiological dysfunctions and may mirror earlier stages of diseases. Several studies have documented that branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) are strongly correlated with obesity, insulin resistance, and coronary artery diseases [6], [7], [8]. Meanwhile, the associations of Glutamic acid (Glu), Glutamine (Gln), and Gln/Glu ratio with DM and CVD were also reported [9]. In addition, some species of lysophosphatidylcholines (LPCs), for example, higher levels of LPC 18:0 (stearoyl) and lower value of LPC 18:1 (oleoyl), LPC 18:2 (linoleoyl) as well as higher values of acetylcarnitine have been found to predict impaired glucose tolerance before the onset of DM [10], [11]. These observations are consistence and raise the possibility that alterations in plasma metabolite levels could presage the onset of DM and CVD and therefore aid in the identification of at risk individuals by adding information over standard clinical markers. However, the usefulness of these metabolites in CVD risk assessment needs to be confirmed and validated on external independent populations with simple and reliable methods. Analytical methods for respective measurement of species of amino acids, carnitine and acylcarnitines, and LPCs have been reported [12], [13], [14], however, it requires separate sample preparations and analyses. It is highly desirable to develop a simple, precise, and reliable method for simultaneous measurement of all the metabolites.

In this study, a liquid chromatography tandem mass spectrometry (LC–MS/MS) method for simultaneous measurement of CVD related metabolic risk factors including BCAAs, AAAs, carnitine, some of the acylcarnitines and LPCs has been established. The relationships between these metabolites and traditional CVD risk factors were analyzed in healthy subjects. The LC–MS/MS method uses small amount of serum samples, requires no sample derivation, separates all the metabolites in 5.5 min, and has high through put. This method has been validated to be simple, precise, and accurate, and can be used in CVD research and studies.

Section snippets

Chemicals and reagents

Standards of amino acids [phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), leucine (Leu), isoleucine (Ile), valine (Val), Gln, Glu], free carnitine, acetylcarnitine, high performance liquid chromatography (HPLC)-grade acetonitrile, ammonium formate and formic acid were obtained from Sigma–Aldrich (St. Louis, MO, USA). Standards of LPCs [palmitoyl (LPC 16:0), LPC 18:0, LPC 18:1], oleoylcarnitine (18:1acylcarnitine) and isotopically labeled internal standards of LPCs (LPC 16:0-D31, LPC

Optimization of LC–MS/MS conditions

The ionization and fragmentation of the amino acids, carnitine, acylcarnitines and LPCs under various MS/MS conditions were tested by injecting each of the standards into the MS/MS system using a syringe pump. The MS/MS conditions were optimized for high signal intensity and detection specificity. The transitions used for the detection of BCAAs and AAAs were the same as in our previous study [15]. The transitions of (M + H)+  (M-NH3)+ was used for both Trp and Gln and (M + H)+  (M-COOH–H2O)+ was

Discussion

Metabolomics, which is designed to quantitatively profile a large number of small molecules in cells or biofluids, is a promising approach to elucidate altered metabolic pathways and discover novel biomarkers in DM and CVD [16]. Recent observations from metabonomic studies have consistently found that BCAAs, AAAs, carnitine, acylcarnitines and some species of LPCs are associated with DM and CVD and are possible risk factors for these metabolic diseases [11], [17]. However, the high throughput

Authors’ conflict of interest disclosure

No authors declared any potential conflicts of interest. The funding organizations played no role in the design of the study, review and interpretation of data, or preparation or approval of the manuscript.

Acknowledgement

This work was supported by the National Natural Science Foundation of China< (81472035, 81171647, and 81201337).

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