Simultaneous quantification of cardiovascular disease related metabolic risk factors using liquid chromatography tandem mass spectrometry in human serum
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).
References (24)
- et al.
National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country years and 2.7 million participants
Lancet
(2011) - et al.
A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance
Cell Metab.
(2009) - et al.
Measurement of free carnitine and acylcarnitines in plasma by HILIC-ESI–MS/MS without derivatization
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
(2013) - et al.
Identification and quantification of phosphatidylcholines containing very-long-chain polyunsaturated fatty acid in bovine and human retina using liquid chromatography/tandem mass spectrometry
J. Chromatogr. A
(2010) - et al.
Validation of the association between a branched chain amino acid metabolite profile and extremes of coronary artery disease in patients referred for cardiac catheterization
Atherosclerosis
(2014) - et al.
An efficient hydrophilic interaction liquid chromatography separation of 7 phospholipid classes based on a diol column
J. Chromatogr. A
(2012) - et al.
Serum lysophosphatidylcholine level is not altered in coronary artery disease
Clin Biochem.
(2012) - et al.
Mortality from heart disease in a cohort of 23,000 patients with insulin-treated diabetes
Diabetologia
(2003) - et al.
The effect of intensive diabetes treatment on resting heart rate in type 1 diabetes: the diabetes control and complications trial/epidemiology of diabetes interventions and complications study
Diabetes Care
(2007) - et al.
Diabetes Prevention Program Research Group. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin
N. Engl. J. Med.
(2002)
‘Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data
Xenobiotica.
Metabolite profiles and the risk of developing diabetes
Nat. Med.
Cited by (19)
Serum glutamate and glutamine-to-glutamate ratio are associated with coronary angiography defined coronary artery disease
2022, Nutrition, Metabolism and Cardiovascular DiseasesCitation Excerpt :We first analyzed the age and gender-adjusted correlations of Glu, Gln and Gln/Glu with conventional CAD risk factors and found that Glu was positively correlated with the known CAD risk factors, such as BMI, FBG, TG, Crea, and UA, and negatively correlated with HDL-C, whereas reverse correlation between Gln/Glu ratio with these risk factors were observed. These results were in agreement with the previous reports [11,17]. Although Gln and Gln/Glu ratio were highly correlated, correlations between Gln and these parameters attenuated compared with those of Gln/Glu.
Development of a novel analytical method for inflammation and immunity-related metabolites in serum based on liquid chromatography tandem mass spectrometry
2021, TalantaCitation Excerpt :Faouder et al. quantified pro-inflammatory and pro-resolving polyunsaturated acids based on LC-MS/MS, which was optimized to finish a rapid separation of 26 metabolites within 8.5 min with very high sensitivity (0.6–155 pg) [14]. Wang et al. established a method for the quantification of cardiovascular disease related metabolites including amino acids and LPCs using LC-MS/MS. The method could efficiently separate these metabolites within 5.5 min with analytical recoveries ranging from 92.8% to 106.9% [15]. All of the studies above demonstrated good performance in the analyses of various inflammation and immunity-related metabolites (IIMs) based on LC-MS. Nevertheless, currently reported studies for metabolite characterization were mainly focused on several metabolites in a specific category or pathway, comprehensive characterization of IIMs enrolled in multiple associated pathways remains lacking.
Detection of Epstein-Barr virus encoded RNA in fixed cells and tissues using CRISPR/Cas-mediated RCasFISH
2021, Analytical BiochemistryCitation Excerpt :Subsequently, we counted a total 112 particles corresponding to RCasFISH probes and found approximately 92% (103/112) of the RCasFISH particles colocalized with the RNAscope particles, indicating our method share similar detection efficiency and resolution with RNAscope. In clinical settings, EBV LMP1 and EBER are markers routinely employed to detect latent EBV infection in patients with infectious mononucleosis and EBV-associated tumors [24–26]. To set up a rapid, precise and flexible in situ sequential labeling protocol that integrates ISH and IHC in FFPE tissue sections for the diagnosis of EBV-associated cancer, we added antigen retrieval steps before hybridization to increase the producibility of IHC and RCasFISH.
A single-run, rapid polarity switching method for simultaneous quantification of cardiovascular disease-related metabolites using liquid chromatography–tandem mass spectrometry
2021, International Journal of Mass SpectrometryMagnetic nanoparticles functionalized with immobilized apolipoprotein antibodies for direct detection of non-high density lipoprotein cholesterol in human serum
2020, Chemical Engineering JournalCitation Excerpt :Serum total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol, apoAI, apoB, and high-sensitivity C-reactive protein (hsCRP) levels were measured using assay kits from Sekisui Medical Technologies (Osaka, Japan) on a Hitachi 7180 chemistry analyzer (Tokyo, Japan). Serum glutamine (Gln) and glutamic acid (Glu) concentrations were measured using our previous MS/MS method [30]. Data were analyzed using SPSS 22 software (IBM, Armonk, NY, USA).