| Objectives:To develop a simple and precise liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of branched-chain amino acids (BCAAs), aromatic amino acids (AAAs), glutamate (Glu), glutamine (GIn), lysophosphatidylcholines (LPCs), carnitine (CO) and several species of acylcarnitine (AcyCN), which may be the possible risk factors for cardiovascular disease (CVD). To analyze the relationships between the metabolites and the traditional CVD risk factors, diabetes mellitus (DM), carotid intima-media thickness (cIMT) and the existence of carotid plaque.Methods:The liquid chromatography and mass spectrometry conditions of each compound were established by using the standards. Serum metabolites were extracted with isopropanol containing 0.1% formic acid, reconstituted with the mobile phase and then analyzed by the LC-MS/MS. The stable isotope labeled internal standards were used for quantification. Linearity, limits of detection (LOD), limits of quantification (LOQ), matrix effect, imprecision and sample stability were evaluated.600 volunteers attending an annual physical examination were recruited. Age, sex, height, weight and blood pressure of the subjects were recorded. Serum metabolites of the volunteers were analyzed using the LC-MS/MS method. Serum glucose, total glyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), apolipoprotein AI (ApoAI) and B (ApoB), high sensitivity C reactive protein (hsCRP) and serum creatinine (Crea) were analyzed by using the reagent kit. Bilateral B-mode carotid artery images for intima-media thickness and carotid plaque were acquired using a SDU-1200 ultrasound imager. Associations between metabolites and CVD risk factors, obesity, DM, cIMT and carotid plaque were analyzed.Results:The LC separation was performed with a hydrophilic interaction liquid chromatography (HILIC) silica column maintained at 20℃. Aliquots of 4 μL of the reconstituted samples were injected onto the column and eluted with a mobile phase of 75% acetonitrile (containing 7.5mmol/L ammonium formate,0.5% formic acid, pH 3.0) at a flow rate of 0.25 ml/min. MS/MS was carried out on an API 4000 triple quadrupole mass spectrometer. The MS detection was performed with positive electrospray ionization (ESI) in multiple reaction monitor (MRM) mode.25 serum metabolites were separated efficiently within 5.5 min. The mean analytical recoveries ranged from 86.07%-107.58%. The intra-run CVs were below 5% for all analytes and less than 10% for the measurement of the amino acids, LPCs, CO and most of AcyCNs. The concentrations of all the metabolites were not significantly changed after a prolonged storage of 6 months at-80℃.BCAAs, phenylalanine (Phe), tryptophan (Trp), Glu were significantly positively, correlated with TG (P<0.001), and negatively correlated with HDL-C and apoAI (P< 0.01)after adjusting for age, sex and body mass index (BMI).In contrast, Gln/Glu ratios were negatively associated with TG, ApoB (P<0.001), and positively correlated with HDL-C and apoAI (P<0.001).Saturated LPCs (16:0 LPC,18:0 LPC) were found to be significantly positively correlated with TC, TG, LDL-C and ApoB (P< 0.001) while unsaturated LPCs (18:1 LPC,18:2 LPC) were positively correlated with HDL-C and apoAI (P< 0.001). The results of univariate analysis or logistic regression analysis showed that BCAAs, AAAs, Glu, CO, propionyl-L-carnitine (C3) and valeryl-L-carnitine (C5) were significantly positively correlated with BMI (P<0.001), and Gln,18:1 LPC, 18:2 LPC were significantly negatively correlated with BMI (P<0.001). The diabetic group had significantly higher levels of BCAAs, Phe, Glu, acetylcarnitine (C2), C5 and lower levels of Gln/Glu than the non-diabetic group (P<0.05). Concentrations of BCAAs (P<0.001) and C5 (P<0.01) were significantly elevated in the increased cIMT group compared to the levels in the control group. Subjects with carotid plaque had significantly higher levels of most of the AcyCNs than those without carotid plaque (P< 0.05). In the logistic regression model, CO and AcyCNs were the sole laboratory factor correlated with carotid plaque (OR=1.897, P<0.05).Conclusions:A method for the simultaneous quantification of serum 25 metabolites including amino acids, LPCs, CO and AcyCNs by.LC-MS/MS has been established. This method was simple, precise, and accurate and can be used as an efficient tool in metobolomics research and CVD risk accessment. Serum concentrations of the metabolites were analyzed in 600 volunteers and significant associations between the metabolites and the traditional CVD risk factors, DM, cIMT and the existence of carotid plaque were observed. These metabolites may be used as potential predictors of CVD. |
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