Application Of Metabolomics In Metabolic Disease And Detection Of Amino Acids

Background:As a branch of system biology,metabolomics method can study the endogenous or exogenous metabolites changes caused by disease or drug.It has been used to detect the changes of metabolic pathways,disease markers for diagnosis or reveal the physiological and pathological state of subjects for years.Metabolomics technology,such as gas chromatography,liquid chromatography,or chromatography-mass,always be used to detect amino acids or lipid,etc.In recent years,the incidence of nonalcoholic fatty liver disease(NAFLD)in the countries around the world continues to increase.While it is always found in patients with obesity,type 2 diabetes and the metabolic syndrome,the non-obese population showed a gradual trend of high morbidity.The pathogenesis of the disease is still unclear.All the methods for diagnosis have shortcomings respectively.No effective drug has been found to treat the disease.Looking for the biomarker for diagnosis,analyzing the change of metabolic pathways and exploring the pathogenesis of NAFLD are very important.This study focused on the following two issues.Objective:1.In the first part,we aim to analyze the serum sample of non-obese patients with NAFLD and healthy controls and screen the significantly differential metabolites between the two groups of metabolites,identify specific biomarker,explore the pathogenesis of the disease and look for potential therapeutic targets.2.To develop a gas chromatography–mass spectrometry(GC–MS)detection method for quantification of the nonpolar amino acids in amniotic fluid of congenital malformation including alanine(Ala),leucine(Leu),isoleucine(Ile),proline(Pro)and phenylalanine(Phe).To compare the different amino acids in amniotic fluid between pregnant women with congenital malformation and normal control and anayze the corresponding pathogenesis.Methods:1.Using the platform of UPLC/Q-TOF – MS combined with pattern recognition methods,we applied metabonomics method to analyze the serum samples of non-obese patients with NAFLD(n = 24)and age,BMI-matched healthy controls(n = 22)to find the changes of metabolic components.By Marker View v1.2.1 software,we pretreat and normalize the experimental raw data.Then,we used Metaboanalyst 3.0 software to carry on the principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA).Based on the OPLS-DA analysis,we constructed the recognition model to separate the two groups.According to very importance projection(VIP)got from SIMCA-P13.0 software and P value in univariate statistical analysis,we screen and identify the distinguishing ions between NAFLD patients and healthy controls.Metaboanalyst 3.0 software is used to analyze the disturbed metabolic pathways.By receiver operating characteristic curves(ROC),we evaluate the clinical diagnostic performance of metabolites.2.After precipitated protein of the 100 ?L supernatant of amniotic fluid by methyl alcohol,the supernatant was dried by nitrogen.The extractions were treated with MSTFA for derivatization.Then gas chromatography-mass spectrometry was used to detect and analyze the amino acids.Results:1.The metabolic pattern of non-obese NAFLD patients and healthy controls can be separated clearly in both negative and positive models.Finally,17 metabolites were detected significantly different between the two groups.These substances include steroid hormones and their derivatives,fatty acid ester,carbohydrate,benzene and its derivatives,uric acid,etc.Compared with healthy controls,patients with NAFLD have higher serum levels of chenodeoxycholic acid,taurocholic acid,l-carnitine,arachidonic acid,proline,gentisic acid,hydroxy butyric acid,leucine,glutamic acid,uric acid,creatinine.Significant decrease in the levels of palmitoleic acid,palm amide,3-phenoxy benzoic acid,betaine.Non-obese patients with NAFLD also showed abnormal metabolic pathways,including in arginine and proline metabolism,D-Glutamine and D-glutamate metabolism,alanine,aspartate and glutamate metabolism,arachidonic acid metabolism.2.This method was proved to be good sensitivity,precision,accuracy and recoveries.Under the optimum testing conditions,the limit of detection(LOD)was 0.12~0.38 mg/L.The calibration curves showed good linearity over the investigated concentration range between 0.5 and 10 mg/L.The recoveries were 91.1 % to 104.4 %.The relative standard deviation of intra and inter-day were 0.8 % to 9.3 %.The developed method was applied to the quantification of 5 nonpolar amino acids in amniotic fluid of 17 pregnant women with congenital malformation and 13 normal control pregnant woman.The contents of leucine and isoleucine decreased in disease group compared to controls.The difference of the other three amino acids between the two groups had no statistical significance.Conclusions:1.Using the metabolomics research method,this study found that there are a variety of abnormal endogenous metabolites in non-obese patients with NALFD.The results of our research will help to understand the pathogenesis of NAFLD and provide evidence for the diagnosis and treatment of the disease.2.The validation results showed that the method was suitable for detection of the amino acids in amniotic fluid and having broad prospect of clinical application.Leucine may participate in the pathogenesis of congenital malformation.