| Objective:To explore the changes of endogenous small molecule metabolites in rats and patients with deep venous thrombosis(DVT)based on metabolomics technique.To screen the potential biomarkers for early diagnosis and forensic identification of DVT.To find out the metabolic pathways induced by DVT in rats and human.Methods:In part 1,the DVT rat model was developed by ligated the inferio vena cava of rats.The rats were randomly divided into three groups(n=10):a control group,a DVT group,and a sham group.HE staining and masson staining were used to valid model.Urine of rats was collected from 48 to 72 h after operating in metabolic cage.Blood was collected and centrifugated to collect serum at 72 h.The metabolomic data of urine and serum obtained from NMR analysis was imported into SIMCA-P 14.1 software.Then the metabolites were screened based on variable importance in the projection(VIP)obtained from OPLS-DA combined with t test.The affected metabolic pathways of rats were identified by the biological function analysis of metabolites.In part 2,85 cases of DVT patients and healthy control were recruited,respectively.Blood was drawn from an antecubital vein in the morning after a 12-h overnight fast and was centrifugated to collect serum.The serum were detected by ~1H NMR and LC-MS,respectively.The original spectra was preprocessed using MestReNova 9.0 software and Compound Discoverer 2.0 software.Then the data was imported into SIMCA-P 14.1software,respectively.The metabolites were screened based on VIPs obtained from OPLS-DA and t test.The affected metabolic pathways of patients were identified by the biological function analysis of metabolites.Results:The first part of the experiment successfully established DVT rat model which was proved successful by the general observation,HE staining and Masson staining.There were significant differences in serum and urine metabolic profiles from the model group,the sham operation group and the control group.11 different metabolites were screened from the serum and urine of the model group,respectively.The affected mainly metabolic pathways in rats were valine,leucine and isoleucine biosynthesis,butyric acid metabolism,pyruvate metabolism,glycolysis or glycogenogenesis,ketone synthesis and degradation,glyoxylic acid and dicarboxylic acid metabolism,galactose metabolism,arginine and proline metabolism,tricarboxylic acid cycle,alanine,aspartic acid and glutamic acid metabolism.A set of serum biomarkers composed of alanine,acetoacetic acid,glucose,O-acetyl glycoprotein,methanol and N-acetyl glycoprotein was obtained through the ROC curve analysis.A set of urine biomarkers were composed of leucine,glutamine,creatine Creatinine,sucrose,3-hydroxybutyric acid,lactic acid,acetone,alpha-ketoglutarate,citric acid and hippuric acid.In the second part,the metabolic profiles of serum in DVT patients and the healthy control person obtained by ~1H NMR and LC-MS have significantly difference.65differential metabolites were screened out from the DVT patients.20 metabolites were obtained by ~1H NMR and 47 metabolites were obtained by LC-MS,in which glutamine and creatine were detected by both analytical methods.The altered metabolic pathways in patients were:synthesis and degradation of ketone bodies,pyruvate metabolism,glutamine and glutamic metabolism,glycine,serine and threonine metabolism,phenylalanine metabolism,butyric acid metabolism,glycolysis and gluconeogenesis,valine,leucine and isoleucine biosynthesis,arginine and proline metabolism,aminoacylated tRNA biosynthesis,propionic acid metabolism,valine,leucine and isoleucine degradation and so on.A set of serum biomarkers composed of 42 metabolites was obtained through the ROC curve analysis.Conclusions:In this study,the metabolic profile in DVT rats and patients’body fluids were investigated based on metabolomics.The metabolites from serum,urine of DVT rats and serum of DVT patients may be the biomarkers for diagnosis of DVT.The altered metabolic pathway including glycolysis,amino acid metabolism,tricarboxylic acid cycle,the decomposition of fat and other energy metabolism were found in the body of DVT.The results provide a foundation and new strategies for the diagnosis and forensic identification of deep venous thrombosis. |
