| Diabetes is a chronic metabolic disease caused by the interaction of genes and environment,and charactered by high morbidity and mortality and multiple complications,and type 2 diabetes accounts for around 90%of all cases of diabetes.To date,the causes of type 2 diabetes are not completely understood and there is a lack of pre-diabetes marker for diagnosis.In order to study the biochemical mechanism in the development of type 2 diabetes and search for potential urinary biomarkers,we established a urine metabolome analysis method based on UHPLC-QTOF-MS,and then which was employed to study the difference of urine metabolomes in different stages of type 2 diabetes.This paper includes the following two parts:First,establishment of urine metabonomic analysis method based on UHPLC-QTOF-MS.Through optimizing chromatographic column,mobile phase conditions and mass spectrometric parameters,a global metabonomic analysis method for urine based on mass spectrometry has been established.This method can cover metabolites of TCA circle,?-oxidation of fatty acids,protein metabolism,nucleic acid metabolism,amino acid metabolism and other metabolic pathways.By acquisition and analysis of the mass spectra of pure chemical standards,a mass spectrum database was established in our laboratory.At the same time,the preparation method,dilution ratio and collection conditions of urine were optimized,which provided a method for the study of urinary metabonomic analysis based on UHPLC-QTOF-MSSecond,the application of the established method to study the urinary metabonomic changes during the onset of type 2 diabetes.We studied the urinary metabonomic changes of normal,pre-diabetes,(including impaired glucose tolerance and impaired fasting glucose)and type 2 diabetes using the analytical method mentioned aboveNormalization is prerequisite for reliability of metabonomic analysis.In this study,two normalization methods,including normalization to creatinine intensity and probabilistic quotient normalization method based on the mode of quotients(mPQN),were used to process the urinary metabonomic data.It was found that results of the two normalization methods were basically the same,which indicated both the two normalization methods were suitable for metabonomic analysis of urinary metabonomic data based on UHPLC-QTOF-MS.In addition,we analyzed urinary metabonomic changes of diabetes in different stages.There were urinary metabolomic fingerprint with different degrees of glucose intolerance,the altered metabolic fingerprint associated mainly with TCA intermediates,branched chain amino acids,aromatic amino acids,glucose and its oxidation products,choline metabolism,4-pyridoxic acid produced by oxidation of vitamin B6,dicarboxylic acid,acylcarnitines,and microbiota-host co-metabolites.The Metabolites with significant inter-group differences was subjected to logistic regression analysis and ROC analyses to establish and to assess the multi-metabolite signature for reliably differentiating IFG,IGT and T2DM from NGT,respectively.The metabolites of betaine,acetyl-l-tyrosine,succinic acid and isobutyrylcarnitine in urine could be used as a combined marker to distinguish the state of type 2 diabetes from the normal state,and the AUC of the model is 0.911(95%CI:0.859-0.962).The other four metabolites,succinic acid,D-Glucuronic acid,L-2-Aminoadipic acid and xanthine,can be used as a combined marker to distinguish impaired glucose tolerance from normal glucose tolerance,and the AUC of the model is 0.780(95%CI:0.692-0.868).The two metabolites of p-cresol sulfate and pimelic acid in urine can be used as a combined marker to distinguish the impaired and normal state of fasting blood glucose,and the AUC of the model is of 0.823(95%CI:0.697-0.949)Compared with blood,Urine is easy to collection,less restriction of volume,and no injury to the human body,and the collection process is less harmful to the individuals Combination of limited urinary metabolites could readily distinguish IGT,IFG and T2DM from NGT.Such metabolite combinations can be used as urinary signatures for prediabetic and diabetic conditions,which probably provides a useful way for early detection of glucose intolerances in large populations,which is of great significance for clinical pre-diagnosis.In summary,we established a urinary metabonomic analysis method based UHPLC-QTOF-MS,and studied urinary metabonomic changes during the onset of type 2 diabetes,providing a new way for the study of clinical diagnostic markers about type 2 diabetes. |
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