Based On Relatively Quantitative Targeted Metabolomics Method To Study The Metabolic Disorder Of Heavy Alcohol Use Rats And Therapeutic Mechanism Of Diazepam

Part 1: Development of mass spectrometry-based relatively quantitative targeted method for amino acids and neurotransmittersObjective: An efficient relatively quantitative targeted analysis approach was developed by applying a HILIC-MS/MS system coupled with the QC-based random forest signal correction(QC-RFSC)algorithm to meet the demand for the analysis of the small molecule metabolites present in biological samples.Method: Serum samples were treated by protein precipitation.The relatively quantitative targeted method was performed by using an Agilent 1200 series HPLC coupled online via electrospray ionization with an Agilent 6410 triple quadrupole mass spectrometer in MRM mode.A ZORBAX HILIC Plus column(4.6 mm × 100 mm,3.5 μm)from Agilent was applied for chromatographic separation.The binary gradient mode was used for the sample analysis.Solvents A and B were water and acetonitrile,respectively,and both contained 20 m M ammonium formate.Agilent Mass Hunter Chemstation software(B.01.03)was used to automate peak integration to obtain the raw data.Stat Target,providing a novel algorithm based on QC-RFSC to correct for unwanted signal drift,was applied to ensure the replicability and accuracy of the data.Multivariate analysis and statistical power analysis were performed by using metaboanalyst.In a proof of concept,this method was also used to perform a metabonomics study for major depression.Results: High-quality relative quantification was achieved without internal standards,and integrated peak areas were successfully used for statistical and pathway analyses.Amino acids and neurotransmitters(dopamine,kynurenic acid,urocanic acid,tryptophan,kynurenine,tyrosine,valine,threonine,serine,alanine,glycine,glutamine,citrulline,GABA,glutamate,aspartate,arginine,ornithine and histidine)in serum samples were simultaneously determined with the newly developed method.To demonstrate the applicability of this method in large-scale analyses,we analyzed the above metabolites in serum from patients with major depression.The serum levels of Glutamate,aspartate,threonine,glycine and alanine were significantly higher,and those of citrulline,kynurenic acid and urocanic acid were significantly lower,in patients with major depression than in controls.Conclusion: We developed an efficient relatively quantitative targeted analysis approach for the determination of small molecule metabolites present in biological samples.Part 2: Acute alcohol exposure markedly inhibit the activity of the Glutamatergic systemObjective: The goal of this study was to investigate the change of various amino acids and neurotransmitters affected by acute alcohol exposure and research the pathophysiology of alcoholism.Methods: The mouse model of acute alcohol exposure were built according to the method for William J.Mc Bride.The hydrophilic interaction liquid chromatography(HILIC/QQQ MS)method was used to determine 15 amino acids and neurotransmitters to find out the different metabolites between the test and control groups.Results: Brain tissue levels of alanine,serine,glycine and Glutamate of the test group were significantly lower than those in the controls.Significantly higher brain tissue tryptophan was observed in the test group(adjusted P values less than 0.05).Conclusion: The results suggest that acute alcohol exposure can significantly change the amino acids and neurotransmitters,such as alanine,serine,glycine,glutamate and tryptophan.Except for the alanine,serine,glycine,glutamate and tryptophan can affect the activity of the Glutamatergic system.We assume that acute alcohol exposure can inhibit the activity of the Glutamatergic system through various pathways.Part 3: The metabolic disorder of heavy alcohol use and therapeutic target for diazepamObjective: The purpose of this research was to investigate the change of various amino acids and neurotransmitters in heavy alcohol use rats,and also the effect of diazepam which is the clinical commonly used drug of replacement therapy for alcohol dependence.Method: The intermittent access to 20% alcohol two-bottle choice drinking procedure was used to induce heavy alcohol use rats.After the model being made,the quantitative targeted method,which has been previously developed,was used to investigate the change of various amino acids and neurotransmitters in heavy alcohol use rats and diazepam treatment groups.Methods Enzyme-linked immunosorbent assay(ELISA)was used to determine BDNF level in the hippocampus.Metabolites that RSD more than 15% were excluded from statistical analysis.Multivariate analysis and statistical power analysis were performed by using metaboanalyst.Principal component analysis was used to visualize the general cluster of QC samples to evaluate data precision.The partial least squares-discriminant analysis model(PLS-DA)was applied to identify the differences in metabolic profiles between groups.The student’s t-test,calculated in Graph Pad Prism 8,was used to determine the significance of each metabolite.The p values were adjusted with Benjamini Hochberg’s false discovery rate,and values 0.05 or below were considered statistically significant.Results: Metabonomics study of heavy alcohol use rats showed that Glutamate(VIP=2.006,P=0.002)and aspartate(VIP=1.990,P=0.003)level were significantly higher than normal control in serum,and Glutamate(VIP=1.600,P=0.001),glycine(VIP=1.506,P=0.004)and alanine(VIP=1.502,P=0.005)level were significantly lower than normal control in hippocampus tissue.In the group of diazepam treatment,we discovered that glutamate(VIP=1.602,P<0.001)and glycine(VIP=1.651,P<0.001),which were found to significantly increase in heavy alcohol use rats,were markedly decreased in serum after continuous administration for two weeks.Furthermore,the serum level of kynurenine also significantly increases after treatment.In the hippocampus,5-hydroxy Tryptamine(VIP=1.776,P<0.001),glycine(VIP=1.506,P=0.004),γ-aminobutyric acid(VIP=1.621,P=0.001)and glutamate(VIP=1.526,P=0.006)were significantly higher after diazepam treatment.Finally,we discovered that BDNF level in the hippocampus was significantly higher in heavy alcohol use rats,but significantly reduced after diazepam treatment.Conclusion: Several amino acids and neurotransmitters abnormalities were observed in serum and hippocampus of heavy alcohol use rats.Especially for glutamate and BDNF,which were significantly decreased in heavy alcohol use rats.They play an important cooperative effect in the regulation of synaptic transmission and neuronal development.We also found that administration of diazepam could reverse the level of glutamate and BDNF.