Metabonomics Study Of Wilson's Disease

Background:Hepatolenticular degeneration disease is an abnormal copper metabolism caused by autosomal recessive inheritance.Excess copper accumulates in important organs of the human body such as brain,liver,cornea and kidney,which impairs the normal function of organs and can be life-threatening.Metabolomics is a high-throughput and high-precision quantitative analysis of metabolite changes in organisms by in vitro factors,looking for changes in the types and quantities of different metabolites,and trying to reveal the interrelationships and laws between living body physiology and pathological changes.It is a new subject after genomics,transcriptomics and proteomics.At present,it is widely used in the screening and development of new drugs,research on the mechanism of drug action and its toxicity evaluation,diagnosis and treatment of diseases and prognosis.At present,there are metabolomics-related studies on animal models of hepatolenticular degeneration disease(ATP7B gene knockout mice and LEC mouse models),and changes in lipid metabolism have been observed,but there is currently no research on metabolomics in patients with hepatolenticular degeneration.Aim:In this study,metabolomics changes in hepatolenticular degeneration were studied by ultra-high performance liquid chromatography-mass spectrometry metabolomics research platform to analyze the significance of differential metabolites in patients with Wilson's disease and to analyze the most meaningful metabolism pathway,further elucidating the possible pathogenic mechanisms of hepatolenticular degeneration.Method:The serum of patients with hepatolenticular degeneration who were hospitalized in the First Hospital of Jilin University from September 2016 to October 2018 and those who were examined at the First Hospital Medical Center of Jilin University were collected.In the case of patients with informed consent,5 ml of fasting venous blood was collected from patients with hepatolenticular degeneration,centrifuged for 5 min within 2 hours,centrifuged at 4000 rpm,and the supernatant was labeled and stored in a-80 °C refrigerator.The specimens were taken out,thawed and pretreated before the experiment,and the samples pretreated by the patient were detected by UHPLC-Q Exactive Orbitrap HRMS,getting the raw data.Data ProcessingThe raw data files were preprocessed and imported into MetaboAnalyst4.0 software for principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA)to screen differential metabolites with variable important projection(VIP)greater than 1.According to the t-test results,the differential metabolites were screened,and the receiver operating characteristic curve(ROC)was established for three differential metabolites with small p-values.The area under the ROC curve(AUC)was evaluated to judge whether the differential metabolite have a medium or above diagnostic value.And conduct metabolic pathway analysis.Result:1.In this study,the PCA and PLS-DA models of the patient group in the treatment of hepatolenticular degeneration and the controls in positive and negative ion mode were successfully constructed.In the PCA model,the difference between the two groups was not obvious.The separation trend increases in the PCA model.2.A total of 35 serum metabolites with significant differences were identified between the patients and the healthy control group.The 35 differential metabolites were Taurine,N-Methylphenylethanolamine,Ubiquinone-1,Benzyl2R-3S-2-methyl-3-hydrox-ybutanoate(uncertain),N1-Acetylspermidine,3-Deoxy-D-manno-octulosonat-e(uncertain,UN),2-Arachidonoy-lglycerol(UN),Hexadecanedioic acid,Ethionine,Dopamine3-O-sulfate,1-Palmitoylglycerophosphocholine,13-Hydroxylupan ine,Chenodeoxyglycocholate,N-Acetylmuramate(UN),Indoleacrylicacid,Methylimida-zoleacetic acid(UN),5-Hydrox-yisourate(UN),Choline phosphate,5-6-Dihydroxy-3-methyl-2-oxo-1-2-dihydroquinoline(UN),Taurochenodeoxycholicacid,1-Pyrroline-4-hydroxy-2-carboxylate,2-Methylserine(UN),I-Urobilinogen,Ecdysone(UN),Coniferylalcohol(UN),N5-Ethyl-L-glutamine,D-2-Amino-hexano-6-lactam(UN)Protoporphyrinogen IX,4-Guanidinobutanoate(UN),Aspartyl-Phenylalanine,Docosahexaenoicacid,2alpha-D-mannosyl-D-glycerate(UN),hydroxy-2-oxo-Heptaned ioicacid(UN),hexadecyl-ethanolamine,6-Acetyl-D-glucose(UN).The above compounds were compared with the online database and were not completely verified by the standard.Therefore,there may be qualitative inaccuracies.So we use UN as a marker.3.According to the ROC curve analysis,it was found that the first three metabolites which having the lowest p value in t-test have good differentiation value between patients and controls.Taurine,N-Methylphenylethanolamine and Ubiquinone-1 might serve as markers between patients and controls.4.Three metabolic pathways with higher pathway impact are Porphyrin and chlorophyll metabolism,Arginine and proline metabolism,Taurine and hypotaurine metabolism.The pathogenesis of hepatolenticular degeneration might be related to the shortage of energy supply,the increase of oxygen free radicals production and the clearance of obstacles,and the abnormal ammonia metabolism.Conclusion:1.Compared with controls,the metabolic profile of patients was altered obviously.2.Taurine,N-Methylphenylethanolamine and Ubiquinone-1 may serves as the marker metabolites in distinguishing patients from controls.3.The pathogenesis of WD may be related to the shortage of energy supply,oxidative stress,and the abnormal ammonia metabolism.