Study On Biomarkers Of Alcoholic Liver Disease And Its Different Stages Based On Metabolomics Technology

Background and objective:At present,with the development of economy and society in China,alcoholic liver disease(ALD)has gradually become the main cause of morbidity and mortality of liver diseases,and it is also a major social health problem.ALD refers to a series of liver diseases,including steatosis,steatohepatitis,liver fibrosis,cirrhosis and even hepatocellular carcinoma.Due to the lack of specific clinical symptoms and diagnostic indicators of ALD,most ALD patients enter the irreversible cirrhosis stage when they see a doctor,so the early diagnosis and treatment of ALD is very necessary for disease prevention and monitoring.Metabonomics is an analytical technology platform developed in recent years,which is widely used in the medical field.It is mainly used in screening disease diagnosis indexes and quantitative detection of small molecular substances.Therefore,metabolomics technology was used to screen the differential metabolites in the early stage of ALD and liver cirrhosis,and to identify and verify the feasibility of these metabolites as diagnostic markers for the early and different stages of ALD.Methods:This study included 128 patients with early liver injury,40 patients with alcoholic liver cirrhosis(ALC),and 40 healthy controls.Patients with early liver injury included 45 cases of ALD,41 cases of non-alcoholic fatty liver disease(NAFLD)and 42 cases of viral liver disease(VLD).Ultra-high performance liquid chromatographyquadrupole mass spectrometry(UHPLC-Q-TOF MS)was used to analyze the differential metabolites in serum samples.The XCMSplus platform and SIMCA software were used to process and analyze the data,and the differential metabolites screened out were identified by XCMS online platform and METLIN database,and the identified compounds were enriched and analyzed by Matebo Analyst online software.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to identify and quantitatively detect some metabolites in the differential metabolic pathway.The correlation between different metabolites and clinical laboratory results was determined by Spearman correlation coefficient.Results:A total of 40 different metabolites were found in three groups of patients with early liver disease and healthy controls,including 5 bile acids(BAs),8 amino acids,18 lipids and 9 other substances.The 40 kinds of differential metabolites formed six significant differential metabolic pathways,including primary BA biosynthesis,biosynthesis of unsaturated fatty acids,fatty acid elongation,arginine biosynthesis,pantothenic acid and coenzyme A biosynthesis,and glutathione metabolism,among which primary BA biosynthesis was the most significant differential metabolic pathway(P=0.007).Therefore,15 kinds of BA profiles were detected,and the results showed that BA profiles had the best discrimination for early ALD patients: compared with the healthy control group,the synthesis of cholic acid(CA)(P< 0.001)and chenodesoxycholic acid(CDCA)(P= 0.005)increased;the combination of CA and CDCA with taurine and glycine increased,including glycocholic acid(GCA)(P= 0.001),glycochenodeoxycholic acid(GCDCA)(P= 0.001)and taurochenodeoxycholic acid(TCDCA)(P= 0.01);ursodeoxycholic acid(UDCA)(P= 0.004),Tauroursodeoxycholic acid(TUDCA)(P= 0.028)and Glycoursodeoxycholic acid(GUDCA)(P= 0.002)in secondary BA also increased significantly;and 12 significantly changed BA profiles can be used as diagnostic markers of ALD(AUC=0.883).The change trend of BA profiles in ALD was similar to that in NAFLD,but the change of BA profiles in ALD was more obvious than that in NAFLD.GCDCA(P= 0.001),GUDCA(P= 0.001),TBA(P= 0.015),primary BA(P= 0.018),secondary BA(P= 0.022)and conjugated BA(P= 0.004)in ALD group were significantly higher than those in NAFLD group.The main difference between ALD group and VLD group was that the change trend of secondary BA was opposite.Compared with VLD group,deoxycholate acid(DCA)(P= 0.009),lithocholic acid(LCA)(P= 0.002),glycine lithocholic acid(GLCA)(P< 0.001)and taurodeoxycholic acid(TDCA)(P= 0.002)in ALD group were all lower.Therefore,early ALD can be distinguished from early liver diseases with other causes by the changes of BA profiles.In order to determine the diagnostic significance of BA profiles in different stages of ALD,the BA profiles in ALC was detected.Compared with early ALD,except for CA,CDCA and DCA,ALC showed no significant differences(P > 0.05),all other BA profiles were significantly increased,and 19 differential BA profiles could be used as a diagnostic indicator for the staging of alcoholic liver fibrosis(AUC=0.868).Conclusion:Based on these results,BA profiles(CA,CDCA,TCA,GCA,TCDCA,GCDCA,LCA,DCA,UDCA,TLCA,GLCA,TDCA,GDCA,TUDCA,GUDCA,TBA,primary BA,secondary BA,free-BA,conjugated BA,G-conjugated BA,T-conjugated BA)are potential indicators in the diagnosis of ALD and evaluation of different stages.