| Background/Objective:It is found that there are more and more patients with liver injury caused by other causes besides viral hepatitis,including drug-induced liver injury,primary biliary cholangitis,autoimmune hepatitis and so on.The clinical manifestations of drug-induced liver injury(DILI),primary biliary cholangitis(PBC)and autoimmune hepatitis(AIH)are similar,which often require differential diagnosis.The gold standard of diagnosis is liver biopsy.Drug-induced liver injury refers to the liver injury caused by drugs or their metabolites,also known as drug-induced hepatitis.It is known that thousands of drugs can cause liver injury or potential hepatotoxicity;many drug excipients,Chinese herbal medicine and health care drugs can also lead to liver injury.At present,the incidence of drug-induced liver injury is reported in China.Acute drug-induced liver injury accounts for about 20% of hospitalized patients with acute liver injury.Drug-induced liver injury has no specific clinical signs or symbols,and its clinical manifestations can range from asymptomatic,mild,non-specific biochemical changes to acute liver failure.Clinical diagnosis is difficult,so it is necessary to study the exact diagnostic indicators of drug-induced liver injury.Primary biliary cholangitis and autoimmune hepatitis are two types of autoimmune liver diseases(ALD),which caused by disorders of the autoimmune system.Diagnosis of primary biliary cholangitis and autoimmune hepatitis is challenging due to a variety of clinical manifestations and laboratory findings.Diagnosis of primary biliary cholangitis and autoimmune hepatitis should be combined with clinical features,liver biochemical abnormalities,autoantibodies and liver histological abnormalities.However,liver biochemical function and autoantibody levels are not the gold standard for diagnosing these two diseases.Because of the invasive nature of liver biopsy it is not easy to be accepted by patients in clinic.In addition,atypical liver histology is easy to confuse the diagnosis.In view of the lack of effective diagnostic markers for drug-induced liver injury,primary biliary cholangitis and autoimmune hepatitis,combined with the increasingly mature metabolomics techniques,including non-target metabolomics(i.e.full contour metabolomics)and target metabolomics,we hope to apply metabolomics to study the pathogenesis of these diseases and to find alternatives(Surrogate)indicators to instead of liver biopsy.We applied the methods of non-target metabonomics and bile acid target metabonomics to explore the metabolomic characteristics of these diseases,and then to explore the potential diagnostic markers of drug induced-liver injury,primary biliary cholangitis and autoimmune hepatitis.Methods:1.Serum samples from 38 patients with drug-induced liver injury and 30 healthy controls were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS).The metabolites and metabolic pathways were identified.The metabolic changes,related metabolic pathways and inter-group differences in different degree of drug-induced liver injury were analyzed.Select reaction monitoring(SRM)was used to quantitatively analyze 15 kinds of target bile acid metabolites.ROC curves were used to identify the metabolites related to the diagnosis of drug-induced liver injury and the degree of drug induced liver injury.2.Serum samples from 54 patients with primary biliary cholangitis,26 patients with autoimmune hepatitis and 39 healthy controls were analyzed by Ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)serum metabolomics.The metabolites and metabolic pathways were identified,and the metabolic changes,metabolic pathways and inter-group differences between primary biliary cholangitis and autoimmune hepatitis were analyzed.Fifteen kinds of target metabolites of bile acids were quantitatively analyzed by SRM,and the differential metabolites related to the diagnosis of primary biliary cholangitis were screened by ROC curve analysis.Result:1.By principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA),six metabolites with different characteristics of drug-induced liver injury were screened.After identification of metabolites and enrichment analysis of metabolic pathways,the levels of glycocholic acid,glycodeoxycholic acid,taurochenodeoxycholic acid,taurocholic acid,linoleamide in drug-induced liver injury increased,the level of lysophosphatidylthanolamine(18:0/0:0)in drug-induced liver injury decreased,ROC curve analysis,AUC was more than 0.9,sensitivity and specificity were more than 80% respectively,can be used as potential biomarker of drug induced liver injury.Compared with the healthy control group,the above substances in the severe liver injury,compared with the mild liver injury group was significant differences,and can be used as a potential biomarker to assess the severity of liver injury.The metabolites of drug-induced liver injury mainly involved in primary bile acid synthesis,fatty acid biosynthesis and glycerol phospholipid metabolism pathway.2.The SRM metabolomic analysis of bile acid in patients with druginduced liver injury showed that with the increase of liver injury,the levels of glycocholic acid,glycochenodeoxycholic acid,glycochenodeoxycholic suophate,taurocholic acid,taurodeoxycholic acid and tauroursodeoxycholic acid in serum of patients with severe DILI were significantly higher than those of healthy controls.The increase of these bile acids is closely related to the severity of liver injury.At the same time,it was found that the serum levels ofdeoxycholic acid,chenodeoxycholic acid and lithocholic acid in patients with severe DILI were significantly lower than those in the healthy control group,with significant differences.The decrease of these bile acid levels was closely related to the severity of liver injury.Elevated glycocholic acid,glycochenodeoxycholic acid and taurodeoxycholic acid which found in non-target metabolomics showed the same trend in target bile acid detection.ROC curve analysis showed that the area under the curve were more than 0.9,sensitivity and specificity were more than 80% respectively.3.By principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA),we found that 17 kinds of substances in patients with primary biliary cholangitis were significantly higher than those in healthy control group,p<0.05;9 kinds of primary biliary cholangitis and healthy control group compared to the decline of metabolites,p<0.05.12 kinds of substances in autoimmune hepatitis and healthy control group compared to the increase of substances,p<0.05;12 kinds of autoimmune hepatitis and healthy control group compared to the decline of substances,p<0.05.Glycocholic acid,glycodeoxycholic sulphate,taurocholic acid,taurodeoxycholic acid,tauroursodeoxycholic acid bilirubin,dihydrosphingosine,4-hydroxydihydrosphingosine,lysophosphatidylcholine(18:3(6Z,9Z,12Z))and lysophosphatidylethanolamine(20:3(11Z,14 Z,17Z)/0:0)were all up-regulated found in patients with primary biliary cholangitis and autoimmune hepatitis.12-ketoxycholic acid,pyroglutamic acid,lactic acid,hypoxanthine,lysophosphatidylethanolamine(0:0/20:2(11Z,14Z))and monoglyceride were all down-regulated.Primary biliary cholangitis and autoimmune hepatitis have similar metabolic changes,i.e.metabolic characteristics.The area under the curve of 12 metabolites in primary biliary cholangitis was greater than 0.8.The first three metabolites were taurocholic acid,tauroursodeoxycholic acid and LysoPC(18:3(6Z,9Z,12Z)).Compared with healthy control group,the sensitivity and specificity of these metabolites were more than 80% and 90%respectively.The area under the curve of 11 metabolites in autoimmune hepatitis was greater than 0.8.The first three metabolites were lysophosphatidylcholine(18:3(6Z,9Z,12Z),lysophosphatidylethanolamine(0:0/20:2(11Z,14Z))and pyroglutamic acid.The sensitivity and specificity of these metabolites were more than 80% respectively.Differential substances in primary biliary cholangitis and autoimmune hepatitis are mainly concentrated in phenylalanine,tyrosine and tryptophan biosynthesis,primary bile acid synthesis and phospholipid metabolism.Most of the metabolites of the two groups were the same,affecting the metabolism of amino acids,bile acids,lipids and energy.4.The SRM mtabonomic analysis of bile acid in patients with primary cholangitis and autoimmune hepatitis showed that all bile acids were elevated compared with healthy control group.Compared with bile acids in patients withprimary biliary cholangitis and autoimmune hepatitis,chenodeoxycholic acid,litholic acid,the sum of taurolithocholic acid and litholithic acid were significant difference between the two groups,with statistical significance.The levels of glycochenodeoxycholic acid,glycochenodeoxycholic acid sulfate,taurodeoxycholic acid and tauro conjugated bile acid increased with the degree of Child-puph grading,which were related to the severity of the disease.Glycochenodeoxycholic acid and taurodeoxycholic acid were the same changes to the liver damage degree with drug-induced liver injury.5.Bile acid SRM metabolomics in drug-induced liver injury,primary biliary cholangitis,autoimmune hepatitis and healthy control group analysis showed that the level of chenodeoxycholic acid in drug-induced liver injury was higher than the other three groups,p<0.05;while the level of lithocholic acid in PBC was higher than the other three groups,p<0.05;the levels Cholic acid and taurolithocholic acid in PBC were higher than the other three groups,p<0.05.Conclusion:1.In this study,metabolomics,including non-target and bile acid target metabolomics,was applied to identify differences in serum metabolites in patients with drug-induced liver injury,primary biliary cholangitis and autoimmune hepatitis.These differences not only existed between the disease group and the healthy control group,but also between the three disease groups.2.We found that glycocholic acid,glycodeoxycholic acid,taurochenodeoxycholic acid,taurocholic acid,linoleamide and lysophosphatidylthanolamine(18:0/0:0)could be used as potential diagnostic indicators for drug-induced liver injury and as candidate markers for severity assessment of drug-induced liver injury.Chenodeoxycholic acid is significantly increased in drug-induced liver injury,which is expected to be a potential marker in the differential diagnosis of primary biliary cholangitis and autoimmune hepatitis.3.We found that most metabolites change characteristics between primary biliary cholangitis and autoimmune hepatitis were similar,and the overall metabolic change trend of enrichment pathway analysis was basically consistent.But in bile acid,lipid metabolites of obvious similarities and differences,metabolites such as glycocholic acid,taurocholic acid and so on elevated,hemolysis phosphatidyl ethanolamine(0:0/20:2(11Z,14Z))decreased,Chenodeoxycholic acid and lithocholic acid have differences between PBC and AIH.These findings are of great significance for the analysis of the similarities and differences in the pathogenesis of the two diseases and the differential diagnosis of the two diseases.4.Lithocholic acid,the sum of lithocholic acid and taurolithocholic acid are significantly increased in primary biliary cholangitis,which is expected to be apotential biomarker for the identification of autoimmune hepatitis and drug-induced liver injury,with good clinical application potential. |
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