GLDH Combined With FECH,HO-1 And GST-α As Biomarkers Of Antituberculosis Drugs Induced Liver Injury

Backgrounds and Objectives:Antituberculosis drugs are easy to cause idiosyncratic drug-induced liver injury(IDILI).The existing biomarkers of liver injury cannot accurately diagnose IDILI in early stage,nor can they judge its prognosis.Finding new biomarkers of liver injury caused by antituberculosis drugs can help to better treat tuberculosis and reduce the occurrence of IDILI and severe adverse prognosis events of IDILI.In order to find potential new biomarkers of liver injury caused by antituberculosis drugs,in this study,an animal experiment and a clinical study were conducted to explore the sensitivity,specificity and prognostic value of glutamate dehydrogenase(GLDH)combined with ferrochelatase(FECH),heme oxygenase-1(HO-1)and glutathione-S-transferase-α(GST-α),as biomarkers of liver injury induced by antituberculosis drugs.Methods:This study is divided into animal experiment part and clinical study part.In the animal experiment,42 SPF male SD rats were reared in a standard barrier environment.After adaptive feeding for one week,they were randomly divided into two groups according to body weight:experimental group(36 rats)and control group(6 rats).Rats in the experimental group were randomly divided into 6 groups as 0,7,14,21,28 and 35 days group,with 6 rats in each group.Experimental group were given isoniazide(INH)100mg/kg/d by intragastric administration.The 35-day group received isoniazid continuously for 28 days,and was fed until the 35 th day.Rats in the control group were given normal saline for 28 days.The body weight,food intake,water intake,sensitivity to external reactions,hair and fecal morphology of the rats were recorded before daily administration.At day 0,7,14,21,28,32 and 35,1m L blood was collected from orbit of all surviving rats to separate serum.24 hours after the last intragastric administration,the rats were weighed,anesthetized,the blood from the heart was collected to separate the serum,the whole liver was dissected to separate,the fresh weight of the liver was weighed,and part of the liver was taken for fixation.The levels of ALT,AST,TBIL,DBIL,TBA,ALP and γ-GT in serum were determined by automatic biochemical analyzer.Isoniazid was added into blank serum in vitro to investigate its interference to the determination of transaminase.HE staining of liver was used to observe and evaluate the histopathological changes of rat liver.Lipid deformation was observed by liver oil red O staining and lipid area was counted.GLDH,FECH,HO-1 and GST-α in serum were determined by enzyme linked immunosorbent assay(ELISA)and ROC curve was drawn to evaluate the potential of each indicator as a biomarker.The clinical study recruited 122 hospitalized tuberculosis patients taking anti-tuberculosis drugs.According to the clinical diagnostic criteria for liver injury caused by anti-tuberculosis drugs,patients meeting the criteria were divided into severe liver injury group(22 cases),patients who did not meet the criteria but had abnormal liver function indicators were divided into mild liver injury group(25 cases),and patients with normal liver function indicators were divided into non-liver injury group(75cases).Collect patient’s medical records and liver function test results.Residual serum was collected after each test of liver function,and the levels of GLDH,FECH,HO-1 and GST-α in serum were determined by ELISA.ROC curve was drawn to evaluate the efficacy of each indicator in the diagnosis of liver injury induced by antituberculosis drugs,and to distinguish the severe liver injury patients from patients with abnormal liver function test.Combined diagnostic test and logistic regression model were used to explore the efficacy of GLDH combined with FECH,HO-1and GST-α in the diagnosis of severe liver injury induced by antituberculosis drugs.Results:1.The rat model of INH-induced liver injury was successfully established.The liver injury of rats showed as follows:(1)decreased weight gain,decreased food intake,decreased hair gloss and dry stool;(2)the liver index was significantly higher than that of the control group;(3)serum ALT level decreased and AST level did not change significantly,but serum ALT level increased significantly on day 28 compared with day 21;(4)serum total bilirubin and total bile acid levels were significantly increased;(5)serum ALP level decreased,γ-GT level did not change significantly;(6)HE staining showed hepatic steatosis,increased inflammatory cell infiltration,hepatocyte enlargement and partial liver cells nuclear pyknosis.(7)Oil red O staining showed that liver steatosis worsened with prolonged administration time.Liver injury in rats was mainly characterized by lipid degeneration and cholestasis,and the severity of injury was mild.Significant recovery of liver injury was observed one week after drug withdrawal.2.In the rat model of INH-induced liver injury,ALT and AST have insufficient diagnostic ability for liver injury.INH can interfere with the determination of serum transaminase and make the determination result of serum transaminase lower than the true value.The degree of interference is related to the concentration of INH,and the normal clinical INH dose has no interference to the determination of transaminase.3.In the rat model of INH-induced liver injury,the areas under ROC curve of serum GLDH and FECH were 0.7692 and 0.7284,respectively,indicating that both of them have potential as biomarkers for the diagnosis of INH-induced liver injury.4.In the INH-induced liver injury rat model,serum levels of FECH,HO-1 and GST-α were significantly increased during liver injury repair.ROC curve analysis showed that FECH,HO-1 and GST-α could effectively identify liver repair,and the combination of the serum levels of FECH,HO-1 and GST-α with injury biomarker GLDH might have prognostic value.5.Clinical studies showed that the area under ROC curve of serum GLDH and FECH for diagnosing liver injury caused by antituberculosis drugs was 0.9124 and 0.6634,respectively,which was statistically significant.The accuracy of GLDH in diagnosing severe liver injury caused by antituberculosis drugs was 82.61%,and the accuracy of GLDH was 82.50% in distinguishing patients with severe liver injury from patients with abnormal liver function test.6.The combination of serum GLDH and FECH significantly improved the diagnostic specificity and accuracy,the diagnostic accuracy was improved to 90.43%,and could compensate for the low positive predictive value(PPV)of GLDH alone.Conclusions:GLDH and FECH have potential as biomarkers for the diagnosis of liver injury induced by anti-tuberculosis drugs.GLDH alone in the diagnosis of liver injury induced by antituberculosis drugs has high sensitivity,but low specificity and low PPV.The combination of GLDH and FECH could significantly improve the specificity,PPV and diagnostic accuracy,and reduce the false positive rate.The combination of GLDH with FECH,HO-1 and GST-α as biomarkers of liver injury induced by antituberculosis drugs may have prognostic value.