Proteomic Signature Of Acute Liver Failure:from Discovery And Verification In A Pig Model To Confirmation In Humans

Background&Aims:Acute liver failure(ALF)is a fatal condition hallmarked by rapid development,which makes timely diagnosis crucial to adopting appropriate treatment strategies and improving outcomes.The present study aimed to describe the dynamic alterations of serum protein expression associated with ALF development and progression,and to seek novel biomarkers of early diagnosis and prognosis in ALF.Methods:Miniature pigs(n=38)were employed to establish ALF models by infusing D-galactosamine(GALN,1.3 g/kg).Detection of protein expression in pooled serum samples(n= 10)at baseline(immediately after GALN administration)and at 36 hours after treatment was analysed using label-free quantitation(LFQ)in combination with shotgun proteomics.Subsequently,the temporal evolution of the expression of serum proteins potentially associated with ALF was investigated in individual pigs(n=8)via parallel reaction monitoring(PRM)-based targeted proteomics.Liver tissue samples were used for pathology and transmission electron microscope detection to confirm the expression of each target protein and ALF-associated mitochondrial changes.Two promising biomarkers,the retinol binding protein 4(RBP4)and fructose-1,6-bisphosphatase 1(FBP1),were further validated using enzyme-linked immunosorbent assays in serum samples from ALF patients(n=34)with various etiological backgrounds.Results:A total of 1589 serum proteins were detected via LFQ.Gene ontology analysis suggested a significant enrichment of differentially expressed proteins related to energy metabolism.Targeted proteomics confirmed that GALN infusion triggered time-dependent changes in the expression of 20 proteins related to energy metabolism.In addition,mitochondrial degradation and downregulation of mitochondrial cytochrome c oxidase 1 and 2 were confirmed in GALN-insulted pig liver.We further validated two novel ALF biomarkers.Serum levels of RBP4,which were found to decrease prior to progression of ALT-related changes in the GALN-insulted pig model,were further confirmed to distinguish between healthy individuals and ALF patients(p<0.001).In addition,FBP1 showed a prognostic value for short-term survival(30 days)equal to that of the Model of End-stage Liver Disease score(area under the receiver operating characteristic curve,0.778),as cumulative survival was significantly lower(p=0.002)in ALF patients with higher FBP1 levels(>16.89 ?g/dL)than in those with lower FBP1 levels.Conclusions:Serum levels of proteins related to energy metabolism increased constantly during ALF,accompanied with mitochondrial injury.RBP4 and FBP1 are promising ALF biomarkers,to be used for early diagnosis and prognosis,respectively.