Study On Metabolic Changes During Liver Regeneration Based On GC-MS And Discovery Of Potential Biomarkers

AimTo clarify the metabolic changes during liver regeneration after partial hepatectomy(PHx)based on Gas Chromatography-Mass Spectrometer(GC-MS)and search for potential biomarkers.To clarify the potential clinical values of the selected metabolic biomarkers.MethodsIn this study,a total of 65 rats were randomly divided into three groups: pre-PHx(normal control group,n =5),PHx group(model group,n =30),and Sham group(Sham operation group,n =30).Rats in PHx group and Sham group were sacrificed at 30min(minutes),6h(hours),24 h,48h,72 h and 168 h after surgery,and biological samples such as blood and liver were collected at each time point(5 rats/group/time point).Liver injury was evaluated via morphologlical changes,ultrastructural changes and biochemical indicators.Liver regeneration was evaluated via liver weight/body weight,and expression of PCNA and Ki67.Plasma was used for metabolomics detection by using GC-MS(Gas Chromatography-Mass Spectrometer).Principal Component Analysis(PCA),and Partial Least Squares Discriminant Analysis,PLS-DA)and orthogonal partial least squares discriminant analysis(Orthogonal Partial Least Squares Discriminant Analysis,OPLS-DA)and other multivariate statistical analysis were used to screen out the different metabolites between PHx group and Sham group,and then analyzed the related metabolic pathways.Then,we further screened out the different metabolites at the different time points.Finally,the metabolic changes during liver regeneration were clarified.The PHx group was divided into three subgroups(30min-6h group,24h-48 h group and 72h-168 h group).Candidate biomarkers related to liver regeneration were identified via subgroup analysis.And the predictive performance of the candidate biomarkers were evaluated via receiver operating characteristic curve(Receiver Operating Characteristic,ROC).And finally,the clinical value of the candidate biomarkers was evaluated in clinical serum samples.ResutsSix hours after PHx,AST(aspartate aminotransferase),ALT(alanine aminotransferase),TBIL(total bilirubin)and ALB(albumin)showed significant changes.At 24 h after PHx,ALT and AST reached the highest levels(55.11±11.69 IU/L和 79.54±31.65 IU/L),and ALB dropped to the lowest(27.86±1.53 g/L).Until 168 h after PHx,ALT、AST、TBIL and ALB gradually returned to the normal range.The liver weight/body weight ratio gradually increased after PHx,and there was no difference between PHx and Sham group at 168h;PCNA+ cells and hepatic expression of PCNA significantly increased at 24 h after PHx(37.90 ±3.04%),reached the highest at 48h(55.02±9.72%),and then began to decline at 72 h and dropped to normal levels at 168h;moreover,consistent with the results of PCNA staining,Ki67+ cells increased at 24 h after PHx(29.27±2.39%),reached the highest at 48h(41.79±12.06%),and then began to decline at 72 h to dropped to normal levels at 168 h.Then,186 metabolites were identified using GC-MS.These metabolites could be separate well between PHx group and Sham group on PCA,PLS-DA and OPLS-DA models.We screened out 34 differentially metabolites,and these metabolites were enriched in arginine and proline metabolism,aminoacyl-t RNA biosynthesis,cysteine and methionine metabolism and other metabolic related pathways.And we found that the metabolic changes were most obvious at PHx-24 h.Moreover,the metabolic changes at different time points after PHx were various.In addition,differentially metabolites were identifed among three sub-groups of PHx(30min-6h,24h-48 h and 72h-168h).Among these,1,5-anhydroglucitol had a good performance in discriminating the 24h-48 h from30min-6h and 72h-168h(The area under curves,AUCs >0.8).Finally,we evaluated the clinical value of 1,5-anhydroglucitol.We found that 1,5-anhydroglucitol was significantly decreased in patients after hepatectomy(105.10±22.42 μg/m L vs 93.03± 17 μ g/m L,p <0.05).The levels of 1,5-anhydroglucitol(1d post-surgery)and platelet count(1d post-surgery)are risk factors for patients’ hospital stay,which could predict whether the hospital stay is >10 days(AUC = 0.833).ConclusionAccording to the proliferation of hepatocytes,liver regeneration after PHx(30min-168h)can be roughly divided into 3 stages: 30min-6h;24h-48 h and 72h-168 h.A total of 34 differentially expressed metabolites were screened between PHx and Sham,mainly enriched in arginine and proline metabolism,aminoacyl-t RNA biosynthesis,cysteine and methionine metabolism and pyrimidine metabolism.Among these differentially expressed metabolites,1,5-anhydroglucitol was identified as a biomarker related to hepatocyte proliferation during liver regeneration,and had certain clinical application value.After hepatectomy,low 1,5-anhydroglucitol was a risk factor for patient’ hospital stay.