A Metabonomic Approach For The Early Prognostic Evaluation Of Experimental Sepsis

ObjectiveEarly prognostic evaluation of sepsis is an attractive strategy to decrease the mortality of septic patients,but presently there are no satisfactory approaches.This study is to establish an early,rapid and efficient metabonomic approach for prognostic evaluation of sepsis,and then to explore some potential biomarkers related with the outcome.MethodsSeventy-five rats were randomly allocated to sham-operated group(n=25) and CLP group(n=50).According to the difference in survival duration during the following six days,CLP rats were divided into surviving(n=23,survival duration exceeding 6 d) and nonsurviving group(n=22,survival duration between 24 h and 6 d),excluding five undesirable ones(dead during the first day).At 12 h after surgery, approximately 1.5 ml blood was drawn using tail shearing method in these groups, then left to clot for 2 hours at room tempreture.The serum was separated by centrifugation at 3000 g for 20 min,and the aliquot was stored at -80℃until metabonomic analysis.After special pretreatments,serum samples were analyzed to acquire metabolic profiles using HPLC/MS and NMR spectroscopy respectively. Principle component analysis(PCA) and orthogonal single collection partial least squares discriminant analysis(OPLS-DA) were employed to visualize the changes in the metabolic profiles of sera from surviving,nonsurviving and sham-operated rats, then to discover potential biomarkers related with the otcome of septic rats.Radial basis function neural network(RBFNN) was employed to build predictive model for prognostic evaluation of sepsisResults Based on HPLC/MS data,PCA allows a clear discrimination of the pathologic characteristics among rats from three groups.The results from metabolin identifiction that the levels of palmitoleic acid,palmitic acid,linoleic acid,oleic acid and stearic acid are lower in septic rats,especially in nonsurvivors.It suggests that these important free fatty acids mainly used for energy metabolism are consumed greatly to raise energy supply for organism and their levers may be related with the prognosis of septic rats.Moreover,the levers of another three identified metabolin,including acid and docosapentaenoic acid are higher in septic rats,especially in survivors.It suggests that the levers of these polyunsaturated fatty acids(PUFA) with anti-inflammatory effect increase greatly against the excessive inflammatory reaction and might be related with the prognosis of septic rats.A RBFNN model for outcome predication was built upon the metabolic profile data from rat sera with the sensitivity of(96.1±3.6)%(n=10) and specificity of(91.0±4.3)%(n=10).Based on NMR spectra data,OPLS-DA also allows a clear discrimination of the pathologic characteristics among rats from three groups.Compared with sham-operated group,marked decrease in formate lever and increases in hydroxybutyrate,lactate,acetate,acetoacetate and alanine levers,were observed in septic rats,especially in nonsurvivors,which might due to the enhanced catabolisms of carbohydrate,protein,fatty acid and nucleic acid.Based on the integrity metabolic profile data from ~1H NMR data,another RBFNN model for outcome predication was constructed with the sensitivity of(91.1±5.6)%(n=10) and specificity of(87.9±2.1)%(n=10).ConclusionsAfter sepsis,the metabolism of carbohydrate,protein,fat and nucleinic acid changes markedly.Both HPLC/MS-based and NMR-based metabonomic approachs combined with pattern recognition permit the overall monitoring for the stress metabolism,and allow accurate outcome prediction of septic rats in the early stage. The proposed approach has advantages of rapid,low-cost and efficiency,and is expected to be applied in clinical prognostic evaluation of septic patients. The analytic range of HPLC/MS is different with NMR spectroscopy.The former lays particular emphasis on those metabolins with molecular mass＞100,such as free fatty acid.The latter emphasizes those metabolins with molecular mass＜100, such as carbohydrate and amino acid.Therefore,HPLC/MS coupled with NMR spectroscopy will allow overall metabolin information related with sepsis,resulting in more accurate evaluation on illness severity of sepsis.