A Metabonomic Approach For The Early Diagnosis And Identification Of Experimental Sepsis Induced By Gram-negative And Gram-positive Bacterium In Rats

ObjectiveEarly diagnosis and identification of sepsis is an attractive strategy to decrease the morbidity and mortality in patients with sepsis. But there are no perfect methods to make a definite diagnosis and distinction early on sepsis presently. This study used cecal ligation and puncture (CLP) and Staphylococcus aureus injection to build Gram-positive and Gram-negative models. Then, metabonomic approach would be used to analyze tendency of metabolin in the models, and establish an early, rapid and efficient strategy for early diagnosis and identification of sepsis, and then to research some potential biomarkers related with the outcome.MethodsSprague-Dawley rats were randomly allocated to normal group (n=30), sham-operated group (n=30), cecal ligation and puncture group (n=30), and Staphylococcus aureus-injected group (n=30). At 12 h after cecal ligated and punctured (18#, puncture twice), approximately 1.5 ml blood was assembled from puncturation of the carotid arteries of Sprague-Dawley rat anesthetized by Chloral Hydrate (10%) in the cecal ligation and puncture group, then left to clot for 2 hours at room temperature. The serum was separated by centrifugation at 3000 g for 20 min, and the aliquot was stored at-80℃until metabonomic analysis. At 4 h after Staphylococcus aureus injected (DNA concentration=1g/kg, volume=3ml),1.5 ml blood was assembled form Staphylococcus aureus-injected group, the samples of collection and pretreatment same as cecal ligation and puncture group. The samples collected time and manner from normal and sham-operated group correspond with cecal ligation and puncture group and Staphylococcus aureus-injected group.After special pretreatments, serum samples were analyzed to acquire metabolic profiles using rapidly-performance liquid chromatography-time of fly-mass spectra (RPLC-Q-TOF-MS) spectroscopy. Multivariate analysis (MVA) and Principle component analysis (PCA) and Orthogonal single collection partial least squares analysis (OPLS) were employed to visualize the changes in the metabolic profiles of sera from normal, sham-operated, CLP, and Staphylococcus aureus-injected rats, then to displayer in score plot and loading plot, and discover potential biomarkers related with the outcome of septic rats.ResultsBased on RPLC-TOF-MS data, PCA (positive and negative model) allows a clear discrimination of the pathologic characteristics among rats. The results from metabolin identification have suggested there are markedly distinctions between normal-control/sham-operated group and cecal ligation and puncture group/ Staphylococcus aureus-injected group. The metabolin from sham-operated group and normal-control group has some distribution in the metabonomic spectrum. The results come from orthogonal single collection partial least squares (OPLS) analysis between normal-control/sham-operated group and cecal ligation and puncture group/ Staphylococcus aureus-injected group have suggest that there are 229 bio-markers in the negative model and 247 bio-markers in the positive model. The 107 bio-markers in the negative model and 110 bio-markers in the positive model would identify to verify with Human Metabolome Database (http://metlin.scripps.edu) and Scripps Center for Mass Spectrometry (http://www.hmdb.ca). ConclusionsBased on RPLC-TOF-MS metabonomic approaches combined with pattern recognition permit the overall monitoring for the stress metabolism, and allow accurate outcome diagnosis and identification of septic rats in the early stage. The proposed approach has advantages of rapid, low-cost and efficiency, and is expected to be applied to septic patients in clinical.