Discovery Of Potential Metabolite Biomarkers In Ascites Of HCC

Objectives New and effective diagnostic methods have been in pressing need as for the diagnosis and therapy of hepatocellular carcinoma. This study, by analyzing the cirrhosis and cirrhosis with Hepatocellular carcinoma (HCC) patients sera metabolite profile, is to discover differential metabolites and explore the molecular mechanism of HCC with cirrhosis representing by these differential metabolites.Methods A HPLC-LTQ Orbitrap XL MS platform was used to analyze the ascites specimens from 30 cirrhosis patients and 26 cirrhosis with HCC patients.20% samples were selected randomly form each group as a testing group not involved in the data analyzing procedures to test the models constructed in the study. Based on the left 80% samples called training group samples, principle component analysis (PCA) and orthogonal partial least square (OPLS) methods were first used to analyze the variation of the metabolite profiles data, then a two-class orthogonal partial least square discriminate analysis (OPLS-DA) models was constructed to discriminate cirrhosis group and cirrhosis with HCC group. After that, the ion variables whose VIP (variable importance in projection) values>1 were selected, in which the variables whose confidence intervals in the VIP plot and coefficient plot covering 0 were excluded. S plot was used to confirmed these selected variables. The models constructed in this study were considered as valuable when both Q2 and R2Y of the model were more than 50%. Metabolites were identified by searching Mass Frontier 6.0, Medlin (www.medlin.scripps.edu), KEGG (http://www.genome.jp/kegg/ligand. html) and HMDB (http://hmdb.ca/) databases by the corresponding exact mass, MS/MS spectrum analyzing and standards validation. The models were validated by testing group and cross-validation.Results The performance of the HPLC-LTQ Orbitrap XL MS platform is drifted with time, which has no effects on this study. The OPLS-DA model based on ascites metabolite profile has good prediction abilities (Q2=79.5%), which was validated by 7 fold cross validation and the correct classification rates on testing group samples are all 100%. In total,37 feature ions were selected, of which 24 metabolites were identified.21 metabolite levels were up-regulated in ascites of cirrhosis with HCC patients, while 3 metabolites levels were down-regulated. Metabolites promoting apoptosis including Phytosphingosine and 13-HETE were up-regulated; 5-Methylthioribose 1-phosphate (MTA) which promotes tumor cell growth and angiogenesis has higher levels in ascites of cirrhosis with HCC patients levels, while 12-oxo-20-dihydroxy-leukotriene level was down-regulated, which has the ability of anti apoptosis.Conclusions (1) The study reports a metabolomic methods in the study of ascites specimen. (2) four potential metabolite biomarkers were discovered, including Phytosphingosine, MTA,13-HETE and Allose. (3) This study indicates that the ascites metabolite changes could reflect apoptosis- and tumor growth-related metabolism.