The Study On Tissue Metabolomites In Hepatocellular Carcinoma Patients

Objectives To discover the potential metabolite biomarker group as an assisting method for the early diagnosis of hepatocellular carcinoma (HCC), metabolite profiles of HCC tissues were analyzed by a high performance liquid chromatography-mass spectrometry platform (HPLC-MS). Free amino acid in plasma and tumor tissue from patients with liver cancer and their correlation with volume of tumor were researched to lay the basis for auxiliary diagnosis, nutritional support of liver cancer and the clinical application of amino acid imbalance solutions.Methods Tumor tissue, transitional tissue and normal liver tissue were sampled from 13 patients with HCC (confirmed by pathology), who were admitted in the liver surgery department of Tianjin Third Central Hospital from Nov,2009 to Jul,2010. The specimens were tested by a HPLS-MS platform after a pretreatment procedure. The raw data were firstly processed by MZmine 2.0 software before being analyzed by SIMCA-P+12.0.1.0 (Sweden Umetrics Company). Orthogonal partial least squares discriminant analysis (OPLS-DA) model were constructed from the tissue metabolite profile data, variable importance in projection (VIP) and VIP confidence of which were then used to select potential biomarkers. Non-parametric test by SPSS 15.0 (SPSS, USA) were used to exclude variables with p>0.05 from these selected potential metabolite biomarkers.13 cases of plasma and tissue specimens were collected from primary liver cancer patients at hepatobiliary surgery department in the third center hospital from November 2009 to July 2010. The amino acids of these specimens were detected respectively using L-8900 Amino Acid Analyzer. Subsequently, combined with pathology and tumor volume of plasma, the correlation between free changes of amino acid with tumor volume were analysed by data analysis software SPSS15.0.Results (1) The OPLS-DA models based on the tissue metabolite profiles is good enough to separate normal tissue, transitional tissue and tumor tissue. (2) 46 metabolite ions were discovered to play important roles in separating different tissues, of which 36 metabolites were identifications. The levels of Lewis X trisaccharide, PC(22:6), Palmitaldehyde and Palmitic acid are lower in both tumor tissue and transitional tissue then in normal liver tissue. Compared with normal liver and transitional tissue, Geranyl-PP, Homoanserine and MG (20:5) levels are decreased in tumor tissue, while 3,4,5-Trimethoxycinnarnic acid, PG levels were increased in tumor tissue. The levels of Aminoacetone,2-Aminomuconic acid, Adenine, NNK, LPA(0:0/20:4),13E-Tetranor-16- carboxy- LTE4,7-Methylguanosine, CPA(16:0/0:0), 7,8-Dihydroneopterin, LPA(16:0/0:0), Taurocholic acid 3-sulfate, Vaccenic acid, TG, Alpha-CEHC, Hemin, PE(14:1/14:0), LPE(22:5), Protoporphyrinogen IX, LPC(17:0), LPC(18:0), L-Aspartic acid,2-Aminobenzoic acid, Oxalosuccinic acid, Palmitic acid, Phenylalanylphenylalanine are increased both in tumor tissue and transitional tissue. (3)Compared with the normal group, plasma free amino acids such as aspartic acid, threonine, isoleucine were significantly reduced(p<0.05) in HCC group; cystine, methionine, tyrosine were significantly increased (p<0.05); Branched-chain amino acids decreased(p<0.05), Aromatic amino acids were increased and BCAA/AAA were reduced(p<0.05).(4)Compared with normal liver tissue? most amino acids levels were significant changed in transitional tissue (P<0.05); compared with transitional tissues, amino acids such as taurine, threonine, glycine, tyrosine, phenylalanine, arginine were elevated in cancer tissues, whereas alanine. valine. leucine. ornithine were reduced. Compared with normal liver tissue, amino acids such as tyrosine, phenylalanine, arginine were progressively rised in transitional tissues and PLC; in contrast, alanine, glycine, valine acid, leucine, ornithine, histidine showed declining trends; others such as taurine, threonine, glutamic acid were decreased in transitional tissue but increased in hepatocellular carcinoma.(5)There was negative correlation between valine (r=-0.812), methionine (r=-0.920), leucine (r=-0.71) tyrosine (r= 0.812), arginine (r=-0.87) in liver tissue and corresponding plasma free amino acids respectively.(6) Free threonine (r=0.90), glutamate (r=0.87), valine (r=0.93), methionine (r=0.86), isoleucine (r=0.95), leucyl Acid (r=0.92), tyrosine (r=0.89), phenylalanine (r=0.84) etc in liver tissue, was positively related to tumor sizeConclusion:This study indicate that 7 metabolites have special change in the development of liver cancer which could be metabolite biomarker including PC/PE, LPA/CPA, Amino acetone, NNK,7-Methylguanosine. The results show that the OPLS-DA model constructed from tissue metabolite profiles can well distinguish the stage of HCC development and successfully discover and identify corresponding metabolite biomarkers of various stages. This study provides new ideas and methods for molecular markers discovery, investigations of liver cancer and liver metabolic pathway and the early diagnosis of HCC.This study shows that due to metabolic disorders, most of amino acids in plasma and tissue are changed; the levels of valine, leucine, methionine, tyrosine in plasma and tissues are on the contrast and were positively correlated with tumor size, and other animo aids such as fine acid, glutamic acid, phenylalanine Dengjun play important roles in the process of HCC development. Liver tissue amino acid analysis in patients could lay a foundation for the diagnosis, nutritional support and clinical therapy.