A Serum Metabonomics Study On Alcohol-and HBV-induced Hepatic Cirrhosis

BackgroundChina is a high incidence area of liver disease, according to epidemiological study of hepatitis B virus infection in 2006 in china, more than 90 million people are suffering from hepatitis B virus infection and about 20 million people become chronic hepatitis B infection. About 10%-20% chronic hepatitis B progress to cirrhosis in 5 year. With the social and economic development, alcohol-induced hepatitis or cirrhosis also is increasing. This is a hazard health problem for people, thus, early diagnosis and pathogenesis of alcohol-induced cirrhosis and HBV-induced cirrhosis are very important.The liver is the most important metabolic organ of the body and the hub of metabolism. The liver pathophysiologic changes, will lead to metabolic network changes. Metabonomics is a new platform of system biology study, which is widely and effectively applied in pharmaceutical industry and clinical diagnosis, it is to study the biological system by examining biological system's (cell, tissue or organism) metabolites changes when perturbed by the stimulus (after the disease or environmental change). Clinically, metabonomics is mainly used in diagnosis, pathogenesis and prognosis. The commonly metabonomics study methods are nuclear magnetic resonance (NMR), gas chromatography mass spectrometry (GC/MS), liquid chromatography mass spectrometry (LC/MS), ultra-performance liquid chromatography mass spectrometry (UPLC/MS) is more efficient, rapid and sensitive compared with other methods, which is more and more used in metabonomics. Metabonomics is a robust clinical research tool.In this study, linear gradient ultra-performance liquid chromatography time of flight mass spectrometry (UPLC/TOF/MS) in positive ion mode was used to analysis serum of patients with alcohol-induced liver cirrhosis and HBV-induced cirrhosis. Obtained data analyzed by principal component analysis (PCA), orthogonal partial least squares cluster analysis (OPLS), Exploring early diagnostic serum biomarkers of alcohol-induced liver cirrhosis and HBV-induced liver cirrhosis.Methods:Serums were collected prospectively from normal control subjects (n=22) and patients with alcoholic cirrhosis (n=18) or hepatitis B virus (HBV)-induced cirrhosis (n=19). Acetonitrile (600μL) was added to the serum (200μL) and vortex mixed for 1 min. Then, the mixture was laid at room temperature for 10 min and centrifuged at 10000rpm for 10 min at 4°C. Aliquots (450μL) of the resulting clear supernatants were placed into the glass inserts of the UPLC vials and were prepared for UPLC/TOFMS analysis. The data were exported and analyzed by principal component analysis (PCA) and partial least squares discrimination analysis (PLS-DA) using SIMCA-P 12.0 (Umetrics AB, Umea, Sweden). Assessment the effectiveness of metabonomics used to the early diagnosis of liver cirrhosis.Result:Establishment serum metabonomic model of early diagnosis of liver cirrhosis based on UPLC/MS. The serums were analyzed by UPLC/MS and data were analyzed by principal component analysis (PCA) and partial least squares discrimination analysis (PLS-DA). Clear separation was achieved among the three groups and between alcohol- and HBV-induced cirrhosis. This suggests that, compared with the healthy controls, the metabolism of patients with liver cirrhosis had greatly changed, but metabolism between alcohol- and HBV-induced cirrhosis patients had some degree of similarity. Components that played important roles in the separation were picked according to the variable importance in the projection (VIP) parameter. Significant differences in the metabonome among the three groups were observed. Metabolites that decreased in the serum of patients with hepatic cirrhosis included lysophosphatidyl cholines (LPCs) (LPC C16:0, LPC C18:0, LPC C18:2, LPC C18:3, LPC C20:3, LPC C20:5), while bile acids (glycocholic acid, glycochenodeoxycholic acid), hypoxanthine, and stearamide increased in the serum of patients with liver cirrhosis. These are considered "common" biomarkers for hepatic cirrhosis. Oleamide and myristamide increased in alcoholic cirrhosis but decreased in HBV-induced cirrhosis. These could be "specific" biomarkers for differential diagnosis between alcohol- and HBV-induced hepatic cirrhosis.Conclusions:In this study, we establish serum metabonomic model of diagnosis of alcohol-induced liver cirrhosis, HBV-induced liver cirrhosis and drug-induced liver injury based on UPLC/MS, combined with multivariate statistical analysis. We found that metabonomics is a powerful tool in the area of clinical research.