Targeted and Global Metabolomics of Hepatocellular Carcinoma and Hepatitis C Using LCMS/MS and GC/MS

Metabolomics focuses on the quantitative and qualitative analysis of small molecule and represents a promising approach for biomarker discovery, monitoring of therapies and diseases pathogeneses. In this thesis, targeted and global mass spectrometry (MS)-based metabolomics is used for discovery of disease biomarkers. MS is a powerful approach for detecting metabolite or small molecules and their altered levels in a variety of biological systems and states. Often, one of a variety of MS methods is used for metabolite profiling. However, each method is sensitive or selective to a specific set of metabolites and typically provides complimentary but often non-overlapping information on the metabolome. The use of multiple MS platforms offers more options for biomarker discovery and avenues for better understanding the pathogenesis by enhancing the pool of altered metabolites. In this work, we report on the identification of serum metabolite markers and development of classification models for distinguishing hepatocellular carcinoma (HCC) and hepatitis C virus (HCV) patients using two MS platforms: GC-MS (gas chromatography-mass spectrometry), and LC-MS/MS (liquid chromatography-triple quadrupole mass spectrometry), individually. Global (GC-MS) and targeted (LC-MS/MS) metabolite profiling were performed on the same set of patient samples. The results provide a promising methodology to distinguish cirrhotic HCV patients, who are at high risk to develop HCC, from those who have already progressed to HCC. The results also provide insights into the altered metabolism between HCC and HCV. The last part of the thesis focuses on the absolute quantitation of metabolites using MS to overcome the technical challenge of accurately measuring metabolite concentrations in blood. It is impractical to use isotope labeled standards for all metabolites as such standards are either not available or are very expensive. To overcome this problem, we investigated the applicability of utilizing heavy 13C-labeled metabolites extracted from commercially available uniformly 13C-labled lyophilized algal cells as internal standards for absolute quantification of plasma metabolites using LC-ESI-MS/MS. This strategy could significantly decrease costs and labor compared to using a suite of individually obtained labeled standards