Untargeted metabolomic analysis using LC-TOF/MS and LC-MS/MS for revealing metabolic alterations linked to alcohol-induced hepatic steatosis in rat serum and plasma

Abstract

Alcohol-induced hepatic steatosis (AHS), an early stage of alcoholic liver disease, is characterized by large amounts of fat deposited in hepatocytes. Although some important biomarkers have been identified, there still is no systematic view of AHS-associated biomarker alteration in vivo. Blood matrix choice is a fundamental consideration for biomarker exploration. In the present study, a reliable untargeted metabolomic method based LC-TOF/MS and LC-MS/MS was developed to (i) interrogate overall differences of endogenous metabolites in rat serum and plasma, and investigate which of the two matrices is more suitable for the discovery of endogenous biomarkers, (ii) analyze metabolic alterations linked to incident AHS. As a result, metabolite level differences between serum and plasma in normal rats were mainly related to 7 peptides, 8 lysophosphatidylcholines, 3 phosphatidylcholines, 2 heterocyclic compounds, 7 polyunsaturated fatty acids and their oxidative fatty acids. Notably, 7 characteristic peptides were observed only in serum. All the altered metabolites were associated with clotting cascade reaction, indicating that serum conceals the changes in metabolome that have occurred in vitro. The results demonstrated that plasma which represented the original properties of the blood sample was a more suitable matrix to explore potential biomarkers. In the plasma untargeted metabolomic study, the metabolic alterations linked to AHS were mainly involved in phospholipid metabolism, amino acids metabolism, fatty acid metabolism, cholesterol metabolism and sphingolipid metabolism. These findings provided a systematic view of metabolic alterations linked to AHS, demonstrating the untargeted metabonomic method was a robust method for examining the molecular mechanisms of disease.