Metabonomic Investigation of Liver Profiles of Nonpolar Metabolites Obtained from Alcohol-Dosed Rats and Mice Using High Mass Accuracy MSⁿ Analysis

Abstract

Alcoholism is a complex disorder that, in man, appears to be genetically influenced, although the underlying genes and molecular pathways are not completely known. Here the intragastric alcohol feeding model in rodents was used together with high mass accuracy LC/MSn analysis to assess the metabonomic changes in nonpolar metabolite profiles for livers from control and alcohol treated rats and mice. Ion signals with a peak area variance of less than 30% (based on repeat analysis of a pooled quality control sample analysed throughout the batch) were submitted to multivariate statistical analysis (using principal components analysis, PCA). PCA revealed robust differences between profiles from control and alcohol-treated animals from both species. The major metabolites seen to differ between control and alcohol-treated animals were identified using high accuracy MSn data and verified using external search engines (http://www.lipidmaps.org; http://www.hmdb.ca; http://www.genome.jp/kegg/) and authentic standards. The main metabolite classes to show major changes in the alcoholic liver-derived samples were fatty acyls, fatty acid ethyl esters, glycerolipids and phosphatidylethanol homologues. Significant metabolites that were up-regulated by alcohol treatment in both rat and mouse livers included fatty acyls, metabolites such as octadecatrienoic acid and eicosapentaenoic acid, a number of fatty acid ethyl esters such as ethyl arachidonate, ethyl docosahexaenoic acid, ethyl linoleate and ethyl oleate and phosphatidylethanol (PEth) homologues (predominantly PEth 18:0/18:2 and PEth 16:0/18:2; PEth homologues are currently considered as potential biomarkers for harmful and prolonged alcohol consumption in man). A number of glycerophospholipids resulted in both up-regulation (m/z 903.7436 [M+H]+ corresponding to a triglyceride) and down-regulation (m/z 667.5296 [M+H]+ corresponding to a diglyceride). Metabolite profiles were broadly similar in both mouse and rat models. However, there were a number of significant differences in the alcohol-treated group particularly in the marked down-regulation of retinol and free cholesterol in the mouse compared to the rat. Unique markers for alcohol treatment included ethyl docosahexaenoic acid. Metabolites were identified with high confidence using predominantly negative ion MSn data for the fatty acyl components to match to www.lipidmaps.org MS and MS/MS databases; interpreting positive ion data needed to take into account possible adduct ions which may confound the identification of other lipid classes. The observed changes in lipid profiles were consistent with alcohol induced liver injury in humans.

Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study

“…Our results, however, identify ethyl oleate (C18:1) as the most abundant FAEE in the plasma and liver of alcohol-fed mice. This fi nding is consistent with work done in other animal models and humans ( 7,42,43 ). Accumulation of FAEE in the liver of alcohol-fed mice may contribute to the development of ALD, and although methods have been developed to detect these toxic lipids, their metabolism and the basis for their cytotoxic effects remain to be elucidated.…”

Global Metabolic Profiling for The Study of Alcohol-Related Disorders

“…The profiling technique that has been most applied for global metabolite profiling in the case of ethanol is LC-MS, particularly the combination of UPLC with TOF-MS [25][26][27][28][29][30][31][32][33], followed by 1 H NMR spectroscopy [34][35][36][37][38][39][40], with a single study that employed GC-MS [41]. In Tables 1 & 2 the metabolite profiling methods by NMR or LC-MS, respectively, are summarized.…”

“…Most of the published work on metabolomics for the study of alcohol-related metabolic dysfunctions relate to the ana lysis of urine or liver samples of rodents (rats or mice of different strains, wild type or transgenic) [25][26][27][28]30,31,[34][35][36][37][38][39]41] with limited reports describing metabolic profiling of brain, serum or plasma [29,30,33,34,[36][37][38]; there are also two recent studies on pancreatic tissue and the GI tract [32,40]. In the majority of studies in rodents, the alcohol was administered via oral gavage or in the animals drinking water [25,29,31,32,34,36,40,41] [26,27,35,37,38].…”

“…In the majority of studies in rodents, the alcohol was administered via oral gavage or in the animals drinking water [25,29,31,32,34,36,40,41] [26,27,35,37,38]. In some cases, the intragastric feeding model was also adopted [28,30], allowing a controlled 24 h a day delivery of ethanol that provides a robust and repeatable model of ethanol exposure.…”

Biomarker discovery of liver diseases using mass spectrometry-based metabolomics.

“…Even though FLD is reversible, it may lead to inflammatory disease such as steatohepatitis and eventually cirrhosis [57]. FLD can occur after drinking large amount of alcohol(acute), or after a period of heavy drinking(chronic Several efforts have been devoted to liver disease to discover metabolite biomarkers using different metabolomics approaches such as GC-MS [61][62][63] and HPLC-MS [64][65][66][67].…”