Investigation of chronic alcohol consumption in rodents via ultra-high-performance liquid chromatography–mass spectrometry based metabolite profiling

doi:10.1016/j.chroma.2012.02.053

Journal of Chromatography A

Volume 1259, 12 October 2012, Pages 128-137

https://doi.org/10.1016/j.chroma.2012.02.053 Get rights and content

Abstract

Alcohol consumption in man, when seen in its extreme form of alcoholism, is a complex and socially disruptive disorder that can result in significant levels of liver injury. Here the rodent “intragastric feeding model” was used together with UHPLC–TOFMS analysis to determine changes in global metabolite profiles for plasma and urine from alcohol treated rats and mice compared to control animals. Multivariate statistical analysis (using principal components analysis, PCA) revealed robust differences between profiles from control and alcohol-treated animals from both species. A large number of metabolites were seen to differ between control and alcohol-treated animals, for both biofluids.

Highlights

► Urine and Plasma from Rodent “intragastric alcohol feeding model” metabolically profiled with UPLC-TOF–MS. ► Scope was to determine changes in the metabolite profiles due to alcohol treatment. ► PCA revealed robust differences between profiles from control and alcohol-treated animals from both species. ► A large number of metabolites were seen to differ between control and alcohol-treated animals, for both biofluids.

Introduction

Alcohol consumption in humans, especially the chronic exposure seen with alcoholism, can result in significant levels of liver injury. In order to gain insights into the biological effects of alcohol disruption much research has been undertaken using animal models, including e.g., the intragastric infusion model [1], [2], [3], [4]. Recently the effects of ethanol exposure to rodents on global endogenous metabolite profiles has become an area of increasing interest with the application of metabonomics/metabolomics approaches based on FT-MS [5], LC–MS [6], [7] and NMR [8], [9] spectroscopy. These studies have highlighted the plethora of changes occurring to the metabolic phenotypes (metabotypes [10]) of organs and biofluids of rats and mice exposed to ethanol. Recently we described the effects of ethanol exposure on the metabolic profiles of liver in rat and mouse following the intragastric feeding of ethanol [7] where profound changes were observed in metabolic profiles, particularly fatty acids, fatty acid ethyl esters, glycerolipids, and phosphatidyl-ethanol homologues. Thus metabolites such as octadecatrienoic acid, eicosapentaenoic acid, ethyl arachidonate, ethyl docosahexaenoic acid, ethyl linoleate, and ethyl oleate as well as phosphatidylethanol (PEth) homologues (including PEth 18:0/18:2 and PEth 16:0/18:2) were up-regulated [7]. Such findings are of considerable interest in developing appropriate animal models for man as e.g., PEth homologues are currently considered as potential biomarkers for harmful, and prolonged, alcohol consumption in humans [11], [12], [13]. Whilst the metabolite profiles observed were broadly similar in both rodent species a number of differences were detected including a marked down-regulation of retinol and free cholesterol in the mouse compared to the rat following alcohol administration. Further to our investigation of the effects of intragastric ethanol administration to rodents, we now present in the current manuscript an examination of the metabolic profiles of blood plasma and urine from both rats and mice (the study on mice included animals treated for two different periods). The final goal of the research was to characterise metabolome alterations following alcohol exposure and to gain new insight in the biochemical mechanism of alcohol toxicity in the animal models.

Section snippets

Chemicals

All solvents used were of HPLC grade and obtained from Fisher Scientific (Loughborough, Leicestershire, UK). Formic acid of analytical grade was also purchased from Fisher Scientific. Ultra Pure Water (18.2 MΩ) was obtained from a Purelab Ultra system from Elga (Marlow, Bucks, UK). All other chemicals, and metabolites used as standards (including taurine, lysine, arginine, glutamine, 4-hydroxy-l-proline, 2-methylglutamic acid, urinc acid, N-acetylglutamine, N-acetylglycine, nicotinic acid,

Data quality

As a first step the validity of the data were investigated by examination of the replicate QC profiles in the data sets as a means of evaluating both retention time and signal intensity repeatability during the analysis (by monitoring a number of peaks along the chromatogram) [14]. Further, the data for the two technical replicates of each test sample were compared in a similar way. Results obtained from both QC and test sample replicates showed high consistency of the profiles. In addition

Discussion

Not unexpectedly, especially when our previous results on the metabolite composition of liver extracts are considered [7], there were large changes in the plasma and urinary metabolomes of mice fed intragastrically with ethanol for periods of 2 and 4 weeks. In addition the alcohol fed mice showed progression between the two dosing periods. Such progressive changes have also been noted in the urinary metabolomes in studies on the Ppara-null mouse [6]. Similarly there were detectable changes in

Conclusions

The results from this preliminary examination of blood plasma and urine from mice treated with alcohol provided LC-TOF–MS profiles that differed greatly from those obtained from control animals, with urine enabling the best differentiation between the groups. Temporal effects were also noted in mice, with ethanol administration for 4 weeks providing stronger effects on the metabolome compared to 2 weeks treatment. For rats plasma was less useful for studying the effects of exposure to ethanol but

Acknowledgements

The authors wish to thank the EU committee for funding through a Transfer of Knowledge Industry-Academia partnership grant (TOK-IAP 29640). H.G. Gika is grateful to the EU Committee for a re-integration grant (ERG 202132) funding her work at the Aristotle University Thessaloniki. Cheng Ji is supported by the US NIH/NIAAA grants R01AA018846-3 and R01AA018612-2 This research has been co-financed by the European Union (European Social Fund–ESF) and Greek national funds through the Operational

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Investigation of chronic alcohol consumption in rodents via ultra-high-performance liquid chromatography–mass spectrometry based metabolite profiling

Abstract

Highlights

Introduction

Section snippets

Chemicals

Data quality

Discussion

Conclusions

Acknowledgements

Toxicol. Appl. Pharmacol.

FEBS Lett.

J. Chromatogr. B

Anal. Biochem.

Am. J. Physiol.

Hepatology

Methods Mol. Biol.

Hepatology

J. Proteome Res.

J. Proteome Res.

Chem. Res. Toxicol.