Hepatic lipid profiling of deer mice fed ethanol using 1H and 31P NMR spectroscopy: A dose-dependent subchronic study

Abstract

Chronic alcohol abuse is a 2nd major cause of liver disease resulting in significant morbidity and mortality. Alcoholic liver disease (ALD) is characterized by a wide spectrum of pathologies starting from fat accumulation (steatosis) in early reversible stage to inflammation with or without fibrosis and cirrhosis in later irreversible stages. Previously, we reported significant steatosis in the livers of hepatic alcohol dehydrogenase (ADH)-deficient (ADH−) vs. hepatic ADH-normal (ADH+) deer mice fed 4% ethanol daily for 2 months [Bhopale et al., 2006, Alcohol 39, 179–188]. However, ADH− deer mice fed 4% ethanol also showed a significant mortality. Therefore, a dose-dependent study was conducted to understand the mechanism and identify lipid(s) involved in the development of ethanol-induced fatty liver. ADH− and ADH+ deer mice fed 1, 2 or 3.5% ethanol daily for 2 months and fatty infiltration in the livers were evaluated by histology and by measuring dry weights of extracted lipids. Lipid metabolomic changes in extracted lipids were determined by proton (1H) and 31phosphorus (31P) nuclear magnetic resonance (NMR) spectroscopy. The NMR data was analyzed by hierarchical clustering (HC) and principle component analyses (PCA) for pattern recognition. Extensive vacuolization by histology and significantly increased dry weights of total lipids found only in the livers of ADH− deer mice fed 3.5% ethanol vs. pair-fed controls suggest a dose-dependent formation of fatty liver in ADH− deer mouse model. Analysis of NMR data of ADH− deer mice fed 3.5% ethanol vs. pair-fed controls shows increases for total cholesterol, esterified cholesterol, fatty acid methyl esters (FAMEs), triacylglycerides and unsaturation, and decreases for free cholesterol, phospholipids and allylic and diallylic protons. Certain classes of neutral lipids (cholesterol esters, fatty acyl chain (-COCH2-) and FAMEs) were also mildly increased in ADH− deer mice fed 1 or 2% ethanol. Only small increases were observed for allylic and diallylic protons, FAMEs and unsaturations in ADH+ deer mice fed 3.5% ethanol vs. pair-fed controls. PCA of NMR data showed increased clustering by gradual separation of ethanol-fed ADH− deer mice groups from their respective pair-fed control groups and corresponding ethanol-fed ADH+ deer mice groups. Our data indicate that dose of ethanol and hepatic ADH deficiency are two key factors involved in initiation and progression of alcoholic fatty liver disease. Further studies on characterization of individual lipid entities and associated metabolic pathways altered in our deer mouse model after different durations of ethanol feeding could be important to delineate mechanism(s) and identify potential biomarker candidate(s) of early stage ALD.

The Hepatic Lipidome: A Gateway to Understanding the Pathogenes is of Alcohol-Induced Fatty Liver

“…47,53,54,55,56,57,58,59 As summarized in Table 1, these publications encompass studies in different species, with different alcohol feeding protocols and methods of analysis. A brief summary of each of these reports is provided below, including information on the analytical method, experimental model, main findings and conclusions.…”

“…The authors compiled a metabolomic profile of the liver of alcohol-exposed zebrafish, including lipids, combining data collected using 1 H-NMR spectroscopy and GC-MS. Similar to the limitations experienced by Fernando et al ., 58 the identification of individual lipids by 1 H-NMR spectroscopy was limited and only changes in broad lipid categories were identified. For example, alcohol exposure was associated with increased hepatic phosphatidylcholine levels.…”

“…also used 1 H and 32 P NMR spectroscopy to study the hepatic lipidome of deer mice fed ethanol. 58 These authors used Lieber-DeCarli liquid diets to feed deer mice alcohol for two months, at a final ethanol concentration of 1%, 2% or 3.5%. Moreover, they also compared the effect of alcohol feeding in the liver of wild-type animals, with deer mice carrying a natural genetic mutation in hepatic alcohol dehydrogenase (ADH), as previously described.…”

Proteomic Profiling of Liver and Plasma in Chronic Ethanol Feeding Model of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice

“…An impaired β oxidation of fatty acids can result in accumulation of lipids in the hepatocytes as reported in ethanol-fed ADH − deer mice (Bhopale et al, 2006; Fernando et al, 2012). However, over expression of enoyl CoA hydratase as found in the livers of ethanol group could be a biological response to compensate an impaired β oxidation of fatty acids.…”

“…Chronic alcohol abuse is also known to impair hepatic alcohol dehydrogenase (ADH), a major enzyme involved in the oxidative metabolism of alcohol (Sharakawi, 1984; Panés et al, 1993; Kaphalia et al, 1996). Published work from our laboratory have shown that chronic ethanol feeding via Lieber–DeCarli liquid diet for 60 days increases hepatic lipids and formation of fatty acid ethyl esters (nonoxidative metabolites of ethanol), and causes notable hepatic injury in ADH − deer mice as compared to normal hepatic ADH (ADH + ) deer mice (Bhopale et al, 2006; Fernando et al, 2012). …”