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Rapid chromatographic method to decipher distinct alterations in lipid classes in NAFLD/NASH.

World journal of hepatology (2013-11-02)
Stephan Laggai, Yvette Simon, Theo Ranssweiler, Alexandra K Kiemer, Sonja M Kessler
ABSTRACT

To establish a simple method to quantify lipid classes in liver diseases and to decipher the lipid profile in p62/IMP2-2/IGF2BP2-2 transgenic mice. Liver-specific overexpression of the insulin-like growth factor 2 mRNA binding protein p62/IMP2-2/IGF2BP2-2 was used as a model for steatosis. Steatohepatitis was induced by feeding a methionine-choline deficient diet. Steatosis was assessed histologically. For thin layer chromatographic analysis, lipids were extracted from freeze-dried tissues by hexane/2-propanol, dried, redissolved, and chromatographically separated by a two-solvent system. Dilution series of lipid standards were chromatographed, detected, and quantified. The detection was performed by either 2',7'-dichlorofluoresceine or a sulfuric acid/ethanol mixture. Histological analyses confirmed steatosis and steatohepatitis development. The extraction, chromatographic, and detection method showed high inter-assay reproducibility and allowed quantification of the different lipid classes. The analyses confirmed an increase of triglycerides and phosphatidylethanolamine and a decrease in phosphatidylcholine in the methionine-choline deficient diet. The method was used for the first time to asses the lipid classes induced in the p62-overexpressing mouse model and showed a significant increase in all detected lipid species with a prominent increase of triglycerides by 2-fold. Interestingly, the ratio of phosphatidylcholine to phosphatidylethanolamine was decreased, as previously suggested as a marker in the progression from steatosis to steatohepatitis. The thin layer chromatography analysis allows a reliable quantification of lipid classes and provides detailed insight into the lipogenic effect of p62.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-α-Lysophosphatidylcholine from egg yolk, ≥99%, Type I, powder
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Non-hydroxy fatty acid ceramide from bovine brain, ≥99%
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3-sn-Phosphatidylethanolamine from bovine brain, Type I, ≥98% (TLC), lyophilized powder
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Stearic acid, analytical standard
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Glyceryl trioleate, ≥99%
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L-α-Phosphatidylcholine, egg yolk, Type XVI-E, ≥99% (TLC), lyophilized powder
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1,2-Diacyl-sn-glycero-3-phospho-L-serine, ≥97% (TLC), from bovine brain, amorphous powder
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Cholesterol, Sigma Grade, ≥99%
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1,3-Diolein, ≥99% (GC)