Original Article
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World J Hepatol. Oct 27, 2013; 5(10): 558-567
Published online Oct 27, 2013. doi: 10.4254/wjh.v5.i10.558
Rapid chromatographic method to decipher distinct alterations in lipid classes in NAFLD/NASH
Stephan Laggai, Yvette Simon, Theo Ranssweiler, Alexandra K Kiemer, Sonja M Kessler
Stephan Laggai, Yvette Simon, Theo Ranssweiler, Alexandra K Kiemer, Sonja M Kessler, Department of Pharmacy, Pharmaceutical Biology, Saarland University, Saarbruecken 66123, Germany
Author contributions: Laggai S, Kiemer AK and Kessler SM designed experiments, analysed data and wrote the manuscript; Kessler SM and Kiemer AK initiated and directed the study; Simon Y and Ranssweiler T designed experiments and participated in data acquisition; Laggai S, Kiemer AK and Kessler SM contributed to revising it critically for important intellectual content; Laggai S, Simon Y, Ranssweiler T, Kiemer AK and Kessler SM contributed to final approval of the version to be published.
Supported by The Graduiertenförderung of Saarland University (Laggai S), an EASL Dame Sheila Sherlock Fellowship (Kessler SM); and by the research committee of Saarland University (61-cl/Anschub2012)
Correspondence to: Alexandra K Kiemer, PhD, Professor of Pharmaceutical Biology, Department of Pharmacy, Pharmaceutical Biology, Saarland University, Campus C2.2, Saarbruecken, 66123, Germany. pharm.bio.kiemer@mx.uni-saarland.de
Telephone: +49-681-30257301 Fax: +49-681-30257302
Received: July 17, 2013
Revised: September 23, 2013
Accepted: October 11, 2013
Published online: October 27, 2013

AIM: 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.

METHODS: 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.

RESULTS: 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.

CONCLUSION: The thin layer chromatography analysis allows a reliable quantification of lipid classes and provides detailed insight into the lipogenic effect of p62.

Keywords: Non alcoholic steatohepatitis, Non alcoholic fatty liver disease, Thin layer chromatography, IMP2/IGF2BP2, p62, Methionine choline deficient diet, Polar lipids, neutral lipids, Phosphatidylcholine/phosphatidylethanolamine ratio, Triglycerides

Core tip: We describe a new method to quantify lipid classes in steatosis/steatohepatitis having advantages over both histology and classical analytical methods. Since lipid classes exert differential pathophysiological actions our method should be of interest for all researchers dealing with mechanisms of steatosis and steatohepatitis. We employ our method to investigate the lipid profile in the steatotic p62 transgenic mouse model. p62 was originally identified as an autoantigen overexpressed in hepatocellular carcinoma patients, its expression correlates with poor prognosis, and it induces steatosis. The interesting lipid profile in p62 transgenic animals suggests that it might advance the step from steatosis towards steatohepatitis.