Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPAR? and Fatty Acid Uptake

Labrie, Marilyne; Lalonde, Simon; Najyb, Ouafa; Thiery, Maxime; Daneault, Caroline; Des Rosiers, Chrisitne; Rassart, Eric; Mounier, Catherine

Fatty acid (FA) profiling FA composition was measured by a modified gas chromatography-mass spectrometry (GC-MS) method, as previously described [48] . Briefly, total lipids were extracted from plasma with a mixture of methyl-tert-butyl ether (MTBE), methanol and water [49] . For liver, pulverized tissues (25mg) were incubated overnight at 4°C in a solution of chloroform/methanol (2:1) containing 0.004% butylated hydroxytoluene (BHT), filtered through gauze and dried under nitrogen gas. Plasma and liver FA were analyzed as their fatty acid methyl derivatives (FAME) after direct transesterification with acetyl chloride/methanol [50] . Injections (2 μL for plasma and 1 μL for liver samples) were performed onto an Agilent 7890B gas chromatograph equipped with a Select FAME CP7420 capillary column (100 m; 250 μm inner diameter; 230 μm thickness) coupled with a 5977A Mass Selective Detector operated in positive chemical ionisation mode using ammonia as reagent gas. FA were identified according to their retention time and m/z, and their concentration was calculated using a mix of internal and external labelled standards added to liver and plasma samples at known concentrations. The concentration of arachidonic acid, calculated using its [ 2 H 8 ]-labeled counterpart as internal standard, is reported as absolute concentration (μM or nmol/mg tissue) and relative to total fatty acid content (%).