Ceramide and lipid analysis

Tissue lipids were extracted using the Folch method described by Löfgren et al.⁵³ with slight modifications. Briefly, tissues samples were dissolved in methanol in 50 mL glass tubes. The mix was sonicated in an ultrasonic processor (Dr. Hielscher, Teltow, Germany) during 15 second, two cycles. Then, dichloromethane (1:2 methanol/dichloromethane) was added and mixed during 20 min. Acetic acid 20 mM (1:3 acetic acid/dichloromethane) was added and samples were again mixed for 20 min. Samples were centrifuged at 2100 rpm, 5 min, 4 °C. Bottom organic phases were transferred to a new glass tube and the methanolic phase was washed with dichloromethane (1:1 dichloromethane/methanol) and mixed for 10 min before centrifugation with the same conditions as described above. The organic phases were collected and mixed and filtered through 0.45 µm, evaporated with nitrogen and weighted. Lipids extracts were maintained at −35 °C. The separation of ceramides of lipid extracts from tissues samples were performed with a HPLC Agilent Technologies, model 1200 (Agilent Technologies, Palo Alto, CA, USA) coupled to an evaporative light scattering detector (ELSD) (SEDERE. SEDEX 85 model, Alfortville Cedex, France) using pre-filtered compressed air as the nebulizing gas at pressure of 350 KPa, temperature of 90 °C and the gain was set at 6. Two columns Zorvax Rx-SIL column (Agilent Technologies, Palo Alto, CA, USA) of 250 mm × 4.5 mm and 5 μm particle size, were coupled in series with a precolumn with the same refill. They were equilibrated at 40 °C. The injection volume was 50 μL at concentration of 5 mg/mL in CH₂Cl₂ using chromatographic solvent gradient as we have previously described⁵⁴.