Ats4 apparatus

Liver samples were extracted with a mixture of chloroformmethanol (2:1, v/v) according to Folch et al. (15) in the presence of 50 mg/l butylated hydroxytoluene to prevent lipid oxidation. Folch extracts were automatically applied on silica gel 60 highperformance TLC (HPTLC) plates (250 µm, 20 × 10 cm; Merck), pretreated with 1•5 % (w/v) boric acid in ethanol (100 %), on a 4-mm band width using ATS4 apparatus (CAMAG). The development was performed first with methanol-pentanechloroform (3:52:45, by vol.), which allowed the separation of NEFA, CHOL and TAG + cholesteryl ester (TAG + CE) on a 50-mm total migration distance and then with diethyl etherpentane (3:97, v/v), which allowed the separation of TAG and CE on a 57-mm total migration distance. The scanning of the plates was carried out using a TLC scanner 3 (CAMAG), operating in the reflectance mode. The plates were scanned at 550 nm after dipping in a solution of copper sulphate (CuSO 4 -H 3 PO 4 -H 2 O, 40 g:20 ml:230 ml by vol.) and heating for 20 min at 180°C. The different classes of neutral lipids (NEFA, CHOL, TAG and CE) were finally identified by comparing their retention factor (R f ) with authentic standards and quantified using calibration curves of the same standards.

Muscle lipids were extracted using a mixture of chloroformmethanol (2:1, v/v) in the presence of butylated hydroxytoluene (50 mg/l). The application of lipid extracts of muscle homogenate on silica gel 60 HPTLC plates (250 µm, 20 × 10 cm; Merck), pre-treated with 1•5 % p/v boric acid in ethanol (100 %), was automatically performed on a 4-mm band width using a CAMAG ATS4 apparatus. Two developments were performed, first with pentane-chloroform-methanol (52:45:3, by volume) on 60-mm migration distance and second with pentane-diethyl ether (97:3 by volume) on 67-mm migration distance, which allowed the separation of neutral lipids. The scanning of the plates was carried out using a CAMAG TLC scanner 3 operating in the reflectance mode. The plates were scanned at 550 nm after dipping in a solution of copper sulphate 640 mM in H 3 PO 4 1•18 M and heating for 20 min at 180°C. The 1-monoacylglycerol (MAG), 1,2-DAG and 1,3-DAG contents were finally identified by comparing their retention factor (R f ) with authentic standards and were quantified using calibration curves of the same standards.

Muscle lipids were extracted using a mixture of chloroformmethanol (2:1, v/v) in the presence of butylated hydroxytoluene (50 mg/l). P was quantified to determine total muscle phospholipids (37) . The application of muscle chloroformmethanol extracts on silica gel 60 HPTLC plates (250 µm, 20 × 10 cm), pre-treated with 2•3 % p/v boric acid in ethanol (100 %), was automatically performed on a 4-mm band width using a CAMAG ATS4 apparatus. The development was performed with methanol-acetic acid-pentane-chloroform (15:10:30:45, by volume), which allowed the separation of phospholipids and neutral lipids on a 60-mm total migration distance. The scanning of the plates was carried out using a CAMAG TLC scanner 3, operating in the reflectance mode. The plates were scanned at 715 nm after dipping in a solution of Blue Spray (1:2:3, v/v/v, Blue Spray-H 2 SO 4 4•2 M-acetone) and heating for 3 min at 55°C. The different classes of phospholipids (phosphatidylcholine (PC); phosphatidylethanolamine (PE); phosphatidylinositol (PI); phosphatidylserine (PS); sphingomyelin (SM); lysophosphatidylcholine (LPC); phosphatidylglycerol (PG); phosphatidic acid (PA) and cardiolipin (CL)) were finally identified by comparing their R f with authentic standards and were quantified using calibration curves of the same standards (38) .