Lipid analysis
Sterol and fatty acids were determined as described by Chalbi et
al. , (2015). A mixture of chloroform-methanol (1:2, 0.75 mL) was added
in an Eppendorf tube to both, microsomal and plasma membrane fractions
(0.5 mL), along with β-cholestanol (20 µL, 0.1 mg
mL-1) used as internal standard for sterol analysis.
Chloroform (CHCl3; 0.25 mL) was added and the mixture
was shaken and centrifuged at 10,000 x g for 6 min. The
CHCl3 layer was retained, evaporated to dryness under
N2 and made up to 100 µL with CHCl3. For
sterol analysis, 20 µL of the CHCl3 extract was placed
in a glass vial (2 mL), evaporated to dryness under N2and acetylated using pyridine (50 µL) and Ac2O (100 µL).
After 2h, the solvents were evaporated under N2, ethyl
acetate was added and the sterols analyzed by GC using an HP5-bonded
capillary column (30 m x 0.25 mm x 0.25 µm) coupled to a flame
ionization detector (FID), with H2 as carrier (1mL
min-1) and a temperature programmed of 120-260°C at
5°C min-1, then 260-280°C at 2°C
min-1, and finally 280-300°C at 6°C
min-1. The injector and detector temperatures were 150
and 320°C, respectively. Bound fatty acids were determined by using
20-II portions of the CHCl3 extract; evaporating them to
dryness under N2, transmethylating with sodium methoxide
(0.5 N) in methanol (0.5 mL) and heating at 30°C for 7 min. The
resultant fatty acids methyl esters were extracted with hexane (1 mL),
evaporated under N2, dissolved in ethyl acetate (20 µL)
and analyzed by GC using an HP5-bonded capillary column (30 m x 0.25 mm
x 0.25 µm), with FID, He as carrier (1 mL min-1) and a temperature
programmed of 150-195°C at 3°C min-1, then 195-220°C
at 2°C min-1, and finally 220-300°C at 6°C
min-1. The injector and detector temperatures were 280
and 300°C, respectively.