SUPPLEMENTARY MATERIALS AND METHODS
Lipid assays
Phosphatidylcholine (PC) and sphingomyelin (SM) contents. Lipids were extracted according
to the method of Folch et al. (20). Dimyristoylphosphatidylcholine (DMPC, Avanti Polar
Lipids) and lauroylsphingomyelin (LSM, Avanti Polar Lipids, Alabaster, AL, USA) were used
as internal standards, and 50 mg/l of butylated hydroxytoluene was added to the solvent. An
aliquot of the chloroform phase was evaporated, and 100 µl of chloroform/methanol (4/1) was
added for liquid chromatography-mass spectrometry (LC-MS). Phospholipid analysis was
performed on a Hypersil Si 2 x 200 mm column (Agilent Technologies, Santa Clara, CA,
USA) with a binary gradient of solvent A (5 mM ammonium acetate in chloroform/methanol,
4/1, v/v), and solvent B (5 mM ammonium acetate in chloroform/methanol/water, 6/3.4/0.6,
v/v/v). Elution was performed at a flow-rate of 0.3 ml/min. Positive electrospray ionization
mass spectrometry was performed on a single quadrupole mass spectrometer (MSD 1100,
Agilent Technologies). The orifice voltage was set at 120 V, the capillary voltage at 3.5 kV,
the drying gas (Nitrogen) flow at 8 l/min and scan range from m/z 400 to 950. Integrated peak
areas from extracted ion chromatograms (EIC) for m/z = 700 to 950 at the retention time (Rt)
of PC, and SM were divided by the EIC for m/z = 679 at the Rt of DMPC, or m/z = 647 at the
Rt of LSM. Levels were determined by comparison of this ratio with a standard curve of
known amounts of phosphatidylcholine and sphingomyelin.
α-tocopherol contents. α-tocopherol was assayed in brain homogenates by GC-MS. The
extraction procedure was conducted as previously described (21). The extract was dissolved in
100 µl N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane (4/1, v/v) (Pierce,
Rockford, IL, USA) and samples were incubated at 60°C for 30 min to form trimethylsilyl
ethers. After evaporation, the residue was dissolved in 100 µl hexane for GC-MS analysis.
GC-MS was performed with the same method and the same instrument as for cholesterol
analysis. Ions at m/z 502 and 460 were used to measure α-tocopherol and tocol, respectively.
α-Tocopherol level was determined by comparison with a standard curve that was obtained
with known amounts of α-tocopherol (Fluka 89550).
Fatty acids contents. Fatty acids were extracted from saponified brain homogenates,
derivatized to pentafluorobenzyl esters, and quantified by GC-MS following the method
described by Arnauld et al. (22). Heptadecanoic acid and tricosanoic acid were used as
internal standards. Chromatography was performed on an HP-5MS fused silica capillary
column (Agilent Technologies) using helium as the carrier gas. The mass spectrometer was
operated under negative chemical ionization mode with methane as reactant gas. A SIM
program was used for mass spectrometry with [M-181]- ions as quantification.
Protein extraction and western blotting
After adjustment to the same protein concentration (5 µg/ml), samples were diluted (1:1) in
Laemmli buffer, boiled for 5 min and loaded on a 10% polyacrylamid gel. After protein
transfer, the membrane was blocked in 5% non-fat dry milk and was incubated in primary
antibody overnight at +4°C, then incubated for 2 h with a horseradish peroxidase-conjugated
secondary antibody. Peroxidase activity was revealed by using enhanced chemiluminescence
reagents (Pierce ECL, Thermo Scientific). Intensity of peroxidase activity was quantified
using an Odyssey Fc quantitative fluorescence imaging system (LI-COR ScienceTec,
Courtaboeuf, France).