Cell or tissue lipids were extracted by the procedures similar to the Folch method [4 (link)]. Chloroform/methanol (2:1, v/v) containing 0.005% butylated hydroxytoluene (as antioxidant) was added (usually 5 ml solvent added to 50–100 μl sample) and mixed vigorously for 1 min then left at 4°C overnight. One ml of 0.9% NaCl was added and mixed again. The chloroform phase containing lipids was collected. The remains were extracted with another 2 ml chloroform. The chloroform was pooled and dried under nitrogen and subjected to methylation. To monitor the recovery rate, the fatty acid C23:0 was added to the samples (usually 1 μg added to 2 mg tissue sample) as an internal standard.
Fatty acid methyl esters were prepared by methods similar to those described previously [5 (link),6 (link)] using BF3/methanol reagent (14% Boron Trifluoride). Lipid sample was mixed with 1 ml hexane in 16 ml glass tubes with Teflon-lined caps. BF3/MeOH reagent (1 ml) was added and the mixture was heated at 90–110°C in a metal block or a sand bath for 1 hour, cooled to room temperature and methyl esters extracted in the hexane phase after addition of 1 ml H2O. Samples were allowed to stand for 20–30 min, and then the upper hexane layer was removed and concentrated under nitrogen.
Fatty acid methyl esters were analyzed by gas chromatography using a fully automated HP5890 system equipped with a flame-ionization detector, as described previously [7 (link)] The chromatography utilized an Omegawax 250 capillary column (30 m × 0.25 mm I.D.). Peaks were identified by comparison with fatty acid standards (Nu-chek-Prep, Elysian, MN), and area and its percentage for each resolved peak were analyzed using a Perkin-Elmer M1 integrator.
Fatty acid methyl esters were prepared by methods similar to those described previously [5 (link),6 (link)] using BF3/methanol reagent (14% Boron Trifluoride). Lipid sample was mixed with 1 ml hexane in 16 ml glass tubes with Teflon-lined caps. BF3/MeOH reagent (1 ml) was added and the mixture was heated at 90–110°C in a metal block or a sand bath for 1 hour, cooled to room temperature and methyl esters extracted in the hexane phase after addition of 1 ml H2O. Samples were allowed to stand for 20–30 min, and then the upper hexane layer was removed and concentrated under nitrogen.
Fatty acid methyl esters were analyzed by gas chromatography using a fully automated HP5890 system equipped with a flame-ionization detector, as described previously [7 (link)] The chromatography utilized an Omegawax 250 capillary column (30 m × 0.25 mm I.D.). Peaks were identified by comparison with fatty acid standards (Nu-chek-Prep, Elysian, MN), and area and its percentage for each resolved peak were analyzed using a Perkin-Elmer M1 integrator.
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