Boron trifluoride

Subjects were required to fast overnight for a minimum of 12 h prior to blood collection, separation of plasma and subsequent freezing of samples at -80°C. Frozen plasma samples were thawed on ice for 30 min and a mixture of chloroform: methanol (2:1 v/v) was added to a 50 μl aliquot and analyzed as described previously [13 (link)]. In brief, free fatty acid C17:0 was used as an internal standard (5 μg of 1 mg/ml stock). Samples were flushed with nitrogen gas prior to storage over night at 4°C. The next day, samples were subjected to a double extraction. The lower organic phase containing lipids were dried down under a gentle stream nitrogen then saponified by KOH in methanol for 1 hour and subsequently methylated by boron trifluoride (14%) for 1 hour. Fatty acid methyl esters (FAME) were quantified as previously described by gas chromatography [14 (link)]. FA peak areas were determined using EZChrom Elite software (Version 3.3.2) [15 (link)]. The internal standard was used to calculate FA concentrations (μmol/L) (S1 Table). The responsiveness of the detector was routinely checked against the composition of a commercial mixture of FAME standards.

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.

Approximately 20 mg of mycelia were used for each lipid extraction. Accurately weighed portions of pulverized mycelium were extracted using the method of Bligh and Dyer [87] (link) under acidified conditions with pentadecanoic acid and heneicosanoic acid added as internal standards. The solvent from the fungal extract was removed under a stream of nitrogen. Lipids were saponified in 1 ml of freshly prepared 5% ethanolic potassium hydroxide at 60°C for 1 h under an argon atmosphere. After cooling, 1 ml of water was added to the samples and non-saponifiable lipids were extracted into 3 ml of hexane. The aqueous layer was acidified with 220 µl of 6 M hydrochloric acid and the fatty acids extracted into 3 ml of hexane. After removing the hexane in a stream of nitrogen, fatty acids were converted to methyl esters by first treating with 1 ml of 0.5 M methanolic sodium hydroxide at 100°C for 5 min under argon followed by 1 ml of 14% methanolic boron trifluoride at 100°C for 5 min under argon [88] . After cooling the sample was mixed with 2 ml of hexane followed by 4 ml of saturated aqueous sodium chloride. After separating the phases, aliquots of the hexane layers were diluted 24-fold with hexane and then analyzed by GC/MS. One µl was injected in the splitless mode onto a 30 m×250 µm DB-WAXETR column (Agilent Technologies, Santa Clara, California) with 0.25 µm film thickness. The temperature program was as follows: 100°C for 2 min, ramp to 200°C at 16°C per min, hold for one min, ramp to 220°C at 4°C per min, hold one min, ramp to 260°C at 10°C per min, and hold for 11 min. Helium was the carrier gas at a constant flow of 1.5 ml/min. The mass spectrometer was operated in positive-ion electron impact mode with interface temperature 260°C, source temperature 200°C, and filament emission 250 µA. Spectra were acquired from m/z 50 to 450 with a scan time of 0.433 s. Lower-boiling fatty acid methyl esters were quantified using the pentadecanoic acid internal standard, whereas higher-boiling methyl esters were quantified using the heneicosanoic acid internal standard.

The sample prepared for methylation was mixed with 5 mL of 0.5 N sodium hydroxide methanol solution, and the sample was saponified for about 10 min under reflux in a water-cooled condenser. When the sample was completely saponified, the solution remained clear. After saponification, the sample was cooled to 75 °C, and 6 mL of a 20% boron trifluoride solution in methanol was added through a condenser. The methylation process was carried out for 5 to 10 min at a temperature of 70–75 °C. Upon completion of methylation, the flask was cooled, and 4–5 mL of n-hexane and 20 mL of distilled water were added. The methyl esters of the fatty acids were extracted twice with n-hexane, and the aqueous methanol layer was separated from the n-hexane layer. The n-hexane layers were combined, dried, and neutralized by adding anhydrous sodium sulfate and sodium carbonate.

The reagents used in this study were N, O-bis(trimethylsilyl) trifluoroacetamide (BSTFA), trimethylsilyl chloride (TMCS), boron trifluoride methanol solution (Sigma-Aldrich, St. Louis, MO, USA), and ethylic ether (JT Baker, Deventer, Holland).