Boron trifluoride methanol

Lipids were extracted from ileum via the Folch Method [32 (link)] and phospholipid fractions were separated by thin layer chromatography (TLC) as previously described [29 (link)]. Briefly, samples were spotted on H-plates (EMD Chemicals, Gibbstown, NJ, USA) to separate phospholipid species. Bands corresponding to lyso-phosphatidylcholine (lyso-PC), sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) were collected and methylated with 14% boron trifluoride-methanol (Fisher Scientific, Mississauga, ON, Canada). Fatty acid methyl esters were separated on a DB-FFAP fused-silica capillary column (15 m, 0.1 m film thickness, 0.1 mm i.d.; Agilent, Mississauga, ON, Canada) and quantified on an Agilent 6890 gas chromatograph. Peaks were identified by retention times of fatty acid methyl ester standards (Nu-Chek-Prep, Elysian, MN, USA) using EZchrom Elite version 3.2.1 software. Fatty acid results were calculated as percent composition or ug/0.1 g tissue with 10 µg of heptadecanoic acid (17:0) added to each fraction for the internal standard.

The reference standards of the fatty acids β-sitosterol, δ-tocopherol, lipopolysaccharide (LPS), CDCl₃, Griess reagent (N5751) and N-nitro-l-arginine-methyl ester (l-NAME) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol, tetrahydrofuran acetonitrile, sodium hydroxide, and boron trifluoride/Methanol were obtained from Fisher Scientific (Pittsburgh, PA, USA). DMEM (Dulbecco’s modified Eagle medium), FBS (fetal bovine serum), trypsin-EDTA, l-glutamine, penicillin/streptomycin were obtained from HyClone (South Logan, UT, USA). The tetrazolium salt (MTT) kit was purchased from Roche Applied Science (Mannheim, Germany). Isoflurane was purchased from DS Pharma Animal Health Co. (Osaka, Japan).