Lipid Profiling of Ileum Samples

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.