Fatty Acid Composition Analysis Protocol

Fatty acid composition in each experimental diet was determined by modifying a previously described protocol (64 (link)). Briefly, 400 mg of each diet sample was reconstituted in a 4:1 (v/v) ethanol/methanol solution + 0.1% (v/v) butylated hydroxytoluene (BHT) and heated 15 min at 55°C in a CEM Mars 6 Xpress microwave digestion system (CEM Corporation, Matthews, NC). Then, 2 mg of extracted fatty acids from each diet sample were converted to fatty acid methyl esters (FAMEs) by treating with 500 µl of toluene and 20 µg of internal standard (methyl-12-tridecenoate), incubating with 2 ml of KOH (0.5 N) at 50°C for 10 min, then subsequently incubating with 3 ml of methanolic HCl (5% [v/v] at 80°C for 10 min to allow base-catalyzed methylation and acid-catalyzed methylation, respectively. Following methylation, 2 ml of HPLC-grade water was added to the samples, and FAMEs were extracted by adding 2 ml of hexane to the samples twice. Extracted FAMEs were dried under nitrogen with an Organomation Multivap Nitrogen Evaporator (Organomation Associates, Berlin, MA). Dried FAMEs were then resuspended in 1 ml of isooctane and kept at -20°C until further analysis.
FAMEs were analyzed by GC-MS as previously described (64 (link)). Briefly, FAMEs in each sample were separated on a Perkin Elmer 680/600 GC-MS (Waltham, MA) outfitted with a HP-88 capillary column (100 m × 0.25 mm inner diameter × 0.2 µm film thickness; Agilent Technologies, Santa Clara, CA). MassLynx^TM (4.1 SCN 714; Waters Corporation, Milford, MA) was used to compare analyte retention time and electron ionization (EI) mass fragmentation to those in the reference standard, which consisted of Supelco 37 Component FAME Mix (Sigma-Aldrich, St. Louis, MO), mead acid, docosatetraenoic acid, ω-3 docosapentaenoic acid (DPA), ω-6 DPA, and palmitelaidic acid (Cayman Chemical, Ann Arbor, MI). FAME analyte peak areas were converted to individual FAME concentrations using a standard curve based on the reference standard and internal standard. For fatty acids with a detected chain length of 10 to 24 carbon atoms, fatty acid content in the diet is reported as percentage (w/w) of total fatty acids quantified (Table 1).