Docosatrienoic acid 22 3n 3 ethyl ester

The fatty acid internal standard, docosatrienoic acid (22:3n-3) ethyl ester, GC reference standard (GLC-462), fish oil ethyl DHA, and microalgal ethyl DHA were purchased from NuChek Prep, Inc (Elysian, MN). Uniformly labeled ¹³C DHA methyl ester was purchased from Cambridge Isotope Laboratories, Inc. For isotopic analysis, the reference materials (USGS70, USGS71, and USGS72) were obtained from Reston Stable Isotope Laboratory-United States (Reston, VA). Boron trifluoride in methanol (14%) was purchased from Sigma-Aldrich. All solvents used were American Chemical Society or HPLC grade and were purchased from either MilliporeSigma (Mississauga, ON, Canada) or Fisher Scientific (Ottawa, ON, Canada).

Lipids were extracted from 300 μl of human plasma, 100 μl of mouse serum, and 50 mg of mouse brain and liver in 2:1 chloroform:methanol by a modified Folch method (41 (link)). Docosatrienoic acid (22:3n-3) ethyl ester (Nu-Chek Prep) was included at 10, 5, and 25 μg, respectively, as internal standard for fatty acid quantification. After homogenization, lipid and aqueous phases were separated with the addition of 0.88% KCl, centrifuged at 500 g for 5 min, and the bottom lipid-containing chloroform phase was isolated. The resulting total lipid extracts for plasma/serum and an aliquot of the liver total lipid extracts were dried under a stream of nitrogen and transesterified with 14% boron trifluoride in methanol for 1 h at 100 °C. The samples were cooled to room temperature, 1 ml of water and hexane were added, vortexed, and centrifuged at 500 g for 5 min. The top hexane layer containing fatty acid methyl esters (FAMEs) were isolated, dried under nitrogen, resuspended in 100–200 μl of heptane, analyzed by GC-FID, recapped, and subsequently analyzed by GC-C-IRMS.