Menhaden

Total lipids were extracted from PBS-washed cell pellets using a methanol/chloroform mixture according to Bligh and Dyer [16] (link). Subsequently, a transesterification was performed by incubating samples with 0.5 N methanolic sodium hydroxide at 100°C for 10 min followed by methanolic boron trifluoride (10% w/w; Supelco, Bellefonte, PA, USA) treatment at 100°C for 5 min. Subsequently, fatty acid methyl esters (FAME) were extracted with n-hexan, separated in a gas chromatograph (GC 17a V3, Shimadzu, Kyoto, Japan) using a fused-silica capillary column with medium polarity (DB 225 MS, 60 m×0.25 mm i.d., 0.25 µm film thickness; Agilent Technologies, Santa Clara, USA) and detected downstream by a flame ionization detector (FID). GC conditions were as previously described [17] (link). Peak area integration was accomplished using GC solution software version 2.3 in comparison to previously measured reference standards (BR2, BR4, and ME93 from Larodan/CPS-Chemie, Aachen, Germany; Menhaden from Sigma-Aldrich, Taufkirchen, Germany; 463 and 674 from Nu-Chek-Prep, Elysian, USA).

Fish oils such as tuna, sardine, Alaska pollock, and pacific saury were obtained from Nippon Suisan Kaisha (Tokyo, Japan), and menhaden, soybean, and coconut oil were purchased from Sigma-Aldrich. DHA ethyl ester and EPA ethyl ester were obtained from Chemport (Taejon, Korea). CPFA-D85E02 EE (85% DHA/2% EPA) was used as the source of DHA, and CPFA-E92D00 EE (92% EPA/0% DHA) was the source of EPA. DHA and EPA contents were adjusted to those of tuna oil: DHA (23%) and/or EPA (7%). All oil and DHA/EPA was stored at −80°C after air purge by N₂ gas.