Menhaden oil

The fatty acid methyl esters (FAME) were determined by acid-catalysed transesterification. Briefly, 300 mg of dried sample from the by-products were weighed to a test tube and 5 mL of a 5% acetyl chloride-methanolic solution was added and then left to react for 1 h in a water bath at 80 °C. Once sample extracts were cooled, 1 mL of Milli-Q water and 2 mL n-heptane were added. After a vortex agitation and centrifugation for 3 min at 3000× g the organic phase was collected and filtered through anhydrous sodium sulphate. Two millilitres of the final extract were analysed in a GC/FID system consisting of a Bruker Scion 456-GC (West Lothian, UK), equipped with an auto-sampler and with split mode of 100:1. The split/splitless injector and a flame ionisation detector were both set at 250 °C. The separation of the FAME was carried out with helium in a capillary column DB-WAX (Agilent Technologies, Santa Clara, CA, USA) (film thickness, 0.25 μm), 30 m × 0.25 mm i. d., using a temperature program for the column starting at 180 °C and increasing to 200 °C at 4 °C/min, holding for 10 min at 200 °C, heating to 210 °C at the same rate and holding at this temperature for 14.5 min. FAME identification was based on their retention time, using a standard mix (PUFA-3, Menhaden oil, Sigma-Aldrich) as a reference. Results were expressed as a percentage.

All the reagents were purchased from Merck (Darmstadt, Germany). GLC-463 mix was from Nu-Check (Elysian, MN, USA), and EPA, DHA, FAME 189-19, menhaden oil, pure triglycerides, and (+)-α-tocopherol were from Sigma-Aldrich (St. Louis, MO, USA).

The fatty acid profile was determined in the samples before and after digestion (bioaccessible fraction). Fatty acid methyl esters (FAME) were prepared by acid‐catalyzed transesterification using the methodology described by Bandarra, Batista, Nunes, Empis, and Christie (1997). Samples were injected into a Varian Star 3800 Cp gas chromatograph (Walnut Creek, CA, USA), equipped with an autosampler with a flame ionization detector at 250°C. FAME were identified by comparing their retention time with those of Sigma‐Aldrich standards (PUFA‐3, Menhaden oil, and PUFA‐1, Marine source from Supelco Analytical). Data in mg/100 g of edible part were calculated using the peak area ratio (% of total fatty acids) and the lipid conversion factors set by a previous study (Weihrauch, Posati, Anderson, & Exler, 1977).

Total lipids from the lipid raft fractions from the hippocampus homogenates were extracted with chloroform/methanol (2:1 v/v) containing butylated hydroxytoluene (0.01%) as the antioxidant, according to Folch et al. (1957) [123 (link)] with small modifications. The organic solvent was evaporated under a stream of nitrogen, and the lipid content determined gravimetrically and stored in fresh solvent at −20 °C until further analysis. Lipids from the lipid rafts were subjected to acid-catalyzed transmethylation using 1 mL of toluene and 2 mL of 1% sulfuric acid (v/v) in methanol for 17 h in a heater at 50 °C. The resultant fatty acid methyl esters (FAMEs) and dimethylacetals (DMAs) were purified by thin layer chromatography (TLC), and quantified using a TRACE GC Ultra (Thermo Fisher Scientific, Waltham, MA, USA) gas chromatograph equipped with a flame ionization detector, with helium as the carrier gas. Individual FAME and DMA peaks were identified by referring to authentic standards (Mix C4-C24 and PUFA n° 3 from menhaden oil (Supelco Inc., Bellefonte, PA, USA).

Fatty acid methyl esters (FAME’s) were prepared by acid-catalyzed transesterification using the methodology described by Bandarra et al. [21 (link)]. Samples were injected into a Varian Star 3800 CP gas chromatograph (Walnut Creek, CA, USA and equipped with an auto sampler with a flame ionization detector at 250 °C. FAME’s were identified by comparing their retention times with those of Sigma–Aldrich standards (PUFA-3, Menhaden oil, and PUFA-1, Marine source from Supelco Analytical).