B1252

Experimental oil was mixed following Liao’s method [18 (link)]. Soybean oil and experimental oil mixture were purchased from a local supermarket and analyzed by gas chromatography (GC). Samples were extracted using a modified Folch method [19 (link)]. Samples was extracted using chloroform/methanol = 2/1 (v/v) for 1 h, and then distilled water was added to separate the liquid. The extract was incubated for 10 min at room temperature and centrifuged at 4 °C and 3000 rpm for 10 min. The lower phase was collected. FA methylation was performed by heating the sample to 88 °C with 14% boron trifluoride/methanol (B1252, Sigma, St. Louis, MO, USA) for 1 h to form FA methyl ether (FAME) and then the solvent was removed. FAME was analyzed using a FOCUS™ GC (Thermo Fisher Scientific, Milan, Italy) equipped with a 30-m × 0.32-mm inner diameter (I.D.) × 0.20-μm df Rtx-2330 column (Restek, Bellefonte, PA, USA) and flame ionization detector.
FA compositions of the soybean oil and experimental oil were analyzed by GC. Results were obtained based on the retention time of the appropriate standard (GLC-455; Supelco, St. Louis, MO, USA), and percentages of the FA profile were calculated based on 16 different FAs, as shown in Table 1.

The protocol used for analysis of the composition of fatty acids in EPA-rich oil capsules and serum was adapted from the methodology described previously (28 (link)). Mouse blood was collected by cardiac puncture 3 days after wound induction. Blood was centrifuged at 4,481g for 15 minutes at 4°C to yield serum. Methanolic NaOH (1 mL) and 25 uL internal standard (1,2-dipentadecanoyl-sn-glycero-3-phosphocholine, Sigma^®, 850350P, diluted to 1mg/mL in isooctane) were added to 150 uL serum or oil capsules. This mixture was heated for 15 minutes. After cooling, 2 mL esterification reagent (boron trifluoride-methanol solution, BF3, Sigma^®, B1252) was added and the samples were heated for 5 minutes. Then, 1 mL isooctane and 5 mL saturated NaCl solution was added. After separation of the phases, the upper phase (250 uL) was collected for analysis [see (15 (link)) for detailed methodology]. Identification of fatty acid peaks was made by comparison with the 37 fatty acid methyl ester calibration standards.