Lipids, fatty acids and hydroxy-fatty acids of Euphausia pacifica

Euphausia pacifica is a good candidate for a resource of marine n-3 PUFA. However, few reports exist of the lipid and fatty acid composition of E. pacifica. To examine the potential of E. pacifica as a resource of marine n-3 PUFA, we analyzed E. pacifica oil. We extracted lipids from E. pacifica harvested from the Pacific Ocean near Sanriku, Japan. Lipid classes of E. pacifica oil were analyzed by TLC-FID and the fatty acid composition of the oil was analyzed by GC/MS. Free fatty acids and hydroxy-fatty acids were analyzed by LC/QTOFMS. The lipid content of E. pacifica ranged from 1.30% to 3.57%. The ratios of triacylglycerols, phosphatidylcholine, phosphatidylethanolamine and free fatty acids in E. pacifica lipids were 5.3–23.0%, 32.6–53.4%, 8.5–25.4% and 2.5–7.0%, respectively. The content of n-3 PUFA in E. pacifica lipids was 38.6–46.5%. We also showed that E. pacifica contains unusual fatty acids and derivatives: C16-PUFAs (9,12-hexadecadienoic acid, 6,9,12-hexadecatrienoic acid and 6,9,12,15-hexadecatetraenoic acid) and hydroxy-PUFAs (8-HETE and 10-HDoHE). E. pacifica is a good resource of marine n-3 PUFA. Moreover, E. pacifica can provide C16-PUFA and hydroxy-PUFAs.

The quantities of free fatty acids detected in E. pacifica are shown in Table 3. The major free fatty acids in E. pacifica were palmitic acid, oleic acid, EPA, and DHA. The content of palmitoleic acid, oleic acid, arachidonic acid and EPA in E. pacifica was high in samples collected on April 24 2017. In a comparison of E. pacifica with E. superba, the palmitoleic acid and arachidonic acid content was clearly higher in E. pacifica, whereas the palmitic acid was higher in E. superba than in E. pacifica. As compared with Balanus rostratus Hoek and Marsupenaeus japonicus, the EPA and DHA content was much higher in E. pacifica.  Table 2. Fatty acid composition in lipids.  Table 4. The content of 8-HEPE gradually increased from February to April. In E. pacifica, the 8-HEPE content was higher than that of 8-HETE and 10-HDoHE. Furthermore, the 8-HEPE content was much higher in E. pacifica than in E. superba, Balanus rostratus Hoek or Marsupenaeus japonicus 8-HEPE, 8-HETE and 10-HDoHE share a common hydroxyl at the n-12 carbon position. To examine whether 8-HEPE is produced from EPA by enzymatic oxidation, we incubated fresh or boiled E. pacifica protein with EPA and determined the amount of 8-HEPE (Fig. 6). We found that 8-HEPE was produced by incubating E. pacifica protein with EPA, and the amount of 8-HEPE produced was significantly decreased by heating E. pacifica protein at 95 °C for 5 min.

Discussion
In this study, we showed that the oil content of E. pacifica was 1.30-3.57%, approximately half of which consisted of phosphatidylcholine and phosphatidylethanolamine. Palmitic acid, EPA, and DHA were the major fatty acids that compromised the E. pacifica oil. The content of n-3 PUFAs was 38.6-46.5% (Table 2). In previous reports, the lipid content of E. pacifica caught near the Ogasawara Coast, Miyagi, and Hokkaido was determined as 0.59-1.75%, 3.34-6.41%, and 1.1-3.2%, respectively, on a wet weight basis 8,11 . In each area, the lipid content of E. pacifica was high in spring (March -May) as compared with any other season. In spring, phospholipids accounted for more than 40% of the lipid content of E pacifica, and the content of n-3 PUFA was more than 29%. These results indicate that E. pacifica lipid composition has remained stable for at least the past 20 years, and that E. pacifica caught in spring is a good resource of the phospholipid form of n-3 PUFA.
We previously reported that E. pacifica contains 8-HEPE 9 . In this study, we demonstrated that E. pacifica also contains 8-HETE and 10-HDoTE (Fig. 5). Prior to our previous study 9 , hydroxy-PUFAs had not been reported in krill. However, prostanoids, oxylipins, cyclooxygenase and lipoxigenase had been reported in crustaceans, and some biological functions have been predicted [15][16][17][18] . Our present results indicate that E. pacifica has a lipoxygenase that can oxidize the n-12 carbon of EPA, AA, and DHA (Fig. 6). The 8-HEPE contents in E. pacifica was increased from February to April (Table 3); thus, it seems that 8-HEPE might contribute to E. pacifica growth and maturation. Identification of the E. pacifica lipoxygenase and biochemical analysis will clarify the biological function of hydroxy-PUFAs. In addition, the identification of 8-HEPE synthetic pathways in E. pacifica might provide a way to produce 8-HEPE artificially.

Conclusions
To meet the continuing increasing demand for marine n-3 PUFA globally, E.pacifica might represent a new resource of the phospholipid form of n-3 PUFA. Moreover, E. pacifica can provide unusual fatty acids, C16-PUFA and hydroxy-PUFAs that cannot be obtained from other resources.

Gas chromatography/mass spectrometry (GC/MS). Triacylglycerol (TAG) and phospholipid (PL)
were esterified by incubation with methanol containing 5% hydrochloric acid at 50 °C for 30 min. Analysis of the fatty acid methyl esters was performed on a gas chromatography (Agilent Technologies 6890 N)/mass spectrometry (Agilent Technologies 5975B) system using with a DB-23 gas chromatography column (60 m × 0.25 mm i.d. and 0.15-µm film thickness [Agilent technologies]). Helium (carrier gas) was passed through the column at a constant linear velocity of 40.0 mL/min, and the split ratio was 50. The initial oven temperature was maintained at 50 °C for 1 min, increased to 175 °C at a rate of 25 °C/min, increased to 230 °C at a rate of 4 °C/min, and then     E. pacifica protein extraction and enzyme reaction. E. pacifica was homogenized in a 3-fold higher volume of Tris-HCl buffer (pH 7.4) on ice and centrifuged at 20,000 g for 10 min at 4 °C. Next, E. pacifica protein in the liquid layer was concentrated by Amicon Ultra 10 K (Merck Millipore). The concentration of E. pacifica protein was measured by Coomassie Plus Reagent (Thermo Scientific), and then 50 µg of E. pacifica protein and 2 nmol EPA were incubated in 20 µL of Tris-HCl (pH7.4) buffer at 20 °C for 1 hour. After the enzymatic reaction, 60 µL of acetonitrile containing 1% of formic acid was added to the samples and mixed. Next, the samples were centrifuged at 20,000 g for 10 min at room temperature. The 8-HEPE concentration of the liquid layer was measured by LC/QTOFMS.