Euphausia pacifica as a source of 8(R)-hydroxy-eicosapentaenoic acid (8R-HEPE), 8(R)-hydroxy-eicosatetraenoic acid (8R-HETE) and 10(R)-hydroxy-docosahexaenoic acid (10R-HDHA)

Figures & data

Figure 1. Structure and m/z of 8-HEPE, 8-HETE, and 10-HDHA.

Table 1. Proportion of lipid class in incubated E. pacifica oil.

	Triacylglycerol	Phosphatidylcholine	Phosphatidylethanolamine	Fatty acid
0 hour	17.2 ± 2.98	23.9 ± 5.18^a	5.3 ± 0.40	8.2 ± 2.03^a
2 hours	16.7 ± 5.63	18.1 ± 2.36^ab	4.7 ± 0.51	12.0 ± 1.76^ab
4 hours	17.2 ± 2.02	18.0 ± 4.36^ab	4.9 ± 0.24	14.2 ± 2.40^ab
6 hours	18.5 ± 3.01	16.3 ± 2.67^ab	4.3 ± 0.67	16.6 ± 2.14^b
8 hours	18.2 ± 4.23	14.6 ± 2.46^b	4.4 ± 0.45	17.5 ± 2.10^bc
24 hours	18.6 ± 4.02	13.1 ± 1.56^b	3.9 ± 0.66	20.6 ± 1.89^c

Values are the mean ± s.d. of three samples. Values marked by different letters were significantly different (p < 0.05).

Table 2. LC/QTOF analysis of PC and fatty acid in incubated E. pacifica oil.

		0 hr	2 hr	4 hr	6 hr	8 hr	24 hr
Phosphatidylcholine	PC 16:0–20:5	1.00	0.83	0.78	0.72	0.71	0.21
	PC 16:0–18:1	1.00	0.93	0.91	0.89	0.89	0.56
	PC 16:0–22:6	1.00	0.87	0.82	0.72	0.64	0.65
	PC 20:5–20:5	1.00	0.59	0.79	0.48	0.71	0.56
	PC 20:5–22:6	1.00	0.84	0.78	0.65	0.71	0.66
	PC 16:0–16:1	1.00	1.13	0.74	0.72	0.75	0.61
	PC 18:1–20:5	1.00	1.06	0.73	0.77	0.67	0.58
	PC 16:0–18:4	1.00	1.14	0.69	1.02	0.29	0.24
	PC 16:0–18:2	1.00	1.18	0.88	0.89	0.78	0.78
	PC 14:0–20:5	1.00	0.68	0.65	0.63	0.67	0.48
Fatty acid	FA 14:0	1.00	1.95	2.86	3.60	4.03	3.73
	FA 16:0	1.00	1.10	1.67	1.89	2.15	2.45
	FA 16:1	1.00	1.62	2.72	2.73	3.28	4.21
	FA18:1	1.00	1.29	1.70	1.82	1.99	2.37
	FA 18:2	1.00	1.54	2.22	2.43	2.63	2.83
	FA 18:4	1.00	1.65	2.62	2.66	3.20	3.15
	FA 20:5	1.00	1.24	1.33	1.29	1.32	1.23
	FA 22:6	1.00	1.47	1.79	1.91	1.99	2.19

Values are indicated by the relative area on the ion chromatograph detected by QTOFMS normalized against the 0 hr sample. The top 10 molecular species of PC according to the height of the peak detected by LC/QTOFMS are shown.

Figure 2. Examination of the conditions of incubation to increase the levels of 8-HEPE, 8-HETE, and 10-HDHA.

Examination of incubation temperature, pH (a) and buffer volume (b). The value corresponding to E. pacifica extract prior to incubation was used as a control. B. The ratio shown in the x-axis refers to the weight of E. pacifica: water volume. A,B. Samples were incubated for 3 h. (c) Examination of incubation time. (a-c) Values are the mean ± s.d. of four samples. * indicate a significant difference compared with the control (p < 0.05). Values marked by different letters were significantly different (p < 0.05).

Table 3. Examination of the concentration of 8-HETE, 8-HEPE, and 10-HDHA using synthetic adsorbent beads. Values are the mean of three examinations.

Name of beads	Extract (mg)	Proportion of 8-HETE (%)	Proportion of 8-HEPE (%)	Proportion of 10-HDHA (%)
HP-20	88	0.01	0.58	0.08
SP850	13	n.d.	2.29	0.37
SP70	28	0.05	1.18	0.17
SP700	10	0.20	5.28	0.92
SP207	8	0.26	5.26	0.87
HP2MGL	2	n.d.	3.74	0.46

Figure 3. Examination of liquid-liquid separation.

The contents of 8-HEPE, EPA and PA in the upper or lower fraction were quantified by LC/QTOFMS. Values are the mean of three examinations.

Table 4. Summary of the purification of 8-HEPE, 8-HETE, and 10-HDHA from 1 kg of E. pacifica.

		#1	#2	#3	#4	#5	#6
E. pacifica	Date of collection	February 25	March 6	April 10	April 4	April 18	April 17
	Lipid content (%)	1.6	1.4	2.7	2.8	3.4	3.4
	Weight (kg)	1.0	1.0	1.0	1.0	1.0	1.0
	8-HEPE (ppm)	5.92	2.90	6.14	12.99	14.00	12.67
	8-HETE (ppm)	0.94	0.19	0.55	1.02	0.88	0.98
	10-HDHA (ppm)	1.26	0.69	0.20	1.04	0.30	0.35
Incubated E. pacifica solution	Volume (L)	2.24	2.14	2.48	2.44	2.40	2.38
	8-HEPE (ppm)	57.90	50.50	152.80	214.10	303.30	272.00
	8-HETE (ppm)	4.90	3.13	6.67	3.89	10.80	10.40
	10-HDHA (ppm)	11.80	6.35	10.69	6.70	12.59	10.75
SP700 Eluted fraction	Weight (g)	1.21	1.20	2.36	1.94	1.57	1.35
	8-HEPE (%)	11.00	7.00	12.60	16.40	15.90	26.93
	8-HETE (%)	0.90	0.38	0.51	0.22	0.41	0.78
	10-HDHA (%)	2.08	0.86	0.77	0.46	0.56	0.82
Lower fraction	Weight (g)	0.26	0.23	0.59	0.42	0.40	0.35
	8-HEPE (%)	49.21	37.85	50.34	68.57	56.25	80.05
	8-HETE (%)	1.80	1.23	1.20	0.90	1.39	1.61
	10-HDHA (%)	3.88	2.86	1.82	1.65	1.76	1.78
Preparative HPLC	8-HEPE (mg)	107.4	140.7	151.7	242.6	273.9	280.1
	8-HETE (mg)	3.0	1.0	2.5	1.7	1.7	2.8
	10-HDHA (mg)	10.5	5.2	6.8	4.6	3.1	3.1

Figure 4. Scheme outlining the purification of 8-HEPE, 8-HETE, and 10-HDHA from 1 kg of E. pacifica.

Values are the mean of six examinations. Chromatograms of preparative HPLC are shown at the bottom. The peaks of 8-HEPE, 8-HETE and 10-HDHA are annotated in the 235 nm chromatogram. The chromatogram shows the peak corresponding to 8-HEPE. An expanded range around the peaks for 10-HDHA and 8-HETE is shown in supplemental Figure S2.

Figure 5. Stereochemical analysis of 8-HETE, 8-HEPE, and 10-HDHA purified from E. pacifica.

The chromatograph of 8-HETE was an extracted ion chromatogram of product ion (m/z = 155.071) generated from precursor ion (m/z = 319.2). The chromatograph of 8-HEPE was an extracted ion chromatogram of product ion (m/z = 155.071) generated from precursor ion (m/z = 317.2). The chromatograph of 10-HDHA was an extracted ion chromatogram of product ion (m/z = 153.092) generated from precursor ion (m/z = 343.2).

Data availability statement

The data that support the findings of this study are available from the corresponding author, H.Y., upon reasonable request.