INTRODUCTION

Krill oil (KO) extracted from crustaceans (Euphausia superba), a shrimp-like marine zooplankton living in the Antarctic, has been recognized as an important source of long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) in the last decade (Tou et al., 2007). KO is a rich source of long-chain n-3 PUFA, found in both the sn-1 and sn-2 positions of phospholipids (mainly phosphatidylcholine (PtdCho), triacylglycerols (TAG), and in free fatty acids (Winther et al., 2011). In contrast to KO, the n-3 PUFA in fish oils (FO) are in the TAG form (Winther et al., 2011; Tou et al., 2007).

Several post-prandial and longer-term human studies have investigated the bioavailability (blood levels) of n-3 PUFA from KO and FO in human trials. The results have been inconsistent (Maki et al., 2009; Ramprasath et al., 2013; Schuchardt et al., 2011; Yurko-Mauro et al., 2015). We have investigated the differences in lipidomic profiles between KO and FO supplementations in a post-prandial study (Sung et al., 2019) and a 30-day study (Sung et al., 2020). The post-prandial study showed that eicosapentaenoic acid (EPA) and DHA from KO were preferentially incorporated into plasma phospholipid fraction, whereas following FO consumption EPA and DHA were partitioned towards the TAG fraction (Sung et al., 2019). The 30-day study reported that 77 molecular species of lipids differed significantly between KO and FO. After KO supplementation, the majority of lipid species that increased more than after FO supplementation contained saturated and monounsaturated fatty acids; in contrast, the majority of lipid species that increased more after FO than KO were those containing n-6 PUFA (Sung et al., 2020). The present study is a secondary analysis of the 30-day study, looking specifically at the incorporation of n-3 PUFA into the phospholipid molecular species at days 15 and 30.

The aim was to determine whether the n-3 PUFA from the KO and FO supplements partitioned into plasma phospholipid molecular species to the same extent, following 30 days of supplementation of seven capsules of KO per day (containing a total of 1.27 g n-3 PUFA) or five capsules of FO per day (total of 1.44 g n-3 PUFA).

MATERIALS AND METHODS Subjects and supplements

This was a randomized crossover study with KO or FO supplementation for 30 days. The supplementation periods were separated by a minimum of 30-day washout (Cicero et al., 2016). During the study period, all participants were instructed to maintain their habitual diet and requested not to consume fish, seafood, or n-3 fortified foods more than once a week. For interventions, participants consumed daily seven 1-g capsules of KO containing 1.27 g of long-chain n-3 PUFA (EPA + DHA + DPAn-3) or five 1-g capsules of FO containing 1.44 g of long-chain n-3 PUFA for 30 days each; these capsule numbers gave the closest possible match for total long-chain n-3 PUFA per group (Sung et al., 2020). Participants were required to attend the clinic at days 0 (baseline), 15, and 30 of each supplementation for blood sample collection. On the evening prior to each clinic visit, participants were required to consume one of the most common low-fat dishes in their diet, avoid drinking alcohol and strenuous physical activities, and fast approximately 10 h overnight. On each study day, standardized procedures were performed where participants arrived at the clinic between 7 and 9 am, and a fasting blood sample (10 ml) was collected via a venepuncture by a qualified practitioner.

A total of 11 healthy women aged between 18 and 50 years with BMI 20–35 (kg/m2), who had not experienced menopause, were recruited through emails to all Victoria University staff and students, flyer advertisements via the Victoria University Nutritional Therapy Teaching Clinic, community centers and local medical practices. Participants were screened for their suitability for the 30-day study as they completed a medical questionnaire, anthropometric measurements, and the electronic PUFA food frequency questionnaire (FFQ) prior to enrolling into the study. Participants were excluded if their daily long-chain n-3 PUFA was more than 500 mg based on results of the electronic PUFA FFQ (Sullivan et al., 2008). Participants were also excluded if they were cigarette smokers; pregnant or lactating; or had heart, liver, kidney, or inflammatory bowel disease, diabetes; or medications interfering with lipid metabolism or lowering blood lipids; allergy to fish or other seafood; or intake of oily fish more than twice a week or supplements including n-3 fatty acids in the past 4 weeks prior to the study.

The study supplements of KO (Swisse Wellness Pty Ltd., Euphausia Superba oil, Victoria, Australia) and FO (Swisse Wellness Pty Ltd., Natural fish oil, Victoria, Australia) were purchased from a local pharmacy and the fatty acid and lipidomic profiles of these oils were analyzed prior to the commencement of the intervention and have been published in full (Sung et al., 2019, 2020).

Ethics approval was obtained from the Victoria University Human Research Ethics Committee (HRE15-031). All experimental procedures were performed in accordance with the Declaration of Helsinki of the World Medical Association. Written informed consent was obtained from all participants prior to the study. This trial was registered with the Australian New Zealand Clinical Trial Registry (ACTRN 12615000472572).

Lipid extraction

Plasma lipids were isolated using a single-phase chloroform: methanol (CHCl3:MeOH) extraction as previously described (Weir et al., 2013). Briefly, plasma samples (10 μl) were extracted in a single-phase extraction with 20 volumes of CHCl3:MeOH (2:1) and 10 μl of an internal standard mix (in CHCl3:MeOH [1:1]) containing between 50 and 1000 pmol each of 23 non-physiological or stable isotope-labeled lipid standards (Weir et al., 2013).

Mass spectrometry and lipid analysis

Lipid analysis was performed by high-performance liquid chromatography electrospray ionization-tandem mass spectrometry (HPLC ESI-MS/MS) using an Agilent 1290 HPLC coupled to an Agilent 6490 triple quadrupole mass spectrometer. The settings of LC ESI-MS/MS were as follows: gas temperature 150°C, gas flow 17 L/min, nozzle pressure 20 psi, sheath gas temperature 200°C, sheath gas flow 10 L/min, capillary voltage 3500 V and nozzle voltage 1000 V. Liquid chromatography was performed on a Zorbax Eclipse Plus C18, 1.8 μm, 50 × 2.1 mm column (Agilent Technologies) using solvents A and B consisting of water:acetonitrile:isopropanol, 50:30:20 and 1:9:90, respectively, both containing 10 mM ammonium formate. The column was heated to 60°C and the autosampler regulated to 25°C. A total of 522 lipid species were analyzed using dynamic multiple reaction monitoring where data were collected for a retention time window specific to each lipid species. Results from the chromatographic data were analyzed using Mass Hunter Quant where relative lipid abundances were calculated by relating each area under the chromatogram for each lipid species to the corresponding internal standard. Correction factors were applied to adjust for different response factors, where these were known. Species that were chromatographically separated were labeled as such (e.g., PC [16:0–22:6] and PC [18:2–20:4]), whereas species that were mixed isomers were given the standard phospholipid notation (e.g. PC[40:8] was a mixture of 20:4/20:4 and 18:2–22:6) (Huynh et al., 2019). Where structural details were sufficient, lipids were manually annotated as containing long-chain n-3 components (i.e., 20:5 EPA, 22:5 DPA, and 22:6 DHA).

Statistical analysis

Statistical analyses were performed to compare the significant effects of the 30-day supplementation on plasma lipid molecular species between the KO and FO supplementation groups. Values are expressed as mean of concentration ± standard error mean (SEM) for 11 participants. The normality of data distribution was checked using D'Agostino & Pearson normality test. Log-transformation of data was carried out where appropriate. Two-way analysis of variance (ANOVA) for repeated measurements was performed to analyze supplementation effect over time (interaction time × supplementation), differences between time points within supplementation and the same time point between the two omega-3 supplementations. All p values were corrected for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR). P < 0.05 was considered significant. The analyses were performed using GraphPad Prism version 7.01.

RESULTS

A total of 11 healthy women completed the 30-day crossover dietary intervention. The KO intervention was associated with significant increases in plasma EPA concentration at days 5, 10, and 30, compared with FO, resulting in a significantly greater net incremental area under the curve for EPA response following KO consumption compared with FO (Sung et al., 2020). For both treatments, the level of plasma DHA increased significantly over the 30 days, but there was no significant difference between treatments.

EPA-molecular species

A total of 21 EPA-containing phospholipid molecular species were detected in the concentration range of 107–60,310 pmol/ml, as shown in Table 1. The majority of these showed significant increases in concentration at day 30 after both KO supplementation (17/21 increased) and FO supplementation (14/21 increased). At day 30, 14 molecular species in the KO group had significantly higher concentrations compared with the FO group. For the remaining six molecular species from the total of 20, there were no significant differences between the two n-3 oil supplementation groups. Eleven of the 14 which were significantly different were ether-phospholipids, while 3/14 were diacyl phospholipids (PC [16:0–20:5]; PC [38:5] [b]; PC [18:2–20:5]) (Table 1). The nine molecular species whose initial concentrations exceeded 500 pmol/ml and which were significantly different between KO and FO are shown in Figure 1. This illustrates that molecular species following KO supplementation showed progressive increases in concentration over the 30 days compared with the FO group, where in most cases the concentration had plateaued by day 15.

TABLE 1. Lipidomic changes in plasma EPA (20:5) phospholipid molecular species over the 30-day krill oil and fish oil supplementation EPA (20:5) molecular species Concentration (pmol/ml) p value Krill oil Fish oil Krill oil Fish oil Krill oil vs. Fish oil Time Treatment Interaction T = 0 T = 15 T = 30 T = 0 T = 15 T = 30 T0:T30 T0:T30 T = 0 T = 15 T = 30 PC (38:5)(a) 16,314 16,624 19,209 15,720 18,518 19,096 0.00 0.00 0.46 0.03 0.89 0.00 0.54 0.09 PC (16:0/20:5) 14,060 48,819 60,310 12,143 48,925 49,923 0.00 0.00 0.62 0.98 0.01 0.00 0.03 0.14 PC (38:5)(b) 9893 24,824 31,028 9354 26,468 26,682 0.00 0.00 0.78 0.40 0.03 0.00 0.22 0.10 PC (O-38:5) 5145 4770 5401 4834 4592 4601 0.33 0.38 0.24 0.50 0.01 0.24 0.04 0.22 PC (O-36:5) 4318 3657 4138 4493 4110 4033 0.46 0.07 0.48 0.07 0.67 0.01 0.41 0.28 PC (P-38:5)(a) 2374 2837 3346 2424 2651 2654 0.00 0.11 0.72 0.19 0.00 0.00 0.02 0.00 PE (P-18:0/20:5) 1560 5487 7895 1583 5537 4806 0.00 0.00 0.97 0.94 0.00 0.00 0.00 0.00 PE (38:5)(a) 1506 1286 1045 1786 1391 1169 0.00 0.00 0.03 0.40 0.33 0.00 0.27 0.56 PE (P-16:0/20:5) 864 4171 4574 807 3001 2454 0.00 0.00 0.68 0.13 0.00 0.00 0.00 0.13 PE (O-38:5)(a) 770 881 1138 685 665 539 0.00 0.20 0.45 0.06 0.00 0.41 0.00 0.01 PE (38:5)(b) 725 1457 1495 735 1474 1373 0.00 0.00 0.94 0.89 0.35 0.00 0.64 0.69 PC (O-40:5) 694 653 741 685 672 662 0.19 0.52 0.79 0.58 0.03 0.31 0.33 0.14 PC (P-16:0/20:5) 688 2221 3079 644 2076 2173 0.00 0.00 0.86 0.55 0.00 0.00 0.01 0.04 PE (P-18:1/20:5)(a) 578 2242 2646 535 1760 1569 0.00 0.00 0.56 0.27 0.01 0.00 0.01 0.19 LPC (20:5 sn1) 457 2036 2505 439 1823 2111 0.00 0.00 0.94 0.38 0.12 0.00 0.14 0.55 PC (18:2–20:5) 320 1186 1544 347 1078 1124 0.00 0.00 0.86 0.46 0.01 0.00 0.10 0.11 PC (P-038:5)(b) 285 504 707 290 499 498 0.00 0.00 0.92 0.92 0.00 0.00 0.05 0.01 PE (16:0–20:5) 165 457 457 165 465 435 0.00 0.00 1.00 0.89 0.67 0.00 0.85 0.92 LPC (20:5 sn2) 148 648 799 147 594 681 0.00 0.00 1.00 0.44 0.11 0.00 0.12 0.50 PE (O-38:5)(b) 141 571 686 131 301 229 0.00 0.02 0.94 0.00 0.00 0.00 0.00 0.00 PE (P-18:1–20:5)(b) 107 324 425 100 297 308 0.00 0.00 0.84 0.47 0.00 0.00 0.03 0.09 Notes: Values are expressed as the mean of plasma EPA (20:5) molecular species concentration (n = 11). Species shaded in bold significantly different between KO and FO at day 30. Two-way analysis of variance for repeated measurements was performed to analyze supplementation effect over time (interaction time × supplementation), the difference between time points within supplementation, and each time point between the two omega-3 supplementation groups. All p values were corrected for multiple comparisons using the Benjamini–Hochberg FDR. Abbreviations: EPA, eicosapentaenoic acid; LPC, lyso-phosphatidylcholine; PC, phosphatidylcholine; PC(O), alkylphosphatidylcholine; PC(P), alkenylphosphatidylcholine; PE, phosphatidylethanolamine; PE(O), alkyl-phosphatidylethanolamine; PE(P), alkenyl-phosphatidylethanolamine; T0, baseline; T15, 15 days; T30, 30 days.

Incorporation of EPA (20:5) into nine molecular species with initial concentrations exceeding 500 pmol/ml following the krill oil (KO, dashed lines) or fish oil (FO, solid line) supplementation over 30 days. Values are expressed as mean ± SEM (pmol/ml) (n = 11). Two-way analysis of variance for repeated measurements was performed to analyze the difference between each time point between the two omega-3 supplementation groups. The asterisks (*,**) in graphs indicate significant differences (p ≤ 0.05, p ≤ 0.01, respectively) between the KO and FO supplementation groups. EPA, eicosapentaenoic acid; PC, phosphatidylcholine; PC(O), alkylphosphatidylcholine; PC(P), alkenylphosphatidylcholine; PE(O), alkylphosphatidylethanolamine; PE(P), alkenylphosphatidylethanolamine

DHA molecular species

A total of 38 DHA-containing phospholipid molecular species were detected in the concentration range of 133–86,864 pmol/ml, as shown in Table 2. The majority of these showed significant increases in concentration at day 30 after both KO supplementation (34/38 increased) and FO supplementation (28/38 increased).

TABLE 2. Lipidomic changes in plasma DHA (22:6) phospholipid molecular species over the 30-day krill oil and fish oil supplementation DHA (22:6) molecular species Concentration (pmol/ml) p value Krill oil Fish oil Krill oil Fish oil Krill oil vs. fish oil Time Supplement Interaction T = 0 T = 15 T = 30 T = 0 T = 15 T = 30 T0:T30 T0:T30 T = 0 T = 15 T = 30 PC (16:0/22:6) 56,421 74,143 84,089 51,481 83,726 86,864 0.00 0.00 0.24 0.03 0.50 0.00 0.42 0.06 PC (18:0/22:6) 11,335 16,397 18,919 11,228 17,858 19,596 0.00 0.00 0.92 0.17 0.52 0.00 0.13 0.57 PE (16:0/22:6) 6043 6148 5208 5100 6575 6346 0.15 0.04 0.11 0.45 0.05 0.20 0.65 0.05 PC (38:6)(a) 5011 8187 9645 5233 7741 7782 0.00 0.00 0.70 0.44 0.00 0.00 0.06 0.05 PE (P-16:0/22:6) 3271 5059 5852 3222 4296 3874 0.00 0.08 0.89 0.04 0.00 0.00 0.00 0.00 PC (18:1/22:6)(a) 3245 3698 3874 3184 3589 4083 0.01 0.00 0.76 0.59 0.31 0.00 0.93 0.49 PE (18:0/22:6) 2610 2975 2803 2351 3187 3111 0.43 0.00 0.29 0.38 0.21 0.02 0.57 0.22 PE (P-18:0/22:6) 2377 3033 3973 2439 3329 3000 0.00 0.07