Sung, Hyunsin (2017) The effect of krill oil supplementation focusing on the incorporation of plasma omega-3 polyunsaturated fatty acids, clinical biomarkers and lipidomic profiles in women. PhD thesis, Victoria University.

Abstract

Circulating lipids play an important role in human physiology and pathophysiology. Lipids, as the major components in various cellular membranes, are involved in homeostatic regulation, particularly in relation to immune function and inflammatory mechanisms. With the growing global prevalence of lifestyle-related diseases, including obesity, diabetes, cardiovascular disorders and cancers, dietary lipids have received a great attention. Long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) have been associated with a broad range of health benefits. The three main LC n-3 PUFA are eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and docosapentaenoic acids (DPA, 22:5n-3). Fish oil and krill oil are currently the most available sources of EPA and DHA as over-the-counter supplements, although other marine sources such as algae oil are also rich in EPA and DHA. Krill oil, derived from Antarctic krill (Euphausia Superba), is rich in EPA and DHA found in phospholipids (predominantly phosphatidylcholine) rather than triacylglycerol (TAG), in which EPA and DHA in fish oil are found. Krill oil also contains astaxanthin, a carotenoid contributing to its red colour which may also have beneficial health effects (Barros et al. 2014a, Pashkow et al. 2008). Despite a number of studies examining the effects of krill oil compared with fish oil on the incorporation of LC n-3 PUFA into different tissues, the outcomes have been conflicting, which might be associated with the different study designs using different chemical forms of fish oil and/or different doses of LC n-3 PUFA, and focusing at different target tissues. The research presented in this thesis consists of nine chapters covering a literature review (Chapter two) and two intervention studies in humans (Chapters four, five, six and seven) which have examined the effect of krill oil compared with fish oil on the incorporation of LC n-3 PUFA into plasma lipid fractions. There were a postprandial and a longer-term (30 days) intervention studies, and both clinical studies were randomised crossover designs involved healthy women (n = 10 and n = 11, respectively). All participants were instructed to maintain the habitual dietary intake and habitual physical activity throughout the interventions. The aim of the postprandial study was to compare the incorporation of LC n-3 PUFA into the plasma and circulating lipids in plasma and chylomicron fractions from five capsules (1 g each) of krill oil compared with five capsules (1g each) of fish oil and 5 g of the olive oil (control) over a 5-hour postprandial period. The second study aimed to investigate the longer-term effect of krill oil supplementation (containing 1,269 mg/d of LC n-3 PUFA including EPA, DHA and DPA) on the plasma LC n-3 PUFA, plasma circulating TAG and inflammatory biomarkers compared with fish oil supplementation (containing the closest possible match to these fatty acids from the capsules, 1,441 mg/d) over a 30-day intervention period. In both studies, lipidomics, was applied to identify the differences in plasma lipid molecular responses between krill oil and fish oil supplementation. Using this technique, a number of plasma lipid classes, and lipid molecular species containing EPA and DHA were identified and quantified. In the 5-hour postprandial study (Chapters four and five), there were no significant differences in the levels of TAG or cholesterol in plasma or chylomicron between the three study oil interventions, although the expected increases in chylomicron TAG were observed in all groups. In comparison to the olive oil, both krill oil (containing 907 mg of LC n-3 PUFA) and fish oil (containing 1,441 mg of LC n-3 PUFA) supplementation significantly increased the level of plasma EPA, which plateaued after three hours; there were no significant differences in the plasma EPA levels between krill oil and fish oil supplementation groups. There were no significant changes in either DHA or DPA between the three groups. Krill oil, with a lower dose of EPA in this study, showed a similar incorporation outcome of EPA into plasma lipids as fish oil. Given that there were 31% less EPA from krill oil, these results indicate a differential extent of incorporation of EPA between krill oil and fish oil, suggesting that EPA from krill oil may be more efficiently incorporated into the plasma than fish oil. The advanced technique for lipidomics was performed by high-performance liquid chromatography-mass spectrometer analysis (HPLC MS/MS), which was able to identify and quantify changes in various lipid molecular species containing LC n-3 PUFA in both the postprandial and the longer-term studies. Therefore, the HPLC MS/MS facilitated a comparison between differences in the individual lipid molecular species between krill oil and fish oil supplementation. A more sensitive setting of HPLC MS/MS was applied to the postprandial data than the longer-term data, based on the settings applied by the research laboratory at Baker Heart and Diabetes Institute where these analyses were conducted. In Chapter five, the postprandial plasma lipidomic changes are reported at hours zero (baseline), 3 and 5. A total of 29 lipid classes (≥ 500 pmol/mL) (for example: TAG, diacylglycerol (DAG), phosphatidylcholine (PC), cholesterol esther (CE)) were identified; six of these including O-linked phosphatidylethanolamine classes had significantly greater the incremental area under the curve from baseline (net iAUC 0-5 h) after krill oil supplementation compared with fish oil supplementation. Over the postprandial period, 56 EPA-containing and 76 DHA-containing molecular species (for example 16:0-20:5-PC, 16:0-18:1-20:5-TAG, 16:0-22:6-PC, 16:0-18:1-22:6-TAG) were significantly increased after both krill oil and fish oil supplementation. There were 33 phospholipid molecular species containing EPA, and 16 of these molecular species, including six ether-phospholipid molecular species had significantly greater increased net iAUC 0-5 h after krill oil than fish oil supplementation. In contrast, for TAG and DAG molecular species containing EPA, seven out of a total of 21 showed significantly increased net iAUC 0-5 h for fish oil compared with krill oil. Put simply, the EPA from krill oil was associated with increases in phospholipid EPA-molecular species, while the EPA from fish oil was associated with increased TAG and DAG EPA-molecular species. There were 49 phospholipid molecular species containing DHA, and 11 of these including six ether-phospholipid molecular species, had significantly greater increased net iAUC 0-5 h after krill oil supplementatin than fish oil supplementation. In a total of 61 AA-containing molecular species (for example 16:0-20:4-PC, 16:1-20:4-DAG) identified, there were 51 phospholipid molecular species containing AA, and seven of these including six ether-phospholipid molecular species, had significantly greater increased net iAUC 0-5 h after krill oil supplementation than fish oil. A novel finding from this postprandial study was that there was a consistent trend that ether-phospholipid classes (O-linked (containing an alkyl bond) or P-linked (containing an alkenyl bond) phosphatidylcholine and phosphatidylethanolamine) were significantly increased after krill oil supplementation, but decreased after fish oil supplementation. Consistently, it was found that EPA- and DHA-containing ether-phosphatidylethanolamines were significantly increased after the krill oil supplementation, but decreased after the fish oil supplementation. While the significance of this finding is not clear, it is worth noting that plasma levels of O- and P-linked phosphatidylethanolamine have been reported to be decreased in a number of disease states including Alzheimer’s disease. Little is known about the origin of these ether-phospholipids in plasma, but the fact that krill oil increased their post-prandial levels and fish oil decreased them is a clear differentiation between these two omega-3 oils. In the longer-term study (Chapters six and seven), EPA, DHA and DPA were significantly increased after both krill oil and fish oil supplementation over the 30-day period (p < 0.001). The main response to the 30-day krill oil supplementation was that the increase of plasma EPA level was significantly greater in the net iAUC 0-30 d than that of fish oil supplementation (p < 0.05). Both krill oil and fish oil significantly reduced plasma TAG over the intervention period (p < 0.05 and p < 0.01, respectively), but no significant differences were observed between the two groups. Over the 30-day intervention period, some plasma pro-inflammatory cytokines including IL-1β, IL-10, IL-4 and IL-5 (p ≤ 0.05) were significantly reduced after krill oil supplementation, while no such changes were found after fish oil supplementation. In Chapter seven, the long-term lipidomic changes (≥ 500 pmol/mL), at days zero (baseline), 15 and 30, are reported. Twenty three EPA-containing and 46 DHA-containing molecular species were significantly increased after both krill oil and fish oil supplementation over the 30-day supplementation period. Among EPA-, DHA-, and DPA-containing molecular species, there were 20 cases of net iAUC 0-30 d significant differences between the two supplementation. Fourteen of these molecular species in phospholipid species, including 12 ether-phospholipid species, had significantly greater increased net iAUC 0-30 d after krill oil than fish oil (p ≤ 0.05) supplementation. Consistently, it was found that EPA- and DHA-containing ether-phospholipid species, including six ether-phosphatidylethanolamines, were significantly increased after the krill oil supplementation, and decreased after the fish oil supplementation. The changes in the ether-phospholipids in the long-term trial were consistent with the changes described in the postprandial trial (Chapter five). These results support strongly the differentiation between krill oil and fish oil although there are still many unanswered questions flowing from this novel finding. What is known about plasmalogens is that they play a role in anti-inflammatory response, which might be linked to the significant decrease in pro-inflammatory cytokines observed in the present study. Overall, both postprandial and longer-term studies demonstrated that EPA from krill oil is efficiently incorporated into plasma, has a similar effect on the plasma TAG-lowering and a greater efficacy on the plasma inflammatory biomarkers when compared with fish oil. No previous studies have investigated plasma lipidomic responses to krill oil and fish oil supplementation in humans. There were significant increases in molecular species containing EPA and DHA following supplementation with krill oil and fish oil over both the postprandial and the longer-term periods. The plasma lipidomic changes of net iAUC over both intervention periods were significantly different between krill oil and fish oil supplementation, particularly for phospholipids (krill oil resulted in a greater increase than fish oil) and TAG (fish oil resulted in a greater increase than krill oil, as described in Chapter five). A novel aspect identified in this study was that krill oil increased ether-phospholipids, particularly ether-linked phosphatidylethanolamine, whereas fish oil decreased ether-phospholipids. The biological relevance of this novel lipidomic finding has yet to be fully explored.

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