BIOAVAILABILITY OF LONG CHAIN OMEGA-3 POLYUNSATURATED FATTY ACIDS FROM PHOSPHOLIPIDRICH HERRING ROE OIL IN MEN AND WOMEN WITH MILDLY ELEVATED TRIACYLGLYCEROLS Alvin Berger, MS, Ph. D, Prof. June 2016

Official Publication title: Cook CM, Hallaråker H, Sæbø P-C, Innis SM, Kelley KM, Sanoshy KD, Berger A, Maki KC. 2016. Bioavailability of long chain omega-3 polyunsaturated fatty acids from phospholipid-rich herring roe oil in men and women with mildly elevated triacylglycerols. PLEFA 10.1016/j.plefa.2016.01.007

June, 2016 Arctic Nutrition | www.arcticnutrition.no/

1

TABLE OF CONTENTS EXECUTIVE SUMMARY ...................................................................................... 3 PURPOSE ............................................................................................................ 3 BACKGROUND .................................................................................................... 3 EXPERIMENTAL DESIGN ................................................................................... 5 KEY FINDINGS..................................................................................................... 5 CONCLUSIONS.................................................................................................... 8 FURTHER WORK................................................................................................. 8 REFERENCES ..................................................................................................... 9 FOR MORE INFORMATION .............................................................................. 11

June, 2016 Arctic Nutrition | www.arcticnutrition.no/

2

EXECUTIVE SUMMARY We previously demonstrated in a Norwegian student population (with high basal levels of Omega 3 intake) that Arctic Nutrition’s MOPL30 product containing DHA: EPA in a 3:1 ratio, partly bound to phospholipids, increased levels of EPA and DHA dramatically versus baseline, in only two weeks [1]. We then demonstrated in a rat study, MOPL30 was rapidly and efficiently taken up by brain, showing the product to be bioavailable and bioaccretable (taken up by tissues) [2]. To expand on these previous results, herein, we evaluated equivalent doses of EPA and DHA as present in MOPL30 or fish oil, in a population with mildly elevated triglycerides. Acute bioavailability (measured as the net incremental area under the curve from 0 to 12h for EPA, DHA, and EPA+DHA in plasma phosphatidylcholine-PC) was significantly higher after MOPL30 versus fish oil (Figure 1). Under our specific experimental conditions, after 2-weeks, both groups equivalently elevated EPA + DHA in PC by 3-fold. Taken together, results from the three studies provides strong evidence MOPL30 is both a highly bioavailable and bioaccretable source of EPA and DHA. The more rapid uptake of EPA and DHA into plasma with MOPL30 versus fish oil, offers specific clinical benefits.

PURPOSE To understand if Arctic Nutrition’s MOPL30 immature Spring herring roe-derived, DHA-, EPA- and choline-rich lipid supplements will be better absorbed (bioavailable; on the basis of Omega 3 polyunsaturated fatty acid [PUFA] incorporation into plasma PC); and could improve blood lipid profiles (important for heart health, obesity, and diabetes), in a population with mildly elevated triglycerides, in comparison to a fish oil, matched for EPA and DHA content.

BACKGROUND Overall benefits of Omega 3s: The health benefits of a higher fish intake containing Omega 3 long-chain PUFAs have been documented in thousands of studies [3]. Heart health benefits of Omega 3 PUFAs, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been attributed to: reductions in fasting triacylglycerol (TAG; or triglyceride); blood pressure lowering; anti-inflammatory and antiarrhythmic (restoring irregular heart beat) benefits; improved insulin sensitivity and vascular endothelial function; and reduced thrombotic (clotting) tendency [4]. Omega 3 PUFAs also benefit cognition, metabolic syndrome, and diabetes, amongst other conditions. DHA is a major structural and signaling molecule in the brain, most touted for its roles in

June, 2016 Arctic Nutrition | www.arcticnutrition.no/

3

cognition and visual function; whereas EPA is most known for its antiinflammatory benefits. New sources of Omega 3s are needed to meet increasing demand: Intake of Omega 3 PUFAs is very low in many countries, which is detrimental to our health and an economic burden for health care systems. As the global intake of Omega 3 PUFAs increases in response to recommendations from numerous health authorities to increase intake, new sources of Omega 3 PUFA are needed, preferably from a source that is potent, sustainable and underutilized. Introducing MOPL30: Immature roe from Spring-spawning Norwegian herring represents such an underutilized source of Omega 3 PUFAs to meet increasing needs. Of the approximately 600,000 tons of herring caught in Norway annually, only a small percentage of herring roe is used for human consumption. Arctic Nutrition’s herring roe product MOPL™30 (also marketed as Romega™ 30) is prepared from this immature herring roe, and has a DHA: EPA ratio of about 3:1. Bioavailability benefit of marine phospholipids: Studies have shown that marine phospholipids (PLs), including krill oil sources, may improve bioavailability of Omega 3 PUFAs, and have additional functional benefits relative to the more common TAG forms of Omega 3s in both animal- and human clinical studies [5-9]. A higher bioavailability could potentially justify a lower intake, and offer improved health benefits. Benefits of marine phospholipids beyond bioavailability: PL forms of Omega 3s are particularly beneficial for psoriasis [5] and gut disease [10], and improving other conditions involving inflammation and immune regulation [11-13]. PL forms of Omega 3s can also affect gene transcripts related to glucose metabolism (with implications for diabetes) more than other forms of Omega 3s [14, 15]; and can inhibit the growth of cancer cells in vitro [16]. Choline is a component of a major PL, known as phosphatidylcholine (PC). Choline is a key nutrient in itself, with vital roles in proper liver function and cognition; it is often under consumed [17]. Marine phospholipids (such as MOPL30) provide choline and increase plasma choline in clinical trials [1, 18]. Krill oil is a beneficial marine phospholipid, but differs structurally from MOPL: Most of the research on marine phospholipids has focused on krill oil. Krill oil has similarities to MOPL30, but EPA predominates over DHA in krill oil (ratio of EPA: DHA is about 2:1, versus a 1:3 ratio in MOPL30), and there are other technical differences. Krill provides the antioxidant astaxanthin, which can be a confounding variable in clinical trials. Another confounding variable with krill oil is the potentially high levels of free fatty acids which positively affects bioavailability and bioaccretion of EPA and DHA [19]. Last, many of the studies with krill oil are confounded by: different doses and ratios of EPA: DHA administered within- and between studies; different lipid pools assessed in different studies; and different June, 2016 Arctic Nutrition | www.arcticnutrition.no/

4

durations of the study [20, 21]. The EPA and DHA in krill oil may be more bioavailable than in fish oils even when doses of EPA and DHA and astaxathin content are matched [9, 22]; but in some studies this increased bioavailability with krill oil over fish oil is not observed [20, 23]. Overall, extreme caution is needed in comparing studies with MOPL30 to krill oil with respect to bioavailability, bioaccretion, and effects on physiological variables for the aforementioned reasons. Benefits observed previously with MOPL30 in humans: We previously demonstrated in healthy young Norwegian students, that after only 2 weeks consumption of MOPL30 containing DHA: EPA in a 3:1 ratio, partly bound to phospholipids, levels of EPA and DHA were dramatically increased, triglycerides decreased, HDL-cholesterol increased, and choline and betaine increased (each gram of MOPL30 provides 42 mg of free choline equivalents) relative to baseline [1]. MOPL30 was thus bioavailable and potentially taken up by tissues, and this could benefit many conditions linked to EPA, DHA, and phospholipid/choline status. This includes heart health, liver health, gut health, skin inflammation, obesity, and diabetes [2]. With respect to skin inflammation, MOPL30, or molecules very similar to MOPL30, have been shown to benefit psoriasis, likely by possessing EPA and DHA but also structural components of the PC molecule, to repair damaged barrier function in cells and tissues [5, 24]. Benefits observed previously with MOPL30 in animals: Herring roe phospholipids have been demonstrated in animal studies to improve plasma lipid profiles and inflammatory parameters; and improve insulin sensitivity [25, 26]. The EPA and DHA in our specific herring roe product MOPL30 was found to be rapidly and efficiently taken up by rat brain regions, demonstrating bioaccretion (tissue uptake) in this critical organ. [2].

EXPERIMENTAL DESIGN Detailed Product description: MOPL30 oil is extracted from Norwegian, Springspawning, herring immature roe, mixed 50/50 with fish oil TAG to adjust viscosity, that overall contains 45% Omega 3 PUFA (sum EPA+n3 DPA+DHA, g/100g product basis) with a DHA: EPA ratio of approximately 3:1. Approximately 30% of EPA and DHA in product are esterified to PL (mainly PC); the rest is esterified to TAG. 32% of total lipids are PL, 89% are PC with minor amounts lysoPC. Experimental Design: We used a randomized, single-dose, single-blind, crossover, active-reference, two-week clinical trial to assess bioavailability of EPA, DHA, and n-3 docosapentaenoic acid (DPA). Lipid measurement were made after a single serving of all 12 capsules at 12 h, and after two weeks daily supplementation (four capules consumed at each of three meals). Thirty-two adults with fasting TAG of 100-399 mg/dL were stratified by sex and age, and June, 2016 Arctic Nutrition | www.arcticnutrition.no/

5

randomly assigned to receive 12 capsules/day containing MOPL30 (628 mg EPA; 1810 mg DHA; 137 mg/day DPA) or TAG-rich fish oil (575 mg EPA; 1843 mg DHA; 259 mg/day DPA) each for a 2-week period separated by a 4-week washout between arms. Relatively large amounts of capsules were used for analytical reasons.

KEY FINDINGS 











Neither supplement, even at the relatively high dose employed, caused adverse gastrointestinal symptoms. Nor were there changes in body weight, blood pressures or heart rate. The lack of reported “fish burps” is important as physician’s report their patients frequently stop consuming fish oil capsules due to burping. Acute bioavailability (assessed as net incremental area under the curve from 0 to 12 h for EPA, DHA, and EPA+DHA in plasma PC; with or without normalization to EPA + DHA intake) were higher after MOPL30 versus fish oil (Figure 1). This suggests a high rate of beneficial absorption for subsequent tissue distribution and tissue uptake of these key fatty acids. After 2-weeks, fasting plasma PC EPA+ DHA (and EPA and DHA separately) were equivalently elevated about 3-fold relative to baseline for both oils. This lack of difference could relate to: the specific population studied; the high dose used; the specific lipid pool examined; or the short 2-week study duration. Another possibility is that more MOPL30 could be taken up into tissues, or at a different rate, relative to fish oil, leaving less EPA and DHA to be measured in plasma [2]. Both supplements equivalently decreased TAG and equivalently increased HDL-C (cholesterol) and LDL-C after 2 weeks. These changes to TAG and HDL-C are highly beneficial for heart health, diabetes, and metabolic syndrome/insulin resistance; and have been reported in many other fish oil and marine phospholipid studies, including those with DHA predominating over EPA, as in MOPL30 [27, 28]. Why did the supplements increase LDL-C? Fish oils, particularly their DHA and DPA components, may increase LDL-C and possibly HDL-C via postulated mechanisms: 1) lowering circulating Apo C-III inhibits lipoprotein lipase, subsequently lowering plasma TAG and increasing VLDL to LDL conversion, thereby increasing LDL-C; 2) the reduction in TAG reduces exchange of TAG for cholesterol ester (via cholesteryl ester transfer protein), having the net effect of increasing HDL-C, but also LDLC [29]. The increase in LDL-C could be more pronounced in populations with elevated TAG, as no increase occurred with a similar MOPL30 dose in young, normotriglyceridemic students [1]. Are the observed increases in LDL-C a concern? No, fish oils are well established to have a cardio-protective benefit, even if they may increase

June, 2016 Arctic Nutrition | www.arcticnutrition.no/

6





(large-sized) LDL-C [30, 31]. Moreover, it is not LDL-C per se that contributes to heart disease, but small-dense sub-particles of LDL (sdLDL) and oxidized LDL (oxLDL) [32], neither of which were measured herein. However, in a recent study with the marine phospholipid krill oil [18], it was similarly reported that LDL-C increased, but the increase came from large LDL particles and not the damaging sdLDL particles [30, 31]. Total-C and non-HDL-C were increased more with MOPL30 than fish oil. It is expected that these parameters would increase since both HDL-C and (likely large) LDL-C particles were increased. Differences in plasma totalC would not relate to the small amount of cholesterol present in MOPL30 (≈10 mg cholesterol/single MOPL30 capsule containing 356 mg oil). By comparison, there are 375 mg cholesterol in two chicken eggs. Total-C/HDL-C was lowered only with the fish oil group and unchanged with MOPL30. This marker is a robust coronary risk parameter in European populations [33] and the lowering with fish oil is desirable.

Figure 1: Over a 12-hour period (4-12 hours after administration), the bioavailability of EPA, DHA, n3 DPA (latter not shown) was higher when these fatty acids were administered partly bound to phospholipids (in MOPL 30 or Romega 30) than when they were bound as triacylgycerol (in fish oil). The amounts of these fatty acids in MOPL30 were normalized to the amounts in fish oil. Statistical significance is indicated by asterisks. This higher acute amount of fatty acids in the plasma with MOPL30, could translate to improved tissue distribution and tissue uptake of these key fatty acids, thus improving some physiological outcomes.

June, 2016 Arctic Nutrition | www.arcticnutrition.no/

7

CONCLUSIONS 



MOPL30 is a well-tolerated, bioavailable, and bioaccretable source of EPA, DHA, and DPA, which outperforms a conventional fish oil matched for EPA and DHA content, in terms of rapid delivery of these fatty acids to the plasma. Over a 2-week period, the two sources were largely equivalent in terms of EPA and DHA levels, and cholesterol parameters assessed.

FURTHER WORK Levels of lysoPC enriched with EPA and DHA (derived endogenously from the PC and other lipids in MOPL30) could be assessed in the future, as this unique lipid source may be a special delivery vehicle for providing DHA to the brain [19, 3439]. It is also highly interesting to know the levels of EPA and DHA in adipose tissue biopsies after consuming MOPL30, since these fatty acids may be deposited in adipose tissue for subsequent uptake by the brain and other tissues [33]. It would be desirable to perform lipoprotein sub-class analysis to better understand the changes to LDL-C and HDL-C observed herein. Last, additional sub-populations, such as diabetics, and obese subjects could be examined in the future, as the decrease in TAG, increase in HDL-C, and favorable increases in EPA and DHA would be quite desirable for these populations. Persons suffering with inflammation, where there are also barrier function perturbations, as in psoriasis and ulcerative colitis, would also particularly benefit from MOPL30. As MOPL30 is oxidatively-stable relative to other fish oils, it may be possible to apply MOPL30 in penetrating topical formulations. Our acute data suggests MOPL30 is rapidly absorbed and equilibrated into plasma PC pools. Thus, MOPL30 could be used to rapidly provide EPA and DHA in clinical conditions such as brain trauma and concussion.

June, 2016 Arctic Nutrition | www.arcticnutrition.no/

8

REFERENCES 1. Bjorndal, B., et al., Phospholipids from herring roe improve plasma lipids and glucose tolerance in healthy, young adults. Lipids Health Dis, 2014. 13: p. 82. 2. Kitson, A.P., et al., Effect of dietary docosahexaenoic acid (DHA) in phospholipids or triglycerides on brain DHA uptake and accretion. J Nutr Biochem, 2016. 33: p. 91-102. 3. Mozaffarian, D. and E.B. Rimm, Fish intake, contaminants, and human health: evaluating the risks and the benefits. JAMA, 2006. 296(15): p. 188599. 4. Harris, W.S., T.D. Dayspring, and T.J. Moran, Omega-3 fatty acids and cardiovascular disease: new developments and applications. Postgrad Med, 2013. 125(6): p. 100-13. 5. Dupont, P., Traitement du psoriasis par la lécithine marine. Phytothérapie, 2006. 1(April): p. 15-22. 6. Burri, L., et al., Marine omega-3 phospholipids: metabolism and biological activities. Int J Mol Sci, 2012. 13(11): p. 15401-19. 7. Davidson, M.H., et al., A novel omega-3 free fatty acid formulation has dramatically improved bioavailability during a low-fat diet compared with omega-3-acid ethyl esters: The ECLIPSE (Epanova((R)) compared to Lovaza((R)) in a pharmacokinetic single-dose evaluation) study. J Clin Lipidol, 2012. 6(6): p. 573-84. 8. Ferramosca, A., L. Conte, and V. Zara, A krill oil supplemented diet reduces the activities of the mitochondrial tricarboxylate carrier and of the cytosolic lipogenic enzymes in rats. J Anim Physiol Anim Nutr (Berl), 2012. 96(2): p. 295-306. 9. Ramprasath, V., et al., Enhanced increase of omega-3 index in healthy individuals with response to 4-week n-3 fatty acid supplementation from krill oil versus fish oil. Lipids Health Dis, 2013. 12: p. 178 doi:10.1186/1476-511X12-178. 10. Stremmel, W., et al., Phosphatidylcholine (lecithin) and the mucus layer: Evidence of therapeutic efficacy in ulcerative colitis? Dig Dis, 2010. 28(3): p. 490-6. 11. Da Boit, M., et al., The Effect of Krill Oil Supplementation on Exercise Performance and Markers of Immune Function. PLoS One, 2015. 10(9): p. e0139174. 12. Skorve, J., et al., Fish oil and krill oil differentially modify the liver and brain lipidome when fed to mice. Lipids Health Dis, 2015. 14: p. 88. 13. Costanzo, M., et al., Krill oil reduces intestinal inflammation by improving epithelial integrity and impairing adherent-invasive Escherichia coli pathogenicity. Dig Liver Dis, 2016. 48(1): p. 34-42. 14. Vigerust, N.F., et al., Krill oil versus fish oil in modulation of inflammation and lipid metabolism in mice transgenic for TNF-alpha. Eur J Nutr, 2012. 15. Burri, L. and L. Johnsen, Krill products: an overview of animal studies. Nutrients, 2015. 7(5): p. 3300-21. June, 2016 Arctic Nutrition | www.arcticnutrition.no/

9

16. Xiao, S.U., et al., The Effect of Krill Oil and n-3 Polyunsaturated Fatty Acids on Human Osteosarcoma Cell Proliferation and Migration. Curr Drug Targets, 2015. 17. Mehedint, M.G. and S.H. Zeisel, Choline's role in maintaining liver function: new evidence for epigenetic mechanisms. Curr Opin Clin Nutr Metab Care, 2013. 16(3): p. 339-45. 18. Berge, R.K., et al., Krill oil reduces plasma triacylglycerol level and improves related lipoprotein particle concentration, fatty acid composition and redox status in healthy young adults - a pilot study. Lipids Health Dis, 2015. 14: p. 163. 19. Chen, C.T., et al., Plasma non-esterified docosahexaenoic acid is the major pool supplying the brain. Sci Rep, 2015. 5: p. 15791. 20. Salem, N., Jr. and C.N. Kuratko, A reexamination of krill oil bioavailability studies. Lipids Health Dis, 2014. 13: p. 137. 21. Ulven, S.M. and K.B. Holven, Comparison of bioavailability of krill oil versus fish oil and health effect. Vasc Health Risk Manag, 2015. 11: p. 511-24. 22. Kohler, A., et al., Bioavailability of fatty acids from krill oil, krill meal and fish oil in healthy subjects--a randomized, single-dose, cross-over trial. Lipids Health Dis, 2015. 14: p. 19. 23. Yurko-Mauro, K., et al., Similar eicosapentaenoic acid and docosahexaenoic acid plasma levels achieved with fish oil or krill oil in a randomized doubleblind four-week bioavailability study. Lipids Health Dis, 2015. 14: p. 99. 24. Sgarbi, C.R., R. Villa, and M.B.L. Franco, Oral supplementation of Omega-3 as an adjunct in the treatment of psoriasis. J Invest Dermatol, 2012. 132: p. SS62. doi:10.1038/jid.2012.298 http://www.nature.com/jid/journal/v132/n2s/pdf/jid2012298a.pdf 42nd Annual European Society for Dermatological Research (ESDR) Meeting, Venice, Italy, 19-22 September 2012 http://www.esdr2012.org/. 25. Higuchi, T., N. Shirai, and H. Suzuki, Effects of dietary herring roe lipids on plasma lipid, glucose, insulin, and adiponectin concentrations in mice. J Agric Food Chem, 2006. 54(10): p. 3750-5. 26. Bjorndal, B., et al., Dietary supplementation of herring roe and milt enhances hepatic fatty acid catabolism in female mice transgenic for hTNFalpha. Eur J Nutr, 2012. 51(6): p. 741-53. 27. Calabresi, L., et al., An omega-3 polyunsaturated fatty acid concentrate increases plasma high-density lipoprotein 2 cholesterol and paraoxonase levels in patients with familial combined hyperlipidemia. Metabolism, 2004. 53(2): p. 153-8. 28. Maki, K.C., et al., Effects of prescription omega-3-acid ethyl esters on fasting lipid profile in subjects with primary hypercholesterolemia. J Cardiovasc Pharmacol, 2011. 57(4): p. 489-94. 29. Browning, L.M., et al., Incorporation of eicosapentaenoic and docosahexaenoic acids into lipid pools when given as supplements providing doses equivalent to typical intakes of oily fish. Am J Clin Nutr, 2012. 96(4): p. 748-58. June, 2016 Arctic Nutrition | www.arcticnutrition.no/

10

30. Kelley, D.S., et al., Docosahexaenoic acid supplementation improves fasting and postprandial lipid profiles in hypertriglyceridemic men. Am J Clin Nutr, 2007. 86(2): p. 324-33. 31. Neff, L.M., et al., Algal docosahexaenoic acid affects plasma lipoprotein particle size distribution in overweight and obese adults. J Nutr, 2011. 141(2): p. 207-13. 32. Mason, R.P., S.C. Sherratt, and R.F. Jacob, Eicosapentaenoic Acid Inhibits Oxidation of ApoB-containing Lipoprotein Particles of Different Size In Vitro When Administered Alone or in Combination with Atorvastatin Active Metabolite Compared With Other Triglyceride-Lowering Agents. J Cardiovasc Pharmacol, 2016. 33. Bazinet, R.P. and S. Laye, Polyunsaturated fatty acids and their metabolites in brain function and disease. Nat Rev Neurosci, 2014. 15(12): p. 771-85. 34. Chen, S. and P.V. Subbaiah, Regioisomers of phosphatidylcholine containing DHA and their potential to deliver DHA to the brain: role of phospholipase specificities. Lipids, 2013. 48(7): p. 675-86. 35. Liu, L., et al., Higher efficacy of dietary DHA provided as a phospholipid than as a triglyceride for brain DHA accretion in neonatal piglets. J Lipid Res, 2014. 55(3): p. 531-9. 36. Alakbarzade, V., et al., A partially inactivating mutation in the sodiumdependent lysophosphatidylcholine transporter MFSD2A causes a non-lethal microcephaly syndrome. Nat Genet, 2015. 47(7): p. 814-7. 37. Guemez-Gamboa, A., et al., Inactivating mutations in MFSD2A, required for omega-3 fatty acid transport in brain, cause a lethal microcephaly syndrome. Nat Genet, 2015. 47(7): p. 809-13. 38. Quek, D.Q., et al., Structural Insights into the Transport Mechanism of the Human Sodium-dependent Lysophosphatidylcholine Transporter MFSD2A. J Biol Chem, 2016. 291(18): p. 9383-94. 39. Wong, B.H., et al., Mfsd2a Is a Transporter for the Essential omega-3 Fatty Acid Docosahexaenoic Acid (DHA) in Eye and Is Important for Photoreceptor Cell Development. J Biol Chem, 2016. 291(20): p. 10501-14.

FOR MORE INFORMATION For more information, please contact [email protected] Copyright Information ©2016 Arctic Nutrition, All Rights Reserved.

June, 2016 Arctic Nutrition | www.arcticnutrition.no/

11