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Did You Know?...The Research Behind Fish Oil Form - EE vs. TG vs. PL

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Summary
The positive effects of long-chain omega-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), found in fish, krill and algae oils for cardiovascular, retinal, and neurological health are substantiated by human clinical trials. Therefore, EPA and DHA are an integral dietary supplement utilized in clinical practice. However, various marketing strategies have resulted in much confusion and misinformation surrounding the effects of molecular form of EPA and DHA provided by commercially available fish oil and krill oil supplements on oral bioavailability, and patient health and safety, overshadowing clinical results from human randomized controlled trials (RCT). Dietary supplements provide omega-3 EPA and DHA as either ethyl ester (EE) or triglyceride (TG) forms from fish oil, or the phospholipid (PL) form from krill oil. While a single-dose 72-hour kinetics study suggests increased bioavailability of PL>TG>EE (1), the physiological and clinical benefits of EPA and DHA are based on chronic consumption. Recently, similar plasma and red blood cell (RBC) levels of EPA + DHA were achieved with EE fish oil, TG fish oil and PL krill oil products when matched for dose and EPA + DHA content over 4-weeks of supplementation, indicating comparable oral bioavailability irrespective of supplement form (2). Currently, the vast majority of human RCT substantiating the clinical efficacy and health benefits of fish oil have used EE fish oil supplements. When considering an omega-3 fatty acid supplement for patient health and safety, total EPA and DHA content, quality, purity and stability should be of utmost importance. Nevertheless, this article will strive to add some clarity to this continuously debated topic of TG vs. EE vs. PL omega-3 fatty acid dietary supplement form. In summary, at this time it is not substantiated by human clinical research that bioavailability of the TG form is more efficient or clinically superior to the EE form.

The Molecular Forms
Triglyceride (TG)
Triglyceride (TG) is the structure of fat that contains three fatty acids (i.e. EPA and DHA) esterified to a glycerol backbone. The triglyceride structure is the naturally occurring form of fatty acids found in fish and the common form found in the human body. Due to the low concentrations of EPA and DHA in its natural TG molecular form, the majority of fish oil supplements undergo molecular distillation.

Ethyl Ester (EE)
Molecular distillation is a manufacturing process used to concentrate EPA and DHA in fish oil. During this process, the glycerol backbone of the TG is replaced by ethanol to significantly increase the EPA and DHA concentration in fish oil. The majority of fish oil supplements sold in North America and globally, as well as utilized in large human RCTs, are ethyl ester (EE) concentrates.

Re-esterified Triglyceride (rTG)
Once molecularly distilled and the desired concentration of EPA and DHA is achieved, the ethanol backbone can be removed and the EPA and DHA fatty acids can be re-esterified to a glycerol backbone, an enzymatic process called glycerolysis. This additional process forming re-esterified triglyceride (rTG) concentrates is a more expensive manufacturing step, and is associated with a higher fish oil product cost.

Phospholipid (PL) from Krill Oil
Krill oil is comprised of both phospholipids (PL) and TG. The primary PL in krill oil is phosphatidylcholine (PC), with 40% of total fatty acids bound to the PC as EPA and DHA (3). However, the PL content in commercially availability krill oil products can vary widely from approximately 19-81% (4).

Absorption and metabolism of natural triglycerides vs. ethyl esters
The digestion of dietary fats, including fish oil, occurs in the small intestine via bile salt emulsification and pancreatic lipase hydrolysis. TG is hydrolyzed to release two free fatty acids (FFA) and a monoglyceride (one fatty acid bonded to glycerol), whereas EE is hydrolyzed to release a FFA plus ethanol. Monoglycerides and FFA associate with bile salts and phospholipids to form micelles that get transported to intestinal enterocytes for absorption. Once inside the intestinal enterocyte, monoglycerides and FFA, whether originally from dietary TG or EE fish oil, are reassembled back into TG. Next, TG as well as cholesterol and fat-soluble vitamins are packaged into chylomicrons for transport of lipids in the circulation.

Studies have shown that the rate of intestinal absorption differs between EE and TG forms (5-7). Pancreatic lipase hydrolyzes EE at a slower rate than TG. However, the slower absorption is equally effective over a 24-hour period, with the EE form providing a sustained uptake of EPA and DHA and steady plasma levels (6).

While the TG form of fish oil provides a monoglyceride substrate that can be used for TG resynthesis in the enterocyte, FFA released by EE obtain a glycerol molecule from other dietary fat sources during digestion for TG resynthesis within the enterocyte. Published human studies have shown that both the TG and EE forms of EPA and DHA from fish oil have better absorption across the intestinal wall when consumed with a meal. Moreover, a high-fat meal is associated with increased absorption as compared with a low-fat meal (8).

When the EE form of fish oil is consumed, the small amount of ethanol that is released during the process of intestinal metabolism is considered insignificant and ethyl esters are safe for human consumption. For example, if 1000 mg of omega-3 fatty acids were consumed in the EE form, this would release approximately 150 mg of ethanol which is approximately 1% the amount of ethanol in a bottle of beer.

Bioavailability
There is ongoing debate regarding the differences in absorption and bioavailability between the molecular forms of EPA and DHA from commercially available omega-3 supplements - EE or TG forms from fish oil or the PL form from krill oil. Evidence from human clinical trials is mixed, as some studies have observed higher bioavailability of rTG form versus EE form (9-11), whereas others have shown equivalent bioavailability between rTG and EE forms (1,2). In addition, some studies suggest that the PL form provided by krill oil has a higher bioavailability than both EE or TG forms provided by fish oil (1, 12, 13). However, these studies failed to match the total concentrations and doses of EPA and DHA supplemented (13), as well as the n-6 content between supplements (12) or the free EPA and DHA content within the oils (1), resulting in questionable conclusions. A reexamination of krill oil bioavailability studies concluded “no greater bioavailability of krill oil versus fish oil and that more carefully controlled human trials must be performed to establish their relative efficacies after chronic administration” (14).

In addition, while a single-dose kinetic study suggests increased bioavailability of krill oil versus fish oil over 72-hours (1), the health benefits of EPA and DHA supplementation substantiated in clinical research are based on chronic consumption versus single-dose intakes. It is therefore cautioned to interpret differences in bioavailability characteristics from single dose or short-term supplementation as clinically relevant for supplements, such as fish oil supplements, which are to be given chronically (14). Furthermore, Arterburn et al. demonstrated that steady state plasma levels of LC n-3-PUFA were achieved after 4-weeks (15).

Recently, Yurko-Mauro et al. investigated the oral bioavailability of the same dose of both EPA + DHA in the form of fish oil-EE versus fish oil-TG versus krill oil-PL in both plasma and RBC over 4-week supplementation using a double-blind, randomized, 3-treatment, multi-dose parallel study (2). Sixty-six healthy adults were supplemented for 28 days with 1.3 g/day of EPA+DHA total (approximately 816 mg/day EPA and 522 mg/day DHA, regardless of formulation) as fish oil-EE, fish oil-TG, or krill oil-PL (6 capsules per day). Plasma and RBC samples were collected at baseline (pre-dose on Day 1) and at 4, 8, 12, 48, 72, 336, and 672 hours (Week 4). Plasma concentrations of EPA+DHA reflect short-terms intake, whereas RBC concentrations of EPA+DHA reflect long-term bioavailability, tissue composition, and EPA+DHA utilization in the body.

By 4-weeks of supplementation, EPA +DHA plasma levels reached a plateau in healthy adults, demonstrating an estimate of steady-state long-chain omega-3 PUFA levels (15). Results showed no significant differences in mean fasting plasma concentrations of EPA+DHA after 4-weeks between the 3 formulations, demonstrating comparable oral bioavailability (2). The results also showed nearly identical bioavailability among the 3 forms at early time-points (<48 hours) of plasma measures, illustrating no better bioavailability of one form versus another when equivalent concentrations of omega-3 fatty acids were administered.

In addition, at 4-weeks the EPA+DHA concentrations in RBCs were not significantly different, providing comparable omega-3 indexes and similar long-term bioavailability after supplementation of the 3 formulations. Since the Omega-3 Index is a recognized marker of cardiac risk (16), the results from this study reveal that the forms of EE or TG from fish oil or PL from krill oil all provide adequate EPA+DHA RBC levels to achieve cardiovascular benefits.

Identity, potency, and purity - Determining the content and safety of fish oil supplements
Ensuring that omega-3 supplements used by patients are both safe and high quality is of utmost importance. In addition, certificate of analysis outlining the measurement of the potency of EPA and DHA, environmental contaminants (i.e. heavy metals, PCBs, pesticides, solvent residues), and oxidation kinetics (i.e. peroxide and anisidine values) for omega-3 oils available on the market should be a priority of suppliers.

When choosing a fish oil product for patients, it is important that label claims for EPA and DHA are met, especially when targeting a specific therapeutic dosage. Unfortunately, some studies of supplements on the commercial market found that more than half of omega-3 dietary supplements did not meet their label claim for EPA and DHA, regardless of their molecular form (17,18). In addition, a quarter of the fish oil supplements exceeded recommended limits for peroxide values (17). Furthermore, krill oil supplements provide a much lower does of EPA+DHA per softgel as compared to fish oil concentrates, therefore, patients may have to take several more krill oil softgels per day to reach therapeutic daily dosages.

Environmental contaminants (i.e. heavy metals and PCBs) and the oxidation kinetics of EPA and DHA omega-3 fatty acids in the form of EE and TG from fish oil and PL from krill oil has been debated. Fish and krill oil processing and manufacturing conditions as well as inclusion of antioxidants (i.e. vitamin E) in dietary supplements impact the stability, purity and quality of the oils. Fish oils are typically manufactured under precise conditions to reduce oxidation and ensure stability (i.e. nitrogen flushing). Since EPA and DHA have a higher number of double bonds, they are susceptible to oxidative degradation. The oxidative quality of fish oil supplements can be assessed by measuring peroxide value (PV), with a maximum acceptable PV of fish oil products <5 meqkg-1 (19). Commercial liquid fish oil products are more susceptible to oxidation due to their exposure to air each time a bottle is opened by a consumer, therefore, specialty bottle caps are sometimes used on commercial liquid fish oil products, and refrigeration once opened as well as consumption within a specified time frame is recommended. TG is more resistant to oxidation than EE (20), therefore, TG form is the preferred commercial liquid fish oil product. However, softgels are typically hermetically sealed protecting the oil, either TG form or EE form, from oxidation, and therefore, are not subject to the same stability concerns as liquid products. Peroxide and anisidine values are one of the most important indicators of the quality/freshness of fish oil before they are manufactured to ensure stability. The most effective way to assess the stability and safety of your fish oil or krill oil product is to request Certificate of Analysis from your fish oil supplier to ensure identity, potency, and purity for the specific product lot number provided to patients.

The Parlor Trick…is it just a marketing trick?
The styrofoam cup (a.k.a. parlor trick) is a common misconception about fish oil in EE form. Unfortunately, these types of tricks confuse the consumer. All forms of fish oil dissolve polystyrene, however, the EE forms does so at a faster rate because EE's polarity is similar to the polarity of polystyrene, and hence, it will react and dissolve polystyrene much faster. Even pure lemon oil dissolves styrofoam at the same rate as EE. Like dissolves like. The gastro-intestinal (GI) tract is not composed of polystyrene found in styrofoam, and EE do not react like this in the GI tract. Both EE and TG are safe and have generally recognized as safe (GRAS) status and are safe for human consumption. So, overall, the Parlor Trick is merely a marketing gimmick and there is absolutely no safety concern with the EE form of fish oil.

References:
  1. Schuchardt JP et al. Incorporation of EPA and DHA into plasma phospholipids in response to different omega-3 fatty acid formulations - a comparative bioavailability study of fish oil vs. krill oil. Lipids Health Dis. 2011;10:145.
  2. Yurko-Mauro K et al. Similar eicosapentaenoic acid and docosahexaenoic acid plasma levels achieved with fish oil or krill oil in a randomized double-blind four-week bioavailability study. Lipids Health Dis. 2015;14:99
  3. Kidd PM. Omega-3 DHA and EPA for cognition, behavior, and mood: clinical findings and structural-functional synergies with cell membrane phospholipids. Altern Med Rev. 2007;12:207-227.
  4. Araujo P et al. Determination and structural elucidation of triacylglycerols in krill oil by chromatographic techniques. Lipids. 2014;49:163-72.
  5. Lawson LD, Hughes BG. Human absorption of fish oil fatty acids as triacylglycerols, free acids, or ethyl esters. Biochem Biophys Res Commun. 1988;152:28-35.
  6. Rupp H et al. Risk stratification by the "EPA+DHA level" and the "EPA/AA ratio" focus on anti-inflammatory and antiarrhythmogenic effects of long-chain omega-3 fatty acids. Herz. 2004;29:673-85.
  7. Krokan HE et al. The enteral bioavailability of eicosapentaenoic acid and docosahexaenoic acid is as good from ethyl esters as from glyceryl esters in spite of lower hydrolytic rates by pancreatic lipase in vitro. Biochim Biophys Acta. 1993:1168;59-67.
  8. Lawson LD, Hughes BG. Absorption of eicosapentaenoic acid and docosahexaenoic acid from fish oil triacylglycerols or fish oil ethyl esters co-ingested with a high-fat meal. Biochem Biophys Res Commun. 1988;156:960-3.
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  11. Schuchardt JP et al. Moderate doses of EPA and DHA from re-esterified triacylglycerols but not from ethyl-esters lower fasting serum triacylglycerols in statin-treated dyslipidemic subjects: Results from a six month randomized controlled trial. Prostaglandins Leukot Essent Fatty Acids. 2011;85:381-6.
  12. Ramprasath VR 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 Dec 5;12:178.
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  18. Kleiner AC et al. A comparison of actual versus stated label amounts of EPA and DHA in commercial omega-3 dietary supplements in the United States. J Sci Food Agric. 2015;95(6):1260-7.
  19. The Global Organization for EPA and DHA (GOED) Voluntary Monograph. Version 5 (Issue Date November 19, 2015).
  20. Yoshii H et al. Autoxidation kinetic analysis of docosahexaenoic acid ethyl ester and docosahexaenoic triglyceride with oxygen sensor. Biosci Biotechnol Biochem. 2002;66:749–753.