17.11.08
Media exposure, scientific findings, and word of mouth have lead to a significant increase in fish oil supplementation over the past 5 years. The popularity of these supplements has also lead to an increased concern over product quality. The term “pharmaceutical quality” is typically associated with higher quality fish oils; however the use of this term is not regulated and can be freely used by any branded fish oil product. Most industry experts associate pharmaceutical quality with products that comply with a fish oil monograph developed by the Council for Responsible Nutrition (CRN). Compliance with the CRN monograph does not necessarily mean that a product is of the highest quality. The CRN fish oil monograph was an important step forward in creating stringent quality and purity standards for fish oil supplements, but there are still some quality parameters it does not address.
One of the most controversial and debated quality issues not addressed by the CRN monograph is the fish oil’s molecular form – Ethyl Esters or Triglycerides.
What are triglycerides?
Triglycerides (TG) are the natural molecular form that fats are typically found in most food sources. For instance, the omega-3 fats present in all fish species are almost exclusively present as TGs1. TGs are fats that are comprised of three fatty acids (i.e. EPA and DHA) linked to a molecule of glycerol. Free fatty acids are rapidly oxidized and therefore the glycerol backbone helps to stabilize the fat molecules and prevent breakdown and oxidation 2.
What are ethyl esters, and how are they produced?
Ethyl esters (EE) are an alternate form of fats that are synthetically derived by reacting free fatty acids with ethanol (alcohol)3. During the processing of some fish oils the fatty acids are cleaved from their natural glycerol backbone then esterified (linked) with a molecule of ethanol. This process, called trans-esterification, results in a product called fish oil EEs, also commonly known as Fatty Acid Ethyl Esters (FAEE)4. The production of EE fish oils is required for the concentration of long chain omega-3s. The fish oil EEs are heated under vacuum in a process referred to as molecular distillation or short path evaporation. The process selectively concentrates the longer chain polyunsaturates resulting in an oil with a higher concentration of both EPA and DHA. This EE concentrate is typically the end product that is sold and marketed as an “omega-3 fish oil concentrate” 3. The proper term for EE fish oils is “semi-synthetic” referring to the fact that both ethanol and fatty acids are natural however the esterification of these two substances is not found in natural food sources of omega-3 fats.
Are all fish oil concentrates ethyl esters?
The vast majority of fish oil concentrates sold globally, including those sold in North America are EEs. Unfortunately, only a very small portion of fish oil concentrates are sold as natural TGs. An additional step in the processing of fish oils can convert EEs back into their natural TG state. This process removes the ethanol backbone and re-esterifies the omega-3 fatty acids to a glycerol backbone.
The process of converting TGs to EEs is necessary from a technical standpoint in the production of fish oil concentrates, however re-converting EEs back to a TG fish oil is a simple matter of quality, efficacy, safety, and cost.
Absorption and metabolism of natural triglycerides vs. ethyl esters
Dietary fish oil is digested in the small intestine by the emulsifying action of bile salts and the hydrolytic activity of pancreatic lipase 1,5. The hydrolysis of a triglyceride (TG) molecule produces two free fatty acids (FFA) and a monoglyceride (one fatty acid combined to glycerol) 1,5. These metabolic products are then absorbed by intestinal enterocytes and reassembled again as TGs 1,5. Carrier molecules called chylomicrons then transport the TGs into the lymphatic channel and finally into the blood 6.
The digestion of EE fish oils is slightly different due to the lack of a glycerol backbone 1. In the small intestine it is again the pancreatic lipase that hydrolyzes the fatty acids from the ethanol backbone however the fatty acid-ethanol bond is up to 50x more resistant to pancreatic lipase as compared to hydrolysis of TGs7,8. The EEs that get hydrolyzed produce free fatty acids plus ethanol. The FFA’s are taken up by the enterocytes and must be reconverted to TGs to be transported in the blood 1. The TG form of fish oil contains its own monoglyceride substrate, where as EE fish oils, coupled to ethanol, do not. EE must therefore obtain a monoglyceride substrate from another source. Without a glycerol or monoglyceride substrate TG re-synthesis is delayed, suggesting that transport to the blood is more efficient in natural TG fish oils in comparison to EEs. Furthermore, this delay of TG re-synthesis in EE fish oils could cause an increase in free fatty acids and subsequent oxidation of those free fatty acids.
Bioavailability of triglycerides vs. ethyl ester fish oils
Numerous studies have assessed the absorption and bioavailability of ethyl ester (EE) fish oils. Most studies have measured the amount of EPA and DHA in blood plasma after ingestion of fatty acids as either TGs or EEs. Although a few studies have found that the absorption rate is similar between the two types of oils, the overall evidence suggests that triglyceride (TG) fish oils are better absorbed in comparison to EEs. Natural TG fish oil results in 50% more plasma EPA and DHA after absorption in comparison to EE oils 9, TG forms of EPA and DHA were shown to be 48% and 36% better absorbed than EE forms 10, EPA incorporation into plasma lipids was found to be considerably smaller and took longer when administered as an EE 11, plasma lipid concentrations of EPA and DHA were significantly higher with daily portions of salmon in comparison to 3 capsules of EE fish oil 12 and in the rat, DHA TG supplementation led to higher plasma and erythrocyte DHA content than did DHA EE 13 and a higher lymphatic recovery of EPA and DHA 14.
One of the causative factors for the poor bioavailability of EE fish oil is a much greater resistance to digestive enzymes. As previously mentioned, during the digestive process, pancreatic lipase enzymes hydrolyse (cleave) the oils to liberate the fatty acids and EE fish oil is much more resistant to this enzymatic process than the natural TG form 7. A recent study assessed the specificity of five lipases towards EPA and DHA in TG and EE forms of fish oil. All of the investigated lipases discriminated against both EPA and DHA more in EE oils than in the natural TG oils. In other words, both EPA and DHA were more easily hydrolysed from a TG than from an EE. EPA and DHA hydrolysis would be further compromised in individuals who suffer from a digestive disorder, such as pancreatic insufficiency. EE fish oils should be avoided in such populations as they would likely cause malabsorption of EPA and DHA. Review of the existing literature provides evidence which suggests that omega-3 fatty acids in the natural form of TGs are more efficiently digested and significantly better incorporated into plasma lipids in comparison to EE forms (see table 1 for additional studies).
Ethyl ester fish oils are less stable, and readily oxidize
Omega-3 fish oils in the form of EEs are much less stable than those in the natural TG form and readily oxidize. The oxidation kinetics of DHA as an EE or as a TG were assessed by measuring the concentration of oxygen found in the head space of a reaction vessel with both TG and EE forms 15. The EE form of DHA was more reactive, and quickly oxidized, demonstrating that EE fish oils are far less stable and more readily produce harmful oxidation products 15. Furthermore, the stability of DHA containing oil in phospholipid, triacylglycerol and EEs form has been assessed 16. After a 10-week oxidation period, the EE DHA oil decayed 33% more rapidly 16.
Ethyl ester fish oil safety
During the digestive process, fish oil EEs are converted back to TGs by intestinal enterocytes 1 which results in the release of ethanol. Although the amount of ethanol released in a typical dose of fish oil is small, those with sensitivities to alcohol or those who are alcoholics should refrain from the consumption of EE fish oils. Young children may also be more vulnerable to the toxic effects of ethanol even in small quantities.
The exact amount of ethanol released from the EE fish oil is dependent on the exact profile of the fatty acids. For a typical 60% omega-3 EE concentrate the amount of ethanol would be approximately 15% by weight (see figure 1).
Figure 1.
Determination of ethanol percentage in ethyl ester fish oils
Fatty acid profile for ethyl ester fish oil: 30% EPA, 20% DHA. Remaining fatty acids assumed to be predominantly 16:0.
Molar mass of ethanol 46.07g/mol
Molar mass of EPA 330.51g/mol
Molar mass of DHA 356.55g/mol
Calculated per 100g of oil
%ethanol from EPA = 30g/330.51g/mol * 46.07 = 4.18g
%ethanol from DHA = 20g/356.55g/mol * 46.07 = 2.58g
%ethanol from 16:0 = 50g/284.48g/mol* 46.07 = 8.09g
Total ethanol = 14.85g or 14.85%
Additional concern exists regarding whether a small portion of EE fish oil is absorbed directly into the body as fatty acid ethyl esters (FAEE)17. Unlike TGs, the presence in the body of FAEEs has been found to potentiate cytotoxicity 17. Several in vitro studies using purified lysosomes 18, purified mitochondria 19 or intact Hep G2 cells 20 have provided evidence for toxicity of FAEE.
Studies in animals have shown that ethanol released into the liver and pancreas can result in severe organ damage 21. Post mortem organ analysis has demonstrated that FAEEs are toxic mediators of ethanol induced cellular injury 22, and have been shown to induce pancreatic injuries when infused in vivo into rats 23. It is possible that efficient FAEE digestion in the GI tract could prevent toxicity 3, but until further studies carefully examine EE oxidation, the potential for direct uptake of FAEE, or EE absorption into the circulation via the stomach, EE fish oils should be consumed with caution.
How can I determine if my fish oil is a natural triglyceride or an ethyl ester?
There is a simple, inexpensive and rapid method to determine if a fish oil is in the TG or EE form by using polystyrene (Styrofoam) cups.
Method:
1. Measure and place 20ml of fish oil in a polystyrene cup. Place the cup on a plate to avoid any mess.
2. Observe the cup after 10 minutes. If the fish oil has leaked significantly through the cup it contains EEs.
Due to their chemical composition, EE fish oils will actually eat straight through the polystyrene cup. This effect will become evident after just a few minutes, however significant leakage is seen after 10 minutes. Natural TG fish oils placed in the same cup will not show leakage after 10 minutes. Natural TG fish oils may show leakage through the cup in very small amounts after 2-3 hours.
Conclusion
Fish oil supplements in the natural TG form offer numerous advantages when compared to those in the EE form; Oils in a TG form are completely safe to consume, are 100% natural, provide increased absorption, and are much more stable. While fish oil EEs are a source of omega-3 fatty acids, research shows that they are not as beneficial to the consumer as fish oils in the natural TG form.
The question remains “why are most fish oil supplements being sold as EEs”? There is only one reason - economics. The process to convert fish oil EEs back to TGs is costly. Bulk oil costs for TG concentrates are typically 30-40% higher than EE concentrates. So to reduce costs and increase profits producers of fish oil supplements choose to use EE concentrates for their product. While some countries have gone as far as banning the sale of EE fish oils, other countries such as the US, Canada, and the UK allow the sale of fish oils in EE form and do not require that the fish oil form is specified on the label, leading consumers to believe the product is natural.
In keeping with Ascenta’s commitment to high quality standards and the exclusive use of natural ingredients, our entire line of omega-3 supplements uses fish oils in the natural TG form.
Table 1: Studies comparing the absorption of triglyceride and ethyl ester Fish Oils
Hansen JB, Olsen JO, Wilsgård L, Lyngmo V, Svensson B. Comparative effects of prolonged intake of highly purified fish oils as ethyl ester or triglyceride on lipids, haemostasis and platelet function in normolipaemic men. Eur J Clin Nutr,47, 497-507.
31 normolipaemic non-obese men (21-47 yrs) were given 4g highly purified omega-3 ethyl ester fatty acids, 4 g corn oil as a placebo, or 12 g n-3 triglycerides for 7 weeks. The daily intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was 2.2 and 1.4 for TG, and 2.2 and 1.2 for EE. Blood samples were collected at week 1, 3 and 7. Comparison of time course incorporation of n-3 fatty acids in plasma phospholipids by repeated measures of variance did no show any difference between the TG and EE n-3 sources. Repeated measure ANOVA did however reveal a significant difference between TG and EE with respect to the incorporation of EPA into plasma cholesterol esters. Argument is made that higher amounts of omenga-3 fatty acid lead to decreased differences between absorptions. Although higher doses of omenga-3 fatty are not always realistic.
Beckermann B, Beneke M, Seitz I. 1990. Comparative bioavailability of eicosapentaenoic acid and docosahexaenoic acid from triglycerides, free fatty acids and ethyl esters in volunteers. Arzneimittelforschung, 40(6):700-4.
The bioavailability of EPA and DHA from triglycerides, free fatty acids and ethyl esters was investigated in 8 female volunteers in a randomized triple cross-over trial with baseline control. EPA/DHA was administered in capsules in form of triglycerides, free fatty acids and ethyl esters. The resulting EPA/DHA plasma levels were determined and evaluated. The mean relative bioavailability of EPA/DHA compared to triglycerides was 186/136% from free fatty acids and 40/48% from ethyl esters. Maximal plasma levels were about 50% higher with free fatty acids and about 50% lower with ethyl esters as compared to triglycerides. The tolerability of the free fatty acids was much worse than that of triglycerides and ethyl esters. The main side effect was eructation.
Krokan HE, Bjerve KS, Mørk E. 1993. 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,1168, 59-67.
Enteral absorption by healthy male volunteers of EPA and DHA from an ethyl ester and natural triglyceride fish oil was found to be similar after intake of equivalent doses however; hydrolysis of natural triglyceride fish oil was more efficient. In spite of the similar serum levels of EPA and DHA obtained in vivo, in vitro hydrolysis by porcine pancreatic lipase of the ethyl ester was 3-fold slower than hydrolysis of a the triglyceride. Under similar conditions release of AA from triglyceride and ethyl ester was essentially similar and approx. 1.5-fold faster than release of EPA and DHA from ethyl esters. There are therefore differences in the rate of hydrolysis of ethyl ester and triglycerides fish oils.
el Boustani S, Colette C, Monnier L, Descomps B, Crastes de Paulet A, Mendy F. (1987). Enteral absorption in man of eicosapentaenoic acid in different chemical forms. Lipids, 10, 711-4.
After administering the equivalent of 1g of EPA in four different chemical forms, the kinetics of EPA incorporation into plasma triglycerides were compared by gas liquid chromatography on a capillary column following separation of the lipid fraction by thin layer chromatography. EPA incorporation into plasma triglycerides was markedly smaller and later when EPA was administered as an ethyl ester rather than as EPA free fatty acid, EPA arginine salt or 1,3-dioctanoyl-2-eicosapentaenoyl glycerol. Our results and the data in the literature are compatible with the hypothesis that the glycerol form of EPA is absorbed with minimum hydrolysis and escapes random distribution between the other positions of the glycerol molecule during the absorption process.
Lawson LD, Hughes BG. (1988). Human absorption of fish oil fatty acids as triacylglycerols, free acids, or ethyl esters. Biochem Biophys Res Commun, 52, 328-35.
As triacylglycerols, eicosapentaenoic acid (1.00 g) and docosahexaenoic acid (0.67 g) were absorbed only 68% and 57% as well as the free acids. The ethyl esters were absorbed only 20% and 21% as well as the free acids. The incomplete absorption of eicosapentaenoic and docosahexaenoic acids from fish oil triacylglycerols correlates well with known in vitro pancreatic lipase activity.
Visioli F, Risé P, Barassi MC, Marangoni F, Galli C. (2003). Dietary intake of fish vs. formulations leads to higher plasma concentrations of n-3 fatty acids. Lipids, 38, 415-8
For 6 weeks, volunteers were given 100 g/d of salmon, or 1 or 3 capsules of ethyl ester fish oil/d. Marked increments in plasma EPA and DHA concentrations (microgram/mg total lipid) and percentages of total fatty acids were recorded at the end of treatment with either omega-3 capsules or salmon. Increments in plasma EPA and DHA concentration after salmon intake were significantly higher than after administration of capsules. The same increments would be obtained with at least two- and nine fold higher doses of EPA and DHA, respectively, if administered with capsules rather than salmon. We provide experimental evidence that natural omega-3 fatty acids from fish are more effectively incorporated into plasma lipids than when administered as capsules.
Valenzuela A, Valenzuela V, San hueza, J, Nieto S. (2005). Effect of supplementation with docosahexaenoic acid ethyl ester and sn-2 docosahexaenyl monoacylglyceride on plasma and erythrocyte fatty acids in rats. Ann Nutr Metab. 49, 49-53.
Female rats received a 40-day supplementation of either DHA ethyl ester or DHA-monoglycerate. Plasma and erythrocyte fatty acid composition were assessed by gas chromatography at day 0 and 40 of supplementation. DHA ethyl ester increased plasma and erythrocyte DHA by 15 and 11.9%, respectively, with no modification of arachidonic acid (AA) content. DHA-monoglycerate supplementation increased plasma and erythrocyte DHA by 24 and 23.8%, respectively, and reduced AA by 5.5 and 3%, respectively. Although this data is done with animals, the authors conclude that in the rat, DHA-monoglycerate supplementation allows a higher plasma and erythrocyte DHA content than DHA-ethyl ester.
Ikeda I, Sasaki E, Yasunami H, Nomiyama S, Nakayama M, Sugano M, Imaizumi K, Yazawa K. (1995). Digestion and lymphatic transport of eicosapentaenoic and docosahexaenoic acids given in the form of triacylglycerol, free acid and ethyl ester in rats. Biochim Biophys Acta; 1259: 297-304.
Lymphatic transport of EPA and DHA with trieicosapentaenoyl glycerol (TriEPA) and tridocosahexaenoyl glycerol (TriDHA) was compared with the transport of ethyl ester and free acid in rats cannulated with thoracic duct. Trioleoylglycerol (TO) served as a control. Lymphatic recovery of EPA and DHA in rats given TriEPA and TriDHA was significantly higher at the first 3 h after the administration compared to those given as free acid or ethyl ester. The 24-h recovery was comparable between triacylglycerol (TAG) and free acid, while it was significantly lower in ethyl ester. The hydrolysis rate of ethyl esters was extremely low even in 6 h incubation with lipase. Although this data is done with animals, the authors conclude that there is less lymphatic recover of EPA and DHA when they are in ethyl ester form.
Nordøy A, Barstad L, Connor WE, Hatcher L. 1991. Absorption of the n-3 eicosapentaenoic and docosahexaenoic acids as ethyl esters and triglycerides by humans. Am J Clin Nutr. 53:1185-90.
Five normolipemic subjects received three test meals. 1) 40g n-3 triglycerides, 2) 28 g n-3 ethyl ester plus 12 g olive oil, 3) 28 g n-3 ethyl ester and 4) 40g olive oil. When equivalent amounts of fat were given, the increase in chylomicrons and plasma triglycerides was similar; n-3 fatty acid contents were also similar after n-3 fatty acid intake as ethyl esters or triglycerides. Ethyl esters alone were well absorbed and produced similar n-3 fatty acid responses in plasma triglycerides and chylomicrons. At 24 h after the n-3 fatty acid-containing meals, the fatty acid plasma concentration of these acids was similar. This study suggests that n-3 fatty acids given as ethyl esters or triglycerides were equally well absorbed. However, the doses of fish oil given were unrealistically high thus one should be hesitant to draw conclusions from such data.
References
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2) Segura R. (1988). Preparation of fatty acid methyl esters by direct transesterification of lipids with aluminum chloride-methanol. J Chromatogr.;441:99-113.
3) Saghir M, Werner J, Laposata M. (1997). Rapid in vivo hydrolysis of fatty acid ethyl esters, toxic nonoxidative ethanol metabolites. Am J Physiol.;273:G184-90.
4) Mogelson S, Pieper SJ, Lange LG. (1984). Thermodynamic bases for fatty acid ethyl ester synthase catalyzed esterification of free fatty acid with ethanol and accumulation of fatty acid ethyl esters. Biochemistry. 1984 Aug 28;23(18):4082-7.
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6) Lambert MS, Botham KM, Mayes PA. (1997). Modification of the fatty acid composition of dietary oils and fats on incorporation into chylomicrons and chylomicron remnants. Br J Nutr.;76:435-45
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8) Yang LY, Kukis A, Myher JJ. (1990). Intestinal absorption of menhaden and rapeseed and their fatty acid methyl and ethyl esters in the rat. Biochem Cell Biol.;68:480-91
9) Beckermann B, Beneke M, Seitz I. (1990). Comparative bioavailability of eicosapentaenoic acid and docosahexaenoic acid from triglycerides, free fatty acids and ethyl esters in volunteers. Arzneimittelforschung; 40(6):700-704.
10) Lawson LD, Hughes BG. (1988). Human absorption of fish oil fatty acids as triacylglycerols, free acids, or ethyl esters. Biochem Biophys Res Commun, 52, 328-335
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13) Valenzuela A, Valenzuela V, Sanhueza J, Nieto S. (2005). Effect of supplementation with docosahexaenoic acid ethyl ester and sn-2 docosahexaenyl monoacylglyceride on plasma and erythrocyte fatty acids in rats. Ann Nutr Metab; 49: 49-53
14) Ikeda I, Sasaki E, Yasunami H, Nomiyama S, Nakayama M, Sugano M, Imaizumi K, Yazawa K. (1995). Digestion and lymphatic transport of eicosapentaenoic and docosahexaenoic acids given in the form of triacylglycerol, free acid and ethyl ester in rats. Biochim Biophys Acta; 1259: 297-304.
15) Yoshii H, Furuta T, Siga H, Moriyama S, Baba T, Maruyama K, Misawa Y, Hata N, Linko P. (2002). Autoxidation kinetic analysis of docosahexaenoic acid ethyl ester and docosahexaenoic triglyceride with oxygen sensor. Biosci Biotechnol Biochem;66:749-753.
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17) Best CA, Laposata M. (2003). Fatty acid ethyl esters: toxic non-oxidative metabolites of ethanol and markers of ethanol intake. Front Biosci; 8: 202-17.
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24) Hansen JB, Olsen JO, Wilsgård L, Lyngmo V, Svensson B. (1993). Comparative effects of prolonged intake of highly purified fish oils as ethyl ester or triglyceride on lipids, homeostasis and platelet function in normolipaemic men. Eur J Clin Nutr;,47: 497-507.
25) Krokan HE, Bjerve KS, Mørk E. (1993). 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; 1168: 59-67.
26) Harris WS, Zucker ML, Dujovne CA. (1988). Omega-3 fatty acids in hypertriglyceridemic patients: triglycerides vs methyl esters. Am J Clin Nutr; 48: 992-997
27) Nordøy A, Barstad L, Connor WE, Hatcher L. (1991). Absorption of the n-3 eicosapentaenoic and docosahexaenoic acids as ethyl esters and triglycerides by humans. Am J Clin Nutr 53:1185-90.
