Fish oil supplementation has increased significantly within the past decade as a result of media exposure, scientific findings, and word of mouth. In fact, fish oil supplements are now the third most frequently used supplements following multivitamin-minerals and calcium-containing supplements  . Much conversation and debate has been generated regarding fish oil quality and bioavailability, in particular with respect to the different molecular forms of commercially available fish oil supplements.
Quality guidelines for fish oil supplements
The Global Organization for EPA and DHA (GOED) is an important proactive and accountable association of the finest manufacturers, marketers, and supporters of EPA and DHA omega-3 fatty acids. GOED sets strict quality and purity standards for the omega-3 business sector and the GOED Voluntary Monograph has served as the definition of quality in the industry since 2002. The Monograph adopts the strictest aspects of quality and safety regulations around the world, including environmental contaminants and oxidation, thus ensuring that products meeting this strict standard are safe and effective.
The GOED Monograph represents an important step forward in the standardization of high quality fish oil but unfortunately, it does not address all quality-related issues. In particular, the Monograph does not differentiate between lipid classes (or molecular forms) of fish oil products. Fish oils have traditionally been marketed in a molecular form called triglycerides (TG). Within the past decade, however, “fish oil” supplements are largely marketed in an alternative molecular form called fatty acid ethyl esters (EE).
What are triglycerides?
Triglycerides are made of three fatty acids (e.g., EPA and DHA) attached to a glycerol backbone; it is the molecular form that makes up virtually all fats and oils in both animal and plant species. The omega-3 fats present in fish are almost exclusively TG .
What are EE?
EE are made of one fatty acid attached to one ethanol molecule; they are a class of lipids that are well characterized with their own monograph in the British Pharmacopoeia . Generally, EE are not found in nature and are therefore created through chemical synthesis.
How are TG and EE different from a molecular standpoint?
EPA and DHA in EE form have different chemical properties than EPA and DHA provided in the natural TG form of fish oil. Both TG and EE are “esterified forms,” which means that an ester link/bond holds the fatty acids onto their backbone; the fatty acids in TG are esterified to a glycerol backbone whereas the fatty acids in EE are esterified to an ethanol (alcohol) backbone. This may seem inconsequential but it is important. According to Bailey’s Industrial Oil and Fat Products (6th Ed.), “Fatty acids in oils and fats are found esterified to glycerol .”Given this definition in a standard reference book on food chemistry and processing technology related to edible oils, EE are not technically oils. TG, on the other hand, which contain three fatty acids attached to a glycerol backbone, meet the above definition and are therefore classified as oils. Labelling EE forms of EPA and DHA as “fish oil” is legally allowed but it could be considered a misnomer since EE are not technically oil.
How are EE produced?
EPA and DHA in EE form are highly refined omega-3 fatty acids that are created by reacting free fatty acids (FFA) with ethanol in a process called trans-esterification. This process involves removing the glycerol backbone of TG fish oil, resulting in FFA and a free glycerol molecule. An ethanol molecule is then attached to each of the FFA, creating EE. The resulting EE allow for the concentration of the omega-3 long chain fatty acids at lower temperatures; this process (molecular distillation) allows for the selective concentration of EPA and DHA to levels greater than found naturally in fish . The resulting EE concentrate of EPA and DHA is subsequently marketed and sold as “fish oil concentrate.”
Are all fish oil concentrates EE?
Manufactured EE concentrates can be converted back to the natural TG form using enzymes in a process called glycerolysis. Food-grade enzymes separate the ethanol molecule from the fatty acid, creating a FFA and a free ethanol molecule. When glycerol is added back into the solution the enzymes then re-esterify the fatty acids back onto a glycerol backbone creating TG oil. These oils are commonly referred to as re-esterfied (or reformed) triglycerides (rTG), which have identical structures to natural TG but with higher concentrations of the desired fatty acids, EPA and DHA.
The process of converting EE back to TG is a costly step and is therefore bypassed by many fish oil manufacturers. In fact, the vast majority of fish oil concentrate softgels sold globally, including those sold in North America, are EE concentrates. Only a small percentage of fish oil concentrate softgels on the market are TG oils. Whereas fish oil softgels are commonly found in EE form, the majority of liquid fish oil products sold in North America are in TG form. Liquid EE products are not widely used because they degrade faster and are therefore characterized by a more intense fishy flavour thus making them unpalatable for consumers.
Metabolism of TG versus EE
While converting concentrated EE fish oil back to its natural TG form increases manufacturing costs, it improves metabolism and bioavailability. All dietary fat (TG) is digested in the small intestine by the action of bile salts and pancreatic lipase. Bile salts break up fat globules into much smaller emulsion droplets, which increase the surface area where lipase can work to liberate two of the three fatty acids from the TG resulting in two FFA and a monoglyceride (one fatty acid attached to glycerol). FFA and monoglycerides then form micelles, which are absorbed by intestinal enterocytes, the absorptive cells lining the intestines. Once inside the enterocyte, the FFA and monoglycerides are reassembled back into TG. Carrier molecules called chylomicrons then transport the TG into the lymphatic channel and finally into the blood (reviewed in ).
The digestion of EE is slightly different because EE lack a glycerol backbone. In the small intestine, EE are emulsified by bile salts and hydrolyzed by pancreatic lipase. This hydrolysis releases the fatty acid from the ethanol backbone resulting in a FFA and an ethanol molecule. While the release of ethanol in the intestine has been expressed as a potential concern regarding the safety of EE, the ethanol release in this process is considered insignificant and EE are safe for consumption in humans . Similar to TG, the FFA liberated from EE are absorbed by enterocytes where they are converted to TG so they can be transported in the blood. This step is straightforward with TG since TG already contain a glycerol molecule that can be used to re-esterify the FFA back to TG within intestinal enterocytes. EE contain ethanol and not glycerol, which means that the FFA must obtain a glycerol molecule from another source (such as dietary fat) within the enterocyte to become transformed into a TG. Once transformed, TG are packaged into chylomicrons that transport the TG into the lymphatic channel and subsequently into the blood.
Metabolism of EE is less efficient than TG. Pancreatic lipase hydrolyses EE to a lesser degree and at a slower rate than TG [7-10]. In fact, it takes pancreatic lipase 10 to 50 times longer to break the fatty acid-ethanol bond found in EE compared to breaking the fatty acid-glycerol bond found in TG . To complicate matters, EPA and DHA hydrolysis may be further compromised in individuals with digestive disorders, such as pancreatic insufficiency. This has been demonstrated in a cystic fibrosis population whereby supplementation with EE fish oil increased the EPA/arachidonic acid ratio 9.8-fold, falling short of the 23-fold increase in healthy subjects . Furthermore, since a glycerol or monoglyceride substrate is absent in EE, TG re-synthesis in the enterocyte is delayed and subsequent transport of EPA and DHA to the blood may be less efficient in EE fish oils than TG fish oils.
Given the metabolism differences between TG and EE, one could conclude that there would be differences in EPA and DHA absorption as well. A comprehensive review of the scientific literature does indeed provide evidence suggesting that fish oil in TG form is more efficiently absorbed than fish oil in EE form [7, 8, 14-16].
Absorption of TG versus EE
There is a debate currently about whether different molecular forms of EPA and DHA are similarly absorbed; it has actually become quite the contentious topic. Many fish studies have been conducted and of course fish contains omega-3 fatty acids in the natural TG form, however, the vast majority of studies demonstrating the clinical efficacy of fish oil have used EE fish oil. Importantly, most of these studies have not directly compared EE versus TG in terms of clinical efficacy or absorption.
There have been a number of studies that have directly compared the absorption or bioavailability of EE and TG oils. Although a small number of these studies have found that absorption is similar between TG and EE oils [9, 17], the majority of research supports significantly increased absorption and bioavailability of EPA and DHA from TG versus EE oils. For example, a small study found that plasma EPA and DHA levels were 50% higher after supplementation with TG fish oil than with EE fish oil . A different study found more dramatic differences with EPA and DHA from TG being 340% and 271% better absorbed, respectively, than those consumed as EE. The authors of that study went so far as concluding that EE “are poorly absorbed in man” due to decreased pancreatic lipase activity . Furthermore, an additional study involving supplementation of fasting volunteers with 1,000 mg of EPA as either TG or EE reported that EPA incorporation into plasma lipids was considerably higher for the TG group compared to the EE group; incorporation of EPA into plasma lipids was also much slower for the EE group. The researchers suggested that this could be a result of low pancreatic lipase during EE digestion, coupled with reduced incorporation of EPA into micelles .
While these smaller studies provide support that EPA and DHA are better absorbed from TG oils than EE oils, some more recent well-designed studies provide strong evidence that TG oils have superior bioavailability. A two-week double-blind placebo-controlled study compared the absorption of different oil preparations in 72 subjects: a TG concentrate; an EE concentrate; a FFA preparation; fish body oil that was obtained from pressing the bodies of fish (considered natural TG fish oil); cod liver oil obtained from pressing the livers of cod fish (considered natural TG fish oil); and a corn placebo oil. Increases in absolute amounts of EPA and DHA in blood lipids were examined and results revealed that bioavailability of EPA and DHA from the TG concentrate was 124% superior compared with natural fish oil while the bioavailability from the EE concentrate was 73% when compared with natural fish oil. In other words, EPA and DHA were significantly better absorbed in the TG concentrate and natural TG fish oil than they were in the EE concentrate. The researchers stated that these results were likely a result of pancreatic lipase activity, which limited the absorption of EPA and DHA from the EE oil .
Another six-month double-blind placebo-controlled trial involving 150 subjects investigated the effect of EPA and DHA given as a TG concentrate, EE concentrate, or a corn placebo oil on the omega-3 index, a measure of red blood cell EPA and DHA levels. After three months, the omega-3 index increased by 186% in the TG group compared to 161% in the EE group, representing a 25% difference. After six months, the omega-3 index increased by 197% in the TG group compared to 171% in the EE group, representing a 26% difference . Furthermore, in a subset of these subjects, fasting serum triacylglycerol levels were found to be significantly reduced from baseline in the TG group (but not the EE group) after three and six months of supplementation . These results are extremely valuable because they suggest that not only are cellular levels of EPA and DHA raised about 25% more effectively when consumed as a TG concentrate versus an EE concentrate but also that the bioavailability difference translates into more desirable biological effects.
Oxidation and stability of TG versus EE
Few studies directly compare oxidation rates of TG and EE fish oil products with similar levels of EPA and DHA. The studies that have been conducted demonstrated that fatty acids in the form of EE oxidize faster than those in TG form. For example, when TG and EE fish oils were incubated at 80°C while bubbling air through them, EE oxidized more rapidly than TG . Additionally, in another study where oxygen concentration was monitored in the headspace of DHA-enriched fish oil stored at various temperatures, results revealed that DHA in EE form oxidized faster than TG . These results were supported by another clinical trial that found oxidation proceeded more rapidly in EE than TG .
Most relevant is a recently conducted study that is currently under review for peer-reviewed publication. This trial assessed oxidation of TG and EE fish oil that had similar amounts of EPA and DHA at 5-60°C. The rate of oxidation, as measured by change in peroxide value and Anisidine value, was found to be lower in TG fish oil than in EE .
How can I determine if my fish oil is a TG or EE?
Fish oil products available in North America do not require labels to state whether or not oil is in TG or EE form. Since this information is not easily accessible, there is a simple, inexpensive, and rapid method to determine if a fish oil supplement is in the TG or EE form by using polystyrene (Styrofoam) cups. Measure and place 20 ml of fish oil in a polystyrene cup, place the cup on a plate to avoid any mess, and observe the cup after 10 minutes. Due to their chemical composition, EE oils will actually dissolve the polystyrene cup. While this effect becomes evident after just a few minutes, significant leakage is seen after 10 minutes. EE oils dissolve polystyrene by dissolving the chemical bonds used to keep the polystyrene from collapsing. When these bonds are broken, the air trapped in the polystyrene escapes causing the structure to collapse. Natural TG fish oils placed in the same cup, on the other hand, will not show leakage after 10 minutes but they may show leakage through the cup in very small amounts after 2-3 hours.
Natural TG fish oil supplements offer numerous advantages over EE fish oil supplements. First, TG oils are the molecular form found naturally in fish and they are more resistant to oxidation than EE oils. Most importantly, however, is the fact that TG fish oils are metabolized and absorbed more efficiently and completely than EE oils, which may increase overall health benefits for consumers.
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