Research on phospholipids and free fatty acids
Research suggests that different forms of long-chain omega-3 fatty acids EPA (eicosapentaenoic acid, or 20:5 n-3) and DHA (docosahexaenoic acid, or 22:6 n-3) possess different bioavailability profiles. With this in mind, today’s omega-3 supplement marketers aggressively market claims of superior bioavailability, comparing one EPA/DHA omega-3 form to another.
Too often, however, they do so without authentic and consistent evidence-based clinical research, published in peer-reviewed journals, to support their bioavailability claims. For the public interested in optimizing EPA/DHA intakes, such misleading or false claims can be both confusing and detrimental. Many products also fail to disclose their specific form of EPA/DHA, further preventing the consumer from doing an
So what does the latest research show? Let’s take a look. This article follows up on an earlier article I wrote, "Omega-3 Bioavailability: Is One Form of Omega-3 More Bioavailable than Another?"1 published in Nutritional Outlook’s November 2011 issue.
First, what is bioavailability? The term bioavailability is closely associated with the concept of “digestibility.” Digestibility, in relation to fat digestion, includes the enzymatic hydrolysis of dietary fat (predominantly in the form of triglycerides) in the small intestine and the absorption of the hydrolyzed by-products (free fatty acids plus monoglycerides) across the gut wall. Traditionally, direct measures of digestion calculate “digestibility coefficients,” or the digestion percentage (efficiency) of various nutrients based on the amount of nutrient consumed relative to that collected in total fecal excretion (i.e., undigested) over a period of a few days. For instance, human studies measuring the digestibility of dietary omega-6 polyunsaturated fatty acids, as linoleic acid, show digestibility efficiencies approaching 90–95%.2 However, no such studies have been reported in human adults using different supplement forms of EPA/DHA omega-3 fatty acids
consumed with meals.
Thus, for the purpose of this article, the bioavailability of EPA/DHA refers to the net rise in blood levels of these fatty acids, over and above the initial baseline levels, following oral ingestion of a fixed amount of EPA and/or DHA for a period of time.
Bioavailability measures depend on numerous factors. In addition to the form and amount of EPA/DHA ingested, the length of a study can influence resulting bioavailability measures. For instance, consider the differences between “acute” and “chronic” studies. An acute study is a short-term study; typically, follow-up blood measurements are taken between 1 to 24 hours following a single dose of EPA/DHA. A chronic study is longer-term. Blood measures are taken before and after an extended duration of three or more weeks of daily intake of supplemental EPA/DHA.
Subjects’ dietary intakes can also affect measurements. For example, it is well known that a diet higher in fat, rather than a low-fat meal, supports better omega-3 bioavailability.1 Earlier literature indicates absorption efficiencies for EPA in the triglyceride or ethyl ester form of 90% and 60%, respectively. Considering the effect of factors such as a high-fat meal, controlled bioavailability trials are designed to require that the omega-3 supplement be taken under the same dietary conditions each day, and at the same time, to reduce confounding variables.
What are the different forms of EPA/DHA? The fatty acid forms most common to dietary supplements are triglycerides, ethyl esters, free fatty acids, and phospholipids.
Triglyceride: Omega-3 fatty acids EPA/DHA, along with other fatty acids, are connected to all three carbons of a glycerol (3-carbon) backbone-hence, the prefix tri in the word triglyceride. The triglyceride form is by far the predominant omega-3 form in our food supply, including in fish and seafood, fish oils, and re-esterified triglyceride supplements wherein the omega-3 content is concentrated following processing of the starting fish oil.
Ethyl Ester: EPA/DHA fatty acids are chemically connected to ethanol using processing technology that allows the ethyl ester forms of EPA/DHA to be concentrated via molecular distillation, or other procedures, so as to produce omega-3 “concentrates.” These ethyl ester concentrates can then be used directly as sources of EPA/DHA in dietary supplements, or can be converted back to the triglyceride form for supplements (“re-esterified” triglycerides).
Phospholipid: EPA, DHA, and other fatty acids are connected to a glycerol (3-carbon) backbone so that two fatty acids are present on two of the carbons, and one of those carbons is associated with the “head group” of the phospholipid (e.g., phosphorylcholine in the case of one type of phospholipid called phosphatidylcholine). Phospholipid forms containing omega-3 fatty acids are usually minor components of most marine oils, but there are some known fish, seafood, and derived oil sources that have levels of EPA/DHA in phospholipid forms that approach the levels present in triglycerides.
Free Fatty Acid: The EPA/DHA fatty acids are not connected to a glycerol backbone as they are in the triglyceride and phospholipid forms, nor are they chemically linked to ethanol such as in the ethyl ester form; hence, the term free. In the vegetable oil industry, “acid value” is measured because it is correlated with the level of free fatty acid in the oil. This level is an indication of the deterioration of the oil due to the breakdown of the triglycerides within to glycerol and free fatty acids. Free fatty acids can be naturally occurring in certain oil sources or can be generated intentionally from triglycerides via oil processing to yield a product enriched in free fatty acids.
Partial Glyceride: Triglyceride forms containing EPA/DHA can be converted by oil-processing technologies to generate partial glyceride forms: a) a diglyceride form containing only two fatty acids (“di”) connected to a glycerol backbone, or b) a monoglyceride form containing only one fatty acid (“mono”) connected to a glycerol backbone.
As previously discussed in the 2011 “Function Follows Form” article, most studies indicate that the “natural” triglyceride form of EPA/DHA-as found in fish, fish oil, or re-esterified triglycerides-is more bioavailable than the ethyl ester form. Studies show bioavailability differences between the two forms, when comparing essentially identical dosages of EPA and DHA, ranging from moderate to an even greater degree in at least one clinical trial. The 2011 article reviewed some of the most significant studies comparing the bioavailability of the triglyceride versus the ethyl ester form. I will not review those same studies again in this article.
Instead, for the present update, I will focus on the most recent studies to emerge in the last two years since the 2011 article published. As it turns out, the most recent updates (mostly acute studies) focus mainly on the bioavailability of the phospholipid and free fatty acid forms of omega-3, with some additional developing research on the partial glyceride form, monoglyceride.
The past two years have seen interest surge in krill oil omega-3 fatty acids. Part of the appeal, and the message conveyed by marketers, is the claim that the EPA/DHA omega-3 fatty acids in krill oil are in a phospholipid form that enables better bioavailability. In fact, however, as discussed previously in my 2011 article, the currently available body of well-controlled, published, chronic studies so far shows that there is very little or no overall difference in the bioavailability of EPA/DHA from krill oil or phospholipids relative to fish oil’s triglyceride form. Much of the research to date, including the most recent acute studies on EPA/DHA phospholipids from sources such as krill oil, have not yet provided reliable evidence showing a superior bioavailability over fish oil triglycerides.
Some new evidence supports this conclusion. A recent acute study in humans from Germany, published in Lipids in Health and Disease,3 found no statistically significant difference in bioavailability between EPA plus DHA in krill oil versus fish oil (triglyceride form or ethyl ester form). Researchers looked at the rise in blood levels of EPA plus DHA in plasma phospholipid in the short interval-up to 72 hours-following ingestion.
Of note, the researchers did report an insignificant trend towards a moderately higher rise in circulating EPA levels with krill oil. However, such is not evidence-based support for a differential bioavailability between krill oil and fish oil, because the results were not statistically significant.
It is, however, noteworthy that the authors found a considerable percentage of the EPA and DHA in krill oil present in the free fatty acid form (22% and 21%, respectively). Much of the omega-3 in krill oil comprises a mixture of forms, including triglycerides, phospholipids, and a considerable amount of free fatty acids.3 According to the researchers, any bioavailability difference for EPA/DHA from krill oil might be due to the fact that a considerable amount of the omega-3 fatty acids in the krill oil are in free fatty acid form-and not due to the fatty acids being in phospholipid form.4
In order to draw further bioavailability comparisons between omega-3 phospholipids and other omega-3 forms, future head-to-head bioavailability studies are needed to pit EPA/DHA in krill oil (containing omega-3 in mixed triglyceride plus phospholipid forms) against fish oil (containing omega-3 in triglyceride form and no phospholipids). The studies should be long-term chronic studies up to several weeks or months in duration, using krill oil supplements that are essentially devoid of EPA/DHA in the free fatty acid form.
Additionally, certain commercial sources claim that krill oil’s phospholipid form may be more bioavailable because phospholipids do not require hydrolysis by digestive enzymes in order to be absorbed. However, I argue that it is well known that orally ingested phospholipids are hydrolyzed by pancreatic enzymes, called phospholipases, in the small intestine, to then release free fatty acids plus lysophospholipids. (Lysophospholipids are phospholipid remnant molecules with one fatty acid missing.)
These free fatty acids and lysophospholipids are then absorbed and re-assembled back to phospholipids in the intestinal cell. The digestion of a triglyceride or phospholipid omega-3 fatty acid form involves breakdown in the small intestine, absorption of the hydrolytic products across the gut wall, and re-conversion back to the parent lipid types within the intestinal cell. Phospholipids are broken down in the digestive tract by enzymatic hydrolysis prior to the passing across the gut wall of their released hydrolytic products containing the fatty acid components. Thus, claiming that omega-3 fatty acids in a phospholipid form are much more bioavailable because they are not hydrolyzed in the digestive tract is not a scientifically valid argument.
Another thing to add concerning phospholipids is that omega-3 fatty acids such as DHA are present in the cell membranes of the brain, retina, and other tissues in a phospholipid form. This phospholipid form includes classes and subclasses of different phospholipid types, such as phosphatidylcholine and phosphatidlyserine. However, it needs to be made clear that intact phospholipid molecules are not considered to be absorbed intact to any significant extent following ingestion via supplementation. They cannot, therefore, be delivered directly to the brain and other tissues.5 Furthermore, there is little evidence that an intact phospholipid molecule, including one containing DHA, can cross the blood-brain barrier to any significant degree. By contrast, however, DHA circulating in the blood in a free fatty acid form following metabolism in the liver or elsewhere can readily cross the blood-brain barrier. This may also be true of DHA in the lysophospholipid form.6 The different DHA-containing phospholipids-including DHA in cellular phosphatidylcholine or phosphatidylserine-then need to be re-synthesized in the brain to support their health functions.
In the supplements market, we see many supplements containing specific phospholipid sub-types (e.g., DHA in a phosphatidylserine form). We anticipate others will also emerge as they undergo research and development.7
As reviewed in my 2011 article, some short-term human studies indicate that EPA/DHA in free fatty acid forms are moderately more bioavailable than those in triglyceride or ethyl ester forms. The suggestion is that enzymatic hydrolyses of omega-3 fatty acids esterified to a glycerol backbone (as in a triglyceride or phospholipid form) or to ethanol (as in an ethyl ester form), are likely not 100% complete in the gut, as required for subsequent absorption. The free fatty acid form, however, does not require such hydrolysis for absorption.
A very recent acute study8 tested the bioavailability of a novel free fatty acid form of EPA/DHA in subjects put on either a low-fat or high-fat diet. Blood measures were taken up to 24 hours following a single dose of omega-3 administered in either free fatty acid form or ethyl ester form. This short-term, acute study used the area under the curve over 24 hours to estimate relative bioavailability following a single ingested dose of EPA/DHA.
When comparing results for subjects on the low-fat diet, the free fatty acid form was found to be more bioavailable than the ethyl ester form. In the high-fat group, however, the bioavailability difference between the two forms was relatively minor, including no difference in the blood levels of omega-3 at 24 hours. This study may indicate an apparent better bioavailability of the free fatty acid omega-3 form over an ethyl ester form, but, again, this is a single-dose acute study and it is not as reliable as a longer-term chronic study would be.
Upon digestive hydrolysis in the small intestine, the triglyceride form of omega-3 fatty acid containing EPA/DHA undergoes breakdown to intermediary diglyceride, and, finally, to the end-product monoglyceride and free fatty acids containing omega-3 and other fatty acids. These form micelles, which are very small, emulsified particles that enhance and facilitate bioavailability. Micelles containing monoglycerides and free fatty acids adhere to the inner wall of the small intestine as part of the absorption process.
Recent studies evaluated the bioavailability of monoglyceride forms containing EPA/DHA and found that the monoglyceride form exhibits a high degree of efficient absorption.9 This is likely due to the fact that the monoglyceride form (which is an absorbable form), when provided directly, bypasses the enzymatic hydrolytic step in the gut that converts dietary or supplemental triglycerides containing omega-3 to the monoglyceride form.
Thanks to its higher bioavailability, the monoglyceride form may offer a clinical advantage to patients struggling with lipid absorption deficits. Such patients lack the ability to readily hydrolyze triglycerides to the monoglycerides and free fatty acids needed for bioavailability of EPA/DHA; thus, they would be able to utilize the monoglyceride form for absorption if the monoglyceride form were provided directly.
In my opinion, stated as well in my 2011 article-and this applies to all types of omega-3 studies-one cannot, and should not, readily extrapolate findings from very short-term, acute studies (with single-dosing designs) and apply the results with the same certainty as one could with longer-term, chronic studies. Studies show that by supplementing with higher levels of EPA/DHA fatty acids on a regular basis, one can reach a new and higher blood status-but this generally only happens after several weeks. Thus, bioavailability studies need to be conducted over extended time periods, for several weeks or longer. Omega-3 bioavailability studies would greatly benefit from longer studies. For instance, a direct comparison of the free fatty acid versus triglyceride form would be of considerable interest for such prolonged, chronic studies.
I strongly recommend relying on well-controlled, chronic, longer-term studies rather than solely relying on single-dose acute studies, including the ones discussed in this article, in order to draw reliable conclusions on the relative bioavailability and clinical efficacy of different forms of EPA/DHA. Acute and chronic studies can complement each other if they indicate similar findings on relative bioavailabilities. For instance, chronic studies have shown a better bioavailability for triglycerides over ethyl esters.1 We should await similar chronic studies before drawing conclusions for EPA/DHA in phospholipid and free fatty acid forms. I also advocate conducting traditional digestibility determinations2 on omega-3 fatty acids from different food and
1. B Holub et al., “Function Follows Form,” Nutritional Outlook, vol. 14, no. 9 (November 2011): 34–40.
2. NT Bendsen et al., “Effect of dietary calcium on fecal fat excretion: a randomized crossover trial,” International Journal of Obesity, vol. 32, no. 12
(December 2008): 1816–1824.
3. JP Schuchardt 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 in Health and Disease, published online August 22, 2011.
4. JP Schuchardt et al., “Bioavailability of long-chain omega-3 fatty acids,” Prostaglandins, Leukotrienes and Essential Fatty Acids (PLEFA), vol. 89, no. 1 (July 2013): 1–8.
5. L Kim et al., “Intestinal absorption of polyunsaturated phosphatidylcholine in the rat,” Hoppe Seylers Z Physiol. Chem., vol. 357, no. 9 (September 1976): 1321–1331.
6. M Picq et al., “DHA metabolism : targeting the brain and lipoxygenation,” Molecular Neurobiology, vol. 42, no. 1 (August 2010): 48–51.
7. PL Wood et al., “Oral bioavailability of the ether lipid plasmalogen precursor, PPI-1011, in the rabbit: a new therapeutic strategy for Alzheimer’s disease,” Lipids in Health and Disease, published online December 5, 2011.
8. MH Davidson et al., “A novel omega-3 free fatty acid formulation has dramatically improved bioavailability during low-fat diet compared with omega-3-acid ethyl esters: The ECLIPSE (Epanova compared to Lovaza in a pharmacokinetic single-dose evaluation) study,” Journal of Clinical Lipidology, vol. 6, no. 6 (November 2012): 573–584.
9. C Cruz-Hernandez et al., “Benefits of structured and free monoacylglycerols to deliver eicosapentaenoic acid (EPA) in a model of lipid malabsorption,” Nutrients, vol. 4, no. 11 (2012): 1781–1793.