Bioavailability etc. of Omega-3 – Nov 2018

A Comprehensive Review of Chemistry, Sources and Bioavailability of Omega-3 Fatty Acids
Mateusz Cholewski Monika Tomczykowa 2© and Michal Tomczyk k michal.tomczyk@umb.edu.pl
Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Biaiystok, ul. Mickiewicza 2a, 15-230 Biaiystok, Poland; matchol.bot@gmail.com
Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Biaiystok, ul. Mickiewicza 2a, 15-230 Biaiystok, Poland; monika.tomczyk@umb.edu.pl

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Omega-3 fatty acids, one of the key building blocks of cell membranes, have been of particular interest to scientists for many years. However, only a small group of the most important omega-3 polyunsaturated fatty acids are considered. This full-length review presents a broad and relatively complete cross-section of knowledge about omega-3 monounsaturated fatty acids, polyunsaturates, and an outline of their modifications. This is important because all these subgroups undoubtedly play an important role in the function of organisms. Some monounsaturated omega-3s are pheromone precursors in insects. Polyunsaturates with a very long chain are commonly found in the central nervous system and mammalian testes, in sponge organisms, and are also immunomodulating agents. Numerous modifications of omega-3 acids are plant hormones. Their chemical structure, chemical binding (in triacylglycerols, phospholipids, and ethyl esters) and bioavailability have been widely discussed indicating a correlation between the last two. Particular attention is paid to the effective methods of supplementation, and a detailed list of sources of omega-3 acids is presented, with meticulous reference to the generally available food. Both the oral and parenteral routes of administration are taken into account, and the omega-3 transport through the blood-brain barrier is mentioned. Having different eating habits in mind, the interactions between food fatty acids intake are discussed. Omega-3 acids are very susceptible to oxidation, and storage conditions often lead to a dramatic increase in this exposure. Therefore, the effect of oxidation on their bioavailability is briefly outlined.

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Bioavailability

Bioavailability is a relative term, which can refer to both the speed of absorption and the quantity of the substance absorbed. The speed can be understood as the rate at which the substance is absorbed from the gastrointestinal tract and reaches the portal system. Absorption of the substance occurs in the gastrointestinal tract only to a certain extent, depending on many factors. The extent of absorption and the speed of substance transport to the portal circulation describe the bioavailability in the narrower sense. Traditionally, bioavailability can also be considered in a broader context, taking into account the amount of substance that reaches the systemic circulation or the place of physiological destiny (activity) [167]. This broader approach is particularly important when considering the effect of metabolic processes and excretion on the transport of substances from the portal circulation. Not all of the absorbed substance reaches the systemic circulation or tissue compartment consistent with the physiological destination. This difference in amount is very important from the point of view of pharmacokinetics and dietary planning.
Fatty acids may be present in the body as free fatty acids, bound to glycerol, to form triacylglycerol (TG), diacylglycerol (DAG) or monoacylglycerol (MAG), or to form a composition of membrane phospholipids. In naturally occurring TG molecules, LC PUFA occupies the second position [167]. In the phospholipids of cell membranes the latter position is competed by EPA and DHA with arachidonic acid, and if necessary, they are released by the enzyme phospholipase A2 and are used to synthesise eicosanoids [136]. Otherwise, in the human brain, VLCFAs (C34-38) are attached to the skeleton of glycerol (glycerol moiety), which in phospholipids are located in the sn-1 position [122].
In fish and fish oils, LC omega-3 PUFAs are mainly found as triacylglycerides and free fatty acids [167,168]. In Krill oil phospholipids are also an important fraction of these fatty acids (30-65% of EPA and DHA), mainly phosphatidylcholine [167,169-171]. EPA and DHA represent approximately 18% and 12%, respectively, of the content of naturally occurring fish oils [167]. However, due to the transesterification process, oil blends often contain much more of both EPA and DHA. This process is related to the substitution of the removed glycerol backbone with ethanol, resulting in the formation of ethylesters (EE), which can then be converted to re-esterified TG (rTG)-ethanol is enzymatically removed, resulting in free fatty acids being released, then attached by enzymes back to the glycerol backbone [167,168]. An example of a drug in which the content of EPA (DHA) is increased as a result of transesterification is Lovaza [168,172]. Another method to increase the content of EPA and DHA has been used in Epanova. Glycerol is removed and replaced with a hydrogen atom, which, in combination with the released fatty acid, forms a carboxylic acid. Ethylesters (of which Lovaza is composed) require the hydrolysis of the ester bond by pancreatic lipase before they release the free fatty acids that can be absorbed in the small intestine. This step is not required by the carboxylic acids. Interestingly, EPA and DHA-EE are also absorbed unchanged. However, this form accounts for < 1% of the total pool of EPA and DHA in circulation after ingestion of omega-3 acids EE [168].
In rTG, LC PUFAs take up not mainly the sn-2 position (which takes place in natural TG), but can also (simultaneously) be bound in the position of sn-1 or sn-3 with equal paradigmicity. rTG particles frequently contain two LC PUFAs—then the probability of binding EPA and DHA in the sn-1 or sn-3 position is higher than in the sn-2 position [167]. According to Schuchardt, [167] binding of LC omega-3 PUFA to glycerol in the sn-1/3 position facilitates the lipase hydrolysis of the bond, thus increasing bioavailability. According to Dyerberg, [173] the presence of MAG and DAG in rTG mixtures increases the absorption of LC PUFAs in the intestine due to the easier formation of micelles. Bandarra [174], in turn, based on the results of his research on hamsters, proves that the location of DHA in the sn-2 position increases the absorption of this acid in the intestine and its incorporation into tissues. However, a certain limitation of Bandarra's study is that the author used a commercially available fish oil, which is known only to be rich in DHA with an unspecified binding site with the glycerol backbone—we do not know what part of DHA is associated in positions other than sn-2.

Methods of Measuring the Bioavailability of Omega-3 Fatty Acids

We can measure omega-3 FAs concentration in plasma, serum, blood cells and lymph. The content of FAs in the plasma reflects the short to medium-long supply of fatty acids in the diet, while the concentration of fatty acids in the blood cells is usually a good indicator of long-term bioavailability [167,175]. As far as the long-chain omega-3 fatty acids are concerned, it is possible to measure many markers that indicate the presence of DHA in a specific form, but only one (the level of phospholipid EPA in plasma) that is useful for determining the level of EPA [167,176]. Admittedly, erythrocyte EPA is a weak dose-dependent indicator of LC omega-3 PUFAs substitution at normal dietary levels, however (sum of), erythrocyte EPA and DHA concentration seems to be, as will be mentioned below, a relatively good indicator of long-term bioavailability and also reflects the content of LC omega-3 PUFAs in non-blood tissues [167,177]. In Browning's study, [177] EPA + DHA-PC (in the case of sudden changes in intake) and platelet/mononuclear cells EPA + DHA (in the case of long-term consumption assessment) were considered biomarkers that best represent the intake of fish with high fat content in a typical UK population (1-4 servings a week).

Chemical Binding

The bioavailability of omega-3 fatty acids can also be expressed by calculating different coefficients, the most important of which seem to be omega-3 index, Cmax and AUCt (area under the (concentration-time) curve), the modification of which is the incremental area under the curve (iAUCt) [169].
The omega-3 index is defined as the proportion of the sum of EPA and DHA content in the total fatty acid content in the erythrocyte (erythrocyte membrane), and is expressed as a percentage [167,178,179]. It is a good indicator of long-term bioavailability (from the last 80-120 days [179]) due to the long lifetime of erythrocytes and their high number in the blood.
Plasma content of EPA and DHA, as well as many other fatty acids, weakly correlates or does not correlate at all with the levels of these acids in the erythrocyte membrane [179]. The omega-3 index is also a good indicator of the incorporation of fatty acids into tissues, and this applies not only to gastrointestinal tissues, but also to the myocardium, liver, and kidney [167,180]. It was established that the supply of long-chain omega-3 fatty acids at the level of 1 g/d may result in an increase in the omega-3 index by two percentage points over eight weeks [181]. The omega-3 index is influenced by many factors, such as smoking, physical activity (the intensity of which decreases the omega-3 index), and genes [178].
The maximum levels of EPA and DHA in plasma (Cmax) can be determined five-nine h after administration, while the persistent levels of EPA and DHA in plasma are achieved within two weeks of daily supplementation. The half-life of EPA and DHA after repeated administration is 37 h and 48 h, respectively [170].
The bioavailability of omega-3 fatty acids varies depending on the type of chemical binding (lipid structure), and can be ranked as follows: PL > rTG > TG > FFA > EE [167,178,179,182,183]. Both EE and rTG are not natural components of dietary oils, but they are created in the process of their chemical modification, which is called transesterification. As a result, highly concentrated oils containing up to 90% of EPA and DHA can be obtained [167]. The hierarchical order presented above does not, however, reflect reality to the extent that some authors would like it to. In their trial Kohler et al. [178] proved that the bioavailability of EPA and DHA in the form of phospholipids does not have to be greater than the bioavailability of these acids in the form of triglycerides—the bioavailability of EPA and DHA in krill meal was comparable to their bioavailability in fish oil, despite a slightly higher total fat content in krill meal. Yurko-Mauro et al. [184] achieved similar results—krill oil containing almost 44% of phospholipids showed bioavailability similar to the bioavailability of fish oil, both in the form of triglycerides and ethyl esters. This indicates the (great) share of non-chemical binding (and total fat content) factors in shaping the bioavailability of omega-3 fatty acids [169,178,185]. One explanation may be the observation of Nordoy, Reference [186], who noticed similarly good absorption of omega-3 fatty acids (including EPA and DHA) in the form of TG and EE if they are administered as a component of fish oil in equivalent amounts. Both krill oil, and to a lesser extent, krill meal and fish oil, which are rich in fat.
Many studies, however, maintain that krill oil, especially when very rich in phospholipids, is characterized by extremely high bioavailability, significantly higher than triglyceride-rich fish oil [170,187-189]. Those also find an understandable explanation. Between the omega-3 PUFAs bound to phospholipids and those linked to triglycerides, there is some difference associated with predestination to a specific type of blood transport and metabolism in the liver. DHA-TG is preferentially assigned to LDL-PL, while DHA-PL is preferentially assigned to HDL-PL. It was also found that omega-3 PUFAs, including DHA, in the form associated with phospholipids, are more intensely embedded in tissues. This is probably due to the better availability of LC n-3 PUFA-PL acids contained in krill oil than those present in fish oil LC n-3 PUFA-TG for liver beta-oxidation pathways [189]. Krill oil inhibits de novo lipogenesis, but enhances fatty oxidation [170].

Brain Transport

Omega-3 acids are incorporated into the cell membrane of many organs and tissues, above all the heart, nervous tissue and retina [167]. Oral supplementation with omega-3 PUFAs increases the content of these acids in the cerebrospinal fluid [190]. Efficient passage of the blood-brain barrier, however, requires carrier particles—in the case of DHA, it is 1-lyso, 2-docosahexaenoyl-glycerophosphocholine (LysoPC-DHA), which increases intracerebral DHA transport up to 10-fold. It is a brain-specific particle and does not facilitate the transport of DHA to the heart, liver or kidney, although detailed studies are required in humans. Carriers (transporting DHA to the brain) with potentially better properties are synthesized, an example of which is obtaining of AceDoPC (1-acetyl,2-docosahexaenoyl-glycerophosphocholine) [191].

Parenteral Administration

Most of the studies, especially those based on humans, which serve to determine the bioavailability of omega-3 acids, apply to their oral administration. It is difficult to fully validate the parenteral administration of omega-3 fatty acids in relation to the healthy population, because this method of supply is reserved mainly for patients undergoing intensive therapy, both adults and preterm infants [142,192,193]. In addition, it is worth noting that parenteral administration of mixtures based on fish oil may lead to biochemical liver damage and even the progression of fibrosis in this organ [194].
Al-Taan et al. [195] conducted a study on 20 patients awaiting the surgical removal of colorectal metastases from liver cancer. These individuals had normal liver function tests and plasma lipid levels within the reference range. The aim of the study was to assess the content of fatty acids in plasma phosphatidylcholine and erythrocytes during and after intravenous infusion of oil emulsion. Phosphatidylcholine (PC) is the main phospholipid that can be found in the circulation (blood) and erythrocyte (membrane) during and shortly after intravenous infusion of the oil emulsion. Parenteral administration of DHA and EPA lipid emulsion allowed a rapid and significant increase in their blood levels (EPA/DHA in plasma PC and EPA in erythrocytes). However, EPA levels returned to their initial values five-12 days after the end of the infusion. Not only Al-Taan, but also Browning [177], suggested a quicker turnover of EPA than DHA in cells, and thus this effect occurs with both oral and intravenous supply, with a different time of administration. The fact of a relatively short infusion of oil emulsion is also significant.

Matrix Effect and Emulsification

Many authors emphasize the key role of the fat content in food in the absorption of omega-3 acids ('the matrix effect' [167,179]) and suggest the necessity of introducing a recommendation to consume formulations containing omega-3 fatty acids with high-fat food [167,178,196]. Schuchardt underscores the lack of the expected cardioprotective effect in the German population supplementing omega-3 PUFAs during breakfast due to the relatively low-fat typical German breakfast [167]. Similarly, American society is in the habit of eating a low-fat breakfast, which in their case contains only 16% of the fat consumed during the day [197]. In addition, the fat content of food is sometimes inversely proportional to the amount of omega-3 fatty acids it contains. For example, high-fat plants may have few omega-3 fatty acids—Entandrophragma angolense—a potential health benefit food source has fat estimated as 59.43% of fresh weight. However, 33.29 weight % of Entandrophragma seed oil contains oleic acid and only 0.2 weight % is alpha-linolenic acid [139,198]. Chia, on the other hand, contains oil in the amount of 27 g per 100 g of seeds, of which 64.04% is ALA and only 14.98% are saturated fatty acids [68]. Soybean contains slightly less fat. However, the high content of linoleic acid and the unfavourable omega-6/omega-3 ratio make it a food of dubious health value, especially because it has been shown that induction of obesity in mice with soybean oil is possible [95,139,199].
The fat contained in the diet stimulates the pancreas to secrete fat-digesting enzymes and the gallbladder to eject bile that contains bile salts, which emulsify fats and activate pancreatic lipase [196]. However, it is recommended that persons with a high cardiovascular risk should reduce the supply of animal fat. On the other hand, substituting animal fat with vegetable fat is not necessarily a good solution, taking into account eating habits, ubiquitous overweight and often low content of omega-3 fatty acids in rich-fat plants. These people benefit from the achievements of nanotechnology, among which Cavazos-Garduno [200] mentions nanoparticles, nanospheres, nanocapsules, solid lipid nanoparticles (SLN), self-emulsifying drug delivery systems (SEDDS) and nanoemulsions, which significantly improve the absorption of fatty acids in a low-fat environment. Research indicates that particles smaller than 0.2 micrometers are absorbed better.
The self-microemulsifying delivery system (SMEDS) contains LC n-3 FA-EE and a number of compounds that are emulsified in the stomach without the need for a high-fat meal [196]. It has been known for many years that emulsified omega-3 acids are characterized by equally good bioavailability in poor and high fat environments [196,201]. Qin analysis [196] indicates that the absorption of DHA + EPA EE contained in SMEDS preparations is 6 times higher than DHA + EPA EE alone. To confirm the effectiveness of SMEDS technology, long-term studies are needed to analyze the embedding process of the above-mentioned fatty acids in the structure of cell membranes, especially erythrocytes.
The benefits of emulsification were also described by Puri, [138]. He used similar self-nanoemulsifying drug delivery systems (SNEDDS).
Absorption of omega-3 fatty acid ethylesters can also be enhanced through the use of Advanced Lipid Technologies (ALT). ALT is, according to the manufacturer, a special lipophilic system that, regardless of the supply of food and the fat content thereof, increases the bioavailability of lipid-based compounds, including omega-3 fatty acid ethyl esters, generating the spontaneous formation of micelles. An example of a preparation equipped with this component is SC401 (DHA and EPA ethylesters + Advanced Lipid Technologies). In the supply of low-fat food, the intake of SC401 was associated with significantly higher values demonstrating the bioavailability of DHA and EPA than Lovaza (nearly 2-fold higher Cmax and 3-fold higher AUC(0-last)).
Free fatty acids are another example of a formula well absorbed with a low fat content in the diet. Epanova bioavailability in conditions of low fat supply is much greater than with Lovaza (AUC(0_t) for Epanova is four times higher than for Lovaza), which in turn, consumed with high-fat food, has similar bioavailability as SC401 accepted in conditions of low food supply [201].
Vegetarians and vegans will also be able to use the emulsification technique in line with their own lifestyle, as the results of the latest research indicate the possibility of using vegetable proteins from pea, lentil, and faba bean as emulsifiers [202]. Moreover, oil-in-water nanoemulsions are constructed using vegetarian oils (algae and flaxseed oil) [202,203].
A very common method of the incorporation of nanoemulsion into food is its transformation, most often into the form of powder, using microencapsulation [204]. Microencapsulation stabilizes the oil mixture, protects it against oxidation, and eliminates the phase difference often present when mixed with food [181]. Microencapsulation of fish oil increases the bioavailability of omega-3 PUFAs to a value very close to the bioavailability of these acids in meals rich in fish oil in liquid form [205]. Emulsification increases the bioavailability of LC omega-3 PUFAs by increasing the efficiency of incorporation into triacylgliceryde-rich lipoproteins [206]. Emulsifiers also modify the expression of genes responsible for the transport of fatty acids in enterocytes [143]. Emulsification (pre-emulsification) increases the absorption of LC PUFAs, including DHA, EPA, and ALA, but does not have the effect on the absorption of fatty acids having shorter chain length and fewer unsaturated bonds [143,207,208].
Of course, emulsification elevates not only the bioavailability of omega-3 acids in the blood, but also in the lymph. Emulsifiers used in food are part of this mechanism. For example, soya lecithin added to flaxseed oil increases the number and size of ALA-rich chylomicrons produced by enterocytes. However, another emulsifier, sodium caseinate, significantly reduces the absorption of ALA in the intestine. This is probably related to the effect on the expression of the FABP2 gene, which is involved in the transport of fatty acids in enterocytes. It was noted that the presence of soy lecithin in the emulsion was accompanied by high expression of this gene, whereas the use of sodium caseinate was associated with a decrease in FABP2 gene expression [143]. This process requires careful research on humans, because it is likely that the effect of emulsifiers on gene expression should be considered during food production, especially when we expect a specific healing effect.

Bioavailability - Conclusions

Opinions about the bioavailability of omega-3 fatty acids are divided.

• Some believe that phospholipid-bound acids are absorbed better, as well as more intensely incorporated into tissues than those associated in triglyceride form, due to the specificity of blood transport and better accessibility of beta-oxidation pathways.
• Others, however, are of the opinion that factors unrelated to the type of chemical binding play a key role, and the fat content in food is decisive.
• In addition, promising effects seem to result from the use of special lipophilic systems and nano(micro)emulsions.
  However, the type of emulsifier used should be taken into account, as some influence the expression of the gene involved in the transport of fatty acids in enterocytes in various ways.
• Fatty acids affect each other's metabolism in the body, which is of dietary importance.
• Several times higher consumption of omega-6 than omega-3 fatty acids is recommended by most researchers.
• Omega-3 fatty acids, like all fatty acids, are highly susceptible to oxidation.

There are methods that disrupt this process and may be used in the production of supplements, but the significance of the process itself for bioavailability is unclear.