Agents of Resolution of Inflammation
Both omega-3 and omega-6 fatty acids are essential fatty acids. This means they cannot be synthesized by the human body and must be part of the diet. But why are they essential? The answer is primarily based on their role in modulating the inflammatory process that is essential for our survival. Inflammation is the foundation of our ability to fight off microbial invasions and to heal from physical injuries. Yet if that inflammatory response is not attenuated completely, the result is low-level chronic inflammation, which is the foundation of many chronic disease conditions.
In other words, our survival requires a therapeutic zone of inflammation that is not too high, but not too low.
Phases of inflammation
There are two distinct phases of the inflammatory process: The initiation and resolution phases (1). Both are active processes that each are modulated by essential fatty acids. Omega-6 fatty acids activate the initiation phase, and omega-3 fatty acids activate the resolution phase. As a consequence, the balance of omega-6 and omega-3 fatty acids in the diet will have a profound impact on the balance of the two phases that constitute the overall inflammatory response.
Synthetic pathways of omega-6 and omega-3 fatty acids
The hormones that control the inflammatory process are long-chain essential fatty acids (greater than 20 carbon atoms in length) whereas most other dietary essential fatty acids consist of shorter-chain (18 carbon atoms in length) omega-6 and omega-3 fatty acids. It is only the longer-chain essential fatty acids that play a role in inflammation as they serve as the substrates to make the hormones that control both the initiation and resolution of inflammation.
The metabolic conversion of these short-chain essential fatty acids into the longer-chain fatty acids necessary to make eicosanoids and resolvins consists of several rate-determining enzymes. Specifically, the rate-limiting enzymes are the delta-5-desaturase and delta-6 desaturase enzymes. These enzymes insert distinct double bonds into the essential fatty acid molecule until it reaches a spatial configuration that can be converted into distinct hormones that control the inflammatory process. These can be either pro-inflammatory eicosanoids coming from the long-chain omega-6 fatty acid arachidonic acid (AA) or pro-resolution resolvins coming from the long-chain omega-3 fatty acids eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).
The metabolic conversion of all three long-chain essential fatty acids (AA, EPA, and DHA) uses many of the same enzymes including the rate-limiting desaturase enzymes. Thus the levels of the omega-6 and omega-3 fatty acids in the diet will a have significant impact on the types of inflammatory hormones that are eventually synthesized. Furthermore, both the rate-limiting enzymes (delta-5 and delta-6 desaturase) are also under dietary control (2-5). In particular, both desaturase enzymes are stimulated by the hormone insulin (stimulated by the levels of carbohydrate in the diet) and are also inhibited by the hormone glucagon (stimulated by the levels of protein in the diet).
The metabolic pathways leading to AA and EPA are essentially the same as shown in Figure 1
Figure 1. Metabolic Pathways for the Synthesis of
Arachidonic Acid (AA) and Eicosapentaenoic acid (EPA).
The further conversion of EPA in DHA is more complicated as shown in Figure 2.
Figure 2. Metabolic Pathways for the Conversion of EPA into DHA
Although there are more steps in the metabolic conversion of EPA into DHA, it is an ongoing process so that DHA levels are always greater than EPA levels in the steady state.
Thus the balance of omega-6 and omega-3 fatty acids in the diet as well as the balance of protein and carbohydrate in the diet will have an impact on the eventual balance of the precursors of pro-inflammatory eicosanoids (coming from AA) and pro-resolution resolvins (coming from EPA and DHA).
Inflammation is like a faucet. Once you turn it on, you have to turn it off. If the faucet is not completely turned off either due to an overactive initiation phase or lack of a sufficient resolution phase, the result will be low-level chronic inflammation or cellular inflammation that will disrupt hormonal signaling pathways and eventually lead to a wide number of chronic conditions associated with inflammation (6).
Short history of inflammation
The cardinal signs of inflammation (really the initiation of inflammation) were described in ancient Roman times. These are heat, swelling, redness, and pain. In the 19th century, the German physician Virchow added loss of function as another cardinal sign of inflammation.
On the other hand, much of our knowledge about resolution is far more recent. Just as there are cardinal signs of inflammation, there are also cardinal signs of resolution. These include the removal of cellular debris from the site of inflammation and the regeneration and repair of damaged tissue. With an appropriate resolution response, loss of function does not occur. In retrospect, the loss of function described by Virchow is the consequence of unresolved resolution of inflammation.
Molecular events in inflammation
The innate immune system is the first responder in the development of inflammation (7). Factors that activate the innate immune system include microbial infections or physical injury. The diet can also activate the innate immune system. The pathway is either through sensors on the cell surface (toll-like receptors or receptors for advanced glycosylated end products or RAGE) or the internal formation of inflammasomes for the clearance of externally damaged cell products brought into the interior of the cell by endocytosis (8).
Regardless of the initiating factor, there are a number of complex events that leads to the migration of neutrophils to the site of inflammation (7,9). A key factor in this initiation phase of inflammation are pro-inflammatory eicosanoids (leukotrienes) that lead the neutrophils to the site of inflammation and also activate the key gene transcription factor nuclear factor kappaB (NF-κB) that accelerates the production of inflammatory cytokines and the COX-2 enzyme to enhance the intensity of the initial inflammatory response (10). This initial migration of neutrophils is followed by the later migration of macrophages into the same site. Whereas neutrophils are short-lived, the macrophages have a longer lifetime. Furthermore, the first wave of macrophages to reach the site of inflammation are activated to pro-inflammatory M1 stage to continue the initiation phase although at a lower intensity than the more highly active neutrophils. Resolvins catalyze the polarization of macrophages from pro-inflammatory M1 phase into the anti-inflammatory M2 phase. With that transition induced by resolvins, tissue repair and regeneration can take place (11).
Time course of inflammation
The time course of inflammation can be seen in Figure 3.
Figure 3. Time course of inflammation and resolution
What is not commonly understood is that anti-inflammatory drugs are also anti-resolution drugs. One can define anti-inflammation as the stoppage of the migration of the neutrophils to the site of inflammation. However, the neutrophils are necessary to initiate the synthesis of resolvins that cause the change in the polarization of the pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Therefore anti-inflammatory drugs can also be seen as anti-resolution drugs. It may be this inhibition of the resolution phase by anti-inflammatory drugs maybe a contributing factor to their side effects upon prolonged use (6).
There is a need for adequate levels of both for EPA and DHA for complete resolution since both fatty acids are the substrates for the production of different types of resolvins as shown in Figure 4.
Figure 4. Biosynthetic Pathways for Resolvin Synthesis
This is because EPA and DHA are the substrates for very different types of resolvins that bind to different receptors and have very different physical actions as shown in Figure 5.
Figure 5. Resolvin Receptors
This is why you need a combination of two to optimize the resolution response.
What is the appropriate intake of omega-3 fatty acids for adequate resolution?
Our knowledge of resolvins that drive resolution of inflammation is very recent compared to the understanding of eicosanoids that drive initiation of inflammation. Since both pro-inflammatory eicosanoids and pro-resolution resolvins are needed for an adequate inflammatory response, the question becomes what is the optimal dietary balance of long-chain dietary omega-6 and long-chain omega-3 fatty acids in the blood?
Estimates of the dietary intake of AA (the building block for pro-inflammatory eicosanoids) and EPA and DHA (the building blocks for pro-resolution resolvins) in Paleolithic populations has been estimated to been a approximately a 1:1 ratio (12). This would make teleological sense as neo-Paleolithic populations were exposed to a greater number of microbial invasions and physical injuries compared to modern man.
Today our largest contributor to inflammation is not microbial invasions or physical injuries, but our diet. This is because of the massive expansion of the intake of omega-6 fatty acids coming from refined vegetable oils (corn, soy, sunflower, safflower, and others). Since refined vegetable oils are now the least expensive source of dietary calories in the world, there increased consumption has dramatically altered the balance of the omega-6 and omega-3 fatty acid precursors for the generation of pro-inflammatory eicosanoids and pro-resolution resolvins. This imbalance is further enhanced by the growing use of refined carbohydrates in the worldwide diet that increases the generation of insulin, which accelerates the conversion of omega-6 fatty acids into AA (2-5). At the same time, there has been a significant decrease in the intake of fish rich in EPA and DHA due to a combination of overfishing and increasing cost. The end result has been a growing mismatch between the precursors initiation and resolution of inflammation on a worldwide basis.
Measuring adequate levels of omega-3s necessary for resolution comes from analysis of their levels in the blood. There are currently two methodologies used to do this. The first is the Omega-3 index, which is the summation of the percentage of EPA and DHA in the total fatty acid content of the blood. It is suggested that an ideal level would be greater than 8% of the total fatty acids consisting of these two omega-3 fatty acids which is similar to that found in the Japanese population (13). Unfortunately this marker tells nothing of the relative strength of the initiation to resolution phases.
The other commonly used marker is the AA/EPA ratio. This provides a better indication of the strength of the two phases of the inflammation process. From a methodological perspective, measuring more than two of the 35 fatty acids commonly found in the blood will give rise to significant errors in reproducibly. The first use of the AA/EPA ratio to demonstrate its reliability as a marker of inflammatory cytokines was demonstrated in 1989 (14).
DHA levels are always higher than EPA levels due to the constant metabolism of EPA into DHA. Therefore as long as the EPA levels are approximately 4% of total fatty acids, then the combined EPA and DHA will always be greater than 8%. Ideally the AA levels in the blood should be between 7 and 9% of the total fatty acids. Therefore a good clinical goal is to keep the AA/EPA ratio between 1.5 and 3. This is the range generally found in the Japanese population (15).
Why Not Eat More Fish?
The easiest way to improve either marker is to simply eat more fish rich in EPA and DHA. The Japanese are able to accomplish this because they are the largest consumers our fish in the world. Unfortunately, many popular fishes are lean and therefore low in omega-3 fatty acids so it requires eating large amounts of them to get adequate levels of omega-3 fatty acids needed for resolution.
However, there are other problems with consuming more fatty fish to increase the levels of omega-3 fatty acids in the blood. These problems are heavy metal and chemical toxin contamination. The larger a predatory fish, the greater the levels of mercury will be found in that fish. Common edible fishes such as swordfish and tuna fall into this category. More insidious is that all fish contain chemical toxins such as polychlorinated biphenyls (PCBs). These are known neurotoxins, carcinogens and endocrine disruptors (16,17). The higher the levels of the omega-3 fatty acids in a fish, the higher the levels of PCBs since these toxins are fat-soluble.
The solution to both problems (low levels of omega-3 fatty acids and high levels of chemical toxins) can be overcome by supplementation with refined omega-3 fatty acid concentrates.
History of omega-3 fatty acid concentrates
The history of fish concentrates started with the use of garum in ancient Roman times. Garum consisted of fermented fish intestines that were used throughout the Roman Empire as prized seasoning by all classes of individuals. The next major advance was the use of fermented cod liver oil that contained about 15% by weight of EPA and DHA. The introduction of heating in iron pots in the 1850s considerably speeded the extraction process of the fish oil from the cod liver. In the 1980s, the use of fish body began to replace cod liver oil for two reasons. One benefit was to increase the EPA and DHA content to approximately 30% of the total fatty acids and the second benefit was the reduction of Vitamin A levels in the final product to reduce potential toxicity of this vitamin.
In the 1990s, manufacturing technology using molecular distillation allowed the development of even higher EPA and DHA concentrates. This is the methodology used to make current prescription drugs consisting of omega-3 fatty acids. This older manufacturing technology has been improved in recent years with the introduction of super critical fluid extraction and improved thermal fractionation techniques to obtain even higher purity omega-3 fatty acid concentrates.
Measuring purity of omega-3 concentrates
Regardless of how the omega-3 fatty acid concentrates are made, their purity must be constantly tested, in particular relative to PCB levels and rancidity of the final product.
It is expensive to measure PCB levels, but necessary because every lot will be different because of natural variability in the fish stock used. Total Oxidation (Totox) is a marker of oil rancidity. This marker is the basis of all edible oil trading worldwide. Because of the high concentrations of EPA and DHA in omega-3 fatty acid concentrates the likelihood of developing rancidity is quite high thus the importance of ensuring adequate anti-oxidant protection to prevent the development of aldehydes and ketones during its stated shelf life.
Since prescription-grade omega-3 fatty acid concentrates developed in the 1990s are now generic products, their published standards can be compared to fish oil products sold in the mass-market (health food grade) and also to the omega-3 fatty acid concentrates (high-performance grade) made by newer technologies now available. The comparison of these general standards that govern oil quality is shown in Table 1.
Table 1. Purity Standards for Omega-3 concentrates
|Type of oil||Upper PCB levels (ppb)||Upper Totox Levels (meq/kg)|
|Health Food grade||90||26|
|Generic prescription grade||50||26|
It can be seen from Table 2 that omega-3 fatty acid concentrates sold by prescription only are not all that different in terms of PCB purity and rancidity from what is sold in a supermarket. However, under newer manufacturing technologies, it is possible to produce non-prescription omega-3 fatty acid concentrates of much higher purity. This is critical for long-term, high-dose applications.
Forms of omega-3 concentrates
There are three distinct forms of omega-3 fatty acid products sold. These are ethyl esters, free fatty acids, and reconstituted triglycerides. All three forms start out as ethyl esters since only this form can be concentrated by existing technologies. Virtually all prescription omega-3 fatty acid products available consist of the ethyl ester format since additional processing to convert them to free fatty acids or reconstituted triglycerides can oxidize the EPA and DHA molecules. As an example one could convert the ethyl ester into the free fatty acid after concentration, but the stability of free fatty acid is less than the ethyl ester. Alternatively, the ethyl ester could be reconstituted into triglyceride molecule. However, this triglyceride requires additional processing and any oxidized fatty acids cannot be removed since the increased molecular weight of the reconstituted triglyceride prevents its distillation.
Furthermore, the configuration of the fatty acids in reconstituted triglycerides is very different than those found in the natural triglyceride configuration. In the natural state, most of the EPA and DHA fatty acids are located primarily on the 2-position of the triglyceride molecule. The lipases in the digestive that convert the natural triglyceride to a monoglyceride for absorption specifically cleave the fatty acids in the 1 and 3 positions of the natural triglyceride thus leaving the EPA and DHA highly concentrated in the 2-position to give it better oxidative stability. In reconstituted triglycerides, all positions have the same concentrations of omega-3 fatty acids so when the reconstituted triglyceride is absorbed approximately 2/3 of the EPA and DHA will have been are released as free fatty acids that are more prone to oxidation. Furthermore, studies have indicated no statistically significant differences of these three forms in terms of absorption (18-21).
Natural fish oils cannot be concentrated or refined. Cod liver oil is rich in PCBs. Another example is krill oil, which is really an odd mixture of free fatty acids and phospholipids that have low in EPA and DHA content. Contrary to marketing statements, the absorption of krill oil is no better than ethyl esters or reconstituted triglycerides (22,23).
Benefits of increasing omega-3 fatty intake
The reason to increase omega-3s in the diet is to increase the precursor pool size to make resolvins thus accelerating resolution. The benefits of this can be shown in a genetically modified fat-1 mouse model in which genes have been inserted that convert omega-6 fatty acids to omega-3 fatty acids thus maintaining the AA/EPA ratio close to 1. Although a similar AA/EPA ratio can be obtained by feeding high-dose omega-3 fatty acid concentrates, the use of fat-1 mice gives a clear indication of the therapeutic benefits of maintaining a low AA/EPA without dietary intervention. In many ways, this fat-1 mice model is relatively immune to a wide range of chronic inflammatory conditions ranging from obesity to diabetes, to auto-immune conditions and neurological conditions compared to its wild-type counterparts (24-29).
It has also been shown in recent clinical studies of using reasonably high-dose omega-3 fatty acids (3-4 grams of EPA and DHA per day) that resolvin levels can be increased (30).
This use of high dose omega-3 fatty acids may be significant as seen in animal models with conditions associated with chronic inflammation. When the AA/EPA ratio is reduced to approximately one as seen in the transgenetic fat-1 mouse or upon supplementation with high-dose omega-3 fatty acids at the level of 100-200 mg/kg body weight per day these conditions are not apparent. This would translate to approximately 7 to 14 grams of EPA and DHA per day in a typical 70 kg human. It should be noted that these levels of daily intake of EPA and DHA are very similar to those estimated to have been consumed by neo-Paleolithic man (12).
Clinical benefits of high dose omega-3 concentrates
Animal models or the use of transgenetic fat-1 mice suggest that the use of high-dose omega-3 fatty acids to lower the AA/EPA ratio can bring the initiation and resolution phases of inflammation into a better balance. Are there clinical studies that support the same hypothesis? The answer appears to be yes, but only if therapeutic levels of EPA and DHA are used to lower the AA/EPA ratio where that balance of initiation and resolution is restored.
It was first demonstrated in 1989 that supplementing the diet of healthy subjects with 5 grams of EPA and DHA per day could reduce the AA/EPA ratio from 21 to 2.5 within 10 weeks. In that same time period the levels of pro-inflammatory cytokines from stimulated leukocytes were significantly reduced. Once the supplementation was discontinued, the AA/EPA ratio rose back to it original starting level as did the levels of the pro-inflammatory cytokines (14)
The largest cardiovascular study ever done (JELIS) took individuals all taking statins and with initially low AA/EPA ratios of 1.6 and lowered them even more with the addition of high levels of EPA. The further reduction of the AA/EPA ratio to 0.8 over a 3 ½ year period demonstrated a 20% reduction in cardiovascular events compared to the control group getting supplements of olive oil that had no effect on the their starting AA/EPA ratio (31). A more recent study has demonstrated that high-dose EPA and DHA (3.4 grams per day) reduced scar tissue formation on the heart muscle surface after a heart attack (32).
Two ocular studies with patients with age-related dry macular degeneration (AMD) have demonstrated significant improvements in visual acuity using between 5 and 7.5 grams of EPA and DHA per day (33,34). Studies with a much lower dose of EPA and DHA demonstrated no benefits suggesting a potential dose-response relationship (35).
Likewise studies using high-dose EPA and DHA (10 to 15 grams of EPA and DHA per day) have demonstrated significant improvement in children with ADHD (36,37). Other neurological conditions such as depression have also benefited from high-dose EPA and DHA supplementation (38,39). Finally in cases of severe brain trauma administration of 15 grams of EPA and DHA per day demonstrated significant improvements in their recovery (40).
Many chronic disease conditions may result from the imbalance of the initiation and resolution phases of inflammation. Thus the diet may represent an important factor in re-establishing a more appropriate balance between initiation and resolution. If so, anti-inflammatory nutrition using therapeutic levels of omega-3 fatty acids may prove a very attractive approach for management of such chronic disease conditions associated with chronic inflammation. It also suggests that maintaining a low AA/EPA ratio in the blood may have significant long-term potential in delaying the advent of chronic disease and therefore substantially reduce health costs in the future.
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