Polyunsaturated Fatty Acids: Health Impacts

In order to encourage discussions on "polyunsaturated fatty acids" and health, the Brazilian branch of the International Life Sciences Institute (ILSI) promoted the XII International Workshop Series on Foods with Functional Properties and/or Health needs consumption impact on cardiovascular diseases and cancer; influence on gene expression; immunological system and inflammation; sources these fatty acids; benefits during and cost and benefit of supplementation; and recommendations of consumption and/or supplementation with these fatty acids are specific to particular groups and still require further studies. With respect to regulations in terms of legislation, each country/authority recommends different content properties and/or health claims. The event generated prospects for research fields, development, and regulation of polyunsaturated fatty acids in the scientific community and industries. pregnancy and breastfeeding; supplementation during pregnancy; maintenance of the skin and epithelial cells of the digestive system; structure of cell membranes; anti-inflammatory action; and maintenance of normal LDL cholesterol level.


INTRODUCTION
Polyunsaturated fatty acids (PUFA) contain more than one unsaturation in their molecules and due to this feature they have the potential to be beneficial to health. The most studied fatty acids are the essential, namely: linoleic acid (LA) and α-linolenic acid (ALA); and those considered long-chain fatty acids: eicosapentaenoic acid (EPA); docosahexaenoic acid (DHA); and arachidonic acid (AA), which can be divided into two classes, omega-3 (w-3, n-3): ALA, DHA, and EPA, and omega-6 (w-6, n-6): LA and AA. The "XII International Workshop Series on Foods with Functional Properties and/or Health Claims for Polyunsaturated Fatty Acids: Impacts on Health" held in São Paulo, Brazil, on 28 and 29 November 2013, addressed the effect of these fatty acids on health, since there may be controversial evidence regarding some topics and scientific discussions of great value.
The goal of the event was to consolidate the knowledge and scientific advances in recent studies addressing the relationship between PUFAs and health. The topics presented were: (a) Overview: what they are and how they function in the body; (b) nutritional needs, dietary reference intakes (DRI) and consumption; (c) natural/industrial sources; (d) gene expression and nutrigenomics; (e) cardiovascular diseases and diabetes; (f) cancer; (g) importance and benefits associated with DHA in pregnancy and childhood; (h) balance between omega-3/omega-6 and aging; (i) effects of omega-3 fatty acids (EPA and DHA) on inflammation biomarkers; (j) cost and benefit of interventions with omega-3; (k) regulatory status in Brazil and in the world. Representatives of academia, industry and regulatory agencies, and experts from Brazil, Chile, Israel, the United States and the United Kingdom participated in the event. The workshop was coordinated by Professor Franco Lajolo from the University of São Paulo, São Paulo, Brazil.

OVERVIEW
Fatty acids participate in various processes in the body, such as the characterization of lipids and plasma levels. They have action on inflammatory processes, the hepatic lipid metabolism, and adipose tissue.
PUFAs have their importance related to their melting point and chemical structure (carbon chain folding, location of methylene carbon, and number of double bonds), and may exert different functions in the body [1,2].
In the 1900s (industrial revolution), there was increase in the consumption of fat and oils, which are essentially composed of saturated fatty acids (SFA) and PUFAs of the omega-6 series. On the other hand, there was a decrease in the consumption of PUFAs of the omega-3 series. This imbalance was associated with circulatory problems [3,4].
Omega-3 and omega-6 PUFAs are essential for the body and compete for desaturase and elongase enzymes originating differentiated series of eicosanoids (prostacyclin, thromboxane, and leukotrienes), which will have specific functions in each tissue type, namely: production and inhibition of platelet aggregation; anti-inflammatory, chemotactic, and vasodilator effect; and uptake of cholesterol from the tissues. It is worth mentioning that polyunsaturated and trans fatty acids may interfere in the mechanism of these enzymes and inactivate the eicosanoids. The vast majority of the eicosanoids derived from omega-6 fatty acids have proinflammatory and proarrhythmic effects and induce fever, pain, bronchoconstriction, proaggregating effect, and vasoconstriction. Eicosanoids derived from omega-3 fatty acids have anti-inflammatory, antiarrhythmic and anti-aggregation effects, and decreased oxidative stress [5,6,7].
The main PUFAs representative of the omega-3 fatty acids are: ALA; EPA; and DHA, and of the omega-6 fatty acids are: LA and AA [4,8].
Omega-3 PUFAs have proven to be beneficial for health. In cardiovascular cases, they inhibit platelet aggregation (anti-thrombotic effect), stimulate vessel dilation, have anti-inflammatory effect, reduce chemotaxis of leukocytes, inhibit the synthesis of triacylglycerides in the liver by inhibiting the secretion of smaller VLDL particles which become larger LDL particles (atherogenic), stimulate the reverse transport of this cholesterol favouring its capture to the liver and its elimination through the bile duct [9].
These fatty acids have great significance in brain development, especially during pregnancy and early life, and they are incorporated into the retina. DHA has active participation in the following brain processes: synaptogenesis; neuronal migration; and neurogenesis. Its importance is related to fluidity in the cell membranes of the central nervous tissue and rods and cones in the retina. It increases light sensitivity of photoreceptors; facilitates the movement of neurons from the ventricular to the peripheral zone (neurogenesis), stimulates preand postnatal development of glia cells, and stimulates synaptogenesis in preformed neurons [10].

NUTRITIONAL NEEDS, DIETARY REFERENCE INTAKES, AND CONSUMPTION (FAMILY BUDGET SURVEY)
The increased consumption of saturated fats is associated with cardiovascular diseases, which are the leading cause of death in the world (30% of worldwide mortality) [

NATURAL/INDUSTRIAL SOURCES -OMEGA-3
Due to scientific evidence of the benefits of omega-3 PUFAs-brain and visual development in children, immunity, eye health, heart health, cognitive development, inflammation, and cancer-consumption can achieve around 1.3 million m 3 /year, considering the current world population and the recommendation for supplementation between 250 and 500 mg/day [18,20].
Marine microorganisms are the main source for consumption and production of encapsulated fish oil. Fish consume these microorganisms and accumulate fatty acids. On the other hand, these microorganisms can be genetically modified with plant genes, fungi, or animals and they produce oil. Fish can be consumed or have their oil extracted. Oils are encapsulated and can be consumed or used for food and drinks enrichment [2,1].
The current scenario demonstrates that fish oil production is declining and other sources are being used in addition to fish, such as algae and microorganisms. Approximately one third of fish products are oil and the species used are mainly anchovy and tuna. The difficulty in using these sources is due to different concentrations of omega-3 PUFAs in each fish species, as well as their individual amount [19,20].
According to the predictions for mass supplementation, there will be a difference of Other possible sources are being commercially evaluated for extraction of oil rich in omega-3 PUFAs from fish and shrimp. Other sources, such as zooplankton, algae, fungi, and plants genetically modified are being developed. Each source has its own peculiarity: fish is the largest source with variety of quality and quantity. As a result of new emerging technologies for oil extraction, fatty acids are found in different forms (phospholipids, triacylglycerides), with highest concentration of >90%, and have the ability to ensure the nutritional demand; shrimp: consumed in the diet has high levels of DHA and omega-3 PUFAs found in various forms and concentrations; however, the demand in the market is relatively small; zooplankton: commonly known as "krill"-successful in the United States and Australia-is original from Antarctica and serves as food for whales. It is particularly found in the form of phospholipids and is the best bioavailable source with EPA/DHA levels of <20% and high EPA concentration; algae: predominant source of omega-3 PUFAs in the market. There are two types of microorganisms and one of them is photosynthetic, naturally found in the form of triacylglycerides.
They produce high concentrations of DHA and EPA. They are more expensive than fish oil and cheaper than krill oil; fungi: currently the greatest production of EPA. They are a commercial possibility, but little used; genetically modified plants: widely studied and little found for marketing. They are potentially promising as new technology, but this source that posses many challenges, such as cost, viability, and sensory [19,21].

GENE EXPRESSION AND NUTRI-GENOMICS
Nutrigenomics is the application of highthroughput genomics tools in nutrition research. Is a "omic" discipline that seeks to understand the relationship between the genes and the nutrients through the cell response to nutrient. This process of cell responses can be divided into steps that include: genomics, transcriptome, proteome and metabolome. All these steps should be known in order to promote health. Brieflly, the cells capture the nutrients and activate a cascade of intracellular reactions, including the activation of transcription factors promoting the gene expression into the DNA (genomics), which transmit this information to the outside of the DNA through ribonucleic acid messengers (mRNA -transcriptome) that produce proteins (proteomics), externalizing the response to nutrientes (metabolome). Nutrients may change any of these steps and the cell response depends on the nutrients captured. Nutrigenomics can promote an increased understanding of how nutrition influences metabolic pathways and homeostatic control, how this regulation is disturbed in the early phase of a diet-related disease and to what extent individual sensitizing genotypes contribute to such diseases. Ultimately, nutrigenomics will allow effective dietary-intervention strategies to recover normal homeostasis and to prevent diet-related diseases The authors previously showed that, in HER2overexpressing breast cancer cells, HER2 is present in a complex equilibrium of preassociated active or inactive homodimers, heterodimers and monomers on the cell membrane. A key factor in HER2 receptor activation is their location in the cell membrane. For the activation and transmission of signals to occur, the HER2 receptors must be located in lipid rafts. Lipid rafts are specific compartments of the cell membrane rich in cholesterol and saturated phospholipids that, when clustered together, function as operating platforms for signaling molecules. To ensure lipid raft synthesis, HER2 promotes the activation of fatty acid synthase (FASN). FASN increases the synthesis of saturated FA frequently used for the lipid raft formation in the cell membrane. This guarantees the proper localization and activation of HER2 homo-or heterodimeric receptors. Therefore, in breast cancer cells, it is possible that the overexpression of HER2 receptors may be accompanied by an increase in lipid raft microdomains in the cell membrane, thereby establishing a vicious cycle of aberrant prosurvival cell signaling [27,28].
The upregulation of saturated FA biogenesis represents a "lipogenic benefit" for the proliferation and survival of breast cancer cells by providing lipid raft components for the proper localization and activation of HER2 in the cell membrane. However, accumulation these lipids in nonadipose tissue promptly stimulates lipolysis and apoptosis and can act as an inhibitory feedback signal for endogenous FA synthesis. On the other hand, these events seem to be avoided in HER2-overexpressing breast carcinoma cells, through the conversion and storage of FAs as triglycerides by peroxisome proliferator-activated receptor gamma (PPARγ). Rather than preventing lipotoxicity, the transcriptional activation of PPARγ increase the expression of genes related to uptake and transport of exogenous FA, contributing to the establishment of lipogenic phenotype in HER2overexpressing cells. Therefore, in these breast cancer cells, upregulation of FASN appears to be a downstream manifestation of an early and common deregulation of upstream regulatory circuits that affect the lipogenic genetic program.
In HER2-overexpressing breast cancer cells, the lipogenic genetic program, in addition to FA synthesis, requires the coordinated expression of genes involved in the following: (a) the conversion and storage of excess saturated FAs (e.g., palmitate) to triglycerides, thereby avoiding lipotoxicity; and (b) the uptake and transport of other exogenous FAs, which are necessary to maintain a constant supply of lipids/lipid precursors, membrane lipid raft production, and lipid-based posttranslational protein modifications in these highly proliferative cells. The treatment of the HER2-overexpressing breast cancer cells with DHA, (a)increase the percentage of DHA in the cell membrane, (b) disrupt the lipid rafts, (c) inhibit signaling pathways initiated by HER-2, (d) decreased lipogenesis gene expression and (e) induce cell apoptosis. According to nutrigenomics analysis these data suggested that DHA might be a useful therapeutic agent for controlling HER2-mediated oncogenesis [27,28].
Therefore, it should be confirmed which nutrients might have potential effects on genetics and usefull in the disease prevention.

IMMUNOLOGICAL SYSTEM AND INFLAMMATIONS
The immunological system features a complex set of many cell types, which produce a large number of responses in order to protect the host from foreign agents, such as bacteria, fungi, viruses, and mutant cells. Firstly, any immune response involves the recognition of the pathogen or other foreign material and, secondly, the preparation of a reaction targeted at eliminating that element. In broad terms, the different types of immune response fall into two categories: non-specific responses (non-adaptive or innate) and immune responses (acquired). An inappropriate immune response may cause diseases such as rheumatoid arthritis and inflammatory bowel disease. The increase in the inflammatory response is present in clinical situations, such as cancer, type 2 diabetes, obesity, and cardiovascular diseases. [29,30,31,32].
Studies have demonstrated that the incorporation of EPA and DHA into phospholipids of mononuclear cells and neutrophils is dosedependent. It is worth mentioning that individuals who had received supplementation with fish oil exhibited increased EPA and DHA concentrations in phospholipids of membranes in mononuclear cells and concomitant reduction in the AA/EPA ratio in the plasma membrane [33,34].
Changes in the constitution of membrane phospholipids directly influence the synthesis of mediators of lipid derivatives such as eicosanoids.
Therefore, omega-3 PUFA supplementation causes a competition between eicosapentaenoic acid (omega-3 PUFA) and arachidonic acid (omega-6 PUFA) as precursors in the synthesis of eicosanoids. This competition favours the synthesis of 3-and 5-series prostaglandins and leukotrienes, respectively, to the detriment of 2-series prostaglandins and thromboxanes, and 4-series leukotrienes, which feature pro-inflammatory properties. Therefore, arachidonic acid is potentially pro-inflammatory, whereas the presence of omega-3 PUFA limits this effect, since 3-series prostaglandins and thromboxanes and 5-series leukotrienes feature reduced pro-inflammatory potential [35,36].
SFAs are involved in molecular mechanisms that trigger inflammatory response in humans. In this context, it is observed that lauric SFA acid can activate the Toll-like receptor 4 (TLR4) and, consequently, the NF-κB signaling pathway. On the other hand, omega-3 PUFAs (EPA and DHA) feature opposite effect, namely, they inhibit the activity of the NF-κB transcription factor, which indicates that this is one of the potential mechanisms of action that explain the antiinflammatory effect of fatty acids (EPA and DHA). It is also worth mentioning that omega-3 and omega-6 PUFAs are also involved in the resolution of inflammation [37,38,39].
Therefore, PUFAs act in the body in three respects: cellular immune function; systemic inflammation; and intestinal mucosa. EPA and DHA can be embedded in the cell membrane as phospholipids and act in "lipids rafts", in addition to influencing signal transduction, activation and inhibition of transcription factors, and modulation of the eicosanoid synthesis; as described in Fig. 1 [40].

CARDIOVASCULAR DISEASES AND DIABETES
The relationship between food and cardiovascular diseases is widespread and occurs through the regulation of cholesterol levels in blood. Functional food, such as omega-3 PUFAs, should be assessed with respect to consumption and supplementation [14,41].
Two studies, one conducted by Jakobsen et al. Possible benefits of omega-3 fatty acids are confirmed through improved lipid profile, decreased blood pressure, platelet aggregation, blood viscosity and circulating pool of catecholamine, and increased endothelium relaxation [14,45]. A recent study conducted by Mozaffarian and Wu [46] with more than 280,000 individuals revealed inverse relationship between fish intake and cardiovascular diseases. Omega-3 PUFA reduces the risk of death from cardiovascular diseases and its consumption should be at least 250 mg/day, or fish at least twice a week.
A study conducted by Burr et al. [47] found that individuals supplemented with fish oil or consuming fish after myocardial infarction exhibited 29% reduction of deaths. A study known as GISSI [48] showed that 11,232 individuals who were followed up for 3.5 years after acute myocardial infarction had had an early benefit. The group supplemented had 21% decrease in mortality in 21% (response in 90 days), 45% in sudden death (120 days), and 30% in cardiovascular mortality (240 days). There was no difference observed between the groups after that period. Yokoyama et al. [49] conducted a similar study with a lower number of individuals submitted to treatment with statins and found no differences between the groups with and without EPA supplementation.
Rizos et al. [50] confirmed that the benefit of omega-3 PUFAs with respect to mortality decreases over the years. This may be due to studies conducted with few events and short follow-up. Medical treatment and prevention of cardiovascular diseases have changed over the years.
A study conducted in 2010 [51] found that there was no difference between sudden death and mortality in the groups with and without omega-3 PUFA supplementation for individuals after myocardial infarction undergoing modern therapy (No. = 3,851).
Another study [52] used margarines and assessed coronary events and cardiovascular diseases in four groups (placebo; diet + EPA/DHA; diet + ALA; and diet + EPA/DHA + ALA) and found no difference between the groups. Rizos et al. [50] conducted a study with 68,680 individuals and found an association between the consumption of omega-3 PUFAs and the reduction in all mortality causes (cardiac death, sudden death, and myocardial infarction). On the other hand, the consumption had no effect on smokers. That same study assessed the reviews and meta-analyses conducted between 1995 and 2012 and found an average association of 0.96%, revealing that omega-3 PUFA had no influence.

CANCER
Omega-3 PUFA, EPA and DHA alter the basic structure of the cell membrane and promote its fluidity and elasticity, thereby changing the Ionic permeability and the organization of proteins in the membrane [53]. It is known that DHA changes the composition of lipid rafts, which can influence signal transduction proteins, which in turn modify the regulation of immune, inflammatory, and tumourigenic responses [54,55].
The inflammatory response is related to various processes, such as structural changes of the cell membrane, resolvines and NF -k B, synthesis of eicosanoids, adhesion molecules, and chemotaxis. Due to the great number of double bonds in n-3 fatty acids present in lipid rafts, they are easily peroxidized by reactive oxygen species. This lipid peroxidation can change the cell cycle of malignant cells. Thus, omega-3 fatty acids can inhibit early phases of tumour growth and decrease their replication progression [56,57].
Specifically, DHA can promote increased apoptosis, decreased cell proliferation and angiogenesis, and inhibition of carcinogenesis. These mechanisms can result in the optimization of the treatment of cachexia, and radio-and chemotherapy [58,59].
The cell membrane of cancer cells has its pattern changed and PUFAs are present in smaller amount. On the other hand, SFA and cholesterol exhibit higher concentration, which increases the rigidity of the cell membrane and hinders lipid peroxidation and the activity of antioxidant enzymes. In other words, cancer cells adapt themselves to reduce the production of free radicals, avoid apoptosis, and promote cancer proliferation [54,60].
A study conducted by Weylandt et al. [61] with mice supplemented with omega-3 PUFAs found that the number and diameter of hepatocellular carcinoma were significantly smaller in the supplemented group than in the control group. MacLean et al. [62] found that cancer prevention may be related to high intake of omega-3 PUFAs; however, studies on prostate cancer have shown that DHA concentrations are directly linked to increased risk of developing the disease and its severity.
Dietary DHA is readily incorporated into the fatty tissue and stimulates the anti-tumour action of anthracyclines and sensitises breast tissue cells to docetaxel. DHA-enriched diets increase tumour regression after irradiation; however, the addition of vitamin E eliminates the sensitivity to irradiation induced by DHA. Due to their easy peroxidation, omega-3 fatty acids are sold in products containing high doses of vitamin E [63].
DHA increases the activity of diverse drugs with different modes of action and its incorporation is different in every individual. High concentrations of this fatty acid during first-line chemotherapy can improve survival of the patient with cancer [64,65,66].
In terminally ill patients, EPA and DHA seem to increase appetite and weight gain, as well as the level of physical activity. Immunomodulatory diets contain omega-3 PUFA and its intake is associated with the reduction in postoperative complications [67,69]. Fig. 2 shows a summary of PUFAs action (DHA, EPA and AA).

Fig. 2. Mechanisms of PUFAs action of cancer
Adapted from Larsson et al. [68] 9. IMPORTANCE AND BENEFITS ASSOCIATED WITH DHA

IN PREGNANCY AND CHILDHOOD
Lipids provide more than 50% of the energy in infants. PUFAs are present in all tissues and cells of the body, and 10% of the dry weight of the brain is composed of DHA [2,70].
There are studies that have been conducted since 1929 addressing the relationship between PUFAs and growth/development of children/babies. DHA is embedded in the membranes of phospholipids through the diet or the metabolism of its precursor (ALA) by the action of desaturase and elongase enzymes [2,71].
DHA is of fundamental importance in the structure and function of the nervous tissue. There is a close relationship between its content in the brain and increased learning and adaptation ability, and it is related to the establishment of neural circuits. This fatty acid increases differentiation and neural growth, synaptogenesis, neurogenesis, and neuroprotection in the brain [70,72]. During pregnancy, the intake of DHA is important for pregnant women, because it allows longer pregnancies, decreases insulin resistance, gestational diabetes, and the risk of post-partum depression. For the baby, it improves visual acuity and colour perception. It can increase by up to 4 points the intelligence coefficient, improves learning and memory ability, and decreases the incidence of attention deficit. Studies conducted with animals and humans have shown that the consumption of DHA and its incorporation has a relationship with psychomotor development, intelligence, and ability to resolve problems [2,70,72].
The DHA Oxford Learning and Behaviour (DOLAB) study conducted by Richardson et al. [70] showed significant improvement in reading comprehension in 12 children aged 7-9 years supplemented with 600 mg/day of DHA from algae. There was improvement within three months and mainly in the group with greater difficulty. Montgomery et al. [72] found that low levels of DHA are associated with poor reading and memorization ability, as well as greater questioning and rebelliousness towards their parents. Lassek and Gaulin [73] observed that levels of DHA in breast milk contributed very significantly to the grades obtained by children in the PISA mathematics test (Programme for International Student Assessment) applied in 28 countries.
DHA levels in breast milk explain more than 20% of the performance variance in an international cognitive test. It is the only variable explained that is not socioeconomic. Its concentration in breast milk is influenced by the consumption of fish. Larger values have been observed in Japan and Philippines, and lower in North American countries and Chile [74].
A study conducted by Nobili et al. [75] assessed children with liver diseases and found that the consumption of DHA significantly decreased the accumulation of fat in the liver and the level of triglycerides after six months, and increased insulin sensitivity. Table 2 shows the recommendations of the Food and Agriculture Organization for PUFA intake in babies and children, and Table 3 show the recommendations for pregnancy and breastfeeding [15].

W3/w6 BALANCE AND AGING
Synaptogenesis has increasing formation during development (children), decreases during the aging process and the loss occurs the other way around. Myelination and neurotransmitters/ hormones behave the same way in the brain and these are the three most important processes that influence synapse that changes the brain chemistry. The decrease in the key factors with age is related to cognition, motor activity, Alzheimer's disease, multiple sclerosis, and Parkinson's disease. Different brain processes mature at different ages. The cortex is mature at nine years of age, the motor function at 15, and the sensory function at five [76].
The nutrients reach the brain through nutrition and the blood-brain barrier. It can absorb EPAs and DHAs directly or metabolize through the precursor ALA. The same enzymes are used for series of omega-3 and omega-6 PUFAs, and the overload of one of them can impair the synthesis of other via [76,77].
The fluidity of the membrane is altered by decreased PUFA and increased cholesterol, which is directly related to the age due to the oxidation of fatty acids and ethanol, the presence of toxic metabolites, and decreased non-toxic steroids and metabolites. Restricted diet, intellectual stimulation, and oxidative stress also influence this fluidity, which is related to neural conduction, ion channels, enzymes in the membrane, and receptors for neurotransmitters. They also interfere in the physiology (thermoregulation, pain, sleep), cognition (learning and memorization), and diseases (Alzheimer's, Parkinson's, epilepsy) [78].  Studies conducted with different LA/ALA ratios showed that 4:1 was the most efficient, since it reversed the cognitive decline induced by neurotoxins and age, and protected from agents that induce epilepsy, stress and inflammation. This ratio is a perfect set of fatty acids that transported by blood reaches the brain intact and may be separated by a specific enzyme [77].
A study conducted by Yehuda et al. [78] with a ALA/LA ratio of 1:4 showed improved sleep in the group supplemented with omega-3 PUFAs, since sleep is a problem during aging. Treatments with essential fatty acids featured benefits, such as improved quality of life and cognition.

EFFECTS OF FATTY ACIDS (EPA AND DHA) ON INFLAMMATION BIOMARKERS
Omega-3 PUFAs are ingested in the diet. Their digestion and absorption occur in the gastrointestinal tract. They are transported and distributed throughout the body via the lymphatic and blood system and deposited in the cell membranes [79].
Bioavailability can be defined in two ways: (a) the speed and the extent to which the substance is absorbed in the intestinal tract and enters the circulation with possible losses occurring in the metabolism and excretion; and (b) the amount of the substance that actually reaches the systemic circulation or the physiological site of its activity. How is it possible to know the amount incorporated and its path and the status of omega-3 PUFAs? [79].
Biomarkers of quick incorporation are nonesterified fatty acids, phospholipids, cholesterol esters, and triglycerides in plasma. On the other hand, biomarkers of slow incorporation are the blood mononuclear cells, platelets, and red blood cells. The assessment of total omega-3 PUFAs in blood can have a great variation due to different times of incorporation [45,80].
The assessment of omega-3 PUFA status can be performed by: (a) the serum levels and the plasma-short-term availability indicating the amount absorbed in the body. The advantage is the assessment of the speed, extent of absorption and influencing factors (composition of the diet), and the disadvantage is the inadequacy of the indicator to assess the extent absorbed in tissues; and (b) levels of EPA and DHA in the membranes of the erythrocyteslong-term availability-and it is possible to assess whether the supplementation is adequate. The advantage is that EPA and DHA concentrations in the membrane of the erythrocyte are well correlated with omega-3 PUFAs concentration in the cell membranes and other tissues (myocardium, liver, and nerves) [81,82]. The definition of omega-3 PUFA content and the sum of EPA and DHA in red blood cells is expressed as a percentage of total fatty acids [83].
The increased consumption of fish is correlated with higher levels of omega-3 PUFAs and supplementation. Individuals with omega-3 PUFA content between 4 and 8% have less likelihood of having a cardiovascular accident and for those with content greater than or equal to 8% this risk is low. Case control studies have shown an inverse relationship between omega-3 PUFA content and the first cardiac event. A study conducted by Aarsetoey et al. [84] found that there is no reduction in cardiovascular disease when there are high levels of omega-3 PUFAs, but, on the other hand, there is no negative correlation. Socioeconomic factors-such as education-influence omega-3 PUFAs levels, the greater this factors, the greater fatty acids concentrations [85].

COST AND BENEFIT OF INTERVENTIONS WITH OMEGA-3
Omega-3 PUFAs bring benefits throughout life in the following stages: pregnancy and childhood (cognitive and visual development); childhood/children/adolescents (cognitive, attention, and visual development), over 50 years old (visual, neurological, cardiovascular, and bone health); and between 20 and 40 years old (women's health, pregnancy, and cardiovascular health). The economic return most quickly reported with supplementation-compared to spending on diseases-refers to cognitive development, prevention of prematurity, prevention of cognitive decline, and prevention of cardiovascular diseases. This way, the following cases should be taken into consideration: Pregnancy -One out of 10 babies are born prematurely around the world (about 15 million per year). Approximately, one million children die every year due to complications caused by prematurity. In addition, premature babies have many problems during growth, such as attention and visual disorders. The investment should be performed in women's health and care provided to the babies during the first year of life. According to studies, supplementation with 600 mg/day of DHA increases 2.87 days the gestational period and 172 g the weight of the newborn. The cost for each premature baby is about 51,600 U.S. dollars per year, whereas the cost of DHA supplementationconsidering a prematurity incidence of 10%, and an odds ratio of 2.69-, would be USD4,608, and would avert costs of USD32,418, which represents a return of 700% [70,86,87,88,89].

Cognitive health in older adults -
According to the Memory Improvement with Docosahexaenoic Acid Study (MIDAS), [90] the supplementation dose assessed in older adults (over 55 years old) was 900 mg/day. Considering that supplementation with 1,000 mg/day of DHA would cost 92.15 U.S. dollars per patient/year, the total amount spent would be two billion U.S. dollars. In this way, supplementing all older adults over 55 years would cost seven billion U.S. dollars and 13 billion U.S. dollars would be saved preventing new cases of Alzheimer's disease, revealing a return of 186% [90,91].
Cardiovascular disease -Supplementation with omega-3 PUFAs decreases the incidence of cardiovascular diseases, reduces all mortality causes-death from cardiac causes and sudden cardiac deathand is also associated with the increase of five years in the survival of cardiac patients. The cost of treatment for cardiovascular diseases in the United States is 100 billion U.S. dollars per year and the expected total cost related to cardiovascular diseases between 2013 and 2020 is 600 billion U.S. dollars. On average, 137,210 events per year or 1.1 million accumulated events related to cardiovascular diseases could be avoided by supplementing all adults over 55 with diagnosed CHD. Hospitals costs of 2.06 billion U.S. dollars a year, with an accumulated saving of 16.46 billion U.S. dollars or net savings of 4 billion dollars could be achieved by this measure from 2013 to 2020 [92][93][94][95][96][97].

REGULATORY STATUS IN BRAZIL AND IN THE WORLD
Several countries have legislation to regulate the information about the contents provided in the packages of processed products. Table 4 shows the criteria related to these claims in several countries [98][99][100][101][102][103][104].
The recommendation for nutritional property established by the FDA is valid until December 2015. After this date, new recommendation that does not provide for such DHA and EPA properties will enter into force [99].
In addition to content claims, packages of food may contain health claims revealing the benefits of omega-3 PUFA supplementation. Each country has different claims and the most common is related to cardiovascular diseases.
The following list describes these regulations in different locations: European Union (2012): [105] "Alphalinolenic acid helps in maintaining normal blood cholesterol levels". Food should be a source of omega-3 fatty acid and inform the consumer that the beneficial effect is obtained with daily intake of 2 g ALA.
"Docosahexaenoic acid helps in maintaining the normal functioning of the brain". Food should contain at least 40 mg of DHA powder per 100 g or 100 kcal and inform the consumer that the beneficial effect is obtained with daily intake of 250 mg DHA.
"Docosahexaenoic acid contributes to the maintenance of normal visual conditions". The food should contain at least 40 mg of DHA powder per 100 g or 100 kcal and inform the consumer that the beneficial effect is obtained with daily intake of 250 mg DHA.
Eicosapentaenoic acid (EPA)/ docosahexaenoic acid (DHA): "Eicosapentaenoic and docosahexaenoic acids contribute to the normal functioning of the heart". Food should be at least a source of omega-3 fatty acids and inform the consumer that the beneficial effect is obtained with daily intake of 250 mg EPA and DHA. European Food Authority -EFSA [106] (available at: http://www.efsa.europa.eu/en/panels/nda.htm?wt rl=01) There is a request from the Commission of Nutrition and Allergy to the Scientific Committee of Health Claims concerning docosahexaenoic and eicosapentaenoic acids related to: development of the brain, eyes and nerves; maintenance of normal brain function; maintenance of normal vision; maintenance of normal heart function; maternal health; Table 4. Nutritional Information about omega-3 PUFAs content in several countries

Source
High content Very high in European Union (1924/2006) [98] Minimum of 0.3 g ALA or 40 mg EPA + DHA per 100 g or 100 kcal.
Minimum of 0.6 g ALA or 80 mg EPA + DHA per 100 g or 100 kcal. FDA [99] Minimum