MARINE LIPID COMPOSITIONS AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to novel marine lipid compositions comprising combinations of omega-3 fatty acid rich functional phospholipids and omega-3 fatty acid rich triglycerides. In addition, food supplements, functional food, drugs and feed products comprising such compositions are provided along with methods of their use.

BACKGROUND OF THE INVENTION Marine lipids such as omega-3 rich triglycerides and omega-3 rich phospholipids can be isolated from a number of different natural sources such as fish, crustaceans, plankton, seals, whales as well as algae using extraction technologies. In addition, they can be prepared industrially using chemical or bio-catalytical methods such as enzyme catalyzed transesterification of crude soy lecithin with fish oil fatty acids [I].

The anti-inflammatory properties of omega-3 fatty acids are well known and the use as an anti-inflammatory agent has been described both for triglycerides and phospholipids [2-3]. Actually, omega-3 fatty acids are famous for their anti-inflammatory properties, and it has been shown that omega-3 fatty acids alleviate the symptoms of a series of autoimmune, atherosclerotic and inflammatory diseases including inflammatory bowel diseases and rheumatoid arthritis [4-6]. Suppression of inflammation has been proposed as one of the strategies to slow down the progress of these diseases. Hence, this invention discloses the effect on marine lipid compositions on the concentration of markers of inflammation such as TNF-α and other cytokines such as interleukin-1/3 and interleukin 6. In addition, since arachidonic acid (AA) is the predominant precursor of the eicosanoid mediators of inflammatory responses (prostaglandins, thromboxanes and leukorrienes), this invention discloses the reduction of AA level and the improvement in the EP A/ AA ratio in different lipid pools in tissues such as in the phospholipids isolated from adipose tissue, heart, testicles, plasma, brain and liver.

The bioavailability of EPA and DHA from fish oil triglycerides have been reported to be high- in healthy adults. However, for certain conditions i.e. pathological conditions such as extrahepatic cholestasis and for pre-term infants the absorption can be low. For example it

was shown that the absorption of DHA from egg lecithin in pre-term infants was 90% compared to 80% from triglycerides [7]. Absorption of long chain PUFA (AA and DHA) is less (75% and 62%, respectively) than the absorption of C 18 PUFA (94%) in pre-term infants [8]. The difference between Cl 8 PUFA and long chain PUFA absorption is likely to become less apparent in older children and adults. SaIa-Vi Ia et al [9-10] investigated the bioavailabilities of DHA-PL and DHA-TG in full term infants and found no differences based on plasma lipid enrichments. Valenzuela et al. [11] supplemented female rats with different forms of DHA including egg yolk PL and single cell algae TG. They found also no difference in absorption of DHA from PL and TG based on plasma lipid enrichments. However, the tissue and milk fat levels were higher in PL-DHA compared to the TG-DHA supplemented rats. These data indicate that although there were no differences in the bioavailability, efficacy with respect to tissue enrichment was higher for PL-DHA compared to TG-DHA. Furthermore, the relative absorption of EPA and DHA ethyl esters (4 g/d) compared to oleic acid calculated from peak concentrations was 94 and 100%, respectively. Estimates of relative absorption based on the area under the concentration curve indicated a relative absorption of 91% for EPA and 93% for DHA [12]. Bioavailability of C18:l, C18:2 and C18:3 in adult humans are close to 100% (note 94%.in preterm infants). Thus the bioavailability of EPA and DHA delivered in different forms is, according to previous, work likely to be over 90%.

SUMMARY OF THE INVENTION

In some embodiments, the invention provides a composition comprising a triglyceride and a phospholipids in a ratio ranging from 1 : 10 to 10: 1 ; said phospholipids having the following structure:

wherein Rl is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is a mixture of H, choline, ethanolamine, inositol and serine, said phospholipid having at least 1% of DH A/EPA, said phospholipids have a concentration of OH in the range of 25-50%. hi further embodiments,, the invention provides a marine lipid composition characterized by providing higher uptake of omega-3 fatty acids into plasma as compared to administration of purified triglycerides, phospholipids, or natural marine phospholipids. In further embodiments, the invention provides a composition characterized by efficiently improving the AA/EPA ratio in plasma phospholipids as compared to administration of purified triglycerides, phospholipids, or natural marine phospholipids. In still other embodiments, the invention is a marine lipid composition characterized by efficiently increasing the concentration of omega-3 fatty acids in tissues as compared to administration of purified triglycerides, phospholipids, or natural marine phospholipids. In still further embodiments, the invention the composition is characterized by reducing the concentration of biomarkers of inflammation as compared to administration of purified triglycerides, phospholipids, or natural marine phospholipids. In other embodiments of the invention, the marine lipid composition is formulated into an animal feed, a food product, a food supplement and a drug.

In some embodiments, the present invention provides a composition comprising phospholipids having the following structure:

wherein Rl is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is a mixture of H, choline, ethanolamine, inositol and serine, said phospholipid having at least 1% of DHA/EPA at positions Rl and/or R2 and from about 20-50% of OH at positions Rl and/or R2. In some embodiments, the composition is acylated in a range from about 55% to about 85%. In some embodiments, the omega-3 fatty acids are selected from the group consisting of EPA, DHA, DPA and α-linolenic acid (ALA). In some embodiments, the composition is

substantially free of organic solvents and volatile organic compounds such as short chain fatty acids, short chain aldehydes and short chain ketones. In some embodiments, the composition has at least 5% of a combination of EPA and DHA esterifϊed. In some embodiments, the composition has at least 10% of a combination of EPA and DHA esterified. In some embodiments, the composition has at least 20% of a combination of EPA and DHA esterified. In some embodiments, the composition has at least 30% of a combination of EPA and DHA esterified. In yet other embodiments, said composition contains from about 5%, 10%, 20% and 30 % EPA/DHA attached to position 1 and/ or position 2. In some embodiments, the composition has a ratio of EPA/DHA ranging from 1:1 to 4:1. In some embodiments, the composition has a ratio of EPA/DHA ranging from 2:1 to 4:1. In some embodiments, the composition is acylated in a range from 60% to 80%. In some embodiments, the composition is acylated in a range from 50% to 75%.

In some embodiments, the composition further comprises a lipid carrier in a ratio of from 1:10 to 10:1 to said phospholipids. In some embodiments, the lipid carrier and said phospholipids are in a ratio of from about 5:1 to 1:5. In some embodiments, the composition comprises from about 20% to about 90% of said phospholipid composition and from about 10% to about 50% of said lipid carrier. The present invention is not limited to any particular lipid carrier. In some embodiments, the lipid carrier is selected from the group consisting of a triglyceride, a diglyceride, an ethyl ester, and a methyl ester and combinations thereof. In some embodiments, the composition provides higher uptake of omega-3 fatty acids into _' plasma as compared to natural marine phospholipids when administered to subjects. In some embodiments, the composition improves the AAJEPA ratio in plasma phospholipids when administered to subjects as compared to natural marine phospholipids. In some embodiments, the composition increases the concentration of omega-3 fatty acids in tissues when administered to subjects as compared to natural marine phospholipids. In some embodiments, the composition reduces the concentration of biomarkers of inflammation when administered to subjects as compared to natural marine phospholipids. In some embodiments, the present invention provides a food product comprising the foregoing compositions. In some embodiments, the present invention provides an animal feed comprising the foregoing compositions. In some embodiments, the present invention provides a food supplement comprising the foregoing compositions. In some embodiments, the present invention provides a pharmaceutical composition comprising the foregoing compositions.

Tn some embodiments, the present invention provides methods of preparing a bioavailable omega-3 fatty acid composition comprising: a) providing a purified phospholipid composition comprising omega-3 fatty acid residues and a purified triglyceride composition comprising omega-3 fatty acid residues; b) combining said phospholipid composition and said triglyceride composition to form a bioavailable omega-3 fatty acid composition. In some embodiments, the bioavailable phospholipid composition is one of the compositions described above. In some embodiments, the methods further comprise the step of encapsulating said bioavailable omega-3 fatty acid composition. In some embodiments, the bioavailable omega-3 fatty acid composition has increased bioavailability as compared to purified triglycerides or phospholipids comprising omega-3 fatty acid residues. In some embodiments, the methods further comprise the step of packaging the bioavailable omega-3 fatty acid composition for use in functional foods. In some embodiments, the methods further comprise the step of assaying the bioavailable omega-3 fatty acid composition for bioavailability. In some embodiments, the methods further comprise administering the bioavailable omega-3 fatty acid composition to a patient. In some embodiments, the present invention provides a food product, animal feed, food supplement or pharmaceutical composition made by the foregoing process.

In some embodiments, the present invention provides methods for reducing symptoms of cognitive dysfunction in a child comprising administering an effective amount of a marine phospholipid composition, wherein said symptoms are selected from the group consisting of ability to complete task, ability to stay on task, ability to follow instructions, ability to complete assignments, psychomotor function, long term memory, short term memory, ability to make a decision, ability to follow through on decision, ability to self-sustain attention, ability to engage in conversations, sensitivity to surroundings, ability to plan, ability to carry out plan, ability to listen, interruptions in social situations, temper tantrums, level/frequency of frustration, level/frequency restlessness, frequency/level fidgeting, ability to exhibit delayed gratification, aggressiveness, demanding behavior/frequency of demanding behavior, sleep patterns, restive sleep, interrupted sleep, awakening behavior, disruptive behavior, ability to exhibit control in social situations, ability to extrapolate information and ability to integrate information. In some embodiments, the child exhibits one or more symptoms of Attention Deficit Hyperactivity Disorder (ADHD), is suspected of having ADHD, or has been diagnosed with ADHD. In some embodiments, the child

exhibits one or more symptoms of autistic spectrum disorder, is suspected of having autistic spectrum disorder, or has been diagnosed with autistic spectrum disorder. In further embodiments, the present invention provides methods of increasing cognitive performance in an aging mammal comprising administering an effective amount of a marine phospholipid composition. In some embodiments, the cognitive performance is selected from the group consisting of memory loss, forgetfulness, short-term memory loss, aphasia, disorientation, disinhibition, and behavioral changes. In some emboidments, the mammal is a human. In some emboidments, the mammal is a pet selected from the group consisting of cats and dogs. In some embodments, the mammal has symptoms of age-associated memmory impairment or decline.

The foregoing methods are not limited to the use of any particular marine phospholipid composition. In some embodiments, the marine phospholipid composition comprises phospholipids having the following structure:

wherein Rl is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is a mixture of H, choline, ethanolamine, inositol and serine, said phospholipid having at least 1% of omega-3 fatty acid moieties at positions Rl and/or R2. In some embodiments, the phospholipid composition comprises from about 20-50% of OH at positions Rl and/or R2. In some embodiments, the phospholipid composition further comprises a lipid carrier. In some embodiments, the phospholipid composition is prepared from natural marine phospholipids isolated from a marine organism. In some embodiments, the phospholipid composition is enzymatically prepared by reacting lecithin with DHA and EPA in the presence of an enzyme. In some embodiments, the lecithin is soybean or egg lecithin. In some embodiments, the omega-3 fatty acid moieties are selected from the group of EPA and DHA and combination thereof. In some embodiments, the effective amount of said

phospholipid composition comprises from about 300 to about 1000 mg omega-3 fatty acids. In some embodiments, the phospholipid composition is administered orally. In some embodiments, the phospholipid composition is provided in a gel capsule or pill.

In some embodiments, the present invention provides methods of treating a subject by administration of a marine phospholipid composition comprising administering a marine phospholipid composition to said subject under conditions such that a desired condition is improved, wherein said conditions is selected from the group consisting of fertility, physical endurance, sports performance, muscle soreness, inflammation, auto-immune stimulation, metabolic syndrome, obesity and type II diabetes. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. The present invention is not limited to any particular marine phospholipid composition. In some embodiments, the marine phospholipid composition comprises phospholipids having the following structure:

wherein Rl is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is a mixture of H, choline, ethanolamine, inositol and serine, said phospholipid having at least 1% of omega-3 fatty acid moieties at positions Rl and/or R2. In some embodiments, the phospholipid composition comprises from about 20-50% of OH at positions Rl and/or R2. In some embodiments, the phospholipid composition is prepared from natural marine phospholipids isolated from a marine organism. In some embodiments, the composition further comprises a lipid carrier. In some embodiments, the phospholipid composition is enzymatically prepared by reacting lecithin with DHA and EPA in the presence of an enzyme. In some embodiments, the lecithin is soybean or egg lecithin. In some embodiments, the omega-3 fatty acid moieties are selected from the group of EPA and DHA and combination thereof. In some embodiments, the effective amount of said phospholipid composition comprises from about 300 to about 1000 mg omega-3 fatty acids. In some embodiments, the

phospholipid composition is administered orally. In some embodiments, the phospholipid composition is provided in a gel capsule or pill. In some embodiments, the human is a male.

In some embodiments, the present invention provides methods for prophylactically treating a subject by administration of a marine phospholipid composition comprising administering a marine phospholipid composition to a subject under conditions such that an undesirable condition is prevented, wherein said undesirable condition is selected from the group consisting of weight gain, infertility, obesity, metabolic syndrome, diabetes type II, mortality in subjects with a high risk of sudden cardiac death, and induction of sustained ventricular tachycardia. In some embodiments, the subject is at risk for developing a condition selected from the group consisting of weight gain, obesity, metabolic syndrome, diabetes type II, mortality in subjects with a high risk of sudden cardiac death, and induction of sustained ventricular tachycardia. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. The present invention is not limited to any particular marine phospholipid composition. In some embodiments, the marine phospholipid composition comprises phospholipids having the following structure:

wherein Rl is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is a mixture of H, choline, ethanolamine, inositol and serine, said phospholipid having at least 1% of omega-3 fatty acid moieties at positions Rl and/or R2. In some embodiments, the phospholipid composition comprises from about 20-50% of OH at positions Rl and/or R2. In some embodiments, the phospholipid composition is prepared from natural marine phospholipids isolated from a marine organism. In some embodiments, the composition further comprises a lipid carrier. In some embodiments, the phospholipid composition is enzymatically prepared by reacting lecithin with DHA and EPA in the presence of an enzyme. In some

embodiments, the lecithin is soybean or egg lecithin. In some embodiments, the omega-3 fatty acid moieties are selected from the group of EPA and DHA and combination thereof. In some embodiments, the effective amount of said phospholipid composition comprises from about 300 to about 1000 mg omega-3 fatty acids. In some embodiments, the phospholipid composition is administered orally. In some embodiments, the phospholipid composition is provided in a gel capsule or pill.

DESCRIPTION OF THE FIGURES

Figure 1. The total amount of EPA consumed during the four-week rat trial (mean ± SE).

Figure 2. Relative EPA (20:5) content of plasma (mean + SE; n = 6).

Figure 3. Relative 20:5 content of red blood cells (mean + SE; n = 5-6).

Figure 4. Relative 20:5 content of monocytes (mean + SE; n = 5-6).

Figure 5. The composite change score for ADHD subjects receiving placebo and omega-3 phospholipids.

Figure 6. The composite change score for healthy subjects receiving omega-3.

Figure 7. Cognitive performance test of aged beagle dogs.

Figure 8. Schematic drawing of the experimental setup.

Figure 9. EPA levels in plasma as a function of hours after one bolus intake of a marine phospholipid composition.

Figure 10. EPA levels in plasma as a function of hours after one bolus intake of a marine phospholipid composition.

Figure 11. ARA levels in plasma as a function of hours after one bolus intake of a marine phospholipid composition.

DEFINITIONS

As used herein, "phospholipid" refers to an organic compound having the following general structure:

wherein Rl is a fatty acid residue or -OH, R2 is a fatty acid residue or -OH, and R3 is a — H or a nitrogen containing compound such as choline (HOCH ₂ CH ₂ N ⁺ (CH _S ) ₃ OH ^" ), ethanolamine (HOCH ₂ CHaNHa), inositol or serine. Rl and R2 cannot simultaneously be OH. When R3 is an -OH, the compound is a diacylglycerophosphate, while when R3 is a nitrogen-containing compound, the compound is a phosphatide such as lecithin, cephalin, phosphatidyl serine or plasmalogen.

The Rl site is herein referred to as position 1 of the phospholipid, the R2 site is herein referred to as position 2 of the phospholipid, and the R3 site is herein referred to as position 3 of the phospholipid.

As used herein, the term omega-3 fatty acid refers to polyunsaturated fatty acids that have the final double bond in the hydrocarbon chain between the third and fourth carbon atoms from the methyl end of the molecule. Non-limiting examples of omega-3 fatty acids include, 5,8,1 1,14,17-eicosapentaenoic acid (EPA), 4,7,10,13,16,19-docosahexanoic acid (DHA) and 7,10,13,16,19-docosapentanoic acid (DPA).

As used herein, the term "bioavailability" refers to the degree and rate at which a substance (as a drug) is absorbed into a living system or is made available at the site of physiological activity.

As used herein, the term "functional food" refers to a food product to which a biologically active supplement has been added.

As used herein, the term "fish oil" refers to any oil obtained from a marine source e.g. tuna oil, seal oil and algae oil.

As used herein, the term "lipase" refers to any enzyme capable of hydrolyzing fatty acid esters

As used herein, the term "food supplement" refers to a food product formulated as a dietary or nutritional supplement to be used as part of a diet.

As used herein, the term "acylation" means fatty acids attached to the phospholipid. 100% acylation means that there are no lyso- or glycerol-phospholipids.

As used herein, the term "normal child" means a child that has not been diagnosed as suffering from a cognitive disorder.

As used herein, the term "at risk child" means a child exhibiting one or more symptoms of a cognitive disorder.

As used herein, the term "cognition" is used to describe that operation of the mind process by which we become aware of objects of thought and perception including all aspects of perceiving, thinking and remembering.

As used herein, the term "psychomotor" refers to motor action directly proceeding from mental activity.

As used herein, the term "extracted marine phospholipid" refers to a composition characterized by being obtained from a natural source such as krill, fish meal, pig brain or eggs.

As used herein, the term "metabolic syndrome" refers a to syndrome marked by the presence of usually three or more of a group of factors (as high blood pressure, abdominal obesity, high triglyceride levels, low HDL levels, and high fasting levels of blood sugar) that are linked to an increased risk of cardiovascular disease and type 2 diabetes.

DETAILED DESCRIPTION OF THE INVENTION

This invention discloses that the uptake/absorption of omega-3 fatty acids attached to phospholipids are dependent on the level of LPL and GPL. Preferably, in order to ensure maximum uptake the level of LPL should be in the range of 15-45% and the level of GPL should be 0%. Furthermore, this invention discloses that the pure PC transesterified with EP A/DH A have a different effect on gene expression in the liver than 40% PC transesterified with EPA/DHA. It is disclosed that the two compositions regulated around 40 genes differently. Furthermore, the invention discloses that the EPA/DHA ratio is important. The treatment containing a EPA/DHA ratio of 2:1 regulated key enzymes involved in the inflammatory response (NF-κB) in a positive way, the treatment containing a EPA/DHA ratio of 1 : 1 did not.

A. Phospholipid Compositions

The present invention describes novel marine lipid compositions comprising an omega-3 containing phospholipid and a triacylglyceride (TG) in a ratio from about 1:10 to 10:1.

Preferably the ratio is in the range of from about 3:1 to 1 :3, more preferably the ratio is in the range of about 1 :2 to 2:1. Preferably, the TG is a fish oil such as tuna oil, herring oil, menhaden oil, cod liver oil and algae oil. However, this invention is not limited to omega-3 containing oils as other TG sources are contemplated such as vegetable oils. The phospholipids in the composition have the following structure:

wherein Rl is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is a mixture of H, choline, ethanolamine, inositol and serine. Attached to position 1 or position 2 are least 1% omega-3 fatty acids, preferably at least 5%, more preferably, at least 10% omega-3 fatty acids, up to about 15%, 20%, 30%, 40%, 50%, or 60% omega-3 fatty acids. The omega-3 fatty acids can be EPA, DHA, DPA or Cl 8:3 (n-3), most preferably the omega-3 fatty acids are EPA and DHA. The phospholipid composition preferably contains OH in position 1 or position 2 in a range of 25% to 50% in order to maximize absorption in- vivo.

In some embodiments, the present invention provides bioavailable and bioefficient omega-3 fatty acids. This invention shows that the novel marine lipid composition disclosed above enhances the uptake of the omega-3 fatty acid in vivo and incorporates omega-3 fatty acids more efficiently into tissues of adult rats than pure fish oil does. An embodiment of the invention is to use the marine lipid composition for efficient increase of omega-3 fatty acids in the liver, brain, adipose tissue, plasma, testicles and heart. Furthermore, this invention also discloses that the marine lipid compositions efficiently reduced the concentration of the pro-inflammatory precursor AA in total lipids and in phospholipids in tissues. It. is disclosed that the concentration of AA in the different lipid pools in the liver, brain, adipose tissue, plasma, testicles and heart can be more efficiently reduced than using fish oil. Hence, the composition can be used to improve the EP A/ AA ratio, which is a bio-marker of silent inflammation. The invention also discloses that the incorporation of the omega-3 fatty acids into monocytes is also more efficient using the claimed marine lipid composition as opposed to the fish oil. Yet another embodiment of the invention is to use the marine lipid composition to reduce chronic and acute inflammation in humans and in animals. Acute inflammation is mediated by granulocytes or polymorphonuclear leukocytes, while chronic

inflammation is mediated by mononuclear cells such as monocytes. Monocytes protect against blood-borne pathogens and moves quickly to sites of infection in the tissues, secreting large amounts of pro-inflammatory prostaglandins. Furthermore, low grade chronic inflammation may be the underlying cause of many life-style related diseases such as obesity, arthritis, diabetes type II, metabolic syndrome, Alzheimer's disease, osteoarthritis, inflammatory bowel disease, allergy and asthma [14]. Hence, the marine lipid composition can be used to treat and prevent diseases linked to chronic inflammation. This invention discloses that the inflammatory response of monocytes harvested from animals in lower in animals treated with the marine lipid composition compared to fish oil. The concentrations of the pro-inflammatory cytokines such as interleukin-1/3, interleukin-6 as well as tumor necrosis factor α (TNF-α) were reduced for the group fed the marine lipid composition compared to fish oil. These cytokines are important markers of real inflammation as for examples 11-1/3 induces fever. 11-6 also induces fever in addition to being linked to the acute phase response. TNF-α is involved in systemic inflammation as well and is released by white blood cells in the case of damage. It has a range of different biological effects such as increasing insulin resistance, stimulating the acute phase response in the liver and affecting the hypothalamus causing appetite suppression and fever.

This invention also discloses that the fatty acid composition of the brain and adipose tissue phospholipids changes after in take of omega-3 fatty acids for 30 days. A significant reduction of the arachidonic acids can be found in the phospholipids in the brain and adipose tissue for the rats given either the EPA- or DHA-rich PL diets (PL 1 and PL 2, respectively). This may affect the inflammatory response in this tissue and thereby have a great impact on cognitive diseases/conditions such as Parkinson's or and Alzheimer's where the inflammatory component is fundamental for the progression of the disease. This invention also discloses that the reduction of ARA is present also in the sn-2 position of the phospholipids in the brain. This is very important as the pro-inflammatory eicosanoids are produced from ARA, which are catalytically hydrolyzed from position 2 on the phospholipid by the action of phospholipase A2. The phospholipase A2 is released after stimuli at the cell wall, it then moves to the nuclear membrane where the hydrolysis of the phospholipid takes place.

In adipose tissue, accumulation of EPA and DHA in both total lipids (table 3) and PLs

(table 8) is substantial when omega-3 supplements were fed and negligible when the control

diet was fed. The increase was more pronounced in total lipids, which mainly consists of triglycerides (99% of fat cell lipid content). This invention demonstrates that omega-3 phospholipids can increase the accumulation of EPA/DHA into adipose tissue. This is important as the adipose tissue can function as a reservoir for these fatty acids. Arachidonic acid concentration in total lipids was higher in omega-3 supplemented animals, showing probably an increase of lipoprotein lipase activity, in agreement with the ability of omega 3 in decreasing plasma TAGs concentration. On the other hand, arachidonic acid levels in adipose tissue PLs were significantly lower in omega-3 supplemented animals than the levels in controls. Peculiar enough, the PL-EPA diet was the most efficient in decreasing arachidonic acid. In addition, the invention discloses that the reduction of ARA is also observed in the sn-2 position of the phospholipids of the omega-3 supplemented animals. This is very important as the pro-inflammatory eicosanoids are produced from ARA, which are catalytically hydrolyzed from position 2 on the phospholipid by the action of phospholipase A2. The phospholipase A2 is released after stimuli at the cell wall, it then moves to the nuclear membrane where the hydrolysis of the phospholipid takes place. The reduction of ARA in position 2 on the phospholipids may affect the inflammatory response in this tissue, which may have practical application in different pathologies of the adipose tissue and in its physiological activity of accumulation and release of fatty acids.

Fatty acid data from brain are well in line with the data from adipose tissue. Also in this tissue, we found a significant decrease of arachidonic acid in PLs, but surprisingly, only with PL-EPA and PL-DHA (table 7). On the other hand, DHA levels in both total lipids and PLs were not influenced by the omega-3 diets, while there was a small but significant increase in EPA levels. Lack of increase in DHA levels is likely to be attributable to the fact that the rats in this study were adults and pass the stage in development where they incorporate DHA in the brain (mainly PE). On the other hand, EPA being present at low concentration has more margin to increase. Furthermore, this may affect the inflammatory response in this tissue, which may have a great impact in such diseases as Parkinson's and Alzheimer's where the inflammatory component is fundamental for the progression of the disease. Positional distribution of arachidonic acid show that the ARA content is reduced for the EPA-PL groups, as stated before this is very important as it influences the proinflammatory eicosanoid production.

In liver, as expected, we found for all omega-3 groups a significant increase of EPA and DHA and decrease of arachidonic acid. No great differences were expected between total lipids and PLs because about 80% of liver total lipids are PLs (table 4, 9 and 14).

Heart total lipids and PLs (table 6 and table 11, respectively) showed a strong increase of EPA and DHA with a concomitant decrease of arachidonic acid when omega-3 supplements were fed. The strong decrease in the omega-6/omega-3 ratio in heart lipids is important considering the possible impact on the anti-inflammatory potential. Observed change in heart tissue fatty acids (increase of fatty acids with 6 or 5 double bonds) also suggests a possible increase in membrane fluidity. This change was most striking in the PL-DHA group where the increase of DHA was significantly higher than the increase in the TG-oil and PL-EPA groups. The fluidity of myocardium cell membrane seems to play an important role in controlling arrhythmia. Ventricular arrhythmia, is one of the main causes of sudden cardiac death. Furthermore, atrial fibrillation is another pathological state with a high incidence and important health consequences.

Testicular long chain PUFAs are of special interest because there is a high rate of production of prostaglandins from the omega-6 PUFA (arachidonic acid mainly) into the semen or seminal fluid. High rate of prostaglandin production does not indicate an active inflammatory process but a stimulus for the uterus smooth muscle to favour male fertility. An omega-3 induced decrease of arachidonic acid as observed in other tissue could be detrimental to the male fertility if it occurred also in testis. Furthermore, testicular tissue has also a high level of DPA (22:5 omega-6), which may serve as a reservoir for arachidonic acid. Arachidonic acid could be formed according to the need, through the retroconversion mechanism in the peroxisomes. A similar mechanism may take place with DHA to form EPA in other tissues. Our data (table 5) show an increase of EPA and DHA and a small decrease of arachidonic acid in the total lipids fraction when omega-3 fatty acids are fed. However, there is no change in arachidonic acid levels in PL when TG-oil and PL-EPA are fed and interestingly a significant increase in the PL-DHA group. Furthermore, DPA n-6 concentration in total lipids was not influenced by omega-3 supplementation but there was a significant increase in DPA in the PL-EPA group (table 5). Overall, these data seem to indicate that the diets with omega-3 did not change the arachidonic and DPA n-6 concentrations in a way that would predict negative effects on male fertility. In contrast, increase in arachidonic acid content of testicular PLs (table 10) when PL-DHA was fed and

increase in DPA when PL-EPA was fed could be interpreted to be positively associated with male fertility.

Another embodiment of the invention is to formulate the marine lipid compositions into a feed product for the purpose of reducing low grade chronic inflammation in animals. It can also be formulated into a food product and given to humans for the same purpose. Furthermore, it can be formulated as a functional food product, as a drug or as food supplement.

In some embodiments, the compositions of this invention are contained in acceptable excipients and/or carriers for oral consumption. The actual form of the carrier, and thus, the compositions itself, is not critical. The carrier may be a liquid, gel, gelcap, capsule, powder, solid tablet (coated or non-coated), tea, or the like. The composition is preferably in the form of a tablet or capsule and most preferably in the form of a hard gelatin capsule. Suitable excipient and/or carriers include maltodextrin, calcium carbonate, dicalcium phosphate, tricalcium phosphate, microcrystalline cellulose, dextrose, rice flour, magnesium stearate, stearic acid, croscarmellose sodium, sodium starch glycolate, crospovidone, sucrose, vegetable gums, lactose, methylcellulose, povidone, carboxymethylcellulose, corn starch, and the like (including mixtures thereof). Preferred carriers include calcium carbonate, magnesium stearate, maltodextrin, and mixtures thereof. The various ingredients and the excipient and/or carrier are mixed and formed into the desired form using conventional techniques. The tablet or capsule of the present invention may be coated with an enteric coating that dissolves at a pH of about 6.0 to 7.0. A suitable enteric coating that dissolves in the small intestine but not in the stomach is cellulose acetate phthalate. Further details on techniques for formulation for and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

In other embodiments, the supplement is provided as a powder or liquid suitable for adding by the consumer to a food or beverage. For example, in some embodiments, the dietary supplement can be administered to an individual in the form of a powder, for instance to be used by mixing into a beverage, or by stirring into a semi-solid food such as a pudding, topping, sauce, puree, cooked cereal, or salad dressing, for instance, or by otherwise adding to a food.

The compositions of the present invention may also be formulated with a number of other compounds. These compounds and substances add to the palatability or sensory perception of the particles (e.g., flavorings and colorings) or improve the nutritional value of the particles (e.g., minerals, vitamins, phytonutrients, antioxidants, etc.).

The dietary supplement may comprise one or more inert ingredients, especially if it is desirable to limit the number of calories added to the diet by the dietary supplement. For example, the dietary supplement of the present invention may also contain optional ingredients including, for example, herbs, vitamins, minerals, enhancers, colorants, sweeteners, flavorants, inert ingredients, and the like. For example, the dietary supplement of the present invention may contain one or more of the following: ascorbates (ascorbic acid, mineral ascorbate salts, rose hips, acerola, and the like), dehydroepiandosterone (DHEA), Fo-Ti or Ho Shu Wu (herb common to traditional Asian treatments), Cat's Claw (ancient herbal ingredient), green tea (polyphenols), inositol, kelp, dulse, bioflavinoids, maltodextrin, nettles, niacin, niacinamide, rosemary, selenium, silica (silicon dioxide, silica gel, horsetail, shavegrass, and the like), spirulina, zinc, and the like. Such optional ingredients may be either naturally occurring or concentrated forms.

In some embodiments, the dietary supplements further comprise vitamins and minerals including, but not limited to, calcium phosphate or acetate, tribasic; potassium phosphate, dibasic; magnesium sulfate or oxide; salt (sodium chloride); potassium chloride or acetate; ascorbic acid; ferric orthophosphate; niacinamide; zinc sulfate or oxide; calcium pantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxine hydrochloride; thiamin mononitrate; folic acid; biotin; chromium chloride or picolonate; potassium iodide; sodium selenate; sodium molybdate; phylloquinone; vitamin D ₃ ; cyanocobalamin; sodium selenite; copper sulfate; vitamin A; vitamin C; inositol; potassium iodide. Suitable dosages for vitamins and minerals may be obtained, for example, by consulting the U.S. RDA guidelines.

In further embodiments, the compositions comprise at least one food flavoring such as acetaldehyde (ethanal), acetoin (acetyl methylcarbinol), anethole (parapropenyl anisole), benzaldehyde (benzoic aldehyde), N-butyric acid (butanoic acid), d- or 1-carvone (carvol), cinnamaldehyde (cinnamic aldehyde), citral (2,6-dimethyloctadien-2,6-al-8, gera-nial, neral), decanal (N-decylaldehyde, capraldehyde, capric aldehyde, caprinaldehyde, aldehyde

C-IO), ethyl acetate, ethyl butyrate, 3 -methyl-3 -phenyl glycidic acid ethyl ester (ethyl-methyl-phenyl-glycidate, strawberry aldehyde, C- 16 aldehyde), ethyl vanillin, geraniol (3,7-dimethyl-2,6 and 3,6-octadien-l-ol), geranyl acetate (geraniol acetate), limonene (d-, 1-, and dl-), linalool (linalol, 3,7-dimethyl-l,6-octadien-3-ol), linalyl acetate (bergamol), methyl anthranilate (methyl-2-aminobenzoate), piperonal

(3,4-methylenedioxy-benzaldehyde, heliotropin), vanillin, alfalfa (Medicago sativa L.), allspice (Pimento, officinalis), ambrette seed {Hibiscus abelmoschus), angelic (Angelica archangelica), Angostura (Galipea officinalis), anise (Pimpinella anisum), star anise (Illicium verum), balm (Melissa officinalis), basil (Ocimum basilicum), bay (Laurus nobilis), calendula (Calendula officinalis), (Anthemis nobilis), capsicum (Capsicum frutescens), caraway (Carum carvi), cardamom (Elettaria cardamomum), cassia, (Cinnamomum cassia), cayenne pepper (Capsicum frutescens), Celery seed (Apium graveolens), chervil (Anthriscus cerefolium), chives (Allium schoenoprasum), coriander (Coriandrum sativum), cumin (Cuminum cyminum), elder flowers (Sambucus canadensis), fennel (Foeniculum vulgare), fenugreek (Trigonella foenum-graecum), ginger [Zingiber officinale), horehound (Marrubium vulgare), horseradish (Armoracia lapathifolia), hyssop (Hyssopus officinalis), lavender (Lavandula officinalis), mace (Myristica fragrans), marjoram (Majorana hortensis), mustard (Brassica nigra, Brassica juncea, Brassica hirta), nutmeg (Myristica fragrans), paprika (Capsicum annuum), black pepper (Piper nigrum), peppermint (Mentha piperita), poppy seed (Papayer somniferum), rosemary (Rosmarinus officinalis), saffron (Crocus sativus), sage (Salvia officinalis), savory (Satureia hortensis, Satureia montana), sesame (Sesamum indicum), spearmint (Mentha spicata), tarragon (Artemisia dracunculus), thyme (Thymus vulgaris, Thymus serpyllum), turmeric (Curcuma longa), vanilla (Vanilla planifolia), zedoary (Curcuma zedoariά), sucrose, glucose, saccharin, sorbitol, mannitol, aspartame. Other suitable flavoring are disclosed in such references as Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing, p. 1288-1300 (1990), and Furia and Pellanca, Fenaroli's Handbook of Flavor Ingredients, The Chemical Rubber Company, Cleveland, Ohio, (1971), known to those skilled in the art.

In other embodiments, the compositions comprise at least one synthetic or natural food coloring (e.g., annatto extract, astaxanthin, beet powder, ultramarine blue, canthaxanthin, caramel, carotenal, beta carotene, carmine, toasted cottonseed flour, ferrous gluconate, ferrous lactate, grape color extract, grape skin extract, iron oxide, fruit juice, vegetable juice,

dried algae meal, tagetes meal, carrot oil, corn endosperm oil, paprika, paprika oleoresin, riboflavin, saffron, tumeric, tumeric and oleoresin).

In still further embodiments, the compositions comprise at least one phytonutrient (e.g., soy isoflavonoids, oligomeric proanthcyanidins, indol-3-carbinol, sulforaphone, fibrous ligands, plant phytosterols, ferulic acid, anthocyanocides, triterpenes, omega 3/6 fatty acids, conjugated fatty acids such as conjugated linoleic acid and conjugated linolenic acid, polyacetylene, quinones, terpenes, cathechins, gallates, and quercitin). Sources of plant phytonutrients include, but are not limited to, soy lecithin, soy isoflavones, brown rice germ, royal jelly, bee propolis, acerola berry juice powder, Japanese green tea, grape seed extract, grape skin extract, carrot juice, bilberry, flaxseed meal, bee pollen, ginkgo biloba, primrose (evening primrose oil), red clover, burdock root, dandelion, parsley, rose hips, milk thistle, ginger, Siberian ginseng, rosemary, curcumin, garlic, lycopene, grapefruit seed extract, spinach, and broccoli.

In still other embodiments, the compositions comprise at least one vitamin (e.g., vitamin A, thiamin (Bl), riboflavin (B2), pyridoxine (B6), cyanocobalamin (B12), biotin, ascorbic acid (vitamin C), retinoic acid (vitamin D), vitamin E, folic acid and other folates, vitamin K, niacin, and pantothenic acid). In some embodiments, the particles comprise at least one mineral (e.g., sodium, potassium, magnesium, calcium, phosphorus, chlorine, iron, zinc, manganese, flourine, copper, molybdenum, chromium, selenium, and iodine). In some particularly preferred embodiments, a dosage of a plurality of particles includes vitamins or minerals in the range of the recommended daily allowance (RDA) as specified by the United States Department of Agriculture. In still other embodiments, the particles comprise an amino acid supplement formula in which at least one amino acid is included (e.g., 1- camitine or tryptophan).

Transesterification of phosphatidylcholine (PC) under solvent free conditions has been performed by Haraldsson et al in 1999 [15], with the results of high incorporation of EPA/DHA and with the following hydrolysis profile PC/LPC/GPC=39/44/17. Extensive hydrolysis and by-product formation is generally considered a problem with transesterification reactions, resulting in low product yields. This invention discloses a process for transesterification of crude soybean lecithin (mixture of PC, PE and PI). In the first step, the lecithin is hydrolyzed using a lipase in the presence of water (pH=8). The use

of a variety of lipases is contemplated, including, but not limited to, Thermomyces Lanuginosus lipase, Rhizomucor miehei lipase, Candida Antarctica lipase, Pseudomonas fluorescence lipase, and Mucor javanicus lipase. The first step takes around 24 hours and results in a product comprising predominantly of lyso-phospholipids and glycerophospholipids such as PC/LPC/GPCKJ/lS/δS. In the second step, free fatty acids are added such as EPA and DHA, however any omega-3 fatty acid is contemplated. Next a strong vacuum is applied to the reaction vessel for 72 hours. However, the reaction length can be varied in order to obtain a composition with the desired amount of phospholipids and lyso-phospholipids. By extending the reaction time beyond 72 hours, a product comprising more than 65% phospholipids can be obtained. Next, a lipid carrier is added to the reaction mixture in order to reduce the viscosity of the solution. The added amount of triglycerides can be 10%, 20%, 30%, 40% or more, it depends on the requested viscosity of the final product. The lipid carrier can be a fish oil such as tuna oil, menhaden oil and herring oil, or any triglyceride, diglyceride, ethyl- or methylester of a fatty acid. In the final step, the product is subjected to a molecular distillation and the free fatty acids are removed, resulting in a final product comprising of phospholipids (lyso-phospholipids and phospholipids) and triglycerides in a ratio of preferably 2:1.

This invention further discloses a process for the enzymatic transesterification/esterification of phospholipids with fatty acids alkyl esters or free fatty acids in an evacuated vessel (B). A reduced pressure is applied to the vessel B (0.001-30 mbar) and water vapor (moisture) is allowed to enter the reaction mixture through a tube from a second vessel (A) (Figure 5 for schematic drawing of the experimental setup). The water in vessel A is heated to 25-30 ⁰ C. By adding moisture to an evacuated reaction vessel the rate of reaction could either be increased of the lipase dosage could be reduced. In addition, the reuse of the enzymes was improved. Finally, a novel marine phospholipid composition was prepared characterized by being acylated in the range of 55%-85%, having at least 5% EPA and/or DHA esterified, having a EPA/DHA ratio of at least 1.

Accordingly, in preferred embodiments, the present invention utilizes a phospholipid, preferably a phosphatide such as lecithin. The present invention is not limited to the use of any particular phospholipid. Indeed, the use of a variety of phospholipids is contemplated. In some embodiments, the phospholipid is a phosphatidic or lysophosphatidic acid. In more preferred embodiments, the phospholipid is a mixture of phosphatides such as

phosphatidylcholine, phosphatidylethnolamine, phosphatidylserine and phosphatidylinositol. The present invention is not limited to the use of any particular source of phospholipids, hi some embodiments, the phospholipids are from soybeans, while in other embodiments, the phospholipids are from eggs. In particularly preferred embodiments, the phospholipids utilized are commercially available, such as Alcolec 40P® from American Lecithin Company Inc (Oxford, CT, USA). The present invention is not limited to the use of any particular enzyme. Indeed, the use of a variety of enzymes is contemplated, including, but not limited to Thermomyces Lanuginosus lipase, Rhizomucor miehei lipase, Candida Antarctica lipase, Pseudomonas fluorescence lipase, and Mucor javanicus lipase. This invention is not limited to any particular fatty acid alkyl ester either. This includes, but not limited to: decanoic acid (10:0), undecanoic acid (11:0), 10-undecanoic acid (11:1), lauric acid (12:0), cis-5-dodecanoic acid (12:1), tridecanoic acid (13:0), myristic acid (14:0), myristoleic acid (cis-9-tetradecenoic acid, 14:1), pentadecanoic acid (15:0), palmitic acid (16:0), palmitoleic acid (cis-9-hexadecenoic acid, 16:1), heptadecanoic acid (17:1), stearic acid (18:0), elaidic acid (trans-9-octadecenoic acid, 18:1), oleic acid (cis-9-octadecanoic acid, 18:1), nonadecanoic acid (19:0), eicosanoic acid (20:0), cis-11-eicosenoic acid (20:1), 11,14-eicosadienoic acid (20:2), heneicosanoic acid (21:0), docosanoic acid (22:0), erucic acid (cis-13-docosenoic acid, 22:1), tricosanoic acid (23:0), tetracosanoic acid (24:0), nervonic acid (24:1), pentacosanoic acid (25:0), hexacosanoic acid (26:0), heptacosanoic acid (27:0), octacosanoic acid (28:0), nonacosanoic acid (29:0), triacosanoic acid (30:0), vaccenic acid (t-11-octadecenoic acid, 18:1), tariric acid (octadec-6-ynoic acid, 18:1), and ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid, 18:1) and ω3, ω6, and α>9 fatty acyl residues such as 9,12,15-octadecatrienoic acid (α-linolenic acid) [18:3, ω3]; 6,9,12,15- octadecatetraenoic acid (stearidonic acid) [18:4, ω3]; 11,14,17-eicosatrienoic acid (dihomo- α-linolenic acid) [20:3, ω3]; 8,11,14,17-eicosatetraenoic acid [20:4, ω3], 5,8,11,14,17- eicosapentaenoic acid [20:5, α>3]; 7,10,13,16,19-docosapentaenoic acid [22:5, α>3]; 4,7,10,13,16,19-docosahexaenoic acid [22:6, ω3];9,12-octadecadienoic acid (linoleic acid) [18:2, ω6]; 6,9,12-octadecatrienoic acid (γ-linolenic acid) [18:3, ω6]; 8,11,14-eicosatrienoic acid (dihomo-γ-linolenic acid) [20:3 ω6]; 5,8,11,14-eicosatetraenoic acid (arachidonic acid) [20:4, 0)6]; 7,10,13,16-docosatetraenoic acid [22:4, ω6]; 4,7,10,13,16-docosapentaenoic acid [22:5, ω6]; 6,9-octadecadienoic acid [18:2, ω9]; 8,11-eicosadienoic acid [20:2, ω9]; 5,8,11-eicosatrienoic acid (Mead acid) [20:3, ω9]; tlθ,cl2 octadecadienoic acid; clθ,tl2 octadecadienoic acid; c9,tl l octadecadienoic acid; and t9,cl l octadecadienoic acid.

Moreover, acyl residues may be conjugated, hydroxylated, epoxidated or hydroxyepoxidated acyl residues.

Marine phospholipids extracted from marine sources have a characteristic smell and taste of rancid fish. The GC profile of the volatiles confirms the presence of these degradation products, such as short chain aldehydes and carboxylic acids. In preferred embodiments, the synthetic marine phospholipid compositions of the present invention are substantially free of volatile organic compounds and are therefore much more suitable as a food supplement for humans and animals. Accordingly, in preferred compositions, the present invention provides synthetic marine phospholipids compositions having high or increased palatability, wherein the high or increased palatability is due to low levels of organic solvents and/or volatile organic compounds. In preferred embodiments, palatability is assayed by feeding the composition to a panel of subjects, preferably human. In more preferred embodiments, the phospholipids compositions have high or increased palatability as compared to naturally extracted marine phospholipids. In other preferred embodiments, the synthetic marine phospholipids compositions of the present invention are safe for oral administration.

B. Uses of Phospholipid Compositions

There is increasing evidence that lack of omega-3 fatty acids is associated with childhood developmental cognitive disorders [20-22]. A number of randomized, controlled trials have now addressed this issue reporting that omega-3 supplementation can reduce behavioral and learning difficulties in both ADHD and dyslexia [22-23]. The treatment should preferably consist not only of a mixture of EPA/DHA, but also include omega-6 fatty acids such as 6,9,12 gamma linolenic acid (GLA)[51]. Fontani at al. [24] recently reported the results of the only randomized controlled trial of omega-3 and cognition in healthy individuals. Significant improvements on tests of sustained attention and inhibition were observed, as well as reduction in self-rated symptoms of anger, anxiety, fatigue, depression and confusion with a corresponding increase in self-rated levels of vigor for the omega-3 group compared to placebo. However, this manuscript suffered from a number of significant limitations that make interpretation of the results difficult. The study did not include a practice test to minimize "learning effects" prior to the baseline assessment, nor were the results from the placebo group reported or any comparison made between placebo and the active treatment. Previously, in order to investigate the effect of a treatment on a

developmental cognitive disorder, behavioral rating scales have been used as the primary outcome measures. Nowadays, many clinical trials of stimulant medication for children with ADHD include cognitive outcome as a co-primary or secondary outcome measure [25]. This is because cognitive dysfunction occurs commonly in children with ADHD, and also there is now substantial evidence that such dysfunction may be responsive to treatment. In addition, cognitive tests may also provide an objective outcome measure and a more direct measurement of the child's brain function than subjective parent or teacher-rated behavioral rating scales.

An embodiment of the invention is to use omega-3 rich phospholipids with EPA/DHA ratio of 2:1 (preferably substantially free of GLA) to reduce the symptoms of cognitive dysfunction in normal children and children with developmental cognitive disorders such as ADHD, dyslexia, dyspraxia and autism. Non-limiting examples of the symptoms that are alleviated are poor long term memory, poor short term memory, inability to make a decision, inability to follow through on a decision, inability to engage in conversations, insensitivity to surroundings and inability to plan a task. This invention discloses that supplementing a child's diet with phospholipid compositions comprising from about 366 mg/d to about 700 mg/d omega-3 per day (EPA:DHA ratio of 2:1) for 12 weeks improves the cognitive function as assessed using conventional rating scales and questionnaires as well as computerized cognitive tests. Previous publications do not disclose the use of omega-3 fatty acids, and in particular phospholipids comprising omega-3 fatty acids, to reduce the symptoms/specific observation criteria that make up the syndrome.

This invention discloses that supplementing a child's diet with phospholipid compositions comprising from about 360 mg to about 700 mg omega-3 per day (EPA:DHA ratio of 2:1) (preferably substantially free of GLA) for 12 weeks results in the improvement in quality of life and quality of health in a child as assessed by the parents using a questionnaire [25]. In the questionnaire, the parent will answer questions related to the child's quality of life, overall health, physical pain, joy over life, ability to concentrate, safety feeling, energy, bodily appearance, ability to gather information, sleep pattern, ability to perform on activities, capacity for school, personal relationships, negative feelings (such as blue mood, despair, anxiety and depression), abdominal discomfort, incidences of constipation, diarrhea, dry mouth, nausea, heartburn, anger, nervousness, binge eating, chest pain, shortness of

breath, blurred vision, tremor, memory loss, drowsiness, fatigue, coordination, mental sharpness, hair, skin, nails, eczema and tendency to sweat.

This invention discloses that after administration of marine phospholipids with a EPA/DHA ratio of 2:1 for one week a number of genes involved in the inflammatory response are regulated in a positive way. Furthermore, it is disclosed that marine phospholipids with EPA/DHA ratio of 1 : 1 do not regulate any genes involved in the inflammatory response. Examples of the proteins regulated by the high EPA phospholipid are the CCAAT/enhancer binding protein (C/EBP), monoglyceride lipase (MgIl), Nuclear Factor-kappaB activating protein (NF-κB AP-I) and Tnf receptor-associated factor 6 (Trafό). C/EBP plays a key role in acute-phase response to inflammatory cytokine IL-6 [26], Trafό positively regulates the biosynthesis of interleukin-6 and interleukin-12, as well as the I-kappaB kinase/NF-kappaB cascade [27] and NF-κB AP-I induce the expression of genes involved in inflammation [28]. Recent research suggests that brain inflammation may be the underlying cause of several cognitive disorders including ASD, aged associated cognitive decline and Alzheimer's disease. Alzheimer's disease is the most common cause of dementia and characterized clinically by progressive cognitive deterioration together with declining activities of daily living and neuropsychiatric symptoms or behavioral changes. Other embodiments of the invention are to use omega-3 rich phospholipids to reduce the symptoms of ASD, aged associated cognitive decline, aged associated memory decline and Alzheimer's disease. Non-limiting examples of the symptoms cognitive decline and Alzheimer's disease are memory loss, forgetfulness, short-term memory loss, aphasia, disorientation, disinhibition, behavioral changes and deterioration of musculature and mobility.

This invention also discloses that the fatty acid composition of the brain lipids and phospholipids changes after in take of omega-3 fatty acids for 30 days. A significant reduction of the arachidonic acids can be found in the phospholipids in the brain for the rats given either the EPA- or DHA-rich PL diets. This may affect the inflammatory response in this tissue and thereby have a great impact on cognitive diseases/conditions such as Parkinson's or and Alzheimer's where the inflammatory component is fundamental for the progression of the disease. This invention also discloses that the reduction of ARA is present also in the sn-2 position of the phospholipids in the brain. This is very important as the pro-inflammatory eicosanoids are produced from ARA, which are catalytically hydrolyzed from position 2 on the phospholipid by the action of phospholipase A2. The

phospholipase A2 is released after stimuli at the cell wall, it then moves to the nuclear membrane where the hydrolysis of the phospholipid takes place.

Previously, it has been disclosed that omega-3 supplementation has an effect on learning ability in aged Wistar rats [29], as well as improving of retinal function [30] and trainability in puppies [31-34]. In addition it has been shown that extracted phospholipids from pig brain can enhance behavior, learning ability and retinal function in old mice [35].

In another embodiment of this invention, omega-3 rich phospholipids are utilized to improved cognitive and/or retinal function in mammals, preferably aging mammals, such as humans, and pets as dog and cats. Aging humans are generally older than about 40, 50, 60, 70 or 80 years old, while aging pets are preferably more than 5, 6, 7, 8, 9, 10, 11, or 12 years old. In some preferred embodiments, phospholipid compositions comprising EPA/DHA in ratio of from about 2:1 are administered in order to improve cognitive function and retinal function. Cognitive function is assessed using the delayed non- matching-to position task (DNMP). The task specifically requires dogs to remember the location of an object for either a short or long delay-period. The test assesses both general cognitive ability, which is indicated by overall performance and memory capacity, which is indicated by performance at long-delays. DNMP is highly sensitive to age and models the early deficits in visuospatial memory developing in mild cognitive impairment as in Alzheimer's disease [36]. The cognitive test data revealed statistically significant differences, with the low dose subjects doing better on the last 5 treatment sessions than they did in baseline testing or on the first five treatment sessions. By contrast, the high dose subjects performed more poorly at the long-delay on the last 5 treatment sessions. In addition, at the short delay, the low and medium dose groups showed substantial improvement on the last test block. These results suggest that the test compound can either improve or impair memory, depending on dose, and that the optimal dose is in the 12 to 26 mg/kg range. The fact that the greatest effect was observed at the short delay is evidence that the treatment produces a global improvement in cognitive functioning, rather than a selective improvement in memory. The ERG analysis revealed statistically significant increases in signal amplitude in the second component of the ERG response in the dark- adapted eye and statistically significant decrease in response latency. The most consistent effect in ERG was an increase in the amplitude of the scoptopic B wave response, which was observed at all three stimulus levels. The scoptopic responses is the response of the

dark adapted eye and is linked to the function of the rods, photoreceptors in the retina of the eye. The B wave response represents the response of post-receptor cells in the retina, predominantly, the bipolar and horizontal cells. The increased B-wave response, therefore, represents enhanced transmission of visual information from the photoreceptors to the second level retinal cells. In this instance, the enhanced response is for dark vision, but the canine eye contains a higher proportion of rods than that of the primate, suggesting that rod vision is of more general importance for the dog than the human. These results are consistent with the suggestion that retinal processing is enhanced by treatment with the test compound.

Another embodiment of the invention is the use of omega-3 rich phospholipids to improve fertility in healthy and asthenozoospermic humans and animals. Testicular long chain PUFAs are of special interest because there is a high rate of production of prostaglandins from the omega-6 PUFA (arachidonic acid mainly) into the semen or seminal fluid. High rate of prostaglandin production does not indicate an active inflammatory process but a stimulus for the uterus smooth muscle to favour male fertility [37]. In addition, arachidonic acid, prostaglandins and leukotrienes have been implicated in mediating the stimulatory actions of luteinizing hormone on testicular steroid synthesis. An omega-3 induced decrease of arachidonic acid as observed in other tissue could be detrimental to the male fertility, if it occurred also in testis. Furthermore, testicular tissue has also a high level of DPA (22:5 omega-6), which may serve as a reservoir for arachidonic acid. Arachidonic acid could be formed according to the need, through the retroconversion mechanism in the peroxisomes. A similar mechanism may take place with DHA to form EPA in other tissues. Data disclosed in this application (table 5) show an increase of EPA and DHA and a small decrease of arachidonic acid in the total lipids fraction when omega-3 fatty acids are fed. However, there is no change in arachidonic acid levels in the phospholipids (PL) when TG- oil are fed and interestingly a significant increase in the PL-EPA (EPA rich phospholipids) and PL-DHA (DHA rich phospholipids) group (table 10). This can also be seen in the sn-2 positional analysis on the phospholipids (table 15) which is very important as prostaglandins are produced from ARA, which are catalytically hydrolyzed from position 2 on the phospholipid by the action of phospholipase A2. The phospholipase A2 is released after stimuli at the cell wall, it then moves to the nuclear membrane where the hydrolysis of the phospholipid takes place. Furthermore, DPA n-6 concentration in total lipids was not influenced by omega-3 supplementation but there was a significant increase in DPA in the

PL-EPA group. Overall, these data show that the diets with omega-3 phospholipids changed the arachidonic and DPA n-6 concentrations in a way that would predict positive effects on male fertility.

In another embodiment, this invention provides methods to reduce inflammation/treat an inflammatory disorder in an animal or a human subject by administering an omega-3 rich phospholipid characterized by having a high EPA/DHA ratio, preferably at least 2:1. This invention discloses that after administering marine phospholipids with a EPA/DHA ratio of 2: 1 for 1 week a number of genes involved in the inflammatory response are regulated in a positive way. Furthermore, it is disclosed that marine phospholipids with EPA/DHA ratio of 1:1 do not regulate any genes involved in the inflammatory response. Examples of the proteins regulated by the high EPA phospholipid are the CCAAT/enhancer binding protein (C/EBP), monoglyceride lipase (MgIl), Nuclear Factor-kappaB activating protein (NF-κB AP-I) and Tnf receptor-associated factor 6 (Trafό). C/EBP plays a key role in acute-phase response to inflammatory cytokine IL-6 [38], Trafό positively regulates the biosynthesis of interleukin-6 and interleukin-12, as well as the I-kappaB kinase/NF-kappaB cascade [39]and NF-κB AP-I induce the expression of genes involved in inflammation [40].

Another embodiment of the invention is the use of marine phospholipids to alleviate muscle soreness/muscle pain after physical exercise/sports activity. Delayed onset muscle soreness normally increases in intensity during the first 24 hours after exercise and peaks before 72 hours [41], and then subsides so that by 5-7 days post exercise it is gone. The discomfort ranges from mild to extreme soreness, which prevents the use of the muscle. The reason for the soreness is damage to the connective and/or contractile tissues and that initiates inflammation [42]. Injury in a muscle causes monocytes to migrate to the area and secrets large amounts of pro-inflammatory prostaglandins. This invention discloses that administration of omega-3 rich phospholipids with EPA:DHA ratio of at least 2:1 for 1 week reduces the expression of enzymes in the inflammatory response such as the expression proteins in the NF- K B pathway. Furthermore, it has been shown that the concentration of DHA in red blood cells after a bolus intake of DHA-PC peaked after 9 hours, whereas similar intake of DHA in the form of triglycerides peaked after 12 hours

[43]. Hence, omega-3 rich phospholipids, preferably with an EPA:DHA ratio of 2:1, are

suitable for a rapid reduction in inflammation, preferably in the area of reducing pain, more preferably in the area of reducing delayed onset muscles soreness after physical exercise.

Another embodiment of the invention is to treat conditions involving inflamed joints such as rheumatoid arthritis and osteoarthritis.

Another embodiment of the invention is the use of omega-3 phospholipids to improve physical performance/endurance e.g. in athletes. Studies have shown that incorporation of omega-3 fatty acids into the membrane of RBCs increase the deformability of RBCs [44] which again facilitates the transport of RBCs through the capillary bed [45]. This effect enhances the oxygen delivery to contracting muscle which may have a benefit on improving physical performance. This invention discloses that mice fed a diet comprising omega-3 rich phospholipids perform better than mice fed omega-3 rich triglycerides i.e. increased submaximal endurance in a treadmill running test.

Another embodiment of the invention is the use of marine phospholipids to prevent obesity and for weight management in humans and animals. This invention discloses that omega-3 rich phospholipids (EPA:DHA ratio of 2:1) regulate several genes linked to lipid metabolism in a positive way such as gamma-butyrobetaine hydroxylase and gunanine nucleotide binding protein. The results show that guanine nucleotide binding protein is down regulated. This results in an increased inhibition of adenylate cyclase (AC). AC catalyzes the conversion of ATP to 3',5'-cyclic AMP (cAMP) and pyrophosphate. cAMP is an important molecule in eukaryotic signal transduction and is responsible for the intracellular mediation of hormonal effects on various cellular processes such as lipid metabolism, membrane transport, and cell proliferation [46]. Furthermore, the level of gamma-butyrobetaine hydroxylase is increased leading to increased biosynthesis of L - carnitine (3-hydroxy-4-N-trimethylaminobutyrate) [47]. Increased carnitine levels result in increased /3-oxidation [48], since carnitine is responsible for transport of fatty acids into a cell's mitochondria. This invention disclose that marine phospholipids can increase β- oxidation of fatty acids in mitochondria which may represents shift in fuel use from glucose and amino acids to fats. Marine phospholipids can therefore be used to prevent weight gain or obesity in combination with a high fat diet.

In yet another embodiment, marine phospholipids are provided as a prophylactic treatment of rapid heart beat (sustained ventricular tachycardia) in patients at high risk of sudden cardiac death. Published data have shown that omega-3 fatty acids reduce cardiovascular mortality [49], and that incidences of ventricular tachycardia can be reduced in patients after infusion of omega-3 fatty acids [50]. Due to the rapid incorporation of marine phospholipids into RBCs [43], phospholipids are more suitable than triglycerides when a rapid/acute/immediate effect is needed. Patients with sustained ventricular tachycarida in patients with a high risk of sudden cardiac death, hence it is likely that marine phospholipids will be more efficient preventing death than fish oil. This invention discloses that hheart total lipids and phospholipids (table 6 and table 11, respectively) showed a strong increase of EPA and DHA with a concomitant decrease of arachidonic acid when omega-3 supplements were fed. The strong decrease in the omega-6/omega-3 ratio in heart lipids is important considering the possible impact on the anti-inflammatory potential. Observed change in heart tissue fatty acids (increase of fatty acids with 6 or 5 double bonds) also suggests a possible increase in membrane fluidity. This change was most striking in the PL-DHA group where the increase of DHA was significantly higher than the increase in the TG-oil and PL-EPA groups. The fluidity of myocardium cell membrane seems to play an important role in controlling arrhythmia. Ventricular arrhythmia, is one of the main causes of sudden cardiac death. Furthermore, atrial fibrillation is another pathological state with a high incidence and important health consequences.

Another embodiment of the invention is the use of marine phospholipids to reduce the symptoms of metabolic syndrome and/or diabetes type II. Metabolic syndrome is considered as a combination of metabolic disorders that increases a subject's risk for cardiovascular disease and type 2 diabetes. The criteria for metabolic syndrome are fasting hyperglycemia, high blood pressure, central obesity, decreased HDL cholesterol, increased triglycerides and elevated uric acid levels. Hence, Zucker diabetic fatty rat can be used to monitor the effect of dietary intervention on the development and progress of metabolic syndrome. This invention discloses that omega-3 phospholipids are superior to omega-3 triglycerides in alleviating insulin resistance, improving cholesterol profile and reducing plasma triglycerides.

In some embodiments, the marine phospholipid compositions are derived from marine organisms such as fish, fish eggs, shrimp, krill, etc. In some embodiments, the marine

phospholipids comprise a mixture of phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidyl inositol (PI), and phosphatidylethanolamine (PE). Indeed, the present invention presents the surprising results that phospholipid compositions comprising a mixture of PC, PS ₅ PI and PE are bioavailable and bioefricient. This results in an important advantage over phospholipid compositions synthesized or containing, for example, pure PS,

PC, or PI which can be expensive and difficult to make. In some embodiments, the methods of the present invention utilize novel marine lipid compositions comprising an omega-3 containing phospholipid and a triacylglyceride (TG) in a ratio from about 1:10 to

10:1. Preferably the ratio is in the range of from about 3:1 to 1:3, more preferably the ratio is in the range of about 1:2 to 2:1. Preferably, the TG is a fish oil such as tuna oil, herring oil, menhaden oil, krill oil, cod liver oil or algae oil. However, this invention is not limited to omega-3 containing oils as other TG sources are contemplated such as vegetable oils. In some embodiments, the phospholipids in the composition have the following structure:

wherein Rl is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is a mixture of H, choline, ethanolamine, inositol and serine. Attached to position 1 or position 2 are least 1% omega-3 fatty acids, preferably at least 5%, more preferably at least 10% omega-3 fatty acids, up to about 15%, 20%, 30%, 40% , 50%, or 60% omega-3 fatty acids. The omega-3 fatty acids can be EPA, DHA, DPA or Cl 8:3 (n-3), most preferably the omega-3 fatty acids are EPA and DHA. The phospholipid composition preferably contains OH in position 1 or position 2 in a range of preferably about 15% to 60%, and more preferably from about 20% to 50% in order to maximize absorption in-vivo.

EXPERIMENTAL

EXAMPLE 1

The difference in bioavailability and bioefficacy between the marine lipid composition of the present invention and a fish oil were investigated in a rat experiment. The rat feed was prepared using AIN-93 except that soybean oil was removed from the feed. The pelleted AIN-93 diet was ground and the marine lipid compositions (PL 1 and PL 2) as well as fish oil (TG oil) and control were added to this ground feed. The marine lipid compositions were prepared using enzymatic (lipase) catalyzed transesterifϊcation of soy lecithin with fish oil fatty acids according to the method described in Example 4, followed by the addition of a triglyceride carrier and short path distillation. The concentration of EPA, DHA and 18:3 n- 3 in the different diets can be seen in the table below (table 1).

Table 1. Amount of different fatty acid in the final feed products

Thirty six newly weaned male Sprague Dawley rats (start weight 168 ± 11 g) were used in the experiment. The rats were initially given low-essential oil rat feed, containing 20 g of sunflower oil and 10 g of flaxseed oil per kg of feed, for one week. After the first week, modified AJN-93 diet powder without the test oil was given to rats ad libitum until the start of the experiment. Feeding of rats was stopped 12 hours before the sampling, 30 days after the start of feeding. Each rat was individually anaesthetized with carbon dioxide, weighed and euthanized with cervical dislocation. Next, blood was sampled and centrifuged to separate plasma and blood cells. Then abdominal skin was removed and 70 ml of sterile Hepes-Hanks was injected into the peritoneal cavity to collect intraperitoneal lymphocytes. The abdomen was gently massaged for about 3 minutes after which the buffer solution was drained and centrifuged in Falcon tubes (200 x g, 10 min) to collect the cells. The cells were resuspended into 1 ml of freezing fluid (10% DMSO, 90% fetal bovine serum) in 1.5 ml Eppendorf tubes. These tubes were then frozen to dry ice temperature for one hour by immersing the tubes in isopropanol placed on dry ice. This enabled a slower freezing rate than by putting the cells directly on dry ice. In the laboratory, the cells were stored overnight at -80 ⁰ C and then stored in liquid nitrogen. The derivatization of the lipids in order to perform gas chromatographic (GC) analysis was carried accordingly to [16]. The

run conditions for the GC were according to [17]. The growth of rats did not differ between the feeding groups (data not shown). The intake of feed, and the intake fatty acids thereof, was monitored by keeping the rats in metabolic cages which allows the measurement of eaten and uneaten portion of feed. The PL 1 test group consumed somewhat less EPA than the TG oil group, whereas the PL 2 and the control group consumed much less EPA than both the PL 1 and the TG oil groups (figure 1). The amount of EPA in plasma varied between the groups and the results are shown figure 2. Even though the estimated intake of EPA was higher in the TG Oil group than the PL 1 group, the area% of EPA measured in plasma for PLl was higher than for TG oil. Indicating a higher bioavailability of EPA from of PL 1 than from the TG oil. Furthermore, this was also observed in the FA profile of the red blood cells and the monocytes (figure 3 and figure 4, respectively). Demonstrating that the PL 1 composition was more efficient in enriching these cells with omega-3 than the TG oil group, hence being more bioeffϊcient than TG.

EXAMPLE 2

The total fatty acid profile for the lipids in the brains (table 2), adipose tissue (table 3), liver (table 4), testicles (table 5) and heart (table 6) were isolated from the rats in example 1. The PL 1 composition increases the DHA content in brain and adipose tissue more than the TG composition. The PL 1 composition increased the EPA content in the adipose tissue more than the TG composition. It is to be observed that the PLl composition increases the EPA/DHA content in the phospholipids and in the total lipids of the different tissues as well as reduces the AA/EPA ratio more than the TG oil composition.

EXAMPLE 3

The fatty acid profile of the phospholipids isolated from the brain (table 7), adipose tissue (table 8), liver (table 9), testicles (table 10) and heart (Table 11) in the rats from example 1 were determined.

EXAMPLE 4

50g of soy lecithin from American Lecithin Company Inc (Oxford, CT, USA), 4Og of TL- IM lipase from Novozymes (Bagsvaerd, Denmark) and 5g of water (adjusted to pH=8 using NaOH) were mixed in a reaction vessel at 5O ⁰ C for 24 hours. Next, 1Og of free fatty acids containing 10% EPA and 50% DHA from Napro Pharma (Brattvaag, Norway) was added, followed by application of vacuum to the reaction vessel. After 72 hours the reaction was terminated and the phospholipid mixture was analyzed using HPLC and GC. The results showed that the relationship between PC/LPC/GPC was 65/35/0, and that the content of EPA and DHA was around 10% and 12%, respectively. Next, 2Og of sardine oil was added to the reaction mixture which comprised of 18% EPA and 12% DHA (relative GC peak area), followed by molecular distillation. The final product contained around 70% acetone insolubles, around 30% triglycerides and traces of free fatty acids.

EXAMPLE 5

Marine phospholipids were prepared using either 40% soy PC (American Lecithin Company Inc, Oxford, CT, USA) (MPLl) according to the method in example 4 or using 96% pure soy PC (Phospholipid GmbH, Kόln, Germany) (MPL2) according to a method described in [19]. Fatty acid content and the level of bi-products are shown in table 17. The MPL treatments consisted of a mixture of phospholipids, lyso-phospholipids and glycerol- phospholipids. Looking only at the PC/LPC/GPC relationship, it was 64/33/2 and 42/40/18 for MPLl and MPL2, respectively. Finally, all three treatments were emulsified into skimmed milk.

Table 17. Composition of the phospholipids used in example 5

18 newly weaned Sprague-Dawley rats were fed the milk emulsions for 1 week. Each rat was placed in its own cage to ensure that they got an even amount of test substance and the milk was consumed by the rat pups ad libitum. After 1 week the experiment was terminated and the rats were decapitated. The animals were kept without food for 24 hours before sampling. Entire livers were collected and frozen immediately using liquid nitrogen (stored at -65°C). Total RNA was isolated from the liver samples according to the Quiagen RNAEasy Midi Kit Protocol. The RNA samples were quantified and quality measured by NanoDrop and Bioanalyzer. The isolated RNA was hybridized onto a gene chip RAE230 2.0 from Affymetrix (Santa Clara, CA, USA). The expression level of each gene was measured using an Affymetrix GeneChip 3000 7G scanner. The results were suitable for all chips except 2 and they were excluded from the trial. The results are based on (log) probe set summary expression measures, computed by RMA, and linear models are fitted using Empirical Bayes methods for borrowing strength across genes (using the Limma package in R). The p-value are adjusted for multiple testing using the Benjamini-Hochberg-method, controlling the False Discovery Rate (FDR), where FDR = the proportion of null- hypotheses of no DE that are falsely rejected.

It was observed that MPLl and MPL2 are biologically different compounds due to the fact that over 40 genes were differentially expressed (table 18).

Table 18 List of genes differentially expressed (DE) by MPLl versus MPL2. The list is sorted according to increasing p-values. SLR: Estimated signal log-ratio (<0: down regulated gene, >0: up regulated gene). Fold change: Estimated fold change corresponding to the parameter (<1: down regulated gene, >1: up regulated gene). Affy fold change:

Estimated fold change using the Affymetrix definition (<-l: down regulated gene,>l: up regulated gene) df: Degrees of freedom (= number of arrays - number of estimated parameters).

MPL2 regulates 401 genes versus the control (table 19). A number of genes listed are involved maintenance of the cell, in transcription and protein synthesis as well as signaling pathways. Others are involved in regulation of metabolism and the inflammatory response such as Tnf receptor-associated factor 6 (Traf6_predicted) (fold change of 0.53), guanine nucleotide binding protein alpha inhibiting 2 (Gnai2) (fold change of 0.6, gamma- butyrobetaine hydroxylase (Bboxl) (fold change of 1.32), monoglyceride lipase (MgH) (fold change 0.52), nuclear NF-kappaB activating protein (fold change 0.65) and CCAAT/enhancer binding protein (C/EBP) (fold change of 0.66).

Table 19. List of genes differentially expressed (DE) by MPL2 versus control. SLR: Estimated signal log-ratio (<0: down regulated gene, >0: up regulated gene). Fold change:

Estimated fold change corresponding to the parameter (<1: down regulated gene, >1: up regulated gene). Affy fold change: Estimated fold change using the Affymetrix definition (<-l: down regulated gene,>l: up regulated gene) df: Degrees of freedom (= number of arrays - number of estimated parameters)

EXAMPLE 6

The effect of dietary supplementation of omega-3 phospholipids for 12 weeks on cognitive function, quality of life and behavioral outcome in children with ADHD have been investigated. Four children with ADHD were recruited, 2 children received a placebo (olive oil), 1 child received omega-3 phospholipids (700 mg/day, EPA:DHA=2:1) and the last child received omega-3 phospholipids (350 mg/day, EPA:DHA=2:1). Inclusion criteria were age (8-12 years), diagnosis of ADHD according to DSM-IV criteria, exhibiting symptoms of essential fatty acid deficiency and permission from parent/guardian. Exclusion criteria were use of dietary supplement containing omega-3 or omega-6 in the previous 6 months, consuming more than 2 fish meals per week, receiving medical treatment for a major health condition such as diabetes, depression or having a bleeding disorder. At baseline, after 4 weeks and after 12 weeks the child's cognitive ability was measured using computerized cognitive tests from Cogstate (Melbourne, Australia) as well as the conventional cognitive test from TEA-Ch [45] and WASI (Wechshler Abbreviated Scales of Intelligence) [46]. The parent/guardian completed a World Health Organization-Quality of life questionnaire [33], symptoms check list, BRIEF (Behavioral Rating Inventory of Executive Function) [47] and Conners' rating scale -revised (SCR-R) [48]. For each subject at each assessment, an average of the standardized scores was computed. The results are shown in figure 5, showing an improvement of the cognitive performance for the children receiving omega-3 phospholipids compared to baseline. None of the children receiving placebo completed the trial.

EXAMPLE 7

The effect of dietary supplementation of omega-3 phospholipids for 12 weeks on cognitive function, quality of life and behavioral outcome in healthy children was investigated. 20 children was recruited 10 children received placebo (olive oil) and 10 children received omega-3 phospholipids (700 mg omega-3/day, EPA:DHA=2:1). Inclusion criteria were male and female age 8-12 years and permission from parent/guardian. Exclusion criteria were use of omega-3 and omega-6 dietary supplement, consuming more than 2 meals of fish per week, receiving medical treatment for any major health conditions such as diabetes, history of traumatic brain injury, symptoms on ADHD according to DSM-IV criteria and bleeding disorders. At baseline, after 4 weeks and after 12 weeks the child's cognitive ability was measured using computerized cognitive tests from Cogstate (Melbourne, Australia) as well as the conventional cognitive test from TEA-Ch [45] and Weschler Abbreviated Scales of Intelligence (WASI) [46]. The parent/guardian completed a World Health Organization-Quality of life questionnaire [33], symptoms check list, BRIEF (Behavioral Rating Inventory of Executive Function) [47] and Conners' rating scale - revised (SCR-R) [48]. For each subject at each assessment, an average of the standardized scores was computed. The results are shown in figure 6, showing an improvement of the cognitive performance for the healthy children receiving omega-3 phospholipids compared to both placebo and baseline. One of the subjects was removed from the data set due to poor response. 3 children did not follow through the study after the initial baseline assessment.

EXAMPLE 8

A dose-finding study aimed to investigate the effect of three dose levels (13 mg/kg, 26 mg/kg and 52 mg/kg) of the omega-3 rich phospholipids on visuospatial memory in aged beagle dogs was performed. 18 beagle dogs of age 7 years were recruited with the absence of any clinical symptoms that could affect the objectives of the study, as determined by a veterinarian. The variable measured at baseline and after 4 week were cognition as measured by the delayed non-matching-to position task (DNMP) [44]. Electroretinography (ERG) is an electrophysical technique which measures the retinal action potentials in response to light stimulation and is used to assess retinal function [49]. DNMP is a test of working memory performance and subjects will receive 10 trials daily. During the baseline phase, all subjects was given 5 cognitive test sessions on a DNMP, which provided a means of assessing visuospatial working memory and establish baseline performance levels on the spatial memory test. The baseline cognitive test performance was then used to assign animals into three cognitively equivalents groups of 6 animals per group. Each trial of the

DNMP task consists of two phases. In the sample, or presentation phase, a red block is presented to the subject over one of three food wells. The subject is required to displace the block and retrieve the food in the well below the block. The block is then removed from view of the subject and a delay is initiated. At the end of the delay, the choice phase begins in which subject is presented with two identical blocks; one over the initial well and a second over one of the two remaining wells. Subjects are required to respond to the novel position to obtain the food reward. A 30-s inter-trial interval will be used to separate each trial. For the present study, all DNMP testing will consist of variable-delayed testing in which delays of 20 or 90 s will occur equally over the 10 daily test trials. The results show an improvement in cognitive function in the aged beagle dogs (Figure 7), especially for the low (13 mg/kg) and intermediate (26 mg/kg) dose using the short delay test. For the long delay test, an improvement was observed for the low dose (13 mg/kg), whereas a reduction of cognitive performance was observed for the high dose (52 mg/kg).

The ERG's were analyzed with repeated measures ANOVA's for both response latency and response amplitude. Each analysis looked at a single variable, with baseline and treatment conditions serving as a within subject variable and dose as a between subject variable. Each of the following 10 variables were examined 1) A wave scotopic at 0 log intensity, 2)A wave scotopic at 1.2 log intensity, 3) A wave photopic at 0 log intensity, 4) A wave photopic at 1.2 log intensity, 5) B wave scotopic at —3 log intensity, 6) B wave scotopic at 1 log intensity, 7) B wave scotopic at 1.2 log intensity and 8)B wave scotopic at 30 hz frequency.

Statistically significant treatment effects in response amplitude were observed in the scotopic response in the B wave at the 0 log ( p=0.02) and in the 1.2 log response

(p=0.0007) . The response at -3 log was marginally significant (p=0.06). These results reflect larger responses under the treatment condition than baseline. The largest effects were observed at the low and high dose. The results also revealed a significant n increase in the amplitude of the scoptopic response to the A wave at 1 and 1.2 log intensities, the treatment effect achieved statistical significance (p=0.04 and p=0.007 respectively). These changes reflected shorter onset latency in the medium dose group.

EXAMPLE 9

18 healthy asthenozoospermic males (sperm motility <50%) are to be recruited in a fertility experiment. Other inclusion criteria are to be age (25-50), lack of ejaculation 2-5 days before sampling as well as a signed consent. Exclusion criteria are to be consumption of omega-3 products, use of carnitine, use of CoQlO, alcohol abuse and moderately severe co- morbid disease. The following variables are to be investigated at baseline and after 12 weeks: sperm motility, sperm count, sperm concentration, sperm morphology, sperm phospholipid fatty acid profile and pH. 6 males are to administer a marine phospholipid consisting of 700 mg/g EPA/DHA (ratio 2:1) daily for 12 weeks. 6 males are to administer olive oil and 6 males are to administer fish oil as described [52] for 12 weeks. After 12 weeks administering marine phospholipids an improvement in sperm motility, sperm count, sperm concentration, sperm morphology is to be found compared to both placebo and fish oil. An increase in DHA level in the sperm phospholipid is to be found compared to placebo and fish oil as well.

EXAMPLE 10

The effect of dietary supplementation of omga-3 phospholipids on the prevention of obesity is to be investigated. 5 rats will be fed a control + high fat diet containing essentially no EPA/DHA for 2 weeks, 5 rats will be fed a marine phospholipid high fat diet and 5 rats will be fed a fish oil high fat diet also for 2 weeks. It is to be found that the weight gain and/or accumulation of adipose tissue are significantly larger in the control high fat diet, compared to other two diets. It is to be found that the weight gain and/or accumulation of adipose tissue is significantly larger in the fish oil high fat diet compared to the marine phospholipid high fat diet.

EXAMPLE 11

The effect of dietary supplementation of omega-3 phospholipids (700 mg omega-3/day, EPA:DHA=2:1) on delayed onset muscle soreness (DOMS) in human subjects is to be investigated. 10 subjects will receive omega-3 phospholipids and 10 subjects will receive fish oil for 30 days prior to exercise. Next, DOMS is to be induced e.g. by 50 maximal isokinetic eccentric elbow flexion contractions. DOMS is to be measured by asking the subjects about pain, swelling and muscle strength as well as measuring typical markers for DOMS and muscle damage such as creatine kinase. It is to be observed that the use of omega-3 phospholipids significantly reduces DOMS compared to fish oil.

EXAMPLE 12

The immediate effect of administration of omega-3 phospholipids in humans with sustained ventricular tachycarida is to be investigated. 10 patients with implanted cardioverter defibrillators and repeated episodes of documented, sustained ventricular tachycardia are to be enrolled in a study. Omega-3 fatty acids 3.8 g are to be infused either in the form of fish oil (N=5) or marine phospholipids (N=5). Sustained ventricular tachycardia is to be induced using paced cycle lengths of variable length in the patients in both groups. It is to be found that the group receiving omega-3 phospholipids have fewer cases of ventricular tachycardia than the group receiving omega-3 fish oil.

EXAMPLE 13

The effect of dietary supplementation of omega-3 phospholipids on physical endurance is to be investigated in a experiment with rats (N=6). Rats will be fed one of two diets containing 20 energy % of fat for 5 to 10 weeks. Control diet containing essentially no EPA/DHA will be supplemented with 10 energy % of olive oil and the test diet will be supplemented with 10 energy % of marine phospholipid. It is to be found that rats consuming the marine phospholipid diet will demonstrate increased submaximal endurance in a treadmill running test.

EXAMPLE 14

The effect of dietary supplementation of omega-3 phospholipids on inflammation is to be investigated in a experiment with rats (n=6-12). The experiment will use a control diet containing essentially no EPA/DHA, a positive control diet containing fish oil and a test diet containing marine phospholipids (EPArDHA; 2:1). The effect of marine phospholipids on the inflammatory process will be examined first by analyzing the fatty acid profile of inflammatory cell membrane phospholipids (e.g. monocytes and macrophages). The effect of omega-3 fatty acids on inflammation is mediated through the link between phospholipid stores of inflammatory cells [43]. Inflammatory cells typically contain a high proportion of arachidonic acid (AA; C20:4 omega-6) and low proportions of omega-3 fatty acids. Dietary supplementation with omega-3 fatty acids decreases the omega-6:omega-3 ratio of the inflammatory cells thus decreasing the inflammatory potential of the inflammatory cells. This is a desirable change in humans with chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and atopic diseases such as asthma. It is to be observed that marine phospholipids reduce omega-6:omega-3 ratio in blood

inflammatory cells such as in monocytes significantly compared to the control and fish oil supplemented diets. In the second stage of the experiment monocytes harvested from the experimental animals will be challenged with lipopolysaccharide (LPS), bacterial cell surface antigen that triggers and inflammatory response in monocytes, and the pro- inflammatory cytokine response (TNFα, IL- lβ, IL-6 and IL-8) will be measured. It is to be observed that marine phospholipids are more effective in reducing inflammatory cytokine production in monocytes than the control and fish oil containing diet.

EXAMPLE 15 The purpose of the study is to differentiate the effect of omega-3 phospholipids, omega-3 triglycerides and control on inflammation, blood lipids, insulin resistance and oxidative stress. Different forms of omega-3 fatty acids are given to Zϋcker diabetic fatty rats (ZDF rats), an animal model relevant to human obesity, for 5 weeks. The omega-3 rich phospholipids were prepared according to the method in example 12. The data is presented in Table 20. It is observed that there are no difference between the treatments on insulin levels and HOMA estimates. However, it is observed that in the phospholipid group are the most efficient formulation in improving plasma glucose levels. Elevated plasma glucose levels are one of the signs/symptoms of metabolic syndrome. Further, it is expected that omega-3 phospholipids are the most efficient formulation in improving blood lipids such as HDL, LDL, triglycerides and free fatty acids, for reducing inflammatory markers such as TNF alfa, IL-I beta, IL-6, IL-10, TGF beta and fibrinogen in plasma, and for reducing markers of oxidative stress such as PUFA hydroperoxides and 15-F2t-Isoprostanes in plasma and tissues (subcutaneous and visceral adipose tissue, liver, brain and heart).

Table 20. The effect of control, omega-3 phospholipids and omega-3 triglycerides on markers of insulin resistance in ZDF rats.

29,864 1,659 0,478 0,467

EXAMPLE 16

The effect of omega-3 rich phospholipids on collagen induced rheumatoid arthritis is to be investigated in a therapeutic animal model. The omega-3 rich phospholipids were prepared according to the method in example 12. Rheumatoid arthritis (RA) is considered to be a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. It is expected that omega-3 phospholipids are more efficient in increasing clinical arthritis scores than omega-3 triglycerides and placebo.

EXAMPLE 17

A marine phospholipid composition containing 8.4 % EPA and 1.2 % DHA was prepared using a crude soybean lecithin as a starting material according to [19]. A marine oil was added to the phospholipid mixture (30% w/w) so that the total level of EPA was 21.9% and for DHA 9.4%. Furthermore, soy lecithin and lyso-phospholipids prepared according to [53] were added to the mixture in variable amounts so that a range of PC/LPC/GPC ratios could be obtained (Table 21). By using this method, all the treatments (MPL1-MPL5) contained exactly the same amount EPA and DHA. Lipid compositions were consumed as a single bolus by adult Sprague-Dawley rats and the appearance of EPA/DHA in blood was measured at different time points from 1 to 12 hours after ingestion. The concentration of EPA/DHA was determined using GC-FID and reported as area percentage. Figures 9 and 10 show that composition MPL2 and MPL3 results in the highest concentration of EPA/DHA in plasma after uptake. Comparing the surface area under each curve it is clear that MPL2 and MPL3 demonstrates a higher bioavailability of EPA/DHA than the other composition MPLl, MPL4 and MPL5.

Table 21. Hydrolysis profile of the compositions tested

EXAMPLE 18

60 g fatty acid ethyl ester consisting of 10% EPA and 50% DHA (FAEE 10-50), obtained from Napro Pharma (Brattvaag, Norway) and 15 g TL-IM obtained from Novozymes

(Bagsvaerd, Denmark) were mixed in an evacuated round bottomed glass flask for 15

minutes. Next, N ₂ was released into the glass flask and the mixture was heated to 65°C. 20 g Alcolec 40P® from American Lecithin Company Inc (Oxford, CT, USA) was then added to the reaction mixture. Alcolec 40P® is a crude soybean phospholipid product containing 40% PC, 26% phosphatidylethanolamine, 11% phosphatidylinositol, 1% phosphatidylserine, 13% phytoglycolipids as well as 14% other phosphatides (w,w). Next, the glass flask was evacuated (20-30 mbar). Finally, a second vessel containing water (3O ⁰ C), was connected to the reaction vessel through a plastic tube (Figure 1). The reduced pressure allowed moisture from the headspace of the second vessel to be added through the reaction mixture continuously. In order to obtain the final product the enzymes were removed by filtration. Finally, a triglyceride carrier was added to the product, followed by removal of the residual free fatty acids and/or esters by short path distillation. In order to analyze the product, the sample was fractionated by HPLC-UV (λ=207 nm) with a silica column and methanol- water (92:8, v/v) as mobile phase. The isolated PC + LPC fraction was then dried under nitrogen prior to derivatization; finally the fatty acid profile was determined by analyzing the derivatives using GC-FID. Furthermore, the relationship between PC, LPC and GPC was determined using HPLC with the method above, except that the UV detector was replaced by an evaporative light scattering detection (ELSD). Integrated ELSD peak areas were reported for PC/LPC/GPC (total 100%); however for simplicity other PL species were not analyzed. The results obtained for example 3 is shown in table 22 below.

Table 22. Results obtained after transesterification using vacuum and water addition

EXAMPLE 19

The enzymes from example 3 were isolated by filtration and the possibility of reuse was determined in the following experiment. 30 g FAEE (10-50), 1O g Alcolec and 15 g used enzymes (equivalent to 7.5 g enzyme because the used enzymes had absorbed product from

the first reaction). The reaction was performed at 65°C and stirred at 200 rpm using a shaker incubator. The transesterified phospholipids were analyzed as in the previous example and the results are shown in table 22 below.

Table 22 Results obtained with reused enzymes using incubator shaker

* ELSD peak area (total 100%). Only peaks relating to PC, LPC and GPC are integrated. **EPA/DHA attached to PC + LPC

EXAMPLE 20 The same conditions as in example 1 were used, except that the amount of lipase was 1O g. The results are shown in table 23.

Table 23. Results obtained with reduced lipase dosage after transesterification using vacuum and water addition

* ELSD peak area (total 100%). Only peaks relating to PC, LPC and GPC are integrated. **EPA/DHA attached to PC + LPC

EXAMPLE 22

The enzymes from example 3 were isolated by filtration and the possibility of reuse was determined in the following experiment. 30 g FAEE (10-50), 10 g Alcolec and 15 g used enzymes (equivalent to 7.5 g enzyme because the used enzymes had absorbed product from the first reaction). The reaction was performed using the same conditions as in example 3. See table 24 below for results.

Table 24. Reuse of enzymes from example 3 using vapor addition into evacuated reaction vessel.

* ELSD peak area (total 100%). Only peaks relating to PC, LPC and GPC are integrated. **EPA/DHA attached the fraction consisting of PC + LPC

EXAMPLE 23

The same conditions as in example 3 are applied, except that the pressure in the reaction vessel is 1 mbar. The results obtained are similar to the results in Table 3, except that the hydrolysis and the acid values are reduced. After 6 days the relationship between PC species is 80/10/0 and the acids value is 40. The incorporation of EPA/DHA is the same.

EXAMPLE 24

The safety of omega-3 rich phospholipids prepared in the presence of chloroform and omega-3 rich phospholipids prepared under solvent free conditions is to be examined by feeding pregnant rats for 1 week. It is to be found that the treatment containing omega-3 rich phospholipids with traces of chloroform will result in damage to the developing fetus than the treatment containing essentially no traces of organic solvents.

References

[I] WO 2006054183

[2] P.C. Calder. Prostaglandins, Leukotrienes and Essential Fatty Acids 2006; 75; 197-202.

[3] US 5,434,183

[4] Alexander JW. Nutrition 14 (1998) 627.

[5] Belluzi A, Boschi S, Brignola C, Munarini A, Cariani G and Miglio F. Am J Clin Nutr

71 (2000) 339.

[6] Kremer JM.. Am J Clin Nutr 71 (2000) 249

[7] VP Carnielli, G. Verlato, F. Pederzini, I. Luijendijk, A. Boerlage, D. Pedrotti and P.

Sauer Am J Clin Nutr 1998;67 ; 97-103.

[8] M. Moya, E. Cortes, M. Juste, J. G. De Dios and A Vera Eur. J. Clin. Nutr. 2001; 55;

755-762. [9] A. SaIa- Vila, A. I. Castello, C. Campoy, M. Rivero, M. Rodriguez-Palermoe and M. C.

Lopez-Sabater. J. Nutr. 2004; 134; 868-873.

[10] A. SaIa- Vila, C. Campoy, A. I. Castellote, FJ. Garrido, M. Rivero, M. Rodriguez-

Palmero and M. C. Lopez-Sabater. Prostaglandins, Leukotrienes and Essential Fatty Acids;

2006; 74; 143-148. [11] A. Valenzuela, S. Nieto, J. Sanhueza, M. J. Nunez and C. Ferrer. Annals of Nutrition &

Metabolism; 2005; 49; 325-332.

[12] J. B.Hansen, S: Grimsgaard, H. Nilsen, A. Nordoy and K.H. Bonaa. Lipids; 1998; 33;

131-138.

[13] US provisional application entitled "Functional Phospholipid Compositions" with serial number 60/798,027 filed 05/05/2006.

[14] P.C Calder, Philip C. Am. J. Clin. Nutr; 2006; 83; 1505S-1519S.

[15] G. G. Haraldsson, A. Thorarensen, JAOCS 75 (1999) 1143-1149.

[16] G. Lepage and CC. Roy; J. Lipid Res; 1986;. 27; 114-120.

[17] T. Moriguchi, S-Y Lim, R. Greiner, W. Lefkowitz, J. Loewke, J. Hoshiba and N. Salem. J. Lipid Res; 2004;. 45; 1437-1445.

[18] H. Salman, M. Bergman, H Bessler, S Alexandrova, B. Beilin, M. Djaldetti. Acta Phys

Scand; 2000; 168, 431-436.

[19] Haraldsson GG and Thorarensen A, JAOCS 75 (1999) 1143.

[20] Richardson AJ and Montgomery P. Pediatrics 115 (2005)1360. [21] Richardson AJ. Lipids. 39 (2004) 1215..

[22] Richardson AJ and Pun BK. Prog Neuropsychopharmacol Biol Psychiatry 26 (2002)

233.

[23] Burgess JR. Attention deficit hyperactivity disorder: observational and interventional studies. In: NIH workshop on omega-3 essential fatty acids and psychiatric disorders, 2-3 September 1998. Bethesda, MD: National Institutes of Health; 1998.

[24] Fontani G, Corradeschi F and Felici A. Eur J Clin Invest. 35 (2005) 691.

[25] Mollica CM, Maruff P and Vance A. Hum Psychopharntacol 19 (2004) 445.

[26] Bannach FG, Gutierrez-Fernandez A, Parmer RJ and Miles LA J Thromb Haemost. 2

(2004) 2205.

[27] Choi Y. AdvExp MedBiol.560 (2005) 77.

[28] Leon KG and Karsan A. Histol Histopathol. 15 (2000) 1303.

[29] Gamoh S, Hashimoto M., Hossain S, Masumura S. Clin Exp Pharmacol Physiol 28

(2001) 266. [30] Filburn CR and Griffin D. Veterinary Therapeutics. 6 (2005) 29.

[31] Mueller RS, Fieseler KV and Fettman MJ. J Small An Pract 45 (2004) 293.

[32] Heinemann KM, Waldron MK, Bigley KE and Bauer JE (2005). Improvement of

Retinal Function in Canine Puppies from Mothers Fed Dietary Long Chain n-3

Polyunsaturated Fatty Acids during Gestation and Lactation (abstract) in: The American College of Veterinary Internal Medicine Meeting Conference Proceedings (ACVIM).

[33] US 20050075399

[34] US 20040028719

[35] Carrie I, Smirnova M, Clement M., De Javal D, Frances H, Bourre JM. Nutr.

Neuroscience 5 (2002) 43 [36] Chan ADF, Nippak P, Murphey H., Ikeda-Douglas C, Muggenberg B, Head E, Cotman

CW, Milgram NW. Behavioral Neuroscience 116 (2002) 443.

[37] Mandl, J. P. Journal of Reproduction and Fertility (1972), 31(2), 263-9.

[38] Bannach FG, Gutierrez-Fernandez A, Parmer RJ and Miles LA. J Thromb Haemost. 2

(2004) 2205. [39] Choi Y. Adv Exp Med Biol.560 (2005) 77.

[40] Leon KG and Karsan A. Histol Histopathol. 15 (2000) 1303,

[41] Armstrong RB. Med Sci. Sports Exerc 22 (1990) 529.

[42] Smith LL. Med. Sci. Sports Exersc 25 (1991) 542.

[43] Lemaitre-Delaunay D, Pachiaudi C, Laville M, Pousin J, Armstrong M and Lagarde M. J. Lipid Res. 40 (1999) 1867.

[44] Andersson A, Nalsen C, Tengblad A, Vessby B. Am J Clin Nutr 76 (2002) 1222.

[45] Bruckner G, Webb P, Greeenwell L Chow C, Richardson D, Atherosclerosis 66 (1987)

237.

[46] Ohyanagi M and Iwasaki T. MoI Cell Biochem. 160-161 (1996) 153. [47] Rebouche CJ. Am JCUn Nutr. 54 (1991) 1147S.

[48] Reda E, D'Iddio S, Nicolai R, Benatti P, Calvani M. Acta Diabetol. 40 (2003) S 106.

[49] Kris-Ethertom PM, Harris WS, Appel LJ, Circulation 106 (2002) 2747.

[50] Screpf R, Limmert T, Weber PC, Theisen K, Sellmayer A. Lancet 363 (2004) 1 141.

[51] Peet M and Stokes C. Drugs 65 (2005) 1051.

[53] Sarney DB, Fregapane G and Vulfson EN. JAOCS 71 (1994) 93.