Title:
Anti-inflammatory properties of marine lipid compositions
Kind Code:
A1


Abstract:
Novel marine lipid compositions comprising triglycerides and omega-3 rich phospholipids are described. The compositions are characterized by providing highly bioavailable omega-3, increased tissue incorporation of omega-3 and reduced concentration of pro-inflammatory cytokines.



Inventors:
Bruheim, Inge (Volda, NO)
Griinari, Mikko (Espoo, FI)
Banni, Sebastiano (Cagliari, IT)
Saebo, Per Christian (Volda, NO)
Fuglseth, Erik (Molde, NO)
Application Number:
11/800229
Publication Date:
02/21/2008
Filing Date:
05/04/2007
Assignee:
Natural ASA (Lysaker, NO)
Primary Class:
Other Classes:
554/78, 514/78
International Classes:
A61K35/12; A61K31/685; A61P29/00; C07F9/02
View Patent Images:



Primary Examiner:
POLANSKY, GREGG
Attorney, Agent or Firm:
Casimir Jones S. C. (440 Science Drive, Suite 203, Madison, WI, 53711, US)
Claims:
1. A composition comprising phospholipids having the following structure: wherein R1 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 R1 and/or R2 and from about 20-50% of OH at positions R1 and/or R2.

2. The composition as claimed in claim 1, wherein said composition is acylated in a range from about 55% to about 85%.

3. The composition as claimed in claim 2, wherein said composition has a ratio of EPA/DHA ranging from 1:1 to 4:1.

4. The composition of claim 2, wherein said composition having a ratio of EPA/DHA ranging from 2:1 to 4:1.

5. The composition of claim 1, wherein said composition further comprises a lipid carrier in a ratio of from 1:10 to 10:1 to said phospholipids.

6. The composition of claim 5, wherein said lipid carrier is selected from the group consisting of a triglyceride, a diglyceride, an ethyl ester, and a methyl ester and combinations thereof.

7. The composition in claim 1, wherein said composition provides higher uptake of omega-3 fatty acids into plasma as compared to natural marine phospholipids.

8. The composition in claim 1, wherein said composition improves the AA/EPA ratio in plasma phospholipids as compared to natural marine phospholipids.

9. The composition in claim 1, wherein said composition increases the concentration of omega-3 fatty acids in tissues as compared to natural marine phospholipids.

10. The composition in claim 1, wherein said composition reduces the concentration of biomarkers of inflammation as compared to natural marine phospholipids.

11. A food product comprising the composition in claim 1.

12. An animal feed comprising the composition in claim 1.

13. A food supplement comprising the composition in claim 1.

14. A pharmaceutical comprising the composition in claim 1.

15. The composition of claim 5, wherein said lipid carrier and said phospholipids are in a ratio of from about 5:1 to 1:5.

16. The composition of claim 5, wherein said composition comprises from about 20% to about 90% of said phospholipid composition and from about 10% to about 50% of said lipid carrier.

17. A method 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.

18. The method of claim 17, further comprising the step of encapsulating said bioavailable omega-3 fatty acid composition.

19. The method of claim 17, wherein said bioavailable omega-3 fatty acid composition has increased bioavailability as compared to purified triglycerides or phospholipids comprising omega-3 fatty acid residues.

20. The method of claim 17, further comprising the step of packaging the bioavailable omega-3 fatty acid composition for use in functional foods.

21. The method of claim 17, further comprising the step of assaying the bioavailable omega-3 fatty acid composition for bioavailability.

22. The method of claim 17, further comprising administering the bioavailable omega-3 fatty acid composition to a patient.

23. A food product comprising a bioavailable omega-3 fatty acid composition made the process of claim 17.

24. An animal feed comprising a bioavailable omega-3 fatty acid composition made the process of claim 17.

25. A food supplement comprising a bioavailable omega-3 fatty acid composition made the process of claim 17.

26. A pharmaceutical comprising a bioavailable omega-3 fatty acid composition made the process of claim 17.

Description:

This application claims the benefit of U.S. Provisional Applications 60/798,026, 60/798,027, and 60/798,030, all filed May 5, 2006, and U.S. Provisional Application 60/872,096, filed Dec. 1, 2006, each of which is incorporated herein by reference in their entirety.

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 [1].

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-1β and interleukin 6. In addition, since arachidonic acid (AA) is the predominant precursor of the eicosanoid mediators of inflammatory responses (prostaglandins, thromboxanes and leukotrienes), this invention discloses the reduction of AA level and the improvement in the EPA/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 C18 PUFA (94%) in pre-term infants [8]. The difference between C18 PUFA and long chain PUFA absorption is likely to become less apparent in older children and adults. Sala-Vila 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 C18:1, 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 R1 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, said phospholipids have a concentration of OH in the range of 25-50%. In 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 R1 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 R1 and/or R2 and from about 20-50% of OH at positions R1 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 esterified. 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 AA/EPA 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.

In 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 embodiments, the mammal is a human. In some embodiments, the mammal is a pet selected from the group consisting of cats and dogs. In some embodiments, the mammal has symptoms of age-associated memory 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 R1 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 R1 and/or R2. In some embodiments, the phospholipid composition comprises from about 20-50% of OH at positions R1 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 R1 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 R1 and/or R2. In some embodiments, the phospholipid composition comprises from about 20-50% of OH at positions R1 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 R1 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 R1 and/or R2. In some embodiments, the phospholipid composition comprises from about 20-50% of OH at positions R1 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

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

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

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

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

FIG. 5. Schematic drawing of experimental set-up.

FIG. 6. EPA levels in plasma as a function of hours after one bolus intake of a marine phospholipid composition.

FIG. 7. EPA levels in plasma as a function of hours after one bolus intake of a marine phospholipid composition.

FIG. 8. 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 R1 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 (HOCH2CH2N+(CH3)3OH), ethanolamine (HOCH2CH2NH2), inositol or serine. R1 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 R1 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, 5,8,11,14,17-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.

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 EPA/DHA 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.

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 R1 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 C18:3 (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 EPA/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-1β, 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 I1-1β induces fever. I1-6 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 pro-inflammatory 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 favor 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 composition contains no traces of organic solvents which is an important property regarding the safety of consuming such compounds. Phospholipids prepared using chemical or enzymatic methods in the presence of organic solvents may contain residual solvents that may be a health hazard. VOC are often co-extracted when marine phospholipids are extracted, such VOC's may contribute to the smell taste of the phospholipids.

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 D3; 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 l-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-10), 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-1-ol), geranyl acetate (geraniol acetate), limonene (d-, l-, and dl-), linalool (linalol, 3,7-dimethyl-1,6-octadien-3-ol), linalyl acetate(bergamol), methyl anthranilate(methyl-2-aminobenzoate), piperonal(3,4-methylenedioxy-benzaldehyde, heliotropin), vanillin, alfalfa (Medicago sativa L.), allspice (Pimenta 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), maroram (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 zedoaria), 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 (B1), 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-carnitine 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/GPC=0/15/85. 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) (FIG. 5 for schematic drawing of the experimental setup). The water in vessel A is heated to 25-30° 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. In 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, Conn., 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 ω9 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, ω3]; 7,10,13,16,19-docosapentaenoic acid [22:5, ω3]; 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, ω6]; 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]; 8,1 1-eicosadienoic acid [20:2, ω9]; 5,8,1 1-eicosatrienoic acid (Mead acid) [20:3, ω9]; t10,c12 octadecadienoic acid; c10,t12 octadecadienoic acid; c9,t11 octadecadienoic acid; and t9,c11 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.

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 transesterification 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
g/100 gg/100 gg/100 gSUM g/100 g
EPADHA18:3n3EPA + DHA + 18:3n3
ControlT4000.260.26
TG OilT10.610.390.241.23
PL 1T20.610.350.261.22
PL 2T30.240.730.261.23

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 AIN-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 anesthetized 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×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° 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 (FIG. 1). The amount of EPA in plasma varied between the groups and the results are shown FIG. 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 PL1 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 (FIG. 3 and FIG. 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 bioefficient 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 increase the EPA content in the adipose tissue more than the TG composition. It is to be observed that the PL1 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.

TABLE 2
Fatty acid profile of the total lipids isolated from the rat brain (mmol/g lipids).
18:118:218:3 n320:3ARAEPA22:422:5 n622:5 n3DHA
TG-oil370120.355.7153.72.268.55.65.0191.4
PL-1399120.337.2160.72.270.27.25.7203.3
PL-2345130.335.7141.61.760.85.83.4190.3
Control363120.305.2174.90.181.47.61.7189.6

TABLE 3
Fatty acid profile of the total lipids isolated from adipose tissue (mmol/g lipids).
18:118:218:3 n320:3ARAEPA22:422:5 n622:5 n3DHA
TG-oil35030640.21.46.321.76.30.911.327.5
PL-150023658.21.97.024.07.01.07.230.1
PL-242053645.72.46.812.86.82.7104.050.9
Control41911328.20.83.80.33.80.626.10.8

TABLE 4
Fatty acid profile of the total lipids in liver (mmol/g lipids).
18:118:218:3 n320:3ARAEPA22:422:5 n622:5 n3DHA
TG-oil304.2514.131.913.2278.9147.82.52.949.9272.8
PL-1264.3501.628.112.8317.3120.22.42.649.8259.0
PL-2226.6462.719.912.3289.080.43.05.530.4263.1
Control359.2493.721.78.9480.99.212.04.514.7145.8

TABLE 5
Fatty acid profile of the total lipids in testicles.
nmoles
FA/mgn3n620:3
lipids18:420:518:318:322:616:120:418:222:520:3n922:418:1
TG-oil3.217.011.71.856.647.6280.1195.16.9285.63.756.2326.8
PL-10.610.89.21.752.435.6294.9210.16.1294.23.461.3348.3
PL-22.46.11.72.056.00.0310.8156.52.4345.36.259.5332.5
Control1.81.11.62.027.20.0335.5162.21.3319.15.878.7330.1

TABLE 6
Fatty acid profile of the total lipids in heart.
nmoles
FA/mgn3n620:3
lipids18:420:518:318:322:616:120:418:222:520:3n922:418:1
TG-oil2.044.720.20.9314.049.7279.6787.94.06.83.0288.9
PL-11.545.025.60.8314.444.6300.4789.04.47.23.3276.6
PL-20.229.19.90.4372.130.1291.0690.39.06.31.54.4237.0
Control0.02.97.20.7209.9495.8756.214.76.22.430.3289.5

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.

TABLE 7
Fatty acid profile of the phospholipids isolated from the brain (mmol/g lipids).
18:118:218:3 n320:3ARAEPA22:422:5 n622:5 n3DHA
TG-oil260.89.90.24.5147.171.972.85.65.3196.8
PL-1289.910.90.14.2115.071.454.74.34.5170.1
PL-2232.62.10.64.2122.381.257.44.40.7175.9
Control288.84.43.5181.600.186.87.30.5213.1

TABLE 8
Fatty acid profile of the phospholipids isolated from
the adipose tissue (mmol/g lipids).
18:118:218:3 n320:3ARAEPA22:422:5 n622:5 n3DHA
TG-oil0.70.90.040.040.450.100.00.00.00.17
PL-10.60.50.040.020.240.060.00.00.00.09
PL-20.50.60.030.040.370.050.00.00.00.13
Control0.50.40.040.030.570.010.00.00.00.04

TABLE 9
Fatty acid profile of the phospholipids isolated from the liver (mmol/g lipids).
18:118:218:3 n320:3ARAEPA22:422:5 n622:5 n3DHA
TG-oil84.2165.44.411.5256.961.61.71.6920.5186.5
PL-1107.2227.64.611.4287.367.92.01.6229.1207.7
PL-2117.0347.76.612.3314.751.72.75.1226.0247.7
Control86.7204.41.67.6393.62.88.03.4111.0125.9

TABLE 10
Fatty acid profile of the phospholipids isolated from the testicles
nmoles FA/mg
lipids20:522:620:418:222:5 n322:5 n622:418:1
TG-oil3.1823.00173.1055.301.55234.2420.50118.11
PL-13.7131.41227.4482.562.05317.9028.27143.41
PL-23.33946.79335.87127.502.04265.3645.48155.71
Control0.43014.08204.2749.390.36161.2035.70118.18

TABLE 11
Fatty acid profile of the phospholipids isolated from the heart.
nmoles20:3
FA/mg lipids20:5n3 18:322:620:418:222:520:3n922:418:1
TG-oil35.06.4286.3240.3877.54.25.13.2119.6
PL-127.56.0261.6205.1754.63.44.22.696.3
PL-220.84.1331.1227.6570.37.95.01.24.1115.7
Control1.63.1187.1348.8505.913.73.21.922.0128.6

TABLE 12
Fatty acid profile of the sn-2 position on the phospholipids isolated from
the adipose tissue
sn-2EPAn3 18:3DHAARA18:2
TG-oil0.060.030.090.240.57
PL-10.090.040.120.290.70
PL-20.040.110.350.60
Control0.000.040.450.49

TABLE 13
Fatty acid profile of the sn-2 position on the phospholipids isolated
from the brain
sn-2EPADHAARA18:222:520:322:418:1
TG-oil1.6164.7123.69.85.03.972.8197.4
PL-11.3146.396.511.63.74.151.8260.2
PL-21.2173.5120.33.24.44.257.4223.4
Control0.1203.0171.64.47.13.281.1258.4

TABLE 14
Fatty acid profile of the sn-2 position of the phospholipids in the liver.
n3n620:3
sn-218:420:518:318:322:616:120:418:222:5n322:520:3n922:418:120:2
TG-oil0.550.22.30.3145.36.9188.6149.518.60.96.40.50.945.12.7
PL-10.460.12.90.4176.70.0235.6193.024.31.28.30.81.677.92.5
PL-20.145.84.20.5212.422.2264.5343.725.63.79.60.62.087.02.5
Control0.02.51.30.4108.631.7330.9204.411.02.76.01.26.462.63.7

TABLE 15
Fatty acid profile of the sn-2 position of the phospholipids in the testis.
sn-220:522:620:418:222:522:418:1
TG-oil2.921.8167.660.7224.420.5109.3
PL-13.428.4210.777.2290.628.2120.3
PL-22.541.6304.7110.4244.145.4137.8
Control0.413.6202.150.9157.235.6113.7

TABLE 16
Fatty acid profile of the sn-2 position of the phospholipids in the heart
sn-220:5n3 18:322:620:418:222:520:320:3 n922:418:1
TG-oil28.65.3215.4164.9741.72.93.23.268.2
PL-122.24.3193.4131.5601.82.12.22.552.5
PL-217.63.4256.7160.4519.55.73.21.24.170.0
Control1.41.9163.7283.9467.08.42.91.921.996.0

EXAMPLE 4

50 g of soy lecithin from American Lecithin Company Inc (Oxford, Conn., USA), 40 g of TL-IM lipase from Novozymes (Bagsvaerd, Denmark) and 5 g of water (adjusted to pH=8 using NaOH) were mixed in a reaction vessel at 50° C. for 24 hours. Next, 10 g 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, 20 g 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

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 [20] were added to the mixture in variable amounts so that a range of PC/LPC/GPC ratios could be obtained (Table 17). 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. FIGS. 6 and 7 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 MPL1, MPL4 and MPL5.

TABLE 17
Hydrolysis profile of the compositions tested
TreatmentMPL1MPL2MPL3MPL4MPL5
PC/LPC/GPC85/15/070/30/055/45/040/60/047/37/16

EXAMPLE 6

Marine phospholipids were prepared using either 40% soy PC (American Lecithin Company Inc, Oxford, Conn., USA) (MPL1) or 96% pure soy PC (Phospholipid GmbH, Köln, Germany) (MPL2) according to a method described by others [4]. Fatty acid content and the level of bi-products are shown in table 18. 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 MPL1 and MPL2, respectively. Finally, all three treatments were emulsified into skimmed milk.

TABLE 18
Composition of the phospholipids used in example 2
PC/LPC/18:2
CompositionGPC(n − 6)18:3 (n − 3)EPADHA
MPL164/33/2 129 mg/g9 mg/g51 mg/g171 mg/g
MPL242/40/18124 mg/g9 mg/g96 mg/g 96 mg/g

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 Rnaesy 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, Calif., 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. Using statistical tools a list of genes expressed differentially between MPL1 versus MPL2 (Table 3) was obtained. 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 MPL1 and MPL2 are biologically different compounds due to the fact that over 40 genes were differentially expressed (table 19).

TABLE 19
List of genes differentially expressed (DE) by MPL1 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 (<−1: down regulated gene, >1: up regulated gene) df:
Degrees of freedom (= number of arrays − number of estimated parameters).
FoldAffy
Gene-nametp-valueFDRSLRchangeFold.changedf
Wiskott-Aldrich syndrome-like8.350.000000.002611.022.022.0214
Similar to osteoclast inhibitory lectin7.400.000000.003921.492.802.8014
neuron-glia-CAM-related cell adhesion−7.140.000000.00392−0.550.68−1.4614
molecule
dehydrodolichyl diphosphate synthase7.090.000000.003920.571.491.4914
(predicted)
casein kinase II, alpha 1 polypeptide7.050.000000.003921.472.772.7714
similar to cisplatin resistance-associated7.030.000000.003920.941.921.9214
overexpressed protein (predicted)
similar to Retinoblastoma-binding protein 27.010.000000.003920.671.591.5914
(RBBP-2)
serine/threonine kinase 25 (STE20 homolog,6.900.000000.003920.431.351.3514
yeast)
Delangin (predicted)6.890.000000.003920.691.611.6114
similar to Hypothetical protein MGC30714−6.800.000000.00401−0.400.76−1.3214
myeloid/lymphoid or mixed-lineage leukemia 56.770.000000.004010.681.601.6014
(trithorax homolog, Drosophila) (predicted)
Radixin6.760.000000.004010.741.671.6714
similar to myocyte enhancer factor 2C6.700.000000.004200.601.521.5214
WD repeat and FYVE domain containing 16.640.000000.004430.801.741.7414
(predicted)
similar to Retinoblastoma-binding protein 26.550.000000.005090.871.821.8214
(RBBP-2)
zinc and ring finger 1 (predicted)−6.500.000000.00528−0.510.70−1.4214
pumilio 1 (Drosophila) (predicted)6.450.000000.005350.591.511.5114
leukocyte receptor cluster (LRC) member 86.440.000000.005351.062.082.0814
(predicted)
Similar to collapsin response mediator protein-−6.400.000000.00542−0.520.70−1.4314
2A
SWI/SNF related, matrix associated, actin6.370.000000.005420.741.671.6714
dependent regulator of chromatin, subfamily a,
member 4
B-cell CLL/lymphoma 7C (predicted)6.370.000000.005420.511.421.4214
synaptic vesicle glycoprotein 2b6.330.000000.005680.971.961.9614
leptin receptor overlapping transcript−6.310.000000.00570−0.580.67−1.5014
Transcribed locus6.220.000010.006580.441.361.3614
similar to mKIAA1321 protein6.200.000010.006580.991.991.9914
retinol binding protein 2, cellular6.180.000010.006600.591.511.5114
similar to Safb2 protein6.160.000010.006600.621.531.5314
similar to Zbtb20 protein6.150.000010.006600.491.401.4014
phosphofructokinase, liver, B-type6.130.000010.006650.671.601.6014
Transcription elongation factor B (SIII),6.100.000010.006680.431.351.3514
polypeptide 3
Echinoderm microtubule associated protein like6.100.000010.006681.002.002.0014
4 (predicted)
DNA topoisomerase I, mitochondrial6.080.000010.006780.611.531.5314
ectonucleoside triphosphate diphosphohydrolase 55.960.000010.008490.741.671.6714
nuclear factor I/X5.940.000010.008590.651.571.5714
WW domain binding protein 45.900.000010.009140.581.501.5014
Acetyl-coenzyme A acetyltransferase 15.840.000010.009670.691.611.6114
similar to THO complex 25.840.000010.009670.741.671.6714

MPL2 regulates 401 genes versus the control (table 20). 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 (Bbox1) (fold change of 1.32), monoglyceride lipase (Mg11) (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). The data listed in table 4 show that a omega-3 rich phospholipid with an EPA/DHA ratio of 2:1 behaves differently compared to placebo. A phospholipid composition with an EPA/DHA ratio o 1:1 did not show any difference versus placebo on gene expression. Consequently, the EPA/DHA ratio is important and should preferably be 2:1.

TABLE 20
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 (<−1: down
regulated gene, >1: up regulated gene) df: Degrees of freedom (= number of arrays −
number of estimated parameters)
FoldAffy
Gene-IDGene nametp-valueFDRSLRchangeFold.changedf
1367588_a_atribosomal protein L13A−5.220.000050.00568−0.440.74−1.3614
1367844_atguanine nucleotide binding−5.470.000030.00417−0.540.69−1.4614
protein, alpha inhibiting 2
1367958_atabl-interactor 1−6.420.000000.00119−0.690.62−1.6214
1367971_atprotein tyrosine phosphatase−6.010.000010.00190−0.360.78−1.2814
4a2
1368057_atATP-binding cassette, sub-−5.060.000070.00688−0.540.69−1.4514
family D (ALD), member 3
1368405_atv-ral simian leukemia viral−4.860.000110.00913−0.440.74−1.3614
oncogene homolog A (ras
related)
1368646_atmitogen-activated protein4.980.000080.007720.701.621.6214
kinase 9
1368649_atdyskeratosis congenita 1,−6.730.000000.00080−0.530.69−1.4414
dyskerin
1368662_atring finger protein 39−7.010.000000.00053−0.610.66−1.5214
1368703_atenigma homolog−5.380.000030.00450−0.760.59−1.7014
1368824_atcaldesmon 1−7.180.000000.00043−1.000.50−2.0014
1368841_attranscription factor 4−4.940.000090.00828−0.380.77−1.3114
1368867_atGERp95−7.830.000000.00019−0.850.56−1.8014
1369094_a_atprotein tyrosine phosphatase,−7.220.000000.00042−0.970.51−1.9614
receptor type, D
1369127_a_atprostaglandin F receptor4.850.000110.009210.451.371.3714
1369174_atSMAD, mothers against DPP−5.190.000050.00581−0.380.77−1.3014
homolog 1 (Drosophila)
1369227_atChoroidermia5.040.000070.007180.471.391.3914
1369249_atprogressive ankylosis homolog5.360.000040.004670.481.391.3914
(mouse)
1369501_atzinc finger protein 2605.170.000050.005950.411.331.3314
1369517_atpleckstrin homology, Sec7 and4.930.000090.008290.481.401.4014
coiled/coil domains 1
1369546_atbutyrobetaine (gamma), 2-4.960.000090.008110.401.321.3214
oxoglutarate dioxygenase 1
(gamma-butyrobetaine
hydroxylase)
1369628_atsynaptic vesicle glycoprotein−7.200.000000.00042−1.110.46−2.1514
2b
1369689_atN-ethylmaleimide sensitive6.220.000010.001550.661.581.5814
fusion protein
1369736_atepithelial membrane protein 15.740.000020.002860.621.541.5414
1369775_atnuclear ubiquitous casein−7.560.000000.00027−0.790.58−1.7314
kinase and cyclin-dependent
kinase substrate
1370184_atcofilin 1−6.070.000010.00178−0.380.77−1.3014
1370260_atadducin 3 (gamma)−5.500.000030.00399−0.760.59−1.7014
1370328_atDickkopf homolog 3 (Xenopus4.800.000120.009640.591.511.5114
laevis)
1370717_atAP1 gamma subunit binding6.000.000010.001920.581.501.5014
protein 1
1370831_atmonoglyceride lipase−5.470.000030.00414−0.940.52−1.9214
1370901_atsimilar to hypothetical protein−4.830.000120.00948−0.340.79−1.2714
MGC36831 (predicted)
1370946_atnuclear factor I/X−10.640.000000.00002−1.170.45−2.2514
1370949_atmyristoylated alanine rich−7.580.000000.00026−1.170.44−2.2614
protein kinase C substrate
1370993_atlaminin, gamma 16.130.000010.001710.631.541.5414
1371034_atone cut domain, family−5.650.000020.00327−1.770.29−3.4014
member 1
1371059_atprotein kinase, cAMP-5.240.000050.005560.481.401.4014
dependent, regulatory, type 2,
alpha
1371345_atmethyl-CpG binding domain−5.320.000040.00491−0.340.79−1.2714
protein 3 (predicted)
1371361_atsimilar to tensin−7.210.000000.00042−0.600.66−1.5114
1371394_x_atsimilar to Ab2-143−5.110.000060.00645−0.630.64−1.5514
1371397_atnitric oxide synthase−5.530.000020.00383−0.340.79−1.2614
interacting protein (predicted)
1371428_at−5.760.000010.00276−0.370.77−1.2914
1371430_atdystroglycan 1−5.460.000030.00417−0.620.65−1.5314
1371432_at−4.950.000090.00811−0.360.78−1.2814
1371452_atbone marrow stromal cell-−5.050.000070.00705−0.460.73−1.3714
derived ubiquitin-like protein
1371573_atribosomal protein L36a−5.900.000010.00221−0.400.76−1.3214
(predicted)
1371589_atUbiquitin-Like 5 Protein−5.280.000040.00518−0.570.68−1.4814
1371590_s_atUbiquitin-Like 5 Protein−4.940.000090.00829−0.390.76−1.3114
1371779_atsorting nexin 6 (predicted)5.640.000020.003290.631.551.5514
1371826_atTranscribed locus−5.580.000020.00359−0.480.72−1.3914
1371896_atgrowth arrest and DNA-−6.020.000010.00189−0.430.74−1.3514
damage-inducible, gamma
interacting protein 1 (predicted)
1371918_atCD99−5.350.000040.00476−0.370.77−1.2914
1372057_atCDNA clone MGC: 124976−6.120.000010.00173−0.380.77−1.3014
IMAGE: 7110947
1372137_atbiogenesis of lysosome-related−6.030.000010.00187−0.410.75−1.3214
organelles complex-1, subunit
1 (predicted)
1372142_atarsA arsenite transporter, ATP-−4.930.000090.00829−0.370.77−1.3014
binding, homolog 1 (bacterial)
(predicted)
1372236_atSimilar to Caspase recruitment−4.900.000100.00871−0.360.78−1.2914
domain protein 4
1372469_atTranscribed locus−4.840.000110.00945−0.360.78−1.2814
1372697_atmitochondrial ribosomal−5.700.000020.00299−0.580.67−1.4914
protein S15
1373031_attripartite motif protein 8−5.130.000060.00628−0.440.74−1.3614
(predicted)
1373105_atinterleukin 1 receptor-like 1−5.010.000080.00742−0.370.77−1.3014
ligand (predicted)
1373135_atsimilar to hypothetical protein−5.300.000040.00503−0.550.68−1.4614
MGC2744
1373206_atsimilar to FAD104 (predicted)6.730.000000.000800.641.561.5614
1373303_atsimilar to mKIAA3013 protein−5.280.000040.00514−0.480.72−1.3914
1373347_atDMT1-associated protein−6.180.000010.00162−0.730.60−1.6614
1373378_atATP/GTP binding protein 15.390.000030.004490.511.421.4214
(predicted)
1373804_atForkhead box P1 (predicted)−5.280.000040.00518−0.590.66−1.5114
1373885_atchromobox homolog 5−5.940.000010.00208−1.040.48−2.0614
(Drosophila HP1a) (predicted)
1374002_at−6.780.000000.00074−0.860.55−1.8214
1374283_atfetal Alzheimer antigen−7.440.000000.00032−0.740.60−1.6714
(predicted)
1374425_attransducin-like enhancer of−4.910.000100.00849−0.400.76−1.3214
split 1, homolog of Drosophila
E(spl) (predicted)
1374509_atSimilar to RIKEN cDNA−5.620.000020.00337−0.470.72−1.3914
1110018O08
1374511_at5.600.000020.003450.551.471.4714
1374657_atTranscribed locus−4.880.000100.00890−0.340.79−1.2714
1374733_atsymplekin (predicted)−5.040.000070.00716−0.360.78−1.2814
1374772_atsimilar to Chromosome 135.180.000050.005810.461.381.3814
open reading frame 21
1374837_atB-cell CLL/lymphoma 7C−8.920.000000.00006−0.710.61−1.6314
(predicted)
1374851_atsimilar to RIKEN cDNA−4.890.000100.00879−0.390.76−1.3114
2810405O22 (predicted)
1374852_athypothetical LOC362592−5.200.000050.00579−0.370.78−1.2914
1375214_atUDP-N-acetyl-alpha-D-−5.310.000040.00500−0.580.67−1.5014
galactosamine:polypeptide N-
acetylgalactosaminyltransferase
2 (predicted)
1375335_atheat shock 90 kDa protein 1,−5.260.000040.00538−0.550.68−1.4614
beta
1375396_atpumilio 1 (Drosophila)−10.050.000000.00003−0.920.53−1.8914
(predicted)
1375421_a_atpraja 2, RING-H2 motif−6.510.000000.00102−0.600.66−1.5214
containing
1375453_at−12.320.000000.00000−1.020.49−2.0214
1375469_atSWI/SNF related, matrix−7.970.000000.00017−0.930.53−1.9014
associated, actin dependent
regulator of chromatin,
subfamily a, member 4
1375533_atvestigial like 4 (Drosophila)−5.300.000040.00505−0.610.66−1.5214
(predicted)
1375548_atsimilar to RIKEN cDNA−5.640.000020.00328−0.580.67−1.5014
4732418C07 (predicted)
1375621_at−7.050.000000.00051−0.960.51−1.9514
1375632_atsimilar to 60S ribosomal−4.850.000110.00921−0.290.82−1.2214
protein L38
1375650_atbromodomain containing 4−6.640.000000.00088−0.480.71−1.4014
(predicted)
1375658_atTranscribed locus−5.000.000080.00756−0.440.74−1.3514
1375696_atinterferon (alpha and beta)4.810.000120.009580.591.511.5114
receptor 1 (predicted)
1375703_atmyeloid/lymphoid or mixed-−10.200.000000.00003−1.020.49−2.0314
lineage leukemia 5 (trithorax
homolog, Drosophila)
(predicted)
1375706_at−5.010.000080.00743−0.490.71−1.4014
1375763_atsimilar to 2700008B19Rik−7.080.000000.00050−0.540.69−1.4514
protein
1375958_at−5.130.000060.00628−0.650.64−1.5714
1376059_at5.330.000040.004830.351.281.2814
1376256_atWD repeat and FYVE domain−9.160.000000.00005−1.100.47−2.1514
containing 1 (predicted)
1376299_atsimilar to Retinoblastoma-−9.220.000000.00005−0.890.54−1.8514
binding protein 2 (RBBP-2)
1376450_attransmembrane protein 5−6.260.000010.00147−0.550.68−1.4614
(predicted)
1376523_atAt rich interactive domain 4A−5.530.000020.00383−0.770.59−1.7014
(Rbp1 like) (predicted)
1376524_athypothetical protein Dd25−6.690.000000.00082−0.660.63−1.5814
1376532_atsimilar to FAD104 (predicted)6.060.000010.001780.561.471.4714
1376728_atTranscribed locus−4.800.000120.00966−0.350.78−1.2714
1376917_atzinc finger protein 292−5.210.000050.00571−0.660.63−1.5814
1376982_atTranscribed locus−5.490.000030.00405−0.450.73−1.3714
1377105_at−6.970.000000.00056−0.890.54−1.8514
1377302_a_atmethylmalonic aciduria−5.100.000060.00660−0.520.70−1.4314
(cobalamin deficiency) type A
(predicted)
1377524_atsimilar to CG18661-PA−5.360.000030.00465−0.430.74−1.3514
(predicted)
1377663_atras homolog gene family,−5.000.000080.00756−0.870.55−1.8214
member E
1377683_atsimilar to hypothetical protein−6.630.000000.00088−0.560.68−1.4714
FLJ13045 (predicted)
1377728_atLOC499567−5.450.000030.00419−1.030.49−2.0414
1377766_atTranscribed locus4.800.000120.009640.371.291.2914
1377899_atsimilar to RIKEN cDNA−4.990.000080.00760−0.460.73−1.3814
2810025M15 (predicted)
1377906_atDEAH (Asp-Glu-Ala-His) box−4.820.000120.00950−0.730.60−1.6614
polypeptide 36 (predicted)
1377914_atserine/arginine repetitive−6.410.000000.00120−0.980.51−1.9714
matrix 1 (predicted)
1378155_atsimilar to KIAA1096 protein−5.680.000020.00313−0.890.54−1.8614
1378163_atTranscribed locus−4.860.000110.00913−0.780.58−1.7114
1378170_atTranscribed locus−5.000.000080.00756−0.920.53−1.9014
1378194_a_atrap2 interacting protein x−4.820.000120.00950−0.720.61−1.6514
1378361_atchromodomain helicase DNA−7.320.000000.00039−0.730.60−1.6614
binding protein 7 (predicted)
1378453_at−4.840.000110.00938−0.740.60−1.6614
1378504_atInsulin-like growth factor I−5.410.000030.00440−0.960.51−1.9514
mRNA, 3′ end of mRNA
1378786_atTranscribed locus, weakly4.890.000100.008790.331.251.2514
similar to NP_780607.2
hypothetical protein
LOC10905 [Mus musculus]
1379062_atsimilar to Expressed sequence−6.600.000000.00090−1.080.47−2.1214
AU019823
1379073_atSimilar to RIKEN cDNA−5.510.000030.00394−0.490.71−1.4014
2310067G05
1379101_atDEAH (Asp-Glu-Ala-His) box−5.550.000020.00375−0.870.55−1.8214
polypeptide 36 (predicted)
1379112_AtAt rich interactive domain 4A−5.700.000020.00299−0.440.74−1.3514
(Rbp1 like) (predicted)
1379232_atTBC1D12: TBC1 domain−6.980.000000.00056−1.400.38−2.6314
family, member 12 (predicted)
1379330_s_atCDNA clone IMAGE: 7316839−4.800.000120.00967−0.360.78−1.2814
1379332_atTranscribed locus, strongly−4.880.000100.00886−0.610.66−1.5214
similar to XP_417265.1
PREDICTED: similar to F-
box-WD40 repeat protein 6
[Gallus gallus]
1379399_atsimilar to cDNA sequence−5.370.000030.00459−0.420.75−1.3414
BC016188 (predicted)
1379457_atneural precursor cell expressed,−5.390.000030.00449−0.560.68−1.4814
developmentally down-
regulated gene 1 (predicted)
1379469_atsimilar to transducin (beta)-like−6.230.000010.00153−0.910.53−1.8814
1 X-linked
1379485_ateukaryotic translation initiation−7.080.000000.00050−1.680.31−3.2114
factor 3, subunit 10 (theta)
(predicted)
1379571_atplakophilin 4 (predicted)−5.420.000000.00436−0.740.60−1.6714
1379578_atsimilar to Zbtb20 protein−8.890.000000.00006−0.710.61−1.6314
1379662_a_atSNF related kinase4.930.000090.008290.361.291.2914
1379715_atsimilar to CG9346-PA−4.930.000090.00829−0.710.61−1.6314
(predicted)
1379826_atsimilar to hypothetical protein−5.950.000010.00208−0.620.65−1.5414
MGC31967
1380008_atsimilar to Neurofilament triplet−5.110.000060.00645−0.600.66−1.5214
H protein (20 kDa
neurofilament protein)
(Neurofilament heavy
polypeptide) (NF-H)
(predicted)
1380060_atDNA topoisomerase I,−5.230.000050.00566−0.530.69−1.4414
mitochondrial
1380062_atmembrane protein,−6.880.000000.00065−0.750.59−1.6814
palmitoylated 6 (MAGUK p55
subfamily member 6)
(predicted)
1380166_atSimilar to hypothetical protein5.630.000020.003330.341.271.2714
FLJ12056
1380371_atdelangin (predicted)−9.370.000000.00005−0.940.52−1.9114
1380446_atmyeloid/lymphoid or mixed-−5.000.000080.00756−0.620.65−1.5414
lineage leukemia (trithorax
homolog, Drosophila);
translocated to, 10 (predicted)
1380503_athypothetical LOC305452−6.070.000010.00178−0.620.65−1.5314
(predicted)
1380728_atSimilar to collapsin response6.090.000010.001780.491.411.4114
mediator protein-2A
1381469_a_atPERQ amino acid rich, with−5.490.000030.00405−0.510.70−1.4314
GYF domain 1 (predicted)
1381525_at−4.820.000120.00952−0.410.75−1.3314
1381542_atUBX domain containing 2−6.150.000010.00171−0.830.56−1.7814
(predicted)
1381548_atgolgi phosphoprotein 4−5.810.000010.00256−0.690.62−1.6114
(predicted)
1381567_athypothetical LOC2943904.970.000080.008000.361.291.2914
(predicted)
1381764_s_atring finger protein 126−5.540.000020.00382−0.510.70−1.4214
(predicted)
1381809_atankyrin repeat domain 11−5.940.000010.00209−1.110.46−2.1714
(predicted)
1381829_at−6.270.000000.00145−1.070.48−2.1014
1381878_atubinuclein 1 (predicted)−5.820.000010.00252−1.180.44−2.2614
1381958_atsimilar to mKIAA0259 protein−6.900.000000.00062−1.270.41−2.4214
1382000_at4.820.000120.009500.411.331.3314
1382009_atTranscribed locus−5.390.000030.00449−0.690.62−1.6214
1382027_atLOC498010−6.280.000000.00144−0.760.59−1.7014
1382056_atsimilar to splicing factor p54−8.130.000000.00016−0.970.51−1.9614
1382109_atnuclear NF-kappaB activating−5.830.000010.00250−0.620.65−1.5314
protein
1382155_at6.370.000000.001260.581.501.5014
1382193_atTranscribed locus−6.070.000010.00178−1.420.37−2.6714
1382306_atAriadne ubiquitin-conjugating6.590.000000.000900.591.501.5014
enzyme E2 binding protein
homolog 1 (Drosophila)
(predicted)
1382307_atprotein phosphatase 1,−4.790.000130.00976−0.470.72−1.3914
regulatory (inhibitor) subunit
12A
1382358_atSimilar to SRY (sex−5.340.000040.00482−0.650.64−1.5714
determining region Y)-box 5
isoform a
1382372_atAryl hydrocarbon receptor−5.070.000070.00680−0.740.60−1.6714
1382430_atsimilar to KIAA1585 protein−5.620.000020.00338−0.580.67−1.5014
(predicted)
1382434_atectonucleoside triphosphate−5.890.000010.00229−0.730.60−1.6614
diphosphohydrolase 5
1382466_atsimilar to RIKEN cDNA−5.430.000030.00433−0.980.51−1.9714
6530403A03 (predicted)
1382551_atsimilar to Intersectin 2 (SH3−6.720.000000.00080−1.410.38−2.6714
domain-containing protein 1B)
(SH3P18) (SH3P18-like
WASP associated protein)
1382558_attranscription factor 3−6.130.000010.00171−0.620.65−1.5414
(predicted)
1382573_atTranscribed locus5.080.000070.006770.381.301.3014
1382584_atsimilar to mKIAA1321 protein−7.220.000000.00042−1.150.45−2.2214
1382620_atankyrin repeat domain 11−9.690.000000.00003−0.950.52−1.9314
(predicted)
1382797_atsimilar to 1500019C06Rik−5.020.000080.00742−0.470.72−1.3914
protein
1382813_atsimilar to RIKEN cDNA−5.360.000040.00467−0.460.73−1.3814
4930444A02 (predicted)
1382862_atTranscribed locus−6.230.000010.00153−1.160.45−2.2314
1382904_atsimilar to hypothetical protein−9.040.000000.00005−0.850.56−1.8014
DKFZp434K1421 (predicted)
1382935_atsimilar to Hypothetical protein−6.540.000000.00097−0.640.64−1.5614
KIAA0141
1382939_attranslocated promoter region−5.180.000050.00581−1.130.46−2.1914
(predicted)
1382957_atsimilar to cisplatin resistance-−8.040.000000.00016−1.080.47−2.1114
associated overexpressed
protein (predicted)
1382960_atTranscribed locus−5.950.000010.00208−0.770.59−1.7014
1382972_atTranscribed locus, strongly5.170.000050.005950.371.291.2914
similar to XP_226713.2
PREDICTED: similar to Src-
associated protein SAW
[Rattus norvegicus]
1383008_atSMC4 structural maintenance−5.190.000050.00581−0.980.51−1.9714
of chromosomes 4-like 1
(yeast) (predicted)
1383040_a_at−5.460.000030.00419−0.470.72−1.3814
1383052_a_at−6.540.000000.00097−0.620.65−1.5314
1383054_at−7.860.000000.00019−0.760.59−1.7014
1383060_atG kinase anchoring protein 1−5.820.000010.00255−0.440.74−1.3514
(predicted)
1383085_atSimilar to Sh3bgrl protein−5.200.000050.00576−0.860.55−1.8114
1383179_atSimilar to hypothetical protein−5.100.000060.00660−0.750.60−1.6814
HSPC129 (predicted)
1383184_atzinc and ring finger 15.030.000070.007310.391.311.3114
(predicated)
1383334_atTranscribed locus−5.370.000030.00461−0.460.73−1.3714
1383455_atglutamyl-prolyl-tRNA−6.800.000000.00072−0.720.61−1.6514
synthetase (predicted)
1383535_atankyrin repeat and SOCS box-6.240.000010.001520.381.301.3014
containing protein 8 (predicted)
1383615_a_atsimilar to HECT domain−6.060.000010.00178−1.080.47−2.1114
containing 1
1383687_at−5.400.000030.00441−0.430.74−1.3414
1383776_atTranscribed locus6.410.000000.001200.621.541.5414
1383786_atTranscribed locus−5.130.000060.00628−0.500.71−1.4114
1383825_atradixin−9.200.000000.00005−1.010.50−2.0114
1383827_attousled-like kinase 1−6.090.000010.00178−1.250.42−2.3714
(predicted)
1384125_atmyeloid/lymphoid or mixed-−5.970.000010.00202−0.510.70−1.4214
lineage leukemia 5 (trithorax
homolog, Drosophila)
(predicted)
1384131_atADP-ribosylation factor-like 66.100.000010.001760.701.631.6314
interacting protein 2 (predicted)
1384146_atSimilar to CD69 antigen (p60,−5.220.000050.00568−1.350.39−2.5514
early T-cell activation antigen)
1384154_atWW domain binding protein 4−5.330.000040.00483−0.520.70−1.4314
1384260_atTranscribed locus−6.360.000000.00126−0.660.63−1.5814
1384263_atATP-binding cassette, sub-−7.270.000000.00040−0.720.61−1.6414
family A (ABC1), member 13
(predicted)
similar to hypothetical protein
MGC33214 (predicted)
1384339_s_atcasein kinase II, alpha 1−8.810.000000.00006−1.830.28−3.5614
polypeptide
1384376_atsimilar to FLJ14281 protein−5.420.000030.00436−0.650.64−1.5714
1384394_at−7.210.000000.00042−0.610.65−1.5314
1384609_a_atsimilar to RIKEN cDNA−6.610.000000.00090−0.910.53−1.8714
B230380D07 (predicted)
1384766_a_atsimilar to PHD finger protein−5.180.00005−0.00581−0.700.61−1.6314
14 isoform 1
1384791_atUDP-GlcNAc:betaGal beta-−5.080.000060.00671−0.750.60−1.6814
1,3-N-
acetylglucosaminyltransferase
1 (predicted)
1384792_atformin binding protein 3−6.700.000000.00082−0.970.51−1.9614
(predicted)
1384857_atA kinase (PRKA) anchor−5.620.000020.00338−1.050.48−2.0714
protein (yotiao) 9
1385006_atalpha thalassemia/mental−4.940.000090.00829−0.450.73−1.3714
retardation syndrome X-linked
homolog (human)
1385038_atsimilar to hedgehog-interacting−9.940.000000.00003−0.800.57−1.7514
protein
1385076_at−5.780.000010.00271−0.570.68−1.4814
1385077_atsimilar to golgi-specific−7.830.000000.00019−1.050.48−2.0714
brefeldin A-resistance guanine
nucleotide exchange factor 1
(predicted)
1385101_a_atUnknown (protein for−5.530.000020.00383−0.970.51−1.9614
MGC: 73017)
1385108_atTranscribed locus−4.830.000110.00946−1.270.42−2.4014
1385240_atWD repeat domain 33−4.810.000120.00958−0.930.52−1.9114
(predicted)
1385320_atsimilar to Pdz-containing−5.260.000040.00530−0.470.72−1.3814
protein
1385407_atTCDD-inducible poly(ADP-−5.460.000030.00417−1.340.39−2.5414
ribose) polymerase (predicted)
1385408_atsimilar to mKIAA0518 protein−5.860.000010.00236−1.310.40−2.4914
1385689_atTranscribed locus−4.830.000110.00948−0.680.62−1.6014
1385852_atCREB binding protein−4.850.000110.00927−0.530.69−1.4414
hypothetical gene supported by
NM_133381
1385931_athook homolog 3−7.300.000000.00040−1.660.32−3.1614
1385999_atYME1-like 1 (S. cerevisiae)−4.800.000120.00964−0.630.64−1.5514
1386191_a_atTranscribed locus5.220.000050.005680.461.371.3714
1386641_atTranscribed locus−5.410.000030.00440−0.970.51−1.9514
1386793_atsimilar to zinc finger protein 61−5.280.000040.00514−0.590.66−1.5114
1387087_atCCAAT/enhancer binding−5.430.000030.00435−0.610.66−1.5214
protein (C/EBP), beta
1387306_a_atearly growth response 24.820.000120.009500.331.261.2614
1387365_atnuclear receptor subfamily 1,−4.860.000110.00913−0.350.78−1.2714
group H, member 3
1387415_a_atsyntaxin binding protein 54.860.000110.009130.401.321.3214
(tomosyn)
1387458_atring finger protein 47.050.000000.000510.751.691.6914
1387664_atATPase, H+ transporting, V15.550.000020.003750.461.381.3814
subunit B, isoform 2
1387757_atliver regeneration p-53 related5.190.000050.005810.511.421.4214
protein
1387760_a_atone cut domain, family−6.120.000010.00171−1.580.34−2.9814
member 1
1387789_atv-ets erythroblastosis virus E26−6.610.000000.00090−0.580.67−1.5014
oncogene like (avian)
1387915_atRatsg2−4.790.000130.00976−0.330.79−1.2614
1387947_atv-maf musculoaponeurotic−5.080.000070.00674−0.800.57−1.7414
fibrosarcoma oncogene family,
protein B (avian)
1388022_a_atdynamin 1-like4.950.000090.008110.431.351.3514
1388059_a_atsolute carrier family 115.750.000010.002800.431.351.3514
(proton-coupled divalent metal
ion transporters), member 2
1388089_a_atring finger protein 45.730.000020.002890.501.411.4114
1388157_atmyristoylated alanine rich−5.760.000010.00276−0.510.70−1.4314
protein kinase C substrate
1388196_atNCK-associated protein 15.310.000040.005000.481.401.4014
1388251_atprotein kinase C, lambda5.210.000050.005710.551.471.4714
1388313_atribosomal protein s25−4.830.000110.00946−0.630.65−1.5414
1388353_atproliferation-associated 2G4,−6.350.000000.00127−0.670.63−1.5914
38 kDa
1388388_atProtein phosphatase 2,−5.320.000040.00487−0.410.75−1.3314
regulatory subunit B (B56),
delta isoform (predicted)
1388396_atserine/threonine kinase 25−5.490.000030.00404−0.340.79−1.2714
(STE20 homolog, yeast)
1388503_atsimilar to CREBBP/EP300−6.060.000010.00178−0.400.76−1.3214
inhibitory protein 1
1388714_atelongation factor RNA−5.880.000010.00229−0.460.73−1.3714
polymerase II (predicted)
1388735_atSimilar to keratin associated4.840.000110.009450.501.411.4114
protein 10-6
1388752_atBCL2-associated transcription−4.810.000120.00958−0.400.76−1.3214
factor 1 (predicted)
1388849_atProtease, serine, 25 (predicted)−5.970.000010.00202−0.500.71−1.4114
1388888_atTranscribed locus5.130.000060.006320.401.321.3214
1389268_atsimilar to DNA polymerase−5.210.000050.00571−0.370.77−1.2914
lambda
1389307_atsimilar to Amyloid beta (A4)−4.910.000100.00854−0.490.71−1.4014
precursor-like protein 1
1389419_atTranscribed locus−6.540.000000.00097−1.300.40−2.4714
1389432_atpre-B-cell leukemia−4.930.000090.00829−0.550.69−1.4614
transcription factor 2
1389444_atTranscribed locus−6.840.000000.00068−1.060.48−2.0914
1389806_atTranscribed locus−7.630.000000.00026−0.540.69−1.4614
1389868_atsimilar to RCK−6.340.000000.00128−1.470.36−2.7614
1389963_atP55 mRNA for p55 protein−5.540.000020.00378−0.440.74−1.3514
1389986_atLOC499304−5.390.000030.00449−2.570.17−5.9314
1389989_atalpha thalassemia/mental−4.930.000090.00829−0.540.69−1.4514
retardation syndrome X-linked
homolog (human)
1389998_atNuclear receptor subfamily 2,−6.030.000010.00187−0.690.62−1.6114
group F, member 2
1390048_atserine/arginine repetitive−5.810.000010.00256−1.040.49−2.0514
matrix 2 (predicted)
1390120_a_atring finger protein 1−5.700.000020.00299−0.360.78−1.2814
1390121_atGLIS family zinc finger 24.810.000120.009580.431.341.3414
(predicted)
1390227_atCDNA clone IMAGE: 7300848−5.910.000010.00219−1.030.49−2.0414
1390360_a_atsimilar to Safb2 protein−4.790.000130.00976−0.480.72−1.3914
1390410_atTranscribed locus−4.790.000120.00973−0.490.71−1.4014
1390436_atAutophagy 7-like (S. cerevisiae)−7.880.000000.00019−1.460.36−2.7614
(predicted)
1390448_atsimilar to 1110065L07Rik5.080.000070.006740.321.251.2514
protein (predicted)
1390454_at4-nitrophenylphosphatase−5.470.000030.00415−0.410.75−1.3314
domain and non-neuronal
SNAP25-like protein homolog
1 (C. elegans) (predicted)
1390576_atTranscribed locus−5.100.000060.00660−0.670.63−1.5914
1390660_atT-box 2 (predicted)5.010.000080.007520.401.321.3214
1390706_atspectrin beta 2−5.550.000020.00376−0.710.61−1.6414
1390739_atsimilar to zinc finger protein−5.510.000030.00395−0.520.70−1.4314
609
similar to zinc finger protein
609
1390777_atsterol-C5-desaturase (fungal−6.590.000000.00090−0.700.61−1.6314
ERG3, delta-5-desaturase)
homolog (S. cerevisae)
1390779_atSimilar to phosphoseryl-tRNA−4.860.000110.00913−0.640.64−1.5614
kinase
1390813_atSimilar to RNA-binding−5.190.000050.00581−0.620.65−1.5414
protein Musashi2-S
1390884_a_atUDP-GlcNAc: betaGal beta-4.870.000100.009040.491.401.4014
1,3-N-
acetylglucosaminyltransferase
7 (predicted)
1391021_atsimilar to KIAA1749 protein−7.550.000000.00027−0.740.60−1.6714
(predicted)
1391170_atsimilar to mKIAA1757 protein−9.210.000000.00005−2.010.25−4.0414
(predicted)
1391222_atsimilar to Nedd4 binding−5.910.000010.00219−0.810.57−1.7614
protein 1 (predicted)
1391297_atREST corepressor 1 (predicted)−5.170.000050.00595−0.920.53−1.8914
1391578_at−8.480.000000.00009−1.110.46−2.1514
1391584_atTranscribed locus6.040.000010.001850.451.371.3714
1391625_atWiskott-Aldrich syndrome-like−10.520.000000.00002−1.280.41−2.4314
(human)
1391669_atprotein tyrosine phosphatase,−6.210.000010.00156−0.820.56−1.7714
receptor type, B (predicted)
1391689_atsimilar to Retinoblastoma-−9.060.000000.00005−1.200.44−2.3014
binding protein 2 (RBBP-2)
1391701_atMYST histone−5.180.000050.00581−0.980.51−1.9714
acetyltransferase (monocytic
leukemia) 3 (predicted)
1391743_atELAV (embryonic lethal,−4.800.000120.00964−1.360.39−2.5814
abnormal vision, Drosophila)-
like 1 (Hu antigen R)
(predicted)
1391830_atcopine VIII (predicted)−5.220.000050.00568−1.080.47−2.1214
1391838_atankyrin repeat domain 11−7.810.000000.00019−1.150.45−2.2314
(predicted)
1391848_atRNA binding motif protein 27−7.020.000000.00053−0.760.59−1.7014
(predicted)
1391968_atSimilar to expressed sequence−4.890.000100.00883−0.690.62−1.6214
AA415817
1392000_atSimilar to PHD finger protein5.010.000080.007420.451.371.3714
14 isoform 1
1392061_atminichromosome maintenance5.340.000040.004820.541.461.4614
deficient 10 (S. cerevisiae)
(predicted)
1392269_attranscriptional regulator,−6.230.000010.00153−1.130.46−2.1914
SIN3A (yeast) (predicted)
1392277_at−7.290.000000.00040−0.480.72−1.4014
1392322_atGTPase, IMAP family member 7−4.830.000120.00948−0.290.82−1.2214
1392472_atsimilar to myocyte enhancer−9.770.000000.00003−0.880.54−1.8414
factor 2C
1392552_atsimilar to transcription−6.150.000010.00169−0.960.51−1.9514
repressor p66 (predicted)
1392564_atmyeloid/lymphoid or mixed-−6.130.000010.00171−0.570.68−1.4814
lineage leukemia 5 (trithorax
homolog, Drosophila)
(predicted)
1392629_a_atsimilar to MADP-1 protein−4.930.000090.00829−0.820.57−1.7714
(predicted)
1392738_atsimilar to KIAA1096 protein−5.880.000010.00231−0.750.59−1.6814
1392825_atLOC499256−5.200.000050.00580−0.930.53−1.9014
1392864_atRho GTPase activating protein−8.050.000000.00016−1.370.39−2.5814
5 (predicted)
1392932_atleukocyte receptor cluster−4.810.000120.00958−0.790.58−1.7314
(LRC) member 8 (predicted)
1392936_atsimilar to RNA binding motif−4.820.000120.00950−0.880.54−1.8514
protein 25
1392984_atcopine III (predicted)−7.830.000000.00019−0.950.52−1.9314
1393151_at5.030.000070.007260.651.571.5714
1393226_atTranscribed locus−4.940.000090.00828−0.730.60−1.6614
1393290_atsimilar to myocyte enhancer−5.650.000020.00327−0.500.71−1.4214
factor 2C
1393322_atTAF15 RNA polymerase II,−6.180.000010.00162−1.000.50−2.0014
TATA box binding protein
(TBP)-associated factor
(predicted)
1393378_at−5.720.000020.00293−0.520.70−1.4314
1393443_a_atsimilar to CGI-112 protein−5.330.000040.00483−0.470.72−1.3914
(predicted)
1393505_x_atsimilar to RIKEN cDNA−7.600.000000.00026−0.690.62−1.6114
B230380D07 (predicted)
1393511_atsimilar to galactose-3-O-5.100.000060.006550.411.331.3314
sulfotransferase 4
1393560_at−4.910.000100.00852−0.510.70−1.4214
1393576_atTranscribed locus−4.820.000120.00950−0.620.65−1.5414
1393593_atsimilar to KIAA0597 protein5.430.000030.004350.571.481.4814
1393639_atmyosin X (predicted)−4.950.000090.00811−0.590.67−1.5014
1393790_atHRAS-like suppressor5.440.000030.004320.441.351.3514
(predicted)
1393798_atalpha thalassemia/mental−5.000.000080.00757−0.840.56−1.7914
retardation syndrome X-linked
homolog (human)
1393804_atsimilar to hypothetical protein−6.790.000000.00073−0.850.56−1.8014
FLJ22490 (predicted)
1393809_atTnf receptor-associated factor 6−8.480.000000.00009−0.900.53−1.8714
(predicted)
1393811_atsimilar to putative repair and−6.080.000010.00178−0.790.58−1.7314
recombination helicase
RAD26L
1393910_atsimilar to Fam13a1 protein−4.850.000110.00921−0.810.57−1.7514
(predicted)
1393981_atsimilar to KIAA0423−5.240.000050.00556−0.570.68−1.4814
(predicted)
1394003_atsimilar to DNA polymerase−5.590.000020.00349−0.590.67−1.5014
epsilon p17 subunit (DNA
polymerase epsilon subunit 3)
(Chromatin accessibility
complex 17) (HuCHRAC17)
(CHRAC-17)
1394220_atSimilar to hypothetical protein5.460.000030.004170.431.341.3414
(predicted)
1394243_atsimilar to spermine synthase−6.110.000010.00175−0.600.66−1.5114
1394436_atsperm associated antigen 9−6.600.000000.00090−0.910.53−1.8814
(predicted)
1394497_atsimilar to TCF7L2 protein−8.030.000000.00016−1.060.48−2.0814
1394594_atTranscribed locus5.090.000060.006710.421.341.3414
1394715_atDicer1, Dcr-1 homolog5.140.000060.006270.541.461.4614
(Drosophila) (predicted)
1394740_at5.410.000030.004400.521.431.4314
1394742_atTranscribed locus−5.730.000020.00289−0.980.51−1.9814
1394746_athect (homologous to the E6-AP−7.320.000000.00039−0.940.52−1.9114
(UBE3A) carboxyl terminus)
domain and RCC1 (CHC1)-like
domain (RLD) 1 (predicted)
1394814_attranslocated promoter region−6.130.000010.00171−0.630.64−1.5514
(predicted)
1394849_atTranscribed locus−5.220.000050.00569−1.610.33−3.0514
1394865_atTransmembrane protein 7−7.850.000000.00019−0.920.53−1.9014
(predicted)
1394965_atenthoprotin5.300.000040.005030.401.321.3214
1394969_atTranscribed locus5.400.000030.004410.391.311.3114
1394985_atearly endosome antigen 1−7.600.000000.00026−1.000.50−2.0014
(predicted)
1395211_s_atsupervillin (predicted)−8.740.000000.00007−0.980.51−1.9714
1395237_ateukaryotic translation initiation−8.310.000000.00012−0.870.55−1.8314
factor 5B
1395264_atsimilar to Rap1-interacting−6.850.000000.00067−0.950.52−1.9314
factor 1
1395331_atsimilar to hypothetical protein4.840.000110.009450.311.241.2414
CL25084 (predicted)
1395338_atleucine-rich PPR-motif5.240.000050.005550.751.681.6814
containing (predicted)
1395516_atsimilar to hypothetical protein−4.890.000100.00883−0.590.66−1.5114
FLJ10154 (predicted)
1395565_atCOP9 signalosome subunit 45.550.000020.003760.401.321.3214
1395610_atsimilar to Hypothetical protein5.660.000020.003250.331.261.2614
MGC30714
1395616_atsimilar to Ab2-008 (predicted)−5.030.000070.00729−0.500.71−1.4214
1395625_atTranscribed locus−6.030.000010.00187−0.760.59−1.7014
1395739_atsimilar to RIKEN cDNA5.050.000070.006980.541.461.4614
C920006C10 (predicted)
1395814_atTranscribed locus−5.090.000060.00663−0.780.58−1.7114
1395976_atsimilar to phosphoinositol 4-−6.370.000000.00126−0.570.67−1.4914
phosphate adaptor protein-2
1395981_athelicase, ATP binding 1−5.760.000010.00276−0.620.65−1.5414
(predicted)
1396036_atRal GEF with PH domain and−6.670.000000.00084−1.040.49−2.0614
SH3 binding motif 2
(predicted)
1396063_atDEK oncogene (DNA binding)−4.820.000120.00952−0.630.65−1.5514
1396100_atsimilar to RIKEN cDNA−5.150.000060.00610−0.560.68−1.4714
2010009L17 (predicted)
1396170_atWW domain binding protein 4−7.780.000000.00020−0.770.59−1.7114
1396187_atHypothetical protein5.140.000060.006220.511.431.4314
LOC606294
1396202_atTranscribed locus4.970.000080.007950.521.441.4414
1396403_at−9.070.000000.00005−1.010.50−2.0214
1396803_atsimilar to THO complex 2−7.090.000000.00050−0.900.54−1.8614
1397203_atPRP4 pre-mRNA processing−6.180.000010.00162−0.670.63−1.5914
factor 4 homolog B (yeast)
(predicted)
1397234_atG patch domain containing 1−5.650.000020.00326−0.490.71−1.4014
(predicted)
1397367_atA disintegrin and5.050.000070.006980.471.381.3814
metalloprotease domain 23
(predicted)
1397508_atsimilar to RIKEN cDNA−5.080.000060.00671−0.620.65−1.5414
2310005B10
1397552_atechinoderm microtubule−8.470.000000.00009−1.390.38−2.6214
associated protein like 4
(predicted)
1397627_atdiaphanous homolog 1−5.070.000070.00680−0.520.70−1.4314
(Drosophila) (predicted)
1397647_atsolute carrier family 255.510.000030.003950.621.541.5414
(mitochondrial carrier;
ornithine transporter) member
15 (predicted)
1397669_atChemokine (C—C motif)5.780.000010.002710.511.431.4314
receptor 6 (predicted)
1397674_ateukaryotic translation initiation−6.440.000000.00116−0.760.59−1.6914
factor 3, subunit 8, 110 kDa
(predicted)
1397676_atSimilar to osteoclast inhibitory−6.680.000000.00084−1.340.39−2.5414
lectin
1397758_atSimilar to choline−4.830.000110.00946−0.380.77−1.3014
phosphotransferase 1;
cholinephosphotransferase 1
alpha;
cholinephosphotransferase 1
1397959_atsimilar to RIKEN cDNA−6.390.000000.00123−1.140.45−2.2014
D130059P03 gene (predicted)
1398311_a_atkinase D-interacting substance5.140.000060.006270.441.361.3614
220
1398351_atUbiquitin specific protease 7−5.600.000020.00349−0.420.75−1.3414
(herpes virus-associated)
(predicted)
1398420_atSimilar to E3 ubiquitin ligase−5.330.000040.00483−0.940.52−1.9214
SMURF2 (predicted)
1398436_atubiquitin specific protease 42−6.360.000000.00126−0.760.59−1.6914
(predicted)
1398486_atCDNA clone MGC: 93990−8.090.000000.00016−1.530.35−2.8914
IMAGE: 7115381
1398522_atsimilar to Ab2-034 (predicted)−4.920.000090.00832−0.510.70−1.4214
1398553_atsimilar to CGI-100-like protein−6.910.000000.00062−1.680.31−3.2014
1398834_atmitogen activated protein−4.940.000090.00828−0.320.80−1.2514
kinase kinase 2
1398926_atprefoldin 1 (predicted)−5.950.000010.00208−0.480.72−1.4014
1398963_atTAF10 RNA polymerase II,−5.420.000030.00436−0.410.75−1.3314
TATA box binding protein
(TBP)-associated factor
(predicted)
1399099_atheterogeneous nuclear−4.940.000090.00829−0.540.69−1.4614
ribonucleoprotein U-like 1
(predicted)
1399140_atTranscribed locus−5.160.000050.00597−0.490.71−1.4014
AFFX-BioB-Biotin synthase−4.890.000100.00879−0.640.64−1.5614
M_atbiotin synthesis, sulfur
insertion?
AFFX-dethiobiotin synthetase−4.920.000090.00834−0.700.62−1.6214
BioDn-5_at
AFFX-r2-Ec-dethiobiotin synthetase−5.410.000030.00440−0.510.70−1.4314
bioD-5_at

EXAMPLE 7

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, N2 was released into the glass flask and the mixture was heated to 65° C. 20 g Alcolec 40P® from American Lecithin Company Inc (Oxford, Conn., 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 (30° C.), was connected to the reaction vessel through a plastic tube (FIG. 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 7 is shown in table 20 below.

TABLE 20
Results obtained after transesterification using vacuum and water addition
Reaction timePC/LPC/GPC*EPA/DHA**Acid value
1 day65/31/43.6/2.643
2 days52/45/35.3/4.755
5 days78/22/05.2/4.665
6 days72/26/26.0/5.375

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

**EPA/DHA attached to PC + LPC

EXAMPLE 8

The enzymes from example 7 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 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 21 below.

TABLE 21
Results obtained with reused enzymes using incubator shaker
Reaction timePC/LPC/GPC*EPA/DHA**Acid value
1 day94/5/10.4/0.555
2 days87/11/20.7/0.776
5 days68/26/61.0/0.885

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

**EPA/DHA attached to PC + LPC

EXAMPLE 9

The same conditions as in example 1 were used, except that the amount of lipase was 10 g. The results are shown in table 22.

TABLE 22
Results obtained with reduced lipase dosage after transesterification
using vacuum and water addition
Reaction timePC/LPC/GPC*EPA/DHA**Acid value
1 day90/10/00.9/0.729
2 days74/24/22.1/1.587
3 days49/27/244.6/4.4102
6 days25/32/436.7/6.9115

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

**EPA/DHA attached to PC + LPC

EXAMPLE 10

The enzymes from example 7 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 23 below for results.

TABLE 23
Reuse of enzymes from example 7
using vapor addition into evacuated reaction vessel.
Reaction timePC/LPC/GPC*EPA/DHA**Acid value
1 day79/17/41.0/0.955
2 days59/31/102.8/2.676
3 days52/34/143.8/3.585
6 days37/43/205.6/5.795

*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 11

The same conditions as in example 7 are applied, except that the pressure in the reaction vessel is 1 mbar. The results obtained are similar to the results in Table 23, 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 12

The safety of omega-3 rich phospholipids prepared in the presence of chloroform and omega-7 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 phospholipds with traces of chloroform will result in damage to the developing fetus than the treatment containing essentially no traces of organic solvents.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

REFERENCES

[1] WO 2006054183

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

[3] U.S. Pat. No. 5,434,183

[4] Alexander J W. 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 J M. Am J Clin Nutr 71 (2000) 249

[7] V P 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. Sala-Vila, A. I. Castello, C. Campoy, M. Rivero, M. Rodriguez-Palermoe and M. C. Lopez-Sabater. J. Nutr. 2004; 134; 868-873.

[10] A. Sala-Vila, C. Campoy, A. I. Castellote, F. J. 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] U.S. provisional application entitled “Functional Phospholipid Compositions” with Ser. No. 60/798,027 filed May 5, 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 C. C. 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 G G and Thorarensen A, JAOCS 75 (1999) 1143.

[20] Samey D B, Fregapane G and Vulfson E N. JAOCS 71 (1994) 93.