US20080044487A1

US20080044487A1 - Anti-inflammatory properties of marine lipid compositions

Info

Publication number: US20080044487A1
Application number: US11/800,229
Authority: US
Inventors: Inge Bruheim; Mikko Griinari; Sebastiano Banni; Per Christian Sæbo; Erik Fuglseth
Original assignee: Natural ASA
Current assignee: Aker Biomarine ASA
Priority date: 2006-05-05
Filing date: 2007-05-04
Publication date: 2008-02-21
Also published as: US11400105B2; US20190070203A1; US20200179416A1; US10117882B2; US20230047556A1; US20100316680A1; US10525068B2

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.

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 g g/100 g g/100 g SUM g/100 g

EPA DHA 18:3n3 EPA + DHA + 18:3n3

Control T4 0 0 0.26 0.26

TG Oil T1 0.61 0.39 0.24 1.23

PL 1 T2 0.61 0.35 0.26 1.22

PL 2 T3 0.24 0.73 0.26 1.23
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).

	18:1	18:2	18:3 n3	20:3	ARA	EPA	22:4	22:5 n6	22:5 n3	DHA

TG-oil	370	12	0.35	5.7	153.7	2.2	68.5	5.6	5.0	191.4
PL-1	399	12	0.33	7.2	160.7	2.2	70.2	7.2	5.7	203.3
PL-2	345	13	0.33	5.7	141.6	1.7	60.8	5.8	3.4	190.3
Control	363	12	0.30	5.2	174.9	0.1	81.4	7.6	1.7	189.6

TABLE 3


Fatty acid profile of the total lipids isolated from adipose tissue (mmol/g lipids).

	18:1	18:2	18:3 n3	20:3	ARA	EPA	22:4	22:5 n6	22:5 n3	DHA

TG-oil	350	306	40.2	1.4	6.3	21.7	6.3	0.9	11.3	27.5
PL-1	500	236	58.2	1.9	7.0	24.0	7.0	1.0	7.2	30.1
PL-2	420	536	45.7	2.4	6.8	12.8	6.8	2.7	104.0	50.9
Control	419	113	28.2	0.8	3.8	0.3	3.8	0.6	26.1	0.8

TABLE 4


Fatty acid profile of the total lipids in liver (mmol/g lipids).

	18:1	18:2	18:3 n3	20:3	ARA	EPA	22:4	22:5 n6	22:5 n3	DHA

TG-oil	304.2	514.1	31.9	13.2	278.9	147.8	2.5	2.9	49.9	272.8
PL-1	264.3	501.6	28.1	12.8	317.3	120.2	2.4	2.6	49.8	259.0
PL-2	226.6	462.7	19.9	12.3	289.0	80.4	3.0	5.5	30.4	263.1
Control	359.2	493.7	21.7	8.9	480.9	9.2	12.0	4.5	14.7	145.8

TABLE 5


Fatty acid profile of the total lipids in testicles.

nmoles

FA/mg

n3

n6

20:3

lipids

18:4

20:5

18:3

22:6

16:1

20:4

18:2

22:5

20:3

n9

22:4

18:1

TG-oil	3.2	17.0	11.7	1.8	56.6	47.6	280.1	195.1	6.9	285.6	3.7	56.2	326.8
PL-1	0.6	10.8	9.2	1.7	52.4	35.6	294.9	210.1	6.1	294.2	3.4	61.3	348.3
PL-2	2.4	6.1	1.7	2.0	56.0	0.0	310.8	156.5	2.4	345.3	6.2	59.5	332.5
Control	1.8	1.1	1.6	2.0	27.2	0.0	335.5	162.2	1.3	319.1	5.8	78.7	330.1

TABLE 6


Fatty acid profile of the total lipids in heart.

nmoles

FA/mg

n3

n6

20:3

lipids

18:4

20:5

18:3

22:6

16:1

20:4

18:2

22:5

20:3

n9

22:4

18:1

TG-oil	2.0	44.7	20.2	0.9	314.0	49.7	279.6	787.9	4.0	6.8		3.0	288.9
PL-1	1.5	45.0	25.6	0.8	314.4	44.6	300.4	789.0	4.4	7.2		3.3	276.6
PL-2	0.2	29.1	9.9	0.4	372.1	30.1	291.0	690.3	9.0	6.3	1.5	4.4	237.0
Control	0.0	2.9	7.2	0.7	209.9		495.8	756.2	14.7	6.2	2.4	30.3	289.5

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).

	18:1	18:2	18:3 n3	20:3	ARA	EPA	22:4	22:5 n6	22:5 n3	DHA

TG-oil	260.8	9.9	0.2	4.5	147.17	1.9	72.8	5.6	5.3	196.8
PL-1	289.9	10.9	0.1	4.2	115.07	1.4	54.7	4.3	4.5	170.1
PL-2	232.6	2.1	0.6	4.2	122.38	1.2	57.4	4.4	0.7	175.9
Control	288.8	4.4		3.5	181.60	0.1	86.8	7.3	0.5	213.1

TABLE 8


Fatty acid profile of the phospholipids isolated from
the adipose tissue (mmol/g lipids).

	18:1	18:2	18:3 n3	20:3	ARA	EPA	22:4	22:5 n6	22:5 n3	DHA

TG-oil	0.7	0.9	0.04	0.04	0.45	0.10	0.0	0.0	0.0	0.17
PL-1	0.6	0.5	0.04	0.02	0.24	0.06	0.0	0.0	0.0	0.09
PL-2	0.5	0.6	0.03	0.04	0.37	0.05	0.0	0.0	0.0	0.13
Control	0.5	0.4	0.04	0.03	0.57	0.01	0.0	0.0	0.0	0.04

TABLE 9


Fatty acid profile of the phospholipids isolated from the liver (mmol/g lipids).

	18:1	18:2	18:3 n3	20:3	ARA	EPA	22:4	22:5 n6	22:5 n3	DHA

TG-oil	84.2	165.4	4.4	11.5	256.9	61.6	1.7	1.69	20.5	186.5
PL-1	107.2	227.6	4.6	11.4	287.3	67.9	2.0	1.62	29.1	207.7
PL-2	117.0	347.7	6.6	12.3	314.7	51.7	2.7	5.12	26.0	247.7
Control	86.7	204.4	1.6	7.6	393.6	2.8	8.0	3.41	11.0	125.9

TABLE 10


Fatty acid profile of the phospholipids isolated from the testicles

nmoles FA/mg
lipids	20:5	22:6	20:4	18:2	22:5 n3	22:5 n6	22:4	18:1

TG-oil	3.18	23.00	173.10	55.30	1.55	234.24	20.50	118.11
PL-1	3.71	31.41	227.44	82.56	2.05	317.90	28.27	143.41
PL-2	3.339	46.79	335.87	127.50	2.04	265.36	45.48	155.71
Control	0.430	14.08	204.27	49.39	0.36	161.20	35.70	118.18

TABLE 11


Fatty acid profile of the phospholipids isolated from the heart.

nmoles								20:3
FA/mg lipids	20:5	n3 18:3	22:6	20:4	18:2	22:5	20:3	n9	22:4	18:1

TG-oil	35.0	6.4	286.3	240.3	877.5	4.2	5.1		3.2	119.6
PL-1	27.5	6.0	261.6	205.1	754.6	3.4	4.2		2.6	96.3
PL-2	20.8	4.1	331.1	227.6	570.3	7.9	5.0	1.2	4.1	115.7
Control	1.6	3.1	187.1	348.8	505.9	13.7	3.2	1.9	22.0	128.6

TABLE 12


Fatty acid profile of the sn-2 position on the phospholipids isolated from
the adipose tissue

	sn-2	EPA	n3 18:3	DHA	ARA	18:2

TG-oil	0.06	0.03	0.09	0.24	0.57
PL-1	0.09	0.04	0.12	0.29	0.70
PL-2	0.04		0.11	0.35	0.60
Control	0.00		0.04	0.45	0.49

TABLE 13


Fatty acid profile of the sn-2 position on the phospholipids isolated
from the brain

sn-2	EPA	DHA	ARA	18:2	22:5	20:3	22:4	18:1

TG-oil	1.6	164.7	123.6	9.8	5.0	3.9	72.8	197.4
PL-1	1.3	146.3	96.5	11.6	3.7	4.1	51.8	260.2
PL-2	1.2	173.5	120.3	3.2	4.4	4.2	57.4	223.4
Control	0.1	203.0	171.6	4.4	7.1	3.2	81.1	258.4

TABLE 14


Fatty acid profile of the sn-2 position of the phospholipids in the liver.

n3

n6

20:3

sn-2

18:4

20:5

18:3

22:6

16:1

20:4

18:2

22:5n3

22:5

20:3

n9

22:4

18:1

20:2

TG-oil	0.5	50.2	2.3	0.3	145.3	6.9	188.6	149.5	18.6	0.9	6.4	0.5	0.9	45.1	2.7
PL-1	0.4	60.1	2.9	0.4	176.7	0.0	235.6	193.0	24.3	1.2	8.3	0.8	1.6	77.9	2.5
PL-2	0.1	45.8	4.2	0.5	212.4	22.2	264.5	343.7	25.6	3.7	9.6	0.6	2.0	87.0	2.5
Control	0.0	2.5	1.3	0.4	108.6	31.7	330.9	204.4	11.0	2.7	6.0	1.2	6.4	62.6	3.7

TABLE 15


Fatty acid profile of the sn-2 position of the phospholipids in the testis.

sn-2	20:5	22:6	20:4	18:2	22:5	22:4	18:1

TG-oil	2.9	21.8	167.6	60.7	224.4	20.5	109.3
PL-1	3.4	28.4	210.7	77.2	290.6	28.2	120.3
PL-2	2.5	41.6	304.7	110.4	244.1	45.4	137.8
Control	0.4	13.6	202.1	50.9	157.2	35.6	113.7

TABLE 16


Fatty acid profile of the sn-2 position of the phospholipids in the heart

sn-2	20:5	n3 18:3	22:6	20:4	18:2	22:5	20:3	20:3 n9	22:4	18:1

TG-oil	28.6	5.3	215.4	164.9	741.7	2.9	3.2		3.2	68.2
PL-1	22.2	4.3	193.4	131.5	601.8	2.1	2.2		2.5	52.5
PL-2	17.6	3.4	256.7	160.4	519.5	5.7	3.2	1.2	4.1	70.0
Control	1.4	1.9	163.7	283.9	467.0	8.4	2.9	1.9	21.9	96.0

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

Treatment MPL1 MPL2 MPL3 MPL4 MPL5

PC/LPC/GPC 85/15/0 70/30/0 55/45/0 40/60/0 47/37/16

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

Composition GPC (n − 6) 18:3 (n − 3) EPA DHA

MPL1 64/33/2 129 mg/g 9 mg/g 51 mg/g 171 mg/g

MPL2 42/40/18 124 mg/g 9 mg/g 96 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).

					Fold	Affy
Gene-name	t	p-value	FDR	SLR	change	Fold.change	df

Wiskott-Aldrich syndrome-like	8.35	0.00000	0.00261	1.02	2.02	2.02	14
Similar to osteoclast inhibitory lectin	7.40	0.00000	0.00392	1.49	2.80	2.80	14
neuron-glia-CAM-related cell adhesion	−7.14	0.00000	0.00392	−0.55	0.68	−1.46	14
molecule
dehydrodolichyl diphosphate synthase	7.09	0.00000	0.00392	0.57	1.49	1.49	14
(predicted)
casein kinase II, alpha 1 polypeptide	7.05	0.00000	0.00392	1.47	2.77	2.77	14
similar to cisplatin resistance-associated	7.03	0.00000	0.00392	0.94	1.92	1.92	14
overexpressed protein (predicted)
similar to Retinoblastoma-binding protein 2	7.01	0.00000	0.00392	0.67	1.59	1.59	14
(RBBP-2)
serine/threonine kinase 25 (STE20 homolog,	6.90	0.00000	0.00392	0.43	1.35	1.35	14
yeast)
Delangin (predicted)	6.89	0.00000	0.00392	0.69	1.61	1.61	14
similar to Hypothetical protein MGC30714	−6.80	0.00000	0.00401	−0.40	0.76	−1.32	14
myeloid/lymphoid or mixed-lineage leukemia 5	6.77	0.00000	0.00401	0.68	1.60	1.60	14
(trithorax homolog, Drosophila) (predicted)
Radixin	6.76	0.00000	0.00401	0.74	1.67	1.67	14
similar to myocyte enhancer factor 2C	6.70	0.00000	0.00420	0.60	1.52	1.52	14
WD repeat and FYVE domain containing 1	6.64	0.00000	0.00443	0.80	1.74	1.74	14
(predicted)
similar to Retinoblastoma-binding protein 2	6.55	0.00000	0.00509	0.87	1.82	1.82	14
(RBBP-2)
zinc and ring finger 1 (predicted)	−6.50	0.00000	0.00528	−0.51	0.70	−1.42	14
pumilio 1 (Drosophila) (predicted)	6.45	0.00000	0.00535	0.59	1.51	1.51	14
leukocyte receptor cluster (LRC) member 8	6.44	0.00000	0.00535	1.06	2.08	2.08	14
(predicted)
Similar to collapsin response mediator protein-	−6.40	0.00000	0.00542	−0.52	0.70	−1.43	14
2A
SWI/SNF related, matrix associated, actin	6.37	0.00000	0.00542	0.74	1.67	1.67	14
dependent regulator of chromatin, subfamily a,
member 4
B-cell CLL/lymphoma 7C (predicted)	6.37	0.00000	0.00542	0.51	1.42	1.42	14
synaptic vesicle glycoprotein 2b	6.33	0.00000	0.00568	0.97	1.96	1.96	14
leptin receptor overlapping transcript	−6.31	0.00000	0.00570	−0.58	0.67	−1.50	14
Transcribed locus	6.22	0.00001	0.00658	0.44	1.36	1.36	14
similar to mKIAA1321 protein	6.20	0.00001	0.00658	0.99	1.99	1.99	14
retinol binding protein 2, cellular	6.18	0.00001	0.00660	0.59	1.51	1.51	14
similar to Safb2 protein	6.16	0.00001	0.00660	0.62	1.53	1.53	14
similar to Zbtb20 protein	6.15	0.00001	0.00660	0.49	1.40	1.40	14
phosphofructokinase, liver, B-type	6.13	0.00001	0.00665	0.67	1.60	1.60	14
Transcription elongation factor B (SIII),	6.10	0.00001	0.00668	0.43	1.35	1.35	14
polypeptide 3
Echinoderm microtubule associated protein like	6.10	0.00001	0.00668	1.00	2.00	2.00	14
4 (predicted)
DNA topoisomerase I, mitochondrial	6.08	0.00001	0.00678	0.61	1.53	1.53	14
ectonucleoside triphosphate diphosphohydrolase 5	5.96	0.00001	0.00849	0.74	1.67	1.67	14
nuclear factor I/X	5.94	0.00001	0.00859	0.65	1.57	1.57	14
WW domain binding protein 4	5.90	0.00001	0.00914	0.58	1.50	1.50	14
Acetyl-coenzyme A acetyltransferase 1	5.84	0.00001	0.00967	0.69	1.61	1.61	14
similar to THO complex 2	5.84	0.00001	0.00967	0.74	1.67	1.67	14

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)

						Fold	Affy
Gene-ID	Gene name	t	p-value	FDR	SLR	change	Fold.change	df

1367588_a_at	ribosomal protein L13A	−5.22	0.00005	0.00568	−0.44	0.74	−1.36	14
1367844_at	guanine nucleotide binding	−5.47	0.00003	0.00417	−0.54	0.69	−1.46	14
	protein, alpha inhibiting 2
1367958_at	abl-interactor 1	−6.42	0.00000	0.00119	−0.69	0.62	−1.62	14
1367971_at	protein tyrosine phosphatase	−6.01	0.00001	0.00190	−0.36	0.78	−1.28	14
	4a2
1368057_at	ATP-binding cassette, sub-	−5.06	0.00007	0.00688	−0.54	0.69	−1.45	14
	family D (ALD), member 3
1368405_at	v-ral simian leukemia viral	−4.86	0.00011	0.00913	−0.44	0.74	−1.36	14
	oncogene homolog A (ras
	related)
1368646_at	mitogen-activated protein	4.98	0.00008	0.00772	0.70	1.62	1.62	14
	kinase 9
1368649_at	dyskeratosis congenita 1,	−6.73	0.00000	0.00080	−0.53	0.69	−1.44	14
	dyskerin
1368662_at	ring finger protein 39	−7.01	0.00000	0.00053	−0.61	0.66	−1.52	14
1368703_at	enigma homolog	−5.38	0.00003	0.00450	−0.76	0.59	−1.70	14
1368824_at	caldesmon 1	−7.18	0.00000	0.00043	−1.00	0.50	−2.00	14
1368841_at	transcription factor 4	−4.94	0.00009	0.00828	−0.38	0.77	−1.31	14
1368867_at	GERp95	−7.83	0.00000	0.00019	−0.85	0.56	−1.80	14
1369094_a_at	protein tyrosine phosphatase,	−7.22	0.00000	0.00042	−0.97	0.51	−1.96	14
	receptor type, D
1369127_a_at	prostaglandin F receptor	4.85	0.00011	0.00921	0.45	1.37	1.37	14
1369174_at	SMAD, mothers against DPP	−5.19	0.00005	0.00581	−0.38	0.77	−1.30	14
	homolog 1 (Drosophila)
1369227_at	Choroidermia	5.04	0.00007	0.00718	0.47	1.39	1.39	14
1369249_at	progressive ankylosis homolog	5.36	0.00004	0.00467	0.48	1.39	1.39	14
	(mouse)
1369501_at	zinc finger protein 260	5.17	0.00005	0.00595	0.41	1.33	1.33	14
1369517_at	pleckstrin homology, Sec7 and	4.93	0.00009	0.00829	0.48	1.40	1.40	14
	coiled/coil domains 1
1369546_at	butyrobetaine (gamma), 2-	4.96	0.00009	0.00811	0.40	1.32	1.32	14
	oxoglutarate dioxygenase 1
	(gamma-butyrobetaine
	hydroxylase)
1369628_at	synaptic vesicle glycoprotein	−7.20	0.00000	0.00042	−1.11	0.46	−2.15	14
	2b
1369689_at	N-ethylmaleimide sensitive	6.22	0.00001	0.00155	0.66	1.58	1.58	14
	fusion protein
1369736_at	epithelial membrane protein 1	5.74	0.00002	0.00286	0.62	1.54	1.54	14
1369775_at	nuclear ubiquitous casein	−7.56	0.00000	0.00027	−0.79	0.58	−1.73	14
	kinase and cyclin-dependent
	kinase substrate
1370184_at	cofilin 1	−6.07	0.00001	0.00178	−0.38	0.77	−1.30	14
1370260_at	adducin 3 (gamma)	−5.50	0.00003	0.00399	−0.76	0.59	−1.70	14
1370328_at	Dickkopf homolog 3 (Xenopus	4.80	0.00012	0.00964	0.59	1.51	1.51	14
	laevis)
1370717_at	AP1 gamma subunit binding	6.00	0.00001	0.00192	0.58	1.50	1.50	14
	protein 1
1370831_at	monoglyceride lipase	−5.47	0.00003	0.00414	−0.94	0.52	−1.92	14
1370901_at	similar to hypothetical protein	−4.83	0.00012	0.00948	−0.34	0.79	−1.27	14
	MGC36831 (predicted)
1370946_at	nuclear factor I/X	−10.64	0.00000	0.00002	−1.17	0.45	−2.25	14
1370949_at	myristoylated alanine rich	−7.58	0.00000	0.00026	−1.17	0.44	−2.26	14
	protein kinase C substrate
1370993_at	laminin, gamma 1	6.13	0.00001	0.00171	0.63	1.54	1.54	14
1371034_at	one cut domain, family	−5.65	0.00002	0.00327	−1.77	0.29	−3.40	14
	member 1
1371059_at	protein kinase, cAMP-	5.24	0.00005	0.00556	0.48	1.40	1.40	14
	dependent, regulatory, type 2,
	alpha
1371345_at	methyl-CpG binding domain	−5.32	0.00004	0.00491	−0.34	0.79	−1.27	14
	protein 3 (predicted)
1371361_at	similar to tensin	−7.21	0.00000	0.00042	−0.60	0.66	−1.51	14
1371394_x_at	similar to Ab2-143	−5.11	0.00006	0.00645	−0.63	0.64	−1.55	14
1371397_at	nitric oxide synthase	−5.53	0.00002	0.00383	−0.34	0.79	−1.26	14
	interacting protein (predicted)
1371428_at		−5.76	0.00001	0.00276	−0.37	0.77	−1.29	14
1371430_at	dystroglycan 1	−5.46	0.00003	0.00417	−0.62	0.65	−1.53	14
1371432_at		−4.95	0.00009	0.00811	−0.36	0.78	−1.28	14
1371452_at	bone marrow stromal cell-	−5.05	0.00007	0.00705	−0.46	0.73	−1.37	14
	derived ubiquitin-like protein
1371573_at	ribosomal protein L36a	−5.90	0.00001	0.00221	−0.40	0.76	−1.32	14
	(predicted)
1371589_at	Ubiquitin-Like 5 Protein	−5.28	0.00004	0.00518	−0.57	0.68	−1.48	14
1371590_s_at	Ubiquitin-Like 5 Protein	−4.94	0.00009	0.00829	−0.39	0.76	−1.31	14
1371779_at	sorting nexin 6 (predicted)	5.64	0.00002	0.00329	0.63	1.55	1.55	14
1371826_at	Transcribed locus	−5.58	0.00002	0.00359	−0.48	0.72	−1.39	14
1371896_at	growth arrest and DNA-	−6.02	0.00001	0.00189	−0.43	0.74	−1.35	14
	damage-inducible, gamma
	interacting protein 1 (predicted)
1371918_at	CD99	−5.35	0.00004	0.00476	−0.37	0.77	−1.29	14
1372057_at	CDNA clone MGC: 124976	−6.12	0.00001	0.00173	−0.38	0.77	−1.30	14
	IMAGE: 7110947
1372137_at	biogenesis of lysosome-related	−6.03	0.00001	0.00187	−0.41	0.75	−1.32	14
	organelles complex-1, subunit
	1 (predicted)
1372142_at	arsA arsenite transporter, ATP-	−4.93	0.00009	0.00829	−0.37	0.77	−1.30	14
	binding, homolog 1 (bacterial)
	(predicted)
1372236_at	Similar to Caspase recruitment	−4.90	0.00010	0.00871	−0.36	0.78	−1.29	14
	domain protein 4
1372469_at	Transcribed locus	−4.84	0.00011	0.00945	−0.36	0.78	−1.28	14
1372697_at	mitochondrial ribosomal	−5.70	0.00002	0.00299	−0.58	0.67	−1.49	14
	protein S15
1373031_at	tripartite motif protein 8	−5.13	0.00006	0.00628	−0.44	0.74	−1.36	14
	(predicted)
1373105_at	interleukin 1 receptor-like 1	−5.01	0.00008	0.00742	−0.37	0.77	−1.30	14
	ligand (predicted)
1373135_at	similar to hypothetical protein	−5.30	0.00004	0.00503	−0.55	0.68	−1.46	14
	MGC2744
1373206_at	similar to FAD104 (predicted)	6.73	0.00000	0.00080	0.64	1.56	1.56	14
1373303_at	similar to mKIAA3013 protein	−5.28	0.00004	0.00514	−0.48	0.72	−1.39	14
1373347_at	DMT1-associated protein	−6.18	0.00001	0.00162	−0.73	0.60	−1.66	14
1373378_at	ATP/GTP binding protein 1	5.39	0.00003	0.00449	0.51	1.42	1.42	14
	(predicted)
1373804_at	Forkhead box P1 (predicted)	−5.28	0.00004	0.00518	−0.59	0.66	−1.51	14
1373885_at	chromobox homolog 5	−5.94	0.00001	0.00208	−1.04	0.48	−2.06	14
	(Drosophila HP1a) (predicted)
1374002_at		−6.78	0.00000	0.00074	−0.86	0.55	−1.82	14
1374283_at	fetal Alzheimer antigen	−7.44	0.00000	0.00032	−0.74	0.60	−1.67	14
	(predicted)
1374425_at	transducin-like enhancer of	−4.91	0.00010	0.00849	−0.40	0.76	−1.32	14
	split 1, homolog of Drosophila
	E(spl) (predicted)
1374509_at	Similar to RIKEN cDNA	−5.62	0.00002	0.00337	−0.47	0.72	−1.39	14
	1110018O08
1374511_at		5.60	0.00002	0.00345	0.55	1.47	1.47	14
1374657_at	Transcribed locus	−4.88	0.00010	0.00890	−0.34	0.79	−1.27	14
1374733_at	symplekin (predicted)	−5.04	0.00007	0.00716	−0.36	0.78	−1.28	14
1374772_at	similar to Chromosome 13	5.18	0.00005	0.00581	0.46	1.38	1.38	14
	open reading frame 21
1374837_at	B-cell CLL/lymphoma 7C	−8.92	0.00000	0.00006	−0.71	0.61	−1.63	14
	(predicted)
1374851_at	similar to RIKEN cDNA	−4.89	0.00010	0.00879	−0.39	0.76	−1.31	14
	2810405O22 (predicted)
1374852_at	hypothetical LOC362592	−5.20	0.00005	0.00579	−0.37	0.78	−1.29	14
1375214_at	UDP-N-acetyl-alpha-D-	−5.31	0.00004	0.00500	−0.58	0.67	−1.50	14
	galactosamine:polypeptide N-
	acetylgalactosaminyltransferase
	2 (predicted)
1375335_at	heat shock 90 kDa protein 1,	−5.26	0.00004	0.00538	−0.55	0.68	−1.46	14
	beta
1375396_at	pumilio 1 (Drosophila)	−10.05	0.00000	0.00003	−0.92	0.53	−1.89	14
	(predicted)
1375421_a_at	praja 2, RING-H2 motif	−6.51	0.00000	0.00102	−0.60	0.66	−1.52	14
	containing
1375453_at		−12.32	0.00000	0.00000	−1.02	0.49	−2.02	14
1375469_at	SWI/SNF related, matrix	−7.97	0.00000	0.00017	−0.93	0.53	−1.90	14
	associated, actin dependent
	regulator of chromatin,
	subfamily a, member 4
1375533_at	vestigial like 4 (Drosophila)	−5.30	0.00004	0.00505	−0.61	0.66	−1.52	14
	(predicted)
1375548_at	similar to RIKEN cDNA	−5.64	0.00002	0.00328	−0.58	0.67	−1.50	14
	4732418C07 (predicted)
1375621_at		−7.05	0.00000	0.00051	−0.96	0.51	−1.95	14
1375632_at	similar to 60S ribosomal	−4.85	0.00011	0.00921	−0.29	0.82	−1.22	14
	protein L38
1375650_at	bromodomain containing 4	−6.64	0.00000	0.00088	−0.48	0.71	−1.40	14
	(predicted)
1375658_at	Transcribed locus	−5.00	0.00008	0.00756	−0.44	0.74	−1.35	14
1375696_at	interferon (alpha and beta)	4.81	0.00012	0.00958	0.59	1.51	1.51	14
	receptor 1 (predicted)
1375703_at	myeloid/lymphoid or mixed-	−10.20	0.00000	0.00003	−1.02	0.49	−2.03	14
	lineage leukemia 5 (trithorax
	homolog, Drosophila)
	(predicted)
1375706_at		−5.01	0.00008	0.00743	−0.49	0.71	−1.40	14
1375763_at	similar to 2700008B19Rik	−7.08	0.00000	0.00050	−0.54	0.69	−1.45	14
	protein
1375958_at		−5.13	0.00006	0.00628	−0.65	0.64	−1.57	14
1376059_at		5.33	0.00004	0.00483	0.35	1.28	1.28	14
1376256_at	WD repeat and FYVE domain	−9.16	0.00000	0.00005	−1.10	0.47	−2.15	14
	containing 1 (predicted)
1376299_at	similar to Retinoblastoma-	−9.22	0.00000	0.00005	−0.89	0.54	−1.85	14
	binding protein 2 (RBBP-2)
1376450_at	transmembrane protein 5	−6.26	0.00001	0.00147	−0.55	0.68	−1.46	14
	(predicted)
1376523_at	At rich interactive domain 4A	−5.53	0.00002	0.00383	−0.77	0.59	−1.70	14
	(Rbp1 like) (predicted)
1376524_at	hypothetical protein Dd25	−6.69	0.00000	0.00082	−0.66	0.63	−1.58	14
1376532_at	similar to FAD104 (predicted)	6.06	0.00001	0.00178	0.56	1.47	1.47	14
1376728_at	Transcribed locus	−4.80	0.00012	0.00966	−0.35	0.78	−1.27	14
1376917_at	zinc finger protein 292	−5.21	0.00005	0.00571	−0.66	0.63	−1.58	14
1376982_at	Transcribed locus	−5.49	0.00003	0.00405	−0.45	0.73	−1.37	14
1377105_at		−6.97	0.00000	0.00056	−0.89	0.54	−1.85	14
1377302_a_at	methylmalonic aciduria	−5.10	0.00006	0.00660	−0.52	0.70	−1.43	14
	(cobalamin deficiency) type A
	(predicted)
1377524_at	similar to CG18661-PA	−5.36	0.00003	0.00465	−0.43	0.74	−1.35	14
	(predicted)
1377663_at	ras homolog gene family,	−5.00	0.00008	0.00756	−0.87	0.55	−1.82	14
	member E
1377683_at	similar to hypothetical protein	−6.63	0.00000	0.00088	−0.56	0.68	−1.47	14
	FLJ13045 (predicted)
1377728_at	LOC499567	−5.45	0.00003	0.00419	−1.03	0.49	−2.04	14
1377766_at	Transcribed locus	4.80	0.00012	0.00964	0.37	1.29	1.29	14
1377899_at	similar to RIKEN cDNA	−4.99	0.00008	0.00760	−0.46	0.73	−1.38	14
	2810025M15 (predicted)
1377906_at	DEAH (Asp-Glu-Ala-His) box	−4.82	0.00012	0.00950	−0.73	0.60	−1.66	14
	polypeptide 36 (predicted)
1377914_at	serine/arginine repetitive	−6.41	0.00000	0.00120	−0.98	0.51	−1.97	14
	matrix 1 (predicted)
1378155_at	similar to KIAA1096 protein	−5.68	0.00002	0.00313	−0.89	0.54	−1.86	14
1378163_at	Transcribed locus	−4.86	0.00011	0.00913	−0.78	0.58	−1.71	14
1378170_at	Transcribed locus	−5.00	0.00008	0.00756	−0.92	0.53	−1.90	14
1378194_a_at	rap2 interacting protein x	−4.82	0.00012	0.00950	−0.72	0.61	−1.65	14
1378361_at	chromodomain helicase DNA	−7.32	0.00000	0.00039	−0.73	0.60	−1.66	14
	binding protein 7 (predicted)
1378453_at		−4.84	0.00011	0.00938	−0.74	0.60	−1.66	14
1378504_at	Insulin-like growth factor I	−5.41	0.00003	0.00440	−0.96	0.51	−1.95	14
	mRNA, 3′ end of mRNA
1378786_at	Transcribed locus, weakly	4.89	0.00010	0.00879	0.33	1.25	1.25	14
	similar to NP_780607.2
	hypothetical protein
	LOC10905 [Mus musculus]
1379062_at	similar to Expressed sequence	−6.60	0.00000	0.00090	−1.08	0.47	−2.12	14
	AU019823
1379073_at	Similar to RIKEN cDNA	−5.51	0.00003	0.00394	−0.49	0.71	−1.40	14
	2310067G05
1379101_at	DEAH (Asp-Glu-Ala-His) box	−5.55	0.00002	0.00375	−0.87	0.55	−1.82	14
	polypeptide 36 (predicted)
1379112_At	At rich interactive domain 4A	−5.70	0.00002	0.00299	−0.44	0.74	−1.35	14
	(Rbp1 like) (predicted)
1379232_at	TBC1D12: TBC1 domain	−6.98	0.00000	0.00056	−1.40	0.38	−2.63	14
	family, member 12 (predicted)
1379330_s_at	CDNA clone IMAGE: 7316839	−4.80	0.00012	0.00967	−0.36	0.78	−1.28	14
1379332_at	Transcribed locus, strongly	−4.88	0.00010	0.00886	−0.61	0.66	−1.52	14
	similar to XP_417265.1
	PREDICTED: similar to F-
	box-WD40 repeat protein 6
	[Gallus gallus]
1379399_at	similar to cDNA sequence	−5.37	0.00003	0.00459	−0.42	0.75	−1.34	14
	BC016188 (predicted)
1379457_at	neural precursor cell expressed,	−5.39	0.00003	0.00449	−0.56	0.68	−1.48	14
	developmentally down-
	regulated gene 1 (predicted)
1379469_at	similar to transducin (beta)-like	−6.23	0.00001	0.00153	−0.91	0.53	−1.88	14
	1 X-linked
1379485_at	eukaryotic translation initiation	−7.08	0.00000	0.00050	−1.68	0.31	−3.21	14
	factor 3, subunit 10 (theta)
	(predicted)
1379571_at	plakophilin 4 (predicted)	−5.42	0.00000	0.00436	−0.74	0.60	−1.67	14
1379578_at	similar to Zbtb20 protein	−8.89	0.00000	0.00006	−0.71	0.61	−1.63	14
1379662_a_at	SNF related kinase	4.93	0.00009	0.00829	0.36	1.29	1.29	14
1379715_at	similar to CG9346-PA	−4.93	0.00009	0.00829	−0.71	0.61	−1.63	14
	(predicted)
1379826_at	similar to hypothetical protein	−5.95	0.00001	0.00208	−0.62	0.65	−1.54	14
	MGC31967
1380008_at	similar to Neurofilament triplet	−5.11	0.00006	0.00645	−0.60	0.66	−1.52	14
	H protein (20 kDa
	neurofilament protein)
	(Neurofilament heavy
	polypeptide) (NF-H)
	(predicted)
1380060_at	DNA topoisomerase I,	−5.23	0.00005	0.00566	−0.53	0.69	−1.44	14
	mitochondrial
1380062_at	membrane protein,	−6.88	0.00000	0.00065	−0.75	0.59	−1.68	14
	palmitoylated 6 (MAGUK p55
	subfamily member 6)
	(predicted)
1380166_at	Similar to hypothetical protein	5.63	0.00002	0.00333	0.34	1.27	1.27	14
	FLJ12056
1380371_at	delangin (predicted)	−9.37	0.00000	0.00005	−0.94	0.52	−1.91	14
1380446_at	myeloid/lymphoid or mixed-	−5.00	0.00008	0.00756	−0.62	0.65	−1.54	14
	lineage leukemia (trithorax
	homolog, Drosophila);
	translocated to, 10 (predicted)
1380503_at	hypothetical LOC305452	−6.07	0.00001	0.00178	−0.62	0.65	−1.53	14
	(predicted)
1380728_at	Similar to collapsin response	6.09	0.00001	0.00178	0.49	1.41	1.41	14
	mediator protein-2A
1381469_a_at	PERQ amino acid rich, with	−5.49	0.00003	0.00405	−0.51	0.70	−1.43	14
	GYF domain 1 (predicted)
1381525_at		−4.82	0.00012	0.00952	−0.41	0.75	−1.33	14
1381542_at	UBX domain containing 2	−6.15	0.00001	0.00171	−0.83	0.56	−1.78	14
	(predicted)
1381548_at	golgi phosphoprotein 4	−5.81	0.00001	0.00256	−0.69	0.62	−1.61	14
	(predicted)
1381567_at	hypothetical LOC294390	4.97	0.00008	0.00800	0.36	1.29	1.29	14
	(predicted)
1381764_s_at	ring finger protein 126	−5.54	0.00002	0.00382	−0.51	0.70	−1.42	14
	(predicted)
1381809_at	ankyrin repeat domain 11	−5.94	0.00001	0.00209	−1.11	0.46	−2.17	14
	(predicted)
1381829_at		−6.27	0.00000	0.00145	−1.07	0.48	−2.10	14
1381878_at	ubinuclein 1 (predicted)	−5.82	0.00001	0.00252	−1.18	0.44	−2.26	14
1381958_at	similar to mKIAA0259 protein	−6.90	0.00000	0.00062	−1.27	0.41	−2.42	14
1382000_at		4.82	0.00012	0.00950	0.41	1.33	1.33	14
1382009_at	Transcribed locus	−5.39	0.00003	0.00449	−0.69	0.62	−1.62	14
1382027_at	LOC498010	−6.28	0.00000	0.00144	−0.76	0.59	−1.70	14
1382056_at	similar to splicing factor p54	−8.13	0.00000	0.00016	−0.97	0.51	−1.96	14
1382109_at	nuclear NF-kappaB activating	−5.83	0.00001	0.00250	−0.62	0.65	−1.53	14
	protein
1382155_at		6.37	0.00000	0.00126	0.58	1.50	1.50	14
1382193_at	Transcribed locus	−6.07	0.00001	0.00178	−1.42	0.37	−2.67	14
1382306_at	Ariadne ubiquitin-conjugating	6.59	0.00000	0.00090	0.59	1.50	1.50	14
	enzyme E2 binding protein
	homolog 1 (Drosophila)
	(predicted)
1382307_at	protein phosphatase 1,	−4.79	0.00013	0.00976	−0.47	0.72	−1.39	14
	regulatory (inhibitor) subunit
	12A
1382358_at	Similar to SRY (sex	−5.34	0.00004	0.00482	−0.65	0.64	−1.57	14
	determining region Y)-box 5
	isoform a
1382372_at	Aryl hydrocarbon receptor	−5.07	0.00007	0.00680	−0.74	0.60	−1.67	14
1382430_at	similar to KIAA1585 protein	−5.62	0.00002	0.00338	−0.58	0.67	−1.50	14
	(predicted)
1382434_at	ectonucleoside triphosphate	−5.89	0.00001	0.00229	−0.73	0.60	−1.66	14
	diphosphohydrolase 5
1382466_at	similar to RIKEN cDNA	−5.43	0.00003	0.00433	−0.98	0.51	−1.97	14
	6530403A03 (predicted)
1382551_at	similar to Intersectin 2 (SH3	−6.72	0.00000	0.00080	−1.41	0.38	−2.67	14
	domain-containing protein 1B)
	(SH3P18) (SH3P18-like
	WASP associated protein)
1382558_at	transcription factor 3	−6.13	0.00001	0.00171	−0.62	0.65	−1.54	14
	(predicted)
1382573_at	Transcribed locus	5.08	0.00007	0.00677	0.38	1.30	1.30	14
1382584_at	similar to mKIAA1321 protein	−7.22	0.00000	0.00042	−1.15	0.45	−2.22	14
1382620_at	ankyrin repeat domain 11	−9.69	0.00000	0.00003	−0.95	0.52	−1.93	14
	(predicted)
1382797_at	similar to 1500019C06Rik	−5.02	0.00008	0.00742	−0.47	0.72	−1.39	14
	protein
1382813_at	similar to RIKEN cDNA	−5.36	0.00004	0.00467	−0.46	0.73	−1.38	14
	4930444A02 (predicted)
1382862_at	Transcribed locus	−6.23	0.00001	0.00153	−1.16	0.45	−2.23	14
1382904_at	similar to hypothetical protein	−9.04	0.00000	0.00005	−0.85	0.56	−1.80	14
	DKFZp434K1421 (predicted)
1382935_at	similar to Hypothetical protein	−6.54	0.00000	0.00097	−0.64	0.64	−1.56	14
	KIAA0141
1382939_at	translocated promoter region	−5.18	0.00005	0.00581	−1.13	0.46	−2.19	14
	(predicted)
1382957_at	similar to cisplatin resistance-	−8.04	0.00000	0.00016	−1.08	0.47	−2.11	14
	associated overexpressed
	protein (predicted)
1382960_at	Transcribed locus	−5.95	0.00001	0.00208	−0.77	0.59	−1.70	14
1382972_at	Transcribed locus, strongly	5.17	0.00005	0.00595	0.37	1.29	1.29	14
	similar to XP_226713.2
	PREDICTED: similar to Src-
	associated protein SAW
	[Rattus norvegicus]
1383008_at	SMC4 structural maintenance	−5.19	0.00005	0.00581	−0.98	0.51	−1.97	14
	of chromosomes 4-like 1
	(yeast) (predicted)
1383040_a_at		−5.46	0.00003	0.00419	−0.47	0.72	−1.38	14
1383052_a_at		−6.54	0.00000	0.00097	−0.62	0.65	−1.53	14
1383054_at		−7.86	0.00000	0.00019	−0.76	0.59	−1.70	14
1383060_at	G kinase anchoring protein 1	−5.82	0.00001	0.00255	−0.44	0.74	−1.35	14
	(predicted)
1383085_at	Similar to Sh3bgrl protein	−5.20	0.00005	0.00576	−0.86	0.55	−1.81	14
1383179_at	Similar to hypothetical protein	−5.10	0.00006	0.00660	−0.75	0.60	−1.68	14
	HSPC129 (predicted)
1383184_at	zinc and ring finger 1	5.03	0.00007	0.00731	0.39	1.31	1.31	14
	(predicated)
1383334_at	Transcribed locus	−5.37	0.00003	0.00461	−0.46	0.73	−1.37	14
1383455_at	glutamyl-prolyl-tRNA	−6.80	0.00000	0.00072	−0.72	0.61	−1.65	14
	synthetase (predicted)
1383535_at	ankyrin repeat and SOCS box-	6.24	0.00001	0.00152	0.38	1.30	1.30	14
	containing protein 8 (predicted)
1383615_a_at	similar to HECT domain	−6.06	0.00001	0.00178	−1.08	0.47	−2.11	14
	containing 1
1383687_at		−5.40	0.00003	0.00441	−0.43	0.74	−1.34	14
1383776_at	Transcribed locus	6.41	0.00000	0.00120	0.62	1.54	1.54	14
1383786_at	Transcribed locus	−5.13	0.00006	0.00628	−0.50	0.71	−1.41	14
1383825_at	radixin	−9.20	0.00000	0.00005	−1.01	0.50	−2.01	14
1383827_at	tousled-like kinase 1	−6.09	0.00001	0.00178	−1.25	0.42	−2.37	14
	(predicted)
1384125_at	myeloid/lymphoid or mixed-	−5.97	0.00001	0.00202	−0.51	0.70	−1.42	14
	lineage leukemia 5 (trithorax
	homolog, Drosophila)
	(predicted)
1384131_at	ADP-ribosylation factor-like 6	6.10	0.00001	0.00176	0.70	1.63	1.63	14
	interacting protein 2 (predicted)
1384146_at	Similar to CD69 antigen (p60,	−5.22	0.00005	0.00568	−1.35	0.39	−2.55	14
	early T-cell activation antigen)
1384154_at	WW domain binding protein 4	−5.33	0.00004	0.00483	−0.52	0.70	−1.43	14
1384260_at	Transcribed locus	−6.36	0.00000	0.00126	−0.66	0.63	−1.58	14
1384263_at	ATP-binding cassette, sub-	−7.27	0.00000	0.00040	−0.72	0.61	−1.64	14
	family A (ABC1), member 13
	(predicted)
	similar to hypothetical protein
	MGC33214 (predicted)
1384339_s_at	casein kinase II, alpha 1	−8.81	0.00000	0.00006	−1.83	0.28	−3.56	14
	polypeptide
1384376_at	similar to FLJ14281 protein	−5.42	0.00003	0.00436	−0.65	0.64	−1.57	14
1384394_at		−7.21	0.00000	0.00042	−0.61	0.65	−1.53	14
1384609_a_at	similar to RIKEN cDNA	−6.61	0.00000	0.00090	−0.91	0.53	−1.87	14
	B230380D07 (predicted)
1384766_a_at	similar to PHD finger protein	−5.18	0.00005	−0.00581	−0.70	0.61	−1.63	14
	14 isoform 1
1384791_at	UDP-GlcNAc:betaGal beta-	−5.08	0.00006	0.00671	−0.75	0.60	−1.68	14
	1,3-N-
	acetylglucosaminyltransferase
	1 (predicted)
1384792_at	formin binding protein 3	−6.70	0.00000	0.00082	−0.97	0.51	−1.96	14
	(predicted)
1384857_at	A kinase (PRKA) anchor	−5.62	0.00002	0.00338	−1.05	0.48	−2.07	14
	protein (yotiao) 9
1385006_at	alpha thalassemia/mental	−4.94	0.00009	0.00829	−0.45	0.73	−1.37	14
	retardation syndrome X-linked
	homolog (human)
1385038_at	similar to hedgehog-interacting	−9.94	0.00000	0.00003	−0.80	0.57	−1.75	14
	protein
1385076_at		−5.78	0.00001	0.00271	−0.57	0.68	−1.48	14
1385077_at	similar to golgi-specific	−7.83	0.00000	0.00019	−1.05	0.48	−2.07	14
	brefeldin A-resistance guanine
	nucleotide exchange factor 1
	(predicted)
1385101_a_at	Unknown (protein for	−5.53	0.00002	0.00383	−0.97	0.51	−1.96	14
	MGC: 73017)
1385108_at	Transcribed locus	−4.83	0.00011	0.00946	−1.27	0.42	−2.40	14
1385240_at	WD repeat domain 33	−4.81	0.00012	0.00958	−0.93	0.52	−1.91	14
	(predicted)
1385320_at	similar to Pdz-containing	−5.26	0.00004	0.00530	−0.47	0.72	−1.38	14
	protein
1385407_at	TCDD-inducible poly(ADP-	−5.46	0.00003	0.00417	−1.34	0.39	−2.54	14
	ribose) polymerase (predicted)
1385408_at	similar to mKIAA0518 protein	−5.86	0.00001	0.00236	−1.31	0.40	−2.49	14
1385689_at	Transcribed locus	−4.83	0.00011	0.00948	−0.68	0.62	−1.60	14
1385852_at	CREB binding protein	−4.85	0.00011	0.00927	−0.53	0.69	−1.44	14
	hypothetical gene supported by
	NM_133381
1385931_at	hook homolog 3	−7.30	0.00000	0.00040	−1.66	0.32	−3.16	14
1385999_at	YME1-like 1 (S. cerevisiae)	−4.80	0.00012	0.00964	−0.63	0.64	−1.55	14
1386191_a_at	Transcribed locus	5.22	0.00005	0.00568	0.46	1.37	1.37	14
1386641_at	Transcribed locus	−5.41	0.00003	0.00440	−0.97	0.51	−1.95	14
1386793_at	similar to zinc finger protein 61	−5.28	0.00004	0.00514	−0.59	0.66	−1.51	14
1387087_at	CCAAT/enhancer binding	−5.43	0.00003	0.00435	−0.61	0.66	−1.52	14
	protein (C/EBP), beta
1387306_a_at	early growth response 2	4.82	0.00012	0.00950	0.33	1.26	1.26	14
1387365_at	nuclear receptor subfamily 1,	−4.86	0.00011	0.00913	−0.35	0.78	−1.27	14
	group H, member 3
1387415_a_at	syntaxin binding protein 5	4.86	0.00011	0.00913	0.40	1.32	1.32	14
	(tomosyn)
1387458_at	ring finger protein 4	7.05	0.00000	0.00051	0.75	1.69	1.69	14
1387664_at	ATPase, H+ transporting, V1	5.55	0.00002	0.00375	0.46	1.38	1.38	14
	subunit B, isoform 2
1387757_at	liver regeneration p-53 related	5.19	0.00005	0.00581	0.51	1.42	1.42	14
	protein
1387760_a_at	one cut domain, family	−6.12	0.00001	0.00171	−1.58	0.34	−2.98	14
	member 1
1387789_at	v-ets erythroblastosis virus E26	−6.61	0.00000	0.00090	−0.58	0.67	−1.50	14
	oncogene like (avian)
1387915_at	Ratsg2	−4.79	0.00013	0.00976	−0.33	0.79	−1.26	14
1387947_at	v-maf musculoaponeurotic	−5.08	0.00007	0.00674	−0.80	0.57	−1.74	14
	fibrosarcoma oncogene family,
	protein B (avian)
1388022_a_at	dynamin 1-like	4.95	0.00009	0.00811	0.43	1.35	1.35	14
1388059_a_at	solute carrier family 11	5.75	0.00001	0.00280	0.43	1.35	1.35	14
	(proton-coupled divalent metal
	ion transporters), member 2
1388089_a_at	ring finger protein 4	5.73	0.00002	0.00289	0.50	1.41	1.41	14
1388157_at	myristoylated alanine rich	−5.76	0.00001	0.00276	−0.51	0.70	−1.43	14
	protein kinase C substrate
1388196_at	NCK-associated protein 1	5.31	0.00004	0.00500	0.48	1.40	1.40	14
1388251_at	protein kinase C, lambda	5.21	0.00005	0.00571	0.55	1.47	1.47	14
1388313_at	ribosomal protein s25	−4.83	0.00011	0.00946	−0.63	0.65	−1.54	14
1388353_at	proliferation-associated 2G4,	−6.35	0.00000	0.00127	−0.67	0.63	−1.59	14
	38 kDa
1388388_at	Protein phosphatase 2,	−5.32	0.00004	0.00487	−0.41	0.75	−1.33	14
	regulatory subunit B (B56),
	delta isoform (predicted)
1388396_at	serine/threonine kinase 25	−5.49	0.00003	0.00404	−0.34	0.79	−1.27	14
	(STE20 homolog, yeast)
1388503_at	similar to CREBBP/EP300	−6.06	0.00001	0.00178	−0.40	0.76	−1.32	14
	inhibitory protein 1
1388714_at	elongation factor RNA	−5.88	0.00001	0.00229	−0.46	0.73	−1.37	14
	polymerase II (predicted)
1388735_at	Similar to keratin associated	4.84	0.00011	0.00945	0.50	1.41	1.41	14
	protein 10-6
1388752_at	BCL2-associated transcription	−4.81	0.00012	0.00958	−0.40	0.76	−1.32	14
	factor 1 (predicted)
1388849_at	Protease, serine, 25 (predicted)	−5.97	0.00001	0.00202	−0.50	0.71	−1.41	14
1388888_at	Transcribed locus	5.13	0.00006	0.00632	0.40	1.32	1.32	14
1389268_at	similar to DNA polymerase	−5.21	0.00005	0.00571	−0.37	0.77	−1.29	14
	lambda
1389307_at	similar to Amyloid beta (A4)	−4.91	0.00010	0.00854	−0.49	0.71	−1.40	14
	precursor-like protein 1
1389419_at	Transcribed locus	−6.54	0.00000	0.00097	−1.30	0.40	−2.47	14
1389432_at	pre-B-cell leukemia	−4.93	0.00009	0.00829	−0.55	0.69	−1.46	14
	transcription factor 2
1389444_at	Transcribed locus	−6.84	0.00000	0.00068	−1.06	0.48	−2.09	14
1389806_at	Transcribed locus	−7.63	0.00000	0.00026	−0.54	0.69	−1.46	14
1389868_at	similar to RCK	−6.34	0.00000	0.00128	−1.47	0.36	−2.76	14
1389963_at	P55 mRNA for p55 protein	−5.54	0.00002	0.00378	−0.44	0.74	−1.35	14
1389986_at	LOC499304	−5.39	0.00003	0.00449	−2.57	0.17	−5.93	14
1389989_at	alpha thalassemia/mental	−4.93	0.00009	0.00829	−0.54	0.69	−1.45	14
	retardation syndrome X-linked
	homolog (human)
1389998_at	Nuclear receptor subfamily 2,	−6.03	0.00001	0.00187	−0.69	0.62	−1.61	14
	group F, member 2
1390048_at	serine/arginine repetitive	−5.81	0.00001	0.00256	−1.04	0.49	−2.05	14
	matrix 2 (predicted)
1390120_a_at	ring finger protein 1	−5.70	0.00002	0.00299	−0.36	0.78	−1.28	14
1390121_at	GLIS family zinc finger 2	4.81	0.00012	0.00958	0.43	1.34	1.34	14
	(predicted)
1390227_at	CDNA clone IMAGE: 7300848	−5.91	0.00001	0.00219	−1.03	0.49	−2.04	14
1390360_a_at	similar to Safb2 protein	−4.79	0.00013	0.00976	−0.48	0.72	−1.39	14
1390410_at	Transcribed locus	−4.79	0.00012	0.00973	−0.49	0.71	−1.40	14
1390436_at	Autophagy 7-like (S. cerevisiae)	−7.88	0.00000	0.00019	−1.46	0.36	−2.76	14
	(predicted)
1390448_at	similar to 1110065L07Rik	5.08	0.00007	0.00674	0.32	1.25	1.25	14
	protein (predicted)
1390454_at	4-nitrophenylphosphatase	−5.47	0.00003	0.00415	−0.41	0.75	−1.33	14
	domain and non-neuronal
	SNAP25-like protein homolog
	1 (C. elegans) (predicted)
1390576_at	Transcribed locus	−5.10	0.00006	0.00660	−0.67	0.63	−1.59	14
1390660_at	T-box 2 (predicted)	5.01	0.00008	0.00752	0.40	1.32	1.32	14
1390706_at	spectrin beta 2	−5.55	0.00002	0.00376	−0.71	0.61	−1.64	14
1390739_at	similar to zinc finger protein	−5.51	0.00003	0.00395	−0.52	0.70	−1.43	14
	609
	similar to zinc finger protein
	609
1390777_at	sterol-C5-desaturase (fungal	−6.59	0.00000	0.00090	−0.70	0.61	−1.63	14
	ERG3, delta-5-desaturase)
	homolog (S. cerevisae)
1390779_at	Similar to phosphoseryl-tRNA	−4.86	0.00011	0.00913	−0.64	0.64	−1.56	14
	kinase
1390813_at	Similar to RNA-binding	−5.19	0.00005	0.00581	−0.62	0.65	−1.54	14
	protein Musashi2-S
1390884_a_at	UDP-GlcNAc: betaGal beta-	4.87	0.00010	0.00904	0.49	1.40	1.40	14
	1,3-N-
	acetylglucosaminyltransferase
	7 (predicted)
1391021_at	similar to KIAA1749 protein	−7.55	0.00000	0.00027	−0.74	0.60	−1.67	14
	(predicted)
1391170_at	similar to mKIAA1757 protein	−9.21	0.00000	0.00005	−2.01	0.25	−4.04	14
	(predicted)
1391222_at	similar to Nedd4 binding	−5.91	0.00001	0.00219	−0.81	0.57	−1.76	14
	protein 1 (predicted)
1391297_at	REST corepressor 1 (predicted)	−5.17	0.00005	0.00595	−0.92	0.53	−1.89	14
1391578_at		−8.48	0.00000	0.00009	−1.11	0.46	−2.15	14
1391584_at	Transcribed locus	6.04	0.00001	0.00185	0.45	1.37	1.37	14
1391625_at	Wiskott-Aldrich syndrome-like	−10.52	0.00000	0.00002	−1.28	0.41	−2.43	14
	(human)
1391669_at	protein tyrosine phosphatase,	−6.21	0.00001	0.00156	−0.82	0.56	−1.77	14
	receptor type, B (predicted)
1391689_at	similar to Retinoblastoma-	−9.06	0.00000	0.00005	−1.20	0.44	−2.30	14
	binding protein 2 (RBBP-2)
1391701_at	MYST histone	−5.18	0.00005	0.00581	−0.98	0.51	−1.97	14
	acetyltransferase (monocytic
	leukemia) 3 (predicted)
1391743_at	ELAV (embryonic lethal,	−4.80	0.00012	0.00964	−1.36	0.39	−2.58	14
	abnormal vision, Drosophila)-
	like 1 (Hu antigen R)
	(predicted)
1391830_at	copine VIII (predicted)	−5.22	0.00005	0.00568	−1.08	0.47	−2.12	14
1391838_at	ankyrin repeat domain 11	−7.81	0.00000	0.00019	−1.15	0.45	−2.23	14
	(predicted)
1391848_at	RNA binding motif protein 27	−7.02	0.00000	0.00053	−0.76	0.59	−1.70	14
	(predicted)
1391968_at	Similar to expressed sequence	−4.89	0.00010	0.00883	−0.69	0.62	−1.62	14
	AA415817
1392000_at	Similar to PHD finger protein	5.01	0.00008	0.00742	0.45	1.37	1.37	14
	14 isoform 1
1392061_at	minichromosome maintenance	5.34	0.00004	0.00482	0.54	1.46	1.46	14
	deficient 10 (S. cerevisiae)
	(predicted)
1392269_at	transcriptional regulator,	−6.23	0.00001	0.00153	−1.13	0.46	−2.19	14
	SIN3A (yeast) (predicted)
1392277_at		−7.29	0.00000	0.00040	−0.48	0.72	−1.40	14
1392322_at	GTPase, IMAP family member 7	−4.83	0.00012	0.00948	−0.29	0.82	−1.22	14
1392472_at	similar to myocyte enhancer	−9.77	0.00000	0.00003	−0.88	0.54	−1.84	14
	factor 2C
1392552_at	similar to transcription	−6.15	0.00001	0.00169	−0.96	0.51	−1.95	14
	repressor p66 (predicted)
1392564_at	myeloid/lymphoid or mixed-	−6.13	0.00001	0.00171	−0.57	0.68	−1.48	14
	lineage leukemia 5 (trithorax
	homolog, Drosophila)
	(predicted)
1392629_a_at	similar to MADP-1 protein	−4.93	0.00009	0.00829	−0.82	0.57	−1.77	14
	(predicted)
1392738_at	similar to KIAA1096 protein	−5.88	0.00001	0.00231	−0.75	0.59	−1.68	14
1392825_at	LOC499256	−5.20	0.00005	0.00580	−0.93	0.53	−1.90	14
1392864_at	Rho GTPase activating protein	−8.05	0.00000	0.00016	−1.37	0.39	−2.58	14
	5 (predicted)
1392932_at	leukocyte receptor cluster	−4.81	0.00012	0.00958	−0.79	0.58	−1.73	14
	(LRC) member 8 (predicted)
1392936_at	similar to RNA binding motif	−4.82	0.00012	0.00950	−0.88	0.54	−1.85	14
	protein 25
1392984_at	copine III (predicted)	−7.83	0.00000	0.00019	−0.95	0.52	−1.93	14
1393151_at		5.03	0.00007	0.00726	0.65	1.57	1.57	14
1393226_at	Transcribed locus	−4.94	0.00009	0.00828	−0.73	0.60	−1.66	14
1393290_at	similar to myocyte enhancer	−5.65	0.00002	0.00327	−0.50	0.71	−1.42	14
	factor 2C
1393322_at	TAF15 RNA polymerase II,	−6.18	0.00001	0.00162	−1.00	0.50	−2.00	14
	TATA box binding protein
	(TBP)-associated factor
	(predicted)
1393378_at		−5.72	0.00002	0.00293	−0.52	0.70	−1.43	14
1393443_a_at	similar to CGI-112 protein	−5.33	0.00004	0.00483	−0.47	0.72	−1.39	14
	(predicted)
1393505_x_at	similar to RIKEN cDNA	−7.60	0.00000	0.00026	−0.69	0.62	−1.61	14
	B230380D07 (predicted)
1393511_at	similar to galactose-3-O-	5.10	0.00006	0.00655	0.41	1.33	1.33	14
	sulfotransferase 4
1393560_at		−4.91	0.00010	0.00852	−0.51	0.70	−1.42	14
1393576_at	Transcribed locus	−4.82	0.00012	0.00950	−0.62	0.65	−1.54	14
1393593_at	similar to KIAA0597 protein	5.43	0.00003	0.00435	0.57	1.48	1.48	14
1393639_at	myosin X (predicted)	−4.95	0.00009	0.00811	−0.59	0.67	−1.50	14
1393790_at	HRAS-like suppressor	5.44	0.00003	0.00432	0.44	1.35	1.35	14
	(predicted)
1393798_at	alpha thalassemia/mental	−5.00	0.00008	0.00757	−0.84	0.56	−1.79	14
	retardation syndrome X-linked
	homolog (human)
1393804_at	similar to hypothetical protein	−6.79	0.00000	0.00073	−0.85	0.56	−1.80	14
	FLJ22490 (predicted)
1393809_at	Tnf receptor-associated factor 6	−8.48	0.00000	0.00009	−0.90	0.53	−1.87	14
	(predicted)
1393811_at	similar to putative repair and	−6.08	0.00001	0.00178	−0.79	0.58	−1.73	14
	recombination helicase
	RAD26L
1393910_at	similar to Fam13a1 protein	−4.85	0.00011	0.00921	−0.81	0.57	−1.75	14
	(predicted)
1393981_at	similar to KIAA0423	−5.24	0.00005	0.00556	−0.57	0.68	−1.48	14
	(predicted)
1394003_at	similar to DNA polymerase	−5.59	0.00002	0.00349	−0.59	0.67	−1.50	14
	epsilon p17 subunit (DNA
	polymerase epsilon subunit 3)
	(Chromatin accessibility
	complex 17) (HuCHRAC17)
	(CHRAC-17)
1394220_at	Similar to hypothetical protein	5.46	0.00003	0.00417	0.43	1.34	1.34	14
	(predicted)
1394243_at	similar to spermine synthase	−6.11	0.00001	0.00175	−0.60	0.66	−1.51	14
1394436_at	sperm associated antigen 9	−6.60	0.00000	0.00090	−0.91	0.53	−1.88	14
	(predicted)
1394497_at	similar to TCF7L2 protein	−8.03	0.00000	0.00016	−1.06	0.48	−2.08	14
1394594_at	Transcribed locus	5.09	0.00006	0.00671	0.42	1.34	1.34	14
1394715_at	Dicer1, Dcr-1 homolog	5.14	0.00006	0.00627	0.54	1.46	1.46	14
	(Drosophila) (predicted)
1394740_at		5.41	0.00003	0.00440	0.52	1.43	1.43	14
1394742_at	Transcribed locus	−5.73	0.00002	0.00289	−0.98	0.51	−1.98	14
1394746_at	hect (homologous to the E6-AP	−7.32	0.00000	0.00039	−0.94	0.52	−1.91	14
	(UBE3A) carboxyl terminus)
	domain and RCC1 (CHC1)-like
	domain (RLD) 1 (predicted)
1394814_at	translocated promoter region	−6.13	0.00001	0.00171	−0.63	0.64	−1.55	14
	(predicted)
1394849_at	Transcribed locus	−5.22	0.00005	0.00569	−1.61	0.33	−3.05	14
1394865_at	Transmembrane protein 7	−7.85	0.00000	0.00019	−0.92	0.53	−1.90	14
	(predicted)
1394965_at	enthoprotin	5.30	0.00004	0.00503	0.40	1.32	1.32	14
1394969_at	Transcribed locus	5.40	0.00003	0.00441	0.39	1.31	1.31	14
1394985_at	early endosome antigen 1	−7.60	0.00000	0.00026	−1.00	0.50	−2.00	14
	(predicted)
1395211_s_at	supervillin (predicted)	−8.74	0.00000	0.00007	−0.98	0.51	−1.97	14
1395237_at	eukaryotic translation initiation	−8.31	0.00000	0.00012	−0.87	0.55	−1.83	14
	factor 5B
1395264_at	similar to Rap1-interacting	−6.85	0.00000	0.00067	−0.95	0.52	−1.93	14
	factor 1
1395331_at	similar to hypothetical protein	4.84	0.00011	0.00945	0.31	1.24	1.24	14
	CL25084 (predicted)
1395338_at	leucine-rich PPR-motif	5.24	0.00005	0.00555	0.75	1.68	1.68	14
	containing (predicted)
1395516_at	similar to hypothetical protein	−4.89	0.00010	0.00883	−0.59	0.66	−1.51	14
	FLJ10154 (predicted)
1395565_at	COP9 signalosome subunit 4	5.55	0.00002	0.00376	0.40	1.32	1.32	14
1395610_at	similar to Hypothetical protein	5.66	0.00002	0.00325	0.33	1.26	1.26	14
	MGC30714
1395616_at	similar to Ab2-008 (predicted)	−5.03	0.00007	0.00729	−0.50	0.71	−1.42	14
1395625_at	Transcribed locus	−6.03	0.00001	0.00187	−0.76	0.59	−1.70	14
1395739_at	similar to RIKEN cDNA	5.05	0.00007	0.00698	0.54	1.46	1.46	14
	C920006C10 (predicted)
1395814_at	Transcribed locus	−5.09	0.00006	0.00663	−0.78	0.58	−1.71	14
1395976_at	similar to phosphoinositol 4-	−6.37	0.00000	0.00126	−0.57	0.67	−1.49	14
	phosphate adaptor protein-2
1395981_at	helicase, ATP binding 1	−5.76	0.00001	0.00276	−0.62	0.65	−1.54	14
	(predicted)
1396036_at	Ral GEF with PH domain and	−6.67	0.00000	0.00084	−1.04	0.49	−2.06	14
	SH3 binding motif 2
	(predicted)
1396063_at	DEK oncogene (DNA binding)	−4.82	0.00012	0.00952	−0.63	0.65	−1.55	14
1396100_at	similar to RIKEN cDNA	−5.15	0.00006	0.00610	−0.56	0.68	−1.47	14
	2010009L17 (predicted)
1396170_at	WW domain binding protein 4	−7.78	0.00000	0.00020	−0.77	0.59	−1.71	14
1396187_at	Hypothetical protein	5.14	0.00006	0.00622	0.51	1.43	1.43	14
	LOC606294
1396202_at	Transcribed locus	4.97	0.00008	0.00795	0.52	1.44	1.44	14
1396403_at		−9.07	0.00000	0.00005	−1.01	0.50	−2.02	14
1396803_at	similar to THO complex 2	−7.09	0.00000	0.00050	−0.90	0.54	−1.86	14
1397203_at	PRP4 pre-mRNA processing	−6.18	0.00001	0.00162	−0.67	0.63	−1.59	14
	factor 4 homolog B (yeast)
	(predicted)
1397234_at	G patch domain containing 1	−5.65	0.00002	0.00326	−0.49	0.71	−1.40	14
	(predicted)
1397367_at	A disintegrin and	5.05	0.00007	0.00698	0.47	1.38	1.38	14
	metalloprotease domain 23
	(predicted)
1397508_at	similar to RIKEN cDNA	−5.08	0.00006	0.00671	−0.62	0.65	−1.54	14
	2310005B10
1397552_at	echinoderm microtubule	−8.47	0.00000	0.00009	−1.39	0.38	−2.62	14
	associated protein like 4
	(predicted)
1397627_at	diaphanous homolog 1	−5.07	0.00007	0.00680	−0.52	0.70	−1.43	14
	(Drosophila) (predicted)
1397647_at	solute carrier family 25	5.51	0.00003	0.00395	0.62	1.54	1.54	14
	(mitochondrial carrier;
	ornithine transporter) member
	15 (predicted)
1397669_at	Chemokine (C—C motif)	5.78	0.00001	0.00271	0.51	1.43	1.43	14
	receptor 6 (predicted)
1397674_at	eukaryotic translation initiation	−6.44	0.00000	0.00116	−0.76	0.59	−1.69	14
	factor 3, subunit 8, 110 kDa
	(predicted)
1397676_at	Similar to osteoclast inhibitory	−6.68	0.00000	0.00084	−1.34	0.39	−2.54	14
	lectin
1397758_at	Similar to choline	−4.83	0.00011	0.00946	−0.38	0.77	−1.30	14
	phosphotransferase 1;
	cholinephosphotransferase 1
	alpha;
	cholinephosphotransferase 1
1397959_at	similar to RIKEN cDNA	−6.39	0.00000	0.00123	−1.14	0.45	−2.20	14
	D130059P03 gene (predicted)
1398311_a_at	kinase D-interacting substance	5.14	0.00006	0.00627	0.44	1.36	1.36	14
	220
1398351_at	Ubiquitin specific protease 7	−5.60	0.00002	0.00349	−0.42	0.75	−1.34	14
	(herpes virus-associated)
	(predicted)
1398420_at	Similar to E3 ubiquitin ligase	−5.33	0.00004	0.00483	−0.94	0.52	−1.92	14
	SMURF2 (predicted)
1398436_at	ubiquitin specific protease 42	−6.36	0.00000	0.00126	−0.76	0.59	−1.69	14
	(predicted)
1398486_at	CDNA clone MGC: 93990	−8.09	0.00000	0.00016	−1.53	0.35	−2.89	14
	IMAGE: 7115381
1398522_at	similar to Ab2-034 (predicted)	−4.92	0.00009	0.00832	−0.51	0.70	−1.42	14
1398553_at	similar to CGI-100-like protein	−6.91	0.00000	0.00062	−1.68	0.31	−3.20	14
1398834_at	mitogen activated protein	−4.94	0.00009	0.00828	−0.32	0.80	−1.25	14
	kinase kinase 2
1398926_at	prefoldin 1 (predicted)	−5.95	0.00001	0.00208	−0.48	0.72	−1.40	14
1398963_at	TAF10 RNA polymerase II,	−5.42	0.00003	0.00436	−0.41	0.75	−1.33	14
	TATA box binding protein
	(TBP)-associated factor
	(predicted)
1399099_at	heterogeneous nuclear	−4.94	0.00009	0.00829	−0.54	0.69	−1.46	14
	ribonucleoprotein U-like 1
	(predicted)
1399140_at	Transcribed locus	−5.16	0.00005	0.00597	−0.49	0.71	−1.40	14
AFFX-BioB-	Biotin synthase	−4.89	0.00010	0.00879	−0.64	0.64	−1.56	14
M_at	biotin synthesis, sulfur
	insertion?
AFFX-	dethiobiotin synthetase	−4.92	0.00009	0.00834	−0.70	0.62	−1.62	14
BioDn-5_at
AFFX-r2-Ec-	dethiobiotin synthetase	−5.41	0.00003	0.00440	−0.51	0.70	−1.43	14
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, N₂was 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 time	PC/LPC/GPC*	EPA/DHA**	Acid value

1 day	65/31/4	3.6/2.6	43
2 days	52/45/3	5.3/4.7	55
5 days	78/22/0	5.2/4.6	65
6 days	72/26/2	6.0/5.3	75

*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 time	PC/LPC/GPC*	EPA/DHA**	Acid value

1 day	94/5/1	0.4/0.5	55
2 days	87/11/2	0.7/0.7	76
5 days	68/26/6	1.0/0.8	85

*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 time	PC/LPC/GPC*	EPA/DHA**	Acid value

1 day	90/10/0	0.9/0.7	29
2 days	74/24/2	2.1/1.5	87
3 days	49/27/24	4.6/4.4	102
6 days	25/32/43	6.7/6.9	115

*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 time	PC/LPC/GPC*	EPA/DHA**	Acid value

1 day	79/17/4	1.0/0.9	55
2 days	59/31/10	2.8/2.6	76
3 days	52/34/14	3.8/3.5	85
6 days	37/43/20	5.6/5.7	95

*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.

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.