TW200412942A - Appetite control method - Google Patents

Abstract

Products, including nutritional products, dietary supplements and formulas, that contain long chain polyunsaturated fatty acids (LCPs or LC-PUFAs), specifically n-3 LCPs like DHA. Also a methods of using such products to control appetite and help treat and/or prevent obesity and conditions of overweight, especially in a pediatric population. Dietary DHA can act centrally as an antagonist of the CB1 receptor in the brain in opposition to the endocannabinoids that increase food intake. This is particularly advantageous when DHA is fed during periods of rapid brain growth such as infancy, childhood and adolescence.

Description

200412942 Rose, description of the invention: [Technical field to which the invention belongs] The present invention relates to nutritional supplements and formula products, which include long-chain polyunsaturated fatty acids (LCPs or LC-PUFAs), especially Xin 3 LCps; and the use of the Products to control appetite and help treat and / or prevent obesity and overweight, especially for the pediatric population. [Previous technology] 1.1 Introduction Over the past 10 years, children's overweight and obesity in Western society have increased significantly. The treatment strategies include increasing physical activity and deliberately limiting calories in order to affect the negative energy balance. Attempts have also been made to intervene in medicine. Prevention strategies emphasize balanced nutrition with physical activity. The present invention tests whether the ingested fat · quality can play an appetite-regulating role through n-6 and n-3 fatty amidin compounds formed in the brain. Endocannabinoids are a group of naturally occurring compounds that exhibit quasi-cannabinol properties, such as: analgesics, over-eating, cognitive deterioration, and exercise control, especially the effects of appetite. Over the past 10 years, endogenous fatty donkey-based derivatives that bind to cannabinoid receptors have been discovered. This receptor is better known as CBiKCB2. The fatty fluorenyl derivatives are a family of compounds N-fluorenylethanolamine (NAEs) and monofluorenyl glycerol (MAGs; Mechoulam et al. '1998). Peanut immersion ethanolamine, 20: 4n-6 ΝΑΕ is made of arachidonic acid and ethanolamine. Recently it has been shown that when injected into diet-restricted mice and pre-fed rats, the food content was increased. Consumption (Hao et al., 2001 and Williams and Kirkham, 2001). 20: 4n-6 MAG is made from 86783 200412942 arachidonic acid and glycerol and has recently been shown to increase food intake when injected into the brain of rats (Kirkham et al., 2002). Other n-6 and n-3 fatty amidyl compounds also bind to the CB receptor, i.e. those containing -20 cleavage and at least 3 double bonds (Mechoulam et al., 1998). Arachidonic acid (AA, 20: 4n-6) and docosahexahexanoic acid (DHA '22: 6n-3) can be used in vivo to desaturate and prolong essential fatty acids, linoleic acid and linolenic acid Process 'to produce' or derived from food. During the "brain growth sprint," animal model studies during this period have shown that changing dietary essential fatty acids and long-chain η-6 and n-3 polyunsaturated fatty acid levels can cause corresponding long-chain n-6 and n- 3 Changes in fatty acids, especially AA and DHA (Ward et al., 1998 and 1999; de la Presa Owens and Innis, 1999 and 2000) ° For piglets fed a formula, a recent study has confirmed that ingestion of AA and DHA will cause Increases in the brain of corresponding n-6 and n-3 NAEs and some monoglycerides (MAGs; Berger et al., 2001). 20 ·· 4n-6 ΝΑΕ through the cannabinoid receptor, CB !, to exert its nerve conduction-like effect has been fully established (Chaperon and Thi6bot, 1999). The CB! Receptor is found throughout the brain, including the hypothalamus, which is important for appetite regulation. * 1.2 Literature overview 1.2.1 The prevalence of obesity Over the past two decades, the number of overweight and obese children and adolescents has increased steadily throughout the United States and many Western countries (Harnack et al., 2000; Schneider, 2000; Onis and Bl6ssner , 2000; Muller et al., 1999; Heird, 2000; Spruijt-Metz et al., 2002). The Centers for Disease Control defined overweight as the weight gain relative to the accepted standard expected weight relative to 86783 200412942 height; and defined obesity as an excess of body lipids relative to the sacroiliac muscle mass (CDC, 2002). Overweight and obesity are generally defined as having a mass index (weight / height 2) between 25 and 29.9, or 30 (CDC, 2002). The prevalence of overweight children (ages 6-17) in 2000 was between 11-24%, with older children having a higher percentage (Schneider, 2000). Overweight children and adolescents often remain overweight or become obese as adults, and therefore increase the risk of co-morbidity, such as type 2 diabetes and cardiovascular disease. Studies have identified major public health issues that are important in addressing childhood obesity prevention. Although many factors contribute to weight gain, leading to overweight or obesity, most research focuses on genetic, cultural, behavioral, and environmental factors, such as socioeconomic status, inactive lifestyles, and lack of physical activity. This study focused on the regulation of food intake by the central nervous system. 1.2.2 Regulation of food intake by the central nervous system The central nervous system plays a major role in regulating appetite and final food consumption. Regardless of the healthy weight of an adult or child, food intake regulation is required. A balance of energy intake and energy expenditure is required. When the balance is reversed by the support of energy intake, the body tends to store the excess energy. Repeated or prolonged excess energy storage can lead to becoming overweight and, if sustained, can lead to obesity. The regulation of food intake is a highly complex process, which is controlled to a large extent by the inferior optic mast in the brain. For midpoint control of energy intake for maintaining body weight, complex integration of neural, endocrine, sensory, and thermoregulatory signals in peripheral and various regions of the brain is required (Williams et al., 2000; Hovel, 2001; van Dijk et al., 2000; Berthoud (2000) Some researchers have moved from studying feeding behavior and satiety to studying the regulation of appetite by the phrenic nervous system (Wllllams et al. 2000; Kaiyala et al. 1995). The hypothalamus plays an important role in regulating energy balance. For example, in the hypothalamus, increasing the level of neuropeptide γ stimulates appetite, and increasing α-black cells stimulates the level of hormones, inhibits eating and leads to weight loss, and when the appetite nerve corresponds to low blood glucose levels, Seems engaged in stimulating eating. 1 Kaiyala et al. (1995), in his research on the balance of sacral ganglia in the box nervous system and its fatty nature, suggested two different series of peripheral bluffing. Short-term meal-related signals and long-term fat-related signals regulate neural pathways in the brain to influence the start and end of meals. (1) Leptin and insulin Theta leptin and insulin are based on information about fat content stored in the body = known hormones supplied to the brain (van Dijk et al. 2_). As such, leptin and insulin help regulate food intake. Prion protein is secreted from the obese cells, peptide hormone. The secretion of prion protein is directly proportional to the fat storage amount. Insulin is also a peptide hormone secreted from pancreatic B cells. It plays a central role in controlling the use and storage of glucose and fat. The amount of insulin secreted at any time is also directly proportional to body fat. Leptin and insulin move through receptors in the hypothalamus in the brain. The fact that the receptor is found in the hypothalamus provides direct evidence of fat and carbohydrate storage, and hints at the role of this hormone in appetite regulation (Berthoud, 2000). (2) Endogenous cannabinoids (End〇cannabin〇ys) have many related neural pathways, hormones and receptors not only with body weight, but also with the maintenance of body fat 86783 200412942. Recent studies have identified fatty acid-derived compounds that are produced in the brain and act on specific receptors known to affect appetite. The compound is an endogenous cannabinoid (ie, an endogenous cannabinoid) and has been shown to play a neuromodulatory role in regulating appetite. The main history of the field is described below, followed by a more detailed description of the two major cannabinoid N-fluorenylethanolamines (NAEs) and monofluorenyl glycerols (MAGs). The active ingredient in marijuana, Δ9-tetrahydrocannabinol (THC), has an appetite stimulating effect and is prescribed by some doctors to help patients maintain weight (Mechoulam and Fride, 2001). Due to the biological effects of Δ9-ΤΗ (3), researchers began to look for endogenous compounds, endocannabinoids. The Δ9-THC was conveyed by a specific cannabinoid receptor called the CB! Receptor. In 1990 Earlier, a group of bioactive fatty amidin compounds showing neuromodulator activity at cannabinoid receptors were identified (Devane et al., 1992; HanuS et al., 1993). Thereafter, another group was identified Cannabinoid G receptors show monoacid glycerol or MAGs with neuromodulation activity (Sugiura et al., 1995; Mechoulam et al., 1995). Recent studies have shown that both the endocannabinol family (NAEs and MAGs) are implicated in The prion protein signal pathway in the hypothalamus (Di Marzo et al., 2001; Mechoulam and Fride, 2001). Prion protein has been shown to inhibit the formation of NAEs and MAGs. In the study of DiMarzo and Colleagues (2001), intravenous injection of peptone Protein, reducing the levels of 20: 4n-6 ΝΑΕ and 20: 4n-6 MAG in the brain (DiMarzo et al., 2001). The results suggest that the interaction between prion protein and endocannabinoids is regulated by Activation of the CB! Receptor in the optic mast, The regulation of food intake. 200 412 942

In the same study, to assess the role of prion protein in the endocannabinoid system, Di Marzo et al. (2001) injected 125 or 250 pg of prion protein intravenously into normal Sprague-Dawley rats. In 30 minutes, compared with the untreated control group, the level of the optic mound content under 20: 4n-6 ΝΑΕ and 20: 4n-6 MAG decreased by 40-50%. In addition, obese Zucker rats lacking prion protein signals showed reduced levels of 20: 4n-6 MAG in the inferior colliculus compared with non-obese Zucker control rats. More observations of leptin-deficient mice show an increase in 20 ·· 4η · 6 MAG or 20: 4n-6 ΝΕΕ or both in the hypothalamus. Therefore, prion protein plays an actual role in the regulation of endogenous cannabinoids. 1.2,3 Identification of endogenous cannabinoids The study of the identification of NAEs as a biological activity using neuromodulatory activity began nearly 100 years ago. Excellent reviews of this research history are available (eg, Mechoulam et al., 1998; Di Marzo et al., 1999; Hillard, 2000; Onaivi et al., 2002), and the history of this research is not repeated in this article.

△ 9-THC and other synthetic cannabinoid agonists have been shown to bind to specific cannabinoid receptors and inhibit the signal pathways of adenylate cyclase and N-type calcium channel G protein. The receptor is typically Called the CB1 & CB2 receptor (Felder et al., 1993). The 031 receptor is mainly found in the brain, and some mRNAs are also expressed in peripheral organs (adrenal, heart, lung, prostate, uterus, ovary, testes, bone marrow, thymus, peach glands and testes). The CB2 receptor is found in cells of the immune system (Buckley et al., 1998). McLaughlin et al. (1994) studied the development of cannabis receptors such as Sprague-Dawley pups, and found that the cannabis-like receptor mRNA appeared as early as 3 days after the pups were born. 86783 -10- 200412942 Subsequently, the identification of CBi receptors in the brain, researchers began to look for the emergence of endogenous ligands in the brain. In 1992, Devane et al. Reported the identification and structural formula of natural brain molecules that bind to cannabinoid receptors (Devane et al., 1992). They found that the group of pig brain extracts contained compounds that bound to the CBi receptor. They named the compound anandamide, and it is now commonly referred to as N-arachidene alcoholamine (20: 4n-6 ΝΑΕ). They purified 20: 4η · 6 NAE to test their ability to inhibit the twitch response of isolated rat vas deferens to test their pharmacological activity of cannabis. This test method is the standard model for studying the mode of action of psychotropic drugs. The structural formula of anandamide is determined by mass spectrometry φ and nuclear magnetic resonance method. 20: The chemical name of 4η-6ΝΑΕ is [5,8,11,14-icosatetraassociate, (N-2-hydroxyethyl)-(al 1-Z)]. Anandamide and its effects are also described in WO 2001/24645 A1 (Nestle, 2001). Since then, several other fatty sulfonyl compounds have also been identified that bind to this type of cannabinol receptor. In 1993, Hanug et al. Identified two other long-chain fatty fluorenylethanolamines that bind to the CBi receptor, high-γ-linoleylethanolamine (20: 3η-6 ΝΑΕ), and 7,10,13,16- Eicosatetraenylethanolamine (22: 4η-6 ΝΑΕ). In 1-995, Sugiura et al. And Mechoulam et al. Isolated different fatty amidine compounds, 2-peanut-impregnated glycerol, or 20: 4n-6 MAG from rat brain and dog intestine, respectively. Activity of cannabis-like receptor agonists. 20: 4n-6 MAG has also been shown to bind to the CB1 & CB2 receptor, and exhibits cannabis-like activity in vitro and in vivo. Although most of the studies on the specific role of endocannabinoids that bind to CBi receptors in the brain are related to 20: 4n-6 ΝΑΕ, 'other fatty amidyl NAEs and MAGs also bind to CBi receptors (Mechoulam et al., 1998) and can play a role in the regulation of the central nervous system in food intake 86783-11-200412942 (Di Marzo et al., 2001; Berger et al., 2001; Kirkham et al., 2002). 1.2.4 Tissue distribution of endocannabinoids 20: 4n-6 ΝΑΕ is found in many species and tissues including rats, pigs, cows, and humans (Schmid et al., 1995; Felder et al., 1996; Kondo et al. , 1998; Bisogno et al., 1999; Schmid et al., 2000). NAEs are found in tissues with CBi receptors, including brain, kidney, spleen, testes, skin, plasma, and uterus. Their concentration in the rat brain ranges from undetected to 29 pmol / g (Mechoulam et al., 1998). Since 20: 4η · 6 MAG binds to both CBi & CB2, it also appears to be a physiologically important and biologically active molecule. They have been found in dog intestines, dogs, pancreas and brain (Mechoulam et al., 1998; Bisogno et al., 1999 ·

Schmid et al., 2000; Kondo et al., 1998) Zhanluo / Chen in the brain was 800 times more anandamide (Suguira and Waku, 2000). 1,2.5 Biosynthesis of NAEs and MAGs The proposed mechanism of NAE biosynthesis includes the transfer of fatty amidyl chain from the position of phosphoryl g @@ sn-1 to _Ca + 2 to primary amines. 'Formation of N-acid phosphonoethanolamine (NAPE) and lysophosphate ester biliary test (pat ^ eeu · and Cravat 2001). NAPE is then hydrolyzed by enzymes such as phospholipase D to produce equivalent NAE and phosphatidic acid. This two reaction is considered to be a close collaboration between the proposed mechanism of MAG biosynthesis and the ναε4 mesh 4 乂 shown to be dependent on Ca + 2 (Mechoulam et al., 1998). Inositol phospholipid-specific phospholipase c causes the release of mono-glycerol and inositol triphosphate, which is then hydrolyzed with Sn_1_di- " glycan lipids to produce MAG (Ameri, 1999). 86783 -12- 200412942 1.2.6 Transfer and degradation of NAEs and MAGs NAEs and MAGs are released from the phospholipid membrane and used to bind to the CBi receptor. They are also rapidly hydrolyzed by membrane-bound enzymes called fatty amidinohydrolase (FAAH) or sometimes 'anandamide [20: 4n-6 ΝΑ] hydrolase' (Patricelli and Cravat, 2001; Goparaju Et al., 1998; Giang and Cravat, 1997). Giuffrida et al. (2001) proposed that 20: 4n-6 NAE and 20: 4n-6 MAG are hydrolyzed in a two-step process, which involves the hydrolysis of enzymes after they are delivered to a degradation site by a specific carrier. Due to its rapid degradation, endocannabinoids are thought to form and be used close to the CBi receptor. The delivery of NAEs and MAGs into cells by the parent carrier is proposed based on the rate of action, temperature, saturation, and matrix selectivity. Special research is needed to better understand how degradation of NAEs and MAGs is regulated. However, most researchers agree that FAAH is a key enzyme for the hydrolysis of endocannabinoid age. FAAH appears to be a total hydrolytic enzyme that acts on many biologically active lipids and esters (Giuffrida et al., 2001). 20 ·· 4n-6 ΝΑΕ is hydrolyzed by FAAH to free peanut immersion acid and ethanolamine. 20: 4η · 6 MAG is decomposed into free arachidonic acid and glycerol by the enzymatic action of FAA ^ i. Although it has not been empirically confirmed, another mechanism for the degradation of MAGs has been proposed, which may be monoglyceride. 1.2.7 Composition of House Fatty Acids and Brain Lipoic Acid Composition Rats (Ward et al., 1998 and 1999; Wainwright et al., 1999) and piglets (de la Presa Owens and Innis, 1999 and 2000; Arbuckle and Innis, 1993) showed that feeding different diets of long-chain n-6 and n-3 fatty acids will lead to different relative levels of long-chain n-6 and n-3 lipids in the brain. 86783-13-200412942 fatty acids. Explicitly 'Different amounts and ratios of dietary essential fatty acids, linoleic acid (18: 2n-6) and linolenic acid (18: 3n-3), and / or their long-chain polyunsaturated fatty acid derivatives, arachidene Acid (20: 4n-6) and docosahexaenoic acid (22: 6n-3), respectively, cause different levels of 20: 4n-6 and 22: 6n-3 content in the cerebral phospholipid membrane. Breast milk and formula-fed infants who died during the first year of life have also been reported to have different levels of 20. 4n-6 and 22: 6n-3 in their brains (Farquharson et al., 1995; Makrides et al., 1994). \ ¥ & ^ et al. (1998) demonstrated the feeding effect related to "dose" when changing the content of 20: 411-6 and 22: 611-3 in rat milk formula. Using a 3x3 design, the young rats from the 5th to the 18th day after birth were fed one of three levels of 20: 4n-6 and 22: 6n-3 with a gastrostomy tube (0%, 0.4% of total fatty acids or 2.4%). The formula takes into account the appropriate content of essential fatty acids, with 10% of the total fatty acids being 18: 2η · 6 and 1% being 18: 3n-3. By the 18th day after birth, the fatty acids in erythrocytes and cerebral phospholipid membranes usually reflect the fatty acid composition of dietary supplements. In addition, when only 20: 4η · 6 or 22: 6n-3 were consumed, the levels of unadded n-6 or n-3 long-chain fatty acids in red blood cells and cerebral phospholipids were lower than those in the relatively unsupplemented winter control group (ie, When supplemented with 20: 4n-6 alone compared with the control group without supplementation, 20 ·· 4n_6 increased and 22 ·· 6n-3 decreased in cerebral phospholipids). In 1999, de la Presa Owens and Innis studied the effects of foods lacking essential fatty acids with 0: 0.2% 20: 411-6 and 0% or 0.16% 22: 611-3 (0.8% of total fatty acids was 18: 2n-6 and 0.05% are 18: 3η · 3). They fed one of the formulas to the piglets from birth to the 18th day after birth and found that the supplement formula increased the cerebral phospholipid membranes by 20: 4η-6 and 22: 6η-3. Piglets fed with fatty acids 86783 -14- 200412942 have lower 20: 4n-6 when compared with piglets fed with appropriate amounts of essential fatty acids (8.3% 18: 2n-6 and 0.8% 18: 3n-3) And 22 ·· 6n-3. 1.2.8 Dietary fatty acids, NAEs and MAGs. As dietary fatty acids appear to affect the levels of 20: 4n-6 and 22 · 6n-3 fatty acids in brain phospholipids, it is reasonable to assume that different diets of n-6 and n-3 Fat causes similar changes in the levels of 20: 4n-6 NAE and 20: 4n-6 MAG in the brain. A study of piglets formulated (Berger et al., 2001) provided initial evidence of this effect. Berger et al. Provided evidence that different levels of dietary 20: 4n-6 and 22: 6n-3 fatty acids increased their equivalent NAEs and some MAGs, as well as other long-chain fatty fluorene-based NAEs and MAGs. During the first 18 days of life, the piglets consumed 0.3% 20 · 4n-6 or 0.2% 22: 6n-3, or 0.3% 20 · 4n-6 and 0.2% 22: 6n-3 Formula. All formulations include sufficient levels of essential fatty acids (15_16% 18: 2n-6 and 1.5% 18: 3n-3 of total fatty acids). They showed that piglet foods containing 20: 4n-6 and 22: 6n-3 increased NAEs and MAGs of long chains n-6 and n-3 in the brain. 20: 4n-6 ΝΑΕ increased by 4 times, 20: 5n-3 JNAE increased by 5 times, 22: 5η-3 and 22: 6η_3 ΝΑΕ increased by 9-10 times; 22: 4, 6 1 ^ person, and 22: 611_3 1 ^ person〇 increased almost twice; although 20: 4n-6 MAG did not increase. They proposed to change the level of NAE precursors, or to provide a matrix for biosynthesis, and dietary fatty acids to regulate the level of NAE. 1.2.9 Injectable NAEs and Ingestion / Aspirin Control There is evidence for growing the body to connect 20: 4n-6 ΝΑΕ with eating behavior. Studies on the feeding behavior of presatisfied rats (Williams and Kirkham, 1999) 86783 -15- 200412942 and studies on the feeding behavior of fasting mice (Hao et al., 2000) reported that after injection of 2: 4n-6 ΝΑΕ, Effect on food intake. Other studies of suckling mice (Fride et al., 2001) and mice with 0 receptor 1 knockout (Marz, et al., 2001) were reported in the CB! Receptor inhibitor (Sr141716A) [(N-VI Hydropyridine-1-yl) _5- (4-chlorophenyl) -1- (2,4-dichlorophenyl) -4-methyl-1-11-pyrazole-3-carboxamide], Reduced food intake. In pre-satiated rats, Williams and Kirkham (1999) investigated whether 20: 4n-6 ΝΑΕ triggers overeating and whether this is related to the specific role of cBi receptors. In the same study, but in the second series of evaluations, 8 rats received subcutaneous injections of specific CBi receptor antagonists before receiving a 20: 4n-6 ΝΑΕ injection at 1.0 mg / kg. 20: 4η-6 NAE of all doses caused significant overeating. The overeating caused by the administration of 20: 4η-6 ΝΑΕ was also blocked by the pretreatment of the CBj # antagonist. The authors propose that the 20: 4n-6 NAE given subcutaneously mimics the effect of the endogenous N • fluorethanolamine system on appetite regulation, which includes the CB i receptor in the hypothalamus. In the model of food restriction, Hao et al. (2001) studied the effect of a low dose of 20: 4η · 6 ΝΑΕ (0 · 001 mg / kg) on food intake response after a 40% heat limit. In their study, inbreeding female BALB / c mice were assigned by Ik machine to either the vehicle group or the 20: 4n-6 ΝΑΕ treatment group. The mice were fed food groups that were weighed before and after feeding, including spilled portions. The mice were fed 2.5 hours a day for 7 days (between 9 am and 12 pm). Ten minutes before feeding, 0.001, 0.7 or 4 mg / kg < 20: 4η-6NAE or the excipient alone was injected intraperitoneally at a volume of 0.1 mL / 10 g body weight. The control group received sufficient heat to maintain weight, while the food restriction group accepted 86783 -16- 200412942 to give the control group 40% of the calories. Restrictions on food continue to high hills of weight or to 15 g or less. This study showed that mice injected with 0.001 mg / kg of 20: 4n-6 NAE consumed significantly more food than the control group. The treatment group with 0.00 mg / kg 20: 4η-6 ΝΑΕ also showed effects of improving cognitive function and reversing severe food restriction. The other two 20: 4η-6 NAE treatment groups did not show any significant changes. The results suggest that the effect of 2 ·· 4η-6 ΝΑΕ on appetite may vary depending on the dose and experimental conditions. The CBl receptor activity appears to be biphasic.

Fride et al. (2001) investigated the effect of blocking cb activity in suckling rats. Mice were injected intraperitoneally with a CBi receptor inhibitor (SR141716A) at 20 mg / kg on the first or second day after birth. Researchers have observed an overwhelming effect on mortality. On the 1st day of birth, injection of the antagonist caused all mice to die on day 4 i 'injection on the 2nd day after birth caused 50% of mice to die. The same study, but different experiments (Fride et al., 2001) mice were injected with 20 mg / kg of antagonist daily from day 2 to day 8 of birth. All mice immediately stopped gaining weight and died on day 8. The combination of Δ9 and HC with this antagonist caused a slight increase in weight after the 8th day. Using 20: 4.0 MAG with the antagonist failed to gain weight or gain life. Researchers concluded from this experiment that "in the early life of rats, the endocannabinoid system played a vital role in breast pumping and growth and development. In the recent study of CB! Stylistic mice, Di Marz〇 et al. Humans (2001) evaluated prion protein and endocannabinoids for maintaining food intake. After fasting, the 1-diploid gene knockout mice and the wild-type control group were injected with an excipient or CBi receptor antagonists. CB 1 receptor knockout mice given excipients ate significantly less than the wild-type control group. CB1 receptor antagonists 86783 -17- 200412942 reduced food intake to the Food intake levels of excipient CBi receptor knockout mice; administration of antagonists to CBi receptor knockout mice resulted in unchanged food intake. The results further provided that endocannabinoids can regulate food intake In summary, studies have shown that different diets of n-6 and n-3 fatty acids affect the composition of n-6 and n-3 translipids in the brain, and related brain n-6 and n-3 NAEs and MAGs. Further, including notes 20: 4η-6 ΝΑΕ studies in rodents confirm the effect on appetite and eating behavior. 1.3 References The following is a list of related references arranged in alphabetical order. Each major description can be found in the background discussion above or elsewhere. .

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Williams G, Harrold JA, and Cutler DJ · The hypothalamus and the regulation of energy homeostasis: lifting the lid on a black box. Proceedings of the Nutritional Society, 59: 385-396? (2000). [Abstract] The present invention has several Aspects. In a first aspect, the present invention includes a method of reducing appetite of a mammal, the method comprising administering an effective amount of long-chain n-3 PUFA to the mammal to reduce the appetite of the mammal. In a second aspect, the invention includes a method of antagonizing the CBi receptor in the brain of a mammal, the method comprising administering an effective amount of a long chain n-3 PUFA to the mammal to inhibit the activity of the CBi receptor in the mammal's brain. In a third aspect, the present invention includes a method for reducing obesity or overweight in a mammalian population, the method comprising administering an effective amount of long-chain n-3 PUFA to at least some members of the population to reverse regulation Appetite of the mammal. In each respect, the preferred long-chain n-3 PUFA is DHA; this can be administered separately from AA. Preferably, the long-chain n-3 PUFA is administered during the growth period. Long chain n-3 PUFAs are preferably administered before or at the same time as appetite stimuli. In each aspect, the preferred effective dosage level for infants is from about 8 to about 396 mg / kg / day, (preferably from about 127 to 165 mg / kg / day); from about 84 to about 11610 mg / day under 15 years of age for adults About 84 to about 15, 8 3 2 mg / day. Better levels are included here. Finally, the present invention includes a method for regulating mammalian appetite, which comprises administering 86783 -29- 200412942 to the mammal in an effective relative amount of long-chain n-3 pUFA and long-chain n-6 PUFA 'to regulate the appetite of grandchildren. . The long-chain n_3 pUFA preferably includes DHA and the long-chain n-6 PUFA preferably includes AA. Preferably, the long chain n-3 PUFA is administered during growth. Long chain n_3 pUFA is preferably administered before or at the same time as the appetite stimulus. In each aspect, the preferred effective dose level is from about 8 to about 396 mg / kg / day (preferably about; ^ to ⑹mg / kg / day); children under 15 years old weigh about 84 to about 11610 mg / day, and adults About 84 to about 15,832 mg / day. Better levels are included here. [Embodiment] Detailed description 2.1 Terms of lipids Fatty acids are important nutritional components. Fatty acids are classified based on the length of carbon chains and their saturation characteristics. Long-chain fatty acids have 16 to 24 or more carbons and are also saturated or unsaturated. In longer fatty acids, there may be one or more unsaturations, which are represented by the terms "unit unsaturation" and "multiple unsaturation," respectively. The present invention is particularly interested in long-chain polyunsaturated fatty acids (LCp's or LC-PUFAs) having 20 or more carbons. According to the nomenclature familiar to biochemical experts, LC-PUFAs are classified according to the number and position of double bonds in fatty acids. Depending on the position of the double bond near the methyl end of the fatty acid, there are two main series or families of LC-PUFAs: although the n_6 series does not have double bonds until the first 6 carbons, the η-3 series contains a double bond on the third carbon. Therefore, peanut humic acid (n A Aπ or n AR Aπ) has a chain length of 20 carbons and 4 double bonds start at the 6th carbon. As a result, it is referred to as "20: 4η-6". Similarly, docosahexacarboxylic acid (`` DHA '') has a chain length of 22 carbons, and 6 double bonds start at the third carbon from the methyl 86783 -30- 200412942 end, so it was designed as "22 : 6n_3 ". In the present invention, aa and dha are particularly important. Other important LCPs are C18 fatty acids, which are precursors in this biosynthetic pathway and are described in U.S. Patent No. 5,223,285. Therefore, linoleic acid (18: 2n-6, "LA,") and intermediate γ · linolenic acid (18: 3n_6 ,,, GLA ") and two high linolenic acid (20: 3ii-6, & quot DHGLA ") is an important precursor to AA (2 ·· 4n-6). Similarly, α-linolenic acid (18 ·· 3n-3 ,,, ALA ,,) and the intermediate octadecyl four Folic acid (18 ·· 4n_3) and EPA (2〇 ·· 5n_3) are important precursors to DHA (22: 6n-3). Fatty acids are common in nature and are lipidated as alcohols to alcohols. Glycerides Are one or more fatty acids and glycerol (1,2,3-glycerol) esters. If only one position of the glycerol backbone molecule is fatty acidized, a "monoglyceride," is produced; If two positions are fatified, a "diglyceride" is produced; if glycerol < all three positions are targeted by a fatty acid g, a triglyceride, or a triglyceride is produced. Phospholipids are a special form of diglycerides in which the third position of the glycerol backbone is bound to nitrogen-containing compounds such as choline, serine amine, inositol, etc. via phosphatase. Glycine triphosphate and phosphorous materials are classified as long-chain or medium-chain based on the fatty acids to which they are attached. The "source" + / of the fatty acid may include any such type of glyceride from a natural or other source. π lipids is a common way to describe fatty acids or oily ingredients. In nutrition, the purpose of the month is to provide energy and essential fatty acids and enhance oil-soluble materials to absorb soil, and biotin. The type of lipids consumed affects most physiologists, such as plasma lipid profiles, lipid composition of cell membranes and organs, and the synthesis of cancer-free velocities I vehicles, such as 86783-31- 200412942, such as prostaglandins and thromboxanes. Other physiological effects of lipids are described in the background. Sources of longer LCPs include dairy products like eggs and cream; seafood oils such as cod, Manhattan fish, sardines, catfish and many other fish; certain animal oils, lard, tallow and microbial oils such as mushroom and algae oils Is described in detail in U.S. Pat. Nos. 5,374,657, 5,550,156, and 5,658,767. In particular, fish oil is a good source of DHA. They are sold in the market as "high ΕΡΑΠ & Πlow ΕΡΑΠ", which has a high DHA: ΕΡΑ ratio, preferably at least 3: 1. Seaweed oil such as dinoflagellates from Dinophyceae net, especially Crypthecodinium cohnii It is also a source of DHA (including DHASCO ™), as shown in U.S. Patent Nos. 5,397,591, 5,407,957, 5,492,938 and 5,711,983. Mortierella, especially M. alpina and Pythiuminsidiosum are good sources of AA, including ARASCO ™, such as U.S. Patent No. No. 5,658,767 and Yamada et al. J. Dispersion Science and Technology, 10 (4 & 5), PP 561-579 (1989) and Shinmen et al., Appl. Microbiol. Biotechnol. 31: 11-16 (1989) Revealed. Of course, the source of LCPs can be developed synthetically, or genetically manipulated by other organisms, especially vegetables and / or oil-bearing plants. Genes for saturating and elongating enzymes have been identified from many organisms, and They can be genetically engineered into plants or other host cells to allow them to produce large amounts of oil-containing LCPs at low cost. Recombinant oils are also encompassed by the present invention. 2.2 Stimulation or Stress On the one hand, the present invention is used in conjunction with environmental stress or stimulation. Research in Rodents 86783 • 32- 200412942 shows that mild to moderate stress leads to increased food intake, although more severe This is not the case for stress (Harris et al., 2000). The effect of stress on food intake depends on the duration of stress, and includes both physical and mental stress. Mild stresses known to cause increased food intake in rats include mandarin tail, Short-term restraint or touch, food restriction, and sleep deprivation. Children with Westernized social experience, intermittent mild stress, are inferred to cause appetite responses. Examples can include irregular meal times, sleep deprivation (Sekme et al., 2000) 2; Buboltz et al., 2001) and expectations of parents who have won in school and / or sports. Children with door locks may experience additional intermittent stress. Stress or stimulation may have the effect of increasing food intake (also In other words, it means the stress or stimulus of "affecting appetite". In the food restriction period before this study, Showed such mild pressure that it caused an appetite response. It seemed to be most obvious after 4 (%) food restriction throughout the night and weakened after fasting all night on day 20. Different Following the R substance restriction example, the 'different appetite response can be explained by the restricted sample size' adaptive response to the fasting / eating paradigm, otherwise the latter (fasting all night) exceeds the low / moderate stress limit. 2.3 Product Types * The dietary fatty acids of the present invention can have many types, including (but not limited to, nutritional products, dietary supplements, medicines, or other products. They can be used at any age, such as infants, children, or adults. In特别 Special right monks when growing up, such as infants and children and adolescents, ^ 〇, J brothers know that the more fat in the diet of the present invention can be incorporated into nutritional "excipients or vehicles," including (but not limited to) FdA's French foot food classification ·· Traditional food, special, pay ~ slave ③ lack of food, dietary supplement 86783 -33- 200412942 agent and food for medicine. The nutrition products of camping products contain large nutrients, gp, fat, protein And carbohydrates, and adjust their relative content according to the age and conditions of the intended user, and ^ includes micronutrients such as vitamins, minerals and trace minerals. Noun, 'nutrition products' include (but are not limited to) the FDA's Legal Food Classification: Traditional foods, foods for special dietary uses, medical foods and infant formulas. Foods for special dietary uses are expected to provide nutrients Supplement the diet or as a single item of the diet to provide special dietary requirements due to physical, physiological, and pathological conditions. "Medical food" is food prepared for consumption or taken orally under the supervision of a doctor, based on The understanding of the scientific principles established by medical evaluation is intended to be used as a special dietary management of diseases or to differentiate nutritional needs. In addition, "dietary supplements" are intended to be taken in the form of tablets, capsules or liquids to supplement the diet, and do not represent traditional use. Food or as a separate meal or diet. 2.3.1 Infant-free infant formula means compliance with the Infant Formula Act (21 usc § 35〇 (a) et. S.). Standards and benchmarks (nutritional formulation), and # 月 望 replaced Or # filled with human milk. Although the formula is used in at least 3 different forms (powder, liquid concentrate and ready-to-eat liquid (" RTF)). It is customary to mention nutritional concentrates on the basis of eating, Therefore, RTF is often described. Of course, other types can be reconstituted or diluted to the same composition according to the manufacturer's instructions, and those who are familiar with this solution can calculate the correlation. Composition for concentration or powder formation. 86783 -34- 200412942 π standard or "term" infant formula means infants who are expected to be born at term as the initial formula for feeding. Protein, fat and carbohydrate components, respectively Provides heat from about 8 to 10, 46 to 50, and 41 to 44%; this heat density range is narrow from about 660 to about 700 Kcal / L (or 19-21Cal / fl.oz.), Usually about 675 Up to 680 (20 Cal / fl.oz.). The caloric distribution of fat, protein and carbohydrate components can be adjusted among infant formula makers at different times. SIMILAC ™ (Ross Products Division, Abbott Laboratories ) ^ ENFAMIL ™ (Mead Johnson Nutritionals) and GOOD STARTTM (Carnation) are examples of term infant formulas. The "'nutrition fortification'" formula means fortified infant formula relative to the π standard "or " term" formula. The original definition of differentiated nutrition fortified formulas was calorie density; the second factor was the concentration of protein. For example, a formula with a caloric density of about 700 kcal / L or more or a protein concentration of about 18 g / L or more can be considered as "nutrition-enhancing π." Nutrition-enhancing formulas also typically include higher amounts of drugs (ie, about 650 mg / L or more). ) And / or phosphorus (ie, about 450 mg / L or more). Examples include similac NEOSURE ™ and Similac Special CareTM formulas. 2.3.3 Drinking supplements. Fillings Dietary supplements are soft gums and capsules with specific nutrients Agents, sanzhai j, bonding agents, liquids, and other dosage forms are usually expected to support the normal structure and function of the body. Dietary supplements can be formulated with appropriate excipients and carriers, much like pharmaceutical products. Soft gels are widely used Used in the pharmaceutical industry, as an oral preparation, it contains many different types of medicines and vitamin products. Soft gums are used in many different sizes and shapes' including round, | 卩, rectangular, tubular and other special 86783 -35- 200412942 2 The shape is star-shaped. The finished capsules or soft gels can be made into various colors, with or without opacifying agent. Soft gels are mainly used to surround liquids, and more particularly for oily solutions, suspensions and emulsions. positive The fillings used are vegetables, animal or mineral oils, liquid hydrocarbons, volatile oils and polyethylene glycols. Soft gelatin capsules can be manufactured using techniques known to those skilled in the art. Wu Guo: Li Di 4,935,243, Nos. 4,817,367 and 4,744,988 describe the soft capsules "preparation of formulas. The manufacturing of variations on the upper bound must be familiar to those skilled in medicine and medicine. Typically, it includes a shell made mainly of gelatin, a plasticizer, and water and the contents of the shell. The filling can be selected from any of a wide variety of materials compatible with the plastic shell. 、, = Speaking of 'gelatin capsule manufacturing system includes three main systems: sheet forming early 7C' ㈣ forming unit and capsule reproduction unit. The melted gelatin is formed into a sheet of a desired thickness. The sheet is inserted between a pair of roll molds, which are installed in a desired die head in a capsule forming unit. For charging: "Ruli" display The filling nozzle is used to release the desired amount of filling liquid between the two gelatin sheets. When the gelatin flakes are brought into contact with each other so that an unfilled capsule is formed, adjust the outflow time, so it is controlled by the die. Chengyi Ao Shao was filled with no filling solution. The formed gelatin capsules were removed from the member by wiping with a roll. Before the material container was filled, the Ming capsule was stored in a large container for storage. Back to the filled The two halves of the capsule and shell can be formed separately and sealed after filling. / The agent is usually composed of the active ingredient, usually a "pharmaceutically acceptable salt," in combination with the month! / 闰 / 月 W and other excipients in the model Medium compression and forming. Details of how to form capsules and lozenges can be obtained from any source, including 86783 -36- 200412942

Remington's Pharmaceutical Sciences, XV version (1975). Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. Describe pharmaceutical acceptable salts in detail in J. Pharmaceutical Science, 1977, 66: 1 et seq., Which is incorporated herein by reference. During the final isolation and purification of the compounds of the present invention, the salts can be prepared on the spot, or each can be prepared by reacting the function of a free base with an appropriate organic acid. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, butyrate Salt, camphor salt, camphor sulfonate, digluconate, glycerol phosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, bromate, broken salt, 2-carboxythioate, lactate, maleate, mesylate, nicotinate, 2-sulfonate, oxalate, pamoate, pectate, Sulfate, 3-phenylpropionate, picrate, trimethylacetate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, P -Tosylate and undecanoate. Nitrogen-containing basic groups can also be quaternized with the agent to form lower alkyl halides, such as / methyl, ethyl, propyl and butyl chlorides, bromides, and chiral compounds; , Such as dimethyl, diethyl, dibutyl and dipentyl sulfate I; long bond functions, such as dodecyl, tetradecyl, cetyl and octadecyl chloride, Bromides and iodides; arylalkyl halides such as benzyl and phenethyl bromide and others. Thus obtain water-soluble or oil-soluble or dispersible products. Examples of acids used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. 86783 -37- 200412942 react a carboxylic acid-containing moiety with an appropriate base, such as a pharmaceutically acceptable metal cation hydroxide, carbonate, hydride carbonate, or with ammonia or an organic, secondary, or tertiary amine During the reaction, the resulting compound can be prepared as an alkali addition salt on the spot during the final isolation and purification. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals, such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like, and non-toxic quaternary ammonia and amine cations Including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like. Other representative organic amines used to form base addition salts include ethylenediamine, ethanolamine, diethanolamine, hexahydropyridine, piperin, and the like. 2.4 doses The content of specific fatty acids in the formulation is typically expressed as a percentage of the total fatty acids. This percentage is multiplied by the absolute concentration of the total fatty acid in the formula or g / i00kCal) to obtain the absolute concentration of the fatty acid (in g / L or g / i00kcal, respectively). The total fatty acid can be estimated to be about 95% of the total fat to calculate the weight of the dried sugar. By heat density. The simple transition from mg / 100 kcal to mg / L & is known to those skilled in the art. According to the present invention, a DHA-enriched nutritional composition can provide less than about 5% of daily caloric intake from a single source eating situation, such as 100% of infant formula, to a conventional light meal. If the formulation is for a newborn, it can be supplemented with some human milk. When the baby grows to about 2 to 4 months, it is often started to supply part of the calories with solid food. The content of the formulation can be reduced according to the percentage of total calorie intake. The nutritional or calorie component of any supplement or medicine is often downsized and underestimated. 86783 -38- 200412942 According to the present invention, it is advantageous to combine DHA dose with mild pressure as mentioned before. In the case of young rats, rats fed with 2.5% DHA of total fatty acids, without ARA content levels (0% or 2.5%) and marginal levels of linoleic acid and α-linolenic acid, were exposed to a shock Appetite mild stress foods eat less than 11% of the diet at weaning within the first 2 hours after the food restriction period. Others have used the same rat milk model to show that dose-related ARA (0, 0.4%, and 2.3% of total fatty acids) and DHA (0, 0.4%, and 2.3%) have different effects on brain growth. Effects of Chain η-6 and n-3 Fatty Acids (Ward et al., 1999) 〇 In this study, the brains of rats fed 0 and 2.5% DHA diets were associated with 22: 6n-3 levels that differed from those by Ward Et al. (1999) reported similar results for 0 and 2.3% DHA diets. Based on the relationship between the level of DHA in the brain and appetite in the linked example, and the relationship between dietary DHA and the level of DHA in the brain (this example and Ward et al. 1999), it is reasonable to expect a reduction of about 5% due to 0.4% dietary DHA Food intake. From a public health point of view, people continue to reduce: _5% of calorie intake can reduce the risk of becoming overweight and obese. Table A: DHA content levels (% of total fatty acids intake) Nutritional products are designed to be better and better. Babies under term 0.10-2.5 0.10-1.0 0.15-0.50 0.10-2.5 0.10-1.0 0.15-0.50 86783 at birth 39 200412942 2-6 months 0.10-2.5 0.10-1 · ° 0.15-0.50 6-12 months 0.10-3.0 0.10-1 · 3 0.15-0.70 Child 1-5 years 0.10-5.0 0.10-2.0 0.30-1.00 5- 15 years old 0.10-5.0 0.10-2.0 0.30-1.00 Adult Adult 0.10-5.0 0.10-2.〇_- 0.30-1.00 The better period of intake of DHA fortified food is when the increase of long bond n-6 and n-3 fatty acids is the most Quick time-that is, in infancy, children and adolescence. The fastest rate of brain growth occurs during infancy. However, brain growth and neural maturity continue until about 12-20 years of age. The content of fatty acids in the adult brain can also be affected by adult food, but it takes a longer time. According to the present invention, from the lack of Table B, it is proposed that there is an effective dose of the component, DHA, regardless of whether it is given as a 2Γ AL · 4 knowledge point for supplements or drugs. Table B: Diet dha for each age group The best intake is best for infants with the best heat consumption per month. 13 Lower range 8 Higher range 396 Birth to 6 months ^ mg / kg / day *** 13 19 8 13 158 79 ** mg / kg / day *** kcal / kg / day 120 90 150 kcal / kg / day 86783 .40- 200412942 Normal 11 11 16 100 Lower range 8 8 13 80 Higher 317 127 63 120 6-12 Month * * * mg / kg / 夭 ^ * 氺 * kcal / kg / day normal 11 11 16 100 lower range 8 8 13 80 higher 380 165 89 120 children 1-5 years old mg / χ *** kcal / days usual 137 137 412 1300 lower range 84 84 253 800 higher 9499 3800 1900 1800 5-15 years old books mg / i *** kcal / days usual 190 190 570 1800 lower range 84 84 253 800 southerly 11610 4644 2322 2200 Adult Adult 氺 氺 氺 mg / i 氺 氺 氺 kcal / day usual 211 211 633 2000 Lower range 84 84 253 800 Higher 15832 6333 3166 3000 So, for example, the effective dosage range for children 1 to 5 years is 84-9499 mg per day, preferably 84-3800 mg per day, and most preferably 252-322 mg per day. The comparable value for adults is 84-15832, preferably 84-6333, and most preferably 253- 86783 -41-200412942 3 166. Please note: Values given by children and adults are in mg / day, infants are in mg / kg / day. Therefore, the comparable value for infants up to about 6 months of age is 8-3 80 mg / kg / day, preferably 8-165 mg / kg / day, and most preferably 13-89 mg / kg / day. With this application, the number range given as "x-y" should be interpreted as "from about X to about y", of course, about "to modify both the \ value and the y value. In addition, this range naturally indicates that an indefinite value between X and y is revealed simply and unambiguously by this range. For example, 0.10-2.5 clearly reveals the values such as 019, 05, 0.823, 1.25, 1.64, 1.999, etc. and the values "about (MO or" about ,, 25. 2.5) 2.5 manufacturing methods to read the general knowledge of this technology Conventional methods can be used to make the liquid and powder nutritional products of the present invention. Three pastes are briefly prepared, mixed together, heat treated, standardized, spray dried (if applicable), packaged and sterilized (if applicable). 2.5.1 Liquid The product first prepares a carbohydrate / mineral paste with hot water, and stirs to an elevated temperature. Then minerals are added. Minerals can include (but not limited to) sodium citrate, sodium chloride, " citric acid Potassium, potassium chloride, magnesium chloride, calcium phosphate, calcium carbonate, potassium iodide, and pre-mixed trace minerals. Carbohydrate sources such as one or more of lactose, corn syrup solids, sucrose, and / or maltodextrin are soluble in water. This forms a carbohydrate solution. A source of dietary fiber, such as soy polysaccharides, can also be added. The completed carbohydrate / mineral paste is stirred at an elevated temperature and placed until mixed with other paste pieces, It is preferably not more than about twelve hours. The base oily mixture is mixed and heated to prepare an oily paste. The base oily 86783 -42- 200412942 mixture typically contains some soybeans, coconut, palm oil, high oil safflower or sunflower oil And medium chain triglyceride combinations. Emulsifiers such as diethyl tartaric acid mono- and di-glycerides, soy mono-, di-glycerides and soy lecithin can be used. Any or all oil-soluble Vitamin A, dE (natural R, R, R type or synthetic) and K can be added separately or made into 4_ ^ Shao points. Betacarotoxin, which functions as an antioxidant in the body, can also be added, like Antlers as stabilizers. Containing specific LCPs, oils important to the present invention (for example, DHA and AA) can be added to oily slurries. The Lcpg must be used with care because they are prone to decomposition and spoilage. What is done The oily slurry is kept under stirring until mixed with other slurry. It is best not to be longer than about 12 hours. Let's prepare the water-like protein 'first heat the water to a suitable elevated temperature while stirring' and then Protein source is stirring Add water to the mix. Typically, the protein source is whole or hydrolyzed milk protein (eg, whey, whey), human or hydrolyzed vegetable protein (eg, soy), free amino acids and their Blends. Generally 'any known source of amine nitrogen can be used in the present invention. The finished protein syrup is kept at elevated temperature during stirring until combined with other syrups', preferably no longer than about 2 Hours. As a substitute, some proteins ..., water-in-protein mixes are not as good as fat-in-protein mixes in emulsions. :: water, protein and carbohydrate / mineral slurry are stirred and mixed, and the resulting dagger water is kept at a relatively high temperature. After a delay (for example, several minutes :) 'Add the oily slurry to the mixed slurry from the previous step while stirring. As the sister said: add to the oil mixture, the LCP oil can be directly added to the A mixture of protein Benito water compounds / minerals and oily pulp. 86783 -43. 200412942 After sufficient stirring to fully combine all the ingredients, adjust the pH of the complete mixture to the desired range. The mixed slurry is then subjected to aeration, ultra-high temperature heat treatment, emulsification and homogenization, and then cooled to a refrigerated temperature. Preferably, after completing the above steps, an appropriate analysis test is performed for quality control. Based on the analysis results of the quality control test, add an appropriate amount of water to one batch and stir and dilute. Aqueous vitamin solutions (including sodium silicate) containing water-soluble vitamins and trace minerals are prepared and added to the pasty mixture while stirring. Individual solutions containing nucleotides were prepared and added to the previous mixing slurry with stirring. The pH value of the final product can be adjusted to achieve the best product stability. The finished product is then filled into appropriate metal, glass or plastic containers and subjected to final sterilization using conventional techniques. Alternatively, the liquid product is aseptically sterilized and filled into a plastic container. 2.5.2 Powder products Carbohydrate / mineral pulps are prepared using the liquid product manufacturing methods previously described. Use the liquid product manufacturing method described above and the following exceptions to prepare oil slurry: 1) emulsifiers (mono-, di-glycerol, egg-lipid fat) and stabilizers (antler) are typically not added to powders, 2) During any subsequent spray-drying process, in addition to betacarotene, other antioxidants such as mixed tocopherol and ascorbyl palmitate can be added to help maintain the oxidative properties of the product and 3) specific to the invention Instead of adding LCPs to the slurry, add them after mixing the slurry. The protein water slurry was prepared by the liquid product manufacturing method described previously. 86783 -44- 200412942 Mix carbohydrate / stone material pulp, protein water slurry, and oil slurry in the same manner as described in the manufacture of tracing products. After adjusting the pH of the completed mixture, LCPs were then added to the mixed slurry while stirring. When the mixture is passed through the tube at a certain rate, it is better to meter in the product slowly (on-line mixing). After aeration, ultra-high temperature treatment, emulsification, and homogenization, the process mixture can be distilled to increase the solids content of the mixture to promote more effective spray drying. The mixture is then spray-dried through a preheater and high-pressure pump using conventional τ mist drying techniques. The spray-dried powder is solidified and then filled into metal or plastic cans or metal foil bags under vacuum, nitrogen or other inert environments. Any variation of the manufacturing process is known or obvious to those skilled in the art. The invention is not intended to be limited to any particular process. All US patent references cited herein are incorporated by reference. Pharmaceutical dosage form Pharmaceutical dosage form can be applied to two types of medicine and food supplement. Those who are familiar with this skill include tablets, capsules, pills, powders, and other dosage forms. The method of making each of these dosage forms is known and is not repeated here except as mentioned in the earlier sections. Example 3.1 Experimental design The plan was to raise young rats artificially with different formulas of n-6 and n-3, and then evaluate the food intake after weaning the silver solid food. One group of rats was fed and tested in February, and the other was in April 2002. The original intention is to combine the results of February and 86783 -45- 200412942 in April, but methodological issues (described below) limit the reliability of some of the results in February. Data for April are reliable and complete. The study on captive feeding and food intake was held in the laboratory of Dr. John Edmond of the University of California, Los Angeles, under the kind care of Rose korsak, which is double-blinded with different composition of rat milk and Feeding group. 3.1.1 The basis of experimental design The model of feeding rats with neonatal gastrostomy feeding formula is designed with 2x2 factor. Rats (Ward et al., 1998 and 1999) and piglets (de la Presa Owens and Innis, 1999 and 2000) previously formulated in previous studies have shown that arachidonic acid (AA) and twenty-two carbons in the brain The level of fatty acids in hexadecanoic acid (DHA) will vary with the dietary levels of AA and DHA (Ward et al., 1998 and 1999; de la Presa Owens and Innis, 1999) or not (de la Presa Owens and Inis, 2000) changes in the levels of their precursors, linoleic acid and linolenic acid. In this study, the dietary levels of AA and DHA were selected from the model of feeding rats with the same gastrostomy. Data on similar levels of DHA published by Ward et al. (1998) and Wainwright et al. (1999). The levels of AA and DHA studied were 0% and 2.5% of total fatty acids, used alone or in combination; the fourth group was not fed AA or DHA (Tables 3.1 and 3.2). The study period was the AA and DHA (feeding) feeding period. Arachidonic acid (20: 4n-6; AA) docosahexaenoic acid 0.0% 2.5% (22: 6n-3; DHA) 2.5% 2.5% Table 3.1 2x2 factor design. Fatty acid percentages are expressed as% of total fatty acids. 86783 -46- 200412942 The design shows the equivalent of a rat milk formula combination without AA, DHA; AA, + DHA; + AA, DHA; + AA, + DHA (see Table 3.2). Formula combination AA DHA no AA, no DHA 0.0% 0.0% no AA, + DHA 0.0% 2.5% + AA, no DHA 2.5% 0.0% + AA, + DHA 2.5% 2.5% Table 3.2 Experimental group shows AA and DHA feeding period Of four different rat milk formula combinations. Fatty acids are expressed as% of total fat. AA, Peanut Dipping Acid (20: 4n-6); DHA, Docosaic Acid (22: 6n_3). The basic formula is designed to contain marginal sufficient levels of linoleic acid and undetectable levels of α-linolenic acid. In the four experimental groups of rats, the levels of AA and DHA in the brain are greatly different. This formulation has been used in studies on rats (Wainwright et al., 1999) and piglets (de la Presa Owens and Innis, 1999 and 2000). 3.1.2 Reference group In addition to the four rat milk_formulation combinations, two reference combinations were also studied. A reference group was a normal infant group, in which the young rats remained in the female rats until the 20th day to obtain their brain tissue. The normal suckling rats were not included in the experimental food intake period. The second suckling rat reference group was used as the experimental design reference group. At the beginning of the experimental food intake period, the pups were removed from the females and the feeding measurements were taken on the 19th and 20th days. The rat continued to milk or be introduced to feed before the food intake period began. 3 · 1 · 3 Statistical Analysis 86783 -47- 200412942 The data results were analyzed using SAS / Stat software, version 8.2 (SAS® Institute, Inc., Cary, NC). Use the two-line, non-interaction mode to evaluate the main effects of AA and DHA. This enables comparison between AA-containing formulations and DHA-containing formulations. Increasing the number of animals in each group makes statistical analysis more effective. Single-line variation analysis with adjusted sample size but no multiple comparisons was used to compare the infant reference group to other groups. The significance level was set at 0.05. The number of animals in each group was chosen between 8 and 16. 3.2 Feeding period 3.2.1 Artificial feeding process Pregnant Sprague-Dawley rats were obtained by Charles River Laboratory (Wilmington, MA) on the 14th day of pregnancy. Placed in a temperature-controlled environment with a 12-hour light / dark cycle. Pups were born on the 21st day of pregnancy within 24 hours. The birthday is set to day 0. On the 6th day after birth, male pups were removed from the females and reared artificially with rat milk formula until the 18th day. This step has been described in detail in the literature by Sonnenberg et al., 1982; Smart et al., 1983 and 1984; and Auestad et al., 1989. Similar steps have been described by Ward et al., 1998 and Wainwright et al., 1999. On the 6th day after birth, the young rats were randomly assigned to one of the four rat milk formula combinations (Table 3.2). The pups were lightly anesthetized, fitted with intragastric cannulas, and individually placed in a free-floating hold to maintain 36% soil. (: In a pin-sized plastic container on a water bath. Individual young rats' cannulae are connected by polyethylene tubes to a needle filled with one of the four experimental rat milk formulas. From day 6 to day 8 The stomach was immersed in a stomach so that the young rat was raised. Using a programmable pump placed in a refrigerator on the table top, the preparation was delivered to the young rat at 20 or 30 minutes per hour depending on the age of the rat. 86783 -48 -200412942 The pump settings are modified daily to deliver a specific amount of rat milk formula to the young rat to support normal growth. The research plan is shown in Table 3.3. 3.2.2 The composition of the rat milk formula is whey in addition to the protein source And cheese powder, as described in the literature (Auestad et al., 1989; Ward et al., 1998), to prepare a rat milk formula (generously provided by the Ross Products Division of Abbott Laboratories). A short preparation consists of lactase, whey and water. The composition is a pre-milk base. Then, a fat mixture (see Table 3.4), lactose, minerals, vitamins, and other nutrients found in rat milk are added to the pre-milk base and mixed using a Polytron homogenizer (see Table 3.5). Fat mixture for rat milk preparation Blended to provide the marginal content of linoleic acid, linolenic acid, and different levels of AA and DHA. Age after birth, days 6-15 16 17 18 19 20 Calorie intake,% of calories required: Rat milk formula (AA and DHA feeding period) 100 80 80 20 0 0 Feed 0 20 20 20 Feeding period,% of diet (unlimited) Feed 100 100 Table 3.3 AA and DHA heat sources during feeding and feeding period. Four rat milk formula combinations The feeding plan of the experiment. The experimental rat milk formula was randomly assigned to the 6th day after birth and fed to the 18th day. The rat milk formula contains: no AA or DHA; no AA, 2.5% DHA; 2.5% AA, no DHA Or 2.5% AA, 2.5% DHA. The feed is a semi-solid food and was introduced to all 4 groups on the 16th day. It was exclusively eaten during the food introduction period. The feed complies with the AIN-93 nutrition recommendation and does not contain AA or DHA 86783 -49- 200412942 No AA + AA No DHA + DHA No DHA + DHA Fat-proof mixture,% of total oil Cocoa oil 67.5 60.0 60.1 52.6 MCT oil 1 32.5 27.5 27.4 22.6 AA oil 2 0.0 0.0 12.5 12.4 DHA oil 3 0.0 12.5 0.0 12.4 Fatty acids, 4% of total fatty acids C8: 0 28.1 14.7 14.3 0.0 C10: 0 20.1 10.5 10.1 0.2 C12: 0 27.7 15.1 13.9 0.9 C14: 0 10.7 12.0 6.1 7.4 C16: 0 5.9 11.1 9.9 15.4 C18: 0 1.7 1.3 6.5 6.3 C22: 0 0.0 0.1 0.8 1.0 C24 : 0 0.0 0.1 0.9 0.9 Total (saturated) 32.1 64.8 62.3 94.2 C16: l 0.0 0.6 0.1 0.7 C18: l 4.0 12.9 8.3 17.6 Total (unsaturated) 4.0 13.4 8.4 18.2 C18: 2n-6 1.1 0.9 4.2 4.0 C18: 3n -6 0.0 0.0 1.7 1.7 C20: 3n-6 0.0 0.0 1.8 1.8 C20: 4n-6 (AA) 0.1 0.0 19.9 20.1 86783 -50- 200412942 Total n-6 1.2 0.9 27.5 27.6 C22: 6n-3 (DHA) 0.0 20.4 0.0 20.3 Total n-3_0.0 20.4 0.0 20.3 Table 3.4 Fatty acid composition of fat mixture, which is used in the preparation of rat milk formula for experiments. Results are expressed as% of total fatty acids. AA, arachidonic acid or C20: 4n-6; DHA, docosahexaenoic acid or C22: 6n-3, iMCT oil, medium chain triglyceride oil. 2AA oil, ARASCOtm & 3DHA oil, DHASCOTM (Martek Biosciences Corp., Columbia, MD), approximately 20% AA and DHA, respectively. 4-fatty acids at concentrations less than 0.5% of total fatty acids are not shown. __ Ingredients __g / 2.5 L_ Protein: cheese 157.5 whey 105.8 water 1987 mixed amino acid 2.425 carbohydrate: lactose 87.5 fat mixture (see table 3.4) 350.0 minerals: calcium carbonate 15.08 calcium gluconate 3.413 calcium chloride 6.95 calcium-free Carbon substance mixture (containing iron) 15.1 86783 -51-200412942 Copper sulfate solution 1 0.0749 Zinc sulfate solution 2 0.2845 Vitamins: Vitamin mixture (Teklad) 10.0 Vitamin mixture (supplemented) 1.375 Others: Meat test 0.1 Inosinic acid 0.175 Ethanolamine 0.0855 Table 3.5 Composition of rat milk formula. List ingredients at 2.5 liters. Copper sulfate solution 1 is 30.9 g CuS04 · 5H20 / L H20; zinc sulfate solution 2 is 379.3 g ZnS04 · 7H20 / L H20 (as described by Auestad et al., 1989). The fatty acid composition of the rat milk formula was determined by gas chromatography. The results are shown in Table 3.6. The target percentages of fatty acids linoleic acid, linolenic acid, AA, and DHA have achieved their concentrations as close to or as close as expected. For a formula raised in February, the AA appeared to be low (1.4% vs. 2.5% of the total fatty acid target). This appears to be due to improper laboratory operations during GC analysis or other experimental errors. The result will probably be close to 2.5%, because the exact same fat mix was used to prepare the formula for both February and April rearing. Other AA-added formulations contain about 2.5% AA as expected. February breeding April breeding No AA + AA No AA + AA No DHA + DHA No DHA + DHA No DHA + DHA No DHA + DHA 06:01 0.6 0.6 0.6 0.5 0.6 0.6 0.6 0.5 86783 -52- 200412942 C8: 0 23.8 21.6 22.6 19.7 24.0 22.4 22.1 20.2 C10: 0 17.2 15.7 16.4 14.7 17.0 15.9 15.7 14.6 C12: 0 29.8 29.3 29.8 27.8 31.0 29.3 29.3 27.7 C14: 0 12.0 12.4 11.9 12.1 12.1 12.2 11.6 11.7 C16: 0 7.8 8.4 7.9 9.2 7.0 7.7 7.6 8.2 C18: 0 2.3 2.3 2.6 3.0 2.1 2.1 2.7 2.7 C18: ln-9 5.1 6.1 5.3 6.8 4.8 5.9 5.4 6.5 C18: 2n-6 1.3 1.3 1.5 1.7 1.3 1.2 1.7 1.6 C20: 4n-6 (AA) 0.0 0.0 1.4 2.4 0.0 0.0 2.5 2.5 C22: 6n-3 (DHA) 0.0 2.3 0.0 2.4 0.0 2.6 0.0 2.6 Table 3.6 AA and DHA feeding period, fatty acid composition of rat milk formula. Results are expressed as% of total fatty acids. AA, arachidonic acid or C20: 4n_6; DHA, docosahexaenoic acid or C 2 2: 6 η-3. Fatty acids less than 0.5% of total fatty acids are not shown. 3 · 2 · 3 Growth Evaluation The captive young rats were weighed each day. Weigh at the beginning and end of AA and DHA feeding periods, and also at the time when the food introduction period will be reported. 3.3 Food introduction milk-free coarse meal (Bioserv inc., Frenchtown, NJ), fat / this product (coconut oil · MCT oil, 70:30, w / w) and water until the viscosity becomes brittle Food feed for the February experiment. Due to the viscosity of the food and feed, the accuracy of the food introduction measurement in the February experiment is questionable. Therefore, small granular foods with the same nutritional composition were prepared (Research Diets Inc., Princeton, NJ) for the April experiment. The pellets are very dense and firm. 86783 -53- Hard, worried that freshly weaned mice will not eat the solid pellets easily. Therefore, the small grains of mashed mashed into Zhizhi 'and mixed with water to form 1/4 " to 1/2, semi-solid spheres are used for food ^ The nutrient profile of the diet of two good feeds conforms to the recommendations of AIN-93 ( Reeves et al., 1993). Fatty acid analysis was performed on food feeds and the results are shown in Table 3.7. On the 16th day, the young rats were guided to feed weaning food. The feed contained 10 g / 100 g wet fat, which was a mixture of coconut oil: MC1 ^ (70:30, w / w). All rat experimental groups were weaned into the same Aa or DHA-free food feed to eliminate the strong and chaotic effects of the special flavors that were designed by trial and error. On the 16th and 17th days, 80% of the daily caloric requirement came from the laboratory rat formula that was assigned ’20% of the caloric requirement came from the food feed (Table 3.3). In a few minutes, younger "April 1 April 2 C6: 03 0.6 1.3 C8: 0 23.6 27.1 C10: 0 17.0 12.0 C12: 0 31.4 31.8 C14: 0 12.2 12.8 C16: 0 7.0 7.1 C18: 0 2.1 7.1 C18: ln-9 4.7 0.9 C18: 2n-6 1.3 0.2 C20: 4n-6 (AA) * 0.0 0.0 C22: 6n-3 (DHA) * 0.0 0.0 Table 3 Fatty acids 86783 -54- 200412942. Results are expressed as% of total fatty acids. The fat mixture was coconut oil: MCT oil (70:30, w / w). The food feed for February is made from non-fat powder. The fat mixture is added to the feed with water to form a crispy viscosity. 2April food feed is made of small grains, containing the same fat mixture; crush the small grains and add a small amount of water to form a 1/4 "to 1/2, feed ball. The fat is equal to or less than 0.5% Not reported, except for those who are indicated by an *. Beginning at 5 pm on the 18th day, the caloric value of the young rats will be limited by 20% of the caloric requirement from rat milk formula and 2G% from the wet feed. The formula Dilute with water to 2G% of the initial heat content. The introduction of water is not restricted to maintain the proper moisture of the animal. At 9 am on day 19, mole rats are stimulated to urinate and then weighed. Stomach cannula Remove and place the young rats in individual cages. The cages contain water bottles and about 15 g of "brittle" feed on a ceramic dish (February experiment) or directly add a "feed ball" to the bottom of the cage (April (Experiment). The cage has a transparent plastic bottom and sides, about 8 inches wide and 12 inches long. It is surrounded by a sloping top with a silk screen and a water bottle. Every 2 hours, a total of 8 hours' weigh the leftover feed To determine the amount of food that has been eaten. Due to the evaporation of food during the introduction period, three feeds are included, the control group, The weight loss was measured. At the end of the food introduction period, the rat was re-weighed. Then the feed was removed from the cage, and the rat was fasted for 18 hours and freely ingested water. At 9 am on the 20th day, the rats were stimulated again The rats were allowed to urinate, seeded, placed in their cages, and supplied with water and about 15 g of pupae. After 2 hours, the amount of food eaten and the final weight of the rats were determined. 3.4 Tissues were collected on day 20 'Food After the introduction period, the young rats were beheaded and sacrificed to obtain a maximum body weight of 86783 -55- 200412942. The brain was removed, weighed, and quickly cooled with liquid nitrogen; east (within 5 minutes). Brain tissues were stored frozen at -70 ° C Box. Blood is collected from the stump of the neck, mixed with heparin, placed on ice, and centrifuged to determine sufficient phase separation to prepare plasma. Plasma is stored at -70 ° C. Brain and plasma samples are taken from UCLA overnight on dry ice. Shipped to Ross Products Division of Abbott Labs, Columbus, Ohio, completely cold rolled upon arrival. Check the samples for damage and signs of melting and immediately transfer to -70 ° C refrigerator for analysis. Plasma samples were obtained, but Not analyzed as this 3.5. Extraction and analysis of lipids 3.5.1 Fat composition of the three lipid moieties in the brain was determined. Phospholipid fatty acid methyl esters were determined in a similar manner to that described by Ward et al. (1999). In addition to HPLC, There are methods of gas chromatography mass spectrometry (GC / MS) and liquid chromatography-mass spectrometry (LC / MS / MS) for measuring MAG and NAE fatty acids (Berger et al., 2001; Kempe et al., 1996) Fontana et al., 1995; Felder et al., 1996; Wang et al., 2001). However, a less expensive and simpler method for measuring this fatty acid in brain tissue has been developed. Total lipids were extracted from rat brains using a typical Folch extraction method for lipid extraction (Folch et al., 1957). The total lipids extracted from each mouse brain were separated into neutral lipids and phospholipids using a silicone cartridge. The high performance liquid chromatography (HPLC) was used to separate the neutral lipid fraction into MAG and NAE fractions. After derivatization into the corresponding fatty acid-resistant methyl ester, the fatty acid composition of the MAG, NAE, and phosphoric acid ester fractions was determined by gas-liquid phase chromatography (GLC). The result of the fat 86783 -56- 200412942 fatty acid composition corresponds to the total fatty acids of the phospholipid membrane in the brain, and the results of the MAG and NAE fatty acid represent the concentration of the fatty amidyl derivative in the rat brain. 3.5.2 Reagents and suppliers Erythramide and docosa tetraenoylethanolamide were obtained from Cayman Chemical Co. (Ann Arbor, M1). Twenty-two carbon dilute si chloride and fatty acid standards were obtained from Nu-Chek Prep, Inc. (Elysan, MN). Ethanolamine and boron trifluoride-methanol complex (BF3) were from Sigma-Aldrich (Milwaukee, WI). Dichloromethane, methanol, chloroform, hexane, ethyl acetate and isopropanol were from Burdick & Jackson (Muskegon, MI), petroleum ether was from Mallinckrodt (Paris, KY) and formic acid was from J.T. Baker (Phillipsburg, NJ). All reagents used are analytical grade. LHPK Shixi gel thin-layer chromatography plate and filter paper were from Whatman (Clifton, NJ). Micropipettes, tubes, and bottles were from VWR Scientific (Bridgeport, NJ). HPLC separation tube (Chromegasphere SI-60, 4.6 X 150 mm, 10 μ, 60A) from ES Industries (Marlton, NJ). 3.5.3 Fatty acid standard A GLC fatty acid standard was prepared. In a short time, the representative mixture of fatty acid methyl esters (-98% purity) is correctly weighed in a specific order over a 100 mL pear-shaped triangular flask with the empty bottle weight removed to determine the proper mixing. After all fatty acid methyl esters have been added and mixed, the bottle is weighed to obtain the final weight of the standard. Add one hundred milligrams of the standard to an ampoule, fill it with nitrogen, seal with a propane flame, and store in a refrigerator at -20 ° C until use. Quantitatively transfer the contents of one Anxun to a 25 mL volumetric flask 86783 -57- 200412942, and dilute to this volume with hexane to prepare a GLC stock standard. The GLC stock standard was diluted 1: 3 (v / v) with hexane to prepare the GLC working standard, and injected into the GLC between 1 and 5 μL. 3.5.4 Internal Standards Two internal standards are required, monoheptadecanoin for the MAG part, and dodecyltriethanolamine (22: 3n-3 ΝΑΕ) for the ΝΑΕ part. A single heptadecane was prepared by properly weighing 100 mg in a 10 mL volumetric flask and diluting to its capacity with chloroform. Docosatrienylethanolamine was prepared as described by Hand et al. In 1993. Conveniently, approximately 100 mg of 222 trichloride chloride is dissolved in 1 mL of dichloromethane. The mixture was then transferred to a test tube. Add 1 mL of ethanolamine solution (20% in dichloromethane) to the test tube at 0 ° C and fill with nitrogen. The tube was thoroughly mixed with shaking every 3 minutes for a total of 15 minutes. Add 8 mL of dichloromethane to a volume of 10 mL. The sample was then mixed thoroughly with 5 mL of water under nitrogen. Centrifuge the sample at 2000 rpm at 20 ° C for 2 minutes to separate the water and organic layers. Aspirate the organic (bottom) layer into a clean test tube and wash with 5 mL of H2O under N2. Centrifuge the sample as described above and aspirate the organic layer into a clean test tube. The combined organic layers were evaporated to dryness under a chemical gas. The sample was then reconstituted in 10.0 mL of chloroform: methanol (1: 1, v / v), dried with N2 gas, sealed, and stored at -20 ° C. Methylation was followed by GLC quantification to determine the concentration of the obtained standard in docosatrienylethanolamine. Extraction of 3 · 5 · 5 samples

Frozen rat brain samples stored at -70 ° C were divided into two hemispheres; one half was used to determine the fatty acids of the phospholipid, MAG, and NAE fractions, and the other half was frozen at -70 ° C. The brain for analysis was transferred to a 50 mL glass centrifuge tube. Add 8 mL of 86783 -58- 200412942, using a Polytron dispersion and mixing system (Kinematica, Switzerland) until well mixed. Wet the homogenizer probe with 2 mL of methanol added directly to the centrifuge tube. Then add 20 mL of chloroform to the sample and shake well to mix. The samples were left undisturbed for at least 1 hour at room temperature. Add the known standard 9.91 pgimonoheptadecanoin and 3.32 pg of dodecadienylethanolamine. Add 6 mL of 0.9% physiological saline and shake well to mix the sample. The sample was then centrifuged at 2000 rpm, 15 ° C for 7 minutes until used (Beckman AllegraTM 6R Centrifuge; Fullerton CA). The organic and aqueous layers were well separated. The chloroform (bottom) layer was aspirated into a clean 30 mL test tube. The samples were then evaporated to dryness under N2 and stored at -20 ° C or reconstituted in 500 pL of chloroform. 3.5.6 Purification of the SEP-PAK Cassette Each sample of the reconstituted brain extract was loaded into a Silica Plus SEP_PAK cassette (Waters / Millipore, Milford, MA) using a disposable glass pipette. The brain extract-containing test tube was washed twice with 500 μΐ of chloroform, and then the chloroform was put into a cassette to confirm that all extracts were transferred to the cassette. Neutral lipids were dissolved in 15 mL of aerosol: methanol (99: 1, v / v), and phospholipids were dissolved in 15 mL of methanol. The neutral lipid eluate was filtered through a needle filter (Gelman Acrodisc® CR PTFE, 0.45 μ or 0.2 μ, 25 mm; Ann Arbor, MI) connected to the bottom of the SEP-PAK cassette. Collect each eluate into a test tube and evaporate to dryness under N 2 gas. 3.5.7 Stepwise separation of high performance chromatography

After each eluate was evaporated to dryness, it was resuspended in 125 μ! ^ Or 300 μί of hexane · isopropanol (IPA; 90: 10, v / v) and injected into Hewlett Packard HPLC 86783 -59- 200412942 (Roseville , CA), the HPLC contains a separation tube of Chromegasphere SI-60 '4.6 χ 150 mm, 10 μ, 60A (ES Industries, Marlton, NJ) and a strip light scattering detector (Alltech ELSD, Deerfield, IL) For separation of MAGs and NAEs. The mobile phase gradient is shown in Table 3.8 (adapted from Liu et al., 1993). A solution containing the internal standards triclosan, monoheptadecanoin, and docosatriolethanolamine was injected before each HPLC run. To confirm the delay time of free acids, MAGs and NAEs. After confirming the delay time, cut off the evaporative light scattering detector and connect the HPLC mobile phase line directly to the partial collector (BioRad, Model 2128; Hercules, CA). ___ Mobile phase% hexane Mixed solvent 1 0.0 98 2 8.0 65 35 8.5 2 98 15.0 2 98 15.1 98 2 98 2 Table 3.8 HPLC mobile phase gradient for separation of fatty acids, monoglycerides and N-fluorenylethanolamine. 1 Hexane: isopropanol: ethyl acetate: 10% formic acid in isopropanol (80: 10: 10: 1, v / v / v / v). The flow rate is 2_0 mL / min. Then, a 250 μm resuspended neutral lipid fraction extract (ie, chloroform · methanol SEP-PAK eluate) was injected into the HPLC separation tube, and the corresponding dissolution time of MAGs and NAEs was collected group. HPLC and MAG fractions collected from each mouse brain sample were evaporated to dryness under N2 gas. 86783 -60- 200412942 3.5.8 Methylation process Re-suspend MAG and NAE fractions in hexane: isopropanol: ethyl acetate (80 ·· 10: 10, ν / ν / ν) and transfer to 2 mL sulphur in a white screw cap bottle. The portion was re-evaporated to dryness under a N2 atmosphere at room temperature. Then, samples containing MAGs, NAEs, and phospholipids were methylated by adding excess boron trifluoride-methanol complex, BF3, under N2. After tightly closing the lid with a Teflon liner, place the sample on a square hot plate at 95 ° C for 20 minutes. The sample was cooled to room temperature and opened carefully. Transfer the MAG and NAE samples to a 15 mL tube using methanol. Then, add 2 mL of 0.9% saline and 4 mL of hexane to the sample and mix thoroughly by shaking. For each sample, use a disposable glass pipette to remove the hexane layer, transfer to a clean 15 mL test tube, and evaporate to dryness under 1 ^ 2 gas. The methylated hexane extracts of MAG and NAE were reconstituted in 100 pL of hexane for GLC analysis of fatty acid composition. The dried phospholipid methylated hexane extract was reconstituted in 10 mL of hexane, and the reconstituted extract was diluted by 50 μί with 150 μE of hexane for GLC analysis of fatty acid composition. 3 · 5 · 9 gas-liquid layer analysis. Hewlett Packard 6890 gas-liquid chromatography (GLC) was used to analyze fatty acid methyl esters. The chromatograph was equipped with a flame ionization detector; and Omegawax320 was coated with Polyethylene glycol lava stone separation tube, 0.32 mm ID X 30 m, and film thickness of 0.25 mm (Supelco, Inc .; Bellefonte, PA). The settings of the gas chromatography device were adjusted to optimal signal sensitivity similar to the conditions described by Ward et al. (1999). Each sample of 5 μΐ was injected into the gas chromatograph using an automatic sample injector (Hewlett Packard 7673A). 86783 -61-200412942 The corresponding fatty acid methyl ester internal standard was co-dissolved to separate individual fatty acids. Relative percentages of total fatty acids have been typically reported in the literature to report amino acid levels in the phospholipid portion of the mouse brain. The specific content of individual NAE fatty acids in lipid extracts in the brain was quantified with methyl docosatrienoate relative to the nae internal standard. Similarly, the content of individual MAG fatty acids in lipid extracts in the brain was quantified with methyl undecanoate relative to the monoglyceride standard. Corresponding fatty acids of MAG and NAE are reported as wet weight of ng / g and gg / g mouse brain, respectively. 4.1 Study combination Two groups of mice were reared artificially, one in February 2002 and the other in April 2000. A total of 64 mice were cannulated each day, and 32 mice were reared each time, with 8 mice per experimental formula group. Several mice were kept in captivity. Dead mice were replaced by male rats from the February study, but were not replaced after the 7th day after birth in the April study. Only rats that were replaced within 24 hours of the first day of the device cannula (day 6 after birth) were included in the final data. Therefore, all the old mice in the last data were kept in captivity from the 6th day to the 18th day after birth for a total of 13 days. Therefore, any dietary fatty acid has the same group of fatty acids in the phospholipid membrane in the brain. A total of 74 mice were cannulated and π died. Table 4.1 shows the number of mice that were housed, cannulated and killed per experimental formula group. It is worth noting that when DHA was not included in the formulation, more rats died than when DHA was included in the formulation (10 mice versus 3 mice, respectively, p = 0.054). 86783 -62-200412942 Rat milk formula group 1 without DHA without ΑΑ + DHA without DHA + ΑΑ + DHA February feeding with casing 11 9 10 9 death 3 1 2 1 April feeding with casing 9 9 9 8 death 3 1 2 0 Combined breeding with cannula 20 18 19 17 Death 6 2 4 1 Table 4.1 Number of mice that were fed, cannulated and dead in February and April. From the 6th to the 16th day after birth, 100% of the calories, 80% of the calories on the 17th and 18th days, and 20% of the calories in the last 18 hours before the introduction of food, the stomach opening tube will contain different amounts of AA And DHA rat milk formula (see Table 3.6). When DHA was not in the formulation, more deaths occurred (ρ = 0.05). No other obvious associations were found between the formulation combinations. AA, peanut dipping acid; DHA, dodecahexaacid. 4.2 Growth data Growth is assessed using consolidated data (February and April) and key data (April). Except for the end of the food introduction experiment (day 20 after birth), when the start of captive breeding (6 or 7 days after birth, data not shown) and end (day 18 after birth), the weight of the rat milk formula group There are no significant differences (Table 4.2). There was a significant difference in 'body weight' between the suckling rat reference group and other experimental formula groups. 86783 -63- 200412942 (ρ < 0.05). The suckling rat reference feeding group was significantly larger than the four formula group on the 8th day. The suckling rat reference normal group was larger than the formula group and the reference silver food group on the 20th day. Significant differences are also seen in brain weight. Rats fed the DHA formula were slightly but significantly less brain weight than mice fed the DHA-free formula (p < 0.05). All brain weights ranged from 1.3 g to 1.4 g. 4.3 Food introduction study A food introduction study began on day 19, during which all rats were given the same diet without restriction (Table 4.3). The results are provided for February and April (combined data) and separately for April (main data), as the measurement of the introduction of the food substantially improved during the feeding period in February and April. There was a significant main effect of feeding AA-containing rat milk formula on food introduction after weaning (Table 4.3). Rats previously fed the formula containing AA ate about 13% more feed than mice previously fed the formula without AA, regardless of the level of DHA. This effect was seen on the 19th day in the combined data, the first 2 hours of the food introduction period, and the first 2 hours on the 19th and 20th days in the main data. There is also a significant main effect of DHA on the food introduction after fe / f milk. The rats previously fed the DHA-containing formula consumed about 1% less feed than the mice previously fed the DHA-free formula, regardless of the level of AA. This effect was seen on day 9 of the combined data and the main data, the first 2 hours of the food introduction period. 86783 • 64- 200412942 ^ 4.2 Ridge! Tellurium climbs to ILfrR ^ + Jingmu tick time. i nest H- 阼 漭 铖 6 昀 铖 16 ^ 100% fried 渰 i-铖 17 沣 1 8 ^ 80% S 沴 ^-铖 19 1 4S Throw 1 8. J > — 20% S »4-洤 远 f 翌 ptf-δ 笫 吟 一: s ΑΑ 沣 DffiA ?, 3 · 6) ^^ IL Private 铖 7 沣 18 outside -δ 20% 渰 i ^ ~ -f m 鹞 瀹 Single 111 > 0-3: Ma Ling-Zhi ^ lingmai 2 > ^ Cold ~ ^ Yin Shen 翱"Zhen Ling > > 涔 01 ^ ~ 卜彻 涔 洳 0-A ^ OVA >»^; (+) Fang 洳 _ ^ _ 涔 米 ^ 施 才 14 ^ 乂-) ^^ $ 14- * :. ^ 1 獬, # Poor ^ 卜 _ s IL; 搽 Ling 洚 Cold String (Fdg) i ^ it 冷 洚 拎 4S 铖 19 ^ 漭 fen 渰 S 牦 Han-Fen Bi Bi Miao 鸩 ♦ Listen. " Let the passengers defy Wei Lingming (11 " 9-15 涔 / Listen) Mushroom l · 『沣 3』, Yushen Umbrella. -^ 沏 3: Fortunately, poor life 9 " 5-8 sounds / listening) Mushroom 111 > 3 b Guest habitat Han 瀹 Cafe-^ 巌 卡 ^ 信 袅 今 ^ 蒜 袅 定 缪 ^ 浼 浼. Mai 7 钤 涔 钤 涔 Jin 蚧 蚧 ^ = * = 詾 75 ^? ≪ 0 * 05 It ’s time to go outside and ask for 3: ^ 冽. meeting? 5. ^^^ 翁 钋, 4 夔 啭 hydrocarbon ^ 表 畤 ^^^ 冽 -twist ^ letter 哳 砟 ~ ^ 巌 14 ^ g Ah ^^^ 铖 l00 > (T + 3 ^) Life umbrella 卩 玲 参 51 铖 20 ^ (-f + 12b?) When the life of the umbrella is 巌 20, g & 20 > H $ 1.34 ± 0.05? 1.35 ± oo4f Pri. 7 · 5 ± 1.7 Pri. 7 · 7 ± 1 ο 43.3 ± 1.6. 44.4 ± 1.4 H] f Jin: 1.32 + 0.0 ^ L37 ± 0_05 Speech L34 ± 0b5 仏 0056 (+) 0035 (-) L42 ± 0b5eL40 ± 0b5— L33 ± oo3. L40 ± oo5 & 1.35 ± oo4. 0.097 (+) 0.026 (-) L42 ± oo5 & L46 ± oo ° p 矣 2 ± 3b 47.3 ± 3.1 private 4.6 ± 2. Private 3.9Η-1.3

Age AA age DHA + DHA

+ AA age DHA + DHA VP1 VP1 vo.l vpl 払 7.1 ± 1_5 51.2 ± 5b * 払 7.1 ± 1.5 V0.1 VP1 vpl vpl 48.2 ± 1.6 * 矣. 2 ± 1.2 *

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Fdg i £ ^ &86; 86783 -65- 200412942 > 4.3 ♦ Take a ^ s- 阼 漭 铖 6 沙 _ 铖 16 ^ 100% stir fry J:-铖 17 涔 18 sand 80% customer coated i-wrinkle 19 ^ Yin sang 51 > ^ Leng Yiyi 诹 漭 18 VB order 20% s ^ -Wayuan f 逦 pt ^ f-δρ > ^: f5! 3 ^ # sAA 涔 DHAS > 3 · 6) ~ Qi IL 蚧Prairin relief. Private 铖 17 沣 100 >-s 20% »i-吟 Yin AA ^ DHA ~ ^ glft read 牮 ila-s Ma Ling-如 Fen Ru Ling 51 51 Leng Bu 诱 Yin 吟.铖 19 ^ 二 泛 ^ 岭 棼 ~ Clan »〇〇 子 众-渰 萍-洙 部 1 滁 滁 族 族 --Bu ^ 窆 呤 岭 参 族» 2 2/0 order. : XANOVA ^^ 脔 玲 AAiLDnA- ^ h 涔 洳 涔 洳; (+) Fang > ^ 涔 涔 米 ^ 施 才 呤 参 3: 茜 »二-) ^ 4 ^. 涔 湃 涔 湃 Λ 族 ^ 玲 棼 艺 族». ^ 1 獬, # Poor bismuth- £ _ sacrifice; Tiao Yin ^ Cold Poverty (2 bar ^ Tiao Yin ^ 43: 铖 19 ^ ^ 漭 踔 华 (nH5l8fcR) ^ iJ 铖 2 窗 # 一 参-拉Juice 巌 +; f 冽 袅 β β garlic to establish a thinner handle. ± SD; ** na-^ 4 quaternary hydrocarbon; AA-oblique 阼 Wei Zun 隳; DHA-b + 卜 chicken external examination 鵁 19 ^ Assistance 2 ^ B order 19 ^ gt ^ 8, Ji order 20 > Zheng Go 2 2 4 order 19 > Zheng Pu 2v ^ Scale 19 ^ rub 4: 8 2 4 order 铖 20 > Yu Kai 2VB order lp3 ± rl 17 · 1 ± 1.3 13 · 1 ± 1 · 2 10.8 ± 1. Ren17 · 2 ± 1 · 6 12/7 ± 1.3 16bo ± 1.7 12 · 3 ± 1 · 5 9 · 6 ± 1 · 6 16bo ± lbo 12 · 0 ± 1 · 2 17 · 7 ± 1.8 12 · 9 ± 2 · 0 12 · 3 ± 1ο 18 · 4 ± 1 · 00 14ο ± 1.5 1Ρ5Η-10 17 · 7 ± 1 · 2 13 · 9 ± 2 」 s00 ± p9 17 · 7 ± 1ο 13 · 8 ± 2 · 1 9.8 at PLA / 100 gft 9.8H-L3 11 · 2 ± 1 · 6 s ^ c

DHA

+ DHA

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+ DHA

»≫ AA

+ AA

na na np 6.8 ± lo lbo— • 8 na na na np np na

Fdg ^ 86783 -66- 200412942 4.4 Results of squamous lipid fatty acids Table 4.4 shows the results of fatty acid content levels of phospholipid membranes in the brain expressed as a percentage of total fatty acids (ie g / 100 g of total fatty acids). In this study, the effects of dietary n-6 and n-3 fatty acids on the composition of n-6 and n-3 fatty acids in the brain were similar to those previously shown in the literature (Ward et al., 1999; de la Presa Owens and Innis, 1999 )similar. There were no significant differences in saturated fatty acids between groups. AA has significant main effects on unsaturated fatty acids (C18 ·· 1 and C20: 1) in cerebral phospholipids. Among the phospholipids, there is a consistent overall effect of reducing the AA of the diet and increasing the content of linoleic acid (C18: 2n-6) and C20: 3n_6 in the diet. For other n-6 phospholipid fatty acids, there is a consistent and comprehensive main effect of increasing dietary AA and reducing dietary DHA by AA and C22 · 4n-6 levels. There are also significant main effects of dietary AA in reducing phospholipid DHA in the brain; likewise, dietary DHA increases DHA in brain phospholipids. 4.5 Results of N-fluorenylethanolamine (NAE) fatty acids The results of η-6 and n-3 NAEs are shown in Table 4.5 and are expressed in ng / g brain. AA has a significant main effect of increasing 20: 4n-6 ΝΑΕ in the brain. AA also has a significant main effect of increasing total η-6 ΝΑΕ in the brain. No other significant main effect was seen. No significant major effect of dietary DHA on n-6 or n-3 fatty acids was seen. Results of 4.6 monofluorinated glycerol (MAG) fatty acids The results of η-6 and n-3 MAG are shown in Table 4.6 and expressed in pg / g brain. Diet AA had no significant effect on n-6 or n-3 MAG. However, DHA has a significant major effect of increasing 22: 6n-3 MAG in the brain and also increasing total n-3 MAG in the brain. 86783 -67- 200412942 Related data In addition to data on the main effects of testing diet AA and DHA, and comparison of ANOVA between the experiment and the infant group, Spearman's correlation was evaluated by computer to determine whether it was in specific NAEs and MAGs (including n- 6 / n-3 ratio) and food introduction. After classifying the data and eliminating possible edge weights, Spearman's correlation was picked. The results are shown in Tables 4.7 and 4.8. Significant positive correlation (r = 0.45, p = 0.03) found at 20: 4n-6 NAE / 22: 6n-3 ΝΑΕ content ratio with food introduction during the first 2 hours on day 19 and food accumulated on days 19 and 20 Introduced (Table 4.7). A significant positive correlation was also seen between MAG fatty acids and food introduction (Table 4 · 8). The ratio of MAG (20: 4n-6 MAG / 22: 6n-3 MAG and n-6 MAG / n-3 MAG) is positively related to food introduction, which was introduced on days 19 and 20 The measurement was performed for 2 hours, and the cumulative food introduction measurement was also performed for 2 hours on the 19th and 20th days (r = 0.42 to 62, ρ = 0.001 to 0.005). There is also a trend between the 22: 6n-3 MAG and the sum of n-3 MAG and the introduction of food (ρ <〇; 1), the introduction of the food includes the first 2 hours of day 19 and the accumulation of the entire food study Food introduction (r = -0.39, ρ = 0.06). 86783 -68- 200412942, 4.4 Ridge tastes abnormal, listen to the muddy surface Miao garlic full ~ garlic indigo groaning one: Yaque. Miao 洳 ^ Weng Jiao Bu Miao fried% > > 钋. Pan-carnitine thin order min_Rong 茸 奇 薛 遄 鸸 冷 # 蚧 Vegetables association with vegetables> 3.6. Mit Ling AAiLDHA-^^ 涔 涔 & AMOVA >^^; (+) ^ > h 涔 铋 bismuth garlic garlic ginseng. ^ Family »二-) 方 ^^% 涔 米 bismuth yilingshen ~ Clan »Two *) ^^ Xiao Ping Xiong 3: Cold ^^; | ^ 渰 aii ^ will make tons of rice. ^ 1 涔, ♦ bismuth-poor ittsIL sound. It Ling (fdgHi cold listening bismuth bark bird cold scale-铖 a 9 ^ JL ^ S: Fen Tu 牦 Han-Fen Li 鸩, <% listening. 0 018: 0 C200 C22: 0 ^ #, ^ 3: C16: ls C18: ls C20: ls 016: 4 n-6 garlic but clsn-6 c20: 2n-6 C20: 3n_6 C20: 4n-6 C22: 4n 丨 6 C22: 5n-6 n-3BS ^ _ C22: 5n-3 C22: 6n-3 1 · 1 ± 0 · 1 16.9H-2 · Private 18 · 5 ± 0 · 5 p3 ± oo plH-pl P7H -S 15 · 3 ± 0.9 1 · 2 ± 0 · 2 2.8 ± ε P6Η-P0 plK-oo ο · 7 ± οο 14_4 ± 1.0 4.4Η-P3 2.8Η-0.5 P2KK-P0 18.4Η-1.2 UH-P1 17 7 ± 0.9 180Η-0.4- SH-PO ρ1 ± οο ιοΗ-0.3 15 · 2 ± 0 · 8 1ο ± 0.2 3ο ± ε P8 ± P1 P2 ± P1 0.9Η-P1 S.2H-P7 Zn2Η-P1 P9 ± P1 P7Ρ-P1 25 · 6 ± ρ6 1 · 1 ± ρ1 18 · 1 ± 14 18 · 4 ± ε P2Η-0.0 ρ1 ± 0 · 0 0 · 9 ± 0 · 3 14 · 2 ± 0 · 2 ρ9 -P1 2 · 9 ± ρ3 0.3 ± οο ο · 1 ± οο ο · 3 ± οο 16 · 1 ± ρ5 6ο ± ρ5 5 · 2 ± 0_8 ρ3 ± οο 13 · 9 ± 1 · 1 UH-P1 17 · 9 ± 0.9 18 · 4 ± ρ3 P2Η-P0 P1 ± 0.0 ΙοΗ-0.3 13.9K-P00 P8 ± ρ2 3ο ± ΟΛ δΗ-οο P1K-P0 P4Η-P0 14 · 4 ± 0 · 7 SH-P2 1 · 0 ± 0 · 1 P2Η- 0.0 2L6 = tp5 * * < 0 · 001 () Λ0.001 (+)

i ± SD ΡΛ1 VP1 VP1 0085 VP1 VP1 νο · 1 ΛΡ001 0007 VP1 * (丨) <0001 * () * (+) * (+) p--Nt vpl vpl vpl vpl &gt; 0,1vpl (丨) vs (_ ) vpl vpl * (+) (丨) vs * (+) * (丨) * (丨) * (丨) uH-0.1 l? ° 3 ± 0bo 18.4KS 0 · 2 ± 0 · 0 plH-oo loHhs 13 6 ± ρ3 p8 ± 0.1 2.8H-P6 p9 ± oo p2 ± oo p6ioo 14,9 ± p 払 4 · 7 ± P1 1.4H-P1 ρ3 ± 0 · 0 19.7H-P5 lbH-pl 18.1H-0.6 1 °° 2 ± £ ο · 3 ± οο P2H-P0 ρ9 ± 0 · 3 14.4 ± 1.0 ro ± 0.2 2.7 ± s LlK-pl 0 · 2 ± 0 · 1 ρ7 ± 0 · 1 14.2H-P9 ί ± ρ2 1.2 ± 0 · 1 Ρ4 ± 0.0 19 · 6 ± P6

3 ^ DHA + DHA

+ AA _DHA + DHA

βχκ Fdg ^^ JL · ^ 86783 -69- 200412942 &gt; 4.5 Provoking Expenses 涔 ί tp N-si ^ pgl Seline 关 Pass +. Miscellaneous rice ^ 5 ^ 巌 when neighbors.奇 寐 懂 鸸 莾-Acoustic ILgs ^^ gg Ma-ANOVA ^^ &gt; Zhailing AAiLDHtA Fried, 涔 洳; (+) ^ &gt;. ^ 涔 梆 涔 梆 ^ 才 才 命 参 夕 族 | € 2 ^^^^^^^^^^^ ^^ »二 *) ^ &gt; ^^ Splash pile ^^. 5. ^^ Two punches ^ oblique splash pile s .i% 涔 rice two IL mud listening bismuth jE fs IL mud; purine (fdg) ^ cold listening敏 it Ling 洚 冷 scale- 铖 19 ^ Agsfen 渰 牦 豸 j ^^ 崎 # 鸩 4:, ^^. C20: 4n-6 C22: 5n 丨 6 n-6〆 ^ C22: 5n 丨 3 022: 6n_3? 3 4N9 ± S5 Private 0.2K-12Λ 83.1H-11.5 1500 ± 14 · 7 39 · 5 ± 7 · 57 55.2 ± 21 · 6 3 £ ± 9.93 33ο ± 17 · Ren 69 · 4 ± 25 · 5 払 1 · 3 ± 23ο 払 7.1Η-9.12 88.4 ± 29 · 5 51 · 7 ± 10 · 8 — ± 3.8 91 · 7—払 2.3Η-14.6 払 6.0 ± 9 · 07 8? ° 3η-15 · 7 55 · 4 ± 20 · 4 Private 3 · 7 ± 25ο 99 · 0 ± 44 · 8 33 · 1 ± 18 · 2 払 8 5 ± 19ο 8L6 ± 32.1 0.008 c + vo.lvo.l vpl 0027 (+) VP 1 3 3 vpl ναι vo.l vo.l pxi pA.i

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+ AA age DHA + DHA

50b ± 29.4 36.7 ± 18 · 3 86.7H-44.3 27bo ± 16.7 Private 6.7 ± 7 · 53 74 · 5 ± 20.7 払 7.2 ± 11.7 2S ± 17.7 77 · 1 ± 26 · 5 33 · 3 ± 7 · 72 払7 · 7 ± 13.5 81 · 0 ± 19 · 2 IL ^, 4r ice dagger 86783 -70- 200412942 &gt; 4.6 book Jin Jin | LH, my sister fvJI 邾 邾 呤 关 关 +.郗 米 ^: ^ 's 巌 14 ^ &gt;卞 ^ Garlic Zhuang. "2 1 Jin 蚧 liang ^ 沴 &gt; 4" yarn 棼 泾. "&Amp; &gt; of 0 &lt; &gt; ^^ 征 玲 &gt; &gt; ^ 0 ^^ 卜. ±. 涔 涔 ^ (()) ^ &gt; ^^ 涔 &gt; ^ Two ^ _ 涔 米 扣 族 A 呤 参 蛉 族 渰 二 ^ 浬 ¾ 蝌 Sfen ^ 衾 逆 二 冲 &gt; 涔 涔 米. ^ 1 涔, ♦ ^^ J £ Bricks IL 涔; it Ling (fdg) ^ Ling hears the cold bird-缉 19 ^^ 3Sfen 渰 声 豸 丄 分 # squat 4,4: ^. N20: 2n-6 n20: 3n-6 n20: 4n-6 022: 5n-6 C22 : 5n-3 022: 6n 丨 3 n 丨 3 ^ 0b59 ± 0b05 0.199 ± oo20 3.386 ± 0.870 p145 ± oo29 3.7oooo ± 0.889 oo20 ± oo23 0.904 ± 0.155 0924 ± 0177 P125 ± P103 P345H- 0.101 3.24 ^ 1 ± 1.03 Private 0.035S.026 3/746 ± 1.102 oo5? Oo36 1.638 ± 0.512 1.688 ± 0.4S3

^ AA i ^ DffiA + DHA 0b32 ± 0b20 0.126 ± 0.039 3_495 ± p940 p324 ± oo97 3.97s ± lo51 oo27 ± oo26 p7soo ± 0.310 0OO15H-0.298 0b54 ± 0b38 0.141 ± oo4 ^ 3.897 ± lo05 oo30 ± oo30 払 123 ± 1ο Private 5 oo23 ± oo23 1.3003 ± ρ413 1.406 ± 0.39〇〇

+ AA age 1.236 ± P435 1.287 ± P460 p124 ± 0b72 0 · 244 ± ρ116 3.471H-1.200 oo78 ± oo33 3.917H-1.39 Private 003719 1.260 ± P566 1.297 ± P588

ANOVAW ^ 涔 AA DHA ILR, ^ Ice Fdg ^^ i ^ &gt; 86783 -71-200412942 ^ 4 · 7ZAE ^ 7JC + ^^ # 2 &gt; ~ spearman ^^^ o oAE-N-sii ^ sl ^ ^ as ^ l ^ # A ^ #? l &gt; Suwei. ^ Ji Leng Xiang 2 &gt; ^ 3 Po ^ Reference 3: Wrinkle 19 沣 20 ^^^ 都 ~ 冷 # 翁 ^. ^ -Here ^. ^ 6311 ^ 11 ^ 今 萦 ^ η 丨 6 NAEs 20: 4η_6 ΝΑΕ n-6 NAEtp n-3 NAEs 22: 6n-3 NAE n-3 NAE ^ · ^ (NAES》 b 20: 4n-6 / 22: 6n -3 n 丨 6A ^ / n-3A ^ 0.341 P336 丨 P95 0.108 P447 P377 ^ i 0.111 P117 P667 PS5 0033 0076 铖 19 &gt; -2νΒ rabbit 0073 丨 0074 -0.160 丨 0089 P271 —0029 P740 0.737 P466 P687 P211 0.897 P207puo- P238 -0.182 P446 P231 P344 P618 P27 Kernel 0.406 0033 0.288

? 2? 2S-r PNV Onionin rs 86783 -72- 200412942 &gt; 4boMAG ^^ 7JC ^^^ # ^; v- ^ spearITmn ^^^^^ CMAG- ^ gl; SL4 '; a- ^ a- sin ^^^ L ^^ Bu Yihe. M fibrin? A &gt; S Park Yu ♦ 铖 19 沣 20 ^^ Huimou ~ Ling Shen 箨 ^. : 2 1! 3 蟊 ^. ^ 6311 ^ 11 Bismuth Today's Monument Dan ^ Mls. η 丨 6 MAGs 20: 4n-6 MAG η 丨 6 MAGe ^ n-3 MAGs 22: 6n-3 MAG n-3 MAG ~ t (MAGS) vrb 20: 4n-6 / 22: 6n-3 n 丨 6 ^ ^ / n-3 ^ · ^ P208 0.221 -0394 丨 P393 P615 P646 phf P342 P310 0063 0063 0.S2 0001 铖 19 Sha-2 22 4 ♦ one 0035 0018 丨 P301 丨 P291 P457 0.424 ^ 0 * 0076 P936 0.162 PI 79 0029 004 払 0.50 0.094 ^ -0396 丨 P389 0.570 P565 铖 20 sand -2 2 4 rabbit r pii pifi 0.660 P670 0061 0066 0005 0005 86783 -73- 200412942 5.0 Discussion and conclusion This is n-6 showing different diets before weaning And n-3 polyunsaturated fatty acids, and the first study on the effect of food introduction after no longer feeding this fatty acid. Regardless of the level of docosahexaenoic acid (DHA), arachidonic acid (AA) was fed from 6 to 18 days after birth, resulting in food restrictions on the 19th and 20th days. 13% increase in food consumption. Similarly, regardless of the AA content, feeding DHA from 6 to 18 days after birth resulted in food consumption reductions of up to 12% on the 19th day after birth. As demonstrated in previous studies (Ward et al., 1998 and 1999; Wainwright et al., 1999), we modified the diet by introducing n-6 and n-3 fatty acids in a model of captive mice, and we modified The fatty acid composition of the brain phospholipid membrane. Since the feeding period coincides with the rapid brain growth period, the captive rat model is an excellent choice for modifying the composition of the cerebral phospholipid membrane. The results of our human phospholipid membrane fatty acids are also consistent with the results of previous studies using similar diets lacking essential fatty acid margins (de la Pres a Owens and Innis, 1999 ^ 2000). ○ We have seen in the brains of rats fed AA-containing formulas20 : Significant increase in 4n-6 NAE (p = 0.008). However, this 40% increase is different from the increase reported by Berger et al. In 2001. Berger and report: Piglets fed 0.2% AA (percent of total fatty acids) increased 4 times 20: 4n-6 ΝΑΕ.

Berger et al. Also reported increases in several n-3 NAEs between 5 and 9 times. We have not shown any statistically significant increase in the level of n-3 NAE, but we did see similar results for MAG fatty acids in the brain as reported by Berger et al. 86783 -74- 200412942 We did not show statistically significant differences in any of the n-6 MAGs, however, we did show a statistically significant increase of 22: 6n-3 MAG, and also the sum of n-3 MAG . There are several differences in the study design that explain why our results differ from those reported by Berger. First, we used a captive rat model containing AA and / or DHA at levels of 0% and 2.5%, while Berger et al. Used small bottles fed with 0.2% AA and 0.16% DHA (% total calories). Pig pattern. Secondly, our formula is deficient in fatty acids. Berger et al. Fed sufficient levels of linoleic acid and linolenic acid, and reported that when sufficient essential fatty acids are present, dietary AA and DHA can only increase n-6 and n-3 NAEs. Content level. Third, we sacrificed the mice immediately after the final food introduction study (ie, decapitated the mice), while Berger et al. Waited 3 to 4 hours after the final formula was fed. Kirkham et al. (2002) recently reported different levels of NAE and MAG in the brain during fasting, eating, and decapitation. They found that the levels of 20 ·· 4n-6 ΝΑΕ and MAG increased after fasting; 20: 4n-6 MAG decreased during the diet, and remained unchanged when compared with the control group during decapitation. The differences in the study design at least partly explain why the dietary effect statement for n-6 and n-3 NAE and MAG fatty acid levels is less than that reported by Berger. Our results do not show a direct relationship between the increase in 20: 4n-6 ΝΑΕ and the introduction of food in the AA diet of the diet, as may be based on the expectations of published literature (Williams et al. 1999; Hao et al. 2000), but This result instead suggests a relationship between the ratio of n-6 and n-3 NAE and MAG fatty acids and food introduction. 20: 4n-6 ΝΑΕ is the endocannabinoid fraction that has the most research on appetite. However, it is reasonable that other endocannabinoids of the n-6 and η_3 families play an appetite-regulating role. Endophytic compounds containing at least 20 carbons and 3 double bonds 86783 -75- 200412942 Mephol exhibits its activity at cannabinoid receptors and 11_3 endogenous cannabinoids. This endogenous cannabinoids show more than n-6 family Different results contain affinity (Mechoulam et al., 1998; Klrkham et al., 2002). Interestingly, we found a significant positive correlation between the diet-induced wn_6 / n-3 NAEs ratio and food introduction, as well as the n-6 / n_3MAG ratio and food introduction. We have also found that dietary D Η A has a significant main effect associated with reduced food introduction. In addition, the correlation between 22: 6n-3 MAG (and the sum of n-3 MAG) and food introduction shows a negative trend (genera), so that when 22: MAG (and n-3 MAGs) increase, food Consumption is reduced. It seems that individual NAE and MAG n-6 and n-3 fatty acids in the brain have less influence on appetite regulation after stimulation than the relative contents of NAE and MAG n-6 and n-3 fatty acids. All our studies have produced important findings about the potential appetite regulation of the central nervous system. Most importantly, we show that dietary n-6 and b-3 fatty acids affect food introduction. Because the different n_6 and n_3 foods were introduced into the food research laboratory and fed with a gastrotomy tube, all rats were fed the same dietary food during the food introduction study, so the observed effect could not be smelled or the food (Feed) other characteristics to illustrate. These observations may have other explanations, such as the release or activity of feeding different foods on hormones known to regulate appetite (eg, insulin; prion protein) and neurotransmitters (eg, serotonin) . However, based on our data, it is reasonable to conclude that the effects observed on food consumption can be mediated by altering the composition of the n-6 and n-3 fatty acids in the phospholipid membrane of the brain, thus changing its ΝΑΕ- And MAG-fatty acid levels. Endogenous NAEs and MAGs act via cannabinoid receptors (CBl). Indeed 86783 -76- 200412942 Li: Adding 20: 4η-6 ΝΑΕ leads to overeating. The effect of dietary DHA on food introduction has not been previously studied, and the association with reduced food introduction has not been expected. Other possibilities for the effects of dietary fatty acids on food introduction need to be assessed, such as the response of prion protein, insulin and other hormones and neurotransmitters (such as sleep deprivation) to food consumption after stimulation the study. The levels of 20: 4n-6 ΝΑΕ and 20: 4n-6 MAG reported in decapitated rats are very similar to those recently reported by Kirkham et al. (2001). With these similarities, it is reasonable to conclude that, compared to the standard GC / MS method, the newly developed method for quantitative large brain MAGs and NAEs is a viable alternative. Conclusion: We first demonstrated that dietary n-6 and n-3 fatty acids may affect the introduction of food by forming specific n-6 and n-3 NAEs and MAGs. Further research is needed to more clearly determine how fatty acids in the diet mediate appetite regulation by the central nervous system. 86783 77-

Claims (1)

4, 4, 10. 10. 11. Application scope: A method for reducing appetite in mammals, which includes administering a long-chain PUFA into the mammalian body, and the PUFA effectively reduces the appetite in the mammal. For example, the method of claiming the scope of the patent application is cleared, wherein the long chain includes DHA with puFA. The method of the second patent in m, wherein the investment of the long bond n_3 pUFA is independent of A A. ~ The method of claiming the scope of patent application, wherein the long-chain puFA is administered before or at the same time as the stimulation of food anger. ★ The method claimed in item VII of the patent, wherein the long bond M puFA is administered to an infant at a content of about 8 to about 396 mg / kg body weight per day. For example, the method in the patent application range, wherein the long-chain n_3 PUFA is administered to a child or an adult at a level of about 84 to about 15,832 mg per day. A method for feeding an appetite of a mammal, which comprises administering a relative content of the mammalian appetite to the mammal, a long bond of n-3 PUFA and a long chain of n-6 pUFA of a certain content. For example, the method of claim 7 in which the long-chain HP leg includes DHA and the long-chain n-6 PLJFa includes ΑΑ. For example, the method of claim 7 in which the long-chain WA is administered before or at the same time as the appetite impact stimulation. The method of claim 7 in which the long-chain M PUPA is administered to an infant at a level of about 8 to about 396 mg / kg body weight per day. For example, the method of claim 7 in which the long-chain W puFA is administered to a child or an adult at a level of about 84 to about 15,832 mg per day. 86783 12. A method for antagonizing the CB! Receptor in the mammal's brain, comprising administering a certain amount of long-chain n_3 PUFA to the mammal, the content of the PUFA can effectively antagonize the CB! Receptor in the mammal's brain active. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. The method of claim 12 in which the long-chain n_3 PUFA includes DHA. For example, the method of claim 12 in which the long-chain n-3 PUFA is administered independently of AA. For example, the method of claim 12 in which the long-chain n-3 PUFA is administered before or at the same time as an appetite stimulus. For example, the method of claim 12 in which the long-chain n-3 PUFA is administered to an infant at a content of about 8 to about 396 mg / kg body weight per Θ. For example, the method of claim 12 in which the long-chain n-3 PUFA is administered to a child or an adult at a content of about 84 to about 15,32 mg per day. A method for reducing the occurrence of obesity or overweight in a mammal, which comprises administering a certain amount of long-chain n_3 PUFA to at least some members of the animal, and the content of the PUFA can effectively negatively regulate the mammal's appetite. For example, the method of claim 18 in the patent scope, wherein the long-chain n-3 PUFA includes DHA. I The method of claim 18 in the patent scope, wherein the investment of the long-chain n-3 PUFA is independent of A A. For example, the method of claim 18, wherein the long-chain n-3 PUFA is administered before or at the same time as the growth stimulus. The method of claim 18, wherein the long-chain n-3 PUFA is administered to an infant at a content of about 8 to about 396 mg / kg body weight per day. For example, the method of claim 18, wherein the long-chain n-3 PUFA is administered to a child or an adult at a level of about 84 to about 15,32 mg. 86783 200412942 柒 Designated representative map: (1) The designated representative map in this case is: (). (2) Brief description of the element representative symbols in this representative figure: 捌 If there is a chemical formula in this case, please disclose the chemical formula that can best show the characteristics of the invention: 86783.doc