CN1717184A - Methods and compositions for providing glutamine - Google Patents

Abstract

Methods and compositions for providing glutamine supplementation to a human by orally administering an effective amount of N-acetyl-L-glutamine or a nutritionally acceptable salt thereof. The N-acetyl L-glutamine or a nutritionally acceptable salt thereof can be incorporated into any liquid composition that is suitable for human consumption. Examples of suitable compositions include aqueous solutions such as for use as oral rehydration solutions and liquid nutritional formulas (including enteral formulas, oral formulas, formulas for adults, formulas for children and formulas for infants). The quantity of N-acetyl-L-glutamine or nutritionally acceptable salt thereof can vary widely but typically, these compositions will contain sufficient N-acetyl-L-glutamine or a nutritionally acceptable salt thereof to provide at least 140 mg of total glutamine per kg of body weight per day.

Description

Be used to provide the method and composition of glutamine

Invention field

The present invention relates to the method that acceptable salt provides glutamine to replenish in N-acetyl group-L-glutamine by oral effective dose or its nutrition.

Background technology

Glutamine is rich in amino acid in the human body.The free amino acid of its occupy-place in skeletal muscle more than 60% and to account for global cycle amino acid whose more than 20%.Glutamine involves a lot of body functions, comprises that gluconeogenesis, nucleotides are synthetic, acid-base balance and other crucial metabolic process.Studies show that glutamine is to duplicate various cells especially intestines and stomach and the employed important metabolism substrate of mucomembranous cell rapidly.Glutamine in vivo can effectively be absorbed in the jejunum (part of small intestine) the people.

Because in fact glutamine can obtain by the institute of body is synthetic in a organized way, so it is not considered to essential amino acid.It is believed that when body is in normal physiological status it can produce the glutamine that is enough to keep body aequum (just consuming the tissue of glutamine).Yet numerous studies show that, under unusual physiological status (just disease and metabolism difficulty), the production of glutamine becomes and is not enough to satisfy the needs of body.Therefore, can more properly regard glutamine as a kind of essential amino acid with good conditionsi.For example, multinomial research is referred to glutamine in the situation of damage of intestines.Souba, W.W.; Smith, R.J.; And Wilmore, D.J.:Glutamine Metabolism by the Intestinal Tract.JPEN 9 (5): 608-617 (1985); Furst, P.; Albers, S and Stehle, P.:Evidence for a nutritional need for glutamine in catabolicpatients.Kidney Intl.36 (Suppl.27): S-287-S-292 (1989); Klimberg, V.S. waits the people: Oral glutamine accelerates healing ofthe small intestine and improves outcome after whole abdominalradiation.Shown that glutamine is the main energy source that is used to cultivate HeLa cell.Reitzer, L.J.; Wice, B.M.; And Kennell, D.:Evidence thatglutamine, not sugar, is the major energy source for culturedHeLa cells.J.Biol.Chem.254 (8): 2669-2676 (1979).In addition, show that also glutamine can preferentially be utilized by tumour cell, cause glutamine quilt loss gradually in the cancer patient.Souba，W.W.：glutamine?and?Cancer.Ann.Surg.218(6)：715-728(1993)。

Past has begun to replenish glutamine in various nutrient formulation food.Replenish and just mean the extra glutamine (can be free amino acid, also can be other form that concentrates relatively, for example gluten after the hydrolysis) of adding in this formula food.As naturally occurring amino acid, glutamine is present in all protein with certain content, thereby it all has certain content in containing any nutrient formulation food of protein.Yet glutamine only contains certain the most naturally occurring protein on a small quantity, therefore in order to prepare the glutamine formula food that content surpasses certain level, must add the glutamine of supplementary form.These replenished in the formula food of glutamine some be the impaired patient of patient, GI function towards those metabolism difficulties (for example the GI function that causes by serious multiple injuries, diarrhoea, inflammatory bowel disease, GI operation impaired, because serious burn or the damage that chemotherapy or radiotherapy cause) on market, have patient's (for example Crohn's disease) of malabsorption symptom and/or acute injury.

Prepare this easiest method that is rich in the supplementing preparation of glutamine and be the hydrolysis by seitan, seitan is characterized in that containing a large amount of glutamine as the mixture of the compound of polypeptide.Because wherein seitan is hydrolyzed fragmented form, so these products are considered to not contain seitan in operation.Yet, use " no seitan " compound that this class is rich in glutamine to have potential danger, this is because seitan is the startup envirment factor of celiaca, has shown that the fractionlet of gliadin has toxic action for the abdominal cavity patient.

Celiaca is a kind of autoimmunity enteropathy, and it is initiated by the cereal that picked-up contains seitan in susceptible individual.Gliadin part in the gluten and the similar protein,alcohol-soluble matter in other cereal are the envirment factors that causes intestinal tract injury.Verified at present, celiaca is that the result that inappropriate T cell is regulated immune response is carried out in picked-up to seitan.This disease is also relevant with the HLA (HLA) in the main histocompatibility complex.And continuing to exist under the condition of seitan, this disease can make self long-term existence.Typical intestinal tract injury is characterised in that absorbability fine hair reduces and the folliculus hyperplasia, and this can be resolved by get rid of the cereal that contains seitan from patient's feed fully.Recently statistics shows, just has nearly 1 may be subjected to this situation and influence in global population in common every 100-300 individuality.The feature of celiaca further also is the complication of a series of obvious obstruction quality of life, and these complication in most cases also can life-threatening, for example mucous membrane lymthoma.

Owing to have above-mentioned medical science advantage, so people take various effort so that glutamine is mixed in the nutrition product.The problem of this effort of frustrating is glutamine stable limited in the aqueous solution.Known free glutamine is degraded in water-bearing media and is formed pyroglutamic acid and glutamic acid.Many studies show that, pyroglutamic acid are the neurotoxins in the rodent body.People such as C.F.deMello: Neurochemical effects of L-pyroglutamic acid.Neurochem.Res.20 (12): 1437-1441 (1995); McGreer, E.G. and Singh, E.:Neurotoxic effects of endogenous materials:quinolinic acid, L-pyroglutamic acid, and thyroid releasinghormone (TRH) .Exp.Neurol.16 (3-4): 410-413 (1984); Rieke, people such as G.K.: L-Pyroglutamate:an alternative neurotoxin for arodent model of Huntington ' s disease.Exp.Neurol.104 (2): 147-154 (1989).When this nutrient formulation food of feed, this degraded had both generated pyroglutamic acid, had also reduced available glutamine content in the body simultaneously.Therefore, free glutamine mainly is confined to the powdery formula food as the purposes in the additional glutamine source in the nutrient source, this powdery formula food can water before on the feed (24-48 hour) rehydration immediately or almost at once, and be preferably in after the rehydration under the freezing condition and store.This class powdery formula food comprises Alitra  (Ross Products Division of AbbottLaboratories), Nu-Immu  (Enjoy Foods) and Vivonex Plus  (Sandoz).These preparations can provide about 25.4,20.1 and 14.5g glutamine/1500 kilocalorie heat (according to analyze) respectively.In addition, people's such as Mawatari European patent application EP 1097646 discloses the purposes of improvement powdered milk composition that contains glutamine and/or contain the peptide of glutamine.Though this series products has important function for patient's health care, powder is thought not to be best by the U.S. most health care department.Owing in a lot of American communities, lack private doctor, so health care department still wishes the nutrient formulation food (RTF) of ready-made feed very much through training.In addition, this class nutrient formulation food must have at least 12 months shelf-life to be accepted by market.Therefore, You Li glutamine is because of limited this class RTF product that is not suitable for of its stability.

The researcher continues to seek the glutamine source that has long-time stability in solution.For example, people such as Guerrant are the United States Patent (USP) 5 of " Stable Glutamine Derivatives forOral and Intravenous Rehydration and Nutrition Therapy " at title, the purposes that alanine-glutamine is played the part of this role is disclosed in 561,111.Generally speaking, people such as Guerrant think can settle the acyl group protecting group on glutamine, but does not provide biological data to prove this opinion.In addition, the document does not provide any relevant guidance that contains the special preparation of the oral of a certain amount of this analog derivative or intravenous compound yet.

As people such as Gandini at " HPLC Determinationof PyroglutamicAcid as a Degradation Product in Parenteral Amino AcidFormulations " Chromatographia, vol.36, pointed among the pp.75-78 (1993), this carelessness is extremely important for the existing formulation problems of this class solution.In this article, the author thinks that in order to solve the problem that glutamine is degraded to pyroglutamic acid, the someone advised the use dipeptides, but problem is to cause like this amino acid content of resulting solution unbalanced qualitatively.The author thinks that also the glutamine derivative is that acetyl group-glutamine has low bioavilability.

It is very relevant with other researcher's of this field work that people such as Gurrant lack biological data.People such as Palmerini give the N-acetyl group-L-glutamine of mouse labelled with radioisotope for oral use." Uptake of Doubly-Labeled N-Acetyl-L-Glutamine inRat Brain and Intestinal Mucosa in Vivo ", Farmaco, vol.36 (7), pp.347-355 (in July, 1981).People such as Palmerini confirm that N-acetyl group L-glutamine (NAQ) strides in the intestines mucous membrane by complete absorption.Because one of primary activity of glutamine is the epithelial cell of nutrition internal organ, so acetyl group functional group will not made those skilled in the art not the source of NAQ as glutamine in the nutrition product by the intestines hydrolysis.This functional group mainly appears in the interior absorption process of amino acid whose intestines.

People such as Magnusson are at " Utilization of IntravenouslyAdministered N-Acetyl-L-Glutamine in Humans " Metabolism; vol.38 (8); suppl.1 (August); N-acetyl group-existing problem of L-glutamine of using in the nutrient formulation food has been discussed among the pp.82-88 (1989), and they find to have discharged the dosage that passes through N-acetyl group-L-glutamine that intravenous gives of 20-40% in urine.People such as Wallace also mentioned the other problem that especially may exist in mouse, they think at last may exist the poor appetite and can not make full use of for example problem of N-acetyl group-(alanine) 2 of acetylizad peptide class.“Uptake?of?acetylated?peptides?from?the?smallintestine?in?sheep?and?their?nutritive?value?inrats”BritishJournal?of?Nutrition，v.80，pp.101-108(1998)。

Summary of the invention

According to the present invention, find that N-acetyl group L-glutamine has the effectiveness as human oral glutamine replenishers.The present inventor finds that the intestinal tissue of human body can utilize the source as N-acetyl group L-glutamine glutamine.Therefore, the plan of N-acetyl group-L-glutamine can being mixed is in the liquid nutritional formula food of human consumption.These compositions have long-term stability, but and can provide N-acetyl group-L-glutamine with the form of human body biological utilisation.This N-acetyl group L-glutamine can give with the form of acceptable salt in acid or its nutrition.According to the early stage achievement in research of finishing on one's body inhuman other animal, above-mentioned discovery is that institute is unforeseeable in advance.

Acceptable salt in N-acetyl group-L-glutamine or its nutrition can be mixed in any one fluid composition that is fit to human consumption.The example of suitable compositions comprises ORS and the liquid nutritional formula food (comprising that intestines formula food, formula of oral food, adult use formula food, pediatric patients formula food and formula food for baby) that aqueous solution is for example oral.The consumption of acceptable salt can random variation in N-acetyl group-L-glutamine or its nutrition, but common described composition should contain acceptable salt in N-acetyl group-L-glutamine of being enough to obtain the total glutamine of 10mg/kg body weight/day at least or its nutrition.

Accompanying drawing is described

Fig. 1 has described the stability of N-acetyl group-L-glutamine under different pH values and environment temperature with diagrammatic form.For the sample of pH5.0 to pH8.0, all values are identical.

Fig. 2 has described after at room temperature preserving the N-acetyl group-L-glutamine aqueous solution 180 days with diagrammatic form, the catabolite that forms in the pH of 2.0-8.0 scope in this solution.

Fig. 3 has described this material of remaining in enteric cavity function as the time add glutamine or N-acetyl group-L-glutamine in the chitling ring that separates after in internal medicine operation described herein test with diagrammatic form.The percentage of the analyte of residual analyte when accounting for time zero is represented.

Fig. 4 has described in internal medicine operation described herein test introducing the glucose that is added behind this material in the chitling ring that separates with diagrammatic form and has remained in amount in the enteric cavity as the function of time.The percentage of the amount of residual glucose when accounting for time zero is represented.

Fig. 5 has described glutamine content (mcg/mL) in the pig loop door blood as the function of time after the administration with diagrammatic form, wherein introduces different material (glucose saline contrast, the dextrose in saline solution of glutamine or the dextrose in saline solution of N-acetyl group-L-glutamine) in the intestines ring of the separation of these pigs.

Fig. 6 has described the content (representing with the mcg/ gram mucous membrane that wets) of glutamine in the chitling jejunal mucous membrane measured after internal medicine operation as herein described test and glutamate with diagrammatic form.

Fig. 7 shows from the health pig of the ENSURE PLUS formula food of having taken food (A, B group) and the electronic transmission micrograph of the enterocyte of the jejunum of the pig of 30 days the EMP of formula food that is supplemented with caseinate (C, D group), glutamine (E, F group) or NAQ (G, H group) of having taken food, and for example cytoplasm gap and lymphocyte phenomenon of osmosis are analyzed clearly to its inflammation signal.

Fig. 8 shows the electronic transmission micrograph of ileum enterocyte of pig that is supplemented with 30 days the EMP of same recipe food of caseinate (C, D group), glutamine (E, F group) or NAQ (G, H group) from the health pig of feed ENSURE PLUS formula food (A, B group) and feed, and for example cytoplasm gap and lymphocyte phenomenon of osmosis are analyzed clearly to its inflammation signal.

Fig. 9 shows different product/compound for the influence that occurs the probability of Apoptosis and inflammation on the celiaca patient's who does not receive treatment mucous membrane.

Figure 10 shows the epithelium TUNEL expression pattern of the mucous membrane sample of accepting N-acetyl-l-glutamine (A) and P5 (B) treatment.

Figure 11 shows the CD25 expression pattern in the chamber, last rim surface zone of the mucous membrane sample of accepting N-acetyl-l-glutamine (A) and P5 (B) treatment.

Describe in detail

The following term that uses in this application has the implication of following appointment:

A) " total glutamine " but refer to total amount from the biological utilisation that is expressed as glutamine in any source or glutamine that can potential utilization. But glutamine and the glutamine source of other biological utilisation, for example N-acetyl group-L-glutamine that it glutamine that provides with free glutamine form may be provided, exist as the glutamine of the part of peptide or whole protein. The accessory substance (such as pyroglutamic acid etc.) that does not comprise glutamine degraded. As the example of this calculating, the following product of describing a kind of supposition.

A kind ofly contain the nutrition product that 60 grams per liters contain the protein system of the slight aminosal of complete sum, it comprises:

I. the free glutamine of 1.1 grams per liters is measured according to method well known to those skilled in the art.

Ii. the mixture of protein that contains the slight hydrolysis of complete sum of 50.0 grams per liter protein, it (for example passes through as people such as Fouques by disclosed method analysis, " Study ofthe Conversion of Asparagine and Glutamine of proteins intoDiaminopropionic and Diaminobutyric Acids Using[Bis (trifluoroacetoxy) iodo] benzene Prior to Amino AcidDetermination. " Analyst, Volume 116, (May), the method of pp 529-531 (1991)), contain 3.4 gram glutamine/100 gram protein.

Iii. the N-of 11.6 grams per liters acetyl group-L-glutamine, it contains the glutamine that 9.0 grams calculate according to following formula:

Therefore, " total glutamine " is the summation in these three kinds of sources, that is: 1.1 gram/L (dissociating)+(3.4g/100g protein * 50g protein/L)+9.0 gram/L (NAQ)=11.8 restrains.

B) " mmole " is meant mM (just 1 mole 1/1000)

C) term " acceptable salt in the nutrition " is meant and is suitable for suitable salt to the acetyl group of the N-in the fluid composition of human oral administration-L-glutamine.Acceptable salt is the salt that substituted by another cation of the hydrogen on the carboxyl wherein in the nutrition of N-acetyl group-L-glutamine.This class salt can in the final separation of N-acetyl group-L-glutamine and purge process, prepare or individually by with carboxyl and suitable alkali for example metal cation hydroxide, carbonate or bicarbonate or prepare with ammoniacal liquor or organic primary amine, secondary amine or reactive tertiary amine.In the nutrition acceptable salt can based on alkali metal or alkaline-earth metal for example lithium, sodium, potassium, calcium, magnesium and aluminium and nontoxic season ammonia and quaternary ammonium cation for example ammonium, tetramethyl-ammonium, tetraethyl ammonium, methylamine, diethylamine, trimethylamine, triethylamine, diethylamine, ethamine, tri-n-butylamine, pyridine, N, accelerine, N-methyl piperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzyl amine, N, N-dibenzyl phenyl amine, 1-Chinese ephedra amine (1-ephenamine) and N, N '-dibenzyl-ethylenediamin.Other the representational organic amine that can be used for forming alkali formula addition salts comprises ethylenediamine, monoethanolamine, diethanol amine, piperidines and piperazine.

D) place that relates to the electrolyte consumption in specification or claims should be interpreted as referring to the ultimate density of electrolyte in oral ORS.Running water contains residual sodium, chlorine etc. usually.In this application, the sodium value is that 40mEq is meant that the whole sodium that are present in the oral ORS equal 40mEq, the existing sodium of water that comprises the sodium of adding and be used for preparing oral ORS.

E) place that relates to digital scope among the application should be understood that it is modified by adjective " approximately ".In addition, any digital scope all should be considered to provide support to the claim that relates to this scope subclass.For example, the scope that discloses 1-10 should be considered to give in specification and claims the random subset in this scope that support (just scope 2-9,3-6,4-5,2.2-3.6,2.1-9.9 etc.) is provided.

The invention provides the method and composition that acceptable salt provides glutamine to replenish to the people in N-acetyl group-L-glutamine by oral effective dose or its nutrition.N-acetyl group-L-the glutamine that is suitable in this nutrient formulation food can utilize well-known conventional chemical synthesizing mean preparation; for example in the presence of suitable base catalyst (for example pyridine), hatch free L-glutamine with acetic anhydride; then synthesize; carry out suitable purifying by recrystallization, like this pure compound of the food-grade that can obtain suiting.In fact, the chemical company of much being proficient in chemistry of amino acids can provide the N-acetyl group-L-glutamine of food-grade (KyowaHakko Kogyo Co for example, Ltd., Tokyo, Japan or Flamma, s.p.a., Italy).Perhaps, also can utilize the suitable food-grade N-acetyl group-L-glutamine of other method (for example microbial fermentation, referring to JP 51038796, JP57001994, JP 57016796) preparation.Acceptable salt is the salt that the hydrogen on its carboxyl is substituted by another cation in the nutrition of N-acetyl group-L-glutamine.This class salt can in the final separation of N-acetyl group-L-glutamine and purge process, prepare or individually by with carboxyl and suitable alkali for example metal cation hydroxide, carbonate or bicarbonate or prepare with ammoniacal liquor or organic primary amine, secondary amine or reactive tertiary amine.In the nutrition acceptable salt can based on alkali metal or alkaline-earth metal for example lithium, sodium, potassium, calcium, magnesium and aluminium and nontoxic season ammonia and quaternary ammonium cation for example ammonium, tetramethyl-ammonium, tetraethyl ammonium, methylamine, diethylamine, trimethylamine, triethylamine, diethylamine, ethamine, tri-n-butylamine, pyridine, N, accelerine, N-methyl piperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzyl amine, N, N-dibenzyl phenyl amine, 1-Chinese ephedra amine (1-ephenamine) and N, N '-dibenzyl-ethylenediamin.Other the representational organic amine that can be used for forming alkali formula addition salts comprises ethylenediamine, monoethanolamine, diethanol amine, piperidines and piperazine.If necessary, can use suitable pharmaceutical grade N-acetyl group-glutamine from Sigma.

Comprise acceptable salt in the N-acetyl group-glutamine of oral effective dose or its nutrition to the method that the people provides glutamine to replenish.Usually, N-acetyl group-L-glutamine can be taken with the form of liquid, for example as the part of oral ORS, sports drink or intestines formula food.

Acceptable salt is preferably in the N-acetyl group-glutamine of effective dose or its nutrition is enough to provide the total glutamine of about 10-50g/sky or at least approximately amount, the amount of the total glutamine of 250mg/kg body weight/day (mg/kg/ days) at least more preferably of the total glutamine of 140mg/kg body weight/day.Total glutamine that N-acetyl group-L-glutamine can be consumed by the patient from about 1-100% every day substantially provides, preferably by from about 10-95%, more preferably approximately total glutamine of consuming substantially every day of the patient of 75-90% provides.

If acceptable salt is as unique source that the glutamine that consumes for the patient is provided in N-acetyl group-L-glutamine or its nutrition, the preferred amounts of acceptable salt is preferably about at least 0.7 mM/kg/ days in N-acetyl group-L-glutamine or its nutrition so.More preferably, acceptable salt can be about at least 1.0 mMs/kg/ days in the N-of effective dose acetyl group-L-glutamine or its nutrition.Especially more preferably the N-of effective dose acetyl group-L-glutamine can be about at least 1.5 mMs/kg/ days.

As mentioned above, total change for the change of consumption that acceptable salt in the needed N-acetyl group of glutamine-L-glutamine of 250mg/kg/ days or its nutrition is provided with the glutamine content in all other protein components that the patient consumed.As a total principle, in order fully to obtain benefit of the present invention, the patient should consume about at least 0.7 to the N-acetyl group-L-glutamine of about 4.0 mMs or its nutrition acceptable salt/kg/ days.Lower consumption may also be useful, and this depends on total glutamine content of other component in the protein system.Generally speaking, should provide enough N-acetyl group-L-glutamine to the patient, its delivering amount is total glutamine/kg body weight/day of about at least 140mg, more preferably about at least total glutamine of 250mg/kg body weight/day.

Can take method to provide glutamine to replenish to adult, children and baby.Term child is meant that the age is 1 years old extremely about 16 years old (being adulthood) people.The term baby is meant and comprises institute's has age less than 1 years old people, also comprises precocious baby and slight preemie.The precocious baby of term is meant the babies that are less than 2500 grams before 37 weeks of pregnancy when being born and/or being born, the baby of the 23-28 week birth that the slight preemie of term is meant in pregnancy.Term as used herein minor comprises children and baby.

Give the concentration of the glutamine equivalent of adult, children and baby's feed can be different.One of its reason is that there are great changes in the demand to heat density under different ambient stresses.The example of this situation is to produce when the intestines nutrition that the patient who for example suffers from severe trauma or precocious baby can only bear minimum volume.In this class situation, the mode of most of nutrition feed outside at first can be by enteron aisle provides.In these cases, indivisible intestines nutrition is acceptable, and this is good for supply glutamine equivalent as much as possible.Therefore, can use acceptable salt in the N-acetyl group-glutamine of high concentration or its nutrition.In Another Application, in order to support organ function, replenish acceptable salt in N-acetyl group-glutamine or its nutrition can for standard infant formula food, can use lower in fact concentration in this case.For glutamine wherein is favourable any situation, can use N-acetyl group-L-glutamine.This class situation comprises at least: promote from gastrointestinal surgery, intestines and stomach resection, small intestine transplantation recover and the wound of other postoperative recover, hungry, acute illness and for example multiple wound of damage, short bowel syndrome, burn, bone-marrow transplantation, AIDS, canker sore, celiaca, Crohn's disease, NEC, internal organs precocity, and prevention or reduce and infect for example pyemic order of severity at random.Glutamine replenish also help the prevention internal organs relevant with some particular treatment (for example chemotherapy or radiotherapy) degenerate or in some oral feed is subjected to the situation of serious restriction (for example extremely precocity) also useful.Also comprise the combination of top described situation in addition.

N-acetyl group of the present invention-L-glutamine can use the liquid solution administration that is suitable for human consumption arbitrarily.For example, can simply N-acetyl group-L-glutamine be dissolved in the water.If necessary, it can be mixed in the process beverage of seasoning to improve its palatability.For example, it can be mixed Kool-Aid or soda water for example in Pepsi or the cola.In preferred embodiments, N-acetyl group-L-glutamine can be mixed sports drink for example among the Gator-Aid.

Yet N-acetyl group-L-glutamine can carry out administration by oral ORS (ORS) or liquid nutritional formula food usually.Mix aqueous solution for example among the ORS the N-acetyl group-L-glutamine consumption can random variation.Usually ORS can contain acceptable salt in N-acetyl group-L-glutamine of at least about 5.0mmole or its nutrition/rise solution, also contains a spot of water, glucose and sodium in addition.More preferably, ORS can contain acceptable salt in about 20 to the about 300mmole/ N-acetyl group-L-glutamine that rise or its nutrition, and more preferably about 25 to about 200mmole.If use liquid for example Kool-Aid or Gator-Aid, then the consumption of N-acetyl group-L-glutamine should be with suitable at the described consumption of ORS.

Oral ORS is well-known in the art.The ORS that is used for the present invention should contain FDA usually for desired all electrolyte that meet its concentration of oral rehydrating formulation of selling in the U.S..Except sodium (Na ⁺), potassium (K ⁺), chlorine (Cl ^-) and citrate ions outside, this oral ORS also contains carbohydrate source, for example glucose, fructose and dextrose.Be typically, this ORS contains water, carbohydrate, sodium ion, potassium ion, chlorion and citrate ions.

Such as is known to persons skilled in the art, the sodium ion amount of using in ORS can random variation.Usually, ORS should contain the sodium of about 30mEq/L to about 95mEq/L.In preferred embodiments, sodium content can be for approximately 30mEq/L be to about 70mEq/L, and more preferably about 40mEq/L is 60mEq/L extremely approximately.Suitable sodium source comprise but be not restricted to sodium chloride, natrium citricum, sodium acid carbonate, sodium carbonate, NaOH, and composition thereof.The employed milliequivalent of this paper (mEq) is meant the ion populations in the solution, and it is measured by the ion concentration in given volume.Measured value is represented with the number of milliequivalent/liter (mEq/L).Use the compound of this numerical value then by the atomic weight that mEq be multiply by inorganic ions, milliequivalent can be converted into milligram divided by inorganic ions.

ORS also should contain the potassium ion source.The consumption of potassium can random variation.Then, as a total principle, ORS should contain the potassium of about 10mEq/L to about 30mEq/L usually.In preferred embodiments, they contain the potassium of about 15mEq/L to about 25mEq/L.Suitable potassium source comprise but be not restricted to potassium citrate, potassium chloride, saleratus, potash, potassium hydroxide, and composition thereof.

ORS also should contain carbohydrate source.As mentioned above, the consumption of used carbohydrate is extremely important.Its consumption must remain on less than about 3%w/w, is more preferably less than about 2.5%w/w.The consumption that is between the extremely about 2.0%w/w of about 3%w/w suits.Excessive carbohydrate can aggravate liquid and the electrolyte solution loss relevant with diarrhoea.

Can use in the prior art employed any carbohydrate in oral ORS.Suitable carbohydrate comprise but be not restricted to simple and complicated carbohydrate, glucose, dextrose, fructose oligosaccharides, fructose and glucose polymer, corn syrup, high-fructose corn syrup, sucrose, maltodextrin, and composition thereof.

ORS also should comprise the alkali source that is used to replace the diarrhoea loss usually.Usually in order to obtain The above results, citrate should be mixed in the oral ORS.Citrate is metabolised to the bicarbonate of equivalent, and it helps to keep acid-base balance.Although citrate is preferred alkali source, also can use any alkali that is impregnated in usually in the ORS.

Such as is known to persons skilled in the art, the consumption of citrate can be different.Usually, citrate is extremely approximately 40mEq/L of about 10mEq/L, and more preferably approximately 20mEq/L most preferably is extremely approximately 35mEq/L of about 25mEq/L to about 40mEq/L.Suitable citrate source comprises but is not restricted to potassium citrate, natrium citricum, citric acid and composition thereof.

ORS also should contain the chloride source usually.Such as is known to persons skilled in the art, muriatic consumption can be different.Usually, it is the chloride of about 30mEq/L to about 80mEq/L that ORS should contain consumption, and more preferably approximately 30mEq/L most preferably is about 30mEq/L to about 70mEq/L to about 75mEq/L.Suitable chloride source comprises but is not restricted to sodium chloride, potassium chloride and composition thereof.

Randomly, stodgy oligosaccharides can be closed and mix among the ORS.Stodgy oligosaccharides brings useful influence can for the microorganism species in the intestines and stomach.They help to suppress for example growth of clostridium difficile of pathogenic organism.These oligosaccharides have optionally promoted the growth of non-pathogenic microorganism species.The oral ORS of this class has been described in the United States Patent (USP) 5,733,759 that is registered in April 5 nineteen ninety-five, once more its content is incorporated herein by reference.Usually, oligosaccharides should be fructose oligosaccharides, inulin for example raftilose or wood sugar oligosaccharides.Its consumption can random variation, can be 1-100 grams per liter aqueous solution, more preferably 3-30 grams per liter aqueous solution.

ORS also should comprise the flavor enhancement that is used to improve its palatability usually, especially for pediatric patients.Flavor enhancement can be covered the saline taste of this aqueous solution.Spendable flavor enhancement comprises but is not restricted to peach, butter peach, cowberry, banana, cherry, tangerine, grape, fruit juice, bubble gum, apple, raspberry and strawberry.Can add artificial sweetener to replenish taste and to cover saline taste.Spendable artificial sweetener comprises that asccharin, nutrition are sweet, asccharin glycosides, acesulfane-K (ace-K) etc.

In order to help to prolong storage period, can add anticorrisive agent.This area professional can select the suitable anticorrisive agent of suitable consumption to realize above-mentioned purpose.Typical preservatives comprises but is not restricted to potassium sorbate and Sodium Benzoate.

Except above-mentioned carbohydrate, ORS can also contain ground rice, perhaps help diarrhea treatment rice in other composition.The a variety of oral ORSs that are supplemented with rice have been described in the existing document.The method of using this class to be supplemented with the oral ORS of rice is that those skilled in the art institute is well-known.The example that this class is supplemented with the oral complex aqueous solution of rice comprises the solution that is described in the U.S. Patent number 5,489,440 that is published on February 6th, 1996.

ORS can adopt the well-known technology of those skilled in the art to prepare.As a total principle, all component dryings can be mixed; Dispersed with stirring is in water; Randomly be heated to suitable temperature then to dissolve all components.Then this ORS is encapsulated sterilization to food-grade standard known in the art.

Such as is known to persons skilled in the art, ORS can be with different formulation administrations, and this depends on patient's preference.For example, if freezing for example with the form of ice lolly, this oral ORS of the easier feed of possible some children.Oral ORS ice lolly is described in detail in U.S. Patent number 5,869, in 459.In order to improve the especially biddability of pediatric patients of patient, oral ORS can also be made the form of gel.The rehydration composition of gelinite is described in the U.S. Patent Application Serial Number 09/368,388 that is registered on August 4th, 1999.This gel also is described in the PCT application number 99/15862.Generally speaking, but aqueous solution can be made the form of flow-gel.Perhaps, also can be made into the gel structure that can oneself support.By suitable gelling agent is mixed in this aqueous solution, can realize above-mentioned purpose.

The suitable gelling agent that is used for this aqueous solution comprises but is not restricted to agar, alginic acid and salt, gluing, gum arabic, tower is breathed out glue, cellulose derivative, curdlan, fermentation natural gum, furcellaran glue, gelatin, gellan gum, Indian gum, guar gum, the A Outa carrageenan, the Ai Er liver moss, card handkerchief carrageenan, konjaku flour, karaya, lime reaches carrageenan, pine glue/arabogalactan, locust bean gum, pectin, tamarind gum, tower draws bean gum, bassora gum, natural and modified starch, xanthans and composition thereof.The usage ratio of described gelling agent is about 0.05 to about 50 w/w %.

As mentioned above, acceptable salt can be by the form administration of liquid nutritional product in N-acetyl group-L-glutamine or its nutrition.N-acetyl group-the glutamine that mixes in the liquid nutritional formula food can random variation, but should meet above-mentioned dosage guide.The consumption that is used for acceptable salt in the N-acetyl group-L-glutamine of liquid nutritional formula food or its nutrition should depend on various factors, comprises whether said preparation provides the amount of most of or main nutrient source, glutamine source that whether said preparation contains other, the formula food that consumes every day and patient's type (its amount of formulation that consumed every day is also influential) of planning to use said preparation.When being included in glutamine in other protein component and merging, in order to obtain total glutamine/kg body weight/day of 140mg at least, this formula food preferably contains acceptable salt in the N-acetyl group-L-glutamine of q.s or its nutrition.The percentage of the protein heat form that provides can also be provided the consumption of acceptable salt in N-acetyl group-L-glutamine or its nutrition.According to this expression way, nutrient formulation food can contain acceptable salt in the N-acetyl group-L-glutamine of about 1 to about 100% protein heat or its nutrition.This percentage is to calculate according to the protein portion of acceptable salt in N-acetyl group-L-glutamine or its nutrition (glutamine part just), does not consider that any heat of being contributed by the part of the nonprotein in the acceptable salt in N-acetyl group-glutamine or its nutrition (just acetic acid esters or salt part) is interior.Preferably, use nutrient formulation food, can contain acceptable salt in N-acetyl group-L-glutamine of being enough to provide about 10 to about 95% protein heat or its nutrition for the adult.If this nutrient formulation food is intended for use pupillary words, N-acetyl group-L-glutamine should exist with the consumption that is enough to provide about 1 to about 12% protein heat so.

The liquid nutritional formula food comprises that Enteral formulations, formula of oral food, adult use formula food, pediatric patients formula food and formula food for baby.For example, intestines formula food and nutrient formulation food are the piths of patient's health care in acute health care institute and long-term care place (being sanatorium).Normally be used to improve the below standard state of nutrition although replenish these formula foods, they also can be used as the long-term unique source of nutrition that uses of ordinary person.Therefore, if this class formula food is in order to satisfy the underfed main purpose of prevention, they must contain the protein, fat, inorganic elements, electrolyte of q.s etc. so.These formula foods are administered to the patient with the form of liquid usually, and this is food because most target patient can not be taken food.Although some patient can the drink-service preparation, also there are a lot of patients to take Enteral formulations by snuffing pipe (NG pipe or feed pipe).

The liquid nutritional formula food contains the protein component that the 14-35% that accounts for the total heat content of this formula food is provided, the lipid composition that the carbohydrate ingredient of the 36-76% that accounts for total heat content is provided and the 6-51% that accounts for total heat content is provided.The liquid nutritional formula food can be adult's formula food, paediatrics formula food or infant formula (can be to adult, pediatric patients or baby use as this aqueous solution).Use for high glutamine, preferred liquid nutrient formulation food can provide most of at least source of nutrition.Certainly, liquid nutritional formula food as herein described also can not be the most of at least source as nutrition, particularly in the situation that those most of non-enteral formula food are not practice standard (in considerable precocious baby, they slowly take off milk with oral feed in the several weeks the earliest that break away from the uterus).At least most of source of term nutrition is meant plans the formula food of feed to be enough to provide the required total amount of heat of the patient that takes this formula food and the amount feed of half at least of nutrition.Falling in this definition is formula food and eating method thereof as the unique source of nutrition, by the patient who takes this formula food is provided required whole total amount of heats and nutrition like this.The amount of institute's calorific requirement and nutrition can be with the patient different and different, this depends on various variablees for example age, body weight and physical condition.Keeping the consumption of the required nutrient formulation food of the heat of appropriate amount and nutrition can be determined by those of ordinary skills, as the heat and nutrition that will mix appropriate amount in this class formula food.For instance, if this formula food for adult use formula food, its protein component can contain described liquid nutritional formula food total heat content about 14 to about 35%; Carbohydrate ingredient can contain described liquid nutritional formula food total heat content about 36 to about 76%; Lipid composition can contain described liquid nutritional formula food total heat content about 6 to about 41%.This nutrient formulation food can be the formula food that is used for the formula food of oral feed or is used for the enteron aisle feed.Give one example again, if said preparation is used preparation for the minor, its protein component can contain described liquid nutritional formula food total heat content about 8 to about 25%; Carbohydrate ingredient can contain described liquid nutritional formula food total heat content about 39 to about 44%; Lipid composition can contain described liquid nutritional formula food total heat content about 45 to about 51%.These scopes are only used for illustrating, and do not mean that and be construed as limiting.

Because odjective cause, this series products should contain acceptable salt in the N-acetyl group-L-glutamine of the only about half of or more consumption that is enough to provide total glutamine content or its nutrition.Perhaps, also the effective dose of acceptable salt in N-acetyl group-L-glutamine or its nutrition can be expressed as the form of mM/1000 kilocalorie.According to this expression way; if the target consumption of glutamine is about 300mg glutamine every day/kg/ days; so for the adult; nutrient formulation food should preferably contain acceptable salt/1000 kilocalorie nutrient formulation food in the N-acetyl group-L-glutamine of about at least 35 mMs or its nutrition; for children, baby or precocious baby (minor), should contain acceptable salt/1000 kilocalorie nutrient formulation food in the N-acetyl group-L-glutamine of about at least 5.0 mMs or its nutrition.More preferably; this class preparation for the adult, should contain about 35 to the N-acetyl group-L-glutamine of about 160 mMs or its nutrition acceptable salt/1000 kilocalorie nutrient formulation food; for children, should contain about 5.0 to the N-acetyl group-L-glutamine of about 32 mMs or its nutrition acceptable salt/1000 kilocalorie nutrient formulation food, for baby or precocious baby, contain about 5.0 to the N-acetyl group-L-glutamine of about 26 mMs or its nutrition acceptable salt/1000 nutrient formulation food.

Except N-acetyl group-glutamine, it is suitable carbohydrate, lipid and the protein of known preparation nutrient formulation food for a person skilled in the art that this nutrient formulation food also should contain.Suitable carbohydrate comprises but is not restricted to the starch of after the hydrolysis that derives from corn, cassava, rice or potato of wax shape or non-waxy form, complete, natural and/or chemical modification; Carbohydrate is glucose, lactose, sucrose, maltose, the corn syrup that is rich in fructose, corn syrup solids and their mixture for example.Maltodextrin is to carry out the polysaccharide that obtains after acid or the enzyme hydrolysis by the starch starch of corn or rice (for example from).Degree according to hydrolysis is carried out classification to it, represents with dextrose equivalent (DE).The DE of used any maltodextrin is preferably and is lower than about 18-20 in this nutrient formulation food.

Suitable lipid comprise but be not restricted to coconut oil, soybean oil, corn oil, olive oil, safflower oil, rich oil safflower oil, MCT oil (median chain triglyceride oil), sunflower oil, rich oil sunflower oil, palm oil, palm olein, Tower rape oil, fish oil, palm-kernel oil, pilchardine, soya-bean oil, cottonseed oil, lecithin, arachidonic acid and DHA the lipid source, and composition thereof.The lipid source of arachidonic acid and DHA comprises but is not limited only to marine flora and fauna oil (Marine oil), egg oil and fungi or algal oil.The fat of a lot of merchandise resourceses is that those skilled in the art are known and can easily obtain.For example, soybean and cupu oil can be by Archer Daniels Midlandof Decatur, and Illinois obtains.Corn, coconut, palm and palm-kernel oil can be by Portland, and the Premier Edible Oils Corporation of Organ obtains.Coconut oil after the fractionation can be by LaGrange, and the Henkel Corporation of Illinois obtains.Rich oil safflower oil and rich oil sunflower oil can be by Eastlake, and the SVO Specialty Products of Ohio obtains.Marine flora and fauna oil can be obtained by the Mochida International of Tokyo.Olive oil can be obtained by the Oils of North Humberside of the U.S..Sunflower and cottonseed oil can be obtained by the Cargil of Minneapolis.Safflower oil can be obtained by the California Oils Corporation of the Richmond in California.

Except the oils of these food-grade, can also mix structured lipid if necessary.Structured lipid is known in the art.The concise and to the point description of relevant structured lipid can be referring to INFORM, Vol..8, and no.10, page 1004, and title is Structured lipids allow fattailoring (in October, 1997).In addition also can be referring to U.S. Patent number 4,871,768.Structured lipid preferably contains the triacylglycerol of medium chain and long-chain fat acid blend on identical glycerol molecule.Structured lipid and the purposes in Enteral formulations thereof also are described in U.S. Patent number 6,194, in 37 and 6,160,007.

Suitable protein source comprises but is not restricted to milk, whey and newborn cut, soybean, rice, meat (for example beef), animals and plants (for example pea, potato), egg (egg is white), gelatin and fish.Suitable native protein source comprise but be not restricted to soybean substrate, milk matrix, casein, lactalbumin, rice protein, beef collagen, pea protein, potato protein, and composition thereof.Suitable protein hydrolysate comprises but is not restricted to soybean protein hydrolysate, caseic hydrolysate, lactalbumin hydrolysate, rice protein hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg plain boiled water hydrolysis products, glutin hydrolysate, the combination of animal/vegetable protein hydrolysate and their mixture.Protein after the hydrolysis (protein hydrolysate) is meant hydrolysis or fragments into shorter peptide fragment and amino acid whose protein.This class range of hydrolysed peptides fragment and the easier digestion of free amino acid.Say that in a broad sense protein just is hydrolyzed after one or more amido link fractures.The fracture of amido link can be for example owing to heating or shear and have a mind to or by mistake occur in the manufacture process.For purposes of this disclosure, the protein of hydrolysis is meant the protein through processing or handled according to the mode of fracture amido link.Can on purpose be hydrolyzed, for example pass through with enzyme or acid treatment native protein.The aminosal that is preferred in this liquid nutritional formula food as herein described is the protein that is hydrolyzed into following degree, wherein the ratio of ammonia nitrogen (AN) and total nitrogen be about 0.1AN than about 1.0TN to about 0.4AN than about 1.0TN, be preferably about 0.25AN than about 1.0TN to about 0.4AN than about 1.0TN.(AN: TN ratio is meant independent protein hydrolysate, does not represent the AN in the final nutrient formulation food: TN ratio).

Protein can also provide with the form of free amino acid.In order to obtain nutritionally more complete and formula food balance, replenish each seed amino acid can for nutrient formulation food.The example of suitable free amino acid comprises but is not restricted to be considered to usually all the free L-amino acid of the part in the protein system, but comprise that especially those are considered to must or be the amino acid of essential amino acid under certain condition from the consideration of nutrition angle, just: tryptophan, tyrosine, serine, methionine, arginine, leucine, valine, lysine, phenylalanine, isoleucine, threonine and histidine.Usually other (nonprotein) amino acid that adds in nutritional product comprises carnitine and tarine.In some cases, amino acid whose D-form be considered to the L-form be suitable on nutrition, can use isomer mixture (for example D, L-methionine) in order to reduce cost.

This nutrient formulation food preferably also contains vitamin and the inorganic elements that consumption is enough to keep patient's daily nutrition needs of taking this formula food.Those skilled in the art considered that nutrient formulation food need strengthen some vitamin and inorganic elements usually and can satisfy daily nutritional need to guarantee them at the product lay up period.Those skilled in the art also think, consider disease that some are potential or the disease that the patient suffered from, and some microcomponent also has potential benefit for the crowd.For example, for example chromium, carnitine, taurine and vitamin E for beneficial for diabetics.Formula food preferably includes but is not restricted to following vitamin and inorganic elements: calcium, phosphorus, sodium, chlorine, magnesium, manganese, iron, copper, zinc, selenium, iodine, chromium, molybdenum, inositol, carnitine, taurine, vitamin A, C, D, E, K and B complex compound and their mixture.

If this nutrient formulation food is intended for use the baby, so concrete nutritional guidelines can be referring to Infant Formula Act, 21 U.S.C.section 350 (a).Nutritional guidelines in this decree are done along with the further research of relevant nutritional requirement and constantly revise.In the claim claimed nutrient formulation food mean comprise contain those not at the formula food of this vitamin listed and inorganic elements interior.

This liquid nutritional formula food can also contain fiber and stabilizing agent.Suitable fiber and/or stabilizing agent source comprise but are not restricted to xanthans; guar gum; gum arabic; karaya; bassora gum; agar; furcellaran glue; gellan gum; locust bean gum; pectin; high and the low pectin of methoxyl content; oat beta-glucan; carrageenan; psyllium; gelatin; microcrystalline cellulose; CMC (sodium carboxymethylcellulose); methylcellulose; hydroxypropyl methylcellulose; hydroxypropyl cellulose; DATEM (the diacetyl tartrate of monoglyceride and diglyceride); dextran; carrageenan; FOS (fructose oligosaccharides); and their mixture.The solubility edible fibers of a lot of merchandise resourceses is available.For example gum arabic, hydrolyzed carboxymethylcellulo, e, melon that natural gum, pectin and methoxyl content height and low pectin can be by the TIC Gums of Maryland Belcamp, and Inc. obtains.Oat beta-glucan can be by the Mountain Lake Specialty Ingredients of Omaha, the Nebraska State, and Inc. obtains.Psyllium can be obtained by the Meer Corporation of the North Bergen of New Jersey, and carrageenan can be obtained by the FMC Corporation of philadelphia, pa.

The fiber that mixes can also be the edible fibers of solubility, and its representative example comprises oat fruit fiber, pea fiber, fibre and soya, cotyledon fiber, sugar beet fiber, cellulose and corn bran.A lot of sources of the edible fibers of solubility also are available.For example.Corn bran can be obtained by the Quaker Oats in Chicago, Illinois; The oat fruit fiber can be obtained by the Canadian Harvest in Cambridge, the Minnesota State; The pea fiber can be obtained by the Woodstone Foods in lake, Canadian Winnipeg; Fibre and soya can be obtained by the Fibrad Group of Maryland LaVale; Cotyledon fiber can be obtained by the Protein Technologies International of Missouri State St.Louis; Sugar beet fiber can be obtained by the Delta Fiber Foods of Minnesota State Minneapolis; Cellulose can be obtained by the James River Corp. of the Saddle Brook of New Jersey.

About fiber and can be referring to people's such as Garleb U.S. Patent number 5,085,883 with its going through of example of mixing in the formula food.

The fiber consumption that is used for this formula food can be different.The fiber of employed particular type is unimportant.Can use any suitable human consumption and in the nutrient formulation food substrate, keep stable fiber.

Except fiber, this nutrient formulation food can also contain compound sugar for example fructose oligosaccharides (FOS) or glucose compound sugar (GOS).Compound sugar is carried out fermenting fast and fully to shorten fatty acid chain by the anerobe that suppresses large intestine.These compound sugar are preferred energy sources of most of Bifidobacterium classes, but for example C.perfringens, difficult sour bacterium or E.coli. do not utilize by pathogenic organisms.

This liquid nutritional formula food can also contain aromatic to improve its palatability.Spendable aromatic comprises but is not restricted to chocolate, vanilla, coffee, peach, butter peach, cowberry, banana, cherry, tangerine, grape, fruit juice, bubble gum, apple, raspberry and strawberry.Can add artificial sweetener to replenish fragrance and to cover saline taste.Spendable artificial sweetener comprises that asccharin, nutrition are sweet, asccharin glycosides, acesulfane-K (ace-K) etc.

The liquid nutritional formula food can adopt the well-known method preparation of those skilled in the art.Known various preparation method.Usually, these methods comprise by one or more solution and one or more carbohydrate, protein, lipid, vitamin and inorganic elements that contains water and form slurry.This slurry is carried out emulsification, homogenize and cooling.Before handling, can in this slurry, add various other solution afterwards or in this two time periods.Formula food after will handling is then sterilized, and can further dilute to supply ready-made feed to use or the liquid form storage to concentrate.If resulting formula food plans to make the liquid of using or concentrating for ready-made feed, can before sterilization, add the water of Sq.

The invention still further relates to a kind of method that alleviates the patient's who suffers from celiaca the interior mucosal inflammation of intestines, it is by using the NAQ that is impregnated in above-mentioned aqueous solution and the liquid nutritional formula food.In embodiment 5, the inventor utilizes TUNEL label monitoring epithelium apoptosis phenomenon, utilizes subcutaneous inflammation in the CD25 monitoring, they have found to induce compound/products a kind of or two kinds of labels, thinks that these compound/products have toxicity to celiaca patient's mucous membrane.Yet N-acetyl group-glutamine has tangible nutritive validity for untreated celiaca patient's slicer.Define this trophism by the improvement situation to the mucous membrane in the epithelial cell particularly, the mucous membrane for other sample in the epithelial cell significantly improves.This beat all nutritional activities of N-acetyl group-glutamine make mucous membrane overall condition with respect in addition negative control (having only medium) for all make moderate progress.

Embodiment

The method for preparing the liquid nutritional formula food

Can prepare the liquid nutritional formula food that falls in claims scope of the present invention by following step.Exemplify these embodiment and be only used for explaining, and should not be understood that restriction.Only otherwise depart from scope of the present invention, other carbohydrate, lipid, protein, stabilizing agent, vitamin and inorganic elements also are operable.

Embodiment 1

Preparation contains the method for the liquid nutritional formula food of N-acetyl group-L-glutamine

The listed feedstock production of use table 1 obtains containing the fluid product of the ready-made feed of confession of N-acetyl group-L-glutamine.The step that is used to prepare this product is as described below.

Table 1: the former material list that is used for the perfuming grass product

The component title	Consumption (/ 1000kg)
		Water	To full dose
Maltodextrin	77.88kg
		Sucrose	52.80kg
Soybean protein hydrolysate	30.11kg
		Fish oil/medium chain structured lipid	16.14kg
Casein sodium	14.74kg
		Fructose oligosaccharides	5.792kg
Tower rape oil	4.482kg
		Soybean oil	4.482kg
45% potassium hydroxide	3.653kg
		Tricalcium phosphate	2.866kg
N-acetyl group-L-glutamine	10.03kg
		The L-arginine	2.425kg
Natrium citricum	2.293kg
		Artificial Carmel	1.500kg
N﹠A vanilla material	1.000kg
		Emulsifying agent	1.076kg
Magnesium phosphate	0.948kg
		Magnesium chloride	0.860kg
Potassium citrate	0.838kg
		Ascorbic acid	0.697kg
The Cobastab chloride	0.474kg
		Gellan gum	0.250kg
Vitamin D, E, K pre-composition 1	0.203kg
		Taurine	0.139kg
Carnitine	0.130kg
		Vitamin E (R, R, R)	0.123kg
Trace inorganic elements pre-composition 2	0.101kg
		Water-soluble microorganism pre-composition 3	0.0882kg
30% bata-carotene	15.5 gram
		Vitamin A (55%)	5.07 gram
KI	0.194 gram
		Sodium selenite	0.132 gram
Vitamin K	0.0617 gram

1. vitamin D, E, K pre-composition are included in cholecalciferol (0.0980 gram), d-alpha-tocopherol yl acetate (55.93 gram) and the vitamin K1 (0.0338 gram) in coconut oil (146.77 gram) carrier.

2. micro-inorganic elements pre-composition (every 1000kg finished product) is carried zinc sulfate (46.3 gram), ferric sulfate (39.2 gram), magnesium sulfate (11.4 gram), copper sulphate (3.89 gram).

3. the water soluble vitamin pre-composition is included in niacinamide (33.07 gram), d-calcium pantothenate (21.43 gram), folic acid (0.742 gram), thiamines chloride HCL (5.47 gram), riboflavin (4.27 gram), pyroxidine HCL (5.26 gram), cobalamin (0.0147 gram) and the biotin (0.644 gram) in dextrose (17.29 gram) carrier.

Step: by preparing three kinds of pre-compositions that mix, mix with marine products vegetable and animals oils/MCT structured lipid then, heat treated, typing, encapsulation and sterilization prepare above-mentioned liquid nutritional product.Describe its preparation method below in detail.

By at first the water of Sq is heated with stirring to about 65 ℃ to about 71 ℃ temperature, prepare the slurry of carbohydrate/inorganic elements.The inorganic elements that under vigorous stirring, adds aequum in the order then: natrium citricum, micro-inorganic elements pre-composition, potassium citrate, magnesium chloride, tricalcium phosphate and KI.Next, under vigorous stirring, in this slurry, add the maltodextrin (Maltring M-100, by Grain ProcessingCorporation of Muscatine, Iowa provides) of aequum, when temperature keeps about 71 ℃, make its dissolving.The sucrose and the fructose oligosaccharides (Nutriflora-PS fructose oligosaccharides powder, by Golden Technologies Companyof Golden, Colorado provides) that under vigorous stirring, add aequum then.Subsequently with the gellan gum (KelcogelW of aequum, by Kelco, Division of Merck and Company Incorporated of San Diego, California provides) do according to 1: 5 (gellan gum/sucrose ratio) with sucrose and mix, under vigorous stirring, be added in the slurry.Next, the sodium selenite that under agitation will be dissolved in the warm water is added in the slurry.Resulting carbohydrate/inorganic elements slurry is about 65 ℃ to about 71 ℃ in temperature and keeps down vigorous stirring to be no more than gas hour that directly it mixes with other slurry.

Mix to about 65 ℃ of following vigorous stirring and to obtain oil and mix thing by the soybean oil of aequum and Tower rape oil are about 55 ℃ in temperature.Under vigorous stirring, add the emulsifying agent of aequum, the diacetyl tartrate (Panodan, by GrindstedProducts Incorporated of New Century, Kansas provides) of single two monoglycerides then, make its dissolving.Stir then and add vitamin D, E, K pre-composition, 55% Retinol Palmitate, D-α-a-tocopherol yl acetate (R, R, R form), vitamin K and 30% beta carotene.Resulting oil mixes thing and is about 55 ℃ in temperature and is no more than 12 hours to about 65 ℃ of following gentle agitation that directly it mixes with other slurry.

By at first the water of Sq is heated with stirring to about 65 ℃ to about 71 ℃ temperature, prepare the slurry of protein in water.Stir and add soybean protein hydrolysate (by MDFoods of Viby J., Denmark provides).Stir the N-acetyl group-L-glutamine (from Ajinomoto) that adds aequum.Add potassium hydroxide solution (45%) and improve pH to about 5.6.Slowly stir adding L-arginine, solution stirs to clarify (pH＞6.2).And then be mixed into the casein sodium (Alanate 167, and by NewZealand Milk Products Incorporated of Santa Rosa, California provides) of the partial hydrolysis of aequum in the slurry.Resulting oil mixes thing and is about 60 ℃ in temperature and is no more than 2 hours to about 71 ℃ of following gentle agitation, up to mixing with other slurry.

Protein slurry in water and oil are mixed thing mix, resulting mixing slurry remain on about 55 ℃ to about 65 ℃ temperature.Place after at least one hour, stir to add carbohydrate/inorganic elements slurry, resulting mixing slurry remain on about 55 ℃ to about 65 ℃ temperature.Then marine flora and fauna oil/MCT structured lipid is stirred and be added in the slurry of this mixing.It is desirable to, marine flora and fauna oil/MCT structured lipid is metered in the product as form of mixtures by conduit according to constant rate of speed.After placement was no less than one minute no more than two hours, should mix slurry according to the methods known in the art deoxidation, ultraviolet high-temperature process, homogenize.Be cooled to then about 1 ℃ to about 7 ℃ temperature, stir to about 7 ℃ temperature at about 1 ℃ and to store.Preferably, after finishing above-mentioned steps, carry out suitable analytical test with the control quality.Based on the analysis result of this quality control, in sample, stir an amount of water of adding and reach final dilution factor (demarcation).

By low amounts of water is heated with stirring to about 43 ℃ to about 66 ℃ temperature; stir then and add following component: ascorbic acid, 45% potassium hydroxide, taurine, water soluble vitamin pre-composition, Cobastab chloride and L-camitine prepare vitamin solution.In above-mentioned mixing slurry, stir then and add this vitamin slurry.

Prepare aromatic solution by in the water of Sq, stirring adding natural and artificial vanilla material and artificial caramel material.In above-mentioned mixing slurry, stir then and add this aromatic slurry.

The pH that can regulate product is to obtain best product stability.Then resulting product is placed suitable container (using the 8oz. metal can in this case) to accept last sterilization (using the distillation sterilization in this case).

Embodiment 2

The stability study of moisture N-acetyl group-L-glutamine

Carry out following research with estimate moisture N-acetyl group-glutamine after heating under the different pH values and be similar to stability in the employed matrix of liquid nutritional type products.

The heat endurance of moisture N-acetyl group-L-glutamine and glutamine

In order to test the stability of moisture N-acetyl group-glutamine after heating, carry out following step.Do not regulate pH, prepare the aqueous solution of about 1mg/mL (being respectively 5.3mM and 6.8mM) of N-acetyl group-L-glutamine (from Sigma, catalog number (Cat.No.) A-9125) and glutamine (from Aldrich, catalog number (Cat.No.) G-320-2).The pH of resulting N-acetyl group-L-glutamine solution is 2.9, and the pH of glutamine solution is 6.0.Solution is at 100 ℃ of Reacti-Therm that use the 4mL conduit with sealing down ^TMThe agitating heating piece heats, corresponding time point of each conduit: 15 minutes, 30 minutes, 1 hour and 2 hours.Sample places frozen water up to cooling after heat block is removed immediately.Every increment an amount of part originally is by 0.45 micron filter (MilliporeMillex)-HV, and 25mm) HPLC mensuration is carried out in filtration.

Use Inertsil  C8,5 microns, 4.6 * 250mm post (from KeystoneScientific, Inc., Bellefonte PA) carries out HPLC and analyzes.Mobile phase is a water, regulates pH with HCl and is 2.2 (when waiting 1mL/ minute).Volume injected is 10 microlitres.Carry out UV detection at the 214nm place.

The result is as shown in table 2.Glutamine is hatched in the process unstable under 2 hours 100 ℃.Boil this pH and be after 6.0 the glutamine solution 1 hour, main catabolite is a pyroglutamic acid.Boil after this glutamine solution 2 hours, pyroglutamic acid remains main catabolite, but also detects glutamic acid.

N-acetyl group-L-glutamine is more stable than glutamine.Identify that by retention time main catabolite is N-acetyl group-glutamic acid; This is identified by mass spectrum (MS) and nuclear magnetic resoance spectrum (NMR) and confirms.Second top of identifying by MS and NMR is 2,6-dioxopiperidine yl acetamide.In N-acetyl group-L-glutamine solution, only in 2 hours sample, detect pyroglutamic acid, and just have extremely low concentration, be 0.2 area percentage.

Table 2: at the aqueous solution of 100 ℃ of heating glutamine and N-acetyl group-L-glutamine

	Glutamine solution (height %)			N-acetyl group-L-glutamine solution (area %)
								Time (minute)	GLN 1	GLU 2	PGA 3	NAQ 4	2，6-DPA 5	PGA 3	NAE 6
0	100.0	---	Do not detect	99.7	---	---	0.3
								30	90.2	---	9.8	98.1	0.6	---	1.2
60	80.0	---	20.0	96.9	1.3	---	1.8
								120	53.6	10.6	35.8	93.4	2.7	0.2	3.7

¹Glutamine; ²Glutamate; ¹Pyroglutamic acid; ⁴N-acetyl group-L-glutamine; ⁵2,6-dioxopiperidine yl acetamide; ⁶N-acetyl group-L-glutamic acid.

Moisture N-acetyl group-L-glutamine and the stability of glutamine under different pH values

For the stability of the aqueous solution under different pH values of measuring N-acetyl group-L-glutamine, carry out following step.Preparing pH is 2.0-8.0 and the N-acetyl group that increases progressively 1 pH unit gradually-L-glutamine aqueous solution.Optionally, before final dilution, use the pH (ultimate density=1mg/mL or 5.3mM N-acetyl group-L-glutamine) of hydrochloric acid or sodium hydrate regulator solution.Unique a glutamine solution is not regulated pH (measured pH=6.0), makes the glutamine that contains 1mg/mL or 6.8mM.All solution sterilize after filtration (Millipore Millex -GS, 25mm, 0.22 micron pore size, aseptic) enter in the Autosampler, at room temperature (17-25 ℃) stores.N-acetyl group-L-glutamine sample is measured on 1-180 days different time points by HPLC.The glutamine sample is measured on similar 1-45 days time point by HPLC.

The stability of finding N-acetyl group-L-glutamine depends on pH.The results are summarized among Fig. 1 and 2.Under all pH values, N-acetyl group-L-glutamine did not demonstrate signs of degradation in 7 days.Under pH5.0-8.0, N-acetyl group-L-glutamine is stable in six months, and the N-acetyl group-L-glutamine above 99.6% remains unchanged.All the time detected catabolite is N-acetyl group-glutamic acid, and it was lower than 0.5% in six months.Under pH4.0, place through six months, all detect N-acetyl group-glutamic acid and 2,6-dioxopiperidin yl acetamide, residual have N-acetyl group-L-glutamine of 97.9%.Under pH3.0, N-acetyl group-L-glutamine was placed through 90 days, reduced to 94.2% at 4th month, reduced to 90.4% at 6th month.In the sample of pH3.0, detect N-acetyl group-glutamic acid and 2,6-dioxopiperidin yl acetamide has roughly suitable concentration, and beginning was about 0.15% at the 15th day, rose to approximately 1% at the 30th day, rose to about 5% at 6th month.At 6th month, detecting pyroglutamic acid was 0.5%.At pH is 2.0 o'clock, and N-acetyl group-L-glutamine was 97.0% at the 15th day, only rose to 55.7% at 6th month.N-acetyl group-glutamic acid is that pH is the main degradation products in 2.0 the sample, is 2.5% at the 15th day, is 37.2%, 2 at 6th month, 6-dioxopiperidin yl acetamide by the 15th day 0.5% rise to 6th month 4.9%.PH is that N-acetyl group-L-glutamine sample of 2.0 o'clock is unique sample that pyroglutamic acid increases that demonstrates: at the 30th day was 0.2%, rose to 2.2% at 6th month.

In glutamine solution (pH6.0), find that this sample places at room temperature that pyroglutamic acid is 0.2% after 3 days.After 45 days, find that it is 3.3%, glutamine is 96.7%.The HPLC analysis result is represented to be recorded in the table 3 with height percentage.

Table 3: the stability in the glutamine aqueous solution that at room temperature pH is 6.0, concentration is 1mg/mL

Analyte	2 days	3 days	7 days	15 days	30 days	45 days
							GLN 1	100.0	99.8	99.5	99.0	98.1	96.7
PGA 2		0.2	0.5	1.0	1.9	3.3

¹Glutamine; ²Pyroglutamic acid.

N-acetyl group-L-glutamine and the stability of glutamine in the liquid nutritional type products

In order to measure the stability of N-acetyl group-L-glutamine in being similar to the used matrix of liquid nutritional type products, carry out following step.Prepare three kinds of analytic products; a kind of N-acetyl group-L-glutamine (N-acetyl group-L-glutamine is from Ajinomoto) that contains; a kind ofly contain glutamine (from Ajinomoto) (theoretical concentration is respectively 16.5mg/mL and 12.8mg/mL; with weight is that unit substitutes partially protein) and control group (Optimental ; Ross Products Division, Abbott Laboratories).Prepare the product that contains N-acetyl group-L-glutamine according to embodiment 1 described step.Prepare the product that contains glutamine according to the method similar, except substituting N-acetyl group-L-glutamine with glutamine (7.79kg) with it.Before distillation sterilization and measure their catabolite afterwards, the distillation sterilization is liquid nutritional formula food processing method commonly used (this paper uses at 128 ℃ and continues 5 minutes down).Product at room temperature (20-22 ℃) stores, and measures the 1st, 2 and catabolite 3 months the time.Glutamine, N-acetyl group-L-glutamine and pyroglutamic acid (if present) are quantized on each method and time point.

In order to analyze glutamine, N-acetyl group-L-glutamine and pyroglutamic acid, sample is filtered according to following method by HPLC.The 5.0mL sample is transferred in the 50mL volumetric flask.Add 20 1M hydrochloric acid, sample is diluted to certain volume with deionized water.(Millipore, Millexg-HV 25mm) filter sample by 0.45 micron filter.Sample carries out HPLC according to the method described above and analyzes (heat endurance part).

Be present in pyroglutamic acid total amount in this protein formulation by following method mensuration, comprise the terminal pyroglutamic acid of free pyroglutamic acid and N-.At first, sample is made aqueous solution form, concentration is about 18g gross protein/L.20 milliliters preparation-obtained sample raw material places 1.5mL nut bottle, add 980 milliliters of fresh enzyme solutions (0.05M Tris, 0.005M dithiothreitol dithio, 0.001M disodium ethylene diamine tetraacetate (EDTA), pH8.0 contains the fructose Glucoamino peptase of Unit 11/mL).Bottle tightly seals, and at room temperature (21-24 ℃) hatched 24 hours.Solution is processed by C-18 SPE cartridge case according to the method that describes in detail below then.In order to measure free pyroglutamic acid, the sample solution that this is initial is diluted to total protein content and is 2-3g/L in deionized water, then by the processing of C-18 SPE cartridge case.

C-18 SPE (SPE) cartridge case (100mg/1mL size) is from Burdick ﹠amp; Jackson, Muskegon, MI.Methyl alcohol with 2 * 5 volumes prepares the SPE cartridge case for using, and uses the washed with de-ionized water of 2 * 5 volumes then.Slowly add the 1mL sample, the material that will pass through is collected in the 1 gram nut bottle.By adding 2 * 500 ml deionized water, mobile phone is finished elution by the volume in the identical vials.Mix eluate, (from Gelman, Ann Arbor MI) filters a part of then sample for 25mm, 0.45 millimeter filter by 0.45 millimeter filter before HPLC analyzes.Used HPLC system has following parameter: pump processor G1312A, auto injection processor G1313A, temperature-adjusting device column compartment processor G1316A, PDAD processor G1315A, peak synthesizer/data processing processor G2170AA, all from Agilent Technologies, Palo Alto, CA. pillar: 6.5 * 150mm ION-310,8 millimeters, from Interaction Chromatography, San Jose, CA.Before the use, system is in advance at mobile phase (5mN H ₂SO ₄) in, under 40 ℃, the 0.3mL/min balance.

In order to analyze, inject 10 milliliters of samples or standard items, pillar uses the mobile phase of 0.3mL/min at 40 ℃ of following wash-outs.Be absorbed in 210nm and 220nm place detection wash-out material by UV.Flowing time is 45 minutes.

By determining unknown concentration of specimens with standard control.The aqueous solution of three kinds of pyroglutamic acids is enough to usually as standard items, promptly 10,20 and 40mg/L (pyroglutamic acid is from Fluka, Milwaukee, WI).

During N-acetyl group-L-glutamine is being sterilized in the nutrient type product or room temperature storage do not demonstrate catabolite in 3 months later on.The results are summarized in the table 4.All detect the small peak corresponding to N-acetyl group-glutamic acid on all time points, but remain approximately identical concentration, this shows do not have the catabolite that can measure for N-acetyl group-glutamic acid.

In the glutamine supplementary, through sterilization steps, glutamine is reduced to 1/3 of initial concentration; Detecting after 2 months does not have glutamine.In this product kind, the concentration that detects pyroglutamic acid is consistent with the conversion fully of glutamine.

Table 4: N-acetyl group-L-glutamine in the contrast liquid nutrient formulation food and glutamine store the stability after 3 months under process and room temperature.

	The product that contains N-acetyl group-L-glutamine	The product that contains glutamine
				Analyte	N-acetyl group-L-glutamine (mmol/L product)	Glutamine (mmol/L product)	Fructose glutamic acid (mmol/L product)
Theoretical value	87.7	87.6	---
				Before the sterilization	92.5	97.8	27.1*
After the sterilization	89.8	34.2	77.5*
				1 month	89.8	13.7	92.9*
2 months	94.1	Trace	79.8**
				3 months	89.3	Do not detect	80.6**

* the response factor according to the glutamine standard items calculates.Do not obtain suitable standard items in the test later stage.

* calculates according to the response factor of pyroglutamic acid standard items.

Embodiment 3

The bioavilability of glutamine and N-acetyl group-L-glutamine

Carry out following research with N-acetyl group-L-glutamine with respect to the bioavilability ratio of glutamine in pig model.The intestinal loop model has used the part of stripped intestines to estimate the absorption and the mechanism of N-acetyl group-L-glutamine and glutamine.This feed model evaluation after feed as usual to the absorbing state of N-acetyl group-L-glutamine and glutamine.

The intestines ring model

In 4 days, make 22 pig adequacy test conditions of raising and train that are weighed as 15-20kg.Arbitrarily satisfy standard pig feed and the water that is fit to these Animal nutrition demands (Nutrient Requirements ofSwine, the 9th edition, 1998, Subcommittee on Swine Nutrition, NationalResearch Council) to the pig feed.At random these animals are divided into C group [6 pigs, take dextrose in saline solution (Braun cat No 622647), 5% glucose, 0.9%NaCl], G organizes [8 pigs, take identical dextrose in saline solution with 8g/l Gln (Sigma cat No G-3126) reinforcement] and N group [8 pigs are taken the identical dextrose in saline solution with 10g/l NAQ (Sigma cat No A-9125) reinforcement].Before operation, allow animal go on a hunger strike 15 hours.Test the same day, weigh, with Stresnil  and penthotal anesthesia to animal.Cut postanesthetic pig open by midfield sagittal cutting operation.In about 1 meter near-space intestines fragment, about 1 meter Treitz ligament is inserted the 125mL analytical solution with the speed of 50-75mL/min.By obtaining intestines infusion solution sample at the 0th, 15,30,60,90,120,150 and 180 minute puncture infusion intestines.Sample is freezing in liquid nitrogen, remains under-80 ℃ up to analyzing.Obtain the portal vein sample in the test tube of anticoagulant by containing at the 0th, 15,30,60,90,120,150 and 180 minute puncture portal vein.Sample remain under 4 ℃ up under 1500xg centrifugal 15 minutes with separated plasma and red blood cell.Blood plasma is freezing up to analyzing at-20 ℃.By obtaining the jugular vein blood sample originally in the test tube that is containing anticoagulant puncture in the 0th, 60,120 and 180 minute.After 3 hours, pig death obtains the mucous membrane sample from 25cm infusion intestines fragment.This part uses ice-cold saline solution fully to wash, and spreads out along its length.By using glass slicker scraping intestines inner surface to remove mucosa removal, in liquid nitrogen, store down then in-80 ℃.

According to the methods below N-acetyl group-L-glutamine is analyzed.For intestines infusion solution sample and plasma sample, with aqueous solution dilution 1: 10 (w/v) of part sample with 0.05% perchloro-acetate (PCA).For the mucous membrane sample, with of the 0.05%PCA aqueous solution homogenize of the wet sample of 0.2mg with 5mL.Centrifugal (15,000xg, 3 minutes, environment temperature) after, sample filters by 0.45 millimeter filter, injects by 2690 Separation Module, PDA monitor and LichroCart 250-4 cartridge case (Purospher RP18e, 250 * 4mm, 5 millimeters) in the HPLC chromatographic system formed.Mobile phase is 2.7 to form by phosphate buffer 0.1M, pH, and flow velocity is 1mL/ minute.Detect the quality of N-acetyl group-L-glutamine at the 210nm place.

According to the methods below glutamine is analyzed.Except sample dilutes 1: 400 (w/v) with the 0.05% PCA aqueous solution, prepare infusion solution sample and the plasma sample that is used for N-acetyl group-L-glutamine intestines analysis according to top method.After sample filtered by 0.45 millimeter filter, 20 ml mixtures were diluted with water to 1mL according to AccQ-Tag method (Waters Corp.) derivation.Sample is cushioned with borate solution simply, with the derivation of 20 milliliters of anti-reactivity materials.After 1 minute, sample is diluted to 1mL, injects the HPLC system, and it is made up of 2690 Separation Module, fluorescence monitor and SupelcoSil LC-18 post (250 * 4mm, 3 milliliters).Mobile phase is that 7.5 and 0.25% triethylamine and 9% acetonitrile are formed by phosphate buffer 0.1M, pH, and flow velocity is 1mL/ minute.Use 250nm excitation wavelength, excite in the 395nm monitoring, finish detection and quantification to glutamate and glutamine.

Use the coupling enzyme assay of having set up that glucose is analyzed.With sample glucose hexokinase and ATP (atriphos) phosphorylation, resulting G-6-P salt is converted into 6-phosphono gluconate with G-6-P salt dehydrogenase simply.During the latter reaction, NAD (nicotine adenine-dinucleotide) is converted into NADH (the reduction form of NAD), and causing increases in the absorption at 340nm place, and it is the part of heavy concentration of glucose in the former state.This determination method can be used as the clinical chemical reagent box by Sigma Chemical Company, St.Louis, and MO (current catalog number 16-20) buys and obtains.

The result

The glutamine or the N-acetyl group-L-glutamine ratio that remain in the intestines inner chamber are introduced the time after the infusion solution.It is closely similar in initial 90 minutes to remain in the residual percentage that is infused into glutamine in the chitling inner chamber that contains equivalent glutamine or N-acetyl group-L-glutamine or N-acetyl group-L-glutamine.Significant difference on the statistics appearred in each group in the time of 120 and 180 minutes.At t _1/2(greatly in the time of 45 minutes), there was no significant difference between glutamine or N-acetyl group-L-glutamine.Fig. 3 has described the glutamine that added or N-acetyl group-L-glutamine with diagrammatic form and has remained in amount in the enteric cavity as the function of the time introduce this material in the chitling ring that exsomatizes in internal medicine operation test described herein after.The percentage of the analyte of residual analyte when accounting for time zero is represented.

Fig. 6 has described the content (representing with the mcg/ gram mucous membrane that wets) of glutamine in the chitling jejunal mucous membrane measured after internal medicine operation as herein described test and glutamate with diagrammatic form.

The glucose ratio that remains in the intestines inner chamber is introduced the infusion solution time afterwards.Difference that there are no significant between C and the G group at any time.Except the 15th minute, there was no significant difference between C and the N group.G and N group difference occurred after the 120th minute time, although by Bonferroni ' s calibration, its unique difference appears at 180 minutes.Fig. 4 has described the glucose that is added with diagrammatic form and has remained in amount in the enteric cavity as the function of the time introduce this material in the isolated pig intestinal loop in internal medicine operation described herein test after.The percentage of the amount of residual glucose when accounting for time zero is represented.

Glutamine in the blood enters the mouth introduce test solution in intestinal loop after.If with results expression is the percentage form of initial concentration, so and (G) and difference occurs between C and N-acetyl group-L-glutamine (N) group (at 90 and 150 minutes, C was to G at control group (C); And at 90,120,150 and 180 minutes, C was to N).Between G and N, there is not significant difference.If structure representation is the form of absolute value,, there is not significant difference between C and the N so except 120 minutes.Consider that together G and N are different from C since 120 minutes to off-test.Fig. 5 has described glutamine content (mcg/mL) in the loop door blood as the function of introducing the time after the test solution in the intestines ring with diagrammatic form.

There is not significant difference aspect glucose in each group loop door blood and glutamine in the peripheral blood or the glucose.In process of the test, just there is negligible difference (a few millionths) at any time in detected complete N-acetyl group-L-glutamine concentration in loop door blood or peripheral blood.

Glutamic acid in the jejunal mucous membrane (GLU) and glutamine (GLN)

High in the glutamate concentration ratio G group in N and C group, in the mucous membrane of N and G group, also demonstrate higher glutamine simultaneously, the G group is higher than the N group basically.Yet, glutamine+glutamate concentration add with to G and N group in similar, this shows that it is suitable adopting these two kinds of foods to send the glutamine carbon skeleton to the mucous membrane metabolic system.In the mucous membrane sample, detect less than complete N-acetyl group-L-glutamine.Fig. 6 has described the content (representing with the mcg/ gram mucous membrane that wets) of glutamine in the chitling jejunal mucous membrane measured after internal medicine operation as herein described test and glutamate with diagrammatic form.

Generally speaking, N-acetyl group-L-glutamine demonstrates the bioavilability similar to glucose, but is lower than glutamine slightly.As if aspect the utilizing after absorption, N-acetyl group-L-glutamine is very similar to glutamine.After being absorbed; N-acetyl group-L-glutamine is rapidly by acyltransferase hydrolysis in the intestines; enter normal glutamine metabolism system, thereby in mucous membrane, obtain the same high concentration of glutamine+glutamate concentration that acquired with feed equivalent glutamine.Excessive glutamine is drained to loop door blood, and resulting concentration is suitable behind the glutamine of glutamine concentration herein and feed equal dose.N-acetyl group-L-glutamine concentration in the loop door blood only has several ppm, and this shows seldom by the complete absorption of blood.The high assimilation ratio and the metabolic mechanism similar to glutamine of N-acetyl group-L-glutamine show to have identical biological action in the various nutritional labelings of organism catabolic phase.

The feed pig model

By specifying the farm that the pig of 15 heavy 15-20kg is provided.Allow these pigs at 2 days endoadaptation laboratory conditions.Standard pig feed and water arbitrarily are provided.After the adaptation, these pigs are divided into the C group at random, and [5 pigs, the standard pig feed of taking adds 3g/kg Cr ₂O ₃, (Merck cat No 1.02483)], G group [5 pigs are taken food C and add 8g/kg Gln, (Ajimoto)] and N group [5 pigs are taken food C and add 10.5g/kg N-acetyl group-L-glutamine, (Flamma)].During experimental study, every animal in each group is taken 1000 gram its corresponding food/skies, is divided into 3 parts of feeds, and water arbitrarily is provided.Take food lasting 5 days of experimental period.

The test same day, weigh to animal, then the morning 7:00 take the food of standard intake (every animal 333g food).Took food back 3 hours, and weighed, take sedative, get blood by puncturing jugular vein to animal.Rapidly pig is cut open by midfield sagittal cutting operation, taken out the content in duodenum, jejunum (about 2 meters Treitz segment) and the ileum (30cm ileocaecal sphineter), freezing in liquid nitrogen, hydrolysis is stored under-80 ℃ up to analysis.Remove liver and kidney sample, incision can observable fat and conjunctive tissue, and is freezing rapidly and be stored under-80 ℃ up to analysis in liquid nitrogen.Method according to describing in the intestines ring test that exsomatizes obtains the intestinal mucosa sample, stores before analyzing.

Analyze glutamine, N-acetyl group-L-glutamine and the chromium oxide (III) of intestinal contents.In order to analyze N-acetyl group-L-glutamine, the intestinal contents after the hydrolysis is dissolved in the aqueous solution of 0.05%PCA with 1: 20 (w/v), carry out HPLC according to the method for in above-mentioned intestines ring, describing then and analyze.

In order to analyze glutamine, the intestinal contents after the hydrolysis is handled, analyze according to the method for in above-mentioned intestines ring, describing then.

Chromium is mixed in the food to obtain reflecting the correction factor of content/kg initial food.In order to analyze chromium oxide (III), carry out following step.The weighing of representational hydrolysis intestinal contents is gone in the nickel crucible, in Muffle furnace.Temperature rises to 500 ℃, continues to keep 2 hours.After the cooling, at about 10 times of weight adding molten mixture (Na of sample ash ₂CO ₃, K ₂CO ₃, KNO ₃, 10: 10: 4w/w/w), fully mixed.Form thin layer from the teeth outwards at the molten mixture that adds additional quantity, on naked light, use gas range fusion 30 minutes, up to obtaining transparent fused mass.Remove crucible from stove, cooling, by with about 20mL water washing pot wall, about 30 minutes of mild heat on hot plate then, thus fully extract fused mass.Abundant when loose when shell, crucible is washed with water four times, all washing lotions are added in the 100mL volumetric flask, are diluted to full volumetric.By utilizing to 0,50,100,200 and 500 milliliter of standard chlorine solution (2.9034g K ₂Cr ₂O ₇/ L is equivalent to 1.5g/L Cr ₂O ₃) analyze the calibration curve that obtains, the absorbance that reads is converted into Cr ₂O ₃The mg number.

The result

The trap data are as shown in table 5 below.The duodenum sample contains chromium oxide (II) content that is not enough to measure.Analysis result can not be calibrated to reflect content/kg initial food.In jejunum contain suitable basically glutamine concentration (in the situation of food G) and N-acetyl group-L-glutamine (in food N), this shows in duodenum and near-space intestines to have similar absorbing state.Yet these foods also contain complete protein, and are indicated as the analysis result of control Food, and the digest of these protein may also produce tangible free glutamine.This shows that the free glutamine content in the initial food was almost absorbed fully before middle ileum.Content to the ilium tip is analyzed, and shows, although the absorption of free glutamine can continue between middle ileum and ilium tip, does not observe the absorption of N-acetyl group-glutamine.Yet all absorption data show that nearly 77% N-acetyl group-L-glutamine is absorbed in this model.

Table 5: as the N-acetyl group-L-glutamine of pig food component and the absorbing state of glutamine

	Glutamine food	N-acetyl group-L-glutamine food	Control Food (C)
				Duodenum	N/D**	N/D	N/D
Middle ileum	10.1±1.9	10.3±2.4	8.8±0.7
				The ilium tip	1.2±0.6	12.8±2.1	2.1±0.7

* for glutamine and control Food, data are meant glutamine (mM/kg initial food).For N-acetyl group-L-glutamine food, data are meant N-acetyl group-L-glutamine (mM/kg initial food).Initial food is (glutamine=54.8 mMs glutamine/kg food, a N-acetyl group-L-glutamine=55.8 mM N-acetyl group-L-glutamine/kg food).

* N/D=does not detect.Chromium oxide (II) value is lower than the numerical value limit of this assay method, does not obtain corrected value.

In the acyltransferase activity in mucous membrane, liver and the kidney-in several tissue of interest (with regard to the importance that nutrition may have) of contrast pig, the measured activity of acyltransferase.Recorded at all test organizations and comprised acyltransferase activity in jejunal mucous membrane, liver and the kidney.Measured concentration is to be 948+300IU/g wet tissue (17.3+7.0IU/mg protein) in jejunal mucous membrane, be 12 in liver, 770+1110IU/g wet tissue (159+30IU/mg protein) is 19 in kidney, 630+3020IU/g wet tissue (302+47IU/mg protein).

Generally speaking, N-acetyl group-L-glutamine mainly is to absorb at duodenum and jejunum top, and above-mentioned position has absorbed at least 77% dosage.Have two main differences between N-acetyl group-L-glutamine and glutamine: N-acetyl group-L-glutamine absorbs saturated appearance early and ileum absorbs lower.

Embodiment 4

N-acetyl group-L-glutamine is for the effect because of the malnutritive damage of intestines that causes

Carry out following research and compare with free glutamine, for the effect of the damage of intestines that causes because of protein-energy malnutrition in the pig to estimate N-acetyl group-L-glutamine.In this research, by specify the farm provide 5 weeks big raise and train pig.These pigs are divided into the 1-2 group at random.(Ross Products Division AbbottLaboratories), continues 30 days to the 3rd group of ad libitum access ENSURE PLUS .In second group, 9 pigs ENSUREPLUS  that also taken food, but only be 20% of first group of daily intake.With second component is 3 groups, 6 every group, all takes calcium caseinate or glutamine or N-acetyl group-L-glutamine and replenishes as daily.At the research initial stage, per day energy and the protein of supplying with to control group are 3300 kilocalories, 138g protein, are 4500 kilocalories, 187g protein during to the research end.In second group, the additional 1.32 gram nitrogen equivalent/skies that add (every day, additional 6.89 gram L-glutamine or 8.87 gram N-acetyl group-L-glutamine or 8.42 restrained casein proteins basically) that provide of caseinate, glutamine and N-acetyl group-L-glutamine.After 30 days, all pigs were gone on a hunger strike 16 hours.Weigh for then these animals, the clothes sedative, anesthesia, last jugular puncture makes its death.

Remove whole small intestine fragment rapidly.Be considered to the jejunum near-end from the long small intestine fragment of the 60cm of Treitz ligament.For jejunum is carried out histologic analysis, select 5cm long segment from the Treitz ligament.The 60cm length of close valvula caeca is considered to the ileum tip.For ileum is carried out histologic analysis, select 5cm long segment from valvula caeca.These intestines fragments are fully washed with ice-cold saline solution, spread out along its length.By using glass slicker scraping intestines inner surface to remove mucosa removal, in liquid nitrogen, store up to carrying out biochemical analysis down then in-80 ℃.

Jejunum and ileal mucous membrane used in 10mM phosphate buffer (pH7.4) protein is carried out in the homogenize of mechanical Potter homogenizer and DNA measures.The enzyme labeling thing of impaired in order to measure, functional and antioxidant system of defense was with mucous membrane homogenate under 3000g centrifugal 10 minutes.Resulting supernatant is used for enzymatic determination.In order to measure total glutathione, the homogenize in 5% trichloroacetic acid of slurry mucous membrane, under 8000g centrifugal 5 minutes.

On above-mentioned sample, carry out biochemical analysis and immune analysis.Adopt respectively the Bradford method (Analytical Biochemistry, Volume 72, pages 248-254,1976) and Labarca and Paigen method (Analytical Biochemistry, Volume 102 (2), pages 344-352,1980) record the concentration of intestinal mucosa and DNA.Because of the damage of intestines degree that malnutrition causes passes through to adopt Goldstein method (R.Goldstein, T.Klein, S.Freier and J.Menczel.American Journal of Clinical Nutrition24:1224-1231,1970) measuring alkaline phosphatase activities estimates.

The activity by measuring glutathione reductase (GR), glutathione transferase (GT) and glutathione peroxidase (GPOX) and the concentration evaluation of nonprotein sulfydryl (mainly being the glutathione (GSH) of reduction) resist the system of defense of oxidative damage.Estimate the activity of glutathione reductase by the method (I.Carlberg and B.Mannervik, Methods inEnzymology, Volume113, pp 484-490,1985) of Carlberg and Mannervik.Adopt people's such as Habig method (W.H.Habig, M.J.Pabst and W.B.Jakoby, Journal of Biological Chemistry.294:7130-7139,1984) to measure glutathione transferase.Method (L.Flohe and W.A.Gunzler by Flohe and Gunzler, Methods in Enzymology, Volume 105, pp 114-121,1984) activity of mensuration glutathione peroxidase, nonprotein sulfhydryl content (being reported as the glutathione equivalent of reduction) is by Anderson method (M.E.Anderson, Methods inEnzymology, 133:548-554,1985) measure.

Such described in detail as follows, according to the stripped IL of people's such as Gautreaux step (M.D.Gautreaux, E.A.Deitch and R.D.Berg, Infection and Immunity62 (7): 2874-2884,1994).Small intestine fragment from jejunum and ileum is respectively separated, and (MO USA) washes away sedative content for PBS, Sigma St.Louis with phosphate buffered saline (PBS).Cutting away can observable Peyer ' s spot, alongst intestines is cut open, is cut into small pieces.In order to separate intestinal epithelial cell, with in the jolting water-bath (per minute shakes 120 times) of these fritters under 37 ℃, containing 5mM dithiotreitol (DTT; Roche Molecular Biochemicals, Indianapolis, IN, USA), 2mM EDTA (Sigma, St.Louis, MO, USA) and 25mM Tris buffer solution (Sigma, St.Louis, Mo, 25ml Hanks balanced salt solution (HBSS USA); Sigma, St.Louis, MO was hatched 30 minutes in USA); Decant supernatant, add fresh HBSS-DTT-EDTA-Tris, repeat above-mentioned incubation step.Merging is from carrying out hatching for twice the epithelial supernatant that contains that obtains, cell is by with containing 5% (v/v) to the insensitive tire intestines of heat blood plasma (Sigma, St.Louis, MO, USA), 20mM HEPES (Sigma, St.Louis, MO, USA), 2mM L-glutamine, 500U penicillin and 100FLg/ml streptomysin (Sigma, St.Louis, MO, rpmi 1640 culture mediums (complete medium) washing USA).By ramenta intestinonun being placed 40ml contain clostridiopetidase A 0.05U/ml, dispase0.30U/ml (Sigma, St.Louis, MO, USA) and deoxyribonuclease I 500U/ml (Roche Molecular Biochemicals, Indianapolis, IN separates obtaining fine layer body lymphocyte (LPL) from the residual precipitate thing in 37 ℃ shake in the water-bath (per minute shakes 120 times) in complete medium USA).Peyer ' s the patch that cuts away places complete medium, cuts with scalpel.Then Peyer ' s the patch of cleaning is handled (reduction incubation time 60 minutes) with clostridiopetidase A according to separating the method that LPL discharges in the Peyer ' s patch lymphocyte (PPL) in front.

To separate the discontinuous Percoll of all types cell process that obtains with Peyer ' s patch by epidermal cell, fine layer body ^TM(Sigma, St.Louis, MO, USA) density gradient centrifugation is with lymphocyte-rich.The Percoll that this is commercially available ^TMSolution obtains waiting the Percoll that oozes with 9% NaCl dilution 9: 10 ^TMSolution obtains 3 kinds of differences with it with the complete medium dilution and is Percoll ^TMThe solution of percentage (75%, 40% and 30%), they are according to using in order of reducing gradually.These cells are suspended in the 4ml complete medium once more remaining 30% part.After centrifugal 20 minutes, remove the interface between 75 and 40% layers under 650g, cell is by centrifugal washing the in the 25ml complete medium.And then cell is suspended in 4ml 40% Percoll ^TMIn, centrifugal under 650g.Collect the cell ball of lymphocyte-rich (IEL, LPL and PPL), by washing with PBS is centrifugal.

Separate the lymphocyte labeling of monoclonal antibody that carries out flow cytometry according to following method that obtains: each lymphocyte of 100 μ l is prepared liquid (2 * 10 ⁶Cel/ml) place the 3-ml test tube of monoclonal antibody (AntiCD1 FITC, Anti CD3s FITC, Anti CD4aPE, AntiCD8a PE, AntiCD1 lb/Mac-1 APC, Anti CD21 APC), in the dark under 4 ℃, hatched 30 minutes with variable concentrations.Cell washs with PBS, and is spherical by centrifugal (500g, 5 minutes) formation, is suspended in once more among the 350ul PBS.

At FACScalibur ^TMFlow cytometer (Becton Dickinson) is gone up pair cell and is prepared cell classification (FACS) analysis that liquid carries out fluorescent activation.For every kind of cell preparation liquid, (the different thiocyanic ester-FITC of fluorescein, phycoerythrin-PE and xenogenesis phycocyanin-APC) are measured non-specific fluorescence by 3 groups of contrasts.

The histologic analysis of small intestine sample is finished by transmission electron microscope(TEM).The sample of jejunum and ileum is fixed in the 0.1mol/L methyl-arsinic acid sodium cushioning liquid of 30g/L glutaraldehyde, pH7.3, and then in the stuck-at-5g/L osmium tetroxide.Sample dewaters in acetone then, plants in Epon 812 resins.Part is with uranyl acetate and the double-deck mark of lead citrate, in Zeiss 902 transmission electron microscope(TEM)s (Zeiss, Oberkochen, Germany) inspection down as thin as a wafer.

Biochemical result

The feed intake is reduced to 20% of control group and causes can not growing fully.The weight average loss 2-3kg that these underfed pigs are total, and control group was at 30 days experimental session growth 18kg.The jejunum of liver weight and every length and mucous membrane weight also obviously alleviate (table 6) because of malnutrition.

Table 6: the liver and the small intestine weight of contrast and protein energy shortage group

	Liver weight (g)	Mucous membrane weight/intestines length (g/cm)
				Jejunum	Ileum
		The control group pig	731.4±26.5			0.092±0.008	0.070±0.007*
Underfed pig	237.9±9.9*			0.035±0.005*	0.025±0.006*

* to the significant difference (p＜0.05) of control group.

With respect to control group (data not shown goes out), the protein content of DNA in the malnutritive pig and mucous membrane unit length obviously low (2-3 is doubly).Yet the ratio of protein/DNA is not influenced by PEM in any intestines fragment.This result shows that in the small intestine of malnutritive pig, whole protein and DNA are synthetic to be weakened.Protein in the intestines (ileum and jejunum) and DNA (ileum) content on the feed in the malnutritive pig replenished of NAQ than the pig kind height of caseinate or glutamine on the feed.This result shows that NAQ has partly protected the building-up process of protein and DNA during malnutrition.

As the activity of the alkaline phosphate fragment of damage of intestines label in malnutritive pig significantly than low in contrast jejunum fragment (data not shown goes out) (2-3 doubly).In the ileum fragment, alkaline phosphate is less to be subjected to underfed the influence.In addition, the malnutritive pig of feed glutamine or NAQ tonic has the high AP activity of pig of having added caseinate than feed in ileum.

Glutathione is the core of whole non-oxidizability system of defense.It is effective free radical street cleaner, and is simultaneously also relevant with other metabolic function, comprises keeping the short altogether factor of the protein sulfhydryl, GT and the GPX that go back ortho states, amino acid transport and protein and DNA synthesizes.Total glutathione concentrations in the intestines fragment of malnutritive pig and control group significantly reduces.Yet than the height in the pig of caseinate or glutamine tonic on the feed, this species diversity does not also constitute significant difference to the GSH content in the intestinal mucosa of the malnutritive pig of NAQ certainly slightly on the feed.

The glutathione transferase of responsible respectively aldehyde detoxifcation of discovery and glutathione reduction and the activity of glutathione reductase in small intestine are because underfed cause all decreases (being again 2-3 times).The reduction of glutathione transfers enzyme activity may be deposited in by aldehydes, epoxides and other product that contains the electrophilic center intestines function is further worsened.Finding that this activity has been added on the feed lessly in the pig of N-acetyl group-L-glutamine is subjected to underfed the influence.The activity of glutathione reductase and glutathione peroxidase in the small intestine fragment because malnutrition has all reduced 2-3 doubly.

Glutathione reductase is also relevant by its oxidised form regeneration with glutathione.Two reduced glutathione molecules of glutathione peroxidase oxidation peroxide that detoxifies.Reduced glutathione has higher trend in the intestinal mucosa of the pig of N-acetyl group-L-glutamine tonic on the feed, this with certain relation arranged in that the sweet peptide peroxidase activity of two-story valley Guang is higher on the same group mutually.

Generally speaking, as if malnutritive detrimental effect for the antioxidant system of defense is more not obvious in the animal than caseinate or glutamine tonic on the feed in the animal replenished of N-acetyl group-L-glutamine on the feed.

Immunology result

Small intestine peyer ' s patch TLC order as malnutritive result reduces to some extent.In ileum, peyer ' s patch TLC order is significantly low than the total number in replenishing N-acetyl group-glutamine or control group in the pig of replenishing caseinate and glutamine.In jejunum, a kind of like this trend also appears, and that peyer ' s patch TLC order is replenishing N-acetyl group-glutamine also significantly than the total height of eye in the pig of replenishing caseinate and glutamine.On the other hand, the lymphocytic total number of jejunum exocuticle is remarkable in the height in the control group in all malnutritive groups.For all experimental group, do not have to there are differences aspect the lymphocyte number of discovery in the fine layer of small intestine.

In all malnutritive groups, the peyer ' s patch lymphocyte of expressing B cell sign thing (CD1 and CD21) low than in the health group is especially in the lymphocytic situation of CD1+.CD21+peyer ' s patch lymphocyte number decreases in ileum, and it significant difference occurs between control group and caseinate and glutamine supplementation group, rather than N-acetyl group-L-glutamine supplementation group.Identical trend in jejunum, also occurs, but do not reach significant difference.CD1lb+peyer ' s patch lymphocyte number reduction in jejunum and ileum also demonstrates a kind of like this trend, and that is exactly that N-acetyl group-L-glutamine supplementation group is lower than caseinate or glutamine supplementation group.

T cell (CD3+ cell) in jejunum and the ileum peyer ' s spot lymphocyte reduces to some extent owing to malnutrition.This minimizing is because assistant (CD4+) and cytotoxin (CD8+) T cell.Yet the minimizing of this T cell has a kind of like this trend, and that is exactly that N-acetyl group-L-glutamine supplementation group is lower than caseinate or glutamine supplementation group.In some situation for example among the CD4+ and CD8+ cell in the ileum, significant difference appears observing between control group and caseinate or the glutamine supplementation group rather than between control group and the N-acetyl group-L-glutamine supplementation group.

As mentioned above, malnutrition has been impelled the sum increase of intraepithelial lymphocyte in the jejunum.This being increased in two class crowd B cells (CD21+) and the T cell (CD3+) all observed.In the B cell, the CD1+ lymphocyte number is significantly higher than other group in N-acetyl group-L-glutamine supplementation group.In the T cell, the inferior crowd of T cytotoxin (CD8+) is significantly higher than control group in the malnutrition group.Yet T assists son (CD4+) inferior crowd in glutamine and N-acetyl group-L-glutamine supplementation group (rather than casein supplementation group) to be significantly higher than control group.This shows that glutamine and N-acetyl group-L-glutamine assist son (CD4+) inferior crowd to have selectively acting for T.For the inferior crowds of all lymphocytes of ileal epithelium endolymph cell, do not observe and have significant difference.

Do not observe aspect fine layer body lymphocyte because the malnutritive significantly important change that occurs.For control group, CD21+ cell (B cell) decreases in the caseinate supplementation group, but this being reduced in glutamine or the N-acetyl group-L-glutamine supplementation group all do not observed.In addition, N-acetyl group-L-glutamine supplementation group rather than glutamine supplementation group significantly are different from the caseinate supplementation group.

Generally speaking; N-acetyl group-L-glutamine supplementation group performance is all good than glutamine or caseinate supplementation group; demonstrate significant difference on the statistics; can reduce because malnutritive and small intestine immunology that cause changes, especially assist aspect total cell number of sub inferior crowd at B and T.

Histology result

From the electronic transmission micrograph of the jejunum enterocyte of health pig and malnutritive pig as shown in Figure 7.In contrast pig (A, B group), the jejunum enterocyte demonstrates the microvillus of rule, narrow space between cells, the nuclear of rule and the globlet cell that contains the high concentration mucoitin.Be supplemented with on the feed in the intestinal mucosa of malnutritive pig of ENSURE PLUS formula food of caseinate (C, D group), demonstrate that serious atrophy phenomenon and microvillus reduce, the space between cells is opened wide, regular nuclear and clear cytoplasm district band with multivesicular body.In this group, the phenomenon that cell detachment and material are clamp-oned the intestines inner chamber also is tangible.Feed is supplemented with in the jejunal mucous membrane (E, F group) of malnutritive pig of ENSURE PLUS formula food of glutamine during malnutrition, demonstrates the microvillus of shortening, the space between cells of expansion and the fractionlet nuclear of rule.In the jejunal mucous membrane of underfed glutamine pig, also find to exist abundant intraepithelial lymphocyte.The pig jejunal mucous membrane (G, H group) that feed is supplemented with the ENSURE PLUS formula food of NAQ more is not subject to the underfed influence of protein ability.In this group, the space between cells that the jejunum enterocyte demonstrates microvillus size, nuclear shape and is close with contrast pig jejunal mucous membrane.Compare with L-glutamine group with underfed caseinate, the intraepithelial lymphocyte phenomenon of osmosis that occurs in the jejunal mucous membrane of underfed NAQ group is rare.Fig. 8 shows the electronic transmission micrograph from the ileum enterocyte of health pig and malnutritive pig.In contrast pig (A, B group), the ileum lymphocyte demonstrates the microvillus of regular distribution, the space between cells with invisible expansion, even and dense cytoplasm and the globlet cell with a large amount of secretory granules.The pig ileal mucous membrane (C, D group) that feed is supplemented with the ENSURE PLUS formula food of caseinate demonstrates that microvillus reduces, expand in some space between cells, have the clear cytoplasm district band in the extrusion of being in of multivesicular body.In this group, wide lymphocyte phenomenon of osmosis also is tangible in the ileal epithelium cell.The pig ileal mucous membrane (E, F group) that feed is supplemented with the ENSUREPLUS formula food of glutamine during malnutrition demonstrates that microvillus reduces, wide space between cells and strong lymphocyte phenomenon of osmosis.The same with jejunal mucous membrane, the pig ileal mucous membrane (G, H group) that feed is supplemented with the ENSURE PLUS formula food of NAQ more is not subject to the influence of protein energy shortage.It demonstrates low microvillus distortion, does not have visible space between cells of expanding, finds rare lymphocyte phenomenon of osmosis and the abundant goblet cell that contains the high-load secretory granules at the enterocyte head portion.

Conclusion

Under normal physiological conditions, between the contribution of the generation of the free radical that oxygen is derived and pair cell antioxidant system thereof, there is stable state.In this research,, cause glutathione to reduce because EMP makes that the intestines balance is broken.In addition, immune response should be EMP and receives weakening greatly in the intestines.

Although aspect the biochemistry and immunology variation that prevent to cause because of the small intestine malnutrition; not observing glutamine has a significant effect; this may be because malnutrition is very serious, but N-acetyl group-L-glutamine has positive role for the order of severity that reduces this variation.

Originally studies show that N-acetyl group-L-glutamine has positive role for small intestine cells, even surpass glutamine.In addition, the electronic transmission micrograph from healthy and malnutritive pig as shown in Figure 7 shows that N-acetyl group-L-glutamine is more effective than glutamine aspect the endo-endothelial layer inflammation on the prevention intestines and stomach.

Embodiment 5

Utilize the celiaca patient's who does not receive treatment tissue culture test to be rich in the toxicity of glutamine preparation for celiaca described herein

The purpose of this research be to estimate several do not contain seitan be rich in the glutamine product for genotoxic potential with pepsin-tryptic seitan preparation, known have pepsin-tryptic seitan preparation and can induce the mucosa infection in the celiaca.In addition also to obtain by seitan be rich in glutamine or the glutamine improved products is studied, whether can induce mucous membrane destruction to estimate the latter to the celiaca patient.

This research is carried out with random fashion.Use the culture model of organizing in abdominal cavity to carry out this research.In order to realize this project purpose, used the celiaca patient's who had not treated small intestine living tissue fragment.

Recruit 12 and be diagnosed as the potential adult patients of not treating the celiaca patient.Their disease by a large amount of anti-tTGs occurring clinical symptoms and the histologic analysis of biopsy of small intestine confirmed.

The description of terminal point is cultivated by related organization

Employing is described in Maiuri, people such as L., and Gastroenterology 110, and the standard method among the 1368-1378. (1996) prepares tissue culture.Simply with small pieces slicer (about 1mm * 1mm) be contained in stainless steel sift in being supplemented with the medium of different compounds, to cultivate 24 hours on the net.Standard positive and negative control in this research, have been adopted.Positive control is gliadin pepsin-trypsase of 1mg/ml.Negative control is independent medium.The ultimate density of employed all test compounds is 40 μ g/ml.Test compounds/product is N-alanyl-glutamine; N-acetyl group-glutamine; From Nutricia, Boca Raton, the Stresson  (P1) of Florida; From Fresenius Kabi, Runcorn, Cheshire, the Reconvan  (P2) of UK; From B.Braun, Bethleham, the NutricompImmun  (P3) of PA; From Hormel Health Labs, Plymouth, the Glutasorb  (P4) of MN; From Novartis, White Plains, the Impact  (P5) of NY; From Ross Products, Columbus, the Optimental  of Ohio adds NAQ (P6); From Ross Products, Columbus, the Optimental  of Ohio adds hydrolyzed wheat seitan (P7).After 24 hours, stop to cultivate, to slicer implant the O.C.T. compound (Tissue Tek, Miles Laboratories, Elkhart, IN, USA) freezing in liquid nitrogen rapidly, be stored in-70 ℃ up to cryogenic absorption.Use cold state to prepare 5 microns sections.

Immunohistochemistry

Use following label carry out this research: TUNEL is used to monitor the epithelium apoptosis, and CD25 is used to monitor the intracutaneous inflammation.Use following reagent according to following step.

Detect the DNA fragment

According to being described in Maiuri, L. do you wait people .DNA fragmentation is a featureof cystic fibrosis epithelial cells:a disease withinappropriate apoptosis? FEBS Letter 408,225-31 (1997) and Maiuri, L. wait people .FAS engagement drives apoptosis of enterocytesof celiac patients.Gut 48, the method among the 418-24 (2001) is measured the DNA fragment in the histotomy.

Detect the CD25+ cell

By immunohistochemistry according to being described in Maiuri, L. wait people .Blockage ofT-cell costimulation inhibits T-cell action in Celiac disease.Gastroenterology 115,564-72. carrying out antigen on freezing histotomy, the method (1998) detects, use mAbs to resist-CD25 (Dako 1: 30), adopt and open the alkaline phosphate dye technology of describing in the document in this identical ginseng according to the front.

For each sample, at least 5 slides are carried out the region between the heart and the diaphragm assessment.Utilization is carried out the specificity check experiment at the mouse IgG or the IgM of inappropriate blood group antigen, analyzes the different culture sample that belongs to same individuality simultaneously.

Morphological analysis

The total number of TUNEL+ enterocyte is meant the percentage of enterocyte.Measurement falls into 1mm ²TCS in the fine layer of CD25+ in the standard area in the scope body.In the back these data are compared.

The result

As shown in Figure 9, these compounds have been induced the different mode that is raised defined epithelial cell damage by TUNEL and CD25 in the chamber, last rim surface zone.Some preparation has been induced increase and apoptotic the inducing that CD25 expresses in last intracutaneous compartment simultaneously.These compounds thereby can be used as positive control (pepsin-trypsase seitan preparation that uses according to 1mg/ml).Other compound has been induced some selectively changing of inducing to the CD25 in epithelial cell apoptosis or the epithelial cell compartment.Other with culture in only being exposed to independent medium (negative control) in viewed apoptosis pattern or CD25 induce as broad as long.The product I mpact that has shown use compound N-acetyl group-glutamine or contain glutamine (p5) in Figure 10 a and b induces the example of epithelial cell apoptosis.Figure 11 a and b have described the pattern of inducing CD25 by same compound.

Conclusion

The result of this research shows that a lot of test compounds have produced significant variation in the celiaca patient's who does not receive treatment small intestine.Some can be induced the epithelial cell apoptosis and promote inflammation in the epidermis, and other compound induce aspect the mucous membrane inflammation more effective.Can induce the compound of two kinds or a kind of label to be considered to have toxicity for celiaca patient's mucous membrane.In these compounds some do not induced any effect, especially compound N-acetyl group-L-glutamine.This compound also has tangible trophism for the celiaca patient's who does not receive treatment living tissue.This nutritive validity be usually expressed as make mucous membrane especially the epithelial cell mucous membrane improve, it obtains obviously to improve for other sample.

This research clearly illustrates that most of test compounds have brought certain variation for the celiaca patient's who does not receive treatment mucous membrane.The most interesting, also be that the most beat all N-of being acetyl group-L-glutamine has nutritional activities.In first embodiment of this product, even with respect to independent medium, it makes the overall condition of mucous membrane make moderate progress.

The front is described the various specific embodiments that fall within the scope of the invention that limits according to claims.These embodiments also do not mean that and limit the scope of the invention to disclosed concrete form.The present invention should cover all various modifications that fall within the spirit and scope of the invention and substitute.

Claims (47)

1. method that provides glutamine to replenish to the people, it comprises acceptable salt in the N-acetyl group L-glutamine of oral effective dose or its nutrition.

2. take acceptable salt in N-acetyl group-L-glutamine of at least 0.7 mM/kg/ days or its nutrition according to the process of claim 1 wherein to described people.

3. take acceptable salt in N-acetyl group-L-glutamine of at least 1.0 moles/kg/ days or its nutrition according to the process of claim 1 wherein to described people.

4. take acceptable salt in N-acetyl group-L-glutamine of at least 1.5 mMs/kg/ days or its nutrition according to the process of claim 1 wherein to described people.

5. according to the method for claim 1, acceptable salt is selected from the wherein said nutrition: lithium, sodium, potassium, calcium, magnesium, and aluminium, ammonium, tetramethyl-ammonium, tetraethyl ammonium, methylamine, diethylamine, trimethylamine, triethylamine, diethylamine, ethamine, tri-n-butylamine, pyridine, N, accelerine, the N-methyl piperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzyl amine, N, N-dibenzyl phenyl ethylamine, 1-Chinese ephedra amine (1-ephenamine), N, N '-dibenzyl-ethylenediamin, ethylenediamine, monoethanolamine, diethanol amine, piperidines, piperazine, and their mixture.

6. be selected from following illness according to the process of claim 1 wherein that described people suffers from: gastrointestinal surgery, intestines and stomach resection, small intestine transplantation, wound after surgery, hunger, serious disease and damage, multiple wound, short bowel syndrome, burn, bone-marrow transplantation, AIDS, portacaval mucositis, cancer, celiaca, Crohn's disease, NEC, internal organs precocity, infection at random, the internal organs relevant with particular treatment are degenerated, oral feed is limited and their combination.

7. aqueous solution, it contains:

A) sodium of every liter of 30mEq to 95mEq;

B) potassium of every liter of 10mEq to 30mEq;

C) citrate of every liter of 10mEq to 40mEq;

D) be less than a kind of carbohydrate of 3.0 w/w %; And

E) the N-acetyl group-L-glutamine of every liter of solution at least 5.0 mMs or the equivalent salt in its nutrition.

8. according to the aqueous solution of claim 7, every liter of solution contains the N-acetyl group-L-glutamine of 20 mM to 300 mMs or the equivalent salt in its nutrition.

9. according to the aqueous solution of claim 7, every liter of solution contains the N-acetyl group-L-glutamine of 25 mM to 200 mMs or the equivalent salt in its nutrition.

10. according to the aqueous solution of claim 7, acceptable salt is selected from the wherein said nutrition: lithium, sodium, potassium, calcium, magnesium, and aluminium, ammonium, tetramethyl-ammonium, tetraethyl ammonium, methylamine, diethylamine, trimethylamine, triethylamine, diethylamine, ethamine, tri-n-butylamine, pyridine, N, accelerine, the N-methyl piperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzyl amine, N, N-dibenzyl phenyl amine, 1-Chinese ephedra amine (l-ephenamine), N, N ＇-dibenzyl-ethylenediamin, ethylenediamine, monoethanolamine, diethanol amine, piperidines, piperazine, and their mixture.

11. according to the aqueous solution of claim 7, wherein said aqueous solution further contains chloride.

12. according to the aqueous solution of claim 7, wherein said carbohydrate is the mixture of dextrose and glucose.

13. according to the aqueous solution of claim 7, wherein said carbohydrate exists with the amount that is less than 3.0 w/w %.

14. according to the aqueous solution of claim 7, wherein said sodium exists with the amount of 30mEq/L to 95mEq/L.

15. according to the aqueous solution of claim 7, wherein said sodium is selected from sodium chloride, natrium citricum, sodium acid carbonate, sodium carbonate, NaOH and composition thereof.

16. according to the aqueous solution of claim 7, wherein said potassium exists with the amount of 10mEq/L to 30mEq/L.

17. according to the aqueous solution of claim 7, wherein said potassium is selected from potassium citrate, potassium chloride, saleratus, potash, potassium hydroxide and composition thereof.

18. according to the aqueous solution of claim 11, wherein said chloride exists with the amount of 30mEq/L to 80mEq/L.

19. according to the aqueous solution of claim 11, wherein said chloride is selected from potassium chloride, sodium chloride and zinc chloride.

20. according to the aqueous solution of claim 7, wherein said citrate exists with the amount of 20mEq/L to 40mEq/L.

21. according to the aqueous solution of claim 7, wherein said citrate is selected from potassium citrate, natrium citricum and citric acid.

22. according to the aqueous solution of claim 7, it further contains at least a flavor enhancement.

23. according to the aqueous solution of claim 7, it further contains at least a artificial sweetener.

24. aqueous solution according to claim 7, it further contains at least a agar that is selected from, alginic acid and salt, acacin, gum arabic, tower is breathed out glue, cellulose derivative, curdlan, fermentation natural gum, furcellaran glue, gelatin, gellan gum, Indian gum, guar gum, the A Outa carrageenan, Ireland liver moss, card handkerchief carrageenan, konjaku flour, karaya, the dish carrageenan, pine glue/arabogalactan, locust bean gum, pectin, tamarind gum, tower draws bean gum, bassora gum, natural and starch modification, the gelling agent of xanthans, its content is enough to support the three-dimensional structure of self.

25. according to the aqueous solution of claim 7, it further contains ground rice.

26. according to the aqueous solution of claim 7, it further contains stodgy oligosaccharides.

27. a liquid nutritional formula food, it contains:

A) protein component, it accounts for the 8-35% of the total heat content of described liquid nutritional formula food;

B) carbohydrate ingredient, it accounts for the 36-76% of the total heat content of described liquid nutritional formula food;

C) lipid composition, it accounts for the 6-51% of the total heat content of described liquid nutritional formula food; And

Account for acceptable salt form in the N-acetyl group-L-glutamine of 1-23% of heat of protein component or its nutrition.

28. adult's liquid nutritional formula food, it contains:

A) protein component, it accounts for the 14-35% of the total heat content of described liquid nutritional formula food;

B) carbohydrate ingredient, it accounts for the 36-76% of the total heat content of described liquid nutritional formula food;

C) lipid composition, it accounts for the 6-51% of the total heat content of described liquid nutritional formula food; And

Acceptable salt/1000 kilocalorie nutrient formulation food in the N-acetyl group L-glutamine of at least 35 mMs or its nutrition.

29. as the nutrient formulation food of definition in the claim 28, wherein said formula food contains acceptable salt/1000 kilocalorie nutrient formulation food in 35 mM to 160 mM N-acetyl group-L-glutamine or its nutrition.

30. a non-adult patient uses the liquid nutritional formula food, it contains:

A) protein component, it contains the 8-25% of the total heat content of described liquid nutritional formula food;

B) carbohydrate ingredient, it accounts for the 39-44% of the total heat content of described liquid nutritional formula food;

C) lipid composition, it accounts for the 45-51% of the total heat content of described liquid nutritional formula food; And

Acceptable salt/1000 kilocalorie nutrient formulation food at least 5.0 mM N-acetyl group L-glutamine or its nutrition.

31. as the nutrient formulation food of definition in the claim 30, wherein said preparation contains acceptable salt/1000 kilocalorie nutrient formulation food in 5.0 mM to 32 mM N-acetyl group-L-glutamine or its nutrition.

32. liquid nutritional formula food according to claim 27, acceptable salt is selected from the wherein said nutrition: lithium, sodium, potassium, calcium, magnesium, and aluminium, ammonium, tetramethyl-ammonium, tetraethyl ammonium, methylamine, diethylamine, trimethylamine, triethylamine, diethylamine, ethamine, tri-n-butylamine, pyridine, N, accelerine, the N-methyl piperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzyl amine, N, N-dibenzyl phenyl amine, 1-Chinese ephedra amine (1-ephenamine), N, N '-dibenzyl-ethylenediamin, ethylenediamine, monoethanolamine, diethanol amine, piperidines, piperazine, and their mixture.

33. according to the liquid nutritional formula food of claim 27, it contains and is less than 1.0g pyroglutamic acid/1500 kilocalorie formula food.

34. according to the liquid nutritional formula food of claim 27, wherein said formula food is adult's formula food, and protein component accounts for the 14-35% of the total heat content of described liquid nutritional formula food; Carbohydrate ingredient accounts for the 36-76% of the total heat content of described liquid nutritional formula food; Lipid composition accounts for the 6-41% of the total heat content of described liquid nutritional formula food; Acceptable salt accounts for the 1-25% of protein heat in this N-acetyl group-L-glutamine or its nutrition.

35. according to the liquid nutritional formula food of claim 27, wherein said formula food is used for the minor, and protein component accounts for the 8-25% of the total heat content of described liquid nutritional formula food; Carbohydrate ingredient accounts for the 39-44% of the total heat content of described liquid nutritional formula food; Lipid composition accounts for the 45-51% of the total heat content of described liquid nutritional formula food; Acceptable salt accounts for the 1-12% of protein heat in this N-acetyl group-L-glutamine or its nutrition.

36. according to the liquid nutritional formula food of claim 27, wherein said liquid nutritional formula food is by oral administration.

37. according to the liquid nutritional formula food of claim 27, wherein said liquid nutritional formula food is by intestinal canal administration.

38. according to the liquid nutritional formula food of claim 27, it further contains vitamin and the inorganic elements that is selected from calcium, phosphorus, sodium, chlorine, magnesium, manganese, iron, copper, zinc, selenium, iodine, chromium, molybdenum, inositol, carnitine, taurine, vitamin A, C, D, E, K and B complex and their mixture.

39. according to the liquid nutritional formula food of claim 27, wherein said lipid composition is selected from lipid source, structured lipid and their mixture of coconut oil, soy sauce, corn oil, olive oil, safflower oil, rich oil safflower oil, MCT oil (median chain triglyceride oil), sunflower oil, rich oil sunflower oil, palm oil, palm olein, Tower rape oil, fish oil, palm-kernel oil, pilchardine, soya-bean oil, cottonseed oil, lecithin, arachidonic acid and DHA.

40. according to the liquid nutritional formula food of claim 27, wherein said protein component contain be selected from soya-bean oil base albumen, the whole protein of the basic albumen of suckling, casein, lactalbumin, rice gluten, beef collagen, pea protein, potato protein and their mixture.

41. according to the liquid nutritional formula food of claim 27, wherein said protein component contains the combination that is selected from soybean protein hydrolysate, caseic hydrolysate, lactalbumin hydrolysate, rice gluten hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg plain boiled water hydrolysis products, glutin hydrolysate, animal/vegetable protein hydrolysate and the aminosal of their mixture.

42. according to the liquid nutritional formula food of claim 27, wherein said protein component contains the free amino acid that is selected from tryptophan, tyrosine, serine, methionine, arginine, leucine, valine, lysine, phenylalanine, isoleucine, threonine, histidine, carnitine, taurine, glycine, alanine, serine cystine, desiodothyroxine asparatate, asparagine glutamatic acid glutamine oxylysine, proline, hydroxy-proline and their mixture.

43. according to the liquid nutritional formula food of claim 27, wherein said carbohydrate ingredient is selected from after the hydrolysis that derives from corn, cassava, rice or potato of wax shape or non-waxy form, complete, natural and starch chemical modification; Carbohydrate is glucose, lactose, sucrose, maltose, the corn syrup that is rich in fructose, corn syrup solids and their mixture for example.

44. one kind by using the method for mucosal inflammation in the intestines that aqueous solution according to claim 7 alleviates described patient to the patient who suffers from celiaca.

45. one kind by using the method for mucosal inflammation in the intestines that liquid nutritional formula food according to claim 27 alleviates described patient to the patient who suffers from celiaca.

46. one kind by using the method for mucosal inflammation in the intestines that liquid nutritional formula food according to claim 28 alleviates described patient to the patient who suffers from celiaca.

47. one kind by using the method for mucosal inflammation in the intestines that liquid nutritional formula food according to claim 30 alleviates described patient to the patient who suffers from celiaca.

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